High-coverage nanopore sequencing of samples from the 1000 Genomes Project to build a comprehensive catalog of human genetic variation.

Fewer than half of individuals with a suspected Mendelian or monogenic condition receive a precise molecular diagnosis after comprehensive clinical genetic testing. Improvements in data quality and costs have heightened interest in using long-read sequencing (LRS) to streamline clinical genomic testing, but the absence of control data sets for variant filtering and prioritization has made tertiary analysis of LRS data challenging. To address this, the 1000 Genomes Project (1KGP) Oxford Nanopore Technologies Sequencing Consortium aims to generate LRS data from at least 800 of the 1KGP samples. Our goal is to use LRS to identify a broader spectrum of variation so we may improve our understanding of normal patterns of human variation. Here, we present data from analysis of the first 100 samples, representing all 5 superpopulations and 19 subpopulations. These samples, sequenced to an average depth of coverage of 37× and sequence read N50 of 54 kbp, have high concordance with previous studies for identifying single nucleotide and indel variants outside of homopolymer regions. Using multiple structural variant (SV) callers, we identify an average of 24,543 high-confidence SVs per genome, including shared and private SVs likely to disrupt gene function as well as pathogenic expansions within disease-associated repeats that were not detected using short reads. Evaluation of methylation signatures revealed expected patterns at known imprinted loci, samples with skewed X-inactivation patterns, and novel differentially methylated regions. All raw sequencing data, processed data, and summary statistics are publicly available, providing a valuable resource for the clinical genetics community to discover pathogenic SVs.

Naturally occurring splice variants dissect the functional domains of BHC80 and emphasize the need for RNA analysis.

Pathogenic PHF21A variation causes PHF21A-related neurodevelopmental disorders (NDDs). Although amorphic alleles, including haploinsufficiency, have been established as a disease mechanism, increasing evidence suggests that missense variants as well as frameshift variants extending the BHC80 carboxyl terminus also cause disease. Expanding on these, we report a proposita with intellectual disability and overgrowth and a novel de novo heterozygous PHF21A splice variant (NM_001352027.3:c.[153+1G>C];[=]) causing skipping of exon 6, which encodes an in-frame BHC80 deletion (p.(Asn30_Gln51del)). This deletion disrupts a predicted leucine zipper domain and implicates this domain in BHC80 function and as a target of variation causing PHF21A-related NDDs. This extension of understanding emphasizes the application of RNA analysis in precision genomic medicine practice.

Generation of tandem alternative splice acceptor sites and CLTC haploinsufficiency: A cause of CLTC-related disorder.

Tandem splice acceptors (NAGNn AG) are a common mechanism of alternative splicing, but variants that are likely to generate or to disrupt tandem splice sites have rarely been reported as disease causing. We identify a pathogenic intron 23 CLTC variant (NM_004859.4:c.[3766-13_3766-5del];[=]) in a propositus with intellectual disability and behavioral problems. By RNAseq analysis of peripheral blood mRNA, this variant generates transcripts using cryptic proximal splice acceptors (NM_004859.4: r.3765_3766insTTCACAGAAAGGAACTAG, and NM_004859.4:r.3765_3766insAAAGGAACTAG). Given that the propositus expresses 38% the level of CLTC transcripts as unaffected controls, these variant transcripts, which encode premature termination codons, likely undergo nonsense mediated mRNA decay (NMD). This is the first functional evidence for CLTC haploinsufficiency as a cause of CLTC-related disorder and the first evidence that the generation of tandem alternative splice sites causes CLTC-related disorder. We suggest that variants creating tandem alternative splice sites are an underreported disease mechanism and that transcriptome-level analysis should be routinely pursued to define the pathogenicity of such variants.

Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in GLS.

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.

Can tandem alternative splicing and evasion of premature termination codon surveillance contribute to attenuated Peutz-Jeghers syndrome?

Alternative use of short distance tandem sites such as NAGNn AG are a common mechanism of alternative splicing; however, single nucleotide variants are rarely reported as likely to generate or to disrupt tandem splice sites. We identify a pathogenic intron 5 STK11 variant (NM_000455.4:c.[735-6A>G];[=]) segregating with the mucocutaneous features but not the hamartomatous polyps of Peutz-Jeghers syndrome in two individuals. By RNAseq analysis of peripheral blood mRNA, this variant was shown to generate a novel and preferentially used tandem proximal splice acceptor (AAGTGAAG). The variant transcript (NM_000455.4:c.734_734 + 1insTGAAG), which encodes a frameshift (p.[Tyr246Glufs*43]) constituted 36%-43% of STK11 transcripts suggesting partial escape from nonsense mediated mRNA decay and translation of a truncated protein. A review of the ClinVar database identified other similar variants. We suggest that nucleotide changes creating or disrupting tandem alternative splice sites are a pertinent disease mechanism and require contextualization for clinical reporting. Additionally, we hypothesize that some pathogenic STK11 variants cause an attenuated phenotype.

JASPAR 2020: update of the open-access database of transcription factor binding profiles.

JASPAR (http://jaspar.genereg.net) is an open-access database of curated, non-redundant transcription factor (TF)-binding profiles stored as position frequency matrices (PFMs) for TFs across multiple species in six taxonomic groups. In this 8th release of JASPAR, the CORE collection has been expanded with 245 new PFMs (169 for vertebrates, 42 for plants, 17 for nematodes, 10 for insects, and 7 for fungi), and 156 PFMs were updated (125 for vertebrates, 28 for plants and 3 for insects). These new profiles represent an 18% expansion compared to the previous release. JASPAR 2020 comes with a novel collection of unvalidated TF-binding profiles for which our curators did not find orthogonal supporting evidence in the literature. This collection has a dedicated web form to engage the community in the curation of unvalidated TF-binding profiles. Moreover, we created a Q&A forum to ease the communication between the user community and JASPAR curators. Finally, we updated the genomic tracks, inference tool, and TF-binding profile similarity clusters. All the data is available through the JASPAR website, its associated RESTful API, and through the JASPAR2020 R/Bioconductor package.

GeneBreaker: Variant simulation to improve the diagnosis of Mendelian rare genetic diseases.

Mendelian rare genetic diseases affect 5%-10% of the population, and with over 5300 genes responsible for ∼7000 different diseases, they are challenging to diagnose. The use of whole-genome sequencing (WGS) has bolstered the diagnosis rate significantly. The effective use of WGS relies on the ability to identify the disrupted gene responsible for disease phenotypes. This process involves genomic variant calling and prioritization, and is the beneficiary of improvements to sequencing technology, variant calling approaches, and increased capacity to prioritize genomic variants with potential pathogenicity. As analysis pipelines continue to improve, careful testing of their efficacy is paramount. However, real-life cases typically emerge anecdotally, and utilization of clinically sensitive and identifiable data for testing pipeline improvements is regulated and limiting. We identified the need for a gene-based variant simulation framework that can create mock rare disease scenarios, utilizing known pathogenic variants or through the creation of novel gene-disrupting variants. To fill this need, we present GeneBreaker, a tool that creates synthetic rare disease cases with utility for benchmarking variant calling approaches, testing the efficacy of variant prioritization, and as an educational mechanism for training diagnostic practitioners in the expanding field of genomic medicine. GeneBreaker is freely available at http://GeneBreaker.cmmt.ubc.ca.

Polyploidy can drive rapid adaptation in yeast.

Polyploidy is observed across the tree of life, yet its influence on evolution remains incompletely understood. Polyploidy, usually whole-genome duplication, is proposed to alter the rate of evolutionary adaptation. This could occur through complex effects on the frequency or fitness of beneficial mutations. For example, in diverse cell types and organisms, immediately after a whole-genome duplication, newly formed polyploids missegregate chromosomes and undergo genetic instability. The instability following whole-genome duplications is thought to provide adaptive mutations in microorganisms and can promote tumorigenesis in mammalian cells. Polyploidy may also affect adaptation independently of beneficial mutations through ploidy-specific changes in cell physiology. Here we perform in vitro evolution experiments to test directly whether polyploidy can accelerate evolutionary adaptation. Compared with haploids and diploids, tetraploids undergo significantly faster adaptation. Mathematical modelling suggests that rapid adaptation of tetraploids is driven by higher rates of beneficial mutations with stronger fitness effects, which is supported by whole-genome sequencing and phenotypic analyses of evolved clones. Chromosome aneuploidy, concerted chromosome loss, and point mutations all provide large fitness gains. We identify several mutations whose beneficial effects are manifest specifically in the tetraploid strains. Together, these results provide direct quantitative evidence that in some environments polyploidy can accelerate evolutionary adaptation.

Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers.

Histone deacetylase inhibitors (HDACIs) are known to alter gene expression by both up- and down-regulation of protein-coding genes in normal and cancer cells. However, the exact regulatory mechanisms of action remain uncharacterized. Here we investigated genome wide dose-dependent epigenetic and transcriptome changes in response to HDACI largazole in a transformed and a non-transformed cell line. Exposure to low nanomolar largazole concentrations (

A trans-acting Variant within the Transcription Factor RIM101 Interacts with Genetic Background to Determine its Regulatory Capacity.

Most genetic variants associated with disease occur within regulatory regions of the genome, underscoring the importance of defining the mechanisms underlying differences in regulation of gene expression between individuals. We discovered a pair of co-regulated, divergently oriented transcripts, AQY2 and ncFRE6, that are expressed in one strain of Saccharomyces cerevisiae, ∑1278b, but not in another, S288c. By combining classical genetics techniques with high-throughput sequencing, we identified a trans-acting single nucleotide polymorphism within the transcription factor RIM101 that causes the background-dependent expression of both transcripts. Subsequent RNA-seq experiments revealed that RIM101 regulates many more targets in S288c than in ∑1278b and that deletion of RIM101 in both backgrounds abrogates the majority of differential expression between the strains. Strikingly, only three transcripts undergo a significant change in expression after swapping RIM101 alleles between backgrounds, implying that the differences in the RIM101 allele lead to a remarkably focused transcriptional response. However, hundreds of RIM101-dependent targets undergo a subtle but consistent shift in expression in the S288c RIM101-swapped strain, but not its ∑1278b counterpart. We conclude that ∑1278b may harbor a variant(s) that buffers against widespread transcriptional dysregulation upon introduction of a non-native RIM101 allele, emphasizing the importance of accounting for genetic background when assessing the impact of a regulatory variant.

The Influence of Polyploidy on the Evolution of Yeast Grown in a Sub-Optimal Carbon Source.

Polyploidization events have occurred during the evolution of many fungi, plant, and animal species and are thought to contribute to speciation and tumorigenesis, however little is known about how ploidy level contributes to adaptation at the molecular level. Here we integrate whole genome sequencing, RNA expression analysis, and relative fitness of ∼100 evolved clones at three ploidy levels. Independent haploid, diploid, and tetraploid populations were grown in a low carbon environment for 250 generations. We demonstrate that the key adaptive mutation in the evolved clones is predicted by a gene expression signature of just five genes. All of the adaptive mutations identified encompass a narrow set of genes, however the tetraploid clones gain a broader spectrum of adaptive mutations than haploid or diploid clones. While many of the adaptive mutations occur in genes that encode proteins with known roles in glucose sensing and transport, we discover mutations in genes with no canonical role in carbon utilization (IPT1 and MOT3), as well as identify novel dominant mutations in glucose signal transducers thought to only accumulate recessive mutations in carbon limited environments (MTH1 and RGT1). We conclude that polyploid cells explore more genotypic and phenotypic space than lower ploidy cells. Our study provides strong evidence for the beneficial role of polyploidization events that occur during the evolution of many species and during tumorigenesis.

Evidence for Association Between Low Frequency Variants in CHRNA6/CHRNB3 and Antisocial Drug Dependence.

Common SNPs in nicotinic acetylcholine receptor genes (CHRN genes) have been associated with drug behaviors and personality traits, but the influence of rare genetic variants is not well characterized. The goal of this project was to identify novel rare variants in CHRN genes in the Center for Antisocial Drug Dependence (CADD) and Genetics of Antisocial Drug Dependence (GADD) samples and to determine if low frequency variants are associated with antisocial drug dependence. Two samples of 114 and 200 individuals were selected using a case/control design including the tails of the phenotypic distribution of antisocial drug dependence. The capture, sequencing, and analysis of all variants in 16 CHRN genes (CHRNA1-7, 9, 10, CHRNB1-4, CHRND, CHRNG, CHRNE) were performed independently for each subject in each sample. Sequencing reads were aligned to the human reference sequence using BWA prior to variant calling with the Genome Analysis ToolKit (GATK). Low frequency variants (minor allele frequency < 0.05) were analyzed using SKAT-O and C-alpha to examine the distribution of rare variants among cases and controls. In our larger sample, the region containing the CHRNA6/CHRNB3 gene cluster was significantly associated with disease status using both SKAT-O and C-alpha (unadjusted p values <0.05). More low frequency variants in the CHRNA6/CHRNB3 gene region were observed in cases compared to controls. These data support a role for genetic variants in CHRN genes and antisocial drug behaviors.

Prolonged Cre expression driven by the α-myosin heavy chain promoter can be cardiotoxic.

Studying the importance of genetic factors in a desired cell type or tissue necessitates the use of precise genetic tools. With the introduction of bacteriophage Cre recombinase/loxP mediated DNA editing and promoter-specific Cre expression, it is feasible to generate conditional knockout mice in which particular genes are disrupted in a cell type-specific manner in vivo. In cardiac myocytes, this is often achieved through α-myosin heavy chain promoter (αMyHC)-driven Cre expression in conjunction with a loxP-site flanked gene of interest. Recent studies in other cell types demonstrate toxicity of Cre expression through induction of DNA damage. However, it is unclear to what extent the traditionally used αMyHC-Cre line [1] may exhibit cardiotoxicity. Further, the genotype of αMyHC-Cre(+/-) is not often included as a control group in cardiac myocyte-specific knockout studies. Here we present evidence that these αMyHC-Cre(+/-) mice show molecular signs of cardiac toxicity by 3months of age and exhibit decreased cardiac function by 6months of age compared to wild-type littermates. Hearts from αMyHC-Cre(+/-) mice also display evidence of fibrosis, inflammation, and DNA damage. Interestingly, some of the early functional changes observed in αMyHC-Cre(+/-) mice are sexually dimorphic. Given the high level of Cre recombinase expression resulting from expression from the αMyHC promoter, we asked if degenerate loxP-like sites naturally exist in the mouse genome and if so, whether they are affected by Cre in the absence of canonical loxP-sites. Using a novel bioinformatics search tool, we identified 619 loxP-like sites with 4 or less mismatches to the canonical loxP-site. 227 sites overlapped with annotated genes and 55 of these genes were expressed in cardiac muscle. Expression of ~26% of the 27 genes tested was disrupted in αMyHC-Cre(+/-) mice indicating potential targeting by Cre. Taken together, these results highlight both the importance of using αMyHC-Cre mice as controls in conditional knockout studies as well as the need for a less cardiotoxic Cre driver for the field.

Quantitative trait locus mapping of acute functional tolerance in the LXS recombinant inbred strains.

BACKGROUND: We previously reported that acute functional tolerance (AFT) to the hypnotic effects of alcohol was significantly correlated with drinking in the dark (DID) in the LXS recombinant inbred panel, but only in mice that had been pretreated with alcohol. Here, we have conducted quantitative trait locus (QTL) mapping for AFT. DNA sequencing of the progenitor ILS and ISS strains and microarray analyses were also conducted to identify candidate genes and functional correlates. METHODS: LXS mice were given either saline or alcohol (5 g/kg) on day 1 and then tested for loss of righting reflex AFT on day 2. QTLs were mapped using standard procedures. Two microarray analyses from brain were conducted: (i) naïve LXS mice and (ii) an alcohol treatment time course in the ILS and ISS. The full genomes of the ILS and ISS were sequenced to a depth of approximately 30×. RESULTS: A significant QTL for AFT in the alcohol pretreatment group was mapped to distal chromosome 4; numerous suggestive QTLs were also mapped. Preference drinking and DID have previously been mapped to the chromosome 4 locus. The credible interval of the significant chromosome 4 QTL spanned 23 Mb and included 716 annotated genes of which 150 had at least 1 nonsynonymous single nucleotide polymorphism or small indel that differed between the ILS and ISS; expression of 48 of the genes was cis-regulated. Enrichment analysis indicated broad functional categories underlying AFT, including proteolysis, transcription regulation, chromatin modification, protein kinase activity, and apoptosis. CONCLUSIONS: The chromosome 4 QTL is a key region containing possibly pleiotropic genes for AFT and drinking behavior. Given that the region contains many viable candidates and a large number of the genes in the interval fall into 1 or more of the enriched functional categories, we postulate that many genes of varying effect size contribute to the observed QTL effect.

Nanopore sequencing of 1000 Genomes Project samples to build a comprehensive catalog of human genetic variation.

Less than half of individuals with a suspected Mendelian condition receive a precise molecular diagnosis after comprehensive clinical genetic testing. Improvements in data quality and costs have heightened interest in using long-read sequencing (LRS) to streamline clinical genomic testing, but the absence of control datasets for variant filtering and prioritization has made tertiary analysis of LRS data challenging. To address this, the 1000 Genomes Project ONT Sequencing Consortium aims to generate LRS data from at least 800 of the 1000 Genomes Project samples. Our goal is to use LRS to identify a broader spectrum of variation so we may improve our understanding of normal patterns of human variation. Here, we present data from analysis of the first 100 samples, representing all 5 superpopulations and 19 subpopulations. These samples, sequenced to an average depth of coverage of 37x and sequence read N50 of 54 kbp, have high concordance with previous studies for identifying single nucleotide and indel variants outside of homopolymer regions. Using multiple structural variant (SV) callers, we identify an average of 24,543 high-confidence SVs per genome, including shared and private SVs likely to disrupt gene function as well as pathogenic expansions within disease-associated repeats that were not detected using short reads. Evaluation of methylation signatures revealed expected patterns at known imprinted loci, samples with skewed X-inactivation patterns, and novel differentially methylated regions. All raw sequencing data, processed data, and summary statistics are publicly available, providing a valuable resource for the clinical genetics community to discover pathogenic SVs.

Human germline heterozygous gain-of-function STAT6 variants cause severe allergic disease.

STAT6 (signal transducer and activator of transcription 6) is a transcription factor that plays a central role in the pathophysiology of allergic inflammation. We have identified 16 patients from 10 families spanning three continents with a profound phenotype of early-life onset allergic immune dysregulation, widespread treatment-resistant atopic dermatitis, hypereosinophilia with esosinophilic gastrointestinal disease, asthma, elevated serum IgE, IgE-mediated food allergies, and anaphylaxis. The cases were either sporadic (seven kindreds) or followed an autosomal dominant inheritance pattern (three kindreds). All patients carried monoallelic rare variants in STAT6 and functional studies established their gain-of-function (GOF) phenotype with sustained STAT6 phosphorylation, increased STAT6 target gene expression, and TH2 skewing. Precision treatment with the anti-IL-4Rα antibody, dupilumab, was highly effective improving both clinical manifestations and immunological biomarkers. This study identifies heterozygous GOF variants in STAT6 as a novel autosomal dominant allergic disorder. We anticipate that our discovery of multiple kindreds with germline STAT6 GOF variants will facilitate the recognition of more affected individuals and the full definition of this new primary atopic disorder.

REViewer: haplotype-resolved visualization of read alignments in and around tandem repeats.

BACKGROUND: Expansions of short tandem repeats are the cause of many neurogenetic disorders including familial amyotrophic lateral sclerosis, Huntington disease, and many others. Multiple methods have been recently developed that can identify repeat expansions in whole genome or exome sequencing data. Despite the widely recognized need for visual assessment of variant calls in clinical settings, current computational tools lack the ability to produce such visualizations for repeat expansions. Expanded repeats are difficult to visualize because they correspond to large insertions relative to the reference genome and involve many misaligning and ambiguously aligning reads. RESULTS: We implemented REViewer, a computational method for visualization of sequencing data in genomic regions containing long repeat expansions and FlipBook, a companion image viewer designed for manual curation of large collections of REViewer images. To generate a read pileup, REViewer reconstructs local haplotype sequences and distributes reads to these haplotypes in a way that is most consistent with the fragment lengths and evenness of read coverage. To create appropriate training materials for onboarding new users, we performed a concordance study involving 12 scientists involved in short tandem repeat research. We used the results of this study to create a user guide that describes the basic principles of using REViewer as well as a guide to the typical features of read pileups that correspond to low confidence repeat genotype calls. Additionally, we demonstrated that REViewer can be used to annotate clinically relevant repeat interruptions by comparing visual assessment results of 44 FMR1 repeat alleles with the results of triplet repeat primed PCR. For 38 of these alleles, the results of visual assessment were consistent with triplet repeat primed PCR. CONCLUSIONS: Read pileup plots generated by REViewer offer an intuitive way to visualize sequencing data in regions containing long repeat expansions. Laboratories can use REViewer and FlipBook to assess the quality of repeat genotype calls as well as to visually detect interruptions or other imperfections in the repeat sequence and the surrounding flanking regions. REViewer and FlipBook are available under open-source licenses at https://github.com/illumina/REViewer and https://github.com/broadinstitute/flipbook respectively.

Exome sequencing enables diagnosis of X-linked hypohidrotic ectodermal dysplasia in patient with eosinophilic esophagitis and severe atopy.

X-linked hypohidrotic ectodermal dysplasia (XLHED) is the most common form of ectodermal dysplasia. Clinical and genetic heterogeneity between different ectodermal dysplasia types and evidence of incomplete penetrance and variable expressivity increase the potential for misdiagnosis. We describe a family with X-linked hypohidrotic ectodermal dysplasia (XLHED) presenting with variable expressivity of symptoms between affected siblings. In addition to the classical signs of hypohidrosis, hypotrichosis and hypodontia, the index patient-a 5 year old boy, also presented with a severe atopy phenotype that was not observed in the other two affected brothers. Exome sequencing in the index and the mother identified a pathogenic nonsense variant in EDA (NM_001399.4: c.766 C>T; p. Gln256Ter). This study highlights how exome sequencing was crucial in establishing a precise molecular diagnosis of XLHED by enabling us to rule out other differential diagnoses including NEMO deficiency syndrome, that was initially presented as a clinical diagnosis to the family.

Adult GAMT deficiency: A literature review and report of two siblings.

Guanidinoacetate methyltransferase (GAMT) deficiency is a creatine deficiency disorder and an inborn error of metabolism presenting with progressive intellectual and neurological deterioration. As most cases are identified and treated in early childhood, adult phenotypes that can help in understanding the natural history of the disorder are rare. We describe two adult cases of GAMT deficiency from a consanguineous family in Pakistan that presented with a history of global developmental delay, cognitive impairments, excessive drooling, behavioral abnormalities, contractures and apparent bone deformities initially presumed to be the reason for abnormal gait. Exome sequencing identified a homozygous nonsense variant in GAMT: NM_000156.5:c.134G>A (p.Trp45*). We also performed a literature review and compiled the genetic and clinical characteristics of all adult cases of GAMT deficiency reported to date. When compared to the adult cases previously reported, the musculoskeletal phenotype and the rapidly progressive nature of neurological and motor decline seen in our patients is striking. This study presents an opportunity to gain insights into the adult presentation of GAMT deficiency and highlights the need for in-depth evaluation and reporting of clinical features to expand our understanding of the phenotypic spectrum.