A base substitution at the splice acceptor site of intron 5 of the COL1A2 gene activates a cryptic splice site within exon 6 and generates abnormal type I procollagen in a patient with Ehlers-Danlos syndrome type VII.

The dermal type I collagen of a patient with Ehlers-Danlos type VIIB (EDS-VIIB) contained normal alpha 2(I) chains and mutant pN-alpha 2(I)' chains in which the amino-terminal propeptide (N-propeptide) remained attached to the alpha 2(I) chain. Similar alpha 2(I) chains were produced by cultured dermal fibroblasts. Amino acid sequencing of tryptic peptides, prepared from the mutant amino-terminal pN-alpha 2(I) CB1' peptide, indicated that five amino acids, including the N-proteinase (the specific proteinase that cleaves the procollagen N-propeptide) cleavage site, had been deleted from the junction of the N-propeptide and the N-telopeptide (the nonhelical domain at the amino-terminus of the alpha chains of fully processed type I polypeptide chains) of the mutant pro-alpha 2(I)' chain. The corresponding 15 nucleotides, which were deleted from approximately half of the alpha 2(I) cDNA polymerase chain reaction products, of the alpha 2(I) cDNA polymerase chain reaction products, were encoded by the +1 to +15 nucleotides of exon 6 of the normal alpha 2(I) gene (COL1A2). These 15 nucleotides were deleted in the splicing of alpha 2(I) pre-mRNA to mRNA as a result of inactivation of the 3' splice site of intron 5 by an AG to AC mutation and the activation of a cryptic AG splice acceptor site corresponding to positions +14 and +15 of exon 6. Loss of the N-proteinase cleavage site explained the persistence of the pN-alpha 2(I)' chains in the dermis and in fibroblast cultures. Collagen production by cultured dermal fibroblasts was doubled, possibly due to reduced feedback inhibition by the N-propeptides. In contrast to previously reported cases of EDS-VIIB, Lys5 of the N-telopeptide was not deleted and appeared to take part in the formation of intramolecular cross-linkages. However, increased collagen solubility and abnormal extraction profiles of the mutant type I collagen molecules indicated that collagen cross-linking was abnormal in the dermis. The proband and her son were heterozygous for the mutation. It is likely that the heterozygous loss of the N-proteinase cleavage site, with persistence of a shortened N-propeptide, was the major factor responsible for the EDS-VIIB phenotype.

Type I collagen is the major fibrillar collagen of dermis, ligament, tendon, and bone (I). The component al (1) and a2(I) chains are synthesized as prepro-a chains. The signal peptides are removed shortly after translation, and the procollagen chains are assembled into triple helical molecules after the post-translational modifications of certain prolyl and lysyl residues. During or shortly after secretion, the amino-and carboxyl-terminal propeptide extensions of the pro-a chains are removed by N-and C-proteinases,' respectively, to yield the mature chains of type I collagen (1).
Genetically determined defects in the processing of the Npropeptide produce the type VI1 variant of the Ehlers-Danlos syndrome (EDS-VII) (2). Three subtypes of EDS-VI1 have been described. Patients with EDS-VIIA and EDS-VIJB have similar clinical phenotypes, with multiple congenital dislocations, generalized joint laxity, and mild laxity and fragility of the skin. EDS-VIIC, which has only been described in animals, is characterized by gross skin fragility referred to as dermatosparaxis ("torn skin") (3).
In EDS-VIIA, the N-propeptide remains attached to the al(1) chains due to loss of the N-proteinase cleavage site with persistence of the composite pN-al(1) chain in some of the type I collagen molecules that are incorporated into the extracellular matrix (4). In one patient, the molecular defect consisted of a heterozygous mutation in which the 3' codon of exon 6 of the COLlAl gene was changed from ATG for methionine 159 to ATA for isoleucine (5). Cultured EDS-VIIA fibroblasts produced approximately 50% normal proal(1) chains, 10% mutant chains containing the amino acid substitution, and 40% mutant chains lacking the 24 amino acids normally encoded by exon 6. The deletion of exon 6encoded sequences from pro-al(1) chains, due to skipping of these sequences in the splicing of pre-mRNA to mRNA, removed the N-proteinase cleavage site and the lysine crosslinking site in the N-telopeptide (5).
In EDS-VIIB, the N-propeptide remains attached to the a2(I) chain because of loss of the N-proteinase cleavage site from the pro-a2(1) chain. Five patients have been shown to have heterozygous mutations of exon 6 or intron 6 of the COLlA2 gene. One patient had the same mutation in the 3' nucleotide of exon 6, as observed in the patient with EDS-The abbreviations used are: N-and C-proteinases, the specific proteinases that cleave the procollagen amino-and carboxyl-terminal propeptides, respectively; EDS, Ehlers-Danlos syndrome; EDS-VII, type VI1 variant of the syndrome; COLIAI and COLIA2, gene locus for al (1) and &(I) chains of type I collagen, respectively; N-propeptide, amino-terminal propeptide; pN-al(1) and pN-aP(I), partially processed type I procollagen chains retaining their N-propeptides; Ntelopeptide, nonhelical domain at the amino terminus of the a chains of fully processed type I polypeptide chains; CNBr, cyanogen bromide; CB, cyanogen bromide peptide; SDS, sodium dodecyl sulfate; bp, base pair(s); PTH, phenylthiohydantoin; PCR, polymerase chain reaction. VIIA (6). It also led to the production of two mutant mRNAs. Three patients had the same base change, A for G, in the highly conserved +1 splice donor site of intron 6 (7-9). Another patient had a substitution of C for T at the +2 position of intron 6 (10). These intronic substitutions altered the highly conserved -GT-splice donor site and led to complete skipping of exon 6 sequences in the splicing of pre-mRNA to mRNA. Exon 6 of the COLlA2 gene encodes 18 amino acids, including the N-proteinase cleavage site and the lysine crosslinking site in the N-telopeptide (11). EDS-VIIC is due to an autosomal recessive deficiency in N-proteinase activity leading to the persistence of the Npropeptides of the pro-al(1) and pro-a2(I) chains (3). No humans have yet been reported with this form of EDS-VII.
We report a patient with EDS-VIIB who had a heterozygous mutation of the splice acceptor site of intron 5 of the COLlA2 gene that resulted in the loss of 15 nucleotides from a2(I) mRNA and five amino acids from pro-a2(I) chains. The deleted sequence corresponded to the +1 to +15 nucleotides of exon 6 of the COLlA2 gene. The N-proteinase cleavage site was deleted, but the lysine cross-linking site was retained within the N-telopeptide of mutant a2(I) chains.

EXPERIMENTAL PROCEDURES~
The proband was a 30-year-old female with congenital dislocations of the hips and severe laxity of other joints. The clinical features were of the type VI1 form of EDS, and it was inherited as an autosomal dominant trait (2). Skin biopsies were obtained from the forearm of the proband. Her son, who was also born with dislocated hips, died suddenly a t 3 months of age. Fibroblast cultures and tissue samples were not available, but formalin-fixed tissue samples, obtained at autopsy 11 years ago, were available for analysis. All samples were collected with approval of the ethics committee of this hospital.

RESULTS
Characteristics of Dermal Collagens-Electrophoresis of sequential NaCl, acetic acid, and guanidine HCl extracts from EDS dermis revealed an additional collagenous band that was not observed in extracts of age-matched normal dermis ( 1). A similar band was also noted in the limited pepsin digest that was undertaken without previous extractions of the dermis. The abnormal component migrated with the characteristics of a mutant pN-aB(1) chain, as reported in patients with EDS-VIIB (12,13). It was designated as a pN-aS(1)' chain. The proportion of the a2(I) chain that was present in the mutant form was 53% in the salt-soluble fraction, 29% in the acetic acid-soluble fraction, 80% in the guanidine-soluble fraction, and 23% in the limited pepsin digest.
Dimeric, trimeric, and higher order cross-linked collagen chains were observed in the extracts of EDS and control dermis (Fig. 1). Differences were observed in the proportions of the dimeric 011 and 012 chains in extracts of the EDS and control dermis. In the acetic acid, guanidine HC1, and pepsin extracts of the control dermis, the 012:0ll ratios were 3.8, 2.3, and 2.6, respectively. In similar EDS extracts, these ratios were reduced to 2.7, 1.5, and 1.6.
In all EDS extracts, the 011 chains migrated normally, but differences were observed in the migration of the 012 chains. In the acetic acid extract, the 012 chains migrated slowly, whereas in the guanidine and pepsin extracts, the 012 band was broad and contained both normally migrating and slowly migrating forms of this dimer. The two forms of the 012 dimer were not resolved sufficiently to enable their proportions to be quantified. A similar abnormal 012 dimer, consisting of a normal al(1) chain and a mutant pN-a2(1)' chain, was reported in the tissues of another case of EDS-VIIB (13). As a result, it was designated 012'.
Sequential extraction solubilized more collagen from EDS than from control dermis (Table I). Acetic acid and guanidine HCl solubilized approximately 2 and 4 times the amount of collagen from EDS dermis, respectively. However, the amount of collagen solubilized by limited pepsin digestion of EDS dermis was only minimally increased.
Characteristics of Collagens Produced by Cultured Fibroblasts-EDS and control fibroblasts produced type I and I11 collagens in short term cultures (Fig. 2). In the EDS samples, the migrations of the al(1) chains and the disulfide-linked al(II1) chains were normal, but in addition to the normally migrating a2(I) chain, there was also a more slowly migrating chain similar to that observed in extracts of dermis. The abnormal chain was designated as a mutant pN-aB(1)' chain.
Total collagen production by the EDS fibroblasts was more than double the control values (Table 11). Both type I and I11 collagen production was increased, with the increase being greater for type I collagen. Approximately equal amounts of the normal and mutant a2(I) chains were produced by EDS fibroblasts (Table 111). Type I procollagen molecules containing the mutant pro-a2(1)' chain appeared to be secreted more efficiently than those containing the normal pro-a2(I) chain.

TABLE I Solubility of dermal collagen
The dermis was defatted in chloroform/methanol, lyophilized, and either extracted sequentially with NaCI, acetic acid, and guanidine HCI or digested with pepsin as described under "Experimental Procedures." The results are expressed as pg of collagen/mg, dry weight, of dermis (34), and the percentage of the collagen extracted is indicated in parentheses.   "Values that were more than 2 standard deviations above the control means.

Normal and mutant &?(I) chain production and secretion by EDS fibroblasts
The production and distribution of normal and mutant a2(I) chains by cultured EDS dermal fibroblasts were determined as described under "Experimental Procedures." The values in parentheses represent the percentage of mutant chains in each sample. The distribution of the normal and mutant a2(I) chains between the cell layer and medium was used to calculate the secretion of type I collagen molecules containing either a normal a2(I) or a mutant ~N-a2(1)' chain. Amino Acid Sequence of Mutant Collagen Peptide-The electrophoretic findings were consistent with a heterozygous mutation involving the deletion of the N-proteinase cleavage site with persistence of the N-propeptide attachment to the arS(1) chain. To investigate this proposal further, we purified the normal and mutant aZ(1) chains and peptides from the medium of long term cultures of EDS fibroblasts.

Normal a2(I) chain
Procollagen was converted to collagen by limited pepsin digestion under conditions that preserved the pN-aP(1)' chains (13). Normal al(1) and 02(I) chains, as well as the pN-(r2(1)' chains were separated from disulfide-linked type I11 collagen chains by gel permeation chromatography (Fig. 3). The normal and mutant a2(I) chains were then resolved from most of the al(1) chains by reverse-phase chromatography ( Fig. 4). Electrophoresis of CNBr peptides showed that the coeluting a2(I) and pN-aZ(1)' chains contained the major aP(1) CB3.5 and CB4 peptides, as well as an abnormal peptide that had an approximate M, of 7,000 (Fig. 5). The migration of the abnormal peptide was similar to the migration of the normal pN-aZ(1) CB1 peptide: and it was therefore designated as a pN-aZ(1) CB1' peptide (13). Incompletely cleaved CNBr peptides were also observed, and the two that were unique to the EDS sample probably contained the pN-aZ(1) CB1' peptide ( Fig. 5) (13).
The pN-aZ(1) CB1' peptide was purified by reverse-phase chromotography (Fig. 6) and digested with thermolysin. The thermolytic peptides were resolved by reverse-phase chromotography (results not shown), and selected peptides were sequenced. Peptide Tm6 revealed the abnormal sequence Leu-Gly-X-Tyr-Asp-Gly. Both glycine and proline were found at position 3, probably due to a contaminant peptide coeluting with peptide Tm6. Neither of the possible sequences occur in normal pro-a2(I) or pro-al(1) chains (11). However, if the unassigned residue was Gly, then the unusual sequence would be produced by a deletion of five amino acids from within the normal pro-a2(1) sequence Le~~~-Gly-Gly-Asn-Phe-AZu-AZu-Gln-Tyr-A~p-Gly'~ '. Such a deletion would remove the normal N-proteinase cleavage site at Ala57-Gln58 and account for the persistence of the pN-aZ(1)' chains in EDS fibroblast cultures and dermis (11). The deleted peptide is normally encoded by the +1 to +15 nucleotides of exon 6 of the COLlA2 gene (10). This proposal was investigated further by cDNA and genomic DNA sequencing.
Characteristics of the Mutant DNA Sequences-First strand cDNA was synthesized from total fibroblast RNA. Amplification of the region of the EDS aS(1) cDNA that spanned the peptide deletion was undertaken, and approximately equal amounts of a normal 229-bp and a mutant 214-bp product were obtained. The larger product contained the normal se- The pN-aP(1) CB1 peptide includes the 57-residue N-propeptide, with its 15 Gly-X-Y triplets and short N-and C-terminal globular domains, the 11-residue N-telopeptide, and the first triplet of the main triple helical domain of the a2(I) chain (11).
The amino acid residues are numbered from the start of the Npropeptide of the pro-a2(1) chain (11). quence, which extended from ProzR of the N-propeptide to T h P of the triple helical domain of the a2(I) chain and was encoded by exon 5 to exon 8 of the COLlA2 gene (Fig. 7). The same sequence was found in all nine normal clones that were sequenced. The smaller product contained the normal sequence, with the exception of a deletion of 15 nucleotides that normally encode Ams4 to Gln", including the N-proteinase cleavage site at Ala"-GlnSR (Fig. 7 ) . The same sequence was found in all nine mutant clones that were sequenced. This finding was consistent with the mutant amino acid sequence obtained from peptide Tm6.
The deletion observed in the abnormal cDNA PCR product corresponded to the +1 to +15 nucleotides of exon 6 of the COLlA2 gene (10,ll). Genomic DNA sequencing was undertaken to determine the precise mutation giving rise to the deletion within the cDNA. A single normal sized product of 195 bp was produced by the PCR using intron 5-and intron 6-specific primers (7). Sequencing of the cloned product revealed normal and mutant sequences that differed at one location (Fig. 8). There was a substitution of G by C at the splice acceptor site of intron 5 (7). Fourteen clones, from two separate PCR reactions, were sequenced, including two normal clones and 12 that contained the substitution of G by C. The +1 to +15 nucleotides of exon 6 corresponded to the a. Mutant Normal A G C T A G C T b. Pro G l y Arg Asp G l y G l u Asp G l y Pro Thr G l y Pro Pro G l y Pro Pro 43

C C C C A C A G G C C C T C C T G G T C C A C C T 3 3 0
Asp G1) EyS G l y V a l G l y Imu G l y Pro :lY Pro Set G l y Len Ikt G l Y 75

I %on 7
Pro Arg G l y Pro Pro G l y Ala Ala G l y Ala Pro G l y Pro G h G l y Phe 91

I %on 8
G l n G l y Pm Ala G l y G l u Pro G l y Glu Pm G l y G l n Thr104  a c t a q t a a c t a a a a a t a t t t t a t a t a t a t a t a t a Asn Phe Ala Ala Gln Tyr Asp Gly sequence deleted from approximately half of the cDNA PCR products. Genomic PCR was also used to determine whether the son of the proband had inherited the EDS mutation. Sixty pg of DNA was obtained from formalin-fixed specimens of spleen, and 6 pg of DNA were from similar specimens of bone. Electrophoresis showed that the extracts contained a mixture of undegraded and partially degraded DNA (Fig. 9). Amplification of total DNA from spleen and bone yielded the expected 195-bp fragment (Fig. 9). Four mutant clones from bone, six mutant clones from spleen, and two normal clones from bone were sequenced. The normal and mutant sequences obtained were the same as those obtained from fibroblast genomic DNA of the proband (results not shown).

a t t t t t t t t t t t t a c t t c t c t a g M C TlT GCT GCT CAG TAT GAT
Spliced Forms of Mutant mRNAs"S1 nuclease cleavage of mRNAcDNA heteroduplexes was used to determine the characteristics of the mRNAs produced by splicing of pre-mRNA in cultured EDS fibroblasts. We observed the expected 138bp protected fragment from the cleavage of heteroduplexes containing mutant mRNA lacking the first 15 nucleotides encoded by exon 6 (Fig. 10). The faster migrating fragments of 137-134 bp were probably generated by excessive digestion of the 138-bp fragment.
A search was also made for other fragments that may have arisen from alternative splicing mechanisms. These included 125-and 122-bp fragments that would have been produced if other AG dinucleotides within the exon 6-encoded sequences were used as cryptic splice acceptor sites and a 99-bp fragment if exon 6 sequences were completely skipped (Fig. 8). However, none of these fragments nor any other sized fragments were observed.
54% of the labeled cDNA probe used to form heteroduplexes

Collagen Mutation in Ehlers-Danlos Syndrome 6365
with EDS mRNA was cleaved by S1 nuclease. As collagen splicing defects may be temperature-sensitive, these analyses were repeated after growing fibroblasts at the lower temperature of 33 "C (6). There was no apparent temperature effect, as 50% of the probe was also cleaved to produce the same cleavage product.
Processing of Procollagen to Collagen-The processing of procollagen to collagen was studied using short term fibroblast cultures to which dextran sulfate was added (14). Under these conditions, the procollagen secreted by normal fibroblasts is rapidly processed, and all of the collagen is in the cell layer. Fully processed al(1) and a2(I) chains and partially processed pN-al(1) and pN-aP(1) chains were observed in the cell layer fractions from EDS and control cultures (Fig. 11). However, processing of the pN-a2(1) chains to a2(I) chains and, to a lesser extent, the processing of pN-al(1) chains to al(1) chains were impaired in EDS cultures. In the control cultures, 8% of the a2(I) chains were in the pN form, whereas in the EDS cultures, 59% were in the pN form. This finding is consistent with the heterozygous nature of the mutation; about half the production consisted of normal pro-a2(1) chains, and the other half consisted of mutants. In control cultures, 20% of the al(1) chains were present in the pN form, whereas in the EDS cultures, 36% were in the pN form. This finding suggested that the removal of the N-propeptide from the pNal(1) chain was retarded in procollagen molecules that contained a mutant pro-a2(1)' chain.
The high molecular weight bands observed in the control sample (Fig. 11) were collagenous, as they were digested with bacterial collagenase. Pro-a chains and PC-a chains were generated following reduction of disulfide bonds with @-mercaptoethanol (results not shown). The high molecular weight bands were, therefore, disulfide-linked procollagens and pCcollagens (14).

DISCUSSION
The proband was shown to have EDS-VIIB due to a heterozygous mutation of the COLlA2 gene. In keeping with previously reported patients, the N-proteinase cleavage site was deleted from mutant pro-a2(1)' mRNA and pro-a2 (1) chains due to anomalous splicing of exon 6 sequences in the conversion of pre-mRNA to mRNA (6)(7)(8)(9)(10). In contrast to previous patients, however, only five rather than all of the 18 amino acids encoded by exon 6 were deleted in the proband. The deleted peptide removed the N-proteinase cleavage site but not the lysine cross-linking site in the N-telopeptide of the a2(I) chain (11).
Pedigree analysis showed autosomal dominant inheritance of the EDS-VIIB clinical phenotype. This inheritance pattern was confirmed by identification of the heterozygous mutation in genomic DNA extracted from the formalin-fixed and paraffin-embedded tissues of the proband's son who was born with dislocated hips and died suddenly at 3 months of age.
The mutation changed the splice acceptor site of intron 5 from -AG-to -AC-, in contrast to other cases of EDS-VIIB in which the mutations involved the 3' nucleotide of exon 6 or the splice donor site of intron 6 of the COLlA2 gene (6)(7)(8)(9)(10). Mutations of the consensus -AG-splice acceptor sequence were shown in human @-globin, dihydrofolate reductase, apolipoprotein E, and arginosuccinate genes to prevent pre-mRNA splicing at the altered site (15)(16)(17)(18). Similarly, a change in the splice acceptor consensus sequence of intron 27 of the COLlA2 gene, from -AG-to -GG-, resulted in the in-frame splicing out of exon 28-encoded sequences in a patient with osteogenesis imperfecta (19). A deletion of 19 bp from the 3' region of intron 10 of the COLlA2 gene removed the consensus -AG-sequence and also produced a splicing defect with an inframe loss of exon 11-encoded sequences in another patient with osteogenesis imperfecta (20). Cryptic splice acceptor sites did not appear to be activated by either COLlA2 mutation.
In the present case of EDS-VIIB, the mutant pro-a2(1)' mRNA lacked 15 nucleotides, and the mutant pN-a2(1)' chains lacked the corresponding five amino acids. These findings indicated that the -AG-dinucleotide sequence at positions +14 and +15 of exon 6 was activated as a cryptic splice acceptor site by cultured EDS fibroblasts. We did not find any evidence, from cDNA PCR products, S1 nuclease cleavage of mRNAcDNA heteroduplexes, or peptide maps, of other spliced products of the mutant allele. In particular, there was no evidence of complete skipping of exon 6 or of the use of other cryptic splice acceptor sites within exon 6. The similar electrophoretic migrations of the pN-aB(1)' chains obtained from fibroblast cultures and dermis suggested that the same cryptic splice acceptor site was used in vivo as in vitro.
The splicing of long pre-mRNAs, such as the pro-a2(1) pre-mRNA, is complex and requires precise localization and correct pairing of cognate 5' and 3' splice sites (21,22). Removal of each intron involves two separate transesterification steps. The first step requires cleavage at the 5' intron-exon junction and yields a free 5' exon. A 2',5'-phosphodiester is formed between the phosphate group at the 5' terminus of the intron and the 2"hydroxyl of an adenosine residue at the branch site to produce an intron loop or "lariat" (21). The second transesterification step joins the exon sequences and displaces the intron, still in the lariat form (22). Most of the mammalian branch points are located within 18-40 nucleotides of the splice acceptor site and have a consensus sequence of -YNC-URAC-, in which Y indicates a pyrimidine, R a purine, and N indicates any base (23). The precise branch point in intron 5 of the pro-a2(1) pre-mRNA was not determined, but it is likely to be at A-57 within the sequence -ACAUGAC-, which has 86% homology with the branch point consensus sequence. This putative branch point in intron 5 is further from the normal 3' splice site than in most mammalian introns (22). However, the long polypyrimidine tract downstream of the branch point would be expected to promote spliceosome as-

Collagen Mutation in Ehlers-Danlos Syndrome
sembly and cleavage at the 5' splice site, even in the absence of the wild type splice acceptor site (22). Recognition of the splice acceptor site may involve a scanning process in which the first -AG-dinucleotide is located downstream of the branch point and polypyrimidine tract (22). In the absence of the wild type consensus sequence, a neighboring "best-fit" splice site is selected, but if a suitable site is not present then exon skipping occurs (24). Such a mechanism may account for the different splicing outcome in our patient when compared with other cases of splice acceptor mutations of the COLlA2 gene. In our patient, the cryptic splice acceptor -AG-site was nearby at positions +14 and +15 of exon 6. In contrast, the cryptic splice acceptor sites in exon 28 in one case of osteogenesis imperfecta and in exon 11 in another case of osteogenesis imperfecta were further away, a t positions +44 and +45, respectively (19,20).
Impaired processing of pN-a2(1) to a2(I) chains, consistent with loss of the N-proteinase cleavage site, was observed in the collagens obtained from the proband's dermis and cultured fibroblasts. To demonstrate the defect using cultured dermal fibroblasts, we either added dextran sulfate to the culture medium to induce processing of procollagen by the N-and Cproteinases or removed the propeptides by limited pepsin cleavage of the telopeptides (14). Both of these techniques produced similar results and confirmed the in uiuo finding of mutant pN-aZ(1)' chains. The studies with dextran sulfate also suggested that cleavage of the N-propeptide of normal pro-al(1) chains was retarded in type I procollagen molecules containing a mutant pro-af(1) chain.
Limited pepsin digestion was also used in previous reports to demonstrate mutant pN-a2(I) chains in patients with EDS-VIIB due to loss of the 18 amino acids normally encoded by exon 6 of the COLlA2 gene (6)(7)(8)(9)(10). In these cases, resistance to limited pepsin cleavage was expected, as the N-telopeptide, with its pepsin-sensitive sites, was deleted. Resistance of the pN-a2(1)' chains to limited pepsin digestion was unexpected in the current case, as only the amino-terminal Gln residue was deleted from the N-telopeptide. However, the peptide deletion disrupted the C-terminal Gly-Asn-Phe triplet of the helical domain of the N-propeptide and resulted in the formation of a new Gly-X-Y triplet consisting of Gly53 of the Npropeptide and Tyr' and Asp3 of the N-telopeptide (29). It is likely that the pepsin-sensitive bonds were inaccessible to cleavage because of conformational changes in the N-telopeptide.
The normal and mutant alleles of the COLlA2 gene were expressed to similar extents by dermal fibroblasts, as approximately equal amounts of normal and mutant a2(I) mRNA and type I procollagen were produced. However, the total production of type I and I11 collagen was increased, as reported in other cases of EDS-VI1 and in dermatosparaxis (4,25). The mechanism accounting for this increase was not determined, but it probably involved reduced feedback inhibition of type I and I11 collagen production by the N-propeptides (26-28). Most of the inhibitory effect resides in the large amino-terminal globular domain of the N-propeptide of the pro-al(1) chain, a domain that is absent in the N-propeptide of the pro-aZ(1) chain (11,27). The amount of free N-propeptide of the pro-al(1) chain available for feedback regulation of collagen synthesis may have been reduced in the fibroblast cultures from the proband. Delayed cleavage of this N-propeptide in molecules containing a mutant pN-a2(1)' chain, as shown in cultures supplemented with dextran sulfate, would reduce its supply. In addition, the cleaved N-propeptide of the pro-al(1) chain may remain attached, by noncovalent bonds, to the uncleaved N-propeptide of the mutant pN-aS(1)' chains (29).
The normal and mutant alleles were probably also expressed equally in uiuo, as approximately equal amounts of the normal and mutant a2(I) chains were observed in the salt-soluble collagen, which is the newly synthesized collagen, extracted from the proband's dermis (1). We did not determine whether the production of type I and I11 collagen was increased in the dermis. Evidence of abnormal intramolecular and intermolecular collagen cross-linking was found in the proband's dermis. Intramolecular cross-linkages form by oxidative deamination of Lys' of the N-telopeptide of the a2(I) chain and Lysg of the N-telopeptide of the al(1) chain by lysyl oxidase, followed by condensation of the resulting allysine residues to form an aldol condensation product (30). The 011 and 012 dimeric chains found in acetic acid and guanidine HC1-soluble collagen are cross-linked in this manner (13). The finding of an abnormal 012 dimer in these extracts from the proband's dermis suggested that Lys5 of the mutant pN-aS(1)' chain had undergone oxidative deamination and formation of an intramolecular cross-link with Lysg of an al(1) chain. In support of this proposal, a similar abnormal 012 dimer was found in pepsin digests but was absent in the acetic acid and guanidine HC1 extracts of tissues from a patient with EDS-VIIB whose mutant pN-a2(I) chains lacked Lys5 due to loss of the 18 amino acids encoded by exon 6 of the COLlA2 gene. These findings also showed that the abnormal 012 dimer in the pepsin digests contained intermolecular cross-linkages that did not involve Lys' of the N-telopeptide. An abnormal 012 dimer was also found in the limited pepsin digests of dermis from the current case, but its component chains probably contained intramolecular and intermolecular cross-linkages. Although our findings indicated that Lys' of the mutant pN-a2(I) chain was able to form cross-linkages, the reduced proportion of total 012:/3ll dimers indicated that they were formed inefficiently.
The increased solubility of the dermal collagen and the differing proportions of the free pN-a2(I)':a2(1) chains in the extracts were also consistent with our proposal that collagen cross-linking was abnormal. The low proportion of mutant chains in the acetic acid extract of the dermis suggests that mutant molecules were degraded soon after secretion unless they were protected by being incorporated into the more stable extracellular matrix. The preferential extraction of mutant molecules from the acetic acid-insoluble matrix by guanidine HC1 is consistent with abnormal cross-linking but may also indicate major deficiencies in fibrillogenesis and collagenmatrix interactions.
The findings in the present study confirm that the human EDS-VI1 phenotype is associated with a heterozygous loss of the N-proteinase cleavage site of the pro-al(1) or pro-a2(1) chains. The clinical phenotype is characteristic, and the diagnosis is easily confirmed by electrophoresis of collagens extracted from the dermis or of collagens prepared from fibroblast cultures. The present study, however, shows that loss of the N-proteinase cleavage site of the pro-a2(1) chain may occur as a result of splicing mutations of intron 5, as well as from the reported mutations involving the 3' nucleotide of exon 6 or the splice donor site of intron 6 of the COLlA2 gene. The present study also shows that the loss of the Nproteinase cleavage site, with persistence of a shortened Npropeptide, is the major factor responsible for the phenotype.