Altered plasma protein profiles in genetic FTD – a GENFI study

Ullg ene al. Molecula Neu odegene a ion (2023) 18:85 h ps://doi.o g/10.1186/s13024-023-00677-6 RESEARCH ARTICLE Open Access © The Au ho (s) 2023. Open Access This a icle is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License, which pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons licence, and indica e i changes we e made. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons licence, unless indica ed o he wise in a c edi line o he ma e ial. I ma e ial is no included in he a icle’s C ea i e Commons licence and you in ended use is no pe mi ed by s a u o y egula ion o exceeds he pe mi ed use, you will need o ob ain pe mission di ec ly om he copy igh holde . To iew a copy o his licence, isi h p://c ea i ecommons.o g/licenses/by/4.0/. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecom‑ mons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed in a c edi line o he da a. Molecula Neu odegene a ion Al e ed plasma p o ein p o iles ingene ic FTD – aGENFI s udy Abbe Ullg en1,2,3†, Linn Öije s ed 1,2,3†, Jennie Olo sson1,4, So ia Be gs öm1,4, Julia Remnes ål1,4, John C. an Swie en5, Lize C. Jiskoo 5, Ha o Seelaa 5, Ba ba a Bo oni6, Raquel Sanchez‑Valle7, Fe min Mo eno8,9, Robe La o ce10, Ma his Syno zik11,12, Daniela Galimbe i13,14, James B. Rowe15, Ma io Masellis16, Ma ia Ca mela Ta aglia17, Elizabe h Finge 18, Rik Vandenbe ghe19,20,21, Alexand e de Mendonça22, Pie o Ti abosch23, Isabel San ana24,25, Simon Ducha me26,27, Ch is R. Bu le 28,29, Alexande Ge ha d30,31, Ma kus O o32, A abella Bouzigues33,34, Lucy Russell33,34, Imogen J. Swi 33,34, Ai ana Sogo b‑Es e e33,34, Ca olin Helle 33,34, Jona han D. Roh e 33,34, Anna Månbe g1,4, Pe e Nilsson1,4, Ca oline G a 1,2,3* and on behal o he Gene ic F on o empo al Demen ia Ini ia i e (GENFI) Abs ac Backg ound Plasma bioma ke s e lec ing he pa hology o on o empo al demen ia would add signi ican alue o clinical p ac ice, o he design and implemen a ion o ea men ials as well as ou unde s anding o disease mechanisms. The aim o his s udy was o explo e he le els o mul iple plasma p o eins in indi iduals om amilies wi h gene ic on o empo al demen ia. Me hods Blood samples om 693 pa icipan s in he GENe ic F on o empo al Demen ia Ini ia i e s udy we e ana‑ lysed using a mul iplexed an ibody a ay a ge ing 158 p o eins. Resul s We ound 13 ele a ed p o eins in symp oma ic mu a ion ca ie s, when compa ing plasma le els om peo‑ ple diagnosed wi h gene ic FTD o heal hy non‑mu a ion con ols and 10 p o eins ha we e ele a ed compa ed o p esymp oma ic mu a ion ca ie s. Conclusion We iden i ied plasma p o eins wi h al e ed le els in symp oma ic mu a ion ca ie s compa ed o non‑ ca ie con ols as well as o p esymp oma ic mu a ion ca ie s. Fu he in es iga ions a e needed o elucida e hei po en ial as luid bioma ke s o he disease p ocess. Keywo ds F on o empo al demen ia, Plasma bioma ke s, GRN, C9o 72, MAPT, Neu odegene a ion Backg ound F on o empo al demen ia (FTD) is a g oup o neu ode- gene a i e diseases whe e he mos common pheno ypes a e beha iou al a ian FTD (b FTD) and p ima y p o- g essi e aphasias (PPA). The e is a g ea he e ogenei y in FTD, bo h in e ms o clinical symp oms, unde lying gene ic causes, and neu opa hological indings. O e he pas yea s, e o has been pu in o explaining he di e - si y by sea ching o luid bioma ke s ha e lec di e en aspec s o FTD [1]. Mos e o s ha e ocused on inding bioma ke s in ce eb ospinal luid (CSF) and a ew p om- ising candida es ha e been ound, such as neu o ilamen ligh chain (NEFL) and neu onal pen axin 2 (NPTX2) [2, 3]. Howe e , he use o CSF bioma ke s is limi ed by he in asi e na u e o he sampling p ocedu e and es ic ed a ailabili y. The e o e, a eliable blood-based bioma ke †Abbe Ullg en and Linn Öije s ed sha ed i s au ho . *Co espondence: Ca oline G a ca oline.g a @ki.se Full lis o au ho in o ma ion is a ailable a he end o he a icle Page 2 o 12 Ullg ene al. Molecula Neu odegene a ion (2023) 18:85 would be ex emely aluable. A well-known blood-based bioma ke in gene ic FTD is p og anulin (GRN), which is educed in indi iduals wi h loss-o - unc ion mu a- ions in he gene wi h he same name [4]. While se um o plasma GRN le els can be used o con i m mu a ions in GRN, hey do no co ela e wi h clinically impo an me ics such as age a onse [4]. P e ious s udies ha e also iden i ied glial ib illa y acidic p o ein (GFAP), au and NEFL as possible plasma-based bioma ke s, whe e GFAP is ele a ed in symp oma ic GRN mu a ion ca i- e s, au is ele a ed in spo adic FTD and in symp oma ic MAPT mu a ion ca ie s, and NEFL is ele a ed in bo h gene ic and spo adic FTD [5–7]. Howe e , none o he p o eins a e speci ic o FTD since inc eased le els ha e been obse ed in o he neu ological diseases [8, 9]. A la ge sc een o plasma p o eins in FTD and Alzheime disease (AD) ound a panel o 12 p o eins ha disc imi- na ed be ween he wo diseases. Howe e , hese p o eins we e associa ed wi h AD pa hology and no di e ences we e ound be ween FTD cases and con ols [10]. Fu - he s udies a e he e o e needed o ind bioma ke s ha a e FTD speci ic. He e, we p esen an explo a o y plasma p o iling s udy o 158 p o eins in 693 pa icipan s in he well-desc ibed gene ic FTD coho . To ou knowledge, a plasma p o- eomic analysis o his magni ude has no been done in gene ic FTD be o e. We aimed o in es iga e di e ences in plasma p o ein le els be ween bo h symp oma ic and p esymp oma ic mu a ion ca ie s compa ed o non- ca ie amily membe s who se e as con ols. Ou ind- ings indica e al e a ions in plasma p o ein le els be ween symp oma ic mu a ion ca ie s and non-ca ie con ols, as well as gene speci ic di e ences in GRN mu a ion ca ie s. Ma e ials andme hods Coho All clinical da a and samples included in he s udy we e collec ed wi hin he Gene ic on o empo al demen ia ini ia i e (GENFI) be ween 2012 and 2019 [11]. Va i- ables included we e age a sampling, sex, mu a ion g oup (symp oma ic mu a ion ca ie s, SMC; p esymp oma ic mu a ion ca ie s, PMC; o non-ca ie con ols, NC), gene ic g oup (ch omosome 9 open eading ame 72, C9o 72; p og anulin, GRN; o mic o ubule associa ed p o ein au, MAPT), clinical pheno ype, and age a onse . In o al, baseline plasma samples om 701 pa icipan s we e collec ed including 141 SMC (63 C9o 72, 50 GRN, and 28 MAPT), 283 PMC (97 C9o 72, 135 GRN and 51 MAPT) and 277 NC. Ca ie s o FTD-causing a ian s in o he genes we e no included. Clinically, he SMC mos equen ly p esen ed wi h b FTD (n = 102), ollowed by PPA (n = 25), FTD wi h concomi an amyo ophic la e al scle osis (ALS) (n = 5) and o he FTD- ela ed pheno ypes (n = 5). Sample collec ion acco ding oGENFI p o ocol Blood samples (n = 701) we e collec ed a 20 di e en si es in Eu ope and Canada in e hylenediamine e aace ic acid (EDTA) ubes. Samples we e cen i uged a 2200 × g o 5min a 22°C and he supe na an plasma was ans- e ed o 0.5ml polyp opylene c yo ubes and s o ed a -80°C un il analysis. Suspension bead a ay assay The plasma samples we e dilu ed and labelled wi h a en- old mola excess o bio in (NHS-PEG4-bio in. 21329, The mo Scien i ic), hea ea ed, and subsequen ly mixed wi h an an ibody suspension bead a ay as desc ibed in de ail p e iously [12, 13]. A s ep a idin conjuga ed luo- opho e (S ep a idin R-Phycoe y h in Conjuga e, In - i ogen) enabled he de ec ion o he p o eins, and he eadou was pe o med on a Flexmap 3D ins umen (Luminex co po a ion). Binding e en s we e displayed as signal in ensi y. Published as well as in e nal unpublished wo k we e used o guide he selec ion o a ge p o eins (n = 163) which was based on p e iously iden i ied p om- ising a ge s, p o eins in ol ed in sugges ed pa hologi- cal p ocesses o neu odegene a ion and p o eins wi h en iched exp ession in b ain compa ed o o he issue [14–17]. The majo i y o he an ibodies (n = 156) we e selec ed om he Human P o ein A las p ojec (www. p o e ina l as. o g) and he emaining se en we e ob ained om o he p o ide s (M067-3 om MBL In e na ional; MA1-70053, PA5-34943, 34–1000 om In i ogen An i- bodies; MAB2037-SP, AF2420, AF3154 om R&D Sys- ems). The mean coe icien o a iance pe 384-well pla e (n = 3) was less han 10%, and 97% o he an ibod- ies had an indi idual coe icien o a iance below 20%. The in e -assay co ela ions we e high ( ho > 0.8 o 154 an ibodies). A e quali y con ol analysis, i e an ibodies we e excluded due o high co ela ion o a nega i e con- ol ( ho > 0.6) esul ing in 158 p o ein a ge s o u he analysis (Supplemen a y Table1). S a is ical analysis Da a p e‑p ocessing All da a p e-p ocessing, analysis and illus a ions we e pe o med in R S udio e sion 2022.2.3.492 using R e - sion 4.2.1 [18]. The da a was no malised in wo s eps o diminish he e ec s o ime delay du ing eadou and po en ial di e ences be ween pla es [19]. P io o s a is- ical analysis, he da a was log2- and z- ans o med ia mean cen ing and uni a iance scaling. Ou lie samples wi h a median p o ein le el h ee s anda d de ia ions highe o lowe han he median o he whole coho Page 3 o 12 Ullg ene al. Molecula Neu odegene a ion (2023) 18:85 we e excluded om he analysis (n = 8 samples emo ed). A esidual adjus men app oach was used o deal wi h he po en ial con ounding e ec o heal hy ageing on p o ein le els [20]. The e ec o heal hy ageing on p o ein le els was es ima ed in he NC ia linea mixed e ec models using p o ein le els as he esponse a iable, age as a ixed e ec and collec ion si e as a andom in e cep (lme , lme4, [21]). Fo each subjec in he o e all coho (including each o he NC, PMC, and SMC g oups), he adjus ed p o ein le els we e hen ob ained h ough he ollowing: whe e P o einadj. is he age adjus ed p o ein le el, P o ein is he o iginal p o ein le el, β is he age-associa ed be a coe icien , Age is he subjec ’s age and Age is he mean age in he en i e coho . Demog aphic s a is ics The pa icipan s’ ages ollowed a no mal dis ibu ion, e alua ed by isual assessmen o no mal p obabili y plo and his og am. Di e ences in age be ween SMC, PMC and NC we e assessed by one-way ANOVA and Tukey’s HSD pos hoc es . Pea son’s Chi-squa ed es was used o in es iga e di e ences in sex be ween SMC, PMC and NC. P- alues below 0.05 we e conside ed signi ican . P o ein p o ile analysis Di e ences in p o ein le els be ween SMC and NC we e examined ia binomial gene alised linea mixed e ec s models using clinical s a us (i.e. SMC o NC) as he esponse a iable, p o ein le els and sex as ixed e ec s P o einadj .=P o ein −β(Age −Age ) wi h a andom in e cep based on collec ion si e (glme , lme4, [21]). One model pe p o ein was buil . The same me hod was used o assess di e ences in p o ein le - els be ween SMC and p esymp oma ic mu a ion ca i- e s (PMC), PMC e sus NC, as well as o analyse gene speci ic di e ences e.g., SMC ca ying a GRN mu a ion (SMC-GRN) s NC. Log2 old changes we e calcula ed by sub ac ing he median log2 ans o med p o ein le - els in NC o PMC om he median log2 ans o med p o ein le els in SMC. In con as , he e ec s o age and sex on he p o ein le els in mu a ion ca ie s as well as in non-ca ie s we e es ima ed ia gene alised linea mixed- e ec s models using p o ein le els as he esponse; age and sex as ixed e ec s wi h a andom in e cep based on collec ion si e (lme , lme4, [21]). P- alues we e calcula ed using he Sa e hwai e’s deg ees o eedom me hod (lme Tes , [22]). Mul iple es ing co ec ions we e made ia he Benjamini–Hochbe g me hod o con olling alse-disco e y a es and an adjus ed p- alue o 0.05 was conside ed signi ican . Only adjus ed p- alues a e epo ed unless clea ly s a ed o he wise. P o ein – p o- ein co ela ions we e calcula ed using Spea man’s ank co ela ion coe icien . P o ein clus e s a e based on hie - a chical clus e ing using Wa d’s clus e ing c i e ion. Resul s Coho In o al, plasma esul s om 693 pa icipan s we e included in he s a is ical analysis, and demog aphic da a o he coho is p esen ed in Table 1. The age was no signi ican ly di e en be ween NC and PMC (p- alue = 0.06), bu SMC we e olde han bo h NC Table 1 Demog aphic da a o he coho a One-way ANOVA (F(2,690) = 106.0, p = 7.13 × 10–41). Di e ences in age be ween NC s SMC and PMC s SMC (Tukey mul iple compa ison pos -hoc es ). No di e ence be ween NC and PMC (p = 0.06) b Pea son’s Chi-squa ed es . Mo e emales in NC and PMC compa ed o SMC (X2(1, N = 413) = 6.1, p = 1.3 × 10–2) and X2(1, N = 417) = 14.9, p = 1.1 × 10–4 espec i ely). No di e ence be ween NC and PMC (X2(1, N = 556) = 2.6, p = 0.1) Non-ca ie s (NC) P esymp oma ic mu a ion ca ie s (PMC) Symp oma ic mu a ion ca ie s (SMC) To al p- alue No. o pa icipan s 276 280 137 693 Age, mean yea s (SD) 47 (14) 45 (12) 63 (9) 49 (14) < 0.001a Females (%) 152 (55) 174 (62) 57 (42) 383 (55) < 0.001b Mu a ed gene (%) 417 (60) GRN 133 (48) 49 (36) C9o 72 96 (34) 62 (45) MAPT 51 (18) 26 (19) Age a onse , mean yea s (SD) GRN 61 (8) C9o 72 60 (9) MAPT 51 (8) Page 4 o 12 Ullg ene al. Molecula Neu odegene a ion (2023) 18:85 and PMC (p- alue = 7.13 × 10–41). The e we e mo e emales in NC (55%) and PMC (62%) compa ed o SMC (42%) (NC s SMC: p- alue = 1.3 × 10–2; PMC s SMC: p- alue = 1.1 × 10–4). Al e ed plasma p o ein le els insymp oma ic mu a ion ca ie s When compa ing plasma p o ein le els in SMC o NC, we ound ha 13 p o eins we e ele a ed in SMC (Fig. 1A, Table 2 and Supplemen a y Fig. 1). In he compa ison be ween SMC e sus PMC, we ound ha 10 p o eins we e ele a ed in SMC (Fig.1B, Table2). The e we e six o e lapping p o eins i.e., six p o eins had ele a ed le els in SMC bo h in he compa ison o NC as well as in he compa ison o he p o ein le els in PMC. An o e iew o how hese p o eins co ela e wi h each o he can be ound in Fig.2. When s a i ying by gene ic g oup, abphilin 3a (RPH3A) was inc eased in SMC-GRN compa ed o NC (p- alue = 1.3 × 10–3, odds a io = 1.915) whe eas p og anulin, as expec ed, was dec eased (p- alue = 9.3 × 10–6, odds a io = 0.152). No p o eins had signi ican ly di e en le els in he com- pa ison be ween SMC-C9 and NC no in he compa i- son be ween SMC-MAPT and NC (da a no shown). Nex , we in es iga ed he co ela ion be ween age and p o ein le els o he al e ed p o eins in mu a ion ca ie s. Among he 17 unique p o eins wi h ele a ed le els in SMC (SMC s NC o SMC s PMC), 13 had a signi ican co ela ion wi h age (Table3). When analysing sex di e ences o he 17 p o- eins ele a ed in SMC, wo p o eins we e ound o be inc eased in men compa ed o women in he mu a ion ca ie s: apolipop o ein E (APOE, p- alue = 1.73 × 10–2, β = 0.267) and apolipop o ein C1 (APOC1, p- alue = 1.73 × 10–2, β = 0.235). No sex di e ences we e ound among he NC o ei he Fig. 1 Volcano plo s o plasma p o ein le els showing ‑log10 (p‑ alues) o he log2( old change) o compa isons be ween di e en g oups. Plasma p o ein le el di e ences be ween A) all SMC and NC, B) all SMC and all PMC. Each p o ein is ep esen ed by a g ay do and a e colou ed ed i he p o ein le els a e inc eased in he SMC compa ed o he compa ison g oup (NC, o PMC). Do ed ho izon al line = adjus ed p‑ alue 0.05, do ed e ical line = log2 old change 0 Page 5 o 12 Ullg ene al. Molecula Neu odegene a ion (2023) 18:85 p o ein (p- alue = 1.49 × 10–1 and p- alue = 7.4 × 10–1, espec i ely). Finally, we explo ed i any o he 158 p o eins included in his s udy we e ound a di e en le els in SMC wi h b FTD compa ed o SMC wi h PPA bu ound no signi i- can di e ences (da a no shown). Plasma p o ein le els in hep esymp oma ic s age We also in es iga ed he possibili y o de ec di e ences in p o ein le els al eady a he p esymp oma ic s age o FTD by i s compa ing PMC o NC and hen s a i y- ing by gene. The only di e ence ound in hese com- pa isons was dec eased le els o GRN in PMC-GRN compa ed o NC (p- alue = 4.44 × 10–3). Howe e , he p o eins neu o ilamen medium chain (NEFM), neu onal pen axin 2 (NPTX2) and chi inase 3 like 1 (CHI3L1) showed ends o being ele a ed in PMC-GRN com- pa ed o NC (unadjus ed p- alue = 3.1 × 10–3, unadjus ed p- alue = 4.8 × 10–3 and unadjus ed p- alue = 4.6 × 10–3, espec i ely), hough hese di e ences we e no signi i- can a e adjus men o mul iple es ing. None o he h ee p o eins showed any co ela ion wi h age when analysed in PMC-GRN alone o in PMC-GRN oge he wi h SMC-GRN (Supplemen a y Table2). NPTX2 was, howe e , ele a ed in SMC compa ed o NC and CHI3L1 was jus abo e he signi icance h eshold in he same compa ison (p- alue = 5.1 × 10–2). Nei he we e signi i- can in he compa ison be ween SMC and PMC (bo h p- alues > 0.3). Discussion We pe o med ex ensi e p o ein p o iling o plasma om a gene ic FTD coho , collec ed wi hin he GENFI s udy. We ound 13 signi ican ly inc eased plasma p o eins in pa ien s wi h gene ic FTD compa ed o non-ca ie con- ols and 10 p o eins ha we e signi ican ly inc eased compa ed o p esymp oma ic mu a ion ca ie s. Six o hese p o eins we e signi ican ly di e en in bo h com- pa isons, indica ing ha hey likely a e associa ed wi h symp om onse a he han he p esence o one o he pa hogenic mu a ions. These six p o eins we e also sig- ni ican ly co ela ed wi h inc eased age in mu a ion ca - ie s, a e co ec ing o heal hy ageing, which u he s eng hens hei associa ion wi h symp om onse . In con as , ou p o eins, inc eased in SMC s NC, we e no co ela ed wi h age, no ele a ed in he SMC s PMC compa ison, sugges ing ha hey may be ele a ed al eady be o e symp om onse . One o hese p o eins NPTX2, is o pa icula in e es . NPTX2, a synap ic p o- ein, which has p e iously been shown o be educed in CSF om pa ien s wi h FTD and is po en ially one o he i s p o ein bioma ke s o become abno mal in gene ic Table 2 Compa ison o plasma p o ein le els in symp oma ic mu a ion ca ie s e sus non‑ca ie s and p esymp oma ic mu a ion ca ie s P o eins wi h s a is ically signi ican di e en plasma le els in he compa ison be ween symp oma ic mu a ion ca ie s (SMC) and non-ca ie s (NC) o in he compa ison be ween SMC and p esymp oma ic mu a ion ca ie s (PMC), including p- alues and odds a ios wi h 95% con idence in e als. All p- alues a e adjus ed o mul iple es ing. Non-signi ican p- alues a e in i alics. An as e isk indica es p o eins wi h signi ican ly di e en plasma le els in bo h compa isons P o ein SMC s NC SMC s PMC p- alue Odds a io p- alue Odds a io RPH3A* 2.68 × 10–2 1.535 (1.228—1.919) 2.27 × 10–21.46 (1.177—1.811) NPTX2 2.93 × 10–2 1.499 (1.2—1.874) 3.52 × 10–1 1.176 (0.964—1.433) XPO5 3.73 × 10–2 1.598 (1.216—2.1) 1.29 × 10–1 1.322 (1.056—1.656) RGS7BP* 3.73 × 10–2 1.504 (1.181 – 1.915) 2.27 × 10–2 1.619 (1.246 – 2.103) APOE* 3.73 × 10–2 1.532 (1.179—1.992) 3.94 × 10–2 1.516 (1.165—1.972) S100A12* 3.73 × 10–2 1.5 (1.173—1.919) 3.94 × 10–2 1.394 (1.126—1.725) GLA 3.87 × 10–2 1.605 (1.194—2.158) 4.25 × 10–1 1.185 (0.938—1.498) APOC1* 4.06 × 10–2 1.627 (1.194—2.218) 3.94 × 10–2 1.651 (1.2—2.271) EIF4ENIF1 4.60 × 10–2 1.503 (1.144—1.976) 3.31 × 10–1 1.264 (0.976—1.636) LCAT 4.60 × 10–2 1.414 (1.128—1.773) 8.46 × 10–2 1.336 (1.075—1.662) C7 4.60 × 10–2 1.49 (1.14—1.948) 3.08 × 10–1 1.277 (0.981—1.663) CHGA 4.60 × 10–2 1.458 (1.132—1.877) 5.96 × 10–2 1.436 (1.111—1.855) ADAMTS1* 5.00 × 10–2 1.404 (1.113—1.77) 3.11 × 10–2 1.564 (1.198—2.04) TFEB 7.47 × 10–2 1.362 (1.082—1.715) 2.27 × 10–2 1.555 (1.229—1.967) LRRFIP2 7.47 × 10–2 1.344 (1.079—1.674) 3.94 × 10–2 1.44 (1.141—1.818) LAMA2 9.31 × 10–2 1.327 (1.057—1.667) 2.27 × 10–2 1.57 (1.219—2.022) IL1B 1.69 × 10–1 1.308 (1.018—1.681) 3.94 × 10–2 1.576 (1.173—2.117) Page 6 o 12 Ullg ene al. Molecula Neu odegene a ion (2023) 18:85 FTD [3, 23, 24]. We ha e, as o ye , no explana ion o why NPTX2 is educed in CSF and ele a ed in plasma, o i he NPTX2 de ec ed in plasma is b ain de i ed. How- e e , inding ele a ed le els o NPTX2 in plasma om SMC sugges s ha NPTX2 could wo k as a plasma-based bioma ke . We ound wo p o eins, GRN and RPH3A, ha di - e ed in SMC-GRN compa ed o NC, while no p o eins we e obse ed a di e en le els in nei he SMC-MAPT no SMC-C9, compa ed o NC. A educ ion o GRN in p og anulin mu a ion ca ie s is o cou se expec ed since all known pa hogenic FTD- ela ed GRN mu a ions lead o haploinsu iciency. On he o he hand, RPH3A was ele a ed in SMC-GRN. RPH3A is in ol ed in p esynap ic esicle a icking and has been implica ed o play a ole in synap ic dys unc ion in o he neu odegene a i e dis- eases [25, 26]. In addi ion o he indings in SMC-GRN, we obse ed some indica ions o di e ences al eady in he p esymp oma ic s ages in GRN mu a ion ca i- e s. While no s a is ically signi ican a e adjus men o mul iple es ing, he di e ences a e s ill no ewo hy since he p o eins, NEFM, NPTX2 and CHI3L1, all ha e been epo ed as bioma ke candida es in CSF [16, 27]. NPTX2 was also ele a ed in all SMC compa ed o NC and CHI3L1 was jus abo e he h eshold o signi icance while nei he o hese wo p o eins we e ele a ed in SMC when compa ed o PMC. Taken oge he his indica es ha hese wo p o eins migh be up egula ed al eady a he p esymp oma ic s age. Howe e , u he s udies a e needed o es ablish i hese p o eins indeed a e ela ed o p esymp oma ic changes in GRN mu a ion ca ie s o i i is a spu ious inding. Biological sex is a known isk ac o o se e al ypes o demen ia, wi h emale sex being a isk ac o o AD and male sex being mo e common in FTD [28, 29]. In ligh o his, we analysed i any o he p o eins iden i ied in he cu en s udy exhibi ed any sex speci ic pa e ns. While we could no de e mine any signi ican in e ac- ions be ween sex and mu a ion s a us o hese p o eins (da a no shown), wo p o eins we e signi ican ly co e- la ed wi h sex in he mu a ion ca ie g oup, bu no in con ols, sugges ing a po en ial biological e ec . We acknowledge se e al limi a ions in his s udy. The ocus was on gene ic FTD and samples om pa ien s Fig. 2 Plo s o p o ein – p o ein co ela ions. P o ein o de is based on hie a chical clus e ing. Co ela ion plo o he 17 p o eins wi h ele a ed le els in symp oma ic mu a ion ca ie s (SMC) compa ed o non‑ca ie s (NC) o p esymp oma ic mu a ion ca ie s (PMC). The colou scale indica es Spea man’s ank co ela ion coe icien om da k blue (‑1) o b igh ed (1) Page 7 o 12 Ullg ene al. Molecula Neu odegene a ion (2023) 18:85 wi h o he neu odegene a i e diseases we e no included in he analysis. Follow-up s udies wi h com- pa isons o o example AD and ALS will elucida e he impo ance o al e ed plasma p o eins in FTD in ela- ion o o he diseases as well as spo adic FTD. The sus- pension bead a ay echnique is a me hod o analysing mul iple p o eins simul aneously, which is use ul in an explo a o y s udy like his. Howe e , a high- h oughpu an ibody-based single-binde assay can ha e educed sensi i i y, which may limi he de ec ion o low abun- dan p o eins and equi e u he alida ion o an ibody speci ici y. In addi ion, we used a a ge ed app oach, and he p o ein analysis was hus limi ed by he p o ein selec ion as well as he a ailabili y o an ibodies. Conclusions To ou knowledge, his is he i s la ge scale plasma p o ein p o iling speci ically in gene ic FTD. A eli- able luid bioma ke could aid o example in diagnos- ing FTD a an ea ly s age o in selec ing indi iduals o upcoming clinical ials. Blood-based bioma ke s would ha e he ad an age o being easy o access and widely a ailable compa ed o CSF-bioma ke s. He e, we ha e p esen ed an explo a o y s udy p o iding p o eins, including a p e ious CSF-bioma ke , ha a e o in e es o u u e in es iga ions as po en ial bioma ke s. Supplemen a y In o ma ion The online e sion con ains supplemen a y ma e ial a ailable a h ps:// doi. o g/ 10. 1186/ s13024‑ 023‑ 00677‑6. Addi ional ile1: Supplemen a y Table1. An ibodies used in he suspension bead a ay plasma analysis. Supplemen a y Table2. P o eins wi h di e en le els in PMC compa ed o NC. Supplemen a y Figu e1. Boxplo s o he 13 p o eins ha di e ed be ween SMC and NC. Sup- plemen a y Figu e2. Boxplo s o he 10 p o eins ha di e ed be ween SMC and PMC. Acknowledgemen s Fi s , we would like o hank all he pa icipan s and hei amilies o con ib‑ u ing o he s udy. We would also like o hank he GENFI esea ch coo dina‑ o s who helped wi h a anging he isi s and he en i e s a o he Human P o ein A las o hei e o s. Gene ic F on o empo al Demen ia Ini ia i e (GENFI) collabo a ion g oup Au ho A ilia ion Sónia A onso Ins i u o Ciencias Nuclea es Aplica‑ das a Saude. Uni e sidade de Coimb a. Coimb a. Po ugal Ma ia Rosa io Almeida Facul y o Medicine. Uni e si y o Coimb a. Coimb a. Po ugal Sa ah Ande l‑S aub Depa men o Neu ology. Uni e si y o Ulm. Ulm. Ge many Ch is in Ande sson Depa men o Clinical Neu oscience. Ka olinska Ins i u e . S ockholm. Sweden Anna An onell Alzheime ’s disease and O he Cogni‑ i e Diso de s Uni . Neu ology Se ice. Hospi al Clínic. Ba celona. Spain And ea A ighi Fondazione IRCCS Ca’ G anda Ospedale Maggio e Poli‑ clinico. Neu odegene a i e Diseases Uni . Milan. I aly; Uni e si y o Milan. Cen o Dino Fe a i. Milan. I aly Mi cea Balasa Alzheime ’s disease and O he Cogni‑ i e Diso de s Uni . Neu ology Se ice. Hospi al Clínic. Ba celona. Spain My iam Ba andia an Cogni i e Diso de s Uni . Depa men o Neu ology. Donos ia Uni e si y Hos‑ pi al. San Sebas ian. Gipuzkoa. Spain; Neu oscience A ea. Biodonos ia Heal h Resea ch Insi u e. San Sebas ian. Gipuzkoa. Spain Nu ia Ba galló Imaging Diagnos ic Cen e . Hospi‑ al Clínic. Ba celona. Spain Roba Ba ha Depa men o Medical Biophysics. The Uni e si y o Wes e n On a io. London. On a io. Canada; Cen e o Func ional and Me abolic Map‑ ping. Roba s Resea ch Ins i u e. The Uni e si y o Wes e n On a io. London. On a io. Canada Benjamin Bende Depa men o Diagnos ic and In e ‑ en ional Neu o adiology. Uni e si y o Tübingen. Tübingen. Ge many Emanuele Bu a i ICGEB T ies e, I aly Luisa Benussi Is i u o di Rico e o e Cu a a Ca a ‑ e e Scien i ico Is i u o Cen o San Gio anni di Dio Fa ebene a elli. B escia. I aly Maxime Be oux Inse m 1172. Lille. F ance Table 3 Co ela ions be ween age and p o ein le els in mu a ion ca ie s Co ela ions be ween p o ein le els and age in all mu a ion ca ie s (MC) including p- alues and be a coe icien s wi h 95% con idence in e als. All p- alues a e adjus ed o mul iple es ing. Non-signi ican p- alues a e in i alics P o ein p‑ alue β RPH3A 9.98 × 10–7 0.019 (0.013—0.026) IL1B 2.16 × 10–4 0.012 (0.006—0.017) RGS7BP 5.35 × 10–4 0.012 (0.006—0.018) TFEB 5.35 × 10–4 0.013 (0.006—0.019) S100A12 1.04 × 10–3 0.013 (0.006—0.02) GLA 4.18 × 10–3 0.01 (0.004—0.016) EIF4ENIF1 5.22 × 10–3 0.008 (0.003—0.014) APOE 5.22 × 10–3 0.009 (0.003—0.015) CHGA 5.22 × 10–3 0.009 (0.003—0.015) LRRFIP2 5.22 × 10–3 0.011 (0.004—0.018) ADAMTS1 5.25 × 10–3 0.009 (0.003—0.015) LAMA2 2.15 × 10–2 0.008 (0.002—0.014) APOC1 3.09 × 10–2 0.006 (0.001—0.011) XPO5 1.19 × 10–1 0.006 (-0.001—0.013) LCAT 1.57 × 10–1 0.005 (-0.002—0.012) NPTX2 1.68 × 10–1 0.005 (-0.002—0.013) C7 3.73 × 10–1 0.003 (-0.003—0.008) Page 8 o 12 Ullg ene al. Molecula Neu odegene a ion (2023) 18:85 Giuliano Bine i Is i u o di Rico e o e Cu a a Ca a ‑ e e Scien i ico Is i u o Cen o San Gio anni di Dio Fa ebene a elli. B escia. I aly Sand a Black Sunnyb ook Heal h Sciences Cen e. Sunnyb ook Resea ch Ins i u e. Uni e ‑ si y o To on o. To on o. Canada Ma ina Bocche a Depa men o Neu odegene a i e Disease. Demen ia Resea ch Cen e. UCL Ins i u e o Neu ology. Queen Squa e. London. UK Se gi Bo ego‑Ecija Alzheime ’s disease and O he Cogni‑ i e Diso de s Uni . Neu ology Se ice. Hospi al Clínic. Ba celona. Spain Jose B as Cen e o Neu odegene a i e Science. Van Andel Ins i u e. G and Rapids. Michigan. MI 49503. USA Rose B u ae s Labo a o y o Cogni i e Neu ology. Depa men o Neu osciences. KU Leu en. Leu en. Belgium Ma a Cañada CITA Alzheime . San Sebas ian. Gipuz‑ koa. Spain Valen ina Can oni Cen e o Neu odegene a i e Diso de s. Neu ology Uni . Depa ‑ men o Clinical and Expe imen al Sciences. Uni e si y o B escia. B escia. 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Canada An onella Albe ici Cen e o Neu odegene a i e Diso de s, Depa men o Clinical and Expe imen al Sciences, Uni e si y o B escia, B escia, I aly Gio gio Giaccone Fondazione IRCCS Is i u o Neu o‑ logico Ca lo Bes a. Milano. I aly Ana Go os idi Neu oscience A ea. Biodonos ia Heal h Resea ch Insi u e. San Sebas ian. Gipuzkoa. Spain Ca oline G ea es Depa men o Neu odegene a i e Disease. Demen ia Resea ch Cen e. UCL Ins i u e o Neu ology. Queen Squa e. London. UK Ri a Gue ei o Cen e o Neu odegene a i e Science. Van Andel Ins i u e. G and Rapids. Michigan. MI 49503. USA Ca olin Helle Depa men o Neu odegene a i e Disease. Demen ia Resea ch Cen e. UCL Ins i u e o Neu ology. Queen Squa e. London. UK Begoña Indakoe xea Cogni i e Diso de s Uni . Depa men o Neu ology. Donos ia Uni e si y Hos‑ pi al. San Sebas ian. Gipuzkoa. Spain; Neu oscience A ea. Biodonos ia Heal h Resea ch Insi u e. San Sebas ian. Gipuzkoa. Spain Vesna Jelic Di ision o Clinical Ge ia ics. Ka olin‑ ska Ins i u e . S ockholm. Sweden Hans‑O o Ka na h Di ision o Neu opsychology. He ie‑ Ins i u e o Clinical B ain Resea ch and Cen e o Neu ology. Uni e si y o Tübingen. Tübingen. Ge many Ron Ke en The Uni e si y Heal h Ne wo k. To on o Rehabili a ion Ins i u e. To on o. Canada G ego y Kuchcinski Uni Lille. F ance Tobias Langhein ich Di ision o Neu oscience and Expe i‑ men al Psychology. Wol son Molecula Imaging Cen e. Uni e si y o Man‑ ches e . Manches e . UK Thibaud Lebou ie Uni Lille. F ance Ma ia João Lei ão Cen e o Neu osciences and Cell Biology. Uni e sidade de Coimb a. Coimb a. Po ugal Page 9 o 12 Ullg ene al. Molecula Neu odegene a ion (2023) 18:85 Albe Lladó Alzheime ’s disease and O he Cogni‑ i e Diso de s Uni . Neu ology Se ice. Hospi al Clínic. Ba celona. Spain Ca olina Ma u a Labo a o y o Language Resea ch. Cen o de Es udos Egas Moniz. Facul y o Medicine. Uni e si y o Lisbon. Lisbon. Po ugal Simon Mead MRC P ion Uni . Depa men o Neu‑ odegene a i e Disease. UCL Ins i u e o Neu ology. Queen Squa e. 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UK Ch is en Shoesmi h Depa men o Clinical Neu ologi‑ cal Sciences. Uni e si y o Wes e n On a io. London. On a io. Canada Miguel Tábuas‑Pe ei a Neu ology Depa men . Cen o Hos‑ pi ala e Uni e si a io de Coimb a. Coimb a. Po ugal; Facul y o Medi‑ cine, Uni e si y o Coimb a, Coimb a, Po ugal Mikel Tain a Neu oscience A ea. Biodonos ia Heal h Resea ch Insi u e. San Sebas ian. Gipuzkoa. Spain Rica do Taipa Neu opa hology Uni and Depa men o Neu ology. Cen o Hospi ala do Po o—Hospi al de San o An ónio. Opo o. Po ugal Da id Tang‑Wai The Uni e si y Heal h Ne wo k. K em‑ bil Resea ch Ins i u e. To on o. Canada Da id L Thomas Neu oimaging Analysis Cen e. Depa men o B ain Repai and Reha‑ bili a ion. UCL Ins i u e o Neu ology. Queen Squa e. London. UK Paul Thompson Di ision o Neu oscience and Expe i‑ men al Psychology. Wol son Molecula Imaging Cen e. Uni e si y o Man‑ ches e . Manches e . UK Håkan Thonbe g Cen e o Alzheime Resea ch. Di i‑ sion o Neu oge ia ics. Ka olin‑ ska Ins i u e . S ockholm. Sweden