Next-Generation Sequencing (NGS) Analysis Illustrates the Phenotypic Variability of Collagen Type IV Nephropathies

**Background:** Collagen type IV is a major component of basement membranes in the kidney, cochlea, and eye. Mutations in COL4A3, COL4A4, and COL4A5 are classically associated with Alport syndrome (AS) and thin basement membrane nephropathy (TBMN). However, recent evidence suggests that the phenotypic spectrum of COL4 mutations may be broader, including focal segmental glomerulosclerosis (FSGS) and cystic kidney disease. This study aimed to characterize unexpected renal phenotypes in patients with COL4 mutations identified through next-generation sequencing (NGS).

**Methods:** A total of 176 adult patients (94 female, 82 male; mean age 40.4 years, range 18–79) with suspected inherited kidney disorders were recruited from a Center for Rare Kidney Diseases. Patients were categorized into cystic disorders (n=104, including 64 with polycystic kidney disease and 40 with Bardet–Biedl syndrome), glomerulonephritis (n=29), renal stones and metabolic diseases (n=5), and tubulopathies (n=38). DNA was extracted from peripheral blood and analyzed using either a kidney-focused NGS panel (Nephroplex, 115 genes, n=107) or clinical exome sequencing (~5000 genes, n=69). Variants were filtered for quality, population frequency (MAF<1%), and pathogenicity using multiple in silico tools (SIFT, FATHMM, MutationAssessor, Polyphen-2, MutationTaster, Provean, MuPRO, PANTHER, PhD-SNP, SNP&GO) and ACMG guidelines. Sanger sequencing confirmed candidate mutations. A multidisciplinary team compared clinical and molecular diagnoses; cases with discrepancies were selected for detailed analysis.

**Key Results:** Disease-causing variants were identified in 83 of 176 patients (47.2%). In 78 of these 83 (94%), the genetic diagnosis confirmed the clinical suspicion. In 5 cases (6%), the genetic result was unexpected; 3 of these 5 harbored COL4 mutations. Case 1: a 43-year-old male with childhood microhematuria, proteinuria (2 g/24h), eGFR 60 mL/min/1.73 m², and biopsy-proven FSGS with IgM and C3 deposits. He and his mother carried a heterozygous c.693+2T>C variant in COL4A4 (likely pathogenic, splice donor loss, SpliceAI delta score 0.89). Case 2: a 49-year-old male with no prior history, presenting with hypertension, eGFR 13 mL/min/1.73 m², sensorineural deafness, and bilateral cortical nephrocalcinosis on CT. He carried a hemizygous c.991G>A (p.Gly331Ser) variant in COL4A5 (pathogenic by multiple in silico tools). Case 3: a 59-year-old male with childhood hematuria, proteinuria since age 31, eGFR 39 mL/min/1.73 m², and multiple renal cysts (subcentimetric right, large left). He and his son carried a heterozygous c.1589G>A (p.Gly530Glu) variant in COL4A4 (likely pathogenic). All three variants were not reported in ClinVar.

**Clinical Implications:** This study demonstrates that COL4A4 and COL4A5 mutations can present with renal phenotypes beyond classic Alport syndrome and TBMN, including familial FSGS, cortical nephrocalcinosis with ESRD, and cystic kidney disease. These findings expand the differential diagnosis for unexplained chronic kidney disease, especially when family history, early-onset hematuria, or extra-renal features (e.g., hearing loss) are present. The authors recommend considering COL4 genetic testing in patients with atypical presentations such as FSGS, cortical calcification, or renal cysts, particularly when standard genetic panels for cystic diseases are negative. The study is limited by the small number of COL4 cases and lack of functional validation for the splice variant. Larger cohorts and mechanistic studies are needed to clarify how specific COL4 mutations lead to diverse renal outcomes.