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1.
Nat Commun ; 12(1): 2901, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006870

RESUMO

Proliferative chronic myelomonocytic leukemia (pCMML), an aggressive CMML subtype, is associated with dismal outcomes. RAS pathway mutations, mainly NRASG12D, define the pCMML phenotype as demonstrated by our exome sequencing, progenitor colony assays and a Vav-Cre-NrasG12D mouse model. Further, these mutations promote CMML transformation to acute myeloid leukemia. Using a multiomics platform and biochemical and molecular studies we show that in pCMML RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic kinases such as polo-like kinase 1 (PLK1). PLK1 transcript levels are shown to be regulated by an unmutated lysine methyl-transferase (KMT2A) resulting in increased promoter monomethylation of lysine 4 of histone 3. Pharmacologic inhibition of PLK1 in RAS mutant patient-derived xenografts, demonstrates the utility of personalized biomarker-driven therapeutics in pCMML.


Assuntos
Proteínas de Ciclo Celular/genética , GTP Fosfo-Hidrolases/genética , Histona-Lisina N-Metiltransferase/genética , Leucemia Mielomonocítica Crônica/genética , Proteínas de Membrana/genética , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Animais , Proteínas de Ciclo Celular/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Estimativa de Kaplan-Meier , Leucemia Mielomonocítica Crônica/metabolismo , Leucemia Mielomonocítica Crônica/terapia , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/genética , Transplante de Células-Tronco/métodos , Transplante Homólogo , Sequenciamento Completo do Exoma/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
2.
Biomed Res Int ; 2021: 9247541, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33959666

RESUMO

Atrioventricular block (AVB) is a leading cause of sudden cardiac death, and most of AVB cases are presented as autosomal dominant. The electrocardiogram of AVB patients presents an abnormal progressive cardiac conduction disorder between atria and ventricles. Transient receptor potential melastatin 4 (TRPM4) is a nonselective Ca2+-activated cation channel gene defined as a novel disease-causing gene of AVB. So far, 47 mutations of TRPM4 have been recorded in Human Gene Mutation Database. The aim of this study was to explore the relationship between TRPM4 mutation and pathogenesis of AVB. We investigated a Chinese family with AVB by whole-exome sequencing. An arrhythmia-related gene filtering strategy was used to analyze the disease-causing mutations. Three different bioinformatics programs were used to predict the effects of the mutation result. A novel mutation of TRPM4 was identified (c.2455C>T/p.R819C) and cosegregated in the affected family members. The three bioinformatics programs predicted that the novel mutation may lead to damage. Our study will contribute to expand the spectrum of TRPM4 mutations and supply accurate genetic testing information for further research and the clinical therapy of AVB.


Assuntos
Bloqueio Atrioventricular/genética , Mutação/genética , Canais de Cátion TRPM/genética , Sequenciamento Completo do Exoma/métodos , Adulto , Idoso , Criança , China , Feminino , Humanos , Masculino
3.
Pan Afr Med J ; 38: 111, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33912281

RESUMO

Millions of patients, with suspected complex neurogenetic disorders, living in resource limited regions around the world have no access to genetic testing despite the rapidly expanding availability and decreasing costs of genetic testing in first world nations. The barriers to increasing availability of genetic testing in resource limited nations are multifactorial but can be attributed, in large part, to a lack of awareness of the power of genetic testing to lead to a rapid, cost-effective, diagnosis that potentially will have profound clinical implications on treatment and patient outcomes. We report our experience with whole exome sequencing (WES) done for the first time in 5 patients of African descent with a suspected neurogenetic disorder living in a resource limited setting on the Eastern Caribbean island of Barbados. A diagnostic pathogenic mutation was found in 3 patients in the SCN1A, STXBP1 and SCN4A, who clinically were diagnosed with Dravet syndrome, Lennox-Gastaut syndrome, paramytonia and seizures respectively. A variant of undetermined significance was found in a patient with global developmental delays, hypotonia, with abnormal eye movements. In one patient WES was non-diagnostic. This result highlights the high yield of WES in carefully selected patients with a neurologic disease and the need for increase access to genetic testing in resource limited settings globally.


Assuntos
Testes Genéticos/métodos , Doenças do Sistema Nervoso/diagnóstico , Sequenciamento Completo do Exoma/métodos , Adulto , Barbados , Criança , Análise Custo-Benefício , Testes Genéticos/economia , Humanos , Lactente , Proteínas Munc18/genética , Mutação , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/fisiopatologia , Sequenciamento Completo do Exoma/economia , Adulto Jovem
4.
Int J Mol Sci ; 22(6)2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33801838

RESUMO

Sudden cardiac death (SCD) is a diagnostic challenge in forensic medicine. In a relatively large proportion of the SCDs, the deaths remain unexplained after autopsy. This challenge is likely caused by unknown disease mechanisms. Changes in DNA methylation have been associated with several heart diseases, but the role of DNA methylation in SCD is unknown. In this study, we investigated DNA methylation in two SCD subtypes, sudden unexplained death (SUD) and sudden unexpected death in epilepsy (SUDEP). We assessed DNA methylation of more than 850,000 positions in cardiac tissue from nine SUD and 14 SUDEP cases using the Illumina Infinium MethylationEPIC BeadChip. In total, six differently methylated regions (DMRs) between the SUD and SUDEP cases were identified. The DMRs were located in proximity to or overlapping genes encoding proteins that are a part of the glutathione S-transferase (GST) superfamily. Whole genome sequencing (WGS) showed that the DNA methylation alterations were not caused by genetic changes, while whole transcriptome sequencing (WTS) showed that DNA methylation was associated with expression levels of the GSTT1 gene. In conclusion, our results indicate that cardiac DNA methylation is similar in SUD and SUDEP, but with regional differential methylation in proximity to GST genes.


Assuntos
Metilação de DNA , Morte Súbita Cardíaca/etiologia , Predisposição Genética para Doença/etiologia , Glutationa Transferase/genética , Sequências Reguladoras de Ácido Nucleico/genética , Morte Súbita Inesperada na Epilepsia/etiologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Predisposição Genética para Doença/genética , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Sequenciamento Completo do Exoma/métodos , Sequenciamento Completo do Genoma/métodos , Adulto Jovem
5.
Nat Commun ; 12(1): 2028, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795686

RESUMO

Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans.


Assuntos
Mutação com Ganho de Função , Hipopituitarismo/genética , Hipotálamo/metabolismo , Hipófise/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Corticotrofos/citologia , Corticotrofos/metabolismo , Displasia Ectodérmica/genética , Facies , Insuficiência de Crescimento/genética , Células HEK293 , Cardiopatias Congênitas/genética , Humanos , Lactente , Sistema de Sinalização das MAP Quinases/genética , Melanotrofos/citologia , Melanotrofos/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sequenciamento Completo do Exoma/métodos
6.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804237

RESUMO

Ataxia in children is a common clinical sign of numerous neurological disorders consisting of impaired coordination of voluntary muscle movement. Its most common form, cerebellar ataxia, describes a heterogeneous array of neurologic conditions with uncountable causes broadly divided as acquired or genetic. Numerous genetic disorders are associated with chronic progressive ataxia, which complicates clinical management, particularly on the diagnostic stage. Advances in omics technologies enable improvements in clinical practice and research, so we proposed a multi-omics approach to aid in the genetic diagnosis and molecular elucidation of an undiagnosed infantile condition of chronic progressive cerebellar ataxia. Using whole-exome sequencing, RNA-seq, and untargeted metabolomics, we identified three clinically relevant mutations (rs141471029, rs191582628 and rs398124292) and an altered metabolic profile in our patient. Two POLR1C diagnostic variants already classified as pathogenic were found, and a diagnosis of hypomyelinating leukodystrophy was achieved. A mutation on the MMACHC gene, known to be associated with methylmalonic aciduria and homocystinuria cblC type, was also found. Additionally, preliminary metabolome analysis revealed alterations in our patient's amino acid, fatty acid and carbohydrate metabolism. Our findings provided a definitive genetic diagnosis reinforcing the association between POLR1C mutations and hypomyelinating leukodystrophy and highlighted the relevance of multi-omics approaches to the disease.


Assuntos
Ataxia Cerebelar/diagnóstico , RNA Polimerases Dirigidas por DNA/genética , Genoma/genética , Oxirredutases/genética , Transcriptoma/genética , Adolescente , Adulto , Ataxia Cerebelar/genética , Ataxia Cerebelar/patologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Metaboloma/genética , Mutação/genética , Linhagem , RNA-Seq , Deficiência de Vitamina B 12/genética , Sequenciamento Completo do Exoma/métodos , Adulto Jovem
7.
Medicine (Baltimore) ; 100(12): e25264, 2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33761726

RESUMO

RATIONALE: Primary peritoneal epithelioid mesothelioma of clear cell type is an extremely rare entity composed of clear cytoplasm. It is challenging to diagnose because of the morphological resemblance to clear cell tumor. PATIENTS CONCERNS: A 69-year-old male patient had swollen lymph nodes in the right inguinal region for 7 months and was constipated for 1 month. DIAGNOSIS: The patient was diagnosed as peritoneal epithelioid mesothelioma of clear cell type based on computed tomography scan, pathology, immunohistochemistry, special staining and whole-exome sequencing. This patient harbored VHL gene alteration in exon 1 and homologous recombination defect (with a score of 45). This finding indicated that this patient might be sensitive to platinum-based therapy and Poly ADP-ribose Polymerase (PARP) inhibitor. This patient carried no microsatellite instability, a low level of tumor mutation burden, and a high extent of intratumoral heterogeneity. Eighteen neoantigens were detected. INTERVENTIONS: The patient received surgery-based multidisciplinary treatment by integrating cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC). HIPEC was administered with docetaxel 120 mg plus cisplatin 120 mg, at 43°C, for 60 minutes. After operation, the patient received intravenous (IV) chemotherapy with docetaxel 60 mg, pemetrexed 750 mg and cisplatin 100 mg, and then intraperitoneal (IP) chemotherapy with docetaxel 40 mg. The patient received interventional therapy of hepatic artery embolization for 5 times. OUTCOMES: Regular follow-up was performed until Oct 14, 2020. The patient died 31.6 months later owing to incomplete intestinal obstruction. LESSONS: Primary peritoneal epithelioid mesothelioma of clear cell type needs to be differentiated from a variety of clear cell tumors. This disease is characterized by specific genetic alteration. Whole-exome sequencing contributes to guide individualized therapy. CRS-HIPEC helps achieve long-term overall survival.


Assuntos
Cisplatino/administração & dosagem , Procedimentos Cirúrgicos de Citorredução/métodos , Docetaxel/administração & dosagem , Pemetrexede/administração & dosagem , Neoplasias Peritoneais , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Idoso , Antineoplásicos/administração & dosagem , Embolização Terapêutica/métodos , Humanos , Imuno-Histoquímica , Masculino , /patologia , /terapia , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/patologia , Neoplasias Peritoneais/fisiopatologia , Neoplasias Peritoneais/terapia , Tomografia Computadorizada por Raios X/métodos , Resultado do Tratamento , Sequenciamento Completo do Exoma/métodos
8.
BMC Neurol ; 21(1): 96, 2021 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-33653295

RESUMO

BACKGROUND: Due to large genetic and phenotypic heterogeneity, the conventional workup for Charcot-Marie-Tooth (CMT) diagnosis is often underpowered, leading to diagnostic delay or even lack of diagnosis. In the present study, we explored how bioinformatics analysis on whole-exome sequencing (WES) data can be used to diagnose patients with CMT disease efficiently. CASE PRESENTATION: The proband is a 29-year-old female presented with a severe amyotrophy and distal skeletal deformity that plagued her family for over 20 years since she was 5-year-old. No other aberrant symptoms were detected in her speaking, hearing, vision, and intelligence. Similar symptoms manifested in her younger brother, while her parents and her older brother showed normal. To uncover the genetic causes of this disease, we performed exome sequencing for the proband and her parents. Subsequent bioinformatics analysis on the KGGSeq platform and further Sanger sequencing identified a novel homozygous GDAP1 nonsense mutation (c.218C > G, p.Ser73*) that responsible for the family. This genetic finding then led to a quick diagnosis of CMT type 4A (CMT4A), confirmed by nerve conduction velocity and electromyography examination of the patients. CONCLUSIONS: The patients with severe muscle atrophy and distal skeletal deformity were caused by a novel homozygous nonsense mutation in GDAP1 (c.218C > G, p.Ser73*), and were diagnosed as CMT4A finally. This study expanded the mutation spectrum of CMT disease and demonstrated how affordable WES could be effectively employed for the clinical diagnosis of unexplained phenotypes.


Assuntos
Doença de Charcot-Marie-Tooth/diagnóstico , Proteínas do Tecido Nervoso/genética , Sequenciamento Completo do Exoma/métodos , Adulto , Grupo com Ancestrais do Continente Asiático , Doença de Charcot-Marie-Tooth/genética , Pré-Escolar , China , Códon sem Sentido , Diagnóstico Tardio , Feminino , Homozigoto , Humanos , Masculino , Atrofia Muscular/genética , Linhagem , Fenótipo , Irmãos
9.
Mol Genet Genomics ; 296(3): 653-663, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33694043

RESUMO

Next generation sequencing tests are used routinely as first-choice tests in the clinic. However, systematic performance comparing the results of exome sequencing as a single test replacing Sanger sequencing of targeted gene(s) is still lacking. Performance comparison data are critically important for clinical case management. In this study, we compared Sanger-sequencing results of 258 genes to those obtained from next generation sequencing (NGS) using two exome-sequencing enrichment kits: Agilent-SureSelectQXT and Illumina-Nextera. Sequencing was performed on leukocytes and buccal-derived DNA from a single individual, and all 258 genes were sequenced a total of 11 times (using different sequencing methods and DNA sources). Sanger sequencing was completed for all exons, including flanking ± 8 bp regions. For the 258 genes, NGS mean coverage was > 20 × for > 98 and > 91% of the regions targeted by SureSelect and Nextera, respectively. Overall, 449 variants were identified in at least one experiment, and 407/449 (90.6%) were detected by all. Of the 42 discordant variants, 23 were determined as true calls, summing-up to a truth set of 430 variants. Sensitivity of true-variant detection was 99% for Sanger sequencing and 97-100% for the NGS experiments. Mean false-positive rates were 3.7E-6 for Sanger sequencing, 2.5E-6 for SureSelect-NGS and 5.2E-6 for Nextera-NGS. Our findings suggest a high overall concordance between Sanger sequencing and NGS performances. Both methods demonstrated false-positive and false-negative calls. High clinical suspicion for a specific diagnosis should, therefore, override negative results of either Sanger sequencing or NGS.


Assuntos
Exoma/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento Completo do Exoma/métodos , DNA/genética , Éxons/genética , Variação Genética/genética , Humanos , Análise de Sequência de DNA/métodos
10.
Nat Commun ; 12(1): 1751, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741915

RESUMO

Malignant Pleural Mesothelioma (MPM) is typically diagnosed 20-50 years after exposure to asbestos and evolves along an unknown evolutionary trajectory. To elucidate this path, we conducted multi-regional exome sequencing of 90 tumour samples from 22 MPMs acquired at surgery. Here we show that exomic intratumour heterogeneity varies widely across the cohort. Phylogenetic tree topology ranges from linear to highly branched, reflecting a steep gradient of genomic instability. Using transfer learning, we detect repeated evolution, resolving 5 clusters that are prognostic, with temporally ordered clonal drivers. BAP1/-3p21 and FBXW7/-chr4 events are always early clonal. In contrast, NF2/-22q events, leading to Hippo pathway inactivation are predominantly late clonal, positively selected, and when subclonal, exhibit parallel evolution indicating an evolutionary constraint. Very late somatic alteration of NF2/22q occurred in one patient 12 years after surgery. Clonal architecture and evolutionary clusters dictate MPM inflammation and immune evasion. These results reveal potentially drugable evolutionary bottlenecking in MPM, and an impact of clonal architecture on shaping the immune landscape, with potential to dictate the clinical response to immune checkpoint inhibition.


Assuntos
Deleção Cromossômica , Neoplasias Pulmonares/genética , Mesotelioma/genética , Mutação , Neoplasias Pleurais/genética , Proteínas Supressoras de Tumor/genética , Células Clonais/metabolismo , Células Clonais/patologia , Análise por Conglomerados , Estudos de Coortes , Humanos , Estimativa de Kaplan-Meier , Prognóstico , Microambiente Tumoral/genética , Proteínas Supressoras de Tumor/classificação , Sequenciamento Completo do Exoma/métodos
11.
Ned Tijdschr Geneeskd ; 1652021 01 21.
Artigo em Holandês | MEDLINE | ID: mdl-33560600

RESUMO

Introduction of new genetic test technologies in the last decade have accelerated genetic diagnosis in many medical specialties and have increased diagnostic yield considerably. SNP-arrays have been established as first tier diagnostic tools, more and more being replaced by next generation sequencing strategies, like targeted genomic panels and whole exome sequencing. We present the diagnostic work-up of a clinical case, a girl with congenital vertebral and rib anomalies. This case illustrates the complexity of genetic tests and the need for knowledge and experience to interpret the results. Intensive collaboration between pediatrician, clinical geneticist and laboratory specialist is mandatory, as is long-term commitment to involve parents in the diagnostic journey .


Assuntos
Anormalidades Múltiplas/diagnóstico , Testes Genéticos/métodos , Costelas/anormalidades , Coluna Vertebral/anormalidades , Sequenciamento Completo do Exoma/métodos , Anormalidades Múltiplas/genética , Criança , Feminino , Humanos
12.
Anticancer Res ; 41(2): 1035-1040, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33517312

RESUMO

BACKGROUND/AIM: The definition of multiple oral cancers is based on the distances between the tumors. However, it is not possible to accurately predict tumor origins based only on clinical criteria. PATIENTS AND METHODS: We performed whole-exome sequencing (WES) to analyze the genetic alterations in five tumors of two patients who underwent surgery in our hospital. RESULTS: In case 1, the distances between tumors on the right mandibular gingiva and buccal mucosa were more than 15 mm, leading to a clinical diagnosis of multiple primary tumors. WES revealed common mutations between tumors, suggesting that the tumors were derived from the same clone. In contrast, in case 2, the distance between tumors on the right side of the tongue was only 10 mm, but the tumors were diagnosed as double primary tumors because their mutations were completely different. CONCLUSION: WES, rather than the available clinical criteria, can clarify the clonal origins of multiple oral cancers.


Assuntos
Células Clonais/patologia , Neoplasias Bucais/diagnóstico , Mutação , Neoplasias Primárias Múltiplas/diagnóstico , Sequenciamento Completo do Exoma/métodos , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Neoplasias Bucais/cirurgia , Metástase Neoplásica , Neoplasias Primárias Múltiplas/genética , Neoplasias Primárias Múltiplas/cirurgia , Especificidade da Espécie
13.
Nat Commun ; 12(1): 518, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483490

RESUMO

Homozygosity mapping is a powerful method for identifying mutations in patients with recessive conditions, especially in consanguineous families or isolated populations. Historically, it has been used in conjunction with genotypes from highly polymorphic markers, such as DNA microsatellites or common SNPs. Traditional software performs rather poorly with data from Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS), which are now extensively used in medical genetics. We develop AutoMap, a tool that is both web-based or downloadable, to allow performing homozygosity mapping directly on VCF (Variant Call Format) calls from WES or WGS projects. Following a training step on WES data from 26 consanguineous families and a validation procedure on a matched cohort, our method shows higher overall performances when compared with eight existing tools. Most importantly, when tested on real cases with negative molecular diagnosis from an internal set, AutoMap detects three gene-disease and multiple variant-disease associations that were previously unrecognized, projecting clear benefits for both molecular diagnosis and research activities in medical genetics.


Assuntos
Mapeamento Cromossômico/métodos , Biologia Computacional/métodos , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Polimorfismo de Nucleotídeo Único , Software , Predisposição Genética para Doença/genética , Genótipo , Homozigoto , Humanos , Internet , Mutação , Reprodutibilidade dos Testes , Sequenciamento Completo do Exoma/métodos
14.
Int Heart J ; 62(1): 127-134, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-33455984

RESUMO

Mutations in the sarcomeric protein filamin C (FLNC) gene have been linked to hypertrophic cardiomyopathy (HCM), as they have been determined to increase the risk of ventricular arrhythmia and sudden death. Thus, in this study, we identified a novel missense mutation of FLNC in a Chinese family with HCM, and, interestingly, a second novel truncating mutation of MYLK2 was discobered in one family member with different phenotype.We performed whole-exome sequencing in a Chinese family with HCM of unknown cause. To determine and confirm the function of a novel mutation of FLNC, we introduced the mutant and wild-type gene into AC16 cells (human cardiomyocytes): we then used western blotting to analyze the expression of FLNC in subcellular fractions, and confocal microscope to observe the subcellular distribution of the protein.As per our findings, we were able to identify a novel missense single nucleotide variant (FLNC c.G5935A [p.A1979T]) in the family, which segregates with the disease. FLNC expression levels were observed to be equivalent in both wild-type and p.A1979T cardiomyocytes. However, the expression of the mutant protein has resulted in cytoplasmic protein aggregations, in contrast to wild-type FLNC, which was distributed in the cytoplasm and did not form aggregates. Unexpectedly, a second truncating mutation, NM_033118:exon8:c.G1138T:p.E380X of the MYLK2 gene, was identified in the mother of the proband with dilated cardiomyopathy, which was not found in other subjects.We then identified the FLNC A1979T mutation as a novel pathogenic variant associated with HCM in a Chinese family as well as a second causal mutation in a family member with a distinct phenotype. The possibility that there is more than one causal mutation in cardiomyopathy warrants clinical attention, especially for patients with atypical clinical features.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Proteínas de Ligação ao Cálcio/genética , Cardiomiopatias/genética , Filaminas/genética , Quinase de Cadeia Leve de Miosina/genética , Adulto , Cardiomiopatias/diagnóstico por imagem , Cardiomiopatias/fisiopatologia , Cardiomiopatia Dilatada/genética , Cardiomiopatia Hipertrófica/genética , Morte Súbita Cardíaca/epidemiologia , Feminino , Predisposição Genética para Doença , Humanos , Mutação de Sentido Incorreto/genética , Miócitos Cardíacos/ultraestrutura , Linhagem , Fenótipo , Fatores de Risco , Fibrilação Ventricular/epidemiologia , Fibrilação Ventricular/mortalidade , Sequenciamento Completo do Exoma/métodos
15.
Gene ; 775: 145431, 2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33444683

RESUMO

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a global epidemic that often progresses to liver cirrhosis and hepatocellular carcinoma. In contrast to most world populations where NAFLD is mostly prevalent among obese, NAFLD among Indians and generally among South and South-East Asians is unique and highly prevalent among individuals who are lean. Genetics of NAFLD in Indian populations is understudied. In this study, we have used an exome-wide approach to identify genetic determinants of hepatic fat content (HFC) in India. METHODS: HFC was measured in 244 participants using Proton magnetic resonance spectroscopy (H1-MRS). Quantitative trait loci (QTL) mapping was done exome-wide, to identify SNPs associated with HFC. The effects of the interaction between adiposity and QTLs on HFC were studied using a regression model. Association of the significant loci with disease severity was studied in 146 NAFLD patients among 244 participants, who underwent liver biopsy. RESULTS: Our study identified 4 significantly associated SNPs (rs738409 and rs2281135 (PNPLA3), rs3761472 (SAMM50), rs17513722 (FAM161A) and rs4788084), with HFC after adjusting for the effects of covariates (p-value < 0.0005). rs738409, rs2281135 (PNPLA3), and rs3761472 (SAMM50) were associated with hepatocyte ballooning, lobular and portal inflammation and non-alcoholic steatohepatitis (NASH) (p-value < 0.05). rs4788048 is an eQTL for IL27 and SULT1A2 genes, both of which are highly expressed in healthy livers and are likely to be involved in NAFLD pathogenesis. CONCLUSIONS: Our study identified the novel association of rs4788084 with HFC, which regulates the expression of IL-27, an immune regulatory gene. We further showed that adiposity affected the HFC, irrespective of the genetic predisposition.


Assuntos
Interleucinas/genética , Gordura Intra-Abdominal/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/patologia , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Exoma/métodos , Adulto , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/genética , Regiões Promotoras Genéticas , Espectroscopia de Prótons por Ressonância Magnética , Índice de Gravidade de Doença , Ultrassonografia
16.
Methods Mol Biol ; 2190: 1-32, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32804359

RESUMO

Mutations in protein-coding regions can lead to large biological changes and are associated with genetic conditions, including cancers and Mendelian diseases, as well as drug resistance. Although whole genome and exome sequencing help to elucidate potential genotype-phenotype correlations, there is a large gap between the identification of new variants and deciphering their molecular consequences. A comprehensive understanding of these mechanistic consequences is crucial to better understand and treat diseases in a more personalized and effective way. This is particularly relevant considering estimates that over 80% of mutations associated with a disease are incorrectly assumed to be causative. A thorough analysis of potential effects of mutations is required to correctly identify the molecular mechanisms of disease and enable the distinction between disease-causing and non-disease-causing variation within a gene. Here we present an overview of our integrative mutation analysis platform, which focuses on refining the current genotype-phenotype correlation methods by using the wealth of protein structural information.


Assuntos
Análise Mutacional de DNA/métodos , Estudos de Associação Genética/métodos , Mutação/genética , Exoma/genética , Genótipo , Humanos , Fenótipo , Sequenciamento Completo do Exoma/métodos
17.
Neurosci Lett ; 744: 135597, 2021 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-33373675

RESUMO

Genetics has an essential role in the development of early-onset Parkinson's disease (EOPD). Consequently, genetic screening is of great significance for the diagnosis and treatment of EOPD. In this study, we reported two EOPD with compound heterozygous in PARKIN detected by whole-exome sequencing (WES) and ligation-dependent probe amplification (MLPA). Two unrelated EOPD patients and their parents were enrolled in this study. Genetic analysis was performed through WES and verified by direct Sanger sequencing. In addition, MLPA was used to detect exon dosage. Detailed clinical manifestations and several scale assessments were collected for genotype and phenotype analysis. Compound heterozygous mutations in PARKIN were identified in both patients. c.735-1G > A and Ex2del were detected in Case A, while G284R (c.850 G > C) and Ex2del were found in Case B. These variants were confirmed to originate from their normal parents. The c.735-1G > A is a novel PARKIN variant, which was predicted to result from disappearing of the acceptor splice site by NetGene2. The G284R is a previously reported pathological mutation and the Ex2del is a hot variant of PARKIN found in the Asian population. The phenotypes of both patients are quite different, the main manifestation of case A is rigidity onset, while the case B starts with tremor and foot dystonia. In the present study, we reported a novel compound heterozygous form of PARKIN consisting of splice variant c. 735-1G > A and Ex2del. Moreover, we also found that tiny differences in genotypes of PARKIN may lead to obvious clinical phenotypic differences.


Assuntos
Variação Genética/genética , Heterozigoto , Doença de Parkinson/diagnóstico , Doença de Parkinson/genética , Ubiquitina-Proteína Ligases/genética , Adulto , Bases de Dados Genéticas , Feminino , Humanos , Linhagem , Sequenciamento Completo do Exoma/métodos
18.
Am J Hum Genet ; 108(1): 100-114, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33352116

RESUMO

Chiari I malformation (CM1), the displacement of the cerebellum through the foramen magnum into the spinal canal, is one of the most common pediatric neurological conditions. Individuals with CM1 can present with neurological symptoms, including severe headaches and sensory or motor deficits, often as a consequence of brainstem compression or syringomyelia (SM). We conducted whole-exome sequencing (WES) on 668 CM1 probands and 232 family members and performed gene-burden and de novo enrichment analyses. A significant enrichment of rare and de novo non-synonymous variants in chromodomain (CHD) genes was observed among individuals with CM1 (combined p = 2.4 × 10-10), including 3 de novo loss-of-function variants in CHD8 (LOF enrichment p = 1.9 × 10-10) and a significant burden of rare transmitted variants in CHD3 (p = 1.8 × 10-6). Overall, individuals with CM1 were found to have significantly increased head circumference (p = 2.6 × 10-9), with many harboring CHD rare variants having macrocephaly. Finally, haploinsufficiency for chd8 in zebrafish led to macrocephaly and posterior hindbrain displacement reminiscent of CM1. These results implicate chromodomain genes and excessive brain growth in CM1 pathogenesis.


Assuntos
Malformação de Arnold-Chiari/genética , Proteínas de Ligação a DNA/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Animais , Malformação de Arnold-Chiari/patologia , Encéfalo/patologia , Estudos de Casos e Controles , Feminino , Haploinsuficiência/genética , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Siringomielia/genética , Sequenciamento Completo do Exoma/métodos , Peixe-Zebra/genética
19.
PLoS One ; 15(12): e0243935, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33332422

RESUMO

BACKGROUND: Recent molecular characterization of urothelial cancer (UC) has suggested potential pathways in which to direct treatment, leading to a host of targeted therapies in development for UC. In parallel, gene expression profiling has demonstrated that high-grade UC is a heterogeneous disease. Prognostic basal-like and luminal-like subtypes have been identified and an accurate transcriptome BASE47 classifier has been developed. However, these phenotypes cannot be broadly investigated due to the lack of a clinically viable diagnostic assay. We sought to develop and evaluate a diagnostic classifier of UC subtype with the goal of accurate classification from clinically available specimens. METHODS: Tumor samples from 52 patients with high-grade UC were profiled for BASE47 genes concurrently by RNAseq as well as NanoString. After design and technical validation of a BASE47 NanoString probeset, results from the RNAseq and NanoString were used to translate diagnostic criteria to the Nanostring platform. Evaluation of repeatability and accuracy was performed to derive a final Nanostring based classifier. Diagnostic classification resulting from the NanoString BASE47 classifier was validated on an independent dataset (n = 30). The training and validation datasets accurately classified 87% and 93% of samples, respectively. RESULTS: Here we have derived a NanoString-platform BASE47 classifier that accurately predicts basal-like and luminal-like subtypes in high grade urothelial cancer. We have further validated our new NanoString BASE47 classifier on an independent dataset and confirmed high accuracy when compared with our original Transcriptome BASE47 classifier. CONCLUSIONS: The NanoString BASE47 classifier provides a faster turnaround time, a lower cost per sample to process, and maintains the accuracy of the original subtype classifier for better clinical implementation.


Assuntos
Biomarcadores Tumorais/genética , Prognóstico , Transcriptoma/genética , Neoplasias da Bexiga Urinária/genética , Idoso , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Análise em Microsséries/métodos , Proteínas de Neoplasias/genética , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/classificação , Neoplasias da Bexiga Urinária/patologia , Urotélio/metabolismo , Urotélio/patologia , Sequenciamento Completo do Exoma/métodos
20.
Medicine (Baltimore) ; 99(51): e23864, 2020 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-33371171

RESUMO

ABSTRACT: Schaaf-Yang syndrome (SYS) is a recently identified disorder caused by a loss-of-function mutation in a maternally imprinted gene, MAGEL2, at 15q11.2q13. Due to its extreme rarity and wide range of clinical severity, clinical suspicion is difficult for a physician. In the current study, its frequency among the Korean pediatric patients with developmental delay (DD) or intellectual disability (ID) was assessed. As the first report of Korean patients with SYS, our study aims to increase the awareness of this condition among the physicians taking care of the pediatric patients with DD/ID and hypotonia.The patients diagnosed with SYS by whole-exome sequencing (WES) among the 460 Korean pediatric patients with DD/ID were included, and their clinical and molecular features were reviewed.Four patients (0.9%) were diagnosed with SYS. Profound DD (4 patients), multiple anomalies including joint contractures and facial dysmorphism (4 patients), generalized hypotonia (3 patients), and severe respiratory difficulty requiring mechanical ventilation (3 patients) were noted in most cases, similar to those in previous reports. Sleep apnea (2 patients), autistic features (2 patients), a high grade of gastroesophageal reflux (1 patient), and seizures (1 patient) were found as well. A total of 3 different truncating MAGEL2 mutations were identified. A previously-reported mutation, to be the most common one, c.1996dupC, was found in 2 patients. The other 2 mutations, c.2217delC and c.3449_3450delTT were novel mutations. As MAGEL2 is maternally imprinted, 2 patients had inherited the MAGEL2 mutation from their respective healthy fathers.SYS is an extremely rare cause of DD/ID. However, hypotonia, joint contractures, profound DD/ID and facial dysmorphism are the suggestive clinical features for SYS. As a maternally imprinted disorder, it should be reminded that SYS may be inherited in form of a mutation from a healthy father.


Assuntos
Síndrome de Prader-Willi/diagnóstico , Proteínas/análise , Pré-Escolar , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/genética , Feminino , Humanos , Lactente , Masculino , Hipotonia Muscular/diagnóstico , Hipotonia Muscular/genética , Síndrome de Prader-Willi/genética , Proteínas/genética , República da Coreia , Sequenciamento Completo do Exoma/métodos
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