Ovine reference materials and assays for prion genetic testing

Abstract

Background: Genetic predisposition to scrapie in sheep is associated with several variations in the peptide sequence of the prion protein gene (PRNP). DNA-based tests for scoring PRNP codons are essential tools for eradicating scrapie and for evaluating rare alleles for increased resistance to disease. In addition to those associated with scrapie, there are dozens more PRNP polymorphisms that may occur in various flocks. If not accounted for, these sites may cause base-pair mismatching with oligonucleotides used in DNA testing. Thus, the fidelity of scrapie genetic testing is enhanced by knowing the position and frequency of PRNP polymorphisms in targeted flocks.

Results: An adaptive DNA sequencing strategy was developed to determine the 771 bp PRNP coding sequence for any sheep and thereby produce a consensus sequence for targeted flocks. The strategy initially accounted for 43 known polymorphisms and facilitates the detection of unknown polymorphisms through an overlapping amplicon design. The strategy was applied to 953 sheep DNAs from multiple breeds in U.S. populations. The samples included two sets of reference sheep: one set for standardizing PRNP genetic testing and another set for discovering polymorphisms, estimating allele frequencies, and determining haplotype phase. DNA sequencing revealed 16 previously unreported polymorphisms, including a L237P variant on the F141 haplotype. Two mass spectrometry multiplex assays were developed to score five codons of interest in U.S. sheep: 112, 136, 141, 154, and 171. Reference tissues, DNA, trace files, and genotypes from this project are publicly available for use without restriction.

Conclusion: Identifying ovine PRNP polymorphisms in targeted flocks is critical for designing efficient scrapie genetic testing systems. Together with reference DNA panels, this information facilitates training, certification, and development of new tests and knowledge that may expedite the eradication of sheep scrapie.

Figure 1

Physical maps of the ovine PRNP coding sequence, polymorphisms, and assay elements. Panel A features include: thick shaded arrow, coding sequence; black arrow, 3' untranslated region of exon 3; hatched arrows, ovine repetitive elements; white numbered vertical rectangles, octapeptide repeats; vertical lines, positions of SNPs; green single headed arrows, PCR amplification and/or sequencing primers (GenBank AY326330). SNP position numbers are distance to the first base of the PRNP start codon. Letters below SNPs are IUB ambiguity codes (R = a/g, Y = c/t, M = a/c, K = g/t, S = c/g, W = a/t, B = c/g/t, H = a/c/t, D = a/g/t) [56]. Red numbers and letters indicate sites affected by nonsynonymous substitutions at codons 112, 136, 154, 171, and 237. PRNP octapeptide repeats at positions 160 to 285 have either five or six repeats (5rep or 6rep). The asterisks denote SNPs not previously reported. MAF histograms correspond to genotypes from approximately 950 sheep available at http://cgemm.louisville.edu/USDA/index.html. Panel B: Map of ovine PRNP and regions targeted for PCR-amplification. PCR amplicons are depicted as open rectangles. The numbers by green arrows are USMARC primers (see Additional File 2). Panel C: Map of 314 bp PCR-amplified fragment for MALDI-TOF MS testing and expanded histogram of MAF. Features include: large bent blue arrows, PCR-amplification primers with mass tags added; horizontal blue arrows, hME extension primers for MALDI-TOF MS testing.

Figure 2

USMARC Sheep Diversity Family Panel version 2.45. The panel is composed of 96 unrelated sires, 96 ewes, and 192 twin offspring.

Figure 3

Comparison of sequence variants in the PrP GPI-SP region. Precursor PrP structural features include: an N-terminus signal peptide (N-SP), a five octapeptide repeat region (5-OR), a hydrophobic region (H), a disulfide bridge (S-S), N-linked glycosylation sites (dots), and a GPI signal peptide (GPI-SP). The residue numbers above the consensus sequence are those for ovine PrP. The peptide cleavage and GPI attachment site is indicated by omega-site zero (ω₀). After synthesis and translocation to the endoplasmic reticulum, a GPI moiety is typically attached to the ω₀ site of wild-type precursor PrP by a transamidation reaction and the last 23 residues are cleaved. The residues associated with familial CJD are shown in bold (M232R [34-37], M232T [33], P238S [38]). For comparison, nonsynonymous substitutions encoded by ovine PRNP are also shown in bold (L237P, this work; P241S [21,57]).

Figure 4

Mass spectrograms of ovine PRNP codons at positions 112, 136, 141, 154, and 171. A single PCR reaction was used to amplify a 336 bp genomic DNA region and the product split for use in two subsequent multiplex hME reactions. Spectral peaks represent singly-charged ions whose mass-to-charge ratio (m/z) was compared with calibrants for mass determination. Spectra feature labels: s and a, sense and antisense analytes produced from respective hME extension primers; p, unincorporated extension primer; ~, peak height clipped to conserve space. Two artifact peaks are produced as a consequence of multiplex design considerations. The first is a g nucleotide "pausing peak" in the codon 141 antisense assay (5530 Da, feature label "1"). The second artifact peak (feature label "2") is a g nucleotide misincorporation/insertion followed by a ddT termination in the codon 141 sense assay, i.e. 5'-[primer]-CGddT-3' (4866 Da). The correct termination product is 5'-[primer]-CddT-3' (4537 Da). This artifact peak at 4866 Da appears sporadically and independent of sample type or quality. Panels A and B: mass spectrograms illustrating the A136V and Q171R heterozygote. Panels C and D: mass spectrograms illustrating the R154H and Q171H heterozygote. Panels E and F: mass spectrograms illustrating the L141F heterozygote. Panels G and H: mass spectrograms illustrating the M112T heterozygote. Panels I and J: mass spectrograms illustrating the A136T and H171K heterozygote. The T136 was a synthetic allele that was added to the primer extension reaction cocktail to reference animal 200665213 (homozygous for A136).