*3.2. Association between the Age of Onset of AD and Age of Onset of Hypothyroidism*

Among individuals with a history of hypothyroidism and a reported age of hypothyroidism onset, we observed a significant difference in the age of hypothyroidism by the age of AD onset (*p* = 0.003; Table 1). More specifically, individuals belonging to the early AD onset group had a significantly earlier age of a diagnosis of hypothyroidism, followed by the typical onset, and then by the late AD onset group. Importantly, at the time of

**and Herminia Diana Rosas 1,2,3**

**Citation:** Lai, F.; Mercaldo, N.D.; Wang, C.M.; Hersch, M.S.; Hersch, G.G.; Rosas, H.D.; Association between Hypothyroidism Onset and Alzheimer Disease Onset in Adults with Down Syndrome. *Brain Sci.* 

https://doi.org/10.3390/xxxxx

Academic Editor: Rebecca Sims

**Publisher's Note:** MDPI stays neu-

Received: 31 July 2021 Accepted: 8 September 2021 Published: 16 September 2021

**2021**, *11*, x.

**Disease Onset in Adults with Down Syndrome** 

**1. Introduction** 

*Article* 

evaluation for AD, the majority of the cohort was euthyroid; there was no significant difference amongst the cohorts with respect to TSH or Free T<sup>4</sup> blood concentrations. \* Correspondence**:** flai@partners.org **Florence Lai 1,2,3,\*, Nathaniel D. Mercaldo 1,2, Cassandra M. Wang 4, Micaela S. Hersch 5, Giovi G. Hersch 6** 

**Association between Hypothyroidism Onset and Alzheimer** 

**Florence Lai 1,2,3,\*, Nathaniel D. Mercaldo 1,2, Cassandra M. Wang 4, Micaela S. Hersch 5, Giovi G. Hersch 6** 

2 Massachusetts General Hospital, Boston, MA 02114, USA

4 Harvard College, Harvard University, Cambridge, MA 02138, USA;

1 Harvard Medical School, Boston, MA 02115, USA; nmercaldo@mgh.harvard.edu (N.D.M.);

5 School of Nursing, Simmons University, Boston, MA 02115, USA; micaela.hersch@simmons.edu 6 College of Arts & Sciences, Boston University, Boston, MA 02215, USA; herschgi@bu.edu

**Disease Onset in Adults with Down Syndrome** 

rosas@helix.mgh.harvard.edu (H.D.R.)

cassandrawang@college.harvard.edu

3 McLean Hospital, Belmont, MA 02478, USA

We subsequently evaluated the association between the age of hypothyroidism onset and age of AD onset. The Spearman correlation between the age of hypothyroidism onset and age of AD onset was 0.43 (95% CI: 0.27, 0.57) (*p* < 0.001). When assuming a linear representation of age of hypothyroidism (*p*anova > 0.05), we observed that for each year increase in the age of hypothyroidism onset, the unadjusted odds of having a later age of AD onset increased by a factor of 1.09 (95% CI: 1.05–1.12). Thus, among patients with hypothyroidism, those who developed it earlier appeared to have an earlier age of AD onset. **Abstract:** Adults with Down syndrome (DS) have an exceptionally high frequency of Alzheimer disease (AD) with a wide variability in onset, from 40 to 70 years of age. Equally prevalent in DS is hypothyroidism. In this study, we sought to quantify the relationship between the two. A total of 232 adults with DS and AD were stratified into three AD onset age groups: early (<47 years), typical (48–59), and late (>59). Among patients with available data, differences in the distributions of demographics, hypothyroidism variables (presence, age of onset), thyroid function tests, thyroid autoantibodies, and APOE genotypes were assessed (e.g., chi-squared, Mann–Whitney tests). Spearman and partial Spearman correlations and ordinal logistic regression models were constructed to **Citation:** Lai, F.; Mercaldo, N.D.; 1 Harvard Medical School, Boston, MA 02115, USA; nmercaldo@mgh.harvard.edu (N.D.M.); rosas@helix.mgh.harvard.edu (H.D.R.) 2 Massachusetts General Hospital, Boston, MA 02114, USA 3 McLean Hospital, Belmont, MA 02478, USA 4 Harvard College, Harvard University, Cambridge, MA 02138, USA; cassandrawang@college.harvard.edu 5 School of Nursing, Simmons University, Boston, MA 02115, USA; micaela.hersch@simmons.edu 6 College of Arts & Sciences, Boston University, Boston, MA 02215, USA; herschgi@bu.edu

Similar findings were observed after accounting for each demographic variable and co-varying medical condition of interest (history of vitamin B12 deficiency: 0.44 (0.25, 0.59); history of OSA: 0.45 (0.26, 0.60); BMI: 0.44 (0.22, 0.62); APOE quantify the association between ages of AD and hypothyroidism onset with and without covariate adjustments. We observed a positive association between the ages of AD and hypothyroidism onset after accounting for APOE-Ɛ4 (correlation: 0.44, 0.24, 0.60; odds ratio: 1.09, 1.05–1.14). However, an early age of hypothyroidism onset and the presence of the APOE-Ɛ4 allele were independently associated with the early age of AD onset. Similar findings were observed when accounting for other factors. Our study provides evidence for the importance of hypothyroidism and associated pathological mechanisms for risk of AD in DS. **Keywords:** Down syndrome; early-onset Alzheimer disease; late-onset Alzheimer disease; hypothyroidism; thyroid autoantibodies; TSH; Free T4; APOE Ɛ4 Wang, C.M.; Hersch, M.S.; Hersch, G.G.; Rosas, H.D.; Association between Hypothyroidism Onset and Alzheimer Disease Onset in Adults with Down Syndrome. *Brain Sci.*  **2021**, *11*, x. https://doi.org/10.3390/xxxxx Academic Editor: Rebecca Sims Received: 31 July 2021 Accepted: 8 September 2021 Published: 16 September 2021 4 status: 0.44 (0.24, 0.60), sex: 0.43 (0.24, 0.58); level of intellectual disability: 0.46 (0.28, 0.61); all *p* < 0.001). There was insufficient evidence to conclude that a non-linear coding of age of hypothyroidism onset and the interaction between age of hypothyroidism onset and each demographic variable improved the model fit of age of AD onset (all *p*anova > 0.05). Thus, we observed that for each year increase in the age of hypothyroidism onset, the adjusted odds of having a later age of AD onset increased by a factor of: 1.09 (95% CI: 1.05–1.12) after adjusting for history of vitamin B12 deficiency, 1.08 (1.05, 1.12) after adjusting for sex, 1.09 (1.05, 1.13) after adjusting for history of OSA, 1.10 (1.05, 1.14) after adjusting for BMI, 1.10 (1.06, 1.14) after adjusting for level of intellectual disability, and 1.09 (1.05, 1.14) after adjusting for APOE \* Correspondence**:** flai@partners.org **Abstract:** Adults with Down syndrome (DS) have an exceptionally high frequency of Alzheimer disease (AD) with a wide variability in onset, from 40 to 70 years of age. Equally prevalent in DS is hypothyroidism. In this study, we sought to quantify the relationship between the two. A total of 232 adults with DS and AD were stratified into three AD onset age groups: early (<47 years), typical (48–59), and late (>59). Among patients with available data, differences in the distributions of demographics, hypothyroidism variables (presence, age of onset), thyroid function tests, thyroid autoantibodies, and APOE genotypes were assessed (e.g., chi-squared, Mann–Whitney tests). Spearman and partial Spearman correlations and ordinal logistic regression models were constructed to quantify the association between ages of AD and hypothyroidism onset with and without covariate adjustments. We observed a positive association between the ages of AD and hypothyroidism onset after accounting for APOE-Ɛ4 (correlation: 0.44, 0.24, 0.60; odds ratio: 1.09, 1.05–1.14). However, an 4 status.

### **Publisher's Note:** MDPI stays neutral with regard to jurisdictional early age of hypothyroidism onset and the presence of the APOE-Ɛ4 allele were independently associated with the early age of AD onset. Similar findings were observed when accounting for other *3.3. Thyroid Autoantibodies*

*Article* 

**and Herminia Diana Rosas 1,2,3**

Adults with Down syndrome (DS) have an especially high risk for developing Alzheimer disease (AD), with onset at least two decades earlier than in the general population [1,2]. Although there is great variation in the age of AD onset, from as early as 40 to as late as 70 or older [3,4], this variability in onset is poorly understood. The leading hypothesis for the pathogenesis of AD in DS has been attributed to overexpression of the gene for amyloid precursor protein (APP) located on the triplicated chromosome 21 [5], although factors other than amyloid likely influence the wide range of AD onset age in those with DS, just as they do in sporadic AD. Some of these factors include not only the claims in published maps and institutional affiliations. **Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article factors. Our study provides evidence for the importance of hypothyroidism and associated pathological mechanisms for risk of AD in DS. **Keywords:** Down syndrome; early-onset Alzheimer disease; late-onset Alzheimer disease; hypothyroidism; thyroid autoantibodies; TSH; Free T4; APOE Ɛ4 To evaluate the potential contribution of autoimmune thyroiditis to the risk of developing AD early, we evaluated TPO antibody levels; these were available for only approximately 41% (96/232) of the entire cohort (Table 3). Among those with a recorded TPO value, we were unable to detect differences in the distribution of TPO values by the age of AD onset (*p* = 0.591). The frequency of an elevated TPO, defined as a level above 9 IU/mL, was present in approximately 33% of the early, 40% of the typical, and 32% of the late AD onset group (*p* = 0.731).

**1. Introduction** 


Apolipoprotein Ɛ4 (APOE Ɛ4) genotype, but other genetic factors, as well as environmental or biological factors and co-existing medical conditions. distributed under the terms and conditions of the Creative Commons At-**Table 3.** Thyroid antibody summaries by age of AD onset group.

*Brain Sci.* **2021**, *11*, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/brainsci Thyroglobulin (Tg) antibody levels were available for approximately 32% (74/232) of the cohort. Among those with a recorded Tg value, values tended to be less than 4 IU/mL, but we were unable to detect differences in the distribution of these values by the age of AD onset (*p* = 0.533).

*Article* 

*Article* 

**2021**, *11*, x.

**2021**, *11*, x.

Received: 31 July 2021

tional affiliations.

### **4. Discussion** 1 Harvard Medical School, Boston, MA 02115, USA; nmercaldo@mgh.harvard.edu (N.D.M.); rosas@helix.mgh.harvard.edu (H.D.R.)

**and Herminia Diana Rosas 1,2,3**

**and Herminia Diana Rosas 1,2,3**

It is well known that both hypothyroidism and AD occur in high frequency in those with DS; however, our study is the first to explore the potential relationship between the age of onset of hypothyroidism and age of onset of AD in DS. Although an earlier age of AD onset in the DS population has been reported to occur, on average, 20 years earlier than in the neurotypical population, it has generally been ascribed to the triplication of APP on chromosome 21. However, our data suggest that the presence of hypothyroidism early in life may also provide its own contribution to risk for AD in DS. This was true even when other co-variates and other medical co-morbidities known to independently contribute to cognitive dysfunction were taken into consideration. Earlier onset of hypothyroidism within the DS population was associated with an even greater risk for the earlier onset of AD. Although the biological mechanisms that might explain this are as yet unknown, one possibility is that thyroid hormone itself may impact the expression of amyloid precursor protein, such that reduced thyroid hormone levels increase APP expression and elevations of pathogenic amyloid [30]. This effect may be even more significant in those with DS who have a much higher amyloid burden. 4 Harvard College, Harvard University, Cambridge, MA 02138, USA; cassandrawang@college.harvard.edu 5 School of Nursing, Simmons University, Boston, MA 02115, USA; micaela.hersch@simmons.edu 6 College of Arts & Sciences, Boston University, Boston, MA 02215, USA; herschgi@bu.edu \* Correspondence**:** flai@partners.org **Abstract:** Adults with Down syndrome (DS) have an exceptionally high frequency of Alzheimer disease (AD) with a wide variability in onset, from 40 to 70 years of age. Equally prevalent in DS is hypothyroidism. In this study, we sought to quantify the relationship between the two. A total of 232 adults with DS and AD were stratified into three AD onset age groups: early (<47 years), typical (48–59), and late (>59). Among patients with available data, differences in the distributions of demographics, hypothyroidism variables (presence, age of onset), thyroid function tests, thyroid autoantibodies, and APOE genotypes were assessed (e.g., chi-squared, Mann–Whitney tests). Spearman and partial Spearman correlations and ordinal logistic regression models were constructed to quantify the association between ages of AD and hypothyroidism onset with and without covariate adjustments. We observed a positive association between the ages of AD and hypothyroidism onset **Citation:** Lai, F.; Mercaldo, N.D.; Wang, C.M.; Hersch, M.S.; Hersch, G.G.; Rosas, H.D.; Association be-2 Massachusetts General Hospital, Boston, MA 02114, USA 3 McLean Hospital, Belmont, MA 02478, USA 4 Harvard College, Harvard University, Cambridge, MA 02138, USA; cassandrawang@college.harvard.edu 5 School of Nursing, Simmons University, Boston, MA 02115, USA; micaela.hersch@simmons.edu 6 College of Arts & Sciences, Boston University, Boston, MA 02215, USA; herschgi@bu.edu \* Correspondence**:** flai@partners.org **Abstract:** Adults with Down syndrome (DS) have an exceptionally high frequency of Alzheimer disease (AD) with a wide variability in onset, from 40 to 70 years of age. Equally prevalent in DS is hypothyroidism. In this study, we sought to quantify the relationship between the two. A total of 232 adults with DS and AD were stratified into three AD onset age groups: early (<47 years), typical (48–59), and late (>59). Among patients with available data, differences in the distributions of demographics, hypothyroidism variables (presence, age of onset), thyroid function tests, thyroid autoantibodies, and APOE genotypes were assessed (e.g., chi-squared, Mann–Whitney tests). Spearman and partial Spearman correlations and ordinal logistic regression models were constructed to **Citation:** Lai, F.; Mercaldo, N.D.; rosas@helix.mgh.harvard.edu (H.D.R.) 2 Massachusetts General Hospital, Boston, MA 02114, USA 3 McLean Hospital, Belmont, MA 02478, USA 4 Harvard College, Harvard University, Cambridge, MA 02138, USA; cassandrawang@college.harvard.edu 5 School of Nursing, Simmons University, Boston, MA 02115, USA; micaela.hersch@simmons.edu 6 College of Arts & Sciences, Boston University, Boston, MA 02215, USA; herschgi@bu.edu \* Correspondence**:** flai@partners.org **Abstract:** Adults with Down syndrome (DS) have an exceptionally high frequency of Alzheimer disease (AD) with a wide variability in onset, from 40 to 70 years of age. Equally prevalent in DS is hypothyroidism. In this study, we sought to quantify the relationship between the two. A total of 232 adults with DS and AD were stratified into three AD onset age groups: early (<47 years), typical (48–59), and late (>59). Among patients with available data, differences in the distributions of demographics, hypothyroidism variables (presence, age of onset), thyroid function tests, thyroid autoantibodies, and APOE genotypes were assessed (e.g., chi-squared, Mann–Whitney tests). Spear-

**Association between Hypothyroidism Onset and Alzheimer** 

**Florence Lai 1,2,3,\*, Nathaniel D. Mercaldo 1,2, Cassandra M. Wang 4, Micaela S. Hersch 5, Giovi G. Hersch 6** 

2 Massachusetts General Hospital, Boston, MA 02114, USA

1 Harvard Medical School, Boston, MA 02115, USA; nmercaldo@mgh.harvard.edu (N.D.M.);

**Disease Onset in Adults with Down Syndrome** 

**Association between Hypothyroidism Onset and Alzheimer** 

**Florence Lai 1,2,3,\*, Nathaniel D. Mercaldo 1,2, Cassandra M. Wang 4, Micaela S. Hersch 5, Giovi G. Hersch 6** 

1 Harvard Medical School, Boston, MA 02115, USA; nmercaldo@mgh.harvard.edu (N.D.M.);

**Disease Onset in Adults with Down Syndrome** 

**Disease Onset in Adults with Down Syndrome** 

*Article* 

**Association between Hypothyroidism Onset and Alzheimer** 

rosas@helix.mgh.harvard.edu (H.D.R.)

**and Herminia Diana Rosas 1,2,3**

3 McLean Hospital, Belmont, MA 02478, USA

As expected, the presence of an APOE after accounting for APOE-Ɛ4 (correlation: 0.44, 0.24, 0.60; odds ratio: 1.09, 1.05–1.14). However, an early age of hypothyroidism onset and the presence of the APOE-Ɛ4 allele were independently associated with the early age of AD onset. Similar findings were observed when accounting for other factors. Our study provides evidence for the importance of hypothyroidism and associated pathological mechanisms for risk of AD in DS. tween Hypothyroidism Onset and Alzheimer Disease Onset in Adults with Down Syndrome. *Brain Sci.*  https://doi.org/10.3390/xxxxx 4 allele was associated with an earlier onset of AD in DS, as has been reported by others [31]; however, we did not detect an interaction between the presence of the APOE quantify the association between ages of AD and hypothyroidism onset with and without covariate adjustments. We observed a positive association between the ages of AD and hypothyroidism onset after accounting for APOE-Ɛ4 (correlation: 0.44, 0.24, 0.60; odds ratio: 1.09, 1.05–1.14). However, an early age of hypothyroidism onset and the presence of the APOE-Ɛ4 allele were independently associated with the early age of AD onset. Similar findings were observed when accounting for other factors. Our study provides evidence for the importance of hypothyroidism and associated patho-Wang, C.M.; Hersch, M.S.; Hersch, G.G.; Rosas, H.D.; Association between Hypothyroidism Onset and Alzheimer Disease Onset in Adults with Down Syndrome. *Brain Sci.*  4 allele and the relationship between the age of onset of AD and age of hypothyroidism onset. Our findings suggest that APOE man and partial Spearman correlations and ordinal logistic regression models were constructed to quantify the association between ages of AD and hypothyroidism onset with and without covariate adjustments. We observed a positive association between the ages of AD and hypothyroidism onset after accounting for APOE-Ɛ4 (correlation: 0.44, 0.24, 0.60; odds ratio: 1.09, 1.05–1.14). However, an early age of hypothyroidism onset and the presence of the APOE-Ɛ4 allele were independently associated with the early age of AD onset. Similar findings were observed when accounting for other **Citation:** Lai, F.; Mercaldo, N.D.; Wang, C.M.; Hersch, M.S.; Hersch, G.G.; Rosas, H.D.; Association between Hypothyroidism Onset and Alzheimer Disease Onset in Adults with Down Syndrome. *Brain Sci.*  4 and early age of hypothyroidism may each contribute independently to the risk for early AD onset in the DS population.

**Keywords:** Down syndrome; early-onset Alzheimer disease; late-onset Alzheimer disease; hypothyroidism; thyroid autoantibodies; TSH; Free T4; APOE Ɛ4 **1. Introduction**  Adults with Down syndrome (DS) have an especially high risk for developing Alzheimer disease (AD), with onset at least two decades earlier than in the general population [1,2]. Although there is great variation in the age of AD onset, from as early as 40 to as late as 70 or older [3,4], this variability in onset is poorly understood. The leading hypothesis for the pathogenesis of AD in DS has been attributed to overexpression of the gene Academic Editor: Rebecca Sims Received: 31 July 2021 Accepted: 8 September 2021 Published: 16 September 2021 **Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. logical mechanisms for risk of AD in DS. **Keywords:** Down syndrome; early-onset Alzheimer disease; late-onset Alzheimer disease; hypothyroidism; thyroid autoantibodies; TSH; Free T4; APOE Ɛ4 **1. Introduction**  Adults with Down syndrome (DS) have an especially high risk for developing Alzheimer disease (AD), with onset at least two decades earlier than in the general population [1,2]. Although there is great variation in the age of AD onset, from as early as 40 to as https://doi.org/10.3390/xxxxx Academic Editor: Rebecca Sims Accepted: 8 September 2021 Published: 16 September 2021 **Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institufactors. Our study provides evidence for the importance of hypothyroidism and associated pathological mechanisms for risk of AD in DS. **Keywords:** Down syndrome; early-onset Alzheimer disease; late-onset Alzheimer disease; hypothyroidism; thyroid autoantibodies; TSH; Free T4; APOE Ɛ4 **1. Introduction**  Adults with Down syndrome (DS) have an especially high risk for developing Alzheimer disease (AD), with onset at least two decades earlier than in the general population **2021**, *11*, x. https://doi.org/10.3390/xxxxx Academic Editor: Rebecca Sims Received: 31 July 2021 Accepted: 8 September 2021 Published: 16 September 2021 **Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institu-In a subset of our cohort for whom results for serum thyroid autoantibodies were available, approximately 40% had thyroid autoantibodies as evidence of autoimmune thyroiditis, consistent with what has been reported previously in DS [32]. A similar frequency of thyroid autoantibodies was present in each of the three age of AD onset groups, suggesting that the presence of autoantibodies did not confer added risk for the development of early AD in the DS population. However, we did not have sufficient sample size to evaluate the potential interaction with the presence of thyroid autoantibodies. Nevertheless, given the comparable distribution and similar blood levels across the three groups, it appears unlikely that thyroid autoantibodies confer added risk for early AD onset in the DS population. Future studies are needed to fully evaluate the potential contribution of autoimmune thyroiditis to AD risk in DS.

*Brain Sci.* **2021**, *11*, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/brainsci for amyloid precursor protein (APP) located on the triplicated chromosome 21 [5], although factors other than amyloid likely influence the wide range of AD onset age in those with DS, just as they do in sporadic AD. Some of these factors include not only the Apolipoprotein Ɛ4 (APOE Ɛ4) genotype, but other genetic factors, as well as environmental or biological factors and co-existing medical conditions. Thyroid dysfunction, including congenital, subclinical, and autoimmune thyroid conditions [6], is a very common medical co-morbidity in individuals with DS. In a large **Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). late as 70 or older [3,4], this variability in onset is poorly understood. The leading hypothesis for the pathogenesis of AD in DS has been attributed to overexpression of the gene for amyloid precursor protein (APP) located on the triplicated chromosome 21 [5], although factors other than amyloid likely influence the wide range of AD onset age in those with DS, just as they do in sporadic AD. Some of these factors include not only the Apolipoprotein Ɛ4 (APOE Ɛ4) genotype, but other genetic factors, as well as environmental or biological factors and co-existing medical conditions. Thyroid dysfunction, including congenital, subclinical, and autoimmune thyroid conditions [6], is a very common medical co-morbidity in individuals with DS. In a large **Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). [1,2]. Although there is great variation in the age of AD onset, from as early as 40 to as late as 70 or older [3,4], this variability in onset is poorly understood. The leading hypothesis for the pathogenesis of AD in DS has been attributed to overexpression of the gene for amyloid precursor protein (APP) located on the triplicated chromosome 21 [5], although factors other than amyloid likely influence the wide range of AD onset age in those with DS, just as they do in sporadic AD. Some of these factors include not only the Apolipoprotein Ɛ4 (APOE Ɛ4) genotype, but other genetic factors, as well as environmental or biological factors and co-existing medical conditions. Thyroid dysfunction, including congenital, subclinical, and autoimmune thyroid conditions [6], is a very common medical co-morbidity in individuals with DS. In a large tional affiliations. **Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). It is important to point out that at the time of an AD diagnosis, a majority of patients in each of the cohorts had TSH levels within the normal ranges, confirming that the majority were euthyroid at the time of AD diagnosis, and that there were no significant differences amongst TSH levels across the groups. Similarly, the overall frequency of hypothyroidism in our sample was consistent with the findings in a large meta-analysis of adults with DS [8], indicating that our cohort is representative of the DS population at large. A diagnosis of hypothyroidism appeared to occur, on average, more than a decade prior to a diagnosis of AD (as defined as the difference between the age of AD onset and age of hypothyroidism onset), irrespective of age of AD onset. Alterations in thyroid function have been associated with a higher risk of developing AD later in life in the neurotypical population [33], suggesting that complex interactions between thyroid hormone, thyroid function and risk for AD may be particularly salient in the DS population.

### *Brain Sci.* **2021**, *11*, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/brainsci **5. Limitations**

*Brain Sci.* **2021**, *11*, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/brainsci The primary limitations of this study include: (1) the retrospective nature of the study design; (2) the possible measurement error associated with ages of AD onset and hypothyroidism onset; and (3) the degree of missingness especially involving the thyroid autoantibody panels. The validity of these results assumes that the covariate missingness mechanism is completely random. Nevertheless, the results of this study need to be studied further in larger (and ideally prospectively collected) datasets.
