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.

A.A Chiodo,A Hockey,W.G Cole

doi:10.1016/s0021-9258(18)42703-2

A.A Chiodo, A Hockey + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)42703-2

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Abstract

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.

Highlights

A Base Substitution at the Splice Acceptor Site of Intron 5 of the COLlA2 Gene Activates a Cryptic Splice Site withinExon 6 and Generates Abnormal Type I Procollagen in a Patient withEhlersDanlos Syndrome Type VII*
Amino acid sequencing of tryptic peptides, prepared from the mutant amino-terminal pN-a2(I) CB1’ peptide, indicated that five amino acids, including the N-proteinase cleavage site, hadbeen deleted from the junction of the N-propeptide and the N-telopeptide of the mutant pro-a2(1)’ chain
The corresponding 15 nucleotides, which were deleted from approximately 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

Summary

RESULTS

Characteristicsof Dermal Collagens-Electrophoresis of sequential NaCl, acetic acid, and guanidine HCl extracts from EDS dermis revealed an additional collagenousband that was not observed in extracts of age-matched normal dermis The abnormal component migrated with the characteristics of a mutantpN-aB(1)chain, as reported in patients with EDS-VIIB (12, 13) It was designated as a pN-aS(1)’ chain. 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’. Characteristics of Collagens Produced by Cultured Fibroblasts-EDS and control fibroblasts produced type I and I11 collagensin short termcultures (Fig. 2).In theEDS 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, therewas a more slowlymigrating chain similar to that observed in extracts of dermis. The dermis was defatted inchloroform/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

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DISCUSSION

Methods