JAK3, JAK2 and GATA1 Mutations in Childhood Acute Megakaryoblastic Leukemia with and without Down Syndrome.
Authors: Hama Asahito , Yagasaki Hiroshi , Muramatsu Hideki , Nishio Nobuhiro , Yoshida Nao , Takahashi Yoshiyuki , Kojima Seiji 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) in children with Down syndrome (DS) exhibits unique characteristics, which includes the predominance of megakaryoblastic leukemia (AMKL). In about 10% of DS infants, transient myeloproliferative disorder (TMD) develops and then spontaneously resolves in most cases. However, 20% of TMD cases develop AMKL within 3 years, suggesting that additional genetic alterations might occur during the progression from TMD to AMKL. GATA1 mutations contribute to both the pathogenesis of TMD and AMKL, suggesting that the acquisition of additional genetic alterations might be necessary for the progression from TMD to AMKL. Recently, activating mutations of the JAK3 gene have been found in each of 16 non-DS and 3 DS-AMKL patients. These results suggest that the activating JAK3 mutations are involved in the pathogenesis of AMKL both with and without DS. In this study, we simultaneously analyzed JAK3, JAK2V617F which plays a critical role in the pathogenesis of myeloproliferative disorders and GATA1 mutations in non-DS children with AMKL and in DS children with AMKL or TMD. The study population included 11 non-DS children with AMKL and 20 AMKL and 6 TMD children with DS. After obtaining informed consent from the parents of the children for the purpose of sample banking and molecular analyses, bone marrow (BM) or peripheral blood (PB) samples were obtained from all of the patients with AMKL and TMD at the time of diagnosis. High-molecular weight DNA was extracted from the samples using standard methods. For the screening of the JAK3 mutations, we amplified genomic DNA that corresponded to all 23 exons of the JAK3 gene by using polymerase chain reaction (PCR) that employed 22 primer pairs. Amplified products were subjected to the denaturing HPLC (DHPLC) analysis using a WAVE Maker System (Transgenomic Inc., San Jose, CA). The DHPLC gradients and temperatures were determined using WAVE Maker System software. When heterozygous profiles were identified after a visual inspection of the chromatograms, amplified products were cloned into the pGEM-T Easy vector (Promega, Madison, WI) and sequenced on a DNA sequencer (310; Applied Biosystems, Foster City, CA) using a BigDye terminator cycle sequencing kit (Applied Biosystems). For screening of GATA1 mutations and JAK2V617F mutations, we amplified the genomic DNA that corresponded to exon 2 of GATA1 and exon12 of JAK2 by using PCR that employed 1 primer pair respectively. Amplified products were cloned into the pGEM-T Easy vector and sequenced on a DNA sequencer using a BigDye terminator cycle sequencing kit. Mutations of JAK3 were found in 2 of the DS-AMKL patients and in 1 DS-TMD patient. A573V and A593T substitutions in the JH2 pseudokinase domain along with Q501H substitutions in the JH3 domain were observed in the DS-AMKL patients and an I87T substitution in the JH7 domain of the receptor-binding domain was observed in DS-TMD. In one of the DS-AMKL patients, A573V and A593T substitutions were found in the same allele. GATA1 mutations were observed in 19 of the 20 DS-AMKL patients, in all of the DS-TMD patients and in 1 non-DS-AMKL patient. JAK2V617F mutation was only found in 1 DS-AMKL patient who had also JAK3 mutation (Q501H). The fact that the JAK3 mutations were found in both the AMKL and TMD patients indicates that this is an early event that occurs during the development of AMKL in DS patients and that other genetic changes may contribute to the development of AMKL.