DNA methylation profiling: comparison of genome-wide sequencing methods and the Infinium Human Methylation 450 Bead Chip.

AIMS: To compare the performance of four sequence-based and one microarray methods for DNA methylation profiling. METHODS: DNA from two cell lines were profiled by reduced representation bisulfite sequencing, methyl capture sequencing (SS-Meth Seq), NimbleGen SeqCapEpi CpGiant(Nimblegen MethSeq), methylated DNA immunoprecipitation (MeDIP) and the Human Methylation 450 Bead Chip (Meth450K). RESULTS & CONCLUSION: Despite differences in genome-wide coverage, high correlation and concordance were observed between different methods. Significant overlap of differentially methylated regions was identified between sequenced-based platforms. MeDIP provided the best coverage for the whole genome and gene body regions, while RRBS and Nimblegen MethSeq were superior for CpGs in CpG islands and promoters. Methylation analyses can be achieved by any of the five methods but understanding their differences may better address the research question being posed.

Relationship of genetic variation in the serotonin transporter gene (SLC6A4) and congenital and acquired cardiovascular diseases.

Recent reports have suggested an association between variation in the serotonin transporter and primary pulmonary hypertension and myocardial infarction. We set out to determine whether these associations were present in a population of patients who underwent SLC6A4 genotyping and to explore whether genetic variation in the serotonin transporter might be also associated with other cardiovascular functional and structural abnormalities. Included were 3473 patients who were genotyped for the SLC6A4 5HTTLPR polymorphism and a subset for rs25531 (n=816) and STin2 (n=819). An association was observed between 5HTTLPR and primary pulmonary hypertension (p=0.0130), anomalies of the cerebrovascular system (p<0.0001), and other anomalies of great veins (p=0.0359). The combined 5HTTLPR and rs25531 genotype was associated with tachycardia (p=0.0123). There was an association of the STin2 genotype with abnormal electrocardiogram (ECG) (p=0.0366) and abnormal cardiac study (0.0311). Overall, these results represent a step toward the understanding of the impact of SLC6A4 variation on cardiovascular pathology.

Clinical risk factors and serotonin transporter gene variants associated with antidepressant-induced mania.

INTRODUCTION: Identifying clinical and genetic risk factors associated with antidepressant-induced mania (AIM) may improve individualized treatment strategies for bipolar depression. METHOD: From 2009 to 2012, bipolar depressed patients, confirmed by DSM-IV-TR-structured interview, were screened for AIM. An AIM+ case was defined as a manic/hypomanic episode within 60 days of starting or changing dose of antidepressant, while an AIM- control was defined as an adequate (≥ 60 days) exposure to an antidepressant with no associated manic/hypomanic episode. 591 subjects (205 AIM+ and 386 AIM-) exposed to an antidepressant and a subset of 545 subjects (191 AIM+ and 354 AIM-) treated with a selective serotonin reuptake inhibitor (SSRI) or serotonin-norepinephrine reuptake inhibitor (SNRI) were used to evaluate the association of AIM with phenotypic clinical risk factors previously published. 295 white subjects (113 AIM+ cases, 182 AIM-controls) were genotyped for 3 SLC6A4 variants: the 5-HTTLPR, single nucleotide polymorphism (SNP) rs25531, and the intron 2 variable number of tandem repeats (VNTR). Tests of association with AIM were performed for each polymorphism and the haplotype. RESULTS: The only clinical risk factors associated with AIM in the overall and the SSRI + SNRI analysis was bipolar I subtype. The S allele of 5-HTTLPR was not significantly associated with AIM; however, a meta-analysis combining this sample with 5 prior studies provided marginal evidence of association (P = .059). The L-A-10 haplotype was associated with a reduced risk of AIM (P = .012). DISCUSSION: Narrowly defined, AIM appears to be at greatest risk for bipolar I patients. Our haplotype analysis of SLC6A4 suggests that future pharmacogenetic studies should not only focus on the SLC6A4 promotor variation but also investigate the role of other variants in the gene.

Adenosinergic regulation of striatal clock gene expression and ethanol intake during constant light.

Circadian rhythm and sleep disruptions occur frequently in individuals with alcohol use disorders (AUD) and present significant barriers to treatment. Recently, a variant of adenosine transporter, equilibrative nucleoside transporter 1 (ENT1), was associated with the co-occurrence of sleep problems and AUD. We have previously shown that mice lacking ENT1 (ENT1 KO) have reduced adenosine levels in the striatum and drink more alcohol compared with wild types (WT). However, it is unknown whether ENT1 deletion disrupts circadian rhythms, which may contribute to alcohol preference in ENT1 KO mice. Here we used these mice to determine whether endogenous adenosine regulates circadian genetic and behavioral rhythms and influences alcohol intake during chronodisruption. We examined circadian locomotor activity in ENT1 KO vs WT littermates and found that ENT1 KO mice were both active earlier and hyperactive compared with WT mice at night. We used real-time PCR and immunohistochemistry to estimate striatal clock gene levels and found that PER2 expression in the striatum was blunted by ENT1 deletion or A2A receptor (A2AR) antagonism. Next, we exposed ENT1 KO and WT mice to constant light (LL) and found further elevation in ethanol intake in ENT1 KO, but not in WT mice, supporting the notion that circadian dysfunction may contribute to increased alcohol intake in ENT1 KO mice. Finally, we showed that A2AR agonist administration normalized PER1 and PER2 expression and circadian locomotor activity in ENT1 KO mice. Together, our results demonstrate that adenosine signaling regulates cellular and behavioral circadian timing and influences alcohol intake during chronodisruption.

Associations between serotonin transporter gene polymorphisms and heat pain perception in adults with chronic pain.

BACKGROUND: The triallelic serotonin transporter gene linked polymorphic region (5-HTTLPR) has been associated with alterations in thermal pain perception. The primary aim of this study was to investigate the associations between heat pain (HP) perception and the triallelic 5-HTTLPR in a large cohort of adults with chronic pain. METHODS: The cohort included 277 adults with chronic pain who met inclusion criteria, and were consecutively admitted to an outpatient pain rehabilitation program from March 2009 through March 2010. Individuals were genotyped for the triallelic 5-HTTLPR (including rs25531) and categorized as high, intermediate, or low expressors of the serotonin transporter. Standardized measures of HP perception were obtained using a validated quantitative sensory test method of levels. RESULTS: The distribution of the high, intermediate, and low expressing genotypes was 61 (22%), 149 (54%) and 67 (24%), respectively. The Hardy-Weinberg P-value was 0.204 which indicated no departure from equilibrium. A significant effect of genotype was observed for values of HP threshold (P = 0.029). Individual group comparisons showed that values of HP threshold were significantly greater in the intermediate compared to the high expressing group (P = 0.009) but not the low expressing group (P > 0.1). In a multiple variable linear regression model, the intermediate group (P = 0.034) and male sex (P = 0.021) were associated with significantly greater values of HP 0.5, but no significant genotype-by-sex interaction effect was observed. CONCLUSIONS: In this study that involved adults with chronic pain, the intermediate triallelic 5-HTTLPR expressing group, but not the low expressing group, was associated with greater HP thresholds compared to the high expressing group.

Association of the PDYN gene with alcohol dependence and the propensity to drink in negative emotional states.

Synthetic κ-opioid receptor (KOR) agonists induce dysphoric and pro-depressive effects and variations in the KOR (OPRK1) and prodynorphin (PDYN) genes have been shown to be associated with alcohol dependence. We genotyped 23 single nucleotide polymorphisms (SNPs) in the PDYN and OPRK1 genes in 816 alcohol-dependent subjects and investigated their association with: (1) negative craving measured by a subscale of the Inventory of Drug Taking Situations; (2) a self-reported history of depression; (3) the intensity of depressive symptoms measured by the Beck Depression Inventory-II. In addition, 13 of the 23 PDYN and OPRK1 SNPs, which were previously genotyped in a set of 1248 controls, were used to evaluate association with alcohol dependence. SNP and haplotype tests of association were performed. Analysis of a haplotype spanning the PDYN gene (rs6045784, rs910080, rs2235751, rs2281285) revealed significant association with alcohol dependence (p = 0.00079) and with negative craving (p = 0.0499). A candidate haplotype containing the PDYN rs2281285-rs1997794 SNPs that was previously associated with alcohol dependence was also associated with negative craving (p = 0.024) and alcohol dependence (p = 0.0008) in this study. A trend for association between depression severity and PDYN variation was detected. No associations of OPRK1 gene variation with alcohol dependence or other studied phenotypes were found. These findings support the hypothesis that sequence variation in the PDYN gene contributes to both alcohol dependence and the induction of negative craving in alcohol-dependent subjects.

CYP2D6*11 and challenges in clinical genotyping of the highly polymorphic CYP2D6 gene.

CYP2D6 is genotyped clinically for prediction of response to tamoxifen, psychotropic drugs and other medications. Phenotype prediction is dependent upon accurate genotyping. The CYP Allele Nomenclature Committee maintains the allelic nomenclature for CYP2D6; however, in some cases, the list of polymorphisms associated with a given allele is incomplete. Clinical laboratories and in vitro diagnostic manufacturers rely upon this nomenclature, in addition to the literature, to infer allelic function and haplotypes and when they design CYP2D6-testing platforms. This article provides more complete sequencing data for the CYP2D6*11 allele and describes the difficulties encountered in genotyping CYP2D6 when incomplete data are available. The CYP Allele Nomenclature Committee should provide clear information about the completeness of the original data used to define each allele.

Correlation of CYP2B6, CYP2C19, ABCC4 and SOD2 genotype with outcomes in allogeneic blood and marrow transplant patients.

CYP2B6, CYP2C19, ABCC4, and SOD2 have been implicated in adverse drug reactions and survival after cyclophosphamide (CPA) treatment. 110 BMT patients who received high dose CPA treatment were genotyped for variants in these genes and the results were correlated with toxicity and relapse. CYP2B6 genotype significantly influenced overall toxicity suggesting active CYP2B6 alleles led to higher rates of overall toxicity. The p.R487C deficiency allele was significantly associated with a lower rate of overall toxicity and a higher rate of relapse. SOD2 rs4880 V16A polymorphism was associated with significantly less CPA-related overall toxicity and significantly lower relapse rates by Kaplan-Meier analysis although the SOD2 finding regarding relapse was not significant when evaluated by the cumulative incidence function.

Frequency of undetected CYP2D6 hybrid genes in clinical samples: impact on phenotype prediction.

Cytochrome P450 2D6 (CYP2D6) is highly polymorphic. CYP2D6-2D7 hybrid genes can be present in samples containing CYP2D6*4 and CYP2D6*10 alleles. CYP2D7-2D6 hybrid genes can be present in samples with duplication signals and in samples with homozygous genotyping results. The frequency of hybrid genes in clinical samples is unknown. We evaluated 1390 samples for undetected hybrid genes by polymerase chain reaction (PCR) amplification, PCR fragment analysis, TaqMan copy number assays, DNA sequencing, and allele-specific primer extension assay. Of 508 CYP2D6*4-containing samples, 109 (21.5%) harbored CYP2D6*68 + *4-like, whereas 9 (1.8%) harbored CYP2D6*4N + *4-like. Of 209 CYP2D6*10-containing samples, 44 (21.1%) were found to have CYP2D6*36 + *10. Of 332 homozygous samples, 4 (1.2%) harbored a single CYP2D7-2D6 hybrid, and of 341 samples with duplication signals, 25 (7.3%) harbored an undetected CYP2D7-2D6 hybrid. Phenotype before and after accurate genotyping was predicted using a method in clinical use. The presence of hybrid genes had no effect on the phenotype prediction of CYP2D6*4- and CYP2D6*10-containing samples. Four of four (100%) homozygous samples containing a CYP2D7-2D6 gene had a change in predicted phenotype, and 23 of 25 (92%) samples with a duplication signal and a CYP2D7-2D6 gene had a change in predicted phenotype. Four novel genes were identified (CYP2D6*13A1 variants 1 and 2, CYP2D6*13G1, and CYP2D6*13G2), and two novel hybrid tandem structures consisting of CYP2D6*13B + *68×2 + *4-like and CYP2D6*13A1 variant 2 + *1×N were observed.

Sex-Specific Regulation of Depression, Anxiety-Like Behaviors and Alcohol Drinking in Mice Lacking ENT1.

OBJECTIVES: Adenosine signaling has been implicated in the pathophysiology of several psychiatric disorders including alcoholism, depression, and anxiety. Adenosine levels are controlled in part by transport across the cell membrane by equilibrative nucleoside transporters (ENTs). Recent evidence showed that a polymorphism in the gene encoding ENT1 is associated with comorbid depression and alcoholism in women. We have previously shown that deletion of ENT1 reduces ethanol intoxication and elevates alcohol intake in mice. Interestingly, ENT1 null mice display decreased anxiety-like behavior compared to wild-type littermates. However, our behavioral studies were performed only in male mice. Here, we extend our research to include female mice, and test the effect of ENT1 knockout on other behavioral correlates of alcohol drinking, including depressive and compulsive behavior, in mice. METHODS: To assess depression-like behavior, we used a forced swim test modified for mice. We examined anxiety-like behavior and locomotor activity in open field chambers, and perseverant behavior using the marble-burying test. Finally, we investigated alcohol consumption and preference in female mice using a two-bottle choice paradigm. RESULTS: ENT1 null mice of both sexes showed reduced immobility time in the forced swim test and increased time in the center of the open field compared to wild-type littermates. ENT1 null mice of both sexes showed similar locomotor activity levels and habituation to the open field chambers. Female ENT1 null mice displayed increased marble-burying compared to female wild-types, but no genotype difference was evident in males. Female ENT1 null mice showed increased ethanol consumption and preference compared to female wild-types. CONCLUSIONS: Our findings suggest that ENT1 contributes to several important behaviors involved in psychiatric disorders. Inhibition of ENT1 may be beneficial in treating depression and anxiety, while enhancement of ENT1 function may reduce compulsive behavior and drinking, particularly in females.

CYP2D6: novel genomic structures and alleles.

OBJECTIVE: CYP2D6 is a polymorphic gene. It has been observed to be deleted, to be duplicated and to undergo recombination events involving the CYP2D7 pseudogene and surrounding sequences. The objective of this study was to discover the genomic structure of CYP2D6 recombinants that interfere with clinical genotyping platforms that are available today. METHODS: Clinical samples containing rare homozygous CYP2D6 alleles, ambiguous readouts, and those with duplication signals and two different alleles were analyzed by long-range PCR amplification of individual genes, PCR fragment analysis, allele-specific primer extension assay, and DNA sequencing to characterize alleles and genomic structure. RESULTS: Novel alleles, genomic structures, and the DNA sequence of these structures are described. Interestingly, in 49 of 50 DNA samples that had CYP2D6 gene duplications or multiplications where two alleles were detected, the chromosome containing the duplication or multiplication had identical tandem alleles. CONCLUSION: Several new CYP2D6 alleles and genomic structures are described which will be useful for CYP2D6 genotyping. The findings suggest that the recombination events responsible for CYP2D6 duplications and multiplications are because of mechanisms other than interchromosomal crossover during meiosis.

Characterization of large rearrangements in autosomal dominant polycystic kidney disease and the PKD1/TSC2 contiguous gene syndrome.

Large DNA rearrangements account for about 8% of disease mutations and are more common in duplicated genomic regions, where they are difficult to detect. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2. PKD1 is located in an intrachromosomally duplicated region. A tuberous sclerosis gene, TSC2, lies immediately adjacent to PKD1 and large deletions can result in the PKD1/TSC2 contiguous gene deletion syndrome. To rapidly identify large rearrangements, a multiplex ligation-dependent probe amplification assay was developed employing base-pair differences between PKD1 and the six pseudogenes to generate PKD1-specific probes. All changes in a set of 25 previously defined deletions in PKD1, PKD2 and PKD1/TSC2 were detected by this assay and we also found 14 new mutations at these loci. About 4% of the ADPKD patients in the CRISP study were found to have gross rearrangements, and these accounted for about a third of base-pair mutation negative families. Sensitivity of the assay showed that about 40% of PKD1/TSC contiguous gene deletion syndrome families contained mosaic cases. Characterization of a family found to be mosaic for a PKD1 deletion is discussed here to illustrate family risk and donor selection considerations. Our assay improves detection levels and the reliability of molecular testing of patients with ADPKD.

Comprehensive molecular diagnostics in autosomal dominant polycystic kidney disease.

Mutation-based molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) is complicated by genetic and allelic heterogeneity, large multi-exon genes, duplication of PKD1, and a high level of unclassified variants (UCV). Present mutation detection levels are 60 to 70%, and PKD1 and PKD2 UCV have not been systematically classified. This study analyzed the uniquely characterized Consortium for Radiologic Imaging Study of PKD (CRISP) ADPKD population by molecular analysis. A cohort of 202 probands was screened by denaturing HPLC, followed by direct sequencing using a clinical test of 121 with no definite mutation (plus controls). A subset was also screened for larger deletions, and reverse transcription-PCR was used to test abnormal splicing. Definite mutations were identified in 127 (62.9%) probands, and all UCV were assessed for their potential pathogenicity. The Grantham Matrix Score was used to score the significance of the substitution and the conservation of the residue in orthologs and defined domains. The likelihood for aberrant splicing and contextual information about the UCV within the patient (including segregation analysis) was used in combination to define a variant score. From this analysis, 44 missense plus two atypical splicing and seven small in-frame changes were defined as probably pathogenic and assigned to a mutation group. Mutations were thus defined in 180 (89.1%) probands: 153 (85.0%) PKD1 and 27 (15.0%) PKD2. The majority were unique to a single family, but recurrent mutations accounted for 30.0% of the total. A total of 190 polymorphic variants were identified in PKD1 (average of 10.1 per patient) and eight in PKD2. Although nondefinite mutation data must be treated with care in the clinical setting, this study shows the potential for molecular diagnostics in ADPKD that is likely to become increasingly important as therapies become available.

Analysis of hypocretin (orexin) antibodies in patients with narcolepsy.

STUDY OBJECTIVES: We tested the hypothesis that patients with narcolepsy have serum antibodies specific for preprohypocretin and its derivatives. DESIGN: We tested sera from strictly diagnosed HLA DQB1*0602-positive narcoleptic patients with cataplexy for evidence of autoantibodies against human preprohypocretin, hypocretin 1 and 2, N-terminal leader and C-terminal peptides of preprohypocretin using enzyme-linked immunosorbent assays (ELISA). These results were compared to samples from nonnarcoleptic psychiatric and sleep apnea controls. Laboratory personnel were blinded to subject status. SETTING: Narcoleptic patients and nonnarcoleptic controls were recruited from the Mayo Clinic facilities in Rochester, Minnesota; Scottsdale, Arizona; and Jacksonville, Florida. Laboratory testing was conducted in the Mayo Psychogenomic Laboratory at the Rochester Mayo Clinic. PARTICIPANTS: A sample of 34 narcoleptic patients and 49 nonnarcoleptic controls. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: ELISA measurements were in optical density. Primary analyses were of the entire narcoleptic and control groups for each potential antigen, and none of the differences reached P values required for significance after Bonferroni adjustment. Secondary analyses by age and sex yielded P values that were significant after Bonferroni adjustment in only 2 cases, but further statistical analyses cast doubt on the veracity of these differences. In all cases where a significant difference was recorded, the hypothesis was not supported because the control optical density reading was higher than the narcoleptic values. CONCLUSIONS: These ELISA assay results do not support the hypothesis that HLA DQB1*0602-positive narcolepsy with cataplexy is associated with serum antibodies against preprohypocretin or its cleavage products.

Studies of humoral immunity to preprohypocretin in human leukocyte antigen DQB1*0602-positive narcoleptic subjects with cataplexy.

BACKGROUND: Canine models for narcolepsy have mutations of the hypocretin receptor 2 gene, and preprohypocretin knockout murine lines exhibit narcoleptic-like behaviors. Human narcolepsy with cataplexy is associated with human leukocyte antigen DQB1*0602 and reduced hypocretin levels in cerebrospinal fluid, suggesting an autoimmune diathesis. We tested the hypothesis that DQB1*0602-positive narcoleptic subjects with cataplexy have immunoglobulin (Ig)G reactive to human preprohypocretin and its cleavage products. METHODS: Serum samples of 41 DQB1*0602-positive narcoleptic subjects with cataplexy and 55 control subjects were studied, as were 19 narcoleptic and 13 control samples of cerebrospinal fluid. We tested for IgG reactive to preprohypocretin and its major cleavage products (including hypocretin 1 and 2), using immunoprecipitation assays (IP), immunofluorescence microscopy (IF) of Chinese hamster ovarian cells expressing preprohypocretin, and Western blots. RESULTS: There was no evidence for IgG reactive to preprohypocretin or its cleavage products in CSF of subjects with narcolepsy as measured by IPs, Western blots, and IF. Although the IP with CSF and the C-terminal peptide showed significant differences by two methods of comparison, the control subjects had higher counts per minute than narcoleptic subjects, which was opposite to our hypothesis. CONCLUSIONS: The hypothesis that DQB1*0602-positive narcoleptic subjects with cataplexy have IgG reactive to preprohypocretin or its cleavage products was not supported.

The metabolism of pyrazoloacridine (NSC 366140) by cytochromes p450 and flavin monooxygenase in human liver microsomes.

Pyrazoloacridine (PZA) is an experimental antitumor agent presently under investigation for treatment of solid tumors on the basis of its unique mechanism of action and selectivity for human solid tumor xenograft in mice. Using capillary electrophoresis coupled with electrospray ionization mass spectrometry, we have identified three oxidative PZA metabolites, 9-desmethyl-PZA, N-demethyl-PZA, and PZA N-oxide. The cytochrome p450 (CYP) isoforms involved in PZA metabolism were characterized by studies with CYP chemical inhibitors, correlation of marker activities for selected CYPs with formation of the metabolites using a human liver panel, and PZA metabolism by cDNA-expressed CYPs. 9-Desmethyl-PZA formation was catalyzed by CYP1A2, whereas N-demethyl-PZA formation was catalyzed by CYP3A4. PZA N-oxide formation was catalyzed by flavin monooxygenase (FMO) rather than CYP, as determined by studies with chemical inhibitors of FMO and metabolism by cDNA-expressed human flavin monooxygenase. After administration of [10b-(14)C]PZA to mice, six urinary metabolites were detected by high-performance liquid chromatography UV and radiochromatograms including 9-desmethyl-PZA, N-demethyl-PZA, and PZA N-oxide. Trace concentrations of 9-desmethyl-PZA and PZA N-oxide were detected in mouse plasma. PZA N-oxide and N-demethyl-PZA were detected in urine from patients after PZA administration. PZA, 9-desmethyl-PZA, and PZA N-oxide inhibited growth of A375 human melanoma cells. IC(50) values were 0.17, 0.11, and 7.0 micro M, respectively, for the three molecules.