四氢生物蝶呤负荷试验诊断四氢生物蝶呤反应性苯丙氨酸羟化酶缺乏症的临床研究

目的探讨四氢生物蝶呤(BH4)反应性苯丙氨酸羟化酶(PAH)缺乏症的临床诊断方法,进一步了解其临床特征,为该型患儿应用BH4药物治疗提供科学依据。方法73例高苯丙氨酸血症(HPA)患儿,男47例,女26例,平均年龄1.93个月。所有患儿都进行口服BH4负荷试验,同时进行尿蝶呤谱分析、红细胞二氢蝶啶还原酶测定。对其中血苯丙氨酸(Phe)浓度<600μmol/L者给予口服Phe BH4联合负荷试验,对部分BH4反应性PAH缺乏症患儿,在普食条件下给予BH4片剂(10～20mg/kg)替代治疗6～7天,观察其疗效。结果(1)在BH4负荷试验中,不同类型HPA患儿的血Phe浓度表现出特征性的曲线改变,22例诊断为经典型苯丙酮尿症(PKU),39例中度PKU,12例四氢生物蝶呤缺乏症;(2)在中度PKU患儿中发现22例(56.4%)对BH4有反应;(3)6例BH4反应性PAH缺乏症患儿以BH410mg/kg治疗6～7天,其中4例血Phe浓度能控制到正常或接近正常治疗水平,另2例BH4需增加到20mg/kg使Phe浓度显著下降。结论在BH4负荷试验中,部分因苯丙氨酸羟化酶缺乏引起的中轻度PKU患儿对BH4有反应性,给予这些患儿BH4治疗可部分或全部替代低苯丙氨酸饮食治疗,拓宽了PKU的治疗方法,有助于提高患儿的生活质量。     

不同类型高苯丙氨酸血症的临床表型

目的探讨不同类型高苯丙氨酸血症（HPA）临床表型,了解HPA患者对低苯丙氨酸（Phe）饮食治疗的反应性。方法HPA患儿44例均口服四氢生物喋呤（BH4）负荷试验（20 mg/kg）或Phe-BH4联合负荷试验,同时进行尿喋呤谱分析、红细胞二氢喋呤还原酶（DHPR）测定。治疗前采用Gesell发育量表进行智力测定并行脑电图检查。予苯丙氨酸羟化酶（PAH）缺乏症患者低Phe饮食治疗,控制血Phe在120~360μmol/L,监测Phe耐受量。结果1.确诊BH4反应性PAH缺乏症患儿12例。男7例,女5例;平均年龄7.8个月;生化代谢表型均为轻度或中度HPA。2.部分BH4缺乏症患儿确诊前经低Phe饮食治疗后血Phe可以控制在较低水平,但仍出现进行性神经损害,脑电图异常率为72.7%。3.BH4反应性PAH缺乏症的发育商明显高于其他HPA;饮食治疗中的Phe耐受量较高。结论BH4反应性PAH缺乏症患者对饮食治疗的反应性更好。早期BH4缺乏症的鉴别诊断非常重要。     

四氢生物蝶呤反应性苯酮尿症的研究进展

四氢生物蝶呤(BH4)反应性苯酮尿症是苯丙氨酸羟化酶(PAH)基因突变导致PAH活性降低,且患者服用BH4后24h内血苯丙氨酸(Phe)浓度较服药前下降30%或以上的一种特殊类型。该文探讨了BH4反应性PAH缺乏症的基因型及其与表型的关系、发生机制、诊断和治疗研究进展。BH4可部分或完全替代低苯丙氨酸饮食治疗,为苯酮尿症个体化治疗提供了新途径。     

四氢生物蝶呤反应性苯丙氨酸羟化酶缺乏症的临床与基因研究

目的 探讨四氢生物蝶呤（tetrahydrobiopterin，BH4）反应性苯丙氨酸羟化酶（phenylalanine hydroxylase，PAH）缺乏症临床表型和基因型的关系。方法 38例高苯丙氨酸血症（hyperphenylalaninemia，HPA）患儿均进行口服BH。负荷试验（20ms／kg）或Phe-BH。联合负荷试验，同时进行尿蝶呤谱分析、红细胞二氢蝶啶还原酶（dihyaropteridine reductase，DHPR）测定。对7例BH4反应性PAH缺乏症患儿采用聚合酶链反应（PCR）和单链构象多态性（single strand conformation polymorphism，SSCP）分析对PAH外显子进行突变筛检，并结合DNA直接测序方法进行突变分析。结果 确诊10例BH4反应性PAH缺乏症患儿，男6例，女4例；平均年龄7．8个月；生化代谢表型均为轻度或中度HPA。7例BH4反应性PAH缺乏症患儿PAH基因型分别为S70del／-、R241C／R243Q、S70del／A389G、Y166X／-、R11lX/-、EX6-96A〉G／R241C和IVS4-1G〉A／R241C。A389G是新发现的突变基因型。结论 BH4反应性PAH缺乏症多表现为轻、中度HPA生化代谢表型，R241C是BH4反应性相关突变基因型中较常见的一种类型。推测S70del可能是一种BH4反应性相关突变类型.     

四氢生物蝶呤反应性苯丙氨酸羟化酶缺乏症

目的：四氢生物蝶呤(BH4)可以使BH4缺乏症病人的血液苯丙氨酸水平正常化,但是对苯丙酮酸尿症(PKU)病人无效。最近在新生儿PKU筛查中发现了对BH4有反应的轻度PKU患者。本研究将探讨BH4和苯丙氨酸羟化酶(PAH)基因突变在对BH4有反应的轻度PKU和轻度高苯丙酸血症(HPA)患者中的作用。方法：对经新生儿PKU筛查中发现的生物蝶呤代谢正常的轻度HPA患者,进行单次(10mg/kg)、4次、1周[20 mg/(kg·d)]的BH4口服负荷试验及长期BH4治疗,评估其对BH4口服负荷试验的反应性。结果：在单剂量BH4口服负荷试验中,典型PKU患者的血苯丙氨酸水平没有降低。在单剂量BH4口服负荷试验中血苯丙氨酸水平下降超过20%的患者,在4次BH4口服负荷试验中下降亦超过20%。1周BH4负荷试验确认在单剂量和4次BH4负荷试验中表现出弱反应性的病人对BH4有反应。许多患轻度PKU和轻度HPA且有R241C基因位点的病人,都对BH4治疗有反应。在无BH4反应性的典型PKU病人中未发现R241C、P407S和A373T基因突变。结论：1周BH4负荷试验用于诊断BH4反应性PAH缺乏症最为有效。等位基因R241C、P407S和A373T与轻度HPA和轻度PKU病人具有H4反应性有关。BH4治疗是针对轻度HPA和轻度PKU的一种新颖、有效的药物治疗,有望代替限制苯丙氨酸饮食的方法。     

二氢蝶啶还原酶缺乏症一例的诊治及基因突变分析

目的 探讨二氢蝶啶还原酶(DHPR)缺乏症的临床诊治和基因突变分析.方法 (1)归纳、总结临床症状、体征,血苯丙氨酸(Phe)浓度;(2)进行Phe(100 mg/kg)+四氢生物蝶呤(BH4)(20 mg/kg)负荷试验、尿蝶呤谱分析及红细胞DHPR测定;(3)进行DHPR基因QDPR突变检测;(4)采用BH4、神经递质前质等治疗,随访疗效.结果 (1)患儿男,生后5个月出现抬头困难、头发黄、抽 搐、肌张力低下.生后1岁6个月Phe 600 μmol/L;(2)负荷试验前血Phe浓度476 μmol/L,Phe负荷3 h上升至1355 μmol/L,BH4负荷24 h血Phe浓度缓慢降至610 μmol/L;(3)尿新蝶呤2.92 mmol/mol肌酐,生物蝶呤7.44 mmol/mol肌酐,生物蝶呤百分比71.79％;(4)DHPR活性(0.27～0.51)nmol/(min·5 mm disc),为正常对照的6.11％～10.60％,诊断为DHPR缺乏症;(5)患儿QDPR基因突变类型为c.515C＞T及c.661C＞T,c.515C＞T突变未见报道;(6)患儿普食下接受BH4(10～20)ms/(kg·d)或联合少量无Phe奶粉、神经递质前质L-DOPD(3～5)ms/(kg·d)(联合carbidopa)、5羟色氨酸(3～5)ms/(kg·d)及叶酸15 mg/d治疗.治疗6个月症状明显改善,抽搐停止、会扶走,血Phe 60 μmol/L.结论 (1)DHPR缺乏症具有肌张力低下等BH4缺乏症共同特点;(2)BH4负荷后血 Phe浓度下降缓慢;尿生物蝶呤增高,DHPR活性低下是确诊依据;(3)c.515C＞T(P172L)是QDPR基因新的致病突变;(4)神经递质前质、叶酸及大剂量BH4(或联合无Phe奶粉)治疗疗效显著.     

二氢蝶啶还原酶缺乏症1例报告

正二氢蝶啶还原酶(DHPR)缺乏是四氢生物蝶呤(BH4)缺乏症病因之一。血DHPR活性明显降低是DHPR缺乏症的生化诊断依据,基因突变分析从分子生物学角度证实了临床诊断的准确性。现总结经基因检测确诊的1例二氢蝶啶还原酶缺乏症,报告如下。1病例资料患儿男,2014-06-26就诊于西北妇女儿童医院。生后21 d因遗传代谢病筛查发现血苯丙氨酸(phenylalanine,Phe)升高而就诊。患儿系第1     

四氢生物蝶呤反应性苯酮尿症的研究进展

四氢生物蝶呤（BH4）反应性苯酮尿症是苯丙氨酸羟化酶（PAH）基因突变导致PAH活性降低，且患者服用BH4后24h内血苯丙氨酸（Vhe）浓度较服药前下降30％或以上的一种特殊类型。该文探讨了BH4反应性PAH缺乏症的基因型及其与表型的关系、发生机制、诊断和治疗研究进展。BH4可部分或完全替代低苯丙氨酸饮食治疗，为苯酮尿症个体化治疗提供了新途径。     

多巴反应性肌张力障碍一家系四氢生物蝶呤代谢与基因研究

目的　探讨多巴反应性肌张力障碍 (DRD)其四氢生物蝶呤 (BH4)代谢及基因突变与临床表型关系。方法　对DRD的一家系 4人进行苯丙氨酸 (Phe)和BH4负荷试验、尿蝶呤谱分析 ,对所有成员进行三磷酸鸟苷环化水解酶 1基因 (GCH1)检测。结果　 3例DRD患儿及母亲平均血Phe浓度、Phe与酪氨酸比值 (Phe/Tyr)在Phe负荷试验 3～ 4h明显高于正常对照组 ,负荷 2h尿生物蝶呤水平上升低于对照组 ;3例患儿服用BH4后上述结果恢复正常。除父亲未检测到基因突变外 ,所有成员GCH1突变类型为IVS5 +3insT。 2例有症状者小剂量左旋多巴 (5 0～ 6 0mg/d)治疗有效。结论　DRD者BH4代谢有不同程度异常 ,临床表型差异较大 ,对原因不明的肌张力障碍者可做DRD的筛查。     

769例高苯丙氨酸血症诊治和基因研究

目的　对 76 9例高苯丙氨酸血症 (HPA)患者进行诊治随访和基因检测。方法　对 76 9例 (新生儿筛查获诊 95例 ,高危筛查获诊 5 0例 ,非筛查获诊 6 2 4例 )患者进行家族史调查 ;采用Guthrie细菌抑制法或定量荧光检测法进行血苯丙氨酸浓度测定 ;采用尿蝶呤分析进行四氢生物蝶呤缺乏症(BH4D)鉴别诊断 ;采用低或无苯丙氨酸奶粉对苯丙酮尿症 (PKU)患者进行治疗 ;评价各型HPA临床疗效 ;应用变性梯度凝胶电泳 (DGGE)及微卫星连锁分析等方法 ,对 10 0例经典型PKU患者进行PKU致病基因突变检测 ;对 2 3个PKU高危家庭进行产前诊断。结果　 (1) 76 9例中 730例为经典型PKU ,2 7例轻型HPA ,12例BH4D。 (2 ) 338例接受了低 /无苯丙氨酸奶粉治疗 ,治疗期间血苯丙氨酸浓度控制达良好和一般的 ,95例新生儿筛查获诊者中占 71 6 % ,非筛查获诊的 2 4 3例中占 5 4 7%。 (3)对治疗者中 112例进行智能测定 ,5 5例经筛查获诊者的智商 (IQ)为 (78± 2 2 )分 ,5 7例非筛查获诊者的IQ为 (6 2± 18)分 ,两组IQ差异有显著意义 (P <0 0 0 1) ;5例接受治疗的BH4D者IQ 70～ 80分。 (4) 72例正常入学者 ,6 6 7%患者学习成绩达中等或以上。 (5 )在 10 0例经典型PKU中发现 5种基因突变(Arg111Ter、Arg2 4 1Cys、Arg2 4 3Glu、IVS6nt     

Evaluation of neonatal BH4 loading test in neonates screened for hyperphenylalaninemia

Background

The outcome in phenylketonuria is related to the early diagnosis and management due to neonatal screening.

Aims

To assess the interest of tetrahydrobiopterin (BH4) loading test and phenylalanine hydroxylase (PAH) genotyping in the management of neonates with hyperphenylalaninemia (HPA).

Study design

We evaluate the effectiveness of a BH4 loading test (20 mg/kg) in ten neonates screened for HPA. We evaluated the time required to reach a target plasma Phenylalanine (Phe) level below 300 µmol/l. We compared these ten BH4-loaded patients to the 10 previous neonates non-loaded with BH4. In all these patients, the PAH genotype was determined.

Results

One loaded patient had biopterin synthesis deficiency and has been retrieved from statistical analysis. All others patients have PAH deficiency. Between the BH4 loaded group (L) and the BH4 non-loaded group (NL), a statistically significant difference was observed in the average time required to reached the target Phe level (13.56 ± 4.30 (L) vs. 20.6 ± 7.59 days (NL) [p < 0.02]). Results of the genotyping from all but one of these 19 patients indicated that among all mutations present in this patient population, there were 4 known PAH mutations associated with BH4 responsiveness (p.R261Q, the p.V388 M, the p.E390G and the p.Y414C). These mutations were found in 4 non-loaded and 6 loaded patients. Two patients had a more than 90% reduction in their plasma Phe level within 24 h after the load. One of these patients had a PTPS deficiency. The other fully responsive patient (p.Y414C and IVS10-11G > A) has been treated with BH4 from birth with an excellent metabolic control for three years now.

Conclusion

BH4 loading test improves the management of HPA. It allows an immediate identification of the children fully responsive to BH4. Our results therefore suggest the incorporation of BH4 loading test in the management of neonates screened for HPA.     

In vivo studies of phenylalanine hydroxylase by phenylalanine breath test: diagnosis of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

Tetrahydrobiopterin (BH4)-responsive phenylalanine hydroxylase (PAH) deficiency is characterized by reduction of blood phenylalanine level after a BH4-loading test. Most cases of BH4-responsive PAH deficiency include mild phenylketonuria (PKU) or mild hyperphenylalaninemia (HPA), but not all patients with mild PKU respond to BH4. We performed the phenylalanine breath test as reliable method to determine the BH4 responsiveness. Phenylalanine breath test quantitatively measures the conversion of L-[1-13C] phenylalanine to 13CO2 and is a noninvasive and rapid test. Twenty Japanese patients with HPA were examined with a dose of 10 mg/kg of 13C-phenylalanine with or without a dose of 10 mg . kg(-1) . d(-1) of BH4 for 3 d. The phenylalanine breath test [cumulative recovery rate (CRR)] could distinguish control subjects (15.4 +/- 1.5%); heterozygotes (10.3 +/- 1.0%); and mild HPA (2.74%), mild PKU (1.13 +/- 0.14%), and classical PKU patients (0.29 +/- 0.14%). The genotypes in mild PKU cases were compound heterozygotes with mild (L52S, R241C, R408Q) and severe mutations, whereas a mild HPA case was homozygote of R241C. CRR correlated inversely with pretreatment phenylalanine levels, indicating the gene dosage effects on PKU. BH4 loading increased CRR from 1.13 +/- 0.14 to 2.95 +/- 1.14% (2.6-fold) in mild PKU and from 2.74 to 7.22% (2.6-fold) in mild HPA. A CRR of 5 to 6% reflected maintenance of appropriate serum phenylalanine level. The phenylalanine breath test is useful for the diagnosis of BH4-responsive PAH deficiency and determination of the optimal dosage of BH4 without increasing blood phenylalanine level.     

Long-term treatment and diagnosis of tetrahydrobiopterin-responsive hyperphenylalaninemia with a mutant phenylalanine hydroxylase gene

A novel therapeutic strategy for phenylketonuria (PKU) has been initiated in Japan. A total of 12 patients who met the criteria for tetrahydrobiopterin (BH(4))-responsive hyperphenylalaninemia (HPA) with a mutant phenylalanine hydroxylase (PAH) (EC 1.14.16.1) gene were recruited at 12 medical centers in Japan between June 1995 and July 2001. Therapeutic efficacy of BH(4) was evaluated in single-dose, four-dose, and 1-wk BH(4) loading tests followed by long-term BH(4) treatment, and also examined in relation to the PAH gene mutations. The endpoints were determined as the percentage decline in serum phenylalanine from initial values after single-dose (>20%), four-dose (>30%), and 1-wk BH(4) (>50%) loading tests. Patients with mild PKU exhibiting decreases in blood phenylalanine concentrations of >20% in the single-dose test also demonstrated decreases of >30% in the four-dose test. The 1-wk test elicited BH(4) responsiveness even in patients with poor responses in the shorter tests. Patients with mild HPA, many of whom carry the R241C allele, responded to BH(4) administration. No clear correlation was noted between the degree of decrease in serum phenylalanine concentrations in the single- or four-dose tests and specific PAH mutations. The 1-wk test (20 mg/kg of BH(4) per day) is the most sensitive test for the diagnosis of BH(4)-responsive PAH deficiency. Responsiveness apparently depends on mutations in the PAH gene causing mild PKU, such as R241C. BH(4) proved to be an effective therapy that may be able to replace or liberalize the phenylalanine-restricted diets for a considerable number of patients with mild PKU.     

Differential diagnosis of hyperphenylalaninaemia by a combined phenylalanine-tetrahydrobiopterin loading test

We describe a new fully reliable method for the differential diagnosis of tetrahydrobiopterin-dependent hyperphenylalaninaemia (HPA). The method comprises the combined phenylalanine (Phe) plus tetrahydrobiopterin (BH₄) oral loading test and enables the selective screening of BH₄ deficiency when pterin analysis is not available or when a clear diagnosis has not been previously made. It should be performed together with the measurement of dihydropteridine reductase (DHPR) activity in blood. The new combined loading test was performed in nine patients with primary HPA, three with classical phenylketonuria (PKU), three with DHPR deficiency, and three with 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency. Three hours after oral Phe loading (100 mg/kg body weight), synthetic BH₄ was administered orally at doses of either 7.5 or 20 mg/kg body weight. Amino acid (Phe and tyrosine) and pterin (neopterin and biopterin) metabolism and kinetics were analysed. By exploiting the decrease in serum Phe 4 and 8h after administration, a clear response was obtained with the higher BH₄ dose (20 mg/kg body weight), allowing detection of all cases of BH₄ deficiency, as well as differentiation of BH₄ synthesis from regeneration defects. Since DHPR deficient patients who were previously shown to be non-responsive to the simple BH₄ loading test gave a positive response, the combined Phe plus BH₄ loading test can be used as a more reliable tool for the differential diagnosis of HPA in these patients. Moreover, it takes advantage of being performed while patients are on a Phe-restricted diet.     

Assessment of tetrahydrobiopterin responsiveness in Turkish hyperphenylalaninemic patients

Tetrahydrobiopterin (BH4) therapy is the latest alternative approach in phenylalanine hydroxylase (PAH) deficiency, and is suggested for a number of hyperphenylalaninemic (HPA) patients with certain mutations. In our unit, therapeutic efficacy of BH4 was evaluated in 20 HPA patients (4 mild HPA, 9 mild phenylketonuria-PKU, 7 moderate PKU) by a single oral dose of BH4. Overall, 60% of the patients responded (45% favorably, 15% partially). All of the mild HPA patients and 55% of mild PKU patients responded to BH4 favorably and an additional 11% of mild PKU patients responded partially. Of 7 moderate PKU patients, 2 responded partially (28%). The genotypes of the patients who responded to BH4 favorably were: DelF39/-, L48S/L48S, R261Q/- (4 patients), A300S/IVS2nt5g > c, A300S/-, E390G/E390G. The genotypes of the patients who exhibited a partial response were: L48S/L48S, R261Q/ R261Q, IVS10nt546/-. We concluded that since there are too many mutations and many patients are compound heterozygote, it is difficult to predict BH4 responsiveness based solely on genotype, especially for the mutations which show inconsistent phenotypes. The best way to identify the patients who are more likely to benefit from BH4 administration is performing BH4 loading test. Long-term BH4 loading test should be performed in classical and moderate PKU patients to confirm that they are not responsive to BH4.     

Tetrahydrobiopterin monotherapy for phenylketonuria patients with common mild mutations

The effect of tetrahydrobiopterin (BH(4)) administration was studied in three infants with BH(4) responsive phenylalanine hydroxylase (PAH) deficiency by correlating different BH(4) doses with plasma phenylalanine levels under defined protein intake.