Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterin-deficient hyperphenylalaninemias using high-throughput targeted sequencing

Genetic diagnostics of phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficient hyperphenylalaninemia (BH4DH) rely on methods that scan for known mutations or on laborious molecular tools that use Sanger sequencing. We have implemented a novel and much more efficient strategy based on high-throughput multiplex-targeted resequencing of four genes (PAH, GCH1, PTS, and QDPR) that, when affected by loss-of-function mutations, cause PKU and BH4DH. We have validated this approach in a cohort of 95 samples with the previously known PAH, GCH1, PTS, and QDPR mutations and one control sample. Pooled barcoded DNA libraries were enriched using a custom NimbleGen SeqCap EZ Choice array and sequenced using a HiSeq2000 sequencer. The combination of several robust bioinformatics tools allowed us to detect all known pathogenic mutations (point mutations, short insertions/deletions, and large genomic rearrangements) in the 95 samples, without detecting spurious calls in these genes in the control sample. We then used the same capture assay in a discovery cohort of 11 uncharacterized HPA patients using a MiSeq sequencer. In addition, we report the precise characterization of the breakpoints of four genomic rearrangements in PAH, including a novel deletion of 899 bp in intron 3. Our study is a proof-of-principle that high-throughput-targeted resequencing is ready to substitute classical molecular methods to perform differential genetic diagnosis of hyperphenylalaninemias, allowing the establishment of specifically tailored treatments a few days after birth.     

Genetic background of hyperphenylalaninemia in Nagasaki,Japan

Phenylketonuria (PKU) and related hyperphenylalaninemia (HPA) are caused by a deficiency in hepatic phenylalanine hydroxylase (PAH). The incidence of PKU in Nagasaki prefecture is higher than that in all parts of Japan (1/15 894 vs 1/120 000). To investigate the genetic background of patients with HPA in Nagasaki prefecture, mutation analysis was done in 14 patients with PKU or mild HPA. Homozygous or compound heterozygous PAH mutations were identified in all the patients. The spectrum of PAH mutations in the cohort was broad and similar to those in all parts of Japan and East Asian countries. R53H is the most common mutation in patients with mild HPA. The present results provide further support for genotype–phenotype correlations in patients with HPA. The high incidence of PKU in Nagasaki, the westernmost part of Japan, might be due to migration of people with PAH mutations from China and Korea, and geographic factors.     

2010-2016年陕西省新生儿疾病筛查治疗及随访结果分析

目的 总结并分析2010-2016年陕西省新生儿疾病筛查中心高苯丙氨酸血症(HPA)、先天性甲状腺功能减低症(CH)的筛查及治疗、随访情况。方法 新生儿出生72 h后采集足跟血,制成干血滤纸片,采用荧光法检测滤纸干血斑中苯丙氨酸(Phe)、促甲状腺素(TSH)的水平,Phe ≥2.0 mg/dl、TSH ≥10 μU/ml为阳性,阳性病人召回复查,采用串联质谱法检测血Phe、酪氨酸(Tyr),化学发光免疫分析技术检测甲状腺功能五项(TSH、T₃、T₄、FT₃、FT₄)而确诊。结果 2010-2016年全省助产机构活产数共2 751 268例,接受筛查2 329 335例,筛查率84.67%;确诊轻度HPA 131例,发病率1/17 781;确诊苯丙酮尿症(PKU) 300例,发病率1/7 764; 确诊CH 966例,发病率1/2 411。确诊后即建立病历档案,分别给予低苯丙氨酸饮食及口服左旋甲状腺素片(L-T₄)替代治疗。通过Gesell发育量表监测发育情况,发现6月龄内开始接受治疗的PKU患儿智商(IQ)水平明显优于6月龄后开始治疗的患儿(P<0.01),筛查确诊后即坚持治疗随访的CH 患儿,其IQ与正常同龄儿无明显差异(P>0.05)。结论 全面开展新生儿疾病筛查,提高筛查覆盖率,尽早规范化开始治疗及长期随访监测,可使PKU、CH患儿避免体格和智力不可逆损害,对改善其预后和生存质量意义重大。     

Repeated adverse fetal outcome in pregnancy complicated by uncontrolled maternal phenylketonuria

Phenylketonuria in pregnancy carries with it an increased risk of spontaneous abortion and development of a fetus that is affected by the maternal phenylketonuria syndrome. This syndrome is characterized by low birthweight, congenital heart disease, microcephaly, childhood growth failure, and cognitive impairment. It is the result of the hyperphenylalaninemia that accompanies the phenylketonuric state, and may therefore be avoided by maintaining maternal serum phenylalanine levels within the normal range. Phenylalanine is an essential amino acid and may be controlled by dietary manipulation. Presented here is a case history of a woman with phenylketonuria who was unable to satisfactorily control her serum phenylalanine levels in each of her three pregnancies. All three children were adversely affected by the fetopathy of the maternal phenylketonuria syndrome, each with evidence of growth failure and impaired neurodevelopment. This patient illustrates the difficulties that may be encountered when providing obstetric care to the woman with phenylketonuria who is not able or not willing to restrict her dietary intake of phenylalanine. The discussion includes consideration of management strategies, including dietary therapy and legal intervention.     

Body mass index rebound and overweight at 8 years of age in hyperphenylalaninaemic children

Aim: To evaluate whether the age at body mass index (BMI) rebound may be associated with overweight at age 8 y in hyperphenylalaninaemic (HPA) children. Methods: A longitudinal observational study including 97 HPA children born 1984-1993 and detected by the National Neonatal Screening programme. Children were followed up at the same institution and evaluated for dietary intakes and anthropometrical parameters from diagnosis up to the age of 8 y. Outcome measure was overweight at age 8 y, defined according to the International Obesity Task Force. The age at BMI rebound, BMI before and at rebound were considered as potential determinants. Familial overweight, breastfeeding and macronutrients intake at age 1 y were considered as confounders. Results: Mean (95% confidence interval [CI]) age at BMI rebound was 5.0 (4.7-5.3) y. At the age of 8 y, 24.7% (95% CI 16.3-33.1%) of children was overweight. Children overweight at the age of 8 y exhibited earlier BMI rebound than non-overweight children (mean difference [95% CI] -2.1 [-2.8 to -1.4] y) and higher BMI from the age of 1 y (mean difference [95% CI] 1.2 [0.9-2.5] kg/m[Formula: See Text]) onward. Overweight was more likely in children with, rather than without, parental overweight (41.0% vs 19.8%). After adjustment for confounders, logistic analysis showed that earlier BMI rebound (odds ratio [OR] 2.4, 95% CI 1.2-4.8) and BMI at age 1 y (OR 2.3, 95%CI 1.1-4.98) were independently associated with overweight at the age of 8 y.

Conclusion: Within the population of this study, overweight at age 8 y was positively associated with early BMI rebound and BMI at age 1 y.     

New insights into tetrahydrobiopterin pharmacodynamics from Pah, a mouse model for compound heterozygous tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

Phenylketonuria (PKU), an autosomal recessive disease with phenylalanine hydroxylase (PAH) deficiency, was recently shown to be a protein misfolding disease with loss-of-function. It can be treated by oral application of the natural PAH cofactor tetrahydrobiopterin (BH₄) that acts as a pharmacological chaperone and rescues enzyme function in vivo. Here we identified Pah^enu1/2 bearing a mild and a severe mutation (V106A/F363S) as a new mouse model for compound heterozygous mild PKU. Although BH₄ treatment has become established in clinical routine, there is substantial lack of knowledge with regard to BH₄ pharmacodynamics and the effect of the genotype on the response to treatment with the natural cofactor. To address these questions we applied an elaborate methodological setup analyzing: (i) blood phenylalanine elimination, (ii) blood phenylalanine/tyrosine ratios, and (iii) kinetics of in vivo phenylalanine oxidation using ¹³C-phenylalanine breath tests. We compared pharmacodynamics in wild-type, Pah^enu1/1, and Pah^enu1/2 mice and observed crucial differences in terms of effect size as well as effect kinetics and dose response. Results from in vivo experiments were substantiated in vitro after overexpression of wild-type, V106A, and F263S in COS-7 cells. Pharmacokinetics did not differ between Pah^enu1/1 and Pah^enu1/2 indicating that the differences in pharmacodynamics were not induced by divergent pharmacokinetic behavior of BH₄. In conclusion, our findings show a significant impact of the genotype on the response to BH₄ in PAH deficient mice. This may lead to important consequences concerning the diagnostic and therapeutic management of patients with PAH deficiency underscoring the need for individualized procedures addressing pharmacodynamic aspects.     

Molecular genetics of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

Mutations in the phenylalanine hydroxylase (PAH) gene result in phenylketonuria (PKU). Tetrahydrobiopterin (BH(4))-responsive hyperphenylalaninemia has been recently described as a variant of PAH deficiency caused by specific mutations in the PAH gene. It has been suggested that BH(4)-responsiveness may be predicted from the corresponding genotypes. Data from BH(4) loading tests indicated an incidence of BH(4)-responsiveness of >40% in the general PKU population and >80% in mild PKU patients. The current project entailed genotype analysis of 315 BH(4)-responsive patients tabulated in the BIOPKUdb database and comparison with the data from the PAHdb locus-specific knowledgebase, as well as with previously published PAH mutations for several European countries, Northern China, and South Korea. We identified 57 mutations, presenting with a substantial residual PAH activity (average approximately 47%), presumed to be associated with BH(4)-responsiveness. More than 89% of patients are found to be compound heterozygotes. The three most common mutations found in >5% of BH(4)-responsive patients are p.A403 V, p.R261Q, and p.Y414C. Using the Hardy-Weinberg formula the predicted average frequency of BH(4)-responsiveness in European populations was calculated to be 55% (range 17-79%, lowest in Baltic countries and Poland and highest in Spain), 57% in Northern China, and 55% for South Korea. The genotype-predicted prevalence of BH(4)-responsiveness was higher than prevalence data obtained from BH(4) loading tests. Inconsistent results were observed for mutations p.L48S, p.I65 T, p.R158Q, p.R261Q, and p.Y414C. Our data suggest that BH(4)-responsiveness may be more common than assumed and to some extent may be predicted or excluded from the patient's genotype.     

以肝功能轻度异常为首发表现的高苯丙氨酸血症1例

高苯丙氨酸血症(hyperphenylalaninemia,HPA)是一种遗传代谢性疾病,以苯丙氨酸羟化酶(phenylalanine hydroxylase,PAH)活性缺乏,致血浆苯丙氨酸浓度升高为特征。该病早期无明显临床症状,随着病情加重会逐渐出现严重神经系统功能障碍,但首发症状为肝功能异常的着实罕见。本文现对1例以肝功能轻度异常为首发表现的HPA患者进行报道,并回顾相关文献,以进一步提高临床医生对此类疾病不典型表现的辨识能力,达到早诊断、早治疗的目的。     

高苯丙氨酸血症1 066例随访分析

【目的】 了解高苯丙氨酸血症(hyperphenylalaminemia, HPA)患者治疗随访及预后。 【方法】 对在本院诊断治疗的1 066例HPA的治疗随访结果进行回顾性分析。 【结果】 1)在1 066例HPA患儿中,共有1 016例为苯丙氨酸羟化酶缺乏症,即经典型苯丙酮尿症(phenylketonuria, PKU),50例为四氢生物蝶呤缺乏症(tetrahydrobiopterin deficiency, BH₄D)。在这些PKU患者中,有369例(34.62%)患儿在新生儿筛查时被确诊并在3个月内开始规律治疗,241例(22.61%)在生后3～12个月内确诊, 456例(42.78%)在1岁后才确诊。2)3个月内筛查治疗的患儿智力发育明显高于非筛查确诊的患儿(96±15, 69±11;t=14.19,P＜0.01﹚。3)3个月后才确诊的经治疗后智力水平也有明显提高﹙46±15,69±11,t=7.13,P＜0.05﹚。4)氢质子磁共振波谱(¹HMRS)检测22例HPA患儿的脑苯丙氨酸(phenylalanine, Phe)浓度表明患儿血、脑Phe浓度与智商均呈负相关关系(r_血=0.505,r_脑=0.647,P<0.01)。 【结论】 对所有HPA患者均应进行鉴别诊断,尽早确诊和治疗, 控制血、脑苯丙氨酸浓度是减少智能落后的有效措施。