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Hereditary spastic paraplegias are a diverse group of degenerative disorders that are clinically categorized as isolated; with involvement of lower limb spasticity, or symptomatic, where spastic paraplegia is complicated by further neurological features. We sought to identify the underlying genetic causes of these disorders in the participating patients. Three consanguineous families with multiple affected members were identified by visiting special schools in the Punjab Province. DNA was extracted from blood samples of the participants. Exome sequencing was performed for selected patients from the three families, and the data were filtered to identify rare homozygous variants. ExomeDepth was used for the delineation of the copy number variants. All patients had varying degrees of intellectual disabilities, poor speech development, spasticity, a wide-based gait or an inability to walk and hypertonia. In family RDHR07, a homozygous deletion involving multiple exons and introns of SPG11 (NC000015.9:g.44894055_449028del) was found and correlated with the phenotype of the patients who had spasticity and other complex movement disorders, but not those who exhibited ataxic or indeterminate symptoms as well. In families ANMD03 and RDFA06, a nonsense variant, c.985C > T;(p.Arg329Ter) in DDHD2 and a frameshift insertionâdeletion variant of AP4B1, c.965-967delACTinsC;p.(Tyr322SerfsTer14), were identified which were homozygous in the patients while the obligate carriers in the respective pedigrees were heterozygous. All variants were ultra-rare with none, or very few carriers identified in the public databases. The three loss of function variants are likely to cause nonsense-mediated decay of the respective transcripts. Our research adds to the genetic variability associated with the SPG11 and AP4B1 variants and emphasizes the genetic heterogeneity of hereditary spastic paraplegia.
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Variações do Número de Cópias de DNA , Linhagem , Paraplegia Espástica Hereditária , Humanos , Masculino , Feminino , Paraplegia Espástica Hereditária/genética , Éxons/genética , Criança , Adolescente , Adulto , Sequenciamento do Exoma , Pré-Escolar , Complexo 4 de Proteínas Adaptadoras/genética , Consanguinidade , Homozigoto , Fenótipo , Adulto Jovem , ProteínasRESUMO
Introduction/background: Bladder exstrophy epispadias complex (BEEC) is a rare congenital anomaly of unknown etiology, although, genetic and environmental factors have been associated with its development. Variants in several genes expressed in the urogenital pathway have been reported as causative for bladder exstrophy in human and murine models. The expansion of next-generation sequencing and molecular genomics has improved our ability to identify the underlying genetic causes of similarly complex diseases and could thus assist with the investigation of the molecular basis of BEEC. Objective: The objective was to identify the presence of rare heterozygous variants in genes previously implicated in bladder exstrophy and correlate them with the presence or absence of bladder regeneration in our study population. Patients and Methods: We present a case series of 12 patients with BEEC who had bladder biopsies performed by pediatric urology during bladder neck reconstruction or bladder augmentation. Cases were classified as "sufficient" or "insufficient" (n = 5 and 7, respectively) based on a bladder volume of greater than or less than 40% of expected bladder size. Control bladder tissue specimens were obtained from patients (n = 6) undergoing biopsies for conditions other than bladder exstrophy. Whole exome sequencing was performed on DNA isolated from the bladder specimens. Based on the hypothesis of de novo mutations, as well as the potential implications of autosomal dominant conditions with incomplete penetrance, each case was evaluated for autosomal dominant variants in a set of genes previously implicated in BEEC. Results: Our review of the literature identified 44 genes that have been implicated in human models of bladder exstrophy. Our whole exome sequencing data analysis identified rare variants in two of these genes among the cases classified as sufficient, and seven variants in five of these genes among the cases classified as insufficient. Conclusion: We identified rare variants in seven previously implicated genes in our BEEC specimens. Additional research is needed to further understand the cellular signaling underlying this potentially genetically heterogeneous embryological condition.
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The dilemma of how to categorize and classify diseases has been debated for centuries. The field of medical genetics has historically approached nosology based on clinical phenotypes observed in patients and families. Advances in genomic sequencing and understanding of genetic contributions to disease often provoke a need to reassess these classifications. The Clinical Genome Resource (ClinGen) has developed frameworks to classify the strength of evidence underlying monogenic gene-disease relationships, variant pathogenicity, and clinical actionability. It is therefore necessary to define the disease entity being evaluated, which can be challenging for genes associated with multiple conditions and/or a broad phenotypic spectrum. We therefore developed criteria to guide "lumping and splitting" decisions and improve consistency in defining monogenic gene-disease relationships. Here, we outline the precuration process, the lumping and splitting guidelines with examples, and describe the implications for clinical diagnosis, informatics, and care management.
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Hereditary deafness is clinically and genetically heterogeneous. We investigated deafness segregating as a recessive trait in two families. Audiological examinations revealed an asymmetric mild to profound hearing loss with childhood or adolescent onset. Exome sequencing of probands identified a homozygous c.475G>A;p.(Glu159Lys) variant of CLDN9 (NM_020982.4) in one family and a homozygous c.370_372dupATC;p.(Ile124dup) CLDN9 variant in an affected individual of a second family. Claudin 9 (CLDN9) is an integral membrane protein and constituent of epithelial bicellular tight junctions (TJs) that form semipermeable, paracellular barriers between inner ear perilymphatic and endolymphatic compartments. Computational structural modeling predicts that substitution of a lysine for glutamic acid p.(Glu159Lys) alters one of two cis-interactions between CLDN9 protomers. The p.(Ile124dup) variant is predicted to locally misfold CLDN9 and mCherry tagged p.(Ile124dup) CLDN9 is not targeted to the HeLa cell membrane. In situ hybridization shows that mouse Cldn9 expression increases from embryonic to postnatal development and persists in adult inner ears coinciding with prominent CLDN9 immunoreactivity in TJs of epithelia outlining the scala media. Together with the Cldn9 deaf mouse and a homozygous frameshift of CLDN9 previously associated with deafness, the two bi-allelic variants of CLDN9 described here point to CLDN9 as a bona fide human deafness gene.
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Claudinas , Surdez , Adolescente , Animais , Criança , Claudinas/genética , Surdez/genética , Células HeLa , Homozigoto , Humanos , Camundongos , Mutação , LinhagemRESUMO
PURPOSE: Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disorder that manifests with fever, early-onset vasculitis, strokes, and hematologic dysfunction. This study aimed to identify disease-causing variants by conventional Sanger and whole exome sequencing in two families suspected to have DADA2 and non-confirmatory genotypes. ADA2 enzymatic assay confirmed the clinical diagnosis of DADA2. Molecular diagnosis was important to accurately identify other family members at risk. METHODS: We used a variety of sequencing technologies, ADA2 enzymatic testing, and molecular methods including qRT-PCR and MLPA. RESULTS: Exome sequencing identified heterozygosity for the known pathogenic variant ADA2: c.1358A>G, p.Tyr453Cys in a 14-year-old female with a history of ischemic strokes, livedo, and vasculitis. No second pathogenic variant could be identified. ADA2 enzymatic testing in combination with quantitative RT-PCR suggested a loss-of-function allele. Subsequent genome sequencing identified a canonical splice site variant, c.-47+2T>C, within the 5'UTR of ADA2. Two of her unaffected siblings were found to carry the same two pathogenic variants. A homozygous 800-bp duplication comprising exon 7 of ADA2 was identified in a 5-year-old female with features consistent with Diamond-Blackfan anemia (DBA). The duplication was missed by Sanger sequencing of ADA2, chromosomal microarray, and exome sequencing but was detected by MLPA in combination with long-read PCR sequencing. The exon 7 duplication was also identified in her non-symptomatic father and younger sister. CONCLUSIONS: ADA2 pathogenic variants may not be detected by conventional sequencing and genetic testing and may require the incorporation of additional diagnostic methods. A definitive molecular diagnosis is crucial for all family members to make informed treatment decisions.
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Adenosina Desaminase/deficiência , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Padrões de Herança , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Penetrância , Adolescente , Adulto , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Criança , Pré-Escolar , Ativação Enzimática , Feminino , Estudos de Associação Genética/métodos , Genótipo , Humanos , Masculino , Mutação , Linhagem , Fenótipo , Análise de Sequência de DNA , Sequenciamento do Exoma , Adulto JovemRESUMO
Hearing loss affects 380 million people worldwide due to environmental or genetic causes. Determining the cause of deafness in individuals without previous family history of hearing loss is challenging and has been relatively unexplored in Pakistan. We investigated the spectrum of genetic variants in hearing loss in a cohort of singleton affected individuals born to consanguineous parents. Twenty-one individuals with moderate to severe hearing loss were recruited. We performed whole-exome sequencing on DNA samples from the participants, which identified seventeen variants in ten known deafness genes and one novel candidate gene. All identified variants were homozygous except for two. Eleven of the variants were novel, including one multi-exonic homozygous deletion in OTOA. A missense variant in ESRRB was implicated for recessively inherited moderate to severe hearing loss. Two individuals were heterozygous for variants in MYO7A and CHD7, respectively, consistent with de novo variants or dominant inheritance with incomplete penetrance as the reason for their hearing loss. Our results indicate that similar to familial cases of deafness, variants in a large number of genes are responsible for moderate to severe hearing loss in sporadic individuals born to consanguineous couples.
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Predisposição Genética para Doença , Variação Genética , Perda Auditiva/genética , Adolescente , Sequência de Aminoácidos , Audiometria de Tons Puros , Criança , Pré-Escolar , Surdez/genética , Estudos de Associação Genética , Heterozigoto , Homozigoto , Humanos , Mutação de Sentido Incorreto , Paquistão , Fenótipo , Sequenciamento do Exoma , Adulto JovemRESUMO
OBJECTIVE: We sought to evaluate the performance of exome sequencing (ES) in determining an underlying genetic etiology for cases of fetal pleural effusions. STUDY DESIGN: We examined a prospective cohort series of fetal pleural effusions visualized sonographically between 1 April 2016 and 31 August 2017. Fetal pleural effusions attributed to twin sharing, anemia, or structural anomalies were excluded, as were all cases with a genetic diagnosis established by karyotype or chromosomal microarray analysis. The remaining cases with pleural effusions of unclear etiology were offered ES. ES was performed by clinical sequencing and/or sequencing under the Baylor-Hopkins Center for Mendelian Genomics' (BHCMG) research platform. All cases were evaluated for novel genes or phenotypic expansion of disease-causing genes. RESULTS: ES was performed on six probands affected by pleural effusions. A pathogenic variant was identified in one case (16.7%). Four additional cases had variants of uncertain significance (VUS) in candidate genes of pathological interest. Neither clinical nor candidate genes were evident in the final case. CONCLUSION: ES should be considered in the evaluation of prenatally detected idiopathic pleural effusions when other diagnostic workup for a genetic etiology has failed.
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Sequenciamento do Exoma , Doenças Fetais/genética , Derrame Pleural/genética , Actinina/genética , Moléculas de Adesão Celular/genética , Estudos de Coortes , Proteínas da Matriz Extracelular/genética , Pestanas/anormalidades , Feminino , Doenças Fetais/diagnóstico por imagem , Fatores de Transcrição Forkhead/genética , Humanos , Linfedema/diagnóstico , Linfedema/genética , Derrame Pleural/diagnóstico por imagem , Gravidez , Estudos Prospectivos , Receptor EphB4/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genéticaRESUMO
Variants of KCNQ4 are one of the most common causes of dominantly inherited nonsyndromic hearing loss. We investigated a consanguineous family in which two individuals had prelignual hearing loss, apparently inherited in a recessive mode. Whole-exome sequencing analyses demonstrated genetic heterogeneity as variants in two different genes segregated with the phenotype in two branches of the family. Members in one branch were homozygous for a pathogenic variant of TMC1. The other two affected individuals were homozygous for a missense pathogenic variant in KCNQ4 c.872C>T; p.(Pro291Leu). These two individuals had prelingual, progressive moderate to severe hearing loss, while a heterozygous carrier had late onset mild hearing loss. Our work demonstrates that p.Pro291L variant is semi-dominantly inherited. This is the first report of semi-dominance of a KCNQ4 variant.
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Surdez/genética , Sequenciamento do Exoma/métodos , Canais de Potássio KCNQ/genética , Mutação de Sentido Incorreto , Idade de Início , Consanguinidade , Feminino , Heterogeneidade Genética , Predisposição Genética para Doença , Humanos , Leucina/genética , Masculino , Linhagem , Prolina/genéticaRESUMO
Identifying genes and variants contributing to rare disease phenotypes and Mendelian conditions informs biology and medicine, yet potential phenotypic consequences for variation of >75% of the ~20,000 annotated genes in the human genome are lacking. Technical advances to assess rare variation genome-wide, particularly exome sequencing (ES), enabled establishment in the United States of the National Institutes of Health (NIH)-supported Centers for Mendelian Genomics (CMGs) and have facilitated collaborative studies resulting in novel "disease gene" discoveries. Pedigree-based genomic studies and rare variant analyses in families with suspected Mendelian conditions have led to the elucidation of hundreds of novel disease genes and highlighted the impact of de novo mutational events, somatic variation underlying nononcologic traits, incompletely penetrant alleles, phenotypes with high locus heterogeneity, and multilocus pathogenic variation. Herein, we highlight CMG collaborative discoveries that have contributed to understanding both rare and common diseases and discuss opportunities for future discovery in single-locus Mendelian disorder genomics. Phenotypic annotation of all human genes; development of bioinformatic tools and analytic methods; exploration of non-Mendelian modes of inheritance including reduced penetrance, multilocus variation, and oligogenic inheritance; construction of allelic series at a locus; enhanced data sharing worldwide; and integration with clinical genomics are explored. Realizing the full contribution of rare disease research to functional annotation of the human genome, and further illuminating human biology and health, will lay the foundation for the Precision Medicine Initiative.
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Doenças Genéticas Inatas/genética , Heterogeneidade Genética , Genoma Humano/genética , Genômica/tendências , Bases de Dados Genéticas , Predisposição Genética para Doença , Humanos , National Institutes of Health (U.S.) , Linhagem , Estados Unidos , Sequenciamento do Exoma/métodosRESUMO
BACKGROUND: Thoracic aortic aneurysms progressively enlarge and predispose to acute aortic dissections. Up to 25% of individuals with thoracic aortic disease harbor an underlying Mendelian pathogenic variant. An evidence-based strategy for selection of genes to test in hereditary thoracic aortic aneurysm and dissection (HTAAD) helps inform family screening and intervention to prevent life-threatening thoracic aortic events. OBJECTIVES: The purpose of this study was to accurately identify genes that predispose to HTAAD using the Clinical Genome Resource (ClinGen) framework. METHODS: We applied the semiquantitative ClinGen framework to assess presumed gene-disease relationships between 53 candidate genes and HTAAD. Genes were classified as causative for HTAAD if they were associated with isolated thoracic aortic disease and were clinically actionable, triggering routine aortic surveillance, intervention, and family cascade screening. All gene-disease assertions were evaluated by a pre-defined curator-expert pair and subsequently discussed with an expert panel. RESULTS: Genes were classified based on the strength of association with HTAAD into 5 categories: definitive (n = 9), strong (n = 2), moderate (n = 4), limited (n = 15), and no reported evidence (n = 23). They were further categorized by severity of associated aortic disease and risk of progression. Eleven genes in the definitive and strong groups were designated as "HTAAD genes" (category A). Eight genes were classified as unlikely to be progressive (category B) and 4 as low risk (category C). The remaining genes were recent genes with an uncertain classification or genes with no evidence of association with HTAAD. CONCLUSIONS: The ClinGen framework is useful to semiquantitatively assess the strength of gene-disease relationships for HTAAD. Gene categories resulting from the curation may inform clinical laboratories in the development, interpretation, and subsequent clinical implications of genetic testing for patients with aortic disease.
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Aneurisma da Aorta Torácica/diagnóstico , Aneurisma da Aorta Torácica/genética , Dissecção Aórtica/diagnóstico , Dissecção Aórtica/genética , Predisposição Genética para Doença/genética , Testes Genéticos/normas , Feminino , Testes Genéticos/métodos , Humanos , Masculino , Reprodutibilidade dos TestesRESUMO
Purpose: The measurement of mutations in pancreatic juice samples collected from the duodenum during endoscopic ultrasound (EUS) may improve the diagnostic evaluation of patients undergoing pancreatic surveillance. Our aim was to evaluate the accuracy of using pancreatic juice mutation concentrations to predict the presence and histologic grade of neoplasia in the pancreas.Experimental Design: Digital next-generation sequencing (NGS) of pancreatic juice DNA using a targeted 12-gene panel was performed on 67 patients undergoing pancreatic evaluation during EUS, including patients with pancreatic ductal adenocarcinoma, patients who subsequently underwent pancreatic resection for precursor lesions, patients undergoing surveillance for their familial/inherited susceptibility to pancreatic cancer, and normal pancreas disease controls.Results: Patients with pancreatic cancer or high-grade dysplasia as their highest grade lesion had significantly higher pancreatic juice mutation concentrations than all other subjects (mean/SD digital NGS score; 46.6 ± 69.7 vs. 6.2 ± 11.6, P = 0.02). Pancreatic juice mutation concentrations distinguished patients with pancreatic cancer or high-grade dysplasia in their resection specimen from all other subjects with 72.2% sensitivity and 89.4% specificity [area under the curve (AUC) = 0.872]. Mutant TP53/SMAD4 concentrations could distinguish patients with pancreatic cancer or high-grade dysplasia in their resection specimen from all other subjects with 61.1% sensitivity and 95.7% specificity (AUC = 0.819). Among 31 high-risk individuals under surveillance, 2 of the 3 individuals with most abnormal pancreatic juice mutation profiles also had the most abnormalities on pancreatic imaging.Conclusions: Pancreatic juice mutation analysis using digital NGS has potential diagnostic utility in the evaluation of patients undergoing pancreatic surveillance. Clin Cancer Res; 24(12); 2963-74. ©2018 AACRSee related commentary by Lipner and Yeh, p. 2713.
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Ácidos Nucleicos Livres , Mutação , Pâncreas/metabolismo , Pâncreas/patologia , Suco Pancreático/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Análise Mutacional de DNA , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Pancreáticas/epidemiologia , Vigilância da População , Curva ROCRESUMO
Purpose Deleterious germline mutations contribute to pancreatic cancer susceptibility and are well documented in families in which multiple members have had pancreatic cancer. Methods To define the prevalence of these germline mutations in patients with apparently sporadic pancreatic cancer, we sequenced 32 genes, including known pancreatic cancer susceptibility genes, in DNA prepared from normal tissue obtained from 854 patients with pancreatic ductal adenocarcinoma, 288 patients with other pancreatic and periampullary neoplasms, and 51 patients with non-neoplastic diseases who underwent pancreatic resection at Johns Hopkins Hospital between 2000 and 2015. Results Thirty-three (3.9%; 95% CI, 3.0% to 5.8%) of 854 patients with pancreatic cancer had a deleterious germline mutation, 31 (3.5%) of which affected known familial pancreatic cancer susceptibility genes: BRCA2 (12 patients), ATM (10 patients), BRCA1 (3 patients), PALB2 (2 patients), MLH1 (2 patients), CDKN2A (1 patient), and TP53 (1 patient). Patients with these germline mutations were younger than those without (mean ± SD, 60.8 ± 10.6 v 65.1 ± 10.5 years; P = .03). Deleterious germline mutations were also found in BUB1B (1) and BUB3 (1). Only three of these 33 patients had reported a family history of pancreatic cancer, and most did not have a cancer family history to suggest an inherited cancer syndrome. Five (1.7%) of 288 patients with other periampullary neoplasms also had a deleterious germline mutation. Conclusion Germline mutations in pancreatic cancer susceptibility genes are commonly identified in patients with pancreatic cancer without a significant family history of cancer. These deleterious pancreatic cancer susceptibility gene mutations, some of which are therapeutically targetable, will be missed if current family history guidelines are the main criteria used to determine the appropriateness of gene testing.
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Carcinoma Ductal Pancreático/genética , Predisposição Genética para Doença/genética , Mutação em Linhagem Germinativa , Neoplasias Pancreáticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/epidemiologia , Carcinoma Ductal Pancreático/terapia , Feminino , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/epidemiologia , Neoplasias Pancreáticas/terapia , Prevalência , Análise de Sequência de DNA , Estados Unidos/epidemiologiaRESUMO
This unit describes a technique for generating exome-enriched sequencing libraries using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. Utilizing commercially available kits, we present a low-input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution-targeted selection for exons, followed by sequencing using the Illumina next-generation short-read sequencing platform. © 2017 by John Wiley & Sons, Inc.
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DNA/genética , Sequenciamento do Exoma , Exoma/genética , Formaldeído , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Inclusão em Parafina , Fixação de Tecidos , Humanos , ParafinaRESUMO
Discovering the genetic basis of a Mendelian phenotype establishes a causal link between genotype and phenotype, making possible carrier and population screening and direct diagnosis. Such discoveries also contribute to our knowledge of gene function, gene regulation, development, and biological mechanisms that can be used for developing new therapeutics. As of February 2015, 2,937 genes underlying 4,163 Mendelian phenotypes have been discovered, but the genes underlying â¼50% (i.e., 3,152) of all known Mendelian phenotypes are still unknown, and many more Mendelian conditions have yet to be recognized. This is a formidable gap in biomedical knowledge. Accordingly, in December 2011, the NIH established the Centers for Mendelian Genomics (CMGs) to provide the collaborative framework and infrastructure necessary for undertaking large-scale whole-exome sequencing and discovery of the genetic variants responsible for Mendelian phenotypes. In partnership with 529 investigators from 261 institutions in 36 countries, the CMGs assessed 18,863 samples from 8,838 families representing 579 known and 470 novel Mendelian phenotypes as of January 2015. This collaborative effort has identified 956 genes, including 375 not previously associated with human health, that underlie a Mendelian phenotype. These results provide insight into study design and analytical strategies, identify novel mechanisms of disease, and reveal the extensive clinical variability of Mendelian phenotypes. Discovering the gene underlying every Mendelian phenotype will require tackling challenges such as worldwide ascertainment and phenotypic characterization of families affected by Mendelian conditions, improvement in sequencing and analytical techniques, and pervasive sharing of phenotypic and genomic data among researchers, clinicians, and families.
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Doenças Genéticas Inatas/genética , Genética Médica/métodos , Genética Médica/tendências , Fenótipo , Proteínas/genética , HumanosRESUMO
Lateral meningocele syndrome (LMS, OMIM%130720), also known as Lehman syndrome, is a very rare skeletal disorder with facial anomalies, hypotonia and meningocele-related neurologic dysfunction. The characteristic lateral meningoceles represent the severe end of the dural ectasia spectrum and are typically most severe in the lower spine. Facial features of LMS include hypertelorism and telecanthus, high arched eyebrows, ptosis, midfacial hypoplasia, micrognathia, high and narrow palate, low-set ears and a hypotonic appearance. Hyperextensibility, hernias and scoliosis reflect a connective tissue abnormality, and aortic dilation, a high-pitched nasal voice, wormian bones and osteolysis may be present. Lateral meningocele syndrome has phenotypic overlap with Hajdu-Cheney syndrome. We performed exome resequencing in five unrelated individuals with LMS and identified heterozygous truncating NOTCH3 mutations. In an additional unrelated individual Sanger sequencing revealed a deleterious variant in the same exon 33. In total, five novel de novo NOTCH3 mutations were identified in six unrelated patients. One had a 26 bp deletion (c.6461_6486del, p.G2154fsTer78), two carried the same single base pair insertion (c.6692_93insC, p.P2231fsTer11), and three individuals had a nonsense point mutation at c.6247A > T (pK2083*), c.6663C > G (p.Y2221*) or c.6732C > A, (p.Y2244*). All mutations cluster into the last coding exon, resulting in premature termination of the protein and truncation of the negative regulatory proline-glutamate-serine-threonine rich PEST domain. Our results suggest that mutant mRNA products escape nonsense mediated decay. The truncated NOTCH3 may cause gain-of-function through decreased clearance of the active intracellular product, resembling NOTCH2 mutations in the clinically related Hajdu-Cheney syndrome and contrasting the NOTCH3 missense mutations causing CADASIL.
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Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Éxons , Meningocele/diagnóstico , Meningocele/genética , Mutação , Receptores Notch/genética , Criança , Pré-Escolar , Análise Mutacional de DNA , Exoma , Fácies , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imageamento por Ressonância Magnética , Masculino , Fenótipo , Receptor Notch3 , Adulto JovemRESUMO
The microRNA-183/96/182 cluster is highly expressed in the retina and other sensory organs. To uncover its in vivo functions in the retina, we generated a knockout mouse model, designated "miR-183C(GT/GT)," using a gene-trap embryonic stem cell clone. We provide evidence that inactivation of the cluster results in early-onset and progressive synaptic defects of the photoreceptors, leading to abnormalities of scotopic and photopic electroretinograms with decreased b-wave amplitude as the primary defect and progressive retinal degeneration. In addition, inactivation of the miR-183/96/182 cluster resulted in global changes in retinal gene expression, with enrichment of genes important for synaptogenesis, synaptic transmission, photoreceptor morphogenesis, and phototransduction, suggesting that the miR-183/96/182 cluster plays important roles in postnatal functional differentiation and synaptic connectivity of photoreceptors.
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MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Degeneração Retiniana/genética , Animais , Modelos Animais de Doenças , Íntrons , Luz/efeitos adversos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Família Multigênica , Neurogênese/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/lesões , Retina/metabolismo , Retina/efeitos da radiação , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Órgãos dos Sentidos/metabolismo , Sinapses/genética , Sinapses/metabolismo , Transmissão Sináptica/genética , Síndrome , Visão Ocular/genéticaRESUMO
Mutations in miRNA genes have been implicated in hearing loss in human families and mice. It is also possible that mutations in miRNA binding sites of inner ear targets alter gene expression levels and lead to hearing loss. To investigate these possibilities we screened predicted target genes of the miR-183 miRNA cluster known to be expressed in the inner ear sensory epithelium. In one Iranian family segregating autosomal recessive non-syndromic hearing loss (ARNSHL), we identified a homozygous variant in a predicted miR-96/182 binding site in the 3'UTR of the RDX (DFNB24) gene. However, in vitro functional studies showed that this site is not a functional target for miR-96/182. We extended our study to include the miR-183 genes themselves and 24 additional predicted target genes of the miRNA-183 cluster. Screening these miRNAs and target sequences in numerous families segregating either autosomal dominant non-syndromic deafness (ADNSHL) or ARNSHL did not identify any potential deafness-causing mutations. These results suggest that mutations disrupting gene regulation by the miR-183 cluster are not a common cause of human hearing loss.
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Surdez/genética , MicroRNAs/genética , Mutação , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas do Citoesqueleto/genética , Primers do DNA/genética , Feminino , Genes Dominantes , Genes Recessivos , Homozigoto , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Família Multigênica , Linhagem , Homologia de Sequência do Ácido NucleicoRESUMO
MicroRNAs (miRNAs), an important class of small regulatory molecules for gene expression, are transcribed by RNA polymerase II. But little is known about the mechanisms that control miRNA expression. Comparing miRNA expression profiles between colon cancer cell line HCT 116 and its derivative, DNA methyltransferase 1 and 3b (DNMT1 and DNMT3b) double knockout cell line, we found that the expression of about 10% miRNAs was regulated by DNA methylation. In addition, neither 5-aza-2'-deoxycytidine treatment nor deletion of DNMT1 alone recapitulated miRNA expression profile seen in the double knockout cell line, suggesting that miRNA expression was tightly controlled by DNA methylation and partial methylation reduction was not sufficient for miRNA reexpression. We also found that HOXA3 and HOXD10 were putative targets of mir-10a, one of the differentially expressed miRNAs that is located in HOX gene cluster.
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Neoplasias do Colo/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Fatores de Transcrição/genética , DNA Metiltransferase 3BRESUMO
Although microRNAs (miRNAs) provide a newly recognized level of regulation of gene expression, the miRNA transcriptome of the retina and the contributions of miRNAs to retinal development and function are largely unknown. To begin to understand the functions of miRNAs in retina, we compared miRNA expression profiles in adult mouse retina, brain, and heart by microarray analysis. Our results show that at least 78 miRNAs are expressed in adult mouse retina, 21 of which are potentially retina-specific. Among these, we identified a polycistronic, sensory organ-specific paralogous miRNA cluster that includes miR-96, miR-182, and miR-183 on mouse chromosome 6qA3 with conservation of synteny to human chromosome 7q32.2. In situ hybridization showed that members of this cluster are expressed in photoreceptors, retinal bipolar and amacrine cells. Consistent with their genomic organization, these miRNAs have a similar expression pattern during development with abundance increasing postnatally and peaking in adult retina. Target prediction and in vitro functional studies showed that MITF, a transcription factor required for the establishment and maintenance of retinal pigmented epithelium, is a direct target of miR-96 and miR-182. Additionally, to identify miRNAs potentially involved in circadian rhythm regulation of the retina, we performed miRNA expression profiling with retinal RNA harvested at noon (Zeitgeber time 5) and midnight (Zeitgeber time 17) and identified a subgroup of 12 miRNAs, including members of the miR-183/96/182 cluster with diurnal variation in expression pattern. Our results suggest that miR-96 and miR-182 are involved in circadian rhythm regulation, perhaps by modulating the expression of adenylyl cyclase VI (ADCY6).