Prader-Willi syndrome genetic subtypes and clinical neuropsychiatric diagnoses in residential care adults.

The historical diagnosis of Prader-Willi syndrome (PWS), a complex genetic disorder, in adults is often achieved by clinical presentation rather than by genetic testing and thus limited genetic subtype-specific psychometric investigations and treatment options. Genetic testing and clinical psychiatric evaluation using Diagnostic and Statistical Manual (DSM)-IV-TR criteria were undertaken on 72 adult residents (34 M; 38 F) from the Prader-Willi Homes of Oconomowoc (PWHO), a specialty PWS group home system. Methylation specific-multiplex ligation probe amplification and high-resolution microarrays were analyzed for methylation status, 15q11-q13 deletions and maternal uniparental disomy 15 (mUPD15). Seventy (33M; 37F) of 72 residents were genetically confirmed and 36 (51%) had Type I or Type II deletions; 29 (42%) with mUPD15 and 5 (7%) with imprinting defects from three separate families. Psychiatric comorbidities were classified as anxiety disorder (38%), excoriation (skin picking) (33%), intermittent explosive disorder ([30%-predominantly among males at 45% compared with females at 16% [OR = 4.3, 95%CI 1.4-13.1, P < 0.008]) and psychotic features (23%). Psychiatric diagnoses did not differ between mUPD15 vs deletion, but a greater number of psychiatric diagnoses were observed for the larger Type I (4.3) vs smaller Type II (3.6) deletions when age was controlled (F = 5.0, P < 0.04). Adults with PWS presented with uniformly higher rates of psychiatric comorbidities which differed by genetic subtype with gender-specific trends.

Examination of Global Methylation and Targeted Imprinted Genes in Prader-Willi Syndrome.

CONTEXT: Methylation changes observed in Prader-Willi syndrome (PWS) may impact global methylation as well as regional methylation status of imprinted genes on chromosome 15 (in cis) or other imprinted obesity-related genes on other chromosomes (in trans) leading to differential effects on gene expression impacting obesity phenotype unique to (PWS). OBJECTIVE: Characterize the global methylation profiles and methylation status for select imprinted genes associated with obesity phenotype in a well-characterized imprinted, obesity-related syndrome (PWS) relative to a cohort of obese and non-obese individuals. DESIGN: Global methylation was assayed using two methodologies: 1) enriched LINE-1 repeat sequences by EpigenDx and 2) ELISA-based immunoassay method sensitive to genomic 5-methylcytosine by Epigentek. Target gene methylation patterns at selected candidate obesity gene loci were determined using methylation-specific PCR. SETTING: Study participants were recruited as part of an ongoing research program on obesity-related genomics and Prader-Willi syndrome. PARTICIPANTS: Individuals with non-syndromic obesity (N=26), leanness (N=26) and PWS (N=39). RESULTS: A detailed characterization of the imprinting status of select target genes within the critical PWS 15q11-q13 genomic region showed enhanced cis but not trans methylation of imprinted genes. No significant differences in global methylation were found between non-syndromic obese, PWS or non-obese controls. INTERVENTION: None. MAIN OUTCOME MEASURES: Percentage methylation and the methylation index. CONCLUSION: The methylation abnormality in PWS due to errors of genomic imprinting effects both upstream and downstream effectors in the 15q11-q13 region showing enhanced cis but not trans methylation of imprinted genes. Obesity in our subject cohorts did not appear to impact global methylation levels using the described methodology.

EVALUATION OF PLASMA SUBSTANCE P AND BETA-ENDORPHIN LEVELS IN CHILDREN WITH PRADER-WILLI SYNDROME.

BACKGROUND: Prader-Willi syndrome (PWS) is a rare obesity-related genetic disorder often caused by a deletion of the chromosome 15q11-q13 region inherited from the father or by maternal disomy 15. Growth hormone deficiency with short stature, hypogonadism, cognitive and behavioral problems, analgesia, decreased gastric motility and decreased ability to vomit with hyperphagia are common in PWS leading to severe obesity in early childhood, if not controlled. Substance P (SP) and beta-endorphin (BE) are neuropeptides involved with centrally and peripherally mediated pain perception, emotional regulation, and gastric motility impacting nausea, emesis and feeding patterns. OBJECTIVE: The goal of this study was to investigate potential mechanisms for PWS symptom development for pain, emotion and gastric motility and plasma levels of substance P and beta-endorphin between PWS and unrelated unaffected children. METHODOLOGY: Plasma samples were collected from 23 Caucasian children with PWS and 18 unrelated, unaffected siblings with an average age of 8.2 ±2.0 years and age range of 5 to 11 years following an overnight fast and neuropeptide substance p and beta-endorphin levels were assessed using Multiplex sandwich immunoassays using the Luminex magnetic-bead based platform. Linear regression analysis was carried out on log-transformed values adjusted for age, sex, and body mass index (BMI). RESULTS: The mean plasma SP (57 ± 23 pg/ml) and BE (592 ± 200 pg/ml) levels in PWS were significantly higher than SP (35 ± 20 pg/ml, F=10.5, P<0.01) and BE (402 ± 162 pg/ml, F=10.8, P<0.01) levels found in unrelated, unaffected siblings suggesting a previously uncharacterized neuroendocrine pathophysiology in PWS. CONCLUSIONS: The increased BE and SP plasma levels relative to unrelated, unaffected siblings may contribute to hyperphagia, abnormal pain sensation and adrenal insufficiency seen in PWS. Increases in SP levels may be modulated by central and/or peripheral actions of BE on opioid, GABA or POMC precursors and may reflect loss of feedback inhibitory control. Further studies are needed to confirm and elucidate the biochemical basis for observed disturbances in neuropeptide levels seen in our study and may impact on the development and persistence of symptoms commonly seen in PWS.

Over-expression of the miRNA cluster at chromosome 14q32 in the alcoholic brain correlates with suppression of predicted target mRNA required for oligodendrocyte proliferation.

We examined miRNA expression from RNA isolated from the frontal cortex (Broadman area 9) of 9 alcoholics (6 males, 3 females, mean age 48 years) and 9 matched controls using both the Affymetrix GeneChip miRNA 2.0 and Human Exon 1.0 ST Arrays to further characterize genetic influences in alcoholism and the effects of alcohol consumption on predicted target mRNA expression. A total of 12 human miRNAs were significantly up-regulated in alcohol dependent subjects (fold change≥1.5, false discovery rate (FDR)≤0.3; p<0.05) compared with controls including a cluster of 4 miRNAs (e.g., miR-377, miR-379) from the maternally expressed 14q32 chromosome region. The status of the up-regulated miRNAs was supported using the high-throughput method of exon microarrays showing decreased predicted mRNA gene target expression as anticipated from the same RNA aliquot. Predicted mRNA targets were involved in cellular adhesion (e.g., THBS2), tissue differentiation (e.g., CHN2), neuronal migration (e.g., NDE1), myelination (e.g., UGT8, CNP) and oligodendrocyte proliferation (e.g., ENPP2, SEMA4D1). Our data support an association of alcoholism with up-regulation of a cluster of miRNAs located in the genomic imprinted domain on chromosome 14q32 with their predicted gene targets involved with oligodendrocyte growth, differentiation and signaling.

Global DNA promoter methylation in frontal cortex of alcoholics and controls.

To determine if ethanol consumption and alcoholism cause global DNA methylation disturbances, we examined alcoholics and controls using methylation specific microarrays to detect all annotated gene and non-coding microRNA promoters and their CpG islands. DNA was isolated and immunoprecipitated from the frontal cortex of 10 alcoholics and 10 age and gender-matched controls then labeled prior to co-hybridization. A modified Kolmogorov-Smirnov test was used to predict differentially enriched regions (peaks) from log-ratio estimates of amplified vs input DNA. More than 180,000 targets were identified for each subject which correlated with >30,000 distinct, integrated peaks or high probability methylation loci. Peaks were mapped to regions near 17,810 separate annotated genes per subject representing hypothetical methylation targets. No global methylation differences were observed between the two subject groups with 80% genetic overlap, but extreme methylation was observed in both groups at specific loci corresponding with known methylated genes (e.g., H19) and potentially other genes of unknown methylation status. Methylation density patterns targeting CpG islands visually correlated with recognized chromosome banding. Our study provides insight into global epigenetic regulation in the human brain in relationship to controls and potentially novel targets for hypothesis generation and follow-up studies of alcoholism.

Plasma cytokine levels in children with autistic disorder and unrelated siblings.

BACKGROUND: The pathogenesis of autistic disorder (AD) is not clearly understood but genetic factors and the immune system have been implicated. Disturbed immunoglobulin levels and autoantibodies to neuronal elements have been reported in AD including cytokines encoded by genes involved with cell proliferation, migration and adhesion but there is a paucity of data comparing cytokine levels in children with AD and unrelated siblings without AD. METHODS: We analyzed 39 plasma cytokines in 99 well-characterized children with AD between 5 and 10 years of age and 40 age and gender matched healthy unrelated siblings without AD under the same clinical assessments, specimen processing and laboratory conditions. Multiplex sandwich immunoassays were used with the Luminex fluorescent-bead based platform. Log-transformed values of the 29 cytokines meeting laboratory criteria for inclusion were analyzed by analysis of covariance with a general linear model adjusting for diagnosis, gender, diagnosis by gender interaction effects, age and days of specimen handling. The Tukey-Kramer post hoc test was used to control for multiple comparisons. RESULTS: Eight of 29 cytokine levels analyzed were significantly lower in children with AD compared with unrelated siblings without the diagnosis of AD. Three of the cytokines are known to be involved with hematopoiesis and five with attraction of T-cells, natural killer cells and monocytes. CONCLUSIONS: Plasma cytokine levels representing chemokines involved in the T-helper cell immune system and hematopoiesis were lower in the children with AD compared with unrelated siblings without AD necessitating further studies to confirm immunological disturbances influencing hematopiesis and antibody production in the children with AD. Linking genes that encode immune related proteins and cytokines are important to study for their impact on critical periods of brain development and function.

Paternal alcoholism predicts the occurrence but not the remission of alcoholic drinking: a 40-year follow-up.

OBJECTIVE: To test the effects of father's alcoholism on the development and remission from alcoholic drinking by age 40. METHOD: Subjects were selected from a Danish birth cohort that included 223 sons of alcoholic fathers (high risk; HR) and 106 matched controls (low risk; LR). Clinical examinations were performed at age 40 (n = 202) by a psychiatrist using structured interviews and DSM-III-R diagnostic criteria. RESULTS: HR subjects were significantly more likely than LR subjects to develop alcohol dependence (31% vs. 16%), but not alcohol abuse (17% vs. 15%). More subjects with alcohol abuse were in remission at age 40 than subjects with alcohol dependence. Risk did not predict remission from either alcohol abuse or alcohol dependence. CONCLUSION: Familial influences may play a stronger role in the development of alcoholism than in the remission or recovery from alcoholism.

Rats prefer cocaine over nicotine in a two-lever self-administration choice test.

Smoking is considered the leading cause of preventable death in the United States, but studies in animals suggest that nicotine is only weakly reinforcing. The maintenance of a dangerous habit by a weakly reinforcing agent has been the topic of some dispute. Using a two-lever "choice" self-administration procedure developed in our laboratory, we evaluated drug preferences as an index of relative reward strength for nicotine versus cocaine in nicotine-trained rats. Rats were initially exposed to each drug separately in single-lever self-administration sessions and then allowed to choose between them in a two-lever choice test session offering both drugs. When offered choices between different nicotine doses [8, 25, and 75 microg/kg/injection (inj), free base], rats responded approximately equally for any dose, regardless of which doses were compared. Rats clearly preferred 267 or 800 microg/kg/inj cocaine hydrochloride to any of the nicotine doses. These results indicate that cocaine has greater reward strength than nicotine and supports previous findings that self-administering rats seek to maximize reward magnitude regardless of the self-administered drug or training history. It is possible that dependence elevates nicotine's reward magnitude or nicotine addiction may rely more importantly upon negative rather than pure positive reinforcement.

Rats choose cocaine over dopamine agonists in a two-lever self-administration preference test.

Rats will self-administer dopamine D(1) and D(2) agonists, alone or in combination. Response rates and patterns for the D(1):D(2) combinations are nearly identical to those induced by cocaine. Here we examine whether rats prefer cocaine over D(1) or D(2) agonists presented alone or in D(1):D(2) combinations. During daily 3-h tests in a two-lever box, cocaine was available at either the right or left lever and the active side was alternated daily. After response rates had stabilized (+/-10% for 2 days), different groups were offered cocaine (800 microg/kg/injection) at one lever and either another dose (267, 1600, or 2400 microg/kg/injection) of cocaine or a dopamine agonist at the other lever. Animals consistently chose the higher of the presented cocaine doses over the low cocaine dose (267 microg). In choices between cocaine and dopamine agonists, the preferred cocaine dose (800 microg) was chosen over doses of the D(1) (SKF 82958) or D(2) ((+)-PHNO) agonist. However, no preference was shown between 800 microg cocaine and D(1):D(2) agonist mixtures, and the high-dose agonist mixture was preferred to the low cocaine dose. These results suggest that neither D(1) nor D(2) agonists alone fully duplicate the reinforcing actions of cocaine, but agonist combinations may approximate cocaine's reinforcement strength.

Restoration of oral all-trans retinoic acid bioavailability after a brief drug holiday.

We evaluated the utility of a 7-day drug holiday in the restoration of chronic dosing all-trans-retinoic acid (tRA) blood levels in 11 non-small cell lung carcinoma patients. Baseline kinetic studies (day 1) were compared to postchronic dosing (day 7) and drug holiday kinetics (day 14). High levels of baseline pharmacokinetic variability and variability in response to prolonged tRA therapy were evident. Median area under the curve (AUC) decreased from a baseline level of 1.2 to 0.69 microg/ml/h (p = 0.03). t (1/2) showed no significant alterations. A near-significant increase in T (lag) (p = 0.08) was noted, which suggests modulation of absorbance parameters. Trends in AUC were strongly correlated with C (max) as was T (max) with T (lag). After a 7-day drug holiday the median AUC significantly increased from the day 7 value to 1.8 microg/ml/h p = 0.01). Since post-drug holiday values for parameters were not statistically different from baseline pharmacokinetic values, this suggests a complete restoration of tRA bioavailability.