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Homozygosity mapping and whole-genome sequencing reveals a deep intronic PROM1 mutation causing cone-rod dystrophy by pseudoexon activation.

ABSTRACT: Several genes have been implicated in the autosomal recessive form of cone-rod dystrophy (CRD), but the majority of cases remain unsolved. We identified a homozygous interval comprising two known genes associated with the autosomal recessive form of CRD, namely RAB28 and PROM1, in a consanguineous family with clinical evidence of CRD. Both genes proved to be mutation negative upon sequencing of exons and canonical splice sites but whole-genome sequencing revealed a private variant located deep in intron 18 of PROM1. In silico and functional analyses of this variant using minigenes as splicing reporters revealed the integration of a pseudoexon in the mutant transcript, thereby leading to a premature termination codon and presumably resulting in a functional null allele. This is the first report of a deep intronic variant that acts as a splicing mutation in PROM1. The detection of such variants escapes the exon-focused techniques typically used in genetic analyses. Sequencing the entire genomic regions of known disease genes might identify more causal mutations in the autosomal recessive form of CRD.

SUBMITTER: Mayer AK

PROVIDER: S-EPMC4755378 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Homozygosity mapping and whole-genome sequencing reveals a deep intronic PROM1 mutation causing cone-rod dystrophy by pseudoexon activation.

Mayer Anja K AK Rohrschneider Klaus K Strom Tim M TM Glöckle Nicola N Kohl Susanne S Wissinger Bernd B Weisschuh Nicole N

European journal of human genetics : EJHG 20150708 3

Several genes have been implicated in the autosomal recessive form of cone-rod dystrophy (CRD), but the majority of cases remain unsolved. We identified a homozygous interval comprising two known genes associated with the autosomal recessive form of CRD, namely RAB28 and PROM1, in a consanguineous family with clinical evidence of CRD. Both genes proved to be mutation negative upon sequencing of exons and canonical splice sites but whole-genome sequencing revealed a private variant located deep i ...[more]

PMID: 26153215

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Project description:To investigate the genetic basis for autosomal recessive cone-rod dystrophy (CRD) in a consanguineous Israeli Jewish family.Patients underwent a detailed ophthalmic evaluation, including eye examination, visual field testing, optical coherence tomography (OCT), and electrophysiological tests, electroretinography (ERG) and visual evoked potential (VEP). Genome-wide homozygosity mapping using a single nucleotide polymorphism (SNP) array was performed to identify homozygous regions shared among two of the affected individuals. Mutation screening of the underlying gene was performed with direct sequencing. In silico and in vitro analyses were used to predict the effect of the identified mutation on splicing.The affected family members are three siblings who have various degrees of progressive visual deterioration, glare, color vision abnormalities, and night vision difficulties. Visual field tests revealed central scotomas of different extension. Cone and rod ERG responses were reduced, with cones more severely affected. Homozygosity mapping revealed several homozygous intervals shared among two of the affected individuals. One included the PROM1 gene. Sequence analysis of the 26 coding exons of PROM1 in one affected individual revealed no mutations in the coding sequence or in intronic splice sites. However, in intron 21, proximate to the intron-exon junction, we observed a homozygous 10 bp deletion between positions -26 and -17 (c.2281-26_-17del). The deletion was linked to a known SNP, c.2281-6C>G. The deletion cosegregated with the disease in the family, and was not detected in public databases or in 101 ethnically-matched control individuals. In silico analysis predicted that this deletion would lead to altered intron 21 splicing. Bioinformatic analysis predicted that a recognition site for the SRSF2 splicing factor is located within the deleted sequence. The in vitro splicing assay demonstrated that c.2281-26_-17del leads to complete exon 22 skipping.A novel and unique intronic mutation of PROM1, underlying autosomal recessive CRD in a consanguineous Israeli family, was found. This report expands the spectrum of pathogenic mutations of PROM1 and further demonstrates the importance of intronic mutations.

Project description:To identify the causative mutation in a canine cone-rod dystrophy (crd3) that segregates as an adult onset disorder in the Glen of Imaal Terrier breed of dog.Glen of Imaal Terriers were ascertained for crd3 phenotype by clinical ophthalmoscopic examination, and in selected cases by electroretinography. Blood samples from affected cases and non-affected controls were collected and used, after DNA extraction, to undertake a genome-wide association study using Affymetrix Version 2 Canine single nucleotide polymorphism chips and 250K Sty Assay protocol. Positional candidate gene analysis was undertaken for genes identified within the peak-association signal region. Retinal morphology of selected crd3-affected dogs was evaluated by light and electron microscopy.A peak association signal exceeding genome-wide significance was identified on canine chromosome 16. Evaluation of genes in this region suggested A Disintegrin And Metalloprotease domain, family member 9 (ADAM9), identified concurrently elsewhere as the cause of human cone-rod dystrophy 9 (CORD9), as a strong positional candidate for canine crd3. Sequence analysis identified a large genomic deletion (over 20 kb) that removed exons 15 and 16 from the ADAM9 transcript, introduced a premature stop, and would remove critical domains from the encoded protein. Light and electron microscopy established that, as in ADAM9 knockout mice, the primary lesion in crd3 appears to be a failure of the apical microvilli of the retinal pigment epithelium to appropriately invest photoreceptor outer segments. By electroretinography, retinal function appears normal in very young crd3-affected dogs, but by 15 months of age, cone dysfunction is present. Subsequently, both rod and cone function degenerate.Identification of this ADAM9 deletion in crd3-affected dogs establishes this canine disease as orthologous to CORD9 in humans, and offers opportunities for further characterization of the disease process, and potential for genetic therapeutic intervention.

Dataset Information

Homozygosity mapping and whole-genome sequencing reveals a deep intronic PROM1 mutation causing cone-rod dystrophy by pseudoexon activation.

Publications

Homozygosity mapping and whole-genome sequencing reveals a deep intronic PROM1 mutation causing cone-rod dystrophy by pseudoexon activation.

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