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Human Molecular Genetics, 2003, Vol. 12, No. 3 217-225
© 2003 Oxford University Press

Tissue-specific RNA surveillance? Nonsense-mediated mRNA decay causes collagen X haploinsufficiency in Schmid metaphyseal chondrodysplasia cartilage

John F. Bateman1,*, Susanna Freddi1, Gary Nattrass2 and Ravi Savarirayan1,3

1Cell and Matrix Biology Research Unit, Murdoch Childrens Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia and 2Department of Orthopaedics and 3Genetics Health Services Victoria, Royal Children's Hospital, Parkville, Victoria 3052, Australia

Received August 21, 2002; Revised November 7, 2002; Accepted November 19, 2002


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations resulting in a premature termination codon (PTC) are a major cause of inherited disorders, and the majority of these mutant RNA transcripts are subjected to nonsense-mediated mRNA decay (NMD). This RNA surveillance results in reduced mutant allele expression, the extent of which can impact on the clinical severity. The molecular mechanisms of NMD in mammalian cells, its relationship to splicing and translation, downstream sequence elements and binding factors remains only partially understood. Currently there is little information on whether the extent of NMD is gene- or tissue-specific, although nonsense mutation inhibition of RNA splicing has been shown to exhibit some tissue and gene specificity in vitro. Schmid metaphyseal chondrodysplasia results from heterozygous mutations in the gene for collagen X (COL10A1), expressed by the hypertrophic chondrocytes of growth plate cartilage. In one patient a PTC mutation has been shown to result in complete NMD and collagen X haploinsufficiency in cartilage. Here we show that, in this patient, and in another with a different collagen X PTC mutation also leading to complete NMD in cartilage, the mutant mRNAs were not subjected to NMD in non-cartilage cells (lymphoblasts and bone cells). These data suggest that novel RNA surveillance mechanisms may exist in cartilage and that tissue specificity of NMD could be of importance in understanding the molecular pathology of nonsense mutations. Furthermore, the demonstration of collagen X haploinsufficiency in the second patient to be studied at the level of tissue expression, confirms that nonsense mutations leading to complete mutant collagen X mRNA degradation in cartilage is an important molecular cause of SMCD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Schmid metaphyseal chondrodysplasia (SMCD; MIM 156500) is an autosomal dominant skeletal dysplasia characterized by short stature, coxa vara, genu varum and a wide irregular growth plate (1). SMCD results from heterozygous mutations in the gene for collagen X, a short-chain collagen whose expression is largely restricted to the hypertrophic chondrocytes of growth plate cartilage (2). The precise role of collagen X in the hypertrophic cartilage has not been defined although several lines of evidence suggest that it can form hexagonal network structures and may be important in regional extracellular matrix organization by interacting with other matrix components (3–6). While studies on SMCD collagen X mutations have demonstrated that collagen X is critical for normal growth plate function and endochondral bone formation, and have defined domains crucial for collagen X trimerization, the limited functional studies on these mutants have not yet revealed the details of the molecular role of collagen X (7).

Collagen X mutations reported to date have been almost exclusively localized to the C-terminal globular NC1 trimerization domain (see reviews 7,8). The NC1 mutations are about equally divided into two mutation types, missense mutations and mutations that introduce premature termination signals (nonsense or frameshift mutations). Because of the inherent difficulties in obtaining mutant cartilage for direct analysis, studies exploring the effect of the mutations on collagen X synthesis and assembly have employed site-directed mutagenesis and in vitro expression. These studies have clearly shown that all the SMCD mutations studied compromise or prevent mutant collagen X trimerization (9–12), consistent with the three-dimensional structure of the of the NC1 trimerization domains (13,14). While such expression studies in cell-free (12) or semi-permeablized cell expression systems (15) have also suggested that mutant collagen could exert a dominant negative effect on normal collagen X assembly, recent studies on transfected cells where accurate collagen X heterotrimer assembly and secretion can occur (16) failed to demonstrate any significant dominant interference by the mutant chains on wild-type collagen X assembly. These data suggested that SMCD missense mutations that prevent assembly result in a functional haploinsufficiency of collagen X.

Furthermore, the direct analysis of cartilage tissue from a SMCD patient with a heterozygous Y632X* premature stop codon mutation revealed that in cartilage the mutant message was fully degraded and the disease resulted from collagen X haploinsufficiency (11). These data suggested that in SMCD patients with nonsense mutations the mutant mRNA may be consistently subject to surveillance and degradation, and the resulting haploinsufficiency might be a common cause of the disorder. Degradation of mRNAs containing mutations that introduce premature termination codons is a common finding in many diseases (17,18). This nonsense-mediated decay (NMD) is an important quality control mechanism, reducing the amount of non-functional mRNA that would produce truncated proteins with the potential to exert severe dominant negative effects. The mechanisms of NMD are not yet fully understood, with evidence for both cytoplasmic and nuclear scanning models (17,19–21).

To further explore the molecular pathogenesis of SMCD, in these studies we present a second SMCD kindred analysed at the molecular and cartilage tissue level. The mutation was a single nucleotide substitution producing a premature stop codon in the NC1 domain (W611X). Analysis of the expression of normal and mutant allele transcripts in growth plate cartilage by RT–PCR, sequencing, primer extension, and the ‘protein truncation test’ all revealed no detectable mutant mRNA in cartilage due to NMD. Our data shows conclusively that the growth plate abnormalities of SMCD can result from haploinsufficiency, a reduction by 50% in collagen X. Importantly, our studies also showed that NMD of two collagen X SMCD mutants, W611X and Y632X, did not occur in the non-cartilage lymphoblast and bone cells, and the NMD was specific for cartilage. These data suggest that there are novel RNA surveillance mechanisms in cartilage, and highlight the importance of specifically studying the effect of collagen X nonsense mutations in cartilage tissue. Measurement of the effect of mutations on NMD in other tissues, or from more accessible cells such as lymphoblasts could lead to erroneous conclusions on the level of mutant expression and molecular basis of the disease in cartilage.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Clinical data
The proband presented at age 7 years with bowing of the legs and short stature. Family history revealed that the father and paternal uncle had been investigated in the past for short stature and bowed legs and that many other family members on the paternal side had a similar body habitus. Clinical examination demonstrated proportionate short stature, increased lumbar lordosis, waddling gait, genu varum and prominence of the knee joints. Radiographs of the skeleton showed generalized metaphyseal widening, sclerosis and irregularity, coxa vara (Fig. 1A) and bowed femora. The remainder of the skeleton was radiographically normal. Based on these clinical and radiographic findings, the diagnosis of Schmid metaphyseal chondrodysplasia was made.



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  		Figure 1. (A) Antero-posterior radiograph of the pelvis showing typical findings of coxa vara with metaphyseal sclerosis and irregularity. (B) The genomic PCR product containing the mismatched fragment from control and propositus was directly sequenced (see Methods for details). Sequencing identified a single G1928 to A base mutation indicated by an arrow in the SMCD sequence. This converts the codon TGG for Trp 611 to a termination codon TAA. (C) Sequence analysis of PCR fragments from control, SMCD genomic DNA and SMCD cartilage RNA. The G–A base mutation is indicated by the arrow, showing that the presence of this mutation is only in the SMCD genomic DNA and not in the mRNA isolated from the SMCD growth plate cartilage.

 
Characterization of the mutation
RNA produced by in vitro transcription of T7 or SP6-driven PCR products spanning the collagen X NC1 domain target region of genomic DNA, was formed into heteroduplexes and scanned for mismatches by RNAse digestion (data not shown). RNA heteroduplexes from controls were not cleaved, but the 784 bp heteroduplexes from affected family members were cleaved at mismatches to give 330 and 450 bp fragments. To identify the base changes resulting in the RNAse cleavage, genomic PCR products containing the mismatch were directly sequenced (Fig. 1B), revealing a heterozygous G to A change at position G1832 in the SMCD patient DNA. This mutation (c.1832G>A) changed the codon TGG for tryptophan at position 611 (W611) to a stop codon, TAG, in one collagen X allele (Fig. 1B). In addition, one unaffected family member also showed RNAse cleavage to produce different sized mismatch fragments to those of the affected family members. Direct sequencing defined this as a novel polymorphism, GTG1815(Val)>GTC(Val) (data not shown).

Mutant expression in growth plate cartilage
To determine the relative levels of the W611X mutant and normal {alpha}1(X) mRNA, three sensitive approaches were used. Firstly, RNA extracted from SMCD cartilage was RT–PCR amplified and directly sequenced (Fig. 1C) and compared to genomic sequence from the SMCD patient and control. Whereas the SMCD genomic sequence clearly shows equal abundance of the G and A at position 1832 of the collagen X cDNA sequence, in the PCR products amplified from cartilage mRNA there was no evidence of the mutant sequence. These data suggested that the mutant mRNA was absent, or at least below the levels of RT–PCR detection. The second approach to detect and quantify mutant mRNA was the use of allele-specific primer extension using cDNA products amplified from growth cartilage mRNA by RT–PCR. The 18mer oligonucleotide used for primer extension analysis annealed 5' and one base short of the mutation (Fig. 2). Extension with radioactive dGTP will detect primer extension of three guanosines from template sequence originating from the normal allele resulting in a labelled 21mer. The use of radioactive dATP will detect specific primer extension by one adenosine from the mutant allele template resulting in a labelled 19mer. This analysis showed the expected extension from templates amplified from the patient's genomic DNA, with extension from both the normal and mutant alleles (Fig. 2, lanes 3 and 4). In contrast, only the 21mer-dGTP primer extension product of the normal allele was detected from RT–PCR templates amplified from the patient's mRNA extracted from cartilage (lanes 5 and 6). Surprisingly, when the illegitimate collagen X mRNA transcripts expressed at low level by non-collagen X producing cells such as lymphoblasts was analyzed by primer extension (Fig. 2, lanes 7 and 8), both normal and mutant transcripts were detected.



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  		Figure 2. Primer extension analysis of PCR-amplified fragments from genomic DNA and from mRNA by RT–PCR was performed on a 2.1 kb RT–PCR fragment generated from mRNA isolated from growth plate cartilage (lanes 5 and 6) and lymphoblasts (lanes 7 and 8) from the patient. Lanes 1 and 2 are a no-DNA control. A 510 bp PCR-amplified genomic fragment spanning the mutant region from the patient was also analysed similarly (lanes 3 and 4). The sequence of the 18mer extension primer with 3' normal and mutant allele sequences is shown at the top of the figure. G* and A* represent radioactive extension of [32P]-guanosine and [32P]-adenosine, respectively. Extension with [32P]-guanosine (lanes 1, 3, 5 and 7) identifies the normal allele that resulted in a radioactively-labelled 21mer, whereas, extension with [32P]-adenosine (lanes 2, 4, 6 and 8) identifies the mutant allele producing a radioactively-labelled 19mer as indicated.

 
To confirm the absence of the mutant mRNA in SMCD cartilage we also used a specific mutation detection method to scan for mRNA containing premature termination mutations, the protein truncation test (PTT) (22–24). In this method, the cDNAs produced by RT–PCR of the mRNA is amplified using forward primers containing a T7-promoter sequence and translation initiation signals such that the resultant products can be transcribed and translated into protein in vitro. Mutations that result in premature termination produce smaller protein products that can be resolved by SDS–PAGE from the protein products produced from the normal allele that does not contain a premature stop codon. When mRNA extracted from SMCD patient cartilage tissue, and from chondrocytes isolated from the SMCD patient cartilage, was analyzed by the PTT (Fig. 3A, lanes 1 and 2) only a single protein band of 49 kDa was translated in vitro, corresponding to the normal full-length PCR product. These data confirmed the sequencing and primer extension data, demonstrating that in patient cartilage tissue or cells there was no evidence of any mRNA expression from the mutant allele containing the premature stop codon.



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  		Figure 3. (A) Protein truncation test (PTT) analysis of SMCD W611X and Y632X. RNA from the W611X SMCD growth plate cartilage (lane 1), chondrocytes (lanes 2 and 3), lymphoblasts (lanes 4 and 5) and bone cells (lanes 6 and 7) were used as a template for RT–PCR/PTT. In lanes 8 and 9 RNA from Y632X bone cells was used as the template. Primers spanning mRNA bases 700–2139 were used to amplify a 1440 bp PCR product which was then transcribed and translated in vitro. The resultant radiolabeled protein products were resolved on 14% SDS/polyacrylamide gels. The full-length PCR product produces a 49 kDa protein band, while the W611X and Y632X mutants produce 41.5 and 44 kDa protein bands, respectively. Each cell line was pre-incubated for 0 (-) or 6 h (+) with 100 µg/ml cycloheximide (CHX) prior to RNA extraction. (B) Primer extension analysis of the 2.1 kb RT–PCR fragment generated from mRNA isolated from patient W611X chondrocytes pre-incubated with (+) or without (-) CHX. Extension with [32P]-guanosine (lanes 1, 3 and 5) identifies the normal allele (21mer) and with [32P]-adenosine (lanes 2, 4 and 6) identifies the mutant allele (19mer). Radioactive incorporation was quantified by phosphor image analysis and the relative abundance of each extension product was calculated by adjusting for nucleotide specific activity and the differential extension of three G's in the normal allele (Wt) product and one A in the mutant allele product (Mut) and is expressed as the percentage of total.

 
mRNA surveillance mechanisms completely remove the nonsense mRNA in cartilage tissue and cells but not in non-cartilage cells
Mutant mRNAs containing premature stop codons are commonly degraded with cells by an ‘mRNA surveillance’ mechanism called nonsense-mediated mRNA decay (NMD) (17,18). In SMCD we have reported (11) another case (Y632X) where there was a total absence of premature stop codon-containing mRNA in cartilage, leading us to conclude that mRNA surveillance in the cartilage cells led to complete mutant mRNA degradation. However this was never directly demonstrated. In order to establish if NMD occurred in the W611X SMCD patient, we used an experimental manipulation to stabilize the mutant mRNA by incubation of the SMCD chondrocytes with cycloheximide prior to mRNA isolation (23,24). In chondrocytes preincubated with cycloheximide (Fig. 3A, lane 3) the PTT revealed two approximately equally abundant bands, representing the full-length normal allele product (49 kDa) and the predicted truncated protein (41.5 kDa) from the stop codon-containing mutant allele PCR product. To accurately determine the level of normal and mutant allele expression in these samples, primer extension analysis was performed (Fig. 3B). Quantitation of this data, taking into account radiolabeled nucleotide specific activity and the different number of nucleotides extended in the normal and mutant alleles, clearly demonstrated that the level of expression of normal and mutant alleles is comparable (46 and 54% of total, respectively) if mRNA surveillance is inhibited (Fig. 3B, lanes 3 and 4). However, the data also show that when NMD is not inhibited by protein synthesis inhibitors the mutant allele mRNA is unable to be detected in cartilage cells (Fig. 3A, lane 2 and Fig. 3B, lanes 1, 2, 5 and 6) and tissue (Fig. 3A, lane 1) from the patient and must be completely degraded by the cartilage mRNA surveillance mechanisms. To explore whether this mutant collagen X NMD is cartilage-specific, we performed the PTT on mRNA isolated from the SMCD patient's lymphoblast and bone cells either with or without pre-treatment with cycloheximide (Fig. 3A, lanes 6–9). Surprisingly, we noted that when the collagen X mRNA that is illegitimately transcribed at low levels by lymphoblasts was analyzed, mutant collagen X mRNA was detected, indicating that NMD of collagen X mRNA did not occur (Fig. 3A, lanes 4–5). The presence of stable mutant mRNA in the patient's lymphoblasts was confirmed by primer extension analysis (Fig. 2, lanes 7–8). Bone cells also only express collagen at illegitimate levels, and in cells from the SMCD patient (W611X) (Fig. 3A, lanes 6–7) and in a previously characterized SMCD mutant (Y632X) where complete NMD of the mutant has been shown in cartilage (lanes 8–9), mutant collagen X NMD did not occur.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
This is the second SMCD patient characterized by a heterozygous collagen X nonsense mutation where the mutant mRNA is not detectable in RNA extracted from the patient's cartilage tissue. In this patient we were able to demonstrate, using isolated chondrocytes incubated with and without cyloheximide, that the lack of apparent expression the mutant allele in the cartilage was due to cellular mutant RNA surveillance that results in nonsense-mediated decay of the W611X mutant mRNA. While in the previously studied patient (Y632X) (11) we did not directly confirm that the suppression of mutant mRNA expression was due to RNA surveillance, in chondrocyte cell cultures stably transfected with a mouse collagen X gene containing a Y632X mutation, we have also demonstrated that cycloheximide-inhibitable NMD occurs (Bateman, Golub and Freddi, unpublished data). Our data clearly demonstrates that in the only two nonsense mutation SMCD patients (W611X and Y632X) studied at the cartilage tissue level, the phenotype results from the expression of half normal levels of collagen X in vivo. Thus it seems very likely that complete NMD and haploinsufficiency is the common, if not sole, consequence of collagen X premature termination mutations in this disease.

Exonic mutations can also cause altered splicing (21,25–27) by interfering with exon splicing signals. In the case of nonsense mutations this has been called nonsense altered splicing (NAS), whereby the exon containing the premature termination codon (PTC) is spliced out (25,27). While there is no bioinformatic evidence for a downstream exon or pseudo-exon in the collagen X gene, which would be a requirement for NAS, it was nonetheless important to directly determine if NAS contributed to the disease phenotype in this patient. NAS is not inhibited by protein synthesis inhibitors (25) and thus if NAS occurred it would reduce the level of mutant allele detected using our primer extension and PTT assays of mRNA isolated from cycloheximide treated chondrocytes. The presence of equal levels of mutant and normal allele mRNA when NMD is inhibited by cycloheximide (Fig. 3A and 3B) effectively rules out NAS and confirms that the mutant allele haploinsufficiency in this patient results from nonsense-mediated mRNA decay.

The mechanistic complexities of NMD are only just being appreciated. The apparently disparate models of NMD that attempt to explain the context of the mutation in relation to the exon–intron structure of the gene, the role of nuclear scanning processes, and the more obvious connection with cytoplasmic protein translation are beginning to coalesce into an integrated model that interconnects pre-RNA splicing, RNA transport from the nucleus and translation in the regulation of NMD (19,20,26,28,29) . In brief, the current model posits that during pre-mRNA splicing a complex of proteins is deposited ~20 nucleotides upstream of exon–exon junctions. This protein complex which is comprised of a range of proteins involved in splicing and mRNA transport, marks the site of intron excision and is called the exon–exon junction complex (EJC). The EJC recruits other proteins, including members of the Upf family, that are crucially involved in determining NMD during an initial, or ‘pioneer’, round of translation. In general, if an in-frame termination codon is upstream of an EJC, it is identified as ‘premature’, whereas since it is unusual to find an intron in the 3' untranslated region of mRNA, the normal stop codon has no downstream EJC and is not targeted for NMD.

Collagen X NMD provides information that extends this model. Firstly, the collagen X nonsense mutations that cause NMD are in the last exon of the gene (exon 3 encoding the NC1 domain) and since these mutations are not upstream of a conventional EJC, they would not be predicted to cause NMD. This suggests that there are sequences in the collagen X gene that can substitute for the information provided by a conventional EJC and elicit NMD. While the two mutations, W611X and Y632X, which are 211 bp and 147 bp upstream of the normal stop codon, both result in complete NMD in cartilage, detailed studies on the relationship between site of the mutation within the exon and the extent of NMD could provide useful new information on how the context of the stop codon is interpreted by the NMD machinery. Another explanation for the NMD of collagen X falling outside the normally accepted rules for the relationship of the exon–exon junction sequence to the mRNA surveillance, is that the process may be mechanistically different for collagen X in cartilage.

The second contribution of collagen X mutations to our understanding of NMD relates to the novel finding of an apparent tissue specificity of the mutant mRNA degradation. In our studies on the two collagen X premature termination mutations expressed by bone cells and lymphoblasts from the patients, incubation with the NMD inhibitor, cyloheximide, did not change the relative levels of the mutant and normal mRNAs, demonstrating that the mutant mRNA was not significantly degraded in these non-cartilage cells. Collagen X expression is restricted to hypertrophic chondrocytes, and the bone cells and lymphoblasts only express collagen X at low basal, or ‘illegitimate’, levels. While it is possible that when collagen X is expressed at such low levels the normal surveillance mechanisms do not function, this seems unlikely since other illegitimate nonsense-containing transcripts, in particular collagens I and VI (24,30) and collagen II (23), have been clearly shown to undergo NMD in lymphoblasts. The alternative hypothesis is that that there is a cartilage-specific mRNA surveillance and nonsense-mediated decay mechanism involved in the degradation of mutant collagen X mRNA. These results raise the exciting possibility of the existence of tissue specificity of RNA surveillance of other genes. The collagen X NMD data presented here do not necessarily conflict with the concept of a generic NMD mechanistic backbone, but suggest that subtle changes in the expression of EJC proteins and other factors may allow an ‘overlay’ of cell- and/or gene-specific regulation of NMD. Some support for subtle NMD response differences is also provided by the regulation of NMD in T-cell receptor-ß genes by the position of the nonsense mutation independent of the accepted rule that an intron must be at least 55 bp downstream (31,32). Since it is becoming clear that pre-RNA splicing, and the decoration of the mRNA with protein complexes at the EJC, is critically integrated into the NMD machinery, and that differential splicing of many genes shows cell and tissue specificity, it may be less controversial than first thought to consider the possibility of tissue specificity in RNA surveillance.

Our studies suggest subtleties of the collagen X RNA surveillance mechanism that will require further detailed studies to fully elucidate, but potentially have the ability to significantly impact on our understanding of the molecular basis of numerous disease-causing mutations. From the immediate pragmatic point of view, this information should cause us to be careful in interpretation of data on nonsense-mediated decay in diagnostically accessible cells such as lymphoblasts. Sole reliance on analysis of mRNA from lymphoblasts (or other tissue source where collagen X is not expressed) would have resulted in the incorrect conclusion that mutant mRNA was expressed and the resulting truncated protein would be available to exert a dominant negative effect on cartilage matrix assembly. Thus it is important to confirm the level of NMD in the affected tissues before we can be confident about the consequence of nonsense-mediated decay on mutant protein expression and the molecular pathology of nonsense mutations in disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell culture
After receiving parental consent and the approval of the Institutional Ethics Committee, a blood sample was collected and a lymphoblastoid cell line was established from the patient's blood sample by transformation with Epstein–Barr virus as described previously (33). A central transphyseal core of each proximal femoral growth plate was obtained at the time of the patient's bilateral proximal femoral valgus osteotomies (performed as treatment for his bilateral coxa vara deformity). Chondrocytes were isolated from cartilage as previously described (34). The cartilage biopsy, was dissected free of bone and of the bulk of the upper cartilage articular and proliferative zones, resulting in a cartilage sample enriched in hypertrophic and prehypertrophic cartilage regions. The cartilage was digested with 2 mg of bacterial collagenase (Worthington CLS2)/ml of DMEM (Invitrogen) containing 5% (v/v) bovine serum, 100 units/ml of penicillin, 100 µg/ml streptomycin, at 37°C for 16 h (34) and filtered through a cell strainer (Becton Dickinson, NJ, USA) to remove undigested material. The released chondrocytes were washed in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) bovine serum and cultured at high density to maintain chondrocyte phenotype (34) for 5 days. Bone samples were also obtained from a second patient with a previously described mutation (Y632X) (11) during orthopaedic procedures. Osteoblast cultures from bone samples, carefully dissected to remove any cartilaginous tissues, were established as previously described (35). Soft tissues and surface cells were removed from the bone samples by digestion with collagenase for 2 h. The bone samples were placed in a culture dish with DMEM, 10% (v/v) bovine serum and antibiotics. Osteoblasts that grew out from the bone chips were subcultured and expanded.

Amplification of COL10A1 genomic sequences
Genomic DNA was extracted from whole blood (36). Primers HX1-T7, 5'-TAATACGACTCACTATAGGGTAAAGGGGATCCAGGAAGTC-3' (bp 1395–1414) and HX6-SP6 5'-ATTTAGGTGACACTATAGAACTTTTCAGCCTACCTCCATA-3' (bp 2139–2120) were used to generate a 784 bp fragment with T7 and SP6 phage promoters into the PCR products. The polymerase chain reactions were carried out using the DNA amplification kit from Perkin Elmer Cetus. The cycling conditions were an initial cycle at 95°C for 2 min, 56°C for 1.5 min and 72°C for 1.5 min followed by 30 cycles at 95°C for 30 s, 56°C for 30 s and 72°C for 30 s. All PCR products were analyzed on 1.5% agarose gels and used for mutation detection.

Mutation detection
Mutation detection was performed using the MisMatch DetectTM II kit (Ambion) as specified by the manufacturer's protocol. Briefly, control and SMCD modified PCR products were transcribed using either T7 or SP6 RNA polymerase to make sense and antisense RNA probes. An equal volume of control and SMCD transcripts were hybridized to generate heteroduplex RNA. The hybridized samples are then treated with diluted RNase solutions which specifically cleave at mismatches in the double-stranded RNA targets. The resultant RNase cleavage products are then resolved on a 2% Nusieve agarose gel.

Sequencing of PCR products
The 784 bp fragment containing the mismatch was purified by agarose electrophoresis and recovered using the Geneclean II kit (Integrated Sciences). Purified DNA was sequenced using the AmplicycleTM Sequencing Kit (Perkin Elmer) and [{alpha}-33P]dATP(2000 Ci/mmol, NEN Research Products).

RNA sequencing and primer extension analysis
Total RNA was extracted from 8 µm transverse frozen-sections from the SMCD growth plate cartilage by using a combination of the Chomczynski method (37) and the RNeasyTM total RNA kit (QIAGEN). The entire coding region of the collagen X was amplified by RT–PCR (Perkin Elmer Cetus) from cDNA produced using random hexamers as primers for reverse transcription and Amplitaq GoldTM (Perkin-Elmer-Cetus). The conditions for cDNA synthesis were those recommended by the manufacturer. Exon 2 primer HX11, 5'-GAGAATATGCTGCCAATACCCT-3' (bp -6–19) and exon 3 primer HX6, 5'-CTTTTCAGCCTACCTCCATA-3' (bp 2139–2120), were used in the PCR step to amplify a 2.1 kb mRNA-specific PCR product. PCR consisted of an initial denaturation at 95°C for 10 min, followed by 35 cycles at 95°C for 30 s, 60°C for 30 s and 72°C for 1 min. The PCR product was then purified by agarose electrophoresis and recovered using the Geneclean II kit. A 510 bp fragment (bp 1630–2129) encompassing the mutation was amplified from the 2.1 kb purified RT–PCR fragment using primers BX1, 5'-CAGGGGGTAACAGGAATGCC-3' (bp 1630–1649) and HX6. The RT–PCR product was then purified and sequenced as outlined in the previous section.

Primer extension analysis was also used to estimate the concentration of normal and mutant message (11). Approximately 5 ng of the 2.1 kb cDNA fragment was used as a template for single nucleotide extension with oligonucleotide HX18 (5'-TGAAAGGGACTCATGTTT-3', bp 1814–1831) that primes one base 5' to the mutation. The reaction was carried out at 95°C for 1 min, 50°C for 2 min and 72°C for 1 min in 10 µl of 10 mM Tris–HCl, pH 8.3 containing 50 mM KCl, 1.5 mM MgCl2, 0.01% (w/v) gelatin, 1 µM primer and 2 µCi of either [{alpha}-32P]-dGTP or [{alpha}-32P]-dATP (NEN Life Science Products), in the absence of non-radioactive dNTPs. The products were analyzed on a 15% (w/v) denaturing polyacrylamide gel containing 7 M urea and the radioactivity of the extended products were quantified using a phosphor-imager (Molecular Dynamics).

RT–PCR/protein truncation test
Prior to RNA extraction, SMCD lymphoblast, cartilage and bone cell cultures were incubated with 100 µg/ml cycloheximide (Sigma Chemical Co., St Louis, MO, USA) for 0 or 6 h (23,24). RNA was isolated using the RNeasyTM total RNA kit. RT–PCR (Perkin-Elmer-Cetus) was performed as outlined previously using primers HX11 and HX6 to amplify the entire 2.1 kb coding region. For cartilage cell RNA, 35 cycles of PCR were used, while for bone cell and lymphoblast RNA, 45 cycles of PCR were required to amplify the low basal levels of collagen X mRNA. For PTT analysis of all three cell types, a 1440 bp fragment (bp 700–2139) was amplified from the 2.1 kb primary RT–PCR product using a T7-modified primer HX5-PTT, 5'-GCTAATACGACTCACTATAGGAACAGACCACCATGAAAGGTGATAGAGGTTTTCC-3' (bp 700–719) and HX6. RT–PCR was performed using Taq polymerase (Amplitaq, Perkin-Elmer-Cetus) and 35 cycles at 95°C for 30 s, 60°C for 30 s and 72°C for 1 min. PCR products were analyzed on 1.2% agarose gels and used for PTT analysis. The T7-modified RT–PCR products were transcribed and translated using the TNTTM T7-coupled reticulocyte lysate system (Promega, Madison, WI, USA) according to the manufacturer's protocol. Reactions were performed in a 12.5 µl reaction volume containing 4 µl of RT–PCR product. The translated protein products were labeled with 10 µCi of translation grade L-[35S]-methionine (1000 Ci/mmol, DuPont NEN, Boston, MA, USA) Prior to electrophoresis samples were diluted in gel loading buffer (2% SDS, 10 mM dithiothrietol), denatured at 65°C for 10 min and were analyzed on 14% (w/v) SDS/polyacrylamide gels with a 4.5% (w/v) stacking gel. Radioactively labeled proteins were detected by fluorography (38).


   ACKNOWLEDGEMENTS
 
The authors would like to thank Dr Shireen Lamandé for valuable discussions during the course of these studies and critical evaluation of the manuscript and Dr Katrina Bell for help with bioinformatic analysis. This work was supported by grants from the National Health and Medical Research Council of Australia and the Murdoch Childrens Research Institute.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +61 393456367; Fax: +61 393457997; Email: bateman{at}cryptic.rch.unimelb.edu.au Back

* Sequences are numbered from the translation initiation codon according to the nomenclature of den Dunnen and Antonarakis (39). Nucleotide +1 is the A of the ATG-translation initiation codon and the initiating methionine is numbered as amino acid +1. Genebank entry NM_000493 was used as the reference sequence. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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