Whole exome sequencing identifies a new ciliary gene in autosomal recessive rod-cone dystrophy

Abstract

Purpose: Autosomal recessive rod-cone dystrophies (arRCD) are a heterogeneous group of retinal diseases with more than forty implicated genes. However, comprehensive targeted next generation sequencing (NGS) still identifies cases with no underlying gene defect and whole exome sequencing (WES) offers, in these cases, a method of choice to hunt for new candidates. Our purpose was to apply WES to identify a novel gene defect in an arRCD case, to screen a large cohort of 338 patients by direct sequencing, to establish its prevalence in arRCD and to evaluate the expression and localization of the respective transcript and protein.

Methods: After exclusion of known gene defects through a comprehensive method (ARVO 2011, abstract # 6605), WES was applied to an arRCD case, his unaffected first-cousin-parents, his unaffected brother and sister. Filtering used available SNP databases, bioinformatic pathogenic prediction programs, retinal transcriptomic databases, co-segregation analysis and Sanger sequencing to select the most likely pathogenic variant. Further Sanger sequencing of the new candidate was performed on 338 unrelated arRCD. Expression studies for mRNA (RT-PCR and in situ hybridization) were performed in retinal sections. In addition, protein immunolocalization was achieved by co-staining against gamma and acetylated alpha tubulins human fibroblasts after in vitro induction of monocilia by serum deprivation.

Results: WES identified a homozygous nonsense change in a novel gene segregating with the disease. Further Sanger sequencing of a large arRCD cohort identified two additional cases with nonsense mutations leading to a prevalence of ~1% of cases in our cohort. This gene had never been associated with photoreceptor diseases. Transcriptomic and bioinformatic databases reported the product of this gene to be associated with the centrosome and expressed in photoreceptors. In situ hybridization studies revealed mRNA expression within the outer nuclear layer of mouse retinal sections. In addition, immunostaining of human monocilia in induced-fibroblasts confirmed protein localization at the basal body.

Conclusions: Whole exome sequencing and further Sanger sequencing of a large cohort of arRCD patients identify a novel candidate encoding a ciliary protein critical for photoreceptor homeostasis. This novel gene defect accounts for 1% of arRCD in the studied cohort.

Keywords: 696 retinal degenerations: hereditary • 539 genetics • 648 photoreceptors