The Section on Connective Tissue Disorders studies the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which primary collagen defects cause skeletal fragility and other significant connective tissue symptoms and then to apply the knowledge gained to the treatment of children with these conditions. An understanding of the interactions of mutant collagen molecules with the normal collagenous and noncollagenous components of matrix will also enhance our understanding of normal bone function and may yield insights applicable to the more common forms of osteoporosis. The section has recently focused on the development of a nonlethal animal model for OI with a classical collagen mutation. We have generated a nonlethal knock-in mouse, the Brittle mouse (Brtl), with a glycine substitution mutation in the a1(I) chain. The mouse will provide an excellent model for pharmacological treatment trials, for approaches to gene therapy suitable for dominant disorders, and for investigations of the skeletal matrix in OI.

The section conducts an integrated program of laboratory and clinical investigation. Children with types III and IV OI form a longitudinal study group. They are enrolled in age-appropriate clinical protocols for treatment with growth hormone or bisphosphonate, for comparative approaches to rehabilitation medicine, or for the natural history of the neurological and pulmonary complications of OI. In turn, skin and bone samples from our patients provide the material for mutation identification and investigations of collagen processing, fibrillogenesis, and osteoblast biology. This arrangement allows us to integrate data in a manner that is unique among OI research programs.

The Brtl Mouse Model for OI
Forlino, Ty, Bergwitz, Dogulu, Marini
We have generated a knock-in murine model for OI that carries a typical OI mutation in type I collagen under the control of the endogenous promoter. We have named the mouse Brittle (Brtl) and have introduced a gly349—›cys substitution into one col1a1 murine allele. The mutation was modeled on the defect present in one of our type IV OI patients. It is typical of the glycine substitution defects that characterize about 85 percent of the known type I collagen mutations.

The nonlethal Brtl mouse is an excellent model for type IV OI. Despite perinatal rib and long bone fractures, the nonlethal Brtl mouse evidences few such fractures after birth. The Brtl mouse is smaller than its wild-type littermates and is distinguished by bowed long bones, a flared rib cage, and a flattened calvarium. Alcian blue and alizarin red staining indicate general undermineralization. Biochemically, the mutant collagen is well expressed, and the a1(I) dimer band has the same electrophoretic mobility as collagen from the child with the same mutation. The substitution has a minimal effect on thermal stability. Histologically, long bones exhibit disorganized trabeculae, the calvarium is thin, and dentinogenesis imperfecta is present. Brtl is thus an excellent model for a wide range of studies, including testing of pharmacological agents, development of gene therapy, and studies of extracellular matrix and osteoblast biology. We are currently engaged in investigations of the effect of the collagen mutation on bone density, biomechanics, and histomorphometry. We are also involved in a treatment trial of bisphosphonate in the Brtl mouse and its wild-type littermates.

Ribozyme Approach to OI Gene Therapy
Bergwitz, Nishioka, Marini
We have taken a mutation suppression approach to gene therapy of the dominant negative connective tissue disorders. Suppression of the expression of the mutant collagen allele would, in principle, transform a structural collagen mutation with severe clinical consequences into a quantitative mutation with mild to undetectable clinical symptoms.

For our agent to achieve allele-specific suppression of the mutant transcript, we are using hammerhead ribozymes. We have previously shown that ribozyme cleavage was completely allele-specific in vitro (Grassi et al., Nucl Acids Res 1997;25:3451-3458). More recently, to demonstrate allele-specific cleavage in cells, we have used fibroblasts from a patient with OI whose mutation itself generates a novel ribozyme cleavage site (Dawson and Marini, 2000). In cells stably transfected with active ribozyme, we observed a 50 percent suppression of mutant collagen mRNA levels, as judged by quantitative competitive PCR. The extent of ribozyme suppression of normal collagen mRNA varied with the particular vector sequences in the ribozyme tail from no to 20 percent suppression. This antisense effect may be based on an altered cycling efficiency of ribozyme off the target transcript.

Study of Collagen Mutations Causing OI and Ehlers-Danlos Syndrome
Cabral, manto, Marini

Figure 5

Dark field microscopy and EM images of (A) normal fibrils (B) fibrils formed by collagen including monomers deleted for telopeptide binding site.

We have reported two interesting mutations in a1(I). A gly76—›glu substitution occurring in severe type III OI is the first nonlethal glutamic acid substitution reported in the a1(I) chain (Cabral et al., 2001). The mutation is illustrative of the markedly different role played by glutamic acid residues in the X position, where they are abundant and contribute to the natural staggered conformation of the collagen helix by interchain hydrogen bonding, and in the Gly position, where their size and charge are highly detrimental.

We have also investigated an exon 41 skipping defect in a1(I). This exon contains the entire sequence that, based on studies with synthetic peptides, is thought to be crucial for telopeptide binding in fibril formation. The absence of the telopeptide binding site from a portion of the collagen monomers results in significantly slower in vitro fibrillogenesis and abnormal fibril morphology (Figure). We showed that the telopeptide binding site is crucial for lateral growth of fibrils, since the resulting proband fibrils have increased length/diameter ratios (Cabral et al., in press).

We direct a portion of our laboratory effort to investigations of collagen mutations causing Ehlers-Danlos syndrome. Recently, mutations in the chains of type V collagen have been recognized as a cause of EDS. About 30 percent of cases of classical EDS have an apparent null allele of a1(V). We delineated a functional null a1(V) allele in EDS II (Bouma et al., 2001). Proband heterotrimer composition was unaltered from a1(V)2 a2(V) despite a1(V) chain haploinsufficiency. Dermal fibrils showed greater heterogeneity, though the same average diameter, than in controls. The null type V allele appears to cause clinical features similar to those seen with structural defects of type V collagen. The features differ from the type I collagen mutations in osteogenesis imperfecta, where null alleles cause a distinctly milder phenotype and may reflect the role of type V collagen in limiting fibril diameter.

Growth Hormone Treatment Trial of OI
Compeggie, Marini
We have been conducting a treatment trial of recombinant growth hormone (rGH) with our longitudinal population of children with types III and IV OI. Twenty-six children were treated with rGH for 12 to 18 months. We conducted the trial in recognition that extreme short stature is one of the cardinal features of osteogenesis imperfecta and has medical as well as social consequences. The mechanism of growth deficiency in OI remains unknown.

Of the 26 children who were treated, 14 increased their linear growth rate by 50 percent or more compared with untreated patients. Those children who had a positive response in terms of linear growth also showed positive changes at the bone level that were not seen in nonresponders. Only the responders experienced a significant increase in vertebral DEXA Z scores and a decrease in long bone fractures. The results of the iliac crest biopsy were particularly interesting. During the treatment year, only the responders achieved significant increases in parameters of bone structure and formation. They evidenced a rise in bone volume as a consequence of greater trabecular number. The bone formation rate was also significantly elevated. Although the cortical width did not increase, the higher number of trabeculae is consistent with a positive change in the quality of the bone rather than in simple bone growth. Nonresponders for linear growth did not have these positive changes in histology. A salient question is why some children did not respond. In this study, the baseline serum C-propeptide (PICP) levels were an excellent predictor of a positive response, suggesting that ability to secrete type I collagen may be a crucial variable and that responsiveness may reside in cell surface interactions with growth factors such as TGF-b.