Genetic Identification And Functional Research Of Hereditary Concomitant Exotropia

Part I: Identification of candidate genes in concomitant exotropia pedigreesObjective: By means of genetic research,the pedigrees and sporadic cases of concomitant exotropia were collected to explore and identify the candidate genes of concomitant exotropia.Methods:1.In this study,58 individuals were enrolled from 11 concomitant exotropia pedigrees(including intermittent exotropia and constant exotropia)which was treated in the Second People’s Hospital of Yunnan Province from January 2016 to December2018.The clinical examination data and 4-5ml peripheral blood/each person was got from the 58 individuals.570 individuals with sporadic concomitant exotropia were enrolled from January 2016 to March 2019.We collected their clinical data,4-5ml peripheral blood/each person,and some of the patients’ extraocular muscles(which removed from the surgically).Also,220 normal individuals were enrolled from July2021 to August 2021.We collected their clinical data,4-5ml peripheral blood/each person.Whole exome sequencing was performed in 28 patients and 11 normal individuals of 11 concomitant exotropia pedigrees by second-generation sequencing.After that,the sequencing data were taken for biological information analyzed,screened and compared.According to the results,all 58 individuals from 11 concomitant exotropia pedigrees were verified by Sanger sequencing and co-isolation analysis.2.According to the results of Sanger sequencing and pedigrees co-isolation analysis,4 candidate genes and mutation sites were selected to perform Sanger sequencing in sporadic concomitant exotropia samples to verify the mutation frequency.3.According to the verification results of sporadic samples,capture and sequencing chips of 3 candidate genes were designed,and 220 patients with sporadic concomitant exotropia were selected for capture and sequencing of whole exons,in order to discover more mutation sites of candidate genes.Results:1.According to pedigrees analysis and sequencing results,all the 11 concomitant exotropia pedigrees enrolled in the study were autosomal dominant inheritance.The results of pedigrees co-isolation analysis revealed 8 potential pathogenic candidate genes(containing 12 mutation sites):(BBS1 c.163G>A(p.V55M);AUTS2c.979C>T(p.R327C);GTDC2 c.1181G>A(p.R394Q);COL4A2c.1328C>G(p.P443R)、 c.2051G>C(p.G684A)、 c.4255A>G(p.M1419V)、 c.4256T>C(p.M1419T);SYNE1 c.1838C>T(p.S613F)、c.6992C>A(p.A2331D);LOXHD1c.6413G>A(p.R2138Q);HERC2 c.10474T>C(p.S3492P)and CDH3 c.2395G>A(p.A799T)).All the 12 mutations are missense mutations.We got 3different mutations sites from COL4A2 in 3 pedigrees,and 2 different mutations sites from SYNE1 in 2 pedigrees.2.We identified BBS1 same mutation site(c.163G>A(p.V55M))in 5 cases among 552 sporadic concomitant exotropia samples;there is no AUTS2 c.979C>T(p.R327C)mutation was found in 496 sporadic samples;there is no GTDC2 c.1181G>A(p.R394Q)mutation was found in 422 sporadic samples.None of the three gene locus mutations was detected in 220 normal people.There were 13 cases of SYNE1 c.1838C>T(p.S613F)mutation was found in 511 sporadic samples,and also14 cases was found in 220 normal samples of the same site mutation.3.Among 220 sporadic concomitant exotropia patients by whole-exon capture sequencing of 3 target genes,we identified 3 other mutations in BBS1(BBS1c.1339G>A(p.A447T)、c.1660A> T(p.S554C)and c.1772C>T(p.A591V)),7 other mutations in AUTS2(AUTS2 c.661-143892T>G 、 c.*637G>A 、 c.*151T>C 、c.2600A>G(p.K867R)、 c.*173＿*176dup、 c.-336 del and c.*1821G>A)and 4 other mutations in GTDC2(GTDC2 c.-346C>T、c.-259G>A、c.484G>A(p.D162N)and c.1195G>A(p.E399K)).None of these mutations was detected in 220 normal individuals.Conclusion(s): In this study,eight genes(BBS1、AUTS2、GTDC2、COL4A2、SYNE1、LOXHD1、HERC2 and CDH3)and their mutation sites were identified for the first time for co-isolation in concomitant exotropia pedigrees,which may be the pathogenic genes causing hereditary concomitant exotropia.Part Ⅱ: The functional and mechanism research of the candidate gene BBS1 in concomitant exotropiaObjective: According to the results of sequencing and identification in concomitant exotropia families and sporadic samples,the candidate gene BBS1 was selected for the next research target.4 mutation sites(BBS1 c.163G>A(p.V55M),BBS1 c.1339G>A(p.A447T),BBS1 c.1660A> T(p.S554C)and BBS1 c.1772C>T(p.A591V)The function and influence of T(p.A591V))were used for explore their functions and effects by studying extraocular muscle samples and in vitro cell function experiments.Methods: 1.RT-q PCR was used to detect and analyze BBS1 RNA in extraocular muscle samples of patients with concomitant exotropia,in order to investigate the expression level of candidate gene BBS1 in different patients with concomitant exotropia.2.The lentivirus vector plasmid with the candidate gene BBS1 wild type and 4 mutation sites was constructed,which was used to extract RNA for quantitative analysis of BBS1 m RNA at transcription level after transfection of 293 T cells and ARPE-19 cells,and the total protein was extracted for Western Blot analysis.To examine the effects of target mutation sites on transcription and translation levels of cultured cells in vitro.3.The ARPE-19 cells which transfected with lentiviral vector plasmid were take into Co-Immunoprecipitation experiment in order to search for other proteins that might interact with the target protein BBS1.According to the results,the functional and mechanism of candidate gene BBS1 that may cause concomitant exotropia was discussed.Results: 1.RT-q PCR analysis of total RNA extracted from external ocular muscle tissues of patients with concomitant exotropia showed no significant difference in the relative expression level of wild-type m RNA between the BBS1 mutant group and the non-BBS1 mutant group(P>0.05).2.The results of RT-q PCR analysis of 293 T cells and ARPE-19 cells transfected with lentivirus vector plasmid of BBS1 wild type and 4 mutant sites showed no significant differences in m RNA relative expression levels between the wild type and mutant groups(P>0.05).Western Blot analysis showed that there was no significant difference in the protein expression levels of BBS1 wild type and mutant groups(P>0.05).3.Co-Immunoprecipitation results showed that BBS4,BBS8 and BBS9 were significantly weaker than wild-type BBS1-WT at BBS1-V55 M sites,and the difference has statistically significant(P<0.05).Conclusion(s): The target mutation site of BBS1 does not trigger changes at the the level of RNA transcription and protein translation,but the BBS1-V55 M weakened its binding to the BBS4,BBS8,and BBS9 proteins in the BBSome complex.BBS1-V55 M may affect the structure and/or function of BBsome through certain mechanisms,and thus induce concomitant exotropia.