Fine Mapping Of Pathogenic Gene In A Porcupine Model Of Spontaneous Syndromic Deafness Associated With Pigmentation Abnormalities

Objective: A porcupine model family of spontaneous syndromic deafness associated with pigmentation abnormalities was established,and its pathogenic gene and mutation site were accurately identified in this study,with the aim of providing theoretical foundation and reference to understand the corresponding molecular pathogenic process of human hereditary deafness.Methods: 1.Establishment of a porcupine model family of spontaneous syndromic deafness associated with pigmentation abnormalities: Four mutant male porcupines were bred with wild-type female porcupines.Then the inherited colony was expanded to establish a hereditary deafness disease model family that can inherit stably,which was detected and compared with the phenotypic characteristics and quantitative ratio of mutant and wild-type individuals in all offsprings.Next,a chi-square test was performed to confirm the mutant phenotype’s inheritance pattern.2.Phenotype identification: Gross appearance examination was conducted to observe and compare the skin,hair(including thorns)and retinal color of mutant individuals and wild-type individuals,and to check whether the mutant individuals had microophthalmic phenotype.Skin and cochlea tissues of mutant and wild-type individuals were collected for pathological observation.Brain stem evoked potential(ABR)was also used to examine and identify auditory function.3.Fine mapping of pathogenic gene and mutation site: Firstly,the Bulk Segregant Analysis(BSA)method was used to screen SNPs or In Dels in porcupine model family.Secondly,wild-type porcupine skin tissue samples were collected for non-parametric transcriptome sequencing,and candidate gene transcripts were annotated and extracted.Then the candidate genes were annotated by bioinformatics method.Finally,the sequence of the mutation site of the candidate gene was amplified by PCR and verified by Sanger sequencing.Results: 1.A porcupine model family of spontaneous syndromic deafness associated with pigmentation abnormalities was successfully created: Four male mutant porcupines of the F0 generation were bred to produce 38 F1 generations,with 21 mutants and 17 wild-types.The separation ratio between mutant traits and wild-type traits was 1.24.It was verified by genetic chi square test that its genetic model was in line with autosomal dominant inheritance.The F1 generation mutant individuals were bred to obtain 3 F2 generation,of which 2 were ominant homozygotes.2.Phenotypic identification results: The skin and hair(including thorns)of heterozygous porcupine with spontaneous pigmentation syndrome deafness mutation have different degrees of abnormal pigmentation,the eye retina is lightly stained,and the skin and hair of homozygous mutant individuals have no pigmentation(complete albinism),and show ocular malformation(microphthalmos);The structure of cochlear stria vascularis of mutant porcupine is loose and collapsed,the internal capillaries are reduced,and the boundary with adjacent tissues is unclear;It was found that wild-type porcupine could elicit distinguishable and repeatable ABR waveforms at full frequency(0.5 k Hz,1k Hz,4 k Hz,8 k Hz,16 k Hz and 32 k Hz),while mutant porcupine could not elicit distinguishable and repeatable ABR waveforms at maximum loud pressure level 120 d B.The results showed that the mutant porcupine had severe sensorineural deafness.3.Fine mapping of pathogenic gene and mutation site: A total of 30931439 SNPs and 3357842 In Dels were obtained by BSA method.The SNP index and indel index in the wild-type pool minus the mutant pool were calculated Δ SNP index and Δ Indel index,and then carry out replacement test.A total of 545 SNPs and In Dels loci reached 99% confidence level.The skin tissue of wild-type porcupine was sequenced by non-parametric transcriptome sequencing,and original data of about 6 Gp were obtained.Four transcription sequences of Mitf gene of porcupine were annotated(Gene Bank entry number: MW840376,MW840377,MW840378 and MW840379),and Blast comparison with human MITF gene transcript,further annotating the exon region of porcupine Mitf.SNPs and In Dels loci of the porcupine Mitf gene sequence screened by BSA method were located according to the annotation results of this gene,and a meaningful mutation site was obtained,Mitf c.875＿877del AGA(Arg217del).Primers were designed according to this site for PCR amplification and Sanger sequencing verification.The results showed that the mutant site was consistent with the porcupine mutant phenotypic traits.Conclusion: In this study,we successfully established a porcupine model family of spontaneous syndromic deafness associated with pigmentation abnormalities whose genetic model is autosomal dominant inheritance;Phenotypic identification showed that the phenotypic characteristics of the model were very similar to those of human Waardenburg syndrome type 2;Moreover,this study accurately identified the pathogenic gene and mutation site of the porcupine model family as Mitf c.875＿877del AGA(Arg217del).The results can provide theoretical basis and reference for the study of the molecular pathogenesis of corresponding human hereditary deafness diseases.