DNA-binding specificities of human transcription factors.

Although the proteins that read the gene regulatory code, transcription factors (TFs), have been largely identified, it is not well known which sequences TFs can recognize. We have analyzed the sequence-specific binding of human TFs using high-throughput SELEX and ChIP sequencing. A total of 830 binding profiles were obtained, describing 239 distinctly different binding specificities. The models represent the majority of human TFs, approximately doubling the coverage compared to existing systematic studies. Our results reveal additional specificity determinants for a large number of factors for which a partial specificity was known, including a commonly observed A- or T-rich stretch that flanks the core motifs. Global analysis of the data revealed that homodimer orientation and spacing preferences, and base-stacking interactions, have a larger role in TF-DNA binding than previously appreciated. We further describe a binding model incorporating these features that is required to understand binding of TFs to DNA.

Deficient H2A.Z deposition is associated with genesis of uterine leiomyoma.

One in four women suffers from uterine leiomyomas (ULs)-benign tumours of the uterine wall, also known as uterine fibroids-at some point in premenopausal life. ULs can cause excessive bleeding, pain and infertility1, and are a common cause of hysterectomy2. They emerge through at least three distinct genetic drivers: mutations in MED12 or FH, or genomic rearrangement of HMGA23. Here we created genome-wide datasets, using DNA, RNA, assay for transposase-accessible chromatin (ATAC), chromatin immunoprecipitation (ChIP) and HiC chromatin immunoprecipitation (HiChIP) sequencing of primary tissues to profoundly understand the genesis of UL. We identified somatic mutations in genes encoding six members of the SRCAP histone-loading complex4, and found that germline mutations in the SRCAP members YEATS4 and ZNHIT1 predispose women to UL. Tumours bearing these mutations showed defective deposition of the histone variant H2A.Z. In ULs, H2A.Z occupancy correlated positively with chromatin accessibility and gene expression, and negatively with DNA methylation, but these correlations were weak in tumours bearing SRCAP complex mutations. In these tumours, open chromatin emerged at transcription start sites where H2A.Z was lost, which was associated with upregulation of genes. Furthermore, YEATS4 defects were associated with abnormal upregulation of bivalent embryonic stem cell genes, as previously shown in mice5. Our work describes a potential mechanism of tumorigenesis-epigenetic instability caused by deficient H2A.Z deposition-and suggests that ULs arise through an aberrant differentiation program driven by deranged chromatin, emanating from a small number of mutually exclusive driver mutations.

Inherited mutations affecting the SRCAP complex are central in moderate-penetrance predisposition to uterine leiomyomas.

Uterine leiomyomas (ULs) are benign smooth muscle tumors that are common in premenopausal women. Somatic alterations in MED12, HMGA2, FH, genes encoding subunits of the SRCAP complex, and genes involved in Cullin 3-RING E3 ligase neddylation are mutually exclusive UL drivers. Established predisposition genes explain only partially the estimated heritability of leiomyomas. Here, we examined loss-of-function variants across 18,899 genes in a cohort of 233,614 White European women, revealing variants in four genes encoding SRCAP complex subunits (YEATS4, ZNHIT1, DMAP1, and ACTL6A) with a significant association to ULs, and YEATS4 and ZNHIT1 strikingly rank first and second, respectively. Positive mutation status was also associated with younger age at diagnosis and hysterectomy. Moderate-penetrance UL risk was largely attributed to rare non-synonymous mutations affecting the SRCAP complex. To examine this disease phenotype more closely, we set out to identify inherited mutations affecting the SRCAP complex in our in-house sample collection of Finnish individuals with ULs (n = 860). We detected one individual with an ACTL6A splice-site mutation, two individuals with a YEATS4 missense mutation, and four individuals with DMAP1 mutations: one splice-site, one nonsense, and two missense variants. These individuals had large and/or multiple ULs, were often diagnosed at an early age, and many had family history of ULs. When a somatic second hit was found, ACTL6A and DMAP1 were silenced in tumors by somatic mutation and YEATS4 by promoter hypermethylation. Decreased H2A.Z staining was observed in the tumors, providing further evidence for the pathogenic nature of the germline mutations. Our results establish inactivation of genes encoding SRCAP complex subunits as a central contributor to moderate-penetrance UL predisposition.

Molecular subclass of uterine fibroids predicts tumor shrinkage in response to ulipristal acetate.

Precision medicine carries great potential for management of all tumor types. The aim of this retrospective study was to investigate if the two most common genetically distinct uterine fibroid subclasses, driven by aberrations in MED12 and HMGA2 genes, respectively, influence response to treatment with the progesterone receptor modulator ulipristal acetate. Changes in diameter and mutation status were derived for 101 uterine fibroids surgically removed after ulipristal acetate treatment. A significant difference in treatment response between the two major subclasses was detected. MED12 mutant fibroids had 4.4 times higher odds of shrinking in response to ulipristal acetate treatment as compared to HMGA2 driven fibroids (95% confidence interval 1.37-13.9; P = 0.013), and in a multivariate analysis molecular subclassification was an independent predictive factor. Compatible with this finding, gene expression and DNA methylation analyses revealed subclass specific differences in progesterone receptor signaling. The work provides a proof-of-principle that uterine fibroid treatment response is influenced by molecular subclass and that the genetic subclasses should be taken into account when evaluating current and future uterine fibroid therapies.

Genome-Wide Association Study Identifies 4 Novel Risk Loci for Small Intestinal Neuroendocrine Tumors Including a Missense Mutation in LGR5.

BACKGROUND & AIMS: Small intestinal neuroendocrine tumor (SI-NET) is a rare disease, but its incidence has increased over the past 4 decades. Understanding the genetic risk factors underlying SI-NETs can help in disease prevention and may provide clinically beneficial markers for diagnosis. Here the results of the largest genome-wide association study of SI-NETs performed to date with 405 cases and 614,666 controls are reported. METHODS: Samples from 307 patients with SI-NETs and 287,137 controls in the FinnGen study were used for the identification of SI-NET risk-associated genetic variants. The results were also meta-analyzed with summary statistics from the UK Biobank (n = 98 patients with SI-NET and n = 327,529 controls). RESULTS: We identified 6 genome-wide significant (P < 5 × 10-8) loci associated with SI-NET risk, of which 4 (near SEMA6A, LGR5, CDKAL1, and FERMT2) are novel and 2 (near LTA4H-ELK and in KIF16B) have been reported previously. Interestingly, the top hit (rs200138614; P = 1.80 × 10-19) was a missense variant (p.Cys712Phe) in the LGR5 gene, a bona-fide marker of adult intestinal stem cells and a potentiator of canonical WNT signaling. The association was validated in an independent Finnish collection of 70 patients with SI-NETs, as well as in the UK Biobank exome sequence data (n = 92 cases and n = 392,814 controls). Overexpression of LGR5 p.Cys712Phe in intestinal organoids abolished the ability of R-Spondin1 to support organoid growth, indicating that the mutation perturbed R-Spondin-LGR5 signaling. CONCLUSIONS: Our study is the largest genome-wide association study to date on SI-NETs and reported 4 new associated genome-wide association study loci, including a novel missense mutation (rs200138614, p.Cys712Phe) in LGR5, a canonical marker of adult intestinal stem cells.

Genetic and Epigenetic Characteristics of Inflammatory Bowel Disease-Associated Colorectal Cancer.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disorder associated with an elevated risk of colorectal cancer (CRC). IBD-associated CRC (IBD-CRC) may represent a distinct pathway of tumorigenesis compared to sporadic CRC (sCRC). Our aim was to comprehensively characterize IBD-associated tumorigenesis integrating multiple high-throughput approaches, and to compare the results with in-house data sets from sCRCs. METHODS: Whole-genome sequencing, single nucleotide polymorphism arrays, RNA sequencing, genome-wide methylation analysis, and immunohistochemistry were performed using fresh-frozen and formalin-fixed tissue samples of tumor and corresponding normal tissues from 31 patients with IBD-CRC. RESULTS: Transcriptome-based tumor subtyping revealed the complete absence of canonical epithelial tumor subtype associated with WNT signaling in IBD-CRCs, dominated instead by mesenchymal stroma-rich subtype. Negative WNT regulators AXIN2 and RNF43 were strongly down-regulated in IBD-CRCs and chromosomal gains at HNF4A, a negative regulator of WNT-induced epithelial-mesenchymal transition (EMT), were less frequent compared to sCRCs. Enrichment of hypomethylation at HNF4α binding sites was detected solely in sCRC genomes. PIGR and OSMR involved in mucosal immunity were dysregulated via epigenetic modifications in IBD-CRCs. Genome-wide analysis showed significant enrichment of noncoding mutations to 5'untranslated region of TP53 in IBD-CRCs. As reported previously, somatic mutations in APC and KRAS were less frequent in IBD-CRCs compared to sCRCs. CONCLUSIONS: Distinct mechanisms of WNT pathway dysregulation skew IBD-CRCs toward mesenchymal tumor subtype, which may affect prognosis and treatment options. Increased OSMR signaling may favor the establishment of mesenchymal tumors in patients with IBD.

No evidence of EMAST in whole genome sequencing data from 248 colorectal cancers.

Microsatellite instability (MSI) is caused by defective DNA mismatch repair (MMR), and manifests as accumulation of small insertions and deletions (indels) in short tandem repeats of the genome. Another form of repeat instability, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), has been suggested to occur in 50% to 60% of colorectal cancer (CRC), of which approximately one quarter are accounted for by MSI. Unlike for MSI, the criteria for defining EMAST is not consensual. EMAST CRCs have been suggested to form a distinct subset of CRCs that has been linked to a higher tumor stage, chronic inflammation, and poor prognosis. EMAST CRCs not exhibiting MSI have been proposed to show instability of di- and trinucleotide repeats in addition to tetranucleotide repeats, but lack instability of mononucleotide repeats. However, previous studies on EMAST have been based on targeted analysis of small sets of marker repeats, often in relatively few samples. To gain insight into tetranucleotide instability on a genome-wide level, we utilized whole genome sequencing data from 227 microsatellite stable (MSS) CRCs, 18 MSI CRCs, 3 POLE-mutated CRCs, and their corresponding normal samples. As expected, we observed tetranucleotide instability in all MSI CRCs, accompanied by instability of mono-, di-, and trinucleotide repeats. Among MSS CRCs, some tumors displayed more microsatellite mutations than others as a continuum, and no distinct subset of tumors with the previously proposed molecular characters of EMAST could be observed. Our results suggest that tetranucleotide repeat mutations in non-MSI CRCs represent stochastic mutation events rather than define a distinct CRC subclass.

Lack of an association between gallstone disease and bilirubin levels with risk of colorectal cancer: a Mendelian randomisation analysis.

BACKGROUND: Epidemiological studies of the relationship between gallstone disease and circulating levels of bilirubin with risk of developing colorectal cancer (CRC) have been inconsistent. To address possible confounding and reverse causation, we examine the relationship between these potential risk factors and CRC using Mendelian randomisation (MR). METHODS: We used two-sample MR to examine the relationship between genetic liability to gallstone disease and circulating levels of bilirubin with CRC in 26,397 patients and 41,481 controls. We calculated the odds ratio per genetically predicted SD unit increase in log bilirubin levels (ORSD) for CRC and tested for a non-zero causal effect of gallstones on CRC. Sensitivity analysis was applied to identify violations of estimator assumptions. RESULTS: No association between either gallstone disease (P value = 0.60) or circulating levels of bilirubin (ORSD = 1.00, 95% confidence interval (CI) = 0.96-1.03, P value = 0.90) with CRC was shown. CONCLUSIONS: Despite the large scale of this study, we found no evidence for a causal relationship between either circulating levels of bilirubin or gallstone disease with risk of developing CRC. While the magnitude of effect suggested by some observational studies can confidently be excluded, we cannot exclude the possibility of smaller effect sizes and non-linear relationships.

Exome-wide somatic mutation characterization of small bowel adenocarcinoma.

Small bowel adenocarcinoma (SBA) is an aggressive disease with limited treatment options. Despite previous studies, its molecular genetic background has remained somewhat elusive. To comprehensively characterize the mutational landscape of this tumor type, and to identify possible targets of treatment, we conducted the first large exome sequencing study on a population-based set of SBA samples from all three small bowel segments. Archival tissue from 106 primary tumors with appropriate clinical information were available for exome sequencing from a patient series consisting of a majority of confirmed SBA cases diagnosed in Finland between the years 2003-2011. Paired-end exome sequencing was performed using Illumina HiSeq 4000, and OncodriveFML was used to identify driver genes from the exome data. We also defined frequently affected cancer signalling pathways and performed the first extensive allelic imbalance (AI) analysis in SBA. Exome data analysis revealed significantly mutated genes previously linked to SBA (TP53, KRAS, APC, SMAD4, and BRAF), recently reported potential driver genes (SOX9, ATM, and ARID2), as well as novel candidate driver genes, such as ACVR2A, ACVR1B, BRCA2, and SMARCA4. We also identified clear mutation hotspot patterns in ERBB2 and BRAF. No BRAF V600E mutations were observed. Additionally, we present a comprehensive mutation signature analysis of SBA, highlighting established signatures 1A, 6, and 17, as well as U2 which is a previously unvalidated signature. Finally, comparison of the three small bowel segments revealed differences in tumor characteristics. This comprehensive work unveils the mutational landscape and most frequently affected genes and pathways in SBA, providing potential therapeutic targets, and novel and more thorough insights into the genetic background of this tumor type.

Exome and immune cell score analyses reveal great variation within synchronous primary colorectal cancers.

BACKGROUND: Approximately 4% of colorectal cancer (CRC) patients have at least two simultaneous cancers in the colon. Due to the shared environment, these synchronous CRCs (SCRCs) provide a unique setting to study colorectal carcinogenesis. Understanding whether these tumours are genetically similar or distinct is essential when designing therapeutic approaches. METHODS: We performed exome sequencing of 47 primary cancers and corresponding normal samples from 23 patients. Additionally, we carried out a comprehensive mutational signature analysis to assess whether tumours had undergone similar mutational processes and the first immune cell score analysis (IS) of SCRC to analyse the interplay between immune cell invasion and mutation profile in both lesions of an individual. RESULTS: The tumour pairs shared only few mutations, favouring different mutations in known CRC genes and signalling pathways and displayed variation in their signature content. Two tumour pairs had discordant mismatch repair statuses. In majority of the pairs, IS varied between primaries. Differences were not explained by any clinicopathological variable or mutation burden. CONCLUSIONS: The study shows major diversity within SCRCs. Rather than rely on data from one tumour, our study highlights the need to evaluate both tumours of a synchronous pair for optimised targeted therapy.

Variation at 2q35 (PNKD and TMBIM1) influences colorectal cancer risk and identifies a pleiotropic effect with inflammatory bowel disease.

To identify new risk loci for colorectal cancer (CRC), we conducted a meta-analysis of seven genome-wide association studies (GWAS) with independent replication, totalling 13 656 CRC cases and 21 667 controls of European ancestry. The combined analysis identified a new risk association for CRC at 2q35 marked by rs992157 (P = 3.15 × 10-8, odds ratio = 1.10, 95% confidence interval = 1.06-1.13), which is intronic to PNKD (paroxysmal non-kinesigenic dyskinesia) and TMBIM1 (transmembrane BAX inhibitor motif containing 1). Intriguingly this susceptibility single-nucleotide polymorphism (SNP) is in strong linkage disequilibrium (r2 = 0.90, D' = 0.96) with the previously discovered GWAS SNP rs2382817 for inflammatory bowel disease (IBD). Following on from this observation we examined for pleiotropy, or shared genetic susceptibility, between CRC and the 200 established IBD risk loci, identifying an additional 11 significant associations (false discovery rate [FDR]) < 0.05). Our findings provide further insight into the biological basis of inherited genetic susceptibility to CRC, and identify risk factors that may influence the development of both CRC and IBD.

Fast motif matching revisited: high-order PWMs, SNPs and indels.

Motivation: While the position weight matrix (PWM) is the most popular model for sequence motifs, there is growing evidence of the usefulness of more advanced models such as first-order Markov representations, and such models are also becoming available in well-known motif databases. There has been lots of research of how to learn these models from training data but the problem of predicting putative sites of the learned motifs by matching the model against new sequences has been given less attention. Moreover, motif site analysis is often concerned about how different variants in the sequence affect the sites. So far, though, the corresponding efficient software tools for motif matching have been lacking. Results: We develop fast motif matching algorithms for the aforementioned tasks. First, we formalize a framework based on high-order position weight matrices for generic representation of motif models with dinucleotide or general q -mer dependencies, and adapt fast PWM matching algorithms to the high-order PWM framework. Second, we show how to incorporate different types of sequence variants , such as SNPs and indels, and their combined effects into efficient PWM matching workflows. Benchmark results show that our algorithms perform well in practice on genome-sized sequence sets and are for multiple motif search much faster than the basic sliding window algorithm. Availability and Implementation: Implementations are available as a part of the MOODS software package under the GNU General Public License v3.0 and the Biopython license ( http://www.cs.helsinki.fi/group/pssmfind ). Contact: janne.h.korhonen@gmail.com.

Mendelian randomisation implicates hyperlipidaemia as a risk factor for colorectal cancer.

While elevated blood cholesterol has been associated with an increased risk of colorectal cancer (CRC) in observational studies, causality is uncertain. Here we apply a Mendelian randomisation (MR) analysis to examine the potential causal relationship between lipid traits and CRC risk. We used single nucleotide polymorphisms (SNPs) associated with blood levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) as instrumental variables (IV). We calculated MR estimates for each risk factor with CRC using SNP-CRC associations from 9,254 cases and 18,386 controls. Genetically predicted higher TC was associated with an elevated risk of CRC (odds ratios (OR) per unit SD increase = 1.46, 95% confidence interval [CI]: 1.20-1.79, p = 1.68 × 10-4 ). The pooled ORs for LDL, HDL, and TG were 1.05 (95% CI: 0.92-1.18, p = 0.49), 0.94 (95% CI: 0.84-1.05, p = 0.27), and 0.98 (95% CI: 0.85-1.12, p = 0.75) respectively. A genetic risk score for 3-hydoxy-3-methylglutaryl-coenzyme A reductase (HMGCR) to mimic the effects of statin therapy was associated with a reduced CRC risk (OR = 0.69, 95% CI: 0.49-0.99, p = 0.046). This study supports a causal relationship between higher levels of TC with CRC risk, and a further rationale for implementing public health strategies to reduce the prevalence of hyperlipidaemia.

Mendelian randomisation analysis strongly implicates adiposity with risk of developing colorectal cancer.

BACKGROUND: Observational studies have associated adiposity with an increased risk of colorectal cancer (CRC). However, such studies do not establish a causal relationship. To minimise bias from confounding we performed a Mendelian randomisation (MR) analysis to examine the relationship between adiposity and CRC. METHODS: We used SNPs associated with adult body mass index (BMI), waist-hip ratio (WHR), childhood obesity and birth weight as instrumental variables in a MR analysis of 9254 CRC cases and 18 386 controls. RESULTS: In the MR analysis, the odds ratios (ORs) of CRC risk per unit increase in BMI, WHR and childhood obesity were 1.23 (95% CI: 1.02-1.49, P=0.033), 1.59 (95% CI: 1.08-2.34, P=0.019) and 1.07 (95% CI: 1.03-1.13, P=0.018), respectively. There was no evidence for association between birth weight and CRC (OR=1.22, 95% CI: 0.89-1.67, P=0.22). Combining these data with a concurrent MR-based analysis for BMI and WHR with CRC risk (totalling to 18 190 cases, 27 617 controls) provided increased support, ORs for BMI and WHR were 1.26 (95% CI: 1.10-1.44, P=7.7 × 10(-4)) and 1.40 (95% CI: 1.14-1.72, P=1.2 × 10(-3)), respectively. CONCLUSIONS: These data provide further evidence for a strong causal relationship between adiposity and the risk of developing CRC highlighting the urgent need for prevention and treatment of adiposity.

Genome-wide analysis of ETS-family DNA-binding in vitro and in vivo.

Members of the large ETS family of transcription factors (TFs) have highly similar DNA-binding domains (DBDs)-yet they have diverse functions and activities in physiology and oncogenesis. Some differences in DNA-binding preferences within this family have been described, but they have not been analysed systematically, and their contributions to targeting remain largely uncharacterized. We report here the DNA-binding profiles for all human and mouse ETS factors, which we generated using two different methods: a high-throughput microwell-based TF DNA-binding specificity assay, and protein-binding microarrays (PBMs). Both approaches reveal that the ETS-binding profiles cluster into four distinct classes, and that all ETS factors linked to cancer, ERG, ETV1, ETV4 and FLI1, fall into just one of these classes. We identify amino-acid residues that are critical for the differences in specificity between all the classes, and confirm the specificities in vivo using chromatin immunoprecipitation followed by sequencing (ChIP-seq) for a member of each class. The results indicate that even relatively small differences in in vitro binding specificity of a TF contribute to site selectivity in vivo.

Identification of candidate oncogenes in human colorectal cancers with microsatellite instability.

Microsatellite instability can be found in approximately 15% of all colorectal cancers. To detect new oncogenes we sequenced the exomes of 25 colorectal tumors and respective healthy colon tissue. Potential mutation hot spots were confirmed in 15 genes; ADAR, DCAF12L2, GLT1D1, ITGA7, MAP1B, MRGPRX4, PSRC1, RANBP2, RPS6KL1, SNCAIP, TCEAL6, TUBB6, WBP5, VEGFB, and ZBTB2; these were validated in 86 tumors with microsatellite instability. ZBTB2, RANBP2, and PSRC1 also were found to contain hot spot mutations in the validation set. The form of ZBTB2 associated with colorectal cancer increased cell proliferation. The mutation hot spots might be used to develop personalized tumor profiling and therapy.

Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities.

The genetic code-the binding specificity of all transfer-RNAs--defines how protein primary structure is determined by DNA sequence. DNA also dictates when and where proteins are expressed, and this information is encoded in a pattern of specific sequence motifs that are recognized by transcription factors. However, the DNA-binding specificity is only known for a small fraction of the approximately 1400 human transcription factors (TFs). We describe here a high-throughput method for analyzing transcription factor binding specificity that is based on systematic evolution of ligands by exponential enrichment (SELEX) and massively parallel sequencing. The method is optimized for analysis of large numbers of TFs in parallel through the use of affinity-tagged proteins, barcoded selection oligonucleotides, and multiplexed sequencing. Data are analyzed by a new bioinformatic platform that uses the hundreds of thousands of sequencing reads obtained to control the quality of the experiments and to generate binding motifs for the TFs. The described technology allows higher throughput and identification of much longer binding profiles than current microarray-based methods. In addition, as our method is based on proteins expressed in mammalian cells, it can also be used to characterize DNA-binding preferences of full-length proteins or proteins requiring post-translational modifications. We validate the method by determining binding specificities of 14 different classes of TFs and by confirming the specificities for NFATC1 and RFX3 using ChIP-seq. Our results reveal unexpected dimeric modes of binding for several factors that were thought to preferentially bind DNA as monomers.

Transposable elements as tissue-specific enhancers in cancers of endodermal lineage.

Transposable elements (TE) are repetitive genomic elements that harbor binding sites for human transcription factors (TF). A regulatory role for TEs has been suggested in embryonal development and diseases such as cancer but systematic investigation of their functions has been limited by their widespread silencing in the genome. Here, we utilize unbiased massively parallel reporter assay data using a whole human genome library to identify TEs with functional enhancer activity in two human cancer types of endodermal lineage, colorectal and liver cancers. We show that the identified TE enhancers are characterized by genomic features associated with active enhancers, such as epigenetic marks and TF binding. Importantly, we identify distinct TE subfamilies that function as tissue-specific enhancers, namely MER11- and LTR12-elements in colon and liver cancers, respectively. These elements are bound by distinct TFs in each cell type, and they have predicted associations to differentially expressed genes. In conclusion, these data demonstrate how different cancer types can utilize distinct TEs as tissue-specific enhancers, paving the way for comprehensive understanding of the role of TEs as bona fide enhancers in the cancer genomes.

Vitamin C boosts DNA demethylation in TET2 germline mutation carriers.

BACKGROUND: Accurate regulation of DNA methylation is necessary for normal cells to differentiate, develop and function. TET2 catalyzes stepwise DNA demethylation in hematopoietic cells. Mutations in the TET2 gene predispose to hematological malignancies by causing DNA methylation overload and aberrant epigenomic landscape. Studies on mice and cell lines show that the function of TET2 is boosted by vitamin C. Thus, by strengthening the demethylation activity of TET2, vitamin C could play a role in the prevention of hematological malignancies in individuals with TET2 dysfunction. We recently identified a family with lymphoma predisposition where a heterozygous truncating germline mutation in TET2 segregated with nodular lymphocyte-predominant Hodgkin lymphoma. The mutation carriers displayed a hypermethylation pattern that was absent in the family members without the mutation. METHODS: In a clinical trial of 1 year, we investigated the effects of oral 1 g/day vitamin C supplementation on DNA methylation by analyzing genome-wide DNA methylation and gene expression patterns from the family members. RESULTS: We show that vitamin C reinforces the DNA demethylation cascade, reduces the proportion of hypermethylated loci and diminishes gene expression differences between TET2 mutation carriers and control individuals. CONCLUSIONS: These results suggest that vitamin C supplementation increases DNA methylation turnover and provide a basis for further work to examine the potential benefits of vitamin C supplementation in individuals with germline and somatic TET2 mutations. TRIAL REGISTRATION: This trial was registered at EudraCT with reference number of 2018-000155-41 (01.04.2019).

Identity-by-descent-based phasing and imputation in founder populations using graphical models.

Accurate knowledge of haplotypes, the combination of alleles co-residing on a single copy of a chromosome, enables powerful gene mapping and sequence imputation methods. Since humans are diploid, haplotypes must be derived from genotypes by a phasing process. In this study, we present a new computational model for haplotype phasing based on pairwise sharing of haplotypes inferred to be Identical-By-Descent (IBD). We apply the Bayesian network based model in a new phasing algorithm, called systematic long-range phasing (SLRP), that can capitalize on the close genetic relationships in isolated founder populations, and show with simulated and real genome-wide genotype data that SLRP substantially reduces the rate of phasing errors compared to previous phasing algorithms. Furthermore, the method accurately identifies regions of IBD, enabling linkage-like studies without pedigrees, and can be used to impute most genotypes with very low error rate.