1 68 2 69 3 70 4 Alzheimer’s & Dementia - (2017) 1-9 71 5 72 6 73 7 74 8 Featured Article 75 9 76 10 Neuropathologic features of TOMM40 ’523 variant on late-life cognitive 77 11 78 12 decline 79 13 80 14 81 15 a,b, c a,d a,b,e f 82 Q11 Lei Yu *, Michael W. Lutz , Jose M. Farfel , Robert S. Wilson , Daniel K. Burns , 16 f g,h a,b,e a,b 83 17 Ann M. Saunders , Philip L. De Jager , Lisa L. Barnes , Julie A. Schneider , 84 18 David A. Bennetta,b 85 19 a 86 Q1 Rush Alzheimer’s Disease Center, Rush University Medical Center, Chicago, IL, USA 20 b 87 21 Department of Neurological Sciences, Rush University Medical Center, Chicago, IL, USA 88 cDepartment of Neurology, Duke University School of Medicine, Durham, NC, USA 22 d 89 23 Discipline of Geriatrics, Faculty of Medicine of the University of Sao Paulo, Brazil 90 eDepartment of Behavioral Sciences, Rush University Medical Center, Chicago, IL, USA 24 f 91 25 Zinfandel Pharmaceuticals, Inc., Research Triangle Park, NC, USA 92 gCenter for Translational and Systems Neuroimmunology, Department of Neurology, Columbia University Medical Center, New York, NY, USA 26 h 93 27 Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA 94 28 95 29 96 30 Abstract Introduction: The study investigated the role of neuropathologies in the relationship between 97 31 TOMM40 ’523 genotype and late-life cognitive decline. 98 32 Methods: Participants were community-dwelling older persons who had annual cognitive assess- 99 33 ments and brain autopsies after death. Genotyping used DNA from peripheral blood or postmortem 100 34 101 brain tissue. Linear mixed models assessed the extent to which the association of ’523 genotype with 35 102 36 cognitive decline is attributable to neuropathologies. 103 ε 37 Results: Relative to 3/3 homozygotes with ’523-S/VL or ’523-VL/VL genotype, both ’523-L 104 38 carriers and ε3/3 homozygotes with ’523-S/S genotype had faster cognitive decline. The association 105 39 of ’523-L with cognitive decline was attenuated and no longer significant after controlling for 106 40 Alzheimer’s and other neuropathologies. By contrast, the association of ’523-S/S was unchanged. 107 41 Discussion: There are two distinct TOMM40 ’523 signals in relation to late-life cognitive decline. 108 42 One signal primarily acts through AD and other common neuropathologies, whereas the other 109 43 operates through a different mechanism. 110 44 Ó 2017 the Alzheimer’s Association. Published by Elsevier Inc. All rights reserved. 111 45 112 46 113 47Q3 Keywords: Neuropathologies; TOMM40 ’523; Late-life cognitive decline 114 48 115 49 116 50 117 51 118 52 1. Introduction 119 53 Apolipoprotein E (APOE) is the best-known susceptibil- 120 54 Q4 121 Dr. Lutz receives consulting fees from Zinfandel Pharmaceuticals, Inc. ity gene for late-onset Alzheimer disease (AD) [1]. The 55 and is supported, in part, by NIH grant 5R01AG040370-03. Dr. Burns is 122 56 an employee of Zinfandel Pharmaceuticals, Inc. Dr. Saunders is the CEO region on chromosome 19 that harbors the APOE gene 123 57 of Zinfandel Pharmaceuticals, Inc. Dr. Saunders is a member of the includes a large haplotype block that contains several other 124 58 Biomarker Committee for the Zinfandel-Takeda Alliance and the genes including apolipoprotein C1 (APOC1) and translocase 125 59 TOMORROW clinical trial. Dr. Schneider has served as a consultant for of outer mitochondrial membrane 40 (TOMM40) [2]. Beside 126 60 127 Navidea Biopharmaceuticals Inc., Eli Lilly Inc., Genetech Inc., and Michael APOE ε alleles, multiple genetic variations within the block 61 J Fox Foundation. Dr. Bennett serves on the adjudication committee for 128 TOMM40 62 AD4833/TOMM40_301 study funded by Takeda Pharmaceuticals USA, Inc. have also been implicated in AD [3–7]. ’523, a 129 63 130 Q2 *Corresponding author. Tel.: ---; Fax: ---. poly-T polymorphism at an intronic region of TOMM40,is 64 E-mail address: [email protected] of particular interest. The variable length of poly-T repeat 131 65 132 66 http://dx.doi.org/10.1016/j.jalz.2017.05.002 133 67 1552-5260/Ó 2017 the Alzheimer’s Association. Published by Elsevier Inc. All rights reserved. 134

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135 at ’523 locus is associated with age at disease onset and and an Anatomical Gift Act were provided by each partici- 202 136 cognition [7–9]. Notably, the variant is in linkage pant. By early January 2017, 1501 participants of European 203 137 204 138 disequilibrium (LD) with APOE genotype. Among ancestry had died, of which 1326 had undergone brain 205 139 Caucasians, APOE ε4 is exclusively linked to the long autopsy (autopsy rate 5 88.3%). The present study focused 206 140 (’523-L) poly-T repeat whereas ε3 can be linked to either on individuals who had genotype data and longitudinal 207 141 short (’523-S) or very long (’523-VL) poly-T repeat. This cognitive assessments (N 5 1114). The mean age at death 208 142 LD structure has two implications. The close linkage be- was 89.4 years (standard deviation [SD] 5 6.4), 66.6% 209 143 ε 5 210 144 tween 4 and ’523-L raises the question whether TOMM40 were females (N 742), and the mean education was 211 145 ’523-L is merely a proxy of ε4 [10]. By contrast, three major 16.3 years (SD 5 3.6). 212 146 ’523 genotypes are present in APOE ε3/3 homozygotes, 213 147 namely ’523-S/S, ’523-S/VL and ’523-VL/VL. An earlier 2.2. APOE and TOMM40 ’523 genotyping 214 148 study found that age at AD onset varies across these three ge- 215 149 216 notypes [11], and we recently reported that ’523-S/S carriers DNA was extracted from peripheral blood and in some 150 cases from frozen postmortem brain tissue. The genotyping 217 151 exhibit faster decline in late-life cognition compared with 218 152 ’523-S/VL or ’523-VL/VL carriers [12]. These findings sug- were performed at Polymorphic DNA Technologies 219 153 gest that the ’523 effect is not fully attributable to the LD (Alameda, CA). The vendor was blinded to all clinical and 220 154 with APOE variants. However, the neurobiologic or patho- neuropathologic information. APOE genotype was based 221 155 222 biologic basis underlying these associations remains unclear. on two polymorphisms (rs429358 and rs7412) at exon 4 of 156 the APOE gene. TOMM40 ’523 refers to rs10524523, a 223 157 Evidence suggests that the ε4 allele is directly involved in 224 158 the pathogenesis of AD via regulating b-amyloid accumula- homopolymer length polymorphism (poly-T) at intron 6 of 225 159 tion, a key neuropathologic feature of the disease [13–15].In the TOMM40 gene (chr19:44,899,792-44,899,826, human 226 160 this study, we aimed to determine whether the ’523 effect genome reference assembly GRCh38/hg38). The ’523 geno- 227 161 228 was related to AD or other neuropathologies among type was determined by the length of poly-T repeat, as 162 previously described [20]. Briefly, a ’523 short allele 229 163 persons with APOE ε3/3. 230 164 To examine the role of common neuropathologies in the (’523-S) has poly-T repeat length less than 20, a long allele 231 165 relationship between TOMM40 ’523 genotype and longitudi- (’523-L) has poly-T repeat length between 20 and 29, and a 232 166 nal cognitive decline, we leveraged cognitive, genetic, and very long allele (’523-VL) has poly-T repeat length of 30 233 167 and above. 234 168 neuropathologic data from a large number of community- 235 169 based older Caucasian Americans who were followed annu- 236 170 ally for up to 21 years and had undergone brain autopsy after 2.3. Annual cognitive assessments 237 171 238 death. We previously reported that AD pathology in general, Participants underwent uniform annual cognitive assess- 172 and b-amyloid in particular, mediates the effect of APOE ε4 239 173 ments for up to 22 years (mean 5 8.3, SD 5 4.5). Cognitive 240 174 with cognitive decline and AD dementia [16,17]. Owing to its performance was assessed using a battery of 17 tests [21]. 241 ε 175 strong linkage with APOE 4, we first confirm that the same Scores from each test were standardized using the baseline 242 176 relationship exists for TOMM40 ’523-L. Then, we test the mean and SD of the two cohorts. The resulting z-scores 243 177 ε 244 hypothesis that among persons with APOE 3/3, the were averaged across the tests to obtain a composite measure 178 ’523-S/S is also associated with measures of AD pathology. 245 179 of global cognition. The composite measure minimizes the 246 180 Failure to find such an association, in contrast to a strong floor and ceiling artifacts that are common for individual 247 ε 181 association between the APOE 4–’523-L haplotype and tests, and similar approach has been applied in many other 248 182 AD pathology, would suggest that the two genes have studies [22–25]. 249 183 separate and relatively independent effects on cognition and 250 184 operate through different pathologic mechanisms. 251 185 2.4. Postmortem neuropathologic evaluations 252 186 253 187 At autopsy, we quantified the burdens of common 254 188 2. Methods age-related neuropathologies including AD, macroscopic 255 189 infarcts, microinfarcts, Lewy bodies, hippocampal 256 190 2.1. Study participants 257 sclerosis, TDP-43, cerebral amyloid angiopathy, athero- Q5 191 b 258 192 Participants came from two ongoing longitudinal cohort sclerosis, and arteriolosclerosis. -amyloid and phosphory- 259 193 studies of aging and dementia, the Religious Orders Study lated PHFtau tangles, two molecular-specific pathologic Q6 260 194 (ROS) [18] and the Rush Memory and Aging Project hallmarks of AD, were assessed in eight brain regions using 261 195 (MAP) [19]. ROS and MAP enroll community-dwelling immunohistochemistry [26]. Percent area positive for 262 196 older persons without known dementia. Participants were b-amyloid was computed for each region using image anal- 263 197 264 198 followed annually for detailed cognitive and clinical assess- ysis and averaged across the regions to obtain a summary 265 199 ments; all agreed to brain donation after death. Both studies measure of b-amyloid load. Density of PHFtau tangles 266 200 were approved by the Institutional Review Board of Rush per mm2 was computed for each region using a stereologi- 267 201 University Medical Center, and written informed consent cal mapping station and averaged to obtain a summary 268

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269 measure of PHFtau tangle density. Chronic macroscopic in- 3. Results 336 270 farcts were recorded during gross examination and verified 337 271 338 histologically [27]. Chronic microinfarcts were identified 3.1. The linkage pattern between APOE and TOMM40 272 ’523 339 273 in a minimum of nine regions using hematoxylin and eosin 340 274 (H&E) staining [28]. The presence of Lewy bodies in Of the 1114 autopsied individuals included in the study, 341 275 neocortical regions was identified using a-synuclein immu- 60.1% were of APOE ε3/3 genotype, 26.6% were ε4 carriers 342 276 nostaining [29]. Hippocampal sclerosis refers to severe ε ε ε ε 343 277 (i.e., 2/4, 3/4, or 4/4) and the rest were 2 carriers 344 278 neuronal loss and astrogliosis of CA1 and/or subiculum (Table 1). The well-known LD between APOE and 345 279 and was determined using H&E staining [30].TDP-43 TOMM40 ’523 was clearly evident (Table 2). Specifically, 346 280 pathology was assessed in five regions using monoclonal APOE ε4 carriers and TOMM40 ’523-L carriers were highly 347 281 antibodies to phosphorylated TDP-43 and was rated on a concordant such that 94.9% of all ε4 carriers had ’523-L and 348 282 four-level scale including no inclusion, inclusion in amyg- ε k 5 349 283 95.3% of all ’523-L carriers had 4 (Cohen’s 0.93, 95% 350 284 dala, inclusions in amygdala and limbic, or inclusions in confidence interval [CI] 5 0.91–0.96). By contrast, three 351 285 amygdala, limbic, and neocortex [31]. Cerebral amyloid major ’523 genotypes were observed in APOE ε3/3 homozy- 352 286 angiopathy was assessed in four neocortical regions using gotes, of which ’523-S/S accounted for 25.5%, ’523-S/VL 353 287 monoclonal antibodies to b-amyloid [32]. Amount of amy- and ’523-VL/VL accounted for 46.7% and 25.7%, respec- 354 288 loid deposition in the vessel walls was scored for each ε 355 289 tively. Notably, the ’523-L allele was also absent from 2 356 290 region, and the average scores across the regions were sum- carriers in this autopsied sample. 357 291 marized into a four-level scale representing none, mild, 358 292 moderate, or severe. Atherosclerosis was assessed in the 359 293 360 circle of Willis during gross examination and arterioloscle- 3.2. TOMM40 ’523 and cognitive decline 294 rosis was assessed in anterior basal ganglia using H&E 361 295 Before examining their association with neuropa- 362 296 staining, and both were rated on a four-level scale of 363 297 none, mild, moderate, or severe [33]. thology, we first confirm that both ’523-L carriers in LD 364 298 with ε4 and the ε3/3 homozygotes with ’523-S/S exhibit 365 299 faster decline in the current sample, which represents a 366 2.5. Statistical analysis 300 subset of autopsied individuals used in our prior report 367 301 368 Frequency tables and Cohn’s k described the linkage [12]. As expected, the results from a linear mixed model 302 ε 369 303 pattern between APOE and TOMM40 ’523 genotypes. Anal- (Table 3 Model A) found that compared with 3/3 homozy- 370 304 ysis of covariance examined the adjusted mean level of gotes with ’523-S/VL or ’523-VL/VL genotype, ’523-L 371 305 continuous pathologic indices by ’523 genotype. For each carriers declined faster in cognition (estimate 520.059, 372 306 of the binary (or ordinal) pathologic indices, logistic regres- standard error [SE] 5 0.009, P , .001). Furthermore, ε3/ 373 307 3 homozygotes with ’523-S/S also had faster decline but 374 308 sion tested the ’523 association with the odds of having the 375 309 corresponding pathology (or the odds of having more severe with a weaker effect that was approximately 40% that of 376 310 pathology). ’523-L (estimate 520.023, SE 5 0.010, P 5 .024). 377 311 To examine the extent to which common neuropathol- 378 312 ogies contribute to the association between ’523 genotype 379 313 Table 1 380 5 Q10 314 with cognitive decline, we applied linear mixed models Basic characteristics of the study participants (N 1114) 381 315 with annual global cognition as the longitudinal outcome. Mean (SD) or N (%) 382 316 The models included a term for time in years before death, 383 Age at death (years) 89.4 (6.4) 317 which estimates the mean rate of cognitive decline. The 384 Female, N (%) 742 (66.6) 318 predictor of main interest was interaction term between 385 319 Education (years) 16.3 (3.6) 386 320 ’523 genotype and time, which estimates the genotype asso- Number of cognitive assessments 8.3 (4.5) 387 321 ciation with cognitive decline. We repeated the models three APOE genotype 388 ε 322 times, (1) without adjustment for neuropathologies; (2) 2/2 6 (0.5) 389 ε 323 2/3 142 (12.8) 390 adjustment for AD pathologies; and (3) adjustment for AD ε 324 2/4 27 (2.4) 391 and other common neuropathologies. If neuropathologies ε 325 3/3 670 (60.1) 392 ε 326 are involved in the association of ’523 genotype with cogni- 3/4 251 (22.5) 393 ε 327 tive decline, we expect that the estimate for the interaction 4/4 18 (1.6) 394 328 term and the corresponding statistical significance would TOMM40 ’523 genotype 395 Short/short (S/S) 199 (17.9) 329 be attenuated after controlling for neuropathologies. 396 Short/long (S/L) 141 (12.7) 330 The analyses were performed using SAS/STAT soft- 397 331 Short/very long (S/VL) 389 (34.9) 398 332 ware, version 9.4 for Linux (SAS Institute Inc., Cary, Long/long (L/L) 18 (1.6) 399 333 NC, USA). Statistical significance was determined at a Long/very long (L/VL) 136 (12.2) 400 334 level of 0.05, and all the models were controlled for age, Very-long/very long (VL/VL) 231 (20.7) 401 335 sex, and education. Abbreviation: SD, standard deviation. 402

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403 Table 2 470 404 Distribution of TOMM40 ’523 genotype by APOE 471 405 472 Frequency row 406 473 percent column 407 474 TOMM40 TOMM40 TOMM40 TOMM40 TOMM40 TOMM40 408 percent ’523-S/S ’523-S/VL ’523-VL/VL ’523-S/L ’523-L/VL ’523-L/L 475 409 APOE ε2* 27 70 51 0 0 0 476 410 18.24 47.30 34.46 0.00 0.00 0.00 477 411 13.57 17.99 22.08 0.00 0.00 0.00 478 412 APOE ε3/3 171 313 172 5 9 0 479 413 25.52 46.72 25.67 0.75 1.34 0.00 480 414 85.93 80.46 74.46 3.55 6.62 0.00 481 415 APOE ε4y 1 6 8 136 127 18 482 416 0.34 2.03 2.70 45.95 42.91 6.08 483 417 0.50 1.54 3.46 96.45 93.38 100.00 484 418 485 419 Abbreviations: S/S, short/short; S/VL, short/very long; VL/VL, very-long/very long; S/L, short/long; L/VL, long/very long; L/L, Long/long. 486 ε ε ε 420 * 2 consists of 2/2 and 2/3. 487 yε ε ε ε 421 4 consists of 2/4, 3/4, and 4/4. 488 422 489 423 490 424 3.3. TOMM40 ’523 and neuropathologies pathologies for ε3/3 homozygotes with ’523-S/S 491 425 (Table 4). 492 426 493 Next, we examine the genotype associations with Similar results were observed in relation to other 427 b 494 428 -amyloid load and PHFtau tangle density. The distribu- common age-related neuropathologies (Table 4). Briefly, 495 429 tions of both indices by ’523 genotypes showed that ’523-L carriers were more likely to have macroscopic 496 430 average level of b-amyloid and PHFtau tangle pathology infarcts (odds ratio [OR] 5 1.45, 95% CI 5 1.07–1.98) 497 431 was noticeably elevated among ’523-L carriers, similar and hippocampal sclerosis (OR 5 1.85, 95% CI 5 1.13– 498 432 APOE ε 499 433 to 4. Burdens of these pathologies were similar 3.02). In addition, they had greater odds of having more 500 434 by the ’523-S/S status (Fig. 1A and 1B). In analysis of advanced TDP-43 pathology (OR 5 2.03, 95% 501 435 covariance models adjusted for demographics, compared CI 5 1.51–2.73) and amyloid angiopathy (OR 5 3.77, 502 436 with the reference group (i.e., ε3/3 homozygotes 95% CI 5 2.84–5.00). Notably, the association with these 503 437 with ’523-S/VL or ’523-VL/VL genotype), the levels non-AD pathologies persisted even after the adjustment for 504 438 505 of b-amyloid load and PHFtau tangle density on average b-amyloid load and PHFtau tangle density. By contrast, we 439 , 506 440 were higher in ’523-L carriers (both P’s .001). By did not find significant association of ’523-S/S with any of 507 441 contrast, we did not observe difference in AD the neuropathologic indices examined (all P’s . .05). 508 442 509 443 510 444 511 445 Table 3 512 446 TOMM40 ’523 genotypes, neuropathologies, and cognitive decline 513 447 514 Model A Model B Model C 448 515 449 Estimate (SE), P Estimate (SE), P Estimate (SE), P 516 450 517 , 451 Age 0.0001 (0.0006), .927 0.0014 (0.0006), .013 0.0027 (0.0006), .001 518 452 Male 0.022 (0.008), .008 0.005 (0.008), .517 0.007 (0.007), .312 519 453 Education 0.0026 (0.0011), .018 0.0021 (0.0009), .028 0.0017 (0.0009), .050 520 2 2 454 Amyloid load - 0.006 (0.003), .051 0.006 (0.003), .074 521 2 , 2 , 455 PHFtau tangle density - 0.038 (0.003), .001 0.033 (0.003), .001 522 2 , 456 Macroscopic infarcts - - 0.024 (0.007), .001 523 457 Microinfarcts - - 0.004 (0.007), .560 524 2 , 458 Lewy bodies - - 0.057 (0.009), .001 525 2 , 459 Hippocampal sclerosis - - 0.041 (0.011), .001 526 2 , 460 TDP-43 - - 0.010 (0.003), .001 527 2 461 CAA - - 0.008 (0.004), .043 528 2 , 462 Atherosclerosis - - 0.017 (0.004), .001 529 2 463 Arteriolosclerosis - - 0.007 (0.004), .048 530 2 2 2 2 464 ’523-S/S 0.023 (0.010), .024 0.019 (0.009), .031 0.022 (0.008), .007 531 2 , 2 2 465 ’523-L 0.059 (0.009), .001 0.024 (0.008), .003 0.011 (0.008), .160 532 466 Abbreviations: CAA, cerebral amyloid angiopathy; SE, standard error. 533 467 NOTE. Estimates in each column were obtained from separate linear mixed models. The estimates came from the interaction terms with time in years before 534 468 death, which refer to the associations of corresponding predictors with annual rate of decline. Model A was controlled for demographics only; Model B was 535 469 controlled for demographics and AD (amyloid and tangle) pathologies; and Model C was controlled for demographics, AD, and other non-AD pathologies. 536

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537 604 538 605 539 606 540 607 541 608 542 609 543 610 544 611 545 612 546 613 547 614 548 615 549 616 550 617 551 618 552 619 553 620 554 621 555 622 556 623 557 624 558 625 559 626 560 627 561 628 562 629 563 630 564 631 565 632 566 633 567 634 568 635 569 636 570 637 571 638 572 639 573 640 574 641 575 642 576 643 577 644 578 645 579 646 580 Fig. 1. Distributions of common neuropathologic indices by TOMM40 ’523 genotypes. Panel A: box plot for b amyloid load; Panel B: box plot for PHFtau tangle 647 581 density; Panel C through Panel I: bar charts for percent participants with cerebral infarcts (C), Lewy bodies (D), hippocampal sclerosis (E), TDP-43 (F), amyloid 648 582 angiopathy (G), atherosclerosis (H), and arteriolosclerosis (I). Abbreviations: CAA, cerebral amyloid angiopathy; L, long; S, short; VL, very long. 649 583 650 584 651 585 652 586 3.4. The role of neuropathologies in TOMM40 ’523 Next, we repeated the model by adding terms for other 653 587 association with cognitive decline non-AD pathologic indices (Table 3 Model C). In addition 654 588 to AD, multiple pathologies including macroscopic in- 655 589 To investigate the role of neuropathologies in farcts, neocortical Lewy bodies, hippocampal sclerosis, 656 590 TOMM40 ’523 association with cognitive decline, we 657 591 TDP-43, amyloid angiopathy, and atherosclerosis were 658 first extended the previous linear mixed model by adding independently associated with faster decline in cognition. 592 terms for b-amyloid load and PHFtau tangle density 659 593 Notably, we observed further attenuation of the ’523-L as- 660 594 (Table 3 Model B). AD pathologies, PHFtau tangle sociation with cognitive decline from the original 20.059 661 595 density in particular, were associated with faster cogni- to 20.011, an 80% reduction such that it no longer reached 662 596 tive decline. After controlling for AD pathologies, while 5 5 663 597 statistical significance (SE 0.008, P .160). By contrast, 664 the association of ’523-L remained significant, the effect the estimate for ’523-S/S remained almost identical; it was 598 size was attenuated by about 60% such that the point 665 599 originally 20.023, and after controlling for AD and other 666 2 2 600 estimate was reduced from 0.059 to 0.024 pathologies, it was 20.022 (SE 5 0.008, P 5 .007). This 667 5 5 601 (SE 0.008, P .003). By contrast, the estimate for strongly suggests that the association of ’523-S/S with 668 602 2 669 ’523-S/S only changed about 17% from 0.023 to decline was not attributable to these pathologies. Fig. 2 603 20.019 (SE 5 0.009, P 5 .031). 670

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671 Table 4 burden of neuropathologies including AD and other com- 738 672 TOMM40 ’523 genotypes and neuropathologies mon non-AD neuropathologies. By contrast, we did not 739 673 740 Neuropathologic outcomes Estimate (SE), P ε 674 observe difference in neuropathologies for 3/3 homozy- 741 675 Amyloid load* gotes with ’523-S/S. Notably, the ’523-L association 742 676 ’523-S/S 0.042 (0.098), .669 with cognitive decline is mediated through common neu- 743 677 ’523-L 0.674 (0.082), ,.001 ropathologies. This is expected due to its strong LD with 744 678 PHFtau tangle density* APOE ε4. On the other hand, the ’523-S/S association 745 679 746 ’523-S/S 0.114 (0.115), .323 among APOE ε3/3 homozygotes is not explained by 680 ’523-L 0.818 (0.096), ,.001 747 681 Macroscopic infarctsy these pathologies, indicating a separate association 748 682 ’523-S/S 20.103 (0.194), .593 signal. These findings offer new insights into the neuro- 749 683 ’523-L 0.373 (0.158), 0.018 pathologic basis underlying the association between 750 y 684 Microinfarcts TOMM40 ’523 and late-life cognitive decline and pro- 751 685 752 ’523-S/S 0.290 (0.192), .131 vide strong evidence that a haplotype within TOMM40 686 ’523-L 0.108 (0.166), .513 753 y ε 687 Lewy bodies is associated with AD independent of APOE 4. Because 754 688 ’523-S/S 20.052 (0.276), .852 of the strong linkage between TOMM40 ’523-L and 755 689 ’523-L 0.331 (0.213), .120 APOE ε4 [10,34], ’523-L carriers almost exclusively 756 y 690 Hippocampal sclerosis have the ε4 allele and vice versa, and less than 3% of 757 691 2 758 ’523-S/S 0.346 (0.370), .350 1114 individuals included in this study are discordant 692 ’523-L 0.615 (0.250), .014 759 693 TDP-43y cases. Consequently, we expect that the relationship be- 760 694 ’523-S/S 0.079 (0.180), .661 tween ’523-L and neuropathologies as well as down- 761 695 ’523-L 0.709 (0.151), ,.001 stream cognitive decline would highly mimic that of 762 y 696 Cerebral amyloid angiopathy ε4. Indeed, we found that ’523-L was strongly associated 763 697 764 ’523-S/S 0.049 (0.167), .769 with multiple neuropathologies including AD. Similar 698 ’523-L 1.327 (0.144), ,.001 765 y ε 699 Atherosclerosis associations have been widely reported for APOE 4 766 700 ’523-S/S 20.181 (0.166), .276 [32,35–38]. Furthermore, we found that ’523-L carriers 767 701 ’523-L 0.054 (0.139), .699 had faster cognitive decline and the association dimin- 768 y 702 Arteriolosclerosis ished after accounting for neuropathologies. This is high- 769 703 2 770 ’523-S/S 0.084 (0.164), .608 ly consistent with our previous observation that the 704 ’523-L 0.096 (0.137), .483 771 705 association of ε4 with cognition and cognitive decline 772 706 Abbreviation: SE, standard error. is also largely attributable to AD and other non-AD pa- 773 707 NOTE. The estimates show the associations of corresponding ’523 geno- 774 types relative to the reference (’523-S/VL or ’523-VL/VL). The estimates thologies [16,17]. Taken together, these findings 708 suggest that ’523-L and ε4shareacommonneuropatho- 775 709 from logistic regression were log odds ratios of having a neuropathology 776 710 (or log odds of having more advanced neuropathology) relative to the refer- logic footprint in relation to cognitive decline. However, 777 711 ence. our study does not inform the extent to which the APOE 778 712 *Estimates in each cell were obtained from separate models of analyses of ε4–’523-L haplotype associations with neuropathologies 779 713 covariance, adjusted for demographics. 780 y and cognitive decline results from APOE ε4, ’523-L, or 714 Estimates in each cell were obtained from separate models of logistic 781 regression, adjusted for demographics. the complete haplotype. 715 ε 782 716 In contrast to APOE 4, three major TOMM40 ’523 783 717 genotypes are present in APOE ε3/3 homozygotes. Previ- 784 718 ous studies show that the risk of clinical diagnosis of AD 785 719 illustrates that the ’523-L association with cognitive and age at onset differ by these ’523 genotypes, though 786 720 decline varied before and after controlling for neuropathol- findings are inconsistent [10,11,20,39,40]. Using data 787 721 ogies, but ’523-S/S did not. 788 722 from the entire ROS and MAP cohorts, both dead and 789 723 alive, we previously reported an association of 790 724 TOMM40 791 4. Discussion ’523-S/S with faster cognitive decline among 725 APOE ε3/3 homozygotes. Here, we expand on prior 792 726 There is evidence that multiple genetic variations in work in an important way. After confirming that the 793 727 794 728 the APOE haplotype block are implicated in AD demen- same association exists among the autopsied subgroup, 795 729 tia susceptibility [3–7]. Using data from more than 1000 we examine its relation to multiple neuropathologies. 796 730 community-dwelling older persons who had died and We show that unlike ’523-L, none of these pathologic 797 731 undergone brain autopsy, we confirmed two distinct asso- indices differ by ’523-S/S status. Consequently, the asso- 798 732 ciation signals at the TOMM40 ’523 locus in relation to ciation of ’523-S/S with cognitive decline is not affected 799 733 800 734 late-life cognitive decline, the clinical hallmark of AD. by any of these pathologies, including AD. Our findings 801 735 We investigated the extent to which AD and other implicate that ’523-S/S represents a risk factor for cogni- 802 736 common age-related neuropathologies contribute to these tive decline separate from ’523-L or APOE ε4; further- 803 737 relationships. We found ’523-L carriers had higher more, the neuropathologic basis of this association also 804

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818 FPO 885 819 = 886 820 887 821 888 web 4C 822 Fig. 2. The effects of TOMM40 ’523 genotypes on annual rate of cognitive decline before and after the adjustment for neuropathologies. On the x-axis, “A: No 889 823 path” refers to the effects estimated from the model adjusted only for demographics; “B: AD” refers to the effect estimated from the model adjusted for de- 890 824 mographics and AD pathologies only; “C: AD and other pathologies” refers to the effect estimated from the full model adjusted for demographics, AD, and 891 825 other non-AD pathologies. Mean estimates 1/21.96 standard error for the effects of ’523-L and ’523-S/S are shown on the y-axis. It is evident that the asso- 892 826 ciation of ’523-L with cognitive decline was attenuated after controlling AD pathology and became not significant after further controlling for other non-AD 893 827 pathologies (left panel). By contrast, the association of ’523-S/S remained essentially unchanged, such that the estimates with and without controlling for pa- 894 828 thologies are similar and all are significantly above zero (right panel). Abbreviation: AD, Alzheimer’s disease. 895 829 896 830 897 831 ε 898 832 differs from that of ’523-L or 4. These results are 22 years help to capture person-specific trajectories of 899 833 somewhat unexpected. However, emerging evidence cognitive change with a high level of fidelity. Limitations 900 834 from large clinical pathologic studies suggests that while are noted. The present study is restricted to older persons 901 835 common neuropathologic burdens such as Alzheimer’s, of European ancestry. The linkage patterns of APOE and 902 836 cerebrovascular, or Lewy body diseases account for a TOMM40 ’523 are known to differ in African Americans; 903 837 904 838 majority of person-specific variation in late-life cognitive therefore, the extent to which these findings are generaliz- 905 839 decline, an appreciable amount of variation remains able to other population is unknown. In addition, both 906 840 unexplained (i.e., residual cognitive decline) [21].The ROS and MAP are voluntary cohorts, and the findings await 907 841 independent association of ’523-S/S with cognitive replications from other longitudinal clinical pathologic 908 842 decline reported here suggests that it accounts for some studies. 909 843 910 844 of this residual decline. In conclusion, through investigating the role of 911 845 The molecular effects of APOE–TOMM40 ’523 Alzheimer and other common neuropathologies in the 912 846 haplotypes remain unclear. Proteins encoded by both relationship between TOMM40 ’523 and late-life cognitive 913 847 genes have been functionally implicated in AD and other decline, the study revealed two distinct association signals. 914 848 neurodegenerative diseases. Although the involvement of The association of TOMM40 ’523-L with cognitive decline 915 849 b 916 850 APOE in -amyloid accumulation and clearance has is primarily mediated by common neuropathologies. By 917 851 been well established [14,41,42], mitochondrial contrast, the association of TOMM40 ’523-S/S is relatively 918 852 dysfunction is also shown to increase the risk for AD independent of these pathologies. 919 853 [43,44]. The mitochondrial protein encoded by TOMM40 920 854 is essential in transporting protein precursors into 921 855 922 TOMM40 856 mitochondria [45,46]. Alterations of Acknowledgments 923 857 expression have been reported in AD, but with 924 858 conflicting results [47,48]. Notably, several studies have The study was funded by National Institute on Aging 925 859 926 Q7 shown a cis-eQTL where ’523-S acts as a repressor to grants (R01AG17917, P30AG10161, RF1AG15819, 860 reduce the gene expression [48,49]. This regulatory R01AG042210) and the Illinois Department of Public 927 861 928 862 function of the ’523 variant has also been reported in Health. The study was also supported in part by an 929 863 human cell culture, where the study demonstrates that unrestricted educational grant from Zinfandel Pharma- Q8 930 864 ’523 is a putative regulatory element that influences the ceuticals, Inc. We thank all ROS and MAP participants 931 865 TOMM40 promoter activity in vitro [50]. for donating their data and biospecimen. We also thank 932 866 To our knowledge, this is the largest study to interrogate all the staff and investigators at the Rush Alzheimer’s 933 867 934 868 the relationship between TOMM40 ’523 with postmortem Disease Center (RADC). Data and biospecimen from 935 869 neuropathologies. Comprehensive postmortem evaluations ROS and MAP for research can be requested through 936 870 quantified multiple neuropathologies that are observed in the RADC Research Resource Sharing Hub (www.radc. 937 871 aging brain. Annual uniform cognitive assessments up to rush.edu). 938

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939 [8] Hayden KM, McEvoy JM, Linnertz C, Attix D, Kuchibhatla M, 1006 940 RESEARCH IN CONTEXT Saunders AM, et al. A homopolymer polymorphism in the 1007 941 TOMM40 gene contributes to cognitive performance in aging. 1008 942 Alzheimers Dement 2012;8:381–8. 1009 943 [9] Johnson SC, La Rue A, Hermann BP, Xu G, Koscik RL, Jonaitis EM, 1010 944 1. Systematic review: Literature reviews via PubMed et al. The effect of TOMM40 poly-T length on gray matter volume and 1011 945 search suggest that TOMM40 ’523 variant is associ- cognition in middle-aged persons with APOE epsilon3/epsilon3 1012 946 ated with late-life cognitive decline and the associ- genotype. Alzheimers Dement 2011;7:456–65. 1013 947 ation is not fully attributable to the linkage [10] Jun G, Vardarajan BN, Buros J, Yu CE, Hawk MV, Dombroski BA, 1014 948 1015 disequilibrium with APOE variant. Yet, the under- et al. Comprehensive search for Alzheimer disease susceptibility 949 loci in the APOE region. Arch Neurol 2012;69:1270–9. 1016 950 lying neuropathologic correlates remain unclear. [11] Crenshaw DG, Gottschalk WK, Lutz MW, Grossman I, Saunders AM, 1017 951 1018 2. Interpretation: Through investigating the role of AD Burke JR, et al. Using genetics to enable studies on the prevention of 952 Alzheimer’s disease. Clin Pharmacol Ther 2013;93:177–85. 1019 953 and other common neuropathologies in the relation- [12] Yu L, Lutz MW, Wilson RS, Burns DK, Roses AD, Saunders AM, et al. 1020 954 ship between TOMM40 ’523 and longitudinal cogni- TOMM400 523 variant and cognitive decline in older persons with 1021 955 tive decline, this study reveals two association APOE ε3/3 genotype. Neurology 2017;88:661–8. 1022 956 APOE [13] Reiman EM, Chen K, Liu X, Bandy D, Yu M, Lee W, et al. Fibrillar 1023 957 signals. The ’523 long allele, in linkage with 1024 ε4, primarily acts through common neuropathol- amyloid-beta burden in cognitively normal people at 3 levels of genetic 958 risk for Alzheimer’s disease. Proc Natl Acad Sci U S A 2009;106:6820–5. 1025 959 ogies, whereas the ’523 short/short genotype among [14] Castellano JM, Kim J, Stewart FR, Jiang H, DeMattos RB, 1026 960 APOE ε3/3 homozygotes represents a separate risk Patterson BW, et al. Human apoE isoforms differentially regulate brain 1027 961 factor that operates through a different mechanism. amyloid-beta peptide clearance. Sci Transl Med 2011;3:89ra57. 1028 962 [15] Schmechel DE, Saunders AM, Strittmatter WJ, Crain BJ, Hulette CM, 1029 963 3. Future directions: This study is restricted to older Joo SH, et al. Increased amyloid beta-peptide deposition in cerebral 1030 964 persons of European ancestry, and the generaliz- cortex as a consequence of apolipoprotein E genotype in late-onset 1031 965 1032 ability of our findings to other populations awaits Alzheimer disease. Proc Natl Acad Sci U S A 1993;90:9649–53. 966 [16] Bennett DA, Wilson RS, Schneider JA, Evans DA, Aggarwal NT, 1033 967 investigation. Future studies are warranted to deter- Arnold SE, et al. Apolipoprotein E epsilon4 allele, AD pathology, 1034 968 mine the neurobiology that drives the association of and the clinical expression of Alzheimer’s disease. Neurology 2003; 1035 969 TOMM40 ’523 with cognitive decline that are not 60:246–52. 1036 970 due to common age-related neuropathologies. [17] Yu L, Boyle PA, Leurgans S, Schneider JA, Bennett DA. Disentangling 1037 971 the effects of age and APOE on neuropathology and late life cognitive 1038 972 decline. Neurobiol Aging 2014;35:819–26. 1039 973 [18] Bennett DA, Schneider JA, Arvanitakis Z, Wilson RS. Overview and 1040 974 findings from the Religious Orders Study. Curr Alzheimer Res 2012; 1041 975 9:628–45. 1042 976 [19] Bennett DA, Schneider JA, Buchman AS, Barnes LL, Boyle PA, 1043 977 References Wilson RS. Overview and findings from the Rush Memory and Aging 1044 978 Project. Curr Alzheimer Res 2012;9:646–63. 1045 979 [1] Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, [20] Caselli RJ, Dueck AC, Huentelman MJ, Lutz MW, Saunders AM, 1046 980 Gaskell PC, Small GW, et al. Gene dose of apolipoprotein E type 4 Reiman EM, et al. Longitudinal modeling of cognitive aging and the 1047 981 allele and the risk of Alzheimer’s disease in late onset families. TOMM40 effect. Alzheimers Dement 2012;8:490–5. 1048 982 Science 1993;261:921–3. [21] Boyle PA, Wilson RS, Yu L, Barr AM, Honer WG, Schneider JA, et al. 1049 983 [2] Yu CE, Seltman H, Peskind ER, Galloway N, Zhou PX, Rosenthal E, Much of late life cognitive decline is not due to common neurodegen- 1050 984 et al. Comprehensive analysis of APOE and selected proximate erative pathologies. Ann Neurol 2013;74:478–89. 1051 985 markers for late-onset Alzheimer’s disease: patterns of linkage [22] Rajan KB, Arvanitakis Z, Lynch EB, McAninch EA, Wilson RS, 1052 986 disequilibrium and disease/marker association. Genomics 2007; Weuve J, et al. Cognitive decline following incident and preexisting 1053 987 89:655–65. diabetes mellitus in a population sample. Neurology 2016;87:1681–7. 1054 988 [3] Parker GR, Cathcart HM, Huang R, Lanham IS, Corder EH, [23] Ossenkoppele R, Zwan MD, Tolboom N, van Assema DM, 1055 989 Poduslo SE. Apolipoprotein gene E4 allele promoter polymorphisms Adriaanse SF, Kloet RW, et al. Amyloid burden and metabolic function 1056 990 as risk factors for Alzheimer’s disease. Psychiatr Genet 2005; in early-onset Alzheimer’s disease: parietal lobe involvement. Brain 1057 991 15:271–5. 2012;135:2115–25. 1058 992 [4] Takei N, Miyashita A, Tsukie T, Arai H, Asada T, Imagawa M, et al. [24] Jack CR Jr, Knopman DS, Weigand SD, Wiste HJ, Vemuri P, 1059 993 Genetic association study on in and around the APOE in late-onset Lowe V, et al. An operational approach to National Institute on 1060 994 Alzheimer disease in Japanese. Genomics 2009;93:441–8. Aging-Alzheimer’s Association criteria for preclinical Alzheimer 1061 995 [5] Harold D, Abraham R, Hollingworth P, Sims R, Gerrish A, disease. Ann Neurol 2012;71:765–75. 1062 996 Hamshere ML, et al. Genome-wide association study identifies vari- [25] Kohler S, Thomas AJ, Barnett NA, O’Brien JT. The pattern and course 1063 997 ants at CLU and PICALM associated with Alzheimer’s disease. Nat of cognitive impairment in late-life depression. Psychol Med 2010; 1064 998 Genet 2009;41:1088–93. 40:591–602. 1065 999 [6] Li H, Wetten S, Li L, St Jean PL, Upmanyu R, Surh L, et al. Candidate [26] Bennett DA, Schneider JA, Wilson RS, Bienias JL, Berry-Kravis E, 1066 1000 single-nucleotide polymorphisms from a genomewide association Arnold SE. Amyloid mediates the association of apolipoprotein E e4 1067 1001 study of Alzheimer disease. Arch Neurol 2008;65:45–53. allele to cognitive function in older people. J Neurol Neurosurg 1068 1002 [7] Roses AD, Lutz MW, Amrine-Madsen H, Saunders AM, Psychiatry 2005;76:1194–9. 1069 1003 Crenshaw DG, Sundseth SS, et al. A TOMM40 variable-length [27] Schneider J, Wilson R, Cochran E, Bienias J, Arnold S, Evans D, et al. 1070 1004 polymorphism predicts the age of late-onset Alzheimer’s disease. Relation of cerebral infarctions to dementia and cognitive function in 1071 1005 Pharmacogenomics J 2010;10:375–84. older persons. Neurology 2003;60:1082–8. 1072

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1073 [28] Arvanitakis Z, Leurgans SE, Barnes LL, Bennett DA, Schneider JA. polymorphisms in TOMM40 in Alzheimer disease. Arch Neurol 1140 1074 Microinfarct pathology, dementia, and cognitive systems. Stroke 2011;68:1013–9. 1141 1075 2011;42:722–7. [40] Li G, Bekris LM, Leong L, Steinbart EJ, Shofer JB, Crane PK, et al. 1142 1076 [29] Schneider JA, Arvanitakis Z, Yu L, Boyle PA, Leurgans SE, TOMM40 intron 6 poly-T length, age at onset, and neuropathology of 1143 1077 Bennett DA. Cognitive impairment, decline and fluctuations in older AD in individuals with APOE 3/3 3.3 Alzheimers Dement 2013;9:554–61. 1144 1078 community-dwelling subjects with Lewy bodies. Brain 2012; [41] Holtzman DM, Bales KR, Tenkova T, Fagan AM, Parsadanian M, 1145 1079 135:3005–14. Sartorius LJ, et al. Apolipoprotein E isoform-dependent amyloid 1146 1080 [30] Nag S, Yu L, Capuano AW, Wilson RS, Leurgans SE, Bennett DA, deposition and neuritic degeneration in a mouse model of Alzheimer’s 1147 1081 et al. Hippocampal sclerosis and TDP-43 pathology in aging and disease. Proc Natl Acad Sci U S A 2000;97:2892–7. 1148 1082 Alzheimer disease. Ann Neurol 2015;77:942–52. [42] Koffie RM, Hashimoto T, Tai HC, Kay KR, Serrano-Pozo A, Joyner D, 1149 1083 [31] Yu L, De Jager PL, Yang J, Trojanowski JQ, Bennett DA, et al. Apolipoprotein E4 effects in Alzheimer’s disease are mediated by 1150 1084 Schneider JA. The TMEM106B locus and TDP-43 pathology in older synaptotoxic oligomeric amyloid-b. Brain 2012;135:2155–68. 1151 1085 persons without FTLD. Neurology 2015;84:927–34. [43] Swerdlow RH, Khan SM. The Alzheimer’s disease mitochondrial 1152 1086 [32] Yu L, Boyle PA, Nag S, Leurgans S, Buchman AS, Wilson RS, et al. cascade hypothesis: an update. Exp Neurol 2009;218:308–15. 1153 1087 APOE and cerebral amyloid angiopathy in community-dwelling older [44] Hirai K, Aliev G, Nunomura A, Fujioka H, Russell RL, Atwood CS, 1154 1088 persons. Neurobiol Aging 2015;36:2946–53. et al. Mitochondrial abnormalities in Alzheimer’s disease. J Neurosci 1155 1089 [33] Arvanitakis Z, Capuano AW, Leurgans SE, Bennett DA, Schneider JA. 2001;21:3017–23. 1156 1090 Relation of cerebral vessel disease to Alzheimer’s disease dementia [45] Hill K, Model K, Ryan MT, Dietmeier K, Martin F, Wagner R, et al. 1157 1091 and cognitive function in elderly people: a cross-sectional study. Tom40 forms the hydrophilic channel of the mitochondrial import 1158 1092 Lancet Neurol 2016;15:934–43. pore for preproteins [see comment]. Nature 1998;395:516–21. 1159 1093 [34] Lutz MW, Crenshaw DG, Saunders AM, Roses AD. Genetic variation [46] Gottschalk WK, Lutz MW, He YT, Saunders AM, Burns DK, 1160 1094 at a single locus and age of onset for Alzheimer’s disease. Alzheimers Roses AD, et al. The Broad Impact of TOM40 on Neurodegenerative 1161 1095 Dement 2010;6:125–31. Diseases in Aging. J Parkinsons Dis Alzheimers Dis 2014;1. Q9 1162 1096 [35] Verghese PB, Castellano JM, Holtzman DM. Apolipoprotein E in [47] Goh LK, Lim WS, Teo S, Vijayaraghavan A, Chan M, Tay L, et al. 1163 1097 Alzheimer’s disease and other neurological disorders. Lancet Neurol TOMM40 alterations in Alzheimer’s disease over a 2-year follow-up 1164 1098 2011;10:241–52. period. J Alzheimers Dis 2015;44:57–61. 1165 1099 [36] Wang QY, Wang WJ, Wu L, Liu L, Han LZ. Meta-analysis of APOE [48] Payton A, Sindrewicz P, Pessoa V, Platt H, Horan M, Ollier W, et al. A 1166 1100 epsilon2/epsilon3/epsilon4 polymorphism and cerebral infarction. J TOMM40 poly-T variant modulates gene expression and is associated 1167 1101 Neural Transm (Vienna) 2013;120:1479–89. with vocabulary ability and decline in nonpathologic aging. Neurobiol 1168 1102 [37] Biffi A, Sonni A, Anderson CD, Kissela B, Jagiella JM, Schmidt H, Aging 2016;39:217.e1–217.e7. 1169 1103 et al. Variants at APOE influence risk of deep and lobar intracerebral [49] Linnertz C, Anderson L, Gottschalk W, Crenshaw D, Lutz MW, 1170 1104 hemorrhage. Ann Neurol 2010;68:934–43. Allen J, et al. The cis-regulatory effect of an Alzheimer’s disease- 1171 1105 [38] Yip AG, McKee AC, Green RC, Wells J, Young H, Cupples LA, et al. associated poly-T locus on expression of TOMM40 and apolipoprotein 1172 1106 APOE, vascular pathology, and the AD brain. Neurology 2005; E genes. Alzheimers Dement 2014;10:541–51. 1173 1107 65:259–65. [50] Bekris LM, Lutz F, Yu CE. Functional analysis of APOE locus genetic 1174 1108 [39] Cruchaga C, Nowotny P, Kauwe JS, Ridge PG, Mayo K, Bertelsen S, variation implicates regional enhancers in the regulation of both 1175 1109 et al. Association and expression analyses with single-nucleotide TOMM40 and APOE. J Hum Genet 2012;57:18–25. 1176 1110 1177 1111 1178 1112 1179 1113 1180 1114 1181 1115 1182 1116 1183 1117 1184 1118 1185 1119 1186 1120 1187 1121 1188 1122 1189 1123 1190 1124 1191 1125 1192 1126 1193 1127 1194 1128 1195 1129 1196 1130 1197 1131 1198 1132 1199 1133 1200 1134 1201 1135 1202 1136 1203 1137 1204 1138 1205 1139 1206

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