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.

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?

H. Acar¹, T. Yakut², T. Çora¹, Ü. Egeli², M. Kaynak¹, S. Yildirim¹;
¹Department of Medical Biology and Genetics, Selçuk University, Medical Faculty, Konya, TURKEY, ²Department of Medical Biology and Genetics, Uludag University, Faculty of Medicine, Bursa, TURKEY.

Translocation carriers have been considered as reproductive failures, either due to spermatogenetic arrest or to unbalanced progeny. During the meiosis, both translocated chromosomes and their normal homologues can lead to unbalanced patterns of segregation for these chromosomes. Analysis of live born offspring or fetuses does not provide accurate information about meiosis segregation products because of early missing of abnormal conception. However, it has been postulated that there is an increased frequency of aneuploidies involving unrelated to the translocation, termed as interchromosomal effect. Therefore, in this study, we aimed to present our primary findings for evaluation of interchromosomal effects of different rearrangements, such as different balanced reciprocal and Robertsonian translocations and inversion, on occurrence of aneuploidies of sperm nuclei of carriers. Interphase sperm-fluorescence in situ hybridization (FISH) analysis was performed by using chromosome specific DNA probes that were not involving in those rearrangements

D. Ehling¹^,2, A. Tauchen¹, T. Schmitt-John¹^,2, J. Weidner², J. Wirth¹^,2;
¹Developmental Biology and Molecular Pathology, University of Bielefeld, Bielefeld, GERMANY, ²Praenadia GmbH, Muenster, GERMANY.

Conventional fluorescence in situ hybridization (FISH) is a useful tool for the detection of human chromosomal abnormalities in prenatal and postnatal diagnostics. The quality of DNA probes contributes to the signal detection and is important for a confident interpretation of FISH results. We have developed high quality FISH probes suitable for the identification of the major trisomies (21, 13, 18) as well as common structural rearrangements such as deletion of 22q11. Most of our BAC clones were isolated by using the REPuter program which allows us to visualize distributions of exact and degenerate repeats with a minimal length of 20 bp. Regions with high gene content and relatively low repeat density were selected and primers were designed for the identification of BAC clones. Furthermore, for improvement of the FISH signals we have assembled BAC contigs and used these as complex probe mixtures. The BAC clones were mapped to different regions of chromosome 21 including the centromeric region of 21q11, the Down syndrome critical regions at 21q22, and subtelomeric region of 21q22.3. Several DNA probes were isolated in the region of 13q32, 18q21, 22q11 and subtelomeric region of 22q13. The probe set extremely facilitates the detection of specific chromosome imbalances on uncultured amniotic fluid cells and metaphase chromosomes and is greatly useful for the identification of partial trisomies, partial monosomies and interstitial deletions.

Gonadal dysgenesis. Presentation of 3 cases and report on the database of a Paediatric Endocrine Unit in Hungary.

M. Dobos¹, G. Fekete¹, J. Szabó¹, R. Schellberg², R. Raff², G. Schwanitz², J. Solyom¹;
¹Semmelweis University, Budapest, HUNGARY, ²Friedrich Wilhelms University Institute of Human Genetics, Bonn, GERMANY.

Gonadal dysgenesis encompasses a heterogeneous group of different chromosomal, gonadal and genital abnormalities characterised by the presence of dysgenetic testes and/or streak gonads, persistence of Müllerian duct structures and a variable degree of genital ambiguity or female phenotype. Clinical, pathological, hormonal and genetic aspects of 24 patients with complete or partial gonadal dysgenesis have been studied. Three patients had complete (pure) gonadal dysgenesis (karyotype: 46,XY). The karyotype of the 21 patients with a clinical diagnosis of partial gonadal dysgenesis (PGD) was 45,X/46,XY (n=10), 46,XY (n=6) or others (n=5). The types of PGD, based on pathological findings of the gonads, were mixed gonadal dysgenesis in 5 cases, dysgenetic male pseudohermaphroditism in 6 cases, bilateral gonadoblastoma in one case (no histological data are available in 9 cases). Genital phenotype was predominantly male (n=8), ambiguous (n=9) or predominantly female (n=4).
We present 3 new cases with testicular intersexuality. The cytogenetic investigations were carried out on both peripheral lymphocyte cultures and on buccal cells by GTG and FISH technics. The karyotype analyses of two patients with partial gonadal dysgenesis showed 45,X/46,XY mosaicism in different proportion of tissues, while the third case proved to be 46, XY/47, XYY mosaic with genital ambiguity. The phenotype and genotype correlations and hormonal results are discussed.

M. Rodríguez de Alba, E. Sánchez-Gutierrez, I. Lorda-Sánchez, C. González-González, C. Gacituaga, F. Infantes, C. Ayuso, C. Ramos;
Department of Genetics. Fundación Jiménez Díaz, Madrid, SPAIN.

The Pierre Robin Sequence (PRS) consists in the association of micrognathia, glossoptosis and cleft soft palate. PRS may present as an isolated (nonsyndromic) feature, generally on a sporadic basis, or may aggregate with additional findings that together define a syndrome, which in turn may itself be found to segregate in the family.
Although no genetic locus is known for nonsyndromic PRS, genetic factors are thought to play a role in this functional and morphological entity. The 2q32 region has been specifically associated with isolated cleft-palate.
We present a father and a daughter carriers of a balanced translocation between chromosomes 2 and 17 (2q32;q24) and with the phenotipic manifestations of the PRS. The paternal grandparents were studied and neither of them presented the traslocation nor the PRS.
HYPOTHESIS: The case presented here suggests the existence of a genetic base not only for the cleft-palate feature but for the nonsyndromic PRS located on 2q32. The absence of other phenotipic manifestations rather ssuggests the idea that in this family there is not a microdeletion but a breakage point in the possible “candidate gene”.

J. J. M. Engelen¹, A. C. Knegt², J. C. M. Albrechts¹, L. Spruyt¹, C. T. R. M. Schrander-Stumpel¹, A. J. H. Hamers¹;
¹Department of Genetics and Celbiology, University Maastricht, Maastricht, NETHERLANDS, ²Department of Clinical Genetics, University of Amsterdam, Amsterdam, NETHERLANDS.

Micro-FISH is a technique that comprises the physical dissection of a GTG-banded chromosome (part), followed by a degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) to amplify the dissected chromosomal material, labelling of the PCR product and subsequently reverse painting. In clinical cytogenetics micro-FISH can be used to characterise marker chromosomes, (de novo) unbalanced translocations and complex chromosome rearrangements.
We present four cases with an unbalanced karyotype due to the presence of (DA/DAPI and NOR negative) marker chromosomes. In a 8-year-old not mentally retarded boy, referred for cytogenetic examination because of growth retardation, a mos 47,XY,+mar[75]/46,XY[25] karyotype was determined. Micro-FISH showed that the minute marker contained the centromere of chromosome 8. Chromosome analysis in a 26-year-old female showed an extra marker chromosome in 50% of her lymphocytes. She was referred because of mental retardation, dysmorphism and obesity. The marker chromosome contained chromosome region 14q10->14q12. The third patient was referred for chromosome analysis at the age of 10 years because of growth retardation. She appeared to be carrier of 2 different marker chromosomes. At the age of 26 years chromosome analysis was repeated. In 47% of the analysed cells both markers were present, in 7% a small marker and in 29% a larger marker chromosome was found. Micro-FISH disclosed that one marker contained chromosome region 19q10->19q13.1, the other marker contained chromosome region 20p10->20p11.2. The fourth marker chromosome was present in 25% of amniotic fluid cells of a woman referred because of advanced maternal age. The contents of this marker chromosome was region 19p10->19p13.1.

A familial translocation t(3;5) identified by chance in a case of essential thrombocythemia (ET)

A. A. Arghir¹, N. M. Berbec², A. Rodewald³, L. Serghei², A. G. Lungeanu⁴;
¹National Institute, Bucharest, ROMANIA, ²"Carol Davila" University of Medicine, Bucharest, ROMANIA, ³Institute for Human Biology, Univesrity, Hamburg, GERMANY, ⁴National Institute"Victor Babes", Bucharest, ROMANIA.

In this paper we report a familial translocation t(3;5)(q26;q21) found by chance in a 72 year old female at the moment of the diagnosis of ET. Because the patient had an excellent treatment outcome and rapidly achieved hematological remission we supposed that this constitutional translocation might not be involved in ET pathogenesis, but rather in the reproduction. This is the reason why we extended the cytogenetic investigations in the patient's offspring: her 52 year old only daughter and two grandchildren (a boy aged 22 and a girl aged 18). All of them are phenotipically normal including hematological values. Using peripheral blood samples for chromosomne slides and GTG-banding followed by FISH with telomere probes and whole chromosome painting ( Appligene Oncor CP5406G, CP5410R and Cytocell, PCM 356) we identified the same translocation t(3;5)(q26;q21)in proband, her daughter and granddaughter, but not in the male offspring. The maternal inheritance of such a large translocation in three generations without striking reproductive failures is unexpected taking into account the involvement of chromosomal rearrangements in meiotic segregation of unbalanced gametes. In order to have a better and precise
characterization of the breakpoints, extensive studies are needed. Molecular description of genomic region flanking the 3q and 5q breakpoints will help us to understand the relationship between this structural rearrangement and proband's hematologic disorder (ET), if any.
Acknowledgements: ANSTI Grant 5195/1999-2001, and VIASAN project 089/2001-2004.

Y. Tarkan-Argüden¹, S. Yilmaz¹, A. Deviren², D. Kuru³, 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.

There are reports on balanced translocations of chromosomes 4 and 7 with different chromosomes with various breakpoints in the parents of abnormal offsprings with unbalanced karyotypes, but we found no reports of t(4;7)(q31;p22) in our search of literature.
We present a family that have a balanced reciprocal translocation between chromosomes 4 and 7.
The proband who was 20 days old, was referred to our department because of clitoris hypertrophy. She had no other abnormalities. Her Na⁺, K⁺, and 17 a OH progesteron levels were, 139 (N:135-145), 5.5 (N:3.5-5), and 16.8 (N:1.05-40.41), respectively. Her father was 37, and her mother was 35 year-old at the time of delivery. The marriage was non-consanguineous. They had a healthy daughter who was 9 year-old. When the proband was reexamined after 6 months, her genitalia was appeared to be normal.
Cytogenetic analysis on peripheral blood cultures of the proband by GTL banding revealed 46,XX,t(4;7)(q31;p22) chromosome constitution. Then we performed cytogenetic analysis on the parents and the sister. Proband’s mother had 46,XX karyotype but we found 46,XY, t(4;7)(q31;p22) in her father and 46,XX,t(4;7)(q31;p22) in her sister. Then two sisters of the father were studied and revealed 46,XX, t(4;7)(q31;p22) karyotypes.

Using of dual-color in situ hybridization for detection of numerical abnormalities in uncultured and cultured leucocytes of radiochemical industrial workers

V. A. Timoshevsky, S. A. Nazarenko;
Institute of Medical Genetics, Tomsk, RUSSIAN FEDERATION.

As a rule the numerical chromosomal aberrations are not taken into account in the genotoxicity testing. However, it is possible that many harmful substances can cause this type of chromosomal alterations. Fluorescence in situ hybridization (FISH) is a powerful technique that allows numerical chromosome aberrations (aneuploidy) to be detected in interphase cells. We used dual-color FISH for detection of aneuploidy in peripheral blood leucocytes of 15 men – radiochemical industrial workers, contacted with complex of radioactive and chemical substances. Analysis of hypo- and hyperploidy was carried out for three chromosomal pairs: 7/12, 11/16 and X/Y using digoxigenin/biotin labeled probes. We found significantly increased frequencies of numerical chromosome abnormalities in uncultured leucocytes of the exposed workers compared to the unexposed controls (10 individuals) for nullisomy Y-chromosomes only. However significant differences were found in cultured lymphocytes for hypodiploidy of chromosomes 11, 12 and hyperdiploidy of chromosomes X and 12. Moreover, a tendency to higher values was observed for hypodiploidy 16 and total frequency of numerical abnormalities in cultured lymphocytes. We conclude that agents of the radiochemical industry can induce aneuploidy in leucocytes and cause the premutagenic lesions, which are expressed during the cultivation of lymphocytes in vitro. It is possible, that some chromosomes have larger liability to nondisjunction and loss. Our data suggest that analysis of numerical aberrations must be carried out in addition to standard cytogenetics tests.

Inverted duplications associated with terminal deletions : the phenotypic impact of this newly recognizable rearrangement.

M. Zollino¹, R. Lecce², F. Tiziano², G. Neri²;
¹Medical Genetics Catholic University, Rome, ITALY, ²Medical Genetics, Catholic University, Rome, ITALY.

Distal inverted duplications associated with a terminal deletion represent a newly recognizable category of chromosome alterations. Genotype-phenotype correlations are unclear.
We observed a dup/del rearrangement in 6 patients presenting with MCA/MR syndrome. It involved chromosomes 4p (3 patients), 5p (one patient), 1q (one patient) and 19q (one patient). The duplication was detected by conventional cytogenetics, the deletion by molecular probes only.
Chromosome 4p. Both the duplication and the deletion did differ in size in individual patients. The deletions, spanning 3.4, 10 and 12 Mb on 4p16, respectively, encompassed the Wolf-Hirschhorn syndrome critical region" (WHSCR)in each occasion. Clinically, all patients presented with a WHS phenotype.
Chromosome 5p. The 5p13.3-p15.1 region was duplicated, the deleted 5p15.1-pter segment was demonstred by FISH to include the "cri du chat" syndrome critical region. The patient presented with a "cri du chat" syndrome phenotype.
Chromosomes 1q and 19q. A very distal duplication was observed in these cases, affecting the 1q42-q44 and 19q13.2-q13.4 regions, respectively. Accordingly, a very small deletion, just including the subtelomeric region, was detected. Clinical signs in both patients were consistent with the respective partial trisomy syndrome phenotype.
We observed that, whenever the deletion included critical regions with strong pleyotropic effects, such as the WHSCR and the "cri du chat" syndrome CR, clinical signs were consistent with a "deletion" phenotype only. A partial trisomy syndrome phenotype was observed in cases with a small subtelomeric deletion.
A proper assessment of the deletion is recommended in dup/del rearrangements.

Mosaic Trisomy 7; An Age Related Or Tissue Specific Finding In Normal Human Tissues

Y. Mehraein, S. Bastian, C. Fromberg, K. D. Zang;
University of the Saarland, Dept. for Human Genetics, Homburg/Saar, GERMANY.

Mosaic trisomy 7 has been found in a variety of tumors, non tumorous lesions, and even apparently normal tissues. Although characteristic in some condition, the meaning and pathological relevance of trisomy 7 is still unclear. Recent investigation has given rise to the question, whether somatic gain of chromosome 7 is rather related to age than to a specific disease.
By FISH we analyzed the chromosome 7 copy number in blood lymphocytes of 30 healthy individuals with normal conventional karyotype selected for three different age groups. FISH was performed as cohybridization of centromeric probes for chromosome 7 and 10; 1000 interphase nuclei each were analyzed.
Furthermore to identify a potential tissue specific increase of trisomy 7 in a group of 12 old individuals (> 80 years) 200 interphase nuclei of three different tissues (blood lymphocytes, hair root , and mucosal cells) each were analyzed comparatively by FISH.
In blood lymphocytes between all groups for the analyzed chromosomes revealing trisomy rates of below 0,5%, respectively, no significant differences were evident; thus an age related increase of trisomy 7 could not be verified for this tissue. In the intraindividual comparison of cells from different tissues , however, while blood lymphocytes and hair root cells showed similar low trisomy 7 rates (on the average 0.8%) in all cases, a variable but significant increase of mosaic trisomy 7 up to 11,5% was identified in mucosal cells of several individuals. An age dependence of the observed tissue specific amplification of chromosome 7 has to be investigated.

Very large pericentric inversion of chromosome 4 giving rise to a rec(4)chromosome and Wolf-Hirschhorn syndrome.

T. Ilus¹, K. Õunap¹, O. Bartsch², H. Varendi³;
¹Medical Genetics Center, Tartu University Clinics, Tartu, ESTONIA, ²Institute of clinical Genetics, Technical University of Dresden, Dresden, GERMANY, ³Department of Pediatrics, Tartu University Clinics, Tartu, ESTONIA.

We report a case of Wolf-Hirschhorn syndrome in a 1.5-year-old boy born to phenotypically normal parents. Cytogenetic analysis revealed a deletion at 4p15.3 in the boy and a large pericentric inversion of chromosome 4 in the father. The family history was unremarkable. The boy had been born at term with low birth weight, 1700g. Our examination at the age of 2 weeks showed marked growth deficiency, microcephaly, hypertelorism, strabismus, epicanthal folds, prominent glabella, cleft lip and palate, downturned "fishlike’ mouth, micrognathia, dysplastic ears, left simian crease and hypospadias. He also had a cardiac defect (pulmonary stenosis). One year old, he showed profound mental retardation and severe seizures that were difficult to treat. The low birth weight and the clinical signs represent typical Wolf-Hirschhorn syndrome.
Standard cytogenetic analysis from peripheral blood lymphocytes indicated a 4p deletion, karyotype 46,XY,del(4)(p15.3). The mother‘s karyotype was normal (46,XX), but the father demonstrated a large inversion of chromosome 4, karyotype 46,XY,inv(4)(p15.3q35). We then confirmed in the proband, using FISH-analysis, submicroscopic distal 4q trisomy(q35-qter) in addition to the terminal 4p deletion(4pter-4p15.3).
FISH karyotypes:
father: fish inv(4)(p15.3q35)(D4S2930/D4S3186+mv::D4Z1+, D4S96+mv)
proband: fish der(4)(qter-q35::p15.3-qter)(D4S2930/D4S3186+mv::D4S96-, D4Z1+, D4S2930/D4S3186+st)pat
Distal 4q duplication is usually not associated with a distinct clinical phenotype, and therefore the predominance of the Wolf-Hirschhorn phenotype was to be expected in this patient. Semicryptic or cryptic large pericentric inversions represent an important cytogenetic mechanism that can give rise to terminal deletion/duplication syndromes. Prenatal diagnosis must be considered in these cases because of the possibility of recombinants, here, dup(4)(p15.3-pter) and del(4)(q35-qter).

The collection of DNA probes for prenatal, postnatal and preimplantation diagnosis by FISH

I. V. Soloviev¹, Y. B. Yurov¹, S. G. Vorsanova², P. Patsalis³, P. Ioannou³;
¹National Center of Mental Health, Moscow, RUSSIAN FEDERATION, ²Institute of Pediatry and Children Surgery, Moscow, RUSSIAN FEDERATION, ³Institute of Neurology and Genetics, Nicosia, CYPRUS.

Creation of representative DNA probe collection is still actual problem in laboratories of Central and Eastern Europe. Availability of different DNA probes could help in introduction of FISH technology in many laboratories, and, therefore, increase the efficiency of cytogenetic diagnosis. We have identified a large set of original cloned DNA sequences, which could be used as DNA probes in molecular-cytogenetic studies. Alphoid plasmid and cosmid clones, containing repetitive elements, hybridized to pericentromeric regions of most part of chromosomes were identified and tested in clinical-cytogenetic studies (Vorsanova et al., 1986, 1991, 1996; Soloviev et al.,1993,1994,1995). We have analyzed large-insertion PAC library, containing more that 110000 genomic clones. We have detected more than 1600 centromeric and 600 telomeric PAC clones. 350 centromeric and 320 telomeric PAC clones were initially analyzed by FISH. Large insertion clones from total genomic human PAC library were identified for most part of telomeric regions. DNA probes with high potential for diagnosis of common human aneuploidies, involving chromosomes 21, 13, 18, X and Y were carefully selected in cytogenetic studies. DNA probes for centromeric and telomeric chromosomal regions are usefull both as markers for cytogenetic diagnosis and as tool for detailed analysis of numerical and structural chromosomal aberrations in pre-, post- and preimplantation diagnosis, including the analysis of fetal cells .Supported by grant COPERNICUS-2.

Peculiarities of late replication of chromosomes of human fetal and chorionic villi cell at 7 and 12 weeks of gestation.

A. V. Vorobyeva, A. A. Pendina, O. G. Chiryaeva, T. V. Kuznetzova;
Ott's Institute of Obstetrics and Gynecology RAMS, St-Petersburg, RUSSIAN FEDERATION.

The pattern of late replicating metaphase chromosomes from 24-hours cultures of CVS and tissues fragments from five human embryos at 7 and 12 weeks gestation was studied. BrdU was added by impulses through 2 hours. Monoclonal anti-BrdU antibodies FITC - conjugated goat anti-mouse antibody were used to identify the BrdU-labeled sites.
Metaphase spreads with markers of late replications were chosen for analyses. The intertissue differences in initiation and termination of replication in pericentric heterochromatin of chromosomes 1, 9 were shown in chorionic villi and embryonic cells. Pericentric heterochromatin of chromosome 16 was shown to be latest replicating segment in both tissues compared to pericentric heterochromatin of chromosomes 1, 9.
At 7 weeks of gestation some G-bands of chromosomes 1, 2, 3, 4, 5, 9, 11, 13, 14, 16 replicated simultaneously with pericentric heterochromatin of chromosomes 1, 9, 16 in chorionic cells, while in embryonic cells they replicated earlier than pericentric heterochromatin.
At 12 weeks of gestation however these segments replicated simultaneously with pericentric heterochromatin of chromosomes 1, 9, and 16 in both tissues. These data suggest that changes in of replication pattern of heterochromatin regions probably reflect differences in their functional status in embryonic and extraembryonic tissues at different stages of embryonic development.

H. Bruyere¹, E. Separovic¹, J. Guscott², T. Pantzar¹;
¹University of British Columbia, Vancouver, BC, CANADA, ²Vancouver General Hospital, Vancouver, BC, CANADA.

A 49-year-old male with hypogonadism and gynecomastia was found to have a 46,XX karyotype with additional chromosomal material on the short arm of a chromosome 15. C-banding and NOR-banding as well as fluorescence in situ hybridization with chromosomes 15 and Y centromeric probes, a Y chromosome short arm unique locus probe and a Y chromosome long arm heterochromatin probe were perform to further characterize this extra material. It was found to contain the Y chromosome short arm and centromere. This case represents a rare cytogenetic Klinefelter syndrome variant, with a diploid complement and a stable dicentric derivative chromosome from an unbalanced translocation involving the long arm of the Y chromosome and the short arm of a chromosome 15.

Molecular studies of small marker chromosomes in patients with 45,X/46,X,+mar mosaicism

J. W. Kim¹, J. M. Kim¹, E. H. Cho¹, J. T. Seo², Y. S. Lee², J. M. Yun², H. M. Ryu³, S. Y. Park¹;
¹Laboratory of Medical Genetics, Samsung Cheil Hospital and Women's Healthcare Center, Seoul, REPUBLIC OF KOREA, ²Department of Urology, Samsung Cheil Hospital and Women's Healthcare Center, Seoul, REPUBLIC OF KOREA, ³Department of Obstetric and Gynecology, Samsung Cheil Hospital and Women's Healthcare Center, Seoul, REPUBLIC OF KOREA.

We have studied the small marker chromosome in four patients with mosaic karyotype 45,X/46,X+mar. Male patients were referred for azoospermia and female for primary amenorrhea. We used fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) to determine the origin and structure of the marker chromosome. FISH studied with SRY/CEPX probe (Vysis) confirmed the Y origin of the marker and detected that four patients showed double SRY signals on both ends of abnormal Y chromosome and one male patient had only one SRY gene. PCR analysis using primers for 11 loci along Y chromosome including SRY, RPS4Y, DYS14 (Yp), DYZ3 (Ycen), sY84 (AZFa), sY129, sY134 (AZFb), sY156, sY254, sY255 (AZFc, DAZ gene) and DYZ1 (Yqh) was investigated. Two male patients were the Yq breakpoint to the region between AZFa and AZFb. In female patient, the genes from SRY to AZFb were positive. In other two patients there was a failure to detect the alpha-satellite (DYZ3) that is an the integral part of the centromere. One of them was the Yp breakpoint between DYS14 and DYZ3, and the other having one SRY signal carried the Yp (SRY, RPS4Y, DYS14) and Yq (sY84, sY129, sY134) except DYZ3 unexpectly. It is assumed that the deletion of distal Yq euchromatin playing an important role in the spermatogenetic process lead only to azoospermia.

L. M. Serghei¹, A. Stana², C. Geormaneanu¹, V. Stoian³, A. Arghir¹, A. G. Lungeanu¹;
¹National Institute"Victor Babes", Bucharest, ROMANIA, ²Filantropia Hospital, Bucharest, ROMANIA, ³Bucharest University, Faculty of Biology, Bucharest, ROMANIA.

Cytogenetic analyses were carried out on 54 cases of primary amenorrhea, using peripheral blood lymphocytes, GTG and CBG-banding. The monosomy X occured in 49 patients with Turner syndrome(TS). Chromosomal sexual assignment was established as 46,XY in three phenotypically females. One of them revealed gonadal tumor when she underwent gonadectomy at the age of 21. The involvement of autosomes in the ethiology of primary amenorrhea came into discussion in two cases;the karyotyping showed the folowing cellular mosaics:[46,XX,+18pter(80%)/45, XX,-14,+18pter(20%)] and [45,X0(60%)/46, X0,mar(?)(40%)], respectively.
At the time patient 1 reffered, she had hormonal-induced intermittent, irregular menses. We attempted to find correlation between the constitutional +18pter and the observed resistance to hormone-replacement therapy. Normal X chromosome pattern was noted both in hypodiploid and diploid cells. Neither the cryptic sex chromosomal abnormalities nor the origin of the 18pter extra material from chromosome 14 are excluded. Regarding patient 2, we mention that monosomy X was present in all 200 examined metaphases, strongly suggesting TS as cause of primary amenorrhea, but the patient lacked Turner stigmata. Moreover, the diploid cells exhibited a marker that appeared to be a dicentric derived from a translocation between 21 and 20 chromosomes. The cytogenetic approach proved once again its value in diagnosis of primary amenorrhea. Our study has to be extended at molecular level, in order to define accurately the provenience and the roles of the markers, especially those derived from autosomes.
Acknowledgements: VIASAN PROJECT NO. 127/2001-2003

M. Zanganeh, R. Karimi-Nejad, T. Nayeb Bagher, S. Voghouie, H. Miri, M. Karimi-Nejad;
Karimi-Nejad Genetics and Pathology Center, Tehran, IRAN (ISLAMIC REPUBLIC OF).

More than 850 bone marrow samples have been analyzed in our center during the past five years. Among 850 patients, 486 (57%) were referred for malignant disorders and 131 cases (15%) for non-malignant disease. 233 casses (28%) didn't have any primary diagnosis at the time samples were taken of which 169 belonged to the first 500 samples. Of the non-malignant cases, 87 were referred for anemia, 43 pre-transplant, 44 post-transplant, and 44 for post transplant study of a malignant disorder. The probable clinical diagnoses for the malignant cases were as follow: 164 (34%) for ALL, 135 (28%) AML, 123(26%) CML, 23(5%) MDS, 21 (4%) lymphoma, 6 (1%) Multiple Myeloma, 6 (1%) MPD and 8 (1%) others.
Chromosomal aberrations were detected in 73 (50%) of the 145 successful cultures of patients without any diagnosis, 69 (54%) of 128 cultures with diagnosis of ALL, 68 (55%) of 123 AML patients, and 80 (73%) of 109 patients suspected for CML. Among 22 conclusive MDS cases, 11 (50%) patients had some chromosomal change, ten (50%) out of 20 lymphoma cases and 3 out of 6 MPD and none of the 4 Multiple myeloma had chromosomal changes.
Among 88 post-transplant cases, 44 (50%) were malignant and 44 (50%) non-malignant. 72 (82%) patients showed donor cell line, 8 (9%) patients showed the recipient cell line and another 8 (9%) patients showed chimerism of donor and recipient cell lines.

N. V. Kovaleva;
Institute of Obstetrics and Gynaecology RAMS, St.Petersburg, RUSSIAN FEDERATION.

Homologous Robertsonian translocations/isochromosomes represent unique model for studying chromosome behavior in the absence of homologous pairing and recombination. Though the number of published cases is small, and the only data available is the sex of the offspring of the dup(21q) carriers, some conclusions on the segregation pattern of both rearranged chromosomes and gonosomes can be made. It was found that 7 male carriers of dup(21q) fathered 13 children with Down syndrome of known sex. 12 of them were males, differing from 1:1 ratio (p=0.0017, binomial test). There was no significant male predominance in cases of maternal transmission (23 males, 16 females). This finding is considered as a direct evidence for NMC of the dup(21q) and X-chromosome. NMC, proven for Drosophila [Grell, 1971], was proposed to explain male excess in Down syndrome (DS) with paternally derived trisomy [Kovaleva NV.Genetika(Russ)1992,28:5-15]. NCM of chromosomes 21 and X would result in production of different gametes: 23,X, 23,Y, 23(+21,-Y), 22,XY(-21), 22,0(-Y), 22,X(-21), 24,Y(+21), 24,XY, also explaining the occurrence of gonosome aneuploidy and double aneuploidy of paternal origin. Further data supporting the NMC hypothesis come from studies on sperm in healthy and infertile men [Griffin et al,1996; Baumgartner et al,1999], in fathers of patients with aneuploidy [Blanco et al,1998; Soares et al,2001] and in carriers of balanced translocations (“interchromosomal effect”) [Morel et al,2001; Pellestor et al, 2001], together with data on elevated risk of progeny with autosomal trisomies in carriers of sex chromosome anomalies (Nivelon-Chevallier et al, 1988; Tarani et al, 1998; Hennebicq et al, 2001).

M. Zamanian¹, S. Tootian¹, 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).

The index case was a female with a lichenoid lesion on her face. She reffered to our center for G banding karyotype because her sons were going to marry and she was worried about her son's future. She was an educated woman and a laboratory technician and there wasn't any sign of mental retardation. Her father had 4 children from her mother and four children from another wife. She had a nephew from her brother and a neice from her sister in law. Both children were mentally retarded and translocation (2,11) were found in the both children.
G banding method was used for karyotypeing of our index and her sons. Translocation(2,11) was detected in the index.We also examined her two sons for translocations and both of them were normal.There are two questions here: 1)Is this type of translaocation a strong cause for mental retardation of the neice and nephew? 2)If yes why the index case didn't have any sign of mental retardation?
It seems that because our procedure cannot detect all the sub bands in a patient's chromosomes. Microdeletion or inversion may be change the phenotype in the cases nephew and neice. FISH or molecular methods can be help to find out the problem in this family.

R. Karimi-Nejad, N. Naghash-pour, F. Azimi, S. Razavi, M. Soleimani, T. Nayeb Bagher, M. Rahimi, S. Voghouie, M. Karimi-Nejad;
Karimi-Nejad Genetics and Pathology Center, Tehran, IRAN (ISLAMIC REPUBLIC OF).

Among 28000 karyotypes of peripheral blood performed in our center for various indications, 35 balanced translocations of chromosomes 13 and 14 have been detected. Of these 35, 26 were referred for history of abortions, IUFDs, offspring with congenital anomalies, and offspring with trisomy 13. Interestingly, 6 cases were 3 first cousin couples where both partners were carriers. The remaining 9 individuals were referred for reasons other than reporductive failure, 1 for mental retardation, another for premarital check up and the other 7 for primary amenorrhea.
Considering that 13 of the 35 translocation carriers were males, 7/23 der(13;14) carriers had primary amenorrhea. Cases referred for primary amenorrhea were aged 12-19 years without history of menstruation, and with one exception (12 years old) they had normal age related secondary sexual characteristic development, and growth.
Overall, 413/28000 cases were referred for primary amenorrhea who did not show related chromosomal changes such as 46,XY and 45,X variants. 10/363 had chromosomal aberrations including pericentric inversion of chromosome 1 and 2, paracentric inversion of chromosome 14 and the other 7 had der(13;14).
We would appreciate to be informed of similar findings and any guidance as to the etiology and possible association our data suggest.

R. Schellberg¹, M. Dobos², G. Fekete², G. Schwanitz¹, R. Raff¹;
¹Institut für Humangenetik, Bonn, GERMANY, ²II. Kinderklinik der Semmelweis Universität, Budapest, HUNGARY.

Mosaicism is caused by postzygotic aberrant mitoses and leads to numerical or structural aberrations or a combination of both. The karyotype of the zygote can be normal or abnormal.
Our investigation group comprises 25 cases of gonosomal mosaicism with a Y-chromosome analysed in at least one cell system: 45,X/46,XY (n=8), 45,X/46,X,idic(Y)-mosaicism (n=7), 45,X/47,XYY-mosaicisms (n=5), 45,X/46,X,del(Y) (n=4) and 45,X/46,X,r(Y) (n=1).
13 patients were phenotypically male, 7 were female and 5 showed intersexual external genitalia.
The patients age at the time of chromosome investigation ranged from the prenatal period up to the age of 36 years.
The most frequent clinical findings were: growth retardation, abnormalities of the external genitalia, kidney malformations and different types of heart defect.
Cytogenetic investigations combined metaphase and interphase analyses with FISH (DNA probes: wcp X and Y; CEP X and Y; Yph 3.4). Between 2 and 5 cell systems per patient were analysed with regard to their origin from different blastodermic layers.
An unequal distribution of the mosaic could be demonstrated in the different cell systems, with no preferential combination.
Patients with dicentric Y-chromosome revealed the highest instability of karyotype. There was no age dependent karyotype changes in our investigation group.
Supported by: DAAD/MÖB, Richard-Winter-Stiftung

S. Cappellacci¹, S. Martinelli², R. Rinaldi¹, E. Martinelli¹, P. Parisi³, B. Mancini², P. Grammatico²^,1;
¹Medical Genetics, San Camillo-Forlanini Hospital, Rome, ITALY, ²Medical Genetics, University "La Sapienza", Rome, ITALY, ³Outpatient Service of Pediatric Neurology S. Camillo-Forlanini Hospital, Rome, ITALY.

We present a case of partial duplication of the long arm of chromosome 12 characterized by FISH techniques using YAC probes.
On physical examination, at 8 years, our patient demonstrated: macrocephaly, flat occiput, long palpebral fissures, long eyelashes, protruding nasal root, anteverted nostrils, large and asymmetric ears, thin lips, light prognathism, malar hypoplasia, short stubby hands and femoral dysplasia. Magnetic resonance imaging showed partial agenesis of the cerebellar vermis and cystic dilatation of the fourth ventricle consistent with Dandy-Walker malformation.
A neurological and behavioural assessment revealed a psychomotor retardation and attention deficit/hyperactivity disorder (ADHD).
Standard cytogenetic analysis showed a partial duplication of the long arm of chromosome 12. Parents’ karyotypes were normal.
To define the extension of the duplicated region we performed FISH analyses by using the following YAC probes (kindly supplied from YAC Screening Centre, Tigem, Milan, Italy): 943B11 (q21; 96.5 cM), 778F6 (q22; 97.6 cM), 850A12 (q22; 99.6 cM), 934C1 (q23; 108 cM), 827A10 (q24.1; 137.5 cM), 910B10 (q24; 154 cM), and 812D10 (q24.3; 161 cM). The analyses evidenced a tandem duplication of the 12q22q24.33 region with the proximal breakpoint located between 96.5 and 97.6 cM and the distal one between 154 and 161 cM resulting in the following karyotype: 46,XY,dup(12)(q22q24.33).
In order to contribute to the identification of the characteristic clinical features associated with 12q partial duplication we reviewed the literature and compared our case with those already described.

M. H. Lee;
Laboratory of Medical Genetics, Samsung Cheil Hospital and Women's Healthcare Center, Seoul, REPUBLIC OF KOREA.

Ring chromosomes are rare abnormalities that typically arise de novo. The usual ring phenotype have been shown to have a wide range of intellectual functioning and congenital anomalies. We found several ring chromosome 4 instabilities in mosaic form with a normal cell line in peripheral blood cells of a 27 year old woman who was referred for infertility and short stature. The results of cytogenetic analysis showed 45,XX,-4/ 46,XX,r(4)/ 46,XX,dic r(4)/ 47,XX,r(4),+r(4)/ 46,XX karyotype in three repeated examinations. FISH analysis executed for precise characterization of the ring chromosome 4 breakpoints using chromosome 4p, 4q telomeric probes demonstrated deletion of the 4p telomere from the ring chromsome 4. Parental karyotypes were normal. After then, she became naturally pregnant and we performed amniocentesis at 16 weeks gestation. The fetal karyotype was normal. The phenotypes of our patient seemed to be related to the ring syndrome by the ring chromosome instability. The present study would be offer information to the long-term consequences of ring chromosome instability on clinical outcome.

A. Lee¹, D. Lie¹, S. Tien², C. Rudduck Sivaswaren²;
¹Division of Medical Sciences, National Cancer Centre, Singapore, SINGAPORE, ²Department of Pathology, Cytogenetics, Singapore General Hospital, Singapore, SINGAPORE.

Background: Translocations and deletions in the chromosome 11q23 region are frequent in hematologic neoplasms such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic lymphocytic leukemia (CLL). We describe here the use of a bacterial artificial chromosome (BAC) probe set from the 11q23 region, applied to these malignancies.
Methods: Four BAC probes from one contig, and two BAC probes from another contig on 11q23 spanning the MLL region were labeled with either biotin or DIG for dual color fluorescence in situ hybridization (FISH) analysis. These probes were applied to cases with hematologic malignancies with known structural abnormalities involving 11q23.
Results: A split signal was detected in one ALL case with t(4;11)(q21;q23), one AML case with t(9;11)(p22;q23) and another AML case with t(11;19)(q23;p13), demonstrating that the probes span the MLL region known to be involved in these translocations.
The probes were further tested on one case of AML with add(11)(q23) and two cases of MDS and two cases of CLL, all with del(11)(q13q23). The additional material on 11q23 and marker chromosomes were found to be amplification of the regions detected by the probe set. Although the MDS and CLL cases cytogenetically appeared to have the same abnormality, the MDS cases had deletion of both contigs, while the CLL cases only had deletion of one contig.
Conclusions: At a molecular level, deletions in our CLL and MDS cases appeared different. This study demonstrates the importance of combining molecular and cytogenetic techniques to further define chromosome abnormalities.

Deletion of abl gene resulting from a recombination of a maternal (3;22;9)(q22;q12;q34.1) translocation in a child with axial hypotonia and dysmorphy.

A. Aboura, P. Labrune, F. Perreaux, V. Poncet, S. Brisset, L. Foix-L'Helias, G. Tachdjian;
Hôpital Antoine Béclère, Clamart, FRANCE.

Small deletions near breakpoints may be an important cause of disease in apparently balanced chromosome rearrangements. We report on chromosomal findings in a boy with axial hypotonia and dysmorphy. He was born after a full-term uneventful pregnancy. He was the third child of healthy unrelated parents. The mother has had nine miscarriages. The child was first admitted at the age of 8 months for gastro-enteritis which was rapidly cured. Physical examination showed moderate axial hypotonia, hypertelorism and bilateral epicanthus, high-arched palate, macroglossia, and antimongoloid palpebral fissures. There was no evidence for visceral malformation. By conventional cytogenetic analysis a reciprocal balanced translocation between chromosomes 3 and 22 was diagnosed. In order to define clearly the breakpoints on the two chromosomes, chromosome painting was used and revealed a complex, apparently balanced translocation t(3;22;9). This translocation was inherited from the mother. Fluorescence in situ hybridization with different locus probes near breakpoints showed a deletion of abl gene located at 9q34.1 in the patient. This deletion was not found in the mother and in the sister carrying the translocation. Deletion of abl gene has been described once in a newborn with a complex cardiac anomaly and carrying a paracentric inversion of the long arm of chromosome 9 (Kleyman et al., Am J Med Genet, 1997).
Our case emphasises that molecular cytogenetic analysis of breakpoints in apparently balanced chromosomal translocations should be systematically carried out in patients with phenotypic abnormalities.

E. Popelinska¹, D. Leznarova², P. Vlasin², P. Kuglik³;
¹Cytogenetic Laboratory, Prenatal Diagnosis Centre, Brno, CZECH REPUBLIC, ²Prenatal Diagnosis Centre, Brno, CZECH REPUBLIC, ³Dept. of Molecular Biology and Genetics, Masaryk University, Brno, CZECH REPUBLIC.

We report a case of prenatally diagnosed mosaicism 45,X/47,XX,+8. Prenatal diagnosis of mosaic trisomy 8 is relatively rare and the occurence of this abnormality in association with monosomy X is very unusual.
Our patient-24 years old primigravida -was reffered for abnormal ultrasound findings [agenesis corp.callosi,heart defect] at 33 t.g. Routine cytogenetic analysis of fetal blood [FB] cells and cultured amniotic fluid [AF] cells was performed. In FB cells the mosaic karyotype 45,X[10]/47,XX,+8[28] we found. All examined cultured AF cells were 45,X.
Results of cytogenetic analysis were compared with fluorescent in situ hybridization. FISH confirmed the mosaic karyotype 45,X/47,XX,+8 in FB cells and in uncultured amniocytes as well as in peripheral blood lymphocytes and buccal epithelial cells of the affected child after delivery. In cultured AF cells trisomy 8 was not found by FISH.
Our findings indicate the difficulties in the prenatal diagnosis of autosomal mosaicism, namely trisomy 8. This chromosomal abnormality can be missed with routine prenatal cytogenetic analysis because of a different tissue-specific distribution of the abnormal cell line.

The usage of FISH-WCP technique under the cytogenetical observation of 120 Chernobyl liquidators with different radiation doses

FISH-WCP technique was introduced in Ukraine for the first time in cytogenetic lab of RCRM in 1999 in the frame of the Ukrainian-American project “Leukemia” for the comparison of different dosimetry methods (including biodosimetry) in Chernobyl liguidators. According to the protocol of the dosimetry part of the Project all cytogenetecal investigations had been fulfilled in blind manner. Method FISH (with directly labeled by Spectrum Orange DNA-probes to chromosomes 1, 2, 4) had been tested on the 120 liquidators of the Chernobyl accident ranging in age from 37 to 73 years in dose range from 10 till > 100 cGy. The data received confirmed that at present FISH-WCP technique can be successfully use in delayed terms following the radiation exposure for the indication of human irradiation and group dosimetry. As regard of the usage FISH method for individual retrospective dosimetry of radiation exposure especially in the range of low doses there are many problems of which needed in further investigations. At present the high and variable background frequencies of complete translocations, strong age effect and essential interindividual variability in the rate of radiation-induced stable aberrations permit the use FISH for estimation and verification of doses in the part of Chernobyl liquidators with assumed radiation exposure more than 25 cGy.
This work had been supported by National Cancer Institute of USA in the frame of Ukrainian-American Project "Leukemia".

J. Lissitsina¹, R. Mikelsaar¹, K. Varb¹, M. Punab²;
¹Institute of General and Molecular Pathology, Tartu, ESTONIA, ²Tartu University Clinicum, Tartu, ESTONIA.

The incidence of chromosomal abnormalities in infertile males varied from 2.2 to 14.3%. The incidence of chromosomal variants was shown to be at 34.5% in infertile men and 10-15% in the general population. Our study was performed to determine the frequency of chromosomal abnormalities and polymorphism in infertile men with azoospermia and severe oligospermia (sperm density <5x10M/mL). Chromosomal analysis of 27 infertile men was performed both from peripheral blood lymphocytes and skin fibroblasts cultures using GTG, C banding, fluorescent in situ hybridization (FISH) and other methods. In 5 (18.5%) cases chromosomal abnormalities were found. There was one case (3.7%) with numerical chromosomal abnormality (47,XXY). Structural abnormalities were revealed in 4 cases (14.8%), from which 3 were mosaics (in 3%-98% of cells). The chromosomal variants were found in 8 (29.6%) patients. In 15 (55.6%) cases the karyotype was normal. We have found higher than given in the literature percentage of chromosomal abnormalities (mainly structural abnormalities), which could be reason of infertility. This finding could be explained partly by small amount of cases. We wish also to stress, that to reveal the low percentage mosaicism, it seems to be necessary to analyze at least 33-50 mitoses. However, the frequency of chromosomal variants in infertile men could support the opinion that chromosomal variants may be associated with reproductive failure in men.

A comparative analysis of G-banding, FISH and RT-PCR for the diagnosis of bcr-abl-positive CML

R. Brill-Zamir, I. Laevski, D. Sahar, R. Gershoni-Baruch;
Rambam Medical Center, Haifa, ISRAEL.

Phildelphia (Ph) chromosome-positive CML, with the bcr-abl gene translocation, has a dismal prognosis. The identification of Ph-positive patients is vitally important because only aggressive therapeutic approaches, such as interferon alpha (IFN-alpha) treatment and allogeneic bone marrow transplantation, may result in long-term disease-free survival. Routine diagnostic methods for detection of the bcr-abl translocation include conventional cytogenetics(G-banding), reverse transcriptase-polymerase chain reaction (RT-PCR), and more recently, fluorescence in situ hybridization (FISH) analysis. Routine cytogenetics . sensitive at the initial diagnosis is unreliable during treatment. RT-PCR analysis is considered the most sensitive tool for the detection of the bcr-abl translocation, and is widely used alone, or in combination with the other methods. FISH analysis is simple, extremely reliable and sensitive. This study compares the efficiency of the three methods for the initial diagnosis of Ph-positive CML, and for detection of minimal residual disease during treatment. Conventional G-banding cytogenetics, FISH with BCR and ABL double-color probes and RT-PCR for detecting Ph-positive CML were undertaken in 22 CML patients undergoing either IFN-alpha treatment or allogeneic bone marrow transplantation (allo-BMT). The results obtained using the three methodological approaches were 100% correlated at diagnosis. Following treatment the cytogenetic analysis becomes technically less feasible and the results less reliable. Whereas, RT-PCR and FISH analysis remain equally and mutually contributive. We thus conclude that the concomitant use of FISH and RT-PCR remain the optimal diagnostic combination for the detection of the bcr-abl translocation.

Trisomy 12 mosaicism in a newborn presenting with chylothorax, facial dysmorphism, genital anomalies and congenital heart disease

V. Klamroth, R. Exeler, I. Kennerknecht, J. Horst;
Westfälische Wilhelms-Universität, Münster, GERMANY.

We present a newborn girl with trisomy 12 mosaicism,[46,XX/47,XX+21]. The girl was born at 39 weeks of gestation after an uneventful pregnancy to healthy parents.
After birth, chylothorax, congenital heart disease (VSD, ASD), muscle hypotonia, facial dysmorphism and ambiguous genitalia were cardinal features.
Chromosome analysis of various tissues revealed trisomy 12 mosaicism (skin 2/60 cells, muscle 3/43 cells, lung tissue 7/37 cells, blood and pleura: normal karyotype).
Complete trisomy 12 in humans is not viable. To our knowledge this is the tenth published case of trisomy 12 mosaicism in a liveborn. Obviously due to the nature of mosaicism, reported cases show a wide spectrum of anomalies ranging from Kartagener syndrome in an otherwise healthy man to multiple congenital abnormalities including complex heart malformations, facial dysmorphism, urogenital abnormalities (renal hypoplasia, ectopia vesicae) and further inner malformations (multiple accessory spleens, pancreatic-spleenic fusion, gallbladder hypoplasia) with neonatal death. No typical mosaic trisomy 12 phenotype can be delineated.
We report the first case of trisomy 12 mosaicism presenting with chylothorax, facial dysmorphism and genital anomalies as cardinal features. We compare our patient with earlier described cases.

Coincidental Structural And Numerical Aberrations Of The Chromosome 15 - Three Different Case Reports

E. Kocarek¹, D. Novotna², P. Balicek³, T. Marikova¹, M. Malikova², A. Zumrova¹, K. Novotna¹, M. Strnad¹, P. Goetz¹;
¹Charles University 2nd Medical School, Prague, CZECH REPUBLIC, ²Motol University Hospital, Institute of Biology and Medical Genetics, Prague, CZECH REPUBLIC, ³University Hospital - Department of Clinical Genetics, Hradec Kralove, CZECH REPUBLIC.

We report three cases of chromosome 15 aberrations. First one is a girl with dysmorphic features, seizures, and a delay of psychomotoric development. Her karyotype was 47,XX,+mar. Result of our FISH analysis was 47,XX,+mar.ish idic(15)(q11-q13)(D15Z1++,D15S10+,PML-). The second case is a 26-years old healthy pregnant woman, whose foetus has karyotype 47,XX,+mar. Our FISH result was 47,XX,+mar.ish dic(15)(D15Z1++,SNRPN-, PML-). The third case report desribes a 2-years old boy with blindness, deafness, dumbness and cryptoorchism.The cytogenetic analysis revealed a karyotype 45,XY,der(21), t(15;21)(q13;q22.3), -15. FISH confirmed the cytogenetic result: 45,XY,der(21), t(15;21)(q13;q22.3),-15.ish der(21)(D13Z1/D21Z1+,D15Z1-, SNRPN-, UBE3A/D15S10-, D21S270/D21S337/D21S55/D21S233+, PML+). The result indicates haploinsufficiency of the critical Prader-Willi/Angelman region in 15q11-q13. The work was supported by grant IGA NE5685-3 (Ministry of Health CR) and research project of the Charles University No.111300003.

Cytogenetic Analysis of 111 Cases of Adult Acute Lymphoblastic Leukaemia in an Asian Population.

C. Rudduck Sivaswaren¹, S. L. Tien²^,1;
¹Department of Pathology, Cytogenetics, Singapore General Hospital, SINGAPORE, ²Department of Haematology, Singapore General Hospital, SINGAPORE.

The majority of cases with acute lymphoblast leukemia (ALL) have abnormal chromosomes. Many of these abnormalities have been shown to be important clinical prognosticators. Cytogenetic analysis is therefore used routinely in management of ALL. In the current study we evaluate the cytogenetic abnormalities seen in a series of cases of adult ALL in an Asian population.
A total of 111 cases of ALL admitted to Singapore General Hospital between 1995 and 2001 were analysed. The age range was 16-81 (46 females, 65 males).
Chromosome preparations were made from bone marrow cells using direct and 24 hour cultures. Karyotypes were constructed in accordance with ISCN nomenclature.
Chromosome abnormalities were seen in 73% of cases a normal karyotype in 21.6% while the remaining 5.4% were unsuccessful.
The most common balanced structural abnormalities were t(9;22)(q34;q11.2) seen in 24 (21.6%) and 1 variant translocation, followed by t(8;14)(q24;q32) in 5 (4.5%) with variant translocations in 4 additional cases. The diagnostically important abnormalities t(4;11)(q21;q23) were seen in 2 cases and der(19)t(1;19)(q23;p13) and it’s balanced form in 3 and 1 case respectively. Deletion of 9q was the most common region of deletion seen in 7 (6.3%) cases.
All the abnormal cases but 3 had structural abnormalities. A hypodiploid karyotype of <46 chromosomes was seen in 10.8 %, pseudodiploid in 41.4%, hyperdiploid of 47-50 in 12.6% and hyperdiploid >50 in 6.3% of cases respectively.
The most frequent numerical gains were for chromosomes 8, 21, 14, 18, while loss of chromosome 9 was the most common loss.

D. Genenviève¹, L. Faivre², S. Lesourd¹, F. Mugneret², D. Heron¹;
¹Département de Génétique, Hôpital de la Pitié-Salpétrière, Paris, FRANCE, ²Service de Génétique, Hôpital d'Enfants, Dijon, FRANCE.

Although 47,XXX, 47,XXY, 47,XYY and 45,X karyotypes are frequent and occur at least 1 in 400 birth, patients with more than one extra sex chromosomes are very rare.
Here we report on two new cases with 48, XXXX (tetrasomy X) karyotype and reviewed the litterature. The pregnancy and delivery were normal in both cases. In case one, neonatal measurements were 2625 g for weight (- 1.5 SD), 46.5 cm for length (- 1.5 SD) and 34 cm for OFC (M). IUGR was noted in case two with 2100g for weight and 43 cm for length at birth. In both cases, facial dysmorphism including bilateral epicathus, synophris, long eyelashes, bulbous nose, long and flat filtrum, small mouth and bilateral radioulnar synostosis was noted. Evolution was marked by mild mental retardation with speech difficulties.
In attempt to well define the tetrasomy X, we reviewed the litterature. To our knowledge, since the first description of an abnormal number of chromosome in cultured lymphocytes reported by Lejeune et al in 1959, only 40 cases with 48,XXXX have been described in the litterature. We found that mental retardation was constant excluding one case with normal intelligence. Other features were particular facial dysmorphism, radioulnar synostosis and similar but non-specific behavioural problems. This syndrome seems to be clinically recognisable.

One event, two cell lines, three chromosomes, four breakpoints: unusual mosaic complex translocation in a patient with oligospermia.

J. M. Dupont¹, F. Baverel¹, M. Savale², A. Lebbar¹, D. Le Tessier¹, C. Patrat³, J. Guibert², D. Rabineau¹;
¹Histologie Embryologie Cytogénétique, Hôpital Cochin, Paris, FRANCE, ²Gynécologie, Hôpital Cochin, Paris, FRANCE, ³Histologie Embryologie Biologie de la Reproduction, Hôpital Cochin, Paris, FRANCE.

Complex chromosomal rearrangements and mosaic reciprocal translocations are rare events in constitutional cytogenetics. We report here on a 52 years old man with variable oligospermia who was referred to our laboratory for cytogenetic examination before AMP procedure. This man has two healthy children from a previous union. GTG and RHG banding revealed a complex translocation between chromosomes 9, 12 and 14 in 20% of the cells from two consecutive peripheral blood sampling. These standard banding techniques led to the proposal of the following chromosomal pattern: 46,XY,t(9;12;14)(q32;q13;q32)/46,XY.
Unexpectedly, FISH revealed a far more complex pattern, with two breakpoints on der(12). This derivative chromosome harbors material from chromosomes 9 and 14 hence correct designation is 46,XY,t(12;14;12;9)(q13;q32;p13;q32).ish t(12;14;12;9)(Tel14q+,wcp14+,wcp12+,wcp9+,Tel 9q+;wcp14+,wcp12+,Tel12q+;wcp9+,wcp12+, Tel 12p+)/46,XY. Careful hematological examination of the patient has been conducted to eliminate a rising malignant process.
During meiosis, these complex rearrangements are expected to form a multivalent from which multiple gametic combination can occur, most of which will be unbalanced. However, if female gametogenesis can accomodate itself to the complexity thrust upon it and produce balanced ovocytes, the rule of the greater vulnerability of spermatogenesis to structural rearrangement applies particularly in the case of complex abnormalities and heterozygote males are often sterile.
In our patient, the presence of a normal cell line may explain the more or less conserved fertility; however, in case of pregnancy following AMP, a prenatal diagnosis should be proposed to the parents as some of the imbalances might be viable.

G. Viot¹, V. Desportes², C. Ozilou³, A. Choiset¹, S. Girard¹, A. Munnich⁴, M. Prieur³, S. P. Romana⁵, M. Vekemans³, C. Turleau⁵;
¹Hopital Saint Vincent de Paul, Paris, FRANCE, ²Hopital Saint vincent de Paul, Paris, FRANCE, ³Hopital Necker-Enfants Malades, Paris, FRANCE, ⁴Hopital Necker- Enfants Malades, Paris, FRANCE, ⁵Hopital Necker-enfants Malades, Paris, FRANCE.

FISH studies using subtelomeric probes allowed us to explain MR/MCA recurrence in two large families with follow-up of 40 and 20 years respectively. Repeated high-resolution karyotypes were performed in both families. In the first family, a sex-linked recessive inheritance was suggested because two related male patients were observed. However a girl with the same clinical abnormalities was born recently. A multiprobe subtelomeric FISH study led to the identification of a familial cryptic translocation t(17;22)(q25;q13.3). A derivative 22 yielding monosomy 22q13 and trisomy 17q25 was observed in all affected patients tested. The translocation was segregated over at least four generations. In the second family, the observation of recurrent malformations and perinatal deaths over several generations suggested the presence of a familial rearrangement. Two cousins, a girl and a boy, with similar clinical findings died at a young age. Recently, photographs of both children were examined again. They showed facial dysmorphism evocative of a 2q27 deletion. FISH studies with chromosome 2 subtelomeric probes confirmed this imbalance and show that it derived from a cryptic familial t(2;17)(q37.3;q25). These observations illustrate that reassessment of " old " genetic files using both molecular cytogenetic tools and new syndromes clinical descriptions can point towards a correct diagnosis. In addition, they emphasise that recurrence of similar MR/MCA findings in different branches of a kinship is highly suggestive of a chromosomal anomaly. Genetic counselling and prenatal diagnosis are now available for both these large families.

T. Martin Denavit¹, D. Sanlaville¹, C. Grillon², V. Malan¹, P. Nizard¹, A. Ardalan¹, L. Burglen³, J. Taillemite¹, M. Portnoï¹;
¹Laboratoire de Cytogénétique Hôpital Saint Antoine, Paris, FRANCE, ²Service de Néonatologie Hôpital Trousseau, Paris, FRANCE, ³Service de Génétique hôpital Trousseau, Paris, FRANCE.

Cat Eye syndrome (CES) is characterized by a variety of congenital defects including ocular coloboma, anal atresia, preauricular tags or pits, heart and kidneys defects, dysmorphic facial features and mental retardation. The penetrance and severity are highly variable. This syndrome is associated with a supernumerary bisatellited dicentric marker arising from an inverted duplication of chromosome 22 classified into 2 types based on the location of the breakpoints. Most common CES chromosomes correspond to the smaller type I.
We describe a female child with a severe phenotype of CES, born at 40 weeks of gestation from healthy parents. An intrauterine growth retardation was diagnosed at 22 weeks of gestation and was confirmed at birth. She developed a severe respiratory distress. She had hypotonia, dysmorphic features and malformations including downslanting palpebral fissures, hypertelorism, ocular coloboma, bilateral aplasia of the external ears, anal stenosis, patent ductus arteriosus. She died at age 1 month. Chromosome analysis showed a karyotype with an extranumerary bisatellited marker in all cells examined. The parents had normal karyotypes. FISH using 14/22 alpha satellite, WCP of chromosome 22 and N25 probes showed that the marker was a dicentric inverted duplication of the short arm and proximal long arm of chromosome 22, symmetrical and localized distally to the Digeorge locus (type II).
The severe phenotype of our patient demonstrated the phenotypic variability which does not seem to be related to the size of the duplication or presence of mosaïcism. This phenotypic variability increases the difficulty of genetic counselling.

E. Ruiz-Casares¹^,2, Z. Tümer¹, M. Bugge¹, N. Henriques-Gil², L. Rodriguez³, F. Lopez³, P. Kroisel⁴, K. Wagner⁴, C. Lundsteen⁵, V. Kalscheuer⁶, N. Tommerup¹;
¹Wilhelm Johannsen Centre for Functional Genome Research, IMBG, Panum Institute, University of Copenhagen, Copenhagen, DENMARK, ²Seccion de Genetica, Universidad San Pablo CEU, Madrid, SPAIN, ³Estudio Colaborativo Español de Malformaciones Congenitas (ECEMC), Madrid, SPAIN, ⁴Institut für Medizinische Biologie und Humangenetik, Karl-Franzens-Universität Graz, Graz, AUSTRIA, ⁵Dept of Clinical Genetics, Rigshospitalet, Copenhagen, DENMARK, ⁶Max-Planck-Institute for Molecular Genetics, Berlin, GERMANY.

As part of an EU-supported project aimed at the clinical, epidemiological and genetic study of cleft-lip-palate (EUROCRAN), we have initiated a search for candidate genes for orofacial clefts by mapping apparently balanced chromosomal breakpoints associated with cleft-lip-palate. The Mendelian Cytogenetics Network database (http://mcndb.imbg.ku.dk) contains information on 75 breakpoints in 25 individuals with oral clefting. Ordered YAC/BAC clones were used to map the breakpoints associated with two of these rearrangements: One involved a 46,XY,ins(9;5)(p22.2;q23.3q33.3) karyotype; the 5q33.3 breakpoint has been narrowed to a 250 kb region. Two overlapping BACs at the 9p22 breakpoint contain a hypothetical unknown gene, and the 5q23.3 breakpoint has been narrowed to a 150 kb region containing three candidate genes, including a putative zinc finger gene. None of these three breakpoints affects chromosomal regions previously known to harbor loci associated with orofacial clefts. The second case displayed an aberrant banding pattern on 7q36, a region previously implicated in congenital malformations affecting midline structures and a region included among those associated with orofacial clefts in more than one case in MCNdb (1p31, 4q21, 6p24, 7q36, 9p13, 16q24, 17q23 and 17p25). Chromosome 7 paint did not suggest the involvement of other chromosomes, the 7q-subtelomeric signals were normal, and high resolution CGH was normal, excluding deletions/duplications larger than ~3 Mb. This supports that the abnormal banding pattern is caused by a small intrachromosomal rearrangement, e.g. an inversion. We will try to solve this by a systematic FISH strategy using pooled BACs from 7q36.

C. Sebaoun, O. Dupuy, C. Borie, N. Bondeux, N. Collot, J. Oury, A. Delezoide, P. L. Eydoux;
Hôpital Robert Debré, Paris, FRANCE.

We report a female fetus with isochromosome 18q showing features of of both trisomy 18 and monosomy 18p. Few cases of this fetal syndrome have been published.
The pregnancy was uneventful, until prenatal sonographic examination at 20 weeks of gestation showed intra-uterine growth retardation (IUGR) and an encephalocele. The parents were non consanguineous, and had no relevant history of genetic diseases. Chromosomal examination of amniotic fluid cells showed an isochromosome 18q in all examined cells (46,XX,i(18q)). The fetus thus had trisomy 18q associated with monosomy 18p. The parents elected termination of pregnancy.
Fetopathologic examination revealed features of monosomy 18p (holoprosencephaly and facial malformations, occipital meningocele, heart and skeletal malformations), and trisomy 18q (microcephaly, short neck with pterygium colli, club feet, ovarian epithelial hypoplasia). In this case, the severity of the phenotype resulted in prenatal recognition of the chromosomal abnormality. Holoprosencephaly is not a common feature in post natal monosomy 18p (16% of all cases). It is thought to result from the deletion of HPE4, a gene encoding TGIF and located at 18q11.3.
Isochromosome 18q is thought to result from centromere fission (monocentric chromosome), or from chromatid exchange (dicentric chromosomes). Isochromosome (18q) is seen as a derivative chromosome; to our knowledge, no cases of supernumerary i(18q) have been reported, probably because tetrasomy 18q is not viable. The fetal features of isochromosome (18q) closely resembles the post natal phenotype, although the fetal syndrome may be more severe.

D. Molina Gomes, V. Nebout, V. Serazin, F. Bru, J. P. Bernard, F. Daikha-Dahmane, F. Vialard, J. Selva;
Fédération de Génétique et Service de Gyneco-Obstétrique. Centre Hospitalier Intercommunal Poissy Saint Germain en Laye et Faculté de Médecine de Paris Ouest, Poissy, FRANCE.

We report a case of partial duplication of the short arm of the chromosome 8 associated with an isolated agenesis of the corpus callosum.
This is the first pregnancy of a young unrelated couple. During the second trimester an isolated agenesis of the corpus callosum was found.
A fetal blood and amniotic fluid sample were performed. The conventional karyotype showed an excess of material on the short arm of chromosome 8 suggesting a duplication. Parental karyotypes were normal. In situ hybridization with the whole chromosome painting of chromosome 8 confirmed the duplication. The couple chosed to interrupt the pregnancy. Anatomo-pathological examination showed a total agenesis of the corpus callosum associated with discrete dysmorphy. Array 300Ô using the GenosensorÔ System was used to confirm the duplication of 8p chromosome. Presently the GenosensorÔ System includes 6 clones in the 8p region. 3 of them, the most centromeric ones, were abnormal detecting a duplication of the (8p11-8p22) region. We have to use BAC clones and FISH in order to precise the breakpoints of this duplicated fragment, and to try to reduce the critical zone of the corpus callosum on chromosome 8p.

Chromosome aberrraton frequency for medical staff exposed to ionizing radiation from open and close sources

S. Tomanovic, D. Mirkovic, B. Djurovic, M. Hrnjak, M. Misovic;
Military Medical Academy, Belgrade, YUGOSLAVIA.

It has been evidenced that ionizing radiation induces microscopicaly detected changes in genetical structure of cell. Analysis of chromosome aberrations give informations about biological effects and biological responses of living organisms to ionizing radiation.
The aim of this paper was to show the results of chromosome aberration frequency analysis for persons occupationaly exposed to ionizing radiation from open and close sources.
We analised 106 health workers in period 1997-1999, and they were categorised into two groups according to the type of radiation they were exposed to: medical workers in nuclear medicine (24) who were exposed to ionizing radiation from open sources, and medical staff in diagnostic radiology (82) who were exposed to ionizing radiation from close sources.
The average duration of occupational exposition was 10.2 years for nuclear medicine staff, and 11.6 years for diagnostic radiology staff.
The results of cytogenetic examinations showed that chromosome aberration frequency was higher for nuclear medicine employers (in 4 persons-16.67%) than for the radiology employers (in 8 persons-8.54%), but there was no statistical significance (p=0.4622).
There was also no diference in chromosome aberration frequency for diferent professions in any of this group.

Testing the ability of the "Geno Sensor System" to measure the DNA copy numbers in 4 cell strains containing cytogenetically mapped abnormalities.

F. Wuilque¹, F. Vialard², D. Molina-Gomes², C. Le Caignec³, V. Serazin², L. Grimaud¹, M. Boceno³, M. V. Senat², J. Roume², J. M. Rival³, Y. Guidicelli², J. Selva²;
¹Adgenix, Voisins le Bretoneux, FRANCE, ²Fédération de Génétique et Service de Gynécologie-Obstétrique- Centre Hospitalier Intercommunal Poissy-Saint Germain en Laye et Faculté de Médecine de Paris Ouest, Poissy, FRANCE, ³CHU Nantes, Nantes, FRANCE.

Microarray bases genomic analysis is a novel technique designed for rapid detection of changes in numbers of human DNA specific sequence copies. The VYSIS GenoSensor™ System contains 287 human loci including single copy telomeric sequences, genes involved in micro-deletion syndromes, single copy sequences near the centromere and different loci reported to be amplified in various human cancers. We tested the ability of this system to measure single copy changes in 4 cell strains [a whole 21 chromosome gain (case 1), 2 deletions (1p deletion (case 2), 4p deletion (case 3)), one duplication 8p (case 4)].
The assay involves sample DNA labelling with Cy3 fluorophore. This is mixed with whole genomic reference DNA labelled with Cy5 fluorophore and co-hybridized to an Array 300™ microarray. After hybridization, target spots are analysed using the GenoSensor™ reader system. The proprietary software automatically identifies each spot and calculates for each target a normalized ratio that indicates the degree of gain or loss of copy number in the sample DNA.
All 21 clones were detected as gained for case 1. For cases 2 and 3 the detected DNA loss was in accordance with that found with other techniques results. For case 4 we confirmed the duplication of 8p, as well.
We now intend to use this system for the diagnosis of unknown changes in DNA copy number which might occur in different fetal or newborn pathologies with a probable genetic etiology where abnormalities have not yet been detected with other techniques.

Meiotic segregation analysis in three infertile patients with balanced reciprocal translocations

J. Puechberty¹, B. Andreo², G. Lefort³, P. Blanchet¹, P. Sarda¹, F. Pellestor²;
¹Genetics Department - Arnaud de Villeneuve Hospital, Montpellier, FRANCE, ²Institut de Génétique Humaine, CNRS, Montpellier, FRANCE, ³Cytogenetics Department - Arnaud de Villeneuve Hospital, Montpellier, FRANCE.

Carriers of balanced reciprocal translocations may present with infertility, recurrent miscarriages, or offsprings with unbalanced karyotype and multiple congenital anomalies. If the risk of chromosomal imbalance may be estimated at birth, the implication of this imbalance for infertility and miscarriages is more difficult to determine. To assess the importance of these chromosomal anomalies during the gametogenesis, we have explored three infertile phenotypically normal men with reciprocal translocations. We performed meiotic segregation analysis of their sperm using FISH and PRINS methods. For patients 1 and 2, respectively with reciprocal translocations t(1;4)(q12;q28) and t(4;5)(q35;q34), more than 10000 spermatozoa were screened. In both cases, alternate segregation (giving phenotypically normal offspring) is the most frequent mode of segregation (68 and 70%) and the most frequent type of imbalance is adjacent 1 segregation (24 and 28%), corresponding to the most expected imbalance at birth. For patient 3 with reciprocal translocation t(1;21)(q11;q21) and with severe oligozoospermia, only 981 spermatozoa (from two ejaculates) could be screened. Alternate segregation is also the most frequent type of segregation (80%) and the two most frequent types of imbalance are adjacent 1 (10%) and 3:1 exchange (7,5%), this latter is responsible for the most expected imbalance at birth. Although alternate segregation is the major type of segregation, these three men have infertility. Likely there are other factors which can participate in the mechanisms of the reproductive failure, possibly from maternal origin as well. Meiotic segregation analysis in sperm is important to adjust a realistic management of the male infertility.

C. Hernando Davalillo¹^,2, P. Grao², A. Balaguer³, E. Triviño², J. Egozcue¹, C. Fuster¹;
¹Dpt. de Biologia Cellular, Fisiologia i Immunologia. Unitat de Biologia. Facultat de Medicina. Universidad Autónoma, Barcelona, SPAIN, ²Departamento de Genética. CERBA Internacional, S.A.E. Sabadell, Barcelona, SPAIN, ³Servicio de Pediatria. Hospital Sant Joan de Reus, Barcelona, SPAIN.

We report an “apparent” balanced de novo complex chromosome reorganization by the combined use of G-banding, FISH and CGH techniques in a two-year-old infant with some features vaguely reminiscent of trisomy 21. Conventional G-banding showed a complex chromosome translocation involving chromosomes 1, 4, 6 and 11. Multicolor FISH (24 colours) confirmed the presence of this complex chromosome translocation. A more complex reorganization was detected using whole chromosome painting probes for the chromosomes involved in this rearrangement. In this case we found two cryptic interstitial translocations in the same derivative chromosome: der(6) t(6;11;4;1;4). In the results obtained by CGH did not show any loss or gain of chromosome material in this patient. Our results indicate that the phenotypic abnormalities observed in this infant, with this “apparent” balanced complex rearrangement, may have resulted from either a small structural loss of material or a functional loss of a gene action.
Acknowledgements. Financial support was given by DGESIC (PB98-0891).