Intra-familiar discordant PKU phenotype explained by mutation analysis in three pedigrees

Classical phenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) are two phenotypes of phenylalanine hydroxylase (PAH) deficiency with different degrees of severity. We have analyzed three families in which classical PKU, MHP and a normal phenotype occurred within each family due to the different combinations of three mutations segregating within the family. Indeed, sequence PAH analysis revealed three different alleles segregating in each family. This report suggests that when discordant phenotypes occur in a family, complete analysis of the PAH gene may be performed in order to support the diagnosis and assist in accurate genetic counseling and patient management. We further support the marked heterogeneity of hyperphenylalaninemia primarily due to allelic heterogeneity at the PAH locus.     

Mutation analysis in hyperphenylalaninemia patients from South Italy

Background

Mutations in the gene encoding phenylalanine hydroxylase (PAH, EC 1.14.16.1) are associated with various degrees of hyperphenylalaninemia (HPA), including classical phenylketonuria (PKU).

Objective

The aim of the study was to determine the mutations responsible for mild forms of HPA and to relate different clinical phenotypes of HPA patients to their PAH genotypes in order to better predict the clinical phenotype and implement optimal dietary therapy and prognosis in newborns with the disease.

Methods

Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by direct DNA sequencing in 30 HPA patients (Phe levels ranging from 2 to 6 mg/dL) from Southern Italy who were identified in a neonatal screening program and a genotype–phenotype correlation was performed.

Results

PAH gene mutation was identified in 39 out of 60 alleles with a mutation detection rate of 65%. Eighteen mutations, 2 undescribed, were observed (13 missense mutations, 1 deletion, 4 splice site mutations). Using the “in vitro” predicted residual activity, a good genotype–phenotype correlation was obtained also in a new mild HPA case, a PAH compound heterozygote, previously undetected.

Conclusion

A marked genetic heterogeneity was found in HPA patients from Southern Italy and a good genotype–phenotype correlation was obtained. Identification of PAH gene mutations responsible for PAH deficiency will therefore be useful in the prediction of biochemical and clinical phenotypes in HPA patients.     

Structural and functional analyses of mutations of the human phenylalanine hydroxylase gene

BACKGROUND: Phenylketonuria (PKU) is an inborn error of metabolism that results from a deficiency of phenylalanine hydroxylase (PAH). We demonstrated PAH mutational spectrum from patients with PKU, including 10 novel and 3 tetrahydrobiopterin (BH(4))-responsive mutations. In this study, 11 PAH missense mutations, including 6 novel mutations (P69S, G103S, L293M, G332V, S391I, A447P) found in our previous study, 2 mutations common in east Asian patients with PKU (R243Q, R413P), and 3 tetrahydrobiopterin (BH(4))-responsive mutations (R53H, R241C, R408Q) have been functionally and structurally analyzed. METHODS: A transient protein overexpression system and an in vitro BH(4)-responsiveness study were used. The effects of PAH missense mutations on the PAH protein structure were also analyzed. To determine the conservation of 12 mutated residues, PAH was aligned using BLAST against full genomic sequences of 221 different species. Model structures of PAH protein and the composite tetramer were constructed using the software program, SHEBA. RESULTS: No PAH activity was detected for some mutants. However, the residual activities associated with other mutants ranged over a wide spectrum. The missense mutations responsive to BH(4) were not highly conserved throughout the 43 species in the multiple sequence alignment that encode PAH. The composite model structure of PAH revealed that dimer stability was reduced in the BH(4)-responsive mutants, whereas tetramer stability remained normal. CONCLUSION: This expression study analyzed PAH mutations and model structures of mutant PAH proteins are proposed. Correlation between the proposed mutant PAH structures and functions are suggested.     

新疆少数民族苯丙酮尿症患者的突变基因分析

目的 探讨新疆少数民族苯丙酮尿症(PKU)患者的基因突变特征,为有针对性的防治策略提供科学依据.方法 采用单链构象多态性分析技术和PCR产物直接测序方法 ,检测12例少数民族PKU患者的苯丙氨酸羟化酶(PAH)基因突变.结果 从12例维吾尔族、回族和哈萨克族PKU患者(24个PAH等位基因)中,检出13种基因突变,包括错义突变8种、无义突变1种、剪切位点突变3种,其中突变频率最高的是EX6-96A＞G和P281L,EX6-96A＞G常见于国内和亚洲地区,P281L多见于欧洲各地.突变频率较高的R243Q、R111X、R176X和F161S 4种突变中,其中R243Q是我国北方地区居首位的突变,R111X处于第3位.而R176X和F161S在世界范围内都极为少见,尤其F161S是具有中国特色的基因突变,是第2次在中国人中发现.结论 新疆少数民族中的PAH突变基因不仅与亚洲黄种人和欧洲及拉美人种表现出密切的联系,而且也存在着明显的差异,形成了自身独立的遗传规律和特点,是我国一个十分特殊的PAH突变基因分布区域.     

Identification of exonic deletions in the PAH gene causing phenylketonuria by MLPA analysis

BACKGROUND: Multiplex ligation probe amplification (MLPA) is a sensitive and efficient technique for molecular diagnosis of diseases involving deletions or duplications of large genomic regions. In phenylketonuria (PKU), most of the mutant alleles correspond to missense mutations and large deletions have been scarcely identified. In this study, we report for the first time the use of MLPA analysis on PKU patients to detect exonic deletions. METHOD: DNA from 22 unrelated PKU patients with an incomplete genetic diagnosis after standard mutation detection analysis were subjected to MLPA analysis. Deletions were confirmed by long-range PCR and sequence analysis. RESULTS: The technique identified two large genomic deletions in the phenylalanine hydroxylase (PAH) gene, of 6.6 kb and 1.8 kb, including exons 3 and 5, respectively. The chromosomal breakpoints were established by long-range PCR and chromosomal walking, confirming the involvement of repetitive sequences in the deletions. CONCLUSION: MLPA may complement routine mutation screening in PKU patients, although, in the sample studied, exonic deletions in the PAH gene do not appear to be a frequent cause of PKU.     

Development of a skin-based metabolic sink for phenylalanine by overexpression of phenylalanine hydroxylase and GTP cyclohydrolase in primary human keratinocytes

Phenylketonuria, PKU, is caused by deficiency of phenylalanine hydroxylase (PAH) resulting in increased levels of phenylalanine in body fluids. PAH requires the non-protein cofactor BH4 and the rate-limiting step in the synthesis of BH4 is GTP cyclohydrolase I (GTP-CH). Here we show that overexpression of the two enzymes PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH4 supplementation. Integration of multiple PAH and GTP-CH transgenes were achieved after optimized retroviral transduction. Phenylalanine clearance was measured ex vivo in primary human keratinocytes cotransduced with PAH and GTP-CH (more than 370 nmol/24 h/106 cells), a level exceeding that of a human liver cell line (HepG2 cells). Cells overexpressing either one of the enzymes alone did not clear significant amounts of phenylalanine. Transfer of the two genes into the same cell was not necessary, since cocultivation of cells transduced separately with PAH and GTP-CH also resulted in phenylalanine clearance. Thus the experiments indicate metabolic cooperation between cells overexpressing PAH and cells overexpressing GTP-CH, possibly due to intercellular transport of synthesized BH4.     

Tetrahydrobiopterin responsiveness in patients with phenylketonuria

OBJECTIVES: To investigate the BH4 response in a group of patients with phenylketonuria (PKU) in order to offer this alternative treatment to the responsive patients. DESIGN AND METHODS: The 24-h-long Phe/BH4 loading test was performed on 64 PKU patients requiring dietary treatment. RESULTS: All patients with mild-PKU and 75% of patients with moderate-PKU were BH4 responsive, while only 11% of classic-PKU patients showed good/partial response (P < 0.0001). The percentages of Phe decrease after the BH4 loading test were significantly different in the three PKU phenotypes (mild PKU: 67.9 +/- 18.7; moderate PKU: 37.4 +/- 16.8; and classical PKU: 21.9 +/- 13.7; ANOVA with Bonferroni correction: P < 0.0001). We report four mutations (P147S, D222G, P275S, and P362T) not previously associated with BH4 responsiveness, all of them combined with mutations with zero predicted residual activity. CONCLUSION: Both the percentage of Phe decrease and the Phe value achieved 24 h after BH4 loading are valuable data in predicting a response. We report four mutations not previously associated with BH4 responsiveness.     

苯丙酮尿症患儿苯丙氨酸羟化酶exon6基因、exon7基因突变的研究

目的 探讨山西省苯丙酮尿症(PKU)患儿的苯丙氨酸羟化酶(PAH)exon6 基因、exon7 基因的突变特征.方法 通过测序及序列比对的方法对56 例PKU 患儿和112 例健康儿童的336 个PAH exon6 基因、exon7 基因进行序列分析,以确定其突变位点、性质和频率.结果 通过序列分析,发现在PKU 患儿和健康儿童中均高频率的出现Q232Q(CAA→CAG)、V245V(GTG→GTA)两种同义突变,其中cDNA 696 位点的频率高达96.2%,cDNA 735 位点的频率高达76.1%.健康儿童的其他序列与Genbank 完全相同.而PKU 患儿的基因序列中还发现了9 种共计37 个突变基因,占全部PAH 突变基因的33.04%.exon 6 仅发现一种突变基因Y204C,突变频率达9.8%;exon 7 中R243Q 的突变率最高,占10.7%,其次为Ivs7 +2 T ＞A,占5.4%,其余的G247V、R252Q、L255S、R261Q、T278I 和E280K 分别占0.9%、0.9%、0.9%、0.9%、2.7%和0.9%.9 种突变都在之前文献中有过报道,其中有7 种错义突变和2 种剪接位点突变.结论 明确了PAH exon6 基因、exon7 基因的突变种类和分布等特征,表明exon6 基因、exon7 基因中Y204C 和R243Q属于山西人群中PAH 基因突变的热点.     

Carcinogenic Effects in a Phenylketonuria Mouse Model

Phenylketonuria (PKU) is a metabolic disorder caused by impaired phenylalanine hydroxylase (PAH). This condition results in hyperphenylalaninemia and elevated levels of abnormal phenylalanine metabolites, among which is phenylacetic acid/phenylacetate (PA). In recent years, PA and its analogs were found to have anticancer activity against a variety of malignancies suggesting the possibility that PKU may offer protection against cancer through chronically elevated levels of PA. We tested this hypothesis in a genetic mouse model of PKU (PAH^enu2) which has a biochemical profile that closely resembles that of human PKU. Plasma levels of phenylalanine in homozygous (HMZ) PAH^enu2 mice were >12-fold those of heterozygous (HTZ) littermates while tyrosine levels were reduced. Phenylketones, including PA, were also markedly elevated to the range seen in the human disease. Mice were subjected to 7,12 dimethylbenz[a]anthracene (DMBA) carcinogenesis, a model which is sensitive to the anticancer effects of the PA derivative 4-chlorophenylacetate (4-CPA). Tumor induction by DMBA was not significantly different between the HTZ and HMZ mice, either in total tumor development or in the type of cancers that arose. HMZ mice were then treated with 4-CPA as positive controls for the anticancer effects of PA and to evaluate its possible effects on phenylalanine metabolism in PKU mice. 4-CPA had no effect on the plasma concentrations of phenylalanine, phenylketones, or tyrosine. Surprisingly, the HMZ mice treated with 4-CPA developed an unexplained neuromuscular syndrome which precluded its use in these animals as an anticancer agent. Together, these studies support the use of PAH^enu2 mice as a model for studying human PKU. Chronically elevated levels of PA in the PAH^enu2 mice were not protective against cancer.     

Metabolic basis of sexual dimorphism in PKU mice after genome-targeted PAH gene therapy.

We have previously reported a transgene delivery system based on phiBT1 bacteriophage integrase that results in targeted insertion of transgenes into mammalian genomes, and its use in the delivery of murine phenylalanine hydroxylase (PAH) complementary DNA (cDNA) into the hepatocytes of male phenylketonuria (PKU) mice, leading to a complete and permanent correction of their hyperphenylalaninemic phenotype. In this study, we report only partial phenotypic correction in female PKU mice, even though hepatic PAH activities in both sexes after gene treatment were similar. Daily injections of tetrahydrobiopterin (BH4), an essential co-factor for phenylalanine hydroxylation, in the gene-treated females led to complete correction of their PKU phenotype. After gonadectomy, serum phenylalanine levels in the gene-treated females were reduced to normal, whereas those in the gene-treated males remained unchanged. The sterile gene-treated PKU mice were subjected to daily sex hormone injections. Whereas the estradiol-treated sterile males developed hyperphenylalaninemia, the dihydrotestosterone-treated sterile females remained normal phenylalaninemic. The results indicate that it is estrogen that suppresses the steady-state levels of BH4 in mouse hepatocytes that became limiting, which is the underlying mechanism for the observed sexual dimorphism in PKU mice after PAH gene treatment. Livers of the PAH gene-corrected PKU mice also appeared normal and without apparent pathologies.     

BH4 responsiveness associated to a PKU mutation with decreased binding affinity for the cofactor

BACKGROUND: Tetrahydrobiopterin (BH4), cofactor of phenylalanine hydroxylase, can be used to treat a subset of phenylketonuria (PKU) patients as it results in a reduction in blood phenylalanine levels. The molecular basis of the response appears to be multifactorial. METHOD: A standard BH4 loading test (20 mg/kg) was performed. Genotyping was performed by DGGE and sequencing analysis. Expression analysis of the D129G mutation was performed in E. coli (expression as fusion protein MBP-PAH) and in a human hepatoma cell line with an N-terminal FLAG epitope. RESULTS: We report the positive response and long-term treatment of a patient functionally hemizygous for the D129G mutation in the phenylalanine hydroxylase gene. Expression in the prokaryotic system revealed partial activity and a decreased binding affinity for BH4 of the mutant protein. In the eukaryotic system the mutant protein shows reduced stability. CONCLUSION: The D129G mutation which confers a BH4-responsive phenotype, has a decreased binding affinity for BH4.     

Low therapeutic threshold for hepatocyte replacement in murine phenylketonuria.

Phenylalanine homeostasis in mammals is primarily controlled by liver phenylalanine hydroxylase (PAH) activity. Inherited PAH deficiency (phenylketonuria or PKU) leads to hyperphenylalaninemia in both mice and humans. A low level of residual liver PAH activity ensures near-normal dietary protein tolerance with normal serum phenylalanine level, but the precise threshold for normal phenylalanine clearance is unknown. We employed hepatocyte transplantation under selective growth conditions to investigate the minimal number of PAH-expressing hepatocytes necessary to prevent hyperphenylalaninemia in mice. Serum phenylalanine levels remained normal in mice exhibiting nearly complete liver repopulation with PAH-deficient hepatocytes (<5% residual wild-type liver PAH activity). Conversely, transplantation of PAH-positive hepatocytes into PAH-deficient Pah(enu2) mice, a model of human PKU, yielded a significant decrease in serum phenylalanine (<700 muM) when liver repopulation exceeded approximately 5%. These data suggest that restoration of phenylalanine homeostasis requires PAH activity in only a minority of hepatocytes.     

Trends in enzyme therapy for phenylketonuria.

Phenylketonuria (PKU) is an inborn error of amino acid metabolism caused by phenylalanine hydroxylase (PAH) deficiency. Dietary treatment has been the cornerstone for controlling systemic phenylalanine (Phe) levels in PKU for the past 4 decades. Over the years, it has become clear that blood Phe concentration needs to be controlled for the life of the patient, a difficult task taking into consideration that the diet becomes very difficult to maintain. Therefore alternative models of therapy are being pursued. This review describes the progress made in enzyme replacement therapy for PKU. Two modalities are discussed, PAH and phenylalanine ammonia-lyase PAH. Developing stable and functional forms of both enzymes has proven difficult, but recent success in producing polyethylene glycol-modified forms of active and stable PAH shows promise.     

Long-term correction of hyperphenylalaninemia by AAV-mediated gene transfer leads to behavioral recovery in phenylketonuria mice

Classical phenylketonuria (PKU) is a metabolic disorder caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). If untreated, accumulation of phenylalanine will damage the developing brain of affected individuals, leading to severe mental retardation. Here, we show that a liver-directed PAH gene transfer brought about long-term correction of hyperphenylalaninemia and behavioral improvement in a mouse model of PKU. A recombinant adeno-associated virus (AAV) vector carrying the murine PAH cDNA was constructed and administered to PAH-deficient mice (strain PAH(enu2)) via the portal vein. Within 2 weeks of treatment, the hyperphenylalaninemic phenotype improved and completely normalized in the animals treated with higher vector doses. The therapeutic effect persisted for 40 weeks in male mice, while serum phenylalanine concentrations in female animals gradually returned to pretreatment levels. Notably, this long-term correction of hyperphenylalaninemia was associated with a reversal of hypoactivity observed in PAH(enu2) mice. While locomotory activity over 24 h and exploratory behavior were significantly decreased in untreated PAH(enu2) mice compared with the age-matched controls, these indices were completely normalized in 12-month-old male PKU mice with lowered serum phenylalanine. These results demonstrate that AAV-mediated liver transduction ameliorated the PKU phenotype, including central nervous system dysfunctions.     

石家庄市苯丙氨酸羟化酶缺乏症的基因突变研究

目的 分析石家庄市新生儿疾病筛查诊治中心确诊的苯丙氨酸羟化酶缺乏症(PAHD)患儿中PAH基因的突变规律及特点,为石家庄市PAHD的产前诊断、治疗提供有力的科学依据.方法 收集在石家庄市新生儿疾病筛查诊治中心确诊的PAHD患儿67例作为研究对象,提取患儿及其父母外周血DNA标本进行二代测序,测序范围包括13个外显子和外...     

高分辨熔解曲线检测苯丙氨酸羟化酶基因突变的临床价值

目的 研究用HRM分析技术检测经典型PKU患者PAH基因突变的临床应用价值。方法采用HRM分析技术对17例经典型PKU患者PAH基因的13个外显子及外显子-内含子交界区域进行检测,并采用DNA测序验证HRM检测结果。同时评价HRM诊断PKU的敏感度和特异度。另外,对其中2个家系的风险胎儿进行产前诊断。结果采用HRM分析技术从17例经典型PKU患者中检出有2个致病突变的患者16例,检出有3个致病突变患者l例。同时检出194V(c.280A＞G)、IVS4nt-1G＞A(c.442-1G＞A)、R158Q(c.473G＞ A)、Q160X(c.478C＞ T)、W187X(c.561G＞ A)、E6nt-96A＞ G(c.611A＞ G)、G239D(c.716G＞ A)、R241C(c.721C＞ T)、R243Q(c.728G＞ A)、G247R (c.739C-＞ C)、G247V(c.740G＞ T)、R261X(c.781C ＞T)、R261Q(c.782G＞ A)、H264R(c.791A ＞G)、F302 fsX39(c.904delT)、E305K(c.913G＞ A)、G312V(c.935G＞ T)、Y356X(c.1068C＞A)、V399V (c.1197A ＞T)、R408Q(c.1223G＞ A)、T418P(c.1252A ＞C)、A434D(c.1301C＞ A) 22种致病突变;Q232Q(c.696G＞A)、V245V(c.735G＞ A)、L385L(c.1155C＞G)3种沉默突变;1种单核苷酸多态点rs2280615(c.402A＞ C)。其中194V(c.280A＞ G)、Q160X(c.478C＞ T)、H264R(c.791A ＞G)、G312V (c.935G＞T)和E305K(c.913G＞ A)为PAH基因新发现的突变。2例风险胎儿经产前诊断,1例未发现致病突变,1例诊断为携带者。HRM突变筛查经典型PKU的敏感度和特异度均为100％,其检测结果与DNA测序结果完全一致。结论HRM分析技术是一种简单、准确、快速、高通量、低成本的遗传学分析方法,可用于经典型PKU的PAH基因突变筛查,并可用于已知父母突变的经典型PKU家系快速产前诊断。     

Testing for Tetrahydrobiopterin Responsiveness in Patients with Hyperphenylalaninemia due to Phenylalanine Hydroxylase Deficiency

Introduction

Pharmacological levels of the phenylalanine hydroxylase enzyme cofactor, tetrahydrobiopterin (BH4), reduce plasma phenylalanine levels in some patients with phenylketonuria (PKU), providing the first pharmacological therapy for PKU. Responsiveness to this therapy must be determined empirically through a BH4 loading test or trial. The authors have analyzed the loading tests currently in use in light of the numerous factors that can influence their results. Sapropterin dihydrochloride is a stable, synthetic form of BH4 approved for treatment of PKU in responsive patients.

Methods

An expert panel identified evidence from published reports of clinical experience. Reports of research involving at least 25 patients and published in English were considered.

Results

In all, 14 studies met both criteria; eight employing the sapropterin dihydrochloride preparation from Schircks Laboratories and six the sapropterin dihydrochloride preparation from Biomarin/Merck Serono.

Conclusion

The arbitrary responsiveness definition of a >30% reduction in blood phenylalanine appears to be a good compromise between sensitivity and specificity for the initial screening test. However, individual patient characteristics should be considered when interpreting results, especially in patients with low baseline phenylalanine levels.     

苯丙氨酸羟化酶缺乏症家系PAH基因新发突变分析

目的 对一个苯丙氨酸羟化酶缺乏症(phenylalanine hydroxylase deficiency,PAHD)家系苯丙氨酸羟化酶(phenylalanine hydroxylase,PAH)基因进行突变位点检测和分析,探讨此家系发病原因。方法 采用二代测序技术(next generation sequencing,NGS)和多重连接探针扩增技术(multiplex ligation-dependent probe amplification,MLPA)对先证者及其父母的静脉血进行PAH基因组测序和外显子缺失、重复分析。结果 先证者找到1个错义突变和1个剪接缺失,分别为:第6外显子c.630T>G和第2外显子c.61-1G>A,这两个变异在人类基因突变数据库(Human Gene Mutation Database,HGMD)中未见报道,根据美国医学遗传学与基因组学学会(America College of Medical Genetics and Genomics, ACMG)指南判定为临床意义未明和疑似致病性变异;信息软件REVEL预测结果为有害和未知。采用MLPA对先证者进行外显子缺失、重复分析显示PAH基因外显子拷贝数未发现异常。结论 PAH基因6号外显子c.630T>G和2号外显子c.61-1G>A可能是该PAHD家系的致病突变。     

Development of pegylated forms of recombinant Rhodosporidium toruloides phenylalanine ammonia-lyase for the treatment of classical phenylketonuria.

Phenylketonuria (PKU) is a metabolic disorder due primarily to mutations in the PAH gene that impair both phenylalanine hydroxylase activity and disposal of l-phenylalanine from the normal diet. Excess phenylalanine is toxic to cognitive development and a low-phenylalanine diet prevents mental retardation, but it is a difficult therapeutic option. Previous studies with recombinant phenylalanine ammonia-lyase, PAL, demonstrated pharmacologic and physiologic proofs of principle for PAL as an alternative therapy for PKU but its immunogenicity was problematic. From a series of formulations of linear and branched polyethylene glycols chemically conjugated to PAL, we have created a parenteral therapeutic agent for PKU treatment. All the pegylated molecules were fully characterized in vitro and the most promising formulations were then tested in vivo in the PKU mouse model. The linear 20-kDa PEG-PAL combination abolished in vivo immunogenicity after repeated challenge while retaining full catabolic activity against phenylalanine, suggesting potential as a novel PKU therapeutic.