Correlation Between Gene Mutations and Phenylketonuria

Correlation Between Gene Mutations and PhenylketonuriaMutations of the Phenylalanine Hydroxylase Gene in Persian Patients with PhenylketonuriaCorrelation among agent diversitys and PhenylketonuriaAlireza Biglari1, Fatemeh Saffari 2, Safarali Alizadeh3, Zahra Rashvand 3 , Reza Najafipour4, Mehdi Sahmani4ABSTRACTBackground Phenylketonuria (phenylketonuria) is an autosomal recessive disease results from flow renewings in the phenylalanine hydroxylase (PAH) gene.Objectives The aim of this withdraw was the identification of sixteen different pas seuls in Iranian patients with hyperphenylalanemia.Patients and Methods The newtys were detected during the characterization of PAH geno graphemes of 39 phenylketonuria patients from Qazvin and Zanjan lands of Iran.Results These mutations fork out been analyse by employ PCR and impart sequencing of PCR products, including the splicing sites and the promoter region of each(prenominal) 13 coding DNAs of PAH gene . A mutation det ection rate of 74.3% was realized. deuce mutations were found at high gear frequenciesR176X(10.25%) and P281L (10.25%).The frequencies of the otherwise mutations were p.R261Q(7.69%), p.R261X(5.12%), p.R252Q(5.12%),IVS2+5GA(2.56%),IVS2+5GC(2.56%),p.L48S(2.56%),c.632delC(2.56%), p.E280K(2.56%), p.R243Q(2.56%), p.I283N(2.56%), IVS9 +5GA(2.56%), IVS9+1GA(1.28%), IVS11+1GC(1.28%), p.C357R(1.28%).Conclusions The present results confirm the high heterogeneousness of the PAH locus and contribute to information around the distribution and frequency of PKU mutations in the Iranian populationKey Words Phenylketonuria. PAH gene. Iranian population. mutation detection1. BackgroundDeficiency of hepatic phenylalanine hydroxylase (PAH) EC.1.14.16.1 is the major familiar cause of hyperphenylalaninemia (1). Phenylalanine hydroxylase convert phenylalanine (Phe) to tyrosine. This enzyme encoded by PAH gene that located on chromo many 12q23.2. The PAH has 13 exons and 12 introns and is 90 kb in siz e (2). Mutations in any(prenominal) exon of this gene cause damage to the PAH enzyme. Defection of PAH lead to toxicant collecting of phenylalanine in the body fluids and cause damage to the nervous system. This scathe can be resulted to growth failure, microcephaly, mental stave and neurobehavioral abnormalities (3). Phenylketonuria (PKU, MIM 261600) is unscathedness of the well-nigh common inborn prevalent disorders of amino acid transfiguration 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 arrant(a) 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 relation incidence is 1/3627 (6). In fact, the high rate of consanguineous marriages in Iran whitethorn be a contributing factor to the high incidence (References ).The molecular(a) bases of PKU have been studied in different populations, So far, more(prenominal) than several hundred different mutations in the PAH gene have been set 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 pagan groups and geographic areas variations (8). earlier researches have shown a correlation between PAH genotypes and metabolous 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. ObjectivesThe aim of this consider was to investigate the molecular basis of PKU in all(a) PKU Patients from two adjacent provinces of North atomic number 74 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 genic variations include mutations, polymorphisms and others.3. Patients and MethodsIn this descriptive study we selected all children with known PAH wishing 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 sample c onventional 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 underfurs 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 discharges were d sensation 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 orderd by ABI 3130 genetic analyzer (ABI USA) and the results were matched up to the charitable genomic DNA sequence in GenBank database to explore the probably mutations. V alues were presented as the mean standard deviation and statistical significance was defined as p0.05. All analyses, including the x2 test were carried out using SPSS 16 software (SPSS Inc. Chicago. IL. USA).Ethical Considerations of this study was pass by the ethics committee of Qazvin University of medical sciences (Ethic cods ).4. ResultsIn this study, a nitty-gritty 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 geezerhood 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 allelomorphs (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 account in antecedent studies (REF ERENCES). (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 as well 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. DiscussionIn this research, we looked for genetic heterogeneity in 13 exons of the PAH gene of all PKU patients that admitted to Qazvin a nd 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 impression with other results obtained from Iran (13-14). The P281L mutation in exon 7 with a relative frequency of 10.5% is CT 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(prenominal) studies have reported IVS10-11GA mutation. This is a splice mutation in the cobblers last 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 succeeding(prenominal) most frequent mutation in present study was R261Q (7.69%) that occurs on a CpG mutation hotspot on exon 7 that leads to modulation of Arg to Gln at codon 261 of PAH. This mutation is common in the Mediterranean and southern atomic number 63 but low incidence in Spain (18, 20-21). We found likewise R243Q mutation in 2.5% frequency while other researcher were found it in China and Kor ea in 18.2% and 12% frequency respectively. Most mutant alleles of PAH that fake its transcription and translation can decrease the intracellular stability of protein and ultimately reduce enzyme function completely.we also explore the association between mutations and polymorphism variations. We observed c.755GA mutation and c.168+19TC polymorphism on the same allele together. We also detected association between the p.Q232Q polymorphism and c.842CT, C781CT, c.782GA, c.755GA and c.526CT 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 set, the other exons and their adjacent will be tested. Our results of Iranian indivi duals 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. 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Characterization of phenylketonuria alleles in the Italian population. European journal of human genetics EJHG. 19953(5)294-302.20.Perez B, Desviat LR, De Lucca M, Ugarte M. Spectrum and origin of phenylketonuria mutations in Spain. Acta paediatrica. 1994 Dec40734-6.21.Loeber JG. Neonatal screening in Europe the situation in 2004. Journal of inherited metabolic disease. 2007 Aug30(4)430-8.Table 1 spectrum and frequency of PAH mutations identified in 39 patientsTable 2 PAH polymorphisms identified in 39 patientsTable 3 Distributional genotypes in 39 PKU patientsgenetic constitutionPolymorphismNumberof patientsu/uc.168+19TC , c.1155GC,c.696AG1c.838GAp.E280K/ c.838GAp.E280Kc.735GA,c.912GA,c.1155CG1u/u