cystic fibrosis ( cf ; mim # 219700 ) is the most common lethal autosomal recessive disorder in caucasian population ; it affects 1 in 2500 newborns among caucasians ( 1 , 2 ) . cystic fibrosis ( cf ) is caused by mutations in cystic fibrosis transmembrane conductance regulator ( cftr ) gene , which encodes a camp- dependent chloride channel that is found at the apical membrane of epithelial cells , including respiratory epithelia and submucosal glands , exocrine pancreas , liver , sweat ducts , and the reproductive tract . cftr is a member of the atp - binding cassette ( abc ) membrane transporter superfamily that includes proteins such as the multiple drug resistance protein ( mdr ) and bacterial periplasmic permeases ( 1 , 3 ) . cftr gene is localized on the long arm of chromosome 7 ( 7q21 - 34 ) , spanning approximately 190 kb of genomic dna . the gene consists of 27 exons and encodes a mature mrna transcript of 6.5 kb that is translated into a 1480 amino acid protein ( 3 ) . to date , more than 1950 mutations ( 4 ) have been identified in cystic fibrosis transmembrane conductance regulator ( cftr ) gene ( 4 ) . many of these mutations are rare in the population and based on the reports only a few affected individuals . in addition to f508 mutation , these mutations vary greatly in their frequency and distribution , but most of them are very rare . only four mutations ( p.g542x , p.n1303k , p.g551d , and p.w1282x ) have overall frequencies greater than 1% ( 5 ) . cf remains as a life - threatening autosomal recessive condition affecting caucasians ( 5 ) . in the us , each year the cf incidence is estimated to be1.2500 to 1.90,000 live births ( 1 ) . in the case of cftr , different patterns of migration and settlement have led to worldwide variation of the mutations cf is rare and less documented among asians ( 1 , 2 ) . the exact incidence of cf is not known but the predictions show the variation of 1.10,000 to 1.350,000 of estimated frequencies . many children with cf in these populations probably remain undiagnosed due to lack of clinical suspicion and proper diagnostic facilities . the objective of this study was to determine the presence of cftr gene mutations ( exons 10 and 17a ) among 27 cf patients in the population of kermanshah province with respiratory and gastrointestinal manifestations similar to those reported in cf patients . adequate and accurate information of cf mutations in particular populations provides information for cf prevention programs applicable via heterozygote screening and prenatal diagnosis ( 6 ) . the identification of mutations and their frequencies are thus critically important for designing gene probes for effective diagnosis of cf in a given population . further , different mutations are associated with varying severity and prognosis management ( 7 ) . among different provinces of iran , so , the molecular characterization of cf in kermanshah province , as a diverse and mainly kurdish populated area in iran , seems a necessity . adequate and accurate information of cf mutations in particular populations provides information for cf prevention programs applicable via heterozygote screening and prenatal diagnosis ( 6 ) . the identification of mutations and their frequencies are thus critically important for designing gene probes for effective diagnosis of cf in a given population . further , different mutations are associated with varying severity and prognosis management ( 7 ) . among different provinces of iran , so , the molecular characterization of cf in kermanshah province , as a diverse and mainly kurdish populated area in iran , seems a necessity . this study has been done in medical genetics laboratory of kermanshah university of medical sciences in iran . the study population included patients attending the pediatric outpatient clinics ( pediatric chest and genetics clinics ) and those who were admitted in the pediatric medicine or surgery wards with a suspected diagnosis of cf . from 2011 to 2012 , 27 unrelated families who had an affected child with cf were referred to the medical genetics lab , kermanshah university of medical science , kermanshah , iran . peripheral blood samples of cf patients ( sweat chloride > 60 mmol / l diagnostic of cf and clinical symptoms characteristic of cf ) were collected in edta tubes . all patients were fully informed that their blood would be used for molecular investigation of cftr gene mutations , and consent was obtained from each patient or his/ her guardians . genomic dna was collected from peripheral whole blood using the " salting out " procedure ( 12 ) . sweat samples were gathered by the macroduct sweat collection system ( wescor , logan , ut ) . localized sweating was stimulated by the iontophoresis of pilocarpine into the skin of the flexor surface of the forearm or thigh . sweat was then collected in micro bore tubing ( macro - duct ) , the amount of sweat was quantitated , and the sample was then analyzed for chloride concentration , using the standard ise method . pcr amplification of exon10 and 17a of cftr gene was performed using gene amp pcr system 9700 ( applied biosystems , usa ) . the primers used in this study were synthesized by operon ( metabion , germany ) . the sequences of primers , listed in 5-3direction are shown in the ( table 1 ) . all samples were sequenced in both the forward and reverse direction using the same primers used in the pcr reactions . pcr products were purified using qiaquick pcr purification kit . for sequencing analysis , samples were analyzed by direct sequencing of exon10 and 17a of cftr gene and their flanking introns in an abi-3130 dna analyzer ( applied biosystems , usa ) . the sequences were compared with the wild - type cftr nucleotide sequence using seqcape software ( applied biosystems ) . all identified sequence changes were confirmed by direct dna sequencing in the reverse direction . from 2011 to 2012 , 27 unrelated families who had an affected child with cf were referred to the medical genetics lab , kermanshah university of medical science , kermanshah , iran . peripheral blood samples of cf patients ( sweat chloride > 60 mmol / l diagnostic of cf and clinical symptoms characteristic of cf ) were collected in edta tubes . all patients were fully informed that their blood would be used for molecular investigation of cftr gene mutations , and consent was obtained from each patient or his/ her guardians . genomic dna was collected from peripheral whole blood using the " salting out " procedure ( 12 ) . sweat samples were gathered by the macroduct sweat collection system ( wescor , logan , ut ) . localized sweating was stimulated by the iontophoresis of pilocarpine into the skin of the flexor surface of the forearm or thigh . sweat was then collected in micro bore tubing ( macro - duct ) , the amount of sweat was quantitated , and the sample was then analyzed for chloride concentration , using the standard ise method . pcr amplification of exon10 and 17a of cftr gene was performed using gene amp pcr system 9700 ( applied biosystems , usa ) . the primers used in this study were synthesized by operon ( metabion , germany ) . the sequences of primers , listed in 5-3direction are shown in the ( table 1 ) . all samples were sequenced in both the forward and reverse direction using the same primers used in the pcr reactions . samples were analyzed by direct sequencing of exon10 and 17a of cftr gene and their flanking introns in an abi-3130 dna analyzer ( applied biosystems , usa ) . the sequences were compared with the wild - type cftr nucleotide sequence using seqcape software ( applied biosystems ) . all identified sequence changes were confirmed by direct dna sequencing in the reverse direction . a total of 27 unrelated patients ( 13 males and 14 females ) aged two months to 19 years originating from kermanshah province were analyzed in this study . 66.7% of the patients were from consanguineous marriage ( mostly between first cousins ) . in our investigation , nearly 80% of the patients had malnutrition , abnormal stools and failure to thrive , 22 cases had been diagnosed with meconium ileus and intestinal obstruction . mutation screening of cftr gene in 54 alleles by sequencing reaction for all common mutations ( exon 10 and exon 17a ) showed that 10 alleles were f508 ( 14.81% ) , s466x ( 1.85% ) and t1036i ( 1.85% ) and also showed 40 alleles ( 74.1% ) with m470v polymorphism . five patients were heterozygous for f508 , one patient was homozygous for f508 and one patient was a compound heterozygous ( f508/s466x ) ( table 2 , figure 1 ) . m470v polymorphism was 63% in homozygote genotype patients and it was 11.1% for the heterozygote genotypes ( figure 1 ) . nucleotide alterations identified by sequencing cftr gene mutations identified as a result of the study ( exon 10 and exon 17a ) a total of 27 unrelated patients ( 13 males and 14 females ) aged two months to 19 years originating from kermanshah province were analyzed in this study . 66.7% of the patients were from consanguineous marriage ( mostly between first cousins ) . in our investigation , nearly 80% of the patients had malnutrition , abnormal stools and failure to thrive , 22 cases had been diagnosed with meconium ileus and intestinal obstruction . mutation screening of cftr gene in 54 alleles by sequencing reaction for all common mutations ( exon 10 and exon 17a ) showed that 10 alleles were f508 ( 14.81% ) , s466x ( 1.85% ) and t1036i ( 1.85% ) and also showed 40 alleles ( 74.1% ) with m470v polymorphism . five patients were heterozygous for f508 , one patient was homozygous for f508 and one patient was a compound heterozygous ( f508/s466x ) ( table 2 , figure 1 ) . m470v polymorphism was 63% in homozygote genotype patients and it was 11.1% for the heterozygote genotypes ( figure 1 ) . nucleotide alterations identified by sequencing cftr gene mutations identified as a result of the study ( exon 10 and exon 17a ) different ethnic groups and tribes live in iran and ethnic / genetic heterogeneity has resulted in a high number of different mutations that account for cf . comparison between different provinces showed that the mutation spectrum differs substantially in types and frequencies ( 810 ) . to evaluate and analyze our results , there is a need for some general facts about the ethnicity of the people who were investigated in this study . this is the first time that such a study is done for kurdish people in islamic republic of iran . the kurdish people or kurds are part of the iranian people native to the middle east , which includes adjacent parts of iran , iraq , syria , and turkey ( 11 ) . it even extends to some other neighboring ethnics such as lorr , turk and persians . this province is located in neighbor of the provinces : hamadan , lorestan , ilam and kurdistan . the west part of this province is along iraqi borders . due to these unique geographical , ethnical and lingual properties of this region , f508 was present on 8 ( 14.81% ) of all 54 cf chromosomes analyzed ( 11.11% heterozygote and 3.7% homozygote ) . although the frequency of f508 among these patients was lower ( 14.81% ) than its frequency in european countries ( table 3 ) , it was still the most frequent mutation among those reported to date in iran ( 810 ) . previous studies representing overall iranian cf patients reported a frequency of 16  18.1% for this mutation ( 9 , 10 ) . f508 is the most common mutation in the world and this mutation accounts for 70% of worldwide ( 7 ) mutated alleles of cf chromosomes but with great variation of frequency from 100% ( faroe islands ) ( 11 ) to 18% ( tunisia ) . most of the mentioned chromosomes share the same but rare haplotype for a one occurrence mutation in a normal population . f508 mutation distribution shows a decreasing frequency in europe . in the case of middle east , ethnic background and consanguinity account for the variation . the preliminary reports on the mutation spectrum in iranian cf children indicate that p.f508del is the most common mutation , accounting for about 1625% of the mutations . identification of the mutations is of great importance for further evaluation , patient counseling and prenatal diagnosis . comparison of the frequency of common cftr mutations ( % ) in the present study , west asia , north africa and indian subcontinent some reports about this mutation ( s466x ) in italy 's northeast , france 's northwest , turkey , greece and india the frequency of this mutation in western asia and north of africa is less than europe . f508 mutation frequency is decreasing from northwest of europe to its southeast , in such a way that this frequency in denmark is 88% ( 28 ) , in england is 74% ( 29 ) , in germany is 71% ( 30 ) , in swiss 71% , in france 69% ( 30 ) , in bulgaria 60% , in spain 53% ( 31 ) , in sweden 52% ( 30 ) , in greece 52% ( 32 ) and in italy 45% ( 30 ) . because of decreasing mutation frequency from northwest to southeast of europe , it is obvious that it is about 24  27% in turkey ( 33 ) and also it is clear that iran 's mutation frequency is similar to turkey 's . this mutation is the most widespread one among iran 's proportional mutations and in contrast to other reported mutations has remarkable percentage in the country . in previous studies , the percentage of homozygote of this mutation has reached to 5.8% . in the present study , this mutation is observed for the first time in iran as compound heterozygous and this should be noted that heterozygote genotype along with mutation of f508 ( 1.85% ) were found in one of the patients . this patient was not born out of consanguinity marriage and this patient showed the symbols of malnutrition , respiratory problems and meconium ileus . in previous studies , in the common wealth of independent states ( cis ) , the mutation of s466x with f508 in the form of compound heterozygous was determined . s466x mutation with homozygote genotype was previously reported in tehran , khorasan , hamedan and markazi provinces . it seems that this mutation exists among many families in different regions of iran and it seems relatively ubiquities in iran . nonetheless , this mutation is very rare worldwide ; for example , the frequency of 0.5% has been reported in serbia and montenegro . there are also some reports about this mutation in italy 's northeast , france 's northwest , turkey , greece and india ( 16 , 17 , 30 , 3234 ) . 
  comparison of the frequency of common cftr mutations , f508 and s466x in iran in exon 17a , t1036i mutation is reported as a heterozygote for the second time in the world . this mutation was reported for the first time in 2006 , in iran ( 8) . this mutation may be connected to low frequency of studied patients and the common tribal marriages in iran and west asian cultures . the frequency of t1036i mutation ( c to t at 3239 ) was 1.85% in this study and it was involved with just one patient ( figure 1 and table 2 ) . the patients had the disorders of respiratory - digestive and meconium ileus and the parents of the patient had no genetic relationship ( 1 ) . polymorphism of m470v is a silent polymorphism which changes one nucleotide and converts the methionine amino acid to valine in the place of 42376223 . however , m470v is not strongly neutral because a study showed that this polymorphism leads to degradation of cl transitions through the cell 's membrane . it has also been showed that whenever m470v polymorphism comes along with another polymorphism mutation that leads to less activation of cftr protein function ( 8 , 35 ) . there are some reports worldwide which show m470v polymorphism with t5 and t7 allele ( 35 ) from tn polymorph placement cause illness in combination ( t repetitions are studied in cf patients and men 's infertility ) ( 36 ) . some reports also evaluated the potential connection of men 's prostate cancer with this polymorphism ( 37 , 38 ) . the effectiveness of this allele in the illness phenotype is not completely clear yet and it deserves more investigations . the incidence of cystic fibrosis is high in case of europeans . medical community 's lack of knowledge of the disease , poor access to medical facilities and health care for cf patients , confounding diagnosis , a high infant mortality rate , and low life expectancy in general could account for the unexplained low cf incidence in some countries with a large proportion of caucasians of european origin . in some cases , intermediate sweat chloride levels may exist in some patients , and this may lead to misdiagnosis of them . the cf frequency might be higher for some isolated populations due to consanguinity . in case of turkey and saudi arabia , hence , cf can be regarded as a prevalent illness even outside europe and united state which may have different mutation spectrums . in this regard , ascertainment of cftr mutation carrier frequencies and the cf incidence are necessary among iranian populations . f508 was present on 8 ( 14.81% ) of all 54 cf chromosomes analyzed ( 11.11% heterozygote and 3.7% homozygote ) . although the frequency of f508 among these patients was lower ( 14.81% ) than its frequency in european countries ( table 3 ) , it was still the most frequent mutation among those reported to date in iran ( 810 ) . previous studies representing overall iranian cf patients reported a frequency of 16  18.1% for this mutation ( 9 , 10 ) . f508 is the most common mutation in the world and this mutation accounts for 70% of worldwide ( 7 ) mutated alleles of cf chromosomes but with great variation of frequency from 100% ( faroe islands ) ( 11 ) to 18% ( tunisia ) . most of the mentioned chromosomes share the same but rare haplotype for a one occurrence mutation in a normal population . f508 mutation distribution shows a decreasing frequency in europe . in the case of middle east , ethnic background and consanguinity account for the variation . the preliminary reports on the mutation spectrum in iranian cf children indicate that p.f508del is the most common mutation , accounting for about 1625% of the mutations . identification of the mutations is of great importance for further evaluation , patient counseling and prenatal diagnosis . comparison of the frequency of common cftr mutations ( % ) in the present study , west asia , north africa and indian subcontinent some reports about this mutation ( s466x ) in italy 's northeast , france 's northwest , turkey , greece and india the frequency of this mutation in western asia and north of africa is less than europe . f508 mutation frequency is decreasing from northwest of europe to its southeast , in such a way that this frequency in denmark is 88% ( 28 ) , in england is 74% ( 29 ) , in germany is 71% ( 30 ) , in swiss 71% , in france 69% ( 30 ) , in bulgaria 60% , in spain 53% ( 31 ) , in sweden 52% ( 30 ) , in greece 52% ( 32 ) and in italy 45% ( 30 ) . because of decreasing mutation frequency from northwest to southeast of europe , it is obvious that it is about 24  27% in turkey ( 33 ) and also it is clear that iran 's mutation frequency is similar to turkey 's . this mutation is the most widespread one among iran 's proportional mutations and in contrast to other reported mutations has remarkable percentage in the country . in previous studies , the percentage of homozygote of this mutation has reached to 5.8% . in the present study , this mutation is observed for the first time in iran as compound heterozygous and this should be noted that heterozygote genotype along with mutation of f508 ( 1.85% ) were found in one of the patients . this patient was not born out of consanguinity marriage and this patient showed the symbols of malnutrition , respiratory problems and meconium ileus . in previous studies , in the common wealth of independent states ( cis ) , the mutation of s466x with f508 in the form of compound heterozygous was determined . s466x mutation with homozygote genotype was previously reported in tehran , khorasan , hamedan and markazi provinces . it seems that this mutation exists among many families in different regions of iran and it seems relatively ubiquities in iran . nonetheless , this mutation is very rare worldwide ; for example , the frequency of 0.5% has been reported in serbia and montenegro . there are also some reports about this mutation in italy 's northeast , france 's northwest , turkey , greece and india ( 16 , 17 , 30 , 3234 ) . in exon 17a , t1036i mutation is reported as a heterozygote for the second time in the world . this mutation was reported for the first time in 2006 , in iran ( 8) . this mutation may be connected to low frequency of studied patients and the common tribal marriages in iran and west asian cultures . the frequency of t1036i mutation ( c to t at 3239 ) was 1.85% in this study and it was involved with just one patient ( figure 1 and table 2 ) . the patients had the disorders of respiratory - digestive and meconium ileus and the parents of the patient had no genetic relationship ( 1 ) . polymorphism of m470v is a silent polymorphism which changes one nucleotide and converts the methionine amino acid to valine in the place of 42376223 . however , m470v is not strongly neutral because a study showed that this polymorphism leads to degradation of cl transitions through the cell 's membrane . it has also been showed that whenever m470v polymorphism comes along with another polymorphism mutation that leads to less activation of cftr protein function ( 8 , 35 ) . there are some reports worldwide which show m470v polymorphism with t5 and t7 allele ( 35 ) from tn polymorph placement cause illness in combination ( t repetitions are studied in cf patients and men 's infertility ) ( 36 ) . some reports also evaluated the potential connection of men 's prostate cancer with this polymorphism ( 37 , 38 ) . the effectiveness of this allele in the illness phenotype is not completely clear yet and it deserves more investigations . the incidence of cystic fibrosis is high in case of europeans . medical community 's lack of knowledge of the disease , poor access to medical facilities and health care for cf patients , confounding diagnosis , a high infant mortality rate , and low life expectancy in general could account for the unexplained low cf incidence in some countries with a large proportion of caucasians of european origin . in some cases , intermediate sweat chloride levels may exist in some patients , and this may lead to misdiagnosis of them . the cf frequency might be higher for some isolated populations due to consanguinity . in case of turkey and saudi arabia , hence , cf can be regarded as a prevalent illness even outside europe and united state which may have different mutation spectrums . in this regard , ascertainment of cftr mutation carrier frequencies and the cf incidence are necessary among iranian populations . frequency of f508 , s466x and t1036i mutations in this study are quite comparable to similar studies in iran and neighboring regions . the remaining mutations and their frequency identification call for a need to design some specific tests to achieve more precision in clinical diagnosis . moreover determination of the full spectrum of mutations in cftr gene in kermanshah province entails the analysis of remaining exons in cf patients . establishment of cf prevention programs by carrier screening and prenatal diagnosis seems to be an essential program in genetic counseling and prenatal care of carrier families .
backgroundcystic fibrosis ( cf ) is the most common genetic disorder with autosomal recessive inheritance among caucasian populations . so far , 
 more than 1950 different mutations were identified in cystic fibrosis transmembrane conductance regulator ( cftr ) gene . 
 cftr gene has 27 exons . 
 the type and distribution of mutations vary widely among different countries and/or ethnic groups . 
 therefore , a comprehensive analysis was performed on exon10 and exon17a of cftr gene in cf patients in the kermanshah province , western iran.methodswe tested 27 patients admitted to the medical genetics laboratory of kermanshah university of medical sciences . 
 the patients were from different cities of kermanshah province . 
 all the patients had the clinical signals and two positive sweat tests . after filling agreement forms and questionnaire , 
 the peripheral blood sampling and dna extraction were done . 
 dna samples were extracted . 
 pcr and sequencing special pcr were done . 
 finally analysis of the results with dna sequencing analysis version 5.2 software was performed.resultscftr mutations analysis identified 4 different mutations in our cf patients . 
 the disease - causing mutations were p.f508del ( f508 ) ( 14.81% ) , p.s466x ( 1.85% ) , and p.t1036i ( 1.85% ) . m470v 
 polymorphism with frequency of 74.1% was found in 23 patients ( 17 homozygous and 6 heterozygous).conclusionthree disease - causing mutations in cf patients in the present study account for approximately 18.51% of mutations . 
 the frequency of p.f508del , the most common mutation was 1618.1% in iranian population . 
 the results of the present study can be applied for genetic counseling , population screening and prenatal diagnosis .