Christiane Alexander

OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28

Autosomal dominant optic atrophy (ADOA) is the most prevalent hereditary optic neuropathy resulting in progressive loss of visual acuity, centrocoecal scotoma and bilateral temporal atrophy of the optic nerve with an onset within the first two decades of life. The predominant locus for this disorder (OPA1; MIM 165500) has been mapped to a 1.4-cM interval on chromosome 3q28–q29 flanked by markers D3S3669 and D3S3562 (ref. 3). We established a PAC contig covering the entire OPA1 candidate region of approximately 1 Mb and a sequence skimming approach allowed us to identify a gene encoding a polypeptide of 960 amino acids with homology to dynamin-related GTPases. The gene comprises 28 coding exons and spans more than 40 kb of genomic sequence. Upon sequence analysis, we identified mutations in seven independent families with ADOA. The mutations include missense and nonsense alterations, deletions and insertions, which all segregate with the disease in these families. Because most mutations probably represent null alleles, dominant inheritance of the disease may result from haploinsufficiency of OPA1. OPA1 is widely expressed and is most abundant in the retina. The presence of consensus signal peptide sequences suggests that the product of the gene OPA1 is targeted to mitochondria and may exert its function in mitochondrial biogenesis and stabilization of mitochondrial membrane integrity.

A major marker for normal tension glaucoma: Association with polymorphisms in the OPA1 gene

Abstract. Normal tension glaucoma (NTG) is a major form of glaucoma, associated with intraocular pressures that are within the statistically normal range of the population. OPA1, the gene responsible for autosomal dominant optic atrophy represents an excellent candidate gene for NTG, as the clinical phenotypes are similar and OPA1 is expressed in the retina and optic nerve. Eighty-three well-characterized NTG patients were screened for mutations in OPA1 by heteroduplex analysis and bi-directional sequencing. Sequences found to be altered in NTG subjects were examined for variations in 100 population controls. A second cohort of 80 NTG patients and 86 population controls was subsequently screened to determine whether the initial findings could be replicated. A single nucleotide polymorphism (SNP) on intervening sequence (IVS) 8 (IVS8 + 4 C/T) was found to be strongly associated with the occurrence of NTG in both cohorts (χ2=7.97, P=0.005 in the first cohort, χ2=9.93, P=0.002 in the second cohort; odds ratio 3.1 (95% CI: 1.8–5.6). A second SNP (IVS8 + 32 T/C) appeared to be associated with disease in the first cohort (χ2=4.71, P=0.030), but this finding could not be replicated in the second cohort. In the combined cohort, the compound at-risk genotype IVS8 + 4 C/T, + 32 T/C was strongly associated with the occurrence of NTG (χ2=22.04, P=0.00001 after correcting for testing four genotypes). These results indicate that polymorphisms in the OPA1 gene are associated with NTG and may be a marker for the disease.

Fine scale mapping places DLG1, the gene encoding hDlg, telomeric to the OPA1 candidate region

Species:Mouse Locus name: methionine synthase or 5-methyltetrahydrofolatehomocysteine methyltransferase Locus symbol:Mtr Map position: proximal–D13Mit1–1.06 cM ± 1.06 SE– Mtr, D13Bir4, D13Bir6–1.06 ± 1.06–D13Abb1e–2.13 ± 1.49–D13Bir7–distal Method of mapping:Mtr was localized by RFLP analysis of 96 animals from an interspecific backcross panel ((C57BL/6JEi × SPRET/Ei)F1 × SPRET/Ei) provided by The Jackson Laboratory, Bar Harbor, Me. (BSS panel) [1]. Database deposit information: The data are available from the Mouse Genome Database, accession number MGD-JNUM-39061. Molecular reagents:A 1095-bp mouse cDNA was obtained by reverse transcription/PCR of mouse liver RNA, with degenerate oligonucleotides based on regions of homology within the methionine synthase sequences of lower organisms. The two primers (D1730 and D1733), as described by Leclerc et al. [2], were successful in amplifying both human and mouse cDNAs. The PCR products from both species were subcloned and sequenced; they showed 89% identity. The mouse cDNA was labeled by random priming and hybridized to Southern blots of EcoRI-digested mouse genomic DNA. Allele detection:Allele detection was performed by RFLP analysis of an EcoRI polymorphism. The C57BL/6J strain has alleles of approximately 13 kb, while theMus spretusstrain has alleles of approximately 9 kb and 4 kb. A constant band of approximately 0.5 kb was seen in both strains. Previously identified homologs: Human MTR has been mapped to chromosomal band 1q43 by fluorescence in situ hybridization [2–4]. Discussion: Methionine synthase (EC 2.1.1.13, 5-methyltetrahydrofolate-homocysteine methyltransferase) catalyzes homocysteine remethylation to methionine, with 5-methyltetrahydrofolate as the methyl donor and methylcobalamin as a cofactor. Nutritional deficiencies and genetic defects in homocysteine metabolism result in varying degrees of hyperhomocysteinemia. Dramatic elevations in plasma and urinary homocysteine levels are associated with the inborn error of metabolism, homocystinuria. Consequent to the recent isolation of the human cDNA for methionine synthase [2–4], two groups of investigators have identified mutations in methionine synthase in homocystinuric patients [2, 5]. Mild elevations in plasma homocysteine are thought to be a risk factor for both vascular disease and neural tube defects [6–8]. A genetic variant in methylenetetrahydrofolate reductase (MTHFR), the enzyme that synthesizes 5-methyltetrahydrofolate for the methioninesynthase reaction, is the most common genetic determinant of hyperhomocysteinemia identified thus far [9]. Mild defects in the methionine synthase reaction are also potential candidates for hyperhomocysteinemia and the associated multifactorial diseases. A common variant has been reported for the human methionine synthase gene, but its physiologic consequences have not yet been determined [2, 4]. The mapping of the human MTR gene to 1q43 and of the mouse gene to proximal Chromosome (Chr) 13 is consistent with previous findings of human/mouse homologies between these 2 chromosomal regions; the human and mouse nidogen genes have been mapped to 1q43 and proximal Chr 13, respectively [10]. Several genes have already been implicated in neural tube defects in mice [11]. Studies involving the mouse methionine synthase gene will be useful in assessing the role of this important enzyme in the development of birth defects and/or vascular disease.