RESUMEN
In a 28-year-old male with a mild mitochondrial myopathy manifesting as exercise intolerance and early signs of cardiomyopathy without muscle weakness or ophthalmoplegia, we identified two novel mutations in the SLC25A4 gene: c.707G>C in exon 3 (p.(R236P)) and c.116_137del in exon 2 (p.(Q39Lfs*14)). Serum lactate levels at rest were elevated (12.7 mM). Both the patient's father and brother were heterozygous carriers of the c.707G>C mutation and were asymptomatic. The second mutation causes a 22 bp deletion leading to a frame shift likely giving rise to a premature stop codon and nonsense-mediated decay (NMD). The segregation of the mutations could not be tested directly as the mother had died before. However, indirect evidence from NMD experiments showed that the two mutations were situated on two different alleles in the patient. This case is unique compared to other previously reported patients with either progressive external ophthalmoplegia (PEO) or clear hypertrophic cardiomyopathy with exercise intolerance and/or muscle weakness carrying recessive mutations leading to a complete absence of the SLC25A4 protein. Most likely in our patient, although severely reduced, SLC25A4 is still partially present and functional.
RESUMEN
BACKGROUND: Mitochondrial disorders are associated with abnormalities of the oxidative phosphorylation (OXPHOS) system and cause significant morbidity and mortality in the population. The extensive clinical and genetic heterogeneity of these disorders due to a broad variety of mutations in several hundreds of candidate genes, encoded by either the mitochondrial DNA (mtDNA) or nuclear DNA (nDNA), impedes a straightforward genetic diagnosis. A new disease gene is presented here, identified in a single Kurdish patient born from consanguineous parents with neonatally fatal Leigh syndrome and complex I deficiency. METHODS AND RESULTS: Using homozygosity mapping and subsequent positional candidate gene analysis, a total region of 255.8 Mb containing 136 possible mitochondrial genes was identified. A pathogenic mutation was found in the complex I subunit encoding the NDUFA9 gene, changing a highly conserved arginine at position 321 to proline. This is the first disease-causing mutation ever reported for NDUFA9. Complex I activity was restored in fibroblasts of the patient by lentiviral transduction with wild type but not mutant NDUFA9, confirming that the mutation causes the complex I deficiency and related disease. CONCLUSIONS: The data show that homozygosity mapping and candidate gene analysis remain an efficient way to detect mutations even in small consanguineous pedigrees with OXPHOS deficiency, especially when the enzyme deficiency in fibroblasts allows appropriate candidate gene selection and functional complementation.
Asunto(s)
Complejo I de Transporte de Electrón/genética , Enfermedad de Leigh/diagnóstico , Enfermedad de Leigh/genética , Mutación Missense , Secuencia de Aminoácidos , Células Cultivadas , Consanguinidad , Análisis Mutacional de ADN , Complejo I de Transporte de Electrón/metabolismo , Resultado Fatal , Estudios de Asociación Genética , Homocigoto , Humanos , Recién Nacido , Imagen por Resonancia Magnética , Masculino , Datos de Secuencia Molecular , NeuroimagenRESUMEN
The mitochondrial DNA (mtDNA) is highly variable, containing large numbers of pathogenic mutations and neutral polymorphisms. The spectrum of homoplasmic mtDNA variation was characterized in 730 subjects and compared with known pathogenic sites. The frequency and distribution of variants in protein coding genes were inversely correlated with conservation at the amino acid level. Analysis of tRNA secondary structures indicated a preference of variants for the loops and some acceptor stem positions. This comprehensive overview of mtDNA variants distinguishes between regions and positions which are likely not critical, mainly conserved regions with pathogenic mutations and essential regions containing no mutations at all.
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Secuencia Conservada , ADN Mitocondrial/genética , Adolescente , Adulto , Anciano , Niño , Preescolar , ADN Mitocondrial/química , Humanos , Lactante , Persona de Mediana Edad , Conformación de Ácido Nucleico , Polimorfismo Genético , ARN de Transferencia/genética , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
BACKGROUND: Leigh syndrome is an early onset, progressive, neurodegenerative disorder with developmental and motor skills regression. Characteristic magnetic resonance imaging abnormalities consist of focal bilateral lesions in the basal ganglia and/or the brainstem. The main cause is a deficiency in oxidative phosphorylation due to mutations in an mtDNA or nuclear oxidative phosphorylation gene. METHODS AND RESULTS: A consanguineous Moroccan family with Leigh syndrome comprise 11 children, three of which are affected. Marker analysis revealed a homozygous region of 11.5 Mb on chromosome 20, containing 111 genes. Eight possible mitochondrial candidate genes were sequenced. Patients were homozygous for an unclassified variant (p.P193L) in the cardiolipin synthase gene (CRLS1). As this variant was present in 20% of a Moroccan control population and enzyme activity was only reduced to 50%, this could not explain the rare clinical phenotype in our family. Patients were also homozygous for an amino acid substitution (p.L159F) in C20orf7, a new complex I assembly factor. Parents were heterozygous and unaffected sibs heterozygous or homozygous wild type. The mutation affects the predicted S-adenosylmethionine (SAM) dependent methyltransferase domain of C20orf7, possibly involved in methylation of NDUFB3 during the assembly process. Blue native gel electrophoresis showed an altered complex I assembly with only 30-40% of mature complex I present in patients and 70-90% in carriers. CONCLUSIONS: A new cause of Leigh syndrome can be a defect in early complex I assembly due to C20orf7 mutations.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Enfermedad de Leigh/enzimología , Enfermedad de Leigh/genética , Metiltransferasas/genética , Proteínas Mitocondriales/genética , Mutación/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Secuencia de Bases , Preescolar , Análisis Mutacional de ADN , Complejo I de Transporte de Electrón/genética , Familia , Femenino , Homocigoto , Humanos , Enfermedad de Leigh/diagnóstico por imagen , Enfermedad de Leigh/metabolismo , Leucocitos Mononucleares/enzimología , Imagen por Resonancia Magnética , Masculino , Metiltransferasas/química , Proteínas Mitocondriales/química , Datos de Secuencia Molecular , Marruecos , Linaje , Tomografía Computarizada por Rayos X , Adulto JovenRESUMEN
BACKGROUND: Mutations in the DNA polymerase-gamma (POLG) gene are a major cause of clinically heterogeneous mitochondrial diseases, associated with mtDNA depletion and multiple deletions. OBJECTIVE: To determine the spectrum of POLG mutations in our Dutch patient cohort, to evaluate the pathogenicity of novel mutations, and to establish genotype-phenotype correlations. RESULTS: The authors identified 64 predominantly recessive mutations in 37 patients from a total of 232 patients, consisting of 23 different mutations. The substitution p.A467T was most frequently observed (n = 23), but was as frequent in childhood cases as in adult cases. Five new pathogenic recessive mutations, p.Lys925ArgfsX42, p.R275X, p.G426S, p.A804T and p.R869Q were identified. The known dominant chronic progressive external ophthalmoplegia (CPEO) mutation p.R943H was for the first time associated with premature ovarian failure as well. In 19 patients the authors identified only a single recessive mutation, or a sequence variant with unclear clinical significance. The data substantiate earlier observations that in POLG patients a fatal status epilepticus and liver failure can be triggered by sodium valproate. It is therefore important to exclude POLG mutations before administering this treatment. CONCLUSION: The clinical features of the patient are the most important features to select putative POLG mutation carriers and not the presence of mtDNA deletions or OXPHOS (oxidative phosphorylation) activity. The authors conclude that POLG mutations are an important cause of heterogeneous mitochondrial pathology and that more accurate genotype-phenotype correlations allow a more rapid genetic diagnosis and improved prognosis for mutation carriers.
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ADN Polimerasa Dirigida por ADN/genética , Mutación , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Niño , Preescolar , Estudios de Cohortes , Simulación por Computador , Análisis Mutacional de ADN , ADN Polimerasa gamma , ADN Mitocondrial/genética , Femenino , Frecuencia de los Genes , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Oftalmoplejía Externa Progresiva Crónica/genética , Linaje , Fenotipo , Insuficiencia Ovárica Primaria/genética , Alineación de SecuenciaRESUMEN
Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease characterized by left ventricular hypertrophy (LVH) predominantly affecting the interventricular septum. Cardiac myosin-binding protein C (cMyBP-C) mutations are common causes of FHC. Gene expression profiling was performed in left ventricles of 9-week-old wild-type mice, heterozygous cMyBP-C KO mice displaying asymmetric septal hypertrophy, and homozygous mice developing eccentric LVH. Knocking out one or two cMyBP-C genes leads primarily to gene expression changes indicating an increased energy demand, activation of the JNK and p38 parts of the MAPK pathway and deactivation of the ERK part, and induction of apoptosis. Altered gene expression for processes related to cardiac structure, contractile proteins, and protein turnover was also identified. Many of the changes were more pronounced in the homozygous KO mice. These alterations point to physiological and pathological adaptations in the prehypertrophic heterozygous KO mice and the hypertrophic homozygous mice.
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Cardiomiopatía Hipertrófica Familiar/metabolismo , Proteínas Portadoras/metabolismo , Trastornos de los Cromosomas/metabolismo , Regulación de la Expresión Génica , Hipertrofia Ventricular Izquierda/metabolismo , Miocardio/metabolismo , Animales , Apoptosis/genética , Cardiomiopatía Hipertrófica Familiar/genética , Cardiomiopatía Hipertrófica Familiar/patología , Proteínas Portadoras/genética , Trastornos de los Cromosomas/genética , Trastornos de los Cromosomas/patología , Quinasas MAP Reguladas por Señal Extracelular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Heterocigoto , Homocigoto , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/patología , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Noqueados , Miocardio/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Tabique Interventricular/metabolismo , Tabique Interventricular/patología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Cardiac hypertrophy is an important risk factor for cardiac morbidity and mortality. To unravel the underlying pathogenic genetic pathways, we hybridized left ventricular RNA from Transverse Aortic Constriction mice at 48 h, 1 week, and 2, 3, and 8 weeks after surgery to microarrays containing a 15K fetal cDNA collection. Key processes involved an early restriction in the expression of metabolic genes, accompanied by increased expression of genes related to growth and reactivation of fetal genes. Most of these genes returned to basal expression levels during the later, compensated hypertrophic phase. Our findings suggest that compensated hypertrophy in these mice is established by rapid adaptation of the heart at the cost of gene expression associated with metabolic activity, with only temporary expression of possible maladaptive processes. Therefore, the transient early changes may reflect a beneficial response to pressure overload, as deterioration of cardiac hemodynamic function or heart failure does not occur.
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Cardiomegalia/genética , Regulación de la Expresión Génica , Animales , Aorta/cirugía , Cardiomegalia/etiología , Modelos Animales de Enfermedad , Metabolismo Energético/genética , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/genética , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Presión VentricularRESUMEN
Screening the mitochondrial DNA of a 64-year-old woman with mitochondrial myopathy revealed 76% of the tRNA(Leu(UUR)) A3302G mutation in muscle. Muscle of her affected son carried 96% mutated mitochondrial DNA. Both patients were biopsied twice, showing isolated complex I deficiency in the son's first biopsy, additional increased (within normal range) complex II + III activities in his second biopsy, combined complex I, II + III deficiency in mothers first biopsy and additional complex IV deficiency in her second biopsy. After a stay in the mountains, the son died of cardiac arrhythmia. The A3302G mutation has been reported before and is associated with mitochondrial myopathy and cardiorespiratory failure. Pathogenesis is explained by abnormal mtRNA processing, which was also reported for the adjacent C3303T mutation associated with cardiomyopathy and/or skeletal myopathy. Our findings suggest that a high mutation load of the A3302G mutation can lead to fatal cardiorespiratory failure, likely triggered by low environmental oxygen pressure and exercise.
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ADN Mitocondrial/genética , Paro Cardíaco/genética , Miopatías Mitocondriales/genética , Mutación , ARN de Transferencia de Leucina/genética , Riesgo , Adulto , Análisis Mutacional de ADN/métodos , Femenino , Paro Cardíaco/etiología , Paro Cardíaco/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Miopatías Mitocondriales/complicaciones , Miopatías Mitocondriales/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologíaRESUMEN
In patients with mitochondrial disease a continuously increasing number of mitochondrial DNA (mtDNA) mutations and polymorphisms have been identified. Most pathogenic mtDNA mutations are heteroplasmic, resulting in heteroduplexes after PCR amplification of mtDNA. To detect these heteroduplexes, we used the technique of denaturing high performance liquid chromatography (DHPLC). The complete mitochondrial genome was amplified in 13 fragments of 1-2 kb, digested in fragments of 90-600 bp and resolved at their optimal melting temperature. The sensitivity of the DHPLC system was high with a lowest detection of 0.5% for the A8344G mutation. The muscle mtDNA from six patients with mitochondrial disease was screened and three mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNA(Leu(UUR)) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNA(Leu(UUR)) gene (80% heteroplasmy) and the third patient with Leigh syndrome carried a T9176C mutation in the ATPase6 gene (93% heteroplasmy). We conclude that DHPLC analysis is a sensitive and specific method to detect heteroplasmic mtDNA mutations. The entire automatic procedure can be completed within 2 days and can also be applied to exclude mtDNA involvement, providing a basis for subsequent investigation of nuclear genes.
Asunto(s)
Cromatografía Líquida de Alta Presión , ADN Mitocondrial/genética , Pruebas Genéticas/métodos , Genoma , Análisis Heterodúplex/métodos , Mutación/genética , Adenosina Trifosfatasas/genética , Secuencia de Bases , Cardiomiopatías/genética , Cardiomiopatías/patología , Núcleo Celular/genética , Análisis Mutacional de ADN/métodos , Cartilla de ADN/genética , Enzimas de Restricción del ADN/metabolismo , Humanos , Enfermedad de Leigh/genética , Enfermedad de Leigh/patología , Mitocondrias Musculares/genética , Mitocondrias Musculares/patología , Miopatías Mitocondriales/genética , Miopatías Mitocondriales/patología , ATPasas de Translocación de Protón Mitocondriales , Distrofias Musculares/genética , Distrofias Musculares/patología , Desnaturalización de Ácido Nucleico , Polimorfismo Genético/genética , Aminoacil-ARN de Transferencia/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Tamaño de la Muestra , Sensibilidad y Especificidad , Temperatura , Factores de TiempoRESUMEN
BACKGROUND: Alport syndrome (AS) is a clinically and genetically heterogeneous renal disorder, predominantly affecting the type IV collagen alpha 3/alpha 4/alpha 5 network of the glomerular basement membrane (GBM). AS can be caused by mutations in any of the three genes encoding these type IV collagen chains. The majority of AS families (85%) are X-linked (XL-AS) involving mutations in the COL4A5 gene. Mutations in the COL4A3 and COL4A4 genes cause autosomal recessive AS (AR-AS), accounting for approximately 14% of the cases. Recently, autosomal dominant AS (AD-AS) was linked to the COL4A3/COL4A4 locus in a large family. METHODS: COL4A3 and COL4A4 cDNAs were generated by nested reverse transcription-polymerase chain reaction and were analyzed by DNA sequence analysis. Denaturating high-performance liquid chromatography (DHPLC) was used for mutation and segregation analysis at the genomic DNA level. RESULTS: In the AD-AS family, a splice site mutation resulting in skipping of exon 21 of the COL4A3 gene was detected. The mutation does not alter the reading frame and is predicted to result in a COL4A3 chain with an internal deletion. CONCLUSION: As the NC domain is intact, this chain may be incorporated and distort the collagen triple helix, thereby causing the dominant effect of the mutation. The finding of a specific COL4A3 mutation in AD-AS completes the spectrum of type IV collagen mutations in all genetic forms of AS.