Charles Curtis

DNA from Buccal Swabs Recruited by Mail: Evaluation of Storage Effects on Long-Term Stability and Suitability for Multiplex Polymerase Chain Reaction Genotyping

We provide details of an inexpensive and rapid method for extraction of DNA from buccal swabs (including samples received through the mail) and from a range of other tissue samples. The procedure we have developed provides amounts of DNA adequate for several thousand polymerase chain reactions (PCRs), and we have validated its potential for long-term storage. Samples stored for >4 years are of comparable concentration and provide as robust PCR templates as those tested immediately after extraction. The availability of this technology is of considerable significance in planning DNA banks from population collections and cohorts.

The serotonin transporter and clozapine response.

SIR – Smeraldi and collaborators reported that genetic variants of the serotonin transporter (5-HTT) gene are associated with levels of response to the antidepressant fluvoxamine. Individuals homozygous for a short allele (480 bp) of a polymorphism in the promoter region (5HTTLPR) of the gene showed poorer response to the antidepressant fluvoxamine than subjects heterozygous or homozygous for the long allele (520 bp). Variation in the 5-HT uptake may also affect antipsychotic action mediated through the serotonergic system. We have previously reported association between polymorphisms in serotonin receptors and response to clozapine, a drug that displays high levels of affinity for them. However, those associations could not fully explain the heterogeneous response to clozapine treatment observed and contribution from other genes has been hypothesised. We have tested the contribution of the 5-HTT genetic variants in determining clozapine response by genotyping two polymorphisms (the biallelic polymorphism in the promoter region of the gene, 5-HTTLPR, studied by Smeraldi and collaborators, and a VNTR in intron 2 of the gene) in a sample of subjects undergoing clozapine treatment. Two hundred and sixty-eight schizophrenic patients were included in this study. The patients were white Caucasians of British origin and were collected in the London, Cambridge and Burnley areas. All patients had a DSM-IIIR or DSM-IV diagnosis of schizophrenia and had shown no improvement after treatment with at least two neuroleptics before undergoing treatment with clozapine. Treatment response was assessed at least 3 months after optimisation of therapeutic doses. Response was evaluated using the Global Assessment Scale (GAS) and an improvement of 20 points was considered as the threshold for appropriate response. According to this criteria the sample included 180 subjects who improved satisfactorily and 88 patients who showed poor response to clozapine treatment. Informed consent was obtained from all the subjects included in the study. The 5-HTT VNTR in intron 2 and the biallelic polymorphism in the linked polymorphic region (5HTTLPR) were genotyped following the methods previously described. The genotyping results are summarised in Table 1. Chi-square tests were calculated for all comparisons using the SPSS (version 8.0) and EpiInfo (version 6) statistical packages. Haplotype analyses were performed using the EH programme (version 1.11). This sample had an 80% power to detect association with an odds ratio of 2.2. No differences were observed in allele or genotype frequencies of the 5-HTT VNTR polymorphism in intron 2 when comparing patients who showed marked improvement with Table 1 Genotype frequencies of the 5-HTT VNTR (intron 2) and the 5-HTTLPR polymorphisms in a sample of clozapinetreated patients

Paraoxonase in Persian Gulf War veterans

Learning ObjectivesSummarize what previous studies have shown about possible causes of low serum paraoxonase (PON1).Recall what this study showed about serum PON1 activity in Gulf War veterans, in veterans deployed elsewhere, and its relation to symptoms.Present possible explanations of group differences in serum PON1 activity. Serum paraoxonase (PON1) is responsible for the metabolism of organophosphates in serum, and PON1 activity is a major determinant of their toxicity in humans. There have been reports linking lowered PON1 activity to physical symptoms after deployment to the Persian Gulf War (PGW) of 1990 to 1991. Therefore, the object of this study was to determine (1) whether PON1 activity was decreased among symptomatic PGW veterans compared with asymptomatic PGW veterans and (2) to determine whether PGW veterans as a whole had lower PON1 activity compared with other military control groups. This was a case control study nested in occupational cohort study of military personnel. Four groups of military personnel were identified from a large epidemiological study of health effects of deployment to the PGW and Bosnia: (1) symptomatic PGW veterans, n = 115; (2) healthy PGW veterans, n = 95; (3) symptomatic Bosnia peacekeeping veterans, n = 52; and (4) symptomatic nondeployed military controls, n = 85. The main outcome measures were PON1 activity and genotype for PON1–55 and -192. We found significant differences in PON1 activity among these four groups, and although the two Gulf groups did not differ in PON1 activity, those deployed to the Gulf had significantly lower PON1 activity compared with the non-PGW groups (median difference = 70.9; 95% CI: 20.2, 121.5, P = 0.012). These differences were not explained by a range of potential confounders, or differences in PON1 coding region polymorphisms. PON1 activity is reduced in PGW veterans compared with military control groups. The effect is independent of ill health in PGW veterans.

In search of genes associated with risk for psychopathic tendencies in children: a two‐stage genome‐wide association study of pooled DNA

BACKGROUND Quantitative genetic data from our group indicates that antisocial behaviour (AB) is strongly heritable when coupled with psychopathic, callous-unemotional (CU) personality traits. We have also demonstrated that the genetic influences for AB and CU overlap considerably. We conducted a genome-wide association scan that capitalises on these findings in an attempt to identify quantitative trait loci (QTLs) that may increase risk for psychopathic tendencies (AB+/CU+). METHODS Teacher ratings at age 7 were used to screen 8374 twins with available DNA samples for individuals that were high vs. low on both AB and CU. In Stage 1, we screened for allele frequency differences in 642,432 autosomal single-nucleotide polymorphisms (SNPs) using the Affymetrix 6.0 GeneChip with pooled DNA for high-scoring (AB+/CU+) versus low-scoring children (N = approximately 300/group). In Stage 2, we tested the 3000 most strongly associated SNPs from Stage 1 for association in the same direction in a second sample of high- versus low-scoring children from the same twin study (18% co-twins). RESULTS Using allele frequencies estimated from pooled DNA, we found suggestive evidence for enrichment of association in the second stage of our two-stage genome-wide association design and focus on reporting the 30 top-ranking SNPs nominally associated with psychopathic tendencies. These SNPs include neurodevelopmental genes such as ROBO2. CONCLUSIONS Although none of the SNPs reached genome-wide statistical significance we have generated a list of SNPs that are potentially associated with psychopathic tendencies, which we believe warrant verification and replication in large independent and clinical samples.

The correlation between reading and mathematics ability at age twelve has a substantial genetic component

Dissecting how genetic and environmental influences impact on learning is helpful for maximizing numeracy and literacy. Here we show, using twin and genome-wide analysis, that there is a substantial genetic component to children’s ability in reading and mathematics, and estimate that around one half of the observed correlation in these traits is due to shared genetic effects (so-called Generalist Genes). Thus, our results highlight the potential role of the learning environment in contributing to differences in a child’s cognitive abilities at age twelve.

An Examination of Polygenic Score Risk Prediction in Individuals With First-Episode Psychosis

BACKGROUND Polygenic risk scores (PRSs) have successfully summarized genome-wide effects of genetic variants in schizophrenia with significant predictive power. In a clinical sample of first-episode psychosis (FEP) patients, we estimated the ability of PRSs to discriminate case-control status and to predict the development of schizophrenia as opposed to other psychoses. METHODS The sample (445 case and 265 control subjects) was genotyped on the Illumina HumanCore Exome BeadChip with an additional 828 control subjects of African ancestry genotyped on the Illumina Multi-Ethnic Genotyping Array. To calculate PRSs, we used the results from the latest Psychiatric Genomics Consortium schizophrenia meta-analysis. We examined the association of PRSs with case-control status and with schizophrenia versus other psychoses in European and African ancestry FEP patients and in a second sample of 248 case subjects with chronic psychosis. RESULTS PRS had good discriminative ability of case-control status in FEP European ancestry individuals (9.4% of the variance explained, p < 10-6), but lower in individuals of African ancestry (R2 = 1.1%, p = .004). Furthermore, PRS distinguished European ancestry case subjects who went on to acquire a schizophrenia diagnosis from those who developed other psychotic disorders (R2 = 9.2%, p = .002). CONCLUSIONS PRS was a powerful predictor of case-control status in a European sample of patients with FEP, even though a large proportion did not have an established diagnosis of schizophrenia at the time of assessment. PRS was significantly different between those case subjects who developed schizophrenia from those who did not, although the discriminative accuracy may not yet be sufficient for clinical utility in FEP.

Pharmacogenetics of antidepressant response: A polygenic approach

Background: Major depressive disorder (MDD) has a high personal and socio‐economic burden and > 60% of patients fail to achieve remission with the first antidepressant. The biological mechanisms behind antidepressant response are only partially known but genetic factors play a relevant role. A combined predictor across genetic variants may be useful to investigate this complex trait. Methods: Polygenic risk scores (PRS) were used to estimate multi‐allelic contribution to: 1) antidepressant efficacy; 2) its overlap with MDD and schizophrenia. We constructed PRS and tested whether these predicted symptom improvement or remission from the GENDEP study (n = 736) to the STAR*D study (n = 1409) and vice‐versa, including the whole sample or only patients treated with escitalopram or citalopram. Using summary statistics from Psychiatric Genomics Consortium for MDD and schizophrenia, we tested whether PRS from these disorders predicted symptom improvement in GENDEP, STAR*D, and five further studies (n = 3756). Results: No significant prediction of antidepressant efficacy was obtained from PRS in GENDEP/STAR*D but this analysis might have been underpowered. There was no evidence of overlap in the genetics of antidepressant response with either MDD or schizophrenia, either in individual studies or a meta‐analysis. Stratifying by antidepressant did not alter the results. Discussion: We identified no significant predictive effect using PRS between pharmacogenetic studies. The genetic liability to MDD or schizophrenia did not predict response to antidepressants, suggesting differences between the genetic component of depression and treatment response. Larger or more homogeneous studies will be necessary to obtain a polygenic predictor of antidepressant response. HIGHLIGHTSAntidepressant efficacy, major depressive disorder and schizophrenia are complex polygenic traits.Genetic overlap has been previously demonstrated among different psychiatric disorders.Polygenic risk scores did not predict antidepressant efficacy between two independent samples.Polygenic risk scores did not show overlap between antidepressant efficacy and major depressive disorder.Polygenic risk scores did not show overlap between antidepressant efficacy and schizophrenia.

Plasma protein biomarkers of Alzheimer’s disease endophenotypes in asymptomatic older twins: early cognitive decline and regional brain volumes

There is great interest in blood-based markers of Alzheimer’s disease (AD), especially in its pre-symptomatic stages. Therefore, we aimed to identify plasma proteins whose levels associate with potential markers of pre-symptomatic AD. We also aimed to characterise confounding by genetics and the effect of genetics on blood proteins in general. Panel-based proteomics was performed using SOMAscan on plasma samples from TwinsUK subjects who are asymptomatic for AD, measuring the level of 1129 proteins. Protein levels were compared with 10-year change in CANTAB-paired associates learning (PAL; n=195), and regional brain volumes (n=34). Replication of proteins associated with regional brain volumes was performed in 254 individuals from the AddNeuroMed cohort. Across all the proteins measured, genetic factors were found to explain ~26% of the variability in blood protein levels on average. The plasma level of the mitogen-activated protein kinase (MAPK) MAPKAPK5 protein was found to positively associate with the 10-year change in CANTAB-PAL in both the individual and twin difference context. The plasma level of protein MAP2K4 was found to suggestively associate negatively (Q<0.1) with the volume of the left entorhinal cortex. Future studies will be needed to assess the specificity of MAPKAPK5 and MAP2K4 to eventual conversion to AD.

Genome-wide association study using family-based cohorts identifies the WLS and CCDC170/ESR1 loci as associated with bone mineral density

BackgroundOsteoporosis is a common and debilitating bone disease that is characterised by a low bone mineral density (BMD), a highly heritable trait. Genome-wide association studies (GWAS) have proven to be very successful in identifying common genetic variants associated with BMD adjusted for age, gender and weight, however a large portion of the genetic variance for this trait remains unexplained. There is evidence to suggest significant genetic correlation between body size traits and BMD. It has also recently been suggested that unintended bias can be introduced as a result of adjusting a phenotype for a correlated trait. We performed a GWAS meta-analysis in two populations (total n = 6,696) using BMD data adjusted for only age and gender, in an attempt to identify genetic variants associated with BMD including those that may have potential pleiotropic effects on BMD and body size traits.ResultsWe observed a single variant, rs2566752, associated with spine BMD at the genome-wide significance level in the meta-analysis (P = 3.36 × 10−09). Logistic regression analysis also revealed an association between rs2566752 and fracture rate in one of our study cohorts (P = 0.017, n = 5,654). This is an intronic variant located in the wntless Wnt ligand secretion mediator (WLS) gene (1p31.3), a known BMD locus which encodes an integral component of the Wnt ligand secretion pathway. Bioinformatics analyses of variants in moderate LD with rs2566752 produced strong evidence for a regulatory role for the variants rs72670452, rs17130567 and rs1430738. Expression quantitative trait locus (eQTL) analysis suggested that the variants rs12568456 and rs17130567 are associated with expression of the WLS gene in whole blood, cerebellum and temporal cortex brain tissue (P = 0.034–1.19 × 10−23). Gene-wide association testing using the VErsatile Gene-based Association Study 2 (VEGAS2) software revealed associations between the coiled-coil domain containing 170 (CCDC170) gene, located adjacent to the oestrogen receptor 1 (ESR1) gene, and BMD at the spine, femoral neck and total hip sites (P = 1.0 × 10−06, 2.0 × 10−06 and 2.0 × 10−06 respectively).ConclusionsGenetic variation at the WLS and CCDC170/ESR1 loci were found to be significantly associated with BMD adjusted for only age and gender at the genome-wide level in this meta-analysis.

Genome-wide association study of response to cognitive–behavioural therapy in children with anxiety disorders

Background Anxiety disorders are common, and cognitive–behavioural therapy (CBT) is a first-line treatment. Candidate gene studies have suggested a genetic basis to treatment response, but findings have been inconsistent. Aims To perform the first genome-wide association study (GWAS) of psychological treatment response in children with anxiety disorders (n = 980). Method Presence and severity of anxiety was assessed using semi-structured interview at baseline, on completion of treatment (post-treatment), and 3 to 12 months after treatment completion (follow-up). DNA was genotyped using the Illumina Human Core Exome-12v1.0 array. Linear mixed models were used to test associations between genetic variants and response (change in symptom severity) immediately post-treatment and at 6-month follow-up. Results No variants passed a genome-wide significance threshold (P = 5 × 10−8) in either analysis. Four variants met criteria for suggestive significance (P<5 × 10−6) in association with response post-treatment, and three variants in the 6-month follow-up analysis. Conclusions This is the first genome-wide therapygenetic study. It suggests no common variants of very high effect underlie response to CBT. Future investigations should maximise power to detect single-variant and polygenic effects by using larger, more homogeneous cohorts.