Analysis Of The Gene Mutations And Clinical Features In Children With Alport Syndrome

BackgroundAlport syndrome (AS) is a hereditary nephritis characterized by hematuria, high-tone sensorineural deafness, ocular abnormalities and progressive nephritis with declining renal function. The X-linked form of the disease (XLAS), accounting for 85% of all cases, results from mutations in the type IV collagen α5 chain gene (COL4A5), and the rarer autosomal modes of inheritance, including autosomal recessive AS (ARAS) and autosomal dominant AS (ADAS), are caused by mutations in COL4A3 or COL4A4 gene, encoding a3 (IV) and a4 (IV) chains, respectively.It was formerly believed that AS is a rare disease, early investigation found that the incidence of AS was 1:5000-10000; 2315 children who underwent renal biopsy in China, the incidence of AS was 1.2%. With the progress of diagnostic techniques and better understanding of genetic disease, reports about AS have become more frequent. AS has no specific cure methods till now, most children will progress to renal disease end-stage.3% AS children accounted for children in renal disease end-stage. Because of great danger of this disease, more clinicians have focused on AS. AS with diverse clinical manifestations is usually insidious in onset and atypical clinical feature will be found in a number of children, so AS may be probably underdiagnosed or misdiagnosed by clinicians. AS patients have poor prognosis and easily progressed to end-stage renal disease, so early diagnosis of AS will be clinically significant.To date, three valuable diagnostic criteria of AS included:ultrastructural changes of the GBM suggestive for AS, Ⅳ collagen a chains is absent from the glomerular or epidermal membrane, COL4A5/3/4 gene mutations. Ⅳ collagen a chains can express normal in basement membrane, which accounted for about 30% AS patients. The mechanism has not been very clear, presumably because some mutations will not make a difference to structure and function of Ⅳ collagen a chain. Early in the disease, glomerular basement membrane may be normal or only characterized by diffuse thinning by electron microscopy. Not all the AS patients are characterized by irregular thinning and thickening of GBM with multi lamination and splitting of the lamina densa. If Ⅳ collagen a chain is dyeing normal or GBM does not appear typical ultrastructural pathological changes, we can’t rule out AS easily. For AS, clinical phenotype, kidney or skin Ⅳ collagen a chain staining and electron microscopy examination only provide auxiliary information for diagnosis. Genetic testing is the gold standard method for making a diagnosis of AS.Because COL4A5/3/4 genes are very large, containing 51,52 and 48 exons, respectively, and there is no mutational hot spots, genetic testing is not extensive. Sanger sequencing technology was considered as the "gold standard" of genetic disease, but it is time-consuming, laborious and expensive, and it is not suitable for AS with huge numbers of pathogenic gene exon. Due to the lower cost and much higher throughput, the next-generation sequencing technology has been widely used in not only detecting the known pathogenic gene mutation, but also discovering new gene mutations. Few national pedigree studies on AS used this technology, beside some case reports or small pedigree studies. Therefore, we collected 30 suspected and confirmed AS children with renal biosy, clinical features and genetic testing data. Our aim was to establish AS individualized diagnosis, further analysis the AS gene mutations and clinical phenotype characteristics, clear AS gene mutation type, enrich AS gene mutation database, and provide basic work for the in-depth study of pathogenesis. In this study, two synonymous mutations c.3246G>A and c.465G>A were detected respectively. It updates our traditional idea of "synonymous mutations cannot cause diseases", and provides the basis for the study of synonymous mutation.ObjectiveTo establish AS individualized diagnosis and further analysis the AS gene mutations and clinical phenotype characteristics.To clear gene mutation type of AS patients, enrich AS gene mutation database, and provide basic work for in-depth study of pathogenesis.Methods30 children with suspected or confirm diagnosis of AS were included in the study, which were from Nanjing General Hospital of Pediatrics between January 2005 and December 2015. All patients were in accordance with AS diagnostic criteria after gene sequencing. We collected clinical data of AS patients and related family members including gender, onset form, eye and ear lesions, kidney damage in patients and other information. We collected renal biopsy information including renal tissue by light microscopy, immunofluorescence, and electron microscopy and IV collagen a chain in GBM. The initial analysis of clinical features was performed. The study protocol was approved by the local ethics committee and parents of AS children agreed and signed informed consent befor collecting 30 AS family members’ peripheral blood samples. We performed whole-exome sequencing of 30 children with suspected or confirmed diagnosis of AS, and Sanger sequencing were validate in their family members. The dbSNP database, human gene mutation database (HMGD), and other public database filter mutations, and SIFT software was used for protein function prediction. By analysis of gene mutations, we aim to establish AS individualized diagnosis. Digenic inheritance in AS was discussed by pedigree analysis. All statistical calculations were performed using SPSS for version 19.0. Results were expressed as mean ± 1 SE. Student’s T test was used to detect significant differences in all quantitative data. Continuous measures were compared using Fisher exact test. P values less than 0.05 deemed significant.Results1. Preliminary analysis of AS clinical features30 cases all have different degrees of hematuria (100%). microscopic haematuria was found in 19 cases (63.33%), including 15 male and 4 female patients.11 cases(36.67%) were reported with gross hematuria, including 5 male and 6 female patients.The degrees of hematuria had no significant differences according to gender CP=0.108). Microscopic haematuria affected people between the ages at onset of 1 month and 10 years, with the median age at 2.86+2.46 years. Gross hematuria was found in patients between the ages at onset of 1 to 6 years, with the median age at 2.55 ±1.29 years. There were no differences in median age at onset (P=0.701).23 cases complicated by proteinuria (76.67%),17 patients were male and 6 patients were female.7 cases had no proteinuria (23.33%),3 patients were male and 4patients were female. Cases were grouped according to 24-hour quantitative protein. Patients in the absence of proteinuria, with Low-grade proteinuria, moderate proteinuria and high-grade proteinuria were respectively included in group A, group B, group C and group D. There were 7 cases (23.33%) in group A, including 3 males and 4 females; 4 cases (13.33%) in group B, including 3 males and 1 female; 15 cases in group C (50%), including 12 males and 3 females; 4 cases (13.33%) in group D, including 2 males and 2 females. Gender did not differ between four groups (P=0.322). Group A with the average age at onset was 2.71±1.11 years; group B with the average age at onset was 4.05 ±2.85 years; group C with the average age at onset was 2.47+2.47 years, group D with the average age at onset was 2.50+0.58 years. The onset age of the children have no statistical difference between four groups (P= 0.621). All 30 children were in Stage 1 CKD. Extrarenal features were not so common in AS patients. Hearing loss was present in 1 male case who was detected bilaterally symmetrical sensorineural hearing loss (6.25%).23 AS children performed kidney biopsy, Renal pathology showed that 13 of them(56.5%) had minimal change disease (MCD), and 10 (43.5%) mesangial proliferative glomerulonephritis (MsPNG).19 patients (82.6%) showed abnormal renal a-chain distribution, and the other 4 patients show normal renal α-chain distribution Extensive lamination and split of glomerular basement membrane dense layers were found in 9 patients (39.1%),and other 2 cases characterized by thin basement membrane disease,6 cases characterized by mesangial proliferative changes,6 cases did not find glomeruli.2 Analysis of gene mutations in children with ASOf these 35 gene mutations which were detected in 30 patients,21 missense mutations,2 synonymous mutations,4 splice-site mutations,3 truncating mutations,2 insertion mutations,4 deletion mutations and 1 compound mutations were accounted for these pathogenic mutations.35 gene mutations were identified:24 in COL4A5,6 in COL4A3 and 5in COL4A4. It was observed that 20 patients inherited the mutation from the mother.8 patients inherited from the father,8 patients carried the novel mutations and 1 patients inherited one mutation from the mother and another mutation from the father by Sanger sequencing. Altogether,31 unique mutations were found. All patients had a final diagnosis of AS. In this study, two synonymous mutations c.3246G>A and c.465G>A were detected respectively, which pathogenicity was ascertained by pedigree study.Patients comprised 26 males and 10 females with XLAS.15 missense mutations (57.69%),2 truncating mutations (7.69%),3 splice-site mutations (13.04%),2 insertion mutations,2 deletion mutations and 2 synonymous mutations were found in XLAS children. The age of patients with different mutation types who progress to ESRD was statistically different (P=0.018). Patients with missense mutation presented slower progression than nonsense mutations, splice site mutation and frame shift mutation.XLAS male patients were earlier than female patients to progress to ESRD, and the difference of onset age was statistically significant (P=0.043).6 patients were found with two mutations in different genes.(member 1,5,9,12,25). But it did not support digenic inheritance in Alport syndrome raised by Mencarelli.Conclusions1. Hematuria is usually the initial symptom in most patients with early age at onset. Extrarenal features were not so common in 30 children with normal renal function. Early renal pathology may show mild and non-specific by light microscopy, mainly MCD. Electron microscopy performance was atypical, and IV collagen a chain immunofluorescence also has limitations. According to the clinical phenotype and renal pathology results, it may be missed or misdiagnosed of AS.2. Genetic testing will complete the individual diagnosis of AS children, and it will contribute to early diagnosis of AS and determining gene mutations.It helps to enrich AS gene mutation database, and provide basic work for the in-depth study of pathogenesis.3. Genetic testing is beneficial to determine the prognosis of children with AS. Prognosis is highly variable in patients with the different inherited types and mutation types.4. The study does not support the digenic inheritance in AS.