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Additional file 2: Fig. S1. Timeline from first analysis, VUS discovery, publication, and diagnosis.

Contains fulltext : 108716.pdf (Publisher’s version ) (Open Access) Usher syndrome (USH) is a clinically and genetically heterogeneous disorder characterized by visual and hearing impairments. Clinically, it is subdivided into three subclasses with nine genes identified so far. In the present study, we investigated whether the currently available Next Generation Sequencing (NGS) technologies are already suitable for molecular diagnostics of USH. We analyzed a total of 12 patients, most of which were negative for previously described mutations in known USH genes upon primer extension-based microarray genotyping. We enriched the NGS template either by whole exome capture or by Long-PCR of the known USH genes. The main NGS sequencing platforms were used: SOLiD for whole exome sequencing, Illumina (Genome Analyzer II) and Roche 454 (GS FLX) for the Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH gene regions displaying an overall coverage higher than 25x, whereas whole exome sequencing yielded a similar coverage for only 50% of those regions. Overall this integrated analysis led to the identification of 11 novel sequence variations in USH genes (2 homozygous and 9 heterozygous) out of 18 detected. However, at least two cases were not genetically solved. Our result highlights the current limitations in the diagnostic use of NGS for USH patients. The limit for whole exome sequencing is linked to the need of a strong coverage and to the correct interpretation of sequence variations with a non obvious, pathogenic role, whereas the targeted approach suffers from the high genetic heterogeneity of USH that may be also caused by the presence of additional causative genes yet to be identified.

Massively parallel sequencing has tremendous diagnostic potential but requires enriched templates for sequencing. Here we report the validation of an arraybased sequence capture method in genetically heterogeneous disorders. The model disorder selected was AR ataxia, using five subjects with known mutations and two unaffected controls. The genomic sequences of seven disease genes, together with two control loci were targeted on a 2-Mb sequence-capture array. After enrichment, the patients' DNA samples were analyzed using one-quarter Roche GS FLX Titanium sequencing run, resulting in an average of 65Mb of sequence data per patient. This was sufficient for an average 25-fold coverage/base in all targeted regions. Enrichment showed high specificity; on average, 80% of uniquely mapped reads were on target. Importantly, this approach enabled automated detection of deletions and hetero- and homozygous point mutations for 6/7 mutant alleles, and greater than 99% accuracy for known SNP variants. Our results also clearly show reduced coverage for sequences in repeat-rich regions, which significantly impacts the reliable detection of genomic variants. Based on these findings we recommend a minimal coverage of 15-fold for diagnostic implementation of this technology. We conclude that massive parallel sequencing of enriched samples enables personalized diagnosis of heterogeneous genetic disorders and qualifies for rapid diagnostic implementation. Refereed/Peer-reviewed

Contains fulltext : 89704.pdf (Publisher’s version ) (Closed access) Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous retinal disorder characterized by abnormal vascularisation of the peripheral retina, often accompanied by retinal detachment. To date, mutations in three genes (FZD4, LRP5, and NDP) have been shown to be causative for FEVR. In two large Dutch pedigrees segregating autosomal-dominant FEVR, genome-wide SNP analysis identified an FEVR locus of approximately 40 Mb on chromosome 7. Microsatellite marker analysis suggested similar at risk haplotypes in patients of both families. To identify the causative gene, we applied next-generation sequencing in the proband of one of the families, by analyzing all exons and intron-exon boundaries of 338 genes, in addition to microRNAs, noncoding RNAs, and other highly conserved genomic regions in the 40 Mb linkage interval. After detailed bioinformatic analysis of the sequence data, prioritization of all detected sequence variants led to three candidates to be considered as the causative genetic defect in this family. One of these variants was an alanine-to-proline substitution in the transmembrane 4 superfamily member 12 protein, encoded by TSPAN12. This protein has very recently been implicated in regulating the development of retinal vasculature, together with the proteins encoded by FZD4, LRP5, and NDP. Sequence analysis of TSPAN12 revealed two mutations segregating in five of 11 FEVR families, indicating that mutations in TSPAN12 are a relatively frequent cause of FEVR. Furthermore, we demonstrate the power of targeted next-generation sequencing technology to identify disease genes in linkage intervals.

Contains fulltext : 89284.pdf (Publisher’s version ) (Closed access) The per-generation mutation rate in humans is high. De novo mutations may compensate for allele loss due to severely reduced fecundity in common neurodevelopmental and psychiatric diseases, explaining a major paradox in evolutionary genetic theory. Here we used a family based exome sequencing approach to test this de novo mutation hypothesis in ten individuals with unexplained mental retardation. We identified and validated unique non-synonymous de novo mutations in nine genes. Six of these, identified in six different individuals, are likely to be pathogenic based on gene function, evolutionary conservation and mutation impact. Our findings provide strong experimental support for a de novo paradigm for mental retardation. Together with de novo copy number variation, de novo point mutations of large effect could explain the majority of all mental retardation cases in the population. 01 december 2010