Wendy M. Hutchison

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.

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.

Isolation and characterisation of the mouse pyruvate dehydrogenase E1α genes

Abstract We have characterized two mouse genes that code for the E1α subunit of pyruvate dehydrogenase (PDH), Phda-1 and Phda-2. The coding regions show a high degree of homology with each other and with the human PDH genes, PDHA1 and PDHA2. Conserved regions include mitochondrial import sequences, phosphorylation sites and a putative TPP binding site. The PDH genes have an analogous chromosomal arrangement to PGK genes in that two isoforms code for a functionally and structurally similar product. Pdha-1 codes for a somatic isoform and maps to the X-chromosome. Pdha-2 is located on an autosome, is intronless and only expressed in spermatogenic cells. Comparison of human and mouse PDH and PGK gene sequences shows that the somatic sequences are more conserved relative to the testis-specific isoforms, and that the mouse PDH E1α genes have experienced a faster rate of DNA change compared to their human counterparts.

Segregation of mutations in arylsulphatase E and correlation with the clinical presentation of chondrodysplasia punctata.

Sixteen males and two females with symmetrical (mild) type of chondrodysplasia punctata were tested for mutations in the X chromosome located arylsulphatase D and E genes. We identified one nonsense and two missense mutations in the arylsulphatase E gene in three males. No mutations were detected in the arylsulphatase D gene. Family studies showed segregation of the mutant genes establishing X linked inheritance for these families. Asymptomatic females and males were found in these studies. The clinical presentation varies not only between unrelated affected males, but also between affected males within the same family. We also conclude that clinical diagnosis of chondrodysplasia punctata in adults can be difficult. Finally, our results indicate that brachytelephalangy is not necessarily a feature of X linked symmetrical chondrodysplasia punctata.

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.

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, Reissners 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.

Polymorphisms in the human X-linked pyruvate dehydrogenase E1α gene

SummaryPyruvate dehydrogenase E1α deficiency is an X-chromosome-linked disorder, often with fatal consequences. We have searched for genetically useful polymorphisms in or near this gene. No restriction fragment length polymorphisms were detected using a battery of 36 different restriction enzymes and probing with a fulllength cDNA fragment, or two single-copy genomic fragments located within intron 8, and 15 kb 3′ of the coding region, respectively. The chemical cleavage method was then applied to the detection of base changes in or near the gene. One polymorphism was found in exon 8 of the coding region. However, no base changes were detected in intron 3 or in the part of intron 8 covered by fragment gB2. Three blocks of microsatellite DNA containing variable numbers of CA-repeats were isolated from the 5′ end of the gene and characterized. Length polymorphisms in these microsatellite DNAs were analysed using the polymerase chain reaction. Although the three loci are tightly linked, the polymorphisms appear not to be in disequilibrium, making them useful markers in linkage studies of the pyruvate dehydrogenase E1α gene. Of 31 females analysed 12 (39%) were heterozygous for at least one length polymorphism of the three (CA)n alleles.

The V368i mutation in Twinkle does not segregate with adPEO

Autosomal dominant progressive external ophthalmoplegia (AdPEO; OMIM 157640) is a mendelian disorder characterized by the presence of multiple deletions of mitochondrial (mt) DNA. Additional clinical features may include exercise intolerance, muscle weakness, peripheral neuropathy, deafness, ataxia, cataracts, hypogonadism, and severe depression. The disease has been linked to missense mutations in three different genes: ANT1, encoding the heart/muscle isoform of the ATP/ADP translocator, C10ORF2, encoding Twinkle, a putative mtDNA helicase, and POLG1, encoding the mtDNA polymerase. We previously have identified 11 missense mutations in the gene encoding Twinkle in 12 adPEO families. Their pathogenic significance was based on absence in ethnically matched controls, occurrence within conserved regions, and segregation with the disease. In particular, we reported a heterozygous G1102A transition leading to a V368I amino acid change in the Twinkle protein sequence of two nuclear families, both originating from Sicily. Although the V368I is a poorly conserved amino acid residue and the V368I change is conservative, the mutation was considered as pathogenic because it was absent in the healthy offspring of both probands, in 130 unrelated Italian control samples, and in 30 additional control samples from the Netherlands. A subsequent analysis of eight patients from Spain with a mitochondrial disorder associated with multiple mtDNA deletions in muscle, showed the presence of a heterozygous V368I change in Twinkle in two unrelated patients, both affected by a mitochondrial myopathy but not by PEO. However, a molecular screening on 55 control individuals from Spain showed the presence of a heterozygous V368I change in 8 unrelated individuals. None of them was affected by either PEO or mitochondrial disease. A screening of 110 additional Italian controls has shown the presence of the V368I change in two unrelated subjects, both free of any symptom of mitochondrial disease. Therefore, we found 10 healthy controls harboring the V368I mutation among 425 healthy individuals from different ethnical backgrounds tested for the mutation, which yields an estimated frequency of 1.2% for this allele in the general population. Furthermore, a screening of POLG1 in one of the two V368I original probands showed the presence of two allelic missense mutations, suggesting that POLG1 indeed could be the disease gene in these cases (M. Zeviani, unpublished data). Likewise, the V368I mutation was found in an Australian patient with PEO and muscular myopathy and was absent in 100 controls from the same area. However, the patient later has been shown to have a well-established pathogenic mtDNA mutation in muscle (H. Dahl, unpublished data). Although there is little doubt that mutations in Twinkle indeed are a cause of familial PEO, on the basis of the above findings, we conclude that the V368I change in Twinkle is a nonpathogenic polymorphism. Centro de Investigación, Hospital 12 de Octubre, Madrid, Fondo de Investigacion Sanitari, Spain; Luisa Mariani Center for the Study of Children’s Mitochondrial Disorders, National Neurological Institute, Milan, Italy; The Murdoch Children’s Research Institute, Melbourne, Australia; and University of Tampere, Tampere, Finland

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 γ (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.