Analysis of GATA1 Mutations in Down Syndrome Infants with Transient Abnormal Myelopoiesis and Clinical Impacts of GATA1 Mutation Types: A Report from the JPLSG TAM-10 Study

Introduction: Approximately 5-10% of neonates with Down syndrome (DS) develop transient abnormal myelopoiesis (TAM). Almost all patients with TAM have GATA1 mutations resulting in the exclusive expression of a truncated protein (GATA1s). Although TAM patients exhibit various hematological abnormalities including circulating blasts, leukocytosis and thrombocytopenia, these abnormalities have been also reported in DS neonates without TAM. Therefore, analysis of GATA1 mutations is very important in the diagnosis of TAM. However, standard procedures to detect GATA1 mutations have not been established.

Most of GATA1 mutations occur within the exon 2 and the surrounding sequences, but types of the mutations are varied, including insertions, deletions, duplications and point mutations. We previously reported that the expression levels of GATA1s were varied depending on types of mutations and might be associated with phenotypes of TAM including white blood cell (WBC) counts at diagnosis and a risk of progression to myeloid leukemia of DS (Kanezaki et al., Blood 2010). However, these findings have not been confirmed by other groups and effects of GATA1 mutation types on other clinical features of TAM have not been investigated.

Patients and Methods: One hundred sixty-seven patients were enrolled in TAM-10 study and blood samples were available in 166 patients. GATA1 mutations were analyzed by Sanger sequencing using genomic DNA and complementary DNA (cDNA) prepared from peripheral blood. Expression patterns of GATA1 mRNA isoforms were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Targeted next-generation sequencing (NGS) were performed for patients in whom GATA1 mutations were not detected by Sanger sequencing. GATA1 mutations were classified into 3 groups according to the predicted consequences, splicing error (SE), loss of the first methionine (LOM) and premature termination codon (PTC). Blood smears were centrally reviewed. Patients whose smears were prepared more than 14 days after the onsets of TAM were excluded from the morphological analyses. Differences in clinical parameters among the 3 mutation groups were analyzed using the Fisher's exact test, Kruskal-Wallis test or Steel-Dwass test.

Results: Mean age at sample collection, WBC count and blast percentage of blood samples were 8 days (range, 0-70 days), 22,100 µ/l (range, 4,400-422,000 µ/l), and 28.5% (range, 0-95%), respectively. GATA1 mutations were identified in 153 of 166 patients (92%) by Sanger sequencing. Although GATA1 mutations were not detected in 13 patients, splicing mutations were suspected in 7 patients because of the lack of the full-length GATA1 mRNA isoforms. In 12 of these 13 patients, blast percentages of the samples were less than 5%. GATA1 mutations were identified after targeted NGS in 10 of 13 patients negative for GATA1 mutations by Sanger sequencing. Of note, splicing mutations were confirmed after targeted NGS in all 7 patients suspected of having splicing mutations by RT-PCR. Collectively, GATA1 mutations were identified in 163 of 166 patients (98%). After exclusion of patients with multiple mutations (n=14) and internal deletion mutations (n=5), 39, 13 and 92 patients were classified into the SE, LOM and PTC groups, respectively. WBC counts at diagnosis were not significantly different among the 3 groups. However, the incidences of eosinophilia (>1,500 µ/l) were significantly different among the 3 groups (P<0.0001) and eosinophilia was more frequent in the SE (14/31, 45%) and LOM (4/11, 36%) groups than in the PTC (6/76, 8%) group (P<0.0001 and P=0.041, respectively). The levels of alanine aminotransferase (ALT) at diagnosis were also different among the 3 groups (P<0.00001) and the difference was statistically significant between the SE (median, 69; range, 11-468) and PTC group (median, 16; range, 3-380; P<0.00001).

Conclusion: These results suggest that Sanger sequencing using cDNA as well as genomic DNA is rapid and sensitive method to detect GATA1 mutations and that targeted NGS is useful for detection of GATA1 mutations in patients with low blast percentages. GATA1 mutation types may affect some clinical features of TAM including the numbers of eosinophils and the levels of ALT. Because estimated expression levels of GATA1s are higher in SE and LOM groups than PTC group, high GATA1s expression might be associated with eosinophilia and increased levels of ALT in TAM.