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1.
BMC Gastroenterol ; 21(1): 339, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34470610

RESUMO

AIM: To discover the novel ATP7B mutations in 103 southern Chinese patients with Wilson disease (WD), and to determine the spectrum and frequency of mutations in the ATP7B gene and genotype-phenotype correlation in a large-scale sample of Chinese WD patients. METHODS: One hundred three WD patients from 101 unrelated families in southern China were enrolled in this study. Genomic DNA was extracted from the peripheral blood. Direct sequencing of all 21 exons within ATP7B was performed. Subsequently, an extensive study of the overall spectrum and frequency of ATP7B mutations and genotype-phenotype correlation was performed in all Chinese patients eligible from the literature, combined with the current southern group. RESULTS: In 103 patients with WD, we identified 48 different mutations (42 missense mutations, 4 nonsense mutations and 2 frameshifts). Of these, 3 mutations had not been previously reported: c.1510_1511insA, c.2233C>A (p.Leu745Met) and c.3824T>C (p.Leu1275Ser). The c.2333G>T (p.Arg778 Leu) at exon 8, was the most common mutation with an allelic frequency of 18.8%, followed by c.2975C>T (p.Pro992Leu) at exon 13, with an allelic frequency of 13.4%. In the comprehensive study, 233 distinct mutations were identified, including 154 missense mutations, 23 nonsense mutations and 56 frameshifts. Eighty-five variants were identified as novel mutations. The c.2333G>T (p.Arg778 Leu) and c.2975C>T (p.Pro992Leu) were the most common mutations, with allelic frequencies of 28.6% and 13.0%, respectively. Exons 8, 12, 13, 16 and 18 were recognised as hotspot exons. Phenotype-genotype correlation analysis suggested that c.2333G>T (p.Arg778 Leu) was significantly associated with lower levels of serum ceruloplasmin (P = 0.034). c.2975C>T (p.Pro992Leu) was correlated with earlier age of disease onset (P = 0.002). Additionally, we found that the c.3809A>G (p.Asn1270Ser) mutation significantly indicated younger onset age (P = 0.012), and the c.3884C>T (p.Ala1295Val) mutation at exon 18 was significantly associated with hepatic presentation (P = 0.048). Moreover, the patients with mixed presentation displayed the initial WD features at an older onset age than the groups with either liver disease or neurological presentation (P = 0.039, P = 0.015, respectively). No significant difference was observed in the presence of KF rings among the three groups with different clinical manifestations. CONCLUSION: In this study, we identified three novel mutations in 103 WD patients from the southern part of China, which could enrich the previously established mutational spectrum of the ATP7B gene. Moreover, we tapped into a large-scale study of a Chinese WD cohort to characterise the overall phenotypic and genotypic spectra and assess the association between genotype and phenotype, which enhances the current knowledge about the population genetics of WD in China.


Assuntos
Proteínas de Transporte de Cátions , ATPases Transportadoras de Cobre , Degeneração Hepatolenticular , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/genética , China , ATPases Transportadoras de Cobre/genética , Análise Mutacional de DNA , Estudos de Associação Genética , Genótipo , Degeneração Hepatolenticular/genética , Humanos , Mutação
2.
J Int Med Res ; 49(8): 3000605211035895, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34433328

RESUMO

OBJECTIVE: Tuberous sclerosis (TSC) is an autosomal dominant disorder, often detected during childhood. We present the results of genetic testing in a newborn with suspected TSC. METHODS: A newborn with no specific clinical manifestations of TSC showed evidence of TSC on magnetic resonance imaging and echocardiography. Next-generation sequencing (NGS) and multiple ligation-dependent probe amplification (MLPA) of the TSC1 and TSC2 gene exons were carried out to confirm the diagnosis. RESULTS: The results of MLPA were negative, but NGS showed a heterozygous mutation in the TSC1 gene comprising insertion of a T residue at c.2165 (exon 17) to c.2166 (exon 17), indicating a loss of function mutation. These results were verified by Sanger sequencing. This genetic change was present in the newborn but the parental genotypes were wild-type, indicating a de novo mutation. CONCLUSIONS: In this case, a case of TSC caused by a heterozygous mutation in the TSC1 gene was confirmed by NGS sequencing. This indicates the suitability of genetic testing for the early diagnosis of clinically rare and difficult-to-diagnose diseases, to guide clinical treatment.


Assuntos
Proteína 1 do Complexo Esclerose Tuberosa , Esclerose Tuberosa , Análise Mutacional de DNA , Diagnóstico Precoce , Humanos , Recém-Nascido , Mutação , Esclerose Tuberosa/diagnóstico por imagem , Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteínas Supressoras de Tumor/genética
3.
Viruses ; 13(7)2021 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-34372543

RESUMO

Human immunodeficiency virus type 2 (HIV-2) accumulates fewer mutations during replication than HIV type 1 (HIV-1). Advanced studies of HIV-2 mutagenesis, however, have historically been confounded by high background error rates in traditional next-generation sequencing techniques. In this study, we describe the adaptation of the previously described maximum-depth sequencing (MDS) technique to studies of both HIV-1 and HIV-2 for the ultra-accurate characterization of viral mutagenesis. We also present the development of a user-friendly Galaxy workflow for the bioinformatic analyses of sequencing data generated using the MDS technique, designed to improve replicability and accessibility to molecular virologists. This adapted MDS technique and analysis pipeline were validated by comparisons with previously published analyses of the frequency and spectra of mutations in HIV-1 and HIV-2 and is readily expandable to studies of viral mutation across the genomes of both viruses. Using this novel sequencing pipeline, we observed that the background error rate was reduced 100-fold over standard Illumina error rates, and 10-fold over traditional unique molecular identifier (UMI)-based sequencing. This technical advancement will allow for the exploration of novel and previously unrecognized sources of viral mutagenesis in both HIV-1 and HIV-2, which will expand our understanding of retroviral diversity and evolution.


Assuntos
HIV-1/genética , HIV-2/genética , Análise de Sequência de DNA/métodos , Biologia Computacional/métodos , Análise Mutacional de DNA/métodos , Genoma Viral/genética , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação/genética , Fluxo de Trabalho
4.
Medicine (Baltimore) ; 100(32): e26911, 2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34397927

RESUMO

RATIONALE: With the recent advancements in molecular biology research, epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have emerged as excellent therapies for patients with EGFR-mutant cancers. However, these patients inevitably develop cross-acquired resistance to EGFR-TKIs. Transformation to small-cell lung cancer (SCLC) is considered a rare resistance mechanism against EGFR-TKI therapy. Here, we report a case of TKI resistance due to SCLC transformation and demonstrate its mechanisms and clinical features. PATIENT CONCERNS: A 54-year-old Chinese man with a history of smoking for 40 years complained of an intermittent cough in March 2019. DIAGNOSIS: Transbronchial lung biopsy was performed on the basal segment of the left lower lobe, which confirmed lung adenocarcinoma. In January 2020, repeat biopsy was performed, and the results of immunohistochemistry (IHC) staining showed TTF-1 (+), CK7 (+), napsin A (+), syn (+), and CD56 (+), with a Ki-67 (+) index 80% of small cell carcinomas. Infiltrating adenocarcinomas and small cell carcinomas were observed. INTERVENTIONS: Icotinib (125 mg thrice daily) was administered as a first-line treatment from June 2019. We subsequently administered a chemotherapy regimen consisting of etoposide (180 mg, days 1-3) plus cisplatin (45 mg, days 1-3) every 3 weeks for 1 cycle after recurrence. As the patient could not tolerate further chemotherapy, he continued taking icotinib orally and received whole-brain radiotherapy 10 times to a total dose of 30 Gy after brain metastases. OUTCOMES: The patient relapsed after successful treatment with icotinib for 9 months. A partial response was achieved after 4 cycles of chemotherapy, and despite the brief success of chemotherapy, our patient exhibited brain metastasis and metastases of the eleventh thoracic spine and the second lumbar vertebra with pathological fracture. The patient eventually died of aggressive cancer progression. LESSONS: Our case highlights the possibility of SCLC transformation from EGFR-mutant adenocarcinoma and the importance of repeat biopsy for drug resistance. Serum neuron-specific enolase levels may also be useful for detecting early SCLC transformation.


Assuntos
Adenocarcinoma de Pulmão/genética , DNA de Neoplasias/genética , Neoplasias Pulmonares/genética , Mutação , Carcinoma de Pequenas Células do Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Biópsia , Transformação Celular Neoplásica , Análise Mutacional de DNA , Receptores ErbB/genética , Receptores ErbB/metabolismo , Evolução Fatal , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Carcinoma de Pequenas Células do Pulmão/metabolismo , Tomografia Computadorizada por Raios X
5.
Crit Rev Oncol Hematol ; 165: 103430, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34339834

RESUMO

Diffuse large B-cell lymphoma (DLBCL), the most common type of Non-Hodgkin lymphoma (NHL), is a highly heterogeneous and aggressive disease. Regardless of this heterogeneity, all patients receive the same first-line therapy, which fails in 30-40 % of patients, who are either refractory or relapse after remission. With the aim of stratifying patients to improve treatment outcome, different clinical and genetic biomarkers have been studied. The present systematic review aimed to identify somatic mutations that could serve as prognosis biomarkers or as therapeutic target mutations in DLBCL. Regarding their role as prognostic markers, mutations in CD58 and TP53 seem the most promising predictors of poor outcome although the combination of different alterations and other prognostic factors could be a more powerful strategy. On the other hand, different approaches regarding targeted therapy have been proposed. Therefore, mutational analysis could help guide treatment choice in DLBCL yet further studies and clinical trials are needed.


Assuntos
Linfoma Difuso de Grandes Células B , Recidiva Local de Neoplasia , Análise Mutacional de DNA , Humanos , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Mutação , Prognóstico
6.
Klin Monbl Augenheilkd ; 238(7): 773-780, 2021 Jul.
Artigo em Inglês, Alemão | MEDLINE | ID: mdl-34376007

RESUMO

Over the last ten years, much has been learnt about the genetic characteristics and genetic evolution of uveal melanoma. It has been shown that uveal melanoma differs fundamentally from non-uveal melanoma and is an independent genetic subtype. Compared to other tumours, uveal melanoma has a low mutational burden. There are recurring chromosomal aberrations with losses of 1p, 6q, 8p and 16q, gains of 6p and 8q, and the presence of monosomy 3. GNAQ, GNA11, PLCB4, CYSLTR2, MAPKAPK5, as well as mutations in BAP1, SF3B1, SRSF2 and EIF1AX, the latter being linked to a higher risk of metastasis, have been identified as significantly mutated genes. In rare cases, a BAP1 germline mutation may also be present. In addition to higher risk of uveal melanoma, this variant is also linked with other tumours. In this case, additional work-up, genetic counselling and screening of family members should be offered. While the knowledge about the genetic characteristics of uveal melanoma is already routinely used for diagnostic and prognostic purposes, targeted genotype-dependent therapy of uveal melanoma is currently still missing.


Assuntos
Melanoma , Ubiquitina Tiolesterase , Neoplasias Uveais , Análise Mutacional de DNA , Humanos , Melanoma/diagnóstico , Melanoma/genética , Mutação , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Neoplasias Uveais/diagnóstico , Neoplasias Uveais/genética
7.
Science ; 373(6558): 1030-1035, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34385354

RESUMO

Biological mechanisms underlying human germline mutations remain largely unknown. We statistically decompose variation in the rate and spectra of mutations along the genome using volume-regularized nonnegative matrix factorization. The analysis of a sequencing dataset (TOPMed) reveals nine processes that explain the variation in mutation properties between loci. We provide a biological interpretation for seven of these processes. We associate one process with bulky DNA lesions that are resolved asymmetrically with respect to transcription and replication. Two processes track direction of replication fork and replication timing, respectively. We identify a mutagenic effect of active demethylation primarily acting in regulatory regions and a mutagenic effect of long interspersed nuclear elements. We localize a mutagenic process specific to oocytes from population sequencing data. This process appears transcriptionally asymmetric.


Assuntos
Genoma Humano , Mutação em Linhagem Germinativa , Algoritmos , Ilhas de CpG , Dano ao DNA , Desmetilação do DNA , Análise Mutacional de DNA , Replicação do DNA , Variação Genética , Células Germinativas , Humanos , Elementos Nucleotídeos Longos e Dispersos , Mutagênese , Oócitos/fisiologia , Transcrição Genética
9.
Nat Commun ; 12(1): 4423, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285218

RESUMO

The Cancer Genome Atlas (TCGA) is one of the largest biorepositories of digital histology. Deep learning (DL) models have been trained on TCGA to predict numerous features directly from histology, including survival, gene expression patterns, and driver mutations. However, we demonstrate that these features vary substantially across tissue submitting sites in TCGA for over 3,000 patients with six cancer subtypes. Additionally, we show that histologic image differences between submitting sites can easily be identified with DL. Site detection remains possible despite commonly used color normalization and augmentation methods, and we quantify the image characteristics constituting this site-specific digital histology signature. We demonstrate that these site-specific signatures lead to biased accuracy for prediction of features including survival, genomic mutations, and tumor stage. Furthermore, ethnicity can also be inferred from site-specific signatures, which must be accounted for to ensure equitable application of DL. These site-specific signatures can lead to overoptimistic estimates of model performance, and we propose a quadratic programming method that abrogates this bias by ensuring models are not trained and validated on samples from the same site.


Assuntos
Biomarcadores Tumorais/análise , Aprendizado Profundo , Processamento de Imagem Assistida por Computador/métodos , Neoplasias/patologia , Manejo de Espécimes/métodos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Análise Mutacional de DNA/métodos , Confiabilidade dos Dados , Perfilação da Expressão Gênica/métodos , Humanos , Mutação , Estadiamento de Neoplasias , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/mortalidade , Medição de Risco/métodos
10.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299376

RESUMO

Redß is a 261 amino acid protein from bacteriophage λ that promotes a single-strand annealing (SSA) reaction for repair of double-stranded DNA (dsDNA) breaks. While there is currently no high-resolution structure available for Redß, models of its DNA binding domain (residues 1-188) have been proposed based on homology with human Rad52, and a crystal structure of its C-terminal domain (CTD, residues 193-261), which binds to λ exonuclease and E. coli single-stranded DNA binding protein (SSB), has been determined. To evaluate these models, the 14 lysine residues of Redß were mutated to alanine, and the variants tested for recombination in vivo and DNA binding and annealing in vitro. Most of the lysines within the DNA binding domain, including K36, K61, K111, K132, K148, K154, and K172, were found to be critical for DNA binding in vitro and recombination in vivo. By contrast, none of the lysines within the CTD, including K214, K245, K251, K253, and K258 were required for DNA binding in vitro, but two, K214 and K253, were critical for recombination in vivo, likely due to their involvement in binding to SSB. K61 was identified as a residue that is critical for DNA annealing, but not for initial ssDNA binding, suggesting a role in binding to the second strand of DNA incorporated into the complex. The K148A variant, which has previously been shown to be defective in oligomer formation, had the lowest affinity for ssDNA, and was the only variant that was completely non-cooperative, suggesting that ssDNA binding is coupled to oligomerization.


Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Lisina/genética , Domínios Proteicos/genética , Proteínas Virais/genética , Células Cultivadas , Análise Mutacional de DNA/métodos , DNA de Cadeia Simples , Escherichia coli/genética , Humanos , Ligação Proteica/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Recombinação Genética/genética
11.
Mol Cell ; 81(14): 2989-3006.e9, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34197737

RESUMO

Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure-specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure-specific nuclease and BLM partner for replication fork restart. We find that elevated EXO5 in tumors correlates with increased mutation loads and poor patient survival, suggesting that EXO5 upregulation has oncogenic potential. Structural, mechanistic, and mutational analyses of EXO5 and EXO5-DNA complexes reveal a single-stranded DNA binding channel with an adjacent ATR phosphorylation motif (T88Q89) that regulates EXO5 nuclease activity and BLM binding identified by mass spectrometric analysis. EXO5 phospho-mimetic mutant rescues the restart defect from EXO5 depletion that decreases fork progression, DNA damage repair, and cell survival. EXO5 depletion furthermore rescues survival of FANCA-deficient cells and indicates EXO5 functions epistatically with SMARCAL1 and BLM. Thus, an EXO5 axis connects ATR and BLM in directing replication fork restart.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Replicação do DNA/genética , DNA/genética , Exonucleases/genética , Instabilidade Genômica/genética , RecQ Helicases/genética , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/genética , DNA Helicases/genética , Análise Mutacional de DNA/métodos , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , Mutação/genética , Oncogenes/genética , Fosforilação/genética , Regulação para Cima/genética
12.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199056

RESUMO

Palmoplantar keratodermas (PPKs) are characterized by thickness of stratum corneum and epidermal hyperkeratosis localized in palms and soles. PPKs can be epidermolytic (EPPK) or non epidermolytic (NEPPK). Specific mutations of keratin 16 (K16) and keratin 1 (K1) have been associated to EPPK, and NEPPK. Cases of mosaicism in PPKs due to somatic keratin mutations have also been described in scientific literature. We evaluated a patient presenting hyperkeratosis localized monolaterally in the right palmar area, characterized by linear yellowish hyperkeratotic lesions following the Blaschko lines. No other relatives of the patient showed any dermatological disease. Light and confocal histological analysis confirmed the presence of epidermolityic hyperkeratosis. Genetic analysis performed demonstrates the heterozygous deletion NM_006121.4:r.274_472del for a total of 198 nucleotides, in KRT1 cDNA obtained by a palmar lesional skin biopsy, corresponding to the protein mutation NP_006112.3:p.Gly71_Gly137del. DNA extracted from peripheral blood lymphocytes did not display the presence of the mutation. These results suggest a somatic mutation causing an alteration in K1 N-terminal variable domain (V1). The deleted sequence involves the ISIS subdomain, containing a lysine residue already described as fundamental for epidermal transglutaminases in the crosslinking of IF cytoskeleton. Moreover, a computational analysis of the wild-type and V1-mutated K1/K10 keratin dimers, suggests an unusual interaction between these keratin filaments. The mutation taster in silico analysis also returned a high probability for a deleterious mutation. These data demonstrate once again the importance of the head domain (V1) of K1 in the formation of a functional keratinocyte cytoskeleton. Moreover, this is a further demonstration of the presence of somatic mutations arising in later stages of the embryogenesis, generating a mosaic phenotype.


Assuntos
Queratina-10/química , Queratina-1/química , Queratina-1/genética , Nevo/etiologia , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência , Neoplasias Cutâneas/etiologia , Sequência de Aminoácidos , Sequência de Bases , Biópsia , Análise Mutacional de DNA , Imunofluorescência , Humanos , Imuno-Histoquímica , Queratina-1/metabolismo , Queratina-10/metabolismo , Modelos Moleculares , Nevo/metabolismo , Nevo/patologia , Conformação Proteica , Multimerização Proteica , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Relação Estrutura-Atividade
13.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202524

RESUMO

Left ventricular non-compaction cardiomyopathy (LVNC) is a rare heart disease, with or without left ventricular dysfunction, which is characterized by a two-layer structure of the myocardium and an increased number of trabeculae. The study of familial forms of LVNC is helpful for risk prediction and genetic counseling of relatives. Here, we present a family consisting of three members with LVNC. Using a next-generation sequencing approach a combination of two (likely) pathogenic nonsense mutations DSG2-p.S363X and TBX20-p.D278X was identified in all three patients. TBX20 encodes the cardiac T-box transcription factor 20. DSG2 encodes desmoglein-2, which is part of the cardiac desmosomes and belongs to the cadherin family. Since the identified nonsense variant (DSG2-p.S363X) is localized in the extracellular domain of DSG2, we performed in vitro cell transfection experiments. These experiments revealed the absence of truncated DSG2 at the plasma membrane, supporting the pathogenic relevance of DSG2-p.S363X. In conclusion, we suggest that in the future, these findings might be helpful for genetic screening and counseling of patients with LVNC.


Assuntos
Cardiomiopatias/diagnóstico , Cardiomiopatias/etiologia , Desmogleína 2/genética , Mutação , Proteínas com Domínio T/genética , Disfunção Ventricular Esquerda/diagnóstico , Disfunção Ventricular Esquerda/etiologia , Adulto , Células Cultivadas , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética/métodos , Predisposição Genética para Doença , Testes de Função Cardíaca , Humanos , Imageamento por Ressonância Magnética/métodos , Linhagem , Avaliação de Sintomas
14.
J Stroke Cerebrovasc Dis ; 30(9): 105997, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34303089

RESUMO

OBJECTIVES: To identify the underlying genetic defect for a consanguineous family with an unusually high number of members affected by cerebral small vessel disease. MATERIALS AND METHODS: A total of 6 individuals, of whom 3 are severely affected, from the family were clinically and radiologically evaluated. SNP genotyping was performed in multiple members to demonstrate genome-wide runs-of-homozygosity. Coding variants in the most likely candidate gene, HTRA1 were explored by Sanger sequencing. Published HTRA1-related phenotypes were extensively reviewed to explore the effect of number of affected alleles on phenotypic expression. RESULTS: Genome-wide homozygosity mapping identified a 3.2 Mbp stretch on chromosome 10q26.3 where HTRA1 gene is located. HTRA1 sequencing revealed an evolutionarily conserved novel homozygous c.824C>T (p.Pro275Leu) mutation, affecting the serine protease domain of HtrA1. Early-onset of cognitive and motor deterioration in homozygotes are in consensus with CARASIL. However, there was a clear phenotypic variability between homozygotes which includes alopecia, a suggested hallmark of CARASIL. All heterozygotes, presenting as CADASIL type 2, had spinal disk degeneration and several neuroimaging findings, including leukoencephalopathy and microhemorrhage despite a lack of severe clinical presentation. CONCLUSION: Here, we clearly demonstrate that CARASIL and CADASIL type 2 are two clinical consequences of the same disorder with different severities thorough the evaluation of the largest collection of homozygotes and heterozygotes segregating in a family. Considering the semi-dominant inheritance of HTRA1-related phenotypes, genetic testing and clinical follow-up must be offered for all members of a family with HTRA1 mutations regardless of symptoms.


Assuntos
Alopecia/genética , CADASIL/genética , Infarto Cerebral/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Leucoencefalopatias/genética , Mutação , Doenças da Coluna Vertebral/genética , Adulto , Idade de Início , Alopecia/diagnóstico , Alopecia/fisiopatologia , CADASIL/diagnóstico , CADASIL/fisiopatologia , Infarto Cerebral/diagnóstico , Infarto Cerebral/fisiopatologia , Consanguinidade , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Hereditariedade , Heterozigoto , Homozigoto , Humanos , Leucoencefalopatias/diagnóstico , Leucoencefalopatias/fisiopatologia , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Índice de Gravidade de Doença , Doenças da Coluna Vertebral/diagnóstico , Doenças da Coluna Vertebral/fisiopatologia
15.
Am J Hum Genet ; 108(7): 1283-1300, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34214447

RESUMO

Most rare clinical missense variants cannot currently be classified as pathogenic or benign. Deficiency in human 5,10-methylenetetrahydrofolate reductase (MTHFR), the most common inherited disorder of folate metabolism, is caused primarily by rare missense variants. Further complicating variant interpretation, variant impacts often depend on environment. An important example of this phenomenon is the MTHFR variant p.Ala222Val (c.665C>T), which is carried by half of all humans and has a phenotypic impact that depends on dietary folate. Here we describe the results of 98,336 variant functional-impact assays, covering nearly all possible MTHFR amino acid substitutions in four folinate environments, each in the presence and absence of p.Ala222Val. The resulting atlas of MTHFR variant effects reveals many complex dependencies on both folinate and p.Ala222Val. MTHFR atlas scores can distinguish pathogenic from benign variants and, among individuals with severe MTHFR deficiency, correlate with age of disease onset. Providing a powerful tool for understanding structure-function relationships, the atlas suggests a role for a disordered loop in retaining cofactor at the active site and identifies variants that enable escape of inhibition by S-adenosylmethionine. Thus, a model based on eight MTHFR variant effect maps illustrates how shifting landscapes of environment- and genetic-background-dependent missense variation can inform our clinical, structural, and functional understanding of MTHFR deficiency.


Assuntos
Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Análise Mutacional de DNA , Diploide , Biblioteca Gênica , Genótipo , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/fisiologia , Saccharomyces cerevisiae/genética
16.
Nat Commun ; 12(1): 4172, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234141

RESUMO

Cell-free DNA (cfDNA) is attractive for many applications, including detecting cancer, identifying the tissue of origin, and monitoring. A fundamental task underlying these applications is SNV calling from cfDNA, which is hindered by the very low tumor content. Thus sensitive and accurate detection of low-frequency mutations (<5%) remains challenging for existing SNV callers. Here we present cfSNV, a method incorporating multi-layer error suppression and hierarchical mutation calling, to address this challenge. Furthermore, by leveraging cfDNA's comprehensive coverage of tumor clonal landscape, cfSNV can profile mutations in subclones. In both simulated and real patient data, cfSNV outperforms existing tools in sensitivity while maintaining high precision. cfSNV enhances the clinical utilities of cfDNA by improving mutation detection performance in medium-depth sequencing data, therefore making Whole-Exome Sequencing a viable option. As an example, we demonstrate that the tumor mutation profile from cfDNA WES data can provide an effective biomarker to predict immunotherapy outcomes.


Assuntos
DNA Tumoral Circulante/genética , Análise Mutacional de DNA/métodos , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias/genética , Sequenciamento Completo do Exoma/métodos , Adulto , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Biópsia , DNA Tumoral Circulante/sangue , Simulação por Computador , Conjuntos de Dados como Assunto , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias/sangue , Neoplasias/tratamento farmacológico , Neoplasias/mortalidade , Polimorfismo de Nucleotídeo Único , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Intervalo Livre de Progressão , Sensibilidade e Especificidade
17.
Nat Commun ; 12(1): 4178, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234147

RESUMO

Mosaic loss of chromosome Y (LOY) in leukocytes is the most common form of clonal mosaicism, caused by dysregulation in cell-cycle and DNA damage response pathways. Previous genetic studies have focussed on identifying common variants associated with LOY, which we now extend to rarer, protein-coding variation using exome sequences from 82,277 male UK Biobank participants. We find that loss of function of two genes-CHEK2 and GIGYF1-reach exome-wide significance. Rare alleles in GIGYF1 have not previously been implicated in any complex trait, but here loss-of-function carriers exhibit six-fold higher susceptibility to LOY (OR = 5.99 [3.04-11.81], p = 1.3 × 10-10). These same alleles are also associated with adverse metabolic health, including higher susceptibility to Type 2 Diabetes (OR = 6.10 [3.51-10.61], p = 1.8 × 10-12), 4 kg higher fat mass (p = 1.3 × 10-4), 2.32 nmol/L lower serum IGF1 levels (p = 1.5 × 10-4) and 4.5 kg lower handgrip strength (p = 4.7 × 10-7) consistent with proposed GIGYF1 enhancement of insulin and IGF-1 receptor signalling. These associations are mirrored by a common variant nearby associated with the expression of GIGYF1. Our observations highlight a potential direct connection between clonal mosaicism and metabolic health.


Assuntos
Proteínas de Transporte/genética , Cromossomos Humanos Y/genética , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Mosaicismo , Adulto , Idoso , Proteínas de Transporte/metabolismo , Estudos de Casos e Controles , Análise Mutacional de DNA , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Estudo de Associação Genômica Ampla , Humanos , Insulina/metabolismo , Leucócitos , Mutação com Perda de Função , Masculino , Pessoa de Meia-Idade , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/genética , Sequenciamento Completo do Exoma
18.
Nat Commun ; 12(1): 4217, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244513

RESUMO

The functional consequences of genetic variants within 5' untranslated regions (UTRs) on a genome-wide scale are poorly understood in disease. Here we develop a high-throughput multi-layer functional genomics method called PLUMAGE (Pooled full-length UTR Multiplex Assay on Gene Expression) to quantify the molecular consequences of somatic 5' UTR mutations in human prostate cancer. We show that 5' UTR mutations can control transcript levels and mRNA translation rates through the creation of DNA binding elements or RNA-based cis-regulatory motifs. We discover that point mutations can simultaneously impact transcript and translation levels of the same gene. We provide evidence that functional 5' UTR mutations in the MAP kinase signaling pathway can upregulate pathway-specific gene expression and are associated with clinical outcomes. Our study reveals the diverse mechanisms by which the mutational landscape of 5' UTRs can co-opt gene expression and demonstrates that single nucleotide alterations within 5' UTRs are functional in cancer.


Assuntos
Regiões 5' não Traduzidas/genética , Análise Mutacional de DNA/métodos , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Neoplasias da Próstata/genética , Linhagem Celular Tumoral , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Mutação Puntual , Próstata/patologia , Neoplasias da Próstata/patologia , Biossíntese de Proteínas/genética , RNA-Seq
19.
Nat Commun ; 12(1): 4254, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253723

RESUMO

Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis.


Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Membrana Celular/metabolismo , Lipoproteínas/biossíntese , Acilação , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Sequência Conservada , Cisteína/metabolismo , Análise Mutacional de DNA , Processamento de Proteína Pós-Traducional , Relação Estrutura-Atividade , Especificidade por Substrato
20.
J Stroke Cerebrovasc Dis ; 30(8): 105674, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34119749

RESUMO

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), which is caused by the Notch3 gene mutation, has its unique clinical and imaging characteristics. Here we present a Chinese family with a novel mutation on exon 10 of Notch3 gene. METHODS: Clinical and MRI data of the three patients in the family during the 7-year follow-up were collected. The CADASIL Scale Score was calculated to evaluate the disease risk of the three patients at their first admission or clinic visit. Five family members underwent genetic test. RESULTS: Genetic test confirmed the diagnosis of CADASIL in this family. A novel mutation of p.C533S on exon 10 of Notch3 gene was detected. The CADASIL score of the proband and her sister was both 17 and that of her brother was 14. CONCLUSIONS: Our report not only expands the mutation spectrum of Notch3 gene in CADASIL, but also shows the distinct heterogeneity of CADASIL patients in the same family with the same mutation.


Assuntos
CADASIL/genética , Mutação de Sentido Incorreto , Receptor Notch3/genética , Adulto , Grupo com Ancestrais do Continente Asiático/genética , CADASIL/diagnóstico , CADASIL/etnologia , China , Análise Mutacional de DNA , Éxons , Feminino , Predisposição Genética para Doença , Hereditariedade , Heterozigoto , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo
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