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.
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