10 years' experience in fragile X testing among mentally retarded individuals in Greece: a molecular and epidemiological approach.

Fragile X syndrome, the second most common genetic cause of mental retardation, is due to the expansion of a trinucleotide repeat (CGG)n within the first exon of the FMR-1 gene. Molecular genetic analysis provides accurate diagnosis and facilitates genetic counselling and prenatal testing. Screening for the fragile X mutation in a sample of 3,888 individuals in Greece is reported: 1,755 children with non-specific mental retardation, 1,733 parents and other family members and 400 normal individuals. Molecular analysis allowed for the identification and characterization of 52 fragile X families confirming the clinical diagnosis in 57 males and 4 females. Sixty-six female carriers (6 mentally retarded) and 4 normal transmitting males were also identified. Four severely retarded males and their mothers carried unmethylated premutations, while a moderately retarded girl had a deletion of approximately equal to 150 bp. Overall sizing of the CGG repeat produced an allele distribution of 6-58 CGG repeats (mean 28-30), similar to that in other Caucasian populations.

Identification of fetal nucleated red blood cells in the maternal circulation during pregnancy using anti-hemoglobin-epsilon antibody.

AIM: To investigate the use of anti-hemoglobin-epsilon antibody in order to identify fetal cells in the maternal circulation during pregnancy. MATERIALS AND METHODS: 48 blood samples were obtained from pregnant women, 26 in the 1st trimester and 22 in the 2nd trimester. Magnetic activated cell sorting was used for fetal cell enrichment followed by immunophenotyping with a monoclonal antibody against hemoglobin-epsilon. FISH with X, Y and 21 chromosome-specific probes was performed in 29 cases. RESULTS: The mean number of epsilon-positive cells was 9.2 (range 2-23) in the 1st trimester, 4.8 (range 3-13) in the 2nd trimester and 22 (range 15-28) in pregnancies with Down syndrome. No significant difference was noted in the number of epsilon-positive nucleated red blood cells (NRBCs) isolated from carriers and noncarriers of beta-thalassemia. FISH analysis was successful in 24 cases. In 4 cases with known male fetuses, an average of 4.7 epsilon-positive cells showed a Y signal. In 4 cases with Down syndrome, all epsilon-positive cells showed 3 signals for chromosome 21. CONCLUSION: Anti-hemoglobin-epsilon antibody has increased specificity for fetal NRBCs and should be preferentially used to improve noninvasive prenatal diagnosis of chromosome abnormalities from fetal cells in maternal blood.

Efficiency of interphase fluorescence in situ hybridization for BCR/ABL on peripheral blood smears for monitoring of CML patients: a comparison with bone marrow findings.

Conventional cytogenetic analysis (CCA) is the standard method for monitoring of the Philadelphia (Ph) chromosome in chronic myeloid leukemia (CML). Evaluation of breakpoint cluster region/abelson murine leukemia (BCR/ABL) fusion using interphase fluorescence in situ hybridization on peripheral blood smears (PB-FISH) might be another approach allowing more frequent and less invasive follow-up investigations. Herein, BCR/ABL fusion gene was assessed on 21 PB smears from 16 CML patients in chronic phase. Results of PB-FISH were compared with those of CCA and interphase FISH on bone marrow aspirates (BM-FISH). PB-FISH analysis was combined with CD3 immunophenotyping that allowed simultaneous investigation of the leukemic status of CD3(+) T lymphocytes and scoring CD3(-) cells for BCR/ABL fusion gene. Moreover, the frequency of BCR/ABL fusion in nonlymphoid PB cells was estimated according to the differential leukocyte counts. The incidence of BCR/ABL(+) fusion signals in CD3(+) T cells of CML patients was 5.3% (SD +/- 1.9) and did not exceed the normal cut-off value of 8%. A significant correlation (P < 0.001) was found between results of PB-FISH and methods of BM analysis (CCA or BM-FISH). Correction of PB-FISH results to include only nonlymphoid or CD3(-) cells reduced the mean of differences and improved agreement between PB-FISH and CCA or BM-FISH methods. The best agreement was noted between CCA and PB-FISH on nonlymphoid cells. On the other hand, results of BM-FISH agreed well with those of PB-FISH on CD3(-) cells. These findings imply that PB-FISH on nonlymphoid or CD3(-) cells is reliable and may replace BM analysis for monitoring of response to treatment in CML patients.

Use of annexin V antibody to identify apoptotic cells during pregnancy.

In a previous study, we demonstrated that apoptosis increased according to gestational age, accounting partly for the presence of free fetal DNA in maternal plasma and serum. Using simultaneous TUNEL assay and FISH analysis, we identified the fetal origin of part of the apoptotic cell population, but very few TUNEL-positive cells showed hybridization signals since they were in a late apoptosis stage and nuclei were destroyed. In the present study, the apoptotic cell population was identified immunocytochemically using Annexin V, a marker of cells in an early stage of apoptosis. The mean apoptosis rate in mononuclear cells isolated from the peripheral blood of 20 pregnant women in the 16th to 19th week of pregnancy with Annexin V was 6.8 +/- 0.5% (range: 4.2-8.1%) compared to 6.14 +/- 0.5% (range: 3.7-6.9%) obtained with ethidium bromide staining. FISH using X and Y chromosome-specific probes was applied in 11 cases known to be carrying male fetuses. Eighty percent of Annexin V+ cells showed hybridization signals, while the proportion of apoptotic cells showing X/Y signals was 7.8% (range: 5-12%). Although our results are still preliminary, it seems that use of Annexin V antibody to detect the apoptotic cell population improves FISH analysis and allows a more accurate estimate of the proportion of fetal cells among the apoptotic cell population.

Detection of fetal NRBCs in maternal blood of pregnant carriers of beta-thalassemia using anti-gamma and anti-epsilon monoclonal antibodies.

Fetal nucleated red blood cells (NRBCs) entering maternal circulation during pregnancy constitute a potential source of material for safe and reliable noninvasive prenatal diagnosis. The increased prevalence of beta-thalassemia mutations in countries like Greece may create a problem, making it difficult to distinguish between NRBCs of fetal or maternal origin. Use of Ab against embryonic hemoglobin epsilon may increase specificity for fetal NRBC detection. In the present study, Ab against embryonic hemoglobin epsilon was used in the first and second trimesters of pregnancy in order to determine if specificity for fetal NRBC detection could be increased.

Evaluation at single cell level of residual Philadelphia negative hemopoietic stem cells in chronic phase CML patients.

In chronic myeloid leukemia, accurate determination of Ph(-) Hemopoietic stem cells (HSC) in peripheral blood (PB), bone marrow (BM) and leukapheresis products is important for the selection of patients for whom mobilization, collection, and autografting of Ph(-) HSC are envisaged. To this effect, the BCR/ABL fusion was assessed at the single cell level in 25 sets of PB and BM samples using dual-color I-FISH in immunophenotyped CD34(+) cells and RT-PCR of individual CFU-GM colonies. In 15 cases found to be 100% Ph(+), the respective BCR/ABL gene was absent in 30% of CD34(+) cells, while the respective transcripts could not be identified in 17% of CFU-GM. The mean percentage of BCR/ABL(-) CD34(+) cells and CFU-GM cells was higher (38% and 29%, respectively) in untreated patients than in treated patients (24% and 7%, respectively). In eight cases with cytogenetic response (CgR), the percentage of Ph(-) metaphases correlated with the level of BCR/ABL(-) colonies in BM and PB and with the proportion of BCR/ABL(-) CD34(+) cells in the BM. Immunophenotyping and FISH was fast, easy, always informative, and quantitative for the BCR/ABL(-) CD34(+) cells. Our results show that (a) at early diagnosis a high frequency of BCR/ABL(-) HSC circulate in the PB and that Ph(-) hematopoiesis is not completely suppressed; (b) although normal clonogenic cells decline rapidly within a few months after diagnosis, appreciable numbers of normal CD34(+) cells survive in chronic phase, especially in patients with CgR.

Improved specificity of NRBC detection in chorionic villus sample supernatant fluids using anti-zeta and anti-epsilon monoclonal antibodies.

OBJECTIVE: Fetal erythrocytes leak from fetal capillaries at the time of chorionic villus sampling (CVS). It has been reported that in approximately 60% of CVS cases fetal nucleated red blood cells (NRBC) can be isolated from the supernatant fluid by immunophenotyping with monoclonal antibody (Ab) against the gamma-chain of fetal hemoglobin and used as an additional source for confirmation of the fetal karyotype. However, the increased prevalence of beta-thalassemia mutations in countries such as Greece results in many pregnant women who produce gamma-positive cells. This makes it difficult to distinguish between the fetal and maternal origin of the NRBC. Use of Abs against embryonic hemoglobin chains zeta and epsilon may increase specificity for fetal NRBC detection. METHODS: Mouse monoclonal Abs against Hb-zeta and Hb-epsilon were used in order to examine if specificity for fetal NRBC detection in CVS supernatant fluids could be improved. 41 samples were studied using anti-zeta and 20 using anti-epsilon monoclonal Abs. RESULTS: Anti-zeta or anti-epsilon positive erythrocytes were, respectively, identified in 52 of 61 CVS samples and anti-zeta or anti-epsilon positive NRBC were present in all cases. The mean number of Hb-positive erythrocytes identified with the anti-zeta Ab was 58 and the mean number of NRBC 29. The mean number of anti-epsilon positive erythrocytes was 30 and of NRBC 23. FISH with X and Y chromosome specific probes was performed in 26 cases and the results were concordant with the CVS karyotype. Statistical analysis using the correlation test showed that anti-zeta and anti-epsilon were more specific for the detection of embryonic NRBCs. CONCLUSIONS: Since embryonic monoclonal Abs show increased specificity, they should be preferentially used for NRBC detection in CVS supernatant fluids. Furthermore, the increased specificity of anti-zeta and anti-epsilon Abs may considerably improve prenatal diagnosis from fetal cells isolated from maternal circulation.

Cohen syndrome: two new cases in siblings.

UNLABELLED: Cohen syndrome is a rare genetic disorder consisting of truncal obesity, hypotonia, mental retardation, characteristic facial appearance and ocular anomalies. Other diagnostic clinical features include narrow hands and feet, low growth parameters, neutropenia and chorioretinal dystrophy. We describe the similarities in the clinical and developmental profile of two siblings with Cohen syndrome, providing evidence for autosomal recessive inheritance in this condition. CONCLUSION: The diagnosis of Cohen syndrome should be suspected in mentally retarded children with the above characteristics. Neutropenia and ocular anomalies with high-grade myopia and chorioretinal dystrophy are also considered important findings and can aid in the clinical diagnosis especially at an early age.

Screening for minor changes in the distal part of the human dystrophin gene in Greek DMD/BMD patients.

The distal part of the human dystrophin gene is characterised by particular features and seems to play an important functional role. Additionally in recent years several data have implicated minor mutations in this gene region in some patients with mental retardation (MR). In order to screen for pathogenic mutations at the distal part of the human dystrophin gene we have used single-strand conformation analysis of products amplified by polymerase chain reaction (PCR-SSCA) in 35 unrelated male Greek DMD/BMD patients with no detectable deletions. Seven patients also had severe mental retardation. Direct sequencing of samples demonstrating a shift of SSCA mobility revealed six different and pathogenic minor changes, five in DMD and one in a BMD patient. Four of the mutations were found in DMD patients with severe MR. Three of these mutations were localised in exon 66, which presents an interesting similarity with part of the 3' end of the genome of eastern equine encephalomyelitis virus (EEEV). The present data from Greek DMD/BMD patients give further information about the phenotypic effects consequent on mutations in exons at the distal part of the human dystrophin gene.

Identification of three polymorphisms in the dystrophin gene.

Three polymorphisms were identified in the dystrophin gene using the polymerase chain reaction (PCR) and single strand conformation analysis (SSCA). Two of them (in intron 3) were reported for the first time while the third (in intron 43) is of interest as it is found mostly in patients with a recombination event in the same region.

Chromosome fragility and predisposition to childhood malignancies.

In order to clarify the possible connection between autosomal folate sensitive Fragile Sites (FS) and genetic susceptibility to haemopoetic disease in children we investigated the frequency and distribution of FS in the Peripheral Blood Lymphocytes (PBL) of 56 children with newly diagnosed and untreated haematologic malignancies and their parents. The incidence was compared with that of 146 normal controls (children and adults). In all patients the Bone Marrow (BM) karyotype was also determined. Heritable FS were detected in 49 patients (87.5%). 20 children had more than one FS and in all cases it was inherited from one of their parents, although there was a significant excess of transmitting mothers. 19 different FS were identified: 14 common, 4 rare and one, 22q11, which has not been previously reported, but it is considered as important as it coincides with the cancer breakpoint resulting in the formation of the Philadelphia (Ph) chromosome. The frequency of FS in the PBL of the patients was significantly higher than in the controls and this increase was independent of any abnormality detected in the malignant cells of the BM. However, patients with an abnormal BM karyotype displayed increased frequency of FS induction as compared to patients with a normal karyotype. In three cases the heritable FS was found to be at or near the breakpoints of the chromosomal rearrangements detected in the malignant cells. The findings are discussed with regard to cancer specific breakpoints, oncogene loci and sites where viral DNA can be inserted to the genome. The results of this study suggest that autosomal folate sensitive FS may increase the risk for haematologic malignancies through a complex mechanism which remains to be clarified.

Awareness and use of prenatal diagnosis among Greek women: a national survey.

The prevention of genetic diseases through prenatal diagnosis depends to a large extent on the awareness and acceptance of available methods by the public. A national survey was conducted among Greek women in order to explore their attitudes towards and their use of prenatal diagnosis in relation to their lifestyle. The survey was originally addressed to 3000 Greek women 18-65 years of age. Using as a criterion having a child 5 years old or younger, 350 women were eligible for the study. It was noted that 52 per cent of the respondents were adequately informed, while 48 per cent had either superficial knowledge of the subject or no knowledge at all. Amniocentesis was the method that most women were familiar with. The majority said that they were informed by their doctors and the media, and 13 per cent of the participants had prenatal diagnosis during a previous pregnancy. Twenty-two per cent of those who were not tested were over 35 years of age at the time of pregnancy. There was a significant positive correlation between awareness and acceptance of prenatal diagnosis, on the one hand, and the social, educational and financial profile of the women, on the other. Women aware of prenatal diagnosis adhered more closely to a healthy lifestyle and lived a family-centred life.

PIP: The prevention of genetic diseases and congenital anomalies through prenatal diagnosis depends on public awareness and acceptance of the available methods. These factors were investigated in 1995 in a national survey of 350 Greek women with a child 5 years of age or younger. In Greece, prenatal testing is available at no charge to all eligible women who request it and has been widely publicized since 1976. 181 women (51.7%) were adequately informed about prenatal diagnosis, while the remaining 169 (48.3%) had superficial knowledge or no knowledge at all. 151 women knew about amniocentesis, 85 were aware of biochemical screening tests, 70 knew about ultrasound scanning, and 29 mentioned chorionic villus sampling. Physicians and the media were the main sources of knowledge about the subject. 47 women (13.4%) had undergone prenatal diagnosis during a previous pregnancy--30 because of advanced maternal age and 10 for "additional safety." 67 women (22%) who had not been tested were over 35 years old at last pregnancy. The reasons given by women eligible for prenatal diagnosis for not having the test were that there was no problem with the pregnancy or the doctor did not recommend it. No respondent cited financial, social, or ethical reasons for not accessing this procedure. Women's awareness of prenatal diagnosis increased with age, educational level, socioeconomic status, family income, and urban residence.

Fetal nucleated erythrocytes (NRBCs) in chorionic villus sample supernatant fluids: an additional source of fetal material for karyotype confirmation.

Fetal erythrocytes leak from the fetal capillaries at the time of chorionic villus removal. The purpose of this study was to determine if fetal nucleated erythrocytes (NRBCs) could be isolated from the chorionic villus sampling (CVS) supernatant fluid and used as an additional source of fetal material in order to confirm the fetal karyotype in cases of CVS mosaicism. One hundred CVS supernatant fluids were studied by simultaneous immunophenotyping, using a mouse antifetal haemoglobin antibody, UCH gamma, combined with fluorescent in situ hybridization (FISH) analysis using X- and Y-specific DNA probes. A chromosome 18 probe was also used in the case of a known male fetus with trisomy 18. Fetal haemoglobin (HbF)-positive cells were identified in 73 supernatant fluids and HbF-positive nucleated cells were present in 60 samples. The number of cells detected per sample showed great variation among the individual samples. FISH analysis was performed in 41 cases. FISH prediction of the fetal gender was concordant with the CVS karyotype in all cases, and the fetal trisomy 18 was correctly verified. In five cases in which Y sequences were detected, a small number of HbF-positive cells with two X signals were also identified; interestingly, in three of the five cases, the mother was a beta-thalassaemia carrier. This technique can be used as a quick and accurate method for the immediate verification of CVS results in cases of mosaicism, thus avoiding second-trimester amniocentesis.

Prenatal diagnosis services in Greece.

The report presents the available prenatal diagnosis (PND) services in Greece. PND for chromosomal anomalies after amniocentesis was initiated in 1976 and became gradually widely accepted. Chorionic villus sampling was introduced in 1983. Approximately 6,500 women undergo PND for chromosomal anomalies annually, most of them for advanced maternal age. 700 cases are also tested prenatally each year for haemoglobinopathies. The study describes the PND services available in the country, the procedures currently used, the impact of prenatal testing on the prevention of genetic diseases and the legal issues concerning PND in Greece.

Mapping dystrophin gene recombinants in Greek DMD/BMD families: low recombination frequencies in the STR region.

A systematic study of 42 Greek DMD/BMD families using 14 polymorphic markers that span the dystrophin gene was performed in order to assess the position and frequency of recombinants in the Greek population and to test whether "hot spots" of recombination and deletions coincide when exclusively studying DMD/BMD families. We report a low percentage of recombination between markers STR44 and STR50; otherwise, the distribution of recombination events in other parts of the gene is largely in agreement with previously published data on Centre d'Etude du Polymorphisme Humaine families. We therefore propose that recombination frequencies and the correlation between recombination and deletion "hot spots" should be evaluated on DMD/BMD families exclusively.

Deletion patterns of Duchenne and Becker muscular dystrophies in Greece.

We present molecular data from 90 Greek boys with Duchenne or Becker muscular dystrophy using cDNA analysis or multiplex PCR or both. Deletions were detected in 63.3% of patients and were mainly clustered in two areas of the gene, one in the 3' and one in the 5' end of the gene (exons 3-19 and 44-53). Almost 17% of deletion breakpoints lay in intron 44 while 29% of deletions have a breakpoint in intron 50. Thus the distribution of deletions in our DMD/BMD patients differs from that previously reported. Furthermore a 1:4.35 proximal:distal ratio was observed in familial cases and a 1:2.45 ratio in isolated ones.

Autosomal folate sensitive fragile sites in normal and mentally retarded individuals in Greece.

The frequencies of autosomal folate sensitive fragile sites were compared in populations of mentally retarded fra(X) negative (N = 220) and normal children (N = 76) in Greece. In addition, the frequency of autosomal fragile sites was studied in 20 known fra(X) children in order to test if the fra(X) syndrome is associated with general chromosome instability. The frequencies of both common and rare autosomal fragile sites did not differ significantly between the mentally retarded and the normal children, although the rate of expression was considerably higher in the retarded group. Autosomal fragile sites were not increased in the fra(X) patients. Fra(3)(p14) was by far the most frequent one in all groups. The frequency of fra(6)(q26) was found to be considerably higher among the mentally retarded children, this difference being almost statistically significant. Further cytogenetic studies of normal and retarded individuals are required in order to elucidate this point further.

Martin-Bell syndrome in Greece, with report of another 47,XXY fragile X patient.

A cytogenetic investigation was carried out among 200 mentally retarded boys in Greece for the detection of the fragile X [fra(X)] syndrome. Thirteen patients were found to carry fra(X) (6.5%). Of those, six boys had a history of familial X-linked mental retardation, two had the phenotype of the Martin-Bell syndrome, four had only mental retardation of unknown etiology, and one was a mentally retarded patient with Klinefelter syndrome. The remaining 187 boys were fra(X) negative. Our findings emphasize the importance of early identification of this syndrome in the diagnosis and prevention, through proper genetic counselling, of mental retardation.

Prenatal diagnosis of chromosomal abnormalities from chorionic biopsy samples: improved success rate using a modified direct method.

Several methods for fetal chromosome analysis using chorionic biopsy samples were compared. A modified direct method for culturing villi was considered to be the method of choice and details are presented of 186 pregnancies tested prenatally. The success rate in obtaining a fetal karyotype with the direct method was 93 per cent. The fetal loss rate in the prenatal series was 4.3 per cent and congenital abnormalities in the babies already born did not differ from the expected incidence.