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Equal expression of type X collagen mRNA from mutant and wild typeCOL10A1 alleles in growth plate cartilage from a patient with metaphyseal chondrodysplasia type Schmid
  1. CARL A GREGORY*,
  2. BERNHARD ZABEL,
  3. MICHAEL E GRANT*,
  4. RAYMOND P BOOT-HANDFORD*,
  5. GILLIAN A WALLIS*
  1. * Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK
  2. Department of Pediatrics, University of Mainz, Germany
  3. Department of Medicine, University of Manchester, Oxford Road, Manchester M13 9PT, UK
  1. Dr Wallis, gwallis{at}fs1.scg.man.ac.uk

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Editor—Type X collagen is a short chain collagen consisting of three α1(X) chains encoded by theCOL10A1 gene. The α1(X) chains are composed of three structurally distinct domains, an amino-terminal globular domain (NC2), a triple helical region, and a carboxyl-terminal globular domain (NC1).1 Type X collagen is predominantly synthesised by the hypertrophic chondrocytes of the vertebrate growth plate but its precise function during development remains unclear.2 To date, 27 naturally occurring mutations within specific regions of COL10A1 have been reported to cause the autosomal dominant human disorder metaphyseal chondrodysplasia type Schmid (MCDS), which is characterised by short stature, a waddling gait, and coxa vara.2 Of these 27COL10A1 mutations, two occur within a single codon and cause single amino acid substitutions at the putative signal sequence cleavage site within NC2,3 12 mutations cause amino acid substitutions that map to two distinct regions of the predicted structure of the NC1 domain,4 and the remaining mutations introduce stop codons or frameshifts plus premature stop codons that affect, at most, 40% of the carboxyl-terminal region of the NC1 domain. No mutations causing MCDS have yet been found altering the collagenous region of type X collagen, and in two unrelated families with MCDS we have not been able to find mutations in the entire coding region of COL10A1 (unpublished data). The probability of all 27 MCDS mutations clustering within the NC1 and NC2 encoding portions of the gene by chance alone is approximately 1 in 7.6 × 108 and for mutations predicted to truncate the α1(X) chains is approximately 1 in 106. This restricted distribution of the COL10A1mutations causing MCDS strongly suggests that these mutations alter specific function(s) of the encoded α1(X) chains.

The molecular mechanism(s) by which mutations inCOL10A1 cause MCDS remain under debate.5 In vitro association of MCDS mutant and normal α1(X) chains has been reported, suggesting that dominant interference may be the underlying molecular mechanism.4 6 These in vitro observations have yet to be proven in vivo primarily because of the difficulty of obtaining sufficient growth plate tissue from patients with MCDS for studies of type X collagen biosynthesis. In contrast to the in vitro data, in the only previously reported investigation of the biosynthesis of type X collagen in growth plate cartilage from a patient with MCDS, it has been shown that mRNA representing the mutant allele (which contained a single base pair substitution that introduced a premature termination codon in the NC1 encoding domain) was not present in the growth plate cartilage biopsy.7 This finding was explained in that mRNA encoding premature termination codons has been shown to be rapidly degraded by the proof reading machinery of the cell in a number of inherited diseases.8 This in vivo data implied that haploinsufficiency is the underlying mutation mechanism causing the MCDS phenotype in this patient and raised the question as to whether other mutations in the COL10A1 NC1 encoding domain may alter mRNA stability and thereby explain the clustering of the mutations in that domain.

To investigate the mechanism of MCDS pathology fully, there is a clear necessity for direct analysis of the hypertrophic chondrocytes and growth plate cartilage in other cases of MCDS. Although samples of growth plate cartilage from MCDS patients are extremely rare, we were fortunate to acquire such tissue from an affected subject who was heterozygous for a single base pair mutation, T1894C, predicted to cause a single amino acid substitution (S600P) in the NC1 domain of type X collagen.5 The patient had a phenotype entirely consistent with MCDS. Length at birth was normal (50 cm) and in the first year of life, the tentative diagnosis was hip dysplasia. In the second year, progressive coxa vara became apparent and at the age of 2½ years the definite diagnosis of MCDS was made. Clinical symptoms included short limbed short stature (80 cm), bowed legs, and waddling gait. Radiological findings consisted of coxa vara and metaphyseal changes including flaring, signs of sclerosis, irregularities, and growth plate widening, which were more severe at the hips than at the knees. Osteotomy was performed to correct the position of the legs and during this operation iliac crest needle biopsies were carried out to obtain material from the growth plate in this area. Informed consent for this procedure was obtained from the parents.

We used the growth plate biopsy to determine whether in this instance mRNA from both the normal and mutant alleles was available for translation. For this purpose, approximately 50 mg of the cartilage was finely ground under liquid nitrogen and total RNA and genomic DNA was extracted using a standard protocol (Trizol, Gibco BRL).9The purified RNA was treated with RNAse free DNAse (Promega) and reverse transcribed in two separate reactions using either oligo-dT or random hexamers (Superscript II reverse transcription kit, Gibco BRL). Two further identical reactions were carried out without the addition of reverse transcriptase to control for the contamination of the RNA by genomic DNA. cDNA generated from both the oligo-dT and random primed reactions were pooled. NC1 encoding genomic DNA and cDNA was amplified using oligonucleotides: sense, CCAGCTCATATGGCAACTAAGGGCCTC (nucleotides 1429-1455) and antisense, GGGGTGTACTCACATTGGAGCCAC (nucleotides 2082-2052). Cycling conditions were 95°C for two minutes, 60°C for two minutes, 72°C for two minutes for 40 cycles. When cDNA was used as a template, a correctly sized 502 bp fragment was amplified (fig 1A, lane 3). This fragment was not detected in control PCR reactions confirming that there was no detectable genomic contamination of the RNA (fig 1A, lane 4). The COL10A1 NC1 encoding region was also amplified from genomic DNA (fig 1A, lane 2). Direct sequencing of the PCR products generated from both genomic DNA and from cDNA detected the wild type and mutant alleles (data not shown) and the PCR fragments representing both alleles were cloned into the T/A vector, pCR 2.1 (InVitrogen).

Figure 1

(A) Ethidium bromide stained agarose gel electrophoresis of PCR products of the COL10A1 NC1 encoding domain generated from genomic DNA, from cDNA prepared by reverse transcription of RNA isolated from the MCDS growth plate tissue, and from RNA processed as for the generation of cDNA, but with no reverse transcriptase in the reaction buffer (the cDNA control). Lane 1, 100 bp markers; lane 2, genomic DNA template; lane 3, cDNA template; lane 4, cDNA control. The position of the 502 bp PCR product representing the NC1 encoding domain is indicated. (B) Ethidium bromide stained PAGE analysis of PCR-amplified NC1 encoding DNA modified to incorporate a restriction endonuclease site in fragments harbouring the T1894C mutation. Lane 1, 100 bp markers; lanes 2-5, undigested PCR products amplified from cDNA, genomic DNA (gDNA), and cloned wild type and mutant (T1894C) alleles; lanes 6-9, the corresponding PCR products digested with AccB71. Bands representing uncut fragments (194 bp) and digestion products (140 bp and 54 bp) are indicated. (C) ASO analysis of slot blots containing PCR amplified NC1 encoding cDNA, genomic DNA (gDNA), and the cloned wild type and mutant alleles. Oligonucleotides complementary to wild type and mutant (T1894C) alleles were hybridised to duplicate filters. For each sample, four slots were loaded containing 0.5 μg (top), 0.25 μg, 0.125 μg, and 0.0625 μg of DNA.

In order to introduce a restriction endonuclease site forAccB7I (CCAN5TGG) into the mutant NC1 encoding DNA, single overlap extension PCR was used as previously described.10 For this purpose, the 502 bp fragments derived from genomic DNA, cDNA, and the cloned normal and mutant alleles were used as templates in PCR reactions with the mutagenic oligonucleotides (nucleotides 1897-2007, sense, 5′ TACCATGGGCATGTGAAAGGG 3′ and antisense, 5′ CCCTTTCACATGCCCATGGTA 3′) and the flanking oligonucleotides (sense nucleotides, 1849-1870, 5′ AGGACTGGAATCTTTACTTGT 3′ and antisense nucleotides, 2027-2048, 5′ CTCATTTTCTGTGAGATCGATGAT 3′), generating a 194 bp fragment. The predicted size of fragments containing the T1894C substitution following cleavage with AccB7I were 140 bp and 54 bp and this was confirmed following digestion of the engineered PCR products generated from the cloned mutant (fig 1B, lane 8) and wild type (fig 1B, lane 9) alleles. Digestion of the engineered PCR products generated from the MCDS cDNA and genomic DNA confirmed the presence of both alleles (fig 1B, lanes 6 and 7, respectively).

To quantify accurately the levels of wild type and mutant encoding type X collagen mRNA in the MCDS tissue, ASO hybridisation analyses were carried out as described previously.11 For this purpose, PCR generated NC1 encoding DNA generated from genomic DNA, cDNA, and the cloned wild type and mutant alleles was alkali denatured and slot blotted. Duplicate filters were hybridised to 32P labelled mutant specific (ATACTATTTTCCATACCACGT) and wild type specific (ATACTATTTTTCATACCACGT) oligonucleotides (nucleotides 1974-1995) and the relative levels of wild type and mutant specific were digitally imaged (fig 1C) and quantified using the phosphoimaging system (Fuji-Bas). No cross hybridisation of the mutant and wild type oligonucleotides was detected when hybridised against the cloned wild type and mutant alleles, respectively. Quantitative analysis of the hybridisation of the mutant and wild type oligonucleotides to the MCDS growth plate cDNA showed that the mutant and wild type alleles were represented in a 1:1 ratio.

In this study, we have therefore shown that mRNA transcribed from both the wild type and mutant COL10A1alleles is available for translation in growth plate cartilage taken from a patient with MCDS. The translation of this mRNA would lead to the synthesis of α1(X) chains, 50% of which would contain a single amino acid substitution, S600P, in the type X collagen NC1 domain. It has been shown in in vitro studies that NC1 domains containing MCDS mutations are able to trimerise with wild type chains4 and lead to the folding of the collagen triple helix.6 These data, together with the clustering of mutations inCOL10A1, can only be rationalised if, in most cases of MCDS, dominant interference of normal type X collagen by MCDS mutant chains is occurring. Thus, the report7 that in one patient with MCDS, mRNA representing the mutant allele (which contained a premature termination codon in the NC1 encoding domain) was not present in a growth plate cartilage biopsy remains a conundrum. If, from the analysis of tissue from further patients with MCDS, mRNA instability caused by nonsense mutations is proven, then it must follow that such nonsense mutations only cause mRNA instability when within a restricted region of NC1, as similar mutations causing MCDS have not been found in other regions of COL10A1. An explanation would then be needed as to why mutations causing mRNA instability have the same restricted distribution as those mutations that do not have that effect. These issues could be resolved if mutations in other regions of COL10A1causing MCDS are identified. More likely, however, is that conclusive data will come from the detailed examination of the biosynthesis of type X collagen in growth plate tissue either from patients with MCDS or from transgenic mice that harbour MCDS mutations.

Acknowledgments

We thank Dr Mike Briggs for his invaluable advice and assistance. This work was funded by the Arthritis Research Campaign, UK.

References

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