thyroid cancer is the most common endocrine neoplasia and accounts for 1% of all human cancers ( 1 ) .
medullary thyroid carcinoma ( mtc ) is a rare neoplasm of the calcitonin - secreting thyroid cells and accounts for 510% of all thyroid cancers .
mtc can occurs as sporadically ( 75% ) or as a part of the autosomal dominantly inherited forms ( 25% ) , ( 2 , 3 ) . the inherited type of mtc can be divided in three clinically distinct forms : multiple endocrine neoplasia type2a ( men2a ) , multiple endocrine neoplasia type2b ( men2b ) , and familial mtc ( fmtc ) ( 4 ) .
men2a is defined by mtc , bilateral pheochromocytoma , and multiple tumors of the parathyroid glands ( parathyroid adenomas , primary hyperparathyroidism or hpt ) , within a single patient or family .
men2b is characterized by the early development of an aggressive form of mtc in all affected individuals ( typically during the first year of life ) , pheochromocytoma , the absence of hyperparathyroidism , and visible physical stigmata such as raised bumps on the lips and tongue , ganglioneuromas of the intestine , marfanoid body habitus with skeletal deformations .
fmtc is characterized by a strong predisposition to mtc in families with a very low incidence of other endocrinopathies related to men2 ( 1 , 5 ) . the molecular pathology of inherited mtcs is constitutive of ret ( rearranged during transfection ) proto - oncogene .
the ret gene is located on chromosome 10q11.2 , consisting of 21 exons and encodes a tyrosine kinase receptor .
this complex plays a critical role in cell proliferation and differentiation of tissues derived from neural crest cells , such as c - cells of thyroid gland ( 5 , 6 ) .
ret is a tyrosine kinase protein and includes three domains ; a large extracellular domain containing a cysteine - rich region and a series of cadherin homology domains , a trans - membrane domain , and an intracellular tyrosine kinase domain , required for ret phosphorylation and downstream signaling ( 5 , 7 ) .
this protein is activated by binding a soluble ligand of the glial cell - line - derived neurotrophic factor ( gdnf ) family and requires a co - receptor of the gdnf family receptors  ( gfr ) ( 8) .
the majority of men2a families are associated with one of the six point mutations in conserved cysteine residues in exon10 ( codons 609 , 611 , 618 , and 620 ) or exon11 ( codons 630 , 634 ) in the extracellular domain of the ret ( 2 , 5 , 810 , 12 ) . in fmtc cases ,
ret mutations are mainly detected in the same six codons as for men2a and also in exon13 ( codon 768 ) , or in exon15 ( codon 891 ) , in the intracellular region of ret ( 5 , 8 , 12 ) .
mutation of codon 918 in ret exon16 in the tyrosine kinase domain , accounts for the vast majority of patients with men2b ( 5 , 8) . for the first time , ret polymorphisms g691s and s904s were described in 1994 ( 13 , 14 ) .
recent studies have found g691s in exon11 , s904s single neucleotide polymorphisms in exon15 of the ret pathway to be associated with the risk of developing sporadic mtc ( smtc ) ( 7 ) .
it seems , two of these ret snps ( g691s and s904s ) may modify the age at onset of mtc tumor in family members ( 5 , 15 ) but fabienne lesueur et al . have rejected this hypothesis ( 7 ) . in the present study , we report the frequency of g691s / s904s haplotype in iranian mtc patients and their relatives .
over the last ten years , one hundred ninety mtc patients , were referred to cellular and molecular research center of shahid beheshti university of medical sciences in order to ret genetic screening .
this study has been approved by the ethics committee of cellular and molecular research center , research institute for endocrine sciences , shahid beheshti university of medical sciences .
a questionnaire was used to collect information about age , sex , and history of neoplasia . if any mutation was found in patients , their first - degree relatives were invited for screening .
121 first - degree relatives from 31 index cases comprised 57 males and 64 females , with meansd age 29.0916.34 years .
genomic dna was extracted from peripheral blood leucocytes using standard salting out / proteinase k method and stored at -20c .
for identification of ret variant 691 ( rs1799939 , codon 691 of exon 11 , ggt > agt , gly > ser ) and 904 ( rs1800863 , codon 904 of exon 15 , tcc > tcg , ser > ser , ) , dna samples were amplified using the polymerase chain reaction ( pcr ) and the specific oligonucleotides primers ( table1 ) .
the exon11 running profile was constituted initial denaturation at 96 c for 1min and , followed by 30 cycles with 96 c for 45s , annealing at 60 c for 1min dependent on the primer sequences , and extension at 72c for 45s , with a final extension at 72 c for 10 min .
sequences of oligonucleotide primers used for pcr amplification of exons 11 , 15 of the ret pcr reaction for exon 15 was constituted initial denaturation at 94c for 3min and , followed by 35 cycles of 94c for 30s , annealing starting at 56c for 1min , 72c for 1min , and a final extension at 72c for 10min .
the amplimers were confirmed for the presence or absence of mutations by direct dna sequencing method ( abi 3100 genetic analyzer and big dye terminator v3.1 cycle sequencing kit , applied biosystems , california , usa ) . to detect the ret mutations and snps , sequences were analyzed by chromas 2.33 software and were also compared with the reference ret gene sequence using web based tool ncbi blast .
over the last ten years , one hundred ninety mtc patients , were referred to cellular and molecular research center of shahid beheshti university of medical sciences in order to ret genetic screening .
this study has been approved by the ethics committee of cellular and molecular research center , research institute for endocrine sciences , shahid beheshti university of medical sciences .
a questionnaire was used to collect information about age , sex , and history of neoplasia . if any mutation was found in patients , their first - degree relatives were invited for screening .
121 first - degree relatives from 31 index cases comprised 57 males and 64 females , with meansd age 29.0916.34 years .
genomic dna was extracted from peripheral blood leucocytes using standard salting out / proteinase k method and stored at -20c .
for identification of ret variant 691 ( rs1799939 , codon 691 of exon 11 , ggt > agt , gly > ser ) and 904 ( rs1800863 , codon 904 of exon 15 , tcc > tcg , ser > ser , ) , dna samples were amplified using the polymerase chain reaction ( pcr ) and the specific oligonucleotides primers ( table1 ) .
the exon11 running profile was constituted initial denaturation at 96 c for 1min and , followed by 30 cycles with 96 c for 45s , annealing at 60 c for 1min dependent on the primer sequences , and extension at 72c for 45s , with a final extension at 72 c for 10 min .
sequences of oligonucleotide primers used for pcr amplification of exons 11 , 15 of the ret pcr reaction for exon 15 was constituted initial denaturation at 94c for 3min and , followed by 35 cycles of 94c for 30s , annealing starting at 56c for 1min , 72c for 1min , and a final extension at 72c for 10min .
the amplimers were confirmed for the presence or absence of mutations by direct dna sequencing method ( abi 3100 genetic analyzer and big dye terminator v3.1 cycle sequencing kit , applied biosystems , california , usa ) .
to detect the ret mutations and snps , sequences were analyzed by chromas 2.33 software and were also compared with the reference ret gene sequence using web based tool ncbi blast .
among 311 participants , 18 dna samples did not have enough quality , so were excluded from the study .
we analyzed germline dna mutations of ret gene in 181 iranian mtc patients and 112 relatives , with a total of 293 members .
the patients included , 33 fmtc , 6 men2a , 2 men2b , 1 pheochromocytoma and 145 apparently smtc cases ( table 2 ) .
the frequency of females in our population was a little higher than males , but this ratio was not significant ( 160 vs. 133 , p > 0.05 ) .
specifically , we found 2 germline polymorphisms of ret gene at codon691 ( ggt > agt , exon 11 , rs1799939 ) that causes glycine to serine amino acid substitution , and codon 904 ( tcc > tcg , exon 15 , rs1800863 ) that does not lead to an ami - no acid alteration .
furthermore , the allele frequencies of each of these snps were similar in all patients and relatives ( 21.5% and 10.75% , respectively ) .
the frequency of patients with medullary thyroid carcinoma as the g691s and s904s variants were in complete linkage disequilibrium , so the results were grouped together and referred to as g691s / s904s haplotype ( fig.1 ) .
the analysis of g691s / s904s ret gene haplotype showed that 104 of 181 ( 57.45% ) mtc patients and 55 of 112 ( 49.1% ) relatives had this haplotype .
in particular , 82 mtcs and 47 relatives were heterozygous and 22 mtcs and 8 relatives were homozygous for the g691s / s904s haplotype ( table 3 ) .
there was not significant correlation between age of diagnosis and the presence of g691s / s904s haplotype .
the frequency of g691s and s904s variants in the population the frequency distribution of g691s and s904s variants among mtc patients and their relatives
in this study , we found two variants of ret gene , g691s ( ggt > agt , exon 11 , rs1799939 ) and s904s ( tcc > tcg , exon 15 , rs1800863 ) that are cosegregated together as a haplotype , suggesting that these polymorphisms are in linkage disequilibrium with each other .
the assessment of the occurrence of two polymorphic changes g691s , and s904s in our population is similar to the study performed by lucieli ceolin et al .
unfortunately , in our study we did not have control group to compared with healthy population , but some studies have demonstrated that the ret variant g691s is more frequent in mtc patients than in the general population ( 16 , 17 ) .
it has also been postulated that such variants are relatively common in the population which may confer a much higher attributable risk in the general population than mutations in high penetrance cancer susceptibility genes ( 5 , 11 ) .
some studies support our data that both g691s and s904s snps are associated with sporadic mtc and men2a ( 5 , 7 , 18 ) .
two studies have shown that this haplotype may modify the age at onset of mtc tumor in family members ( 5 , 15 ) , however , this hypothesis is controversial ( 7 ) . the ret - g691s polymorphism has been suggested as a genetic modifier in men2a ( 15 ) .
overall , these data strengthen the potential role of the ret - g691s polymorphism in mtc .
one of the probable mechanisms is that the g691s polymorphism seems to enhance ret on - cogenicity of other distinct mutations affecting ret ( 1820 ) , on the other hands , bases exchange in the dna molecule could create a new alternative splicing site , leading to the synthesis of a truncated protein , erroneous ligand binding , micro - rna binding , change of structure and mrna stability as well as a number of copies , and also the change in the secondary structure of proteins ( 16 ) .
the ret gene mutations are a critical factor in patient management , e.g. to decide on total or partial thyroidectomy or the age at which the child should be subject to surgery ( 6 ) .
ret gene mutations are very important in mtc patient management , especially in decision making for total or partial thyroidectomy or the age at which the child should be subject to surgery . in the case of inherited mutations ,
those who test negative for germline mutations will be reassured ; those who test positive for germline mutations will be screened for calcitonin levels and will be offered a prophylactic thyroidectomy depending on the complete clinical and biochemical picture .
this data showed the frequency of g691s / s904s haplotype among iranian mtc patients and their family , for the first time .
even though g691s / s904s haplotype are not considered as oncogenic mutations at this time , its functional role should be investigated .
further researches on this issue can contribute toward clarifying the prevalence of this haplotype and its probable modifying effect on the phenotypic characteristic of mtc .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors .
 abstractbackgroundmedullary thyroid carcinoma ( mtc ) occurs in both sporadic ( 75% ) and hereditary ( 25% ) forms . 
 the missense mutations of the rearranged during transfection ( ret ) proto - oncogene in mtc development have been well demonstrated . 
 the aim of this study was to investigate frequency of g691s / s904s haplotype in mtc patients and their relatives.methodsin this research 293 participants were studied , including 181 patients ( 102 female , 79 male ) and 112 their relatives ( 58 female , 54 male ) . 
 genomic dna was extracted from peripheral blood leucocytes using the standard salting out / proteinase k method . 
 nucleotide change detection was performed using pcr and direct dna sequencing methods.resultsaccording to dna sequencing results , 159 individuals ( 104 patients , 55 relatives ) had both g691s ( rs1799939 ) missense mutation in exon11 and s904s ( rs1800863 ) synonymous mutation in exon 15 of ret proto - oncogene . 
 the allele frequency of g691s / s904s haplotype was 21.15% in patients and 10.75% in their relatives.conclusionthe obtained data showed the frequency of g691s / s904s ret gene haplotype among iranian mtc patients and their relatives . 
 the g691s and s904s nucleotide changes were in complete linkage disequilibrium , so the results were grouped together and referred to as g691s / s904s haplotype . 
 further analysis is need to demonstrate the association between this haplotype and mtc development . 
Introduction
Material and Methods
Samples
Genomic DNA extraction
PCR assay
Bioinformatics and Statistical analysis
Results
Discussion
Conclusion
Ethical considerations