Abstract
Objective: To analyze the association of polymorphisms in the tau gene with pathologically confirmed corticobasal degeneration (CBD).
Background: The authors previously described an extended tau haplotype (H1) that covers the human tau gene and is associated with the development of progressive supranuclear palsy (PSP). The authors now extend this analysis to CBD, a neurodegenerative condition with clinical and neuropathologic similarities to PSP. Like PSP, CBD is associated with accumulation of aggregates containing the 4-repeat isoforms of tau. Because of difficulty in diagnosis of CBD, the authors only analyzed cases with pathologically confirmed CBD.
Methods: The authors collected 57 unrelated, neuropathologically confirmed cases of CBD. Tau sequencing in these cases failed to show the presence of pathogenic mutations. Polymorphisms that spanned the tau gene were analyzed in all CBD cases and controls.
Results: Analyzing tau polymorphisms in CBD cases showed that the frequency of H1 and H1/H1 was significantly increased when analyzing all cases and when separating by country of origin. H1 frequency in all CBD cases was 0.921, compared with a control frequency of 0.766 (X2 = 9.1, p = 0.00255 [1df], OR 3.56 [8.43 > CI 95% > 1.53]). The H1/H1 frequency was also significantly higher at 0.842 compared with 0.596 in age-matched controls (X2 = 17.42, p = 0.00016, 2df), OR 3.61 [7.05 > CI 95% > 1.85]).
Conclusions: The CBD tau association described here suggests that PSP and CBD share a similar cause, although the pathogenic mechanism behind the two diseases leads to a different clinical and pathologic phenotype.
Corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) are disorders clinically characterized by parkinsonism and neurodegeneration.1-7⇓⇓⇓⇓⇓⇓ Pathologically the two disorders are distinct, although the abnormal tau protein selectively deposited in the brain tissue in both CBD and PSP are isoforms with four microtubule binding repeat domains.8-10⇓⇓ The clinical diagnosis of CBD is often difficult; many cases have overlapping features with other disorders such as PSP, frontotemporal dementia (FTD), and cerebrovascular disorders.11-13⇓⇓ The clinical features of CBD include asymmetric extrapyramidal signs, parkinsonism, alien limb, ideomotor apraxia, and focal myoclonus followed by cognitive impairment. CBD patients show normal saccadic velocity but increased latency of saccades14; they also may show oculomotor apraxia. In particular, CBD 135 is frequently misdiagnosed as PSP and vice versa, making genetic association studies difficult in a clinically diagnosed CBD series5,6,11⇓⇓; hence, in this study we included only cases with pathologically confirmed CBD.
Neuropathologically, CBD is distinguished from PSP and other dementia by several important features. The gross brain examination usually shows narrowing of cortical gyri, most marked in pre- and post-central regions associated with widening of sulci, greatest severity contralateral to the side of motor onset. Most pathology in CBD is in the cerebrum, whereas the basal ganglia, diencephalons, and brainstem are mainly targets of PSP. Histologically, there are ballooned neurons, neuronal loss, astrocytosis, and tau-positive neuronal and glial inclusions. The most characteristic neuronal tau pathology in CBD is numerous and widespread wispy, fine filamentous inclusions within neuronal cell bodies, whereas affected neurons in PSP have compact, dense filamentous aggregates characteristic of globose neurofibrillary tangles.9,10⇓
Six major protein isoforms of tau are found in the adult human brain. These are generated by alternative splicing of exons 2, 3, and 10.15-17⇓⇓ Exons 9 through 12 encode four microtubule-binding domains that are imperfect repeats of 31 or 32 residues. Alternative splicing of exon 10 generates isoforms with either 4 (exon 10+: ‘4-repeat tau’) or 3 (exon 10-: ‘3-repeat tau’) microtubule binding domains. The neurofibrillary tangles that are observed in CBD and PSP consist of straight filaments that contain predominantly 4-repeat tau protein isoforms, whereas the neurofibrillary tangles consisting of paired helical filaments found in AD contain all six major tau isoforms (4-repeat and 3-repeat).2,15⇓
Mutations in the tau gene are associated with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17).17 In this context, we have recently shown that mutations in the 5′ splice site of tau exon 10 increase the incorporation of this exon into tau mRNA and thus increase the proportion of 4-repeat tau isoforms.18 In affected families, this increase is associated with the formation of 4-repeat tau containing neurofibrillary tangles.19 This observation shows that control of this alternative splicing event is critical and that disregulation can result in tangle formation and neurodegeneration.18,19,20,22⇓⇓⇓ The FTDP-17 families are clinically similar to CBD; there also are rare families that are neuropathologically similar to CBD11; one such family has been reported with a tau exon 10 splice site (+16) mutation.22 This emphasizes the importance of sequencing tau exons 9 through 13 in familial CBD.
The genetics of CBD had not been studied until recently, because the disease is usually sporadic in occurrence. However, the discovery of mutations in the tau gene leading to FTDP-17 and previous reports of the association between a tau extended haplotype and PSP23-28⇓⇓⇓⇓⇓ led us to investigate CBD.
We have previously identified in the tau gene a series of single nucleotide polymorphisms (SNP) in exons 1, 2, 3, 9 (3 SNP), 11, and 13 and a large deletion in intron 10, which were present in both controls and patients. Analysis of the occurrence of these polymorphisms indicated that they are in complete disequilibrium with each other.28 Thus, they define two extended haplotypes (designated H1 and H2) that cover the entire tau gene (∼100kb). We have not identified a single recombinant event in these haplotypes in any of the unrelated patient and control individuals tested (>500 total). These haplotypes are therefore clearly ancient in the white population. Multiple rare polymorphisms are present only on the more common H1, suggesting that these arose by independent mutagenic events after the establishment of this haplotype. In addition, the dinucleotide polymorphism (a0), shown previously to be associated with PSP, is inherited with the two extended haplotypes. Dinucleotide polymorphism alleles a0 (11 repeats), a1 (12 repeats), and a2 (13 repeats) are inherited with the H1, whereas the a3 (14 repeats) and a4 (15 repeats) alleles are inherited with the H2.
Because of the difficulty in making a definitive clinical diagnosis of CBD, we collected brain tissue from 57 neuropathologically confirmed cases of CBD. Most of the neuropathologically confirmed cases of CBD also presented clinically with features consistent with CBD, although a minority of cases presented with the clinical diagnosis of PSP, FTD, primary progressive aphasia, and one case with myoclonic rigidity. We also have seen a number of clinically diagnosed cases of CBD that have alternative diagnoses after neuropathologic examination.6,7⇓ In the tau gene, exons 9 through 13 were sequenced in 43 cases in which frozen tissues was available, and in 10 of these cases the entire coding region was sequenced. Exons 9 through 13 were analyzed because mutations that affect these tau exons have been shown to yield tauopathies that can resemble CBD both clinically and neuropathologically. Most tau mutations are present in exon 10.18 In cases in which only formalin-fixed, wet, or paraffin-embedded tissue was available, only poor quality DNA could be obtained. In these cases, only the tau dinucleotide TG repeat polymorphism was analyzed. Here we investigate these polymorphisms and their association with CBD as a step toward defining the precise mechanism of genetic susceptibility for this disease.
Methods.
Samples.
Brain tissue from a total of 57 cases of CBD was obtained from the United States (30 cases), Canada (14 cases), and the United Kingdom (13 cases). The diagnosis of CBD in each case had been confirmed fully by pathologic examination of brain tissue, including immunocytochemical analysis by a neuropathologist with an interest in CBD (United States: D.D., I.L., and E.B.; Canada: C.B. and D.G.M.; United Kingdom: P.L. and D.M.).
The neuropathologic criteria (no publication has as yet defined this) used for the diagnosis of CBD are as follows: 1) cortical neuronal loss and gliosis with swollen neurons and typical corticobasal inclusions; 2) variable cell loss and gliosis in the striatum, globus pallidum, thalamus, nigra, amygdala, hippocampus, and dentate. There are abundant cortical and white matter argyrophilic threads and ballooned and phosphorylated neurofilament-positive neurons. Immunocytochemisty was used in all cases. This is particularly important to define the subcortical tau pathology that is essential in the diagnosis of CBD. Different antibodies were used in the diagnosis of CBD according to the neuropathologist involved. The main tau antibodies used were tau AT8 and PHF-1 (provided by Peter Davies, Albert Einstein College of Medicine). Neuronal and glial tau pathology were present in all cases. This consisted of neuronal granular deposits, tangles, coils, neurophil threads in the cortex, and profiles in the white matter and astrocytic and oligodendrocyte coils. Cortical involvement was bilateral and widespread, sparing the occipital cortex.
DNA was extracted from frozen brain in 43 cases (Promega DNA extraction kit, Madison, WI). When only formalin-fixed, wet, or paraffin-embedded tissue was available (14 cases), DNA was extracted from the cerebellum by use of an intensive extraction procedure.29
PCR and sequencing.
Tau exons and flanking intronic sequences were amplified from genomic DNA from individuals with primers previously described.16-18⇓⇓ Each 50-μL reaction contained 25 ng DNA, 20 pmol of each primer, 0.2 mmol/L dNTP, 1 unit of Taq polymerase (Qiagen, Valencia, CA) and the addition of 20% Q-solution (Qiagen). Amplification conditions were 35 cycles of 94 °C for 30 seconds, 60 to 50 °C touchdown annealing for 30 seconds, and 72 °C for 45 seconds with a final extension of 72 °C for 10 minutes. All PCR products to be sequenced were purified by using the Qiaquick PCR Purification Kit (Qiagen). For each exon, 100 ng product was sequenced in both directions by using the Big Dye kit (Perkin Elmer, Foster City, CA) and the relevant PCR primers. Sequencing was performed on an ABI377 Prism automated sequencer and analyzed by using Factura 2.1 and Sequence Navigator software (Perkin Elmer).
Genotyping and polymorphism analysis.
Previously reported polymorphisms23,28⇓ in exons 1, 2, 3, 9 (3 SNP), 11, and 13 and a large deletion in intron 10 were analyzed by PCR amplification over the polymorphic region followed by digestion of the product with the diagnostic restriction enzyme (PCR-RFLP).28 In the PSP series and for some CBD cases, the genotypes for polymorphisms in exons 9 and 11 were determined from the sequence analysis. All samples were genotyped for the intronic dinucleotide repeat polymorphism by PCR by using a tet-labeled forward primer followed by analysis on the ABI377 using Genotyper software (Perkin Elmer).
Results.
Association of tau haplotypes with CBD.
Sequence analysis of exons 9 through 13 (43 cases) and the entire tau gene (10 of these cases) failed to show the presence of pathogenic mutations. This confirms the findings of a previous community-based study30 that indicated that highly penetrant tau mutations are absent in sporadic CBD. This highlights the importance of family history in the identification of tauopathy cases with tau mutations. The tau polymorphisms analyzed in CBD were the dinucleotide TG repeat marker and SNP (see Methods) spanning the whole gene that have previously been shown to be associated with PSP. The haplotype and genotype data were identical for the polymorphisms analyzed with no recombination (tables 1 and 2⇓). A highly significant overrepresentation of H1 and H1/H1 were observed in the CBD cases compared with controls. Aged white U.S.A. controls (n = 145, mean age 63 years) and U.K. white control cases (n = 75, mean age 68 years) were used for comparison. The tau polymorphism data in the control groups was in Hardy–Weinberg equilibrium. Each of the SNP across the tau gene displayed evidence of association with CBD in that H1 and H1H1 were significantly overrepresented in all three populations compared with controls (see tables 1 and 2⇓): All international CBD cases compared with controls H1, X2 = 9.1, p = 0.00255, 1df and H1H1, X2 = 17.42, p = 0.00016, 2 df. The OR, with the inheritance of H1, was 3.56 (8.43 > CI 95% > 1.53), and H1H1 was calculated to be 3.61 (7.05 > CI 95% > 1.85) when all CBD cases were analyzed. Each of these SNP and the dinucleotide TG repeat polymorphism are in complete disequilibrium with each other in the CBD series. This disequilibrium has previously been identified in our PSP and control populations.28
Tau haplotype frequency in neuropathologically confirmed corticobasal degeneration (CBD) cases and controls
Tau genotype frequency in neuropathologically confirmed corticobasal degeneration (CBD) cases and controls
Discussion.
Previous studies have reported an association between a dinucleotide polymorphism in the tau gene and PSP.23-28⇓⇓⇓⇓⇓ These association results reflect the broader association between PSP and an extended haplotype that encompasses the entire tau gene.28 Mutations in the tau gene lead to the development of FTDP-17 cases, which are clinically and pathologically similar to PSP. This suggests that the associated haplotype is functionally relevant in the pathogenesis of PSP. Like PSP, CBD is a sporadic late-onset extrapyramidal syndrome characterized by selective 4-repeat tau deposition.
We therefore speculated that a similar association with the H1 and H1/H1 of tau also might be observed for CBD. The significant clinical overlap of CBD with other movement disorders made it mandatory that the tau sequence and haplotypes were determined in only pathologically proven CBD cases. This is the first report to look at a substantial pathologic series of CBD cases, and we have demonstrated a highly significant association with H1 and H1/H1. In our study, the criterion for inclusion was a diagnosis of pathologically confirmed CBD. No selection bias toward H1 occurred, because all available cases were analyzed. Previous studies looking at clinically diagnosed CBD have reached contrasting conclusions. One study failed to show an association between CBD and the tau haplotype, whereas in a second recent study on 18 cases of CBD (two pathologically confirmed), a significant association with the H1 was observed.27,31⇓ The discrepancy with the prior association studies is almost certainly attributable to the use of clinically diagnosed cases of CBD that at necropsy have different neuropathologic diagnoses.
In this study, pathologically confirmed CBD displayed a highly significant association with tau H1 and H1/H1. Thus, CBD shares a common tau haplotype with PSP. The 57 CBD cases showed no recombination between H1 and H2 haplotypes over the entire length of the tau gene, which is consistent with previous reports on the absence of recombination between H1 and H2 in PSP and control individuals. These data are essentially identical to those we and others have previously reported for the association between PSP and tau (CBD H1/H1 genotype 84.2% versus 87.5% in PSP). No pathogenic tau mutations were observed in our CBD series, confirming the rarity of these mutations in a sporadic non-AD dementia population.30 The extremely rare nature of CBD and the fact that multiple international centers were involved in the collection of the samples makes matching cases and controls extremely difficult. However, despite this, when the cases from separate centers were analyzed independently (with the relevant controls), U.S.A., Canada, and U.K. CBD cases all showed a significant association with H1 and H1/H1 genotype.
Taken together, these data therefore strongly support the view that PSP and CBD are genetically, as well as pathologically, related diseases. Although there may be risk factors that cause the divergent clinical presentations, it is likely that both disorders share a common pathogenic mechanism that involves tau dysfunction.
Acknowledgments
Supported by the Wellcome Trust, the Mayo Foundation, the Robert and Clarice Smith Fellowship (to M.H.), the Medical Research Council UK, the National Institute on Aging, and the Progessive Supranuclear Palsy (Europe) Association for supporting this work.
Acknowledgment
The authors thank the Harvard Brain Bank, the AD Brain Bank at the Institute of Psychiatry, and the PD Brain Bank at the Institute of Neurology for brain tissue.
- Received July 27, 2000.
- Accepted February 24, 2001.
References
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