Cell type signatures in cell-free DNA fragmentation profiles reveal disease biology.

Circulating cell-free DNA (cfDNA) fragments have characteristics that are specific to the cell types that release them. Current methods for cfDNA deconvolution typically use disease tailored marker selection in a limited number of bulk tissues or cell lines. Here, we utilize single cell transcriptome data as a comprehensive cellular reference set for disease-agnostic cfDNA cell-of-origin analysis. We correlate cfDNA-inferred nucleosome spacing with gene expression to rank the relative contribution of over 490 cell types to plasma cfDNA. In 744 healthy individuals and patients, we uncover cell type signatures in support of emerging disease paradigms in oncology and prenatal care. We train predictive models that can differentiate patients with colorectal cancer (84.7%), early-stage breast cancer (90.1%), multiple myeloma (AUC 95.0%), and preeclampsia (88.3%) from matched controls. Importantly, our approach performs well in ultra-low coverage cfDNA datasets and can be readily transferred to diverse clinical settings for the expansion of liquid biopsy.

Cell-free DNA methylation-based preeclampsia prediction: A journey to improve maternal health.

Presymptomatic prediction of preeclampsia (PE) is crucial to enable early prophylactic treatment. Current screening tools are either complex or lack predictive value. We recently demonstrated that cell-free DNA methylation can be leveraged to predict early-onset PE in 57% at a 10% false positive rate. Importantly, this minimally invasive screening test can be implemented as an add-on to current widespread noninvasive prenatal aneuploidy screening. Here, we highlight the pitfalls and promising prospects of this research.

Cell-free DNA methylome analysis for early preeclampsia prediction.

Preeclampsia (PE) is a leading cause for peripartal morbidity, especially if developing early in gestation. To enable prophylaxis in the prevention of PE, pregnancies at risk of PE must be identified early-in the first trimester. To identify at-risk pregnancies we profiled methylomes of plasma-derived, cell-free DNA from 498 pregnant women, of whom about one-third developed early-onset PE. We detected DNA methylation differences between control and PE pregnancies that enabled risk stratification at PE diagnosis but also presymptomatically, at around 12 weeks of gestation (range 9-14 weeks). The first-trimester risk prediction model was validated in an external cohort collected from two centers (area under the curve (AUC) = 0.75) and integrated with routinely available maternal risk factors (AUC = 0.85). The combined risk score correctly predicted 72% of patients with early-onset PE at 80% specificity. These preliminary results suggest that cell-free DNA methylation profiling is a promising tool for presymptomatic PE risk assessment, and has the potential to improve treatment and follow-up in the obstetric clinic.

Oxidative Bisulfite Sequencing: An Experimental and Computational Protocol.

Bisulfite sequencing (BS-seq) remains the gold standard technique to quantitively map DNA methylation at a single-base resolution. However, BS-seq cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Oxidative bisulfite sequencing (oxBS-seq) was one of the first techniques that enabled absolute quantification of 5mC and 5hmC at single-base resolution. OxBS-seq uses chemical oxidation of 5hmC prior to bisulfite treatment to provide a direct readout of 5mC; comparison with BS-seq data can then be used to infer 5hmC levels. Here we describe in detail an updated version of our laboratory's oxBS-seq protocol, which uses potassium perruthenate (KRuO4) as an oxidant. We also describe a bioinformatics pipeline designed to handle Illumina short read sequencing data from whole-genome oxBS-seq.

DNA methylation repels binding of hypoxia-inducible transcription factors to maintain tumor immunotolerance.

BACKGROUND: Hypoxia is pervasive in cancer and other diseases. Cells sense and adapt to hypoxia by activating hypoxia-inducible transcription factors (HIFs), but it is still an outstanding question why cell types differ in their transcriptional response to hypoxia. RESULTS: We report that HIFs fail to bind CpG dinucleotides that are methylated in their consensus binding sequence, both in in vitro biochemical binding assays and in vivo studies of differentially methylated isogenic cell lines. Based on in silico structural modeling, we show that 5-methylcytosine indeed causes steric hindrance in the HIF binding pocket. A model wherein cell-type-specific methylation landscapes, as laid down by the differential expression and binding of other transcription factors under normoxia, control cell-type-specific hypoxia responses is observed. We also discover ectopic HIF binding sites in repeat regions which are normally methylated. Genetic and pharmacological DNA demethylation, but also cancer-associated DNA hypomethylation, expose these binding sites, inducing HIF-dependent expression of cryptic transcripts. In line with such cryptic transcripts being more prone to cause double-stranded RNA and viral mimicry, we observe low DNA methylation and high cryptic transcript expression in tumors with high immune checkpoint expression, but not in tumors with low immune checkpoint expression, where they would compromise tumor immunotolerance. In a low-immunogenic tumor model, DNA demethylation upregulates cryptic transcript expression in a HIF-dependent manner, causing immune activation and reducing tumor growth. CONCLUSIONS: Our data elucidate the mechanism underlying cell-type-specific responses to hypoxia and suggest DNA methylation and hypoxia to underlie tumor immunotolerance.

Heterozygous loss-of-function variants of MEIS2 cause a triad of palatal defects, congenital heart defects, and intellectual disability.

Deletions on chromosome 15q14 are a known chromosomal cause of cleft palate, typically co-occurring with intellectual disability, facial dysmorphism, and congenital heart defects. The identification of patients with loss-of-function variants in MEIS2, a gene within this deletion, suggests that these features are attributed to haploinsufficiency of MEIS2. To further delineate the phenotypic spectrum of the MEIS2-related syndrome, we collected 23 previously unreported patients with either a de novo sequence variant in MEIS2 (9 patients), or a 15q14 microdeletion affecting MEIS2 (14 patients). All but one de novo MEIS2 variant were identified by whole-exome sequencing. One variant was found by targeted sequencing of MEIS2 in a girl with a clinical suspicion of this syndrome. In addition to the triad of palatal defects, heart defects, and developmental delay, heterozygous loss of MEIS2 results in recurrent facial features, including thin and arched eyebrows, short alae nasi, and thin vermillion. Genotype-phenotype comparison between patients with 15q14 deletions and patients with sequence variants or intragenic deletions within MEIS2, showed a higher prevalence of moderate-to-severe intellectual disability in the former group, advocating for an independent locus for psychomotor development neighboring MEIS2.