History of Finding Genes and Mutations Causing Inherited Retinal Diseases.

This is a brief history of the work by many investigators throughout the world to find genes and mutations causing inherited retinal diseases (IRDs). It largely covers 40 years, from the late-1980s through today. Perhaps the best reason to study history is to better understand the present. The "present" for IRDs is exceptionally complex. Mutations in hundreds of genes are known to cause IRDs; tens of thousands of disease-causing mutations have been reported; clinical consequences are highly variable, even within the same family; and genetic testing, counseling, and clinical care are highly advanced but technically challenging. The aim of this review is to account for how we have come to know and understand, at least partly, this complexity.

Overcoming the Challenges to Clinical Development of X-Linked Retinitis Pigmentosa Therapies: Proceedings of an Expert Panel.

X-linked retinitis pigmentosa (XLRP) is a rare inherited retinal disease manifesting as impaired night vision and peripheral vision loss that progresses to legal blindness. Although several trials of ocular gene therapy for XLRP have been conducted or are in progress, there is currently no approved treatment. In July 2022, the Foundation Fighting Blindness convened an expert panel to examine relevant research and make recommendations for overcoming the challenges and capitalizing on the opportunities in conducting clinical trials of RPGR-targeted therapy for XLRP. Data presented concerned RPGR structure and mutation types known to cause XLRP, RPGR mutation-associated retinal phenotype diversity, patterns in genotype/phenotype relationships, disease onset and progression from natural history studies, and the various functional and structural tests used to monitor disease progression. Panel recommendations include considerations, such as genetic screening and other factors that can impact clinical trial inclusion criteria, the influence of age on defining and stratifying participant cohorts, the importance of conducting natural history studies early in clinical development programs, and the merits and drawbacks of available tests for measuring treatment outcomes. We recognize the need to work with regulators to adopt clinically meaningful end points that would best determine the efficacy of a trial. Given the promise of RPGR-targeted gene therapy for XLRP and the difficulties encountered in phase III clinical trials to date, we hope these recommendations will help speed progress to finding a cure. Translational Relevance: Examination of relevant data and recommendations for the successful clinical development of gene therapies for RPGR-associated XLRP.

Identification of a novel large multigene deletion and a frameshift indel in PDE6B as the underlying cause of early-onset recessive rod-cone degeneration.

A family, with two affected identical twins with early-onset recessive inherited retinal degeneration, was analyzed to determine the underlying genetic cause of pathology. Exome sequencing revealed a rare and previously reported causative variant (c.1923_1969delinsTCTGGG; p.Asn643Glyfs*29) in the PDE6B gene in the affected twins and their unaffected father. Further investigation, using genome sequencing, identified a novel ∼7.5-kb deletion (Chr 4:670,405-677,862del) encompassing the ATP5ME gene, part of the 5' UTR of MYL5, and a 378-bp (Chr 4:670,405-670,782) region from the 3' UTR of PDE6B in the affected twins and their unaffected mother. Both variants segregated with disease in the family. Analysis of the relative expression of PDE6B, in peripheral blood cells, also revealed a significantly lower level of PDE6B transcript in affected siblings compared to a normal control. PDE6B is associated with recessive rod-cone degeneration and autosomal dominant congenital stationary night blindness. Ophthalmic evaluation of these patients showed night blindness, fundus abnormalities, and peripheral vision loss, which are consistent with PDE6B-associated recessive retinal degeneration. These findings suggest that the loss of PDE6B transcript resulting from the compound heterozygous pathogenic variants is the underlying cause of recessive rod-cone degeneration in the study family.

Mutations in the Kinesin-2 Motor KIF3B Cause an Autosomal-Dominant Ciliopathy.

Kinesin-2 enables ciliary assembly and maintenance as an anterograde intraflagellar transport (IFT) motor. Molecular motor activity is driven by a heterotrimeric complex comprised of KIF3A and KIF3B or KIF3C plus one non-motor subunit, KIFAP3. Using exome sequencing, we identified heterozygous KIF3B variants in two unrelated families with hallmark ciliopathy phenotypes. In the first family, the proband presents with hepatic fibrosis, retinitis pigmentosa, and postaxial polydactyly; he harbors a de novo c.748G>C (p.Glu250Gln) variant affecting the kinesin motor domain encoded by KIF3B. The second family is a six-generation pedigree affected predominantly by retinitis pigmentosa. Affected individuals carry a heterozygous c.1568T>C (p.Leu523Pro) KIF3B variant segregating in an autosomal-dominant pattern. We observed a significant increase in primary cilia length in vitro in the context of either of the two mutations while variant KIF3B proteins retained stability indistinguishable from wild type. Furthermore, we tested the effects of KIF3B mutant mRNA expression in the developing zebrafish retina. In the presence of either missense variant, rhodopsin was sequestered to the photoreceptor rod inner segment layer with a concomitant increase in photoreceptor cilia length. Notably, impaired rhodopsin trafficking is also characteristic of recessive KIF3B models as exemplified by an early-onset, autosomal-recessive, progressive retinal degeneration in Bengal cats; we identified a c.1000G>A (p.Ala334Thr) KIF3B variant by genome-wide association study and whole-genome sequencing. Together, our genetic, cell-based, and in vivo modeling data delineate an autosomal-dominant syndromic retinal ciliopathy in humans and suggest that multiple KIF3B pathomechanisms can impair kinesin-driven ciliary transport in the photoreceptor.

X-Chromosome Inactivation Is a Biomarker of Clinical Severity in Female Carriers of RPGR-Associated X-Linked Retinitis Pigmentosa.

PURPOSE: X-linked retinitis pigmentosa can manifest in female carriers with widely variable severity, whereas others remain unaffected. The contribution of X-chromosome inactivation (XCI) to phenotypic variation has been postulated but not demonstrated. Furthermore, the impact of genotype and genetic modifiers has been demonstrated in affected males but has not been well established in female carriers. The purpose of this study was to describe the scope of clinical phenotype in female carriers with mutations in RPGR and quantify the contribution of genotype, genetic modifiers, and XCI to phenotypic severity. DESIGN: Cohort study. PARTICIPANTS: Seventy-seven female carriers with RPGR mutations from 41 pedigrees. METHODS: Coding single nucleotide polymorphisms were sequenced in candidate genetic modifier genes encoding known RPGR-interacting proteins. X-chromosome inactivation ratios were determined in genomic DNA isolated from blood (n = 42) and saliva (n = 20) using methylation status of X-linked polymorphic repeats. These genetic data were compared with disease severity based on quantitative clinical parameters. MAIN OUTCOME MEASURES: Visual acuity, Humphrey visual field (HVF) results, full-field electroretinography results, and dark adaptation. RESULTS: Most individuals at all ages were mildly affected or unaffected, whereas those who progressed to moderate or severe vision loss were older than 30 years. RPGR genotype was not associated with clinical severity. The D1264N variant in RPGRIP1L was associated with more severe disease. Skewed XCI toward inactivation of the normal RPGR allele was associated with more severe disease. The XCI ratio in both blood and saliva was a predictor of visual function as measured by HVF diameter, rod amplitude, flicker amplitude, and flicker implicit time. For carriers with extreme XCI skewing of 80:20 or more, 57% were affected severely compared with 8% for those with XCI of less than 80:20 (P = 0.002). CONCLUSIONS: Female carriers with mutations in RPGR demonstrate widely variable clinical severity. X-chromosome inactivation ratios correlate with clinical severity and may serve as a predictor of clinically significant disease. Because RPGR gene therapy trials are underway, a future imperative exists to determine which carriers require intervention and when to intervene. X-chromosome inactivation analysis may be useful for identifying candidates for early intervention.

Detection of Large Structural Variants Causing Inherited Retinal Diseases.

Current application of next-generation sequencing (NGS) leads to detection of the underlying disease-causing gene and mutation or mutations in from 60% to 85% of patients with inherited retinal diseases (IRDs), depending on the methods used, disease type, and population tested. In a cohort of 320 families with autosomal dominant retinitis pigmentosa (adRP), we have detected the mutation in 82% of cases using a variety of methods, leaving more than 50 families with "elusive" disease genotypes. All of the remaining families have been screened for mutations in known IRD genes using retinal-targeted-capture NGS, and most have been tested by whole-exome NGS. Linkage mapping has been conducted in several large families. In one of these families, with DNA samples from ten affected family members and six unaffected, linking members, we observed substantial maximum two-point LOD scores for linkage to both chromosomes 2 and 4. Subsequent 10X Genomics Chromium™ sequencing, which facilitates linked-read, phase-known chromosomal analysis, revealed a balanced translocation of the q terminus arms of chromosomes 2 and 4 involving 35 Mb and 73 Mb of 2 and 4, respectively. The balanced translocation is present in all affected family members and absent from all unaffected individuals. Family histories suggest multiple miscarriages are associated with the translocation. The breakpoint on chromosome 4 is within or 5' to the LRAT gene, whereas the chromosome 2 break is in a gene-poor region. We conclude that the balanced translocation is the cause of adRP in this family, which may lead to dysregulation of the LRAT gene. Since multiple miscarriages are a hallmark of balanced translocations, this possibility should be considered in evaluating family histories. Further, large structural variants, which are not easily detected by conventional sequencing methods, may account for a significant fraction of the remaining unsolved families.

Time Course of Disease Progression of PRPF31-mediated Retinitis Pigmentosa.

PURPOSE: Variants in PRPF31, a splicing factor, are a common cause of autosomal dominant retinitis pigmentosa (RP). Deleterious variants are thought to cause disease by haploinsufficiency. In anticipation of upcoming replacement gene therapy trials, we present the phenotype and clinical progression of a large cohort of patients with PRPF31-mediated RP. DESIGN: Cross-sectional with retrospective review. METHODS: A total of 26 patients with RP and 5 asymptomatic individuals, all with deleterious variants in PRPF31 (from 13 families), were selected from our database of patients followed longitudinally. Ages ranged from 9 to 77 years (mean 47 years), with an average follow-up time of 16 years. All patients underwent ophthalmic examination including psychophysical tests, electrophysiology, and imaging. All available records were reviewed retrospectively. Additionally, all patients were contacted, and all available patients (n = 7) were examined in an additional prospective follow-up visit. RESULTS: Age of onset ranged from 6 to 71 years, without apparent relationship to specific variant. Two adults (aged 42 and 77 years) and 3 teenaged children were found to harbor a mutation with no evidence of RP. In those with RP, visual field area (spot size III) declined exponentially at a rate of 8.1% per year of disease duration (P < .001, 95% confidence interval [CI] 5.6-10.6), cone electroretinogram amplitude declined exponentially at a rate of 7.3% per year of disease duration (P < .001, 95% CI 5.4-9.1), and ellipsoid zone area declined exponentially at a rate of 5.4% per year of disease duration (P < .001, 95% CI 3.7-7.1). CONCLUSIONS: PRPF31-mediated retinitis pigmentosa is characterized by a variable age of onset. Once disease develops, it follows a predictable exponential time course.

Molecular Findings in Families with an Initial Diagnose of Autosomal Dominant Retinitis Pigmentosa (adRP).

Genetic testing of probands in families with an initial diagnosis of autosomal dominant retinitis pigmentosa (adRP) usually confirms the diagnosis, but there are exceptions. We report results of genetic testing in a large cohort of adRP families with an emphasis on exceptional cases including X-linked RP with affected females; homozygous affected individuals in families with heterozygous, dominant disease; and independently segregating mutations in the same family. Genetic testing was conducted in more than 700 families with a provisional or probable diagnosis of adRP. Exceptions to the proposed mode of inheritance were extracted from our comprehensive patient and family database. In a subset of 300 well-characterized families with a probable diagnosis of adRP, 195 (70%) have dominant mutations in known adRP genes but 25 (8%) have X-linked mutations, 3 (1%) have multiple segregating mutations, and 3 (1%) have dominant-acting mutations in genes previously associated with recessive disease. It is currently possible to determine the underlying disease-causing gene and mutation in approximately 80% of families with an initial diagnosis of adRP, but 10% of "adRP" families have a variant mode of inheritance. Informed genetic diagnosis requires close collaboration between clinicians, genetic counselors, and laboratory scientists.

Molecular Defects of the Disease-Causing Human Arrestin-1 C147F Mutant.

Purpose: The purpose of this study was to identify the molecular defect in the disease-causing human arrestin-1 C147F mutant. Methods: The binding of wild-type (WT) human arrestin-1 and several mutants with substitutions in position 147 (including C147F, which causes dominant retinitis pigmentosa in humans) to phosphorylated and unphosphorylated light-activated rhodopsin was determined. Thermal stability of WT and mutant human arrestin-1, as well as unfolded protein response in 661W cells, were also evaluated. Results: WT human arrestin-1 was selective for phosphorylated light-activated rhodopsin. Substitutions of Cys-147 with smaller side chain residues, Ala or Val, did not substantially affect binding selectivity, whereas residues with bulky side chains in the position 147 (Ile, Leu, and disease-causing Phe) greatly increased the binding to unphosphorylated rhodopsin. Functional survival of mutant proteins with bulky substitutions at physiological and elevated temperature was also compromised. C147F mutant induced unfolded protein response in cultured cells. Conclusions: Bulky Phe substitution of Cys-147 in human arrestin-1 likely causes rod degeneration due to reduced stability of the protein, which induces unfolded protein response in expressing cells.

Next-generation sequencing to solve complex inherited retinal dystrophy: A case series of multiple genes contributing to disease in extended families.

PURPOSE: With recent availability of next-generation sequencing (NGS), it is becoming more common to pursue disease-targeted panel testing rather than traditional sequential gene-by-gene dideoxy sequencing. In this report, we describe using NGS to identify multiple disease-causing mutations that contribute concurrently or independently to retinal dystrophy in three relatively small families. METHODS: Family members underwent comprehensive visual function evaluations, and genetic counseling including a detailed family history. A preliminary genetic inheritance pattern was assigned and updated as additional family members were tested. Family 1 (FAM1) and Family 2 (FAM2) were clinically diagnosed with retinitis pigmentosa (RP) and had a suspected autosomal dominant pedigree with non-penetrance (n.p.). Family 3 (FAM3) consisted of a large family with a diagnosis of RP and an overall dominant pedigree, but the proband had phenotypically cone-rod dystrophy. Initial genetic analysis was performed on one family member with traditional Sanger single gene sequencing and/or panel-based testing, and ultimately, retinal gene-targeted NGS was required to identify the underlying cause of disease for individuals within the three families. Results obtained in these families necessitated further genetic and clinical testing of additional family members to determine the complex genetic and phenotypic etiology of each family. RESULTS: Genetic testing of FAM1 (n = 4 affected; 1 n.p.) identified a dominant mutation in RP1 (p.Arg677Ter) that was present for two of the four affected individuals but absent in the proband and the presumed non-penetrant individual. Retinal gene-targeted NGS in the fourth affected family member revealed compound heterozygous mutations in USH2A (p. Cys419Phe, p.Glu767Serfs*21). Genetic testing of FAM2 (n = 3 affected; 1 n.p.) identified three retinal dystrophy genes (PRPH2, PRPF8, and USH2A) with disease-causing mutations in varying combinations among the affected family members. Genetic testing of FAM3 (n = 7 affected) identified a mutation in PRPH2 (p.Pro216Leu) tracking with disease in six of the seven affected individuals. Additional retinal gene-targeted NGS testing determined that the proband also harbored a multiple exon deletion in the CRX gene likely accounting for her cone-rod phenotype; her son harbored only the mutation in CRX, not the familial mutation in PRPH2. CONCLUSIONS: Multiple genes contributing to the retinal dystrophy genotypes within a family were discovered using retinal gene-targeted NGS. Families with noted examples of phenotypic variation or apparent non-penetrant individuals may offer a clue to suspect complex inheritance. Furthermore, this finding underscores that caution should be taken when attributing a single gene disease-causing mutation (or inheritance pattern) to a family as a whole. Identification of a disease-causing mutation in a proband, even with a clear inheritance pattern in hand, may not be sufficient for targeted, known mutation analysis in other family members.

A Novel Dominant Mutation in SAG, the Arrestin-1 Gene, Is a Common Cause of Retinitis Pigmentosa in Hispanic Families in the Southwestern United States.

Purpose: To identify the causes of autosomal dominant retinitis pigmentosa (adRP) in a cohort of families without mutations in known adRP genes and consequently to characterize a novel dominant-acting missense mutation in SAG. Methods: Patients underwent ophthalmologic testing and were screened for mutations using targeted-capture and whole-exome next-generation sequencing. Confirmation and additional screening were done by Sanger sequencing. Haplotypes segregating with the mutation were determined using short tandem repeat and single nucleotide variant polymorphisms. Genealogies were established by interviews of family members. Results: Eight families in a cohort of 300 adRP families, and four additional families, were found to have a novel heterozygous mutation in the SAG gene, c.440G>T; p.Cys147Phe. Patients exhibited symptoms of retinitis pigmentosa and none showed symptoms characteristic of Oguchi disease. All families are of Hispanic descent and most were ascertained in Texas or California. A single haplotype including the SAG mutation was identified in all families. The mutation dramatically alters a conserved amino acid, is extremely rare in global databases, and was not found in 4000+ exomes from Hispanic controls. Molecular modeling based on the crystal structure of bovine arrestin-1 predicts protein misfolding/instability. Conclusions: This is the first dominant-acting mutation identified in SAG, a founder mutation possibly originating in Mexico several centuries ago. The phenotype is clearly adRP and is distinct from the previously reported phenotypes of recessive null mutations, that is, Oguchi disease and recessive RP. The mutation accounts for 3% of the 300 families in the adRP Cohort and 36% of Hispanic families in this cohort.

Diagnosis of a mild peroxisomal phenotype with next-generation sequencing.

Peroxisomal biogenesis disorders (PBD) are caused by mutations in PEX genes, and are typically diagnosed with biochemical testing in plasma followed by confirmatory testing. Here we report the unusual diagnostic path of a child homozygous for PEX1 p.G843D. The patient presented with sensorineural hearing loss, pigmentary retinopathy, and normal intellect. After testing for Usher syndrome was negative, he was found to have PBD through a research sequencing panel. When evaluating a patient with hearing loss and pigmentary retinopathy, mild PBD should be on the differential regardless of cognitive function.

North Carolina macular dystrophy (MCDR1) caused by a novel tandem duplication of the PRDM13 gene.

PURPOSE: To identify the underlying cause of disease in a large family with North Carolina macular dystrophy (NCMD). METHODS: A large four-generation family (RFS355) with an autosomal dominant form of NCMD was ascertained. Family members underwent comprehensive visual function evaluations. Blood or saliva from six affected family members and three unaffected spouses was collected and DNA tested for linkage to the MCDR1 locus on chromosome 6q12. Three affected family members and two unaffected spouses underwent whole exome sequencing (WES) and subsequently, custom capture of the linkage region followed by next-generation sequencing (NGS). Standard PCR and dideoxy sequencing were used to further characterize the mutation. RESULTS: Of the 12 eyes examined in six affected individuals, all but two had Gass grade 3 macular degeneration features. Large central excavation of the retinal and choroid layers, referred to as a macular caldera, was seen in an age-independent manner in the grade 3 eyes. The calderas are unique to affected individuals with MCDR1. Genome-wide linkage mapping and haplotype analysis of markers from the chromosome 6q region were consistent with linkage to the MCDR1 locus. Whole exome sequencing and custom-capture NGS failed to reveal any rare coding variants segregating with the phenotype. Analysis of the custom-capture NGS sequencing data for copy number variants uncovered a tandem duplication of approximately 60 kb on chromosome 6q. This region contains two genes, CCNC and PRDM13. The duplication creates a partial copy of CCNC and a complete copy of PRDM13. The duplication was found in all affected members of the family and is not present in any unaffected members. The duplication was not seen in 200 ethnically matched normal chromosomes. CONCLUSIONS: The cause of disease in the original family with MCDR1 and several others has been recently reported to be dysregulation of the PRDM13 gene, caused by either single base substitutions in a DNase 1 hypersensitive site upstream of the CCNC and PRDM13 genes or a tandem duplication of the PRDM13 gene. The duplication found in the RFS355 family is distinct from the previously reported duplication and provides additional support that dysregulation of PRDM13, not CCNC, is the cause of NCMD mapped to the MCDR1 locus.

Multimodal Imaging in Wagner Syndrome.

Wagner syndrome is a rare vitreoretinopathy described in a limited number of families. Here the authors describe four cases of suspected Wagner syndrome. All four cases had depressed rod and cone function on electroretinography, outer retinal disruption on spectral-domain optical coherence tomography, and constricted central visual fields with smaller isopter testing. Fundus autofluorescence performed in one patient highlighted a perivascular pattern to chorioretinal atrophy. Two patients had a history of uveitis with active cystoid macular edema. The diagnosis of Wagner syndrome was supported in three cases with genetic testing for VCAN mutations, whereas the other case harbored a variation of unknown significance in VCAN that may have been nonpathogenic. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:574-579.].

De Novo Occurrence of a Variant in ARL3 and Apparent Autosomal Dominant Transmission of Retinitis Pigmentosa.

BACKGROUND: Retinitis pigmentosa is a phenotype with diverse genetic causes. Due to this genetic heterogeneity, genome-wide identification and analysis of protein-altering DNA variants by exome sequencing is a powerful tool for novel variant and disease gene discovery. In this study, exome sequencing analysis was used to search for potentially causal DNA variants in a two-generation pedigree with apparent dominant retinitis pigmentosa. METHODS: Variant identification and analysis of three affected members (mother and two affected offspring) was performed via exome sequencing. Parental samples of the index case were used to establish inheritance. Follow-up testing of 94 additional retinitis pigmentosa pedigrees was performed via retrospective analysis or Sanger sequencing. RESULTS AND CONCLUSIONS: A total of 136 high quality coding variants in 123 genes were identified which are consistent with autosomal dominant disease. Of these, one of the strongest genetic and functional candidates is a c.269A>G (p.Tyr90Cys) variant in ARL3. Follow-up testing established that this variant occurred de novo in the index case. No additional putative causal variants in ARL3 were identified in the follow-up cohort, suggesting that if ARL3 variants can cause adRP it is an extremely rare phenomenon.

Autosomal Dominant Retinal Dystrophies Caused by a Founder Splice Site Mutation, c.828+3A>T, in PRPH2 and Protein Haplotypes in trans as Modifiers.

PURPOSE: We determined the phenotypic variation, disease progression, and potential modifiers of autosomal dominant retinal dystrophies caused by a splice site founder mutation, c.828+3A>T, in the PRPH2 gene. METHODS: A total of 62 individuals (19 families) harboring the PRPH2 c.828+3A>T mutation, had phenotype analysis by fundus appearance, electrophysiology, and visual fields. The PRPH2 haplotypes in trans were sequenced for potential modifying variants and generalized estimating equations (GEE) used for statistical analysis. RESULTS: Several distinct phenotypes caused by the PRPH2 c.828+3A>T mutation were observed and fell into two clinical categories: Group I (N = 44) with mild pattern dystrophies (PD) and Group II (N = 18) with more severe cone-rod dystrophy (CRD), retinitis pigmentosa (RP), and central areolar chorioretinal dystrophy (CACD). The PRPH2 Gln304-Lys310-Asp338 protein haplotype in trans was found in Group I only (29.6% vs. 0%), whereas the Glu304-Lys310-Gly338 haplotype was predominant in Group II (94.4% vs. 70.4%). Generalized estimating equations analysis for PD versus the CRD/CACD/RP phenotypes in individuals over 43 years alone with the PRPH2 haplotypes in trans and age as predictors, adjusted for correlation within families, confirmed a significant effect of haplotype on severity (P = 0.03) with an estimated odds ratio of 7.16 (95% confidence interval [CI] = [2.8, 18.4]). CONCLUSIONS: The PRPH2 c.828+3A>T mutation results in multiple distinct phenotypes likely modified by protein haplotypes in trans; the odds of having the CACD/RP-like phenotype (versus the PD phenotype) are 7.16 times greater with a Glu304-Lys310-Gly338 haplotype in trans. Further functional studies of the modifying haplotypes in trans and PRPH2 splice variants may offer therapeutic targets.

Identification of a Novel Gene on 10q22.1 Causing Autosomal Dominant Retinitis Pigmentosa (adRP).

Whole-genome linkage mapping identified a region on chromosome 10q21.3-q22.1 with a maximum LOD score of 3.0 at 0 % recombination in a six-generation family with autosomal dominant retinitis pigmentosa (adRP). All known adRP genes and X-linked RP genes were excluded in the family by a combination of methods. Whole-exome next-generation sequencing revealed a missense mutation in hexokinase 1, HK1 c.2539G > A, p.Glu847Lys, tracking with disease in all affected family members. One severely-affected male is homozygous for this region by linkage analysis and has two copies of the mutation. No other potential mutations were detected in the linkage region nor were any candidates identified elsewhere in the genome. Subsequent testing detected the same mutation in four additional, unrelated adRP families, for a total of five mutations in 404 probands tested (1.2 %). Of the five families, three are from the Acadian population in Louisiana, one is French Canadian and one is Sicilian. Haplotype analysis of the affected chromosome in each family and the homozygous individual revealed a rare, shared haplotype of 450 kb, suggesting an ancient founder mutation. HK1 is a widely-expressed gene, with multiple, abundant retinal transcripts, coding for hexokinase 1. Hexokinase catalyzes phosphorylation of glucose to glusose-6-phospate, the first step in glycolysis. The Glu847Lys mutation is in a highly-conserved site, outside of the active site or known functional sites.

Founder Effect of a c.828+3A>T Splice Site Mutation in Peripherin 2 (PRPH2) Causing Autosomal Dominant Retinal Dystrophies.

IMPORTANCE: Screening for splice site mutation c.828+3A>T in the peripherin 2 (PRPH2) gene should be a high priority in families with highly variable retinal dystrophies. The correction of missplicing is a potential therapeutic target. OBJECTIVE: To determine the prevalence, genetic origin, and molecular mechanism of a donor c.828+3A>T mutation in the PRPH2 (peripherin 2, retinal degeneration slow) gene in individuals with retinal dystrophies. DESIGN, SETTING, AND PARTICIPANTS: Case-control study that took place at the University of Texas Health Science Center, the University of Iowa, and the Retina Foundation of the Southwest, from January 1, 1987, to August 1, 2014, including affected individuals from 200 families with a diagnosis of autosomal dominant retinitis pigmentosa, 35 families with unspecified macular dystrophies, and 116 families with pattern dystrophy. Participants were screened for the c.828+3A>T mutation by restriction-enzyme digest, single-strand conformational polymorphism screening, or bidirectional sequencing. Haplotypes of polymorphic markers flanking the PRPH2 locus and sequence variants within the gene were determined by denaturing gel electrophoresis or automated capillary-based cycle sequencing. The effect of the splice site mutation on the PRPH2 transcript was analyzed using NetGene2, a splice prediction program and by the reverse transcription polymerase chain reaction of illegitimate transcripts from peripheral white blood cells. MAIN OUTCOMES AND MEASURES: Results of testing for splice site mutation, haplotypes, and alternate transcripts. RESULTS: The PRPH2 mutation was found in 97 individuals of 19 independently ascertained families with a clinical diagnosis of retinitis pigmentosa, macular dystrophy, and/or pattern dystrophy. All affected individuals also shared a rare haplotype of approximately 644 kilobase pairs containing the c.828+3A>T mutation, which extends from the short tandem repeat polymorphism D6S282 to c.1013G>A (rs434102, a single-nucleotide polymorphism) in exon 3 of PRPH2, suggesting this mutation is from a common ancestor and is a founder mutation. It has a prevalence of 2% in families diagnosed as having autosomal dominant retinitis pigmentosa and 10% in families with variable clinical diagnosis of pattern, macular, and retinal dystrophies. Individuals with the c.828+3A>T mutation expressed a PRPH2 transcript not found in control participants and that was consistent with abnormal splicing. CONCLUSIONS AND RELEVANCE: The PRPH2 c.828+3A>T splice site mutation is a frequent cause of inherited retinal dystrophies and is owing to the founder effect. The likely cause of disease is the missplicing of the PRPH2 message that results in a truncated protein product. Identifying the genetic etiology assists in more accurate management and possible future therapeutic options.

Genes and Mutations Causing Autosomal Dominant Retinitis Pigmentosa.

Retinitis pigmentosa (RP) has a prevalence of approximately one in 4000; 25%-30% of these cases are autosomal dominant retinitis pigmentosa (adRP). Like other forms of inherited retinal disease, adRP is exceptionally heterogeneous. Mutations in more than 25 genes are known to cause adRP, more than 1000 mutations have been reported in these genes, clinical findings are highly variable, and there is considerable overlap with other types of inherited disease. Currently, it is possible to detect disease-causing mutations in 50%-75% of adRP families in select populations. Genetic diagnosis of adRP has advantages over other forms of RP because segregation of disease in families is a useful tool for identifying and confirming potentially pathogenic variants, but there are disadvantages too. In addition to identifying the cause of disease in the remaining 25% of adRP families, a central challenge is reconciling clinical diagnosis, family history, and molecular findings in patients and families.

A dominant mutation in hexokinase 1 (HK1) causes retinitis pigmentosa.

PURPOSE: To identify the cause of retinitis pigmentosa (RP) in UTAD003, a large, six-generation Louisiana family with autosomal dominant retinitis pigmentosa (adRP). METHODS: A series of strategies, including candidate gene screening, linkage exclusion, genome-wide linkage mapping, and whole-exome next-generation sequencing, was used to identify a mutation in a novel disease gene on chromosome 10q22.1. Probands from an additional 404 retinal degeneration families were subsequently screened for mutations in this gene. RESULTS: Exome sequencing in UTAD003 led to identification of a single, novel coding variant (c.2539G>A, p.Glu847Lys) in hexokinase 1 (HK1) present in all affected individuals and absent from normal controls. One affected family member carries two copies of the mutation and has an unusually severe form of disease, consistent with homozygosity for this mutation. Screening of additional adRP probands identified four other families (American, Canadian, and Sicilian) with the same mutation and a similar range of phenotypes. The families share a rare 450-kilobase haplotype containing the mutation, suggesting a founder mutation among otherwise unrelated families. CONCLUSIONS: We identified an HK1 mutation in five adRP families. Hexokinase 1 catalyzes phosphorylation of glucose to glucose-6-phosphate. HK1 is expressed in retina, with two abundant isoforms expressed at similar levels. The Glu847Lys mutation is located at a highly conserved position in the protein, outside the catalytic domains. We hypothesize that the effect of this mutation is limited to the retina, as no systemic abnormalities in glycolysis were detected. Prevalence of the HK1 mutation in our cohort of RP families is 1%.