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Use of the 46/1 haplotype to model JAK2V617F clonal architecture in PV patients: clonal
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evolution and impact of IFNα treatment.
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Hasan Salma 1,2,3, Bruno Cassinat 4, Nathalie Droin 1,2,3, Jean Pierre Le Couedic 1,2,3, Fabrizia
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Favale
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Dosquet 4, Christine Chomienne 4,6, Eric Solary 1,2,3, Jean Luc Villeval 1,2,3, Nicole Casadevall
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1,7
1,2,3
, Barbara Monte-Mor
1,2,3
, Catherine Lacout
1,2,3
, Michaela Fontenay5, Christine
, Jean Jacques Kiladjian 8, William Vainchenker 1,2, 3*, Isabelle Plo 1,2,3*
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SUPPLEMENTAL METHODS:
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Patients.
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We studied samples from twelve 46/1 heterozygous individuals: 9 patients with a JAK2V617F-
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positive PV and 3 JAK2 wild-type (WT) hemochromatosis. 15 patients were studied during
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PEGASYS (peginterferon alpha 2a) therapy at a weekly dose ranging from 67.5 g to 180 g.
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Repeated samples were obtained during their clinical survey. Informed consent was obtained
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from each of them in accordance with the Declaration of Helsinki.
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Cell purification and culture.
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CD34+ and CD3+ cells were isolated from mononuclear cells by immunomagnetic enrichment
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and the following fraction (CD90+CD34+CD38-, CD34+CD38-, CD34+CD38+) were sorted,
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cloned at 1 cell /well and cultured in presence of a cocktail of human recombinant cytokines. 1
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Fourteen days later, individual colonies were plucked and lysed.
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Nucleic acid extraction.
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Genomic DNA from granulocytes, CD34+, CD3+ cells was isolated using DNA QiaAmp Kit
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(Qiagen, Courtaboeuf, France). Colonies were lysed with proteinase K and 0.2% Tween 20
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(Sigma) at 65° for 50 minutes and 95°C for 15 minutes.
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Next generation sequencing (NGS).
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DNA sample (CD34+ cells or granulocytes) was amplified with two sets of primers tagged
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with a unique and identical bar code to generate a library (JAK2_S3, JAK2_AS3;
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rs12343861_S1, rs12343861_AS1). Amplicons were sequenced using ion torrent PGM to
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quantify the JAK2V617F and rs12343867 allele burdens (average 10 000 reads). For patients
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prior treatment with IFNα, genes (DNMT3A, TET2, ASXL1, SUZ12, EZH2, ZRSR2, SF3B1
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(exon 14), RUNX1, IDH1/2, U2AF1 (except exon 2) were amplified using ampliseq
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technology and libraries were sequenced using ion torrent PGM with a 318 chip (average 100
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reads)
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Basis for the formula for the frequency of homozygous and heterozygous cells:
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The method can only be applied to patients heterozygous for the 46/1 haplotype that is
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associated with acquisition of JAK2V617F mutation. It is based on the precise determination of
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the 9pUPD level by measuring the level of a heterozygous SNP, from the 46/1 haplotype, and
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thus to calculate the frequency of the JAK2V617F homozygous clone. Subsequently, the
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JAK2V617F burden is determined to calculate the frequency of wild type JAK2 and JAK2V617F
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heterozygous clone.
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- 1) How to calculate JAK2V617F/V617F cells:
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As selected patients display germline heterozygous for 46/1 haplotype (rs12343867)
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 they should present 50% rs12343867 allele burden.
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As rs12343867 and JAK2V617F recombinate simultaneously, rs12343867 is used as a tag to
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follow mitotic recombination.
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Examples:
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If a patient presents 100% rs12343867 allele burden; it means that all cells have recombined
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JAK2V617F (i.e. 100% JAK2V617F/V617F cells).
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If a patient present now 75% rs12343867 allele burden; it means that half of cells have
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recombined JAK2V617F (i.e. 50% JAK2V617F/V617F cells).
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=>Formula is: The % of JAK2V617F/V617F cells =(rs12343867 allele burden -50%) x 2
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For these calculations we need to quantify global rs12343867 allele burden by Taqman allelic
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discrimination PCR or by NGS which is more accurate to quantify small differences.
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2) How to calculate JAK2V617F/WT cells:
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As we know now the % of JAK2V617F/V617F cells,
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Using the JAK2V617F allele burden,
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=> it possible to determine the JAK2V617F/WT cells.
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Example: If a patient present 75% rs12343867 allele burden =>50% JAK2V617F/V617F cells (see
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above) and we detect a 60% global JAK2V617F allele burden.
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Calculations are 60% global JAK2V617F allele burden - 50% JAK2V617F/V617F cells
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=> We find 10% JAK2V617F allele burden that represents 20% of JAK2V617F/WT cells.
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=>Formula is: the % of JAK2V617F/WT cells = [% of JAK2V617F allele burden
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JAK2V617F/V617F cells) x 2
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For these calculations we need to quantify global JAK2V617F allele burden by Taqman allelic
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discrimination PCR or NGS that is more accurate.
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- % of
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3) How to calculate JAK2WT/WT cells:
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As we know now the % of JAK2V617F/V617F cells and the % of JAK2V617F/WT cells, we can
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easily deduce the % of JAK2WT/WT cells.
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=>Formula is: the % of JAK2WT/WT cells = 100 – (% of JAK2V617F/V617F + % of JAK2V617F/WT
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cells)
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JAK2V617F and rs12343867 quantification by real-time polymerase chain reaction.
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TaqMan allelic discrimination assays were used to determine the allele burdens of JAK2V617F 2
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and rs12343867 (C_31941689 assay) (ABI 7500, Applied biosystem, Invitrogen). The region
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encompassing JAK2V617F and rs12343867 SNP was pre-amplified by PCR (with JAK2_S2
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and JAK2_AS2) for single colony DNAs (Table S1) and used to determine the mutational
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status of each clone. For controls, DNA from WT, heterozygous and homozygous JAK2V617F
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and rs12343867 patients were used. Therefore, we quantified allele burdens for granulocytes,
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CD34+ and CD3+ cells with the measurement of angles as shown in Figure S3. Of note,
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colonies genotyping only display heterozygous or homozygous rs12343867 and wild-type,
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heterozygous or homozygous JAK2V617F.
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SUPPLEMENTAL FIGURES :
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Figure S1: 46/1 allele is not responsible for frequent homologous recombination event:
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CD34+ cells from hemochromatosis patients were cloned at one cell/well and cultured for 14
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days in serum-free medium in the presence of cytokines. DNA was extracted from each
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colony and was subjected to rs12343867 C or T allele discrimination (representative from 3
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patients). Black circles stand for positive controls and crosses stand for colonies from the
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hemochromatosis patient. Black squares represent H2O.
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Figure S2: JAK2V617F clonal architecture in PV patients in HSC compartments.
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Either CD34+CD38+ or CD34+CD38- or CD90+CD34+CD38- cells from PV patients were
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cloned and cultured for 14 days in serum-free medium in the presence of cytokines. DNA was
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extracted from each colony (average of 100 colonies for CD34+CD38+ and CD34+CD38- and
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62 colonies for CD90+CD34+CD38-) and was subjected to JAK2 Taqman allelelic
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discrimination. The observed values of WT, JAK2V617F/WT and JAK2V617F/V617F clones were
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represented.
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Figure S3: Representation of rs12343867 and JAK2V617F allele discrimination from
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one patient.
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Allele burdens were quantified for rs12343867 and JAK2V617F from granulocytes, CD34+,
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CD3+ cells or CD34+CD38+ colonies with the measurement of angles. Of note, colonies
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genotyping display either heterozygous or homozygous rs12343867 and wild-type or
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heterozygous or homozygous JAK2V617F. Positive standards are indicated by filled circles.
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Figure S4: Sequencing by NGS of mutated genes associated with JAK2V617F in patients
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before IFNα treatment
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DNAs from patients before IFNα treatment were sequenced by NGS on DNMT3A, TET2,
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ASXL1, SUZ12, EZH2, ZRSR2, SF3B1 (exon 14), RUNX1, IDH1/2, U2AF1 (except exon 2).
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Three novel unannotated variants of TET2 were found. Two of them were located in exon 11
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leading to an amino acid change in the DSBH domain. ND stands for not determined.
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SUPPLEMENTAL REFERENCES
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Itzykson R, Kosmider O, Renneville A, Morabito M, Preudhomme C, Berthon C et
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2.
Dupont S, Masse A, James C, Teyssandier I, Lecluse Y, Larbret F et al. The JAK2
617V>F mutation triggers erythropoietin hypersensitivity and terminal erythroid
amplification in primary cells from patients with polycythemia vera. Blood 2007;
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