The etiology of liver disease remains elusive in some adults presenting with severe hepatic dysfunction.

Tumor invasion can be induced by changes in gene expression that alter cell phenotype. The transcription factor ΔNp63α promotes basal-like breast cancer (BLBC) migration by inducing the expression of the mesenchymal genes Slug and Axl, which confers cells with a hybrid epithelial/mesenchymal state. However, the extent of the ΔNp63α regulated genes that support invasive behavior is not known. Here, using gene expression analysis, ChIP-seq, and functional testing, we find that ΔNp63α promotes BLBC motility by inducing the expression of the atypical cadherin FAT2, the vesicular binding protein SNCA, the carbonic anhydrase CA12, the lipid binding protein CPNE8 and the kinase NEK1, along with Slug and Axl. Notably, lung squamous cell carcinoma migration also required ΔNp63α dependent FAT2 and Slug expression, demonstrating that ΔNp63α promotes migration in multiple tumor types by inducing mesenchymal and non-mesenchymal genes. ΔNp63α activation of FAT2 and Slug influenced E-cadherin localization to cell-cell contacts, which can restrict spontaneous cell movement. Moreover, live-imaging of spheroids in organotypic culture demonstrated that ΔNp63α, FAT2 and Slug were essential for the extension of cellular protrusions that initiate collective invasion. Importantly, ΔNp63α is co-expressed with FAT2 and Slug in patient tumors and the elevated expression of ΔNp63α, FAT2 and Slug correlated with poor patient outcome. Together, these results reveal how ΔNp63α promotes cell migration by directly inducing the expression of a cohort of genes with distinct cellular functions and suggest that FAT2 is a new regulator of collective invasion that may influence patient outcome.

Lipoprotein(a) (Lp(a)) is an important causal cardiovascular risk factor, with serum Lp(a) levels predicting atherosclerotic heart disease and genetic determinants of Lp(a) levels showing association with myocardial infarction. Lp(a) levels vary widely between populations, with African-derived populations having nearly 2-fold higher Lp(a) levels than European Americans. We investigated the genetic basis of this difference in 4464 African Americans from the Jackson Heart Study (JHS) using a panel of up to 1447 ancestry informative markers, allowing us to accurately estimate the African ancestry proportion of each individual at each position in the genome. In an unbiased genome-wide admixture scan for frequency-differentiated genetic determinants of Lp(a) level, we found a convincing peak (LOD = 13.6) at 6q25.3, which spans the LPA locus. Dense fine-mapping of the LPA locus identified a number of strongly associated, common biallelic SNPs, a subset of which can account for up to 7% of the variation in Lp(a) level, as well as >70% of the African-European population differences in Lp(a) level. We replicated the association of the most strongly associated SNP, rs9457951 (p = 6 × 10(-22), 27% change in Lp(a) per allele, ∼5% of Lp(a) variance explained in JHS), in 1,726 African Americans from the Dallas Heart Study and found an even stronger association after adjustment for the kringle(IV) repeat copy number. Despite the strong association with Lp(a) levels, we find no association of any LPA SNP with incident coronary heart disease in 3,225 African Americans from the Atherosclerosis Risk in Communities Study.

Nonalcoholic fatty liver disease (NAFLD) is a rising cause of hepatocellular carcinoma (HCC). We examined whether inherited pathogenic variants in candidate genes (n = 181) were enriched in patients with NAFLD-HCC. To this end, we resequenced peripheral blood DNA of 142 NAFLD-HCC, 59 NAFLD with advanced fibrosis, and 50 controls, and considered 404 healthy individuals from 1000 G. Pathogenic variants were defined according to ClinVar, likely pathogenic as rare variants predicted to alter protein activity. In NAFLD-HCC patients, we detected an enrichment in pathogenic (p = 0.024), and likely pathogenic variants (p = 1.9*10-6), particularly in APOB (p = 0.047). APOB variants were associated with lower circulating triglycerides and higher HDL cholesterol (p < 0.01). A genetic risk score predicted NAFLD-HCC (OR 4.96, 3.29-7.55; p = 5.1*10-16), outperforming the diagnostic accuracy of common genetic risk variants, and of clinical risk factors (p < 0.05). In conclusion, rare pathogenic variants in genes involved in liver disease and cancer predisposition are associated with NAFLD-HCC development.

Association signals in GWAS are usually prioritized solely by p values. Here, we attempt to improve the power of GWAS by using a weighted false discovery rate control procedure to detect associations of low-frequency variants with effect sizes similar to or even larger than those of common variants. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to test for association with fasting glucose levels in the Atherosclerosis Risk in Communities Study (ARIC) population. In addition to finding several previously identified sequence variations, we identified a low-frequency variant (rs1209523; minor allele frequency = 0.043) near FOXA2 that was associated with fasting glucose levels in European Americans (EAs) (n = 7428, p value = 1.3 x 10(-5)). The association between rs1209523 and glucose levels was also significant in African Americans (AAs) (n = 2029, p value = 6.7 x 10(-3)) of the ARIC and was confirmed by replication in both EAs and AAs of the Dallas Heart Study (n = 963 and 1571, respectively; p values = 5.3 x 10(-3) and 5.8 x 10(-4), respectively) and in EAs of the Cooper Center Longitudinal Study (n = 2862; p value = 1.6 x 10(-2)). A meta-analysis of these five populations yielded an estimated effect size of -1.31 mg/dl per minor allele (p value = 2.2 x 10(-11)). This study reveals that there is a cache of less-frequent variants in GWAS arrays that can be identified via analytical approaches accounting for allele frequencies.

The prevalence of obesity (body mass index (BMI) > or =30 kg/m(2)) is higher in African Americans than in European Americans, even after adjustment for socioeconomic factors, suggesting that genetic factors may explain some of the difference. To identify genetic loci influencing BMI, we carried out a pooled analysis of genome-wide admixture mapping scans in 15,280 African Americans from 14 epidemiologic studies. Samples were genotyped at a median of 1,411 ancestry-informative markers. After adjusting for age, sex, and study, BMI was analyzed both as a dichotomized (top 20% versus bottom 20%) and a continuous trait. We found that a higher percentage of European ancestry was significantly correlated with lower BMI (rho = -0.042, P = 1.6x10(-7)). In the dichotomized analysis, we detected two loci on chromosome X as associated with increased African ancestry: the first at Xq25 (locus-specific LOD = 5.94; genome-wide score = 3.22; case-control Z = -3.94); and the second at Xq13.1 (locus-specific LOD = 2.22; case-control Z = -4.62). Quantitative analysis identified a third locus at 5q13.3 where higher BMI was highly significantly associated with greater European ancestry (locus-specific LOD = 6.27; genome-wide score = 3.46). Further mapping studies with dense sets of markers will be necessary to identify the alleles in these regions of chromosomes X and 5 that may be associated with variation in BMI.

Telomerase is an enzyme that catalyzes the addition of nucleotides on the ends of chromosomes. Rare loss of function mutations in the gene that encodes the protein component of telomerase (TERT) have been described in patients with idiopathic pulmonary fibrosis (IPF). Here we examine the telomere lengths and pulmonary fibrosis phenotype seen in multiple kindreds with heterozygous TERT mutations.

Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem of unknown etiology that varies in prevalence among ancestry groups. To identify genetic variants contributing to differences in hepatic fat content, we carried out a genome-wide association scan of nonsynonymous sequence variations (n = 9,229) in a population comprising Hispanic, African American and European American individuals. An allele in PNPLA3 (rs738409[G], encoding I148M) was strongly associated with increased hepatic fat levels (P = 5.9 x 10(-10)) and with hepatic inflammation (P = 3.7 x 10(-4)). The allele was most common in Hispanics, the group most susceptible to NAFLD; hepatic fat content was more than twofold higher in PNPLA3 rs738409[G] homozygotes than in noncarriers. Resequencing revealed another allele of PNPLA3 (rs6006460[T], encoding S453I) that was associated with lower hepatic fat content in African Americans, the group at lowest risk of NAFLD. Thus, variation in PNPLA3 contributes to ancestry-related and inter-individual differences in hepatic fat content and susceptibility to NAFLD.

Fuchs' endothelial corneal dystrophy (FECD) is the most common repeat expansion disorder. FECD impacts 4% of U.S. population and is the leading indication for corneal transplantation. Most cases are caused by an expanded intronic CUG tract in the TCF4 gene that forms nuclear foci, sequesters splicing factors and impairs splicing. We investigated the sense and antisense RNA landscape at the FECD gene and find that the sense-expanded repeat transcript is the predominant species in patient corneas. In patient tissue, sense foci number were negatively correlated with age and showed no correlation with sex. Each endothelial cell has ∼2 sense foci and each foci is single RNA molecule. We designed antisense oligonucleotides (ASOs) to target the mutant-repetitive RNA and demonstrated potent inhibition of foci in patient-derived cells. Ex vivo treatment of FECD human corneas effectively inhibits foci and reverses pathological changes in splicing. FECD has the potential to be a model for treating many trinucleotide repeat diseases and targeting the TCF4 expansion with ASOs represents a promising therapeutic strategy to prevent and treat FECD.

The translocation of effectors into the host cell through type 3 secretion systems (T3SS) is a sophisticated strategy employed by pathogenic bacteria to subvert host responses and facilitate colonization. Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) utilize the Tir and EspFu (also known as TccP) effectors to remodel the host cytoskeleton, culminating in the formation of attaching and effacing (AE) lesions on enterocytes. While some EPEC strains require tyrosine phosphorylation of Tir and recruitment of the host Nck to trigger actin polymerization, EHEC and certain EPEC strains, whose Tir is not phosphorylated, rely on the effector EspFu for efficient actin remodeling. Here, we investigated the role played by Tir-Nck and Tir-EspFu actin polymerization pathways during the infection of epithelial cells, as well as the host transcriptional response to the AE lesion formation induced by EPEC. We found that EspFu-mediated actin assembly promotes bacterial attachment and epithelial colonization more efficiently than Tir-Nck. Moreover, we showed that both actin polymerization mechanisms can activate inflammatory pathways and reverse the anti-inflammatory response induced by EPEC in epithelial cells. However, this activity is remarkably more evident in infections with EspFu-expressing EPEC strains. This study demonstrates the complex interactions between effector-mediated actin remodeling and inflammation. Different strains carry different combinations of these two effectors, highlighting the plasticity of pathogenic E. coli enteric infections.IMPORTANCE EPEC is among the leading causes of diarrheal disease worldwide. The colonization of the gut mucosa by EPEC results in actin pedestal formation at the site of bacterial attachment. These pedestals are referred to as attaching and effacing (AE) lesions. Here, we exploit the different molecular mechanisms used by EPEC to induce AE lesions on epithelial cells, showing that the effector EspFu is associated with increased bacterial attachment and enhanced epithelial colonization compared to the Tir-Nck pathway. Moreover, we also showed that actin pedestal formation can counterbalance the anti-inflammatory activity induced by EPEC, especially when driven by EspFu. Collectively, our findings provide new insights into virulence mechanisms employed by EPEC to colonize epithelial cells, as well as the host response to this enteric pathogen.

How genetic defects trigger the molecular changes that cause late-onset disease is important for understanding disease progression and therapeutic development. Fuchs' endothelial corneal dystrophy (FECD) is an RNA-mediated disease caused by a trinucleotide CTG expansion in an intron within the TCF4 gene. The mutant intronic CUG RNA is present at one-two copies per cell, posing a challenge to understand how a rare RNA can cause disease. Late-onset FECD is a uniquely advantageous model for studying how RNA triggers disease because: (i) Affected tissue is routinely removed during surgery; (ii) The expanded CTG mutation is one of the most prevalent disease-causing mutations, making it possible to obtain pre-symptomatic tissue from eye bank donors to probe how gene expression changes precede disease; and (iii) The affected tissue is a homogeneous single cell monolayer, facilitating accurate transcriptome analysis. Here, we use RNA sequencing (RNAseq) to compare tissue from individuals who are pre-symptomatic (Pre_S) to tissue from patients with late stage FECD (FECD_REP). The abundance of mutant repeat intronic RNA in Pre_S and FECD_REP tissue is elevated due to increased half-life in a corneal cells. In Pre_S tissue, changes in splicing and extracellular matrix gene expression foreshadow the changes observed in advanced disease and predict the activation of the fibrosis pathway and immune system seen in late-stage patients. The absolute magnitude of splicing changes is similar in pre-symptomatic and late stage tissue. Our data identify gene candidates for early drivers of disease and biomarkers that may represent diagnostic and therapeutic targets for FECD. We conclude that changes in alternative splicing and gene expression are observable decades prior to the diagnosis of late-onset trinucleotide repeat disease.

Fatty liver disease (FLD) is a growing health issue with burdening unmet clinical needs. FLD has a genetic component but, despite the common variants already identified, there is still a missing heritability component. Using a candidate gene approach, we identify a locus (rs71519934) at the Pleckstrin and Sec7 domain-containing 3 (PSD3) gene resulting in a leucine to threonine substitution at position 186 of the protein (L186T) that reduces susceptibility to the entire spectrum of FLD in individuals at risk. PSD3 downregulation by short interfering RNA reduces intracellular lipid content in primary human hepatocytes cultured in two and three dimensions, and in human and rodent hepatoma cells. Consistent with this, Psd3 downregulation by antisense oligonucleotides in vivo protects against FLD in mice fed a non-alcoholic steatohepatitis-inducing diet. Thus, translating these results to humans, PSD3 downregulation might be a future therapeutic option for treating FLD.

Gata4, Hand2, Mef2c, and Tbx5 (GHMT) can reprogram transduced fibroblasts into induced pacemaker-like myocytes (iPMs), but the underlying mechanisms remain obscure. Here, we explore the role of Hand2 in iPM formation by using a combination of transcriptome, genome, and biochemical assays. We found many shared transcriptional signatures between iPMs and the endogenous sinoatrial node (SAN), yet key regulatory networks remain missing. We demonstrate that Hand2 augments chromatin accessibility at loci involved in sarcomere organization, electrical coupling, and membrane depolarization. Focusing on an established cardiac Hand2 cistrome, we observe selective reorganization of chromatin accessibility to promote pacemaker-specific gene expression. Moreover, we identify a Hand2 cardiac subtype diversity (CSD) domain through biochemical analysis of the N terminus. By integrating our RNA-seq and ATAC-seq datasets, we highlight desmosome organization as a hallmark feature of iPM formation. Collectively, our results illuminate Hand2-dependent mechanisms that may guide future efforts to rationally improve iPM formation.

Preaxial polydactyly (PPD) is a common congenital hand malformation classified into four subtypes (PPD I-IV). Variants in the zone of polarizing activity regulatory sequence (ZRS) within intron 5 of the LMBR1 gene are linked to most PPD types. However, the genes responsible for PPD I and the underlying mechanisms are unknown.

Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.

Despite considerable progress in elucidating the molecular basis of various progeroid syndromes, some rare patients remain unexplained.

27-Hydroxycholesterol (27-HC) is the most abundant oxysterol that increases the risk of breast cancer progression. However, little is known about epigenetic regulation of 27-HC metabolism and its role in breast tumor initiation. Using genetic mouse mammary tumor and human breast cancer models, we showed here that the histone reader ZMYND8 was selectively expressed in breast cancer stem cells (BCSCs) and promoted epithelial-mesenchymal transition (EMT), BCSC maintenance and self-renewal, and oncogenic transformation through its epigenetic functions, leading to breast tumor initiation. Mechanistically, ZMYND8 was a master transcriptional regulator of 27-HC metabolism. It increased cholesterol biosynthesis and oxidation but blocked cholesterol efflux and 27-HC catabolism, leading to accumulation of 27-HC in BCSCs. Consequently, 27-HC promoted EMT, oncogenic transformation, and tumor initiation through activation of liver X receptor. These findings reveal that ZMYND8 is an epigenetic booster that drives breast tumor initiation through metabolic reprogramming.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge, and effective tracking requires rapid return of results. Surveillance of variants is typically performed by whole genome sequencing (WGS), which can be financially prohibitive and requires specialized equipment and bioinformatic expertise. Genotyping approaches are rapid methods for monitoring SARS-CoV-2 variants but require continuous adaptation. Fragment analysis may represent an approach for improved SARS-CoV-2 variant detection.

Diabetes mellitus (DM) in children and adolescents is typically caused by type 1 DM, followed by type 2 DM and maturity-onset diabetes of the young (MODY). We report an unusual Asian Indian family in which three members presented with DM at ages 15, 20, and 30, but not fitting the typical clinical picture of type 1 DM, type 2 DM, or MODY. The primary objective was to elucidate the molecular genetic basis of DM in this family.

Cholera toxin (CT) is the etiological agent of cholera. Here we report that multiple classes of fucosylated glycoconjugates function in CT binding and intoxication of intestinal epithelial cells. In Colo205 cells, knockout of B3GNT5, the enzyme required for synthesis of lacto- and neolacto-series glycosphingolipids (GSLs), reduces CT binding but sensitizes cells to intoxication. Overexpressing B3GNT5 to generate more fucosylated GSLs confers protection against intoxication, indicating that fucosylated GSLs act as decoy receptors for CT. Knockout (KO) of B3GALT5 causes increased production of fucosylated O-linked and N-linked glycoproteins, and leads to increased CT binding and intoxication. Knockout of B3GNT5 in B3GALT5 KO cells eliminates production of fucosylated GSLs but increases intoxication, identifying fucosylated glycoproteins as functional receptors for CT. These findings provide insight into molecular determinants regulating CT sensitivity of host cells.