ABSTRACTS
ESHG - Posters: P 5 Cytogenetics
P0315
Molecular Cytogenetic Study of Cases With Short Stature
N. A. Meguid 1, A. Mahmoud 1, A. Khalil 1,
M. Amer 2, A. Dardir 1, H. Atteya 1;
1National Research Centre, Human Genetics Department, Cairo, EGYPT, 2Faculty
of Science, Cairo University, Cairo, EGYPT.
Among 150 cases with short stature and delayed puberty referred to the
outpatient clinic of Human Genetics Department, National Research Centre,
during two years period, we selected 25 Egyptian girls with phenotype far more
severely affected than expected in Turner syndrome.
Initially, the karyotype in some cases was thought to be 45,X with a
chromosome marker. However, re-examination with FISH probes showed 14 subjects
with 45,X/46,X r(X) karyotype. Seven subjects with 46,XisoX(q) karyotype; 2
subjects with 45,X karyotype; and 2 subjects with 46,XX karyotype. Tiny ring X
was present in 5 cases, and inactivation was proved by molecular cytogenetic
techniques. The clinical picture of cases with ring (X) chromosome includes
mental retardation in 8 patients (the non-verbal I.Q. tends to be lower than
the verbal I.Q.) and learning disability in 3 cases. Dysmorphic features are
found in 3 cases and limb anomalies in 5 cases.
Our results showed that the severe phenotype was present in the cases with
tiny ring (X) chromosomes suggesting mutation in the X chromosome inactivation
pathway and that the inability of these rings to inactivate was responsible
for the severe phenotypes. We confirm the advent of in situ hybridization with
chromosome specific DNA probes in identifying small structurally abnormal
chromosomes.
P0316
Pure partial trisomy 4q caused by a tandem direct duplication
dup(4)(q27qter)
H. Elghezal 1, H. Sennana Sendi 1, M. Griba 1,
S. Ibala Romdhan 1, K. Monastiri 2, A. Saad 1;
1Service de Cytogénétique. CHU Farhat Hached, Sousse, TUNISIA, 2Service
de Pediatrie. Hopital Fatouma Bourguiba, Monastir, TUNISIA.
We report a 3 year old boy with a de novo direct tandem duplication
dup(4)(q27qter) confirmed by FISH using whole chromosome 4 painting probe. Our
patient showed a particular dysmorphic phenotype, an epilepsy, a
sensorioneural deafness and a moderate mental retardation. Magnetic resonance
imagine of the brain showed a dilated cerebral ventricles. No other visceral
malformation was detected.
Few cases of pure partial trisomy 4q was described. According to this new case
and previously published data, we support that the renal malformation obseved
in meny cases of 4q trisomy is related to a region proximal to 4q27,
neurosensorial deafness is related to 4q31q33 region and suggest that trisomy
of the 4q33q35 region is associated with minor clinical effect.
P0317
Effect of hydroxyurea and catalase on chromosomal damage and G2 arrest in
Fanconi Anemia lymphoblasts from groups FA-A, FA-B, FA-C, FA-D1 and FA-E.
A. Carnevale, B. Molina, R. Ortiz, L. Gomez, L. Legorreta, M. L.
Velazco, S. Frías;
Instituto Nacional de Pediatría, DF, MEXICO.
Fanconi Anemia(FA) is heterogeneous, and 8 complementation groups have been
discovered: FA-A,B,C,D1,D2,E,F,G. We showed that in lymphoblasts FA-A and B
damaged with mitomycin C(MMC), Hydroxyurea(HU), added in G2, produces
potentiation with a striking increase of chromosomal aberrations(CA). Here, we
investigated whether this potentiation is due to dNTP pool depletion or free
radicals (FR) produced by HU. Normal lymphoblastoid cell cultures and from
FA-A,B,C,D1 and E were grown. Half cultures were treated with 10un/ml MMC for
24 hours; during G2 phase all cultures were treated with HU 2mM, and half were
treated with Catalase 0.6 mg/ml which eliminates FR. Cells were processed to
analyze: a) CA in 50 cells per treatment per cell line; b) Proportion of cells
in G2/M by flow cytometry. Cultures were done in triplicate, data were
compared by varianza and Tukey or Tamhane test and Z of proportions. Increased
CA were found in all cell lines, but MMC-HU potentiation was observed only in
FA-A (300%) and FA-B (100%). Catalase added to FA-A and B diminished CA
frequency to 5-10%; however, CA frequency never returned to that MMC-induced.
These data suggest that MMC-HU potentiation in FA-A and B is through
alteration of dNTP pool. The proportion of G2 cells increased in MMC-treated
cultures and decreased with HU in all FA cell lines, indicating that the
restriction point of G2 is normal, and HU reduces the proportion of G2 cells,
possibly because cells fail to arrive to G2. All data suggest an abnormal
postreplicative repair in FA-A and B.
P0318
FISH at complicated cytogenetic cases
M. Nemeckova;
GENNET CZ s.r.o., Prague, CZECH REPUBLIC.
Three cases of complicated chromosomal rearrangements elucidated by FISH are
presented:
-Familial isodicentric supernumerary chromosome 15 and familial inverted
chromosome 18 at families with reproductive failure.
-Pseudodicentric chromosome 18 at fetus with Edwards syndrome.
Role of FISH as the adjunct of classical pre- and postnatal cytogenetics in
routine practice is discussed.
P0319
Blepharophimosis Is The Most Constant Clinical Feature In 14qter
Microdeletion Syndrome
O. Rittinger 1, G. Kronberger 1, C. Fauth 2;
1Landeskliniken Salzburg, Salzburg, AUSTRIA, 2Institut
für Humangenetik, TU Muenchen, Muenchen, GERMANY.
Mental retardation is a distressing disorder affecting approximately 3% of the
population. Among these, cytogenetic anomalies explain about 30% of patients
with more severe mental retardation. Using conventional methods, detection of
subtle structural aberrations is clearly limited to 6-10Mb. According to Flint
et al.(Nat Genet,1995) a substantial percentage of mental retardation is
caused by subtelomeric abnormalities. A couple of methods is now available to
establish diagnosis of submicoscopic deletions or more complex rearrangements.
We observed a young lady in which the dysmorphic phenotype in spite of a
normal karyotype remained highly suggestive for a chromosomal aberration: the
most impressive feature was the orbital region with blepharophimosis,
epicanthal folds, puffy eyelids, a long shaped face with a pointed chin and
small ears. The hands were small with tapering fingers, body length was at the
90th percentile range, no microcephaly was observed. Hypertrichosis was seen
only in the first few years. Mental disability was in the mild range, with
surprisingly good speech development. Finally, diagnosis was done at the age
of 12 years using a commercial multitelomere FISH-kit (Cytocell). It revealed
a subtelomeric de novo deletion on chromosome 14q. This result in the patient
and the normal parental karyotype were confirmed by a new strategy recently
described by Fauth et al. (Hum Genet,2001). Considering the results of other
groups we suggest blepharophimosis to be the most constant and impressive
clinical feature of this rare condition and we recommend therefore to rule out
14qter deletion in all patients with equivocal blepharophimosis syndromes.
P0320
FISH assessment of sperm aneuploidy frequencies in ICSI patients with severe
oligoasthenotetraozoospermia (OAT)
N. Ditzel 1, L. Jelinkova 1, B. Gläser 2,
E. Strehler 1, N. Reeka 1, G. Speit 2, K. Sterzik 1;
1Christian-Lauritzen-Institut, Ulm, GERMANY, 2University
of Ulm, Department of Human Genetics, Ulm, GERMANY.
The aim of this study was to examine chromosomes in sperm by
fluorescence-in-situ-hybridisation (FISH) with considerable differences in
disomy frequency for the chromosomes 13, 16 and 21.
On date 4 patients with oligoasthenotetraozoospermia were involved in our
study. FISH procedure was made by standard protocol, using probes for
chromosome 13 and 21 (locus-specific probes) and a probe for chromosome 16
(centromeric satellite probe). For each patient 2000 sperms were analysed.
In the patients, the incidence of chromosome 13 and 16 disomy ranged from
0.08% to 0.21%, and from 0.05% to 0.13% respectively. In the case of
chromosome 21 we observed substantially higher rate of disomy in one of our 4
patients. In three patients, the 21 disomy ranged from 0.1% to 0.29%, while in
the fourth patient, the 21 aneuploidy reached 4.1%.
Our results support the hypothesis of positive correlation between OAT and
higher disomy rates. However, this correlation is not linear and absolute,
among patients with severe OAT we can observe also high proportion of men with
normal range of disomy. Only one of our patients had a substantially higher
rate of 21 disomy than men with normal sperm quality. The other three patients
had normal disomy rates of chromosomes 13, 16 and 21 even suffering from OAT.
According to our results and also results from literature, we recommend a more
intensive prenatal control of pregnancies, originating from the ICSI method.
The study will be continued and we will include more data in the poster.
P0321
Molecular cytogenetic analysis of a constitutional de novo interstitial
deletion of chromosome 12 (del(12)(p12.3p12.1)) in a boy with developmental
delay and minor anomalies
B. Gläser 1, E. Rossier 1, G. Barbi 1,
L. Delle Chiaie 2, C. Blank 3, W. Vogel 1, H.
Kehrer-Sawatzki 1;
1University of Ulm, Department of Human Genetics, Ulm, GERMANY, 2University
of Ulm, Department of Gynecology and Obstetrics, Ulm, GERMANY, 3University
of Ulm, Department of Pediatrics, Ulm, GERMANY.
We describe the case of a 6-month-old boy with psychomotor retardation,
craniofacial dysmorphism, cleft lip and palate, as well as hearing and visual
impairment. Analysis of G-banded metaphase chromosomes from the propositus
revealed the presence of an interstitial deletion of the short arm of
chromosome 12 (del(12)(p12.3p12.1)). To define the deletion extent on the
molecular cytogenetic level, we hybridized BAC clones mapped to band 12p11,
12p12 and 12p13, respectively, to metaphase chromosomes of the proband. Our
FISH results demonstrate that the deletion on chromosome 12 spans the region
flanked by BACs RP11-174G6 (12p12.3) and RP11-325D10 (12p12.1). As deduced
from the map position of these BAC clones in the Ensembl contigs, the deletion
encompasses about 12.5 Mb. According to the Ensembl database, the deletion is
flanked by markers D12S1832 and G62375.
Up to now seven patients with de novo interstitial deletions involving bands
12p12 have been described in the literature. Some of this patients had a
number of clinical symptoms in common with our patient, like psychomotor
retardation, microcephaly and dysmorphic facial features. Diverse
cardiovascular anomalies and eye abnormalities were also observed in several
cases, but all these features are equally found associated with other
chromosomal aberrations. It is currently unknown, if among rare structural
chromosome aberrations like these interstitial deletions within 12p breakpoint
cluster regions can also be found as is the case in the more frequent category
of microdeletions responsible for microdeletion syndroms like for example
DiGeorge and Prader Willi syndrome.
P0322
Tandem duplication of the NF1 gene detected by high-resolution FISH in
17q11.2 region
P. Riva, C. Gervasini, A. Bentivegna, M. Venturin, L. Corrado, L.
Larizza;
Department of Biology and Genetics, Medical Faculty - University of Milan,
Milan, ITALY.
The gene responsible for Neurofibromatosis type 1 (NF1), which has been mapped
to 17q11.2, has one of the highest observed mutation rates. We have previously
shown by means of high resolution FISH that a number of the loci flanking the
NF1 gene are duplicated, in line with the previous identification of NF1 REPs
by other groups. We here report on a direct tandem duplication of the NF1 gene
identified in 17q11.2 by means of high-resolution FISH. FISH on stretched
chromosomes using locus-specific probes revealed the duplication of most NF1
gene from the promoter to 3’UTR, but with at least the lack of exon 22.
Fiber FISH using PACs/BACs specific for the NF1 gene, including the 5’UTR
and 3’UTR and flanking regions, visualized the direct tandem duplication of
NF1 and showed the similar, but not identical genomic organization of the
duplicon copies. A duplicated NF1 gene was also found at orthologous
chromosomes loci in chimpanzee and gorilla, suggesting that the duplication
occurred before the divergence of great apes. We hypothesize that the NF1
intrachromosomal duplication may contribute to the high whole gene mutation
rate by gene conversion, an unlikely mechanism in the case of NF1 pseudogenes
containing only limited portions of the NF1 gene. The functional activity of
the NF1 copy remains to be investigated. Detection of the NF1 duplicon by
high-resolution FISH may pave the way to filling up the gaps in the human
genomic sequence of the pericentromeric 17q11.2 region.
P0323
A case of Wolf-Hirschhorn syndrome diagnosed by FISH
E. Braha 1, O. Bartsch 2, M. Volosciuc 1,
C. Gavrilovici 1, M. Covic 1;
1University of Medicine and Pharmacy 'Gr. T. Popa', Iasi, ROMANIA, 2Institute
for Clinical Genetics, Technical University, Dresden, GERMANY.
The Wolf-Hirschhorn syndrome (WHS) is caused by partial deletion of chromosome
4p. In a subset of cases the deletion may be so small that it may escape
detection by standard chromosome analysis. The syndrome is characterized by
severe growth and psychomotor retardation, microcephaly, 'Greek helmet' facies
and closure defects .
We report on a 4-month-old girl whose clinical signs strongly suggested WHS.
She is the product of the third pregnancy of young healthy non-consanguineous
parents. The parents had had a miscarriage at 4.5 months gestation and a child
who had died one month old.with cleft lip and palate and other congenital
malformations.
Clinical findings at the age of four months included severe postnatal growth
retardation, microcephaly, dysmorphic facies (hypertelorism, iris coloboma,
cleft palate, micrognathia), heart defect , right renal agenesis and
functional neurological abnormalities .
Chromosomal analysis by G-banding at 450 - 550 bands resolution indicated a
normal 46,XX karyotype. Because clinical signs were very suggestive of WHS,
FISH studies were performed using two DNA probes for chromosome 4p16, one BAC
19G2 and one from Vysis Inc. The patient demonstrated a typical 4p16 deletion
spanning both probes.
To our knowledge, this may represent the first case of a FISH diagnosis
established in North-Eastern Romania. The diagnosis was possible by
collaboration with the Molecular Cytogenetic Unit at the Dresden University.
On the basis of the reproductive history of the parents, it makes sense to
expect a cytogenetically cryptic balanced chromosomal rearrangement in this
family. Parental FISH testing is presently in working.
P0324
Ring autosomes database of National Registry of Chromosomal Abnormalities:
the study of mitotic instability of constitutional ring chromosomes
A. D. Polityko, N. Rumyantseva, T. Egorova, I. Pisarik, O. Khurs;
Institute for Hereditary Diseases, Minsk, BELARUS.
Twelve patients with ring chromosomes 4, 13, 15, 18, 21 and 22 in
constitutional karyotype were registered in Belarus National Registry of
Chromosomal Abnormalities among the individuals who were cytogenetically
studied in Republic Genetic Center during 1983-2001 years. In theory,
phenotype abnormalities of ring chromosome carriers are associated with loss
of chromosomal material. However, in part of cases telomere pairing without
deletion of important genetic material takes place in ring formation, and the
mitotic ring instability can cause the phenotypic anomalies, independently
what chromosome is involved. The phenotype of such a “general ring syndrome”
consists of growth failure without malformations, few or no minor anomalies,
mild-moderate mental retardation.
We studied the mitotic behavior of various size ring chromosomes in non-mosaic
karyotypes
- to examine the supposition that the size of ring influences on it stability
and
- to evaluate the correlation between ring size and instability of somatic
cells.
Cytogenetic analysis showed dicentric rings of twice the size, smaller rings,
three- and tetracentric rings, double size and single size rings
simultaneously, two single size rings, poliploid (three-, tetra- and
pentaploid) metaphases, metaphases with 45 chromosomes without ring and other
abnormalities. Mitotic instability of ring chromosomes in vitro as well as the
lability of rings in vivo may lead to high cellular death rate and interfere
with normal development. Our data further support the suggestion that larger
ring configurations are more unstable than smaller ring chromosomes. More
essential contribution of larger rings instability to formation of «general
ring syndrome» is discussed.
P0325
Alternative mechanisms in the pathogenesis of true hermaphroditism
S. H. Kofman-Alfaro 1 ,2, G. Queipo 3,
R. Peña 4, K. Nieto 5, L. M. Dorantes 4, L.
Eraña 4, A. L. Jimenez 3, D. Soderlund 6, E.
Lieberman 7, A. Radillo 3, J. C. Zenteno 3;
1Hospital General de Mexico, Mexico DF, MEXICO, 2Universidad
Nacional Autonoma de Mexico, Mexico DF, MEXICO, 3Hospital General de
Mexico, Mexico, MEXICO, 4HIM"Federico Gómez", Mexico,
MEXICO, 5Omnilab, Mexico, MEXICO, 6UIM Biologia del
desarrollo CMN-SXXI, Mexico, MEXICO, 7Departamento de Investigacion
en Genetica Humana, Mexico, MEXICO.
True hermaphroditism (TH) shows genetic heterogeneity and several genetic
abnormalities have been associated with the dual gonadal development: point
mutations in the SRY gene in 46, XY patients, trisomy for chromosome 22 and
hidden mosaicism for a Y chromosome or Y sequences in 46, XX patients. We
performed cytogenetic and molecular analyses of Y sequences in DNA from blood
leukocytes and gonadal tissue in 12 Mexican TH. The karyotypes did not
revealed chromosome abnormalities. Nine cases showed a 46, XX karyotype and
eight of them were SRY negative in leukocytic and gonadal DNA. In case 1, also
a 46, XX TH, PCR and FISH analysis performed in an ovotestis homogenate and
ovarian region revealed the presence of SRY, indicating a gonadal mosaicism.
Other Y sequences (PABY, ZFY, Ycen, Yqh) tested in all 46, XX TH including
patient 1, were negative in both tissues. In-patient 4, PCR revealed the
presence of Ycen and Yqh and absence of Yp sequences in leukocytic, ovotestis
and fibroblastic DNA. FISH analysis confirmed the presence of a Y mosaicism
and each Y positive cell showed two X centromeres, demonstrating a second cell
line with 47, XX del (Y) (p?). In 3 patients with a second cell line with a Y
chromosome (patients 10-12), all Y sequences tested were amplified. Sequence
analyses in the 4 SRY positive cases (patients 1, 10-12) was normal, excluding
SRY mutations. We confirm that the presence of hidden Y mosaicism must be
discarded in the molecular study of TH.
P0326
Mosaic case of Patau Syndrome with karyotype
46,XXt(13;13)/45,XXt(13;15)/46,XX
L. Kartashova, N. Nikitina, E. Nikolaeva, V. Sorokina, O. Bushueva, L.
Ponomareva;
Medico-Genetic Center, Ekaterinburg, RUSSIAN FEDERATION.
The index patient – a 8,5-year-old girl - was the second child in the family
(the first child was a healthy boy). She was born when her mother was 21 years
old at 36 weeks of gestation.Her weight was 3250g, length – 57cm. Her early
milestones of development were retarded.The girl sat at 10 months, walked
without any aid by 18 months and said her first words at the age of 2,5 years.
At the age of 8,5 years her height was 138 cm, weight – 25 kg, cranial
circumference – 52,5cm.
Dysmorphic features included: hypotelorism, enophtalmos, progenia, pro-minent
sharpended nose, deformed ears. Speech and mental development were retarded.
Her behavior was characterized by inappropriate smiles.
The proband’s karyotype was: 46,XX t(13;13)/45,XX t(13;15)/46,XX.
Her mother had a normal 46,XX karyotype. Her father’s karyotype was unknown.
P0327
A natural equivalent to human satellite DNA-based artificial chromosome
persists over 140 years, in a three-generation family
G. Hadlaczky 1 ,2, G. Bujdosó 3 ,4,
É. Koltai 5, G. Schwanitz 6, E. Csonka 7;
1Biological Research Center, Hungarian Academy of Sciences, Szeged,
HUNGARY, 2Chromos Molecular Systems Inc.,, Burnaby, BC, CANADA, 3Semmelweis
Medical School, Department of Forensic Medicine,, Budapest, HUNGARY, 4Research
Unit sponsored by the Hungarian Academy of Sciences, Budapest, HUNGARY, 5Semmelweis
Medical School, Department of Forensic Medicine, Budapest, HUNGARY, 6Institute
of Human Genetics, University of Bonn, Bonn, GERMANY, 7Institute of
Genetics,Biological Research Center, Hungarian Academy of Sciences, Szeged,
HUNGARY.
Human artificial chromosomes represent an attractive tool for gene therapy.
Recently, we demonstrated that human satellite DNA-based artificial
chromosomes (hSATACs) could be generated via amplification-dependent de novo
chromosome formation induced by integration of exogenous DNA sequences into
the centromeric/rDNA regions of human acrocentric chromosomes. Human SATACs
have been successfully constructed in different cell types from exogenous DNA,
and “neutral” endogenous sequences of the short arm of chromosome #15.
These artificially generated accessory chromosomes carry predictable DNA
sequences and they contain defined genetic information. We suggested that
hSATACs composed of rDNA and non-coding satellite DNA sequences that lack
transcription units for undesired and unknown genes can be regarded as
genetically “neutral” and hence are prototypes of safe or low risk
artificial chromosome vectors.
Here, we report the existence and characterization of a natural equivalent to
human satellite DNA-based artificial chromosomes. This small stable accessory
chromosome derived from the centromeric/short arm region of human chr #15 is
carried by healthy members of a family, and remained apparently unchanged at
least in three generations. The stable persistence of this natural accessory
chromosome supports the assumption that hSATACs derived from the
centromeric/short arm region of chr #15 may be suitable long-term gene
expression platforms without detrimental consequences.
P0328
Paternal origin of der(X)t(X;6) in a girl with trisomy 6p
I. Petkovic 1, I. Barisic 1, R. Bago 2,
S. Hecimovic 2;
1University Children's Hospital Zagreb, Zagreb, CROATIA, 2Ruder
Boskovic Institute, Zagreb, CROATIA.
Here we report on the girl with trisomy 6p due to unbalanced t(X;6) and
unusual mosaicism involving abnormalities of chromosomes 6 and 10 in the
mother. Our patient is 6-year-old girl with moderate mental retardation, short
stature, failure to thrive and mild facial dysmorphism. The chromosome
analysis of the proband showed a 46,X,der(X)t(X;6)(q22; p11) karyotype. The
derived X was late replicating in all investigated cells with variable
spreading of X chromosome inactivation onto translocated 6p. The normal
karyotype was observed in the father, while the mother presented 46,XX/ 46,XX,
der(10)t(6;10)(p11;p11). The mother is a mosaic with unbalanced t(6;10) in
4,7% of cells. To the best of our knowledge, this unusual mosaicism has not
been reported yet. We suggest that chromosome constitution in the mother is
due to postzygotic recombination involving chromosome 6 and 10 at S/G2 phase
of the cell cycle. In order to understand the mechanism of formation of der(X)
in the proband we performed DNA polymorphism analysis. The molecular analysis
revealed that chromosomes X and 6 involved in the rearrangement are of
paternal origin. This work was supported by grant from Ministry of Science and
Technology Republic of Croatia ( project no. TP-01/072-01).
P0329
Cytogenetic, FISH and molecular analysis of the stable dicentric X
chromosome
I. Petkovic 1, I. Barisic 1, R. Bago 2, I.
Sansovic 1;
1University Children's Hospital Zagreb, Zagreb, CROATIA, 2Ruder
Boskovic Institute, Zagreb, CROATIA.
We present the results of cytogenetic and molecular study in a 5-years old
girl with mild dismorphism, grow retardation and structural rearrangement of X
chromosome. Both parents presented normal karyotype. Chromosome analysis
revealed one normal and one aberrant X chromosome. The rearranged X is a large
submetacentric with one primary constriction and two blocks of C-staining. It
is composed entirely of the X chromosome material. Two mosaic cell lines were
detected by interphase FISH with X chromosome specific centromeric probe.
Three signals were observed in 84.5% and one signal in 15.5% of interphase
cells. Replication analysis showed the normal X always early replicating while
the dicentric was late replicating in all investigated cells. Molecular
analysis using polymorphic DNA markers revealed that the dicentric is of
paternal origin. Based on this study the karyotype of the patient is
45,X/46,X,psu idic(X)(q22.3). We suggest that dicentric is the results of
postzygotic isolocal break in both chromatids of the paternal X chromosome,
subsequent rejoining of broken ends, followed by inactivation of one
centromere. These results point out to the importance of combined cytogenetic,
FISH and molecular study in order to elucidate the mechanisms of formation of
chromosomal abnormalities. This work was supported by grant from Ministry of
Science and Technology Republic of Croatia ( project no. TP-01/072-01).
P0330
Terminal deletion 4q in a patient with complex chromosomal rearrangement and
characteristic phenotype
M. Czako, E. Morava, B. Cser, J. Karteszi, G. Kosztolanyi;
Dep. Med. Genetics and Child Development, Pécs, HUNGARY.
We report on a 2 year old patient, who was born at term with low birth weight,
brachycephaly, low set ears, full periorbital region, curved eyebrows,
strabism, depressed nasal bridge, upturned nose, high arched palate and
micrognathia. He had overlapping fingers as observed in trisomy 18, but no
cardiac anomalies. He had severe postnatal growth retardation and profoundly
delayed psychomotor development. Routine cytogenetic analysis suggested a
balanced translocation between chromosomes 6;14 and a possible deletion of the
short arm of chromosome 4. FISH studies were performed showing a complex
rearrangement between chromosomes 4, 6 and 14. Distal segment of chromosome 6
(breakpoint q16) was translocated to the long arm of chromosome 4 (breakpoint
q26). A part of the long arm of chromosome 4, distal to the breakpoint, was
translocated to the short arm of chromosome 14. Studies for the
Wolf-Hirschhorn locus detected two signals 46,XY,t(4;6),t(4;14),4qter-. Since
no signal was detected by FISH with 4q terminal specific probe on the
derivative 14, we assume a fourth break at the distal part of the long arm of
chromosome 4 leading to a terminal deletion, and suggest that the long arm
segment of chromosome 4 was translocated by the terminal broken end. Thus,
this complex rearrangement resulted in a 4q terminal deletion. The
phenotypical features were comparable to that of the previously described
patients with ࡄq- syndrome’. Our observation contributes to the
phenotypic spectrum of the rare ࡄq- syndrome’.
P0331
Monozygotic twins with a trisomy 11p due to an unbalanced
translocation.
D. Marcus-Soekarman 1, G. Hamers 1, S. Velzeboer 2,
J. Nijhuis 3, C. de Die-Smulders 1, C. Schrander-Stumpel 1,
J. Engelen 1;
1Department of Genetics, University Hospital, Maastricht,
NETHERLANDS, 2Department of Pediatrics, University Hospital,
Maastricht, NETHERLANDS, 3Department of Obstetrics, University
Hospital, Maastricht, NETHERLANDS.
A pregnancy, complicated by a twin transfusion syndrome (TTS), resulted in the
birth of female twins by cesarean section in the 30th gestational week. Since
both twins showed dysmorphic features, in one more pronounced than the other,
karyotyping of blood samples was done in both. This showed a 46, XX,
der(15)/46, XX karyotype in both.
Fluorescent in situ hybridisation (FISH) with a panel of probes identified the
additional material on the short arm of chromosome 15 as to be derived from
the short arm of chromosome 11.
Since at that time, the twins showed discrepant phenotypes which we felt could
not be explained by the TTS alone, FISH with an 11p-probe was performed on a
buccal smear and a urine sample of both twins. This showed three signals in
part of the cells with the most dysmorphic twin having a larger proportion of
abnormal cells, both in buccal and bladder cells.
Meanwhile, DNA-investigations on blood samples of the children and their
parents had confirmed that the twins were monozygotic.
Clinical data and results of the chromosomal investigations will be presented
in detail. The children show features of a phenotype as described by
Slavonitek et al (1). Mosaicism for unbalanced structural chromosomal
abnormalities in live-born infants is a rare observation and its origin in
these twins will be discussed.
1. Slavonitek, A., Gaunt, L., Donnai, D. Paternally inherited duplications of
11p15.5 and Beckwith-Wiedemann syndrome. J.Med.Genet. 1997;34:819-826
P0332
Prognostic impact of cytogenetics in patients with B-CLL
L. Sindelarova 1, K. Michalova 1 ,2, Z.
Zemanova 1, J. Brezinova 2, S. Kurkova 2, E. Cmunt 3;
1Center of Oncocytogenetics, General Faculty Hospital and 1st Medical
Faculty of Charles University, Prague, CZECH REPUBLIC, 2Institute of
Haematology and Blood Transfusion, Prague, CZECH REPUBLIC, 31st
Medical Department, General Faculty Hospital and 1st Medical Faculty of Charles
University, Prague, CZECH REPUBLIC.
B-chronic lymphocytic leukemia (B-CLL), the most common adult leukemia in
Western countries is characterized by clonal chromosomal abnormalities
detected in almost 50% of studied patients. The most frequent are deletions
13q14, trisomy 12, deletions 17p13 and deletions 11q22-q23.
During the last four years we performed dual color I-FISH on bone marrow
and/or blood smears of 178 patients (114 males, 64 females, mean age 64,1
years) with B-CLL.
Trisomy 12 was found in 34 of them (19%), deletion 13q14 was analysed in 162
patients and proved in 85 of them (52.5%). Deletion 17p13 was found in 32
(19.6%) patients out of 163 examined. By probe LSI MLL for 11q23 region we
studied 56 patients. Although this gene is not included in pathogenesis of
B-CLL, deletion was proved in 10 (17.8%) of them. All DNA probes were from
VYSISTM and cut-off level 2.5% was established on controls (standard deviation
not exceed 0.5%). Recent studies revealed that deletions of chromosome bands
11q22-q23 including the ATM gene locus are one of the most common chromosomal
aberrations in B-CLL. We will retrospectively evaluate the incidence of the
deletion in our patients with ATM/FDX probe.
Correlations of the molecular-cytogenetic findings with the immunophenotype,
clinical course will be presented and prognostic impact will be discussed.
This work was supported by grant IGA MZ CR NC 6470-3/2001 and CEZ J 13/98
111100004
P0333
A case with double minute chromosome carrying chromosome's 8 centromere
amplicons.
S. Kokkinou, S. Diamantopoulou, E. Meidanis;
Sismanoglio General Hospital, Marousi, GREECE.
Double minute chromosomes (dms) are extensively associated with cancer cells.
There are few reports of their presence in the peripheral cells of normal
individuals. We report the existence of a dm chromosome in the peripheral
blood lymphocytes of a 31 years old young lady, without any type of malignancy
or any history of prior exposure to mutagenic agents. The patient had a
leukopenia due to an anemia of vitamin B12 deficiency. The bone marrow
aspiration and tephrine biopsy was not diagnostic for an MDS (FAB
classification). She is followed cytogenetically for a period of five years.
Peripheral blood samples were cultured accordingly to standard techniques.
Chromosome preparations were stained with GTG banding technique. The karyotype
was 47,XX (dm included in 50 analyzed metaphases). The dm was studied in
detail with FISH. We used a specific for 8q24 region (LSI-VYSIS), that
contains c-myc oncogene, a specific for MLL gene at 11q23 (LSI-VYSIS), and a
centromeric for chromosome 8 (cep8-VYSIS). The visible dm was a centromere of
chromosome 8 in 100% of the metaphase spreads and in 500 counted interphase
nuclei as well. To the best of our knowledge this is the first case in which
on a dm c-myc oncogene or MLLgene is not amplified and it contains centromer's
8 amplicons. We believe that: (1) the patient has an unusual type of MDS, (2)
the amplified genes on that extra centromere of chromosome 8 propably give a
favorable advantage to her mild and stable clinical course.
P0334
A solution for bloody amnios ?
D. Blake, P. Perrin;
MCL, Medizinische Laboratorien, Düdingen, SWITZERLAND.
The presence of blood in amniotic fluids is classically associated with a
reduced number of colonies and an increased culture time. In a prospective 1
year study, 42 bloody amniotic fluids were treated with a commercially
available selective lysing solution (VitalyseÔ,
BioErgonomics, Inc.). All samples were run in parallel: half of the
independent culture vessels were treated with VitalyseÔ
prior to culturing, half were cultured using standard operating procedure. For
both techniques, we compared the number of colonies per cover slip and number
of days in culture. One case failed with both sets of cultures. In the
remaining cases, the data collected, although not statistically significant,
indicate an increase (53%) in the average number of colonies produced (9.9 vs.
6.5) and a decrease (6%) in number of days in culture (9.7 vs. 10.4) after
treatment with VitalyseÔ. Additionally, no
difference in metaphase quality was observed. These data argue in favor of the
safety and benefits of VitalyseÔ solution. Further
prospective studies to support these findings are currently underway.
P0335
Cytogenetic and FISH analyses of masked and complex Philadelphia chromosome
translocations in chronic myeloid leukemia.
F. Morel 1, A. Herry 1, M. J. Le Bris 1,
N. Guéganic 1, M. F. Scoazec 1, P. Morice 2, J.
F. Abgrall 3, P. Bourquard 2, C. Berthou 4, M. De
Braekeleer 1;
1Service de Cytogénétique, Cytologie et Biologie de la
Reproduction, UBO & CHU, Brest, FRANCE, 2Service d'Hématologie,
CH Yves Le Foll, St-Brieuc, FRANCE, 3Service d'Hématologie
biologique, CHU, Brest, FRANCE, 4Service d'Hématologie clinique,
CHU, Brest, FRANCE.
Bone marrow samples from 112 patients with chronic myeloid leukemia (CML) were
investigated using cytogenetic methods. Fluorescent in situ hybridization
(FISH) with whole chromosome paints and Vysis BCR-ABL ES probe was used to
confirm and/or complete the findings. Eight variant Philadelphia chromosome
(Ph) translocations were identified. Three-way Ph translocations were found in
seven patients. Chromosome 4 was involved in two cases and chromosomes 3, 11,
14, 17, and 16 in one case each; in the latter patient, a ring chromosome of
the translocated 9 was found (r(9)t(9;16;22)). The eighth patient had a
five-way Ph translocation t(2;9;16;22;22); it involved the fusion of the 3'ADN
sequence of the ABL oncogene with the 5'DNA sequence of the BCR gene on the Ph
chromosome and the insertion of the 5'end of ABL in the other chromosome 22.
The BCR/ABL fusion gene was detected on the Ph chromosome in all 8 cases but 2
also presented a deletion of the 5' ABL region on the derivative chromosome 9.
A masked Ph chromosome was identified among the 112 patients; it involved the
insertion of ABL into BCR on an apparently normal chromosome 22, resulting in
a fusion ABL-BCR gene. In conclusion, FISH analyses allowed, not only a more
accurate characterization of these complex translocations with subtle
abnormalities and the identification of cryptic rearrangements, but also the
recognition of the deletion of the 5' ABL region, which is now regarded by
some workers to be of bad prognosis.
P0336
MLL rearrangements in balanced and unbalanced 11q aberrations in patients
with hematological malignancies
J. Brezinova 1, Z. Zemanova 2, S. Kurkova 1,
L. Sindelarova 2, J. Sajdova 1, K. Michalova 1 ,2;
1Institute of Hematology and Blood Transfusion, Prague, CZECH
REPUBLIC, 2Center of Oncocytogenetics, General Faculty Hospital and
1st Medical Faculty of Charles University, Prague, CZECH REPUBLIC.
Structural rearrangements involving chromosome band 11q23 have been observed
in various hematological malignancies and the great majority of chromosomal
changes involve the MLL gene. More than 50 different chromosome bands have
been found to be implicated in these rearrangements, and identification of MLL
together with its localization can be easily done by FISH with specific DNA
probes.
We investigated rearrangements of MLL gene in 14 patients with different
hematological malignancies (9 myeloid, 5 lymphoid), whose bone marrow cells
contained in three cases balanced and in eleven cases unbalanced 11q
aberrations including translocations, deletions and insertions. FISH with dual
colour locus specific probe for MLL gene (VYSIS) proved in those with balanced
aberrations the rearrangement of MLL gene in one case, in two cases MLL gene
was translocated on the partner chromosome without rearrangement. In the
cohort of patients with unbalanced 11q translocations the deletion of MLL gene
was proved in three cases, rearrangement of MLL gene in three cases, in
another three cases MLL gene was translocated on the partner chromosome
without any change detectable by FISH and in two cases MLL gene remained on
band 11q23. Whole chromosome painting probes (CAMBIO) and multicolour FISH
(mFISH - MetaSystems) were used for identification of chromosomes involved in
translocations.
Correlation of clinical characteristics, outcome and survival of patients
according to diagnoses and various types of 11q rearrangements will be
discussed.
This work was supported by grants IGA MZCR NC/6675-3 and GACR 301-01-0200.
P0337
Clinical implications of del(20q) in patients with hematologic myeloid
disorders
S. Kurkova 1, J. Brezinova 1, Z. Zemanova 2,
L. Sindelarova 2, J. Cermak 1, M. Siskova 2, R.
Neuwirtova 2, K. Michalova 1 ,2;
1Institute of Hematology and Blood Transfusion, Prague, CZECH
REPUBLIC, 2Center of Oncocytogenetics, General Faculty Hospital and
1st Medical Faculty of Charles University, Prague, CZECH REPUBLIC.
Deletion of the long arm of chromosome 20 represents the most common
chromosomal abnormality associated with the myeloproliferative syndrome (MPS).
This aberration can be also found in bone marrow cells of the patients with
other myeloid malignancies including myelodysplastic syndromes (MDS) and acute
myeloid leukemia. Deleted part of 20q is rather small and its extent and
breakpoints on the long arms of chromosome 20 can be identified by
fluorescence in situ hybridization (FISH) with specific DNA probes.
We report findings in bone marrow cells of 34 patients (19 males, 15 females,
mean age 65 years, range 21-89 years) with malignant myeloid diseases with
deletion 20q determined by classical cytogenetic techniques. All cases were
studied by FISH using locus specific probes for band 20q12 (LSI D20S108,
VYSIS). According to the results of both cytogenetic analyses this cohort of
patients was divided into two groups: in the first one the patients with
deletion 20q as a single aberration (20 patients) were presented, in the
second group were the cases with deletion 20q and other chromosomal changes
(14 patients). We correlated hematologic findings with the results of
cytogenetic examinations and time of survival in both groups of the patients.
The prognostic value of deletion del(20q) in our cohort in comparison to those
published in literature will be presented. Deletion del(20q) as a single
aberration is a sign of better prognosis for the patients with MPS and/or MDS.
This work was supported by grants IGA MZCR NC/6675-3 and GACR 301 01 0200.
P0338
Importance of genetic investigation in couples involved in "in vitro
fertilisation"(IVF)
P. Capkova 1, K. Adamova 1, A. Santava 2,
R. Vrtel 1, J. Kolarova 2, I. Oborna 3, A. Sobek 4,
J. Santavy 2;
1Institute of Medical Genetics and Foetal Medicine, University
Hospital of Palacky University Olomouc, Olomouc, CZECH REPUBLIC, 2Faculty
of Medicine, Palacky University Olomouc, Olomouc, CZECH REPUBLIC, 3Clinic
of Gynaecology and Obstetrics, University Hospital of Palacky University
Olomouc, Olomouc, CZECH REPUBLIC, 4Fertimed, Private Centre of
Assisted Reproduction, Olomouc, CZECH REPUBLIC.
PROBLEM: The aim of study was to evaluate the contribution of chromosomal
abnormalities and gene mutations (AZF and CFTR) in cases of decreased
fertility.
RESULTS: The frequency of chromosomal aberrations in examined group was 6,5 %
(29/444). We detected 7 (1,5 %) cases of balanced chromosomal rearrangements,
2 (0,45 %) cases of cytogenetic deletion of Y chromosome, 1 (0,23 %) case of
inversion, 1 (0,23 %) case of marker chromosome, 13 (3,0 %) cases of gonosomal
mosaicism and 5 (1,13 %) of gonosomal aneuploidies. The prevalence of AZF
deletion at azoospermic men in this group was 3,91 % (5/128) and CFTR gene
mutation was 4,79 % (7/146).
In a small group of pregnant patients after IVF included in our study the
frequency of chromosomal abnormalities was 18,6 %.
CONCLUSION: High number of infertile couples is affected by chromosomal
aberrations. It is suggested that chromosomal analyses should be performed
before IVF treatment. Incidence of AZF a CFTR gene mutations was also frequent
in studied group and for that reason the screening of both AZF deletion and
CFTR gene mutation is recommended in azoospermic men.
P0339
Prenatally diagnosed chromosome abnormalities and ultrasound markers: A
report of 537 cases
H. Hichri, H. Sennana Sendi, H. El Ghezal, M. Gribaa, S. Ibala
Romdhana, A. Saâd;
Laboratoire de Cytogénétique et de Biologie de la Réproduction, CHU Farhat
Hachad, Sousse, TUNISIA.
Fetuses with chromosomal abnormalities are usually characterized by specific,
minor or major, multiple anomalies accepted as sonographic markers of
chromosomopathies.
Fetal karyotyping was performed for 537 patients because of fetal
abnormalities on ultrasonography. The fetal karyotype was abnormal in 8.94% of
cases (48/537). There were: autosomal aneuploidy in 26 cases (4.84%),
structural rearrangements in 9 cases(1.67%), sex aneuploidy in 8 cases
(1.49%), triploidy in 3 cases (0.56%) and marker chromosomes in 2
cases(0.37%).Unbalanced structural anomalies were frequently associated with
intrauterine growth retardation (4.13%). Within the group of single
sonographic anomaly, the most frequent chromosomal disorders were trisomies 21
and 18 (3.17%). Among fetuses with multiple sonographic anomalies, Turner's
syndrome was detected in 3.06%, trisomy 21 or trisomy 18 in 2.04%. Chromosomal
abnormalities were detected in malformations affecting:
-nervous system : 5.67%
-gastrointestinal system : 7.14%
-renal system :10.4%
-chest : 13.72%
-neck : 16.67%
-abdomen : 30.77%
-skeleton : 8.19%
-hydrops : 8.33%
Minor fetal anomalies also serve as ultrasound markers of fetal aneuploidy,
the most specific is nucal translucency. It was associated with chromosomal
anomalies in 13.3% of cases.
P0340
DNA analysis of Y - chromosomal sequences from different tissues in Turner
syndrome patients.
R. Vodicka 1, R. Vrtel 1, J. Zapletalova 2,
A. Santava 1, K. Adamova 1;
1Institute of Medical Genetics and Foetal Medicine, University
Hospital of Palacky University, Olomouc, CZECH REPUBLIC, 2Department
of Pediatrics, University Hospital of Palacky University, Olomouc, CZECH
REPUBLIC.
Introduction
Incidence of Turner syndrome (TS) is about 1: 2500 of liveborn girls in the
Czech Republic. Chromosome Y sequences having the physiological function in
males can promote the development of gonadoblastoma in undifferentiated gonads
of TS patients. The risk of tumourgenesis is about 30%.
Methods
Samples of 124 Czech TS patients were examined by polymerase chain reaction
(PCR) and quantitative fluorescent PCR (QF PCR) in 4 loci. Y - positive cases
were furthermore tested using fluorescent in situ hybridisation (FISH).
Results
Tests of peripheral blood by:
PCR in loci DYZ3 4,8%, AMGY 3,2%, SRY 3,9%, PABY 4% and
QF PCR in loci DYZ3 13,7%, AMGY 5,6%.
Detection of Y sequences from paraffin - embedded gonadoblastoma tissue in
DYZ3 and TSPY loci from 2 samples. One sample was positive in both loci.
FISH with Y centromeric probe was performed on following sections: two samples
from gonadoblastoma tissues and one sample from undifferentiated gonad. One
sample was a mosaic 1 in 20. The others showed only sporadic positive signal.
Conclusion
The majority of hidden mosaicism is not detectable by conventional cytogenetic
methods. The classical PCR combined with the detection of products on agarose
gel (ethidium bromide stained) was quite unsuitable for mosaic determination.
QF PCR is the most sensitive and the most precise method for the assessment of
Y chromosome mosaicism in patients with Turner syndrome. It enables the most
effective selection of persons under the risk of gonadoblastoma development.
P0341
Distal trisomy 14q and aorto-pulmonary window : a case report
Y. Perrin 1, M. C. Addor 1, N. Sekarski 2,
D. F. Schorderet 1;
1Division of Medical Genetics, Lausanne, SWITZERLAND, 2Department
of pediatrics, Lausanne, SWITZERLAND.
We report on a newborn female with partial trisomy 14q. She was born at term
after an uneventful pregnancy as the first child of an unrelated couple. Her
birth weigth was 2830g, length 47cm, head circumference 30cm. At birth she was
noted to have an unusual face, a 3/6 systolic murmur and was admitted to the
pediatric department. She had a large anterior fontanel, hypertelorism, broad
nasal bridge, high arched palate, micrognathism, low set ears, short neck,
overriding fingers, rocker-bottom feet. She also presented hypotonia,
opisthotonos, left side earing impairment, corneal epitheliopathy due to her
lagophthalmia. An echocardiogram performed on the 3rd day of life showed a
large aorto-pulmonary window with significant left to right shunt which
required surgical closure at one month. At two months, signs of virilisation
were noticed. Endocrinologic and radiologic investigation concluded to side
effects of medication.
Cytogenetic analysis was done. Initial investigation demonstrated additional
material on chromosome 3. Fluorescent in situ hybridization showed that this
material originated from chromosome 14. Her caryotype was:
46,XX,der(3)t(p25;q24). Studies of her parents' chromosomes revealed a
reciprocal paternal balanced translocation between chromosomes 3 and 14. His
caryotype was: 46,XY,t(3;14)(p25;q24).
We compare her clinical features with the 8 other trisomy 14q24->qter
published.
P0342
Characterization of two small supernumerary marker chromosomes (SMC) by
acro/cenM-FISH - first case with partial hexasomy 15pter->15q13
A. Heller 1, B. Albrecht 2, A. Nietzel 1,
H. Starke 1, F. von Eggeling 1, U. Claussen 1, T.
Liehr 1;
1Institute of Human Genetics and Anthropology, Jena, GERMANY, 2Institute
of Human Genetics, Essen, GERMANY.
Cytogenetic analysis performed in a three year old girl resulted in a
karyotype 48,XX,+2mar[25/25]. She presented the following clinical features:
severe mental retardation, microcephaly, postaxial hexadactyly plus a
pachygyry. Such small SMCs often are uneasy to characterize in standard
cytogenetic or molecular cytogenetic approaches. Recently, we developed a
probe set, using all human centromeric probes labeled in different colors,
allowing the simultaneous characterization and identification of all
chromosomes by their centromeric region (Nietzel et al., 2001, Hum Genet, 108,
199-204). The technique, called cenM-FISH has been extended by the
introduction of an additional probe specific for the short arm of all human
acrocentric chromosomes called midi54 (described in Mrasek et al., 2001,
Cytogenet Cell Genet, 93, 242-248). This acro/cenM-FISH probe set revealed in
the present case, that the both SMC were identical derivatives of chromosomes
15 with two specific signals for the centromere 15 specific and the midi54
probe. Additional FISH experiments using the high resolution multicolor
banding (MCB) technique and probes specific for the Prader-Willi/Angelman
syndrome region (SNRPN and D15S10), respectively, characterized the derivative
chromosomes as dicentric iso-chromosomes i(15)(pter->q13::q13->pter). To
the best of our knowledge, this is the first case with partial hexasomy
15pter->15q13. Studies to clarify the origin of the derivatives
(UPD-analysis) are in progress. In summary, acro/cenM-FISH is a very useful
approach for the one step identification of all human chromosomes by their
centromeres and acrocentric p-arms. Supported by Herbert Quandt Stiftung der
VARTA AG, Wilhelm Sander-Stiftung (99.105.1) and EU (ICA2-CT-2000-10012 and
QLRT-1999-31590).
P0343
Cytogenetic studies in lymphocyte cultures from 3 members of a family
affected with Werner's Syndrome
B. Porto 1, O. Pereira 2, I. Malheiro 1;
1Lab. Citogenética, Instituto Ciências Biomédicas Abel Salazar,
Porto, PORTUGAL, 2Serviço de Dermatologia, Hospital Geral de Stº
António, Porto, PORTUGAL.
Werner's Syndrome (WS), a rare autosomal disorder arising as a consequence of
mutations in a gene coding for a protein member of Rec Q family of DNA
helicases (WRN), is characterized by premature aging exhibiting chromosome
instability and predisposition to cancer. In cell lines derived from WS
patients both stable and unstable chromosome aberrations had already been
detected, and recently it has been shown that WS cells have a slower rate of
repair associated with DNA damage induced in S-phase. In the present work we
performed PHA-stimulated lymphocyte cultures, with and without induction with
diepoxybutane (DEB), from two brothers with WS, another healthy sister and 6
healthy controls. The purpose was to analyse the frequency and pattern of
chromosome instability in patients from the same family. Our results showed a
higher frequency of random chromosome aberrations in all members of the
family, compared with controls; however, no consistent pattern of
chromosome-specific aneuploidies and structural aberrations was detected among
the 3 members of the family, supporting the hypothesis that chromosome
instability is related with the disease but the selection of the chromosome
aberrations involved is not genetically determined. This study also revealed
that the repair associated with DNA damage induced by DEB is not significantly
affected in the 3 members of the family.
P0344
Supernumerary marker 22 chromosome: Clinical, cytogenetic and molecular
analysis of five patients.
I. Lopez Pajares 1, A. Delicado 1, P. Lapunzina 1,
M. Mori 1, M. de Torres 1, A. Sanz 2;
1Hospital Universitario La Paz, Madrid, SPAIN, 2Hospital
de la Princesa, Madrid, SPAIN.
Extra structurally abnormal microchromosomes are a heterogeneous group of
chromosomes also known as markers or small supernumerary chromosomes . In most
cases, the extra chromosome is derivated from an acrocentric chromosome.Thus,
patients have duplications, or in some cases triplications of the chromosomal
segments included in the extra.
Patients, material and methods : We have studied five patients with an extra
marker 22 chromosome. All patients were evaluated with GTL, CBG, NOR-Ag
banding, fluorescence in situ hybridation (FISH) using several probes and
microsatellite anlaysis with four different markers.
Results: Four patients had typical clinical signs and symptoms associated to
the Cat eye syndrome (CES), showed peculiar face, abnormal ears, anal
stenosis/atresia and some degree of mental retardation, ranging from mild to
moderate. Iris coloboma, a clinical finding usually observed in CES, have not
been found in none of our patients.One of them had a complex congenital heart
defect and died at age of three months.Cytogenetic studies showed that one
child had mosaicism for the marker chromosome, and the remaining four children
had 47 chromosomes in all evaluated metaphases. Parents were all
cytogenetically normal.
There was heterogeneity on the FISH and microsatellite results demonstrating
that the duplicated segment varied in size in each patient.
Comments: Our results in five patients with extra satellited 22 marker
chromosome showed that the phenotype in these patients is characteristic of
CES and that iris coloboma may be not present in a percentage of patients.
P0345
Chromosome 9p- deletion syndrome: 3 "de novo" cases
N. Oliva Teles, M. Reis Lima, L. Ramos, G. Lima, F. P. Oliveira, J.
Aguiar, M. Pinto;
Instituto de Genética Médica Jacinto Magalhães, Porto, PORTUGAL.
The 9p- deletion syndrome was characterized for the first time in 1973. The
main clinical features of this syndrome are dysmorphic facial features
(trigonocephaly, midface hypoplasia, upward-slanting palpebral fissures and a
long philtrum) and mental retardation.
The authors present the clinical description and the cytogenetic findings in
three patients, aged 1, 4 and 43 years, with “de novo” deletions in the
short arm of chromosome 9 (in 2 cases, 9p22 and 9p23 in the other). Apart from
the mental retardation, the reasons for referral were, respectively,
Pierre-Robin syndrome with low weight and height, severe facial dysmorphic
features and apparent Down syndrome.
It is important to note that, although the symptoms are typical and diagnosis
may be suspected at birth, most paediatricians are not aware of this syndrome;
therefore, no provisional diagnosis or detailed clinical description are given
to the cytogenetics laboratory in the majority of cases. The clinical details,
together with high resolution banding patterns (especially if the breakpoint
region is very close to the 9p telomere) are the best combination to avoid the
underestimation of this syndrome.
P0346
A new probe set for the characterization of centromere-near
rearrangements
H. Starke 1, A. Weise 2, A. Nietzel 2,
A. Heller 2, U. Claussen 2, T. Liehr 2;
1(1) Institute of Human Genetics and Anthropology, Jena, GERMANY, 2Institute
of Human Genetics and Anthropology, Jena, GERMANY.
A variety of FISH approaches have been developed in the last decade, covering
the entire human genome in multiple ways. Nonetheless, there is still a
chromosomal region which is not well investigated with the presently available
probe sets: the pericentric region. This region is of special interest when
small supernumerary marker chromosomes (sSMC) shall be characterized. Neither
whole nor partial chromosome painting (wcp and pcp) probes are informative if
centromere-near euchromatic material is present on a sSMC. This question can
be answered best when hybridizing centromere-near probes like BACs. At present
we are working on 24 probe sets, each for one human chromosome, which consists
of a centromere specific satellite probe, one centromere-near BAC in q and p
(excluding the acrocentric chromosomes) and arm-specific pcp probes. These
probes are used after characterizing the sSMC by cenM-FISH (Nietzel etal.,
2001, HumGenet, 108, 199-204). Up to now two cases with cat eye syndrome (CES)
chromosomes [inv dup(22)(q11.2)] and three sSMC derived from #2, #16 and #22,
respectively, have been characterized by centromere-near BAC probes. As
expected, centromere-near euchromatic material was present in the
CES-chromosomes. For the derivative #2 q-arm specific centromere-near material
could be detected, while the derivatives #16 and #22 consisted only of
centromere and heterochromatin. Additionally, a clinical case with a normal
karyotype apart from the very small pericentric inversion could be detected
using the chromosome 2 specific peri-centromere probe set. In summary, we
present a probe set useful to characterize any kind of centromere-near
rearrangement. Supported by EU (QLRT-1999-31590)
P0347
Role of chromosome abnormalities in recurrent In-Vitro Fertilization
implantation failure
I. Yehudai 1, H. Bar-El 1, Z. Borochowitz 1,
R. Diukman 2, H. Dar 1 ,3;
1Bnai-Zion Medical Center, Haifa, ISRAEL, 2Carmel Medical
Center, Haifa, ISRAEL, 3Technion - Faculty of Medicine, Haifa,
ISRAEL.
The association between recurrent miscarriges and parental chromosome
anbormalities has been well documented. Much less is known concerning the role
of parental chromosome abnormalities in recurrent in-vitro fertilization (IVF)
implantation failures.
The aim of this study was to evaluate the contribution of chromosome
abnormalities as potential cause of IVF implantation failures.
Two hundred and forty four consecutive patients (122 couples) who had at least
3 attempts of embryos transferred without achieving clinical pregnancy, were
evaluated for chromosome abnormalities. Five hundred and twenty six
consecutive patients (263 couples) who had experienced 3 or more miscarriges,
and 2032 consecutive antenatal chromosome diagnoses referred because of
advanced maternal age or maternal anxiety (unbalanced karyotypes associated
with mental retardation were excluded), served as control groups.
Chromosome abnormalities were detected in 5/244 patients with IVF failures
(4.1% of the couples), in 10/526 patients with recurrent miscarriges (3.8% of
the couples), and in 5/2032 (0.25%) antenatal diagnoses.
The chromosome abnormalities detected in the IVF failures group included 2
reciprocal translocations, one case of 46,XYY, one case of 46,X,del(X)(p22),
and one case of 46,XY(96%)/45,X(4%) mosaicism.
The prevalence of chromosome abnormalities among the IVF patients was
significantly higher than in the antenatal group (p<0.001), and it was
similar to the prevalence of chromosome abnormalities detected in the
recurrent miscarriges group.
The results of the study suggest that chromosome analysis should be considered
as part of the routine investigation of patients with recurrent IVF
implantation failures.
P0348
Unusual inverted duplication of chromosome 15 with terminal deletion.
R. Genesio 1 ,2, A. Borghese 1, D. De Brasi 3,
P. Di Micco 3, P. Di Costanzo 3, A. Conti 1, D.
Paladini 4, A. Loffredo 4, P. Martinelli 4, L.
Nitsch 1;
1Cytogenetics and Prenatal Diagnosis - University of Naples, Naples,
ITALY, 2BioGeM, Naples, ITALY, 3Dpt.of Pediatrics -
University of Naples, Naples, ITALY, 4Dpt. Obstetrics and Gynecology
- University of Naples, Naples, ITALY.
We report here the case of a female premature newborn with multiple severe
congenital defects. Ultrasonographic examination at 26 weeks of gestation
evidenced growth retardation, cardiac and kidney abnormalities, arthrogryposis
and foot deformity. These anomalies were all confirmed at birth. Moreover,
facial dysmorphism, left-hand deformity, dislocation of the hip and
hypoplastic corpus callosum were found. Cytogenetic investigation performed by
high resolution G banding on amniocytes, fetal blood and skin fibroblasts
indicated a possible duplication of 15q. FISH analysis was performed with a
painting probe for chromosome 15, with a telomeric probe and with specific 15q
probes, to identify and delimit the duplicated region. An inverted duplication
of the q14-26.2 region was assessed. The deletion of q26.3 was also
demonstrated. The resulting karyotype was: 46, XX, inv dup
del(15)(pter->q26.2::q26.2->q14). The parents’ karyotypes were normal.
The phenotypic alterations of the proband are only in part superimposable to
those described in the literature for analogous cases. This could be due to
the extent of the duplication and/or to the monosomy of a portion of q26.
Chromosomal aberrations involving the human chromosome 15 (most often the
q11-13 region) have been frequently reported in the literature. It has been
suggested that duplicons with opposite orientation may predispose to inverted
duplications and that distal deletion and a single copy region between the
duplicated regions are then present. We are currently investigating the
hypothesis that the rearrangement described here might be related to the
recently discovered duplicons in the 15q26 region.
P0349
Asynchronous replication of bi-allelically expressed loci: A new phenomenon
in turner syndrome
O. Reish 1, R. Gal 1, L. Gaber 2, C.
Sher 1, Z. Bistritzer 1, A. Amiel 2;
1Assaf Harofeh, Zerifin, ISRAEL, 2Meir Medical Center,
Kefar Saba, ISRAEL.
Purpose: Transcriptional activity of genes is related to their replication
timing; alleles showing the common biallelic mode of expression replicate
synchronously, whereas those with a monoallelic mode of expression replicate
asynchronously. Here we determined the level of synchronization in replication
timing of alleles in subjects with Turner syndrome. Methods: We used
FISH for 3 loci not linked to X chromosome, in lymphocytes derived from 12
controls, 3 individuals with Turner and 4 with mosaic Turner syndrome. Results:
In cells derived from controls, each pair of alleles replicated
synchronously; yet these same alleles replicated asynchronously in cells
monosomic for X chromosome derived from Turner and mosaic Turner patients.
When the level of 45,X0 was low in the mosaic samples, the replication pattern
of the 46,XX cells was normal. However, in samples with a high level of
mosaicism, a significantly increased asynchronous replication was detected in
the 46,XX cells. Conclusion: An altered temporal replication control in
Turner syndrome may suggest an epigenetic mechanism involved in this condition
affecting the aneuploid and euploid cells.
P0350
Cytogenetic analysis of 179 infertile Iranian women.
F. Mortezapour 1, L. Seyed Mortaz 1, F. Razazian 1,
B. Hashemi 1, H. Sayar 1, S. Joghehdost 1 ,2,
M. Houshmand 1 ,2;
1Blood transfusion organization, Tehran, IRAN (ISLAMIC REPUBLIC OF), 2National
Research Center for Genetic Engineering and Biotechnology, Tehran, IRAN (ISLAMIC
REPUBLIC OF).
Turner syndrome in adult is typically presented by primary
amenorreha,infantile genitlia and failure of secondary sexual development.
Gonadal dysgenesis resulting in primary infertility is one of the most common
features of turner syndrome. Nevertheless, menstruation and pregnancy have
been recorded in a few 45,X subjects. But whether on not the fertility is
associated with a 46,XX cell line the germ cells is not known. We have
investigated 218 female patients which were referred to our lab for primary
amenorrhea or secondary amenorrhea or for R/O Turner syndrome, 35 cases showed
an abnormal cytogenetic analysis. The most common chromosol abnormality was
45,X(18/35). The other karyotypes consist of: pseu/idic(X)(q22);
del(15)(q11);add(X)(q28);r(X);iso(X)(q10);4 cases of mosaicism
(45,X/46,XX/47,XXX);46X,iso(X)(q10)/45X;45X/46x;del(X)(q22.2); 2 cases of
inv(9)(p11,q13). The latter cases will present a normal variant in human.
P0351
A New Case of Mosaic Supernumerary Ring Chromosome 8 Syndrome
S. Yilmaz 1, A. Deviren 2, D. Kuru 3, Y.
Tarkan-Argüden 1, B. Karaman 4, A. Yüksel 3, S.
Hacihanefioglu 1;
1Istanbul University, Cerrahpasa Medical School, Div.of Biomedical
Sciences, Dept.of Genetics, Istanbul, TURKEY, 2Istanbul University,
Genetics and Teratology Research Center (GETAM), Istanbul, TURKEY, 3Istanbul
University, Cerrahpasa Medical School, Div. of Biomedical Sciences, Dept.of
Medical Biology, Istanbul, TURKEY, 4Istanbul University, Child Health
Institute, Medical Genetics Division, Istanbul, TURKEY.
We described a de novo mos r(8) by conventional cytogenetic and FISH
tecniques. The patient was referred because of neuromotor growth retardation
and dysmorphic facial findings. He was a 15 year-old male, with a height of
157 cm (10th centile), and weight of 37.5 kg (<3rd centile), head
circumference 55 cm (75th centile). His born weight was 1750 gr (<3rd
centile), and length was 50 cm (50th centile). He had long and expressionless
face with a prominent maxilla, everted lips, streched lingual frenulum which
had been cut afterwards. He had big low-set ears, epicanthus, prominent root
of nose, plagiocephaly, pterigium coli, camptodactyly, deep plantar creasis,
overlapping of toes, hammer big toes and cryptorchidism. He had elongated thin
trunk with narrow shoulders. There were cafe-au-lait on dorsal region and.
Radiographic findings showed a mild kyphoscoliosis on dorsal vertebras, spina
bifida on L4-L5. Magnetic rezonans Imaging (MRI) study revealed hypolasia of
splenium region of corpus callosum and cavum septum pellicidum abnormalities.
His IQ level was 67, he had poor speech and language development, poor
awareness of necessary life skills, and had autistic-like behaviours with
shyness and stubbornnness. We performed cytogenetic analysis on peripheral
blood cultures by GTL banding and found 47,XY,+r[29]/46,XY[33]. We applied
FISH and obtained signals on the ring chromosome with 8 painting and 8 alpha
satellite probes. Oncor (Gaithersburg) digoxigenin-labelled Coatosome probes
and Vysis were used for FISH analysis. The resulting karyotype after FISH
analysis was mos 47,XY,+r. ish r(8)(wcp8+;D8Z1+).
P0352
Pure partial trisomy for long arm of chromosome 9
R. Airinei 1, I. Dimofte 2, A. Popa 2,
D. P. Balaban 3, L. C. Enache 4, V. Broasca Madar 5;
1Faculty of Medicine, Constanta, ROMANIA, 2Medical
Genetics Department, Faculty of Medicine, Constanta, ROMANIA, 3Biochemistry
Department, Faculty of Medicine, Constanta, ROMANIA, 4Cell and
Molecular Biology Deparment, UMF "Gr.T.Popa", Iasi, ROMANIA, 5Management
and Public Health Department, Faculty of Medicine, Constanta, ROMANIA.
A case of a 4-year-old boy with trisomy of the long arm of the chromosome 9 is
described (46,XY, der(9), t(9;9) (q32:q12)).
The trisomy is probably the result of a translocation of the long arm of the
chromosome from one homologue to the other in a prenatal gonad.
The clinical features of the child which severe developmental retardation,
bird-like facies, tapered fingers, and flexion contracture of the legs are
similar to those of the few cases described of trisomy of the whole
chromosome.
P0353
Paracentric inversion of chromosome 1 - inv(1)(p31.2p35.2) - in a large
family from northern Finland
J. Leisti, E. Kinnunen-Siira, K. Haapala, R. Herva, H. Kokkonen;
Oulu University Hospital, Oulu, FINLAND.
We describe a large family in which a paracentric chromosomal inversion was
first detected in the early 80´s in a 1-year-old boy with severe mental
retardation, infantile spasms and multiple congenital anomalies. He had
inherited the inversion in an unbalanced form from the mother, who was carrier
of inv(1)(p31.2p35.2) and who had had several spontaneous abortions.
Subsequently this inversion has been independantly ascertained in six
individuals of whom four were studied for developmental delay and dysmorphic
features. In them no light microscopic differences could be seen at
prometaphase level, when compared with the inversions of their healthy
parents. This prompted us to search for the origins of the carriers of the
inversion; with the help of church records a founder couple born in 1760 and
1763 could be detected, whose descendants all the inv(1) carriers are. - The
cytogenetic, clinical and genealogical data of the family will be presented
and the role of the inversion in the family and the population will be
discussed.
P0354
Diagnosis of low rate mosaic trisomy 21 by FISH in a girl with inherited
balanced translocation 6;15
M. Kocova;
Pediatric Clinic, Medical Faculty, Skopje, THE FORMER YUGOSLAV REPUBLIC OF
MACEDONIA.
FISH can help in the diagnosis of mosaic chromosomal abnormalities. Here we
present a girl diagnosed with low rate mosaicism for trisomy 21 utilizing this
method.
Case report: A 12-year girl presented due to schooling problems. She was a
student in the 6th grade of elementary school, very good in literature and
writing, however she had poor grades in math and logical sciences. She had
several pneumonias and additional lobe in right lung has been diagnosed.
Otherwise, she grew normally, and the puberty occurred at an age of 11 years.
Physically she had some of the features of Down syndrome, e.g. flat nasal
bridge, epicantus, low set years, brachicephalia. However, she had a very well
developed speech, with significant eloquence, and no suspicion of Down
syndrome occurred previously.
Karyotype detected balanced translocation 6p; 7q, inherited from her father.
No other chromosomal abnormality was found by karyotyping. FISH was performed
on the chromosome spreads from blood lymphocytes. Out of several thousands of
cells, trisomy 21 appeared in three.
Low rate mosaicism in our patient confirms that the proportion of trisomic
cells influences some of the features of Down syndrome including the speech
development. FISH can help to detect small number of trisomic cells in cases
with unusual presentations of the syndrome.
P0355
Partial monosomy 22q11 associated with velo-cardio-cutaneus syndrome due to a
de novo translocation (15; 22) (p11; q11)
E. Sukarova-Angelovska, M. Kocova, M. Krstevska-Konstantinova, G.
Ubovic, V. Anastasovska;
University Children Hospital, Skopje, THE FORMER YUGOSLAV REPUBLIC OF
MACEDONIA.
Reciprocal translocations are said to be a common finding in the cytogenetic
laboratories. In most of the cases there isn’t a loss of material when a
breakage between two chromosomes occurs. A balanced career doesn’t have any
pathological signs. Sometimes during these breaks a certain amount of genetic
material has been lost, or the break occurs in a specific region for some
syndrome. Then the career expresses more or less dysmorphic signs, mental
retardation, or organic lesions.
A 2 year old boy approached to the clinic because of motor delay and
dysmorphic features. It was a second child of young and unrelated parents. The
pregnancy was normal; the baby was a full-term neonate, with birth weight and
length under 3rd percentile. The boy had mild brachycephaly, flat facies,
hypertelorizm, up-slanted palpebral fissures, squared nasal root, big ears,
micrognatia, and swallowing difficulties due to velopharyngeal incompetence.
Also the boy presented heart defect, crypyorchidism, inguinal hernia, slender
hands with clinodactily of fifth finger. Neurological findings revealed
hypotonia, motor and mental delay. Chromosomal finding represents
translocation between chromosomes 15 and 22, 46, XY, t (15; 22) (p11; q11)
with a breakpoint in a VCFS site. Analysis of the mother’s chromosomes
showed huge satellites on chromosomes 15 and 22, which may be the cause of the
breakage and translocation of chromosomes in the child. When reciprocal
translocation occurs in a specific region, it produces deletions of specific
genes that lead to a certain phenotype of a well known syndrome.
P0356
Pseudohermaphroditism, etiopathogenesis and classification
F. Razazian 1, S. Soleimani 1, F. Nassiri 1,
B. Hashemi 1, H. Sayar 1, S. Joghehdost 1 ,2,
M. Houshmand 1 ,2;
1Blood transfusion organization, Tehran, IRAN (ISLAMIC REPUBLIC OF), 2National
Research Center for Genetic Engineering and Biotechnology, Tehran, IRAN (ISLAMIC
REPUBLIC OF).
Advance in experimental endocrinology, biochemistry, genetics, and molecular
biology have all contributed to our understanding of the process of human sex
differentiation in the last 5 year. Based on the recognition of the underlying
anomaly in the process of sexual differentiation intersex disorders may be
devided into abnormal gonadal determination and abnormal genital
differentiation [including male and female pseudohermaphrodism(MPH &
FPH)]. Abnormal gonadal determination is mainly dependent on sex chromosomal
defects that can be detected by cytogenetic analysis or by the DNA probes for
genes located on the Y chromosome. Individuals with ambiguous genitalia but
two differentiated testis are called MPH. Females with ambiguous external
genitalia but normal ovaries and normal internal genitalia are called FPH. The
XX males may be divided into 3 subgroups:1) 46,XX males with SRY gene, 2)46,XX
males without the SRY gene and 3)46,XX/46,XY mosaics. The insidence of 1 in
20.000 to 30.000 males with a 46,XX karyotype have been reported. We have
investigated 586 infertile patients who was referred to our labfrom 1997 until
2002, we have found 39 cases (39/586)with sex reversal which was more common
in the male group as male pseudohermaphrodism(24/39). The main reason of
referal in the MPH group was azoospermia & in the FPH group consists of
primary amenorrhea. Cytogenetic analysis is the main method for diagnosis of
sex reversal as a cause of infertility, but for determining the etiology of
this pattern we need molecular technology to study the SRY gene.
P0357
Evaluation of 355 azoospermics Iranian patients by cytogeneic method.
M. Rahnama 1, S. Tootian 1, M. Zamanian 1,
B. Hashemi 1, H. Sayar 1, S. Joghehdost 1 ,2,
M. Houshmand 1 ,2;
1Blood transfusion organization, Tehran, IRAN (ISLAMIC REPUBLIC OF), 2National
Research Center for Genetic Engineering and Biotechnology, Tehran, IRAN (ISLAMIC
REPUBLIC OF).
spermatogenesis in human is a complex, dynamic process which the duration
takes 70 days. The onset of release of spermatozoa occurs at about 13.5 years
of age and continuing through out life into the eighth and ninth decades. In
quantitative terms the out put of spermatozoa in an average adult man is 1500
to 2500 million sperm commonly present in a single ejaculate. Total absence of
sperm from the semen is called azoospermia. Sperm concentration of less than
20 million/ml are classified as oligospermic. We have investigated 417
azoospermic patients in our center and a constitutional chromosomal aberration
were diagnosed in 110/417(26.4%). Whereas the 47,XXY chromosome complement was
the commonest (71/110),the following abnormal karyotypes were also
found:46,XX; del(Y)(q11);48,XXYY; inv(Y)(pqq.2,q11.2); t(2,12); t(12,22);
t(13q,14q); 45,X/46,XY; 47,XXY/46,XY and 6 patients had inv(9)(p11q13) but the
pattern have been observed as a normal variant in human. We have found 4
patients with complex structural and aneuploidy abnormalities
(46,X,idic(Y)(q11.31)/45,X; 47,XXY/48XXY+mar/48XXXY; 47,XXY, inv(9)(p11q13);
46,X,del(Y)(q11.21)/45,X). Pooled data from the literature showed that the
frequency of chromosomal abnormalities is higher in azoospermic than in
infertile. We have observed 26.4% chromosomal abnormality in azoospermia and
20% in infertile men, that is compatible with the data from literature.
P0358
Chromosome deletions in 13q: a review of 11 new cases
C. Candeias, M. Mota Freitas, N. Oliva Teles, M. L. Fonseca Silva, M.
C. Ribeiro, G. Lima, F. P. Oliveira, F. Pinto, M. Pinto;
Instituto de Genética Médica Jacinto Magalhães, Porto, PORTUGAL.
Partial deletions of the long arm of chromosome 13, del(13q), are uncommon
chromosome abnormalities in which a portion of the long arm is missing. The
range and severity of the symptoms may vary greatly, depending upon the size
and location of the absent segment.
The authors present 11 cases of "de novo" 13q deletions: del
(13)(q31) - 1case; del (13)(q32) - 4 cases; del (13)(q22.3) - 1 case; r(13) -
4 cases; mosaic r(13) - 1 case. All these patients were referred for
cytogenetic studies due to multiple congenital abnormalities, ranging from
mild to severe.
A correlation phenotype/karyotype will be established and compared with the
reported literature.
P0359
Identification of supernumerary marker chromosome 20 in 3 patients.
M. Pierluigi 1, S. Cavani 1, M. Malacarne 1,
F. Faravelli 1, L. Dalpra' 2, E. Sala 2, N. Villa 2,
D. Gaveglio 2, M. Silengo 3, G. B. Ferrero 3, E.
Frate 4, F. Dagna Bricarelli 1;
1Laboratorio di Genetica Umana, Ospedali Galliera, Genova, ITALY, 2Laboratorio
di Citogenetica, Ospedale S. Gerardo, Monza, ITALY, 3Dipartimento di
Pediatria, Univerità di Torino, Torino, ITALY, 4Ambulatorio di
Genetica Medica, Azienda ULSS 9-TV, ITALY.
Marker chromosomes (MCs) are supernumerary structurally abnormal chromosomes
in which no part can be identified by cytogenetic banding techniques (ISNC
1995). They are detected with a frequency of 1:1,660-1:1,040 at amniocentesis,
1:5,555 in liveborn individuals and 1:330 in mentally retarded patients. MCs
are classified as de novo or familial, satellited or non-satellited and mosaic
or non-mosaic. In some families, MCs are transmitted through several
generations, apparently with no associated abnormalities, whereas other MCs
carriers may present serious clinical symptoms, such as mental retardation,
dysmorphic features and malformations.
Several syndromes associated with MCs are known. Mosaic i(12p) causes the
Pallister-Killian syndrome; an inv dup(22) is found in the "cat eye
syndrome" and an i(18p) syndrome has also been described.
MCs can now be identified by molecular cytogenetic methods but limited data
currently do not permit consistent phenotype-genotype correlation to be made.
In addition, variation in the size and parental origin of the marker can
influence the outcome.
The occurence of a supernumerary marker chromosome 20 is rare and no common
phenotype has been established. So far, there are 11 cases reported with an
extra marker (20) and their phenotype varies from normal to abnormal.
We describe 3 further patients with MCs derived from chromosome 20 and
identified by FISH. These MCs have been sized with BACs probes and clinical
and cytogenetic findings are compared with results of case reports published
to date.
This study was supported by Telethon Italia (grant E.0827)
P0360
Scanning probe microscopy studies on human metaphase chromosomes
M. Oberringer, B. Heinz, A. Englisch, H. Gao, U. Hartmann;
Institute of Experimental Physics, Saarbrücken, GERMANY.
A better knowledge of biochemical and structural properties of human
chromosomes is important for cytogenetic investigations and diagnostics. Since
conventional fluorescence microscopy has reached its limit due to the
restricted optical resolution (0,2µm), we are currently working on the
indroduction of different scanning probe microscopy techniques in this field:
Atomic Force Microscopy (AFM) and Scanning Near-field Optical Microscopy
(SNOM).
The topography of GTG-banded human metaphase chromosomes was evaluated by AFM
and compared to the standard banding pattern according to the international
system for human cytogenetic nomenclature (ISCN 1995), which is based on the
optical properties of the chromosomes. A better resolution of the chromosomal
surface in general and of the bands themselves was acchieved by applying AFM.
Another approach to make the chromosomes more accessible to DNA-probes during
fluorescence in situ hybridization (FISH) and to improve the visualization of
their structure consists of various biochemical treatments. We were using the
protein cleaving enzyme trypsin and the polyanion heparin additionally to well
established procedures like for example HCl-incubation to remove the
DNA-binding proteins, histones as well as non-histones. The structural effects
of these treatments were visualized by AFM and the binding structure of
target- and probe-DNA after FISH was investigated by SNOM.
P0361
The cytogenetic analysis in female patients with amenorrhea and sexualization
troubles
E. V. Gorduza, T. Angheloni, C. Bujoran, O. Stoica, M. Volosciuc, E.
Braha, M. Covic;
University of Medicine and Pharmacy, Iasi, ROMANIA.
The aim of our study is to analyse the relationship between clinical diagnosis
and chromosome studies in female with amenorrhea or sexualization troubles. We
divided the lot of 197 patients in 11 groups, on clinical basis. In Turner
syndrome cases we found: 45,X (41) 45,X/46,XX (23) 46,XX (23) 46,XX/47,XXX (2)
45,X/46,X,r(X) (1) 45,X/46,XY (1) 45,X/46,XX/46,XY (1) 46,XY (1). Karyotypes
for primary amenorrhea cases resulted in: 45,X/46,XX(5) 46,XX(25)
46,XX/47,XXX(1) 45,X/46,X,i(Xq)(1) 46,XX/47,XXX(1) 46,XY(5). In secondary
amenorrhea cases we found: 46,XX (9) 45,X/47,XXX(1). Karyotype founded in
Morris syndrome (4) and delayed puberty (1) phenotypes: 46,XY. In Rokytansky
syndrome cases (13) we found 46,XX karyotype. In short stature cases we found:
46,XX (1) 45,X/47,XXX(1) 45,X/46,XX/47,XXX (1). The clinical cases with triplo
X (2) was confirmed: 46,XX/47,XXX (1) 45,X/46,XX/47,XXX (1). In cases with
Noonan syndrome we found: 46,XX (3), 45,X (1), 45,X/46,XX (1). In ovarian
dysgenesia cases we found: 45,X (2) 45,X/46,XX (2) 46,XX (2) 47,XXX (1) 46,XY
(1) 46,XX/47,XXY (1). In cases with clinical suspicion of female
pseudohermaphroditism we found: 46,XX (10) 45,X/46,XX (1) 45,X/46,XY (1) 46,XY
(1). In intersexuality cases we found: 46,XX (8) 46,XY (3) 49,XXXXY (2) 45,X
(1) 45,X/46,XX (1) 45,X/46,XY(1) 46,XX/46,XY (1). Our studies results confirm
the concordance between clinical aspects and cytogenetics results in Turner,
Rokytansky and Morris syndromes, but difficulties in clinical diagnosis exist
in females with isolated amenorrhea or ovarian dysgenesia. Instead, in
intersexuality and pseudohermaphroditisms we found a discordance. In
conclusion, improving of clinical examination and cytogenetic confirmation are
needed in patients with sexualization troubles.
P0362
Results of chromosome studies in 664 Iranian couples with the history of
recurrent early pregnancy loss.
Y. Shafeghati 1 ,2, M. Karimi Nejad 2,
M. Zangeneh 2, R. Karimi Nejad 2, Q. Baba Mohammadi 2,
N. Almadani 2, N. Almadani 2, F. Afroozan 2;
1Univerity of Welfare Science and Rehabilitation, Tehran, IRAN
(ISLAMIC REPUBLIC OF), 2Karimi Nejad Genetics Center, Tehran, IRAN
(ISLAMIC REPUBLIC OF).
Approximately 15 to 20 per cent of recognized pregnancies end in a
first-trimester spontaneous abortion.An estimated 0.4 to 0.5 per cent of them
have 3 or more consecutive spontaneous abortions. This group of women are
categorized as having " habitual abortion". More than 50 percent of
spontaneous first trimester abortion specimens that undergo karyotyping are
found to have a chromosomal abnormality, which is the major cause for
spontaneous abortion.
Karyotype studies of couples with two or more spontaneous abortions revealed
in 2.78 to 3.4 per cent of the cases that one of the couples was a balanced
reciprocal translocation carrier. By high resolution banding technique a
slightly higher rate (4.76 per cent) was detected.
1328 persons (664 couples) referred to our Genetics Center with the hisorty of
two or more consecutive spontaneous abortions for chromosome studies. We found
major chromosome abnormalities in 35 cases (5.27 per cent). The most common
type of abnormalities was translocation, in 7 cases the mosaicism of sex
chromosome abnormalities has been detected. In one case Yq inversion, one case
46,XY male pseudohermaphroditism, and another single case of pericenteric
inversion of chromosome No 1 were the final diagnoses. On mosaic cases the
most common type, was the mosaic Turner syndrome (4 cases), followed by the
mosaic Klinefelter syndrome with 2 cases. We found only 1 case with mosaic
trisomy 13.
We were confronted with 68 cases that showed minor chromosome abnormalities
(10.2 per cent). The most common anomaly was pericentric inversion of
chromosome No.9 (17 cases).
P0363
Is tumourigenesis triggered by chronic inflammation?
M. Jennewein 1, M. Oberringer 2;
1Department of Trauma-, Hand- and Reconstructive Surgery, Homburg,
GERMANY, 2Institute of Experimental Physics, Saarbrücken,
GERMANY.
During the last years, investigators aimed at the identification of new
dignostic and prognostic features of tumourigenesis by correlating cytogenetic
data with the histopathological stage of tumour. Several tumours (e.g.
esophagus, colon, lung) were characterized to be preceeded by chronic
inflammation.
In an effort to evaluate if there was a relationship between chronic
inflammation and tumour development we determined tumour indicating parameters
like polyploidization, centrosome hyperamplification, multipolar mitoses and
the state of p53 during inflammation in humans and mice.
In inflammatory tissue of human and rodent wounds we detected an increased
tetra- and polyploidization rate, accompanied by centrosome hyperamplification
in human wounds. Tetraploid cells seem to be advantageous during times of
unfavourable circumstances, as they are during inflammation. Their number
decreased at the end of the healing process during scar formation, coordinated
by an intact p53.
In inflammatory tissue of the bronchus, we also detected tetra- and aneuploid
cells as well as centrosome hyperamplification, both increasing with the grade
of inflammation. Since tetraploidy is defined as an intermediate during
tumourigenesis, a decrease of this population, which we could see after
transient inflammation in the human wound, is necessary to prevent a malignant
development. However chronic inflammation, as in the bronchus, promotes
aneuploidization and malignancy because the unfavourable conditions are
persistent and expose the cells to prolonged stress.
We could also show, that an enhanced number of centrosomes plays a critical
role during the process of segregating the chromosomes into normal euploid
(diploid) cells as well as into aneuploid ones.
P0364
Detection of cerbB-2 gene amplification in bilharzial bladder cancer using
fluorescence in situ hybridization
H. W. Mostafa 1, M. S. Aly 1, H. M. Khaled 2;
1Faculty of Science, Cairo University (Beni Suef branch), Cairo,
EGYPT, 2National Cancer Institute, Cairo, EGYPT.
Gene amplification are common in many different tumor types and may confer
diagnostic, prognostic, or therapeutic information for patient management.
Amplification and overstatement of the cerbB-2 gene occurs in different types
of cancer. Fluorescence in situ hybridization (FISH) represents the newest
methodological approach for testing for this genetic alteration.
In this study, FISH is used in a series of archival human bilharzial bladder
cancer specimens to evaluate for the presence of cerbB-2 gene alterations in
the most common malignant tumor in Egypt and some other countries. The study
included 41 cases, 32 males and 9 females. Twenty-one cases had squamous cell
carcinoma, 17 had transitional cell carcinoma, 2 had adenocarcinoma, and one
case had undifferentiated carcinoma. After performing radical cystectomy to
these patients, stage p3b was the commonest lesion being present in 22 cases,
while p3a lesions occurred in 9 cases, p2 in 6, and p1 in one case. Pathologic
tumor stage could not be assessed in 3 cases. Pelvic nodal affection occurred
in 12 out of 36 (33.3 %) in whom pelvic nodes were examined.
Our data demonstrate that 16 of 41 tumor samples (36.6%) show evidence of true
cerbB-2 gene amplification. Of the remaining samples, 21 (53.4%) show no gene
amplification and 4 (10 %) fall into the borderline category with a ratio
between one and two cerbB-2 genes/cell relative to chromosome 17 centromeres.
Our data indicate a possible role of the cerbB-2 gene in the development of
aggressive behavior in bilharzial bladder tumors.
P0365
Micronucleus Frequencies in Buccal Mucosa,Urothelial Cells and Peripheral
Blood Lymphocytes of Smokers.
S. Demirel, A. G. Zamani, G. H. Durakbasi, A. Acar;
Department of Medical Genetics,Meram Medical Faculty,Selcuk University, Konya,
TURKEY.
The micronucleus (MN) assay in human peripheral blood and exfoliated cells has
been widely used to detect genotoxic effects of environmental mutagens,
infectious agents and hereditary diseases. In the present study, we aimed to
determine the genetic toxicity of cigarette smoking. MN assay was performed on
buccal mucosa, urothelial cells and periheral blood lymphocyte samples
obtained from 15 healthy male smokers ( > 5 pack-years) and 15 male
non-smoker controls who had not been exposed to any known genotoxic agent. It
was found that, the mean value (+/- SD) of MN frequency in oral mucosa cells
from smokers and controls were 1,208 ± 0,220 % and 0,264±0,105 %, in
urothelial exfoliative cells 1,292±0,280 % and 0,120±0,088 %, in peripheral
blood lymphocytes 1,534±0,234 % and 0,386±0,124 %, respectively. The mean MN
frequencies in the buccal mucosa, urothelial exfoliative cells, and peripheral
blood lymphocytes were significantly higher in smokers than in those of
controls ( p< 0,0005). Our data suggest that chromosomal damage in these
tissues were induced by cigarette smoking.
P0366
Chromosomal aberrations in Cis and Ta bilharzial bladder cancer
M. S. Aly 1, H. M. Khaled 2, N. Mokhtar 2;
1Faculty of Science, Cairo University (Beni Suef branch), Cairo,
EGYPT, 2National Cancer Institute, Cairo University, Cairo,
EGYPT.
Carcinoma of the bladder is the most prevalent cancer in Egypt and most
African countries.At the National Cancer Institute, Cairo, it constitutes
30.3% of all cancers.The median age at diagnosis is 46 years,with male
preponderance of 5:1.It has several unique clinical, epidemiological, and
histological characteristics suggesting that it is a distinct entity from
bladder cancer seen in Western countries.Genetic alterations in bilharzial
related bladder cancer have been studied infrequently, and specially in the
advanced sittings i.e. T3 and T4 stages.The objective of this study was to
extend establishing the base line cytogentic profile of this type of
malignancy to carcinoma in situ stages. For this purpose, FISH was applied to
interphase nuclei of frozen-stored samples with biotinylated repetitive DNA
probes specific for all chromosomes to detect numerical chromosome changes in
25 patients presenting with carcinoma in situ of bladder. Twenty four cases
had transitional cell carcinoma and one case had squamous cell carcinoma. Six
out of 24 TCC cases had diploid chromosome count with all the probes.
Numerical chromosome aberrations were detected in 18 cases (75%).In 8 cases, a
loss of chromosome 9 was observed.In one case, an additional loss of
chromosome 17 was detected.One case demonstrated a loss of chromosome 17,
whereas another two cases showed a gain of chromosome 7. Loss of chromosome Y
was observed in 9 of the 22 male cases studied (40.9%).The only case with SCC
had normal diploid chromosome count with all the probes used. A theory of
bilharzial bladder cancer pathogenesis is suggested.
P0367
Partial Deletion 18p in a Patient with Psoriasis Vulgaris
R. Mikelsaar 1, K. Muru 1, K. Varb 1, A.
Kulla 2, A. Süvari 3;
1Institute of General and Molecular Pathology, University of Tartu,
Tartu, ESTONIA, 2Department of Pathology, Tartu University Clinics,
Tartu, ESTONIA, 3Viljandi Children Aid and Social Center, Viljandi,
ESTONIA.
Deletion 18p (18p-) is characterized by variety of clinical features including
mental retardation, facial abnormalities and skeletal anomalies. Skin
abnormalities are rarely reported. There are no cases of psoriasis vulgaris in
patients with the 18p- syndrome. Psoriasis vulgaris is a chronic inflammatory
skin disease that affects about 3% of the caucasian population. The psoriasis
susceptibility loci have been suggested within many chromosomes (1p, 1q, 2p,
6p, 17q etc.). We describe the first case of psoriasis vulgaris in a male with
18p- syndrome. Examination at the age 27 years showed psoriatic red scaly
patches on the skin of his head and forearms. He was moderately mentally
retarded and had dysmorphic features: short stature, downslanted palpebral
fissures, exophthalmus, prognathism, kyphosis, brachydactyly, micropenis etc.
Cytogenetic analyses using GTG banding revealed partial deletion 18p in all
cells studied. FISH showing two fluorescent signals with WCP18 DNA probe and
one signal with 18p subtelomeric DNA probe on metaphases confirmed the
finding. Karyotype is 46,XY,del(18)(:p11.23-qter). As psoriasis vulgaris has
not been previously described in patients with 18p- syndrome, one might argue
that they have not reached an age to be able to determine whether they are
affected with psoriasis or not. Follow-up examination of these patients will
be necessary. The coexistence of psoriasis vulgaris and 18p- in our patient is
possibly an association, but it might also be a causal connection and may be
helpful in the localization of an additional susceptibility locus for
psoriasis in the region of 18p.
P0368
Case report of a de novo duplication 14q11.2-q21.2
B. Oehl-Jaschkowitz 1, M. G. Shamdeen 2, S. Reichardt 1,
V. Kalscheuer 3, K. D. Zang 1, T. Martin 1;
1Institute of Human Genetics, Homburg, GERMANY, 2Centre
for Paediatric and Juvenile Medicine, The Saarland University Hospital, Homburg,
GERMANY, 3Max-Planck-Institute for Molecular Genetics, Berlin,
GERMANY.
We present the case of a 10-month-old girl, second child of nonconsanguineous
healthy parents, with developmental delay, growth retardation (height and head
circumference 3 rd percentile), several dysmorphic stigmata and
severe West syndrome.
Conventional chromosomal banding analysis led to the suspicion of one
derivative chromosome 14. A whole chromosome paint for chromosome 14 gave a
homogeneous staining, excluding an interchromosomal rearrangement. CGH
revealed a gain of chromosomal material of the region 14q11-21. FISH analysis
with region specific YACs confirmed the duplication. The duplication spans
from 14q11.2 to 14q21.2. and accounts for approximately 16Mb. Chromosomal
analysis of both parents showed normal karyotypes. Even a minor 14q
duplication of one parent was excluded by FISH. There is no published case of
proximal duplication 14q with exactly the same breakpoints. Thus, it is
conceivable that not all clinical features of our patient are concordant to
other patients with proximal duplication 14q. Especially West syndrome has yet
not been described in this context.
Methodologically our case confirms that CGH is a useful tool in molecular
cytogenetics particularly for the detection of small chromosomal
rearrangements not concerning the subtelomeres for which a specific diagnostic
is available.
P0369
The shape, length and banding pattern of human interphase chromosomes
U. Claussen 1, J. Lemke 1, J. Claussen 1,
I. Chudoba 2, T. Liehr 1, P. Muehlig 3, K.
Sperling 4;
1Institute of Human Genetics and Anthropology, Jena, GERMANY, 2MetaSystems,
Altlussheim, GERMANY, 3Institute of Molecular Biotechnology, Jena,
GERMANY, 4Institute of Human Genetics, Berlin, GERMANY.
Interphase chromosomes analysed with currently available techniques do not
present any recognizable structures such as bands, centromeres, telomeres, or
specific shapes. Microirradiation experiments and molecular cytogenetic
investigations with whole chromosome paints and region specific
microdissection probes have confirmed a territorial organization of
chromosomes in interphase nuclei. Until now, however, their structure is not
well understood. Using laser scanning microscopic examination and the
high-resolution DNA-based multicolour banding (MCB) technique, we have
generated a banding pattern and have determined the length of human chromosome
5 in lymphocyte interphase nuclei, and in nuclei of HeLa cells arrested at
different phases of the cell cycle. The shape and MCB pattern of chromosome 5
in interphase nuclei is similar to that of metaphase chromosome 5 at all
stages of the cell cycle. The length of the chromosome axis is comparable to
that of a metaphase chromosome at a 600-band resolution. Therefore, the
concept of chromosome condensation during mitosis has to be reassessed.
Interphase chromosome banding can be used to identify chromosome aberrations
and opens new fields in cytogenetic analysis.
P0371
Using chromosomal microdissection for production of specific molecular
probes.
M. Zawada 1, A. Pienkowska 2, C. Schelling 3;
1Institute of Human Genetics, Polish Academy of Sciences, Poznan,
POLAND, 2Academy of Agricultural Sciences, Poznan, POLAND, 3Swiss
Federal Institute of Technology, Zurich, SWITZERLAND.
The microdissection of chromosomes is the technique for production high
specific molecular probes, which are necessary for diagnostic fluorescence in
situ hybridization (FISH) with structural and numeral abnormal chromosomes,
including marker chromosomes. The specificity of these probes is connected to
identification of chromosomal aberration in precise clinical case, which
diagnosis by using commercial probes is very expensive and time-consuming. To
this time we prepared microdissection of marker chromosomes from human and
translocated chromosome X from dog. We prepared also probes from X and Y
bovine chromosomes for diagnosis of aberration of these chromosomes. All FISH
experiments showed the hybridization signals and allowed for identification of
studied cases. From our experience we know that microdissection of chromosomes
is irreplaceable tool for obtain of probes more adequate for the specific
clinical case than commercial probes.
This work was supported by grant from Committee for Science Research No
6PO6D02821.
P0372
Micronuclei Induction in Rat Embryonic Blood Cells Following Exposure to
Different Modes of Electromagnetic Fields
M. Aksoy 1, H. Acar 1, K. Karabulut 2,
A. Salbacak 2, I. Uysal 2, M. Kaynak 1;
1Department of Medical Biology and Genetics, Selcuk University,
Medical Faculty, Konya, TURKEY, 2Department of Anatomy, Selcuk
University, Medical Faculty, Konya, TURKEY.
In modern society, human population have been exposed to different modes of
electromagnetic fields (EMFs). Many researchers have been conducted on whether
cancer risks may be associated with such exposures. It has known that low
frequency EMFs don't produce enough energy to damage DNA. However,
epidomiological studies suggest that EMFs have been associated with increased
incidence of cancer risk and must be considered as a potential genotoxic
agent. In this study, genotoxic effects of different modes of magnetic fields
were investigated by using the micronucleus assay. For this, rat embryos were
exposed to different modes(5,10,20,30mGA) of EMF for 48 hours in culture
between 9.5 and 11.5 days of embryological development in which early
organogenesis takes place.After culturing the embryos,their blood samples were
collected by removing the visceral yolk sac and the embryo in RPMI-1040
medium.The sample was directly processed for micronucleus assay.Results from
control and experimental groups were compared statiscally.We did not observe
any significant difference between the control and experimental groups
(p>0.05),at these low modes of EMF. Further experiments will be carried out
in order to examine the genotoxic effects of higher doses of EMF on rat
embryonic growth and development.
P0373
A complex sex chromosome abnormality associated with short stature and
hypogonadism
M. Volosciuc 1, E. Braha 1, C. Bujoreanu 2,
M. Covic 2;
1University of Medicine and Pharmacy "Gr.T.Popa" Iasi,
Iasi, ROMANIA, 2University of Medicine and Pharmacy
"Gr.T.Popa", Iasi, ROMANIA.
Feminine hypogonadism is characterized by delay puberty, absent of the sexual
characters and primary amenorrhea. In some cases these findings are due by
gonadal dysgenesis which are based a numerical or structural chromosome
anomalies.
We reported a 15 years 2 months old girl with proportionate short stature,
thin body, delay puberty (primary amenorrhea, B1, PH3). Supplementary features
were excessive pigmented nevi (on face and thorax), narrow, hyperconvex nails
and thumbs with radial angulation.
The pregnancy and delivery were uneventful and the proposita was born at term
with 2300g weight. She is the first child of young healthy non-consanguineous
couple. Both parents have healthy children from others marriages.
Sex chromatin showed two Barr bodies. Other investigations performed are
showing a mean intelligence (IQ= 106), high levels of FSH and LH and the
presence of uterus with bilateral polycystic ovaries (echographic exam of
pelvis).
G-banded chromosomal analysis (with an average resolution of 450-550 bands)
revealed a 46,XX/47,XXX/45,X/46,X, +mar karyotype.
The patient combine clinical features from different gonadal dysgenesis
(Turner syndrome, triplo X). The karyotype could explain miscellaneous
features. The origin of marker presently being arranged for and results, if
any, will be shown at the meeting
P0374
Investigation of chromosomal aberrations in hepatocellular carcinoma in Egypt
by fluorescence in situ hybridization
H. El-Gebaly 1, M. S. Aly 1, H. M. Khaled 2;
1Faculty of Science, Cairo University (Beni Suef branch), Cairo,
EGYPT, 2National Cancer Institute, Cairo, EGYPT.
Hepatocellular carcinoma (HCC) is a very common and highly malignant tumor,
associated mainly with chronic viral hepatitis, cirrhosis of any cause,
aflatoxin exposure and ethanol consumption. Cytogenetic analysis on HCC has
been limited because of poor hepatocyte growth in vitro. Conventional
cytogenetic studies have demonstrated frequent abnormalities of specific
chromosomes in hepatocellular carcinoma. Molecular cytogenetic approaches have
applied only rarely in the characterization of hepatocellular carcinoma (HCC).
The main aim in this study was to evaluate genetic aberrations of different
chromosomes in HCC.
The study included 30 patients with hepatocellualr carcinoma who have been
diagnosed and treated at National Cancer Institute, Cairo University during
the period 1997-1998. The charts of the patients were reviewed to retrieve
their clinico-pathologic data. They were 20 males and 10 females with a M/F
ration of 2. Their ages ranged between 14 years and 80 years (median 55
years).
Interphase cytogenetics by fluorescence in situ hybridization with the use of
a panel of centromere-associated DNA probes for chromosomes 1, 2, 3, 4, 5, 6,
7, 8, 9 and 10 were performed on paraffin-embedded HCC specimens. Numerical
abnormalities of chromosomes 1, 4, and 6 were found in 7, 15 and 12 cases,
respectively. Trisomies of chromosomes 8 and 9 were found in 6, 9 cases
respectively. Gain and/or loss of more than one chromosome were detected in 27
of 30 cases. Gains and losses of DNA found in this study probably involve
oncogenes and tumor suppressor genes that play a role in the puzzle of
hepatocarcinogenesis.
P0375
The effects of diagnostic ultrasound exposure on the meiotic prophase in rats
oocytes.
E. Lapina, O. Krasnikova, T. Kuznetzova, V. Baranov;
Ott's Institute of Obstetrics and Gynecology, RAMS, St-Petersburg, RUSSIAN
FEDERATION.
Over the past 30 years ultrasound has become a widely used tool in obstetrics
diagnostic. The safety of obstetric ultrasonography was proved with standard
teratologycal methods. Recently a number of biological effects have been
observed following diagnostic ultrasound exposure in various experimental
systems (Tarantal, Hendrickx, 1989; Jensh et. al, 1995; Newnham et. al.,
1993). Taken into account a widespread application of ultrasound in prenatal
medicine and obstetrics its effect on oogenesis in mammalian fetuses deserves
special attention.
The subjects were 19 albino rats. The proportion of meiotic prophase stages in
the fetal ovaries on the 21st day of development were studied after 30 minutes
exposure to diagnostic ultrasound (intensity <100mW/cm2, frequency 5MHz) of
female rats on the 17th day of pregnancy (group 1). The control pregnant rats
were assigned to two other groups: group 2 - the sham-irradiated group that
was treated in the same way except for irradiation (n=5); group 3 - untreated
group (n=5). Most cells in all groups (56,44±8,33
- in group 1; 63,66±6,03 - in group 2; 69,68±10,8
- in group 3) demonstrated zygotene-pachytene figures. No differences were
found in distribution of meiotic prophase stages in the groups. Our data
demonstrate that diagnostic ultrasound exposure has not significant influence
on the developmental schedule of oocytes maturation.
P0376
Analysis of meiotic prophase in normal and abnormal human female
fetuses
O. Krasnikova, E. Lapina, T. Kuznetzova;
Ott's Institute of Obstetrics and Gynecology, St-Petersburg, RUSSIAN
FEDERATION.
Mammalian oocytes at meiotic prophase stage demonstrate a remarkable
sensibility to different mutagenic factors (Kolomiez et. al., 1992), therefore
the investigation of exogenous and endogenous factors effect on human germ
cells dynamics is of specially importance.
The 19 human female fetuses on 19-26 weeks of gestation were assigned to 4
groups respective to abortion reasons. The distribution of meiotic prophase
stages in the oocytes was studied on 76 cytogenetic preparations.
In fetuses on 19-20 weeks of gestation (n=12) meiotic figures were represented
by predominantly zygotene stage cells. In spontaneous abortion the amount of
diplotene-dictiotene cells was statistically lower (t=2,94 P>95%) compared
to the control group (social reasons abortion).
During the 23-26 weeks of gestation in malformations fetuses with normal
karyotype and (n=4) dictiotene stage predominate, while in aberrant karyotype
group (n=3) the amount of cells on this stage was 10 times less.
The decrease in amount of cells at the end of prophase 1 can be attributed to
partial degeneration of oocytes during the preceding stages.
The differences in distribution of oocyte meiosis stages in the embryos of the
same age suggest the existence of different factors that influence on meiotic
prophase in human oogenesis
P0377
Balanced chromosomal anomalies found in persons with reproductive
failures
A. R. M. Stana 1, A. G. Lungeanu 2, D.
Pelinescu-Onciul 3;
1Filantropia Hospital, Titu Maiorescu University, Bucharest, ROMANIA,
2National Institute"Victor Babes", Bucharest, ROMANIA, 3Filantropia
Hospital, Bucharest, ROMANIA.
Sometimes in the field of human reproduction we forget about the necessity of
cytogenetically examination of infertile couples. We found some cildless
couples that have already been treating their infertility for years. In three
of such couples we identified one partner as a carrier of a constitutional
chromosomal anomaly. From them two cases were certified as familial
rearrangements. First, a pericentric inversion of chromosome 5 in a
normospermic man whose wife, after seven spontaneous abortions, finally gave
birth to a healthy boy who inherited the same karyotype from his father. The
second one, a maternal translocation t(13;18) in a woman detected after eight
years period of sterility and two spontaneous abortions. Another balanced
translocation t(5;13) without familial data was found in a normospermic man
whose wife had four repeated unsuccessful pregnancies. When the pacient learnt
about the diagnosis he did not want to cooperate any longer, so we could not
contact other members of his family. Our results point out the value of the
cytogenetically screening of the couples with reproductive failures as well as
the emotional and familial aspects of the genetic counselling.
P0378
Molecular cytogenetic characterization of disseminated tumor cells in renal
cell carcinomas
R. Gangnus;
Institut für Humangenetik der TU München, Munich, GERMANY.
Bone marrow is the most important secondary organ for the dissemination of
epithelial cells in tumor patients. Several recent studies demonstrated that
the detection of such disseminated cells may represent an independent
prognostic factor in several tumor entities. However, in urologic tumors the
role of disseminated epithelial cells has up to date not been established.
Furthermore, a detailed characterization of the genome of these rare events
remains an arduous task. The aims of our study include the development of new
molecular cytogenetic methods for a detailed characterization of disseminated
cells and their application on bone marrow of patients with urologic tumors,
such as renal cell carcinomas and bladder cancer.
Our molecular cytogenetic strategy employs in a first step an enrichment of
disseminated cytokeratin-positive cells using established protocols (e.g.
magnetic beads). In a second step, disseminated cells are analyzed either by
multicolor interphase-FISH and/or by single cell CGH. Multicolor
interphase-FISH is done by using simultaneously at least seven different
DNA-probes, each labeled in a different color.
RCC was chosen as a model tumor entity for two reasons: firstly, our knowledge
about cytogenetic rearrangements in this tumor entity is fairly advanced.
Secondly, chromosomal rearrangements have a limited complexity .
First applications of this strategy demonstrating the feasibility for a high
resolution characterization of the genome of rare cells will be shown. This
strategy should allow to gain new insights into genetic changes involved in
tumor progression and in early dissemination. The application of these methods
is currently also extended to bladder cancer.
P0379
Karyological analysis of immature germ cells in sperm from men with impaired
spermatogenesis.
I. Fedorova 1 ,2, J. Loginova 1, L.
Petrova 1, O. Chiryaeva 1, T. Kuznetzova 1;
1Ott’s Institute of Obstetrics and Gynecology, St-Petersburg,
RUSSIAN FEDERATION, 2St-Petersburg State University, St-Petersburg,
RUSSIAN FEDERATION.
The diagnosis of male infertility is based on analysis of ejaculate and testis
biopsy. Due to definite limitations of testis biopsy analysis of ejaculate is
usually more preferable. Semenograme allows to obtain information about mature
germ cells: concentration, motility and morphological characteristic of
spermatozoa. However, more detailed information about spermatogenesis might be
obtained by analysis of ejaculated immature germ cells using the method called
Quantitative Karyological Analysis of Immature Spermatogenic Cells (QKAISC)
(Kurilo,1993). This method permits to determine the relative portion of germ
cells at different stages and the stage of spermatogenesis block.
The samples of semen from 4 control subject and 25 patients with impaired
spermatogenesis were analysed using QKAISC technique. The partial block of
spermatogenesis at the prepachytene stages was detected in 2 patients with
47,XXY, but in patient with 46,XY/47,XXY spermatogenesis block was detected
after MI division. The partial arrest of spermatogenesis at the prepachytene,
pachytene and diplotene stages was detected in 2 patients with 46,XY t(9;13)
and 46,XY t(Y;5). In 8 46,XY patients with azoospermia the increased number of
degenerated spermatogeneous cells and somatic cells was detected. In 5 46,XY
patients with oligoastenoteratospermia and oligoastenospermia the partial
block of spermatogenesis was detected after MI division with increased number
of degenerated spermatogenic cells. There were no difference between control
subjects and patients with normal and minor impairments of semen
characteristics. These results demonstrate the significance of QKAISC
technique in performing on complex examination of ejaculate from patients with
infertility.
Kurilo et al. Probl. Repr.(rus) 1995. v.3. p.33
P0380
Interstitial deletion in 10q11.2 : a nonpathogenic euchromatic deletion
M. Gregoire 1, V. Bourdon 1, F. Rousselet 1,
D. Guillet-May 2, J. Hubert 3, P. Jonveaux 1;
1Laboratoire de Génétique-EA 3441, CHU, Nancy, FRANCE, 2Service
d'Assistance Médicale à la Procréation Maternité Régionale, Nancy, FRANCE, 3Service
d'Urologie, CHU, Nancy, FRANCE.
Euchromatic autosomal imbalance detectable at the cytogenetic level is usually
associated with mental retardation and congenital abnormality. However,
exceptional families have been reported in which cytogenetically visible
deletions are segregating without detectable phenotypic effect. We report on a
family in which a young man was referred for chromosomal analysis for
infertility associated with azoospermia. A complex chromosomal rearrangement
was observed, comprising a reciprocal translocation with a deletion :
46,XY,der(10) del(10) (q11.2q11.2) t(4 ;10)(p15.1 ;q11.1). Blood from his
brother and their mother was cultured and the same chromosomal abnormality was
found in both. FISH study with specific whole chromosome painting probes and
comparative genomic hybridization were performed both in the index case and
his relatives, confirming the complex rearrangement. Reciprocal translocations
may be associated with male infertility, as an insurmontable obstacle to cell
division in the spematocyte, resulting in azoospermia.(oogenesis is apparently
less vulnerable). To our knowledge, only one case of this deletion have been
reported in the literature, without phenotypic consequences. Based on these
data, deletion of band 10q11.2 appears to have no phenotypic consequences.
P0381
Sex reversal secondary to Xp functional disomy including DAX1 gene by
t(X;Y)(p21.2;p11.3)
D. Sanlaville 1, F. Vialard 1, J. Gekas 2,
L. Vue-Droit 3, A. Corré 4, B. Devauchelle 2, A.
Ardalan 1, V. Malan 1, T. Martin-Denavit 1, P.
Nizard 1, F. Thépot 2, J. Taillemite 1, M.
Portnoï 1;
1Hôpital Saint Antoine, Paris, FRANCE, 2Hôpital
d'Amiens, Amiens, FRANCE, 3Hôpital de Saint Quentin, Saint Quentin,
FRANCE, 4Hôpital Trousseau, Paris, FRANCE.
Translocations involving the short arms of the X and Y chromosomes are
extremely rare. They result from a recombinaison in the paternal germline. The
most frequent are reported in XX males and rarely in XY females, resulting
from SRY gene transfer, leading to complete sex reversal. Male-to-female sex
reversal has been also observed in individuals with partial duplication of Xp
and an intact SRY gene. The dosage sensitive sex-reversal is due to
duplication of the DAX1 gene in Xp21.2. We describe a 7 month-old phenotypic
female child with severe psychomotor retardation, growth retardation,
dysmorphic features, cleft palate, cardiac and cerebral anomalies. The
patient's karyotype was 46,X,+ mar in all cells examined. The karyotype of
parents were normal. FISH techniques provided evidence that the derivative
chromosome was a der(Y) resulting from the transposition of Xp material,
including Xp21, onto the terminal short arm of an Y chromosome. No deletion of
the Y chromosome was detected. The child's karyotype was: 46,X der
(Y)t(X;Y)(p21.2;p11.3). Abnormal clinical findings of our patient is due to
funtional disomy for Xp21.2-pter segment, because the Xp translocated portion
is active. Her complete sex reversal results from the presence of two active
copies of DAX1 gene. The phenotype of our patient is similar to previous
reports of genetic males carrying a partial duplication of Xp. To our
knowledge, this is only the second report of a t(X;Y) resulting from the
transposition of Xp to the short arm of the Y chromosome. Genes involved in
human sex determination are discussed.
P0382
Are There Interchromosomal Effects of Chromosomal Rearrangements on
Occurrence of Aneuploidy in Sperm Nuclei of Carriers?
H. Acar 1, T. Yakut 2, T. Çora 1, Ü.
Egeli 2, M. Kaynak 1, S. Yildirim 1;
1Department of Medical Biology and Genetics, Selçuk University,
Medical Faculty, Konya, TURKEY, 2Department of Medical Biology and
Genetics, Uludag University, Faculty of Medicine, Bursa, TURKEY.
Translocation carriers have been considered as reproductive failures, either
due to spermatogenetic arrest or to unbalanced progeny. During the meiosis,
both translocated chromosomes and their normal homologues can lead to
unbalanced patterns of segregation for these chromosomes. Analysis of live
born offspring or fetuses does not provide accurate information about meiosis
segregation products because of early missing of abnormal conception. However,
it has been postulated that there is an increased frequency of aneuploidies
involving unrelated to the translocation, termed as interchromosomal effect.
Therefore, in this study, we aimed to present our primary findings for
evaluation of interchromosomal effects of different rearrangements, such as
different balanced reciprocal and Robertsonian translocations and inversion,
on occurrence of aneuploidies of sperm nuclei of carriers. Interphase
sperm-fluorescence in situ hybridization (FISH) analysis was performed by
using chromosome specific DNA probes that were not involving in those
rearrangements
P0383
High quality BAC FISH probes for the detection of chromosome imbalances
D. Ehling 1 ,2, A. Tauchen 1, T.
Schmitt-John 1 ,2, J. Weidner 2, J. Wirth 1 ,2;
1Developmental Biology and Molecular Pathology, University of
Bielefeld, Bielefeld, GERMANY, 2Praenadia GmbH, Muenster,
GERMANY.
Conventional fluorescence in situ hybridization (FISH) is a useful tool for
the detection of human chromosomal abnormalities in prenatal and postnatal
diagnostics. The quality of DNA probes contributes to the signal detection and
is important for a confident interpretation of FISH results. We have developed
high quality FISH probes suitable for the identification of the major
trisomies (21, 13, 18) as well as common structural rearrangements such as
deletion of 22q11. Most of our BAC clones were isolated by using the REPuter
program which allows us to visualize distributions of exact and degenerate
repeats with a minimal length of 20 bp. Regions with high gene content and
relatively low repeat density were selected and primers were designed for the
identification of BAC clones. Furthermore, for improvement of the FISH signals
we have assembled BAC contigs and used these as complex probe mixtures. The
BAC clones were mapped to different regions of chromosome 21 including the
centromeric region of 21q11, the Down syndrome critical regions at 21q22, and
subtelomeric region of 21q22.3. Several DNA probes were isolated in the region
of 13q32, 18q21, 22q11 and subtelomeric region of 22q13. The probe set
extremely facilitates the detection of specific chromosome imbalances on
uncultured amniotic fluid cells and metaphase chromosomes and is greatly
useful for the identification of partial trisomies, partial monosomies and
interstitial deletions.
P0384
Gonadal dysgenesis. Presentation of 3 cases and report on the database of a
Paediatric Endocrine Unit in Hungary.
M. Dobos 1, G. Fekete 1, J. Szabó 1, R.
Schellberg 2, R. Raff 2, G. Schwanitz 2, J. Solyom 1;
1Semmelweis University, Budapest, HUNGARY, 2Friedrich
Wilhelms University Institute of Human Genetics, Bonn, GERMANY.
Gonadal dysgenesis encompasses a heterogeneous group of different chromosomal,
gonadal and genital abnormalities characterised by the presence of dysgenetic
testes and/or streak gonads, persistence of Müllerian duct structures and a
variable degree of genital ambiguity or female phenotype. Clinical,
pathological, hormonal and genetic aspects of 24 patients with complete or
partial gonadal dysgenesis have been studied. Three patients had complete
(pure) gonadal dysgenesis (karyotype: 46,XY). The karyotype of the 21 patients
with a clinical diagnosis of partial gonadal dysgenesis (PGD) was 45,X/46,XY
(n=10), 46,XY (n=6) or others (n=5). The types of PGD, based on pathological
findings of the gonads, were mixed gonadal dysgenesis in 5 cases, dysgenetic
male pseudohermaphroditism in 6 cases, bilateral gonadoblastoma in one case
(no histological data are available in 9 cases). Genital phenotype was
predominantly male (n=8), ambiguous (n=9) or predominantly female (n=4).
We present 3 new cases with testicular intersexuality. The cytogenetic
investigations were carried out on both peripheral lymphocyte cultures and on
buccal cells by GTG and FISH technics. The karyotype analyses of two patients
with partial gonadal dysgenesis showed 45,X/46,XY mosaicism in different
proportion of tissues, while the third case proved to be 46, XY/47, XYY mosaic
with genital ambiguity. The phenotype and genotype correlations and hormonal
results are discussed.
P0385
Segregation of Pierre Robin Sequence with a 2;17 (q32;q24)
Translocation
M. Rodríguez de Alba, E. Sánchez-Gutierrez, I. Lorda-Sánchez, C.
González-González, C. Gacituaga, F. Infantes, C. Ayuso, C. Ramos;
Department of Genetics. Fundación Jiménez Díaz, Madrid, SPAIN.
The Pierre Robin Sequence (PRS) consists in the association of micrognathia,
glossoptosis and cleft soft palate. PRS may present as an isolated
(nonsyndromic) feature, generally on a sporadic basis, or may aggregate with
additional findings that together define a syndrome, which in turn may itself
be found to segregate in the family.
Although no genetic locus is known for nonsyndromic PRS, genetic factors are
thought to play a role in this functional and morphological entity. The 2q32
region has been specifically associated with isolated cleft-palate.
We present a father and a daughter carriers of a balanced translocation
between chromosomes 2 and 17 (2q32;q24) and with the phenotipic manifestations
of the PRS. The paternal grandparents were studied and neither of them
presented the traslocation nor the PRS.
HYPOTHESIS: The case presented here suggests the existence of a genetic base
not only for the cleft-palate feature but for the nonsyndromic PRS located on
2q32. The absence of other phenotipic manifestations rather ssuggests the idea
that in this family there is not a microdeletion but a breakage point in the
possible “candidate gene”.
P0386
Identification of marker chromosomes using microdissection and FISH
J. J. M. Engelen 1, A. C. Knegt 2, J. C. M.
Albrechts 1, L. Spruyt 1, C. T. R. M. Schrander-Stumpel 1,
A. J. H. Hamers 1;
1Department of Genetics and Celbiology, University Maastricht,
Maastricht, NETHERLANDS, 2Department of Clinical Genetics, University
of Amsterdam, Amsterdam, NETHERLANDS.
Micro-FISH is a technique that comprises the physical dissection of a
GTG-banded chromosome (part), followed by a degenerate oligonucleotide
primed-polymerase chain reaction (DOP-PCR) to amplify the dissected
chromosomal material, labelling of the PCR product and subsequently reverse
painting. In clinical cytogenetics micro-FISH can be used to characterise
marker chromosomes, (de novo) unbalanced translocations and complex chromosome
rearrangements.
We present four cases with an unbalanced karyotype due to the presence of
(DA/DAPI and NOR negative) marker chromosomes. In a 8-year-old not mentally
retarded boy, referred for cytogenetic examination because of growth
retardation, a mos 47,XY,+mar[75]/46,XY[25] karyotype was determined.
Micro-FISH showed that the minute marker contained the centromere of
chromosome 8. Chromosome analysis in a 26-year-old female showed an extra
marker chromosome in 50% of her lymphocytes. She was referred because of
mental retardation, dysmorphism and obesity. The marker chromosome contained
chromosome region 14q10->14q12. The third patient was referred for
chromosome analysis at the age of 10 years because of growth retardation. She
appeared to be carrier of 2 different marker chromosomes. At the age of 26
years chromosome analysis was repeated. In 47% of the analysed cells both
markers were present, in 7% a small marker and in 29% a larger marker
chromosome was found. Micro-FISH disclosed that one marker contained
chromosome region 19q10->19q13.1, the other marker contained chromosome
region 20p10->20p11.2. The fourth marker chromosome was present in 25% of
amniotic fluid cells of a woman referred because of advanced maternal age. The
contents of this marker chromosome was region 19p10->19p13.1.
P0387
A familial translocation t(3;5) identified by chance in a case of essential
thrombocythemia (ET)
A. A. Arghir 1, N. M. Berbec 2, A. Rodewald 3,
L. Serghei 2, A. G. Lungeanu 4;
1National Institute, Bucharest, ROMANIA, 2"Carol
Davila" University of Medicine, Bucharest, ROMANIA, 3Institute
for Human Biology, Univesrity, Hamburg, GERMANY, 4National
Institute"Victor Babes", Bucharest, ROMANIA.
In this paper we report a familial translocation t(3;5)(q26;q21) found by
chance in a 72 year old female at the moment of the diagnosis of ET. Because
the patient had an excellent treatment outcome and rapidly achieved
hematological remission we supposed that this constitutional translocation
might not be involved in ET pathogenesis, but rather in the reproduction. This
is the reason why we extended the cytogenetic investigations in the patient's
offspring: her 52 year old only daughter and two grandchildren (a boy aged 22
and a girl aged 18). All of them are phenotipically normal including
hematological values. Using peripheral blood samples for chromosomne slides
and GTG-banding followed by FISH with telomere probes and whole chromosome
painting ( Appligene Oncor CP5406G, CP5410R and Cytocell, PCM 356) we
identified the same translocation t(3;5)(q26;q21)in proband, her daughter and
granddaughter, but not in the male offspring. The maternal inheritance of such
a large translocation in three generations without striking reproductive
failures is unexpected taking into account the involvement of chromosomal
rearrangements in meiotic segregation of unbalanced gametes. In order to have
a better and precise
characterization of the breakpoints, extensive studies are needed. Molecular
description of genomic region flanking the 3q and 5q breakpoints will help us
to understand the relationship between this structural rearrangement and
proband's hematologic disorder (ET), if any.
Acknowledgements: ANSTI Grant 5195/1999-2001, and VIASAN project
089/2001-2004.
P0388
A Family With Reciprocal 4;7 Translocation
Y. Tarkan-Argüden 1, S. Yilmaz 1, A. Deviren 2,
D. Kuru 3, A. Yüksel 3, S. Hacihanefioglu 1;
1Istanbul University, Cerrahpasa Medical School, Div.of Biomedical
Sciences, Dept.of Genetics, Istanbul, TURKEY, 2Istanbul University,
Genetics and Teratology Research Center (GETAM), Istanbul, TURKEY, 3Istanbul
University, Cerrahpasa Medical School, Div.of Biomedical Sciences, Dept.of
Medical Biology, Istanbul, TURKEY.
There are reports on balanced translocations of chromosomes 4 and 7 with
different chromosomes with various breakpoints in the parents of abnormal
offsprings with unbalanced karyotypes, but we found no reports of
t(4;7)(q31;p22) in our search of literature.
We present a family that have a balanced reciprocal translocation between
chromosomes 4 and 7.
The proband who was 20 days old, was referred to our department because of
clitoris hypertrophy. She had no other abnormalities. Her Na +, K +,
and 17 a OH progesteron levels were, 139
(N:135-145), 5.5 (N:3.5-5), and 16.8 (N:1.05-40.41), respectively. Her father
was 37, and her mother was 35 year-old at the time of delivery. The marriage
was non-consanguineous. They had a healthy daughter who was 9 year-old. When
the proband was reexamined after 6 months, her genitalia was appeared to be
normal.
Cytogenetic analysis on peripheral blood cultures of the proband by GTL
banding revealed 46,XX,t(4;7)(q31;p22) chromosome constitution. Then we
performed cytogenetic analysis on the parents and the sister. Proband’s
mother had 46,XX karyotype but we found 46,XY, t(4;7)(q31;p22) in her father
and 46,XX,t(4;7)(q31;p22) in her sister. Then two sisters of the father were
studied and revealed 46,XX, t(4;7)(q31;p22) karyotypes.
P0389
Using of dual-color in situ hybridization for detection of numerical
abnormalities in uncultured and cultured leucocytes of radiochemical industrial
workers
V. A. Timoshevsky, S. A. Nazarenko;
Institute of Medical Genetics, Tomsk, RUSSIAN FEDERATION.
As a rule the numerical chromosomal aberrations are not taken into account in
the genotoxicity testing. However, it is possible that many harmful substances
can cause this type of chromosomal alterations. Fluorescence in situ
hybridization (FISH) is a powerful technique that allows numerical chromosome
aberrations (aneuploidy) to be detected in interphase cells. We used
dual-color FISH for detection of aneuploidy in peripheral blood leucocytes of
15 men – radiochemical industrial workers, contacted with complex of
radioactive and chemical substances. Analysis of hypo- and hyperploidy was
carried out for three chromosomal pairs: 7/12, 11/16 and X/Y using
digoxigenin/biotin labeled probes. We found significantly increased
frequencies of numerical chromosome abnormalities in uncultured leucocytes of
the exposed workers compared to the unexposed controls (10 individuals) for
nullisomy Y-chromosomes only. However significant differences were found in
cultured lymphocytes for hypodiploidy of chromosomes 11, 12 and hyperdiploidy
of chromosomes X and 12. Moreover, a tendency to higher values was observed
for hypodiploidy 16 and total frequency of numerical abnormalities in cultured
lymphocytes. We conclude that agents of the radiochemical industry can induce
aneuploidy in leucocytes and cause the premutagenic lesions, which are
expressed during the cultivation of lymphocytes in vitro. It is possible, that
some chromosomes have larger liability to nondisjunction and loss. Our data
suggest that analysis of numerical aberrations must be carried out in addition
to standard cytogenetics tests.
P0390
Inverted duplications associated with terminal deletions : the phenotypic
impact of this newly recognizable rearrangement.
M. Zollino 1, R. Lecce 2, F. Tiziano 2,
G. Neri 2;
1Medical Genetics Catholic University, Rome, ITALY, 2Medical
Genetics, Catholic University, Rome, ITALY.
Distal inverted duplications associated with a terminal deletion represent a
newly recognizable category of chromosome alterations. Genotype-phenotype
correlations are unclear.
We observed a dup/del rearrangement in 6 patients presenting with MCA/MR
syndrome. It involved chromosomes 4p (3 patients), 5p (one patient), 1q (one
patient) and 19q (one patient). The duplication was detected by conventional
cytogenetics, the deletion by molecular probes only.
Chromosome 4p. Both the duplication and the deletion did differ in size in
individual patients. The deletions, spanning 3.4, 10 and 12 Mb on 4p16,
respectively, encompassed the Wolf-Hirschhorn syndrome critical region"
(WHSCR)in each occasion. Clinically, all patients presented with a WHS
phenotype.
Chromosome 5p. The 5p13.3-p15.1 region was duplicated, the deleted 5p15.1-pter
segment was demonstred by FISH to include the "cri du chat" syndrome
critical region. The patient presented with a "cri du chat" syndrome
phenotype.
Chromosomes 1q and 19q. A very distal duplication was observed in these cases,
affecting the 1q42-q44 and 19q13.2-q13.4 regions, respectively. Accordingly, a
very small deletion, just including the subtelomeric region, was detected.
Clinical signs in both patients were consistent with the respective partial
trisomy syndrome phenotype.
We observed that, whenever the deletion included critical regions with strong
pleyotropic effects, such as the WHSCR and the "cri du chat"
syndrome CR, clinical signs were consistent with a "deletion"
phenotype only. A partial trisomy syndrome phenotype was observed in cases
with a small subtelomeric deletion.
A proper assessment of the deletion is recommended in dup/del rearrangements.
P0391
Mosaic Trisomy 7; An Age Related Or Tissue Specific Finding In Normal Human
Tissues
Y. Mehraein, S. Bastian, C. Fromberg, K. D. Zang;
University of the Saarland, Dept. for Human Genetics, Homburg/Saar,
GERMANY.
Mosaic trisomy 7 has been found in a variety of tumors, non tumorous lesions,
and even apparently normal tissues. Although characteristic in some condition,
the meaning and pathological relevance of trisomy 7 is still unclear. Recent
investigation has given rise to the question, whether somatic gain of
chromosome 7 is rather related to age than to a specific disease.
By FISH we analyzed the chromosome 7 copy number in blood lymphocytes of 30
healthy individuals with normal conventional karyotype selected for three
different age groups. FISH was performed as cohybridization of centromeric
probes for chromosome 7 and 10; 1000 interphase nuclei each were analyzed.
Furthermore to identify a potential tissue specific increase of trisomy 7 in a
group of 12 old individuals (> 80 years) 200 interphase nuclei of three
different tissues (blood lymphocytes, hair root , and mucosal cells) each were
analyzed comparatively by FISH.
In blood lymphocytes between all groups for the analyzed chromosomes revealing
trisomy rates of below 0,5%, respectively, no significant differences were
evident; thus an age related increase of trisomy 7 could not be verified for
this tissue. In the intraindividual comparison of cells from different tissues
, however, while blood lymphocytes and hair root cells showed similar low
trisomy 7 rates (on the average 0.8%) in all cases, a variable but significant
increase of mosaic trisomy 7 up to 11,5% was identified in mucosal cells of
several individuals. An age dependence of the observed tissue specific
amplification of chromosome 7 has to be investigated.
P0392
Very large pericentric inversion of chromosome 4 giving rise to a
rec(4)chromosome and Wolf-Hirschhorn syndrome.
T. Ilus 1, K. Õunap 1, O. Bartsch 2, H.
Varendi 3;
1Medical Genetics Center, Tartu University Clinics, Tartu, ESTONIA, 2Institute
of clinical Genetics, Technical University of Dresden, Dresden, GERMANY, 3Department
of Pediatrics, Tartu University Clinics, Tartu, ESTONIA.
We report a case of Wolf-Hirschhorn syndrome in a 1.5-year-old boy born to
phenotypically normal parents. Cytogenetic analysis revealed a deletion at
4p15.3 in the boy and a large pericentric inversion of chromosome 4 in the
father. The family history was unremarkable. The boy had been born at term
with low birth weight, 1700g. Our examination at the age of 2 weeks showed
marked growth deficiency, microcephaly, hypertelorism, strabismus, epicanthal
folds, prominent glabella, cleft lip and palate, downturned "fishlike’
mouth, micrognathia, dysplastic ears, left simian crease and hypospadias. He
also had a cardiac defect (pulmonary stenosis). One year old, he showed
profound mental retardation and severe seizures that were difficult to treat.
The low birth weight and the clinical signs represent typical Wolf-Hirschhorn
syndrome.
Standard cytogenetic analysis from peripheral blood lymphocytes indicated a 4p
deletion, karyotype 46,XY,del(4)(p15.3). The mother‘s karyotype was normal
(46,XX), but the father demonstrated a large inversion of chromosome 4,
karyotype 46,XY,inv(4)(p15.3q35). We then confirmed in the proband, using
FISH-analysis, submicroscopic distal 4q trisomy(q35-qter) in addition to the
terminal 4p deletion(4pter-4p15.3).
FISH karyotypes:
father: fish inv(4)(p15.3q35)(D4S2930/D4S3186+mv::D4Z1+, D4S96+mv)
proband: fish der(4)(qter-q35::p15.3-qter)(D4S2930/D4S3186+mv::D4S96-, D4Z1+,
D4S2930/D4S3186+st)pat
Distal 4q duplication is usually not associated with a distinct clinical
phenotype, and therefore the predominance of the Wolf-Hirschhorn phenotype was
to be expected in this patient. Semicryptic or cryptic large pericentric
inversions represent an important cytogenetic mechanism that can give rise to
terminal deletion/duplication syndromes. Prenatal diagnosis must be considered
in these cases because of the possibility of recombinants, here,
dup(4)(p15.3-pter) and del(4)(q35-qter).
P0393
The collection of DNA probes for prenatal, postnatal and preimplantation
diagnosis by FISH
I. V. Soloviev 1, Y. B. Yurov 1, S. G. Vorsanova 2,
P. Patsalis 3, P. Ioannou 3;
1National Center of Mental Health, Moscow, RUSSIAN FEDERATION, 2Institute
of Pediatry and Children Surgery, Moscow, RUSSIAN FEDERATION, 3Institute
of Neurology and Genetics, Nicosia, CYPRUS.
Creation of representative DNA probe collection is still actual problem in
laboratories of Central and Eastern Europe. Availability of different DNA
probes could help in introduction of FISH technology in many laboratories,
and, therefore, increase the efficiency of cytogenetic diagnosis. We have
identified a large set of original cloned DNA sequences, which could be used
as DNA probes in molecular-cytogenetic studies. Alphoid plasmid and cosmid
clones, containing repetitive elements, hybridized to pericentromeric regions
of most part of chromosomes were identified and tested in clinical-cytogenetic
studies (Vorsanova et al., 1986, 1991, 1996; Soloviev et al.,1993,1994,1995).
We have analyzed large-insertion PAC library, containing more that 110000
genomic clones. We have detected more than 1600 centromeric and 600 telomeric
PAC clones. 350 centromeric and 320 telomeric PAC clones were initially
analyzed by FISH. Large insertion clones from total genomic human PAC library
were identified for most part of telomeric regions. DNA probes with high
potential for diagnosis of common human aneuploidies, involving chromosomes
21, 13, 18, X and Y were carefully selected in cytogenetic studies. DNA probes
for centromeric and telomeric chromosomal regions are usefull both as markers
for cytogenetic diagnosis and as tool for detailed analysis of numerical and
structural chromosomal aberrations in pre-, post- and preimplantation
diagnosis, including the analysis of fetal cells .Supported by grant
COPERNICUS-2.
P0394
Peculiarities of late replication of chromosomes of human fetal and chorionic
villi cell at 7 and 12 weeks of gestation.
A. V. Vorobyeva, A. A. Pendina, O. G. Chiryaeva, T. V. Kuznetzova;
Ott's Institute of Obstetrics and Gynecology RAMS, St-Petersburg, RUSSIAN
FEDERATION.
The pattern of late replicating metaphase chromosomes from 24-hours cultures
of CVS and tissues fragments from five human embryos at 7 and 12 weeks
gestation was studied. BrdU was added by impulses through 2 hours. Monoclonal
anti-BrdU antibodies FITC - conjugated goat anti-mouse antibody were used to
identify the BrdU-labeled sites.
Metaphase spreads with markers of late replications were chosen for analyses.
The intertissue differences in initiation and termination of replication in
pericentric heterochromatin of chromosomes 1, 9 were shown in chorionic villi
and embryonic cells. Pericentric heterochromatin of chromosome 16 was shown to
be latest replicating segment in both tissues compared to pericentric
heterochromatin of chromosomes 1, 9.
At 7 weeks of gestation some G-bands of chromosomes 1, 2, 3, 4, 5, 9, 11, 13,
14, 16 replicated simultaneously with pericentric heterochromatin of
chromosomes 1, 9, 16 in chorionic cells, while in embryonic cells they
replicated earlier than pericentric heterochromatin.
At 12 weeks of gestation however these segments replicated simultaneously with
pericentric heterochromatin of chromosomes 1, 9, and 16 in both tissues. These
data suggest that changes in of replication pattern of heterochromatin regions
probably reflect differences in their functional status in embryonic and
extraembryonic tissues at different stages of embryonic development.
P0395
Rare cytogenetic Klinefelter syndrome variant
H. Bruyere 1, E. Separovic 1, J. Guscott 2,
T. Pantzar 1;
1University of British Columbia, Vancouver, BC, CANADA, 2Vancouver
General Hospital, Vancouver, BC, CANADA.
A 49-year-old male with hypogonadism and gynecomastia was found to have a
46,XX karyotype with additional chromosomal material on the short arm of a
chromosome 15. C-banding and NOR-banding as well as fluorescence in situ
hybridization with chromosomes 15 and Y centromeric probes, a Y chromosome
short arm unique locus probe and a Y chromosome long arm heterochromatin probe
were perform to further characterize this extra material. It was found to
contain the Y chromosome short arm and centromere. This case represents a rare
cytogenetic Klinefelter syndrome variant, with a diploid complement and a
stable dicentric derivative chromosome from an unbalanced translocation
involving the long arm of the Y chromosome and the short arm of a chromosome
15.
P0396
Molecular studies of small marker chromosomes in patients with 45,X/46,X,+mar
mosaicism
J. W. Kim 1, J. M. Kim 1, E. H. Cho 1,
J. T. Seo 2, Y. S. Lee 2, J. M. Yun 2, H. M. Ryu 3,
S. Y. Park 1;
1Laboratory of Medical Genetics, Samsung Cheil Hospital and Women's
Healthcare Center, Seoul, REPUBLIC OF KOREA, 2Department of Urology,
Samsung Cheil Hospital and Women's Healthcare Center, Seoul, REPUBLIC OF KOREA, 3Department
of Obstetric and Gynecology, Samsung Cheil Hospital and Women's Healthcare
Center, Seoul, REPUBLIC OF KOREA.
We have studied the small marker chromosome in four patients with mosaic
karyotype 45,X/46,X+mar. Male patients were referred for azoospermia and
female for primary amenorrhea. We used fluorescent in situ hybridization
(FISH) and polymerase chain reaction (PCR) to determine the origin and
structure of the marker chromosome. FISH studied with SRY/CEPX probe (Vysis)
confirmed the Y origin of the marker and detected that four patients showed
double SRY signals on both ends of abnormal Y chromosome and one male patient
had only one SRY gene. PCR analysis using primers for 11 loci along Y
chromosome including SRY, RPS4Y, DYS14 (Yp), DYZ3 (Ycen), sY84 (AZFa), sY129,
sY134 (AZFb), sY156, sY254, sY255 (AZFc, DAZ gene) and DYZ1 (Yqh) was
investigated. Two male patients were the Yq breakpoint to the region between
AZFa and AZFb. In female patient, the genes from SRY to AZFb were positive. In
other two patients there was a failure to detect the alpha-satellite (DYZ3)
that is an the integral part of the centromere. One of them was the Yp
breakpoint between DYS14 and DYZ3, and the other having one SRY signal carried
the Yp (SRY, RPS4Y, DYS14) and Yq (sY84, sY129, sY134) except DYZ3 unexpectly.
It is assumed that the deletion of distal Yq euchromatin playing an important
role in the spermatogenetic process lead only to azoospermia.
P0397
The chromosomal abnormalities value in primary amenorrhea etiology.
L. M. Serghei 1, A. Stana 2, C. Geormaneanu 1,
V. Stoian 3, A. Arghir 1, A. G. Lungeanu 1;
1National Institute"Victor Babes", Bucharest, ROMANIA, 2Filantropia
Hospital, Bucharest, ROMANIA, 3Bucharest University, Faculty of
Biology, Bucharest, ROMANIA.
Cytogenetic analyses were carried out on 54 cases of primary amenorrhea, using
peripheral blood lymphocytes, GTG and CBG-banding. The monosomy X occured in
49 patients with Turner syndrome(TS). Chromosomal sexual assignment was
established as 46,XY in three phenotypically females. One of them revealed
gonadal tumor when she underwent gonadectomy at the age of 21. The involvement
of autosomes in the ethiology of primary amenorrhea came into discussion in
two cases;the karyotyping showed the folowing cellular
mosaics:[46,XX,+18pter(80%)/45, XX,-14,+18pter(20%)] and [45,X0(60%)/46,
X0,mar(?)(40%)], respectively.
At the time patient 1 reffered, she had hormonal-induced intermittent,
irregular menses. We attempted to find correlation between the constitutional
+18pter and the observed resistance to hormone-replacement therapy. Normal X
chromosome pattern was noted both in hypodiploid and diploid cells. Neither
the cryptic sex chromosomal abnormalities nor the origin of the 18pter extra
material from chromosome 14 are excluded. Regarding patient 2, we mention that
monosomy X was present in all 200 examined metaphases, strongly suggesting TS
as cause of primary amenorrhea, but the patient lacked Turner stigmata.
Moreover, the diploid cells exhibited a marker that appeared to be a dicentric
derived from a translocation between 21 and 20 chromosomes. The cytogenetic
approach proved once again its value in diagnosis of primary amenorrhea. Our
study has to be extended at molecular level, in order to define accurately the
provenience and the roles of the markers, especially those derived from
autosomes.
Acknowledgements: VIASAN PROJECT NO. 127/2001-2003
P0398
Result of Cytogenetic Study of 850 Bone Marrow Samples
M. Zanganeh, R. Karimi-Nejad, T. Nayeb Bagher, S. Voghouie, H. Miri,
M. Karimi-Nejad;
Karimi-Nejad Genetics and Pathology Center, Tehran, IRAN (ISLAMIC REPUBLIC
OF).
More than 850 bone marrow samples have been analyzed in our center during the
past five years. Among 850 patients, 486 (57%) were referred for malignant
disorders and 131 cases (15%) for non-malignant disease. 233 casses (28%)
didn't have any primary diagnosis at the time samples were taken of which 169
belonged to the first 500 samples. Of the non-malignant cases, 87 were
referred for anemia, 43 pre-transplant, 44 post-transplant, and 44 for post
transplant study of a malignant disorder. The probable clinical diagnoses for
the malignant cases were as follow: 164 (34%) for ALL, 135 (28%) AML, 123(26%)
CML, 23(5%) MDS, 21 (4%) lymphoma, 6 (1%) Multiple Myeloma, 6 (1%) MPD and 8
(1%) others.
Chromosomal aberrations were detected in 73 (50%) of the 145 successful
cultures of patients without any diagnosis, 69 (54%) of 128 cultures with
diagnosis of ALL, 68 (55%) of 123 AML patients, and 80 (73%) of 109 patients
suspected for CML. Among 22 conclusive MDS cases, 11 (50%) patients had some
chromosomal change, ten (50%) out of 20 lymphoma cases and 3 out of 6 MPD and
none of the 4 Multiple myeloma had chromosomal changes.
Among 88 post-transplant cases, 44 (50%) were malignant and 44 (50%)
non-malignant. 72 (82%) patients showed donor cell line, 8 (9%) patients
showed the recipient cell line and another 8 (9%) patients showed chimerism of
donor and recipient cell lines.
P0399
Evidence for nonhomologous meiotic co-orientation (NMC) in man
N. V. Kovaleva;
Institute of Obstetrics and Gynaecology RAMS, St.Petersburg, RUSSIAN
FEDERATION.
Homologous Robertsonian translocations/isochromosomes represent unique model
for studying chromosome behavior in the absence of homologous pairing and
recombination. Though the number of published cases is small, and the only
data available is the sex of the offspring of the dup(21q) carriers, some
conclusions on the segregation pattern of both rearranged chromosomes and
gonosomes can be made. It was found that 7 male carriers of dup(21q) fathered
13 children with Down syndrome of known sex. 12 of them were males, differing
from 1:1 ratio (p=0.0017, binomial test). There was no significant male
predominance in cases of maternal transmission (23 males, 16 females). This
finding is considered as a direct evidence for NMC of the dup(21q) and
X-chromosome. NMC, proven for Drosophila [Grell, 1971], was proposed to
explain male excess in Down syndrome (DS) with paternally derived trisomy
[Kovaleva NV.Genetika(Russ)1992,28:5-15]. NCM of chromosomes 21 and X would
result in production of different gametes: 23,X, 23,Y, 23(+21,-Y), 22,XY(-21),
22,0(-Y), 22,X(-21), 24,Y(+21), 24,XY, also explaining the occurrence of
gonosome aneuploidy and double aneuploidy of paternal origin. Further data
supporting the NMC hypothesis come from studies on sperm in healthy and
infertile men [Griffin et al,1996; Baumgartner et al,1999], in fathers of
patients with aneuploidy [Blanco et al,1998; Soares et al,2001] and in
carriers of balanced translocations (“interchromosomal effect”) [Morel et
al,2001; Pellestor et al, 2001], together with data on elevated risk of
progeny with autosomal trisomies in carriers of sex chromosome anomalies
(Nivelon-Chevallier et al, 1988; Tarani et al, 1998; Hennebicq et al, 2001).
P0400
One karyotype with two features,a report from Iran.
M. Zamanian 1, S. Tootian 1, S. Joghehdost 1 ,2,
M. Houshmand 1 ,2;
1Blood transfusion organization, Tehran, IRAN (ISLAMIC REPUBLIC OF), 2National
Research Center for Genetic Engineering and Biotechnology, Tehran, IRAN (ISLAMIC
REPUBLIC OF).
The index case was a female with a lichenoid lesion on her face. She reffered
to our center for G banding karyotype because her sons were going to marry and
she was worried about her son's future. She was an educated woman and a
laboratory technician and there wasn't any sign of mental retardation. Her
father had 4 children from her mother and four children from another wife. She
had a nephew from her brother and a neice from her sister in law. Both
children were mentally retarded and translocation (2,11) were found in the
both children.
G banding method was used for karyotypeing of our index and her sons.
Translocation(2,11) was detected in the index.We also examined her two sons
for translocations and both of them were normal.There are two questions here:
1)Is this type of translaocation a strong cause for mental retardation of the
neice and nephew? 2)If yes why the index case didn't have any sign of mental
retardation?
It seems that because our procedure cannot detect all the sub bands in a
patient's chromosomes. Microdeletion or inversion may be change the phenotype
in the cases nephew and neice. FISH or molecular methods can be help to find
out the problem in this family.
P0401
Association of der(13;14) with primary amenorrhea
R. Karimi-Nejad, N. Naghash-pour, F. Azimi, S. Razavi, M. Soleimani,
T. Nayeb Bagher, M. Rahimi, S. Voghouie, M. Karimi-Nejad;
Karimi-Nejad Genetics and Pathology Center, Tehran, IRAN (ISLAMIC REPUBLIC
OF).
Among 28000 karyotypes of peripheral blood performed in our center for various
indications, 35 balanced translocations of chromosomes 13 and 14 have been
detected. Of these 35, 26 were referred for history of abortions, IUFDs,
offspring with congenital anomalies, and offspring with trisomy 13.
Interestingly, 6 cases were 3 first cousin couples where both partners were
carriers. The remaining 9 individuals were referred for reasons other than
reporductive failure, 1 for mental retardation, another for premarital check
up and the other 7 for primary amenorrhea.
Considering that 13 of the 35 translocation carriers were males, 7/23
der(13;14) carriers had primary amenorrhea. Cases referred for primary
amenorrhea were aged 12-19 years without history of menstruation, and with one
exception (12 years old) they had normal age related secondary sexual
characteristic development, and growth.
Overall, 413/28000 cases were referred for primary amenorrhea who did not show
related chromosomal changes such as 46,XY and 45,X variants. 10/363 had
chromosomal aberrations including pericentric inversion of chromosome 1 and 2,
paracentric inversion of chromosome 14 and the other 7 had der(13;14).
We would appreciate to be informed of similar findings and any guidance as to
the etiology and possible association our data suggest.
P0402
Gonosomal Mosaicisms derived from a XY-Zygote
R. Schellberg 1, M. Dobos 2, G. Fekete 2,
G. Schwanitz 1, R. Raff 1;
1Institut für Humangenetik, Bonn, GERMANY, 2II.
Kinderklinik der Semmelweis Universität, Budapest, HUNGARY.
Mosaicism is caused by postzygotic aberrant mitoses and leads to numerical or
structural aberrations or a combination of both. The karyotype of the zygote
can be normal or abnormal.
Our investigation group comprises 25 cases of gonosomal mosaicism with a
Y-chromosome analysed in at least one cell system: 45,X/46,XY (n=8),
45,X/46,X,idic(Y)-mosaicism (n=7), 45,X/47,XYY-mosaicisms (n=5),
45,X/46,X,del(Y) (n=4) and 45,X/46,X,r(Y) (n=1).
13 patients were phenotypically male, 7 were female and 5 showed intersexual
external genitalia.
The patients age at the time of chromosome investigation ranged from the
prenatal period up to the age of 36 years.
The most frequent clinical findings were: growth retardation, abnormalities of
the external genitalia, kidney malformations and different types of heart
defect.
Cytogenetic investigations combined metaphase and interphase analyses with
FISH (DNA probes: wcp X and Y; CEP X and Y; Yph 3.4). Between 2 and 5 cell
systems per patient were analysed with regard to their origin from different
blastodermic layers.
An unequal distribution of the mosaic could be demonstrated in the different
cell systems, with no preferential combination.
Patients with dicentric Y-chromosome revealed the highest instability of
karyotype. There was no age dependent karyotype changes in our investigation
group.
Supported by: DAAD/MÖB, Richard-Winter-Stiftung
P0403
12q22q24.33 Duplication: case report and review of the literature
S. Cappellacci 1, S. Martinelli 2, R. Rinaldi 1,
E. Martinelli 1, P. Parisi 3, B. Mancini 2, P.
Grammatico 2 ,1;
1Medical Genetics, San Camillo-Forlanini Hospital, Rome, ITALY, 2Medical
Genetics, University "La Sapienza", Rome, ITALY, 3Outpatient
Service of Pediatric Neurology S. Camillo-Forlanini Hospital, Rome, ITALY.
We present a case of partial duplication of the long arm of chromosome 12
characterized by FISH techniques using YAC probes.
On physical examination, at 8 years, our patient demonstrated: macrocephaly,
flat occiput, long palpebral fissures, long eyelashes, protruding nasal root,
anteverted nostrils, large and asymmetric ears, thin lips, light prognathism,
malar hypoplasia, short stubby hands and femoral dysplasia. Magnetic resonance
imaging showed partial agenesis of the cerebellar vermis and cystic dilatation
of the fourth ventricle consistent with Dandy-Walker malformation.
A neurological and behavioural assessment revealed a psychomotor retardation
and attention deficit/hyperactivity disorder (ADHD).
Standard cytogenetic analysis showed a partial duplication of the long arm of
chromosome 12. Parents’ karyotypes were normal.
To define the extension of the duplicated region we performed FISH analyses by
using the following YAC probes (kindly supplied from YAC Screening Centre,
Tigem, Milan, Italy): 943B11 (q21; 96.5 cM), 778F6 (q22; 97.6 cM), 850A12
(q22; 99.6 cM), 934C1 (q23; 108 cM), 827A10 (q24.1; 137.5 cM), 910B10 (q24;
154 cM), and 812D10 (q24.3; 161 cM). The analyses evidenced a tandem
duplication of the 12q22q24.33 region with the proximal breakpoint located
between 96.5 and 97.6 cM and the distal one between 154 and 161 cM resulting
in the following karyotype: 46,XY,dup(12)(q22q24.33).
In order to contribute to the identification of the characteristic clinical
features associated with 12q partial duplication we reviewed the literature
and compared our case with those already described.
P0404
Ring chromosome 4 associated chromosome instability
M. H. Lee;
Laboratory of Medical Genetics, Samsung Cheil Hospital and Women's Healthcare
Center, Seoul, REPUBLIC OF KOREA.
Ring chromosomes are rare abnormalities that typically arise de novo. The
usual ring phenotype have been shown to have a wide range of intellectual
functioning and congenital anomalies. We found several ring chromosome 4
instabilities in mosaic form with a normal cell line in peripheral blood cells
of a 27 year old woman who was referred for infertility and short stature. The
results of cytogenetic analysis showed 45,XX,-4/ 46,XX,r(4)/ 46,XX,dic r(4)/
47,XX,r(4),+r(4)/ 46,XX karyotype in three repeated examinations. FISH
analysis executed for precise characterization of the ring chromosome 4
breakpoints using chromosome 4p, 4q telomeric probes demonstrated deletion of
the 4p telomere from the ring chromsome 4. Parental karyotypes were normal.
After then, she became naturally pregnant and we performed amniocentesis at 16
weeks gestation. The fetal karyotype was normal. The phenotypes of our patient
seemed to be related to the ring syndrome by the ring chromosome instability.
The present study would be offer information to the long-term consequences of
ring chromosome instability on clinical outcome.
P0405
A novel BAC probe set for the analysis of hematological malignancies
A. Lee 1, D. Lie 1, S. Tien 2, C.
Rudduck Sivaswaren 2;
1Division of Medical Sciences, National Cancer Centre, Singapore,
SINGAPORE, 2Department of Pathology, Cytogenetics, Singapore General
Hospital, Singapore, SINGAPORE.
Background: Translocations and deletions in the chromosome 11q23 region are
frequent in hematologic neoplasms such as acute lymphoblastic leukemia (ALL),
acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic
lymphocytic leukemia (CLL). We describe here the use of a bacterial artificial
chromosome (BAC) probe set from the 11q23 region, applied to these
malignancies.
Methods: Four BAC probes from one contig, and two BAC probes from another
contig on 11q23 spanning the MLL region were labeled with either biotin or DIG
for dual color fluorescence in situ hybridization (FISH) analysis. These
probes were applied to cases with hematologic malignancies with known
structural abnormalities involving 11q23.
Results: A split signal was detected in one ALL case with t(4;11)(q21;q23),
one AML case with t(9;11)(p22;q23) and another AML case with
t(11;19)(q23;p13), demonstrating that the probes span the MLL region known to
be involved in these translocations.
The probes were further tested on one case of AML with add(11)(q23) and two
cases of MDS and two cases of CLL, all with del(11)(q13q23). The additional
material on 11q23 and marker chromosomes were found to be amplification of the
regions detected by the probe set. Although the MDS and CLL cases
cytogenetically appeared to have the same abnormality, the MDS cases had
deletion of both contigs, while the CLL cases only had deletion of one contig.
Conclusions: At a molecular level, deletions in our CLL and MDS cases appeared
different. This study demonstrates the importance of combining molecular and
cytogenetic techniques to further define chromosome abnormalities.
P0406
Deletion of abl gene resulting from a recombination of a maternal
(3;22;9)(q22;q12;q34.1) translocation in a child with axial hypotonia and
dysmorphy.
A. Aboura, P. Labrune, F. Perreaux, V. Poncet, S. Brisset, L.
Foix-L'Helias, G. Tachdjian;
Hôpital Antoine Béclère, Clamart, FRANCE.
Small deletions near breakpoints may be an important cause of disease in
apparently balanced chromosome rearrangements. We report on chromosomal
findings in a boy with axial hypotonia and dysmorphy. He was born after a
full-term uneventful pregnancy. He was the third child of healthy unrelated
parents. The mother has had nine miscarriages. The child was first admitted at
the age of 8 months for gastro-enteritis which was rapidly cured. Physical
examination showed moderate axial hypotonia, hypertelorism and bilateral
epicanthus, high-arched palate, macroglossia, and antimongoloid palpebral
fissures. There was no evidence for visceral malformation. By conventional
cytogenetic analysis a reciprocal balanced translocation between chromosomes 3
and 22 was diagnosed. In order to define clearly the breakpoints on the two
chromosomes, chromosome painting was used and revealed a complex, apparently
balanced translocation t(3;22;9). This translocation was inherited from the
mother. Fluorescence in situ hybridization with different locus probes near
breakpoints showed a deletion of abl gene located at 9q34.1 in the patient.
This deletion was not found in the mother and in the sister carrying the
translocation. Deletion of abl gene has been described once in a newborn with
a complex cardiac anomaly and carrying a paracentric inversion of the long arm
of chromosome 9 (Kleyman et al., Am J Med Genet, 1997).
Our case emphasises that molecular cytogenetic analysis of breakpoints in
apparently balanced chromosomal translocations should be systematically
carried out in patients with phenotypic abnormalities.
P0407
Prenatal Diagnosis of 45,X/47,XX,+8 mosaicism
E. Popelinska 1, D. Leznarova 2, P. Vlasin 2,
P. Kuglik 3;
1Cytogenetic Laboratory, Prenatal Diagnosis Centre, Brno, CZECH
REPUBLIC, 2Prenatal Diagnosis Centre, Brno, CZECH REPUBLIC, 3Dept.
of Molecular Biology and Genetics, Masaryk University, Brno, CZECH
REPUBLIC.
We report a case of prenatally diagnosed mosaicism 45,X/47,XX,+8. Prenatal
diagnosis of mosaic trisomy 8 is relatively rare and the occurence of this
abnormality in association with monosomy X is very unusual.
Our patient-24 years old primigravida -was reffered for abnormal ultrasound
findings [agenesis corp.callosi,heart defect] at 33 t.g. Routine cytogenetic
analysis of fetal blood [FB] cells and cultured amniotic fluid [AF] cells was
performed. In FB cells the mosaic karyotype 45,X[10]/47,XX,+8[28] we found.
All examined cultured AF cells were 45,X.
Results of cytogenetic analysis were compared with fluorescent in situ
hybridization. FISH confirmed the mosaic karyotype 45,X/47,XX,+8 in FB cells
and in uncultured amniocytes as well as in peripheral blood lymphocytes and
buccal epithelial cells of the affected child after delivery. In cultured AF
cells trisomy 8 was not found by FISH.
Our findings indicate the difficulties in the prenatal diagnosis of autosomal
mosaicism, namely trisomy 8. This chromosomal abnormality can be missed with
routine prenatal cytogenetic analysis because of a different tissue-specific
distribution of the abnormal cell line.
P0408
The usage of FISH-WCP technique under the cytogenetical observation of 120
Chernobyl liquidators with different radiation doses
M. A. Pilinskaya;
Research Center for Radiation Medicine, Kiev, UKRAINE.
FISH-WCP technique was introduced in Ukraine for the first time in cytogenetic
lab of RCRM in 1999 in the frame of the Ukrainian-American project “Leukemia”
for the comparison of different dosimetry methods (including biodosimetry) in
Chernobyl liguidators. According to the protocol of the dosimetry part of the
Project all cytogenetecal investigations had been fulfilled in blind manner.
Method FISH (with directly labeled by Spectrum Orange DNA-probes to
chromosomes 1, 2, 4) had been tested on the 120 liquidators of the Chernobyl
accident ranging in age from 37 to 73 years in dose range from 10 till >
100 cGy. The data received confirmed that at present FISH-WCP technique can be
successfully use in delayed terms following the radiation exposure for the
indication of human irradiation and group dosimetry. As regard of the usage
FISH method for individual retrospective dosimetry of radiation exposure
especially in the range of low doses there are many problems of which needed
in further investigations. At present the high and variable background
frequencies of complete translocations, strong age effect and essential
interindividual variability in the rate of radiation-induced stable
aberrations permit the use FISH for estimation and verification of doses in
the part of Chernobyl liquidators with assumed radiation exposure more than 25
cGy.
This work had been supported by National Cancer Institute of USA in the frame
of Ukrainian-American Project "Leukemia".
P0409
Chromosomal analyses in infertile men.
J. Lissitsina 1, R. Mikelsaar 1, K. Varb 1,
M. Punab 2;
1Institute of General and Molecular Pathology, Tartu, ESTONIA, 2Tartu
University Clinicum, Tartu, ESTONIA.
The incidence of chromosomal abnormalities in infertile males varied from 2.2
to 14.3%. The incidence of chromosomal variants was shown to be at 34.5% in
infertile men and 10-15% in the general population. Our study was performed to
determine the frequency of chromosomal abnormalities and polymorphism in
infertile men with azoospermia and severe oligospermia (sperm density
<5x10M/mL). Chromosomal analysis of 27 infertile men was performed both
from peripheral blood lymphocytes and skin fibroblasts cultures using GTG, C
banding, fluorescent in situ hybridization (FISH) and other methods. In 5
(18.5%) cases chromosomal abnormalities were found. There was one case (3.7%)
with numerical chromosomal abnormality (47,XXY). Structural abnormalities were
revealed in 4 cases (14.8%), from which 3 were mosaics (in 3%-98% of cells).
The chromosomal variants were found in 8 (29.6%) patients. In 15 (55.6%) cases
the karyotype was normal. We have found higher than given in the literature
percentage of chromosomal abnormalities (mainly structural abnormalities),
which could be reason of infertility. This finding could be explained partly
by small amount of cases. We wish also to stress, that to reveal the low
percentage mosaicism, it seems to be necessary to analyze at least 33-50
mitoses. However, the frequency of chromosomal variants in infertile men could
support the opinion that chromosomal variants may be associated with
reproductive failure in men.
P0410
A comparative analysis of G-banding, FISH and RT-PCR for the diagnosis of
bcr-abl-positive CML
R. Brill-Zamir, I. Laevski, D. Sahar, R. Gershoni-Baruch;
Rambam Medical Center, Haifa, ISRAEL.
Phildelphia (Ph) chromosome-positive CML, with the bcr-abl gene translocation,
has a dismal prognosis. The identification of Ph-positive patients is vitally
important because only aggressive therapeutic approaches, such as interferon
alpha (IFN-alpha) treatment and allogeneic bone marrow transplantation, may
result in long-term disease-free survival. Routine diagnostic methods for
detection of the bcr-abl translocation include conventional
cytogenetics(G-banding), reverse transcriptase-polymerase chain reaction
(RT-PCR), and more recently, fluorescence in situ hybridization (FISH)
analysis. Routine cytogenetics . sensitive at the initial diagnosis is
unreliable during treatment. RT-PCR analysis is considered the most sensitive
tool for the detection of the bcr-abl translocation, and is widely used alone,
or in combination with the other methods. FISH analysis is simple, extremely
reliable and sensitive. This study compares the efficiency of the three
methods for the initial diagnosis of Ph-positive CML, and for detection of
minimal residual disease during treatment. Conventional G-banding
cytogenetics, FISH with BCR and ABL double-color probes and RT-PCR for
detecting Ph-positive CML were undertaken in 22 CML patients undergoing either
IFN-alpha treatment or allogeneic bone marrow transplantation (allo-BMT). The
results obtained using the three methodological approaches were 100%
correlated at diagnosis. Following treatment the cytogenetic analysis becomes
technically less feasible and the results less reliable. Whereas, RT-PCR and
FISH analysis remain equally and mutually contributive. We thus conclude that
the concomitant use of FISH and RT-PCR remain the optimal diagnostic
combination for the detection of the bcr-abl translocation.
P0411
Trisomy 12 mosaicism in a newborn presenting with chylothorax, facial
dysmorphism, genital anomalies and congenital heart disease
V. Klamroth, R. Exeler, I. Kennerknecht, J. Horst;
Westfälische Wilhelms-Universität, Münster, GERMANY.
We present a newborn girl with trisomy 12 mosaicism,[46,XX/47,XX+21]. The girl
was born at 39 weeks of gestation after an uneventful pregnancy to healthy
parents.
After birth, chylothorax, congenital heart disease (VSD, ASD), muscle
hypotonia, facial dysmorphism and ambiguous genitalia were cardinal features.
Chromosome analysis of various tissues revealed trisomy 12 mosaicism (skin
2/60 cells, muscle 3/43 cells, lung tissue 7/37 cells, blood and pleura:
normal karyotype).
Complete trisomy 12 in humans is not viable. To our knowledge this is the
tenth published case of trisomy 12 mosaicism in a liveborn. Obviously due to
the nature of mosaicism, reported cases show a wide spectrum of anomalies
ranging from Kartagener syndrome in an otherwise healthy man to multiple
congenital abnormalities including complex heart malformations, facial
dysmorphism, urogenital abnormalities (renal hypoplasia, ectopia vesicae) and
further inner malformations (multiple accessory spleens, pancreatic-spleenic
fusion, gallbladder hypoplasia) with neonatal death. No typical mosaic trisomy
12 phenotype can be delineated.
We report the first case of trisomy 12 mosaicism presenting with chylothorax,
facial dysmorphism and genital anomalies as cardinal features. We compare our
patient with earlier described cases.
P0412
Coincidental Structural And Numerical Aberrations Of The Chromosome 15 -
Three Different Case Reports
E. Kocarek 1, D. Novotna 2, P. Balicek 3,
T. Marikova 1, M. Malikova 2, A. Zumrova 1, K.
Novotna 1, M. Strnad 1, P. Goetz 1;
1Charles University 2nd Medical School, Prague, CZECH REPUBLIC, 2Motol
University Hospital, Institute of Biology and Medical Genetics, Prague, CZECH
REPUBLIC, 3University Hospital - Department of Clinical Genetics,
Hradec Kralove, CZECH REPUBLIC.
We report three cases of chromosome 15 aberrations. First one is a girl with
dysmorphic features, seizures, and a delay of psychomotoric development. Her
karyotype was 47,XX,+mar. Result of our FISH analysis was 47,XX,+mar.ish
idic(15)(q11-q13)(D15Z1++,D15S10+,PML-). The second case is a 26-years old
healthy pregnant woman, whose foetus has karyotype 47,XX,+mar. Our FISH result
was 47,XX,+mar.ish dic(15)(D15Z1++,SNRPN-, PML-). The third case report
desribes a 2-years old boy with blindness, deafness, dumbness and
cryptoorchism.The cytogenetic analysis revealed a karyotype 45,XY,der(21),
t(15;21)(q13;q22.3), -15. FISH confirmed the cytogenetic result:
45,XY,der(21), t(15;21)(q13;q22.3),-15.ish der(21)(D13Z1/D21Z1+,D15Z1-,
SNRPN-, UBE3A/D15S10-, D21S270/D21S337/D21S55/D21S233+, PML+). The result
indicates haploinsufficiency of the critical Prader-Willi/Angelman region in
15q11-q13. The work was supported by grant IGA NE5685-3 (Ministry of Health
CR) and research project of the Charles University No.111300003.
P0413
Cytogenetic Analysis of 111 Cases of Adult Acute Lymphoblastic Leukaemia in
an Asian Population.
C. Rudduck Sivaswaren 1, S. L. Tien 2 ,1;
1Department of Pathology, Cytogenetics, Singapore General Hospital,
SINGAPORE, 2Department of Haematology, Singapore General Hospital,
SINGAPORE.
The majority of cases with acute lymphoblast leukemia (ALL) have abnormal
chromosomes. Many of these abnormalities have been shown to be important
clinical prognosticators. Cytogenetic analysis is therefore used routinely in
management of ALL. In the current study we evaluate the cytogenetic
abnormalities seen in a series of cases of adult ALL in an Asian population.
A total of 111 cases of ALL admitted to Singapore General Hospital between
1995 and 2001 were analysed. The age range was 16-81 (46 females, 65 males).
Chromosome preparations were made from bone marrow cells using direct and 24
hour cultures. Karyotypes were constructed in accordance with ISCN
nomenclature.
Chromosome abnormalities were seen in 73% of cases a normal karyotype in 21.6%
while the remaining 5.4% were unsuccessful.
The most common balanced structural abnormalities were t(9;22)(q34;q11.2) seen
in 24 (21.6%) and 1 variant translocation, followed by t(8;14)(q24;q32) in 5
(4.5%) with variant translocations in 4 additional cases. The diagnostically
important abnormalities t(4;11)(q21;q23) were seen in 2 cases and
der(19)t(1;19)(q23;p13) and it’s balanced form in 3 and 1 case respectively.
Deletion of 9q was the most common region of deletion seen in 7 (6.3%) cases.
All the abnormal cases but 3 had structural abnormalities. A hypodiploid
karyotype of <46 chromosomes was seen in 10.8 %, pseudodiploid in 41.4%,
hyperdiploid of 47-50 in 12.6% and hyperdiploid >50 in 6.3% of cases
respectively.
The most frequent numerical gains were for chromosomes 8, 21, 14, 18, while
loss of chromosome 9 was the most common loss.
P0414
two new cases with 48, XXXX chromosome and review of the litterature.
D. Genenviève 1, L. Faivre 2, S. Lesourd 1,
F. Mugneret 2, D. Heron 1;
1Département de Génétique, Hôpital de la Pitié-Salpétrière,
Paris, FRANCE, 2Service de Génétique, Hôpital d'Enfants, Dijon,
FRANCE.
Although 47,XXX, 47,XXY, 47,XYY and 45,X karyotypes are frequent and occur at
least 1 in 400 birth, patients with more than one extra sex chromosomes are
very rare.
Here we report on two new cases with 48, XXXX (tetrasomy X) karyotype and
reviewed the litterature. The pregnancy and delivery were normal in both
cases. In case one, neonatal measurements were 2625 g for weight (- 1.5 SD),
46.5 cm for length (- 1.5 SD) and 34 cm for OFC (M). IUGR was noted in case
two with 2100g for weight and 43 cm for length at birth. In both cases, facial
dysmorphism including bilateral epicathus, synophris, long eyelashes, bulbous
nose, long and flat filtrum, small mouth and bilateral radioulnar synostosis
was noted. Evolution was marked by mild mental retardation with speech
difficulties.
In attempt to well define the tetrasomy X, we reviewed the litterature. To our
knowledge, since the first description of an abnormal number of chromosome in
cultured lymphocytes reported by Lejeune et al in 1959, only 40 cases with
48,XXXX have been described in the litterature. We found that mental
retardation was constant excluding one case with normal intelligence. Other
features were particular facial dysmorphism, radioulnar synostosis and similar
but non-specific behavioural problems. This syndrome seems to be clinically
recognisable.
P0415
One event, two cell lines, three chromosomes, four breakpoints: unusual
mosaic complex translocation in a patient with oligospermia.
J. M. Dupont 1, F. Baverel 1, M. Savale 2,
A. Lebbar 1, D. Le Tessier 1, C. Patrat 3, J.
Guibert 2, D. Rabineau 1;
1Histologie Embryologie Cytogénétique, Hôpital Cochin, Paris,
FRANCE, 2Gynécologie, Hôpital Cochin, Paris, FRANCE, 3Histologie
Embryologie Biologie de la Reproduction, Hôpital Cochin, Paris, FRANCE.
Complex chromosomal rearrangements and mosaic reciprocal translocations are
rare events in constitutional cytogenetics. We report here on a 52 years old
man with variable oligospermia who was referred to our laboratory for
cytogenetic examination before AMP procedure. This man has two healthy
children from a previous union. GTG and RHG banding revealed a complex
translocation between chromosomes 9, 12 and 14 in 20% of the cells from two
consecutive peripheral blood sampling. These standard banding techniques led
to the proposal of the following chromosomal pattern:
46,XY,t(9;12;14)(q32;q13;q32)/46,XY.
Unexpectedly, FISH revealed a far more complex pattern, with two breakpoints
on der(12). This derivative chromosome harbors material from chromosomes 9 and
14 hence correct designation is 46,XY,t(12;14;12;9)(q13;q32;p13;q32).ish
t(12;14;12;9)(Tel14q+,wcp14+,wcp12+,wcp9+,Tel
9q+;wcp14+,wcp12+,Tel12q+;wcp9+,wcp12+, Tel 12p+)/46,XY. Careful hematological
examination of the patient has been conducted to eliminate a rising malignant
process.
During meiosis, these complex rearrangements are expected to form a
multivalent from which multiple gametic combination can occur, most of which
will be unbalanced. However, if female gametogenesis can accomodate itself to
the complexity thrust upon it and produce balanced ovocytes, the rule of the
greater vulnerability of spermatogenesis to structural rearrangement applies
particularly in the case of complex abnormalities and heterozygote males are
often sterile.
In our patient, the presence of a normal cell line may explain the more or
less conserved fertility; however, in case of pregnancy following AMP, a
prenatal diagnosis should be proposed to the parents as some of the imbalances
might be viable.
P0416
From Mendelian inheritance to telomeres ...
G. Viot 1, V. Desportes 2, C. Ozilou 3,
A. Choiset 1, S. Girard 1, A. Munnich 4, M. Prieur 3,
S. P. Romana 5, M. Vekemans 3, C. Turleau 5;
1Hopital Saint Vincent de Paul, Paris, FRANCE, 2Hopital
Saint vincent de Paul, Paris, FRANCE, 3Hopital Necker-Enfants
Malades, Paris, FRANCE, 4Hopital Necker- Enfants Malades, Paris,
FRANCE, 5Hopital Necker-enfants Malades, Paris, FRANCE.
FISH studies using subtelomeric probes allowed us to explain MR/MCA recurrence
in two large families with follow-up of 40 and 20 years respectively. Repeated
high-resolution karyotypes were performed in both families. In the first
family, a sex-linked recessive inheritance was suggested because two related
male patients were observed. However a girl with the same clinical
abnormalities was born recently. A multiprobe subtelomeric FISH study led to
the identification of a familial cryptic translocation t(17;22)(q25;q13.3). A
derivative 22 yielding monosomy 22q13 and trisomy 17q25 was observed in all
affected patients tested. The translocation was segregated over at least four
generations. In the second family, the observation of recurrent malformations
and perinatal deaths over several generations suggested the presence of a
familial rearrangement. Two cousins, a girl and a boy, with similar clinical
findings died at a young age. Recently, photographs of both children were
examined again. They showed facial dysmorphism evocative of a 2q27 deletion.
FISH studies with chromosome 2 subtelomeric probes confirmed this imbalance
and show that it derived from a cryptic familial t(2;17)(q37.3;q25). These
observations illustrate that reassessment of " old " genetic files
using both molecular cytogenetic tools and new syndromes clinical descriptions
can point towards a correct diagnosis. In addition, they emphasise that
recurrence of similar MR/MCA findings in different branches of a kinship is
highly suggestive of a chromosomal anomaly. Genetic counselling and prenatal
diagnosis are now available for both these large families.
P0417
Cat eye syndrome associated with a severe phenotype
T. Martin Denavit 1, D. Sanlaville 1, C. Grillon 2,
V. Malan 1, P. Nizard 1, A. Ardalan 1, L. Burglen 3,
J. Taillemite 1, M. Portnoï 1;
1Laboratoire de Cytogénétique Hôpital Saint Antoine, Paris,
FRANCE, 2Service de Néonatologie Hôpital Trousseau, Paris, FRANCE, 3Service
de Génétique hôpital Trousseau, Paris, FRANCE.
Cat Eye syndrome (CES) is characterized by a variety of congenital defects
including ocular coloboma, anal atresia, preauricular tags or pits, heart and
kidneys defects, dysmorphic facial features and mental retardation. The
penetrance and severity are highly variable. This syndrome is associated with
a supernumerary bisatellited dicentric marker arising from an inverted
duplication of chromosome 22 classified into 2 types based on the location of
the breakpoints. Most common CES chromosomes correspond to the smaller type I.
We describe a female child with a severe phenotype of CES, born at 40 weeks of
gestation from healthy parents. An intrauterine growth retardation was
diagnosed at 22 weeks of gestation and was confirmed at birth. She developed a
severe respiratory distress. She had hypotonia, dysmorphic features and
malformations including downslanting palpebral fissures, hypertelorism, ocular
coloboma, bilateral aplasia of the external ears, anal stenosis, patent ductus
arteriosus. She died at age 1 month. Chromosome analysis showed a karyotype
with an extranumerary bisatellited marker in all cells examined. The parents
had normal karyotypes. FISH using 14/22 alpha satellite, WCP of chromosome 22
and N25 probes showed that the marker was a dicentric inverted duplication of
the short arm and proximal long arm of chromosome 22, symmetrical and
localized distally to the Digeorge locus (type II).
The severe phenotype of our patient demonstrated the phenotypic variability
which does not seem to be related to the size of the duplication or presence
of mosaïcism. This phenotypic variability increases the difficulty of genetic
counselling.
P0418
Mapping of chromosomal breakpoints associated with orofacial clefts.
E. Ruiz-Casares 1 ,2, Z. Tümer 1, M.
Bugge 1, N. Henriques-Gil 2, L. Rodriguez 3, F.
Lopez 3, P. Kroisel 4, K. Wagner 4, C. Lundsteen 5,
V. Kalscheuer 6, N. Tommerup 1;
1Wilhelm Johannsen Centre for Functional Genome Research, IMBG, Panum
Institute, University of Copenhagen, Copenhagen, DENMARK, 2Seccion de
Genetica, Universidad San Pablo CEU, Madrid, SPAIN, 3Estudio
Colaborativo Español de Malformaciones Congenitas (ECEMC), Madrid, SPAIN, 4Institut
für Medizinische Biologie und Humangenetik, Karl-Franzens-Universität Graz,
Graz, AUSTRIA, 5Dept of Clinical Genetics, Rigshospitalet,
Copenhagen, DENMARK, 6Max-Planck-Institute for Molecular Genetics,
Berlin, GERMANY.
As part of an EU-supported project aimed at the clinical, epidemiological and
genetic study of cleft-lip-palate (EUROCRAN), we have initiated a search for
candidate genes for orofacial clefts by mapping apparently balanced
chromosomal breakpoints associated with cleft-lip-palate. The Mendelian
Cytogenetics Network database (http://mcndb.imbg.ku.dk) contains information
on 75 breakpoints in 25 individuals with oral clefting. Ordered YAC/BAC clones
were used to map the breakpoints associated with two of these rearrangements:
One involved a 46,XY,ins(9;5)(p22.2;q23.3q33.3) karyotype; the 5q33.3
breakpoint has been narrowed to a 250 kb region. Two overlapping BACs at the
9p22 breakpoint contain a hypothetical unknown gene, and the 5q23.3 breakpoint
has been narrowed to a 150 kb region containing three candidate genes,
including a putative zinc finger gene. None of these three breakpoints affects
chromosomal regions previously known to harbor loci associated with orofacial
clefts. The second case displayed an aberrant banding pattern on 7q36, a
region previously implicated in congenital malformations affecting midline
structures and a region included among those associated with orofacial clefts
in more than one case in MCNdb (1p31, 4q21, 6p24, 7q36, 9p13, 16q24, 17q23 and
17p25). Chromosome 7 paint did not suggest the involvement of other
chromosomes, the 7q-subtelomeric signals were normal, and high resolution CGH
was normal, excluding deletions/duplications larger than ~3 Mb. This supports
that the abnormal banding pattern is caused by a small intrachromosomal
rearrangement, e.g. an inversion. We will try to solve this by a systematic
FISH strategy using pooled BACs from 7q36.
P0419
Fetal isochromosome 18q: a case report and review of the literature.
C. Sebaoun, O. Dupuy, C. Borie, N. Bondeux, N. Collot, J. Oury, A.
Delezoide, P. L. Eydoux;
Hôpital Robert Debré, Paris, FRANCE.
We report a female fetus with isochromosome 18q showing features of of both
trisomy 18 and monosomy 18p. Few cases of this fetal syndrome have been
published.
The pregnancy was uneventful, until prenatal sonographic examination at 20
weeks of gestation showed intra-uterine growth retardation (IUGR) and an
encephalocele. The parents were non consanguineous, and had no relevant
history of genetic diseases. Chromosomal examination of amniotic fluid cells
showed an isochromosome 18q in all examined cells (46,XX,i(18q)). The fetus
thus had trisomy 18q associated with monosomy 18p. The parents elected
termination of pregnancy.
Fetopathologic examination revealed features of monosomy 18p
(holoprosencephaly and facial malformations, occipital meningocele, heart and
skeletal malformations), and trisomy 18q (microcephaly, short neck with
pterygium colli, club feet, ovarian epithelial hypoplasia). In this case, the
severity of the phenotype resulted in prenatal recognition of the chromosomal
abnormality. Holoprosencephaly is not a common feature in post natal monosomy
18p (16% of all cases). It is thought to result from the deletion of HPE4, a
gene encoding TGIF and located at 18q11.3.
Isochromosome 18q is thought to result from centromere fission (monocentric
chromosome), or from chromatid exchange (dicentric chromosomes). Isochromosome
(18q) is seen as a derivative chromosome; to our knowledge, no cases of
supernumerary i(18q) have been reported, probably because tetrasomy 18q is not
viable. The fetal features of isochromosome (18q) closely resembles the post
natal phenotype, although the fetal syndrome may be more severe.
P0420
Isolated Agenesis of the Corpus Callosum and 8p Duplication
D. Molina Gomes, V. Nebout, V. Serazin, F. Bru, J. P. Bernard, F.
Daikha-Dahmane, F. Vialard, J. Selva;
Fédération de Génétique et Service de Gyneco-Obstétrique. Centre
Hospitalier Intercommunal Poissy Saint Germain en Laye et Faculté de Médecine
de Paris Ouest, Poissy, FRANCE.
We report a case of partial duplication of the short arm of the chromosome 8
associated with an isolated agenesis of the corpus callosum.
This is the first pregnancy of a young unrelated couple. During the second
trimester an isolated agenesis of the corpus callosum was found.
A fetal blood and amniotic fluid sample were performed. The conventional
karyotype showed an excess of material on the short arm of chromosome 8
suggesting a duplication. Parental karyotypes were normal. In situ
hybridization with the whole chromosome painting of chromosome 8 confirmed the
duplication. The couple chosed to interrupt the pregnancy.
Anatomo-pathological examination showed a total agenesis of the corpus
callosum associated with discrete dysmorphy. Array 300Ô
using the GenosensorÔ System was used to confirm
the duplication of 8p chromosome. Presently the GenosensorÔ
System includes 6 clones in the 8p region. 3 of them, the most centromeric
ones, were abnormal detecting a duplication of the (8p11-8p22) region. We have
to use BAC clones and FISH in order to precise the breakpoints of this
duplicated fragment, and to try to reduce the critical zone of the corpus
callosum on chromosome 8p.
P0421
Chromosome aberrraton frequency for medical staff exposed to ionizing
radiation from open and close sources
S. Tomanovic, D. Mirkovic, B. Djurovic, M. Hrnjak, M. Misovic;
Military Medical Academy, Belgrade, YUGOSLAVIA.
It has been evidenced that ionizing radiation induces microscopicaly detected
changes in genetical structure of cell. Analysis of chromosome aberrations
give informations about biological effects and biological responses of living
organisms to ionizing radiation.
The aim of this paper was to show the results of chromosome aberration
frequency analysis for persons occupationaly exposed to ionizing radiation
from open and close sources.
We analised 106 health workers in period 1997-1999, and they were categorised
into two groups according to the type of radiation they were exposed to:
medical workers in nuclear medicine (24) who were exposed to ionizing
radiation from open sources, and medical staff in diagnostic radiology (82)
who were exposed to ionizing radiation from close sources.
The average duration of occupational exposition was 10.2 years for nuclear
medicine staff, and 11.6 years for diagnostic radiology staff.
The results of cytogenetic examinations showed that chromosome aberration
frequency was higher for nuclear medicine employers (in 4 persons-16.67%) than
for the radiology employers (in 8 persons-8.54%), but there was no statistical
significance (p=0.4622).
There was also no diference in chromosome aberration frequency for diferent
professions in any of this group.
P0422
Testing the ability of the "Geno Sensor System" to measure the DNA
copy numbers in 4 cell strains containing cytogenetically mapped
abnormalities.
F. Wuilque 1, F. Vialard 2, D. Molina-Gomes 2,
C. Le Caignec 3, V. Serazin 2, L. Grimaud 1, M.
Boceno 3, M. V. Senat 2, J. Roume 2, J. M. Rival 3,
Y. Guidicelli 2, J. Selva 2;
1Adgenix, Voisins le Bretoneux, FRANCE, 2Fédération de
Génétique et Service de Gynécologie-Obstétrique- Centre Hospitalier
Intercommunal Poissy-Saint Germain en Laye et Faculté de Médecine de Paris
Ouest, Poissy, FRANCE, 3CHU Nantes, Nantes, FRANCE.
Microarray bases genomic analysis is a novel technique designed for rapid
detection of changes in numbers of human DNA specific sequence copies. The
VYSIS GenoSensor™ System contains 287 human loci including single copy
telomeric sequences, genes involved in micro-deletion syndromes, single copy
sequences near the centromere and different loci reported to be amplified in
various human cancers. We tested the ability of this system to measure single
copy changes in 4 cell strains [a whole 21 chromosome gain (case 1), 2
deletions (1p deletion (case 2), 4p deletion (case 3)), one duplication 8p
(case 4)].
The assay involves sample DNA labelling with Cy3 fluorophore. This is mixed
with whole genomic reference DNA labelled with Cy5 fluorophore and
co-hybridized to an Array 300™ microarray. After hybridization, target spots
are analysed using the GenoSensor™ reader system. The proprietary software
automatically identifies each spot and calculates for each target a normalized
ratio that indicates the degree of gain or loss of copy number in the sample
DNA.
All 21 clones were detected as gained for case 1. For cases 2 and 3 the
detected DNA loss was in accordance with that found with other techniques
results. For case 4 we confirmed the duplication of 8p, as well.
We now intend to use this system for the diagnosis of unknown changes in DNA
copy number which might occur in different fetal or newborn pathologies with a
probable genetic etiology where abnormalities have not yet been detected with
other techniques.
P0423
Meiotic segregation analysis in three infertile patients with balanced
reciprocal translocations
J. Puechberty 1, B. Andreo 2, G. Lefort 3,
P. Blanchet 1, P. Sarda 1, F. Pellestor 2;
1Genetics Department - Arnaud de Villeneuve Hospital, Montpellier,
FRANCE, 2Institut de Génétique Humaine, CNRS, Montpellier, FRANCE, 3Cytogenetics
Department - Arnaud de Villeneuve Hospital, Montpellier, FRANCE.
Carriers of balanced reciprocal translocations may present with infertility,
recurrent miscarriages, or offsprings with unbalanced karyotype and multiple
congenital anomalies. If the risk of chromosomal imbalance may be estimated at
birth, the implication of this imbalance for infertility and miscarriages is
more difficult to determine. To assess the importance of these chromosomal
anomalies during the gametogenesis, we have explored three infertile
phenotypically normal men with reciprocal translocations. We performed meiotic
segregation analysis of their sperm using FISH and PRINS methods. For patients
1 and 2, respectively with reciprocal translocations t(1;4)(q12;q28) and
t(4;5)(q35;q34), more than 10000 spermatozoa were screened. In both cases,
alternate segregation (giving phenotypically normal offspring) is the most
frequent mode of segregation (68 and 70%) and the most frequent type of
imbalance is adjacent 1 segregation (24 and 28%), corresponding to the most
expected imbalance at birth. For patient 3 with reciprocal translocation
t(1;21)(q11;q21) and with severe oligozoospermia, only 981 spermatozoa (from
two ejaculates) could be screened. Alternate segregation is also the most
frequent type of segregation (80%) and the two most frequent types of
imbalance are adjacent 1 (10%) and 3:1 exchange (7,5%), this latter is
responsible for the most expected imbalance at birth. Although alternate
segregation is the major type of segregation, these three men have
infertility. Likely there are other factors which can participate in the
mechanisms of the reproductive failure, possibly from maternal origin as well.
Meiotic segregation analysis in sperm is important to adjust a realistic
management of the male infertility.
P0424
Detection of a partially cryptic complex chromosome translocation by
FISH
C. Hernando Davalillo 1 ,2, P. Grao 2,
A. Balaguer 3, E. Triviño 2, J. Egozcue 1, C.
Fuster 1;
1Dpt. de Biologia Cellular, Fisiologia i Immunologia. Unitat de
Biologia. Facultat de Medicina. Universidad Autónoma, Barcelona, SPAIN, 2Departamento
de Genética. CERBA Internacional, S.A.E. Sabadell, Barcelona, SPAIN, 3Servicio
de Pediatria. Hospital Sant Joan de Reus, Barcelona, SPAIN.
We report an “apparent” balanced de novo complex chromosome
reorganization by the combined use of G-banding, FISH and CGH techniques in a
two-year-old infant with some features vaguely reminiscent of trisomy 21.
Conventional G-banding showed a complex chromosome translocation involving
chromosomes 1, 4, 6 and 11. Multicolor FISH (24 colours) confirmed the
presence of this complex chromosome translocation. A more complex
reorganization was detected using whole chromosome painting probes for the
chromosomes involved in this rearrangement. In this case we found two cryptic
interstitial translocations in the same derivative chromosome: der(6)
t(6;11;4;1;4). In the results obtained by CGH did not show any loss or gain of
chromosome material in this patient. Our results indicate that the phenotypic
abnormalities observed in this infant, with this “apparent” balanced
complex rearrangement, may have resulted from either a small structural loss
of material or a functional loss of a gene action.
Acknowledgements. Financial support was given by DGESIC (PB98-0891).