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.
Humans ; Nephritis, Hereditary/pathology* ; Hematuria/complications* ; Retrospective Studies ; Collagen Type IV/genetics* ; Genotype ; Mutation

OBJECTIVE: To analyze the clinical phenotype and genetic characteristics of a patient with Alport syndrome. METHODS: A patient with Alport syndrome who had visited the First Affiliated Hospital of Zhengzhou University in November 2020 was selected as the study subject. Clinical data of the patient were collected. High-throughput sequencing was carried out to detect potential variant of the COL4A3, COL4A4 and COL4A5 genes, and Sanger sequencing was carried out for verification of candidate variants in the family. RESULTS: The main clinical manifestations of the patient included hematuria, proteinuria, and impaired hearing. Audiometric testing suggested symmetrical cochlear sensory neural hearing loss on both sides. Renal biopsy revealed mild mesangial proliferative glomerulonephritis. Genetic testing revealed that the patient has harbored compound heterozygous variants of the COL4A4 gene, namely c.940G>A (p.Gly314Ser) and c.3773G>A (p.Gly1258Asp), which were respectively inherited from her father and mother. Neither variant has been reported before, and were predicted to be pathogenic based on the guidelines from the American College of Medical Genetics and Genomics. CONCLUSION The c.940G>A (p.Gly314Ser) and c.3773G>A (p.Gly1258Asp) compound heterozygous variants of the COL4A4 gene probably underlay the Alport syndrome in this patient. Above finding has enriched the mutational spectrum of the COL4A4 gene.
Female ; Humans ; Nephritis, Hereditary/genetics* ; Hematuria ; Genetic Testing ; Genomics ; Hearing ; Collagen Type IV/genetics*

OBJECTIVE: To explore the genetic basis for a patient with Alport syndrome (AS) and confirm the existence of a splicing variant. METHODS: An AS patient diagnosed at the Affiliated Hospital of Inner Mongolia Medical University on January 8, 2021 for significant proteinuria and occult hematuria was selected as the study subject. Clinical data was collected. Peripheral blood samples were collected for the extraction of genomic DNA. Whole exome sequencing and Sanger sequencing were carried out to identify potential genetic variants. An in vitro experiment was also conducted to verify the abnormal mRNA splicing. Bioinformatic software was used to analyze the conservation of amino acids of the variant sites and simulate the 3D structure of the variant collagen IV protein. Immunofluorescence and immunohistochemistry were carried out on renal tissues from the patient to confirm the presence of AS kidney injury. RESULTS: The patient, a 21-year-old male, had a 24-hour urine protein of 3.53 g/24 h, which fulfilled the diagnostic criteria for proteinuria. His blood uric acid has also increased to 491 μmol/L. DNA sequencing revealed that he has harbored a c.835-9T>A splice variant of the COL4A5 gene, which was not found in either of his parents. In vitro experiment confirmed that the variant has removed 57 bp from the exon 15 of the mRNA of the COL4A5 gene. The deletion may cause loss of amino acid residues from positions 279 to 297, which in turn may affect the stability of the secondary structure of the α5 chain encoded by the COL4A5 gene. The amino acids are conserved across various species. The result of homology modeling indicated that the trimerization of Col-IV with the mutated α5 chain could be achieved, however, the 3D structure was severely distorted. The AS kidney damage was confirmed through immunofluorescence assays. Based on the guidelines from the American College of Medical Genetics and Genomics, the c.835-9T>A variant was classified as likely pathogenic (PVS1_Moderate+PS3_Moderate+PM2_Supporting+PS2+PP3+PP4). CONCLUSION The c.835-9T>A variant of the COL4A5 gene probably underlay the AS in this patient. In vitro experiment has confirmed the abnormal splicing caused by the variant. Histopathological examination of the kidney tissue has provided in vivo evidence for its pathogenicity. Above finding has expanded the mutational spectrum of the COL4A5 gene.
Humans ; Male ; Young Adult ; Amino Acids ; China ; Collagen Type IV/genetics* ; Exons ; Nephritis, Hereditary/genetics* ; RNA Splicing

OBJECTIVE: To analysis variants of COL4A5 gene in two Chinese pedigrees affected with Alport syndrome (AS) and provide prenatal diagnosis for them. METHODS: Two unrelated ethnic Han Chinese pedigrees who had visited the First Affiliated Hospital of Zhengzhou University respectively in September 2018 and January 2020 were selected as the study subjects. Clinical data were collected, and genomic DNA was extracted from peripheral venous blood and amniotic fluid samples for genetic testing. Following next generation sequencing, candidate variants of the COL4A5 gene were verified by Sanger sequencing and bioinformatic analysis. The gender of the fetuses was determined by the presence of sex-determining region on Y (SRY). RESULTS: Genetic testing revealed that the proband and a fetus from pedigree 1 had both harbored a c.2723G>A (p.Gly908Glu) variant in exon 32 of the COL4A5 gene, whilst the proband and a fetus from pedigree 2 had both harbored a c.3817G>A (p.Gly1273Asp) variant in exon 44 of the COL4A5 gene. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were classified as likely pathogenic (PP2+PM2_Supporting). Following exclusion of maternal contamination, PCR amplification of the SRY region indicated that both fetuses were males. CONCLUSION The c.2723G>A (p.Gly908Glu) and c.3817G>A (p.Gly1273Asp) variants of the COL4A5 gene probably underlay the AS in the two pedigrees. Detection of the SRY region can reliably identify the fetal sex, which is conducive to the prenatal diagnosis. Above results have also enriched the mutational spectrum of the COL4A5 gene and provided a reference for correlating the genotype and phenotype of the AS.
Female ; Humans ; Male ; Pregnancy ; Collagen Type IV/genetics* ; East Asian People ; Genetic Testing ; Nephritis, Hereditary/genetics* ; Pedigree ; Prenatal Diagnosis

Humans ; Nephritis, Hereditary/genetics* ; Astigmatism ; Mutation ; Collagen Type IV/genetics*

Epidermolysis bullosa (EB) is a group of clinically and genetically heterogeneous diseases characterized by trauma-induced mucocutaneous fragility and blister formation. Here, we investigated five Chinese families with EB, and eight variants including a novel nonsense variant (c.47G>A, p.W16*) in LAMA3, a known recurrent variant (c.74C>T, p.P25L) in KRT5, 2 novel (c.2531T>A, p.V844E; c.6811_6814del, p.R2271fs) and 4 known (c.6187C>T, p.R2063W; c.7097G>A, p.G2366D; c.8569G>T, p.E2857*; c.3625_3635del, p.S1209fs) variants in COL7A1 were detected. Notably, this study identified a nonsense variant in LAMA3 that causes EB within the Chinese population and revealed that this variant resulted in a reduction in LAMA3 mRNA and protein expression levels by nonsense-mediated mRNA decay. Our study expands the mutation spectra of Chinese patients with EB.
Humans ; Asian People/genetics* ; China ; Collagen Type VII/genetics* ; Epidermolysis Bullosa/genetics* ; Epidermolysis Bullosa Dystrophica/genetics* ; Keratin-5/genetics* ; Mutation ; Pedigree ; Laminin/genetics*

Objective: To investigate the expression and function of collagen type ⅩⅦ α1 (COL17α1) in aging mouse skin and its effect on the stemness and proliferation of human epidermal stem cells (ESCs), and to explore the mechanism of related microRNA (miR) in intervening the expression of COL17α1 of human ESC. Methods: The method of experimental research was used. Twelve 2-month-old (young) and twelve 24-month-old (aged) male C57BL/6J mice were selected, and full-thickness skin samples from their upper back were taken for follow-up detection. After hematoxylin-eosin staining of the full-thickness skin samples of young mice and aged mice, the structure of the epidermis was observed and the thickness of the epidermis was measured; the morphology of epidermal basement membrane and hemidesmosomes were observed by transmission electron microscopy, and the hemidesmosomes were counted; the mRNA and protein expressions of COL17α1 were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively, and the protein expression and distribution of COL17α1 was observed and detected by immunofluorescence method. The fresh foreskin tissue discarded after surgery was obtained from 3 healthy men aged 20-30 years who underwent circumcision at the Fourth Medical Center of PLA General Hospital, ESCs were extracted and well-grown cells were wsed for follow-up experiments. According to the random number table (the same grouping method below), ESCs were divided into blank control group, transfection reagent control group, empty vector plasmid group, and COL17α1 knockdown plasmid group with corresponding treatment. After 48 hours of culture, the mRNA expression of COL17α1 was detected by real-time fluorescent quantitative RT-PCR, the protein expressions of COL17α1 and cytokeratin 14 (CK14) were detected by Western blotting, and the cell proliferation level was detected by cell counting kit 8. miRs that might act on the 3' non-coding region of COL17α1 mRNA were screened through DIANA, miRTarBase, miRNAMap, TargetScan, and microRNA databases. The ESCs were divided into negative control group transfected with miR mimic negative control and each miR mimic group transfected with each of the previously screened miR mimics. Forty-eight hours after transfection, the protein expression of COL17α1 was detected by Western blotting. Based on the sequencing data set GSE114006 in Gene Expression Omnibus (GEO), the GEO2R tool was used to statistically analyze the expression of the previously screened miRs that could cause the reduction of COL17α1 protein expression in the skin of 30 young (18-25 years old) and 30 elderly (>70 years old) human skins. The full-thickness skin samples of young mice and aged mice were taken, and the expressions of increased miRs in the aforementioned aged human skin were detected by real-time fluorescent quantitative RT-PCR. Two batches of human ESCs were taken, the first batch was divided into COL17α1 wild type+miR-203b-3p negative control group and COL17α1 wild type+miR-203b-3p mimic group, and the second batch was divided into COL17α1 mutant+miR-203b-3p negative control group and COL17α1 mutant+miR-203b-3p mimic group. Each group of ESC was transfected with corresponding sequences respectively. Forty-eight hours later, the luciferase reporter gene detection kit was used to detect the gene expression level of COL17α1. The number of samples in the tissue experiment was 6, and the number of samples in the cell experiment was 3. Data were statistically analyzed with independent sample t test, one-way analysis of variance, least significant difference test or Dunnett's test, Mann-Whitney U test or Kruskal-Wallis H test. Results: Compared with those of young mice, the boundary between the epidermis and the dermis of the aged mice skin was blurred and the cell layers were less, and the thickness of epidermis was significantly thinner (Z=-2.88, P<0.01); the morphology of basement membrane was discontinuous, with less unevenly distributed hemidesmosomes at the epidermis-dermis junction, and the number of hemidesmosomes was significantly reduced (Z=-2.91, P<0.01); the mRNA and protein expression levels of COL17α1 in the skin of aged mice were significantly decreased (with t values of 10.61 and 6.85, respectively, P<0.01). Compared with those of young mice, the protein expression of COL17α1 in the basal layer of epidermis and the bulb of hair follicle in the skin of aged mice was significantly decreased (Z=-2.24, P<0.05). After 48 hours of culture, the protein expression levels of COL17α1 in ESCs of blank control group, transfection reagent control group, empty vector plasmid group, and COL17α1 knockdown plasmid group were 1.00±0.27, 1.12±0.21, 1.13±0.23, and 0.42±0.18, respectively. Compared with those of blank control group, the mRNA and protein expression levels of COL17α1, the protein expression level of CK14, and the proliferation level of ESCs in transfection reagent control group and empty vector plasmid group did not change significantly (P>0.05), while these indexes in COL17α1 knockdown plasmid group were significantly decreased (P<0.05 or P<0.01). miR-203a-3p, miR-203b-3p, miR-512-5p, miR-124-3p, miR-28-5p, miR-590-3p, and miR-329-5p might bind to the 3' non-coding region of COL17α1 mRNA. Forty-eight hours after transfection, compared with 1.000±0.224 in negative control group, the protein expression level of COL17α1 in ESCs of miR-329-5p mimic group, miR-203b-3p mimic group, and miR-203a-3p mimic group decreased significantly (0.516±0.188, 0.170±0.025, and 0.235±0.025, with t values of 3.17, 5.43, and 5.07, respectively, P<0.05 or P<0.01). Only the expression level of miR-203b-3p in the skin of the elderly was significantly higher than that of the young (t=3.27, P<0.01). The expression level of miR-203b-3p in the skin of aged mice was significantly higher than that of young mice (Z=-2.88, P<0.01). Forty-eight hours after transfection, the gene expression level of COL17α1 in ESCs of COL17α1 wild type+miR-203b-3p mimic group was significantly lower than that of COL17α1 wild type+miR-203b-3p negative control group (t=7.66, P<0.01). The gene expression level of COL17α1 in ESCs of COL17α1 mutant+miR-203b-3p mimic group was similar to that of COL17α1 mutant+miR-203b-3p negative control group (P>0.05). Conclusions: The mRNA and protein expression levels of COL17α1 decrease with age increasing in mice, which may lead to the detachment of mouse ESC from the epidermal basement membrane. Decreased expression of COL17α1 can inhibit the expression of CK14 and ESC proliferation, which may be responsible for the thinning of the epidermis and slower wound healing in aged human skin. The increased expression of miR-203b-3p in aged mouse skin can target and bind to the 3' non-coding region of COL17α1 mRNA, hindering the post-transcriptional translation process, thus resulting in decreased COL17α1 protein expression.
Adolescent ; Adult ; Aged ; Animals ; Autoantigens ; Humans ; Keratin-14 ; Male ; Mice ; Mice, Inbred C57BL ; MicroRNAs/genetics* ; Non-Fibrillar Collagens/pharmacology* ; Polyesters ; RNA, Messenger ; Skin Aging ; Stem Cells ; Young Adult

OBJECTIVE: To explore the genetic basis for two Chinese pedigrees affected with Alport syndrome. METHODS: Potential variants of the COL4A5 gene were screened by next generation sequencing (NGS). Candidate variants were verified by Sanger sequencing of other members from the pedigrees as well as 100 healthy controls. ClustalX 2.1 win was used to analyze the conservation of amino acid sequences. SWISS-MODEL was used for assessing the influence of variations on the protein structure. RESULTS: Two heterozygous missense variants of the COL4A5 gene, namely c.2210G>A (p.Gly737Asp) and c.3799G>A (p.Gly1267Ser), were respectively identified in the affected individuals from the two pedigrees but not among the 100 healthy controls. Neither variant was reported previously. CONCLUSION The c.2210G>A (p.Gly737Asp) and c.3799G>A (p.Gly1267Ser) variants of the COL4A5 gene probably underlay the Alport syndrome in these pedigrees. Above finding has enriched the spectrum of COL4A5 gene variants and provided a basis for genetic counseling and prenatal diagnosis for the families.
Pregnancy ; Female ; Humans ; Nephritis, Hereditary/genetics* ; Pedigree ; Collagen Type IV/genetics* ; Mutation ; China

Collagen Type IV/genetics* ; Genetic Testing ; Humans ; Nephritis, Hereditary/therapy*

OBJECTIVE: To perform gene mutation analysis in a Chinese pedigree with dystrophic epidermolysis bullosa pruriginosa (DEB-Pr), and explore phetotype, genotype, and genotypes-phenotypes relationship of DEB-Pr. METHODS: Potential variants of the COL7A1 gene were detected by skin targeted sequencing panel and verified by Sanger sequencing. The pathogenicity of the variation was analyzed. RESULTS: Compound heterozygous variants, c.4128delT and c.8234G>A, were detected in the COL7A1 gene of the two patients. The c.4128delT(p.Pro1376fs) variant was derived from their mother and unreported previously. According to the American College of Medical Genetics and Genomics Standards and Guidelines, it was suggested to be a pathogenic mutation. The c.8234G>A(p.Arg2745Gln) variant was derived from their father, and possibly is a pathogenic variation. CONCLUSION In this study, the compound heterozygous variants of c.4128delT(p.Pro1376fs) and c.8234G>A(p.Arg2745Gln) of the COL7A1 gene probably underlies the disease in this patient and his sister. And our study expands the database on mutations of DEB-Pr.
Collagen Type VII/genetics* ; Epidermolysis Bullosa Dystrophica/genetics* ; Female ; Humans ; Male ; Mutation ; Pedigree ; Phenotype