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MOLECULAR SCREENING OF FXI DEFICIENCY GENE FRAGMENT IN NILI-RAVI BUFFALO BULLS OF PUNJAB, PAKISTAN.

Byline: K. Zahra, M. Y. Zahoor, M. Imran, K. Ashraf, A. Nadeem, I. Rashid, Habib-ur-Rehman, K. Hassan, M. Younas, M. Akhtar and W. Shehzad

Keywords: recessive traits, FXI deficiency, Nili-Ravi buffalos.

INTRODUCTION

Livestock capacity building is imperative to meet the future demand of burgeoning populations in terms of food and livelihood in agriculture based countries. The input of livestock in food production delivers essential protein sources. The success of livestock farming is linked to increased production via improving animal health. With proper monitoring of disease dynamics in livestock, the losses of valuable breeding animals can be reduced. Among various health concerns in buffalos, genetic disorders are of apprehension as the diagnosis and treatment is difficult. Genetic disorders adversely affect wild and domesticated animal populations both qualitatively and quantitatively and they can turn out to be notable economic concern (Albarella et al., 2017). Rudimentary data is available on the congenital defects in Bubalus bubalis. With the focus on livestock genetics, existing information can be updated through targeted research projects on key species.

Congenital bleeding disorders had been identified in dairy cattle either as functional and structural defects in hemostatic pathways. Although, biochemical assays are present for many disorders but these can overlap positive and negative predictive values of carrier detection. FXI deficiency is one such disorder which has been extensively reported in cattle breeds worldwide. Factor XI is a plasma glycoprotein involved in the intrinsic pathway of hemostasis (Gentry and Black, 1980; Gentry, 1984). It generates additional thrombin by a positive feedback mechanism. First described in humans by Rosenthal (1953), the deficiency was also identified in other mammals. Human FXI gene is located on chromosome 4 which was first traced by Asakai et al. (1987) and murine FXI gene resides on chromosome number 8. In cattle, FXI gene is located on chromosome 27 (NCBI RefSeq: NC_007328.3).

The total size of the gene is 19150 bp in length (from nucleotide 17607721 to 17626871) and it comprises 15 exons and 14 introns (Kumar et al., 2011). FXI deficiency in Holstein cattle is concomitant with a 76 bp segment insertion [AT(A)28TAAAG(A)26GGAAATAATAATTCA] within exon 12. This insertion ends up into a stop codon that results in non-functional and inactive protein with a missing functional protease domain encoded by exons 13, 14 and 15. Structurally, FXI is a homodimer; each subunit contains four apple domains (which form a disk structure with binding sites for platelets, HMW kininogen, and factor IX) and a protease domain (Gailani and Smith, 2009). In Japanese black cattle, a mutation in exon 9 was also linked to the disease. Same was reported for Wagyu cattle (www.wagyuinternational.com). The animals heterozygous of the trait appear to be normal phenotypically, while homozygotes exhibit hemophilia-like disorder referred to as hemophilia C in humans.

The incidental bleeding manifestations can present during horning, elective surgery or chance injury. Occasionally, affected calves may continue to bleed from the umbilical cord, or during dehorning, injections and castration and bleeding may be fatal in some cases (Brush et al., 1987). Symptomatic cows may have hints of blood in colostrum and milk; which also became the basis of disease identification in United Kingdom. The affected animals can become anemic susceptible to pneumonia, mastitis and metritis. The heterozygotes have low calving and survival rates with high morbidity and mortality (Liptrap et al., 1987; Marron et al., 2004). In cattle, FXI deficiency was first reported in US Holsteins (Kociba et al., 1969). Since then, further studies have been reported from United Kingdom (Brush et al., 1975), Canada (Gentry and Ross, 1993), Japan (Kuneida et al., 2005) and many other countries of the world.

The accurate genetic analyses provide reliable measure of disease phenotype, even in the prospective progeny. The DNA-based detection tests make comprehension of phenotype-genotype correlates more convenient than the conventional methods and enhance the accuracy of carrier identification. To date, there were few reports available on bovine inherited disorders from Pakistan which targeted Bos taurus and Bubalus bubalis (Nasreen et al., 2009; Mehmood et al., 2011, Imran et al., 2012). No work has been done previously on FXI gene in Pakistan. In line with this, the current study was planned to identify the FXI deficient sires among Nili-Ravi buffalos of Pakistan through molecular based screening.

MATERIALS AND METHODS

A total of 152 healthy breeding buffalo bulls (Nili-Ravi breed) were randomly selected for this cross-sectional study. Blood samples (10 mL each) collected from the jugular vein were preserved in EDTA containing vacutainers to avoid blood clotting. Phlebotomy was carried out under standard veterinary regimes with prior permission from the ethical review committee for sampling. Whole genomic extraction was carried out by organic method (Sambrook and Russel, 2001) and DNA yield was quantified by 0.8% agarose gel and spectrophotometer (NanoDrop 2000C, Thermo Fisher Scientific, Massachusetts, USA). The genetic screening of FXI gene was done by amplifying the gene fragment containing an insertion mutation; the primers sequence was as reported by Marron et al. (2009). The PCR reaction was optimized using touchdown PCR strategy (-1 AdegC decrease for first 10 cycles), followed by gradient PCR. The optimized PCR conditions were followed for further FXI gene amplification.

The final reaction mixture contained 1x PCR buffer, 1.5 mM MgCl2, 400 uM of each dNTPs, 1 unit of Taq DNA polymerase (Thermo Fisher Scientific, Massachusetts, USA), 0.4 uM each of forward (5'-CCCACTGGCTAGGAATCGTT-3') and reverse (5'-CAAGGCAATGTCATATCCAC-3') primers and 50 ng/uL of genomic DNA. The final volume of reaction mixture was adjusted to 25uL. Temperature profile of the thermocycler (Bio-Rad, California, USA) was as follows: initial denaturation at 95 AdegC for 5 minutes, 35 cycles of 94 AdegC for 1.5 minutes, annealing of primers at 55 AdegC for 1 minute and extension at 72 AdegC for 2 minutes, followed by a final extension at 72 AdegC for 10 minutes. The PCR amplicons were analyzed on 2% agarose gel. The precipitated PCR products were subjected to sequencing PCR and resolved on Genetic Analyzer 3130 (Applied Biosystems, CA, USA).

The DNA sequence chromatogram data were analyzed by FinchTVA(r) and the pair-wise homology analysis was done by NCBI-BLAST (https://www.ncbi.nlm.nih.gov/BLAST/), while, multiple sequence alignment was carried out by Clustal Omega software (Sievers et al. 2011).

RESULTS AND DISCUSSION

A total of 152 Nili-Ravi buffalo bulls from Punjab, Pakistan were screened for FXI deficiency. EBV data and pedigrees of all the bulls were recorded at the start of study. Phenotypically, all the bulls were healthy and no bleeding episodes were recorded during blood collection. DNA PCR amplification generated a 244 bp sequence of the FXI gene (Exon 12) from Nili-Ravi buffalo bulls (Figure 1). The sequence of the amplicon was aligned using NCBI-BLAST (https://www.ncbi.nlm.nih.gov/BLAST/) which revealed 98% homology with the FXI gene of Bos taurus followed by Bubalus bubalis. The results of this study provided evidence that there were no heterozygous bulls among the studied lot. The sequence of final 244 bp Nili-Ravi FXI gene fragment was analyzed for single nucleotide polymorphisms (SNPs) compared with that of FXI sequences of Bos taurus and Bubalus bubalis (Pandharpuri buffalo breed; NCBI_003104605.1).

A transversion of adenine into thymine at position 1782 (c.1782A>T) resulted into a silent conversion of glutamine into Asparagine (Figure 3). FXI deficiency was initially studied by Kociba et al. (1969) in American cattle with complete absence of coagulation protein. Later on, Marron et al. (2004) investigated and confirmed that a 76 bp insertion mutation in the FXI gene had truncated some essential functional part of the protein in Holstein cattle leading to hereditary FXI deficiency. They investigated a total of 419 animals and detected five carriers with 1.2% frequency of mutant alleles. Since then, various studies on incidence of FXI deficiency in dairy cattle breeds such as Holstein, Friesian and their cross-breds have been reported from different countries.

Kociba et al. (1969), Robinson et al. (1997) and Marron et al. (2004) described this disorder in Holsteins of USA, Gentry et al. (1975) from that of Canada, Brush et al. (1975) in those of England with high incidence. Citek et al. (2008) studied FXI deficiency in Czech Republic but the incidence was low; a single carrier out of 309 animals was found. From Poland, Gurgul et al. (2009) reported the carriers of this disease in 3 out of 128 studied cows. They also reported that this disorder was associated with udder health in cattle. Studies from Turkey also reported the occurrence of thisdisorder. Meydan et al. (2009) found 4 carriers among 225 Holsteins in Turkey with 0.9% mutant allele frequency. In another report by Meydan et al. (2010), the mutant allele frequency was 0.006%, with 4 being carrier out of 350 cattle studied. Similarly, Oner et al. (2010) studied the incidence of FXI carriers in Turkey. Among 170 studied animals, 2 carriers were detected with 0.06% mutant allele frequency.

Karsli et al. (2011) studied 504 cattle and they detected 0.4% FXI carriers. Akyuz et al. (2012) estimated 0.85% carrier frequency in 161 Holstein bulls from Anatolia, Turkey. Agaoglu et al. (2015) reported 1.8% carrier frequency of Factor XI deficiency in Holsteins in Burdur, Turkey. Avanus and Altinel (2016) studied the FXI deficiency in Thrace region of Turkey. No carriers were found among 287 Holsteins studied by them. Studies from Asian countries also identified the occurrence of this hereditary disease in their livestock. Kuneida et al. (2005) studied the complete coding region of the FXI gene in Japanese black cattle. Ghanem et al. (2005) found 5 carriers out of 500 studied cows in Japan (0.5% mutant allele frequency).Watanabe et al. (2006) reported high incidence of FXI deficiency in stunted Japanese black cattle. The incidence of heterozygosity was up to 50% and that of homozygosity was 5.2%.

Ohba et al. (2008) surveyed the Japanese cattle and out of 123 animals (both dams and sires), 7 were detected as homozygous for the mutant allele. The frequency of mutants was higher up to 26.4%. In contrast, Eydivandi et al., (2011) investigated the FXI gene frequency in Iranian cattle and out of 330 animals none were found to be carriers. Also, Bagheri et al. (2012) reported no carriers in Khuzestan buffalo breed of Iran out of 300 studied buffalos. Similarly, in a study by Siswanti et al. (2014), the incidence of FXI deficiency was studied in Indonesian Bali cattle. Among 325 samples, no carriers were detected. India harbors large populations of Bos taurus and Bubalus bubalis. FXI deficiency was reported from India in Bos taurus but the incidence was low. Mukhopadhaya et al. (2006) studied 307 cows and 259 water buffaloes and found no carrier.

In another study, Patel et al. (2007) carried out a thorough investigation on 1001 Indian cows and buffaloes of various breeds; the FXI deficient cattle were detected with 0.6% mutant frequency. Patel and Patel (2014) reviewed that the FXI deficiency gene had been extensively studied in Indian Holstein cattle. In a recent study by Mondal et al. (2016), Indian Sahiwal bulls were tested for FXI deficiency. They screened a total of 120 bulls with 0.025% mutant allele frequency. The results of these studies from Asian countries were in accordance with the findings of current study, as no carriers were detected in native Pakistani buffalo breed. It is noteworthy that no carriers of FXI deficiency were detected in Bubalus bubalis to date. The results of our study are concordant with the findings of Patel et al. (2007). They reported no carriers from India in Bubalus bubalis (Surti, Jaffarabadi and Murrah buffalo breeds).

In the current study, a silent nucleotide change was observed in the FXI gene of Nili-Ravi buffalo. There was a transversion of adenine into thymine (c.1782A>T) which resulted into an amino acid change of glutamine into Asparagine (Figure 3). The Similarly, Azad et al. (2010) screened 200 crossbred Karan Fries in Haryana, India and found no carriers. However, a non-synonymous nucleotide change (GaA) was observed at position 105. Phenotypically, no bleeding episodes were reported in the study animals. Factor XI deficiency disorder is a rare hematological disorder inherited in an autosomal recessive manner. Such disorders may express phenotypically in homozygous form, thus, in most of the cases carrier animals are difficult to identify unless the disease appears itself in affected homozygous animals. As discussed by Ramesha et al. (2017), there was very low incidence of FXI deficiency in Poland, Turkey, Iran, and India (0.006%-0.6%) which is in agreement with our findings.

It is also recommended that the screening for carrier identification for the recessive disorders should be extensive with increased sample size. Congenital genetic disorders significantly limit the success of selective breeding programs as the phenotypic identification of carrier animals is not possible until appearance of affected animals among the subsequent populations (Al-Haggar, 2013). It becomes difficult to distinguish between the FXI deficient normal and carrier bulls phenotypically, but molecular screening is convenient as normal and carrier bulls can be identified by the position of amplicons on agarose gel; as the amplicon of normal animal is of 244 bp and that of carrier animal is 322 bp in size because of an insertion of 76 bp fragment in mutated animals. Thus, this test can be commercialized for the farmers at large.

In order to avoid the dissemination of deleterious alleles from prospective sires or dams, it becomes inevitable to reduce the disease burden by genetic screening of breeding animals.

Acknowledgements: The authors are grateful to the Punjab Agricultural Research Board (PARB project No. 492) and the Higher Education Commission (HEC) for their financial support of the study.

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