Jeremy Veenstra-VanderWeele

Pharmacogenetics and the serotonin system: initial studies and future directions.

Serotonin (5-hydroxytryptamine, 5-HT) appears to play a role in the pathophysiology of a range of neuropsychiatric disorders, and serotonergic agents are of central importance in neuropharmacology. Genes encoding various components of the 5-HT system are being studied as risk factors in depression, schizophrenia, obsessive-compulsive disorder, aggression, alcoholism, and autism. Recently, pharmacogenetic research has begun to examine possible genetic influences on therapeutic response to drugs affecting the serotonin system. Genes regulating the synthesis (TPH), storage (VMAT2), membrane uptake (HTT), and metabolism (MAOA) of 5-HT, as well as a number of 5-HT receptors (HTR1A, HTR1B, HTR2A, HTR2C, and HTR5A), have been studied and this initial research is reviewed here. After a brief introduction to serotonin neurobiology and a general discussion of appropriate genetic methodology, each of the major 5-HT-related genes and their encoded proteins are reviewed in turn. For each gene, relevant polymorphisms and research on functional variants are discussed; following brief reviews of the disorder or trait association and linkage studies, pharmacogenetic studies performed to date are covered. The critical and manifold roles of the serotonin system, the great abundance of targets within the system, the wide range of serotonergic agents-available and in development-and the promising preliminary results suggest that the serotonin system offers a particularly rich area for pharmacogenetic research.

Transmission disequilibrium testing of arginine vasopressin receptor 1A (AVPR1A) polymorphisms in autism.

Impairment in social reciprocity is a central component of autism. In preclinical studies, arginine vasopressin (AVP) has been shown to increase a range of social behaviors, including affiliation and attachment, via the V1a receptor (AVPR1A) in the brain. Both the behavioral effects of AVP and the neural distribution of the V1a receptor vary greatly across mammalian species. This difference in regional receptor expression as well as differences in social behavior may result from a highly variable repetitive sequence in the 5′ flanking region of the V1a gene (AVPR1A). Given this comparative evidence for a role in inter-species variation in social behavior, we explored whether within our own species, variation in the human AVPR1A may contribute to individual variations in social behavior, with autism representing an extreme form of social impairment. We genotyped two microsatellite polymorphisms from the 5′ flanking region of AVPR1A for 115 autism trios and found nominally significant transmission disequilibrium between autism and one of the microsatellite markers by Multiallelic Transmission/Disequilibrium test (MTDT) that was not significant after Bonferroni correction. We also screened approximately 2 kb of the 5′ flanking region and the coding region and identified 10 single nucleotide polymorphisms.

Molecular genetics of autism spectrum disorder.

We are on the brink of exciting discoveries into the molecular genetic underpinnings of autism spectrum disorder. Overwhelming evidence of genetic involvement coupled with increased societal attention to the disorder has drawn in more researchers and more research funding. Autism is a strongly genetic yet strikingly complex disorder, in which evidence from different cases supports chromosomal disorders, rare single gene mutations, and multiplicative effects of common gene variants. With more and more interesting yet sometimes divergent findings emerging every year, it is tempting to view these initial molecular studies as so much noise, but the data have also started to coalesce in certain areas. In particular, recent studies in families with autism spectrum disorder have identified uncommon occurrences of a novel genetic syndrome caused by disruptions of the NLGN4 gene on chromosome Xp22. Previous work had identified another uncommon syndrome that is caused by maternal duplications of the chromosome 15q11–13 region. We highlight other converging findings, point toward those areas most likely to yield results, and emphasize the contributions of multiple approaches to identifying the genes of interest.

Association studies of serotonin system candidate genes in early-onset obsessive-compulsive disorder.

BACKGROUND Family-based evidence for association at serotonin system genes SLC6A4, HTR1B, HTR2A, and brain-derived neurotrophic factor (BDNF) has been previously reported in obsessive-compulsive disorder (OCD). Early-onset OCD is a more familial form of the disorder. METHODS We used the transmission-disequilibrium test of association at common polymorphisms in each of these genes in 54 parent-child trios ascertained through probands with early-onset OCD. RESULTS No evidence for association was detected at any of the polymorphisms in the entire set of subjects. Nominally significant association was found at the HTR2A rs6311 polymorphism in subjects with tic disorder and OCD (p = .05), replicating a previous finding in Tourette syndrome and OCD. Nominally significant association was also found for the SLC6A4 HT transporter gene-linked polymorphic region (5-HTTLPR) polymorphism for female subjects (p = .03). Neither association would remain significant after statistical correction for multiple testing. Despite no individual study reporting replication, a pooled analysis of five replication studies of the SLC6A4 5-HTTLPR polymorphism supports association (p = .02). CONCLUSIONS Low power across individual association studies in OCD may lead to a false acceptance of the null hypothesis. Accumulation of evidence from multiple studies will be necessary to evaluate the potential role for these genes in contributing to susceptibility to OCD.

Genomic organization of the SLC1A1 /EAAC1 gene and mutation screening in early-onset obsessive-compulsive disorder

The first genome scan conducted in early-onset obsessive-compulsive disorder used a non-parametric analysis to identify a peak in a region of chromosome 9 containing the gene SLC1A1, which codes for the neuronal and epithelial glutamate transporter EAAC1. Interaction between the glutamatergic and serotonergic systems within the striatum suggests EAAC1 as a functional candidate in OCD as well. We determined the genomic organization of SLC1A1 primarily by using primers designed from cDNA sequence to amplify from adaptor-ligated genomic DNA restriction fragments. In order to confirm SLC1A1 as a positional candidate in early-onset OCD, common single nucleotide polymorphisms (SNPs) were identified that enabled mapping of SLC1A1 within the region of the lod score peak. Based on the linkage evidence, the coding region was sequenced in the probands of the seven families included in the genome scan. No evidence was found for a functional mutation, but several SNPs were identified. Capillary electrophoresis SSCP typing of a haplotype consisting of two common SNPs within EAAC1 revealed no significant linkage disequilibrium.

Genome-wide association study identifies ITGB3 as a QTL for whole blood serotonin

Serotonin has been implicated in common disorders involving the central nervous, gastrointestinal, cardiovascular, and pulmonary systems. We describe the first genome-wide screen to identify quantitative trait loci (QTLs) influencing whole blood serotonin in 567 members of a single large pedigree, using a novel association-based mapping approach. We identified an association between the β3 integrin (ITGB3) Leu33Pro polymorphism on 17q21 and whole blood serotonin levels (P-value=9.8 × 10−5). This variant explained the evidence for linkage in this region when included as a covariate in the linkage analysis (change in LOD from 1.87 to 0.16), indicating that ITGB3 may be an important serotonin QTL.

Genetics of childhood disorders: XLVI. Autism, Part 5: Genetics of autism

Autism is one of the most heritable complex genetic disorders in psychiatry. Despite this high heritability, autism has a heterogeneous etiology, with multiple genes and chromosomal regions likely to be involved. Scientists are using both indirect and direct approaches to identify autism susceptibility genes. Indirect approaches include the characterization of less complex genetic disorders that share some of the symptoms of autism, including Rett syndrome or fragile X syndrome, in the hope that these analyses will provide clues to the more complex disorder of autism (see recent reviews in this space, February and May 2000). Direct approaches include three overlapping methodologies to identify genes or regions of interest in autism: chromosomal methods, such as karyotyping and fluorescence in situ hybridization (FISH); linkage studies, such as genome screens in affected sibling pairs; and gene association studies, including candidate gene studies. These approaches are yielding preliminary findings that are reviewed here. While no specific gene variant has been identified and confirmed that contributes to the expression of autism, it is very likely that several will be confirmed over the next decade. Twin and sibling studies demonstrate heritability in autism. The monozygotic twin of a patient with autism, who shares nearly 100% of nuclear DNA, has an approximately 60% chance of having autism, while the concordance for an autism spectrum disorder is greater than 90%. The dizygotic twin of a patient, who shares 50% of the genes, has approximately the same risk as a sibling, about 4.5%. A member of the general population has approximately 0.2% chance of having autism. Genetics of Childhood Disorders: XLVI. Autism, Part 5: Genetics of Autism

Mutation screening of the UBE3A/E6-AP gene in autistic disorder

Previous reports of individuals with autistic disorder with maternal duplications of 15q11–q13,1–11 the Prader-Willi/Angelman syndrome region, suggest this area as a source of candidate genes in autistic disorder. Maternal truncation mutations in UBE3A, which encodes for E6-AP ubiquitin-protein ligase, have been shown to cause Angelman syndrome,12,13 which can also result from the absence of maternal chromosomal material from this region. Despite showing no evidence for imprinting in other tissues, this gene was recently discovered to be preferentially maternally expressed in human brain14,15 and expressed solely from the murine maternal chromosome in the hippocampus and cerebellar Purkinje cells,16 regions implicated in the neuropathology of autism.17–20 Based on this evidence, the coding region and a putative promoter region were sequenced in ten autistic subjects. Several polymorphisms were detected, but no evidence was found for a functional mutation. Evidence for likely altered regulation of UBE3A expression in maternal 15q11–q13 duplications suggests further investigation of the regulatory regions of this gene in autistic disorder.

High throughput fluorescent CE-SSCP SNP genotyping.

Large numbers of single nucleotide polymorphisms (SNPs) are being identified by several laboratories for the purpose of developing dense genetic maps. Single-strand conformation polymorphism (SSCP) analysis has been widely used as a method for detecting novel sequence variations in PCR products. Differences in migration of single-stranded DNA can be used not only to find mutations, but to genotype SNPs in large sample populations. Using PCR with fluorescent labeling and automated capillary electrophoresis SSCP (CE-SSCP), we have developed a panel of 15 functional candidate SNPs. With an automated single capillary instrument, relatively rapid and low cost CE-SSCP SNP genotyping using currently available technology is feasible for 135 000 genotypes per year. With parallel multiple array capillary electrophoresis, more genotypes per year may be attainable.

Coupling of optimized multiplex PCR and automated capillary electrophoresis for efficient genome-wide searches

▼Genome-wide searches for linkage have come into frequent use to dissect complex genetic diseases and traits (Ref. 1, 2, 3, 4). The coelectrophoresis of dinucleotide repeat markers using multiple fluorescent labels has allowed investigators to efficiently reach the 10 cM resolution necessary to effectively screen the genome for linkage (Ref. 5, 6, 7). Despite the increased efficiency of coelectrophoresis, however, singleplex amplification of each marker remains a slow and cumbersome process. No one has described multiplex PCR conditions for an entire linkage mapping set. So, in order to increase throughput and decrease error rates, multiplex PCR was optimized for each of the three colors in each of the 28 panels of the ABI PRISM Linkage Mapping Set version 1(Perkin Elmer). These conditions should translate well for use with Linkage Mapping Set version 2, since 52% of the optimized multiplexes consist only of primer sets that were not replaced in the second version. Another 36% have only one primer set that was replaced, while the remaining 12% will probably not be useful. Figure 1 shows the typical progressions as primer concentrations within one multiplexed color are optimized. Amplification was carried out using 0.2 Units of AmpliTaq Gold DNA Polymerase (Perkin Elmer) in 10 mM Tris-HCl, 50 mM KCl, 2.5 mM MgCl2, and 250 μM of each dNTP for a total volume of 5 μL. Thermocycling was performed on a Perkin Elmer GeneAmp PCR System 9600(Perkin Elmer) using an initial hot start at 95 ◦C for 12 minutes, followed by 10 cycles of 94 ◦C for 15 seconds, 55 ◦C for 15 seconds, and 72 ◦C for 30 seconds, then 20 cycles of 89 ◦C for 15 seconds, 55 ◦C for 15 seconds, and 72 ◦C for 30 seconds, and a final extension step of 72 ◦C for 30 minutes. Primer concentrations were varied as shown above the position of each marker. Separation was carried out using capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer (Perkin Elmer). The figure shows the electropherogram of only one of the four subjects used to optimize multiplex conditions. The multiplex shown is Panel 24 [TET], which proved quite difficult to optimize, finally requiring separation into two multiplexes. The bottom two electropherograms correspond to the multiplex of the two markers that failed to amplify within the larger multiplex. D17S928 overlapped with invariant nonspecific products of the multiplex, but this did not interfere with genotyping when all markers in the panel were pooled and electrophoresed. Two panels were run separately for singleplex and then multiplex PCR. The results are presented in (Table 1). Singleplex PCRs were optimized by modifying annealing temperatures and MgCl2 concentration. A comparison between the two methods shows a clear advantage for multiplex PCR in the ratio of marker standard deviation with average peak height, which corresponds to more even peak heights. The difference between the overall between markers standard deviation for the multiplexed markers, 530, and the between markers standard deviation within each multiplex PCR, 309, is the direct result of pooling four tubes of PCR products. By extrapolation, most of the higher standard deviation, relative to average peak height, for singleplex PCR