papillary thyroid carcinoma ( ptc ) is the most common form of thyroid malignancy , which generally has a good prognosis and accounts for approximately 8085% of all thyroid carcinomas [ 13 ] .
however , this type of cancer may cause distant metastasis and be more aggressive in older patients .
established risk factors for ptc include ionizing radiation , positive family history , and thyroid nodular disease ; but these factors do not appear to account for the increasing incidence of ptc .
studies have shown that age , gender , hashimoto 's thyroiditis , thyroid - stimulating hormone concentrations , solitary nodularity , and anti - thyroglobulin antibodies positivity are known risk factors for ptc development [ 7 , 8 ] .
other studies have proposed that genetic factors may also contribute to the risk of ptc [ 9 , 10 ] .
gender difference in incidence , aggressiveness , and prognosis has been well - established in thyroid cancer .
the incidence of thyroid cancer has been reported to be three to five times more frequent in women , and this gender difference is particularly obvious for women of reproductive age .
gender disparity in thyroid cancer has also been known to be specific to the histologic subtype of thyroid cancer , with the more commonly differentiated thyroid cancer of follicular cell origin including ptc in women .
however , the potential reason for this disparity is poorly understood . genetic analysis such as
single - nucleotide polymorphism analysis has been suggested to be helpful in better understanding the molecular basis for gender disparity in thyroid and other cancers .
the cellular mesenchymal - epithelial transition ( met ) factor is a plasma membrane tyrosine kinase receptor that has low activity in normal tissues but is dysregulated in many tumor types .
the dysregulated activation of met kinase may correlate with the aggressiveness of tumor growth and metastasis .
the role of met mutation in human cancer was first established in papillary renal carcinoma .
mutations of the met protooncogene have also been described in several other types of human cancers and were suggested to correlate with tumor metastasis [ 1719 ] .
therefore , the present study was designed to investigate the association of met single - nucleotide polymorphisms ( snps ) with the gender disparity of thyroid tumors , as well as the metastasis and prognosis of ptc in the chinese population .
the study subjects comprised 858 patients with ptc ( 43.7  9.17 , 208 males/650 females ) , 556 patients with nodular goiter ( ng , 48.6  9.94 , 131 males/425 females ) , and 896 population - based normal controls ( nc , 43.7  9.06 , 219 males/677 females ) .
all patients were recruited from the china - japan union hospital of jilin university during august 2012 to december 2014 .
healthy individuals were collected from the first hospital of jilin university during the same period .
patients with ptc and ng were diagnosed according to the revised american thyroid association management guidelines for patients with thyroid nodules and differentiated thyroid cancers .
age- and gender - matched control subjects were from the general population , who were free from thyroid diseases , diabetes , and other endocrine system diseases .
this study was reviewed and approved by the medical ethics committee of the school of public health , jilin university ; and written informed consent was obtained from all participants .
two tag snps of the gene , rs1621 and rs6566 , were obtained through haploview 4.2 ( population : chb , r square cutoff : 0.8 , maf cutoff : 0.1 , and d = 1 ) .
blood samples were collected from all participants , and the whole dna genome was isolated using a dna extraction kit ( beijing kangwei century biotech co. , ltd . , china ) according to standard protocols .
the concentration and purity of the dna samples were determined by a uv-260 spectrophotometer ( shimadzu , kyoto , japan ) .
snp genotyping was carried out using the sequenom massarray platform ( san diego , ca , usa ) .
primer sequences used were as follows : rs1621-f : acgttggatgaccctgagcagaactttgtg , rs1621-r : acgttggatggtaacctacaccacatgcac , and rs6566-f : acgttggatgctggcaataaccactatcag ; rs6566-r : acgttggatgactgaatggtacttcgtatg .
polymerase chain reaction ( pcr ) analysis was performed with taq dna polymerase ( tiangen biotech co. , ltd . ,
pcr condition was 94c for 15 minutes to perform a hot - start , followed by denaturing at 94c for 20 seconds , annealing at 56c for 30 seconds , extension at 72c for one minute for 45 cycles , and incubation at 72c for three minutes .
then , shrimp alkaline phosphatase ( sap ) reaction was performed by incubating the pcr product with sap ( sequenom , inc . , san diego , ca , usa ) at 37c for 40 minutes , followed by inactivation at 85c for five minutes .
the iplex extension reaction was performed at 94c for 30 seconds and for five seconds , followed by 40 cycles at 52c for five seconds and five cycles at 80c for five seconds and at 72c for three minutes .
the product was desalted by the addition of resin in a 384-dimple plate , mixed , resuspended , and centrifuged to separate the extension products from the resin .
the completed products were analyzed using the massarray typer software version 4.0 ( sequenom , usa ) .
all statistical analyses were conducted using the  genetics  and  dgc.genetics  packages running in the r software environment ( version 3.0.2 ) .
the interaction between the met gene and gender was examined by logistic regression using an additive model .
there was no apparent difference with respect to gender among the three groups ( p = 0.991 ) .
the age of patients in the ng group was comparatively older than the age of patients in the ptc and nc groups ( p < 0.001 ) , while there was no significant difference between the ptc and nc groups ( p = 0.947 ) .
the allele and genotype frequencies of met in the ptc , ng , and nc groups are listed in table 1 . met snps ( rs1621 and rs6566 )
interactions between met snps ( rs1621 and rs6566 ) and ptc were analyzed by stratifying the patients according to gender .
the met snp rs1621 genotype was significantly associated with ptc in female subjects ( p = 0.037 ) , but not in their male counterparts ( p > 0.05 ) , as shown in table 2 .
there was no significant association between rs6566 and ptc in both male and female genders ( p > 0.05 ) .
therefore , the genotype frequency of rs1621 was analyzed in the female and male part of the ptc , ng , and nc groups , respectively .
multiple comparison adjustments were made using bonferroni correction (  = 0.017 = 0.05/3 ) . among female patients ,
the rs1621 ag genotype was significantly higher in patients with ptc when compared with normal controls , while the frequency of genotypes aa and gg was comparatively lower ( p = 0.01 ) .
there was no significant difference in rs1621 genotype distribution between the ng and nc groups or between the ptc and ng groups ( p = 0.908 and p = 0.078 , resp . ) .
differences in rs1621 genotype frequencies in female patients in each group were assessed by an additive model of logistic regression using the major allele as a reference .
ag genotype revealed an increasing risk of ptc in female patients ( or : 1.465 , 95% ci : 1.1181.92 ) , as shown in table 3 , indicating an interaction between gender and met snp ( rs1621 ) in ptc patients .
further analysis of the association between the genotype frequencies of met snp ( rs1621 ) and ptc metastasis or prognosis of patients did not reveal a significant correlation in both male and female patients ( table 4 ) .
the presence of adenosine ( a ) at snp rs1621 has been reported to increase the risk of cancer development .
snp rs1621 in the seed - matching sequence of met has been suggested to affect the activity of mir-199a , which mediates the downregulation of the met gene through targeting the 3-utr .
furthermore , snp rs1621 was also selected to investigate its effect on breast cancer risk .
snp rs6566 , located in met , has also been investigated for gastric cancer risk . in this study , these two tag snps ( rs1621 and rs6566 ) were obtained by haploview 4.2 ( population : chb , r square cutoff : 0.8 , maf cutoff : 0.1 , and d = 1 ) .
our study revealed a significant gender difference in the genotype frequencies of met snp rs1621 among patients with ptc and ng and nc .
multiple comparisons between groups further confirmed the statistical difference in rs1621 genotype frequency for female patients in the ptc and nc groups .
genotype ag in rs1621 increased the risk of ptc in female patients , while there was no significant difference between groups for male patients .
a higher ag genotype frequency was the significant risk factor for ptc in female patients , compared with male patients .
however , no significant association was found between rs1621 genotype frequency and the metastasis and prognosis of ptc in both male and female patients .
disparity between genders in incidence , disease aggressiveness , therapy responsiveness , and prognosis has long been observed in a variety of gender - nonspecific cancers . as the most common cancer of the endocrine system ,
thyroid cancer has a well - established gender disparity in incidence , aggressiveness , and prognosis .
it has been reported to be the seventh most common malignancy in women , but not among the most common 15 cancers in men .
reproductive , menstrual , and environmental factors have been hypothesized to account for this gender - specific disparity .
however , other studies have also indicated that dietary , environmental , and reproductive factors , as well as frequent activating somatic mutations , do not appear to contribute to this difference in ptc , the most common type of thyroid cancer .
female gender hormones have been suggested to play a crucial role in the carcinogenesis of cancers , while hormonal exposure did not affect the innate characteristics of the tumor .
gender disparity in cancers has been postulated to be due to yet unidentified molecular factors .
the identification of these factors may help us better understand the biological behavior of cancers , which is essential in the development of effective strategies for cancer diagnosis and treatment .
the met protooncogene encodes a tyrosine kinase receptor that is known to influence cell invasion .
it has been demonstrated to be significantly overexpressed in ptc   and has been suggested to play an important role in ptc invasion and metastasis .
met mutations have been reported in various types of cancers and were found to be correlated with tumor metastasis [ 1719 , 35 ] .
however , other studies also revealed low mutation frequencies   or the mutation was not correlated with the progression of the disease . in thyroid cancer ,
met snps rs1621 and rs6566 were reported in our study , and stratification analysis according to gender revealed an increased risk of ptc in female patients with the ag genotype in rs1621 , which has been previously reported to correlate with chronic rhinosinusitis .
met single - nucleotide variants have been identified in ptc with or without distant metastases and were suggested to correlate with the aggressive behavior of the disease . therefore , the association between rs1621 and tumor metastasis , as well as the prognosis of patients , was analyzed in ptc patients .
our results revealed that there was no obvious correlation between rs1621 and the metastasis and prognosis of ptc patients in either the male or female gender .
further studies are needed to uncover the gender disparity of met snp rs1621 in ptc patients . in conclusion
, our study revealed that the polymorphism of snp locus rs1621 in the met gene may be associated with gender disparity in ptc .
higher ag genotypes in rs1621 were correlated with ptc in female patients , but not in their male counterparts . however , snp rs1621 was not correlated with metastasis and prognosis in both male and female ptc patients .
further studies are needed to better clarify the association between met polymorphism and gender disparity in ptc and its potential mechanisms .
 to investigate the association of met snps with gender disparity in thyroid tumors , as well as the metastasis and prognosis of patients , 858 patients with papillary thyroid carcinoma ( ptc ) , 556 patients with nodular goiter , and 896 population - based normal controls were recruited . 
 the genotyping of met snps was carried out using the sequenom massarray system . 
 the distribution of met snps ( rs1621 and rs6566 ) was different among groups . 
 gender stratification analysis revealed a significant association between the rs1621 genotype and ptc in female patients ( p = 0.037 ) , but not in male patients ( p > 0.05 ) . 
 for female patients , the rs1621 ag genotype was significantly higher in patients with ptc than in normal controls ( p = 0.01 ) and revealed an increasing risk of ptc ( or : 1.465 , 95% ci : 1.1181.92 ) . 
 however , association analysis of the rs1621 genotype with metastasis and prognosis revealed no significant correlation in both male and female patients . 
 the findings of our study showed that polymorphism of snp locus rs1621 in met gene may be associated with gender disparity in ptc . 
 higher ag genotypes in rs1621 were correlated with ptc in female patients , but not in male patients . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion