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Genotype of PRNP: Buffaloes

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INVESTIGATION OF PRNP PROMOTER AND INTRON 1INDEL POLYMORPHIMS OF ANATOLIAN, MURRAH AND CROSSBRED BUFFALOES

Yalçın YAMAN1*, Cemal ÜN2, Orhan KARADAÄž1

ABSTRACT

As a direct public health threatening disease, Bovine Spongiform Encephalopathy (BSE) of the cattleprobablymost important disease among other Transmissible Spongiform Encephalopathies (TSE). It can be transmitted to human and cause to a new variant of the Creutzfeldt–Jakob disease (CJD). Unlike CJD, variant CJD (vCJD) has unusually affected younger people. It is known that prion protein coding gene (PRNP) plays a major role in the TSE susceptibility or resistance. Recent researches demonstrated that insertion/ deletion (indel) polymorphisms within promoter and intron 1 region of thePRNPrelated to BSE susceptibility in cattle. In contrast to cattle, BSE never been reported in water buffalo, hence, PRNP polymorphisms may be an explanation for buffalo resistance to BSE. The aim of this study was to evaluate allele, genotype and haplotype frequencies of the PRNP promoter and intron 1 indel polymorphism in Anatolian, Murrah and Anatolian X Murrah crossbred buffaloes. According to the our findings,there were no deletion alleles at two mentioned loci. All studied animals were monomorphic and have carried ins/ins haplotypes which are considered as the most resistant genotype to BSE.

Key words: Buffalo, BSE, PRNP, Promoter, Intron 1, Indel

  1. Introduction:

Normal cellular prion proteins (PrPC) whichare coded by PRNP gene (McKintosh 2003) have some known functions in the organism such as “cellular trafficking, metabolism of the copper uptake, protection against oxidative stress, cell adhesion, differentiation, signaling and cell survival”(reviewed by Martins et al. 2002). After a posttranslational process PrPC converted into abnormal isoform called “prion”or PrPSC during which it acquires high beta sheet content and gain partly resistance to protease. Prions are defined as transmissible infectious particles that are lack of nucleic acid and composed ofmisfolded cellular prion protein. Prions cause incurable neurodegenerative disorders, also known as Transmissible Spongiform Encephalopathy (TSE) include Creutzfeldt–Jakob disease (CJD), Gerstmann– Straussler–Scheinker (GSS) disease, kuru and fatal familial insomnia in humans, Bovine Spongiform Encephalopathy (BSE) in cattle and scrapie insheep(Prusiner 1991, Prusiner 1997, Weissmann et al. 2002). Like other TSEs, BSE cause to fatal, irreversible neurodegeneration in the Central Nervous System (CNS) and the disease manifested by dementia and/or ataxia in cattle (Sander et all. 2004). BSE can be transmitted to humans and causes a new variant of CJD (vCJD) which have affected to unusually youngerpeople. Furthermore, it is shown that BSE also transmitted to sheep by whole blood transfusion. Similarity ofsheep-BSE experimental model and human vCJD pathogenesisget raised the concerns about thatBSE canspread between species(Hill et al. 1997, Bruce et al. 1997, Houston et al. 2000).

PRNPgene is of a key role in the pathogenesis of prion diseases, for example PRNP polymorphism at codon 129 for familial CJD and Gerstmann-Sträussler syndrome (GSS) in humans (Collinge et al. 1991, Palmers et al. 1991) also polymorphisms at codons 136, 154 and 171 for scrapie in sheep are related to disease susceptibility(Goldmann et al. 1990, Hunter et al. 1994, Clouscard et al. 1995, Hunter et al. 1997). More recently, significant relation with PRNP polymorphisms and BSEsusceptibility was defined in cattle. It is reported 23 bp indel within the promoter and 12bp indel within the intron1 of the PRNP gene associated with varying susceptibility to BSE (Sander et al. 2004, Sander et al. 2005, Juling et al. 2006).

To morphological and geographical aspect Anatolian water buffalo considered belonging to river type and Mediterranean breed as all European buffaloes included same type and breed (Soysal et al. 2005, Borghese and Mazzi 2005). Recently a karyotyping analysis study revealed that having25 pairs ofchromosome number (2n=50), Anatolian water buffalo is the same type with the river buffalo as genetically too (Ün et al. 2014). Since 1999 a improving project has been conducted by crossbreeding with Murrah buffaloes imported from Bulgaria and currently in progress at Sheep Research Station of Bandirma district in the Turkish province of Balikesir.

Up to now noTSE was identified in Buffaloes(Di Guardo 2014). It is not clear if the buffaloes are genetically resistant to the TSEsor not. Although numerous studies have been conducted in order to determine promoter and intron 1 polymorphism of cattle PRNP(Haase et al. 2007, Kerber et al. 2008, Muramatsu et al. 2008, Ün et al. 2008) there are fewreports regarding water buffalo PRNP Indel polymorphism. There is one other research conducted on Anatolian buffalo PRNP indel polymorphism, but as we know no study on Murrah buffalo.

The aim of this study was to genotype of PRNP according to 23bp and 12 bp indel polymorphism of Anatolian, Murrah and crossbred buffaloes, expand the data for the PRNP genotype of the Anatolian water buffalo and compare allele/genotype frequencies with those in other buffalo breeds.

  1. Materials and Methods:

2. 1. Animals

Total 195water buffalo consisted 89 Anatolian, 20 Murrah and 86 Murrah X Anatolian crossbreds were studied. Because of the number of the purebred Murrah buffaloes were not sufficientrelatively, the F1, G1 and G2 level Murrah x Anatolian crossbred which some of their ancestors culled before the sample collection were included the study due to the detection of all possible in/del polymorphisms. These animals have been raised in the Sheep Research Station of General Directorate of Agricultural Research and Policies (TAGEM) of Republic of Turkey Ministry of Food, Agriculture and Livestock.

2. 2. DNA isolation and PCR

10 ml sterilized tubes with EDTA were used for whole blood collection. Blood samples were collected under aseptic conditions from V. jugulars and stored in -20 0C until laboratory working. The genomic DNA was extracted from whole blood samples using commercial kits. DNA quantification was made using Qubit device. To amplify promoter and intron regions of the PRNP gene two separate PCR reaction was conducted using the primers designed for cattle (Juling et al. 2006).

The PCR mixture was consisted 1 U Taq DNA polymerase (Fermantas Life Sciences, Burlington, Canada), 2. 5 mÓŽ10X PCR buffer [750 mÓŽ Tris-HCl (pH 8. 0), 200 mÓŽ (NH4)2SO4, 0. 1% Tween 20)], 1. 5 mÓŽ MgCl2, 50–100 ng genomic DNA, 100 µÓŽ dNTP (Fermantas Life Sciences) and 10 pmol of each primer and H2O to a final volume of the 25 µl. Reaction condition was as follows; 95 C for 5 min; 32 cycles of 94 0C for 45 s, 58 0C for 45 s, 72 0C for 45 s; and a final extension at 72 0C for 7 min. PCR products were separated on 3% agarose gel, stained with safe stain (NBS Biologicals, England)and visualized under UV light. Gel electrophoresisbanding patterns were expected to be either 191 (+) or 168 (-) for indel 23bp of promoter and either 215 (+) or 203bp (-) for 12bp indel of the intron 1 region.

2. 3. Statistical Analysis

Genotypic and allelic frequencies of the PRNP promoter and intron 1 variants were estimated via direct counting. In order to check whether the population in Hardy–Weinberg equilibrium or not the PopGene32 software (Yeh et al. 2000) was used.

  1. Results and discussion

In the study, all buffaloes were detected as monomorphic for both loci. There were no deletion allele and both of two loci were mixed as +/+ 23bp and +/+12bp for all buffaloes. Neither 23bp nor 12 indel allele frequencies were consistent Hardy-Weinberd equation (HWE, df=1, P< 0,001). From 1970 to 2008, there has been a dramatic decrease in the buffalo population of approximately 93 percent in Turkey (Sarıözkan 2011). The loss of genetic diversity and deviation from HWE might result from such a population bottleneck.

In another study carried out on Anatolian Water buffaloes PRNP indel allele frequencies were found to be %92 and %8 for 23 bpindel, %86 and %14 for 12bp indel respectively (Oztabak et al. 2009). Sample collection for that research was made from entirely unrelated buffaloes which reared in different provinces in Turkey. When comparing with those results obtained by Oztabak et. al, it can be considered that the inbreeding coefficient of our animals might have increased along the years. It may be another additional reason whydeletion alleles have disappeared.

It was demonstrated that nucleotide changes in non-coding regions of the PRNP gene have an influence on prion protein expression levels(Sander et al. 2005, Kashkevich et al. 2007, Msalya et al. 2011) and it has been hypothesized that PRNP promoter polymorphisms including intron 1 region, which is contributing to promoter activity might change PRNP expression level, thus, could modify the susceptibility and/or incubation timeof the BSE disease(Sander et al. 2005).

     

Promoter-23 bp

Ä°ntron 1-12 bp

 

Country

Breed

n

Ä°n (+)

Del (-)

Ä°n (+)

Ä°n/Del

References

Indonesia

River Buffalo

14

100

0

100

0

Uchida et al. (2014)

Thai

River Buffalo

45

53

47

84

16

Pakistan

Nili Buffalo

66

94

6

86

14

Imrana et al. (2012)

Ravi Buffalo

39

97

3

83

17

Azikheli Buffalo

20

100

0

95

5

Kundhi Buffalo

34

97

3

88

12

Nili Ravi Buffalo

122

94

6

87

13

Germany

Nehir Buffalo

11

100

0

100

0

Kobak et al. (2014)

Poland

Nehir Buffalo

29

100

0

100

0

Turkey

Anatolian Buffalo

106

92

8

86

14

Oztabak et al. (2009)

Turkey

Anatolian Buffalo

89

100

0

100

0

This study

Murrah Buffalo

20

100

0

100

0

Anatolian X Murrah

86

100

0

100

0

It is reported that the cattle which are carrying 23bp ins / 12 bp ins haplotype are clearly the most resistant individuals (Sander et al. 2004, Sander et al. 2005, Juling et al. 2006). While some researchers have found the stronger effect at 23 bp promoter polymorphism (Sander et al. 2004 and Haase et al. 2007), however, others have argued that 12bp intron 1 polymorphism has a major effect on BSE susceptibility(Juling et al. 2006, Kashkevich et al. 2007). As shown in the table 1, 23bp promoter deletion allele frequencies of other buffalo breeds found between 0 to 0,08 even 0,47 in Thai water buffaloes, likewise 12bp intron 1 deletion allele frequencies varying between 0 to 0. 17. In the animals included the our study there was no deletion allele at the both regions. Whether promoter or intron1 deletions have stronger association with BSE susceptibility, in both case Anatolian and Murrah water buffaloes seem to be more resistant to BSE comparing most of the other buffaloes breed

Water buffaloPRNPindel frequencies mostly different from cattle (Oztabak et al. 2009, Imrana et al. 2012, Uchida et al. 2014, Kobak et al. 2014). Since 1986 when the first recognition of BSE in UK, it is estimated that several million cattle exposed to infection (SMÄ°TH, P. G. and BRADLEY, R. 2003),beside this, buffaloes have likely consumed the prion contaminated feed. (Aidaros 2003). Taking into account that there is no report about buffalo TSE, these genetic differences may be the probable reason of the resistance of water buffaloes to BSE (Di Guardo 2014). On the other hand, aside from relation between PRNP indel polymorphism and BSE susceptibility in cattle it is reported that breed specific differences could be exist (Haase et al. 2007). It can be assumed that same differences could be exist between buffalo breeds too. This and other relevant studies(Oztabak et al. 2009, Imrana et al. 2012, Kobak et al. 2014,Uchida et al. 2014) indicate that PRNP promoter and intron 1 region in water buffaloes mostly consists of insertion alleles therefore further investigations on PRNP gene, including ORF and 3’ UTR regions will contribute to our understanding of genetic fundamentals of the Bovidae Prion diseases.


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