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 (APRT1, APRTQ0, and APRTJ) that are clearly distinguishable from each other. From five genetically heterozygous cell lines with two different genotypes (APRT1/APRTQ0 and APRT1/APRTJ), 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 (APRTJ or APRTQ0) and an artificially produced mutation in the other previously functional allele (APRT1). The present procedure provided the only diagnostic method for Japanese-type APRT heterozygotes (APRT1/APRT*J).