Association between TBXT rs2305089 polymorphism and chordoma in Iranian patients identified by a developed T‐ARMS‐PCR assay

Abstract Background Chordoma is a locally aggressive bone tumor with a high capability of recurrence. Because chordoma often occurs at critical locations next to neurovascular structures, there is an urgent need to introduce validated biomarkers. T‐box transcription factor T (TBXT; OMIM: 601397) plays an important role in the pathogenesis and survival of chordoma cells. Methods Herein, we aimed to show whether rs2305089 polymorphism is correlated with chordoma in the Iranian population. In order to detect rs2305089, tetra‐primer amplification refractory mutation system‐polymerase chain reaction (T‐ARMS‐PCR) was used. In total, 19 chordoma patients and 108 normal healthy individuals were recruited and screened using T‐ARMS‐PCR. The results were subsequently validated by Sanger sequencing. Results The genotype distributions and allele frequencies were significantly different among the patient and healthy groups (p‐value <0.05). The A allele of rs2305089 showed a significant positive association with chordoma risk (p‐value <0.05). DNA sequencing verified the T‐ARMS‐PCR results as well. This study demonstrated the association between TBXT rs2305089 and chordoma in an Iranian population using a simple, accurate, and cost‐effective T‐ARMS‐PCR assay. Conclusions Our results were in line with those of previous studies showing that TBXT rs2305089 is associated with chordoma development. We also developed an efficient T‐ARMS‐PCR assay to determine the genotype of rs2305089.


| INTRODUC TI ON
Chordoma (OMIM: 215400) is a rare primary bone tumor that originated from notochord remnants-a rod-like structure that supports embryo development-in the axial skeleton. [1][2][3] Chordoma usually progresses slowly, but its locally aggressive nature can affect the patients' quality of life. 4 This kind of cancer often occurs sporadically, but some rare familial cases have also been documented. 5 The estimated overall incidence of chordoma in the general population is 0.08 per 100,000 live births. 4 This cancer affects males more frequently than females (2:1). 6 Although chordoma's occurrence has been reported from infancy to senescence, it often occurs in the sixth decade of patients' life. 2 Furthermore, its occurrence tends to be higher in Caucasians than in African-Americans (4:1). 2 The first-line strategies to treat primary chordoma are still based on surgical resection followed by radiotherapy, although these strategies result in a median overall survival of 7.7 years. 7,8 Moreover, chordoma, as a mildly aggressive cancer, often affects the adjacent vital tissues before becoming symptomatic-thus, completely resecting all cancer-related tissues is demanding. In short, there is an urgent need to introduce safe biomarkers (e.g., susceptible genetic loci) to better manage patients as accurately as possible.
TBXT (T-box transcription factor T; OMIM: 601397) has been introduced as one of the most important genes in embryonic development. This gene is exclusively expressed during the early stages of development and is silenced in most developed tissues, except differentiated tissues of the testis and some parts of the thyroid. 9,10 TBXT-also known as the T gene or brachyury-functions as an embryonic nuclear transcription factor in mesoderm formation and differentiation. This protein binds to the palindromic T-site of DNA using its N-terminus (T-box). 11 Interestingly, Tbxt −/− mice die during the embryonic period due to multiple mesodermal abnormalities. 11 The TBXT is located on the 6q27 region and is associated with susceptibility to chordoma and neural tube defects. 5,9,12,13 For example, the homozygous H171R was observed in four members of three unrelated consanguineous families with sacral agenesis and vertebral anomalies. 14 Germline duplication of TBXT was also observed in the familial type of chordoma. 5 Moreover, the silencing of TBXT in chordoma cell line U-CH1 resulted in decreased cell proliferation. 12 Pillay et al. indicated a strong association between chordoma risk and the common nonsynonymous single-nucleotide polymorphism (SNP) rs2305089 in Europeans. 1 Rs2305089 (NM_003181.4; c.530G>A: p. Gly177Asp) is located at exon 4 of TBXT. The researchers also indicated a higher mRNA expression of TBXT in patients with AA genotype than in GA sporadic chordoma patients without TBXT gene duplication. 1 The higher susceptibility imputed to the A allele to chordoma was later confirmed by Kelley et al.

in American and
Canadian individuals. 3 On the contrary, a study using 65 skull-based chordoma patients and 120 healthy individuals among the Chinese population showed that rs2305089 was not significantly correlated with chordoma risk. 15 Since the reported association of rs2305089 may be linked to 'population-specific' backgrounds, it is imperative to check this association in different ethnic populations.
Hence, finding disorder-associated alleles/genotypes is important to reduce the burden of genetic disorders and take steps toward turning 'generalized' medicine into 'personalized' medicine. Although using high-throughput approaches (e.g., genome-wide association studies, TaqMan genotyping assay, and whole-exome sequencing) is highly suggested for genetic association study, they are not costaffordable for limited resources. Such techniques, in some cases, can be replaced by inexpensive, simple, and quick ones, such as tetraprimer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR). T-ARMS-PCR amplifies the specific target template by only two sets of primers, including inner and outer ones.
The outer primers are non-allele-specific and simply amplify the target sequences of the main template, whereas the inner primers are allele-specific ones whose 3′-ends embrace the polymorphic nucleotides.
The present study investigated the association between TBXT rs2305089 polymorphism and chordoma in an Iranian population.
Herein, we also aimed to develop a reliable, low-cost, and simple T-ARMS-PCR method to ease the genotyping of this polymorphism.

| Study participants
In order to determine whether rs2305089 is associated with chor-

| DNA Extraction
A blood sample of approximately 10 ml was collected from each participant, and their DNA samples were isolated using a standard salting-out procedure. 16,17 The quality and quantity of the DNA samples were determined with a 1.5% agarose gel and NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Clayton, VIC, Australia), respectively.

| Primer designing and T-ARMS-PCR
The DNA sequence of the TBXT gene was retrieved from the NCBI database according to Human Genome Reference hg19 (NM_003181.4). Subsequently, primers were designed for selected SNP using Primer1 software (Table 1). 18 The specificity of each primer was assessed using 'NCBI Primer BLAST' (https://www.ncbi. nlm.nih.gov/tools/ prime r-blast/) and in-silico PCR of UCSC genome browser (https://genome.ucsc.edu/cgi-bin/hgPcr).
T-ARMS-PCR was performed in a total volume of 20 μl, includ-

| Sanger sequencing
Sanger sequencing was used to validate the T-ASMS-PCR results. To this end, 15 samples showing the GG, GA, and AA genotype by T-ARMS-PCR were amplified and sequenced. To amplify the template, we used the same outer primer sets as mentioned above.

| Statistical Analysis
The statistical analysis was performed using SPSS software version 24.0 (SPSS Inc.,). A chi-square (χ 2 ) test was used to compare the allele and genotype frequencies between patients and control groups. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using χ 2 test. p-values <0.05 were considered statistically significant.

| Demographic data
The median (IQR) ages of patients and control participants were 52.0 (16-80) years and 47.0 (30-83) years, respectively. The patient group comprised of six females and 13 males, while the control group consisted of 55 females and 53 males. The patient-gender distribution was compatible with those reported in previous reports. 6 We could not find any statistically significant differences between the two groups in terms of their age (p-value =0.4) and gender (p-value =0.1). The most important clinical findings detected in the patients are put forth in Table 2.

| T-ARMS-PCR
A T-ARMS-PCR electrophoretogram of rs2305089 is shown in Figure 1A. The GA genotype showed three bands (262 bp, 587 bp, and 810 bp), while the GG genotype showed two bands (262 bp and 810 bp). Also, the AA genotype showed two bands (587 bp and 810 bp).
The genotype distributions and allele frequencies of rs2305089 in patients and control participants are summarized in Table 3.  (Table 3).

| DISCUSS ION
TBXT gene encodes an embryonic nuclear transcription factor that is necessary for proper mesoderm formation and differentiation. As an evolutionarily conserved gene ( Figure 1C,1D), TBXT controls the development of the notochord and is then silenced during later developmental stages (e.g., in the human fetus at ~12 weeks). Therefore, the notochord recedes prenatally. Furthermore, TBXT can promote the epithelial-mesenchymal transition. 7 Also, to some extent, it controls the cell cycle and biological behavior of cancer cells. 22 Herein, we unveiled a significant association between chordoma and the presence of the AA genotype of rs2305089 in the   One of the limitations of our study is the limited number of patients that in turn can be attributed to this fact that chordoma is a rare disease. Our data were fully consistent with the previous findings, showing that this study has adequate power to arrive at a conclusion. Another limitation is that we did not determine the effects of rs2305089 on the expression of TBXT or other downstream genes. Further investigations should be conducted to determine the RNA and protein levels of TBXT in the patients.

| CON CLUS IONS
T-ARMS-PCR is a sensitive, specific, and cost-effective technique for SNP genotyping. We indicated a significant association

ACK N OWLED G M ENTS
We appreciate all the study subjects and their families for participating in this research. This work was supported by Iran University of Medical Sciences, Tehran, Iran (Grant Number: 98-3-49-15990).