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 vitro. Due 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. One goal is to create an experimental approach for determining the effects of alleles with potential functional significance. To measure """"""""downstream"""""""" 5HT5A receptor-mediated effects that are mediated through activation of adenyclyclase, we have developed a reporter gene construct incorporating a cyclic AMP response element (CRE) promoter flanking the coding sequence for a red-shifted variant of Green Fluorescent Protein (GFP), which allows one to follow gene expression in living cells as well as providing quantitative data when fluorescence intensity is monitored. CRE elements are binding sites for the cyclic AMP binding protein (CREB) transcription factor. Differentiated PC12 cells express D1 receptors, which will be used as a positive control for CREB activation. CREB activation will then be determined by monitoring GFP production, which can also be performed in living cells by monitoring the effects of dopamine or serotonin on dopamine D1 and 5HT2A receptor co-expressing cell lines using the CREB-GFP reporter construct. Results of these experiments will provide additional justification for examining molecular, pharmacological, and behavioral studies in variant expressing 5T5A receptor subunit """"""""knock-in"""""""" transgenic mice. 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 vivo. The 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. Frontal cognitive function is critical in alcoholism and other substance abuse, where impulse control may be impaired, in SZ, where specific deficits have been identified, and in behavioral disinhibition syndrome following head injury. A common (allele frequency=0.42) polymorphism which affects COMT enzyme activity was linked to frontal cortical function in several datasets with widely differing baseline performances. Gene dosage relationship to performance on the Wisconsin Card Sort pas seen in controls, head injured patients, and SZs. There was also a significant relationship to performance in well sibs of SZs and there was TDT linkage to SZ. These studies were followed by metabolic brain imaging (BOLD, MRI) during a working memory task which accesses functions of the frontal lobe. Both dorsolateral frontal cortex and the functionally related anterior cingulate showed a significant relationship to COMT genotype and, as predicted, the Val158 allele correlated with cortical insufficienty during the working memory task.
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