American Journal of Medical Genetics (Neuropsychiatric Genetics) 60:550-553 (1995)

Evidence for Apolipoprotein E €4 Association in Early-Onset Alzheimer’s Patients With Late-Onset Relatives

Jordi Pbrez-Tur, Dominique Campion, Maria Martinez, Alexis Brice, Sandrine Tardieu, Didier Hannequin, Yves Agid, Andrb Delacourte, Franqoise Clerget-Darpoux, Marie-Christine Chartier-Harlin, and the French Alzheimer’s Disease Collaborative Study Group Unite 422 INSERM (J.P.-T., A.D., M-C.C.-H.), Lille, Unite 155 INSERM (D.C., M.M., F.C.-D.) and Unit6289 INSERM (A.B., S.T., Y.A.), Paris, CHU (D.H.), Rouen, France

Recently several reports have extended the apolipoprotein E (AF’OE) €4 association found in late-onset Alzheimer’s disease (LOAD) patients to early-onset (EO) AD pa- tients. We have studied this question in a large population of 119 EOAD patients (on- set 560 years) in which family history was carefully assessed and in 109 controls. We show that the APOE €4 allele frequency is in- creased only in the subset of patients who belong to families where LOAD secondary cases are present. Our sampling scheme per- mits us to demonstrate that, for an individ- ual, bearing at least one €4 allele increases both the risk of AD before age 60 and the probability of belonging to a family with late-onset affected subjects. Our results sug- gest that a subset of EOAD cases shares a common determinism with LOAD cases. 0 1995 Wiley-Liss, Inc.

KEY WORDS: association study, sporadic and familial AD cases, family history of dementia

INTRODUCTION Currently, 3 genetic loci have been identified that

predispose to Alzheimer’s disease (AD): the APP gene on chromosome 21 [Goate et al., 19911, a yet unidenti- fied gene on chromosome 14 [Schellenberg et al., 19921

Received for publication September 20, 1994; revision received March 23, 1995.

Address reprint requests to Dr. M.-C. Chartier-Harlin, Unit6 422-INSERM, Place de Verdun, F59045 Lille Cedex, France.

The French Alzheimer’s Disease Collaborative Study Group is Michel Bellis, Bruno Dubois, Yves Goas, Catherine Thomas-An- terion, Agnes Jaillard-Serradt, Frangois Ledoze, Jacques Mallet, Agnes Michon, Florence Pasquier, Michele Puel, and Marie- Agnes Zimmerman.

0 1995 Wiley-Liss, Inc.

and the apolipoprotein E gene (APOE gene, apoE pro- tein) on chromosome 19 [Strittmatter et al., 1993a1. This latter has three major alleles, €2, €3, €4, which are found in the general population. The three apoE poly- morphic proteins differ by a single amino acid substi- tution E2 (Cys”’, Cys”’), E3 (Cys1l2, Arg”’), and E4 (Arg”’, Arg”’). Strittmatter and colleagues have shown that the APOE gene located in chromosome 19q13.2 was a susceptibility gene for the clinical expression of AD. The E4 isoform of apoE has been identified as a fac- tor increasing the risk for both early-onset (EO) and late-onset (L0)AD of sporadic and familial type [Corder et al., 1993; Mayeux et al., 1993; Saunders et al., 1993; Strittmatter et al., 1993; Chartier-Harlin e t al., 1994; Okuizumi et al., 1994; van Duijn et al., 19941. The abundant confirmation of these important data in the literature attest to the critical role of APOE €4 in AD. Interestingly, van Duijn and collaborators [19941 stud- ied a large EOAD population in order to “examine effect modification of the association by family history of dementia” and reported a “higher frequency of the e4 allele in patients with positive family history than those with negative history.” In this study, we analysed the APOE €4 allele frequency in a large EOAD popula- tion who was further classified regarding to the distri- bution of age at onset among secondary cases. We obtain evidence for the association with the €4 allele in one familial EOAD subgroup.

POPULATION AND METHODS Population

One hundred nineteen unrelated Caucasian AD pa- tients of French origin with onset 560 years (mean age at onset of 54.4 t 4.8 years, range 37-60) were re- cruited from consecutive admission in several hospitals [Campion et al., 19951. All the patients fulfilled the NINCDS-ADRDA criteria for probable AD. The control population was composed of 109 persons matched for place of birth and age who shared the same environ- ment (i.e., the spouses of the proband, who usually agreed to participate to the study).

APOE and Early-Onset AD 551

obtained in the 119 unrelated EOAD patients (onset 560 years) and in 109 healthy controls are presented in Table 11. APOE allele distribution was significantly dif- ferent in controls and patients (2 = 13.57, P = 0.0009, with 2df) the €4 allele being higher in patients (28%) than in controls (16%). The increase of €4 allele fre- quency was a t the expenses of both €2 and €3 alleles. When the €4 alleles were excluded from the pool of alleles, the €2 allele frequency was slightly more de- creased than €3, but the difference was not statistically significant (P = 0.052). In controls, the allelic frequen- cies found for the three APOE alleles were similar to those published in the literature. No €21~2 homozygous was observed in both groups. Only 3 control individuals were typed €21~4, while this genotype was absent in the patient group. Both genotypes €31~4 and €41~4 were sig- nificantly increased among EOAD patients ( x 2 = 14.93, P = 0.003 with 4df).

The 3 subgroups of probands (sporadic, familial type 1, and familial type 2 cases) were not homogeneous ei- ther in APOE allele frequencies ( x 2 = 16.19, P = 0.0031 with 4df) or genotype distributions (x2 = 15.76, P = 0.014 with 6df). The frequency of €4 allele was 20% in sporadic patients, 19% in familial type 1 probands, and 45% in familial type 2 probands. With respect to the al- lele frequency found in controls (16%), the €4 allele was only significantly increased in type 2 patients. Con- servely, APOE allele and genotype frequencies were not significantly different between sporadic and familial type 1 cases and controls.

The odds ratios for EOAD according to the number of €4 allele carried by subjects are shown in Table 111. The association between AD and APOE €4 allele was statis- tically significant only for type 2 probands. In the cor- responding group the odds ratio for the association of AD with one or two €4 alleles was 4.8 (95% CI [2.2&10.651. The risk was higher for bearers of two €4 copies than for €4 heterozygotes. Analysis of the gene dosage in type 2 probands revealed no correlation between age at onset and the €4 allele copy number. The average age a t on- set for subjects with two APOE €4 alleles [ ~ 4 / ~ 4 (n = 9)], one APOE €4 allele [ ~ 4 / x (n = 1611 or without APOE €4 allele [XK (n = 13)] was 56.8 2 2.8, 54.2 2 4.9, 54.0 2 4.9, respectively. The same calculation performed in the other subgroups of patients led to the same negative re- sult (data not shown). I t is possible that the truncation of the age a t onset distribution of our population may have limited the statistical power to detect such an effect.

DISCUSSION In this report, we compared a population of 119 un-

related EOAD patients (onset 5 6 0 years) to a popula- tion of 109 healthy controls matched for age and places of birth. We demonstrated a significant excess of APOE €4 allele in the the whole EOAD population. This excess was a t the expense of both €3 and €2. Among the 3 groups of patients, heterogeneity for APOE alleles and genotypes distributions is significant.

For sporadic EOAD probands, there is only a modest increase in the €4 allele frequency as compared to con- trols. Interestingly, van Djuin et al. [19941 have de-

For all patients detailed data on family history of de- mentia were collected on 3 sibships (the proband’s sib- ship and each parental sibship). A particular attempt was made to examine all living relatives and multiple informants were used for deceased or unavailable rela- tives. This information was used to define two groups of probands: 1. Sixty-three probands presented no family history of

dementia and constituted the sporadic subgroup (mean onset among probands: 54.7 2 3.1)

2. Fifty-six probands belonged to families displaying secondary cases among parents and/or second- degree relatives. These probands were identified as familial probands. In this group, the distribution of the age a t onset among relatives was examined and allowed to distinguish 2 types of familial probands (types 1 and 2) as defined in Campion et al. [1995]. The familial type 1 probands belonged to pedigrees where all secondary cases had an onset before age 60 (n = 18, mean onset among probands: 49.7 2 6.0, mean onset among relatives: 50.3 ? 6.1). The re- maining familial type 2 probands belonged to pedi- grees where LOAD (i.e., onset >60) secondary cases were present (mean onset among probands = 56.1 2 3.1; mean onset among affected relatives = 74.4 2 8.3). Table I shows the stratification of our 119 EOAD cases with respect to this classification.

APOE Genotype Genomic DNA was obtained from peripheral blood

cells and APOE alleles €2, €3, and €4 were determined using a polymerase chain reaction based strategy as de- scribed [Hixson and Vernier, 19901. The primers were designed to amplify a DNA fragment of 244 bp contain- ing the APOE polymorphism. The amplification prod- ucts were digested with HhaI, subjected to electro- phoresis on a 10% non-denaturing polyacrylamide gel, and then stained with ethidium bromide.

Statistical Analysis Allele frequencies were determined by counting the

number of each allele and by expressing i t as a propor- tion of the allele total number. The distributions of APOE genotypes in patients and controls were com- pared by a x2 test. A Mann-Whitney U-test was used to compare the mean ages at onset between the different subpopulations. Odds ratios (OR) were calculated and computed with 95% confidence intervals.

RESULTS The allelic frequencies and genotype distributions

TABLE I. Sex and Age a t Onset of EOAD Population

Sex Age a t onset Men Women (mean 2 S.D.)

Isolated 21 42 54.7 2 4.4 Familial type 1 8 10 49.7 t 6.0 Familial type 2 14 24 56.1 5 3.1 All 43 76 54.4 i: 4.8

552 P6rez-Tur et al.

TABLE 11. Genotypes and Allele Frequencies in EOAD and Control Populations

Genotypes n €2/€3 23 €3/€3 33 €2/€4 24 €3/€4 34 €4/€4 44

Alleles Chrom €2 €3 €4

AD cases

Controls 109

14 64

3 25

3 218

7.8% 76.6% 15.6%

All 119"

7 58 0

42 12

23Sb 2.9%

69.3% 27.7%

Isolated 63" 4

36 0

21 2

126"" 3.2%

77.0% 19.8%

Familial type 1 18* 1

11 0 5 1

2.8% 77.8% 19.4%

36""

Familial type 2 38"

2 11 0

16 9

76"" 2.6%

52.6% 44.7%

~~ ~

"AD versus controls, P = 0.003 ( x 2 = 14.93 with 4 dfl. 'AD versus controls, P = 0,00091 ( x 2 = 13.57 with 2 dfl. *P = 0.014, ( x 2 = 15.76 with 6dfl. Isolated versus controls, x 2 = 1.22 with 2df,P = nonsignificant.,Familial type 1 versus controls, xa = 0.46 with Zdf, P = non significant. Familial type 2 versus controls, x1 = 24.01 with 2df, p = 10 ,7 .

**P = 0.0031, (x' = 16.19 with ldo . Isolated versus controls, X a = 1.01 with ldf, P = non significant. Familial type 1 versus controls, xL = 0.34 with ldf, P = nonsignificant. Familial type 2 verslis controls, 2 = 26.92 with ldf , P =

lation of EOAD patients, whereas in other reports [Mayeux et al., 1993; Chartier-Harlin e t al., 1994; Okuizumi et al., 19941 evidence for association in spo- radic cases was found. These different results may be due to the definition of "a sporadic case," which has no intrinsic meaning but depends, indeed, on the familial information. In contrast to most of the studies, the in- formation was extended to the second degree relatives in our study. Limiting the information to the first de- gree relatives would have led us to include 12 out of 38 type 2 probands in the sporadic group. In view of the imperfection inherent in the definition of sporadic dis- ease, we would suggest it is likely that there is no asso- ciation between genuinely sporadic EOAD and apoE.

In the group of type 1 patients, with familial age at onset below 60, there is no evidence for the role of APOE €4 allele. Currently, we have some information concerning the genetic defects present in our familial type 1 probands. Exons 16 and 17 APP mutations [Goate et al., 1991; Murrel e t al., 1991; Chartier-Harlin et al., 1991; Mullan et al., 19921 were not found among any of these patients [Carter et al., 1992; Brice et al., 19931. Conversely, both linkage [Campion et al., 19951 and association studies [Brice et al., 19951 have sug- gested that segregation of AD within a large proportion of the families of these probands is determined by a pathological mutation located on chromosome 14 [Schellenberg et al., 19921 These results are in agree- ment with a previous report suggesting that apoE is not involved in the development of the disease within

EOAD probands from families with chromosome 14 encoded AD [Van Broeckhoven et al., 19941.

The main finding of this study is the considerable in- crease of the €4 allele frequency among familial type 2 probands. Indeed, 66% had at least one allele €4 and 24% were homozygous €41~4. The interpretation is that for an individual, even before age 60, APOE €4 allele favours the onset of AD but the mean age a t onset is greater than 60 when considering his first and second degree relatives. van Duijn et al. [1994] concluded that APOE €4 is associated to EOAD when the family is compatible with dominant inheritance. Note that our type 1 probands and several of our type 2 probands be- long to families compatible with dominant inheritance but that the association with APOE €4 is restricted to type 2 probands [Campion et al., 19951. In other words, if we characterised the families of type 1 and type 2 probands by mean age of onset of affected members (re- spectively, 560 and >60), the association concerns only the second type of families. This is in agreement with the previous studies on LOAD. Our sampling scheme permits us to demonstrate that, for an individual, bear- ing a t least one €4 allele increases both the risk of AD before age 60 and the probability of belonging to a fam- ily with late-onset affected subjects. Our results sug- gest that a subset of EOAD cases shares a common de- terminism with LOAD cases.

Finally, since the APOE €4 association in EOAD probands is restricted to EOAD patients with LO af- fected relatives, the association observed in the whole

TABLE 111. Odds Ratios for EOAD Cases According to the Number of €4 Alleles (X)

€4 allele copies All AD cases Isolated Familial type 1 Familial type 2

X = l 1.80 [1.01-3.221" 1.46 [0.74-2.851 1.16 [0.38-3.551 3.43 [1.47-8.021" x 2 1 2.09 [1.21-3.631" 1.45 rO.21-8.101 1.26 [0.43-3.651 4.84 [2.20-10.65]:': X = 2 4.80 [1.30-17.74]* 1.30 rO.21-8.101 2.17 [0.21-22.571 18.10 [4.30-75.411"

~ ~~

*OR >1 for a P value of 0.05. 95% confidence intervals are in brackets.

WOE and Early-Onset AD 553

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668-671.

fected relatives, the association observed in the whole EOAD population is only to this subgroup. Our data in- dicate that this type of EOAD patients may be etiologi- cally related to LOAD patients. However, the €4 allele is not a sufficient condition to develop EOAD. Although the strength of the association in this particular sub- group is impressive, it remains true, however, that 34% of such probands did not carry this risk factor, indicat- ing that other factors are involved.

ACKNOWLEDGMENTS We are indebted to all patients and clinicians who

participated in our research. We thank Dr. Christine van Broekhoven for pre-publication data, and Dr L. Bu6e for critical reading of this manuscript. This re- search was funded by an INSERM Network “492002,” CNAMTS “998558,” and by a CHRU de Lille “9306” grant. J.P.-T. is a recipient of an INSERM fellowship. D.C. received a fellowship from “Fondation Pierre Louis pour 1’Epidemiologie du Viellissement.”

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