新生儿苯丙酮尿症、先天性甲状腺功能低下筛查分析(附38286例报告)

目的探讨新生儿苯丙酮尿症及先天性甲状腺功能低下筛查的应用价值。方法选择2012年8月至2013年8月38 286例新生儿,对其干血样进行新生儿疾病筛查。苯丙酮尿症筛查指标为血苯丙氨酸(phe),实验室方法用免疫荧光分析法。先天性甲状腺功能低下筛查指标为血促甲状腺素(TSH),实验室方法用时间分辨免疫荧光分析法。结果苯丙酮尿症筛查38 286例,初筛阳性130例,阳性率3.395‰;确诊9例,确诊率69.230‰;发病率0.235‰。先天性甲状腺功能低下筛查38 286例,初筛阳性60例,初筛阳性率1.567‰,确11例,初筛阳性确诊率183.333‰,发病率0.287‰。结论开展新生儿疾病筛查对降低出生缺陷,提高我国人口素质有重要意义。

TWENTY-ONE PKU OUT OF 358767 NEWBORNS——THE INCIDENCE OF PKU IN SHANGHAI,CHINA

A study on incidence of phenylketonuria (PKU) by neonatal screening was done in Shanghai. Twenty-one cases of PKU out of 358767 newborns screened during 7 years were comfirmed with the incidence of about 1/17000. This incidence is very comparable with that in the western countries but much higher than that in Japan.

Application of Short Tandem Repeat in Prenatal Diagnosis for Phenmylketonuria during the First Trimester

Objective : To find a simple and rapid way far the prenatal diagnosis of phenyUce-tonuria (PKU) during the first trimester in order to prevent inborn PKU patients as early as possible. Methods :DNA was extracted respectively from the Mood sampleps of 9 families' members and chori-onic tissues of 9 embryoes by cliorionic vittus sampling (CVS). The independent short tandem repeat (STR) alleles of members in 9 families with classic form of PKU were analyzed and prenatal diagnosis were conducted using polymerase chain reaction (PCR) together with denaturing gradient gel elec-trophoresis(DGGE)and silver dyeing. Results-.We identified 1 embryo with PKU, 2 normal individuals and 5 carriers among 9 subjects. Conclusion: Prenatal diagnosis for PKU by STR is available in the first trimester. This procedure was promising and would be widely used in Chinese population.

<i>PAH</i>mutational spectrum: still expanding

Phenylketonuria (PKU, MIM 261600) is the most common inborn error of amino acid metabolism. To date, a total of more than 500 mutations have been associated with the disease. In this report, the novel p.Glu182Lys mutation, found in a Portuguese family in combination with the previously reported p.Leu 348Val, is presented and its putative deleterious impact discussed.

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.

High Fischer ratio oligopeptides in food:sources,functions and application prospects

High Fischer ratio oligopeptides(HFROs)are a group of oligopeptides containing high levels of branched-chain amino acids(BCAA)and low levels of aromatic amino acids(AAA).HFROs have received a lot of attention as they are believed to have significant physiological activities,including antioxidant,liver damage repair,anti-fatigue,anti-tumor and energy supply to the body.HFROs are available from a wide range of sources and both plant and animal proteins can be used to prepare HFROs but the physiological tolerability and rejection of special populations needs to be considered.Enzymatic hydrolysis is the most common method for the preparation of HFROs,but optimization of the separation and purification process is still needed in the future.Diseases caused by disruptions in the balance of BCAA and AAA in the blood,such as hepatic encephalopathy,can be treated by supplementing HFROs with drugs or food.In addition,HFROs are able to reduce fatigue feedback and assist in the treatment of phenylketonuria at the molecular nutrient level.The aim of this review is to review recent research on HFROs and provide new perspectives on the high value use of crops and the development of novel functional and special medical purpose foods.

Expression of phenylalanine ammonia lyase as an intracellularly free and extracellularly cell surface-immobilized enzyme on a gut microbe as a live biotherapeutic for phenylketonuria

Phenylketonuria(PKU),a disease resulting in the disability to degrade phenylalanine(Phe)is an inborn error with a 1 in 10,000 morbidity rate on average around the world which leads to neurotoxicity.As an potential alternative to a protein-restricted diet,oral intake of engineered probiotics degrading Phe inside the body is a promising treatment,currently at clinical stage II(Isabella,et al.,2018).However,limited transmembrane transport of Phe is a bottleneck to further improvement of the probiotic’s activity.Here,we achieved simultaneous degradation of Phe both intracellularly and extracellularly by expressing genes encoding the Phe-metabolizing enzyme phenylalanine ammonia lyase(PAL)as an intracellularly free and a cell surface-immobilized enzyme in Escherichia coli Nissle 1917(EcN)which overcomes the transportation problem.The metabolic engineering strategy was also combined with strengthening of Phe transportation,transportation of PAL-catalyzed trans-cinnamic acid and fixation of released ammonia.Administration of our final synthetic strain TYS8500 with PAL both displayed on the cell surface and expressed inside the cell to the Pah^(F263S)PKU mouse model reduced blood Phe concentration by 44.4%compared to the control Ec N,independent of dietary protein intake.TYS8500 shows great potential in future applications for PKU therapy.

Screening for tetrahydrobiopterin deficiency among hyperphenylalaninemia patients in Southern China

Abstract Objectives To assess the incidence of tetrahydrobiopterin (BH4)deficiency among patients with hyperphenylalaninemia (HPA) in southern Chinese and evaluate clinical outcome and gene mutations in tetrahydrobiopterin deficient patients.Methods Urinary neopterin (N) and biopterin (B) was analyzed in 87 patients with hyperphenylalaninemia by high-performance liquid chromatography. Further combined loading tests with phenylalanine(Phe) (100*!mg/kg) and tetrahydrobiopterin (BH4) (7.5*!mg/kg) were performed in suspected patients with abnormal urinary pterin profiles. Gene mutation analysis was performed for patients with BH4 deficiency and their parents. BH4 deficient patients were treated with BH4 and neurotransmitter precursors after diagnosis. Blood phenylalanine levels, clinical symptoms and mental development were followed up.Results Eleven patients were diagnosed as having BH4 deficiency caused by 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency. The incidence of tetrahydrobiopterin (BH4) deficiency among patients with hyperphenylalaninemia (HPA) in southern Chinese was 10%. Combined loading tests with phenylalanine and oral BH4 were done in 4 of 11 patients and their phenylalanine levels were decreased to normal 4-6h after BH4 administration. Four different mutations (P87S, N52S, D96N and G144R) in the PTPS gene were detected in 5 families. Five PTPS-deficient patients were treated with synthetic BH4, neurotransmitter precursors (L-dopa plus carbidopa, and 5-hydroxytryptophan). They had satisfactory physical and mental development after treatment. One patient with partial PTPS deficiency had normal growth and mental development without treatment. Conclusions Our results emphasize that screening for BH4 deficiency should be carried out in all patients with hyperphenylalaninemia in order to minimize the misdiagnosis. Patients with BH4 deficiency should be treated early with BH4 and a combination of neurotransmitter precursors.

Newborn screening in Zhejiang, China

Background It has been 11 years since newborn screening started in Zhejiang in 1999.The aim of this study was to analyze and summarize the status of newborn screening in Zhejiang from 1999 to 2009.Methods Blood samples were collected from the heels of newborns 72 hours after birth.We have conducted laboratory tests that the congenital hypothyroidism （CH） and circulating levels of thyroid-stimulating hormone （TSH） was detected.Blood phenylalanine （Phe） was detected for phenylketonuria （PKU）.Dissociation-enhanced lanthanide fluorescent immunoassay （DELFIA） was used for detection.Results From 1999 to 2009,3875228 newborns were screened and 2309 cases were confirmed as CH and 155 cases were confirmed as PKU.The incidence of CH and PKU were 1：1678 and 1：25 001 respectively.Conclusion In 11 years,the Zhejiang newborn screening center screened more than 3.8 million newboms,and helped more than 2000 CH and PKU patients to obtain early treatment in order to prevent physical disability and mental retardation.

Phenylketonuria in Hong Kong Chinese： a call for hyperphenylalaninemia newborn screening in the Special Administrative Region, China

Hyperphenylalaninemia is one of the commonest inborn errors of metabolism affecting approximately 1 in 15 000 live births. Among Chinese, BH4 deficiency leading to hyperphenylalaninemia is much commoner than in Caucasians. Exact diagnosis is important for the treatment and genetic counseling. In 2000, newborn screening for phenylketonuria is mandatory by law in China throughout the whole country. However, it is not yet included in the newborn screening program of the Hong Kong Special Administrative Region, China. Published data on hyperphenylalaninemia among Hong Kong Chinese are largely lacking. We report a 1-year-old Hong Kong Chinese girl with severe 6-pyruvoyl-tetrahydropterin synthase （PTPS） deficiency. The patient presented with infantile hypotonia and was misdiagnosed as cerebral palsy. She had very mild hyperphenylalaninemia （95 IJmol/L）, significantly high phenylalnine-to-tyrosine ratio （3.1）, and ele~,ated prolactin of 1109 m lU/L. Genetic analysis confirmed a homozygous known disease-causing mutation PTS NM_000317.1： c.259C〉T; NP_000308.1： p.P87S in the proband. In our local experience, while the estimated prevalence of hyperphenylalaninemia due to PTPS deficiency was reported to be 1 in 29 542 live births, not a single case of phenylalanine hydroxylase deficiency has been reported. Furthermore, there is a general lack of awareness of inherited metabolic diseases in the community as well as among the medical professionals. Very often, a low index of clinical suspicion will lead to delay in diagnosis, multiple unnecessary and costly investigations, prolonged morbidity and anxiety to the family affected. We strongly recommend that expanded newborn screening for hyperphenylalaninemia should be implemented for every baby born in the Hong Kong Special Administrative Region, China.

Multiplex Allele-specific Polymerase Chain Reaction in the Diagnosis of Phenylketonuria

Point mutation is an important molecular mechanism leading to human geneticdiseases and remains one of the focal areas of research in gene diagnosis. This notereports a simple and rapid method for the detection of known mutant phenylketonuria(PKU)alleles-Multiplex Allele-specific Polymerase Chain Reaction (MASPCR) whichcan identify several point mutations at the same time.

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%.

A NEW SILENT MUTATION FOUND IN THE CHINESE PAH LOCUS AND ITS ROLE IN THE PRENATAL DIAGNOSIS OF PHENYLKETONURIA

A silent mutation or sequence polymorphism, A to T substitution at codon 399 in exon11 of the PAH gene from a Chinese PKU patient, was found by sequence analysis. The fre-quencies of this new mutation in normal and abnormal (PKU) genes were 0.005 and 0.09,respectively, based on the analyses of 100 normal individuals and 39 PKU patients usingDNA amplification with polymerase chain reaction (PCR) and oligonucleotide hybridizationmethods. This silent mutation can be used as a "genetic marker" for PKU prenatal diagno-sis. Recently, a fetus at risk for PKU, who could not be completely predicted by RFLPslinkage analysis, was prenatally diagnosed with this genetic marker.

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.