COL4A1 and COL4A2 mutations and disease: insights into pathogenic mechanisms and potential therapeutic targets

Human Molecular Genetics, 2012, Vol. 21, Review Issue 1 doi:10.1093/hmg/dds346 Advance Access published on August 21, 2012 R97–R110 COL4A1 and COL4A2 mutations and disease: insights into pathogenic mechanisms and potential therapeutic targets Debbie S. Kuo1, Cassandre Labelle-Dumais1 and Douglas B. Gould1,2,3,∗ 1 Department of Ophthalmology, 2Department of Anatomy and 3Institute for Human Genetics, UCSF School of Medicine, San Francisco, CA 94143, USA Received July 25, 2012; Revised and Accepted August 14, 2012 TYPE IV COLLAGENS The type IV collagens are encoded by three pairs of paralogous genes [collagen type IV alpha 1 (COL4A1) through COL4A6]. COL4A1 and COL4A2 are highly conserved across species and their protein products are present in almost all basement membranes, whereas COL4A3 through COL4A6 are more spatially and temporally restricted (1). The proteins encoded by these six genes associate nonrandomly into three distinct heterotrimers in vivo: a1a1a2, a3a4a5 and a5a5a6 (2 – 4). Mutations in COL4A3, COL4A4 and COL4A5 cause Alport Syndrome—a pleiotropic disease affecting the retina, cochlea and kidney that often results in end-stage renal disease (5). Large deletions involving the adjacent COL4A5 and COL4A6 genes are reported to cause diffuse leiomyomatosis (6). Here, we review emerging developments regarding the biology and pathogenic mechanisms underlying COL4A1- and COL4A2-associated diseases. COL4A1 (NM_001845) and COL4A2 (NM_001846) comprise 52 and 48 exons, respectively, and are arranged head to head on opposite strands of human Chromosome 13 (13q34). The two genes are separated by 127 nucleotides containing a shared bi-directional promoter that requires additional elements to control tissue specificity and the level and ratio of expression (Fig. 1) (7). Murine Col4a1 (NM_009931) and Col4a2 (NM_009932) are located on chromosome 8 (5.0 cM) in a similar genomic organization (8,9). COL4A1 and COL4A2 mRNAs are subject to post-transcriptional control, including regulation by a family of microRNAs that down-regulate their expression (10– 16) and other microRNAs that indirectly regulate collagen synthesis (17,18). The Caenorhabditis elegans COL4A2 ortholog has a developmentally regulated, alternatively spliced isoform (19). Alternatively spliced COL4A1 and COL4A2 isoforms are predicted in humans and mice. One in particular (ENST00000397198) omits amino acids 498 – 848 which encompass an angiogenesis regulatory domain, putative integrin-binding sites and a region containing an interesting class of mutations in human patients (20) (see below); however, there is currently little empirical evidence to support the existence of alternative splicing in vivo. COL4A1 and COL4A2 proteins contain three major domains: an amino-terminal 7S domain, a central triple-helix-forming (collagenous) domain and a carboxyterminal non-collagenous (NC1) domain (Fig. 1). The 7S domain participates in inter-molecular cross-linking and macromolecular organization. The collagenous domain constitutes the majority of the protein and consists of long stretches of (Gly-X-Y)n repeats where X and Y are variable amino ∗ To whom correspondence should be addressed at: 10 Koret Way, Room K235, San Francisco, CA 94143-0730, USA. Tel: +1 4154763592; Fax: +1 4154760336; Email: # The Author 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Heterotrimers composed of collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) constitute one of the most abundant components of nearly all basement membranes. Accordingly, mutations in COL4A1 or COL4A2 are pleiotropic and contribute to a broad spectrum of disorders, including myopathy, glaucoma and hemorrhagic stroke. Here, we summarize the contributions of COL4A1 and COL4A2 mutations in human disease, integrate knowledge gained from model organisms and evaluate the implications for pathogenic mechanisms and therapeutic approaches. R98 Human Molecular Genetics, 2012, Vol. 21, Review Issue 1 Figure 1. Distribution of COL4A1 and COL4A2 mutations in schematics of human and mouse proteins. The Col4a1 and Col4a2 genes are transcribed from a shared, bidirectional promoter. Mature proteins are composed of three distinct domains: 7S, collagenous and non-collagenous (NC1). Mutations identified in humans and in mice are indicated above and below the schematics, respectively, with mutations causing HANAC Syndrome (hereditary angiopathy with nephropathy, aneurysms and muscle cramps) shown in red. Probable pathogenic human mutations, defined as displaying an unambiguous familial inheritance pattern, are in bold while other putative pathogenic human mutations are in plain text. BIOSYNTHESIS OF a1a1a2 HETEROTRIMERS COL4A1 and COL4A2 are translated at the rough endoplasmic reticulum (ER) where nascent peptides interact with ER resident proteins to ensure proper folding, post-translational modification and heterotrimer assembly (Fig. 2A). NC1 domains are folded and stabilized by intra-molecular crosslinks formed by protein disulfide isomerase (PDI) before determining the register of the triple helix and initiating heterotrimer formation with one COL4A2 and two COL4A1 peptides (a1a1a2) (3,23). Prior to triple helix formation, the individual peptides of the trimeric complex undergo several post-translational modifications, including hydroxylation of prolyl and lysyl residues and N-linked and O-linked glycosylation. Hydroxylation of proline to hydroxyproline (Hyp) is critical for triple helix stabilization. Without Hyp, melting temperatures of triple helices are near physiological temperatures. Hydroxylation of Y-position prolines to 4-Hyp is catalyzed by prolyl 4-hydroxylase (P4H). In mammals, P4H is an a2b2 tetramer in which PDI is the b-subunit and the a-subunit posses the substrate recognition domain and the enzymatic active site. Vertebrates have three a-subunit isoforms, P4HA1-3, suggesting that there may be redundancy or substrate specificity. Caenorhabditis elegans lacking P4H activity or mice that are deficient for P4ha1 have basement membranes that lack type IV collagen and rupture easily, leading to lethality (24– 26) (Table 1). Mice deficient for P4ha2 have no obvious phenotype (27) and mice deficient for P4ha3 have not been reported. Vertebrates also have three prolyl 3-hydroxylase (P3H) isoforms, P3H1-3, that catalyze the less common modification of X-position proline to 3-Hyp. Mutations in P3H1 (officially LEPRECAN 1: leucine proline-enriched proteoglycan) cause severe autosomal recessive osteogenesis imperfecta type VIII (28). The P3H2 gene (LEPREL1: leprecan-like 1) appears to encode the P3H responsible for modifying type IV collagens (29) and is expresse (...truncated)