Telomeres are nucleoprotein structures present at the ends of eukaryotic chromosomes that play a central role in guarding the integrity of the genome by protecting chromosome ends from degradation and fusion. Length regulation is central to telomere function. To broaden our knowledge about the mechanisms that control telomere length, we have carried out a systematic examination of Ϸ4,800 haploid deletion mutants of Saccharomyces cerevisiae for telomere-length alterations. By using this screen, we have identified >150 candidate genes not previously known to affect telomere length. In two-thirds of the identified mutants, short telomeres were observed; whereas in one-third, telomeres were lengthened. The genes identified are very diverse in their functions, but certain categories, including DNA and RNA metabolism, chromatin modification, and vacuolar traffic, are overrepresented. Our results greatly enlarge the number of known genes that affect telomere metabolism and will provide insights into how telomere function is linked to many other cellular processes.

ObjectiveLimb‐girdle muscular dystrophies (LGMDs), one of the most heterogeneous neuromuscular disorders (NMDs), involves predominantly proximal‐muscle weakness with >30 genes associated with different subtypes. The clinical‐genetic overlap among subtypes and with other NMDs complicate disease‐subtype identification lengthening diagnostic process, increases overall costs hindering treatment/clinical‐trial recruitment. Currently seven LGMD clinical trials are active but still no gene‐therapy‐related treatment is available. Till‐date no nation‐wide large‐scale LGMD sequencing program was performed. Our objectives were to understand LGMD genetic basis, different subtypes’ relative prevalence across US and investigate underlying disease mechanisms.MethodsA total of 4656 patients with clinically suspected‐LGMD across US were recruited to conduct next‐generation sequencing (NGS)‐based gene‐panel testing during June‐2015 to June‐2017 in CLIA‐CAP‐certified Emory‐Genetics‐Laboratory. Thirty‐five LGMD‐subtypes‐associated or LGMD‐like other NMD‐associated genes were investigated. Main outcomes were diagnostic yield, gene‐variant spectrum, and LGMD subtypes’ prevalence in a large US LGMD‐suspected population.ResultsMolecular diagnosis was established in 27% (1259 cases; 95% CI, 26–29%) of the patients with major contributing genes to LGMD phenotypes being: CAPN3(17%), DYSF(16%), FKRP(9%) and ANO5(7%). We observed an increased prevalence of genetically confirmed late‐onset Pompe disease, DNAJB6‐associated LGMD subtype1E and CAPN3‐associated autosomal‐dominant LGMDs. Interestingly, we identified a high prevalence of patients with pathogenic variants in more than one LGMD gene suggesting possible synergistic heterozygosity/digenic/multigenic contribution to disease presentation/progression that needs consideration as a part of diagnostic modality.InterpretationOverall, this study has improved our understanding of the relative prevalence of different LGMD subtypes, their respective genetic etiology, and the changing paradigm of their inheritance modes and novel mechanisms that will allow for improved timely treatment, management, and enrolment of molecularly diagnosed individuals in clinical trials.

Non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ), and microhomology-mediated replication-dependent recombination (MMRDR) have all been put forward as mechanisms to explain DNA rearrangements associated with genomic disorders. However, many nonrecurrent rearrangements in humans remain unexplained. To further investigate the mutation mechanisms of these copy number variations (CNVs), we performed breakpoint mapping analysis for 62 clinical cases with intragenic deletions in the human DMD gene (50 cases) and other known diseasecausing genes (one PCCB, one IVD, one DBT, three PAH, one STK11, one HEXB, three DBT, one HRPT1, and one EMD cases). While repetitive elements were found in only four individual cases, three involving DMD and one HEXB gene, microhomologies (2-10 bp) were observed at breakpoint junctions in 56% and insertions ranging from 1 to 48 bp were seen in 16 of the total 62 cases. Among these insertions, we observed evidence for tandem repetitions of short segments (5-20 bp) of reference sequence proximal to the breakpoints in six individual DMD cases (six repeats in one, four repeats in three, two repeats in one, and one repeat in one case), strongly indicating attempts by the replication machinery to surpass the stalled replication fork. We provide evidence of a novel template slippage event during replication rescue. With a deeper insight into the complex process of replication and its rescue during origin failure, brought forward by recent studies, we propose a hypothesis based on aberrant firing of replication origins to explain intragenic nonrecurrent rearrangements within genes, including the DMD gene.

After this review, 17 VUS (37%; 7 in ACADM, 9 in GALT, and 1 in PAH) were reclassified from uncertain (6 to benign or likely benign and 11 to pathogenic or likely pathogenic). We identified common types of missing information that would have helped make a definitive classification and categorized this information by ease and cost to obtain.Genet Med 19 1, 77-82.

The diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing. Our laboratory uses a standard methylation-sensitive PCR (MSP) to target the differentially methylated SNRPN gene to test for Prader-Willi syndrome (PWS) and Angelman syndrome. One patient, a 27-month-old female, who lacked the classical clinical features of PWS, but had a molecular diagnosis of PWS by MSP by another laboratory, had repeat testing in our laboratory. Testing by MSP in our laboratory also identified an apparent loss of the unmethylated paternal allele, consistent with a diagnosis of PWS. Confirmatory testing using Southern blot analysis with a methylation-sensitive restriction enzyme showed a normal pattern of methylation, detecting both the methylated maternal and unmethylated paternal alleles. To investigate these discrepant results, we amplified and sequenced the SNRPN locus in this patient and identified a single nucleotide change within the binding site for the unmethylated DNA-specific primer. These results indicate this nucleotide change led to allelic dropout in the MSP analysis, yielding the false-positive result. Subsequently, MSP analysis using an alternate primer set that was developed by our laboratory detected both methylated and unmethylated alleles. These findings illustrate that allelic dropout due to the presence of rare polymorphisms can cause false-positive results in commonly used MSP assays and lead to molecular misdiagnosis.