Genetic analysis of patients with leukocyte adhesion deficiency: Genomic sequencing reveals otherwise undetectable mutations

Objective. The aim of this study was to analyze mutations in DNA from patients with leukocyte adhesion deficiency (LAD), an immunodeficiency caused by absence of the β2 subunit (CD18) of the leukocyte integrins LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150,95 (CD11c/CD18), and CR4 (CD11d/CD18). Methods. We developed genomic DNA PCR sequencing to detect mutations not only in exons but also in introns. Results. Eight LAD patients were analyzed, of which five had homozygous mutations, i.e., a 0.8-kb deletion, a branchpoint mutation in intron 5 causing mRNA missplicing, a nonsense mutation, and two missense mutations. Four of these mutations are novel. We cotransfected the two mutant CD18 proteins with normal CD11a, b, or c in COS cells. This resulted in absence of all three β2 integrins on the surface of cells transfected with CD18252Arg. However, CD18593Cys supported some LFA-1 and p150,95 formation in COS cells. The other three patients were compound heterozygotes in which only one allele had previously been characterized, because the other alleles were undetectable at the cDNA level. We identified the unknown mutations as a novel two-nucleotide deletion, a nonsense mutation, and a single nucleotide deletion. Conclusion. Our method allows identification of mutations in CD18 from genomic DNA. This opens the possibility of early prenatal diagnosis of LAD and reliable carrier detection.