Fatal familial insomnia, a prion disease with a mutation at codon 178 of the prion protein gene

Abstract

Background: We previously described two members of a family affected by an apparently genetically determined fatal disease characterized clinically by progressive insomnia, dysautonomia, and motor signs and characterized pathologically by severe atrophy of the anterior ventral and mediodorsal thalamic nuclei. Five other family members who died of this disease, which we termed "fatal familial insomnia," had broader neuropathologic changes suggesting that fatal familial insomnia could be a prion disease.

Methods: We used antibodies to prion protein (PrP) to perform dot and Western blot analyses, with and without proteinase K, on brain tissue obtained at autopsy from two patients with fatal familial insomnia, three patients with sporadic Creutzfeldt-Jakob disease, and six control subjects. The coding region of the PrP gene was amplified and sequenced in the samples from the two patients with fatal familial insomnia. Restriction-enzyme analysis was carried out with amplified PrP DNA from 33 members of the kindred.

Results: Protease-resistant PrP was found in both patients with fatal familial insomnia, but the size and number of protease-resistant fragments differed from those in Creutzfeldt-Jakob disease. In the family with fatal familial insomnia, all 4 affected members and 11 of the 29 unaffected members had a point mutation in PrP codon 178 that results in the substitution of asparagine for aspartic acid and elimination of the Tth111 I restriction site. Linkage analysis showed a close relation between the point mutation and the disease (maximal lod score, 3.4 when theta was zero).

Conclusions: Fatal familial insomnia is a prion disease with a mutation in codon 178 of the PrP gene, but the disease phenotype seems to differ from that of previously described kindreds with the same point mutation.

Figure 1.. Pedigree of a Kindred with Fatal Familial Insomnia.

Squares denote male subjects, circles female subjects, diamonds subjects of either sex, and slashed symbols dead subjects. Stippled symbols denote subjects possibly affected according to nonmedical records; hatched symbols, subjects probably affected according to medical records; solid symbols, subjects proved to be affected, by histologic examination; and symbols with asterisks, subjects examined clinically in the present study. The sixth generation, which had no affected members, is not shown.

Figure 2.. Dot Blots Demonstrating Protease-Resistant PrP in Two Subjects with Fatal Familial Insomnia.

Dots representing samples of brain tissue were treated with proteinase K and subjected to immunoreaction with antiserum R073.5 Tissue samples were obtained from the basal ganglia (B), thalamus (Th), and cerebellum (C) and from the occipital (O), parietal (P), temporal (T), and frontal (F) cortical areas of Subjects IV-37 and V-58 and a control; cerebral cortical tissue from a subject with sporadic Creutzfeldt-Jakob disease (CJD) was used as a positive control. The immunoreaction was stronger and was present in more brain regions in Subject V-58 than in Subject IV-37; it was very strong in the subject with Creutzfeldt-Jakob disease and absent in the control.

Figure 3.. Western Blots Showing the Size of the PrP Fragments Generated by Treatment with Proteinase K.

The blots are those of a control, three subjects with sporadic Creutzfeldt-Jakob disease (1, 2, and 3), and two subjects with fatal familial insomnia (IV-37 and V-58). Molecular-weight markers are expressed in kilodaltons, at left. Plus signs and minus signs denote treatment with proteinase K and no treatment, respectively. The protease-resistant fragments from the subjects with fatal familial insomnia differ in size from those from the subjects with Creutzfeldt-Jakob disease.

Figure 4.. Autoradiograph of Sequencing Gel from Part of the PrP Coding Region in the Mutant and Normal Alleles of Subject V-58.

The three bands that identify the three base pairs of codon 178 in the two alleles are indicated by arrowheads. The GAC→AAC mutation that leads to the substitution of asparagine for aspartic acid can be detected by comparing codon 178 in the two alleles. The same findings were made in Subjects IV-16 and IV-37. The DNA bases are indicated at the top of the figure.

Figure 5.. Tth 111 I Digestion of the 800-bp Amplified PrP Coding Region in Samples from Six Members of the Kindred with Fatal Familial Insomnia.

Three bands of 800, 556, and 244 bp are present in members carrying the GAO→AAC mutation, which abolishes the Tth 111 I restriction site in one of the two alleles (lanes 1, 2, and 3). Only two fragments of 556 and 244 bp are present in members without the mutation (lanes 4, 5, and 6), since both alleles are completely digested (these members are not identified, to protect confidentiality). Lane 7 represents DNA markers — i.e., the products of Bg/l-digested and H/nfl-digested pBR322 DNA.