Promoter hypermethylation analysis in myelodysplastic syndromes: diagnostic & prognostic implication.

BACKGROUND & OBJECTIVE: Myelodysplastic syndromes (MDS) are a heterogenous group of haematopoietic stem cell disorders that are multifactorial in their aetiology. Unique genetic alterations in combinations or in isolation account for a small fraction of MDS suggesting the epigenetic hypermethylation as a possible leading cause for MDS and its transformation to acute myelocytic leukaemia (AML). Therefore, in this study, promoter hypermethylation status of key cell cycle regulators was assessed as markers in MDS patients and association of hypermethylation with clinical progression of disease was also studied. METHODS: Promoter hypermethylation analysis of five tumour associated genes namely p16, p15, MGMT, hMLH1 and E-cadherin were done for 41 MDS patient samples with its various subtype. The hypermethylation analysis was done by using semi-nested multiplex PCR. RESULTS: Eighty per cent of (33/41) of the MDS samples were found to be methylated in any one of the four genes (p16, p15, MGMT and E-cadherin). The p15 methylation was found to be the most frequent 61 per cent (25/41), E-cadherin was methylated in 39 per cent (16/41) and p16 in 37 per cent (15/41) of the cases. MGMT gene showed a low 5 per cent (2/41) methylation whereas hMLH1 gene was not methylated in any one of the samples analysed. INTERPRETATION & CONCLUSION: Differential rate of methylation of the four genes (p16, p15, MGMT and E-cadherin) was observed in MDS samples. All the samples analysed showed the absence of a methylator phenotype in MDS. The methylation frequency of all these genes increased with the clinical severity of the MDS subtypes. Therefore, hypermethylation may be used as a diagnostic and prognostic tool in ascertaining the clinical severity of MDS.

Polymorphism in ADH and MTHFR genes in oral squamous cell carcinoma of Indians.

OBJECTIVES: Alcohol consumption is known to increase the risk for several cellular disorders like oral cancer. The risk may be reinforced by polymorphism in genes like alcohol dehydrogenase. Therefore, this study is designed to asses the polymorphic status in ADH1B (formerly ADH2), ADH1C (formerly ADH3) and MTHFR genes in order to correlate the susceptibility to oral squamous cell carcinoma (OSCC). SUBJECTS AND METHODS: DNA from 126 OSCC samples were amplified using primers for ADH1B, ADH1C and MTHFR genes. The amplicons were analyzed for ADH1B*1, ADH1C*2 and MTHFR C677T allelic polymorphism by restriction digestion using appropriate enzymes. RESULTS: ADH1B*1/*1 genotype in cancer patients who were heavy drinkers showed a negligible risk association with an odds ratio of 1.62; 95% CI = 1.08-2.14. In OSCC patients, ADH1C*2/*2 genotypes showed a relatively higher risk (odds ratio 2.65; 95% CI = 1.78-3.53) in heavy drinkers and a less significant risk (1.6; 95% CI = 1.15-2.03) in moderate drinkers and negligible risk in light drinkers (1.23; 95% CI = 0.77-1.63). In contrast, MTHFR 677TT genotype showed a high risk association for OSCC in heavy drinkers (odds ratio 3.0; 95% CI = 2.02-4.0). Interestingly, the combination of ADH1B*1/*1/ MTHFR 677TT genotypes in alcoholic cancer patients showed a high risk (odds ratio 4.16; 95% CI = 2.78-5.53). A similar risk (odds ratio 4.16; 95% CI = 1.18-5.53) was shown by ADH1B*1/*2/*2/*2MTHFR 677TT genotype combination. The ADH1C*2/*2 /MTHFR 677TT genotype combination showed the maximum risk (odds ratio 20; 95% CI = 13.45-26.64) in the heavy drinker group. This combination showed a high risk in moderate drinkers (odds ratio 5.88; 95% CI = 4.24-7.50) and relatively lower risk in light drinkers (odds ratio 2.77; 95% CI = 1.74-3.68). CONCLUSIONS: The ADH1C*2/*2/MTHFR 677TT genotype combination appears to be more susceptible for OSCC, since it showed a 20-fold increase in risk in heavy drinkers and a 5.9- and 2.8-fold increase in risk respectively in moderate drinkers and light drinkers. This study suggests the association of ADH1C*2/*2/MTHFR 677TT genotype combination as a risk factor for OSCC in alcoholics.

A 5-month, randomized, placebo-controlled trial of galantamine in AD. The Galantamine USA-10 Study Group.

OBJECTIVE: To investigate the efficacy and tolerability of galantamine, using a slow dose escalation schedule of up to 8 weeks, in 978 patients with mild to moderate AD. METHODS: A 5-month multicenter, placebo-controlled, double-blind trial. Following a 4-week placebo run-in, patients were randomized to one of four treatment arms: placebo or galantamine escalated to final maintenance doses of 8, 16, or 24 mg/day. Outcome measures included the cognitive subscale of the AD Assessment Scale (ADAS-cog), the Clinician's Interview-Based Impression of Change plus Caregiver Input (CIBIC-plus), the AD Cooperative Study Activities of Daily Living inventory, and the Neuropsychiatric Inventory. Standard safety evaluations and adverse event monitoring were carried out. RESULTS: After 5 months, the galantamine-placebo differences on ADAS-cog were 3.3 points for the 16 mg/day group and 3.6 points for the 24 mg/day group (p < 0.001 versus placebo, both doses). Compared with placebo, the galantamine 16- and 24-mg/day groups also had a significantly better outcome on CIBIC-plus, activities of daily living, and behavioral symptoms. Treatment discontinuations due to adverse events were low in all galantamine groups (6 to 10%) and comparable with the discontinuation rate in the placebo group (7%). The incidence of adverse events in the galantamine groups, notably gastrointestinal symptoms, was low and most adverse events were mild. CONCLUSIONS: Galantamine 16 and 24 mg/day significantly benefits the cognitive, functional, and behavioral symptoms of AD as compared with placebo. Slow dose escalation appears to enhance the tolerability of galantamine, minimizing the incidence and severity of adverse events.

Identifying dementia in the primary care practice.

BACKGROUND: The purpose of this study was to evaluate the utility (i.e., positive and negative predictive value) of the 7 Minute Screen in identifying patients with probable Alzheimer's disease (AD) in a primary care practice. A second objective was to estimate the number of undiagnosed AD patients in a typical primary care practice. METHODS: One hundred thirty-seven successive admissions (96%) of patients over the age of 60 to a primary care practice over a 53-day period who completed informed consent documents were administered the 7 Minute Screen. All patients who screened positive (n = 13) and a random sample of those who screened negative (n = 26) returned for full diagnostic evaluation. Positive predictive value (PPV) and negative predictive value (NPV) of the 7 Minute Screen were determined using the criterion standard of clinical diagnosis established by examination, history, and laboratory studies. Test-retest reliability and time for administration were also determined. RESULTS: Of the 137 patients evaluated, 13 screened positive and 124 screened negative. Eleven of the 13 patients who screened positive were willing to return to the primary care practice for follow-up evaluation. A random sample of 26 patients who screened negative all agreed to return for follow-up evaluation. Of the 11 patients who screened positive and who returned for evaluation, 10 were subsequently diagnosed with probable AD. The remaining patient was diagnosed with mixed dementia. The caregivers of the two patients who refused to return were contacted and both indicated that the patients were having significant cognitive problems as verified by an activities of daily living scale. Of the 26 patients who screened negative, 25 were judged to be cognitively normal and the 26th was judged to have mild cognitive impairment. DISCUSSION: In successive admissions of patients over the age of 60 in a primary care practice, the 7 Minute Screen showed a PPV of 91% and an NPV of 96% in identifying patients who were subsequently identified with AD or other dementing disorder. These data suggest that this may be a useful instrument in identifying patients who should undergo diagnostic evaluation for AD and other dementing disorders. Additionally, extrapolation from the data in this practice suggests that there may be between 75 and 100 AD patients in the typical primary care practice, many of whom may not be diagnosed.

Correlational analysis of five commonly used measures of mental status/functional abilities in patients with Alzheimer disease.

There are multiple assessment techniques to help clinicians diagnose, stage, and measure the rate of progression in Alzheimer disease (AD). We analyzed retrospectively the relationship between scores on commonly used scales and tests (Mini-Mental State, the Blessed Information-Memory-Concentration Test, the Alzheimer's Disease Assessment Scale--cognitive portion, the Activities of Daily Living Scale, and the Global Deterioration Scale) of 100 successive admissions to a memory clinic. Patients were included in the study if they were diagnosed subsequently with probable AD and if all five measures were administered in the same day. Regression analysis yielded 20 linear equations that allowed for conversion between test scores on any two instruments. With the exception of the Activities of Daily Living Scale (intercorrelation range with the other four instruments, r = 0.56-0.66), intercorrelations were generally high (r = 0.81-0.87). The results of this study should provide a clinically useful tool for converting test scores on five commonly used dementia screening/rating instruments.

Five-year retention of the classically conditioned eyeblink response in young adult, middle-aged, and older humans.

Human participants who 5 years earlier participated in studies of acquisition of the classically conditioned eyeblink response to a tone conditioned stimulus (CS) and an air puff unconditioned stimulus (UCS) returned to the laboratory to test for retention of the conditioned response (CR). Retention consisted of 20 tone CS-alone presentations. Young adult participants (23-31 years of age at the time of retention testing) showed good retention of the CR (45%), middle-aged participants (45-52 years) showed reduced retention (28%), and older participants (69-78 years) showed little evidence of retention (< 5%). Retention testing was followed by reacquisition of the CR in which the CS and the UCS were again paired. The ability to reacquire the CR also showed a decline with age. The data suggest that the CR can be retained over long intervals and that the degree of retention is age dependent.

Recognition of Alzheimer's disease: the 7 Minute Screen.

BACKGROUND AND OBJECTIVES: Because Alzheimer's disease (AD) tends to be underdiagnosed, we developed a brief neurocognitive screening battery to identify AD patients. The 7 Minute Screen consists of four individual tests (orientation, memory, clock drawing, verbal fluency). The screen can be rapidly administered and scored and therefore may be appropriate for use in the primary care setting. This study determined the validity and reliability of the 7 Minute Screen in distinguishing patients with AD from healthy controls. METHODS: The 7 Minute Screen was administered to 60 consecutive referrals to a memory disorders clinic who were subsequently diagnosed with probable AD and to 60 community-dwelling individuals. Analysis of the combined scores on the four individual tests was used to determine the probability of dementia in each subject. We also evaluated test-retest and inter-rater reliability, as well as the time required to administer the battery. RESULTS: When compared with the normal subjects, the patients with AD were significantly more impaired on each of the four tests included in the 7 Minute Screen. When the four tests were combined into a logistic regression model, the battery correctly diagnosed 92% of the patients with AD and 96% of the normal subjects. The battery performed equally well when only patients with mild and very mild AD were included. Mean time for administration and scoring was 7 minutes 42 seconds. CONCLUSIONS: The 7 Minute Screen is a reliable and valid instrument for identifying patients with AD. It appears to be a potentially useful tool for identifying patients with AD in a primary care setting.

A 7 minute neurocognitive screening battery highly sensitive to Alzheimer's disease.

OBJECTIVE: To determine the validity and reliability of a rapidly administered neurocognitive screening battery consisting of 4 brief tests (Enhanced Cued Recall, Temporal Orientation, Verbal Fluency, and Clock Drawing) to distinguish between patients with probable Alzheimer's disease (AD) and healthy control subjects. SUBJECTS: Sixty successive referrals to the Memory Disorders Clinic at Southwestern Vermont Medical Center, Bennington, who were diagnosed as having probable AD and 60 community-dwelling volunteers of comparable age, sex distribution, and education. DESIGN: Interrater and test-retest reliability, intergroup comparisons between patients with AD and control subjects on the 4 individual tests, and determination of probability of dementia for patients with AD and control subjects using the entire battery of tests. SETTING: Outpatient care. MAIN OUTCOME MEASURE: Comparison of the probability of dementia on the 7 Minute Screen with the criterion standard of clinical diagnosis established by examination and laboratory studies. SECONDARY OUTCOME MEASURES: Test-retest and interrater reliability (correlation coefficients), time for administration. RESULTS: Mean time of administration was 7 minutes 42 seconds. Mean scores for patients with AD and control subjects on all 4 individual tests were significantly different (for each, P<.001). When the 4 tests were combined in a logistic regression, the battery had a sensitivity of 100% and a specificity of 100%. A series of 1000 repeated random samples of 30 patients with AD and 30 control subjects taken from the overall sample of 60 patients with AD and 60 control subjects had a mean sensitivity of 92% and a mean specificity of 96%. The battery was equally sensitive to patients with mild AD as demonstrated by correctly classifying all 13 patients with AD using Mini-Mental State Examination scores of 24 or higher. Neither age nor education was a statistically significant factor when added as a covariate. Test-retest reliabilities for individual tests ranged from 0.83 to 0.93. Test-retest reliability for the entire battery was 0.91. Interrater reliability for the entire battery was 0.92. CONCLUSIONS: The 7 Minute Screen appears highly sensitive to AD and may be useful in helping to make initial distinctions between patients experiencing cognitive changes related to the normal aging process and those experiencing cognitive deficits related to dementing disorders such as AD. It has reasonable interrater and test-retest reliability, can be administered in a brief period, and requires no clinical judgment and minimal training.

Classic conditioning in aged rabbits: delay, trace, and long-delay conditioning.

Young (0.5 years) and aged (2+, 3+, and 4+ years) rabbits underwent acquisition of the classically conditioned nictitating membrane response in a delay (500-ms conditioned stimulus [CS], 400-ms interstimulus interval [ISI]), long-delay (1,000-ms CS, 900-ms ISI), or trace (500-ms CS, 400-ms stimulus-free period) paradigm. Collapsing across age groups, there is a general tendency for animals to acquire trace conditioning more slowly than delay conditioning. Collapsing across conditioning paradigms, there is a general tendency for aged animals to acquire more slowly than younger animals. Of greater significance, however, are the age differences in the different conditioning paradigms. In the delay and long-delay paradigms, significant conditioning deficits first appeared in the 4(+)-year-old group. In the trace conditioning paradigm, significant conditioning deficits became apparent in the 2(+)-year-old animals.

Nimodipine facilitates retention of the classically conditioned nictitating membrane response in aged rabbits over long retention intervals.

Aged rabbits initially underwent 18 days of acquisition of the classically conditioned nictitating membrane response (NMR) using a tone conditioned stimulus (CS) and an air puff unconditioned stimulus (UCS). They were then treated with a low or high dose of nimodipine or a vehicle for 90 days. During this time no further CS-UCS pairings were presented. They underwent testing for retention of the conditioned response (CR) at 30 and 90 days. Retention testing consisted of 20 presentations of the CS alone. Rabbits in the control condition retained 46.4% of their predrug levels of conditioned responding and rabbits receiving the low dose of nimodipine retained 37.3% of their predrug levels after 30 days. After 90 days, retention in these animals declined to 8.1% and 14.1%, respectively. In contrast, rabbits receiving the high dose of nimodipine retained 85% of their predrug learning at 30 days with little decline at 90 days (77.1%). Nonassociative factors such as sensitivity to the CS or UCS could not explain these effects.

Classical conditioning in patients with Alzheimer's disease: a multiday study.

Previous studies demonstrated that patients with Alzheimer's disease (AD) do not acquire the classically conditioned eyeblink response. These studies, however, were only tested over a single conditioning session and, hence, raise the question of whether AD patients are capable of acquiring the response if sufficient training is given. This question may be of some importance whether AD patients can ultimately acquire the response has implications for the underlying neurobiological deficit in disrupted conditioning in AD. This study tested AD patients and age-matched controls over 4 days. As in previous studies, AD patients performed significantly worse than controls on Day 1, but by Day 4, they were not significantly different from controls. Subsequent testing indicated that these effects were not due to nonassociative variables such as changes in sensitivity to stimuli or disruption of the motor response. Also, it was reported that neither AD patients nor controls showed any evidence of acquisition in an explicitly unpaired paradigm, suggesting that neither pseudoconditioning nor sensitization is contributory. Data are discussed in terms of the possible role of the hippocampus in mediating conditioning deficits in AD patients.

Age-related deficits in retention of the classically conditioned nictitating membrane response in rabbits.

Young and aged rabbits underwent classical conditioning of the nictitating membrane response (NMR) to a tone conditioned stimulus (CS) and a corneal airpuff unconditioned stimulus (UCS) for 18 consecutive days. Rabbits were then returned to their home cages for a 90-day period in which they received no further conditioning, but they were handled daily. On Day 91 they underwent retention testing during which the CS alone was presented 20 times. This was immediately followed by reacquisition in which the CS and UCS were again paired for 100 trials. Reacquisition was repeated on the following day. As in previous studies, aged rabbits acquired the conditioned response (CR) more slowly than young rabbits; however, by the end of acquisition, both groups reached similar asymptotic levels. Retention of the CR was significantly lower for aged than young rabbits. Reacquisition was also retarded in aged vs. young rabbits. Nonassociative factors, such as sensitivity to the stimuli or general health, could not account for these differences. Data are discussed in terms of using retention of the conditioned eyeblink response as a model system for studying age-related memory deficits.

A 30-week randomized controlled trial of high-dose tacrine in patients with Alzheimer's disease. The Tacrine Study Group.

OBJECTIVE: To evaluate the efficacy and safety of high-dose tacrine hydrochloride over 30 weeks in patients with probable Alzheimer's disease. DESIGN: A 30-week randomized, double-blind, placebo-controlled, parallel-group trial. SETTING: Outpatients at 33 US centers. PATIENTS: Men and women at least 50 years of age with mild to moderate Alzheimer's disease and otherwise in good health. INTERVENTIONS: Group 1 received placebo; group 2 received 40 mg/d of tacrine for 6 weeks, then 80 mg/d for 24 weeks; groups 3 and 4 received 40 mg/d of tacrine for 6 weeks, 80 mg/d for 6 weeks, and 120 mg/d for 6 weeks. Group 3 remained on a dosage of 120 mg/d for a total of 18 weeks; after 6 weeks at 120 mg/d, group 4 titrated to 160 mg/d for the last 12 weeks. PRIMARY OUTCOME MEASURES: Clinician Interview-Based Impression (CIBI), Alzheimer's Disease Assessment Scale--Cognitive subscale (ADAS-Cog), and Final Comprehensive Consensus Assessment (FCCA). RESULTS: A total of 663 patients entered the study; 653 patients were included in an intent-to-treat (ITT) analysis; 263 had evaluable data at 30 weeks. The results of the ITT analysis revealed significant (P < or = .05) dose-response trends and between-group comparisons on CIBI and ADAS-Cog. In evaluable patients, significant dose-response trends were observed for all three primary measures (P < or = .001). Significant differences in favor of 160 mg/d of tacrine vs placebo were observed on the CIBI (P < or = .002) and ADAS-Cog and FCCA (P < or = .001), as well as caregiver-global and quality-of-life assessments (P < or = .05). On the CIBI, 23% and 42% of tacrine-treated patients in the ITT and evaluable-patient populations, respectively, were rated improved compared with 17% and 18% of placebo patients, respectively. The primary reasons for withdrawal of tacrine-treated patients were asymptomatic liver transaminase elevations (28%) and gastrointestinal complaints (16%). These adverse events were reversible on discontinuation of treatment, and many patients were able to restart tacrine. CONCLUSIONS: Tacrine produced statistically significant, dose-related improvements on objective performance-based tests, clinician- and caregiver-rated global evaluations, and measures of quality of life. There was no evidence that the large number of patient withdrawals biased the overall conclusions of the study.