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Genetic Disorders of Bone and Extracellular Matrix

• Joan C. Marini, MD, PhD, Chief, Section on Heritable Disorders of Bone and Extracellular Matrix
• Chi-Wing Chow, PhD, Staff Scientist
• Ying Liu, PhD, Biologist
• Heeseog Kang, PhD, Postdoctoral Fellow
• Smriti Aryal, PhD, Visiting Fellow
• Robert D. Carlson, BS, Postbaccalaureate Intramural Research Training Award Fellow
• TzeYin Tang, BS, Postbaccalaureate Intramural Research Training Award Fellow
• Ashita S. Vadlamudi, BS, Postbaccalaureate Intramural Research Training Award Fellow

In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders known as osteogenesis imperfecta (OI). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and other connective tissue symptoms and then to apply this knowledge to the treatment of children with these conditions. Recently, we identified several genes among the long-sought causes of recessive OI. Discoveries of defects in collagen modification have generated a new paradigm for collagen-related disorders of matrix. We established that structural defects in collagen cause dominant OI while deficiency of proteins that interact with OI for folding, post-translational modification, or processing cause recessive OI. Our challenge now is to understand the cellular and biochemical mechanisms of recessive OI. We also generated a knock-in murine model for OI with a classical collagen mutation as well as a murine model for recessive type IX OI and X-linked type XVIII OI and are using these models to study disease pathogenesis and the skeletal matrix of OI, the effects of pharmacological therapies, and approaches to gene therapy. Our clinical studies involve children with types III and IV OI, who form a longitudinal study group enrolled in age-appropriate clinical protocols for the treatment of their condition.

Mechanism of rare forms of osteogenesis imperfecta

OI type V is caused by a recurrent dominant mutation (c.-14C→T) in IFITM5, which encodes BRIL, a bone-restricted interferon-induced transmembrane protein–like protein most strongly expressed in osteoblasts, which plays a role in mineralization. Patients with type V OI have distinctive clinical manifestations with overactive bone mineralization and mesh-like lamellation on bone histology. We identified eight patients with the recurrent type V OI mutation and investigated their osteoblasts in culture. The mutant Bril transcripts and protein were stable and expressed at levels comparable to control. Both early and late markers of osteoblast differentiation are elevated in type V OI osteoblasts, including the osteoblast differentiation factor Runx2, alkaline phosphatase, bone sialoprotein, osteopontin, and osteocalcin. Mineralization was also elevated in type V OI osteoblasts, which occurs despite the seemingly paradoxical reduction in transcripts for type I collagen in mid to late differentiation. The studies demonstrated that a gain-of-function mechanism underlies type V OI and establish its collagen-related defect. We completed collaborative experiments with the labs of Nadja Fratzl-Zelman and Frank Rauch, focusing on the mineralization of bone tissue from patients with type V OI. qBEI of bone from untreated patients was used to determine BMDD (bone mineral density distribution) and lacunar density. As in collagen-based OI and consistent with the elevated mineralization seen in type V OI osteoblasts, the bone tissue mineralization was also significantly increased. The osteocyte lacunar density is elevated in association with immature mesh-like bone structure.

Recessive null mutations in SERPINF1, which encodes pigment epithelium–derived factor (PEDF), cause OI type VI. PEDF is well known as a potent anti-angiogenic factor. Type VI OI patients have no serum PEDF, elevated alkaline phosphatase (ALPL) as children, and bone histology with broad unmineralized osteoid and a fish-scale pattern. At first, types V and VI OI appear unconnected. However, we identified a patient with severe atypical type VI OI, whose osteoblasts displayed minimal secretion of PEDF, but whose SERPINF1 sequences were normal despite typical type VI OI bone histology. Surprisingly, exome sequencing on this proband and family members yielded a de novo mutation in IFITM5 in one proband allele, causing a p.S40L substitution in the BRIL intracellular domain. The IFITM5 transcript and BRIL protein level were normal in proband fibroblasts and osteoblasts. SERPINF1 expression and PEDF secretion were reduced in proband osteoblasts. In contrast, osteoblasts from a typical case of type V OI have elevated SERPINF1 expression and PEDF secretion during osteoblast differentiation. Together, the data suggest that BRIL and PEDF have a relationship that connects the genes for types V and VI OI and their roles in bone mineralization.

The endoplasmic reticulum (ER)–resident procollagen 3-hydroxylation complex is responsible for the 3-hydroxylation of type I collagen alpha1(I) chains. Deficiency of components of the collagen P3H (prolyl 3-hydroxylase) complex causes recessive OI. Thus, deficiency in CRTAP (cartilage-associated protein) causes type VII OI, while deficiency in P3H1 causes type VIII OI. Types VII and VIII OI are severe-to-lethal bone dysplasias that are clinically nearly indistinguishable because the two proteins are mutually protective.

For type VIII OI, we investigated bone and osteoblasts with collaborators Nadja Fratzl-Zelman and Cathleen Raggio. Collagen has near-absent 3-hydroxylation from both bone and dermis, demonstrating that P3H1 is the unique enzyme responsible for collagen 3-hydroxylation. Bone histomorphometry revealed reduced cortical width, very thin trabecular with patches of increased osteoid, although the overall osteoid surface was normal. Quantitative backscattered electron imaging (qBEI) showed increased mineralization of cortical and trabecular bone, as is typical of other OI types. However, the proportion of bone with low mineralization was higher in Type VIII bone than type VII, consistent with patchy osteoid in type VIII but not type VII.

The third member of the complex, Cyclophilin B (CyPB), encoded by PPIB, is an ER–resident peptidyl-prolyl cis-trans isomerase (PPIase), which functions both independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is the major PPIase catalyzing collagen folding. We characterized the first patient with deficiency of PPIB, which causes recessively inherited Type IX OI. Our group generated a Ppib knock-out (KO) mouse model that recapitulates the Type IX OI phenotype. Collagen from KO mouse tissue and cells is nearly lacking 3-hydroxylation. Intracellular collagen folding occurs more slowly in CyPB-null cells, supporting the enzyme's role as the rate-limiting step of folding. However, treatment of KO cells with the cyclophilin inhibitor cyclosporin A caused further delay in folding, providing support for the existence of a further collagen PPIase. We found that CyPB supports collagen lysyl hydroxylase (LH1) activity, demonstrating significantly reduced hydroxylation of the helical crosslinking residue K87, which directly affects both the extent and type of collagen intermolecular crosslinks in bone tissue. However, CyPB deficiency results in increased hydroxylation at telopeptide crosslinking sites in tendon, with moderate increase in glycosylation. CyPB interacts with all three LHs and has a site-specific impact on tissue organization. For example, the type I collagen in dentin of KO mice is under-hydroxylated in a different pattern than in bone, resulting in dentin defects. Also, in the brains of KO mice, absence of the chaperone CyPB increases aggregation of the prior protein PrP. Thus, post-translational modification if critically important in connective tissue formation.

Recessive type XIV OI is a moderately severe bone dysplasia caused by null mutations in TMEM38B, which encodes TRIC-B. TRIC-B forms a monovalent cation channel in the ER membrane that is thought to counterbalance IP₃R (inositol triphosphate receptor)–mediated calcium release from the ER to the cytoplasm. Using fibroblasts and osteoblasts from three independent probands, we found that TRIC-B was undetectable in their cells. Although TRIC-B is not a calcium channel, together with our collaborators Yoshi Yamada and Josh Zimmerberg, we showed that absence of TRIC-B results in reduced calcium flux from the ER and abnormal store-operated calcium entry, although ER steady-state calcium is normal. As expected, the disturbed calcium flux causes ER stress along the PERK pathway (involved in the unfolded protein response) and increased BiP, a regulator of ER stress. Disruption of intracellular calcium dynamics also alters the expression and activity of several collagen-modifying enzymes and chaperones in the ER. As a result, lysyl hydroxylation of the collagen helix by LH1 is reduced by 30% even though LH1 levels are increased and, conversely, hydroxylation of the telopeptide lysine by LH2 is increased, despite reduced levels of its PPIase FKBP65. Procollagen chain assembly is also delayed, likely through sequestration of protein disulfide isomerase (PDI) by calreticulin. A substantial proportion of the resulting misfolded collagen is retained in the cells, and any secreted lower-stability forms are not incorporated into matrix. These data support a role for TRIC-B in calcium homeostasis and directly connect TMEM38B defects to the collagen-related paradigm of OI. Other ER pathways are likely also disrupted by abnormal calcium flux in these cells, and redundancy for potassium and calcium flux likely modulates the impact of these defects on skeletal development.

In collaboration with Vorasuk Shotelersuk and Cecilia Giunta, we identified a new OI causative gene on the X-chromosome. This is the first type of OI with X-linked inheritance, and it causes a moderate to severe bone dysplasia with pre- and postnatal fractures of ribs and long bone, bowing of long bones, low bone density, kyphoscoliosis and pectal deformities, and short stature. The affected individuals have missense mutations in MBTPS2, which encodes the protein S2P. S2P is a transmembrane protein in the Golgi and is a critical component of regulated membrane proteolysis (RIP). In RIP, regulatory proteins are transported from the ER membrane to the Golgi in times of cell stress or sterol depletion, where they are sequentially cleaved by S1P/S2P to release activated N-terminal fragments that enter the nucleus and activate gene transcription. Mutant S2P protein is stable but has impaired RIP functioning, with deficient cleavage of the ER–stress transducers OASIS, ATF6, and SREBP. Furthermore, hydroxylation of the collagen residue K87 is reduced by half in proband bone, consistent with reduced LH in proband osteoblasts. Reduced collagen crosslinks presumably undermine bone strength. Also, proband osteoblasts have broadly defective differentiation. The mutations in MBTPS2 demonstrate that RIP plays a fundamental role in bone development as well as in cholesterol metabolism.

C-propeptide cleavage site mutations increase bone mineralization.

Type I procollagen is processed to mature collagen by the removal of both N- and C-terminal propeptides. The C-propeptide is cleaved at the Ala-Asp peptide bond between the telopeptide and the C-propeptide of each chain by C-proteinase/BMP-1. Probands with substitutions at any of the four cleavage site residues have a High Bone Mass form of OI, first reported by our lab in collaboration with Katarina Lindahl's group. The patients have elevated bone density DEXA Z-scores and, on bone histology, patchy unmineralized osteoid. The processing of the C-propeptide from collagen secreted by proband cells is delayed. We investigated mineralization with BMDD to show that, in the alpha2(I) cleavage site mutation, the bone had a uniformly higher mineral density, while in the alpha1(I) mutation, the average mineral density was markedly heterogeneous, with areas of either very high or low bone density.

To investigate the role of the C-propeptide in bone mineralization and developmental progression, we developed a knock-in murine model with a COL1A1 cleavage site mutation. Bone collagen fibrils showed a "barbed-wire" appearance consistent with the presence of the pC-collagen that was detected in extracts of bone from mutant mice, and with impaired collagen processing in vitro. Impaired C-propeptide processing affects skeletal size and biomechanics. The mice are smaller than wild-type litter mates. Their femora exhibit extreme brittleness on mechanical testing, as well as reduced fracture load. BMDD measurement on femora from 2, 6, and 12-month-old mice show significantly increased mineralization compared with wild-type (WT), which continues to increase in HBM (high bone mass) mice even after WT mineralization plateaus at 6 months. Serum markers of bone remodeling, PINP and TRAP, are significantly increased in HBM mice. Osteocyte density is reduced but lacunar area is increased. We are currently investigating osteoblasts, osteocytes and osteoclast in HBM mice.

Insights from the Brtl mouse model for OI

The Brtl mouse model for OI, generated by the Marini lab, is a knock-in mouse that contains a Gly349Cys substitution in the alpha1(I) chain. Brtl was modeled on a type IV OI child and accurately reproduces features of type IV OI. Brtl has provided important insights into both potential OI treatments and the mechanism of OI. In a treatment trial of the bisphosphonate alendronate in Brtl and wild-type (WT) littermates, bone density, bone volume, and trabecular number improved with treatment, as did load-to-fracture. However, detrimental side effects such as retained mineralized cartilage, reduced material properties, and altered osteoblast morphology occurred with treatment. The results reinforce the conclusion of the pediatric trial to limit the duration of bisphosphonate treatment.

We also collaborated with Kenneth Kozloff's group to investigate a potential anabolic therapy, sclerostin antibody (Scl-Ab), which stimulates osteoblasts via the canonical Wnt pathway. Scl-Ab stimulated bone formation in young Brtl mice and increased bone mass and load-to-fracture. Treatment with Scl-Ab caused no detrimental change in Brtl bone material properties. Nano-indentation studies indicating unchanged mineralization showed that the hyper-mineralization of bisphosphonate treatment did not occur. In addition, Scl-AB was successfully anabolic in adult Brtl mice, and may be a therapy for adult patients who have fewer treatment options. Because Scl-AB is a short-acting drug, we recently investigated sequential Scl-AB/bisphosphonate treatment. The study showed that administration of a single dose of bisphosphonate after Scl-AB cessation preserved anabolic gains from the Scl-AB treatment.

A third therapeutic trial involving Brtl approached allele-specific silencing of the col1a1 mutation, undertaken in collaboration with Antonella Forlino. Specific small interfering RNAs (siRNA) were evaluated ex vivo in Brtl fibroblasts for their effect on collagen transcripts and protein. A preferential reduction in mutant transcripts by about half was associated with a 40% decrease in mutant collagen chain.

Brtl also provided important information about the cytoskeletal organization in OI osteoblasts and their potential role in the phenotypic variability of OI. Abnormal cytoskeletal organization was demonstrated to occur only in lethal pups. Comparison of lethal and surviving Brtl pups' skin/bone and bone/skin hybrid networks highlighted three proteins involved in cytoskeletal organization: vimentin, stathmin, and coffin-1. The alterations were shown to affect osteoblast proliferation, collagen deposition, integrin, and TGF-beta signaling. The data open the possibility that cytoskeletal elements may be novel treatment targets for OI.

Two basic insights have emerged from Brtl studies. The hyper-mineralization of OI bone was previously thought to be a passive process. Altered levels for osteocyte transcripts involved in bone mineralization, such as Dmp1 and Sost1, demonstrated, however, the presence of an actively directed component. Second, the osteoclast is important to the OI phenotype, with elevated numbers and TRAP (tartrate-resistant acid phosphatase) staining of osteoclasts and precursors. Co-culture experiments with Brtl and WT mesenchymal stem cells (MSCs) and osteoclast precursors yielded elevated osteoclast numbers from WT or Brtl precursors grown with Brtl MSCs, but not with WT MSCs. The results indicate that an osteoblast product is necessary and sufficient for elevated osteoclast numbers and could provide an important target for treatment of OI.

Natural history and bisphosphonate treatment of children with types III and IV OI

We recently published the cardiopulmonary aspects of our natural history study on types III and IV OI. Longitudinal evaluations were completed in 23 children with type III OI and 23 children with type IV OI, who had pulmonary function tests every 1–2 years. Compared with size-matched children, our patients showed a significant decline over time in pulmonary function, including lung volumes and flow rates. The decline was worse in the 36 children with scoliosis (average curve 25 degrees) but also occurred in 20 participants without scoliosis, who had declining function with restrictive disease, suggesting that the pulmonary dysfunction of OI is attributable to a primary defect in the lung related to structurally abnormal collagen. The studies are important because pulmonary issues are the most prevalent cause of morbidity and mortality in OI. Affected individuals should seek anticipatory evaluation and treatment.

Currently, OI–specific growth curves are not available, despite the fact that short stature is one of the cardinal features of OI. We assembled our longitudinal growth data on 100 children with type III and IV OI to generate sex- and type-specific growth curves for OI. The data show that gender and OI type have significant effects on height in OI, but not the collagen chain in which the causative mutation is located. Boys are taller than girls and type IV OI boys and girls are taller than type III. Weight differs by OI type, but not by gender or mutant collagen chain. Interestingly, head circumference does not differ by gender, OI type, or collagen mutation. Imposition of OI height curves on standard CDC curves reveals an overlapping of type III and IV percentiles and the absence of a growth spurt in type III OI. Standard growth curves for OI will be of great value to primary caregivers and families and will provide a baseline for treatment trials.

Our randomized controlled trial of bisphosphonate in children with types III and IV OI was the first randomized bisphosphonate trial for OI in the United States. It examined direct skeletal and secondary gains reported in uncontrolled trials. For skeletal outcomes, we found increased BMD (bone mineral density) Z-scores and improved vertebral geometry. We noted that vertebral BMD improvement tapered off after two years' treatment. Our treatment group did not experience fewer long-bone fractures, coinciding with the lack of improvement or equivocal improvement in fractures in other controlled trials. The BEMB controlled trial did not support the claims for improvement in ambulation level, lower-extremity strength, or alleviation of pain, suggesting these were placebo effects in observational trials. Our current recommendation is for treatment for 2–3 years, with subsequent follow-up of bone status. We are now engaged in a dose-comparison trial, comparing the dose from our first trial with a lower dose, achieved by increasing the cycle interval at the same dose/kg/cycle. Given the decade-long half-life and side effects of bisphosphonate on normal as well as dysplastic bone, including decline in the quality of bone material, it is important to determine the lowest cumulative dose that will provide vertebral benefits. Preliminary analysis indicates that OI children obtain comparable benefits from lower and higher pamidronate doses.

OI Mutation Consortium

The BEMB assembled and leads an international consortium of connective tissue laboratories for the compilation and analysis of a database of mutations in type I collagen that cause OI. When the first analysis of the database was published in 2007, it listed over 830 mutations, including 682 glycine substitutions and 150 splice-site defects. Genotype-phenotype modeling revealed distinct functions for each alpha chain of type I collagen, including the occurrence of exclusively lethal mutations in the Major Ligand Binding Regions (MLBR) of the alpha1(I) chain on the collagen monomer and the overlapping of the regularly spaced clusters of lethal mutations along the alpha2(I) chain with the proteoglycan binding sites on the collagen fibril. The modeling for alpha2(I) supports the Regional Model first proposed by the BEMB 20 years ago, which now correctly predicts 86% of clinical outcomes. The Consortium Database has provided the basis for others to model functional domains for interaction of fibrils with cells and matrix. The Consortium Database now contains over 1,570 mutations from nine international laboratories.

Additional Funding

• NICHD DIR Director's Award

Publications

1. Forlino A, Marini JC. Osteogenesis Imperfecta: new forms reveal novel gene functions in bone development and unexpected protein-protein interactions. Lancet 387 2015 387:1657-1671.
2. Lindert U, Cabral WA, Ausavarat S, Tongkobpetch S, Ludin K, Barnes AM, Yeetong P, Weis M, Krabichler B, Srichomthong C, Makareeva E, Janecke A, Kennerknect I, Leikin S, Rothlisberger B, Rohrbach M, Eyre DR, Suphapecetiporn K, Giunta C, Marini JC, Shotelersuk V. MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-osteogenesis imperfecta. Nat Commun 2016 7:11920.
3. Cabral WA, Ishikawa M, Garten M, Makareeva EN, Sargent BM, Weis MA, Barnes AM, Webb EA, Shaw NJ, Ala-Kokko L, Lacbawan FL, Högler W, Leikin S, Blank PS, Zimmerberg J, Erye DR, Yamada Y, Marini JC. Absence of the ER cation channel TMEM38B/TRIC-B disrupts intracellular calcium homeostasis and dysregulates collagen synthesis in recessive osteogenesis imperfecta. PloS Genetics 2016 12(7):e1006156.
4. Fratzl-Zelman N, Barnes AM, Weis MA, Carter E, Hefferan TE, Perino G, Chang W, Smith PA, Roschger P, Klaushofer K, Glorieux F, Eyre DR, Raggio C, Rauch F, Marini JC. Non-lethal type VIII osteogenesis imperfecta has elevated bone matrix mineralization. J Clin Endorinol Metab 2016 101(9):3516-3525.
5. Webb EA, Balasubramanian M, Fratzl-Zelamn N, Wayne WA, Titheradge H, Alsaedi A, Saraff V, Cole T, Stewart S, Crabtree N, Sargent BM, Gamsjaeger S, Paschalis EP, Rischger P, Klaushofer K, Marini JC, Shaw NJ, Högler W. Skeletal and bone material phenotype associated with osteogenesis imperfect due to mutations in TMEM38B suggests a novel patho-mechanism leading to increased bone fragility in humans. J Clin Endocrinol Metab 2017 102(6):2019-2028.

Collaborators

• Patricia Becerra, PhD, Laboratory of Retinal Cell and Molecular Biology, NEI, Bethesda, MD
• Paul Coucke, PhD, Universitair Ziekenhuis Gent, Ghent, Belgium
• Anne De Paepe, MD, PhD, Universitair Ziekenhuis Gent, Ghent, Belgium
• David Eyre, PhD, University of Washington, Seattle, WA
• Antonella Forlino, PhD, Università degli Studi di Pavia, Pavia, Italy
• Nadja Fratzl-Zelman, PhD, Ludwig Boltzmann-Institut für Osteologie, Hanusch Krankenhaus der WGKK und Unfallkrankenhaus Meidling, Vienna, Austria
• Cecilia Giunta, PhD, Kinderspital Zürich, Zürich, Switzerland
• Wolfgang Högler, MD, DSc, FRCPCH, Birmingham Children's Hospital NHS Foundation Trust, Birmingham, United Kingdom
• Kenneth Kozloff, PhD, University of Michigan, Ann Arbor, MI
• Sergey Leikin, PhD, Section on Physical Biochemistry, NICHD, Bethesda, MD
• Katarina Lindahl, MD, Uppsala Universitet, Uppsala, Sweden
• Joseph Orgel, PhD, Illinois Institute of Technology, Chicago, IL
• Scott Paul, MD, Rehabilitation Medicine, NIH Clinical Center, Bethesda, MD
• Cathleen L. Raggio, MD, Weill Medical College of Cornell University, New York, NY
• Frank Rauch, MD, Shriners Hospital for Children, Montreal, Canada
• James San Antonio, PhD, Jefferson University, Philadelphia, PA
• Vorasuk Shotelersuk, MD, FABMG, King Chulalongkorn Memorial Hospital, Bangkok, Thailand
• Brant Weinstein, PhD, Section on Vertebrate Organogenesis, NICHD, Bethesda, MD
• Yoshi Yamada, PhD, Molecular Biology Section, NIDCR, Bethesda, MD
• Mitsuo Yamauchi, PhD, University of North Carolina, Chapel Hill, NC
• Joshua Zimmerberg, MD, PhD, Section on Cellular and Membrane Biophysics, NICHD, Bethesda, MD
• The OI Mutation Consortium, NICHD, Bethesda, MD

Contact

For more information, email marinij@mail.nih.gov.

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