Familial Myeloproliferative Neoplasms Research Articles

Prospective genetic germline evaluation in a consecutive group of adult patients aged

Relevance of germline (GL) predisposition in myelodysplastic syndromes (MDSs) was stressed in both 2022 WHO and International Consensus classifications, but its incidence is probably underestimated, especially in young adult patients. We selected a cohort of 31 consecutive de novo MDS patients with unusual young age (<60 years). We performed exome sequencing (ES) on DNA extracted from noninvasive sources (peripheral blood and saliva), filtering for a panel of 344 genes specifically tailored for detecting GL variants related to clonal and nonclonal cytopenia. We observed at least one high- or low-confidence GL MDS variant in 7/31 (22.6%) and 9/31 (29.0%) of cases, respectively. Four of 31 patients (12.9%) confirmed having established MDS/AML predisposing disorders. We found heterozygous variants in genes involved in DNA repair/cancer predisposition (ATM, ATR, FANCM, PARN, BRCA1, BRCA2, CHEK2, MSH2) in 9/31 (29.0%) casesand variants affecting ribosome biogenesis (SBDS), hematopoietic stem cell (GATA2), and megakaryocyte (ANKRD26) differentiation in single cases. Two cases had variants in RBBP6, a gene previously described exclusively in familial myeloproliferative neoplasms. Lastly, four cases had variants in genes related to inherited anemias (CUBN and PIEZO1 genes). Our results showed that "young" MDS patients aged 40-60 years carried reported and unreported GL variants with an unexpectedly high proportion, and these events co-occurred with somatic mutations recurrent in myeloid neoplasms. We explored the "no man's land" of the young adult MDS cases adopting a practical and scalable diagnostic tool, capable to detect GL variants avoiding invasive methods.

Characterization of myeloproliferative neoplasms in the paediatric and young adult population.

The aim of this study was to compare the genomic features and clinical outcomes between paediatric and young adult patients (PAYA, <40 years) and older adults (OA, ≥40 years) with myeloproliferative neoplasms (MPN) to gain insight into pathogenesis, disease prognosis and management. Of 630 MPN patients, 171 (27%) were PAYA with an average age at diagnosis of 31 years. Females were more prevalent in PAYA than OA (71% vs 58%; p=0.002), and PAYA more frequently presented with essential thrombocytosis (ET) at diagnosis (67% vs 39%; p < 0.001). The presence of a JAK2 somatic mutation was higher in OA (80.4% vs 64.3%; p < 0.001), while a CALR mutation or lack of any traditional driver mutation was more common in PAYA (20.5% vs 10.5%; p=0.001, 8.8% vs 3.7%; p=0.01 respectively). Venous thrombosis was more common in PAYA compared to OA (19.8% vs 10.7%; p=0.002). PAYA had a higher prevalence of familial MPN and familial cancer predisposition, and two PAYA patients harboured pathogenic germline JAK2 lesions. PAYA demonstrated longer survival from diagnosis than OA (median not reached vs 13 years), while disease transformation was less frequent (19.3% vs 37.9%).

Recurrent germline variant in ATM associated with familial myeloproliferative neoplasms.

Genetic predisposition (familial risk) in the myeloproliferative neoplasms (MPNs) is more common than the risk observed in most other cancers, including breast, prostate, and colon. Up to 10% of MPNs are considered to be familial. Recent genome-wide association studies have identified genomic loci associated with an MPN diagnosis. However, the identification of variants with functional contributions to the development of MPN remains limited. In this study, we have included 630 MPN patients and whole genome sequencing was performed in 64 individuals with familial MPN to uncover recurrent germline predisposition variants. Both targeted and unbiased filtering of single nucleotide variants (SNVs) was performed, with a comparison to 218 individuals with MPN unselected for familial status. This approach identified an ATM L2307F SNV occurring in nearly 8% of individuals with familial MPN. Structural protein modeling of this variant suggested stabilization of inactive ATM dimer, and alteration of the endogenous ATM locus in a human myeloid cell line resulted in decreased phosphorylation of the downstream tumor suppressor CHEK2. These results implicate ATM, and the DNA-damage response pathway, in predisposition to MPN.

Germline Testing for Patients with Familial MPN: A Single-Center Experience

Hereditary hematologic malignancies (HMs) are caused by germline variants that may be shared between multiple family members. Genetic testing and counseling in this cohort can provide surveillance for at-risk family members, screen potential bone marrow donors and inform therapeutic decision-making. In contrast to defined syndromes that predispose individuals to acute leukemia, the hereditary nature of myeloproliferative neoplasms (MPNs) remains understudied and is often ignored in hereditary hematology workup. MPNs, including essential thrombocythemia (ET), polycythemia rubra vera (PRV), primary myelofibrosis (PMF), chronic myelomonocytic leukemia (CMML) and hereditary erythrocytosis/thrombocytosis, often present sporadically in patients with no family history. However, at least 7.6% of all patients with apparently sporadic myeloproliferative disorders are reported to have a family history of MPN. MPNs are commonly associated with somatic mutations in driver genes JAK2, CALR, and MPL. While no single-gene familial MPN has been formally defined, germline pathogenic variants in these driver genes have been reported in some families. Additionally, variants in EGLN1/EPAS1 and duplications of the 14q32.2 genomic region have been described in multiple cases of familial MPNs. Finally ETV6 and SH2B3, genes classically associated with other inherited myeloid and lymphoid HMs, have some overlap with MPN presentation. Overall, familial clustering of MPNs has been observed in small case and large cohort studies, with a higher incidence of MPNs in first-degree relatives (FDRs) of affected patients. The Princess Margaret Cancer Centre (PM) is the largest leukemia center in Canada and has seen over 3000 adult patients with a HM between 2015-2021, including 664 patients diagnosed with a MPN. Here, we present our single-center experience in a small cohort of adult MPN patients referred during this period for hereditary hematology workup. Germline testing is primarily based on family history or suspicious findings on blood/bone marrow next generation sequencing (NGS), but testing may also be initiated based on recent literature and team discussion. Germline analysis was performed on DNA extracted from skin fibroblasts according to our genetic testing workflow. Since hereditary MPN testing is not fully characterized and some MPN genes have overlap with other HMs, testing was performed on genes related to erythrocytosis, inherited bone marrow failure syndromes, and/or myeloid malignancies, with recent patients receiving a panel comprehensive of all three. A total of 14 patients (7 male, 7 female; age range 35-74y, median 46y) diagnosed with a MPN (5 ET, 3 MF, 2 PRV, 3 erythrocytosis, 1 PRV/MF,) were referred to genetics for germline analysis (Table 1). 13/14 (93%) patients were referred with a positive family history of HM: 10/14 (71%) had at least one FDR with a HM, nine of whom had at least one FDR with a shared clinical presentation. Three patients only had a second or third degree family history of HM, and one patient had no family history of HM. Out of seven patients with a JAK2 mutation on blood/bone marrow, none were germline. On average, the time between MPN diagnosis and referral to genetics was six years (range 0-18 years). Overall, 2/14 (14%) patients referred had at least one actionable result, where actionability is defined by the presence of a pathogenic variant in any gene associated with an increased cancer risk. Of these two positive cases, only one (7% of all referrals) occurred in a gene associated with HMs: EGLN1. This patient was diagnosed with EGLN1-familial erythrocytosis. A second patient with no family history who was tested due to biallelic MPL mutations seen on blood/bone marrow NGS was found to be positive for ATM, a gene associated with other hereditary cancer syndromes. In addition, 2/14 (14%) patients had incidental findings associated with carrier status for an autosomal recessive disorder. 10/14 (71%) patients were found to have at least one variant of uncertain significance. Notably, while we saw strong familial patterns in MPN families, germline testing did not always detect a hereditary component; 8/9 patients with a first-degree family history of MPN had no actionable variants. While the hereditary nature of MPNs should be considered in clinical analysis, larger cohort studies are needed to determine stronger clinical markers of germline inheritance. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Contribution of JAK2 46/1 Haplotype in the Predisposition to Myeloproliferative Neoplasms.

Haplotype 46/1 (GGCC) consists of a set of genetic variations distributed along chromosome 9p.24.1, which extend from the Janus Kinase 2 gene to Insulin like 4. Marked by four jointly inherited variants (rs3780367, rs10974944, rs12343867, and rs1159782), this haplotype has a strong association with the development of BCR-ABL1-negative myeloproliferative neoplasms (MPNs) because it precedes the acquisition of the JAK2V617F variant, a common genetic alteration in individuals with these hematological malignancies. It is also described as one of the factors that increases the risk of familial MPNs by more than five times, 46/1 is associated with events related to inflammatory dysregulation, splenomegaly, splanchnic vein thrombosis, Budd–Chiari syndrome, increases in RBC count, platelets, leukocytes, hematocrit, and hemoglobin, which are characteristic of MPNs, as well as other findings that are still being elucidated and which are of great interest for the etiopathological understanding of these hematological neoplasms. Considering these factors, the present review aims to describe the main findings and discussions involving the 46/1 haplotype, and highlights the molecular and immunological aspects and their relevance as a tool for clinical practice and investigation of familial cases.

CHST15 gene germline mutation is associated with the development of familial myeloproliferative neoplasms and higher transformation risk

Herein, we describe the clinical and hematological features of three genetically related families predisposed to myeloproliferative neoplasms (MPNs). Using whole-exome sequencing, we identified a c.1367delG mutation(p.Arg456fs) in CHST15 (NM_001270764), a gene encoding a type II transmembraneglycoproteinthat acts as a sulfotransferase and participates in the biosynthesis of chondroitin sulfate E, in germline and somatic cells in familial MPN. CHST15defects caused an increased JAK2V617F allele burden and upregulated p-Stat3 activity,leading to an increase in the proliferative and prodifferentiation potential of transgenic HEL cells. We demonstrated that mutant CHST15 is able to coimmmunoprecipitate the JAK2 protein,suggesting the presence of a CHST15-JAK2-Stat3 signaling axis in familial MPN. Gene expression profiling showed that the FREM1, IFI27 and C4B_2 genes are overexpressed in familial MPN, suggesting the activation of an “inflammatory response-extracellular matrix-immune regulation” signaling network in the CHST15 mutation background.We thus concluded that CHST15 is a novel gene that predisposes to familial MPN and increases the probability of disease development or transformation.

Whole Genome Sequencing Uncovers a Rare Germline Variant in ATM Associated with Familial Myeloproliferative Neoplasms

Introduction: Myeloproliferative neoplasms (MPN) are sporadic diseases characterized by a somatic driver mutation in the JAK2, CALR or MPL gene. Although it is generally considered a sporadic disease, approximately 10% of MPN cases display familial clustering, and there is a 5 to 7-fold increased risk of developing an MPN among first degree relatives of MPN patients. In contrast to other myeloid malignancies, investigation of large pedigrees with familial clustering of MPN has failed to identify high-risk predisposition genes relevant to the general MPN population. Genome wide association studies (GWAS) have identified common, low penetrance risk alleles for MPN predisposition in multiple genes including JAK2, TERT, TET2, ATM and SH2B3. In order to identify novel germline predisposition variants in MPN, an unbiased whole genome sequencing (WGS) approach was utilized to examine genomic structure and germline variations in a cohort of individuals with familial MPN.Methods: The study cohort was comprised of 67 individuals with familial MPN enrolled in a prospective research registry at Johns Hopkins Hospital. Familial MPN was defined as a diagnosis of MPN in an individual with a family history of MPN or related myeloid malignancy (myelodysplastic syndrome and chronic myelomonocytic leukemia) in a first or second degree relative. Neutrophil genomic DNA was subjected to WGS using Illumina HiSeq platform and sequenced to 60x depth. We performed germline variant calling using HaplotypeCaller and following the GATK best practices. The variants detected were further enriched for germline by allele frequency 40-60% or >90% and presence in the gnomAD database. Non-synonymous coding variants that occurred in the study cohort at a statistically higher frequency that in the general population (gnomAD) were selected for further analysis. Prediction of variant deleteriousness was assessed by 4 algorithms (Provean, SIFT, Polyphen-2, CADD).Results: Filtering of 32,788 non-synonymous, likely germline variants produced 148 that occurred at a higher frequency in our cohort than in the general population (p < 0.01, Fisher's exact test). Of these, 29 were predicted to be pathogenic in 3 out of 4 algorithms. Five unrelated individuals were found to harbor a heterozygous p.Leu2307Phe variant in the ATM gene (chr11:108326169 C>T). The clinical characteristics of these individuals are presented in Table 1. The structure prediction of ATM indicates that Ser2306 is a potential phosphorylation site of protein kinase A, suggesting that the methionine-aromatic bond between M2026 and the mutated F2307 may block the phosphorylation of S2306 (Figure 1).Conclusions: We identified a rare ATM germline variant (chr11:108326169 C>T; p.Leu2307Phe) present in 5 individuals with familial MPN. ATM is involved in DNA damage repair and important in the maintenance of genomic integrity. Heterozygous germline variants in ATM are known to predispose to multiple cancer types, including breast, prostate, pancreatic and melanoma. Further, common polymorphisms in ATM have been found to be associated with the MPN phenotype via GWAS. Our data suggests that this variant may impact ATM activation and its function in DNA damage repair, and functional studies are in progress. These data implicate a rare germline ATM variant as a novel risk factor for development of MPN. [Display omitted] DisclosuresHourigan: Sellas: Research Funding.

Germline variants in DNA repair genes, including BRCA1/2, may cause familial myeloproliferative neoplasms

AbstractThe molecular causes of myeloproliferative neoplasms (MPNs) have not yet been fully elucidated. Approximately 7% to 8% of the patients carry predisposing genetic germline variants that lead to driver mutations, which enhance JAK-STAT signaling. To identify additional predisposing genetic germline variants, we performed whole-exome sequencing in 5 families, each with parent-child or sibling pairs affected by MPNs and carrying the somatic JAK2 V617F mutation. In 4 families, we detected rare germline variants in known tumor predisposition genes of the DNA repair pathway, including the highly penetrant BRCA1 and BRCA2 genes. The identification of an underlying hereditary tumor predisposition is of major relevance for the individual patients as well as for their families in the context of therapeutic options and preventive care. Two patients with essential thrombocythemia or polycythemia vera experienced progression to acute myeloid leukemia, which may suggest a high risk of leukemic transformation in these familial MPNs. Our study demonstrates the relevance of genetic germline diagnostics in elucidating the causes of MPNs and suggests novel therapeutic options (eg, PARP inhibitors) in MPNs. Furthermore, we uncover a broader tumor spectrum upon the detection of a germline mutation in genes of the DNA repair pathway.

Germline RBBP6 Mutations In Myeloproliferative Neoplasms

The pathogenesis of myeloproliferative neoplasms (MPN) is determined by the acquisition of somatic mutations in several genes, such as JAK2, MPL, and TET2. Familial clustering of MPN is observed in 5-10% of cases following predominantly autosomal dominant inheritance with incomplete penetrance. Familial MPN patients carry somatically acquired mutations in JAK2, MPL and other genes similar to sporadic cases. It is thought that the germline mutations in familial cases of MPN predispose to acquisition of somatic mutations which in turn drive the disease. No gene has been implicated in familial MPN predisposition so far. The JAK2haplotype predisposing to MPN at population level does not explain familial MPN cases.We studied a family with five MPN cases in four generations displaying an autosomal dominant pattern of inheritance with reduced penetrance. DNA was available from three MPN cases in the family. One of the patients carried a JAK2-V617F mutation and a deletion on chromosome 22q, while an MPL-W515L mutation was detected in another affected member of the family. The third patient did not have JAK2 or MPLmutations, but carried three chromosomal aberrations on chromosomes 2p, 7q and 15q. Chromosomal aberrations were detected using Affymetrix SNP 6.0 microarrays. The genotypic data from SNP 6.0 arrays was used to perform high-resolution nonparametric linkage analysis. In order to identify the germline mutation predisposing to MPN in the family we performed whole exome sequencing of the three affected members using Illumina next-generation sequencing technology. The mutations identified from exome sequencing were combined with the linkage analysis data.We identified a germline mutation in the RBBP6 gene that segregates with the MPN phenotype in the studied pedigree. Further screening in familial and sporadic MPN cases identified several other germline RBBP6mutations in 5% of the familial and 0.6% of the sporadic MPN cases. Familial MPN cases mostly had the diagnosis of primary myelofibrosis. All the detected mutations cluster in putative p53-binding region of the protein, suggesting a possible link of the mutations and p53 function.The functional effect of the mutations was assessed in a doxycycline-inducible HEK-flp-in cell line system. Upon doxycyline induction, the wild-type and mutant RBBP6 constructs were over-expressed and the consequent functional effect of mutation was assessed using RNA sequencing and global proteomic analyses. The combination of the two datasets yielded 109 genes/proteins that were significantly changed in both the RNA sequencing and the global proteomic results when comparing wild-type to mutant RBBP6, 87 of which were downregulated by the mutation, while 22 were upregulated. Several important cell cycle and apoptosis regulators were found among the deregulated genes, including TP53, BAX, BID and USP7 (downregulated) and CDK6 (upregulated). KEGG pathway analysis of the downregulated genes showed significant enrichment for the p53 and the apoptosis pathways.We have identified germline mutations in RBBP6 that predispose to the development of MPN and are enriched in the families. Functional studies have shown that the mutations likely affect the p53 pathway and apoptosis and thus increase the risk of acquiring somatic mutations that lead to MPN. Our study implicates RBBP6 in MPN susceptibility and suggests that RBBP6 is an important cancer-associated gene. Disclosures:No relevant conflicts of interest to declare.

Novel germline mutation KMT2A G3131S confers genetic susceptibility to familial myeloproliferative neoplasms.

The current study analyzed the clinical and genetic characteristics of a family with familial myeloproliferative neoplasms (MPNs). Whole-exome sequencing was conducted, and a germline heterozygous mutation in lysine methyltransferase 2A (KMT2A, also known as MLL1), G3131S (c.9391G > A, p.Gly3131Ser, rs150804738), was identified. Somatic DNA and germline DNA were collected from 8 family members, 120 healthy donors (somatic DNA), and 30 healthy donors (germline DNA). Using Sanger sequencing, the KMT2A G3131S mutation was analyzed. Four individuals, the proband (II-1), his sister (patient II-2), and family members II-3 and III-1 (somatic DNA and germline DNA), tested positive for the KMT2A G3131S mutation. We did not observe the KMT2A G3131S mutation in healthy donors (somatic DNA and germline DNA), indicating that this is not a SNP. Bioinformatics analysis of KMT2A G3131S suggested that protein structure changes could be caused by this mutation. To further elucidate the function of KMT2A G3131S, the CRISPR-Cas9 technique was applied to generate a KMT2A G3131S heterozygous K562 cell line. The colony formation potency, apoptosis, and cell cycle of KMT2A G3131S mutant K562 cells were analyzed. The results demonstrated that KMT2A G3131S mutant K562 cells showed increased proliferation and colony formation ability. Immunophenotyping was performed using flow cytometry to analyze the surface marker expression of gene-edited KMT2A G3131S mutant K562 cells. A significant increase in CD11b and mild increases in CD61 and CD235a were observed in KMT2A G3131S mutant K562 cells, suggesting that the KMT2A G3131S mutant could cause an increase in myeloproliferation. May-Giemsa staining showed that the morphological changes in KMT2A G3131S mutant K562 cells were consistent with the flow cytometry analysis. To verify which downstream genes were affected by the KMT2A G3131S mutant, we performed real-time PCR to evaluate the expression of previously reported KMT2A-related genes and found that C-MYB expression was significantly decreased. Western blotting was applied to investigate the expression of Kmt2a and C-myb proteins, and the results showed that in KMT2A G3131S mutant K562 cells, the expression of C-myb was decreased. Our findings suggested that KMT2A G3131S could affect the myeloproliferation of K562 cells and decrease C-myb expression. In conclusion, KMT2A G3131S could be considered a novel genetic susceptibility gene in familial MPN.

Germline ERBB2 Variants Associate with Myeloproliferative Neoplasms

Background: The myeloproliferative neoplasms (MPN) and myelodysplastic syndrome (MDS) are usually sporadic diseases, however, familial cases are well-described. Approximately 10% of MPN cases display familial clustering, and there is a 5-7 fold increased risk of developing an MPN among first degree relatives of MPN patients. While familial MDS appears to be less common, identification of multiple predisposition genes has led to the incorporation of this entity into the most recent WHO classification of myeloid neoplasms. Identification of predisposing germline mutations in the myeloid malignancies has led to a better understanding of the pathophysiology of these diseases and improved clinical care. However, many familial MDS and MPN cases exist in which the inherited genetic lesion is unknown. Our recent investigation of a multigenerational family with inherited MDS/AML marked by erythroid hyperplasia identified a missense variant in ERBB3 that co-segregated with the disease phenotype (1). In this study, we investigated a kindred with an autosomal dominant familial cancer syndrome characterized by both MPN and solid tumor malignancies.Methods: Whole exome sequencing was performed via the Baylor-Hopkins Center for Mendelian Genomics at Center for Inherited Disease Research at Johns Hopkins University. Saliva or peripheral blood samples were used as source DNA. Sequence analysis and variant calling/filtering was performed as previously described (1). Targeted sequencing of the ERBB genes was performed via MiSeq using an Illumina TruSeq Custom Amplicon (v1.5) designed to sequence the entire open reading frame of selected genes. Analysis was performed using Illumina BaseSpace TruSeq applications. Germline variants were identified via variant allele frequency and heterozygous, nonsynonymous variants with a minor allele frequency < 0.01 and a depth > 40x were prioritized.Results: The proband was diagnosed with a JAK2 V617F positive MPN at age 37. Subsequent personal cancer history included melanoma and renal carcinoma. Family history was significant for ovarian cancer and melanoma in her mother, MPN and melanoma in her maternal aunt, and bladder cancer in her maternal uncle (Figure 1A). Exome sequencing of the three family members affected with melanoma was performed in order to identify the predisposing inherited variant in this family. Sequence analysis produced 71 candidate rare, heterozygous, nonsynonymous single nucleotide variants (SNV), including one in the gene ERBB2 . Genotyping of 2 additional family members for the ERBB2 c.3182T>C;p.L1061P SNV established that it co-segregates with disease (Figure 1A). To further investigate the prevalence of rare germline ERBB2 and ERBB3 variants in both MPN and MDS, we performed targeted sequencing of these genes in two discovery cohorts: 1) 96 cases of MPN, of which 63 had a 1st or 2nd degree relative with a family history of MPN and 2) 19 cases of erythroid MDS/AML. Five out of 96 (5.2%) cases of MPN were found to have ERBB2 variants, while 3 of 96 (3.1%) had ERBB3 variants. Of the 19 erythroid MDS/AML cases, no ERBB2 variants were identified, while 1 had an ERBB3 variant. Further, one MPN case with a germline ERBB2 c.G3250T:p.D1084Y variant had a family history of breast cancer in her mother, who also carried this variant (Figure 1B). We analyzed a validation cohort of 630 cases of hematologic malignancies (approximately 20% MPN) sequenced as part of their clinical evaluation at the Sidney Kimmel Cancer Center. Similar to the findings in our discovery cohorts, rare germline ERBB2 variants were enriched in MPN, found in approximately 5% of MPN compared to only 1.8% of all other myeloid disorder diagnoses. In contrast, germline ERBB3 variants were present in 1.6% of MPN compared to 1.8% of the other myeloid disorders (Table).Conclusions: We have identified germline ERBB2 variants that co-segregate with disease in two kindreds with familial cancer syndromes manifesting as a mixed MPN and solid tumor phenotype. In addition, we show that germline ERBB2 and ERBB3 variants are present in patients with hematologic malignancies at a higher incidence than expected in the general population, and ERBB2 variants are associated with an MPN phenotype. Together, these data implicate these genes in the predisposition to myeloid malignancy as well as other familial cancer syndromes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical Analysis of 269 Ph Chromosome-Negative Myeloproliferative Neoplasms Patients Stratified by Age

To investigate the difference of clinical characteristics between young patients(age≤40 years old) and middle-older patients(age>40 years old) with the myeloproliferative neoplasms（MPN）. The clinical data (gene mutations， peripheral blood routine examinations， imaging examination and past history) of 269 MPN patients was collected and analyzed. In essential thrombocythemia (ET) group, the proportion of triple-negative type in young patients was higher than that in middle-older group, while the peripheral white blood cell（WBC） and platelets（PLT） counts in the first visit were lower. In polycythemia vera (PV) group, the total detection rate of JAK2V617F (80.65%) was lower than that of other research reports. Young patients with PV showed the lower JAK2V617F rate and lower WBC count, compared with the middle-older aged patients. Both CALR and MPL mutations were not found in PV patients. There was only 1 primary myelofibrosis (PMF) patient aged <40 years old. 91.67% of the patients merged splenomegaly and this rate was higher than that of ET or PV patients. It was found that there were a diagnosed familial MPN family and an undiagnosed family， and the youngest patient was only 8 years old. The second-generation gene sequencing detection for them was not carried out. Age is an important reference index in the assessment of risks. The MPN patients with different age and types show much difference in gene mutations, peripheral blood cell counts, thrombotic events and sizes of spleen. The onset ages of patients with familial MPN trends to be generational younger.

Germline Mutations Predispose to Familial Myeloproliferative Neoplasms

Background: Myeloproliferative neoplasms (MPNs), commonly polycythemia vera (PV), primary myelofibrosis (PMF) and essential thrombocythemia (ET), are a group of malignant hematologic disorders characterized by proliferation and accumulation of terminally differentiated blood cells of erythroid, myeloid and megakaryocytic origin (Cazzola, Blood 2014).Somatic driver mutations, frequently JAK2V617F, MPLW515L/K or mutant CALR in sporadic MPNs affect signaling by the thrombopoietin (THPO) and/or the erythropoietin (EPO) receptor (Pickman, PLoS 2016). MPL is the receptor of THPO and JAK2 is the key downstream mediator of THPO/MPL signaling (Nangalia NEJM 2013). In familial MPNs (families with more than 1 member developing a MPN) a germline mutation predisposes to disease development. These mutations can reveal pathways or processes involved in the pathogenesis of the disease, independent of, or cooperating with, the somatic driver mutations (Rumi, JCO 2007). We identified 3 putative predisposing germline mutations in 1 family, 3 affected generations developed MPN (PMF, ET and PV): RBSN frameshift,JAK2R340Q &amp; PRKAA2Q425P missense variants. RBSN encodes Rabenosyn-5 protein (RBSN) which plays a role in early growth-factor receptor clathrin-dependent endocytosis (Nielsen, J Cell Biol 2000) (Fig.1).We discovered that wild type (WT) Rabenosyn-5 regulates cell surface MPL levels in hematopoietic progenitor cells and predict that MPL levels on the cell surface are negatively associated with WT Rabenosyn-5 levels. We hypothesize that the RBSN mutation predisposes to MPN development in this family, leading to abnormal activation of THPO signaling by reducing MPL turnover. Methods: Peripheral blood DNA from patient samples were analyzed for potential putative mutations using Agilent SureSelect Human ALL Exon V5+UTRs exome capture kit followed by parallel sequencing with Illumina HiSeq 2000. In vitro studies involved using the BaF3 system and CRISPR-Cas9 gene editing technique. We generated BaF3-MPL (BaF3 cells transduced with human CD4-MPL fusion) cell lines that rely on IL3 or human THPO cytokines for growth &amp; proliferation (Fig.2&amp;3). We transduced RBSN into the BaF3-MPL cell lines and produced RBSN heterozygous (HT) and homozygous (HM) mutants. We first generated LentiCRISPRV2GFP-mRBSN vector by inserting RBSN specific guide RNA into the LentiCRISPRV2GFP vector and generated lentivirus expressing both Cas9 and guide RNA by co-transfecting 293T cells with LentiCRISPRV2GFP vector and viral packaging vectors. The final virus was used to infect BaF3-MPL cells. Transduced cells were purified by FACS to select GFP+ cells and individual cells were grown in culture. RBSN gene mutations were examined using PCR and sequenced accordingly. To study whether the RBSN mutation alters the surface MPL levels, we stained BaF3-MPL, HT-RBSN-BaF3 and HM-RBSN-BaF3 cells with APC-CD4 antibody and the surface levels of MPL were detected by FACS by comparing the mean fluorescence intensity (MFI) of APC. Results: We identified several colonies with either HT-RBSN-BaF3 or HM-RBSN-BaF3. The guide RNA we designed specifically targeted the 12th exon of the RBSN gene which best recapitulates the mutations observed in our patients, thus, the frameshift mutations in HT-RBSN-BaF3 and HM-RBSN-BaF3 cell lines we predict will make truncated forms of RBSN which will be verified by Western Blotting for future experiments. Surface levels of MPL in the HT-RBSN-BaF3 and HM-RBSN-BaF3 cells are greater than that in BaF3-MPL cells, suggesting RBSN negatively regulates THPO-MPL signaling by regulating the cell surface MPL levels (Fig. 4). Conclusions: RBSN is a regulator of receptor trafficking, which mediates the fusion of the endocytosed receptor to the early endosome and then lysosome for degradation. We propose that genetic inactivation of RBSN might prevent the endocytosed MPL from endosome-degradation and enhance the reuse of MPL by recycling. Future experiments are planned to include in vivo studies to study whether inactivation of RBSN promoted MPN-like disease development into NSG mice and transducing our established cell lines to label individual cell compartments to study THPO-stimulated signaling. Our findings, if confirmed, will further clarify aspects of familial and somatic MPN pathogenesis and may inform efforts to devise new therapeutic and diagnostic strategies for this disease complex. Disclosures No relevant conflicts of interest to declare.

ATG2B/Gskip in De Novo Acute Myeloid Leukemia (AML): High Prevalence of Germline Predisposition in French West Indies and Potential Role of Overexpression in Acquired AML

In 2015, a germline copy number variation of chromosome 14 (CNVdup14) including ATG2B and GSKIP genes was described as a predisposition genetic factor responsible of familial myeloproliferative neoplasms from French West Indies (Saliba et al, Nat Gen 2015), frequently progressing to AML. In this study, we looked at the presence of this CNVdup14 in a cohort of Caribbean islands patients (pts) with non-secondary aggressive hematological malignancies (HM). We also studied the expression of ATG2B and GSKIP genes in a cohort of acquired AML pts.This is a retrospective multicenter study of adults Caribbean islands pts treated at Gustave Roussy Cancer Center (Villejuif, France) and at the French West Indian hospitals (Martinique and Guadeloupe) between May 2000 and May 2018. We included pts with AML, acute undifferentiated leukemia (AUL), acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LL). Pts with personal history of myeloproliferative or myelodysplastic syndromes before the onset of aggressive HM were not included in this study. The presence of the CNVdup14 was carried out by PCR analyses in all the pts.For the second part of the study, expression of ATG2B and GSKIP genes were assessed in newly diagnosed de novo AML pts with normal karyotype or trisomy 8 (samples from the GOELAMSTHEQUE) by quantitative RT-PCR and expressed as relative expression PPIA/HPRT/H2A.Z.One hundred pts were analyzed. Median age was 52 years (IQ 40-62) with male predominance (61%). Fifty eight pts came from Martinique, 42 pts from the rest of the Caribbean islands (including 28, 4, 3, 2 pts from Guadeloupe, Haiti, Saint Martin, Dominican Republic, respectively). Seventy eight pts had AML. Among them, according to revised MRC cytogenetic classification, 11 (14%) were favorable, 47 (60%) intermediate and 20 (26%) adverse. Seventeen pts had ALL, 3 LL, and 2 AUL. On the entire cohort, all except nine pts were treated with intensive chemotherapy, 80 reached complete remission, 29 relapsed, 46 pts died. Thirty two pts received hematopoietic stem cell transplantation (HSCT).Six pts were positive for the CNVdup14 by PCR (confirmed by SNP array in the 5 pts with leftover DNA available). All had an AML (no pts with favorable AML) and were originated from Martinique. These pts represented 14% of the 43 AML from Martinique in our cohort (17% if we excluded favorable AML). One was known to be part of an ATG2B/GSKIP family, 2 pts had no familial history of myeloid malignancies and 2 new families were discovered. Median white blood cell, hemoglobin and platelets counts were 17.7 G/l (IQ 6.7-48), 8.15 g/dl (6.9-9.7) and 58 G/l (20-132), respectively. Median age at AML diagnosis was 49 years (34-55), 3/6 (50%) had extramedullary localization compared to 11/78 (14%) for others AML pts. Karyotype was normal for 4, or showed a monosomy 7 for 2 pts. NGS panel showed distinct abnormalities compared to the entire cohort (Fig A). None had JAK2, MPL, CALR, P53, RUNX1, DNMT3A, FLT3-ITD mutations. All harbored an epigenetic and/or spliceosome mutation (IDH n=3, TET2 n=3, ASLX1 n=3, SRSF2 n=3). Five out of the six pts received intensive treatment and 4 achieved complete remission. Two received HSCT, 2 relapsed and 4 died.Median overall survival (OS) of the entire cohort was 35.7 months (22.5-89.5) and progression free survival (PFS) 27.6 months (15.6 -56.1). As CNVdup14 pts had AML only, we next evaluated survival according to the predisposition status in the AML cohort. Pts with CNVdup14 had a median OS and PFS of 19 (6.5-29) and 11.4 (6.5-29) months, respectively, compared with 52.6 (22.9-100.2) and 30 months (15.6-60) in CNV wild-type counterparts (PFS Fig B).We next evaluated ATG2B and GSKIP expression in a cohort of 46 random de novo AML pts (GOELAMS-LAM-IR-2006 multicenter trial). Median expression of ATG2B and GSKIP were 4.8 (2.6-12.9) and 5.3 (0.3-5.1) respectively. No CNVdup14 was detected. Interestingly we found a correlation between the two genes expression (Pearson Correlation Coefficients 0.55 and linear regression p< 0.001, Fig C). Expression of ATG2B and GSKIP was also correlated with leukocytosis (p=0.003 and p=0.07) (Fig D). We found no impact on OS and PFS.For the first time, we described a high percentage of the germline CNVdup14 in de novo AML pts from Martinique (14%). Moreover evaluation of ATG2B and GSKIP expression suggested that the role of theses 2 genes in leukemogenesis is not limited to pts with the CNVdup14. [Display omitted] Disclosuresde Botton:Agios: Research Funding; Celgene: Honoraria, Research Funding. Benabelali:CERBA laboratory: Employment.

Familial MPN Predisposition.

Chronic myeloproliferative neoplasms (MPN) characteristically arise from a somatic mutation in the pluripotent hematopoietic stem cell, and most common recurring mutations are in the JAK2, CALR, and cMPL genes. However, these mutations are not founder mutations, but mainly drive the disease phenotype and a pre-existing germline predisposition has been long speculated, but has not been clearly defined to date. Genome-wide association studies in family clusters of MPN have identified a number of genetic variants that are associated with increased germline risk for developing clonal MPN. The strongest association discovered so far is the presence of JAK2 46/1 haplotype, and subsequently, many studies have found additional variants in other genes, most notably in TERT gene. However, these still account for a small fraction of familial MPN, and more in-depth studies including whole genome sequencing are needed to gain better insight into familial genetic predisposition of clonal MPNs.

Advances in understanding the pathogenesis of familial myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are generally acquired as a result of a somatic stem cell mutation leading to clonal expansion of myeloid precursors. In addition to sporadic cases, familial MPN occurs when one or several MPN affect different relatives of the same family. MPN driver mutations (JAK2, CALR, MPL) are somatically acquired also in familial cases, so a genetic predisposition to acquire one of the MPN driver mutations would be inherited, even though the causative germline mutations underlying familial MPN remain largely unknown. Recently some germline variants [ATG2B and GSKIP duplication, RBBP6 mutations, SH2B3 (LNK) mutations], which can cause familial MPN, have been reported but these mutations are rare and do not explain most familial cases. Patients with familial MPN show the same clinical features and suffer the same complications as those with sporadic disease. This review aims to offer up-to-date information regarding the genetics of familial MPN.

Clinical Exome Sequencing Unravels New Disease Causing Mutations in Myeloproliferative Neoplasms (MPNs): A Pilot Study in Patients from the State of Qatar

In Qatar, a cluster of ET familial cases (25 families from 5 major tribes) has been documented. This observation represents a unique interest for the scientific community worldwide, as the cultural traditions of tribal marriages between 1stcousins is expected to lead to the preservation of a limited genetic pool (giving rise to founder effects). CES was employed to analyze a 3 probands from three unrelated families (S1, S2, S3). All 6 patients were Qatari belonging to consanguineous families with a positive family history of MPNs. CES confirmed the diagnosis in the 3 probands: two patients (S1 and S2) were classified as Essential Thrombocythemia (ET), one patient (S3) as unclassified MPNs. In the other 3 subjects (S4, S4, S6), several shared deleterious mutations and polymorphisms were uncovered. CES identified 10 novel mutations in known genes as well as 7 novel candidate genes and 2 (6%) previously reported mutations in the three probands (S1, S2, S3) who's diagnosis was confirmed . S1: a confirmed diagnosis and phenotype of ET (JAK2 and THPO), S2: a confirmed diagnosis and phenotype of ET (JAK2 and MPL) and S3: a confirmed diagnosis of U-MPNs (TERT, ATM, NUMA1 and ASXL) The mutations/variants were classified according to ACMG recommendations into 5 classes. The number of mutations/variants detected in each class is as follows: Class 1 - Previously reported as disease-causing: 6 Class 2 - Previously unreported, but of the type which can cause the disorder: 3 Class 3 - Previously unreported, may or may not be the cause of the disorder (Variant of uncertain clinical significance): 48 Class 4 - Previously unreported but likely neutral: 1 Class 5 - Previously reported as neutral: 3 CES identified a total of 61 interesting variants which occurred in 50 genes, 13 were recurrently. The vast majority of the prioritized variants were missense 50(82%); the remaining were frameshift (small insertions and deletions) 10(16%) or affecting splicing sites 1(2%). The most interesting finding is the presence of 10 novel mutations in known genes related to MPNs as well as 7 variants in novel candidate genes in the three probands who's diagnosis was confirmed via CES. This study presents novel data that contributes to understanding the high level of genetic complexity in MPNs Qatari patients. It has identified new variants in cancer-related genes potentially involved in the pathogenesis of the disease Our study of familial MPNs led to the identification of new disease-causing mutations and variants, some previously described as either germline or somatic. A clinical cohort of 3 Qatari probands suspected to have MPNs as well as three family members with a positive family history of MPNs presented at National Center for Cancer Care (NCCCR) S1) is a female who presented with high hemoglobin (Hb) level, red blood cells (RBCs) count, leukocytosis with neutrophilia, very high platelets and normal erythropoietin (EPO); subject 2 (S2) is a female who presented with normal Hb, RBCs count, slightly high WBCs and high platelets; subject 3 (S3) is a male who presented with high Hb, normal WBCs and EPO 3 family members, subject 4 -a mother presented with normal Hb level, sustained high RBCs count and slightly high WBCs count, subject 5 a daughter presented with unexplained sustained erythrocytosis, leukocytosis (absolute neutrophilia, for more than 2 years Chronic headache, normal MRI brain and no progression to cancer no Ischemic events, subject 6- healthy daughter with normal CBC indicesThe pattern of mutations identified was unique. In this study 10 novel mutations in known genes already implicated in the pathogenesis of MPNs were detected including THPO, CBL, ASXL1, MPL (2x), BCL2, MYO18B, MTOR, PDGFRB, TERT. Mutations/variants were detected in 9 genes not previously associated with MPNs suggesting potential novel gene associations. Pathogenic/likely pathogenic mutations, uncertain variants and neutral/likely neutral variants were identified using the CES approach. A complex combination of mutations was observed in our cohort of subjects. For example in subject 1 (S1), an ET patient, the following mutations were observed: THPO S184R, ITGA2 R76Q, JAK2 V617F, CBL E196G, CBFB G180S, FANCA S858R, ASXL1 T600P fs103. The following detailed analysis of each mutation reveals that all these mutations could potentially play a role in the pathogenesis of MPNs Disclosures No relevant conflicts of interest to declare.

Germline duplication of ATG2B and GSKIP predisposes to familial myeloid malignancies.

No major predisposition gene for familial myeloproliferative neoplasms (MPN) has been identified. Here we demonstrate that the autosomal dominant transmission of a 700-kb duplication in four genetically related families predisposes to myeloid malignancies, including MPN, frequently progressing to leukemia. Using induced pluripotent stem cells and primary cells, we demonstrate that overexpression of ATG2B and GSKIP enhances hematopoietic progenitor differentiation, including of megakaryocytes, by increasing progenitor sensitivity to thrombopoietin (TPO). ATG2B and GSKIP cooperate with acquired JAK2, MPL and CALR mutations during MPN development. Thus, the germline duplication may change the fitness of cells harboring signaling pathway mutations and increases the probability of disease development.

The Drosophila retinoblastoma binding protein 6 family member has two isoforms and is potentially involved in embryonic patterning.

The human retinoblastoma binding protein 6 (RBBP6) is implicated in esophageal, lung, hepatocellular and colon cancers. Furthermore, RBBP6 was identified as a strong marker for colon cancer prognosis and as a predisposing factor in familial myeloproliferative neoplasms. Functionally, the mammalian protein interacts with p53 and enhances the activity of Mdm2, the prototypical negative regulator of p53. However, since RBBP6 (known as PACT in mice) exists in multiple isoforms and pact−/− mice exhibit a more severe phenotype than mdm2−/− mutants, it must possess some Mdm2-independent functions. The function of the invertebrate homologue is poorly understood. This is complicated by the absence of the Mdm2 gene in both Drosophila and Caenorhabditis elegans. We have experimentally identified the promoter region of Snama, the Drosophila homologue, analyzed potential transcription factor binding sites and confirmed the existence of an additional isoform. Using band shift and co-immunoprecipitation assays combined with mass spectrometry, we found evidence that this gene may be regulated by, amongst others, DREF, which regulates hundreds of genes related to cell proliferation. The potential transcription factors for Snama fall into distinct functional groups, including anteroposterior embryonic patterning and nucleic acid metabolism. Significantly, previous work in mice shows that pact−/− induces an anteroposterior phenotype in embryos when rescued by simultaneous deletion of p53. Taken together, these observations indicate the significance of RBBP6 proteins in carcinogenesis and in developmental defects.

Predisposition to Myeloproliferative Neoplasms

Familial forms of myeloproliferative neoplasms (MPN) and genetic contribution to sporadic cases of MPN have long been recognized. In the majority of cases, familial MPN is inherited as an autosomal dominant trait. The penetrance varies from around 20% to up to 100% in some pedigrees. We can distinguish two types of familial MPN. Type 1 has high penetrance, polyclonal hematopoiesis and hyperproliferation of a single hematopoietic lineage, caused by mutations in a single gene and usually manifesting at birth or early childhood. Examples are mutations in the genes for the erythropoietin receptor, thrombopoietin or its receptor, MPL. The type 2 familial MPNs are characterized by clonal hematopoiesis, low penetrance and manifestation beginning in most cases later in adult life. These type 2 familial MPNs are classical examples of inherited predisposition to a clonal malignant disease, in which acquired somatic mutations in hematopoietic cells are required for disease manifestation. Affected family members typically display the same acquired driver mutations in the genes for JAK2 (JAK2-V617F or JAK2-exon12), MPL (MPL-W515), or calreticulin (CALR) as patients with sporadic MPN. The mutated genes and the mechanism of how these inherited germline mutations predispose to MPN have not yet been elucidated. The search for these germline mutations has been hampered by the low penetrance of MPN manifestation and the rare occurrence of pedigrees that are large enough for genetic studies. Furthermore, the few candidate gene mutations that have been identified to date do not map to one gene locus and the function of the candidate genes does not fall into a common category. Two models of how the germline predisposition interacts with acquired driver mutations can be considered. First, the germline mutation may increase the mutation rate for gene mutations in JAK2, MPL, and CALR. Second, the germline mutation functionally synergizes with mutations in JAK2, MPL, and CALR and promotes disease initiation. The current state of our studies and studies in other laboratories will be discussed. Disclosures Skoda: Novartis: Consultancy; Sanofi: Consultancy.