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1.
Circ Genom Precis Med ; 14(4): e003300, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34319147

RESUMO

BACKGROUND: Alterations in electrocardiographic (ECG) intervals are well-known markers for arrhythmia and sudden cardiac death (SCD) risk. While the genetics of arrhythmia syndromes have been studied, relations between electrocardiographic intervals and rare genetic variation at a population level are poorly understood. METHODS: Using a discovery sample of 29 000 individuals with whole-genome sequencing from Trans-Omics in Precision Medicine and replication in nearly 100 000 with whole-exome sequencing from the UK Biobank and MyCode, we examined associations between low-frequency and rare coding variants with 5 routinely measured electrocardiographic traits (RR, P-wave, PR, and QRS intervals and corrected QT interval). RESULTS: We found that rare variants associated with population-based electrocardiographic intervals identify established monogenic SCD genes (KCNQ1, KCNH2, and SCN5A), a controversial monogenic SCD gene (KCNE1), and novel genes (PAM and MFGE8) involved in cardiac conduction. Loss-of-function and pathogenic SCN5A variants, carried by 0.1% of individuals, were associated with a nearly 6-fold increased odds of the first-degree atrioventricular block (P=8.4×10-5). Similar variants in KCNQ1 and KCNH2 (0.2% of individuals) were associated with a 23-fold increased odds of marked corrected QT interval prolongation (P=4×10-25), a marker of SCD risk. Incomplete penetrance of such deleterious variation was common as over 70% of carriers had normal electrocardiographic intervals. CONCLUSIONS: Our findings indicate that large-scale high-depth sequence data and electrocardiographic analysis identifies monogenic arrhythmia susceptibility genes and rare variants with large effects. Known pathogenic variation in conventional arrhythmia and SCD genes exhibited incomplete penetrance and accounted for only a small fraction of marked electrocardiographic interval prolongation.

2.
J Cell Sci ; 134(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34155518

RESUMO

Mutations in CEP290 (also known as NPHP6), a large multidomain coiled coil protein, are associated with multiple cilia-associated syndromes. Over 130 CEP290 mutations have been linked to a wide spectrum of human ciliopathies, raising the question of how mutations in a single gene cause different disease syndromes. In zebrafish, the expressivity of cep290 deficiencies were linked to the type of genetic ablation: acute cep290 morpholino knockdown caused severe cilia-related phenotypes, whereas deficiencies in a CRISPR/Cas9 genetic mutant were restricted to photoreceptor defects. Here, we show that milder phenotypes in genetic mutants were associated with the upregulation of genes encoding the cilia-associated small GTPases arl3, arl13b and unc119b. Upregulation of UNC119b was also observed in urine-derived renal epithelial cells from human Joubert syndrome CEP290 patients. Ectopic expression of arl3, arl13b and unc119b in cep290 morphant zebrafish embryos rescued Kupffer's vesicle cilia and partially rescued photoreceptor outer segment defects. The results suggest that genetic compensation by upregulation of genes involved in a common subcellular process, lipidated protein trafficking to cilia, may be a conserved mechanism contributing to genotype-phenotype variations observed in CEP290 deficiencies. This article has an associated First Person interview with the first author of the paper.


Assuntos
Antígenos de Neoplasias , Proteínas de Ciclo Celular , Cílios , Proteínas do Citoesqueleto , Proteínas Monoméricas de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos de Neoplasias/genética , Proteínas de Ciclo Celular/genética , Cílios/genética , Cílios/metabolismo , Proteínas do Citoesqueleto/genética , Humanos , Proteínas Associadas aos Microtúbulos , Mutação/genética , Regulação para Cima/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
3.
Genet Med ; 23(9): 1689-1696, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33976420

RESUMO

PURPOSE: To evaluate the diagnostic yield and clinical relevance of clinical genome sequencing (cGS) as a first genetic test for patients with suspected monogenic disorders. METHODS: We conducted a prospective randomized study with pediatric and adult patients recruited from genetics clinics at Massachusetts General Hospital who were undergoing planned genetic testing. Participants were randomized into two groups: standard-of-care genetic testing (SOC) only or SOC and cGS. RESULTS: Two hundred four participants were enrolled, 202 were randomized to one of the intervention arms, and 99 received cGS. In total, cGS returned 16 molecular diagnoses that fully or partially explained the indication for testing in 16 individuals (16.2% of the cohort, 95% confidence interval [CI] 8.9-23.4%), which was not significantly different from SOC (18.2%, 95% CI 10.6-25.8%, P = 0.71). An additional eight molecular diagnoses reported by cGS had uncertain relevance to the participant's phenotype. Nevertheless, referring providers considered 20/24 total cGS molecular diagnoses (83%) to be explanatory for clinical features or worthy of additional workup. CONCLUSION: cGS is technically suitable as a first genetic test. In our cohort, diagnostic yield was not significantly different from SOC. Further studies addressing other variant types and implementation challenges are needed to support feasibility and utility of broad-scale cGS adoption.

4.
J Mol Diagn ; 23(5): 589-598, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33631351

RESUMO

Diagnostic laboratories gather phenotypic data through requisition forms, but there is no consensus as to which data are essential for variant interpretation. The ClinGen Cardiomyopathy Variant Curation Expert Panel defined a phenotypic data set for hypertrophic cardiomyopathy (HCM) variant interpretation, with the goal of standardizing requisition forms. Phenotypic data elements listed on requisition forms from nine leading cardiomyopathy testing laboratories were compiled to assess divergence in data collection. A pilot of 50 HCM cases was implemented to determine the feasibility of harmonizing data collection. Laboratory directors were surveyed to gauge potential for adoption of a minimal data set. Wide divergence was observed in the phenotypic data fields in requisition forms. The 50-case pilot showed that although demographics and assertion of a clinical diagnosis of HCM had 86% to 98% completion, specific phenotypic features, such as degree of left ventricular hypertrophy, ejection fraction, and suspected syndromic disease, were completed only 24% to 44% of the time. Nine data elements were deemed essential for variant classification by the expert panel. Participating laboratories unanimously expressed a willingness to adopt these data elements in their requisition forms. This study demonstrates the value of comparing and sharing best practices through an expert group, such as the ClinGen Program, to enhance variant interpretation, providing a foundation for leveraging cumulative case-level data in public databases and ultimately improving patient care.

6.
Eur J Med Genet ; 61(10): 621-626, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29655801

RESUMO

Given that all forms of Usher syndrome (USH) present with hearing loss in advance of retinal disease, the syndromic nature of the disorder is rarely appreciated when critical management decisions are being made. As a result, molecular diagnostics are crucial in guiding the management of USH patients. While 11 genes have been associated with USH, the USH2A gene is one of the largest contributors. Approximately 20% of suspected USH probands that undergo USH2A sequencing at our laboratory receive an inconclusive result due to the identification of a monoallelic disease-causing variant in USH2A. Many studies suggest that intragenic deletions and duplications represent an important USH2A variant type that can be missed by sequencing assays if supplemental algorithms or testing methods are not applied. To gain a comprehensive view of the contribution of USH2A CNVs to USH, we conducted prospective and retrospective screening in 700 hearing loss probands. Fourteen individuals with 11 unique USH2A CNVs are reported, including one pathogenic multi-exon duplication. Additionally, we mapped deletion breakpoints and performed a meta-analysis of USH2A CNVs to evaluate recurrence and underlying mechanisms. This analysis revealed breakpoint grouping within three introns, raising the possibility of CNV-susceptible regions within the gene. Overall, our data highlight the diversity of pathogenic CNVs in this gene, demonstrating that the comprehensive, high-resolution USH2A CNV analysis methods employed here are essential components of clinical genetic testing for USH.


Assuntos
Variações do Número de Cópias de DNA , Proteínas da Matriz Extracelular/genética , Síndromes de Usher/genética , Pontos de Quebra do Cromossomo , Humanos , Íntrons
7.
Cilia ; 7: 2, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29568513

RESUMO

Background: Primary cilia mediate signal transduction by acting as an organizing scaffold for receptors, signalling proteins and ion channels. Ciliated olfactory sensory neurons (OSNs) organize olfactory receptors and ion channels on cilia and generate a calcium influx as a primary signal in odourant detection. In the zebrafish olfactory placode, ciliated OSNs and microvillus OSNs constitute the major OSN cell types with distinct odourant sensitivity. Methods: Using transgenic expression of the calcium biosensor GCaMP5 in OSNs, we analysed sensory cilia-dependent odour responses in live zebrafish, at individual cell resolution. oval/ift88 mutant and ift172 knockdown zebrafish were compared with wild-type siblings to establish ciliated OSN sensitivity to different classes of odourants. Results: oval/ift88 mutant and ift172 knockdown zebrafish showed fewer and severely shortened OSN cilia without a reduction in OSN number. The fraction of responding OSNs and response amplitudes to bile acids and food odour, both sensed by ciliated OSNs, were significantly reduced in ift88 mutants and ift172-deficient embryos, while the amino acids responses were not significantly changed. Conclusions: Our approach presents a quantitative model for studying sensory cilia signalling using zebrafish OSNs. Our results also implicate ift172-deficiency as a novel cause of hyposmia, a reduced sense of smell, highlighting the value of directly assaying sensory cilia signalling in vivo and supporting the idea that hyposmia can be used as a diagnostic indicator of ciliopathies.

8.
Genet Med ; 19(11): 1245-1252, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28471438

RESUMO

PurposeArrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart disease. Clinical follow-up of incidental findings in ARVC-associated genes is recommended. We aimed to determine the prevalence of disease thus ascertained.MethodsIndividuals (n = 30,716) underwent exome sequencing. Variants in PKP2, DSG2, DSC2, DSP, JUP, TMEM43, or TGFß3 that were database-listed as pathogenic or likely pathogenic were identified and evidence-reviewed. For subjects with putative loss-of-function (pLOF) variants or variants of uncertain significance (VUS), electronic health records (EHR) were reviewed for ARVC diagnosis, diagnostic criteria, and International Classification of Diseases (ICD-9) codes.ResultsEighteen subjects had pLOF variants; none of these had an EHR diagnosis of ARVC. Of 14 patients with an electrocardiogram, one had a minor diagnostic criterion; the rest were normal. A total of 184 subjects had VUS, none of whom had an ARVC diagnosis. The proportion of subjects with VUS with major (4%) or minor (13%) electrocardiogram diagnostic criteria did not differ from that of variant-negative controls. ICD-9 codes showed no difference in defibrillator use, electrophysiologic abnormalities or nonischemic cardiomyopathies in patients with pLOF or VUSs compared with controls.ConclusionpLOF variants in an unselected cohort were not associated with ARVC phenotypes based on EHR review. The negative predictive value of EHR review remains uncertain.


Assuntos
Displasia Arritmogênica Ventricular Direita/genética , Exoma , Variação Genética , Análise de Sequência de DNA , Adulto , Displasia Arritmogênica Ventricular Direita/epidemiologia , Estudos de Coortes , Registros Eletrônicos de Saúde , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Prevalência
9.
Science ; 354(6319)2016 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-28008009

RESUMO

The DiscovEHR collaboration between the Regeneron Genetics Center and Geisinger Health System couples high-throughput sequencing to an integrated health care system using longitudinal electronic health records (EHRs). We sequenced the exomes of 50,726 adult participants in the DiscovEHR study to identify ~4.2 million rare single-nucleotide variants and insertion/deletion events, of which ~176,000 are predicted to result in a loss of gene function. Linking these data to EHR-derived clinical phenotypes, we find clinical associations supporting therapeutic targets, including genes encoding drug targets for lipid lowering, and identify previously unidentified rare alleles associated with lipid levels and other blood level traits. About 3.5% of individuals harbor deleterious variants in 76 clinically actionable genes. The DiscovEHR data set provides a blueprint for large-scale precision medicine initiatives and genomics-guided therapeutic discovery.


Assuntos
Prestação Integrada de Cuidados de Saúde , Doença/genética , Registros Eletrônicos de Saúde , Exoma/genética , Sequenciamento de Nucleotídeos em Larga Escala , Adulto , Desenho de Fármacos , Frequência do Gene , Genômica , Humanos , Hipolipemiantes/farmacologia , Mutação INDEL , Lipídeos/sangue , Terapia de Alvo Molecular , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA
10.
Mol Biol Cell ; 25(12): 1836-44, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24743595

RESUMO

Glutamylation is a functionally important tubulin posttranslational modification enriched on stable microtubules of neuronal axons, mitotic spindles, centrioles, and cilia. In vertebrates, balanced activities of tubulin glutamyl ligase and cytoplasmic carboxypeptidase deglutamylase enzymes maintain organelle- and cell type-specific tubulin glutamylation patterns. Tubulin glutamylation in cilia is regulated via restricted subcellular localization or expression of tubulin glutamyl ligases (ttlls) and nonenzymatic proteins, including the zebrafish TPR repeat protein Fleer/Ift70. Here we analyze the expression patterns of ccp deglutamylase genes during zebrafish development and the effects of ccp gene knockdown on cilia formation, morphology, and tubulin glutamylation. The deglutamylases ccp2, ccp5, and ccp6 are expressed in ciliated cells, whereas ccp1 expression is restricted to the nervous system. Only ccp5 knockdown increases cilia tubulin glutamylation, induces ciliopathy phenotypes, including axis curvature, hydrocephalus, and pronephric cysts, and disrupts multicilia motility, suggesting that Ccp5 is the principal tubulin deglutamylase that maintains functional levels of cilia tubulin glutamylation. The ability of ccp5 knockdown to restore cilia tubulin glutamylation in fleer/ift70 mutants and rescue pronephric multicilia formation in both fleer- and ift88-deficient zebrafish indicates that tubulin glutamylation is a key driver of ciliogenesis.


Assuntos
Carboxipeptidases/fisiologia , Cílios/fisiologia , Ácido Glutâmico/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Animais , Técnicas de Silenciamento de Genes , Larva/citologia , Larva/enzimologia , Microtúbulos/metabolismo , Pronefro/citologia , Pronefro/enzimologia , Processamento de Proteína Pós-Traducional , Peixe-Zebra
11.
Am J Hum Genet ; 93(4): 672-86, 2013 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-24094744

RESUMO

Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.


Assuntos
Transtornos da Motilidade Ciliar/genética , Glicoproteínas/genética , Síndrome de Kartagener/genética , Peixe-Zebra/genética , Animais , Chlamydomonas/genética , Cílios/genética , Análise Mutacional de DNA/métodos , Dineínas/genética , Feminino , Humanos , Masculino , Mutação , Fases de Leitura Aberta , Planárias/genética , Proteoma/genética
12.
Methods Enzymol ; 525: 219-44, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23522472

RESUMO

Zebrafish are ideally suited for analysis of genes required for ciliogenesis and cilia function. Combining genetic manipulation with high quality in vivo imaging, zebrafish embryos provide a high-throughput system for annotation of the cilia proteome. The specific advantages of the system are the availability of cilia mutants, the ability to target genes of unknown function using antisense methods, the feasibility of observing cilia in living embryos, and the ability to image fixed cilia in wholemount at high resolution. Techniques are described for analysis of mutants, gene knockdown using antisense morpholino oligos, visualizing cilia and cilia orientation in wholemount zebrafish embryos, live imaging cilia, and electron microscopy of zebrafish cilia.


Assuntos
Cílios/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Cílios/fisiologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peixe-Zebra
13.
Nat Genet ; 44(6): 714-9, 2012 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-22581229

RESUMO

Cilia are essential for fertilization, respiratory clearance, cerebrospinal fluid circulation and establishing laterality. Cilia motility defects cause primary ciliary dyskinesia (PCD, MIM244400), a disorder affecting 1:15,000-30,000 births. Cilia motility requires the assembly of multisubunit dynein arms that drive ciliary bending. Despite progress in understanding the genetic basis of PCD, mutations remain to be identified for several PCD-linked loci. Here we show that the zebrafish cilia paralysis mutant schmalhans (smh(tn222)) encodes the coiled-coil domain containing 103 protein (Ccdc103), a foxj1a-regulated gene product. Screening 146 unrelated PCD families identified individuals in six families with reduced outer dynein arms who carried mutations in CCDC103. Dynein arm assembly in smh mutant zebrafish was rescued by wild-type but not mutant human CCDC103. Chlamydomonas Ccdc103/Pr46b functions as a tightly bound, axoneme-associated protein. These results identify Ccdc103 as a dynein arm attachment factor that causes primary ciliary dyskinesia when mutated.


Assuntos
Dineínas/metabolismo , Síndrome de Kartagener/genética , Animais , Cílios/metabolismo , Feminino , Humanos , Masculino , Mutação , Linhagem , Peixe-Zebra
14.
Genome Res ; 22(8): 1541-8, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22555591

RESUMO

Genetic mapping of mutations in model systems has facilitated the identification of genes contributing to fundamental biological processes including human diseases. However, this approach has historically required the prior characterization of informative markers. Here we report a fast and cost-effective method for genetic mapping using next-generation sequencing that combines single nucleotide polymorphism discovery, mutation localization, and potential identification of causal sequence variants. In contrast to prior approaches, we have developed a hidden Markov model to narrowly define the mutation area by inferring recombination breakpoints of chromosomes in the mutant pool. In addition, we created an interactive online software resource to facilitate automated analysis of sequencing data and demonstrate its utility in the zebrafish and mouse models. Our novel methodology and online tools will make next-generation sequencing an easily applicable resource for mutation mapping in all model systems.


Assuntos
Análise Mutacional de DNA/métodos , Software , Peixe-Zebra/genética , Alelos , Animais , Mapeamento Cromossômico/métodos , Cromossomos/genética , Cruzamentos Genéticos , Feminino , Frequência do Gene , Genômica/métodos , Homozigoto , Masculino , Cadeias de Markov , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Fatores de Tempo
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