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.

Long-term follow-up of a family with dominant X-linked retinitis pigmentosa.

PURPOSE: To document the progression of disease in male and female members of a previously described family with X-linked dominant retinitis pigmentosa (RP) caused by a de novo insertion after nucleotide 173 in exon ORF15 of RPGR. METHODS: The clinical records of 19 members of family UTAD054 were reviewed. Their evaluations consisted of confirmation of family history, standardised electroretinograms (ERGs), Goldmann visual fields, and periodic ophthalmological examinations over a 23-year period. RESULTS: Male members of family UTAD054 had non-recordable to barely recordable ERGs from early childhood. The males showed contracted central fields and developed more severe retinopathy than the females. The female members showed a disease onset delayed to teenage years, recordable but diminishing photopic and scotopic ERG amplitudes in a cone-rod pattern, progressive loss and often asymmetric visual fields, and diffuse atrophic retinopathy with fewer pigment deposits compared with males. CONCLUSIONS: This insertion mutation in the RPGR exon ORF15 is associated with a RP phenotype that severely affects males early and females by 30 years of age, and is highly penetrant in female members. Families with dominant-acting RPGR mutations may be mistaken to have an autosomal mode of inheritance resulting in an incorrect prediction of recurrence risk and prognosis. Broader recognition of X-linked RP forms with dominant inheritance is necessary to facilitate appropriate counselling of these patients.

Splice site mutation in the peripherin/RDS gene associated with pattern dystrophy of the retina.

PURPOSE: To report the phenotype and genotype of a splice site mutation at intron 2 of the peripherin/RDS gene in four half-siblings with pattern dystrophy of the retina. DESIGN: Experimental study. METHODS: In four siblings with a common mother and three separate fathers, complete ophthalmic examination, pedigree, electrophysiologic testing, and fluorescein angiography studies were obtained. Genomic DNA from serum lymphocytes was isolated and used as a template for primers specific for the cone homeobox gene (CRX), rhodopsin (RHO), and peripherin/RDS genes to conduct single stranded conformational analysis and cycle sequencing. RESULTS: The pedigree of four affected siblings suggested probable autosomal dominance transmission of pattern dystrophy. In the four siblings, best corrected visual acuity ranged from 20/20 to 20/80 by Snellen chart. Clinical findings included discrete, localized degenerative changes of the macular retinal pigment epithelium in all patients, with subclassification foveal. One patient exhibited pigment clumping within the atrophic areas. Another patient exhibited yellow flecks diffusely in the macula. Fluorescein angiographic findings included central hypofluorescence with a surrounding rim of hyperfluorescence that corresponded to the observed fundus lesions and window defects. There was a range of electroretinography (ERG) and electrooculography (EOG) findings. One patient demonstrated both cone and rod dysfunction on ERG testing and another demonstrated decreased rod function. EOG testing was normal in two patients and mildly diminished in one patient. DNA sequencing identified a point mutation in intron 2 of the peripherin/RDS gene, consisting of an A to T change at 1068+3, present in all four affected patients. CONCLUSIONS: Four siblings with pattern dystrophy of the retina presented a splice site mutation in the peripherin/RDS gene.

Comparative analysis of aryl-hydrocarbon receptor interacting protein-like 1 (Aipl1), a gene associated with inherited retinal disease in humans.

Mutations in AIPL1 cause Leber congenital amaurosis (LCA), the most severe form of inherited blindness in children; however, the function of this protein in normal vision remains unknown. To determine amino acid subsequences likely to be important for function, we have compared the protein sequence of several species. Sequence conservation is highest across the three Aipl1 tetratricopeptide (TPR) motifs and extends across the protein, except for a proline-rich amino acid sequence present only at the C-terminus of primate Aipl1. The length of the proline-rich region varies within primates; however, the length differences between human and primate Aipl1 do not correlate with evolutionary distance. These observations reinforce the importance of the TPR domains for function, the similarity of Aipl1 to a family of proteins that act as molecular chaperones, and the importance of comparative sequencing data for determination of whether AIPL1 sequence variants in patients are likely to cause retinopathy.

Autosomal dominant retinal degeneration and bone loss in patients with a 12-bp deletion in the CRX gene.

PURPOSE: To define the phenotypic expression of a deletion in the gene encoding the transcription factor CRX in a large, seven-generation, white family. METHODS: Fourteen affected individuals, all heterozygous for the Leu146del12 mutation in the cone-rod homeobox gene (CRX), and four nonaffected relatives from the same family were examined with visual function tests, and 10 underwent bone mineral density (BMD) measurement. RESULTS: The ability of the mutated CRX protein to transactivate rhodopsin promoter was decreased by approximately 25%, and its ability to react synergistically with neural retinal leucine zipper (NRL) was reduced by more than 30%. The affected members of the family had an autosomal dominant ocular condition most closely resembling Leber congenital amaurosis (LCA) with severe visual impairment at an early age. Depending on age, affected members showed varying degrees of significant visual acuity loss, elevated dark-adaptation thresholds, significantly reduced cone and rod electroretinogram (ERG) amplitudes, and progressive constriction of the visual fields, in most cases leading to complete blindness. Six affected members had reduced levels of BMD in the spine and the hip (osteopenia). Four affected female members who were receiving long-term hormonal replacement therapy (HRT) demonstrated normal values of BMD. CONCLUSIONS: This large deletion of the CRX gene is associated with a severe form of autosomal dominant retinal degeneration. Affected members not receiving HRT showed reduced BMD (osteopenia). This phenotype may reflect the abnormal influence of mutant CRX on both retinal and pineal development.

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.

Remapping of the RP15 locus for X-linked cone-rod degeneration to Xp11.4-p21.1, and identification of a de novo insertion in the RPGR exon ORF15.

X-linked forms of retinitis pigmentosa (XLRP) are among the most severe, because of their early onset, often leading to significant vision loss before the 4th decade. Previously, the RP15 locus was assigned to Xp22, by linkage analysis of a single pedigree with "X-linked dominant cone-rod degeneration." After clinical reevaluation of a female in this pedigree identified her as affected, we remapped the disease to a 19.5-cM interval (DXS1219-DXS993) at Xp11.4-p21.1. This new interval overlapped both RP3 (RPGR) and COD1. Sequencing of the previously published exons of RPGR revealed no mutations, but a de novo insertion was detected in the new RPGR exon, ORF15. The identification of an RPGR mutation in a family with a severe form of cone and rod degeneration suggests that RPGR mutations may encompass a broader phenotypic spectrum than has previously been recognized in "typical" retinitis pigmentosa.

Visual phenotype in patients with Arg41Gln and ala196+1bp mutations in the CRX gene.

Our aim was to describe the visual function characteristics of affected members from two unrelated families with different dominant mutations in the CRX gene. Standard full-field ERGs and high-intensity a-wave series were obtained. In addition, in most subjects, dark-adapted (DA) thresholds, color vision function (arrangement tests), and static perimetry were assessed. A point mutation in codon 41 of the CRX gene (Arg41Gln) was identified in family members from the RFS087 family who were tested on several occasions since 1983. Depending on age, affected members showed varying degrees of acuity loss, normal or slightly elevated DA thresholds, reduced cone a- and b-wave amplitudes, normal or minimally delayed cone b-wave implicit times, and normal rod and cone phototransduction gain parameters. An insertion mutation (Ala196+1bp) was found in two members of another family (RFS014). Affected members showed reduced visual acuity, normal or slightly elevated DA thresholds, relatively preserved rod ERG and substantially reduced or undetectable cone ERG, and normal rod phototransduction gain parameters. The Arg41Gln was associated with a late-onset, slowly progressing mild form of cone-rod dystrophy with cone loss but preserved rod and cone sensitivity until later in life. The Ala196+1bp mutation was associated with an early-onset, severe form of cone-rod dystrophy similar to that described in the original CORD2 family (Evans et al., Arch Ophthalmol 1995;113:195-201).

Prevalence of AIPL1 mutations in inherited retinal degenerative disease.

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophy and the most frequent cause of inherited blindness in children. LCA is usually inherited in an autosomal recessive fashion, although rare dominant cases have been reported. One form of LCA, LCA4, maps to chromosome 17p13 and is genetically distinct from other forms of LCA. We recently identified the gene associated with LCA4, AIPL1 (aryl-hydrocarbon interacting protein-like 1) and identified three mutations that were the cause of blindness in five families with LCA. In this study, AIPL1 was screened for mutations in 512 unrelated probands with a range of retinal degenerative diseases to determine if AIPL1 mutations cause other forms of inherited retinal degeneration and to determine the relative contribution of AIPL1 mutations to inherited retinal disorders in populations worldwide. We identified 11 LCA families whose retinal disorder is caused by homozygous or compound heterozygous AIPL1 mutations. We also identified affected individuals in two apparently dominant families, diagnosed with juvenile retinitis pigmentosa or dominant cone-rod dystrophy, respectively, who are heterozygous for a 12-bp AIPL1 deletion. Our results suggest that AIPL1 mutations cause approximately 7% of LCA worldwide and may cause dominant retinopathy.

Evaluation of human diacylglycerol kinase(iota), DGKI, a homolog of Drosophila rdgA, in inherited retinopathy mapping to 7q.

PURPOSE: To determine the genomic organization of diacylglycerol kinase(iota) and to test whether defects in this gene are present in individuals affected with autosomal dominant retinitis pigmentosa (adRP). Diacylglycerol kinase(iota) has been mapped to the RP10 locus on 7q and shows 49% sequence similarity to the Drosophila DGK2 rdgA gene. Since mutations in the DGK2 rdgA gene cause photoreceptor degeneration in Drosophila, it is possible that mutations in diacylglycerol kinase(iota) could be responsible for human retinal degeneration. METHODS: DNA sequence from genomic clones containing diacylglycerol kinase(iota) was compared with the cDNA sequence to identify intron/exon boundaries. Single-strand conformational analysis and PCR product sequencing were used to screen members of one family previously mapped to the RP10 locus and 47 small unmapped families with autosomal dominant retinitis pigmentosa. RESULTS: Diacylglycerol kinase(iota) is divided into 35 exons with the initiation codon being present in exon 2. Mutational analysis found a missense change (Lys153Phe) in three adRP families; however, it did not segregate with disease in one of the families. Silent substitutions were seen in codons 865 and 875. Intronic variation was detected in the amplifications of exons 3,5,18, 28, and 32; these do not affect splice site consensus sequences. Typing of a polymorphic variant detected in intron 31 in members of the RP10 family gave a LOD score of -4.2 at 0% recombination. CONCLUSIONS: No evidence of disease-associated mutations was found in any of the samples tested. Based on the linkage data and mutation screening, diacylglycerol kinase(iota) is excluded as a candidate for the RP10 form of adRP and cannot be a frequent cause of other forms of adRP. Mutations in diacylglycerol kinase(iota) may yet be the cause of recessive forms of retinal degeneration in humans, either in homozygous or compound heterozygous forms. The data provided here will permit testing of this hypothesis.

Mutations in a new photoreceptor-pineal gene on 17p cause Leber congenital amaurosis.

Leber congenital amaurosis (LCA, MIM 204000) accounts for at least 5% of all inherited retinal disease and is the most severe inherited retinopathy with the earliest age of onset. Individuals affected with LCA are diagnosed at birth or in the first few months of life with severely impaired vision or blindness, nystagmus and an abnormal or flat electroretinogram (ERG). Mutations in GUCY2D (ref. 3), RPE65 (ref. 4) and CRX (ref. 5) are known to cause LCA, but one study identified disease-causing GUCY2D mutations in only 8 of 15 families whose LCA locus maps to 17p13.1 (ref. 3), suggesting another LCA locus might be located on 17p13.1. Confirming this prediction, the LCA in one Pakistani family mapped to 17p13.1, between D17S849 and D17S960-a region that excludes GUCY2D. The LCA in this family has been designated LCA4 (ref. 6). We describe here a new photoreceptor/pineal-expressed gene, AIPL1 (encoding aryl-hydrocarbon interacting protein-like 1), that maps within the LCA4 candidate region and whose protein contains three tetratricopeptide (TPR) motifs, consistent with nuclear transport or chaperone activity. A homozygous nonsense mutation at codon 278 is present in all affected members of the original LCA4 family. AIPL1 mutations may cause approximately 20% of recessive LCA, as disease-causing mutations were identified in 3 of 14 LCA families not tested previously for linkage.

Mutations in the RP1 gene causing autosomal dominant retinitis pigmentosa.

Retinitis pigmentosa is a genetically heterogeneous form of retinal degeneration that affects approximately 1 in 3500 people worldwide. Recently we identified the gene responsible for the RP1 form of autosomal dominant retinitis pigmentosa (adRP) at 8q11-12 and found two different nonsense mutations in three families previously mapped to 8q. The RP1 gene is an unusually large protein, 2156 amino acids in length, but is comprised of four exons only. To determine the frequency and range of mutations in RP1 we screened probands from 56 large adRP families for mutations in the entire gene. After preliminary results indicated that mutations seem to cluster in a 442 nucleotide segment of exon 4, an additional 194 probands with adRP and 409 probands with other degenerative retinal diseases were tested for mutations in this region alone. We identified eight different disease-causing mutations in 17 of the 250 adRP probands tested. All of these mutations are either nonsense or frameshift mutations and lead to a severely truncated protein. Two of the eight different mutations, Arg677X and a 5 bp deletion of nucleotides 2280-2284, were reported previously, while the remaining six mutations are novel. We also identified two rare missense changes in two other families, one new polymorphic amino acid substitution, one silent substitution and a rare variant in the 5'-untranslated region that is not associated with disease. Based on this study, mutations in RP1 appear to cause at least 7% (17/250) of adRP. The 5 bp deletion of nucleotides 2280-2284 and the Arg677X nonsense mutation account for 59% (10/17) of these mutations. Further studies will determine whether missense changes in the RP1 gene are associated with disease, whether mutations in other regions of RP1 can cause forms of retinal disease other than adRP and whether the background variation in either the mutated or wild-type RP1 allele plays a role in the disease phenotype.

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.

Localization of retina/pineal-expressed sequences: identification of novel candidate genes for inherited retinal disorders.

More than 100 genes causing inherited retinal diseases have been mapped to chromosomal locations, but less than half of these genes have been cloned. Mutations in many retina/pineal-specific genes are known to cause inherited retinal diseases. Examples include mutations in arrestin, rhodopsin kinase, and the cone-rod homeobox gene, CRX. To identify additional candidate genes for inherited retinal disorders, novel retina/pineal-expressed EST clusters were identified from the TIGR Human Gene Index database and mapped to specific chromosomal sites. After known human gene sequences were excluded, and repeat sequences were masked, 26 novel retina and pineal gland cDNA clusters were identified. The retinal expression of each novel EST cluster was confirmed by PCR assay of a retinal cDNA library, and each cluster was localized in the genome using the GeneBridge 4.0 radiation hybrid panel. In silico expression data from the TIGR database suggest that these EST clusters are retina/pineal-specific or predominantly expressed in these tissues. This combination of database analysis and laboratory investigation has localized several EST clusters that are potential candidates for genes causing inherited retinopathy.

Identifying and mapping novel retinal-expressed ESTs from humans.

PURPOSE: The goal of this study was to develop efficient methods to identify tissue-specific expressed sequence tags (ESTs) and to map their locations in the human genome. Through a combination of database analysis and laboratory investigation, unique retina-specific ESTs were identified and mapped as candidate genes for inherited retinal diseases. METHODS: DNA sequences from retina-specific EST clusters were obtained from the TIGR Human Gene Index Database. Further processing of the EST sequence data was necessary to ensure that each EST cluster represented a novel, non-redundant mapping candidate. Processing involved screening for homologies to known genes and proteins using BLAST, excluding known human gene sequences and repeat sequences, and developing primers for PCR amplification of the gene encoding each cDNA cluster from genomic DNA. The EST clusters were mapped using the GeneBridge 4.0 Radiation Hybrid Mapping Panel with standard PCR conditions. RESULTS: A total of 83 retinal-expressed EST clusters were examined as potential novel, non-redundant mapping candidates. Fifty-five clusters were mapped successfully and their locations compared to the locations of known retinal disease genes. Fourteen EST clusters localize to candidate regions for inherited retinal diseases. CONCLUSIONS: This pilot study developed methodology for mapping uniquely expressed retinal ESTs and for identifying potential candidate genes for inherited retinal disorders. Despite the overall success, several complicating factors contributed to the high failure rate (33%) for mapping EST-clustered sequences. These include redundancy in the sequence data, widely dispersed sequences, ambiguous nucleotides within the sequences, the possibility of amplifying through introns and the presence of repetitive elements within the sequence. However, the combination of database analysis and laboratory mapping is a powerful method for identification of candidate genes for inherited diseases.

A range of clinical phenotypes associated with mutations in CRX, a photoreceptor transcription-factor gene.

Mutations in the retinal-expressed gene CRX (cone-rod homeobox gene) have been associated with dominant cone-rod dystrophy and with de novo Leber congenital amaurosis. However, CRX is a transcription factor for several retinal genes, including the opsins and the gene for interphotoreceptor retinoid binding protein. Because loss of CRX function could alter the expression of a number of other retinal proteins, we screened for mutations in the CRX gene in probands with a range of degenerative retinal diseases. Of the 294 unrelated individuals screened, we identified four CRX mutations in families with clinical diagnoses of autosomal dominant cone-rod dystrophy, late-onset dominant retinitis pigmentosa, or dominant congenital Leber amaurosis (early-onset retinitis pigmentosa), and we identified four additional benign sequence variants. These findings imply that CRX mutations may be associated with a wide range of clinical phenotypes, including congenital retinal dystrophy (Leber) and progressive diseases such as cone-rod dystrophy or retinitis pigmentosa, with a wide range of onset.

Linkage mapping of Thiel-Behnke corneal dystrophy (CDB2) to chromosome 10q23-q24.

Corneal dystrophy of the anterior basement membrane is a heterogeneous set of diseases characterized by painful, recurrent, bilateral erosions of the cornea, which often result in significant visual impairment. There are several similar but clinically distinct forms of anterior basement membrane/Bowman's membrane disease, including two autosomal dominant forms, Reis-Bücklers and Thiel-Behnke corneal dystrophy. Genes causing autosomal, nonsyndromic corneal dystrophy have been mapped to human chromosomes 1p, 5q, 12q, 16q, 17p, and 20p. Using microsatellite markers closely linked to the known corneal dystrophy loci, we excluded linkage between the known sites and the disease locus in a large, four-generation family with Thiel-Behnke corneal dystrophy. A genome-wide search using a panel of microsatellite markers demonstrated a maximum two-point lod score of 4.0 at 0% recombination between the disease locus in this family and the marker D10S1239, which maps to 10q23-q24. Testing with additional microsatellite markers from 10q places the disease locus between D10S677 and D10S1671, a distance of approximately 12.0 cM, with a maximum multipoint lod score of 5.5. Based on this evidence, we have identified another locus (CDB2) for corneal dystrophy of the anterior basement membrane/Bowman's membrane, Thiel-Behnke type, further demonstrating the exceptional genetic and phenotypic heterogeneity of these diseases.

Molecular analysis of the human vitamin D binding protein (group specific component, Gc) in tuberous sclerosis complex (TSC).

Group specific component (Gc) is an abundant plasma protein whose functional role is not clearly established. Gc protein is synthesised in the liver and is known to bind vitamin D, vitamin D metabolites, and G actin; Gc protein is also implicated in macrophage activation. Several polymorphic electrophoretic variants of Gc protein are found in all human populations; the most common alleles are Gc-1f, Gc-1s, and Gc-2. In previous studies, Gc allele frequencies, determined using isoelectric focusing or immunofixation or both, were significantly different in patients with tuberous sclerosis complex (TSC) from matched controls, with an excess of Gc-2 in patients. Linkage association between Gc and TSC is unlikely since the Gc locus maps to chromosome 4q12, whereas the two common forms of TSC map to 9q34 and 16p13.1, respectively. However, a direct cause and effect relationship between Gc protein and TSC symptoms is possible. To investigate further the relationship between the Gc locus and TSC, two Gc restriction site polymorphisms, HaeIII and StyI, were typed in 43 unrelated white subjects with TSC. The frequencies of the restriction site polymorphisms in the TSC patients did not differ from those in control populations. Therefore a direct association between Gc type and TSC is unlikely. The previously reported association was either spurious or the result of typing errors in plasma from subjects with underlying abnormalities in plasma proteins.