Localization The Disease-associated Genes Of Keratoconus In Chinese Han Population

Background:Keratoconus is a corneal ectasia disease,characterized by progressive central corneal thinning,protruding partly as conical shape,thus developing high myopia and irregular astigmatism.Glasses and appropriate corneal contact lens are used for relieving symptom at early stage,while corneal transplantation surgery are needed when it comes to severe decreased vision.It is reported that the prevalence and incidence rates assume higher among Asians,with a more serious manifestation and an earlier onset age,than the Caucasian population.However,studies conducted by Asians are rare,and the exact etiology remains unclear.With the coming of the Precision Medicine and Targeted therapy era and roaring development of sequencing technology,gene theory plays a more important role during the several theories.There are totally more than 40 genes have been localized and cloned worldwide,but,to our regret,keratoconus shows high genetic heterogeneity,that is,same mutation can cause different symptom,likewise,same symptom can be caused by different mutations.Neither of any gene can be account for the vast majority of cases.Given to aforementioned reasons,we conduct this research to find keratoconus-related genes in Chinese Han population,enlarge mutation spectrum and replicate previous results,hoping to provide theoretical foundation for early diagnosis and targeted therapy.Methods:Totally,there are 53 sporadic primary keratoconus patients and 100 unrelated population-matched controls are included in this study.Following thorough inquiry,negative family histories were taken,each participate underwent a comprehensive ocular and systemic evaluation by the same experienced doctor.Genomic DNA samples were extracted from peripheral blood.Candidate genes were screened by next-generation sequencing in KC patients and were further identified by Sanger sequencing.Positive loci were screened in healthy controls subsequently.Computational algorithms are used to predict the effect of amino acid substitution on the corresponding protein.Results:76 variants were detected by next-generation sequencing in keratoconus coho rt.After removing known single nucleotide polymorphisms,loci met following co nditions:（i）with minor allele frequency（MAF）<0.1%（according to data from the May 2012 release of the 1KG project and dbSNP database）;（ii）absence of the res ults of whole-exome sequencing（WES）data performed in 220 Chinese Han popul ation without other ocular disorders were subsequently screened by Sanger seque ncing to affirm the results.Except one false-positive loci,there are 14 new varia nts were finally predicted to be potentially related with keratoconus,including 1d eletion mutation（COL4A4:c.3636₃637de1:p.R1212fs）;1 insertion mutation（TGFBI:c.624+7->A）;12 missense mutations（ZNF469:c.2059G>A:p.E687K;c.2137C>A:p.P 713T;c.3466G>A:p.A1156T;c.3749C>T:p.P1250L;c.4300G>A:p.D1434N;c.4684G>A:p.E1562K;c.7262G>A:p.R2421H;FNDC3B:c.455C>T:p.P152L;LOX:c.95C>T:p.P32L;MPDZ:c.28G>A:p.A10T;COL5A1:c.1372C>T:p.P458S;TIMP3:c.476C>T:p.S159F）.All of the aforementioned variants were heterozygous without the occurrenc e in healthy controls.Conclusion:1.This is the first study which screens so many keratoconus-related genes using next-generation sequencing technology.2.Fourteen new variants in eight genes were detected after further identifying,which enlarges the mutation spectrum.3.This study confirmed that the keratoconus is genetically heterozygous,provided new breakthrough for pathogenesis and theoretical foundation for early diagnosis and targeted therapy.