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PURPOSE: To identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). METHODS: We coupled phenotyping with exome or genome sequencing of 467 probands (550 affected and 1108 total individuals) with genetically unsolved oCCDDs, integrating analyses of pedigrees, human and animal model phenotypes, and de novo variants to identify rare candidate single nucleotide variants, insertion/deletions, and structural variants disrupting protein-coding regions. Prioritized variants were classified for pathogenicity and evaluated for genotype/phenotype correlations. RESULTS: Analyses elucidated phenotypic subgroups, identified pathogenic/likely pathogenic variant(s) in 43/467 probands (9.2%), and prioritized variants of uncertain significance in 70/467 additional probands (15.0%). These included known and novel variants in established oCCDD genes, genes associated with syndromes that sometimes include oCCDDs (e.g., MYH10, KIF21B, TGFBR2, TUBB6), genes that fit the syndromic component of the phenotype but had no prior oCCDD association (e.g., CDK13, TGFB2), genes with no reported association with oCCDDs or the syndromic phenotypes (e.g., TUBA4A, KIF5C, CTNNA1, KLB, FGF21), and genes associated with oCCDD phenocopies that had resulted in misdiagnoses. CONCLUSION: This study suggests that unsolved oCCDDs are clinically and genetically heterogeneous disorders often overlapping other Mendelian conditions and nominates many candidates for future replication and functional studies.
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Penetrance is the probability that an individual with a pathogenic genetic variant develops a specific disease. Knowing the penetrance of variants for monogenic disorders is important for counseling of individuals. Until recently, estimates of penetrance have largely relied on affected individuals and their at-risk family members being clinically referred for genetic testing, a 'phenotype-first' approach. This approach substantially overestimates the penetrance of variants because of ascertainment bias. The recent availability of whole-genome sequencing data in individuals from very-large-scale population-based cohorts now allows 'genotype-first' estimates of penetrance for many conditions. Although this type of population-based study can underestimate penetrance owing to recruitment biases, it provides more accurate estimates of penetrance for secondary or incidental findings. Here, we provide guidance for the conduct of penetrance studies to ensure that robust genotypes and phenotypes are used to accurately estimate penetrance of variants and groups of similarly annotated variants from population-based studies.
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Incomplete penetrance, or absence of disease phenotype in an individual with a disease-associated variant, is a major challenge in variant interpretation. Studying individuals with apparent incomplete penetrance can shed light on underlying drivers of altered phenotype penetrance. Here, we investigate clinically relevant variants from ClinVar in 807,162 individuals from the Genome Aggregation Database (gnomAD), demonstrating improved representation in gnomAD version 4. We then conduct a comprehensive case-by-case assessment of 734 predicted loss of function variants (pLoF) in 77 genes associated with severe, early-onset, highly penetrant haploinsufficient disease. We identified explanations for the presumed lack of disease manifestation in 701 of the variants (95%). Individuals with unexplained lack of disease manifestation in this set of disorders rarely occur, underscoring the need and power of deep case-by-case assessment presented here to minimize false assignments of disease risk, particularly in unaffected individuals with higher rates of secondary properties that result in rescue.
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BACKGROUND: Genetic variants that cause rare disorders may remain elusive even after expansive testing, such as exome sequencing. The diagnostic yield of genome sequencing, particularly after a negative evaluation, remains poorly defined. METHODS: We sequenced and analyzed the genomes of families with diverse phenotypes who were suspected to have a rare monogenic disease and for whom genetic testing had not revealed a diagnosis, as well as the genomes of a replication cohort at an independent clinical center. RESULTS: We sequenced the genomes of 822 families (744 in the initial cohort and 78 in the replication cohort) and made a molecular diagnosis in 218 of 744 families (29.3%). Of the 218 families, 61 (28.0%) - 8.2% of families in the initial cohort - had variants that required genome sequencing for identification, including coding variants, intronic variants, small structural variants, copy-neutral inversions, complex rearrangements, and tandem repeat expansions. Most families in which a molecular diagnosis was made after previous nondiagnostic exome sequencing (63.5%) had variants that could be detected by reanalysis of the exome-sequence data (53.4%) or by additional analytic methods, such as copy-number variant calling, to exome-sequence data (10.8%). We obtained similar results in the replication cohort: in 33% of the families in which a molecular diagnosis was made, or 8% of the cohort, genome sequencing was required, which showed the applicability of these findings to both research and clinical environments. CONCLUSIONS: The diagnostic yield of genome sequencing in a large, diverse research cohort and in a small clinical cohort of persons who had previously undergone genetic testing was approximately 8% and included several types of pathogenic variation that had not previously been detected by means of exome sequencing or other techniques. (Funded by the National Human Genome Research Institute and others.).
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Variación Genética , Enfermedades Raras , Secuenciación Completa del Genoma , Femenino , Humanos , Masculino , Estudios de Cohortes , Exoma , Secuenciación del Exoma , Enfermedades Genéticas Congénitas/diagnóstico , Enfermedades Genéticas Congénitas/etnología , Enfermedades Genéticas Congénitas/genética , Pruebas Genéticas , Genoma Humano , Fenotipo , Enfermedades Raras/diagnóstico , Enfermedades Raras/etnología , Enfermedades Raras/genética , Análisis de Secuencia de ADN , Niño , Adolescente , Adulto Joven , AdultoRESUMEN
Human pluripotent stem (hPS) cells can, in theory, be differentiated into any cell type, making them a powerful in vitro model for human biology. Recent technological advances have facilitated large-scale hPS cell studies that allow investigation of the genetic regulation of molecular phenotypes and their contribution to high-order phenotypes such as human disease. Integrating hPS cells with single-cell sequencing makes identifying context-dependent genetic effects during cell development or upon experimental manipulation possible. Here we discuss how the intersection of stem cell biology, population genetics and cellular genomics can help resolve the functional consequences of human genetic variation. We examine the critical challenges of integrating these fields and approaches to scaling them cost-effectively and practically. We highlight two areas of human biology that can particularly benefit from population-scale hPS cell studies, elucidating mechanisms underlying complex disease risk loci and evaluating relationships between common genetic variation and pharmacotherapeutic phenotypes.
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Genética de Población , Genómica , Humanos , Enfermedad/genética , Variación Genética , Genómica/métodos , Fenotipo , Células Madre Pluripotentes , Análisis de la Célula Individual/métodosRESUMEN
Understanding the genetic basis of gene expression can help us understand the molecular underpinnings of human traits and disease. Expression quantitative trait locus (eQTL) mapping can help in studying this relationship but have been shown to be very cell-type specific, motivating the use of single-cell RNA sequencing and single-cell eQTLs to obtain a more granular view of genetic regulation. Current methods for single-cell eQTL mapping either rely on the "pseudobulk" approach and traditional pipelines for bulk transcriptomics or do not scale well to large datasets. Here, we propose SAIGE-QTL, a robust and scalable tool that can directly map eQTLs using single-cell profiles without needing aggregation at the pseudobulk level. Additionally, SAIGE-QTL allows for testing the effects of less frequent/rare genetic variation through set-based tests, which is traditionally excluded from eQTL mapping studies. We evaluate the performance of SAIGE-QTL on both real and simulated data and demonstrate the improved power for eQTL mapping over existing pipelines.
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Purpose: To identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). Methods: We coupled phenotyping with exome or genome sequencing of 467 pedigrees with genetically unsolved oCCDDs, integrating analyses of pedigrees, human and animal model phenotypes, and de novo variants to identify rare candidate single nucleotide variants, insertion/deletions, and structural variants disrupting protein-coding regions. Prioritized variants were classified for pathogenicity and evaluated for genotype/phenotype correlations. Results: Analyses elucidated phenotypic subgroups, identified pathogenic/likely pathogenic variant(s) in 43/467 probands (9.2%), and prioritized variants of uncertain significance in 70/467 additional probands (15.0%). These included known and novel variants in established oCCDD genes, genes associated with syndromes that sometimes include oCCDDs (e.g., MYH10, KIF21B, TGFBR2, TUBB6), genes that fit the syndromic component of the phenotype but had no prior oCCDD association (e.g., CDK13, TGFB2), genes with no reported association with oCCDDs or the syndromic phenotypes (e.g., TUBA4A, KIF5C, CTNNA1, KLB, FGF21), and genes associated with oCCDD phenocopies that had resulted in misdiagnoses. Conclusion: This study suggests that unsolved oCCDDs are clinically and genetically heterogeneous disorders often overlapping other Mendelian conditions and nominates many candidates for future replication and functional studies.
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Missense variants can have a range of functional impacts depending on factors such as the specific amino acid substitution and location within the gene. To interpret their deleteriousness, studies have sought to identify regions within genes that are specifically intolerant of missense variation 1-12 . Here, we leverage the patterns of rare missense variation in 125,748 individuals in the Genome Aggregation Database (gnomAD) 13 against a null mutational model to identify transcripts that display regional differences in missense constraint. Missense-depleted regions are enriched for ClinVar 14 pathogenic variants, de novo missense variants from individuals with neurodevelopmental disorders (NDDs) 15,16 , and complex trait heritability. Following ClinGen calibration recommendations for the ACMG/AMP guidelines, we establish that regions with less than 20% of their expected missense variation achieve moderate support for pathogenicity. We create a missense deleteriousness metric (MPC) that incorporates regional constraint and outperforms other deleteriousness scores at stratifying case and control de novo missense variation, with a strong enrichment in NDDs. These results provide additional tools to aid in missense variant interpretation.
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BACKGROUND: Untranslated regions (UTRs) are important mediators of post-transcriptional regulation. The length of UTRs and the composition of regulatory elements within them are known to vary substantially across genes, but little is known about the reasons for this variation in humans. Here, we set out to determine whether this variation, specifically in 5'UTRs, correlates with gene dosage sensitivity. RESULTS: We investigate 5'UTR length, the number of alternative transcription start sites, the potential for alternative splicing, the number and type of upstream open reading frames (uORFs) and the propensity of 5'UTRs to form secondary structures. We explore how these elements vary by gene tolerance to loss-of-function (LoF; using the LOEUF metric), and in genes where changes in dosage are known to cause disease. We show that LOEUF correlates with 5'UTR length and complexity. Genes that are most intolerant to LoF have longer 5'UTRs, greater TSS diversity, and more upstream regulatory elements than their LoF tolerant counterparts. We show that these differences are evident in disease gene-sets, but not in recessive developmental disorder genes where LoF of a single allele is tolerated. CONCLUSIONS: Our results confirm the importance of post-transcriptional regulation through 5'UTRs in tight regulation of mRNA and protein levels, particularly for genes where changes in dosage are deleterious and lead to disease. Finally, to support gene-based investigation we release a web-based browser tool, VuTR, that supports exploration of the composition of individual 5'UTRs and the impact of genetic variation within them.
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Regiones no Traducidas 5' , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , Humanos , Dosificación de Gen , Regulación de la Expresión Génica , Sitio de Iniciación de la Transcripción , Empalme Alternativo , Conformación de Ácido NucleicoRESUMEN
OBJECTIVE: Most families with heritable neuromuscular disorders do not receive a molecular diagnosis. Here we evaluate diagnostic utility of exome, genome, RNA sequencing, and protein studies and provide evidence-based recommendations for their integration into practice. METHODS: In total, 247 families with suspected monogenic neuromuscular disorders who remained without a genetic diagnosis after standard diagnostic investigations underwent research-led massively parallel sequencing: neuromuscular disorder gene panel, exome, genome, and/or RNA sequencing to identify causal variants. Protein and RNA studies were also deployed when required. RESULTS: Integration of exome sequencing and auxiliary genome, RNA and/or protein studies identified causal or likely causal variants in 62% (152 out of 247) of families. Exome sequencing alone informed 55% (83 out of 152) of diagnoses, with remaining diagnoses (45%; 69 out of 152) requiring genome sequencing, RNA and/or protein studies to identify variants and/or support pathogenicity. Arrestingly, novel disease genes accounted for <4% (6 out of 152) of diagnoses while 36.2% of solved families (55 out of 152) harbored at least one splice-altering or structural variant in a known neuromuscular disorder gene. We posit that contemporary neuromuscular disorder gene-panel sequencing could likely provide 66% (100 out of 152) of our diagnoses today. INTERPRETATION: Our results emphasize thorough clinical phenotyping to enable deep scrutiny of all rare genetic variation in phenotypically consistent genes. Post-exome auxiliary investigations extended our diagnostic yield by 81% overall (34-62%). We present a diagnostic algorithm that details deployment of genomic and auxiliary investigations to obtain these diagnoses today most effectively. We hope this provides a practical guide for clinicians as they gain greater access to clinical genome and transcriptome sequencing.
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Secuenciación del Exoma , Enfermedades Neuromusculares , Humanos , Enfermedades Neuromusculares/genética , Enfermedades Neuromusculares/diagnóstico , Masculino , Femenino , Adulto , Análisis de Secuencia de ARN/métodos , Niño , Adolescente , Exoma/genética , Persona de Mediana Edad , Adulto Joven , Preescolar , Secuenciación de Nucleótidos de Alto Rendimiento , Lactante , Pruebas Genéticas/métodosRESUMEN
Genes encoding long non-coding RNAs (lncRNAs) comprise a large fraction of the human genome, yet haploinsufficiency of a lncRNA has not been shown to cause a Mendelian disease. CHASERR is a highly conserved human lncRNA adjacent to CHD2-a coding gene in which de novo loss-of-function variants cause developmental and epileptic encephalopathy. Here we report three unrelated individuals each harboring an ultra-rare heterozygous de novo deletion in the CHASERR locus. We report similarities in severe developmental delay, facial dysmorphisms, and cerebral dysmyelination in these individuals, distinguishing them from the phenotypic spectrum of CHD2 haploinsufficiency. We demonstrate reduced CHASERR mRNA expression and corresponding increased CHD2 mRNA and protein in whole blood and patient-derived cell lines-specifically increased expression of the CHD2 allele in cis with the CHASERR deletion, as predicted from a prior mouse model of Chaserr haploinsufficiency. We show for the first time that de novo structural variants facilitated by Alu-mediated non-allelic homologous recombination led to deletion of a non-coding element (the lncRNA CHASERR) to cause a rare syndromic neurodevelopmental disorder. We also demonstrate that CHD2 has bidirectional dosage sensitivity in human disease. This work highlights the need to carefully evaluate other lncRNAs, particularly those upstream of genes associated with Mendelian disorders.
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Short tandem repeats (STRs) are highly polymorphic sequences throughout the human genome that are composed of repeated copies of a 1-6-bp motif. Over 1 million variable STR loci are known, some of which regulate gene expression and influence complex traits, such as height. Moreover, variants in at least 60 STR loci cause genetic disorders, including Huntington disease and fragile X syndrome. Accurately identifying and genotyping STR variants is challenging, in particular mapping short reads to repetitive regions and inferring expanded repeat lengths. Recent advances in sequencing technology and computational tools for STR genotyping from sequencing data promise to help overcome this challenge and solve genetically unresolved cases and the 'missing heritability' of polygenic traits. Here, we compare STR genotyping methods, analytical tools and their applications to understand the effect of STR variation on health and disease. We identify emergent opportunities to refine genotyping and quality-control approaches as well as to integrate STRs into variant-calling workflows and large cohort analyses.
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Genoma Humano , Repeticiones de Microsatélite , Humanos , Repeticiones de Microsatélite/genética , Análisis de Secuencia de ADN/métodos , Técnicas de Genotipaje/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , GenotipoRESUMEN
Recessive diseases arise when both copies of a gene are impacted by a damaging genetic variant. When a patient carries two potentially causal variants in a gene, accurate diagnosis requires determining that these variants occur on different copies of the chromosome (that is, are in trans) rather than on the same copy (that is, in cis). However, current approaches for determining phase, beyond parental testing, are limited in clinical settings. Here we developed a strategy for inferring phase for rare variant pairs within genes, leveraging genotypes observed in the Genome Aggregation Database (v2, n = 125,748 exomes). Our approach estimates phase with 96% accuracy, both in trio data and in patients with Mendelian conditions and presumed causal compound heterozygous variants. We provide a public resource of phasing estimates for coding variants and counts per gene of rare variants in trans that can aid interpretation of rare co-occurring variants in the context of recessive disease.
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Exoma , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Exoma/genética , Secuenciación del Exoma , GenotipoRESUMEN
The depletion of disruptive variation caused by purifying natural selection (constraint) has been widely used to investigate protein-coding genes underlying human disorders1-4, but attempts to assess constraint for non-protein-coding regions have proved more difficult. Here we aggregate, process and release a dataset of 76,156 human genomes from the Genome Aggregation Database (gnomAD)-the largest public open-access human genome allele frequency reference dataset-and use it to build a genomic constraint map for the whole genome (genomic non-coding constraint of haploinsufficient variation (Gnocchi)). We present a refined mutational model that incorporates local sequence context and regional genomic features to detect depletions of variation. As expected, the average constraint for protein-coding sequences is stronger than that for non-coding regions. Within the non-coding genome, constrained regions are enriched for known regulatory elements and variants that are implicated in complex human diseases and traits, facilitating the triangulation of biological annotation, disease association and natural selection to non-coding DNA analysis. More constrained regulatory elements tend to regulate more constrained protein-coding genes, which in turn suggests that non-coding constraint can aid the identification of constrained genes that are as yet unrecognized by current gene constraint metrics. We demonstrate that this genome-wide constraint map improves the identification and interpretation of functional human genetic variation.
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Genoma Humano , Genómica , Modelos Genéticos , Mutación , Humanos , Acceso a la Información , Bases de Datos Genéticas , Conjuntos de Datos como Asunto , Frecuencia de los Genes , Genoma Humano/genética , Mutación/genética , Selección GenéticaRESUMEN
Predicted loss of function (pLoF) variants are often highly deleterious and play an important role in disease biology, but many pLoF variants may not result in loss of function (LoF). Here we present a framework that advances interpretation of pLoF variants in research and clinical settings by considering three categories of LoF evasion: (1) predicted rescue by secondary sequence properties, (2) uncertain biological relevance, and (3) potential technical artifacts. We also provide recommendations on adjustments to ACMG/AMP guidelines' PVS1 criterion. Applying this framework to all high-confidence pLoF variants in 22 genes associated with autosomal-recessive disease from the Genome Aggregation Database (gnomAD v.2.1.1) revealed predicted LoF evasion or potential artifacts in 27.3% (304/1,113) of variants. The major reasons were location in the last exon, in a homopolymer repeat, in a low proportion expressed across transcripts (pext) scored region, or the presence of cryptic in-frame splice rescues. Variants predicted to evade LoF or to be potential artifacts were enriched for ClinVar benign variants. PVS1 was downgraded in 99.4% (162/163) of pLoF variants predicted as likely not LoF/not LoF, with 17.2% (28/163) downgraded as a result of our framework, adding to previous guidelines. Variant pathogenicity was affected (mostly from likely pathogenic to VUS) in 20 (71.4%) of these 28 variants. This framework guides assessment of pLoF variants beyond standard annotation pipelines and substantially reduces false positive rates, which is key to ensure accurate LoF variant prediction in both a research and clinical setting.
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Patrón de Herencia , Humanos , Exones , IncertidumbreRESUMEN
Short-read genome sequencing (GS) holds the promise of becoming the primary diagnostic approach for the assessment of autism spectrum disorder (ASD) and fetal structural anomalies (FSAs). However, few studies have comprehensively evaluated its performance against current standard-of-care diagnostic tests: karyotype, chromosomal microarray (CMA), and exome sequencing (ES). To assess the clinical utility of GS, we compared its diagnostic yield against these three tests in 1,612 quartet families including an individual with ASD and in 295 prenatal families. Our GS analytic framework identified a diagnostic variant in 7.8% of ASD probands, almost 2-fold more than CMA (4.3%) and 3-fold more than ES (2.7%). However, when we systematically captured copy-number variants (CNVs) from the exome data, the diagnostic yield of ES (7.4%) was brought much closer to, but did not surpass, GS. Similarly, we estimated that GS could achieve an overall diagnostic yield of 46.1% in unselected FSAs, representing a 17.2% increased yield over karyotype, 14.1% over CMA, and 4.1% over ES with CNV calling or 36.1% increase without CNV discovery. Overall, GS provided an added diagnostic yield of 0.4% and 0.8% beyond the combination of all three standard-of-care tests in ASD and FSAs, respectively. This corresponded to nine GS unique diagnostic variants, including sequence variants in exons not captured by ES, structural variants (SVs) inaccessible to existing standard-of-care tests, and SVs where the resolution of GS changed variant classification. Overall, this large-scale evaluation demonstrated that GS significantly outperforms each individual standard-of-care test while also outperforming the combination of all three tests, thus warranting consideration as the first-tier diagnostic approach for the assessment of ASD and FSAs.
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Trastorno del Espectro Autista , Femenino , Embarazo , Humanos , Trastorno del Espectro Autista/diagnóstico , Trastorno del Espectro Autista/genética , Primer Trimestre del Embarazo , Ultrasonografía Prenatal , Mapeo Cromosómico , ExomaRESUMEN
Understanding the penetrance of pathogenic variants identified as secondary findings (SFs) is of paramount importance with the growing availability of genetic testing. We estimated penetrance through large-scale analyses of individuals referred for diagnostic sequencing for hypertrophic cardiomyopathy (HCM; 10,400 affected individuals, 1,332 variants) and dilated cardiomyopathy (DCM; 2,564 affected individuals, 663 variants), using a cross-sectional approach comparing allele frequencies against reference populations (293,226 participants from UK Biobank and gnomAD). We generated updated prevalence estimates for HCM (1:543) and DCM (1:220). In aggregate, the penetrance by late adulthood of rare, pathogenic variants (23% for HCM, 35% for DCM) and likely pathogenic variants (7% for HCM, 10% for DCM) was substantial for dominant cardiomyopathy (CM). Penetrance was significantly higher for variant subgroups annotated as loss of function or ultra-rare and for males compared to females for variants in HCM-associated genes. We estimated variant-specific penetrance for 316 recurrent variants most likely to be identified as SFs (found in 51% of HCM- and 17% of DCM-affected individuals). 49 variants were observed at least ten times (14% of affected individuals) in HCM-associated genes. Median penetrance was 14.6% (±14.4% SD). We explore estimates of penetrance by age, sex, and ancestry and simulate the impact of including future cohorts. This dataset reports penetrance of individual variants at scale and will inform the management of individuals undergoing genetic screening for SFs. While most variants had low penetrance and the costs and harms of screening are unclear, some individuals with highly penetrant variants may benefit from SFs.
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Cardiomiopatías , Cardiomiopatía Dilatada , Cardiomiopatía Hipertrófica , Femenino , Masculino , Humanos , Adulto , Penetrancia , Cardiomiopatías/genética , Cardiomiopatía Dilatada/genética , Frecuencia de los GenesRESUMEN
Genetic association studies have made significant contributions to our understanding of the etiology of neurodevelopmental disorders (NDDs). However, these studies rarely focused on the African continent. The NeuroDev Project aims to address this diversity gap through detailed phenotypic and genetic characterization of children with NDDs from Kenya and South Africa. We present results from NeuroDev's first year of data collection, including phenotype data from 206 cases and clinical genetic analyses of 99 parent-child trios. Most cases met criteria for global developmental delay/intellectual disability (GDD/ID, 80.3%). Approximately half of the children with GDD/ID also met criteria for autism. Analysis of exome-sequencing data identified a pathogenic or likely pathogenic variant in 13 (17%) of the 75 cases from South Africa and 9 (38%) of the 24 cases from Kenya. Data from the trio pilot are publicly available, and the NeuroDev Project will continue to develop resources for the global genetics community.