Familial Myeloproliferative Neoplasms Research Articles

Germ-Line and Somatic Mutations in Familial Myeloproliferative Neoplasms (MPNs). a Pilot Study

Background: Familial MPNs are uncommon disorders that, like sporadic cases, are characterized by clonal hematopoiesis and presence of somatic mutations, e.g. JAK2, CALR, MPL and occasionally TET2. There is little information, however, about germ-line mutations in these families that may explain the low penetrance hereditary predisposition.Methods: We studied five families with MPNs, each with at least 2 affected members. After obtaining an informed consent, clinical data was obtained from the patients’ electronic medical records. Blood and buccal samples were collected from patients and unaffected relatives. Exome sequencing was performed on the blood DNA samples using Agilent SureSelect Human All Exon V5+UTRs exome capture kit followed by massively parallel sequencing with Illumina HiSeq 2000. Sanger sequencing was then done on both the blood and buccal swab DNA samples to validate selected gene variants and to differentiate the nature of those variants (germ line or somatic).Results: The 5 families participating in this study had the following diagnoses: 1. Mother: polycythemia vera (PV); son: essential thrombocythemia (ET), 2. Mother: primary myelofibrosis (MF); daughter: unclassifiable MPN (UMPN), 3. Father: PV; son: PV, 4. Sister: MF; sister: MF, 5. Two aunts: MF; niece; UMPN. Six patients were positive for JAK2, V617F mutation. Blood and buccal samples were collected from 5 patients and 4 relatives. In all 5 families, the pro-band was younger at the time of diagnosis than his/her affected relatives. The clinical course of the MPNs appeared to be similar to the sporadic form.Exome sequencing revealed TET2 mutations in 2 probands. In addition, novel non-synonymous mutations in several candidate genes, KMT2D, KMT2C, NBEAL1, NBEAL2, AHNAK2, RNF213, were identified in the blood samples from the patients but not their unaffected relatives. These include two novel KMT2D mutations in two unrelated families. These 2 mutations were also found in the matching buccal swab samples, indicating that they are germ line mutations.Discussion: KMT2D and KMT2C mutations have been previously identified as somatic mutations in lymphoid malignancies, including non-Hodgkin’s lymphomas (Morin 2011), and as germ line compound heterozygote mutations in infant MLL and ALL (Valentine 2014). About 32% of diffuse large cell lymphoma and 89% of follicular lymphoma have somatic mutations of KMT2D. NBEAL2 germ line mutations are associated with familial gray platelet syndrome, where some patients have myelofibrosis (Gunay-Aygun 2011). To our knowledge, this is the first report describing germ line mutations in familial MPNs. The possible role of these mutations in predisposition to MPN will be discussed. Studies on additional families with MPNs are planned. DisclosuresNo relevant conflicts of interest to declare.

Targeted Exome Sequencing Identifies Novel Mutations in Familial Myeloproliferative Neoplasms Patients in the State of Qatar

Background:Myeloproliferative Neoplasms (MPNs) are clonal hematopoietic disorders characterized by excessive proliferation of one or more myeloid cell lineages. Philadelphia negative MPNs include Polycythemia Vera (PV), Essential Thrombocytosis (ET) & Primary Myelofibrosis (PMF). MPNs are associated with the presence JAK2 V617F mutation in 95% of PV & 50% of ET & PMF patients. Several molecular techniques such as RQ-PCR, HRM & Sequencing are currently used to detect common mutations. However, there are still significant numbers of MPNs that are negative to the most common genetic anomalies & many mutations are still unknown. The advent of Next Generation Sequencing (NGS) gives the opportunity to study relevant mutations in several genes.Aim:Utilizing NGS to identify potential genetic anomalies causing familial MPNs patients in Qatar.Methods:6 MPNs patients from consanguineous families & 5 healthy individuals were consented into the study & peripheral blood samples were collected. gDNA was extracted & used for multiplex PCR amplification of amplicons targeting cancer associated mutations in 28 key genes (JAK2, MPL, THPO, CBL, LNK, SH2B3, NF1, SOCS1/2/3, TP53, NRAS/KRAS, NF1, IDH1/2, EZH2, ASXL1, TET2, ATM, KIT, RB, TP53, IKZF1, RUNX1, PDGFRB, TERT & CALR) using the Ion AmpliSeq Kit. NGS was performed via the Ion Torrent using the 318 chip & data was analyzed with the Torrent Suite Software. Mutation details were obtained from COSMIC database. A hg 19 sequence was used as reference. The confirmation of NGS data was performed using RQ-PCR or Sequencing.Results:11 samples were successfully sequenced, with a mean depth of 1500 reads & the FASTQC plugin indicated good quality sequencing metrics. JAK2 V617F, JAK2 exon 12-15 & MPL (S505N, W515 L/K) negative samples tested before via RQ-PCR, HRM & sequencing were called negative by NGS.NGS identified novel deleterious mutations in MPNs patients. Out of 6 familial cases, 5 patients (P1- P5) were ET & 1 patient (P6) was PV. P1 had JAK2 V617F, ASXL1 T600P, CBFB G180S, THPO S184R &ITGA2R76Q, P2 had JAK2 V617F, MPL A554G & ATM F582L, the other three Patients (P3, P4 & P5) had CLAR K385fs*47 & one PV patient (P6) had TYK2 E1163G, ASXL1 P808H, PDGFRB P4L & TERT G300fs.Among the patients & healthy individuals, mutations/SNVs such as MPL P106L, K553N, SH2B3 L476F, ATM F1036F KIT N564S & TET2 T730R were also foundDiscussion & conclusion:Initial screening of known common genes (JAK2 V617F, JAK2 exon 12-15 & MPL W515 L/K) mutations did not reveal the causative mutations in 3% of 180 PV patients, 52% of 200 ET patients & 77% of 20 PMF patients.In this study, several deleterious somatic & germ-line mutations & SNVs were identified using Targeted Exome Sequencing approach.A complex combination of mutations in JAK2, THPO, ITGA2 & MPL genes occurred in ET patients & coexistence of several oncogenic events in TYK2, ASXL1, PDGFRB & TERT occurred in PV patient. This finding may also suggest that the MPNs phenotype may depend on presence of other mutations.It is worth mentioning that the presence of ATM variant in P2 is associated with increased risk of CLL.Somatic CALR type-2 mutation was identified in 3 ET (nonmutated JAK2 or MPL) patients. This mutation is 5-bp TTGTC insertion in exon 9 that generates a mutant protein with a novel C-terminal (p.K385fs*47).In patients & healthy individuals, a heterozygous germ-line mutation in exon 3 of the MPL gene (MPL P106L) has been observed. it has previously been described as a rare autosomal-dominant disorder. However, this mutation is considered to be frequent in Arabic populations, leading to severe thrombocytosis in homozygotes & occasionally to mild thrombocytosis in heterozygotes. In addition, several unreported variants of uncertain significance were identified.Our preliminary results suggested that MPNs patients in Qatar have several potential disease- associated variants & mutations. Evidences show that there exists a possibility of the disease arising out of the accumulation of genetic alterations & not as the consequence of a single genetic-hit event. This could possibly be due to the high rate of consanguineous marriages in Qatar i.e. the "Founder Effect".Our results recommended carrying out WES to explore & identify mutations which will be crucial to characterize many cases of MPNs with unknown molecular causes, gain a deep understanding of genotype-phenotype correlations & MPNs pathogenesis. DisclosuresAl-Dewik:Qatar National Research Fund: Patents & Royalties, Research Funding. Yassin:Qatar National research fund: Patents & Royalties, Research Funding.

Common germline variation at the TERT locus contributes to familial clustering of myeloproliferative neoplasms.

The C allele of the rs2736100 single nucleotide polymorphism located in the second intron of the TERT gene has recently been identified as a susceptibility factor for myeloproliferative neoplasms (MPN) in the Icelandic population. Here, we evaluate the role of TERT rs2736100_C in sporadic and familial MPN in the context of the previously identified JAK2 GGCC predisposition haplotype. We have confirmed the TERT rs2736100_C association in a large cohort of Italian sporadic MPN patients. The risk conferred by TERT rs2736100_C is present in all molecular and diagnostic MPN subtypes. TERT rs2736100_C and JAK2 GGCC are independently predisposing to MPN and have an additive effect on disease risk, together explaining a large fraction of the population attributable fraction (PAF = 73.06%). We found TERT rs2736100_C significantly enriched (P = 0.0090) in familial MPN compared to sporadic MPN, suggesting that low-penetrance variants may be responsible for a substantial part of familial clustering in MPN. Am. J. Hematol. 89:1107–1110, 2014. © 2014 The Authors. American Journal of Hematology published by Wiley Periodicals, Inc.

Molecular heterogeneity of familial myeloproliferative neoplasms revealed by analysis of the commonly acquired JAK2, CALR and MPL mutations.

The myeloproliferative neoplasms (MPN) are clonal, hematological malignancies that include polycythemia vera, essential thrombocythemia and primary myelofibrosis. While most cases of MPN are sporadic in nature, a familial pattern of inheritance is well recognised. The phenotype and status of the commonly acquired JAK2 V617F, CALR exon 9 and MPL W515L/K mutations in affected individuals from a consecutive series of ten familial MPN (FMPN) kindred are described. Affected individuals display the classical MPN phenotypes together with one kindred identified suggestive of hereditary thrombocytosis. In affected patients the JAK2 V617F mutation is the most commonly acquired followed by CALR exon nine mutations with no MPL W515L/K mutations detected. The JAK2 V617F and CALR exon 9 mutations appear to occur at approximately the same frequency in FMPN as in the sporadic forms of these diseases. The familial nature of MPN may often be overlooked and accordingly more common than previously considered. Characterisation of these FMPN kindred may allow for the investigation of molecular events that contribute to this inheritance.

Looking for CALR mutations in familial myeloproliferative neoplasms

Looking for CALR mutations in familial myeloproliferative neoplasms

A novel germline JAK2 mutation in familial myeloproliferative neoplasms

A novel germline <i>JAK2</i> mutation in familial myeloproliferative neoplasms

Long term follow up of 93 families with myeloproliferative neoplasms: life expectancy and implications of JAK2V617F in the occurrence of complications

The long-term evolution of familial myeloproliferative neoplasms was studied in 93 families with 227 subjects including 97 with polycythemia vera (PV), 105 essential thrombocythemia (ET), 14 primary myelofibrosis (PMF) and 11 chronic myeloid leukemia (CML). In PV patients, with 12years of median follow-up, overall survival was 83% at 10years and 37% at 20years. A high JAK2(V617F) allele burden was correlated with the transformation to myelofibrosis (p<0.0001), but not with the transformation to acute leukemia. Among the 105 ET, with 8years of median follow-up, overall survival was 83% at 10years and 57% at 20years. Progression to acute leukemia and progression to myelofibrosis were 10% and 13%. There was a trend toward a more frequent evolution to myelofibrosis when the JAK2(V617F) mutated allele burden was >50% (p=0.09), but not to AML. Hematologic transformation of the MPN was responsible for 69% of the deaths, cerebral stroke for 7% and 4% died of myocardial infarction. Eleven JAK2(V617F) mutated patients developed 13 deep splanchnic thromboses in PV and ET. Finally whereas patients with familial PV and ET have a comparable prognosis to non-familial MPN, the JAK2(V617F) mutation was associated with a more frequent occurrence of thrombosis in the entire population.

Biomarkers in cancer: is ‘omices’ the way to go

Biomarkers in cancer: is ‘omices’ the way to go

The role of the JAK2 GGCC haplotype and the TET2 gene in familial myeloproliferative neoplasms

Myeloproliferative neoplasms constitute a group of diverse chronic myeloid malignancies that share pathogenic features such as acquired mutations in the JAK2, TET2, CBL and MPL genes. There are recent reports that a JAK2 gene haplotype (GGCC or 46/1) confers susceptibility to JAK2 mutation-positive myeloproliferative neoplasms. The aim of this study was to examine the role of the JAK2 GGCC haplotype and germline mutations of TET2, CBL and MPL in familial myeloproliferative neoplasms. We investigated patients with familial (n=88) or sporadic (n=684) myeloproliferative neoplasms, and a control population (n=203) from the same demographic area in Italy. Association analysis was performed using tagged single nucleotide polymorphisms (rs10974944 and rs12343867) of the JAK2 haplotype. Sequence analysis of TET2, CBL and MPL was conducted in the 88 patients with familial myeloproliferative neoplasms. Association analysis revealed no difference in haplotype frequency between familial and sporadic cases of myeloproliferative neoplasms (P=0.6529). No germline mutations in TET2, CBL or MPL that segregate with the disease phenotype were identified. As we observed variability in somatic mutations in the affected members of a pedigree with myeloproliferative neoplasms, we postulated that somatic mutagenesis is increased in familial myeloproliferative neoplasms. Accordingly, we compared the incidence of malignant disorders between sporadic and familial patients. Although the overall incidence of malignant disorders did not differ significantly between cases of familial and sporadic myeloproliferative neoplasms, malignancies were more frequent in patients with familial disease aged between 50 to 70 years (P=0.0198) than in patients in the same age range with sporadic myeloproliferative neoplasms. We conclude that the JAK2 GGCC haplotype and germline mutations of TET2, CBL or MPL do not explain familial clustering of myeloproliferative neoplasms. As we observed an increased frequency of malignant disorders in patients with familial myeloproliferative neoplasms, we hypothesize that the germline genetic lesions that underlie familial clustering of myeloproliferative neoplasms predispose to somatic mutagenesis that is not restricted to myeloid hematopoietic cells but cause an increase in overall carcinogenesis.

Long-Term Follow-up of 94 Families with Myeloproliferative Neoplasms: Implications of the V617F JAK2 mutation, Life Expectancy and Complication Rates

AbstractAbstract 1978To study the evolution and prognosis in familial cases of myeloproliferative neoplasms (MPN), we collected clinical data and blood samples from 94 families defined by the presence of at least two affected subjects with one MPN. A total of 228 subjects including 99 polycythemia vera (PV), 104 essential thrombocythemia (ET), 14 primary myelofibrosis (PMF) and 11 chronic myeloid leukaemia (CML) were recruited. Occurrence was vertical in 63 families involving 2 generations in 48, 3 generations in 13, and two other families implicating respectively 4 and 5 generations. The distribution of MPN was most compatible with autosomal dominant inheritance. Phenotypic spectrum within families was either homogenous (56/94) or heterogeneous (35/94) and the main association (21/35) was PV and ET. Clinical and haematological data of affected subjects at diagnosis were similar to sporadic MPN. The V617F JAK2 mutation was found in 87% of patients with PV, in 59% of patients with ET, in 64% with PMF and in none patient with CML. 99 PV patients were studied (median age, 54 years; median follow-up, 12 years). Overall survival was 83% at 10 years and 37% at 20 years. At time of analysis 53% were alive; death was due to the hematologic malignancy in 74% of cases. Progression to acute leukemia and secondary myelofibrosis risks were respectively 21% and 20%, after a median time of 14 years (5,4-32,5) and 13 years (4,7-31). No difference in frequency of transformation in acute leukemia was observed between the JAK2 positive (23%) and JAK2 negative (20%) PV patients. Interestingly, none of JAK2-negative PV patients progressed to myelofibrosis, whereas 13 (20%) of JAK2-positive patients suffered from this adverse evolution. A high JAK2 allele burden was correlated with transformation to myelofibrosis. Of 49 patients with < 50% JAK2 mutated allele burden, only 2 (5%) developed myelofibrosis, whereas of 23 patients with ≥ 50% allele burden, 11 (48%) developed this complication (p<0,0001). Thrombosis occurred in 42% of PV patients, arterial in 19 (50%) cases, venous in 16 (42%), and 2 patients suffered both arterial and venous thrombosis. Hemorrhagic manifestations occurred in 2% of cases. Among the 104 ET (median age, 45 years; median follow-up, 8 years), overall survival was 83% at 10 years and 57% at 20 years. At time of analysis 83% were alive, 70% of deaths were related to the MPN. The risks of progression to acute leukemia and myelofibrosis were 10% and 13%, after a median time of 7,4 years (0,8-24) and 16 years (2,5-20,5) respectively. In particular, two non-related ET patients evolved rapidly after diagnosis, developing acute leukemia within the first year. We found no difference in frequency of transformation to acute leukemia or myelofibrosis according to JAK2 status. 32% ET patients suffered from thrombosis, arterial in 18 (60%) of cases and venous in 10(33%). Hemorrhagic manifestations occurred in 3% of cases. In the 14 PMF patients (median age, 53 years; median follow-up, 8 years), overall survival was 46% at 10 years. Transformation risk to acute leukemia was 23% with a median time of 26 months (1-50). Considering the whole population, occurrence of thrombosis was correlated with JAK2 mutational status, with 18% of any thrombosis in the JAK2-negative group and 37% in the JAK2-positive group (p=0.009). Among these, 12 (26%) patients developed deep venous thrombosis such as portal venous thrombosis or Budd-Chiari syndrome. In conclusion, the analysis of these 94 familial myeloproliferative neoplasms cases highlights that the presence of JAK2 mutation is associated with transformation to myelofibrosis for familial PV patients, and with a more frequent occurrence of thrombosis in the entire population. Patients with familial PV have a comparable prognosis to non familial PV. But, in our cohort, there seems to be an excess mortality of familial ET patients when compared to sporadic ET without clear correlation with JAK2 mutational status. Other genetic events involved in familial myeloproliferative neoplasms could explain this phenomenon. [Display omitted] Disclosures:No relevant conflicts of interest to declare.