Prioritizing Variants in Complete Hereditary Breast and Ovarian Cancer (HBOC) Genes in Patients Lacking known BRCA Mutations.

BRCA1 and BRCA2 testing for Hereditary breast and ovarian cancer (HBOC) does not identify all pathogenic variants. Sequencing of 20 complete genes in HBOC patients with uninformative test results (N = 287), including non-coding and flanking sequences of ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD51B, STK11, TP53, and XRCC2, identified 38,372 unique variants. We apply information theory (IT) to predict and prioritize non-coding variants of uncertain significance (VUS) in regulatory, coding, and intronic regions based on changes in binding sites in these genes. Besides mRNA splicing, IT provides a common framework to evaluate potential affinity changes in transcription factor (TFBSs), splicing regulatory (SRBSs), and RNA-binding protein (RBBSs) binding sites following mutation. We prioritized variants affecting the strengths of 10 splice sites (4 natural, 6 cryptic), 148 SRBS, 36 TFBS, and 31 RBBS. Three variants were also prioritized based on their predicted effects on mRNA secondary (2°) structure, and 17 for pseudoexon activation. Additionally, 4 frameshift, 2 in-frame deletions, and 5 stop-gain mutations were identified. When combined with pedigree information, complete gene sequence analysis can focus attention on a limited set of variants in a wide spectrum of functional mutation types for downstream functional and co-segregation analysis. This article is protected by copyright. All rights reserved

A Unified Analytic Framework for Prioritization of Non-Coding Variants of Uncertain Significance in Heritable Breast and Ovarian Cancer

Background Sequencing of both healthy and disease singletons yields many novel and low frequency variants of uncertain significance (VUS). Complete gene and genome sequencing by next generation sequencing (NGS) significantly increases the number of VUS detected. While prior studies have emphasized protein coding variants, non-coding sequence variants have also been proven to significantly contribute to high penetrance disorders, such as hereditary breast and ovarian cancer (HBOC). We present a strategy for analyzing different functional classes of non-coding variants based on information theory (IT) and prioritizing patients with large intragenic deletions. Methods We captured and enriched for coding and non-coding variants in genes known to harbor mutations that increase HBOC risk. Custom oligonucleotide baits spanning the complete coding, non-coding, and intergenic regions 10 kb up- and downstream of ATM, BRCA1, BRCA2, CDH1, CHEK2, PALB2, and TP53 were synthesized for solution hybridization enrichment. Unique and divergent repetitive sequences were sequenced in 102 high-risk, anonymized patients without identified mutations in BRCA1/2. Aside from protein coding and copy number changes, IT-based sequence analysis was used to identify and prioritize pathogenic non-coding variants that occurred within sequence elements predicted to be recognized by proteins or protein complexes involved in mRNA splicing, transcription, and untranslated region (UTR) binding and structure. This approach was supplemented by in silico and laboratory analysis of UTR structure. Results 15,311 unique variants were identified, of which 245 occurred in coding regions. With the unified IT-framework, 132 variants were identified and 87 functionally significant VUS were further prioritized. An intragenic 32.1 kb interval in BRCA2 that was likely hemizygous was detected in one patient. We also identified 4 stop-gain variants and 3 reading-frame altering exonic insertions/deletions (indels). Conclusions We have presented a strategy for complete gene sequence analysis followed by a unified framework for interpreting non-coding variants that may affect gene expression. This approach distills large numbers of variants detected by NGS to a limited set of variants prioritized as potential deleterious changes

The expanding large-spored Metschnikowia clade: Metschnikowia matae sp. nov., a yeast species with two varieties from the Brazilian Atlantic Forest

Fifty-two yeast isolates from flowers and associated nitidulid beetles of the Brazilian Atlantic Forest (Mata Atlântica) region were found to represent a new species in the large-spored Metschnikowia clade. The species is heterothallic, haploid, and allogamous, and produces asci with two aciculate ascospores that can reach 80 μm in length, as is typical in the clade. Analysis of sequences of the ribosomal RNA gene cluster indicates that the new species is closely related to Metschnikowia lochheadii, which ranges across Central America to northern Brazil, occurs as an adventive species in Hawaii, but is rarely found in central Brazil. The species is not readily distinguishable from relatives based on morphology or growth responses, but is well delineated from M. lochheadii on reproductive isolation. Based on an intron splice site PCR screen, we selected 26 isolates for further study. The sequence of the region that includes the complete internal transcribed spacer/5.8S rRNA gene segment as well as the D1/D2 domains of the large subunit rRNA gene contained three polymorphic segments and 14 haplotypes were identified. Of these, a single divergent isolate from the southernmost of four sampled localities exhibited diminished mating success when crossed with others. We describe two varieties, Metschnikowia matae var. matae sp. nov. var. nov. (type UFMG-CM-Y395T, CBS 13986T, NRRL Y-63736T; allotype UFMG-CM-Y391A, CBS 13987A, NRRL Y-63735A) and Metschnikowia matae var. maris sp. nov. var. nov. (type UFMG-CM-Y397T, CBS 13985T, NRRL Y-63737T). We also report on the discovery of the h + mating type of Candida ipomoeae and transfer of the species to Metschnikowia ipomoeae comb. nov. (allotype UWOPS 12-660.1A, CBS 13988A, NRRL Y-63738A)108753763CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE MINAS GERAIS - FAPEMIG141563/2011-7RDP-00094-1

Abnormal glycogen chain length pattern, not hyperphosphorylation, is critical in Lafora disease

12 p.-7 fig. Nitschke, Felix et al.Lafora disease (LD) is a fatal progressive epilepsy essentially caused by loss-of-function mutations in the glycogen phosphatase laforin or the ubiquitin E3 ligase malin. Glycogen in LD is hyperphosphorylated and poorly hydrosoluble. It precipitates and accumulates into neurotoxic Lafora bodies (LBs). The leading LD hypothesis that hyperphosphorylation causes the insolubility was recently challenged by the observation that phosphatase-inactive laforin rescues the laforin-deficient LD mouse model, apparently through correction of a general autophagy impairment. We were for the first time able to quantify brain glycogen phosphate. We also measured glycogen content and chain lengths, LBs, and autophagy markers in several laforin- or malin-deficient mouse lines expressing phosphatase-inactive laforin. We find that: (i) in laforindeficient mice, phosphatase-inactive laforin corrects glycogen chain lengths, and not hyperphosphorylation, which leads to correction of glycogen amounts and prevention of LBs; (ii) in malin-deficient mice, phosphatase-inactive laforin confers no correction; (iii) general impairment of autophagy is not necessary in LD. We conclude that laforin’s principle function is to control glycogen chain lengths, in a malin-dependent fashion, and that loss of this control underlies LD.This work was supported by families and friends of the Chelsea’s Hope Lafora Disease Research Fund, Associazione Italiana Lafora (AILA), France-Lafora, the Milana and Tatjana Gajic Lafora Disease Foundation, Genome Canada, the Ontario Brain Institute (OBI) and The National Institute of Neurological Disorders and Stroke of the National Institutes of Health (NIH) under award number P01 NS097197. Mitchell A. Sullivan was supported by an NHMRC CJ Martin Fellowship (GNT1092451).Peer reviewe

Skeletal Muscle Glycogen Chain Length Correlates with Insolubility in Mouse Models of Polyglucosan-Associated Neurodegenerative Diseases

Lafora disease (LD)and adult polyglucosan body disease (APBD)are glycogen storage diseases characterized by a pathogenic buildup of insoluble glycogen. Mechanisms causing glycogen insolubility are poorly understood. Here, in two mouse models of LD (Epm2a and Epm2b )and one of APBD (Gbe1 ), the separation of soluble and insoluble muscle glycogen is described, enabling separate analysis of each fraction. Total glycogen is increased in LD and APBD mice, which, together with abnormal chain length and molecule size distributions, is largely if not fully attributed to insoluble glycogen. Soluble glycogen consists of molecules with distinct chain length distributions and differential corresponding solubility, providing a mechanistic link between soluble and insoluble glycogen in vivo. Phosphorylation states differ across glycogen fractions and mouse models, demonstrating that hyperphosphorylation is not a basic feature of insoluble glycogen. Lastly, model-specific variances in protein and activity levels of key glycogen synthesis enzymes suggest uninvestigated regulatory mechanisms. EPM2A, EPM2B, or GBE1 deficiency causes insoluble glycogen accumulation and neurodegenerative diseases. Sullivan et al. show that these defects do not impair the construction of WT-like soluble glycogen. Demonstrating varying chain length distributions and correlating precipitation propensity among WT-glycogen molecules, a mechanistic explanation emerges for the structural characteristics of insoluble glycogen

GYS1 or PPP1R3C deficiency rescues murine adult polyglucosan body disease

Objective: Adult polyglucosan body disease (APBD) is an adult-onset neurological variant of glycogen storage disease type IV. APBD is caused by recessive mutations in the glycogen branching enzyme gene, and the consequent accumulation of poorly branched glycogen aggregates called polyglucosan bodies in the nervous system. There are presently no treatments for APBD. Here, we test whether downregulation of glycogen synthesis is therapeutic in a mouse model of the disease. Methods: We characterized the effects of knocking out two pro-glycogenic proteins in an APBD mouse model. APBD mice were crossed with mice deficient in glycogen synthase (GYS1), or mice deficient in protein phosphatase 1 regulatory subunit 3C (PPP1R3C), a protein involved in the activation of GYS1. Phenotypic and histological parameters were analyzed and glycogen was quantified. Results: APBD mice deficient in GYS1 or PPP1R3C demonstrated improvements in life span, morphology, and behavioral assays of neuromuscular function. Histological analysis revealed a reduction in polyglucosan body accumulation and of astro- and micro-gliosis in the brains of GYS1- and PPP1R3C-deficient APBD mice. Brain glycogen quantification confirmed the reduction in abnormal glycogen accumulation. Analysis of skeletal muscle, heart, and liver found that GYS1 deficiency reduced polyglucosan body accumulation in all three tissues and PPP1R3C knockout reduced skeletal muscle polyglucosan bodies. Interpretation: GYS1 and PPP1R3C are effective therapeutic targets in the APBD mouse model. These findings represent a critical step toward the development of a treatment for APBD and potentially other glycogen storage disease type IV patients