Gene Mutation Analysis And Genotype-phenotype Correlation Of Type Ⅳ Collagen Related Diseases

Objective: Both Alport syndrome（AS）and thin-basement-membrane nephropathy（TBMN）are type IV collagen related diseases,however,the prognosis of the two diseases is very different.For children or women,it is difficult to distinguish them between clinical manifestations and pathological changes.Therefore,the discovery of the new gene mutations could expand the COL4 An gene mutation spectrum and is significant for distinction and prenatal diagnosis.This study explored the characteristics of the COL4 An gene mutation and analyzed the relationship between genotype and phenotype of 6 families with type IV collagen related diseases.Methods: The clinical manifestations and pathological features of 16 patients in 6 families were described.The COL4 An gene of 6 families was screened by next generation sequencing（NGS）,and the suspected candidate mutations were verified by Sanger sequencing.Three online programs（Poly Phen-2,SIFT and Mutation Taster）were employed to predict pathogenicity of the missense variants detected by the NGS analysis.Moreover,for amino acid substitutions,multiple sequence alignments through Vector NTI Advance 10-Align were used to evaluate evolutionary conservation.Potential splicing mutations were analyzed by the Berkeley Drosophila Genome Project（BDGP）.RNA was extracted from the skin cells and kidney tissue cells of some patients to verify the splicing products.For one detected splicing variant,we adopted T-A cloning to explore and verify the proportion of different products.We also tied to prove that whether there was inactivation of the X chromosome in two female patients of the same family.Result:（1）By next generation sequencing,nine of the new COL4 An gene mutations were identified in 16 patients.They were COL4A3-c.1697G>T（p.Gly566Val）,COL4A3-c.2687T>C（p.Phe896Ser）,COL4A3-c.2990G>A（p.Gly997Glu）,COL4A4-c.1715G>C（p.Gly572Ala）,COL4A4-c.3289+3A>G,COL4A5-c.1424-4C>G（p.Lys474_Gly475ins Val）,COL4A5-c.2777G>T（p.Gly926Val）,COL4A5-c.4298-2 A>T,COL4A5-c.1552G>A（p.Gly518Arg）.Among of them,three gene mutations（c.1697G>T & c.2687T>C & c.2990G>A）might be associated with thin-basement-membrane nephropathy,two mutations（c.1715G>C & c.3289+3A>G）might be associated with autosomal recessive Alport syndrome,and other four mutations（c.1424-4C>G &c.2777G>T & c.4298-2 A>T & c.1552G>A）might be related to X-linked Alport syndrome.Among these 9 novel mutations,6 were missense mutations and 3 ones could lead splicing errors.（2）In silico analysis by three different software programs（SIFT,Poly Phen-2 and Mutation-Taster）showed that the 5 new missense mutations found in this study were disease causing（c.1697G>T,c.2990G>A,c.1715G>C,c.2777G>T and c.1552G>A）.The rest missense mutation c.2687T>C was a benign mutation.In addition,the missense mutation pathogenicity scoring system indicated that c.2687T>C and c.1715G>C were moderately pathogenic,while c.1697G>T,c.2990G>A,c.2777G>T and c.1552G>A were highly pathogenic.Multiple sequence alignments showed that all of them were highly conservative except c.2687T>C.Therefore,we could conclude that COL4A3-c.2687T>C（p.Phe896Ser）was suspected to be a SNP.（3）The mutation c.3289+3A>G was a splicing mutation in intron 34 of COL4A4 gene which can lead to the deletion of the entire exon 34.The mutation c.1424-4C>G in the intron 21 of COL4A5 activates a potential splicing site in intron 21,which lead to a insertion of three bases TAG into the front of exon 22（c.1423₁424ins TAG;p.Lys474_Gly475ins Val）.c.4298-2A>T in intron 47 of COL4A5,as the canonical splice acceptor site,lead to a complex splicing pattern with the formation of two transcripts.One was the product of skipping of entire exon 48,and the other one was transcript of skipping exon 48 with insertion of a 152-bp fragment of a cryptic exon derived from intron 47.（4）The transcripts of the mutation c.4298-2 A>T were inserted into the p GEMT Easy vector.Sequencing results showed that approximately 20%（10/48）of the c DNA products only missed the whole exon 48,and 30%（14/48）displayed lack of the exon 48 but with insertion of a cryptic exon.（5）Two female patients in family X3 did not have a skewed inactivation of the Xchromosome,and patient IIc was a compound heterozygote.Result: We found a total of 8 mutations of the COL4 An gene and one suspected SNP,which expanded the gene mutation database.It was noteworthy that we reported the first Chinese female patient with X linked Alport syndrome caused by a compound heterozygous mutation.