Liddle综合征表型和基因型关系分析及基于中国北方汉族人群药物基因组学的华法林维持剂量公式建立

【作者】 龚玲；

【导师】 惠汝太；

【作者基本信息】 北京协和医学院 ， 内科学， 2014， 博士

【摘要】 背景：Liddle综合征是一种常染色体显性遗传疾病,由上皮钠通道的基因突变导致。已报道的Liddle综合征患者中,绝大多数均因错义突变或移码框架突变而导致位于上皮钠通道p或γ亚基羧基末端保守的富含脯氨基酸的PY模体发生缺失或改变。Liddle综合征的典型表现为早发的低钾性高血压,并伴随血浆低肾素活性和低醛固酮水平,但临床上,不同患者血压水平、血浆钾浓度和其它临床特征均表现出很大的表型异质性。目标：通过两个Liddle综合征中国家庭分析基因型与表型的关系。方法：收集疑似Liddle综合征的两个患者及其家庭成员的临床资料,并对编码上皮钠通道β亚基的SCNN1B基因和编码γ亚基的SCNN1G基因羧基端进行测序。其中一个家庭除了高血压和低血钾外,曾有家庭成员发生猝死,所以对此家庭成员进行了额外的428个心血管疾病相关基因的全外显子测序。检测到上皮钠通道基因突变后,与既往研究中携带同样突变的患者表型进行比较和分析。结果：先证者1所在家庭中,先证者1及其姐姐、父亲均检出杂合的无义突变βR566X。他们都有高血压低血钾,但程度较轻,血压也可通过钙通道阻滞剂联合上皮钠通道阻滞剂很好地控制。在既往的4个对βR566X的报道中,其中2个报道中的携带者表型较轻,与本文类似；但另外2个报道中携带者却有严重的表型,包括早发卒中和死亡等。在先证者2家系中所发现的杂合βR597PfrX607移码框架突变则带来了严重恶性的表型,包括难以控制的高血压、低钾、严重的靶器官损害,并且先证者2及其父亲均在20余岁时发生猝死。但在既往的报道中pR597PfrX607突变仅引起中等程度的表型,患者血压可用上皮钠通道阻滞剂单药控制,并且无早发恶性心血管事件家族史。在另外进行外显子扫描的428个心血管疾病相关的基因中,除了SCNN1B外,一些错义突变或移码框架突变也被发现,但它们大多同样存在于先证者2正常表型的兄长。先证者2独有的几个突变所在基因尚无功能实验或者清晰的相关性实验证明其与血压相关。结论：即使携带同样的突变,Liddle综合征的表型变异也很显著。同样的突变导致不同表型的机制值得更深入的研究,对调节因素进行干预可能改善疾病的病程和预后。背景：目前华法林仍然是预防和治疗血栓栓塞的主要口服抗凝药,但由于其治疗窗狭窄、个体反应差异大,对华法林治疗剂量的控制仍然面临挑战。华法林的作用受到很多因素的影响,包括性别、年龄、体表面积、服用的其它药物和食物等。同时华法林的作用也受到基因因素的调控。既往已经有许多研究表明基因CYP2C9和VKORCl内的单核酸多态性位点与华法林剂量相关,基因CYP4F2中的位点rs2108622(V433M)也曾被报道与华法林剂量相关。美国食品和药物管理局已明确提出,“患者的CYP2C9和VKORC1基因型,可以协助选择华法林起始剂量”。既往研究已经在中国人中建立了一些预测华法林剂量的公式,但普遍样本量小,仅有的两个较大的中国汉族人群华法林维持剂量的研究分别在中国的南部和中部展开。目的：本研究旨在中国北方居住人群中评估影响华法林维持剂量的基因并建立一个整合基因和常见干扰因素的公式用于预测华法林维持剂量。方法：顺序纳入800个因心脏瓣膜置换术需要长期华法林维持治疗的患者,对他们进行了23个相关单核苷酸多态性位点的基因分型。首先检测这些单核苷酸多态性位点与华法林维持剂量的关系,在先证队列(总体队列的70%人群)中,基于基因型与华法林维持剂量的关系,建立一个整合了常见非基因因素的维持剂量计算公式,并在验证队列(总体队列的30%人群)中对公式进行评估。结果：基因VKORC1和CYP2C9的单核苷酸多态性位点显示出与华法林剂量的显著相关性。基因CYP4F2中rs2108622位点在单因素分析中与华法林剂量关联未达显著意义(P=0.152),但最终的多因素回归模型中P值变为0.01。总的来说,在初始队列(n=551)中,华法林剂量在不同程度上被多个因素影响,VKORC1rs7294(27.3%),CYP2C9*3(7.0%),体表面积(4.2%),年龄(2.7%),目标INR值(1.4%),CYP4F2rs2108622(0.7%),氨碘酮使用(0.6%),糖尿病(0.6%)以及地高辛使用(0.5%),总共约45.1%的华法林剂量变异可由以上因素解释。在验证队列(n=236)中,实际维持剂量与预测剂量显著相关(r=0.609,P<0.001)。另外,相较于中国南方公式和中部公式,我们的公式对中国北方人群华法林维持剂量的预测最为准确。结论：本研究建立的公式可能帮助中国北方患者更有效地进行个体华法林剂量管理。更多还原

【Abstract】 Background:Liddle syndrome is an autosomal-dominant inheritance disease, caused by mutations in an epithelial sodium channel (ENaC). Most reported mutations in patients with Liddle syndrome are either missense or frameshift mutations that either delete or alter the conserved proline-rich PY motif (PPPXY sequence) localized at the C-terminal ends of the β and γ subunits of ENaC. Typical Liddle syndrome is characterized by early onset of hypokalemic hypertension with low plasma renin activity (PRA) and low serum aldosterone level, but patients with Liddle syndrome show marked phenotypic variation in blood pressure, serum potassium and other clinical manifestations.Objective:Analyzed the correlation of genotype-phenotype in two Chinese families with Liddle syndrome.Methods:The sequence of C-terminus of SCNN1B and SCNN1G were screened in the two families with likely Liddle syndrome, and there clinical information were collected. In addition to hypertension and hypokalemia, one of the two pedigrees had sudden death in their family members, so the exons of428reported genes-related to cardiovascular diseases were screened as well in the family. We also compared the phenotypes among the patients carrying the identical mutation in our study and in previous studiesResults:A heterozygous PR566X nonsense mutation was found in the proband-1in the first pedigree, and the proband’s sister and father. They showed mild phenotype with hypertension and hypokalemia, and the blood pressure was under control by calcium channel bloker and ENac blocker. Two of the four previous studies report that the mutation causes severe phenotype, even die of stroke in their thirties, while the other two families showed a mild clinical phenotype. In contrast, a heterozygous pR597PfrX607frameshift mutation was identified in the proband-2in the second pedigree, showing malignant phenotype including resistant hypertension, hypokalemia, and sever target organ damagement. Both the proband-2and the proband-2’s father had sudden death in their twenties. But the same mutation has been related to moderate phenotype in previous studies, in which blood pressure could been controlled by ENaC blocker and no early maglinant cardiovascular events. By screening of the exons in428cardiovascular disease-related genes, some missense and frameshift mutations were found other than SCNN1B, but most of them were also detected in the proband’s brother, who was a genetically unaffected member with normal phenotype. Genes with mutation-unique to the proband-2has not been associated with blood pressure by either function study or convincable association study.Conclusion:The phenotypes of Liddle syndrome are varied significantly even with the same mutation. The mechanisms why the same mutation causes very different phenotype need to be explored because intervention of these modifiers may change the disease course and prognosis accordingly. Background:Warfarin has remained the mainstay of oral anticoagulant therapy for the treatment and prevention of thromboembolism. However, management of warfarin therapy is challenging because of its narrow therapeutic index and wide inter-individual variability. Effect of warfarin could be influenced by many factors, incluing gender, age, body surface area, concominant drugs and foods. Also genetic variants could affect warfarin, many studies have shown that single nucleotide polymorphisms (SNPs) within CYP2C9and VKORC1genes are related to warfarin dose requirement. CYP4F2rs2108622(V433M) was found to be associated with warfarin dose in3independent Caucasian cohorts. Food and Drug Administration has changed the drug label for warfarin and include the statement,"The patient’s CYP2C9and VKORC1genotype information, when available, can assist in selection of the starting dose." Previous studies constructed many algorithms to predict the warfarin maintenance dose in Chinese individuals. However, they frequently used relatively small populations (<400subjects) except in two studies where one included845from Southern China, and the other included641from Central ChinaObjectives:The objective of the current study was to assess these genetic determinants of the warfarin maintenance dose and to construct an algorithm integrating common interference factors to predict the dose in a large population who lived in Northern China.Methods:This study enrolled800consenting patients with heart-valve replacements who were receiving long-term warfarin maintenance therapy,23related SNPs were genotyped. First, the associations of these SNPs with the warfarin maintenance dose were tested. Second, on the basis of genotypes associated with the warfarin maintenance dose, an algorithm integrating common non-genetic factors was constructed to predict the dose in the derivation cohort (70%of the whole cohort), and was assessed in the validation cohort (30%of the whole cohort).Results:Only VKORC1and CYP2C9SNPs were observed to be significantly associated with warfarin dose. The relationship between gene CYP4F2rs2108622and warfarin dose was not significant in the univariate analysis(P=0.152), however, the P-value was0.01in the final multiple regression model. Generally, in the derivation cohort (n=551), warfarin dose variability was influenced, in decreasing order, by VKORC1rs7294(27.3%), CYP2C9*3(7.0%), body surface area(4.2%), age(2.7%), target INR(1.4%), CYP4F2rs2108622(0.7%), amiodarone use(0.6%), diabetes mellitus(0.6%), and digoxin use(0.5%), which account for45.1%of the warfarin dose variability. In the validation cohort (n=236), the actual maintenance dose was significantly correlated with predicted dose (r=0.609, P<0.001). Furthermore, our algorithm demonstrated the best predictor in northern Chinese people compared with the other two algorithms derived from southern Chinese and central Chinese populations.Conclusions:Our algorithm could improve the personalized management of warfarin use in Northern Chinese patients.更多还原