TWINGEN : protocol for an observational clinical biobank recall and biomarker cohort study to identify Finnish individuals with high risk of Alzheimer’s disease

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Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ TWINGEN : protocol for an observational clinical biobank recall and biomarker cohort study to identify Finnish individuals with high risk of Alzheimer’s disease © 2024 the Authors Published version Vuoksimaa, Eero; Saari, Toni T; Aaltonen, Aino; Aaltonen, Sari; Herukka, SannaKaisa; Iso-Markku, Paula; Kokkola, Tarja; Kyttälä, Aija; Kärkkäinen, Sari; Liedes, Hilkka; Ollikainen, Miina; Palviainen, Teemu; Ruotsalainen, Ilona; Toivola, Auli; Urjansson, Mia; Vasankari, Tommi; Vähä-Ypyä, Henri; Forsberg, Markus M; Hiltunen, Mikko; Jalanko, Anu; Kälviäinen, Reetta; Kuopio, Teijo; Lähteenmäki, Jaakko; Nyberg, Pia; Männikkö, Minna; Serpi, Raisa; Siltanen, Sanna; Palotie, Aarno; Kaprio, Jaakko; Runz, Heiko; Julkunen, Valtteri; FinnGen Vuoksimaa, Eero, Saari, Toni T, Aaltonen, Aino, Aaltonen, Sari, Herukka, Sanna-Kaisa, IsoMarkku, Paula, Kokkola, Tarja, Kyttälä, Aija, Kärkkäinen, Sari, Liedes, Hilkka, Ollikainen, Miina, Palviainen, Teemu, Ruotsalainen, Ilona, Toivola, Auli, Urjansson, Mia, Vasankari, Tommi, VähäYpyä, Henri, Forsberg, Markus M, Hiltunen, Mikko, Jalanko, Anu, Kälviäinen, Reetta, Kuopio, Teijo, Lähteenmäki, Jaakko, Nyberg, Pia, Männikkö, Minna, Serpi, Raisa, Siltanen, Sanna, Palotie, Aarno, Kaprio, Jaakko, Runz, Heiko, Julkunen, Valtteri, FinnGen. (2024). TWINGEN : protocol for an observational clinical biobank recall and biomarker cohort study to identify Finnish individuals with high risk of Alzheimer’s disease. BMJ Open, 14(6), Article e081947. https://doi.org/10.1136/bmjopen-2023-081947 2024 1 VuoksimaaE, etal. BMJ Open 2024;14:e081947. doi:10.1136/bmjopen-2023-081947 Open access TWINGEN: protocol for an observational clinical biobank recall and biomarker cohort study to identify Finnish individuals with high risk of Alzheimer’s disease Eero Vuoksimaa ,1 Toni T Saari ,1 Aino Aaltonen,1 Sari Aaltonen ,1 SannaKaisa Herukka ,2,3 Paula IsoMarkku ,1 Tarja Kokkola ,2 Aija Kyttälä,4 Sari Kärkkäinen,2 Hilkka Liedes,1,5 Miina Ollikainen,1,6 Teemu Palviainen,1 Ilona Ruotsalainen,1,7 Auli Toivola,4 Mia Urjansson,1 Tommi Vasankari ,8,9 Henri Vähä-Ypyä,8 Markus M Forsberg,10,11 Mikko Hiltunen ,2 Anu Jalanko,1 Reetta Kälviäinen ,2,3 Teijo Kuopio,12 Jaakko Lähteenmäki,7 Pia Nyberg,13,14 Minna Männikkö ,15 Raisa Serpi,13 Sanna Siltanen,16 Aarno Palotie ,1,17,18 Jaakko Kaprio ,1 Heiko Runz ,1,19 Valtteri Julkunen,2,3 FinnGen To cite: VuoksimaaE, SaariTT, AaltonenA, etal. TWINGEN: protocol for an observational clinical biobank recall and biomarker cohort study to identify Finnish individuals with high risk of Alzheimer’s disease. BMJ Open 2024;14:e081947. doi:10.1136/ bmjopen-2023-081947 ►Prepublication history and additional supplemental material for this paper are available online. To view these files, please visit the journal online (https://doi.org/10.1136/ bmjopen-2023-081947). EV and TTS contributed equally. Received 10 November 2023 Accepted 09 May 2024 For numbered affiliations see end of article. Correspondence to Dr Toni T Saari; toni. saari@ helsinki. fi Protocol © Author(s) (or their employer(s)) 2024. Reuse permitted under CC BY. Published by BMJ. ABSTRACT Introduction A better understanding of the earliest stages of Alzheimer’s disease (AD) could expedite the development or administration of treatments. Large population biobanks hold the promise to identify individuals at an elevated risk of AD and related dementias based on health registry information. Here, we establish the protocol for an observational clinical recall and biomarker study called TWINGEN with the aim to identify individuals at high risk of AD by assessing cognition, health and ADrelated biomarkers. Suitable candidates were identified and invited to participate in the new study among THL Biobank donors according to TWINGEN study criteria. Methods and analysis A multicentre study (n=800) to obtain bloodbased biomarkers, telephoneadministered and webbased memory and cognitive parameters, questionnaire information on lifestyle, health and psychological factors, and accelerometer data for measures of physical activity, sedentary behaviour and sleep. A subcohort is being asked to participate in an inperson neuropsychological assessment (n=200) and wear an Oura ring (n=50). All participants in the TWINGEN study have genomewide genotyping data and up to 48 years of followup data from the populationbased older Finnish Twin Cohort (FTC) study of the University of Helsinki. The data collected in TWINGEN will be returned to THL Biobank from where it can later be requested for other biobank studies such as FinnGen that supported TWINGEN. Ethics and dissemination This recall study consists of FTC/THL Biobank/FinnGen participants whose data were acquired in accordance with the Finnish Biobank Act. The recruitment protocols followed the biobank protocols approved by Finnish Medicines Agency. The TWINGEN study plan was approved by the Ethics Committee of Hospital District of Helsinki and Uusimaa (number 16831/2022). THL Biobank approved the research plan with the permission no: THLBB2022_83. INTRODUCTION Alzheimer’s disease (AD)—the most common cause of dementia—is characterised by pathological accumulation of betaamyloid (Aβ) and tau in the brain.1 As populations age, STRENGTHS AND LIMITATIONS OF THIS STUDY ⇒A large sample of individuals is recruited from a representative biobank database. ⇒Using health registry information, we exclude those with documented Alzheimer’s disease (AD) or other neurological or psychiatric diseases that can affect cognition. Prescreening limits the sending of unnecessary invitations and saves costs. ⇒Participants have up to 48 years of followup questionnaire and clinical data from the Finnish Twin Cohort study and these data can be combined with multifaceted Finnish health registry information. Previous genotype data is available in the biobank from all TWINGEN study participants. ⇒We assess the feasibility of remote cognitive testing and blood samples in largescale screening of AD risk, translating to the requirements of intervention trials and clinical practice. ⇒Limitations of the study are a lack of gold standard biomarkers (cerebrospinal fluid, positron emission tomography imaging) and neurological examinations. Protected by copyright. on June 12, 2024 at Jyvaskylan Ylioposto Kirjasto/Kausijulkaisut.http://bmjopen.bmj.com/BMJ Open: first published as 10.1136/bmjopen-2023-081947 on 12 June 2024. Downloaded from 2VuoksimaaE, etal. BMJ Open 2024;14:e081947. doi:10.1136/bmjopen-2023-081947 Open access the prevalence of dementia is projected to nearly double every two decades1 2 and AD and related dementias are becoming one of the most common causes of death in many countries (20% of deaths in Finland ranking it as the third most common cause of death).3 The AD process starts up to 20–30 years before the diagnosis, so intervention trials targeted at preclinical or prodromal stages of AD are of high priority, but timeconsuming and costly with screenfailure rates of 78%–88%.4 In clinical practice, AD is diagnosed mainly based on the clinical phenotype, episodic memory impairment being the cognitive hallmark, while in research there has been a shift from clinical diagnosis to biological classification independent of the cognitive status.5 Evidence of diagnostic properties of different bloodbased biomarkers is rapidly accumulating,6 but populationbased studies are still scarce. In one populationbased study, 11% of older adults (median age 74 years) without dementia were found to have Aβ pathology using bloodbased biomarkers.7 Our earlier pilot study explored the feasibility of FinnGen, a nationwide Finnish biobank study8 in recruiting individuals with AD to an observational study via the biobank.9 Our protocol included bloodbased biomarkers and remote cognitive assessment, approaches that are suggested to improve the recruitment of participants for AD trials.10–12 Another FinnGen study showed that biobank participants could be recontacted for additional data collection on a larger scale as well.13 The full potential of population biobank datasets lies in the large cohorts of undiagnosed individuals who are at the risk of developing AD in near future and might be suitable for targeted screening for early diagnostics and interventional trials encompassing both pharmacological treatments and lifestyle interventions. After showing in our pilot study that recall of biobank participants with AD to a clinical study including multimodal (remote and inperson) cognitive assessment and bloodbased biomarker analyses is feasible,9 we modified the protocol of the pilot study to target cognitively unimpaired older adults (based on health registry data) in the current study. Additionally, we augmented the assessment battery with passive technology for measuring physical activity, sedentary behaviour and sleep. This protocol paper describes TWINGEN, a populationbased followup study investigating the utility of easily implementable methods for assessing the risk of AD. We aim to conduct a proofofprinciple study for using biobank registries as a platform for recruiting participants suitable for clinical trials, particularly in diseases where recruitment and screening have generally been challenging. We also focus on the remote cognitive assessment methods and bloodbased biomarkers of AD. The study also aims to enrich existing biobank data derived from a longstanding prospective twin study with cognitive and lifestyle measures. The research setting is unique as it uses the prescreening and recall option based on the data of the biobank in combination with long preceding populationbased followup data from the twin study. METHODS AND ANALYSIS Study participant selection The target group of the TWINGEN study are individuals who have participated in the older Finnish Twin Cohort (FTC) study of the University of Helsinki (UH), and whose samples and data have been transferred to THL Biobank in 2018. The FTC was chosen as the primary target of this study because it is a populationbased followup study with up to 48 years of previous comprehensive health data available. Combining the historical data with newly collected samples would allow building of longitudinal trajectories of various lifestyle and health factors to latelife cognitive decline. The main selection criteria in the biobank were previous participation in FTC, age (65–85), place of current residence in Finland, Finnish as the first language and no known diagnosis affecting cognition in biobank records. The selection of eligible FTC study participants was done through THL Biobank and data collection was carried out by the UH for those living in the greater Helsinki area or surrounding regions and by regional biobanks and Turku University of Applied Science based on the residency of the participant. Selection also included participation in the FinnGen nationwide biobank research study for two reasons.8 First, FinnGen supported the collection of the TWINGEN cohort with the aim to enrich phenotype information. Second, the collected TWINGEN data are to be returned to THL Biobank, from where they can later be requested for the FinnGen study and combined with its extensive gene and health register data. Thus far, FinnGen has produced genotype data from ca. 500 000 biobank donors of all Finnish biobanks to perform largescale genome and health research. In the following sections, we describe each of the data sources and the study protocol of TWINGEN. The older FTC study The older FTC study from the UH is a populationbased study that includes all Finnish samesex twins born before 1958 and living in Finland at the start of the study in 1974 (figure 1).14 The baseline survey was conducted in 1975 via postal questionnaire and 27 750 individuals participated with an 89% participation rate. Followup questionnaires were sent in 1981 (n=24 684 with an 84% response rate), 1990 for those born in 1930 or later (n=12 502; 77%) and in 2011–2012 for those born in 1945–1957 (n=8410; 72%).15 Those born in 1938–1944 have also participated in MEMTWIN II study (n=1772) that used telephone interview to assess cognition.16 Some of the twins born in 1945–1957 have participated in Essential Hypertension EPIgenetics study (EHEPI, n=445).17 The MEMTWIN II and EHEPI study participants are the primary groups of interest in TWINGEN because they have either earlier Protected by copyright. on June 12, 2024 at Jyvaskylan Ylioposto Kirjasto/Kausijulkaisut.http://bmjopen.bmj.com/BMJ Open: first published as 10.1136/bmjopen-2023-081947 on 12 June 2024. Downloaded from 3 VuoksimaaE, etal. BMJ Open 2024;14:e081947. doi:10.1136/bmjopen-2023-081947 Open access Figure 1 Flowchart of study participant selection. EHEpi, Essential Hypertension EPIgenetics. Protected by copyright. on June 12, 2024 at Jyvaskylan Ylioposto Kirjasto/Kausijulkaisut.http://bmjopen.bmj.com/BMJ Open: first published as 10.1136/bmjopen-2023-081947 on 12 June 2024. Downloaded from 4VuoksimaaE, etal. BMJ Open 2024;14:e081947. doi:10.1136/bmjopen-2023-081947 Open access cognitive (MEMTWIN II; table 1) or multiomics data (EHEPI; online supplemental table 1) available. To achieve the target number of 800 participants, the biobank selection was expanded to the twins born in 1945–1952 who had not participated in MEMTWIN II or EHEPI. Most of the invited twins were from samesex pairs, but we also invited twins from oppositesex pairs included in the older FTC study in year 1995–1996, when they replied to a brief health questionnaire (figure 1). We prioritised invitations to participants who lived closest to one of the six study sites. All TWINGEN participants from samesex twin pairs have longitudinal questionnaire data on health and healthrelated behaviours from years 1975, 1981 and 1990, and those born in 1945–1957 have data also from year 2011 to 2012. An overview of longitudinal data available for the MEMTWIN II and EHEPI and other TWINGEN participants are presented in table 2. Necessary inclusion criteria for TWINGEN were available DNA sample in THL Biobank and not having any of the exclusion criteria (table 3). Additional references for studies using the older FTC data are found in online supplemental table 2. Recall procedure The results of a THL Biobank feasibility assessment indicated that there were 6053 individuals aged 65–85 who have participated in the FTC of UH and have data in THL Biobank. Approximately 1000 individuals were excluded due to our exclusion criteria of AD and other neurological or psychiatric diseases that can affect cognition (figure 1; table 3). Furthermore, the genotype data of approximately 100 individuals have been verified by FinnGen, but these data have not been returned to their respective biobanks at the time of the feasibility assessment; thus, these individuals are excluded from the pool of potentially eligible participants. The target sample size of 800 participants was chosen as it fits the timeframe and resources of the study and is sufficiently large for stratifying participants at varying risks for AD.7 As a biobank recall study, TWINGEN participants are contacted by THL Biobank by an invitation letter. The invitation letter includes information about the individuals’ prior participation to the older FTC study and the transfer to and storage of samples and data at THL Biobank, information about the participation in the FinnGen study via the biobank, and information about the new TWINGEN study. The invitation letter also contains two separate consent forms: one for the participation in the TWINGEN study and the biobank consent for THL Biobank. The consent for THL Biobank is needed to store the new samples and data obtained in TWINGEN to THL Biobank and to confirm the biobank participation with a written biobank consent, which is the primary basis for storing samples and data into a biobank. After receiving consent forms, UH research staff contacts the potential participants to verify that the individuals have understood the purpose and procedures of the study. Additionally, as the health registry data available in the biobank is not up to date but reflects status at the end of year 2022, research staff verifies (via telephone) that the exclusion criteria are not met. Eligibility of each individual is assessed independently of their cotwin’s vital status or eligibility. Table 1 Cognitive measures used in the TWINGEN study Inperson neuropsychological assessment Telephone (TELE/ TICSm3) Webbased computerised assessment (cCOG) Memory and learning CERAD Word List Learning Word List Learning Episodic Memory Learning CERAD Word List Recall Word List Recall Episodic Memory Recall CERAD Word List Recognition Episodic Memory Recognition CERAD Constructional Praxis Recall WMSIII Logical Memory Story A WMSIII Logical Memory Story A Recall Executive function Trail Making TestB Modified Trail Making TestB Stroop Interference Stroop Setshifting Visuospatial skills and visuoconstruction CERAD Constructional Praxis Fragmented Letters CERAD Clock Drawing Test Language skills and fluency CERAD Naming Test Similarities CERAD Semantic Fluency Semantic Fluency Processing speed Trail Making TestA Modified Trail Making TestA Stroop Word Reading Stroop Colour Naming Global cognition CERAD MMSE TELE Global Score cCOG Global Score TICS Global Score TICSm3 Global Score MEMTWINII participants have prior TELE/TICSm data. CERADnb, Consortium to Establish a Registry for Alzheimer’s Diseaseneuropsychological battery; MMSE, MiniMental State Examination; TELE, telephone assessment for dementia; TICS, Telephone Interview for Cognitive Status; TICSm, modified Telephone Interview for Cognitive Status; TICSm3, TICS m including three learning trials in the Word List Learning; WMSIII, Wechsler Memory Scale 3rd edition. Protected by copyright. on June 12, 2024 at Jyvaskylan Ylioposto Kirjasto/Kausijulkaisut.http://bmjopen.bmj.com/BMJ Open: first published as 10.1136/bmjopen-2023-081947 on 12 June 2024. Downloaded from 5 VuoksimaaE, etal. BMJ Open 2024;14:e081947. doi:10.1136/bmjopen-2023-081947 Open access Table 2 Previous data from the participants recruited in TWINGEN Baseline assessment (1975) First followup (1981) Second followup (1990) Third followup (2011–2012) MEMTWINII (2013– 2017)* EHEpi (2012–2015) Primary study reference Education x x Vuoksimaa et al, 2016b Chronic or serious illness x x x x x x Kaprio et al, 201914 Physical activity x x x x x Piirtola et al, 2017 Smoking x x x x x Kaprio and Koskenvuo 1988 Alcohol use x x x x x Sipilä et al, 2016 Sleep x x x x Kaprio et al, 201914 Medications x x x x x x Huang et al, 2018 Anthropometrics (weight, height) x x x x x Piirtola et al, 2017 Dietary habits x x Kaprio et al, 201914 Blood pressure x x x x x IsoMarkku et al, 2021 Cholesterol x x x IsoMarkku et al, 2021 Diabetes x x x x x IsoMarkku et al, 2021 Subjective memory complaints x Life satisfaction x x x x KoivumaaHonkanen et al, 2000 Loneliness x x x x KoivumaaHonkanen et al, 2000 Social support x x Romanov et al, 2003 Cognition (TELE/TICSm) x Lindgren et al, 201916 Depressive symptoms BDI CESD CESD Saari et al, 202346 Personality (EPI) x x x Rose et al, 198850 Accelerometer (physical activity) x Waller et al, 2019 Multiomics data x Drouard et al, 2022 DNA x x Kaprio et al, 201914 See online supplemental file 3 for full reference information and additional references on previous studies using older Finnish Twin Cohort study data. *Third followup assessments for those born in 1938–1944. BDI, Beck Depression Inventory; CESD, Center for Epidemiological StudiesDepression scale; EPI, Eysenck Personality Inventory; LS, life satisfaction; TELE, telephone assessment for dementia; TICSm, modified Telephone Interview for Cognitive Status. Protected by copyright. on June 12, 2024 at Jyvaskylan Ylioposto Kirjasto/Kausijulkaisut.http://bmjopen.bmj.com/BMJ Open: first published as 10.1136/bmjopen-2023-081947 on 12 June 2024. Downloaded from 6VuoksimaaE, etal. BMJ Open 2024;14:e081947. doi:10.1136/bmjopen-2023-081947 Open access Data collection Data collection started on 29 March 2023 and is planned to be completed by the end of 2023. Data collection is conducted in six locations across Finland: at the Institute for Molecular Medicine Finland (FIMM), UH in Helsinki, biobanks in four locations across Finland: in Jyväskylä (Central Finland biobank), Kuopio (Biobank of Eastern Finland), Oulu (Arctic Biobank and Biobank Borealis of Northern Finland) and Tampere (Finnish Clinical Biobank Tampere) and at the clinical laboratory of Turku University of Applied Sciences. Protocol for all participants includes telephoneadministered and computeradministered cognitive testing, blooddraw and selfreport questionnaire. Additionally, a waistworn accelerometer will be given to participants who are willing to complete 1 week athome measurement. Furthermore, individuals living in the greater Helsinki area are invited to participate in inperson neuropsychological testing (target sample size n=200) and measurement of weight, height, waist circumference and blood pressure. In Helsinki, we also provide Oura rings for 50 participants who are willing and able to wear the ring and use the associated mobile app. After participating in the study, participants receive a report based on their performance in telephone interviewbased and computerised tests of cognition and about their physical activity. Data cleaning, feedback to participants and transfer of data to THL biobank are estimated to be completed by the end of June 2024. A detailed description of the data collections is provided later. Telephone interview for assessing cognitive status and function Two validated telephoneadministered cognitive screening instruments are used: a telephone assessment for dementia (TELE)18 and the modified Telephone Interview for Cognitive Status (TICSm).19 TELE and TICS have been translated to Finnish and adapted to Finnish culture20 and both have been used in the older FTC study. TICSm, a modified version of TICS includes an additional delayed free recall of the 10word list and has been used in the MEMTWIN II substudy.21 We further modified the TICSm by including three learning trials of the 10word list and this instrument is later referred to as TICSm3.9 We also included semantic fluency. Three trial word list task yields immediate and delayed free recall measures of episodic memory. Semantic fluency score is the number of animals named in 1 min (table 1). In the telephone interview, the participants are also asked about their functional abilities regarding household maintenance, ambulation, shopping, dressing and undressing, use of mobility aids, memory problems and possible visits to the doctor regarding memory problems. Telephone interviews were conducted by trained study nurses or psychologists. Computerised web-based cognitive testing Webbased cCOG tool created by Combinostics (Tampere, Finland) is used for computerised cognitive testing.22 It includes six subtests: Episodic Memory (learning and recall), Reaction Time, Modified Trail Making A and B and Distorted Letters. The tasks measure visual processing, memory, processing speed, attention and executive function (table 1). In addition to cognitive tests, cCOG includes background questions (education) and a 7item questionnaire designed to assess probable dementia with Lewy bodies.23 The test battery takes about 25 min to complete and is performed via a keyboard and a mouse or a touchscreen device. In-person neuropsychological tests The participants in Helsinki study site undergo an inperson neuropsychological assessment, with a target sample size of 200 individuals. The minimum sample size to detect medium correlations (r=0.3) between cognitive measures with a significance level=0.05 and power=0.8 is n=85, thus, the target sample size of 200 individuals with inperson cognitive assessment is adequate for examining correlations between inperson and remote cognitive measures. The larger sample size is expected to be adequate for factor analyses of the neuropsychological battery. The inperson neuropsychological assessment consists of the Consortium to Establish a Registry for Alzheimer’s Diseaseneuropsychological battery (CERADnb)24 and tests measuring executive functions, processing speed and episodic memory. The CERADnb includes Table 3 Exclusion criteria for the TWINGEN study ICD10 code Explanation G30 and F00 Any variant of Alzheimer’s disease or any dementia relating to Alzheimer’s disease, dementia with Lewy bodies, frontotemporal dementia and mixed dementia F01F03 Dementia, any aetiology G20 Parkinson’s disease G35 Multiple sclerosis I60, I61 and I63 Intracerebral haemorrhage, subarachnoid haemorrhage or ischaemic stroke and their subcategories S06.1–S06.7 Traumatic brain injuries other than concussion (S06.0) F20 Schizophrenia F31 Bipolar disorder F33, F32.1–F32.3, F34 Recurrent depression, moderate or severe depression and longlasting mood disorders F60 Personality disorders F10 All diagnoses relating to excessive usage of alcohol F11–F19 Intoxication because of opioids, cannabinoids, sedative medication, cocaine or hallucinogens F70–F73 Different stages of intellectual disabilities Protected by copyright. on June 12, 2024 at Jyvaskylan Ylioposto Kirjasto/Kausijulkaisut.http://bmjopen.bmj.com/BMJ Open: first published as 10.1136/bmjopen-2023-081947 on 12 June 2024. Downloaded from 7 VuoksimaaE, etal. BMJ Open 2024;14:e081947. doi:10.1136/bmjopen-2023-081947 Open access MiniMental State Examination and Semantic Fluency, abbreviated Boston Naming Test, Word List Learning, Recall and Recognition, and Constructional Praxis (Copy and Recall). The Finnish version of the CERADnb also includes Clock Drawing test.25 Finnish education adjusted cutoffs are available for total score and for each subtest.26 In addition to the CERADnb, we include the following tests: Logical Memory Story A from the Wechsler Memory Scale 3rd edition,27 Trail Making Test A and B28 and Stroop test.29 The version of Stroop used in this study is the 40item version used in the FINGER study30 with an additional fourth ‘setshifting’ condition whereby the task is to name the colour of ink (as in the classical Stroop condition) or to read out the colourword when the word is inside a rectangle. Neuropsychological tests are administered by trained psychologists. A summary of key cognitive measures of all three modalities are presented in table 1. Blood sample A venous blood sample is drawn from the inside of the elbow or alternatively from the back of the hand. A total of six tubes are collected: three BD Vacutainer K2EDTA (10/10 mL) tubes, two BD Vacutainer SSTII Advanceserum gel tubes (10/8.5 mL) and one BD PAXgene Blood RNA (7/2.5 mL) tube (online supplemental figure 1). Processing will be done immediately after the samples have been taken. Serum tubes are allowed to clot at least 30 min (max. 60 min) before separating. EDTAplasma tubes do not clot but two EDTA tubes are also let sit for the same time as serum because of the easier workflow. Serum and two EDTAplasma tubes are centrifuged (1500g) for 10 min and the supernatants are pooled within serum and plasma. Samples are apportioned into 0.5 mL aliquots and stored at −20°C. One EDTA tube and PAXgene RNAtube will be stored as a whole blood at −20°C. Serum, plasma and RNA samples final storage temperature is at −80°C. One of the 0.5 mL EDTAplasma aliquot will be sent to University of Eastern Finland for biomarker analysis. RNA, EDTA whole blood samples, and half of the serum and plasma aliquots are dedicated to the twin study, while the other half will be available for research via THL Biobank. Blood-based AD biomarkers The primary bloodbased biomarkers include phosphorylatedtau181 (ptau181), phosphorylatedtau217 (ptau217), Aβ142/40, glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL); all measured using Simoa HDX Analyzer (Quanterix, Billerica, Massachusetts, USA). Plasma ptau181 levels are quantified using Simoa ptau181 Advantage V2.1 Kit (Ref# 104111, Quanterix),31 Aβ1–40, Aβ1–42, GFAP and NfL levels using Simoa Neurology 4Plex E Advantage Kit (Ref# 103670, Quanterix),32 and ptau217 levels using ALZpath Simoa pTau217 v2 Assay Kit (Ref# 104371, Quanterix).33 Prior to analyses, EDTA plasma samples are thawed, mixed and centrifuged (10 000×g, 5 min, +20°C). These biomarkers are determined at the Biomarker Laboratory of University of Eastern Finland. Apolipoprotein E genotype and polygenic risk scores Apolipoprotein E (APOE) status (ε4carrier vs noncarrier and number of ε4alleles) is defined by two singlenucleotide polymorphisms, rs429358 and rs7412, in chromosome 1934 and polygenic risk score (PRS; with and without APOE) of AD is based on the Bellenguez et al35 or newer metaanalysis if available. Genetic data will be used to calculate also PRS’s for diseases and traits (such as cardiovascular disease and educational attainment) that are related to risk and protective factors of dementia.36 Accelerometer-measured physical activity, sedentary behaviour and sleep A triaxial accelerometer (UKK RM42, UKK Terveyspalvelut Oy, Tampere, Finland) is used to monitor participants’ daily physical activity, sedentary behaviour and sleep for 7 consecutive days (table 4).37 In addition to total time spent in physical activity of different intensities and sedentary behaviour, we will also measure number of bouts and length of the bouts of physical activity and sedentary behaviour. The participants receive the devices during their inperson visit at FIMM or by mail if participating in other location. Participants are asked to wear the accelerometer on the hip during waking hours and on the Table 4 Core UKK RM42 accelerometer and Oura ring parameters UKK RM42 accelerometer Oura ring Target n=800 Target n=50 Participants from all sites Participants from Helsinki site Wearing time=1 week Wearing time=2 weeks Physical activity parameters Light physical activity Low intensity activity Moderate physical activity Medium intensity activity Vigorous physical activity High intensity activity Total physical activity Total physical activity Number of steps Number of steps Standing time Sedentary behaviour parameters Lying time Inactive time Reclining time Resting time Sitting time Number of breaks during sedentary time Sleep parameters Total sleep time Total sleep time Restless sleep time Restless sleep percentage Restful sleep time Total amount of deep sleep Total amount of rapid eye movement sleep Total amount of light sleep Protected by copyright. on June 12, 2024 at Jyvaskylan Ylioposto Kirjasto/Kausijulkaisut.http://bmjopen.bmj.com/BMJ Open: first published as 10.1136/bmjopen-2023-081947 on 12 June 2024. Downloaded from 8VuoksimaaE, etal. BMJ Open 2024;14:e081947. doi:10.1136/bmjopen-2023-081947 Open access wrist during sleep. At least a 4day monitoring period with a minimum of 10 hours weartime a day will be required for the adequate accelerometer data collection.38 The UKK RM42 device and its closely related counterpart Hookie AM 20 accelerometer have been used in samples with over 18 000 Finnish 18–85 years old adults.37 39 Thus, the UKK RM42 accelerometer is usable in the TWINGEN sample of 65–85 years old, and we will get an opportunity to compare the measurements against normative data of Finnish adults. The analyses of raw acceleration data of the UKK RM42 are based on validated algorithms; the technical details related to the recording and analysing of raw acceleration data are given elsewhere.40–42 Oura-measured physical activity, sedentary behaviour and sleep Oura ring (Gen3 Heritage, Ōura Health Ltd., Oulu, Finland) will be given to 50 participants in Helsinki study collection site. The number of participants asked to wear an Oura ring was an experimental pilot within our larger study. The target sample of 50 was purposed to evaluate the feasibility of a measurement requiring a smart phone app in older adults. First, participants use a ringsize kit to determine optimal ring size and then they receive the ring either at their inperson visit or by mail. Participants are asked to wear the ring (width: 7.9 mm, thickness: 2.55 mm, weight: 4–6 g) in any finger for 2 weeks during day and night, except when charging the ring every 4–6 days (20–80 min to fully charge). To monitor participant’s sleep, sedentary behaviour and physical activity (table 4), the Oura ring uses infrared photoplethysmography sensors, negative temperature coefficient sensor and 3D accelerometer. Participants receive written instructions on using the Oura ring and downloading the Oura mobile application, through which they can access their own data. If necessary, the research staff provides phone guidance for both using the ring and installing the application. The data from Oura ring is transferred to participant’s Oura application when opening the application and to a cloud server. Data collection will be monitored from Oura cloud server and participants’ will be sent a reminder if there are no data from previous 2 days. The Oura ring’s sleep stage detection algorithm (wake, light nonrapid eye movement (NREM) sleep, deep NREM sleep, rapid eye movement sleep) has been validated against polysomnography and it showed 80%–96% accuracy, 74%–82% sensitivity and 79%–98% specificity.43 Furthermore, moderatetovigorous intensity physical activity and step count of Oura ring has shown strong correlations with accelerometermeasured corresponding values.44 Questionnaire Participants are given a 16page selfreport questionnaire that includes many of the same measures as in previous questionnaires in years 1975, 1981, 1990 and 2011 (table 2). Questions cover anthropometrics, demographics, social relationships, chronotype, health (general, cardiovascular, dementia, memory, medications, vision, hearing, balance and mobility) and healthrelated behaviour including sleep, physical activity, smoking and alcohol use. Psychological wellbeing scales included in the questionnaire are: 8item Center for Epidemiologic Studies Depression scale45 46; 7item Purpose in Life subscale from Ryff’s Scales of Psychological WellBeing47 48; Extraversion (9 items) and Neuroticism (10 items) from the short version of Eysenck Personality Inventory49 50; and fouritem life satisfaction scale derived from questionnaires by Allardt.51 52 Patient and public involvement statement None. Aims, data analysis and future directions In addition to the overarching aim of assessing the feasibility of biobank recall in the context of preclinical AD, we also have more focused research questions (see online supplemental figure 2 for an overview of aims and associated data). In the initial stage of TWINGEN, we cannot make clinical or research diagnoses of AD, however, we will be able to follow these individuals through national health registry information that are compiled in FinnGen. Registrybased data will allow to predict progression to AD. Possible followup visits can also include gold standard measures for diagnosing AD, such as cerebrospinal fluid or positron emission tomography imaging. By combining all data, we aim to stratify our participants in subgroups of low, intermediate and high AD risk based on genetic, biomarker, cognitive, lifestyle and symptom data. The stratification will be based on a combination of percentiles or cutoffs (eg, Ashton et al33) for bloodbased biomarkers, cutoffs for cognitive impairment in CERAD,26 cCOG22 and TELE/TICS,16 lifestyle risk scores and subjective memory complaints. We also aim to use neuropsychological criteria for mild cognitive impairment classification where −1 SD performance in at least two tests are required independent of subjective memory complaints.53 54 Biomarkers and cognitive data allow to derive subgroups based on biomarker profile and cognitive stating included in the AT(N) framework.5 Additionally, we will use APOE status and PRS for genetic risk profiling although genetics are not included in the AT(N) framework. The stratification approaches are potentially useful for improving participant selection for AD drug and intervention trials. These methods would also be valuable in clinical settings where noninvasive and widely available tools for evaluating the presence of AD pathology underlying cognitive symptoms is important, especially once diseasemodifying treatments become available. TWINGEN will also establish a baseline cohort that can be used in followup studies with neuroimaging and cerebrospinal fluid biomarkers. We aim to assess the comparability of inperson, telephonebased and computerised cognitive assessments using correlation analysis of total scores and tests of different modalities assessing the same cognitive Protected by copyright. on June 12, 2024 at Jyvaskylan Ylioposto Kirjasto/Kausijulkaisut.http://bmjopen.bmj.com/BMJ Open: first published as 10.1136/bmjopen-2023-081947 on 12 June 2024. Downloaded from