Homozygosity and Heterozygosity for Null Col5a2 Alleles Produce Embryonic Lethality and a Novel Classic Ehlers-Danlos Syndrome–Related Phenotype

Abstract

Null alleles for the COL5A1 gene and missense mutations for COL5A1 or the COL5A2 gene underlie cases of classic Ehlers-Danlos syndrome, characterized by fragile, hyperextensible skin and hypermobile joints. However, no classic Ehlers-Danlos syndrome case has yet been associated with COL5A2 null alleles, and phenotypes that might result from such alleles are unknown. We describe mice with null alleles for the Col5a2. Col5a2 À/À homozygosity is embryonic lethal at approximately 12 days post conception. Unlike previously described mice null for Col5a1, which die at 10.5 days post conception and virtually lack collagen fibrils, Col5a2 À/À embryos have readily detectable collagen fibrils, thicker than in wild-type controls. Differences in Col5a2 À/À and Col5a1 À/À fibril formation and embryonic survival suggest that a1(V) 3 homotrimers, a rare collagen V isoform that occurs in the absence of sufficient levels of a2(V) chains, serve functional roles that partially compensate for loss of the most common collagen V isoform. Col5a2 þ/À adults have skin with marked hyperextensibility and reduced tensile strength at high strain but not at low strain. Col5a2 þ/À adults also have aortas with increased compliance and reduced tensile strength. Results thus suggest that COL5A2 þ/À humans, although unlikely to present with frank classic Ehlers-Danlos syndrome, are likely to have fragile connective tissues with increased susceptibility to trauma and certain chronic pathologic conditions. Collagen V is a low-abundance fibrillar collagen widely distributed in vertebrate tissues as a1(V) 2 a2(V) heterotrimers, 1 which are incorporated into growing fibrils with the more abundant collagen I and involved in regulating the geometry and tensile strength of the resulting collagen I/V heterotypic fibrils. 2,3 Mutations in the genes encoding either the a1(V) 4 or a2(V) 5 chain can result in the human heritable connective tissue disorder classic Ehlers-Danlos syndrome (cEDS), clinical hallmarks of which include skin hyperextensibility, atrophic scarring, and joint hypermobility, with patients also often presenting with easy bruising and bleeding. 6 At the molecular level, the collagen fibrils of cEDS skin have variability in diameter not seen in normal skin and include large diameter collagen fibril aggregates with abnormal cauliflower-like shapes when viewed in cross section. 6 Deficits in the tensile strength of cEDS collagen fibrils are inferred from the hyperextensibility and fragility of cEDS skin and the hypermobility of cEDS joints.Most cEDS cases that have been characterized at the molecular level are heterozygous for null alleles of the a1(V) chain gene COL5A1, 7 resulting in the deposition of haploinsufficient levels of normal collagen V in tissues, with excess a2(V) chains unable to form stable triple helical molecules or be incorporated into the extracellular matrix (ECM). 8

Deficits in Col5a2 Expression Result in Novel Skin and Adipose Abnormalities and Predisposition to Aortic Aneurysms and Dissections

“…Unlike heterozygous Col5a1 þ/À adult mice, which exhibit hyperextensible skin at both high and low stress, and on electron microscopic examination display large-diameter dermal aggregates of collagen fibrils with cauliflower-like contours, 14 cardinal features of cEDS, 12 adult heterozygous Col5a2 þ/À mice exhibited hyperextensible skin at high, but not low, stress and dermal collagen fibrils with relatively mild abnormalities in contour and diameter. 16 Together, these data suggested that human COL5A2 þ/À heterozygotes, although likely more susceptible to skin tearing/wounding than normal, might not present with frank cEDS. This conclusion, together with the finding that aortas of Col5a2 þ/À heterozygous mice have reduced tensile strength and increased elasticity, raised the question of the clinical ramifications of the COL5A2 þ/À genotype in human populations.…”

“…12 To determine in vivo effects of reduced or absent a2(V) chains, we recently generated and characterized mice with constitutively null Col5a2 alleles. 16 Homozygosity for the null Col5a2 allele yielded embryonic lethality at approximately 12 days after conception, 16 approximately 2 days later than the embryonic lethality previously described for homozygous null Col5a1 À/À mice, 11 although embryos of both genotypes had similar cardiovascular signs, suggesting deficits in early cardiovascular integrity. However, although Col5a1 À/À embryos lacked identifiable mesenchymal collagen fibrils, 11 Col5a2 À/À embryos had identifiable mesenchymal collagen fibrils, albeit with abnormally large diameters and abnormal configurations.…”

“…This conclusion, together with the finding that aortas of Col5a2 þ/À heterozygous mice have reduced tensile strength and increased elasticity, raised the question of the clinical ramifications of the COL5A2 þ/À genotype in human populations. 16 In the current report, we further elucidated the roles of a2(V) collagen chain in adult tissues and pathology, in part by using a conditional-null mouse model with floxed Col5a2 alleles that avoids embryonic lethality and allows study of the effects of postnatal Col5a2 ablation. Data presented herein show the a2(V) chain to play important roles in growth and that sufficiently low a2(V) chain levels yield skin that, like that of cEDS patients, 19 has delayed and aberrant wound healing, has greatly reduced breaking strength/tensile strength, and contains the large cauliflower-like collagen fibril aggregates pathognomic of cEDS.…”

“…However, although Col5a1 À/À embryos lacked identifiable mesenchymal collagen fibrils, 11 Col5a2 À/À embryos had identifiable mesenchymal collagen fibrils, albeit with abnormally large diameters and abnormal configurations. 16 Differences in collagen fibrils and survival time between the two types of homozygous null embryos suggested that aberrant a1(V) 3 homotrimers, which are formed by a1(V) chains in the absence of a2(V) chains, 17,18 can functionally compensate, in part, for loss of a1(V) 2 a2(V) heterotrimers.…”

The triple helix of collagens – an ancient protein structure that enabled animal multicellularity and tissue evolution

“…Embryonic lethality at E10-E12 (Wenstrup et al, 2004;Park et al, 2015) VI (G) Bethlem myopathy 1 (Scacheri et al, 2002;Pepe et al, 1999;Pan et al, 1998;Lucioli et al, 2005;Lampe et al, 2005;Jöbsis et al, 1996;Baker et al, 2007) (G) Ullrich congenital muscular dystrophy 1 (Lampe et al, 2005;Kawahara et al, 2007;Giusti et al, 2005;Baker et al, 2005) Dystonia 27 (Zech et al, 2015) Abnormalities in morphology of l muscle, impaired skeletal muscle contractility, myopathy (Bonaldo et al, 1998) VII (G) Epidermolysis bullosa dystrophica, multiple types (Shinkuma, 2015;Fine et al, 1989;Christiano et al, 1993Christiano et al, , 1994Christiano et al, , 1995Christiano et al, , 1996Woodley et al, 2004) Postnatal lethality within the first week from complications of blistering (Heinonen et al, 1999) VIII (N) Corneal dystrophies (Biswas et al, 2001) Dysgenesis of the anterior segment of the eye (Hopfer et al, 2005) IX (G) Multiple epiphyseal dysplasia 2, 3 and 6 (Paassilta et al, 1999;Jeong et al, 2014;Holden et al, 1999;Czarny-Ratajczak et al, 2001) Stickler Syndrome 4 and 5 (Van Camp et al, 2006;Baker et al, 2011) (N) Intervertebral disc disease (Paassilta et al, 2001;Annunen et al, 1999b) one, two or all three distinct chains of the triple helix in different combinations to directly bind a molecule ( Fig. 2B; modes 1-3).…”