Whole-exome Sequencing Identifies A Causative Gene Of X-linked Dominant High Myopia And The Establishment Of Human Gene Variant Database-LOVD

Part I Whole-exome sequencing identifies a causative gene of X-linked dominant high myopiaBackground:High myopia is an extreme form of myopia and is usually defined by the presence of an ocular axial length longer than26mm or a refractive error of greater than-6.00diopters （D）. The prevalence of high myopia is1%-2%in China. The pathogenesis of high myopia is complex and remains unclear. The environmental and genetic factors both contribute to the myopia, but the genetic factors play an important role in the development of high myopia. Twenty-one chromosomal loci or genes associated with high myopia have been identified up to date. Next generation sequencing technologies have recently been applied to the discovery of the genetic causes of human diseases. Whole exome sequencing （WES） focuses on the protein-coding regions. It reduces the cost and has been the most efficient option. WES has identified many genes for Mendelian diseases successfully since2010. This provides a new approach to identify the causative gene and study the mechanism of the hereditary high myopia.Methods:Three affected individuals from the same family with high myopia were performed the whole exome sequencing. The candidate causative genes and variants were screened in accordance with the prioritization strategies. Sanger sequencing was carried out to validate the variants for the available family members, and to further narrow down the candidate variants. The remaining candidate variants were analyzed by bioinformatics tools for the potential pathogenic variants and genes. mRNA relative level expression patterns of the candidate gene in mouse different tissues were studied by using reverse transcription PCR （RT-PCR）. The causative gene was validated by linkage analysis.Results:Nine candidate variants were identified by whole exome sequencing and variants prioritization strategies. Three variants on X chromosome remained as potential pathogenic variants after sequencing validation analysis on all the available family members. A nonsense mutation c.228T>A p.Tyr76*in A3gene was considered as the pathogenic variant by bioinformatics tools. This variant was not found in at least600normal individuals. The RT-PCR results showed that gene A3was highly expressed in mouse eye tissues. It has been reported that gene A3expressed in retina cone cell and participated the deactivation of the phototransduction cascade. Linkage analysis showed that the A3gene was co-segregate with high grade myopia phenotype in the family, supporting the pathogenicity of the gene. A3gene deficient larval zebrafish revealed that gene A3deficiency resulted in prolonged ERG b-wave amplitude recovery times. This phenomenon was consistent with the ERG measurements data in the high myopia individuals.Conclusion:High myopia disease has distinct genetic heterogeneity, including many inheritance modes and genes. In this study, we reported the first case of X-linked dominant high myopia and identified A3gene as the possible causative gene. The combination of bioinformatics tools, linkage analysis and whole exome sequencing is a powerful strategy for identifying gene responsible for Mendelian disorders. Part â…¡ The establishment of human gene variant database-LOVDHuman Variome Project （HVP） is an international consortium of clinicians, geneticist, scientist and researchers in bioinformatics around the world. Its purpose of HVP is to build gene specific database, collect and share all genetic variants from published papers and research institutions, analyze the relations between genotype and phenotype, and explore the molecular pathogenesis. The project aims to contribute to the human genetic disease prevention and gene diagnostics. The HVP consortium expects to establish the country nodes and creates the database to encourage the collection, curation and sharing of all genetic variations affecting human diseases. The LOVD （Leiden Open source Variation Database） is a flexible, freely available tool for gene-centered collection and display of DNA variations. The LOVD platform recommended by the HVP has been accepted by many institutions in the world. LOVD platform was used to build the human variation database in China node （http://www.genomed.org/LOVD2/）. Currently,96genes were created with a collection of a total of14859variants, including9224unique variants. Pathogenic variants and likely pathogenic variants account for about37%of data. The indels and substitute variants are the most common variants which spread out different exons of those genes. However, pathogenic variants may concentrate in limited exons of a gene. For example the exon30-40in the MYH7gene and exonl-7in the CDC73gene. A change c.1032G>A in the last base of exon6in the KCNQ1gene does not alter the coded alanine （p.Ala344Ala）, but instead affects the mRNA transcript. A deep intronic variation c.903+469T>C in the MTRR gene is the most frequent pathogenic variation causing the cblE type of homocystinuria. Besides, there are some pathogenic variations in the5’-UTR and3’-UTR region, such as c.-3G>C in BRCA1gene and c.1277₁278delTAA in GLA gene. In conclusion, LOVD can be used to study the hot spots, variant types, frequency, genetic mode, ethic difference, relation between genotype and phenotype etc. The database will also provide a great resource to researchers, geneticist and clinicians, while doing the genetic test and variants interpretation.