Mutation Analysis Of PAH Gene In Patients With PKU And Prenatal Diagnosis In Han Population From Northern China

Phenylketonuria(PKU, MIM# 261600) is the most common inborn error of amino acid metabolism disorders due to autosomal recessive inheritance. It was mainly caused by the mutation of phenylalanine hydroxylase(PAH, 612349)gene, resulted in the activity of phenylalanine hydroxylase(PAH) decreased or absence. Failure to convert phenylalanine(Phe) to tyrosine lead to accumulation of phenylalanine in the serum. Untreated phenylketonuria is associated with progressive intellectual impairment, accompanied by a constellation of additional symptoms, which include eczematous rash, autism, seizures, and motor deficits. Neonatal screening of PKU is an important method to find hyperphenylalaninemia(HPA). HPA is a genetically heterogeneous disorder, including the deficiency of phenylalanine hydroxylase and tetrahydrobiopterin deficiency(BH4D), and the therapy are completely different. So prompt screening and accurate diagnosis have significant means for PKU patients.The human PAH gene, located on chromosome 12q23.2,consists of 13 exons spanning 90 kb, which encodes a monomer protein with 452 amino acids. PAH gene analysis is the golden standard, directly detected gene mutation. The PAH gene mutation distribution have significant regional and racial genetic heterogeneity characteristics. The the study on PAH gene mutation spectrum is to provide an important basis for rapid and efficient gene diagnosis, genetic consultation of families of patients and prognostic evaluation of future PKU cases, and prenatal diagnosis of PKU in Chinese region and nation, and it is crucial to further explore the relationship between genotype and phenotype.PKU is a remediable hereditary disease can be treated by restricted-phenylalanine diet, and early diagnosis and prompt intervention undoubtedly allowed more than 90% children to reach normal level of intellectual development. However, the expensive costs, unstable efficacy and lower compliance still cause some children to suffer from the disease. Prenatal diagnosis is the main means for prevention PKU child birth, especially for the families that have given birth to PKU patients. However, prenatal diagnosis is not possible by PAH enzyme from amniotic fluid assay because the enzyme is only expressed by the liver. Prenatal diagnosis of PKU is feasible only by molecular studies. Cloned human phenylalanine hydroxylase gene allows prenatal diagnosis by DNA analysis deducing phenotype of the fetus.PAH gene mutation in Chinese people exist hot spots, furthermore over 60% are point mutation. Melting curve analysis-based PCR assay technology can be effectively to detect gene point mutation, providing rapid diagnosis platform. But we must obtain the characteristics of PAH gene mutation spectrum of Chinese people as a precondition,and screening the hot spots for desiging probe to improve the efficiency of diagnosis. We performed melting curve analysis-based PCR assay for PKU diagnosis using real time PCR. The system has characteristic with fastness, efficiency, high throughput, lowcost. Materials and methods 1 PKU PedigreesA total number of 655 cases Han PKU families in Northern China without genetic relationship were recruited in this study from January 2008 to June 2014. All of the patients were excluded tetrahydrobiopterin deficiency through BH4 loading test and urinary pterin analysis combined with clinical symptoms.118 at-risk fetuses(5 families experienced twice diagnosis, as in the line of a twin pregnancy) in 112 families were received prenatal genetic diagnosis. 107 familes were core families, and the other 5 probands were deceased in the families when his relatives come to the hospital. 2 Sanger sequencing and analysisWe designed 13 pairs of primers of PAH gene, including 13 exons, exon/intron boundaries, according to the principle of primers sequence design for PCR amplification. We used direct bidirectional Sanger sequencing method to detect the PCR products. The sequencing results were compared with the PAH c.DNA sequencing of Genebank by Chromas software, and to find the gene mutation. The variation sites of all patients’ sequences were aimed to the sites to detect the parents. 3 MLPAWe detected the large deletion/duplication of PAH gene by MLPA method in PKU patients, in whom only one or no mutation was identified in the PAH gene after Sanger sequencing. 4 Melting curve analysis-based PCR assayWe adopt melting curve analysis-based PCR assay to detect the common 26 kinds of PAH gene mutation. 5 STR analysisThe three STR sites PAH-STR、PAH-26、PAH-32 were selected to linkage analysis for prenatal genetic fetus. The primers for PCR were designed according to the literature,5 and the upstream primer 5 ’end was tagged fluorescein FAM. PCR reaction was routine. PCR products 5μL after degeneration was directly analyzed for fragment analysis on the ABI 3130-xl genetic analyzer. 6 Nomenclature and Validation of mutationThe potential mutations were compared with the known disease-causing mutations deposited in disease databases, including the Human Gene Mutation Database( HGMD professional 2014.3)( http://www.biobase-international. com/ product/hgmd)and the mutation database for the human phenylalanine hydroxylase gene(http://www.pahdb.mcgill.ca/). We finally obtained 136 known mutations and 39 novel potential mutations. We excluded the SNP by query thousands database, db SNP database and hapmap database further. The novel mutations were named according to the international gene mutation nomenclature system(http://www.hgvs.org/ mutnomen nomenclature). 7 Bioinformatic analysis of novel sequence variationThe Evolutionary conservation of a non-synonymous variant was investigated with protein sequence alignment generated by Clustal W(http://www.ebi.ac.uk/ clustalw/) and compared with that presented by the Ensembl Database(http://www. ensembl. org). The functional consequences of the missense variants were predicted using Polymorphism Phenotyping(Poly Phen-2). Protein Variation Effect Analyzer(PROVEAN) scores, which predict whether a protein sequence variation affects protein function, were also determined(http://provean.jcvi.org/about.php). 8 Prenatal TestingPAH gene sequencing combined with linkage analysis were performed on the fetuses, at the same time Maternal pollution from fetal samples and paternity was confirmed by using Power Plex 16 HS System Kit. 9 Follow upUmbilical cord blood was collected at birth for genetic analysis, and urine organic acid analysis using tandem mass spectrometry(MS/MS) was detected concurrently after birth. Developmental assessment of the newborn was performed with age. Results 1 The distribution of PAH gene mutations in patientsA total number of 1266 mutant alleles were detected in the 1310 PAH alleles of 655 PKU pedigrees, the allele mutation detection rate was 96.64%.613 patients have double mutant alleles, 40 patients carrying a single allele mutation, 2 patients were not detected mutant allele.534(81.5%) patients carrying compound heterozygous mutations, 63(9.6%) patients carrying homozygous mutation, 40(6.1%) patients carrying a single heterozygous mutations, 16(2.4%) patients carrying 3 mutations, 2(0.3%)patients were not detected mutations.We detected the corresponding mutation sites of parents’ samples, and found 2 patients carried de-novel mutation, the phylogenetic relationship was identified to be biological, all mutations of other patients were inherited from their parents.The discovery of 3 mutation sites in 16 families were confirmed that the mutations all came from parents after the parents sequencing, and one of the parents was carried 2 mutation sites on the same allele. 2 Types and characteristics of PAH gene mutationA spectrum of 175 PAH gene mutations were detected in 655 PKU families(Table 2), which involved 7 types of mutation, including missense mutations(108, 61.8%), splicing mutations(34,19.4%), nonsense mutations(15,8.6%), small deletions(10,5.7%), arge deletions(6,l 3.4%), insertion mutation(1,0.57%), indel mutation(1,0.57%).The mutation frequency of p.R243Q(17.70%) was the highest of 1266 detected alleles, followed by EX6-96A>G(8.27%), p.V399V(6.40%), p.R53H(4.68%), p.Y356X(4.68%), p.R241C(4.60%), p.R413P(4.60%), p.R111X(4.37%), c.442-1G>A(3.43%), the above 9 mutations were accounted for 58.73% of all mutant alleles. 3 MLPAPositive We detected the large deletion/duplication of PAH gene by MLPA method in 53 PKU patients, in whom only one or no mutation was identified in the PAH gene after Sanger sequencing. In 13 of the 53 patients were detected exonic deletions. 4 Melting curve analysisThe detection and accuracy of were 100% aim at 26 PAH gene sites of the kit. 5 The bioinformatics analysis results of novel mutation sequence variations of PAH geneThe amino acid sequences of PAH protein in human, chimpanzee, zebrafish, rodents and lizards were compared and analyzed, and found that the novel variants sites of PAH gene are highly conserved in all species. Combination of PROVEAN prediction, Poly Phen-2, the results showed that the majority of the novel sequence variations may be deleterious mutation rather than polymorphism. 6 Prenatal diagnosisThe Prenatal fetus in 107 families were detected by PAH gene mutation analysis combined with short STR linkage analysis method, the other 5 families used only PAH mutation analysis as the proband deceased(Table 5).112 of 118 fetuses were obtained definitive diagnosis by PAH gene mutation analysis, as diagnostic rate was 91.5%. In addition,the 5 families carrying one mutant allel failed diagnosis by PAH gene mutation analysis but succeed combined with STR linkage analysis.Among the 118 prenatal genotype fetuses, 31 carried compound heterozygous alleles, Analysis of the aborted tissue confirmed that these fetuses carried two mutant PAH alleles. 58 fetuses were heterozygous carrying one mutant allele and 29 fetuses did not carry any detectable mutant allele presumably normal. 7 Genetic Counseling and follow-upFollowing genetic counselling, the 30 parents of these fetuses chose to terminate the pregnancy, whose futus carried compound heterogeneous mutations with the proband. The result were confirmed by analysing aborted tissue. But one couple chose to continue the pregnancy, and the concentration of serum Phe of the neonate was 1650μmol/L(27.5mg/dl)by neonatal screening. 87 carriers and normal fetus continued pregnancy,among whom 64 had been normal born passing neonatal screening, the rest 23 are still in pregnancy. Conclusions1.The constitution of PAH gene mutation in Han population of northern China were distinctly different from other ethnic groups, The construction of PAH gene mutation database in PKU patients of northern China may lay the foundation for gene diagnosis, prenatal diagnosis and population screening.2.PAH gene mutation analysis combined with STR linkage analysis approach can provide rapid and accurate prenatal diagnosis for PKU pedigree.3.39 novel mutations enrich human gene mutation database.4. Melting curve analysis-based PCR assay is an rapid, accurate and economic method to detect PKU.