The Deafness-Associated Mitochondrial DNA Mutation at Position 7445, Which Affects tRNASer(UCN) Precursor Processing, Has Long-Range Effects on NADH Dehydrogenase Subunit ND6 Gene Expression

The pathogenetic mechanism of the deafness-associated mitochondrial DNA (mtDNA) T7445C mutation has been investigated in several lymphoblastoid cell lines from members of a New Zealand pedigree exhibiting the mutation in homoplasmic form and from control individuals. We show here that the mutation flanks the 3' end of the tRNASer(UCN) gene sequence and affects the rate but not the sites of processing of the tRNA precursor. This causes an average reduction of ~70% in the tRNASer(UCN) level and a decrease of ~45% in protein synthesis rate in the cell lines analyzed. The data show a sharp threshold in the capacity of tRNASer(UCN) to support the wild-type protein synthesis rate, which corresponds to ~40% of the control level of this tRNA. Strikingly, a 7445 mutation-associated marked reduction has been observed in the level of the mRNA for the NADH dehydrogenase (complex I) ND6 subunit gene, which is located ~7 kbp upstream and is cotranscribed with the tRNASer(UCN) gene, with strong evidence pointing to a mechanistic link with the tRNA precursor processing defect. Such reduction significantly affects the rate of synthesis of the ND6 subunit and plays a determinant role in the deafness-associated respiratory phenotype of the mutant cell lines. In particular, it accounts for their specific, very significant decrease in glutamate- or malate-dependent O2 consumption. Furthermore, several homoplasmic mtDNA mutations affecting subunits of NADH dehydrogenase may play a synergistic role in the establishment of the respiratory phenotype of the mutant cells

MRPS18CP2 alleles and DEFA3 absence as putative chromosome 8p23.1 modifiers of hearing loss due to mtDNA mutation A1555G in the 12S rRNA gene

<p>Abstract</p> <p>Background</p> <p>Mitochondrial DNA (mtDNA) mutations account for at least 5% of cases of postlingual, nonsyndromic hearing impairment. Among them, mutation A1555G is frequently found associated with aminoglycoside-induced and/or nonsyndromic hearing loss in families presenting with extremely variable clinical phenotypes. Biochemical and genetic data have suggested that nuclear background is the main factor involved in modulating the phenotypic expression of mutation A1555G. However, although a major nuclear modifying locus was located on chromosome 8p23.1 and regardless intensive screening of the region, the gene involved has not been identified.</p> <p>Methods</p> <p>With the aim to gain insights into the factors that determine the phenotypic expression of A1555G mutation, we have analysed in detail different genetic and genomic elements on 8p23.1 region (<it>DEFA3 </it>gene absence, <it>CLDN23 </it>gene and <it>MRPS18CP2 </it>pseudogene) in a group of 213 A1555G carriers.</p> <p>Results</p> <p>Family based association studies identified a positive association for a polymorphism on <it>MRPS18CP2 </it>and an overrepresentation of <it>DEFA3 </it>gene absence in the deaf group of A1555G carriers.</p> <p>Conclusion</p> <p>Although none of the factors analysed seem to have a major contribution to the phenotype, our findings provide further evidences of the involvement of 8p23.1 region as a modifying locus for A1555G 12S rRNA gene mutation.</p

Construction of an ~700-kb transcript map around the Familial Mediterranean Fever locus on human chromosome 16p13.3

We used a combination of cDNA selection, exon amplification, and computational prediction from genomic sequence to isolate transcribed sequences from genomic DNA surrounding the familial Mediterranean fever (FMF) locus. Eighty-seven kb of genomic DNA around D16S3370, a marker showing a high degree of linkage disequilibrium with FMF, was sequenced to completion, and the sequence annotated. A transcript map reflecting the minimal number of genes encoded within the ∼700 kb of genomic DNA surrounding the FMF locus was assembled. This map consists of 27 genes with discreet messages detectable on Northerns, in addition to three olfactory-receptor genes, a cluster of 18 tRNA genes, and two putative transcriptional units that have typical intron–exon splice junctions yet do not detect messages on Northerns. Four of the transcripts are identical to genes described previously, seven have been independently identified by the French FMF Consortium, and the others are novel. Six related zinc-finger genes, a cluster of tRNAs, and three olfactory receptors account for the majority of transcribed sequences isolated from a 315-kb FMF central region (betweenD16S468/D16S3070 and cosmid 377A12). Interspersed among them are several genes that may be important in inflammation. This transcript map not only has permitted the identification of the FMF gene (MEFV), but also has provided us an opportunity to probe the structural and functional features of this region of chromosome 16.Michael Centola, Xiaoguang Chen, Raman Sood, Zuoming Deng, Ivona Aksentijevich, Trevor Blake, Darrell O. Ricke, Xiang Chen, Geryl Wood, Nurit Zaks, Neil Richards, David Krizman, Elizabeth Mansfield, Sinoula Apostolou, Jingmei Liu, Neta Shafran, Anil Vedula, Melanie Hamon, Andrea Cercek, Tanaz Kahan, Deborah Gumucio, David F. Callen, Robert I. Richards, Robert K. Moyzis, Norman A. Doggett, Francis S. Collins, P. Paul Liu, Nathan Fischel-Ghodsian and Daniel L. Kastne

Nuclear background determines biochemical phenotype in the deafness-associated mitochondrial 12S rRNA mutation

The pathogenetic mechanism of the human mitochondrial 12S rRNA gene mutation at position 1555, associated with non-syndromic deafness and aminoglycoside-induced deafness, has been investigated in 33 transformants obtained by transferring mitochondria from lymphoblastoid cell lines into human mitochondrial DNA (mtDNA)-less (ρ°206) cells. In this nearly constant nuclear background, 15 transformants derived from five symptomatic individuals from a large Arab-Israeli family, carrying this mutation in homoplasmic form, exhibited significant decreases compared with nine control transformants in the rate of growth in a medium containing galactose instead of glucose, as well as in the rates of mitochondrial protein synthesis and of substrate-dependent respiration. Most significantly, these decreases were very similar to those observed in nine transformants derived from three asymptomatic members of the family. This result in transmitochondrial cybrids is in contrast to the differences in the same parameters previously demonstrated between the original lymphoblastoid cell lines derived from the symptomatic and asymptomatic members of the Arab-Israeli family. In addition, the intragroup variability in biochemical dysfunction among the lymphoblastoid cell lines from different symptomatic or asymptomatic or control individuals was significantly reduced in the derived mitochondrial transformants carrying the same nuclear background. These observations provide strong genetic and biochemical evidence in support of the idea that the nuclear background plays a determinant role in the phenotypic manifestation of the non-syndromic deafness associated with the A1555G mutation

A biochemical basis for the inherited susceptibility to aminoglycoside ototoxicity

The A1555 G mutation in mitochondrial 12S rRNA has been found to be associated with non-syndromic deafness and aminoglycoside-induced deafness. The sensitivity to the aminoglycoside paromomycin has been analyzed in lymphoblastoid cell lines derived from five deaf individuals and five hearing individuals from an Arab-Israeli family carrying the A1555G mutation, and three married-in controls from the same family. Exposure to a high concentration of paromomycin (2 mg/ml), which caused an 8% average increase in doubling time (DT) in the control cell lines, produced higher average DT increases (49 and 47%) in the A1555G mutation-carrying cell lines derived from symptomatic and asymptomatic individuals, respectively. The ratios of translation rates in the presence and absence of paromomycin, which reflected the effect of the drug on mitochondrial protein synthesis, were significantly decreased in the cell lines derived from symptomatic and asymptomatic individuals (by 30 and 28% on average, respectively), compared with the ratios in the control cell lines. These ratios showed, in both groups of mutant cell lines, a significant negative correlation with the ratios of DTs in the presence and absence of the antibiotic. These results have provided the first direct evidence that the mitochondrial 12S rRNA carrying the A1555G mutation is the main target of aminoglycosides. They suggest that these antibiotics exert their detrimental effect through an alteration of mitochondrial protein synthesis, which exacerbates the inherent defect caused by the mutation, reducing the overall translation rate down to and below the minimal level required for normal cellular function (40–50%)

Partial activity is seen with many substitutions of highly conserved active site residues in human Pseudouridine synthase 1

Pseudouridine synthase 1 (Pus1p) is an enzyme that converts uridine to Pseudouridine (Ψ) in tRNA and other RNAs in eukaryotes. The active site of Pus1p is composed of stretches of amino acids that are highly conserved and it is hypothesized that mutation of select residues would impair the enzyme's ability to catalyze the formation of Ψ. However, most mutagenesis studies have been confined to substitution of the catalytic aspartate, which invariably results in an inactive enzyme in all Ψ synthases tested. To determine the requirements for particular amino acids at certain absolutely conserved positions in Pus1p, three residues (R116, Y173, R267) that correspond to amino acids known to compose the active site of TruA, a bacterial Ψ synthase that is homologous to Pus1p, were mutated in human Pus1p (hPus1p). The effects of those mutations were determined with three different in vitro assays of pseudouridylation and several tRNA substrates. Surprisingly, it was found that each of these components of the hPus1p active site could tolerate certain amino acid substitutions and in fact most mutants exhibited some activity. The most active mutants retained near wild-type activity at positions 27 or 28 in the substrate tRNA, but activity was greatly reduced or absent at other positions in tRNA readily modified by wild-type hPus1p

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

MRPS18CP2 alleles and DEFA3 absence as putative chromosome 8p23.1 modifiers of hearing loss due to mtDNA mutation A1555G in the 12S rRNA gene

Background: Mitochondrial DNA (mtDNA) mutations account for at least 5% of cases of postlingual, nonsyndromic hearing impairment. Among them, mutation A1555G is frequently found associated with aminoglycoside-induced and/or nonsyndromic hearing loss in families presenting with extremely variable clinical phenotypes. Biochemical and genetic data have suggested that nuclear background is the main factor involved in modulating the phenotypic expression of mutation A1555G. However, although a major nuclear modifying locus was located on chromosome 8p23.1 and regardless intensive screening of the region, the gene involved has not been identified. Methods: With the aim to gain insights into the factors that determine the phenotypic expression of A1555G mutation, we have analysed in detail different genetic and genomic elements on 8p23.1 region (DEFA3 gene absence, CLDN23 gene and MRPS18CP2 pseudogene) in a group of 213 A1555G carriers./n/nResults/nFamily based association studies identified a positive association for a polymorphism on MRPS18CP2 and an overrepresentation of DEFA3 gene absence in the deaf group of A1555G carriers. Conclusion: Although none of the factors analysed seem to have a major contribution to the phenotype, our findings provide further evidences of the involvement of 8p23.1 region as a modifying locus for A1555G 12S rRNA gene mutation.This work was supported by "La Marató de TV3" (993610), "Instituto de Salud Carlos III, FIS-ISCIII" (G03/203, PI052347 and CIBER-CB06/02/0058) and "Generalitat de Catalunya" (2005SGR00008). The Spanish National Genotyping Center (CeGen) is founded by "Genoma España". EB is recipient of a fellowship from "Generalitat de Catalunya" (2003FI00066). JMM was supported by the CRG under project SAF2002-00799 (Spanish Ministry of Science and Education), and by a fellowship of the Danone Institute. NFG acknowledges support from NIH/NIDCD grant RO1DC01402

Biochemical characterization of the mitochondrial tRNA(Ser(UCN)) T7511C mutation associated with nonsyndromic deafness

We report here the biochemical characterization of the deafness-associated mitochondrial tRNA(Ser(UCN)) T7511C mutation, in conjunction with homoplasmic ND1 T3308C and tRNA(Ala) T5655C mutations using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from an African family into human mtDNA-less (ρ°) cells. Three cybrids derived from an affected matrilineal relative carrying the homoplasmic T7511C mutation, exhibited ∼75% decrease in the tRNA(Ser(UCN)) level, compared with three control cybrids. This amount of reduction in the tRNA(Ser(UCN)) level is below a proposed threshold to support a normal rate of mitochondrial protein synthesis in lymphoblastoid cell lines. This defect is likely a primary contributor to ∼52% reduction in the rate of mitochondrial protein synthesis and marked defects in respiration and growth properties in galactose-containing medium. Interestingly, the T5655C mutation produces ∼50% reduction in the tRNA(Ala) level in mutant cells. Strikingly, the T3308C mutation causes a significant decrease both in the amount of ND1 mRNA and co-transcribed tRNA(Leu(UUR)) in mutant cells. Thus, mitochondrial dysfunctions caused by the T5655C and T3308C mutations may modulate the phenotypic manifestation of the T7511C mutation. These observations imply that a combination of the T7511C mutation with two mtDNA mutations accounts for the high penetrance of deafness in this family