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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.

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.

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. High hyperploidy is a common feature in childhood B-cell precursor acute lymphoblastic leukemia. Here, the authors perform proteogenomic and Hi-C analyses of this leukemia and the ETV6/RUNX1 subtype and show that CTCF and cohesin expression are low in hyperdiploid cases and transcriptional dysregulation in relation to topologically associating domain borders in some of these cases.

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-β 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.

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. GSE33784, GSE33867.

Bilateral renal agenesis/hypoplasia/dysplasia (BRAHD) is a relatively common, lethal malformation in humans. Established clinical risk factors include maternal insulin dependent diabetes mellitus and male sex of the fetus. In the majority of cases, no specific etiology can be established, although teratogenic, syndromal and single gene causes can be assigned to some cases. 45 unrelated fetuses, stillbirths or infants with lethal BRAHD were ascertained through a single regional paediatric pathology service (male:female 34:11 or 3.1:1). The previously reported phenotypic overlaps with VACTERL, caudal dysgenesis, hemifacial microsomia and Müllerian defects were confirmed. A new finding is that 16/45 (35.6%; m:f 13:3 or 4.3:1) BRAHD cases had one or more extrarenal malformations indicative of a disoder of laterality determination including; incomplete lobulation of right lung (seven cases), malrotation of the gut (seven cases) and persistence of the left superior vena cava (five cases). One such case with multiple laterality defects and sirelomelia was found to have a de novo apparently balanced reciprocal translocation 46,XY,t(2;6)(p22.3;q12). Translocation breakpoint mapping was performed by interphase fluorescent in-situ hybridization (FISH) using nuclei extracted from archival tissue sections in both this case and an isolated bilateral renal agenesis case associated with a de novo 46,XY,t(1;2)(q41;p25.3). Both t(2;6) breakpoints mapped to gene-free regions with no strong evidence of cis-regulatory potential. Ten genes localized within 500 kb of the t(1;2) breakpoints. Wholemount in-situ expression analyses of the mouse orthologs of these genes in embryonic mouse kidneys showed strong expression of Esrrg, encoding a nuclear steroid hormone receptor. Immunohistochemical analysis showed that Esrrg was restricted to proximal ductal tissue within the embryonic kidney. The previously unreported association of BRAHD with laterality defects suggests that renal agenesis may share a common etiology with heterotaxy in some cases. Translocation breakpoint mapping identified ESRRG as a plausible candidate gene for BRAHD.

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.

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.

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.

Bilateral renal agenesis/hypoplasia/dysplasia (BRAHD) is a relatively common, lethal malformation in humans. Established clinical risk factors include maternal insulin dependent diabetes mellitus and male sex of the fetus. In the majority of cases, no specific etiology can be established, although teratogenic, syndromal and single gene causes can be assigned to some cases. 45 unrelated fetuses, stillbirths or infants with lethal BRAHD were ascertained through a single regional paediatric pathology service (male:female 34:11 or 3.1:1). The previously reported phenotypic overlaps with VACTERL, caudal dysgenesis, hemifacial microsomia and Müllerian defects were confirmed. A new finding is that 16/45 (35.6%; m:f 13:3 or 4.3:1) BRAHD cases had one or more extrarenal malformations indicative of a disoder of laterality determination including; incomplete lobulation of right lung (seven cases), malrotation of the gut (seven cases) and persistence of the left superior vena cava (five cases). One such case with multiple laterality defects and sirelomelia was found to have a de novo apparently balanced reciprocal translocation 46,XY,t(2;6)(p22.3;q12). Translocation breakpoint mapping was performed by interphase fluorescent in-situ hybridization (FISH) using nuclei extracted from archival tissue sections in both this case and an isolated bilateral renal agenesis case associated with a de novo 46,XY,t(1;2)(q41;p25.3). Both t(2;6) breakpoints mapped to gene-free regions with no strong evidence of cis-regulatory potential. Ten genes localized within 500 kb of the t(1;2) breakpoints. Wholemount in-situ expression analyses of the mouse orthologs of these genes in embryonic mouse kidneys showed strong expression of Esrrg, encoding a nuclear steroid hormone receptor. Immunohistochemical analysis showed that Esrrg was restricted to proximal ductal tissue within the embryonic kidney. The previously unreported association of BRAHD with laterality defects suggests that renal agenesis may share a common etiology with heterotaxy in some cases. Translocation breakpoint mapping identified ESRRG as a plausible candidate gene for BRAHD.