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Splicing Variation Analysis Of Hmbs Gene In Acute Intermittent Porphyria In China And Pathogenicity Validation Of Two Non-classical Regional Splicing Variations

Posted on:2023-11-24Degree:MasterType:Thesis
Country:ChinaCandidate:J J WangFull Text:PDF
GTID:2544306794462424Subject:Endocrine
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Objective:Acute intermittent porphyria(AIP,OMIM 176000)is a rare autosomal dominant disorder caused by defects in hydroxymethylbilane synthase(HMBS).HMBS gene diagnosis is the gold standard for the diagnosis of the disease,and it is very important for the diagnosis of patients with acute attack,the guidance and genetic counseling of patients with latent disease.In the past,the detection and interpretation of gene variation paid more attention to amino acid changes caused by exon variation,but less attention to splicing abnormalities caused by exon and intron.In recent years,more and more evidences show that splicing variation plays an important role in the pathogenesis of diseases.The interpretation of pathogenicity of variant can guide clinical decision making correctly.Therefore,by analyzing the splicing variation of HMBS genes in China and conducting in vitro functional experiments on the pathogenicity of non-classical splicing variation,this study provides reliable scientific evidence for gene diagnosis and genetic counseling of AIP,and provides molecular basis and new ideas for the pathogenesis and treatment of AIP in the future.Methods:Pubmed database,Embase database,CNKI,Wanfang Medical Science and other databases were used to search Chinese literatures related to splicing variations in HMBS genes(as of September 2021).For the two non-classical sites of splicing variation(c.33+5G>A and c.88-4_-16 del AAGTCTCTACCCG)to verify the pathogenicity: Firstly,the pathogenicity of non-classical Splicing variants was predicted by Splicing related bioinformatics software(Human Splicing Finder,Splice Site Prediction by Neural Network software,Net Gene2 Server software).Then,minigene assay was performed to clone the target gene fragment with mutation sites,and then the wild-type and mutant recombinant vectors were constructed.After transfection into HEK293 T and MCF-7cells,RNA was extracted and c DNA reverse transcription was carried out.And electrophoresis and sequencing technology were used to verify the influence of different sites in non-classical regions on splicing.Results:1.A total of 26 splicing variation sites were retrieved from 7 literatures reported in China,among which 4 sites were repeated variation sites(c.33+5G>A、 c.88-1G>C、c.88-2A>G and c.913-2A>G).Splicing variation was found in all introns except intron No.4,No.6,No.8,No.9 and No.12 of HMBS genes,and There were 6 variants in intron 2and 5 variants in intron 11.Fifteen of them were splicing variations located in the classical region,of which 12 were receptor sites.2.The prediction results of two software for variant c.33+5G>A indicated that the original donor site was destroyed,suggesting that shearing might be affected.Results of its minigene assay showed an abnormal transcript that retained 91 bp on the left side of intron 1,and its splicing patterns was Exon1(33bp)-▽Intron1(91bp)-exon2(54bp).Protein primary structure analysis showed that protein translation was terminated prematurely(p.(E12Vfs*71)).3.Two software of variant c.88-4_-16 del AAGTCTCTACCCG suggested that the original receptor sites were destroyed by mutation,suggesting that shearing was affected.Results of its minigene assay showed an abnormal transcript that lossed 15 bp at the head of intron 3,and its splicing patterns was Exon2(54bp)-△Exon3(58bp).Primary structure analysis of the protein showed that it resulted in loss of amino acids(p.(L88_Q102del)).Conclusion:1.Among the HMBS gene variations reported in China,there are 26 splicing variation sites,and intron 2 and intron 11 are high frequency variation introns.Most of the splicing variation sites are in the classical region,and the receptor sites are more common.2.The variant c.33+5G>A produced abnormal transcripts with intron retention.The variant c.88-4_-16 del AAGTCTCTACCCG showed abnormal transcripts producing exon deletion.The non-classical sites in the above two HMBS gene variants all caused the abnormal splicing,suggesting c.33+5G>A and c.88-4_-16 del AAGTCTCTACCCG were pathogenic.
Keywords/Search Tags:Acute intermittent porphyria, HMBS gene, Splicing variant, Minigene assay
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