Hum Genet (1991) 87 : 167-172

9 Springer-Verlag1991

Cysteine in the triple helical domain of the proa2(1) chain of type-I collagen in nonlethal forms of osteogenesis imperfecta Daniel H . Cohn I and Peter H . Byers 2

1Division of Medical Genetics, SSB-3, Cedars-SinaiMedicalCenter, 8700 BeverlyBoulevard, Los Angeles, CA 90048, and Department of Pediatrics, Schoolof Medicine, Universityof Californiaat Los Angeles, Los Angeles, California,USA 2Department of Pathologyand Department of Medicine and Center for Inherited Disease, Universityof Washington, Seattle, WA 98195, USA Received June 29, 1990 / Revised August 1, 1990 Summary. To determine if some individuals with deforming varieties of osteogenesis imperfecta (OI) carry point mutations in the COL1A2 gene of type-I collagen, we examined collagens synthesized by cell strains from affected individuals for the presence of cysteine in the triple helical domain of the a2(I) chain, a domain from which it is normally excluded. We identified 4 individuals out of 60 whose cells synthesized a population of a2(I) chains with a cysteine residue in the triple helix. The clinical differences among the affected individuals and the heterogeneity in the locations of the cysteine residues suggest that the position of the substitution within the chain is important in determining the clinical phenotype. These data confirm that individuals with nonlethal OI may commonly harbor defects in the COLIA2 gene, and suggest that many of the defects are substitutions for glycine residues in the a2(I) triple helical domain.

Introduction

Osteogenesis imperfecta (OI) is a heterogeneous connective tissue disorder characterized by bone fragility (Sillence et al. 1979, 1984; Smith et al. 1983). The clinical spectrum of the disease ranges from a phenotype that is lethal in the perinatal period (OI type II) to very mild, and in some cases subclinical phenotypes (OI type IV and OI type I; Sillence et al. 1979). Most forms of OI result from defects in the synthesis or structure of the chains of type-I collagen (Byers and Bonadio 1985; Byers 1989). Two major classes of type-I collagen gene mutations have been observed in individuals with OI phenotypes: mutations that affect the amount of type-I collagen synthesized (quantitative defects) and mutations that alter Offprint requests to: D.H. Cohn, Division of Medical Genetics, SSB-3, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA

the structure of type-I collagen chains (qualitative defects). More than 85% of those affected with OI are heterozygous for a dominant mutation in either the COL1A1 or COL1A2 genes that encode the proal(I) and proa2(I) chains of type-I procollagen, respectively (Wenstrup et al. 1990a). The mildest form of OI, OI type I, generally results from mutations that decrease the amount of type-I procollagen synthesized but do not alter the structure of the molecules secreted (Barsh et al. 1982; Rowe et al. 1985; Wenstrup et al. 1990b; Willing et al. 1990; also see Byers 1989 for review). In contrast, OI type II, OI type III, and OI type IV generally result from mutations that alter the structure of the type-I procollagen synthesized (Steinmann et al. 1984; Bonadio and Byers 1985; Wenstrup et al. 1986, 1990b; Byers et al. 1988). Three classes of mutations (point mutations, splicing mutations, and multiexon deletions or insertions) are the most commonly encountered types. Of these, point mutations, which generally result in substitutions for glycine residues in the triple helical domain of either the cd(I) or a2(I) chain, are the most frequent (reviewed by Byers 1990). Analysis of families demonstrated linkage of the mildto-moderate OI-type-IV phenotype to polymorphic markers in the COL1A2 gene in most families (Sykes et al, 1986, 1990; Tsipouras et al. 1984), but the OI-type-IV phenotype can result from mutations in the COLIA1 gene (deVries and deWet 1986; Marini et al. 1989). Biochemical and molecular studies have demonstrated that single exon deletions in the a2(I) chain (Kuivaniemi et al. 1988a; Wenstrup et al. 1990a) or point mutations that result in substitution for a glycine residue in the triple helical domain of a2(I) (e.g., arginine for glycine at position 1012; Wenstrup et al. 1988) can produce the OItype-IV phenotype. The experimental evidence that substitution for a triple helical glycine residue in the a2(I) chain can produce mild to moderately severe OI phenotypes led us to try to determine if deforming varieties of OI could commonly result from point mutations in the COLIA2 gene. Cys-

168 t e i n e is e x c l u d e d f r o m t h e t r i p l e helix o f the a l ( I ) a n d ct2(I) chains o f t y p e - I c o l l a g e n a n d is a m o n g the a m i n o acid substitutions t h a t c o u l d result f r o m single n u c l e o t i d e c h a n g e s in glycine c o d o n s . W e i d e n t i f i e d 4 cell strains o u t o f 60 cell strains f r o m i n d i v i d u a l s with n o n l e t h a l O I p h e n o t y p e s t h a t c o n t a i n e d c y s t e i n e in t h e triple helix o f t h e a 2 ( I ) chain. T h e s e d a t a i n d i c a t e t h a t m u t a t i o n s that r e s u l t in s u b s t i t u t i o n s for glycine r e s i d u e s in the t r i p l e helical d o m a i n o f t h e a 2 ( I ) chain a r e r e s p o n s i b l e for some deforming varieties of OI. The data further argue t h a t t h e d o m a i n o f t h e cL2(I) c h a i n in which t h e substitution occurs is an i m p o r t a n t d e t e r m i n a n t o f the clinical phenotype.

Materials and methods Clinical summary Patient A (reference number 86-148). The proband is the second of two affected daughters born to an affected mother. The mother (86-149) is the only daughter of an affected father (86-150) who, on the basis of clinical information, was the first affected individual in his family. The proband's birthweight was 61b 13 oz, length was 18 in., and the left hip was dislocated at birth. Fracture history is significant for fractures of the left tibia at 2 years of age and the left humerus at 3 and again at 8 years of age. The affected sister (86147) was 6 lb 2 oz at birth and 18 in. long. At 3 years of age she suffered a fracture of the hip, and at 4 fractured the left femur. The femur was fractured in the same place 2 months later and again 1 year later and was surgically repaired by rodding. In the family the phenotype is characterized by mild short stature and moderate fracture frequency with little deformity. Patient B (85-037). The patient is the only affected son of an affected father (85-038) from a large family with a history of OI. Birthweight was 41b l o z , length was 16 in., the calvarium was noted to be soft with a large anterior fontanelle, and the femurs and tibias were markedly bowed. At 2 weeks of age a skeletal survey showed generalized demineralization, bowing of the femurs, rib deformities, and abnormalities of the metaphyses of the long bones. Disease in this family is similar to that in family A but more variable, with some obligate carriers having no history of fractures and other affected individuals with progressive deforming disease. However, even the most severely affected individuals are more mildly affected than is patient C (see below). Patient C (86-185). The patient is the first child of clinically normal parents. Birth weight was 4 lb 6 oz, length 16.4 in., a wide anterior fontanelle was noted, eyes were prominent, and there was marked shortness and bowing of the upper and lower limbs. Radiographs showed marked generalized demineralization and Wormian bones. Bowing of the radii and femurs and fractures of the left tibia and fibula were also apparent. Disease in the affected individual is severe, and falls between OI type IV and OI type III. Patient D (86-044). This individual was initially ascertained because he had fathered two infants with lethal OI born to separate spouses. He had had no fractures and was 5'3" tall. All other male family members were above 5'8" in height and none had a significant fracture history. Cells from the parents of the identified individual and from his three siblings were also analyzed. No cells were available from either affected infant.

Growth and labeling of cells Dermal punch biopsies were obtained and fibroblasts grown in Dulbecco-Vogt Modified Eagle Medium (DMEM, Grand Island Biologicals) using previously described conditions (Bonadio and Byers 1985). Fibroblastic cells from chorionic villus biopsies were

grown in Chang medium. Proteins were labeled by incubation of the cells with 2,3,4,5-[3H]proline or [35S]cysteine (Amersham) under previously described conditions (Bonadio and Byers 1985; Bonadio et al. 1985). Proteins from the medium were harvested in the presence of protease inhibitors and concentrated by ethanol precipitation in the presence of carrier collagen (Sigma). Digestion of procollagen with pepsin (Boehringer-Mannheim) was carried out for 16h at 4~ in 0.5M acetic acid adjusted to pH 2.0 with HC1. The reaction was stopped with pepstatin and the samples lyophilized. Following dialysis into the appropriate buffer, collagens were cleaved with mammalian collagenase (Stricklin et al. 1978), generously provided by Eugene Bauer, Stanford University.

Analysis of collagens Collagens were separated under nonreducing conditions in 5% polyacrylamide slab gels containing sodium dodecyl sulfate (SDSPAGE; Laemmli 1970) and 2M urea. Collagens cleaved with mammalian collagenase were separated under the same conditions in 10% polyacrylamide gels. Collagens separated in one dimension were cleaved in the gel with cyanogen bromide and the resulting peptides were then separated in a second dimension gel (Bonadio et al. 1985).

Results Identification and local&ation of cysteine in the ~2(I) chain Cell strains f r o m 60 i n d i v i d u a l s with n o n l e t h a l f o r m s of O I w e r e s e l e c t e d for s t u d y if t h e y s y n t h e s i z e d a n d sec r e t e d b o t h a n o r m a l a n d an o v e r m o d i f i e d p o p u l a t i o n o f t y p e - I p r o c o l l a g e n m o l e c u l e s . T h e cells w e r e l a b e l e d with [35S]cysteine, the c y s t e i n e - c o n t a i n i n g p r o p e p t i d e s f r o m t h e chains o f the t y p e - I p r o c o l l a g e n s w e r e r e m o v e d by digestion with p e p s i n , a n d the a chains f r o m the m e d i u m w e r e s e p a r a t e d b y S D S - P A G E . O f t h e 60 cell strains 4 s y n t h e s i z e d a p e p s i n - r e s i s t a n t p r o t e i n l a b e l e d with [35S]cysteine that m i g r a t e d in the p o s i t i o n e x p e c t e d for an o v e r m o d i f i e d cL2(I) chain (Fig. 1). T o confirm that the l a b e l e d p r o t e i n for e a c h cell strain c o r r e s p o n d e d to the ct2(I) chain a n d to d e t e r m i n e t h e app r o x i m a t e l o c a t i o n o f t h e cysteine r e s i d u e , we i d e n t i f i e d the ~2(I) c y a n o g e n b r o m i d e p e p t i d e in which the [35S]cys-

Fig. 1. Analysis of pepsin-digested collagens from the medium of fibroblasts derived from individuals with nonlethal osteogenesis imperfecta (OI). Cell strains were labeled with [35S]cysteine. A [3H]proline-labeled control marks the positions of the a(I) and u2(I) chains. Lane 2 is a negative control using a cell strain derived from a normal individual

169

Fig. 2A-C. Localization of the cysteine residues within the ct2(I) chain. A Cyanogen bromide (CB) peptides of cysteine-containing a2(I) chains. [35S]cysteine-labeled collagens were separated in a first dimension, lanes were resected from the gel and collagens were digested in the gel slice with CNBr and separated in a second dimension. [3H]proline-labeled collagens processed in parallel were used to mark the positions of the a2(I) CB peptide fragments. Arrows indicate the positions of the cysteine-containing CB peptide fragments. B Digestion of cysteine-labeled collagens with mammalian collagenase. [3H]proline-labeled collagens processed in parallel mark the positions of the al(I) and tt2(I) collagenase fragments identified to the left of the figure. C Locations of the cysteine residues for all four cell strains are summarized on the line diagram

-

"86- ] 85

86-14586-148 ~t Iv

A

B

Fig, 3A-D. Pedigrees of the individuals identified as carrying a cysteine substitution in the a2(I) chain. Arrows mark the probands. Filled-in symbols indicate individuals known to have OI. Hatched symbols indicate individuals thought to have OI based on descriptive evidence. Asterisks indicate individuals whose cells were assayed for cysteine-containing collagens

teine label was located (Fig. 2A). For the cell strain from patient C the cysteine was placed within a2CB4 (triple helical residues 6-327) and in the other 3 cell strains into a2CB3-5 (residues 357-1014). Digestion of cysteine-labeled collagens with m a m m a l i a n collagenase to cleave the a chains into the A fragment (residue 1-775) and B

C

D

fragment (776-1014) placed the cysteine within the A fragment in the latter 3 cell strains (Fig. 2B). Family studies Pedigrees for each of the probands are shown in Fig. 3 (see Materials and methods for a complete clinical description). All affected m e m b e r s of the families of patients A and B (Fig. 3B) contained the a2(I) cysteine (see pedigrees). Cells derived f r o m the parents of patient C (Fig. 3C) did not contain the a2(I) cysteine, and molecules synthesized by parental cells were not overmodified (data not shown). Thus the affected individual carries a new dominant mutation.

170

Fig. 4. Prenatal diagnosis for family D (proband is 86-044). Labeled collagens are shown. [3H]proline-labeled control (lane 1) marks the positions of the ctl(I) and ct2(I) chains. The remaining lanes contain [35S]cysteine-labeledcollagens secreted into the medium from a fibroblast control (lane 2), chorionic villus sample control (lane 3), prenatal diagnosis chorionic villus sample (lane 4), fibroblasts from individual 86-044 (lane 5), and fibroblasts from the pregnant mother (lane 6). The arrow marks the cysteine-labeled ct2(I) chain synthesized by cells from the affected father

The only clinical manifestation of OI in patient D (Fig. 3D) is that he is of short stature. H e was ascertained because he has fathered two children with the perinatal lethal form of OI (OI type II), each with a different spouse. We find the occurrence of lethal OI in this pedigree paradoxical. Cells were not cultured from either infant so we do not know if the infants inherited the cysteine-containing allele from their father. The parents of patient D are clinically normal and their cells do not synthesize the cysteine-containing a2(I) chain (data not shown), indicating that his mutation is new. Prenatal diagnosis Shortly after identification of the abnormal presence of cysteine in the triple helical domain of ~2(I) in collagens synthesized by cells from patient D we were asked to perform prenatal diagnosis and agreed to assay collagens synthesized by chorionic villus cells from the fetus bisied at 10 weeks gestation. Cells were labeled with ]cysteine and pepsin-digested collagens from the medium were analyzed by S D S - P A G E (Fig. 4). Cells from the fetus were indistinguishable from control cells, indicating that it had not inherited the cysteine-encoding allele from the father. On the basis of this result and the absence of overmodification of type-I collagen molecules (data not shown), we concluded that it was unlikely that the infant had OI. Because we had been unable to study cells from either previously affected infant, we could not exclude the possibility that the lethal phenotype was independent of the cysteine substitution, but felt this was unlikely. Ultrasound examinations at 16 and 24 weeks gestation showed no evidence of bone deformity and a normal male infant was delivered at term.

Discussion Linkage studies, direct biochemical evidence, and D N A sequence information have all indicated that nonlethal OI can result from mutations in the COL1A2 gene of

type-I collagen. The present study was initiated as a general way to screen cell strains from individuals with this phenotype for a specific type of mutation in the gene. By labeling cells with [35S]cysteine, we identified four individuals whose cells synthesized type-I procollagen that contained cysteine in the triple helical domain of the ~2(I) chain, a region from which it is normally excluded. Our results suggest that many individuals with nonlethal forms of OI have point mutations in the COL1A2 gene. In selecting the cell strains for study, we assumed that overmodification was primarily produced by mutations that resulted in substitution for glycine within the triple helical domain of one of the chains of type-I procollagen. If deforming varieties of OI are all produced by mutations that result in substitutions for glycine in the ct2(I) chain, then correcting for codon usage (deWet et al. 1987; Kuivaniemi et al. 1988b), 12.8% should be substitutions by cysteine, if nucleotide substitutions are random. In the 60 cell strains screened, the observed frequency was 4/60 or 6.7%. We think the observed frequency indicates that many individuals with deforming varieties of OI do have mutations in the COL1A2 gene that encode substitutions for glycine residues in the triple helical domain. Beyond statistical variation, the basis of the difference between the observed and expected frequency can be explained by the known exceptions to the principal assumption. First, some mutations may not be substitutions for glycine. In two individuals with OI-type-IV small deletions in the ~2(I) chains have been described (Kuivaniemi et al. 1988a; Wenstrup et al. 1990a), and cell strains from both synthesize an overmodified population of type-I collagen molecules. Second, deforming nonlethal OI can result from mutations in the C O L I A 1 gene, which encodes the p r o a l ( I ) chain (Starman et al. 1990; Marini et al. 1989). For example, the phenotype in patient C is similar to that of an individual heterozygous for substitution of cysteine for glycine at residue 526 in the triple helix of the a l ( I ) chain (Starman et al. 1990). Finally, for perinatal lethal OI, in which previous work has shown that the phenotype often is a consequence of point mutations that produce substitutions for glycine in the a l ( I ) chain, the frequencies of cysteine and arginine substitutions are lower than expected based on random mutation (P.H. Byers, unpublished observation). We think this may reflect a bias toward mutations at the second position of the glycine codons. The finding of a specific biochemical phenotype in these four individuals facilitates risk assessment and prenatal diagnosis in their families. Because amniotic fluid cells synthesize no ~2(I) chains (Crouch and Bornstein 1978), analysis of chorionic villus cells, which synthesize a fibroblastlike repertoire of collagens (Byers and Starman 1990), is the procedure of choice for prenatal diagnosis of mutations that affect type I procollagen synthesis and structure. Our finding of an ~2(I) cysteine substitution in patient C suggests that other individuals with phenotypes on the border between OI type IV and OI type III may carry mutations in the C O L I A 2 gene. The relatively milder phenotype seen in the families of patients A and B,

171 with m o r e c a r b o x y l - t e r m i n a l l o c a t i o n s o f c y s t e i n e resid u e s , suggests t h a t t h e l o c a t i o n o f t h e m u t a t i o n in t h e chain m a y b e i m p o r t a n t in d e t e r m i n i n g p h e n o t y p e . F u r t h e r m o r e , if t h e m u t a t i o n s r e s u l t in s u b s t i t u t i o n s for glycine r e s i d u e s in t h e t r i p l e helix, t h e p a t t e r n o f i n c r e a s ingly m i l d O I p h e n o t y p e s o b s e r v e d as cysteine-for-glycine s u b s t i t u t i o n s in t h e t r i p l e h e l i c a l d o m a i n o f t h e a l ( I ) c h a i n a r e m o v e d f r o m t h e c a r b o x y l - to t h e a m i n o - t e r m i n u s ( S t a r m a n et al. 1990) is n o t o b s e r v e d for t h e a 2 ( I ) chain. F o r p a t i e n t D , t h e l e t h a l p h e n o t y p e s e e n in t w o o f his c h i l d r e n suggests t h a t h e t e r o z y g o s i t y for t h e m u t a t i o n is l e t h a l , a n d t h a t t h e m i l d p h e n o t y p e s e e n in t h e p a t i e n t m a y t h e r e f o r e b e d u e to s o m a t i c m o s a i c i s m ( C o h n et al. 1990; W a l l i s et al. 1990). It will b e i m p o r t a n t to localize p r e c i s e l y t h e c y s t e i n e r e s i d u e s in t h e s e f o u r p a tients b y c h a r a c t e r i z i n g the m u t a t i o n s a n d to c h a r a c t e r i z e a d d i t i o n a l m u t a t i o n s b e f o r e t h e r e l a t i o n s h i p b e t w e e n loc a t i o n o f t h e m u t a t i o n a n d p h e n o t y p e b e c o m e s clear.

Acknowledgements. We thank C. Reiner and K. Braun for technical assistance, R. Wenstrup and B. Starman for helpful discussions, and Alasdair Hunter, Haynes Robinson, Ute Ochs, and Eugene Hoyme for referral of families. Original investigations supported in part by grants from the National Institutes of Health (AR21557, AR07713, and AR39837), a Clinical Research Grant (6-298) from the March of Dimes Birth Defects Foundation, an Arthritis Investigator Award from the Arthritis Foundation, and funds from the Michael Geisman Memorial Fund of the Osteogenesis Imperfecta Foundation.

References Barsh GS, David KE, Byers PH (1982) Type I osteogenesis imperfecta: a non-functional allele for proctl(I) chains of type I procollagen. Proc Natl Acad Sci USA 79 : 3838-3842 Bonadio JF, Byers PH (1985) Subtle structural alterations in the chains of type I procollagen produce osteogenesis imperfeeta type II. Nature 316:363-366 Bonadio J, Holbrook KA, Gelinas RE, Jacob J, Byers PH (1985) Altered triple helical structure of type I procollagen in lethal perinatal osteogenesis imperfecta. J Biol Chem 260 : 1734-1742 Byers PH (1989) Disorders of collagen biosynthesis and structure. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of inherited disease. McGraw-Hill, New York, pp 2805 -2842 Byers PH (1990) Brittle bones - fragile molecules: disorders of collagen gene structure and expression. Trends Genet 6: 293-300 Byers PH, Bonadio JF (1985) Molecular basis of clinical heterogeneity in osteogenesis imperfecta. In: Lloyd JK, Scriver CR (eds) Genetic and metabolic disease in pediatrics. Butterworths, London, pp 56-90 Byers PH, Starman BJ (1990) Prenatal diagnosis of osteogenesis imperfecta by analysis of collagen synthesized by cells cultured from chorionic villus biopsies. Submitted Byers PH, Tsipouras P, Bonadio JF, Starman B J, Schwartz RC (1988) Perinatal lethal osteogenesis imperfecta (OI type II); a biochemically heterogeneous disorder usually due to new mutations in the genes for type I collagen. Am J Hum Genet 42: 237 -248 Cohn DH, Starman B J, Blumberg B, Byers PH (1990) Recurrence of lethal osteogenesis imperfecta due to parental mosaicism for a dominant mutation in a human type I collagen gene. Am J Hum Genet 46: 591-601 Crouch E, Bornstein P (1978) Collagen synthesis by human amniotic fluid cells in culture: characterization of a procollagen with

three identical pro alpha-l(I) chains. Biochemistry 17:54995509 deVries WN, deWet WJ (1986) The molecular defect in an autosoreal dominant form of osteogenesis imperfecta: synthesis of type I procollagen containing cysteine in the triple-helical domain of pro-ul(I) chains. J Biol Chem 261 : 9056-9064 deWet W, Bernhard M, Benson-Chanda V, Chu M-L, Dickson L, Weil D, Ramirez F (1987) Organization of the human proa2(I) collagen gene. J Biol Chem 262 : 16032-16036 Kuivaniemi H, Sabol C, Tromp G, Sippola-Thiele M, Prockop DJ (1988a) A 19 base pair deletion in the procL2(I) gene of type I procollagen that causes in-frame RNA splicing from exon 10 to exon 12 in a proband with atypical osteogenesis imperfecta and in his asymptomatic mother. J Biol Chem 263:11407-11413 Kuivaniemi H, Tromp G, Chu M-L, Prockop DJ (1988b) Structure of a full-length cDNA clone for the prepro alpha 2(I) chain of human type I procollagen. Comparison with the chicken gene confirms unusual patterns of gene conservation. Biochem J 252 : 633-640 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 : 680-685 Marini JC, Grange DK, Gottesman GS, Lewis MB, Koeplin D A (1989) Osteogenesis imperfecta type IV. Detection of a point mutation in one alpha 1(I) collagen allele (COL1A1) by RNA/ RNA hybrid analysis. J Biol Chem 264:11893-11900 Rowe D, Shapiro J, Poirier M, Schlesinger S (1985) Diminished type I collagen synthesis and reduced alpha 1(I) collagen messenger RNA in cultured fibroblasts from patients with dominantly inherited (type I) osteogenesis imperfecta. J Clin Invest 76 : 604-611 Sillence DO, Senn AS, Danks DM (1979) Genetic heterogeneity in osteogenesis imperfecta. J Med Genet 16:101-116 Sillence DO, Barlow KK, Garber AP, Hall JG, Rimoin DL (1984) Osteogenesis imperfecta type II. Delineation of the phenotype with reference to genetic heterogeneity. Am J Med Genet 17 : 407-423 Smith R, Francis MJO, Houghton GF (1983) Brittle bone disease: osteogenesis imperfecta. Butterworths, London Starman B J, Eyre D, Charbonneau H, Harrylock M, Weis MA, Weiss L, Graham JM Jr, Byers PH (1990) Osteogenesis imperfecta" the position of the substitution for glycine by cysteine in the triple helical domain of the proal(I) chain of type I collagen determines the clinical phenotype. J Clin Invest 84:1206-1214 Steinmann B, Rao VH, Vogel A, Bruckner P, Gitzelman R, Byers PH (1984) Cysteine in the triple-helical domain of one allelic product of the ul(I) gene of type I collagen produces a lethal form of osteogenesis imperfecta. J Biol Chem 259:1112911138 Stricklin GP, Eisin AZ, Bauer EA, Jeffrey JJ (1978) Human skin fibroblast collagenase: chemical properties of precursor and active forms. Biochemistry 17 : 2331-2337 Sykes BC, Ogilvie DJ, Wordsworth BP, Anderson DJ, Jones N (1986) Osteogenesis imperfecta is linked to both type I collagen structural genes. Lancet II:69-72 Sykes B, Ogilvie D, Wordsworth P, Wallis G, Mathew C, Beighton P, Nicholls A, Pope FM, Thompson E, Tsipouras P, Schwartz R, Jensson O, Arnason A, Borresen A-L, Heiberg A, Frey D, Steinmann B (1990) Consistent linkage of dominantly inherited osteogenesis imperfecta to the type I collagen loci: COLIA1 and COLIA2. Am J Hum Genet 46 : 293-307 Tsipouras P, Borreson AL, Dickson LA, Berg K, Prockop D J, Ramirez F (1984) Molecular heterogeneity in the mild autosoreal dominant forms of osteogenesis imperfecta. Am J Hum Genet 36:1172-1179 Wallis GA, Starman BJ, Zinn AB, Byers PH (1990) Variable expression of osteogenesis imperfecta in a nuclear family is explained by somatic mosaicism for a lethal point mutation in the al(I) gene (COLIA1) of type I collagen in a parent. Am J Hum Genet 46 : 1034-1040 Wenstrup RJ, Tsipouras P, Byers PH (1986) Osteogenesis imperfecta type IV: biochemical confirmation of genetic linkage to

172 the proct2(I) gene of type I collagen. J Clin Invest 78 : 14491455 Wenstrup RJ, Cohn DH, Cohen T, Byers PH (1988) Arginine for glycine substitution in the triple helical domain of the products of one c~2(I)collagen allele (COL1A2) produces the osteogenesis imperfecta type IV phenoype. J Biol Chem 263 : 7734-7740 Wenstrup RJ, Shrago A, Phillips C, Byers P, Cohn D (1990a) Osteogenesis imperfecta type IV: analysis for mutations in u2(I) chains of type I collagen by u2(I) specific cDNA synthesis and polymerase chain reacton. Ann NY Acad Sci 580 : 546-548

Note added in proof. The mutations in all four families have been shown to result in substitution of cysteine for a triple helical glycine residue. In families A and B, the glycine at residue 646 of the helix is substituted, while in family C the substitution is for glycine 259 [Wenstrup R J, Shrago-Howe AW, Lever LW, Phillips CL, Byers PH, Cohn DH (1991) The effects of different cysteine for glycine substitutions within c~2(I) chains. J Biol Chem 266: 2590-2594]. The mutation in family D results in substitution of cysteine for glycine 472 of the helix [Edwards MJ, Byers PH, Cohn DH (1990) Mild osteogenesis imperfecta produced by somatic mosaicism for a lethal mutation in a type I collagen gene. Am J Hum Genet 47 : A215].

Wenstrup RJ, Willing MC, Starman BJ, Byers PH (1990b) Distinct biochemical phenotypes predict clinical severity in nonlethal variants of osteogenesis imperfecta. Am J Hum Genet 46 : 975-982 Willing MC, Cohn DH, Byers PH (1990) Frameshift mutation near the 3' end of the COL1A1 gene of type I collagen predicts an elongated proal(I) chain and results in osteogenesis imperrecta type I. J Clin Invest 85 : 282-290