IntroductionThe nuanced roles of neuropilin (NRP) isoforms, NRP1 and NRP2, have attracted considerable scientific interest regarding cancer progression. Their differential expressions across various cancer types are specific to NRP isoforms which are shown in a cancer type-dependent manner. It accounts for the different mechanisms involved, driven by a co-expression of gene-sets associated with overexpressed NRP1 or NRP2. Their different expressions on tumour-associated macrophages (TAMs) with disparate markers are associated with the heterogenous tumour microenvironment (TME) through their plasticity and pro-tumorigenic activities.MethodsSingle-cell RNA sequencing (scRNA-seq) analyses were performed on tumours from clear cell Renal Cell Carcinoma (ccRCC) and skin cutaneous melanoma (SKCM) which exhibit the highest expressions of NRP1 and NRP2, respectively. Datasets were processed using established bioinformatics pipelines, including clustering algorithms, to determine cellular heterogeneity and quantify NRP isoform expression within distinct macrophage populations. Using differential gene expression analysis (DEGA) alongside co-enrichment studies, we explored gene-sets associated with NRP1 or NRP2 overexpression in TAMs.ResultsOur analysis revealed a marked upregulation of NRP1 in TAMs isolated from ccRCC and elevated NRP2 expression in SKCM-derived TAMs. Both NRP1+ and NRP2+ macrophages showed an M2-like polarisation characterised by immune suppression and extracellular matrix degradation. Coupled with the previously uncharacterised NRP isoform specific- subpopulations within these cancers identified by DEGA, co-enrichment analyses demonstrated that the upregulation of gene-sets associated with NRP1 is associated with angiogenesis and tumour progression through VEGF signalling, while gene-sets with NRP2 showed dual functionality in the TME-dependent manner. Their distinct roles in regulating macrophage plasticity, tumour invasion, and metastasis were highlighted.DiscussionThese findings underscore distinct isoform-specific mechanisms by which NRP1 and NRP2 contribute to TAM-mediated cancer progression. This study aims to establish a foundation for future research, leading to biological experiments with focused gene-sets derived from our findings. This approach can contribute to the development of immunomodulatory strategies targeting specific NRP isoforms in macrophages, tailored to individual cancer types and abnormal expressions of those gene markers, potentially offering a more effective therapeutic approach compared to broad-spectrum NRP inhibition strategies.

Aim & methods: To investigate peripheral blood methylation episignatures in KMT2B-related dystonia (DYT-KMT2B), the authors undertook genome-wide methylation profiling of ∼2 M CpGs using a next-generation sequencing-based assay and compared the findings with those in controls and patients with KMT2D-related Kabuki syndrome type 1 (KS1). Results: A total of 1812 significantly differentially methylated CpG positions (false discovery rate < 0.05) were detected in DYT-KMT2B samples compared with controls. Multi-dimensional scaling analysis showed that the 10 DYT-KMT2B samples clustered together and separately from 29 controls and 10 with pathogenic variants in KMT2D. The authors found that most differentially methylated CpG positions were specific to one disorder and that all (DYT-KMT2B) and most (Kabuki syndrome type 1) methylation alterations in CpG islands were gain of methylation events. Conclusion: Using sensitive methylation profiling methodology, the authors replicated recent reports of a methylation episignature for DYT-KMT2B. These findings will facilitate the development of episignature-based assays to improve diagnostic accuracy.

HNRNPU encodes a multifunctional RNA-binding protein that plays critical roles in regulating pre-mRNA splicing, mRNA stability, and translation. Aberrant expression and dysregulation of HNRNPU have been implicated in various human diseases, including cancers and neurological disorders. We applied a next generation sequencing based assay (EPIC-NGS) to investigate genome-wide methylation profiling for >2 M CpGs for 7 individuals with a neurodevelopmental disorder associated with HNRNPU germline pathogenic loss-of-function variants. Compared to healthy individuals, 227 HNRNPU-associated differentially methylated positions were detected. Both hyper- and hypomethylation alterations were identified but the former predominated. The identification of a methylation episignature for HNRNPU-associated neurodevelopmental disorder (NDD) implicates HNPRNPU-related chromatin alterations in the aetiopathogenesis of this disorder and suggests that episignature profiling should have clinical utility as a predictor for the pathogenicity of HNRNPU variants of uncertain significance. The detection of a methylation episignaure for HNRNPU-associated NDD is consistent with a recent report of a methylation episignature for HNRNPK-associated NDD.

BackgroundImprinting disorders (ImpDis) comprise diseases which are caused by aberrant regulation of monoallelically and parent-of-origin-dependent expressed genes. A characteristic molecular change in ImpDis patients is aberrant methylation signatures at disease-specific loci, without an obvious DNA change at the specific differentially methylated region (DMR). However, there is a growing number of reports on multilocus imprinting disturbances (MLIDs), i.e. aberrant methylation at different DMRs in the same patient. These MLIDs account for a significant number of patients with specific ImpDis, and several reports indicate a central role of pathogenic maternal effect variants in their aetiology by affecting the maturation of the oocyte and the early embryo. Though several studies on the prevalence and the molecular causes of MLID have been conducted, homogeneous datasets comprising both genomic and methylation data are still lacking.ResultsBased on a cohort of 36 MLID patients, we here present both methylation data obtained from next-generation sequencing (NGS, ImprintSeq) approaches and whole-exome sequencing (WES). The compilation of methylation data did not reveal a disease-specific MLID episignature, and a predisposition for the phenotypic modification was not obvious as well. In fact, this lack of epigenotype-phenotype correlation might be related to the mosaic distribution of imprinting defects and their functional relevance in specific tissues.ConclusionsDue to the higher sensitivity of NGS-based approaches, we suggest that ImprintSeq might be offered at reference centres in case of ImpDis patients with unusual phenotypes but MLID negative by conventional tests. By WES, additional MLID causes than the already known maternal effect variants could not be identified, neither in the patients nor in the maternal exomes. In cases with negative WES results, it is currently unclear to what extent either environmental factors or undetected genetic variants contribute to MLID.

Germline pathogenic variants in two genes encoding the lysine-specific histone methyltransferase genes SETD1A and SETD2 are associated with neurodevelopmental disorders (NDDs) characterized by developmental delay and congenital anomalies. The SETD1A and SETD2 gene products play a critical role in chromatin-mediated regulation of gene expression. Specific methylation episignatures have been detected for a range of chromatin gene-related NDDs and have impacted clinical practice by improving the interpretation of variant pathogenicity. To investigate if SETD1A and/or SETD2-related NDDs are associated with a detectable episignature, we undertook targeted genome-wide methylation profiling of > 2 M CpGs using a next-generation sequencing-based assay. A comparison of methylation profiles in patients with SETD1A variants (n = 6) did not reveal evidence of a strong methylation episignature. A review of the clinical and genetic features of the SETD2 patient group revealed that, as reported previously, there were phenotypic differences between patients with truncating mutations (n = 4, Luscan-Lumish syndrome; MIM:616831) and those with missense codon 1740 variants [p.Arg1740Trp (n = 4) and p.Arg1740Gln (n = 2)]. Both SETD2 subgroups demonstrated a methylation episignature, which was characterized by hypomethylation and hypermethylation events, respectively. Within the codon 1740 subgroup, both the methylation changes and clinical phenotype were more severe in those with p.Arg1740Trp variants. We also noted that two of 10 cases with a SETD2-NDD had developed a neoplasm. These findings reveal novel epigenotype-genotype-phenotype correlations in SETD2-NDDs and predict a gain-of-function mechanism for SETD2 codon 1740 pathogenic variants. (C) The Author(s) 2023. Published by Oxford University Press.

PURPOSE:Disruptions of genomic imprinting are associated with congenital imprinting disorders (CIDs) and other disease states, including cancer. CIDs are most often associated with altered methylation at imprinted differentially methylated regions (iDMRs). In some cases, multiple iDMRs are affected causing multilocus imprinting disturbances (MLIDs). The availability of accurate, quantitative, and scalable high-throughput methods to interrogate multiple iDMRs simultaneously would enhance clinical diagnostics and research. METHODS:We report the development of a custom targeted methylation sequencing panel that covered most relevant 63 iDMRs for CIDs and the detection of MLIDs. We tested it in 70 healthy controls and 147 individuals with CIDs. We distinguished loss and gain of methylation per differentially methylated region and classified high and moderate methylation alterations. RESULTS:Across a range of CIDs with a variety of molecular mechanisms, ImprintSeq performed at 98.4% sensitivity, 99.9% specificity, and 99.9% accuracy (when compared with previous diagnostic testing). ImprintSeq was highly sensitive for detecting MLIDs and enabled diagnostic criteria for MLID to be proposed. In a child with extreme MLID profile a probable genetic cause was identified. CONCLUSION:ImprintSeq provides a novel assay for clinical diagnostic and research studies of CIDs, MLIDs, and the role of disordered imprinting in human disease states.