NXT-EYE: integrative strategy and identification of hereditary blindness

01 January 2013 → 31 December 2016
Regional and community funding: IWT/VLAIO
Research disciplines
  • Medical and health sciences
    • Ophthalmology and optometry
    • Ophthalmology and optometry
    • Ophthalmology and optometry
hereditary blindness hereditary retinal disorders
Project description

Inherited retinal diseases (IRD) are a major cause of early-onset blindness, having an overall prevalence of ~1/3,000. In addition to the wide spectrum of different clinical subtypes, IRD are also characterized by a tremendous genetic heterogeneity. Up to now, molecular genetic studies have identified 256 genes listed in RetNet that are associated with IRD, complicating the establishment of a molecular genetic diagnosis. Recent genetic studies in IRD based on targeted next-generation sequencing, whole exome sequencing (WES), and whole genome sequencing (WGS) show detection rates of 50-70%, leaving still 30-50% of patients without molecular genetic diagnosis. This study not only contributed to the expansion of the IRD mutation spectrum in previously identified disease genes, but more importantly also revealed different types of hidden genetic variation in IRD, such as rare novel disease genes, non-coding variation interfering with the expression of a candidate IRD disease gene, and copy number variations (CNVs).

An integrative approach consisting of identity-by-descent (IBD) mapping, targeted gene screening, and WES in a cohort of 126 independent IRD patients of consanguineous origin revealed an underlying molecular genetic defect in known IRD genes in 74% of patients. A total of 92 mutations in 45 different genes were found, including 48 novel mutations. In a separate study we showed that a nonsense mutation in FAM161A, c.1309A>T, p.(Arg437*), is a founder mutation, underlying approximately 2% of autosomal recessive retinitis pigmentosa (AR RP) cases in Dutch, Belgian, and German populations . The age of onset of the retinal dystrophy appears variable in these patients, but progression can be steep, with almost complete loss of central vision later in life.

WES revealed mutations in six rare novel candidate IRD genes: CEP162, EML4, and PATJ in AR RP patients, ERICH6 in Senior-Loken syndrome, FBN2 in enhanced S-cone syndrome, and DRAM2 in a macular phenotype. Finally, single nucleotide polymorphism chip analysis in a patient with AR cerebellar atrophy revealed the presence of a large deletion disrupting the GRID2 gene. While most of these novel IRD genes still need further functional characterization, the role of GRID2 and DRAM2 in retina was further analyzed in the context of this thesis. The identification of GRID2 as an underlying disease gene of AR cerebellar ataxia associated with retinal dystrophy expanded the clinical spectrum of GRID2 deletion mutants. We demonstrated GRID2 expression and localization in human and murine retina, supporting a so far unknown role of GRID2 in retina. In a collaborative study four additional families were found with a mutation in the DRAM2 gene and DRAM2 was shown to play a role in the initation of autophagy. Murine retinal localization in the photoreceptor inner segments and at the apical surface of retinal pigment epithelial cells suggests a role in the renewal and recycling of photoreceptor outer segments to preserve visual function.

IBD mapping combined with targeted candidate gene screening in a family with an unusual AR RP phenotype revealed a known homozygous missense mutation c.448G>A, p.(Glu150Lys) in the RHO gene. Interestingly, four non-coding homozygous variants were found in two SAMD7 genomic regions relevant for binding of the retinal transcription factor CRX (CRX-bound regions, CBRs). Luciferase assays and electroporation of mouse retinal explants with reporter constructs of wild type and variant SAMD7 CBRs showed significantly decreased SAMD7 reporter activity for the variant construct compared to the wild type sequence, suggesting a cis-regulatory effect on SAMD7 expression. As Samd7 is a recently identified Crx-regulated transcriptional repressor in retina, we hypothesized that these SAMD7 variants might contribute to the retinal phenotype observed here, characterized by unusual, recognizable pigment deposits, differing from the classic spicular intraretinal pigmentation observed in other individuals homozygous for p.(Glu150Lys), and typically associated with RP in general.

Finally, a third type of IRD-associated hidden genetic variation that we studied in this project are CNVs. We mapped the genomic landscape of all RetNet genes in order to identify and prioritize those genes susceptible to CNV formation. Hereto, RetNet genes underwent an assessment of genomic features and of CNV occurrence in the Database of Genomic Variants (DGV) and in literature, revealing 1,345 literature-reported CNVs. Correlation analysis between rankings of genomic features and CNV occurrence demonstrated the strongest correlation between gene size and CNV occurrence in RetNet genes. Apart from this, we identified and delineated 30 CNVs in 10 RetNet genes (BEST1, EYS, KCNV2, MERTK, OPA1, PCDH15, PDE6G, PRPH2, SPATA7, and USH2A) in cases with IRD. Thirteen of these are novel, including a deletion in PDE6G, in which no CNVs have been reported before. Three of these, two in EYS and one in PCDH15, affect non-coding, putative cis-regulatory regions. The identified CNVs were characterized at nucleotide level to reveal the putative underlying mechanism, using Targeted Locus Amplification (TLA). This is a recently described technique based on the crosslinking of physically proximal sequences, that we applied for the first time on extracted genomic DNA to unravel six complex CNVs in a hypothesis-neutral manner. In addition, we designed a customized microarray, arrEYE, for high-resolution CNV analysis of known and candidate IRD genes and retina-expressed noncoding RNAs. The application of arrEYE on a test cohort of 57 IRD patients revealed novel CNVs in USH2A and RCBTB1, and the first reported CNV in HGSNAT, further supporting its recently identified role in non-syndromic IRD.