phenylketonuria ( pku ; omim 261600 ) is defined as an autosomal recessive genetic inborn error of phenylalanine ( phe ) metabolism ( 1 ) .
insufficiency in the hepatic specific enzyme , phenylalanine- 4-hydroxylase ( pah ) ( ec 1.14.16.1 ) leads to hyperphenylalaninemia that is associated with the pku ( 1 ) .
deficiency of the pah enzyme results in the elevation of phe concentration in blood and biological fluids that is approximately above 2 mg / dl ( 120 mol / l ) in the pre - treatment condition ( 1 - 4 ) .
the prevalence of pku among patients institutionalized for mental retardation varies from 1% to 3% ( 4 - 6 ) .
the frequency of pku varies from high incidence in turkey ( about 1 in 2600 births ) to low incidence in japan ( about 1 in 125000 births ) ( 7 ) .
overall , the incidence of pku among caucasians is about 1 in 10,000 , giving a carrier frequency of about 1 in 50 to 1 in 70 ( 7 ) .
the incidence of pku in iranian population has been expected at 1 in 3627 live births ( 8) .
genetic overall diversity in iranian populations is very high and comparable to the other populations from the south caucasus region , anatolia and europe ( 9 ) .
it has been shown that iranian azerbaijanis with a population of about 15 to 20 million are more related to the georgians in comparison to other iranian groups ( 9 ) .
the finding of derenko et al ( 2013 ) is based on maternal genetic structure on the mitochondrial dna studies ( 9 ) . however , this result may change based on paternal genetic structure and y - chromosome tracing .
west azerbaijan province with a population of about 3 million is in north - west of iran and closely related to turks .
regarding to a relatively high incidence of pku alleles in iranian population as well as high rate of consanguineous marriages in iran ( 10 ) , this study was carried out for mutation analysis of the pah gene in west azerbaijan province of iran .
this study was performed regarding the ethical guideline in human genetic research as described by ethics committee of urmia university of medical sciences ( west - azerbaijan , urmia , iran ) and was conducted in accordance with declaration of helsinki .
pku patients were diagnosed and sequentially selected among patients referred to motahari hospital of urmia university of medical sciences .
a total of 109 individuals from 40 pku families including 40 pku patients ( 16 males and 24 females ) and their parents entered in the study .
all cases were diagnosed by a pediatric neurologist in the department of pediatrics at the university hospital of motahary ( urmia , iran ) .
medical data recordings , and tests assessments were carried out by the same physician for all cases using criteria for diagnosis of pku ( 7,11 ) .
an informed written consent from the parents of the kids for research study , 3 to 5 ml whole blood was taken from patients and their parents and collected in ethylenediaminetetraacetic acid ( edta ) tube .
genomic dna was isolated from blood samples using  salting out method ( 12 ) .
a total of 218 alleles from109 individuals from 40 pku families ( 40 pku patients and their parents ) were tested for ivs10 - 11 , s67p , r261q , r252w , ivs11nt-1 g > c , r408q , q232q , r243q , 364delg , l333f , 261x , i65 t , and r408w mutations in the pah gene using restriction fragment length polymorphism - polymerase chain reaction ( rflp - pcr ) as described previously ( 13,14 ) ( table 1 ) .
tested mutations in phenylketonuria families from west azerbaijan province of iran pcr reaction was performed in a 25 l solution containing 50 ng dna , 1x reaction buffer , 10 pmol of each primer , 200 mol of dntps , 0.3 unit of taq dna polymerase , and 1.5 mmol mgcl2 ( genefanavaran , tehran , iran ) .
a 10 to 15 l of the pcr products were examined for the presence or absence of tested mutations by restriction digestion at 37 c for 2 hr according to the manufacturer s instructions .
2%-3% agarose gel containing ethidium bromide was used for electrophoresis of pcr amplicons and digested fragments . presence or absence of a mutation
contains the highest number of mutations ( 87 out of 567 ) ( 15.34% ) ( pahdb ; http://www.mcgill.ca/pahdb ) .
the identified mutations on the exon 7 of the pah gene were confirmed with the direct sequencing in an abi 730xl dna analyzer ( applied biosystems ) using the primer sequences of 5actaccaaaggtctcctagtgcct3 and 5ctacacaactagcctgtggaccag3 ( 15 ) .
the frequencies of ivs10 - 11 , s67p , r261q , r252w , ivs11nt-1 g > c , r408q , and q232q mutations were 28(35 ) , 17(21.25 ) , 15(18.75 ) , 3(3.75 ) , 3(3.75 ) , 2(2.5 ) , and 1(1.25 ) , in case group and 51(23.4 ) , 31(14.2 ) , 27(12.4 ) , 6(2.75 ) , 6(2.75 ) , 4(1.83 ) , and 2(0.92 ) in total group , respectively . observed mutations in the patients were inherited from their parents .
the mutations of r243q , 364delg , l333f , 261x , i65 t , and r408w were not found in this study . of the alleles studied , the most frequent mutation was ivs10nt546 ( 35% ) .
seven mutations represent approximately 86.25% and 83% of pku chromosomes analyzed in cases and total groups , respectively .
our analysis showed that 37.5% ( 15/40 ) and 62.5% ( 25/40 ) of the cases have homozygote and compound heterozygote genotypes regarding the studied mutations ( table 3 ) .
the most common mutations of ivs10 - 11 , s67p , and r261q can be as a result of the high rate of consanguineous marriages .
the frequencies of missense , splice , and silent mutations were 37 ( 46.25 ) , 31 ( 38.75 ) , and 1 ( 1.25 ) , in cases and 68 ( 31.18 ) , 57 ( 26.15 ) , and 2 ( 0.92 ) in total groups , respectively .
mutation analysis in the phenylalanine hydroxylase gene of 40 cases and total ( patients and parents ) groups in west azerbaijan province of iran the allele frequency was based on 218 alleles including mutant ( 153/218 ) and normal ( 65/218 ) alleles in total ( patients and parents ) group the allele frequency was based on 80 mutant alleles in case group ; the mutation detection rate was 86.25% ( 69 out of 80 pku chromosomes ) and 83% ( 127 out of 153 pku chromosomes ) in case and total ( patients and parents ) groups in the west azerbaijani population .
f : frequency distribution of homozygote and compound heterozygote genotypes in the west azerbaijani among phenylketonuria patients detection of ivs10nt-11 ( c.1066 - 11g > a ) and ivs11nt1 g > c ( c.1199 + 1g >
c ) mutations in six samples pcr products ( 357 bp ) were digested with ddei .
the presence of ivs11nt1 g > c mutation naturally produces restriction site for ddei enzyme .
individuals homozygous for normal sequence regarding ivs11nt1 g > c mutation show a single un - cut band of 357 bp .
individuals homozygous for ivs11nt1 g > c mutation show two bands of 244- and 113- bp .
individuals heterozygous for ivs11nt1 g > c mutation show three bands of 357- , 244- and 113- bp . as well as , individuals homozygous for normal sequence regarding ivs10nt-11 mutation show a single un - cut band of 357 bp .
individuals heterozygous for ivs10nt-11 ( c.1066 - 11g > a ) mutation show three bands of 357- , 261- and 96- bp lane m : 50 bp marker ( fermentas ) . lanes 1 : homozygous for ivs10nt-11 g > a mutation .
lanes 4 and 6 : heterozygous for ivs10nt-11 g > a mutation detection of r261q mutation in 7 samples .
individuals homozygous for r261q mutation show a single un - cut band of 285 bp .
individuals heterozygous for r261q mutation show two bands of 285 and 123 bp lane m : 50 bp marker ( fermentas )
lane 7 : homozygous for r261q mutation pku nucleotide sequences of pku mutation of c.782g > a ( p.r261q ) of exon 7 of the pah gene in a sample detection of r252w mutation in 8 samples .
individuals homozygous for r252w mutation show a single un - cut band of 285 bp .
individuals heterozygous for r252w mutation show three bands of 285 , 162 and 123 bp lane m : 50 bp marker ( fermentas ) .
lanes 5 - 8 : without r252w mutation detection of s67p mutation in 11 samples .
the presence of s67p mutation removes restriction site for xbai enzyme individuals homozygous for s67p mutation show a single un - cut band of 463 bp .
individuals heterozygous for s67p mutation show three bands of 463 , 348 and 115 bp lane m : 50 bp marker ( fermentas ) .
the tested mutations were chosen based on similarity between population in the west azerbaijan province and the mediterranean groups .
the ivs10nt-11g > a mutation with systematic name of c.1066 - 11g > a also known by the trivial name ivs10nt546 g > a , is the most common mediterranean pku mutation ( 1 ) .
dworniczak et al ( 1991 ) reported transition of g to a at location 546 in intron 10 of the pah gene ( 1 ) .
this mutation activates a splicing site and leads to insertion of nine nucleotides between exons 10 and 11 during splicing .
ivs10nt546g > a is the major cause of pku in parts of southern and southeastern europe , mainly in turkey .
several investigations have been conducted to study the spectrum of pku causing mutations in various groups ( 8 , 11 , 13 , 16 - 23 ) .
interestingly , the frequency of ivs10nt-11 mutation in our cases is higher compared to other reports ( 8 , 11 , 13 , 16 - 23 ) .
the high rate of consanguineous marriages ( 47.5% ) is a contributing reason to this high prevalence in tested families .
it has been suggested that the world - wide distribution of ivs10nt546 mutation has turkish origin with expansion in different geographic regions ( 7 ) . in this study , it was established that the ivs10 - 11 mutation has the highest frequency in the pku patients among the iranian azerbaijanis , and it can be considered for molecular diagnosis in this population .
the findings of the present study may be a sign of close familial link between west azerbaijanis and turks , which is consistent with the historical as well as geographical relations between west azerbaijan and turkey .
the second most common mutation identified in our investigation , s67p ( c.199t > c ) , is a missense mutation with low frequency in other populations ( 4 ) .
unexpectedly , the mutation of s67p with 25% frequency seemed to be explained by the high rate of consanguineous marriages in the tested group ( 47.5% ) .
the third most common mutation identified in our investigation , r261q ( c.782g > a ) , is a mediterranean missense mutation and occurs on a cpg dinucleotide on exon 7 in the pah gene .
this mutation results in conversion of arggln at codon 261 and is the second most frequent mutation in turks ( 19 ) .
the remaining mutations of r252w ( c.754c > t ) , ivs11nt-1 g > c ( c.1199 + 1g > c ) , r408q ( c.1223g > a ) , and q232q ( c.696a / g ) account for 12.47% of the identifiable mutations .
this study indicated high level of heterogeneity of the pah gene in pku families in the west azerbaijan , and thus further investigation should be carried out .
mutation analysis in the pah gene can be used in carrier detection , prenatal diagnosis and prevent the incidence of pku phenotypes in the west azerbaijani population .
the results of this study would be useful for biomedicine and molecular anthropology studies by tracing the anthropological characters of the population regarding autosomal chromosomes .
this study had some limitations including the small sample size and poor quality of medical records .
exon 11 and its intronic regions carry the most prevalent mutant alleles in pku families in the west azerbaijan .
screening of ivs10nt-11 g > a mediterranean mutation should be tested for detection of possible mutations of pah in the west azerbaijan ( iran ) .
this report reveals the genetic heterogeneity in the west azerbaijani pku population with a high frequency of ivs10nt-11 g > a mutation ( 35% ) that shows similarity to turks .
 objective(s):phenylketonuria ( pku ) is a genetic inborn error of phenylalanine ( phe ) metabolism resulting from insufficiency in the hepatic enzyme , phenylalanine hydroxylase ( pah ) , which leads to elevated levels of phe in the blood . 
 the present study was carried out for mutation analysis of the pah gene in west azerbaijan province of iran.materials and methods : a total of 218 alleles from 40 pku families were studied using restriction fragment length polymorphism - polymerase chain reaction ( rflp - pcr ) method.results:the frequencies of ivs10 - 11 , s67p , r261q , r252w , ivs11nt-1 g > c , r408q , and q232q mutations were 28(35 ) , 17(21.25 ) , 15(18.75 ) , 3(3.75 ) , 3(3.75 ) , 2(2.5 ) , and 1(1.25 ) , in cases group , and 51(23.4 ) , 31(14.2 ) , 27(12.4 ) , 6(2.75 ) , 6(2.75 ) , 4(1.83 ) , and 2(0.92 ) in total group , respectively . 
 the mutations of r243q , 364delg , l333f , 261x , i65 t , and r408w were not detected in our samples.conclusion:it can be concluded that the ivs10 - 11 mutation has the highest frequency in the tested population . to our knowledge , this report is the first in its own kind and provides better understanding of the genetic heterogeneity , the origin and distributions of pah mutations in west azerbaijan province of iran . 
Introduction
Materials and Methods
Results
Discussion
Conclusion