A mutation in the Golgi Qb-SNARE gene GOSR2 causes progressive myoclonus epilepsy with early ataxia

Abstract

The progressive myoclonus epilepsies (PMEs) are a group of predominantly recessive disorders that present with action myoclonus, tonic-clonic seizures, and progressive neurological decline. Many PMEs have similar clinical presentations yet are genetically heterogeneous, making accurate diagnosis difficult. A locus for PME was mapped in a consanguineous family with a single affected individual to chromosome 17q21. An identical-by-descent, homozygous mutation in GOSR2 (c.430G>T, p.Gly144Trp), a Golgi vesicle transport gene, was identified in this patient and in four apparently unrelated individuals. A comparison of the phenotypes in these patients defined a clinically distinct PME syndrome characterized by early-onset ataxia, action myoclonus by age 6, scoliosis, and mildly elevated serum creatine kinase. This p.Gly144Trp mutation is equivalent to a loss of function and results in failure of GOSR2 protein to localize to the cis-Golgi.

Figure 1

Identification of GOSR2 Mutation (A) Graph showing the computed LOD scores for chromosome 17. A single peak, of width 9 cM, with the maximum possible LOD score of 1.93 (indicated by the red highlighting) suggests linkage to a 9.5 Mbp region on chromosome 17 (indicated by the red box on the ideogram). The tile path of regions targeted on the array is indicated by the black shaded regions below the ideogram of chromosome 17. Sequence coverage, calculated as the number of reads per bp of the linkage interval, is indicated in red in the lower graph. (B) Pedigrees of five families with affected females and males (indicated by black circles and squares, respectively). Alleles carried by each individual are indicated; only affected individuals are homozygous for the GOSR2 c.430G>T mutation. Below the pedigrees are sizes of products in bp generated by amplification by PCR of microsatellite markers flanking GOSR2 (indicated on the left) for each affected individual. The red lettering indicates haplotypes observed in more than one individual. (C) The three isoforms of GOSR2 (A, B, and C) are shown by exon structure in the context of genomic DNA (upper half) and the domain structure of the corresponding translated protein products (lower half). Exons are indicated by boxes, and introns are shown as thin lines. Black shading of the exons indicates the open reading frame, whereas gray shading shows untranslated regions. Isoforms A, B, and C correspond to NM_004287.3, NM_054022, and NM_001014511.1, respectively. On the protein structures, the coiled-coil (CC), Q-SNARE (as indicated), and transmembrane (TM) domains are shown. (D) A CLUSTALW alignment of orthologs of GOSR2 from multiple species. The orthologs were identified by using the homologene database, tblastn search of the NCBI nonredundant sequence database, and the SNARE database. Species names and amino acid residue ranges are labeled on the left of the alignment. Identical residues are indicated by the gray background. Gly144 and the corresponding residue in other species are indicated by the black arrowhead; the glutamine residue from which the Q-SNARE gets its name is indicated by the white arrowhead. We performed alignments by using the EBI CLUSTALW server.

Figure 2

GOSR2 Mutation, p.Gly144Trp, Functional Analysis (A–D) Example images taken by indirect immunofluorescent confocal microscopy of cultured primary human fibroblast cell lines derived from an unaffected female control (A and C) and case 1 (B and D). Cells were grown on glass coverslips, fixed with 4% paraformaldehyde, and then permeabilized with 0.2% saponin in phosphate buffered saline (PBS). The cells were then stained with an antibody specific to GOSR2 (mouse pAB, Abnova, Taipei City, Taiwan) (A–D; red channel) and costained with either the Golgi specific marker GOLGA2 (rabbit pAB, Abnova) (A and B) or the endoplasmic reticulum marker PDI (rabbit pAB, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (C and D; green channel). Cells were embedded in Mowiol containing 4', 6-diamidino-2-phenylindole (DAPI; blue channel) to stain nuclei and Dabco (Sigma Aldrich, Steinheim, Germany) as an antifading agent. Note that only the control cell line has evidence of costaining of GOLGA2 with GOSR2 (A; indicated by the yellow color). (E) Bos1 p.Gly176Trp cannot substitute bos1. Yeast strain BY4742 strain was transformed with pRS316-bos1 and the resulting strain transformed with bos1 deletion cassettes constructed with PCR. 5-fluoroorotic acid (5-FOA) is converted by Ura3 (present in pRS316-bos1) to 5-fluorouracil, which is toxic for the cell. We used negative selection with 5-FOA to confirm the bos1 deletion (Δbos1) strain. The pRS315-bos1 and pRS315-bos1-Gly176 vectors allow expression of the wild-type and mutant bos1 under negative selection conditions. Serial dilutions (1:10) of the indicated strains were spotted on SC-Leu/Ura and 5-FOA plates and incubated at 30°C for 48 hr. Only cells that lack a functional bos1 gene after negative selection for the pRS316-bos1 vector on the 5-FOA plates failed to thrive. Results indicate that cells transformed with pRS315-bos1-Gly176Trp are equivalent to the Δbos1 strain.