Human prion diseases may be acquired as infectious diseases, they may be inherited in an autosomal dominant fashion or occur sporadically. Mutations and polymorphisms in the sequence of the coding region of the prion protein gene (PRNP) have been established as an important factor in all of these three types of prion diseases. Therefore, a total of 578 patients with suspect prion diseases referred to the German Creutzfeldt-Jakob disease (CJD) surveillance unit over a period of 4.5 years have been examined for mutations and polymorphisms in the coding region of PRNP. We found 40 cases with a missense mutation previously reported as pathogenic. Amongst these, the aspartate to asparagine change at codon 178 (D178N) was the most common mutation. All of these cases carried the D178N mutation in coupling with methionine at codon 129, resulting in the typical fatal familial insomnia (FFI) genotype. Most cases with pathogenic mutations were not found in the group of clinically "probable" cases according to established clinical criteria, supporting the notion that inherited prion diseases often exhibit atypical features. Two novel missense mutations (T188R and P238S) and several silent polymorphisms were found, demonstrating the quality of our screening procedure based on a modified version of the single-stranded conformational polymorphism technique. In "definite" CJD cases with no pathogenic mutation, the patients clinically classified as "probable" were mostly homozygous for methionine at the common polymorphism at codon 129, whereas there was a marked over-representation of patients homozygous for valine amongst those clinically classified as "possible". This large study on suspect cases of human prion diseases in Germany clearly shows that PRNP genetics is essential for a comprehensive analysis of prion diseases.

The gene for agouti signaling protein (ASIP) is centrally involved in the expression of coat color traits in animals. The Mangalitza pig breed is characterized by a black-and-tan phenotype with black dorsal pigmentation and yellow or white ventral pigmentation. We investigated a Mangalitza x Piétrain cross and observed a coat color segregation pattern in the F2 generation that can be explained by virtue of two alleles at the MC1R locus and two alleles at the ASIP locus. Complete linkage of the black-and-tan phenotype to microsatellite alleles at the ASIP locus on SSC 17q21 was observed. Corroborated by the knowledge of similar mouse coat color mutants, it seems therefore conceivable that the black-and-tan pigmentation of Mangalitza pigs is caused by an ASIP allele a(t), which is recessive to the wild-type allele A. Toward positional cloning of the a(t) mutation, a 200-kb genomic BAC/PAC contig of this chromosomal region has been constructed and subsequently sequenced. Full-length ASIP cDNAs obtained by RACE differed in their 5' untranslated regions, whereas they shared a common open reading frame. Comparative sequencing of all ASIP exons and ASIP cDNAs between Mangalitza and Piétrain pigs did not reveal any differences associated with the coat color phenotype. Relative qRT-PCR analyses showed different dorsoventral skin expression intensities of the five ASIP transcripts in black-and-tan Mangalitza. The a(t) mutation is therefore probably a regulatory ASIP mutation that alters its dorsoventral expression pattern.

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