A cysteine for glycine substitution at position 175 in an alpha 1 (I) chain of type I collagen produces a clinically heterogeneous form of osteogenesis imperfecta.

The molecular basis for Osteogenesis Imperfecta in a large kindred with a highly variable phenotype was identified by sequencing the mutant pro alpha 1 (I) protein, cDNA and genomic DNA from the proband. Fibroblasts from different affected individuals all synthesize both normal Type I procollagen molecules and abnormal Type I procollagen molecules in which one or both pro alpha 1 (I) chain(s) contain a cysteine residue within the triple helical domain. Protein studies of the proband localized the mutant cysteine residue to the alpha 1 (I) CB 8 peptide. We now report that cysteine has replaced glycine at triple helical residue 175 disrupting the invariant Gly-X-Y structural motif required for perfect triple helix formation. The consequences include post-translational overmodification, decreased thermal stability, and delayed secretion of mutant molecules. The highly variable phenotype in the present kindred cannot be explained solely on the basis of the cysteine for glycine substitution but will require further exploration.

Repeated helical epitopes of defined amino acid sequence in human type III collagen identified by monoclonal antibodies.

Two monoclonal antibodies, designated 1F8 (IgG1) and 5B10 (IgG1), have been produced in mice against native human type III collagen. These antibodies were highly type and species specific, recognizing the triple helical domain of type III as tested by ELISA. Immunofluorescence studies using each of these antibodies resulted in a fibrous staining pattern in human skin dermis. Immunogold electron microscopy resulted in a periodic distribution of gold particulates along banded collagen fibrils. Assuming that the total contour length of pepsin digested type III collagen is 300 nm, measurements of antibody-antigen complexes visualized by rotary shadowing revealed that each antibody bound at the same two sites: one approximately at the middle of the helix (153 nm from the N-terminus), the other at a site one-quarter the triple helical length from the N-terminus (75 nm). That the one-quarter binding site was closest to the N-terminus was determined by antibody incubation following tadpole collagenase treatment, which results in a larger, N-terminus containing fragment (binding antibody) and a smaller C-terminus containing fragment (not binding antibody). Located at each antibody binding epitope is a sequence of 10 amino acids: Gly-Ala-Hyp-Gly-Leu-Arg-Gly-Gly-Ala-Gly. Renatured cyanogen bromide-cleaved(CB)-peptides, CB4 and CB8, containing these repeated sequences reacted with each antibody, whereas other renatured type III CB-peptides were unreactive as determined by Western blotting analysis and ELISA. This was further confirmed by inhibition tests using a 10 residue synthetic peptide of identical sequence, which yielded 20-30% inhibition of antibody binding to native type III collagen at 4 degrees C. However, no inhibition was noted at higher temperature. These results indicate that both monoclonal antibodies recognize a specific helical conformation of 10 or slightly fewer residues in the three identical polypeptide chains comprising type III collagen.

Characterization of a COL1A1 splicing defect in a case of Ehlers-Danlos syndrome type VII: further evidence of molecular homogeneity.

A child affected by the type VII form of Ehlers-Danlos syndrome (EDS VII) was shown to have a heterozygous structural defect in the amino-terminus of pro-alpha 1(I) collagen. As a result, type I procollagen trimers containing defective subunits are not converted to mature collagen molecules. To identify the cause of the protein abnormality, specifically primed cDNAs and genomic DNA were PCR amplified and sequenced. This analysis disclosed that the protein structural defect is caused by a single base substitution (A for G) at position -1 of the splice donor site of intron 6 of the pro-alpha 1(I) collagen gene (COL1A1). The affected allele produces (a) transcripts lacking exon 6 sequences and (b), in lesser amount, normally spliced transcripts. Furthermore, the rate of exon 6 skipping is temperature dependent, for it appears to decrease substantially when the patient's fibroblasts are incubated at 31 degrees C. These findings are similar to those we previously reported for other unrelated EDS VII cases and, therefore, reemphasize the molecular homogeneity of this rare connective tissue disorder.

Linkage of Marfan syndrome and a phenotypically related disorder to two different fibrillin genes.

Marfan syndrome (MFS), one of the most common genetic disorders of connective tissue, is characterized by skeletal, cardiovascular and ocular abnormalities. The incidence of the disease is about 1 in 20,000, with life expectancy severely reduced because of cardiovascular complications. As the underlying defect is unknown, MFS diagnosis is based solely on clinical criteria. Certain phenotypic features of MFS are also shared by other conditions, which may be genetically distinct entities although part of a clinical continuum. Immunohistochemical studies have implicated fibrillin, a major component of elastin-associated microfibrils, in MFS aetiology. Genetic linkage analysis with random probes has independently localized the MFS locus to chromosome 15. Here we report that these two experimental approaches converge with the cloning and mapping of the fibrillin gene to chromosome 15q15-21, and with the establishment of linkage to MFS. We also isolated a second fibrillin gene and mapped it to chromosome 5q23-31. We linked this novel gene to a condition, congenital contractural arachnodactyly, that shares some of the features of MFS. Thus, the cosegregation of two related genes with two related syndromes implies that fibrillin mutations are likely to be responsible for different MFS phenotypes.

Ehlers-Danlos syndrome type IV: diagnosis and therapy of associated bowel perforation.

Ehlers-Danlos syndrome type IV is a heritable disease of type III collagen metabolism. This diagnosis is suspected in a patient with a combination of clinical manifestations and family history, but it is confirmed only by culture of the patient's skin fibroblasts and demonstration of a defect in type III collagen metabolism. The disease may rarely present with spontaneous colonic perforation, a complication traditionally treated by primary closure of the perforated segment and creation of an end colostomy. Attempts at bowel reanastomosis have often resulted in repeated colon perforations. We present the first patient with Ehlers-Danlos type IV syndrome to develop a colon perforation proximal to an end colostomy, and discuss the surgical strategy to prevent recurrences of this and other postoperative complications associated with the syndrome.

Immunohistologic abnormalities of the microfibrillar-fiber system in the Marfan syndrome.

BACKGROUND: Indirect-immunofluorescence studies of skin and cultured dermal fibroblasts from patients with the Marfan syndrome demonstrate apparent deficiency of one element of connective tissue--the microfibrillar-fiber system--in assays using specific antibodies against fibrillin, a major microfibrillar protein. This study was designed to test whether these immunostaining abnormalities are consistent and diagnostic features of the disease. METHODS: We studied patients with either the Marfan syndrome or various other inherited connective-tissue disorders and normal subjects according to a single-blind protocol in which coded samples of skin, fibroblast cultures, or both were analyzed without knowledge of the clinical diagnosis and classified as "Marfan" or "non-Marfan" before the sample codes were broken. RESULTS: Of the 27 patients with the Marfan syndrome, 24 were correctly identified by the decreased content of microfibrillar fibers in their skin, cultured fibroblasts, or both; in contrast, 19 of 25 patients with other heritable disorders of connective tissue and all 13 normal subjects were correctly classified as "non-Marfan" by these assays (P less than 0.001). CONCLUSIONS: These results document consistent, relatively specific abnormalities of microfibrillar fibers in the Marfan syndrome. The biomechanical incompetence of these structural elements, due to quantitative or qualitative abnormalities, may account for the pleiotropic clinical manifestations of the disease. Therefore, various defects in the expression, structure, assembly, or degradation of the constituent structural glycoprotein (or glycoproteins) of microfibrils may be implicated in the causation of the Marfan syndrome.

Segmental amplification of the entire helical and telopeptide regions of the cDNA for human alpha 1 (I) collagen.

Type I collagen is the site of several common genetic diseases and therefore, the diagnosis of mutational defects occurring therein is of considerable importance. By the polymerase chain reaction amplification of a series of seven overlapping segments, we show that the entire helical and telopeptide regions of the human alpha 1 (I) collagen cDNA can be cloned for sequencing. Unlike all other means of identifying collagen mutations, including protein sequencing and electrophoretic analysis, RNase A hybrid analysis and chemical cleavage of DNA or RNA heteroduplexes, the technique presented is capable of identifying all mutations and polymorphisms without false negative results.

In vivo and in vitro noncovalent association of excised alpha 1 (I) amino-terminal propeptides with mutant pN alpha 2(I) collagen chains in native mutant collagen in a case of Ehlers-Danlos syndrome, type VII.

The cause of the Ehlers-Danlos syndrome Type VII (EDS VII) is considered to be defective removal of the amino-terminal propeptide (N-propeptide) of Type I procollagen due to deficiency of procollagen N-proteinase, the enzyme responsible for the normal proteolytic excision of this precursor-specific domain. Molecules retaining the N-propeptide (pN-collagen molecules) are thought to cause defective fibrillogenesis and cross-linking which eventuate in dramatic joint laxity and joint dislocations, the clinical hallmark of this variety of EDS. Recent studies demonstrate that some EDS VII patients harbor small deletions of either the pro-alpha 1(I) or pro-alpha 2(I) chain of Type I procollagen. We have found an 18-amino acid deletion (due to exon outsplicing) in a mutant pro-alpha 2(I) chain from such a patient. The deleted peptide is the junctional segment (N-telopeptide) linking the alpha 2(I) N-propeptide and major triple helical domains; loss of this short segment results in union of these latter domains and produces a shortened pN alpha 2(I) chain. Directly extracted tissue collagen and pepsin-digested fibroblast collagen contain this mutant pN alpha 2(I) chain and normal alpha 1(I) chains, but not pN alpha 1(I) chains, indicating that the relatively larger alpha 1(I) N-propeptide is excised from the related alpha 1(I) chains. The fate of this alpha 1(I) N-propeptide was unclear and therefore whether or not the intact N-propeptide was, in fact, retained in native mutant collagen was also unclear. In this paper, we describe morphologic, chemical, and immunochemical studies which indicate that the alpha 1(I) N-propeptide is retained in noncovalent association with the mutant pN alpha 2(I) chain in native mutant collagen molecules both in vivo and in vitro. In both instances, the alpha 1(I) N-propeptides are proteolytically cleaved from the related alpha 1(I) chains. These data suggest that retention of a partially cleaved, but essentially intact N-propeptide in mutant collagen may play a role in the pathogenesis of this disease.

Cosegregation of elastin-associated microfibrillar abnormalities with the Marfan phenotype in families.

The Marfan syndrome is a serious heritable connective-tissue disorder characterized primarily by ocular, cardiovascular, and musculoskeletal abnormalities but also involving multiple other tissues and organs of the body. Inherited as an autosomal dominant disorder, the etiology and pathogenesis of the Marfan syndrome are presently unknown. We have documented consistent apparent deficient content of elastin-associated microfibrillar fibers by indirect immunofluorescent (IF) studies of Marfan skin, as well as deficient accumulation of related fibrous materials in cultures of Marfan fibroblasts as compared with normal controls and patients with other heritable disorders of connective tissue. These data have suggested that abnormalities in the microfibrillar component of elastic-fiber systems may have a role in the etiology and pathogenesis of the Marfan syndrome. In the present study, we have analyzed the IF staining patterns of skin and fibroblast cultures from Marfan syndrome patients and normal first-degree relatives in nine Marfan kindreds. Three of these families had at least one affected individual in each of 2 generations, permitting intergenerational comparison of IF patterns. Six kindreds had one or more affected individuals in a single generation, making comparisons between siblings and/or parent-child possible. In all cases, IF abnormalities cosegregated with the Marfan phenotype and all nonaffected family members were normal. Within family groups containing more than one affected individual, the IF staining patterns were similar between affected patients. These data provide further confirmation of consistent and relatively specific deficiency of microfibrillar fibers in Marfan syndrome.

Unilateral microfibrillar abnormalities in a case of asymmetric Marfan syndrome.

The Marfan syndrome is a dominantly inherited connective-tissue disorder characterized by ocular, cardiovascular, and musculoskeletal abnormalities. Although the underlying biochemical and molecular defect(s) of this pleiotropic disease is currently unknown, we have consistently observed apparent diminished content of elastin-associated microfibrillar fibers accumulating in skin, or produced by cultured fibroblasts, from patients with the Marfan syndrome and have documented the cosegregation of these immunofluorescent abnormalities of microfibrillar fibers with the Marfan syndrome phenotype in family studies. Recently, an unusual patient has been described with unilateral phenotypic features of the Marfan syndrome, providing an unique opportunity to compare microfibrillar fibers and other connective-tissue components between the affected and nonaffected sides. In the present report, we demonstrate striking differences in apparent content of microfibrillar fibers, as determined by indirect immunofluorescence of skin and fibroblast cultures, that are revealed when multiple homologous samples derived from different sides of the patient's body are compared. In contrast, no differences in apparent content of type III collagen or in the biosynthesis and apparent structure of types I and III (pro)collagens were found. HLA types and chromosome heteromorphisms were identical in fibroblasts from both sides of the body, eliminating the formal possibility of chimerism and suggesting that a postzygotic mutation accounts for the asymmetric manifestation of the Marfan syndrome in this patient. The observation of striking decreases in microfibrillar fibers on the affected side of the body provides further evidence that abnormalities of this component of the elastic fiber system may be central to the pathogenesis and possibly the etiology of the Marfan syndrome.

Glycine to serine substitution in the triple helical domain of pro-alpha 1 (II) collagen results in a lethal perinatal form of short-limbed dwarfism.

Previous biochemical studies on cartilage tissue from a proband with Type II achondrogenesis-hypochondrogenesis (Godfrey, M., and Hollister, D. W. (1988) Am. J. Hum. Genet. 43, 904-913) indicated heterozygosity for a structural abnormality in the triple helical domain of pro-alpha 1 (II) collagen. Here we demonstrate that the mutation in the type II procollagen gene is a single base change that converts the codon for glycine (GGC) at amino acid 943 of the alpha 1 (II) chain to a codon for serine (AGC). The substitution disrupts the invariant Gly-X-Y structural motif necessary for perfect triple helix formation and leads to extensive overmodification, intracellular retention, and reduced secretion of type II collagen. These findings confirm the proposal that new dominant mutations in the type II procollagen gene may account for some cases of Type II achondrogenesis-hypochondrogenesis. Since recent studies (Lee, B., Vissing, H., Ramirez, F., Rogers, D., and Rimoin, D. (1989) Science 244, 978-980) have identified a dominantly inherited type II procollagen gene deletion in a non-lethal form of skeletal dysplasia, namely spondyloepiphyseal dysplasia, the data more generally demonstrate that different type II procollagen gene mutations eventuate in a wide and diverse spectrum of clinical phenotypes.

Types of collagen in breast capsules.

Representative sections of the capsules that envelop surgically implanted silicone were analyzed for their collagen content. The specimens were biopsied (as incidental procedures) as the opportunities arose. All four classes of capsules as codified clinically and those surrounding tissue expanders are represented. The ages of the capsules ranged from 6 weeks to 13 years. Eighteen specimens were examined by slab gel electrophoresis. Type I collagen predominated in all of the electrophoretic patterns. Type III collagen was consistently present in lesser concentrations. Type V collagen was consistently present as a small fraction. There was no evident correlation between the concentrations of the component collagens and the clinically assessed class of the capsules or of those from around tissue expanders, but there was a correlation to the age of the capsules. The electrophoretic patterns of the collagens of this study are indistinguishable from the (previously reported) analyses of (cutaneous) scar.

Temperature-dependent expression of a collagen splicing defect in the fibroblasts of a patient with Ehlers-Danlos syndrome type VII.

In this article we report the characterization of the molecular lesion in a patient with Ehlers-Danlos syndrome Type VII and provide evidence that a de novo substitution of the last nucleotide of exon 6 in one allele of the pro-alpha 2(I) collagen gene produces normally spliced mRNA and transcripts from which exon 6 sequences have been outspliced as well. Unexpectedly, the expression of the alternative splicing was found to be temperature-dependent, for missplicing in cellula is effectively abolished at 31 degrees C and gradually increases to 100% at 39 degrees C. In contrast, in a similar patient harboring a substitution in the obligatory GT dinucleotide of the 5' splice site of intron 6, complete outsplicing of exon 6 sequences was found at all temperatures.

Decreased thermal denaturation temperature of osteogenesis imperfecta mutant collagen is independent of post-translational overmodifications of lysine and hydroxylysine.

Fibroblasts from many patients with osteogenesis imperfecta (OI) synthesize and secrete Type I collagen which is both overmodified and exhibits a decreased thermal denaturation temperature. We have examined the relationship between overmodification and decreased melting temperature in several favorable OI mutants by selectively inhibiting lysyl hydroxylase activity with the drug Minoxidil and comparing the melting profiles of the resultant undermodified collagen with untreated control. Minoxidil treatment causes an appreciable decrease in hydroxylysine with compensatory increases in lysine content, and the delayed sodium dodecyl sulfate-polyacrylamide gel electrophoretic mobility of the overmodified collagen chains becomes normal. However, the decreased melting temperature was unchanged from untreated OI control. When unhydroxylated collagen produced by normal control and OI fibroblasts incubated with alpha,alpha'-dipyridyl was examined, mutant OI molecules melted at a lower temperature than control. These data indicate that the decreased thermal denaturation temperature of OI mutant collagen is independent of post-translational overmodification of lysine or hydroxylysine. Presumably, substitutions for glycine in the Gly-X-Y structural motif distort the helix and produce lower melting temperatures by presently unknown mechanisms.

Diastrophic dysplasia: evidence against a defect of type II collagen.

A description of an abnormal segment-long-spacing crystallite (SLS) pattern has been reported for type II collagen from patients with diastrophic dysplasia (Stanescu et al., 1982 a), a disorder that is characterized by large collagen fibrils in the cartilage matrix. The abnormal SLS consisted of an altered electron density between bands 42 and 45, which was interpreted as an abnormality in the type II collagen molecule. It was suggested that the type II collagen is abnormal in diastrophic dysplasia. We have examined SLS of type II collagen from two patients with diastrophic dysplasia and found the SLS patterns to be identical with that of control type II SLS in almost all micrographs. In a few micrographs of diastrophic SLS, crystallites exhibiting the pattern reported by Stanescu et al. were seen. However, the abnormally patterned crystallites always consisted of dimers that were overlapped at the COOH ends in such a way that an electron dense band of one crystallite was positioned between bands 42 and 45 of the second crystallite, apparently creating the abnormal pattern. The abnormal SLS pattern seen in these cases of diastrophic dysplasia appears to be the result of overlapping crystallites and may not be the result of an intrinsic abnormality of type II collagen. We have constructed histograms of the collagen fibril diameters in diastrophic cartilage. While they are larger than normal collagen fibrils, this by itself does not indicate an abnormality of type II collagen. We have shown that large fibrils such as these can be obtained from normal type II collagen when the structure of the cartilage is disrupted by extraction with guanidine.(ABSTRACT TRUNCATED AT 250 WORDS)

A cysteine for glycine substitution at position 1017 in an alpha 1(I) chain of type I collagen in a patient with mild dominantly inherited osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heterogeneous group of inherited diseases of connective tissue manifested primarily by excessive fracturing of bone but associated with other abnormalities such as blue sclera, thin skin, herniae, ligamentous laxity, reduced stature and hearing loss. We report here molecular studies on a patient with the mild, dominantly inherited, variety of OI (OI type I) previously shown (Nicholls et al., 1984) to be heterozygous for an abnormal alpha 1(I) chain of type I collagen which contained cysteine near the carboxyl terminus (Steinmann et al., 1986). The cognate alpha 1(I) mRNA region was selected for generation of cDNAs which were subsequently amplified by the polymerase chain reaction (PCR), cloned and sequenced. Two sequences were obtained, one of which corresponded to the normal allele, and the other of which harbored a G to T transversion and resulted in a cysteine for glycine substitution. This is the first single amino acid substitution found in type I OI. Surprisingly, the mutation occurs just outside the triple-helical region of the alpha 1(I) chain, a result that accounts for the strikingly different phenotypic and molecular consequences of this mutation as compared with similar cysteine for glycine substitutions within the same region. The PCR appears to be a useful approach for elucidation of structural mutations in collagen chains.

Type II achondrogenesis-hypochondrogenesis: morphologic and immunohistopathologic studies.

A 32-wk-gestation female with type II achondrogenesis-hypochondrogenesis has been studied. The clinical features were typical, and radiographs revealed short ribs, hypoplastic ilia, absence of ossification of sacrum, pubis, ischia, tali, calcanei, and many vertebral bodies; the long bones were short with mild metaphyseal flaring. The femoral cylinder index was 6.3. Comparison with previous cases placed the patient toward the mild end of the achondrogenesis-hypochondrogenesis spectrum (Whitley-Gorlin prototype IV). Light microscopy revealed hypercellular cartilage with decreased matrix traversed by numerous fibrous vascular canals. The growth plate was markedly abnormal. Ultrastructural studies revealed prominently dilated rough endoplasmic reticulum containing a fine granular material with occasional fibrils in all chondrocytes. Immunohistologic studies indicated irregular large areas of cartilage matrix staining with monoclonal antibody to human type III collagen. The relative intensity of matrix staining for type II collagen appeared diminished. More striking, however, were intense focal accumulations of type II collagen within small rounded perinuclear structures of most chondrocytes but not other cell types. These results strongly suggest intracellular retention of type II collagen within vacuolar structures, probably within the dilated rough endoplasmic reticulum observed in all chondrocytes by electron microscopy (EM), and imply the presence of an abnormal, poorly secreted type II collagen molecule. Biochemical studies (see companion paper) suggest that this patient had a new dominant lethal disorder caused by a structural abnormality of type II collagen.

Type II achondrogenesis-hypochondrogenesis: identification of abnormal type II collagen.

We have extended the study of a mild case of type II achondrogenesis-hypochondrogenesis to include biochemical analyses of cartilage, bone, and the collagens produced by dermal fibroblasts. Type I collagen extracted from bone and types I and III collagen produced by dermal fibroblasts were normal, as was the hexosamine ratio of cartilage proteoglycans. Hyaline cartilage, however, contained approximately equal amounts of types I and II collagen and decreased amounts of type XI collagen. Unlike the normal SDS-PAGE mobility. Two-dimensional SDS-PAGE revealed extensive overmodification of all type II cyanogen bromide peptides in a pattern consistent with heterozygosity for an abnormal pro alpha 1(II) chain which impaired the assembly and/or folding of type II collagen. This interpretation implies that dominant mutations of the COL2A1 gene may cause type II achondrogenesis-hypochondrogenesis. More generally, emerging data implicating defects of type II collagen in the type II achondrogenesis-hypochondrogenesis-spondyloepiphyseal dysplasia congenita spectrum and in the Kniest-Stickler syndrome spectrum suggest that diverse mutations of this gene may be associated with widely differing phenotypic outcome.