Aris N. Economides

Some primates are immune to infection by most African trypanosome parasites due to apolipoprotein L-1 (APOL1), a primate-specific ion channel-forming protein. Our long-term objective has been to reduce African trypanosomiasis in livestock by genetic bioengineering of cattle with primate APOL1 . To select which primate APOL1 , we analyzed Papio ssp. APOL1 proteins and found that Papio hamadryas APOL1 was a strong candidate for transgenic animal production based on its trypanosome-killing capacity, ion channel properties, and stability. We generated seven transgenic murine lines based on the P. hamadryas APOL1 sequence and used these mice to investigate the level of APOL1 expression required for trypanosome immunity in vivo. We challenged the murine lines with three human and four livestock trypanosome isolates. P. hamadryas APOL1 provided protection against all of the human and three of the livestock trypanosome isolates, though not against Trypanosoma vivax despite the logical hypothesis that APOL1 plays a role in primate immunity to that parasite. Occasionally, lower APOL1 expression in heterozygote mice selected for the emergence of APOL1 resistant parasites in several trypanosome spp. Alarmingly, these resistant parasites were also resistant to high levels of APOL1 in homozygous mice, indicating an increase in virulence. A more-highly expressed chimeric APOL1 transgene encoding Homo sapiens APOL1 with the P. hamadryas APOL1 C-terminus was more effectively protective in heterozygote mice; however, we could not produce homozygous mice, suggesting endogenous toxicity to the mice. Together, these data bear relevance to our long-term objective to generate transgenic APOL1 cattle, the feasibility of which is discussed.

Ligands in the transforming growth factor β (TGF-β) family [activins, Bone Morphogenetic Proteins (BMPs), and TGF-βs] signal by bringing together two type I and two type II receptors. Activin receptor-like kinase-2 (ALK2) is the only type I receptor among the seven TGF-β type I receptors that interacts with both activin and BMP ligands. With BMPs, ALK2 acts as a signaling receptor to activate small mothers against decapentaplegic 1 (SMAD1)/5/8 signaling. Alternatively, with activins, such as Activin A (ActA), ALK2 forms nonsignaling complexes that negatively regulate ALK2 and ActA signaling. To gain insight into how ALK2 interacts with two distinct classes of ligands, we resolved the cryoelectron microscopy structure of ALK2 in complex with the type II receptor, ActRIIB, and the ligand, BMP6, in parallel with the corresponding structure with ALK3 for direct comparison. These structures demonstrate that ALK2 and ALK3 utilize different mechanisms to interact with BMP6 at the wrist interface, with ALK2 relying on BMP6 glycosylation and ALK3 relying on a salt bridge. Modeling of ALK2:ActA reveals that binding relies on ActA’s fingertip region, mirroring the interaction of ActA with its other receptor, ALK4. Our results demonstrate that ALK2 is a “hybrid” receptor that incorporates features of BMP type I receptors such as ALK3 at the wrist interface and an activin type I receptor such as ALK4 at the fingertip.

β-Thalassemia is a genetic disorder arising from mutations in the β-globin gene, leading to ineffective erythropoiesis and iron overload. Ineffective erythropoiesis, a hallmark of β-thalassemia, is an important driver of iron overload, which contributes to liver fibrosis, diabetes, and cardiac disease. Iron homeostasis is regulated by the hormone hepcidin; BMP6/hemojuvelin–mediated (BMP6/HJV-mediated) signaling induces hepatic hepcidin expression via SMAD1/5, with transmembrane serine protease 6 (TMPRSS6) being a negative regulator of HJV. Individuals with loss-of-function mutations in the TMPRSS6 gene show increased circulating hepcidin and iron-refractory iron-deficiency anemia, suggesting that blocking TMPRSS6 may be a viable strategy to elevate hepcidin levels in β-thalassemia. We generated a human mAb (REGN7999) that inhibits TMPRSS6. In an Hbb ^th3/+ mouse model of β-thalassemia, REGN7999 treatment led to significant reductions in liver iron, reduced ineffective erythropoiesis, and showed improvements in RBC health, running distance during forced exercise, and bone density. In a phase I, doubleblind, randomized, placebo-controlled study in healthy human volunteers (NCT05481333), REGN7999 increased serum hepcidin and reduced serum iron with an acceptable tolerability profile. Our results suggest that, by both reducing iron and improving RBC function, inhibition of TMPRSS6 by REGN7999 may offer a therapy for iron overload and impaired erythropoiesis in β-thalassemia.

Major depressive disorder (MDD) is a leading cause of disability worldwide. Risk for MDD is heritable, and the genetic structure of founder populations enables investigation of rare susceptibility alleles with large effect. In an extended Old Order Mennonite family cohort, we identified a rare missense variant in GPR156 (c.1599G>T, p.Glu533Asp) associated with a two-fold increase in the relative risk of MDD. GPR156 is an orphan G protein–coupled receptor localized in the medial habenula, a region implicated in mood regulation. Insertion of a human sequence containing c.1599G>T into the murine Gpr156 locus induced medial habenula hyperactivity and abnormal stress-related behaviors. This work reveals a human variant that is associated with depression, implicates GPR156 as a target for mood regulation, and introduces informative murine models for investigating the pathophysiology and treatment of affective disorders.

Ligands in the TGFβ family (activins, BMPs and TGFβs), signal by bringing together two type I and two type II receptors. ALK2 is the only type I receptor among the seven TGFβ type I receptors that interacts with both activin and BMP ligands. With BMPs, ALK2 acts as a signaling receptor to activate SMAD1/5/8 signaling. Alternatively, with activins, such as Activin A (ActA), ALK2 forms nonsignaling complexes that negatively regulate ALK2 and ActA signaling. To gain insight into how ALK2 interacts with two distinct classes of ligands, we resolved the cryoelectron microscopy structure of ALK2 in complex with the type II receptor, ActRIIB, and the ligand, BMP6, in parallel with the corresponding structure with ALK3 for direct comparison. These structures demonstrate that ALK2 and ALK3 utilize different mechanisms to interact with BMP6 at the wrist interface, with ALK2 relying on BMP6 glycosylation and ALK3 relying on a salt bridge. Modeling of ALK2:ActA reveals that binding relies on ActA’s fingertip region, mirroring the interaction of ActA with its other receptor, ALK4. Our results demonstrate that ALK2 is a ‘hybrid’ receptor that incorporates features of BMP type I receptors such as ALK3 at the wrist interface, and an activin type I receptor such as ALK4 at the fingertip. Significance: ALK2 interacts with two different classes of TGFβ ligands: the BMPs and the activins. With BMP ligands, ALK2 signals to play roles in bone modeling, while with activins, ALK2 negatively regulates signaling by forming nonsignaling complexes. We demonstrate that ALK2 binds to favored ligand, BMP6, by utilizing the ligand wrist-helix stabilized by a glycosylation, rather than a charged interaction observed with similar receptor, ALK3. We also show that ALK2 interaction with activin ligand, ActA, is reliant on a single interaction at the other side of the receptor – the fingertip interface. Thus, this study elucidates first structure of ALK2 in complex with a ligand and provides the molecular insight into how ALK2 is able to bind to BMP and activin ligands.

Recent studies have revealed a role for zinc in insulin secretion and glucose homeostasis. Randomized placebo-controlled zinc supplementation trials have demonstrated improved glycemic traits in patients with type II diabetes (T2D). Moreover, rare loss-of-function variants in the zinc efflux transporter SLC30A8 reduce T2D risk. Despite this accumulated evidence, a mechanistic understanding of how zinc influences systemic glucose homeostasis and consequently T2D risk remains unclear. To further explore the relationship between zinc and metabolic traits, we searched the exome database of the Regeneron Genetics Center-Geisinger Health System DiscovEHR cohort for genes that regulate zinc levels and associate with changes in metabolic traits. We then explored our main finding using in vitro and in vivo models. We identified rare loss-of-function (LOF) variants (MAF Solute Carrier Family 39, Member 5 (SLC39A5) associated with increased circulating zinc (p=4.9 × 10^-4). Trans-ancestry meta-analysis across four studies exhibited a nominal association of SLC39A5 LOF variants with decreased T2D risk. To explore the mechanisms underlying these associations, we generated mice lacking Slc39a5. Slc39a5^-/- mice display improved liver function and reduced hyperglycemia when challenged with congenital or diet-induced obesity. These improvements result from elevated hepatic zinc levels and concomitant activation of hepatic AMPK and AKT signaling, in part due to zinc-mediated inhibition of hepatic protein phosphatase activity. Furthermore, under conditions of diet-induced non-alcoholic steatohepatitis (NASH), Slc39a5^-/- mice display significantly attenuated fibrosis and inflammation. Taken together, these results suggest SLC39A5 as a potential therapeutic target for non-alcoholic fatty liver disease (NAFLD) due to metabolic derangements including T2D.

The genetic factors of stroke in South Asians are largely unexplored. Exome-wide sequencing and association analysis (ExWAS) in 75 K Pakistanis identified NM_000435.3(NOTCH3):c.3691 C > T, encoding the missense amino acid substitution p.Arg1231Cys, enriched in South Asians (alternate allele frequency = 0.58% compared to 0.019% in Western Europeans), and associated with subcortical hemorrhagic stroke [odds ratio (OR) = 3.39, 95% confidence interval (CI) = [2.26, 5.10], p = 3.87 × 10⁻⁹), and all strokes (OR [CI] = 2.30 [1.77, 3.01], p = 7.79 × 10⁻¹⁰). NOTCH3 p.Arg231Cys was strongly associated with white matter hyperintensity on MRI in United Kingdom Biobank (UKB) participants (effect [95% CI] in SD units = 1.1 [0.61, 1.5], p = 3.0 × 10⁻⁶). The variant is attributable for approximately 2.0% of hemorrhagic strokes and 1.1% of all strokes in South Asians. These findings highlight the value of diversity in genetic studies and have major implications for genomic medicine and therapeutic development in South Asian populations.

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare disorder, characterized by progressive heterotopic ossification (HO) and painful soft-tissue inflammatory flare-ups. This was a post-hoc analysis from a phase 2 (NCT03188666) trial in which adults with FOP received intravenous anti-activin A antibody garetosmab 10 mg/kg or placebo every 4 weeks over 28 weeks (Period 1), followed by a 28-week open-label treatment and extension (Period 2 and 3). Here we describe flare-ups, their relationship to new HO lesions, and the impact of garetosmab on flare-ups. Volume of new HO lesions was measured by computed tomography. Patient-reported flare-ups were defined by any two of: new onset of pain, swelling, joint stiffness, decrease in movement, or perceived presence of HO. Flare-ups were experienced by 71% (17/24) of placebo-treated patients, 59% (10/17) of whom developed a new HO lesion irrespective of flare-up location; 24% of flare-ups location-matched new HO lesions. Twenty-nine new HO lesions occurred in the placebo cohort by week 28, of which 12 (41%) occurred in the same location as new or ongoing flare-ups. A higher volume of newly formed heterotopic bone (week 28) occurred in placebo-treated patients who had experienced a prior flare-up versus those without (median [Q1:Q3] of 16.6 [12.0:31.1] cm³ versus 3.2 cm³). Garetosmab was previously shown to decrease patient-reported flare-up frequency in Period 1; here, garetosmab reduced the median (Q1:Q3) duration of patient reported flares (15.0 [6.0:82.0] versus 48.0 [15.0:1.00] days) and severity of flare-ups versus placebo. Frequency of corticosteroid use was numerically reduced in those treated with garetosmab (40.0%) versus placebo (58.3%). In this analysis, 71% of placebo-treated adults with FOP experienced flare-ups over 28 weeks, which were associated with an increased volume of newly formed heterotopic bone. Garetosmab reduced the severity and duration of flare-ups with effects sustained during the entire trial. Fibrodysplasia ossificans progressiva (FOP) is a very rare genetic disorder caused by mutations in ACVR1, a gene that encodes for a receptor. In FOP, the mutated receptor is uniquely activated by activin A, a protein that binds ACVR1 (but does not normally activate it). When FOP-mutant ACVR1 is activated by activin A, this causes new bone to form in places where it does not usually develop. More specifically, in FOP, soft tissues (such as skeletal muscles) and connective tissues (such as tendons and ligaments) are gradually replaced by bone outside of the normal skeleton—a process referred to as heterotopic ossification (HO). In people with FOP, the build-up of bone impacts their mobility. Additionally, people with FOP also experience flare-ups, which are painful swellings of the soft tissues. This analysis investigated flare-up events, the relationship of flare-ups to new HO lesions, and the impact of garetosmab on flare-ups during a clinical trial that enrolled people with FOP. Garetosmab is a monoclonal antibody that binds to activin A and blocks it from activating the faulty receptor, hence stopping new heterotopic bone from forming. In this study, approximately half of the patients randomly received placebo and the other half randomly received garetosmab, the study drug. Of the people who received placebo, 71% experienced flare-ups and 59% percent of those who had flare-ups developed a new HO lesion irrespective of flare-up location. We previously reported that garetosmab decreases patient-reported flare-up frequency. In this study, we show that garetosmab also reduces the duration and severity of flare-ups, as well as the frequency of corticosteroid use with the treatment effect maintained for the entire trial.

Coronavirus disease 2019 (COVID-19) and influenza are respiratory illnesses caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses, respectively. Both diseases share symptoms and clinical risk factors¹, but the extent to which these conditions have a common genetic etiology is unknown. This is partly because host genetic risk factors are well characterized for COVID-19 but not for influenza, with the largest published genome-wide association studies for these conditions including >2 million individuals² and about 1,000 individuals^3–6, respectively. Shared genetic risk factors could point to targets to prevent or treat both infections. Through a genetic study of 18,334 cases with a positive test for influenza and 276,295 controls, we show that published COVID-19 risk variants are not associated with influenza. Furthermore, we discovered and replicated an association between influenza infection and noncoding variants in B3GALT5 and ST6GAL1, neither of which was associated with COVID-19. In vitro small interfering RNA knockdown of ST6GAL1—an enzyme that adds sialic acid to the cell surface, which is used for viral entry—reduced influenza infectivity by 57%. These results mirror the observation that variants that downregulate ACE2, the SARS-CoV-2 receptor, protect against COVID-19 (ref. ⁷). Collectively, these findings highlight downregulation of key cell surface receptors used for viral entry as treatment opportunities to prevent COVID-19 and influenza.

BMP/TGFß family ligands act mainly as factors that differentially initiate Smad1/5/8 or Smad2/3 signaling via heterotetrameric complexes comprised of two type I and two type II receptors (IIR). ActA (ActA) stands out as it activates Smad2/3 signaling through type I receptor ACVR1B, whereas it generates non-signaling complexes (NSCs) with ACVR1. In fibrodysplasia ossificans progressiva (FOP), a genetic disorder caused by missense mutations in ACVR1 (ACVR1.FOP), ACVR1.FOP•ActA•IIR complexes activate Smad1/5 signaling, mimicking those formed with BMPs. As the NSCs that ActA forms with ACVR1 are stoichiometrically identical with signaling complexes formed with ACVR1.FOP, we explored how NSCs differ from their signaling counterparts. We show that ACVR1•ActA•IIR complexes rapidly traffic to lysosomes, where their constituent components are degraded, reducing the cell’s responsiveness to BMPs along with ActA’s availability. This property is specific to ActA as Activin B, AB, and AC do not form lysosomal trafficking complexes with ACVR1, but rather remain on the surface as NSCs. Lysosomal trafficking and degradation of ACVR1•ActA•IIR complexes is a novel regulatory mechanism of BMP/TGFß signaling whose physiological roles remain largely unexplored.