苯丙氨酸的正常代谢需要PAH和BH4共同参与( 图 1 )。PAH是苯丙氨酸羟化为酪氨酸的限速酶，在肝脏中代谢苯丙氨酸，受 PAH 基因调控。人PAH在细胞质中以二聚体或活性四聚体形式存在，两者间的平衡依赖于细胞内酸碱度。PAH的结构改变和基因突变会影响自身的酶活性。PAH蛋白分为三个功能区域：调控域(regulatory domain)、催化域(catalytic domain)和寡聚域(oligomerization domain) 。通常来说，底物苯丙氨酸与四聚体PAH调控域相结合，可以激活PAH，同时调控域Ser16的磷酸化能强化两者的结合 。BH4与PAH催化域相结合，可以稳定PAH的三维结构，防止蛋白水解和错误折叠

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BH4不仅是PAH的辅酶，也是酪氨酸羟化酶(tyrosine hydroxylase, TH)和色氨酸羟化酶(tryptophane hydroxylase, TPH)的辅酶，其合成代谢中任何一种合成酶或还原酶缺乏均可导致BH4缺乏，从而降低PAH、TH和TPH的活性，进一步影响脑内神经递质(如多巴胺、5-羟色胺)的合成，导致患者出现严重的神经系统症状 。BH4的合成需鸟苷三磷酸环化水解酶(GTP cyclohydrolase，GTPCH)、PTPS和墨蝶呤还原酶(sepiapterin reductase，SR)参与；其代谢产物通过蝶呤- 4α-甲醇胺脱水酶(pterin- 4α-carbinolamine dehydratase，PCD)和DHPR再生为BH4

PAH缺乏症和BH4缺乏症的致病基因分别是PAH和BH4合成代谢中的相关基因。PAH缺乏症和BH4缺乏症在我国的分布具有一定的地域特征。来自上海、徐州、广西和南京等地区筛查数据的统计分析结果显示 ，800多例HPA患者中PAH缺乏症占91.8%，BH4缺乏症占8.2%；而PAH缺乏症患者中73.7%是苯丙酮尿症患者，26.3%为轻度HPA患者。Zhu等 报道346例PAH缺乏症患者中，经典型苯丙酮尿症172例(49.7%)，轻度苯丙酮尿症156例(45.1%)，轻度HPA 18例(5.2%)。

1953年Jervis 发现PAH的功能缺陷是苯丙酮尿症的特征性表现。20世纪80年代，人 PAH 基因的互补DNA逐渐被克隆和验证 。1986年DiLella等 通过PCR扩增和放射性同位素标记的寡核苷酸特异性杂交在丹麦人群中发现了PAH致病的第一个突变体c.1315+1G>A；之后不久又确定了第二种突变c.1222C>T(p.R408W) 。1992年Konecki等 PAH 全基因。目前 PAH 基因数据库(PAHvdb，)共记录有1024余种突变，外显子3、6、7和11上的突变所占的比例较高，分别为10.1%、14.3%、12.6%和9.5%，突变类型包括58.0%错义突变，8.0%无义突变(包括片段缺失引起蛋白翻译终止)，13.2%剪接突变，14.0%由插入或缺失引起移码突变，3.8%同义突变( 图 2 )。共835个突变位点位于PAH蛋白结构上，其中72.1%的突变发生在催化域，21.6%的突变位于调控域，6.3%的突变位于寡聚域

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Li等 用二代测序技术对我国796例苯丙酮尿症患者进行了检测，结果发现了 PAH 基因上的194个位点突变，其中76.33%突变位点位于外显子上；Liu等 在655例苯丙酮尿症患者中发现174种 PAH 基因突变，包括22个新突变。目前，PAHvdb中记录了200多个在中国人群中发现的 PAH 基因突变，其中82.5%突变位于外显子上，主要分布于外显子3、6、7、11上(占总突变47.5%)，与 图 2 分布基本相符；其他内含子突变仅31个，5′非翻译区端突变4个。我国PAH缺乏症患者的基因突变主要有：p.R243Q、p.R241C、p.Y204C、p.Y356*、p.R111*、p.R413P、p.R408Q、c.442-1G>A和p.V399V等，其中p.R241C在中国台湾人群中较为常见

BH4缺乏症由 PTS 、 QDPR 、 GCH1 、 PCBD1 和 SPR 基因调控。数据显示，全球1118例BH4缺乏症患者中，PTPS、DHPR、SR、GTPCH和PCD缺陷患者的比例分别约为62%、27%、5%、3%和3% 。亚洲人群的BH4缺乏症主要是PTPS缺陷，中东和土耳其人群主要是DHPR缺陷 GCH1 和 SPR 基因的大多数突变体与肌张力障碍相关，为多巴反应型(Dopa-responsive)

我国人群中BH4缺乏症的主要致病基因为 PTS 和 QDPR 。Liu等 报道约86%的BH4缺乏症患者属于PTPS缺陷。Ye等 分析了256例BH4缺乏症患者，发现其中240例为PTPS缺陷，6例为DHPR缺陷；在143例患者的基因分析中，分别发现了33个 PTS 突变位点，5个 QDPR 突变位点和1个 GCH1 突变位点。中国人群 PTS 基因常见的突变位点有：p.N52S、p.P87S、p.D96N、p.K91R、c.84-291A>G/p.Y27Rfs*17、p.T106M、p.V56M、p.T67M、p.L127F和c.118-121del/p.F40Gfs*17等 。中国南方患者常见的突变为p.N52S，北方患者常见的突变为p.D96N和p.K91R，而p.P87S和c.84-291A>G在南北方各占一半 。迄今已有353例中国BH4缺乏症患者记录在BIODEFdb()中。

近年，Anikster等 通过全外显子测序，在6例非亲缘关系的HPA患者中鉴定了新基因 DNAJC12 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_021800.2","term_id":"41406092","term_text":"NM_021800.2"}} NM_021800.2 )上的纯合致病缺失和突变：c.298-968_503-2603del、c.215G>C/p.R72P和c.158-2A>T，突变导致患者PAH酶活性降低。van Spronsen等 在5例HPA患者中发现了三个新位点：c.85delC/p.Q29Kfs*38、c.596G>T/p.*199Lext*42和c.214C>T/p.R72*，再次验证了 DNAJC12 是HPA的新致病基因。 DNAJC12 可以与PAH、酪氨酸羟化酶和色氨酸羟化酶2产生相互作用，从而导致HPA和神经递质缺乏症

PAH缺乏症基因型与表型的相关性是多层次的。根据BIOPKUdb数据库() 的统计分析，PAH缺乏症患者中54.8%为经典型苯丙酮尿症，27.4%为轻度苯丙酮尿症，17.8%为轻度HPA，其中76%的苯丙酮尿症患者基因型为复合杂合突变 。PAH缺乏症的基因型在不同人群中分布不同，代谢表型也存在较大差异。据报道，东欧国家PAH缺乏症的基因型中c.1222C>T(p.R408W)突变体十分常见，其主要表型是经典型苯丙酮尿症；而在欧洲南部，大部分PAH缺乏症的基因型为具有大量酶活性残留的轻度亚型，如c.782G>A(p.R261Q)，因此轻度苯丙酮尿症更常见

另外，一些 PAH 基因突变通过影响其蛋白氨基酸序列、蛋白结构或稳定性水平，决定残留的PAH活性，因此 PAH 基因突变与酶活性、BH4反应性有一定相关性。

基因突变影响残留PAH酶的活性。已知97个残留PAH酶活性的突变中 ，c.202A>G(p.R68G)不影响PAH酶活性，c.569T>C(p.V190A)和c.1247C>A(p.P416Q)分别增加残留PAH酶活性至110%和111%。欧洲常见经典型苯丙酮尿症的突变位点c.1222C>T(p.R408W)、c.1066-11G>A和c.842C>T(p.P281L)对应的残留酶活性均小于10%，而c.1241A>G(p.Y414C)和c.782G>A(p.R261Q)对应的酶活性分别为正常的57%和44%。但有些突变在体外的残留酶活性有重大差异，如p.I65T和p.Y414C。基因型的残留酶活性并不是两个位点体外残留酶活性的简单加减，如复合杂合基因型c.[1222C>T]:[1241A>G]、c.[1066-11G>A]:[1241A>G]的残留PAH酶活性为正常的30%左右。目前，国际上可根据计算基因型相对应的等位基因表型值来预测患者的临床表型，此种方法对基因型为纯合子或半合子的患者临床表型预测的准确率可达90%以上，但对基因型是复合杂合突变预测的准确率仅有77.9% 。基因型、酶活性和临床表型有一定关联：基因型c.[1169A>G]:[1169A>G]的残留酶活性为65%，携带者基本是轻度HPA患者；但也有例外，如基因型c.[754C>T]:[754C>T]的残留酶活性为1%，携带者可能为经典型苯丙酮尿症、轻度苯丙酮尿症或轻度HPA患者 。中国PAH缺乏症人群中常见的基因位点，如c.721C>T(p.R241C)、c.728G>A(p.R243Q)和c.1223G>A(p.R408Q)，其残留酶活性分别为25%、14%和46%，这三个突变体在我国经典型苯丙酮尿症、轻度苯丙酮尿症和轻度HPA患者中均有分布。

BH4的反应性现象最初由Kure等 在日本苯丙酮尿症患者中发现，并分别在1730例和557例PAH缺乏症患者的临床资料中证实 ，从此开启了使用BH4治疗BH4反应性PAH缺乏症的新篇章。轻度HPA和将近78.8%的轻度苯丙酮尿症患者呈BH4反应性，而经典型苯丙酮尿症患者中仅约34.2%患者呈BH4反应性 ，可见BH4反应性在一定程度上是表型依赖的。不同国家经典型苯丙酮尿症患者对BH4的反应性有所不同。Matalon等 研究显示，美国约50%经典型苯丙酮尿症患者对BH4有反应性；澳大利亚新南威尔士地区经典型苯丙酮尿症患者仅有7%对BH4有反应性 ；而邹卉等 研究表明，我国经典型苯丙酮尿症患者约18.1%为BH4反应性。Trunzo等 报道低残留酶活性突变体对应的表型往往严重且没有BH4反应性，如位点p.L249P，仅有7%的PAH酶活性，患者表型为经典型苯丙酮尿症且没有BH4反应性；而高残留酶活性突变体，如位点p.T380M，有28%的PAH酶活性，97%的患者的表型是轻度HPA，且83%的患者呈BH4反应性。在对中国PAH缺乏症患者的BH4反应性研究中，叶军等 发现中国苯丙酮尿症患者BH4反应性中最常见p.R241C突变类型；Tao等 对我国165例苯丙酮尿症患者进行了BH4反应性研究，发现24.24%呈BH4反应性，尤其是携带p.R241C的患者，另外p.L98V和c.353-2A>T也与BH4反应性相关。Zhu等 在94例中国PAH缺乏症患者中发现48例(51%)为BH4反应性，多数是轻度苯丙酮尿症和轻度HPA患者。迄今，已明确50个突变位点与BH4反应性相关，常见位点如：c.782G>A(p.R261Q)、c.1241A>G(p.Y414C)、c.1066-11G>A、c.143T>C(p.L48S)等。国内外同行在BH4治疗苯丙酮尿症患者研究中均发现轻度苯丙酮尿症和轻度HPA患者对BH4的反应性较高，治疗效果明显 。基因型可以部分预测BH4反应性，但单独用基因型来预测个体的BH4反应性仍需谨慎。

BH4缺乏症根据临床表现可以分为三种类型：①中枢型(严重型)：以脑脊液中神经递质代谢产物浓度低和严重的神经系统症状为特征；②周围型(温和型)：脑脊液中神经递质代谢产物的水平正常；③短暂型：表现为一过性的新生儿HPA 。BH4缺乏症的严重程度受多种酶活性影响。目前共有495个BH4缺乏症的相关突变记录在BH4缺乏症及儿童神经递质缺乏症基因数据库(PNDdb，)中 PTS 、 QDPR 、 PCDB1 基因突变会导致HAP，而 GCH1 和 SPR 基因突变主要是导致多巴反应性肌张力障碍和神经递质缺乏症。

我国BH4缺乏症患者主要为PTPS缺乏症，约占96%，其次是DHPR缺乏症患者，约占2.4% 。对四川、郑州、上海等地区筛查数据的统计分析结果显示，PTPS缺陷症患者中基因型为复杂杂合型的占82.4%，纯合型占16.2% 。但在伊朗地区，PTPS缺乏症患者基因型为纯合型的比例高达94.7%，而复杂杂合型仅占5.3% 。Liu等 的研究表明，携带有c.166G>A突变的基因型例如c.[166G>A:73C>G]、c.[317C>T:166G>A]可能与温和型PTPS缺乏症有关，而携带有c.73C>G、c.155A>G、c.226C>T、c.259C>T、c.286G>A突变的基因型例如c.[155A>G:155A>G]、c.[259C>T:155A>G]、c.[259C>T:169_171delGTG]则与严重型PTPS缺乏症相关。 DHPR 基因有83种基因突变，其主要的发病人群位于中东和土耳其。在我国，DHPR缺乏症患者基因型为复杂杂合型的比例约为86% 。目前共有16例中国患者记录在BIODEF数据库()中，所有患者皆为中枢型BH4缺乏症。

HPA是我国遗传代谢病防控的典型，是研究基因型与表型关联的理想疾病模型，但其表型高度异质性的致病机制依然十分复杂。目前已经有超过100种突变通过生物化学研究确定了 PAH 致病作用，有利于更好地了解突变和PAH缺乏症之间的生物化学关系。但由于PAH常以四聚体的形式存在，且大多数PAH缺乏症患者带有不同突变的复合杂合子，两个不同突变的蛋白相互作用可能会导致与其作为单独的纯合突变时不同的BH4反应和不同的蛋白正向或负向作用，甚至会导致单体的错误折叠，从而使表型更严重。血脑屏障与中性大分子氨基酸也增加了表型的复杂性。基因信息与表型数据的结合将有助于医生更快地诊断疾病类型，制订个性化诊疗方案，HPA的防治也将取得更大进步。

Funding Statement

国家重点研发计划（2017YFC1001703）；浙江省医药卫生重大科技项目（WKJ-ZJ-1704）；浙江省卫生高层次人才培养工程（2016-6）

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