Alpha-thalassemia intellectual disability: variable phenotypic expression among males with a recurrent nonsense mutation - c.109C>T (p.R37X).

Alpha-thalassemia intellectual disability, one of the recognizable X-linked disability syndromes, is characterized by short stature, microcephaly, distinctive facies, hypotonic appearance, cardiac and genital anomalies, and marked skewing of X-inactivation in female carriers. With the advent of next generation sequencing, mutations have been identified that result in less severe phenotypes lacking one or more of these phenotypic manifestations. Here we report five unrelated kindreds in which a c.109C>T (p.R37X) mutation segregates with a variable but overall milder phenotype. The distinctive facial appearance of alpha-thalassemia intellectual disability was present in only one of the 18 affected males evaluated beyond the age of puberty, although suggestive facial appearance was present in several during infancy or early childhood. Although the responsible genetic alteration is a nonsense mutation in exon 2 of ATRX, the phenotype appears to be partially rescued by the production of alternative transcripts and/or other molecular mechanisms.

Skewed X-chromosome inactivation in Greek women with idiopathic recurrent miscarriage.

OBJECTIVES: To investigate if skewed X-chromosome inactivation (XCI) is associated with unexplained recurrent miscarriage (RM) in Greek women. METHODS: This was a prospective case-control study. A methylation-sensitive assay was used to investigate the X-inactivation pattern of women with unexplained RM and controls. RESULTS: Fifty-six of the 74 patients (75.7%) and 55 of 80 controls (68.8%) were informative. Among the informative cases, 6/56 (10.7%) women showed extreme XCI (>90%) and among the informative controls, 2/55 (3.6%) showed extreme XCI. CONCLUSIONS: In the present study, women with unexplained RM showed a statistically nonsignificant increase in skewed XCI prevalence (10.7%) compared with control women (3.6%; p = 0.271).

Age and origin of major Smith-Lemli-Opitz syndrome (SLOS) mutations in European populations.

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) (MIM 270 400) is an autosomal recessive multiple congenital anomalies/mental retardation syndrome caused by mutations in the Delta7-sterol reductase (DHCR7, E.C.1.3.1.21) gene. The prevalence of SLOS has been estimated to range between 1:15000 and 1:60000 in populations of European origin. METHODS AND RESULTS: We have analysed the frequency, origin, and age of DHCR7 mutations in European populations. In 263 SLOS patients 10 common alleles (c.964-1G>C, p.Trp151X, p.Thr93Met, p.Val326Leu, p.Arg352Trp, p.Arg404Cys, p.Phe302Leu, p.Leu157Pro, p.Gly410Ser, p.Arg445Gln) were found to constitute approximately 80% of disease-causing mutations. As reported before, the mutational spectra differed significantly between populations, and frequency peaks of common mutations were observed in North-West (c.964-1G>C), North-East (p.Trp151X, p.Val326Leu) and Southern Europe (p.Thr93Met). SLOS was virtually absent from Finland. The analysis of nearly 8000 alleles from 10 different European populations confirmed a geographical distribution of DHCR7 mutations as reported in previous studies. The common Null mutations in Northern Europe (combined ca. 1:70) occurred at a much higher frequency than expected from the reported prevalence of SLOS. In contrast the most common mutation in Mediterranean SLOS patients (p.Thr93Met) had a low population frequency. Haplotypes were constructed for SLOS chromosomes, and for wild-type chromosomes of African and European origins using eight cSNPs in the DHCR7 gene. The DHCR7 orthologue was sequenced in eight chimpanzees (Pan troglodytes) and three microsatellites were analysed in 50 of the SLOS families in order to estimate the age of the three major SLOS-causing mutations. CONCLUSIONS: The results indicate a time of first appearance of c.964-1G>C and p.Trp151X some 3000 years ago in North-West and North-East Europe, respectively. The p.Thr93Met mutations on the J haplotype has probably first arisen approximately 6000 years ago in the Eastern Mediterranean. Together, it appears that a combination of founder effects, recurrent mutations, and drift have shaped the present frequency distribution of DHCR7 mutations in Europe.

Lack of association of birth size with polymorphisms of two imprinted genes, IGF2R and GRB10.

Little is known about the determinants of birth size variability among individuals. Maternal and nutritional factors have been studied, but familial clustering suggests genetic factors as well. As a first step in testing this hypothesis, we examined common sequence variants in IGF2R and GRB10, two genes involved in the regulation of growth and subject to parental imprinting. The IGF2R gene was scanned with five polymorphisms spanning the coding and 3'-UTR for possible association with birth size in a set of 97 normal newborns in Greece. In addition, a silent SNP in GRB10 exon 2 was similarly tested as an exploratory first step. Birth weight and length were compared between groups of newborns divided according to which allele they had received from heterozygous parents. No significant differences were found between alleles in either gene, examined either by parental origin or in aggregate. Thus, we found no evidence that IGF2R variants modulate intrauterine growth within the normal range. If such variants exist in GRB10, they are not in linkage disequilibrium with the marker studied.

Refractory photosensitive epilepsy associated with a complex rearrangement of chromosome 2.

We describe the relevant clinical and therapeutic parameters in a single patient with a complex chromosome 2 abnormality presenting with refractory myoclonic photosensitive epilepsy. FISH technology using yeast artificial chromosomes (YACs) was employed to determine breakage points, microdeletions and inversions on the affected chromosome. In this patient with refractory photosensitive epilepsy, 12 breakpoints and one small inversion were identified on the abnormal chromosome 2. Our data can be used in further genetic studies on the exact location and identification of photosensitivity genes.

Unusual chromosomal mosaicism in Wolf-Hirschhorn syndrome: del(4)(p16)/der(4)(qter-q31.3::pter-qter).

We report on the unusual cytogenetic findings in a girl with moderate mental retardation and a mosaic karyotype 46,XX,del(4)(p16)/46,XX,der(4)(qter-q31.3::pter-qter). The facial features observed in the child initially did not suggest the diagnosis of Wolf-Hirschhorn syndrome (WHS), but the distinct facial gestalt became obvious at prepubertal age. Fluorescence in situ hybridization (FISH) analysis with different probes that map to 4p and 4q helped to clarify the karyotype. We discuss the mechanism of appearance of this unusual type of mosaicism, which has not been reported before.

Xp22.3; Yq11.2 chromosome translocation and its clinical manifestations.

We report clinical and molecular investigations in a boy with karyotype 46,Y,der(X)t(X;Y)(qter-->p22.3::q11.21-->qter) and his mother with karyotype 46,X,der(X)t(X;Y)(qter-->p22.3::q11.21-->qter). Haplo-insufficiency for the Xp22.3-->pter chromosomal region in the boy resulted in postnatal growth retardation, developmental delay, partial ichthyosis and facial dysmorphism, but normal external genitals. His mother has a normal phenotype with normal stature and gonadal function but borderline intelligence. FISH-analysis showed a duplication of the Y-heterochromatin probe in the proband and a deletion of the Y933D4 probe in his mother. Molecular investigations situated the Xp22.3 breakpoint between DXS278 and the KAL gene and the Yq11.21 breakpoint between the DYS391 and DYS390 in the proband and his mother. X-inactivation study was performed by analysis of the polymorphic CAG-repeat in the androgen-receptor gene as described showing a normal random (40% versus 60%) inactivation pattern in the mother. The manifestations in male and female with loss of the Xp22.3-->pter and gain of the Yq11.21-->qter chromosomal region are discussed.

Dysmorphology and mental retardation: molecular cytogenetic studies in dysmorphic mentally retarded patients.

In an institutionalised population of 471 mentally retarded adult residents (436 males and 35 females), 18 patients (16 males and 2 females) with dysmorphic features were selected to perform FISH studies by using subtelomeric probes to discover cryptic terminal deletions or duplications, undetectable with standard banding techniques. In the 13 investigated patients, no abnormalities were found with a selected battery of subtelomeric probes. The results of cryptic chromosomal rearrangement studies are variable but the frequency of positive diagnostic findings seems to be lower than previously expected.

Molecular cytogenetic characterization and origin of two de novo duplication 9p cases.

We report on two additional cases with duplication of 9p, minor with facial anomalies and developmental delay. Using fluorescence in situ hybridization and single-copy probes, we showed that the first case was a direct duplication, whereas the second case was inverted. The extent of the direct duplication was defined as 9p12 --> p24 by microdissection and microcloning of the aberrant chromosome and subsequent chromosome-specific comparative genomic hybridization. DNA polymorphism analysis with eight microsatellite markers revealed that the origin of the dup(9p) was maternal in the first case, whereas it was paternal in the second.

Analysis of chromosomal aberrations in breast cancer by comparative genomic hybridization (CGH). Correlation with histoprognostic variables and c-erbB-2 immunoexpression.

The establishment of additional powerful prognostic markers in breast cancer patients is of unquestionable importance given that breast cancer is characterized by morphologic, biologic and genetic heterogeneity. In the present study we analyzed 8 primary invasive breast carcinomas by comparative genomic hybridization (CGH) in order to find and map the DNA copy number changes occurring in these tumors. Furthermore, in order to evaluate the potential prognostic significance, we compared these genetic changes with other histo- and immunopathologic prognostic variables, such as tumor type, tumor grade, lymph node status, estrogen receptors content and c-erbB-2 oncoprotein expression. All the studied cases showed a wide variety of gains and losses of chromosomal regions or arms distributed among 16 chromosomes with an average number of 6.12 aberrations per case. Although several genetic changes appeared to be common, none was unique or consistent in all the studied cases. The most consistent regions of gain were on 1q, 20q and 8q while the most common regions of loss on 3p and 6q. Accumulation of chromosomal changes were more frequently found in high grade ductal breast carcinomas with overexpression of c-erbB-2 oncoprotein in both lymph node-negative and lymph node-positive patients, whose tumors were positive for estrogen receptors. If any of these genetic changes identified by CGH in breast cancer patients carry prognostic information, regardless of stage or other factors predictive of biologic behavior, further investigation is needed.

Association of estrogen receptor gene polymorphisms with endometriosis.

OBJECTIVE: To explore the association of the estrogen receptor two-allele (point) polymorphism and multiallele (microsatellite) polymorphism with endometriosis. DESIGN: Case-control study. SETTING: Genetics and Endoscopy Unit, Department of Obstetrics and Gynecology, Ioannina University HOSPITAL, Ioannina, Greece. PATIENT(S): Fifty-seven women with surgically and histologically diagnosed endometriosis of stages I-IV. INTERVENTION(S): Diagnostic laparoscopy. MAIN OUTCOME MEASURE(S): Frequency and distribution of the estrogen receptor gene polymorphisms. RESULT(S): There was a statistically significant difference between the patients and the controls in the frequency of the two-allele Pvu II polymorphism (0.72 vs. 0.49) and in the median repeats of the (TA)n multiallele polymorphism (15 vs. 20 repeats). In both groups, linkage was found between the fewer (TA)n repeats (range, 12-19) and the positive Pvu II polymorphism. CONCLUSION(S): The variability of the estrogen receptor gene likely contributes to the pathogenesis of endometriosis.

Molecular screening of fragile X (FRAXA) and FRAXE mental retardation syndromes in the Hellenic population of Greece and Cyprus: incidence, genetic variation, and stability.

This study presents the first large, population-based molecular investigation of the fragile X (FRAXA) and FRAXE mental retardation syndromes in the Hellenic populations of Greece and Cyprus. The aims of this population screening were to determine the prevalence of FRAXA and FRAXE syndromes among idiopathic mentally retarded (IMR) individuals, to estimate the incidence in the general population, and to investigate the molecular mechanism of instability and expansion of the FMR1-repeat. Ten FRAXA patients were identified to have either the full mutation (eight) or premutation (two) from a Hellenic population of 866 unrelated IMR individuals (611 males and 255 females, age range 3-25 years). No FRAXE patients were identified among the 611 IMR males. The incidence of FRAXA in the Hellenic population of Cyprus is estimated at 1 in 4,246 males. The repeat sites from the FMR1 and FMR2 alleles were accurately determined and showed similar distribution and frequencies with other population studies. The analysis of AGG interspersion within the FMR1-repeat in normal males revealed long, pure CGG repeats within the "gray zone" as well as variation within the 3' end showing polarity of instability. This finding supports the hypothesis that the AGG interspersion and the length of the pure repeat are major factors in determining allele stability. Analysis of FRAXAC1, DXS548, and FRAXAC2 identified particular alleles and haplotypes to have a significant association with either gray zone alleles or alleles >15 pure CGG repeats. We hypothesize that this subgroup of alleles and haplotypes are associated with long pure CGGs (>15 CGG) or 35 repeats and, having shared an evolutionary past, would have the tendency to expand.

Fragile X premutations and (TA)n estrogen receptor polymorphism in women with ovarian dysfunction.

We studied five groups of women with ovarian dysfunction for the CGG expansion in FMR1 and a (TA)n polymorphism in the estrogen receptor gene: a) poor responders to ovarian stimulation as part of in vitro fertilization (n = 13); b) women with familial premature ovarian failure (POF) (n = 7); c) sporadic cases with POF (n = 16); d) FRAXA premutation carriers with POF (n = 7); and e) FRAXA premutation carriers without POF (n = 9). FRAXA premutation was found in one woman with familial POF. A significant association of familial POF and FRAXA premutation carriers with POF having low copy of the (TA)n polymorphism as compared to controls was observed. Our preliminary data suggest a potential role of the estrogen receptor in POF, and it may influence the variable age of menopause of the FRAXA premutation carriers.

Effect of Percoll gradient and swim-up preparation on the chromomycin A3 staining of normal and abnormal semen samples.

The protamination of spermatozoa recovered by Percoll gradient and swim-up was investigated by means of chromomycin A3 (CMA3) staining. A total of 34 semen samples from patients undergoing IVF treatment were divided in two groups: normal (A) and oligoasthenozoospermic (B). Samples were divided in fractions, subjected to both techniques of preparation, and stained by CMA3. The percentage of CMA3 positive spermatozoa recovered by Percoll was comparable to swim-up in the normal group. In the abnormal group Percoll resulted in significantly lower CMA3 percentage. It is concluded that the degree of protamination in recovered spermatozoa is influenced by the technique of preparation.

Variation in the number of FMR1 microsatellite repeats in three subgroups of the Hellenic population.

Microsatellites have been used for human evolution and origin studies by comparing their frequency, diversity, and allele size. In this study we report the analysis of three microsatellite loci, FMR1 CGG and flanking DXS548 and FRAXAC2, in three separate groups of the Hellenic population: Athens, representing the general Hellenic population; Epirus (northwest Greece); and Cyprus. Significant variations in frequency and diversity were found in the three groups. Compared with Athens, Epirus had a tendency for longer alleles and a higher heterozygosity for DXS548. Cyprus had a frequency of CGG alleles similar to Athens but a low heterozygosity and a limited number of alleles at DXS548 and FRAXAC2. Allele differences of microsatellite loci not only are present in remote populations but also are evident between groups belonging to the same population. Microsatellite analysis could be a useful tool for identifying the origin of the founder chromosomes in intra-population studies and the time elapsed from the establishment of each population subgroup.

FRAXA and FRAXE prevalence in patients with nonspecific mental retardation in the Hellenic population.

Mutations at FRAXA and FRAXE loci are due to expansions of a CGG trinucleotide repeat and are characterized by mental retardation. Here we report a pilot screening survey by means of cytogenetic and molecular methods of 433 unrelated retarded individuals and their parents of Hellenic origin coming from various parts of Greece and Cyprus. The purpose of the study was to estimate the frequency of FRAXA mutation in individuals with nonspecific mental retardation without family history and phenotypic stigmata in the Hellenic population. Five FRAXA-positive children (1.15%) were identified, of whom four were found to carry a full mutation and one a premutation. Furthermore we present preliminary data on a screening of FRAXE mutation frequency. We screened 257 male patients with nonspecific mental retardation, finding none positive for FRAXE mutation.

Infertility and multiple urogenital abnormalities in a male with mosaic 46,XY/45,XO/47,XXY karyotype and mixed phenotype.

We hereby present a rare case of a 46,XY/45,XO/47,XXY mosaic male patient with a predominance of the XY cell line. The patient, who exhibited phenotypic stigmata of both XO gonadal dysgenesis and Klinefelter syndromes, suffered from infertility and multiple urogenital abnormalities, as our investigation revealed.

Increased nuchal translucency thickness in a fetus at risk for beta-thalassaemia.

In Greece and other Mediterranean countries up to 10% of the population are carriers of beta-thalassaemia and this is the most common indication for chorion villus sampling (CVS): Cytogenetic analysis of the samples is not carried out routinely, but is confined only to women aged 35 years or more. In this report we present a case that illustrates how the measurement of fetal nuchal translucency may be useful in selecting the cases where in addition to the DNA analysis for beta-thalassaemia the samples can be tested for chromosomal defects.