Heterozygous COL4A3 Research Articles

Synergistic toxicity of compound heterozygous mutations in the COL4A3 gene causes end-stage renal disease in A large family of Alport syndrome

Alport syndrome (AS) is a genetic disorder characterized by kidney disease and hearing/vision abnormalities, resulting from mutations in the COL4A3, COL4A4, or COL4A5 genes. While numerous mutations have been identified in AS cases, the precise molecular mechanisms, particularly for compound mutations, remain under investigation. This study investigated the molecular mechanisms of AS in a proband with end-stage kidney disease (ESKD) using whole exome sequencing, which identified two compound heterozygous COL4A3 missense mutations: NM_000091.5:c.1354G > A (p.G452R) and NM_000091.5:c.4793 T > G (p.L1598R). Sixteen family members of the proband were genotyped, and further analyses, including in silico structural prediction, molecular docking, and in vitro co-immunoprecipitation assays, revealed that the p.G452R mutation disrupted the collagen triple helical structure, associated with hematuria in carriers, while the p.L1598R mutation interfered with the interaction between the NC1 domains of COL4A3 and COL4A4 proteins, crucial for collagen trimerization. These findings demonstrate a synergistic loss-of-function effect of the two mutations, contributing to the AS pathogenesis in the proband, and emphasize the importance of genetic screening and personalized treatment strategies for AS.

Increased prevalence of kidney cysts in individuals carrying heterozygous COL4A3 or COL4A4 pathogenic variants.

Clinical variability among individuals with heterozygous pathogenic/likely pathogenic (P/LP) variants in the COL4A3/COL4A4 genes (also called autosomal dominant Alport syndrome or COL4A3/COL4A4-related disorder) is huge; many individuals are asymptomatic or show microhematuria, while others may develop proteinuria and chronic kidney disease (CKD). The prevalence of simple kidney cysts (KC) in the general population varies according to age, and patients with advanced CKD are prone to have them. A possible association between heterozygous COL4A3, COL4A4 and COL4A5 P/LP variants and KC has been described in small cohorts. The presence of KC in a multicenter cohort of individuals with heterozygous P/LP variants in the COL4A3/COL4A4 genes is assessed in this study. We evaluated the presence of KC by ultrasound in 157 individuals with P/LP variants in COL4A3 (40.7%) or COL4A4 (53.5%) without kidney replacement therapy. The association between presence of KC and age, proteinuria, estimated glomerular filtration rate (eGFR) and causative gene was analyzed. Prevalence of KC was compared with historical case series in the general population. Half of the individuals with P/LP variants in COL4A3/COL4A4 showed KC, which is a significantly higher percentage than in the general population. Only 3.8% (6/157) had cystic nephromegaly. Age and eGFR showed an association with the presence of KC (P<.001). No association was found between KC and proteinuria, sex or causative gene. Individuals with COL4A3/COL4A4 P/LP variants are prone to develop KC more frequently than the general population, and their presence is related to age and to eGFR. Neither proteinuria, sex nor the causative gene influences the presence of KC in these individuals.

#6036 CYSTIC KIDNEY PHENOTYPE IS A FREQUENT, AGE- AND EGFR- DEPENDENT FINDING IN ALPORT SYNDROME

Abstract Background and Aims Alport Syndrome (AS), the most common genetic glomerular disease, due to pathogenic variants in COL4A3-COL4A5 genes, includes three main forms: X-linked AS (XLAS, COL4A5); autosomal recessive (ARAS, biallelic pathogenic variants in COL4A3 or COL4A4); autosomal dominant (ADAS; heterozygous COL4A3 or COL4A4 variant). An expanded phenotypic spectrum of AS has been described, including recent reports of multiple kidney cysts in affected patients. The aim of the study was to evaluate the prevalence and characteristics of renal cysts in a cohort of adult patients with AS. Method This retrospective study included subjects with AS followed at the Outpatient Nephrogenetic Clinic of Spedali Civili di Brescia (Italy) from 2002 to 2022 and with at least one available renal imaging study (Ultrasonography/CT scan). Genetic testing was performed by using a next generation sequencing multi-gene panel for kidney disease. Cystic phenotype was defined as the presence of ≥3 cysts in each kidney. The prevalence of renal cystic phenotype was compared between patients (pts) with AS and a group of age- and eGFR-matched pts with sporadic IgA nephropathy (IgA-N). Demographic and clinical features were compared between pts with or without cystic phenotype. Logistic regression was performed to test whether sex, age, type of variant and eGFR (CKD-EPI formula) were independently associated with the cystic phenotype. Results A total of 96 AS pts were studied. The pattern of inheritance was AD in the majority of the cohort (56%), XL in 25%, AR in 2% and unknown in the remaining 17%. The cystic phenotype was observed in 36 pts (38%). When compared to a matched IgA-N cohort (n = 79), the cystic phenotype was significantly more common in AS (42% in AS; 19% in IgA-N; p = 0,002). The majority of AS pts with cystic phenotype showed normal/reduced sized kidneys and multiple cortical and/or parapelvic renal cysts. Increased total kidney volume, in keeping with primary cystic kidney disease, was observed in three patients; however, pathogenic variants in known cysto-genes were excluded. At the time of renal imaging, AS pts with cystic phenotype were older and had a more marked reduction in kidney function than their non-cystic counterparts (Table 1). When stratifying patients based on age or eGFR, the prevalence of the cystic phenotype gradually increased in parallel with older age and declining eGFR (Figure 1). Independent predictors of the cystic phenotype were age (HR 1.96 [95% CI 1.31-2.94] per 10 years; p = 0.001) and eGFR (HR 0.75 [95% CI 0.64-0.89] per 10 ml/min/1.73m2;p = 0.001). Serial longitudinal ultrasounds were available for 15 cystic pts, among whom the cystic phenotype was first observed from 40 years of age onwards, at an overall median age of 59 (IQR 46–65). Conclusion Our data show that multiple bilateral kidney cysts, with no increase in kidney size, are frequently found in AS patients. The cystic phenotype is associated with older age and eGFR decline, suggesting that it can reflect the severity and/or duration of CKD. However, the higher frequency of cystic phenotype in AS than IgAN, the role of the collagen IV α3 to α5 chains in the basement membranes in the glomerulus and distal tubule, and the occurrence of kidney cysts in a canine model of ADAS, support a possible pathogenetic link between type IV collagen mutations and cystogenesis in AS. Finding kidney cysts should not discourage from considering the diagnosis of AS, particularly in adult patients and in the presence of familial CKD. Future prospective studies will be needed to shed light on the prognostic implications of the cystic phenotype and possible genotype-phenotype associations.

Prevalence Estimates of Predicted Pathogenic COL4A3-COL4A5 Variants in a Population Sequencing Database and Their Implications for Alport Syndrome.

The reported prevalence of Alport syndrome varies from one in 5000 to one in 53,000 individuals. This study estimated the frequencies of predicted pathogenic COL4A3-COL4A5 variants in sequencing databases of populations without known kidney disease. Predicted pathogenic variants were identified using filtering steps based on the ACMG/AMP criteria, which considered collagen IV α3-α5 position 1 Gly to be critical domains. The population frequencies of predicted pathogenic COL4A3-COL4A5 variants were then determined per mean number of sequenced alleles. Population frequencies for compound heterozygous and digenic combinations were calculated from the results for heterozygous variants. COL4A3-COL4A5 variants resulting in position 1 Gly substitutions were confirmed to be associated with hematuria (for each, P<0.001). Predicted pathogenic COL4A5 variants were found in at least one in 2320 individuals. p.(Gly624Asp) represented nearly half (16 of 33, 48%) of the variants in Europeans. Most COL4A5 variants (54 of 59, 92%) had a biochemical feature that potentially mitigated the clinical effect. The predicted pathogenic heterozygous COL4A3 and COL4A4 variants affected one in 106 of the population, consistent with the finding of thin basement membrane nephropathy in normal donor kidney biopsy specimens. Predicted pathogenic compound heterozygous variants occurred in one in 88,866 individuals, and digenic variants in at least one in 44,793. The population frequencies for Alport syndrome are suggested by the frequencies of predicted pathogenic COL4A3-COL4A5 variants, but must be adjusted for the disease penetrance of individual variants and for the likelihood of already diagnosed disease and non-Gly substitutions. Disease penetrance may depend on other genetic and environmental factors.

NPHS2 gene polymorphism aggravates renal damage caused by focal segmental glomerulosclerosis with COL4A3 mutation.

Focal segmental glomerulosclerosis (FSGS), a type of primary glomerular disease, is the leading cause of end-stage renal disease (ESRD). Several studies have revealed that certain single-gene mutations are involved in the pathogenesis of FSGS; however, the main cause of FSGS has not been fully elucidated. Homozygous mutations in the glomerular basement membrane gene can lead to early renal failure, while heterozygous carriers develop renal failure symptoms late. Here, molecular genetic analysis of clinical information collected from clinical reports and medical records was performed. Results revealed that nephrosis 2 (NPHS2) gene polymorphism aggravated renal damage in three FSGS families with heterozygous COL4A3 mutation, leading to early renal failure in index patients. Our findings suggest that COL4A3 and NPHS2 may have a synergistic effect on renal injury caused by FSGS. Further analysis of the glomerular filtration barrier could help assess the cause of kidney damage. Moreover, a detailed analysis of the glomerular basement membrane-related genes and podocyte structural proteins may help us better understand FSGS pathogenesis and provide insights into the prognosis and treatment of hereditary glomerulonephropathy.

Deciphering the pathogenesis of the COL4-related hematuric nephritis: A genotype/phenotype study.

BackgroundAlport syndrome (ATS) is a hereditary progressive hematuric nephropathy associated with sensorineural deafness and ocular abnormalities, which is caused by mutations in the COL4A5 gene (X‐linked ATS) and in two autosomal genes, COL4A4 and COL4A3, responsible of both recessive ATS and, when present in heterozygosity, of a spectrum of phenotypes ranging from isolated hematuria to frank renal disease.MethodsRetrospective analysis of the clinical and genetic features of 76 patients from 34 unrelated ATS families (11 with mutations in COL4A5, 11 in COL4A3, and 12 in COL4A4) and genotype/phenotype correlation for the COL4A3/COL4A4 heterozygotes (34 patients from 14 families).ResultsEight (24%) of the 34 heterozygous COL4A3 and COL4A4 carriers developed renal failure at a mean age of 57 years, with a significantly lower risk than hemizygous COL4A5 or double heterozygous COL4A3/COL4A4 carriers (p < 0.01), but not different from that of the heterozygous COL4A5 females (p = 0.6). Heterozygous carriers of frameshift/splicing variants in COL4A3/COL4A4 presented a higher risk of developing renal failure than those with missense variants in the glycine domains (p = 0.015).ConclusionThe renal functional prognosis of patients with COL4A3/COL4A4‐positive ATS recapitulates that of the X‐linked ATS forms, with differences between heterozygous vs. double heterozygous patients and between carriers of loss‐of‐function vs. missense variants.

Type IV Collagen Mutations in Familial IgA Nephropathy

Type IV Collagen Mutations in Familial IgA Nephropathy

COL4A3 mutations cause focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a histologically identifiable glomerular injury often leading to proteinuria and renal failure. To identify its causal genes, whole-exome sequencing and Sanger sequencing were performed on a large Chinese cohort that comprised 40 FSGS families, 50 sporadic FSGS patients, 9 independent autosomal recessive Alport's syndrome (ARAS) patients, and 190 ethnically matched healthy controls. Patients with extrarenal manifestations, indicating systemic diseases or other known hereditary renal diseases, were excluded. Heterozygous COL4A3 mutations were identified in five (12.5%) FSGS families and one (2%) sporadic FSGS patient. All identified mutations disrupted highly conserved protein sequences and none of them was found in either public databases or the 190 healthy controls. Of the FSGS patients with heterozygous COL4A3 mutations, segmental thinning of the glomerular base membrane (GBM) was only detected in the patient with electronic microscopy examination results available. Five ARAS patients (55.6%) had homozygous or compound-heterozygous mutations in COL4A3 or COL4A4. Serious changes in the GBM, hearing loss, and ocular abnormalities were found in 100%, 80%, and 40% of the ARAS patients, respectively. Overall, a new subgroup of FSGS patients resulting from heterozygous COL4A3 mutations was identified. The mutations are relatively frequent in families diagnosed with inherited forms of FSGS. Thus, we suggest screening for COL4A3 mutations in familial FSGS patients.

Novel heterozygous COL4A3 mutation in a family with late-onset ESRD

Thin basement membrane nephropathy (TBMN) and Alport syndrome (ATS) are genetically heterogeneous conditions characterized by structural abnormalities in the glomerular basement membrane (GBM). TBMN presents with hematuria, minimal proteinuria, and normal renal function. Although TBMN is an autosomal dominant disease (COL4A3 and COL4A4), ATS can be inherited X-linked (COL4A5), autosomal recessive, or autosomal dominant (both COL4A3 and COL4A4). The clinical course of TBMN is usually benign, whereas ATS typically results in end-stage renal disease (ESRD). Nevertheless, there is a broad spectrum of clinical phenotypes caused by mutations in COL4A3 or COL4A4. We report an Italian family who presented with hematuria and mild proteinuria. Mutational analysis showed a novel heterozygous mutation p.G291E in exon 15 of the COL4A3 gene. Many different mutations in COL4A3 and COL4A4 that cause TBMN have already been identified, but most genetic variability in these genes has been found to cause autosomal ATS. A valid genotype-phenotype correlation for TBMN or ATS is not yet known. Therefore, it is important to identify new mutations by direct sequencing to clarify their clinical importance, to assess the prognosis of the disease, and to avoid renal biopsy.

Chronic Renal Failure and Shortened Lifespan in COL4A3+/− Mice

A heterozygous mutation in autosomal Alport genes COL4A3 and COL4A4 can be found in 20 to 50% of individuals with familial benign hematuria and diffuse glomerular basement membrane thinning (thin basement membrane nephropathy [TBMN]). Approximately 1% of humans are heterozygous carriers of mutations in the autosomal Alport genes and at risk for developing renal failure as a result of TBMN. The incidence and pathogenesis of renal failure in heterozygous COL4A3/4 mutation carriers is still unclear and was examined further in this study using COL4A3 knockout mice. In heterozygous COL4A3(+/-) mice lifespan, hematuria and renal function (serum urea and proteinuria) were monitored during a period of 3 yr, and renal tissue was examined by light and electron microscopy, immunohistochemistry, and Western blot. Lifespan of COL4A3(+/-) mice was found to be significantly shorter than in healthy controls (21.7 versus 30.3 mo). Persistent glomerular hematuria was detected starting in week 9; proteinuria of > 0.1 g/L started after 3 mo of life and increased to > 3 g/L after 24 mo. The glomerular basement membrane was significantly thinned (167 versus 200 nm in wild type) in 30-wk-old mice, coinciding with focal glomerulosclerosis, tubulointerstitial fibrosis, and increased levels of TGF-beta and connective tissue growth factor. The renal phenotype in COL4A3(+/-) mice resembled the clinical and histopathologic phenotype of human cases of TBMN with concomitant progression to chronic renal failure. Therefore, the COL4A3(+/-) mouse model will help in the understanding of the pathogenesis of TBMN in humans and in the evaluation of potential therapies.

The Clinical Features of Thin Basement Membrane Nephropathy

Thin basement membrane nephropathy (TBMN) is a common, lifelong condition affecting the kidneys that is characterized by microscopic glomerular hematuria, minimal or no proteinuria, and normal renal function. It often is discovered incidentally, and usually has an excellent prognosis. Many cases are familial and show autosomal-dominant inheritance. The defining characteristic is a glomerular basement membrane (GBM) that is thinned to about half its normal thickness on ultrastructural examination of the renal biopsy specimen. However, occasionally patients with TBMN develop marked proteinuria or renal impairment. It is unclear whether individuals with TBMN and impaired renal function represent part of the spectrum of TBMN associated with heterozygous COL4A3 or COL4A4 mutations, or if their disease is caused by mutations of other genes, or whether it is caused by a second coexistent renal lesion or is misdiagnosed Alport syndrome.

Structure of the human type IV collagen gene COL4A3 and mutations in autosomal Alport syndrome.

Mutations in either the COL4A3 or the COL4A4 genes, encoding the alpha3 and alpha4 chains of type IV collagen, are responsible for the autosomal-recessive form of Alport syndrome, a progressive hematuric nephropathy characterized by glomerular basement membrane abnormalities. Reported here are the complete COL4A3 exon-intron structure and a comprehensive screen for mutations of the 52 COL4A3 exons in 41 unrelated patients diagnosed as having autosomal Alport syndrome. This resulted in the identification of 21 mutations that are expected to be causative. Furthermore, it is shown that heterozygous COL4A3 missense mutations, when symptomatic, can be associated with a broad range of phenotypes, from familial benign hematuria to the complete features of Alport syndrome nephropathy.