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1.
Transl Psychiatry ; 11(1): 1, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33414379

RESUMO

PIDD1 encodes p53-Induced Death Domain protein 1, which acts as a sensor surveilling centrosome numbers and p53 activity in mammalian cells. Early results also suggest a role in DNA damage response where PIDD1 may act as a cell-fate switch, through interaction with RIP1 and NEMO/IKKg, activating NF-κB signaling for survival, or as an apoptosis-inducing protein by activating caspase-2. Biallelic truncating mutations in CRADD-the protein bridging PIDD1 and caspase-2-have been reported in intellectual disability (ID), and in a form of lissencephaly. Here, we identified five families with ID from Iran, Pakistan, and India, with four different biallelic mutations in PIDD1, all disrupting the Death Domain (DD), through which PIDD1 interacts with CRADD or RIP1. Nonsense mutations Gln863* and Arg637* directly disrupt the DD, as does a missense mutation, Arg815Trp. A homozygous splice mutation in the fifth family is predicted to disrupt splicing upstream of the DD, as confirmed using an exon trap. In HEK293 cells, we show that both Gln863* and Arg815Trp mutants fail to co-localize with CRADD, leading to its aggregation and mis-localization, and fail to co-precipitate CRADD. Using genome-edited cell lines, we show that these three PIDD1 mutations all cause loss of PIDDosome function. Pidd1 null mice show decreased anxiety, but no motor abnormalities. Together this indicates that PIDD1 mutations in humans may cause ID (and possibly lissencephaly) either through gain of function or secondarily, due to altered scaffolding properties, while complete loss of PIDD1, as modeled in mice, may be well tolerated or is compensated for.


Assuntos
Proteína Adaptadora de Sinalização CRADD , Deficiência Intelectual , Animais , Proteína Adaptadora de Sinalização CRADD/genética , Proteína Adaptadora de Sinalização CRADD/metabolismo , Caspase 2/genética , Caspase 2/metabolismo , Domínio de Morte , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Células HEK293 , Humanos , Índia , Deficiência Intelectual/genética , Camundongos , Mutação
2.
Genes (Basel) ; 13(1)2021 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-35052391

RESUMO

Nucleolin (NCL/C23; OMIM: 164035) is a major nucleolar protein that plays a critical role in multiple processes, including ribosome assembly and maturation, chromatin decondensation, and pre-rRNA transcription. Due to its diverse functions, nucleolin has frequently been implicated in pathological processes, including cancer and viral infection. We recently identified a de novo frameshifting indel mutation of NCL, p.Gly664Glufs*70, through whole-exome sequencing of autism spectrum disorder trios. Through the transfection of constructs encoding either a wild-type human nucleolin or a mutant nucleolin with the same C-terminal sequence predicted for the autism proband, and by using co-localization with the nucleophosmin (NPM; B23) protein, we have shown that the nucleolin mutation leads to mislocalization of the NCL protein from the nucleolus to the nucleoplasm. Moreover, a construct with a nonsense mutation at the same residue, p.Gly664*, shows a very similar effect on the location of the NCL protein, thus confirming the presence of a predicted nucleolar location signal in this region of the NCL protein. Real-time fluorescence recovery experiments show significant changes in the kinetics and mobility of mutant NCL protein in the nucleoplasm of HEK293Tcells. Several other studies also report de novoNCL mutations in ASD or neurodevelopmental disorders. The altered mislocalization and dynamics of mutant NCL (p.G664Glufs*70/p.G664*) may have relevance to the etiopathlogy of NCL-related ASD and other neurodevelopmental phenotypes.


Assuntos
Transtorno do Espectro Autista/patologia , Nucléolo Celular/metabolismo , Heterozigoto , Mutação , Fosfoproteínas/genética , Proteínas de Ligação a RNA/genética , Transtorno do Espectro Autista/genética , Células HEK293 , Humanos , Masculino , Nucleolina
3.
J Mol Diagn ; 21(3): 437-448, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30731207

RESUMO

Inherited cardiomyopathies (ICs) are a major cause of heart disease. Given their marked clinical and genetic heterogeneity, the content and clinical utility of IC multi-gene panels has been the topic of continuous debate. Our genetics diagnostic laboratory has been providing clinical diagnostic testing for ICs since 2012. We began by testing nine genes and expanded our panel by fivefold in 2015. Here, we describe the implementation of a cost-effective next-generation sequencing (NGS)-based assay for testing of IC genes, including a protocol that minimizes the amount of Sanger sequencing required to confirm variants identified by NGS, which reduces the cost and time of testing. The NGS assay was developed for the simultaneous analysis of 45 IC genes and was assessed for the impact of panel expansion on variant detection, turnaround time, and cost of testing in a cohort of 993 patients. The assay led to a considerable reduction in test cost and turnaround time. However, only a marginal increase was observed in the diagnostic yield, whereas the rate of inconclusive findings increased considerably. These findings suggest that the ongoing evaluation of gene content and monitoring of clinical utility for multi-gene tests are essential to achieve maximum clinical utility of multi-gene tests in a publicly funded health care setting.


Assuntos
Cardiomiopatias/diagnóstico , Cardiomiopatias/genética , Atenção à Saúde , Testes Genéticos , Padrões de Herança/genética , Técnicas de Diagnóstico Molecular , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA/normas
4.
J Med Genet ; 56(6): 408-412, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30242101

RESUMO

BACKGROUND: Advances in molecular technologies and in-silico variant prediction tools offer wide-ranging opportunities in diagnostic settings, yet they also present with significant limitations. OBJECTIVE: Here, we contextualise the limitations of next-generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA) and in-silico prediction tools routinely used by diagnostic laboratories by reviewing specific experiences from our diagnostic laboratory. METHODS: We investigated discordant annotations and/or incorrect variant 'callings' in exons of 56 genes constituting our cardiomyopathy and connective tissue disorder NGS panels. Discordant variants and segmental duplications (SD) were queried using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool and the University of California Santa Cruz genome browser, respectively, to identify regions of high homology. Discrepant variant analyses by in-silico models were re-evaluated using updated file entries. RESULTS: We observed a 5% error rate in MYH7 variant 'calling' using MLPA, which resulted from >90% homology of the MYH7 probe-binding site to MYH6. SDs were detected in TTN, PKP2 and MYLK. SDs in MYLK presented the highest risk (15.7%) of incorrect variant 'calling'. The inaccurate 'callings' and discrepant in-silico predictions were resolved following detailed investigation into the source of error. CONCLUSION: Recognising the limitations described here may help avoid incorrect diagnoses and leverage the power of new molecular technologies in diagnostic settings.


Assuntos
Técnicas de Diagnóstico Molecular , Medicina Molecular , Alelos , Biologia Computacional/métodos , Gerenciamento Clínico , Duplicação Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Medicina Molecular/métodos , Medicina Molecular/normas , Anotação de Sequência Molecular
5.
Genet Med ; 20(3): 294-302, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28726806

RESUMO

PurposeThe purpose of this study was to develop a national program for Canadian diagnostic laboratories to compare DNA-variant interpretations and resolve discordant-variant classifications using the BRCA1 and BRCA2 genes as a case study.MethodsBRCA1 and BRCA2 variant data were uploaded and shared through the Canadian Open Genetics Repository (COGR; http://www.opengenetics.ca). A total of 5,554 variant observations were submitted; classification differences were identified and comparison reports were sent to participating laboratories. Each site had the opportunity to reclassify variants. The data were analyzed before and after the comparison report process to track concordant- or discordant-variant classifications by three different models.ResultsVariant-discordance rates varied by classification model: 38.9% of variants were discordant when using a five-tier model, 26.7% with a three-tier model, and 5.0% with a two-tier model. After the comparison report process, the proportion of discordant variants dropped to 30.7% with the five-tier model, to 14.2% with the three-tier model, and to 0.9% using the two-tier model.ConclusionWe present a Canadian interinstitutional quality improvement program for DNA-variant interpretations. Sharing of variant knowledge by clinical diagnostic laboratories will allow clinicians and patients to make more informed decisions and lead to better patient outcomes.


Assuntos
Confiabilidade dos Dados , Testes Genéticos/normas , Disseminação de Informação , Melhoria de Qualidade , Canadá , Tomada de Decisão Clínica , Bases de Dados Genéticas , Genes BRCA1 , Genes BRCA2 , Aconselhamento Genético , Testes Genéticos/métodos , Variação Genética , Programas Governamentais , Humanos , Reprodutibilidade dos Testes , Fluxo de Trabalho
6.
Acta Neuropathol ; 134(6): 889-904, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28685322

RESUMO

X-linked myotubular myopathy (XLMTM), a severe congenital myopathy, is caused by mutations in the MTM1 gene located on the X chromosome. A majority of affected males die in the early postnatal period, whereas female carriers are believed to be usually asymptomatic. Nevertheless, several affected females have been reported. To assess the phenotypic and pathological spectra of carrier females and to delineate diagnostic clues, we characterized 17 new unrelated affected females and performed a detailed comparison with previously reported cases at the clinical, muscle imaging, histological, ultrastructural and molecular levels. Taken together, the analysis of this large cohort of 43 cases highlights a wide spectrum of clinical severity ranging from severe neonatal and generalized weakness, similar to XLMTM male, to milder adult forms. Several females show a decline in respiratory function. Asymmetric weakness is a noteworthy frequent specific feature potentially correlated to an increased prevalence of highly skewed X inactivation. Asymmetry of growth was also noted. Other diagnostic clues include facial weakness, ptosis and ophthalmoplegia, skeletal and joint abnormalities, and histopathological signs that are hallmarks of centronuclear myopathy such as centralized nuclei and necklace fibers. The histopathological findings also demonstrate a general disorganization of muscle structure in addition to these specific hallmarks. Thus, MTM1 mutations in carrier females define a specific myopathy, which may be independent of the presence of an XLMTM male in the family. As several of the reported affected females carry large heterozygous MTM1 deletions not detectable by Sanger sequencing, and as milder phenotypes present as adult-onset limb-girdle myopathy, the prevalence of this myopathy is likely to be greatly underestimated. This report should aid diagnosis and thus the clinical management and genetic counseling of MTM1 carrier females. Furthermore, the clinical and pathological history of this cohort may be useful for therapeutic projects in males with XLMTM, as it illustrates the spectrum of possible evolution of the disease in patients surviving long term.


Assuntos
Heterozigoto , Mutação , Miopatias Congênitas Estruturais/diagnóstico , Proteínas Tirosina Fosfatases não Receptoras/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Estudos de Coortes , Diagnóstico Diferencial , Feminino , Humanos , Pessoa de Meia-Idade , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/fisiopatologia , Fenótipo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Índice de Gravidade de Doença
7.
Brain ; 140(1): 37-48, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27816943

RESUMO

Congenital myopathies define a heterogeneous group of neuromuscular diseases with neonatal or childhood hypotonia and muscle weakness. The genetic cause is still unknown in many patients, precluding genetic counselling and better understanding of the physiopathology. To identify novel genetic causes of congenital myopathies, exome sequencing was performed in three consanguineous families. We identified two homozygous frameshift mutations and a homozygous nonsense mutation in the mitogen-activated protein triple kinase ZAK. In total, six affected patients carry these mutations. Reverse transcription polymerase chain reaction and transcriptome analyses suggested nonsense mRNA decay as a main impact of mutations. The patients demonstrated a generalized slowly progressive muscle weakness accompanied by decreased vital capacities. A combination of proximal contractures with distal joint hyperlaxity is a distinct feature in one family. The low endurance and compound muscle action potential amplitude were strongly ameliorated on treatment with anticholinesterase inhibitor in another patient. Common histopathological features encompassed fibre size variation, predominance of type 1 fibre and centralized nuclei. A peculiar subsarcolemmal accumulation of mitochondria pointing towards the centre of the fibre was a novel histological hallmark in one family. These findings will improve the molecular diagnosis of congenital myopathies and implicate the mitogen-activated protein kinase (MAPK) signalling as a novel pathway altered in these rare myopathies.


Assuntos
Fibras Musculares de Contração Rápida/patologia , Fibras Musculares de Contração Lenta/patologia , Miopatias Congênitas Estruturais , Proteínas Quinases/genética , Adulto , Consanguinidade , Exoma , Feminino , Humanos , MAP Quinase Quinase Quinases , Masculino , Mutação , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/fisiopatologia , Linhagem
8.
Psychiatr Genet ; 26(2): 66-73, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26529358

RESUMO

Non-syndromic autosomal recessive intellectual disability (ID) is a genetically heterogeneous disorder with more than 50 mutated genes to date. ID is characterized by deficits in memory skills and language development with difficulty in learning, problem solving, and adaptive behaviors, and affects ∼ 1% of the population. For detection of disease-causing mutations in such a heterogeneous disorder, homozygosity mapping together with exome sequencing is a powerful approach, as almost all known genes can be assessed simultaneously in a high-throughput manner. In this study, a hemizygous c.786C>G:p.Ile262Met in the testis specific protein Y-encoded-like 2 (TSPYL2) gene and a homozygous c.11335G>A:p.Asp3779Asn in the low-density lipoprotein receptor-related protein 2 (LRP2) gene were detected after genome-wide genotyping and exome sequencing in a consanguineous Pakistani family with two boys with mild ID. Mutations in the LRP2 gene have previously been reported in patients with Donnai-Barrow and Stickler syndromes. LRP2 has also been associated with a 2q locus for autism (AUTS5). The TSPYL2 variant is not listed in any single-nucleotide polymorphism databases, and the LRP2 variant was absent in 400 ethnically matched healthy control chromosomes, and is not listed in single-nucleotide polymorphism databases as a common polymorphism. The LRP2 mutation identified here is located in one of the low-density lipoprotein-receptor class A domains, which is a cysteine-rich repeat that plays a central role in mammalian cholesterol metabolism, suggesting that alteration of cholesterol processing pathway can contribute to ID.


Assuntos
Deficiência Intelectual/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteínas Nucleares/genética , Povo Asiático , Proteínas de Ligação a DNA , Exoma , Feminino , Genes Recessivos , Ligação Genética , Homozigoto , Humanos , Masculino , Mutação de Sentido Incorreto , Paquistão , Linhagem
9.
Hum Mol Genet ; 24(20): 5697-710, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26206890

RESUMO

Histamine (HA) acts as a neurotransmitter in the brain, which participates in the regulation of many biological processes including inflammation, gastric acid secretion and neuromodulation. The enzyme histamine N-methyltransferase (HNMT) inactivates HA by transferring a methyl group from S-adenosyl-l-methionine to HA, and is the only well-known pathway for termination of neurotransmission actions of HA in mammalian central nervous system. We performed autozygosity mapping followed by targeted exome sequencing and identified two homozygous HNMT alterations, p.Gly60Asp and p.Leu208Pro, in patients affected with nonsyndromic autosomal recessive intellectual disability from two unrelated consanguineous families of Turkish and Kurdish ancestry, respectively. We verified the complete absence of a functional HNMT in patients using in vitro toxicology assay. Using mutant and wild-type DNA constructs as well as in silico protein modeling, we confirmed that p.Gly60Asp disrupts the enzymatic activity of the protein, and that p.Leu208Pro results in reduced protein stability, resulting in decreased HA inactivation. Our results highlight the importance of inclusion of HNMT for genetic testing of individuals presenting with intellectual disability.


Assuntos
Genes Recessivos , Histamina N-Metiltransferase/genética , Deficiência Intelectual/genética , Mutação de Sentido Incorreto , Adolescente , Adulto , Sequência de Aminoácidos , Domínio Catalítico , Criança , Pré-Escolar , Simulação por Computador , Análise Mutacional de DNA , Exoma , Feminino , Histamina N-Metiltransferase/metabolismo , Humanos , Lactente , Deficiência Intelectual/enzimologia , Iraque , Masculino , Dados de Sequência Molecular , Linhagem , Alinhamento de Sequência , Turquia , População Branca/genética
10.
PeerJ ; 3: e796, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25780760

RESUMO

Background. Most genetic disorders are caused by single nucleotide variations (SNVs) or small insertion/deletions (indels). High throughput sequencing has broadened the catalogue of human variation, including common polymorphisms, rare variations or disease causing mutations. However, identifying one variation among hundreds or thousands of others is still a complex task for biologists, geneticists and clinicians. Results. We have developed VaRank, a command-line tool for the ranking of genetic variants detected by high-throughput sequencing. VaRank scores and prioritizes variants annotated either by Alamut Batch or SnpEff. A barcode allows users to quickly view the presence/absence of variants (with homozygote/heterozygote status) in analyzed samples. VaRank supports the commonly used VCF input format for variants analysis thus allowing it to be easily integrated into NGS bioinformatics analysis pipelines. VaRank has been successfully applied to disease-gene identification as well as to molecular diagnostics setup for several hundred patients. Conclusions. VaRank is implemented in Tcl/Tk, a scripting language which is platform-independent but has been tested only on Unix environment. The source code is available under the GNU GPL, and together with sample data and detailed documentation can be downloaded from http://www.lbgi.fr/VaRank/.

11.
Hum Mol Genet ; 24(11): 3172-80, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25701870

RESUMO

There are two known mRNA degradation pathways, 3' to 5' and 5' to 3'. We identified likely pathogenic variants in two genes involved in these two pathways in individuals with intellectual disability. In a large family with multiple branches, we identified biallelic variants in DCPS in three affected individuals; a splice site variant (c.636+1G>A) that results in an in-frame insertion of 45 nucleotides and a missense variant (c.947C>T; p.Thr316Met). DCPS decaps the cap structure generated by 3' to 5' exonucleolytic degradation of mRNA. In vitro decapping assays showed an ablation of decapping function for both variants in DCPS. In another family, we identified a homozygous mutation (c.161T>C; p.Phe54Ser) in EDC3 in two affected children. EDC3 stimulates DCP2, which decaps mRNAs at the beginning of the 5' to 3' degradation pathway. In vitro decapping assays showed that altered EDC3 is unable to enhance DCP2 decapping at low concentrations and even inhibits DCP2 decapping at high concentration. We show that individuals with biallelic mutations in these genes of seemingly central functions are viable and that these possibly lead to impairment of neurological functions linking mRNA decapping to normal cognition. Our results further affirm an emerging theme linking aberrant mRNA metabolism to neurological defects.


Assuntos
Endorribonucleases/genética , Deficiência Intelectual/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Adolescente , Criança , Consanguinidade , Endorribonucleases/química , Endorribonucleases/metabolismo , Feminino , Genes Recessivos , Estudos de Associação Genética , Humanos , Masculino , Mutação de Sentido Incorreto , Linhagem , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento Pós-Transcricional do RNA , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Adulto Jovem
12.
Am J Hum Genet ; 95(6): 721-8, 2014 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-25480035

RESUMO

Dendritic spines represent the major site of neuronal activity in the brain; they serve as the receiving point for neurotransmitters and undergo rapid activity-dependent morphological changes that correlate with learning and memory. Using a combination of homozygosity mapping and next-generation sequencing in two consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability, we identified truncating mutations in formin 2 (FMN2), encoding a protein that belongs to the formin family of actin cytoskeleton nucleation factors and is highly expressed in the maturing brain. We found that FMN2 localizes to punctae along dendrites and that germline inactivation of mouse Fmn2 resulted in animals with decreased spine density; such mice were previously demonstrated to have a conditioned fear-learning defect. Furthermore, patient neural cells derived from induced pluripotent stem cells showed correlated decreased synaptic density. Thus, FMN2 mutations link intellectual disability either directly or indirectly to the regulation of actin-mediated synaptic spine density.


Assuntos
Transtornos Cromossômicos/genética , Deficiência Intelectual/genética , Proteínas dos Microfilamentos/genética , Proteínas Nucleares/genética , Deleção de Sequência , Adolescente , Adulto , Sequência de Bases , Transtornos Cromossômicos/fisiopatologia , Estudos de Coortes , Consanguinidade , Egito , Exoma/genética , Feminino , Forminas , Genes Recessivos , Ligação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Deficiência Intelectual/fisiopatologia , Masculino , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Paquistão , Linhagem , Análise de Sequência de DNA
13.
Brain ; 137(Pt 12): 3160-70, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25260562

RESUMO

Centronuclear myopathies are congenital muscle disorders characterized by type I myofibre predominance and an increased number of muscle fibres with nuclear centralization. The severe neonatal X-linked form is due to mutations in MTM1, autosomal recessive centronuclear myopathy with neonatal or childhood onset results from mutations in BIN1 (amphiphysin 2), and dominant cases were previously associated to mutations in DNM2 (dynamin 2). Our aim was to determine the genetic basis and physiopathology of patients with mild dominant centronuclear myopathy without mutations in DNM2. We hence established and characterized a homogeneous cohort of nine patients from five families with a progressive adult-onset centronuclear myopathy without facial weakness, including three sporadic cases and two families with dominant disease inheritance. All patients had similar histological and ultrastructural features involving type I fibre predominance and hypotrophy, as well as prominent nuclear centralization and clustering. We identified heterozygous BIN1 mutations in all patients and the molecular diagnosis was complemented by functional analyses. Two mutations in the N-terminal amphipathic helix strongly decreased the membrane-deforming properties of amphiphysin 2 and three stop-loss mutations resulted in a stable protein containing 52 supernumerary amino acids. Immunolabelling experiments revealed abnormal central accumulation of dynamin 2, caveolin-3, and the autophagic marker p62, and general membrane alterations of the triad, the sarcolemma, and the basal lamina as potential pathological mechanisms. In conclusion, we identified BIN1 as the second gene for dominant centronuclear myopathy. Our data provide the evidence that specific BIN1 mutations can cause either recessive or dominant centronuclear myopathy and that both disorders involve different pathomechanisms.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Mutação/genética , Miopatias Congênitas Estruturais/genética , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Adulto , Idade de Início , Dinamina II/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo
14.
Hum Genet ; 133(11): 1419-29, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25098561

RESUMO

Mirror movements (MRMV) are involuntary movements on one side of the body that mirror voluntary movements on the opposite side. Congenital mirror movement disorder is a rare, typically autosomal-dominant disorder, although it has been suspected that some sporadic cases may be due to recessive inheritance. Using a linkage analysis and a candidate gene approach, two genes have been implicated in congenital MRMV disorder to date: DCC on 18q21.2 (MRMV1), which encodes a netrin receptor, and RAD51 on 15q15.1 (MRMV2), which is involved in the maintenance of genomic integrity. Here, we describe a large consanguineous Pakistani family with 11 cases of congenital MRMV disorder reported across five generations, with autosomal recessive inheritance likely. Sanger sequencing of DCC and RAD51 did not identify a mutation. We then employed microarray genotyping and autozygosity mapping to identify a shared region of homozygosity-by-descent among the affected individuals. We identified a large autozygous region of ~3.3 Mb on chromosome 22q13.1 (Chr22:36605976-39904648). We used Sanger sequencing to exclude several candidate genes within this region, including DMC1 and NPTXR. Whole exome sequencing was employed, and identified a splice site mutation in the dynein axonemal light chain 4 gene, DNAL4. This splice site change leads to skipping of exon 3, and omission of 28 amino acids from DNAL4 protein. Linkage analysis using Simwalk2 gives a maximum Lod score of 6.197 at this locus. Whether or how DNAL4 function may relate to the function of DCC or RAD51 is not known. Also, there is no suggestion of primary ciliary dyskinesis, situs inversus, or defective sperm in affected family members, which might be anticipated given a putative role for DNAL4 in axonemal-based dynein complexes. We suggest that DNAL4 plays a role in the cytoplasmic dynein complex for netrin-1-directed retrograde transport, and in commissural neurons of the corpus callosum in particular. This, in turn, could lead to faulty cross-brain wiring, resulting in MRMV.


Assuntos
Dineínas do Axonema/genética , Cromossomos Humanos Par 22/genética , Transtornos dos Movimentos/genética , Adolescente , Adulto , Sequência de Aminoácidos , Criança , Pré-Escolar , Consanguinidade , Variações do Número de Cópias de DNA , Ligação Genética , Genótipo , Homozigoto , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Transtornos dos Movimentos/congênito , Mutação , Paquistão , Linhagem , Splicing de RNA , Alinhamento de Sequência , Análise de Sequência de DNA , Adulto Jovem
15.
Hum Genet ; 133(8): 975-84, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24623383

RESUMO

In this study, we have performed autozygosity mapping on a large consanguineous Pakistani family segregating with intellectual disability. We identified two large regions of homozygosity-by-descent (HBD) on 16q12.2-q21 and 16q24.1-q24.3. Whole exome sequencing (WES) was performed on an affected individual from the family, but initially, no obvious mutation was detected. However, three genes within the HBD regions that were not fully captured during the WES were Sanger sequenced and we identified a five base pair deletion (actually six base pairs deleted plus one base pair inserted) in exon 7 of the gene FBXO31. The variant segregated completely in the family, in recessive fashion giving a LOD score of 3.95. This variant leads to a frameshift and a premature stop codon and truncation of the FBXO31 protein, p.(Cys283Asnfs*81). Quantification of mRNA and protein expression suggests that nonsense-mediated mRNA decay also contributes to the loss of FBXO31 protein in affected individuals. FBXO31 functions as a centrosomal E3 ubiquitin ligase, in association with SKP1 and Cullin-1, involved in ubiquitination of proteins targeted for degradation. The FBXO31/SKP1/Cullin1 complex is important for neuronal morphogenesis and axonal identity. FBXO31 also plays a role in dendrite growth and neuronal migration in developing cerebellar cortex. Our finding adds further evidence of the involvement of disruption of the protein ubiquitination pathway in intellectual disability.


Assuntos
Cromossomos Humanos Par 16/genética , Proteínas F-Box/genética , Genes Recessivos , Deficiência Intelectual/genética , Deleção de Sequência , Proteínas Supressoras de Tumor/genética , Western Blotting , Mapeamento Cromossômico , Consanguinidade , Feminino , Mutação da Fase de Leitura/genética , Homozigoto , Humanos , Técnicas Imunoenzimáticas , Deficiência Intelectual/patologia , Masculino , Paquistão , Linhagem , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Neurology ; 81(14): 1205-14, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23975875

RESUMO

OBJECTIVE: To identify causative genes for centronuclear myopathies (CNM), a heterogeneous group of rare inherited muscle disorders that often present in infancy or early life with weakness and hypotonia, using next-generation sequencing of whole exomes and genomes. METHODS: Whole-exome or -genome sequencing was performed in a cohort of 29 unrelated patients with clinicopathologic diagnoses of CNM or related myopathy depleted for cases with mutations of MTM1, DNM2, and BIN1. Immunofluorescence analyses on muscle biopsies, splicing assays, and gel electrophoresis of patient muscle proteins were performed to determine the molecular consequences of mutations of interest. RESULTS: Autosomal recessive compound heterozygous truncating mutations of the titin gene, TTN, were identified in 5 individuals. Biochemical analyses demonstrated increased titin degradation and truncated titin proteins in patient muscles, establishing the impact of the mutations. CONCLUSIONS: Our study identifies truncating TTN mutations as a cause of congenital myopathy that is reported as CNM. Unlike the classic CNM genes that are all involved in excitation-contraction coupling at the triad, TTN encodes the giant sarcomeric protein titin, which forms a myofibrillar backbone for the components of the contractile machinery. This study expands the phenotypic spectrum associated with TTN mutations and indicates that TTN mutation analysis should be considered in cases of possible CNM without mutations in the classic CNM genes.


Assuntos
Conectina/genética , Miopatias Congênitas Estruturais/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Dinamina II/genética , Feminino , Genes Recessivos/genética , Humanos , Masculino , Mutação/genética , Proteínas Nucleares/genética , Fenótipo , Proteínas Tirosina Fosfatases não Receptoras/genética , Método Simples-Cego , Proteínas Supressoras de Tumor/genética , Adulto Jovem
17.
PLoS One ; 8(6): e67527, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23826317

RESUMO

Congenital myopathies are severe muscle disorders affecting adults as well as children in all populations. The diagnosis of congenital myopathies is constrained by strong clinical and genetic heterogeneity. Moreover, the majority of patients present with unspecific histological features, precluding purposive molecular diagnosis and demonstrating the need for an alternative and more efficient diagnostic approach. We used exome sequencing complemented by histological and ultrastructural analysis of muscle biopsies to identify the causative mutations in eight patients with clinically different skeletal muscle pathologies, ranging from a fatal neonatal myopathy to a mild and slowly progressive myopathy with adult onset. We identified RYR1 (ryanodine receptor) mutations in six patients and NEB (nebulin) mutations in two patients. We found novel missense and nonsense mutations, unraveled small insertions/deletions and confirmed their impact on splicing and mRNA/protein stability. Histological and ultrastructural findings of the muscle biopsies of the patients validated the exome sequencing results. We provide the evidence that an integrated strategy combining exome sequencing with clinical and histopathological investigations overcomes the limitations of the individual approaches to allow a fast and efficient diagnosis, accelerating the patient's access to a better healthcare and disease management. This is of particular interest for the diagnosis of congenital myopathies, which involve very large genes like RYR1 and NEB as well as genetic and phenotypic heterogeneity.


Assuntos
Doenças Musculares/congênito , Doenças Musculares/diagnóstico , Adulto , Sequência de Bases , Biópsia , Análise Mutacional de DNA , Exoma/genética , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Músculos/patologia , Músculos/ultraestrutura , Doenças Musculares/genética , Mutação/genética , Linhagem , Fenótipo , Análise de Sequência de DNA
18.
PLoS Genet ; 9(6): e1003430, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23754947

RESUMO

Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Processamento Alternativo/genética , Músculo Esquelético/patologia , Doenças Musculares/genética , Miopatias Congênitas Estruturais/genética , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Animais , Sequência de Bases , Cães , Éxons/genética , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Doenças Musculares/veterinária , Especificidade de Órgãos , Sítios de Splice de RNA/genética
19.
Acta Neuropathol ; 125(2): 173-85, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23224362

RESUMO

Neuromuscular disorders (NMD) such as neuropathy or myopathy are rare and often severe inherited disorders, affecting muscle and/or nerves with neonatal, childhood or adulthood onset, with considerable burden for the patients, their families and public health systems. Genetic and clinical heterogeneity, unspecific clinical features, unidentified genes and the implication of large and/or several genes requiring complementary methods are the main drawbacks in routine molecular diagnosis, leading to increased turnaround time and delay in the molecular validation of the diagnosis. The application of massively parallel sequencing, also called next generation sequencing, as a routine diagnostic strategy could lead to a rapid screening and fast identification of mutations in rare genetic disorders like NMD. This review aims to summarize and to discuss recent advances in the genetic diagnosis of neuromuscular disorders, and more generally monogenic diseases, fostered by massively parallel sequencing. We remind the challenges and benefit of obtaining an accurate genetic diagnosis, introduce the massively parallel sequencing technology and its novel applications in diagnosis of patients, prenatal diagnosis and carrier detection, and discuss the limitations and necessary improvements. Massively parallel sequencing synergizes with clinical and pathological investigations into an integrated diagnosis approach. Clinicians and pathologists are crucial in patient selection and interpretation of data, and persons trained in data management and analysis need to be integrated to the diagnosis pipeline. Massively parallel sequencing for mutation identification is expected to greatly improve diagnosis, genetic counseling and patient management.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/tendências , Doenças Neuromusculares/diagnóstico , Doenças Neuromusculares/genética , Heterogeneidade Genética , Humanos , Mutação/genética , Patologia Molecular , Análise de Sequência de DNA
20.
Acta Neuropathol ; 124(2): 273-83, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22526018

RESUMO

Inherited neuromuscular disorders (NMD) are chronic genetic diseases posing a significant burden on patients and the health care system. Despite tremendous research and clinical efforts, the molecular causes remain unknown for nearly half of the patients, due to genetic heterogeneity and conventional molecular diagnosis based on a gene-by-gene approach. We aimed to test next generation sequencing (NGS) as an efficient and cost-effective strategy to accelerate patient diagnosis. We designed a capture library to target the coding and splice site sequences of all known NMD genes and used NGS and DNA multiplexing to retrieve the pathogenic mutations in patients with heterogeneous NMD with or without known mutations. We retrieved all known mutations, including point mutations and small indels, intronic and exonic mutations, and a large deletion in a patient with Duchenne muscular dystrophy, validating the sensitivity and reproducibility of this strategy on a heterogeneous subset of NMD with different genetic inheritance. Most pathogenic mutations were ranked on top in our blind bioinformatic pipeline. Following the same strategy, we characterized probable TTN, RYR1 and COL6A3 mutations in several patients without previous molecular diagnosis. The cost was less than conventional testing for a single large gene. With appropriate adaptations, this strategy could be implemented into a routine genetic diagnosis set-up as a first screening approach to detect most kind of mutations, potentially before the need of more invasive and specific clinical investigations. An earlier genetic diagnosis should provide improved disease management and higher quality genetic counseling, and ease access to therapy or inclusion into therapeutic trials.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Doenças Neuromusculares/diagnóstico , Análise de Sequência de DNA , Bases de Dados Genéticas , Humanos , Doenças Neuromusculares/genética , Doenças Neuromusculares/metabolismo , Reprodutibilidade dos Testes
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