Alport Syndrome With a Rare Collagen Type IV Alpha-4 (COL4A4) Gene Mutation: A Case Report.

Alport syndrome (AS) is a rare, progressive hereditary kidney disease characterized by sensorineural hearing loss and visual abnormalities. It is caused by a mutation in the collagen type IV alpha-4 (COL4A4) gene, which produces type IV collagen, and often manifests in individuals with hematuria, proteinuria, edema, and hypertension. Here, we present a case of AS in a 15-year-old boy with a COL4A4 gene mutation, with renal and extrarenal findings. The patient presented with subnephrotic proteinuria and microscopic hematuria, autoimmune hypothyroidism, and keratoconus. Light microscopy examination of renal biopsy revealed three globally sclerosed renal cortical parenchyma areas with periglomerular fibrosis, and electron microscopy showed variable thickness of glomerular basement membrane with festooned appearance, as well as splitting of lamina densa giving basket weave and criss-cross pattern.

The Importance of Copy Number Variant Analysis in Patients with Monogenic Kidney Disease.

Introduction: Genetic testing can reveal monogenic causes of kidney diseases, offering diagnostic, therapeutic, and prognostic benefits. Although single nucleotide variants (SNVs) and copy number variants (CNVs) can result in kidney disease, CNV analysis is not always included in genetic testing. Methods: We investigated the diagnostic value of CNV analysis in 2432 patients with kidney disease genetically tested at the University Medical Centre Utrecht between 2014 and May 2022. We combined previous diagnostic testing results, encompassing SNVs and CNVs, with newly acquired results based on retrospective CNV analysis. The reported yield considers both the American College of Medical Genetics and Genomics (ACMG) classification and whether the genotype actually results in disease. Results: We report a diagnostic yield of at least 23% for our complete diagnostic cohort. The total diagnostic yield based solely on CNVs was 2.4%. The overall contribution of CNV analysis, defined as the proportion of positive genetic tests requiring CNV analysis, was 10.5% and varied among different disease subcategories, with the highest impact seen in congenital anomalies of the kidney and urinary tract (CAKUT) and chronic kidney disease at a young age. We highlight the efficiency of exome-based CNV calling, which reduces the need for additional diagnostic tests. Furthermore, a complex structural variant, likely a COL4A4 founder variant, was identified. Additional findings unrelated to kidney diseases were reported in a small percentage of cases. Conclusion: In summary, this study demonstrates the substantial diagnostic value of CNV analysis, providing insights into its contribution to the diagnostic yield and advocating for its routine inclusion in genetic testing of patients with kidney disease.

First patient diagnosed with lipoprotein glomerulopathy and Alport syndrome.

Alport syndrome (AS) is one of the most common inherited kidney disorders, involving pathogenic variants of COL4A3, COL4A4 and COL4A5 genes that lead to disruption of the normal structure of collagen IV protein through improper chain or heterotrimer folding or degradation of heterotrimer components. Lipoprotein glomerulopathy (LPG) is an autosomal dominant disease involving APOE gene mutations disturbing lipoprotein metabolism. We report the first case with both AS and LPG in an 11-year-old girl. The patient presented with blepharedema, and decreased vision. Laboratory examinations showed hematemesis, proteinuria, hypoproteinemia, hyperlipidemia and progressive renal failure. Renal biopsy showed the changes of LPG and AS. Whole-exome sequencing (WES) identified two pathogenic variants, c.127C > T in exon 3 of APOE gene, and c.930 + 1G > A in exon 15 of COL4A4 gene. We emphasize the importance of early completion of renal biopsy and WES for early diagnosis of LPG and AS.

Prevalence of Mendelian Kidney Disease Among Patients With High-Risk APOL1 Genotypes Undergoing Commercial Genetic Testing in the United States.

Introduction: Among individuals with high-risk APOL1 genotypes, the lifetime risk of developing kidney failure is ∼15%, indicating that other genetic variants or nongenetic modifiers likely contribute substantially to an individual patient's risk of progressive kidney disease. Here, we estimate the prevalence and distribution of Mendelian kidney diseases among patients with high-risk APOL1 genotypes undergoing commercial genetic testing in the United States. Methods: We analyzed clinical exome sequencing data from 15,181 individuals undergoing commercial genetic testing for Mendelian kidney disease in the United States from 2020 to 2021. We identified patients with high-risk APOL1 genotypes by the presence of G1/G1, G1/G2, or G2/G2 alleles. Patients carrying single risk APOL1 alleles were identified as G1/G0, G2/G0; the remainder of patients were G0/G0. We estimated the prevalence and distribution of Mendelian kidney disease stratified by APOL1 genotype and genetically predicted ancestry. Results: Of 15,181 patients, 3119 had genetic testing results consistent with a molecular diagnosis of Mendelian kidney disease (20.5%). Of 15,181 patients, 1035 (6.8%) had high-risk APOL1 genotypes. Among patients with recent genomic African ancestry, the prevalence of Mendelian kidney diseases was lower in those with high-risk APOL1 genotypes (9.6%; n = 91/944) compared with single risk APOL1 allele carriers (13.6%; n = 198/1453) and those with G0/G0 APOL1 genotypes (16.6%; n = 213/1281). Among patients with Mendelian kidney disease and recent genomic African ancestry, we observed differences in the prevalence of pathogenic/likely pathogenic variants in PKD1 (19.8% in high-risk vs. 30.2% in low-risk genotypes), and COL4A4 (24.2% in high-risk vs. 10.5% in low-risk genotypes). Conclusion: In this selected population of patients undergoing clinical genetic testing, we found evidence of Mendelian kidney disease in ∼10% patients with high-risk APOL1 genotypes.

Alport Syndrome: Clinical Utility of Early Genetic Diagnosis in Children.

Alport syndrome (AS) is a hereditary glomerulopathy due to pathogenic variants in COL4A3, COL4A4, and COL4A5. Treatment with Renin-Angiotensin-Aldosterone System (RAAS) inhibitors can delay progression to end stage renal disease (ESRD). From 2018 until today, we performed Whole Exome Sequencing (WES) in 19 patients with AS phenotype with or without positive family history. Fourteen of these patients were children. Genetic testing was extended to family members at risk. All patients received a genetic diagnosis of AS: five X-linked AS (XLAS) males, five X-linked AS (XLAS) females, six autosomal dominant AS (ADAS), and one autosomal recessive AS (ARAS). After cascade screening four XLAS males and eight XLAS females, six ADAS and three ARAS heterozygotes were added to our initial results. Fifteen patients were eligible to start treatment with RAAS inhibitors after their diagnosis. All XLAS female patients, ARAS heterozygotes, and ADAS have been advised to be followed up, so that therapeutic intervention can begin in the presence of microalbuminuria. Genetic diagnosis of AS ensures early therapeutic intervention and appropriate follow up to delay progression to chronic kidney disease, especially in thet pediatric population.

Genetic study of Alport syndrome in Tunisia.

BACKGROUND: Alport syndrome is a genetic disorder affecting the kidneys, ears, and eyes, causing chronic kidney disease, sensorineural hearing loss, and ocular abnormalities. It results from pathogenic variants in the COL4A3, COL4A4, or COL4A5 genes, with different inheritance patterns: X-linked from COL4A5 variants, autosomal recessive from homozygous variants in COL4A3 or COL4A4, digenic from variants in both COL4A3 and COL4A4, and autosomal dominant from heterozygous variants in COL4A3 or COL4A4. METHODS: We analyzed 45 patients with Alport syndrome from 11 Tunisian families to determine their clinical and genetic characteristics. Clinical data were collected retrospectively, and whole-exome sequencing was conducted on one patient from each family. Sanger sequencing validated pathogenic variants, and cascade screening extended the analysis to 53 individuals. RESULTS: We identified nine likely pathogenic variants among 11 index cases: six novel and three known variations. Of these, five were in COL4A3, and four were in COL4A5, with variants including frameshift, nonsense, missense, and alternative splicing. Most variations affected the Gly-XY codon. Among the 45 clinically identified siblings, 30 tested positive for Alport syndrome. The cascade screening identified 3 additional affected individuals, 10 unaffected siblings, and 10 unaffected parents. The mode of inheritance was autosomal recessive in six families and X-linked in four families. CONCLUSIONS: This study is the first to screen the mutational spectrum of Alport syndrome in Tunisia. It reveals novel pathogenic variants and suggests that autosomal recessive inheritance may be more common in the Tunisian population than X-linked inheritance, contrary to existing literature.

Four novel mutations identified in the COL4A3, COL4A4 and COL4A5 genes in 10 families with Alport syndrome.

BACKGROUND: Alport syndrome (AS) is an inherited nephropathy caused by mutations in the type IV collagen genes. It is clinically characterized by damage to the eyes, ears and kidneys. Diagnosis of AS is hampered by its atypical clinical picture, particularly when the typical features, include persistent hematuria and microscopic changes in the glomerular basement membrane (GBM), are the only clinical manifestations in the patient. METHODS: We screened 10 families with suspected AS using whole exome sequencing (WES) and analyzed the harmfulness, conservation, and protein structure changes of mutated genes. In further, we performed in vitro functional analysis of two missense mutations in the COL4A5 gene (c.2359G > C, p.G787R and c.2605G > A, p.G869R). RESULTS: We identified 11 pathogenic variants in the type IV collagen genes (COL4A3, COL4A4 and COL4A5). These pathogenic variants include eight missense mutations, two nonsense mutations and one frameshift mutation. Notably, Family 2 had digenic mutations in the COL4A3 (p.G1170A) and UMOD genes (p.M229K). Family 3 had a digenic missense mutation (p.G997E) in COL4A3 and a frameshift mutation (p.P502L fs*151) in COL4A4. To our knowledge, four of the 11 mutations are novel mutations. In addition, we found that COL4A5 mutation relation mRNA levels were significantly decreased in HEK 293 T cell compared to control, while the cellular localization remained the same. CONCLUSIONS: Our research expands the spectrum of COL4A3-5 pathogenic variants, which is helpful for clinical and scientific research.

Reclassification of Genetic Testing Results: A Case Report Demonstrating the Need for Structured Re-Evaluation of Genetic Findings.

Rationale: Alport Syndrome (AS) is a progressive genetic condition characterized by chronic kidney disease (CKD), hearing loss, and eye abnormalities. It is caused by mutations in the genes COL4A3, COL4A4, and COL4A5. Heterozygous mutations in COL4A4 and COL4A3 cause autosomal dominant Alport Syndrome (ADAS), and a spectrum of phenotypes ranging from asymptomatic hematuria to CKD, with variable extra-renal features. In the past, heterozygous mutations in these genes were thought to be benign, however recent studies show that about 30% of patients can progress to CKD, and 15% can progress to end stage kidney disease (ESKD). Presenting Concerns: We present a case of a woman who was noted to have microscopic hematuria pre-living kidney donation. Genetic testing revealed a heterozygous variant of uncertain significance (VUS) in the COL4A4 gene. VUSs are medically nonactionable findings and data show that VUSs can be detected in 41% of all patients who undergo clinical genetic testing. VUSs frustrate clinicians and patients alike. Although they cannot be used in medical decision-making, data suggest that reanalysis can result in the reclassification of a VUS over time. Diagnosis: Post-donation, the index patient had a higher than anticipated rise in serum creatinine, raising a concern for possible intrinsic kidney disease. Kidney biopsy was deemed high risk in the setting of a unilateral kidney thereby limiting possible diagnostic intervention to determine the cause of disease. Intervention: Re-evaluation of prior genetic testing results and reassessment of the previously identified VUS in COL4A4 was performed 5-years post-donation. These analyses, along with the addition of new phenotypic data and extended pedigree data, resulted in the reclassification of the previously identified VUS to a likely pathogenic variant. Outcomes: This case demonstrates the importance of structured, periodic re-evaluation of genetic testing results. With the ever-changing landscape of genetics in medicine, the interpretation of a VUS can be dynamic and therefore warrant caution in living kidney donor evaluations. Studies have shown that about 10% of VUSs can be upgraded to a pathogenic classification after an 18- to 36-month interval. Structured re-evaluation of genomic testing results has not yet been integrated into clinical practice and poses a unique challenge in living kidney donation. Novel findings: This case report highlights the variability of the ADAS phenotype caused by pathogenic heterozygous variants in the type 4 collagen genes. It supports the nomenclature change from a benign hematuria phenotype to ADAS, particularly when additional risk factors such as proteinuria, focal segmental glomerulosclerosis or glomerular basement membrane changes on kidney biopsy are present, or as in this case, evidence of disease in other family members.

Justification: Le syndrome d'Alport (SA) est une maladie génétique progressive caractérisée par une insuffisance rénale chronique (IRC), une perte auditive et des anomalies oculaires. La maladie est causée par des mutations dans les gènes COL4A3, COL4A4 et COL4A5. Des mutations hétérozygotes dans les gènes COL4A4 et COL4A3 provoquent le syndrome d'Alport autosomique dominant (SAAD) et un specter de phénotypes allant de l'hématurie asymptomatique à l'IRC, avec des caractéristiques extrarénales variables. Dans le passé, les mutations hétérozygotes de ces gènes étaient considérées comme bénignes, mais des études récentes montrent qu'environ 30 % des patients peuvent progresser vers l'IRC et 15 % vers l'insuffisance rénale terminale (IRT). Présentation du cas: Nous présentons le cas d'une femme chez qui on avait observé une hématurie microscopique avant un don vivant de rein. Les tests génétiques ont révélé un variant hétérozygote de signification incertaine dans le gène COL4A4. Les variants de signification incertaine (VSI) sont des résultats qui ne peuvent être utilisés médicalement et les données montrent qu'ils peuvent être détectés chez 41 % des patients qui subissent des tests génétiques cliniques. Les VSI sont frustrants tant pour les cliniciens que pour les patients. Bien qu'ils ne puissent pas être utilisés dans la prise de décisions médicales, les données suggèrent que leur réanalyse pourrait entraîner leur reclassification au fil du temps. Diagnostic: Après le don, ce cas index a présenté une élévation de la créatinine sérique plus importante que prévu, ce qui a soulevé une préoccupation quant à la présence d'une néphropathie intrinsèque. La biopsie rénale a été jugée à haut risque dans le contexte de rein unilatéral, ce qui a limité la possible intervention diagnostique pour déterminer la cause de la maladie. Intervention: La réévaluation des résultats des tests génétiques antérieurs et du VSI précédemment identifié dans COL4A4 a été réalisée 5 ans après le don. Ces analyses, ainsi que l'ajout de nouvelles données phénotypiques et de données généalogiques étendues, ont abouti à la reclassification du VSI précédemment identifié en variant probablement pathogène. Résultats: Ce cas illustre l'importance de réévaluer de façon structurée et périodique les résultats des tests génétiques. La génétique étant en constante évolution en médecine, l'interprétation d'un VSI peut être dynamique et, ainsi, justifier la prudence dans les évaluations des donneurs de reins vivants. Des études ont montré qu'environ 10 % des VSI peuvent être reclassés comme pathogènes après 18 à 36 mois. La réévaluation structurée des résultats des tests génomiques, qui n'a pas encore été intégrée dans la pratique clinique, pose un défi unique dans le contexte d'un don vivant de reins. Principales observations: Ce rapport de cas met en évidence la variabilité du phénotype du SAAD causé par des variants hétérozygotes pathogènes dans les gènes du collagène de type 4. Il soutient un changement de nomenclature du phénotype d'hématurie bénigne en SAAD, en particulier en présence de facteurs de risque supplémentaires tels que la protéinurie et la glomérulosclérose segmentaire et focale, de modifications de la membrane basale glomérulaire sur la biopsie rénale ou, comme dans ce cas, de preuves de la maladie chez d'autres membres de la famille.

Genetic diagnosis of Alport syndrome in 16 Chinese families.

BACKGROUND: Alport syndrome (AS) is a genetically heterogeneous disorder resulting from mutations in the collagen IV genes COL4A3, COL4A4, and COL4A5. The genetic diagnosis of AS is very important to make precise diagnosis and achieve optimal outcomes. METHODS: In this study, 16 Chinese families with suspected AS were recruited after pedigree analysis, and the clinical presentations were analyzed by a nephrologist. The genetic diagnosis was performed by whole-exome sequencing (WES) and the disease-causing variants were confirmed by Sanger sequencing. RESULTS: The cohort of probands included seven men and nine women, with a mean age of 19.9 years. Pathological analysis showed slight-to-moderate mesangial proliferation, and thin basement membrane was the main findings. Pathogenic variants were revealed by WES in each family, and the co-segregation with renal presentation was confirmed by PCR. In addition, RT-PCR analysis showed that the intronic variant led to aberrant splicing. CONCLUSION: Our findings expand the spectrum of AS gene variation, which will inform genetic diagnosis and add to the theoretical basis for the prevention of AS.

Clinical Significance of the Cystic Phenotype in Alport Syndrome.

RATIONALE & OBJECTIVE: Alport syndrome (AS) is the most common genetic glomerular disease caused by mutations that affect type IV collagen. However, the clinical characteristics and significance of AS with kidney cysts are not well defined. This study investigated the prevalence and clinical significance of cystic kidney phenotype in AS. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: One hundred-eight patients with AS and a comparison cohort of 79 patients with IgA nephropathy (IgAN). Clinical, genetic, and imaging data were collected from medical records. EXPOSURE: Cystic kidney phenotype evaluated by ultrasonography and defined as the presence of≥3 cysts in each kidney; demographic characteristics and estimated glomerular filtration rate (eGFR) at disease onset. OUTCOME: Cystic kidney phenotype in the AS and IgAN cohorts; time to chronic kidney disease (CKD) stage 3b and longitudinal changes in eGFR in the AS cohort. ANALYTICAL APPROACH: Logistic regression analysis to test independent strengths of associations of clinical/demographic features with the binary outcome of cystic phenotype. Survival analysis for the outcome of reaching CKD stage 3b and linear mixed models for changes in eGFR over time in the AS cohort. RESULTS: We studied 108 patients with AS; 76 (70%) had a genetic diagnosis. Autosomal dominant AS was prevalent, accounting for 68% of patients with a genetic diagnosis. Cystic kidney phenotype was observed in 38% of patients with AS and was associated with normal-sized kidneys in all but 3 patients, who showed increased total kidney volume, mimicking autosomal dominant polycystic kidney disease. The prevalence of cystic kidney phenotype was significantly higher in patients with AS when compared with the group of patients with IgAN (42% vs 19%; P=0.002). Patients with the cystic kidney phenotype were older and had more marked reduction in eGFR than patients without cystic changes. Among patients with AS, the cystic phenotype was associated with older age and a faster decline eGFR. LIMITATIONS: Retrospective, single-center study. CONCLUSIONS: Cystic kidney phenotype is a common finding in AS. The cystic kidney phenotype is a common finding in AS, suggesting a possible role in cystogenesis for the genetic variants that cause this disease. PLAIN-LANGUAGE SUMMARY: Hematuria is the classic renal presentation of Alport syndrome (AS), a hereditary glomerulopathy caused by pathogenic variants of the COL4A3-5 genes. An atypical kidney cystic phenotype has been rarely reported in individuals with these variants. To determine the prevalence of kidney cysts, we performed abdominal ultrasonography in a large group of patients with AS and a comparison group of patients with another glomerular kidney disease, IgA nephropathy (IgAN). Multiple kidney cysts, usually with normal kidney volume, were found in 38% of patients with AS. A few patients' kidney volumes were large enough to mimic a different hereditary cystic kidney disease, autosomal dominant polycystic kidney disease. The overall prevalence of kidney cysts in AS was more than double that observed in the well-matched comparison group with IgAN. These findings emphasize the high prevalence of cystic kidney phenotype in AS, suggesting a likely association between the genetic variants that cause this disease and the development of kidney cysts.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Is there a dominant-negative effect in individuals with heterozygous disease-causing variants in COL4A3/COL4A4?

Alport syndrome (AS) shows a broad phenotypic spectrum ranging from isolated microscopic hematuria (MH) to end-stage kidney disease (ESKD). Monoallelic disease-causing variants in COL4A3/COL4A4 have been associated with autosomal dominant AS (ADAS) and biallelic variants with autosomal recessive AS (ARAS). The aim of this study was to analyze clinical and genetic data regarding a possible genotype-phenotype correlation in individuals with disease-causing variants in COL4A3/COL4A4. Eighty-nine individuals carrying at least one COL4A3/COL4A4 variant classified as (likely) pathogenic according to the American College of Medical Genetics guidelines and current amendments were recruited. Clinical data concerning the prevalence and age of first reported manifestation of MH, proteinuria, ESKD, and extrarenal manifestations were collected. Individuals with monoallelic non-truncating variants reported a significantly higher prevalence and earlier diagnosis of MH and proteinuria than individuals with monoallelic truncating variants. Individuals with biallelic variants were more severely affected than those with monoallelic variants. Those with biallelic truncating variants were more severely affected than those with compound heterozygous non-truncating/truncating variants or individuals with biallelic non-truncating variants. In this study an association of heterozygous non-truncating COL4A3/COL4A4 variants with a more severe phenotype in comparison to truncating variants could be shown indicating a potential dominant-negative effect as an explanation for this observation. The results for individuals with ARAS support the, still scarce, data in the literature.

The multifaceted roles of COL4A4 in lung adenocarcinoma: An integrated bioinformatics and experimental study.

BACKGROUND: Abnormal expression of collagen IV subunits has been reported in cancers, but the significance is not clear. No study has reported the significance of COL4A4 in lung adenocarcinoma (LUAD). METHODS: COL4A4 expression data, single-cell sequencing data and clinical data were downloaded from public databases. A range of bioinformatics and experimental methods were adopted to analyze the association of COL4A4 expression with clinical parameters, tumor microenvironment (TME), drug resistance and immunotherapy response, and to investigate the roles and underlying mechanism of COL4A4 in LUAD. RESULTS: COL4A4 is differentially expressed in most of cancers analyzed, being associated with prognosis, tumor stemness, immune checkpoint gene expression and TME parameters. In LUAD, COL4A4 expression is down-regulated and associated with various TME parameters, response to immunotherapy and drug resistance. LUAD patients with lower COL4A4 have worse prognosis. Knockdown of COL4A4 significantly inhibited the expression of cell-cycle associated genes, and the expression and activation of signaling pathways including JAK/STAT3, p38, and ERK pathways, and induced quiescence in LUAD cells. Besides, it significantly induced the expression of a range of bioactive molecule genes that have been shown to have critical roles in TME remodeling and immune regulation. CONCLUSIONS: COL4A4 is implicated in the pathogenesis of cancers including LUAD. Its function may be multifaceted. It can modulate the activity of LUAD cells, TME remodeling and tumor stemness, thus affecting the pathological process of LUAD. COL4A4 may be a prognostic molecular marker and a potential therapeutic target.

Potential Founder Variants in COL4A4 Identified in Bukharian Jews Linked to Autosomal Dominant and Autosomal Recessive Alport Syndrome.

BACKGROUND: Alport syndrome is a hereditary disorder caused by pathogenic variants in the COL4A gene, which can be inherited in an autosomal recessive, dominant, or X-linked pattern. In the Bukharian Jewish population, no founder pathogenic variant has been reported in COL4A4. METHODS: The cohort included 38 patients from 22 Bukharian Jewish families with suspected Alport syndrome who were referred the nephrogenetics clinic between 2012 and 2022. The study collected demographic, clinical, and genetic data from electronic medical records, which were used to evaluate the molecular basis of the disease using Sanger sequencing, and next-generation sequencing. RESULTS: Molecular diagnosis was confirmed in 20/38 patients, with each patient having at least one of the three disease-causing COL4A4 variants detected: c.338GA (p.Gly1008Arg), and c.871-6T>C. In addition, two patients were obligate carriers. Overall, there were 17 heterozygotes, 2 compound heterozygotes, and 3 homozygotes. Each variant was detected in more than one unrelated family. All patients had hematuria with/without proteinuria at referral, and the youngest patient with proteinuria (age 5 years) was homozygous for the c.338G>A variant. End-stage renal disease was diagnosed in two patients at the age of 38 years, a compound heterozygote for c.338G>A and c.871-6T>C. Hearing deterioration was detected in three patients, the youngest aged 40 years, all of whom were heterozygous for c.338G>A. CONCLUSION: This study unveils three novel disease-causing variants, c.3022G>A, c.871-6T>C, and c.338G>A, in the COL4A4 gene that are recurrent among Jews of Bukharian ancestry, and cause Alport syndrome in both dominant and recessive autosomal inheritance patterns.

Abnormal mRNA Splicing Effect of COL4A3 to COL4A5 Unclassified Variants.

Introduction: Genetic diagnosis of Alport syndrome (AS), which results from pathogenic variants in COL4A3, COL4A4, or COL4A5 genes, is hindered by large numbers of unclassified variants detected using next-generation sequencing (NGS). We examined the impact on splicing of variants of uncertain significance in COL4A3 to COL4A5. Methods: Nine unrelated patients with clinical diagnosis or suspicion of AS were enrolled according to the criteria. Their clinical and genetic data were collected. Blood and urine samples were obtained from the patients and their family members. Sanger sequencing was used to confirm the 9 COL4A3 to COL4A5 unclassified variants identified by NGS. COL4A3 to COL4A5 mRNAs from urine were analyzed using targeted reverse transcription polymerase chain reaction and direct sequencing. Results: Nine COL4A3 to COL4A5 unclassified variants were found to alter mRNAs splicing. Skipping of an exon or an exon fragment was induced by variants COL4A3 c.828+5G>A; COL4A4 c.3506-13_3528del; and COL4A5 c.451A>G (p. [Ile151Val]), c.2042-9 T>G, c.2689 G>C (p. [Glu897Gln]) and c.1033-10_1033-2delGGTAATAAA. Retention of an intron fragment was caused by variants COL4A3 c.3211-30G＞T, and COL4A5 c.4316-20T>A and c.1033-10 G>A, respectively. The 9 families in this study obtained genetic diagnosis of AS, including 3 with autosomal recessive AS and 6 with X-linked AS. Conclusions: Our findings demonstrate that urine mRNA analysis facilitates the identification of abnormal splicing of unclassified variants in Alport genes, which provides evidence of routine use of RNA analysis to improve genetic diagnosis of AS.

Phenotyping of a novel COL4A4 and novel GLA variant in a patient presenting with microhematuria and mildly impaired kidney function: a case report.

We describe the case of a 44-year-old male patient with a longstanding history of microhematuria and mildly impaired kidney function (CKD G2A1). The family history disclosed three females who also had microhematuria. Genetic testing by whole exome sequencing revealed two novel variants in COL4A4 (NM_000092.5: c.1181G>T, NP_000083.3: p.Gly394Val, heterozygous, likely pathogenic; Alport syndrome, OMIM# 141200, 203780) and GLA (NM_000169.3: c.460A>G, NP_000160.1: p.Ile154Val, hemizygous, variant of uncertain significance; Fabry disease, OMIM# 301500), respectively. Extensive phenotyping revealed no biochemical or clinical evidence for the presence of Fabry disease. Thus, the GLA c.460A>G, p.Ile154Val, is to be classified as a benign variant, whereas the COL4A4 c.1181G>T, p.Gly394Val confirms the diagnosis of autosomal dominant Alport syndrome in this patient.

The same heterozygous Col4A4 mutation triggered different renal pathological changes in Chinese family members.

Background: Mutations in the collagen components of the glomerular basement membrane (GBM) often lead to hereditary glomerulonephritis. Previous studies have identified that autosomal dominant mutations of Col4A3, Col4A4 or Col4A5 are associated with thin basement membrane nephropathy (TBMN), Alport syndrome and other hereditary kidney diseases. However, the genetic mutations underlying other glomerulonephritis types have not been elucidated. Methods: In this study, we investigated a Chinese family with hereditary nephritis using the methods of genetic sequencing and renal biopsy. Genomic DNA was extracted from peripheral blood of the proband and her sister, and subsequently was performed genetic sequencing. They were found to have the similar mutation sites. Other family members were then validated using Sanger sequencing. The proband and her sister underwent renal puncture biopsies, and experienced pathologists performed PAS, Masson, immunofluorescence, and immunoelectron microscopic staining of the kidney tissue sections. Results: Through genetic sequencing analysis, we detected a novel heterozygous frameshift mutation c.1826delC in the COL4A4 (NM_000092.4) gene coding region, and 1 hybrid missense variation c.86G>A (p. R29Q) was also detected in the TNXB (NM_019105.6) gene coding region in several members of this Chinese family. Interestingly, we found that the same mutations caused different clinical features and distinct pathological changes in individual family members, which confirmed that pathological and genetic testing are crucial for the diagnosis and treatment of hereditary kidney diseases. Conclusion: In this study, we found a novel heterozygous mutation in Col4A4 and co-mutations of the TNXB gene in this Chinese family. Our study indicated that the same Col4A4 mutated variants produced different pathological and clinical changes in different family members. This discovery may provide novel insights into the study of hereditary kidney disease. In addition, new genetic biology techniques and renal biopsy of individual family members are essential.

Kidney Disease Associated With Mono-allelic COL4A3 and COL4A4 Variants: A Case Series of 17 Families.

Rationale & Objective: Mono-allelic variants in COL4A3 and COL4A4 (COL4A3/COL4A4) have been identified in a spectrum of glomerular basement membrane nephropathies, including thin basement membrane nephropathy and autosomal dominant Alport syndrome. With the increasing use of next generation sequencing, mono-allelic COL4A3/COL4A4 variants are detected more frequently, but phenotypic heterogeneity impedes counseling. We aimed to investigate the phenotypic spectrum, kidney biopsy results, and segregation patterns of patients with mono-allelic COL4A3/COL4A4 variants identified by whole exome sequencing. Study Design: Case series. Setting & Participants: We evaluated clinical and pathologic characteristics of 17 Dutch index patients with mono-allelic variants in COL4A3/COL4A4 detected by diagnostic whole exome sequencing and 25 affected family members with variants confirmed by Sanger sequencing. Results: Eight different mono-allelic COL4A3/COL4A4 variants were identified across members of 11 families, comprising 7 glycine substituted missense variants and 1 frameshift variant. All index patients had microscopic hematuria at clinical presentation (median age 43 years) and 14 had (micro)albuminuria/proteinuria. All family members showed co-segregation of the variant with at least hematuria. At end of follow-up of all 42 individuals (median age 54 years), 16/42 patients had kidney function impairment, of whom 6 had kidney failure. Reports of kidney biopsies of 14 patients described thin basement membrane nephropathy, focal segmental glomerulosclerosis, minimal change lesions, and Alport syndrome. Electron microscopy images of 7 patients showed a significantly thinner glomerular basement membrane compared with images of patients with idiopathic focal segmental glomerulosclerosis and other hereditary glomerular diseases. No genotype-phenotype correlations could be established. Limitations: Retrospective design, ascertainment bias toward severe kidney phenotypes, and familial hematuria. Conclusions: This study confirms the wide phenotypic spectrum associated with mono-allelic COL4A3/COL4A4 variants, extending from isolated microscopic hematuria to kidney failure with high intra- and interfamilial variability.

Protective and pathogenic role of collagen subtypes genes COL4A3 and COL4A4 polymorphisms in the onset of keratoconus in South-Asian Pakistani cohort.

Collagen sub-types have an important role in corneal structure and are reported to be an important genetic predictor for keratoconus (KC) development, therefore we assessed the association of collagen subtypes by screening non-synonymous polymorphisms of COL4A3 and COL4A4 in South-Asian (Pakistani) patients. Methods: A total of 257 KC sporadic cases, gender and ethnicity matched 253 control individuals were screened for three non-synonymous single nucleotide polymorphisms (SNPs) rs55703767and rs10178458 in COL4A3 and rs2229814 and one synonymous SNP rs2228555 in COL4A4. The genotyping was done by Competitive Allele specific polymerase chain reaction (PCR) and the data were analyzed statistically. Results: Among the studied SNPs, the COL4A3 rs55703767 GT genotype (dominant model (DM): odds ratio (OR) = 0.243, (95 %CI) = 0.16-0.36, p=>0.0001), and allele-G (OR = 0.35, 95 %CI = 0.26-0.48, p < 0.000)), showed protective association against KC development. While COL4A3 rs10178458 CT genotype (DM: OR = 2.11(95 %CI = 1.16-3.85), COL4A4 rs2229814 TT genotype (RM: OR = 147.778(95 %CI = 20.401-1070.439), (p > 0.05) and allele-T (OR = 2.351(95 %CI = 1.826-3.028), (p > 0.05); COL4A4 rs2228555 AG genotype (DM: OR = 2.370(95 %CI = 1.594-3.524) (<0.0001) and GG genotype (RM: OR = 2.347(95 %CI = 1.587-3.472), (p < 0.0001); and allele-G (OR = 2.024(95 %CI = 1.577-2.597), (p > 0.0001) were observed to be disease associated. Conclusion: COL4A3 rs10178458 and COL4A4 SNPs rs2229814 and rs2228555 were found to be pathogenic for KC, whereas COL4A3 rs55703767 was found to play a protective role against KC development in South-Asian (Pakistani) Cohort.

Mutation Analysis of Thin Basement Membrane Nephropathy.

Thin basement membrane nephropathy (TBMN) is characterized by the observation of microhematuria and a thin glomerular basement membrane on kidney biopsy specimens. Its main cause is heterozygous mutations of COL4A3 or COL4A4, which also cause late-onset focal segmental glomerulosclerosis (FSGS) or autosomal dominant Alport syndrome (ADAS). Thirteen TBMN cases were analyzed using Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and exome sequencing. Ten heterozygous variants were detected in COL4A3 or COL4A4 in nine patients via Sanger sequencing, three of which were novel variants. The diagnostic rate of "likely pathogenic" or "pathogenic" under the American College of Medical Genetics and Genomics guidelines was 53.8% (7 out of 13 patients). There were eight single nucleotide variants, seven of which were glycine substitutions in the collagenous domain, one of which was a splice-site single nucleotide variant, and two of which were deletion variants. One patient had digenic variants in COL4A3 and COL4A4. While MLPA analyses showed negative results, exome sequencing identified three heterozygous variants in causative genes of FSGS in four patients with no apparent variants on Sanger sequencing. Since patients with heterozygous mutations of COL4A3 or COL4A4 showed a wide spectrum of disease from TBMN to ADAS, careful follow-up will be necessary for these patients.