Characterization of serotonin transporter gene (SLC6A4) polymorphisms and its association with drug dependence in a Jordanian Arab population.

Drug dependence is a pattern of repeated self-administration of a drug, which can result in tolerance, withdrawal and compulsive drug-taking behaviour. It has been recently suggested that 5-HTTLPR (LL/LS/SS) variants and rs25531 (A/G) polymorphism in the serotonin transporter gene (SLC6A4) may play a role in drug dependence. The current study aimed to (1) identify allelic, haplotypic and genotypic frequencies of the 5-HTTLPR variants and rs25531 polymorphisms of SLC6A4 gene in drug and nondrug-dependent Jordanian Arab population and (2) determine whether there is an association of these variants in a drug-dependent population from the same area. Jordanian drug male addicts of Arab descent (n = 192) meeting the Diagnostic and Statistical Manual of Mental Disorders - Fourth edition criteria for drug dependence and 230 healthy male controls from an ethnically homogenous Jordanian Arab population were examined. Genotyping was performed using the restriction fragment length polymorphism-polymerase chain reaction-based method to genotype the 5-HTTLPR variants and detect the A/G polymorphism at position rs25531. The biallelic analysis revealed that the frequency of 5-HTTLPR (LL/LS/SS) genotypes was statistically significant different between drug-dependent individuals and controls (χ (2) (2, N = 422), p = 0.04). Drug-dependent subjects had a higher frequency of 'L' allele. However, using the triallelic approach, the estimated frequency of haplotypes (SA , SG , LA and LG ) and phased genotypes (LA /LA , LA /SA , LA /LG , SA /SA and SA /SG) did not show significant association with drug dependence (χ (2) (3, N = 422), p = 0.53 and χ (2) (4, N = 422), p = 0.06, respectively). This study suggests a putative role of the 5-HTTLPR for drug dependence in the Jordanian Nationals of Arab ancestry.

Genetics of psychiatric disorders in the GWAS era: an update on schizophrenia.

The influence of genetic factors in the development of schizophrenia has been convincingly demonstrated by family, twin, and adoption studies. The statistical construct of heritability is generally used for estimating the liability due to genetic factors. Heritability estimates for schizophrenia are reported to be between 60 and 80 %. Due to the technical achievements in whole genome-wide association studies, dissection of the underlying genetic factors was intensified recently, resulting in the conclusion that schizophrenia is essentially a polygenic, complex disorder. Most likely more than 100 genes, each with small effect size, contribute to disease risk. A most recent multi-stage genome-wide association study (Ripke et al. in Nat Genet 2013) identified 22 risk loci and estimated that 8,300 independent single-nucleotide polymorphisms contributed to the risk accounting collectively for 32 % in liability. In addition to this polygenic, complex inheritance, there is also strong indication that in some patients a deletion or insertion of a larger chromosomal region [so-called copy number variation (CNV)] might play a crucial role in pathogenesis. This could be specifically important in sporadic cases with schizophrenia, since a higher frequency of de novo mutations has been associated with these CNVs. Further studies, combining much larger sample sizes as well as application of newer technology, such as deep sequencing technologies will be necessary in order to obtain a more comprehensive understanding of the genetic foundations of schizophrenia.

Schizophrenia risk conferred by rare protein-truncating variants is conserved across diverse human populations.

Schizophrenia (SCZ) is a chronic mental illness and among the most debilitating conditions encountered in medical practice. A recent landmark SCZ study of the protein-coding regions of the genome identified a causal role for ten genes and a concentration of rare variant signals in evolutionarily constrained genes1. This recent study-and most other large-scale human genetics studies-was mainly composed of individuals of European (EUR) ancestry, and the generalizability of the findings in non-EUR populations remains unclear. To address this gap, we designed a custom sequencing panel of 161 genes selected based on the current knowledge of SCZ genetics and sequenced a new cohort of 11,580 SCZ cases and 10,555 controls of diverse ancestries. Replicating earlier work, we found that cases carried a significantly higher burden of rare protein-truncating variants (PTVs) among evolutionarily constrained genes (odds ratio = 1.48; P = 5.4 × 10-6). In meta-analyses with existing datasets totaling up to 35,828 cases and 107,877 controls, this excess burden was largely consistent across five ancestral populations. Two genes (SRRM2 and AKAP11) were newly implicated as SCZ risk genes, and one gene (PCLO) was identified as shared by individuals with SCZ and those with autism. Overall, our results lend robust support to the rare allelic spectrum of the genetic architecture of SCZ being conserved across diverse human populations.

Multi-trait genome-wide association study of opioid addiction: OPRM1 and beyond.

Opioid addiction (OA) is moderately heritable, yet only rs1799971, the A118G variant in OPRM1, has been identified as a genome-wide significant association with OA and independently replicated. We applied genomic structural equation modeling to conduct a GWAS of the new Genetics of Opioid Addiction Consortium (GENOA) data together with published studies (Psychiatric Genomics Consortium, Million Veteran Program, and Partners Health), comprising 23,367 cases and effective sample size of 88,114 individuals of European ancestry. Genetic correlations among the various OA phenotypes were uniformly high (rg > 0.9). We observed the strongest evidence to date for OPRM1: lead SNP rs9478500 (p = 2.56 × 10-9). Gene-based analyses identified novel genome-wide significant associations with PPP6C and FURIN. Variants within these loci appear to be pleiotropic for addiction and related traits.

A family-based association study of DNA sequence variants in GRM7 with schizophrenia in an Indonesian population.

We previously reported genome-wide significant linkage to chromosome 3p in a sib-pair family sample from Indonesia. A promising candidate gene within the linked region is the metabotropic glutamate receptor subtype 7 (GRM7), involved in glutamatergic neurotransmission. We genotyped 18 single nucleotide polymorphisms in GRM7 in the sample of 124 Indonesian sib-pair families that had provided the significant linkage finding. Transmission disequilibrium analysis revealed nominally significant transmission distortion of rs17031835 in intron 1 of GRM7 (p=0.004, before correction for multiple testing), along with haplotypes containing rs17031835. No other single marker was found to be significantly associated with schizophrenia in our sample. The results from our study provide support for the idea that glutamatergic neurotransmission and specifically the GRM7 gene might be relevant to the development of schizophrenia. Further studies supporting this finding are warranted.

Association of CamK2A genetic variants with transition time from occasional to regular heroin use in a sample of heroin-dependent individuals.

OBJECTIVES: Susceptibility to heroin dependence is strongly influenced by genetic factors with heritability estimates as high as 0.7. A number of genes, as well as environmental factors, are likely to contribute to its etiology. Not all individuals who have ever tried heroin at some stage during their lifetime become dependent on heroin. It has been suggested that genetic factors might be more important in the transition stage to heroin dependence rather than in environmental exposures and experimenting with heroin. As the features of substance dependence and memory formation have been found to be strikingly similar, we have focused on a key enzyme involved in long-term potentiation and synaptic plasticity, namely the calcium-dependent/calmodulin-dependent protein kinase IIα (CAMKIIa). We hypothesized, that CamK2A genetic variation may play a role in the transition from occasional to regular heroin use. MATERIALS AND METHODS: Using quantitative trait association analysis, we addressed this hypothesis by correlating the self-reported time interval between occasional and regular heroin use with the frequency of 12 single nucleotide polymorphisms located within the genomic region of the CamK2A gene. A sample of 570 Caucasian patients was available for analysis. RESULTS: Single marker association analysis (rs10066581, P=0.007), as well as haplotype analysis (global P=0.005), suggested an association with the quantitative trait 'time interval from occasional to regular heroin use.' CONCLUSION: Our results propose that genetic variants located in the genomic region of the CamK2A gene may be involved in transition time from occasional to regular heroin use.

Comparative genetic architectures of schizophrenia in East Asian and European populations.

Schizophrenia is a debilitating psychiatric disorder with approximately 1% lifetime risk globally. Large-scale schizophrenia genetic studies have reported primarily on European ancestry samples, potentially missing important biological insights. Here, we report the largest study to date of East Asian participants (22,778 schizophrenia cases and 35,362 controls), identifying 21 genome-wide-significant associations in 19 genetic loci. Common genetic variants that confer risk for schizophrenia have highly similar effects between East Asian and European ancestries (genetic correlation = 0.98 ± 0.03), indicating that the genetic basis of schizophrenia and its biology are broadly shared across populations. A fixed-effect meta-analysis including individuals from East Asian and European ancestries identified 208 significant associations in 176 genetic loci (53 novel). Trans-ancestry fine-mapping reduced the sets of candidate causal variants in 44 loci. Polygenic risk scores had reduced performance when transferred across ancestries, highlighting the importance of including sufficient samples of major ancestral groups to ensure their generalizability across populations.

DNA sequence variants in the metabotropic glutamate receptor 3 and risk to schizophrenia: an association study.

OBJECTIVES: Recent studies investigating the association of DNA variants in the metabotropic glutamate receptor gene (GRM3) with schizophrenia susceptibility revealed conflicting results. In this study, we focused on DNA sequence variants, for which association was reported and attempted to replicate association with schizophrenia or with cognitive deficits known to be present in patients with schizophrenia. METHODS: A sample of 242 families with affected offspring and five single nucleotide markers located in the genomic region of GRM3 has been used to replicate association with schizophrenia. In addition, results of neuropsychological tests, trail making test B and the Stroop color-naming task were available for a subgroup of these families (N=88) and an independent sample of 148 patients with schizophrenia. Correlation of these measurements with genotypic data was performed using analysis of variance. RESULTS: No statistical evidence for association with schizophrenia or correlation with cognitive deficits as measured by the trail making test B or the Stroop color-naming task and the five DNA sequence variants could be detected. A trend towards association with schizophrenia was revealed for a single marker (rs2237562, P=0.056) and for 2-marker and 3-marker haplotypes containing this variant. CONCLUSIONS: Our study of DNA sequence variants in the GRM3 gene did not provide further support for genetic association with schizophrenia or for correlation with cognitive deficits.

Nicotine self-administration in mice is associated with rates of nicotine inactivation by CYP2A5.

RATIONALE: Cyp2a5, the mouse homologue of human CYP2A6, encodes for the enzyme responsible for the primary metabolism of nicotine. Variation in human CYP2A6 activity can alter the amount smoked such as number of cigarettes smoked per day and smoking intensity. Different mouse strains self-administer different amounts of oral nicotine and quantitative trait loci analyses in mice suggested that Cyp2a5 may be involved in differential nicotine consumption behaviors. OBJECTIVES: The goal of this study was to examine whether in vivo nicotine consumption levels were associated with CYP2A5 protein levels and in vitro nicotine metabolism in mice. METHODS: F2 mice propagated from high (C57Bl/6) and low (St/bJ) nicotine consuming mice were analyzed for CYP2A5 hepatic protein levels and in vitro nicotine metabolizing activity. RESULTS: We found that F2 male high-nicotine (n=8; 25.1+/-1.2 microg nicotine/day) consumers had more CYP2A5 protein, compared to low (n=11; 3.8+/-1.4 microg nicotine/day) consumers (10.2+/-1.0 vs 6.5+/-1.3 CYP2A5 units). High consumers also metabolized nicotine faster than the low consumers (6 microM: 0.18+/-0.04 vs 0.14+/-0.07; 30 microM: 0.36+/- 0.06 vs 0.26+/-0.13; 60 microM: 0.49+/-0.05 vs 0.32+/-0.17 nmol/min/mg). In contrast, female high- (25.1+/-2.1 microg nicotine/day) and low-nicotine (4.7+/-1.4 microg nicotine/day) consumers did not show pronounced differences in nicotine metabolism or CYP2A4/5 protein levels; this is consistent with other studies of sex differences in response to nicotine. CONCLUSIONS: These data suggested that among male F2 mice, increased nicotine self-administration is associated with increased rates of nicotine metabolism, most likely, as a result of greater CYP2A5 protein levels.

Further evidence for association of variants in the AKT1 gene with schizophrenia in a sample of European sib-pair families.

BACKGROUND: Involvement of AKT signaling pathways in schizophrenia has been recently suggested, on the basis of several lines of evidence. In addition to impairment of protein-levels and phosphorylation levels in the pathway, association of DNA sequence variants in the AKT1 gene with schizophrenia has been detected in a family sample. METHODS: We investigated the reported association of DNA sequence variants in the AKT1 gene in a sample of 79 sib-pair families with schizophrenia using the five single nucleotide polymorphisms (SNPs) of the original study and two additional SNPs in the neighborhood of the SNP for which association had been reported. RESULTS: We obtained statistical significance for single markers (p = .002) and multilocus haplotypes (p = .0013) located in the same region as has been reported in the previous study. CONCLUSIONS: The replication of association of variants in the AKT1 gene in a family sample with similar ethnical background as in the original study adds further evidence for involvement of AKT1 in development of schizophrenic disorders.

Association of DNA polymorphisms in the synaptic vesicular amine transporter gene (SLC18A2) with alcohol and nicotine dependence.

The brain synaptic vesicular amine transporter SLCA18A2 is a key component for the uptake of monoamines like dopamine or serotonin into vesicles. We have analyzed seven DNA polymorphisms located in the genomic region of SLC18A2 for association with alcohol- and nicotine dependence, using a family-based design. Our sample comprised 131 families with alcohol-dependent offspring and 96 families with at least one nicotine-dependent offspring. For the alcohol-dependent sample, we found statistical significant association for two single markers (rs363387, P=0.03; rs363333, P=0.0066) as well as for several haplotypes (minimal P=0.0038). When the sample with alcohol dependence was stratified according to gender, we observed increased association for the male subgroup (rs363387, P=0.0011). None of the markers showed association in the sample of families with nicotine dependence. However, analysis of a combined sample of alcohol and nicotine-dependent families resulted in single markers as well as several haplotypes showing statistical significant association with substance dependence (minimal P=0.0044). We conclude that DNA polymorphisms located in SLC18A2 might contribute to the development of substance dependence.

Association of specific haplotypes of D2 dopamine receptor gene with vulnerability to heroin dependence in 2 distinct populations.

CONTEXT: Dopamine receptor-mediated pathways play critical roles in the mechanism of addiction. However, associations of the D(2) dopamine receptor gene (DRD2) with substance abuse are controversial. OBJECTIVE: To determine whether susceptibility sites resided at DRD2. DESIGN: Haplotype-based case-control analysis of 2 distinct populations using 10 single nucleotide polymorphisms (SNPs) with heroin dependence. SETTING: Universities of Mainz and Bonn, Germany, and 3 local hospitals in southwestern China. Patients Cases and control subjects recruited from China (486 cases, 313 controls) and Germany (471 cases, 192 controls). INTERVENTIONS: Genotyping for 10 SNPs by 5'-exonuclease fluorescence assays. The D' value of linkage disequilibrium and haplotypes were generated by the expectation-maximization algorithm. MAIN OUTCOME MEASURES: Genotype, allele, and haplotype frequencies were compared between cases and controls by chi(2) tests constructed for each population. An additional 32 SNPs randomly distributed in the genome were genotyped for detecting population admixture in the 2 populations. RESULTS: A haplotype block of 25.8 kilobases (kb) was defined by 8 SNPs extending from SNP3 (TaqIB) at the 5' end to SNP10 site (TaqIA) located 10 kb distal to the 3' end of the gene. Within this block, specific haplotype cluster A (carrying TaqIB1 allele) was associated with a high risk of heroin dependence in Chinese patients (P = 1.425 x 10(-22); odds ratio, 52.80; 95% confidence interval, 7.290-382.5 for 8-SNP analysis). A putative recombination "hot spot" was found near SNP6 (intron 6 ins/del G), creating 2 new daughter haplotypes that were associated with a lower risk of heroin dependence in Germans (P = 1.94 x 10(-11) for 8-SNP analysis). There was no evidence of population stratification in either population. CONCLUSIONS: These results strongly support a role of DRD2 as a susceptibility gene with heroin dependence in Chinese patients and was associated with low risk of heroin dependence in Germans.

Cis-Expression Quantitative Trait Loci Mapping Reveals Replicable Associations with Heroin Addiction in OPRM1.

BACKGROUND: No opioid receptor, mu 1 (OPRM1) gene polymorphisms, including the functional single nucleotide polymorphism (SNP) rs1799971, have been conclusively associated with heroin/other opioid addiction, despite their biological plausibility. We used evidence of polymorphisms altering OPRM1 expression in normal human brain tissue to nominate and then test associations with heroin addiction. METHODS: We tested 103 OPRM1 SNPs for association with OPRM1 messenger RNA expression in prefrontal cortex from 224 European Americans and African Americans of the BrainCloud cohort. We then tested the 16 putative cis-expression quantitative trait loci (cis-eQTL) SNPs for association with heroin addiction in the Urban Health Study and two replication cohorts, totaling 16,729 European Americans, African Americans, and Australians of European ancestry. RESULTS: Four putative cis-eQTL SNPs were significantly associated with heroin addiction in the Urban Health Study (smallest p = 8.9 × 10(-5)): rs9478495, rs3778150, rs9384169, and rs562859. Rs3778150, located in OPRM1 intron 1, was significantly replicated (p = 6.3 × 10(-5)). Meta-analysis across all case-control cohorts resulted in p = 4.3 × 10(-8): the rs3778150-C allele (frequency = 16%-19%) being associated with increased heroin addiction risk. Importantly, the functional SNP allele rs1799971-A was associated with heroin addiction only in the presence of rs3778150-C (p = 1.48 × 10(-6) for rs1799971-A/rs3778150-C and p = .79 for rs1799971-A/rs3778150-T haplotypes). Lastly, replication was observed for six other intron 1 SNPs that had prior suggestive associations with heroin addiction (smallest p = 2.7 × 10(-8) for rs3823010). CONCLUSIONS: Our findings show that common OPRM1 intron 1 SNPs have replicable associations with heroin addiction. The haplotype structure of rs3778150 and nearby SNPs may underlie the inconsistent associations between rs1799971 and heroin addiction.

Serotonin transporter promoter and intron 2 polymorphisms: relationship between allelic variants and gene expression.

BACKGROUND: Two polymorphic regions of serotonin transporter (5-HTT) gene: a 44 base pair (bp) insertion/deletion in the promoter region (5-HTTLPR) and a 17 bp variable number of tandem repeats in second intron (VNTR-2), seem to modulate the gene's transcription in allele-dependent manner. METHODS: We have earlier demonstrated association with 5-HTT gene in families multiply affected by schizophrenia. Here, we investigated separate and combined effects of VNTR-2 and 5-HTTLPR on the rate of peripheral 5-HTT transcription in a sample of offspring from those families. Relative 5-HTT mRNA levels were determined in 53 permanent lymphoblast cell lines by semiquantitative real-time polymerase chain reaction using beta-actin as reference. RESULTS: Since the low-expressing alleles (short [S], 10) appeared to act dominantly, genotypes were grouped as "high-expressing" (long [L]/L, 12/12) versus "low-expressing" (S, 10). At both loci, nonsignificant differences in 5-HTT mRNA levels ( approximately 30%) were observed between "high"- and "low-expressing" genotypes. In order to search for the potential combined effect of 5-HTTLPR and VNTR-2, levels of 5-HTT mRNA were compared among three groups of samples having "low-expressing" genotype at none, one, or both loci. Increase in number of "low-expressing" genotypes significantly reduced relative 5-HTT gene expression (p <.02). CONCLUSIONS: Our results indicate weak individual influence, but possible combined effect, of 5-HTTLPR and VNTR-2 polymorphisms on 5-HTT gene expression.

Investigation of linkage and association/linkage disequilibrium of HLA A-, DQA1-, DQB1-, and DRB1-alleles in 69 sib-pair- and 89 trio-families with schizophrenia.

The hypothesis that HLA antigens confer susceptibility to schizophrenic disorders has been tested by studying linkage and association in a family sample with 69 sib-pair families. Suggestive evidence for linkage was obtained by nonparametric multipoint LOD score analysis with a maximum around DQB CAR (P = 0.0004), a microsatellite marker that is in linkage disequilibrium with the HLA antigen DQB1. Spurious evidence for negative association as calculated by the transmission disequilibrium test was found for HLA- DRB1*11 (chi-square = 11.72, corrected P value = 0.03) and for the haplotype DQB1*301-DQA1*501-DRB1*11 (chi-square = 11.3, corrected P value = 0.043). No evidence of association with these alleles was obtained in a sample of 89 trios with schizophrenic offspring and parents. Our results are not in favor of a direct involvement of the HLA system in development of schizophrenia, but are compatible with the possible existence of a susceptibility gene in the MHC region at chromosome 6p 21.31.

Association of tumor necrosis factor alpha gene -G308A polymorphism with schizophrenia.

BACKGROUND: Tumor necrosis factor alpha (TNFalpha), a cytokine involved in inflammatory processes, has been implicated in the pathophysiology of schizophrenia. The chromosomal location in the major histocompatibility complex (MHC) region on 6p21.1-21.3, a region with evidence for linkage, suggests a role in susceptibility to schizophrenia. Association of the minor (A) allele of the -G308A TNFalpha gene polymorphism with schizophrenia has been reported [Mol. Psychiatry 6 (2001) 79]. METHODS: Association of the -G308A TNFalpha gene and the lymphotoxin alpha (LTalpha)+A252G gene polymorphisms with schizophrenia was studied in 79 sib pair families with linkage in the MHC region and in 128 trio families using the transmission disequilibrium test (TDT). RESULTS: Weak association of the common G allele was detected for TNFalpha -G308A in both samples independently with borderline significance in the sib pair families (0.064) and with a nominally significant value of P=0.022 in the trio families. Combining both samples produced P=0.003, while LTalpha+A252G, located approximately 2-3 kb distally, revealed P=0.03 and the two locus haplotype yielded a P value of 0.001. CONCLUSION: Our data suggests association of the common G allele of the -G308A TNFalpha gene polymorphism with schizophrenia in a sample of 207 families. However, linkage disequilibrium with a different allele of the TNFalpha gene or another gene in the MHC region cannot be excluded.

Introducing a new recruitment approach to sample collection for genetic association studies in opioid dependence.

OBJECTIVE: In a modified case-control association study we tested the assumption that two polymorphisms (A(118)G in exon 1 and IVS2+31 in intron 2) of the human mu-opioid receptor gene (OPRM1) confer susceptibility to opioid dependence. METHODS: In contrast to classical case-control studies both groups, opioid dependent cases and non-opioid dependent controls were recruited from individuals who have had access to drugs including opioids and who had been sentenced for violation of the "Dangerous Drugs Act" in Germany. RESULTS: For the two allelic variants of OPRM1 under study we did not find evidence for association with opioid dependence. CONCLUSIONS ;Despite absence of association we think that this recruitment approach introduced here, is useful since it putatively offers a more adequate matching for case-control association studies of opioid dependent individuals.

Genetics of complex psychiatric disorders: scientific foundations.

Advances in genetic analysis as well as progress in the Human Genome Sequencing Project have raised the hope to elucidate the molecular basis of mental disorders on the DNA level. In the present review we describe and discuss the basic concepts which are currently applied for the genetic approach in order to find DNA variations associated with a disorder. A prerequisite for such studies is the confirmed evidence for genetic transmission established by family-, twin-, and adoption studies. We describe the study design and discuss the mode of inheritance for complex genetic traits in comparison to monogenic, Mendelian traits. Recombination as a basis for linkage analysis is explained and its implication for classical LOD score analysis in families, as well as for affected sib-pair analysis and for analysis of linkage disequilibrium is discussed. Moreover, we describe types of markers and maps used in these studies. We explain and discuss the association approach in conjunction with the Human Genome Project. Finally, we stress possible implications for diagnosis, prevention and new therapeutic approaches.

Association of rs1344706 in the ZNF804A gene with schizophrenia in a case/control sample from Indonesia.

BACKGROUND: Association of rs1344706 in the ZNF804A gene (2q32.1) with schizophrenia was first reported in a genome wide scan conducted in a sample of 479 cases and replicated in 6666 cases. Subsequently, evidence by replication was obtained in several samples with European- and Asian ancestral background. METHODS: We report ascertainment, clinical characterization, quality control, and determination of ancestral background of a case control sample from Indonesia, comprising 1067 cases and 1111 ancestry matched controls. Genotyping was performed using a fluorescence-based allelic discrimination assay (TaqMan SNP genotyping assay) and a newly designed PCR-RFLP assay for confirmation of rs1344706 genotypes. RESULTS: We confirmed association of the T-allele of rs1344706 with schizophrenia in a newly ascertained sample from Indonesia with Southeast Asian ancestral background (P=0.019, OR=1.155, 95%, CI 1.025-1.301). In addition, we studied several SNPs in the vicinity of rs1344706, for which nominally significant results had been reported. None of the association P values of the additional SNPs exceeded that of rs1344706. CONCLUSION: We provide additional evidence for association of the ZNF804A gene with schizophrenia. We conclude that rs1344706 or a yet unknown polymorphism in linkage disequilibrium is also involved in conferring susceptibility to schizophrenia in samples with different (Asian) ancestral backgrounds.