Mutation in the gene encoding ubiquitin ligase LRSAM1 in patients with Charcot-Marie-Tooth disease

Abstract

Charcot-Marie-Tooth disease (CMT) represents a family of related sensorimotor neuropathies. We studied a large family from a rural eastern Canadian community, with multiple individuals suffering from a condition clinically most similar to autosomal recessive axonal CMT, or AR-CMT2. Homozygosity mapping with high-density SNP genotyping of six affected individuals from the family excluded 23 known genes for various subtypes of CMT and instead identified a single homozygous region on chromosome 9, at 122,423,730-129,841,977 Mbp, shared identical by state in all six affected individuals. A homozygous pathogenic variant was identified in the gene encoding leucine rich repeat and sterile alpha motif 1 (LRSAM1) by direct DNA sequencing of genes within the region in affected DNA samples. The single nucleotide change mutates an intronic consensus acceptor splicing site from AG to AA. Direct analysis of RNA from patient blood demonstrated aberrant splicing of the affected exon, causing an obligatory frameshift and premature truncation of the protein. Western blotting of immortalized cells from a homozygous patient showed complete absence of detectable protein, consistent with the splice site defect. LRSAM1 plays a role in membrane vesicle fusion during viral maturation and for proper adhesion of neuronal cells in culture. Other ubiquitin ligases play documented roles in neurodegenerative diseases. LRSAM1 is a strong candidate for the causal gene for the genetic disorder in our kindred.

Figure 1. Maritime family with Charcot-Marie-Tooth and genetic mapping.

(A) Pedigree, with affected patients shaded in black, sampled individuals have four digit id numbers directly below symbols, proband 1675 is indicated with black arrowhead. Sequenced individuals have mutation status indicated (wild type is GG, homozygous mutant is AA, heterozygous mutant is AG). (B) Homozygous haplotype (HH) analysis. Map of the RCHH intervals shared by 5 patients identified by Homozygosity Haplotype algorithm with a cutoff of 3.0 cM.

Figure 2. Sequence showing mutation in genomic and cDNA of affected patient.

(A) Mutation of splice acceptor site AG of LRSAM1 exon 24 (25 in alternative isoform 3) to dinucleotide AA in genomic DNA of patient 1702. Upper to lower panels: translation of coding exon; virtual chromatogram of consensus genomic sequence forward direction; sequence chromatogram of affected patient reverse direction; virtual chromatogram of consensus genomic sequence reverse direction. Red arrow points to homozygous mutation. (B) Sequence of cDNA from RNA of patient 1702 showing aberrant splice site utilization and frameshift of encoded protein. Upper panel, sequence chromatogram of correctly spliced cDNA from exon 24 to 25 (per isoform 3); lower panel, sequence chromatogram of incorrectly spliced cDNA from affected patient. Two base deletion caused by splicing interior to exon 25 (red arrow). (C) Western blot of LRSAM1 protein in cells cultured from patient homozygous for LRSAM1 mutation. EBV-transformed B- lymphocytes from control or patient 1675 were extracted and Western blotted with anti-LRSAM1 antibody. Left, anti-LRSAM1; center, anti-actin; right, Fast green total protein stain.