Screening of potential novel candidate genes in schwannomatosis patients.

Schwannomatosis comprises a group of hereditary tumor predisposition syndromes characterized by, usually benign, multiple nerve sheath tumors, which frequently cause severe pain that does not typically respond to drug treatments. The most common schwannomatosis-associated gene is NF2, but SMARCB1 and LZTR1 are also associated. There are still many cases in which no pathogenic variants (PVs) have been identified, suggesting the existence of as yet unidentified genetic risk factors. In this study, we performed extended genetic screening of 75 unrelated schwannomatosis patients without identified germline PVs in NF2, LZTR1, or SMARCB1. Screening of the coding region of DGCR8, COQ6, CDKN2A, and CDKN2B was carried out, based on previous reports that point to these genes as potential candidate genes for schwannomatosis. Deletions or duplications in CDKN2A, CDKN2B, and adjacent chromosome 9 region were assessed by multiplex ligation-dependent probe amplification analysis. Sequencing analysis of a patient with multiple schwannomas and melanomas identified a novel duplication in the coding region of CDKN2A, disrupting both p14ARF and p16INK4a. Our results suggest that none of these genes are major contributors to schwannomatosis risk but the possibility remains that they may have a role in more complex mechanisms for tumor predisposition.

Enhanced diagnostic yield in Meckel-Gruber and Joubert syndrome through exome sequencing supplemented with split-read mapping.

BACKGROUND: The widespread adoption of high-throughput sequencing technologies by genetic diagnostic laboratories has enabled significant expansion of their testing portfolios. Rare autosomal recessive conditions have been a particular focus of many new services. Here we report a cohort of 26 patients referred for genetic analysis of Joubert (JBTS) and Meckel-Gruber (MKS) syndromes, two clinically and genetically heterogeneous neurodevelopmental conditions that define a phenotypic spectrum, with MKS at the severe end. METHODS: Exome sequencing was performed for all cases, using Agilent SureSelect v5 reagents and Illumina paired-end sequencing. For two cases medium-coverage (9×) whole genome sequencing was subsequently undertaken. RESULTS: Using a standard analysis pipeline for the detection of single nucleotide and small insertion or deletion variants, molecular diagnoses were confirmed in 12 cases (4%). Seeking to determine whether our cohort harboured pathogenic copy number variants (CNV), in JBTS- or MKS-associated genes, targeted comparative read-depth analysis was performed using FishingCNV. These analyses identified a putative intragenic AHI1 deletion that included three exons spanning at least 3.4 kb and an intergenic MPP4 to TMEM237 deletion that included exons spanning at least 21.5 kb. Whole genome sequencing enabled confirmation of the deletion-containing alleles and precise characterisation of the mutation breakpoints at nucleotide resolution. These data were validated following development of PCR-based assays that could be subsequently used for "cascade" screening and/or prenatal diagnosis. CONCLUSIONS: Our investigations expand the AHI1 and TMEM237 mutation spectrum and highlight the importance of performing CNV screening of disease-associated genes. We demonstrate a robust increasingly cost-effective CNV detection workflow that is applicable to all MKS/JBTS referrals.

Extensive gene conversion at the PMS2 DNA mismatch repair locus.

Mutations of the PMS2 DNA repair gene predispose to a characteristic range of malignancies, with either childhood onset (when both alleles are mutated) or a partially penetrant adult onset (if heterozygous). These mutations have been difficult to detect, due to interference from a family of pseudogenes located on chromosome 7. One of these, the PMS2CL pseudogene, lies within a 100-kb inverted duplication (inv dup), 700 kb centromeric to PMS2 itself on 7p22. Here, we show that the reference genomic sequences cannot be relied upon to distinguish PMS2 from PMS2CL, because of sequence transfer between the two loci. The 7p22 inv dup occurred prior to the divergence of modern ape species (15 million years ago [Mya]), but has undergone extensive sequence homogenization. This process appears to be ongoing, since there is considerable allelic diversity within the duplicated region, much of it derived from sequence exchange between PMS2 and PMS2CL. This sequence diversity can result in both false-positive and false-negative mutation analysis at this locus. Great caution is still needed in the design and interpretation of PMS2 mutation screens.

The common TMC1 mutation c.100C>T (p.Arg34X) is not a significant cause of deafness in British Asians.

TMC1, a second-tier deafness gene below GJB2, is an appreciable cause of recessive nonsyndromic hearing loss (DFNB7/11) in North Africa, the Middle East, and parts of South Asia. Additionally, a single founder mutation, c.100C>T (p.Arg34X), dominates the TMC1 mutation spectrum. We investigated the frequency of TMC1 c.100C>T in a large set of British Asians with hearing loss, collectively a group with high prevalence of genetic deafness and limited routine clinical testing options beyond GJB2, on a candidate basis. An estimate of 0.21% (95% confidence interval, 0.04%-1.18%) was gained, indicating no significant enrichment in our set. Identification of the common non-GJB2 deafness genes and mutations in British Asian communities would require data from autozygosity mapping and/or massively parallel sequencing of gene panels.

Low prevalence of DFNB1 (connexin 26) mutations in British Pakistani children with non-syndromic sensorineural hearing loss.

OBJECTIVE: To determine the clinical sensitivity of DFNB1 genetic testing (analysis of the connexin 26 gene GJB2) for non-syndromic sensorineural hearing loss (SNHL) in British Pakistani children and extend to a comparison with British White children and literature data. DESIGN: Retrospective cohort study. SETTING: City of Bradford, UK. PATIENTS: Overall, 177 children (152 families) were eligible; 147 children (123 families) were British Pakistani, and 30 children (29 families) were British White. INTERVENTIONS: DFNB1 testing was offered. MAIN OUTCOME MEASURES: Detection rate for pathogenic bi-allelic GJB2 mutations. RESULTS: DFNB1 testing yielded positive results in 6.9% British Pakistani families compared with 15.4% British White families. Of 65 British Pakistani children tested (from 58 families), five children (from four families) were found to be homozygous for the common South Asian GJB2 mutation p.Trp24X. Of 14 British White children tested (from 13 families), bi-allelic pathogenic GJB2 mutations were seen in two children (from two families). CONCLUSIONS: The contribution of DFNB1 to non-syndromic SNHL in the Bradford British Pakistani children appears to be low when compared with a White peer group and White populations in general. The high prevalence of genetic deafness in this community, attributed to family structure and immigration history, points to a dilution effect in favour of other recessive deafness genes/loci.