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1.
Maturitas ; 141: 9-19, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33036707

RESUMO

Ovarian deficiency, including premature ovarian insufficiency (POI) and diminished ovarian reserve (DOR), represents one of the main causes of female infertility. POI is a genetically heterogeneous condition but current understanding of its genetic basis is far from complete, with the cause remaining unknown in the majority of patients. The genes that regulate DOR have been reported but the genetic basis of DOR has not been explored in depth. Both conditions are likely to lie along a continuum of degrees of decrease in ovarian reserve. We performed genomic analysis via whole exome sequencing (WES) followed by in silico analyses and functional experiments to investigate the genetic cause of ovarian deficiency in ten affected women. We achieved diagnoses for three of them, including the identification of novel variants in STAG3, GDF9, and FANCM. We identified potentially causative FSHR variants in another patient. This is the second report of biallelic GDF9 and FANCM variants, and, combined with functional support, validates these genes as bone fide autosomal recessive "POI genes". We also identified new candidate genes, NRIP1, XPO1, and MACF1. These genes have been linked to ovarian function in mouse, pig, and zebrafish respectively, but never in humans. In the case of NRIP1, we provide functional support for the deleterious nature of the variant via SUMOylation and luciferase/ß-galactosidase reporter assays. Our study provides multiple insights into the genetic basis of POI/DOR. We have further elucidated the involvement of GDF9, FANCM, STAG3 and FSHR in POI pathogenesis, and propose new candidate genes, NRIP1, XPO1, and MACF1, which should be the focus of future studies.


Assuntos
Carioferinas/genética , Proteínas dos Microfilamentos/genética , Proteína 1 de Interação com Receptor Nuclear/genética , Reserva Ovariana/genética , Insuficiência Ovariana Primária/genética , Receptores Citoplasmáticos e Nucleares/genética , Adolescente , Proteínas de Ciclo Celular/genética , DNA Helicases/genética , Feminino , Genômica , Fator 9 de Diferenciação de Crescimento/genética , Humanos , Infertilidade Feminina , Menopausa Precoce/genética , Doenças Ovarianas , Sequenciamento Completo do Exoma , Adulto Jovem
2.
Mol Hum Reprod ; 26(9): 665-677, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32634216

RESUMO

Infertility, a global problem affecting up to 15% of couples, can have varied causes ranging from natural ageing to the pathological development or function of the reproductive organs. One form of female infertility is premature ovarian insufficiency (POI), affecting up to 1 in 100 women and characterised by amenorrhoea and elevated FSH before the age of 40. POI can have a genetic basis, with over 50 causative genes identified. Non-obstructive azoospermia (NOA), a form of male infertility characterised by the absence of sperm in semen, has an incidence of 1% and is similarly heterogeneous. The genetic basis of male and female infertility is poorly understood with the majority of cases having no known cause. Here, we study a case of familial infertility including a proband with POI and her brother with NOA. We performed whole-exome sequencing (WES) and identified a homozygous STAG3 missense variant that segregated with infertility. STAG3 encodes a component of the meiosis cohesin complex required for sister chromatid separation. We report the first pathogenic homozygous missense variant in STAG3 and the first STAG3 variant associated with both male and female infertility. We also demonstrate limitations of WES for the analysis of homologous DNA sequences, with this variant being ambiguous or missed by independent WES protocols and its homozygosity only being established via long-range nested PCR.

3.
Hum Genet ; 139(10): 1325-1343, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32399598

RESUMO

Perrault syndrome is a rare heterogeneous condition characterised by sensorineural hearing loss and premature ovarian insufficiency. Additional neuromuscular pathology is observed in some patients. There are six genes in which variants are known to cause Perrault syndrome; however, these explain only a minority of cases. We investigated the genetic cause of Perrault syndrome in seven affected individuals from five different families, successfully identifying the cause in four patients. This included previously reported and novel causative variants in known Perrault syndrome genes, CLPP and LARS2, involved in mitochondrial proteolysis and mitochondrial translation, respectively. For the first time, we show that pathogenic variants in PEX6 can present clinically as Perrault syndrome. PEX6 encodes a peroxisomal biogenesis factor, and we demonstrate evidence of peroxisomal dysfunction in patient serum. This study consolidates the clinical overlap between Perrault syndrome and peroxisomal disorders, and highlights the need to consider ovarian function in individuals with atypical/mild peroxisomal disorders. The remaining patients had variants in candidate genes such as TFAM, involved in mtDNA transcription, replication, and packaging, and GGPS1 involved in mevalonate/coenzyme Q10 biosynthesis and whose enzymatic product is required for mouse folliculogenesis. This genomic study highlights the diverse molecular landscape of this poorly understood syndrome.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Aminoacil-tRNA Sintetases/genética , Proteínas de Ligação a DNA/genética , Dimetilaliltranstransferase/genética , Endopeptidase Clp/genética , Farnesiltranstransferase/genética , Predisposição Genética para Doença , Geraniltranstransferase/genética , Disgenesia Gonadal 46 XX/genética , Perda Auditiva Neurossensorial/genética , Proteínas Mitocondriais/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Sequência de Bases , Criança , DNA Mitocondrial/genética , Feminino , Expressão Gênica , Disgenesia Gonadal 46 XX/diagnóstico , Disgenesia Gonadal 46 XX/patologia , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Ovário/metabolismo , Ovário/patologia , Linhagem , Peroxissomos/metabolismo , Peroxissomos/patologia
4.
Maturitas ; 131: 78-86, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31787151

RESUMO

Ovarian deficiency, including diminished ovarian reserve and premature ovarian insufficiency, represents one of the main causes of female infertility. Little is known of the genetic basis of diminished ovarian reserve, while premature ovarian insufficiency often has a genetic basis, with genes affecting various processes. NR5A1 is a key gene required for gonadal function, and variants are associated with a wide phenotypic spectrum of disorders of sexual development, and are found in 0.26-8% of patients with premature ovarian insufficiency. As there is some debate about the extent of involvement of NR5A1 in the pathogenesis of ovarian deficiency, we performed an in-depth analysis of NR5A1 variants detected in a cohort of 142 patients with premature ovarian insufficiency, diminished ovarian reserve, or unexplained infertility associated with normal ovarian function. We identified rare non-synonymous protein-altering variants in 2.8 % of women with ovarian deficiency and no such variants in our small cohort of women with infertility but normal ovarian function. We observed previously reported variants associated with premature ovarian insufficiency in patients with diminished ovarian reserve, highlighting a genetic relationship between these conditions. We confirmed functional impairment resulting from a p.Val15Met variant, detected for the first time in a patient with premature ovarian insufficiency. The remaining variants were associated with preserved transcriptional activity and localization of NR5A1, indicating that rare NR5A1 variants may be incorrectly curated if functional studies are not undertaken, and/or that NR5A1 variants may have only a subtle impact on protein function and/or confer risk of ovarian deficiency via oligogenic inheritance.


Assuntos
Infertilidade Feminina/genética , Menopausa Precoce/genética , Reserva Ovariana , Insuficiência Ovariana Primária/genética , Fator Esteroidogênico 1/genética , Adulto , Grupo com Ancestrais do Continente Africano , Alelos , Estudos de Coortes , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Infertilidade Feminina/etnologia , Menopausa Precoce/etnologia , Mutação , Insuficiência Ovariana Primária/etnologia
5.
Cytogenet Genome Res ; 159(4): 201-207, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31865337

RESUMO

Genetic factors are responsible for 15% of male infertility conditions. Numerical and structural chromosomal anomalies (related to the Y chromosome or to the autosomes) are validated genetic factors leading to spermatogenic quantitative defects with a frequency depending on the severity of the phenotype. The most frequent structural chromosomal rearrangements of autosomes are translocations and inversions, whereas dicentric chromosomes involving autosomes are rare. We report a man bearing a pseudodicentric chromosome (9;21) and presenting with oligozoospermia. Extensive cytogenetic analyses were necessary to determine the precise nature of the derivative chromosome and to discount the presence of interstitial telomeric sequences. Defects in spermatogenesis and abnormal segregation at meiosis for existing spermatozoa are proposed and are the likely cause of the reproductive phenotype of the patient.


Assuntos
Cromossomos/genética , Oligospermia/genética , Translocação Genética/genética , Adulto , Inversão Cromossômica/genética , Humanos , Masculino , Espermatogênese/genética , Espermatozoides/fisiologia
6.
Sci Rep ; 9(1): 15433, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31659207

RESUMO

Personalized medicine approaches are increasingly sought for diseases with a heritable component. Systemic lupus erythematosus (SLE) is the prototypic autoimmune disease resulting from loss of immunologic tolerance, but the genetic basis of SLE remains incompletely understood. Genome wide association studies (GWAS) identify regions associated with disease, based on common single nucleotide polymorphisms (SNPs) within them, but these SNPs may simply be markers in linkage disequilibrium with other, causative mutations. Here we use an hierarchical screening approach for prediction and testing of true functional variants within regions identified in GWAS; this involved bioinformatic identification of putative regulatory elements within close proximity to SLE SNPs, screening those regions for potentially causative mutations by high resolution melt analysis, and functional validation using reporter assays. Using this approach, we screened 15 SLE associated loci in 143 SLE patients, identifying 7 new variants including 5 SNPs and 2 insertions. Reporter assays revealed that the 5 SNPs were functional, altering enhancer activity. One novel variant was linked to the relatively well characterized rs9888739 SNP at the ITGAM locus, and may explain some of the SLE heritability at this site. Our study demonstrates that non-coding regulatory elements can contain private sequence variants affecting gene expression, which may explain part of the heritability of SLE.


Assuntos
Predisposição Genética para Doença , Desequilíbrio de Ligação , Lúpus Eritematoso Sistêmico/genética , Polimorfismo de Nucleotídeo Único , Sequências Reguladoras de Ácido Nucleico , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino
7.
Hum Mutat ; 40(7): 886-892, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30924587

RESUMO

Premature ovarian insufficiency involves amenorrhea and elevated follicle-stimulating hormone before age 40, and its genetic basis is poorly understood. Here, we study 13 premature ovarian insufficiency (POI) patients using whole-exome sequencing. We identify PREPL and TP63 causative variants, and variants in other potentially novel POI genes. PREPL deficiency is a known cause of syndromic POI, matching the patients' phenotype. A role for TP63 in ovarian biology has previously been proposed but variants have been described in multiorgan syndromes, and not isolated POI. One patient with isolated POI harbored a de novo nonsense TP63 variant in the terminal exon and an unrelated patient had a different nonsense variant in the same exon. These variants interfere with the repression domain while leaving the activation domain intact. We expand the phenotypic spectrum of TP63-related disorders, provide a new genotype:phenotype correlation for TP63 and identify a new genetic cause of isolated POI.


Assuntos
Códon sem Sentido , Insuficiência Ovariana Primária/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Feminino , Predisposição Genética para Doença , Humanos , Linhagem , Domínios Proteicos , Serina Endopeptidases/genética , Fatores de Transcrição/química , Proteínas Supressoras de Tumor/química , Sequenciamento Completo do Exoma/métodos
8.
Eur J Hum Genet ; 26(9): 1319-1328, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29706645

RESUMO

Next-generation sequencing (NGS) is increasingly being used in a clinical setting for the molecular diagnosis of patients with heterogeneous disorders, such as premature ovarian insufficiency (POI). We performed NGS of ~1000 candidate genes in four unrelated patients with POI. We discovered the genetic cause of "isolated" POI in two cases, both of which had causative variants in surprising genes. In the first case, a homozygous nonsense variant in NBN was causative. Recessive function-altering NBN variants typically cause Nijmegen breakage syndrome characterized by microcephaly, cancer predisposition, and immunodeficiency, none of which are evident in the patient. At a cellular level, we found evidence of chromosomal instability. In the second case, compound heterozygous variants in EIF2B2 were causative. Recessive EIF2B2 function-altering variants usually cause leukoencephalopathy with episodic decline. Subsequent MRI revealed subclinical neurological abnormalities. These cases demonstrate that variants in NBN and EIF2B2, which usually cause severe syndromes, can cause apparently isolated POI, and that (1) NGS can precede clinical diagnosis and guide patient management, (2) NGS can redefine the phenotypic spectrum of syndromes, and (3) NGS may make unanticipated diagnoses that must be sensitively communicated to patients. Although there is rigorous debate about the handling of secondary/incidental findings using NGS, there is little discussion of the management of causative pleiotropic gene variants that have broader implications than that for which genetic studies were sought.


Assuntos
Pleiotropia Genética , Testes Genéticos/métodos , Insuficiência Ovariana Primária/genética , Adolescente , Proteínas de Ciclo Celular/genética , Códon sem Sentido , Fator de Iniciação 2B em Eucariotos/genética , Feminino , Humanos , Proteínas Nucleares/genética , Insuficiência Ovariana Primária/patologia , Análise de Sequência de DNA/métodos , Adulto Jovem
9.
J Hum Genet ; 63(5): 691-698, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29540854

RESUMO

Ovarian reserve represents the number of available follicles/oocytes within ovaries and it can be assessed by follicle stimulating hormone levels, anti-Müllerian hormone levels, and/or antral follicle count determined by ultrasounds. A low ovarian reserve is defined by an abnormal ovarian reserve test. This condition can be considered premature if it occurs before the age of 40, leading to premature ovarian insufficiency. Despite the growing knowledge concerning the genetic basis of ovarian deficiency, the majority of cases remain without a genetic diagnosis. Although 22q11.2 deletions and duplications have been associated with genitourinary malformations, ovarian deficiency is not a commonly reported feature. We report here four patients bearing a 22q11.2 rearrangement, identified during the clinical assessment of their low ovarian reserve or premature ovarian insufficiency, and discuss the molecular basis of the ovarian defects.


Assuntos
Cromossomos Humanos Par 22 , Reserva Ovariana/genética , Insuficiência Ovariana Primária/diagnóstico , Insuficiência Ovariana Primária/genética , Translocação Genética , Adulto , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Feminino , Humanos , Hibridização in Situ Fluorescente , Cariótipo , Fenótipo
10.
Brain ; 140(6): 1595-1610, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28549128

RESUMO

Although mitochondrial disorders are clinically heterogeneous, they frequently involve the central nervous system and are among the most common neurogenetic disorders. Identifying the causal genes has benefited enormously from advances in high-throughput sequencing technologies; however, once the defect is known, researchers face the challenge of deciphering the underlying disease mechanism. Here we characterize large biallelic deletions in the region encoding the ATAD3C, ATAD3B and ATAD3A genes. Although high homology complicates genomic analysis of the ATAD3 defects, they can be identified by targeted analysis of standard single nucleotide polymorphism array and whole exome sequencing data. We report deletions that generate chimeric ATAD3B/ATAD3A fusion genes in individuals from four unrelated families with fatal congenital pontocerebellar hypoplasia, whereas a case with genomic rearrangements affecting the ATAD3C/ATAD3B genes on one allele and ATAD3B/ATAD3A genes on the other displays later-onset encephalopathy with cerebellar atrophy, ataxia and dystonia. Fibroblasts from affected individuals display mitochondrial DNA abnormalities, associated with multiple indicators of altered cholesterol metabolism. Moreover, drug-induced perturbations of cholesterol homeostasis cause mitochondrial DNA disorganization in control cells, while mitochondrial DNA aggregation in the genetic cholesterol trafficking disorder Niemann-Pick type C disease further corroborates the interdependence of mitochondrial DNA organization and cholesterol. These data demonstrate the integration of mitochondria in cellular cholesterol homeostasis, in which ATAD3 plays a critical role. The dual problem of perturbed cholesterol metabolism and mitochondrial dysfunction could be widespread in neurological and neurodegenerative diseases.


Assuntos
Adenosina Trifosfatases/genética , Cerebelo/anormalidades , DNA Mitocondrial/genética , Proteínas de Membrana/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Malformações do Sistema Nervoso/genética , ATPases Associadas a Diversas Atividades Celulares , Adulto , Cerebelo/diagnóstico por imagem , Cerebelo/fisiopatologia , Consanguinidade , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/fisiopatologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Doenças Mitocondriais/diagnóstico por imagem , Doenças Mitocondriais/fisiopatologia , Malformações do Sistema Nervoso/diagnóstico por imagem , Malformações do Sistema Nervoso/fisiopatologia
11.
Endocr Rev ; 37(6): 609-635, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27690531

RESUMO

Premature ovarian insufficiency (POI) is one form of female infertility, defined by loss of ovarian activity before the age of 40 and characterized by amenorrhea (primary or secondary) with raised gonadotropins and low estradiol. POI affects up to one in 100 females, including one in 1000 before the age of 30. Substantial evidence suggests a genetic basis for POI; however, the majority of cases remain unexplained, indicating that genes likely to be associated with this condition are yet to be discovered. This review discusses the current knowledge of the genetic basis of POI. We highlight genes typically known to cause syndromic POI that can be responsible for isolated POI. The role of mouse models in understanding POI pathogenesis is discussed, and a thorough list of candidate POI genes is provided. Identifying a genetic basis for POI has multiple advantages, such as enabling the identification of presymptomatic family members who can be offered counseling and cryopreservation of eggs before depletion, enabling personalized treatment based on the cause of an individual's condition, and providing better understanding of disease mechanisms that ultimately aid the development of improved treatments.


Assuntos
Insuficiência Ovariana Primária/genética , Animais , Feminino , Humanos , Insuficiência Ovariana Primária/diagnóstico , Insuficiência Ovariana Primária/terapia
12.
Am J Hum Genet ; 94(2): 209-22, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24462369

RESUMO

Leigh syndrome (LS) is a severe neurodegenerative disorder with characteristic bilateral lesions, typically in the brainstem and basal ganglia. It usually presents in infancy and is genetically heterogeneous, but most individuals with mitochondrial complex IV (or cytochrome c oxidase) deficiency have mutations in the biogenesis factor SURF1. We studied eight complex IV-deficient LS individuals from six families of Lebanese origin. They differed from individuals with SURF1 mutations in having seizures as a prominent feature. Complementation analysis suggested they had mutation(s) in the same gene but targeted massively parallel sequencing (MPS) of 1,034 genes encoding known mitochondrial proteins failed to identify a likely candidate. Linkage and haplotype analyses mapped the location of the gene to chromosome 19 and targeted MPS of the linkage region identified a homozygous c.3G>C (p.Met1?) mutation in C19orf79. Abolishing the initiation codon could potentially still allow initiation at a downstream methionine residue but we showed that this would not result in a functional protein. We confirmed that mutation of this gene was causative by lentiviral-mediated phenotypic correction. C19orf79 was recently renamed PET100 and predicted to encode a complex IV biogenesis factor. We showed that it is located in the mitochondrial inner membrane and forms a ∼300 kDa subcomplex with complex IV subunits. Previous proteomic analyses of mitochondria had overlooked PET100 because its small size was below the cutoff for annotating bona fide proteins. The mutation was estimated to have arisen at least 520 years ago, explaining how the families could have different religions and different geographic origins within Lebanon.


Assuntos
Deficiência de Citocromo-c Oxidase/etnologia , Deficiência de Citocromo-c Oxidase/genética , Efeito Fundador , Doença de Leigh/etnologia , Doença de Leigh/genética , Proteínas Mitocondriais/genética , Cromossomos Humanos Par 19/genética , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Deficiência de Citocromo-c Oxidase/complicações , DNA Mitocondrial/genética , DNA Mitocondrial/isolamento & purificação , Feminino , Teste de Complementação Genética , Ligação Genética , Estudo de Associação Genômica Ampla , Haplótipos , Homozigoto , Humanos , Lactente , Líbano , Doença de Leigh/complicações , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Linhagem , Polimorfismo de Nucleotídeo Único , Proteômica , Análise de Sequência de DNA
13.
Hum Mol Genet ; 22(22): 4460-73, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23814038

RESUMO

Iron-sulfur clusters (ISCs) are important prosthetic groups that define the functions of many proteins. Proteins with ISCs (called iron-sulfur or Fe-S proteins) are present in mitochondria, the cytosol, the endoplasmic reticulum and the nucleus. They participate in various biological pathways including oxidative phosphorylation (OXPHOS), the citric acid cycle, iron homeostasis, heme biosynthesis and DNA repair. Here, we report a homozygous mutation in LYRM4 in two patients with combined OXPHOS deficiency. LYRM4 encodes the ISD11 protein, which forms a complex with, and stabilizes, the sulfur donor NFS1. The homozygous mutation (c.203G>T, p.R68L) was identified via massively parallel sequencing of >1000 mitochondrial genes (MitoExome sequencing) in a patient with deficiency of complexes I, II and III in muscle and liver. These three complexes contain ISCs. Sanger sequencing identified the same mutation in his similarly affected cousin, who had a more severe phenotype and died while a neonate. Complex IV was also deficient in her skeletal muscle. Several other Fe-S proteins were also affected in both patients, including the aconitases and ferrochelatase. Mutant ISD11 only partially complemented for an ISD11 deletion in yeast. Our in vitro studies showed that the l-cysteine desulfurase activity of NFS1 was barely present when co-expressed with mutant ISD11. Our findings are consistent with a defect in the early step of ISC assembly affecting a broad variety of Fe-S proteins. The differences in biochemical and clinical features between the two patients may relate to limited availability of cysteine in the newborn period and suggest a potential approach to therapy.


Assuntos
Proteínas Reguladoras do Ferro/genética , Proteínas com Ferro-Enxofre/deficiência , Mitocôndrias/genética , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Sequência de Aminoácidos , Transporte de Elétrons , Feminino , Genes Mitocondriais , Homozigoto , Humanos , Recém-Nascido , Proteínas Reguladoras do Ferro/química , Proteínas Reguladoras do Ferro/metabolismo , Proteínas com Ferro-Enxofre/genética , Proteínas com Ferro-Enxofre/metabolismo , Fígado/metabolismo , Masculino , Mitocôndrias/metabolismo , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Músculos/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Sensíveis a N-Etilmaleimida/genética , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Fosforilação Oxidativa , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Alinhamento de Sequência , Análise de Sequência de DNA
14.
PLoS Genet ; 9(12): e1004034, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24385928

RESUMO

Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.


Assuntos
Citocromos b/biossíntese , Complexo III da Cadeia de Transporte de Elétrons/genética , Proteínas de Membrana/genética , Doenças Mitocondriais/genética , Consanguinidade , Citocromos b/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Homozigoto , Humanos , Proteínas de Membrana/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/patologia , Doenças Mitocondriais/terapia , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Fosforilação Oxidativa , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Sci Transl Med ; 4(118): 118ra10, 2012 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-22277967

RESUMO

Advances in next-generation sequencing (NGS) promise to facilitate diagnosis of inherited disorders. Although in research settings NGS has pinpointed causal alleles using segregation in large families, the key challenge for clinical diagnosis is application to single individuals. To explore its diagnostic use, we performed targeted NGS in 42 unrelated infants with clinical and biochemical evidence of mitochondrial oxidative phosphorylation disease. These devastating mitochondrial disorders are characterized by phenotypic and genetic heterogeneity, with more than 100 causal genes identified to date. We performed "MitoExome" sequencing of the mitochondrial DNA (mtDNA) and exons of ~1000 nuclear genes encoding mitochondrial proteins and prioritized rare mutations predicted to disrupt function. Because patients and healthy control individuals harbored a comparable number of such heterozygous alleles, we could not prioritize dominant-acting genes. However, patients showed a fivefold enrichment of genes with two such mutations that could underlie recessive disease. In total, 23 of 42 (55%) patients harbored such recessive genes or pathogenic mtDNA variants. Firm diagnoses were enabled in 10 patients (24%) who had mutations in genes previously linked to disease. Thirteen patients (31%) had mutations in nuclear genes not previously linked to disease. The pathogenicity of two such genes, NDUFB3 and AGK, was supported by complementation studies and evidence from multiple patients, respectively. The results underscore the potential and challenges of deploying NGS in clinical settings.


Assuntos
Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Análise de Sequência de DNA/métodos , Sequência de Aminoácidos , Sequência de Bases , Estudos de Casos e Controles , Núcleo Celular/genética , Criança , Pré-Escolar , DNA Mitocondrial/genética , Complexo I de Transporte de Elétrons/genética , Exoma/genética , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Genes Mitocondriais/genética , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Doenças Mitocondriais/enzimologia , Miopatias Mitocondriais/genética , Dados de Sequência Molecular , Mutação/genética , Fosforilação Oxidativa , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Reprodutibilidade dos Testes
16.
Hum Mutat ; 33(2): 411-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22072591

RESUMO

Next-generation sequencing (NGS) is transitioning from being a research tool to being used in routine genetic diagnostics, where a major challenge is distinguishing which of many sequence variants in an individual are truly pathogenic. We describe some limitations of in silico analyses of NGS data that emphasize the need for experimental confirmation. Using NGS, we recently identified an apparently homozygous missense mutation in NUBPL in a patient with mitochondrial complex I deficiency. Causality was established via lentiviral correction studies with wild-type NUBPL cDNA. NGS data, however, provided an incomplete understanding of the genetic abnormality. We show that the maternal allele carries an unbalanced inversion, while the paternal allele carries a branch-site mutation in addition to the missense mutation. We demonstrate that the branch-site mutation, which is present in approximately one of 120 control chromosomes, likely contributes to pathogenicity and may be one of the most common autosomal mutations causing mitochondrial dysfunction. Had these analyses not been performed following NGS, the original missense mutation may be incorrectly annotated as pathogenic and a potentially common pathogenic variant not detected. It is important that locus-specific databases contain accurate information on pathogenic variation. NGS data, therefore, require rigorous experimental follow-up to confirm mutation pathogenicity.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas Mitocondriais/genética , Técnicas de Diagnóstico Molecular , Mutação , Alelos , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Complexo I de Transporte de Elétrons/deficiência , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ordem dos Genes , Humanos , Íntrons , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Estabilidade Proteica , Sítios de Splice de RNA , Alinhamento de Sequência
17.
J Mol Biol ; 414(3): 413-26, 2011 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-22019594

RESUMO

The assembly of complex I (NADH-ubiquinone oxidoreductase) is a complicated process, requiring the integration of 45 subunits encoded by both nuclear and mitochondrial DNAs into a structure of approximately 1 MDa. A number of "assembly factors" that aid complex I biogenesis have recently been described, including C8orf38. This protein was identified as an assembly factor by its evolutionary conservation in organisms containing complex I and by a C8orf38 mutation in a patient presenting with Leigh syndrome and isolated complex I deficiency. In this report, we have undertaken the characterization of C8orf38 and its role in complex I assembly. Analysis of mitochondria from fibroblasts of a patient harboring a C8orf38 mutation showed almost undetectable levels of steady-state complex I and defective biogenesis of the mtDNA-encoded subunit ND1. Complementation with wild-type C8orf38 restored the levels of both ND1 and complex I, confirming the C8orf38 mutation as the cause of the complex I defect in the patient. In the absence of ND1 in patient cells, early- and mid-stage intermediate complexes were still formed; however, assembly of late-stage intermediates was impaired, indicating a convergence point in the assembly process. While C8orf38 appears to behave at a step in complex I biogenesis similar to that of the assembly factor C20orf7, complementation studies showed that both proteins are required for ND1 synthesis/stabilization. We conclude that C8orf38 is a crucial factor required for the translation and/or integration of ND1 into an early-stage assembly intermediate and that mutation of C8orf38 disrupts the initial stages of complex I biogenesis.


Assuntos
DNA Mitocondrial/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Mutação , Animais , Células COS , Chlorocebus aethiops , Fibroblastos/metabolismo , Células HEK293 , Humanos , Doença de Leigh/genética , Lentivirus/genética , Biossíntese de Proteínas , Isoformas de Proteínas , Estrutura Terciária de Proteína
18.
Cell Metab ; 14(3): 428-34, 2011 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-21907147

RESUMO

The metazoan mitochondrial translation machinery is unusual in having a single tRNA(Met) that fulfills the dual role of the initiator and elongator tRNA(Met). A portion of the Met-tRNA(Met) pool is formylated by mitochondrial methionyl-tRNA formyltransferase (MTFMT) to generate N-formylmethionine-tRNA(Met) (fMet-tRNA(met)), which is used for translation initiation; however, the requirement of formylation for initiation in human mitochondria is still under debate. Using targeted sequencing of the mtDNA and nuclear exons encoding the mitochondrial proteome (MitoExome), we identified compound heterozygous mutations in MTFMT in two unrelated children presenting with Leigh syndrome and combined OXPHOS deficiency. Patient fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of MTFMT. Furthermore, patient fibroblasts have dramatically reduced fMet-tRNA(Met) levels and an abnormal formylation profile of mitochondrially translated COX1. Our findings demonstrate that MTFMT is critical for efficient human mitochondrial translation and reveal a human disorder of Met-tRNA(Met) formylation.


Assuntos
Ciclo-Oxigenase 1/metabolismo , DNA Mitocondrial/química , Fibroblastos/metabolismo , Doença de Leigh/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Biossíntese de Proteínas , RNA de Transferência de Metionina/metabolismo , Células Cultivadas , Criança , Ciclo-Oxigenase 1/genética , DNA Mitocondrial/genética , Fibroblastos/patologia , Heterozigoto , Humanos , Hidroximetil e Formil Transferases , Immunoblotting , Doença de Leigh/metabolismo , Doença de Leigh/patologia , Lentivirus , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mutação , Biossíntese de Proteínas/genética , Análise de Sequência de DNA , Transdução Genética , Vírion
19.
IUBMB Life ; 63(9): 669-77, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21766414

RESUMO

Disorders of oxidative phosphorylation (OXPHOS) have a birth prevalence of ∼1/5,000 and are the most common inborn errors of metabolism. The most common OXPHOS disorder is complex I deficiency. Patients with complex I deficiency present with variable symptoms, such as muscle weakness, cardiomyopathy, developmental delay or regression, blindness, seizures, failure to thrive, liver dysfunction or ataxia. Molecular diagnosis of patients with complex I deficiency is a challenging task due to the clinical heterogeneity of patients and the large number of candidate disease genes, both nuclear-encoded and mitochondrial DNA (mtDNA)-encoded. In this review, we have thoroughly surveyed the literature to identify 149 patients described with both isolated complex I deficiency and pathogenic mutations within nuclear genes. In total, 115 different pathogenic mutations have been reported in 22 different nuclear genes encoding complex I subunits or assembly factors, highlighting the allelic and locus heterogeneity of this disorder. Missense mutations predominate in genes encoding core subunits and some assembly factors while null-type mutations are common in the genes encoding supernumerary subunits and other assembly factors. Despite developments in molecular technology, many patients do not receive molecular diagnosis and no gene has yet been identified that accounts for more than 5% of cases, suggesting that there are likely many disease genes that await discovery.


Assuntos
Complexo I de Transporte de Elétrons/deficiência , Genes/genética , Erros Inatos do Metabolismo/genética , Doenças Mitocondriais/genética , Biologia Computacional , DNA Mitocondrial/genética , Humanos , Mutação/genética , Subunidades Proteicas/genética
20.
Eur J Hum Genet ; 19(7): 769-75, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21364701

RESUMO

Defects of the mitochondrial respiratory chain are associated with a diverse spectrum of clinical phenotypes, and may be caused by mutations in either the nuclear or the mitochondrial genome (mitochondrial DNA (mtDNA)). Isolated complex I deficiency is the most common enzyme defect in mitochondrial disorders, particularly in children in whom family history is often consistent with sporadic or autosomal recessive inheritance, implicating a nuclear genetic cause. In contrast, although a number of recurrent, pathogenic mtDNA mutations have been described, historically, these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency from 101 families. Pathogenic mtDNA mutations were found in 29 of 101 probands (29%), 21 in MTND subunit genes and 8 in mtDNA tRNA genes. Nuclear gene defects were inferred in 38 of 101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. The median age at onset was higher in mtDNA patients (12 months) than in patients with a nuclear gene defect (3 months). However, considerable overlap existed, with onset varying from 0 to >60 months in both groups. Our findings confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children. In the absence of parental consanguinity, we recommend whole mitochondrial genome sequencing as a key approach to elucidate the underlying molecular genetic abnormality.


Assuntos
DNA Mitocondrial/genética , Complexo I de Transporte de Elétrons/deficiência , Complexo I de Transporte de Elétrons/genética , Doenças Mitocondriais/genética , Mutação/genética , Adolescente , Adulto , Idade de Início , Núcleo Celular/genética , Criança , Pré-Escolar , Ativação Enzimática/genética , Humanos , Lactente , Recém-Nascido , Padrões de Herança/genética , Doenças Mitocondriais/mortalidade , Fenótipo , Análise de Sobrevida , Adulto Jovem
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