Pathogenic Mutation Detection and Analysis of NGS Sequencing Data for Several Ophthalmic Diseases


Student thesis: Doctoral Thesis

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Award date10 Jun 2021


Ophthalmic diseases such as macular degeneration, glaucoma, and cataracts can cause vision problems. Most ophthalmic diseases are caused by genetic changes, known as hereditary ophthalmic diseases. Hereditary ophthalmic disease is one of the main causes of blindness in children and adolescents. Hereditary ophthalmic disease affects people of all ages and involves a variety spectrum of diseases, including developmental eye defects, corneal dystrophy, and retinal dystrophy, and inherited optic neuropathy, accompanied by non-syndromic or multisystemic syndrome forms. Unfortunately, some eye diseases are not easily detected in the early stage. Hereditary ophthalmic diseases have significant clinical and genetic heterogeneity. The inheritance pattern follows the Mendelian inheritance pattern, including autosomal dominant (AD), autosomal recessive (AR), X-linked (XL), and mitochondrial inheritance. Hereditary ophthalmic diseases can also have a variety of different clinical phenotypes, and there are some similarities and intersects among different disease phenotypes. The identification of pathogenic mutations can help the clinical diagnosis, especially for those patients with typical clinical phenotypes that doctors are difficult to determine by conventional means. However, limited by traditional methods, it is difficult to discover pathogenic genes on a large scale, and it cannot be applied to the study of small families. Due to the limited number of genes and their pathogenic loci and the limitations of technology, the detection rate of existing clinical tests is inefficient, which puts forward an urgent requirement for researchers and clinicians studying hereditary ophthalmic diseases to search for pathogenic genes by more feasible and efficient methods. 

To study pathogenic genes for hereditary ophthalmic diseases with high efficiency. we establish bioinformatics analysis progress and strategy suitable for NGS sequencing data. The NGS sequencing data bioinformatics analysis mainly includes mapping, mutation detection, annotation, and downstream function analysis, etc. Each step has embedded more efficient and accurate software with a highly automated analysis process in the face of large sample size data. We apply this progress and strategy to study several hereditary ophthalmic diseases including retinitis pigmentosa (RP), hereditary optic neuropathy, Familial exudative vitreoretinopathy (FEVR), and corneal dystrophies (CD). 

Ethnicity-specific population genetic data is necessary to ascertain aetiological variants or delineating genetic characteristics of the genome. Here, we used panel-based targeted exome sequencing to generate distinct high-quality variants for 8,400 Chinese participants with hereditary ophthalmic disease with an average detection rate of over 50%, much higher than reported: 1) in the study of a large cohort of suspected retinitis pigmentosa, a total of 1243 Chinese patients (611 female, 632 male) were recruited, after bioinformatics analysis 72.08% of patients received a causative mutations diagnosis, 517 variants (212 previously reported and 305 novel) spanning 76 genes were identified. Moreover, different genes associated with different onset ages and subgroups with different onset ages showed a diverse mutation spectrum. 2) A total of 177 families (537 subjects) and 164 sporadic patients were recruited in the mtDNA combined targeted exon sequencing study of suspected optic neuropathy. The overall detection rate reached 40.2% (168/418), including 78.6% (132/168) of patients with LHON-related mtDNA mutations and 23.8% (40/168) of patients with ntDNA mutations. Among the samples with ntDNA mutations, three pathogenic genes, OPA1, OPA3 and TMEM126A, accounted for 21.4% (36/168), 2.4% (4/168) and 1.2% (2/168), respectively. Haplogroup analysis showed that 34.5% (29/84) and 28.6% (24/84) of patients with m.11778G>A belonged to haplogroups D and M, and the frequency of subhaplogroups D4, D5 and M7 was higher. 3) FEVR is a group of vitreoretinopathy characterized by high clinical and genetic heterogeneity. We detected a novel start codon mutation in a patient, but asymptomatic sisters and mothers with the same mutation showed typical FEVR characteristics with Peripheral retinal vascularization. A similar start codon TSPAN12 c.2T>C (p.M1T) was reported in a proband in another study, and his asymptomatic father showed typical FEVR characteristics of peripheral retinal vascularization. 4) A large IEDs cohort was recruited, including 69 clinically diagnosed corneal dystrophies patients, and other types of eye disease patients and healthy family members as controls. The mutation spectrum of 22 CD-related genes was evaluated, 21 distinct pathogenic or likely pathogenic mutations were identified, and the overall detection rate was 81.16% (n=56/69). The three detected etiological genes (TGFBI, CHTS6 and SLC4A11), accounted for 91.07%, 7.14% and 1.79% of diagnosed cases, respectively. Among 56 CDs patients with positive detected mutations, the recurrent TGFBI mutations were p.R124H, p.R555W, p.R124C, p.R555Q, p.R124L, and the proportions were 32.14%, 19.64%, 14.29%, 10.71%, and 3.57%, respectively. The recurrent CHTS6 mutations were p.Y358H, p.R140X, p.R205W, and the proportions were 25.00%, 21.43%, and 14.29%, respectively. In the current study, we provide baseline data based on large-scale hereditary ophthalmic disease participants, combined with multiple genomic frequency databases, deleterious assessment, genotype-phenotype association analysis, and family co-segregation analysis, generate distinct high-quality variants within 792 hereditary ophthalmic disease-related genes. We systematically delineated the variations spectrum of hereditary ophthalmic disease-related genes and evaluated the frequency and pathogenicity of all distinct high-quality variants. Furthermore, the genetic characteristics of De novo mutations, founder mutations, and hot spot mutations, digenic mutations were depicted from the genetic data of the Chinese ethnic population. Finally, the genotype-phenotype correlation is explained in terms of genomic characteristics and clinical manifestations. Our research results show that unified analysis of genetic data from large hereditary ophthalmic disease phenotypic heterogeneity cohorts was a powerful genetic discovery approach, which helps us to systematically understand the mutation spectrum and genetic characteristics of hereditary ophthalmic disease-related genes in specific populations.