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.

Tissue-specific genotype-phenotype correlations among USH2A-related disorders in the RUSH2A study.

We assessed genotype-phenotype correlations among the visual, auditory, and olfactory phenotypes of 127 participants with Usher syndrome (USH2) (n =80) or nonsyndromic autosomal recessive retinitis pigmentosa (ARRP) (n = 47) due to USH2A variants, using clinical data and molecular diagnostics from the Rate of Progression in USH2A Related Retinal Degeneration (RUSH2A) study. USH2A truncating alleles were associated with USH2 and had a dose-dependent effect on hearing loss severity with no effect on visual loss severity within the USH2 subgroup. A group of missense alleles in an interfibronectin domain appeared to be hypomorphic in ARRP. These alleles were associated with later age of onset, larger visual field area, better sensitivity thresholds, and better electroretinographic responses. No effect of genotype on the severity of olfactory deficits was observed. This study unveils a unique, tissue-specific USH2A allelic hierarchy with important prognostic implications for patient counseling and treatment trial endpoints. These findings may inform clinical care or research approaches in others with allelic disorders or pleiotropic phenotypes.

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.

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.

SeqCNV: a novel method for identification of copy number variations in targeted next-generation sequencing data.

BACKGROUND: Targeted next-generation sequencing (NGS) has been widely used as a cost-effective way to identify the genetic basis of human disorders. Copy number variations (CNVs) contribute significantly to human genomic variability, some of which can lead to disease. However, effective detection of CNVs from targeted capture sequencing data remains challenging. RESULTS: Here we present SeqCNV, a novel CNV calling method designed to use capture NGS data. SeqCNV extracts the read depth information and utilizes the maximum penalized likelihood estimation (MPLE) model to identify the copy number ratio and CNV boundary. We applied SeqCNV to both bacterial artificial clone (BAC) and human patient NGS data to identify CNVs. These CNVs were validated by array comparative genomic hybridization (aCGH). CONCLUSIONS: SeqCNV is able to robustly identify CNVs of different size using capture NGS data. Compared with other CNV-calling methods, SeqCNV shows a significant improvement in both sensitivity and specificity.

Exome-based mapping and variant prioritization for inherited Mendelian disorders.

Exome sequencing in families affected by rare genetic disorders has the potential to rapidly identify new disease genes (genes in which mutations cause disease), but the identification of a single causal mutation among thousands of variants remains a significant challenge. We developed a scoring algorithm to prioritize potential causal variants within a family according to segregation with the phenotype, population frequency, predicted effect, and gene expression in the tissue(s) of interest. To narrow the search space in families with multiple affected individuals, we also developed two complementary approaches to exome-based mapping of autosomal-dominant disorders. One approach identifies segments of maximum identity by descent among affected individuals; the other nominates regions on the basis of shared rare variants and the absence of homozygous differences between affected individuals. We showcase our methods by using exome sequence data from families affected by autosomal-dominant retinitis pigmentosa (adRP), a rare disorder characterized by night blindness and progressive vision loss. We performed exome capture and sequencing on 91 samples representing 24 families affected by probable adRP but lacking common disease-causing mutations. Eight of 24 families (33%) were revealed to harbor high-scoring, most likely pathogenic (by clinical assessment) mutations affecting known RP genes. Analysis of the remaining 17 families identified candidate variants in a number of interesting genes, some of which have withstood further segregation testing in extended pedigrees. To empower the search for Mendelian-disease genes in family-based sequencing studies, we implemented them in a cross-platform-compatible software package, MendelScan, which is freely available to the research community.

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.

Molecular findings from 537 individuals with inherited retinal disease.

BACKGROUND: Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous set of disorders, for which diagnostic second-generation sequencing (next-generation sequencing, NGS) services have been developed worldwide. METHODS: We present the molecular findings of 537 individuals referred to a 105-gene diagnostic NGS test for IRDs. We assess the diagnostic yield, the spectrum of clinical referrals, the variant analysis burden and the genetic heterogeneity of IRD. We retrospectively analyse disease-causing variants, including an assessment of variant frequency in Exome Aggregation Consortium (ExAC). RESULTS: Individuals were referred from 10 clinically distinct classifications of IRD. Of the 4542 variants clinically analysed, we have reported 402 mutations as a cause or a potential cause of disease in 62 of the 105 genes surveyed. These variants account or likely account for the clinical diagnosis of IRD in 51% of the 537 referred individuals. 144 potentially disease-causing mutations were identified as novel at the time of clinical analysis, and we further demonstrate the segregation of known disease-causing variants among individuals with IRD. We show that clinically analysed variants indicated as rare in dbSNP and the Exome Variant Server remain rare in ExAC, and that genes discovered as a cause of IRD in the post-NGS era are rare causes of IRD in a population of clinically surveyed individuals. CONCLUSIONS: Our findings illustrate the continued powerful utility of custom-gene panel diagnostic NGS tests for IRD in the clinic, but suggest clear future avenues for increasing diagnostic yields.

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.

The Role of X-Chromosome Inactivation in Retinal Development and Disease.

The expression of X-linked genes is equalized between males and females in mammalian species through X-Chromosome inactivation (XCI). Every cell in a female mammalian embryo randomly chooses one X Chromosome for epigenetic silencing at the 8-16 cell stage, resulting in a Gaussian distribution of XCI ratios with a peak at 50:50. At the tail extremes of this distribution, X-linked recessive mutations can manifest in disease in female carriers if the mutant allele is disproportionately active. The role of XCI skewing, if any, in X-linked retinal disease is still unknown, although many have speculated that such skewing accounts for phenotypic variation in female carriers of X-linked retinitis pigmentosa (XlRP). Some investigators have used clinical findings such as tapetal-like reflex, pigmentary changes, and multifocal ERG parameters to approximate XCI patches in the retina. These studies are limited by small cohorts and the relative inaccessibility of retinal tissue for genetic and epigenetic analysis. Although blood has been used as a proxy for other tissues in determining XCI ratios, blood XCI skews with age out of proportion to other tissues and may not accurately reflect retinal XCI ratios. Future investigations in determining retinal XCI ratios and the contribution of XCI to phenotype could potentially impact prognosis for female carriers of X-linked retinal disease.

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.

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

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.

Application of next-generation sequencing to identify genes and mutations causing autosomal dominant retinitis pigmentosa (adRP).

The goal of our research is to identify genes and mutations causing autosomal dominant retinitis pigmentosa (adRP). For this purpose we established a cohort of more than 250 independently ascertained families with adRP in the Houston Laboratory for Molecular Diagnosis of Inherited Eye Diseases. Affected members of each family were screened for disease-causing mutations in genes and gene regions that are commonly associated with adRP. By this approach, we detected mutations in 65 % of the families, leaving 85 families that are likely to harbor mutations outside of the "common" regions or in novel genes. Of these, 32 families were tested by several types of next-generation sequencing (NGS), including (a) targeted polymerase chain reaction (PCR) NGS, (b) whole exome NGS, and (c) targeted retinal-capture NGS. We detected mutations in 11 of these families (31 %) bringing the total detected in the adRP cohort to 70 %. Several large families have also been tested for linkage using Afymetrix single nucleotide polymorphism (SNP) arrays.

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 the small nuclear riboprotein 200 kDa gene (SNRNP200) cause 1.6% of autosomal dominant retinitis pigmentosa.

PURPOSE: The purpose of this project was to determine the spectrum and frequency of mutations in the small nuclear riboprotein 200 kDa gene (SNRNP200) that cause autosomal dominant retinitis pigmentosa (adRP). METHODS: A well-characterized adRP cohort of 251 families was tested for mutations in the exons and intron/exon junctions of SNRNP200 using fluorescent dideoxy sequencing. An additional 21 adRP families from the eyeGENE® Network were tested for possible mutations. Bioinformatic and segregation analysis was performed on novel variants. RESULTS: SNRNP200 mutations were identified in seven of the families tested. Two previously reported mutations, p.Arg681Cys and p.Ser1087Leu, were found in two families each. One family had the previously reported p.Arg681His mutation. Two novel SNRNP200 variants, p.Pro682Ser and p.Ala542Val, were also identified in one family each. Bioinformatic and segregation analyses suggested that these novel variants are likely to be pathogenic. Clinical examination of patients with SNRNP200 mutations showed a wide range of clinical symptoms and severity, including one instance of non-penetrance. CONCLUSIONS: Mutations in SNRNP200 caused 1.6% of disease in our adRP cohort. Pathogenic mutations were found primarily in exons 16 and 25, but the novel p.Ala542Val mutation in exon 13 suggests that variation in other genetic regions is also responsible for causing dominant disease. SNRNP200 mutations were associated with a wide range of clinical symptoms similar to those of individuals with other splice-factor gene mutations.

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.

Autosomal-dominant retinitis pigmentosa caused by a mutation in SNRNP200, a gene required for unwinding of U4/U6 snRNAs.

Mutations in genes associated with the U4/U6-U5 small nuclear ribonucleoprotein (snRNP) complex of the spliceosome are implicated in autosomal-dominant retinitis pigmentosa (adRP), a group of progressive retinal degenerative disorders leading to visual impairment, loss of visual field, and even blindness. We recently assigned a locus (RP33) for adRP to 2cen-q12.1, a region that harbors the SNRNP200 gene encoding hBrr2, another U4/U6-U5 snRNP component that is required for unwinding of U4/U6 snRNAs during spliceosome activation and for disassembly of the spliceosome. Here, we report the identification of a missense mutation, c.3260C>T (p.S1087L), in exon 25 of the SNRNP200 gene in an RP33-linked family. The c.3260C>T substitution showed complete cosegregation with the retinitis pigmentosa (RP) phenotype over four generations, but was absent in a panel of 400 controls. The p.S1087L mutation and p.R1090L, another adRP-associated allele, reside in the "ratchet" helix of the first of two Sec63 domains implicated in the directionality and processivity of nucleic acid unwinding. Indeed, marked defects in U4/U6 unwinding, but not U4/U6-U5 snRNP assembly, were observed in budding yeast for the analogous mutations (N1104L and R1107L) of the corresponding Brr2p residues. The linkage of hBrr2 to adRP suggests that the mechanism of pathogenesis for splicing-factor-related RP may fundamentally derive from a defect in hBrr2-dependent RNA unwinding and a consequent defect in spliceosome activation.

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.