Combining homozygosity mapping with exome capture: SDCCAG8 and retinal-renal ciliopathy

http://www.nature.com/ng/journal/v42/n10/full/ng.662.html

Combining homozygosity mapping with exome capture

The finding that most known NPHP-RC genes caused the disorder only in a small number of cases (<1%)⁹necessitated the ability to map and identify disease genes in single families. We therefore developed a strategy that combines homozygosity mapping in single families¹³ with exon capture and consecutive massively parallel sequencing¹⁴. Using the NimbleGen 385K platform, we designed a ciliopathy candidate exon capture array, which contains oligonucleotides that interrogate ~13,000 exons from the 'UCSC Gene' annotation (see URLs) of 828 NPHP-RC candidate genes. Candidate genes were derived from ciliopathy animal models, from the photoreceptor sensory cilia proteome¹⁵ and from other candidate sources¹⁶(Supplementary Tables 1–3).

Because exon capture with subsequent massively parallel sequencing yields too many variants from normal reference sequence (VRSs) to make a safe call regarding the disease-causing mutation¹⁴, we devised a strategy of a priori reduction of VRSs (Supplementary Table 1). These a priori restriction criteria consisted of: (i) capturing only ~13,000 ciliopathy candidate exons instead of all ~180,000 exons from the collaborative consensus coding sequence (CCDS) project (~15-fold reduction; Supplementary Table 1); (ii) evaluating coding SNPs, splice variants and indels only (as other variants will be difficult to interpret); (iii) removing VRSs from a database of innocuous SNPs (dbSNP130; 2.3-fold reduction); (iv) evaluating only within the mapped homozygous candidate region of an individual or family (~20-fold reduction); and (v) preferentially evaluating truncating mutations (~4-fold reduction). This approach allowed us to reduce the number of VRSs by an average of ~2,760-fold and led to the identification of the disease-causing gene in 3 out of 5 attempts (Supplementary Table 1). We discovered homozygous mutations in the known NPHP-RC genes AHI1 (family A2045) and INVS (family A128; Supplementary Table 1). More importantly, we discovered a homozygous mutation in SDCCAG8 as a new cause of NPHP-RC (Supplementary Table 1).