Mutation Spectrum Of Retnet Genes In Probands With Retinitis Pigmentosa And Associated Diseases

Hereditary retinal degeneration is a kind of common and highly heterogeneous eye disease leading to blindness. There are no effective therapies available. Retinitis pigmentosa(RP) is the most common hereditary retinal degenerations. Leber congenital amaurosis(LCA) is the most severe form of hereditary retinal degenerations. To date, about 253 genes associated with hereditary retinal degeneration were listed in Ret Net(https://www.sph.uth.edu/Ret Net). Of the 253 genes, 82 were associated with RP. The genetic diagnosis for these disease is in difficulty because the causative genes have a great quantity, and the genetics and phenotypes of hereditary retinal degeneration is high heterogeneous. The shortage of sequencing technology limits the reseaches to the single gene in the families with inherited retinal dystrophies. Systematic analysis of all genes responsible for retinal dystrophies in a large number of patients with various inherited retinal dystrophy is not available in a longtime. Along with the rapid development of high-throughput sequencing, the individual genomic information is also increasing dramatically. By virtue of increased complexity, this shift in causative mutations identifying has been accompanied by new challenges in sequence interpretation. This study aimed to comprehensively analyze the variants in all of Ret Net genes in the patients with common hereditary retinal degeneration and provide a systemic overview of the molecular etiology of diseases. The analysis of massive datasets produced with high-throughput sequencing may found the less likely pathogenic genes and variants ignored by previous studies. These findings suggest the further genetic studies should pay attention to these genes.This study provided a comprehensive analysis of mutations in Ret Net genes in RP based on the whole exome sequencing dataset of our group. On the other hand, a series studies summary the mutation specturm of Ret Net genes in LCA, and futher analyzed variants inseveral gene variants. Potential pathogeneic mutations were identified in about 60% of probands with RP and LCA. This study summarized the mutation spectrum and frequency in these two diseases and suggests the causative gene of Alstr?m syndrome, ALMS1, should be regarded as a candidate causative gene in patients diagnosed with isolated early-onset severe retinal dystrophy. This study provided an overview of the problems in the detection of causative mutations based on the data of whole exome sequencing in about thousand cases with hereditary retinal degenerations, including the variants with allele frequencies exceeding 1% were found by comparing our whole exome sequencing database with Human Gene Mutation Database, the variants predicted to be damaging and detected in multiple disease groups, and the candidate damaging variants did not co-segregate with the diseases. These findings suggested analysis of causative mutations by high-throughput sequencing needs to be treated with caution, combined with clinical phenotypes, and analyzed in multiple aspects. The analysis of the known genes not only supplied the clues for discovering novel causative genes of these hereditary ocular diseases, but also provided foundation for developing the genetic diagnosis, the prevention and gene therapy of these diseases.Chapter 1 Mutation spectrum analysis in Ret Net genes in probands with retinitis pigmentosaPurpose: To provide a comprehensive analysis of the spectrum and frequency of all Ret Net genes in 157 Chinese probands with RP. Moreover, to analyze the mutations in genes associated with systemic syndromes accompanied with ocular diseases in probands lacked identified mutations. This study aimed to summarize the problems and solutions in the analysis of causative mutations in hereditary ocular disease based on high-throughput sequencing.Methods: The variants of 191 Ret Net(accessed on January, 2014) genes(62 known genes responsible for RP and 129 genes associated with other forms of hereditary retinal dystrophy) were selected from the dataset of whole exome sequencing performed on the genomic DNA samples of 157 probands with RP. The candidate variants were filtered by bioinformatics analysis, including frequency in database, silicon prediction, analysis of available family members and controls, and confirmed by Sanger sequencing. In conjunction with the data from our previous studies, a series of studies provide a comprehensive analysis of the spectrum and frequency of all Ret Net genes in 157 Chinese probands with RP. The mutations in genes associated with syndromes accompanied with abnormal ocular signs were further analyzed in probands without identified mutations in known Ret Net genes.Results: Putative pathogenic mutations in Ret Net genes were identified in 57.3%(90/157) of probands with RP by whole exome sequencing. Mutations in the 90 families consisted of 31(34.4 %, 31/90) families with heterozygous mutations in the genes that are associated with autosomal dominaint diseases, 41(45.6 %, 41/90) families with homozygous(10) or compound heterozygous(31) mutations in the genes that are associated with autosomal recessive diseases, and 18(14 %, 18/90) families with hemizygous mutations in the genes that are associated with X-linked diseases.A total of 27 variants in 24 genes reported to be associated with systemic diseases(12) and ocular genetic diseases(12) with allele frequencies exceeding 1% were found by comparing our whole exome sequencing database with Human Gene Mutation Database in the analysis of whole exome sequencing data of 828 probans with hereditary ocular diseases. Variants in 14 genes associated with autosomal dominant retinal diseases in Ret Net and predicted to be damaging by bioinformatic analysis were detected in multiple disease groups. Candidatedamaging variants in known retinal disease genes did not co-segregate with the diseases in 16 additional families.Conclusions: Mutations in all known genes associated with retinal degeneration could be identified in 57.3% of 157 patients with RP. A comprehensive analysis of all known genes associated retinal degeneration not only expanded the mutation spectrum and frequency of these genes in Chinese patients with RP, but also provided an overview of the molecular etiology of RP in Chinese patients. The precisely analysis of the known genes also supplied the foundation and clues for discovering novel causative RP genes. This study summarized the problems in analysis of causative mutations by whole exome sequencing and listed the less likely pathogenic genes and mutations in Ret Net genes. These findings suggest that the analysis of causative mutations by high-throughput sequencing need to be combined with clinical phenotypes and analyzed in multiple aspects.Chapter 2 Mutation spectrum analysis in Ret Net genes in probands with retinitis pigmentosa related diseasePurpose: This study aimed to provide a comprehensive analysis of the spectrum and frequency of all known genes associated with retinal dystrophy in a total of 159 Chinese probands with LCA. Furthermore, to identify new LCA causative genes. In addition, mutations in MFRP have been reported to cause an ophthalmic syndrome characterized by posterior microphthalmia, high hyperopia, and retinitis pigmentosa. The purpose of this study was to detect MFRP mutations in 46 unrelated Chinese probands with high hyperopia.Methods: To analyze and summarize the spectrum and frequency of all Ret Net genes in a total of 159 Chinese probands with LCA based on whole exome sequencing and Sanger sequencig. To large the sample size because frequent homozygous or compound heterozygous null mutations in ALMS1 were revealed. Sanger sequencing, co-segregation analysis and analysis of normal individuals was performed in the families with ALMS1 null mutations detected in 1220 families with various hereditary ocular diseases. The other detailed systemic manifestations were evaluated in the available patients in the follow-up examinations. Genomic DNA from 42 probands was initially analyzed by whole exome sequencing. MFRP variants were confirmed by Sanger sequencing. The coding sequence of MFRP for four additional probands was also analyzed by Sanger sequencing. Candidate MFRP variants were further validated in available family members and 192 normal individuals.Results: Overall, a total of 118 putative pathogenic mutations from 22 Ret Net genes were identified in 56.6%(90/159) of probands. These mutations were harbored in 13 LCAassociated genes and in nine genes related to other forms of retinal dystrophy. The mutations in 45 out of 72(62.5%) probands were detected by whole exome sequencing alone. The genes with high mutant frequency were GUCY2D(10.7%), CRB1(7.5%), CEP290(6.9%), RPGRIP1(5.0%), IQCB1(4.4%), ALMS1(3.8%), and CRX(3.8%). Multi-steps analysis identified a total of 13 null mutations in ALMS1 in 11 probands initial dignosed with Leber congenital amaurosis or early-onset cone-rod dystrophy. Causative null mutations in ALMS1 were detected in 11 of 215(5.1%) probands with Leber congenital amaurosis and cone-rod dystrophy. Follow-up examinations revealed absent or mild systemic manifestations of Alstr?m syndrome in 9 of 15 patients in 11 families. Potential pathogenic compound heterozygous mutations were detected in three of the 46 probands with high hyperopia. The clinical data revealed that all patients in this study had high hyperopia of ≥+13.50 dioptersand an eye axial length of ≤16.78 mm. Electroretinography showed normal responses in a patient with missense mutations and reduced rod responses in another patient with missense and truncation mutations in whom optical coherence tomography showed developmental cystoid macular degeneration in both eyes.Conclusions: A series of mutational analyses suggests that all Ret Net genes account for 56.6% of Chinese patients with LCA. A comprehensive molecular genetic analysis of Chinese patients with LCA provides an overview of the spectrum and frequency of ethno-specific mutations of all known genes. This study suggests that ALMS1 should be regarded as a candidate causative gene in patients diagnosed with isolated LCA and early-onset severe CORD. The current study expands our knowledge of the mutation spectrum of MFRP and its associated phenotypes. To our knowledge, this is the first report of MFRP mutations in a Chinese cohort.