Whole Exome Sequencing Identifies a New Splicing Factor Gene Causative of X-linked Spinocerebellar Ataxia

Abstract

Purpose: The genetic basis of X-linked Spinocerebellar Ataxia (SCA-X5: OMIM 300703) remains unclear, although previous linkage analysis identified a candidate locus on Xq25-q27.1. We identified a large family with a phenotype consistent with SCA-X5. We aimed to use whole exome sequencing (WES) to identify the causative gene, characterize the phenotype and the spatiotemporal expression of the protein.

Methods: Detailed ophthalmic and neurological examinations were performed. Eye movement recordings were used to assess eye movement abnormalities using a series of fixation and smooth pursuit tasks. MRI, nerve conduction studies, electromyography and muscle biopsies were obtained from affected subjects. WES was performed on a large three-generation family (6 affected). We identified rare variants by focusing on protein-altering and splice-site changes with an allele frequency <1% in SNP databases and absent from 56 local exomes of unaffected subjects. Mutation was verified using sanger sequencing. Spatiotemporal expression was characterized using immunohistochemistry in human embryonic brain.

Results: Five subjects had variable vertical and horizontal conjugate nystagmus with an increasing slow phase velocity. All affected subjects had saccadic smooth pursuit, ataxia and mild learning difficulties. Four subjects had abnormal head posture and strabismus. Affected females had macro-square wave jerks. Visual acuity ranged from 0.1-0.4 logMAR and stereopsis ranged between 110” of arc to no detectable stereopsis. MRI studies revealed a variable degree of cerebellar vermian atrophy, which was more severe in the male subject (hemizygous mutation) compared to the females (heterozygous mutation). Neuromuscular tests and investigations were normal.<br /> Variant filtering from WES dataset identified a splicing factor gene, SRPK3 at Xq28, mutation of which was causative of SCA-X5. The mutation co-segregated with the phenotype and was absent in the control exomes and SNP databases. Expression was noted in the developing cerebellum, with intense staining in the adjacent choroid plexus epithelium.

Conclusions: For the first time we have identified the gene, SRPK3, mutation of which is causative of SCA-X5. We have shown the detailed phenotypical characteristics associated with SCA-X5, with females less severely affected. We have also characterized the spatiotemporal expression of this protein in the developing brain.