Copy number gain at Xp22.31 includes complex duplication rearrangements and recurrent triplications.

Genomic instability is a feature of the human Xp22.31 region wherein deletions are associated with X-linked ichthyosis, mental retardation and attention deficit hyperactivity disorder. A putative homologous recombination hotspot motif is enriched in low copy repeats that mediate recurrent deletion at this locus. To date, few efforts have focused on copy number gain at Xp22.31. However, clinical testing revealed a high incidence of duplication of Xp22.31 in subjects ascertained and referred with neurobehavioral phenotypes. We systematically studied 61 unrelated subjects with rearrangements revealing gain in copy number, using multiple molecular assays. We detected not only the anticipated recurrent and simple nonrecurrent duplications, but also unexpectedly identified recurrent triplications and other complex rearrangements. Breakpoint analyses enabled us to surmise the mechanisms for many of these rearrangements. The clinical significance of the recurrent duplications and triplications were assessed using different approaches. We cannot find any evidence to support pathogenicity of the Xp22.31 duplication. However, our data suggest that the Xp22.31 duplication may serve as a risk factor for abnormal phenotypes. Our findings highlight the need for more robust Xp22.31 triplication detection in that such further gain may be more penetrant than the duplications. Our findings reveal the distribution of different mechanisms for genomic duplication rearrangements at a given locus, and provide insights into aspects of strand exchange events between paralogous sequences in the human genome.

Detection of clinically relevant exonic copy-number changes by array CGH.

Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy-number variation (CNV). However, intragenic deletions or duplications--those including genomic intervals of a size smaller than a gene--have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy-number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom-designed, exon-targeted oligonucleotide array to detect intragenic copy-number changes in patients with various clinical phenotypes.

Insertional translocation detected using FISH confirmation of array-comparative genomic hybridization (aCGH) results.

Insertional translocations (ITs) are rare events that require at least three breaks in the chromosomes involved and thus qualify as complex chromosomal rearrangements (CCR). In the current study, we identified 40 ITs from approximately 18,000 clinical cases (1:500) using array-comparative genomic hybridization (aCGH) in conjunction with fluorescence in situ hybridization (FISH) confirmation of the aCGH findings, and parental follow-up studies. Both submicroscopic and microscopically visible IT events were detected. They were divided into three major categories: (1) simple intrachromosomal and interchromosomal IT resulting in pure segmental trisomy, (2) complex IT involving more than one abnormality, (3) deletion inherited from a parent with a balanced IT resulting in pure segmental monosomy. Of the cases in which follow-up parental studies were available, over half showed inheritance from an apparently unaffected parent carrying the same unbalanced rearrangement detected in the propositi, thus decreasing the likelihood that these IT events are clinically relevant. Nevertheless, we identified six cases in which small submicroscopic events were detected involving known disease-associated genes/genomic segments and are likely to be pathogenic. We recommend that copy number gains detected by clinical aCGH analysis should be confirmed using FISH analysis whenever possible in order to determine the physical location of the duplicated segment. We hypothesize that the increased use of aCGH in the clinic will demonstrate that IT occurs more frequently than previously considered but can identify genomic rearrangements with unclear clinical significance.

Phenotypic spectrum and genotype-phenotype correlations of NRXN1 exon deletions.

Copy number variants (CNVs) and intragenic rearrangements of the NRXN1 (neurexin 1) gene are associated with a wide spectrum of developmental and neuropsychiatric disorders, including intellectual disability, speech delay, autism spectrum disorders (ASDs), hypotonia and schizophrenia. We performed a detailed clinical and molecular characterization of 24 patients who underwent clinical microarray analysis and had intragenic deletions of NRXN1. Seventeen of these deletions involved exons of NRXN1, whereas seven deleted intronic sequences only. The patients with exonic deletions manifested developmental delay/intellectual disability (93%), infantile hypotonia (59%) and ASDs (56%). Congenital malformations and dysmorphic features appeared infrequently and inconsistently among this population of patients with NRXN1 deletions. The more C-terminal deletions, including those affecting the ß isoform of neurexin 1, manifested increased head size and a high frequency of seizure disorder (88%) when compared with N-terminal deletions of NRXN1.

Phenotypic manifestations of copy number variation in chromosome 16p13.11.

The widespread clinical utilization of array comparative genome hybridization, has led to the unraveling of many new copy number variations (CNVs). Although some of these CNVs are clearly pathogenic, the phenotypic consequences of others, such as those in 16p13.11 remain unclear. Whereas deletions of 16p13.11 have been associated with multiple congenital anomalies, the relevance of duplications of the region is still being debated. We report detailed clinical and molecular characterization of 10 patients with duplication and 4 patients with deletion of 16p13.11. We found that patients with duplication of the region have varied clinical features including behavioral abnormalities, cognitive impairment, congenital heart defects and skeletal manifestations, such as hypermobility, craniosynostosis and polydactyly. These features were incompletely penetrant. Patients with deletion of the region presented with microcephaly, developmental delay and behavioral abnormalities as previously described. The CNVs were of varying sizes and were likely mediated by non-allelic homologous recombination between low copy repeats. Our findings expand the repertoire of clinical features observed in patients with CNV in 16p13.11 and strengthen the hypothesis that this is a dosage sensitive region with clinical relevance.

Rapid prenatal diagnosis using uncultured amniocytes and oligonucleotide array CGH.

OBJECTIVE: Oligonucleotide-based array comparative genomic hybridization (array CGH) is an established method for detecting chromosomal abnormalities. Here, we explored the feasibility of using DNA extracted from uncultured amniocytes in amniotic fluid for array CGH on an oligonucleotide array platform. METHODS: Fifteen fetuses from 14 ongoing pregnancies were studied by array CGH on targeted oligonucleotide arrays with DNA isolated from direct amniotic fluid using a modified DNA extraction protocol. RESULTS: High-quality array CGH results were obtained for 13 samples with suboptimal but interpretable results in only 2 samples due to limited DNA amounts. Array CGH using whole genome amplification (WGA) of DNA for the two cases with limited DNA was successful, and results were consistent with those from unamplified DNA. For another five samples, the results of array CGH with amplified DNA matched those with unamplified DNA. Chromosome analysis was performed for 14 cases and was consistent with array CGH results. CONCLUSION: This study demonstrates the feasibility of prenatal genetic diagnosis using oligonucleotide array CGH analysis for direct analysis of amniocytes without culturing cells. The use of oligonucleotide arrays increases the sensitivity and accuracy of detection over previous bacterial artificial chromosome (BAC)-based arrays. Furthermore, the direct analysis allows for rapid array CGH results and shorter reporting time.