Chapter 16 - OSTEOGENESIS IMPERFECTA by Joan Marini, M.D., Ph.D., Chief, Bone & Extracellular Matrix Branch/NICHD/NIH March 1, 2010 TO OBTAIN A COMPLETE DOWNLOAD OF THIS CHAPTER IN PDF OR WORD FORMAT, CLICK HERE	Contents Contributors Search

Introduction

Osteogenesis imperfecta (OI), also known as Brittle Bone Disease, is a heritable disorder of connective tissue. Its hallmark feature is bone fragility, with a tendency to fracture from minimal trauma or from the work of bearing weight against gravity. In the more severe forms of the disorder, the bones are deformed as well as fragile. Most individuals with OI have significant physical handicaps. Affected persons also exhibit an array of associated features, including short stature, macrocephaly, blue sclerae, dentinogenesis imperfecta, hearing loss and neurological and pulmonary complications. There is no preferential distribution of osteogenesis imperfecta by gender, race, or ethnic group.

Historically, osteogenesis imperfecta has been viewed as an autosomal dominant disorder of type I collagen, the major protein component in the extracellular matrix of bone. In the past several years, this paradigm of OI has undergone a major shift with the identification of autosomal recessive forms. Although recessive OI is not due to defects in collagen, its etiology in a collagen-modification complex is collagen-related. Recessive OI is due to deficiency of any one of the components of the endoplasmic reticulum-resident collagen proplyl 3-hydroxylation complex. OI, regardless of etiology, requires clinical management and genetic analysis. The incidence of forms of OI recognizable at birth is 1/16-20,000, with about equal incidence of mild forms that are not recognizable until later in life. (1)OI and Marfan’s Syndrome share the distinction of being the most common heritable connective tissue disorders.

Clinical classification and phenotype

David Sillence formulated the classification currently in common use for osteogenesis imperfecta in 1979. (2)Since type I collagen defects were not known to cause OI at that time, the Sillence Classification is an artificial grouping based on clinical and radiographic features. The clinical spectrum of OI ranges from perinatal lethal to a mild form that can present in middle-aged adults as premature osteoporosis. The original Sillence Classification included types I thru IV; however, in the past decade five additional types (V thru IX) have been identified using histological and molecular findings. (3-5)Types I to IV of OI have autosomal dominate (AD) inheritance. Although conventionally thought of as AD disorder, the discovery of types VI, VII, VIII, and IX revealed that some forms of OI are autosomal recessive.

	Table 1. Classification of Osteogenesis Imperfecta by Type1
OI Type	Clinical Features	Inheritance
I	Normal stature, little or no deformity, blue sclerae, hearing loss in 50% of families Dentinogenesis imperfecta is rare and may distinguish a subset.	AD2
II	Lethal in the perinatal period; minimal calvarial mineralization, beaded ribs, compressed femurs, marked long bone deformity, platyspondyly	AD (new mutations) Parental mosaicism
III	Progressively deforming bones, usually with moderate deformity at birth. Scleral hue varies, often lightening with age. Dentinogenesis imperfecta common, hearing loss common. Stature very short.	AD Parental mosaicism
IV	Mild to moderate bone deformity and variable short stature; dentinogenesis imperfecta is common and hearing loss occurs in some families. White or blue sclerae.	AD Parental mosaicism
Non-Collagenous Types of OI – Cause Unknown
V	Phenotypically indistinguishable from type IV OI. Distinctive histology of irregular arrangement or meshlike appearance of lamellae. Also have triad of hypertrophic callus formation, dense metaphyseal bands, and ossification of the interosseus membranes of the forearm. Normal type I collagen; no mutations detected.	AD
VI	Phenotypically indistinguishable from type IV OI. Diagnosed on basis of unique histological features. Elevated alkaline phosphatase activity. “Fish-scale” appearance of bone under the microscope.	Unknown
Recessive OI - Defects in Collagen Prolyl 3-hydroxilation Complex Component
VII	Severe or lethal bone dysplasia similar to type II & III. Small head circumference, exophthalmos, white or light blue sclera .	AR
VIII	Severe or lethal bone dysplasia similar to type II & III. West African origin	AR
IX	Moderate to severe bone dysplasia similar to types IV or III OI. White sclera	AR
1Modified from Sillence et al., 1979 2AD = autosomal dominant; AR = autosomal recessive

Patients with type I OI have a distinctly milder form of the disease, which is generally not detectable at birth. Patients with type I OI tend to present with early osteoporosis; DEXA z-scores range from –1 to –3. Patients may have their first fracture in the pre-school years, for example when attaining ambulation. They may also have a series of fractures in the pre-pubertal years due to mild trauma. Fractures generally decrease dramatically in the post-pubertal years. Patients with type I OI have normally modeled bone and may have mild bowing of long bones and minimal central vertebral compressions. They are often a few inches shorter than same gender relatives. Leg length may be disproportionately short. Type I is divided into A and B subtypes based on the presence or absence of dentinogenesis imperfecta (DI). (6)Blue scleral hue is a defining feature in the Sillence classification, though in actuality it may be present or absent. These patients are expected to be spontaneous ambulators, but may have some mild delay of gross motor skills. They can be expected to have a full life span, limited only by greater vulnerability to accidental trauma.

Type II OI is the perinatal lethal form. Infants may be stillborn; if they survive birth, they usually die in the first two months of life. (7)Some infants with type II OI may live for as long as a year, but eventually do succumb to multiple pneumonias or respiratory insufficiency. The limbs, especially the legs, are short with severe bowing deformities. Most often the legs are abducted into the classic “frog leg position”. The cranium is relatively large for the trunk and is very poorly ossified. The anterior fontanelle is large, and often extends frontally to the forehead and laterally along the sagittal suture. The posterior fontanelle is often open as well. The presence of two enlarged fontanelles frequently results in ossification only along the lateral plates and for a fingertip breadth at the crown. The infants tend to have flat triangular facies with a small beaked nose and dark blue-gray sclerae. The thorax is usually deformed with a narrow apex. Radiographic examination reveals multiple in utero fractures in various stages of healing. There may be beads of callus on the ribs, which are quite gracile. The long bones are very osteoporotic with minimal to no cortex. Upper extremity long bone morphology is better than that of the lower extremities. The lower long bones are crumpled as well as fractured and are abnormally modeled, with a cylindrical shape. Thus, the defect in type I collagen affects the development as well as the mineralization of the skeleton.

Type III OI, also known as the Progressive Deforming type (1), is the most severe form of OI compatible with survival beyond infancy and is severely handicapping. Individuals with type III OI can have a full life span, however, a significant proportion succumb to respiratory or neurological complications, either during childhood or in early to middle adult years. The long bones of individuals with type III OI are soft as well as fragile and can have bowing deformities of 70-90°, caused either by the tension of normal muscle on the bone, or from angulated healing of fractures. Long bones have a cylindrical shape with more modeling of the metaphysis than in type II; by late childhood there is often exaggerated metaphyseal flaring accompanied by a slender diaphysis. (8)An additional finding in the metaphysis and epiphysis of lower limb long bones are so called “popcorn” calcifications caused by disorganization around the growth plate. More than half of the individuals with type III OI develop this radiographic change between the ages of 4 to 14 years with resolution of popcorn calcifications when epiphysis close. (9)Fractures can occur from activities of daily living; there may be hundreds of fractures in a lifetime. DEXA z-scores are in the range of –5 to –7 SD. Body proportions are better preserved than in type II OI, with less shortening of the extremities relative to the trunk. The calvarium is almost always relatively macrocephalic for the body and frequently measures greater than 95% for age, though occasionally children will have a normal or smaller than average HC for age. The midface is flat with frontal bossing and DI is common. Children with type III OI almost always develop chest wall abnormalities; pectus carinatum is more frequent and less detrimental to pulmonary status than pectus excavatum. Virtually all children with OI type III will also develop significant scolisis. Without aggressive intervention, these individuals will be wheelchair bound.

Type IV OI is the moderately severe type. The skeletons of these individuals are brittle, not soft. On average, people with type IV OI have dozens of fractures. Most fractures occur either prior to puberty or beyond middle age, with the intervening years relatively protected by sex steroids. Popcorn calcifications have been reported as a radiographic change associated with type IV OI; however, it does not occur as frequently as seen in type III. (9)Individuals are significantly osteoporotic, with DEXA z-scores in the range of –3 to –5 SD. With medical intervention these individuals can expect to be community ambulators and have an essentially normal life span. Body proportions approach normal, although the legs are still short for the trunk and the cranium is relatively macrocephalic. Like type I OI individuals with type IV are divided into types A and B by the Sillence’ classification, based on the presence or absence of dentinogenesis imperfecta. (6)Vertebral compressions in childhood and laxity of paraspinal muscles may lead to significant scoliosis.

In many ways type V OI is clinically indistinguishable from type IV because both types present with frequent fractures, moderate deformity, ligamentous laxity, tendency to bruise easily and infrequent loss of mobility. Clinical, histological and molecular differences exist, however, that distinguish type V from IV. A clinical feature unique to type V OI is severely limited pronation/supination of forearms. In addition, individuals with type V do not have blue sclera or dentinogenesis imperfecta. In type V, the distinctive histology is an irregular arrangement or a meshlike appearance of the lamellae. Patients also have a triad of hypertrophic callus, dense metaphyseal bands and ossification of the interosseus membranes of the forearm. The type I collagen protein of these patients has normal electrophoretic mobility, and no mutations have been detected in type I collagen genes (COL1A1 and COL1A2). (10)The cause of type V OI is unknown.

Type VI OI, like type V, is similar to type IV. Characteristics of individuals with type VI OI include short stature, ligamentous laxity, white or faintly blue sclera and no DI. First fractures in type VI OI occur when affected individuals are infants or toddlers and the frequency of fractures is greater than seen in type IV. Deformity caused by long bone fractures can be moderate to severe, often necessitating support devices for ambulation or wheelchairs to maintain mobility. Type VI OI is distinguished from type IV solely on histology and molecular criteria. (11)Bone histology includes “fish-scale” pattern of the lamellae, and decreased mineralized bone volume secondary to increased osteoid volume. This bone mineralization defect is a defining attribute of Type VI OI. Currently, the molecular defect of type VI remains elusive; although, it likely due to autosomal recessive defect.

Type VII OI is a lethal/severe recessive chondrosseus dysplasia. Fractures and limb deformities are present at birth. Radiographically, long bones are severely undertubulated. Infants with type VII may develop respiratory insufficiency in the neonatal and postnatal periods and frequently die as a result of the underlying problem (i.e., pulmonary anatomical anomalies or infectious disease). (12)Distinctive features of type VII OI include small or normal head circumference, exophthalmia, white or light blue sclera, and rhizomelia. Type I collagen genes are normal in type VII OI. Type VII OI is caused by null mutations in CRTAP. This gene encodes the cartilage-associated protein (CRTAP), which functions as the helper-protein in the collagen prolyl 3-hydroxylation complex. Deficiency of this protein affects post-translational modification of both bone (type I collagen) and cartilage (type II collagen). The index pedigree from Quebec (13)first described for type VII OI has a hypomorphic defect in CRTAP (14)and a correspondingly milder phenotype with rhizomelia, coax vera and white sclerae, more similar to dominant type IV OI in skeletal severity. These children have moderate growth deficiency. They attain ambulation without assistive devices.

Type VIII is also a severe/lethal autosomal recessive form of OI. In this type, it is the enzymatic component of the collagen prolyl 3-hydroxylation complex, P3H1 (encoded by LEPRE1), which is deficient. (15-17)Phenotypic characteristics overlap the dominant types II and severe type III OI, but have the distinguishing features of white sclerae, undertubulated long bones and normal to small head circumference. Like type VII OI, rhizomelia is a distinct feature of type VIII. Some individuals with type VIII OI have lived into their second or third decade (currently, the oldest known individual in early 20’s). Their physical exam is notable for extreme short stature, severe osteoporosis (DEXA z-scores of -6 or -7), and popcorn calcifications during the growing years. The most frequently identified LEPRE1mutation is a West African founder mutation that also occurs in Afro-Caribbeans and African-Americans. Homozygosity for the West African allele has been lethal by 3 months of age.

Type IX OI completes the set of recessive OI types caused by deficiency of components of the collagen prolyl 3-hydroxylation complex. In this type, individuals have deficiency of the third component of the complex, cyclophilin B (encoded by PPIB). (4, 5)These individuals have a distinctive phenotype compared to types VII/VIII in that they do not have rhizomelia; although, they share the white sclera of recessive OI. The total absence of cyclophilin B (CyPB) due to a mutation in the start codon causes moderately severe OI, overlapping dominant type IV OI in skeletal severity. (4)Their osteoporosis is also moderately severe, with DEXA z-scores in the -2 to -3 range. They have attained community ambulation after osteotomy procedures. They have moderate short stature and may or may not have vertebral compressions. In other cases, the presence of misfolded CyPB (5)interferes with function of the 3-hydroxylation complex and causes severe OI.

Secondary features of OI

Scleral hue

Scleral hue is a defining feature of the Sillence classification, with blue sclerae in type I OI, white sclerae in type IV. This resulted in the grouping of children with inconsistent skeletal features. We consider scleral hue a secondary, not a defining, feature. Most people with type I OI have blue sclerae, but some will have white sclerae. Many persons with types III and IV OI will have blue sclerae. Blue sclerae have also been reported in types V and VI OI.

The bluish tinge may result from decreased scleral thickness. (18)However, it can also occur with normal thickness. In this case, tissues with different proteoglycan compositions, and therefore different hydration, may cause the blue tinge by their reflection of wavelengths of color.

Growth

Short stature is the most prevalent secondary feature of OI. Children with types III and IV OI fall off normal growth curves by one year of age, entering a plateau phase with flat or slow growth that lasts until age 4-5 years. After age five years, children with type IV OI often grow either parallel to the normal growth curve or with a moderately decreased slope. However, they cannot make up for the loss of height incurred during the plateau phase, so final stature approximates that of an early teenager. Children with type III OI have increased growth rates after the plateau phase, but the slope is always less than that of the normal curve. Final adult stature is typically in the range of a prepubertal child and can be that of a 5-7 year old. (19)Individuals with type I OI grow parallel to the normal growth curve and final height is usually a few inches shorter than same gender relatives.

The cause of short stature in OI is not clear. Short stature is not caused by fractures or premature closure of growth plates. The recessive types of OI, with extreme short stature caused by deficiency of proteins that function in both cartilage and bone, have called attention to OI as a chondrosseus dysplasia. Short stature in dominant types of OI may be related to defective transitioning at the junction of the growth plate and bone, although this remains to be demonstrated.

Hearing Loss

A majority of adults with osteogenesis imperfecta have functionally significant hearing loss related to combined conductive and sensorineural deficits. (20)Molecular studies have revealed that hearing loss is not related to OI types or to location of mutation in COL1A1 or COL1A2. (21)In most cases, deficits are detectable only on audiology examination in childhood and the teen years; functional loss does not occur until the twenties. A study of hearing loss in Finnish children with OI reported an incidence of 6.7% with loss greater than 20 dB (22); this is comparable to the 7.7% detected in the NIH pediatric OI population. (23)Most pediatric hearing loss is detected between ages 5-9 years; some children may require hearing aids. For adults, the hearing deficits are very similar to those found in otosclerosis.

When hearing loss exceeds the compensation of hearing aids, surgical interventions may be used. Stapedectomy can give satisfactory long-term results; however, this surgery should not be undertaken routinely. The fragility of the small bones of the ear results in a significant percentage of unsatisfactory long term hearing restoration. (24)Given the rarity of OI and surgical complications in OI (i.e., middle ear anatomic anomalies and tendency for profuse bleeding), surgical outcomes may be better at medical facilities experienced with stapes surgery and hearing loss due to OI. (25)Insertion of cochlear implants has been reported in a few case studies (26); however, this data is limited. The implants have resulted in a short-term improvement in hearing ability, but long term hearing restoration remains unknown. (27)

Pulmonary Complications

Cardiopulmonary complications of osteogenesis imperfecta are the major cause of mortality directly related to the disorder. Infants with type II OI die of respiratory insufficiency or pneumonias. Children with type III OI develop vertebral collapse and kyphoscoliosis, which contribute to restrictive lung disease. These skeletal features, as well as the inactivity associated with wheelchair mobility, predispose them to multiple pneumonias. Lung disease may progress to cor pulmonale in middle age. Pulmonary function should be evaluated every few years, starting in childhood, to facilitate early management with bronchodilators. The need for chronic oxygen may arise as early as adolescence but most frequently occurs in the forties and fifties.

Neurological Complications

Osteogenesis imperfecta is frequently associated with either relative or absolute macrocephaly. Between ages 2-3 years, the child’s head circumference may rapidly cross percentile lines for age. Prominence of sulci and ventriculomegaly are not associated with intellectual deficit. There is a high frequency of basilar invagination (BI) in patients with severe osteogenesis imperfecta. BI generally progresses slowly in childhood; radiologic evidence for BI may be present for years before symptoms are present. Children should be screened by CT every 2-3 yrs, and followed annually by MRI if radiographic signs of BI develop. Favorable outcomes have been obtained by delaying surgical intervention until the patient experiences severe headaches as well as long tract signs. Typical clinical features of BI include headaches, dysphagia, ataxia and changes in facial sensation that, if not treated, can progress to rapid neurologic decline and/or respiratory distress. (28)As patients become symptomatic they should be followed in centers (University of Iowa, Johns Hopkins) with experience in performing anterior ventral decompression with occipitocervical fusion in OI patients. (29, 30)

Diagnostic Work-up and Differential Diagnosis

Crucial elements of the diagnostic work-up focus on the skeletal system. The physical exam includes measurements of length and head circumference, as well as notations on body proportions, including upper segment: lower segment ratio and arm span. In addition, the segmental lengths of each limb are measured to detect asymmetry. Individuals with OI frequently have relatively long arm span for length and a shortened lower segment (pubis to floor). Sclerae may be blue or blue-gray and teeth may have dentinogenesis imperfecta, with opalescent or yellow-brown enamel. In the thorax, the spine should be examined for scoliosis and the rib cage for flare and/or pectus carinatum or excavatum. In an infant, the size of the fontanels should be noted. Also essential is a careful family pedigree, with inquiries about fractures, hearing loss, dentinogenesis imperfecta, adult height,racial background and consanguinity.

Radiographic examination consists of a selective skeletal survey. AP and lateral views of the long bones are examined for significant osteoporosis, bowing, healing fractures, metaphyseal flare and the sharpness of the growth plate. AP and lateral views of the spine are examined for scoliosis, vertebral compressions, and sharpness of the vertebral endplates. Rhizomelia is suggestive of recessive types of OI, although it occurs more commonly in chondrodystrophies. A lateral view of the skull should also be obtained to detect wormian bones.

It is essential to obtain a DEXA of the lumbar vertebral bodies for a relatively quantitative assessment of the individual’s osteoporosis. Since the bone matrix in types II, III, IV, VII and VIII OI is qualitatively abnormal, the DEXA z-score reflects the structural arrangement of the mineral as well as the quantity and therefore is not a straightforward quantitative measurement.

Differential diagnosis varies with the severity of OI and age of the patient. On prenatal ultrasound, severe OI may be confused with thanatophoric dysplasia, achondrogenesis type I, or campomelic dysplasia, all of which demonstrate relatively large heads and short limbs. Type III OI may need to be distinguished from infantile hypophosphatasia, which presents with severe osteoporosis and micromelia. Hypophosphatasia results in low serum alkaline phosphatase and increased inorganic pyrophosphate, while in OI, serum alkaline phosphatase is normal or increased. Type IV and more severe type I OI may be confused with primary juvenile osteoporosis or other secondary causes of osteoporosis in childhood, such as hypogonadism of malignancy. Some cases may require collagen studies or bone histology to make a definitive diagnosis. The major differential diagnosis with type I OI is child abuse. Molecular and biochemical studies of collagen and of the components of the collagen prolyl 3-hydroxylation complexcan complement decreased BMD and other skeletal features of OI, as necessary.

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Collagen mutations and genotype-phenotype correlation

The majority (80-90%) of OI causing mutations occur in the genes that code for the two chains that comprise type I collagen, the major protein of the extracellular matrix of bone, skin and tendon. (31)Type I collagen is a heterotrimer composed of two copies of the α1 chain, encoded by the COL1A1 gene on chromosome 17, and one copy of the α2 chain, encoded by COL1A2 on chromosome 7. The two alpha chains are similar in sequence organization; they are composed of 338 uninterrupted repeats of the sequence Gly-X-Y, where gly is glycine, X is often proline and Y is often hydroxyproline. A glycine residue in every third position along the chain is crucial for helix formation; glycine’s small size allows it to be tucked into the sterically constricted internal aspect of the helix. The collagen genes are organized with each exon coding for the helical region beginning with a glycine codon and ending with codon for a Y position; therefore the skipping of a helical exon does not cause a frameshift in the collagen transcript.

Recently, 832 independent mutations in both chains of type I collagen have been described in OI patients. (32)One general correlation between genotype and phenotype has emerged. Type I OI, the mild form, is caused by quantitative defects in collagen. Only half the normal amount of collagen is produced but all the collagen produced is structurally normal. This is almost always due to a null allele of COL1A1. (33)On the other hand, types II, III and IV OI, the clinically significant forms, are caused by structural defects in either of the type I collagen chains. About 80% of these structural mutations cause the substitution of another amino acid, with a charged, polar or bulky side chain, for one of the obligatory glycine residues occurring in every third position along the chain. Glycine substitution mutations temporarily block helix formation and cause overmodification (glycosylation) of the chains of the trimer. About 20% of structural mutations are single exon skipping defects, which are incorporated into the trimer because the frame of the transcript remains intact. (32)Essentially all of the collagen mutations are dominant negative mutations. They excrete their effects by being secreted and incorporate into the matrix, causing a weakened higher order structure.

For structural mutations of type I collagen, the relationship between genotype and phenotype has been elusive. A lethal mutation is more likely to be located in the α1 chain, in which about 35.6% of known glycine substitutions cause lethal OI, than in the α2 chain, in which only 19% are lethal. (32)Nonetheless, both chains contain substantial numbers of mutations causing the full range of the OI phenotype. The two chains have different patterns of lethal and non-lethal mutations along the helical region, supporting different roles for the two chains in matrix. Lethal and non-lethal clusters alternate along the α2(I) chain. (34)The clusters are quite evenly spaced, and appear to play a role in regularly repeating interactions of collagen with non-collagenous matrix molecules. When the alignment of cluster boundaries was compared to the clinical outcome of mutations, the cluster boundaries correctly predicted the phenotype of 86% of α2(I) mutations. (32)In the α1(I) chain, the mutations may disrupt the stability of the collagen helix itself. (32)Two regions of uninterrupted lethal mutations in the carboxyl end of α1(I) coincide with the major ligand binding region (MLBR) for integrins, fibronectin, and COMP. (32)

The phenotype-genotype relationship in OI is complicated by multiple examples of variable expression. Individuals with the same genotype have a different phenotype, an interesting feature of many dominant disorders. In the α1(I) chain, there are several dozen sites with examples of extreme variable expression of the same mutation; these glycine substitutions are found in both lethal and non-lethal forms of OI. A more frequent occurrence in both chains is substantial variation in severity between family members or unrelated individuals with the same mutation. For example, phenotype can range from type III to IV OI. One explanation for this interesting feature may be the existence of discrete modifying genes. Understanding modifying factors may provide new approaches to treatment.

Recently generated mouse models including the Brtl (brittle), Amish, and Aga2 (abnormal gait 2) mice have shed new light on the pathophysiology, modifying factors and treatment of OI. The Brtl mouse is a knock-in model for type IV OI. (35)It contains a classic glycine substitution at α1(I) G349C, which causes dominant negative OI. The Brtl mouse reproduces the phenotype, histology, biochemistry and biomechanics of the disorder. It also has variable phenotypic expression, which may lead to an understanding of modifying factors. Clinically relevant findings elucidated with the Brtl mouse model include postpubertal improvement in bone matrix material properties (36), the imbalance between decreased osteoblast function and increased osteoclasts precursors as a potential lead to novel OI therapies (37), and the concomitant beneficial and detrimental effects of cumulative bisphosphonates exposure. (38)A knock-in mouse model for the α2(I) chain has recently been published. (39)It recapitulates the mutation found in a large Amish pedigree that causes a Gly610Cys substitution, hence its designation as the Amish mouse. Long bones of the Amish mouse are less fragile than those of Brtl. The human pedigree with the Gly610Cys substitution has a wide range of phenotypic variability. Crossing the murine mutations into different genetic backgrounds demonstrated that whole bone fracture susceptibility was influenced by factors reflected in the size and shape of bone, and will be useful for the identification of genetic modifiers. Finally, the Aga 2 mouse has a dominant mutation located in the terminal C-propeptide that was created using an N-ethyl-N-nitrosourea mutagenesis strategy. (40)Like the Brtl mouse model, the Aga2 phenotype has a perinatal lethal and a severe surviving form. This mouse will provide important insight into the special mechanism of OI caused by mutations in the C-propeptide. Since the C-propeptide is removed before collagen is incorporated into matrix, it is not clear why mutations in this region should cause moderate to lethal OI. In Aga2 osteoblasts, the intracellular retention of abnormal collagen chains has been shown to induce the Unfolded Protein Response (UPR) and result in cellular apoptosis.

In addition, there is a naturally occurring mouse model for type III OI, the oim mouse. (41)This mouse is atypical of OI with collagen structural mutations in that it has recessive inheritance. The collagen defect is a mutation in the α2(I) chain that prevents incorporation into heterotrimer and leads to the production of an α1(I) homotrimer. The histomorphometry of the oim mouse differs from that seen in classical dominant OI, limiting the utility of this model. (42)More importantly, individuals with α1(I) homotrimer caused by null-mutations in the amino end of the a2(I) chain have been shown to have Ehlers/Danlos Syndrome but not OI. (43, 44)Since the bonedysplasia of oim cannot be directly attributed to the presence of homotrimer, it is impossible to meaningfully interpret oim investigations.

Recessive OI and genotype-phenotype correlation

Approximately 10-15% of individuals who have a phenotype characteristic of OI do not have a defect in the collagen genes COL1A1 or COL1A2. Molecular and biochemical defects have recently been identified in types VII, VIII and IX OI; specifically, each type has a defect which causes deficiency of one the components of the collagen prolyl 3-hydroxylation complex. Although 3-hydroxylation of Pro986 in type I collagen had been known to occur for almost three decades (45), its importance to bone formation had not been appreciated. The new understanding of recessive OI as a deficiency of this ER-resident collagen modification complex shifted the paradigm for collagen-related bone dysplasias. (3)

LEPRE1,CRTAPand PPIBare the three genes that encode the components of the collagen prolyl 3-hydroxylation complex, prolyl 3-hydroxylase 1 (P3H1), cartilage–associated protein (CRTAP) and cyclophilini B (CyPB), respectively. The proteins form a 1:1:1 complex in the endoplasmic reticulum. (46)The complex binds collagen post-translationally and hydroxylates a single residue, proline 986, on each α1(I) chain. In normal collagen, over 90% of Pro986 residues are 3-hydroxylated. The importance of the collagen prolyl 3-hydroxylation complex for bone development became clear during investigation of the Crtapknock-out mouse. These mice have severe osteopenia, rhizomelia and later develope kyphosis. In addition these mice lacked 3-hydroxylation of Proline986 on both chains of α1 (I) and α1(II) collagen chains. (14)

Likewise individuals with recessive OI have defects in CRTAP (i.e., type VII) and LEPRE1 (i.e., type VIII) genes that result in either absent or severely reduced α1(I) Pro986 3-hydroxylation, respectively. Null mutations in CRTAP or P3H1 result in lethal/severe OI (3)with white sclerae, undertubulation of long bones, rhizomelia and short metacarpals. The type I collagen of these individuals lacks Pro986 hydroxylation. Surprisingly, the collagen that lacks Pro986 hydroxylation is overmodified by Prolyl 4-hydroxylase (P4H) and lysyl hydroxylases (LOH), proteins that modify proline and lysine residues along the length of the helical region of both alpha chains. Excess modification of the helix indicates that folding of the helix has been delayed. Interestingly, the phenotype as well as the collagen biochemical findings of CRTAP and LEPRE1 null mutations is indistinguishable. The basis of this similarity is the mutual protection of CRTAP and P3H1 in the modification complex. (47)Cells with a null mutation in either gene are missing both proteins; restoration of the genetically deficient protein restores both proteins. Thus, null mutations in either gene cause absence of the complex from the cell. Finally, the most common LEPRE1 mutation identified to date is a founder mutation from West Africa (IVS5+2G>T) that has also been identified in African Americans. (15)

Recently, mutations causing deficiency of the third component of the collagen prolyl 3-hydroxylation complex, CyPB have been identified and designated type IX OI. (4, 5)One mutation in the start codon of CyPB causes a total absence of CyPB. These individuals have distinct phenotype and collagen biochemistry compared to types VII/VIII. They have moderatedly severe OI with moderate growth deficiency and are ambulatory with orthopedic intervention. While they share the white sclerae of recessive OI, they do not have rhizomelia. Biochemically, they have normal 3-hydroxylation of Pro986, consistent with persistence of the CRTAP/P3H1 complex in the absence of CyPB. More surprisingly, they do not have excess modification of their collagen helix, suggesting that CyPB is not the rate-limiting peptidly-prolyl isomerase. Other mutations which apparently cause misfolded CyPB presumably interfere with the function of the 3-hydroxylation complex. Like types VII and VIII OI, these CyPB mutations are associated with decreased Pro986 hydroxylation and delayed collagen folding.

Genetic counseling and rationale for collagen studies

Genetic counseling and collagen studies are essential components of complete care for individuals who have OI. With the recent identification of recessive OI, genetic counseling for OI has become more complex. More than half of individuals with autosomal dominant OI have a family history of OI. In a Finish survey (22), about 65 percent of individuals with OI were in families in which a prior generation was affected and the remaining 35 percent represented new mutations in a type 1 collagen gene. In contrast, individuals with autosomal recessive OI seldom have a family history. Collagen sequencing is essential to accurate counseling, given the overlap in phenotypic manifestations. Virtually all type I collagen mutations have dominant inheritance. If no collagen mutation is identified, abnormal collagen biochemistry can point to defects in CRTAPor LEPRE1. PPIB defects will rely on sequencing for detection, since collagen biochemistry is normal.

In autosomal dominant OI, a severe presentation is likely to be the result of a spontaneous mutation that occurred at or around conception; the affected individual is likely to be the first affected person in the family. The parents of a child with a de novomutation are at no increased risk of recurrence compared to the general population. However, genetic testing of both child and parents is required to determine whether the OI is inherited from a mosaic parent (see below), which occurs in 5-10% of new cases and increases the risk of recurrence. Individuals who are affected with dominant OI have a 50% risk of transmission with each pregnancy.

Genetic counseling for autosomal recessive OI is challenging given the limited carrier information about these newly identified OI types. Certainly, parental consanguinity increases the risk that a child may have recessive OI. However, recent data has shown that the carrier frequency for type VIII OI among contemporary West Africans is over 1%; among African Americans about 1/200-300 individuals are carriers. (48)Currently the carrier frequency of Type VII OI is not known. Because both types VII/VIII can present as lethal OI and be incorrectly assumed to be type II OI, the genes for type I collagen frequently are not sequenced leading to the missed diagnosis of recessive OI and parental carrier status. The parents of a child with recessive OI have a 25% risk of recurrence.

Parental Mosaicism

In some families, clinically unaffected parents will have more than one child with dominant OI. This occurs because one parent is a mosaic carrier of the mutation. Presumably, the mutation occurred during the parent’s fetal development; that parent then has both a normal and a mutant cell population. The proportion of mutant cells and their distribution in somatic and germline tissues depends on the timing of the mutation and the distribution of cells arising from the first mutant cell. (49)The frequency of occurrence of mosaic parents is relatively high in OI. Empirically, 5-10% of unaffected couples whose child has dominant OI will be at risk of recurrence. For those couples in which one member is a mosaic carrier the recurrence risk may be as high as 50%, equivalent to the fully heterozygous state. To date, all mosaic parents have been detectable by examination of leukocyte DNA for the mutation present in their child. The mutation may also be detectable in fibroblast, hair bulb and germ cells.

Prenatal Diagnosis

For the first case of moderate to severe OI in a family, prenatal diagnosis will probably occur during ultrasound at 18 to 24 weeks’ gestation. (50)Given the severity of recessive OI caused by null mutations in CRTAPor LEPRE1and their clinical overlap with types II and III OI, the first case of recessive OI in a pedigree can be expected to be diagnosed in the same timeframe by ultrasound.

Detecting recurrence of dominant OI prenatally is easiest if the exact collagen mutation in the affected child is known. In that case, a potential mutation in the current pregnancy can be detected early and with confidence. Cultured chorionic villi cells (CVS) can be used for DNA or RNA extraction and detection by either PCR and restriction enzyme digestion or sequencing. CVS can also be used for biochemical analysis if the known mutation causes significant collagen protein overmodification. (49)Amniocentesis is only appropriate for molecular diagnosis via RNA or DNA analysis. Biochemical analysis of amniocytes is complicated by the overproduction of α1(I) chains; the excess chains form homotrimers, which are overmodified and co-migrate with overmodified heterotrimers, potentially causing a false-positive test result. (49)

Early detection of recurrence of recessive OI should be based on detection of the mutation identified in the first affected child. At this time, there are no data available on expression of the components of the 3-hydroxylation complex in CVS or amniocytes. Thus, analysis of DNA by sequencing or restriction digestion will be required.

Collagen analysis is also useful when the diagnosis is equivocal. A positive collagen biochemical study can counteract charges of child abuse in mild cases, although the absence of a positive study still leaves a substantial possibility (about 25%) of a false negative result. False negative biochemical tests occur with most mutations in the amino-quarter of the alpha chains, which is also a region where almost all mutations are non-lethal. (51)A positive collagen analysis can also settle subtle distinctions between type IV OI and idiopathic juvenile osteoporosis.

From a research standpoint, each new collagen mutation delineated in OI provides information about genotype-phenotype relationships either directly or by making the cells containing that particular mutation available for studies of mechanism at the level of bone matrix. Further, mutations may vary in response to different therapeutic approaches. Determination of mutations that cause OI may allow investigators to understand which drugs or therapies will be helpful for different individuals.

Therapeutic approaches

Conventional

Conventional management of osteogenesis imperfecta involves intensive physical rehabilitation, supplemented with orthopedic intervention as needed. Many parents and physicians place undue importance on the number of fractures sustained by children with OI. Fracture number may not be as important in judging the severity of the disorder as the degree of trauma needed to cause a fracture. In general, children with type III OI sustain fractures from more trivial trauma than those with type IV OI. In addition, they tend to have more fractures in arms and ribs than occur in type IV. Fractures, in addition to long bone deformity, can lead to significant physical handicap.

The goal of physical rehabilitation for children with OI is to promote and maintain optimal functioning in all aspects of life. This is best accomplished by a program combining early intervention, muscle strengthening and aerobic conditioning. Early intervention should include correct positioning of the infant. Proper head support to help avoid torticollis and neutral alignment of the femora are essential. (52)Custom molded seats can help with lower extremity alignment as well as head and spine positioning. (52)Gross motor skills are delayed in OI, mostly because of muscle weakness. This can be addressed with isotonic strengthening exercises of the deltoids and biceps in the upper extremity and the gluteus maximus and medius and trunk extensors in the lower extremity. Strengthening of these muscle groups will ensure that children are able to lift their limbs against gravity and transfer independently. (53)

In patients with potential, protected ambulation should be initiated as early as possible. This frequently requires a combination of surgical correction and physical therapy.

Individuals with OI should be under the care of an orthopedic surgeon with experience in the management of this disorder. Fractures should be evaluated with standard x-rays and should be managed with reduction and realignment, as needed, to prevent loss of function. Cast immobilization should be monitored to minimize any worsening of osteoporosis and muscle weakness. The decision to intervene surgically must take into account functional as well as skeletal status. Appropriate goals for surgery are to correct bowing to enhance ambulation potential and to interrupt a cycle of fracturing and refracturing. The classical surgical procedure was developed by Sofield and Millar, with multiple osteotomies, realignment of the long bone sections and fixation with intramedullary rods. Indications for this procedure include long bone angulation of greater than 40°, functional valgus or varus deformity which interferes with gait, or more than two fractures in the same bone in a 6-month period. Both elongating (Bailey-Dubow and Fassier-Duval) and non-elongating (Rush) rods are currently used for intramedullary fixation. Elongating rods have the advantage of extension with growth, but have a high rate of migration from OI bone. (54)The complication rate is similar for the two types of extensible rods, so choice of rod is best based on surgical experience and preference. (55)Rush rods have less migration potential but need revision as the child outgrows them. In general, intramedullary rods induce significant cortical atrophy through mechanical unloading, especially in the diaphysis. The least stiff and smallest diameter rod possible should be utilized. Current intramedullary rodding procedures necessitate smaller incisions and, therefore, reduce pain and improve healing time after surgery.

Significant scoliosis is a feature of most type III and some type IV OI. Severe scoliosis does not correlate with number of collapsed vertebrae; however, it may be related to ligamentous laxity. Since resultant thoracic deformities can lead to pulmonary compromise, routine attention to the OI spine is warranted. (56)Scoliosis in OI does not respond to management with Milwaukee bracing. Spinal fusion with Harrington rod placement can provide stabilization and some correction to prevent pulmonary complications, but will not fully correct the curve. For best results, corrective surgery should occur when the curvature is less than 60°.

Rarely, long-leg bracing may be indicated to provide support for weak muscles, control joint alignment and improve upright balance. Stabilizing the pelvic girdle and controlling the knees helps facilitate independent movement. Braces do not provide protection per se against fractures. Instead, bracing support promotes increased independent activity that may actually put the child at risk of incurring additional fractures. However, the advantages of increased independence and higher functional level tend to outweigh any increased fracture risk.

Pharmacological Therapy

In the last decade, the potential of bisphosphonate treatment has caused great excitement in the OI patient community and has generated a rush to treatment. These drugs are synthetic analogs of pyrophosphate; their mechanism of action involves the inhibition of bone resorption. Bisphosphonates are deposited on the bone surface and are ingested by osteoclasts, inducing apoptosis. Because they inhibit bone resorption, these drugs have been used to treat malignancies with bony metastases, most commonly breast cancer. In the oncology context, their ability to attenuate the need for major pain medications has been noted, although the duration of this effect was limited in controlled trials. There is also extensive experience with these compounds in treatment of post-menopausal osteoporosis. Only limited knowledge about treatment of patients with structurally abnormal bone matrix has been gathered.

When used in patients with OI, bisphosphonates would presumably not affect the deposition of abnormal collagen into matrix. Thus, patients might have quantitatively more bone after treatment, but it would not be more structurally normal than before drug administration. Uncontrolled studies of pamidronate use in children, teenagers and infants with OI reported not only increased vertebral DEXA and geometry and long bone fractures, but also improved muscle strength, mobility and bone pain. (57, 58)Anecdotal use of the drug has been widely associated with decreased bone pain, especially in the spine, and increased endurance. However, controlled trials (59-62), while they have demonstrated the expected increase in vertebral bone density and, more importantly, in vertebral height and area, have not shown an improvement in motor function, strength, or self-reported pain. No controlled trial reported a decreased incidence of long-bone fractures, although two studies obtained downward trends and two reported decreased relative risks when fractures were modeled for initial BMD, gender and OI type using unspecified models. In fact, the lack of improvement in fracture rates in the controlled double-blind trial of alendronate led the FDA to specify a labeling change for the drug to indicate that no change in fracture or pain incidence occurred with treatment and that alendronate was not indicated for the treatment of OI. (63)The equivocal improvement in fractures in children is illuminated by data from bisphosphonate treatment of the Brtl mouse. (37)Treatment increases bone volume and load to fracture of murine femora, but concomitantly decreases material strength and elastic modulus. Femurs become, ironically, more brittle after prolonged treatment and bands of mineralized cartilage create matrix discontinuities that decrease bone quality. Prolonged treatment also alters osteoblast morphology. In patients, the detrimental effects of bisphosphonate came to medical and public attention with reports of osteonecrosis of the jaw. This complication has not been seen in OI and appears to require the extremely high doses used as adjuvant to chemotherapy. However, the greatest concern in children with OI is oversuppression of bone modeling and remodeling and worsening of bone quality. Long-term treatment, even at standard doses, interferes with bone remodeling and can be detected as metaphyseal undertubulation. (64, 65)Anecdotal reports from surgeons describe treated bone as “rock-hard” and “crumbly”, providing insight into paradoxical increases in fractures in some treated patients. Long-term suppression of bone turnover leads to accumulated micro-damage (microcracks) in bone (66)that may underlie the decrease in material strength.

The beneficial effects of bisphosphonate treatment for vertebral geometry and strength justify its use in OI, although a long-term follow-up study has not been conducted to determine whether treatment results in a decrease in scoliosis in treated OI children. The question now is, given the balance of bone benefits and detriments, how long should OI children be treated and what cumulative dose should they receive? Study protocols that involved children with types III and IV OI demonstrated that gains in BMD (60, 67), cortical width and cancellous bone volume (67)occur with a 2-4 year course of treatment. Thus, a compelling argument can be made for limiting treatment to several years (the NIH plan limits to 3 years, with a maximum of 3 mg/kg/yr delivered in q3 or q6m cycles) with the lowest effective dose of a bisphosphonate that restores vertebral geometry. This treatment phase should be followed by careful monitoring of vertebral geometry, long-bone fractures and DEXA before a decision to initiate a sound round of treatments is made.

The use of growth hormone to ameliorate the cardinal feature of short stature in types III and IV OI is still under active investigation. Approximately half of the 49 children studied to this point have achieved a sustained increase in linear growth of 50% or more over baseline growth rate. (68)Most responders (about 70%) had moderate type IV OI, and higher baseline PICP values. In addition, responders had increased bone formation and density. Trials of growth hormone in children with severe OI and short stature are therefore warranted in an effort to increase final adult stature and further explore the direct effect on bone. Patients who respond to growth hormone have increased BMD and improved bone histology (BV/TV).

Gene therapy

Gene therapy of a dominant negative disorder such as osteogenesis imperfecta is not amenable to the replacement approach being employed for recessive enzyme disorders. Dominant negative disorders are disorders of commission; the mutant collagen is synthesized, secreted from the cell and incorporated into matrix, where it actively participates in weakening the structure. Therefore, researchers have used approaches that either suppress expression of mutant collagen or replace mutant cells with donated bone cell progenitors.

The first approach to mutation suppression is modeled on type I OI, in which individuals have a null allele, make half the normal amount of collagen and have mild disease. Specific suppression of expression of the mutant allele, by hammerhead ribozymes, for example, would transform the recipient biochemically from type II, III or IV OI into type I. (69)Although this suppression is complete and specific in vitro, and substantial (50%) and highly selective (90%) in cells, the successful application to animal models is still in development.

The second approach attempts to replicate the natural example of mosaic carriers, who have a substantial proportion of cells heterozygous for the collagen mutation but are clinically normal. They demonstrate that the presence of a substantial burden of mutant cells can be below the threshold of clinical disease. Studies of osteoblasts from mosaic carriers of type III and IV OI have shown that 40-75% of cells are mutant, setting the threshold for minimal symptoms at 30-40% normal cells. (70)Transplantation studies using murine models have evaluated the potential of mesenchymal stem cells to treat OI. Progenitor cells have been demonstrated to engraft at low levels in oim. (71, 72)Most encouraging have been transplantation studies of adult GFP+ bone marrow into Brtl pups in utero. Despite low engraftment of bone (about 2%), transplantation eliminated the perinatal lethality of Brtl mice and improved the biomechanical properties of femora in 2-month old treated Brtl mice. (73)However, other murine transplantation studies have indicated a limited regenerative capacity of transplanted cells beyond 6 months. (74)A single human fetus received in utero transplantation of fetal mesenchymal stem-cells; engraftment (0.3%) could still be demonstrated in bone at age 9 months. Evaluation of clinical outcome was complicated by treatment in infancy with bisphosphonate but the child had sustained fractures and had significant growth deficiency. (75)Bone marrow transplantation of OI children with marrow-derived mesenchymal cells claimed transient improvement in growth, total body mineral content and fractures (76), but the methodology of these studies were controversial. (77)

A final approach is a variant on cell transplantation and involves gene targeting of mutant COL1A1 and COL1A2 using adeno-associated vectors in adult mesenchymal stem cells (MSC). This has been successful in less than half of 1% of cells with a COL1A1 or COL1A2 mutation, and the production of normal collagen by these targeted cells has been demonstrated. This approach could be potentially valuable for individuals with OI who are past early childhood. However, issues with low targeting success and random integration need to be solved before this approach is suitable for clinical trials. (78, 79)