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1.
Am J Hum Genet ; 110(9): 1454-1469, 2023 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-37595579

RESUMEN

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.


Asunto(s)
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 , Exoma
2.
Nature ; 581(7809): 444-451, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32461652

RESUMEN

Structural variants (SVs) rearrange large segments of DNA1 and can have profound consequences in evolution and human disease2,3. As national biobanks, disease-association studies, and clinical genetic testing have grown increasingly reliant on genome sequencing, population references such as the Genome Aggregation Database (gnomAD)4 have become integral in the interpretation of single-nucleotide variants (SNVs)5. However, there are no reference maps of SVs from high-coverage genome sequencing comparable to those for SNVs. Here we present a reference of sequence-resolved SVs constructed from 14,891 genomes across diverse global populations (54% non-European) in gnomAD. We discovered a rich and complex landscape of 433,371 SVs, from which we estimate that SVs are responsible for 25-29% of all rare protein-truncating events per genome. We found strong correlations between natural selection against damaging SNVs and rare SVs that disrupt or duplicate protein-coding sequence, which suggests that genes that are highly intolerant to loss-of-function are also sensitive to increased dosage6. We also uncovered modest selection against noncoding SVs in cis-regulatory elements, although selection against protein-truncating SVs was stronger than all noncoding effects. Finally, we identified very large (over one megabase), rare SVs in 3.9% of samples, and estimate that 0.13% of individuals may carry an SV that meets the existing criteria for clinically important incidental findings7. This SV resource is freely distributed via the gnomAD browser8 and will have broad utility in population genetics, disease-association studies, and diagnostic screening.


Asunto(s)
Enfermedad/genética , Variación Genética , Genética Médica/normas , Genética de Población/normas , Genoma Humano/genética , Femenino , Pruebas Genéticas , Técnicas de Genotipaje , Humanos , Masculino , Persona de Mediana Edad , Mutación , Polimorfismo de Nucleótido Simple/genética , Grupos Raciales/genética , Estándares de Referencia , Selección Genética , Secuenciación Completa del Genoma
5.
Genet Med ; 23(9): 1715-1725, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34054129

RESUMEN

PURPOSE: To investigate the effect of PLXNA1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and to functionally characterize the zebrafish homologs plxna1a and plxna1b during development. METHODS: We assembled ten patients from seven families with biallelic or de novo PLXNA1 variants. We describe genotype-phenotype correlations, investigated the variants by structural modeling, and used Morpholino knockdown experiments in zebrafish to characterize the embryonic role of plxna1a and plxna1b. RESULTS: Shared phenotypic features among patients include global developmental delay (9/10), brain anomalies (6/10), and eye anomalies (7/10). Notably, seizures were predominantly reported in patients with monoallelic variants. Structural modeling of missense variants in PLXNA1 suggests distortion in the native protein. Our zebrafish studies enforce an embryonic role of plxna1a and plxna1b in the development of the central nervous system and the eye. CONCLUSION: We propose that different biallelic and monoallelic variants in PLXNA1 result in a novel neurodevelopmental syndrome mainly comprising developmental delay, brain, and eye anomalies. We hypothesize that biallelic variants in the extracellular Plexin-A1 domains lead to impaired dimerization or lack of receptor molecules, whereas monoallelic variants in the intracellular Plexin-A1 domains might impair downstream signaling through a dominant-negative effect.


Asunto(s)
Anomalías del Ojo , Trastornos del Neurodesarrollo , Animales , Anomalías del Ojo/genética , Estudios de Asociación Genética , Humanos , Proteínas del Tejido Nervioso/genética , Trastornos del Neurodesarrollo/genética , Fenotipo , Receptores de Superficie Celular , Pez Cebra/genética
6.
Genet Med ; 22(9): 1478-1488, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32528171

RESUMEN

PURPOSE: Several hundred genetic muscle diseases have been described, all of which are rare. Their clinical and genetic heterogeneity means that a genetic diagnosis is challenging. We established an international consortium, MYO-SEQ, to aid the work-ups of muscle disease patients and to better understand disease etiology. METHODS: Exome sequencing was applied to 1001 undiagnosed patients recruited from more than 40 neuromuscular disease referral centers; standardized phenotypic information was collected for each patient. Exomes were examined for variants in 429 genes associated with muscle conditions. RESULTS: We identified suspected pathogenic variants in 52% of patients across 87 genes. We detected 401 novel variants, 116 of which were recurrent. Variants in CAPN3, DYSF, ANO5, DMD, RYR1, TTN, COL6A2, and SGCA collectively accounted for over half of the solved cases; while variants in newer disease genes, such as BVES and POGLUT1, were also found. The remaining well-characterized unsolved patients (48%) need further investigation. CONCLUSION: Using our unique infrastructure, we developed a pathway to expedite muscle disease diagnoses. Our data suggest that exome sequencing should be used for pathogenic variant detection in patients with suspected genetic muscle diseases, focusing first on the most common disease genes described here, and subsequently in rarer and newly characterized disease genes.


Asunto(s)
Exoma , Distrofia Muscular de Cinturas , Anoctaminas , Exoma/genética , Glucosiltransferasas , Humanos , Distrofia Muscular de Cinturas/genética , Secuenciación del Exoma
7.
Genet Med ; 21(3): 694-704, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30072743

RESUMEN

PURPOSE: With the advent of gene therapies for inherited retinal degenerations (IRDs), genetic diagnostics will have an increasing role in clinical decision-making. Yet the genetic cause of disease cannot be identified using exon-based sequencing for a significant portion of patients. We hypothesized that noncoding pathogenic variants contribute significantly to the genetic causality of IRDs and evaluated patients with single coding pathogenic variants in RPGRIP1 to test this hypothesis. METHODS: IRD families underwent targeted panel sequencing. Unsolved cases were explored by exome and genome sequencing looking for additional pathogenic variants. Candidate pathogenic variants were then validated by Sanger sequencing, quantitative polymerase chain reaction, and in vitro splicing assays in two cell lines analyzed through amplicon sequencing. RESULTS: Among 1722 families, 3 had biallelic loss-of-function pathogenic variants in RPGRIP1 while 7 had a single disruptive coding pathogenic variants. Exome and genome sequencing revealed potential noncoding pathogenic variants in these 7 families. In 6, the noncoding pathogenic variants were shown to lead to loss of function in vitro. CONCLUSION: Noncoding pathogenic variants were identified in 6 of 7 families with single coding pathogenic variants in RPGRIP1. The results suggest that noncoding pathogenic variants contribute significantly to the genetic causality of IRDs and RPGRIP1-mediated IRDs are more common than previously thought.


Asunto(s)
ADN Intergénico/genética , Proteínas/genética , Degeneración Retiniana/genética , Adulto , Mapeo Cromosómico , Proteínas del Citoesqueleto , Análisis Mutacional de ADN/métodos , ADN Intergénico/fisiología , Femenino , Células HEK293 , Humanos , Masculino , Mutación , Linaje , Proteínas/fisiología , Degeneración Retiniana/etiología , Secuenciación del Exoma/métodos , Secuenciación Completa del Genoma/métodos
8.
Hum Mutat ; 39(12): 1827-1834, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30240502

RESUMEN

Rare disease investigators constantly face challenges in identifying additional cases to build evidence for gene-disease causality. The Matchmaker Exchange (MME) addresses this limitation by providing a mechanism for matching patients across genomic centers via a federated network. The MME has revolutionized searching for additional cases by making it possible to query across institutional boundaries, so that what was once a laborious and manual process of contacting researchers is now automated and computable. However, while the MME network is beginning to scale, the growth of additional nodes is limited by the lack of easy-to-use solutions that can be implemented by any rare disease database owner, even one without significant software engineering resources. Here, we describe matchbox, which is an open-source, platform-independent, portable bridge between any given rare disease genomic center and the MME network, which has already led to novel gene discoveries. We also describe how matchbox greatly reduces the barrier to participation by overcoming challenges for new databases to join the MME.


Asunto(s)
Almacenamiento y Recuperación de la Información/métodos , Selección de Paciente , Enfermedades Raras/genética , Acceso a la Información , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Difusión de la Información/métodos , Fenotipo , Programas Informáticos , Navegador Web
9.
Physiol Genomics ; 50(11): 929-939, 2018 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-30345904

RESUMEN

Next-generation sequencing is commonly used to screen for pathogenic mutations in families with Mendelian disorders, but due to the pace of discoveries, gaps have widened for some diseases between genetic and pathophysiological knowledge. We recruited and analyzed 16 families with limb-girdle muscular dystrophy (LGMD) of Arab descent from Saudi Arabia and Sudan who did not have confirmed genetic diagnoses. The analysis included both traditional and next-generation sequencing approaches. Cellular and metabolic studies were performed on Pyroxd1 siRNA C2C12 myoblasts and controls. Pathogenic mutations were identified in eight of the 16 families. One Sudanese family of Arab descent residing in Saudi Arabia harbored a homozygous c.464A>G, p.Asn155Ser mutation in PYROXD1, a gene recently reported in association with myofibrillar myopathy and whose protein product reduces thiol residues. Pyroxd1 deficiency in murine C2C12 myoblasts yielded evidence for impairments of cellular proliferation, migration, and differentiation, while CG10721 (Pyroxd1 fly homolog) knockdown in Drosophila yielded a lethal phenotype. Further investigations indicated that Pyroxd1 does not localize to mitochondria, yet Pyroxd1 deficiency is associated with decreased cellular respiration. This study identified pathogenic mutations in half of the LGMD families from the cohort, including one in PYROXD1. Developmental impairments were demonstrated in vitro for Pyroxd1 deficiency and in vivo for CG10721 deficiency, with reduced metabolic activity in vitro for Pyroxd1 deficiency.


Asunto(s)
Distrofia Muscular de Cinturas/genética , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Adulto , Animales , Animales Modificados Genéticamente , Respiración de la Célula/genética , Células Cultivadas , Drosophila , Proteínas de Drosophila/genética , Femenino , Humanos , Masculino , Ratones , Mitocondrias Musculares/genética , Mitocondrias Musculares/metabolismo , Mitocondrias Musculares/patología , Distrofia Muscular de Cinturas/patología , Mioblastos/patología , Linaje , Arabia Saudita , Sudán
10.
Am J Hum Genet ; 97(1): 99-110, 2015 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-26119818

RESUMEN

Ablepharon macrostomia syndrome (AMS) and Barber-Say syndrome (BSS) are rare congenital ectodermal dysplasias characterized by similar clinical features. To establish the genetic basis of AMS and BSS, we performed extensive clinical phenotyping, whole exome and candidate gene sequencing, and functional validations. We identified a recurrent de novo mutation in TWIST2 in seven independent AMS-affected families, as well as another recurrent de novo mutation affecting the same amino acid in ten independent BSS-affected families. Moreover, a genotype-phenotype correlation was observed, because the two syndromes differed based solely upon the nature of the substituting amino acid: a lysine at TWIST2 residue 75 resulted in AMS, whereas a glutamine or alanine yielded BSS. TWIST2 encodes a basic helix-loop-helix transcription factor that regulates the development of mesenchymal tissues. All identified mutations fell in the basic domain of TWIST2 and altered the DNA-binding pattern of Flag-TWIST2 in HeLa cells. Comparison of wild-type and mutant TWIST2 expressed in zebrafish identified abnormal developmental phenotypes and widespread transcriptome changes. Our results suggest that autosomal-dominant TWIST2 mutations cause AMS or BSS by inducing protean effects on the transcription factor's DNA binding.


Asunto(s)
Anomalías Múltiples/genética , Anomalías del Ojo/genética , Enfermedades de los Párpados/genética , Hirsutismo/genética , Hipertelorismo/genética , Hipertricosis/genética , Macrostomía/genética , Modelos Moleculares , Fenotipo , Proteínas Represoras/genética , Anomalías Cutáneas/genética , Proteína 1 Relacionada con Twist/genética , Anomalías Múltiples/patología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Inmunoprecipitación de Cromatina , Exoma/genética , Anomalías del Ojo/patología , Enfermedades de los Párpados/patología , Células HeLa , Hirsutismo/patología , Humanos , Hipertelorismo/patología , Hipertricosis/patología , Macrostomía/patología , Microscopía Electrónica , Datos de Secuencia Molecular , Mutación Missense/genética , Conformación Proteica , Proteínas Represoras/química , Análisis de Secuencia de ADN , Anomalías Cutáneas/patología , Proteína 1 Relacionada con Twist/química , Pez Cebra
11.
J Hum Genet ; 62(2): 243-252, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-27708273

RESUMEN

The current study characterizes a cohort of limb-girdle muscular dystrophy (LGMD) in the United States using whole-exome sequencing. Fifty-five families affected by LGMD were recruited using an institutionally approved protocol. Exome sequencing was performed on probands and selected parental samples. Pathogenic mutations and cosegregation patterns were confirmed by Sanger sequencing. Twenty-two families (40%) had novel and previously reported pathogenic mutations, primarily in LGMD genes, and also in genes for Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital myopathy, myofibrillar myopathy, inclusion body myopathy and Pompe disease. One family was diagnosed via clinical testing. Dominant mutations were identified in COL6A1, COL6A3, FLNC, LMNA, RYR1, SMCHD1 and VCP, recessive mutations in ANO5, CAPN3, GAA, LAMA2, SGCA and SGCG, and X-linked mutations in DMD. A previously reported variant in DMD was confirmed to be benign. Exome sequencing is a powerful diagnostic tool for LGMD. Despite careful phenotypic screening, pathogenic mutations were found in other muscle disease genes, largely accounting for the increased sensitivity of exome sequencing. Our experience suggests that broad sequencing panels are useful for these analyses because of the phenotypic overlap of many neuromuscular conditions. The confirmation of a benign DMD variant illustrates the potential of exome sequencing to help determine pathogenicity.


Asunto(s)
Exoma/genética , Pruebas Genéticas/métodos , Distrofia Muscular de Cinturas/diagnóstico , Distrofia Muscular de Cinturas/genética , Secuencia de Bases , Miopatías Distales/diagnóstico , Miopatías Distales/genética , Femenino , Enfermedad del Almacenamiento de Glucógeno Tipo II/diagnóstico , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Humanos , Masculino , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular Facioescapulohumeral/diagnóstico , Distrofia Muscular Facioescapulohumeral/genética , Mutación/genética , Miopatías Estructurales Congénitas/diagnóstico , Miopatías Estructurales Congénitas/genética , Análisis de Secuencia de ADN/métodos , Estados Unidos
12.
Genet Med ; 18(12): 1303-1307, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27253732

RESUMEN

PURPOSE: Using single-nucleotide polymorphism (SNP) chip and exome sequence data from individuals participating in the National Institutes of Health (NIH) Undiagnosed Diseases Program (UDP), we evaluated the number and therapeutic informativeness of incidental pharmacogenetic variants. METHODS: Pharmacogenomics Knowledgebase (PharmGKB) annotated sequence variants were identified in 1,101 individuals. Medication records of participants were used to identify individuals prescribed medications with a genetic variant that might alter efficacy. RESULTS: A total of 395 sequence variants, including 19 PharmGKB 1A and 1B variants, were identified in SNP chip sequence data, and 388 variants, including 21 PharmGKB 1A and 1B variants, were identified in the exome sequence data. Nine participants had incidental pharmacogenetic variants associated with altered efficacy of a prescribed medication. CONCLUSIONS: Despite the small size of the NIH UDP patient cohort, we identified pharmacogenetic incidental findings potentially useful for guiding therapy. Consequently, groups conducting clinical genomic studies might consider reporting of pharmacogenetic incidental findings.Genet Med 18 12, 1303-1307.


Asunto(s)
Exoma/genética , Genómica , Farmacogenética , Polimorfismo de Nucleótido Simple/genética , Humanos , Hallazgos Incidentales , National Institutes of Health (U.S.) , Estados Unidos
13.
Genet Med ; 18(6): 608-17, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26562225

RESUMEN

PURPOSE: Medical diagnosis and molecular or biochemical confirmation typically rely on the knowledge of the clinician. Although this is very difficult in extremely rare diseases, we hypothesized that the recording of patient phenotypes in Human Phenotype Ontology (HPO) terms and computationally ranking putative disease-associated sequence variants improves diagnosis, particularly for patients with atypical clinical profiles. METHODS: Using simulated exomes and the National Institutes of Health Undiagnosed Diseases Program (UDP) patient cohort and associated exome sequence, we tested our hypothesis using Exomiser. Exomiser ranks candidate variants based on patient phenotype similarity to (i) known disease-gene phenotypes, (ii) model organism phenotypes of candidate orthologs, and (iii) phenotypes of protein-protein association neighbors. RESULTS: Benchmarking showed Exomiser ranked the causal variant as the top hit in 97% of known disease-gene associations and ranked the correct seeded variant in up to 87% when detectable disease-gene associations were unavailable. Using UDP data, Exomiser ranked the causative variant(s) within the top 10 variants for 11 previously diagnosed variants and achieved a diagnosis for 4 of 23 cases undiagnosed by clinical evaluation. CONCLUSION: Structured phenotyping of patients and computational analysis are effective adjuncts for diagnosing patients with genetic disorders.Genet Med 18 6, 608-617.


Asunto(s)
Secuenciación del Exoma/métodos , Exoma/genética , Enfermedades Raras/genética , Enfermedades Raras/fisiopatología , Animales , Biología Computacional , Bases de Datos Genéticas , Modelos Animales de Enfermedad , Estudios de Asociación Genética , Variación Genética , Humanos , Ratones , National Institutes of Health (U.S.) , Pacientes , Fenotipo , Enfermedades Raras/diagnóstico , Enfermedades Raras/epidemiología , Estados Unidos , Pez Cebra
14.
Am J Med Genet A ; 170(12): 3106-3114, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27513830

RESUMEN

Failure to thrive arises as a complication of a heterogeneous group of disorders. We describe two female siblings with spastic paraplegia and global developmental delay but also, atypically for the HSPs, poor weight gain classified as failure to thrive. After extensive clinical and biochemical investigations failed to identify the etiology, we used exome sequencing to identify biallelic UNC80 mutations (NM_032504.1:c.[3983-3_3994delinsA];[2431C>T]. The paternally inherited NM_032504.1:c.3983-3_3994delinsA is predicted to encode p.Ser1328Argfs*19 and the maternally inherited NM_032504.1:c.2431C>T is predicted to encode p.Arg811*. No UNC80 mRNA was detectable in patient cultured skin fibroblasts, suggesting UNC80 loss of function by nonsense mediated mRNA decay. Further supporting the UNC80 mutations as causative of these siblings' disorder, biallelic mutations in UNC80 have recently been described among individuals with an overlapping phenotype. This report expands the disease spectrum associated with UNC80 mutations. © 2016 Wiley Periodicals, Inc.


Asunto(s)
Proteínas Portadoras/genética , Discapacidades del Desarrollo/genética , Insuficiencia de Crecimiento/genética , Proteínas de la Membrana/genética , Paraplejía/genética , Niño , Preescolar , Discapacidades del Desarrollo/complicaciones , Discapacidades del Desarrollo/fisiopatología , Exoma/genética , Insuficiencia de Crecimiento/complicaciones , Insuficiencia de Crecimiento/fisiopatología , Femenino , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Paraplejía/complicaciones , Paraplejía/fisiopatología , Estabilidad del ARN/genética , Hermanos
15.
Ann Clin Transl Neurol ; 11(5): 1250-1266, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38544359

RESUMEN

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.


Asunto(s)
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/métodos
16.
Neurol Genet ; 7(1): e554, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33977140

RESUMEN

OBJECTIVE: To describe the diagnostic utility of whole-genome sequencing and RNA studies in boys with suspected dystrophinopathy, for whom multiplex ligation-dependent probe amplification and exomic parallel sequencing failed to yield a genetic diagnosis, and to use remnant normal DMD splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia. METHODS: Exome, genome, and/or muscle RNA sequencing was performed for 7 males with elevated creatine kinase. PCR of muscle-derived complementary DNA (cDNA) studied consequences for DMD premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle. RESULTS: Splice-altering intronic single nucleotide variants or structural rearrangements in DMD were identified in all 7 families. Four individuals, with abnormal splicing causing a premature stop codon and nonsense-mediated decay, expressed remnant levels of normally spliced DMD mRNA. Quantitative Western blot enabled correlation of wild-type dystrophin and clinical severity, with 0%-5% dystrophin conferring a Duchenne phenotype, 10% ± 2% a Becker phenotype, and 15% ± 2% dystrophin associated with myalgia without manifesting weakness. CONCLUSIONS: Whole-genome sequencing relied heavily on RNA studies to identify DMD splice-altering variants. Short-read RNA sequencing was regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant DMD mRNA. PCR of muscle cDNA provided a simple, yet informative approach. Highly relevant to genetic therapies for dystrophinopathies, our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy, with the collective conclusion that a fractional increase in levels of normal dystrophin between 5% and 20% is clinically significant.

17.
Skelet Muscle ; 8(1): 23, 2018 07 30.
Artículo en Inglés | MEDLINE | ID: mdl-30060766

RESUMEN

BACKGROUND: Dystroglycanopathies are a clinically and genetically heterogeneous group of disorders that are typically characterised by limb-girdle muscle weakness. Mutations in 18 different genes have been associated with dystroglycanopathies, the encoded proteins of which typically modulate the binding of α-dystroglycan to extracellular matrix ligands by altering its glycosylation. This results in a disruption of the structural integrity of the myocyte, ultimately leading to muscle degeneration. METHODS: Deep phenotypic information was gathered using the PhenoTips online software for 1001 patients with unexplained limb-girdle muscle weakness from 43 different centres across 21 European and Middle Eastern countries. Whole-exome sequencing with at least 250 ng DNA was completed using an Illumina exome capture and a 38 Mb baited target. Genes known to be associated with dystroglycanopathies were analysed for disease-causing variants. RESULTS: Suspected pathogenic variants were detected in DPM3, ISPD, POMT1 and FKTN in one patient each, in POMK in two patients, in GMPPB in three patients, in FKRP in eight patients and in POMT2 in ten patients. This indicated a frequency of 2.7% for the disease group within the cohort of 1001 patients with unexplained limb-girdle muscle weakness. The phenotypes of the 27 patients were highly variable, yet with a fundamental presentation of proximal muscle weakness and elevated serum creatine kinase. CONCLUSIONS: Overall, we have identified 27 patients with suspected pathogenic variants in dystroglycanopathy-associated genes. We present evidence for the genetic and phenotypic diversity of the dystroglycanopathies as a disease group, while also highlighting the advantage of incorporating next-generation sequencing into the diagnostic pathway of rare diseases.


Asunto(s)
Variación Genética , Distrofia Muscular de Cinturas/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Distroglicanos/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Glicosilación , Heterocigoto , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Proteínas Musculares/genética , Distrofia Muscular de Cinturas/metabolismo , Mutación , Fenotipo , Secuenciación del Exoma/métodos , Adulto Joven
18.
Front Med (Lausanne) ; 4: 62, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28603714

RESUMEN

Traditionally, the use of genomic information for personalized medical decisions relies on prior discovery and validation of genotype-phenotype associations. This approach constrains care for patients presenting with undescribed problems. The National Institutes of Health (NIH) Undiagnosed Diseases Program (UDP) hypothesized that defining disease as maladaptation to an ecological niche allows delineation of a logical framework to diagnose and evaluate such patients. Herein, we present the philosophical bases, methodologies, and processes implemented by the NIH UDP. The NIH UDP incorporated use of the Human Phenotype Ontology, developed a genomic alignment strategy cognizant of parental genotypes, pursued agnostic biochemical analyses, implemented functional validation, and established virtual villages of global experts. This systematic approach provided a foundation for the diagnostic or non-diagnostic answers provided to patients and serves as a paradigm for scalable translational research.

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