ORIGINAL CONTRIBUTION Genetic Association of a C With Late-Onset Alzheimer Disease

Ulrich Finckh, MD; Heinz von der Kammer, PhD; Joachim Velden; Tiana Michel, MS; Barbara Andresen; Amy Deng; Jun Zhang; Tomas Mu¨ller-Thomsen, MD; Kathrin Zuchowski; Gunnar Menzer; Ulrike Mann, MD; Andreas Papassotiropoulos, MD; Reinhard Heun, MD; Jan Zurdel, MD; Frederik Holst; Luisa Benussi, PhD; Gabriela Stoppe, MD; Jochen Reiss, PhD; Andre´ R. Miserez, MD; Hannes B. Staehelin, MD; G. William Rebeck, PhD; Bradley T. Hyman, MD, PhD; Giuliano Binetti, MD; Christoph Hock, MD; John H. Growdon, MD; Roger M. Nitsch, MD

Objective: To determine whether the cystatin C gene Measures: Molecular testing of the KspI polymorphisms (CST3) is genetically associated with late-onset Alzhei- in the 5Ј flanking region and 1 of CST3 and the apolipo- mer disease (AD). E (APOE) genotype. Mini–Mental State Examina- tion scores for both patients with AD and control subjects. Design: A case-control study with 2 independent study populations of patients with AD and age-matched, cog- Results: Homozygosity for haplotype B of CST3 was sig- nitively normal control subjects. nificantly associated with late-onset AD in both study popu- lations, with an odds ratio of 3.8 (95% confidence inter- Setting: The Alzheimer’s Disease Research Unit at the val, 1.56-9.25) in the combined data set; heterozygosity was University Hospital Hamburg-Eppendorf, Hamburg, Ger- not associated with an increased risk. The odds ratios for many, for the initial study (n=260). For the indepen- CST3 B/B increased from 2.6 in those younger than 75 years dent multicenter study (n=647), an international con- to 8.8 for those aged 75 years and older. The association sortium that included the Massachusetts Alzheimer’s of CST3 B/B with AD was independent of APOE ⑀4; both Disease Research Center at the Massachusetts General genotypes independently reduced disease-free survival. Hospital, Boston; the Scientific Institute for Research and Patient Care, Brescia, Italy; and Alzheimer’s research units Conclusions: CST3 is a susceptibility gene for late- at the Universities of Basel and Zurich, Switzerland, and onset AD, especially in patients aged 75 years and older. Bonn, Goettingen, and Hamburg, Germany. To our knowledge, CST3 B is the first autosomal reces- sive risk allele in late-onset AD. Participants: Five hundred seventeen patients with AD and 390 control subjects. Arch Neurol. 2000;57:1579-1583

LZHEIMER DISEASE (AD) is ments and inhibits cathepsin activities the major cause of demen- within the endosomal-lysosomal sys- tia in the elderly. Familial tem.16 In the brain, cystatin C is synthe- forms of early-onset AD sized by neurons, astrocytes, and cho- are caused by heterozy- roid plexus cells,17-21 and its levels increase gous in the encoding the in response to injury, including ische- A 20,22-25 ␤- precursor protein (APP) and the mia, axotomy, or surgery. Neuro- presenilins PS1 and PS2, but the etiology nal concentrations of cystatin C are in- of the much more prevalent forms of late- creased in AD, and cystatin C reportedly onset AD is unknown.1-6 Several suscep- colocalizes with ␤-amyloid in arteriolar tibility genes were reported to influence walls.26 The physiological function of cys- the risk of developing AD; of these, the ef- tatin C as a inhibitor is regu- fect of the apolipoprotein E gene (APOE) lated either by dimerization or by endo- is now uniformly accepted, whereas ad- proteolysis mediated by cathepsin D, ditional putative genetic risk factors in- which is also increased in early endo- 27 cluding ␣2-macroglobulin, interleukin 6, somes in AD brains. interleukin 1, and cathepsin D are matter Cystatin C is also an amyloidogenic of intense debate.7-14 protein that accumulates in cortical Cystatin C is an endogenous protein- vessels in hereditary cerebral hemor- The affiliations of the authors ase inhibitor of the cathepsins B, H, L, and rhage with of both the Dutch appear in the acknowledgment S.15 Cystatin C is a secretory protein, but and Icelandic types caused by mutations section at the end of the article. it also reaches endocytic cellular compart- of the APP gene or the cystatin C gene

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CST3 SUBJECTS AND METHODS –157 –72 ATG +73 Primer 1206R

SUBJECTS 1 2 3 Primer 024 We conducted molecular genetic studies on 2 inde- A Haplotype pendent study populations: an initial sample (n=260 G A G participants), and a multicenter sample (n=647 participants). The clinical diagnoses of AD were made 1 3 34 according to the NINCDS-ADRDA criteria ; 84% of our B Haplotype patients had Mini–Mental State Examination (MMSE) C C A scores lower than 24, similar to previous studies of AD.35 The control subjects were not demented, and had 2 MMSE scores of 27 or higher. All participants in this B study were Caucasian. Informed consent was ob- A MAGPLRAPLLLLAILAVALAVSPA G SSPGKPPRLV tained from all participants, and the local human stud- T ies committees approved the study protocol. The initial sample included 110 patients with AD A, Allelic variants of CST3. There are 3 polymorphic KspI restriction sites (X) (aged 73.1±8.9 years [mean ± SD]; 79 women) and within 230 base pairs of the 5Ј flanking region and exon 1; they are in strong 150 nondemented control subjects (aged 65.2±14.6 linkage disequilibrium. These 3 polymorphisms in the CST3 gene result in years [mean ± SD]; 66 women) from the Alzheimer’s only 2 commonly found human haplotypes, called CST3 A (nucleotides G, A, and G at positions −157, −72 and +73) and CST3 B (C, C, and A at these Disease Research Unit at the University Hospital Ham- positions), respectively. B, sequence of the cystatin C signal burg-Eppendorf, Germany. An independent hypoth- . Alanine to threonine variation at the −2 position residue of signal esis-testing sample was collected according to identi- peptidase cleavage. cal standardized criteria by an international multi- institutional consortium that included AD research 29,30 units in the United States, Italy, Germany, and Swit- penultimate position of the signal peptide. Because zerland. This sample consisted of 407 patients with AD of linkage disequilibrium, these 3 polymorphisms in the (aged 75.0±9.5 years [mean ± SD]; 284 women) and CST3 gene result in only 2 commonly found human hap- 240 age-matched nondemented controls (aged lotypes, called CST3 A (nucleotides G, A, and G at po- 75.1±6.3 years [mean ± SD]; 139 women). sitions −157, −72 and +73) and CST3 B (C, C, and A at these positions), respectively (Figure).31 In this study, GENOTYPING we determined whether any of the CST3 genotypes are associated with a risk for late-onset AD. Evidence for an Preparations of DNA and polymerase chain re- association of CST3 genotypes with AD has very re- actions (PCRs) were performed following standard 32,33 protocols.29 The 318– (bp) PCR product cently been presented in abstract form. generated with primers 024F (TGGGAGGGACGAGGCGTTCC) RESULTS and 1206R (TCCATGGGGCCTCCCACCAG) was designed to cover all 3 polymorphic KspI sites INITIAL STUDY described above. Digestion with KspI generated 3 frag- ments of 41, 226, and 51 bp in size for haplotype A, or There was a significant association between AD and CST3 2 2 fragments of 127 and 191 bp for haplotype B. These genotype (␹ 2=6.67; 2-sided Fisher exact test, P=.04) banding patterns allowed us to determine the phase of (Table 1). The borderline higher frequency of allele B the polymorphisms. Among the 907 samples geno- (FB) in patients (25%) than in controls (18%) (Pearson typed in this study, there was no case of aberrant band- ␹2=3.75; P=.05) was due to an excess of B/B homozy- ing pattern in this assay, confirming that no other hap- gotes in the patients with AD (9.1%) compared with the lotypes defined by these 3 polymorphic sites were control subjects (2.0%). The proportion of A/B hetero- present in our study population. In addition, we con- zygotes in the patient and control groups was almost iden- firmed these haplotypes by direct sequencing of PCR tical. These data suggested an odds ratio (OR) for AD of products from subjects with the respective genotypes A/A, A/B, or B/B. APOE was routinely genotyped by us- 4.9 (95% confidence interval [CI], 1.32-18.25) in asso- ing a standard PCR- and restriction-based protocol.36 ciation with the homozygous genotype B/B. As ex- The observed CST3 and APOE genotype counts did not pected, there was also a highly significant association be- significantly deviate from those expected under the tween APOE genotype and AD. Of the patients with AD, Hardy Weinberg equilibrium of patients and controls 13.6% were homozygous for the ⑀4 allele, compared with in the initial, the multicenter, or the combined samples. 2.7% of the control subjects; 50.0% of patients with AD were heterozygous ⑀3/⑀4or⑀2/⑀4 (control subjects, 29.3%), and 36.4% of the patients with AD had no ⑀4 al- 2 lele, vs 68% of the controls (␹ 2=29.2; PϽ.001). Non- (CST3), respectively.28 CST3 maps to conditional logistic regression analysis revealed a com- 20p11.2; it contains 3 , and 2 KspI polymor- parably strong effect of CST3 B/B on the individual risk phisms are known in the 5Ј untranslated sequence, com- for AD as the presence of an APOE ⑀4 allele (Table 2). bined with an additional KspI polymorphism that re- Nonconditional logistic regression analyses revealed sig- sults in a threonine for alanine substitution at the nificant effects on the risk for AD of APOE ⑀4, CST3 B/B,

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©2000 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 Table 1. CST3 Genotypes in Patients With Alzheimer Disease and Control Subjects in 2 Independent Study Populations*

Initial Multicenter Combined

Genotype AD 110 Control 150 AD 407 Control 240 AD 517 Control 390 CST3 A/A 65 (59.1) 99 (66.0) 257 (63.1) 161 (67.1) 322 (62.3) 260 (66.7) CST3 A/B 35 (31.8) 48 (32.0) 131 (32.2) 76 (31.7) 166 (32.1) 124 (31.8) CST3 B/B 10 (9.1) 3 (2.0) 19 (4.7) 3 (1.3) 29 (5.6) 6 (1.5)

*AD indicates patients with Alzheimer disease. Data are number of participants (percentage of the total number of participants) within the group.

Table 2. Nonconditional Logistic Regression Analysis Table 3. Odds Ratios for Alzheimer Disease Findings for the Effects of APOE ⑀4, CST3 B/B, Sex, in Association With CST3 or APOE Genotypes and Age on Risk of Alzheimer Disease* Calculated From the Combined Sample*

Variable B SE P † Odds 95% Confidence Genotype Ratio Interval P † Initial CST3 B/B 1.796 0.745 .016 CST3 B/B 3.80 1.56-9.25 .001 APOE ⑀4 positive 1.560 0.302 Ͻ.001 APOE ⑀4 positive 3.60 2.71-4.79 Ͻ.001 Sex 1.027 0.302 Ͻ.001 APOE ⑀4/⑀4 6.91 3.12-15.32 Ͻ.001 Age 0.056 0.014 Ͻ.001 APOE ⑀3/⑀4or⑀2/⑀4‡ 3.10 2.30-4.16 Ͻ.001 CST3 B/B in APOE ⑀4 negative 3.07 1.16-8.12 .02 Multicenter CST3 B/B or APOE ⑀4/⑀4 5.71 3.13-10.40 Ͻ.001 CST3 B/B 1.4386 0.6499 .03 APOE ⑀4 positive 1.4222 0.1872 Ͻ.001 The combined sample includes 517 patients and 390 controls. Sex 0.5800 0.1805 .001 * †Fisher exact test. Age (age-matched) ‡APOE ⑀4 homozygotes excluded. Combined CST3 B/B 1.466 0.152 .002 APOE ⑀4 positive 1.363 0.476 Ͻ.001 Sex 0.725 0.152 Ͻ.001 sion analysis revealed no significant interaction be- Age 0.030 0.007 Ͻ.001 tween APOE and CST3.

*Age is analyzed per year of age. B indicates risks for Alzheimer disease. †Fisher exact test. COMBINED SAMPLE Analysis of the combined sample of 907 participants con- sex, and age, but we did not find evidence for signifi- firmed that CST3 was significantly associated with AD, cant interactions between APOE and CST3. with an OR of 3.8 (95% CI, 1.56-9.25; P=.001). In the same sample, the ORs for APOE ⑀4 heterozygosity and MULTICENTER STUDY homozygosity were 3.09 and 6.91, respectively (Table 3). CST3 and APOE independently affected the risk for AD, There were no significant differences in CST3 allele fre- because the ORs for AD in association with CST3 B/B were quencies in the independently collected control and pa- similar in APOE ⑀4–negative (3.07; 95% CI, 1.16-8.12; tient samples obtained by the multicenter consortium: P=.02) and APOE ⑀4–positive participants. In addition The FB in patients from the United States, Italy, Ger- to the independent risks of developing AD, these 2 risk many, and Switzerland were 0.24, 0.20, 0.21, and 0.21, factors combined lowered the age of disease onset. Of all 2 respectively (␹ 3=0.96; P=.81) and those in control sub- patients with AD, 2.9% carried 2 CST3 B alleles as well jects were 0.13, 0.19, 0.18, and 0.17, respectively as at least 1 APOE ⑀4 allele, compared with none of the 2 2 (␹ 3=1.14; P=.77). Moreover, the expected association 390 control subjects (␹ 1=11.51; 2-sided Fisher exact test, between APOE genotype and AD was confirmed in the PϽ.001). These patients with AD with both risk factors samples from each center (2-sided Fisher exact test, PՅ had a mean±SD age of onset of 69.1±9.5 years com- .004; df=2, for each center). Together, these character- pared with 75.0±9.5 years in the overall patient sample. istics allowed us to combine the samples and gain suffi- Results of Kaplan-Meier survival analyses revealed cient statistical power to test the hypotheses suggested that CST3 B/B reduced mean disease-free survival from by the results of the initial study. There was a signifi- 77 years (SE=0; 95% CI, 76-78 years) in CST3 A/A and 2 cant association between CST3 B/B and AD (␹ 1=5.37; A/B patients with AD to 73 years (SE=2; 95% CI, 70-76 2-sided Fisher exact test, P=.02). The statistically non- years; log rank P=.05; df=1). Parallel analyses con- significantly higher FB in the patients (21%) than in con- firmed the known effects of APOE on mean disease-free trols (17%) (Pearson ␹2=2.61; P=.11) was again due to survival; in this sample, 80 years in ⑀4–negative (SE=1; an excess of homozygous carriers of the B allele in the 95% CI, 78-81 years), 73 years in ⑀4–heterozygous (SE=1; AD group (4.7%) compared with the control subjects 95% CI, 72-75 years), and 70 years in ⑀4–homozygous (1.3%). The OR for AD in association with B/B in the mul- patients with AD (SE=1; 95% CI, 68-72 years) (log rank ticenter sample was 3.87 (95% CI, 1.13-13.21). Regres- PϽ.001; df=2).

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©2000 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 COMMENT terminal hydrophobic signal sequence that is removed during synthesis. The KspI polymorphism in CST3 re- The results of this study establish a genetic association sults in an amino acid exchange from alanine to threo- between CST3 B/B and AD. There was an excess of the nine at the –2 position for signal peptidase cleavage (Fig- CST3 B/B genotype among patients with AD compared ure, B). This variation alters the hydrophobicity profile with control subjects in 2 independent study popula- of the signal sequence, and it reduces its ratio of pre- tions. CST3 B/B was present in 4.7% to 9.1% of our pa- dicted ␣-helix to ␤-sheet contents by approximately 42%. tients with AD compared with 10% to 14% APOE ⑀4/⑀4 This variation could be associated with changes in se- in this study. In addition, CST3 B/B significantly re- cretory processing of cystatin C, but our data do not pro- duced the average disease-free survival by 4 years. Both vide information whether such changes are disease- effects seemed to be independent of APOE genotype. To- related or the polymorphism tested in this study is in gether with the absence of CST3 B/B in cognitively nor- linkage disequilibrium with another disease-related poly- mal control subjects older than 74 years, these data in- morphism upstream or downstream of the analyzed se- dicate that CST3 B/B is a risk factor for late-onset AD. quence. Although statistically significant in this association study, Cystatin C is increased in AD brains, along with its it may be difficult to detect effects of this magnitude with high-affinity substrate cathepsin S that is known to cleave microsatellite markers in whole genome scans of 300 to APP into ␤-amyloid peptide (A␤)–containing derivatives 600 sib pairs. This difficulty may explain the reported in vitro, and to increase A␤ generation in tissue cul- absence of positive LOD scores at chromosome 20p11.2, ture.41,42 Cystatin C inhibits cathepsins with a profile that the genomic region of CST3.37 is similar to that of the inhibitor E-64, Of the susceptibility genes reported to influence the which is known to differentially affect ␥40- and ␥42- risk of developing AD, the effects of the APOE ⑀4 allele secretase processing of APP.15,43 Further studies are re- are best known and accepted. Carrying the ⑀4 allele in- quired to test whether cystatin C has similar activities on creases the risk of developing AD and lowers the age of APP processing, A␤ generation, and amyloid deposition, onset in a dose-dependent fashion, whereas the and whether these differ among the 2 allelic variants. ⑀2 allele may protect against AD, or at least delay its on- The Icelandic form of hereditary cerebral hemor- set. The mechanisms by which APOE affects AD are un- rhage with amyloidosis (HCHWA-I) is caused by a leu- known, although there is an ⑀4 dose-dependent in- cine-to-glutamine at position 68 in cystatin C.44,45 crease in brain amyloid deposits,38 and amyloid plaques As a result of this mutation, cystatin C amyloid aggre- are reduced in transgenic mice lacking APOE.39 APOE gates more readily than the wild-type form.46 The genotypes do not influence the rate of cognitive decline HCHWA-I is characterized by recurrent and pre- once dementia sets in.40 Because the influence of APOE mature death before age 40 years and is associated with is so pervasive in AD, we determined the APOE geno- the deposition in brain blood vessels of cystatin C amy- types of our patients and control subjects to distinguish loid. This disease is clearly different from late-onset AD APOE from CST3 effects. In doing so, we confirmed in dementia in which there is no point mutation in cys- our populations’ prior reports that the APOE ⑀4 allele was tatin C at position 68. Instead, our genotype data pro- associated with sporadic late-onset AD, and that APOE vide evidence for a contribution of CST3 B/B as a ge- ⑀4 shortened the mean dementia-free survival time. We netic risk factor to the multifactorial etiology of late- also documented that the association of the CST3 B/B onset AD. Whether CST3 exerts this effect by influencing genotype with sporadic late-onset AD was independent amyloid deposition, the inflammatory response, or by of APOE, as indicated by a similar OR of CST3 B/B for some other mechanism remains to be determined. AD in APOE ⑀4–negative subjects compared with APOE ⑀4–positive participants. Because 2.9% of our patients with Accepted for publication August 4, 2000. AD but none of the control subjects were APOE ⑀4– From the Departments of Human Genetics (Drs Finckh positive CST3 B/B carriers, additive effects of APOE and and Zurdel and Messrs Menzer and Holst) and Psychiatry CST3 cannot be excluded in this group of patients. (Drs Mu¨ller-Thomsen and Mann and Ms Zuchowski), Uni- In contrast to APOE ⑀4, the heterozygous B allele versity Hospital Hamburg-Eppendorf, Hamburg- did not reduce mean age of onset or survival. It is there- Eppendorf, Germany; the Center for Molecular Neurobiol- fore possible that the effects of the CST3 B allele on the ogy, University of Hamburg, Hamburg, Germany (Drs von risk of AD followed a recessive pattern, suggesting that der Kammer, Mu¨ller-Thomsen, and Nitsch, Mr Velden, Mss CST3 B may represent a recessive risk allele for late- Michel, Andresen, Zhang, and Zuchowski); the Depart- onset AD. In the participants of this study, the OR of CST3 ment of Neurology, Massachusetts General Hospital, Bos- B/B for AD increased with advancing age from 2.59 at ages ton, Mass (Drs Rebeck, Hyman, and Growdon and Ms Deng); younger than 75 years to 8.78 in the age group 75 years the Department of Psychiatry, University of Bonn, Bonn, Ger- and older, suggesting that the risk of AD attributable to many (Drs Papassotiropoulos and Heun); the Departments CST3 B/B reaches its maximum at an older age than the of Psychiatry and Human Genetics, University of Goettin- risk attributable to APOE ⑀4, and that CST3 B/B is a risk gen, Goettingen, Germany (Drs Stoppe and Reiss); the Uni- factor particularly for late-onset AD. This pattern differs versity of Basel, Basel, Switzerland (Drs Miserez, Staehe- from the effects of APOE, which peak in the seventh and lin, and Hock); the Scientific Institute for Research and Patient eighth decades of life and then decline thereafter. Care, Brescia, Italy (Drs Benussi and Binetti); and the Di- The role of cystatin C in the pathogenesis of AD is vision of Psychiatry Research, University of Zurich, Zurich, unknown. Cystatin C is initially synthesized with an N- Switzerland (Drs Hock and Nitsch and Ms Michel).

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©2000 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 This study was supported in part by grants from the 24. Miyake T, Gahara Y, Nakayama M, Yamada H, Uwabe K, Kitamura T. Up- Alzheimer’s Association, Chicago, Ill (IIGR-99-1755); Tele- regulation of cystatin C by microglia in the rat facial nucleus following axotomy. Brain Res Mol. 1996;37:273-282. thon-Italy, Rome (E1084); and the National Institute on Ag- 25. Katakai K, Shinoda M, Kabeya K, et al. Changes in distribution of cystatin C, apo- ing, Bethesda, Md (P50 AG05134). lipoprotein E and ferritin in rat hypothalamus after hypophysectomy. J Neuro- Reprints: Roger M. Nitsch, MD, Division of Psychia- endocrinol. 1997;9:247-253. try Research, University of Zurich, August Forel Str 1, 8008 26. Vinters HV, Nishimura GS, Secor DL, Pardridge WM. 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