Genetic Association and Linkage Analysis of the Apolipoprotein CII Locus and F-m-ilial Alzheimer’s Disease Gerard D. Schellenberg, PhD,“ Michael Boehnke, PhD,I Ellen M. Wijsman, PhD,$ Deborah K. Moore, PhD,’ George M. Martin, MD,6 and Thomas D. Bird, MDll

We previously reported a genetic association between the 3.5 kb (F) Taq I restriction fragment length polymorphism allele of the apolipoprotein CII gene on chromosome 19 and familial Alzheimer’s disease. Here, we report an additional analysis of this association performed on an expanded and better defined data set of 23 families with familial Alzheimer’s disease. The F allele frequency in affected family members in the expanded set was 0.62 k 0.06 (mean k standard error, n = 51 subjects), which differed significantly from a frequency of 0.39 -+ 0.02 (n = 226) for unrelated control subjects ( Z = 3.75,p < 0.0002). These results are consistent with our previous findings and suggest an association between the F allele of apolipoprotein CII and familial Alzheimer’s disease. When the apolipoprotein CII locus was tested for linkage to familial Alzheimer’s disease, LOD scores summed for the complete group of families were negative and close linkage was excluded. Close linkage was also excluded for early-onset families (mean onset age 5 60 years), but small positive LOD scores were obtained for late-onset kindreds. Schellenberg G D , Boehnke M, Wijsman EM, Moore DK, Martin GM, Bird TD. Genetic association and linkage analysis of the apolipoprotein CII locus and familial Alzheimer’s disease. Ann Neurol 1992;31:223-227

The role of genetic factors in the occurrence of Alzheimer’s disease (AD) is well documented. Studies of families of AD probands have repeatedly shown that a family history of A D is a significant risk factor for developing the disease (e.g., [l]). In some rare families, a clear pattern of inheritance is observed (e.g., [2, 33,

From the Divisions of “Neurology RG-27 and SMedicai Genetics GG-19, Department of Medicine, and QDepanmentof Pathology SM-30, University of Washington, Seattle, WA; tDepartment of Biostatistics, School of Public Health, University of Michigan, Ann Arbor, MI; and //Divisionof Neurology, Veterans Administration Medical Center, Seattle, WA. Received Aug 21, 1990, and in revised form Jul 10, 1991. Accepted for publication Jul 14, 1991. Address correspondence to Dr Schellenberg,Division of Neurology RG-27, University of Washington, Seattle, WA 98195.

suggesting that a single dominant gene can cause AD. Recently, Goate and colleagues [4] identified an Alzheimer precursor protein (APP) gene mutation that appears responsible for familial AD (FAD) in a subset of early-onset kindreds [4, 53. The finding of this mutation confirms that in at least some families, FAD is inherited as an autosomal dominant trait. This mutation is not found in many other early-onset kindreds and has not been observed in any late-onset families or patients with sporadic A D C63. In other families, clustering of patients with AD is less dramatic, suggesting that if a dominant gene is responsible for AD, penetrance is incomplete. Alternatively, in these families, multiple genes may contribute to the occurrence of AD. Finally, in the families of most probands, no additional patients with A D are found {l} and, thus, in the most common form of AD, inheritance may not play a major role in pathogenesis. Resolving the mode of inheritance for A D is complicated by fact that the onset of AD typically occurs late in life, and competing causes of death may mask the true extent of familial clustering of the disease. Recent attempts to locate the genes responsible for FAD have focused on both early- and late-onset kindreds with multiple affected members in several generations. Suggestive linkage analysis evidence for an early-onset FAD locus near the centromere of chromosome 21 has been reported [7}. Although this study included a family with the APP,,- mutation, the positive chromosome 2 1 linkage results may indicate the presence of an additional FAD gene centromeric to the APP gene. In other family groups, the same region and the APP gene have been excluded r7-91. More recently, linkage evidence for a late-onset FAD locus on chromosome 17 has been described 173. We previously reported an association between an allele of the apolipoprotein CII (Apo CII) gene (chromosome 19) and FAD in patients from 10 kindreds [lo]. Here, we report new analyses of this genetic association based on an expanded cohort of families. We also report results of linkage analysis between Apo CII and FAD.

Materials and Methods Families The characteristics of the 23 families used in this study are outlined in Table 1 and are described in more detail elsewhere 12,3, 6, lo}. All sampled individuals were examined by a neurologist and associated diagnostic personnel as previously described [2, 31. Confounding disorders such as depression and multi-infarct dementia were carefully excluded. For deceased individuals, medical records were obtained whenever possible to corroborate family history information. For this study, informed consent was obtained from each subject or next-of-kin with approval of the University of Washington Human Subjects Review Committee. Autopies were obtained for at least 1 patient in all families except the HD kindred, a Volga German family (2, 31. The previous

Copyright 0 1992 by the American Neurological Association

223

Tabh 1. Characteristls

of

FAD Kindreds

~

Number of Subjects Genotyped"

Family

Number of Affecteds Geiiotyped

Volga German kindreds R 29 W 4 E 12

HD H HI3 KS

* 5.1 (17) * 4 5 (4) 57 * 5.0 ( 5 ) 57 t 8 5 (14) 57 * 1.1 ( 5 ) 60 * 7.5 (18) 51 54

7

2 25 16 Non-Volga German early-onset kindreds L 20 KG 11 V 4

60 3 SNW

60 5 5.9 (10)

3

3 17

Late-onset kindreds 2 TwHos BH 2 CK 3

P T WLA

RR CSF MI BMH JR Totals

Mean Age-of-Onset t SD (n)

1

41 I 5 . 1 (12) 44 1.1 ( 5 )

2 2 4

51

* 46 * 4.1 (7) 48 * 6.7 (19) 2.7 (7)

* * *

61 2.8 (2) 64 f 0.7 (2) 65 8.3 (3) 67 5.1 (4) 68 t 6.6 (5) 68 t 4.9 ( 3 ) 69 t 4.6 ( 4 ) 7 0 6.1 (4) 70 4.6 (4) 76 9.9 (2) 78 f 2.3 ( 3 )

2 2 2 2

3 2 3 6 6 3 5 5 170

?z

2

3 2 2

* * *

2 1

1 51

Ethnic Origin Voiga Volga Volga Volga Volga Volga Volga

German German German German German German German

German British English ( ? ) French-Canadian RussianiJewish FrenchiGerman British Lithuanian English Norwegian Swedish Black Sea German Danish English ( ? ) English English

"Subjects genotyped were affected individuals, unaffected blood relatives at risk for FAD, and spouses. Genorypc data for the spourcs were used for the linkage analysis but n o t the association analysis. Late-onset fiamilies with FAD are defined as those having an onset mean o f morc than 60 years. FAD = familial Alzheimer's disease.

analysis of Apo CII genotypes included familes BK and GCSA, which were (omitted from the present study. The GCSA kindred was subsequently found to have GerstmannStraussler-Scheinker syndrome [ I I]. Further autopsy data from the BK family showed numerous neurofibrillary tangles but no plaques or detcccable amyloid deposits [3]. This family also had an unusually long duration of disease 131. These results were not entirely consistent with A D and, thus, this kindred was not used in the present analysis. Of the 8 remaining families from the original study, 4 have been expanded. In the V, SNW, L, and P families, 1 addiitional affected patient each has been sampled. Included in the current sample are 7 Volga German kindreds who share a common ethnic origin. FAD in I-hesefamilies is thought to be inherited from a common genetic founder [2]. In the original study, only 1 affected patient from 1 Volga German family (the H kindred) was analyzed.

Assorzation Ana4ysis Estimation of allele frequencies for patients with IFAD was performed as described previously 110, 121. For many of the families used, genotype data were obtained for multiple affected and at-risk subjects. Because blood relatives share genes and are not independent, determination of allele frequencies by allele cocnting is inappropriate. Therefore, we

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calculated the probability of the family data by considering not only the marker and disease phenotype, but also the family relationships using the computer program MENDEL [lo, 121.

Linkage A nalysir LOD scores were calculated using the computer program LIPED modified to handle up to 10 alleles with a cumulative normal age-of-onset correction as previously described [8 1. Autosomal dominant inheritance with age-dependent penctrance was assumed. Apo CII and FAD were assumed to be in linkage equilibrium. The age curve was constructed as a cumulative normal distribution using either the overall sample mean of M = 65.66 years or family-specific means, and an overall standard deviation of 10.87 years. [.OD scores were also calculated by setting the penetrancc of the AD which effectively results in ignoring the AD genotype to phenotypes of the unaffected, but at-risk, subjects.

ls,

Results We previously reported a genetic association between the F (3.5 kb) Taq I restriction fragment iength polymorphism allele of Apo CII and FAD. In that study, genotypes for 21 patients with A D from 10 kindreds

Table 2. Apo Cll Allele Frequency Estimates F Allele Frequency

* SE (da

Population

Affected (Demented)

Unaffected (Not Demented)

Combined

All FAD Families Volga German families Early-onset (NVG) families All early-onset ( N V G + VG) Late-onset families New FAD families Normal control subjects

0.62 5 O.OGb ( 5 1 ) 0.65 0.10‘ ( 1 8 ) 0.70 k O.lOd ( 1 2 ) 0.67 2 0.07‘ (30) 0.54 5 0.09‘(21) 0.65 +- 0.079 ( 3 3 )

0.45 2 0.05 (119) 0.47 2 0.07 (64) 0.44 2 0.09 (36) 0.46 2 0.05 (100) 0.39 0.10 (19) 0.49 +- 0.06 (85)

0.49 2 0.04 (170) 0.51 t 0.07 (82) 0.52 2 0.08 ( 4 8 ) 0.51 0.05 (130) 0.45 0.08 (40) 0.52 0.05 (118) 0.39 2 0.02h (226)

*

*

* * *

’n is the number of subjects genotyped. The unaffected group includes blood relatives at risk for familial Alzheimer’s disease (FAD) but does not include spouses. Genotypes for the apolipoprotein (Apo) CII Taq I restriction fragment length polymorphism site were determined as previously described [ 101. Early-onset families with FAD are arbitrarily defined as those with family mean age-of-onset of 60 years or less. The Volga German kindreds as a group have an onset mean age of 57 years bZ = 3.75, p < 0.0002 compared with combined controls. ‘2 = 2.61, p < 0.01 compared with combined controls. dZ = 2.69, p < 0.008 compared with combined controls. ‘2 = 3.77, p < 0.0002 compared with combined controls. ‘2 = 1.60, 0.10 < p < 0.15 compared with combined controls. 8 2 = 2.89, p < 0.004 compared with combined controls. hFrom [ 10, 151.

VG = Volga German; N V G = non-Volga German.

were compared with data for 226 normal control subjects. The estimated F allele frequency for the patients with FAD was 0.64 k 0.08 (mean -t standard error) which differed significantly from that for the control group (0.39 0.02; Z = 2.87, p < 0.005). To test these original findings, we have reexamined this genetic association in an expanded cohort of 23 families with FAD, which includes 5 1 affected patients and 123 relatives at risk for AD. Results of the association analysis of the expanded Apo CIl genotype data set are shown in Table 2. The estimated F allele frequency for affected patients was 0.62 0.06, which was significantly different from the allele frequency in the 226 normal control subjects ( Z = 3.75, p < 0.0002). The increased level of significance of this study compared with our previous results [lo] is due to the larger number of individuals sampled. Similar results were obtained when the analysis was restricted to subgroups consisting of the Volga German families, non-Volga German early-onset kindreds, or all early-onset families (see Table 2). Also, when only new families not in our previous report were analyzed, the allele frequency for the affected FAD group was significantly different from that for controls (see Table 2). For late-onset families, the estimated F allele frequency for affected patients (0.54 0.09) was intermediate between control subjects and affected patients in the other groups of families analyzed (see Table 2). The allele frequency difference between control subjects and late-onset affected patients, however, was not statistically significant. We also tested for genetic linkage of the Apo CII gene to FAD assuming an autosomal dominant mode

*

*

of inheritance with age-dependent penetrance (Table 3). LOD scores for the group as a whole and for most of the different subgroups were negative. Tight linkage of FAD to Apo CII could be excluded (LOD 5 -2.0) for the entire cohort of families and for each of the subgroups except the late-onset kindreds. For the latter subgroup, small positive LOD scores were obtained that did not reach statistical significance. Some of the evidence against linkage comes from obligate recombinants between Apo CII and FAD in the L, 603, and SNW families. When the data were analyzed assuming 1% penetrance, which eliminates assumptions about the nature of age-dependent penetrance, close linkage to Apo CII was again formally excluded for the group as a whole and for each of the subgroups except the late-onset families, which yielded small positive LOD scores. These data do not exclude more distant linkage nor do they necessarily exclude linkage if there is within group heterogeneity. This group of families and the subgroups also do not show linkage to genetic markers for the proximal region of the long arm of chromosome 21 183.

Discussion The data in Table 2 are consistent with our original observation of a genetic association between the F allele of the Apo CII locus and FAD. The validity of the observed association depends on the match between the control population and the population from which the FAD kindreds originated. If the F allele frequency differs between the 2 populations, or if frequencies from mixed populations are pooled, a false-positive re-

Brief Communication: Schellenberg et d: Apo CII and Familial Alzheimer’s Disease

225

Table 3. LOD Scoresfor Linkage of APo C I I

to

FAD"

___ Recombination Fraction

Group ( N u m b e r of Families)

0.001

All ( 2 1) Volga G e r m a n (7) Early-onset, NVG ( 5 )

All early-onset, N V G

+ VG i12)

Late-onset ( 9 )

-21.11 (-4.35) - 10.70 (-2.01) .- 11.11 ((-3.35) -- 2 1.80 (-5.36) 0.69 (0.42 )

0.05 - 10.38

(-2.62) - 5.90 ( - 1.22) -5.17 (-1.76) - 11.06 (-2.99) 0.68 (0.37)

(0)

.__

0.10

0.15

0.20

0.30

-6.73 (-1.49) -4.00 (-0.80) -3.36 (-1.01) - 7.35

-4.41 (-0.83) -2.73 (-0.53) -2.21 (-0.56) -4.94 (-1.10) 0.53 (0.27 )

- 2.83 (-0.44)

- 1.05 (-0.08) - 0.82 (-0.18) - 0.44 (-0.01) - 1.27

(-1.81)

0.62 (0.32)

-

1.85

(-0.37) - 1.41 (-0.28) -3.26 (-0.65) 0.43 ( 0 . 21)

(-0.19) 0.2 1 (0.11 )

0.40 -- 0.30

(0.00) --0.33 (--0.08)

0.04 (0.05) - 0.36

--

(-0.03) 0.06 I 0.0 3 )

"OD scores were computed using haplotypes constructed from data for the Taq I restriction fragment length polymorphism s ~ t eand the apolipoprotein (Apol C11 minisatellite polymorphism described by Weber and May [ 171. Families B H and TwHOS (Table 1) were not included in the linkage analysis because only a limited number of subjects were available for sampling. A gene frequency of 0.01 and cumulative normal age-of-onset correction was used to compute EOD scores. LOD scores in parentheses were computed assuming l ( ' f penetrance. FAD

=

tamilial Alzheimer's disease; N V G

=

non-Volga German; VG = Volga German

sult could be obtained. Although the F allele frequency estimates do vary in different ethnic populations [ 131 and the possibility of a type I error cannot be excluded, the allele frequency value used in the above analysis is consistent with reported estimates of the F allele frequency in Caucasians which range from 0.36 [I01 to 0.44 [13--16} including an estimate of 0.44 for a German control population { 161. The linkage data appear to exclude Apo CII and closely linked genes such as Apo E and Apo CI as candidates for a major gene responsible for autosomal dominant FAD. T h e F allele could be in linkage disequilibrium with a penetrance modifying allele, however, possibly affecting age of onset. The presence of an F allele is clearly not required for FAD, as some affected patients are S/S homotygous. 14n alternate hypothesis is that in at least some kindreds, a multigenic mode of inheritance may be responsible for FAD and an autosomal dominant model inappropriate. T h e reported association and the small positive LOD scores for the late-onset families are particularly interesting in light of the recent report of positive linkage results for late-onset families for this region of chromosome 19 {9}. Although the late-onset group LO11 scores in Table 3 are nor of sufficient magnitude to confirm the reported linkage, these data are consistent with the chromosome 19 localization of a late-onset FAD gene or susceptibility locus as reported by Pericak-Vance and colleagues {9]. The late-onset families used in this study do not show positive linkage results for chromosome 21 markers ( [ S } , see also [9}).T h e identification of a FAD mutation in the APP gene {4, si), the positive linkage data for early-onset {7) and late-onset kindreds 191 o n chromosomes 2 1 and 19, respectively, and the genetic association data reported here indicate that FAD is genetically heterogeneous.

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Vol 31 No 2 February 1992

Supported by National Institutes of Health Grants AG OS 136 (Alzheimer's Disease Research Center of the University of Washington. G.M.M), HG00376 and HG00290 (M B.), GM 15251 (Veterans Administration Research Funds, T.D.B.),the American I-lealrh Assistance Foundation (G.D.S), the French Foundation (G.D.S.).and the Kennedy Fluid Research Fund (G.D.S.). G. D. S., T. D. B., and G. M. M. are affiliates of the Child Development and Mental Retardation Center at the University of Washington (Seartie. WA). G. D. S., T. D. B., D. K. hl., and G. M. M. are members o f the University of Washington Alzheimer's Disease Research Center. We thank L. J. Anderson, S. Odahl, and E. N. Loomis for excellent technical assistance, E. Nemens and H . Lipe for obtainmg blood samples and medical records, and S. Fredell and M.Muna for cell culture work.

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