Defects in COL1A1 or COL1A2 cause autosomal dominant OI. OI features include bone fragility and deformity, as well as short stature, dentinogenosis imperfecta (DI) and hearing loss. Blue/grey sclerae and wormian bones are frequent, but not uniform, findings. In addition, there is overlap between the features of individuals with different Sillence types.
The Sillence classification included 4 types based on clinical, radiographic, and genetic criteria11, 12
. Although proposed before collagen defects were identified in OI, it remains useful in an updated form which accounts for new gene defects or distinctive histomorphometry ().
The classification shown in this review and elsewhere13
designates the original 4 Sillence types entirely for mutations in COL1A1
. It separates the novel OI types based on the gene in which the mutation occurs and the general function of that gene (collagen prolyl 3-hydroxylation, collagen chaperone, etc). This classification succinctly communicates the genetic defect and general phenotypic severity, while also generating homogenous groupings for therapeutic approaches and basic investigations of disease mechanism. Several versions of an alternative classification have been proposed14, 15
, in which the recessive types are folded into the original Sillence numeration based on clinical phenotype; these classifications vary in whether the histologically defined types V and VI are retained or also classified clinically. The alternative classification results in children with defects in the same gene (ie LEPRE1)
being classified as different types because of clinical variability (ie type II for lethal cases, or III for severe survivors), which is likely to confuse genetic counselling.
OI Type I, the mildest form, has a triad of features: fractures, blue sclera, and hearing loss. Fractures often begin with ambulation and decrease after puberty. These individuals have minimal bone deformity, near normal stature and rarely have DI.
OI Type II is perinatal lethal. Affected infants have short, bowed long bones with crumpling from in utero
fractures, blue/grey sclerae, and a large, soft cranium. Radiographs reveal undertubulated long bones. The most common cause of death is respiratory failure, associated with small thorax, rib fractures, pneumonia, and perhaps with intrinsic collagen-related abnormalities of lung tissue16, 17
OI Type III (progressive deforming) is the most severe, non-lethal form. Affected individuals may sustain hundreds of fractures. Most have triangular facies, frontal bossing, blue/grey sclerae, DI, vertebral compressions and scoliosis. Many have platybasia or basilar invagination. They have extremely short stature; about half have “popcorn” formation (sclerotic lines seen on radiographs representing growth plate fragmentation) at femoral growth plates18
OI Type IV (moderately severe) has a broad phenotypic range overlapping types I and III. Affected individuals incur dozens of long bone fractures but most achieve ambulation. Scleral hue, DI, basilar impression, hearing loss and final stature are variable.
Type I procollagen is a heterotrimer, composed of two pro!1(I) and one pro!2(I) chains, flanked by globular pro-domains at both the amino (N-) and carboxyl (C-) termini (). Glycine is obligatory at every third helical residue of collagen because of spatial constraints inside the triple helix19
. Procollagen is extensively hydroxylated and glycosylated post-translationally (Box 1
Mechanisms contributing to autosomal dominant OI bone dysplasia: from mutant type I collagen gene to bone defect
Box 1 COL1A1
transcripts are translated in the ER into proα1(I) and proα2(I) chains, consisting of a central left handed triple helical domain (1014 aa) flanked by N- and C-terminal globular domains. The terminal C-noncollagenous (C-NC) domain of the proα chains contains interchain disulfide bonds and is important for the assembly and alignment of two α1(I) and one α2(I) chains210
. Proper chain recognition and heterotrimer assembly through the C-NC alignment region is supported by interactions with ER-resident molecular chaperones such as immunoglobulin-heavy-chain binding protein (BiP), Serpinh1 (HSP47), peptidyl prolyl cis-trans
isomerases and the recently identified prolyl 3-hydroxylation complex98, 211, 212
. The assembled trimer then folds the helical domain from C- to N-termini. Proper folding of the triple helical domain requires the presence of a glycine residue at every third amino acid because of steric constrains in the interior aspect of the helix. The helical portions of the collagen chains are subject to a series of post-translational modifications during folding, until the chains become inaccessible in the tight helical configuration. These include prolyl 4-hydroxylation and lysyl hydroxylation of approximately half of proline and one-quarter of lysine residues along the length of the helical region of each chain, catalyzed by prolyl 4-hydroxylase (P4H) and lysyl hydroxylase, respectively, followed by hydroxylysyl glycosylation, catalyzed by glucosyl/galactosyl transferases. Specific proline residues, α1(I)Pro986 and α2(I)Pro707, are fully or partially 3-hydroxylated by the prolyl 3-hydroxylation complex (CRTAP/P3H1/CyPB). After procollagen is secreted into the pericellular space, the terminal propeptides are removed by specific N- and C-proteinases20
. The mature triple helical collagen molecules participate in a higher order structure in the extracellular matrix, the heterotypic fibril. In the fibril, type I collagen is aligned in a quarter-staggered array, yielding D-period banding with overlap and gap regions. Collagen fibrils are stabilized by formation of covalent cross-links between the telopeptides and adjacent domains of collagen molecules, which are catalyzed by lysyl oxidase. Fibrils interact with non-collageneous proteins, bind soluble factors such as growth factors and cytokines, which regulate cell functions, and constitute the scaffold for mineral deposition24
General principles have emerged for genotype-phenotype correlations in dominant OI. The molecular defect in type I OI is a null COL1A1
allele due to frameshifts or PTCs, causing reduced synthesis of structurally normal collagen. Also, splice site defects often lead to alternative splicing with subsequent PTCs. The phenotypes of null COL1A1
alleles and mild helical substitutions may overlap21, 22
. We propose that type I OI should be limited to cases with type I collagen haploinsufficiency, including those individuals whose haploinsufficiency is associated with a moderate clinical outcome. The overwhelming majority of patients with a type I OI phenotype have a null COL1A1
allele. The occasional individual with a collagen structural mutation and a very mild phenotype will be designated type IV OI. In this approach, type I OI is a clinically and biochemically homogenous grouping, as well as the only dominant OI form in which structurally abnormal collagen is not present.
Types II – IV OI are caused by defects in type I collagen structure, most commonly glycine substitutions (80%) and splice site mutations (20%)2
. Glycine substitutions delay helix folding, leading to post-translational overmodification (Box 1
). The OI Mutation Consortium examined 832 mutations in Types II-IV OI, representing substitutions at ~ 44% of glycine residues2
. In both α-chains, substitutions in the N-terminus are non-lethal (). Overall, 36% of COL1A1
glycine substitutions had a lethal outcome, especially those with charged or branched side chains. Substitutions in two α1(I) Major Ligand Binding Regions (MLBR 2 and 3) are exclusively lethal, suggesting collagen-NCP interactions in matrix are essential to bone formation (). Most COL1A2
glycine substitutions are non-lethal (81%). The α2(I) lethal substitutions occur in eight regularly spaced clusters, aligning with proteoglycan binding sites in the collagen fibril ()2, 23, 24
. The different patterns of lethality in α1(I) and α2(I) indicate each chain plays a different role in matrix organization. Also, substitutions at over 40 glycine residues result in both lethal and non-lethal forms of OI2
, supporting the importance of modifying factors25, 26
Distribution of lethal and non-lethal glycine substitutions causing OI along the type I collagen monomer and fibril
Detailed comparison of collagen quantitative and structural mutations with OI phenotype found higher lumbar spine areal BMD, greater cortical width and lower bone turnover parameters in type I OI27
. Furthermore, BMD and histomorphometry of patients with non-lethal OI did not correlate with the α-chain containing the mutation, mutation location in the chain, or the substituting residue, suggesting other factors are crucial for outcome severity.
Rare mutations affecting procollagen processing sites or chain register lead to distinctive variants of OI. The procollagen N- and C-propeptides are cleaved by specific propeptidases in the pericellular space (Box 1
). Glycine substitutions in the first 90 residues of the α1(I) helical region disrupt a stable N-anchor domain and prevent or delay N-propeptide removal. The pN-collagen is incorporated into matrix, decreases fibril size and causes a phenotype with characteristics of both OI and Ehlers-Danlos Syndrome (EDS)28, 29
. Disruption of N-propeptide processing by helical defects in α2(I) also leads to OI/EDS30–34
For C-propeptide processing, substitutions at the cleavage site Asp-Ala residues result in mild OI with increases in vertebral DXA z-scores and bone mineralization that are counterintuitive for OI, due to accelerated mineralization35
. Substitutions in the proα1(I) or proα2(I) C-propeptide have broad phenotypic variability, causing types II-IV OI, though the majority are mild or lethal36–39
. Since α-chains align at the C-terminal end, these mutations delay chain incorporation and helix formation. However, the C-propeptide is not normally incorporated into collagen fibrils, leaving the mechanism of these OI cases unclear.
Small triplet deletion or duplication mutations shift the register of α-chains in the helix. Although the Gly-X-Y sequence is maintained, salt bridges are disrupted by misalignment of X and Y residues between chains. These cases are severe or lethal, and have delayed collagen folding40, 41
. The register shift can propagate to the end of the helix, and impact N-propeptide cleavage. Interestingly, substitutions for Y-position residues may also propagate a register shift nearly the full length of the collagen helix, interfering with N-propeptide processing and causing variable phenotypes including mild OI, hyperextensibility and Caffey Disease, a transient infantile cortical hyperostosis42–44
. Several pedigrees with autosomal dominant Caffey Disease have been shown to have the same COL1A1 R836C (p.R1014C) Y-position change44–46
, associated with self-resolving inflammation and subperiosteal new bone formation with reduced penetrance in infancy. The hyperostosis may be the consequence of the mutation disrupting binding of a ligand, such as IL-2, to collagen and causing increased susceptibility to periosteal injury during infancy44
Understanding the Disease Mechanism: from gene to tissue
Almost all cases of dominant OI have low bone mass and increased skeletal fragility47
. Histomorphometry of OI iliac crests revealed decreased trabecular and cancellous bone volume, increased osteoblast and osteoclast surface, and an overall increase in bone formation rate per bone surface. However, deposition of new bone at the single osteoblast level (MAR) is reduced, and is not compensated by the increased cell number48
. Interestingly, FT-IR and qBEI both revealed elevated bone matrix mineralization. These data support the occurrence of a common defect in OI bone downstream from the collagen quantitative and qualitative mutations, altering bone cell function and the modelling/remodelling mechanisms which normally maintain bone homeostasis27, 48, 49
A variety of murine models for OI are now available for investigation of OI mechanism and pilot treatment studies (). Mov13 mice have a null Col1a1
allele caused by a proviral insertion and model type I OI50, 51
. The oim/oim mouse phenotypically resembles type III OI, although its recessive inheritance is atypical for collagen mutations. A spontaneous single nucleotide deletion in the oim Col1a2
C-propeptide prevents α2(I) incorporation into collagen52
. However, the resulting α1(I) homotrimer does not account for the severe OI phenotype (see Gene and Protein Defects
, below). More recent OI models were generated with knock-in technology or ENU mutagenesis. Knock-in Brtl53
and G610C OI (Amish)26
mice have classical glycine substitutions in α1(I) or α2(I) respectively, leading to phenotypes representative of type IV OI. Aga2 mice were generated by ENU mutagenesis; they have a proα1(I) C-propeptide mutation causing a type III OI phenotype54
. Murine OI models provide direct access to intact long bone and tissues such as lung which are not available from patients; they provide large numbers of samples with the same mutation for studies. These models already play an important role in piloting therapy approaches. In Brtl and oim, cell transplantation has led to positive changes in mechanical properties despite low levels of cellular uptake into bone55–57
. In the same mouse models treated with bisphosphonates, direct access to whole femora revealed both beneficial and potentially detrimental effects58, 59
; RANKL inhibition has also been piloted in oim60, 61
. Of equal importance, murine OI models have provided insight into basic mechanism, including elevated osteoclast function (Brtl and oim)62, 63
, varability of expression (Brtl and Amish)25, 26, 64
, ER Stress54, 65
and apoptosis (Aga2)54
, which provides new targets for therapy.
Factors contributing to the Mechanism of OI
The mechanisms of classical OI encompass the gene mutation, the collagen alteration, and dysfunction at the cellular, matrix (ECM) and tissue levels (). The composition and organization of matrix influences the presence of growth factors and cytokines important for proliferation and differentiation of bone cells66
, as well as matrix mineralization, which confers bone stiffness.
Gene and protein defects
The type I collagen biosynthetic pathway has been extensively reviewed67
and a brief description is provided in Box 1
. The matrix insufficiency of type I OI results from a PTC in the COL1A1
transcript, which activates NMD, reducing mutant transcripts and leading to the synthesis of half the amount of normal collagen. Absence of α1(I) chains is not compatible with life, as demonstrated by embryonic lethality in the homozygous Mov13 mice50
Homozygous null mutations in COL1A2
lead to a range of phenotypes. Those associated with NMD and loss-of-function lead to assembly of α1(I) homotrimer. Clinically, this causes mild EDS with hypermobility in childhood and cardiac valve disease in adulthood, rather than OI68
. In contrast, both one patient with OI69
, and the severe oim/oim mouse () have a deletion in the α2(I) C-propeptide, which does not result in NMD. They produce normal levels of COL1A2
transcripts, which are translated into α2(I) chains that cannot incorporate into collagen. Since α1(I) homotrimer alone does not lead to OI, the intracellular accumulation of mutant α2(I) chains (see Intracellular Stress
, below) may cause the skeletal dysplasia.
Glycine substitutions delay collagen folding and result in overmodified collagen, which may compromise secretion and/or processing2, 70
. Substituting residues disrupt non-covalent bonds, causing local unwinding. Certain substituting residues have greater lethality, as do substitutions in clusters along α2(I), and in α1(I) MLBRs ()2
. Although the overmodification gradient does not correlate with clinical severity in α1(I), empirical rules correctly assign most lethal or non-lethal outcomes71
. In addition, the overmodification of structurally normal collagen in recessive OI (Recessive OI
, below) raises the possibility that excess hydroxylation and glycosylation have a direct detrimental role in matrix.
Misfolded collagen chains in the ER activate the Unfolded Protein Response (UPR), triggering synthesis of chaperones to assist collagen folding or, alternatively, increasing mutant protein degradation72
. Cellular response varies depending on the type of collagen mutation (). Collagen with triple helical mutations is removed by autophagy, as are collagen aggregates in cells lacking HSP4773
. In Aga2 cells, ER-retention of mutant collagen increases expression of chaperones BiP and HSP47, apoptosis-inducing transcription factor Gadd153/CHOP and activation of caspase-3 dependent apoptosis54
. In calvaria of Brtl+/−
perinatal lethal pups, relative intracellular retention of helices with one mutant chain65
is associated with increased expression of Gadd153/CHOP, but normal BiP expression, suggesting collagen misfolding activates the UPR through a BiP-independent response25
. Finally, C-propeptide mutations that impair trimer assembly result in increased BiP expression, retrotranslocation of the misfolded proα chains into the cytosol and degradation via the proteasomal ER-associated degradation (ERAD) pathway74, 75
Compromised ECM Structure and Mineralization
In OI types II-IV, the mixture of normal and mutant α-chains results in matrix heterogeneity and may contribute to the generally greater severity of α1(I) defects, since heterozygous α1(I) defects yield helices with two, one or no mutant chains, while α2(I) defects result in two helix compositions. In Brtl mice, homozygosity for the mutant allele leads to matrix homogeneity and, unexpectedly, to a less severe phenotype, suggesting this feature impacts bone properties76
The association of lethal OI with MLBRs in α1(I) monomers or proteoglycan binding sites on fibrils for α2(I)2, 23, 24
most likely reflects compromised interactions of NCPs with fibrils (). The NCP composition of matrix is altered secondarily in OI, which is also likely to impact bone properties. Cultured OI osteoblasts synthesize reduced amounts of osteonectin and proteoglycans, and increased amounts of fibronectin, thrombospondin and hyaluronan77, 78
. Thrombospondin and decorin bind growth factors, while decorin and fibronectin are important for fibrillogenesis.
The normal D-periodic spacing of fibrils generates gap and overlap regions, which are important for mineral nucleation and collagen cross-links and NCP interactions, respectively24
. In Brtl+/−
bone matrix, the collagen fibril D-period has significantly greater variability in spacing than in wild-type littermates79
. The abnormal structure of heterotypic fibrils could affect the type and amount of mineral deposited by increasing the density of nucleation sites80, 81
; OI matrix also contains abnormal levels of NCPs82
known to regulate crystal deposition and growth83
. Elevated mineral content has been demonstrated by FT-IR and BMDD in OI bone with collagen quantitative and structural defects, and is also found in murine models81, 84
. The elevated mineral content and loss of mineralization heterogeneity contribute to the fragility of OI bone85, 86
, possibly through loss of ductility.
Cell-Cell, Cell-Matrix Interactions
Cellular interactions with abnormal matrix and compromised osteoblast development influence signalling between osteoblasts and osteoclasts, increasing bone remodelling and exacerbating the bone weakness caused by the primary collagen change (). Osteoblasts sense osteocyte apoptosis via gap junctions, and receive negative feedback from osteocytes through sclerostin87,88
. Osteoblasts then trigger osteoclast maturation and recruitment87
. Ultrastructural examination of OI bone revealed increased numbers of osteocytes and multiple osteocytes in some lacunae89
. In the Brtl mouse, osteoclast numbers are elevated in femora, uncoupled from osteoblast numbers. Brtl osteoclast precursors from marrow are larger, more numerous and more intensely TRAP stained than in wild-type62
. The RANKL/OPG ratio is normal in Brtl bone, so other soluble factors triggered by the abnormal matrix may increase osteoclast development. In the oim/oim mouse, elevation of the RANKL/OPG ratio and higher expression of TNF-α were detected in sorted immature osteoblasts, supporting cell-cell signalling as a key aspect of elevated bone turnover in OI63
Cross-links in collagen fibrils are important for preosteoblast maturation90
. In OI, collagen located at the surface of fibrils had fewer cross-links than in the fibril interior.91
Contact with cross-link deficient matrix by OI bone cell populations could contribute to impaired osteoblast maturation and increased osteoclast recruitment. Also, the effects of collagen heterogeneity in dominant OI could be mediated in part by abnormal cross-linking.