wilms tumor ( wt ) , also known as nephroblastoma , is the most frequent renal tumor occurring in children aged 0 to 15 years , especially among those younger than 6 years .
wt accounts for roughly 6% of all childhood cancers ; 5% of whom had bilateral involvement , and 1% of whom had a family history .
thanks to the prospective clinical trials performed by the international society of pediatric oncology  renal tumor study group ( siop - rtsg , europe ) and the children s oncology group ( cog , formerly nwtsg , north america ) , most patients ( 85% ) affected by wt can be successfully cured .
however , some studies released surveys reporting that there are still a few children who may have a poor prognosis or relapse with current therapies , 50% of whom will die despite intensive re - treatment .
it has been proven that genetic variants are strongly associated with the development of wt .
mutations in wt1 or epigenetic defects on chromosome 11p15 contribute to the major genetic susceptibility locus .
previous studies have reported several non - wt1 loci with smaller effects on the genetic predisposition for wt , including p53 gene , insulin - like growth factor - ii gene , ctnnb1 gene , and h19 . however
, less than half of wt cases are attributable to known genes that are associated with wt risk ; therefore , the exact pathogenesis of most wt cases remains unknown and identification of novel genetic loci is urgently needed .
the reversion - inducing cysteine - rich protein with kazal motifs ( reck ) gene , known as a transformation suppressor gene , can restrain tumor invasion and metastasis through suppression of matrix metalloproteinases ( mmps ) , including mmp-4 and mmp-9 .
mmps proteolytically degrade extracellular matrix proteins , which is critical for tumor metastasis and invasion .
reck is expressed in a number of normal tissues , but it appears to be down - regulated in several types of cancers , including esophageal cancer , breast cancer , lung cancer , colorectal cancer , osteosarcoma , gastric carcinoma , prostatic cancer , oral cancer , pancreatic cancer , and cholangiocarcinoma .
moreover , clinical investigations have demonstrated that high expression of reck in tumor tissues usually contributes to increasing survival rates and reducing tumor invasion . moreover , hawthorm et al .  
analyzed wt using single - nucleotide polymorphism ( snp ) mapping array - based comparative genomic hybridization and found chromosomal deletion in 9q ( reck gene locates on 9q13-p12 ) .
although it is well documented that reck has an impact on metastasis and prognosis of human cancers , reck gene snps in wt susceptibility and clinical features remains poorly characterized . to obtain adequate power for assessing the potential association , we chose snps with minor allele frequencies of > 5% .
furthermore , rs10972727 and rs11788747 snps both were previously identified to be potentially associated with several types of human cancers , including oral cancer , non - small cell lung cancer , and hepatocellular carcinoma .
therefore , the purpose of our study was to determine whether the 2 snps ( rs10972727 and rs11788747 ) in reck gene were associated with susceptibility to wt in chinese children .
this case - control study received approval from the human research ethics board of jinan children s hospital .
a total of 291study participants  97 wt children and 194 healthy controls based on an age - sex - matched pair with 1 : 2 ratio  were identified at the jinan children s hospital .
all the participants were han chinese . written informed consent was obtained from parents of each participant before inclusion in the study .
there were 41 male and 56 female patients , and the mean age at the time of surgery was 3.3 years .
the patients histopathological types and cancer grading were determined according to classification of renal tumor of childhood defined by siop - rtsg .
accordingly , 43 ( 44.3% ) out of 97 cases were characterized as wt stage i , 35 ( 36.1% ) as stage ii , 12 ( 12.4% ) as stage iii , and 7 ( 7.2% ) as stage iv . according to the prognostic group ,
moreover , no case had bilateral wt . according to the histologic type , there were 26 cases with blastemal type wt , 8 with diffuse anaplasia , 9 with focal anaplasia , 17 with epithelial , and 37 with mixed .
genomic dna was extracted by qiaamp dna blood mini kits ( qiagen , valencia , ca ) according to the instructions of the manufacturer .
reck genotype polymorphisms ( rs10972727 and rs11788747 ) were determined by polymerase chain reaction restriction fragment length polymorphism ( pcr - rflp ) method .
pcr was carried out in a reaction mixture of 10 l containing 58 l dna template , 0.5 u of taq biocatalysts , 1.8 mmol / l mg2 + , 2.4 l dntps ( promega , madison , wi ) , and 200 nm of each primer .
the 2 snps were genotyped according to the methods described by chung et al . .
primers for rs10972727 were 5-gtagaagaagtgactgatcc-3 and 5-atctgactccgaagataacc-3. the primers for the rs11788747 were 5-ttctatcaggtcatggaaca-3 and 5-tgcggttaagactggagaag-3. the pcr cycling conditions were : initial denaturation at 94c for 5 min , followed by 35 cycles of 1 min at 94c , at 60c for 30 s , and at 72c for 1min , with a final extension at 72c for 10 min . to verify results from pcr - ctpp , approximately 10% of the sample analysis was a duplication and each genotype was determined by the dna sequence analysis .
, hardy - weinberg equilibrium was analyzed to compare the observed and expected genotype frequencies using the standard  test or fisher s exact test .
the differences in distributions of genotypes and alleles between cases and controls were assessed by  tests .
the estimated genotype - specific risks are presented as odds ratios ( ors ) . in all cases ,
this case - control study received approval from the human research ethics board of jinan children s hospital .
a total of 291study participants  97 wt children and 194 healthy controls based on an age - sex - matched pair with 1 : 2 ratio  were identified at the jinan children s hospital .
all the participants were han chinese . written informed consent was obtained from parents of each participant before inclusion in the study .
there were 41 male and 56 female patients , and the mean age at the time of surgery was 3.3 years .
the patients histopathological types and cancer grading were determined according to classification of renal tumor of childhood defined by siop - rtsg .
accordingly , 43 ( 44.3% ) out of 97 cases were characterized as wt stage i , 35 ( 36.1% ) as stage ii , 12 ( 12.4% ) as stage iii , and 7 ( 7.2% ) as stage iv . according to the prognostic group ,
moreover , no case had bilateral wt . according to the histologic type , there were 26 cases with blastemal type wt , 8 with diffuse anaplasia , 9 with focal anaplasia , 17 with epithelial , and 37 with mixed .
genomic dna was extracted by qiaamp dna blood mini kits ( qiagen , valencia , ca ) according to the instructions of the manufacturer .
reck genotype polymorphisms ( rs10972727 and rs11788747 ) were determined by polymerase chain reaction restriction fragment length polymorphism ( pcr - rflp ) method .
pcr was carried out in a reaction mixture of 10 l containing 58 l dna template , 0.5 u of taq biocatalysts , 1.8 mmol / l mg2 + , 2.4 l dntps ( promega , madison , wi ) , and 200 nm of each primer .
the 2 snps were genotyped according to the methods described by chung et al . .
primers for rs10972727 were 5-gtagaagaagtgactgatcc-3 and 5-atctgactccgaagataacc-3. the primers for the rs11788747 were 5-ttctatcaggtcatggaaca-3 and 5-tgcggttaagactggagaag-3. the pcr cycling conditions were : initial denaturation at 94c for 5 min , followed by 35 cycles of 1 min at 94c , at 60c for 30 s , and at 72c for 1min , with a final extension at 72c for 10 min . to verify results from pcr - ctpp ,
approximately 10% of the sample analysis was a duplication and each genotype was determined by the dna sequence analysis .
, hardy - weinberg equilibrium was analyzed to compare the observed and expected genotype frequencies using the standard  test or fisher s exact test .
the differences in distributions of genotypes and alleles between cases and controls were assessed by  tests .
the estimated genotype - specific risks are presented as odds ratios ( ors ) . in all cases ,
five out of 97 cases had a family history of wt , 6 cases had relapse , and 16 had metastasis . in the present study ,
distributions of genotypes in cases and controls were in agreement with hardy - weinberg equilibrium ( rs10972727 , p=0.349 and p=0.121 ; and rs11788747 , p=0.519 and p=0.123 , respectively ) .
table 2 shows the genotype and allele frequencies of the 2 snps ( rs10972727 and rs11788747 ) in reck gene .
no significant difference between cases and controls was observed for distribution of rs10972727 genotype , rs11788747 genotype , and rs11788747 allele .
however , we found that the allele g of rs11788747 snp was significantly associated with an increased risk of wt ( or=0.7 , 95%ci : 0.450.99 ; p=0.042 ) .
moreover , although when taking the aa genotype as a reference , we found that ag + gg genotype was not statistically significantly associated with the risk of wt , there was a pronounced trend that carriers of 1 variant combined genotype had an increasing risk of wt ( or=0.6 , 95%ci : 0.381.03 ; p=0.065 ) .
further analysis was conducted to investigate the associations between distribution of the genotype and allele of rs11788747 polymorphisms and clinicopathological features of wt patients . as shown in table 3 ,
although there was no significant difference in distribution of the genotype , allele g carriers with advanced tumor stage ( p=0.026 ) had an increased risk of wt , as did those who had metastasis ( p=0.002 ) .
table 2 shows the genotype and allele frequencies of the 2 snps ( rs10972727 and rs11788747 ) in reck gene .
no significant difference between cases and controls was observed for distribution of rs10972727 genotype , rs11788747 genotype , and rs11788747 allele .
however , we found that the allele g of rs11788747 snp was significantly associated with an increased risk of wt ( or=0.7 , 95%ci : 0.450.99 ; p=0.042 ) .
moreover , although when taking the aa genotype as a reference , we found that ag + gg genotype was not statistically significantly associated with the risk of wt , there was a pronounced trend that carriers of 1 variant combined genotype had an increasing risk of wt ( or=0.6 , 95%ci : 0.381.03 ; p=0.065 ) .
further analysis was conducted to investigate the associations between distribution of the genotype and allele of rs11788747 polymorphisms and clinicopathological features of wt patients . as shown in table 3 ,
although there was no significant difference in distribution of the genotype , allele g carriers with advanced tumor stage ( p=0.026 ) had an increased risk of wt , as did those who had metastasis ( p=0.002 ) .
the findings of this novel study provide evidence of the effects of snps of reck on wt susceptibility and clinicopathologic status association .
various lines of evidence have found that tumor stage and metastasis are the risk factors for development of wt . in the current study
, we explored the association of reck gene and wt risk , and subsequently analyzed the effects of combinations of functionally related polymorphisms and clinicopathological features of wt patients .
to the best of our knowledge , this is the first study examining the association of reck gene polymorphism with wt risk , although several previous studies have reported on its association with several other types of human cancers . as the most frequent renal tumor occurring in children
, although wt has a high survival rate , 25% of wt children may have a poor prognosis or relapse with current therapies , 50% of whom will die despite intensive re - treatment .
therefore , it is urgent for us to identify novel genetic loci that might be associated with wt risk .
it has been proven that the igf pathway plays a role in development of wt .
the igf pathway is a complex signaling system stimulated by insulin - like growth factors ( igfs ) , which can be produced by almost any tissue in the body and has an important effect on growth , development , and survival in many different cell types .
overexpression of igf2 , activating the insulin signaling pathway , results in disorder of protein synthesis , cell cycle and cell growth , and blocks apoptosis .
furthermore , yamamoto et al .   indicated that reduced reck could increase igf2 expression .
the interaction between reck gene and igf gene might have a potential influence on susceptibility to wt .
a study by huang et al .   demonstrated down - regulation of reck gene expression in patients with wt .
therefore , we conducted this study to determine whether reck gene is associated with wt risk . in the current study , our findings suggest that mutant of rs10972727 in reck had an impact on increasing wt risk .
in addition , allele g carriers with advanced tumor stage involving metastasis had an increasing risk of wt .
advanced tumor stage and metastasis are responsible for patient mortality in most tumors [ 3739 ] .
found the level of reck expression determined the prognosis of pancreatic cancer and tumors , with overexpression of reck significantly decreasing invasiveness compared with reck - negative tumors , revealing the potential value of reck as a prognostic molecular marker for pancreatic cancer .
zhang et al .   reported that silenced reck gene was associated with poor survival in hepatocellular carcinoma .
.   showed that those who carried rs10814325 with at least 1 c allele had a higher risk of hepatocellular carcinoma .
, after a follow - up of salivary adenoid cystic carcinoma patients , revealed that expression of reck and mmp-2 gene were correlated with tumor progression . in our study , we found a correlation between rs10972727 in reck and tumor stage and metastasis .
however , the exact physiological mechanism by which reck influences progression of wt remains unclear .
firstly , the primary weakness of this study is that the small sample size may affect the power in statistical analysis .
secondly , the results were not replicated in additional individuals , and this might contribute to potential false - positive errors . moreover , the method used to select potentially functional snps as targets by using a web - based tool might have led to some positive or negative errors .
finally , we did not perform a functional study to further reveal the mechanism by which the genetic polymorphisms in reck affect wt risk .
data from the present data indicate that rs10972727 in reck gene is not associated with wt risk in chinese children , but the association between mutant g of rs11788747 in reck and wt risk was shown .
g carriers with advanced tumor stage or with metastasis might have an increased risk of wt .
therefore , further research is necessary to reveal the mechanism by which the genetic polymorphisms in reck affect the wt risk .
 backgroundwilms tumor ( wt ) is the most common malignant renal tumor in children . 
 previous studies suggested the reversion - inducing , cysteine - rich protein with kazal motifs ( reck ) down - regulation might have a role in numerous human cancers . 
 the current study was done to investigate the associations of reck single - nucleotide polymorphisms ( snps ) with the wt susceptibility in chinese children.material/methodswe analyzed 2 snps ( rs10972727and rs11788747 ) in a total of 97 wt children and 194 healthy matched controls ( 1:2 ratio ) by real - time pcr and pcr - rflp genotyping analysis.resultswe found that the g allele of rs11788747 in the reck gene was significantly associated with wt in chinese children ( or=0.7 , 95% ci : 0.450.99 ; p=0.042 ) ; as with another snp rs10972727 , however , no statistically significant difference was detected . 
 further analysis showed there was also a statistically significant difference in genotype frequencies between terminal tumor stage ( p=0.026 ) and metastatic groups ( p=0.002).conclusionsthe present data indicate that there is a significant association between mutant g of rs11788747 in reck and wt risk . 
 g carriers with advanced tumor stage or with metastasis might have an increased risk of wt . 
Background
Material and Methods
Study participants and ethical statement
Genotyping of RECK
Statistical analysis
Results
Association of the 2 SNPs with the risk of WT
Stratification analysis
Discussion
Conclusions