Yelena Bykhovskaya

Missense Mutation in Pseudouridine Synthase 1 (PUS1) Causes Mitochondrial Myopathy and Sideroblastic Anemia (MLASA)

Mitochondrial myopathy and sideroblastic anemia (MLASA) is a rare, autosomal recessive oxidative phosphorylation disorder specific to skeletal muscle and bone marrow. Linkage analysis and homozygosity testing of two families with MLASA localized the candidate region to 1.2 Mb on 12q24.33. Sequence analysis of each of the six known genes in this region, as well as four putative genes with expression in bone marrow or muscle, identified a homozygous missense mutation in the pseudouridine synthase 1 gene (PUS1) in all patients with MLASA from these families. The mutation is the only amino acid coding change in these 10 genes that is not a known polymorphism, and it is not found in 934 controls. The amino acid change affects a highly conserved amino acid, and appears to be in the catalytic center of the protein, PUS1p. PUS1 is widely expressed, and quantitative expression analysis of RNAs from liver, brain, heart, bone marrow, and skeletal muscle showed elevated levels of expression in skeletal muscle and brain. We propose deficient pseudouridylation of mitochondrial tRNAs as an etiology of MLASA. Identification of the pathophysiologic pathways of the mutation in these families may shed light on the tissue specificity of oxidative phosphorylation disorders.

Mutation in TRMU Related to Transfer RNA Modification Modulates the Phenotypic Expression of the Deafness-Associated Mitochondrial 12S Ribosomal RNA Mutations

The human mitochondrial 12S ribosomal RNA (rRNA) A1555G mutation has been associated with aminoglycoside-induced and nonsyndromic deafness in many families worldwide. Our previous investigation revealed that the A1555G mutation is a primary factor underlying the development of deafness but is not sufficient to produce a deafness phenotype. However, it has been proposed that nuclear-modifier genes modulate the phenotypic manifestation of the A1555G mutation. Here, we identified the nuclear-modifier gene TRMU, which encodes a highly conserved mitochondrial protein related to transfer RNA (tRNA) modification. Genotyping analysis of TRMU in 613 subjects from 1 Arab-Israeli kindred, 210 European (Italian pedigrees and Spanish pedigrees) families, and 31 Chinese pedigrees carrying the A1555G or the C1494T mutation revealed a missense mutation (G28T) altering an invariant amino acid residue (A10S) in the evolutionarily conserved N-terminal region of the TRMU protein. Interestingly, all 18 Arab-Israeli/Italian-Spanish matrilineal relatives carrying both the TRMU A10S and 12S rRNA A1555G mutations exhibited prelingual profound deafness. Functional analysis showed that this mutation did not affect importation of TRMU precursors into mitochondria. However, the homozygous A10S mutation leads to a marked failure in mitochondrial tRNA metabolisms, specifically reducing the steady-state levels of mitochondrial tRNA. As a consequence, these defects contribute to the impairment of mitochondrial-protein synthesis. Resultant biochemical defects aggravate the mitochondrial dysfunction associated with the A1555G mutation, exceeding the threshold for expressing the deafness phenotype. These findings indicate that the mutated TRMU, acting as a modifier factor, modulates the phenotypic manifestation of the deafness-associated 12S rRNA mutations.

Genome-wide association analyses identify multiple loci associated with central corneal thickness and keratoconus

Central corneal thickness (CCT) is associated with eye conditions including keratoconus and glaucoma. We performed a meta-analysis on >20,000 individuals in European and Asian populations that identified 16 new loci associated with CCT at genome-wide significance (P < 5 × 10−8). We further showed that 2 CCT-associated loci, FOXO1 and FNDC3B, conferred relatively large risks for keratoconus in 2 cohorts with 874 cases and 6,085 controls (rs2721051 near FOXO1 had odds ratio (OR) = 1.62, 95% confidence interval (CI) = 1.4–1.88, P = 2.7 × 10−10, and rs4894535 in FNDC3B had OR = 1.47, 95% CI = 1.29–1.68, P = 4.9 × 10−9). FNDC3B was also associated with primary open-angle glaucoma (P = 5.6 × 10−4; tested in 3 cohorts with 2,979 cases and 7,399 controls). Further analyses implicate the collagen and extracellular matrix pathways in the regulation of CCT.

A nuclear-mitochondrial DNA interaction affecting hearing impairment in mice

The pathophysiologic pathways and clinical expression of mitochondrial DNA (mtDNA) mutations are not well understood. This is mainly the result of the heteroplasmic nature of most pathogenic mtDNA mutations and of the absence of clinically relevant animal models with mtDNA mutations. mtDNA mutations predisposing to hearing impairment in humans are generally homoplasmic, yet some individuals with these mutations have severe hearing loss, whereas their maternal relatives with the identical mtDNA mutation have normal hearing. Epidemiologic, biochemical and genetic data indicate that nuclear genes are often the main determinants of these differences in phenotype. To identify a mouse model for maternally inherited hearing loss, we screened reciprocal backcrosses of three inbred mouse strains, A/J, NOD/LtJ and SKH2/J, with age-related hearing loss (AHL). In the (A/J×CAST/Ei)×A/J backcross, mtDNA derived from the A/J strain exerted a significant detrimental effect on hearing when compared with mtDNA from the CAST/Ei strain. This effect was not seen in the (NOD/LtJ ×CAST/Ei)×NOD/LtJ and (SKH2/J×CAST/Ei)×SKH2/J backcrosses. Genotyping revealed that this effect was seen only in mice homozygous for the A/J allele at the Ahl locus on mouse chromosome 10. Sequencing of the mitochondrial genome in the three inbred strains revealed a single nucleotide insertion in the tRNA-Arg gene (mt-Tr) as the probable mediator of the mitochondrial effect. This is the first mouse model with a naturally occurring mtDNA mutation affecting a clinical phenotype, and it provides an experimental model to dissect the pathophysiologic processes connecting mtDNA mutations to hearing loss.

Inherited susceptibility to aminoglycoside ototoxicity: genetic heterogeneity and clinical implications.

PURPOSE Aminoglycoside-induced ototoxicity appears to have a genetic susceptibility in some individuals, and the A1555G mutation in the mitochondrial 12S ribosomal RNA gene has been shown to be responsible for this susceptibility in all familial cases. An Italian family with 5 family members who became deaf after aminoglycoside exposure presented to us, and molecular analysis excluded the A1555G mutation. The purpose of this study is to identify the molecular basis for the aminoglycoside susceptibility in this family. PATIENTS AND METHODS Two sisters and three of their children developed severe to profound high-frequency hearing loss after aminoglycoside exposure. DNA was extracted from the blood of these individuals and their unaffected relatives, and analyzed for mitochondrial DNA mutations. The region around nucleotide 961 was also cloned and individual clones were sequenced. RESULTS Sequencing of the 12S ribosomal RNA gene revealed a thymidine deletion at position 961, with a complex pattern of sequence around this mutation. Sequencing of individual clones around the 961 mutation demonstrated a varying number of inserted cytosines in different mitochondrial molecules. CONCLUSION This family establishes the nucleotide 961 thymidine deletion associated with a varying number of inserted cytosines in the mitochondrial 12S ribosomal RNA gene as the second pathogenic mutation that can predispose to aminoglycoside ototoxicity. It demonstrates the clinical relevance of taking a family history before administering aminoglycosides to any patient. In addition, it would be desirable for sporadic patients with aminoglycoside-induced hearing loss to be screened with molecular tests for the presence of the 1555 and 961 mutations. Such screening could significantly decrease the prevalence of aminoglycoside-induced hearing loss.

Mitochondrial A7445G mutation in two pedigrees with palmoplantar keratoderma and deafness

A New Zealand and a Scottish pedigree with maternally inherited sensorineural deafness were both previously shown to carry a heteroplasmic A7445G mutation in the mitochondrial genome. More detailed clinical examination of the New Zealand family showed that the hearing loss was progressive, with the severity of the overall loss and the frequencies most affected differing markedly between individuals of similar age, and showed that many relatives also had palmoplantar keratoderma. Review of the literature demonstrated three other large families with presumed autosomal dominant inheritance of palmoplantar keratoderma and hearing loss. In a United Kingdom pedigree the syndrome was transmitted by female and male parents, an inheritance pattern which made mitochondrial inheritance unlikely; however, in a Turkish and a Japanese pedigree the affected individuals were all maternally related. Subsequent analysis of the Japanese pedigree documented the same A7445G mitochondrial mutation as was previously found in the New Zealand and Scottish pedigrees. Other mitochondrial sequence variants previously reported in the New Zealand or Scottish pedigrees were absent from the Japanese pedigree which suggests that the A7445G mutation arose independently in all three pedigrees. To our knowledge palmoplantar keratoderma has not previously been associated with mitochondrial defects; however, the current findings suggest that the A7445G mutation is associated not only with progressive hearing loss but also with palmoplantar keratoderma. The penetrance and expressivity of both symptoms varied considerably between individuals in the Scottish and New Zealand Studies which suggests that additional environmental and/or genetic factors are involved.

Candidate Locus for a Nuclear Modifier Gene for Maternally Inherited Deafness

Maternally inherited deafness associated with the A1555G mutation in the mitochondrial 12S ribosomal RNA (rRNA) gene appears to require additional environmental or genetic changes for phenotypic expression. Aminoglycosides have been identified as one such environmental factor. In one large Arab-Israeli pedigree with congenital hearing loss in some of the family members with the A1555G mutation and with no exposure to aminoglycosides, biochemical evidence has suggested the role of nuclear modifier gene(s), but a genomewide search has indicated the absence of a single major locus having such an effect. Thus it has been concluded that the penetrance of the mitochondrial mutation appears to depend on additive effects of several nuclear genes. We have now investigated 10 multiplex Spanish and Italian families with 35 members with the A1555G mutation and sensorineural deafness. Parametric analysis of a genomewide screen again failed to identify significant evidence for linkage to a single autosomal locus. However, nonparametric analysis supported the role of the chromosomal region around marker D8S277. The combined maximized allele-sharing LOD score of 3.1 in Arab-Israeli/Spanish/Italian families represents a highly suggestive linkage result. We suggest that this region should be considered a candidate for containing the first human nuclear modifier gene for a mitochondrial DNA disorder. The locus operates in Arab-Israeli, Spanish, and Italian families, resulting in the deafness phenotype on a background of the mitochondrial A1555G mutation. No obvious candidate genes are located in this region.

Temporal bone analysis of patients with presbycusis reveals high frequency of mitochondrial mutations

Presbycusis is a histologically and genetically heterogenous group of disorders, which lead to progressive, primarily sensorineural hearing loss with aging. Acquired mitochondrial DNA defects have been proposed as important determinants of aging, particularly in neuro-muscular tissues. The spiral ganglion and membranous labyrinth from archival temporal bones of 5 patients with presbycusis were examined for mutations within the mitochondrially-encoded cytochrome oxidase II gene. When compared to controls, results indicate that mitochondrial mutations in the peripheral auditory system occur commonly with age-related hearing loss, that there is great individual variability in both quantity and location of mutation accumulation, and that at least a proportion of presbycusis patients have a highly significant load of mutations in auditory tissue. This work supports the hypothesis that acquired mitochondrial mutations are a determinant of hearing loss in a subgroup of presbycusis patients.

Evidence for complex nuclear inheritance in a pedigree with nonsyndromic deafness due to a homoplasmic mitochondrial mutation

The relationship between mitochondrial genotype and clinical phenotype is complicated in most instances by the heteroplasmic nature of pathogenic mitochondrial mutations. We have previously shown that maternally inherited hearing loss in a large Arab-Israeli kindred is due to the homoplasmic A1555G mutation in the mitochondrial 12S ribosomal RNA gene [Prezant et al., 1993: Nat Genet 4:289-294]. Family members with this mutation have phenotypes ranging from profound hearing loss to completely normal hearing, and we have shown that there is genetic and biochemical evidence for nuclear gene involvement in this family [Bu et al., 1993: Genet Epidemiol 9:27-44; Guan et al., 1996: Hum Mol Genet 5:963-971]. To identify such a nuclear locus, two candidate genes were excluded through linkage analysis and sequencing, and a genome-wide linkage search in family members who all have the identical homoplasmic mitochondrial mutation, but differ in their hearing status, was performed. In two stages a total of 560 polymorphic genetic markers was genotyped, and the data were analyzed under model-dependent and model-free assumptions. No chromosomal region was identified as a major contributor to the phenotypic expression of the mitochondrial mutation. Thus, in this simplified paradigm of a homoplasmic mitochondrial mutation in a single kindred who all live in the similar environment of a small village, the penetrance of the mitochondrial mutation appears to depend on the interaction of multiple nuclear genes.

Hearing loss due to the mitochondrial A1555G mutation in Italian families

Six Italian families with familial nonsyndromic hearing loss consistent with a maternal pattern of inheritance were analyzed for mitochondrial mutations. The three known mitochondrial mutations associated with nonsyndromic hearing loss were investigated by polymerase chain reaction amplification, followed by restriction fragment length analysis or DNA sequencing. The A7445G mutation and C7472 insertion were not present in either of the families, but the A1555G mutation in the 12S rRNA gene was identified in homoplasmic form in two of the families. In one of the families the onset of hearing loss is congenital, while in the other it starts later in life. The families are from different regions of Italy, and mitochondrial haplotype analysis showed that the mutation arose independently in these two families. This suggests that the A1555G mutation may not be an uncommon cause of hearing loss in Italians, and is clinically important because maternal hearing relatives of patients with the A1555G mutation are at risk for aminoglycoside induced deafness. We discuss potential reasons for the normal phenotype in some relatives with the mutation, and the different onset of hearing loss in the two families.