A candidate gene for autoimmune myasthenia gravis

Abstract

Objective: We sought to identify a causative mutation in a previously reported kindred with parental consanguinity and 5 of 10 siblings with adult-onset autoimmune myasthenia gravis.

Methods: We performed genome-wide homozygosity mapping, and sequenced all known genes in the one region of extended homozygosity. Quantitative and allele-specific reverse transcriptase PCR (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 in a heterozygote.

Results: A region of shared homozygosity at chromosome 13q13.3-13q14.11 was found in 4 affected siblings and 1 unaffected sibling. 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 expression of ENOX1 decreased to about 20% of normal levels in lymphoblastoid cells from individuals homozygous for the variant and to about 50% in 2 unaffected heterozygotes. Allele-specific RT-PCR showed a 55%-60% reduction in the level of the variant transcript in heterozygous cells due to reduced mRNA stability.

Conclusion: These results indicate that this sequence variant in ENOX1 may contribute to the familial autoimmune myasthenia in these patients.

Figure 1. Region of shared homozygosity on chromosome 13 found by linkage analysis using microsatellite markers and single nucleotide polymorphism arrays

A partial pedigree of the kindred shows the haplotype results with microsatellite markers. Haplotypes were reconstructed manually according to the most parsimonious requirement for recombination; the markers are ordered, from top to bottom, from p terminal to q terminal. The box indicates the shared homozygous region on chromosome 13. The homozygous region is between markers D13S219 and D13S326. Note that the parents' haplotype information has been inferred. Age at disease onset is indicated to the left of symbols.

Figure 2. Quantitative reverse transcriptase PCR assays of the ENOX1 transcript levels (y-axis) in the family members and unrelated controls

The results are the mean of 3 replicates done in triplicate experiments relative to HGUSB, and are normalized to the mean of the 2 unrelated controls using the 2-ΔΔCt method. Note that the homozygous affected siblings have about 20% of the normal ENOX1 transcript levels and the heterozygous unaffected individuals have about 50% of the normal levels. Values represent mean ± SEM; ***p < 0.001.