SNP FUNCTION--IN VITRO AND IN VIVO

LNG是一个开创性的实验室，研究脑基因多态性与体外功能的关系，以及通过脑成像和与NIMH合作获得的体内功能测量。多态性/功能关联与已知的递质和蛋白质的分子神经生物学一致，包括纹状体多巴胺转运蛋白基因型/多巴胺转运蛋白密度，中脑核血清素转运蛋白基因型/血清素转运蛋白密度，额叶COMT基因型/代谢活性。功能变异是定位克隆的终点，为连锁研究选择最合适的表型、候选基因假设检验和提高连锁的先验概率提供了宝贵的工具。LNG正在筛选非编码序列对转录和RNA加工的影响，我们正在筛选gpcr的某些编码变体，以改变配体亲和力，信号转导和受体下调。在体外，由于神经适应过程，功能显著的等位基因的表型效应可能很难辨别，除非在细胞表达系统中，检测各种等位基因的遗传背景是相同的。为此，我们使用了瞬时转染的cos-7（小鼠肾脏）细胞和稳定转染的CHO-K1（中国仓鼠卵巢）细胞，目前正在使用瞬时转染的HEK（人类胚胎肾脏）细胞来比较人类HTR5a Pro15和Ser15等位基因。5HT1A是中缝核5 -羟色胺神经元上的一种体树突自身受体，在边缘系统中也以最高的密度在突触后表达。药物行为学研究揭示了5HT1A在激惹攻击中的重要作用。我们在胞外结构域的氨基端检测到两个罕见的氨基酸取代。Gly22Ser[罕见等位基因0.002]在3名芬兰白种人中被观察到。Ile28Val[罕见等位基因0.005]存在于各种人群中。体外表达研究未发现这些等位基因对配体结合或转导有影响。由于同源的b2肾上腺素能受体的两个天然氨基末端取代会影响下调，因此我们探索了这些取代对受体下调的影响。暴露于激动剂8-OH DPAT 24小时后，Ser22等位基因对下调受体无效。我们在5HT2A受体的细胞内环中检测到两个错义替换：Ala447Val[等位基因频率0.007]和His452Tyr[等位基因频率0.093]。由于Tyr452的高频率和真实的5HT2A在血小板中的表达，我们能够比较8个His452/Tyr452杂合子和8个匹配性别、年龄和诊断的His452/His452纯合子的His452和Tyr452等位基因的功能特性。10 mM血清素后，His452/Tyr452杂合子的钙动员减少，细胞内刺激水平的衰减延长。这些观察结果在瞬时转染的细胞中得到了重复。我们检测到的5HT2C Cys23Ser变异的等位基因频率为0.13。变异氨基酸残基再次被认为位于氨基端胞外结构域。HTR2C是x链的；因此，13%的雄性与Ser23半合子，87%的雄性与Cys23半合子。这两种等位基因分别在两种高度不同的细胞环境中表达：cos-7肾细胞和爪蟾卵母细胞。在两种表达系统中，Ser23等位基因对MCPP和5HT的亲和力在cos-7细胞中均降低。我们检测到的其他改变配体亲和力的变异是常见的阿片样物质OPRM1受体变异Asn40Asp，以及改变亲和力和转导的DRD2多巴胺多态性Ser311Cys。体内研究了与焦虑和体外转录改变相关的血清素转运体[SLCA4]多态性的功能。如果SLCA4与焦虑联系的机制是改变转录，一个重要的验证步骤将是证明多态性对人脑中血清素转运体密度的影响。B-CIT SPECT成像用于研究多巴胺转运体（纹状体中可见）和血清素转运体（中脑中定量）的基因型/转运体密度。这些研究由A. Heinz领导并与NIMH合作；液化天然气是遗传成分。首先，我们发现多巴胺转运体等位基因与多巴胺转运体密度有显著关系。这种关联的方向与多巴胺转运体等位基因与多动症的关联以及与降低DAT密度相关的等位基因与可卡因诱导的偏执的关系是一致的。接下来，在42名酗酒者和对照组中发现SLCA4与血清素转运体密度有关。以基因型和诊断为预测变量，采用双向方差分析评估中脑血清素转运体密度。在对照中，较低的转录s等位基因确实与较低的转运体密度相关。在酗酒者中，基因型与转运体密度没有关系。我们推测酗酒者在转运体功能上有持续的变化，例如由于酒精诱导的血清素释放或戒断的影响。我们和其他人发现了精神分裂症的TDT与功能性COMT多态性Val158Met的联系，但我们将这一发现扩展到执行认知表现和体内脑代谢活动。在75名对照组、184名精神分裂症患者和222名精神分裂症患者的兄弟姐妹中，威斯康星卡片分类的表现与COMT基因型进行了比较，结果发现精神分裂症患者和对照组的持久性错误都存在显著的等位基因剂量关系。对额叶代谢活动的研究直接扩展了这一发现。基因型个体在N-back任务期间使用血氧水平依赖[BOLD] fMRI进行评估，该任务访问这些前额叶认知功能。正如预测的那样，在这项记忆任务中，Val158等位基因与额叶代谢活动的增加有关，这与皮层效率下降的假设相一致。因此，Val158等位基因似乎损害了相关的前额叶功能，可能是精神分裂症和其他涉及认知执行功能的疾病的易感基因。

LNG is a groundbreaking laboratory for studies relating brain gene polymorphisms to in vitro function as well as in vivo functional measures obtained by brain imaging, and through collaborations with NIMH. The polymorphism/function associations are congruent with the known molecular neurobiology of the transmitters and proteins and include dopamine transporter genotype/dopamine transporter density in striatum, serotonin transporter genotype/serotonin transporter density in raphe nucleus, and COMT genotype/metabolic activity in frontal lobe. Functional variants are the endgame of positional cloning and offer an invaluable tool for selecting the most appropriate phenotypes for linkage studies, for candidate gene hypothesis testing and for improving the prior probability of linkage. LNG is screeening noncoding sequences for effects on transcription and RNA processing, and we are screening certain coding variants of GPCRs for altered ligand affinity, signal transduction and receptor downregulation.In vitroDue to neuroadaptive processes, phenotypic effects of functionally significant alleles may be difficult to discern except in cellular expression systems in which the genetic background on which the various alleles are assayed is identical. We have used transiently transfected cos-7 [mouse kidney] cells and stably transfected CHO-K1 [Chinese hamster ovary] cells for this purpose and are currently using transiently transfected HEK [human embryonic kidney] cells to compare the human HTR5a Pro15 and Ser15 alleles.5HT1A is a somatodendritic autoreceptor on serotonin neurons in the raphe nuclei and is also expressed postsynaptically with highest densities in the limbic system. Pharmacobehavioral studies have revealed a significant role for 5HT1A in irritable aggression. We detected two rare amino acid substitutions in the amino-terminal, extracellular domain. Gly22Ser [rare allele 0.002] was observed in three Finnish Caucasians. Ile28Val [rare allele 0.005] is present in various populations. In vitro expression studies detected no effect of these alleles on ligand binding or transduction. The effect of these substitutions on receptor down-regulation was explored because two naturally occurring amino terminal substitutions of the homologous b2 adrenergic receptor affect down-regulation. The Ser22 allele was ineffective in down-regulating the receptor via 24 hr exposure to the agonist 8-OH DPAT. We detected two missense substitutions in an intracellular loop of the 5HT2A receptor: Ala447Val [allele frequency 0.007] and His452Tyr [allele frequency 0.093. Due to the high frequency of Tyr452 and the expression of authentic 5HT2A in platelets, we were able to compare the functional properties of the His452 and Tyr452 alleles in eight His452/Tyr452 heterozygotes to eight His452/His452 homozygotes matched for sex, age and diagnosis. After 10 mM serotonin, calcium mobilization was reduced in His452/Tyr452 heterozygotes and the decay of stimulated intracellular levels was prolonged. These observations were replicated in transiently transfected cells. A 5HT2C Cys23Ser variant we detected has an allele frequency of 0.13. The variant amino acid residue is again thought to be located in the amino terminal extracellular domain. HTR2C is X-linked; therefore, 13% of males are hemizygous for Ser23 and 87% are hemizygous for Cys23. Both alleles were individually expressed in two highly distinctive cellular environments: cos-7 kidney cells and Xenopus oocytes. In both expression systems, the Ser23 allele showed diminished affinity for MCPP and affinity for 5HT was diminished in cos-7 cells. Other variants which we have detected which alter ligand affinity are the common m opioid OPRM1 receptor variant Asn40Asp, and the DRD2 dopamine polymorphism Ser311Cys, which alters affinity and transduction.In vivoThe functionality of the serotonin transporter [SLCA4] polymorphism which is associated with anxiety and alters in vitro transcription was pursued in vivo. If the mechanism of the SLCA4 linkage to anxiety was to alter transcription, an important validating step would be to demonstrate an effect of the polymorphism on serotonin transporter density in human brain. B-CIT SPECT imaging was used for genotype/transporter density studies of both the dopamine transporter [visualized in striatum] and serotonin transporter [quantitated in midbrain]. These studies were led by A. Heinz and were collaborative with NIMH; LNG was the genetics component. First, we found a significant relationship of dopamine transporter allele to dopamine transporter density. The direction of this association was congruent with dopamine transporter allele associations to ADHD and with the relationship of the allele associated with reduced DAT density to cocaine-induced paranoia. Next SLCA4 was found to be related to serotonin transporter density in 42 alcoholics and controls. Serotonin transporter density in midbrain was evaluated in a two-way ANOVA with genotype and diagnosis as predictor variables. In controls, the lower transcribing s allele was indeed associated with lower transporter density. In alcoholics, there was no relationship of genotype to transporter density. We speculate that alcoholics have sustained changes in transporter function, for example due to alcohol-induced serotonin release or effects of withdrawal.We and others detected TDT linkage of schizophrenia to the functional COMT polymorphism Val158Met, but we have extended this finding to executive cognitive performance and in vivo brain metabolic activity. Wisconsin Card Sort performance was evaluated versus COMT genotype in 75 controls, 184 schizophrenics, and 222 siblings of schizophrenics, with the result that a remarkable allele-dosage relationship was found to perseverative errors in both schizophrenia patients and controls. This finding was directly expanded by a study of frontal lobe metabolic activity. Genotyped individuals were evaluated using blood oxygen level dependent [BOLD] fMRI during the N-back task, which accesses these prefrontal cognitive functions. As predicted, during this memory task the Val158 allele was associated with increased metabolic activity in frontal lobe - consistent with the hypothesis of diminished cortical efficiency. Thus, the Val158 allele appears to compromise relevant prefrontal function and may be a susceptibility gene for schizophrenia and other diseases involving cognitive executive functions.