Atypical cases of phenylketonuria

Conclusion Neonatal HPA can be caused by deficiency of PAH or of its cofactor. At present, conventional methods are not able to delineate the molecular basis of the mutations in PKU patients. DNA analysis might in future visualize the different genotypes, but might not solve the problem of therapeutic decision.All infants with HPA should be screened for THB deficiency. Diagnostic tools are now available for the recognition of these variants among hyperphenylalaninemic infants. The most important question-which infants can achieve normal development if treated early-remains tobe answered. Efforts have to be directed toward better characterization of individual residual capacity to synthesize THB and toward and definition of protocols for the follow-up of neurotransmitter replacement therapy.Abbreviations PKU phenylketonuria - HPA hyperphenylalaninemia - PAH phenylalanine hydroxylase - DHPR dihydropteridine reductase - BS biopterin synthetase - THB tetrahydrobiopterin - GTP guanosine triphosphate - GTP-ch GTP-cyclohydrolase - N/B ratio neopterin/biopterin ratio     

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。     

Phenylalanine hydroxylase expression in liver of a fetus with phenylketonuria

The expression and activity of phenylalanine hydroxylase was studied in the liver of a fetus aborted after prenatal diagnosis of phenylketonuria. No phenylalanine hydroxylase enzymatic activity or immunoreactive protein was detectable in the PKU liver specimen, though both enzymatic activity and immunoreactive protein were detectable in control specimens of similar gestational age. Phenylalanine hydroxylase messenger RNA of normal size was present in the PKU fetal liver at normal abundance. These results confirm the genetic diagnosis of PKU in this fetus and indicate that the mutations in this fetus affect translation or stability of the phenylalanine hydroxylase protein.     

PKU and NON-PKU Hyperphenylalaninemia: Differentiation, Indication for Therapy and Therapeutic Results

The differential diagnosis of phenylalanine hydroxylase deficiency (PAHD) by biochemical methods is difficult. Using standardized oral protein loading or the intravenous deuterated phenylalanine (phe) load in 46 patients with PAHD, three groups could be distinguished: 1) Phenylketonuria (PKU) with plasma phe levels over 20 mg% under the protein load and with residual activities of less than 1% of normal; 2) mild PKU with plasma phe of 10–20 mg% and residual enzyme activities of 1–3%; 3) Non-PKU hyperphenylalaninemia with plasma phe levels of less than 10 mg% and residual enzyme activities of more than 3%. Psychomotor development in 32 untreated patients with PAHD showed that there is a high risk of brain damage for all patients with in vivo residual activities of less than 3% of normal. Restriction fragment length polymorphism (RFLP) haplotypes at the phenylalanine hydroxylase (PAH) locus in 60 German patients with PAHD showed that 90% of the mutant alleles are confined to four distinct haplotypes. Using an oligonucleotide probe for the splicing mutation associated with mutant haplotype 3 a close association between the mutation and the haplotype 3 has been observed. There is also an association between haplotypes 2 and 3 and PKU patients with residual enzyme activities of less than 1%. However, in only 37% of our patients with PAHD could a direct diagnosis of the mutations in the PAH gene be made. More knowledge of other mutations in the PAH gene is necessary to differentiate patients with PAHD on the DNA level.     

Malignant hyperphenylalaninemia--clinical features, biochemical findings, and experience with administration of biopterins

Four cases of malignant hyperphenylalaninemia (MHPA) are described. Pretreatment serum phenylalanine levels were 1.5, 3.0, 2.4, and 0.9 mmoles/l. Dihydropteridine reductase (DHPR) deficiency was proven in one patient by assays on cultured fibroblastic cells and was presumed in her sibling and in another deceased patient whose parents' fibroblastic cells show approximately 50% of normal enzyme activity. DHPR and phenylalanine hydroxylase deficiency were excluded by assays on liver obtained at autopsy in the 4th patient. Parenteral administration of tetrahydrobiopterin (BH4) corrected the hyperphenylalaninemia and increased the levels of catecholamines and 5-hydroxy-indoles in the one patient studied in life, but BH4 did not reach the cerebrospinal fluid. A 3-wk course of BH4 therapy had no clinical effect. Oral biopterin was absorbed and excreted in the urine, but did not alter the serum phenylalanine level. The frequency of MHPA in Australia was estimated as 7 in 258 patients with phenylketonuria.     

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。     

The PKU mouse project: its history, potential and implications

To produce genetic-based animal models for the human disease phenylketonuria (PKU), we treated mice with the powerful germline mutagen ethylnitrosourea and screened the progeny of these animals for the symptom hyperphenylalaninemia (HPH). Six independent mutant strains have been produced to date that exhibit heritable HPH. The first mutation isolated was found to cause a reduced level of GTP-cyclohydrolase I activity and, as such, yields a model for tetrahydrobiopterin-dependent HPH. The next two mutations have yet to be fully characterized but cause syndromes that appear distinct from any PKU or HPH syndromes yet reported for humans and they are allelic. Next we isolated a mutation that caused a marked reduction in hepatic phenylalanine hydroxylase activity levels. The enzyme deficiency was not sufficient to cause a PKU syndrome but instead produced a mild HPH syndrome. This strain played an instrumental role, however, in the identification of two additional mutant strains that appear to model human PKU very accurately in the laboratory mouse. These latter strains have levels of HPH very similar to human PKU patients, exhibit a phenylalanine-dependent hypopigmentation, and have reproductive difficulties that resemble human maternal PKU.     

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目的探讨四氢生物蝶呤反应性苯丙氨酸羟化酶(PAH)缺乏症这一新的临床变异型在高苯丙氨酸血症(HPA)中的发生率,并进一步了解其PAH基因突变类型。方法2001年1月至2004年12月,上海第二医科大学附属新华医院收治的106例中位年龄2个月(0.5～59个月)的HPA患儿纳入研究。所有患儿做辅酶的羟生物蝶呤(BH4,20mg/kg)或联合血苯丙氨酸(Phe,100mg/kg)负荷试验、尿蝶呤谱分析及红细胞二氢蝶啶还原酶活性(DHPR)测定。以口服BH4后24h内血Phe浓度下降30%以上且排除BH4缺乏症为诊断标准。对13例BH4反应性PAH缺乏症患儿进行PAH基因突变检测。结果106例HPA者中41例(38.7%)为BH4反应性的PAH缺乏症。血Phe浓度在服用BH4前为(816±431)μmol/L,服BH4后24h内降至(267±198)μmol/L,反应下降率(67±19)%。41例尿蝶呤及DHPR活性均正常。106例中轻度HPA(18例)其BH4反应性PAH缺乏症发生率为61.1%(11/18)。13例做PAH基因突变检测,R241C为最多见突变类型(43.8%)。结论61.1%轻度HPA(11/18)及42.4%(28/66)轻度PKU对BH4有较大反应性。BH4负荷试验是该病有效而简便的鉴别诊断方法。     

Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency.

Serum phenylalanine concentrations decreased in 4 patients with hyperphenylalaninemia after loading with tetrahydrobiopterin. There were no abnormalities in urinary pteridine excretion or in dihydropteridine reductase activity. However, mutations were detected in the phenylalanine hydroxylase gene, suggesting a novel subtype of phenylalanine hydroxylase deficiency that may respond to treatment with cofactor supplementation.     

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.     

Relation between genotype and phenotype in Swedish phenylketonuria and hyperphenylalaninemia patients

Phenylketonuria (PKU) and hyperphenylalaninemia (HPA) are caused mostly by an inherited (autosomal recessive) deficiency in hepatic phenylalanine hydroxylase (PAH) activity. More than 50 PAH mutations have ben reported. The goal of the present study was to examine the molecular basis for the clinical heterogeneity of Swedish PKU and HPA patients. Mutations were identified through allele-specific oligonucleotide hybridization or DNA sequencing on 128 of the 176 mutant alleles (73%). Three mutations (R408W, Y414C and IVS12) together accounted for 56% of all mutant alleles and ten relatively infrequent mutations were found on another 17% of all mutant alleles. Patients from 50 of the 88 families (57%) had identified mutations in both PAH genes and allowed use to compare the clinical effects of different combinations of PAH mutations. The in vitro activity of all of these mutations, including the newly identified G272X and L364, have been tested in a eukaryotic expression system. There was a strong relationship between the average in vitro PAH activity of the two mutant enzymes and both the phenylalanine tolerance and the neonatal pretreatment serum phenylalanine concentration. This confirms previous observations in Danish and German PKU patients that disease phenotype is a consequence of the nature of the mutations at the PAH locus and not significantly influenced by other loci. The sample population in the previous study did not, however, include mild HPA patients, and the observed correlation is thus restricted to severe and moderate mutant alleles. Since a comparatively high proportion of the Swedish patients were mildly affected, we have provided additional evidence that this correlation is valid throughout a continuous spectrum of clinical varieties. PAH genotyping could therefore help predict prognosis of a recently diagnosed PKU or HPA child.     

Mutations in the phenylalanine hydroxylase gene: genetic determinants for the phenotypic variability of hyperphenylalaninemia

Phenylalanine hydroxylase (PAH) deficiency is heterogeneous disease at the phenotype level. The spectrum of clinical and metabolic phenotypes spans from the potential pathogenic disease classical phenylketonuria (PKU) to the benign condition non-PKU hyperphenylalaninemia (non-PKU HPA). This review provides an introduction to the clinical variants of PAH deficiency, and summarizes our attempts to define the disease at the molecular level and to relate mutation genotype to clinical outcome. Complete genotype determination in a large number of patients with PAH-deficient hyperphenylalaninemia demonstrates that clinical heterogeneity can be explained by a multiplicity of mutations in the PAH gene. Some combinations of mutations are associated with phenylalanine levels fluctuating around the border between PKU and non-PKU HPA. However, certain mutations seem always to cause non-PKU HPA irrespective of the mutation on the second allele and can, therefore, unambiguously be designated as being associated with the non-PKU HPA phenotype. Our results suggest that mutation analysis in newborns presenting with hyperphenylalaninemia can be used for rapid and highly efficient differential diagnosis of PAH deficiency, and for predicting the severity of the disease. These possibilities may facilitate and optimize the management of hyperphenylalaninemia and thereby improve prognosis.     

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.     

Incidence of Neonatal Hyperphenylalaninemia in Fars Province,South Iran

Objective

Phenylalanine hydroxylase or its cofactor, tetrahydrobiopterin (BH₄), deficiency causes accumulation of phenylalanine in body fluids and central nervous system. Considering the fact that hyperphenylalaninemia is a preventable cause of mental retardation in infants, the objective of this study was to determine the incidence of congenital hyperphenylalaninemia in Fars province, south of Iran.

Methods

In a period of one year from November 2007 to November 2008 blood samples were withdrawn from all newborns born in Fars province for measurement of serum phenylalanine. The samples with a serum level of≥ 2 mg/dl were referred to pediatric endocrine clinic for confirmation and determination of the type of hyperphenylalaninemia by quantitive serum phenylalanine measurements by using High-Pressure liquid chromatography (HPLC) method.

Findings

Nine out of 76966 newborns had a serum phenylalanine level≥2mg/dl, of which 8 cases were confirmed by HPLC. The incidence of the disease was 1:10000. The incidence of mild hyperphenylalaninemia and phenylketonuria (PKU) among the patients was 62.5% and 37.5% respectively and the incidence of BH₄ deficiency was 1/76966.

Conclusion

These findings indicate a high incidence of hyperphenylalaninemia, in the newborns from Fars province. The high incidence makes a comprehensive screening program for management of the disease necessary.     

Gene therapy for phenylketonuria

Classical phenylketonuria (PKU) is an autosomal recessive disorder caused by a deficiency of hepatic phenylalanine hydroxylase (PAH). Three different vector systems have been developed to examine the potential of somatic gene therapy for the treatment of PKU. Recombinant retroviral vectors and DNA/protein complexes can efficiently transduce PAH-deiicient hepatocytes in vitro, but their present phenylketonuria, retrovirus application is limited by their low transduction efficiency in vivo. In contrast, infusion of a recombinant adenoviral vector expressing the human PAH cDNA into the portal circulation of PAH-deficient mice restores 10-80% of normal hepatic PAH activity and completely normalizes serum phenylalanine levels. At present, this effect is transient and re-administration has no further effect. However, this result suggests that PKU can be completely corrected by somatic gene therapy as more persistent vectors are developed.     

Electroencephalographic findings in children with phenylketonuria.

The electroencephalographic findings of 90 patients with phenylketonuria (PKU) who were observed in one clinic were critically reviewed. Seventy-three percent of patients with classical PKU that was diagnosed and treated early (group 1) were found to have normal EEGs, 23% had mild background abnormalities, and 4% had paroxysmal discharges. Only 31% of the patients in whom PKU was diagnosed after 6 months of age (group 2) had normal EEGs, while 24% had background abnormalities and 45% had paroxysmal discharges. Of the patients with atypical PKU (group 3), 62% had normal EEGs and 38% showed background abnormalities. No major changes in EEG patterns were noted with initiation or termination of the low phenylalanine diet. There was no correlation between the degree of dietary control, EEG findings, and intellectual performance in group 1.     

Absent phenylalanine hydroxylase activity without phenylketonuria

A male infant is described who never manifested phenylketonuria even though phenylalanine hydroxylase activity was undetectable in liver tissue. Plasma phenylalanine were elevated in the range typical of PKU patients when the baby was at breast and declined with institution of a low phenylalanine diet. Physical and psychomotor development were normal with the baby on the latter treatment. The results indicate that the absence of phenylketonuria does not rule out phenylalanine hydroxylase activity.Supported by grants CRC RR 00246 from the National Institute of Health and 1-638 from the National Foundation March of Dimes. Dr. Yudkoff is a recipient of grant 140-1977 from the Daland Fund of the American Philosophical Society     

Transient Neonatal Hyperphenylalaninemia due to Immature Development of 7, 8-Dihydrobiopterin Synthesis

We describe a case with transient neonatal hyperphenylalaninemia, possibly due to immature biosynthesis of 7, 8-dihydrobiopterin (BH₂). In the neonatal period his blood phenylalanine levels were high and the patterns of urinary pterins were similar to those found in defective synthesis of BH₂, which results in a type of hyperphenylalaninemia. But the laboratory data became almost normal after three months. Transient hyperphenylalaninemia has generally been thought to be caused by the delayed maturation of phenylalanine hydroxylase. But it is suggested that some cases with transient neonatal hyperphenylalaninemia are due to immaturity of BH₂ cofactor biosynthesis rather than that of phenylalanine hydroxylase.     

Combined tetrahydrobiopterin-phenylalanine loading test in the detection of partially defective biopterin synthesis

Deficiency in the synthesis of biopterin causes neonatal hyperphenylalaninemia. We report a 10-year-old girl of normal appearance with a partial defect in biopterin synthesis, normal intelligence and normal serum phenylalanine levels (95 mol/l) (1.6 mg/dl). During her 1st year of life srum phenylalanine levels were 250 mol/l (4 mg/dl) and phenylalanine loading performed at 6 months and 1 year of age was not followed by an increase in serum tyrosine. At 9 years of age she had developed a severely abnormal EEG with focal spike activity but no observable clinical abnormalities. Determination of urinary pterins showed abnormal low levels of biopterin and high levels of neopterin. Phenylalanine loading combined with oral administration of tetrahydrobiopterin (BH₄) was followed by a normal increase in serum tyrosine and a normal decrease in serum phenylalanine.Considering the importance of BH₄ for the synthesis of dopamine, catecholamines, and serotonin we suggest that these cases should be followed carefully. If neurological symptoms appear, e.g., epilepsy, it may be worthwhile to consider treatment with BH₄ and neurotransmitter precursors.