Pseudouridine synthase 1 deficient mice, a model for Mitochondrial Myopathy with Sideroblastic Anemia, exhibit muscle morphology and physiology alterations.

Mitochondrial myopathy with lactic acidosis and sideroblastic anemia (MLASA) is an oxidative phosphorylation disorder, with primary clinical manifestations of myopathic exercise intolerance and a macrocytic sideroblastic anemia. One cause of MLASA is recessive mutations in PUS1, which encodes pseudouridine (Ψ) synthase 1 (Pus1p). Here we describe a mouse model of MLASA due to mutations in PUS1. As expected, certain Ψ modifications were missing in cytoplasmic and mitochondrial tRNAs from Pus1(-/-) animals. Pus1(-/-) mice were born at the expected Mendelian frequency and were non-dysmorphic. At 14 weeks the mutants displayed reduced exercise capacity. Examination of tibialis anterior (TA) muscle morphology and histochemistry demonstrated an increase in the cross sectional area and proportion of myosin heavy chain (MHC) IIB and low succinate dehydrogenase (SDH) expressing myofibers, without a change in the size of MHC IIA positive or high SDH myofibers. Cytochrome c oxidase activity was significantly reduced in extracts from red gastrocnemius muscle from Pus1(-/-) mice. Transmission electron microscopy on red gastrocnemius muscle demonstrated that Pus1(-/-) mice also had lower intermyofibrillar mitochondrial density and smaller mitochondria. Collectively, these results suggest that alterations in muscle metabolism related to mitochondrial content and oxidative capacity may account for the reduced exercise capacity in Pus1(-/-) mice.

Phenotypic expression of maternally inherited deafness is affected by RNA modification and cytoplasmic ribosomal proteins.

The homoplasmic mitochondrial A1555G mutation in the 12S rRNA gene leads to a mitochondrial translation disorder associated with deafness. The absence of disease in non-cochlear tissues in all patients, and in the cochlea in some patients, is not well understood. We used a system-based approach, including whole genome expression and biological function analysis, to elucidate the pathways underlying tissue specificity and clinical severity of this condition. Levels of over 48K RNA transcripts from EBV-transformed lymphoblasts of deaf and hearing individuals with the A1555G mutation and controls were obtained. Differentially expressed transcripts were functionally grouped using gene set enrichment analysis. Over 50 RNA binding proteins were differentially expressed between deaf and hearing individuals with the A1555G mutation (P-value of 2.56E-7), confirming previous genetic data implicating this pathway in the determination of the severity of hearing loss. Unexpectedly, the majority of cytoplasmic ribosomal genes were up-regulated in a coordinated fashion in individuals with the A1555G mutation versus controls (P-value of 3.91E-135). This finding was verified through real time RT-PCR, and through measuring of protein levels by flow cytometry. Analysis of expression levels of other differentially expressed genes suggests that this coordinated over-expression of cytoplasmic ribosomal proteins might occur through the Myc/Max pathway. We propose that expression levels of RNA binding proteins help determine the severity of the cochlear phenotype, and that coordinated up-regulation of the cytoplasmic translation apparatus operates as a compensation mechanism in unaffected tissues of patients with maternal deafness associated with the A1555G mutation.

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 (Psi) 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 Psi. However, most mutagenesis studies have been confined to substitution of the catalytic aspartate, which invariably results in an inactive enzyme in all Psi 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 Psi 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.

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. RESULTS: Family 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.

Pleiotropic effects and compensation mechanisms determine tissue specificity in mitochondrial myopathy and sideroblastic anemia (MLASA).

The tissue specificity of mitochondrial diseases is poorly understood. Recently, tissue-specific quantitative differences of the components of the mitochondrial translation system have been found to correlate with disease presentation in fatal hepatopathy caused by mutations in mitochondrial translation factor EFG1. MLASA is an autosomal recessive inherited progressive oxidative phosphorylation disorder that affects muscle and erythroid cells. The disease is caused by the homozygous point mutation C656T (R116W) in the catalytic domain of the pseudouridylate synthase 1 (PUS1) gene, which leads to a complete lack of pseudouridylation at the expected sites in mitochondrial and cytoplasmic tRNAs. Despite the presence of these altered tRNAs, most tissues are unaffected, and even in muscle and erythroid cells the disease phenotype only slowly emerges over the course of years. In order to elucidate intracellular pathways through which the homozygous mutation leads to tissue-restricted phenotype, we performed microarray expression analysis of EBV-transformed lymphoblasts from MLASA patients, heterozygous parents, and controls using human Beadchip microarray with 47,296 transcripts. Genes coding for proteins involved in DNA transcription and its regulation, and metal binding proteins, demonstrated major differences in expression between patients and all other individuals with normal phenotype. Genes coding for ribosomal proteins differed significantly between individual with at least one copy of the mutated PUS1 gene and controls. These findings indicate that the lack of tRNA pseudouridylation can be overcome by compensatory changes in levels of ribosomal proteins, and that the disease phenotype in affected tissues is likely due to pleiotropic effects of PUS1p on non-tRNA molecules involved in DNA transcription and iron metabolism. Similar combinations of mechanisms may play a role in the tissue specificity of other mitochondrial disorders.

Pus3p- and Pus1p-dependent pseudouridylation of steroid receptor RNA activator controls a functional switch that regulates nuclear receptor signaling.

It was previously shown that mouse Pus1p (mPus1p), a pseudouridine synthase (PUS) known to modify certain transfer RNAs (tRNAs), can also bind with nuclear receptors (NRs) and function as a coactivator through pseudouridylation and likely activation of an RNA coactivator called steroid receptor RNA activator (SRA). Use of cell extract devoid of human Pus1p activity derived from patients with mitochondrial myopathy and sideroblastic anemia, however, still showed SRA-modifying activity suggesting that other PUS(s) can also target this coactivator. Here, we show that related mPus3p, which has a different tRNA specificity than mPus1p, also serves as a NR coactivator. However, in contrast to mPus1p, it does not stimulate sex steroid receptor activity, which is likely due to lack of binding to this class of NRs. As expected from their tRNA activities, in vitro pseudouridylation assays show that mPus3p and mPus1p modify different positions in SRA, although some may be commonly targeted. Interestingly, the order in which these enzymes modify SRA determines the total number of pseudouridines. mPus3p and SRA are mainly cytoplasmic; however, mPus3p and SRA are also localized in distinct nuclear subcompartments. Finally, we identified an in vivo modified position in SRA, U206, which is likely a common target for both mPus1p and mPus3p. When U206 is mutated to A, SRA becomes hyperpseudouridylated in vitro, and it acquires dominant-negative activity in vivo. Thus, Pus1p- and Pus3p-dependent pseudouridylation of SRA is a highly complex posttranscriptional mechanism that controls a coactivator-corepressor switch in SRA with major consequences for NR signaling.

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.

Human TRMU encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase is a putative nuclear modifier gene for the phenotypic expression of the deafness-associated 12S rRNA mutations.

Nuclear modifier genes have been proposed to modulate the phenotypic manifestation of human mitochondrial 12S rRNA A1491G mutation associated with deafness in many families world-wide. Here we identified and characterized the putative nuclear modifier gene TRMU encoding a highly conserved mitochondrial protein related to tRNA modification. A 1937bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The human TRMU gene containing 11 exons encodes a 421 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs. TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, kidney, and brain. Immunofluorescence analysis of human 143B cells expressing TRMU-GFP fusion protein demonstrated that the human Trmu localizes and functions in mitochondrion. Furthermore, we show that in families with the deafness-associated 12S rRNA A1491G mutation there is highly suggestive linkage and linkage disequilibrium between microsatellite markers adjacent to TRMU and the presence of deafness. These observations suggest that human TRMU may modulate the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA mutations.

Cu/Zn superoxide dismutase and age-related hearing loss.

Mice, in which the genetics can be manipulated and the life span is relatively short, enable evaluation of the effects of specific gene expression on cochlear degeneration over time. Antioxidant enzymes such as Cu/Zn superoxide dismutase (SOD1) protect cells from toxic, reactive oxygen species and may be involved in age-related degeneration. The effects of SOD1 deletion and over-expression on the cochlea were examined in Sod1-null mice, Sod1 transgenic mice and in age- and genetics-matched controls. Auditory brainstem responses (ABR) were measured and cochleae were histologically examined. The absence of SOD1 resulted in hearing loss at an earlier age than in wildtype or heterozygous mice. The cochleae of the null mice had severe spiral ganglion cell degeneration at 7-9 months of age. The stria vascularis in the aged, null mice was thinner than in the heterozygous or wildtype mice. Over-expression of SOD1 did not protect against hearing loss except at 24 months of age. In conclusion, SOD1 seems important for survival of cochlear neurons and the stria vascularis, however even half the amount is sufficient and an over abundance does not provide much protection from age-related hearing loss.

Mitochondrial myopathy, sideroblastic anemia, and lactic acidosis: an autosomal recessive syndrome in Persian Jews caused by a mutation in the PUS1 gene.

We report the seventh case of autosomal recessive inherited mitochondrial myopathy, lactic acidosis, and sideroblastic anemia The patient, a product of consanguineous Persian Jews, had the association of mental retardation, dysmorphic features, lactic acidosis, myopathy, and sideroblastic anemia. Muscle biopsy demonstrated low activity of complexes 1 and 4 of the respiratory chain. Electron microscopy revealed paracrystalline inclusions in most mitochondria. Southern blot of the mitochondrial DNA did not show any large-scale rearrangements. The patient was found to be homozygous for the 656C-->T mutation in the pseudouridine synthase 1 gene (PUS1). Mitochondrial myopathy, lactic acidosis, and sideroblastic anemia is an oxidative phosphorylation disorder causing sideroblastic anemia, myopathy, and, in some cases, mental retardation that is due to mutations in the nuclear-encoded PUS1 gene. This finding provides additional evidence that mitochondrial ribonucleic acid modification impacts the phenotypic expression of oxidative phosphorylation disorders.

Mitochondrial myopathy and sideroblastic anemia (MLASA): missense mutation in the pseudouridine synthase 1 (PUS1) gene is associated with the loss of tRNA pseudouridylation.

A missense mutation in the PUS1 gene affecting a highly conserved amino acid has been associated with mitochondrial myopathy and sideroblastic anemia (MLASA), a rare autosomal recessive oxidative phosphorylation disorder. The PUS1 gene encodes the enzyme pseudouridine synthase 1 (Pus1p) that is known to pseudouridylate tRNAs in other species. Total RNA was isolated from lymphoblastoid cell lines established from patients, parents, unaffected siblings, and unrelated controls, and the tRNAs were assayed for the presence of pseudouridine (Psi) at the expected positions. Mitochondrial and cytoplasmic tRNAs from MLASA patients are lacking modification at sites normally modified by Pus1p, whereas tRNAs from controls, unaffected siblings, or parents all have Psi at these positions. In addition, there was no Pus1p activity in an extract made from a cell line derived from a patient with MLASA. Immunohistochemical staining of Pus1p in cell lines showed nuclear, cytoplasmic, and mitochondrial distribution of the protein, and there is no difference in staining between patients and unaffected family members. MLASA is thus associated with absent or greatly reduced tRNA pseudouridylation at specific sites, implicating this pathway in its molecular pathogenesis.

Genetic factors in aminoglycoside toxicity.

Ototoxicity is the major irreversible toxicity of aminoglycosides, and it occurs both in a dose-dependent and idiosyncratic fashion. The idiosyncratic pathway is presumably due to genetic predispositions, and an inherited mutation in the mitochondrial 12S ribosomal RNA gene that predisposes carriers to aminoglycoside ototoxicity was identified in 1993. Up to a third of patients with aminoglycoside ototoxicity carry this mutation. Two other mutations in the same mitochondrial gene affect a small minority of additional patients. Thus, the prevention of aminoglycoside-induced ototoxicity through family history and molecular diagnosis is possible in many cases. It is the challenge of genomic medicine to translate this more than a decade-old knowledge into clinical practice.

Biochemical characterization of the deafness-associated mitochondrial tRNASer(UCN) A7445G mutation in osteosarcoma cell cybrids.

The deafness-associated A7445G mutation in the precursor of mitochondrial tRNA(Ser(UCN)) has been identified in several pedigrees from different ethnic backgrounds. To determine the role of nuclear background in the biochemical manifestation associated with the A7445G mutation, we performed a biochemical characterization of this mutation using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a New Zealand family into human osteosarcoma mtDNA-less (rho(0)) cells. Compared with three control cybrids, three cybrids derived from an affected matrilineal relative carrying the homoplasmic A7445G mutation exhibited approximately 38-57% decrease in the steady-state level of tRNA(Ser(UCN)), which is less reduced levels than in lymphoblastoid cells in the previous study. Furthermore, approximately 22% reduction in the level of aminoacylation of tRNA(Ser(UCN)) was observed in the mutant cybrid cells. Interestingly, approximately 60-63% decrease of steady-state level of ND6 gene, which belongs to the same precursor as that of tRNA(Ser(UCN)), in cybrid cell lines carrying the A7445G mutation, is more than that observed in lymphoblastoid cells. These observations strongly point out a mechanistic link between the processing defect of the tRNA(Ser(UCN)) precursor and decreased stability of ND6 mRNA precursor. These results also imply the influence of nuclear background on the biochemical phenotype associated with the A7445G mutation.

Phenotype of non-syndromic deafness associated with the mitochondrial A1555G mutation is modulated by mitochondrial RNA modifying enzymes MTO1 and GTPBP3.

Phenotypic expression of the deafness-associated mitochondrial A1555G mutation in the 12S rRNA gene is influenced by aminoglycosides and complex inheritance of nuclear-encoded modifier genes. The position of a major nuclear modifier gene has been localized to chromosome 8p23.1, but the identification of this gene has remained elusive. Recently, we identified a second modifier gene, mitochondrial transcription factor B1 (TFB1M), involved in mitochondrial rRNA modification. In the present study, we tested three genes involved in mitochondrial tRNA or rRNA modification, and two genes associated with non-syndromic deafness, for linkage and linkage disequilibrium (LD) in 214 DNA samples from Spanish, Italian, and Arab-Israeli families with maternally inherited non-syndromic hearing loss. The multipoint non-parametric linkage analysis and transmission disequilibrium test testing were done using all families combined as well as divided based on linkage to the chromosome 8 locus and ethnicity. Two genes, MTO1 and GTPBP3, showed strongly suggestive linkage and significant LD results. Since both genes, as well as TFB1M, are involved in the process of mitochondrial RNA modification, it appears that the modification of mitochondrial RNA is an important regulatory pathway in the phenotypic expression of the deafness-associated mitochondrial A1555G mutation. This conclusion was supported by comparing linkage results of simulated genotypes with actual results for the four genes involved in mitochondrial RNA modification.

Lipoprotein lipase locus and progression of atherosclerosis in coronary-artery bypass grafts.

PURPOSE: Our aim was to test whether polymorphisms in the lipoprotein lipase (LPL) gene were associated with the progression of atherosclerosis in grafts examined in the Post-Coronary Artery Bypass Graft Trial (Post-CABG Trial). METHODS: 843 subjects in the post-CABG trial were genotyped for the LPL-D9N, N291S, PvuII, (TTTA)n, and HindIII polymorphisms. Associations between genotype and angiographically measured progression of atherosclerosis in grafts, medical history, and family history were examined. RESULTS: Greater progression of atherosclerosis was observed in subjects with LPL-HindIII 2/2 (56% versus 42% of those with other LPL HindIII genotypes, P = 0.025) and with LPL (TTTA)n 4/4 (63% versus 43% of those with other (TTTA)n genotypes, P = 0.020). Mantel-Haenszel analysis yielded an odds ratio of 1.84 for the effect of LPL HindIII 2/2 genotype on the progression of atherosclerosis in grafts (P = 0.015) and demonstrated that the effect of genotype on progression was of the same magnitude as, but independent of, the effect of drug treatment. CONCLUSION: The LPL-HindIII 2/2 genotype is a marker for genetic variation in the 3'-end of LPL that acts as an independent risk factor for the progression of atherosclerosis in grafts examined in the Post-CABG Trial.

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.

Gene responsible for mitochondrial myopathy and sideroblastic anemia (MSA) maps to chromosome 12q24.33.

Mitochondrial myopathy and sideroblastic anemia (MSA) is a rare autosomal recessive disorder of oxidative phosphorylation and iron metabolism. Individuals with MSA present with weakness and anemia in late childhood and may become dependent on blood transfusions. Recently, we reported affected sibling pairs from a Jewish-Iranian kindred living in the US [Casas and Fischel-Ghodsian, 2003]. A genome scan and fine mapping of DNA from this family revealed homozygous alleles in the affected individuals, and a multipoint logarithm of the odds (lod) score of 3.3, within 2.3 mb of chromosome 12q24.33. Previously, Inbal et al. [1995: Am J Med Genet 55:372-378] described siblings with a similar clinical phenotype who lived in Israel but originated from the same Iranian town as the US family. Focused analysis of DNA from the Israeli family confirmed the presence of identical, homozygous alleles in the affected of the US and Israeli families within 1.2 mb of chromosome 12q24.33. Combined multipoint linkage analysis revealed a maximum lod score of 5.41 at the 132 cM position of chromosome 12. Therefore, in these two families of Jewish-Iranian descent, a disease gene for MSA maps to a 1.2 mb region of chromosome 12q24.33. This region contains 6 well described genes (SFRS8, MMP17, ULK1, PUS1, EP400, and GALNT9) and at least 15 additional putative transcripts. The known genes are expressed in multiple tissues and lack a function specific to mitochondria, making none an obvious candidate. The eventual identification of the disease gene in MSA is expected to provide insight into the tissue specificity and phenotypic variability of mitochondrial disease.

Mitochondrial myopathy and sideroblastic anemia (MSA) is a rare autosomal recessive disorder of oxidative phosphorylation and iron metabolism. Individuals with MSA present with weakness and anemia in late childhood and may become dependent on blood transfusions. Recently, we reported affected sibling pairs from a Jewish-Iranian kindred living in the US [Casas and Fischel-Ghodsian, 2003]. A genome scan and fine mapping of DNA from this family revealed homozygous alleles in the affected individuals, and a multipoint logarithm of the odds (lod) score of 3.3, within 2.3 mb of chromosome 12q24.33. Previously, Inbal et al. [1995: Am J Med Genet 55:372-378] described siblings with a similar clinical phenotype who lived in Israel but originated from the same Iranian town as the US family. Focused analysis of DNA from the Israeli family confirmed the presence of identical, homozygous alleles in the affected of the US and Israeli families within 1.2 mb of chromosome 12q24.33. Combined multipoint linkage analysis revealed a maximum lod score of 5.41 at the 132 cM position of chromosome 12. Therefore, in these two families of Jewish-Iranian descent, a disease gene for MSA maps to a 1.2 mb region of chromosome 12q24.33. This region contains 6 well described genes (SFRS8, MMP17, ULK1, PUS1, EP400, and GALNT9) and at least 15 additional putative transcripts. The known genes are expressed in multiple tissues and lack a function specific to mitochondria, making none an obvious candidate. The eventual identification of the disease gene in MSA is expected to provide insight into the tissue specificity and phenotypic variability of mitochondrial disease.

Human mitochondrial transcription factor B1 as a modifier gene for hearing loss associated with the mitochondrial A1555G mutation.

Phenotypic expression of the deafness-associated homoplasmic A1555G mutation in the mitochondrial 12S rRNA gene varies from profound congenital hearing loss to normal hearing. It has been shown that this variability in clinical expression in most patients is due to the complex inheritance of multiple nuclear-encoded modifier genes. Human mitochondrial transcription factor B1 (TFB1M) has been proposed as a candidate for being such a modifier, since it methylates adenine residues in the adjacent loop of the A1555G mutation in the 12S rRNA gene. Polymorphic markers within and adjacent to the TFB1M gene were genotyped in 214 individuals from 41 multiplex families with the A1555G mutation of Spanish, Italian, and Arab-Israeli origin. Multipoint non-parametric linkage analysis of all families combined revealed an NPL score of 1.7 (P = 0.05), and a Lod score of 1.4 (P = 0.04). Linkage disequilibrium by the Transmission Disequilibrium Test at D6S1577, a microsatellite adjacent to TFB1M, showed preferential non-transmission of an allele to affected individuals with chi2 = 8.76; P = 0.003. Sequence analysis of the coding region of the gene and testing of all intragenic SNPs did not reveal a putative causative mutation. These data provide suggestive evidence that TFB1M is a nuclear-encoded modifier gene for phenotypic expression of the A1555G mutation, and that the effect may occur through a regulatory or splicing mutation.

Mitochondrial ribosomal proteins: candidate genes for mitochondrial disease.

Most of the energy requirement for cell growth, differentiation, and development is met by the mitochondria in the form of ATP produced by the process of oxidative phosphorylation. Human mitochondrial DNA encodes a total of 13 proteins, all of which are essential for oxidative phosphorylation. The mRNAs for these proteins are translated on mitochondrial ribosomes. Recently, the genes for human mitochondrial ribosomal proteins (MRPs) have been identified. In this review, we summarize their refined chromosomal location. It is well known that mutations in the mitochondrial translation system, i.e., ribosomal RNA and transfer RNA cause various pathologies. In this review, we suggest possible associations between clinical conditions and MRPs based on coincidence of genetic map data and chromosomal location. These MRPs may be candidate genes for the clinical condition or may act as modifiers of existing known gene mutations (mt-tRNA, mt-rRNA, etc.).

Genetic influences on the increase in blood pressure with age in normotensive subjects in Barbados.

The authors tested the single and combined effects of nuclear and mitochondrial DNA genotypes on the phenotypes of systolic blood pressure (SBP) and weight, and their changes over 5 years in normotensive subjects living in Barbados. The nuclear genotypes were gender (Y chromosome), haptoglobin (HP), and group specific component (Gc). A mitochondrial genotype was chosen as a marker for maternal lineage. Baseline clinic SBP and weight (N=78), 24-hour SBP (N=28) were measured. Five years later, clinic SBP and weight were measured again in 28 participants. Male participants generally had higher pressures than female participants. The HP genotype was associated with 5 of the 8 SBP phenotypes. The haptoglobin-1 (HP1) allele was associated with higher clinic (P=.024) and evening SBP at baseline (P=.020). The effect of HP1 appears to be dominant. Haptoglobin-2 (HP2) was associated with the increase in weight over 5 years (P=.002). Group specific component (Gc) genotype was associated with 6 of the 8 SBP phenotypes. The Gc polymorphism 2 was associated with higher 24-hour SBP, sleep SBP (midnight-6 AM), afternoon SBP (noon-6 PM) and evening SBP (6 PM to midnight). Furthermore, we found a significant association between the haptoglobin/mt-DNA and Gc/mt-DNA polymorphisms with SBP between 6 PM and midnight (P=.009 and P=.011, respectively). The 5-year changes in SBP were significantly associated with the haptoglobin/mt-DNA and Gc/mt-DNA polymorphisms (P=.005 and P=.011, respectively). Multivariate analysis for genetic effects on change in weight and change in BP suggested the rise in BP, but was not suggestive of change in weight. Furthermore, multivariate analysis was associated with Gc, but not Haptoglobin genotype. In normotensive subjects of African descent living in Barbados, the increase in blood pressure with age is significantly influenced by both nuclear and mitochondrial genotypes that are more common in African derived populations.