Detection of brain somatic variation in epilepsy-associated developmental lesions

Epilepsy-associated developmental lesions, including malformations of cortical development and low-grade developmental tumors, represent a major cause of drug-resistant seizures requiring surgical intervention in children. Brain-restricted somatic mosaicism has been implicated in the genetic etiology of these lesions; however, many contributory genes remain unidentified. We enrolled 50 children undergoing epilepsy surgery into a translational research study. We performed exome and RNA-sequencing of resected brain tissue samples to identify somatic variation. We uncovered candidate disease-causing somatic variation affecting 28 patients (56%), as well as candidate germline variants affecting 4 patients (8%). We confirmed somatic findings using high-depth targeted DNA sequencing. In agreement with previous studies, we identified somatic variation affecting SLC35A2 and MTOR pathway genes in patients with focal cortical dysplasia. Somatic gains of chromosome 1q were detected in 30% (3 of 10) Type I FCD patients. Somatic variation of MAPK pathway genes (i.e., FGFR1, FGFR2, BRAF, KRAS) was associated with low-grade epilepsy-associated developmental tumors. Somatic structural variation accounted for over one-half of epilepsy-associated tumor diagnoses. Sampling across multiple anatomic regions revealed that somatic variant allele fractions vary widely within epileptogenic tissue. Finally, we identified putative disease-causing variants in genes (EEF2, NAV2, PTPN11) not yet associated with focal cortical dysplasia. These results further elucidate the genetic basis of structural brain abnormalities leading to focal epilepsy in children and point to new candidate disease genes.

Somatic Variation as an Incidental Finding in the Pediatric Next Generation Sequencing Era

The methodologic approach used in next-generation sequencing (NGS) affords a high depth of coverage in genomic analysis. Inherent in the nature of genomic testing, there exists potential for identifying genomic findings that are incidental or secondary to the indication for clinical testing, with the frequency dependent on the breadth of analysis and the tissue sample under study. The interpretation and management of clinically meaningful incidental genomic findings is a pressing issue particularly in the pediatric population. Our study describes a 16-month old male who presented with Dandy-Walker malformation, metopic craniosynostosis and developmental delay. Clinical exome sequencing (ES) trio analysis revealed the presence of two variants in the proband. The first was a de novo variant in the PPP2R1A gene (c.773G>A, p.Arg258His), which is associated with autosomal dominant (AD) intellectual disability, accounting for the proband's clinical phenotype. The second was a recurrent hotspot variant in the CBL gene (c.1111T>C, p.Tyr371His), which was present at a variant allele fraction of 11%, consistent with somatic variation in the peripheral blood sample. Germline pathogenic variants in CBL are associated with AD Noonan syndrome-like disorder with or without juvenile myelomonocytic leukemia (JMML). Molecular analyses using a different tissue source, buccal epithelial cells, suggest that the CBL alteration may represent a clonal population of cells restricted to leukocytes. This report highlights the laboratory methodologic and interpretative processes and clinical considerations in the setting of acquired variation detected during clinical ES in a pediatric patient.

Elevated transcription of transposable elements is accompanied by het-siRNA-driven de novo DNA methylation in grapevine embryogenic callus

Background Somatic variation is a valuable source of trait diversity in clonally propagated crops. In grapevine, which has been clonally propagated worldwide for centuries, important phenotypes such as white berry colour are the result of genetic changes caused by transposable elements. Additionally, epiallele formation may play a role in determining geo-specific (‘terroir’) differences in grapes and thus ultimately in wine. This genomic plasticity might be co-opted for crop improvement via somatic embryogenesis, but that depends on a species-specific understanding of the epigenetic regulation of transposable element (TE) expression and silencing in these cultures. For this reason, we used whole-genome bisulphite sequencing, mRNA sequencing and small RNA sequencing to study the epigenetic status and expression of TEs in embryogenic callus, in comparison with leaf tissue. Results We found that compared with leaf tissue, grapevine embryogenic callus cultures accumulate relatively high genome-wide CHH methylation, particularly across heterochromatic regions. This de novo methylation is associated with an abundance of transcripts from highly replicated TE families, as well as corresponding 24 nt heterochromatic siRNAs. Methylation in the TE-specific CHG context was relatively low over TEs located within genes, and the expression of TE loci within genes was highly correlated with the expression of those genes. Conclusions This multi-‘omics analysis of grapevine embryogenic callus in comparison with leaf tissues reveals a high level of genome-wide transcription of TEs accompanied by RNA-dependent DNA methylation of these sequences in trans. This provides insight into the genomic conditions underlying somaclonal variation and epiallele formation in plants regenerated from embryogenic cultures, which is an important consideration when using these tissues for plant propagation and genetic improvement.

m6A RNA Methylation Regulator YTHDF1 Correlated With Immune Microenvironment Predicts Clinical Outcomes and Therapeutic Efficacy in Breast Cancer

Objective: Increasing evidence highlights the roles of N6-methyladenosine (m6A) and its regulators in oncogenesis. Herein, this study observed the associations of m6A regulators with breast cancer.Methods: RNA-seq profiles of breast cancer were retrieved from the Cancer Genome Atlas (TCGA) database. The expression of m6A regulators was analyzed in tumor and normal tissues. Their expression correlations were analyzed by Spearson test. Overall survival (OS) analysis of these regulators was then presented. Gene set enrichment analysis (GSEA) was performed in high and low YTHDF1 expression groups. The correlations of YTHDF1 expression with immune cells and tumor mutation burden (TMB) were calculated in breast cancer samples. Somatic variation was assessed in high and low YTHDF1 expression groups.Results: Most of m6A regulators were abnormally expressed in breast cancer compared to normal tissues. At the mRNA levels, there were closely relationships between them. Among them, YTHDF1 up-regulation was significantly related to undesirable prognosis (p = 0.025). GSEA results showed that high YTHDF1 expression was associated with cancer-related pathways. Furthermore, YTHDF1 expression was significantly correlated with T cells CD4 memory activated, NK cells activated, monocytes, and macrophages. There were higher TMB scores in YTHDF1 up-regulation group than its down-regulation group. Missense mutation and non-sense mutation were the most frequent mutation types.Conclusion: Our findings suggested that dysregulated m6A regulator YTHDF1 was predictive of survival outcomes as well as response to immunotherapy of breast cancer, and were closely related to immune microenvironment.

Genomic Mosaicism Formed by Somatic Variation in the Aging and Diseased Brain

Over the past 20 years, analyses of single brain cell genomes have revealed that the brain is composed of cells with myriad distinct genomes: the brain is a genomic mosaic, generated by a host of DNA sequence-altering processes that occur somatically and do not affect the germline. As such, these sequence changes are not heritable. Some processes appear to occur during neurogenesis, when cells are mitotic, whereas others may also function in post-mitotic cells. Here, we review multiple forms of DNA sequence alterations that have now been documented: aneuploidies and aneusomies, smaller copy number variations (CNVs), somatic repeat expansions, retrotransposons, genomic cDNAs (gencDNAs) associated with somatic gene recombination (SGR), and single nucleotide variations (SNVs). A catch-all term of DNA content variation (DCV) has also been used to describe the overall phenomenon, which can include multiple forms within a single cell’s genome. A requisite step in the analyses of genomic mosaicism is ongoing technology development, which is also discussed. Genomic mosaicism alters one of the most stable biological molecules, DNA, which may have many repercussions, ranging from normal functions including effects of aging, to creating dysfunction that occurs in neurodegenerative and other brain diseases, most of which show sporadic presentation, unlinked to causal, heritable genes.

Assessment of Epigenetic and Phenotypic Variation in Populus nigra Regenerated via Sequential Regeneration

Somatic variation has been demonstrated in tissue culture regenerated plants of many species. In the genus Populus, phenotypic variation caused by changes in 5-methylcytosine within the plant genome have been reported. To date, the phenotypic and epigenetic stability of plants regenerated from sequential regeneration has not been tested in trees. In this study, we detected DNA methylation of CCGG sites in regenerated plants of five generations in Populus nigra using methylation-sensitive amplified polymorphisms, and evaluated their growth performance and physiological traits. About 10.86–26.80% of CCGG sites in the regenerated plant genome were demethylated and 5.50–8.45% were methylated, resulting in significantly lower DNA methylation levels among all regenerated plants than among donor plants. We detected a significant difference in methylation levels between first regeneration regenerated plants (G1) and those of the other four generations (G2–G5); there were no significant differences among the four later generations. Therefore, the dramatic decrease in DNA methylation levels occurred only in the first and second poplar regenerations; levels then stabilized later in the regeneration process, indicating that two regeneration events were sufficient to change the methylation statuses of almost all CCGG sites sensitive to regeneration. Differences in growth and physiological traits were observed between regenerated plants and donor plants, but were significant only among plants of certain generations. Significant correlations were detected between methylation level and transpiration rate, net photosynthetic rate, peroxidase activity, and instant water utilization efficiency, indicating the involvement of epigenetic regulation in this unpredictable phenotypic variation.

Assessment of the gene mosaicism burden in blood and its implications for immune disorders

There are increasing evidences showing the contribution of somatic genetic variants to non-cancer diseases. However, their detection using massive parallel sequencing methods still has important limitations. In addition, the relative importance and dynamics of somatic variation in healthy tissues are not fully understood. We performed high-depth whole-exome sequencing in 16 samples from patients with a previously determined pathogenic somatic variant for a primary immunodeficiency and tested different variant callers detection ability. Subsequently, we explored the load of somatic variants in the whole blood of these individuals and validated it by amplicon-based deep sequencing. Variant callers allowing low frequency read thresholds were able to detect most of the variants, even at very low frequencies in the tissue. The genetic load of somatic coding variants detectable in whole blood is low, ranging from 1 to 2 variants in our dataset, except for one case with 17 variants compatible with clonal haematopoiesis under genetic drift. Because of the ability we demonstrated to detect this type of genetic variation, and its relevant role in disorders such as primary immunodeficiencies, we suggest considering this model of gene mosaicism in future genetic studies and considering revisiting previous massive parallel sequencing data in patients with negative results.

Somatic PIK3R1 variation as a cause of vascular malformations and overgrowth

Purpose Somatic activating variants in the PI3K-AKT pathway cause vascular malformations with and without overgrowth. We previously reported an individual with capillary and lymphatic malformation harboring a pathogenic somatic variant in PIK3R1, which encodes three PI3K complex regulatory subunits. Here, we investigate PIK3R1 in a large cohort with vascular anomalies and identify an additional 16 individuals with somatic mosaic variants in PIK3R1. Methods Affected tissue from individuals with vascular lesions and overgrowth recruited from a multisite collaborative network was studied. Next-generation sequencing targeting coding regions of cell-signaling and cancer-associated genes was performed followed by assessment of variant pathogenicity. Results The phenotypic and variant spectrum associated with somatic variation in PIK3R1 is reported herein. Variants occurred in the inter-SH2 or N-terminal SH2 domains of all three PIK3R1 protein products. Phenotypic features overlapped those of the PIK3CA-related overgrowth spectrum (PROS). These overlapping features included mixed vascular malformations, sandal toe gap deformity with macrodactyly, lymphatic malformations, venous ectasias, and overgrowth of soft tissue or bone. Conclusion Somatic PIK3R1 variants sharing attributes with cancer-associated variants cause complex vascular malformations and overgrowth. The PIK3R1-associated phenotypic spectrum overlaps with PROS. These data extend understanding of the diverse phenotypic spectrum attributable to genetic variation in the PI3K-AKT pathway.

Fifty generations of amitosis: tracing asymmetric allele segregation in polyploid cells with single-cell DNA sequencing

Amitosis is a widespread form of unbalanced nuclear division whose biomedical and evolutionary significance remain unclear. Traditionally, insights into the genetics of amitosis are acquired by assessing the rate of phenotypic assortment. The phenotypic diversification of heterozygous clones during successive cell divisions reveals the random segregation of alleles to daughter nuclei. Though powerful, this experimental approach relies on the availability of phenotypic markers. Here, we present an approach that overcomes the requirement for phenotypic assortment. Leveraging Paramecium tetraurelia, a unicellular eukaryote with nuclear dimorphism and a highly polyploid somatic nucleus, we use single-cell whole-genome sequencing to track the assortment of developmentally acquired somatic DNA variants. Accounting for genome representation biases, we measure the effect of amitosis on allele segregation across the first ~50 amitotic divisions post self-fertilization and compare our empirical findings with theoretical predictions estimated via mathematical modeling. In line with our simulations, we show that amitosis in P. tetraurelia produces measurable but modest levels of somatic assortment. In forgoing the requirement for phenotypic assortment and employing developmental, environmentally induced somatic variation as molecular markers, our work provides a new powerful approach to investigate the consequences of amitosis in polyploid cells.

Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

Recent advances in throughput and accuracy mean that the Oxford Nanopore Technologies PromethION platform is a now a viable solution for genome sequencing. Much of the validation of bioinformatic tools for this long-read data has focussed on calling germline variants (including structural variants). Somatic variants are outnumbered many-fold by germline variants and their detection is further complicated by the effects of tumour purity/subclonality. Here, we evaluate the extent to which Nanopore sequencing enables detection and analysis of somatic variation. We do this through sequencing tumour and germline genomes for a patient with diffuse B-cell lymphoma and comparing results with 150 bp short-read sequencing of the same samples. Calling germline single nucleotide variants (SNVs) from specific chromosomes of the long-read data achieved good specificity and sensitivity. However, results of somatic SNV calling highlight the need for the development of specialised joint calling algorithms. We find the comparative genome-wide performance of different tools varies significantly between structural variant types, and suggest long reads are especially advantageous for calling large somatic deletions and duplications. Finally, we highlight the utility of long reads for phasing clinically relevant variants, confirming that a somatic 1.6 Mb deletion and a p.(Arg249Met) mutation involving TP53 are oriented in trans.