Polymorphisms in genes CTSB , CTSD , CAPN 2 , KLK 1 and TGFB 1 not associated with susceptibility to atypical or classical ovine scrapie

In the present study polymorphisms in the genes cathepsin B (CTSB), cathepsin D (CTSD), calpain, large polypeptide L2 (CAPN2), kallikrein 1 (KLK1) and transforming growth factor β1 (TGFB1) were investigated for association with scrapie susceptibility in sheep. Therefore single nucleotide polymorphisms in the respective genes were identified and examined for a potential impact on the gene function with different computer programs. Samples of 72 atypical and 104 classical scrapie cases as well as of 443 clinically healthy flock mates were genotyped by PCR-based screening methods. Neither allele frequencies nor genotype frequencies showed significant differences between scrapie positive sheep and control animals in any of the investigated genes.


Introduction
It has been indicated by several studies on quantitative trait loci (QTLs) in mouse and sheep that scrapie susceptibility is influenced by further gene loci besides the well known effects of prion protein (PrP) gene (PRNP) variants (LLOYD et al. 2001, MORENO et al. 2008, MORENO et al. 2003, STEPHENSON et al. 2000).This is interesting, especially in the case of atypical scrapie, where association between PrP haplotypes and susceptibility to the disease is not as stringent as in classical scrapie (LÜHKEN et al. 2007, MADEC et al. 2004, MOUM et al. 2005, ORGE et al. 2004).
The results of ARNOLD et al. (1995) and SUPATTAPONE et al. (1999SUPATTAPONE et al. ( , 2001) ) give evidence for a potential involvement of the endosomal cysteine protease cathepsin B (CTSB) in degradation of the abnormal prion protein isoform (PrP Sc ), which is underlined by the investigations of LUHR et al. (2004aLUHR et al. ( , 2004b) ) who found out that degradation of PrP Sc in scrapie infected neurons is carried out by cysteine proteases at a sour pH and that addition of a selective CTSB inhibitor leads to a significant increase of the PrP Sc content.
Concerning the lysosomal aspartate protease cathepsin D (CTSD), several expression studies reported an elevated concentration of the respective enzyme within the course of scrapie infection in mice (BROWN et al. 2004, RIEMER et al. 2004, XIANG et al. 2004).Furthermore, in brains of sporadic Creutzfeldt-Jakob disease patients a co-localisation of CTSD with intra-and perineural PrP Sc deposits was observed (KOVACS et al. 2007).
The ubiquitous cysteinprotease calpain 2 (CAPN2) as well might play a role for scrapie susceptibility, as the inhibition of calpain prevented the accumulation of PrP Sc in scrapie infected mouse brain cells and resulted in reduced prion titers (YADAVALLI et al. 2004).Here the elevation of C2, a cleavage product of PrP Sc , was most efficiently suppressed by calpain inhibitor IV, an inhibitor of calpain 2 (ANGLIKER et al. 1992, YADAVALLI et al. 2004).In this context the content of C1, a fragment of PrP C which is present in the healthy brain, recovered to a normal level (YADAVALLI et al. 2004).MORENO et al. (2003) found a QTL for scrapie resistance on mouse chromosome 7, in which murine paralogues of the kallikrein 1 gene (KLK1) as well as the transforming growth factor beta gene (TGFB1) are located.Results of HOU et al. (2005) andIWADATE et al. (2003) indicated that the serine protease KLK1 stimulates the release of insulin like growth factor 1 (IGF1), which plays a neuroprotective role in neurodegenerative disorders like Alzheimer disease (DORÉ et al. 1997).This is of interest as in the course of a scrapie infection the binding of IGF1 to its receptor is decreased (öSTLUND et al. 2001).
For TGFB1 BOCHE et al. (2006) claim a protective role with regard to transmissible spongiform encephalopathies.Furthermore in several models for experimental prion disorders elevated levels of TGFB1 have been detected (BAKER et al. 1999, WALSH et al. 2001).

Materials and methods
Samples from 72 atypical and 104 classical scrapie positive sheep as well as 443 clinical healthy flock mates who served as control group were examined.Initially scrapie cases were identified by Bio-Rad Platelia rapid test, Prionics Check Western Blot test, or by Enfer TSE test.Scrapie diagnosis was confirmed and completed with the scrapie type by either SAFimmunoblotting, IHC or both methods (BUSCHMANN et al. 2004, GRETZSCHEL et al. 2005).
DNA extraction was performed as described earlier (LÜHKEN et al. 2004).For sequence analysis primers were chosen from GenBank sequences of cattle and sheep and PCR was performed (Table 1).Sequence analysis was outcared at the ABI PRISM 377 sequencer (Applied Biosystems, Darmstadt, Germany) according to manufacturer's instructions.and 10 % (v/v) DMSO CAPN2 AGT GGG AAA CCG GCT AAA GT (f) 3  Standard PCR conditions 6 57 / 35 / 40 TGC TGG AGT AAG GTC CCA AC (r) 3  and 10 % (v/v) DMSO KLK1 CCT GTT TGA GGA CGA AGA CAC (f) 4  Standard PCR conditions 6 , 65 / 80 / 40 GGT GTA GAC GGA GGG CTT ATT (r) 4  25 nmol MgO(Ac)2 added TGFB1 AGA ACT GCT GTG TTC GTC AGC TC (f) 5  Standard PCR conditions 6 64 / 60 / 40 GTT GGA CAA CTG CTC CAC CTT G (r) 5  and 5 % (v/v) DMSO 1 NM_174 031 (Bta), 2 AF164 143 (Oar), 3 XM_864 105 (Bta), 4 AY290 705 (Bta), 5 NM_001009400 (Oar), Bta=Bos taurus, Oar=Ovis aries, 6 40-50 pmol of each primer, 30-90 ng template DNA, 1-1,75 U Taq DNA polymerase, 640-800 µM dNTPs in 1 fold reaction buffer supplied by the manufacturer in a total volume of 50 µl Identified sequence variants in genes CTSB, CTSD and KLK1 were genotyped in scapie positive and control animals by PCR-restriction fragment length polymorphism (RFLP) analysis with restriction enzymes LweI, BseYI and BsmFI respectively (Table 2).The single nucleotide polymorphism (SNP) in TGFB1 was typed by PCR-RFLP after generation of an amplification created restriction site (ACRS) (HALIASSOS et al. 1989) with enzyme BseGI (Table 2).For typing of the SNP in the CAPN2 gene two allele specific PCRs were carried out, both containing the respective specific primer and the corresponding reverse primer each and additionally an extra forward primer to create a smaller control fragment (Table 2).As in some cases the allele specific PCR failed to detect the wildtype allele (G) in heterozygous animals, all probes assumed to be homozygous for TT were double checked by nested-PCR-RFLP with the enzyme MvaI (Table 2).Allele and genotype frequencies were calculated for the scrapie positive sheep (atypical and classical) and their healthy controls (from flocks affected by atypical or classical scrapie respectively) separately and compared using the chi square test and Fisher´s exact test (SAS v. 8  Ann.temp.=Annealingtemperature, Elong.t.=Elongation time, No. cycles=Number of cycles, 1 specific forward primer (f) used for amplification of the allele T and corresponding reverse primer (r), 2 specific forward primer (f) used for amplification of the allele G and corresponding reverse primer (r), 3 forward primer (f) set in both reactions to create a control fragment with the respective reverse primer, 4 outer primer, 5 inner primer, 6 base pair mismatches are indicated by lower case letters, 7 15 pmol of each primer, 7 -10 ng template DNA, 0,3-0,6 U Taq DNA polymerase, 640-800 µM dNTPs in a 1 fold reaction buffer supplied by the manufacturer in a total volume of 15 µl, 8 two independent reactions for amplification of the T allele, G allele respectively, 9 24 pmol of respective specific forward and corresponding reverse primer each, 12 pmol of extra forward primer to create control fragment, 8 ng template DNA, 0,5 U Taq DNA polymerase, 800 µM dNTPs, 10 % (v/v) DMSO in a 1 fold reaction buffer supplied by the manufacturer in a total volume of 12 µl, 10 10 pmol of each outer primer, 0,35 U Taq DNA polymerase, 800 µM dNTPs, 10 % (v/v) DMSO in a 1 fold reaction buffer in a total volume of 10 µl, 11 16 pmol of each inner primer, 0,5 U Taq DNA polymerase, 800 µM dNTPs, 10 % (v/v) DMSO in a 1 fold reaction buffer supplied by the manufacturer in a total volume of 16 µl

Results and discussion
Sequence analyses led to the identification of several SNPs in the investigated genes, of which one per gene was used for the association analysis (Table 3).At position 12 of CTSD exon IX a silent mutation (C>T) was detected.In case of the C allele we detected a potential exon splicing enhancer (ESE) (GGC CGG G) at this location.The location near the exon-intron boundary is typical for exonic splice control elements (MAJEWSKI and OTT 2002).In case of the T allele the score for this ESE drops below the threshold.As ESEs are of great importance for correct splicing, it is possible that the destruction of the respective ESE causes an exon skipping of exon IX as it has been described for a number of silent mutations in other genes before (CARTEGNI et al. 2002).
In ovine CAPN2 the promoter and part of the first exon were investigated.Computer analysis revealed that the whole region spanned a CpG island including two G/C boxes.In the promoter region at position 191 in front of exon I a single nucleotide polymorphism (G>T) was detected.In case of the G allele a potential binding site (TGC CCG GGG TGC T) for a transcription repressor was identified, which could not be detected for the mutated T allele as the conserved »GGGG« core is essential for it's binding activity (BRESLIN et al. 2002).At position 391 of CTSB intron 9, position 392 of KLK1 intron 4 and position 206 in front of exon VII in intron 6, respectively, a single nucleotide polymorphism (C>T), (C>T), (G>T), respectively, was detected.No evidence could be found for any of these mutations to have a potential impact on the gene function.Significant differences between scrapie positive sheep and control animals were identified neither in allele nor in genotype frequencies for any of the investigated genes.
In contrast to the genes analyzed above, significant differences had been observed between PRNP genotype frequencies of the classical and of the atypical scrapie positive sheep and their healthy flock mates in a previous study (LÜHKEN et al. 2007), which included the samples of the present study.German sheep breeds differ in their PRNP genotype frequencies (DRöGEMÜLLER et al. 2001); thus the breed was considered when samples from healthy flock mates were selected in order to minimize stratification effects.However, any of the five analyzed genes is located on chromosome 13, where PRNP has been mapped (LÜHKEN et al. 2006).Therefore no genetic linkage between PRNP and the analyzed genes could have affected the results of the association analysis.In fact, the population was found to be completely in Hardy Weinberg equilibrium for all analyzed SNPs.
In conclusion, no evidence for an association of SNPs in the genes CTSB, CTSD, CAPN2, KLK1 and TGFB1 with scrapie susceptibility could be detected.Further studies may show if other sequence variants in these candidate genes have an impact on the trait under study.

Table 1
Primers and PCR conditions used for sequence analysis of CTSB, CTSD, CAPN2, KLK1 and TGFB1 Für die Sequenzanalyse von CTSB, CTSD, CAPN2, KLK1 und TGFB1 verwendete Primer und PCR-Bedingungen (CARTHARIUS et al. 2005orphisms in exons or promoter regions of the respective genes were examined for a potential impact on the gene function by the use of computer programs MatInspector(CARTHARIUS et al. 2005), ESEfinder 3.0 (SMITH et al. 2006), and  Sequence Manipulation Suite: CpG Islands (STOTHARD 2000), respectively.
(HARDY 1908)te Inc., Cary, NC, USA).Furthermore in respect to every SNP the Hardy-Weinberg equilibrium in the test population was estimated(HARDY 1908).