In vivo regulation of phenylalanine hydroxylase in the genetic mutant hph-1 mouse model

The hph-1 mouse has low liver activity of GTP cyclohydrolase 1, the rate limiting enzyme in the biosynthesis of tetrahydrobiopterin (BH₄). BH₄ is the cofactor for phenylalanine hydroxylase (PAH) and in the early stages of life the hph-1 mouse is hyperphenylalaninemic. At approximately 15 days after birth the blood phenylalanine levels normalize. During this period the animals provide an in vivo model which can be used to study the regulatory effects of phenylalanine on PAH, and for related pediatric metabolic disease in humans; from birth to youth. We therefore, examined; liver PAH activity using BH₄ and 6-methyltetrahydropterin (6MPH₄) as cofactor; PAH total enzyme concentration by Western blotting using the PH8 antibody, and PAH state of phosphorylation using the PH7 antibody from 4 to 18 days after birth. The findings were compared to the wild type animals that are not hyperphenylalaninemic during this period. PAH (6MPH₄) activity and total protein (PH8 antibody) rose steadily in the hph-1 mice. In control mice, both activity and total protein fluctuated. The degree of phosphorylation of PAH in the mutants and the state of activation (as measured by the 6MPH₄/BH₄ activity ratio) increased as phenylalanine levels rose, and decreased when they fell. Similar patterns were not seen in the control animals. These studies provide in vivo evidence that phenylalanine concentration regulates the activity of PAH in the hph-1 mouse and that this acts via a mechanism that includes phosphorylation of the PAH molecule. The kinetic values (K_m and V_max) for mouse PAH are also reported.     

云南苯丙氨酸羟化酶基因点突变及外显子5内新突变Y166X(C→G)的鉴定

目的研究云南苯丙氨酸羟化酶基因点突变分布概况,以提高该地区苯丙酮尿症（ＰＫＵ）的基因诊断率。方法应用ＰＣＲ－ＡＳＯ探针斑点杂交,ＰＣＲ－ＳＳＣＰ、ＡＳＰＣＲ和ＤＮＡ直接测序等技术,对１３／１４名云南籍ＰＫＵ患儿的苯丙氨酸羟化酶（ＰＡＨ）基因外显子４、５、６、７、１０、１１和１２进行了鉴定分析。结果共检出５种错义突变：Ｒ２４３Ｑ（５／２６）、Ｙ２０４Ｃ（３／２８）、Ｒ４１３Ｐ（２／２８）、Ｔ４１８Ｐ（１／２８）及Ｇ２４７Ｖ（１／２６）,３种无义突变Ｙ１６６Ｘ（Ｃ→Ｇ）（２／２６）、Ｗ３２６Ｘ（１／２８）及Ｙ３５６Ｘ（２／２６）和１种静止突变Ｖ３９９Ｖ（２／２６）。经检索国际ＰＡＨ基因突变数据库,其中Ｙ１６６Ｘ（Ｃ→Ｇ）为首次发现的新突变。结论云南藉ＰＫＵ与中国北方人群有相类似的最常见的ＰＡＨ基因突变类型（Ｒ２４３Ｑ、Ｙ２０４Ｃ、Ｙ３５６Ｘ、Ｖ３９９Ｖ和Ｒ４１３Ｐ）,但与南方人群ＰＡＨ基因突变特点则不同。该发现有助于提高云南ＰＫＵ的基因诊断率,并对我国ＰＡＨ基因突变不同地区及人群的分布、起源研究有参考价值。     

大庆地区新生儿苯丙酮尿症苯丙氨酸筛查切值的研究

目的探讨大庆地区新生儿苯丙氨酸（phenylalanine,Phe）测定时新生儿苯丙酮尿症筛查切值。方法以大庆地区出生的新生儿为筛查对象,于出生后48~72h,充分哺乳后采取足跟血,应用定量酶法检测标本中苯丙氨酸含量。结果2004年1月至2005年12月两年期间共筛查20214例,确定大庆市新生儿苯丙酮尿症的筛查切值为2.60mg/dl。结论新生儿足跟血Phe切值的确定为大庆市新生儿苯丙酮尿症的筛查工作提供依据,早发现早治疗可避免患儿的智力障碍,对提高我国人口素质有着十分重要的意义。     

Information processing in patients with early and continuously-treated phenylketonuria

A total of 33 patients with early and continuously-treated phenylketonuria (PKU) between 7 and 16 years of age and 33 matched controls participated in a study examining perceptual, central, and response-related mechanisms of information processing. The specific mechanisms studied were: perceptual filtering, memory search, response selection, response execution, and motor presetting. In addition, groups were compared on mean intelligence level and task oriented behaviour. The performance of the PKU patients practically matched that of the controls on all three tasks, suggesting that PKU patients who are continuously maintained on a well-controlled phenylalanine-restricted diet are not impaired in the elementary mechanisms of information processing. Furthermore, groups did not differ in mean IQ or task-oriented behaviour.     

In vivo assessment of mutations in the phenylalanine hydroxylase gene by phenylalanine loading: Characterization of seven common mutations

Abstract Mutations in the gene encoding phenylalanine hydroxylase (PAH) cause persistent hyperphenyl-alaninaemia. To date, more than 200 point mutations and microdeletions have been characterized. Each mutation has a particular quantitative effect on enzyme activity and recessive expression of different mutant alleles results in a marked interindividual heterogeneity of metabolic and clinical phenotypes. In this paper we demonstrate how a simple clinical test can be used to evaluate the correlation between mutation genotype and phenylalanine metabolism. In hyperphenylalaninaemic patients with known PAH mutation genotype, we have investigated phenylalanine turnover in vivo by measuring the ability to eliminate a test dose ofl-phenyl-alanine. All patients could be considered functionally hemizygous for one of their mutant alleles by carrying on the other allele a mutation that is known to completely abolish PAH activity and encode a peptide with no immunoreactivity. Seven mutations (R408W, IVS-12nt1, R261Q, G46S, Y414C, A104D, and D415N) were characterized by oral phenylalanine loading, each mutation being represented by at least three patients. The elimination profile determined for a 3-day period provides a measure to compare residual activity of the mutant proteins and to assign each mutation to a particular metabolic phenotype. The established relation between genotype and phenotype may enable prediction of the severity of the disease by genotype determination in the newborn period. This will aid in the management of hyperphenylalaninaemia and may improve prognosis.Conclusion The possibility of predicting the residual enzyme activity by DNA analysis performed already in the newborn period allows the prompt implementation of a diet that is adjusted to the degree of PAH deficiency. This may improve management and prognosis of hyperphenylalaninaemia.     

Phenylketonuria and some aspects of emotional development

Early dietary treatment of phenylketonuria (PKU) prevents intellectual retardation and gross neurological impairment although not all neuropsychological problems. This study investigates to what extent the illness and its treatment imposes a burden on emotional development of early-treated PKU patients and on rearing practices of their parents. It is concluded that in early-treated PKU it is particularly difficult to find constructive ways to adapt to the situation for children as well as for parents. As coping with PKU and its treatment is so complicated, paediatric control should be combined with psychocounselling.