Whole Exome Sequencing Searches For The Suspicious Pathogenic Gene ADAMTS9 Of Adolescent Idiopathic Scoliosis

ObjectiveWhole exome sequencing was used to screen the causative genes of AIS pedigree and the causative genes were verified in other pedigree and sporadic AIS populations.The expression levels of pathogenic genes were compared between AIS case group and normal control group to initially explore its possible pathogenic mechanism.Methods1)Collecting DNA in the peripheral blood from hospitalized or re-examined AIS patients and patients admitted to hospital without other known orthopaedic diseases due to trauma from June 2016 to January2018 in spinal surgery department at the first affiliated hospital of university of China.Besides,lymphocytes in the peripheral blood of AIS pedigree and normal controls were also collected;2)Whole exome sequencing was used to screen the AIS pathogenic genes in family I.The objects sequenced were ?:5(normal),?:6(AIS)and ?:7(AIS),combined with the screening ways of disease-causing genes reported in relevant studies,screening for common mutations in ?:6 and ?:7,and removing the same mutations in ?:5,filtering the candidate genes which were cosegregated in the family I,and the final screened genes were evaluated by software informatics prediction analysis and speciesconservation analysis;Targeted sequencing technology was used to screen the AIS pathogenic genes in family II and sporadic AIS population;3)Using EB virus to transfect B lymphocytes and making immortalized B lymphocytes,extracting total RNA,the expression levels of the causative gene between the AIS patients and control group were evaluated by RT-PCR.ResultsUnder the compounded heterozygous inheritance pattern,7mutations of three genes shared by ?:6 and ?:7(excluding ?:5)were found,but none of these mutations could cosegregate in the heterozygous inheritance model.Under the homozygous inheritance pattern,there were12 common mutations in ?:6 and ?:7(excluding ?:5),and ADAMTS9 c.625C>T(homozygous)were successfully filtered and cosegregated in family I.The software informatics prediction suggests that this site mutation is detrimental and the species conservation analysis suggests that the site is highly conserved;Targeting ADAMTS9 gene in the AIS member of family II.Then the compound heterozygous mutations ADAMTS9 c.625C>T(heterozygous)+ADAMTS9 c.4811G>A(heterozygous)were successfully screened,which achieved disease co-segregation of the family II members under compound heterozygous pattern;In 35 sporadic cases,one of the sporadic patients also present a compound heterozygous mutation: ADAMTS9 c.625C> T +ADAMTS9c.4811G>A.The mutations of ADAMTS9 c.625C>T(homozygous)andADAMTS9 c.4811G>A(heterozygous)were not found in 94 cases of normal control,but in a normal case,ADAMTS9 c.625C>T(heterozygous)was found.After statistical analysis,we found that the verification rate of ADAMTS9 gene mutation in AIS was significantly higher than that of the normal control group(p<0.05).There were any mutations using sanger sequencing of reported genes.RT-PCR showed that the ADAMTS9 expression levels of the AIS members in the two pedigrees were higher than the normal controls(p<0.01).Conclusion1.ADAMTS9 may be the pathogenic gene of AIS;2.The overexpression of ADAMTS9 may be involved in the pathogenesis of AIS.