HIGH- RESOLUTION MELTING (HRM) CURVE ANALYSIS: NEW APPROACH USED TO DETECT BLAD AND DUMPS IN URUGUAYAN HOLSTEIN BREED

María Teresa Teresa Federici,Rody Artigas,Fernando Dutra,Carolina Briano,Andrea Branda Sica,Silvia Llambí

doi:10.5380/avs.v23i4.62578

Abstract

The widespread use of artificial insemination has allowed the expansion of genetic progress. However, it also brought consequences such as the expansion of lethal hereditary diseases and the increase in inbreeding. The object of this study was to establish a fast and sensitive molecular assay to detect bovine leukocyte adhesion deficiency (BLAD) and deficiency of uridine monophosphate synthase (DUMPS) carriers in Uruguayan Holstein cattle by means of high resolution melting (HRM) curve analysis. By testing previously confirmed carrier and non-carrier animals, we set up a rapid, simple, and inexpensive diagnostic test using PCR followed by HRM curve analysis. The PCR-HRM genotyping method was effective for the discrimination of BLAD and DUMPS homozygous genotypes, and the BLAD heterozygous genotype. We conclude that the PCR-HRM assay is a robust, reliable, and economical tool for the detection of these mutations in the Holstein breed, which may be implemented in genetic selection programs.

Highlights

The widespread use of artificial insemination has allowed the expansion of genetic progress
Bovine leukocyte adhesion deficiency (BLAD) (OMIA 000595-9913) is a lethal autosomal recessive disease that affects the Holstein cattle caused by a single point mutation (A/G) at nucleotide 383 of CD18 gene of the chromosome 1 (Angerholm et al, 2007, OMIA 0005959913); this results in a single amino acid change at aminoacid 128 in the B subunit (CD18) of the (β) integrin (Radi et al, 2001) that leads to a greatly reduced expression of heterodimeric (β2) integrin adhesion molecules on leukocytes
The deficiency of uridine monophosphate synthase (DUMPS) (OMIA 0005959913) is another recessive genetic disorder of Holstein cattle characterized by decreased blood activity of the uridine monophosphate synthase (UMPS) enzyme, which leads to embryonic death during the first two months of gestation, more services per calving, and longer than normal calving intervals are required

Summary

Introduction

The widespread use of artificial insemination has allowed the expansion of genetic progress. Bovine leukocyte adhesion deficiency (BLAD) (OMIA 000595-9913) is a lethal autosomal recessive disease that affects the Holstein cattle caused by a single point mutation (A/G) at nucleotide 383 of CD18 gene of the chromosome 1 (Angerholm et al, 2007, OMIA 0005959913); this results in a single amino acid change at aminoacid 128 in the B subunit (CD18) of the (β) integrin (Radi et al, 2001) that leads to a greatly reduced expression of heterodimeric (β2) integrin adhesion molecules on leukocytes This mutation, that causes inadequate mucosal immunity and death before one year of age due to severe and recurrent mucosal infections, has been detected in many countries such as Germany, Denmark, Netherlands, Austria, United Kingdom, Japan, Switzerland, France, Taiwan, Poland, Brazil, Korea, Iran, Turkey, India (Adamov et al, 2014), and Uruguay (Kelly et al, 2010, Branda Sica et al., 2016, Branda Sica et al, 2018). No carrier animals were found in Poland, Iran, India, and Turkey (Kumar et al, 2010); the mutant allele was detected in other studies carried out in the USA, Europe, Argentine and Taiwan (Kumar et al, 2010, Patel et al, 2006)

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Results