Context.— The only genetic locus universally accepted to be important as a risk
factor for late-onset Alzheimer disease (AD) is the apolipoprotein E (APOE) locus on chromosome 19. However, this locus does
not account for all the risk in late-onset disease, and a recent report has
suggested a second locus on chromosome 12p11-12.
Objective.— To look for evidence of linkage on chromosome 12 and to test for the
presence of the new locus in an independent sample of familial late-onset
Design.— Retrospective cohort study. As part of a 20-centimorgan genome screen
(approximately equal to 200 markers), we tested a series of 18 genetic markers
on chromosome 12 and carried out multipoint, nonparametric lod score and exclusion
Setting.— Clinic populations in the continental United States selected from the
National Institute of Mental Health AD Genetics Consortium.
Patients.— We selected samples for DNA analysis from affected sibling pairs, 497
subjects from 230 families with 2 or more affected individuals with probable
or definite AD with onset ages older than 60 years (mean±SD, 75±6
years). Within the families, we used the 2 probable or definitely affected
individuals. In families with more than 2 such cases available, we used all
of them; in families with only 2 such cases in which unaffected individuals
were available, we also sampled the oldest unaffected individual and used
genotype data from this unaffected individual to check for nonpaternity and
Main Outcome Measure.— Presence of linkage or locus on chromosome 12.
Results.— Although linkage analyses confirmed the presence of a genetic susceptibility
factor at the APOE locus in these families with late-onset
AD, we were unable to confirm the presence of a locus close to the marker D12S1042. A moderate lod score (1.91) was found near D12S98 close to the α2-macroglobulin locus
in the affected pairs in which both members did not possess an APOE ∊4 allele.
Conclusions.— APOE remains the only locus established to
be a risk factor for late-onset AD. We were unable to confirm that a locus
on chromosome 12p11-12 has a major effect on risk for late-onset AD, although
an effect smaller than that for APOE cannot be excluded.
LATE-ONSET Alzheimer disease (AD) shows familial clustering1 but does not show a clear mode of inheritance. The
only genetic locus universally accepted as an important risk factor for late-onset
AD is the apolipoprotein E (APOE) locus on chromosome
19.2 A proportion of this familial clustering
is accounted for by genetic variability at the APOE
locus. The APOE ∊4 allele is positively associated
with disease and the APOE2 allele is negatively associated
However, the APOE locus accounts for, at most, about
half of the genetic risk of developing the disease.3,7
Thus, other genes or risk factors must account for the remaining genetic risk
for developing disease.
Several strategies can be used to define these other genetic risk factors:
(1) the analysis of large pedigrees with late-onset disease; however, few
suitable pedigrees have been ascertained8;
(2) the genetic analysis of population isolates with disease; again, few such
isolates have been reported9; (3) the use of
association studies between alleles of candidate genes and disease; and (4)
the application of genome search strategies using large numbers of sibling
Association studies have the advantage of simplicity and speed since
all that is required is the availability of a case-control series of DNA samples
that can be tested for the presence of particular alleles. Furthermore, association
studies can detect relatively small increases in risk associated with particular
alleles. However, since the identification of the APOE
locus, a large number of positive-association studies have been reported for
AD, but none of these have been consistently confirmed. These failures to
confirm associations may reflect different etiologies in different populations,
type I statistical errors (a particular problem because multiple testing is
always involved), or linkage disequilibrium between the tested polymorphism
and the functional polymorphism (with this disequilibrium not present in all
populations). In addition, association studies are sensitive to population
substructure and unnoticed ethnic differences between cases and controls.
Furthermore, association studies currently cannot detect new genes implicated
in disease etiology; they can only be used to test existing "candidate" genes.
Genome search strategies have the disadvantage that they are considerably
less sensitive than association studies and require large amounts of work
in terms of sample collection, data generation, and data analysis. However,
these studies have the potential to identify previously unsuspected loci.
Given the uncertainty of the published disease associations, we initiated
a genome search for other loci. During this process, Pericak-Vance and colleagues10 published a report of a locus on chromosome 12, and
we tried to replicate this finding. A second reason for us to start the genome
search on chromosome 12 is the fact that we and others have found associations
between AD and the LRP gene (located on chromosome
encodes a brain-expressed APOE receptor. In addition,
the ligand for this receptor, α2-macroglobulin, is also encoded
on this chromosome.
We selected families from those collected by the the National Institute
of Mental Health AD Genetics Consortium.7 From
within this family series, we selected 230 families based on the following
criteria: at least 2 affected siblings with probable or definite AD with onset
ages of more than 60 years, sampled and available for genotyping. Within the
families, we genotyped the probable or definitely affected siblings. For families
with more than 2 affected siblings (definite or probable) available, we used
all of them; in families with only 2 affected siblings but in which unaffected
individuals were available, we also sampled the oldest unaffected individual,
so that the genotype data from this unaffected individual could be used to
check for genotyping errors (eg, data suggesting more than 4 parental chromosomes).
Comparison of the structure of our family series with that used by Pericak-Vance
et al10 is shown in Table 1, although we restricted our analysis to sibling pairs.
Generation of Marker Data
The microsatellite polymorphisms were detected by polymerase chain reaction.
The forward primers were labeled at the 5‘ end with a 6-carboxyfluorescein
known as 6-FAM, a tetrachlorinated analogue, or a hexachlorinated analogue
(Perkin Elmer Systems, San Francisco, Calif). The total 5-µL reaction
contained a 50-µg DNA genomic template, 3.5-mol/L end-labeled forward
primer and unlabeled reverse primer, 0.2 mol/L each of dextroadenosine triphosphate,
dextroguanosine 5‘-triphosphate, dextrocytidine 5‘-triphosphate,
dextroribothymide 5‘-triphosphate, 1 unit of Taq DNA polymerase (Promega),
and 1-µL 5× buffer (7.5-mmol/L magnesium chloride, 250-mmol/L
potassium chloride, 50-mmol/L Tris hydrochloride [pH 8.3]). All reaction cocktails
were distributed evenly to a 96-well Falcon assay plate using a Beckman-1000
workstation (Beckman Instruments, San Francisco, Calif). Polymerase chain
reactions were carried out on Hybaid OmniGene thermal cyclers (Hybaid, London,
England) using the following cycling conditions: initial denaturation at 96°C
for 4 minutes, followed by optimized (22-30) cycles of 94°C for 1 minute,
optimized annealing temperature (54°C-60°C) for 1 minute, and 72°C
for 45 seconds, and a final extension of 5 minutes at 72°C. The polymerase
chain reaction products were then diluted at least 8-fold using a Beckman-1000
workstation. A measurement of 0.8 µL of diluted polymerase chain reaction
product was mixed with 2.5 µL of deionized formamide, 0.4 µL of
internal lane standard TAMRA-350 (Perkin Elmer Systems), and 0.5 µL
of blue dye, denatured at 97°C for 5 minutes, rapidly cooled on ice, and
was then electrophoresed on a 6% denaturing polyacrylamide gel and the alleles
were detected on a 373–automated DNA sequencer (Perkin Elmer Systems,
San Francisco, Calif) using GeneScan 2.1 (Perkin Elmer Systems). Two Centre
Erute Polymorphisme Humaine individuals (133101,133102) and 1 local DNA were
loaded on every gel as controls. All alleles were initially assigned and genotyped
semiautomatically using Genotyper 2.0 (Perkin Elmer Systems) without any information
of phenotype. Laboratory personnel were masked to the phenotypic status of
Two-point and multipoint-affected sibling pair analysis was performed
on the entire set of genotyped sibships (292 affected pairs) using MAPMAKER/SIBS.14 This program does not permit analysis of other affected
pairings. In addition, the data set was divided into 2 subsamples, each of
which was analyzed separately using MAPMAKER/SIBS.14
The first group consisted of sibling pairs who both possessed at least 1 APOE ∊4 allele (162 affected pairs), and the second
group consisted of sibling pairs in which neither member possessed an APOE ∊4 allele (63 affected pairs). For example, a
quartet of affected siblings whose APOE genotypes
are APOE3/APOE3, APOE3/APOE3, APOE3/APOE4, and APOE3/APOE4 would contribute
1 pair to each group.
A multipoint exclusion map also was obtained for the entire sample using
MAPMAKER/SIBS. For the purposes of this analysis, the disease-susceptibility
model was parameterized in terms of λS,
the relative risk to siblings of a case.15
A number of values of λS ranging
from 1.2 to 2.0 were tested.
Two-point lod scores between markers and disease are shown in Table 2. Multipoint affected sibling pair
analysis on the entire data set (292 sibling pairs), with lod scores, is shown
in Figure 1. The maximum lod score
obtained was 0.89 at D12S98, corresponding to a chromosome-wide P value of .21. If the multiple testing arising from the APOE ∊4 positive and negative analyses is included,
the P value increases to .48. These P values were simulated from our actual sample, using the observed
marker allele frequencies and the marker map used in the analyses and thus
should not be conservative. (A lod score of zero, suggesting no excess of
allele sharing between affected family members, was obtained in the region
in which Pericak-Vance and colleagues10 found
their highest lod scores.)
We attempted to detect possible epistatic effects between APOE and a susceptibility locus on chromosome 12 by splitting the data
set into 2 subsets. The first portion contained sibling pairs who both possessed
at least 1 APOE ∊4 allele (162 pairs) and is
denoted in Figure 1 by "APOE ∊4 both positive." The other subset contained sibling pairs
with both members APOE ∊4 negative (63 pairs),
and is denoted in Figure 1 by "APOE ∊4 both negative." As shown in Figure 1, neither group had a significant lod score close to D12S1042. However, the subset of affected pairs who were
both APOE ∊4 negative had a multipoint lod of
1.91 at D12S98, corresponding to a chromosome-wide P value of approximately .09 (allowing for multiple testing).
The α2-macroglobulin gene is between D12S98 and D12S397 at this peak, and Blacker and
colleagues7 have recently reported an association
between this gene and AD.16 These data, thus,
would support the notion that α2-macroglobulin is an AD locus.
The results of the multipoint exclusion analysis performed on the entire
data set are shown in Figure 2.
For this analysis, the disease-susceptibility model was parameterized in terms
of the relative risk to siblings of a case, λ-S.15 Assuming the frequency of APOE ∊4 in the general population is 0.15, the relative
risk to ∊4 heterozygotes is 4, and the relative risk to ∊4 homozygotes
is 103, then λS for
the APOE locus is approximately 1.4. As shown in Figure 2, when λS is 1.4, a region extending from D12S310
to 12 centimorgans distal of D12S1292 is excluded
(lod score <−2.0). (This region includes D12S1042, the locus to which Pericak-Vance and colleagues10
found their strongest evidence of linkage.)
To replicate the analysis of Pericak-Vance and colleagues,10
we selected from our sample those families with at least 3 sampled affected
individuals (see Table 1 for the
structure of these "large pedigrees") and analyzed these separately. The lod
scores given by each locus analyzed separately are shown in Table 2. The highest multipoint lod scores were 0.47 at D12S98 and 0.44 at D12S395. This is consistent
with the results obtained from the entire sample, although the lod score is
reduced, as would be expected from the reduction in sample size. In this analysis,
multiply affected families are not greatly different from the others in terms
of their evidence for linkage. However, our series of large families (at least
3 sampled individuals) contained half the number of sampled cousin and avuncular
pairs than the data sets in the previous report. Thus, it remains possible
that some difference in ascertainment between the 2 series could account for
the discrepancies in the results.
Our findings should not be interpreted as indicating that there is no
AD-risk gene close to D12S1042, since, as with the
failures to repeat the observations of genetic associations, it remains possible
that there is such a locus that is important in a few families including a
proportion of those ascertained by Pericak-Vance and colleagues.10
However, it is unlikely that such a locus accounts for a large proportion
of cases of AD, since we were not able to detect evidence of linkage in our
entire data set. However, we were able to detect evidence of linkage to a
marker near the APOE locus using a dinucleotide repeat
marker (D19S412). This marker gave a lod score of
1.1 on its own and 2.1 when used in a multipoint with APOE. These data, not surprisingly, resemble those reported by Blacker
et al,7 since they consist of a largely overlapping
series of samples. Furthermore, the sample we have used here was sufficiently
powerful to exclude (lod <−2) a locus of similar effect size to that
of APOE from the region in which Pericak-Vance and
colleagues10 found their most significant results.
It is therefore unlikely that the difference between our results and those
of Pericak-Vance and colleagues10 is due to
lack of power in our sample. We did obtain a moderate lod score (P=.09) in the subset of affected pairs where both members were APOE ∊4 negative, suggesting the possible involvement
of a locus acting heterogeneously with APOE ∊4.This
locus is exactly at the α2-macroglobulin locus, which Blacker
and colleagues7 have recently suggested is
associated with AD.16 This locus is more than
20 centimorgans from the region implicated by Pericak-Vance and colleagues.10
The LRP gene is close to D12S398, but we are not able to confirm or refute the inference from
which implicate this gene in the etiology of the disease. Our data suggest
that LRP is not a gene of large effect. However,
given the poor genetic resolution of these types of linkage studies, it remains
possible that the LRP gene is within the "linked"
region in the data set. If the α2-macroglobulin is a predisposing
locus for AD,16 it will be important to examine
the genetics of both LRP and its other ligands.
A serious problem in genome scans of complex disorders is the assessment
of the true level of significance associated with a screen in which multiple
tests are performed on a single data set. The problem is that such studies
attempt to resolve 2 complex issues with a single analysis: (1) determination
of the inheritance characteristics of the locus (ie, mode of inheritance,
age-dependent penetrance, "phenocopy" or misdiagnosis rate, disease allele
frequency, interaction with other [APOE] loci, etc)
and (2) identification of the location of this putative gene. This is quite
unlike maximum likelihood methods of analysis in simple Mendelian disorders
in which the inheritance characteristics can be estimated to a high enough
degree of certainty so as not to interfere with the localization. Modification
of the analysis parameters or choosing a variety of linkage analyses and approaches
amounts to multiple testing from a statistical perspective. Although multiple
testing is a legitimate part of trying to determine the inheritance characteristics
of the phenotype, it means that lod scores derived from such analyses cannot
be thought of as equivalent to the lod scores in simple disorders.
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