TWO NOVEL MUTATIONS IN PHENYLALANINE HYDROXYLASE GENE AND IN VITRO EXPRESSION ANALYSIS ON MUTATION ARG252GLN

We report novel mutations in exon 7 of human phenylalanine hydroxylase (PAH) gene of phenylketonuria (PKU ) in southern Chinese, analysed by using PCR-DGGE (denaturing gradient gel electrophoresis ), solid phase DNA sequencing and Ih vliro expression. One of the 2 novel mutations, IVS6nt1, is an intron-exon Junctional mutation which results a splicing defect in mRNA. Arg252Gln is another novel mutation with residual PAH activity only 24 % compared to wild type in in vitro mutagenesis and expression in Cos-1 cell. Other 3 known mutations and polymorphism including Arg241Cys, Arg243Gln and Val245Val(GTG to GTA) together with these novel mutations composed the mutatlonal profile of exon 7 in the PAH gene of PKUs in this populations.

MUTATIONS IDENTIFIED IN EXON 7 OFPHENYLALANINE HYDROXYLASE GENE IN CHINESE

Exon 7 of the l’henylalan1ne hydroxylase (PAH) gene was analyzed in 15 chlldren affected wlth classicphenylketonL1rla (PKU) from northern Chlna by uslng PCRxsingle strand conformation polymorphism(PCR-SSCP) technique and DNA direct sequencing. Six missense mutatlons (l. e. R2413Q. R 241H, G247V,1,2 19H, F2541;lnd G257V )and one silent rnutatlon (V245v ) were identified. The latter three missense mu-tations were demonstrated as novel mltations in comparison with the PAH mutation database. one missense mt1tation (R241 H) was flrst dowumeTlted in Chinese. our results showed populatlon ancl reglon tllffer-ences in the PAH mutation clistribution. and suggest that there is more thfln one founding population forPKU in China. The fincling of novel mutations will enhence the molecular diagnosis of PKU.

A novel large deletion(exons 12,13)and a missense mutation(p.G46R)in the PAH in a Japanese patient with phenylketonuria

Background:Phenylketonuria(PKU)is caused by a defect in phenylalanine hydroxylase(PAH).More than 500 mutations have been reported for the gene encoding PAH.However,approximately l%-5%of these include large deletions and large duplications that cannot be detected by conventional methods.Methods:In this report we tried to fully characterize a PAH-deficient patient.The patient was a 2-year-old Japanese boy who was diagnosed with classical PKU at the time of neonatal screening,which was confirmed by the tetrahydrobiopterin-loading test.PCR-related direct sequencing and multiplex ligation-dependent probe amplification(MLPA)were used to analyze of the PAH of the patient.Results:Using PCR-related direct sequencing method,we could detect only a heterozygous novel missense mutation:p.136G>C(p.G46R).A second mutation was detected by MLPA.The patient was heterozygous for a novel large deletion of exons 12 and 13:c.1200-?_1359+?del(EX12_13del).For genetic counseling,an accurate genetic diagnosis is often necessary.Conclusions:Through a combination of MLPA and conventional methods,the success rate of PAH mutation identification can be close to 100%.

Analysis of EX5del4232ins268 and EX5del955 PAH gene mutations in Ukrainian patients with phenylketonuria

Phenylketonuria(PKU)is an autosomal recessive metabolic disorder caused by deficiency of phenylalanine hydroxylase(PAH).The major molecular defects causing PKU are missense mutations of PAH gene.Large deletions of exon 5(EX5del955 and EX5del4232ins)were first reported by the Czech study and were later found also in the Polish,Slovak,Slovenian and Italian PKU-patients.These observations demonstrate the existence of a common subset of this mutation predominantly among Central European populations of Slavic descent.That is why we suggest that EX5del1955 and EX5del4232ins268 mutations might be frequent causes of PKU in Ukrainian patients.EX5del955 and EX5del4232ins268 mutations were analyzed in 106 unrelated PKU patients negative for PAH gene mutations on one or both alleles from our previous analysis.The simultaneous detection of EX5del4232ins268 and EX5del955 mutations was performed by PCR amplification of mutant alleles.EX5del955 mutation was not detected in the Ukrainian patients.This relative alleles frequency of EX5del4232ins268 mutation in the Ukrainian PKU population was determined as 1,66%.Our findings can be the one more evidence of Central European Slavic origin of EX5del4232ins268 mutation,suggested previously.This finding is important for the improvement of DNA diagnosis necessary for the management of PKU patients from Ukraine.