APOE) *E4 allele and Alzheimer's disease is very strong, the effect of the *E4 allele on cognitive decline in the general population is more equivocal. A cross-sectional study on the lifespan effects of the *E4 allele [*E4 allele on cognitive performance at ages 20–24, 40–44 or 60–64 years.While the evidence of an association between the apolipoprotein E  was significantly poorer for *E4 carriers become vulnerable to greater cognitive decline in the presence of other risk factors at 65–69 years of age.It is possible that  E4 allele (APOE *E4) is associated with increased risk of Alzheimer's Disease (AD). Forty to forty-five percent of individuals diagnosed with Alzheimer's type dementia have at least one *E4 allele, compared with approximately 15% of the American population . SNP-specific primers and probes were designed by ABI (TaqMan genotyping assays) and assays were performed according to the manufacturer's instructions in 5 μl total volumes in 384-well plates. The polymorphisms distinguish the *E2 allele from the *E3 and *E4 alleles at amino acid position 158 (NCBI rs7412) and the *E4 allele from the *E2 and *E3 alleles at amino acid position 112 (NCBI rs429358).Genomic DNA was extracted from cheek swabs using Qiagen DNA Blood kits . To identify the six Cfo1 restriction fragment length analysis, following PCR amplification of part of the APOE gene [TaqMan real-time polymerase chain reaction assays (PCR) were performed in an ABI 7900 HT machine, using a cycling program of: 95°C for 10 min; 40 cycles of 95°C for 15 sec and 60°C for 1 min. Six positive controls, one for each genotype, and one negative control (water) were included in each plate and were consistently called correctly. The genotypes of the six positive controls were confirmed by POE gene .rs7412: χ2 = 1.631, df = 1, p > .20, n = 2,281; rs429358: χ2 = 0.002, df = 1, p > .97, n = 2,281) and Wave 2 . For the purposes of this analysis, participants were classified into three groups: *E4-/*E4- , *E4+/*E4- (heterozygous for *E4) and *E4+/*E4+ (homozygous for *E4).Allelic frequencies were consistent with Hardy-Weinberg expectations for the PATH 60+ age cohort for both Wave 1 , secondary and tertiary educational attainment. Responses to these questions were coded into a single measure corresponding to the number of years of education. For the purposes of the analyses, education was then categorized into four groups: 0–12 years, 13 years , 14–15 years, and 16 years or more.Participants were classified as having head trauma if they responded positively to a question asking whether they had ever had a serious head injury that had caused them to become unconscious, at either Wave 1 or Wave 2 .Past alcohol consumption was measured using two items regarding the frequency and amount of alcohol consumption at the time the participant was drinking at their highest level. Respondents with potentially hazardous or harmful levels of drinking were identified as those who averaged more than 14 standard drinks per week. Current alcohol consumption was measured with the Alcohol Use Disorders Identification Test (AUDIT) . RespondIntelligence was estimated using lexical decision performance on the Spot-the-Word Test Version A (STW), which asks participants to choose the real words from 60 pairs of words and nonsense words .History of stroke was ascertained using one question, "Have you ever suffered a stroke, ministroke or TIA (Transient Ischemic Attack)?"Blood pressure was measured twice during the interview. Readings for diastolic and for systolic pressure were averaged for each interview. Hypertension was defined as having a mean systolic blood pressure ≥ 140, a mean diastolic blood pressure ≥ 90 at Wave 1 or as taking medication for hypertension.Mental speed was measured with the Symbol-Digit Modalities Test, which asks the participant to substitute as many digits for symbols as possible in 90 seconds .Reaction time (RT) was tested using a small box held with both hands, with left and right buttons at the top to be depressed by the index fingers . There wImmediate and delayed recall were assessed with the first trial of the California Verbal Learning Test , which iWorking memory was assessed with the Digits Backwards subtest of the Wechsler Memory Scale , which pThe 11 item Mini-Mental State Examination (MMSE)  was admiCognitive change scores were calculated for each test by subtracting Wave 1 scores from Wave 2 scores.At each wave, participants who scored below a predetermined cut-off on a screening battery were assessed for clinical diagnoses. Participants from the full cohort were selected for clinical assessment if they had any of the following: (1) a Mini-Mental State Examination (MMSE) score ≤ 25; (2) a score below the 5th percentile score on immediate or delayed recall of the California Verbal Learning Test , or (3) a score below the 5th percentile score for Wave 1 on two or more of the following tests: Symbol-Digit Modalities Test (<33), Purdue Pegboard with both hands  or reaction time .The clinical assessment in the PATH study has been described previously . It invoAPOE genotype, other risk factors, and APOE genotype by risk factor interactions on cognitive decline for each of the seven cognitive tasks. Multivariate ANOVA models for each cognitive change score were constructed, simultaneously including APOE genotype and any risk factors found to predict cognitive decline in association with genotype. Participants who did not complete a particular cognitive test at either Wave 1 or Wave 2 were excluded from the bivariate and multivariate analyses for that test. Sample sizes for bivariate and multivariate models were n = 1953 for SDMT, n = 1961 for immediate recall, n = 1961 for delayed recall, n = 1941 for digits backwards, n = 1953 for MMSE, n = 1877 for simple RT, and n = 1860 for choice RT. The reduced samples were due to a combination of missing premorbid intelligence data (24 cases) and missing cognitive test data (range: 36–137 cases). Analyses were repeated with the exclusion of those who were found to have any of the clinical diagnoses at either measurement point (n = 126). A significance level of .05 was used for individual analyses.A series of bivariate models were examined for the effect of *E4 non-carriers, 510 heterozygous for *E4 (25%) and 38 (2%) homozygous for *E4.The genotyping results for this sample have been described previously in Jorm et al. . In the APOE genotype. The p-values in Table APOE genotype categories, the difference was not significant when age was treated as a continuous variable . With the exception of RT, negative change scores indicate poorer performance at Wave 2. *E4 status did not increase the risk of head injury, problem drinking, stroke or current hypertension. There were no significant effects of APOE genotype on gender, education or premorbid intelligence.Tables t1953 = -8.14, p < .0001); Immediate Recall ; Delayed Recall ; Simple RT ; Choice RT . MMSE deterioration was marginally significant . Digits Backward performance improved . The observed effect sizes (Cohen's d) of the changes in the cognitive variables were .11 for SDMT, .14 for immediate recall, .07 for delayed recall, .07 for digits backwards, .05 for MMSE, .44 for simple RT and .23 for choice RT. There was one effect of APOE genotype on cognitive performance. At Wave 2, those homozygous for *E4 performed more poorly on the MMSE . APOE genotype did not significantly predict change in performance on any of the cognitive tests.As was expected, cognitive test scores declined significantly over the 4 year period: SDMT , head injury (SDMT) and premorbid intelligence (MMSE). In addition, the main effect of APOE genotype was significant for SDMT scores when adjusting for head injury and for digits backwards scores when adjusting for premorbid intelligence. No significant interaction effects of APOE with gender, current or past hazardous drinking, stroke or hypertension emerged. These five predictors were subsequently omitted from multivariate models.Bivariate ANOVA models tested the interaction of APOE was found to be significantly associated with performance on SDMT and digits backwards. Interaction effects consistent with the bivariate analyses were also present for genotype and education , head injury (SDMT and MMSE) and premorbid intelligence (MMSE). All of the significant effect sizes were very small, with η2 ranging from 0.003 to 0.009. R2 for the models were also very small, ranging from 0.001 to 0.01.Table APOE heterozygotes and homozygotes combined for simplicity. The interaction between APOE genotype and education was significant for both immediate and delayed recall change scores  tended to show more decline on the recall tasks than those with less education (0–15 years). There was a greater decrease in MMSE scores among *E4 non-carriers who experienced head injury . The main effect of APOE genotype on MMSE at Wave 2 remained significant. The multivariate model excluding participants with a clinical diagnosis is shown in Table The analyses were rerun excluding the participants with a clinical diagnosis (n = 126). The proportion of participants with a diagnosis did not differ significantly across genotypes  reported that the *E4 allele was associated with faster decline on four cognitive domains over 6 years, with the effect greatest for memory [*E4 allele in an representative sample followed for 7 years, with the youngest aged 77 at follow-up. These findings remained when those with mild cognitive impairment were excluded. Similarly, Small, Basun and Backman [*E4 compared to non-carriers within a sample of participants without dementia aged over 80 years. The weaker findings in our study are likely to due to the younger age of the participants who were mostly aged in their mid sixties.As expected, given the age of our sample, the findings of the present study are generally weaker than those from other population studies examining the effects of genotype. These studies usually report relatively robust associations between r memory . Hofer er memory  reported Backman  reportedThe present study specifically sought to determine whether the young old age group might be at a stage when the effect of the APOE genotype 'kicked in'. Our findings are consistent with the effects of APOE emerging at this stage in those vulnerable as a result of the presence of an additional risk factor. We found interactions of genotype with education, intelligence and head injury, but not with high alcohol use, stroke or cardiovascular risk. Earlier longitudinal work reported the effects of genotype emerging in midlife in at risk individuals (those with a first degree relative with AD) ,12 and uAPOE *E4 and low prevalence risk factors such as head injury, stroke and hazardous alcohol use [APOE status .APOE were present primarily for the MMSE. Reflecting its use as a screen for dementia, this test features a number of items that reflect orientation in time and location. It is assessing impairment at a relatively high threshold. However, a characteristic of the MMSE is its ceiling effect. This means that it is easier to register decline, but hard to show improvement. Models which recognize ceiling effects (effectively considering the scores to be censored above) involve very strong assumptions. We took the approach of including only decliners – those showing a decline on the MMSE – and repeating the analyses. The findings remained unchanged. We also examined the proportion of those decliners as a function of MMSE status, and found similar results. One clear implication is that these analyses warrant replication in four years when an additional wave of data will be available which tracks participants over a longer period into middle old age.Whether these significant effects came about because of artifacts associated with psychometric properties of the tests used must be considered. The effects of APOE *E4 in association with risk factors may begin to emerge after 60–65 years of age. The findings are consistent with biochemical research [APOE in the young old may only emerge in interaction with other risk factors and on tests sensitive to cognitive load [On balance, our interpretation of these findings is that effects of research , and witive load ,12. We pAPOE *E4 status does not influence level of cognitive functioning for individuals aged 20–64 years. This suggested that cognitive aging processes are influenced by other unknown or uninvestigated factors at this age range or that the effect of APOE emerges at detectable magnitudes after 64 years of age. Four years later, follow-up data suggest that APOE *E4 is associated with poorer cognitive performance, as measured by the MMSE, and may influence the rate of cognitive decline in interaction with risk factors such as previous head injury or low education. It is possible that *E4 carriers appear to become vulnerable to greater cognitive decline in the presence of other risk factors at this age.Our earlier study provided evidence that The authors declare that they have no competing interests.HC drafted the manuscript and contributed to the design of the study; PJB drafted the manuscript and performed the analysis; AJM contributed to the design of the study, contributed to the analysis and revised the manuscript; AFJ revised the manuscript and contributed to the design of the study; HAM revised the manuscript and assisted with the APOE genotyping; KAM revised the manuscript and assisted with the APOE genotyping; KJA revised the manuscript and contributed to the design of the study; PSS revised the manuscript and contributed to the design of the study; SE assisted with the APOE genotyping; all authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Dizziness in general practice is very common, especially in elderly patients. The empirical evidence for diagnostic tests in the evaluation of dizziness is scarce. Aim of our study was to determine which set of diagnostic tests should be part of a diagnostic protocol for evaluating dizziness in elderly patients in general practice.We conducted a Delphi procedure with a panel of 16 national and international experts of all relevant medical specialities in the field of dizziness. A selection of 36 diagnostic tests, based on a systematic review and practice guidelines, was presented to the panel. Each test was described extensively, and data on test characteristics and methodological quality  were presented. The threshold for in- or exclusion of a diagnostic test was set at an agreement of 70%.During three rounds 21 diagnostic tests were selected, concerning patient history (4 items), physical examination (11 items), and additional tests (6 items). Five tests were excluded, although they are recommended by existing practice guidelines on dizziness. Two tests were included, although several practice guidelines question their diagnostic value. Two more tests were included that have never been recommended by practice guidelines on dizziness.In this study we successfully combined empirical evidence with expert opinion for the development of a set of diagnostic tests for evaluating dizziness in elderly patients. This comprehensive set of tests will be evaluated in a cross-sectional diagnostic study. Dizziness is very common, especially in elderly patients. -3 In 200Although practice guidelines recommend the use of several diagnostic tests in the evaluation of dizziness, these recommendations are mainly expert-based. Many authors have reported on tests used for diagnosing dizziness, but few studies investigated the diagnostic accuracy of these tests. Often the methodological quality of these studies was poor. In addition, all diagnostic accuracy studies were carried out in secondary or tertiary care settings. Therefore these results cannot be straightforward extrapolated to a primary care setting. Furthermore, none of these studies included an elderly population, although the prevalence of dizziness as well as the risk of more serious pathology increases with age .The aim of our study was to determine which set of diagnostic tests should be part of a diagnostic protocol for evaluating dizziness in elderly patients in general practice. This set of tests will be evaluated in a cross-sectional diagnostic study. Because the empirical evidence is scarce and guidelines are contradictory, we have chosen to conduct a Delphi procedure. During this procedure we combined empirical evidence with expert opinion, in order to create a solid base for a future guideline on dizziness.To identify potentially relevant diagnostic tests for dizziness in general practice we performed a sensitive search in PubMed, EMBASE, PsychINFO, CINAHL and Gerolit, from database inception to February 2005 . Two reviewers (OM and JD) independently selected potentially relevant studies on title and abstract (selection criteria: Appendix 1). From the initial 17,950 hits, 228 studies on diagnostic tests for dizziness possibly feasible in primary care were retrieved for full-text reading. One hundred and eighteen studies were excluded after full-text reading. The methodological quality of the remaining 110 studies was assessed with the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) by two couples of reviewers  were removed from the initial list. Based on the identified diagnostic studies and practice guidelines we constituted an initial list of 36 diagnostic tests: 4 elements of patient history, 21 elements of physical examination, and 11 additional tests.A group of 24 national and international experts on dizziness  were invited to participate in the Delphi procedure. Selection of experts was based on publications , or participation in the development of a guideline on dizziness, syncope, or vertigo.The Delphi method, developed by the Rand Corporation in the 1950s, is a method for eliciting consensus opinions from experts. CharacteWe conducted our Delphi procedure via e-mail. In the first round each participant received the list of 36 potential diagnostic tests, with background information on every test, and an overview of the corresponding empirical evidence (as assessed with the QUADAS-tool). The participants also received background information on the search for evidence, the Delphi procedure, the abstracts of relevant diagnostic studies, and a summary of nine practice guidelines concerning 'dizziness', 'syncope' or 'vertigo'. -24 The pIf a participant deemed that another diagnostic test should be added, he or she could note this on the score form with a motivation and a brief description of the test.Tests on which at least 70% of the participants agreed with each other were either included in the protocol or deleted from the list. Tests on which no agreement had been reached were presented in the second round. In this round participants received information on the percentage agreement per test in the first round, motivations for rejection, a summary of comments of the participants, and their own score compared with the group score. The participants had to indicate for each remaining test if it should be incorporated in the diagnostic protocol with a short motivation. This round allowed participants to change their score in view of the group's response.We planned to carry out additional rounds if necessary. We scheduled four weeks for each Delphi round, two weeks for the participants to complete the score form, and two weeks to interpret the results and to incorporate them into the subsequent round's score form.E-mail invitations explaining the study were sent to 24 experts, of which 16 agreed to participate. Reasons for non-participation were lack of time (n = 5), lack of expertise (n = 1), or unknown (n = 2). Information on the participants is presented in Table The results of the Delphi procedure are presented in Table The level of agreement in the first round for recommended tests compared to non-recommended tests was 78.5% vs. 71.1%.In the second round, 17 tests  were assessed. This resulted in three included tests, five excluded tests, and nine tests on which no agreement could be reached. In total, 19 tests were included after 2 rounds.Participants changed their opinion on average almost three times out of 17 (17.3%), varying from zero to six times.Taking into consideration the comments of the participants, and the limited change in group scores of the nine tests on which no agreement had been reached, we deemed an additional voting round not fruitful. The remaining nine tests were therefore summarized  and discussed in a consensus meeting of the research group, after which a draft diagnostic protocol was constructed. Three of the nine tests were added to the draft protocol: the Orthostatic hypotension test (OHT), the Semmes-Weinstein Monofilament Test (SWMT), and the Patient Health Questionnaire (PHQ). Motives for adding the OHT (63% agreement after the second Delphi round) were the frequent application of this test in daily practice , the high prevalence of orthostatic hypotension in elderly people, and the strong request of several panel members to include this test. Motives for adding the SWMT (58% agreement) were the lack of somatosensory tests on the list, the user-friendliness, and the fact that GPs are already familiar with this test (as part of diabetes care). The PHQ (69% agreement) was added to the draft protocol, because various studies suggest that psychiatric disorders may play a causative or contributory role in dizziness. OR "Syncope" [MeSH] OR "Vertigo" [MeSH]) AND ("guideline" [Publication Type] OR "practice guideline" [Publication Type])II. Dutch internet sites*- Care4Cure - Dutch Association of Insurance Medicine - Dutch Association of Nursing Home Physicians - Dutch College of General Practitioners - Dutch Institute for Healthcare Improvement (CBO) - Huisarts en Wetenschap - Nederlands Tijdschrift voor Geneeskunde - Spreekuurassistent - The Netherlands Society of Cardiology (NVVC) - The Netherlands Society of Neurology (NVN) - The Netherlands Society of Occupational Medicin (NVAB) - The Netherlands Society for Otorhinolaryngology and Cervico-Facial Surgery III. International internet sites*- Bandolier - Clinical Evidence - Clinical Knowledge Summaries - The Cochrane Library - DARE - Das Ärztliche Zentrum für Qualität in der Medizin - Evidence-Based Medicine Guidelines - Guidelines Finder - The Guidelines International Network - National electronic Library for Health (NHS), - National Guideline Clearinghouse - National Institute for Health and Clinical Excellence - New Zealand Guidelines Group - Scientific Society of Flemish General Practitioners, WVVH - Scottisch Intercollegiate Guidelines Network (SIGN) - SUMSearch - The Swedish Council on Technology Assessment in Health Care - Trip Database - UpToDate *: If an internet site contained hyperlinks to other sites with possible information about practice guidelines on dizziness, syncope, or vertigo, these sites were also visited.The pre-publication history for this paper can be accessed here:


F1,58 = 26.24, p = 0.047, corrected). Under spontaneous avoidance perfusion increments were observed in several areas that were deactivated by the task, including the perigenual medial prefrontal cortex. Furthermore, individual differences in executive capacity were not associated with positive bias. These findings suggest that spontaneous positive cognitive emotion regulation in health may result from processes that, while actively suppressing emotionally salient information, differ from those associated with effortful and directed control.Even in the presence of negative information, healthy human beings display an optimistic tendency when thinking of past success and future chances, giving a positive bias to everyday's cognition. The tendency to actively select positive thoughts suggests the existence of a mechanism to exclude negative content, raising the issue of its dependence on mechanisms like those of effortful control. Using perfusion imaging, we examined how brain activations differed according to whether participants were left to prefer positive thoughts spontaneously, or followed an explicit instruction to the same effect, finding a widespread dissociation of brain perfusion patterns. Under spontaneous processing of emotional material, recruitment of areas associated with effortful attention, such as the dorsolateral prefrontal cortex, was reduced relative to instructed avoidance of negative material ( An aspect of healthy mental functioning is the positive connotation of thoughts arising spontaneously or occasioned by external stimuli, as is demonstrated by the reversal of this positive bias in depression Because it is active even if negative stimuli are present Outside the field of emotion regulation, however, examples of positive bias are known that appear to arise automatically when evaluating choices or risks scrambled sentences task). When legal sentences with different meanings are possible given the words in the set, a choice is implicitly made among the alternative solutions to the task. For example, “bright the very dismal looks future” may be unscrambled to form a negative thought, “the future looks very dismal,” or a positive thought, “the future looks very bright.” When the alternative sentences have an affective meaning, as in this example, healthy participants are remarkably selective and spontaneously tend to avoid the pessimistic version, forming 70–80% positive sentences even if the existence of emotional words is not mentioned in the explanation of the task Positive bias in cognitions is demonstrable by asking participants to assemble sentences from a set of scrambled words , and emotional . Negative words were significantly more arousing than positive words (see spontaneous group); a second group, in contrast, was explicitly instructed to avoid forming negative sentences (instructed group). Both groups formed sentences under the neutral control and affective condition. We reasoned that, if the effortful control account is correct, a recruitment of brain areas associated with executive processes must be observed in both the spontaneous and instructed groups when confronted with emotional ambiguous material, since avoiding negative sentences would place similar increased demands on the executive attentional network relative to the neutral condition, where sentence formation is unconstrained We undertook a direct comparison of spontaneous and intentional avoidance of negative content in the scrambled sentences task using a neuroimaging technique that allows quantitative estimation of perfusion levels  of participants using a complex memory span test OSPAN . WorkingBy contrasting spontaneous and voluntary forms of avoidance of negative content, we show that areas associated with executive control are recruited less under spontaneous response. Furthermore, we provide evidence for the dissociation between the neural substrates of spontaneous and voluntary emotion regulation. We anticipate that the identification of these spontaneous mechanisms of emotion regulation will improve our understanding of psychiatric disorders of affect.z = −11, p<0.001) as well as in the instructed group (0.5% negative sentences). In the spontaneous group, depressiveness scores were associated with rates of negative sentences , even if these scores were all low and in the normal range. In contrast with the prediction of the effortful control hypothesis, the association between WMC and avoidance of negative sentences in the spontaneous group was not significant . However, WMC scores did predict the proportion of well-formed sentences (negative or positive) formed in the task , both in the spontaneous (z = 3.5) and in the instructed group (z = 2.7). Rates of negative sentences formed during scanning and in the pre-scan test were highly correlated across subjects .As anticipated, participants avoided negative sentences in the spontaneous group  and instruction (explicit instruction or spontaneous avoidance of negative sentences), against the baseline perfusion at rest. This preliminary contrast revealed the activation of areas known to be involved in attentional and linguistic processing x, y, z: 44, 4, 52, F1,58 = 26.24, p = 0.047, corrected at voxel level for the whole volume). Further analysis revealed that the interaction was due to reduced perfusion in the emotional relative to the neutral condition in spontaneous participants , while in instructed participants perfusion increased slightly . This same interaction extended posteriorly on the left to the superior parietal areas activated by the task  and instruction (explicit instruction vs. spontaneous avoidance of negative sentences), using the areas of task activation and deactivation as an interpretive guide , reachinthe task .x, y, z: −64, −10, 28, BA43, t = 4.73, p<0.001), the perigenual medial prefrontal cortex . These activations were more marked in the spontaneous than in the instructed group, as these same areas appear in the interaction between groups and condition in x, y, z: −8, 38, 6, F1,58 = 9.05 [t = −3.01], p = 0.004; insula/prefrontal operculum, x, y, z: −64, −20, 30, F1,58 = 10.20 [t = −3.19], p = 0.002; ITG, x, y, z: −50, −24, −22, F1,58 = 8.23 [t = −2.87], p = 0.006, all uncorrected). Furthermore, these activations were prevalently localized to areas that were deactivated by the main task , and present as a trend in the perigenual MPFC .To further investigate the mechanism of spontaneous control we undertook an exploratory analysis of changes in perfusion in the spontaneous group, contrasting exposure to emotional with neutral material, and using the areas of task activation and deactivation detected by perfusion imaging to aid interpretation  online. x, y, z: −48, 22, −10, t = 5.37, p = 0.01, corrected for the whole volume), and when the spontaneous group was exposed to emotional material . Analysis of the interaction of these areas indicated that these small activations were slightly larger in the spontaneous group . A similar pattern was present on the right .The left ventrolateral prefrontal cortex (VLPFC) was an exception to this pattern . This arOutside the dorsal attentional network, instructed participants showed increased perfusion levels in the presence of emotional material in the occipital/calcarine cortex, and, in the medial wall, the cuneus, precuneus and posterior cingulate  online.Two independent sets of findings presented here support the notion that spontaneous avoidance of negative content reflected mechanisms distinct from explicit control, as predicted by the affect heuristic model of spontaneous avoidance. Firstly, when spontaneously avoiding negative sentences, there was less recruitment of executive attentional areas when exposed to emotional ambiguous material (where avoidance of negative content occurred) than to neutral material (in which no avoidance was required). In contrast, instructed avoidance increased this recruitment, most notably in DLPFC/FEF, an area involved in effortful attending to the visual environment in conformance to current goals and expectations Secondly, working memory capacity had little influence on the rate of the spontaneous avoidance of negative sentences. This was not due to the failure of working memory capacity scores to account for variance in performance, since working memory capacity was associated with the number of sentences formed overall. Hence, these scores were associated with the participants' success in carrying out the task deliberately attended to, but not their success in the spontaneous avoidance of negative sentences.Neuroimaging studies of control of emotional content have investigated the effect of the presence of emotional information while its role as target or distractor is defined by an explicit instruction While this study was primarily designed to answer the question of whether the effortful control hypothesis adequately accounts for spontaneous forms of emotion regulation, it is natural to query the data about the pattern of changes induced by emotional material in the spontaneous group, in view of the information it may provide on how a model of spontaneous regulation may look like. While many of the changes observed in the spontaneous group may have been induced by the presence of emotional stimuli as such, a specific mechanism must have been active to influence the direction of thoughts without recurring to voluntary control. Remarkably, in the spontaneous group very little increased recruitment induced by the presence of emotional words was present in the areas activated by the task, especially compared to the extent of activation of the dorsal attentional network associated with the explicit instruction.Two systems emerged in this study as candidate neural correlates of response modulation in spontaneous avoidance. The first was given by the piece-meal reversal of the typical pattern of activations and deactivations associated with focussed execution of a task relative to an unconstrained resting baseline The second were the areas in VLPFC that were more recruited in the presence of emotional material also during spontaneous control, and in this respect dissociated from more dorsal areas in the prefrontal cortex. This dissociation parallels the known functional dissociation of their right-hemisphere homologues in studies of spatial attention, in which dorsal areas implement voluntary and effortful attentional process, while a ventral network is involved in attentional reorienting according to behavioural relevance and irrespective of perceptual salience As a whole, the modulation of activations and deactivations associated with instructed and spontaneous avoidance suggests a partial transfer of processing resources from a dorsal to a ventral network of cortical areas. The relative recruitment of ventral areas deactivated by the task during spontaneous avoidance is consistent with current models of the function of the default mode network emphasizing its role in spontaneous z = 0.06), age , CES-D (z = −0.01) or OSPAN scores .All participants were recruited through local announcements and gave written informed consent. The study was conducted at the Department of Psychiatry at the University of Ulm, Germany, in accordance with the Declaration of Helsinki and was formally approved by the Ethical Review Board of the University of Ulm. All subjects had normal or corrected-to-normal vision and were right-handed German native speakers. Ongoing medical, neurological, or psychiatric disease (including depression), and current use of psychotropic medication were excluded through an individualized interview with an experienced clinician (R.V. or P.B.). Depressiveness was assessed with a computerized German Version of the Centre for Epidemiologic Studies Depression Scale CES-D, . ParticiThe scrambled sentences task was presented on two occasions: before the scan, to collect data on cognitive bias comparable to those of ref. In the scan version, the original task was modified in two respects. First, to avoid the artefacts possibly produced by speaking out loud or writing, participants were instructed to build the sentences in mind and press a button to confirm that a sentence had been formed. Scrambled sentences were presented through goggles masking the whole field of vision . Time allowed to form sentences was 3 seconds per trial, previously determined as the 95% percentile of button presses indicating completion of sentences in a preliminary pilot study, resulting in 22 scrambled sentence presentations in each 2.5 minutes-long block. This maintained a mild time pressure on the completion of sentences, and kept participants focused on the task during the block, making the block as homogenous as possible as required by the perfusion design F1,999 = 9.26, p = 0.002).To characterize the emotional salience of the words used in the task, a preliminary study was conducted on a separate sample of 19 student volunteers, recruited similarly to participants in the study. Participants were asked to rate the words in the emotional sets for arousal, using a standard visual analogue rating scale http://www.fil.ion.ucl.ac.uk) for realignment and stereotactic normalization to an EPI template . Reconstruction of cerebral blood flow (CBF) values was obtained using software implementing equation (1) of ref. Data were collected at the Department of Psychiatry at the University of Ulm, Germany. All magnetic resonance imaging (MRI) data were obtained with a 3-Tesla Magnetom Allegra  MRI system equipped with a head volume coil. Images were individually screened to exclude pathology. A continuous arterial spin-labelling technique www.r-project.org, Vienna, Austria), which was also used for the tests of the previous section.The proportion of negative sentences, or the proportion of sentences formed overall, was entered in a generalized linear model (logistic regression) with age, sex, and OSPAN score as explanatory variables. The model was fit with the freely available statistical software R  installed on a machine equipped with a 64-bit Athlon processor  running Windows XP . For the generation of random numbers, the ‘MATLAB5 generator’ was used. http://brainvis.wustl.edu/wiki/index.php). The remaining figures are overlays of axial or sagittal slice of a template brain, generated with the freely available application MRIcroN http://www.cabiatl.com/mricro/mricron/index.html).For the whole brain analysis, an explicit mask was obtained by thresholding the Table S1Main contrast task vs. resting baseline. (DOC)Click here for additional data file.Table S2Interaction valence×instruction. (DOC)Click here for additional data file.Table S3Simple contrast emotional vs. neutral in spontaneous group. (DOC)Click here for additional data file.Table S4Simple contrast emotional vs. neutral in instructed group. (DOC)Click here for additional data file.


The s allele of the 5-hydroxytryptamine transporter-linked promoter region (5-HTTLPR) polymorphism of the serotonin transporter gene has been found to be associated with neuroticism-related traits, affective temperaments and response to selective serotonin reuptake inhibitor (SSRI) treatment. The aim of the current study was to develop a psychometric tool that could at least partially substitute for laboratory testing and could predict the presence of the s allele.The study included 138 women of Caucasian origin, mean 32.20 ± 1.02 years old. All subjects completed the Hungarian standardised version of the Temperament Evaluation of the Memphis, Pisa, Paris, and San Diego Autoquestionnaire (TEMPS-A) instrument and were genotyped for 5-HTTLPR using PCR. The statistical analysis included the calculation of the Index of Discrimination (D), Discriminant Function Analysis, creation of scales on the basis of the above and then item analysis and calculation of sensitivity and specificity.Four indices were eventually developed, but their psychometric properties were relatively poor and their joint application did not improve the outcome.We could not create a scale that predicts the 5-HTTLPR genotype with sufficient sensitivity and specificity, therefore we could not substitute a psychometric scale for laboratory genetic testing in predicting genotype, and also possibly affective disorder characterisation and treatment. The s allele of the 5-hydroxytryptamine transporter-linked promoter region (5-HTTLPR) polymorphism of the serotonin transporter gene has been shown to be significantly associated with both unipolar, bipolar and subthreshold forms of affective disorder -8 and alIn the present paper we attempted to delineate and validate a scale based on the TEMPS-A questionnaire to predict the presence of the 5-HTTLPR s allele scale with a different and more rigorous approach.Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) Axis I psychiatric disorders were excluded.The study population included 138 psychiatrically healthy unrelated Hungarian women of Caucasian origin. All participants were aged between 18-64 years; the mean age of our subjects was 32.20 ± 1.02 years. All subjects were screened for neurological and psychiatric disorders using the standardised Hungarian version of the MINI International Neuropsychiatric Interview [The study protocol was reviewed and approved by the Scientific and Research Ethics Committee of the Scientific Health Council of Hungary in charge of genetic experimentation concerning human subjects. All subjects gave written informed consent before participating in the study.All subjects completed the Hungarian standardised version of the TEMPS-A questionnaire that measures affective temperaments on five scales, the depressive, cyclothymic, irritable, anxious and hyperthymic temperaments ,21,22.All subjects were genotyped for 5-HTTLPR by PCR. PCR amplification of 5HTTLPR was performed on genomic DNA extracted from buccal cells , and 5HTAll statistical analyses were carried out using Statistica 7.0 for Windows . In all cases we analysed our data according to the additive model , according to the dominant model , and according to the recessive model .The first step included the calculation of the equivalent of the degree of difficulty  as a meaDiscriminant function analysis was also used in order to obtain two additional indices that could help in separating groups. All the items with D above 15 were included in this type of analysis.Item analysis was performed, and the value of Cronbach's α for each scale was calculated. The sensitivity (Sn) and Specificity (Sp) were also calculated.2 = 0.38934, P = 0.8231).In all, 19 (13.76%) subjects carried the ss genotype, 50 (36.23%) the ll and 69 (50%) sl genotype. A total of 88 subjects (63.77%) carried the s allele while 50 subjects (36.23%) did not carry the s allele. The frequency of the s allele in our sample was 38.77% which parallels the results of earlier studies and is representative of the Caucasian population . The disP > 0.05) and they also did not differ concerning all the TEMPS-A subscales . However, post hoc comparisons indicated a significant difference in case of the Depressive, Anxious, Cyclothymic and Irritable subscales. When considering the presence or absence of the s allele (ss and sl combined vs the ll) then the difference concerning the TEMPS-A subscales was significant  and concerned all individual subscales except the Hyperthymic . The descriptive statistics are shown in Table The various genotype groups  did not differ in age  and specificity (Sp) at various cut-off levels for the two scales is shown in Table All scales and indices correlated moderately but significantly with all TEMPS-A subscales Table .In the present work we attempted to extract a scale from the TEMPS-A questionnaire that would predict the presence of the s allele of the 5-HTTLPR with satisfactory sensitivity and specificity. However, although several items discriminate between the different genotype groups to a high degree, no scale compiling these items showed high sensitivity and specificity with respect to the presence of the s allele. Even the combination of the scales that were derived cannot improve the poor classification outcome.To understand the nature of psychometric disorders and to make more efficient treatment possible, we must not only view these disorders as complex entities in the context of their social, cultural, neurochemical and genetic determinants, but we should also be able to decompose psychiatric disorders into smaller and better characterisable components. The concept of endophenotypes was introduced to aim at identifying and characterising small, atomic phenomena that correspond to an accurately characterisable biochemical process or marker, such as a genetic polymorphism, and which is at the same time highly relevant in the manifestation of psychological phenomena or psychiatric disorders. There is an expanding effort to identify traits and temperaments related to the development of psychiatric illnesses and associate them with genetic factors. Studies have attempted to link psychological traits as measured by psychometric scales with a given polymorphism. Our approach in this case was different: based on an association we had already described between the 5-HTTLPR s allele and several affective temperaments measured by TEMPS-A ,26, we aIn a previous paper we attempted to solve the task of delineating a psychometric scale to predict presence of the s allele by selecting the items which differentiated between the different genotype groups using analysis of variance (ANOVA) and performing a subsequent item analysis . In the The role of genetic factors in the background of personality, vulnerability and consequently psychiatric disorders has gained more recognition and wider acceptance in modern times. It is well accepted that the 5-HTTLPR s allele has a profound role in determining the emergence of neuroticism-related personality traits -12,27 anIn interpreting our results and drawing our conclusions, several limiting factors must be taken into consideration. First of all, our sample was relatively small; studies using larger samples would detect minor differences to a greater accuracy. Also, our sample consisted entirely of women. Further studies are needed to investigate the possibility of extracting a psychometric scale for predicting the s allele in men and in a mixed-gender general study population.Genetic polymorphisms influence not only the emergence of psychiatric diseases but also the pharmacotherapeutic response of these disorders to treatment. Although genetic polymorphisms only mildly contribute to such phenotypical alterations, they may be taken into account when selecting a pharmacological agent. A scale closely related to a given polymorphism may thus be a useful clinical tool, however, the development of such a scale needs further research.The authors declare that they have no competing interests.XG conceived the study, gathered and managed the data, performed the genetic analysis, participated in the statistical analysis and wrote the paper. KNF conceived the study, carried out the literature search and analysis and participated in writing the paper. ZR participated in analysing the data and writing the paper. AL participated in the genetic and statistical analysis. HSA participated in designing the study, analysing the data and writing the paper. GB participated in designing the study, analysing the data and writing the paper. All authors read and approved the final manuscript.


In 1998, mandatory folic acid fortification of white flour and select cereal grain products was implemented in Canada with the intention to increase dietary folate intakes of reproducing women. Folic acid fortification has produced a dramatic increase in blood folate concentrations among reproductive age women, and a reduction in neural tube defect (NTD)-affected pregnancies. In response to improved blood folate concentrations, many health care professionals are asking whether a folic acid supplement is necessary for NTD prevention among women with high blood folate values, and how reliably high RBC folate concentrations predict folate intakes shown in randomized controlled trials to be protective against NTDs. The objective of this study was to determine how predictive blood folate concentrations and folate intakes are of each other in a sample of well-educated lactating Canadian women exposed to high levels of synthetic folate.The relationship between blood folate concentrations and dietary folate intakes, determined by weighed food records, were assessed in a sample of predominantly university-educated lactating women (32 ± 4 yr) at 4-(n = 53) and 16-wk postpartum (n = 55).P < 0.001) and RBC  folate concentrations at 16-wk postpartum. Plasma and RBC folate values at 16-wk postpartum correctly identified the quartile of folate intake of only 26 of 55 (47%) and 18 of 55 (33%) of subjects, respectively. The mean RBC folate concentration of women consuming 151–410 μg/d of synthetic folate (2nd quartile of intake) did not differ from that of women consuming >410 μg/d (3rd and 4th quartile).Median blood folate concentrations of all participants were well above plasma and RBC folate cut-off levels indicative of deficiency  and all, except for 2 subjects, were above the cut-off for NTD-risk reduction (>906 nmol/L). Only modest associations existed between total folate intakes and plasma (r = 0.46, Folate intakes, estimated by food composition tables, and blood folate concentrations are not predictive of each other in Canadian lactating women exposed to high levels of folate. Synthetic intakes > 151–410 μg/d in these women produced little additional benefit in terms of maximizing RBC content. More studies are needed to examine the relationship between blood folate concentration and NTD risk. Until data from such studies are available, women planning a pregnancy should continue to consume a daily folic acid supplement of 400 μg. Folic acid supplementation during the periconceptional period is effective in reducing the risk of neural tube defects (NTDs) -5. Howevrd trimester of pregnancy in a sample of well-educated Canadian women -5-methyltetrahydrofolate ([6S]-5-methylTHF) versus folic acid during lactation (n = 69). A detailed description of the study design, procedures and information on the precision and reproducibility of laboratory procedures can be found elsewhere . BrieflyAt baseline (36 wk gestation), women were enrolled and a self-administered questionnaire was given to collect information on household income and education level. Participants were then given instructions on the completion of 3-d weighed food records and blood samples were collected. Three-d weighed food records and additional blood samples were collected at 4- and 16-wk postpartum (± 1 wk).S]-5-methylTHF or folic acid) or a placebo within one week of delivery. Subjects were instructed to consume the study capsules daily until 16-wk postpartum and to avoid consuming any other folate-containing supplement during the intervention period. In addition all subjects received a daily folate-free multivitamin and mineral supplement, which contained 1 mg vitamin B6, 3 μg vitamin B12 and 4 mg ferrous fumarate .In the overall study, women were assigned to receive either a folate supplement . Dietary folate intakes were then tabulated from the diet records using Health Canada's Canadian Nutrient File (CNF), version 2001 b  which haSAS for WINDOWS  was used for statistical analyses and a probability level of 5% was chosen as the level of statistical significance. The large intra-subject variance in folate intakes were removed using SIDE: Software for Intake Distribution Estimation . The SIDE program operates within SAS for WINDOWS, and generates an estimate of subjects' usual (long-term average) intake by using the distribution of their observed intakes and partially removing the day-to-day variation in individuals' intakes.r) were generated between plasma and RBC folate concentrations and folate intakes . In addition, the association between RBC folate concentrations at 16-wk postpartum and synthetic folate intake (by quartile assignment for each) was investigated using a one-way analysis of variance and Tukey multiple comparison procedures. RBC folate concentrations measured at study recruitment (36 wk gestation) and the form of folate supplement consumed  during the intervention were entered as covariates in these latter analyses.Blood folate concentrations were normalized using logarithmic transformations. Partial correlations  dietary folate intake  and (2) blood folate concentration (plasma and RBC folate) of each subject. When dietary and blood values were assigned to the same quartile, it was concluded that the blood folate value for that subject correctly classified their dietary folate intake. Dietary folate intakes misclassified by only one quartile were considered closely classified, and those misclassified by two or more quartiles were considered misclassified. If no association existed between the assigned quartile of blood folate concentration and dietary folate intake, one would expect 25% of participants to be correctly classified, 37.5% to be closely classified and 37.5% to be misclassified. The actual number of subjects assigned to each quartile was then compared with expected numbers using a chi-square goodness of fit test with 2 degrees of freedom.Total dietary folate intakes are expressed in the manuscript, except where indicated, as "Dietary Folate Equivalents"  to adjust for the greater bioavailability of synthetic  folate relative to endogenous sources . For easOnly those subjects from the overall study who completed 3-d weighed food records and had provided a concomitant blood sample for folate analysis were included in the present analyses . The mean age of the participants at enrollment was 32.2 ± 3.8 y (mean ± SD). Participants were well-educated (51 of 55 had completed college or university) and predominantly from households with incomes > $75,000 per annum. In Ontario, the mean household income in 2001 was estimated be $60,500 . PrenataMedian blood folate concentrations during lactation were well above plasma and RBC folate cut-off levels indicative of deficiency   (Table 1P < 0.001) were found between total (μg DFE) and synthetic folate intakes (μg) and plasma folate concentrations at 4- and 16-wk postpartum  during the periconceptional period compared to those that were not ,3-5. GivP < 0.05) and 16-wk  postpartum were found. The strength of these associations are very similar to those recently reported between dietary folate intake, as assessed by an abbreviated folate-targeted food/supplement screening tool, and RBC folate concentration, in a sample of women from California  [P < 0.01) [Contrary to our original hypothesis, the strength of the association between dietary folate intake, determined by food composition tables, and blood folate concentrations has not improved post-folic acid fortification of the food supply. In the present study, statistically significant correlations between dietary folate intake, as determined by 3-d weighed food records, and RBC folate, an indicator of NTD risk, at 4-  . Neither < 0.01) .There are at least two likely explanations for our observations. First, the relationship between folate intake and blood folate concentration was no longer linear among lactating women in our study who for the most part had been chronically exposed to high levels of synthetic folate Figure . Second,P < 0.001) [Regardless of the poor predictive ability of blood folate measures in assessing relative dietary folate intake in our study, it is clear that RBC folate concentrations in this group of affluent, well-educated lactating women are generally consistent with a reduction in risk of NTD (> 906 nmol/L) as proposed by Daly and colleagues . Using d< 0.001) . In fact< 0.001) ,34.As reported elsewhere, however, approximately one-third of this sample of lactating women had dietary folate intakes (folate endogenous to food + folic acid as a fortificant) below their estimated average requirement . AssuminWhile these data are among the first to specifically assess how predictive blood folate concentrations and folate intakes are of each other post-folic acid fortification of the food supply, in addition to the quality of food composition tables, there are other limitations in estimating folate intake that must be acknowledged. Specifically, many women, particularly those that are overweight or concerned about their body weight, may under-report their dietary intake -45. In aFinally, it should be noted that most lactating women in this study, on the advice of their primary care physician, consumed a 1000 μg/d supplement of folic acid during pregnancy. Many used the same folate-containing supplement prior to conception for NTD-prevention. While the life span of a RBC is ~120 days and hence by 16-wk lactation should reflect dietary and supplemental folate intakes during lactation, the contribution of liver folate stores amassed prior to parturition in meeting the nutritional requirements beyond 16 weeks of lactation could be significant. The absence of an improvement in the association between blood folate concentrations and folate intake between 4 and 16 weeks lactation could reflect this phenomenon. While the incremental increase in liver folate stores secondary to ingestion of 1000 μg/d of supplemental folic acid during pregnancy is not known, visceral folate stores in the form of long-lived pteroylpolyglutamate pools can be converted to pteroylmonoglutamates which can then be released into circulation ,47.In this sample of affluent well-nourished lactating Canadian women chronically exposed to high levels of folate, plasma and RBC folate concentrations were poor predictors of relative folate intake, estimated by food composition tables generally, and synthetic folic acid intake specifically. Synthetic intakes > 151–410 μg/d in these women produced little additional benefit in terms of maximizing RBC content. More studies are needed to examine the relationship between blood folate concentration and NTD risk. Until data from such studies are available, women planning a pregnancy should continue to consume a daily folic acid supplement of 400 μg.The author(s) declare that they have no competing interests.DLO and LAH were responsible for the design of the study and wrote the manuscript. LAH and KLS recruited the subjects and were responsible for the sample collection, laboratory analysis and statistical analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Lysosomal storage diseases (LSD) are inherited disorders caused by deficiency of lysosomal enzymes in which early diagnosis is essential to provide timely treatment. This study reports interval values for the activity of lysosomal enzymes that are deficient in Mucopolysaccharidosis type I, Fabry, Gaucher and Pompe disease, using dried blood spots on filter paper (DBS) samples in a Brazilian population.Reference activity values were obtained from healthy volunteers samples for alpha-galactosidase A (4.57 ± 1.37 umol/L/h), beta-glucosidase (3.06 ± 0.99 umol/L/h), alpha-glucosidase , alpha-iduronidase (3.45 ± 1.21 umol/L/h) and beta-galactosidase (14.09 ± 4.36 umol/L/h).Reference values of five lysosomal enzymes were determined for a Brazilian population sample. However, as our results differ from other laboratories, it highlights the importance of establishing specific reference values for each center. Lysosomal storage diseases (LSD) comprise a group of more than 40 inherited disorders caused by deficiency of specific lysosomal hydrolases, which results in the accumulation of different macromolecules in the lysosome, leading to cell dysfunction and progressive clinical manifestations ,2.Most LSD are inherited in an autosomal recessive manner, with the exception of Hunter, Fabry and Danon diseases that are X-linked. As a group, LSD have an incidence of about 1 per 7700 live births .For some LSD as Fabry, Gaucher and Pompe diseases  and Mucopolysaccharidosis type I (MPS I), enzyme replacement therapy has been widely used as a treatment option, improving the quality of life and prognosis of these patients. Since those diseases are all progressive, early diagnosis is an essential tool for a successful treatment .Biochemical diagnosis of LSD is performed by determination of enzymatic activities in different biological fluids ), using fluorimetry, immunocapture and mass spectrometry assays -8. One oSeveral studies have demonstrated the feasibility of using DBS to diagnose LSD -11, but Laboratório de Erros Inatos do Metabolismo (LEIM) from Universidade Federal de São Paulo (UNIFESP). Research protocols and consent forms as well as the overall investigation were ethically and scientifically approved by the Medical Research and Ethical Committee of UNIFESP (0384/05).This study was performed at ® 903 filter paper. Samples collected at UNIFESP were dried at room temperature for at least 4 hours and stored at the same day at 4°C in sealed plastic bags with desiccant (to prevent hydration) until analysis. Those collected at other locations were prepared and dried in the same conditions (detailed instructions were sent to those centers), sealed in plastic bags and sent to the laboratory in a maximal interval of 5 days, where they were stored at 4°C. Only high quality DBS, evaluated by their macroscopic aspect  and biochemically by βgal activity [Brazilian healthy volunteers (HV) between the ages of 18 and 80 and without a known genetic or chronic disease were selected for our study. Samples were collected from 164 individuals . Blood specimens were collected by venipuncture into heparin tubes and spotted on Whatmanactivity  were useDBS enzyme activity assays were adapted from the methods described by Chamoles and coworkers ,13-17. αReaction buffers, fluorogenic substrates, additional reagents and incubation time used for each DBS assay are described in Table DBS samples from two healthy individuals have been used as internal controls in every reaction as well as a DBS from a confirmed patient. Within-assay coefficients of variation (CVs) were calculated from an internal control sample analyzed in all assays.t test was performed to compare mean activity values from both genders in HV group. The level of significance for statistical analysis was set at 0.05. All analyses were performed using the software MINITAB® 15.1.1.0.Data are expressed as mean ± standard deviation (SD). The parametric Student's βgal activity was analyzed in HV to establish its reference interval for our population and the range was 7.85 and 22.15 μmol/L blood/h (percentile 5 and 95 respectively). βgal was used as control enzyme, and DBS samples with βgal activity lower or higher than the normal range have not been included in this study, as they were considered inappropriate. The mean, standard deviation and interval values of the different enzymatic activities are described in Table et al.: 5.50) [Our results differ from enzymatic activity values previously described by Chamoles and coworkers, especially for αidua activity  ,13-17. R.: 5.50) . HoweverWithin-assay CVs of internal controls were around 10%, interassay CVs did not exceed 20% and imprecision data are in accordance with other related studies ,15,19,20Different biological materials have been used to diagnose LSD patients, but recently several studies have described the advantages of DBS ,19,20. TAlthough we did not evaluate newborns in this study, it is known that this group has a higher range of enzyme activity ,8. ThereIt is also extremely important to emphasize that in order to guarantee the quality of diagnosis, blood collection and DBS samples preparation must be done carefully, and the determination of a control enzyme, such as βgal or hexosaminidase should be performed as a quality control tool ,12,14.Although it has been demonstrated that these enzymes activities are maintained stable on DBS samples, we suggest that a sample from a healthy volunteer should be sent to the laboratory for analysis along with the samples of clinically suspected patients, which could point out any unexpected decrease of the enzymes activities during transportation ,9. ThereWe believe that the determination of enzyme activity using DBS samples is the best option for screening patients with LSD, especially in high risk groups. This alternative has several advantages that outweigh an initial evaluation of suspected subjects using DBS samples. However, as some of our results differ from other laboratories, it highlights the importance of establishing specific reference values for each reference center.The establishment of reference values for lysosomal enzyme activities on DBS in a Brazilian population may facilitate the use of this test as a tool for LSD diagnosis in our country, and even enable the implantation of a newborn screening program.The authors declare that they have no competing interests.KBM, MDBR and VGP participated in the collection of samples, determination of enzyme activities, analysis and interpretation of data and drafting the manuscript. AMM helped with samples collection and the design of the study. VD participated in the design of the study, its coordination and critically revised the manuscript. All authors have read and approved the final manuscript version.


Multiple imputation (MI) provides an effective approach to handle missing covariate data within prognostic modelling studies, as it can properly account for the missing data uncertainty. The multiply imputed datasets are each analysed using standard prognostic modelling techniques to obtain the estimates of interest. The estimates from each imputed dataset are then combined into one overall estimate and variance, incorporating both the within and between imputation variability. Rubin's rules for combining these multiply imputed estimates are based on asymptotic theory. The resulting combined estimates may be more accurate if the posterior distribution of the population parameter of interest is better approximated by the normal distribution. However, the normality assumption may not be appropriate for all the parameters of interest when analysing prognostic modelling studies, such as predicted survival probabilities and model performance measures.Guidelines for combining the estimates of interest when analysing prognostic modelling studies are provided. A literature review is performed to identify current practice for combining such estimates in prognostic modelling studies.Methods for combining all reported estimates after MI were not well reported in the current literature. Rubin's rules without applying any transformations were the standard approach used, when any method was stated.The proposed simple guidelines for combining estimates after MI may lead to a wider and more appropriate use of MI in future prognostic modelling studies. Prognostic models play an important role in the clinical decision making process as they help clinicians to determine the most appropriate management of patients. A good prognostic model can provide an insight into the relationship between the outcome of patients and known patient and disease characteristics ,2.m (>1) values to give m imputed datasets. Previously, three to five imputations were considered sufficient to give reasonable efficiency provided that the fraction of missing information is not excessive . Whe. WheQ0: k regression estimates are a specific value, zero say. A significance level for testing the hypothesis that the combined MI estimate, Qo is provided in Table k, as can result from fitting categorical variables in the regression model.In the context of prognostic modelling, it is useful to test the global null hypothesis that all χ2 statistics, associated with testing a null hypothesis of Ho : Q = Qo, e.g. a regression coefficient is zero or all regression coefficients are zero  instead of being bounded by zero and infinity and is generally used to obtain a confidence interval [The complementary log-log transformation for the predicted survival probability at particular time-points gives a possible range of  instead of the survivorship estimate being bounded by zero and one, and is often used to determine reasonable confidence intervals . A suitainterval . Estimatinterval . Alterna2 statistic [m estimates may provide a more appropriate reflection of the distribution of the values over the imputed datasets, as reported by Clark and Altman [2 statistics and by Clark and Altman [When considering model performance measures, the imputation model should be more general than the prognostic models being investigated, as the performance measures are more sensitive to the choice of imputation model and therefore may produce more bias than seen in the regression parameter estimates from the prognostic model. If one is willing to accept the large sample approximation to normality for the proportion of variance explained measures, e.g. Nagelkerke's Rtatistic , the c-itatistic . The medd Altman  and Sinhd Altman  for the d Altman  for the d Altman  could pr® bibliographic software of all articles published before June 2008 that used multiple imputation techniques and a survival analysis to obtain a prognostic model. Methodological papers were excluded. The aim of the review was to identify how estimates of the parameters of interest in prognostic modelling studies have been combined after performing MI in the published literature.A literature search was performed within the PubMed  and Web of ScienceSixteen non-methodological articles were identified. The MI techniques reported were varied with no overall consensus on technique or statistical software. The number of imputations ranged from five to 10000, with the majority of studies using five or ten imputations. The amount of missingness reported also varied from studies with relatively little missing data  to thoseIn seven articles, no mention of how the estimates of interest were combined after MI was given. Clark et al.  reportedWith the advances in computer technologies and software, MI is becoming more accessible. MI has been performed prior to the analysis of several prognostic modelling studies, e.g. ,31. Few 2, from a linear regression model fitted to normally distributed data can be considered as a squared correlation coefficient and can be transformed by taking the square root and then applying Fisher's Z transformation as for the correlation coefficient. However whether this approach would apply to R2 measures from a survival regression model that may be affected by censored observations as arises in survival analysis is debatable and therefore robust methods are recommended here.This paper has suggested guidelines for combining multiply imputed estimates that are of interest when a survival model is fitted to a dataset and suitable performance measures and predicted survival probabilities are required for summarising the model Table . These pIn this paper, model performance measures were calculated within each imputed dataset using the constructed prognostic model for that dataset and then combined to give an overall multiply imputed measure. The performance of a prognostic model derived using a development sample will also need to be externally validated using an independent dataset , but misThe review of current practice highlighted deficiencies in the reporting of how the multiply imputed estimates given in the published articles were obtained. Thus, it is recommended that future studies include a more thorough description of the methods used to combine all estimates after MI.The ability to use MI methods that are readily available in standard statistical software and apply simple rules to combine the estimates of interest rather than requiring problem specific programmes makes MI more accessible to practising statistician. We hope that this may lead to a more widespread and appropriate use of MI in future prognostic modelling studies and improved comparability of the obtained estimates between studies.MI: multiple imputation; m: number of imputations; SE: standard error.The authors declare that they have no competing interests.All authors made substantial contributions to the ideas presented in this manuscript. AM participated in the conception of this research, the methodological content, the design, coordination and analysis of the literature review and drafted the manuscript. DGA was involved in the conception and design of the study and helped in the writing of the manuscript. RLH participated in the design and methodological content of this study and in the revision of the manuscript. PR contributed to the methodological content of this research and the revision of the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/9/57/prepub


Previous studies have suggested a probable association between the polymorphism of a microsatellite locus located in the promoter of IGF1 (Insulin-like growth factor 1) gene and the serum level of IGF1, as well as many age-related diseases. Based on these results, we hypothesized that this polymorphism may influence longevity in humans. We performed an association study in a Han Chinese population to test this hypothesis.We recruited 493 elderly Han Chinese individuals  and 425 young individuals (controls) from Dujiangyan . The genotype distributions and allele frequencies of the microsatellite site in the elderly and control groups were compared by chi square test.p = 0.011). As the difference was not significant when corrected by Bonferroni method, we speculate that the 18/21 genotype can not be functional in longevity; however, it may link with the real functional loci as there is a long haplotype block embracing the microsatellite locus.Our results suggested that there was no association between the microsatellite polymorphism and longevity in our Han Chinese population. However, there were more male persons with 18/21 genotype in elderly group than that in control group (11.11 vs. 5.45%, There was no association between polymorphism of the microsatellite in promoter of IGF1 gene and longevity in our study. Future association studies containing the long haplotype block are deserved and can test our speculation of the potential linkage of 18/21 genotype and functional loci. E.coli, yeast, drosophila, mice and humans [IGF1 (Insulin-like growth factor 1) plays a number of important roles in the human body. It is involved in physiological processes such as growth, development, and metabolism . Meanwhid humans -7. Elucid humans .Serum IGF1 levels appear to influence susceptibility to disease, and therefore longevity. For example, people with a high level of circulating IGF1 are more susceptible to cancers, while a low level of circulating IGF1 is a risk factor for cardiovascular diseases, premature atherogenesis, and diabetes . FurtherA cytosine-adenine (CA) repeat polymorphism in IGF1 gene promoter region (1 kilo base pairs upstream from the transcription start site)  has beenTo our knowledge there have been no reports on the association between the microsatellite polymorphism and longevity in the Han Chinese people. In this study, we conducted an association study in a Han Chinese population to investigate the potential function of this microsatellite locus in predicting longevity.A total of 493 elderly individuals  from Dujiangyan  agreed to participate in this study. Ages were authenticated by the official certification of the Fifth Nation Census in China as well as by the inference of the number of generations, and the memories of these, their offspring and other local elderly people. The age was confirmed when the certification and the inference were consistent. We also recruited 425 healthy, local individuals between the ages of 32 - 73 years to act as a control group. This control group included 110 spouses of the children of the elderly participants. The subjects' information is listed in Table All participants indicated informed consent by signing a form after they had been given a clear explanation of the potential risks of the study. This research was approved by the Ethics Committee on human experimentation of Kunming Institute of Zoology, Chinese Academy of Sciences and relevant bodies in Dujiangyan. The study was done in accordance with the Declaration of Helsinki and subsequent amendments.-1), 0.5 μL BSA , 0.5 μL forward primer (10 pmol·μL-1), 0.5 μL reverse primer (10 pmol·μL-1), 0.125 μL rTaq polymerase  and distilled water to a total volume of 25 microliter (μL). Thermocycling parameters included an initial denaturation of 5 min. at 95°C; 35 cycles of: 95°C for 30 s, 62°C for 1 min. and 72°C for 1 min.; and a final extension at 72°C for 10 min. PCR products were denatured and size fractionated on 6% denaturing polyacrylamide gel run on an ABI 3730 automated sequencer . Alleles were read and scored by GeneMapper V4.0 software (ABI Perkin Elmer) using Liz500 as size standard. Alleles were verified artificially twice by two different persons. The number of repeats contained in alleles of different length was confirmed by cloning and sequencing two differently sized PCR products . We replicated PCR amplication and genotyping of 30% of the samples, ensuring that replicates were performed by a different researcher. Ambiguous results were confirmed by another PCR and fragment analysis.DNA was extracted from white blood cells in whole blood by the standard phenol/chloroform method. Primers for PCR followed those used in previous study . The forp value was and considering p <0.05 to be significant. We used a Bonferroni correction to correct for multiple tests of the same data.A test of Hardy Weinberg Equilibrium (HWE) was performed using Genepop 3.4 . The proTo explore the potential function of this microsatellite locus, we categorized the whole population into three genotype groups depending on owning 2, 1, or no copies of major alleles (19- and 21-repeat): 19/19 (21/21), 19/non (21/non), and non/non. We performed chi square test to compare 1) each genotype to the sum of the other two, 2) the frequencies of three genotypes, and 3) the distribution of major allele (19(21)-allele vs. non) in elderly and control groups in order to detect any potential functions of the major alleles.p > 0.05).In this study, we examined the microsatellite polymorphism in IGF1 gene promoter in 493 elderly individuals  and 425 control individuals . The length range of the microsatellite locus was 17 to 23 repeats , and the most common alleles are 19-repeat (39.12%) and 21-repeat (26.14%). The genotype distribution followed Hardy-Weinberg Equilibrium (HWE) in both elderly and control groups, as well as when stratified by gender , even when stratified by gender. The results of comparison of the distribution of common alleles (19- and 21-repeat) in both groups are listed in Table p = 0.031). After Bonferroni correction, the result was no longer significant.All alleles and their frequencies are listed in Table p = 0.011; Table To investigate potential causes of the different frequencies of the genotype 21/non in male participants, we compared the distributions of all possible genotypes in the male elderly and male control groups. Altogether, we found six genotypes which included one allele with 21 repeats: 17/21, 18/21, 19/21, 20/21, 21/22, and 21/23 (The number represents how many repeats there is in the microsatellite locus in this genotype). A chi square test showed a significant difference in the distribution of the 18/21 genotype in male elderly and male control groups  to define categories. As stated in the introduction, the common 19-repeat allele is thought to be related to serum IGF1 levels and many age-related diseases, although the results are equivocal. The contradiction between studies can be explained by different population size, different ethnicities in different studies, and small effect of this polymorphism in complex diseases. Similarly, longevity is a complex trait, influenced by the additive effects of many genetic variations (not to mention stochasticity). Thus an association study may simply not have sufficient power to detect significant correlations between the 19-repeat and longevity.p = 0.011) and other genotypes. However, Bonferroni corrections increased the p values past the cut-off point for statistical significance. Based on these results, the 21-repeat can not be a potential functional allele for male longevity. The 18/21 genotype may still be linked to real functional variations, because in a recent study, a long haplotype block including this microsatellite region was reported [We initially found a significant difference between the 21-repeat/non genotype frequencies in male elderly (42.06%) and control (32.18%) groups, as well as between the 18/21 genotype  deserves future studies.p = 0.011). Future studies including an increased sample size and more loci in the long haplotype block will enable us to test the hypothesized linkage between the 18/21 genotype and functional loci.In this study, no association was discovered between the microsatellite site in IGF1 gene promoter and longevity in a Han Chinese population. In males, the 18/21 genotype was more frequent in the elderly group than in the control group (11.11 vs. 5.45%, The authors declare that they have no competing interests.LX and YPZ designed the research and wrote the manuscript. LX conducted the genotyping and statistical analysis. YG assisted in revising the manuscript. YG, SL, JY and LyX collected the blood samples. All authors read the paper and approved the final version.