Comprehensive Genotyping and Phenotyping Analysis of GUCY2D-Associated Rod- and Cone-Dominated Dystrophies.

PURPOSE: To describe the genetic and clinical spectrum of GUCY2D-associated retinopathies and to accurately establish their prevalence in a large cohort of patients. DESIGN: Retrospective case series. METHODS: Institutional study of 47 patients from 27 unrelated families with retinal dystrophies carrying disease-causing GUCY2D variants from the Fundación Jiménez Díaz hospital dataset of 8000 patients. Patients underwent ophthalmological examination and molecular testing by Sanger or exome sequencing approaches. Statistical and principal component analyses were performed to determine genotype-phenotype correlations. RESULTS: Four clinically different associated phenotypes were identified: 66.7% of families with cone/cone-rod dystrophy, 22.2% with Leber congenital amaurosis, 7.4% with early-onset retinitis pigmentosa, and 3.7% with congenital night blindness. Twenty-three disease-causing GUCY2D variants were identified, including 6 novel variants. Biallelic variants accounted for 28% of patients, whereas most carried dominant alleles associated with cone/cone-rod dystrophy. The disease onset had statistically significant differences according to the functional variant effect. Patients carrying GUCY2D variants were projected into 3 subgroups by allelic combination, disease onset, and presence of nystagmus or night blindness. In contrast to patients with the most severe phenotype of Leber congenital amaurosis, 7 patients with biallelic GUCY2D had a later and milder rod form with night blindness in infancy as the first symptom. CONCLUSIONS: This study represents the largest GUCY2D cohort in which 4 distinctly different phenotypes were identified, including rare intermediate presentations of rod-dominated retinopathies. We established that GUCY2D is linked to about 1% of approximately 3000 molecularly characterized families of our cohort. All of these findings are critical for defining cohorts for inclusion in future clinical trials.

Application of multicolour reflectance imaging for the characterisation of inherited retinal disorders.

AIM: To describe the role of multicolour reflectance images (MCI) in the phenotypic diagnosis of inherited retinal disorders (IRDs). METHODS: A retrospective review of consecutive patients affected by IRDs examined with MCI techniques from January to December 2019 at a tertiary care referral centre. All patients had MCI, fundus autofluorescence and optical coherence tomography taken at the same time point. The ability of each modality to highlight clinical features was assessed. Lesions' size was also measured and compared among imaging modalities. RESULTS: Thirty eyes of 15 patients were included in the study, 6 males and 9 females, with a mean age of 44 years (range: 19-57.5). The most frequent clinical diagnosis were: pattern dystrophies, and late-onset retinal degeneration. Next-generation or Sanger sequencing analysis was carried out in all patients. Blue and green reflectance were relevant in highlighting peripheral mottling in fundus albipunctatus, pseudoreticular drusen in late-onset retinal degeneration, parafoveal hyperreflective area in bull's eye maculopathy and crystals in Bietti's crystalline dystrophy. Likewise, it is to mention the ability of infrared reflectance to detect hyperreflective patches in posterior pole in neurofibromatosis type 1 and retinal changes in pattern dystrophies and cone dystrophies. CONCLUSION: Multicolour imaging technique enables the detection of clinical features that could be overlooked by other imaging modalities, allowing accurate phenotypic characterisation of IRDs and guiding genetic diagnose, and may become a meaningful monitoring tool for future treatments.

An evaluation of pipelines for DNA variant detection can guide a reanalysis protocol to increase the diagnostic ratio of genetic diseases.

Clinical exome (CE) sequencing has become a first-tier diagnostic test for hereditary diseases; however, its diagnostic rate is around 30-50%. In this study, we aimed to increase the diagnostic yield of CE using a custom reanalysis algorithm. Sequencing data were available for three cohorts using two commercial protocols applied as part of the diagnostic process. Using these cohorts, we compared the performance of general and clinically relevant variant calling and the efficacy of an in-house bioinformatic protocol (FJD-pipeline) in detecting causal variants as compared to commercial protocols. On the whole, the FJD-pipeline detected 99.74% of the causal variants identified by the commercial protocol in previously solved cases. In the unsolved cases, FJD-pipeline detects more INDELs and non-exonic variants, and is able to increase the diagnostic yield in 2.5% and 3.2% in the re-analysis of 78 cancer and 62 cardiovascular cases. These results were considered to design a reanalysis, filtering and prioritization algorithm that was tested by reassessing 68 inconclusive cases of monoallelic autosomal recessive retinal dystrophies increasing the diagnosis by 4.4%. In conclusion, a guided NGS reanalysis of unsolved cases increases the diagnostic yield in genetic disorders, making it a useful diagnostic tool in medical genetics.

Apparent but unconfirmed digenism in an Iranian consanguineous family with syndromic Retinal Disease.

BACKGROUND: Bardet-Biedl syndrome is an autosomal recessive disease characterized by rod-cone dystrophy, postaxial polydactyly, kidney defects, obesity, mental retardation and hypogonadism. Here, we report different genotypes in two Bardet-Biedl syndrome affected sisters with a different clinical phenotype regarding severity. MATERIALS AND METHODS: The proband of the family was examined by Next Generation Sequencing (NGS) using clinical exome and filtering by syndromic and non-syndromic genes associated with retinal dystrophies. RESULTS: Targeted NGS revealed two novel variants in the MKKS and CEP290 genes in homozygosis state in the proband. Segregation analysis revealed the presence of the same MKKS homozygous variant in her younger affected sister but not the CEP290 variant. Both sisters presented different clinical manifestation, at different ages, with a more severe renal and retinal defect in the case of the sister carrying mutations in both genes. Another unaffected sister showed only homozygosity for the CEP290 variant, thus supporting the non-pathogenic role of this mutation in BBS phenotype. CONCLUSIONS: In this study, NGS proved to be a powerful and efficient sequencing method to identify causal variants in different genes. However, it remarks the importance of the segregation analysis and clinical information to establish the pathogenicity of new variants. The two affected sisters present different genotypes and clinical manifestation, suggesting that the novel CEP290 variant could be acting as a modifier, making the phenotype more severe in the sister homozygote for MKKS and CEP290 genes. On the other hand, the difference in the age of both sisters highlight the important role of monitoring disease progression also to confirm the modifier role of genetic variants.