Gene therapy for adenosine deaminase severe combined immune deficiency-An unexpected journey of four decades.

Severe combined immune deficiency due to adenosine deaminase deficiency (ADA SCID) is an inborn error of immunity with pan-lymphopenia, due to accumulated cytotoxic adenine metabolites. ADA SCID has been treated using gene therapy with a normal human ADA gene added to autologous hematopoietic stem cells (HSC) for over 30 years. Iterative improvements in vector design, HSC processing methods, and clinical HSC transplant procedures have led nearly all ADA SCID gene therapy patients to achieve consistently beneficial immune restoration with stable engraftment of ADA gene-corrected HSC over the duration of observation (as long as 20 years). One gene therapy for ADA SCID is approved by the European Medicines Agency (EMA) in the European Union (EU) and another is being advanced to licensure in the U.S. and U.K. Despite the clear-cut benefits and safety of this curative gene and cell therapy, it remains challenging to achieve sustained availability and access, especially for rare disorders like ADA SCID.

Treatment with Elapegademase Restores Immunity in Infants with Adenosine Deaminase Deficient Severe Combined Immunodeficiency.

PURPOSE: Patients with adenosine deaminase 1 deficient severe combined immunodeficiency (ADA-SCID) are initially treated with enzyme replacement therapy (ERT) with polyethylene glycol-modified (PEGylated) ADA while awaiting definitive treatment with hematopoietic stem cell transplant (HSCT) or gene therapy. Beginning in 1990, ERT was performed with PEGylated bovine intestinal ADA (ADAGEN®). In 2019, a PEGylated recombinant bovine ADA (Revcovi®) replaced ADAGEN following studies in older patients previously treated with ADAGEN for many years. There are limited longitudinal data on ERT-naïve newborns treated with Revcovi. METHODS: We report our clinical experience with Revcovi as initial bridge therapy in three newly diagnosed infants with ADA-SCID, along with comprehensive biochemical and immunologic data. RESULTS: Revcovi was initiated at twice weekly dosing (0.2 mg/kg intramuscularly), and monitored by following plasma ADA activity and the concentration of total deoxyadenosine nucleotides (dAXP) in erythrocytes. All patients rapidly achieved a biochemically effective level of plasma ADA activity, and red cell dAXP were eliminated within 2-3 months. Two patients reconstituted B-cells and NK-cells within the first month of ERT, followed by naive T-cells one month later. The third patient reconstituted all lymphocyte subsets within the first month of ERT. One patient experienced declining lymphocyte counts with improvement following Revcovi dose escalation. Two patients developed early, self-resolving thrombocytosis, but no thromboembolic events occurred. CONCLUSION: Revcovi was safe and effective as initial therapy to restore immune function in these newly diagnosed infants with ADA-SCID, however, time course and degree of reconstitution varied. Revcovi dose may need to be optimized based on immune reconstitution, clinical status, and biochemical data.

ADA2b acts to positively regulate blue light-mediated photomorphogenesis in Arabidopsis.

Cryptochromes (CRYs) act as blue light photoreceptors to regulate various plant physiological processes including photomorphogenesis and repair of DNA double strand breaks (DSBs). ADA2b is a conserved transcription co-activator that is involved in multiple plant developmental processes. It is known that ADA2b interacts with CRYs to mediate blue light-promoted DSBs repair. Whether ADA2b may participate in CRYs-mediated photomorphogenesis is unknown. Here we show that ADA2b acts to inhibit hypocotyl elongation and hypocotyl cell elongation in blue light. We found that the SWIRM domain-containing C-terminus mediates the blue light-dependent interaction of ADA2b with CRYs in blue light. Moreover, ADA2b and CRYs act to co-regulate the expression of hypocotyl elongation-related genes in blue light. Based on previous studies and these results, we propose that ADA2b plays dual functions in blue light-mediated DNA damage repair and photomorphogenesis.

Dual release of daptomycin and BMP-2 from a composite of ß-TCP ceramic and ADA gelatin.

BACKGROUND: Antibiotic-containing carrier systems are one option that offers the advantage of releasing active ingredients over a longer period of time. In vitro sustained drug release from a carrier system consisting of microporous ß-TCP ceramic and alginate has been reported in previous works. Alginate dialdehyde (ADA) gelatin gel showed both better mechanical properties when loaded into a ß-TCP ceramic and higher biodegradability than pure alginate. METHODS: Dual release of daptomycin and BMP-2 was measured on days 1, 2, 3, 6, 9, 14, 21, and 28 by HPLC and ELISA. After release, the microbial efficacy of the daptomycin was verified and the biocompatibility of the composite was tested in cell culture. RESULTS: Daptomycin and the model compound FITC protein A (n = 30) were released from the composite over 28 days. A Daptomycin release above the minimum inhibitory concentration (MIC) by day 9 and a burst release of 71.7 ± 5.9% were observed in the loaded ceramics. Low concentrations of BMP-2 were released from the loaded ceramics over 28 days.

Neurological phenotype of adenosine deaminase 2 deficient patients: a cohort study.

BACKGROUND AND PURPOSE: Patients with adenosine deaminase 2 (ADA2) deficiency can present with various neurological manifestations due to vasculopathies and autoinflammation. These include ischaemic and hemorrhagic stroke, but less clearly defined neurological symptoms have also been reported. METHODS: In this cohort study, patients with confirmed ADA2 deficiency from seven university hospitals in the Netherlands were included. The frequency and recurrence rates of neurological manifestations before and after initiation of tumor necrosis factor α (TNF-α) inhibiting therapy were analyzed. RESULTS: Twenty-nine patients were included with a median age at presentation of 5 years (interquartile range 1-17). Neurological manifestations occurred in 19/29 (66%) patients and were the presenting symptom in 9/29 (31%) patients. Transient ischaemic attack (TIA)/ischaemic stroke occurred in 12/29 (41%) patients and was the presenting symptom in 8/29 (28%) patients. In total, 25 TIAs/ischaemic strokes occurred in 12 patients, one after initiation of TNF-α inhibiting therapy and one whilst switching between TNF-α inhibitors. None was large-vessel occlusion stroke. Two hemorrhagic strokes occurred: one aneurysmatic subarachnoid hemorrhage and one spontaneous intracerebral hemorrhage. Most neurological symptoms, including cranial nerve deficits, vertigo, ataxia and seizures, were caused by TIAs/ischaemic strokes and seldom recurred after initiation of TNF-α inhibiting therapy. CONCLUSIONS: Neurological manifestations, especially TIA/ischaemic stroke, are common in patients with ADA2 deficiency and frequently are the presenting symptom. Because it is a treatable cause of young stroke, for which antiplatelet and anticoagulant therapy are considered contraindicated, awareness amongst neurologists and pediatricians is important. Screening for ADA2 deficiency in young patients with small-vessel ischaemic stroke without an identified cause should be considered.

A High Threshold of Biotherapeutic Aggregate Numbers is Needed to Induce an Immunogenic Response In Vitro, In Vivo, and in the Clinic.

BACKGROUND AND PURPOSE: There is concern that subvisible aggregates in biotherapeutic drug products pose a risk to patient safety. We investigated the threshold of biotherapeutic aggregates needed to induce immunogenic responses. METHODS AND RESULTS: Highly aggregated samples were tested in cell-based assays and induced cellular responses in a manner that depended on the number of particles. The threshold of immune activation varied by disease state (cancer, rheumatoid arthritis, allergy), concomitant therapies, and particle number. Compared to healthy donors, disease state patients showed an equal or lower response at the late phase (7 days), suggesting they may not have a higher risk of responding to aggregates. Xeno-het mice were used to assess the threshold of immune activation in vivo. Although highly aggregated samples (~ 1,600,000 particles/mL) induced a weak and transient immunogenic response in mice, a 100-fold dilution of this sample (~ 16,000 particles/mL) did not induce immunogenicity. To confirm this result, subvisible particles (up to ~ 18,000 particles/mL, containing aggregates and silicone oil droplets) produced under representative administration practices (created upon infusion of a drug product through an IV catheter) did not induce a response in cell-based assays or appear to increase the rate of adverse events or immunogenicity during phase 3 clinical trials. CONCLUSION: The ability of biotherapeutic aggregates to elicit an immune response in vitro, in vivo, and in the clinic depends on high numbers of particles. This suggests that there is a high threshold for aggregates to induce an immunogenic response which is well beyond that seen in standard biotherapeutic drug products.

Human ADA2 Deficiency: Ten Years Later.

PURPOSE OF REVIEW: In this review, an update is provided on the current knowledge and pending questions about human adenosine deaminase type 2 deficiency. Patients have vasculitis, immunodeficiency and some have bone marrow failure. Although the condition was described ten years ago, the pathophysiology is incompletely understood RECENT FINDINGS: Endothelial instability due to increased proinflammatory macrophage development is key to the pathophysiology. However, the physiological role of ADA2 is a topic of debate as it is hypothesized that ADA2 fulfils an intracellular role. Increasing our knowledge is urgently needed to design better treatments for the bone marrow failure. Indeed, TNFi treatment has been successful in treating DADA2, except for the bone marrow failure. Major advances have been made in our understanding of DADA2. More research is needed into the physiological role of ADA2.

Gibberellin Signaling through RGA Suppresses GCN5 Effects on Arabidopsis Developmental Stages.

Histone acetyltransferases (HATs) modify the amino-terminal tails of the core histone proteins via acetylation, regulating chromatin structure and transcription. GENERAL CONTROL NON-DEREPRESSIBLE 5 (GCN5) is a HAT that specifically acetylates H3K14 residues. GCN5 has been associated with cell division and differentiation, meristem function, root, stem, foliar, and floral development, and plant environmental response. The flowers of gcn5 plants display a reduced stamen length and exhibit male sterility relative to the wild-type plants. We show that these effects may arise from gibberellin (GA)-signaling defects. The signaling pathway of bioactive GAs depends on the proteolysis of their repressors, DELLA proteins. The repressor GA (RGA) DELLA protein represses plant growth, inflorescence, and flower and seed development. Our molecular data indicate that GCN5 is required for the activation and H3K14 acetylation of genes involved in the late stages of GA biosynthesis and catabolism. We studied the genetic interaction of the RGA and GCN5; the RGA can partially suppress GCN5 action during the whole plant life cycle. The reduced elongation of the stamen filament of gcn5-6 mutants is reversed in the rga-t2;gcn5-6 double mutants. RGAs suppress the GCN5 effect on the gene expression and histone acetylation of GA catabolism and GA signaling. Interestingly, the RGA and RGL2 do not suppress ADA2b function, suggesting that ADA2b acts downstream of GA signaling and is distinct from GCN5 activity. In conclusion, we propose that the action of GCN5 on stamen elongation is partially mediated by RGA and GA signaling.

Development and Validation of an ADA-Tolerant Assay for Quantification of an Exatecan-Based ADC in Monkey Plasma.

BACKGROUND: The development of an anti-drug antibody (ADA)-tolerant pharmacokinetic (PK) assay is important when the drug exposure is irrelevant to toxicity in the presence of ADA. We aimed to develop and validate an ADA-tolerant assay for an exatecan-based antibody-drug conjugate (ADC) in monkey plasma. RESULTS: The assay tolerated 5.00 µg/mL of ADA at 12 µg/mL of ADC. Its accuracy and precision results satisfied the acceptance criteria. Furthermore, the assay was free from hook and matrix effects and exhibited good dilutional linearity. Additionally, the ADC in plasma samples was stable under different storage conditions. METHOD: An ADA-tolerant ADC assay was configured with an anti-payload antibody for capture, and a drug-target protein combined with a horseradish peroxidase (HRP)-labeled antibody against a drug-target-protein tag for detection. Samples were firstly acidified to dissociate drug and ADA complexes, and to convert the carboxylate form to the lactone form of exatecan molecules; then, the ADAs in the samples were removed with a naked antibody-coated microplate. The treated samples were further incubated with coated anti-payload antibody and captured ADC molecules were quantified by the detection reagent. The developed assay was optimized and validated against regulatory guidelines. CONCLUSIONS: The assay met both methodological and sample-related ADA tolerance requirements, and was applicable to a nonclinical study in cynomolgus monkeys.

PEGylated Recombinant Adenosine Deaminase Maintains Detoxification and Lymphocyte Counts in Patients with ADA-SCID.

PURPOSE: Metabolic detoxification with enzyme replacement therapy (ERT) promotes immune recovery in patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency (ADA-SCID). Elapegademase is a PEGylated recombinant bovine ADA ERT developed to replace the now-discontinued bovine-derived pegademase. This study was a 1-way crossover from pegademase to elapegademase in 7 patients with ADA-SCID to assess efficacy and safety outcomes for elapegademase. METHODS: After once-weekly pegademase dosage was adjusted to achieve therapeutic metabolic detoxification and trough ADA activity, patients transitioned to a bioequivalent dose of elapegademase. Maintenance of metabolic detoxification and adequate ADA activity were evaluated periodically. RESULTS: One patient withdrew after 2 doses of an early elapegademase formulation due to injection-site pain caused by EDTA. The 6 remaining patients completed 71-216 weeks of elapegademase therapy with a formulation that did not contain EDTA. In these patients, elapegademase improved ADA activity compared with pegademase and maintained metabolic detoxification. Total lymphocyte counts increased for all completer patients from between 1.2- and 2.1-fold at the end of study compared with baseline. Elapegademase had a comparable safety profile to pegademase; no patient developed a severe infectious complication. Three patients had transient, non-neutralizing antibodies to pegademase, elapegademase, and/or polyethylene glycol ≤ 47 weeks of treatment without effect on trough plasma ADA activity or trough erythrocyte deoxyadenosine nucleotide levels. CONCLUSION: Elapegademase was safe, well tolerated, achieved stable trough plasma ADA activity with weekly dosing, was effective in maintaining metabolic detoxification, and was associated with maintenance or improvements in lymphocyte counts compared with pegademase therapy in patients with ADA-SCID.

The transcription factor FgStuA regulates virulence and mycotoxin biosynthesis via recruiting the SAGA complex in Fusarium graminearum.

The conserved Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex controls eukaryotic transcription by modifying acetylation of histones. However, the mechanisms for this complex in regulating the transcription of target-specific genes remain largely unknown in phytopathogenic fungi. A filamentous fungal-specific transcription factor FgStuA was identified to interact with the SAGA complex physically. The coordinative mechanisms of FgStuA with the SAGA complex in regulating secondary metabolism and virulence were investigated in Fusarium graminearum with genetic, biochemical and molecular techniques. The transcription factor FgStuA binds to a 7-bp cis-element (BVTGCAK) of its target gene promoter. Under mycotoxin deoxynivalenol (DON) induction conditions, FgStuA recruits the SAGA complex into the promoter of TRI6, a core regulator of the DON biosynthesis gene cluster, leading to enhanced transcription of TRI6. During this process, we found that FgStuA is subject to acetylation by the SAGA complex, and acetylation of FgStuA plays a critical role for its enrichment in the TRI6 promoter. In addition, FgStuA together with the SAGA complex modulates fungal virulence. This study uncovers a novel regulatory mechanism of a transcription factor, which recruits and interacts with the SAGA complex to activate specific gene expression in pathogenic fungi.

Early onset is an indication of the severity of DADA2 disease.

OBJECTIVE: To find indicators of disease severity and factors of early remission in patients with deficiency of adenosine deaminase 2 (DADA2). METHODS: We enrolled six DADA2 patients from six families. Direct sequencing of adenosine deaminase 2 gene (ADA2) was performed by Sanger analysis. A literature review was conducted for articles regarding paediatric DADA2. RESULTS: We found that more organs were involved in early-onset (≤1 year of age) than in late-onset (>1 year of age) DADA2 patients had high level inflammatory responses, such as elevated ESR, SF, serum amyloid A and CRP. Disease severity was not significantly different from missense and frameshift mutation. Early administration of TNF inhibitor might result in better remission and reduce recurrence. In the literature, four articles describing 51 paediatric DADA2 patients were identified. We also found that fever, stroke, peripheral nervous system involvement, hypogammaglobulinaemia and hypertension were more frequent in early onset DADA2 patients. CONCLUSION: Early-onset DADA2 may be more severe. Early administration of TNF inhibitor can effectively reduce recurrence and quickly alleviate the disease.

Research on the improvement effect of Saposhnikovia divaricata (Trucz.) Schischk on rheumatoid arthritis based on the "component-target-pathway" association.

OBJECTIVE: To investigate the therapeutic effect and mechanism of the traditional Chinese medicine Saposhnikovia divaricata (Trucz.) Schischk in rats with complete Freund's adjuvant-induced rheumatoid arthritis (RA). METHODS: The chemical targets and RA targets of Saposhnikovia divaricata (Trucz.) Schischk were acquired by the network pharmacological method. The complete Freund's adjuvant-induced rat RA model was used to further explore the mechanism of Saposhnikovia divaricata (Trucz.) Schischk in improving RA. Pathological changes in the volume of toes, body weight and synovial tissues of joints as well as serum inflammatory factor levels before and after the intervention of Saposhnikovia divaricata (Trucz.) Schischk were investigated. The key metabolic pathways were screened by correlations between metabolites and key targets. Finally, a quantitative analysis of key targets and metabolites was experimentally validated. RESULTS: Saposhnikovia divaricata (Trucz.) Schischk administration increased body weight, mitigated foot swelling and downregulated inflammatory cytokine levels in model rats. The histopathology showed that treatment with Saposhnikovia divaricata (Trucz.) Schischk can induce inflammatory cell infiltration and synovial hyperplasia and obviously reduce cartilage injuries, thus improving arthritis symptoms in rats. According to the network pharmacology-metabonomics association analysis results, the purine metabolic signaling pathway might be the key pathway for RA intervention with Saposhnikovia divaricata (Trucz.) Schischk. Targeted metabonomics, Western blotting (WB) and reverse transcription-polymerase chain reaction (RT‒PCR) assays showed that the recombinant adenosine deaminase (ADA) mRNA expression level and metabolic level of inosine in Saposhnikovia divaricata (Trucz.) Schischk administration group were lower than those of the model group. This reflected that Saposhnikovia divaricata (Trucz.) Schischk could improve RA by downregulating ADA mRNA expression levels and the metabolic level of inosine in the purine signaling pathway. CONCLUSION: Based on the "component-disease-target" association analysis, this study concludes that Saposhnikovia divaricata (Trucz.) Schischk improves complete Freund's adjuvant-induced RA symptoms in rats mainly by downregulating ADA mRNA expression levels in the purine metabolic signaling pathway, mitigating foot swelling, improving the levels of serum inflammatory factors (IL-1ß, IL-6 and TNF-α), and decreasing the ADA protein expression level to intervene in purine metabolism.

Phenolic compounds as potential adenosine deaminase inhibitors: molecular docking and dynamics simulation coupled with MM-GBSA calculations.

Adenosine deaminase (ADA) is a Zn2+-containing enzyme that catalyzes the irreversible deamination of adenosine to inosine or deoxyadenosine to deoxyinosine. In addition to this enzymatic function, ADA mediates cell-to-cell interactions involved in lymphocyte co-stimulation or endothelial activation. ADA is implicated in cardiovascular pathologies such as atherosclerosis and certain types of cancers, including lymphoma and leukemia. To date, only two drugs (pentostatin and cladribine) have been approved for the treatment of hairy cell leukemia. In search of natural ADA inhibitors, we demonstrated the binding of selected phenolic compounds to the active site of ADA using molecular docking and molecular dynamics simulation. Our results show that phenolic compounds (chlorogenic acid, quercetin, and hyperoside) stabilized the ADA complex by forming persistent interactions with the catalytically essential Zn2+ ion. Furthermore, MM-GBSA ligand binding affinity calculations revealed that hyperoside had a comparable binding energy score (ΔG = - 46.56 ± 8.26 kcal/mol) to that of the cocrystal ligand in the ADA crystal structure (PDB ID: 1O5R) (ΔG = - 51.97 ± 4.70 kcal/mol). Similarly, chlorogenic acid exhibited a binding energy score (ΔG = - 18.76 ± 4.60 kcal/mol) comparable to those of the two approved ADA inhibitor drugs pentostatin (ΔG = - 14.54 ± 2.25 kcal/mol) and cladribine (ΔG = - 25.52 ± 4.10 kcal/mol) while quercetin was found to have modest binding affinity (ΔG = - 8.85 ± 7.32 kcal/mol). This study provides insights into the possible inhibitory potential of these phenolic compounds against ADA.

Pleural fluid adenosine deaminase to serum C-reactive protein ratio for diagnosing tuberculous pleural effusion.

BACKGROUND: Tuberculous pleural effusion (TPE) and malignant pleural effusion (MPE) may occasionally show similar cytological and biochemical picture including ADA. In such cases, differentiating TPE and MPE is challenging and needs histopathology of pleural tissue which may involve invasive procedures. The present study aims to evaluate the diagnostic accuracy of pleural fluid ADA to serum CRP (ADA/CRP) ratio to discriminate between tuberculous and malignant pleural effusion. In addition, we investigated whether the ratio ADA/CRP adds diagnostic value to ADA. METHODS: This cross-sectional study was conducted in the National Institute of Diseases of the Chest and Hospital (NIDCH), Mohakhali, Dhaka, from July 2021 to February 2022 on diagnosed patients of TPE and malignant pleural effusion MPE. A receiver operating characteristic curve (ROC) was constructed for identifying TPE. The added value of the ADA/CRP ratio to ADA was evaluated using the net reclassification improvement (NRI) and integrated discrimination improvement (IDI). A value of p < 0.05 was considered statistically significant for all tests. RESULTS: Fifty-nine patients were enrolled in this study, of which 31 had TPE, and 28 had MPE. Pleural fluid ADA to serum CRP ratio and pleural fluid ADA level was significantly higher in patients with TPE, but there was no significant difference in serum CRP levels between patients with TPE and MPE. At cut off value of > 1.25, pleural fluid ADA to serum CRP ratio had a sensitivity of 93.8%, specificity of 85.2%, and positive and negative predictive values were 88.2% and 92% respectively, in the diagnosis of TPE and area under ROC curve (AUC) was 0.94. The NRI and IDI analyses revealed added diagnostic value of ADA/CRP to ADA. CONCLUSION: This study shows that the ADA/CRP ratio improves the diagnostic usefulness of ADA for TPE.

Cordycepin: A review of strategies to improve the bioavailability and efficacy.

Cordycepin is a bioactive compound extracted from Cordyceps militaris. As a natural antibiotic, cordycepin has a wide variety of pharmacological effects. Unfortunately, this highly effective natural antibiotic is proved to undergo rapid deamination by adenosine deaminase (ADA) in vivo and, as a consequence, its half-life is shortened and bioavailability is decreased. Therefore, it is of critical importance to work out ways to slow down the deamination so as to increase its bioavailability and efficacy. This study reviews recent researches on a series of aspects of cordycepin such as the bioactive molecule's pharmacological action, metabolism and transformation as well as the underlying mechanism, pharmacokinetics and, particularly, the methods for reducing the degradation to improve the bioavailability and efficacy. It is drawn that there are three methods that can be applied to improve the bioavailability and efficacy: to co-administrate an ADA inhibitor and cordycepin, to develop more effective derivatives via structural modification, and to apply new drug delivery systems. The new knowledge can help optimize the application of the highly potent natural antibiotic-cordycepin and develop novel therapeutic strategies.

TNF inhibition in vasculitis management in adenosine deaminase 2 deficiency (DADA2).

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited autoinflammatory disorder caused by a loss of functional ADA2 protein. TNF inhibition (TNFi) has proven to be highly effective in treating inflammatory manifestations. OBJECTIVE: We sought to explore the pathophysiology and the underlying mechanisms of TNF-inhibitor response in these patients. METHODS: We performed Sanger sequencing of the ADA2 gene. We used flow cytometry, intracellular cytokine staining, transcriptome analysis, immunohistochemistry, and cell differentiation experiments to define an inflammatory signature in patients with DADA2 and studied their response to TNF-inhibitor treatment. RESULTS: We demonstrated increased inflammatory signals and overproduction of cytokines mediated by IFN and nuclear factor kappa B pathways in patients' primary cells. Treatment with TNFi led to reduction in inflammation, rescued the skewed differentiation toward the proinflammatory M1 macrophage subset, and restored integrity of endothelial cells in blood vessels. We also report 8 novel disease-associated variants in 7 patients with DADA2. CONCLUSIONS: Our data explore the cellular mechanism underlying effective treatment with TNFi therapies in DADA2. DADA2 vasculitis is strongly related to the presence of activated myeloid cells, and the endothelial cell damage is rescued with anti-TNF treatment.

Comprehensive analysis of ADA2 genetic variants and estimation of carrier frequency driven by a function-based approach.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is an autoinflammatory disease caused by deleterious ADA2 variants. The frequency of these variants in the general population, and hence the expected disease prevalence, remain unknown. OBJECTIVE: We aimed to characterize the functional impact and carrier frequency of ADA2 variants. METHODS: We performed functional studies and in silico analysis on 163 ADA2 variants, including DADA2-associated variants and population variants identified in the Genome Aggregation Database. We estimated the carrier rate using the aggregate frequency of deleterious variants. RESULTS: Functional studies of ADA2 variants revealed that 77 (91%) of 85 of DADA2-associated variants reduced ADA2 enzymatic function by >75%. Analysis of 100 ADA2 variants in the database showed a full spectrum of impact on ADA2 function, rather than a dichotomy of benign versus deleterious variants. We found several in silico algorithms that effectively predicted the impact of ADA2 variants with high sensitivity and specificity, and confirmed a correlation between the residual function of ADA2 variants in vitro and the plasma ADA2 activity of individuals carrying these variants (n = 45; r = 0.649; P < .0001). Using <25% residual enzymatic activity as the cutoff to define potential pathogenicity, integration of our results with the database population data revealed an estimated carrier frequency of at least 1 in 236 individuals, corresponding to an expected DADA2 disease prevalence of ~1 in 222,000 individuals. CONCLUSIONS: Functional annotation guides the interpretation of ADA2 variants to create a framework that enables estimation of DADA2 carrier frequency and disease prevalence.

Endogenous Digitalis-like Factors as a Key Molecule in the Pathophysiology of Pregnancy-Induced Hypertension and a Potential Therapeutic Target in Preeclampsia.

Preeclampsia (PE), the most severe presentation of hypertensive disorders of pregnancy, is the major cause of morbidity and mortality linked to pregnancy, affecting both mother and fetus. Despite advances in prophylaxis and managing PE, delivery of the fetus remains the only causative treatment available. Focus on complex pathophysiology brought the potential for new treatment options, and more conservative options allowing reduction of feto-maternal complications and sequelae are being investigated. Endogenous digitalis-like factors, which have been linked to the pathogenesis of preeclampsia since the mid-1980s, have been shown to play a role in the pathogenesis of various cardiovascular diseases, including congestive heart failure and chronic renal disease. Elevated levels of EDLF have been described in pregnancy complicated by hypertensive disorders and are currently being investigated as a therapeutic target in the context of a possible breakthrough in managing preeclampsia. This review summarizes mechanisms implicating EDLFs in the pathogenesis of preeclampsia and evidence for their potential role in treating this doubly life-threatening disease.

The Americans with Disabilities Act, addiction, and recovery.

BACKGROUND: The Americans with Disabilities Act (ADA) is sweeping civil rights legislation that affords people with disabilities equality of opportunity, economic self-sufficiency, independent living, and full participation in daily life. The protections of the ADA extend to individuals with alcohol and substance abuse disorders who are in recovery. OBJECTIVE: The objective of this article is to understand the ADA's definition of disability and how it applies to addiction and recovery. The reader will recognize how the ADA applies to people with addiction to alcohol and those in recovery from substance abuse. The article will describe the rights and responsibilities employers and employees have in the three stages of employment. METHOD: The material in this presentation was developed based on the authors' research, training, education, knowledge, and skill of the ADA. RESULTS: Individuals in recovery are often unaware of their civil rights under the ADA. The ADA ensures that people with disabilities, including those with alcohol use disorder and substance use disorders, have the same rights and opportunities in the workplace. CONCLUSION: The COVID-19 pandemic has resulted in an increase in increased rates of alcoholism and substance abuse. Individuals living with addiction are unaware of the employment protections they may have under the ADA. Title I of the ADA guarantees employment protections to ensure equality in the workplace for people with disabilities. A clearer understanding of the law will ensure that job seekers and employees are treated in an ethical, valued, and courteous way when disclosing disability and the need for accommodation.