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.

Loss of the Y chromosome PAR2 region and additional rearrangements in two familial cases of satellited Y chromosomes: cytogenetic and molecular analysis.

Two cases of rare structural aberrations of the Y chromosome were detected: a del(Y) (q12) chromosome in a child with mild dysmorphic features, obesity and psychomotor delay, and two identical satellited Y chromosomes (Yqs) in a normal twin, which were originally observed during routine prenatal diagnosis. In both cases a Yqs chromosome was detected in the father which had arisen from a reciprocal translocation involving the short arm of chromosome 15 and the heterochromatin of the long arm of the Y chromosome (Yqh). Cytogenetic and molecular studies demonstrated that in the reciprocal product of chromosomes 15 and Y PAR2 could not be detected, showing that PAR2 had been deleted. It is discussed whether the translocation of the short arm of an acrocentric chromosome to the heterochromatin of the long arm of the Y chromosome causes instability of this region which results either in loss of genetic material or interference with the normal mechanism of disjunction.

Measurement of locus copy number by hybridisation with amplifiable probes.

Despite its fundamental importance in genome analysis, it is only recently that systematic approaches have been developed to assess copy number at specific genetic loci, or to examine genomic DNA for submicro-scopic deletions of unknown location. In this report we show that short probes can be recovered and amplified quantitatively following hybridisation to genomic DNA. This simple observation forms the basis of a new approach to determining locus copy number in complex genomes. The power and specificity of multiplex amplifiable probe hybridisation is demonstrated by the simultaneous assessment of copy number at a set of 40 human loci, including detection of deletions causing Duchenne muscular dystrophy and Prader-Willi/Angelman syndromes. Assembly of other probe sets will allow novel, technically simple approaches to a wide variety of genetic analyses, including the potential for extension to high resolution genome-wide screens for deletions and amplifications.

Screening for subtelomeric chromosome abnormalities in children with idiopathic mental retardation using multiprobe telomeric FISH and the new MAPH telomeric assay.

Subtelomeric chromosomal abnormalities are emerging as an important cause of human genetic disorders. The scope of this investigation was to screen a selected group of children with idiopathic mental retardation for subtelomeric anomalies using the multiprobe telomeric FISH method and also to develop and test a new assay, the MAPH telomeric assay, in the same group of patients. The new MAPH telomeric assay uses the recently published MAPH methodology that permits the measurement of locus copy number by hybridisation with a specifically designed set of probes located at the end of human chromosomes. Seventy patients with idiopathic mental retardation have been screened using the established multiprobe telomeric FISH assay and the new MAPH telomeric assay, for all telomeres. One patient with de novo 8p subtelomeric deletion was identified. The new MAPH telomeric assay confirmed the same results in both normal and abnormal samples. This is the first description of the use of MAPH methodology to detect chromosomal imbalances near the telomeres in idiopathic mentally retarded patients. The new MAPH telomeric assay offers a new, fast, accurate and cost effective diagnostic tool to detect chromosomal imbalances near telomeres in mentally retarded patients, as well as the characterisation of known chromosomal abnormalities, spontaneous recurrent miscarriages, infertility, hematological malignancies, preimplantation genetic diagnosis, and other fields of clinical and research interests.

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.

Genetic variation and intergenerational FMR1 CGG-repeat stability in 100 unrelated three-generation families from the normal population.

In order to identify genetic factors governing expansion of the CGG repeat in the FMR1 gene and to determine what predisposes or causes a normal stable allele to change to an unstable premutation allele, it is essential to study and understand the basis of normal variation. The aim of this study was to investigate genetic variation and intergenerational stability of the FMR1 CGG-repeat region in 100 unrelated three-generation families from the general population (651 meioses). The number of CGG-repeats in the FMR1 gene was determined in all 750 individuals from the 100 families (a total of 1,132 X-chromosomes), and the allele frequencies and variability were analyzed. Thirty-six different alleles (12-60 repeats) were seen with 30 (45.8%) as the most common allele; overall female heterozygosity was 73%. Most (>96%) of the normal array lengths were less than 40 repeats. Fifteen families with at least one allele equal to or greater than 40 repeats (40-60) were identified; in one of these families there was an increase of one triplet repeat during transmission from a mother to son. These findings, together with future molecular analyses, may provide data to test proposed models that attempt to explain the mutational process and the population dynamics of the triplet repeat region of the FMR1 gene, including the transition from normal to unstable alleles, or to test other putative cis-acting sequences that may be involved with instability in the FMR1 gene.

Cytogenetic and fragile X molecular testing of individuals with mental retardation of unknown etiology.

The aim of this program was to investigate the patients with Mental Retardation Of Unknown Etiology (MROUE), on the island of Cyprus. The MROUE patients were examined cytogenetically for gross chromosomal abnormalities, and by molecular methods for the Fragile X syndrome pathology. Specialized physicians examined all institutionalized or non institutionalized patients throughout Cyprus. Cytogenetic analysis was carried out on 105 individuals, six of which showed various chromosomal aberrations. PCR and Southern blot analysis were carried out on 170 patients referred for exclusion of the Fragile X syndrome. Three patients had positive findings. Although the number of cases elucidated with this general approach was not spectacular, it allowed the resolution of a few clinically equivocal cases, to the satisfaction of the clinicians and, most importantly, the relatives involved. We believe that such screening programs should continue until all cases are thoroughly examined, thus providing definite genetic counseling and psychological support, at least in those cases that are clearly resolved. Equally important is the prospect for prevention through prenatal diagnostic programs, that are already available for such conditions.

Clinical and molecular description of the prenatal diagnosis of a fetus with a maternally inherited microduplication 22q11.2 of 2.5 Mb.

Microduplications of 22q11.2 have been recently characterized as a new genomic duplication syndrome showing an extremely variable phenotype ranging from normal or mild learning disability to multiple congenital defects and sharing some overlapping features with DiGeorge/Velocardiofacial syndrome (DGS/VCFS). We report on the prenatal diagnosis of a 22q11.2 microduplication in a fetus with normal development that was referred for chromosomal analysis at 17 weeks of gestation because of advanced maternal age. Pregnancy was the result of an IVF-ICSI attempt after 4 years of infertility, mainly due to severe oligoasthenoteratospermia of the father. Amniocentesis was undertaken and cytogenetic analysis revealed an apparently normal male karyotype. Multiple Ligation-dependent Probe Amplification (MLPA) revealed a microduplication in the 22q11.2 chromosome region. Parental analysis showed that the 22q11.2 microduplication has been inherited from the otherwise healthy mother. Analysis with high resolution array-CGH showed that the size of the microduplication is 2.5 Mb and revealed the genes that are duplicated, including the TBX1 gene. The parents elected to continue with the pregnancy and the infant is now five months old and shows normal development.

A prenatally ascertained, maternally inherited 14.8 Mb duplication of chromosomal bands Xq13.2-q21.31 associated with multiple congenital abnormalities in a male fetus.

Duplications of the X chromosome are rare cytogenetic findings, and have been associated with an abnormal phenotype in the male offspring of apparently normal or near normal female carriers. We report on the prenatal diagnosis of a duplication on the long arm of chromosome X from chromosomal band Xq13.2 to q21.31 in a male fetus with increased nuchal translucency in the first trimester and polyhydramnios at 22 weeks of gestation. Amniocentesis was undertaken and cytogenetic analysis revealed additional chromosomal material in the long arm of chromosome X at position Xq13. Analysis with high resolution array CGH revealed the additional material is in fact a duplication of the region Xq13.2-q21.13. The duplication is 14.8 Mb in size and includes fourteen genes: SLC16A2, KIAA2022, ABCB7, ZDHHC15, ATRX, MAGT1, ATP7A, PGK1, TBX22, BRWD3, POU3F4, ZNF711, POF1B and CHM. Analysis of the parents revealed the mother to be a carrier of the same duplication. After elected termination of the pregnancy at 28 weeks a detailed autopsy of the fetus allowed for genotype-phenotype correlations.

A nation-based population screening for azoospermia factor deletions in Greek-Cypriot patients with severe spermatogenic failure and normal fertile controls, using a specific study and experimental design.

Y chromosome microdeletions in the azoospermia factor (AZF) locus have been associated with spermatogenic failure. The frequency of AZF deletions is estimated to be about 10-18% in subgroups of idiopathic azoospermia and severe oligospermia, whereas the deletion frequency is estimated to be about 1.5-10.6% in the general population. Patient selection criteria as well as experimental and study design are the major factors that influence the deletion frequency. We designed a nation-based population screening with a well-defined study and experimental criteria to answer, first, what is the deletion frequency in a study population of high risk for Y deletion in the Greek-Cypriot origin and second, if there are any differences in the deletion frequency in the investigated specific subgroup of patients from different geographic/ethnic origin. Eighty Greek-Cypriot patients who met the selection criteria were included in this study as well as 50 fertile control males. The sample size is quite large when compared with the size of the population. All samples were collected from all districts of the island of Cyprus as the population is of the same religious, geographic and ethnic origin. All patients and controls had detailed clinical information and at least two semen-analysis reports based on World Health Organization standards. Samples with abnormal karyotypes, obstructive azoospermia or oligospermia with >2 x 106/mL were excluded from this study. The experimental design required a referral team and laboratory to undertake the responsibility to collect all the samples, all clinical and laboratory information, isolate DNA and carry out all tests, data analysis and interpretation. In our study, Y chromosome microdeletions have been found in patients with spermatogenic failure. Under the specific patient selection criteria and experimental design, the overall frequency is 5%, while among azoospermic patients it is 12.5%. In the subgroups of patients with idiopathic cause it is 5.9% and in idiopathic azoospermia it is 14.3%. No variation in the overall deletion frequency or the specific subgroups deletion frequency were found, as compared with frequencies found in patients from different geographic/ethnic origin.

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.

Molecular genetics of Turner syndrome: correlation with clinical phenotype and response to growth hormone therapy.

To correlate the origin of the retained X in Turner syndrome with phenotype, pre-treatment height and response to recombinant human growth hormone (rhGH) therapy, systematic clinical assessment and molecular studies were carried out in 33 Greek children with Turner syndrome and their parents including 18 children with 45,X and 15 with X-mosaicism. Microsatellite markers on X chromosomes (DXS101 and DXS337) revealed that the intact X was paternal (Xp) in 15/30 and maternal (Xm) in 15/30 children, while 3/33 families were non-informative. No significant relationship was found between parental origin of the retained X and birth weight/length/gestational age, blepharoptosis, pterygium colli, webbed neck, low hairline, abnormal ears, lymphoedema, short 4th metacarpal, shield chest, widely spaced nipples, cubitus valgus, pigmented naevi, streak gonads, and cardiovascular/renal anomalies. With regard to the children's pre-treatment height, there was a significant correlation with maternal height and target height in both Xm and Xp groups. No differences were found between Xm and Xp groups and the improvement of growth velocity (GV) during the first and second year of rhGH administration, while for both groups GV significantly improved with rhGH by the end of the first and the second year. To our knowledge, this is the first attempt to correlate the parental origin of Turner syndrome with the response to rhGH therapy.