Emerenciano M, Letter to the Editor, Leukemia

Emerenciano M, Letter to the Editor, Leukemia

Supplementary Information accompanies the paper on the Leukemia website ()

Methods

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) from BM cells of the patient, as well as from leukemic cell lines, and peripheral blood cells of healthy individuals. From 1 μg of total RNA, cDNA was synthesized in 20 μl of reaction mixture containing random primers by using the reverse transcriptase enzyme (Invitrogen, Carlsbad, CA, USA). Two microliters of cDNA solution were amplified by PCR in a 25 μl reaction buffer containing each primer for the NUP98-HOXD13 fusion transcript and wild-type HOXD13 transcript, as described.1,2 PCR amplification was carried out using a GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, CA, USA), and PCR products were separated by electrophoresis through a 1.5% bromide stained agarose gel.

Identification of the genomic NUP98-HOXD13 patient-specific fusion

High molecular weight DNA was isolated from mononuclear cells of diagnostic sample using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany). The DNA sample was treated and analyzed as described. 3 Briefly, 1µg of genomic DNA was digested with restriction enzymes and re-ligated to form DNA circles before LDI-PCR using NUP98-specific primers (NUP98.F8 and NUP98.R2 in Supplemental Table 1). Restriction polymorphic PCR amplimers were isolated from the gel and subjected to DNA sequence analyses to obtain the patient-specific fusion sequence.

Detection of genomic NUP98-HOXD13 fusion in the blood spot

A nested-PCR assay was established using primer pairs: NUP98.F1 and HOXD13.R1 (837 bp); NUP98.NF1 and HOXD13.NR1a (214 bp); NUP98.NF1 and HOXD13.NR1b (130 bp) (Supplemental Table 1). A 1/8 portion of Guthrie card was washed twice in 1 ml water (30 min each wash) to remove PCR inhibitors,4 air-dried and used directly in PCR. We followed all described recommendations to perform the backtracking assay in order to establish the sensitivity of the designed primer pairs and to avoid any kind of contamination.5 Briefly, optimal PCR conditions as well as sensitivity evaluation was established for each specific primer pair using an 1:10 dilution series of leukemic patient DNA sample into the DNA of a healthy individual. We included negative controls in all PCR reactions (blank, DNA from leukemia cell line and from PB of healthy individuals). First PCR reaction was run with the external primer pairs and 1 μl of this PCR product was used as a template into a second PCR reaction containing internal primers pair (NUP98.NF1 and either HOXD13.NR1a or NR1b). Sensitivity of 10 pg of patient DNA was achieved after nested reactions (Supplemental Figure 1), and the neonatal sample was tested.

Semiquantitative assessment of the size of the NUP98-HOXD13 clone at birth

DNA was isolated from seven pieces of the Guthrie card spot using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) according to the protocol for isolation of genomic DNA from dried blood. Proteinase K digestion was performed for 12h at 56 ºC to achieve an optimal DNA yield. The proportion of preleukemic cells on Guthrie cards was calculated by tritation PCR experiment. Genomic DNA from diagnosis sample and from Guthrie card was diluted into the DNA of a healthy individual to obtain 1 to 100 000 diploid genome copies. Simultaneously, NUP98-HOXD13 plasmid copies (1 to 100 000) were used to detect NUP98-HOXD13 sequence by PCR experiments. The estimative of the size of the clone at birth was then performed by a direct comparison between the plasmid, diagnostic and the Guthrie card serial dilutions (Figure 2).

Detection of FLT3, NRAS, KRAS, and PTPN11 mutations

Internal tandem duplication (ITD) and point mutations of the Fms-like tyrosine kinase 3 (FLT3) gene were analyzed as previously reported.6Exon 1 of the KRAS gene was amplified using primers described,7 and subsequently digested with either BstNI or PflMI (Biolabs, New England, MA) to address the presence of mutations in codons 12 and 13, respectively. The digested PCR fragments were visualized on a 3% agarose gel. Exon 1 of the NRAS and exon 3 of PTPN11 genes were amplified by PCR, using primers described previously,8,9 and PCR products were subjected to direct sequencing.

DNA and cDNA sequencing

The resulting NUP98-HOXD13 RT-PCR and PCR products and amplified exons of NRAS and PTPN11 were subjected to direct sequencing from both directions on an ABI 3130 automatic DNA sequencer with BigDye™ Primer v3.0 Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequencing data analyses were performed using BioEdit Sequence Alignment Editor Copyright 1997-2007. The BLAST search was used to identify NUP98-HOXD13 chimeric fusion transcript and genomic breakpoint, and point mutations of above referred genes (NCBI, Bethesda, MD, USA).

References

1 Arai Y, Kyo T, Miwa H, Arai K, Kamada N, Kita K et al. Heterogenous fusion transcripts involving the NUP98 gene and HOXD13 gene activation in a case of acute myeloid leukemia with the t(2;11)(q31;p15) translocation. Leukemia 2000; 14: 1621-1629.

2 Shimada H, Arai Y, Sekiguchi S, Ishii T, Tanitsu S, Sasaki M. Generation of the NUP98-HOXD13 fusion transcript by a rare translocation, t(2;11)(q31;p15), in a case of infant leukaemia. Br J Haematol 2000; 110: 210-213.

3 Meyer C, Kowarz E, Hofmann J, Renneville A, Zuna J, Trka J et al. New insights to the MLL recombinome of acute leukemias. Leukemia 2009; 23: 1490-1499.

4 Makowski GS, Davis EL, Aslanzadeh J, Hopfer SM. Enhanced direct amplification of Guthrie card DNA following selective elution of PCR inhibitors. Nucleic Acids Res 1995; 23: 3788-3789.

5 Wiemels J, Kang M, Greaves M. Backtracking of leukemic clones to birth. Methods Mol Biol 2009; 538: 7-27.

6 Emerenciano M, Menezes J, Vasquez ML, Zalcberg I, Thuler LC, Pombo-de-Oliveira MS. Clinical relevance of FLT3 gene abnormalities in Brazilian patients with infant leukemia. Leuk Lymphoma 2008; 49: 2291-2297.

7 Bornholdt J, Hansen J, Steiniche T, Dictor M, Antonsen A, Wolff H et al. K-ras mutations in sinonasal cancers in relation to wood dust exposure. BMC Cancer 2008; 8: 53.

8 Liang DC, Shih LY, Fu JF, Li HY, Wang HI, Hung IJ et al. K-Ras mutations and N-Ras mutations in childhood acute leukemias with or without mixed-lineage leukemia gene rearrangements. Cancer 2006; 106: 950-956.

9 Tartaglia M, Kalidas K, Shaw A, Song X, Musat DL, van dB, I et al. PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity. Am J Hum Genet 2002; 70: 1555-1563.