Multifocal epithelial tumors and field cancerization from loss of mesenchymal CSL signaling.

It is currently unclear whether tissue changes surrounding multifocal epithelial tumors are a cause or consequence of cancer. Here, we provide evidence that loss of mesenchymal Notch/CSL signaling causes tissue alterations, including stromal atrophy and inflammation, which precede and are potent triggers for epithelial tumors. Mice carrying a mesenchymal-specific deletion of CSL/RBP-Jκ, a key Notch effector, exhibit spontaneous multifocal keratinocyte tumors that develop after dermal atrophy and inflammation. CSL-deficient dermal fibroblasts promote increased tumor cell proliferation through upregulation of c-Jun and c-Fos expression and consequently higher levels of diffusible growth factors, inflammatory cytokines, and matrix-remodeling enzymes. In human skin samples, stromal fields adjacent to multifocal premalignant actinic keratosis lesions exhibit decreased Notch/CSL signaling and associated molecular changes. Importantly, these changes in gene expression are also induced by UVA, a known environmental cause of cutaneous field cancerization and skin cancer.

A Potential Contributory Role for Ciliary Dysfunction in the 16p11.2 600 kb BP4-BP5 Pathology.

The 16p11.2 600 kb copy-number variants (CNVs) are associated with mirror phenotypes on BMI, head circumference, and brain volume and represent frequent genetic lesions in autism spectrum disorders (ASDs) and schizophrenia. Here we interrogated the transcriptome of individuals carrying reciprocal 16p11.2 CNVs. Transcript perturbations correlated with clinical endophenotypes and were enriched for genes associated with ASDs, abnormalities of head size, and ciliopathies. Ciliary gene expression was also perturbed in orthologous mouse models, raising the possibility that ciliary dysfunction contributes to 16p11.2 pathologies. In support of this hypothesis, we found structural ciliary defects in the CA1 hippocampal region of 16p11.2 duplication mice. Moreover, by using an established zebrafish model, we show genetic interaction between KCTD13, a key driver of the mirrored neuroanatomical phenotypes of the 16p11.2 CNV, and ciliopathy-associated genes. Overexpression of BBS7 rescues head size and neuroanatomical defects of kctd13 morphants, whereas suppression or overexpression of CEP290 rescues phenotypes induced by KCTD13 under- or overexpression, respectively. Our data suggest that dysregulation of ciliopathy genes contributes to the clinical phenotypes of these CNVs.

Mirror extreme BMI phenotypes associated with gene dosage at the chromosome 16p11.2 locus.

Both obesity and being underweight have been associated with increased mortality. Underweight, defined as a body mass index (BMI) ≤ 18.5 kg per m(2) in adults and ≤ -2 standard deviations from the mean in children, is the main sign of a series of heterogeneous clinical conditions including failure to thrive, feeding and eating disorder and/or anorexia nervosa. In contrast to obesity, few genetic variants underlying these clinical conditions have been reported. We previously showed that hemizygosity of a ∼600-kilobase (kb) region on the short arm of chromosome 16 causes a highly penetrant form of obesity that is often associated with hyperphagia and intellectual disabilities. Here we show that the corresponding reciprocal duplication is associated with being underweight. We identified 138 duplication carriers (including 132 novel cases and 108 unrelated carriers) from individuals clinically referred for developmental or intellectual disabilities (DD/ID) or psychiatric disorders, or recruited from population-based cohorts. These carriers show significantly reduced postnatal weight and BMI. Half of the boys younger than five years are underweight with a probable diagnosis of failure to thrive, whereas adult duplication carriers have an 8.3-fold increased risk of being clinically underweight. We observe a trend towards increased severity in males, as well as a depletion of male carriers among non-medically ascertained cases. These features are associated with an unusually high frequency of selective and restrictive eating behaviours and a significant reduction in head circumference. Each of the observed phenotypes is the converse of one reported in carriers of deletions at this locus. The phenotypes correlate with changes in transcript levels for genes mapping within the duplication but not in flanking regions. The reciprocal impact of these 16p11.2 copy-number variants indicates that severe obesity and being underweight could have mirror aetiologies, possibly through contrasting effects on energy balance.

The impact of chromosomal rearrangements on regulation of gene expression.

The effects that coding region single-nucleotide polymorphisms or mutations have on gene expression have been well documented, predominantly owing to their association with disease. The effects of structural chromosomal rearrangements are also receiving increasing attention with the development of new techniques that allow accurate, high-resolution data, whether genomic interaction or transcriptome data, to be generated right down to the single-cell level. Over the past 18 months, these advances in experimental techniques have been used to further confirm and delineate the substantial effects that chromosome rearrangements can have on the regulation of gene expression and provide evidence of direct links between the two.

Topographic analysis of pancreatic cancer by TMA and digital spatial profiling reveals biological complexity with potential therapeutic implications.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal human malignancies. Tissue microarrays (TMA) are an established method of high throughput biomarker interrogation in tissues but may not capture histological features of cancer with potential biological relevance. Topographic TMAs (T-TMAs) representing pathophysiological hallmarks of cancer were constructed from representative, retrospective PDAC diagnostic material, including 72 individual core tissue samples. The T-TMA was interrogated with tissue hybridization-based experiments to confirm the accuracy of the topographic sampling, expression of pro-tumourigenic and immune mediators of cancer, totalling more than 750 individual biomarker analyses. A custom designed Next Generation Sequencing (NGS) panel and a spatial distribution-specific transcriptomic evaluation were also employed. The morphological choice of the pathophysiological hallmarks of cancer was confirmed by protein-specific expression. Quantitative analysis identified topography-specific patterns of expression in the IDO/TGF-ß axis; with a heterogeneous relationship of inflammation and desmoplasia across hallmark areas and a general but variable protein and gene expression of c-MET. NGS results highlighted underlying genetic heterogeneity within samples, which may have a confounding influence on the expression of a particular biomarker. T-TMAs, integrated with quantitative biomarker digital scoring, are useful tools to identify hallmark specific expression of biomarkers in pancreatic cancer.

Esrrg functions in early branch generation of the ureteric bud and is essential for normal development of the renal papilla.

Congenital anomalies of the kidney and urinary tract (CAKUTs) are common disorders of human development affecting the renal parechyma, renal pelvis, ureter, bladder and urethra; they show evidence of shared genetic aetiology, although the molecular basis of this remains unknown in the majority of cases. Breakpoint mapping of a de novo, apparently balanced, reciprocal translocation associated with bilateral renal agenesis has implicated the gene encoding the nuclear steroid hormone receptor ESRRG as a candidate gene for CAKUT. Here we show that the Esrrg protein is detected throughout early ureteric ducts as cytoplasmic/sub-membranous staining; with nuclear localization seen in developing nephrons. In 14.5-16.5 dpc (days post-conception) mouse embryos, Esrrg localizes to the subset of ductal tissue within the kidney, liver and lung. The renal ductal expression becomes localized to renal papilla by 18.5 dpc. Perturbation of function was performed in embryonic mouse kidney culture using pooled siRNA to induce knock-down and a specific small-molecule agonist to induce aberrant activation of Esrrg. Both resulted in severe abnormality of early branching events of the ureteric duct. Mouse embryos with a targeted inactivation of Esrrg on both alleles (Esrrg(-/-)) showed agenesis of the renal papilla but normal development of the cortex and remaining medulla. Taken together, these results suggest that Esrrg is required for early branching events of the ureteric duct that occur prior to the onset of nephrogenesis. These findings confirm ESRRG as a strong candidate gene for CAKUT.

The effect of translocation-induced nuclear reorganization on gene expression.

Translocations are known to affect the expression of genes at the breakpoints and, in the case of unbalanced translocations, alter the gene copy number. However, a comprehensive understanding of the functional impact of this class of variation is lacking. Here, we have studied the effect of balanced chromosomal rearrangements on gene expression by comparing the transcriptomes of cell lines from controls and individuals with the t(11;22)(q23;q11) translocation. The number of differentially expressed transcripts between translocation-carrying and control cohorts is significantly higher than that observed between control samples alone, suggesting that balanced rearrangements have a greater effect on gene expression than normal variation. Many of the affected genes are located along the length of the derived chromosome 11. We show that this chromosome is concomitantly altered in its spatial organization, occupying a more central position in the nucleus than its nonrearranged counterpart. Derivative 22-mapping chromosome 22 genes, on the other hand, remain in their usual environment. Our results are consistent with recent studies that experimentally altered nuclear organization, and indicated that nuclear position plays a functional role in regulating the expression of some genes in mammalian cells. Our study suggests that chromosomal translocations can result in hitherto unforeseen, large-scale changes in gene expression that are the consequence of alterations in normal chromosome territory positioning. This has consequences for the patterns of gene expression change seen during tumorigenesis-associated genome instability and during the karyotype changes that lead to speciation.

A 600 kb deletion syndrome at 16p11.2 leads to energy imbalance and neuropsychiatric disorders.

BACKGROUND: The recurrent ~600 kb 16p11.2 BP4-BP5 deletion is among the most frequent known genetic aetiologies of autism spectrum disorder (ASD) and related neurodevelopmental disorders. OBJECTIVE: To define the medical, neuropsychological, and behavioural phenotypes in carriers of this deletion. METHODS: We collected clinical data on 285 deletion carriers and performed detailed evaluations on 72 carriers and 68 intrafamilial non-carrier controls. RESULTS: When compared to intrafamilial controls, full scale intelligence quotient (FSIQ) is two standard deviations lower in carriers, and there is no difference between carriers referred for neurodevelopmental disorders and carriers identified through cascade family testing. Verbal IQ (mean 74) is lower than non-verbal IQ (mean 83) and a majority of carriers require speech therapy. Over 80% of individuals exhibit psychiatric disorders including ASD, which is present in 15% of the paediatric carriers. Increase in head circumference (HC) during infancy is similar to the HC and brain growth patterns observed in idiopathic ASD. Obesity, a major comorbidity present in 50% of the carriers by the age of 7 years, does not correlate with FSIQ or any behavioural trait. Seizures are present in 24% of carriers and occur independently of other symptoms. Malformations are infrequently found, confirming only a few of the previously reported associations. CONCLUSIONS: The 16p11.2 deletion impacts in a quantitative and independent manner FSIQ, behaviour and body mass index, possibly through direct influences on neural circuitry. Although non-specific, these features are clinically significant and reproducible. Lastly, this study demonstrates the necessity of studying large patient cohorts ascertained through multiple methods to characterise the clinical consequences of rare variants involved in common diseases.

Immunoglobulin/T Cell Receptor Capture Strategy for Comprehensive Immunogenetics.

In the era of genomic medicine, targeted next generation sequencing strategies (NGS) are becoming increasingly adopted by clinical molecular diagnostic laboratories to identify genetic diagnostic and prognostic biomarkers in hemato-oncology. We describe the EuroClonality-NGS DNA Capture (EuroClonality-NDC) assay, which is designed to simultaneously detect B and T cell clonal rearrangements, translocations, copy number alterations, and sequence variants. The accompanying validated bioinformatics pipeline enables production of an integrated report. The combination of the laboratory protocol and bioinformatics pipeline in the EuroClonality-NDC minimizes the potential for human error, reduces economic costs compared to current molecular testing strategies, and should improve diagnostic outcomes.

Side effects of genome structural changes.

The first extensive catalog of structural human variation was recently released. It showed that large stretches of genomic DNA that vary considerably in copy number were extremely abundant. Thus it is conceivable that they play a major role in functional variation. Consistently, genomic insertions and deletions were shown to contribute to phenotypic differences by modifying not only the expression levels of genes within the aneuploid segments but also of normal copy-number neighboring genes. In this report, we review the possible mechanisms behind this latter effect.

LINE retrotransposons characterize mammalian tissue-specific and evolutionarily dynamic regulatory regions.

BACKGROUND: To investigate the mechanisms driving regulatory evolution across tissues, we experimentally mapped promoters, enhancers, and gene expression in the liver, brain, muscle, and testis from ten diverse mammals. RESULTS: The regulatory landscape around genes included both tissue-shared and tissue-specific regulatory regions, where tissue-specific promoters and enhancers evolved most rapidly. Genomic regions switching between promoters and enhancers were more common across species, and less common across tissues within a single species. Long Interspersed Nuclear Elements (LINEs) played recurrent evolutionary roles: LINE L1s were associated with tissue-specific regulatory regions, whereas more ancient LINE L2s were associated with tissue-shared regulatory regions and with those switching between promoter and enhancer signatures across species. CONCLUSIONS: Our analyses of the tissue-specificity and evolutionary stability among promoters and enhancers reveal how specific LINE families have helped shape the dynamic mammalian regulome.

The Potential of Digital Image Analysis to Determine Tumor Cell Content in Biobanked Formalin-Fixed, Paraffin-Embedded Tissue Samples.

Introduction: Best practices dictate that biobanks ensure accurate determination of tumor content before supplying formalin-fixed, paraffin-embedded (FFPE) tissue samples to researchers for nucleic acid extraction and downstream molecular testing. It is advisable that trained and competent individuals, who understand the requirements of the downstream molecular tests, perform the microscopic morphological examination. However, the special skills, time, and costs associated with these assessments can be prohibitive, especially in large case cohorts requiring extensive pathological review. Determination of tumor content reliably by digital image analysis (DIA) could represent a significant advantage if validated, utilized, and deployed by biobanks. Materials and Methods: Whole slide digital scanned images of colorectal, lung, and breast cancer specimens were created. The scanned images were imported into the DIA software QuPath and digital annotations were completed by biobank technicians, under the direction of trained histopathology senior scientists. Automated cell detection was conducted and tumor epithelial cells were classified and quantified. Results: DIA scores were highly concordant with the manual assessment for 376 of 435 samples (86%). A detailed review of discordant cases indicated digital scores had a higher accuracy than the manual estimation. Conclusion: Automated digital quantification has the potential to replace visual estimations with reduced subjectivity and increased reliability compared with manual tumor estimations. We recommend the use of DIA by biobanks involved in provision of FFPE tissue samples, especially in large research studies requiring high volumes of cases to be analyzed.

Detection of Structural Variants in Circulating Cell-Free DNA from Sarcoma Patients Using Next Generation Sequencing.

Circulating tumour DNA (ctDNA) analysis using next generation sequencing (NGS) is being implemented in clinical practice for treatment stratification and disease monitoring. However, using ctDNA to detect structural variants, a common occurrence in sarcoma, can be challenging. Here, we use a sarcoma-specific targeted NGS panel to identify translocations and copy number variants in a cohort of 12 tissue specimens and matched circulating cell-free DNA (cfDNA) from soft tissue sarcoma patients, including alveolar rhabdomyosarcoma (n = 2), Ewing's Sarcoma (n = 2), synovial sarcoma (n = 2), extraskeletal myxoid chondrosarcoma (n = 1), clear cell sarcoma (n = 1), undifferentiated round cell sarcoma (n = 1), myxoid liposarcoma (n = 1), alveolar soft part cell sarcoma (n = 1) and dedifferentiated liposarcoma (n = 1). Structural variants were detected in 11/12 (91.6%) and 6/12 (50%) of tissue and plasma samples, respectively. Structural variants were detected in cfDNA at variant allele frequencies >0.2% with an average sequencing depth of 1026×. The results from this cohort show clinical potential for using NGS in ctDNA to aid in the diagnosis and clinical monitoring of sarcomas and warrant additional studies in larger cohorts.

Proteogenomics and Hi-C reveal transcriptional dysregulation in high hyperdiploid childhood acute lymphoblastic leukemia.

Hyperdiploidy, i.e. gain of whole chromosomes, is one of the most common genetic features of childhood acute lymphoblastic leukemia (ALL), but its pathogenetic impact is poorly understood. Here, we report a proteogenomic analysis on matched datasets from genomic profiling, RNA-sequencing, and mass spectrometry-based analysis of >8,000 genes and proteins as well as Hi-C of primary patient samples from hyperdiploid and ETV6/RUNX1-positive pediatric ALL. We show that CTCF and cohesin, which are master regulators of chromatin architecture, display low expression in hyperdiploid ALL. In line with this, a general genome-wide dysregulation of gene expression in relation to topologically associating domain (TAD) borders were seen in the hyperdiploid group. Furthermore, Hi-C of a limited number of hyperdiploid childhood ALL cases revealed that 2/4 cases displayed a clear loss of TAD boundary strength and 3/4 showed reduced insulation at TAD borders, with putative leukemogenic effects.

NF-κB-Dependent Lymphoid Enhancer Co-option Promotes Renal Carcinoma Metastasis.

Metastases, the spread of cancer cells to distant organs, cause the majority of cancer-related deaths. Few metastasis-specific driver mutations have been identified, suggesting aberrant gene regulation as a source of metastatic traits. However, how metastatic gene expression programs arise is poorly understood. Here, using human-derived metastasis models of renal cancer, we identify transcriptional enhancers that promote metastatic carcinoma progression. Specific enhancers and enhancer clusters are activated in metastatic cancer cell populations, and the associated gene expression patterns are predictive of poor patient outcome in clinical samples. We find that the renal cancer metastasis-associated enhancer complement consists of multiple coactivated tissue-specific enhancer modules. Specifically, we identify and functionally characterize a coregulatory enhancer cluster, activated by the renal cancer driver HIF2A and an NF-κB-driven lymphoid element, as a mediator of metastasis in vivo We conclude that oncogenic pathways can acquire metastatic phenotypes through cross-lineage co-option of physiologic epigenetic enhancer states.Significance: Renal cancer is associated with significant mortality due to metastasis. We show that in metastatic renal cancer, functionally important metastasis genes are activated via co-option of gene regulatory enhancer modules from distant developmental lineages, thus providing clues to the origins of metastatic cancer. Cancer Discov; 8(7); 850-65. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 781.

Hi-C as a tool for precise detection and characterisation of chromosomal rearrangements and copy number variation in human tumours.

Chromosomal rearrangements occur constitutionally in the general population and somatically in the majority of cancers. Detection of balanced rearrangements, such as reciprocal translocations and inversions, is troublesome, which is particularly detrimental in oncology where rearrangements play diagnostic and prognostic roles. Here we describe the use of Hi-C as a tool for detection of both balanced and unbalanced chromosomal rearrangements in primary human tumour samples, with the potential to define chromosome breakpoints to bp resolution. In addition, we show copy number profiles can also be obtained from the same data, all at a significantly lower cost than standard sequencing approaches.

A diminutive chromosome 21 centromere in acute lymphoblastic leukemia.

A chance observation of a tiny constitutional variant for the centromere of chromosome 21 in two patients with acute lymphoblastic leukemia (ALL), suggested a possible correlation with the cytogenetic findings in their leukemic cells. Interphase FISH revealed three 13/21 centromeric signals and a single MLL signal in the blast cells of each patient. Metaphase FISH with dual-color application of whole-chromosome paint (wcp) and centromeric probes for chromosome 21 showed two copies of chromosome 21, one with a tiny centromeric signal which corresponded to the invisible centromere in the interphase cells. Patient 2700 had a normal karyotype in his bone marrow at diagnosis. All metaphases from his stimulated peripheral blood also had the tiny chromosome 21 centromere, proving it to be a constitutional variant. Patient 3314 showed the abnormal karyotype 46,XY,inv(1)(p?q?),del(11)(q?),del(12)(p?),inc in his bone marrow. Interphase FISH revealed only one copy each of the ABL and ETV6 genes, in addition to the loss of the MLL signal. The question arises, is there an association between the diminutive centromeric signals for chromosome 21 and the chromosomal instability demonstrated by the deletions of key genes from the leukemic blasts of these two patients?

Genetic Analysis of 'PAX6-Negative' Individuals with Aniridia or Gillespie Syndrome.

We report molecular genetic analysis of 42 affected individuals referred with a diagnosis of aniridia who previously screened as negative for intragenic PAX6 mutations. Of these 42, the diagnoses were 31 individuals with aniridia and 11 individuals referred with a diagnosis of Gillespie syndrome (iris hypoplasia, ataxia and mild to moderate developmental delay). Array-based comparative genomic hybridization identified six whole gene deletions: four encompassing PAX6 and two encompassing FOXC1. Six deletions with plausible cis-regulatory effects were identified: five that were 3' (telomeric) to PAX6 and one within a gene desert 5' (telomeric) to PITX2. Sequence analysis of the FOXC1 and PITX2 coding regions identified two plausibly pathogenic de novo FOXC1 missense mutations (p.Pro79Thr and p.Leu101Pro). No intragenic mutations were detected in PITX2. FISH mapping in an individual with Gillespie-like syndrome with an apparently balanced X;11 reciprocal translocation revealed disruption of a gene at each breakpoint: ARHGAP6 on the X chromosome and PHF21A on chromosome 11. In the other individuals with Gillespie syndrome no mutations were identified in either of these genes, or in HCCS which lies close to the Xp breakpoint. Disruption of PHF21A has previously been implicated in the causation of intellectual disability (but not aniridia). Plausibly causative mutations were identified in 15 out of 42 individuals (12/32 aniridia; 3/11 Gillespie syndrome). Fourteen of these mutations presented in the known aniridia genes; PAX6, FOXC1 and PITX2. The large number of individuals in the cohort with no mutation identified suggests greater locus heterogeneity may exist in both isolated and syndromic aniridia than was previously appreciated.

Structural variation-associated expression changes are paralleled by chromatin architecture modifications.

Copy number variants (CNVs) influence the expression of genes that map not only within the rearrangement, but also to its flanks. To assess the possible mechanism(s) underlying this "neighboring effect", we compared intrachromosomal interactions and histone modifications in cell lines of patients affected by genomic disorders and control individuals. Using chromosome conformation capture (4C-seq), we observed that a set of genes flanking the Williams-Beuren Syndrome critical region (WBSCR) were often looping together. The newly identified interacting genes include AUTS2, mutations of which are associated with autism and intellectual disabilities. Deletion of the WBSCR disrupts the expression of this group of flanking genes, as well as long-range interactions between them and the rearranged interval. We also pinpointed concomitant changes in histone modifications between samples. We conclude that large genomic rearrangements can lead to chromatin conformation changes that extend far away from the structural variant, thereby possibly modulating expression globally and modifying the phenotype. GEO SERIES ACCESSION NUMBER: GSE33784, GSE33867.

Structural variation and its effect on expression.

Structural variation, whether it is caused by copy number variants or present in a balanced form, such as reciprocal translocations and inversions, can have a profound and dramatic effect on the expression of genes mapping within and close to the rearrangement, as well as affecting others genome wide. These effects can be caused by altering the copy number of one or more genes or regulatory elements (dosage effect) or from physical disruption of links between regulatory elements and their associated gene or genes, resulting in perturbation of expression. Similarly, large-scale structural variants can result in genome-wide expression changes by altering the positions that chromosomes occupy within the nucleus, potentially disrupting not only local cis interactions, but also trans interactions that occur throughout the genome. Structural variation is, therefore, a significant factor in the study of gene expression and is discussed here in more detail.