Search Results (548)

Gene therapy is undergoing continued clinical translation and technological development. This progress has been marked by regulatory approvals and broadened therapeutic indications across genetic, metabolic, and oncologic diseases and disorders. The field has evolved over decades from early viral-mediated gene addition to approaches capable of targeted editing, regulation, or replacement of genetic information. These systems include base and prime editors, epigenetic modulators, CRISPR-Cas, RNA therapeutics and programmable integration platforms. When paired with increasingly sophisticated viral and nonviral delivery strategies, these technologies enable greater control over tissue targeting, duration of activity, and therapeutic exposure. Recent clinical successes, including approved ex vivo CRISPR-based therapies for hemoglobinopathies, in vivo CRISPR editing for transthyretin amyloidosis, and emerging clinical applications of base and prime editing, provide growing clinical evidence for the feasibility of genetic medicines. However, technological advancement has also made platform selection increasingly complex. Therapeutic performance is determined not by editing efficiency alone, but by the interaction among genetic precision, temporal control, dosage tunability, delivery efficiency, durability, and disease-specific safety requirements. A molecularly efficient platform may still have limited therapeutic value if it cannot reach the disease-relevant cell population at sufficient and safe exposure. In this review, we examine recent technological and clinical advances in genetic medicine with particular emphasis on developments during the past approximately five years. We propose a multidimensional framework in which gene therapy platforms are evaluated according to three intrinsic properties—genetic precision, temporal control, and dosage tunability—while delivery, clinical maturity, and disease context act as major translational constraints. This framework highlights that no single platform is universally optimal; rather, successful therapeutic design depends on matching the biological characteristics of the intervention to the requirements of the disease and target tissue. Remaining challenges in extrahepatic delivery, genomic safety, immunogenicity, manufacturing, and long-term monitoring remain important determinants of broader clinical implementation. Full article

The cardiovascular actions of testosterone remain controversial, particularly regarding the safety of testosterone replacement therapy (TTh) in hypogonadal men. We investigated the effects of sustained supraphysiological testosterone exposure on aortic atherogenesis and cardiac remodelling in the testicular feminised (Tfm) mouse, a model of functional androgen receptor (AR) deficiency. Male Tfm mice and AR-intact XY littermate controls were fed a cholesterol-enriched diet for 28 weeks and received fortnightly intramuscular injections of saline or supraphysiological testosterone, alone or in combination with fulvestrant (oestrogen receptor antagonist) or anastrozole (aromatase inhibitor). Aortic lipid deposition was quantified by Oil Red O staining, while cardiac remodelling was assessed by heart weight, cardiomyocyte cross-sectional area and myocardial gene expression. Supraphysiological testosterone significantly reduced aortic fatty streak formation in Tfm mice compared with saline-treated controls (1.25 ± 0.36% vs. 2.85 ± 0.37%, p < 0.01), an effect preserved following fulvestrant or anastrozole treatment, consistent with mechanisms that do not require classical AR or oestrogen receptor signalling. No additional reduction in aortic lipid deposition was observed in AR-intact XY littermates. In contrast, supraphysiological testosterone increased heart weight, cardiomyocyte size and expression of hypertrophic markers exclusively in XY mice, with no evidence of cardiac remodelling in Tfm mice. Collectively, these findings demonstrate divergent tissue-specific cardiovascular actions of testosterone, whereby supraphysiological exposure promotes AR-dependent cardiac remodelling without conferring additional vascular benefit, supporting maintenance of physiological testosterone concentrations during TTh. Full article

The hepatocyte growth factor (HGF)/c-Met signaling pathway plays a central role in cellular proliferation, survival, migration, invasion, angiogenesis, and therapeutic resistance. Aberrant c-Met activation, driven by gene amplification, overexpression, activating mutations, exon 14-skipping alterations, or ligand-dependent stimulation, drives the development and progression of many solid tumors, positioning c-Met as a key target for anticancer drug development. The clinical effectiveness of c-Met-targeted treatments such as crizotinib, capmatinib, tepotinib, savolitinib, and cabozantinib has confirmed c-Met as a viable oncogenic driver for therapy, leading to the development of various next-generation inhibitors with different structures. This review provides a comprehensive perspective on small-molecule c-Met inhibitors from the perspectives of medicinal chemistry and structure-based drug design, encompassing approved drugs, investigational agents, natural-product-inspired leads, and emerging multitarget anticancer therapeutics. Particular emphasis is given to the principles of molecular recognition that govern c-Met inhibition. This includes the structure of the kinase domain, interactions at the ATP-binding site, recognition of the hinge region, and the different binding modes of Type I, Type II, and allosteric inhibitors. The design, synthesis, biological evaluation, and structure–activity relationships of diverse heterocyclic scaffolds that have shaped c-Met inhibitor discovery are critically analyzed. Key medicinal chemistry strategies, including scaffold hopping, bioisosteric replacement, conformational optimization, molecular hybridization, and multitarget pharmacophore integration, are discussed in the context of potency, selectivity, resistance management, and drug-like properties. Particular attention is given to the integration of structural biology, molecular docking, binding-mode analysis, and structure-guided optimization approaches that have enabled the development of potent c-Met-directed inhibitors. In addition, recent advances in dual- and multitarget agents that simultaneously modulate c-Met and complementary therapeutic targets, including VEGFR-2, EGFR, AXL, MER, PARP1, CDK2, and tubulin, are highlighted as promising strategies for overcoming pathway redundancy and acquired resistance. This review summarizes contemporary structure-based and medicinal chemistry principles underlying c-Met inhibitor discovery, critically evaluates the relationship between biochemical potency and therapeutic efficacy, and provides a framework for the rational design of next-generation c-Met-targeted and multitarget anticancer agents. Full article

Mutations in SLC26A4, which encodes the anion transporter pendrin, represent one of the most common genetic causes of hereditary hearing loss, including Pendred syndrome and DFNB4. Pendrin plays a critical role in maintaining ion homeostasis within the inner ear, particularly in the endolymphatic sac (ES), making it an important therapeutic target for gene replacement strategies. However, efficient delivery of therapeutic genes to relevant inner ear structures remains a major challenge for clinical translation. In this study, we evaluated the inner ear transduction profile of AAV8BP2, an engineered AAV8-derived capsid, in comparison with AAV2.7m8 and AAV8 in mice. Neonatal mice received posterior semicircular canal (PSCC) injections at postnatal day 0 (P0), and viral transduction in the cochlea and ES was assessed at P7. To examine age-dependent differences in viral transduction, additional experiments were performed in adult mice injected at P21 and analyzed at P28. We also compared three surgical delivery routes for inner ear gene transfer: PSCC injection, round window membrane (RWM) injection, and RWM injection combined with PSCC fenestration. AAV8BP2 effectively transduced the ES in both neonatal and adult mice and showed greater GFP expression in the spiral prominence than AAV8 following neonatal administration. Among the three delivery routes evaluated in adult mice, PSCC injection achieved the highest ES transduction while maintaining cochlear hair cell transduction comparable to that achieved with RWM-based approaches. Together, these findings define the relative transduction profiles of the tested AAV capsids and delivery routes and provide a basis for selecting vector-delivery route combinations for SLC26A4-targeted inner ear gene therapy. Full article

Despite major advances in lipid-lowering therapies, a significant unmet need remains, particularly for patients with homozygous familial hypercholesterolemia (HoFH), severe heterozygous familial hypercholesterolemia (HeFH), and those who fail to achieve guideline-recommended LDL-C targets. Nucleic acid-based therapeutics have emerged as a transformative approach for treating hypercholesterolemia. Antisense oligonucleotides and small interfering RNAs (siRNAs) have demonstrated durable hepatic gene silencing and have led to approved therapies, while gene replacement and in vivo genome-editing strategies offer the potential for long-lasting, and possibly one-time, interventions. In parallel, microRNAs (miRNAs) have attracted increasing interest because of their ability to coordinately regulate multiple genes involved in lipoprotein metabolism, cholesterol transport, and lipid homeostasis. Human genetic studies further support the importance of miRNA-mediated regulation, exemplified by a rare ~2.5 kb deletion in the distal LDLR 3′UTR (“del2.5”) that disrupts miRNA-binding sites and is associated with lifelong low LDL-C levels. This review summarizes recent advances, mechanisms of action, clinical progress, and remaining challenges across antisense oligonucleotides, siRNAs, gene therapy, genome editing, and emerging miRNA-based therapeutics for hypercholesterolemia. As an example of the latter approach, the liver-directed miR-30c analog C2 has demonstrated preclinical activity by coordinately reducing hepatic lipoprotein secretion and lipogenesis while enhancing cholesterol elimination, resulting in reduced LDL-C and atherosclerosis. However, it must be noted that these findings remain preclinical, and further optimization of delivery, pharmacokinetics, safety, and long-term efficacy will be required before clinical evaluation. Continued advances in RNA chemistry, targeted delivery, and genome engineering are expected to further expand the therapeutic landscape for dyslipidemia and cardiovascular disease. Full article

Cystic fibrosis is a genetic disorder caused by pathogenic variants in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene and may be complicated by hypokalemic hypochloremic metabolic alkalosis (HHMA), resulting from excessive electrolyte loss through sweat. This retrospective study aimed to clinically characterize individuals with cystic fibrosis who developed HHMA, characterize their CFTR genetic profile and compare it with that of other individuals with cystic fibrosis followed at a Brazilian tertiary referral center, and contextualize these findings within the available national and international literature. Among 257 individuals with cystic fibrosis followed at a tertiary referral center between 2014 and 2024, 41 (15.9%) developed HHMA. Most patients were male (29; 70.7%), the median age at the first episode was approximately four months, and episodes occurred predominantly during summer. The biochemical profile was characterized by metabolic alkalosis, hyponatremia, hypokalemia, and hypochloremia. The F508del variant was the most common allele [F508del homozygous, 9 (22.0%); F508del heterozygous, 21 (51.2%); without the F508del variant, 11 (26.8%)], particularly among patients with recurrent episodes, which occurred in 9/11 (81.8%) cases. The most frequent clinical manifestations were dehydration, cough, and vomiting, although some patients presented with minimal symptoms. Management primarily consisted of intravenous and oral hydration, electrolyte replacement, and antibiotic therapy when indicated, with no HHMA-related deaths observed. The characterization of the CFTR genetic profile and its comparison with the broader cystic fibrosis population followed at the reference center provide additional insights into the genetic background of HHMA in a genetically diverse population. Taken together with findings from the national and international literature, these results indicate that HHMA is a relatively frequent and potentially recurrent complication of cystic fibrosis, particularly during early life, and may be influenced by both genetic and environmental factors. Early recognition, systematic electrolyte monitoring, and appropriate salt and fluid replacement may prevent severe complications and improve clinical management. Full article

Mesenchymal stromal cells (MSCs) have emerged as a promising therapeutic platform for central nervous system disorders, including neurodegenerative diseases and lysosomal storage disorders (LSDs). This review examines MSC mechanisms of action—paracrine activity, immunomodulation, antioxidant effects, TFEB-mediated autophagy regulation, and enzymatic cross-correction in LSDs—while critically assessing translational challenges. We provide a comparative analysis of MSC sources, administration routes, dosing regimens, and safety profiles, with emphasis on hemocompatibility and thrombotic risks. The evidence base for MSC efficacy in amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and LSDs is systematically reviewed, highlighting both promising signals and limitations. MSC-derived extracellular vesicles are discussed as a cell-free alternative with improved safety and potential blood–brain barrier interaction. We propose an individualized monitoring framework integrating clinical scales, biomarkers, and neuroimaging. Despite preclinical promise, the field faces major hurdles: product standardization, optimal dosing, and the need for large, randomized controlled trials. The most rational path forward lies in combination strategies—MSCs as adjuncts to gene or enzyme replacement therapy—and engineered platforms for sustained delivery. This review provides a roadmap for translational decision-making and identifies critical gaps that must be addressed before MSC-based therapies can be integrated into routine neurological practice. Full article

Recombinant adeno-associated virus (rAAV) is a preferred vector in gene therapy, although high production costs inhibit widespread adoption. The most common approach for rAAV production involves transfection of HEK293 cells with three plasmids: pTransgene, pRep/Cap and pHelper. Producing sufficient amounts of these plasmids accounts for up to 40% of total batch costs. Initially, this work aimed to increase plasmid yields by replacing the backbones. While this approach increased pHelper yields, pRep/Cap and pTransgene yields were unaffected. A possible reason was identified: pTransgene contains inverted terminal repeat (ITR) sequences that are essential for rAAV production. ITRs have strong secondary structures (including hairpin loops termed B and C arms) that likely interfere with plasmid production. Therefore, targeted deletions were performed within the ITRs. Partial deletions in both the B and C arms of the ITR were most beneficial, as both plasmid yield and transgene expression increased. Importantly, partial deletions did not reduce rAAV yield, as had been previously observed when the B and C arms were fully deleted. In summary, we report a 140% increase in pHelper plasmid production, while the most successful ITR variant increased pTransgene plasmid yields by 57% and transgene expression by 28%, without reducing rAAV yields or transduction efficiency. Full article

Background/Objectives: Achondroplasia is the most common skeletal dysplasia and the leading genetic cause of disproportionate short stature. Although its biological basis involves gain-of-function variants in the FGFR3 gene, achondroplasia is a lifelong multisystem disorder associated with neurological, respiratory, orthopedic, otolaryngological, cardiovascular, oral, functional, and psychosocial complications. This narrative review aims to synthesize the evidence on developmental and adaptive functioning, age-specific healthcare needs, multidisciplinary service delivery, transition to adult care, psychosocial well-being, caregiver burden, and patient- and family-centered outcomes in achondroplasia across the lifespan. Methods: A narrative literature review was conducted using PubMed/MEDLINE, Scopus, Web of Science Core Collection, and CINAHL, with Google Scholar used as a supplementary source. Studies published between January 2010 and July 2026 were considered, together with earlier clinically relevant reports. Evidence addressing prenatal and postnatal diagnosis, age-specific manifestations, neurological and respiratory complications, orthopedic and otolaryngological care, cardiometabolic risk, growth monitoring, multidisciplinary management, transition to adult services, disease-modifying therapy, quality of life, and caregiver burden was evaluated. Results: The clinical priorities of achondroplasia change substantially across the lifespan. Infancy is characterized by an increased risk of foramen magnum stenosis, cervicomedullary compression, hypotonia, and sleep-disordered breathing, whereas orthopedic deformities, chronic pain, reduced mobility, spinal stenosis, hearing impairment, obesity, and cardiovascular risk become increasingly relevant during later childhood, adolescence, and adulthood. Early diagnosis, condition-specific imaging, neurological and respiratory surveillance, growth monitoring, and coordinated specialist care are essential for preventing severe complications. Vosoritide has introduced a disease-modifying therapeutic option, but it does not replace comprehensive clinical surveillance, rehabilitation, orthopedic care, psychosocial support, or shared decision-making. Functional limitations, environmental barriers, treatment burden, and caregiver stress contribute substantially to reduced quality of life. Conclusions: Achondroplasia should be managed as a lifelong multisystem condition rather than solely as a disorder of short stature. Standardized surveillance, multidisciplinary coordination, planned transition to adult care, and patient- and family-centered management are essential for improving function, autonomy, long-term health outcomes, and quality of life. Full article

Hemodialysis (HD) is the most widely used renal replacement therapy for patients with end-stage renal disease (ESRD) and is frequently accompanied by long-term complications that impair quality of life, including metabolic and inflammatory disturbances. Growing evidence suggests that these complications may be linked to alterations in the gut microbiota; however, microbial composition and interaction patterns in HD patients remain incompletely characterized. In this exploratory, cross-sectional study, high throughput 16S rRNA gene sequencing was used to profile the fecal microbiota of patients undergoing hemodialysis and of healthy controls. The objective was to characterize associations between hemodialysis and gut microbial composition, ecological network organization, and predicted functional potential. Comparative analyses revealed significant differences in bacterial community structure and microbial networks in the HD cohort. Both gender and dialysis vintage were associated with variation in specific taxa, including increased detection of the Synergistetes phylum, particularly among male patients and those undergoing long-term HD. Associations were also observed between clinical and demographic factors and the relative abundance of several short-chain fatty acid-associated taxa, including members of the Lachnospiraceae and Ruminococcaceae families and the genus Bifidobacterium. Predicted functional potential (PICRUSt2) indicated distinct microbial metabolic profiles in HD patients compared with controls, particularly in pathways related to carbohydrate, nucleotide, and amino acid metabolism, with additional variation according to dialysis vintage. Overall, these findings provide an exploratory characterization of structural, compositional, and predicted functional alterations of the gut microbiota associated with hemodialysis. Although the modest cohort size precludes definitive conclusions, the results support the rationale for larger, longitudinal studies investigating microbiota-derived biomarkers and host–microbiome interactions in ESRD. Full article

Bietti crystalline dystrophy like retinal degeneration (BCD) is an inherited retinal degenerative disease caused by defects in the CYP4V2 gene. Recombinant AAV-based gene replacement therapy holds promise for this disease, yet reliable enzymatic activity assays for product potency evaluation remain urgently needed. This study established and validated a method for determining the enzymatic activity of CYP4V2 based on the bioluminescent substrate Luciferin-MultiCYP.HEK293-AAVR cells were seeded at 1 × 10⁴ cells/well and transduced with rAAV-hCYP4V2 across an MOI gradient (1.17 × 10³–2.4 × 10⁶). After incubating at 37 °C for 72 h, medium was replaced with Opti-MEM containing 20 µM Luciferin-MultiCYP and incubated for 90 min ± 30 min. Next, cell supernatants were mixed with luciferase reagent, and bioluminescence was measured. Methodological validation results revealed that the method has good specificity, with an accuracy recovery rate of 100.94% ± 2.84% (RSD = 2.81%). Repeatability (GCV%) was 4.21%, and intermediate precision (RSD) was 6.16%. Linearity was good within the MOI range of 1.17 × 10³ to 2.4 × 10⁶ (R² = 0.9902 ± 0.0061, n = 9). Lastly, the method was successfully applied to activity testing the products of three AAV serotypes (AAV8, AAV2/8, and AAV2). This bioluminescent method meets regulatory potency assay requirements and serves as an effective QC tool for process development, batch release, and stability evaluation of rAAV-hCYP4V2 products. Full article

Hypophosphatasia (HPP) encompasses a group of inherited metabolic bone disorders characterized by defective skeletal mineralization and variable clinical severity in childhood. Substantial allelic heterogeneity contributes to a broad pediatric clinical spectrum, ranging from life-threatening perinatal disease to milder phenotypes characterized by chronic functional impairment. Historically, management relied primarily on supportive interventions aimed at sustaining survival, without modifying the underlying enzymatic defect. The introduction of enzyme replacement therapy (ERT) with asfotase alfa has fundamentally altered the natural history of pediatric HPP by supplementing deficient alkaline phosphatase activity at sites of active mineralization, thereby improving skeletal integrity, enhancing survival in severe forms, and supporting long-term functional gains. This therapeutic shift has redirected clinical priorities from survival alone toward sustained functional development and health-related quality of life. Nevertheless, variability in disease expression and therapeutic response persists, reflecting both diagnostic timing and the molecular heterogeneity of ALPL variants, whose phenotypic consequences cannot be predicted with complete certainty. Growing recognition of the importance of early diagnosis has prompted exploratory efforts toward systematic identification strategies, including neonatal screening initiatives reported in selected populations, which suggest the potential for earlier therapeutic intervention during active skeletal development. Together, these considerations highlight pediatric HPP as a model of precision-oriented management in rare metabolic bone disease, where timely diagnosis and targeted enzyme replacement must be aligned with long-term, multidisciplinary care to optimize outcomes. Full article

Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that is caused in most cases by pathogenic variants in MECP2, the gene encoding methyl-CpG-binding protein 2 (MeCP2). Despite substantial progress in the development of gene therapy, restoring MECP2 expression remains challenging because MeCP2 is highly dosage-sensitive. Both deficiency and excessive expression of this protein are associated with severe neurological abnormalities. This makes simple viral vector-mediated replacement of MECP2 potentially unsafe and underscores the need for multilayered systems that control transgene expression. This review discusses current and emerging strategies for regulating MeCP2 expression in RTT, with an emphasis on non-coding RNA-based and epigenetic mechanisms. Particular attention is given to the limitations of conventional AAV-mediated gene therapy, the use of cell-specific and endogenous promoters, miRNA-regulated elements, autoregulatory systems, and post-transcriptional control of MECP2 expression. Strategies for reactivating the inactive X chromosome are also discussed, including XIST-dependent regulation and epigenome editing. In addition, the review considers CRISPR-mediated regulation, selective epigenetic activation, and combined therapeutic platforms that integrate viral delivery, RNA-dependent post-transcriptional control, and endogenous gene regulation. Overall, clinically applicable gene therapy for RTT will likely need to move beyond simple MECP2 replacement and instead rely on precise cell- and dose-dependent regulation of its expression. Non-coding RNA and epigenetic mechanisms represent important layers of such control and may contribute to the development of safer gene therapy strategies for RTT. Full article

Cardiac disease is a leading cause of morbidity and early death across several lysosomal storage disorders (LSDs); however, the cardiomyopathies of Fabry, Pompe, and Danon disease are still largely treated as separate, substrate-specific disorders. We argue that they are better understood as variations on a single theme: the breakdown of the autophagy–lysosome system within cardiomyocytes. In the healthy heart, this system clears damaged proteins and organelles and is regulated by mTORC1 and the master regulator TFEB. Once lysosomal degradation or autophagosome–lysosome fusion fails, undegraded substrates and defective mitochondria accumulate, driving hypertrophy, interstitial fibrosis, and conduction disease. Danon disease, resulting from the loss of LAMP2, is the clearest example of a primary defect in autophagic flux, whereas the glycogen storage of Pompe disease and the globotriaosylceramide accumulation of Fabry disease impair flux through different upstream mechanisms that converge on the same downstream injury. The same framework extends to other storage disorders with cardiac involvement, such as mucopolysaccharidosis (MPS). We trace this shared pathobiology from molecule to bedside, examine biomarkers that reflect lysosomal and autophagic dysfunction rather than storage alone, and re-examine treatment in that light: why enzyme replacement therapy corrects substrate accumulation but leaves much of the autophagic and mitochondrial damage unresolved, and why gene therapy—particularly AAV9-LAMP2B for Danon disease—together with autophagy- and TFEB-directed strategies may help close that gap. Viewing these disorders through a single mechanistic lens reshapes how we monitor them and where future therapies should be directed. Full article

Background and Objectives: Fabry disease (FD) is an X-linked lysosomal storage disorder caused by pathogenic GLA gene variants, leading to progressive multi-organ damage including end-stage renal disease. Although dialysis patients represent a high-risk population for undiagnosed FD, data from Türkiye using genetic analysis as the primary screening method remain limited. This study aimed to determine FD prevalence among hemodialysis patients across multiple centers in Türkiye and to perform cascade family screening of confirmed cases. Materials and Methods: This multicenter cross-sectional study screened 1359 adult hemodialysis patients across 8 centers in Ankara, Türkiye, using complete GLA gene sequencing. Variants were classified per American College of Medical Genetics and Genomics criteria. Patients with pathogenic variants underwent confirmatory biochemical testing (α-galactosidase A activity and plasma lyso-Gb3). Cascade screening was performed for confirmed index cases. Results: Among 1359 patients (mean age 62.3 ± 14.3 years; 38.5% female), GLA variants were identified in 12 (0.88%): 8 benign/likely benign (including 7 p.D313Y pseudodeficiency alleles), 2 unclassified variants, 1 variant of uncertain significance, and 1 confirmed classic FD (prevalence: 0.07%; 95% CI: 0.002–0.41%). Cascade screening of the index patient identified 6 carriers among 10 relatives tested (60% yield). Three of 7 carriers (43%) were initiated on enzyme replacement therapy. Conclusions: Among 1359 hemodialysis patients, GLA gene sequencing identified 12 variants (0.88%), yet only one was confirmed as a disease-causing mutation responsible for end-stage renal disease (prevalence: 0.07%). The remaining variants comprised polymorphisms, likely benign pseudodeficiency alleles and variants of uncertain significance; most of which would not have been detected by enzyme-based screening alone, as enzyme activity was normal in these carriers. Cascade screening of the single confirmed index case yielded 6 carriers among 10 relatives tested (60%), including one hemizygous male with classic FD on hemodialysis, and three carriers were initiated on enzyme replacement therapy. These findings demonstrate that GLA gene analysis is a valuable instrument for screening in dialysis populations, as it captures the full variant spectrum while enabling rigorous distinction between the overall GLA variant carrier rate and the true disease prevalence defined by variants causing end-stage renal disease. Future screening studies should report prevalence based exclusively on confirmed disease-causing variants rather than total variant counts, which have inflated prevalence estimates in prior literature. Full article