Diagnosis of heterozygous states for adenine phosphoribosyltransferase deficiency based on detection of in vivo somatic mutants in blood T cells: application to screening of heterozygotes.

An accurate diagnosis of heterozygotes for autosomal recessive disorders with unknown mutations can be difficult. Using a unique phenomenon occurring in vivo, we designed a method for the diagnosis of heterozygotes for adenine phosphoribosyltransferase (APRT) deficiency which makes way for a qualitative distinction between normal and heterozygous subjects. We cultured peripheral blood mononuclear cells with 2,6-diaminopurine, an APRT-dependent cytotoxin, to search for in vivo mutational cells. Fifteen putative heterozygotes examined were found to possess such mutant cells at rather high frequencies; thus, a false negative diagnosis is unlikely. The analysis of genomic DNA in 82 resistant clones from two of the heterozygotes clarified that 64 (78%) had lost the germinally intact alleles. Thirteen members of APRT-deficient families were examined; eight proved to be heterozygotes. Among 425 individuals from two separate residential areas of Japan, two heterozygotes were found. The authenticity of the heterozygosity was validated by two separate methods for the two heterozygotes; hence, a false positive diagnosis can be ruled out. Our data showed a calculated heterozygote frequency of 0.47% (95% confidence limits; 0.05%-1.7%), a value compatible with that (1.2%) calculated from data concerning the incidence of 2,8-dihydroxyadenine urolithiasis. This novel genetic approach for identifying heterozygotes is now being tested to search for other enzyme deficiencies in humans.