Silviu Alin Bacanu

An Abundance of Rare Functional Variants in 202 Drug Target Genes Sequenced in 14,002 People

A Deep Look Into Our Genes Recent debates have focused on the degree of genetic variation and its impact upon health at the genomic level in humans (see the Perspective by Casals and Bertranpetit). Tennessen et al. (p. 64, published online 17 May), looking at all of the protein-coding genes in the human genome, and Nelson et al. (p. 100, published online 17 May), looking at genes that encode drug targets, address this question through deep sequencing efforts on samples from multiple individuals. The findings suggest that most human variation is rare, not shared between populations, and that rare variants are likely to play a role in human health. A pharmacogenomics analysis shows how challenging it will be to associate rare variants with phenotypes. Rare genetic variants contribute to complex disease risk; however, the abundance of rare variants in human populations remains unknown. We explored this spectrum of variation by sequencing 202 genes encoding drug targets in 14,002 individuals. We find rare variants are abundant (1 every 17 bases) and geographically localized, so that even with large sample sizes, rare variant catalogs will be largely incomplete. We used the observed patterns of variation to estimate population growth parameters, the proportion of variants in a given frequency class that are putatively deleterious, and mutation rates for each gene. We conclude that because of rapid population growth and weak purifying selection, human populations harbor an abundance of rare variants, many of which are deleterious and have relevance to understanding disease risk.

Genome-Wide Gene-Environment Study Identifies Glutamate Receptor Gene GRIN2A as a Parkinson's Disease Modifier Gene via Interaction with Coffee

Our aim was to identify genes that influence the inverse association of coffee with the risk of developing Parkinsons disease (PD). We used genome-wide genotype data and lifetime caffeinated-coffee-consumption data on 1,458 persons with PD and 931 without PD from the NeuroGenetics Research Consortium (NGRC), and we performed a genome-wide association and interaction study (GWAIS), testing each SNPs main-effect plus its interaction with coffee, adjusting for sex, age, and two principal components. We then stratified subjects as heavy or light coffee-drinkers and performed genome-wide association study (GWAS) in each group. We replicated the most significant SNP. Finally, we imputed the NGRC dataset, increasing genomic coverage to examine the region of interest in detail. The primary analyses (GWAIS, GWAS, Replication) were performed using genotyped data. In GWAIS, the most significant signal came from rs4998386 and the neighboring SNPs in GRIN2A. GRIN2A encodes an NMDA-glutamate-receptor subunit and regulates excitatory neurotransmission in the brain. Achieving P2df = 10−6, GRIN2A surpassed all known PD susceptibility genes in significance in the GWAIS. In stratified GWAS, the GRIN2A signal was present in heavy coffee-drinkers (OR = 0.43; P = 6×10−7) but not in light coffee-drinkers. The a priori Replication hypothesis that “Among heavy coffee-drinkers, rs4998386_T carriers have lower PD risk than rs4998386_CC carriers” was confirmed: ORReplication = 0.59, PReplication = 10−3; ORPooled = 0.51, PPooled = 7×10−8. Compared to light coffee-drinkers with rs4998386_CC genotype, heavy coffee-drinkers with rs4998386_CC genotype had 18% lower risk (P = 3×10−3), whereas heavy coffee-drinkers with rs4998386_TC genotype had 59% lower risk (P = 6×10−13). Imputation revealed a block of SNPs that achieved P2df<5×10−8 in GWAIS, and OR = 0.41, P = 3×10−8 in heavy coffee-drinkers. This study is proof of concept that inclusion of environmental factors can help identify genes that are missed in GWAS. Both adenosine antagonists (caffeine-like) and glutamate antagonists (GRIN2A-related) are being tested in clinical trials for treatment of PD. GRIN2A may be a useful pharmacogenetic marker for subdividing individuals in clinical trials to determine which medications might work best for which patients.

Significant Linkage on Chromosome 10p in Families with Bulimia Nervosa

Bulimia nervosa (BN) is strongly familial, and additive genetic effects appear to contribute substantially to the observed familiality. In turn, behavioral components of BN, such as self-induced vomiting, are reliably measured and heritable. To identify regions of the genome harboring genetic variants conferring susceptibility to BN, we conducted a linkage analysis of multiplex families with eating disorders that were identified through a proband with BN. Linkage analysis of the entire sample of 308 families yielded a double peak, with the highest nonparametric multipoint maximum LOD score (MLS), of 2.92, on chromosome 10. Given the high heritability of self-induced vomiting and the reliability with which it can be measured, we performed linkage analysis in a subset (n=133) of families in which at least two affected relatives reported a symptom pattern that included self-induced vomiting. The highest MLS (3.39) observed was on chromosome 10, between markers D10S1430 and D10S1423. These results provide evidence of the presence of a susceptibility locus for BN on chromosome 10p. Using simulations, we demonstrate that both of these scores, 2.92 and 3.39, meet the widely accepted criterion for genomewide significance. Another region on 14q meets the criterion for genomewide suggestive linkage, with MLSs of 1.97 (full sample) and 1.75 (subset) at 62 centimorgans from p-ter.

Association of multiple DRD2 polymorphisms with anorexia nervosa.

To investigate whether the dopaminergic system plays a role in the etiology of anorexia nervosa (AN) via the dopamine D2 receptor, we investigated association and transmission disequilibrium at seven single-nucleotide polymorphisms (SNPs) spanning about 75 kbp of the gene DRD2. We studied 191 probands with a DSM-IV diagnosis of AN, 457 parents and affected relatives with a DSM-IV eating disorder diagnosis, and 98 unrelated, female, normal weight controls. The −141 C/− insertion/deletion (−141 Indel), previously shown to affect DRD2 transcription efficiency, and multiple exon seven polymorphisms, one of which has previously been shown to affect DRD2 transcript stability, exhibited statistically significant association with diagnosis in haplotype transmission disequilibrium and in haplotype case : control analyses. Significant linkage disequilibrium between the −141 Indel and two exon seven SNPs (939Y and 957Y) was observed over a distance of >50 kbp in the AN probands but not in the controls. Genetically transmitted variation in D2 dopamine receptor expression mediated by functional polymorphisms affecting transcription and translation efficiency may play a role in vulnerability to AN.

Heritability of fearful-anxious endophenotypes in infant rhesus macaques: a preliminary report

BACKGROUND Research efforts to discover the genetic underpinnings of anxiety and depression is challenging because of the etiologic heterogeneity inherent to these disorders. These efforts might be aided by the study of related behavioral phenotypes in model organisms, such as monkeys. METHODS Eighty-five rhesus monkeys (Macaca mulatta) from the Oregon National Primate Research Center were drawn from a standard matriarchal colony and tested for behavioral response in four testing paradigms designed to elicit fearful-anxious reactions. Heritabilities were estimated using variance component-based quantitative genetic analyses with much of the genetic information arising from paternal half-sibs. RESULTS Individual behaviors reflecting increased distress responses (e.g., vocalizations and teeth grinding) and behavioral inhibition (e.g., latency to leave mother, latency to inspect novel fruit) showed significant heritability, even though a small number of monkeys were assessed. Exploratory factor analyses identified seven clusters of behaviors across tests, some of which were found to be heritable. CONCLUSIONS These results indicate that several specific fearful-anxious behaviors in infant rhesus monkeys are heritable within this colony. Accordingly, these phenotypes, which are believed to represent the genetic liability for anxiety and depression, are good candidates for further genetic investigation in this population.

Long-term comparison of tacrolimus- and cyclosporine-induced nephrotoxicity in pediatric heart-transplant recipients

Nephrotoxicity is an adverse effect of cyclosporine and tacrolimus. Studies comparing their long‐term nephrotoxicities are lacking. This study evaluates the nephrotoxicity of these agents over a 7‐year period following heart transplantation. Pediatric heart‐transplant recipients receiving cyclosporine or tacrolimus as primary immunosuppression were evaluated at two centers from 1982 to 1998. Data collected included serum creatinine, height and weight prior to transplantation, at 1 and 6 months and 1 years post transplantation, and at yearly intervals thereafter. Creatinine clearance was calculated and compared between the two groups. Glomerular filtration rate was measured using Tc‐99 m diethylenetriaminepentacetic acid. In total, 123 patients were evaluated. Demographic data of the two groups were comparable. Creatinine clearance demonstrated a steady decline. This decline did not differ statistically between the two groups: tacrolimus 98.9 and 90.7 mL/min/1.73 m2 at 1 month and 5 years, respectively; cyclosporine 110.7 and 81.7 mL/min/1.73 m2 at 1 month and 5 years, respectively. Four patients developed end‐stage renal failure. Calculated creatinine clearance consistently overestimated glomerular filtration rate, the latter being greater than 2 standard deviations below the mean normal in 38% of patients. We conclude that the nephrotoxicities of tacrolimus and cyclosporine are comparable over the medium‐ to long‐term in pediatric heart‐transplant recipients.

Linkage analysis of anorexia and bulimia nervosa cohorts using selected behavioral phenotypes as quantitative traits or covariates

To increase the likelihood of finding genetic variation conferring liability to eating disorders, we measured over 100 attributes thought to be related to liability to eating disorders on affected individuals from multiplex families and two cohorts: one recruited through a proband with anorexia nervosa (AN; AN cohort); the other recruited through a proband with bulimia nervosa (BN; BN cohort). By a multilayer decision process based on expert evaluation and statistical analysis, six traits were selected for linkage analysis (1): obsessionality (OBS), age at menarche (MENAR), and anxiety (ANX) for quantitative trait locus (QTL) linkage analysis; and lifetime minimum body mass index (BMI), concern over mistakes (CM), and food‐related obsessions (OBF) for covariate‐based linkage analysis. The BN cohort produced the largest linkage signals: for QTL linkage analysis, four suggestive signals: (for MENAR, at 10p13; for ANX, at 1q31.1, 4q35.2, and 8q13.1); for covariate‐based linkage analyses, both significant and suggestive linkages (for BMI, one significant [4q21.1] and three suggestive [3p23, 10p13, 5p15.3]; for CM, two significant [16p13.3, 14q21.1] and three suggestive [4p15.33, 8q11.23, 10p11.21]; and for OBF, one significant [14q21.1] and five suggestive [4p16.1, 10p13.1, 8q11.23, 16p13.3, 18p11.31]). Results from the AN cohort were far less compelling: for QTL linkage analysis, two suggestive signals (for OBS at 6q21 and for ANX at 9p21.3); for covariate‐based linkage analysis, five suggestive signals (for BMI at 4q13.1, for CM at 11p11.2 and 17q25.1, and for OBF at 17q25.1 and 15q26.2). Overlap between the two cohorts was minimal for substantial linkage signals.

Psychosocial mediators of racial differences in nighttime blood pressure dipping among normotensive adults.

Shallow declines in nocturnal compared with diurnal blood pressure (BP dipping) have been associated with cardiovascular disease. In U.S. samples, Blacks demonstrate less BP dipping compared with Whites. In a sample of 60 Black and 60 White normotensive adults we examined stress, social integration (including parental status), social support, and hostility as potential mediators of the association between race and BP dipping. The effect of race on diastolic BP dipping was partially mediated by parental status. The effect of race on heart rate dipping was partially mediated by stressful life events. No psychosocial factors mediated the relation between race and systolic BP dipping. Although psychosocial factors appear to account for some of the observed racial differences in nocturnal blood pressure decline, our data suggest that these differences cannot be attributed entirely to covarying psychosocial effects.

Psychotic Symptoms in Alzheimer Disease: Evidence for Subtypes

OBJECTIVE Psychotic symptoms in Alzheimer disease (AD) identify a phenotype with distinct neurobiology and genetic architecture. The authors investigated whether AD with psychosis is homogeneous or is a composite of subtypes. METHODS Authors performed factor and cluster analyses of the psychotic-symptom items of the CERAD Behavioral Rating Scale in 188 probable and possible AD subjects who have displayed at least one psychotic symptom. RESULTS Exploratory factor analysis resulted in a one-factor solution that comprised misidentification delusions, auditory and visual hallucinations, and the misidentification of people. Persecutory delusions were also frequently present and were independent of the misidentification/hallucination factor. Cluster analysis yielded similar results. CONCLUSION Misidentification/hallucinations and persecutory delusions may identify two subtypes of psychosis in AD. Longitudinal study is needed to determine whether these proposed subtypes remain stable and independent over time or merge into a single group over the course of illness.

Deriving behavioural phenotypes in an international, multi-centre study of eating disorders

BACKGROUND An international, multi-site study funded by the Price Foundation has collected 237 affected relative pairs to identify potential genetic factors that may contribute to the pathogenesis of anorexia nervosa (AN). The current report utilized this sample to derive phenotypes from the personality and behavioural traits assessed in a large number of individuals with eating disorders. METHODS Multivariate analytical techniques were used to characterize the relationships among personality (e.g. trait anxiety, perfectionism, harm avoidance, novelty seeking) and behavioural traits (obsessions and compulsions) in individuals with eating disorders (primarily AN; N = 348) and assess the effectiveness of these traits in classifying subjects into diagnostic subtypes. RESULTS Factor analysis revealed that the most influential factor was one of trait anxiety, harm avoidance, perfectionism, obsessive-compulsive behaviours, and diminished self-directedness, although the precise nature of this factor differed slightly across sites. Discriminant analysis was used to evaluate the utility of these personality/behavioural factors in predicting subdiagnosis. Overall, the misclassification rate was 34%; however, there was an 80% rate of accurate classification of those individuals with a diagnosis of restricting-type AN. CONCLUSIONS Trait anxiety, harm avoidance, perfectionism, obsessive-compulsive behaviours and diminished self-directedness may best be conceptualized as parts of the same underlying construct among individuals with eating disorders, particularly AN. These personality and behavioural traits were also found to be of some utility in classifying eating disordered individuals into diagnostic subgroups, particularly those with restricting-type AN. We expect these phenotypic findings to benefit our ongoing search for genetic loci underlying the liability to eating disorders.