Allelic heterogeneity contributes to variability in ocular dysgenesis, myopathy and brain malformations caused by Col4a1 and Col4a2 mutations

Debbie S. Kuo 1 2 Cassandre Labelle-Dumais 1 2 Mao Mao 1 2 Marion Jeanne 1 2 William B. Kauffman 1 2 Jennifer Allen 1 2 Jack Favor 0 Douglas B. Gould 1 2 0 Institute of Human Genetics , Helmholtz Zentrum Mu nchen, Neuherberg D-85764, Germany 1 Department of Anatomy, Institute for Human Genetics, UCSF School of Medicine , San Francisco, CA 94143, USA 2 Department of Ophthalmology Collagen type IV alpha 1 and 2 (COL4A1 and COL4A2) are present in nearly all basement membranes. COL4A1 and COL4A2 mutations are pleiotropic, affecting multiple organ systems to differing degrees, and both genetic-context and environmental factors influence this variable expressivity. Here, we report important phenotypic and molecular differences in an allelic series of Col4a1 and Col4a2 mutant mice that are on a uniform genetic background. We evaluated three organs commonly affected by COL4A1 and COL4A2 mutations and discovered allelic heterogeneity in the penetrance and severity of ocular dysgenesis, myopathy and brain malformations. Similarly, we show allelic heterogeneity in COL4A1 and COL4A2 biosynthesis. While most mutations that we examined caused increased intracellular and decreased extracellular COL4A1 and COL4A2, we identified three mutations with distinct biosynthetic signatures. Reduced temperature or presence of 4-phenylbutyrate ameliorated biosynthetic defects in primary cell lines derived from mutant mice. Together, our data demonstrate the effects and clinical implications of allelic heterogeneity in Col4a1- and Col4a2-related diseases. Understanding allelic differences will be valuable for increasing prognostic accuracy and for the development of therapeutic interventions that consider the nature of the molecular cause in patients with COL4A1 and COL4A2 mutations. - INTRODUCTION Collagen type IV alpha 1, COL4A1 (MIM 120130), and its binding partner COL4A2 (MIM 120090) are major constituents of nearly all basement membranes. COL4A1 and COL4A2 each contain a long, triple-helix-forming, collagenous domain flanked by a short 7S domain at the amino terminus and a globular, non-collagenous (NC1) domain at the carboxy terminus. Two COL4A1 peptides and one COL4A2 peptide associate via their NC1 domains (1,2) and assemble into heterotrimers within the endoplasmic reticulum (ER) before being secreted into the extracellular space. Heterotrimers polymerize to form a collagen IV network that is essential to the development, structure and function of normal tissues. Consistent with the widespread distribution of these proteins, COL4A1 and COL4A2 mutations are pleiotropic and cause a broad spectrum of disorders affecting multiple organs, including the brain, eyes, kidneys and muscles, both in humans and in mice (3 6). Importantly, the severity of pathology is influenced by the genetic-context and environmental factors (7 9). Notably, we To whom correspondence should be addressed at: University of California, San Francisco, Department of Ophthalmology, 10 Koret Way, San Francisco, CA 94143, USA. Tel: +1 4154763592; Fax: +1 4154760336; Email: The authors wish it to be known that, in their opinion, the first two authors should be regarded as co-first authors. have shown previously that the presence and severity of Col4a1-induced ocular and muscular defects in mice is geneticcontext dependent (8,9). In addition, accumulating evidence suggests that allelic heterogeneity also contributes to the variable expressivity of COL4A1 and COL4A2 mutations. Six families presenting with a syndrome referred to as HANAC (hereditary angiopathy, nephropathy aneurysms and cramps) have COL4A1 mutations clustered within 31 amino acids (10). This raises the possibility that COL4A1 mutations that affect specific functional domains may lead to distinct clinical outcomes (11). Furthermore, the specific nature of a mutation within a given protein domain may influence its biosynthetic consequences. Of note, there is little or no tolerance for amino acids other than glycine at every third position of the collagen triple helix; however, there is evidence that all glycine mutations are not functionally equivalent (12,13). In this study, we investigate the contribution of allelic differences to phenotypic variability and to COL4A1 and COL4A2 biosynthesis using a series of Col4a1 and Col4a2 mutant mouse lines on a uniform C57BL/6J genetic background. We show that different Col4a1 and Col4a2 mutations have distinct molecular consequences that lead to ocular, cerebral and myopathic phenotypes of variable severity and penetrance and may reflect mechanistic heterogeneity. Understanding the role of allelic heterogeneity could provide valuable insight into COL4A1- and COL4A2-associated disorders. Moreover, allelic differences may reflect important functional subdomains in COL4A1 and COL4A2 and reveal distinct pathogenic mechanisms. Both of these could provide prognostic information to allow for better patient counseling and guide the development of targeted therapeutic interventions. Col4a1 and Col4a2 mutations cause highly penetrant anterior and posterior ocular dysgenesis To investigate if allelic heterogeneity contributes to phenotypic variability, we analyzed an allelic series of Col4a1 and Col4a2 mutant mice comprising a splice site mutation and seven glycine missense mutations within the triple-helix-forming domains (six in COL4A1, one in COL4A2), and one missense mutation in the globular NC1 domain of COL4A1 (Fig. 1A). We crossed each mutation onto a uniform, C57BL/6J genetic background and evaluated the effects of allelic differences on three organs that are commonly affected in patients with COL4A1 and COL4A2 mutations. COL4A1 and COL4A2 mutations cause ocular defects including anterior segment dysgenesis (ASD) and optic nerve hypoplasia (ONH) in patients and mice. We have shown previously that Col4a1+/Dex41 mice have severe ASD and ONH when maintained on a C57BL/6J background (8). To determine if allelic differences influence ocular manifestations resulting from Col4a1 and Col4a2 mutations, we evaluated ASD and ONH phenotypes in our allelic series using slit lamp examination and histological analysis, respectively. All Col4a1 and Col4a2 mutations caused ASD involving the cornea (cloudiness, scarring, vascularization and iridocorneal adhesions), iris (large tortuous vessels, pigment dispersion), pupil (open, irregularly shaped, eccentric), lens (cataract, persistent pupillary membrane) and anterior chamber (enlargement) that were not observed in Col4a1+/+ mice (Fig. 1B G). Abnormal iris vasculature was the most highly penetrant ocular phenotype observed amongst all Col4a1 and Col4a2 mutants. The expressivity of every mutation was variable and although the majority of mutant eyes displayed at least one abnormal feature, 5 out of 24 Col4a1+/S1582P eyes, 1 out of 22 Col4a1+/G658D eyes and 1 out of 22 Col4a1+/G912V eyes were without any obvious defects on exam. Since mice from each mutant strain are genetically i (...truncated)