GENETIC DISORDERS OF BONE AND EXTRACELLULAR MATRIX
, Head, Section on Connective Tissue Disorders
Weizhong Chang, PhD, Staff Scientist
Antonella Forlino, PhD, Guest Scientist
Thomas Uveges, PhD, Postdoctoral Fellow
Mona Abukhaled, MSN, CRNP, Senior Research Assistant
Wayne A. Cabral, AB, Chemist
Aileen M. Barnes, MS, Research Associate
Deborah Lyu, BS, Postbaccalaureate Fellow
Identification of genetic cause of type VII OI
Structural defects of the heterotrimeric type I collagen molecule are well known to cause OI, a dominant bone disorder. In 1979, researchers postulated a severe recessive form of OI. More recently, investigators have noted that some patients with clinical OI do not have defects detected in the type I collagen genes during sequencing. Such patients may be divided into those with abnormal collagen biochemistry and those with collagen chains demonstrating normal electrophoretic migration. We hypothesized that the cause of recessive OI with abnormal collagen biochemistry and normal collagen gene sequence would involve a gene(s) whose products interact with type I collagen.
Post-translational modifications of collagen are required for efficient folding, secretion, and fibril assembly, including 4-hydroxylation of proline residues, hydroxylation of lysine residues, and subsequent glycosylation of some hydroxylysines. Collagen is also modified by 3-hydroxylation of a single residue in the α1(I) chain at Pro986, although the function of this specific modification has yet to be elucidated. Morello and colleagues (Morello et al., Cell 2006;127:291) have generated a knockout mouse for Cartilage-Associated Protein (CRTAP) with recessive bone dysplasia characterized by defective osteoid formation and severe osteoporosis. The Bachinger laboratory also showed that CRTAP forms a complex in the endoplasmic reticulum with cyclophilin B and prolyl 3-hydroxylase 1 (P3H1)/leprecan (encoded by LEPRE1) that interacts with and 3-hydroxylates collagen. These discoveries made CRTAP a suitable candidate for recessive OI.
Our cell repository contained cells from 10 patients who have overmodified collagen biochemistry yet lack a mutation in either of the type I collagen genes. Of these 10 cases, we identified three patients with null mutations for CRTAP. Their clinical findings overlap with those of lethal type II OI but demonstrate distinctive features, including white sclerae. We screened patients for null mutations by real-time RT-PCR (reverse transcriptase polymerase chain reaction) using primary fibroblast mRNA. Sequencing of the exons and surrounding intronic regions of the CRTAP gene identified homozygous or compound heterozygous mutations in patient genomic DNA, resulting in altered splicing and introduction of premature termination codons in transcripts. All three CRTAP null patients had a severe recessive form of OI, which is lethal in the first year of life. Their long bones were extremely osteoporotic and deformed and demonstrated prenatal fractures and a tubular shape attributable to abnormal modeling. In addition, they had a narrow thorax with several prenatal rib fractures. The mutations identified in CRTAP include a homozygous IVS1+1G→C splicing mutation, a homozygous Gln276Stop in exon 4, and a compound heterozygote of a 16 nt duplication in exon 1 in one allele and a Met1Ile in the other allele. We showed that parents of CRTAP probands are heterozygous carriers. Probands demonstrate an absence of CRTAP protein on Western blot and of Pro986 hydroxylation on mass spectrometry analysis.
Barnes AM, Chang W, Morello R, Cabral WA, Weis MA, Eyre DR, Leikin S, Makareeva E, Kuznetsova N, Uveges TE, Ashok A, Flor AW, Mulvihill JJ, Wilson PL, Sundaram UT, Lee B, Marini JC. Deficiency of cartilage-associated protein in recessive lethal osteogenesis imperfecta. N Engl J Med 2006;355:2757-64.
Identification of the genetic cause of type VIII OI
Given that CRTAP forms a complex in the endoplasmic reticulum with cyclophilin B and prolyl 3-hydroxylase 1 (P3H1)/leprecan (encoded by LEPRE1), which interacts with and 3-hydroxylates collagen, and that CRTAP defects caused recessive OI in 3 of 10 patients with normal collagen genes but biochemically abnormal collagen, mutations in the other proteins in the complex were possible causes of the remaining seven cases.
In fact, we identified null LEPRE1 mutations in the remaining seven cases. These probands had bone dysplasia with features that overlapped with those of lethal type II OI and severe type III OI, but also with distinctive features, including white sclerae, as found in CRTAP defects. We screened patients for null mutations by real-time RT-PCR using primary fibroblast mRNA. Sequencing of the exons and surrounding intronic regions of the LEPRE1 gene identified homozygous or compound heterozygous mutations in patient genomic DNA, resulting in altered splicing and introduction of premature termination codons in transcripts. The seven null LEPRE1 patients had severe to lethal recessive bone dysplasia, characterized by shortened long bones and generalized bone disorganization, and extremely low bone mineral density (BMD) (L1-L4 DEXA z-score = -7). Five of the seven LEPRE1 patients had a common mutant allele, IVS5+1G→T, that apparently originated in West Africa and is present in African Americans. Other LEPRE1 mutations included IVS7+91G→A and IVS9+1G→T splicing mutations, a 1 nt deletion in exon 3, a 7 nt insertion in exon 9, an 11 nt deletion in exon 14, and a homozygous Tyr552Stop in exon 11. Parents of LEPRE1 probands were heterozygous carriers. Furthermore, we demonstrated that P3H1 protein was absent from our patient fibroblasts on Western blots. Using mass spectrometry of collagen tryptic peptides, we determined that the patients’ type I collagen evidenced severely reduced or completely abolished 3-hydroxylation of the α1(I) Pro986 residue. In contrast, the extent of overhydroxylation of proband collagens by lysyl hydroxylase was comparable to that of collagens containing a structural defect in the carboxyl terminus of the helical region, consistent with delayed folding of the collagen. Results of differential scanning calorimetry of patient collagens were consistent with an increased post-translational modification in the absence of a primary structural defect, with patient collagens showing a 1°C increase in thermal stability compared with normal control collagen. In these probands, collagen secretion is moderately delayed, but total collagen secretion is increased. The recessive null mutations of CRTAP and LEPRE1 result in a novel metabolic disorder of bone and demonstrate that the 3-hydroxylation complex is crucial for normal bone development. Our work has generated a new paradigm for genetic disorders of type I collagen, with dominant OI attributable to defects in the COL1A1 or COL1A2 genes and recessive OI attributable to deficiency of components in the complex that modifies collagen.
Cabral W, Chang W, Barnes AM, Weis MA, Scott MA, Leikin S, Makareeva E, Kuznetsova N, Rosenbaum K, Tifft C, Bulas D, Kozma C, Smith P, Eyre D, Marini JC. Prolyl 3-hydroxylase 1 deficiency causes a recessive metabolic bone disorder resembling lethal/severe osteogenesis imperfecta. Nat Genet 2007;39:359-65.
Marini JC, Cabral WA, Barnes AM, Chang W. Components of the collagen prolyl 3-hydroxylation complex are crucial for normal bone development. Cell Cycle 2007;6:1675-81.
Insights from Brtl mouse model for OI
We continue to investigate the Brtl mouse model for OI in order to understand the disorder’s pathological and cellular mechanisms. We first approached the secretion, matrix incorporation, and interactions of collagen molecules with one and two mutant alpha 1 chains. Collagen with no mutant chains or one mutant chain would be expected to constitute 25 and 50 percent, respectively, of matrix collagen content; however, we detected reduced content of molecules with one mutant chain and an increase in the proportion with no mutant chains. We demonstrated selective cellular retention of molecules containing one mutant chain compared with molecules without or with two mutant chains. Collagens were incorporated into matrix in proportion to their presence in media, pointing to the cell as the site of discrimination. In Brtl fibroblasts, the endoplasmic reticulum was engorged, suggesting that the cells were undergoing endoplasmic reticulum stress. The reactive sulfhydryl group in collagen with one mutant chain is exposed to solvent and may form aberrant S-S bonds with other intracellular or extracellular proteins.
We also studied the basis for the phenotypic variability in Brtl, given that it models the phenotypic variability seen in human α1(I) mutations. Brtl has two discrete phenotypes; about 30 percent of pups die within hours of birth, whereas the survivors have a moderately severe skeletal defect. We used microarrary and two-dimensional proteomics for complementary studies of calvarial bone from pups with the lethal and surviving phenotype. We found that Gadd153 expression was two- to three-fold higher in mice with the lethal phenotype than in surviving and wild-type littermates. We detected the increase in Gadd153 expression only in bone tissue—not in skin or lung. Furthermore, Western blot showed increased Gadd153 in calvarial protein extracts. Gadd153 is a member of the C/EBP family and can pair with CEBPβ or α to inhibit osteoblast differentiation and to promote cellular apoptosis, respectively. Conversely, we found both expression (1.6-fold) and protein level (doubled) of the αB-crystallin chain to be higher in the surviving Brtl pups. The role of αB crystallin contrasts with that of Gadd153. αB crystallin is a small heat shock protein known to confer resistance to apoptosis. The data suggest that apoptosis is the primary factor in the phenotypic variability of Brtl mice. We are currently exploring the dimerization partners of Gadd153 in Brtl osteoblasts in culture.
Forlino A, Kuznetsova NV, Marini JC, Leikin S. Selective retention and degradation of molecules with a single mutant alpha (I) chain in the Brtl IV mouse model of OI. Matrix Biol 2007;26:604-14.
Forlino A, Tani C, Rossi A, Lupi A, Campari E, Gualeni B, Bianchi L, Armini A, Cetta G, Bini L, Marini JC. Differential expression of both extracellular and intracellular proteins is involved in the lethal or nonlethal phenotypic variation of BrtlIV, a murine model for osteogenesis imperfecta. Proteomics 2007;7:1877-91.
The Consortium for Collagen Mutations in OI
The BEMB led an international consortium of connective tissue laboratories in assembling and analyzing a database of structural mutations in type I collagen causing OI. The consortium assembled over 830 mutations, including 682 glycine substitutions and 150 splice site defects. Genotype–phenotype modeling revealed different functional relationships for each chain of type I collagen. Glycine substitutions in the α1 chain have a generally more severe outcome, with 36 percent of substitutions resulting in a lethal phenotype. Substitutions by residues with charged or branched side chains have a lethal outcome in the majority of cases. Mutations in the amino quarter of the chain are non-lethal—even those involving residues with charged or branched side chains. We observed two stretches of exclusively lethal mutations in the carboxyl quarter of the chain. The two regions coincide with the Major Ligand Binding Regions (MLBRs) of several ligands on the collagen monomer, including integrins and fibronection. Glycine substitutions in the α2 chain have a more moderate outcome on average, with fewer than 20 percent of occurrences resulting in lethality. Substitutions by residues with charged side chains are predominantly lethal, as in α1, but valine, with a branched side chain, is lethal in 17 percent of occurrences in α2 (as compared with 73 percent in α1). As for α1, occurrences in the amino third of the chain are non-lethal. Thereafter, the lethal mutations occur in eight clusters that are regularly spaced along the chain. The distribution of lethal mutations in α2 continues to follow the pattern we previously described in the Regional Model for this chain, correctly predicting the phenotype of 86 percent of cases in α2 (Marini et al., J Biol Chem 1993;268:2667; Wang et al., J Biol Chem 1993;268:25162). The lethal regions coincide with the binding regions for matrix proteoglycans on the collagen fibril. Finally, splice site mutations lead to mild OI in a minority of cases. Most splice site mutations, even those in invariant positions, lead to significant dysplasia, suggesting that use of an alternative donor or acceptor generates translatable products that can be incorporated into matrix. Most mutations that lead to simple exon skipping have a severe or lethal outcome. The modeling provides a testable hypothesis for the mechanisms of OI.
Marini JC, Forlino A, Cabral WA, Barnes AM, San Antonio JD, Milgrom S, Hyland JC, Korkko J, Prockop DJ, De Paepe A, Couke P, Symoens S, Glorieux FH, Roughley PJ, Lund AM, Kuurila-Svahn K, Hartikka H, Cohn DH, Krakow D, Mottes M, Schwarze U, Chen D, Yang K, Kuslich C, Troendle J, Dalgleish R, Byers PH. Consortium for osteogenesis imperfecta mutations in the helical domain of type I collagen: regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans. Hum Mutat 2007;28:209-21.
Bisphosphonate treatment of children with types III and IV OI
We undertook the first randomized controlled trial of bisphosphonate in children with types III and IV OI. The aim was to test both primary skeletal gains (increased bone density and decreased fractures) and secondary gains (improved functional level and muscle strength and decreased pain) reported in observational trials. Children in the treatment group received pamidronate for 18 to 23 months and experienced improvement in vertebral parameters, including bone mineral density (BMD) z-scores, central vertebral height, and vertebral area. However, the increment in vertebral BMD tapered off after one to two years of treatment, and the treatment group did not experience a decrease in long-bone fractures. No functional effect was seen from bisphosphonate treatment, including no significant change in ambulation level, lower-extremity strength, or pain. Some patients reported increased endurance or decreased back pain, but most reported no perceptible changes. Hence, the changes previously reported in these parameters appear to have been a placebo effect in the uncontrolled trials. The data from our controlled trial were in accord with three other controlled trials conducted in the same time frame. We are now recommending that treatment of children with types III and IV OI with pamidronate be limited to one to two (or at most three) years, with subsequent follow-up of bone status. Furthermore, we are currently engaged in a dose comparison trial, using the dose from our first trial and a lower dose. The trial has now entered the data analysis phase; our hypothesis posits that the children will gain a comparable benefit from the lower dose with fewer detrimental effects.
Letocha AD, Cintas HL, Troendle JF, Reynolds JC, Cann CE, Chernoff EJ, Hill SC, Gerber LH, Marini JC. Controlled trial of pamidronate in children with type III and IV osteogenesis imperfecta confirms vertebral gains but not short-term functional improvement. J Bone Miner Res 2005;20:977-86.
Marini JC. Should children with osteogenesis imperfecta be treated with bisphosphonates? Nat Clin Pract Endrocrinol Metab 2006;2:14-5.
COLLABORATORS
David Eyre, PhD, University of Washington, Seattle, WA
Steven Goldstein, PhD, Universiy of Michigan, Ann Arbor, MI
Sergey Leikin, PhD, Section on Physical Biochemistry, NICHD, Bethesda, MD
Scott Paul, MD, Rehabilitation Medicine, NIH Clinical Center, Bethesda, MD
The OI Mutation Consortium
For further information, contact marinij@mail.nih.gov.
