Germline Haplotypes Research Articles

Generative haplotype prediction outperforms statistical methods for small variant detection in next-generation sequencing data.

Detection of germline variants in next-generation sequencing data is an essential component of modern genomics analysis. Variant detection tools typically rely on statistical algorithms such as de Bruijn graphs or Hidden Markov models, and are often coupled with heuristic techniques and thresholds to maximize accuracy. Despite significant progress in recent years, current methods still generate thousands of false-positive detections in a typical human whole genome, creating a significant manual review burden. We introduce a new approach that replaces the handcrafted statistical techniques of previous methods with a single deep generative model. Using a standard transformer-based encoder and double-decoder architecture, our model learns to construct diploid germline haplotypes in a generative fashion identical to modern large language models. We train our model on 37 whole genome sequences from Genome-in-a-Bottle samples, and demonstrate that our method learns to produce accurate haplotypes with correct phase and genotype for all classes of small variants. We compare our method, called Jenever, to FreeBayes, GATK HaplotypeCaller, Clair3, and DeepVariant, and demonstrate that our method has superior overall accuracy compared to other methods. At F1-maximizing quality thresholds, our model delivers the highest sensitivity, precision, and the fewest genotyping errors for insertion and deletion variants. For single nucleotide variants, our model demonstrates the highest sensitivity but at somewhat lower precision, and achieves the highest overall F1 score among all callers we tested. Jenever is implemented as a python-based command line tool. Source code is available at https://github.com/ARUP-NGS/jenever/.

Abstract 7407: Melanoma clonal subline analysis reveals genetic factors driving intra-tumor heterogeneity

Abstract Melanoma is the most invasive skin cancer caused by the malignant melanocytes. The use of immune checkpoint blockade (ICB) improved the survival rate in advanced melanoma. Yet, the response rate to ICB varies across patients due to the highly heterogeneous nature of melanoma. Recent studies reported genomic and epigenetic factors contributing to the therapeutic response. Identifying these factors involved in clonal evolution in melanoma is a key to better understanding the tumor progression and divergence in the therapy response. To study melanoma heterogeneity, we generated twenty-four clonal sublines, each derived from a single cell isolated from a parental cell line derived from the M4 mouse melanoma model. DNA prepared from each subline and a spleen from a healthy mouse were subjected to long-read sequencing. Importantly, long-read sequencing allows direct detection of methylation states, small variants, and structural variants in the same run. The results thus provide excellent means to study genetic and epigenetic factors in clonal evolution. Each of these sublines was also implanted in distinct mice for survival and tumor growth analysis and further functional evaluation. We developed Severus, a structural variation (SV) caller for long reads, which works with single (germline), paired (tumor-normal), and multiple samples (e.g., multi-site or time series). Severus takes advantage of improved phasing in long-read sequencing, attributes somatic variants to germline haplotype, and builds a haplotype-specific breakpoint graph that is used to cluster multi-break rearrangements and represent the derived chromosomal structure. We benchmarked Severus using multiplatform validated COLO829 truthset and CHM1/CHM13 haploid genomes, and Severus outperformed all other methods in terms of recall and precision. The structural variation (SV) analysis using Severus with all the sublines showed enrichment of possibly clonal SVs in chr4, chr11, and mainly in chr13. Further investigation revealed SVs in chr13 were not distributed across the chromosome but clustered in certain regions. Analysis of somatic SNVs in chr13 corroborates the localized hypermutation profile, which indicates a possible kataegis. Interestingly, a loss of the chr13 copy affected by kataegis in one of the sublines coincides with a better survival rate, as compared to other sublines, in an allograft melanoma mouse model, which further supports the association between the kataegis and tumor progression. We also identified other clonal and subclonal events, including the homozygous deletion of Cdkn2a in most of the sublines, associated with their aggressiveness in vivo. Our analysis allowed us to identify clonal and subclonal genetic and epigenetic factors involved in tumor progression and evolution in a melanoma model, which can potentially translate to human disease. Citation Format: Ayse Keskus, Anton Goretsky, Yuelin Liu, Xiwen Cui, Tanveer Ahmad, Eva Perez Guijarro, Asher Bryant, Erin Malloy, Salem Malikic, Glenn Merlino, Chi-Ping Day, Cenk Sahinalp, Mikhail Kolmogorov. Melanoma clonal subline analysis reveals genetic factors driving intra-tumor heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7407.

Haplotype-resolved germline and somatic alterations in renal medullary carcinomas

BackgroundRenal medullary carcinomas (RMCs) are rare kidney cancers that occur in adolescents and young adults of African ancestry. Although RMC is associated with the sickle cell trait and somatic loss of the tumor suppressor, SMARCB1, the ancestral origins of RMC remain unknown. Further, characterization of structural variants (SVs) involving SMARCB1 in RMC remains limited.MethodsWe used linked-read genome sequencing to reconstruct germline and somatic haplotypes in 15 unrelated patients with RMC registered on the Children’s Oncology Group (COG) AREN03B2 study between 2006 and 2017 or from our prior study. We performed fine-mapping of the HBB locus and assessed the germline for cancer predisposition genes. Subsequently, we assessed the tumor samples for mutations outside of SMARCB1 and integrated RNA sequencing to interrogate the structural variants at the SMARCB1 locus.ResultsWe find that the haplotype of the sickle cell mutation in patients with RMC originated from three geographical regions in Africa. In addition, fine-mapping of the HBB locus identified the sickle cell mutation as the sole candidate variant. We further identify that the SMARCB1 structural variants are characterized by blunt or 1-bp homology events.ConclusionsOur findings suggest that RMC does not arise from a single founder population and that the HbS allele is a strong candidate germline allele which confers risk for RMC. Furthermore, we find that the SVs that disrupt SMARCB1 function are likely repaired by non-homologous end-joining. These findings highlight how haplotype-based analyses using linked-read genome sequencing can be applied to identify potential risk variants in small and rare disease cohorts and provide nucleotide resolution to structural variants.

A unified haplotype-based method for accurate and comprehensive variant calling.

Almost all haplotype-based variant callers were designed specifically for detecting common germline variation in diploid populations, and give suboptimal results in other scenarios. Here we present Octopus, a variant caller that uses a polymorphic Bayesian genotyping model capable of modeling sequencing data from a range of experimental designs within a unified haplotype-aware framework. Octopus combines sequencing reads and prior information to phase-called genotypes of arbitrary ploidy, including those with somatic mutations. We show that Octopus accurately calls germline variants in individuals, including single nucleotide variants, indels and small complex replacements such as microinversions. Using a synthetic tumor data set derived from clean sequencing data from a sample with known germline haplotypes and observed mutations in a large cohort of tumor samples, we show that Octopus is more sensitive to low-frequency somatic variation, yet calls considerably fewer false positives than other methods. Octopus also outputs realigned evidence BAM files to aid validation and interpretation.

Next generation sequencing of BRCA genes in glioblastoma multiform Egyptian patients: a pilot study

Background Germ line mutations of BRCA1 and BRCA2 were correlated with a variety of cancer Authors aimed to use next-generation sequencing (NGS) to detect BRCA1 and BRCA2 germ line mutations in glioblastoma multiform (GBM) Egyptian patients. Materials and methods Genomic DNA was extracted from six GBM cases, amplified using Ion AmpliSeq BRCA1 and BRCA2 panel. DNA libraries were pooled, barcoded and finally sequenced using Ion Torrent Personal Genome Machine sequencer. Results BRCA1 the previously reported rs1799966, rs1799950, rs16941 were found in five cases and they are in a linkage disequilibrium forming two distinct haplotypes, which might support their role in cancer predisposition. Out of the 18 reported variants in BRCA2, three denovo mutations were detected which leads to frame shift. Conclusion Further studies on large number of GBM patients and control cases to determine BRCA1 and BRCA2 germline mutations and haplotypes; diagnostic and prognostic role are encouraged.

Genome sequencing analysis of blood cells identifies germline haplotypes strongly associated with drug resistance in osteosarcoma patients

BackgroundOsteosarcoma is the most common malignant bone tumor in children. Survival remains poor among histologically poor responders, and there is a need to identify them at diagnosis to avoid delivering ineffective therapy. Genetic variation contributes to a wide range of response and toxicity related to chemotherapy. The aim of this study is to use sequencing of blood cells to identify germline haplotypes strongly associated with drug resistance in osteosarcoma patients.MethodsWe used sequencing data from two patient datasets, from Inova Hospital and the NCI TARGET. We explored the effect of mutation hotspots, in the form of haplotypes, associated with relapse outcome. We then mapped the single nucleotide polymorphisms (SNPs) in these haplotypes to genes and pathways. We also performed a targeted analysis of mutations in Drug Metabolizing Enzymes and Transporter (DMET) genes associated with tumor necrosis and survival.ResultsWe found intronic and intergenic hotspot regions from 26 genes common to both the TARGET and INOVA datasets significantly associated with relapse outcome. Among significant results were mutations in genes belonging to AKR enzyme family, cell-cell adhesion biological process and the PI3K pathways; as well as variants in SLC22 family associated with both tumor necrosis and overall survival. The SNPs from our results were confirmed using Sanger sequencing. Our results included known as well as novel SNPs and haplotypes in genes associated with drug resistance.ConclusionWe show that combining next generation sequencing data from multiple datasets and defined clinical data can better identify relevant pathway associations and clinically actionable variants, as well as provide insights into drug response mechanisms.

Common germline haplotypes and genotypes identified in BRCA2 exon 11 of dogs with mammary tumours and histopathological analyses.

The canine BRCA2 is a tumor supressor gene which encodes the BRCA2 protein, involved in DNA repair through interaction with the RAD51 recombinase. This process is mediated by eigth BRC repeats that are encoded by BRCA2 exon 11. Two variants corresponding to human mutations in human BRC3 repeat have been reported in canine BRC3 repeat. In addition, other variants have also been described in canine BRCA2 exon 11. Considering the importance of polymorphisms in human BRCA2 to breast cancer development, this study aimed to investigate the frequency of variants in BRCA2 exon 11 in 48 blood and tissue DNA samples from bitches with canine mammary tumors (CMT), as well as, to analyze tumor stage and histopathological features. Seven Single Nucleotide Polymorphisms (SNPs) were identified, three of which were evaluated as possibily or probably deleterious variant. Interestingly, almost all the 22 mammary tumors (except one) which presented a clinical staging equal to or greater than III carried at least one mutant allele of these three variants. Besides that, no statistically significant correlation was observed between any of the reported SNPs in heterozygosis or homozygosis and either dogs data (such as breed, age or disease stage) or mammary tumors histopathological characteristics. A total of 97.9% of bitches had one to three polymorphisms of the seven identified in this study, which suggests a possibly correlation between the canine BRCA2 exon 11 polymorphisms and mammary carcinogenesis.

Abstract 5320: Novel computational methods reveal subtle clonal mosaicism

Abstract Recent genome-wide surveys have revealed age-related genetic mosaicism and established a link between clonal mosaicism and risk of hematological and solid-tumor cancers (Laurie et al, 2012, Nat Genet, 44:642; Jacobs et al, 2012, Nat Genet, 44:651; Forsberg et al, 2012, AJHG, 90:217). Existing methods to identify chromosomal aberrations leading to mosaic allelic imbalance (AI) rely on the detection of increased variance of the estimated within-sample allele frequencies at heterozygous loci, ignoring the dependence among allele frequencies in regions of an AI-inducing event, such as a deletion or copy-neutral loss of heterozygosity (LOH), expected from the loss or gain of a chunk of an inherited chromosome. As a result, they break down when the proportion of DNA from an aberrant source is below 10-15%. Here we present a suite of new methods (hapLOH, J-LOH, hapLOHseq) that harness the important information contained in the germline haplotypes to help distinguish signal from noise, accommodate multiple samples of different tumor purities (to deconvolute aberration type and tumor purity) and infer co-occurrence of aberrations in heterogeneous mixtures. For example, our initial method, hapLOH (Vattathil & Scheet, 2013, Gen Res 23:152), can identify 10 Mb regions with just 4% of the signal coming from the tumor genome in data simulated from a series of experiments from a 317K SNP microarray. Application of hapLOH to data analyzed by Laurie et al (2012) indicates that we have substantially greater sensitivity to detect clonal mosaicism, particularly events harbored in a small proportion of cells (< 5-10%). We identify more than twice as many mosaic events, demonstrating the potential role for our methods to aid in profiling cancer risk. Our suite of tools is designed for both microarray and next-generation sequencing data and is available at scheet.org. Citation Format: Paul Scheet, Rui Xia, F. Anthony San Lucas, Christina Hahn, Jerry Fowler, Selina Vattathil. Novel computational methods reveal subtle clonal mosaicism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5320. doi:10.1158/1538-7445.AM2014-5320

Identification of allelic imbalance with a statistical model for subtle genomic mosaicism.

Genetic heterogeneity in a mixed sample of tumor and normal DNA can confound characterization of the tumor genome. Numerous computational methods have been proposed to detect aberrations in DNA samples from tumor and normal tissue mixtures. Most of these require tumor purities to be at least 10–15%. Here, we present a statistical model to capture information, contained in the individual's germline haplotypes, about expected patterns in the B allele frequencies from SNP microarrays while fully modeling their magnitude, the first such model for SNP microarray data. Our model consists of a pair of hidden Markov models—one for the germline and one for the tumor genome—which, conditional on the observed array data and patterns of population haplotype variation, have a dependence structure induced by the relative imbalance of an individual's inherited haplotypes. Together, these hidden Markov models offer a powerful approach for dealing with mixtures of DNA where the main component represents the germline, thus suggesting natural applications for the characterization of primary clones when stromal contamination is extremely high, and for identifying lesions in rare subclones of a tumor when tumor purity is sufficient to characterize the primary lesions. Our joint model for germline haplotypes and acquired DNA aberration is flexible, allowing a large number of chromosomal alterations, including balanced and imbalanced losses and gains, copy-neutral loss-of-heterozygosity (LOH) and tetraploidy. We found our model (which we term J-LOH) to be superior for localizing rare aberrations in a simulated 3% mixture sample. More generally, our model provides a framework for full integration of the germline and tumor genomes to deal more effectively with missing or uncertain features, and thus extract maximal information from difficult scenarios where existing methods fail.

Prevalence Of The JAK2V617F Mutation and Associated Risk Haplotype and Determination Of Demographic and Lifestyle Risk Factors In The US Population, Nhanes 1999-2002

BackgroundIn 2008, the WHO released new diagnostic criteria for polycythemia vera (PV), which included testing for the JAK2V617F mutation. The JAK2V617F mutation is associated with PV and other myeloproliferative neoplasms (MPN) and is present in approximately 97% of all PV cases. A germ-line haplotype, referred to as 46/1, has been reported to be associated with acquisition of JAK2V617F. The exact hierarchical position of JAK2V617F and the risk haplotype in the pathogenesis and phenotypic variability of the MPNs remains unclear. Since the current literature on the prevalence of the JAK2V617F mutation in non-MPN populations is limited, the objective of our study was to determine the JAK2V617F prevalence and the 46/1 risk haplotype in the general, non-institutionalized US population. We evaluated demographic information, lifestyle factors, and body burden levels of environmental contaminants to determine if they correlated with JAK2-status. MethodsThe NHANES surveys are conducted by the National Center for Health Statistics (NCHS) at the Centers for Disease Control and Prevention (CDC). In order to obtain a representative sample of the civilian non-institutionalized US population, the surveys consisted of a complex multistage sampling design including questionnaire and examination components, as well as the use of blood specimens for a wide variety of analytical purposes. Consent for use of blood specimens for genetic research purposes is permitted in participants aged 20 years and greater. DNA was collected from 7,839 participants (NHANES, 1999-2002). To detect JAK2V617F, real-time quantitative polymerase chain reaction (qPCR) was used for screening. All JAK2V617F(+) samples were confirmed by an independent laboratory. Two tagged single-nucleotide polymorphism (SNPs), rs12340895 and rs12343867, were genotyped using allelic discrimination. The risk haplotype was empirically determined in JAK2V617F(+) specimens using mutation specific amplification followed by single allele sequence based typing. The mutation and risk haplotype prevalence was determined and frequencies calculated using race/ethnicity, age, and sex as variables. ResultsThe JAK2V617F mutation prevalence was 0.62% in the US general population. Age was the only demographic factor that was significantly associated with the JAK2V617F mutation (p-value = 0.0015). Of the 65 participants who tested positive for JAK2V617F, 10% were between the ages 20-39, 32% were 40-59, and 58% were 60 years and older. Diagnosis of a blood cancer, leukemia, or other related cancer was not reported in any of the JAK2V617F(+) participants. However, PV and other MPNs were not designated as reportable cancers until 2001, and the questionnaire was administered in 1999-2002. After adjusting for race/ethnicity and age, exposure to environmental contaminants was not found to be associated with the JAK2V617F mutation. The haplotype tagging SNP analysis showed a significant association with JAK2V617F(+) participants versus the JAK2V617F (-) participants. However, when the haplotype was empirically determined with single allele sequencing of the informative heterozygous JAK2V617F(+) participants (N=40), the mutation was detected on the 46/1 haplotype in only 55% of the JAK2V617F(+) chromosomes, which was lower than previously reported in MPN populations. The JAK2V617F mutation was also detected on the common non-risk haplotype (T-C) in 25% of the chromosomes and on less common haplotypes (C-C, T-G) in 21% of the chromosomes. ConclusionsThis is the only study to examine the prevalence of JAK2V617F in the US general population. When analyzing a large population-based data set, the analysis showed that the JAK2V617F mutation is present in persons who did not report having an MPN. The incidence of the JAK2V617F was directly correlated with age, and was consistent with the known age of onset of MPNs. Whether this group of individuals has a pre-clinical form of an MPN or the detection of JAKV617F is a transient phenomenon remains unknown. The frequency of the two haplotype-tagging SNPs in the normal population was similar to that previously reported. The risk haplotype however, was not more commonly seen associated with JAK2V617F, indicating that the JAK2V617F may not arise preferentially on the 46/1 risk haplotype. These studies provide important epidemiological information about the occurrence of JAK2V617F in “normal” individuals. Disclosures:No relevant conflicts of interest to declare.

A Germline Variant in the TERT Gene Is a Novel Predisposition Allele Associated with Myeloproliferative Neoplasms

AbstractAbstract 707 Background:Myeloproliferative neoplasms (MPNs) are disorders that result in unregulated overproduction of one or more myeloid blood cell types by the bone marrow. Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) comprise the three classic MPNs. The somatic JAK2 V617F mutation is present in 95% of PV patients, and 50–60% of ET and PMF patients. Past work has identified a germline haplotype in JAK2 associated with risk of developing a V617F-positive MPN (Kilpivaara et al; Olcaydu et al; Jones et al, Nat. Genet., 2009). Methods:We recruited a web-based participatory cohort of individuals with MPNs to better understand the genetic basis of these conditions. Participation in this MPN research initiative included free genotyping, and participants provided informed consent and responses to surveys online, under a protocol approved by the external AAHRPP-accredited IRB, Ethical & Independent Review Services. We enrolled and collected saliva samples from more than 700 participants with the following self-reported diagnoses: systemic mastocytosis (n=142), ET (n=145), PV (n=137), PMF (n=50), chronic myelogenous leukemia (n=108), and 97 with overlapping diagnoses (e.g. ET or PV with PMF). Participants were genotyped using a derivative of the Illumina HumanOmniExpress BeadChip with additional custom content, including probes for the JAK2 V617F variant. We performed a genome-wide association study (GWAS) of classic MPN diagnoses (at least one of ET, PV, or MF), using 407 cases from this MPN research initiative and 94,037 controls drawn from the broader 23andMe research participant community. Association was assessed by logistic regression adjusted for age, gender, and 5 principal components to model ancestry. Results:We replicated the known association between germline variation in JAK2 and classic MPNs (rs12340895: odds ratio=2.5, P=7.4 × 10−35). We also identified a novel genome-wide-significant association in TERT, or telomerase reverse transcriptase (rs2853677: odds ratio=1.6 per G allele, P=6.3 × 10−11). The association is consistent with an additive model on the logistic scale (odds ratio=2.6 for 2 versus 0 G alleles). Another variant in TERT, rs2736100, has previously been associated with several non-hematological cancers and with red blood cell count, and our lead SNP is in linkage disequilibrium with TERT variant rs2736100 (r2=0.54). While no other loci reached the threshold of genome-wide significance (P<5.0 × 10−8), we saw a suggestive association with a germline missense variant (F858L) in ATM, or ataxia telangiectasia mutated gene (rs1800056: odds ratio=2.6 per C allele, P=1.4 × 10−5). This variant has also been reported to be associated with breast cancer and chronic lymphocytic leukemia.The rs2853677 TERT variant was associated with each of ET, PV, and PMF, with similar effect sizes. It was not associated with JAK2 V617F mutation status within the MPN cohort (odds ratio=1.0, P=1.0), while the germline JAK2 variant was preferentially associated with V617F-positive MPNs (odds ratio=2.1, P=3.0 × 10−6). The high-risk allele of rs2853677 was also predictive of V617F status among 23andMe participants who had not been recruited into the MPN community (odds ratio=1.6, P=0.0012). These results suggest that while variation in TERT facilitates either acquisition or proliferation of V617F-positive myeloid cells, it also predisposes to MPNs through a V617F-independent mechanism. Conclusion:We have identified a germline variant in TERT which is a new predisposition allele for classic MPNs. These results demonstrate the potential for web-based recruitment methods to contribute to genetic research for uncommon diseases. Disclosures:Hinds:23andMe: Employment, Equity Ownership, Patents & Royalties. Barnholt:23andMe, Inc.: Employment. Zehnder:23andMe, Inc.: Unpaid advisor and collaborator Other. Kiefer:23andMe, Inc.: Employment. Do:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Eriksson:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Mountain:23andMe, Inc.: Consultancy, Employment, Honoraria, Patents & Royalties, Research Funding. Francke:23andMe, Inc.: Employment, Honoraria, Research Funding; Locus Development: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Tung:23andMe, Inc.: Employment. Mesa:23andMe, Inc: Unpaid advisor and collaborator Other. Gotlib:23andMe, Inc.: Unpaid advisor and collaborator Other.

Haplotype-based profiling of subtle allelic imbalance with SNP arrays.

Due to limitations of surgical dissection and tumor heterogeneity, tumor samples collected for cancer genomics studies are often heavily diluted with normal tissue or contain subpopulations of cells harboring important aberrations. Methods for profiling tumor-associated allelic imbalance in such scenarios break down at aberrant cell proportions of 10%–15% and below. Here, we present an approach that offers a vast improvement for detection of subtle allelic imbalance, or low proportions of cells harboring aberrant allelic ratio among nonaberrant cells, in unpaired tumor samples using SNP microarrays. We leverage the expected pattern of allele-specific intensity ratios determined by an individual's germline haplotypes, information that has been ignored in existing approaches. We demonstrate our method on real and simulated data from the CRL-2324 breast cancer cell line genotyped on the Illumina 370K array. Assuming a 5 million SNP array, we can detect the presence of aberrant cells in proportions lower than 0.25% in the breast cancer sample, approaching the sensitivity of some minimal residual disease assays. Further, we apply a hidden Markov model to identify copy-neutral LOH (loss of heterozygosity) events as short as 11 Mb in mixtures of only 4% tumor using 370K data. We anticipate our approach will offer a new paradigm for genomic profiling of heterogeneous samples.

Genetic and Functional Investigation of Germline JAK2 Alleles That Predispose to Myeloproliferative Neoplasms

Abstract 124A somatic activating mutation in the pseudokinase domain of JAK2 (JAK2V617F) is found in the majority of patients with myeloproliferative neoplams (MPN). Using a genome-wide approach, we and others identified a germline haplotype in the JAK2 locus (rs10974944) that predisposes to the development of JAK2V617F-positive MPN. Importantly, this haplotype is associated with in cis acquisition of the somatic JAK2 mutation. An extended linkage disequilibrium block of 300kb is observed at this locus and others have reported an association between single nucleotide polymorphisms (SNPs) within this haplotype and risk of inflammatory bowel disease consistent with increased JAK-STAT signaling in patients who carry this risk haplotype. The mechanism by which this germline locus contributes to MPN pathogenesis has not been delineated. We hypothesized that the identified allele heightens the risk of developing MPN by either a) increasing the mutational rate at the JAK2 locus, or b) imparting a selective advantage on cells that acquire the somatic mutation through increased JAK2 expression.To address the mutational hypothesis, we performed targeted, high coverage, next-generation sequencing of the entire haplotype and of the entire JAK2 locus in 12 patients homozygous for the risk allele, and in 12 patients without the risk allele. Importantly we did not note an increased rate of somatic mutations (coding or noncoding) in patients homozygous for the risk haplotype. In addition, we expanded our GWAS to include 200 additional cases genotyped using the Illumina 1,000,000 SNP genotyping array. The number of SNPs did not significantly differ between the risk haplotype and non-risk haplotype, further suggesting that there is no increase in mutability attributable to the risk genotype. By constructing a phylogenetic tree, we found that the risk haplotype is ancestral to modern humans and demonstrates evidence of ancestral positive selection, although there was no evidence of recent selection at this locus. Taken together these data suggest that the JAK2 MPN risk hapolotype does not increase the mutational rate at this locus.We next investigated whether the risk allele affects JAK2 expression in hematopoietic cells. We compared the relative abundance of an exonic SNP within the haplotype using matched genomic DNA and cDNA from 8 MPN patients heterozygous for the risk allele. In each case we found that the risk allele was more highly expressed in cDNA compared to the non-risk allele despite similar allelic ratios in genomic DNA. The results suggest an increase in allele-specific expression of JAK2 associated with the JAK2 risk haplotype. We annotated all germline variants in cis with the JAK2 risk haplotype using next generation sequencing data of the entire JAK2 haplotype from MPN patients and from the 1000 Genomes project. We then used Encode ChIP-seq data and the ConSite web-based transcription factor binding prediction model to identify SNPs within the JAK2 haplotype that affect transcription factor binding. We identified a SNP within the JAK2 promoter region, rs1887428, as a potential causative allele because it is significantly associated with MPN (p=9.11E-11) and c-Fos/c-Jun is predicted to preferentially bind to the risk allele. In order to determine if this preferential transcription factor binding leads to a haplotype-specific increase in expression of JAK2, we performed luciferase assays in cells expressing reporter constructs with the two different alleles at rs1887428. Importantly, this demonstrated increased transcriptional activity in cells containing the risk allele at rs1887428, suggesting that enhanced transcription factor binding at rs1887428 may lead to increased JAK2 expression and confer a selective advantage on cells containing the risk haplotype. The effects of allelic variation at rs1887428 on JAK2 expression in hematopoietic cells will be presented.Taken together, our data suggests that the JAK2 MPN risk haplotype contributes to MPN pathogenesis through allele-specific transcription factor binding and JAK2 expression, which increases the selective advantage of JAK2 mutations arising on the risk haplotype. This study provides insight into how predisposing loci increase the predisposition to MPN and to other hematopoietic malignancies. Disclosures:No relevant conflicts of interest to declare.

Advances in understanding and management of polycythemia vera

Polycythemia vera is a relatively common myeloproliferative neoplasm (MPN) molecularly defined by the presence of mutations in the janus kinase (JAK2) gene. Yet, many aspects of pathogenesis remain to be ascertained and no effective treatment for curing the disease or preventing major cardiovascular events and progression to myelofibrosis or acute leukemia exists. The objective of this report is to review recent advances in the field and discuss the potential of novel therapeutic approaches. The discovery of a specific germline haplotype in JAK2 provided an explanation for the well known phenomenon of familial clustering of MPN. Clinical trials with JAK2 inhibitors, either specific or not, have been initiated and first results are available. However, the expectation that these drugs could selectively target mutant cells and cause a molecular remission, similar to the experience with imatinib in chronic myelogenous leukemia, has been largely unmet. With the aim to develop common tools for clinicians involved in conventional and experimental therapies, a set of criteria for evaluating response to treatment have been developed. The last couple of years have witnessed significant improvements in understanding polycythemia vera biology and management, and the activation or completion of novel trials are expected to provide further information to improve the treatment.

The myeloproliferative neoplasm-associated JAK2 46/1 haplotype is not overrepresented in chronic myelogenous leukemia

Dear Editor, Philadelphia-chromosome negative chronic myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis [1]. In 2005, a point mutation in JAK2 (JAK2V617F) was shown to occur in almost all patients with PV and in ≈60% of ET or PMF [2]. A genetic predisposition to developing MPNs has been long suggested by finding clusters of MPNs in kindreds as well as by recent results of a large registry-based epidemiological study performed in Sweden. In this analysis, almost 25,000 relatives of 11,039 patients with MPNs were evaluated. There was a significantly increased relative risk (RR) of developing MPN among first-degree relatives in the order of 5.7, 7.4, and 7.5 for PV, ET, and not otherwise specified MPN, respectively, compatible with a model of autosomal recessive inheritance [2]. While an increased RR for PMF could not be demonstrated, possibly because of the relatively low number of cases observed, an almost statistically significantly higher RR of developing Ph’-positive chronic myelogenous leukemia (CML) was found (RR 1.9, 95% confidence interval 0.9–3.8, P=0.09). Recently, the genetic basis for predisposition to MPNs has been partially clarified by the discovery that the JAK2V617F mutation is acquired preferentially on a specific constitutional JAK2 haplotype. This haplotype, which is called 46/1 or GGCC, is a common, very low penetrance predisposition allele estimated to account for 50% of the population-attributable risk of developing a MPN. In three independent studies, 46/1 has been identified as a germline haplotype block that is strongly associated (3 to 4 odds ratio) with JAK2V617Fmutated MPN [3–5]. Subsequent studies showed association of this haplotype also with JAK2 exon 12-mutated PV [6], JAK2-wild type ET [7], and MPLW515-mutated PMF or ET [8], suggesting that the haplotype increases the risk of developing an MPN regardless of the acquisition of the V617F mutation. However, in another recent study, no such association with MPL mutations was reported [9]. We therefore asked whether the increased risk of developing CML in MPN relatives found in the epidemiological study could be similarly attributed to an increased prevalence of the 46/1 haplotype compared with control subjects. This study reports on two different patients series with Ph’-positive CML collected in Florence (Italy) and in Mannheim (Germany) and analyzed independently using two 46/1 tag SNPs. In the Italian series, the 46/1 tag rs12343867 (with the “C” allele standing for 46/1 haplotype) was analyzed by allelic discrimination using Applied Biosystems SNP genotyping assay; in the German series, the rs12340895 SNP (with “G” allele standing for 46/1 haplotype) was analyzed in Salisbury laboratory by pyrosequencing as described [3]. As control populations, we used 235 healthy volunteers living in Florence for the A. Spolverini : L. Pieri :A. M. Vannucchi (*) UF di Ematologia, Dipartimento di Area Critica Medico-Chirurgica, Universita di Firenze, Istituto Toscano Tumori, Florence, Italy e-mail: amvannucchi@unifi.it

JAK2 V617F and beyond: role of genetics and aberrant signaling in the pathogenesis of myeloproliferative neoplasms

Dysregulated signaling is a hallmark of chronic myeloproliferative neoplasms (MPNs), as evidenced by the identification of the activating JAK2 V617F somatic mutation in almost all patients with polycythemia vera (PV) and 50–60% of essential thrombocythemia and primary myelofibrosis patients. These disorders are clinically distinct, raising the question of how a single mutation can result in such phenotypic diversity. Mouse models have demonstrated that the level of JAK2 V617F expression can modulate the phenotype, and clinical studies of JAK2 V617F allele burden have reported similar findings. It has also been hypothesized that one or more pre-JAK2 V617F events may modify the MPN phenotype. However, the molecular basis of JAK2 V617F-negative essential thrombocythemia and primary myelofibrosis remains largely unexplained. Mutations in the TET2 gene have been identified in both JAK2 V617F-positive and -negative MPNs and other myeloid neoplasms, but their functional and clinical significance have yet to be clarified. In addition, recent reports have identified a specific germline haplotype that increases the predisposition to MPNs. The role of inhibitory pathways (e.g., SOCS and LNK) in regulating JAK–STAT signaling in MPNs is being increasingly recognized. The implications of these findings and their clinical relevance are the focus of this article.

Abstract 89: BamBam: Simultaneous processing of tumor and germline sequencing data

Abstract With the release of multiple fully-sequenced tumor and matched normal genomes from The Cancer Genome Atlas (TCGA), there is great need for tools that can efficiently analyze these enormous datasets. To this end, we developed BamBam, a method that interfaces with the SAMtools library to simultaneously analyze a patient's tumor and germline genomes using short-read alignments stored in SAM/BAM-formatted files. BamBam enables an efficient calculation of regional copy number, loss of heterozygosity (LOH), and the discovery of both somatic and germline sequence variants and structural rearrangements. Chromosomal regions that experience single copy loss or copy neutral LOH are used to phase heterozygous sites and construct germline haplotypes. We used BamBam to analyze the short-read sequencing data of several glioblastoma multiforme and ovarian serous carcinoma tumors produced by the TCGA genome sequencing centers at the Broad Institute and Washington University in St. Louis. In each tumor, BamBam identified somatic homozygous and heterozygous small indels and single nucleotide variants, inferred regions exhibiting large-scale loss of heterozygosity, estimated relative copy number, and identified sites of structural variation. The intra- and inter-chromosomal rearrangements we infer are often found at the boundary of copy number alterations, implicating homology-driven mechanisms. We identified variants shared by more than one tumor as well as events restricted to individual tumors. Strikingly, in each tumor processed thus far, distinct regions of localized chromosomal instability were observed. The inferred haplotypes share substantial regions of identity with the germline haplotypes of the other TCGA samples and phased SNPs from samples of 11 HapMap populations, indicating they have high accuracy. BamBam is a computationally efficient method for surveying large sequencing datasets to produce a set of high-quality genomic events that occur within each tumor relative to its germline. These results provide a glimpse into the chromosomal dynamics of tumors, improving our understanding of these tumors’ final states and the events that led to them. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 89.

A JAK2 Germline Genetic Variation Is Associated with Shortened Survival in Primary Myelofibrosis: Clinical and JAK2V617F Allele Burden Correlates of rs12343867 SNP Genotype in 132 Cases.

Abstract 2895Poster Board II-871 Background:Recent studies have reported a significant association between JAK2V617F acquisition and a specific JAK2 germline haplotype; rs12343867 (located at intron 14 of the JAK2 gene) is one of several SNPs that tag this haplotype. We performed rs12343867 SNP analysis in a consecutive cohort of patients with primary myelofibrosis (PMF) in order to study the clinical and laboratory correlates of the particular JAK2 haplotype and its effect on survival. Methods:Study patients were recruited form the Mayo Clinic database for PMF. Molecular studies were performed on DNA extracted from stored bone marrow. rs12343867 SNP genotyping was performed by a commercially available Taqman assay. Quantitative JAK2V617F analysis was done according to previously published methods. Standard statistical methods were used to test significance of associations between SNP genotype and other variables. Survival analysis was performed by the Kaplan-Meier method and comparisons made by the log-rank test. Cox regression model was used for multivariable analysis. Results:132 patients with PMF were studied (median age 62, range 28-82; 82 males). Risk distribution according to the International Prognostic Scoring System (IPSS) for PMF were low in 39 (29%) patients, intermediate-1 in 41 (31%), intermediate-2 in 25 (20%) and high in 27 (20%). 77 (58%) patients were JAK2V617F positive; median mutant allele burden was 26% (range 1-85) and 21 patients displayed > 50% mutant allele burden.The rs12343867 genotype distributions were C/C 22%, C/T 44% and T/T 34% . The corresponding figures in JAK2V617F positive/negative cases were 31%/9%, 38%/53%, 31%/38% (p=0.01). Among the 21 patients with > 50% JAK2V617F allele burden, 14 (67%) displayed the C/C allele, 2 C/T and 5 T/T. The specific SNP genotype was not significantly affected by age (p=0.33), sex (p=0.46), leukocyte count (p=0.39), platelet count (p=0.09), or IPSS risk category (p=0.63).Patients were followed for a median of 54 months and during this period 73 (55%) deaths were documented. The T/T SNP genotype was significantly associated with shortened survival, compared to either C/C or C/T (p=0.001; Figure). Multivariable analysis showed this association to be independent of IPSS, JAK2V617F mutational status, age or sex. The adverse prognostic effect of the T/T genotype was also apparent when JAK2V617F negative (p=0.01) or positive (p=0.06) cases were analyzed separately. Conversely, JAK2 mutational status or allele burden did not affect survival in patients with T/T or C/C allele. Conclusion:The current study illustrates the non-random haplotype distribution of JAK2V617F in patients with PMF. More importantly, a non-JAK2 haplotype, tagged by the rs12343867 T allele, was associated with inferior survival that is not accounted for by IPSS or JAK2V617F mutational status. These findings, together with previous observations regarding shortened survival associated with low JAK2V617F allele burden, suggest the presence of molecular events in PMF that are more aggressive than JAK2V617F and not necessarily linked to the JAK2 haplotype. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

JAK2 Haplotype SNP Analysis in Essential Thrombocythemia: Clinical and JAK2V617F Allele Burden Correlates in 226 Patients.

Abstract 2893Poster Board II-869 Background:A specific JAK2 germline haplotype has been associated with the occurrence of JAK2V617F in myeloproliferative neoplasms (MPN). Several SNPs, including rs12343867, tag this haplotype. We performed rs12343867 SNP analysis in a consecutive cohort of patients with essential thrombocythemia (ET) in order to study the clinical and JAK2V617F allele burden correlates of the particular SNP genotype. Methods:Study patients were recruited form the Mayo Clinic database for ET. Molecular studies were performed on DNA extracted from stored bone marrow. rs12343867 SNP genotyping was performed by a commercially available Taqman assay. Quantitative JAK2V617F analysis was done according to previously published methods. Standard statistical methods were used to test significance of associations between SNP genotype and other variables. Survival analysis was performed by the Kaplan-Meier method and comparisons made by the log-rank test. Cox regression model was used for multivariable analysis. Results:226 patients with ET were studied (median age 57, range 14-91; 145 females). Median hemoglobin, leukocyte and platelet counts at initial diagnosis were 13.8 g/dL, 9.7 × 109/L and 1000 × 109/L, respectively. 118 patients (52%) were JAK2V617F positive and among them, 90 (76%) displayed JAK2V617F allele burden of < 10% (low allele burden group) and the median (range) mutant allele burden in the remaining 28 patients (higher allele burden group) was 12% (12-68). As expected, JAK2V617F-positive cases, compared to their mutation-negative counterparts, were older (p=0.006) and displayed higher hemoglobin (p=0.0001) and leukocyte (p<0.0001) counts and lower platelet (0.03) count.The rs12343867 genotype distributions were C/C 15%, C/T 53% and T/T 33% (expected frequency in the HapMap-CEU listed control population is ∼ 6%, 46% and 48%, respectively). The corresponding figures in JAK2V617F positive/negative cases were 17%%/11%, 53%/53%, 30%/36% (p=0.29). Unlike the case with JAK2V617F mutational status, rs12343867 SNP genotype did not correlate with age (p=0.69), hemoglobin level (p=0.47), leukocyte count (p=0.25) or platelet count (p=0.33).Patients were followed for a median of 7.4 years and during this period 56 (25%) deaths were documented. rs12343867 SNP genotype did not correlate with survival (Figure). Similarly, survival did not correlate with the presence or absence of JAK2V617F. However, patients with higher JAK2V617F allele burden displayed a shorter survival with borderline significance, during multivariable analysis that included age as a covariate. Conclusion:The current study shows the JAK2 haplotype enrichment in a large series of ET patients. However, the particular phenomenon was not significantly different between JAK2V617F positive and negative cases, which suggests specific haplotype association with the disease rather than the mutation. Consistent with this contention, rs12343867 genotype did not correlate with clinical or laboratory variables that typically associate with JAK2V617F mutational status. Furthermore, unlike the case with primary myelofibrosis, rs12343867 SNP genotype did not correlate with survival, an observation that is not surprising considering the lower level of complexity in molecular pathogenesis in ET compared to PMF. [Display omitted] Disclosures:Pardanani:TargeGen: Research Funding; Cytopia: Research Funding. Off Label Use: Data from ongoing clinical trial will be presented. Tefferi:TargeGen: Research Funding.

JAK2 germline genetic variation affects disease susceptibility in primary myelofibrosis regardless of V617F mutational status: nullizygosity for the JAK2 46/1 haplotype is associated with inferior survival

A common JAK2 germline haplotype (46/1) has been associated with JAK2V617F (VF)-positive myeloproliferative neoplasms. The rs12343867 SNP (C/T) tags this haplotype. A total of 130 patients (77 VF-positive) with primary myelofibrosis (PMF) were analyzed for this informative SNP, using bone marrow-derived DNA. The observed 46/1 C allele frequencies in VF-positive (50%) and VF-negative (36%) patients were both significantly higher than expected in population controls (P<0.01). Genotype distributions in VF-positive/VF-negative patients were CC 31%/9%, CT 38%/53% and TT 31%/38% (P=0.01). CC genotype/C-allele frequencies in patients with <20% VF mutation burden (12%/37%) were similar (P=0.95) to those seen in VF-negative patients (9%/36%), but were significantly lower (P<0.01) than those seen in the presence of >50% mutation burden ( approximately 67%/71%). The rs12343867 genotype did not correlate with the International Prognostic Scoring System (IPSS) score or karyotype. Unexpectedly, the TT genotype was associated with shortened survival (P<0.01), which was not accounted for by IPSS score or VF allele burden. We conclude that JAK2 germline genetic variation affects disease susceptibility, and possibly survival, in PMF, regardless of VF mutational status. Allelic distortion from acquired uniparental disomy contributes to the appearance of a more pronounced effect on disease susceptibility in VF-positive patients, when studying clonally affected tissue.