phenylketonuria ( pku ) is an autosomal recessive inborn error of phenylalanine metabolism , which is caused by mutation in phenylalanine hydroxylase ( pah ) gene .
most of the pah mutations are missense mutations ( 67% ) , which are followed by small or large deletions ( 13% ) .
we reported a patient with classic pku and his parents harboring a large deletion in exon 3 ( ex3del4765 ) of pah gene .
this finding may help improve early detection , differential diagnosis , genetic counseling , and even treatment of patients with pku .
phenylketonuria ( pku ) is caused due to various genetic alterations of phenylalanine hydroxylase ( pah ) enzyme , which is involved in conversion of l - phenylalanine ( l - phe ) to l - tyrosine ( l - tyr ) in phenylalanine metabolic pathway .
the classic pah deficiency is considered , when the serum concentration of unchanged phenylalanine ( phe ) crosses the level of 1200-mol / l ( 1 ) . the phe concentration in the range of 600 to
1200-mol / l is diagnosed as mild pku , and values less than 600-mol / l are classified as hyperphenylalaninemia ( hpa).when the treatment program of a patient with pku did not start at the early weeks of neonatal period developmental delay , mental retardation and microcephaly are caused by accumulation of toxic byproducts of phe within its metabolic pathway .
pah gene is located on chromosome 12q23.2 , spanning 171 kbp and includes 13 exons , which encode a polypeptide of 452 amino acids ( 2 ) .
more than 520 various pathogenic mutations have been reported in all 13 exons of pah gene which could be found in the pah mutation analysis consortium database .
missense mutations constitute 67% of the total pah mutations , which could result in different clinical manifestations based on their effects on structure and function of pah enzyme .
small or large exon deletions are the second frequent genetic alterations of pah gene , which comprises 13% of the total mutations listed in pah mutation database ( http:// www.pahdb.mcgill.ca ) .
although complete deletion of exon 3 of pah gene has been previously reported in european patients , to our knowledge , it has not been identified among asian population , especially in iranian patients with pku yet .
this report aimed to describe the same but the first iranian patient with classic pku who was homozygous for deletion of pah exon 3 .
a 15-day - old male was referred to genomic research center in summer 2014 in tehran due to high level of blood phe detected in the routine national framework of pku neonatal screening ( table 1 ) .
his parents were first cousins with no family history of pku ( figure 1 ) . due to persistent high level of phenylalanine in blood ( 1404-mol / l ) ,
molecular genetic analysis was performed to confirm pku diagnosis in the child . after obtaining approval from the medical ethics committee of shahid beheshti university of medical sciences , and informed consent from patient family , peripheral blood samples from the patient and
extracted genomic dna was amplified by polymerase chain reaction ( pcr ) technique by abi geneamp 9700 device , using pah gene primers amplifying exons 1 - 13 and exon - intron boundaries .
then , pcr products were sequenced using the abi prism 3130 genetic analyzer ( applied biosystems , foster city , ca , usa ) ( primer sequences and pcr programs are available upon request ) . during pcr of pah exons in the patient ,
further analysis was conducted by designing two primers within the flanking introns of exon 3 ( introns 2 and 3 ) . in normal individuals ,
the segment size between two primers is 5885 bps ( which does not amplify in regular pcr ) . in our patient ,
a 1120 bps segment was amplified ( figure 2 ) that demonstrated a large deletion of 4765 bps ( g.21560 - 26324del4765 ) containing exon 3 , and results were confirmed by sanger sequencing ( figure 3 ) too . as a result ,
pku is an autosomal recessive metabolic disorder caused by various molecular alterations in either pah gene or the gene encoding its cofactor , tetrahydrobiopterin ( bh4 ) .
multiple pathogenic and nonpathogenic variations have been reported in all 13 exons of pah gene .
deletions constitute 13% of all kinds of mutations identified in pah gene , however large exon deletions comprise less than 7% of all the deletions .
we reported a new case of 4765 bps deletion ( ex3del4765 ) encompassing the entire exon 3 of pah gene , which has been never described in asian and iranian patients .
this deletion initially was identified in patients with pku from czech republic by performing mlpa ( multiplex ligation - dependent probe amplification ) and long range pcr .
also , it has been reported in patients with pku of other european populations such as italian , polish , and slovene , which could be an example of founder effect ( 3 - 5 ) .
ex3del4765 is produced as a result of intra - chromosomal , unequal homologous recombination between alu - alu repeats , which is the major cause of large genomic rearrangements .
alu repeats are interspersed repetitive elements comprising 10% of the total human genome , and are found in untranslated regions , introns , and even interagenic regions of genome ( 6 ) .
exon 3 of pah gene is involved in encoding the regulatory domain of pah enzyme ( 7 ) , which lies at the n - terminal of the protein .
regulatory domain spans within the 1/3 of the total length of the n - terminal of pah protein and contains an auto - regulatory sequence ( ars ) .
ars plays a fundamental role in the prevention of substrate from binding to enzyme active site ( 8) .
ars also modulates the entrance of substrate , bh4 , and catecholamine inhibitors to control the phe activation .
regulatory domain facilitates access of both substrate and cofactor through interaction with active site of catalytic domain , therefore either partial or complete deletion of it , could interfere with its proper function ( 9).considering the direct effects of various pathogenic mutations on the pah enzyme activity and the serum level of phe , accurate identification of the pah gene mutations could have a determining role in treatment schedule of pku patients . despite the lack of enough investigations , it is demonstrated that some of the pah gene mutations are more responsive to bh4 therapy ( 10 , 11 ) .
moreover , determination of specific pah gene mutation in patients can lead to early initiation of phe - free regimen , which can prevent future severe neurological complications . ascertaining the precise type of underlying molecular defect in pah gene , particularly those occurring in regulatory domain , not only confirms the diagnosis , but also could help health care providers to afford the best treatment program .
in addition , identification of the mutation type could help family genetic counseling , especially in the prenatal diagnosis ( pnd ) of future pregnancies and this is one of the strong points of our study . finally , discovering the deletion of exon 3 in pah gene in iranian population could assist us in better molecular diagnosis of patients with pku and their family members .
 introduction : phenylketonuria ( pku ) is an autosomal recessive inborn error of phenylalanine metabolism , which is caused by mutation in phenylalanine hydroxylase ( pah ) gene . 
 most of the pah mutations are missense mutations ( 67% ) , which are followed by small or large deletions ( 13%).case presentation : we reported a patient with classic pku and his parents harboring a large deletion in exon 3 ( ex3del4765 ) of pah gene . 
 this is the first case report of ex3del4765 in asian patients with pku.conclusions:this finding may help improve early detection , differential diagnosis , genetic counseling , and even treatment of patients with pku . 