The mutation sites were directly examined by sequencing of crude PCR-amplified segments of genome. As shown in Figure 2A, Q3SA exhibits the mutations identified in the diagnostic genotyping originally provided for subject JHU_Q3 (Figure 1A). Each mutated location was clearly heterozygous in Q3SA, as evidenced by the mixed trace from the unpurified PCR product (highlighted in yellow). CAR3 exhibited the c.217_218 delGA mutation, which is consistent with a parental relationship to JHU_Q3. Q3SC, on the other hand, is missing the c.217_218 delGA mutation, because only wild-type sequence is visible in the PCR products. The c.7792 C > T mutation is present in both Q3SA and Q3SC. The same pattern of mutations appears in sequenced, PCR-amplified segments of cDNA (not shown), suggesting that both alleles are transcribed. These results predict that Q3SC carries one allele capable of encoding a full-length ATM protein, while Q3SA does not.
