Progressive supranuclear gaze palsy is in linkage disequilibrium with theτ and not the α-synuclein gene

Progressive supranuclear palsy (PSP) is a late-onset atypical parkinsonian disorder that is characterized by supranuclear vertical gaze palsy, postural instability, rigidity, and dementia, with the variable presence of pyramidal and cerebellar signs.¹ In typical cases, neurofibrillary tangles (NFT) and neuropil threads are present in at least the pallidum, subthalamic nucleus, substantia nigra, or pons. Amyloid deposits and neuritic plaques are notably absent.² Some investigators believe that the aberrant forms of the microtubule-associated protein, tau (τ), that are found in the NFT of patients with PSP are caused by alterations in the expression of the τ gene.^3,4 Common biochemical or genetic factors are also implicated in Parkinson's disease and PSP because of the clinical similarities and the involvement of the dopaminergic systems in both disorders. A gene that lies in the candidate region on chromosome 4q21-q23 for familial Parkinson's disease, humanα-synuclein (SNCA), mediates processes that involve the exocytosis of synaptic vesicles and neuronal cell death.^5-7 These overlapping neuropathologic, biochemical, and clinical factors among patients with Alzheimer's disease, Parkinson's disease, and PSP were considerations in our investigation of the involvement of the τ or SNCA genes in PSP.

The rarity of familial PSP suggests that most cases of PSP are sporadic or that the genealogic information obtained from affected families is imprecise. The late onset of the disease and the lack of the clinical recognition of atypical PSP cases are factors that may hinder accurate phenotypic assignment. Several reports indicate that a familial subset of PSP is recessively inherited^8-10 but at least one family demonstrates a dominant mode of transmission.¹¹ To evaluate a hereditary etiology for PSP, we hypothesized that the occurrence of specific τ or SNCA alleles on the same haplotype was not independent of the PSP disease locus in the Caucasian population. This deviation from independence, termed linkage disequilibrium, is a population genetic phenomenon that is useful in gene mapping.¹² We conducted this study to determine if the τ or SNCA candidate genes were in linkage disequilibrium with PSP.

Methods. Clinical studies. Informed consent was obtained before all genetic studies. This study was approved by the Institutional Review Board at the National Institute of Neurological Disorders and Stroke (NINDS). Individuals were classified as having PSP by the guidelines established by the NINDS and the Society for PSP.² There was no evidence of consanguinity or a family history of PSP in affected patients. All patients with PSP were Caucasian. To determine the frequency of the different τ and SNCA alleles, the DNA samples for the control group were chosen from unrelated Caucasian individuals from the Centre d'Etude Polymorphisme Humain (CEPH) reference panel and unaffected spouses.

Genomic DNA analysis. High molecular weight genomic DNA was isolated from whole-blood lysate by serial phenol/chloroform extractions followed by isopropanol precipitation by methods described previously.¹³ Allele status was determined by the polymerase chain reaction (PCR) using oligonucleotide primers flanking the dinucleotide repeat regions of the τ and SNCA genes. The sense 23-mer primer for amplifying the τ dinucleotide repeat was 5′-GCCTCGCAAATTGCTGGGATTAC-3′ and the 23-mer anti-sense primer was 5′-AGGTGACTGGGTAGAGACAGAGC-3′. The sense 25-mer primer for amplifying the SNCA dinucleotide repeat was 5′-TGCAATAGAGTAGACAAAAGGATGG-3′ and the 22-mer anti-sense primer was 5′-ACATGACTGGCCCAAGATTAAC-3′. Fifty nanograms of genomic DNA were used 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 (10µCi/µL, Amersham, Arlington Heights, IL), 50 mM KCl, 10 mM Tris HCl (pH = 8.3), 0.01% gelatin, and 0.5 U Taq DNA polymerase (Perkin Elmer-Cetus, Foster City, CA). Reactions were performed in a 96-well microtiter plate and amplification was carried out for 30 cycles (45 seconds denaturation at 95°C, 45 seconds annealing at 55°C, and 45 seconds extension at 72°C) on a GeneE thermocycler (Techne Inc., Princeton, NJ). The last extension step was 7 minutes at 72°C. Four µL of the reaction mixture were electrophoresed on an EASIgel [6.0% acrylamide/0.3% bis-acrylamide (7 M urea; Scotlab, Strathclyde, Scotland)]. Radioactive PCR products were visualized by autoradiograms.

Statistical analyses. For each polymorphism, the allele and genotype distribution in the group of individuals with PSP were compared with those in the control group by chi-square analysis. To search for linkage disequilibrium between the PSP disease allele and marker alleles, we used the case-control option of the Estimating Haplotype frequencies program (EH) to test for linkage equilibrium.¹²

Results. The average age of subjects in the control group was 61.5 ± 6.8 years (age ± SD); of patients with PSP, 64.9± 6.5 years. The figure shows the four polymorphic alleles in the τ gene and the three polymorphic alleles found in the SNCA gene. Table 1 shows the frequency distribution of the alleles for τ and SNCA polymorphisms in patients with PSP and in control subjects. When the control subjects and individuals with PSP were divided into two groups according to the presence of the τ a₁a₁ genotype or the τ a₁ allele, the chi-square p value was significant at <0.01. The odds ratio for an association between PSP and the τ a₁a₁ genotype was 4.35 and the odds ratio for an association between PSP and the τ a₁ allele was 3.55. Chi-square analysis of the SNCA genotypes or alleles did not demonstrate significant differences between the control subjects and the patients with PSP.

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Figure. Representative 6% polyacrylamide gels demonstrating (A) the four polymorphic alleles in the tau (τ) gene and(B) the three polymorphic alleles found in the human α-synuclein gene(SNCA). In figure A, the a₁a₁ τ genotype is shown in lanes 1 and 6, the a₁a₂ τ genotype in lane 10, the a₁a₃ τ genotypes in lanes 2, 3, 4, 8, 9, and 11, the a₁a₄ τ genotypes in lanes 5 and 7, and the a₂a₃ τ genotype in lane 12. Infigure B, the a₁a₂ SNCA genotype is shown in lanes 2, 3, 5, and 8, the a₂a₂ SNCA genotypes in lanes 1 and 4, the a₁a₃ SNCA genotype in lane 7, and the a₂a₃ SNCA genotype in lane 6.

The case-control EH program, using a model of dominant inheritance with varying penetrances and gene frequencies, found no significant evidence for linkage disequilibrium between PSP and the τ or SNCA marker alleles. When a model using recessive inheritance with complete penetrance was employed, evidence for linkage disequilibrium was found between PSP and the τ marker alleles (table 2) but not between PSP and the SNCA marker alleles.

Discussion. The term "linkage disequilibrium" is defined by the preferential association of a particular disease allele with a specific allele at a nearby locus more frequently than is expected by chance alone. We found an overrepresentation of the τ a₁a₁ genotype and the gt a₁ allele in our PSP patient population and documented linkage disequilibrium between the disease locus and the τ alleles using a disease model of recessive inheritance. An overrepresentation of the τ a₁ allele was previously reported by Conrad et al.³ in a different group of patients with PSP compared with groups of individuals with Alzheimer's disease and the parkinsonism-dementia complex of Guam. Although Conrad et al.³ did not test for linkage equilibrium using the EH program, calculations using their data also show significant evidence for linkage disequilibrium between the disease locus and the τ alleles using a recessive inheritance model.

In contrast with autosomal dominant familial Parkinson's disease,⁵ the data from our PSP population and other similar PSP populations³ suggest that PSP is a recessive disorder that does not map to the Parkinson's disease SNCA candidate gene on chromosome 4q21-q23. The mode of inheritance of PSP is controversial, but the strict clinical criteria² that we used to diagnose PSP clearly distinguishes our patients from the clinically heterogeneous individuals in the single family with dominantly inherited PSP.¹¹ Our case-control study suggests an association between the τ a₁a₁ genotype and the τ a₁ allele and the PSP disease gene, but this finding does not establish a causal relationship between τ and PSP. Further studies assessing the segregation of specific τ gene mutations in patients with PSP are needed to demonstrate convincingly that the neuropathology in PSP is caused by alterations in the expression of the τ gene.