COL4A5 genotypes and clinical characteristics of children with Alport syndrome / 中国当代儿科杂志

OBJECTIVES@#To investigate the genotypes of the pathogenic gene COL4A5 and the characteristics of clinical phenotypes in children with Alport syndrome (AS).@*METHODS@#A retrospective analysis was performed for the genetic testing results and clinical data of 19 AS children with COL4A5 gene mutations.@*RESULTS@#Among the 19 children with AS caused by COL4A5 gene mutations, 1 (5%) carried a new mutation of the COL4A5 gene, i.e., c.3372A>G(p.P1124=) and presented with AS coexisting with IgA vasculitis nephritis; 3 children (16%) had large fragment deletion of the COL4A5 gene, among whom 2 children (case 7 had a new mutation site of loss51-53) had gross hematuria and albuminuria at the onset, and 1 child (case 13 had a new mutation site of loss3-53) only had microscopic hematuria, while the other 15 children (79%) had common clinical phenotypes of AS, among whom 7 carried new mutations of the COL4A5 gene. Among all 19 children, 3 children (16%) who carried COL4A5 gene mutations also had COL4A4 gene mutations, and 1 child (5%) had COL4A3 gene mutations. Among these children with double gene mutations, 2 had gross hematuria and proteinuria at the onset.@*CONCLUSIONS@#This study expands the genotype and phenotype spectrums of the pathogenic gene COL4A5 for AS. Children with large fragment deletion of the COL4A5 gene or double gene mutations of COL4A5 with COL4A3 or COL4A4 tend to have more serious clinical manifestations.

Analysis and literature review of COL4A5 mutation mosaicism in male patients with X-linked Alport syndrome / 中华肾脏病杂志

Objective:To report four male COL4A5 mutation mosaicism patients with X-linked Alport syndrome, and to provide evidence for diagnosis, genetic counseling, and reproduction in the respective families and improve our knowledge of mosaicism in Alport syndrome. Methods:Suspected male mosaic patients for COL4A5 who met the following criteria: clinical diagnosis of Alport syndrome, harbored COL4A5 mutations detected using next generation sequencing or Sanger sequencing, heterozygosity for the mutant and normal COL4A5 alleles in the DNA demonstrated by Sanger sequencing, registered in the on-line registry of hereditary kidney diseases, and admitted to Peking University First Hospital during the period of April 2018 to April 2019 were enrolled. Clinical data and karyotypes were retrospectively analyzed. Genetic DNA isolated from multiple tissues was analyzed for COL4A5 gene mutations by using PCR and Sanger sequencing. Related literatures published in PubMed, CNKI and Wanfang databases were reviewed. Results:Four COL4A5 somatic and germline mosaic male patients with Alport syndrome were included in the study. Patient 1 was characterized by hematuria and proteinuria. His karyotype of peripheral blood was normal. COL4A5 c.3455-1G>A mosaicism was detected in multiple tissues (peripheral blood, saliva and urine). Patient 2 presented with hematuria and microalbuminuria. His karyotype of peripheral blood was normal. COL4A5 c.4994+1G>A mosaicism was detected in multiple tissues (peripheral blood, saliva and skin fibroblasts). Patients 3 showed hematuria without proteinuria. COL4A5 c.3535G>A mosaicism was found in genomic DNA of peripheral blood and hair. Laboratory and physical examinations of patient 4 showed hematuria and normal renal function, without proteinuria, megasoma or small testes. COL4A5 c.3106G>A mosaicism was detected in genomic DNA of skin fibroblasts. Although without karyotype analysis due to unavailable specimens, 47,XXY or 46,XY/47,XXY mosaicism was not considered according to the reproductive history and lack of clinical manifestations of megasoma and small testes in patients 3 and 4. Renal disease in 8 published male cases with mosaicism for COL4A5 was affected by mutant allelic fractions and genotype. Conclusions:Compared with hemizygous males with X-linked Alport syndrome, the renal phenotype of mosaic males was milder, and associated with mutant allelic fractions and mutation type.

Detection of large deletions in X linked Alport syndrome using competitive multiplex fluorescence polymerase chain reaction / 北京大学学报(医学版)

Objective:To evaluate the ability of multiplex competitive fluorescence polymerase chain reaction in detection of large deletion and duplication genotypes of X-linked Alport syndrome.Methods:Clinical diagnosis of X-linked Alport syndrome was based on either abnormal staining of type Ⅳ collagen α5 chain in the epidermal basement membrane alone or with abnormal staining of type Ⅳ collagen α5 chain in the glomerular basement membrane and Bowman's capsule/ultrastructural changes in the glomerular basement membrane typical of Alport syndrome.A total of 20 unrelated Chinese patients (13 males and 7 females) clinically diagnosed as X-linked Alport syndrome were included in the study.Their genotypes were unknown.Control subjects included a male patient with other renal disease and two patients who had large deletions in COL4A5 gene detected by multiplex ligation-dependent probe amplification.Genomic DNA was isolated from peripheral blood leukocytes in all the participants.Multiplex competitive fluorescence polymerase chain reaction was used to coamplify 53 exons of COL4A5 gene and four reference genes in a single reaction.When a deletion removed exon 1 of COL4A5 gene was identified,the same method was used to coamplify the first 4 exons of COL4A5 and COL4A6 genes,a promoter shared by COL4A5 and COL4A6 genes,and three reference genes in a single reaction.Any copy number loss suggested by this method was verified by electrophoresis of corresponding polymerase chain reaction amplified products or DNA sequencing to exclude possible DNA variations in the primer regions.Results:Genotypes of two positive controls identified by multiplex competitive fluorescence polymerase chain reaction were consistent with those detected by multiplex ligation-dependent probe amplification.Deletions were identified in 6 of the 20 patients,including two large deletions removing the 5'part of both COL4A5 and COL4A6 genes with the breakpoint located in the second intron of COL4A6,two large deletions removing more than 30 exons of COL4A5 gene,one large deletion removing at least 1 exon of COL4A5 gene,and one small deletion involving 13 bps.No duplication was found.Conclusion:Our results show that multiplex competitive fluorescence polymerase chain reaction is a good alternative to classical techniques for large deletion genotyping in X-linked Alport syndrome.

Diagnosis of a boy with sporadic Alport syndrome using next generation sequencing and literature review / 中华实用儿科临床杂志

Objective To study the feasibility of testing three disease-causing genes for Alport syndrome,COL4A3,COL4A4 and COL4AS,in diagnosing patients with sporadic Alport syndrome by using targeted capture and next generation sequencing.Methods The clinical data of a 9-year-old boy suspected with Alport syndrome were collected.Genomic DNA was extracted using standard procedures from the peripheral blood leukocytes of the patient and his parents,respectively.Targeted capture and next generation sequencing and Sanger sequencing were applied to analyze the mutations in the 3 disease-causing genes.Clinical data of cases reported already with autosomal recessive Alport syndrome caused by the mutations in the COL4A4 gene were summarized.Results The patient was presented with neither family history of hematuria nor chronic renal failure.Haematuria and proteinuria were found at the age of 1 year.The patient presented with episodes of macrohaematuria and gradually developed nephrotic-level proteinuria.At the age of 8 years 7 months,bilateral sensorineural hearing loss was diagnosed.So a probable diagnosis of Alport syndrome was postulated.A compound heterozygous pathogenic mutations of 3578-1G ＞ A and 3967 C ＞ T(Q1323X) were identified in the COL4A4 gene in the patient.The mutation of 3578-1G ＞ A was inherited from his father,and the mutation of Q1323X from his mother.The patient was decisively diagnosed with autosomal recessive Alport syndrome.Conclusions The test of COL4A3,COL4A4 and COL4A5 genes can help diagnose patients with sporadic Alport syndrome by using targeted capture and next generation sequencing.

Novel deletion mutation of type-Ⅳcollagen in a Chinese family with Alport syndrome / 医学研究生学报

Objective Alport syndrome is one of the diseases that may lead to the end-stage renal disease ( ESRD) in chil-dren, and the methods for its diagnosis and treatment remain quite limited.This study aimed to investigate the clinical and genetic di-agnosis of a Chinese family with hematuria companied by genetic nephritis. Methods We analyzed the renal pathology of 7 patients in a family, performed immunofluorescence staining of type-Ⅳcollagen in the nephridial and skin tissue, conducted gene sequencing i-dentification using the exon sequence method, and examined the blood and urine samples from the patients. Results Renal patholo-gy manifested mesenterium hyperplasia in the index patient, with IgM+under the light microscope, no thickening or thinning under the electromicroscope, and no absence of type-Ⅳcollagen on immunofluorescence analysis.Mutation of c.1365_1373del TCCAGGCCC (p.Pro456_Pro458del3) was observed in exon 21 of the COL4A5 gene.Only 1682 amino acids were found in the mutated protein as compared with 1685 in the wild type. Conclusion This is the first case of Alport syndrome induced by gene deletion mutation ever reported in China and abroad.There are many female patients in this family, all with a high risk of reproduction failure.Antepartal gene diagnosis or genetic diagnosis before embryo transfer may contribute to the prevention of the disease.

A Exclusive Mapping Analysis on the Gene Associated with X-linked Dominant Inherited Nephritis in One Pedigree / 中国医师杂志

Objective To study the characteristics of mutations of gene COL4A5 encoding type Ⅳ collagen ? 5 chain in one chinese pedigree with X-linked dominant inherited nephritis (Alport's syndrome,AS). Methods Genomic DNA was extracted from 35 members of the pedigree of Alport's syndrome. All of 51 exons of COL4A5 gene were amplified by PCR with the primers synthesized according to the published intron sequences of COL4A5. The PCR products were further analyzed by agarose gel electrophoresis and single strand conformation polymorphism (SSCP). The PCR products showing polymorphism were directly sequenced. Results PCR-SSCP analysis showed that 17 PCR products had abnormal mobility of single strand DNA. DNA sequencing analysis revealed 9 suspicious mutations. But these suspicious mutations were not be confirmed by inverse sequencing analysis. Conclusion The exon mutation of COL4A5 gene of this pedigree did not be found, and the mutations of COL4A5 gene may locate in the its introns.