Polymorphism of Riboavin Transporter (RFVT) Gene in Patients with Esophageal Cancer in Hakka Population in Southern China
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Polymorphism of Riboflavin Transporter (RFVT)
Gene in Patients with Esophageal Cancer in Hakka
Population in Southern China
Tao Li ( litaoli23@163.com )
Medical College of Jiaying University
Wanqin Hu
Medical University of jiaying University
Minli Zheng
medical college of jiaying university
Bo Qiu
medical college of jiaying university
Yuhui Yang
medical college of jiaying university
Research
Keywords: Riboflavin transporter, esophageal cancer, polymorphism,Hakka
Posted Date: July 19th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-709003/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Page 1/13Abstract
Background: The aim of this study was to examine the human riboflavin transporter (RFVT) gene
polymorphism in Hakka esophageal cancer patients in Guangdong Province and to explore its
relationship with esophageal cancer.
Methods: We used matrix-assisted laser desorption ionization flight time mass spectrometry to genotype
RFVT1 rs346821 and RFVT2 rs13042395 in 211 Hakka esophageal cancer patients and 216 healthy
controls in Meizhou.
Results: There were polymorphisms in the rs346821 and rs13042395 in the two groups of Hakka; G/A
polymorphism in the RFVT1 gene rs346821; three genotypes GG, AG, and AA of RFVT rs346821 were
found in the Meizhou Hakka population, with distribution frequencies in the esophageal cancer group at
61.61, 36.02, and 2.37% respectively; the frequencies of allele G and A were 79.62 and 20.38%. The
distribution frequencies of the three genotypes in the control subjects were 54.17, 40.28, and 5.55%
respectively; the frequencies of allele G and A were 74.31 and 25.69%. There was a significant difference
observed in the binary logistic analysis; the occurence of AG and AA genotypes in the Hakka population
were 2.424 times and 1.922 times higher than that of the GG genotype. Bioinformatics analysis revealed
that RFVT1 rs346821 had a missense mutation, and the corresponding amino acid was changed from
alanine to valine, resulting in a change in its protein structure.
Conclusions: The study concluded that RFVT1 rs346821 polymorphism might be associated with the
genetic susceptibility of the Hakka population to esophageal cancer. The variation in the protein structure
resulting from the variation of RFVT1 rs346821 might have a significant influence on the occurrence and
development of esophageal cancer in the Hakka population.
Introduction
Esophageal cancer is one of the most common malignant tumors of the digestive tract and is the sixth
leading cause of cancer deaths[1–3]. There were more than 572,000 people newly diagnosed with
esophageal cancer and 508,585 deaths globally in 2018[4]. Esophageal cancer is also one of the most
commonly diagnosed and fatal types of cancer in Asia, especially in East Asia [5]. Among them, China
has the highest incidence of esophageal cancer, with 234,624 cases of esophageal cancer incidents and
212,586 deaths recorded in 2017[6], mostly in northern China[7–10], which has caused a heavy economic
burden to Chinese people, especially in rural areas[11].
Esophageal cancer is a complex disease with various causes.In addition to alcohol, tobacco and intake
of pickled vegetables[12–14], genetic polymorphism is also closely associated with the occurrence of
esophageal cancer[2, 15–21].The RFVT gene is a riboflavin transporter and riboflavin deficiency is one of
the important risk factors for esophageal cancer[22–24]. The association of riboflavin transporter with
susceptibility to esophageal cancer is however, no clear.
Page 2/13Hakka is a sub-family of Chinese Han population and also widely distribute in Southeast Asia. Located in
the northeast of Guangdong Province, China, the Meizhou region is known as the famous center of Hakka
World, with a total area of 15,876 square kilometers and a population of 5.43 million. More than 95% of
the residents living in Meizhou are Hakkas, who show lots of unique features in diet, life style, culture,
language, and environment [25]. Esophageal cancer was one of the major diseases in the regions
populated by the Hakka population. However, the relationship between RFVT gene polymorphism and
susceptibility to esophageal cancer in this population, needs evaluation. This study is designed to
evaluate the distribution of RFVT1 (rs346821) and RFVT2 (rs13042395) genotypes in this population and
explore the relationship between the polymorphism and the susceptibility to esophageal cancer.
1. Material And Methods
1.1 Clinical data
Newly diagnosed esophageal cancer patients (classified according to the ICD-151 standard) were
recruited between July 2019 and December 2020 at the Affiliated Hospital of Medical College of Jiaying
University and were approved by the hospital ethics committee. We included 211 esophageal cancer
patients: 147 males and 64 females aged 35 to 80 years (average age, 60.44±9.33 years). All patients
underwent endoscopy or pathological examination for clear diagnosis. We randomly selected 216 healthy
subjects with non-digestive diseases and who were cancer-free and were of the same age. The control
group included 136 males and 80 females aged 19 to 79 years (average age, 59.44±11.87 years). The
patients in both groups had patrilineal and matrilineal Hakka ancestry and they had been living in the
Meizhou municipality jurisdiction for 3 generations or more (including 3 generations).
1.2 Genomic DNA extraction
Venous blood (2 ml) anticoagulated using heparin sodium was collected from each esophageal cancer
patient or healthy control. A DNA extraction kit (Promega, USA) was used to extract DNA, according to the
manufacturer’s instructions. The DNA was stored at -80℃ until further analysis.
1.3 Locus selection and primer design
RFVT1 (rs346821) and RFVT2 (rs13042395) gene polymorphisms were selected, based on the gene
target sequence and associated polymorphic loci. Polymerase chain reaction (PCR) primers were
designed using the Sequenom Assay Design 3.1 software (Sequenom,Inc, Delaware, USA). The primers
were amplified and extended using multiplex PCR, specific for this locus. The primers used are as follows:
RFVT1 rs346821: forward: 5’- GGG CAA ACA GGA AAA GCT CT-3’ and reverse: 5’-AGG AGC AGA GGA GGA
AGA GA -3’; RFVT2 rs13042395: forward: 5’- CCC AGC CCA AGA AAG TGA AG -3’; reverse: 5’- ACC AGC
GCT CAA CAG ATA GA-3’.The primers were subjected to mass spectrometry using matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to evaluate the consistency
between the actual and theoretical molecular weights, to ascertain the purity of primers for the
Page 3/13experimental requirements. Characteristic mass spectra of the RFVT1 (rs346821) and RFVT2 (rs13042395) genotypes are shown in Figure.S1. 1.4 Gene analysis Purified genomic DNA samples were diluted and added to a 384-well plate with the designed primers. The PCR reaction consisted of: 1U Taq polymerase, 20-50 ng genomic DNA, 2.5 pmol PCR primers, and 2.5mM deoxyribonucleotide triphosphate (dNTP). PCR amplification conditions were 95°C for 2 min, 45 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 60 s. The remaining dNTP was removed by adding 0.3 U alkaline phosphatase. EXTEND MIX (2 µl) was added to complete the single-base extension process. The reaction conditions were 94°C for 5 s, 52°C for 5 s, 40 external cycles, and 80°Cfor 5 s×5 internal cycles, 72°C for 3 min; the products were stored at 4°C. Following resin desalination, the reaction products were spotted into SpectroCHIP chips (Sequenom) with an automatic spotting instrument and were analyzed using MALDI-TOF-MS. The mass spectra peak was detected using Typer 4.0 software; based on which the target locus genotypes of each sample were interpreted. 1.5 Comprehensive bioinformatics analysis NCBI database was used for analyzing rs346821 C>T point mutation and confirming the corresponding change in the amino acid sequence. The amino acid sequence of RFVT1 from different species was obtained from NCBI database, and the conservation of the mutation site was analyzed. 1.6 Protein model prediction I-TASSER open server was used to predict the protein structure. PYMOL was used to visualize the details of the protein structure. 1.7 Statistical analysis SPSS 21.0 was used for statistical analysis. Hardy-Weinberg equilibrium was tested; the genotype and allele frequency of the patient and control groups were analyzed using the chi-square test. A binary logistic regression analysis was used to evaluate the association between the polymorphic loci and esophageal cancer susceptibility with the odds ratio (OR) and 95% confidence interval (95% CI) as the relative risk. A P value of
groups was not statistically different (Table.S1). Distribution of the age in esophageal cancer group
showed a normal distribution. The proportion of patients aged between 55 and 65 years was the highest,
and the peak incidence occurred at around 60 years of age, and then decreased with age. The ratio of
male to female patients was 2:1 (Table.S2).
2.2 Genotype frequency distribution of RFVT1 (SLC52A1)
rs346821 and RFVT2 (C20orf54) rs13042395 in patients
and controls
The GG, GA, AA polymorphisms were present in rs346821 and CC, CT, and TT polymorphisms were
present in the rs13042395 in the Meizhou Hakka population (Table 3). The distribution frequencies of
rs346821 genotype GG, GA, and AA polymorphisms in the esophageal cancer group were 61.61%, 36.02%,
and 2.37%, respectively; while those in the healthy control group were 54.17%, 40.28%, and 5.55%,
respectively. Binary logistic regression analysis showed that the risk of developing esophageal cancer for
subjects carrying the GA, AA genotype was higher than that for subjects carrying the GG genotype (OR =
2.424, 95% CI = 1.254–4.686; OR = 1.922, 95% CI = 1.005–3.674). Therefore, the risk of esophageal cancer
in the Hakka population carrying the RFVT1 gene rs346821 locus GA and AA genotype was higher. The
distribution frequencies of rs13042395 genotype CC, CT, and TT polymorphisms in the esophageal
cancer group were 32.23%, 28.44%, and 39.33%%, respectively; while that in the healthy control group
were 28.70%, 25.46%, and 45.84%, respectively (Table 1). There was no significant difference in the
frequency between the two groups.
Table 1
Distribution of the rs346821 and rs13042395 genotypes in esophageal cancer group and healthy
control group
Gene locus Genotypes esophageal cancer healthy OR 95%CL ༰
control
(n = 211) (n = 216)
rs346821 GG 130(61.61) 117(54.17) 1
GA 76(36.02) 87(40.28) 2.424 1.254ཞ4.686 0.008
AA 5(2.37) 12(5.55) 1.922 1.005ཞ3.674 0.048
rs13042395 CC 68(32.23) 62(28.70) 1
CT 60(28.44) 55(25.46) 1.005 0.671ཞ1.506 0.979
TT 83(39.33) 99(45.84) 0.769 0.572ཞ1.032 0.08
Page 5/132.3 Allele frequency distribution of the rs346821 and
rs13042395 in the esophageal cancer and the healthy
control groups
The A and G allele frequencies of rs346821 in the esophageal cancer group were 20.38% and 79.62%,
and those in healthy control group were 46.45% and 53.55%, respectively. There was no statistically
significant difference in allele frequency between the two groups (chi-square = 1.377, P = 0.241). The C
and T allele frequencies of rs13042395 in the esophageal cancer group were 46.45 and 53.55%, and
those in healthy control group were 41.44% and 58.56%, respectively. The differences in allele frequencies
between the two groups were not statistically significant (chi-square = 0.901, P = 0.342) (Table 2). These
showed that the distribution of alleles of rs346821 and rs13042395 was not statistically different
between the esophageal cancer and the healthy control groups.
Table 2
Distribution of the rs346821 and rs13042395 alleles in esophageal cancer group
and healthy control group
Gene locus Alleles esophageal cancer healthy X2 ༰
control
(n = 211) (n = 216)
rs346821 A 86(20.38) 111(25.69) 1.377 0.241
G 336(79.62) 321(74.31)
rs13042395 C 196(46.45) 179(41.44) 0.901 0.342
T 226(53.55) 253(58.56)
Page 6/13Table 3
Logistic analysis of age and gender of RFVT1 (rs346821) genotype in esophageal cancer group
and healthy control group
Variables esophageal cancer (n = 211) healthy control (n = 216) X2 P
GG AG AA GG AG AA
Gender
Male 88 56 3 72 56 8 0.739 0.691
Female 42 20 2 45 31 4
Age(years)
≥60 65 34 2 50 45 8 0.518 0.7722.5 Conservative analysis and protein model prediction of
RFVT1 rs346821
We further analyzed the conservation of RFVT1 rs346821 and predicted its protein model. The results
showed that the alanine at position 271 was significant conserved by BLAST (Fig. 1A). By using the I-
TASSER public server, we predicted the protein structure and the protein structure was visualized using
PyMOL. When alanine was replaced by valine, a small hydrophilic polar amino acid was replaced by the
large hydrophobic aromatic amino acid, and the change of this domain influences the function of the
RFVT1 protein (Fig. 1B).
3. Discussion
Riboflavin is involved in the metabolism of fat, amino acid, vitamin, and carbohydrates and plays an
important role in cell proliferation and angiogenesis. It maintains homeostasis. Riboflavin deficiency is a
major risk factor for esophageal cancer. Proper supplementation of riboflavin could significantly reduce
the prevalence and recurrence of esophageal cancer and improve the prognosis of esophageal cancer
patients[26, 27]. However, the effect of dietary riboflavin supplementation varied among individuals,
suggesting an interaction between environment and genetic factors in esophageal cancer. A Genome-
Wide Association Study (GWAS) revealed that the Chinese RFVT2 gene was a susceptible gene in the
Han, Hazaks, and Uygur esophageal cancer patients among China’s population; rs13042395 was the
common susceptibility locus between the two types of esophageal cancer[28]. The other RFVT2 loci, such
as 1172 C > A and 1246A > G, are also associated with esophageal cancer[29]. However, there are also
suggestions that it may not contribute to risk of ESCC in Iranians [30]. Riboflavin transporter-1 (RFVT-1) is
a plasma membrane protein, encoded by the SLC52A1 gene, which can transport vitamin B2 (riboflavin,
RF) into cells. Therefore, it plays a role in controlling the intracellular homeostasis of RF. Currently, the
relationship between RFVT1 rs346821 and RFVT2 rs13042395 and esophageal cancer in Hakka
population is still unclear.
The Hakkas are a branch of the Chinese Han population who speak Hakka and have a relatively unique
genetic background and diet in the Han branch. Studies on the association of gene polymorphisms with
susceptibility to esophageal cancer in the Hakka population have been inconclusive. This study showed
that the average age of patients with esophageal cancer in this area was between 55 and 65 years of age,
with a peak of 60 years. The prevalence of esophageal cancer was higher in male patients when
compared to that in female patients, with the ratio of male to female at 1:2, which was consistent with
earlier reports.
Analysis of RFVT1 (rs346821) and RFVT2 (rs13042395) genotypes showed that there were
polymorphisms at rs346821 (A/G) and rs13042395(C/T) in the Hakka population. Among them, there
were GG, AG, and AA polymorphisms of rs346821 gene in the Hakka population in Meizhou. The
Page 8/13frequencies of distribution of rs346821 genotype among esophageal cancer patients were 61.61, 36.02,
and 2.37 %, respectively; while those in the controls were 54.17, 40.28, and 5.55%, respectively. Subjects
with the GA genotype (OR = 2.424, 95% CI = 1.254ཞ4.686) and AA genotype (OR = 1.922, 95% CI =
1.005ཞ3.674) had a higher risk of developing esophageal cancer, when compared to those carrying the
GG genotype. The risk of esophageal cancer was higher in the Hakka people, who carried the GA and AA
genotypes of RFVT1 rs346821. The distribution frequencies of rs13042395 genotypes was not
significantly different between the esophageal cancer and the control groups, indicating that the
rs13042395 genotypes might not be associated with esophageal cancer in the Hakka population. There
were no significant differences in the distribution frequency of the alleles and the stratified analysis,
based on age and gender, of the RFVT1 (rs346821) and RFVT2 (rs13042395) genes between the
esophageal cancer and the control groups. The results of this study are consistent with most studies[30–
32], but there are also studies suggesting that rs13042395 genotype is associated with esophageal
cancer in Chinese Han population[33].This could possibly be attributed to the relatively unique genetic
background of the Hakka population in the Han branch. The Hakka are most similar to the Han, but they
also show the characteristics of the Shes nationality, who speak the Miao language in China, which is
different from that of the southern Han, who speak the Dong Tai language[34]. The results of this study
further confirm that differences in genetic polymorphism are associated with different populations, races,
and regions.
In this study, a c.812C > T (p.Ala271Val) variant in rs346821 was identified. The mutation at this site was
a missense mutation, resulting in the mutation of alanine to valine, and the alanine at position 271 was
highly conserved among 6 vertebrates. The alanine to valine substitution is likely to significantly impact
the functionality of RFVT1, as this is a change from a small hydrophilic polar amino acid to a large
hydrophobic aromatic amino acid.
4. Conclusions
In conclusion, RFVT1 (rs346821) polymorphism might be associated with esophageal cancer
susceptibility in the Hakka population in Guangdong Province. c.812C > T (p.Ala271Val) variant in
rs346821 might affect the function of RFVT1 by altering the protein structure, which might influence the
occurrence of esophageal cancer in the Hakka population. This is significant for the diagnosis of
esophageal cancer in Hakka population. Genetic-mutation animal model construction and functional
studies would further elucidate the pathogenesis of esophageal cancer in hakka population.
Abbreviations
RFVT
riboflavin transporter; OR:odds ratio; CI:confidence interval; PCR:polymerase chain reaction.
Declarations
Page 9/13Acknowledgements
We thank Dr. Mingyuan Wang for his support in bioinformatics in this study.
Authors’ contributions
Tao Li and Bo Qiu contributed to the conception and designed the study. Wanqin Hu and Minli Zheng
collected the data and carried out the experiment. Yuhui Yang edited the manuscript. All authors read and
approved the final manuscript.
Funding
This research was supported by grants from the science and technology Foundation of Guangdong
Province (NO: 2013B021800084).
Availability of data and materials
The datasets supporting the conclusion of this article are included within the article.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no conflict of interest.
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Page 12/13Figures
Figure 1
Conservative analysis and protein model prediction of RFVT1 rs346821. A.Conservation analysis of the
alanine at position 271 (p.F271) among 6 vertebrates. B.Cartoon representationof the model structure of
RFVT1 by PyMOL.
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