A parallel SNP array study of genomic aberrations associated with mental retardation in patients and general population in Estonia.

The increasing use of whole-genome array screening has revealed the important role of DNA copy-number variations in the pathogenesis of neurodevelopmental disorders and several recurrent genomic disorders have been defined during recent years. However, some variants considered to be pathogenic have also been observed in phenotypically normal individuals. This underlines the importance of further characterization of genomic variants with potentially variable expressivity in both patient and general population cohorts to clarify their phenotypic consequence. In this study whole-genome SNP arrays were used to investigate genomic rearrangements in 77 Estonian families with idiopathic mental retardation. In addition to this family-based approach, phenotype and genotype data from a cohort of 1000 individuals in the general population were used for accurate interpretation of aberrations found in mental retardation patients. Relevant structural aberrations were detected in 18 of the families analyzed (23%). Fifteen of those were in genomic regions where clinical significance has previously been established. In 3 families, 4 novel aberrations associated with intellectual disability were detected in chromosome regions 2p25.1-p24.3, 3p12.1-p11.2, 7p21.2-p21.1 and Xq28. Carriers of imbalances in 15q13.3, 16p11.2 and Xp22.31 were identified among reference individuals, affirming the variable phenotypic consequence of rare variants in some genomic regions considered as pathogenic.

Mutations in the guanine nucleotide exchange factor gene IQSEC2 cause nonsyndromic intellectual disability.

The first family identified as having a nonsyndromic intellectual disability was mapped in 1988. Here we show that a mutation of IQSEC2, encoding a guanine nucleotide exchange factor for the ADP-ribosylation factor family of small GTPases, caused this disorder. In addition to MRX1, IQSEC2 mutations were identified in three other families with X-linked intellectual disability. This discovery was made possible by systematic and unbiased X chromosome exome resequencing.

5.9 Mb microdeletion in chromosome band 17q22-q23.2 associated with tracheo-esophageal fistula and conductive hearing loss.

Only eight cases involving deletions of chromosome 17 in the region q22-q24 have been reported previously. We describe an additional case, a 7-year-old boy with profound mental retardation, severe microcephaly, facial dysmorphism, symphalangism, contractures of large joints, hyperopia, strabismus, bilateral conductive hearing loss, genital abnormality, psoriasis vulgaris and tracheo-esophageal fistula. Analysis with whole-genome SNP genotyping assay detected a 5.9 Mb deletion in chromosome band 17q22-q23.2 with breakpoints between 48,200,000-48,300,000 bp and 54,200,000-54,300,000 bp (according to NCBI 36). The aberration was confirmed by real-time quantitative PCR analysis. Haploinsufficiency of NOG gene has been implicated in the development of conductive hearing loss, skeletal anomalies including symphalangism, contractures of joints, and hyperopia in our patient and may also contribute to the development of tracheo-esophageal fistula and/or esophageal atresia.

Prevalence of the fragile X syndrome among Estonian mentally retarded and the entire children's population.

The aim of this study is to establish the prevalence of fragile X syndrome among Estonian mentally retarded and also among the entire children's population born during the years 1984-2005. The study group consisted of 516 patients (448 boys and 68 girls) who were screened for full mutations in the FMR1 gene during the period 1997-2006. Fourteen boys (2.7%) were found with full mutations of the total mentally retarded individuals tested (3.1% of mentally retarded boys); the full mutation was not detected among girls. The live-birth prevalence of full mutation among boys was 1:13 947. The overall live-birth prevalence of fragile X syndrome was 1:27 115. It was found that the prevalence of fragile X syndrome among mentally retarded individuals in Estonia was the same as in previous studies, but the live-birth prevalence of fragile X syndrome among boys was significantly lower.

Array-MAPH: a methodology for the detection of locus copy-number changes in complex genomes.

High-throughput genome-wide screening methods to detect subtle genomic imbalances are extremely important for diagnostic genetics and genomics. Here, we provide a detailed protocol for a microarray-based technique, applying the principle of multiplex amplifiable probe hybridization (MAPH). Methodology and software have been developed for designing unique PCR-amplifiable sequences (400-600 bp) covering any genomic region of interest. These sequences are amplified, cloned and spotted onto arrays (targets). A mixture of the same sequences (probes) is hybridized to genomic DNA immobilized on a membrane. Bound probes are recovered and quantitatively amplified by PCR, labeled and hybridized to the array. The procedure can be completed in 4-5 working days, excluding microarray preparation. Unlike array-comparative genomic hybridization (array-CGH), test DNA of specifically reduced complexity is hybridized to an array of identical small amplifiable target sequences, resulting in increased hybridization specificity and higher potential for increasing resolution. Array-MAPH can be used for detection of small-scale copy-number changes in complex genomes, leading to genotype-phenotype correlations and the discovery of new genes.

Girl with partial Turner syndrome and absence epilepsy.

This report describes a 16-year-old girl with short stature (-5 standard deviations), normal puberty, panic attacks, absence epilepsy, some stigmata of Turner syndrome, and a Madelung deformity. Routine chromosomal analysis revealed a female karyotype with one abnormal chromosome X, with the suspicion of additional material on the short arm. With fluorescent in situ hybridization and array-multiplex amplifiable probe hybridization methodology, a complex aberration was detected, with a deletion of the distal part of Xp22.33 (including the short-stature homeobox gene) and a duplication of Xp22.32-p22.12 proximal to the deleted segment. The deletion in our patient involves the Xp22.33 region. Two genes in this region may contribute to the patient's phenotype: short-stature homeobox, and visuospatial/perceptual abilities. The duplication in our patient involves the Xp22.12-p22.32 region, which, according to the Online Mendelian Inheritance in Man database, contains at least 93 genes, 49 of which are of unknown function. It is difficult to conjecture which gene overexpression in this region may have contributed to the phenotype of our patient. To our knowledge, this small, complex chromosome X aberration was not described previously.

Screening of 20 patients with X-linked mental retardation using chromosome X-specific array-MAPH.

The rapid advancement of high-resolution DNA copy number assessment methods revealed the significant contribution of submicroscopic genetic imbalances to abnormal phenotypes, including mental retardation. In order to detect submicroscopic genetic imbalances, we have screened 20 families with X-linked mental retardation (XLMR) using a chromosome X-specific array-MAPH platform with median resolution of 238kb. Among the 20 families, 18 were experimental, as they were not previously screened with any microarray method, and two were blind controls with known aberrations, as they were previously screened by array-CGH. This study presents the first clinical application of chromosome X-specific array-MAPH methodology. The screening of 20 affected males from 20 unrelated XLMR families resulted in the detection of an unknown deletion, spanning a region of 7-23kb. Family studies and population screening demonstrated that the detected deletion is an unknown rare copy number variant. One of the control samples, carrying approximately 6-Mb duplication was correctly identified, moreover it was found to be interrupted by a previously unknown 19kb region of normal copy number. The second control 50kb deletion was not identified, as this particular region was not covered by array-MAPH probes. This study demonstrates that the chromosome X-specific array-MAPH platform is a valuable tool for screening patients with XLMR, or other X-linked disorders, and emerges the need for introducing new high-resolution screening methods for the detection of genetic imbalances.

Detection of small genomic imbalances using microarray-based multiplex amplifiable probe hybridization.

Array-based genome-wide screening methods were recently introduced to clinical practice in order to detect small genomic imbalances that may cause severe genetic disorders. The continuous advancement of such methods plays an extremely important role in diagnostic genetics and medical genomics. We have modified and adapted the original multiplex amplifiable probe hybridization (MAPH) to a novel microarray format providing an important new diagnostic tool for detection of small size copy-number changes in any locus of human genome. Here, we describe the new array-MAPH diagnostic method and show proof of concept through fabrication, interrogation and validation of a human chromosome X-specific array. We have developed new bioinformatic tools and methodology for designing and producing amplifiable hybridization probes (200-600 bp) for array-MAPH. We designed 558 chromosome X-specific probes with median spacing 238 kb and 107 autosomal probes, which were spotted onto microarrays. DNA samples from normal individuals and patients with known and unknown chromosome X aberrations were analyzed for validation. Array-MAPH detected exactly the same deletions and duplications in blind studies, as well as other unknown small size deletions showing its accuracy and sensitivity. All results were confirmed by fluorescence in situ hybridization and probe-specific PCR. Array-MAPH is a new microarray-based diagnostic tool for the detection of small-scale copy-number changes in complex genomes, which may be useful for genotype-phenotype correlations, identification of new genes, studying genetic variation and provision of genetic services.