Early onset posterior subscapular cataract in a series of myotonic dystrophy type 2 patients.

PurposeEarly onset posterior subscapular cataract (<50 years of age) is a characteristic feature of myotonic dystrophy type 2 (DM2). Nevertheless, despite being operated at a young age, many patients remain undiagnosed for years. The purpose of this study was to assess the prevalence of early onset posterior subscapular cataract as a presenting symptom of the disease in a cohort of patients with DM2.Patients and methodsWe retrospectively reviewed medical records of DM2 patients followed in our institution for the presence of early onset posterior subscapular cataract, of any secondary causes of cataract, of the age of onset of muscle weakness and of final disease diagnosis.ResultsTwenty-eight patients were studied. Nine patients (32.1%) had presented early onset posterior subscapular cataract at a median age of 43 years (IQR=36-46) and seven (25%) reported it was the presenting sign. No patient was referred for neuromuscular evaluation due to the occurrence of early onset cataract. Median delay between cataract onset and referral for neuromuscular evaluation was 10 years (IQR=6.0-19.5) and final DM2 diagnosis was achieved after a median of 16 years (IQR=6.5-19.5).ConclusionThis study shows that early onset posterior subscapular cataract was the first symptom of the disease in 25% of our DM2 patients. Nevertheless, none was suspected of having cataract in the context of DM2, and referral for neuromuscular evaluation was made after a long delay and usually following the appearance of other symptoms. Ophthalmologists can be the first physicians encountering these patients and should have a low threshold for referring them for neuromuscular evaluation.

Aberrant microRNA expression in tumor mycosis fungoides.

Herein, miRNA candidates relevant to mycosis fungoides were investigated to provide data on the molecular mechanisms underlying the pathogenesis of the disease. The miRNA expression profile of skin biopsies from patients with tumor stage MF (tMF) and normal donors was compared using miRNA microarrays. Overall, 154 miRNAs were found differentially expressed between tMF and the control cohort with the majority of them being up-regulated (57 %). Among the upregulated miRNAs, miR-3177, miR-514b-3p, miR-1267, and miR-1282 were exclusively detected in 70 % of tMF. Additional upregulated miRNAs included miR-34a, miR-29a, let-7a*, and miR-210, while miR-200c* was identified among the downregulated ones. Quantitative real-time polymerase chain reaction was used to further investigate the expression profiles of miR-34a and miR-29a and validated the overexpression of miR-34a. Enrichment studies revealed that the target genes of the differentially expressed miRNAs were important in several cancer-related signaling pathways. The overlapping relationship of the target genes among tMF, Sezary syndrome, and atopic dermatitis revealed several common and disease-specific genes. Collectively, our study modulated miR-34a as a candidate oncogenic molecule and miR-29a as a putative tumor suppressor highlighting their promising potential in the molecular pathogenesis of tMF.

miR-15a and miR-24-1 as putative prognostic microRNA signatures for pediatric pilocytic astrocytomas and ependymomas.

In the current setting, we attempted to verify and validate miRNA candidates relevant to pediatric primary brain tumor progression and outcome, in order to provide data regarding the identification of novel prognostic biomarkers. Overall, 26 resected brain tumors were studied from children diagnosed with pilocytic astrocytomas (PAs) (n = 19) and ependymomas (EPs) (n = 7). As controls, deceased children who underwent autopsy and were not present with any brain malignancy were used. The experimental approach included microarrays covering 1211 miRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate the expression profiles of miR-15a and miR-24-1. The multiparameter analyses were performed with MATLAB. Matching differentially expressed miRNAs were detected in both PAs and EPs, following distinct comparisons with the control cohort; however, in several cases, they exhibited tissue-specific expression profiles. On correlations between miRNA expression and EP progression or outcome, miR-15a and miR-24-1 were found upregulated in EP relapsed and EP deceased cases when compared to EP clinical remission cases and EP survivors, respectively. Taken together, following several distinct associations between miRNA expression and diverse clinical parameters, the current study repeatedly highlighted miR-15a and miR-24-1 as candidate oncogenic molecules associated with inferior prognosis in children diagnosed with ependymoma.

Recurrent copy number variations as risk factors for autism spectrum disorders: analysis of the clinical implications.

Chromosomal microarray analysis (CMA) is currently considered a first-tier diagnostic assay for the investigation of autism spectrum disorders (ASD), developmental delay and intellectual disability of unknown etiology. High-resolution arrays were utilized for the identification of copy number variations (CNVs) in 195 ASD patients of Greek origin (126 males, 69 females). CMA resulted in the detection of 65 CNVs, excluding the known polymorphic copy number polymorphisms also found in the Database of Genomic Variants, for 51/195 patients (26.1%). Parental DNA testing in 20/51 patients revealed that 17 CNVs were de novo, 6 paternal and 3 of maternal origin. The majority of the 65 CNVs were deletions (66.1%), of which 5 on the X-chromosome while the duplications, of which 7 on the X-chromosome, were rarer (22/65, 33.8%). Fifty-one CNVs from a total of 65, reported for our cohort of ASD patients, were of diagnostic significance and well described in the literature while 14 CNVs (8 losses, 6 gains) were characterized as variants of unknown significance and need further investigation. Among the 51 patients, 39 carried one CNV, 10 carried two CNVs and 2 carried three CNVs. The use of CMA, its clinical validity and utility was assessed.

Single-cell high resolution melting analysis: A novel, generic, pre-implantation genetic diagnosis (PGD) method applied to cystic fibrosis (HRMA CF-PGD).

BACKGROUND: Institutions offering CF-PGD face the challenge of developing and optimizing single cell genotyping protocols that should cover for the extremely heterogeneous CF mutation spectrum. Here we report the development and successful clinical application of a generic CF-PGD protocol to facilitate direct detection of any CFTR nucleotide variation(s) by HRMA and simultaneous confirmation of diagnosis through haplotype analysis. METHODS: A multiplex PCR was optimized supporting co-amplification of any CFTR exon-region, along with 6 closely linked STRs. Single cell genotypes were established through HRM analysis following melting of the 2nd round PCR products and were confirmed by STR haplotype analysis of the 1st PCR products. The protocol was validated pre-clinically, by testing 208 single lymphocytes, isolated from whole blood samples from 4 validation family trios. Fifteen PGD cycles were performed and 103 embryos were biopsied. RESULTS: In 15 clinical PGD cycles, genotypes were achieved in 88/93 (94.6%) embryo biopsy samples, of which 57/88 (64.8%) were deemed genetically suitable for embryo transfer. Amplification failed at all loci for 10/103 blastomeres biopsied from poor quality embryos. Six clinical pregnancies were achieved (2 twin, 4 singletons). PGD genotypes were confirmed following conventional amniocentesis or chorionic villus sampling in all achieved pregnancies. CONCLUSIONS: The single cell HRMA CF-PGD protocol described herein is a flexible, generic, low cost and robust genotyping method, which facilitates the analysis of any CFTR genotype combination. Single-cell HRMA can be beneficial to other clinical settings, for example the detection of single nucleotide variants in single cells derived from clinical tumor samples.

Dysmorphology services: a snapshot of current practices and a vision for the future.

Dysmorphology concerns the recognition and management of rare, multiple anomaly syndromes. Genomic technologies and software for gestalt recognition will re-shape dysmorphology services. In order to reflect on a model of the service in the post-genomic era, we compared the utility of dysmorphology consultations in two Mediterranean cities, Athens, Greece and Afula, Israel (MDS), the Manchester Centre for Genomic Medicine, a UK service with dysmorphology expertise (UKDS) and the DYSCERNE, digital service (DDS). We show that it is more likely that chromosome microarray analysis will be performed if suggested in the UKDS rather than in the MDS; this, most probably reflects the difference of access to genetic testing following funding limitations in the MDS. We also show that in terms of achieved diagnosis, the first visit to a dysmorphology clinic is more significant than a follow-up. We show that a confirmed syndrome diagnosis significantly decreases the requests for other, non-genetic, laboratory investigations. Conversely, it increases the requests for reviews by other specialists and, most significantly (t-test: 8.244), it increases further requests for screening for possible associated complications. This is the first demonstration of the demands, on a health service, following the diagnosis of a dysmorphic condition.

Clinical and molecular description of a fetus in prenatal diagnosis with a rare de novo ring 10 and deletions of 12.59 Mb in 10p15.3-p14 and 4.22 Mb in 10q26.3.

Ring chromosomes are rare cytogenetic findings and are mostly associated with an abnormal phenotype. We report on the prenatal diagnosis of a ring chromosome 10 in a fetus in which talipes equinovarus was incidentally found during routine obstetric ultrasound at 22 weeks of gestation. Amniocentesis was undertaken and cytogenetic analysis revealed a de novo non-mosaic apparently stable ring chromosome 10 replacing one of the two homologs. Multiplex Ligation-dependent Probe Amplification (MLPA) revealed subtelomeric deletions in both the short and long arm of chromosome 10. Analysis with high resolution micro-array based comparative genomic hybridization (array-CGH), defined the ring chromosome as del 10p15.3-p14 (12.59 Mb in size) and del 10q26.3 (4.22 Mb in size) and revealed the genes that are deleted. After elected termination of the pregnancy at 27th week of gestation a detailed autopsy of the fetus allowed for genotype-phenotype correlations. To our knowledge, this is the first case of a de novo ring chromosome 10 which is reported during prenatal diagnosis and is thoroughly investigated with array CGH and autopsy study.

Long term follow up of a woman with classic form of Ehlers-Danlos syndrome associated with rare manifestations and review of the literature.

We present the case of a 46 year-old woman with the classic type of Ehlers-Danlos syndrome who developed the rare manifestations of colon diverticula and mitral valve prolapse. We emphasize on clinical features and complications associated to this type of the syndrome, which gradually developed in our patient. A review of the literature referring to the epidemiology, molecular basis and manifestations of the classic type Ehlers-Danlos syndrome is also discussed.

Characterization of 23 small supernumerary marker chromosomes detected at pre-natal diagnosis: The value of fluorescence in situ hybridization.

Small supernumerary marker chromosomes (sSMCs) cannot be identified or characterized unambiguously by conventional cytogenetic banding techniques. Until recently, the large variety of marker chromosomes, as well as the limitations in their identification, have presented a diagnostic problem. In order to determine the origin of sSMCs, we used a variety of fluorescence in situ hybridization (FISH) methods, including centromere-specific multicolor FISH, acrocentric specific multicolor FISH, subcentromere-specific multicolor FISH and multicolor FISH with whole chromosome paint probes. Moreover, uniparental disomy testing was in all cases attempted. From a total of 28,000 pre-natal samples from four diagnostic genetics laboratories in Greece, 23 (0.082%) supernumerary marker chromosomes were detected. The mean maternal age was 36.2 years (range 27-43) and the mean gestational age at which amniocentesis was performed was 18.5 weeks (range 16-23). Eighteen markers were de novo and 5 markers were inherited. Molecular cytogenetic methods were applied to determine the chromosomal origin and composition of the sSMC. In total, 17 markers were derived from acrocentric chromosomes (14, 15, 21 and 22) and 6 markers were non-acrocentric, derived from chromosomes 9, 16, 18, 20 and Y. Uniparental disomy was not detected in any of the cases studied. With regard to pregnancy outcome, 13 pregnancies resulted in normal healthy neonates, while 10 pregnancies were terminated due to ultrasound abnormalities. A total of 23 marker chromosomes from 28,000 pre-natal samples (0.082%) were identified. Molecular cytogenetic techniques provided valuable information on the chromosomal origin and composition of all the sSMCs. Especially in cases with normal ultrasound, the FISH results rendered genetic counseling possible in a category of cases previously considered a diagnostic problem. Abnormal outcome was observed in 10 cases (43,5%), 7 of which showed abnormal ultrasound findings. New technologies, such as array-comparative genomic hybridization, should be used in future genotype-phenotype correlation studies, although the high mosaicism rate poses a problem.

Expanding CEP290 mutational spectrum in ciliopathies.

Ciliopathies are an expanding group of rare conditions characterized by multiorgan involvement, that are caused by mutations in genes encoding for proteins of the primary cilium or its apparatus. Among these genes, CEP290 bears an intriguing allelic spectrum, being commonly mutated in Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), Senior-Loken syndrome and isolated Leber congenital amaurosis (LCA). Although these conditions are recessively inherited, in a subset of patients only one CEP290 mutation could be detected. To assess whether genomic rearrangements involving the CEP290 gene could represent a possible mutational mechanism in these cases, exon dosage analysis on genomic DNA was performed in two groups of CEP290 heterozygous patients, including five JSRD/MKS cases and four LCA, respectively. In one JSRD patient, we identified a large heterozygous deletion encompassing CEP290 C-terminus that resulted in marked reduction of mRNA expression. No copy number alterations were identified in the remaining probands. The present work expands the CEP290 genotypic spectrum to include multiexon deletions. Although this mechanism does not appear to be frequent, screening for genomic rearrangements should be considered in patients in whom a single CEP290 mutated allele was identified.

Unilateral microtia in an infant with trisomy 18 mosaicism.

Trisomy 18 is the second most frequent autosomal aneuploidy, after Down's syndrome, in humans. It causes severe congenital abnormalities and mental retardation although phenotypic features, clinical manifestations and prognosis vary occasionally. In cases oftrisomy 18 mosaicism, as in every chromosomal mosaicism, the spectrum of clinical characteristics extends from pathological to almost normal. We report a 9 months old female infant who has been referred to the Genetics Department for evaluation because of unilateral severe microtia, aplasia of mastoid abscess and hemifacial palsy and inlet type intraventricular defect with pulmonary hypertension. Chromosomal investigation revealed a mosaic trisomy 18 [46,XX/47,XX+18] in proportion of 52% and 48% respectively. Microtia/anotia is present in 1.46-4.36/10,000 live births in the general population while the combination of microtia/anotia with trisomy 18 has been reported in very few cases in the relevant bibliography.

Saethre-Chotzen syndrome with severe developmental delay associated with deletion of chromosomic region 7p15 --> pter.

Saethre-Chotzen syndrome represents one of the most common types of craniosynostosis inherited as an autosomal dominant disorder while sporadic cases have also been reported. It is characterized by high penetrance and variable expressivity, leading to difficulties in clinical diagnosis. Some patients, who exhibit most of the diagnostic criteria of Saethre-Chotzen syndrome, have structural abnormalities of chromosome 7. The case of a 4 year old boy with notable dysmorphic features compatible with Saethre-Chotzen syndrome and severe developmental delay is described. Conventional and molecular cytogenetic analysis of peripheral blood samples from the patient and his parents revealed partial monosomy of chromosomal region 7p15 --> pter de novo. The TWIST gene, located on chromosome 7p21.1, is thought to be a negative transcriptional regulator involved in osteoblast differentiation and maturation and it is thought that haploinsufficiency of the gene can cause the disorder. The diagnosis of Saethre-Chotzen syndrome and the identification of the chromosomal abnormality in the patient facilitated genetic counseling of the family.

Spontaneous ovulation in a true hermaphrodite with normal male phenotype and a rare 46,XX/47,XXY Klinefelter's mosaic karyotype.

BACKGROUND: Most true hermaphrodite patients--characterized by the presence of both ovarian and testicular tissue--demonstrate ambiguous genitalia and are diagnosed at birth, most commonly bearing a 46,XX karyotype. PATIENT AND METHODS: We report on a 13-year-old boy presenting with left scrotal hemorrhage. He had a left inguinal hernia, a palpable testis in the right, normal male external genitalia and significant gynecomastia. During operation, the left gonad and adjacent tissue were removed for histological examination, which revealed the presence of a normal ovary, rich in follicles and a ruptured corpus luteum, suggestive of spontaneous ovulation, with a normal ipsilateral adnexa and semi-uterus. Biopsy of the right gonad revealed a dysgenetic testicle. Endocrinological assessment postoperatively depicted high FSH, pubertal testosterone and low estradiol levels. Cytogenetic analysis in peripheral blood lymphocytes and FISH of the right gonad revealed a 46,XX (70-60%)/47,XXY (30-40%) karyotype, respectively, while molecular analysis verified the presence of SRY and azoospermia factor genes. CONCLUSION: The importance of full histological, cytogenetic and molecular investigation and of interdisciplinary approach in every single patient with sex differentiation disorders is highlighted by this rare case of spontaneous ovulation in a true hermaphrodite with normal male external genitalia and Klinefelter mosaicism.

Steroid hormones polymorphisms and cholelithiasis in Greek population.

BACKGROUND: Genetic variation in genes involved in steroid biosynthesis, metabolism and signal transduction have been suggested to play a role in gallstone disease. METHODS: To elucidate the possible role of genetic variation in the estrogen receptors alpha and beta (ER-alpha, ER-beta) and androgen receptor (AR) genes in breast cancer risk, the -1174(TA)n, c.1092+3607(CA)(n) and c.172(CAG)n repeat polymorphisms of the three genes were studied. A case-control cohort of 99 patients with cholelithiasis and 179 controls were used. RESULTS: No significant difference was observed in the frequency distribution of -1174(TA)(0-26) in the ER-alpha gene between patients and controls, while a significant difference was observed in the frequency distribution of repeat polymorphism c.1092+3607(CA)5-27 and c.172(CAG)5-32 in the ER-beta gene and AR gene, respectively (P< or =0.001 and P=0.05, respectively). A significant difference was observed in the repeat genotype distribution (SS, SL, LL) in the (CA)n of the ER-beta gene (P<0.0001) and in the (CAG)n of the AR gene (P< or =0.0001). A significantly decreased odds ratio for cholelithiasis risk was observed in individuals having the SL and LL genotype for ER-beta gene compared with SS genotype (OR=0.212; 95% CI 0.105-0.426; P<0.0001 and OR=0.042; 95% CI 0.018-0.097, respectively) and LL genotype for AR gene (OR=0.622; 95% CI 0.345-1.121; P=0.114 and OR=0.287; 95% CI 0.151-0.543, P<0.0001, respectively). This protective effect of SL and LL genotypes for ER-beta and LL for AR gene remained evident (P<0.0001 for all of them) even after adjustment for various risk factors. CONCLUSIONS: In conclusion an association for cholelithiasis risk between short alleles for both c.1092+3607(CA)5-27 and c.172(CAG)5-32 repeat polymorphisms of the ER-beta and AR was found in individuals of Greek descent.

Identification of nucleated red blood cells in maternal circulation: a second step in screening for fetal aneuploidies and pregnancy complications.

BACKGROUND: Identification of fetal nucleated red blood cells (NRBCs) in maternal circulation can facilitate non-invasive prenatal diagnosis, but technical difficulties still exist. An increase in the number of circulating NRBCs, however, could indicate fetal aneuploidies or pregnancy complications. MATERIALS AND METHODS: The number of NRBCs was determined from 20 mL peripheral blood in 351 women in the second trimester of pregnancy after isolation by magnetic cell sorting (MACS) with anti-CD71 antibody and identification with May-Grunwald/Giemsa staining. RESULTS: An average of eight NRBCs (range 1-12) were identified among 282 women with chromosomally normal fetuses. In cases known to carry aneuploid fetuses the mean number was 35 (range 7-113), but when the fetus had trisomy 21 (n = 17) an average of 71 NRBCs were identified. Among 26 carriers of beta-thalassemia, 42 NRBCs (range 22-158) were isolated. In pregnancies with abnormal Doppler findings in both uterine arteries (n = 20), 15 NRBCs (range 2-75) were isolated. CONCLUSION: Determining the number of NRBCs in maternal circulation could represent an additional screening step for fetal aneuploidies, as long as the anemic status of the mother is taken into consideration. However, more cases with abnormal Doppler results must be investigated before this test is used for in the prediction of pregnancy complications.

Clinical manifestations in 17 Greek patients with Goldenhar syndrome.

Goldenhar (GS) syndrome is a well-recognised developmental disorder involving first and second branchial arches and characterized by considerable phenotypic variability. The present study presents clinical data on the morphologic features, hearing, ophthalmologic, orthopaedic, neurological, cardiovascular, genitourinary and gastrointestinal evaluation of 17 Greek patients (one pair of monozygotic twins) aged 20 days to 23 years with the clinical diagnosis of GS and with a normal karyotype. The most consistent findings were auricular defects (94%), followed by facial (76%) and ocular anomalies (65%), 70% unilateral, mainly right-sided. In the majority of our patients (90%) mandibular hypoplasia was ipsilateral to the dysplastic ear or the most severely affected ear in bilateral cases. Hearing loss, mainly conductive, was noted in 76% of our patients. Skeletal defects were evident in 23%, while cardiovascular, genitourinary and gastrointestinal in 18%, 23% and 12% respectively. The most frequent neurological manifestation was facial nerve paralysis (12%), while the incidence of mental retardation was higher (23%) than reported in the literature, presumably attributed to the severe hearing and vision loss. In a pair of monozygotic twins of our study discordance of clinical findings was noted. Precise evaluation of GS patients and multidisciplinary care management is necessary to avoid possible complications of many systems and to offer appropriate genetic counselling to the family.

Detection of 22q11.2 deletion among 139 patients with Di George/Velocardiofacial syndrome features.

Cytogenetic and FISH analysis was performed in 139 patients to detect the pathognomonic of Di George/ Velocardiofacial syndrome (DGS/VFCS) deletion 22q11.2. An abnormal karyotype was revealed in 2/139 cases (47, XXY and 46, XX, 2p+). A deletion was found in 17/139 (12.2%) patients (14 males/ 3 females), inherited in 3 (2 maternal and 1 paternal). Patients with 22q11.2 deletion exhibited facial dysmorphic features (82%), congenital heart defects (70%), immunological problems (47%), multiple congenital anomalies (64%), hypocalcemia (47%), mental retardation/learning difficulties (35%), cleft palate/velopharyngeal insufficiency (23.5%), seizures/hypotonia (23%) and growth retardation (12%). Among 56/139 patients with detailed available clinical data, the 22q11.2 deletion was confirmed in all cases with hypocalcemia and in over half of the cases with multiple congenital anomalies, immunological problems and hypotonia/seizures (70%, 60% and 57%, respectively). Genetic reevaluation of 39 patients without the 22q11.2 deletion contributed to the classification of 14 (37%) under different syndromes, emphasizing the need for stricter referral criteria.

Gilbert syndrome associated with beta-thalassemia.

The authors investigated whether the considerable variability in serum bilirubin levels (STB) found in transfusion-dependent beta-thalassemia, beta-thal intermedia, and heterozygous beta-thalassemia individuals could be related to the coexistence of Gilbert syndrome (GS). The promoter region [A(TA)nTAA] of the bilirubin UDP-glucuronosyltransferase gene (UGT1A1) was analyzed in a total of 128 beta-thalassemia individuals (108 transfusion-dependent beta-thal patients, 20 very mild beta-thal intermedia) and in 33 beta-thal heterozygotes. The control group consisted of 70 healthy children with no history of anemia. The frequency of GS genotype (TA)7/(TA)7 did not differ significantly between the groups studied. A significant difference was observed between serum bilirubin levels (STB) and GS genotypes (TA)7/(TA)7 and (TA)6/(TA)7 and also between (TA)7/(TA)7 and (TA)6/(TA)6 for all groups examined. These results confirm that the (TA)7/(TA)7 GS genotype is one of the factors accounting for the hyperbilirubinemia observed in beta-thalassemia major, intermedia, and heterozygous individuals.

Origin of nondisjunction in trisomy 8 and trisomy 8 mosaicism.

Causes of chromosomal nondisjunction is one of the remaining unanswered questions in human genetics. In order to increase our understanding of the mechanisms underlying nondisjunction we have performed a molecular study on trisomy 8 and trisomy 8 mosaicism. We report the results on analyses of 26 probands (and parents) using 19 microsatellite DNA markers mapping along the length of chromosome 8. The 26 cases represented 20 live births, four spontaneous abortions, and two prenatal diagnoses (CVS). The results of the nondisjunction studies show that 20 cases (13 maternal, 7 paternal) were probably due to mitotic (postzygotic) duplication as reduction to homozygosity of all informative markers was observed and as no third allele was ever detected. Only two cases from spontaneous abortions were due to maternal meiotic nondisjunction. In four cases we were not able to detect the extra chromosome due to a low level of mosaicism. These results are in contrast to the common autosomal trisomies (including mosaics), where the majority of cases are due to errors in maternal meiosis.

Detection and incidence of cryptic Y chromosome sequences in Turner syndrome patients.

The presence of Y chromosome sequences in Turner syndrome (TS) patients may predispose them to gonadoblastoma formation with an estimated risk of 15-25%. The aim of this study was to determine the presence and the incidence of cryptic Y chromosome material in the genome of TS patients. The methodology involved a combination of polymerase chain reaction (PCR) and nested PCR followed by Southern blot analysis of three genes the sex determining region Y (SRY), testis specific protein Y encoded (TSPY) and RNA binding motif protein (RBM) (previously designated as YRRM) and nine additional STSs spanning all seven intervals of the Y chromosome. The methodology has a high sensitivity as it detects one 46,XY cell among 10(5) 46,XX cells. Reliability was ensured by taking several precautions to avoid false positive results. We report the results of screening 50 TS patients and the identification of cryptic Y chromosome material in 12 (24%) of them. Karyotypes were divided in four groups: 5 (23.8%) patients out of the 21 TS patients which have the 45,X karyotype (group A) also have cryptic Y sequences; none (0%) of the 7 patients who have karyotypes with anomalies on one of the X chromosomes have Y mosaicism (group B); 1 (6.3%) of the 16 patients with a mosaic karyotype have Y material (group C); and 6 (100%) out of 6 patients with a supernumerary marker chromosome (SMC) have Y chromosome sequences (group D). Nine of the 12 patients positive for cryptic Y material were recalled for a repeat study. Following new DNA extraction, molecular analysis was repeated and, in conjunction with fluorescent in situ hybridization (FISH) analysis using the Y centromeric specific probe Yc-2, confirmed the initial positive DNA findings. This study used a reliable and sensitive methodology to identify the presence of Y chromosome material in TS patients thus providing not only a better estimate of a patient's risk in developing either gonadoblastoma or another form of gonadal tumor but also the overall incidence of cryptic Y mosaicism.