GATA1 mutations can be clinically silent at birth. Analysis of GATA1 mutations in sequential genomic DNA samples in AMKL patients and healthy DS neonates. (A-B) Sequential genomic DNA samples from patients L12 and L13 (A), and L14 (B) were used as a template to amplify radiolabeled GATA1 exon 2. PCR products were then separated on a polyacrylamide gel. Wt indicates the position of the wild-type GATA1 exon 2 PCR product. DNA samples used were neonatal blood spot DNA (BG); diagnostic AMKL DNA (Diag); and remission DNA sample. Patient L14 presented with TMD at birth, and this sample was used in the analysis. Positions of DNA size markers (in base pairs) are indicated on the left. (C) Genomic DNA from 21 unselected healthy DS neonates and one non-DS control (N) was used as template to amplify radiolabeled GATA1 exon 2. PCR products were then separated on polyacrylamide gel; 2 of 21 (N5 and N6) samples tested had detectable mutations (D-E). (D) Neonate N5 has a mutation in GATA1 exon 2, 218 ins 1, detected by direct sequencing of GATA1 exon 2 PCR product amplified from genomic DNA. Note that there are 2 sequences in the chromatogram indicative of 2 GATA1 PCR products, wild type and mutant. The minor trace sequence corresponds to the mutant product, reflecting its lower abundance relative to wild type. (E) Neonate N6 has a mutation in GATA1 exon 2, 302 ins 11 (dup291-301). The mutant-sized GATA1 exon 2 product (C) was excised from the gel and cloned, and sequence of the cloned product is shown. Wild-type GATA1 exon 2 sequence is illustrated for comparison. The predicted consequences of mutations in both N5 and N6 are shown. In N5, mutation leads to a stop codon before codon 84. In N6, mutation is predicted to terminate at codon 140.