Experiment Study On Phenylalanine Hydroxylase Gene Of Shanxi Province

Objects:Eighty-five cases are ages 15 days to 7 years old children that diagnosed with Phenylketonuria come from Shanxi MCH newborn screening centers and clinics, Datong MCH, Changzhi MCH, and Yuncheng MCH.from, among them 48 are male and 37 are female. Phenylalanine concentrations of initial blood testing is from 251μmol / L to-2621μmol / L. All patients underwent a blood-dihydro pteridine reduction, urinary neopterin analysis and tetrahydrobiopterin deficiency load test,and excludes tetrahydrobiopterin deficiency. All tests got the informed consents of their parents or guardians.Phenylketonuria can treat a few genetic diseases, and its incidence rate in China is1/11572. A majority of patients suffer from this disease because of the gene mutations of phenylalanine hydroxylase(PAH), the gene mutations lead to the reduction or even loss of PAH, and the phenylalanine, which cannot be converted to tyrosine, instead of accumulating in large enough amounts in the body.At present, the diagnosis of children patients with phenylketonuria in domestic or abroad is time-consuming and tedious, what’s more it is only appropriate for infants but could do nothing for the prenatal diagnosis. Therefore, based on the studies of the Phenylalanine hydroxylase gene mutation spots of patients with phenylketonuria in Shanxi Province, their distribution and frequency of occurrence, this paper aims to explore a new way of phenylketonuria gene diagnosis and prenatal diagnosis so as to further enrich PAH gene mutation spectrum in China.Methods:1. 85 Cases of typical PKU children patients from September 2008 to May 2013 are collected in all regions of Shanxi Province. PCR-RFLP method is applied for the measurement of PCR amplification of 3rd, 7th, and 11 th exons in typical phenylketonuria patients. Appropriate endonuclease is also used to make endonuclease mutation spots analysis.2. HRM(high resolution melting curve) is applied in the detection of mutation spots of 5th, 6th, 7th, 11 th, 12 th exons in patients with classic phenylketonuria.3.This paper does the in vitro amplification and sequencing of the 13 exon of PAH genes in 85 typical PKU child patients, and then compares with normal PAH gene sequences. Finally, after statistical analysis, the author gets all the genetic mutation spots information of PAH genes in PKU patients in Shanxi Province.Results:1. PCR-RFLP method is applied for the measurement of amplification of 3rd, 7th,and 11 th exons of PAH genes in 85 typical phenylketonuria patients as well as their parents. And then by sequencing, the test results are totally coincide with the sequencing results;2. In this paper, High Resolution Melting(HRM) is used as initial screening method to analyze the mutation of 5th, 6th, 7th, 11 th, and 12 th exons of PAH genes and their exon-intron junction mutations in 85 PKU patients. The study shows that the PCR-HRM sequencing result is completely coincide with that PCR result of 5th, 6th, 7th,11 th, and 12 th exons.3. By studying on the 13 exons of PAH genes in 85 phenylketonuria children patients, this paper arrived at a new finding that is L11 fs X7, P69 H, L98 V, T372 R and IVS11-1G>C are five mutational sites. In addition, this paper worked out that R243 Q and Y204 C are the mutational sites of Shanxi Province.Conclusions:1.PCR-RFLP method has advantages in good repeatability, high specificity and accuracy rate and provides a simple, fast, and accurate genetic analysis for PAH gene research;2. This study confirms that HRM technology is an easy, accurate, fast, high-flux,and low-cost genetic analysis method, not only for the use of PAH gene mutation screening, but especially applicable for prenatal diagnosis with parents of pedigrees PKU mutations.3. By studying on the 13 exons of PAH genes in 85 phenylketonuria children patients, this paper not only provides some certain experimental data for PAH gene mutation in PKU patients in Shanxi Province but contributes to database of PKU patients study in China.4. This study enriched the Chinese PAH gene mutation spectrum.