Nathan Fischel-Ghodsian

A genome‐wide search identifies potential new susceptibility loci for Crohn's disease

Chronic inflammatory bowel disease (IBD) presents as two major clinical forms, Crohns disease (CD) and ulcerative colitis (UC). Genetic epidemiological studies and animal models suggest that inherited factors play significant roles in the susceptibility to both forms of IBD. From four genome-wide scans, putative susceptibility loci on chromosome 16 (IBD1 for CD), and on chromosomes 1, 3, 4, 6, 7, 10, and 12 for IBD, have been identified. Several other groups, including ours, have confirmed linkage to the loci on chromosomes 12 and 16. The aim of this study is to identify other potential susceptibility loci for CD with a genome-wide search approach. In our sample of 222 individuals from 46 families (20 Jewish and 26 non-Jewish), with a total of 65 sibpairs diagnosed with CD, we observed a novel locus with suggestive linkage [multipoint logarithm of the odds score (Mlod) > 2] at chromosome 14q11.2 (Mlod = 2.8, p = 0.0002). In addition, suggestive linkage was observed in our Jewish families at chromosome 17q21-q23 (Mlod = 2.1, p = 0.01) and chromosome 5q33-q35 (Mlod = 2.2, p = 0.0003). The syntenic regions of the latter locus are mapped within two putative loci on mouse chromosomes 11 and 18, which were identified in a mouse IBD model induced by dextran sulfate sodium (29). Our preliminary results provide potential evidence for several susceptibility loci contributing to the risk of CD. The observation of man-mouse synteny may accelerate the identification of CD susceptibility gene(s) on human chromosome 5.

Mitochondrial deafness mutations reviewed.

The first molecular defect for nonsyndromic hearing loss was identified in 1993, and was a mitochondrial mutation. Since then a number of inherited mitochondrial DNA (mtDNA) mutations have been implicated in hearing loss, and acquired mtDNA mutations have been proposed as one of the causes of the hearing loss associated with aging, presbyacusis. These molecular findings have raised as many questions as they have answered, however, since the pathophysiology between the mutations and the clinical phenotype remains poorly understood. This mini‐review will, after a short background review of mitochondrial genetics, (1) outline the different mtDNA mutations associated with inherited syndromic, nonsyndromic, and ototoxic hearing loss, (2) summarize the data on acquired mtDNA mutations and their possible association with presbyacusis, (3) describe the biochemical consequences of the inherited mtDNA mutations, (4) suggest the clinical implications of the identification of these mutations, and (5) discuss the penetrance and tissue specificity of the hearing associated mtDNA mutations. Hum Mutat 13:261–270, 1999.

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.

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

ABSTRACT 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.

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.

Stable length polymorphism of up to 260 kb at the tip of the short arm of human chromosome 16

We have completed a long-range restriction map of the terminal region of the short arm of human chromosome 16 (16p13.3) by physically linking a distal genetic locus (alpha-globin) with two recently isolated probes to telomere-associated repeats (TelBam3.4 and TelBam-11). Comparison of 47 chromosomes has revealed major polymorphic length variation in this region: we have identified three alleles in which the alpha-globin genes lie 170 kb, 350 kb, or 430 kb from the telemere. The two most common alleles contain different terminal segments, starting 145 kb distal to the alpha-globin genes. Beyond this boundary these alleles are nonhomologous, yet each contains sequences related to other (different) chromosome termini. This chromosome size polymorphism has probably arisen by occasional exchanges between the subtelomeric regions of nonhomologous chromosomes; analogous length variation is likely to be present at other human telomeres.

Mitochondrial Gene Mutation Is a Significant Predisposing Factor in Aminoglycoside Ototoxicity

PURPOSE Aminoglycoside-induced deafness caused by mutations in the mitochondrial 12S ribosomal RNA gene has been described in a number of Asian patients. The purpose of the current study is to analyze ethnically diverse patients in the United States with hearing loss after aminoglycoside exposure for presence or absence of these mitochondrial DNA mutations, and establish the frequency and clinical presentation associated with them. PATIENTS AND METHODS Clinical histories, medical records, and blood samples were obtained from 41 unrelated American individuals with hearing loss after aminoglycoside exposure. DNA was extracted from the blood of these individuals, amplified by the polymerase chain reaction, and analyzed for mitochondrial ribosomal RNA gene mutations by allele-specific oligonucleotide hybridization, restriction fragment length polymorphism analysis, and sequencing. RESULTS The nucleotide 1555 A-->G mutation was identified in 7 of 41 individuals (17%). None of the other known mutations was found. The ethnic origin of the individuals with predisposing mutations included Caucasians, Hispanics, and Asians. Four of the 7 patients with the 1555 A-->G mutation had a family history of aminoglycoside-induced ototoxicity. Particularly unexpected was the late onset of hearing loss in 3 of these patients, years after the aminoglycoside exposure. The 12S ribosomal RNA gene was sequenced in these patients, and a second sequence change that could be responsible for the milder phenotype was detected in 1 of the 3 patients. CONCLUSION These findings imply that a significant proportion of patients with aminoglycoside-induced ototoxicity harbor mutations in the 12S rRNA gene, which can be detected by DNA screening. Also, the majority of these hearing losses could have been easily prevented by the simple taking of a clinical history. In these individuals, a genetic susceptibility to the ototoxic effects of aminoglycosides can be diagnosed, and deafness can be prevented in maternal relatives by avoidance of these antibiotics.

Mitochondrial ribosomal RNA gene mutation in a patient with sporadic aminoglycoside ototoxicity

PURPOSE Aminoglycoside-induced deafness has been described in a number of Chinese pedigrees. In nearly all of these families, affected individuals were related through the maternal side. Because mitochondrial DNA is transmitted exclusively through mothers, it had been speculated that a mutation in the mitochondrial DNA might predispose these maternally related family members to aminoglycoside ototoxicity. Recently, we analyzed three such families with multiple cases of ototoxic deafness and identified a pathogenic mutation in the mitochondrial 12S ribosomal RNA gene at nucleotide position 1555. The purpose of the current study is to analyze individuals with no family history of deafness, who had severe hearing loss after aminoglycoside exposure, for presence or absence of this particular mitochondrial DNA mutation. MATERIALS AND METHODS Blood was obtained from 36 Chinese individuals who became deaf after aminoglycoside exposure and had no family history of deafness. The DNA of these individuals was extracted, amplified by the polymerase chain reaction, and analyzed for the mitochondrial ribosomal RNA gene mutation by allele-specific oligonucleotide hybridization and Southern blot analysis. RESULTS In one of these 36 sporadic cases, we identified the nucleotide 1555 A-->G mutation in the mitochondrial genome. CONCLUSION This finding implies that a small proportion of individuals at risk for aminoglycoside ototoxicity harbor the specific mitochondrial DNA mutation identified in the familial cases. In these individuals, a genetic susceptibility to the ototoxic effects of aminoglycosides can be diagnosed, and deafness can be prevented in maternal relatives by avoiding the use of these antibiotics.

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.