Study design, ethics statement, and participant recruitment

This was a retrospective study of a population for which informed consent was obtained as described below. The study data were accessed on September 29, 2021. All data were fully anonymized prior to access. Written informed consent to obtain data from their medical records and biomarker data used in the research was obtained before participation from the nearest caregiver or, if possible, from participants with DS. The study was approved by the University Ethics Committee of Kyoto Prefectural University of Medicine (KPUM), Kyoto, Japan (RBMR-C-1226), and by the Research Ethics Committee of Tokyo Metropolitan Kita Medical and Rehabilitation Center for the Disabled (number: 2019–22). Informed consent was obtained from patients in the DS group when possible and from their nearest relatives. This study was designed and conducted in accordance with the Declaration of Helsinki. We enrolled 56 patients with DS (DS group) registered for DS in the KPUM, Hananoki Medical Welfare Center, and Tokyo Metropolitan Kita Medical and Rehabilitation Center for the Disabled between February 2013 and December 2020. To set a reference value for p-tau, 33 healthy controls (control group) were enrolled from another KPUM registry. Participants in the control group provided written informed consent prior to participation. The registry protocols were approved by the medical ethics committee at KPUM (approval number: ERB-G-12). Control participants were not selected from among the family members of the DS group.

Plasma samples and clinical data collection

Plasma samples were obtained via venous puncture and 8 mL of blood was collected in EDTA-containing tubes. After collection, the plasma was separated by centrifugation for 10 min at 3,000 rpm and placed in polypropylene vials. Fresh samples obtained from the enrolled participants were immediately stored at -80°C until analysis. The DS group were completed by their carers [22]. For most participants, the DSQIID was completed after consultation with their parents. For participants whose parents died or were aged out, the DSQIID was completed by a sibling or staff member at their residential facility. If the DSQIID was evaluated more than once, the results of the questionnaire with the shortest lag time from blood collection were used. DSQIID results, examined before or more than two years after blood collection, were not used for the study. Part 1 of the DSQIID questionnaire, which contained written answers and a few simple questions aimed at estimating the best abilities of the patients, was not included in the analysis. The total score in Parts 2 and 3 and the sub-score of each item represented as zero (the problems existing from before or there are no problems) or one (new symptoms appearing or worsening existing problems) were used for the following analysis.

Diagnosis of DS-AD

It is difficult to diagnose DS-AD based on certain criteria because the intellectual disability of individuals with DS varies greatly from person to person. General criteria for the diagnosis of dementia are not currently specified for people with intellectual disabilities. Studies on DS-AD have used a method in which the diagnosis of DS-AD is made through consensus meetings in a team consisting of dementia specialists, family members or caregivers, and nurses [23]. For example, in a previous study of DS cohorts in Barcelona, London, Kentucky, Cambridge, and Munich, clinical dementia status was determined individually for each participant at a Consensus Case Conference, where these discussions included at least two clinicians with longstanding expertise in evaluating dementia in DS and included a review of the medical and psychiatric history and findings from the neurological exam, interviews with caregivers or family members, and the participant’s performance in the neuropsychological evaluation, taking into consideration the participants’ baseline intelligence quotient, medical and psychiatric conditions, and any major life events [24]. This method conforms to the Consensus Recommendation proposed by Moran et al., which excludes the possibility of cognitive decline due to medical illnesses, environmental changes, or psychological stressors [25]. Based on the above situation in this field and the pathological finding that AD pathology rarely occurs in patients with DS at the age of 30 years or younger [26], in this study, we reviewed the medical records retrospectively to confirm that 1) participants were 30 years or older, 2) the family member or caregiver has complained of a persistent decline in acquired daily living skills for at least 6 months, 3) no factors could explain current symptoms in interviews with the patient and family regarding the psychological impact and changes in living environment which might cause depression or psychiatric symptoms, physical examination, blood and urine tests (not including AD biomarkers) and 4) no lesions on head imaging tests could explain the current symptoms (e.g. hemorrhage, tumor, or infarction). In consensus meetings involving two or more neurologists, a diagnosis of DS-AD was made if the above four criteria were met. (authors: TK, CT, MO, MS, FKM, TO).

Measurement of p-tau and apolipoprotein E typing

The plasma p-tau concentration was measured using the single-molecule immunoarray (Simoa) method with reagents from a single lot using the Simoa P-tau181 assay on an HD-1 Simoa analyzer according to the manufacturer’s protocol (Quanterix, Lexington, MA, USA). All samples were analyzed in duplicate on one occasion.

The apolipoprotein E (ApoE) haplotype was determined by genotyping using the Invader assay or DNA microarray method undertaken by BML Inc. (Tokyo, Japan) or PreMedica Inc. (Tokyo, Japan), or phenotyping by isoelectric electrophoresis for plasma [27] undertaken by BML Inc. (Tokyo, Japan).

Statistics

The–Mann Whitney U test was used to compare two independent groups. Fisher’s exact test was used to evaluate the significance of categorical variables. Univariate correlation analysis was performed using the Spearman’s rank correlation coefficient (Spearman’s test). The difference in the slopes of the regression lines was determined by the significance of the interaction term in the analysis of covariance (ANCOVA). (SPSS ver. 23, IBM Japan Ltd, Tokyo, Japan) Multiple regression analysis was performed with plasma phosphorylated tau concentration after log-transformed as the dependent variable and DSQIID sub-items and age as explanatory variables. SPSS ver. 23 (IBM Japan Ltd., Tokyo, Japan) was used for the multivariate analyses. The normality test for the residuals of multiple regression analysis was performed using the D’Agostino-Pearson omnibus normality test and the Shapiro-Wilk normality test. The above analyses were performed using the GraphPad Prism software (version 9.0; San Diego, CA, USA). The level of significance was set at P<0.05.

Age dependent elevation of plasma p-tau in DS with and without apolipoprotein E allele

As summarized in the upper part of Table 1 and S1 Table, no age or sex differences were observed between the DS and Control groups. None of the participants had chronic kidney disease or a history of stroke/myocardial infarction, which are major confounding factors for plasma p-tau measurement [28]. Apolipoprotein E (ApoE) genotypes or phenotypes were determined in 54 individuals among the 56 enrolled participants with DS, of which 3 had ApoE2/3, 40 had ApoE3/3, and 11 had ApoE3/4. No individual had ApoE2/2, ApoE2/4, or ApoE4/4 mutations. The allele frequencies of ApoE2, ApoE3, and ApoE4 were 2.8%, 87%, and 10.2% respectively. Four participants were diagnosed with DS-AD according to the definitions provided in the methods. The plasma p-tau levels were significantly higher in the DS group than in the control group. When the participants were categorized by age into young (aged16-25 years: n = 9 in control and n = 21 in DS), middle-aged (aged 26–42 years: n = 15 in control and n = 23 in DS), and old cases (over aged older than 43 years: n = 9 in control and n = 12 in DS) as followed the age categories used in previous biomarker studies in this cohort [29, 30], the difference of plasma p-tau between the groups was negligible in the young (P = 0.1355), became significant in the middle-aged (P = 0.0011), and was clearly observed with little overlap in the old cases (P = 0.0001) in Mann-Whitney’s U test (Fig 1A). As expected, plasma p-tau levels were strongly correlated with age in the DS group, whereas no significant relationship was observed in the control group using Spearman’s test (rs = 0.6725, P<0.0001 for the DS group and rs = -0.08301, P = 0.6460 for the control group).

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Fig 1.

(A) Scatter plots of plasma p-tau levels in the DS and Control groups were shown. The participants were categorized by age into young into young (aged 16–26 years), middle-aged (aged 26–42 years), and old cases (aged older than 43 years). Black circles indicate DS-AD cases diagnosed according to the definitions given in the Method. Bars indicate median values. P values obtained by Man-Whitney’s U test were indicated in the columns. (B) Scatter plots regarding relationship between plasma p-tau level and age in the DS group were shown. Individuals with DS were dichotomized into ApoE4(+) (indicated by white circles with dots) and the ApoE4(-) (indicated by white or black circles) groups according to the presence of at least one ApoE4 allele. Black circles indicate DS-AD cases. At the time of plasma collection, there were no patients in the ApoE4(+) group diagnosed with DS-AD. The dashed line for the ApoE4(+) group and solid line for the ApoE4(-) group were regression curves.

https://doi.org/10.1371/journal.pone.0311878.g001

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Table 1. Demographics of the participants.

https://doi.org/10.1371/journal.pone.0311878.t001

We dichotomized individuals with DS into ApoE4(+) and the ApoE4(-) groups according to the presence of at least one ApoE4 allele. No age or sex differences were found between these groups (P = 0.182 for age, P = 0.594 for sex in Mann-Whitney’s U test). As shown in Fig 1B, both groups showed significant age-dependent elevation of plasma p-tau levels in Spearman’s test (rs = 0.8153, P = 0.0015 for the ApoE4(+) group and rs = 0.6263, P<0.0001 for the ApoE4(-) group). We set the cutoff level for plasma p-tau at 1.52 pg/ml based on the calculation of the average value (0.72 pg/ml) + 2 × standard deviation (0.40 pg/ml) in the control group. Based on this cutoff, 12 participants with DS aged > 30 years, the youngest age at which amyloid pathology can be detected in DS [2], were identified to have abnormally elevated plasma p-tau levels. Furthermore, the plasma p-tau levels in four patients clinically diagnosed with DS-AD were well above the cutoff value. When focusing on the impact of the presence of the ApoE4 allele on such p-tau elevation in relatively younger ages of 40 to 50 years, four of the eight cases belonged to the ApoE4(+) group, while all four cases with abnormal p-tau values aged over 50 years were in the ApoE4(-) group. This promotive effect of the age-dependent elevation of p-tau by carrying the ApoE4 allele in DS was also suggested by the fact that the slope of the regression line between age and normalized (log-transformed) p-tau was significantly steeper in the ApoE4(+) group than in the ApoE4(-) group (the interaction term between age and group was significant: P<0.001 by ANCOVA). Note: Although no patients in the ApoE4(+) group were diagnosed with DS-AD at the time of plasma collection, four ApoE4(+) patients with elevated p-tau levels were confirmed to develop DS-AD within the next 2–3 years.

Correlation analysis between DSQIID total / sub-item scores and plasma p-tau

We obtained valid responses to the DSQIID questionnaire from 43 individuals with DS among the 56 enrolled participants. The age, sex, total scores of DSQIID parts 2 and part3 (DSQIID total score), and levels of plasma p-tau in the participants are summarized in the lower part of Table 1 and S1 Table. The age-dependent elevation in p-tau levels remained statistically significant in these 43 individuals (rs = 0.7230, P<0.0001 by Spearman’s test). The DSQIID total scores tended to be positively associated with age; however, this association was not statistically significant (rs = 0.2652, P = 0.0857 by Spearman’s test).

We summarized the relationship between the scores (zero or one) of each sub-item of the DSQIID, age, and plasma p-tau levels (Table 2). Univariate analysis using Spearman’s test revealed that eight subitems of the DSQIID were significantly correlated with age. All these significant correlations were positive; that is, scoring on the sub-items was related to older age. Fourteen sub-items of the DSQIID significantly correlated with plasma p-tau levels. All these significant correlations were also positive; that is, scoring on the sub-items was related to higher p-tau. As expected from the strong positive correlation between age and p-tau level in the DS group, seven of the 14 questions positively correlated with p-tau levels and showed a significant correlation between score and age. However, these significant correlations between p-tau and the sub-item scores disappeared after adjusting for age in the multiple regression analysis. Similarly, as shown at the bottom of the table, there was a significant positive correlation between the DSQIID total scores and plasma p-tau levels in the univariate analysis using Spearman’s test, but this significant association disappeared after age adjustment. Interestingly, one sub-items, "Generally, appears more forgetful,” appeared significant negative correlations with plasma p-tau after age adjustment (P = 0.020, β = -0.224), i.e., scoring on this sub-item was related to lower p-tau suggesting absence of AD pathology.

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Table 2. Correlation between sub-items of DSQIID, age, and plasma p-tau levels.

https://doi.org/10.1371/journal.pone.0311878.t002

According to the original DSQIID paper, Deb et al. conducted a forced four-factor analysis of the questions in Part 2 of the questionnaire and extracted the following four characteristics; 1. Memory / confusion, 2. Feelings of insecurity, 3. Sleep problems, 4. Behaviour problems [15]. We then sub-totaled the items scored in the questionnaire with respect to these four components and conducted univariate and multivariate analyses with age and p-tau levels in the same manner as above (Table 3). In this analysis, the scores of subcomponents 1 (memory/confusion) and 2 (feelings of insecurity) were correlated with plasma p-tau levels, but these correlations disappeared after age adjustment.

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Table 3. Correlation between scores of sub-components of DSQIID part 2, age, and plasma p-tau levels.

https://doi.org/10.1371/journal.pone.0311878.t003

The study enrolled 30 (54%) individuals of relatively young adult Down syndrome younger than 30 years of age, who are generally assumed to have no amyloid pathology [2]. Furthermore, the usefulness of the DSQIID in relatively young patients has not yet been validated. Considering the fact that amyloid pathology in DS appears after the age of 30 years and that previous DSQIID validation studies have been conducted on DS participants over 20 or 40 years of age, the same analyses as those in Tables 2 and 3 were conducted on DS participants over 30 years of age. Although the number of question items and subcomponents for which significant differences were detected was reduced owing to the smaller number of participants in the analysis, similar results were observed in the analysis that included all participants. There was no positive significant correlation between sub-items / sub-components scores and plasma p-tau in multivariate analysis after age-adjustment, while two sub-items,” Talks to self" and "Generally, appears more forgetful,” appeared significant negative correlations with plasma p-tau after age-adjustment with reproducibility in participants aged 30 years and older. (P = 0.023, β = -0.339 and P = 0.047, β = -0.302, respectively) (S2A and S3 Tables). Another issue is the time lag between DSQIID and blood collection. In general, changes in plasma p-tau over time are as small as 4.5% per year [31], even in cases of dementia; therefore, the effect of this time lag on the current results is expected to be limited. However, the study included six cases in which the interval between the DSQIID and p-tau evaluation was > 1 year. The time lag between the DSQIID evaluation and blood collection in these six cases may not be negligible. Thus, we reanalyzed the data after excluding these six cases. The same results were obtained after the exclusion of these six cases. There was no positive significant correlation between sub-items / sub-components scores and plasma p-tau in multivariate analysis after age-adjustment, while two sub-items,” Talks to self" and "Generally, appears more forgetful,” appeared significant negative correlations with plasma p-tau after age-adjustment (P = 0.030, β = -0.491 and P = 0.049, β = -0.351, respectively) (S2B Table).

The major findings of this study are as follows

First, plasma p-tau levels increased in an age-dependent manner, reaching an abnormal cutoff value in individuals over 40 years of age in the DS group. All the patients clinically diagnosed with DS-AD exhibited elevated plasma p-tau levels. These findings correspond with those of recent biomarker studies, including ours, and align with previous pathological and clinical observations that DS-AD typically becomes evident in patients in their 40s [2, 5, 6, 11, 12]. The finding that the slope of the regression line between age and p-tau was steeper in the ApoE4(+) group is also consistent with recent reports indicating that individuals with DS carrying the ApoE4 allele present with AD at younger ages than those without the ApoE4 allele [32]. Considering that elevated plasma p181-tau levels are specifically observed in MCI due to AD and the early phase of AD, but not in other types of neurodegenerative diseases such as vascular dementia or frontotemporal dementia [21, 33], these results validate our assumption that plasma p-tau concentrations can be used as a marker for detecting the development of DS-AD.

Second, the total scores of the DSQIID and the scores on 14 of the 53 sub-items were positively correlated with plasma levels of p-tau; however, none of these correlations remained significant after age adjustment. Considering the following two facts: seven of these 14 sub-item scores showing correlation with p-tau levels were also positively correlated with age, and DSQIID total scores tended to be positively associated with age, the total scores and some sub-scores in the DSQIID likely reflect, to some extent, age-related behavioral changes in individuals with DS. However, the lack of correlation between plasma p-tau and DSQIID total scores or any sub-items after age adjustment raises doubts about the previous assumption that DSQIID can effectively screen for DS-AD. Moreover, one sub-item, "Generally appears more forgetful," showed significant negative correlations with plasma p-tau after age adjustment in this study. However, it should be noted that this significant finding is only part of the exploratory analysis for hypothesis generation, as multiple analyses were conducted during the testing of each question item. Nevertheless, this unexpected result suggests that there is room for improvement in the DSQIID for detecting DS-AD. Although we cannot dismiss the potential confounding effect of the substantial number of young individuals with DS enrolled in this study, results similar to those obtained from participants of all ages were confirmed in the sub-analyses, from which DS participants younger than 30 years were excluded. In this study, we calculated the scores of the four sub-components in DSQIID part 2 and examined the age-adjusted correlations between these sub-component scores and p-tau levels; however, we could not identify any sub-components associated with elevated p-tau levels. Therefore, to enhance the ability of the DSQIID to effectively detect the development of DS-AD, we could not propose strategies for weighing specific subcomponents based on the results of this study. In the future, when revising this questionnaire for greater effectiveness, eliminating questions that are less relevant to DS-AD and incorporating symptoms that are more specific to DS-AD, such as myoclonus [34], may be considered.

We acknowledge that the small sample size was a significant limitation of this study, which may have weakened the statistical power to detect an association between the DSQIIS subscores and p-tau levels. Moreover, although plasma p-tau is the most reliable blood-based marker for detecting DS-AD, the lack of imaging biomarkers such as amyloid and tau PET is another limitation of this study. Future research will need to conduct large-scale case-control studies utilizing both fluid and imaging biomarkers to validate the relationship between DSQIID total and sub-item scores and DS-AD.