Correlation Between Gene Mutations and Phenylketonuria Essay

Correlation Between Gene Mutations and Phenylketonuria Essay

Correlation Between Gene Mutations and Phenylketonuria Essay

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.Correlation Between Gene Mutations and Phenylketonuria Essay

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%).Correlation Between Gene Mutations and Phenylketonuria Essay

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

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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 ).Correlation Between Gene Mutations and Phenylketonuria Essay

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. 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.Correlation Between Gene Mutations and Phenylketonuria Essay

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).Correlation Between Gene Mutations and Phenylketonuria Essay

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).Correlation Between Gene Mutations and Phenylketonuria Essay

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.L385L, p.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.Correlation Between Gene Mutations and Phenylketonuria Essay

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).Correlation Between Gene Mutations and Phenylketonuria Essay 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.Correlation Between Gene Mutations and Phenylketonuria Essay

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).Correlation Between Gene Mutations and Phenylketonuria Essay

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]

Phenylketonuria (PKU; MIM 261600) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH; EC PAH is a hepatic enzyme that catalyses the hydroxylation of L-Phenylalanine (L-Phe) to L-tyrosine using tetrahydrobiopterin (BH4) as a cofactor.Correlation Between Gene Mutations and Phenylketonuria Essay

The human PAH gene, which is located on chromosome 12q, consists of 13 exons spanning 90 kb. To date, more than 520 different mutations in the PAH gene have been characterized in PKU patients and recorded in the PAH Mutation Analysis Consortium Database ( Although most of these mutations are detectable by sequence analysis, with a detection rate of > 95%, large intragenic deletions or duplications cannot be identified using this method. Rare cases of large genomic deletions involving one or more exons of the PAH gene have been described (Sullivan et al., 1989; Avigad et al., 1990; Guldberg et al., 1993, 1997; Okano et al., 1994; Bosco et al., 1996; Zschocke et al., 1999; Gable et al., 2003).Correlation Between Gene Mutations and Phenylketonuria Essay

Recently, multiplex ligation-dependent probe amplification (MLPA) has been used as a sensitive and efficient method for the detection of large deletions and duplications (Schouten et al., 2002). Using MLPA, several studies have identified large deletions and duplications of the PAH gene in PKU patients lacking PAH mutations in one or both alleles, based on sequence analyses (Desviat et al., 2006; Kozak et al., 2006; Birk Moller et al., 2007). However, deletions or duplications of the PAH gene have been detected in fewer than half of the patients tested, and the estimated allelic frequency of exon deletions have been reported as < 3% in a Western population (Desviat et al., 2006; Kozak et al., 2006; Birk Moller et al., 2007).Correlation Between Gene Mutations and Phenylketonuria Essay

In a previous study, only 86% of the mutant alleles were identified by sequence analysis of the entire PAH coding region, suggesting that large deletions or duplication are frequent causes of PKU in Koreans (Lee et al., 2004). To elucidate this hypothesis, we performed both sequencing and gene dosage analyses of the PAH gene in 33 unrelated Korean PKU patients. After identifying a recurrent large deletion that encompasses exons 4 and 5, the deletion breakpoint was analyzed by chromosomal walking. In addition, the relationship between genotype and phenotype, e.g., BH4 responsiveness, was also evaluated.Correlation Between Gene Mutations and Phenylketonuria Essay

Materials and Methods
Thirty-three unrelated patients with PAH deficiency were recruited from the Korean PKU family support group. Most of these patients were identified during neonatal screening, and PAH deficiency was diagnosed by conventional biochemical methods. Biochemical diagnosis was based on measurement of the plasma phenylalanine concentration before starting a phenylalanine-restricted diet (Table 1). Disease severity was classified as follows: classical PKU (≥ 1,200 µM); variant PKU (600-1,200 µM), and mild hyperphenylalaninemia (MHP; < 600 µM). Urinary pterin analyses and dihydropteridine reductase (DHPR) assays were performed to exclude 6-pyruvoyltetrahydropterin synthase (PTPS) deficiencies.

Table 1 Biochemical characteristics and PAH mutations in Korean patients with PKU. Abbreviations: NI, Not informed; PKU, Phenylketonuria; MHP, Mild hyperphenylalaninemia aNBS blood spots, reference range: 19-164 µM; bReference range: < 2.0; cPlasma phenylalanine concentration was measured before starting a phenylalanine-restricted diet, Reference range : 54.9-103.1 µM; dReference range : 0.05-0.19 µM
Full size table
For the BH4 loading test, patients who were not on a phenylalanine-restricted diet were administered BH4 orally at 20 mg/kg (for patients < 36 months of age) or 7.5 mg/kg (> 36 months of age). The blood phenylalanine levels were measured before and at 1, 2, 4, 6, 8, 12, and 24 h after BH4 administration. The BH4 loading test was considered positive when the initial plasma phenylalanine concentration was decreased by at least 40% after 12 h.Correlation Between Gene Mutations and Phenylketonuria Essay

Mutation analysis
After obtaining informed consent from the parents, blood samples were collected from the patients and their family members. Genomic DNA was isolated from peripheral blood leukocytes using the Wizard Genomic DNA Purification kit (Promega, Madison, WI)(Lee et al., 2006). All 13 PAH exons and their flanking intronic sequences were amplified by PCR using appropriate primers designed by the authors (sequences available upon request) and a thermal cycler (Model 9700; Applied Biosystems, Foster City, CA). Five microliters of amplification product were treated with 10 U shrimp alkaline phosphatase and 2 U exonuclease I (USB Corp., Cleveland, OH), and direct sequencing was then performed using the BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) in the ABI Prism 3100 genetic analyzer (Applied Biosystems). Potential novel mutations were defined as being distinct from those in the PAHdb database ( and the mutations reported previously in PubMed (

MLPA analysis
MLPA was carried out for PKU patients who lacked PAH mutations on one or both alleles, based on previous sequencing analyses. The SALSA PO55 PAH MLPA kit (MRC Holland, Amsterdam, The Netherlands) contains 25 sets of probes: 13 PAH-specific sets, and the remainder comprises control standard probes from other human genes. The assay was carried out in 200-µl tubes in a thermal cycler (Model 9700; Applied Biosystems) according to the manufacturer’s instructions. Briefly, a total of 200 ng of genomic DNA from each subject was diluted in 5 µl of TE buffer and denatured at 98℃ for 5 min. MLPA buffer and probe mix (1.5 µl of each) were then added, and the probes were annealed to the target genomic DNA by heating at 95℃ for 1 min, followed by incubation at 60℃ for 16 h. Thirty-two microliters of Ligase-65 mix were added to each sample, and the annealed probes were ligated at 54℃ for 15 min, followed by inactivation at 98℃ for 5 min. Ten microliters of the ligation reaction were removed for multiplex amplification using a pair of common primers, of which one was labeled with the fluorescent dye 6-FAM. Taq polymerase was added to the PCR (total volume of 50 µl) at 60℃, followed by 36 cycles of 95℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min, and a final extension step of 72℃ for 20 min. Between 0.5 µl and 0.75 µl of each reaction was mixed with 0.5 µl of TAMRA-labeled internal size standard and 12.5 µl deionized formamide, and used for fragment analysis on the ABI-3100 capillary sequencer (Applied Biosystems). The conditions used for the fragment analysis were: polymer POP-4 in a 36-cm capillary; run temperature, 60℃; injection voltage, 15 kV; injection time, 3-5 s; run voltage, 15 kV; run time, 24 min. The obtained data were analyzed using the Genescan 3.1.2 software. The peak heights were normalized, and exon deletions were adjudged when the sample peak height was less than 65% of the control peak height.Correlation Between Gene Mutations and Phenylketonuria Essay

Long-range PCR
Long-range PCR using the AccuPower TLA PCR premix (Bioneer, Daejeon, Korea) was performed for those patients who had a deletion of exons 5 and 6. By chromosomal walking with different pairs of primers, the smallest PCR amplicon (expected size: 12,522 bp) was detected using the primer pair: F23 (5′-tcgaactcgcaagtttgttg-3′) and R6 (3′-cagctggagaggattgaagg-5′). The primer sequences were based on the intronic sequences of the PAH gene obtained from the Ensembl Genome Browser (

Molecular modeling
To investigate the effects of the new PAH mutations on the structure of PAH, we used the crystal structure designed with the PAHdb system ( Models of the PAH monomer mutants were generated with the Biopolymer module of the Insight II modeling software (Molecular Simulations, San Diego, CA). The side-chain conformation of the PAH mutant was determined using the Auto-rotamer command in the Biopolymer module of the Insight II package. The three-dimensional model structure of PAH was visualized with PYMOL ( Between Gene Mutations and Phenylketonuria Essay