CAPRINE BETA-MANNOSIDASE - SEQUENCING AND CHARACTERIZATION OF THE CDNA AND IDENTIFICATION OF THE MOLECULAR DEFECT OF CAPRINE BETA-MANNOSIDOSIS

The complete sequence of the caprine beta-mannosidase cDNA coding regi on has been determined, and a mutation that is associated with caprine beta-mannosidosis has been identified. Reverse transcriptase-polymera se chain reactions were performed using primers based on bovine and, l ater, goat cDNA sequences to produce an overlapping series of amplicon s covering the entire coding region. The composite cDNA codes for an 8 79-amino-acid peptide that has four potential N-glycosylation sites. C omparison of the caprine and bovine cDNAs reveals that 96.3% of the nu cleotides and 95.2% of the deduced amino acids are identical. A single -base deletion at position 1398 of the coding sequence was identified in the cDNA isolated from a goat affected with beta-mannosidosis. This deletion results in a shift in the reading frame and a premature term ination of translation, yielding a deduced peptide of 481 amino acids. An assay, developed to determine the presence or absence of this muta tion, confirmed that animals affected with beta-mannosidosis were homo zygous for the mutation and that obligate carriers in a caprine beta-m annosidosis colony were heterozygous. This assay accurately distinguis hed between mutation carrier and noncarrier goats and was used for pre natal diagnosis using DNA collected from fetal fluids. The assay also confirmed chimerism in a goat with an atypically mild beta-mannosidosi s phenotype. Thus, this application enables assessment of the efficacy of engraftment of hematopoietic stem cells after prenatal transfer fr om donor sources. (C) 1996 Academic Press, Inc.