RESUMO
Objective To provide genetic counselling for a pregnant with phenylketonuria(PKU) family history.To provide prenatal diagnosis for the pregnant of the pedigree,followed by identifying of the pathogenic mutation of the proband and the genotype of the other family members.Methods Sanger sequencing was performed to detect the phenylalanine hydroxylase(PAH)gene pathogenic mutation of the patient.Both sequencing and haplotype of the short tandem repeats(STR)site in intron 3 were analyzed for the fetus, whose mother was the aunt of the patient.Results Compound heterozygote mutation of PAH gene,IVS4-1G>A /c.770G>T was identified for the proband,which inherited from his father and mother respectively.The aunt of the patient was a carrier of the IVS 4-1G>A heterozygote mutation,whose husband was identified c.827T>A heterozygote mutation.Prenatal diagnosis disclosed that the fetus inherited the paternal c.827T >A mutation, and the haplotype of the PAH gene was different from the patient. Conclusion According to the counselling of autosomal recessive disorder,for the partner of a carrier,it is suggested that mutation detection should be performed to exclude the possibility of being a carrier too, and then the risk of the offspring can be evaluated precisely.(Chin J Lab Med,2018,41:312-315)
RESUMO
Objective To perform PCR site-directed mutagenesis of nine novel PAH gene mutations (Y154H, R157I, Y206C, G247R, D282G, G346R, S349A, A389G, R400K) identified in northern Chinese and construct mutant recombinant plasmids of PAH gene. Methods 1) Every mutant recombinant plasmid was constructed according to the site of the mutation localized in functional domain of PAH gene and the related clinic phenotype of patients with the gene mutation. 2) Using the wild-type PAH expression vector as a templet, the mutant recombinant plasmids were directly amplified by PCR with Platinium Taq DNA polymerase and nine pairs of primers which were designed according to the human PAH cDNA sequence and the requirement for site-directed mutagenesis technology. 3) The positive strains were selected by Amp resistant test, PCR and restriction endonuclease analysis. The Mva Ⅰ, Mva Ⅰ, Hind Ⅲ, Rsa Ⅰ, Rsa Ⅰ sites exist in the sequences near the mutant sites of S349A, D282G, G247R, Y206C, Y154H, respectively, but not in the related sequence of wild-type PAH expression vector. Restriction endonuclease digestion could be directly used in identifying the mutant sites. However, the amplification created restriction site (ACRS) analysis was supplied in the followed identification of R157I, G346R, A389G, R400K. Finally the sequences of mutant recombinant plasmids of PAH gene were confirmed by DNA sequence analysis. Results Every sequence analysis showed that the mutant nucleic acids were introduced at the expected sites of PAH gene, suggesting that the mutant recombinant plasmids of PAH gene were constructed successfully. Conclusion PCR site-directed mutagenesis is accurate and highly efficient. The successfully mutagenized plasmids of PAH gene lay the foundation for the functional analysis of phenylalanine hydroxylase in mammalian cell system.