Sara J. Bowne

Prevalence of mutations causing retinitis pigmentosa and other inherited retinopathies

Inherited retinopathies are a genetically and phenotypically heterogeneous group of diseases affecting approximately one in 2000 individuals worldwide. For the past 10 years, the Laboratory for Molecular Diagnosis of Inherited Eye Diseases (LMDIED) at the University of Texas‐Houston Health Science Center has screened subjects ascertained in the United States and Canada for mutations in genes causing dominant and recessive autosomal retinopathies. A combination of single strand conformational analysis (SSCA) and direct sequencing of five genes (rhodopsin, peripherin/RDS, RP1, CRX, and AIPL1) identified the disease‐causing mutation in approximately one‐third of subjects with autosomal dominant retinitis pigmentosa (adRP) or with autosomal dominant cone‐rod dystrophy (adCORD). In addition, the causative mutation was identified in 15% of subjects with Leber congenital amaurosis (LCA). Overall, we report identification of the causative mutation in 105 of 506 (21%) of unrelated subjects (probands) tested; we report five previously unreported mutations in rhodopsin, two in peripherin/RDS, and one previously unreported mutation in the cone‐rod homeobox gene, CRX. Based on this large survey, the prevalence of disease‐causing mutations in each of these genes within specific disease categories is estimated. These data are useful in estimating the frequency of specific mutations and in selecting individuals and families for mutation‐specific studies. Hum Mutat 17:42–51, 2001.

Genes and mutations causing retinitis pigmentosa.

Retinitis pigmentosa (RP) is a heterogeneous set of inherited retinopathies with many disease‐causing genes, many known mutations, and highly varied clinical consequences. Progress in finding treatments is dependent on determining the genes and mutations causing these diseases, which includes both gene discovery and mutation screening in affected individuals and families. Despite the complexity, substantial progress has been made in finding RP genes and mutations. Depending on the type of RP, and the technology used, it is possible to detect mutations in 30–80% of cases. One of the most powerful approaches to genetic testing is high‐throughput ‘deep sequencing’, that is, next‐generation sequencing (NGS). NGS has identified several novel RP genes but a substantial fraction of previously unsolved cases have mutations in genes that are known causes of retinal disease but not necessarily RP. Apparent discrepancy between the molecular defect and clinical findings may warrant reevaluation of patients and families. In this review, we summarize the current approaches to gene discovery and mutation detection for RP, and indicate pitfalls and unsolved problems. Similar considerations apply to other forms of inherited retinal disease.

Mutations in a novel retina-specific gene cause autosomal dominant retinitis pigmentosa.

Inherited retinal diseases are a common cause of visual impairment in children and young adults, often resulting in severe loss of vision in later life. The most frequent form of inherited retinopathy is retinitis pigmentosa (RP), with an approximate incidence of 1 in 3,500 individuals worldwide. RP is characterized by night blindness and progressive degeneration of the midperipheral retina, accompanied by bone spicule-like pigmentary deposits and a reduced or absent electroretinogram (ERG). The disease process culminates in severe reduction of visual fields or blindness. RP is genetically heterogeneous, with autosomal dominant, autosomal recessive and X-linked forms. Here we have identified two mutations in a novel retina-specific gene from chromosome 8q that cause the RP1 form of autosomal dominant RP in three unrelated families. The protein encoded by this gene is 2,156 amino acids and its function is currently unknown, although the amino terminus has similarity to that of the doublecortin protein, whose gene (DCX) has been implicated in lissencephaly in humans. Two families have a nonsense mutation in codon 677 of this gene (Arg677stop), whereas the third family has a nonsense mutation in codon 679 (Gln679stop). In one family, two individuals homozygous for the mutant gene have more severe retinal disease compared with heterozygotes.

Next generation sequencing-based molecular diagnosis of retinitis pigmentosa: identification of a novel genotype-phenotype correlation and clinical refinements

Abstract Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical diagnosis of RP due to its high genetic and clinical heterogeneity. Using a gene capture panel that covers 163 of the currently known retinal disease genes, including 48 RP genes, we performed a comprehensive molecular screening in a collection of 123 RP unsettled probands from a wide variety of ethnic backgrounds, including 113 unrelated simplex and 10 autosomal recessive RP (arRP) cases. As a result, 61 mutations were identified in 45 probands, including 38 novel pathogenic alleles. Interestingly, we observed that phenotype and genotype were not in full agreement in 21 probands. Among them, eight probands were clinically reassessed, resulting in refinement of clinical diagnoses for six of these patients. Finally, recessive mutations in CLN3 were identified in five retinal degeneration patients, including four RP probands and one cone-rod dystrophy patient, suggesting that CLN3 is a novel non-syndromic retinal disease gene. Collectively, our results underscore that, due to the high molecular and clinical heterogeneity of RP, comprehensive screening of all retinal disease genes is effective in identifying novel pathogenic mutations and provides an opportunity to discover new genotype-phenotype correlations. Information gained from this genetic screening will directly aid in patient diagnosis, prognosis, and treatment, as well as allowing appropriate family planning and counseling.

A dominant mutation in RPE65 identified by whole-exome sequencing causes retinitis pigmentosa with choroidal involvement

Linkage testing using Affymetrix 6.0 SNP Arrays mapped the disease locus in TCD-G, an Irish family with autosomal dominant retinitis pigmentosa (adRP), to an 8.8 Mb region on 1p31. Of 50 known genes in the region, 11 candidates, including RPE65 and PDE4B, were sequenced using di-deoxy capillary electrophoresis. Simultaneously, a subset of family members was analyzed using Agilent SureSelect All Exome capture, followed by sequencing on an Illumina GAIIx platform. Candidate gene and exome sequencing resulted in the identification of an Asp477Gly mutation in exon 13 of the RPE65 gene tracking with the disease in TCD-G. All coding exons of genes not sequenced to sufficient depth by next generation sequencing were sequenced by di-deoxy sequencing. No other potential disease-causing variants were found to segregate with disease in TCD-G. The Asp477Gly mutation was not present in Irish controls, but was found in a second Irish family provisionally diagnosed with choroideremia, bringing the combined maximum two-point LOD score to 5.3. Mutations in RPE65 are a known cause of recessive Leber congenital amaurosis (LCA) and recessive RP, but no dominant mutations have been reported. Protein modeling suggests that the Asp477Gly mutation may destabilize protein folding, and mutant RPE65 protein migrates marginally faster on SDS-PAGE, compared with wild type. Gene therapy for LCA patients with RPE65 mutations has shown great promise, raising the possibility of related therapies for dominant-acting mutations in this gene.

A splice-site mutation in a retina-specific exon of BBS8 causes nonsyndromic retinitis pigmentosa.

Tissue-specific alternative splicing is an important mechanism for providing spatiotemporal protein diversity. Here we show that an in-frame splice mutation in BBS8, one of the genes involved in pleiotropic Bardet-Biedl syndrome (BBS), is sufficient to cause nonsyndromic retinitis pigmentosa (RP). A genome-wide scan of a consanguineous RP pedigree mapped the trait to a 5.6 Mb region; subsequent systematic sequencing of candidate transcripts identified a homozygous splice-site mutation in a previously unknown BBS8 exon. The allele segregated with the disorder, was absent from controls, was completely invariant across evolution, and was predicted to lead to the elimination of a 10 amino acid sequence from the protein. Subsequent studies showed the exon to be expressed exclusively in the retina and enriched significantly in the photoreceptor layer. Importantly, we found this exon to represent the major BBS8 mRNA species in the mammalian photoreceptor, suggesting that the encoded 10 amino acids play a pivotal role in the function of BBS8 in this organ. Understanding the role of this additional sequence might therefore inform the mechanism of retinal degeneration in patients with syndromic BBS or other related ciliopathies.

Mutations in a BTB-Kelch Protein, KLHL7, Cause Autosomal-Dominant Retinitis Pigmentosa

Retinitis pigmentosa (RP) refers to a genetically heterogeneous group of progressive neurodegenerative diseases that result in dysfunction and/or death of rod and cone photoreceptors in the retina. So far, 18 genes have been identified for autosomal-dominant (ad) RP. Here, we describe an adRP locus (RP42) at chromosome 7p15 through linkage analysis in a six-generation Scandinavian family and identify a disease-causing mutation, c.449G-->A (p.S150N), in exon 6 of the KLHL7 gene. Mutation screening of KLHL7 in 502 retinopathy probands has revealed three different missense mutations in six independent families. KLHL7 is widely expressed, including expression in rod photoreceptors, and encodes a 75 kDa protein of the BTB-Kelch subfamily within the BTB superfamily. BTB-Kelch proteins have been implicated in ubiquitination through Cullin E3 ligases. Notably, all three putative disease-causing KLHL7 mutations are within a conserved BACK domain; homology modeling suggests that mutant amino acid side chains can potentially fill the cleft between two helices, thereby affecting the ubiquitination complexes. Mutations in an identical region of another BTB-Kelch protein, gigaxonin, have previously been associated with giant axonal neuropathy. Our studies suggest an additional role of the ubiquitin-proteasome protein-degradation pathway in maintaining neuronal health and in disease.

Allelic Heterogeneity and Genetic Modifier Loci Contribute to Clinical Variation in Males with X-Linked Retinitis Pigmentosa Due to RPGR Mutations

Mutations in RPGR account for over 70% of X-linked retinitis pigmentosa (XlRP), characterized by retinal degeneration and eventual blindness. The clinical consequences of RPGR mutations are highly varied, even among individuals with the same mutation: males demonstrate a wide range of clinical severity, and female carriers may or may not be affected. This study describes the phenotypic diversity in a cohort of 98 affected males from 56 families with RPGR mutations, and demonstrates the contribution of genetic factors (i.e., allelic heterogeneity and genetic modifiers) to this diversity. Patients were categorized as grade 1 (mild), 2 (moderate) or 3 (severe) according to specific clinical criteria. Patient DNAs were genotyped for coding SNPs in 4 candidate modifier genes with products known to interact with RPGR protein: RPGRIP1, RPGRIP1L, CEP290, and IQCB1. Family-based association testing was performed using PLINK. A wide range of clinical severity was observed both between and within families. Patients with mutations in exons 1–14 were more severely affected than those with ORF15 mutations, and patients with predicted null alleles were more severely affected than those predicted to make RPGR protein. Two SNPs showed association with severe disease: the minor allele (N) of I393N in IQCB1 (p = 0.044) and the common allele (R) of R744Q in RPGRIP1L (p = 0.049). These data demonstrate that allelic heterogeneity contributes to phenotypic diversity in XlRP and suggest that this may depend on the presence or absence of RPGR protein. In addition, common variants in 2 proteins known to interact with RPGR are associated with severe disease in this cohort.

Essential and Synergistic Roles of RP1 and RP1L1 in Rod Photoreceptor Axoneme and Retinitis Pigmentosa

Retinitis pigmentosa 1 (RP1) is a common inherited retinopathy with variable onset and severity. The RP1 gene encodes a photoreceptor-specific, microtubule-associated ciliary protein containing the doublecortin (DCX) domain. Here we show that another photoreceptor-specific Rp1-like protein (Rp1L1) in mice is also localized to the axoneme of outer segments (OSs) and connecting cilia in rod photoreceptors, overlapping with Rp1. Rp1L1−/− mice display scattered OS disorganization, reduced electroretinogram amplitudes, and progressive photoreceptor degeneration, less severe and slower than in Rp1−/− mice. In single rods of Rp1L1−/−, photosensitivity is reduced, similar to that of Rp1−/−. While individual heterozygotes are normal, double heterozygotes of Rp1 and Rp1L1 exhibit abnormal OS morphology and reduced single rod photosensitivity and dark currents. The electroretinogram amplitudes of double heterozygotes are more reduced than those of individual heterozygotes combined. In support, Rp1L1 interacts with Rp1 in transfected cells and in retina pull-down experiments. Interestingly, phototransduction kinetics are normal in single rods and whole retinas of individual or double Rp1 and Rp1L1 mutant mice. Together, Rp1 and Rp1L1 play essential and synergistic roles in affecting photosensitivity and OS morphogenesis of rod photoreceptors. Our findings suggest that mutations in RP1L1 could underlie retinopathy or modify RP1 disease expression in humans.

IMP Dehydrogenase Type 1 Associates with Polyribosomes Translating Rhodopsin mRNA

IMP dehydrogenase (IMPDH) catalyzes the pivotal step in guanine nucleotide biosynthesis. Here we show that both IMPDH type 1 (IMPDH1) and IMPDH type 2 are associated with polyribosomes, suggesting that these housekeeping proteins have an unanticipated role in translation regulation. This interaction is mediated by the subdomain, a region of disputed function that is the site of mutations that cause retinal degeneration. The retinal isoforms of IMPDH1 also associate with polyribosomes. The most common disease-causing mutation, D226N, disrupts the polyribosome association of at least one retinal IMPDH1 isoform. Finally, we find that IMPDH1 is associated with polyribosomes containing rhodopsin mRNA. Because any perturbation of rhodopsin expression can trigger apoptosis in photoreceptor cells, these observations suggest a likely pathological mechanism for IMPDH1-mediated hereditary blindness. We propose that IMPDH coordinates the translation of a set of mRNAs, perhaps by modulating localization or degradation.