Studies Of Hereditary Neurological Disease: Disease Gene Identification

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The purpose of this research program is to investigate the causes of hereditary neurological diseases, with the goal of developing effective treatments for these disorders. A genetic outreach program allows the identification and characterization of patients and families with hereditary neurological diseases. Specific research accomplishments in the past year include (1) the evaluation of a candidate gene for familial autoimmune myasthenia gravis, (2) Charcot-Marie-Tooth disease type 2C (CMT2C), and (3) a new form of hereditary spastic paraplegia mapped to chromosome 19. Myasthenia gravis is usually sporadic. However, familial cases suggest a genetic predisposition. We sought to identify a causative mutation in a previously reported Italian-American kindred with parental consanguinity and five out of ten siblings affected by adult-onset autoimmune myasthenia gravis. We performed genome-wide homozygosity mapping, and sequenced all genes in the one region of extended homozygosity. Quantitative and allele specific RT-PCR were performed on a candidate gene to determine the RNA expression level in affected siblings and controls and the relative abundance of the wild-type and mutant alleles. A region of shared homozygosity at chromosome 13q13.3-13q14.11 was found in four affected subjects and one unaffected individual. A homozygous single nucleotide variant was found in the 3'-untranslated region of the ecto-NADH oxidase 1 gene (ENOX1). No other variants likely to be pathogenic were found in genes in this region or elsewhere. The ENOX1 sequence variant was not found in 764 controls. Quantitative RT-PCR showed that the expression of ENOX1 is decreased to about 20% normal levels in lymphoblastoid cells from individuals who are homozygous for the variant and to about 50% in two unaffected heterozygotes. Allele specific RT-PCR showed a 55-60% reduction in the transcript level of the variant in heterozygote cells due to reduced mRNA stability. These results indicate that this sequence variant in ENOX1 may contribute to the late onset myasthenia in these patients. To our knowledge, this is the first single-gene defect to be associated with autoimmune myasthenia. CMT2C is an autosomal dominant neuropathy characterized by limb, diaphragm, and laryngeal muscle weakness. Two unrelated families with CMT2C showed significant linkage to chromosome 12q24.11. All genes in this region were sequenced and heterozygous missense mutations were identified in the TRPV4 gene causing the amino acid substitutions R269C and R269H. TRPV4 is a well known member of the TRP superfamily of cation channels. In TRPV4-transfected cells, the CMT2C mutations caused marked cellular toxicity and increased constitutive and activated channel currents. Mutations in TRPV4 were previously associated with skeletal dysplasias. Our findings indicate that TRPV4 mutations can also cause a degenerative disorder of peripheral nerves. The CMT2C mutations lie in a distinct region of the TRPV4 ankyrin repeats, suggesting that this striking phenotypic variability may be due to differential effects on regulatory protein-protein interactions. We subsequently investigated interacting proteins and used exome-sequencing looked for a causative mutation in a CMT2C family in which we had found no TRPV4 mutation. The exome analysis revealed a novel TRPV4 mutation in this family, R186Q, which had been missed by Sanger sequencing due to a rare polymorphism at the primer site. We identified a family in Mali with two sisters affected by spastic paraplegia. In addition to spasticity and weakness of the lower limbs, the patients had marked atrophy of the distal upper extremities. Homozygosity mapping using single nucleotide polymorphism arrays showed that the sisters shared a region of extended homozygosity at chromosome 19p13.11-q12 that was not shared by controls. These findings indicate a clinically and genetically distinct form of hereditary spastic paraplegia with amyotrophy, designated SPG43. We subsequently evaluated candidate genes in the critical region by exome sequencing, and identified one gene, C19orf12, with a sequence variant not found in controls. The variant is associated with aberrant localization of the recombinant protein in transfected cells. Mutation in the protein product of this gene, which we have named senfaganin, may be a novel cause of hereditary spastic paraparesis.