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TAU as a Susceptibility Gene for Amyotropic Lateral Sclerosis–Parkinsonism Dementia Complex of Guam
Parvoneh Poorkaj, PhD;
Debby Tsuang, MD;
Ellen Wijsman, PhD;
Ellen Steinbart, MA;
Ralph M. Garruto, PhD;
Ulla-Katrina Craig, PhD;
Nicola H. Chapman, PhD;
Leojean Anderson, BS;
Thomas D. Bird, MD;
Chris C. Plato, PhD;
Daniel P. Perl, MD;
Wigbert Weiderholt, MD;
Douglas Galasko, MD;
Gerard D. Schellenberg, PhD
Arch Neurol. 2001;58:1871-1878.
ABSTRACT
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Background A Guam variant of amyotrophic
lateral sclerosis (ALS-G) and parkinsonism dementia complex (PDC-G) are
found in the Chamorro people of Guam. Both disorders have overlapping
neuropathologic findings, with neurofibrillary tangles in spinal cord
and brain. The cause of ALS-G–PDC-G is unknown, although inheritance
and environment appear important. Because neurofibrillary tangles
containing tau protein are present in ALS-G–PDC-G, and
because mutations in the tau gene (TAU) cause autosomal
dominant frontotemporal dementia, TAU was examined as a
candidate gene for ALS-G–PDC-G.
Methods TAU was evaluated by DNA sequence
analysis in subjects with ALS-G–PDC-G, by linkage analysis of
TAU polymorphisms in an extended pedigree from the village of
Umatac, and by evaluation of linkage disequilibrium with polymorphic
markers flanking and within TAU.
Results Linkage disequilibrium between
ALS-G–PDC-G and the TAU polymorphism CA3662 was observed. For
this 2-allele system, PDC and ALS cases were significantly less likely
than Guamanian controls to have the 1 allele (4.9% and 2% vs 11.5%,
respectively; Fisher exact P = .007).
DNA sequence analysis of TAU coding regions did not
demonstrate a mutation responsible for ALS-G–PDC-G. Analysis of
TAU genotypes in an extended pedigree of subjects from Umatac
showed obligate recombinants between TAU and ALS-G–PDC-G.
Linkage analysis of the Umatac pedigree indicates that TAU is
not the major gene for ALS-G–PDC-G.
Conclusions The genetic association between ALS-G–PDC-G
implicates TAU in the genetic susceptibility to ALS-G–PDC-G.
TAU may be a modifying gene increasing risk for ALS-G–PDC-G
in the presence of another, as yet, unidentified gene.
INTRODUCTION
A UNIQUE FORM of amyotrophic
lateral sclerosis (ALS) exists in the Chamorro people of the western
Pacific island of Guam.1 Guam ALS (ALS-G) is clinically
similar to typical ALS found in other populations,2 as both
disorders have neuropathologic changes in the spinal cord with
characteristic degeneration of the upper and lower motor neurons.
However, unlike typical ALS, in ALS-G, neurofibrillary tangles (NFTs)
occur in the hippocampus, entorhinal cortex, and
neocortex.2, 3, 4 A second disorder in the same population is
parkinsonism dementia complex (PDC-G), an extrapyramidal syndrome with
cognitive decline.5, 6 In PDC-G, as in ALS-G, extensive
neocortical and hippocampal NFT pathology occurs with some spinal cord
degeneration. About 5% of affected subjects have both ALS-G and PDC-G.
Because of the clinical and neuropathologic similarities between ALS-G
and PDC-G, and because both are found exclusively among Western Pacific
groups including the Chamorro population, these 2 disorders may be
different manifestations of a single disease.
The prevalence of ALS-G on Guam, when first described in the
early 1950s,7, 8 was substantially higher than that of
ALS in white populations (50-80 per 100 000 vs 1-6 per
100 000, respectively).7, 8 Similar
prevalence rates were subsequently observed for PDC-G.9 The
highest prevalence of ALS-G on Guam was in the small southern coastal
village of Umatac (250 per
100 000).7, 8, 10, 11 In subsequent decades,
the prevalence of both of these disorders declined, although neither
has completely disappeared. Estimates of ALS-G prevalence for the 1970s
and 1980s range from 30 per 100 00011 to a low of
less than 5 per 100 000.12 Likewise, the prevalence
of PDC-G has declined, but to a lesser extent.9, 13 During
this same period, the age at onset for ALS-G increased from 47.6 to
51.9 years, and for PDC-G, from 42.1 to 52.2 years.14
Ongoing studies indicate that PDC-G is still common on
Guam, but ALS-G is quite rare.
Although the cause of ALS-G–PDC-G is unknown, the disease clusters in
families and, therefore, inheritance may contribute to ALS-G–PDC-G
susceptibility.5, 7, 10, 15, 16, 17 The change in incidence and
onset age suggests that gene-environment interactions are
important.
The NFTs in ALS-G–PDC-G are bundles of paired helical filaments
composed of aggregated hyperphosphorylated tau. These NFTs are
ultrastructurally and biochemically indistinguishable from NFTs in
Alzheimer disease18, 19, 20 and numerous other neurodegenerative
disorders. Mutations in TAU, the gene that encodes tau
protein, cause frontotemporal dementia with parkinsonism chromosome 17
type (FTDP-17).9, 21, 22 This is an autosomal
dominant disease with clinical and neuropathologic features that
overlap with ALS-G–PDC-G.9, 23 Thus, TAU is a
candidate gene for ALS-G–PDC-G. Some FTDP-17 TAU mutations
are missense changes that alter the interactions of tau with
microtubules.24, 25 Other mutations affect TAU
alternative splicing regulation and result in a change in the ratio of
the tau isoforms produced.21, 26, 27, 28 Mutations affecting
splicing
include both missense and silent mutations in
TAU exon 10, and intronic mutations in the sequence closely
flanking this exon.21, 26 In 1 autosomal dominant FTDP-17
family (the hereditary dysphasic dementia [HDDD2] kindred), genetic
linkage analysis conclusively identifies the TAU region of
chromosome 17 as the location of the responsible gene.29
However, no mutation has been identified in this family despite
extensive sequence analysis of all coding regions and intronic
sequences surrounding each exon. Presumably, the mutation is in
regulatory sequences either deep within an intron or in flanking
regulatory sequences. Genetic changes in TAU also contribute
to susceptibility to progressive supranuclear palsy
(PSP). A positive genetic association between nonfamilial PSP
and a polymorphism in intron (I) 9 (I9) of TAU was
found30 and has been confirmed in multiple
studies.31, 32, 33 Again, as with the HDDD2 family, no PSP
susceptibility allele has been identified in coding regions
or in flanking intronic sequences, and the susceptibility site is
presumed to be in a regulatory sequence.
To investigate the role of TAU in ALS-G–PDC-G, we studied
Chamorro subjects from the entire island of Guam and subjects from the
village of Umatac.10 Subjects from Umatac were studied
because of the high incidence of ALS-G–PDC-G in this village.
TAU was evaluated as a candidate gene for ALS-G–PDC-G by DNA
sequence analysis of coding regions and closely flanking intronic
regions. Because some TAU mutations or susceptibility sites
may be in cryptic regulatory sequences, we also performed linkage and
association studies. The DNA sequence analysis and linkage analysis
show that TAU mutations are not the major cause of
ALS-G–PDC-G. However, association studies show that a TAU
variant(s) confers susceptibility to
ALS-G–PDC-G.
SUBJECTS AND METHODS
CHARACTERIZATION OF SUBJECTS
The ALS-G and PDC-G cases and controls were ascertained from the
University of Guam, Mangilao, registry, recruitment at Guam Memorial
Hospital, Oka, Tamining, and from the National Institute of
Neurological and Communicative Disorders and Stroke Guam Intramural
Research Program that operated from 1956 to 1983. Subjects from the
current study were examined by a trained research assistant who
obtained a medical history and performed a brief examination that
included neurologic screening and cognition testing with the Cognitive
Abilities Screening Instrument (CASI). The CASI was pilot
tested on Guam to ensure cultural fairness, to assess the extent of
variation due to age and education, and to develop cutoff points for
screening. Information was collected on risk factors, family history,
medication use, medical and neurologic history, cognitive and motor
symptoms, and functional performance (of activities of daily
living). All subjects who failed screening with the CASI or
whose history or brief examination suggested neurologic disease
underwent detailed evaluation. In the more extensive evaluation, a
neurologist examined mental status and performed a structured
neurologic examination that included the Unified Parkinson's Disease
Rating Scale. A psychometrist administered a standardized test battery
for which normative data have been obtained from elderly Chamorro
subjects who lacked significant medical or neurologic diagnoses.
Laboratory data such as blood test results were reviewed. Neuroimaging
studies were recommended (and could be ordered by personal physicians)
if subjects had unusual clinical pictures or focal neurologic findings.
Consensus diagnoses were made by 3 neurologists, who reviewed all
clinical information. Diagnoses were made at 2 levels: descriptive
syndromes (such as dementia, parkinsonism, ALS, stroke, or other
conditions) and suspected etiologic diagnosis (such as ALS-G or
PDC-G). Standard clinical diagnostic criteria were used wherever possible. Parkinsonism arising after neuroleptic
exposure or in severe stages of dementia was considered to be
secondary. The diagnosis of PDC-G required the insidious onset and
gradual progression of primary parkinsonism and dementia. Modifying
factors such as stroke or other brain diseases were taken into account.
Dementia was diagnosed with criteria of the Diagnostic and
Statistical Manual of Mental Disorders, Fourth
Edition.34 The pure dementia syndrome on Guam resembled
Alzheimer disease clinically, and many patients met standard criteria
for probable Alzheimer disease. Subjects from the National Institute of
Neurological and Communicative Disorders and Stroke studies were all
cases of clinical and autopsy-documented ALS-G and/or PDC-G. Ethnic
Chamorro controls were diagnosed as being normal if they had a CASI
score of 80 or higher, were functionally independent, and lacked
motor weakness, tremor, or gait difficulty on brief screening. Subjects
whose CASI scores were below 80 were designated controls if detailed
neurologic and psychometric evaluation did not show evidence of
dementia, mild cognitive impairment, or a neurologic disorder. Genomic
DNA was prepared as previously described.35, 36 DNA was
either from archived frozen brain samples from previous studies or from
fresh blood samples obtained by the ongoing project.
GENOTYPING SHORT TANDEM REPEAT POLYMORPHISM
MARKERS FOR TAU
Subjects were genotyped for a previously described short tandem repeat
polymorphism (STRP) Tau-CA30 and 3 new STRPs in or near
TAU (Figure
1). The STRPs located 5' and 3'
to TAU were identified by searching for dinucleotide repeat
sequences in the bacterial artificial chromosome clones HCIT104N19
(Genbank accession No. AC003662) and HRPC843B9 (Genbank accession No.
AC004139), which contain TAU 5' and 3' flanking sequences,
respectively. The STRP CA3662 was amplified with the primer pair
3662-23F 5' GGC CGG TAA GAG ATC AGC AAA C and 3662-23R 5' AAC AGC AAG
CAG TAT ATA CC, yielding a product of approximately 230 base pairs (bp)
in size. The STRP Tau-GT was amplified with the primer pair GTF 5' CTT
CAC TCT CGA CTG CAG C and GTR 5' GAC AGC GGA TTT CAG ATT CGG, yielding
a product approximately 262 bp in size. The STRP CA4139 was amplified
with the primer pair 4139 CA1F 5' CGA GAT GGT ACC ACT GCA CTC C and
4139 CA1BR 5' CTG ATA GCA TGT CTT CTA GAA C, yielding a product
approximately 140 bp in size. Genotypes were determined by previously
described methods.37
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Figure 1.
Genomic organization of TAU, adjacent genes, and short tandem
repeat polymorphism sites. Gene and short tandem repeat polymorphism
locations are based on a 400-kilobase (kb) contig assembled from
bacterial artificial chromosome sequences (accession numbers AC004139
and AC003662) and P1 artificial chromosome clone sequences (accession
number AC091628). Genes are represented by the large open
arrows, and direction of transcription (5' to 3') is indicated by the
direction of the arrow. CRFR is the corticotropin-releasing
factor receptor, and KIAA1267 is a gene with an unknown
function. Lengths of each gene and intergenic regions are given at the
bottom. CA3662 is 23 913 base pairs (bp) from the transcription
start site of TAU. Tau-GT is 350 bp 3' after the end of E1.
Tau-CA, in I9, is 6346 bp from the end of E8. CA4139 is 13 563
bp from the end of the 3' untranslated region of TAU and is in KIAA1267.
Both CRFR and KIAA1267 extend past
the end of the contig described.
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TAU MUTATION ANALYSIS
Six ALS-G and 5 PDC-G cases were used for TAU mutational
analysis. Six of the sequenced cases were documented by autopsy,
including 1 case each of ALS-G and PDC-G from Umatac. Both strands of
the gene were sequenced for all coding exons (exons 0, 1-4, 4a, and
5-13), including at least 7 bp of flanking intronic DNA, 50 bp of the
5' untranslated region, and 70 bp of the 3' UTR. Primer pairs for
amplification and sequencing were previously described,38
with the exception of the following new primer sets: exon 10 (10GF 5'
GTC AGT GTG GCC GAA CAC and 10CR 5' GGC TAC ATT CAC CCA GAG G) and exon
11 (11EF 5' TGC TTC TCA TTG AGT TAC ACC and 11ER 5' TTG TCT TGG GCA GCA
TGG CC).
DNA sequence analysis identified three 2-allele polymorphic sites in
subjects with ALS-G–PDC-G. One, in I9, is a 2-allele C/A polymorphism
47 bp (I9-47) from the 3' end of the intron. The I9-47 polymorphism was
genotyped by the following restriction digest assay. A 313-bp fragment
was amplified by polymerase chain reaction using primers 10CR (5' GCT
ACA TTC ACC CAG AGG) and 10F (5' AAG TGG AGG CGT* CCT TGC GGC CAA GC),
where the underlined GC differs from the normal-sequence AG found in
TAU. This sequence change creates a BglI site when a
C is present at the base followed by the asterisk, but not when an A is
present. Digestion with BglI produces 287- and 26-bp fragments
for the C allele and an undigested 313-bp allele for the
A allele. Two additional variable sites, one in I9
(I9-176) and another in I11 (I11 + 90) were genotyped by
DNA sequence analysis as described herein.
STATISTICAL ANALYSES
Tests for possible frequency differences between cases and controls or
between observed and expected frequencies were performed with
 2 tests. Because previous results demonstrated that
combining alleles to collapse cells into a 2 x 2
contingency table can give spurious results,39, 40 all
contingency table analyses were performed on full
2 x m contingency tables, where m is
the number of alleles, haplotypes, or other groups being compared. A
Fisher exact test was performed for the STRP, and exact P
values were estimated. Pairwise tests were performed when the 3
populations differed (ALS-G, PDC-G, and Guam controls).
Linkage analysis was performed for single markers plus the disease by
means of a disease allele frequency of 0.01 and a dominant mode of
inheritance with a 0% sporadic rate. Age-dependence penetrance for the
carrier genotypes was assumed by means of a cumulative normal function
with a mean of 49.4 years and a variance of 96.2 years. These values
corresponded to the mean and variance observed in the affected members
of the entire 1167-subject Umatac pedigree. Linkage analysis of the
full pedigree with its many inbreeding loops was impossible for
computational reasons. Linkage analysis of the largest subcomponent of
the pedigree that both traced back to a single founder couple and that
could be feasibly analyzed was performed by means of the program
FASTLINK,41 version 4.1 (Rockefeller linkage analysis
software; available at: http://linkage.rockefeller.edu/soft/list.html), which implements
computationally efficient methods for automatic loop breaking.42 For a single marker on this subcomponent of the
pedigree, the programs Unknown and M link (within the FASTLINK
software) took 11 hours and 11.35 days to run, respectively, with the
use of a Digital Alpha XP1000 500-MHz workstation (Compaq
Computer Corporation, Houston, Tex).
RESULTS
GENETIC ASSOCIATION STUDIES
TAU was first evaluated as a candidate gene for ALS-G–PDC-G
by comparing STRP allele frequencies of Chamorro cases with those
of Chamorro controls. Cases were 49 subjects with ALS-G (41 with
autopsy
confirmation), 86 subjects with PDC-G (49 with autopsy
confirmation), and 3 subjects with ALS-G–PDC-G (2 with autopsy
confirmation). Chamorro controls (n = 78) were
healthy nondemented subjects with no family history of ALS-G–PDC-G.
Subjects for the genetic association studies were from the entire
island of Guam, except that subjects from Umatac were not used. Four
STRP sites were used including 2 that are within TAU (Tau-GT
and Tau-CA) and 2 that closely flank the 5' and 3' end of TAU
(CA3662 and CA4139, respectively; Figure 1). Allele and
genotype frequencies for CA3662 and Tau-CA are in Table
1 and Table
2. Allele frequencies for CA3662
significantly differed between ALS-G, PDC-G, and controls
(P = .007 among the 3 groups), where there was
an underrepresentation of the rare allele (allele 1) and excess of the
common allele in both case groups when compared with controls (Table
1). Pairwise tests performed between PDC-G cases and controls
and ALS-G cases and controls were also significant
(P = .04). These results demonstrate
that both ALS-G and PDC-G have an association with a TAU
allelic variant.
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Table 1. CA3662 Allele and Genotype Frequencies*
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Table 2. Tau-CA Allele and Genotype Frequencies*
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There was no statistically significant difference in allele frequencies
for the Tau-CA (Table 2), Tau-GT, or CA4139
(P = .09 and P = .27,
respectively; data not shown) when Chamorro subjects with ALS-G and
PDC-G were compared with Chamorro control subjects. The lack of
association between ALS-G–PDC-G and these sites indicates either a
lack of power, or that the regions of TAU tested by these
markers are not in linkage disequilibrium with the disease.
DNA SEQUENCE ANALYSIS OF TAU IN SUBJECTS WITH ALS-G–PDC-G
The genetic association results suggest that TAU could
be either a susceptibility allele or a major gene for ALS-G–PDC-G. To
identify potential TAU mutations responsible for ALS-G–PDC-G,
all coding exons with some flanking intronic regions and portions of
the 3' and 5' untranslated regions were sequenced for 6 subjects with
ALS-G and 5 subjects with PDC-G, including 1 case of each type from
Umatac. No sequence variants were identified in coding regions. Three
sequence variants were found in intronic regions. Two sites were in I9,
a G/A polymorphism at -176 (numbers are relative to the first
nucleotide of E10) and a C/A polymorphism at -47. The third
polymorphic site was a G/A polymorphism in I11 at +90 nucleotide (after
exon 11). To determine whether these variable sites are
potential mutations or benign polymorphisms, Chamorro controls were
also examined for these 3 sites. For each site, both alleles were also
found in Chamorro and white control subjects, indicating that these
variants are not causative mutations.
SEGREGATION OF TAU STRP MARKERS IN THE UMATAC PEDIGREE
Subjects for family linkage analysis were from Umatac (Table
3, Figure 2). As
described in 1969,10 the familial relationships for the
entire village of Umatac dating back to 1830 could be represented as a
single pedigree of 1450 people in 262 sibships over 8 generations,
including 53 subjects affected with ALS-G–PDC-G. Subsequently,
additional subjects with ALS-G–PDC-G were ascertained, including the
16 described in Table 3. This includes 2 PDC-G cases and 1 dementia
case identified by this study in the past 3 years. Figure 2 represents
only the portion of the pedigree containing affected subjects for whom
DNA was available. Also included are relatives needed to connect the
sampled affected subjects. For each sampled affected subject, parental
relationships, shown as different colored lines, were traced back to 6
couples who could not be connected in the earliest known generations
(not shown). The complexity of the pedigree reflects the
inbred nature of this village. Because the present Umatac population
was descended from a small number of original families (8 in 1830) and
the community was genetically isolated, and because most
of the affected subjects are concentrated in a
subset of the Umatac families,10 ALS-G–PDC-G in this
village may be the result of a genetic founder. The identification of
new cases of PDC-G demonstrates that this disease has not disappeared
from Umatac.
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Table 3. Clinical Characteristics of Sampled Affected Individuals From Umatac, Guam*
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Figure 2. Partial pedigree from Umatac, Guam. A small portion of the entire
Umatac pedigree is presented, including 33 affected individuals: 18
with amyotrophic lateral sclerosis (ALS), 13 with parkinsonism dementia
complex (PDC), 1 with ALS-PDC, and 1 with dementia (Table 3).
The portion of the pedigree presented is based on the more extensive
pedigree published previously.10 The pedigree shown
includes all individuals needed to connect the affected subjects who
were sampled. The sampled subjects with the Guam variant of ALS-PDC are
descended from 6 couples who could not be connected in previous
generations by means of records dating to the 1830s. Bloodlines from
these 6 couples are shown in different colors. Individual 719 was a
spouse who does not appear to be related to others in the complete
village pedigree. Genotypes for CA4139 and Tau-GT are shown for
affected subjects, with the former shown above the latter. An asterisk
indicates that DNA is available from a subject. An L indicates that the
subject was used in the linkage analysis; ND, not determined. The
subset of the pedigree used included 48 subjects and 4 inbreeding
loops.
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Fourteen affected Umatac individuals (8 PDC-G cases, 5 ALS-G cases, and
1 dementia case) were genotyped for the 4 TAU STRP sites
(Figure 2). Genotypes for the Tau-GT and CA4139 STRP markers
are displayed on the pedigree. Multiple discordant genotypes are
present between affected subjects for both disease types, indicating
obligate recombinants between TAU and both ALS-G and PDC-G.
For example, ALS-G subjects 286 and 184 are 4/6 and
2/5, respectively, for CA4139 and thus do not share a common
genotype for TAU. Both are descended from the same 2 couples
(couple 1/2 pink and couple 040/041 orange). For CA4139,
PDC-G subjects 500 and 538 (genotypes 2/6 and 2/4,
respectively) are discordant with subject 721 (genotype 5/7),
even though subjects 500 and 721 are descendents of couple 4/3
(blue). Assuming a simple major gene model of inheritance,
these discordances indicate obligate recombination events between
TAU and ALS-G–PDC-G.
Linkage analysis of the Umatac pedigree was also performed with tau-GT,
the most informative STRP in TAU. Because of the extensive
inbreeding in this pedigree, only a subset of the sampled subjects
could be included in the analysis (Figure 2). All resulting
lod scores were negative, with a lod score of -0.025 at a
recombination fraction of 0. Thus, the pattern of marker genotypes
among cases combined with the linkage analysis indicates that
TAU is not the major gene responsible for ALS-G–PDC-G in the
Umatac kindred.
COMMENT
In this study, TAU was evaluated as a candidate gene for
ALS-G–PDC-G. The underlying hypothesis for this work is that
inheritance contributes to susceptibility to this disease complex. The
following lines of evidence support a genetic hypothesis. First, close
relatives of affected subjects are at greater risk for ALS-G–PDC-G
than are close relatives of controls, and approximately 40% of
probands have an affected relative.7, 8, 17 Second, families
have been described with multiple affected subjects in 2 or more
generations.17 The most dramatic of these pedigrees
consists of subjects from Umatac, where the disease can be traced back
4 generations (Figure 2), 10 a pattern consistent with a
genetic cause. Third, segregation analysis of an ALS-G–PDC-G registry
of patients and relatives rejected an environmental factor–only
hypothesis.43 However, an inheritance-only model is also
not consistent with how ALS-G–PDC-G occurs. An environmental
hypothesis is supported by the fact that, for both ALS-G and PDC-G,
during the past 30 to 40 years, the age at onset has
increased13, 14 and the incidence
decreased,12, 44 suggesting a change in exposure to an
environmental factor. Candidates for nongenetic factors include
aluminum deposition,45, 46 excess aluminum absorption caused
by low dietary calcium,47 and
2-amino-3-(methylamino)-propanoic acid, a neurotoxin found in
traditional foods,48 although the evidence that these
factors are important in causing ALS-G–PDC-G is
equivocal.49, 50, 51 The most parsimonious hypothesis is
that ALS-G–PDC-G is the result of interaction between genetic and
environmental susceptibility factors, and exposure to this unknown
environmental factor has decreased in the past 30 to 40 years.
The mode of inheritance of susceptibility to ALS-G–PDC-G is difficult
to predict. Visual inspection of the Umatac pedigree (Figure
1)10 suggests autosomal dominant inheritance with
near-complete penetrance in males and reduced penetrance in females.
Recessive inheritance is unlikely, since the mean inbreeding
coefficient was similar for Umatac sibships with and without affected
subjects.10 Formal segregation analysis of probands and
close relatives for subjects in a registry from the entire island
rejected both dominant and recessive models, but was consistent with a
2-allele additive major locus model.43 However, the
penetrance of this major locus is low, with a maximum liability of 0.1
for homozygous carriers. Certainly, more complex models are possible,
and a major gene hypothesis can also include modifier genes.
The linkage disequilibrium results with CA3662 demonstrate that
TAU is a susceptibility gene for the disease. Although the
results from the other 3 sites tested were not significant, data from
Tau-CA are intriguing (Table 2). For this site, the
142 allele (also called the Ao allele) was previously
reported to be elevated in white patients with PSP, with a frequency of
0.98 in cases and 0.75 in white controls, making it the high-risk
allele for PSP.30 For Chamorro subjects, both cases and
controls had high frequencies of the 142 allele, comparable
with that of white patients with PSP. Allele 142 frequencies
were higher in ALS-G–PDC-G cases than Chamorro controls, but this
difference did not reach statistical significance. Interestingly,
Japanese control subjects also have a high 142 allele frequency of
0.9832 to 1.0,52 suggesting
that Chamorros are more similar to Asians than
to whites. Previous work53 attempting to identify a genetic
association between ALS-G–PDC-G and TAU-related polymorphisms
failed to detect the linkage disequilibrium observed here. This is not
surprising, since the markers used in the previous study were not in
proximity to TAU. None of the markers previously used are in
the genomic sequence available for TAU (Figure 1), indicating
that these markers are at least 70 kb upstream of the 5' end of
TAU and 200 kb downstream from the 3' end of the gene. Regions
of linkage disequilibrium do not typically extend over distances this
great, and thus, failure to detect disequilibrium with distant markers
does not exclude the involvement of TAU in ALS-G–PDC-G. Also,
the sample size used in the previous study was small (23 subjects with
PDC-G, 19 control subjects, and no subjects with ALS-G) compared with
the subject panel used here (Table 1 and Table 2). 53
Although the genetic association studies indicate that TAU
could be a susceptibility gene for ALS-G–PDC-G, TAU does not
appear to be the major gene responsible for the disease. Direct DNA
sequencing of all TAU coding exons present in brain tau
isoforms did not demonstrate any mutations or polymorphisms, either in
controls or in affected subjects, consistent with DNA sequencing
studies by others.53 However, absence of variants in these
exons does not exclude TAU as a major gene or a modifying
susceptibility gene, because genetic variation in noncoding regions not
directly adjacent to exons can cause disease. This has clearly been
demonstrated for the HDDD2 family, where linkage analysis has clearly
demonstrated that TAU is the major gene responsible for
disease,29 yet no mutation in exons or in closely flanking
intronic regions are known for this family. Another example is PSP,
where TAU is clearly a modifying locus as demonstrated by
linkage disequilibrium analysis,30 yet, as for HDDD2, no
coding or closely flanking intron variants are known. For both PSP and
the HDDD2, presumably sequence variants in noncoding regulatory regions
that control gene expression or alternative splicing are pathogenic.
TAU regulatory mutations are known, and these cause FTDP-17 by
affecting alternative splicing of exon 10. These mutations are located
in intron 10, close to the 3' end of TAU exon
10,21, 22, 27 and in exon 10.54 Since regulation
of exon 10 alternative splicing is complex and not completely
understood,54 additional cis-acting sequences may
exist elsewhere in the gene, and these regulatory elements are
candidate locations for mutations and susceptibility sites. Regulatory
sites that control other aspects of TAU gene regulation
scattered throughout the gene are also potential locations for a
mutation-susceptibility site. Thus, the fact that there are no
mutations or polymorphisms associated with ALS-G–PDC-G in
TAU coding regions does not exclude this gene from being
involved in Guam neurodegenerative disease.
The pattern of TAU segregation in the Umatac pedigree is
also not consistent with TAU being the major gene for
ALS-G–PDC-G (Figure 2). Numerous obligate recombinants
demonstrate that no specific TAU allele is required to develop
ALS-G–PDC-G, though a specific TAU allele increases
susceptibility to the disease. This conclusion is based in part on the
hypothesis that all cases of ALS-G–PDC-G are the result of the same
inherited allele(s) or mutation. This is a reasonable assumption
considering that this is a rare disease found only among the Chamorros
from the Mariana Islands, Japanese from the Kii Peninsula in
Japan,55 and
the isolated Auyu and Jakai people of West New
Guinea,56 although no autopsy information is available for
the latter population. In addition, the Chamorro population, originally
50 000 to 150 000 at the time of the first contact with
Europeans in 1521, was reduced to approximately 2500 in 1830s and
further reduced by a smallpox epidemic in 1856.57 This
population history suggests that ALS-G–PDC-G may come from a single or
small number of genetic founders, particularly in the high-incidence
village of Umatac.
The linkage disequilibrium results presented here indicate there may be
a polymorphic site(s) in or closely linked to TAU that
increases risk for ALS-G–PDC-G. The polymorphisms tested are unlikely
to be the actual pathogenic site. Presently, it is not possible to
predict the actual location of the critical TAU susceptibility
site. Because of intrinsic properties of linkage disequilibrium, lack
of significant results for a particular marker does not exclude the
region containing that marker. Thus, even though significant results
were obtained with a marker 5' to TAU, presently the entire
gene must be considered for the susceptibility site. The critical
regulatory sequence affected may be within an intron or in flanking
regions on either side of the gene. Since the flanking genes,
CRFR and KIAA1267, are unrelated to TAU in
terms of sequence homology or obvious predicted function, TAU
is not part of a cluster of related genes. Thus, the susceptibility
site is part of the TAU gene including flanking regulatory
sites most likely located between CRFR and KIAA1267.
Since the entire gene is 130 kb and flanking regions include 50 kb 5'
to the gene and 3 kb 3' to the gene, significant additional sequencing
comparing affected subjects with controls is required. Identification
of the major gene(s) for ALS-G–PDC-G will require linkage analysis of
ALS-G–PDC-G in large families such as the Umatac pedigree.
AUTHOR INFORMATION
Accepted for publication May 4, 2001.
This study was supported by grant PO10135316 from the National
Institute on Aging, National Institutes of Health, Bethesda, Md, and by
the Department of Veterans Affairs, Washington, DC.
We thank the subjects who contributed immensely to this work. We also
thank the staff at the University of Guam for assistance in this work.
From the Geriatric Research Education Clinical Center (Drs Poorkaj,
Bird, and Schellenberg and Mss Steinbart and Anderson) and Mental
Illness Research Education Clinical Center (Dr Tsuang), Veterans
Affairs Puget Sound Health Care System, Seattle Division, Seattle,
Wash; Division of Gerontology and Geriatric Medicine (Dr Poorkaj) and
Departments of Psychiatry and Behavioral Sciences (Dr Tsuang),
Biostatistics (Drs Wijsman and Chapman), Medicine (Division of Medical
Genetics) (Drs Wijsman and Bird), Neurology (Drs Bird and
Schellenberg), and Pharmacology (Dr Schellenberg), University of
Washington, Seattle; Departments of Anthropology and Biological
Sciences, Binghamton University, State University of New York,
Binghamton, NY (Dr Garruto); National Institute of Neurological
Disorders and Stroke, National Institutes of Health, Bethesda, Md (Dr
Garruto); Department of Public Health, University of Guam, Mangilao (Dr
Craig); Neurosciences Department, University of San Diego, San Diego,
Calif (Drs Plato, Weiderholt, and Galasko); and Departments of
Pathology and Psychology, Mount Sinai School of Medicine, New York, NY
(Dr Perl)
Corresponding author and reprints: Gerard D. Schellenberg, PhD,
GRECC 182-B, Veterans Affairs Puget Sound Health Care System, 1660
S Columbian Way, Seattle, WA 98108 (e-mail: zachdad{at}u.washington.edu).
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