ESHG Posters 5

N. A. Meguid¹, A. Mahmoud¹, A. Khalil¹, M. Amer², A. Dardir¹, H. Atteya¹;
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.

H. Elghezal¹, H. Sennana Sendi¹, M. Griba¹, S. Ibala Romdhan¹, K. Monastiri², A. Saad¹;
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.

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.

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.

Blepharophimosis Is The Most Constant Clinical Feature In 14qter Microdeletion Syndrome

O. Rittinger¹, G. Kronberger¹, C. Fauth²;
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.

FISH assessment of sperm aneuploidy frequencies in ICSI patients with severe oligoasthenotetraozoospermia (OAT)

N. Ditzel¹, L. Jelinkova¹, B. Gläser², E. Strehler¹, N. Reeka¹, G. Speit², K. Sterzik¹;
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.

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. Gläser¹, E. Rossier¹, G. Barbi¹, L. Delle Chiaie², C. Blank³, W. Vogel¹, H. Kehrer-Sawatzki¹;
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.

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.

E. Braha¹, O. Bartsch², M. Volosciuc¹, C. Gavrilovici¹, M. Covic¹;
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.

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.

S. H. Kofman-Alfaro¹^,2, G. Queipo³, R. Peña⁴, K. Nieto⁵, L. M. Dorantes⁴, L. Eraña⁴, A. L. Jimenez³, D. Soderlund⁶, E. Lieberman⁷, A. Radillo³, J. C. Zenteno³;
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.

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.

A natural equivalent to human satellite DNA-based artificial chromosome persists over 140 years, in a three-generation family

G. Hadlaczky¹^,2, G. Bujdosó³^,4, É. Koltai⁵, G. Schwanitz⁶, E. Csonka⁷;
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.

I. Petkovic¹, I. Barisic¹, R. Bago², S. Hecimovic²;
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).

I. Petkovic¹, I. Barisic¹, R. Bago², I. Sansovic¹;
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).

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’.

D. Marcus-Soekarman¹, G. Hamers¹, S. Velzeboer², J. Nijhuis³, C. de Die-Smulders¹, C. Schrander-Stumpel¹, J. Engelen¹;
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

L. Sindelarova¹, K. Michalova¹^,2, Z. Zemanova¹, J. Brezinova², S. Kurkova², E. Cmunt³;
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

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.

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.

Cytogenetic and FISH analyses of masked and complex Philadelphia chromosome translocations in chronic myeloid leukemia.

F. Morel¹, A. Herry¹, M. J. Le Bris¹, N. Guéganic¹, M. F. Scoazec¹, P. Morice², J. F. Abgrall³, P. Bourquard², C. Berthou⁴, M. De Braekeleer¹;
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.

MLL rearrangements in balanced and unbalanced 11q aberrations in patients with hematological malignancies

J. Brezinova¹, Z. Zemanova², S. Kurkova¹, L. Sindelarova², J. Sajdova¹, K. Michalova¹^,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.

Clinical implications of del(20q) in patients with hematologic myeloid disorders

S. Kurkova¹, J. Brezinova¹, Z. Zemanova², L. Sindelarova², J. Cermak¹, M. Siskova², R. Neuwirtova², K. Michalova¹^,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.

Importance of genetic investigation in couples involved in "in vitro fertilisation"(IVF)

P. Capkova¹, K. Adamova¹, A. Santava², R. Vrtel¹, J. Kolarova², I. Oborna³, A. Sobek⁴, J. Santavy²;
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.

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.

DNA analysis of Y - chromosomal sequences from different tissues in Turner syndrome patients.

R. Vodicka¹, R. Vrtel¹, J. Zapletalova², A. Santava¹, K. Adamova¹;
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.

Y. Perrin¹, M. C. Addor¹, N. Sekarski², D. F. Schorderet¹;
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.

Characterization of two small supernumerary marker chromosomes (SMC) by acro/cenM-FISH - first case with partial hexasomy 15pter->15q13

A. Heller¹, B. Albrecht², A. Nietzel¹, H. Starke¹, F. von Eggeling¹, U. Claussen¹, T. Liehr¹;
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).

Cytogenetic studies in lymphocyte cultures from 3 members of a family affected with Werner's Syndrome

B. Porto¹, O. Pereira², I. Malheiro¹;
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.

Supernumerary marker 22 chromosome: Clinical, cytogenetic and molecular analysis of five patients.

I. Lopez Pajares¹, A. Delicado¹, P. Lapunzina¹, M. Mori¹, M. de Torres¹, A. Sanz²;
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.

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.

H. Starke¹, A. Weise², A. Nietzel², A. Heller², U. Claussen², T. Liehr²;
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)

Role of chromosome abnormalities in recurrent In-Vitro Fertilization implantation failure

I. Yehudai¹, H. Bar-El¹, Z. Borochowitz¹, R. Diukman², H. Dar¹^,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.

R. Genesio¹^,2, A. Borghese¹, D. De Brasi³, P. Di Micco³, P. Di Costanzo³, A. Conti¹, D. Paladini⁴, A. Loffredo⁴, P. Martinelli⁴, L. Nitsch¹;
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.

Asynchronous replication of bi-allelically expressed loci: A new phenomenon in turner syndrome

O. Reish¹, R. Gal¹, L. Gaber², C. Sher¹, Z. Bistritzer¹, A. Amiel²;
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.

F. Mortezapour¹, L. Seyed Mortaz¹, F. Razazian¹, B. Hashemi¹, H. Sayar¹, S. Joghehdost¹^,2, M. Houshmand¹^,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.

S. Yilmaz¹, A. Deviren², D. Kuru³, Y. Tarkan-Argüden¹, B. Karaman⁴, A. Yüksel³, S. Hacihanefioglu¹;
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+).

R. Airinei¹, I. Dimofte², A. Popa², D. P. Balaban³, L. C. Enache⁴, V. Broasca Madar⁵;
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.

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.

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.

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.

F. Razazian¹, S. Soleimani¹, F. Nassiri¹, B. Hashemi¹, H. Sayar¹, S. Joghehdost¹^,2, M. Houshmand¹^,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.

M. Rahnama¹, S. Tootian¹, M. Zamanian¹, B. Hashemi¹, H. Sayar¹, S. Joghehdost¹^,2, M. Houshmand¹^,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.

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.

M. Pierluigi¹, S. Cavani¹, M. Malacarne¹, F. Faravelli¹, L. Dalpra'², E. Sala², N. Villa², D. Gaveglio², M. Silengo³, G. B. Ferrero³, E. Frate⁴, F. Dagna Bricarelli¹;
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)

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.

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.

Results of chromosome studies in 664 Iranian couples with the history of recurrent early pregnancy loss.

Y. Shafeghati¹^,2, M. Karimi Nejad², M. Zangeneh², R. Karimi Nejad², Q. Baba Mohammadi², N. Almadani², N. Almadani², F. Afroozan²;
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).

M. Jennewein¹, M. Oberringer²;
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.

Detection of cerbB-2 gene amplification in bilharzial bladder cancer using fluorescence in situ hybridization

H. W. Mostafa¹, M. S. Aly¹, H. M. Khaled²;
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.

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.

M. S. Aly¹, H. M. Khaled², N. Mokhtar²;
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.

R. Mikelsaar¹, K. Muru¹, K. Varb¹, A. Kulla², A. Süvari³;
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.

B. Oehl-Jaschkowitz¹, M. G. Shamdeen², S. Reichardt¹, V. Kalscheuer³, K. D. Zang¹, T. Martin¹;
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.

U. Claussen¹, J. Lemke¹, J. Claussen¹, I. Chudoba², T. Liehr¹, P. Muehlig³, K. Sperling⁴;
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.

M. Zawada¹, A. Pienkowska², C. Schelling³;
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.

Micronuclei Induction in Rat Embryonic Blood Cells Following Exposure to Different Modes of Electromagnetic Fields

M. Aksoy¹, H. Acar¹, K. Karabulut², A. Salbacak², I. Uysal², M. Kaynak¹;
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.

A complex sex chromosome abnormality associated with short stature and hypogonadism

M. Volosciuc¹, E. Braha¹, C. Bujoreanu², M. Covic²;
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

Investigation of chromosomal aberrations in hepatocellular carcinoma in Egypt by fluorescence in situ hybridization

H. El-Gebaly¹, M. S. Aly¹, H. M. Khaled²;
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.

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.

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

A. R. M. Stana¹, A. G. Lungeanu², D. Pelinescu-Onciul³;
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.

Molecular cytogenetic characterization of disseminated tumor cells in renal cell carcinomas

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.

Karyological analysis of immature germ cells in sperm from men with impaired spermatogenesis.

I. Fedorova¹^,2, J. Loginova¹, L. Petrova¹, O. Chiryaeva¹, T. Kuznetzova¹;
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

M. Gregoire¹, V. Bourdon¹, F. Rousselet¹, D. Guillet-May², J. Hubert³, P. Jonveaux¹;
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.

Sex reversal secondary to Xp functional disomy including DAX1 gene by t(X;Y)(p21.2;p11.3)

D. Sanlaville¹, F. Vialard¹, J. Gekas², L. Vue-Droit³, A. Corré⁴, B. Devauchelle², A. Ardalan¹, V. Malan¹, T. Martin-Denavit¹, P. Nizard¹, F. Thépot², J. Taillemite¹, M. Portnoï¹;
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.

Are There Interchromosomal Effects of Chromosomal Rearrangements on Occurrence of Aneuploidy in Sperm Nuclei of Carriers?