Screening And Research Of Rare Variants In The Coding Region Of ABCA1 Gene In Patients With Primary Open-angle Glaucoma

Objective: Primary open-angle glaucoma?POAG?is a leading cause of irreversible blindness worldwide.The ABCA1 gene is one of the susceptibility genes of POAG.In this study,we explored the rare nonsynonymous variants in the coding region of ABCA1 gene in patients with POAG.Methods: From March 2017 to March 2018,a total of 398 POAG patients and198 healthy people were recruited from the Department of Ophthalmology and Visual Science,Eye and Ear Nose Throat?ENT?Hospital.Rare variant analysis of the ABCA1 gene was conducted by whole exome sequencing?WES?data of these subjects.The rare variants of ABCA1 gene screened in POAG patients were verified by Sanger sequencing,and the frequency was test in healthy subjects.Bioinformatics analysis and statistical analysis were performed on these rare variants of ABCA1 gene.Then these ABCA1 rare variants were classified according to the standards and guidelines of American College of Medical Genetics and Genomics?ACMG?.Results: 1.The results of WES: A total of 21 rare nonsynonymous variants?minor allele frequency,MAF<0.01?were detected in the coding regions of ABCA1 gene in 27 subjects of the 398 POAG.All of these 21 variants are heterozygous,including 19 missense variants and one stopgain variant?c.2725C>T?p.Arg909X??and one frameshift insert variant?c.1507₁508insGAGGT?p.Glu503GlyfsX7??.And there were four novel variants were detected: c.4310C>A?p.Thr1437Asn?,c.3772G>T?p.Asp1258Tyr?,c.775A>G?p.Lys259Glu?and c.1507₁508insGAGGT?p.Glu503 Gly fsX7?.2.The results of Sanger sequencing: Of the 21 ABCA1 rare variants detected in POAG patients,one was not verified?c.1486C>T?p.Arg496Trp??by Sanger sequencing,which was detected in one sample.And the other variants were all verified by Sanger sequencing.3.Comparison of the test results of ABCA1 gene between POAG patients and healthy people: In 398 POAG patients,the detection rate of the ABCA1 rare variants was 6.5%?26/398?,while in the 198 healthy people,five rare nonsynonymousvariants were detected in the ABCA1 gene in five subjects respectively?2.5%?.The detection rate is significantly different between the POAG group and the healthy control group.The rare variants of ABCA1 gene was a risk factor for POAG?P<0.05,OR=2.70?.4.The results of bioinformatics analysis: Multiple amino acid sequence alignment of ABCA1 amino acid sequences showed that there were 12 variants took place at highly conserved regions.And there were seven variants are predicted to be harmful by software.The region with the most variants is the extracellular domain of the ABCA1 protein.According to the standards and guidelines of ACMG,there were eight likely pathogenic rare variants: c.3772G>T?p.Asp1258Tyr?,c.1507₁508ins GAGGT?p.Glu503GlyfsX7?,c.2725C>T?p.Arg909X?,c.2518T>C?p.Trp840Arg?,c.2471C>T?p.Ser824Leu?,c.2014C>T?p.Arg672Trp?,c.1826C>T?p.Thr609Met?and c.1040A>G?p.Tyr347Cys?.And the other 12 rare variants were classified as uncertain significance.Conclusion: 1.In this study,the detection rate is significantly different between the POAG group and the healthy control group.It suggested that there were some relationship between POAG and rare nonsynonymous variants in ABCA1.2.In this study,rare variants ofABCA1 gene were detected in POAG patients,including four novel variants,which expand the variation spectrum of the ABCA1 gene.