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Correlation Between Gene Mutations and Phenylketonuria

Mutations of the Phenylalanine Hydroxylase Gene in Iranian Patients with Phenylketonuria

Correlation between gene mutations and Phenylketonuria

Alireza Biglari1, Fatemeh Saffari 2, Safarali Alizadeh3, Zahra Rashvand 3 , Reza Najafipour4, Mehdi Sahmani4


Background: Phenylketonuria (PKU) is an autosomal recessive disease results from point mutations in the phenylalanine hydroxylase (PAH) gene.

Objectives: The aim of this study was the identification of sixteen different mutations in Iranian patients with hyperphenylalanemia.

Patients and Methods: The mutations were detected during the characterization of PAH genotypes of 39 PKU patients from Qazvin and Zanjan provinces of Iran.

Results: These mutations have been analyzed by using PCR and direct sequencing of PCR products, including the splicing sites and the promoter region of all 13 exons of PAH gene . A mutation detection rate of 74.3% was realized. Two mutations were found at high frequencies:R176X(10.25%) and P281L (10.25%).The frequencies of the other mutations were: p.R261Q(7.69%), p.R261X(5.12%), p.R252Q(5.12%),IVS2+5G>A(2.56%),IVS2+5G>C(2.56%),p.L48S(2.56%),c.632delC(2.56%), p.E280K(2.56%), p.R243Q(2.56%), p.I283N(2.56%), IVS9 +5G>A(2.56%), IVS9+1G>A(1.28%), IVS11+1G>C(1.28%), p.C357R(1.28%).

Conclusions: The present results confirm the high heterogeneity of the PAH locus and contribute to information about the distribution and frequency of PKU mutations in the Iranian population

Key Words: Phenylketonuria. PAH gene. Iranian population. mutation detection

1. Background

Deficiency of hepatic phenylalanine hydroxylase (PAH) [EC.] is the major frequent cause of hyperphenylalaninemia (1). Phenylalanine hydroxylase convert phenylalanine (Phe) to tyrosine. This enzyme encoded by PAH gene that located on chromosome 12q23.2. The PAH has 13 exons and 12 introns and is 90 kb in size (2). Mutations in any exon of this gene cause damage to the PAH enzyme. Defection of PAH lead to toxic accumulation of phenylalanine in the body fluids and cause damage to the nervous system. This injury can be resulted to growth failure, microcephaly, mental retardation and neurobehavioral abnormalities (3). Phenylketonuria (PKU, MIM# 261600) is one of the most common inborn prevalent disorders of amino acid metabolism characterized by a defect in the hepatic PAH and subsequently phenylalanine accumulation in body fluids (4). According to blood phenylalanine (Phe) levels, PKU has been classified as mild PKU, mild hyperphenylalaninemia (MHP) and classical PKU. Classical PKU is the most severe form of this disorder. Phenylalanine restricted dietary treatment prevents the neurotoxic complications of Phe and its metabolites if it is implemented at an early age (5). The prevalence of PKU varies worldwide. In Caucasians, the prevalence is about 1/10000 live births (5) while Iranian population incidence is 1/3627 (6). In fact, the high rate of consanguineous marriages in Iran may be a contributing factor to the high incidence (References ).

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The molecular bases of PKU have been studied in different populations, So far, more than several hundred different mutations in the PAH gene have been identified by the PAH Mutation Analysis Consortium in the PKU patients. These mutations have been listed in the PAH mutation Analysis Consortium database (http:// www. Pahb.mcgill.ca). The most frequently occurring type of PAH gene mutations are missense (7). The mutations of PAH gene demonstrate considerable ethnic groups and geographic areas variations (8). Previous researches have shown a correlation between PAH genotypes and metabolic phenotypes in PKU patients. Those studies have demonstrated the phenotypic relations of particular mutation combinations (9-11). Mutation analysis of a given population can be useful for the better understanding functional aspects of mutant proteins and the relationship between genotype and phenotype.

2. Objectives

The aim of this study was to investigate the molecular basis of PKU in all PKU Patients from two adjacent provinces of North West of IRAN; Qazvin and Zanjan. For this purpose, all 13 exons of the PAH gene of all patients were analyzed using direct sequencing for detecting of any genetic variations include mutations, polymorphisms and others.

3. Patients and Methods:

In this descriptive study; we selected all children with known PAH deficiency living in Qazvin and Zanjan provinces. Thirty-nine unrelated children were enrolled after obtaining informed consents from the parents. All selected cases have several grades of mental retardation except few patients who have diagnosed during neonatal screening exam. Before molecular studies, The PAH activity and phenylalanine concentration of all patient serum samples were measured by standard conventional biochemical methods. The blood phenylalanine concentration ≥20 mg/dl was clinical criteria for classical PKUdiagnosis (12).

Genomic DNA was extracted from whole blood samples using Qiagen DNA purification kit (Valencia, CA, USA). Specific primers for all 13 exons of PAH gene were designed by primer 3 software and verified them by NCBI database. The PCR conditions for all exons were set experimentally based on each primer specifity. The primers and their reaction specificaions were summarized in table 4. The PCR tests were done by Verity ABI thermal cycler (ABI, USA). PCR products were electrophoresed in 2% agarose gel and visualized after staining by gel red nucleic acid stain (Biotiom; U.S.A). For scanning PAH gene in order to finding any variation in the 13 exons, all amplicons were sequenced by ABI 3130 genetic analyzer (ABI; USA) and the results were matched up to the human genomic DNA sequence in GenBank database to explore the probably mutations. Values were presented as the mean ± standard deviation and statistical significance was defined as p<0.05. All analyses, including the x2– test were carried out using SPSS 16 software (SPSS Inc. Chicago. IL. USA).

Ethical Considerations of this study was approved by the ethics committee of Qazvin University of medical sciences (Ethic cods; ).

4. Results

In this study, a total of 39 PKU patients were subjected to scanning PAH gene heterogeneity from Qazvin and Zanjan province. Among 39 patients, 24 come from the Qazvin province, 15 from the Zanjan. The subjects have 1 month to 10 years old. The serum phenylalanine concentration of all patients was ≥20 mg/dl.

By whole genome sequencing method, 16 different mutations were found in 78 mutant alleles (Diagnostic efficiency 74.3%). The mutations included eight missense mutations (50%), five splice mutations (31%), two nonsense mutations (12.5%) and one deletion (6.25%). All of the mutations were reported in previous studies (REFERENCES). (Table 1). Exon 7, 6, 2 and the flanking intronic regions consist 85.5% of the mutant alleles. The most frequent of mutations were p.R176X and p.P281L by 10.5% frequency followed by p.R261Q (7.69%), p.R261X and p.R252Q (5.12%) which consist nearly 40% of all mutations. The p.R261X and p.R252Q Mutations were less frequent. All other mutations had frequencies less than 3%. Among the 39 unrelated families studied, 20 (51.2%) were homozygote, 6 (15.3%) heterozygote and 2 (5.12 %) were compound heterozygote and 11 (28.2%) were no PKU causing mutations. In addition, the p.L385Lp.Q232Q and p.V245V polymorphisms also were detected in our study with the frequency of 84%, 51% and 17% respectively. These polymorphisms were seen the highest prevalence in PAH gene at other reports (Table 2). Table 3 are shown genotypes of 39 PKU patients too.

5. Discussion

In this research, we looked for genetic heterogeneity in 13 exons of the PAH gene of all PKU patients that admitted to Qazvin and Zanjan University of medical sciences health systems in order to finding causative PKU disease genetic factor. From this experiment, 29 of 39 PKU patients were found to contain the mutation in one or more exons of PAH gene. Our analysis of the homozygosity of the mutations were nearly similar to that observed in northwestern Iranian populations (13). The majority of the recognized mutations were situated in the catalytic domains (143-410 amino acid), and some of them (P281L, R252W) were located in the cofactor binding regions. The most common mutation in our subjects was P281L. These data have the same opinion with other results obtained from Iran (13-14). The P281L mutation in exon 7 with a relative frequency of 10.5% is C→T substitution that lead to conversation of Pro to Leu at codon 281 of PAH gene. The another more frequent mutation in our study was p.R176X (10.25%) which is similar to data obtained from another study at Khorasan Razavi region (14). Previous study on the genotype / phenotype association demonstrated generally a positive correlation between R176 X mutation and classic phenotype (15). Several studies have reported IVS10-11G>A mutation. This is a splice mutation in the end of intron 10 that observed with a high incidence in Mediterranean region, Brazil and some area of Iran including East Azarbaijan, Semnan, Khorasan Razavi and Hamadan (16-19). However this mutation was not found in the present study. The virtual absence of this mutation in our study may reflect the regional variability of populations. The next most frequent mutation in present study was R261Q (7.69%) that occurs on a CpG mutation hotspot on exon 7 that leads to conversion of Arg to Gln at codon 261 of PAH. This mutation is common in the Mediterranean and southern Europe but low incidence in Spain (18, 20-21). We found also R243Q mutation in 2.5% frequency while other researcher were found it in China and Korea in 18.2% and 12% frequency respectively. Most mutant alleles of PAH that manipulate its transcription and translation can decrease the intracellular stability of protein and finally reduce enzyme function completely.

we also explore the association between mutations and polymorphism variations. We observed c.755G>A mutation and c.168+19T>C polymorphism on the same allele together. We also detected association between the p.Q232Q polymorphism and c.842C>T, C781C>T, c.782G>A, c.755G>A and c.526C>T mutations that occurred on the same allele in cis form. similar association have been reported in the previous study (14). In our study, the most mutant alleles were located on exon 7 and 6 (73%). Other studies in Iranian population were reported agreement results with our findings (14, 16).

Thereby to plan detection strategy; the samples will be screened first for mutations in these regions. If mutations were not identified, the other exons and their adjacent will be tested. Our results of Iranian individuals with PKU confirm a heterogeneous spectrum of mutations, displaying different ethnic and geographical origins. Moreover, our findings were slightly different from other ethnic groups. These findings can be useful to genotype/phenotype relationship in patients and provide future some ability to confirmatory diagnostic testing, prognosis and predict severity of PKU patients. [V1]



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2.Santos LL, Fonseca CG, Starling AL, Januario JN, Aguiar MJ, Peixoto MG, et al. Variations in genotype-phenotype correlations in phenylketonuria patients. Genetics and molecular research : GMR. 2010;9(1):1-8.

3.Zhang J, Meng J, Zhai X, Fang G, Gao J, Shi M, et al. [Identification of novel mutations in the phenylalanine hydroxylase gene of classical phenylketonuria]. Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics. 2005 Apr;22(2):134-7.

4.Williams RA, Mamotte CD, Burnett JR. Phenylketonuria: an inborn error of phenylalanine metabolism. The Clinical biochemist Reviews / Australian Association of Clinical Biochemists. 2008 Feb;29(1):31-41.

5.Olsson GM, Montgomery SM, Alm J. Family conditions and dietary control in phenylketonuria. Journal of inherited metabolic disease. 2007 Oct;30(5):708-15.

6.Koochmeshgi J, Bagheri A, Hosseini-Mazinani SM. Incidence of phenylketonuria in Iran estimated from consanguineous marriages. Journal of inherited metabolic disease. 2002 Feb;25(1):80-1.

7.Scriver CR. The PAH gene, phenylketonuria, and a paradigm shift. Human mutation. 2007 Sep;28(9):831-45.

8.Zschocke J. Phenylketonuria mutations in Europe. Human mutation. 2003 Apr;21(4):345-56.

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16.Zare-Karizi S, Hosseini-Mazinani SM, Khazaei-Koohpar Z, Seifati SM, Shahsavan-Behboodi B, Akbari MT, et al. Mutation spectrum of phenylketonuria in Iranian population. Molecular genetics and metabolism. 2011 Jan;102(1):29-32.

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Table 1 spectrum and frequency of PAH mutations identified in 39 patients

Systematic name

(DNA level)

Trivial name

(protein effect)

Location Mutation type Number of alleles Frequency (%)
c.168+5G>A IVS2+5G>A Intron 2 Splicing 2 2.56%
c.168+5G>C IVS2+5G>C Intron 2 Splicing 2 2.56%
c.143T>C p.L48S Exon2 Missense 2 2.56%
c.526C>T p.R176X Exon 6 Nonsense 8 10.25%
c.632delC p.P211>Hfs Exon6 deletion 2 2.56%
c.838G>A p.E280K Exon7 Missense 2 2.56%
c.782G>A p.R261Q Exon7 Missense 6 7.69%
c.842C>T p.P281L Exon7 Missense 8 10.25%
c.781C>T p.R261X Exon7 Nonsense 4 5.12%
c.755G>A p.R252Q Exon7 Missense 4 5.12%
c.728G>A p.R243Q Exon7 Missense 2 2.56%
c.848T>A p.I283N Exon8 Missense 2 2.56%
c.969+1G>A IVS9+1G>A Intron 9 Splicing 1 1.28%
c.969+5G>A IVS9 +5G>A Intron 9 Splicing 2 2.56%
c.1199+1G>C IVS11+1G>C Intron 11 Splicing 1 1.28%
c.1069T>C p.C357R Exon11 Missense 1 1.28%
Number of alleles identified 49

Table 2 PAH polymorphisms identified in 39 patients

Systematic name

(DNA level)

Trivial name

(protein effect)

Location Number of




c.696A>G p.Q232Q Exon 6 40 51.28%
c.735G>A p.V245V Exon7 14 17.9%
c.912G>A p.Q304Q Exon8 2 2.56%
c.1155G>C p.L385L Exon11 66 84.61%fr
c.168+19T>C IVS2+19T>C Intron2 5 6.4%
c.-71A>C 5-UTR 5-UTR 4 5.1%
c.843T>A p.P281P Exon8 2 2.56%
IVS3-22C>T c.353-22C>T Intron 3 2 2.56%
Number of alleles identified 135

Table 3 Distributional genotypes in 39 PKU patients

Genotype Polymorphism Number

of patients

u/u c.168+19T>C , c.1155G>C,c.696A>G 1
c.838G>Ap.E280K/ c.838G>Ap.E280K c.735G>A,c.912G>A,c.1155C>G 1


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