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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.

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.

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.

Purpose: We determined the fraction of families in a well-characterized cohort with a provisional diagnosis of autosomal dominant retinitis pigmentosa (adRP) that have disease-causing mutations in the X-linked retinitis pigmentosa GTPase regulator (RPGR) gene or the retinitis pigmentosa 2 (RP2) gene., Methods: Families with a provisional clinical diagnosis of adRP, and a pedigree consistent with adRP but no male-to-male transmission were selected from a cohort of 258 families, and tested for mutations in the RPGR and RP2 genes with di-deoxy sequencing. To facilitate testing of RPGR in "adRP" families that had no male members available for testing, the repetitive and purine-rich ORF15 of RPGR was subcloned and sequenced in heterozygous female subjects from 16 unrelated families., Results: Direct sequencing of RPGR and RP2 allowed for identification of a disease-causing mutation in 21 families. Of these "adRP" families 19 had RPGR mutations, and two had RP2 mutations. Subcloning and sequencing of ORF15 of RPGR in female subjects identified one additional RPGR mutation. Of the 22 mutations identified, 15 have been reported previously., Conclusions: These data show that 8.5% (22 in 258) of families thought to have adRP truly have X-linked retinitis pigmentosa (XLRP). These results have substantive implications for calculation of recurrence risk, genetic counseling, and potential treatment options, and illustrate the importance of screening families with a provisional diagnosis of autosomal inheritance and no male-to-male transmission for mutations in X-linked genes. Mutations in RPGR are one of the most common causes of all forms of retinitis pigmentosa.

Molecular diagnostics for patients with retinitis pigmentosa (RP) has been hampered by extreme genetic and clinical heterogeneity, with 52 causative genes known to date. Here, we developed a comprehensive next-generation sequencing (NGS) approach for the clinical molecular diagnostics of RP. All known inherited retinal disease genes (n = 111) were captured and simultaneously analyzed using NGS in 100 RP patients without a molecular diagnosis. A systematic data analysis pipeline was developed and validated to prioritize and predict the pathogenicity of all genetic variants identified in each patient, which enabled us to reduce the number of potential pathogenic variants from approximately 1,200 to zero to nine per patient. Subsequent segregation analysis and in silico predictions of pathogenicity resulted in a molecular diagnosis in 36 RP patients, comprising 27 recessive, six dominant, and three X-linked cases. Intriguingly, De novo mutations were present in at least three out of 28 isolated cases with causative mutations. This study demonstrates the enormous potential and clinical utility of NGS in molecular diagnosis of genetically heterogeneous diseases such as RP. De novo dominant mutations appear to play a significant role in patients with isolated RP, having major implications for genetic counselling., (© 2012 Wiley Periodicals, Inc.)

Objectives: To assess the phenotype of patients with X-linked retinitis pigmentosa (XLRP) with RP2 mutations and to correlate the findings with their genotype., Methods: Six hundred eleven patients with RP were screened for RP2 mutations. From this screen, 18 patients with RP2 mutations were evaluated clinically with standardized electroretinography, Goldmann visual fields, and ocular examinations. In addition, 7 well-documented cases from the literature were used to augment genotype-phenotype correlations., Results: Of 11 boys younger than 12 years, 10 (91%) had macular involvement and 9 (82%) had best-corrected visual acuity worse than 20/50. Two boys from different families (aged 8 and 12 years) displayed a choroideremia-like fundus, and 9 boys (82%) were myopic (mean error, -7.97 diopters [D]). Of 10 patients with electroretinography data, 9 demonstrated severe rod-cone dysfunction. All 3 female carriers had macular atrophy in 1 or both eyes and were myopic (mean, -6.23 D). All 9 nonsense and frameshift and 5 of 7 missense mutations (71%) resulted in severe clinical presentations., Conclusions: Screening of the RP2 gene should be prioritized in patients younger than 16 years characterized by X-linked inheritance, decreased best-corrected visual acuity (eg, >20/40), high myopia, and early-onset macular atrophy. Patients exhibiting a choroideremia-like fundus without choroideremia gene mutations should also be screened for RP2 mutations., Clinical Relevance: An identifiable phenotype for RP2-XLRP aids in clinical diagnosis and targeted genetic screening.

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.

Objective: To report discordant phenotypes, resulting from the same mutation in exon ORF15 (GenBank AF286472) of the retinitis pigmentosa GTPase regulator gene (RPGR) (GenBank U57629), in 2 presumed dizygotic twin brothers with X-linked retinal disease., Methods: The 2 brothers underwent complete ophthalmic examination that included best-corrected visual acuity, slitlamp biomicroscopy, and detailed fundus examination. Visual field recording using Goldmann kinetic perimetry and a full-field electroretinogram were also obtained in both patients. Mutational screening was performed for RPGR because of an X-linked pattern of inheritance indicated by pedigree analysis., Results: One brother had a phenotypic expression of cone-rod dystrophy, while the other exhibited X-linked retinitis pigmentosa. A 1-nucleotide deletion was identified in the 3' region of exon ORF15 of RPGR (ORF15 + 1339delA)., Conclusions: An identical mutation in RPGR-ORF15 manifested distinct clinical phenotypes in individuals of the same family. Our data provide strong evidence in support of additional modifier genes that can produce diverse disease phenotypes in patients with RPGR mutations., Clinical Relevance: The clinical observation of different retinal phenotypes in a family with the same mutation in exon ORF15 of RPGR implicates the potential importance of modifier genes for the phenotypic expression of this form of X-linked retinal disease.

Purpose: To investigate macular photoreceptor structure in patients with inherited retinal degeneration using high-resolution images and to correlate the findings with clinical phenotypes and genetic mutations., Methods: Adaptive optics scanning laser ophthalmoscopy (AOSLO) images of photoreceptors were obtained in 16 eyes: five with retinitis pigmentosa (RP), three with cone-rod dystrophy (CRD), and eight without retinal disease. A quadratic model was used to illustrate cone spacing as a function of retinal eccentricity. Cone spacing at 1 degrees eccentricity was compared with standard measures of central visual function, including best-corrected visual acuity (BCVA), foveal threshold, and multifocal electroretinogram (mfERG) amplitude and timing. Intervisit variations were studied in one patient with RP and one patient with CRD. Screening of candidate disease genes identified mutations in two patients, one with RP (a rhodopsin mutation) and the other with CRD (a novel RPGR-ORF15 mutation)., Results: Cone spacing values were significantly different from normal for patients with RP (P = 0.01) and CRD (P < 0.0001) and demonstrated a statistically significant correlation with foveal threshold (P = 0.0003), BCVA (P = 0.01), and mfERG amplitude (P = 0.008). Although many RP patients showed normal cone spacing within 1 degrees of fixation, cones could not be unambiguously identified in several retinal regions. Cone spacing increased in all CRD patients, even those with early disease. Little variation was observed in cone spacing measured during two sessions fewer than 8 days apart., Conclusions: AOSLO images can be used to study macular cones with high resolution in patients with retinal degeneration. The authors present the first report of cone structure in vivo in patients with mutations in rhodopsin and RPGR-ORF15 and show that macular cones display distinct characteristics, depending on the underlying disease. AOSLO imaging, therefore, can provide new insight into possible mechanisms of cone vision loss in patients with retinal degeneration.