Identification Of Pathogenic Gene Mutations In Chinese Alport Syndrome Patients

Objective:Alport syndrome is an inherit kidney disease ascribable to type Ⅳ collagen related genes COL4A3,COL4A4 and COL4A5 which presents with clinical phenotypic heterogeneity and without hot spot mutation regions.In this research the largest cohort of Chinese Alport syndrome patient was created to investigate the clinical feature and mutation spectrum of Chinese patient which will provide new insight of potential pathogenic genes and pathogenesis.Methods:1、This study was aim to conduct a Chinese Alport syndrome clinical cohort by biopsy proven evidence.The clinical data,biopsy results and follow-up outcome were compared.2、The mutations of patients were detected by using whole exome sequencing.Different types of inheritance pattern and mutation were calculated.3、Identification of special mutations were performed in those Alport patients without gene mutations by using whole genome sequencing to explore the possibility of uncommon types of mutations.4、Laminin related genes were screened in Alport patients and immunostaining analysis of laminin-α5 was performed in patients with laminin related gene mutations to determine whether the mutation will affect protein expression.5、Podocyte related genes were screened to determine whether mutations which lead to podocyte injury could cause Alport like phenotypes.Results:1、Clinical data showed male patients presented worse phenotypes compared to females,including earlier disease onset,larger proportions accompanied by hearing loss and greater possibility progressed to ESRD.Combined with biopsy findings,patients with abnormal immunostaining of type Ⅳ collagen suffered a worse prognosis in contrast to patients with normal staining.Mutation detection in COL4A3/A4/A5 gene revealed X-linked patients with COL4A5 gene mutations were less than previous thought.Approximately one third of patients carried COL4A3/A4 gene mutations.2、Copy number variations were detected by whole genome sequencing instead of whole exome sequencing,indicating the existence of special types of mutations.Hypomorphic mutation in LAMA5 gene were identified in Alport syndrome pedigree and resulted in reduced expression of laminin-α5 in GBM.LAMA5 could act as a modifier which lead to aggravate the Alport phenotype.3、Mutations in NPHS1 and MYO1E gene were also identified.A synonymous mutation was found,demonstrating that even when protein sequence was not affected some mutation could cause disease onset by influencing splicing.Based on the previous report that coinheritance of COL4A5 and MYO1E mutations accentuate the severity of disease,we found that MYO1E mutation alone could give rise to GBM ultrastructure changes like Alport syndrome.These results indicated that besides COL4A3/A4 gene mutations,other genes especially podocyte related genes mutation should be accentuated.Conclusions:1、Clinically male patients,patients with hearing loss and abnormal staining of type Ⅳ collagen should be paid more attention for a better prognosis.The percentage of X-linked Alport patients is approaching 60 and the proportion of patients autosomal inheritance pattern is higher than previously thought which should be emphasized.2、Unique types of COL4A3/A4 gene mutations could be detected by suitable sequencing technics.Assumption of different NGS approaches should be considered when the molecular diagnosis of the patient is negative.3、Apart from type Ⅳ collagen related gene mutations,laminin and podocyte gene mutations were identified in some patients which could exacerbate the phenotypes or cause a Alport-like phenotype.