| Type XV collagen: Complete structures of the human COL15A1 and mouse Col15a1 genes, location of type XV collagen protein in mature and developing mouse tissues, and generation of mice expressing truncated type XV collagen | ||
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The modern medicine benefits from the animal models of various human diseases in a number of ways. First of all, they are valuable in the study of the consequences of gene mutations and the biochemical and patho-physiological mechanisms underlying the disease phenotype. Secondly, they are valuable in the testing of potential therapies for corresponding human diseases. The animal models can be classified into two categories based on the “origin” of the mutation, namely to naturally occurring ones and man-made ones. Previously, mutations were caused by exposing animals to x-rays or mutagenic agents, with uncontrollable results. Controlled mutagenesis was achieved as the techniques enabling the genetic manipulation of mice were developed, and is especially powerful in defining the consequences of mutations and functions of new proteins. In the 1980’s, the first transgenic mice were generated by random insertion of a gene into the genome. Only few years later, the techniques were further developed to enable targeting of the gene construct into a specific location of the genome, i.e. the generation of so called knock-out mice or targeted mice (for reviews, see Palmiter & Brinster, 1986; Jaenisch, 1988). Both of these techniques have been successfully used in collagen research. The random insertion of mutated collagen genes into the mouse genome has proven especially useful as the consequences of the expression of mutant collagen chains mimic the situation in patients with collagen mutations due to the dominant-negative effect (see above). Indeed, in a number of mouse lines, the phenotypes of various human diseases have been reproduced (see 2.6.2.). The power of these techniques is illustrated in the number of mouse lines with genetically engineered mutations, which exhibit phenotypes that have not yet been identified in humans, or in those cases where the mouse model preceded the identification of the human disease and eventually helped in identifying the gene mutations responsible. The next two sections and two tables will briefly summarize the currently available naturally occurring and genetically engineered animal models of the collagen genes.
There are several animal models whose phenotypic and biochemical manifestations resemble certain human diseases and which have subsequently been identified as resulting from mutations in the collagen genes (see Table 4). These include three mouse models for diseases affecting cartilage and bone. The oim mice, with a mutation in the Col1a2 gene, have a phenotype similar to the moderate and severe forms of human OI (Chipman et al., 1993). The Dmm mice, with a mutation in the Col2a1 gene, have disproportionate micromelia and other symptoms similar to the human variant of chondrodysplasia, Stickler syndrome (Pace et al., 1997), while the cho mice with a mutation in the Col11a1 gene, develop clinical manifestations similar to the lethal form of human chondrodysplasia (Li et al., 1995a). Furthermore, three canine models exist for hereditary nephritis, differing in their mode of inheritance. Samoyed dogs having a mutation in the gene encoding the α5(IV) chain serve as an animal model for human X-linked Alport syndrome (AS) (Zheng et al., 1994). The hereditary nephritis in bull terrier and English cocker spaniel display autosomal dominant and autosomal recessive modes of inheritance, respectively, and serve as animal models for these forms of human AS, although the underlying genetic defect has not yet been confirmed yet (Hood et al., 1995; Lees et al., 1998). Recently, Nielsen et al. (2000) reported a mutation in the gene encoding the α1(X) chain in domestic pigs that causes dwarfism and growth plate abnormalities similar to those seen in human Schmid metaphyseal chondrodysplasia (SMCD).
Table 4. Animal models with spontaneous mutations for collagen diseases*.
| Locus | Species | Mutation | Mutant phenotype |
|---|---|---|---|
| oim | Mouse | Single base deletion in Col1a2 | Absence of α2(I) collagen in skin and bone, progressive skeletal deformities, fractures, osteopenia |
| dmm | Mouse | 3 nucleotide deletion in Col2a1 | Homozygotes die at birth and display reduced collagen II content, shortened long bones and spine, growth plate abnormalities; heterozygotes are viable, but dwarfed. |
| cho | Mouse | Single base deletion in Col11a1 | Absence of α(XI) collagen in cartilage, abnormally thick collagen fibrils, and disorganized growth plate leading to disproportionate dwarfism, short snout, and cleft palate in homozygotes. |
| Col4a5 | Dog | Gly-to stop codon substitution in Col4a5 | X-linked inheritance. Proteinuria and hematuria leading to progressive renal failure and death at 8 to 15 months of age in affected males. |
| ND | Dog | ND | Autosomal dominant inheritance. Proteinuria and hematuria leading to progressive renal failure and death at variable ages. Anterior lenticonus in some affected dogs. Normal hearing. |
| ND | Dog | ND | Autosomal recessive inheritance. Proteinuria and juvenile-onset chronic renal failure and death at 8 to 27 months of age. |
| Col10a1 | Pig | Gly→Arg substitution | Metaphyseal chondrodysplasia of long bones leading to dwarfism and skeletal defects. |
| *For references, see text. | |||
To date, 12 transgenic mouse lines generated by random insertion of normal or mutated collagen genes and 12 lines generated by a targeted mutation in these genes have been reported in literature (reviewed by Aszodi et al., 1998; Myllyharju & Kivirikko, 2001). The phenotypic consequences of genetically engineered collagen mutations in mice and their equivalent human diseases are summarized in Table 5. Some engineered mutations were modeled after known human disease-causing mutations, such as the in-frame deletions of Col1a1 (Khillan et al., 1991) and Col2a1 (Vandenberg et al., 1991), which mimicked sporadic deletions of lethal OI variant, and the Gly-to-Ser substitution in Col2a1 (Maddox et al., 1997), which corresponded to lethal human chondrodysplasia. Both of these resulted in the reproduction of the same disease phenotypes in mouse. The dominant nature of many collagen mutations in human diseases (see above) is also evident in engineered mouse mutations, as the dominant-negative mutations lead to more severe phenotypes than null mutations. For example, the transgenic mice expressing mutant type X collagen develop a more severe skeletal phenotype (Jacenko et al., 1993) than mice lacking type X collagen (Kwan et al, 1997; Gress & Jacenko, 2000), which were first reported as unsymptomatic (Rosati et al., 1994). Similarly, in two mouse models for degenerative joint disease, the phenotype was more obvious with the dominant-negative mutation of collagen IX than with the null allele (Nakata et al., 1993; Fässler et al., 1994), although as later demonstrated, both approaches led to the functional knock-out of the entire type IX collagen molecule (Hagg et al., 1997, and see below).
The phenotypic severity correlates with the level of the transgene expression in some lines, indicating a certain threshold requirement. For example, mice expressing high levels of truncated collagen I or II develop a lethal phenotype of OI or chondrodysplasia, whereas lower expression levels of these transgenes are associated with osteoporotic or osteoarthritic phenotypes (Khillan et al., 1991; Vandenberg et al., 1991; Helminen et al., 1993; Pereira et al., 1993 and 1994). Moreover, in some lines, extensive breeding resulted in phenotypic variability and incomplete penetrance, which may be an inherent property of the expression of mutated collagen genes (Helminen et al., 1993; Pereira et al., 1993 and 1994). Whereas in most knock-out lines the heterozygotes were asymptomatic, in Col2a1 and Col6a1 lines the heterozygotes displayed the same although milder phenotype than homozygotes. This indicated haploinsufficiency in the gene function (Bonaldo et al., 1998; Li et al., 1995b), a phenomenon also seen in the corresponding human diseases (Richards et al., 2000; Lamandé et al., 1998).
Besides being important models for human diseases, genetically engineered mice have often provided further inside into protein function. Hagg et al. (1997) showed that the inactivation of one chain of heterotrimeric collagen, α1(IX), leads to the functional knock-out of the entire collagen molecule despite normal synthesis of α2(IX) and α3(IX) chains, thus providing the first in vivo evidence of the essential role of α1(IX) in molecular assembly. The consequences of the depletion of Col2a1 demonstrated that a well-organized cartilage matrix is required for the formation of the epiphyseal growth plate of long bones, but it is not essential for the initial mineralization of long bones, the synthesis of periosteal bone, or formation of bone cavities (Li et al., 1995b). The lack of Col3a1 disturbed collagen I fibrillogenesis, resulting in defective development and functional failure in the cardiovascular system and other organs, indicating an essential role for collagen III in fibrillogenesis (Liu et al., 1997). The targeted mutation of Col5a1 provided the first genetic evidence of a regulatory role for collagen V during matrix assembly (Andrikopoulos et al., 1995). Furthermore, the consequences of the Col13a1 mutation and the lack of Col15a1 in mice both provided the first functional evidence for these recently identified widely expressed collagens (Eklund et al., 2001; Sund et al., 2001a). Analogously to that seen in the mouse models of Col5a1 (Andrikopoulos et al., 1995), Col9a1 (Nakata et al., 1993), and Col10a1 (Jacenko et al., 1993), where the phenotypic analysis of the mice preceded the identification of the human mutations, it has been suggested that the consequences of the mutations in Col13a1 and Col15a1 will most likely correlate with as yet unidentified disease phenotypes in humans (Eklund et al., 2001; Sund et al., 2001a).
Table 5. Genetically engineered mutations in collagen genes in mouse.
| Generated mutation | Mutant phenotype | Human disease | References |
|---|---|---|---|
| Transgenic mice created via random insertion of gene construct | |||
| Mov-13, retroviral insertion into the first intron of Col1a1 (consequence, null allele) | Fetal lethality of homozygotes at 12-14 days post coitum (dpc) due to bleedingHeterozygotes viable; reduced collagen I content, decreased mechanical strength of long bones and hearing loss; no fractures | OI mild form | Schnieke et al., 1983;Löhler et al., 1984;Bonadio et al., 1990 |
| Gly859→Cys substitution in Col1a1 | Perinatal lethality of founders; bones poorly mineralized, ribs short and wavy, long bones short and broad | OI severe form | Stacey et al., 1988 |
| Central in-frame deletion of 41 exons of Col1a1 | Perinatal lethal phenotype; fractures of ribs and short bones in mice expressing high levels of the transgene, fractures and decreased collagen and mineral content in mice expressing moderate levels of the transgene | OI severe formOsteoporosis | Khillan et al., 1991; Pereira et al., 1993; Pereira et al., 1994 |
| Gly85→Cys substitution in Col2a1 | Perinatal lethality of heterozygotes; short limbs and trunk, craniofacial abnormalities, cleft palate, disorganization of growth plate | Spondyloepiphyseal dysplasia (SED) | Garofalo et al., 1991 |
| Central in-frame deletion of 12 exons of COL2A1 | Lethality of homozygotes; portion of heterozygotes are perinatally lethal featuring dwarfism, cranial bulge, cleft palate, and delayed mineralization, the rest develop degenerative changes in articular cartilage with age | Chondrodysplasia,Osteoarthritis | Vandenberg et al., 1991Helminen et al., 1993 |
| 15 amino acid deletion in Col2a1 | Perinatal lethality and skeletal deformities characterized by short-limbed dwarfism, cartilage hypoplasia, cleft palate, disorganization of growth plate in heterozygotes expressing high levels of transgene; milder phenotype associated with low transgene expression levels | SED | Metsäranta et al., 1992 |
| Overexpression of Col2a1 | Perinatal lethality in mice expressing high levels of transgene; abnormally thick collagen fibrils | Chondrodysplasia | Garofalo et al., 1993 |
| Gly574→Ser substitution in Col2a1 | Perinatal lethality in 25-50 % of offspring; short-limbed phenotype obvious from 16.5 dpc onwards; delayed ossification, abnormal growth plate architecture, reduced density of collagen fibrils in the matrix | Hypochondrogenesis | Maddox et al., 1997 |
| Large central in-frame deletion of Col9a1 | Mild proportionate dwarfism and eye abnormalities in homozygotes, degenerative changes in articular cartilage also in heterozygotes | Chondrodysplasia, osteoarthritis | Nakata et al., 1993 |
| Central in-frame deletion of 21- or 293-amino acids of chicken Col10a1 | Skeletal deformities including mild dwarfism, neck lordosis, and thoracolumbar kyphosis, compression of hypertrophic zone in growth plate, leucocyte deficiency, lymphopeniaCraniofacial deformities | Spondylometaphyseal dysplasia, metaphyseal chondrodysplasia | Jacenko et al., 1993 Chung et al., 1997 |
| In-frame deletion of Col12a1 | Loss of ordered architecture of periodontal ligament and skin matrix | - | Reichenberger et al., 2000 |
| 90 amino acids in-frame deletion of Col13a1 | Fetal lethality of homozygotes at 10.5 dpc or 13.5 dpc due to the lack of placental formation, or cardiovascular defects; decreased microvessel formation in CNS and trigeminal ganglion | - | Sund et al., 2001 |
| Mice with targeted mutations | |||
| Null mutation of Col2a1 | Perinatal lethality of homozygotes; cleft palate, abnormal cartilage, disorganized growth plate, no endochondrial bone or epiphyseal growth plate in long bones, lack of bone marrow Mild phenotype with minimal cartilagous changes in heterozygotes | Achondrogenesis Stickler syndrome | Li et al., 1995 |
| Null mutation of Col3a1 | Shortened life span of homozygotes (6 months), only 10 % survive to adulthood; death due to rupture of large vessels Reduced collagen I fibril content and variable diameter; heterozygotes appear normal | EDS | Liu et al., 1997 |
| Null mutation of Col4a3 | Postnatal lethality of null mutants at ~ 14 weeks of age due to renal failure, progressive glomerulonephritis with proteinuria, and microhematuria | Autosomal AS | Cosgrove et al., 1996 |
| Deletion of exon 6 in Col5a1 | Death of homozygotes within 48 h or between 2-21 days post partum (after birth) due to respiratory distress caused by severe spinal deformities; skin and eye abnormalities associated with disorganized dermal and corneal collagen fibers; heterozygotes normal. | EDS | Andrikopouloset al., 1995 |
| Null mutation of Col6a1 | Histological features of myopathy, such as fiber necrosis, phacocytosis, variation in the diameter, and increased regeneration; homozygotes more severely affected than heterozygotes | Bethlem myopathy | Bonaldo et al., 1998 |
| Null mutation of Col7a1 | Death of homozygotes within 2 weeks post partum due to complications of severe skin blistering; absence of anchoring fibrils; heterozygotes appear normal | Dystrophic EB | Heinonen et al., 1999 |
| Null mutation of Col9a1 | Degenerative changes in articular cartilage in homozygotes; age of onset 4 months; heterozygotes appear normal | Multiple epiphyseal dysplasia | Fässler et al., 1994 |
| Null mutation of Col10a1 | No gross alterations in skeletal development, mild skeletal phenotype characterized by abnormal distribution of cartilage matrix components, altered bone content, and coxa vara; growth plate compressions and altered hematopoiesis, perinatal lethality of 11 % of homozygotes within 3 weeks | Schmid metaphyseal chondrodysplasia(SMCD) | Gress & Jacenko, 2000;Kwan et al., 1997;Rosati et al., 1994 |
| Targeted disruption of Col11a2 | Hearing loss, loss of organization of the collagen fibrils in tectorial membrane | Non-syndromic hearing loss | McGuirt et al., 1999 |
| Targeted deletion of N-terminal 96 amino acids of Col13a1 | Progressive muscular atrophy in homozygotes, histological changes of muscle fibers such as sarcolemmal invaginations, uneven or partially detached BM, disorganized myofilaments, accumulation and enlargement of mitochondria | - | Kvist et al., 2001 |
| Null mutation of Col15a1 | Mild skeletal myopathy with increased sensitivity to exercise induced muscle damage, cardiovascular defects, such as collapsed capillaries and diminished inotrophic response | - | Eklund et al., 2001 |
| Null mutation of Col18a1 | Ocular abnormalities such as delayed regression of hyaloid vessels, abnormal retinal vasculature, and separation of the vitreal matrix from the inner limiting membrane | Knobloch syndrome | Fukai et al.submitted |
Recently, Rani et al. (1999) demonstrated a rescue of the lethal phenotype of Col2a1 null mice (Li et al., 1995b) by introducing a human COL2A1 gene into the mouse genome, indicating that the human collagen II can substitute for mouse protein. Furthermore, the cartilage of these mice is essentially “humanized”, thus these animals present a unique model to investigate the pathological effects of COL2A1 mutations. Although many successful examples encourage the use of genetic engineering in the elucidation of collagen function, it has certain limitations. One of them is the functional redundancy of the targeted gene or compensation by other gene products, which may explain the lack of severe phenotypes e.g. in collagen X and collagen XVIII knock-outs (Rosati et al., 1994; Fukai, N., Eklund, L., Marneros, A.G., Oh, S.P., Keene, D.R., Tamarkin, L., Li, E., Pihlajaniemi, T., & Olsen B.R., submitted). Accordingly, there may be many unpublished mouse lines with no apparent phenotype.