Identification Of Pathogenic Genes And Multi-omics Study Of Townes-brocks Syndrome

Objective:Townes-Brocks Syndrome(TBS)is a rare autosomal dominant disease with a worldwide incidence rate of 1-9/1,000,000.Up to now,a total of 8 Chinese TBS cases have been reported.TBS can cause dysplasia of multiple systems.Major features include imperforate anus or anal stenosis,dysplastic ears in(overfolded superior helices,microtia)and typical thumb malformations(preaxial polydactyly,triphalangeal thumbs,hypoplastic thumbs)without hypoplasia of the radius.Minor features include sensorineural and/or conductive hearing impairment,foot malformation,renal malformation with or without renal malformation dysfunction,genitourinary malformation and congenital heart disease.The phenotypic heterogeneity of TBS is very significant.Multiple malformations and incomplete penetrance make it difficult to reach early clinical diagnosis.The SALL1 gene is the main pathogenic gene of TBS,but its specific pathogenic molecular pathway remains unclear.Relying on China’s precious genetic resources,our research group,together with more than 50 basic research and clinical professional teams in the field of auditory research,established the "China deafness genetics Consortium"(CDGC)in 2013,and organized the collection of 23927 deafness samples and 7 regional matching data covering 41 nationalities in 31 provinces and autonomous regions of China,In 202 normal control samples,20394 cases of hereditary deafness were detected.Based on the massive gene data of CDGC large-scale deafness cohort,the purposes of this study are: 1)to achieve accurate diagnosis of TBS patients in CDGC large-scale deafness cohort by combining clinical phenotype data and molecular genetic data,and draw complete clinical phenotype spectrum of TBS and SALL1 gene mutation map by combining with public database information;2)to conduct genotype-phenotype correlation analysis based on complete clinical phenotype spectrum of TBS and SALL1 gene mutation spectrum;3)to explore the molecular regulatory network of TBS caused by SALL1 mutations through deep clinical phenotype measurement and multiomics study.Method:By clinical phenotype analysis combined with molecular genetic study,we preliminarily screened and accurately diagnosed suspected TBS patients in CDGC large deafness cohort.We used the targeted capture kit HHL-785 of 785 deafness related genes for highthroughput next-generation sequencing to detect genetic variation.We enrolled suspected patients with 2 or more major features of TBS,and performed large-scale screening and bioinformatics analysis on all cases with heterozygous SALL1 mutations in the gene mutation database of CDGC cohort.According to the expert guidelines of American College of Medical Genetics and Genomics(ACMG),we analyzed the pathogenicity of candidate SALL1 candidate mutations and identified the patients with SALL1 pathogenic mutation.Through the comprehensive analysis of clinical phenotype screening results and gene data screening results,we identified and accurately diagnosed of TBS patients in CDGC cohort.In this study,we reviewed all the reported mutation sites,mutation types,genotypes and phenotypes of SALL1 from1998 to January 2021,and analyzed the genotype-phenotype correlation between the reported cases and diagnosed cases in present study.In order to explore the molecular pathogenesis of TBS caused by SALL1 mutation,we introduced the advanced idea of phenotypic group research.For the first time,clinical phenotypic depth measurement,multi-omics data collection and bioinformatics analysis of family members of patients with TBS and normal people were carried out,RNA-seq and Label-free technics were used for transcriptomics and proteomics study.In this study,family HL-023253 with two TBS patients was selected.Five family members of three generations were evaluated by audiology,radiology and blood biochemistry tests.Peripheral blood samples of seven family members were collected for whole blood RNA-seq and plasma proteomics.We divided the patients and healthy family members into the experimental group and the control group.Principal component analysis(PCA),cluster analysis(cluster)and stepwise linear regression analysis were used to analyze the RNA or protein molecules with absolute value of log(FC)greater than 1 and p value or adj.p value less than 0.05 in the experimental group.Differentially expressed genes(DEGs)and differentially expressed proteins(DEPs)were identified,and then IPA(ingenuity pathway)was used to explore the upstream and downstream genes,related diseases and functions and related molecular networks of DEGs or DEPs,combined with the comprehensive analysis of clinical deep phenotype measurement data,we explored the molecular pathway and related mechanism of SALL1 mutation leading to TBS.Results:Combined with clinical phenotype data and molecular genetic test results,we identified 10 TBS patients from 6 families in large CDGC deafness cohort.5 patients and 9 patients met the clinical and genetic criteria of TBS,and we found a patient with typical TBS phenotypes but didn’t detect any mutations in SALL1 exons or any SALL1 copy number variations.In this study,6 pathogenic variants of SALL1 were identified,among which c.826C>T was reported by previous studies,and the other 5 were novel mutations,including c.1489C>T(p.Gln497*),c.1499＿1500del(p.Lys500Argfs*15),c.1393C>T(p.Gln465*),c.3207 dup C(p.Asn1070Glnfs*32)and c.1341＿1347del C AAACAC(p.Phe447Leufs*44).By deep measurement of clinical phenotypes in TBS patients and healthy family members in family HL-023253,we found some unreported clinical manifestations,such as hyperuricemia without renal structural malformations,subclinical hypothyroidism and abnormal vestibular function,in addition to common clinical phenotypes such as external ear deformity and anal atresia.Whether these newly discovered clinical manifestations are related to SALL1 mutations,more cases and relevant researchse are needed.At present,it is not possible to distinguish which symptoms are related to SALL1 mutation,and which patients with TBS have combined other diseases unrelated to TBS disease.PCA and cluster analysis showed that although SALL1 does not expresse in peripheral blood,there were significant differences between the whole blood transcriptomic and plasma proteomic profiles in TBS patients and normal family members.IPA analysis showed that DEGs and DEPs were significantly enriched in many cancer-related molecular networks.The deep mining of these data will give suggestions to the molecular network and related pathogenesis of SALL1 transcription factor regulations.Conclusion:In this study,we systematically mapped the current clinical phenotype spectrum and SALL1 gene mutation spectrum of TBS,and conducted genotype-phenotype association analysis,and comprehensively combed the correlation of SALL1 mutation sites,mutational type,genotype and phenotype severity.Deep clinical phenotype measurement further found phenotypes that may be related to TBS,suggesting the specificity of clinical manifestations of TBS and the importance of vestibular function test,auditory test,renal function monitoring and uric acid level monitoring.It is the first time to find significant differences in whole-blood transcriptomic and serum proteomic profiles between TBS patients and healthy family members,as well as the association with cancer molecular pathways by multi omics data analysis.It suggests that blood,as an easily available tissue,may indicate the regulatory effect of SALL1 gene pathogenic variations on downstream molecular network,which provides a basis for the subsequent work on for TBS related prognostic indicators and the molecular mechanism of SALL1 leading to TBS.It is important for accurate clinical diagnosis of TBS,reducing recurrence risk and exploring treatment approaches.