Long-term follow-up of the human phenotype in three siblings with cone dystrophy associated with a homozygous p.G461R mutation of KCNV2.

PURPOSE: To provide an up to 14-year overview of the early ocular phenotype in siblings with a homozygous p.G461R mutation in the KCNV2 gene. METHODS: Two brothers and their sister were followed-up clinically from ages 5 years, 4 years, and 2 months, respectively, including complete ophthalmological examinations. Goldmann visual fields, two-color-threshold (2CT) perimetry, color vision testing, optical coherence tomography (OCT), fundus autofluorescence (FAF), and Ganzfeld electroretinograms (ERGs) were performed according to age-related capabilities. Genetic analyses included whole genome linkage analysis, homozygosity mapping, and candidate gene sequencing. RESULTS: All three siblings were homozygous for the p.G461R mutation. At 5 months, the younger brother had no nystagmus and Teller-acuity of 3.2 cyc/deg. At older age, all three presented nystagmus, increased light sensitivity, reduced color discrimination, and relative central scotomas. Visual acuities ranged from 20/200 to 20/70. The macula developed minor irregularities of the RPE, thinning in optical coherence tomography, and a ring of increased FAF. Scotopic (rod) sensitivity was reduced by 2 log and photopic sensitivity by 1 log in two-color-threshold perimetry. ERG responses were markedly delayed. Photopic amplitudes were severely reduced. Scotopic b-waves rose steeply with flash intensity, but for the standard flash supernormal amplitudes were only reached in the girl. CONCLUSIONS: FAF was similar to that in cone-rod dystrophy. Although cone dysfunction was accompanied by rod dysfunction, and scotopic ERGs in patient 2 deteriorated, no patient demonstrated any unequivocal sign of rod degeneration. Grossly delayed b-waves with a steep response-versus-intensity relationship rather than supernormal amplitudes should remind clinicians of this specific condition.

A comprehensive clinical and biochemical functional study of a novel RPE65 hypomorphic mutation.

PURPOSE: Later onset and progression of retinal dystrophy occur with some RPE65 missense mutations. The functional consequences of the novel P25L RPE65 mutation was correlated with its early-childhood phenotype and compared with other pathogenic missense mutations. METHODS: In addition to typical clinical tests, fundus autofluorescence (FAF), optical coherence tomography (OCT), and two-color threshold perimetry (2CTP) were measured. RPE65 mutations were screened by SSCP and direct sequencing. Isomerase activity of mutant RPE65 was assayed in 293F cells and quantified by HPLC analysis of retinoids. RESULTS: A very mild phenotype was detected in a now 7-year-old boy homozygous for the P25L mutation in RPE65. Although abnormal dark adaptation was noticed early, best corrected visual acuity was 20/20 at age 5 years and 20/30 at age 7 years. Nystagmus was absent. Cone electroretinogram (ERG) was measurable, rod ERG severely reduced, and FAF very low. 2CTP detected mainly cone-mediated responses in scotopic conditions, and light-adapted cone responses were approximately 1.5 log units below normal. High-resolution spectral domain OCT revealed morphologic changes. Isomerase activity in 293F cells transfected with RPE65/P25L was reduced to 7.7% of wild-type RPE65-transfected cells, whereas RPE65/L22P-transfected cells had 13.5%. CONCLUSIONS: The mild clinical phenotype observed is consistent with the residual activity of a severely hypomorphic mutant RPE65. Reduction to <10% of wild-type RPE65 activity by homozygous P25L correlates with almost complete rod function loss and cone amplitude reduction. Functional survival of cones is possible in patients with residual RPE65 isomerase activity. This patient should profit most from gene therapy.

Identification of novel mutations in X-linked retinitis pigmentosa families and implications for diagnostic testing.

PURPOSE: The goal of this study was to identify mutations in X-chromosomal genes associated with retinitis pigmentosa (RP) in patients from Germany, The Netherlands, Denmark, and Switzerland. METHODS: In addition to all coding exons of RP2, exons 1 through 15, 9a, ORF15, 15a and 15b of RPGR were screened for mutations. PCR products were amplified from genomic DNA extracted from blood samples and analyzed by direct sequencing. In one family with apparently dominant inheritance of RP, linkage analysis identified an interval on the X chromosome containing RPGR, and mutation screening revealed a pathogenic variant in this gene. Patients of this family were examined clinically and by X-inactivation studies. RESULTS: This study included 141 RP families with possible X-chromosomal inheritance. In total, we identified 46 families with pathogenic sequence alterations in RPGR and RP2, of which 17 mutations have not been described previously. Two of the novel mutations represent the most 3'-terminal pathogenic sequence variants in RPGR and RP2 reported to date. In exon ORF15 of RPGR, we found eight novel and 14 known mutations. All lead to a disruption of open reading frame. Of the families with suggested X-chromosomal inheritance, 35% showed mutations in ORF15. In addition, we found five novel mutations in other exons of RPGR and four in RP2. Deletions in ORF15 of RPGR were identified in three families in which female carriers showed variable manifestation of the phenotype. Furthermore, an ORF15 mutation was found in an RP patient who additionally carries a 6.4 kbp deletion downstream of the coding region of exon ORF15. We did not identify mutations in 39 sporadic male cases from Switzerland. CONCLUSIONS: RPGR mutations were confirmed to be the most frequent cause of RP in families with an X-chromosomal inheritance pattern. We propose a screening strategy to provide molecular diagnostics in these families.