Comparative Analysis of Genomic DNA (gDNA) and Complementary DNA (cDNA) for the Identification of GATA1 Variants by Sanger Sequencing ^†

Introduction: Variants in GATA1 gene represent an early and critical event in Transient Abnormal Myelopoiesis (TAM) and in the progression to Myeloid Leukemia associated with Down syndrome (ML-DS). Most of these alterations are frameshift or nonsense variants within exon 2, directly affecting translation, altering hematopoietic cell proliferation and differentiation balance. Methodology: An analysis of 21 cases (approved by the Institutional Research Ethics Committee under protocol No. 7478993) with suspected TAM or ML-DS was based on samples processed at the Children’s Hospital of Brasília. For each case, both genomic DNA (gDNA) and complementary DNA (cDNA) were extracted from the same specimen and sequenced by Sanger methodology. Different primers for amplification of exon 2 of GATA1 in gDNA and cDNA were used. Results: Comparing gDNA and cDNA revealed that cDNA sequencing provided superior sensitivity in samples with <20% blasts (10/21 cases), where in 70% (7/10) of cases, gDNA failed to detect the mutation, which was identified only in cDNA. Among the 21 cDNA samples analyzed, 42.8% exhibited exclusively mutant transcripts, while the remaining demonstrated concomitant expression of wild-type and mutant transcripts. These findings highlight the capacity of cDNA analysis to reveal transcriptional predominance of mutant alleles and to detect variants not captured at the genomic sequencing. Conclusions: Correlating DNA and cDNA by Sanger sequencing proved to be a highly effective strategy for increasing sensitivity and for characterizing the impact of GATA1 variants. However, cDNA may have limitations in detecting variants in splice sites and regulatory regions. Transcript analysis not only improves diagnostic sensibility but also allows the identification of phenotypes with exclusive mutant expression.