Abstract
Objective: To identify genetic mutations in the coding regions and the splice-donor sites of the tau gene on chromosome 17q in individuals with progressive supranuclear palsy (PSP).
Background: Several studies provide evidence for linkage disequilibrium between a PSP disease gene and allelic variants of the tau gene. However, a causative mutation has not been identified.
Methods: We designed a study to search for genetic mutations in 15 coding regions of the tau gene including the splice-donor sites in 22 patients with PSP by comparing the mobility shifts on single-strand conformation analysis with those of age-matched controls. Fragments with altered migration were sequenced directly and compared for differences in nucleotide composition. Restriction enzyme digests were used to confirm single base-pair substitutions.
Results: Significant differences in mobility shifts were found in exons 1, 4A, and 8 between affected individuals and age-matched controls. All individuals with PSP had a common extended haplotype characterized by a homozygous polymorphism in the 5′ splice site untranslated region of exon 1, two missense mutations in exon 4A (Asp285Asn, Ala289Val), and a nonsense mutation in the 5′ splice site of exon 8.
Conclusions: This study demonstrates that 22 unrelated progressive supranuclear palsy (PSP) patients have four identical sequence variants within the tau gene that are not present in 24 age-matched controls. Although the functional significance of these results on tau protein expression is unknown, the presence of this “susceptibility” haplotype in individuals may place them at risk for developing PSP.
The salient clinical features of progressive supranuclear palsy (PSP) are parkinsonian signs (bradykinesia, postural instability, rigidity, frequent falls, and variable tremor) accompanied by vertical supranuclear palsy and a poor response to levodopa therapy. In typical cases of PSP, aberrant forms of the microtubule-associated protein tau precipitate in subcortical neurons and glia as straight neurofibrillary filaments.1,2 The first evidence that a disease gene for PSP was at or near the genetic locus for tau was provided by linkage disequilibrium studies performed on unrelated individuals by Conrad et al.3 in autopsied cases and by Higgins et al.4 in clinically ascertained cases. These findings were confirmed in European populations from the United Kingdom and Italy5,6 but not Japan.7
The genomic structure of the human tau gene is organized into 16 coding regions of more than 100 kilobases of DNA on chromosome 17q21.8 The first coding region (designated as exon −1 or 0) is not translated. Exons 1, 4, 5, 7, 9, 11, 12, and 13/14 are consistently expressed in neural tissue but regulated expression of exons 2, 3, 4A, 6, 8, and 10 are partially responsible for the production of different tau protein isoforms. In the adult human cortex, exons 4A, 6, and 8 are not expressed8,9 but exons 2, 3, and 10 are alternatively spliced to yield six distinct tau protein isoforms that range from 352 to 441 amino acids. These isoforms differ by the presence of three or four tandem repeats of 31 or 32 amino acids in the carboxyl-terminal region in conjunction with no, one, or two amino acid inserts of 0, 29, or 58 amino acids in the amino-terminal part.10-15 In PSP, the tau protein exists in a hyperphosphorylated state and consists only of the four tandem repeat isoforms.16
Besides PSP, other neurodegenerative diseases, such as AD, frontotemporal dementia and parkinsonism (FTDP), ALS–parkinsonism dementia complex of Guam, and corticobasal degeneration (CBD), also demonstrate abnormal tau neuropathology.1 On this basis, these disorders may be categorized as “tauopathies,” although the ultrastructural and antigenic properties of tau differ among these clinical entities. The hypothesis that specific genetic mutations could result in a tauopathy was substantiated by the discovery of 5′ splice site and missense mutations in exons 9, 10, and 13 of the tau gene in families with dominantly inherited FTDP linked to chromosome 17q (FTDP-17).17-21 One particular missense mutation in exon 13 of the tau gene (R406W) was found in a family with atypical PSP,17,22 but this mutation was not found in 25 unrelated individuals with PSP or in six individuals with another tauopathy, CBD.23 Other investigators have sequenced exons 9 through 13 of the tau gene in PSP patients and evaluated other polymorphisms but were unable to identify a causative mutation for PSP in these regions.24
In the current study, we used a case-control strategy to compare differences in tau exon mobility shifts on single-strand conformation polymorphism analysis (SSCP) between unrelated individuals with PSP and age-matched controls. We report the results of these analyses and identify at least four variants within the tau gene that cosegregate with a PSP disease phenotype.
Methods.
Clinical testing.
Informed consent was obtained from all individuals before clinical and genetic testing. The Institutional Review Board of the New York State Department of Health approved this study. Twenty-two individuals were classified with PSP by the guidelines established by the National Institute of Neurological Disorders and Stroke and the Society for PSP.25 There was no evidence of consanguinity or family history of PSP in affected patients. Twenty-four unrelated individuals matched for age and race were used as controls.
Genomic DNA extraction and SSCP analysis.
High molecular weight genomic DNA was isolated from whole-blood lysate by phenol-chloroform extraction and isopropanol precipitation. The intronic oligonucleotide primers that were used to amplify the tau exons are described in on-line table E1 (see the on-line version of Neurology: www.neurology.org). PCR for each reaction contained 50 ng of genomic DNA in a total volume of 15 μL with 0.8 μM of each primer, 1.5 mM MgCl2, 200 μM dGTP, 200 μM dATP, 200 μM dTTP, 24 μM dCTP, 0.1 μL of [α-32P]dCTP (10 microCuries/μL, Amersham, Arlington Heights, IL), 50 mM KCl, 10 mM Tris HCl (pH = 8.3), 0.01% gelatin, and 0.5 U of AmpliTaq (Perkin Elmer-Cetus, Foster City, CA). Reactions were performed in a 96-well microtiter plate and amplification was carried out for 30 cycles with 45 seconds denaturation at 95° C, 45 seconds annealing at 55° C or 60° C (see on-line version of Neurology, table E1), and 45 seconds extension at 72 °C on a Genius thermocycler (Techne Inc., Princeton, NJ). The last extension step was 7 minutes at 72 °C. The samples were mixed with 20 μL of a solution containing 98% formamide, 0.5 M EDTA, 0.025% xylene cyanol, and 0.025% bromophenol blue, and denatured at 95 °C for 5 minutes. The samples were cooled on ice for 10 minutes, and then four μL of the reaction mixture was loaded on a 30 × 40-centimeter MDE gel (FMC, Rockland, ME). Electrophoresis was performed at 50 Watts for 4 to 8 hours at 4 °C. The gel was transferred to filter paper and exposed to x-ray film at −80 °C for 12 to 24 hours.
DNA sequencing.
Ten nanograms of PCR products were directly sequenced using the dideoxy AmpliTaq.FS cycle sequencing kit (Perkin Elmer-Cetus) and analyzed on the Applied Biosystems Model 377 DNA sequencer. All sequence variants were confirmed manually by using the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit and electrophoresed on RapidGel-GTG-8% polyacrylamide matrix (USB Corporation, Cleveland, OH). Forward and reverse primers were used to sequence the PCR products. The presence of altered restriction enzyme sites was detected by using 1 μg of the PCR products in a reaction with the appropriate restriction enzyme (New England Biolabs, Beverly, MA) by methods described by the manufacturer. The digested products were electrophoresed on a 3.0% agarose gel.
Results.
The phenotype of study participants.
All normal individuals and patients with PSP were white. There were more male PSP study participants (72%) than female (28%). Both sexes were equally represented in the control group. The ages of subjects in all study groups were similar (Controls, mean ± SD 65 ± 6 years, range 56 to 76, n = 24; PSP, 67 ± 8, 52 to 82, n = 22). The age at disease onset (63 ± 7; 50 to 75) was also similar to the age of control subjects. Nineteen patients met the previously published criteria25 for “probable” PSP that included a gradual progressive clinical course with an onset at age 40 or later, vertical supranuclear gaze palsy, prominent postural instability with falls within the first year of the disease, and no evidence of other diseases. The diagnosis of PSP was confirmed at autopsy in three patients.
SSCP analysis.
Eighteen regions of the tau coding sequences including their splice site junctions were analyzed in 22 individuals with clinically ascertained PSP and 24 age-matched normal controls. There were no differences in mobility shifts between affected individuals and controls in exons 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, and 13. Three different mobility shift patterns were found in exon 14. In exon 14, 20 controls (83%) and two affected individuals (9%) had pattern 1; one control (4%) and no affected individuals (0%) had pattern 2; and three controls (13%) and 20 affected individuals (91%) had pattern 3. The mobility shifts in exons 1, 4A, and 8 were identical in all PSP patients.
Identification of tau gene variants in PSP.
We identified base pair differences between normal and affected individuals by directly sequencing the PCR products that were used to generate the mobility shifts on SSCP. The table shows the nucleotides in exons 1, 4A, and 8 that differed between PSP patients and controls. Nucleotide base pair substitutions in exons 4A and 8 were evaluated by direct sequencing and confirmed by restriction enzyme digestion with BsgI and DdeI. A homozygous polymorphism in the 5′ splice site untranslated region (UTR) of exon 1 was confirmed in triplicate in all individuals by forward and reverse manual sequencing (figure). All the PSP patients (n = 22) had a common haplotype comprised of four polymorphisms in exons 1, 4A, and 8 (“B” polymorphisms) (see table). All these PSP patients lacked the “A” polymorphisms that were found in the majority of the age-matched controls (n = 24). Only one control had two B polymorphisms in exon 1 and exon 8. Seven different normal individuals had a haplotype that contained one of the B polymorphisms. The PSP haplotype consisting of all four B polymorphisms was not identified in any of the normal individuals in the age-matched control population.
Tau gene variants in progressive supranuclear palsy (PSP)
Figure. A polymorphism in the tau 5′ splice site untranslated region of exon 1. (a) The manual sequence of a normal individual demonstrating a G/A heterozygous polymorphism in the consensus sequence CAGGTGA G/A C. (b) The manual sequence of an individual with progressive supranuclear palsy demonstrating an A/A homozygous state at the same position.
Discussion.
This study provides evidence that a unique, common tau haplotype cosegregates with a PSP phenotype in a small group of unrelated individuals with the classic signs of the disease. Although confirmation in a larger population is needed, genetic variants of the tau gene may predispose individuals to develop the neuropathologic features of PSP. Further studies will determine whether these allelic variants cause disturbances in the interactions of the tau protein with microtubules. In contrast to the tau mutations in FTDP-17 that affect the microtubule binding domains of the C-terminus of the tau protein (encoded by exons 9 to 12), the PSP variants that we describe are found in domains that may interact with the neural plasma membrane.9,26,27 Amino acid changes in this region may affect the ability of the tau protein to bridge microtubules to this membrane.28
Except for the Asp285Asn polymorphism reported by Poorkaj et al.,18 the three other tau polymorphisms in the table have not been reported previously. The allele frequency distribution for the Asp285Asn polymorphism is different than that reported by Poorkaj et al.18 because these investigators estimated the allele frequencies based on an evaluation of 92 randomly chosen white subjects. In our study, we used an age-matched population as a control rather than a random sample because of the late age at disease onset in our case population.
Because transcripts from exons 4A or 88,12 are not found in adult human cerebral cortex, the polymorphism in the 5′ splice site UTR of exon 1 may influence the expression of tau in PSP patients. However, the significance of the exon 4A and 8 variants cannot be dismissed because the precise expression patterns of these exons at the major sites of PSP pathology (the pallidum, subthalamic nucleus, substantia nigra, or pons) are not well defined. Interestingly, interspecies differences in the expression of exon 8 may underlie a predilection to develop neurofibrillary degeneration.28 Even though the polymorphism in the 5′ splice site UTR of exon 1 may cause inefficient or aberrant splicing of exon 1, the presence of this polymorphism in normal age-matched individuals (table) suggests that it is not directly responsible for the formation of straight neurofibrillary filaments.
One hypothesis, based on the unlikely probability of affected individuals inheriting an identical haplotype, is that all or some of the tau polymorphisms must be present in an individual in order to develop PSP. In two different neurologic illnesses, Creutzfeldt-Jakob disease and fatal familial insomnia, the pathogenic mutation at codon 178 in the PRNP gene on chromosome 20 defines both disorders but a polymorphism at codon 129 determines the phenotype.29 Similarly, the presence of particular tau polymorphisms may define a susceptibility to develop PSP but the absence of some or all of the polymorphisms may not result in the same phenotype or disease susceptibility. An alternative hypothesis is that these tau polymorphisms may be in linkage disequilibrium with another mutation in a nearby gene on chromosome 17q or with an undefined mutation within the tau gene. Further studies evaluating the expression of the tau variants reported in this study are needed to confirm the role, if any, these changes have in tau interactions. Efforts to study the consequences of this common PSP haplotype and to study other exon and intron tau sequence variants will lead to a better understanding of the pathogenesis of PSP in general and the formation of straight neurofibrillary filaments in particular.
Acknowledgments
Supported by a grant from the Margaret Parker Research Fund for Progressive Supranuclear Palsy and by the Society for Progressive Supranuclear Palsy.
Acknowledgment
The authors thank the staff of the Molecular Genetics Core Facility at the David Axelrod Institute, Wadsworth Center, in Albany, NY, for providing the automated DNA sequencing services in this study.
- Received May 7, 1999.
- Accepted July 27, 1999.
References
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