A Study Of Alport Syndrome Family In Western China And Its New Mutation In COL4A5 Gene

Objective: To sequence COL4A5 gene in 1 case of AS-associated children and their families by exon capture-secondary gene sequencing（NGS）technology.The COL4A5 mutant gene was found in the Pumbed database of cinvar,HGMD and COL4A5 mutation databases.Search,no reports were found.The Minigene method was applied to the new mutation in COL4A5 to study its genetic significance.Enrich the AS syndrome COL4A5 gene mutation database to explain the genetic significance of this new mutation.Methods:A detailed history of 10 members of a family of Alport syndrome,detailed genetic mapping,pedigree genetic mapping,whole-exon second-generation sequencing technology gene sequencing and analysis;and the construction of Mini-COL4A5 gene,transfected in HEK293 T cells Mini-COL4A5 gene plasmid（Mini-COL4A5-WT/MiniCOL4A5-Mut）,fluorescent control plasmid（FG12）;Trizol extracted RNA for RT-PCR and electrophoresis;Mini-COL4A5-WT group and Mini-COL4A5-Mut group The RTPCR product T clone was sequenced.Results:The splicing mutations in the COL4A5 gene were found in the positive samples of the probands: NM₀00495: c.1588-2A>G.NM₀00495:c.1588-2A>G mutations are distributed in the family’s proband: hemizygous mutation,the mother of the proband: the codeshift heterozygous,the mutant proband’s daughter: the transplanted heterozygous mutation,the proband The grandmother: the heterozygous mutation of the frameshift,the proband of the proband: a hemizygous mutation.NM₀00495: c.1588-2A>G mutation to the COL4A5 gene The first inferior base in the intron sequence immediately upstream of base 1588（ie,intron 23）was abruptly changed from A to G.Predicting this variation by Mutation Taster software affects protein function.The first-generation RT-PCR product of the Mini-COL4A5-WT group was completely matched with the minigene sequence of COL4A5,and matched with the CDS protein coding region of COL4A5（EXON23-EXON24-EXON25）.The sequencing of the RTPCR product of the Mini-COL4A5-Mut group was sequenced.The minigene sequence of COL4A5 is 10 bp less than the 5’ end of COL4A5 gene exon 24（EXON24）,compared to the CDS protein coding region of COL4A5（EXON23-EXON24-EXON25）at 5 of the COL4A5 gene exon 24（EXON24）’The deletion occurred at the end;the Mini-COL4A5-Mut group RT-PCR product T clone sequence 4 of the 7 T clones were exon 24（EXON24）with 5 bp deletion at the 5’ end and 1 exon 24（EXON24）The 5’ end lacks 76 bp and the two are exon 24（EXON24）hops.Conclusion: 1.COL4A5 gene mutation: NM₀00495: c.1588-2A>G（the intron sequence immediately upstream of the base 1588 of the COL4A5 gene,ie,the second base of the intron in the 23 rd intron is abruptly changed from A to G）The genetic pattern in the family of the proband is consistent with the X-linked dominant inheritance pattern of Alport syndrome 2.COL4A5 gene mutation: NM₀00495: c.1588-2A>G Search for "COL4A5" AND "c.1588-2A>G" in the clinvar,HGMD,COL4A5 mutation database and Pumbed database.COL4A5 gene mutation: NM₀00495: c.1588-2A>G is a new mutation 3.According to the ACMG genetic variation classification criteria and guidelines,this mutation is classified as a suspected pathogenic mutation;4.COL4A5 gene mutation: NM₀00495: c.1588-2A> G,there is no change in the wild type splice site（SD）,but the wild type splice site is changed by position（SA）,which will make Subsequent translations produce frameshift mutations.