Association with menopausal hormone therapy and asymptomatic gallstones in US women in the third National Health and Nutrition Examination Study.

15% of US adults have gallstones, most of which are clinically "silent". Several studies show that menopausal hormone therapy (MHT) increases symptomatic gallstones and cholecystectomy risk. MHT use may be contraindicated in women with gallstones and population studies may be biased by "confounding by contraindication" while the true association between MHT and gallstones remains underestimated. We sought to examine whether MHT use was associated with asymptomatic gallstones using instrumental variable (IV) analysis to account for confounding by contraindication. We used 2018 postmenopausal women from the Third National Health and Nutrition Examination Survey to estimate associations of MHT use with asymptomatic gallstones. A traditional logistic regression analysis was compared to instrumental variable (IV) analysis to account for confounding by contraindication. 12% of women with asymptomatic gallstones and 25% of women without gallstones were current MHT users (P < 0.001). The traditional analysis suggested a decreased odds of asymptomatic gallstones in current versus never users (OR 0.58, 95% CI 0.37, 0.89), but increased odds (OR 1.51, 95% CI 0.44, 5.16) in the IV analysis. The traditional analysis consistently underestimated the odds of asymptomatic gallstones with MHT use compared to the IV analysis. Accounting for confounding by contraindication, we found a suggestive, though imprecise, positive association between MHT use and asymptomatic gallstones among postmenopausal women. Failure to consider contraindication can produce incorrect results.

Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes.

Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19.

Geographical distribution of cystic fibrosis carriers as population genetic determinant of COVID-19 spread and fatality in 37 countries.

COVID-19 has shown a relevant heterogeneity in spread and fatality among countries together with a significant variability in its clinical presentation, indicating that host genetic factors may influence COVID-19 pathogenicity. Indeed, subjects carrying single pathogenic variants of the Cystic Fibrosis (CF) Transmembrane Conductance Regulator (CFTR) gene - i.e. CF carriers - are more susceptible to respiratory tract infections and are more likely to undergo severe COVID-19 with higher risk of 14-day mortality. Given that CF carrier prevalence varies among ethnicities and nations, an ecological study in 37 countries was conducted, in order to determine to what extent the diverse CF carrier geographical distribution may have affected COVID-19 spread and fatality during the first pandemic wave. The CF prevalence in countries, as indicator of the geographical distribution of CF carriers, significantly correlated in a direct manner with both COVID-19 prevalence and its Case Fatality Rate (CFR). In a regression study weighted for the number of tests performed, COVID-19 prevalence positively correlated with CF prevalence, while CFR correlated with population percentage older than 65-year, cancer and CF prevalence. Multivariate regression model also confirmed COVID-19 CFR to be associated with CF prevalence, after adjusting for elderly, cancer prevalence, and weighting for the number of tests performed. This study suggests a putative contribution of population genetics of CFTR in understanding the spatial distribution of COVID-19 spread and fatality.

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males.

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor.

Common, low-frequency, rare, and ultra-rare coding variants contribute to COVID-19 severity.

The combined impact of common and rare exonic variants in COVID-19 host genetics is currently insufficiently understood. Here, common and rare variants from whole-exome sequencing data of about 4000 SARS-CoV-2-positive individuals were used to define an interpretable machine-learning model for predicting COVID-19 severity. First, variants were converted into separate sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. The Boolean features selected by these logistic models were combined into an Integrated PolyGenic Score that offers a synthetic and interpretable index for describing the contribution of host genetics in COVID-19 severity, as demonstrated through testing in several independent cohorts. Selected features belong to ultra-rare, rare, low-frequency, and common variants, including those in linkage disequilibrium with known GWAS loci. Noteworthily, around one quarter of the selected genes are sex-specific. Pathway analysis of the selected genes associated with COVID-19 severity reflected the multi-organ nature of the disease. The proposed model might provide useful information for developing diagnostics and therapeutics, while also being able to guide bedside disease management.

Menopausal hormone therapy and risk of biliary tract cancers.

BACKGROUND AND AIMS: Gallbladder cancer (GBC) has a female predominance, whereas the other biliary tract cancers (BTCs) have a male predominance, suggesting that sex hormones may be involved in carcinogenesis. We sought to evaluate the association between menopausal hormone therapy (MHT) and the risk of BTC in women. APPROACH AND RESULTS: This nested case-control study was conducted in the UK Clinical Practice Research Datalink. Cases diagnosed between 1990 and 2017 with incident primary cancers of the gallbladder (GBC), cholangiocarcinoma (CCA), ampulla of Vater (AVC), and mixed type were matched to 5 controls on birth year, diagnosis year, and years in the general practice using incidence density sampling. Conditional logistic regression was used to calculate ORs and 95% CIs for associations between MHT use and BTC type. The sample consisted of 1,682 BTC cases (483 GBC, 870 CCA, 105 AVC, and 224 mixed) and 8,419 matched controls with a mean age of 73 (SD, 11) years. Combined formulations (estrogen-progesterone) were associated with an increased GBC risk (OR, 1.97; 95% CI, 1.08, 3.59). Orally administered MHT was associated with an increased GBC risk (OR, 2.28; 95% CI, 1.24, 4.17). Estrogen-only formulations (OR, 0.59; 95% CI, 0.34, 0.93) and cream or suppository administrations (OR, 0.57; 95% CI, 0.34, 0.95) were associated with decreased CCA risk. The number of prescriptions, dose, duration of use, and time since last use were not associated with GBC or CCA risk. MHT use was not associated with risk of AVC or mixed cancer. CONCLUSIONS: Combination MHT formulations and oral administrations were associated with increased GBC risk, whereas estrogen-only formulations were associated with a lower CCA risk. MHT formulation and administration should be carefully considered when prescribing.

Severe COVID-19 in Hospitalized Carriers of Single CFTR Pathogenic Variants.

The clinical presentation of COVID-19 is extremely heterogeneous, ranging from asymptomatic to severely ill patients. Thus, host genetic factors may be involved in determining disease presentation and progression. Given that carriers of single cystic fibrosis (CF)-causing variants of the CFTR gene-CF-carriers-are more susceptible to respiratory tract infections, our aim was to determine their likelihood of undergoing severe COVID-19. We implemented a cohort study of 874 individuals diagnosed with COVID-19, during the first pandemic wave in Italy. Whole exome sequencing was performed and validated CF-causing variants were identified. Forty subjects (16 females and 24 males) were found to be CF-carriers. Among mechanically ventilated patients, CF-carriers were more represented (8.7%) and they were significantly (p < 0.05) younger (mean age 51 years) compared to noncarriers (mean age 61.42 years). Furthermore, in the whole cohort, the age of male CF-carriers was lower, compared to noncarriers (p < 0.05). CF-carriers had a relative risk of presenting an abnormal inflammatory response (CRP ≥ 20 mg/dL) of 1.69 (p < 0.05) and their hazard ratio of death at day 14 was 3.10 (p < 0.05) in a multivariate regression model, adjusted for age, sex and comorbidities. In conclusion, CF-carriers are more susceptible to the severe form of COVID-19, showing also higher risk of 14-day death.

Liver X receptor ß regulates bile volume and the expression of aquaporins and cystic fibrosis transmembrane conductance regulator in the gallbladder.

The gallbladder is considered an important organ in maintaining digestive and metabolic homeostasis. Given that therapeutic options for gallbladder diseases are often limited to cholecystectomy, understanding gallbladder pathophysiology is essential in developing novel therapeutic strategies. Since liver X receptor ß (LXRß), an oxysterol-activated transcription factor, is strongly expressed in gallbladder cholangiocytes, the aim was to investigate LXRß physiological function in the gallbladder. Thus, we studied the gallbladders of WT and LXRß-/- male mice using immunohistochemistry, electron microscopy, qRT-PCR, bile duct cannulation, bile and blood biochemistry, and duodenal pH measurements. LXRß-/- mice presented a large gallbladder bile volume with high duodenal mRNA levels of the vasoactive intestinal polypeptide (VIP), a strong mediator of gallbladder relaxation. LXRß-/- gallbladders showed low mRNA and protein expression of Aquaporin-1, Aquaporin-8, and cystic fibrosis transmembrane conductance regulator (CFTR). A cystic fibrosis-resembling phenotype was evident in the liver showing high serum cholestatic markers and the presence of reactive cholangiocytes. For LXRß being a transcription factor, we identified eight putative binding sites of LXR on the promoter and enhancer of the Cftr gene, suggesting Cftr as a novel LXRß regulated gene. In conclusion, LXRß was recognized as a regulator of gallbladder bile volume through multiple mechanisms involving CFTR and aquaporins.NEW & NOTEWORTHY This report reveals a novel and specific role of the nuclear receptor liver X receptor ß (LXRß) in controlling biliary tree pathophysiology. LXRß-/- mice have high gallbladder bile volume and are affected by a cholangiopathy that resembles cystic fibrosis. We found LXRß to regulate the expression of both aquaporins water channels and the cystic fibrosis transmembrane conductance regulator. This opens a new field in biliary tree pathophysiology, enlightening a possible transcription factor controlling CFTR expression.

Shorter androgen receptor polyQ alleles protect against life-threatening COVID-19 disease in European males.

BACKGROUND: While SARS-CoV-2 similarly infects men and women, COVID-19 outcome is less favorable in men. Variability in COVID-19 severity may be explained by differences in the host genome. METHODS: We compared poly-amino acids variability from WES data in severely affected COVID-19 patients versus SARS-CoV-2 PCR-positive oligo-asymptomatic subjects. FINDINGS: Shorter polyQ alleles (≤22) in the androgen receptor (AR) conferred protection against severe outcome in COVID-19 in the first tested cohort (both males and females) of 638 Italian subjects. The association between long polyQ alleles (≥23) and severe clinical outcome (p = 0.024) was also validated in an independent cohort of Spanish men <60 years of age (p = 0.014). Testosterone was higher in subjects with AR long-polyQ, possibly indicating receptor resistance (p = 0.042 Mann-Whitney U test). Inappropriately low serum testosterone level among carriers of the long-polyQ alleles (p = 0.0004 Mann-Whitney U test) predicted the need for intensive care in COVID-19 infected men. In agreement with the known anti-inflammatory action of testosterone, patients with long-polyQ and age ≥60 years had increased levels of CRP (p = 0.018, not accounting for multiple testing). INTERPRETATION: We identify the first genetic polymorphism that appears to predispose some men to develop more severe disease. Failure of the endocrine feedback to overcome AR signaling defects by increasing testosterone levels during the infection leads to the polyQ tract becoming dominant to serum testosterone levels for the clinical outcome. These results may contribute to designing reliable clinical and public health measures and provide a rationale to test testosterone as adjuvant therapy in men with COVID-19 expressing long AR polyQ repeats. FUNDING: MIUR project "Dipartimenti di Eccellenza 2018-2020" to Department of Medical Biotechnologies University of Siena, Italy (Italian D.L. n.18 March 17, 2020) and "Bando Ricerca COVID-19 Toscana" project to Azienda Ospedaliero-Universitaria Senese. Private donors for COVID-19 research and charity funds from Intesa San Paolo.

Retinal and optic nerve degeneration in liver X receptor ß knockout mice.

The retina is an extension of the brain. Like the brain, neurodegeneration of the retina occurs with age and is the cause of several retinal diseases including optic neuritis, macular degeneration, and glaucoma. Liver X receptors (LXRs) are expressed in the brain where they play a key role in maintenance of cerebrospinal fluid and the health of dopaminergic neurons. Herein, we report that LXRs are expressed in the retina and optic nerve and that loss of LXRß, but not LXRα, leads to loss of ganglion cells in the retina. In the retina of LXRß-/- mice, there is an increase in amyloid A4 and deposition of beta-amyloid (Aß) aggregates but no change in the level of apoptosis or autophagy in the ganglion cells and no activation of microglia or astrocytes. However, in the optic nerve there is a loss of aquaporin 4 (AQP4) in astrocytes and an increase in activation of microglia. Since loss of AQP4 and microglial activation in the optic nerve precedes the loss of ganglion cells, and accumulation of Aß in the retina, the cause of the neuronal loss appears to be optic nerve degeneration. In patients with optic neuritis there are frequently AQP4 autoantibodies which block the function of AQP4. LXRß-/- mouse is another model of optic neuritis in which AQP4 antibodies are not detectable, but AQP4 function is lost because of reduction in its expression.

HIV-1 viral protein R (Vpr) induces fatty liver in mice via LXRα and PPARα dysregulation: implications for HIV-specific pathogenesis of NAFLD.

HIV patients develop hepatic steatosis. We investigated hepatic steatosis in transgenic mice expressing the HIV-1 accessory protein Vpr (Vpr-Tg) in liver and adipose tissues, and WT mice infused with synthetic Vpr. Vpr-Tg mice developed increased liver triglyceride content and elevated ALT, bilirubin and alkaline phosphatase due to three hepatic defects: 1.6-fold accelerated de novo lipogenesis (DNL), 45% slower fatty acid ß-oxidation, and 40% decreased VLDL-triglyceride export. Accelerated hepatic DNL was due to coactivation by Vpr of liver X receptor-α (LXRα) with increased expression of its lipogenic targets Srebp1c, Chrebp, Lpk, Dgat, Fasn and Scd1, and intranuclear SREBP1c and ChREBP. Vpr enhanced association of LXRα with Lxrα and Srebp1c promoters, increased LXRE-LXRα binding, and broadly altered hepatic expression of LXRα-regulated lipid metabolic genes. Diminished hepatic fatty acid ß-oxidation was associated with decreased mRNA expression of Pparα and its targets Cpt1, Aox, Lcad, Ehhadh, Hsd10 and Acaa2, and blunted VLDL export with decreased expression of Mttp and its product microsomal triglyceride transfer protein. With our previous findings that Vpr circulates in HIV patients (including those with undetectable plasma HIV-1 RNA), co-regulates the glucocorticoid receptor and PPARγ and transduces hepatocytes, these data indicate a potential role for Vpr in HIV-associated fatty liver disease.

Antiproliferative effects and mechanisms of liver X receptor ligands in pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) is difficult to detect early and is often resistant to standard chemotherapeutic options, contributing to extremely poor disease outcomes. Members of the nuclear receptor superfamily carry out essential biological functions such as hormone signaling and are successfully targeted in the treatment of endocrine-related malignancies. Liver X receptors (LXRs) are nuclear receptors that regulate cholesterol homeostasis, lipid metabolism, and inflammation, and LXR agonists have been developed to regulate LXR function in these processes. Intriguingly, these compounds also exhibit antiproliferative activity in diverse types of cancer cells. In this study, LXR agonist treatments disrupted proliferation, cell-cycle progression, and colony-formation of PDAC cells. At the molecular level, treatments downregulated expression of proteins involved in cell cycle progression and growth factor signaling. Microarray experiments further revealed changes in expression profiles of multiple gene networks involved in biological processes and pathways essential for cell growth and proliferation following LXR activation. These results establish the antiproliferative effects of LXR agonists and potential mechanisms of action in PDAC cells and provide evidence for their potential application in the prevention and treatment of PDAC.