Development and analytical validation of a 25-gene next generation sequencing panel that includes the BRCA1 and BRCA2 genes to assess hereditary cancer risk.

BACKGROUND: Germline DNA mutations that increase the susceptibility of a patient to certain cancers have been identified in various genes, and patients can be screened for mutations in these genes to assess their level of risk for developing cancer. Traditional methods using Sanger sequencing focus on small groups of genes and therefore are unable to screen for numerous genes from several patients simultaneously. The goal of the present study was to validate a 25-gene panel to assess genetic risk for cancer in 8 different tissues using next generation sequencing (NGS) techniques. METHODS: Twenty-five genes associated with hereditary cancer syndromes were selected for development of a panel to screen for risk of these cancers using NGS. In an initial technical assessment, NGS results for BRCA1 and BRCA2 were compared with Sanger sequencing in 1864 anonymized DNA samples from patients who had undergone previous clinical testing. Next, the entire gene panel was validated using parallel NGS and Sanger sequencing in 100 anonymized DNA samples. Large rearrangement analysis was validated using NGS, microarray comparative genomic hybridization (CGH), and multiplex ligation-dependent probe amplification analyses (MLPA). RESULTS: NGS identified 15,877 sequence variants, while Sanger sequencing identified 15,878 in the BRCA1 and BRCA2 comparison study of the same regions. Based on these results, the NGS process was refined prior to the validation of the full gene panel. In the validation study, NGS and Sanger sequencing were 100% concordant for the 3,923 collective variants across all genes for an analytical sensitivity of the NGS assay of >99.92% (lower limit of 95% confidence interval). NGS, microarray CGH and MLPA correctly identified all expected positive and negative large rearrangement results for the 25-gene panel. CONCLUSION: This study provides a thorough validation of the 25-gene NGS panel and indicates that this analysis tool can be used to collect clinically significant information related to risk of developing hereditary cancers.

The genome of the domesticated apple (Malus × domestica Borkh.).

We report a high-quality draft genome sequence of the domesticated apple (Malus × domestica). We show that a relatively recent (>50 million years ago) genome-wide duplication (GWD) has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae. Traces of older GWDs partly support the monophyly of the ancestral paleohexaploidy of eudicots. Phylogenetic reconstruction of Pyreae and the genus Malus, relative to major Rosaceae taxa, identified the progenitor of the cultivated apple as M. sieversii. Expansion of gene families reported to be involved in fruit development may explain formation of the pome, a Pyreae-specific false fruit that develops by proliferation of the basal part of the sepals, the receptacle. In apple, a subclade of MADS-box genes, normally involved in flower and fruit development, is expanded to include 15 members, as are other gene families involved in Rosaceae-specific metabolism, such as transport and assimilation of sorbitol.

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety.

BACKGROUND: Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented. PRINCIPAL FINDINGS: We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before). CONCLUSIONS: Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.

Residues in the first transmembrane domain of the Caenorhabditis elegans GABA(A) receptor confer sensitivity to the neurosteroid pregnenolone sulfate.

The GABA(A) receptor is a target of endogenous and synthetic neurosteroids. Little is known about the residues required for neurosteroid action on GABA(A) receptors. We have investigated pregnenolone sulfate (PS) inhibition of the Caenorhabditis elegans UNC-49 GABA receptor, a close homolog of the mammalian GABA(A) receptor. The UNC-49 locus encodes two GABA receptor subunits, UNC-49B and UNC-49C. UNC-49C is sensitive to PS but UNC-49B is not sensitive. By analyzing chimeric receptors and receptors containing site-directed mutations, we identified two regions required for PS inhibition. Four residues in the first transmembrane domain are required for the majority of the sensitivity to PS, but a charged extracellular residue at the end of the M2 helix also plays a role. Strikingly, mutation of one additional M1 residue reverses the effect of PS from an inhibitor to an enhancer of receptor function. Mutating the M1 domain had little effect on sensitivity to the inhibitor picrotoxin, suggesting that these residues may mediate neurosteroid action specifically, and not allosteric regulation in general.