Noninvasive Discrimination of Low and High-risk Pancreatic Intraductal Papillary Mucinous Neoplasms.

OBJECTIVE: To propose a noninvasive diagnostic approach, which allows reliable distinction between low- and high-risk pancreatic intraductal papillary mucinous neoplasms (IPMNs). BACKGROUND: IPMNs are identifiable precursor lesions of pancreatic cancer, of which surgical resection is warranted prior to the development of invasive carcinoma, but low-grade IPMNs should not be unnecessarily resected. However, diagnostic tools that preoperatively enable accurate risk stratification of IPMNs are missing. METHODS: This single-center, retrospective cohort study included 56 patients who underwent surgical resection for IPMN including 18 low-risk (low-grade) and 38 high-risk (high-grade/invasive carcinoma) IPMNs, from whom clinical features and serum samples were prospectively obtained. An antibody microarray platform was used to analyze the serum proteome. Based on serum markers and selected clinical characteristics support vector machine models were constructed to predict the risk of IPMN malignancy. RESULTS: A serum protein signature discriminating low- and high-risk IPMN patients was identified. Combinations of established clinical features and the newly identified serum biomarkers correctly distinguished low- and high-risk IPMNs in 93% on 1000-fold cross-validation. CONCLUSIONS: This study highlights the synergistic predictive value of combining a novel serum protein signature with conventional clinical characteristics to risk-stratify IPMN patients. If these findings are supported by larger validation studies, they might enable more rational decision-making in clinical management of IPMN patients in conjunction with clinical guidelines.

Contribution of copy-number variation to Down syndrome-associated atrioventricular septal defects.

PURPOSE: The goal of this study was to identify the contribution of large copy-number variants to Down syndrome-associated atrioventricular septal defects, the risk for which in the trisomic population is 2,000-fold more as compared with that of the general disomic population. METHODS: Genome-wide copy-number variant analysis was performed on 452 individuals with Down syndrome (210 cases with complete atrioventricular septal defects; 242 controls with structurally normal hearts) using Affymetrix SNP 6.0 arrays, making this the largest heart study conducted to date on a trisomic background. RESULTS: Large, common copy-number variants with substantial effect sizes (OR > 2.0) do not account for the increased risk observed in Down syndrome-associated atrioventricular septal defects. By contrast, cases had a greater burden of large, rare deletions (P < 0.01) and intersected more genes (P < 0.007) as compared with controls. We also observed a suggestive enrichment of deletions intersecting ciliome genes in cases as compared with controls. CONCLUSION: Our data provide strong evidence that large, rare deletions increase the risk of Down syndrome-associated atrioventricular septal defects, whereas large, common copy-number variants do not appear to increase the risk of Down syndrome-associated atrioventricular septal defects. The genetic architecture of atrioventricular septal defects is complex and multifactorial in nature.

Excess variants in AFF2 detected by massively parallel sequencing of males with autism spectrum disorder.

Autism spectrum disorder (ASD) is a heterogeneous disorder with substantial heritability, most of which is unexplained. ASD has a population prevalence of one percent and affects four times as many males as females. Patients with fragile X E (FRAXE) intellectual disability, which is caused by a silencing of the X-linked gene AFF2, display a number of ASD-like phenotypes. Duplications and deletions at the AFF2 locus have also been reported in cases with moderate intellectual disability and ASD. We hypothesized that other rare X-linked sequence variants at the AFF2 locus might contribute to ASD. We sequenced the AFF2 genomic region in 202 male ASD probands and found that 2.5% of males sequenced had missense mutations at highly conserved evolutionary sites. When compared with the frequency of missense mutations in 5545 X chromosomes from unaffected controls, we saw a statistically significant enrichment in patients with ASD (OR: 4.9; P < 0.014). In addition, we identified rare AFF2 3' UTR variants at conserved sites which alter gene expression in a luciferase assay. These data suggest that rare variation in AFF2 may be a previously unrecognized ASD susceptibility locus and may help explain some of the male excess of ASD.

Exome sequencing identifies a novel FOXP3 mutation in a 2-generation family with inflammatory bowel disease.

OBJECTIVES: Inflammatory bowel disease (IBD) is heritable, but a total of 163 variants commonly implicated in IBD pathogenesis account for only 25% of the heritability. Rare, highly penetrant genetic variants may also explain mendelian forms of IBD and some of the missing heritability. To test the hypothesis that rare loss-of-function mutations can be causative, we performed whole exome sequencing (WES) on 5 members of a 2-generation family of European ancestry presenting with an early-onset and atypical form of IBD. METHODS: WES was performed for all of the 5 family members; the mother and 3 male offspring were affected, whereas the father was unaffected. Mapping, annotation, and filtering criteria were used to reduce candidate variants. For functional testing we performed forkhead box P3 (FOXP3) staining and a T-cell suppression assay. RESULTS: We identified a novel missense variant in exon 6 of the X-linked FOXP3 gene. The c.694A>C substitution in FOXP3 results in a cysteine-to-glycine change at the protein position 232 that is completely conserved among all vertebrates. This variant (heterozygous in the mother and hemizygous in all 3 affected sons) did not impair FOXP3 protein expression, but significantly reduced the ability of the host's T regulatory cells to suppress an inappropriate autoimmune response. The variant results in a milder immune dysregulation, polyendocrinopathy, enteropathy, and X-linked phenotype with early-onset IBD. CONCLUSIONS: Our study illustrates the successful application of WES for making a definitive molecular diagnosis in a case of multiply affected families, with atypical IBD-like phenotype. Our results also have important implications for disease biology and disease-directed therapeutic development.

Prenatal alcohol exposure is associated with changes in placental gene co-expression networks.

Alcohol consumption during pregnancy can result in a range of adverse postnatal outcomes among exposed children. However, identifying at-risk children is challenging given the difficulty to confirm prenatal alcohol exposure and the lack of early diagnostic tools. Placental surveys present an important opportunity to uncover early biomarkers to identify those at risk. Here, we report the first transcriptome-wide evaluation to comprehensively evaluate human placental pathways altered by fetal alcohol exposure. In a prospective longitudinal birth cohort in Cape Town, South Africa, we performed bulk tissue RNAseq in placenta samples from 32 women reporting heavy drinking during pregnancy and 30 abstainers/light drinkers. Weighted gene co-expression network analysis (WGCNA) and differential gene expression analysis were performed to assess associations between fetal alcohol exposure and placental gene expression patterns at a network-wide and single gene level, respectively. The results revealed altered expression in genes related to erythropoiesis and angiogenesis, which are implicated in established postnatal phenotypes related to alcohol exposure, including disruptions in iron homeostasis, growth, and neurodevelopment. The reported findings provide insights into the molecular pathways affected by prenatal alcohol exposure and highlight the potential of placental biomarkers for detecting and understanding the effects of alcohol on fetal development.

Evolution of the immune landscape during progression of pancreatic intraductal papillary mucinous neoplasms to invasive cancer.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMNs) are precursor lesions of pancreatic cancer, which is characterized by an immunosuppressive microenvironment. Yet, the spatial distribution of the immune infiltrate and how it changes during IPMN progression is just beginning to be understood. METHODS: We obtained tissue samples from patients who underwent pancreatic surgery for IPMN, and performed comprehensive immunohistochemical analyses to investigate the clinical significance, composition and spatial organization of the immune microenvironment during progression of IPMNs. Survival analysis of pancreatic cancer patients was stratified by tumour infiltrating immune cell subtypes. FINDINGS: The immune microenvironment evolves from a diverse T cell mixture, comprising CD8+ T cells, Th/c1 and Th/c2 as major players combined with Th9, Th/c17, Th22, and Treg cells in low-grade IPMN, to a Treg dominated immunosuppressive state in invasive pancreatic cancer. Organized lymphoid clusters formed in IPMN surrounding stroma and accumulated immunosuppressive cell types during tumour progression. Survival of pancreatic cancer patients correlated with Th2 signatures in the tumour microenvironment. INTERPRETATION: The major change with regards to T cell composition during IPMN progression occurs at the step of tissue invasion, indicating that malignant transformation only occurs when tumour immune surveillance is overcome. This suggests that novel immunotherapies that would boost spontaneous antitumor immunity at premalignant states could prevent pancreatic cancer development. FUNDING: The present work was supported by German Cancer Aid grants (70,112,720 and 70,113,167) to S. R., and the Olympia Morata Programme of the Medical Faculty of Heidelberg University to S. R.

ATRX, DAXX or MEN1 mutant pancreatic neuroendocrine tumors are a distinct alpha-cell signature subgroup.

The commonly mutated genes in pancreatic neuroendocrine tumors (PanNETs) are ATRX, DAXX, and MEN1. We genotyped 64 PanNETs and found 58% carry ATRX, DAXX, and MEN1 mutations (A-D-M mutant PanNETs) and this correlates with a worse clinical outcome than tumors carrying the wild-type alleles of all three genes (A-D-M WT PanNETs). We performed RNA sequencing and DNA-methylation analysis to reveal two distinct subgroups with one consisting entirely of A-D-M mutant PanNETs. Two genes differentiating A-D-M mutant from A-D-M WT PanNETs were high ARX and low PDX1 gene expression with PDX1 promoter hyper-methylation in the A-D-M mutant PanNETs. Moreover, A-D-M mutant PanNETs had a gene expression signature related to that of alpha-cells (FDR q-value < 0.009) of pancreatic islets including increased expression of HNF1A and its transcriptional target genes. This gene expression profile suggests that A-D-M mutant PanNETs originate from or transdifferentiate into a distinct cell type similar to alpha cells.

Atrx inactivation drives disease-defining phenotypes in glioma cells of origin through global epigenomic remodeling.

Mutational inactivation of the SWI/SNF chromatin regulator ATRX occurs frequently in gliomas, the most common primary brain tumors. Whether and how ATRX deficiency promotes oncogenesis by epigenomic dysregulation remains unclear, despite its recent implication in both genomic instability and telomere dysfunction. Here we report that Atrx loss recapitulates characteristic disease phenotypes and molecular features in putative glioma cells of origin, inducing cellular motility although also shifting differentiation state and potential toward an astrocytic rather than neuronal histiogenic profile. Moreover, Atrx deficiency drives widespread shifts in chromatin accessibility, histone composition, and transcription in a distribution almost entirely restricted to genomic sites normally bound by the protein. Finally, direct gene targets of Atrx that mediate specific Atrx-deficient phenotypes in vitro exhibit similarly selective misexpression in ATRX-mutant human gliomas. These findings demonstrate that ATRX deficiency and its epigenomic sequelae are sufficient to induce disease-defining oncogenic phenotypes in appropriate cellular and molecular contexts.

Integrated Genomic Analysis of Hürthle Cell Cancer Reveals Oncogenic Drivers, Recurrent Mitochondrial Mutations, and Unique Chromosomal Landscapes.

The molecular foundations of Hürthle cell carcinoma (HCC) are poorly understood. Here we describe a comprehensive genomic characterization of 56 primary HCC tumors that span the spectrum of tumor behavior. We elucidate the mutational profile and driver mutations and show that these tumors exhibit a wide range of recurrent mutations. Notably, we report a high number of disruptive mutations to both protein-coding and tRNA-encoding regions of the mitochondrial genome. We reveal unique chromosomal landscapes that involve whole-chromosomal duplications of chromosomes 5 and 7 and widespread loss of heterozygosity arising from haploidization and copy-number-neutral uniparental disomy. We also identify fusion genes and disrupted signaling pathways that may drive disease pathogenesis.

Integrated Genomics for Pinpointing Survival Loci within Arm-Level Somatic Copy Number Alterations.

The identification of driver loci underlying arm-level somatic copy number alterations (SCNAs) in cancer has remained challenging and incomplete. Here, we assess the relative impact and present a detailed landscape of arm-level SCNAs in 10,985 patient samples across 33 cancer types from The Cancer Genome Atlas (TCGA). Furthermore, using chromosome 9p loss in lower grade glioma (LGG) as a model, we employ a unique multi-tiered genomic dissection strategy using 540 patients from three independent LGG datasets to identify genetic loci that govern tumor aggressiveness and poor survival. This comprehensive approach uncovered several 9p loss-specific prognostic markers, validated existing ones, and redefined the impact of CDKN2A loss in LGG.

Dissecting Allele Architecture of Early Onset IBD Using High-Density Genotyping.

BACKGROUND: The inflammatory bowel diseases (IBD) are common, complex disorders in which genetic and environmental factors are believed to interact leading to chronic inflammatory responses against the gut microbiota. Earlier genetic studies performed in mostly adult population of European descent identified 163 loci affecting IBD risk, but most have relatively modest effect sizes, and altogether explain only ~20% of the genetic susceptibility. Pediatric onset represents about 25% of overall incident cases in IBD, characterized by distinct disease physiology, course and risks. The goal of this study is to compare the allelic architecture of early onset IBD with adult onset in population of European descent. METHODS: We performed a fine mapping association study of early onset IBD using high-density Immunochip genotyping on 1008 pediatric-onset IBD cases (801 Crohn's disease; 121 ulcerative colitis and 86 IBD undetermined) and 1633 healthy controls. Of the 158 SNP genotypes obtained (out of the 163 identified in adult onset), this study replicated 4% (5 SNPs out of 136) of the SNPs identified in the Crohn's disease (CD) cases and 0.8% (1 SNP out of 128) in the ulcerative colitis (UC) cases. Replicated SNPs implicated the well known NOD2 and IL23R. The point estimate for the odds ratio (ORs) for NOD2 was above and outside the confidence intervals reported in adult onset. A polygenic liability score weakly predicted the age of onset for a larger collection of CD cases (p< 0.03, R2= 0.007), but not for the smaller number of UC cases. CONCLUSIONS: The allelic architecture of common susceptibility variants for early onset IBD is similar to that of adult onset. This immunochip genotyping study failed to identify additional common variants that may explain the distinct phenotype that characterize early onset IBD. A comprehensive dissection of genetic loci is necessary to further characterize the genetic architecture of early onset IBD.

Tandem repeats and G-rich sequences are enriched at human CNV breakpoints.

Chromosome breakage in germline and somatic genomes gives rise to copy number variation (CNV) responsible for genomic disorders and tumorigenesis. DNA sequence is known to play an important role in breakage at chromosome fragile sites; however, the sequences susceptible to double-strand breaks (DSBs) underlying CNV formation are largely unknown. Here we analyze 140 germline CNV breakpoints from 116 individuals to identify DNA sequences enriched at breakpoint loci compared to 2800 simulated control regions. We find that, overall, CNV breakpoints are enriched in tandem repeats and sequences predicted to form G-quadruplexes. G-rich repeats are overrepresented at terminal deletion breakpoints, which may be important for the addition of a new telomere. Interstitial deletions and duplication breakpoints are enriched in Alu repeats that in some cases mediate non-allelic homologous recombination (NAHR) between the two sides of the rearrangement. CNV breakpoints are enriched in certain classes of repeats that may play a role in DNA secondary structure, DSB susceptibility and/or DNA replication errors.