Genotype-phenotype correlations in pathology caused by collagen type IV alpha 1 and 2 mutations

Abstract

COL4A1 and COL4A2 are extracellular matrix proteins that form heterotrimers and are present in nearly all basement membranes in every organ. In the past decade, COL4A1 and COL4A2 mutations have been identified to cause a multi-system disorder for which penetrance and severity of constituent phenotypes can greatly vary. Here, we compare the outcomes of more than 100 mutations identified in patients and data from a murine allelic series to explore the presence of genotype-phenotype correlations - many of which are shared among other types of collagen. We find that there is a frequency bias for COL4A1 over COL4A2 mutations and that glycine (Gly) substitutions within the triple helical domain are the most common class of mutations. Glycine is most often replaced by a charged amino acid, however the position of the mutation, and not the properties of the substituting amino acid, appears to have a greater influence on disease severity. Moreover, the impact of position is not straightforward. Observations from a murine allelic series suggest that mutations in the NC1 domain may result in relatively mild phenotypes via a 'quantitative' mechanism similar to other types of collagens, however, this effect was not apparent in human reports. Importantly, other position-dependent effects had differential impacts depending on the phenotype of interest. For example, the severity of cerebrovascular disease correlated with an amino-to-carboxy severity gradient for triple-helical glycine substitutions whereas the penetrance and severity of myopathy and nephropathy appear to involve a functional sub-domain(s). Greater understanding of genotype-phenotype correlations and the interaction of consequences of different mutations will be important for patient prognosis and care and for developing mechanism-based therapeutics to treat individual components of this emerging syndrome.

Figure 1. Summary of reported phenotypes for murine Col4a1 and Col4a2 mutations

(A) Schematic diagram illustrating murine mutations reported in the literature. (B) List of Col4a1 and Col4a2 mutations and the phenotypes that have been reported for each.

Figure 2. Allelic heterogeneity for heterotrimer biosynthesis, intracerebral hemorrhage and myopathy

(A) Schematic diagram illustrating the mutations in the allelic series. All mutations are in the triple helical domain, except COL4A1^S1582P, which is in the C-terminal NC1 domain. COL4A1^Δex41 is a splice site mutation causing deletion of exon 41. (B) Western blot quantification demonstrating that the mutation in the NC1 domain (COL4A1^S1582P) had relatively low levels intracellular heterotrimer accumulation and that there is a position-dependent graded severity of heterotrimer accumulation for mutations within the triple helical domain whereby mutations nearer the NC1 domain had higher levels of heterotrimer accumulation. (C) Quantification of intracerebral hemorrhage revealed that COL4A1^Δex41 mutation leads to the most severe phenotype and that point mutations in the triple helical domain nearer the C-terminus tended to cause more (or more severe) hemorrhages. (D) Quantification of non-peripheral nuclei revealed that the Col4a1^G394V mutation, which is in an integrin-binding domain, causes the most severe myopathy. For all groups, n>5. Comparisons between wild-type (Col4a1^+/+) and mutant mice were performed using Student’s t-test; *, p<0.05. **, p<0.01. For comparison among the different strains, a one-way ANOVA followed by a Tukey’s post hoc test was performed; †, p<0.05. Panels A, B and D are modified from Kuo et al. (2014) and Panel C is modified from Jeanne et al. (2015) .

Figure 3. Relative measures of hematuria and albuminuria in mice from an allelic series of Col4a1 and Col4a2 mutations

We collected urine and measured levels of hematuria and albuminuria using colorimetric dipsticks and a clinical chemistry autoanalyzer, respectively (described in Gould et al. (2006) ). A). The proportion of animals of each genotype with no, mild, moderate or severe hematuria are indicated as percentages. Sample sizes for both each genotype are indicated at the top. B) We measured albuminuria by calculating the ratio of albumin to creatinine (mg/g) and indicated the proportion of animals of each genotype with no, mild (<30), moderate (30–60) or severe (>60) albuminuria as a percentage. Sample sizes for both each genotype are indicated at the top.