Mutational spectrum at GATA1 provides insights into mutagenesis and leukemogenesis in Down syndrome

Abstract

Down syndrome (DS) children have a unique genetic susceptibility to develop leukemia, in particular, acute megakaryocytic leukemia (AMkL) associated with somatic GATA1 mutations. The study of this genetic susceptibility with the use of DS as a model of leukemogenesis has broad applicability to the understanding of leukemia in children overall. On the basis of the role of GATA1 mutations in DS AMkL, we analyzed the mutational spectrum of GATA1 mutations to begin elucidating possible mechanisms by which these sequence alterations arise. Mutational analysis revealed a predominance of small insertion/deletion, duplication, and base substitution mutations, including G:C>T:A, G:C>A:T, and A:T>G:C. This mutational spectrum points to potential oxidative stress and aberrant folate metabolism secondary to genes on chromosome 21 (eg, cystathionine-beta-synthase, superoxide dismutase) as potential causes of GATA1 mutations. Furthermore, DNA repair capacity evaluated in DS and non-DS patient samples provided evidence that the base excision repair pathway is compromised in DS tissues, suggesting that inability to repair DNA damage also may play a critical role in the unique susceptibility of DS children to develop leukemia. A model of leukemogenesis in DS is proposed in which mutagenesis is driven by cystathionine-beta-synthase overexpression and altered folate homeostasis that becomes fixed as the ability to repair DNA damage is compromised.

Figure 1

Schematic of GATA1/exon 2 mutations in DS AMkL and TMD patients. Sites of GATA1/exon 2 mutations in DS AMkL and TMD patients are shown. DNA sequence for exon 2 is shown in 3 lines of nucleotide sequence, with nucleotide 1 as the first translated nucleotide of GATA1. Ins/del/dup sites are indicated below the line of the target sequence and identified by patient number. indicating sites of deletion are inserted immediately 5′ to the deleted base. above the line of sequence indicate sites of base substitution and are identified by patient number. More complete information is provided in Table 1. *Identical mutation observed in one person presenting first with TMD (#1) and subsequently with AMkL (#7).

Figure 2

Reduced expression of BER genes in DS patient samples. RNA was isolated and reverse transcribed from blast cells obtained from DS and non-DS (nonDS) patients as described in “Clinical TMD and AMkL samples.” Median gene expression levels were determined by quantitative real-time RT-PCR and normalized to 18S expression. (A) UDG expression. DS TMD vs non-DS AMkL, P < .01; DS AMkL vs non-DS AMkL, P = .1. (B) DNA polymerase β expression. DS TMD vs non-DS AMkL, P = .02; DS AMkL vs non-DS AMkL, P = .07. DNA polymerase β expression in 1 DS AMkL patient sample fell below the level of detection and was excluded from analysis.

Figure 3

Altered BER and increased oxidative stress in DS fetal liver. (A) UDG expression. RNA was isolated and reverse transcribed from DS and non-DS (NDS) fetal liver tissues as described in “Fetal liver tissue.” Median gene expression levels were determined by quantitative real-time RT-PCR and normalized to GAPDH expression. (B) Uracil accumulation in DNA. DNA was isolated from DS and non-DS fetal tissue and analyzed for uracil incorporation as described in “Uracil detection.” Relative uracil levels were determined and median values are presented. (C) DNA polymerase β expression. RNA was isolated and reverse transcribed from DS and non-DS fetal liver tissue as described in “Base excision repair activity.” Median gene expression levels were determined by quantitative real-time RT-PCR and normalized to GAPDH expression. (D) Base excision repair activity. Nuclear proteins were isolated, and BER capacity was determined as described in “Methods.” BER capacity is calculated as the percentage of probe repaired (16-mer/30-mer), and median values are presented. (E) F₂ isoprostane detection. F₂ isoprostanes were measured by gas chromatography–mass spectrometry in fetal liver tissues as described in “Fetal liver tissue.” Median values are presented.

Figure 4

Model: Trisomy 21 accelerates CBS activity and drives mutagenesis of GATA1 toward TMD/AMkL. This model proposes that the mutational spectrum at GATA1 can be explained by increased CBS activity, occurring as a combined result of both copy number increase and an adaptive response to oxidative stress in DS. Bold letters indicate enzymes known to be up-regulated in DS. Bold arrows indicate the primary direction of the reaction. The box around 5-meTHF depicts the trapping of this metabolite in this fully reduced form. CBS indicates cytathionine-β-synthase; DHF, dihydrofolate; dTMP, deoxythymidinemonophosphate; dUMP, deoxyruidinemonophosphate; GSH, reduced glutathione; GSSG, oxidized glutathione; 8-OHdG, 8-hydroxyguanosine; PLP, pyridoxal-l-phosphate; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; SOD, Cu/Zn superoxide dismutase; 5-meTHF, 5-methyltetrahydrofolate; 5,10-meTHF, 5,10-methyltetrahydrofolate; THF, tetrahydrofolate.