The tau gene A0 allele and progressive supranuclear palsy

Individuals.

The clinical diagnosis of PSP (years since disease onset: 2 to 8) was made according to the recommended international criteria.17 A total of 28 asymptomatic PSP family members were included in the study (age 65 ± 7.23 years, range 55 to 80; n = 28). The clinical diagnosis of PD patients (age 59.77 ± 11.63 years, range 44 to 81, n = 18; mean follow-up after disease onset = 4.5 years) was performed according to criteria of the United Kingdom Parkinson’s Disease Society Brain Tissue Bank.18 The pathologic diagnosis of PSP brains was made in accordance with the recent international recommendations.7,8 Control DNA was obtained from healthy individuals (age 52.1 ± 9.98 years, range 39 to 75; n = 79) with no evidence of neurologic diseases in their families who were studied at the Department of Genetics, “Fundación Jiménez Díaz,” Madrid. All individuals in this study were white.

Amplification and identification of tau polymorphism.

Genomic DNA from patients and controls was obtained either from whole-blood lysate or brain samples using standard techniques. Allelotyping was performed by polymerase chain reaction (PCR) using the oligonucleotides described by Conrad et al.12 PCR was performed in a 10-μL reaction mixture containing 100 ng of genomic DNA as template, 10 mM Tris-HCl pH 8.0, 50 mM KCl, 1.5 mM MgCl₂, 0.2 μM each primer, 200 μM dNTPs, 1 unit of Taq DNA polymerase (Boehringer Ingelheim, Germany), and 0.07 μL of [α-³²P]dCTP (10 mCi/μL; Amersham, Arlington Heights, IL). Reactions were performed in a 96-well microtiter plate, and the amplification conditions comprised 30 cycles at 94 °C for 1 minute, annealing at 55 °C for 1 minute, and extension at 72 °C for 1 minute. The final products, after denaturation at 95 °C for 5 minutes, were subjected to 6% polyacrylamide/8 M urea gel electrophoresis. Extension products were detected by autoradiography using Kodak XAR-5 radiographic film.

Chromosome 17 linkage analysis.

Genomic search of chromosome 17 was performed in two PSP white families, a large one from Spain previously described by de Yébenes et al.5 and a small unreported one from the United States (see pedigrees, figure 1). The following short tandem repeat polymorphisms, distributed throughout the chromosome at a mean distance of 15.59 cM, were examined: D17S849, D17S796, D17S1303, D17S122, D17S1288, D17S579, D17S806, D17S809, D17S1290, D17S795, D17S801, D17S836, D17S784, and D17S928. The sense primers were end labeled using adenosine triphosphate polynucleotide kinase with [α-³²P]dCTP. A total of 200 ng of endogenous DNA were used for PCR (30 cycles, denaturation at 94 °C for 1 minute; annealing at 60 °C for 1 minute and extension at 60 °C for 1 minute) in a final volume of a 10-μL mixture containing the sense and antisense primers, 0.5 units of Taq DNA polymerase, and 1.0 mM MgCl₂. After amplification, the different alleles were identified in autoradiographs after electrophoresis in 6% polyacrylamide gel.

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Figure 1. Family trees of two families with progressive supranuclear palsy (PSP). The clinical and pathologic features of some members were previously reported.5

Data analysis.

Linkage analysis was performed with the Mlink program of the LINKAGE package, version 5.2.19 Multipoint analysis was performed with VITESSE version 1.0.20 Analysis of the frequencies of polymorphisms of the tau gene in PSP, other diseases, and controls was performed by the chi-square test. Differences were considered significant when p < 0.05.

Sequence analysis.

Sequencing of the coding exons of the tau gene was performed on both strands from genomic DNA amplified using the primers and conditions described previously.21 This only included exons expressed in the brain (1 to 5, 7, and 9 to 13). Briefly, purified PCR products were sequenced using the rhodamine dye terminator kit from Perkin-Elmer (Foster City, CA). The reactions were resolved using an automated DNA sequencer (ABI377, Perkin-Elmer), and the sequences were analyzed using PolyPhred (Nickerson, US).

Reverse transcriptase-polymerase chain reaction (RT-PCR).

The expression of exon 10 from the tau gene was studied according to Montejo et al.22 Total RNA was prepared from different sections of two normal brains (controls), three AD brains, and four PSP brains using the reagent RNAzol (Tel-Test, Inc., US) and following the supplier’s protocol. Reverse transcription was performed using the first cDNA synthesis kit (Pharmacia, Piscataway, NJ) on 5 μg of brain RNA with oligo(dT) primer. PCR was performed with the oligonucleotides A15 (5′-GCGAATTCAATGCCAGACCTG-3′) and A16 (5′-GCGGATCCCAAACCCTGCTTGG-3′). The amplification conditions comprised 30 cycles at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds, and extension at 72 °C for 1 minute. PCR products were analyzed on agarose gel.

Prevalence of the A0 allele of the tau gene in individuals with PSP.

The distribution of the four polymorphic alleles in the tau gene was studied in patients with familial PSP, their asymptomatic relatives, PSP asymptomatic family members, patients with sporadic PSP, and controls free of neurologic disorders. Table 1 shows an over-representation of the tau genotype A0/A0 and the A0 allele in both familial and sporadic PSP patients compared with controls (p = 0.001, odds ratio 17.03, chi-square test). The high frequency of the A0 allele and the A0/A0 homozygous individuals was, however, not restricted to PSP patients but also occurred in their asymptomatic PSP relatives (p = 0.008, chi-square test), suggesting that this genotype may not be specific for familial PSP. Consistent with these results, the prevalence of the A0/A0 genotype is also greater in individuals with the pathologic diagnosis of PSP than it is in those with the pathologic diagnosis of AD, other neurologic disorders, or absence of neurologic disease, as shown in table 1.

In patients with PD the association between the A0/A0 genotype and the A0 allele with the disease was not significant (p = 0.09, odds ratio 2.86; and p = 0.23, odds ratio 1.86, respectively, chi-square test). There was, however, a trend that does not allow us to disregard the importance of the A0 allele in PD.

Linkage analysis of multiple markers in the region of the tau gene and other areas of chromosome 17.

We tested for linkage in our PSP families with 14 polymorphic markers located throughout chromosome 17. The lod scores of two-point linkage analysis are shown in table 2. Negative lod scores are found for markers D17S1288 and D17S579 located in the vicinity of the cytogenetic region 17q21, where the tau gene is located, as well as in most areas of chromosome 17, with the exception of the distal telomere, where a positive but low lod score was found. We also analyzed our results by multipoint linkage study, as shown in figure 2. This analysis excluded linkage to this area.

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Figure 2. Multipoint lod score analysis of familial progressive supranuclear palsy with polymorphic markers of chromosome 17.

In addition, we analyzed linkage of tau intronic polymorphism with PSP in two families. When the data of these two families were pooled, linkage was excluded for both autosomal dominant and autosomal recessive models of inheritance. If the families were analyzed individually, linkage was excluded in family 2 with the model of dominant inheritance, but we obtained a noninformative lod score of −1.44 with the model of recessive inheritance. In the case of family 1, linkage was excluded under the model of recessive inheritance, and it was noninformative due to its small size (lod score 0.1) under the model of dominant inheritance.

Sequence of the tau gene in pathologically confirmed PSP cases.

We performed sequence analysis of two A0/A0 homozygous PSP patients belonging to the large family (family 2) and two A0/A0 sporadic patients. All were confirmed by pathologic diagnosis. Sequencing of the coding exons of the tau gene failed to detect any change that could account for the appearance of the disease.

Expression of tau mRNA containing exon 10 in PSP brain.

To study whether the disease affects neuronal groups that only express a particular tau isoform, we analyzed, at the cDNA level, the expression pattern of the alternatively spliced exon 10 in each case. We performed the molecular analysis by RT-PCR from the brains of PSP or AD patients and from controls. Our results indicate similar levels of expression in samples from different brain regions from a PSP patient, although a slightly higher proportion of four-repeat cDNA tau isoform was found in the cortex (figure 3A). Because the increase in messenger RNA containing exon 10 would increase the proportion of tau containing four microtubule-binding repeats, we also quantified, by densitometry, the proportion of tau cDNA expressing exon 10 for the samples indicated in figure 3, B and C. The quantified expression of four-repeat tau isoform, represented as a percentage of the total tau (three- plus four-repeat isoforms) in two samples from two brains with different disorders was the following: AD, 20 ± 4 and 31 ± 7; PSP, 23 ± 6 and 28 ± 8; control, 21 ± 3 and 24 ± 5 (see figure 3B). These data do not suggest dramatic differences among the tested samples. However, there is a slight increase in the proportion of four-repeat tau in one AD and in one PSP brain. Although these analyses are only from two samples, it suggests that there is not a clear increase of four-repeat tau in PSP. Nevertheless, to test whether the different polymorphisms modify the expression of exon 10, samples from two regions of an AD patient (A3/A3) and from a PSP patient (A0/A0) were compared by RT-PCR and quantified (see figure 3C). The expression of four-repeat tau isoform from PSP cortex was 33 ± 7 and from basal ganglia was 30 ± 5, whereas the expression in patients with AD were 21 ± 4 from cortex and 20 ± 3 from basal ganglia. These differences were too small to have pathologic significance.

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Figure 3. (A) Expression of tau isoforms in different regions of the brain from a patient with progressive supranuclear palsy (PSP). Reverse-transcriptase polymerase chain reaction was performed to characterize the tau RNA transcripts from cortex (a), globus pallidus (b), and caudate nucleus (c). The electrophoretic mobility of the cDNA expressing exon 10 (4 tubulin-binding motifs) (4R) or not expressing exon 10 (3 tubulin-binding motifs) (3R) are indicated as electrophoretic mobility markers. (B) Expression of tau isoforms in two isoforms in two samples from controls (c, c′) and AD (a, a′) and PSP (p, p′) patients from the temporal gyrus. (C) Expression of tau isoforms from the same AD patient in panel B (A3/A3) from cortex (a) and basal ganglia (a′) and from the same PSP patient (A0/A0) from cortex (p) and basal ganglia (p′). 3R and 4R are as in panel A, and M indicates electrophoretic mobility markers.

Thus, the above results indicate that there is not a clear correlation between the presence of alleles A0 and A3 and the expression of tau with three- or four-repeat isoforms, as determined by analyzing their cDNAs, although a slight increase in four-repeat tau cDNA was observed in the A0 compared with the A3 allele.