4. Results
SNP analysis revealed nineteen regions of LCSH over 5 Mb in length in individual V:11, five of which were above 20 Mb long, and a total of fourteen regions of LCSH in individual V:12 over 5 Mb, including three above 20 Mb (Figure 2). Of the identified LCSH regions, six were common between the affected siblings (Table 1) and thus likely to harbour the locus for their recessively-inherited cerebellar ataxia. No clinically significant copy number changes were detected in either case.
Figure 2  Regions of long contiguous stretches of homozygosity (LCSH) over 5 Mb in length identified by SNP analysis in individuals V:11 (right track) and V:12 (left track). Chromosomes 1 to 12 are depicted in order from top left to right; chromosomes 13 to 22, X and Y are depicted in order from bottom left to right. The arrowed region on chromosome 13 shows the location of the SACS gene, contained within the LCSHs of indicated sizes.
microarrays-04-00490-t001_Table 1 Table 1  Regions of LCSH common to the affected siblings, as detected by SNP analysis.   A search of OMIM genes associated with ataxia and located within the regions of LCSH common to both affected siblings yielded seven candidate genes (Table 2); however, there was no single gene for which full phenotypic concordance could be seen.
microarrays-04-00490-t002_Table 2 Table 2  Candidate ataxia genes located in the regions of LCSH common to both affected siblings, identified by searching OMIM genes for the specific clinical feature “ataxia”. Abbreviations: AR, autosomal recessive; AR, autosomal dominant.   Rather than screen candidate ataxia genes found in the shared LCSH regions, we undertook a complementary WES study, with variant filtering as described (Figure 3), which yielded three possible variants (Table 3). The variant most concordant with the phenotype, most likely to be clinically significant, and located in one of the LCSH regions common to both affected siblings, was the SACS gene variant c.7962T>G p.(Tyr2654*). Sanger-based sequencing confirmed the apparent homozygosity of this variant in the two affected siblings (Figure 4).
microarrays-04-00490-t003_Table 3 Table 3  WES prioritised variants. Variant filtering was performed using an in-house bioinformatics pipeline and Variant Studio software (Illumina). * Data obtained from the ExAC database [11]. Abbreviations: Het, heterozygous; Hom, homozygous; MAF, minor allele frequency.
Figure 3  The pipeline used to select variants of potential clinical interest from exome sequencing. The right arm incorporates the homozygosity data obtained by SNP array; the left arm does not incorporate this information. * Based on Variant Studio annotation, OMIM, NCBI.
Figure 4  Sequence electropherograms showing the bidirectional traces of a region of exon 10 of the SACS gene. Apparent homozygosity for the variant c.7962T>G p.(Tyr2654 *) is seen in the two affected siblings (panels A and B representing V:11 and V:12, respectively). The variant was not detected in an unaffected sibling (panel C).   The nonsense variant, c.7962T>G p.(Tyr2654 *), has not, to our knowledge, been previously reported in the literature or mutation databases (Leiden Open Variation Database; LOVD; www.lovd.nl/3.0/home, Human Gene Mutation Database Professional; HGMD® Pro) [12]; but, given that it results in premature termination of mRNA translation, it is predicted to be pathogenic. The Mutalyzer 2.0.13 website [13] predicts that this variant produces an amino acid sequence which is 58% of the length of that coded by the reference SACS gene transcript (Refseq accession number: NM_014363.4).