COL4A2 mutations impair COL4A1 and COL4A2 secretion and cause hemorrhagic stroke

Abstract

Collagen, type IV, alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers and are abundant components of basement membranes, including those of the cerebral vasculature. COL4A1 mutations are an increasingly recognized cause of multisystem disorders, including highly penetrant cerebrovascular disease and intracerebral hemorrhage (ICH). Because COL4A1 and COL4A2 are structurally and functionally associated, we hypothesized that variants in COL4A2 would also cause ICH. We sequence COL4A2 in 96 patients with ICH and identify three rare, nonsynonymous coding variants in four patients that are not present in a cohort of 144 ICH-free individuals. All three variants change evolutionarily conserved amino acids. Using a cellular assay, we show that these putative mutations cause intracellular accumulation of COL4A1 and COL4A2 at the expense of their secretion, which supports their pathogenecity. Furthermore, we show that Col4a2 mutant mice also have completely penetrant ICH and that mutations in mouse and human lead to retention of COL4A1 and COL4A2 within the endoplasmic reticulum (ER). Importantly, two of the three putative mutations found in patients trigger ER stress and activate the unfolded protein response. The identification of putative COL4A2 mutations that might contribute to ICH in human patients provides insight into the pathogenic mechanisms of this disease. Our data suggest that COL4A2 mutations impair COL4A1 and COL4A2 secretion and can also result in cytotoxicity. Finally, our findings suggest that, collectively, mutations in COL4A1 and COL4A2 contribute to sporadic cases of ICH.

Figure 1

Six Nonsynonymous Rare Variants Identified in Patients Result in Missense Changes in Amino Acids that Are Highly Conserved across Species (A) Electropherograms of genomic DNA from patients (top panels) or control individuals (lower panels) showing coding variants in patients. (B) Multispecies alignment of COL4A2 orthologs shows that most of the rare coding variants identified in patients change highly conserved amino acids.

Figure 2

Functional Assay Reveals that COL4A2^E1123G, COL4A2^Q1150K, and COL4A2^A1690T Impair COL4A2 and COL4A1 Secretion Ratios of extracellular to intracellular COL4A2 (A) or COL4A1 (B) from cells expressing different COL4A2 alleles were determined by immunoblot analysis and are expressed as a percent relative to control (NM_001846.2) COL4A2 cDNA (mean of five independent experiments ± standard error of the mean [SEM]). The highly polymorphic common variant COL4A2^P718S was used as a presumptive negative control, and the pathogenic mutation COL4A2^G1389R was used as a positive control of the functional assay. The variants COL4A2^V192F, COL4A2^K701R, or COL4A2^R1109Q have no functional consequences on the biosynthesis of COL4A1/COL4A2 heterotrimers. The COL4A2^E1123G, COL4A2^Q1150K, and COL4A2^A1690T variants significantly reduce the extracellular to intracellular ratio of COL4A2 and COL4A1, suggesting that they are pathogenic mutations (^∗p < 0.05, ^∗∗p < 0.01). The control cDNA and a common polymorphism are shown in light gray, the known pathogenic variant is shown in black, and the rare variants identified in the patient cohort are shown in dark gray.

Figure 3

Col4a2 Mutant Mice Have Intracerebral Hemorrhages and Retention of COL4A1/A2 Heterotrimers within the ER (A) Prussian Blue staining of brains from Col4a2^+/G646D mutant mice revealed multifocal ICHs in all mutant mice (n = 5) and none in control mice (C57BL/6J Col4a1^+/+; Col4a2^+/+, n = 7, data not shown). The extent of the ICH appeared much less severe than ICH detected in Col4a1^+/Δex40 mutant mice. (B) To compare the severity of ICH between the two mutant lines, we measured the proportional area of ICH in brain sections at regularly spaced intervals (mean ± SEM). Control (C57BL/6J) mice never had detectable ICH and ICHs in Col4a2^+/G646D mutant mice were much less severe than those observed in Col4a1^+/Δex40 mutant mice. (C and D) Immunofluorescent labeling of COL4A1 or COL4A2 (rat H11 or rat H22 [1:200], respectively, with AlexaFluor488 anti-rat IgG [1:500], Invitrogen; green) and the ER marker PDI (mouse ID3 [1:500], Stressgen with AlexaFluor594 anti-mouse IgG [1:500], Invitrogen; red) was performed in MEFs. MEFs were grown on glass coverslips, serum-deprived and treated with ascorbic acid (50 μg/ml) for 24 hr, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton in PBS. Cells were then mounted with Mowiol containing 4′, 6-diamidino-2-phenylindole (DAPI) to label nuclei. Colabeling with PDI and anti-COL4A1 (C) or anti-COL4A2 (D) in control (C57BL/6J Col4a1^+/+; Col4a2^+/+) cells showed considerable COL4A1/A2 labeling that did not colocalize with PDI and therefore was not within the ER. In contrast, in Col4a1^+/Δex40 or Col4a2^+/G646D mutant cells all of the detectable COL4A1 and COL4A2 labeling was colocalized with the ER marker, indicating ER retention of the COL4A1 and COL4A2 proteins. All animal experiments were done with the approval of the UCSF institutional animal care and use committee.

Figure 4

Putative Human Mutations, COL4A2^E1123G, COL4A2^Q1150K, and COL4A2^A1690T Encode Proteins that Are Retained within the Endoplasmic Reticulum Immunofluorescent labeling of COL4A2 (green; as described in Figure 3 legend) was performed in cells expressing control COL4A2 cDNA or different COL4A2 variants. (A) In cells transfected with control COL4A2 cDNA, COL4A2 (green) is detected in the ER where it colocalized with PDI (red) and in post-ER vesicles (arrow). (D) In cells transfected with the pathogenic mutation COL4A2^G1389R, all of the COL4A2 labeling was perinuclear and colocalized with PDI consistent with ER retention. Similarly, in cells transfected with the three putative mutations (B, C, and E), COL4A2 colocalized with PDI in a tight perinuclear pattern. (E) A nontransfected cell (red) that does not express the COL4A2^A1690T variant labeled with PDI and demonstrates a more disperse ER network compared to the transfected cell expressing the putative mutation (yellow).

Figure 5

Putative Human Mutations, COL4A2^E1123G and COL4A2^Q1150K, Trigger ER Stress and Activation of the UPR Quantitative RT-PCRs for ER stress and UPR markers were performed on cells stably transfected with control COL4A2 cDNA or different COL4A2 variants that expressed similar levels of COL4A2, COL4A1, and collagen-binding chaperone HSP47 (top panels). For analysis, cells were serum-deprived and treated with ascorbic acid (50 μg/ml) for 24 hr before being harvested. Total RNAs were isolated from each clone with RNeasy mini kit (QIAGEN) and used for the generation of cDNA with iScript Supermix (Bio-Rad). RT-qPCRs were performed with SsoFast EvaGreen supermix (Bio-Rad) on the equivalent of 20 ng of RNA. Presence of ER stress and UPR activation were investigated with the expression levels of the ER chaperone BIP, two ER-resident transducers of the UPR PERK and ATF6, the downstream transcription factor ATF4 and the UPR target gene CHOP (lower panel). The common variant COL4A2^P718S and the pathogenic mutation COL4A2^G1389R were used as controls. Levels of expression are expressed relative to control (mean of four independent clones ± SEM). ER stress and UPR activation were detected in cells expressing the putative mutant COL4A2^E1123G and COL4A2^Q1150K (^∗p < 0.05, ^∗∗p < 0.01).