Studies of type I collagen in osteogenesis imperfecta

Studies of type I collagen in osteogenesis imperfecta M a t t h e w J. Edwards, MB,BS, a n d John M. G r a h a m , Jr., MD, ScD From the Medical Genetics and Birth Defects Center, Ahmanson Pediatric Center, Cedars-Sinai Medical Center, University of California at Los Angeles School of Medicine We used the results of skin fibroblast type I c o l l a g e n analysis to improve the accuracy of diagnosis and g e n e t i c counseling for six patients with osteogenesis imperfecta. The fibroblasts of two patients with osteogenesis imperfecta type I synthesized a r e d u c e d quantity of qualitatively normal type I p r o c o l l a g e n . Another patient with osteogenesis imperfecta type I had two populations of type I c o l l a g e n molecules, one a p p a r e n t l y normal and the other with a substitution of cysteine for glycine in the triple helical domain. Three sporadic cases with osteogenesis imperfecta types II, III, a n d IV were studied; in e a c h p r o b a n d a normal and an abnormal overmodified p o p u l a t i o n of type I c o l l a g e n m o l e c u l e s were demonstrated, and parental c o l l a g e n s were normal in the two a v a i l a b l e patients. These results i n d i c a t e d that the probands were heterozygous for new dominant mutations and assisted our g e n e t i c counseling, especially in osteogenesis imperfecta types II and III, which were formerly b e l i e v e d to be inherited in an autosomal recessive fashion. The results c o u l d not e x c l u d e parental germ line mosaicism for a new dominant mutation, which has resulted in recurrence in siblings of some patients with osteogenesis imperfecta, so prenatal d i a g n o sis was therefore offered for future pregnancies. Analysis of chorionic villus cell c o l l a g e n may facilitate antenatal diagnosis in selected cases, a n d the study of a larger number of patients may allow correlation of the b i o c h e m i c a l defects with the natural history and prognosis. (J PEDIATR1990;117:67-72)

Abnormalities of type I collagen in osteogenesis imperfecta have been documented for several years. 1 This heritable disorder of bone is divided into four types (Table) on the basis of clinical criteria. 2 0 I has been linked to the genes for type I collagen,35 and most patients in whom collagens have been studied have quantitatively or qualitatively abnormal

type I collagen.68 Type I collagen is a major bone protein and is also synthesized by cultured skin fibroblasts and chorionic villus cells, allowing biochemical confirmation of OI by skin biopsy and potentially by chorionic villus biopsy.l Type I collagen is a heterotrimer; each mature molecule consists of a triple helix of two cq peptide chains, encoded CB

Supported in part by the SHARE Child Disabilities Center, the UCLA Intercampus Training Program, grant 08243 from the National Institutes of Health and the New Hampshire Genetic ServicesProgram, New Hampshire Divisionof Public Health Services. Support for fellowship training provided also by the Maternal and Child Health Department of Dartmouth Medical School. Submitted for publication Nov. 29, 1989; accepted Feb. 7, 1990. Reprint requests: J. M. Graham, Jr., MD, ScD, Director, Clinical Genetics and Dysmorphology,Cedars-Sinai Medical Center, 444 So. San Vicente Blvd., Los Angeles, CA 90048.

9/20/20032

COLIA1 COL1A2

OI

Cyanogen bromide Gene for the o~1 chain of type l collagen Gene for the t~2 chain of type I collagen Osteogenesis imperfecta

by the COL1A1 gene on chromosome 17q21-22 and one t2'2 chain, encoded by the COL1A2 gene on chromosome 7q2122. After transcription and translation, the pro-c~chains are hydroxylated and glycosylated (modified) to a limited extent until they associate and wind into the triple helix; the winding process starts at the carboxyl terminal end and

67

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E d w a r d s and Graham

The Journal o f Pediatrics July 1990

Overmodification

-

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k=.t ~-'.~..

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Case 5, OI Type III III

propagates toward the amino terminal end. N o r m a l structure and function depend on the presence of glycine at every third residue in the triple helical domain, which consists of 1014 amino acids. Most patients with qualitative type l collagen abnormalities studied at the molecular level have had one of a variety of point mutations that have resulted in a substitution for glycine in the triple helix. Substitution for glycine in one or more a chains is believed to retard helix formation past the substituted residue, allowing overmodification of the still-unwound parts of all chains in the molecule, including those with the normal amino acid sequence (Fig. 1, A). 1 The overmodified chains migrate more slowly than normal chains when analyzed by gel electrophoresis and may alter the physical or chemical properties of the molecule in vivo. 1 Electrophoresis of the peptide fragments resulting from cyanogen bromide cleavage of the c~ chains at methionyl residues ~defines the extent of overmodification within the chains and, by inference, the location of the structural defect in the protein (Fig. 1, B). In addition to overmodification, changes in charge, chain length, efficiency of secretion, or thermal stability may also be detected. 912 Study at the D N A level has demonstrated point mutations, deletions, and insertions. 1 In this report we illustrate how analysis of the amount and structure of type I collagen synthesized by cultured skin fibroblasts assisted in the clinical diagnosis of OI and the genetic counseling of six families.

Case 4, OI Type II

11

METHODS

Case 6, OI Type IV t

Case 3, OI Type I

B Fig. I. A, Triple helical winding of c~chains of two type I collagen molecules. Triple helical regions (middle section o f molecules), amino terminal (left) and carboxy terminal right) propeptides, not to scale. Unbroken lines indicate al chains; broken line, a2 chain. Upper molecule: Open square depicts glycine substitution near carboxy terminal end of one al chain, which possibly results in delay of triple helical winding and overmodification (increased glycosyl and galactosyl side groups) of all chain segments amino terminal to substitution. Lower molecule: Substitution closer to amino terminal end results in smaller overmodified segment length, indicated by arrows. Gal, Galactose; Glc, glucose; Man, mannose; NAc, N-acetyl. (Modified from Prockop D J, Kivirikko KI. N Engl J Med t984;3t1:377. Reprinted by permission of The New England Journal o f Medicine.) B, Extent of triple helical abnormality in four patients with structural type I collagen defects (not to scale). Numbers near c~ chains indicate major peptides observed by electrophoresis after cleavage at methionine residues with CB. Horizontal arrows depict number of overmodified cq chain CB peptides that migrate slowly on electrophoresis. Vertical arrow indicates position of cysteine for glycine substitution at amino acid residue 94 of triple helical domain of some ~1 chains in case 3.

W e collected skin biopsy specimens measuring about 3 m m in each dimension from the velar surface of the forearm. The specimens were sliced and cultured at 37 ~ C in M c C o y medium with 20% fetal calf serum. At confluence (usually after about 3 weeks), subcultures were initiated, and when these were confluent they were delivered in T25 flasks at room temperature by overnight mail to the reference laboratory of Dr. P, H. Byers, Department of Pathology, University of Washington, Seattle, where biochemical analyses of collagen were performed. These included incubation of fibroblasts with [3H]proline or [35S]cysteine, extraction of the medium and cell layer procollagens and collagens, electrophoresis of pro-a chains under reducing and nonreducing conditions in 5% acrylamide slab gels containing sodium dodecyl sulfate, and CB mapping as previously described.9, 13 The methods used to demonstrate the presence and location of cysteine in the triple helical domain in patient 3 and her family have been described elsewhere. 13 CASE REPORTS Patient 1: Ol type I. A 6-month-old girl was seen for confirmation of the clinical diagnosis of OI and for counseling of her parents as to prognosis and recurrence risk. Vaginal delivery by the vertex at term was complicated by several rib fractures. At birth

Volume 117 Number 1, Part I

Studies o f type I collagen in OI

69

rr

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Fig. 2. Pedigree of patient 2: OI type 1. Circles, Females; squares, males;filled symbols, family members with OI; open symbols, no known Of; numbers inside triangles, size of sibship (sex unspecified); slash through symbol, deceased; arrow indicates proband.

Table. Clinical classification o f osteogenesis i m p e r f e c t a Fractures, deformity

Dentinogenesis imperfecta

Sclerae

Deafness

Inheritance

I lI

Mild Lethal

Rare Common

Blue Blue

50%

III

Severe, progressive

Common

Variable

Common

IV

Moderate

Common

Normal/gray

Occasional

AD New dominant mutation (AR) New dominant mutation/AR AD

01 type

Data from of Byers PH (In: Scriver CR, Bcaudet AL, Sly MS, Valle D, eds. The metabolic basis of inherited disease. New York: McGraw-Hill, 1989) and Sillence DO, Senn A, Danks DM (J Med Genet 1979;16:101-6). AD, Autosomal dominant; AR, autosomal recessive (infrequent).

she weighed 3.4 kg and measured 50 cm in length and 34.5 cm in head circumference (all near the 50th percentile). Her 23-year-old primiparous mother and 24-year-old father were healthy and unrelated to each other, and had no family history of OI. The proband had blue sclerae, mild tibial bowing, mild frontal bossing, and normal growth and development. Cultured skin fibroblast collagen analysis revealed that her type I collagen and that of her parents migrated normally on electrophoresis. The proband's cells synthesized a reduced quantity of type I collagen compared with the amount of type III collagen. The parents' type 1 collagen was synthesized in normal amounts, suggesting that their daughter had a new dominant mutation. We concluded that the recurrence risk was negligible. Patient 2:O1 type I. A 32-year-old woman sought genetic counseling because of a family history of OI (Fig. 2) and because of her history of frequent fractures of long bones, blue sclerac, mild bowing of the femurs and tibias with normal stature, scoliosis, opalescent teeth, and conductive deafness appearing in the third decade of life. Although she perceived her skeletal problems to be relatively mild, some family members were more severely affected. Because of her deafness, she planned to adopt a child rather than accept a 50% risk of bearing an offspring affected by deafness. Her brother (IV 29, Fig. 2) previously thought that he had OI and limited his

activity because he had had a single greenstick fracture of the wrist as a child. He had normal teeth, stature, and scleral hue. Cultured skin fibroblast collagen analysis revealed that type I collagen produced by the patient migrated normally on electrophoresis but was synthesized in reduced amounts. A family linkage study with the use of probes for the COL1A1 gene was Offered, but the potentially informative relatives declined to participate. Collagen produced by her brother's skin fibroblasts was normal in quantity and quality, allowing us to reassure him that be was unaffected. Patient 3: Ol type I. A 22-year-old woman was referred for genetic counseling after a radiologic diagnosis of OI was made when hand radiograms showed osteoporosis after she had fractured a finger. She had led an active athletic life despite several fractures of long bones and phalanges. She had a family history of frequent fractures and bluish sclerae, which resolved during childhood. Neither the proband nor other affected family members had blue scterae as adults, skeletal deformity, deafness, or dentinogenesis imperfecta. Her adult height was 160.5 em (40th percentile), and results of her physical examination were normal besides the fractured phalanx. Fibroblasts of the patient and her affected mother, sister, and brother produced a population of type I collagen molecules that migrated normally on electrophoresis and another that migrated slowly. Further analysis 13 disclosed a substitution of cys-

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Edwards and Graham

Fig. 3. Patient 4: OI type 11. Neonatal radiograph showing severe generalized osteoporosis, thickened beaded ribs, and crumpled, bent, and thickened long bones.

teine for glycine at residue 94 near the amino terminal end of the triple helix. We estimated a risk to her offspring of 50% and offered to study future pregnancies by electrophoresis of collagens produced by cultured chorionic villus cells. Patient 4: Ol type II. A newborn boy with no family history of OI was delivered by cesarean section at 38 weeks of gestational age because of prolonged labor and weighed 2.61 kg (t0th percentile) at birth. His mother was 21 and his father 24 years of age at the time of delivery. He had severe bowing of long bones, deep blue sclerae, frontal bossing, and craniotabes. Radiographs (Fig. 3) showed severe generalized ostcoporosis, crumpled femora, beaded ribs, and multiple wormian bones in the skull. He died of respiratory failure at 12 days of age. Analysis of the cultured skin fibroblast collagen revealed two populations of type I procollagen molecules: one electrophoretically normal and the other abnormal, migrating slowly. The abnormal molecules were not present in the extracellular medium but were retained in the cell layer, suggesting that severe abnormality prevented their secretion. Cyanogen bromide peptides situated amino-terminal to residues 400 to 550 (~I-CB peptide 3, Fig. 1, B) migrated abnormally slowly on electrophoresis. The parents' collagen was not analyzed; results of their physical examinations were normal. We estimated a recurrence risk of the order of 4% to 7% because there was no known consanguinity.10, 14 We offered chorionic villus biopsy for collagen analysis and fetal ultrasonography for future pregnancies. Patient 5: OI type III. A boy with no known family history of OI or consanguinity was the 3.95 kg product (90th percentile) of a 38-

The Journal of Pediatrics July 1990

week gestation delivered of a 34-year-old mother and 31-year-old father by cesarean section after prenatal diagnosis of OI. Antenatal ultrasonography at 15 weeks of gestational age showed short, thick, bowed femurs and tibias. He had multiple fractures at birth, light gray sclerae, and severe progressive long bone and skull deformities. Neonatal radiographs showed severe osteoporosis, multiple fractures, and no beading of the ribs. His teeth were opalescent, and he had inguinal herniae and communicating hydrocephalus without symptoms of increased intracranial pressure. At 13 months he was unable to roll or sit, and frequent fractures of tubular bones continued. Cultured skin fibroblast collagen analysis was similar to that in patient 4 except that the abnormal a chains were overmodified from between residues 551-816 (CB peptide 7) to the amino-terminal end of the triple helical domain (Fig. 1, B). The parents' collagen analyses were normal. We offered an empiric recurrence risk estimate of 4% to 5%, because parental germ line mosaicism could not be excluded. 14 Patient 6: OI type IV. A 2-year-old boy was born at 38 weeks of gestational age by cesarean section for fetal distress, with a birth weight of 3.26 kg (50th percentile) and length of 53 cm (90th percentile). His mother was 29 years old, his father 30 years old, and there was no relevant family history. He had a fracture of the humerus at 4 months of age and had subsequently fractured toes, pelvis, and lumbar spine. He had gray sclerae, opalescent teeth, triangular face, mild frontal bossing, and no long bone deformity or deafness. Cultured skin fibroblast analysis revealed a normal and an abnormal population of type I collagen chains. In the abnormal molecules, CB peptide 8 (Fig. i, B) migrated slowly on electrophoresis, indicating overmodification at the amino-terminal ends. Results of the parents' collagen analyses were normal. As with patient 5, we estimated a low recurrence risk. We could not exclude parental germline mosaicism, although this has not been reported in OI type IV. DISCUSSION Genetic counseling may be simple in families with OI types I and IV segregating in an autosomal d o m i n a n t fashion. In contrast, the majority of cases of types II and III are sporadic a n d recurrence risks m a y be uncertain without collagen analysis. 1~ Observations of parental consanguinity, sibships with more t h a n one affected child, and phenotypically unaffected parents originally led to the impression t h a t OI types II and III m i g h t be autosomal recessive disorders. However, an empiric recurrence risk for sporadic cases of OI type II of 6% to 7%, r a t h e r t h a n the expected 25%, was later found. This discrepancy has been explained by biochemical studies of fibroblasts from sporadic cases, which usually d e m o n s t r a t e d heterozygosity for new d o m i n a n t mutations, with two populations of type 1 collagen m o l e c u l e s - - a normal one encoded by the normal allele and a slowly migrating population' as a result of the OI allele. T h e parents' fibroblasts have usually produced only n o r m a l molecules, and p a r e n t a l germline mosaicism m a y explain the few observed recurrences. 1~ Mosaicism in sperm for a COLIA1 m u t a t i o n was found in a m a n who had

Volume 117 Number 1, Part 1

a normal phenotype; he had two children with lethal OI type lI by different partners. 15 Germline mosaicism has been found in some other autosomal dominant disorders, and although its incidence is unknown in the other types of OI, it should be kept in mind as a possible cause of recurrence in families with sporadic cases. There are as many as five subgroups of Ol type II, and some of the cases may be recessive.10, 16 A recurrence risk of up to 25% has been estimated for consanguineous parents of a child with OI type I1 or III and a risk of 4.4% if there is no consanguinity. 14 Analysis of the type I collagen produced by the cultured skin fibroblasts of our patients assisted in confirmation of the clinical diagnosis. In two families the biochemical results were compatible with the presence of a nonfunctional COL1A1 allele, iv In four of the six patients the presence of a normal and a structurally abnormal population of type I collagen molecules indicated heterozygosity for dominant disease alleles. The parents' normal type I collagen studies in patients 1, 5, and 6 suggested that the OI genes were due to new mutations. The biochemical findings allowed us to estimate a recurrence risk for the sporadic cases with OI type II and III, which was lower than that based on an assumption of autosomal recessive inheritance. We could not exclude the possibility of parental germline mosaicism and offered biochemical analysis of chorionic villus tissue for future pregnancies. In families who already have a child with an identified qualitative defect, analysis of type I collagen produced by fetal cells obtained by chorionic villus biopsy could possibly allow earlier prenatal diagnosis than mid-second trimester ultrasound examination of the fetus. Linkage study with the use of type I collagen gene probes and D N A from chorionic villus cells Is or amniotic fluid cells offers an alternative if the family is informative. Amniocentesis and analysis of collagen Synthesized by amniotic fluid cells has been performed in conjunction with fetal ultrasonography, w Fetal amniocytes produce some type I collagen in the form of al trimers; cells obtained at amniocentesis may therefore show no abnormality in families with a2 chain defects, 2~ and al chain defects causing overmodification may be masked because al homotrimers are normally modified more than normal type I heterotrimers. In OI type II, fetal ultrasonography may detect fetal skeletal fractures and deformity at 15 to 17 weeks gestation, as early as amniocentesis. 21,22 Milder ultrasonographic changes in OI type III may not be detectable until 19 to 22 weeks, 23, 24 although patient 5 was affected severely enough to have a diagnosis at 15 weeks. In OI types I and IV, fractures or deformity may never develop in some cases. 25 Some general correlations between the biochemical and clinical phenotypes have been proposed, 1 but they are limited by known exceptions. In O l type I there is usually no

Studies o f type I collagen in OI

71

qualitative biochemical abnormality but reduced amounts of electrophoretically normal type I collagen are synthesized. 26,27 Osteogenesis imperfecta type I has been linked to the COL1A1 gene in most families, 3, 28 although linkage to COL1A2 has been reported. 29 The phenotype does not predict the chain carrying the primary defect (or vice versa) with certainty, l~ 11, 3o-33 There may be considerable variability of expression within families even when all affected members appear to have the same collagen defect. 34 Type III OI may present the greatest difficulty in counseling: it may or may not be associated with abnormality of type I collagen or with linkage to the type I collagen genes, kindreds with autosomal recessive transmission have been reported, and recessive types may predominate in some communities.25,35, 36 The study of more cases may allow further correlation of the biochemical defect with the clinical classification, naturai history and prognosis. We thank Drs. Daniel H. Cohn and Peter H. Byers for review of the manuscript and many valuable suggestions. Dr. Byers performed the collagen analyses at the Department of Pathology, University of Washington, Seattle. REFERENCES

1. Byers PH. Disorders of collagen biosynthcsis and structure. In: Striver CR, Beaudet AL, Sly WS, Valle D, cds. The metabolic basis of inherited disease. New York: McGraw-Hill, 1989: 2814-24. 2. Sillence DO, Senn A, Danks DM. Genetic heterogeneity in osteogenesis imperfccta. J Med Genet 1979;16:101-6. 3. Sykes B, Ogilvie D, Wordsworth P, Anderson J, Jones N. Osteogenesis imperfccta is linked to both type I collagen structural genes. Lancet 1986;2:69-72. 4. Tsipouras P, Myers JC, Ramirez F, Prockop DJ. Rcstriction fragment length polymorphism associated with the proa2(I) 9gene of human typc I procollagen: application to a family with an autosomal dominant form of ostcogenesis imperfecta. J Clin Invest 1983;72:1262-7. 5. Falk CT, Schwartz RC, Ramirez F, Tsipouras P. Use of molccular haplotypes specific for the human proa2(I) collagen gene in linkage analysis of the mild dominant forms of osteogenesis imperfccta. Am J Hum Genet 1986;38:269-79. 6. Byers PH, Bonadio JF. The molecular basis of clinical heterogeneity in osteogenesis imperfecta. In: Lloyd J, Scriver CR, eds. Metabolic and gcnetic diseases in pediatrics. London: Butterworths, 1985:56-90. 7. Cheah KSE. Collagen genes and inherited connective tissue discase. Biochem J 1985;229:287-303. 8. Prockop D J, Kivirikko KI. Heritable diseases of collagen. N Engl J Med 1984;311;376-86. 9. Bonadio JF, Byers PH. Subtle structural alterations in the chains of type l procollagen produce osteogenesis irnperfecta type II. Nature 1985;316:363-6. 10. Byers PH, Tsipouras P, Bonadio J, Starman BJ, Schwartz RC. Perinatal lethal osteogenesis imperfecta (OI type II): a biochemically heterogeneous disorder usually due to new mutations in the genes for type ! collagen. Am J Hum Genet 1988;42:237-48.

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11. Willing MC, Cohn DH, Starman B, Holbrook KA, Greenberg CR, Byers PH. Heterozygosity for a large deletion in the cr collagen gene has a dramatic effect on type I collagen secretion and produces perinatal lethal osteogenesis imperfecta. J Biol Chem 1988;263:8398-404. 12. Kuivaniemi H, Sabol C, Tromp G, Sippola-Thiele M, Proekop DJ. A 19 base pair deletion in the pro-o~2(I) gene of type l procollagen that causes in-frame RNA splicing from exon 10 to exon 12 in a proband with atypical osteogenesis imperfecta and in his asymptomatie mother. J Biol Chem 1988;263:1140713. 13. Starman BJ, Eyre D, Charbonneau H, et al. Osteogenesis imperfecta: the position of substitution for glycine by cysteine in the triple helical domain of the procd(I) chains of type I collagen determines the clinical phenotype. J Clin Invest 1989; 84:1206-14. 14. Thompson EM, Young ID, Hall CM, Pembrey ME. Recurrence risks and prognosis in severe sporadic osteogenesis imperfecta. J Med Genet 1987;24:390-405. t5. Cohn DH, Starman BJ, Blumberg B, Byers PH. Recurrence of lethal osteogenesis imperfecta due to parental mosaicism for a dominant mutation in a human type I collagen gene (COLIA1). Am J Hum Genet 1990;46:591-601. 16. Sillenee DO, Barlow KK, Garber AP, Hall JG, Rimoin DL. Osteogenesis imperfeeta type II delineation of the phenotype with reference to genetic heterogeneity. Am J Med Genet 1984;17:407-23. 17. Genovese C, Rowe DW. Analysis of cytoplasmic and nuclear messenger RNA in fibroblasts from patients with type 1 osteogenesis imperfecta. Methods Enzymol 1987;t45:223-35. 18. Tsipouras P, Schwartz RC, Goldberg JD, Berkowitz RL, Ramirez F. prenatal prediction of osteogenesis imperfeeta (OI type IV): excIusion of inheritance using a collagen gene probe. J Med Genet 1987;24:406-9. 19. Shapiro JE, Phillips JA Ill, Byers PH, et al. Prenatal diagnosis of lethal perinatal osteogenesis imperfecta (OI type II). J PEDIATR 1982;100:127-33. 20. Crouch E, Bornstein P. Collagen synthesis by human amniotic fluid cells in culture: characterization of a procollagen with three identical procd (I) chains. Biochemistry 1978; 17:5499509. 21. Elejalde BR, de Elejalde MM. Prenatal diagnosis of perinatally lethal osteogenesis imperfecta. Am J Med Genet 1983; 14:353-9. 22. Brons JT, van der Harten H, Wladimiroff JW, et al. Prenatal ultrasonographic diagnosis of osteogenesis imperfecta. Am J Obstet Gynecol 1988;159:176-81. 23. Aylsworth AS, Seeds JW, Guilford WB, Burns CB, Washburn DB. Prenatal diagnosis of a severe deforming type of osteogenesis imperfecta. Am J Med Genet 1984;19:707-14. 24. Robinson LP, W orthen N J, Lachman RS, Adomian GE, Ri-

The Journal of Pediatrics July 1990

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moin DL. Prenatal diagnosis of osteogenesis imperfecta type III. Prenat Diagn 1987;7:7-15. Williams EM, Nicholls AC, Daw SCM, et al. Phenotypical features of an unique Irish family with severe autosomal recessive osteogenesis imperfecta. Clin Genet 1989;35:181-90. Barsh GS, David KE, Byers PH. Type I osteogenesis imperfecta: a nonfunctional allele for procd(I) chains of type I procollagen. Proc Natt Acad Sci USA 1982;79:3838-42. Rowe DW, Shapiro JR, Poirier M, Schlesinger S. 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 1985;76:604-11. Tsipouras P, Schwartz RC. Dominantly inherited osteogenesis imperfecta (Ot types I and IV) is genetically linked to the COL1A1 and COL1A2 genes of type I collagen [Abstract]. Pediatr Res 1987;21:294A. Wallis G, Beighton P, Boyd C, Mathew CG. Mutations linked to the pro cd(I) collagen gene are responsible for several cases of osteogenesis imperfecta type I. J Med Genet 1986; 23: 411-6. Wenstrup R J, Cohn DH, Cohen T, Byers PH. 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 phenotype. J Biol Chem 1988;263: 7734-40. Cohn DH, Apone S, Eyre DR, et al. Substitution of cysteine for glycine within the carboxyl-terminal telopeptide of the cdchain of type I collagen produces mild osteogenesis imperfecta. J Biol Chem 1988;263:14605-7. Tenni R, Cetta G, Dyne K, et al. Type I procollagen in the severe nonlethal form of osteogenesis imperfecta. Defective proa l (I)chains in a patient with abnormal proteoglycan metabolism and mineral deposits in the dermis. Hum Genet 1988; 79:245-50. Baldwin CT, Constantinou CD, Dumars KW, Prockop DJ. A single base mutation that converts glycine 907 of the cd(I) chain of type I procollagen to aspartate in a lethal variant of osteogenesis imperfecta: the single amino acid substitution near the carboxyl terminus destabilizes the whole triple helix. J Biol Chem 1989;264:3002-6. Superti-Furga A, Pistone F, Romano C, Steinman B. Clinical variability of osteogenesis imperfecta linked to COL1 A2 and associated with a structural defect in the type i collagen molecule. J Med Genet 1989;26:358-62. Sillence DO, Barlow KK, Cole WG, Dietrich S, Garber AP, Rimoin DL. Osteogenesis imperfecta type IlI: delineation of the phenotype with reference to genetic heterogeneity. Am J Med Genet 1986;23:821-32. Viljoen D, Beighton P. Osteogenesis imperfecta type III: an ancient mutation in Africa? Am J Med Genet 1987;27:907-12.