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Abstract High bone resorption by the osteoclast results in osteoporosis, a disease affecting 40% of women after the menopause. Calcitonin, used to treat osteoporosis, inhibits bone resorption via receptors located on the osteoclasts. Two alleles of the calcitonin receptor gene ( CTR) exist: a base mutation T→C in the third intra-cellular C-terminal domain changes a proline (CCG) at position 447 to a leucine (CTG). We therefore studied the distribution of these alleles in a cohort of 215 post-menopausal Caucasian women suffering or not from osteoporotic fractures.

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The region of interest within the point mutation was amplified by PCR and screened for single strand conformation polymorphism. This work was followed by DNA sequencing of the fragments amplified. We found that bone mineral density (BMD) at the femoral neck was significantly higher in heterozygous subjects with the Rr genotype compared with the homozygous leucine (RR) and homozygous proline (rr) genotypes. Also, a decreased fracture risk was observed in heterozygote subjects. In conclusion, our results suggest that polymorphism of CTR could be associated with osteoporotic fractures and BMD in a population of post-menopausal women. CTR heterozygotes could produce both alleles of the receptor.

The heterozygous advantage effect of Rr subjects could explain their protection against osteoporosis: higher bone density and decreased fracture risk. Establishing the genotype of the CTR gene in post-menopausal women could be of value in evaluating their risk of developing fractures.

Introduction Osteoporosis is a severe disease considering its high prevalence and the economic burden of femoral neck fractures. This disease has a strong heritability component and is under polygenic control. Indeed, a relationship between a mutation of the vitamin D receptor and low bone mass (,) has been reported, but the results were not reproducible (,). Also, polymorphism at the SP1 site of the collagen type I promoter is associated with osteoporosis.

These two genes are implicated in osteoblastic bone formation. However, biochemical data show that age-related bone loss and fracture development are associated with increased bone resorption (,). One of the candidate genes of osteoporosis is the calcitonin gene. Calcitonin is a hormone implicated in bone resorption and acts through specific receptors present in large numbers in the osteoclasts (,). This hormone decreases bone resorption and is therefore used to treat osteoporosis.

The calcitonin receptor is a member of the seven transmembrane receptor family and has recently been sequenced and cloned in several species. Three isoforms of the mRNA of the human calcitonin receptor, resulting from alternative splicing , have been described. We previously reported a point mutation polymorphism (T→C) in the 3′-region of the calcitonin receptor gene ( CTR) which induced a Pro→Leu shift in the third intracellular domain of the protein.

The aim of this study was to determine if this polymorphism of the calcitonin receptor gene was associated with osteoporotic fractures. We used single strand conformation polymorphism (SSCP) to study the distribution of the alleles of this receptor in 215 post-menopausal women. The women we studied had no osteoporotic fractures ( n=123) or presented one or more osteoporotic fractures ( n = 92) of wrist or vertebrae. Results reports the clinical data for the two groups of post-menopausal women we studied. Patients with osteoporotic fractures were older, smaller, thinner and menopause onset was earlier. Their bone mineral density (BMD) was lower compared with patients without fractures: femoral neck (0.689 ± 0.10 versus 0.807 ± 0.12 g/cm 2, P. Observed and expected number of patients with the different genotypes in each group, with or without fractures We have studied genomic DNA obtained from the 215 post-menopausal women by PCR-SSCP.

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Displays the pattern of migration of some patients on the SSCP gels. We identified four bands of interest; the additional bands (, and ) corresponded to another migration conformation, as confirmed by sequencing. The distribution of the genotypes is reported in. The distribution of the genotypes in both groups did not differ from the expected Hardy-Weinberg equilibrium for patients with fractures, but patients without fractures had an excess of heterozygotes.

The numbers of patients with or without osteoporotic fractures for the three genotypes are reported in. Compared with the RR and rr homozygotes, the prevalence of Rr heterozygotes was lower in the group with osteoporotic fractures (χ 2 = 6.63, P 60 years presenting with vertebral fractures, measurement of BMD at the lumbar spine is subject to several artefacts in comparison with measurements at the femoral neck, which are more reliable. Indeed, in our population femoral neck BMD was negatively correlated with the number of fractures ( r = −0.44, P. CTR polymorphism is at amino acid 447 where a T→C mutation changes a leucine to a proline.

PCR amplification of genomic DNA was carried out using specific primers bordering the polymorphic 447 site. (A) Autoradio-graphy of SSCP gel. Lanes a-f, DNA from individuals with the leucine allele (bands 1 and 3, additional bands 5 and 6); lanes i and j, DNA from patients with the proline allele (bands 2 and 4 and sometimes additional band 1); lanes g, h and k, DNA from patients with the two alleles; lane l, DNA from the TT cell line with the two alleles. ND, non-denatured DNA. (B) Partial sequence of PCR products amplified from patients homozygous for proline or leucine alleles. CTR polymorphism is at amino acid 447 where a T→C mutation changes a leucine to a proline.

PCR amplification of genomic DNA was carried out using specific primers bordering the polymorphic 447 site. (A) Autoradio-graphy of SSCP gel. Lanes a-f, DNA from individuals with the leucine allele (bands 1 and 3, additional bands 5 and 6); lanes i and j, DNA from patients with the proline allele (bands 2 and 4 and sometimes additional band 1); lanes g, h and k, DNA from patients with the two alleles; lane l, DNA from the TT cell line with the two alleles. ND, non-denatured DNA. (B) Partial sequence of PCR products amplified from patients homozygous for proline or leucine alleles. A polymorphic CTR site is related to BMD. BMD at the femoral neck and lumbar spine in 199 subjects according to their genotype.

Results are expressed as the means ± SEM. Results were analyzed by covariance analysis to correct for the effects of age, weight, height, age of menopause and duration of hormone replacement therapy. P indicates the level of significance between the two means (Fisher's LSD test). This analysis confirmed the existence of a significantly higher BMD value at the femoral neck in Rr versus RR and rr subjects and at the lumbar spine in Rr versus RR subjects. A polymorphic CTR site is related to BMD. BMD at the femoral neck and lumbar spine in 199 subjects according to their genotype. Results are expressed as the means ± SEM.

Results were analyzed by covariance analysis to correct for the effects of age, weight, height, age of menopause and duration of hormone replacement therapy. P indicates the level of significance between the two means (Fisher's LSD test). This analysis confirmed the existence of a significantly higher BMD value at the femoral neck in Rr versus RR and rr subjects and at the lumbar spine in Rr versus RR subjects. Clinical data for the 215 post-menopausal women studied for each CTR genotype We also determined if the two alleles were transcribed and expressed. Reverse transcription of RNA extracted from cell lines showed that T47D (RR) and HL60 (rr) transcribed the leucine and the proline allele, respectively. In the TT cell line (Rr), both alleles were transcribed, as PCR isoforms of the cDNA of the calcitonin receptor containing either leucine or proline were detected after reverse transcription.

Discussion These results confirm the existence of two allelic forms of the human calcitonin receptor gene differing by a single base mutation changing a proline to a leucine in the peptidic sequence of hCTR. The distribution of calcitonin receptor alleles in a sample of the Japanese population is quite different from that we observed in our Caucasian population.

In Japan the proline homozygote (rr) is the most frequent genotype (70%), while in our sample proline homozygotes were rare (6.5%). The risk of vertebral fractures is higher in the Japanese population. It is interesting to note that the frequency of heterozygotes in the Japanese population is much lower (20%) than in the population we studied (41%) and could perhaps account for the increase in risk of vertebral fractures in this population. A recent work in a sample of post-menopausal Italian women without osteoporotic fractures show a similar distribution of the CTR genotypes, the rr genotype representing almost 20% of the population. No significant differences in BMD were observed between the three genotypes by ANOVA analysis.

Our results clearly show that the subjects who are proline/ leucine heterozygotes have a higher bone density than the proline or leucine homozygotes. Moreover, these women have a strikingly lower incidence of vertebral fractures and fracture risk. The data on the genotypes according to fracture were compatible with an advantage of heterozygotes. This last hypothesis is supported by the low BMD in both homozygous groups compared with the heterozygotes. A further argument is the high conservation of the proline residue in all species studied with the exception of the human isoform. The absence of the proline residue could alter the secondary structure of the calcitonin receptor, more so as two other proline residues border this region. Progressive truncation of the C-terminal tail of the porcine receptor greatly decreased internalization of the ligand-receptor complex and reduced the magnitude of adenylate cyclase responses.

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This underlines the importance of the CTR C-terminal domain and suggests that the proline/leucine mutation could alter receptor biological activity. Finally, heterozygotes could produce both alleles of the receptor, resulting in an advantage as compared with homozygotes, especially as this genetic effect accounts for 13% of the total variance of BMD in covariance analysis. However, these results will need to be confirmed on a much larger series of patients as rr homozygotes are rare ( n = 14). Several mRNA isoforms of hCTR can be generated by alternative splicing of one specific gene.

The two main isoforms reported differ by the presence of a 16 amino acid insertion in the first intracytoplasmic domain. Both stimulate adenylate cyclase but only the truncated one is able to activate the inositol phosphate pathway. This diversity of mRNA receptor isoforms is independent of the allelic polymorphism we report here, since the same splicing events could arise on the two alleles, as assessed by sequences so far reported. The two mRNA isoforms of hCTR are expressed in the homozygous RR T47D cell line. HL60 cells, which are rr homozygotes, also express both isoforms. Therefore, there is no clear relationship between the occurrence of these mRNA isoforms and the polymorphism we describe.

Calcitonin's physiological role in age-related bone loss and osteoporosis is controversial. However, it is the only protein which binds specifically to the osteoclast membrane and has a well-demonstrated direct anti-resorptive activity. Our results add an important point to the debate because a mutation of the receptor for this hormone, in a region of likely functional significance, is associated with fracture and low bone mass. Furthermore, the association of a mutation in the receptor for this hormone with osteoporosis fits with the concept that bone loss with age and fractures are associated with high resorption. The higher bone density and decreased fracture risk in patients heterozygous for the CTR polymorphism could also be due to interaction of the receptor gene with other genes implicated in osteoporosis. Finally, our results suggest that the calcitonin receptor gene is one of the candidate genes for osteoporosis. Materials and Methods Clinical subjects We studied 215 post-menopausal French women of Caucasian origin at our clinic.

All the subjects gave their informed consent for the study. The women were either volunteers, part of an epidemiological cohort ( n = 143) or referred for vertebral fractures ( n = 72). All the women completed an osteoporosis-oriented questionnaire (including a dietary questionnaire) and had a spine X-ray. Vertebral crush fractures were assessed according to the Genant criteria. The bone density was measured in 199 patients at the lumbar spine and the femoral neck with a Lunar DPX-L (Madison, WI). Among the volunteers of the epidemiological cohort, 20 had presented with an osteoporotic fracture (wrist or vertebrae). We therefore had a group of 123 women (mean age 65 ± 6, 50–81) without osteoporotic fractures.

Of the sample 31% had received hormone replacement therapy (HRT) for 2.4 ± 4.8 years. According to the WHO definition of osteoporosis based on bone density, 4.1% of them had osteoporosis, 52.1% osteopenia and 43.8% were normal.

Among the 92 women with osteoporotic fractures, 75 had presented with crushed vertebrae and 17 with wrist fractures. The average number of crushed vertebrae was 3.3 ± 1.5.

The weight and height, and age of menopause were lower in women with fractures compared with women without fractures. Tissue culture TT cell line (medullary thyroid carcinoma cell line). TT cells were grown in RPMI 1640 medium with 10 mM HEPES, 6 mM glutamine supplemented with 15% fetal calf serum (FCS). Medium was changed every 2 days until confluence. HL 60 (promyelocytic cell line) and T47D (breast carcinoma cell line) cell lines.

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The cells were cultured in RPMI 1640 in the presence of 2 mM glutamine, penicillin/streptomycin supplemented with 10% FCS. Cells were seeded at a concentration of 250 000 cells/ml. Medium was changed every 2 days. Cells were grown at 37°C in 5% CO 2. Biochemistry DNA extraction. DNA extraction from the patients and the TT, HL 60 and T47D cell lines was carried out using Trizol solution (Life Technologies).

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RNA extraction. Total RNA was extracted from TT and HL60 cells using the guanidine isothiocyanate/chloroform method. Reverse transcription of RNA. An aliquot of 1 µg total RNA extracted from the cell lines was reversed transcribed (Superscript II; Life Technologies) using an oligo(dT) primer for 10 min at 23°C and extension was carried out at 42°C for 50 min. PCR amplification of the cDNA. Aliquots of 200 ng cDNA were first denaturated for 5 min at 950 C. The amplification reaction was carried out using Taq polymerase (Gibco BRL) for 30 cycles: 94°C, 30 s; 60°C 30 s; 72°C 30 s.

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The reaction was terminated by 5 min elongation at 72°C. PCR-SSCP analysis of the calcitonin receptor gene. We searched for the proline/leucine mutation by PCR-SSCP on DNA extracted from cell lines or from whole blood. PCR amplification was performed using two oligonucleotides primers (Genosys) (forward, 5′-ATTCAGTGGAACCAGCTTG-3′; reverse 5′-GATGGCTCAGTGATCACGAT-3′) located on each side of the base pair mutation.

33PdCTP (2 µCi, 2000 Ci/mmol; Dupont de Nemours-NEN) was included. Aliquots of 200 ng genomic DNA were first denaturated for 5 min at 95°C. The amplification reaction was carried out using Taq polymerase (Gibco BRL) for 30 cycles: 940C, 1 min; 60 oC 1 min; 72 oC 2 min. The reaction was terminated by 5 min elongation at 72°C.

The denaturated amplification products were separated overnight by gel electro-phoresis (6% polyacrylamide, 10% glycerol, non-denaturing gel) at room temperature. DNA sequencing of PCR products. After vacuum drying of the gel, autoradiography was performed for 24–48 h. The bands revealed in the SSCP gels were extracted, re-amplified by PCR using the same oligonucleotides primers, purified and sequenced (Double Strand Sequencing kit; Life Technologies).

Samples from 100 patients picked at random were also subjected to PCR using the same primers we used for SSCP and digested overnight with AluI. Fragments were separated by agarose gel electrophoresis and stained with ethidium bromide.

Identical genotypes were observed with both methods. Statistical analysis Results are expressed as means ± SD and were analyzed using variance analysis. Bone density measurements were corrected for age, weight, height, age of menopause and duration of HRT by covariance analysis. Frequency of alleles and fractures were analyzed using the χ 2 distribution.

Acknowledgements This worked was supported by a GENSET fellowship to M.F. And by an individual grant from the French Ministry of Research and Education to J.L.F.