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1.
Nature ; 630(8017): 752-761, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38867045

RESUMEN

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases1,2. Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired3,4. However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing.


Asunto(s)
Disparidad de Par Base , Daño del ADN , ADN de Cadena Simple , Análisis de Secuencia de ADN , Imagen Individual de Molécula , Humanos , Envejecimiento/genética , Desaminasas APOBEC/genética , Desaminasas APOBEC/metabolismo , Disparidad de Par Base/genética , Citidina Desaminasa/metabolismo , Citidina Desaminasa/genética , Citosina/metabolismo , Desaminación , Daño del ADN/genética , Reparación de la Incompatibilidad de ADN/genética , Replicación del ADN/genética , ADN de Cadena Simple/genética , Genoma Mitocondrial/genética , Mutación , Neoplasias/genética , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/normas , Imagen Individual de Molécula/métodos , Masculino , Femenino
2.
Hum Mol Genet ; 32(9): 1552-1564, 2023 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-36611016

RESUMEN

Congenital myasthenic syndrome (CMS) is a heterogeneous condition associated with 34 different genes, including SLC5A7, which encodes the high-affinity choline transporter 1 (CHT1). CHT1 is expressed in presynaptic neurons of the neuromuscular junction where it uses the inward sodium gradient to reuptake choline. Biallelic CHT1 mutations often lead to neonatal lethality, and less commonly to non-lethal motor weakness and developmental delays. Here, we report detailed biochemical characterization of two novel mutations in CHT1, p.I294T and p.D349N, which we identified in an 11-year-old patient with a history of neonatal respiratory distress, and subsequent hypotonia and global developmental delay. Heterologous expression of each CHT1 mutant in human embryonic kidney cells showed two different mechanisms of reduced protein function. The p.I294T CHT1 mutant transporter function was detectable, but its abundance and half-life were significantly reduced. In contrast, the p.D349N CHT1 mutant was abundantly expressed at the cell membrane, but transporter function was absent. The residual function of the p.I294T CHT1 mutant may explain the non-lethal form of CMS in this patient, and the divergent mechanisms of reduced CHT1 function that we identified may guide future functional studies of the CHT1 myasthenic syndrome. Based on these in vitro studies that provided a diagnosis, treatment with cholinesterase inhibitor together with physical and occupational therapy significantly improved the patient's strength and quality of life.


Asunto(s)
Proteínas Mutantes , Mutación , Síndromes Miasténicos Congénitos , Simportadores , Síndromes Miasténicos Congénitos/tratamiento farmacológico , Síndromes Miasténicos Congénitos/genética , Síndromes Miasténicos Congénitos/metabolismo , Síndromes Miasténicos Congénitos/rehabilitación , Humanos , Masculino , Niño , Células HEK293 , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Semivida , Membrana Celular/metabolismo , Transporte de Proteínas , Estaurosporina/farmacología , Bromuro de Piridostigmina/uso terapéutico , Calidad de Vida , Simportadores/química , Simportadores/genética , Simportadores/metabolismo
3.
bioRxiv ; 2023 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-36824744

RESUMEN

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other genetic diseases1-4. Almost all of these mosaic mutations begin as nucleotide mismatches or damage in only one of the two strands of the DNA prior to becoming double-strand mutations if unrepaired or misrepaired5. However, current DNA sequencing technologies cannot resolve these initial single-strand events. Here, we developed a single-molecule, long-read sequencing method that achieves single-molecule fidelity for single-base substitutions when present in either one or both strands of the DNA. It also detects single-strand cytosine deamination events, a common type of DNA damage. We profiled 110 samples from diverse tissues, including from individuals with cancer-predisposition syndromes, and define the first single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumors deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples deficient in only polymerase proofreading. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. Since the double-strand DNA mutations interrogated by prior studies are only the endpoint of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable new studies of how mutations arise in a variety of contexts, especially in cancer and aging.

4.
Nat Commun ; 14(1): 3090, 2023 05 29.
Artículo en Inglés | MEDLINE | ID: mdl-37248219

RESUMEN

Long-read HiFi genome sequencing allows for accurate detection and direct phasing of single nucleotide variants, indels, and structural variants. Recent algorithmic development enables simultaneous detection of CpG methylation for analysis of regulatory element activity directly in HiFi reads. We present a comprehensive haplotype resolved 5-base HiFi genome sequencing dataset from a rare disease cohort of 276 samples in 152 families to identify rare (~0.5%) hypermethylation events. We find that 80% of these events are allele-specific and predicted to cause loss of regulatory element activity. We demonstrate heritability of extreme hypermethylation including rare cis variants associated with short (~200 bp) and large hypermethylation events (>1 kb), respectively. We identify repeat expansions in proximal promoters predicting allelic gene silencing via hypermethylation and demonstrate allelic transcriptional events downstream. On average 30-40 rare hypermethylation tiles overlap rare disease genes per patient, providing indications for variation prioritization including a previously undiagnosed pathogenic allele in DIP2B causing global developmental delay. We propose that use of HiFi genome sequencing in unsolved rare disease cases will allow detection of unconventional diseases alleles due to loss of regulatory element activity.


Asunto(s)
Metilación de ADN , Enfermedades Raras , Humanos , Haplotipos , Enfermedades Raras/genética , Metilación de ADN/genética , Análisis de Secuencia de ADN , Secuencia de Bases , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas del Tejido Nervioso/genética
5.
J Prim Care Community Health ; 12: 21501327211046734, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34583568

RESUMEN

INTRODUCTION: Primary care physicians (PCPs) are considered the gatekeepers of genetic services, but they often underutilize or inappropriately utilize such services, leading to lack of early treatment, incorrect diagnoses, and unnecessary procedures. This study aims to delineate PCP referral patterns, including the frequency of, motivators for, and barriers to genetic referrals and testing in the present landscape of genomics. METHODS: A 34-item online survey was distributed to PCPs in the United States (US). PCP demographics, practice characteristics, and referral patterns, motivators, and barriers were analyzed. Six hypothetical clinical scenarios included in the survey also were presented to a cohort of clinical geneticists. We calculated PCPs' rates of ordering genetic tests and of referral to genetics services in the past year. Rates and responses to clinical scenarios were compared based on respondents' personal and practice characteristics. RESULTS: A total of 95 PCPs and 25 clinical geneticists participated. Among the PCPs, 79% reported referring and 50% reported ordering genetic testing in the last year. PCPs with genetic counselors (GCs) in their clinic referred at significantly higher rates than those without (P = .008). White PCPs referred at significantly higher rates compared to Black or African American PCPs (P = .009). The most commonly reported motivators for referring patients to genetic services were preference for specialist coordination, lack of knowledge, and family's desire for risk information. The most commonly reported barriers were patient refusal, provider concerns about costs to patients, and uncertainty of when a genetic referral is appropriate. In response to clinical scenarios, clinical geneticists were in agreement about the need for genetic testing or referral for 2 of the scenarios. For these 2 scenarios, only 48% and 71% of PCPs indicated that they would offer genetic testing or referral, respectively. CONCLUSIONS: Responses to clinical scenarios suggest that it is not clear to PCPs when referrals or testing are needed. Collaboration with GCs is one approach to reducing barriers to and improving PCPs' utilization of genetic services. Clear guidelines from clinical geneticists may help facilitate appropriate use of genetics services by PCPs. Additional research is needed to further describe barriers that PCPs face in genetic testing/referrals.


Asunto(s)
Médicos de Atención Primaria , Derivación y Consulta , Personal de Salud , Humanos , Pautas de la Práctica en Medicina , Atención Primaria de Salud , Encuestas y Cuestionarios , Estados Unidos
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