Selection of human cells having two different types of mutations in individual cells (genetic/artificial mutants). Application to the diagnosis of the heterozygous state for a type of adenine phosphoribosyltransferase deficiency.

We have previously reported the establishment and characterization of B cell lines from patients and family members with various types of adenine phosphoribosyltransferase (APRT) deficiencies. These cell lines contain, at the APRT locus, three different alleles (APRT*1, APRT*Q0, and APRT*J) that are clearly distinguishable from each other. From five genetically heterozygous cell lines with two different genotypes (APRT*1/APRTQ0 and APRT*1/APRT*J), we have selected 48 clones resistant to 2,6-diaminopurine. Resistance to this adenine analogue is a characteristic of cells having defects in both of the APRT alleles in individual cells. The mutant clones from a cell line from a complete-type heterozygote had APRT activities close to zero (mean = 0.04 nmol/min per milligram protein) in the cell extracts, while 15 clones from four cell lines from the four Japanese-type heterozygotes had significant enzyme activities (mean = 3.88 nmol/min per milligram protein). Kinetic studies on two of the mutants from two Japanese-type heterozygous cell lines have shown that affinity to substrate 5-phosphoribosyl-1-pyrophosphate was reduced, indicating that APRT in those clones reflected the characteristics of the Japanese-type enzyme. The data presented here indicate that clones we obtained are genetic/artificial mutants, each having a genetic mutation in a single allele (APRT*J or APRT*Q0) and an artificially produced mutation in the other previously functional allele (APRT*1). The present procedure provided the only diagnostic method for Japanese-type APRT heterozygotes (APRT*1/APRT*J).