Guy G. Hoffman

Null Alleles of the COL5A1 Gene of Type V Collagen Are a Cause of the Classical Forms of Ehlers-Danlos Syndrome (Types I and II)

Ehlers-Danlos syndrome (EDS) types I and II, which comprise the classical variety, are well characterized from the clinical perspective, but it has been difficult to identify the molecular basis of the disorder in the majority of affected individuals. Several explanations for this failure to detect mutations have been proposed, including genetic heterogeneity, failure of allele expression, and technical difficulties. Genetic heterogeneity has been confirmed as an explanation for such failure, since causative mutations have been identified in the COL5A1, COL5A2, and tenascin X genes and since they have been inferred in the COL1A2 gene. Nonetheless, in the majority of families with autosomal dominant inheritance of EDS, there appears to be linkage to loci that contain the COL5A1 or COL5A2 genes. To determine whether allele-product instability could explain failure to identify some mutations, we analyzed polymorphic variants in the COL5A1 gene in 16 individuals, and we examined mRNA for the expression of both alleles and for alterations in splicing. We found a splice-site mutation in a single individual, and we determined that, in six individuals, the mRNA from one COL5A1 allele either was not expressed or was very unstable. We identified small insertions or deletions in five of these cell strains, but we could not identify the mutation in the sixth individual. Thus, although as many as one-half of the mutations that give rise to EDS types I and II are likely to lie in the COL5A1 gene, a significant portion of them result in very low levels of mRNA from the mutant allele, as a consequence of nonsense-mediated mRNA decay.

Strategy for identification of sequence variants in COL7A1 and a novel 2-bp deletion mutation in recessive dystrophic epidermolysis bullosa

The diagnostic hallmark of the dystrophic forms of epidermolysis bullosa (DEB), a group of heritable blistering skin diseases, is abnormalities in the anchoring fibrils at the dermal‐epidermal basement membrane zone. Since type VII collagen is the major, if not the exclusive, component of the anchoring fibrils, the corresponding gene (COL7A1) is the candidate gene in DEB. Recent cloning of the type VII collagen cDNA and elucidation of the exon‐intron organization of the gene have provided the basis for us to develop a novel strategy for identification of sequence variants in COL7A1. Optimization of 72 balanced primer pairs corresponding to flanking intronic sequences allowed PCR amplification of all 118 exons directly from genomic DNA. The PCR products were examined by heteroduplex analysis followed by comparative nucleotide sequencing. More than 100 sequence variants have been identified thus far in COL7A1 using this method, some of which are single base pair polymorphisms and many of which are pathogenetic mutations contributing to the blistering phenotype in DEB. The comprehensive method described is useful for rapid, reliable, and sensitive detection of sequence variants in COL7A1. We demonstrate the utility of this novel strategy in mutation detection and prenatal exclusion of RDEB in a consanguineous family at risk for recurrence. Hum Mutat 10:408–414, 1997.

Order of Intron Removal Influences Multiple Splice Outcomes, Including a Two-Exon Skip, in a COL5A1 Acceptor-Site Mutation That Results in Abnormal Pro-α1(V) N-Propeptides and Ehlers-Danlos Syndrome Type I

Ehlers-Danlos syndrome (EDS) type I (the classical variety) is a dominantly inherited, genetically heterogeneous connective-tissue disorder. Mutations in the COL5A1 and COL5A2 genes, which encode type V collagen, have been identified in several individuals. Most mutations affect either the triple-helical domain of the protein or the expression of one COL5A1 allele. We identified a novel splice-acceptor mutation (IVS4-2A-->G) in the N-propeptide-encoding region of COL5A1, in one patient with EDS type I. The outcome of this mutation was complex: In the major product, both exons 5 and 6 were skipped; other products included a small amount in which only exon 5 was skipped and an even smaller amount in which cryptic acceptor sites within exon 5 were used. All products were in frame. Pro-alpha1(V) chains with abnormal N-propeptides were secreted and were incorporated into extracellular matrix, and the mutation resulted in dramatic alterations in collagen fibril structure. The two-exon skip occurred in transcripts in which intron 5 was removed rapidly relative to introns 4 and 6, leaving a large (270 nt) composite exon that can be skipped in its entirety. The transcripts in which only exon 5 was skipped were derived from those in which intron 6 was removed prior to intron 5. The use of cryptic acceptor sites in exon 5 occurred in transcripts in which intron 4 was removed subsequent to introns 5 and 6. These findings suggest that the order of intron removal plays an important role in the outcome of splice-site mutations and provide a model that explains why multiple products derive from a mutation at a single splice site.

Procollagen C Proteinase Enhancer 1 Genes Are Important Determinants of the Mechanical Properties and Geometry of Bone and the Ultrastructure of Connective Tissues

ABSTRACT Procollagen C proteinases (pCPs) cleave type I to III procollagen C propeptides as a necessary step in assembling the major fibrous components of vertebrate extracellular matrix. The protein PCOLCE1 (procollagen C proteinase enhancer 1) is not a proteinase but can enhance the activity of pCPs ∼10-fold in vitro and has reported roles in inhibiting other proteinases and in growth control. Here we have generated mice with null alleles of the PCOLCE1 gene, Pcolce, to ascertain in vivo roles. Although Pcolce− / − mice are viable and fertile, Pcolce − / − male, but not female, long bones are more massive and have altered geometries that increase resistance to loading, compared to wild type. Mechanical testing indicated inferior material properties of Pcolce − / − male long bone, apparently compensated for by the adaptive changes in bone geometry. Male and female Pcolce − / − vertebrae both appeared to compensate for inferior material properties with thickened and more numerous trabeculae and had a uniquely altered morphology in deposited mineral. Ultrastructurally, Pcolce − / − mice had profoundly abnormal collagen fibrils in both mineralized and nonmineralized tissues. In Pcolce − / − tendon, 100% of collagen fibrils had deranged morphologies, indicating marked functional effects in this tissue. Thus, PCOLCE1 is an important determinant of bone mechanical properties and geometry and of collagen fibril morphology in mammals, and the human PCOLCE1 gene is identified as a candidate for phenotypes with defects in such attributes in humans.

Infectious and noninfectious recombinant clones of the provirus of SNV differ in cellular DNA and are apparently the same in viral DNA

Ten clones of Charon 4A containing proviruses of spleen necrosis virus, an avian retrovirus, and flanking chicken DNA sequences were isolated and characterized. Some clones gave rise to progeny with viral DNA sequences deleted or duplicated, probably as a result of crossing-over in the 600 bp terminal redundancy in viral DNA. The cellular sequences are different in each clone, indicating that all the proviruses are integrated in different sites in cellular DNA. Six clones are infectious and four are not. All the infectious molecules containing a provirus are of a similar size and are smaller than the noninfectious molecules containing a provirus. The viral DNA is not apparently different in eight clones, but two clones, one infectious and one noninfectious, lack two restriction sites each. Large changes in proviral DNA therefore do not seem responsible for the lack of infectivity of some clones. These results are consistent with the hypothesis that neighboring cellular DNA sequences control proviral expression (infectivity).

bmp1 and mini fin are functionally redundant in regulating formation of the zebrafish dorsoventral axis.

Drosophila metalloproteinase Tolloid (TLD) is responsible for cleaving the antagonist Short gastrulation (SOG), thereby regulating signaling by the bone morphogenetic protein (BMP) Decapentaplegic (DPP). In mice there are four TLD-related proteinases, two of which, BMP1 and mammalian Tolloid-like 1 (mTLL1), are responsible for cleaving the SOG orthologue Chordin, thereby regulating signaling by DPP orthologues BMP2 and 4. However, although TLD mutations markedly dorsalize Drosophila embryos, mice doubly homozygous null for BMP1 and mTLL1 genes are not dorsalized in early development. Only a single TLD-related proteinase has previously been reported for zebrafish, and mutation of the zebrafish TLD gene (mini fin) results only in mild dorsalization, manifested by loss of the most ventral cell types of the tail. Here we identify and map the zebrafish BMP1 gene bmp1. Knockdown of BMP1 expression results in a mild tail phenotype. However, simultaneous knockdown of mini fin and bmp1 results in severe dorsalization resembling the Swirl (swr) and Snailhouse (snh) phenotypes; caused by defects in major zebrafish ventralizing genes bmp2b and bmp7, respectively. We conclude that bmp1 and mfn gene products functionally overlap and are together responsible for a key portion of the Chordin processing activity necessary to formation of the zebrafish dorsoventral axis.

Localization of the human collagen gene COL7A1 to 3p21.3 by fluorescence in situ hybridization

An 8-kb genomic probe, containing 34 collagen-encoding exons, was localized to 3p21.3 by fluorescence in situ hybridization. The genomic probe encoded a previously uncharacterized carboxyl terminal portion of the alpha 1(VII) collagen chain. This mapping result confirms the previous assignment of the alpha 1(VII) gene (COL7A1) to 3p21 and offers a finer subregional localization than was previously available.

Zebrafish chordin-like and chordin are functionally redundant in regulating patterning of the dorsoventral axis.

Chordin is the prototype of a group of cysteine-rich domain-containing proteins that bind and modulate signaling of various TGFbeta-like ligands. Chordin-like 1 and 2 (CHL1 and 2) are two members of this group that have been described in human, mouse, and chick. However, in vivo roles for CHL1 and 2 in early development are unknown due to lack of loss-of-function analysis. Here we identify and characterize zebrafish, Danio rerio, CHL (Chl). The chl gene is on a region of chromosome 21 syntenic with the area of murine chromosome 7 bearing the CHL2 gene. Inability to identify a separate zebrafish gene corresponding to the mammalian CHL1 gene suggests that Chl may serve roles in zebrafish distributed between CHL1 and CHL2 in other species. Chl is a maternal factor that is also zygotically expressed later in development and has spatiotemporal expression patterns that differ from but overlap those of zebrafish chordin (Chd), suggesting differences but also possible overlap in developmental roles of the two proteins. Chl, like Chd, dorsalizes embryos upon overexpression and is cleaved by BMP1, which antagonizes this activity. Loss-of-function experiments demonstrate that Chl serves as a BMP antagonist with functions that overlap and are redundant with those of Chd in forming the dorsoventral axis.

Characterization of the Six Zebrafish Clade B Fibrillar Procollagen Genes, with Evidence for Evolutionarily Conserved Alternative Splicing within the pro-α1(V) C-propeptide

Genes for tetrapod fibrillar procollagen chains can be divided into two clades, A and B, based on sequence homologies and differences in protein domain and gene structures. Although the major fibrillar collagen types I-III comprise only clade A chains, the minor fibrillar collagen types V and XI comprise both clade A chains and the clade B chains pro-alpha1(V), pro-alpha3(V), pro-alpha1(XI) and pro-alpha2(XI), in which defects can underlie various genetic connective tissue disorders. Here we characterize the clade B procollagen chains of zebrafish. We demonstrate that in contrast to the four tetrapod clade B chains, zebrafish have six clade B chains, designated here as pro-alpha1(V), pro-alpha3(V)a and b, pro-alpha1(XI)a and b, and pro-alpha2(XI), based on synteny, sequence homologies, and features of protein domain and gene structures. Spatiotemporal expression patterns are described, as are conserved and non-conserved features that provide insights into the function and evolution of the clade B chain types. Such features include differential alternative splicing of NH(2)-terminal globular sequences and the first case of a non-triple helical imperfection in the COL1 domain of a clade B, or clade A, fibrillar procollagen chain. Evidence is also provided for previously unknown and evolutionarily conserved alternative splicing within the pro-alpha1(V) C-propeptide, which may affect selectivity of collagen type V/XI chain associations in species ranging from zebrafish to human. Data presented herein provide insights into the nature of clade B procollagen chains and should facilitate their study in the zebrafish model system.

Absence of Apparent Disease Causing Mutations in COL5A3 in 13 Patients With Hypermobility Ehlers-Danlos Syndrome

Collagen V is a minor fibrillar collagen, broadly distributed as a1(V)2a2(V) heterotrimers [Fessler and Fessler, 1987] that are incorporated into collagen I fibrils, and which regulate the shapes and diameters of collagen I/V heterotypic fibrils [Birk et al., 1988, 1990]. Defects in the COL5A1 and COL5A2 genes underlie at least half of the cases of classic Ehlers–Danlos syndrome (EDS) [Toriello et al., 1996; Wenstrup et al., 1996; Michalickova et al., 1998; Richards et al., 1998; Schwarze et al., 2000; Malfait and De Paepe, 2005]. Collagen V is also found in the form of a relatively uncharacterized a1(V)a2(V)a3(V) heterotrimer, with a limited tissue distribution. Involvement of COL5A1 and COL5A2 defects in classic EDS, andexpressionofa3(V) chains in joint capsule, skin [Brown et al., 1978], and developing ligaments [Imamura et al., 2000] suggested COL5A3 as a candidate locus for the most common form of EDS, the hypermobility type (h-EDS). A candidate locus has yet to be identified for the vast majority of h-EDS cases [Malfait et al., 2006]. Thus, we examined the COL5A3 locus and its products in 13 patients with h-EDS, identified using the previously specified diagnostic criteria [Beighton et al., 1998]. The features of the individual patients are described in Table I. This research was reviewed and approved by the ethics committee of the Hospital for Sick Children. Primer sets (supporting information Table SI may be found in the online version of this article) were designed for amplification and sequencing of COL5A3 genomic DNA and cDNA. RT-PCR primers were designed to allow detection of exon skipping. Dermal fibroblast cultures, established from patients and cultured as described [Chan and Cole, 1991], were grown to confluence prior to RNA and DNA preparation with TRIzol (Invitrogen, Carlsbad, CA). RNA was reverse-transcribed with Superscript II (Invitrogen). Conditions for RT-PCR were 948C for 3 min followed by 35 cycles of 948C for 20 sec, 628C for 40 sec, 728C for 2 min, and final extension at 728C for 10 min. Conditions for amplifying genomic DNA were 948C for 3 min followed by 35 cycles of 948C for 30 sec, 608C for 30 sec, 728C for 3 min, and final extension at 728C for10 min. PCRemployedPromega Taq polymerase. PCR amplimers were purified from 0.8% agarose electrophoresis gels, and direct sequenced using the ABI PRISM BigDye Terminator kit (Applied Biosystems, Foster City, CA). RT-PCR successfully produced COL5A3 cDNA from dermal fibroblasts, although a3(V) expression has not previously been reported for this cell type. Sequencing of cDNA and genomic DNA from the 13 patients with h-EDS showed no apparent diseasecausing mutations typically associated with heritable collagen diseases, such as premature stop codons, exon skipping, or substitutions for obligatory glycines in Gly-X-Y triplets of the major triple helical (COL1) domain [Michalickova et al., 1998; Schwarze et al., 2000; Myllyharju and Kivirikko, 2001]. Analysis of COL5A3 coding sequences identified 11 single nucleotide polymorphisms (SNPs). Two of the 11 SNPs identified in this