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Col!. Res. Vo!. 1/1981 p. 227-234. Osteogenesis Imperfecta: Promising Beginnings and Continuing Challenges DA VID W. HOLLISTER Division of Medical Genetics Departments of Pediatrics and Medicine Harbor-UCLA Medical Center Torrance, California 90509 Key Words: Osteogenesis Imperfecta, collagen, collagen diseases, collagen structure, collagen metabolism, bone diseases Osteogenesis Imperfecta (01) is a complex, heterogeneous group of inherited diseases characterized by pathologie fragility of bone leading to fractures. The clinical spectrum of 01 is extraordinary, ranging from death in utero with literally hun"dreds of fractures, through dwarfism associated with grotesque malformations of the limbs, to mild deformity without significant growth failure. Some indi- viduals with OIhave an essentially normal phenotype with only a mild increased incidence of bone fractures. In addition to bone fragility and fractures, the skeletal manifestations include defective oe deficient ossification, multiple Wormian bones, osteoporosis, ben ding and curvature of long bones not related to fractures, and epiphyseal and metaphyseal radiolucencies and streaking. It has long been recognized that 01 is a generalized heritable disorder of connective tissue with protean extra-skeletal manifestation. These include blue sclera, opalescent teeth (dentinogenesis imperfecta), thin skin, easy bruisability, herniae, deafness, joint laxity, cardiac valvular incompetence, and various other consequences of fragility of the soft tissues (McKusick, 1972). Amid such clinical diversity, it is hardly surprising that a large number of pathologie and biochemical abnormalities have been recorded in 01. This group of diseases has been regarded as due to disorders of collagen (McKusick, 1972), and in recent years, substantial evidence has been adduced in support of this postulate, although other evidence implicating noncollagen bone pro teins (Dickin- son et al., 1975) and glycosaminoglycans (Cetta et a!., 1977; DiFerrante et al., 1978) has been reported. At present, the available information suggests that most, if not all, varieties of 01 are due to primary defects of collagen. These emerging insights have arisen from our understanding of the biochemistry and metabolism of the collagens and from the application of new technology to the study of 01. Recent progress in the classification of these diseases now offers the possibility of phenotype-biochemical correlations and easier comparisons between the various reported patients and their abnormalities. We now seem poised on the threshold of substantial new insights into these disorders. Not only will it be possible to describe in sophisticated detail alterations in structure, biosynthesis and assembly

Osteogenesis Imperfecta: Promising Beginnings and Continuing Challenges

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Col!. Res. Vo!. 1/1981 p. 227-234.

Osteogenesis Imperfecta: Promising Beginnings and Continuing Challenges

DA VID W. HOLLISTER

Division of Medical Genetics Departments of Pediatrics and Medicine Harbor-UCLA Medical Center Torrance, California 90509

Key Words: Osteogenesis Imperfecta, collagen, collagen diseases, collagen structure, collagen metabolism, bone diseases

Osteogenesis Imperfecta (01) is a complex, heterogeneous group of inherited diseases characterized by pathologie fragility of bone leading to fractures. The clinical spectrum of 01 is extraordinary, ranging from death in utero with literally hun"dreds of fractures, through dwarfism associated with grotesque malformations of the limbs, to mild deformity without significant growth failure. Some indi­viduals with OIhave an essentially normal phenotype with only a mild increased incidence of bone fractures. In addition to bone fragility and fractures, the skeletal manifestations include defective oe deficient ossification, multiple Wormian bones, osteoporosis, ben ding and curvature of long bones not related to fractures, and epiphyseal and metaphyseal radiolucencies and streaking. It has long been recognized that 01 is a generalized heritable disorder of connective tissue with protean extra-skeletal manifestation. These include blue sclera, opalescent teeth (dentinogenesis imperfecta), thin skin, easy bruisability, herniae, deafness, joint laxity, cardiac valvular incompetence, and various other consequences of fragility of the soft tissues (McKusick, 1972).

Amid such clinical diversity, it is hardly surprising that a large number of pathologie and biochemical abnormalities have been recorded in 01. This group of diseases has been regarded as due to disorders of collagen (McKusick, 1972), and in recent years, substantial evidence has been adduced in support of this postulate, although other evidence implicating noncollagen bone pro teins (Dickin­son et al., 1975) and glycosaminoglycans (Cetta et a!., 1977; DiFerrante et al., 1978) has been reported. At present, the available information suggests that most, if not all, varieties of 01 are due to primary defects of collagen. These emerging insights have arisen from our understanding of the biochemistry and metabolism of the collagens and from the application of new technology to the study of 01. Recent progress in the classification of these diseases now offers the possibility of phenotype-biochemical correlations and easier comparisons between the various reported patients and their abnormalities. We now seem poised on the threshold of substantial new insights into these disorders. Not only will it be possible to describe in sophisticated detail alterations in structure, biosynthesis and assembly

228 David W. Hollister

of skeletal collagens, but the advent of recombinant DNA technology as applied to the collagens ofters the bright promise of eventually understanding the altera­tions in structure and regulation of collagen genes (Prockop, 1980). These advances will ofter the potential for useful therapeutic intervention, genetic counseling and prenatal diagnosis for patients and relatives. Of more general biologie import, observations of the consequences of specific aberrations of skeletal collagens will help define the specific role of the collagens in skeletal morphogenesis, growth, calcification and ossification, and remodeling.

Nosology

Numerous attempts have been made to provide a satisfactory classification scheme for 01 (Seedorf, 1949; Cocchi, 1964; Ibsen, 1967; see Sillence et al., 1979a for review). The recent classification of Sillence (Sillence et al., 1979b) is based upon clinical and radiographie features, observed patterns of genetic transmission, natural history and has been adopted by a number of authors. Since this classi­fication ofters a shorthand for the clinical description of many patients, it will be briefly described and used in this article.

Type I 01 is most prevalent and exhibits autosomal dominant inheritance. Fractures may be present at birth, but usually commence during childhood. Severe deformity is unusual, although mild short stature is common. The ocular sclera are blue throughout life, and presenile hearing loss is common.

Type II 01 is lethai in utero or in neontallife and is characterized by extremely deficient ossification, large numbers of fractures leading to a continuously "beaded" appearance of the ribs and crumpled long bones. Severe tissue fragility may lead to avulsion of body parts during delivery. The inheritance is autosomal recessive.

Type III 01 is a recessive disease characterized by extreme bony fragility, multiple fractures, severe growth failure and progressive skeletal deformity which may reach grotesque proportions. The bluish sclera become progressively less blue or normal with age.

Type IV 01 shares many features of the Type III disease, but is inherited as an autosomal dominant with considerable variability in bone fragility, dwarfing and deformity.

Subclassification of these groups according to the absence or presence of dentinogenesis imperfecta has been proposed (Sillence, 1980).

The present classification does not exhaust the diversity exhibited by 01. For example, 01 inherited in an X-linked pattern has been observed (Feingold, 1979), and 01 syndromes with features of the Ehlers-Danlos syndrome or Marfan syndrome have been recorded (Meigel et al., 1974; Biering and Iverson, 1955). Yet other 01 syndromes with distinctive features have been reported (Saint-Martin et al., 1979; Buyse and Bull, 1978). Further, previously delineated inherited con­ditions share some features of 01 and may be inadvertantly misclassified. A possible re cent example is a patient with "an arthropathic form of 01" (Penttinen et al., 1980) who appears to have Winchester syndrome (Hollister et al., 1974). Yet another variety of 01, "camptomelic (bent limb) 01", has recently been studied (see below) (Burgeson et al., 1980).

Osteogenesis Imperfeeta 229

The evidence for collagenous abnormalities in 01 derives from both com­positional studies of various 01 connective tissues and biosynthetic studies which have examined quantitative and qualitative aspects of newly made collagens.

Compositional Studies

Skin This organ is affected in 01, usually being described as thin and fragile. The collagen conte nt per unit surface area or per dry weight is decreased, and the apparent content of different collagen types is alte red in certain types oi OI. Stevenson and colleagues (Stevenson et al., 1970) found only 40-90 % of control hydroxyproline per mm2 of skin in 4 unclassified cases of 01. Francis, Smith and Bauze (Francis et al., 1974) found significantly decreased skin collagen conte nt in 10 cases of 01 Type I, but normal values in 6 cases of probable 01 Type III/IV. The "polymeric" collagen in the latter cases was excessively soluble in basic solution suggesting a defect in cross-linking. One case of probable 01 Type J contained 75 Ofo of control skin collagen whereas a case of 01 Type III/IV had normal dermal collagen content (Cetta, 1977). Sykes and colleagues (Sykes et al., 1977) found an increased a1(III) /a1(1) ratio in 8 of 9 patients with Type I 01 and 3 of 5 patients with 01 III/IV and suggested a possible reduction of Type I collagen accumulation in skin. Skin from a case of Type II 01 contained an apparent excess of a1(III) as compared to a1(1) or a2(1) (Trelstad et al., 1977).

Bone Compositional studies of Type I collagen from 01 bone have revealed variable degrees of overhydroxylation, and one unusual patient exhibited marked underhydroxylation of lysine residues. Type III collagen has been found in 01 bone but not normal bone, and excessive amounts of Type V collagen have been observed in 01 Type II bone.

Pepsin-extracted bone collagen from two cases of dominantly inherited 01 with blue sclera (probable 01 Type I) was comparable in amino acid composition to age-matched controls, but exhibited slight increases in hydroxyproline and hydroxylysine with compensatory decreases in proline and lysine. Electrophoretic analysis was unremarkable and revealed no Type III collagen, and CNBr peptide profiles were comparable to control (Fujii and Tanzer, 1977).

Müller and colleagues found synthesis of Type III collagen in organ culture oE bone from a case of 01 with blue sclera and demonstrated accumulations of Type III by identification of Type III SLS crystallites in pepsin-extracted bone collagen. Immunofluorescent staining of Type III collagen in nonfibrous "holes" in compact bone was also observed. Similar studies of normal bone failed to reveal the presence of Type III collagen within normal bone (Müller et al., 1977).

Bone collagen (femoral and calvarium) from a case of 01 Type II (lethai perinatal) contained a twofold elevation of hydroxylysine residues of both a1(I) and a2(1) chains with a corresponding decrease in lysine. Carbohydrate analysis revealed a two fold increase of galactose and glucose. No Type III collagen was identified by carboxymethylcellulose chromatography (Trelstad et al., 1977). Recent studies, however, have identified Type III collagen by the technique of interrupted electrophoresis with pepsin-solubilized bone collagens from two cases of Type II 01, but not from normal bone collagens. In addition to Type III collagen, excessive amounts of Type V collagen were found as compared with the small amount found in normal bone (Pope et al., 1980a). Burgeson has

230 David W. Hollister

examined pepsin-extracted collagens from femora of 6 cases of 01 Type II and observed similar elevated contents of Type V collagen (Burgeson and Hollister, 1980). Pathologically, such bone exhibits severely deficient osteoid deposition, accounting for the diminished amounts of Type I collagen recovered. The affected bone is also markedly hypercellular (Sillence, 1980). It will be of interest to determine if the excessive accumulation of Type V is simply a reflection of this hypercellularity or, alternatively, if Type V is a constituent of the defective osteoid matrix. An unusual variety of 01 associated with intrauterine fracturcs, severe bending of long bones (camptomelia), and poor ossification of the skull with multiple Wormian bones, has recently been studied (Burgeson et al., 1980). Bone collagen contained only 30 % of normal levels of hydroxylysine. The relationship of this condition to Ehlers-Danlos Type VI is presently unc1ear, but recent evidence from Krane's group (Krane et al., 1980) suggests the possibility of differential expression of lysyl hydroxyl ase activity in different tissues.

Cross-link Studies

Skin Fibroblast cell layer collagens from 3 patients with an autosomal domi­nant variety of 01 contained 3-14 tim es greater amounts of the reduced cross­link, dihydroxylysinonorleucine, but comparable levels of hydroxylysinonorleucine as compared with age-matched control cultures. Only slight increases in content of hydroxylysine or glycosylated hydroxylysine were observed (Lindberg et al., 1979).

Bane Borohydride reduction of pepsin-extracted bone collagen from 2 patients with probable 01 Type I revealed increased levels of the major bone cross-link, dihydroxylysinonarleucine, and increased levels of the reduced aldehyde, di­hydroxynorleucine (1.3-1.6 times age-matched controls) (Fujii and Tanzer, 1977).

Biosynthetic Studies

Fibroblast cultures have been exploited as a model system for 01 studies because of the availability of dermal tissues and the numerous qualitative and quantitative studies which can be performed on radiolabeled newly synthesized procollagens and collagens. An additional advantage is the extensive normative information about collagen metabolism in this cell type. Since skin is abnormal in 01, such studies might be expected to reveal pathogenetic defects. The assump­tion, however, that fibroblasts adequately reflect collagen metabolism in other connective tissue cells such as osteoblasts requires experimental verification.

Fibroblast studies in 01 have revealed alterations in collagen type synthesis and produced evidence for partial or complete absence of the a2(I) chain in some patients. Reduced collagen production and delayed secretion of Type I procollagen have been observed, and evidence has been developed implying structural abnormality of Type I procollagen.

An unusual patient with features of both Marfan syndrome and 01, whose parents were consanguineous, has been recorded (Meigel et al., 1974). Carboxy­methylcellulose chromatograms of pepsin-digested radioactive media collagen from fibroblast cultures revealed a double a1(I) peak and absence of a2(I) chains;

Osteogenesis Imperfeeta 231

similar analysis oE cell layer proteins revealed a similar absence of a2(I). Curiously, later passages of these fibroblasts produced sm all amounts of a2(I) chains although never as much as control cultures. Restudy of the same patient, however, revealed normal synthesis of a2(I) and the presence of a single al(I) peak, and increased amounts of Type III collagen (Müller et al., 1975). Recently, an absence of a2(I) chains in pepsin-digested secreted collagens (and possible broadening of the al (I) band in electrophoretograms) was observed in a case of "moderately severe" 01 (Nicholls et al., 1979). However, in vitra translation of tibroblast mRNA indicates synthesis of the a2(I) chain, a rather surprising result (Rowe, 1980). Apparently this case did not exhibit a Marfanoid phenotype or radiographic changes similar to those described in Meigel's case. The parents were consangineous, and their fibroblasts produced ab out onehalf as much a2(I) chains as normal (Pope and Nicholls, 1980b). Other a2(I) abnormalities have been seen. Byers and colleagues (Byers et al., 1980) have observed synthesis of an apparently abnormal a2(I) chain by fibroblasts from a case of 01 Type 1. These various reported abnormalities of the a2(I) chain are reminiscent of the recent finding of an abnormal a2 chain in a case of Marfan syndrome (Scheck et al., 1979).

Skin fibroblasts from a case of Type II 01 were found to synthesize an increased amount of Type III collagen, apparently due to decreased synthesis of Type I collagen (penttinen et al., 1975). Evidence that this cell strain produces less collagen than normal (with a normal degradation rate) has recently been recorded (Steinmann et al., 1979). Delayed secretion, dilation of rough endoplasmic reti­culum, and accumulation of Type I pro collagen within cultured cells as judged by immunofluorescent studies has been found in skin fibroblasts from a patient with OI Type II (Byers et al., 1980).

Extensive studies of collagen and pro collagens synthesized by fibroblasts from a case of probable 01 Type IU have revealed normal circular dichroism spectra, normal melting curves, and normal resistance to pepsin proteolysis of Type land III procollagens. The Type I procollagen, but not the pepsin-digested product, ex­hibited an unusual tendency to aggregate as judged by centrifugation through suc­rose gradients (Peltonen et al., 1980a). Type I procollagen, but not Type III, in­corporated 2-3 times more tritiated mannose than control normal procollagen. The carboxy-terminal propeptide was found to be the site of excessive mannosyla­tion, and this fragment, like the parent molecule, exhibited a tendency to aggre­gate. Kinetic studies revealed a delayed secretion of Type I procollagen from the OI cells (Peltonen et al., 1980b).

The re cent observation of an increased Type III/Type I ratio in the biosynthetic­secreted products of fibroblasts from an "arthropathic" form of 01 is of interest (penttinen et al., 1980). The patient exhibited severe osteoporosis and progressive periarticular bone loss (osteolysis) associated with subsequent joint fusions similar to patients with the Winchester syndrome (Hollister et al., 1973). Progressive loss of bone substance might be anticipated if bone collagen was susceptible to non­specific proteolysis.

Implication and Future Directions

What have we learned from recent clinical and biochemical studies of OI? Which experimental paths are likely to be fruitful in future studies? A nu mb er

232 Dayid W. Hollister

of observations emerge. It is apparent that no classification scheme for 01 based solely upon clinical and genetic data is, or can be, completely satisfactory in the absence of information regarding the fundamental molecular abnormality. Such schemes should be regarded as temporary expedients to be modified or discarded when further data becomes available. Nevertheless, such classification serves an extremely valuable purpose in guiding further investigations and in permitting comparison with previously reported cases of 01. For this reason, patients should be assigned, if possible, into a clinical classification. However, classification not­withstanding, considerable eHorts should be made to document and publish the clinical manifestations and mo des of inheritance of study patients in order that specific phenotype-biochemical abnormality correlations may be made indepen­dent of any classification.

Type I 01 (in the Sillen ce classification) appears to be due to diminished pro­duction and accumulation of Type I collagen in tissues inherited in a dominant fashion. Indirect evidence for abnormalities such as gene deletion, and gene de­fects causing decreased transcription, RNA processing or chain translation might be obtained by biosynthetic ratios of al(I) and a2(I) chains comparable to the classical studies of thalassemia. Such defects not reflected by structural alterations of procollagen would seem best directly approached by recombinant DNA tech­nology. Careful evaluation of Type I procollagen may reveal structural alterations of the propeptides which impede synthesis and secretion, or render the biosynthe­tic product unable to assemble properly, and hence be degraded protease sensiti­vity of the collagen should also be considered.

Type II 01 could be due to delayed and defective production of Type I pro­collagen, perhaps because of structural defects in propeptides or helical portions of the molecule. The synthesis of other collagen types suggests a selective defect in Type I synthesis. Since this is a recessive condition, each parent would contri­bute one allele for a defective Type I chain, and therefore, biochemical studies of the obligate carriers would be informative. Kinetic studies of synthesis coupled with structural studies of procollagen seem indicated. Other possibilities are per­haps equally feasible.

Types III and IV 01 may represent any of the possibilities discussed above, and at least one ca se appears to be due to overmannosylation of the COOH­terminal propeptide with diminished Type I secretion (Peltonen et al., 1980b). Presumably, the excess mannose is a reflection of a structural defect in one of the pro a chains. Since these patients are more severely affected than Type I 01, it might be expected that the molecular abnormalities will be more marked. EM studies of affected tissues may be extremely useful in suggesting intracellular ac­cumulation and delayed secretion as manifested by dilation of rough endoplasmic reticulum.

The manifest complexities of collagen metabolism virtually assure that a wide variety of defects will ultimately be discovered in 01. The tools are at hand, or under active development for the successful approach to these diseases. Of per­haps more importance, the conviction that 01 is due to collagenous defects is now firm, and therefore, the future direction is more clear. We may anticipate substantial progress in the next decade toward the understanding of this fascinat­ing group of disorders.

Osteogenesis Imperfecta 233

References

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Burgeson, R. E., Hollister, D. W., Lachman, R. S., and Rimoin, D. L.: Camptomelic Osteogenesis Imperfecta: Hydroxylysine-deficient bone collagen - manuscript in pre­paration, 1980.

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234 David W. Hollister

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David W. Hollister, M.D. Division of Medical Genetics Departments of Pediatrics and Medicine Harbor·ULCA Medical Center 1000 West Carson Street Torrance, Cali­fornia 90509, USA.