Faculty Bibliography

Unveiling the transcriptome of human blastocysts can provide a wealth of important information regarding early embryonic ontology. Comparing the mRNA production of embryos with normal and abnormal karyotypes allows for a deeper understanding of the protein pathways leading to viability and aberrant fetal development. In addition, identifying transcripts specific for normal or abnormal chromosome copy number could aid in the search for secreted substances that could be used to non-invasively identify embryos best suited for IVF embryo transfer. Using RNA-seq, we characterized the transcriptome of 71 normally developing human blastocysts that were karyotypically normal vs. trisomic or monosomic. Every monosomy and trisomy of the autosomal and sex chromosomes were evaluated, mostly in duplicate. We first mapped the transcriptome of three normal embryos and found that a common core of more than 3,000 genes is expressed in all embryos. These genes represent pathways related to actively dividing cells, such as ribosome biogenesis and function, spliceosome, oxidative phosphorylation, cell cycle and metabolic pathways. We then compared transcriptome profiles of aneuploid embryos to those of normal embryos. We observed that non-viable embryos had a large number of dysregulated genes, some showing a hundred-fold difference in expression. On the contrary, sex chromosome abnormalities, XO and XXX displayed transcriptomes more closely mimicking those embryos with 23 normal chromosome pairs. Intriguingly, we identified a set of commonly deregulated genes in the majority of both trisomies and monosomies. This is the first paper demonstrating a comprehensive transcriptome delineation of karyotypic abnormalities found in the human pre-implantation embryo. We believe that this information will contribute to the development of new pre-implantation genetic screening methods as well as a better understanding of the underlying developmental abnormalities of abnormal embryos, fetuses and children.

Purpose: Acrylamide, a probable human carcinogen (Group 2A), is ubiquitously present in the human environment. The compound is found in heated starchy foods, coffee, as well as cigarette smoke. Humans are also exposed to acrylamide occupationally. The carcinogenicity of acrylamide has been attributed to the effects of glycidamide, its reactive and mutagenic epoxide metabolite shown to be an inducer of rodent tumors at various anatomical sites. Covalent adducts with haemoglobin were reported in humans exposed to acrylamide, and DNA adducts in experimental systems. Methods: In order to characterize the pre-mutagenic DNA lesions and global mutation spectra induced by acrylamide and glycidamide, we combined DNA-adduct and wholeexome sequencing analyses in an established exposureclonal immortalization system based on mouse embryonic fibroblasts. Results: Sequencing and computational analysis of cell clones immortalized after exposure revealed a unique mutational signature of glycidamide, characterized by predominant T:A>A:T transversions, T:A>C:G transitions and C:G>A:T transversions in specific trinucleotide contexts, and with significant transcription strand bias. Computational interrogation of the human pan-cancer genome sequencing data (PCAWG data set) using experimentally derived glycidamide signature revealed the presence of the glycidamide signature in lung adenocarcinomas, lung squamous cell carcinomas and liver cancer, manifesting as a sub-component of the COSMIC tobacco smoking-related signature 4, as well as possibly associated with dietary intake of acrylamide. Conclusions: Our study demonstrates how the controlled experimental characterization of specific genetic damage associated with glycidamide exposure facilitates identifying corresponding patterns in cancer genome data, thereby underscoring how mutational signature laboratory experimentation contributes to the elucidation of cancer causation

An unbiased platelet transcriptome profile identified ATP binding cassette subfamily C member 4 (ABCC4) as a novel mediator of platelet activity in virologically suppressed human immunodeficiency virus (HIV)-infected subjects on antiretroviral therapy. Using ex vivo and in vitro cellular and molecular assays we demonstrated that ABCC4 regulated platelet activation by altering granule release and cyclic nucleotide homeostasis through a cAMP-protein kinase A (PKA)-mediated mechanism. Platelet ABCC4 inhibition attenuated platelet activation and effector cell function by reducing the release of inflammatory mediators, such as sphingosine-1-phosphate. ABCC4 inhibition may represent a novel antithrombotic strategy in HIV-infected subjects on antiretroviral therapy.

BACKGROUND: Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. METHODS: We analyzed pediatric and adult pineoblastoma samples (n=23) using integrated genomic studies, including genome-wide DNA methylation profiling, whole-exome or whole-genome sequencing, and whole-transcriptome analysis. RESULTS: Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower grade pineal tumors and normal pineal gland. Recurrent somatic mutations were found in genes involved in PKA-and NF-kappaB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expression of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain. Whole-transcriptome analysis showed that homozygous loss of DROSHA led to distinct changes in RNA expression profile. Disruption of the DROSHA locus in human neural stem cells using the CRISPR/Cas9 system, led to decrease of the DROSHA protein, and massive loss of miRNAs. CONCLUSION: We identified recurrent homozygous deletions of DROSHA in pineoblastoma, suggesting that different mechanisms disrupting miRNA processing are involved in the pathogenesis of familial versus sporadic pineoblastoma. Furthermore, a novel microduplication of PDE4DIP leading to upregulation of DUF1220 protein suggests DUF1220 as a novel oncogenic driver in pineoblastoma

Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. Here, we analyzed pediatric and adult pineoblastoma samples (n = 23) using a combination of genome-wide DNA methylation profiling and whole-exome sequencing or whole-genome sequencing. Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower-grade pineal tumors and normal pineal gland. Recurrent variants were found in genes involved in PKA- and NF-κB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expresion of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain.

BACKGROUND:In lung cancer, upregulation of the PI3K pathway is an early event that contributes to cell proliferation, survival, and tissue invasion. Upregulation of this pathway was recently described as associated with enrichment of the lower airways with bacteria identified as oral commensals. We hypothesize that host-microbe interactions in the lower airways of subjects with lung cancer affect known cancer pathways. METHODS:Airway brushes were collected prospectively from subjects with lung nodules at time of diagnostic bronchoscopy, including 39 subjects with final lung cancer diagnoses and 36 subjects with non-cancer diagnosis. Additionally, samples from 10 healthy control subjects were included. 16S rRNA gene amplicon sequencing and paired transcriptome sequencing (RNAseq) were performed on all airway samples. In addition, an in vitro model with airway epithelial cells exposed to bacteria/bacterial products was performed. RESULTS:The composition of the lower airway transcriptome in the cancer patients was significantly different from the controls, which included upregulation of ERK and PI3K signaling pathways. The lower airways of lung cancer patients were enriched for oral taxa (Streptococcus and Veillonella), which was associated with upregulation of the ERK and PI3K signaling pathways. In vitro exposure of airway epithelial cells to Veillonella, Prevotella, and Streptococcus led to upregulation of these same signaling pathways. CONCLUSIONS:The data presented here shows that several transcriptomic signatures previously identified as relevant to lung cancer pathogenesis are associated with enrichment of the lower airway microbiota with oral commensals.

Objective: To characterize the gut microbiota and host gene interactions in Reactive Arthritis (ReA) and postinfectious SpA. Methods: 32 patients with ReA and 32 controls with preceding Gastrointestinal (GI) and/or Genitourinary (GU) infections that did not develop arthritiswere prospectively recruited in Guatemala, a highly prevalent geographic region for this disease. Clinical variables, HLA status and 16S ribosomal RNA gene sequencing of intestinal microbiota were analysed. Results: Subjects with ReA showed no significant differences from controls in gut bacterial richness, alpha or beta diversity. However there was a higher abundance of Erwinia and Pseudomonas, and increased prevalence of typical enteropathogens associated with ReA. In Fact, at least one enteropathogen was present in 71.9% of ReA subjects vs 46.8% of controls (p<0.05). Subjects with ultrasound evidence of enthesitiswere enriched with Campylobacter,while subjects with Uveitis and radiographic sacroiliitis were enriched with Erwinia and unclassified Ruminococcaceae, respectively. Both were enriched in Dialister (log LDA>2). Host genetics, particularly HLA-A24 were associated with differences in gut microbiota diversity irrespective of disease status. We identified several co-occurring taxa that were also predictive of HLA-A24 status, including Ruminococcaceae-Rikenellaceae-Coriobacteriacea and Prevotellaceaeunclassified Sphingobacteriales-Elusimicrobiaceae. Conclusion: This is the first culture-independent study characterizing the gut microbial community of ReA. Although bacterial factors correlated with disease presence and clinical features of ReA, host genetics also appeared to be a major independent driver of intestinal community composition. Understanding of these gut microbiota host-genetic relationships may further clarify the pathogenesis of ReA and related Spondyloarthritis

Background: Microglandular adenosis (MGA) is a rare borderline lesion of the breast characterized by an infiltrative collection of small glands that characteristically lack a myoepithelial cell layer. MGA is associated with invasive carcinoma in 20-30% of cases, and has been proposed as a non-obligate precursor to basal-like breast cancers. Somatic TP53 mutation of MGA and its associated carcinoma has been previously reported. We identified a case of triple negative metaplastic carcinoma with mesenchymal differentiation with morphologic and immunohistochemical evidence of progression from MGA to atypical MGA (AMGA), carcinoma in situ (CIS) and invasive carcinoma. We performed laser microdissection and whole exome sequencing of each four components (MGA, AMGA, CIS and cancer) with a matched benign sample to characterize the mutational landscape of these foci. Design: We selected a case of a metaplastic carcinoma with mesenchymal differentiation in juxtaposition to foci of MGA, AMGA and CIS. Immunohistochemically, all four foci were negative for estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (her2) and positive for S-100. The distinct morphologic components and a matched control lymph node were separately microdissected from formalin-fixed paraffin embedded blocks. DNA extraction was performed and subjected to whole-exome sequencing on Illumina HiSeq platform. The results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. Singlenucleotide and small indel somatic variants were called with MuTect2. Copy number profiles were calculated using Control-FREEC. Clonal populations were identified and quantified using PyClone. Results: Sequencing data resulted in mean coverage of 96-134X of targeted exome regions. Our results found a recurrent stop-gain R213* TP53 mutation in MGA, atypical MGA, CIS and metaplastic carcinoma. In addition, through variant allele frequency analysis, we identified two putative clonal clusters shared by all foci indicating a common molecular signature that is preserved in the morphologic spectrum of MGA-AMGA-CIS-metaplastic carcinoma. Conclusions: MGA is a molecularly advanced lesion with somatic mutation of TP53. We postulate that TP53 is an early event in the progression of MGA through AMGA, CIS and its associated metaplastic carcinoma. We report significant genetic overlap between MGA and its associated cancer

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality in young adults. The exact mechanisms are unknown but death often follows a generalized tonic-clonic seizure. Proposed mechanisms include seizure-related respiratory, cardiac, autonomic, and arousal dysfunction. Genetic drivers underlying SUDEP risk are largely unknown. To identify potential SUDEP risk genes, we compared whole-exome sequences (WES) derived from formalin-fixed paraffin embedded surgical brain specimens of eight epilepsy patients who died from SUDEP with seven living controls matched for age at surgery, sex, year of surgery and lobe of resection. We compared identified variants from both groups filtering known polymorphisms from publicly available data as well as scanned for epilepsy and candidate SUDEP genes. In the SUDEP cohort, we identified mutually exclusive variants in genes involved in µ-opiod signaling, gamma-aminobutyric acid (GABA) and glutamate-mediated synaptic signaling, includingARRB2,ITPR1,GABRR2,SSTR5,GRIK1,CTNAP2,GRM8,GNAI2andGRIK5. In SUDEP patients we also identified variants in genes associated with cardiac arrhythmia, includingKCNMB1,KCNIP1,DPP6,JUP,F2, andTUBA3D, which were not present in living epilepsy controls. Our data shows that genomic analysis of brain tissue resected for seizure control can identify potential genetic biomarkers of SUDEP risk.