Mutation of C20orf7 disrupts complex I assembly and causes lethal neonatal mitochondrial disease.

Complex I (NADH:ubiquinone oxidoreductase) is the first and largest multimeric complex of the mitochondrial respiratory chain. Human complex I comprises seven subunits encoded by mitochondrial DNA and 38 nuclear-encoded subunits that are assembled together in a process that is only partially understood. To date, mutations causing complex I deficiency have been described in all 14 core subunits, five supernumerary subunits, and four assembly factors. We describe complex I deficiency caused by mutation of the putative complex I assembly factor C20orf7. A candidate region for a lethal neonatal form of complex I deficiency was identified by homozygosity mapping of an Egyptian family with one affected child and two affected pregnancies predicted by enzyme-based prenatal diagnosis. The region was confirmed by microcell-mediated chromosome transfer, and 11 candidate genes encoding potential mitochondrial proteins were sequenced. A homozygous missense mutation in C20orf7 segregated with disease in the family. We show that C20orf7 is peripherally associated with the matrix face of the mitochondrial inner membrane and that silencing its expression with RNAi decreases complex I activity. C20orf7 patient fibroblasts showed an almost complete absence of complex I holoenzyme and were defective at an early stage of complex I assembly, but in a manner distinct from the assembly defects caused by mutations in the assembly factor NDUFAF1. Our results indicate that C20orf7 is crucial in the assembly of complex I and that mutations in C20orf7 cause mitochondrial disease.

Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder with a population frequency of approximately 1 in 10,000. The most common epigenetic defect in BWS is a loss of methylation (LOM) at the 11p15.5 imprinting centre, KCNQ1OT1 TSS-DMR, and affects 50% of cases. We hypothesised that genetic factors linked to folate metabolism may play a role in BWS predisposition via effects on methylation maintenance at KCNQ1OT1 TSS-DMR. RESULTS: Single nucleotide variants (SNVs) in the folate pathway affecting methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR), 5-methyltetrahydrofolate-homocysteine S-methyltransferase (MTR), cystathionine beta-synthase (CBS) and methionine adenosyltransferase (MAT1A) were examined in 55 BWS patients with KCNQ1OT1 TSS-DMR LOM and in 100 unaffected cases. MTHFR rs1801133: C>T was more prevalent in BWS with KCNQ1OT1 TSS-DMR LOM (p < 0.017); however, the relationship was not significant when the Bonferroni correction for multiple testing was applied (significance, p = 0.0036). None of the remaining 13 SNVs were significantly different in the two populations tested. The DNMT1 locus was screened in 53 BWS cases, and three rare missense variants were identified in each of three patients: rs138841970: C>T, rs150331990: A>G and rs757460628: G>A encoding NP_001124295 p.Arg136Cys, p.His1118Arg and p.Arg1223His, respectively. These variants have population frequencies of less than 1 in 1000 and were absent from 100 control cases. Functional characterization using a hemimethylated DNA trapping assay revealed a reduced methyltransferase activity relative to wild-type DNMT1 for each variant ranging from 40 to 70% reduction in activity. CONCLUSIONS: This study is the first to examine folate pathway genetics in BWS and to identify rare DNMT1 missense variants in affected individuals. Our data suggests that reduced DNMT1 activity could affect maintenance of methylation at KCNQ1OT1 TSS-DMR in some cases of BWS, possibly via a maternal effect in the early embryo. Larger cohort studies are warranted to further interrogate the relationship between impaired MTHFR enzymatic activity attributable to MTHFR rs1801133: C>T, dietary folate intake and BWS.

Detection of mutations in genes associated with hearing loss using a microarray-based approach.

Knowing the etiology of hearing loss in a person has implications for counseling and management of the condition. More than 50% of cases of early onset, nonsyndromic sensorineural hearing loss are attributable to genetic factors. However, deafness is a genetically heterogeneous condition and it is therefore currently not economically and practically feasible to screen for mutations in all known deafness genes. We have developed a microarray-based hybridization biochip assay for the detection of known mutations. The current version of the hearing loss biochip detects nine common mutations in the connexin 26 gene, four mutations in the pendrin gene, one mutation in the usherin gene, and one mutation in mitochondrial DNA. The biochip was validated using DNA from 250 people with apparent nonsyndromic, moderate to profound sensorineural hearing loss. The hearing loss biochip detected with 100% accuracy the mutations it was designed for. No false-positives or false-negative results were seen. The biochip can easily be expanded to test for additional mutations in genes associated with hearing impairment or other genetic conditions.

Clinical and molecular features of encephalomyopathy due to the A3302G mutation in the mitochondrial tRNA(Leu(UUR)) gene.

BACKGROUND: The mitochondrial DNA mutation A3302G in the tRNA(Leu(UUR)) gene causes respiratory chain complex I deficiency. The main clinical feature appears to be a progressive mitochondrial myopathy with proximal muscle weakness. OBJECTIVE: To report on clinical and molecular features in 4 novel patients with the A3302G mutation. DESIGN: Case reports. PATIENTS: Four patients (3 of whom are from the same family) with a myopathy caused by the A3302G mitochondrial DNA mutation. MAIN OUTCOME MEASURE: Identification of the A3302G mutation by DNA sequencing. RESULTS: All 4 patients had an adult-onset progressive mitochondrial myopathy with proximal muscle weakness, resulting in exercise intolerance. In 2 unrelated patients, upper limb reflexes were absent with preservation of at least some lower limb reflexes. Other features including hearing loss, recurrent headaches, ptosis, progressive external ophthalmoplegia, and depression were present. CONCLUSION: While the dominant clinical features of the A3302G mutation were exercise intolerance and proximal muscle weakness, other features of mitochondrial encephalomyopathies, previously not described for this mutation, were present.

Clinical and molecular features of adPEO due to mutations in the Twinkle gene.

We have analyzed Twinkle, the causative gene for autosomal dominant progressive external ophthalmoplegia (adPEO) on chromosome 10, in 11 Australian autosomal dominant progressive external ophthalmoplegia families of Caucasian origin, and investigated whether there are distinct molecular and clinical features associated with mutations in this gene. We found two new mutations in Twinkle, in 3 of the 11 pedigrees examined. One resides in the linker region of this gene while the other is in the primase domain. Both regions are highly conserved between species. Multiple deletions in the mtDNA from muscle are not always prominent and there are significant variations in the clinical presentation within and between families with mutations in the Twinkle gene. Therefore, genotype/phenotype predictions are difficult. No mutations were found in adenine nucleotide translocator 1 (ANT1), another known adPEO causative gene, in four of the seven remaining families investigated. Thus, Twinkle appears to be the most common gene associated with adPEO in Australian families.

Etiology and audiological outcomes at 3 years for 364 children in Australia.

Hearing loss is an etiologically heterogeneous trait with differences in the age of onset, severity and site of lesion. It is caused by a combination of genetic and/or environmental factors. A longitudinal study to examine the efficacy of early intervention for improving child outcomes is ongoing in Australia. To determine the cause of hearing loss in these children we undertook molecular testing of perinatal "Guthrie" blood spots of children whose hearing loss was either detected via newborn hearing screening or detected later in infancy. We analyzed the GJB2 and SLC26A4 genes for the presence of mutations, screened for the mitochondrial DNA (mtDNA) A1555G mutation, and screened for congenital CMV infection in DNA isolated from dried newborn blood spots. Results were obtained from 364 children. We established etiology for 60% of children. One or two known GJB2 mutations were present in 82 children. Twenty-four children had one or two known SLC26A4 mutations. GJB2 or SLC26A4 changes with unknown consequences on hearing were found in 32 children. The A1555G mutation was found in one child, and CMV infection was detected in 28 children. Auditory neuropathy spectrum disorder was confirmed in 26 children whose DNA evaluations were negative. A secondary objective was to investigate the relationship between etiology and audiological outcomes over the first 3 years of life. Regression analysis was used to investigate the relationship between hearing levels and etiology. Data analysis does not support the existence of differential effects of etiology on degree of hearing loss or on progressiveness of hearing loss.

Levodopa response in Parkinsonism with multiple mitochondrial DNA deletions.

We report a patient with an autosomal dominant chronic progressive external ophthalmoplegia phenotype associated with multiple mtDNA deletions in muscle from a family in which linkage analysis excluded mutations in DNA polymerase gamma (POLG), adenine nucleotide translocase (ANT-1) or C10orf2 (Twinkle). She presented with prominent Parkinsonism characterized by prolonged benefit from levodopa (L-dopa) and the later development of L-dopa induced dyskinesias and motor fluctuations. Thus L-dopa responsiveness, L-dopa induced dyskinesias and motor fluctuations may also occur in atypical Parkinsonism of mitochondrial disease, just as they may in multiple system atrophy.

Molecular characterization and expression of maternally expressed gene 3 (Meg3/Gtl2) RNA in the mouse inner ear.

The pathways responsible for sound perception in the cochlea involve the coordinated and regulated expression of hundreds of genes. By using microarray analysis, we identified several transcripts enriched in the inner ear, including the maternally expressed gene 3 (Meg3/Gtl2), an imprinted noncoding RNA. Real-time PCR analysis demonstrated that Meg3/Gtl2 was highly expressed in the cochlea, brain, and eye. Molecular studies revealed the presence of several Meg3/Gtl2 RNA splice variants in the mouse cochlea, brain, and eye. In situ hybridizations showed intense Meg3/Gtl2 RNA staining in the nuclei of type I spiral ganglion cells and in cerebellum near the dorsal vestibular region of the cochlea. In embryonic mouse head sections, Meg3/Gtl2 RNA expression was observed in the otocyst, brain, eye, cartilage, connective tissue, and muscle. Meg3/Gtl2 RNA expression increased in the developing otocyst and localized to the spiral ganglion, stria vascularis, Reissner's membrane, and greater epithelial ridge (GER) in the cochlear duct. RT-PCR analysis performed on cell lines derived from the organ of Corti, representing neural, supporting, and hair cells, showed significantly elevated levels of Meg3/Gtl2 expression in differentiated neural cells. We propose that Meg3/Gtl2 RNA functions as a noncoding regulatory RNA in the inner ear and that it plays a role in pattern specification and differentiation of cells during otocyst development, as well as in the maintenance of a number of terminally differentiated cochlear cell types.