Clinicopathological Analysis Of Patients With Alport Syndrome And Detection Of Mutations In COL4A5 Gene

Alport syndrome(AS) is a inherited disease characterized by progressive renal failure,with or without sensorineural deafness,lenticonus or retinal lesion.It's a basement embrane disease induced by mutations of genes encoding for the a3～a6 chain of typeⅣcollagen(COL4A3～COL4A6).Mutations in COL4A5 gene,encoding for the a5 chain of typeⅣcollagen is responsible for XLAS,whereas the genes of COIAA3 and COL4A4 on chromosome 2,are involved in the autosomal forms of AS,including autosomal recessive inheritance(ARAS) and autosomal dominant inheritance(ADAS).AS is not rare in the inherited kidney disease,According to the data of America,gene frequency of AS is about 1:5000～10000,and accounts for 1.6 to 4 percent of patients examined by renal biopsy at abroad,whereas 0.73 to 1.2 percent at home.AS is clinically heterogeneous,typical patients have progressive renal failure, hematuria,proteinuria and accompany with hearing loss and/or lenticonus lesion.But not all symptoms present in all patients,some patients just have microscopic hematuria and/or proteinuria.There are also differences of clinical features in patients with AS at home and abroad.Some studies show clinical features of AS at home are heavier proteinuria,higher incidence of deafness and eye abnormal,and there are more juvenile patients.Therefore,it's significant to screen AS patients at home that helps to accumulate clinical features of the disease.Meanwhile,detection of COL4A5 gene in AS patients benefits to know pathogenesis of the disease in molecular level and the relationship of genotype and clinical features.Furthermore,it's a basis of prenatal diagnosis and gene therapy.In this study,the clinical screening was carried out and clinicopathological characteristics were analyzed in confirmed and likely cases.We accumulated clinical features of these patients and found an effective mean of screening.On the above basis,we investigated a XLAS family and detected the mutation of COL4A5 gene in index case and his mother.Methods1.We screened patients in the hospital and Formulated conditions for screening, including abnormality of silver staining under light microscopy.Meanwhile,we carried out indirect immunofluorescence staining of theⅣcollagenαchain of GBM and electron microscopy in suspected patients to clarify the diagnosis. Diagnosis standards were established according to the Flinter's standard and the result of indirect immunofluorescene inαchain of typeⅣcollagen of GBM.We also analyzed the clinicopathological features of confirmed and likely AS cases in this study.2.We investigated a XLAS family and mapped the family pattern.Meanwhile,we collected the clinical features and specimen of members in this family.3.Peripheral blood leukocyte genomic DNA was extracted from blood of index case and his mother.We used PCR-DNA direct sequencing methods to detect mutations of COL4A5 gene.Results1.108 patients were selected for AS screening,5 confirmed and 4 likely cases with AS were diagnosed,accounting for 4.6 percent and 3.7 percent of the total number of screening,respectively.All cases of confirmed and likely AS have a family history of hematuria,and were generally poor response to symptomatic treatment. 5 confirmed cases showed clinical characteristics of early age at onset,severe impairment of renal function,combined with proteinuria,and a high proportion of deafness.Wheares,4 likely cases were normal in renal function,three female likely cases showed persistent abnormal urine test.2.7 confirmed or likely cases was diagnosed by renal biopsy as mild mesangial proliferative glomerulonephritis,minimal change and focal segmental sclerosis, and all of them have the characteristics of abnormal coloring PASM under light microscopy;There were 21 abnormal cases of PASM staining in 98 patients,who underwent renal biopsy.7 confirmed or likely cases were general negative in common immunofluorescence,iFI results of GBMⅣcollagen suggested that male patients were negative,wheares female patients were not continuous inα3andα5 chain staining;Most cases have uneven thickness change in GBM under electron microscopy,2 cases with layered,torn change,and one female likely case showed diffuse thinning of GBM.3.A family survey was carried out according to a proband of AS.The results showed that there were 10 AS cases in the family,distributing in four generations;Among of them,there were 6 female and 4 male patients;4 male patients were early onset, earlier entering the ESRD,and three of them had died of uremia;Only 2 female cases occurred renal failure,and were light clinical symptoms.Male patients were non-genetic to the mass of men,wheares female patients were genetic to the mass of both men and women.The survey also found a rare clinical case of double narrow kidneies,and the case had normal renal function in a long-term.4.Sequencing of COL4A5 gene in prohand showed that C base had been replaced by T base in the 5222 base-point(C5222T) of exon 51,therefore,the 1674 condon translated from the arginine codon into a stop codon(CGA→UGA,R1674Term); the prohand's mother had the T and C heterozygous bases in 5222 base-point.These results had been verified by two-way repeated sequencing,and there were no base replacement in 5222 base-point in normal controls.Conclusions1.The establishment of the screening conditions for AS could be clinically effective in AS screening;The confirmed and likely AS cases provided clinical pathological data of patients in the region.2.The pathological feature of abnormalities of PASM under light microscopy is an effective parameter of screening the patients with AS.3.The family investigated in this study was a juvenile XLAS family.The base replacement of 5222 base point of exon 51 in the COL4A5 gene detected in this study is gene mutation,rather than polymorphism,which induced the clinical features of the family and was reported for the first time.