Type I Collagen Triplet Duplication Mutation in Lethal Osteogenesis Imperfecta Shifts Register of Alpha Chains Throughout the Helix, and Disrupts Incorporation of Mutant Helices into Fibrils and Extracellular Matrix

triple helix. A rare deletion of one or two Gly-X-Y triplets. These mutations shift the register of collagen chains with respect to each other in the helix but do not interrupt the triplet sequence, and they have severe clinical consequences. We investigated the effect of shifting the register of the collagen helix by a single Gly-X-Y triplet on collagen assembly, stability and incorporation into fibrils and matrix. These studies utilized a triplet duplication in COL1A1 exon 44 which occurred in the cDNA and gDNA of two siblings with lethal OI. The normal allele encodes three identical Gly-Ala-Hyp triplets at aa 868-876, while the mutant allele encodes four. The register shift delays helix formation, causing overmodification. Differential scanning calorimetry yielded a decrease in Tm of 2 ° C for helices with one mutant chain, and a 6 ° C decrease in helices with two mutant chains. An in vitro binary co-processing assay of N-proteinase cleavage demonstrated that procollagen with the triplet duplication has slower N-propeptide cleavage than in normal controls or procollagen with pro α 1(I)G832S, G898S or G997S substitutions, showing that the register shift persists through the entire helix. The register shift disrupts incorporation of mutant collagen into fibrils and matrix. Proband fibrils formed inefficiently in vitro and contained only normal helices and helices with a single mutant chain. to incorporate into extracellular matrix deposited in culture by post-confluent control and proband fibroblasts. Matrices were collected at 24 hour intervals and collagens were extracted by pepsin digestion. Samples were analyzed by 6% SDS-urea-PAGE and quantitated by densitometry of autoradiograms. The experiment was done in triplicate; representative gels are shown.


INTRODUCTION
Osteogenesis imperfecta (OI) is an autosomal dominant disorder of connective tissue. Its most significant clinical feature is skeletal fragility, causing the bones of affected individuals to be susceptible to fracture from minimal trauma or non-traumatic impact (1). Other symptoms of OI include short stature, blue sclerae, joint laxity, dentinogenesis imperfecta and hearing loss (2).
The severity of OI varies widely, ranging from perinatal lethal to barely detectable, as delineated by the Sillence classification (3).
The full clinical spectrum of OI is caused by defects in the structure or synthesis of type I collagen, the most abundant protein of the extracellular matrix of bone, skin and tendon (4,5).
Defects in COL1A1 which result in the synthesis of half the normal amount of collagen cause the mildest form of the disease (OI type I) (6). The clinically significant forms of OI (OI types II, III and IV) are caused by structural defects in either the α1(I) or α2(I) chains. Over 250 such mutations have now been delineated in individuals with OI (7,8). The overwhelming majority (about 85%) are point mutations which result in the substitution of the glycine residue in a typical collagen tripeptide, Gly-X-Y, by another amino acid. Substitution mutations are thought to exert a detrimental effect on collagen function because their side chains are larger than that of glycine and cause local interference with the folding of the triple helix (9). A smaller fraction (about 10%) of collagen mutations result in single exon skipping. These mutations maintain the Gly-X-Y triplet pattern, but may cause local looping out of chains in the triple helix (4). An even less common set of structural mutations is located in the C-terminal propeptide. Since the C-propeptide is cleaved from the mature collagen molecule before incorporation into fibrils, the mutant region of the chain is not incorporated into matrix. Instead, they are thought to exert their effect by delaying the incorporation of the mutant chains into collagen trimer (10).
An additional rare and interesting group of mutations consists of deletions or duplications of the codons for one or two Gly-X-Y triplets. Only ten cases of single triplet deletion or duplication have been reported (11)(12)(13)(14), including 5 deletions and 3 duplications in the α1(I) chain and 2 deletions in the α2(I) chain. There are also four cases involving deletion or duplication of two triplets, all in the α2(I) chain (14,15). These mutations are of special interest because they must disrupt collagen functioning by a quite different mechanism than that initiated by glycine substitutions. A priori, one might have expected mild functional defects from mutations that shift the Gly-X-Y register of the collagen helix by a single triplet unit, rather than interrupt helix folding in the manner of a glycine substitution. In fact, small register shifts cause a lethal or severe phenotype. Determinations of helix stability and procollagen processing were reported for two lethal cases with deletion of one of the three Gly-Ala-Hyp triplets at aa 868-876 in αl(I) exon 44 (11,12). These deletions decreased collagen helix Tm by only 0-1 °C.
Processing of proband collagen by pericellular enzymes and purified N-proteinase was indistinguishable from normal, as was cleavage by vertebral collagenase. The processing data led investigators to propose that there was limited propagation of the register shift toward the Nterminal end of the procollagen trimer.
We report here a single triplet duplication in α1(I)E44 in siblings with lethal type II OI.
Determinations of thermal stability and N-protease cleavage indicate that the register shift is propagated the full length of the collagen helix. In vitro fibrillogenesis and matrix deposition studies demonstrate that presence of the register shift impairs incorporation into fibrils and cross-linking into matrix. These studies provide new insight into the mechanisms of register shift mutations in collagen disorders.

Clinical Cases -
The probands were the male and female offspring of a 22 yr old G2P1 mother and a 25 yr old father, born at 32 and 37 weeks gestation, respectively. Prenatal ultrasound at 18-22 wks gestation detected a short limbed skeletal dysplasia in each child. The male child was delivered vaginally with forceps due to breech presentation. Weight was appropriate for age (2013 gms) but crown to heel length was 38 cm (50% for 28 wks gestation). At delivery, he had a soft skull with an anterior laceration , draining blood and CSF, a narrow chest and bowed extremities. He died one hour after birth. The female child was born by SVD. Birth weight was appropriate for age (2770 gms) but length was short (43 cm; 50% for 32 wks gestation).
Deformities noted at birth included a soft cranium with mineralized bone only on lateral portions of the skull, blue sclerae, a high arched palate and a narrow chest. Extremities had rhizomelic shortening and bowing and were abducted into an extreme frog-legged position.
Radiograms showed multiple fractures of ribs and all long bones. The infant died at age one month of respiratory insufficiency.
Mutation Detection and Sequencing -Fibroblast RNA was isolated using Tri-Reagent Matrix Deposition Confluent fibroblasts were stimulated every other day for 9 days with 100 µg/ml ascorbic acid, then incubated for 24 h with 260 µCi/ml [ 3 H]-proline in serum-free medium.
Procollagens in media were precipitated with ammonium sulfate. Matrix collagens were serially extracted, as described (22). In brief, newly synthesized collagens were extracted for 24 h with neutral salt (0.15 M NaCl in 50 mM Tris-HCl, pH 7.5). Collagens with acid-labile crosslinks were extracted for 24 h with 0.5 M acetic acid. Collagens with mature crosslinks were extracted by pepsin digestion (0.1 mg/ml) for 24 h. All matrix fractions were precipited with 2M NaCl.
Matrix Chase -Confluent fibroblasts were stimulated every other day for 9 days with 100 µg/ml ascorbic acid, incubated for 48 h with 260 µCi/ml of [ 3 H]-proline in serum-free medium, then chased with fresh DMEM containing 10% fetal bovine serum and 10 mM non-radioactive proline. Individual cultures were harvested at 24 h intervals for 5 days and the matrix layer was processed with protease inhibitors, as described (22). Matrix extracts were resuspended in 0.5 M acetic acid and digested overnight with pepsin. Collagens were precipitated with 2M NaCl. Experiments were repeated in triplicate.

Preparation of fluorescent labelled procollagen -
Preparation of full-length collagen by N-and C-proteinase cleavage -Ammonium sulfate procollagen precipitate was doped with 10% Cy5-labelled procollagen. The mixture was chromatographed on two 1.6 x 5 cm columns of DEAE cellulose (DE52, Whatman) as described (26)(27)(28). Aliquots from fibers and supernatant were labelled with Cy5 and analyzed on 3-8% Tris-Acetate or 4-12% Bis-Tris mini-gels (Invitrogen). RT-PCR screening of α1(I) cDNA localized the mismatch to exon 44 ( Fig. 2A). Both normal and more slowly migrating products were detected in the probands' cDNA. The more slowly migrating product was faintly visible in the mother's sample. This electrophoretically slower product was shown to be a heteroduplex of normal and mutant fragments. The small fraction of mutant α1(I) transcripts in maternal cells can be easily visualized because of the sensitivity of heteroduplex analysis for structurally distinct products. The localization of the collagen mutation to exon 44 was confirmed by PCR amplification of genomic DNA (Fig. 2B).

Collagen Protein Analysis
Sequencing of subclones of proband cDNA exons 43-45 and proband and mother gDNA intron 43-intron 44 revealed the same relatively unusual type of collagen mutation, confirming the mother as a mosaic carrier. The mutant allele has a 9-bp insertion (5'-GGT GCT CCT-3'), coding exactly for an extra Gly-Ala-Hyp triplet (Fig. 3). The insertion is a duplication in a highly repetitive region. The normal allele has two identical 9-bp sequences coding for aa 868-873, and an adjacent 9-bp that differs by only one nucleotide. The mutant allele has three of the identical 9-bp units.
Effect of register shift on collagen thermal stability -Differential scanning calorimetry thermograms of proband collagen (OI-INS) were done at acidic and neutral pH (Fig. 4). Since mutant collagen has three species, α1 2 α2, α1(ins)α1α2, and α1(ins) 2 α2, up to three different peaks may be expected on thermograms. All three peaks are clearly visible at acidic pH, Matrix deposition -The incorporation of proband and control collagen into matrix was compared by serial extractions of the matrix deposited by cultured cells (Fig. 6). Matrix chase -A pulse-chase experiment examined the stability of collagen deposited in matrix by cultured proband and control cells. Matrix stability was not significantly altered (Fig.   7). The proband's normal and overmodified α1(I) chains could not be quantitated separately, but an equivalent proportion of overmodified α1(I) chains is visible in each proband samples.

DISCUSSION
We have described here a novel single triplet duplication in the type I collagen α1(I) chain and its functional consequences for helix formation and fibrillogenesis. The mutation occurs in two siblings with lethal type II osteogenesis imperfecta. Their mother is a mosaic carrier with a low percentage of heterozygous fibroblasts and leukocytes, 10% and 15%, respectively (Cabral and Marini, unpublished data). Her clinical history and physical exam are entirely normal .
The mutant COL1A1 allele has a 9-bp duplication in exon 44, which has a highly repetitive sequence. In the normal allele, there are two consecutive 5'-GGT GCT CCT-3' units at nt 3255-3272, followed by 9 bp that differ by a single nucleotide, 5'-GGT GCC CCT-3'. These The delay in cross-linking of the triplet duplication is not seen with α2(I)∆E16 collagen (36) ,which causes a larger 6 triplet register shift and is more likely to realign by "looping out" of the normal chains than to propagate the register shift along the full helix (Cabral and Marini, unpublished data).
Proband mature matrix has a turnover that is comparable to normal, reflecting both its  in both probands' (lanes 4,5) and, to a lesser extent, in the mother's samples (lane 6). B, screening of genomic DNA from control and parental leukocytes and proband fibroblasts.