四氢生物蝶呤（BH4）缺乏症（tetrahydro biopterin deficiency），是高苯丙氨酸血症的一种亚型，是一种常染色体遗传性疾病，与苯丙酮尿症（PKU）同属于高苯丙氨酸血症（HPA），是截至21世纪初为止已确认的5000～6000种人类的罕见遗传代谢病之一，主要对人的神经系统造成损害，导致患儿出现智力低下、癫痫等症状。四氢生物蝶呤缺乏症通过对新生儿进行疾病筛查，检出高苯丙氨酸血症后可得到进一步确诊。在中国大陆地区，新生儿高苯丙氨酸血症的发病率约为1.1万分之一；而在高苯丙氨酸血症中约有近两成是四氢生物蝶呤缺乏症。按这个发病率计算，在中国每年约2000万新生儿中可能患四氢生物蝶呤缺乏症的为100~200例。由于四氢生物蝶呤缺乏症分经典型和非经典型两种，因此早期很容易误诊。
GTP环化水解酶1与四氢生物蝶呤合成途径
GTP环化水解酶I(GCHl)是合成四氢生物蝶呤的关键酶。
GTP cyclohydrolase I (GTPCH), encoding by gene GCH1, is the rate-limiting enzyme in the synthesis of tetrahydrobiopterin (BH4). BH4 is the critical co-factor for enzyme phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), tryptophan hydroxylases (TPHs), nitric oxide synthases (NOSs) and alkylglycerol mono-oxygenase (AGMO) .
今天阅读的文献来自广州市妇女儿童医疗中心遗传与内分泌科，关于四氢生物蝶呤代谢病与运动障碍的小鼠模型制备。这个研究设计理念来自于在临床中发现异常基因，但可贵的是，进行了动物实验验证，完善了动物实验模型，同时又有了对临床有提示作用的新发现。
四氢生物蝶呤代谢病与运动障碍的小鼠模型
A novel GTPCH deficiency mouse model exhibiting tetrahydrobiopterin-related metabolic disturbance and infancy-onset motor impairments.
背景：GTP-环水解酶I（GTPCH）缺乏影响四氢生物蝶呤的合成，引起苯丙氨酸分解代谢、神经递质合成、一氧化氮生成异常，导致多种代谢疾病。尽管四氢生物喋呤和神经递质前体左旋多巴可以改善病情，但仍有部分患者存在残余运动和精神缺陷。一个理想的GTPCH缺乏症动物模型可模拟临床症状，特别是运动损伤，但是在机制和治疗研究中，至今仍然没有建立这样的模型。
BACKGROUND:
GTP cyclohydrolase I (GTPCH) deficiency could impair the synthesis of tetrahydrobiopterin and causes metabolic diseases involving phenylalanine catabolism, neurotransmitter synthesis, nitric oxide production and so on. Though improvements could be achieved by tetrahydrobiopterin and neurotransmitter precursor levodopa supplementation, residual motor and mental deficits remain in some patients. An appropriate GTPCH deficiency animal model with clinical symptoms, especially the motor impairments, is still not available for mechanism and therapy studies yet.
目的和方法：
为了研究从患有严重婴儿期开始的多巴反应性运动障碍的患者身上识别出的杂合子GTPCH错义突变p.leu117arg是否是致病因素，并建立一个临床相关的GTPCH缺陷小鼠模型，研究者使用CRISPR/CAS9技术产生了一个类似错义突变的小鼠突变。研究者对杂合子和纯合子突变体进行了一系列的鉴定实验。
OBJECTIVES AND METHODS:
To investigate whether the heterozygous GTPCH missense mutation p.Leu117Arg identified from a patient with severe infancy-onset dopa-responsive motor impairments is causative and establish a clinical relevant GTPCH deficiency mouse model, we generated a mouse mutant mimicking this missense mutation using the CRISPR/Cas9 technology. Series of characterization experiments on the heterozygous and homozygous mutants were conducted.
结果：
突变株中GTPCH的表达无明显变化，但纯合子突变株的酶活性受损。在纯合子突变体的肝脏和大脑中观察到BH4减少和苯丙氨酸积累。大脑的代谢紊乱比肝脏严重。在纯合子突变体的大脑中观察到神经递质多巴胺、去甲肾上腺素和血清素的显著降低。活产纯合子突变体表现出与患者所观察到的疾病症状相似的婴儿期开始运动和发声缺陷，而在年轻的杂合子突变体小鼠中没有观察到明显的症状。用左甲状腺素治疗，纯合子突变体的存活率得到改善，但没有完全挽救。
RESULTS:
The expressions of GTPCH were not significantly changed in the mutants, but the enzyme activities were impaired in the homozygous mutants. BH4 reduction and phenylalanine accumulation were observed both in the liver and brain of the homozygous mutants. Severer metabolic disturbance occurred in the brain than in the liver. Significant reduction of neurotransmitter dopamine, norepinephrine and serotonin was observed in the brains of homozygous mutants. Live-born homozygous mutants exhibited infancy-onset motor and vocalization deficits similar to the disease symptoms observed in the patient, while no obvious symptoms were observed in the young heterozygous mutant mice. With benserazide-levodopa treatment, survival of the homozygous mutants was improved but not completely rescued.
结论：
GTPCH p.leu117arg错义突变有害，可隐性引起四氢生物蝶呤、苯丙氨酸和神经递质代谢紊乱和婴儿期发作的运动功能障碍。该研究团队建立了活体出生的GTPCH缺乏小鼠模型，其表现出明显的运动损伤。肝脏和大脑对GTPCH缺乏的反应中观察到的BH4减少和苯丙氨酸积累的不同程度，为大脑对GTPCH缺乏的易感性提供了新视点。
CONCLUSIONS:
The GTPCH p.Leu117Arg missense mutation is deleterious and could cause tetrahydrobiopterin, phenylalanine and neurotransmitter metabolic disturbances and infancy-onset motor dysfunctions recessively. This is the first GTPCH deficiency mouse model which could be live-born and exhibits significant motor impairments. The different extents of BH4 reduction and phenylalanine accumulation observed between liver and brain in response to GTPCH deficiency gives potential new insights into the vulnerability of brain to GTPCH deficiency.
