A de novo G to T transversion in a pro-alpha 1 (I) collagen gene for a moderate case of osteogenesis imperfecta. Substitution of cysteine for glycine 178 in the triple helical domain

Cultured fibroblasts from a patient affected with a moderate form of osteogenesis imperfecta were defective for the synthesis of type I collagen molecules; about half of the alpha 1(I) chains contained a cysteine residue in the triple helical domain and a disulfide link formed when two mutant alpha 1(I) chains were incorporated into a type I collagen heterotrimer. The proband's parents were clinically and biochemically normal. The cysteine was localized within peptide alpha 1(I)CB8 between residues 170 and 200 of the triple helical domain using a chemical procedure with 2-nitro-5-thiocyanobenzoic acid (Tenni, R., Rossi, A., Valli, M., Mottes, M., Pignatti, P. F., and Cetta, G. (1990) Matrix 10, 20-26). Type I procollagen heterotrimers containing either one or two mutant chains showed (i) a slight abnormality in secretion from cells; (ii) a low degree of post-translational overmodifications; (iii) the same, but lower than normal, thermal stability. Total RNA was isolated from the proband's dermal fibroblast cultures, and cDNAs for pro-alpha 1(I) were prepared d using total RNA. A portion of cDNA, coding for the region encompassing residues 119-193 of alpha 1(I) triple helical domain, was amplified by polymerase chain reaction. A single base pair mismatch was identified by chemical cleavage of DNA.DNA heteroduplexes, indicating a possible substitution of a guanine in the triplet coding for glycine 178 or 181. The same unique mismatch was detected by chemical cleavage in about one-half of the molecules in heteroduplexes formed between patient's pro-alpha 1(I) mRNAs and a normal cDNA probe. The amplified products were cloned and sequenced, confirming the heterozygous nature of the patient and demonstrating the presence and the location of a missense mutation; a single T for G substitution was found in the first base of the triplet coding for residue 178 of alpha 1(I) triple helical domain, leading to a cysteine for glycine substitution. Allele-specific oligonucleotide hybridization to amplified DNA confirmed a de novo point mutation in the proband's genome. The findings in this patient are in accord with the phenotypic gradient model, which correlates the localization of the structural defect with the clinical outcome of osteogenesis imperfecta. The mutant protein has some properties that differ from the caused by the cysteine for glycine 175 substitution, suggesting a direct influence of the neighboring amino acids on the effects of the mutation.     

Lethal perinatal osteogenesis imperfecta due to the substitution of arginine for glycine at residue 391 of the alpha 1(I) chain of type I collagen

A baby with the lethal perinatal form of osteogenesis imperfecta was shown to have a structural defect in the alpha 1(I) chain of type I procollagen. Normal and mutant alpha 1(I) CB8 cyanogen bromide peptides, from the helical part of the alpha 1(I) chains, were purified from bone. Amino acid sequencing of tryptic peptides derived from the mutant alpha 1(I) CB8 peptide showed that the glycine residue at position 391 of the alpha 1(I) chain had been replaced by an arginine residue. This substitution accounted for the more basic charged form of this peptide that was observed on two-dimensional electrophoresis of the collagen peptides obtained from the tissues. The substitution was associated with increased enzymatic hydroxylation of lysine residues in the alpha 1(I) CB8 and the adjoining CB3 peptides but not in the carboxyl-terminal CB6 and CB7 peptides. This finding suggested that the sequence abnormality had interfered with the propagation of the triple helix across the mutant region. The abnormal collagen was not incorporated into the more insoluble fraction of bone collagen. The baby appeared to be heterozygous for the sequence abnormality and as the parents did not show any evidence of the defect it is likely that the baby had a new mutation of one allele of the pro-alpha 1(I) gene. The amino acid substitution could result from a single nucleotide mutation in the codon GGC (glycine) to produce the codon CGC (arginine).     

Detection of point mutations in type I collagen by RNase digestion of RNA/RNA hybrids.

We have developed a strategy for the detection, localization and sequence determination of point mutations in the mRNA coding for the alpha 1(I) and alpha 2(I) chains of type I collagen. Point mutations are detected by RNase A cleavage of mismatches in RNA/RNA hybrids. The mRNAs coding for the fibrillar collagens present special problems for hybrid analysis because of their large size and their GC-rich and repetitive sequences. We have generated a series of overlapping antisense riboprobes covering the entire pro alpha 1(I) and pro alpha 2(I) mRNAs. Uniformly labelled normal antisense riboprobes are hybridized with the total fibroblast RNA of patients with possible mutations in type I collagen. Mismatches in the resulting RNA/RNA hybrids are cleaved with RNase A and the labelled riboprobe cleavage products are examined electrophoretically. The sensitivity and specificity of the system were demonstrated by the detection and localization of a known point mutation in the codon for alpha 1(I) glycine 988 (1). DNA for sequencing the mutations localized by hybrid analysis may be obtained by either (1) generation of a fibroblast cDNA library and isolation of both alleles by plaque screening, or (2) a more rapid method using first strand cDNA synthesis from poly (A+)-mRNA, followed by PCR amplification of the mutation-containing region of the DNA/RNA hybrid. This strategy for detection and isolation has wide application not only for mutations causing connective tissue disorders, but also for mutations in other large and repetitive genes. We have used this strategy for the detection and sequencing of a point mutation in alpha 2(I) mRNA associated with a case of lethal osteogenesis imperfecta. The G----A point mutation in the codon for alpha 2(I) glycine residue 805 results in the substitution of an aspartic acid at this position and is consistent with the proband's collagen protein data.     

Altered triple helical structure of type I procollagen in lethal perinatal osteogenesis imperfecta

Cultured dermal fibroblasts from an infant with the lethal perinatal form of osteogenesis imperfecta (type II) synthesize normal and abnormal forms of type I procollagen. The abnormal type I procollagen molecules are excessively modified during their intracellular stay, have a lower than normal melting transition temperature, are secreted at a reduced rate, and form abnormally thin collagen fibrils in the extracellular matrix in vitro. Overmodification of the abnormal type I procollagen molecules was limited to the NH2-terminal three-fourths of the triple helical domain. Two-dimensional mapping of modified and unmodified alpha chains of type I collagen demonstrated neither charge alterations nor large insertions or deletions in the region of alpha 1(I) and alpha 2(I) in which overmodification begins. Both the structure and function of type I procollagen synthesized by cells from the parents of this infant were normal. The simplest interpretation of the results of this study is that the osteogenesis imperfecta phenotype arose from a new dominant mutation in one of the genes encoding the chains of type I procollagen. Given the requirement for glycine in every third position of the triple helical domain, the mutation may represent a single amino acid substitution for a glycine residue. These findings demonstrate further heterogeneity in the biochemical basis of osteogenesis imperfecta type II and suggest that the nature and location of mutations in type I procollagen may determine phenotypic variation.     

Sequence of normal canine COL1A1 cDNA and identification of a heterozygous alpha1(I) collagen Gly208Ala mutation in a severe case of canine osteogenesis imperfecta

The sequence of canine COL1A1 cDNA was determined from four overlapping COL1A1 RT-PCR products generated from canine fibroblast RNA. In the translated region, nucleotide identity between canine and human COL1A1 cDNA was 93.2%, although the canine sequence lacked nucleotides 204 to 215 in the region coding for the N-propeptide. Amino acid identity was 97.7%. Total RNA and type I collagen were collected from cultured skin fibroblasts of a 12-week-old male golden retriever with pathologic fractures suggestive of osteogenesis imperfecta (OI) and dentinogenesis imperfecta. Sequential, overlapping approximately 1,000-bp fragments of COL1A1 and COL1A2 cDNA were each amplified by RT-PCR using primers containing 5' T7 polymerase sites. These PCR products were transcribed with T7 RNA polymerase, hybridized into RNA duplexes, and cleaved at mismatch sites with RNase. The proband had an unique cleavage pattern for the fragment of COL1A1 mRNA spanning nucleotides 709 to 1,531. Sequence analysis identified a G to C point mutation for nucleotide 1,276, predicting a codon change from glycine (GGA) to alanine (GCA) for amino acid 208. This change disrupts the normal Gly-X-Y pattern of the collagen triple helix. Restriction enzyme digestion of the RT-PCR product was consistent with a heterozygous COL1A1 mutation. Type I collagen was labeled with 3H-proline, salt precipitated, and analyzed by SDS-PAGE. Pepsin digested alpha chains were over-hydroxylated, and procollagen processing was delayed. Thus, canine and human OI appear homologous in terms of clinical presentation, etiology, and pathogenesis.     

A novel Gly to Arg substitution at position 388 of the alpha1 chain of type I collagen in lethal form of osteogenesis imperfecta

Cultured skin fibroblasts from a proband with a lethal form of osteogenesis imperfecta produce two forms of type I collagen chains, with normal and delayed electrophoretic migration; collagen of the proband's mother was normal. Peptide mapping experiments localized the structural defect in the proband to alpha1(I) CB8 peptide in which residues 123 to 402 are spaned. Direct sequencing of amplified cDNA covering this region revealed a G to A single base change in one allele of the alpha1(I) chain, that converted glycine 388 to arginine. Restriction enzyme digestion of the RT-PCR product was consistent with a heterozygous COL1A1 mutation. The novel mutation conforms to the linear gradient of clinical severity for the alpha1(I) chain and results in reduced thermal stability by 3 degrees C and intracellular retention of abnormal molecules.     

A 9-base pair deletion in COL1A1 in a lethal variant of osteogenesis imperfecta

A proband with lethal osteogenesis imperfecta has been investigated for the causative defect at the levels of collagen protein, mRNA, and DNA. Analysis of type I collagen synthesized by the proband's fibroblasts showed excessive post-translational modification of alpha 1(I) chains along the entire length of the helix. Oververmodification of alpha chains could be prevented by incubation of the cells at 30 rather than 37 degrees C, and the thermal stability of the triple helix, as determined by protease digestion, was normal. RNase A cleavage of RNA:RNA hybrids formed between the proband's mRNA and antisense RNA derived from normal pro-alpha 1(I) chain cDNA clones was used to locate an abnormality to exon 43 of the proband's pro-alpha 1(I) collagen gene (COL1A1). The nucleotide sequence of the corresponding gene region showed, in one allele, the deletion of 9 base pairs, not present in either parent, within a repeating sequence of exon 43. The mutation causes the loss of one of three consecutive Gly-Ala-Pro triplets at positions 868-876, but does not otherwise disrupt the Gly-X-Y sequence. Procollagen processing in fibroblast cultures and susceptibility of the mutant collagen I to cleavage with vertebrate collagenase were normal, indicating that the slippage of collagen chains by one Gly-X-Y triplet does not abolish amino-propeptidase and collagenase cleavage sites. How the mutation produces the lethal osteogenesis imperfecta phenotype is not entirely clear; the data suggest that the interaction of alpha chains immediately prior to helix formation may be affected.     

Substitution of cysteine for glycine within the carboxyl-terminal telopeptide of the alpha 1 chain of type I collagen produces mild osteogenesis imperfecta

We have characterized a mutation that produces mild, dominantly inherited osteogenesis imperfecta. Half of the alpha 1 (I) chains of type I collagen synthesized by cells from an affected individual contain a cysteine residue in the 196-residue carboxyl-terminal cyanogen bromide peptide of the triple-helical domain (Steinmann, B., Nicholls, A., and Pope, F. M. (1986) J. Biol. Chem. 261, 8958-8964). Unexpectedly, sequence determined from a proteolytic fragment of the alpha 1 (I) chain derived from procollagen molecules synthesized in the presence of both [3H]proline and [35S]cysteine indicated that the cysteine is located at the third residue carboxyl-terminal to the triple-helical domain, normally a glycine. The nucleotide sequence of a fragment amplified from genomic DNA confirmed the location of the cysteine residue and showed that the mutation was a single nucleotide change in one COL1A1 allele. This represents a new class of mutations, point mutations outside the triple-helical domain of the chains of type I collagen, that produce the osteogenesis imperfecta phenotype.     

RNA sequence analysis of a perinatal lethal osteogenesis imperfecta mutation

The perinatal lethal form of osteogenesis imperfecta often results from mutations which disrupt stable assembly, delay secretion, and cause excessive posttranslational modification of type I procollagen molecules. One such mutation was efficiently characterized by an indirect method of RNA sequence analysis. The mutation initially was localized in procollagen by mapping the distribution of abnormal posttranslational modification within the triple helical domain of mutant molecules. Total RNA was isolated from osteogenesis imperfecta cells in culture, cDNA was synthesized using alpha 1(I) and alpha 2(I) specific primers, and fragments of cDNA suspected to harbor the mutation were amplified by the polymerase chain reaction technique and then cloned in M13 vectors. Sequence analysis of the amplified cDNA revealed a new, heterozygous Gly----Val substitution at residue 256 of the triple helical domain of alpha 1(I) chains produced by the perinatal lethal osteogenesis imperfecta cells. The nature and location of the mutation were confirmed by sequence analysis of amplified genomic DNA. A Gly----Val substitution has not previously been associated with the lethal form of osteogenesis imperfecta, and this mutation has the most amino-terminal location within the alpha 1(I) chain triple helical domain reported to date.     

The effects of different cysteine for glycine substitutions within alpha 2(I) chains. Evidence of distinct structural domains within the type I collagen triple helix

Affected individuals from two apparently distinct, mild osteogenesis imperfecta families were heterozygous for a G to T transition in the COL1A2 gene that resulted in cysteine for glycine substitutions at position 646 in the alpha 2(I) chain of type I collagen. A child with a moderately severe form of osteogenesis imperfecta was heterozygous for a G to T transition that resulted in a substitution of cysteine for glycine at position 259 in the COL1A2 gene. Type I collagen molecules containing an alpha 2(I) chain with cysteine at position 259 denaturated at a lower temperature than molecules containing an alpha 2(I) chain with cysteine at position 646. In contrast to cysteine for glycine substitutions in the alpha 1(I) chain, the severity of the osteogenesis imperfecta phenotype is not directly proportional to the distance of the mutation from the amino-terminal end of the triple helix. These findings could be explained if the type I collagen triple helix contains discontinuous domains that differ in their contributions to maintaining helix stability.     

A frameshift mutation results in a truncated nonfunctional carboxyl-terminal pro alpha 1(I) propeptide of type I collagen in osteogenesis imperfecta

A codon frameshift mutation caused by a single base (U) insertion after base pair 4088 of prepro alpha 1(I) mRNA of type I procollagen was identified in a baby with lethal perinatal osteogenesis imperfecta. The mutation was identified in fibroblast RNA by a new method that allows the direct detection of mismatched bases by chemical modification and cleavage in heteroduplexes formed between mRNA and control cDNA probes. The region of mismatches was specifically amplified by the polymerase chain reaction and sequenced. The heterozygous mutation in the amplified cDNA most likely resulted from a T insertion in exon 49 of COL1A1. The frameshift resulted in a truncated pro alpha 1(I) carboxyl-terminal propeptide in which the amino acid sequence was abnormal from Val1146 to the carboxyl terminus. The propeptide lacked Asn1187, which normally carries an N-linked oligosaccharide unit, and was more basic than the normal propeptide. The distribution of cysteines was altered and the mutant propeptide was unable to form normal interchain disulfide bonds. Some of the mutant pro alpha 1(I)' chains were incorporated into type I procollagen molecules but resulted in abnormal helix formation with over-hydroxylation of lysine residues, increased degradation, and poor secretion. Only normal type I collagen was incorporated into the extracellular matrix in vivo resulting in a tissue type I collagen content approximately 20% of that of control (Bateman, J. F., Chan, D., Mascara, T., Rogers, J. G., and Cole, W. G. (1986) Biochem. J. 240, 699-708).     

The molecular defect in an autosomal dominant form of osteogenesis imperfecta. Synthesis of type I procollagen containing cysteine in the triple-helical domain of pro-alpha 1(I) chains

Synthesis of procollagen was examined in skin fibroblasts from a patient with a moderately severe autosomal dominant form of osteogenesis imperfecta. Proteolytic removal of the propeptide regions of newly synthesized procollagen, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, revealed the presence of type I collagen in which two alpha 1(I) chains were linked through interchain disulfide bonds. Fragmentation of the disulfide-bonded alpha 1(I) dimers with vertebrate collagenase and cyanogen bromide demonstrated the presence of a cysteine residue in alpha 1(I)CB8, a fragment containing amino acid residues 124-402 of the alpha 1(I) collagen chain. Cysteine residues are not normally found in the triple-helical domain of type I collagen chains. The heterozygous nature of the molecular defect resulted in the formation of three kinds of type I trimers: a normal type with normal pro-alpha(I) chains, a type I trimer with one mutant pro-alpha 1(I) chain and two normal chains, and a type I trimer containing two mutant pro-alpha 1(I) chains and one normal pro-alpha 2(I) chain. The presence of one or two mutant pro-alpha 1(I) chains in trimers of type I procollagen was found to reduce the thermal stability of the protein by 2.5 and 1 degree C, respectively. In addition to post-translational overmodification, procollagen containing one mutant pro-alpha 1(I) chain was also cleared more slowly from cultured fibroblasts. The most likely explanation for these disruptive changes in the physical stability and secretion of the mutant procollagen is that a cysteine residue is substituted for a glycine in half of the pro-alpha 1(I) chains synthesized by the patient's fibroblasts.     

A structural mutation of the collagen alpha 1(I)CB7 peptide in lethal perinatal osteogenesis imperfecta

Structurally abnormal type I collagen was identified in the dermis, bone, and cultured fibroblasts obtained from a baby with lethal perinatal osteogenesis imperfecta. Two-dimensional gel electrophoresis of the CNBr peptides demonstrated that the alpha 1(I)CB7 peptide from the alpha 1(I)-chain of type I collagen existed in a normal form and a mutant form with a more basic charge distribution. This heterozygous peptide defect was not detected in the collagens from either parent. The defect was localized to a 224-residue region at the NH2 terminus of the alpha 1(I)CB7 peptide by mammalian collagenase digestion. Analysis of unhydroxylated collagens produced in cell culture indicated that the mutant alpha 1(I)CB7 migrated faster on electrophoresis suggesting that the abnormality may be a small deletion or a mutation that alters sodium dodecyl sulfate binding. The post-translational hydroxylation of lysine residues was increased in the CB7 peptide and also in peptides CB3 and CB8 which are toward the NH2 terminus of the alpha 1(I)-chain. The COOH-terminal CB6 peptide was normally hydroxylated. These findings support the proposal that the lysine overhydroxylation resulted from a perturbation of helix propagation from the COOH to NH2 terminus of the collagen trimer caused by the structural defect in alpha 1(I)CB7.     

Collagen defects in lethal perinatal osteogenesis imperfecta.

Quantitative and qualitative abnormalities of collagen were observed in tissues and fibroblast cultures from 17 consecutive cases of lethal perinatal osteogenesis imperfecta (OI). The content of type I collagen was reduced in OI dermis and bone and the content of type III collagen was also reduced in the dermis. Normal bone contained 99.3% type I and 0.7% type V collagen whereas OI bone contained a lower proportion of type I, a greater proportion of type V and a significant amount of type III collagen. The type III and V collagens appeared to be structurally normal. In contrast, abnormal type I collagen chains, which migrated slowly on electrophoresis, were observed in all babies with OI. Cultured fibroblasts from five babies produced a mixture of normal and abnormal type I collagens; the abnormal collagen was not secreted in two cases and was slowly secreted in the others. Fibroblasts from 12 babies produced only abnormal type I collagens and they were also secreted slowly. The slower electrophoretic migration of the abnormal chains was due to enzymic overmodification of the lysine residues. The distribution of the cyanogen bromide peptides containing the overmodified residues was used to localize the underlying structural abnormalities to three regions of the type I procollagen chains. These regions included the carboxy-propeptide of the pro alpha 1(I)-chain, the helical alpha 1(I) CB7 peptide and the helical alpha 1(I) CB8 and CB3 peptides. In one baby a basic charge mutation was observed in the alpha 1(I) CB7 peptide and in another baby a basic charge mutation was observed in the alpha 1(I) CB8 peptide. The primary defects in lethal perinatal OI appear to reside in the type I collagen chains. Type III and V collagens did not appear to compensate for the deficiency of type I collagen in the tissues.     

Clinical variability of osteogenesis imperfecta reflecting molecular heterogeneity: cysteine substitutions in the alpha 1(I) collagen chain producing lethal and mild forms

We have examined the collagenous proteins extracted from skin and produced by skin fibroblast cultures from the members of a family with mild dominant osteogenesis imperfecta (OI type I). The two affected patients, mother and son, produce two populations of alpha 1(I) chains of type I collagen, one chain being normal, the other containing a cysteine within the triple-helical domain. Both forms can be incorporated into triple-helical molecules with an alpha 2(I) chain. When two mutant alpha (I) chains are incorporated into the same molecule, a disulfide bonded dimer is produced. We have characterized these chains by sodium dodecyl sulfate-gel electrophoresis and CNBr-peptide mapping and by measuring a number of biosynthetic and physical variables. The cysteine was localized to the COOH-terminal peptide alpha (I) CB6. Molecules containing the mutant chains are stable, have a normal denaturation temperature, are secreted normally, and have normal levels of post-translational modification of lysyl residues and intracellular degradation. We have compared and contrasted these observations with those made in a patient with lethal osteogenesis imperfecta in which there was a cysteine substitution in alpha 1(I) CB6 (Steinmann, B., Rao, V. H., Vogel, A., Bruckner, P., Gitzelmann, R., and Byers, P. H. (1984) J. Biol. Chem 259, 11129-11138) and have concluded that the mutation in the present family occurs in the X or Y position of a Gly-X-Y repeating unit of collagen and not in the glycine position shown for the previous patient (Cohn, D. H., Byers, P. H., Steinmann, B, and Gelinas, R. E. (1986) Proc. Natl. Acad. Sci. U. S. A., in press.     

Substitution of arginine for glycine 325 in the collagen alpha 5 (IV) chain associated with X-linked Alport syndrome: characterization of the mutation by direct sequencing of PCR-amplified lymphoblast cDNA fragments.

A large kindred with adult-type X-linked Alport syndrome was studied with regard to a defect in the recently described COL4A5 collagen gene. Southern blot analysis with COL4A5 cDNA probes showed loss of a MspI restriction site. Direct sequencing of cDNA amplified from lymphoblast mRNA demonstrated a single-base substitution converting a glycine codon to arginine at position 325 in the alpha 5 chain of type IV collagen. The triple-helical collagenous domain of alpha 5(IV), characterized by a Gly-X-Y repeat sequence, is interrupted 22 times by noncollagenous sequences. The mutation creates an additional interruption in the Gly-X-Y repeat motif, between interruptions 4 and 5. It is interesting that such glycine substitutions inside the COL1A1 or COL1A2 genes have been associated with many cases of osteogenesis imperfecta. This gly325-to-arg substitution presumably alters the triple-helix formation, and, in turn, modifies the ultrastructural and functional characteristics of the type IV collagen network inside the glomerular basement membrane.     

Incorporation of type I collagen molecules that contain a mutant alpha 2(I) chain (Gly580-->Asp) into bone matrix in a lethal case of osteogenesis imperfecta.

To understand more directly the tissue defect in osteogenesis imperfecta (OI), bone matrix was analyzed from an infant with lethal OI (type II) of defined mutation (collagen alpha 2(I)Gly580-->Asp). Pepsin-solubilized alpha 1(I) and alpha 2(I) chains and derived CNBr-peptides migrated more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis compared with normal human controls. The peptide alpha 2(I)CB3,5, predicted to contain the mutation site, ran as a retarded doublet band and was purified by high performance liquid chromatography and digested with V8 protease. Two peptides with amino-terminal sequences beginning at residue 576 of the alpha 2(I) chain were isolated. One had the normal sequence. The other differed in that aspartic acid replaced glycine at residue 580 as predicted from cDNA analysis, and in having an unhydroxylated proline at residue 579. From yields on microsequencing and the relative intensities of the two forms of alpha 2(I)CB3,5 on SDS-polyacrylamide gel electrophoresis, the ratio of mutant to normal alpha 2(I) chains in the infant's bone matrix was 0.7/1. Although the effects of an efficient incorporation of mutant chains on the properties of the bone matrix are unknown, it may be that in this OI case the tissue abnormalities result more from the presence of mutant protein than from an underexpression of matrix.     

Characterization of point mutations in the collagen COL1A1 and COL1A2 genes causing lethal perinatal osteogenesis imperfecta

Type I collagen mutations in a group of patients with lethal perinatal osteogenesis imperfecta were identified in fibroblast RNA by a new method which can detect, by chemical modification and cleavage, single mismatched bases in heteroduplexes formed between mRNA and normal cDNA probes. Control cDNA probes spanning the area of the pro-alpha 1(I) and pro-alpha 2(I) chains likely to contain the mutations were radioactively labeled and used to form heteroduplexes with total patient RNA. Treatment of these heteroduplexes with hydroxylamine followed by cleavage of the cDNA strand at reactive bases by piperidine identified mismatches in the pro-alpha 1(I) cDNA in four patients. In the fifth patient a mismatch was detected in the pro-alpha 2(I) cDNA. To characterize these mutations the regions containing the mismatches were amplified by the polymerase chain reaction, cloned, and sequenced. All were heterozygous single base mutations which led to the substitution of glycine residues in the helical region of the pro-alpha-chains. The substitutions were pro-alpha 1(I) Gly973 and Gly1006 to Val, Gly928 to Ala, Gly976 to Arg, and pro-alpha 2(I) Gly865 to Ser. These mutations emphasize the importance of the Gly-X-Y repeating amino acid sequence for normal collagen helix formation and function in the extracellular matrix.