Molecular genetic analysis of retinitis pigmentosa in Indonesia using genome-wide homozygosity mapping

Abstract

Purpose: Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous retinal disorder. Despite tremendous knowledge about the genes involved in RP, little is known about the genetic causes of RP in Indonesia. Here, we aim to identify the molecular genetic causes underlying RP in a small cohort of Indonesian patients, using genome-wide homozygosity mapping.

Methods: DNA samples from affected and healthy individuals from 14 Indonesian families segregating autosomal recessive, X-linked, or isolated RP were collected. Homozygosity mapping was conducted using Illumina 6k or Affymetrix 5.0 single nucleotide polymorphism (SNP) arrays. Known autosomal recessive RP (arRP) genes residing in homozygous regions and X-linked RP genes were sequenced for mutations.

Results: In ten out of the 14 families, homozygous regions were identified that contained genes known to be involved in the pathogenesis of RP. Sequence analysis of these genes revealed seven novel homozygous mutations in ATP-binding cassette, sub-family A, member 4 (ABCA4), crumbs homolog 1 (CRB1), eyes shut homolog (Drosophila) (EYS), c-mer proto-oncogene tyrosine kinase (MERTK), nuclear receptor subfamily 2, group E, member 3 (NR2E3) and phosphodiesterase 6A, cGMP-specific, rod, alpha (PDE6A), all segregating in the respective families. No mutations were identified in the X-linked genes retinitis pigmentosa GTPase regulator (RPGR) and retinitis pigmentosa 2 (X-linked recessive; RP2).

Conclusions: Homozygosity mapping is a powerful tool to identify the genetic defects underlying RP in the Indonesian population. Compared to studies involving patients from other populations, the same genes appear to be implicated in the etiology of recessive RP in Indonesia, although all mutations that were discovered are novel and as such may be unique for this population.

Figure 1

Overview of the pedigree structure of the Indonesian families participating in this study. Affected individuals are indicated with filled symbols, whereas unaffected relatives are indicated by open symbols. Symbols with a slash depict deceased individuals. Probands are indicated with arrows, and individuals that were genotyped on genome-wide SNP arrays are marked with asterisks. Upon the identification of mutations in the probands (gene and mutation indicated below the pedigree), segregation analysis was performed in all available relatives, the results of which are indicated with M (mutated allele) or + (wild-type allele).

Figure 2

Overview of linkage plots for the three consanguineous Indonesian families. The graphs depict the linkage analysis results for three consanguineous Indonesian families, W09–0041 (A), W09–0042 (B) and W09–0046 (C) that were analyzed with Illumina 6k arrays. The highest LOD-score peaks were identified using EasyLinkage software. Peaks that correspond to the regions harboring the genes in which mutations were identified, are indicated with red arrows.

Figure 3

Molecular genetic analysis of MERTK in family W09–0038. A: In the upper panel, PCR analysis of exon 15 of MERTK is shown. Exon 15 was not amplified in the two affected individuals of family W09–0038. All relatives and their position in the pedigree are indicated above the electropherogram. Lower panel: after identification of the breakpoints of the complex rearrangement, PCR primers were designed to amplify a product spanning the deletion. A PCR product indicating the presence of a rearrangement is observed in all individuals demonstrating that the unaffected family members are heterozygous carriers of the deletion. B: Schematic representation of the complex rearrangement in MERTK. A deletion of a genomic region containing exon 15 is accompanied by a duplication and an inversion event.

Figure 4

Sequence comparison of amino acids mutated in Indonesian RP families. The mutated and flanking amino acids, from orthologous and homologous protein sequences, for A: CRB1 B: NR2E3 and C: EYS. The arrows indicate the position of the mutated amino acid residue in the alignment. Residues that are conserved in all protein sequences are depicted in white on a black background, whereas residues that are conserved in more than 50% of the analyzed sequences are indicated in black on a gray background.