Germline transmission of LNKE208Q variant in a family with myeloproliferative neoplasms
Authors: Giuseppe Gaetano Loscocco, Carmela Mannarelli, Annalisa Pacilli, Tiziana Fanelli, Giada Rotunno, Francesca Gesullo, Giuditta Corbizi‐Fattori, Alessandro Maria Vannucchi, Paola Guglielmelli
Journal: American Journal of Hematology (2016)

To the Editor: With the aim to discover new mutations associated with familial forms of myeloproliferative neoplasms (MPN) 1, 2, we performed amplicon target resequencing, using the Ion Torrent PGM platform, in a total of 26 members from five families with at least two members affected by MPN. We employed a panel of 23 myeloid neoplasms-associated genes, including the entire coding DNA sequence (CDS) of JAK2, EZH2, SOCS1, SOCS2, SOCS3, STAT1, STAT3, STAT5, TET2, NRAS, KRAS and specific coding regions of JAK1 (exons 14,20-22), JAK3 (exons 12, 14,17-19), ASXL1 (exon 12), IDH1 (exon 4), IDH2 (exon 4), LNK (exons 2-4), CBL (exons 8,9), NFE2 (exon 3), DNMT3A (exons 18-23), SF3B1 (exons 13-16), U2AF1 (exons 2,6,8), and hot spot for SRSF2 (codon P95). In one family (Fig. 1, panel A), we detected a heterozygous LNKE208Q mutation in two affected members, one (F1) diagnosed as primary myelofibrosis (PMF) at the age of 54, while the second (F2), F1's daughter, had been diagnosed as essential thrombocythemia (ET; both according to 2008 WHO criteria) at the age of 37. The PMF patient had a concurrent CALR Type 1 mutation (52-bp deletion, p.L367fs*46), with a variant allele (VA) burden of 55% (as measured by capillary gel electrophoresis) while a JAK2V617F mutation with a VA burden of 27% (by qRT-PCR) was found in the ET patient. The NGS target mutation analysis showed that the PMF patient also presented somatic mutations in ASXL1 (R965X, VA 13%), TET2 (Y867H, VA 63%), NRAS (G12D, VA 11%), and KRAS (A146T, VA 9%), while no additional mutation was detected in the patient with ET. Panel A: Pedigree of the family with germline LNKE208Q variant. Panel B: Sanger sequencing of purified neutrophils (middle chromatogram) and in vitro expanded CD3+ cells (lower chromatogram) from patients F2. Since LNKE208Q mutation has been reported to occur very infrequently as acquired somatic mutation in some patients with so called idiopathic erythrocytosis 3 and in essential thrombocythemia 4, but not yet described in association with other driver mutations such as JAK2V617F and CALR 5, we asked whether the LNK mutation in these subjects could rather represent a germline variant. To this end, we prepared highly (>90%) purified CD3+ cells after in-vitro expansion from both affected subjects and determined that they expressed LNKE208Q mutation in a heterozygous form (Fig. 1, panel B); the preparation of CD3+ cells resulted negative for JAK2 and CALR mutations, although a minimal contamination by myeloid cells could not be excluded in the F1 sample because of the detection of trace mutated CALR sequences. The LNKE208Q variant has not been reported as a SNP in large databases of healthy controls (db SNP, International Genome Sample Resource (IGSR) data portal) while its somatic nature has been reported by Oh et al. (COSM1235390) 4; however, to exclude it might represent a rare variant in the Italian population, we genotyped 100 normal subjects from the same geographical area as the two affected family members, and did not detect it. Then, we genotyped for this variant five additional relatives, and found three of them to be positive for LNKE208Q (Fig. 1). Two of these (F4, F5) were sons of the ET patient, the other one (F3) was the son of the PMF patient (F1), and all three presented normal blood values. They were all negative for JAK2V617F, CALR and MPL mutations; they also did not harbor anyone mutated gene of the 23 analyzed in the NGS panel. We also genotyped all members of this family for the most common predisposition SNPs known to associate with MPN, including the JAK2 46/1 haplotype, TERT, MECOM, THRB-RARB, and HBS1L-MYB 6. Only the 46/1 haplotype strongly correlated with LNKE208Q, while no correlation was observed for the other SNPs (not shown in detail). Finally, we examined an additional 54 MPN patients with a familial history of MPN, as well as 200 patients each with sporadic PV, ET, and PMF. Overall, we found a heterozygous LNKE208Q mutation in two additional cases of JAK2V617F mutated MPN (one PV case with a JAK2V617F VA burden of 80% and one ET case with a JAK2V617F VA burden of 15%) as well as in one ET "triple negative" patient (i.e., wild type for JAK2, CALR, and MPL), all apparently sporadic since all denied a familial history of hematological disorders. Analysis of the CD3+ cell sample from PV patient revealed the LNKE208Q variant in a heterozygous status, suggesting its germline origin. Unfortunately, none of these patients consented to genotype other family members. In summary, we report here the occurrence of LNKE208Q mutation as a germline variant in a family with two cases of MPN. It is noteworthy that an overt MPN phenotype developed only in subjects where the LNKE208Q mutation was associated with a known driver mutation, such as JAK2V617F and CALR. This is in line with experimental data indicating that this mutation, located in the protein PH domain, has weak effects in promoting autonomous progenitor cell proliferation. Gotlib et al. showed that E208Q mutated cells generated a cytokine-specific STAT activation profiles only in response to TPO only, but not G-CSF 4. Thus, one could argue that the LNKE208Q variant per se is not strong enough to induce a MPN phenotype, as suggested by the three additional family members showing with this variant but no MPN phenotype. The fact that this mutation was not detected in 54 patients with a history of familial MPN would indicate that it represents a private germline mutation in this kindred; however, we suspect that at least one additional patient, harboring the JAK2V617F mutation at high mutation burden and the LNKE208Q as heterozygous variant, might indeed constitute a similar case, although we were unable to investigate other family members. Finally, it remains to be elucidated whether the LNKE208Q variant represents a true predisposing factor for the acquisition of one canonical driver mutation that seems necessary for a full born MPN phenotype. PG and AMV provided samples, designed research, interpreted results, and wrote the manuscript; GGL collected clinical data, performed research, and contributed to manuscript drafting; CM, AP, FT, GR, FG, GCF performed research. A complete list of AGIMM investigators is available at http://www.progettoagimm.it.