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1.
Am J Hum Genet ; 105(3): 493-508, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31447100

RESUMO

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

2.
Am J Hum Genet ; 2018 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-30503518

RESUMO

FUK encodes fucokinase, the only enzyme capable of converting L-fucose to fucose-1-phosphate, which will ultimately be used for synthesizing GDP-fucose, the donor substrate for all fucosyltransferases. Although it is essential for fucose salvage, this pathway is thought to make only a minor contribution to the total amount of GDP-fucose. A second pathway, the major de novo pathway, involves conversion of GDP-mannose to GDP-fucose. Here we describe two unrelated individuals who have pathogenic variants in FUK and who presented with severe developmental delays, encephalopathy, intractable seizures, and hypotonia. The first individual was compound heterozygous for c.667T>C (p.Ser223Pro) and c.2047C>T (p.Arg683Cys), and the second individual was homozygous for c.2980A>C (p.Lys994Gln). Skin fibroblasts from the first individual confirmed the variants as loss of function and showed significant decreases in total GDP-[3H] fucose and [3H] fucose-1-phosphate. There was also a decrease in the incorporation of [5,6-3H]-fucose into fucosylated glycoproteins. Lys994 has previously been shown to be an important site for ubiquitin conjugation. Here, we show that loss-of-function variants in FUK cause a congenital glycosylation disorder characterized by a defective fucose-salvage pathway.

3.
Hum Mutat ; 39(11): 1569-1580, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30311390

RESUMO

The ClinGen Inborn Errors of Metabolism Working Group was tasked with creating a comprehensive, standardized knowledge base of genes and variants for metabolic diseases. Phenylalanine hydroxylase (PAH) deficiency was chosen to pilot development of the Working Group's standards and guidelines. A PAH variant curation expert panel (VCEP) was created to facilitate this process. Following ACMG-AMP variant interpretation guidelines, we present the development of these standards in the context of PAH variant curation and interpretation. Existing ACMG-AMP rules were adjusted based on disease (6) or strength (5) or both (2). Disease adjustments include allele frequency thresholds, functional assay thresholds, and phenotype-specific guidelines. Our validation of PAH-specific variant interpretation guidelines is presented using 85 variants. The PAH VCEP interpretations were concordant with existing interpretations in ClinVar for 69 variants (81%). Development of biocurator tools and standards are also described. Using the PAH-specific ACMG-AMP guidelines, 714 PAH variants have been curated and will be submitted to ClinVar. We also discuss strategies and challenges in applying ACMG-AMP guidelines to autosomal recessive metabolic disease, and the curation of variants in these genes.

4.
Genet Med ; 2018 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-30181607

RESUMO

The Clinical Genome Resource (ClinGen) is supported by the National Institutes of Health (NIH) to develop expertly curated and freely accessible resources defining the clinical relevance of genes and variants for use in precision medicine and research. To facilitate expert input, ClinGen has formed Clinical Domain Working Groups (CDWGs) to leverage the collective knowledge of clinicians, laboratory diagnosticians, and researchers. In the initial phase of ClinGen, CDWGs were launched in the cardiovascular, hereditary cancer, and inborn errors of metabolism clinical fields. These early CDWGs established the infrastructure necessary to implement standardized processes developed or adopted by ClinGen working groups for the interpretation of gene-disease associations and variant pathogenicity, and provided a sustainable model for the formation of future disease-focused curation groups. The establishment of CDWGs requires recruitment of international experts to broadly represent the interests of their field and ensure that assertions made are reliable and widely accepted. Building on the successes, challenges, and trade-offs made in establishing the original CDWGs, ClinGen has developed standard operating procedures for the development of CDWGs in new clinical domains, while maximizing efforts to scale up curation and facilitate involvement of external groups who wish to utilize ClinGen methods and infrastructure for expert curation.

5.
Cell Rep ; 23(4): 1005-1019, 2018 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-29694881

RESUMO

Mitochondria shape cytosolic calcium ([Ca2+]c) transients and utilize the mitochondrial Ca2+ ([Ca2+]m) in exchange for bioenergetics output. Conversely, dysregulated [Ca2+]c causes [Ca2+]m overload and induces permeability transition pore and cell death. Ablation of MCU-mediated Ca2+ uptake exhibited elevated [Ca2+]c and failed to prevent stress-induced cell death. The mechanisms for these effects remain elusive. Here, we report that mitochondria undergo a cytosolic Ca2+-induced shape change that is distinct from mitochondrial fission and swelling. [Ca2+]c elevation, but not MCU-mediated Ca2+ uptake, appears to be essential for the process we term mitochondrial shape transition (MiST). MiST is mediated by the mitochondrial protein Miro1 through its EF-hand domain 1 in multiple cell types. Moreover, Ca2+-dependent disruption of Miro1/KIF5B/tubulin complex is determined by Miro1 EF1 domain. Functionally, Miro1-dependent MiST is essential for autophagy/mitophagy that is attenuated in Miro1 EF1 mutants. Thus, Miro1 is a cytosolic Ca2+ sensor that decodes metazoan Ca2+ signals as MiST.

7.
JAMA Pediatr ; 171(12): e173438, 2017 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-28973083

RESUMO

Importance: While congenital malformations and genetic diseases are a leading cause of early infant death, to our knowledge, the contribution of single-gene disorders in this group is undetermined. Objective: To determine the diagnostic yield and use of clinical exome sequencing in critically ill infants. Design, Setting, and Participants: Clinical exome sequencing was performed for 278 unrelated infants within the first 100 days of life who were admitted to Texas Children's Hospital in Houston, Texas, during a 5-year period between December 2011 and January 2017. Exome sequencing types included proband exome, trio exome, and critical trio exome, a rapid genomic assay for seriously ill infants. Main Outcomes and Measures: Indications for testing, diagnostic yield of clinical exome sequencing, turnaround time, molecular findings, patient age at diagnosis, and effect on medical management among a group of critically ill infants who were suspected to have genetic disorders. Results: The mean (SEM) age for infants participating in the study was 28.5 (1.7) days; of these, the mean (SEM) age was 29.0 (2.2) days for infants undergoing proband exome sequencing, 31.5 (3.9) days for trio exome, and 22.7 (3.9) days for critical trio exome. Clinical indications for exome sequencing included a range of medical concerns. Overall, a molecular diagnosis was achieved in 102 infants (36.7%) by clinical exome sequencing, with relatively low yield for cardiovascular abnormalities. The diagnosis affected medical management for 53 infants (52.0%) and had a substantial effect on informed redirection of care, initiation of new subspecialist care, medication/dietary modifications, and furthering life-saving procedures in select patients. Critical trio exome sequencing revealed a molecular diagnosis in 32 of 63 infants (50.8%) at a mean (SEM) of 33.1 (5.6) days of life with a mean (SEM) turnaround time of 13.0 (0.4) days. Clinical care was altered by the diagnosis in 23 of 32 patients (71.9%). The diagnostic yield, patient age at diagnosis, and medical effect in the group that underwent critical trio exome sequencing were significantly different compared with the group who underwent regular exome testing. For deceased infants (n = 81), genetic disorders were molecularly diagnosed in 39 (48.1%) by exome sequencing, with implications for recurrence risk counseling. Conclusions and Relevance: Exome sequencing is a powerful tool for the diagnostic evaluation of critically ill infants with suspected monogenic disorders in the neonatal and pediatric intensive care units and its use has a notable effect on clinical decision making.


Assuntos
Doenças Genéticas Inatas/diagnóstico , Unidades de Terapia Intensiva Pediátrica , Sequenciamento Completo do Exoma/métodos , Adulto , Cuidados Críticos/métodos , Gerenciamento Clínico , Exoma , Aconselhamento Genético/métodos , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Humanos , Lactente , Cuidado do Lactente/métodos , Recém-Nascido , Tempo de Internação/estatística & dados numéricos , Estudos Retrospectivos , Texas
8.
Circ Cardiovasc Genet ; 10(4)2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28798025

RESUMO

BACKGROUND: Left ventricular noncompaction (LVNC) is a genetically and phenotypically heterogeneous disease and, although increasingly recognized in clinical practice, there is a lack of widely accepted diagnostic criteria. We sought to identify novel genetic causes of LVNC and describe genotype-phenotype correlations. METHODS AND RESULTS: A total of 190 patients from 174 families with left ventricular hypertrabeculation (LVHT) or LVNC were referred for cardiac magnetic resonance and whole-exome sequencing. A total of 425 control individuals were included to identify variants of interest (VOIs). We found an excess of 138 VOIs in 102 (59%) unrelated patients in 54 previously identified LVNC or other known cardiomyopathy genes. VOIs were found in 68 of 90 probands with LVNC and 34 of 84 probands with LVHT (76% and 40%, respectively; P<0.001). We identified 0, 1, and ≥2 VOIs in 72, 74, and 28 probands, respectively. We found increasing number of VOIs in a patient strongly correlated with several markers of disease severity, including ratio of noncompacted to compacted myocardium (P<0.001) and left ventricular ejection fraction (P=0.01). The presence of sarcomeric gene mutations was associated with increased occurrence of late gadolinium enhancement (P=0.004). CONCLUSIONS: LVHT and LVNC likely represent a continuum of genotypic disease with differences in severity and variable phenotype explained, in part, by the number of VOIs and whether mutations are present in sarcomeric or nonsarcomeric genes. Presence of VOIs is common in patients with LVHT. Our findings expand the current clinical and genetic diagnostic approaches for patients with LVHT and LVNC.


Assuntos
Estudos de Associação Genética , Disfunção Ventricular Esquerda/diagnóstico , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Adulto , Idoso , Miosinas Cardíacas/genética , Proteínas de Transporte/genética , Criança , Conectina/genética , Feminino , Variação Genética , Ventrículos do Coração/fisiopatologia , Humanos , Proteínas com Domínio LIM/genética , Imagem Cinética por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/genética , Miocárdio/patologia , Cadeias Pesadas de Miosina/genética , Estudos Prospectivos , Índice de Gravidade de Doença , Tropomiosina/genética , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/patologia , Adulto Jovem
9.
Mol Genet Metab ; 122(1-2): 60-66, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28693988

RESUMO

INTRODUCTION: Cobalamin C disease is a multisystemic disease with variable manifestations and age of onset. Genotype-phenotype correlations are well-recognized in this disorder. Here, we present a large cohort of individuals with cobalamin C disease, several of whom are heterozygous for the c.482G>A pathogenic variant (p.Arg161Gln). We compared clinical characteristics of individuals with this pathogenic variant to those who do not have this variant. To our knowledge, this study represents the largest single cohort of individuals with the c.482G>A (p.Arg161Gln) pathogenic variant. METHODS: A retrospective chart review of 27 individuals from 21 families with cobalamin C disease who are followed at our facility was conducted. RESULTS: 13 individuals (48%) are compound heterozygous with the c.482G>A (p.Arg161Gln) on one allele and a second pathogenic variant on the other allele. Individuals with the c.482G>A (p.Arg161Gln) pathogenic variant had later onset of symptoms and easier metabolic control. Moreover, they had milder biochemical abnormalities at presentation which likely contributed to the observation that 4 individuals (31%) in this group were missed by newborn screening. CONCLUSION: The c.482G>A (p.Arg161Gln) pathogenic variant is associated with milder disease. These individuals may not receive a timely diagnosis as they may not be identified on newborn screening or because of unrecognized, late onset symptoms. Despite the milder presentation, significant complications can occur, especially if treatment is delayed.


Assuntos
Gerenciamento Clínico , Variação Genética , Homocistinúria/genética , Deficiência de Vitamina B 12/congênito , Adolescente , Adulto , Alelos , Proteínas de Transporte/genética , Criança , Pré-Escolar , Feminino , Seguimentos , Estudos de Associação Genética , Genótipo , Heterozigoto , Homocistinúria/diagnóstico , Homocistinúria/terapia , Humanos , Hidroxocobalamina/administração & dosagem , Hidroxocobalamina/uso terapêutico , Lactente , Recém-Nascido , Masculino , Mutação , Triagem Neonatal , Fenótipo , Estudos Retrospectivos , Deficiência de Vitamina B 12/diagnóstico , Deficiência de Vitamina B 12/genética , Deficiência de Vitamina B 12/terapia , Adulto Jovem
10.
Int J Mol Sci ; 18(7)2017 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-28704930

RESUMO

Mitochondrial dysfunction plays a central role in the neuropathology associated with status epilepticus (SE) and is implicated in the development of epilepsy. While excitotoxic mechanisms are well-known mediators affecting mitochondrial health following SE, whether hyperactivation of poly(ADP-ribose) polymerase-1 (PARP-1) also contributes to SE-induced mitochondrial dysfunction remains to be examined. Here we first evaluated the temporal evolution of poly-ADP-ribosylated protein levels in hippocampus following kainic acid-induced SE as a marker for PARP-1 activity, and found that PARP-1 was hyperactive at 24 h following SE. We evaluated oxidative metabolism and found decreased NAD⁺ levels by enzymatic cycling, and impaired NAD⁺-dependent mitochondrial respiration as measured by polarography at 24 h following SE. Stereological estimation showed significant cell loss in the hippocampal CA1 and CA3 subregions 72 h following SE. PARP-1 inhibition using N-(6-Oxo-5,6-dihydro-phenanthridin-2-yl)- N,N-dimethylacetamide (PJ-34) in vivo administration was associated with preserved NAD⁺ levels and NAD⁺-dependent mitochondrial respiration, and improved CA1 neuronal survival. These findings suggest that PARP-1 hyperactivation contributes to SE-associated mitochondrial dysfunction and CA1 hippocampal damage. The deleterious effects of PARP-1 hyperactivation on mitochondrial respiration are in part mediated through intracellular NAD⁺ depletion. Therefore, modulating PARP-1 activity may represent a potential therapeutic target to preserve intracellular energetics and mitochondrial function following SE.


Assuntos
Hipocampo/metabolismo , Hipocampo/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/metabolismo , Neurônios/patologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Estado Epiléptico/metabolismo , Estado Epiléptico/patologia , Animais , Western Blotting , Eletroencefalografia , Ratos , Ratos Sprague-Dawley
11.
Am J Hum Genet ; 100(6): 907-925, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28575647

RESUMO

Yin and yang 1 (YY1) is a well-known zinc-finger transcription factor with crucial roles in normal development and malignancy. YY1 acts both as a repressor and as an activator of gene expression. We have identified 23 individuals with de novo mutations or deletions of YY1 and phenotypic features that define a syndrome of cognitive impairment, behavioral alterations, intrauterine growth restriction, feeding problems, and various congenital malformations. Our combined clinical and molecular data define "YY1 syndrome" as a haploinsufficiency syndrome. Through immunoprecipitation of YY1-bound chromatin from affected individuals' cells with antibodies recognizing both ends of the protein, we show that YY1 deletions and missense mutations lead to a global loss of YY1 binding with a preferential retention at high-occupancy sites. Finally, we uncover a widespread loss of H3K27 acetylation in particular on the YY1-bound enhancers, underscoring a crucial role for YY1 in enhancer regulation. Collectively, these results define a clinical syndrome caused by haploinsufficiency of YY1 through dysregulation of key transcriptional regulators.


Assuntos
Cromatina/metabolismo , Haploinsuficiência/genética , Deficiência Intelectual/genética , Transcrição Genética , Fator de Transcrição YY1/genética , Acetilação , Adolescente , Sequência de Bases , Pré-Escolar , Imunoprecipitação da Cromatina , Estudos de Coortes , Elementos Facilitadores Genéticos/genética , Feminino , Ontologia Genética , Haplótipos/genética , Hemizigoto , Histonas/metabolismo , Humanos , Linfócitos/metabolismo , Masculino , Metilação , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Ligação Proteica/genética , Domínios Proteicos , Fator de Transcrição YY1/química
13.
Biochim Biophys Acta Mol Basis Dis ; 1863(6): 1539-1555, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28215579

RESUMO

The maintenance of mitochondrial DNA (mtDNA) depends on a number of nuclear gene-encoded proteins including a battery of enzymes forming the replisome needed to synthesize mtDNA. These enzymes need to be in balanced quantities to function properly that is in part achieved by exchanging intramitochondrial contents through mitochondrial fusion. In addition, mtDNA synthesis requires a balanced supply of nucleotides that is achieved by nucleotide recycling inside the mitochondria and import from the cytosol. Mitochondrial DNA maintenance defects (MDMDs) are a group of diseases caused by pathogenic variants in the nuclear genes involved in mtDNA maintenance resulting in impaired mtDNA synthesis leading to quantitative (mtDNA depletion) and qualitative (multiple mtDNA deletions) defects in mtDNA. Defective mtDNA leads to organ dysfunction due to insufficient mtDNA-encoded protein synthesis, resulting in an inadequate energy production to meet the needs of affected organs. MDMDs are inherited as autosomal recessive or dominant traits, and are associated with a broad phenotypic spectrum ranging from mild adult-onset ophthalmoplegia to severe infantile fatal hepatic failure. To date, pathogenic variants in 20 nuclear genes known to be crucial for mtDNA maintenance have been linked to MDMDs, including genes encoding enzymes of mtDNA replication machinery (POLG, POLG2, TWNK, TFAM, RNASEH1, MGME1, and DNA2), genes encoding proteins that function in maintaining a balanced mitochondrial nucleotide pool (TK2, DGUOK, SUCLG1, SUCLA2, ABAT, RRM2B, TYMP, SLC25A4, AGK, and MPV17), and genes encoding proteins involved in mitochondrial fusion (OPA1, MFN2, and FBXL4).


Assuntos
Dano ao DNA , DNA Mitocondrial , Doenças Mitocondriais , Proteínas Mitocondriais , Animais , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo
14.
Am J Hum Genet ; 100(2): 352-363, 2017 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-28132691

RESUMO

Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological importance of PSMD12 as a scaffolding subunit in proteasome function during development and neurogenesis in particular; they enable the definition of a neurodevelopmental disorder due to PSMD12 variants, expanding the phenotypic spectrum of UPS-dependent disorders.


Assuntos
Transtornos do Neurodesenvolvimento/genética , Complexo de Endopeptidases do Proteassoma/genética , Adolescente , Animais , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Deleção de Genes , Humanos , Lactente , Deficiência Intelectual/genética , Masculino , Microcefalia/genética , Polimorfismo de Nucleotídeo Único , Peixe-Zebra/genética
15.
JIMD Rep ; 35: 33-37, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27900673

RESUMO

We report three patients with elevations of propionylcarnitine (C3), one without elevations of 2-methylcitrate and 3-hydroxypropionate in urine organic acid analysis, and the other two showing only mild elevations, all of whom were subsequently confirmed to have propionic acidemia by molecular analysis of PCCA and PCCB genes. To date, they have had a mild clinical course. These cases illustrate the importance of considering high C3 as the only biochemical abnormality in a diagnosis of propionic acidemia. Since mild C3 elevations may be overlooked and considered non-diagnostic in isolation, we advise considering a diagnosis of propionic acidemia even in the absence of significant elevations 2-methylcitrate or 3-hydroxypropionate in urine organic acid analysis.

16.
Genet Med ; 19(4): 412-420, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27657687

RESUMO

PURPOSE: To investigate the utility of whole-exome sequencing (WES) to define a molecular diagnosis for patients clinically diagnosed with congenital anomalies of kidney and urinary tract (CAKUT). METHODS: WES was performed in 62 families with CAKUT. WES data were analyzed for single-nucleotide variants (SNVs) in 35 known CAKUT genes, putatively deleterious sequence changes in new candidate genes, and potentially disease-associated copy-number variants (CNVs). RESULTS: In approximately 5% of families, pathogenic SNVs were identified in PAX2, HNF1B, and EYA1. Observed phenotypes in these families expand the current understanding about the role of these genes in CAKUT. Four pathogenic CNVs were also identified using two CNV detection tools. In addition, we found one deleterious de novo SNV in FOXP1 among the 62 families with CAKUT. The clinical database of the Baylor Miraca Genetics laboratory was queried and seven additional unrelated individuals with novel de novo SNVs in FOXP1 were identified. Six of these eight individuals with FOXP1 SNVs have syndromic urinary tract defects, implicating this gene in urinary tract development. CONCLUSION: We conclude that WES can be used to identify molecular etiology (SNVs, CNVs) in a subset of individuals with CAKUT. WES can also help identify novel CAKUT genes.Genet Med 19 4, 412-420.


Assuntos
Variações do Número de Cópias de DNA , Predisposição Genética para Doença/genética , Anormalidades Urogenitais/diagnóstico , Refluxo Vesicoureteral/diagnóstico , Sequenciamento Completo do Exoma/métodos , Adolescente , Criança , Pré-Escolar , Feminino , Fatores de Transcrição Forkhead/genética , Fator 1-beta Nuclear de Hepatócito/genética , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas Nucleares/genética , Fator de Transcrição PAX2/genética , Linhagem , Polimorfismo de Nucleotídeo Único , Proteínas Tirosina Fosfatases/genética , Proteínas Repressoras/genética , Anormalidades Urogenitais/genética , Refluxo Vesicoureteral/genética , Adulto Jovem
17.
Mol Genet Metab ; 119(1-2): 68-74, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27484306

RESUMO

Deficiency of the TCA cycle enzyme Succinyl-CoA Synthetase/Ligase (SCS), due to pathogenic variants in subunits encoded by SUCLG1 and SUCLA2, causes mitochondrial encephalomyopathy, methylmalonic acidemia, and mitochondrial DNA (mtDNA) depletion. In this study, we report an 11year old patient who presented with truncal ataxia, chorea, hypotonia, bilateral sensorineural hearing loss and preserved cognition. Whole exome sequencing identified a heterozygous known pathogenic variant and a heterozygous novel missense variant of uncertain clinical significance (VUS) in SUCLG1. To validate the suspected pathogenicity of the novel VUS, molecular and biochemical analyses were performed using primary skin fibroblasts from the patient. The patient's cells lack the SUCLG1 protein, with significantly reduced levels of SUCLA2 and SUCLG2 protein. This leads to essentially undetectable SCS enzyme activity, mtDNA depletion, and cellular respiration defects. These abnormal phenotypes are rescued upon ectopic expression of wild-type SUCLG1 in the patient's fibroblasts, thus functionally confirming the pathogenic nature of the SUCLG1 VUS identified in this patient and expanding the phenotypic spectrum for SUCLG1 deficiency.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Succinato-CoA Ligases/genética , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Criança , DNA Mitocondrial/genética , Feminino , Predisposição Genética para Doença , Genótipo , Heterozigoto , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação de Sentido Incorreto , Succinato-CoA Ligases/metabolismo , Sequenciamento Completo do Exoma
18.
J Mol Diagn ; 18(3): 446-453, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26944031

RESUMO

The identification of mosaicism is important in establishing a disease diagnosis, assessing recurrence risk, and genetic counseling. Next-generation sequencing (NGS) with deep sequence coverage enhances sensitivity and allows for accurate quantification of the level of mosaicism. NGS identifies low-level mosaicism that would be undetectable by conventional Sanger sequencing. A customized DNA probe library was used for capturing targeted genes, followed by deep NGS analysis. The mean coverage depth per base was approximately 800×. The NGS sequence data were analyzed for single-nucleotide variants and copy number variations. Mosaic mutations in 10 cases/families were detected and confirmed by NGS analysis. Mosaicism was identified for autosomal dominant (JAG1, COL3A1), autosomal recessive (PYGM), and X-linked (PHKA2, PDHA1, OTC, and SLC6A8) disorders. The mosaicism was identified either in one or more tissues from the probands or in a parent of an affected child. When analyzing data from patients with unusual testing results or inheritance patterns, it is important to further evaluate the possibility of mosaicism. Deep NGS analysis not only provides insights into the spectrum of mosaic mutations but also underlines the importance of the detection of mosaicism as an integral part of clinical molecular diagnosis and genetic counseling.


Assuntos
Análise Mutacional de DNA , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Mosaicismo , Mutação , Adolescente , Adulto , Sequência de Bases , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Análise Mutacional de DNA/métodos , Feminino , Genes Dominantes , Genes Recessivos , Genes Ligados ao Cromossomo X , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Lactente , Masculino , Adulto Jovem
19.
Mol Genet Metab ; 117(4): 407-12, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26851065

RESUMO

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most frequent maternally inherited mitochondrial disorders. The pathogenesis of this syndrome is not fully understood and believed to result from several interacting mechanisms including impaired mitochondrial energy production, microvasculature angiopathy, and nitric oxide (NO) deficiency. NO deficiency in MELAS syndrome is likely to be multifactorial in origin with the decreased availability of the NO precursors, arginine and citrulline, playing a major role. In this study we used stable isotope infusion techniques to assess NO production in children with MELAS syndrome and healthy pediatric controls. We also assessed the effect of oral arginine and citrulline supplementations on NO production in children with MELAS syndrome. When compared to control subjects, children with MELAS syndrome were found to have lower NO production, arginine flux, plasma arginine, and citrulline flux. In children with MELAS syndrome, arginine supplementation resulted in increased NO production, arginine flux, and arginine concentration. Citrulline supplementation resulted in a greater increase of these parameters. Additionally, citrulline supplementation was associated with a robust increase in citrulline concentration and flux and de novo arginine synthesis rate. The greater effect of citrulline in increasing NO production is due to its greater ability to increase arginine availability particularly in the intracellular compartment in which NO synthesis takes place. This study, which is the first one to assess NO metabolism in children with mitochondrial diseases, adds more evidence to the notion that NO deficiency occurs in MELAS syndrome, suggests a better effect for citrulline because of its greater role as NO precursor, and indicates that impaired NO production occurs in children as well as adults with MELAS syndrome. Thus, the initiation of treatment with NO precursors may be beneficial earlier in life. Controlled clinical trials to assess the therapeutic effects of arginine and citrulline on clinical complications of MELAS syndrome are needed.


Assuntos
Arginina/administração & dosagem , Citrulina/administração & dosagem , Suplementos Nutricionais , Síndrome MELAS/dietoterapia , Síndrome MELAS/metabolismo , Óxido Nítrico/biossíntese , Adolescente , Arginina/farmacocinética , Estudos de Casos e Controles , Criança , Pré-Escolar , Citrulina/farmacocinética , Feminino , Humanos , Síndrome MELAS/diagnóstico , Masculino , Resultado do Tratamento
20.
Mol Genet Metab ; 116(3): 139-45, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26385305

RESUMO

Very long chain acyl-coA dehydrogenase deficiency (VLCADD) is an autosomal recessive inborn error of fatty acid oxidation detected by newborn screening (NBS). Follow-up molecular analyses are often required to clarify VLCADD-suggestive NBS results, but to date the outcome of these studies are not well described for the general screen-positive population. In the following study, we report the molecular findings for 693 unrelated patients that sequentially received Sanger sequence analysis of ACADVL as a result of a positive NBS for VLCADD. Highlighting the variable molecular underpinnings of this disorder, we identified 94 different pathogenic ACADVL variants (40 novel), as well as 134 variants of unknown clinical significance (VUSs). Evidence for the pathogenicity of a subset of recurrent VUSs was provided using multiple in silico analyses. Surprisingly, the most frequent finding in our cohort was carrier status, 57% all individuals had a single pathogenic variant or VUS. This result was further supported by follow-up array and/or acylcarnitine analysis that failed to provide evidence of a second pathogenic allele. Notably, exon-targeted array analysis of 131 individuals screen positive for VLCADD failed to identify copy number changes in ACADVL thus suggesting this test has a low yield in the setting of NBS follow-up. While no genotype was common, the c.848T>C (p.V283A) pathogenic variant was clearly the most frequent; at least one copy was found in ~10% of all individuals with a positive NBS. Clinical and biochemical data for seven unrelated patients homozygous for the p.V283A allele suggests that it results in a mild phenotype that responds well to standard treatment, but hypoglycemia can occur. Collectively, our data illustrate the molecular heterogeneity of VLCADD and provide novel insight into the outcomes of NBS for this disorder.


Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Acil-CoA Desidrogenase de Cadeia Longa/genética , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/genética , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Doenças Musculares/diagnóstico , Doenças Musculares/genética , Triagem Neonatal , Alelos , Carnitina/análogos & derivados , Simulação por Computador , Éxons , Feminino , Triagem de Portadores Genéticos , Genótipo , Humanos , Hipoglicemia/etiologia , Recém-Nascido , Masculino , Mutação de Sentido Incorreto , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA , Espectrometria de Massas em Tandem , Estados Unidos
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