Genetic Basis of Type IV Collagen Disorders of the Kidney

Abstract

The glomerular basement membrane is a vital component of the filtration barrier of the kidney and is primarily composed of a highly structured matrix of type IV collagen. Specific isoforms of type IV collagen, the α3(IV), α4(IV), and α5(IV) isoforms, assemble into trimers that are required for normal glomerular basement membrane function. Disruption or alteration in these isoforms leads to breakdown of the glomerular basement membrane structure and function and can lead to progressive CKD known as Alport syndrome. However, there is wide variability in phenotype among patients with mutations affecting type IV collagen that depends on a complex interplay of sex, genotype, and X-chromosome inactivation. This article reviews the genetic basis of collagen disorders of the kidney as well as potential treatments for these conditions, including direct alteration of the DNA, RNA therapies, and manipulation of collagen proteins.

Introduction

The glomerular basement membrane (GBM) is a vital component of the filtration barrier of the kidney and is primarily composed of a highly structured matrix of type IV collagen, laminin, nidogens, agrin, and perlecan (1). Disruption or alterations in these components lead to breakdown of the GBM structure and function and can lead to progressive CKD. However, there is wide variability in phenotype among patients with mutations affecting type IV collagen that depends on a complex interplay of sex, genotype, and X-chromosome inactivation. This article reviews the genetic basis of type IV collagen disorders of the kidney as well as currently available treatments and potential future genomic treatments for these conditions.

Type IV Collagen

Six genes, COL4A1–COL4A6, encode six isoforms of type IV collagen, α1(IV) to α6(IV). The genes are arranged in three pairs, COL4A1–COL4A2, COL4A3–COL4A4, and COL4A5–COL4A6, situated in a head-to-head orientation on chromosomes 13, 2, and X, respectively. The α(IV) isoforms share structural features, including an amino-terminal sequence of approximately 25 amino acids (7S), a collagenous domain of approximately 1400 amino acids containing multiple Gly-X-Y repeats where X and Y represent nonglycine amino acids, and a carboxy-terminal (NC1) domain of approximately 230 amino acids. Type IV collagen isoforms self-assemble in the endoplasmic reticulum to form triple helices in a very specific stoichiometry. The presence of glycine at every third residue in the collagenous domain is required for assembly of the triple helix. Three heterotrimers that occur in mammalian basement membranes have been described: α1-α1-α2(IV), α3-α4-α5(IV), and α5-α5-α6(IV) (2,3). The α1-α1-α2(IV) network is predominant in the developing GBM until the capillary loop stage, when it is substantially replaced by an α3-α4-α5(IV) network (4). If any of the α3(IV), α4(IV), or α5(IV) isoforms are absent due to severe mutations (truncating mutations, for example), then the other type IV collagen isoforms are degraded, and no α3α4α5(IV) heterotrimers are deposited in the GBM, leading to Alport syndrome (2). In these patients, the α1-α1-α2(IV) network persists, increasing susceptibility to proteolytic degradation and leading to progressive deterioration of the GBM and CKD (4). Milder mutations, generally missense mutations affecting the glycine residues in the collagenous domain that are involved in triple-helix formation, may lead to abnormally folded trimers that are either degraded or lead to formation of an abnormal α3-α4-α5(IV) GBM matrix. Patients with COL4A5 variants who express the α3-α4-α5(IV) network in the GBM have a slower progression of kidney disease (median age of kidney failure >50 years) compared with those patients where the α3-α4-α5(IV) network is absent (median age of kidney failure 29 years) (5). Patients with Alport syndrome may also exhibit sensorineural hearing loss due to the dysfunction of the α3-α4-α5(IV) network in the cochlea as well as ocular findings, such as anterior lenticonus, due to presence of the α3-α4-α5(IV) network in the lens of the eye.

Variants in type IV collagen genes are distributed throughout each gene with no specific hot spots. Over 1500 unique variants have been reported in COL4A5, and over 500 each have been reported in COL4A3 and COL4A4 (6). For COL4A5, these are primarily missense substitutions in 43% (33% in regions encoding glycine within the collagenous domain and 10% other); 34% nonsense mutations (both direct and downstream); 23% splicing variants; 14% small deletions; 7% rearrangements or copy number variants; and small numbers of duplications, insertions, and indels (7). Both the phenotypic heterogeneity of Alport syndrome and the slow progression of the phenotype over decades make assigning pathogenicity using the American College of Medical Genetics and Genomics guidelines potentially problematic (8). This has been addressed for variants in COL4A5 by the development of variant databases, but phenotype genotype correlation data lag behind for variants in COL4A3 and COL4A4 (9,10). This will be addressed by the Clinical Genome Resource Variant Curation Expert Panels, an international collaboration aimed at resolving discrepancies in variant interpretation (9).

Alport Syndrome: Pathogenesis and Current Treatments

If the α3-α4-α5(IV) trimer network does not form in the GBM during the capillary loop stage of glomerular development, the α1-α1-α2(IV) network persists. Compared with the α3-α4-α5(IV) network, basement membranes containing the α1-α1-α2(IV) network are less crosslinked and more susceptible to proteolysis by matrix metalloproteases (2,11). The GBM with predominantly α1-α1-α2(IV) network is also more distensible, leading to biomechanical strain on the GBM affecting the adjacent endothelial cells and podocytes. Alport mice exposed to hypertension with increased biomechanical strain on the GBM demonstrate increased expression of matrix metalloproteinases and inflammatory cytokines compared with wild-type mice (12). In addition, biomechanical strain induces endothelin-1 expression in endothelial cells in animal models of Alport syndrome (13,14). Activation of endothelin type A receptors on mesangial cells leads to mesangial filopodial invasion of the GBM with deposition of aberrant laminins, including laminin 211, that can be blocked with endothelin receptor antagonists (13,15). Podocyte-derived invasions into the GBM in Alport mice have also been described; however, the inciting trigger is unknown (16). Finally, aberrant signaling between the abnormal GBM and the podocyte via integrins also likely plays a role in Alport pathogenesis. Alport mice that have had integrin-α1 or integrin-α2 genes additionally knocked out demonstrate reduced matrix deposition, improved life span, and reduced expression of matrix metalloproteases (17,18). Thus, the thickening of the GBM in Alport syndrome over time appears to be due to increased deposition of matrix from both podocyte and mesangial cell origin triggered by biomechanical strain and aberrant signaling between the GBM and adherent cells (Figure 1).

Figure 1.

Pathogenesis of Alport syndrome kidney disease progression. GBM, glomerular basement membrane.

Current standard of care for patients with Alport syndrome includes inhibition of the renin-angiotensin-aldosterone system to reduce the biomechanical strain on the abnormal GBM. Treatment of mouse models of Alport syndrome with ACE inhibitors doubles the life span of treated animals (19). Retrospective registry studies have also shown an association between improved kidney outcomes and ACE inhibition. A study from the European Alport Registry included 283 patients with Alport syndrome and demonstrated that time to kidney failure was longer in patients who were treated with ACE inhibitors, and the benefit was greatest in those who started treatment earlier (20). These findings were confirmed in a second cohort of Japanese patients with Alport syndrome where those treated with ACE inhibitors had a median age of kidney failure of >50 years, whereas those not treated had a median age of kidney failure of 28 years (5). A prospective study of children with Alport syndrome showed a trend toward delay in progression of proteinuria in children who were treated with ramipril at very early stages of disease (urine albumin <300 mg/g creatinine or isolated hematuria) (21). Given these findings, current treatment recommendations suggest initiation of ACE inhibitors at the time of diagnosis in men or boys with X-linked Alport syndrome and all patients with autosomal recessive Alport syndrome and at initial development of albuminuria in women or girls with X-linked Alport syndrome or all patients with autosomal dominant Alport syndrome (22).

Several other novel drugs are currently in clinical trials to treat Alport kidney disease, primarily targeting later fibrosis signaling pathways. Lademirsen, an inhibitor of microRNA-21 (miRNA-21), is currently being tested in a phase 2 randomized controlled trial in patients with Alport syndrome at high risk of progression (NCT02855268). Bardoxolone is an anti-inflammatory agent that acts via activation of Nrf-2 and inhibition of NF-κB to increase eGFR. Studies in diabetic kidney disease demonstrate an increase in eGFR but were halted early due to a higher risk of hospitalization and death from heart failure (23). Bardoxolone is currently being tested in phase 2/3 randomized controlled trials in patients with Alport syndrome with careful screening to minimize risk of cardiovascular disease (NCT03019185).

Alport Syndrome: Clinical Correlation

Classic Alport syndrome is estimated to occur in 1:50,000 live births (24). Prevalence of milder forms of the disease (heterozygous mutation in COL4A3 and COL4A4) is unknown. X-linked Alport syndrome, caused by mutations in COL4A5 on the X chromosome, accounts for 70%–80% of patients with Alport syndrome. Men with X-linked Alport syndrome invariably develop kidney failure, and their rate of kidney disease progression is strongly influenced by genotype. In a European registry cohort, survival analysis demonstrated that large deletions and nonsense mutations conferred a 90% probability of kidney failure before age 30, compared with a 70% risk with splice site mutations and a 50% risk with missense mutations (25). Similar genotype-phenotype correlations were reported in a Japanese cohort with median age of kidney failure of 18 years for patients with nonsense mutations and 40 years for patients with missense mutations (5). The position of a glycine substitution may also affect the phenotype, as 5′ glycine missense mutations are associated with a more severe phenotype than 3′ glycine mutations (26). The number of side-chain carbon atoms in the substituting amino acid also influences the phenotype associated with a glycine substitution (27).

Women with heterozygous mutations in COL4A5 have a wide spectrum of disease from microscopic hematuria alone to kidney failure (28). In a large cohort of women with X-linked Alport syndrome, the risk of kidney failure was 12% by age 45, 30% by age 60, and 40% by age 80 (29). The explanation for the wide variability in outcomes for women with X-linked Alport syndrome is unclear but is determined at least in part by X inactivation (30). The α3-α4-α5(IV) heterotrimer is present in the GBM in a mosaic pattern due to random X inactivation during fetal development. If by random chance more of the mutant COL4A5 is expressed, kidney outcomes are worse.

Homozygous or compound heterozygous mutations in COL4A3 and COL4A4 cause autosomal recessive Alport syndrome, which accounts for approximately 5% of patients with Alport syndrome. Kidney outcomes in autosomal recessive Alport syndrome are similar to those in men with X-linked Alport syndrome (31). Individuals with heterozygous mutations in COL4A3 or COL4A4 also demonstrate a wide spectrum of disease from microscopic hematuria alone to progressive kidney disease and kidney failure and are categorized as autosomal dominant Alport syndrome (32). Previously, these patients may have been classified as having “thin basement membrane disease” on the basis of biopsy; however, a recent consensus report recommended including all patients with heterozygous mutations in COL4A3 or COL4A4 under the umbrella of Alport syndrome given the similarities in GBM abnormalities and risk of progression requiring ongoing monitoring (32). It has been increasingly recognized that autosomal dominant Alport syndrome accounts for a larger percentage of patients with Alport syndrome than previously recognized, up to 19%–31% of affected patients (33,34).

Digenic inheritance in Alport syndrome has also been described, including patients with dual COL4A3 and COL4A4 variants and patients with COL4A5 and COL4A4/3 variants in both cis and trans configurations (35,36). Sequencing of the coding exons of all three type IV collagen genes is important for diagnosis, even if the inheritance pattern seems clear by pedigree analysis.

Heterozygous mutations in COL4A3 and COL4A4 also may manifest as FSGS with or without classic GBM findings of thinning or thickening with lamellation. This association was first described in 2007 in a cohort of patients from Cyprus and has been reported numerous times since then (37 –39). Type IV collagen mutations are among the most common genetic mutations, identified in up to 31% of adults with familial FSGS (39) and up to 10% of a cohort of predominantly sporadic FSGS (40). It is unclear why some patients with heterozygous COL4A3 or COL4A4 mutations develop classic Alport syndrome kidney phenotype, whereas others exhibit FSGS.

Type IV Collagen Mutations in Other Glomerular Disease

Type IV collagen mutations are also frequently found in patients with CKD who were previously unknown to have Alport syndrome. In a study from Columbia University, 3% of patients with CKD who underwent whole-exome sequencing had pathogenic variants in COL4A3 (0.8%), COL4A4 (0.6%), or COL4A5 (1%), the majority of whom did not have a diagnosis of Alport syndrome (41). This finding highlights the problem of underdiagnosis of patients with type IV collagen mutations.

Type IV collagen mutations may also contribute to pathogenesis in other glomerular disorders. Thin basement membranes have been observed on kidney biopsy in patients with familial IgA nephropathy (42). Pathogenic variants in COL4A3–5 were identified in nine of 46 families with familial IgA nephropathy (43). In a genome-wide association study of over 19,000 patients with diabetic nephropathy, a common missense variant that encodes a tyrosine in place of aspartic acid at position 326 of the COL4A3 protein was identified that was protective against the development of diabetic kidney disease (44). It was hypothesized that the baseline thinning of the GBM associated with this variant prevented the GBM thickening that occurs in diabetic kidney disease and was thus protective. However, in a smaller study of nine individuals with diabetic kidney disease associated with maturity-onset diabetes in the young (MODY), variants in COL4A3 were associated with a more severe kidney phenotype (45). We are just beginning to understand the role of type IV collagen mutations in patients with not only classic Alport syndrome but other glomerular disorders as well.

Alport Syndrome Treatments: Future Genomic Strategies

Current treatments for Alport syndrome only slow the progression of kidney disease, and most patients will still require kidney transplantation; thus, there is an unmet need for novel, curative treatments. Understanding the cellular and molecular biology of the GBM opens up several avenues of genomic therapy for Alport syndrome, including gene editing, gene therapy, RNA therapy, and chaperone therapy (Figure 2).

Figure 2.

Potential genomic treatments for Alport syndrome.COL4A3, COL4A4, and COL4A5 are transcribed into RNA in the nucleus. The RNA is then translated into type IV collagen protein isoforms. These type IV collagen isomers self-assemble into trimers in the endoplasmic reticulum. After they are processed into trimers, they are secreted into the GBM. There are a number of potential targets for rectification of this process in the setting of pathogenic variants in type IV collagen.

Gene Editing

Gene editing targets disease-causing genes to permanently correct, remove, or replace a gene and, thus, cure the disease. Despite significant advances in ex vivo genome editing, it has been used in only a small number of human trials, largely due to concerns about off-target effects, which could disrupt gene function.

Stem cell transplantation is a less precise approach to gene editing and has been tried in animal models of Alport syndrome with promising results, including improvements in kidney function (46). Although mature podocytes are thought to be incapable of replication, bone marrow may act as a podocyte progenitor niche, enabling some regeneration of the GBM (47). Y chromosomes have been demonstrated in the podocytes of male (XY) recipients of female (XX) kidneys, suggesting that some limited regeneration is possible (48). Although patients with GBM disease have had improvement in proteinuria following bone marrow transplants as part of leukemia treatment through recruitment of podocytes and partial expression of α3(IV) chains, this approach is not suitable for the majority of patients (49). Care must be taken with interpretation of stem cell–based experimental results in kidney disease because injection of amniotic fluid stem cells into COL4A5 (−/−) mice before the onset of proteinuria has been shown to delay interstitial fibrosis and glomerular sclerosis, reduce the decline in kidney function, and prolong survival without podocyte differentiation, likely by reducing fibrosis (50). Bone marrow transplants carry a significant mortality risk however; thus, they cannot be considered a safe treatment for Alport syndrome at this time (51).

With experimental evidence showing it is possible to correct a clinically significant proportion of COL4A3 and COL4A5 variants in podocyte cell lines (52), and human trials for Hemophilia-B using infused CRISPR/Cas9-corrected patient-specific stem cells underway (NCT03728322), it is likely that human trials for Alport syndrome will occur in the next decade. Challenges to this approach include difficulties in manipulation of the podocyte in vivo.

Another approach to gene editing that may be explored in Alport syndrome is X-chromosome reactivation. X-chromosome inactivation occurs in early development and ensures that female XX cells have similar expression to male XY cells. In each cell, one X chromosome is randomly epigenetically silenced and is referred to as the inactive X chromosome (Xi), in a process initiated by a molecule called Xist (53). Because the phenotype of women with X-linked Alport syndrome can vary from hematuria to kidney impairment depending on lyonization of the X chromosome, a potential treatment of severe X-linked Alport syndrome in women could be reactivation of the healthy copy of the COL4A5 gene on the Xi. A number of factors have been identified that are involved in this pathway, and the use of small molecule inhibitors targeted at these pathways has been shown to reactivate Xi in mouse models of Rett syndrome (54). Human Genotype-Tissue Expression analysis shows COL4A5 escapes Xi in the brain, although the effect of this is not understood (55). Biallelic expression of some X-linked genes in women has been shown to contribute to a portion of their reduced cancer incidence when compared with men. Thus, Xi remains experimental at this point in time due to significant concerns about off-target effects.

Gene Therapy

In gene therapy, the effect of a mutation is offset by inserting a corrected copy of the gene into the body using a vehicle while the disease-related genes remain in the genome. If the normal gene replaces the mutant allele, then transformed cells proliferate and produce enough normal protein to restore a healthy phenotype. Effective delivery depends on a vehicle, such as a virus or nanoparticle, and an accessible tissue compartment. Thus far, it has been most successful for eye, blood, or bone marrow disease (56).

The feasibility of gene therapy has been shown in mouse models of Alport syndrome with an inducible transgene system, where secretion of α3α4α5(IV) heterotrimers by podocytes into a preformed Alport GBM was effective at restoring the missing collagen IV network, reducing proteinuria, slowing disease progression, and increasing survival (57). Adenovirus-mediated gene transfer into kidney glomeruli has also been demonstrated in mouse models of Alport syndrome (58). However, inducing expression of COL4A3 in mice using an endothelial cell–specific inducible transgenic system does not result in restoration of the α3α4α5(IV) heterotrimer or resolution of the Alport phenotype (59). Successful delivery of COL4A5 into swine kidney by an adenovirus vector has been reported with deposition of α5(IV) into the GBM (60). Unfortunately, this technique required direct infusion of vector into the renal artery, which is not feasible for translation to widespread human application.

Although CRISPR/Cas9 gene editing in the kidney has not yet moved beyond proof of concept, it may be that ocular gene therapy will be utilized at an earlier time point than kidney targeting with delivery of the viral vector directly into the eye (61), where clinical trials and licensed gene replacement therapy are further advanced (62).

Chaperone Therapy

Missense mutations can result in the production of misfolded proteins, which are retained in the endoplasmic reticulum, the quality control center of the cell. This leads to endoplasmic reticulum stress, unfolded protein response activation, and increased cellular apoptosis. Chaperone therapy uses small molecules to unfold abnormal proteins, enabling them to escape the endoplasmic reticulum and be utilized by the cell.

Examples of these agents in human use include Lumacaftor (63), which binds directly to F508del-CFTR correcting its mislocalization, ameliorating the phenotype, and transforming the outlook for people with cystic fibrosis, and Migalastat (64), which stabilizes mutant forms of α-Gal and has shown promise as an alternative to enzyme replacement for patients with Fabry disease.

Mouse models and human kidney biopsy samples of COL4A3 disease have shown unfolded protein response activation (65) as part of the pathogenesis of Alport syndrome. Treatment of cellular models of Alport syndrome with the chaperone sodium-4-phenylbutyrate has shown reduced endoplasmic reticulum stress (66) and may facilitate α5(IV) extracellular transport (67). Future clinical trials targeting Alport phenotypes that lead to unfolded protein response may result in improved outcomes when started early in disease.

RNA Therapy

There are currently three approaches to targeting RNA for treatment of disease: single-stranded antisense oligonucleotides (ASOs), short stretches of DNA that prevent mRNA from being translated into a protein; RNA interference, small interfering RNAs (siRNAs) degrade mRNA and prevent it from being translated into protein and miRNAs, small noncoding RNAs whose functions include post-transcriptional regulation of gene expression; and RNA vaccines, introducing mRNA into the body reprograming the cell to produce a specific protein.

Exon skipping therapy using an ASO has been investigated as a therapy for a small group of patients with a specific variant in COL4A5. Genotype-phenotype correlation data show that some individuals with COL4A5 variants experience a milder clinical course, raising the question of whether exon skipping could nudge the genetic code toward a milder phenotype. For example, published data have shown that specific COL4A5 gene splice site mutations with in-frame deletions showed a good kidney prognosis when compared with an out-of-frame deletion group (68). On the basis of these data, Yamamura et al. (69) have developed an exon-skipping therapy using an ASO targeting truncating variants in exon 21 of the COL4A5 gene.

In patients with truncating variants in COL4A5, the α5(IV) chain will terminate at the stop codon, and the NC1 domain is missing. In contrast, exon-skipping therapy will replace the truncating variant with an in-frame deletion variant at the transcript level, and the NC1 domain is not lost, leading to the formation of the trimer and restoration of the GBM. Mice treated with this protocol had α3α4α5(IV) triple-helix formation with clinical and pathologic improvements, including expression of the α5(IV) chain on GBM and tubular basement membrane with prolonged survival time. Exon skipping does not aim to “cure” the disease but induces a frameshift that leads to the expression of a milder phenotype. The future potential for this approach includes establishing mutant mouse models for other exons that could be targeted by an ASO and will rely on large genomic datasets of individuals with pathogenic variants in COL4A5. There may be significant risk with this approach given that benign hypomorphic variants in gnomAD, such as COL4A5 c.1871G>A, p.(Gly624Asp), have also been implicated in the development of a severe disease (70).

RNA interference can use miRNA, a small, noncoding RNA molecule whose functions include post-transcriptional regulation of gene expression. There are a number of miRNAs that are thought to play a role in the progression of CKD, with several shown to be upregulated in fibrotic kidney biopsies compared with healthy tissue or animal models of human injury. miRNA-21 is upregulated in models of mouse and human kidney disease. In mouse models of Alport syndrome, it is elevated in the kidneys prior to the development of histologic abnormalities and appears to contribute to the pathogenesis of disease by reducing TGF-β–induced fibrogenesis and inflammation (71).

Anti-RNA oligonucleotides can be delivered via subcutaneous injection and act to suppress translation of miRNA. Following injection, the oligonucleotides are found in high concentration in the kidney particularly in the proximal tubular cells, with diseased glomerular cells concentrating the oligonucleotides far more avidly than the healthy glomerulus. In mouse models of Alport syndrome, anti–miRNA-21 oligonucleotides delivered as a weekly injection significantly reduced disease progression and increased life expectancy by 50% (72,73). These agents are currently in clinical trials in humans (see above).

Dominantly inherited conditions could be treated by using specific siRNA molecules to silence the mutated gene or protein, supplying a normal copy of the gene to take over and produce a sufficient amount of the healthy protein. Animal studies have shown promise for this approach in Huntington disease using siRNA to target the degradation of abnormal huntingtin protein (74), and human clinical trials are planned.

Several new coronavirus disease 2019 vaccines involve an intramuscular injection of an mRNA molecule containing the instructions to make a severe acute respiratory syndrome coronavirus 2 spike protein, triggering an immune response and leading to immunity from coronavirus disease 2019 (75). The global drive to develop this vaccine has moved this field of research forward significantly. At the time of writing, large-scale vaccination programs are underway, and although there are not RNA-based therapies for Alport syndrome at present, this field of research is likely to yield benefits for patients with monogenic diseases over the next decade.

In the 30 years since the discovery of mutations in COL4A5 as the cause of Alport syndrome (76), huge strides have been made in understanding of the structure and function of the GBM, as well as the genomic features that affect the course of disease. A number of exciting potential treatments for Alport syndrome are in the pipeline with the aim to slow, and eventually cure, this progressive disease.

Disclosures

C. Quinlan reports employment with The Murdoch Children’s Research Institute, The Royal Children’s Hospital, and the University of Melbourne and serving as section editor for Nephrology. M.N. Rheault reports employment with the University of Minnesota; receiving research funding from Advicenne, Reata, Travere, and Sanofi; and serving on the Alport Syndrome Foundation Medical Advisory Board, NephJC (501c3) Board of Directors, and Pediatric Nephrology Research Consortium (501c3) Steering Committee.

Funding

None.