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Updated listing of mutation map at the human phenylalanine locus among Egyptian population

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The Egyptian Journal of Medical Human Genetics, Vol. 7, (1):15–22, May 2006 ... Updated Listing of Mutation Map at the Human ... Rabah M Shawky, 1 Nasser A Elhawary, 2 Heba H Elsedafy, 1 Solaf M Elsayed, 1 ... 1 Department of Pediatrics, Faculty of
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  The Egyptian Journal of Medical Human Genetics, Vol. 7, No. 1, May 2006 15 Updated Listing of Mutation Map at the Human Phenylalanine Locus among Egyptian Population Rabah M Shawky, 1  Nasser A Elhawary, 2  Solaf M Elsayed, 1 Hosam Abdel-Hamid, 2  Heba H Elsedafy, 11  Department of Pediatrics, Faculty of Medicine, and 2  Medical Genetics Center,  AinShams University, Cairo, Tel. (+2)0101088473, E-mail: nasgenet@hotmail.com Abstract:  Phenylketonuria is the most common inborn error of amino acid metabolism in Egypt with a relatively higher incidence of 1:7,500. Unrelated fifty-one PKU probands were selected from the database-records at the Medical Genetics Center, AinShams University-Cairo. We analyzed the DNA samples using polymerase chain reaction (PCR) combined with restriction enzyme assays, or allele specific oligonucleotide (ASO) testing and direct sequencing to detect 10 PAH gene mutations in exons 2, 3, 6, 7 and 11. We interestingly identified a novel missense CpG site R243P mutation. Moreover, three new known mutations L48S, delEX3 and Y277D unreeled in the Egyptian population. The ten detected mutations covered 58% representing 47 positive chromosomes. The most common mutation was represented by IVS10nt546 (10.8%), while the total missense mutations in our sample group account for the majority of mutations 40%.The high heterozygosity of the mutant PAH locus in Egypt suggests that multiple founder events would explain the presence of hyperphenylalaninemia in Egypt.Further studies will however be necessary to fully exploit the potential of PAH gene analysis to reconstruct the genetic history of PKU in Egypt in context with migrations among European and Mediterranean populations. Egypt J Med Hum Genet 7(1):15–22 Introduction: Phenylketonuria (PKU,MIM #261600) is the most common inborn error of amino acid metabolism in Mediterranean basin with an inci-dence of 1:10,000 to 1:4,400. It is caused by an autosomal recessive deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). To prevent mental retardation due to the neurotoxic effects of high levels of phenylalanine and pathological meta-bolites, patients with PKU must be treated early in their life with a low-L-phenylalanine diet depending on the severity of their clinical phenotype. 1 The PKU-associated hyperphenyl-alaninemia phenotype is highly vari-able, primarily due to a great allelic heterogeneity at the PAH   locus. Phenylalanine hydroxylase ( PAH  ) mutation map reported 512 different disease-causing mutations (http://  Updated PAH gene mutations in Egypt, Elhawary et al, 16 www.pahdb.mcgill.ca) (March, 2006). The severity of the neonate PKU can be predicted based on the patient's genotype and knowledge of its corre-lation with the clinical phenotype. (2,3) Significant variation in the spectrum and prevalence rates of mutations in the PAH   gene in different populations make it important to define the PKU mutation profile in a district popu-lation.Our collaborative joint with OASI institute molecular geneticist staff (Troina, Italy) since 1992 screened a pilot sample of PKU Egyptian families promoting a fruitful preliminary study to identify the PAH   gene abnor-malities. (4-6)  An extension to the previ-ous results was carried out using the newly banked genomic DNA samples.In this study, we have extended our study to the newly banked genomic DNA samples to cover the most common mutation-hotspot exons 2, 6, 7, 11, in addition to exon 3 for 40 un-related PKU Egyptian families. Subjects and Methods  : Patients  The present study was recruited on 51 unrelated Egyptian PKU pa-tients (17 females and 34 males). The PKU probands were selected from the database-records at the Medical Genetics Center, AinShams University (ASMGC). Psychological and devel-opmental assessment of PKU patients was done on a yearly basis between the ages of 1 and 16 y using variety of tests that examine IQ, motor-graphic ability, adaptation, educational achiev-ements, and emotional development. The families investigated are a part of a cohort of 100 Egyptian families with PKU that recently underwent genetic and molecular analysis using denatur-ing high performance liquid chromato-graphy (DHPLC) (Elhawary et al, in preparation). All patients were pre-senting with blood phenylalanine lev-els persis tently above 150 μmol/l and primarily diagnosed based on clinical criteria and investigations. Thirty-two patients (80%) were consanguineous. DNA analysis Genomic DNA was extracted from blood lymphocytes using MiniPrep DNA kit (Qiagen, Valencia, CA). In some cases, DNA was prepared in situ by gentle scraping the buccal mucosa for 30 s using a cytobrush. (7)  The cells obtained were treated directly with diluted NaOH solution, heated, and neutralized with Tris-Cl, pH 8.0. A 2.5-  l volume of buccal cells or Guthrie DNA typically sufficed for PCR amplification . PCR amplification The five exons (2, 3, 6, 7, and 11) and their exon-flanking intronic se-quences of the PAH   gene amplified by PCR. The sequences of oligo-nucleotide primers for PCR were designed in accordance with the literature, (8)  without the GC-clamp tails at the 5'-end (Table 1). Reaction mixtures were made in a total volume of 25-  μ  l containing 0.5  μ  g of genomic DNA, 2.5 pmol of each primer, 0.25 m M   of each dNTP (Roche, GmbH), the reaction buffer 2.5-5 m M   MgCl 2 , 20 m M   Tris-HCl, pH 8.4, and 50 m M  KCl and Taq  DNA polymerase (Bio-ron, GmbH). The reaction mixturewas incubated at 94°C for 4 min, then 30 from 55 to 62°C depending on  The Egyptian Journal of Medical Human Genetics, Vol. 7, No. 1, May 2006 17 Table 1. Oligonucleotide amplification primers and PCR conditions ExonPrimer sequenceT a*  (°C)Size(bp)2 F: 5'-GAGGTTTAACAGGAATGAATTGCT-3'R: 5'-TCCTGTGTTCTTTTCATTGC-3'58264 3 F: 5'-GCCTGCGTTAGTTCCTGTGA-3'R: 5'-CTTATGTTGCAAAATTCCTC-3'55267 6 F: 5'-CCGACTCCCTCTGCTAACCT-3'R: 5'-CAATCCTCCCCCAACTTTCT-3'57326 7 F:5'-GGTGATGAGCTTTTAGTTTTCTTTC-3'R: 5'-AGCAAATGAACCCAAACCTC-3'62263 11 F: 5'-TGAGAGAAGGGCACAAATG-3'R: 5'-GCCAACCACCCACAGATGAG-3'60301* The annealing temperatures (T a ) were optimized toamplify the selected exons using the Gradient Thermal Cycler (Hybaid, USA). the amplified fragment (Table 1), and 72°C for 45 s, then followed by a final elongation of 7 min at 72°C in a PCRExpress Thermocycler (Hybaid, USA). An aliquot of each PCR mixture was analyzed by 1.4% traditional agarose gel chromatography. Enzymatic digestion assays  An 8-10  μ  l aliquot of the amplicons was added to 5 U of the appropriate enzyme (New England Biolabs, USA and Fermantas, GmbH) in the buffer, in a total reaction volume of 25  μ  l (Table 2). All enzymatic reactions were incubated at 37°C for 15-17 h. Products were analyzed by electro-phoresis on 3% MetaPhor gel (BMA, Bioproducts, USA) and characteri-zation of alleles are summarized in table 2. When available, parental DNA samples were used to validate the mutations detected by enzymatic restriction assays to confirm trans configurations in compound hetero-zygosity from hemizygosity. Novel mutations were defined by exclusion from the pahdb (http://www.pahdb.mcgill.ca) and previous reported mutations on PubMed (www.ncbi.nlm.nih.gov/PubMed/). Results  : Novel PAH mutation  Among our sample patients, we in-terestingly identified a sole novel mutation, R243P as a missense CpG site mutation in a frequency of 2% (3/102). (9)  This new mutation was de-tected by denaturing gradient gel elec-trophoresis (DHPLC) (WAVE™ sys-tem, Transgenomic Inc, San Jose,USA ) and confirmed by direct sequencing. Updated PAH gene mutations The overall strategy for genotyping  Updated PAH gene mutations in Egypt, Elhawary et al, 18 Table 2 .Mutations detected by restriction digestion, appropriate enzymes and detection of normal and mutant PAH alleles used in this study MutationsEnzymeDetectionNA (bp) a MA (bp)  b L48S  Mls I c 112/152264EX3 del  − 267Del d E221G  Nco I326229/97V245V  Alu I154 e /69/31/9125/69/31/29/9R252W  Ava I177/86263R261Q  Hinf  I116/147263G272ter   Bam HI115/148263Y277D  Xmi I c 163/100263IVS10nt546  Dde I301222/79 a  Normal allele (NA) showed that a mutation generates a diagnostic restriction site.  b Mutant allele (MA) showed that the mutation ablates a natural restriction site. c  Mls I and  Xmi I enzymes (Fermantas, GmbH) were efficiently used instead of   the traditional  Bal I and  Acc I enzymes (New England Biolabs, USA). d Amplification of exon 3 was carried out in the presence of an internal primer set. The deletion mutation was confirmed by direct sequencing. e The change of GTG>GTA (n>m) creates another  Alu I recognition site characterized by a 125-bp band. The fragments 29/31-bp could not be distinct from each other on traditional agarose gel, but a 9-bp could not be observed. involved the panel of 102 unrelated Egyptian PKU alleles. In this study, the scanning of eight mutations was carried out by the methods of direct DNA diagnosis using the naturally occurring restriction sites in case of L48S, E221G, V245V, R252W, R261Q, G272ter, Y277D  and IVS10nt546  mu-tations in addition to the silent V245V mutation. In addition, four IVS2nt5g-c, R176X, I224T and R243P mutations were identified via  direct sequencing.  A large deletion within the PAH gene of exon 3 was also detected by traditional PCR and confirmed by allele specific oligonucleotide (ASO) testing. These mutations were confined to exons 2, 6, 7 and 11 being had the high frequency mutations region in Egyptian population. Our study was also extended to include exon 3 looking for large exon deletions.We interestingly found three new known mutations (10)  L48S (Konecki et al. 1991), delEX3 [Avigad et al. 1990] and Y277D [Labrune et al. 1991] that have not been previously reported in the Egyptian population in relative frequencies of 10% (5/50), 3.3%  The Egyptian Journal of Medical Human Genetics, Vol. 7, No. 1, May 2006 19 (2/60), and 5.8% (3/52), respectively. Besides additional six PAH muta-tions (10)  E221G [Konecki et al 1991], V245V [Dwornicczak et al 1990], R252W [Okano et al 1990, Abadie et al 1989], R261Q [Abadie et al 1989], G272ter [Melle et al 1991] and IVS10-nt546 [Dwornicczak et al 1991] were screened. All these ten examined mutations covered 57.7% representing 47 positive chromosomes of the total mutations in our Egyptian samples (Table 3). The frequency of the IVS10nt546 mutation (10.4%) represented the most common mutation in our Egyp-tian sample followed by R261Q (6.4%) and L48S (6.3%) (Table 2). It is note to worthy that the previously described IVS2nt5g>c and R176X mutations (11)  were only detected as compound heterozygotes in 2 PKU patients in 2 genetic counseling PKU families through direct sequencing. In addition, we could detect the previous described I224T mutation, (5)  in exon 6 via direct sequencing. Table 3. Molecular analysis results of the PAH   gene identified in Egyptian  population Mutation nameSystematic name(DNA level)Characters of mutationLocationNo. of posit-ives/tested alleles (%)Ref* L48Sc.143T>CMissenseExon 25/80 ( 6.3 )(10)IVS2nt5g-c**c.168+5g>cSplicingIntron 22/50 (4.0)(11)DelEx3-2.5 kbc.169? −352+?del DeletionExon 32/80 ( 2.5 )(10)R176X**c.526C>T*NonsenseExon 62/80 (2.5)(11)I224Tc.671T>CMissenseExon 62/80 (2.5)(5)R243P***c.728G>C*MissenseExon 73/102 ( 2.0 )(9)V245Vc.735G>ASilentExon 711/74 ( 14.9 )(10)R261Qc.782G>A*MissenseExon 76/94 ( 6.4 )(10)Y277Dc.829T>GMissenseExon 73/52 (5.8)(10)IVS10nt546c.1066-11g>aSplicingIntron 1111/102 ( 10.8 )(10) Total47 (57.7%) * Phenylalanine Hydroxylase Mutation Analysis Consortium Database (March,2006); *= CpG site.** The IVS2nt5g-c and R176X mutations were previously described in the Egyptian PKU patients using direct sequencing by Effat et al. (11) ***This new mutation was described using DHPLC and direct sequencing by Elhawary and Shawky. (9)  It has been recently submitted to the PAH   gene map (www.pahdb.mcgill.ca).
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