Identification of a null allele in genetic tests for bovine leukocyte adhesion deficiency in Pakistani Bos indicus × Bos taurus cattle

Two clinically healthy mature Pakistani Bos indicus × Bos taurus cattle were genotyped as homozygous affected for the lethal immunodeficiency disorder bovine leukocyte adhesion deficiency (BLAD) using previously described PCR-RFLP based DNA tests which was confirmed by sequencing. Sequencing of Bos taurus and B. indicus × B. taurus genomic DNA surrounding the disease causing mutation (c.383A > G) in the ITGB2 gene identified numerous variations in exonic and intronic regions within and between species, including substantial variation in primer annealing sites for three PCR-RFLP tests for one of the B. indicus allelic variants. These variations in the primer annealing sites resulted in a null allele in the DNA tests causing the misdiagnosis of some heterozygous B. taurus × B. indicus cattle to be classified as homozygous affected. New primers were designed and a modified test was developed which simultaneously identified the disease mutation and the Pakistani B. indicus allelic variant associated with the null allele in the previous test.