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Type IV Collagen Mutations in Familial IgA Nephropathy

channel that it activates. However, this work highlights the fact that the permeability factor of MCD may not be solely a glomerular one (allowing the leakage of albumin through the glomerular filtration barrier), but also a systemic one, allowing the leakage of sodium in the interstitial space, possibly through the activation of an endothelial sodium channel by proteases. In conclusion, this original work shows, for the first time, an increased endothelial permeability induced by sera from patients with MCD, allowing the passage of sodium through the endothelium, probably participating in the constitution of edema, and reversed by amiloride and by aprotinin.

channel that it activates. However, this work highlights the fact that the permeability factor of MCD may not be solely a glomerular one (allowing the leakage of albumin through the glomerular filtration barrier), but also a systemic one, allowing the leakage of sodium in the interstitial space, possibly through the activation of an endothelial sodium channel by proteases.
In conclusion, this original work shows, for the first time, an increased endothelial permeability induced by sera from patients with MCD, allowing the passage of sodium through the endothelium, probably participating in the constitution of edema, and reversed by amiloride and by aprotinin.

DISCLOSURE
All the authors declared no competing interests.

SUPPLEMENTARY MATERIAL
Supplementary File (Word) Supplementary Material and Methods. Table S1. Patient characteristics. Figure S1. Sera from MCD patients do not modify the permeability of HUVEC to high molecular weight molecules in vitro. Figure S2. The increase in HUVEC permeability is not due to paracellular pathway modifications. Figure S3. The increase of the HUVEC permeability for low molecular weight molecules in vitro is not linked to the Caveolin 1 pathway. Figure S4. Type IV Collagen Mutations in Familial IgA Nephropathy I gA nephropathy (IgAN) is a leading cause of chronic glomerulonephritis, and exhibits highly heterogeneous clinical and pathological features 1,2 . Although IgAN classically presents as a young adult with macroscopic hematuria accompanying an upper respiratory infection or gastrointestinal illness, patients can present with isolated microscopic hematuria, mild proteinuria, and/or hypertension. 1,2 Diagnosis is based on renal biopsy, with characteristic features including mesangial hypercellularity and IgA-dominant deposits in the glomerular mesangium; however, diverse findings can be seen on light and electron microscopy. 1,2 The prevalence of IgAN varies with ethnicity, and correspondingly follows a geographic gradient, being found most commonly in East Asians, followed by Europeans, and rarely among individuals of African descent. 1,S1 Both such ancestry-specific variation and familial clustering of disease support that hereditary factors contribute meaningfully to the pathogenesis of IgAN; yet, although genome-wide association studies have identified many risk loci for sporadic forms of IgAN, the genetic basis of familial disease remains largely unresolved. 1,S2 Interestingly, manifestations of IgAN, including microscopic hematuria and diffuse glomerular basement thinning, 3 can overlap considerably with those of type IV collagen-associated nephropathy, which includes Alport syndrome (AS) and thin basement membrane disease (TBMD) and results from mutations in the COL4A3, COL4A4, and COL4A5 genes. Moreover, prior genome-wide linkage scans of familial IgAN have detected significant signals at the chromosome 2q36 region, 4 which encompasses the COL4A3/A4 locus, further supporting that in some cases, type IV collagen mutations may be associated with IgAN. To investigate this hypothesis, we retrospectively analyzed the exome sequence (ES) data of 46 familial IgAN cases for putatively pathogenic COL4A3-5 variants.
We report notable ES results from 12 of these 46 familial cases. These 12 families were ascertained through a proband with biopsy-proven IgAN who was referred for evaluation of familial IgAN for having at least 1 other family member with known IgAN, chronic kidney disease of undetermined etiology, or hematuria ( Figure 1). IgAN was diagnosed based on a kidney biopsy specimen showing mesangial expansion and/or proliferation with IgA-dominant deposits. Electron microscopy was available for 1 case. We excluded from analysis individuals with a personal and/or family history of clinical features potentially consistent with type IV collagen-associated nephropathy, including hearing loss, and visual impairment ( Table S1). Using consensus guidelines for diagnostic sequence interpretation for COL4A3-5 variants, 5 we identified Pathogenic or Likely Pathogenic variants segregating in 9 families (Table 1, S3-S15 Figure 1, Supplementary  Table S2). In an additional 3 families, we found segregating rare variants ultimately classified as variants of uncertain significance due to insufficient evidence for pathogenicity (Supplementary Table S3, Supplementary Figure S1). Of the 9 families with Pathogenic or Likely Pathogenic variants, 6 (67%) demonstrated autosomal dominant inheritance, with 2 harboring heterozygous variants in COL4A3 and 4 in COL4A4; the remaining 3 showed X-linked inheritance, with variants in COL4A5. Substitution (missense) mutations affecting highly conserved glycine residues in the triple helical collagenous domain were noted in 7 of the 9 families (78%); the other 2 families (22%) had frameshift variants. Five of the 11 variants had been previously reported to be pathogenic in patients clinically diagnosed with AS or TBMD (Table 1). Incomplete penetrance was observed among 3 (PED1, PED2, and PED8) of the 6 families with autosomal disease; in contrast, nephropathy was fully penetrant among families with COL4A5 variants (Figure 1).
Our study builds on prior reports of an expanded phenotypic spectrum among individuals harboring putatively pathogenic type IV collagen mutations. Although traditionally associated with AS and TBMD, such variants are now being detected among individuals clinically diagnosed with other nephropathies, such as focal segmental glomerulosclerosis (FSGS), and among cases with undiagnosed disease. 6,7,S16,S17 Moreover, several recent case reports have noted putatively pathogenic mutations in these genes among individuals with familial hematuric nephropathy initially diagnosed as IgAN. S18-S21 To date, we have found putatively pathogenic COL4A3-5 mutations in 9 of the 46 familial IgAN cases (20%), a yield similar to that from assessments of patients clinically diagnosed with familial focal segmental glomerulosclerosis for mutations in these genes. 7,S17 As for focal segmental glomerulosclerosis, the majority of cases showed autosomal dominant inheritance, with heterozygous mutations in COL4A3 and COL4A4 accounting for 67% (6 of 9) of the positive cases. 7,S17 The greater phenotypic variability noted for autosomal dominant versus X-linked pedigrees, with incomplete penetrance observed in 50% (3 of 6) families with heterozygous COL4A3 or COL4A4 variants, is also consistent with prior studies. 5 The role of type collagen IV mutations in IgAN pathogenesis is unclear. These findings may reflect the limitations of traditional clinical disease classifications, especially for more complex phenotypes. Kidney diseases have traditionally been classified on the basis of clinical symptomatology and histopathology. However, as many nephropathies can have nonspecific and/or heterogenous presentations, each of these can overlap considerably between clinical disease subtypes. Thus, although type IV collagen-associated nephropathy is classically characterized by progressive hematuric renal disease, hearing impairment, and ophthalmologic anomalies, patients may present with isolated hematuria and/or proteinuria, which can be seen across many different types of glomerulopathy, including IgAN. 5 Similarly, albeit consistent with a diagnosis of type IV collagen-associated nephropathy, the histopathologic findings of glomerular basement membrane thinning, splitting, and lamellation have been noted in IgAN patient biopsy specimens, 8,S22,S23 and IgAN has been found concurrently with TBMD. S24 Thus, type IV collagen mutations may serve as modifying factors for IgAN. Alternatively, based on autopsy series and donor biopsy studies showing mesangial IgA deposition in as many as 16% of asymptomatic individuals, the detection of IgA deposits in the probands may be coincidental. 9,S25 Although detailed histopathology was unavailable for the majority of our cases, we were able to obtain renal biopsy data for 2 probands: 1 individual from the PED10 family, who was hemizygous for the COL4A5 p.G784R missense variant, and the other from the PED7 family, who was heterozygous for the COL4A4 p.G807Vfs*62 frameshift variant (Supplementary Figure S2). Interestingly, in both cases, glomerular basement membrane thinning was noted, although no clear-cut lamellation or basket-weaving was observed. Given with the observed phenotypic overlap and previous detection of significant linkage signals at the COL4A3/A4 locus in familial IgAN, 4 our findings encourage additional investigation into a potential shared pathogenesis between these 2 disorders, including among nonfamilial cases. Our study has notable strengths and important limitations. To our knowledge, our investigation represents the largest-scale report to date of type IV collagen variants in familial IgAN. In our analysis, we not only applied detailed, disease-specific criteria to identify putatively pathogenic COL4A3/4/5 variants 5 from ES data, but also obtained DNA samples from family members and performed segregation analysis, further supporting their pathogenicity. However, because our ES analysis was retrospective, we had incomplete clinical data for the families assessed, thereby limiting our ability to examine genotypeÀphenotype relationships. In addition, our study has the technical limitations of ES-based analysis, including the inability to investigate noncoding variants and a low sensitivity for detecting copynumber variation (e.g., exonic deletions), both of which have been found as causal variants for type IV collagen-associated nephropathy. 5 Thus, our findings may underestimate the true prevalence of putatively pathogenic type IV collagen variants in familial IgAN. Future studies integrating genomic and phenotypic data from large, ethnically diverse cohorts of all-cause chronic kidney disease case patients and population controls will support a greater understanding of the phenotypic spectrum and longer-term clinical outcomes associated with type IV collagen mutations, thereby informing diagnostic genetic testing and personalized management for individuals with nephropathy.

DISCLOSURE
All the authors declared no competing interests.

ACKNOWLEDGMENTS
This work was supported by National Institutes of Health grants 1F30DK116473 and R01DK082753. We thank all the study participants and physicians for contributing to this effort.

SUPPLEMENTARY MATERIAL
Supplementary File (PDF) Supplementary Methods. Table S1. Additional clinical and genetic data for the 12 families segregating type IV collagen variants. Table S2. Pathogenic and likely pathogenic variants identified with supporting ACMG classification criteria. Table S3. Variants of uncertain significance (VUS) detected segregating in 3 families. Figure S1. Pedigrees of the 3 families with segregating variants of uncertain significance (VUS). Figure S2. Biopsy data of (A) PED10 and (B) PED7 cases. Supplementary References.