Learning and Recall in Subjects at Genetic Risk for Alzheimer's Disease

There is great interest in studying cognitive function in subjects at risk for Alzheimer's disease (AD).¹ The apolipoprotein E E4 (APOE E4) allele is a risk factor for the development of AD, and subjects carrying the E4 allele have an earlier onset of AD.² Deficits in learning and delayed recall are reported to be among the most sensitive clinical markers of the early stages of AD.^3–6 For example, a 13-year prospective study of 1,045 subjects reported that measures of verbal memory were predictive of the development of AD.⁵ Measures of delayed recall are currently being used as one of the criteria to select individuals for dementia prevention trials.

The California Verbal Learning Test (CVLT) is a psychometric assessment designed to measure list learning and verbal memory ability.^7,8 Unlike tests that ask a subject to recall stories or remember certain tasks, the CVLT does not provide a semantic or procedural context in which the subject can organize the information presented. Thus it measures explicit or episodic memory⁹ in adolescent or adult subjects of any age. In addition to simple recall indices, CVLT results can reveal the underlying mechanisms that lead to changes in memory performance, including learning strategies, error types, and learning processes.^10,11 CVLT scores correlate highly with both the Verbal Comprehension Index of the Wechsler Adult Intelligence Scale-III and the Auditory Memory and General Memory indices of the Wechsler Memory Scale-III.¹²

Normative CVLT data have been presented on healthy young,¹³ depressed,¹⁴ and elderly individuals.¹¹ Paolo et al.¹¹ studied a large sample of elderly subjects (N=212, age range 55–94) and examined norms broken down by gender, age, and education, but not by APOE genotype. Studies of normative data on list-learning tests show that gender accounts for a considerable portion of the variance, with age and education accounting for much less variance in the prediction of cognitive performance.¹⁵ Although the APOE E4 allele itself is not necessarily associated with poorer memory performance,^16,17 some studies have demonstrated that seemingly normal individuals with the E4 allele who are on the verge of developing AD show memory deficits as much as two years before the onset of dementia.^18,19

The purpose of this study was to compare CVLT performance cross-sectionally in an elderly sample prospectively stratified by two risk factors for dementia: APOE and family history.

METHODS

Patient Population

Normal elderly subjects ages 55 and older were recruited from a registry of elderly volunteers interested in research, and by newspaper and flyer advertisement, and gave written informed consent. The primary goal of the study was to examine the effects of APOE and family history status on memory and brain chemistry as measured by magnetic resonance (MR) spectroscopy. Respondents underwent a screening and a brain magnetic resonance imaging scan to rule out significant neurological and psychiatric disorders. The MR scans are currently still being analyzed. All subjects possessed adequate vision and hearing, although some wore glasses and/or hearing aids. Individuals with dementia, depression, and past strokes were excluded from the study. Subjects with pacemakers or other metallic implants that prohibited them from undergoing an MRI were also excluded. Within the pool of subjects meeting our initial study criteria, emphasis was placed on recruiting equal numbers of subjects with and without the APOE E4 allele, and with and without a positive family history of dementia. Our goal was to recruit at least 25 subjects in each of the above four cells, with a target of about 40 per cell, so that we would have sufficient sample sizes in each cell to make comparisons of the memory and brain measures. This stratification required a two-step recruitment because the APOE alleles and FH are not evenly distributed in the population and a random sample would have resulted in far fewer subjects with E4 allele and very few subjects E4+/FH– or vice versa. A sample size of 160 was our target. APOE genotyping was performed with DNA extracted from buffy coat, using polymerase chain reaction (PCR) amplification and the HhaI restriction digest method of Hixson and Vernier.²⁰

Assessment Instruments

During each visit an experienced rater administered the Mini-Mental State Examination (MMSE), to screen for cognitive impairment, and the CVLT, a measure of list learning and verbal memory. The rater then scored the CVLT with computer software designed by the test's developers.⁷

The CVLT centers around a 16-item list, composed of four semantic categories of four words each. Adjacent words on the list are from different categories. Subjects may adopt a serial learning strategy by simply memorizing the words in order, or a semantic learning strategy by assigning the words to their respective semantic categories. The fact that the words are not prototypic examples of their categories increases the difficulty (for example, slacks is in the clothing category, but pants is not).

During a CVLT administration, the main 16-item list (List A) is presented five times, generating a summary learning measure (the sum of scores of the five trials), a recall consistency measure (the same words remembered across the trials), a learning rate (the number of words remembered plotted against trial number), and learning strategy measures (semantic and serial clustering ratios, and the percentage of words recalled from the primacy, middle, and recency regions). A second, different list (List B) designed to interfere with List A is then presented. Afterwards, the subject is asked to name the items from List A freely (short-delay free recall) and by category (short-delay cued recall). The subject then is distracted for 20 minutes, after which he or she is asked again to name the List A words in any order (long-delay free recall), and by category (long-delay cued recall). During any of the recall stages, the subject may name the same word more than twice (a perseveration) or name a word that was not on the original list (an intrusion). Finally, the administrator tests recognition memory by reading a longer list and asking the subject whether each word was on List A or not, generating measures of correct answers (recognition hits), identification of words absent from the list (false positives), and the ability to tell apart valid list words from nonlist words (discriminability).

From these data, scoring software generates a list of contrast measures that indicate the rate of forgetting across various stages during the test. We did not use the last contrast measure, percentage change from discriminability to long-delayed free recall. All in all, one CVLT administration generates 28 indices of verbal memory, learning ability, learning strategy, and forgetfulness.

Statistical Methods

To test the effects of demographic variables, subjects were divided into two groups around the median age, 65; two groups based on gender; three groups based on education level (completed high school education or less, completed or attempted college education, and schooling beyond college); two groups based on the presence or absence of a family history of dementia; and two groups based on the presence (E2/E4, n=4; E3/E4, n=59; E4/E4, n=9) or absence (E2/E2, n=0; E2/E3, n=9; E3/E3, n=72) of the APOE E4 allele.

In accordance with the findings of Small et al.¹⁹ and Bondi et al.,¹⁰ we also hypothesized that subjects without E4 alleles would have higher scores than participants with at least one E4 allele. Age, education, and gender effects were also predicted from the literature. General linear models, t-tests, and chi-square tests on SAS software were done. All analyses were two-tailed.

All of our subjects were elderly and nondemented. Because any group differences were likely to be small, a testwise alpha level of 0.05 was used to determine significance for the a priori hypotheses.

RESULTS

In total, 153 subjects (77 males and 76 females) participated in the study. The age range was 55 to 85, with a mean (±SD) of 66.7±7 years. The mean number of years of education was 15.6±3 and the mean MMSE score was 28.4±1. Of all the subjects, 87 were family history negative (FH–) and 66 were family history positive (FH+). In addition, 81 had no APOE E4 (APOE E4–) alleles, and 72 had either one or two APOE E4 alleles (APOE E4+).

Subjects with ages greater than 65 years demonstrated a greater decline from List A Trial 1 to List B (t=2.1, df=152, P<0.05) and a greater increase from the short delayed free recall measure to the long delayed free recall measure (t=–2.0, df=152, P<0.05) than younger subjects. The effect of age as a continuous variable on CVLT performance was also examined. Both the summary learning measure (F=4.8, df=1,152, P<0.05) and the percent recall from the recency region (F=4.1, df=1,152, P<0.05) declined with age.

Women scored higher than men on every recall measure, including the summary learning measure (t=4.4, df=152, P<0.001). Women also used semantic clustering more than men (t=2.5, df=152, P<0.05) and were better at discriminating words (t=2.3, df=152, P<0.05) and rejecting false positives (t=–2.5, df=152, P<0.05) on the recognition portion of the test. CVLT performance among the education groups differed significantly on only one measure, percent recall from the recency region (F=4.3, df=2,152, P<0.05).

Table 1 displays CVLT scores grouped by APOE E4 and FH status. There were no significant differences on any measures between the APOE E4 and non-APOE E4 groups. FH+ subjects were younger than FH– subjects (Table 1). The only significant CVLT difference between the FH– and FH+ groups was on List B scores (t=2.6, df=151, P<0.05). Table 2 summarizes test performance for subjects grouped by both FH and APOE status. There were no differences overall. The only variable that demonstrated a significant difference was once again List B, on which the E4–/FH– group performed better than the E4+/FH+ group (t=2.6, df=93, P<0.05).

We then used a nonhierarchical linear model to examine the effects of age, gender, education, and genetic status on CVLT measures. After adjusting for the effects of the other variables, the effect of age or E4 status was not significant on any of the recall indices. The effect of gender remained significant (P<0.01) in this model for all recall indices as well as four derived measures. Five recall measures, including the summary learning measure (F=3.8, df=2, 152, P<0.05), the short delayed free recall measure (F=3.1, df=2, 152, P<0.05), and the long delayed free recall measure (F=4.0, df=2, 152, P<0.05) showed significant differences by education status. Female gender and higher education levels were related to better performance.

DISCUSSION

We present CVLT data for a normal, nondemented elderly population stratified by two genetic risk factors for AD. This is also the first report to systematically examine the effects of genetic risk status on CVLT performance in a relatively large aging sample. Aside from the CVLT manual,⁷ only one study, Paolo et al.,¹¹ has presented normative CVLT data for elderly persons. Our findings confirm this report. Examining age as a continuous variable, Paolo et al. reported an age effect on all eight CVLT recall indices. Although we found a statistically significant negative age effect only on the CVLT summary learning measure, Paolo and colleagues' group was older (mean age 70.6±7 years) than ours (65.8±7 years). Since we had a more limited age range, we analyzed our data with age split at age 75 (not shown), and these analyses tended to confirm the findings by Paolo et al. Like those authors, we found that women consistently scored higher than men on both the recall and recognition measures. Likewise, we confirmed their findings that higher education is associated with better performance on many recall indices. Thus, these findings emphasize that the CVLT scores in elderly subjects, like many other cognitive tests, must also be interpreted in the context of age, education, and gender.

This study extends the report of Paolo et al. by also simultaneously examining the effects of family history of dementia and APOE genotype, using a planned stratified recruitment design. In our population, subjects with a family history of dementia performed worse on only one CVLT measure (List B), and subjects with at least one E4 allele did not perform worse on any measures than those without such risk factors. List B is a distraction list, and hence the clinical significance of this finding is puzzling. It could reflect a spurious finding (due to multiple comparisons) or could reflect a true subtle difference in cognitive capacity. In a prior study, Bondi et al.¹⁰ reported that E4-positive elderly subjects tested worse on nine different CVLT measures. Bondi and colleagues' subject pool was smaller (17 E4+, 35 E4– in Bondi; 72 E4+, 81 E4– in our study) and 6 years older on average (mean age 71 in Bondi study; mean age 66 in our study). Age differences may explain both our results, since E4-positive subjects would be expected to perform worse if tested closer to the onset of dementia. Alternatively, our sample consisted of healthy volunteers and the differences may reflect a sampling effect.

There is interest in using tests of word learning and word recall to detect the early stages of dementia. In this respect, our results provide additional data that may enhance the interpretation of these tests in cognitively impaired patients. However, there are also some limitations to this report. We selected a group of subjects from an aging center's registry of volunteers and by advertisement. Our sample size may have been too small to detect subtle differences, and the higher education level may also have obscured genetic differences on test scores. We ran multiple analyses and did not adjust for multiple comparisons. Most of these analyses were preplanned, and the P-values reported should be interpreted with that in mind. The strengths of this study are the careful selection of subjects and the availability of APOE genotyping. These results must be interpreted accordingly.

In summary, age, gender, and educational level have significant, but variable, effects on the CVLT. Neither family history of dementia nor APOE E4 status had a detectable consistent effect on any learning or memory measure. This finding may reflect either the relative insensitivity of the CVLT or the fact that memory impairments in at-risk subjects may not occur until the person is much closer to the onset of AD. Community-based studies of subjects followed longitudinally will be needed to examine these issues further.

ACKNOWLEDGMENTS

This work was supported by the Paul Beeson award from the American Federation for Aging Research and the Novartis Scholar award to Dr. Doraiswamy.

TABLE 1. California Verbal Learning Test (CVLT) scores by presence or absence of APOE E4 alleles and family history (FH)

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TABLE 2. California Verbal Learning Test (CVLT) scores across four groups broken down by APOE E4 status and family history (FH)

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