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Transient receptor potential melastatin 3 (TRPM3) channels are activated by heat, and chemical ligands such as pregnenolone sulphate (PregS) and CIM0216. Here, we show that activation of receptors coupled to heterotrimeric Gi/o proteins inhibits TRPM3 channels. This inhibition was alleviated by co-expression of proteins that bind the βγ subunits of heterotrimeric G-proteins (Gβγ). Co-expression of Gβγ, but not constitutively active Gαi or Gαo, inhibited TRPM3 currents. TRPM3 co-immunoprecipitated with Gβ, and purified Gβγ proteins applied to excised inside-out patches inhibited TRPM3 currents, indicating a direct effect. Baclofen and somatostatin, agonists of Gi-coupled receptors, inhibited Ca2+ signals induced by PregS and CIM0216 in mouse dorsal root ganglion (DRG) neurons. The GABAB receptor agonist baclofen also inhibited inward currents induced by CIM0216 in DRG neurons, and nocifensive responses elicited by this TRPM3 agonist in mice. Our data uncover a novel signaling mechanism regulating TRPM3 channels.

Body fat distribution is a major, heritable risk factor for cardiometabolic disease, independent of overall adiposity. Using exome-sequencing in 618,375 individuals (including 160,058 non-Europeans) from the UK, Sweden and Mexico, we identify 16 genes associated with fat distribution at exome-wide significance. We show 6-fold larger effect for fat-distribution associated rare coding variants compared with fine-mapped common alleles, enrichment for genes expressed in adipose tissue and causal genes for partial lipodystrophies, and evidence of sex-dimorphism. We describe an association with favorable fat distribution ( p  = 1.8 × 10 −09 ), favorable metabolic profile and protection from type 2 diabetes (~28% lower odds; p  = 0.004) for heterozygous protein-truncating mutations in INHBE , which encodes a circulating growth factor of the activin family, highly and specifically expressed in hepatocytes. Our results suggest that inhibin βE is a liver-expressed negative regulator of adipose storage whose blockade may be beneficial in fat distribution-associated metabolic disease. Fat distribution is associated with cardiometabolic disease, although it has been less well studied than overall obesity. In a multiancestry exome-sequencing study, the authors identified predicted loss-of-function mutations in INHBE associated with favorable fat distribution and protection from type 2 diabetes.

Screening 92,445 subjects in the Geisinger-Regeneron DiscovEHR cohort, we identified 5 patients heterozygous for nonsense mutations causing early terminations at Glu307 or Leu328 on the C-terminus of melanocortin 4 receptor (MC4R). Two Q307Ter carriers are severely obese (BMI > 40), while one is overweight (BMI > 25). One L328Ter carrier is overweight and the other is lean. Pedigree analysis for two Q307Ter carriers shows segregation of the variant with higher BMI. Functionally, MC4R(Q307Ter) eliminated receptor surface expression and signaling, while MC4R(L328Ter) functioned like the wild-type receptor. MC4R(Q307Ter) is therefore a loss of function (LOF) variant and the region between the two truncation sites identified in our patients is critical to MC4R function. Truncating MC4R at various C-terminal positions between these two variant sites, we find that cysteine318 sits at a critical junction for receptor trafficking and function. We show that MC4R is lipid modified at cysteine318 and cysteine319. Therefore, truncation early in the MC4R C-terminus results in haploinsufficiency in humans while truncation after the first lipid-modification site is well tolerated. MC4R haploinsufficiency clearly segregates with higher BMI; however, severe obesity is not fully penetrant even in MC4R LOF carriers, suggesting critical roles for environmental and lifestyle factors in MC4R monogenic obesity.

Purpose Genetic variation in MC1R is a main determinant of red hair color (RHC) phenotype and confers susceptibility to skin disorders. Methods We assessed the effects and function of MC1R variants identified in our clinical cohort of 135,947 participants with available exome sequencing using phenome-wide association scan (PheWAS). Expression and function of several variants were evaluated. Results We found 24 nonsense and 215 missense variants in MC1R . Many common missense MC1R variants are strongly associated with skin disorders including skin cancer; however, each variant shows different penetrance and expressivity. Severity of skin phenotype was well correlated with the magnitude of functional defect measured as receptor expression and α-MSH stimulated cAMP production. Remarkably, MC1R deletions and nonsense variants are only weakly associated with milder skin phenotypes. Conclusion Our comprehensive assessment of all MC1R variants in a large cohort clearly establish that individuals with some missense variants are more susceptible to severe skin disorders than those with MC1R deletions or nonsense variants.

Understanding mechanisms of hepatocellular damage may lead to new treatments for liver disease, and genome-wide association studies (GWAS) of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum activities have proven useful for investigating liver biology. Here we report 100 loci associating with both enzymes, using GWAS across 411,048 subjects in the UK Biobank. The rare missense variant SLC30A10 Thr95Ile (rs188273166) associates with the largest elevation of both enzymes, and this association replicates in the DiscovEHR study. SLC30A10 excretes manganese from the liver to the bile duct, and rare homozygous loss of function causes the syndrome hypermanganesemia with dystonia-1 (HMNDYT1) which involves cirrhosis. Consistent with hematological symptoms of hypermanganesemia, SLC30A10 Thr95Ile carriers have increased hematocrit and risk of iron deficiency anemia. Carriers also have increased risk of extrahepatic bile duct cancer. These results suggest that genetic variation in SLC30A10 adversely affects more individuals than patients with diagnosed HMNDYT1. Circulating liver enzymes, like alanine aminotransferase (ALT) and aspartate aminotransferase (AST), are highly heritable and predictive of disease. Here, the authors perform a genome-wide association study on ALT and AST, revealing a rare variant in SLC30A10 associated with elevated ALT and AST.

Background/ObjectivesMelanocortin-4 receptor (MC4R) plays an essential role in food intake and energy homeostasis. More than 170 MC4R variants have been described over the past two decades, with conflicting reports regarding the prevalence and phenotypic effects of these variants in diverse cohorts. To determine the frequency of MC4R variants in large cohort of different ancestries, we evaluated the MC4R coding region for 20,537 eMERGE participants with sequencing data plus additional 77,454 independent individuals with genome-wide genotyping data at this locus.Subjects/MethodsThe sequencing data were obtained from the eMERGE phase III study, in which multisample variant call format calls have been generated, curated, and annotated. In addition to penetrance estimation using body mass index (BMI) as a binary outcome, GWAS and PheWAS were performed using median BMI in linear regression analyses. All results were adjusted for principal components, age, sex, and sites of genotyping.ResultsTargeted sequencing data of MC4R revealed 125 coding variants in 1839 eMERGE participants including 30 unreported coding variants that were predicted to be functionally damaging. Highly penetrant unreported variants included (L325I, E308K, D298N, S270F, F261L, T248A, D111V, and Y80F) in which seven participants had obesity class III defined as BMI ≥ 40 kg/m2. In GWAS analysis, in addition to known risk haplotype upstream of MC4R (best variant rs6567160 (P = 5.36 × 10−25, Beta = 0.37), a novel rare haplotype was detected which was protective against obesity and encompassed the V103I variant with known gain-of-function properties (P = 6.23 × 10−08, Beta = −0.62). PheWAS analyses extended this protective effect of V103I to type 2 diabetes, diabetic nephropathy, and chronic renal failure independent of BMI.ConclusionsMC4R screening in a large eMERGE cohort confirmed many previous findings, extend the MC4R pleotropic effects, and discovered additional MC4R rare alleles that probably contribute to obesity.

Empirical data on conditions that increase risk of coronavirus disease 2019 (COVID-19) progression are needed to identify high risk individuals. We performed a comprehensive quantitative assessment of pre-existing clinical phenotypes associated with COVID-19-related hospitalization. Phenome-wide association study (PheWAS) of SARS-CoV-2-positive patients from an integrated health system (Geisinger) with system-level outpatient/inpatient COVID-19 testing capacity and retrospective electronic health record (EHR) data to assess pre-COVID-19 pandemic clinical phenotypes associated with hospital admission (hospitalization). Of 12,971 individuals tested for SARS-CoV-2 with sufficient pre-COVID-19 pandemic EHR data at Geisinger, 1604 were SARS-CoV-2 positive and 354 required hospitalization. We identified 21 clinical phenotypes in 5 disease categories meeting phenome-wide significance (P<1.60x10-4), including: six kidney phenotypes, e.g. end stage renal disease or stage 5 CKD (OR = 11.07, p = 1.96x10-8), six cardiovascular phenotypes, e.g. congestive heart failure (OR = 3.8, p = 3.24x10-5), five respiratory phenotypes, e.g. chronic airway obstruction (OR = 2.54, p = 3.71x10-5), and three metabolic phenotypes, e.g. type 2 diabetes (OR = 1.80, p = 7.51x10-5). Additional analyses defining CKD based on estimated glomerular filtration rate, confirmed high risk of hospitalization associated with pre-existing stage 4 CKD (OR 2.90, 95% CI: 1.47, 5.74), stage 5 CKD/dialysis (OR 8.83, 95% CI: 2.76, 28.27), and kidney transplant (OR 14.98, 95% CI: 2.77, 80.8) but not stage 3 CKD (OR 1.03, 95% CI: 0.71, 1.48). This study provides quantitative estimates of the contribution of pre-existing clinical phenotypes to COVID-19 hospitalization and highlights kidney disorders as the strongest factors associated with hospitalization in an integrated US healthcare system.

Our GWAS of hematuria in the UK Biobank identified 6 loci, some of which overlap with loci for albuminuria suggesting pleiotropy. Since clinical syndromes are often defined by combinations of traits, generating a combined phenotype can improve power to detect loci influencing multiple characteristics. Thus the composite trait of hematuria and albuminuria was chosen to enrich for glomerular pathologies. Cases had both hematuria defined by ICD codes and albuminuria defined as uACR > 3 mg/mmol. Controls had neither an ICD code for hematuria nor an uACR > 3 mg/mmol. 2429 cases and 343,509 controls from the UK Biobank were included. eGFR was lower in cases compared to controls, with the exception of the comparison in females using CKD-EPI after age adjustment. Variants at 4 loci met genome-wide significance with the following nearest genes: COL4A4 , TRIM27 , ETV1 and CUBN . TRIM27 is part of the extended MHC locus. All loci with the exception of ETV1 were replicated in the Geisinger MyCode cohort. The previous GWAS of hematuria reported COL4A3 - COL4A4 variants and HLA-B *0801 within MHC , which is in linkage disequilibrium with the TRIM27 variant (D′ = 0.59). TRIM27 is highly expressed in the tubules. Additional loci included a coding sequence variant in CUBN (p.Ala2914Val, MAF = 0.014 (A), p  = 3.29E−8, OR = 2.09, 95% CI = 1.61–2.72). Overall, GWAS for the composite trait of hematuria and albuminuria identified 4 loci, 2 of which were not previously identified in a GWAS of hematuria.

Background Nonalcoholic fatty liver disease (NAFLD) is a prevalent complication of extreme obesity. Loading of the liver with fat can progress to inflammation and fibrosis including cirrhosis. The molecular factors involved in the progression from simple steatosis to fibrosis remain poorly understood. Methods Gene expression profiling using microarray, PCR array, and RNA sequencing was performed on RNA from liver biopsy tissue from patients with extreme obesity. Patients were grouped based on histological findings including normal liver histology with no steatosis, lobular inflammation, or fibrosis, and grades 1, 2, 3, and 4 fibrosis with coexistent steatosis and lobular inflammation. Validation of expression was conducted using quantitative PCR. Serum analysis was performed using ELISA. Expression analysis of hepatocytes and hepatic stellate cells in response to lipid loading were conducted in vitro using quantitative PCR and ELISA. Results Three orthogonal methods to profile human liver biopsy RNA each identified the chemokine CCL20 (CC chemokine ligand 20 or MIP-3 alpha) gene as one of the most up-regulated transcripts in NAFLD fibrosis relative to normal histology, validated in a replication group. CCL20 protein levels in serum measured in 224 NAFLD patients were increased in severe fibrosis (p < 0.001), with moderate correlation of hepatic transcript levels and serum levels. Expression of CCL20, but not its cognate receptor CC chemokine receptor 6, was significantly (p < 0.001) increased in response to fatty acid loading in LX-2 hepatic stellate cells, with relative increases greater than those in HepG2 hepatocyte cells. Conclusions These results suggest that expression of CCL20, an important inflammatory mediator, is increased in NAFLD fibrosis. CCL20 serves as a chemoattractant molecule for immature dendritic cells, which have been shown to produce many of the inflammatory molecules that mediate liver fibrosis. These data also point to hepatic stellate cells as a key cell type that may respond to lipid loading of the liver.