Search Results (82)

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a widespread enzymopathy affecting approximately 500 million individuals that represents a significant global health issue. Among the more than 230 identified mutations in the G6PD gene, six class A variants—G6PD Utrecht (Pro409Ser), G6PD Suwalki (Pro409Arg), G6PD Merlo (Pro409Gln), G6PD Kawasaki (Gly410Ala), G6PD Shinagawa (Gly410Asp), and G6PD Riverside (Gly410Cys)—are located in the beta-loop near the NADP⁺ binding site. These mutations are of particular interest due to their association with severe hematologic phenotypes, including chronic hemolytic anemia, as well as their proposed role in the allosteric regulation of G6PD multimerization. This study presents a comprehensive biochemical and functional characterization of these clinically relevant G6PD variants. The variant enzymes were cloned, expressed, and purified for characterization. Kinetic parameters and thermal stability assays, complemented by molecular dynamics simulations (MDS), were employed to elucidate the structural impacts of the mutations. Our results demonstrate that these mutations significantly impair protein function, characterized by reduced affinity for glucose-6-phosphate (G6P) and NADP⁺, as well as altered thermal stability compared with wild-type G6PD. MDS revealed that point mutations in the βN- and βM-sheets in the NADP⁺_s region propagate subtle conformational changes, ultimately affecting the NADP⁺c region and the G6P binding cavity. Furthermore, secondary structure element analyses of the simulation data showed that Pro409 and Gly410 point mutations propagate several changes around residues 195–210 (G6P binding site) and 380–400 (NADP⁺_s), explaining their effect on overall catalytic performance. These findings enhance our understanding of the molecular mechanisms underlying G6PD deficiency and its clinical implications, providing a foundation for future therapeutic strategies aimed at mitigating the effects of these variants. Full article

Background/Objectives: Glycogen storage disease type II, also known as Pompe disease (PD), is a rare autosomal recessive genetic disorder triggered by a deficiency in lysosomal acid α-glucosidase (GAA). Recently, we discovered two deleterious missense variants of the GAA gene, c.1799G>A (p.Arg600His) (a pathogenic mutation) and c.55G>A (p.Val19Met), in a domestic short-haired cat with PD. This study aimed to design genotyping assays for these two variants and ascertain their allele frequencies in Japanese cat populations. Methods: We developed fluorescent probe-based real-time polymerase chain reaction assays to genotype the c.1799G>A and c.55G>A variants. A total of 738 cats, comprising 99 purebred cats from 20 breeds and 540 mixed-breed cats, were screened using these assays. Results: Genotyping assays clearly differentiated all known genotypes of the two variants. None of the 738 cats tested carried the c.1799G>A variant. However, we identified cats with c.55G/A and c.55A/A genotypes in the purebred (A allele frequency: 0.081) and mixed-breed cats (0.473). A significant difference (p < 0.001) was observed in the A allele frequency between the two groups. Conclusions: The c.1799G>A mutation appears rare in cat populations, suggesting it may be confined to specific pedigree Japanese mixed-breed cats. The c.55G>A variant was detected in purebred and mixed-breed cats, suggesting that it may not be directly linked to feline PD. However, additional studies are required to elucidate the precise relationship between this variant and cardiac function. Genotyping assays will serve as valuable tools for diagnosing and genotyping feline PD. Full article

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzymatic disorder, affects over 500 million people worldwide and is often linked to exercise intolerance due to oxidative stress, but its true impact on physical performance remains unclear. This study aimed to evaluate the physiological and metabolic effects of G6PD deficiency on endurance capacity. Using humanized mice carrying the African G6PD variant [V68M; N126D] (hG6PD_A−), we show that despite reduced pentose phosphate pathway activity, these mice exhibit a 10.8% increase in treadmill critical speed (CS)—suggesting enhanced endurance capacity. Multi-omics profiling across red blood cells, plasma, skeletal muscle, spleen, kidney, and liver reveals metabolic adaptations, including elevated glycolysis, fatty acid oxidation, and increased mitochondrial activity, alongside heightened oxidative phosphorylation in muscle and accelerated red blood cell turnover in the spleen and liver. These findings indicate that systemic metabolic reprogramming may offset antioxidant deficiencies, potentially conferring a performance advantage. Given that G6PD deficiency affects up to 13% of African Americans and is associated with cardiovascular health disparities, our results challenge conventional exercise restrictions and highlight the need for personalized exercise guidelines for affected individuals. Full article

TREM2 (triggering receptor expressed on myeloid cells 2) is a membrane-bound receptor primarily expressed on microglia in the central nervous system (CNS). TREM2 plays a crucial role in regulating immune responses, phagocytosis, lipid metabolism, and inflammation. Mutations in the TREM2 gene have been linked to various neurodegenerative diseases, including Alzheimer’s disease (AD), frontotemporal dementia (FTD), Parkinson’s disease (PD), and Nasu–Hakola disease (NHD). These mutations are suggested to impair microglial activation and reduce the ability to clear amyloid aggregates, leading to exacerbated neuroinflammatory responses and accelerating disease progression. This review provides an overview of TREM2 structure, functions, and known pathogenic variants—including Arg47His, Arg62His, His157Tyr, Tyr38Cys, and Thr66Met. Furthermore, the molecular and cellular consequences of TREM2 mutations are introduced, such as impaired ligand binding, altered protein folding and trafficking, enhanced TREM2 shedding, and dysregulated inflammatory signaling. We also highlight recent advances in therapeutic strategies aimed at modulating TREM2 signaling. These include monoclonal antibodies (e.g., AL002, CGX101), small molecule agonists, and gene/cell-based therapies that seek to restore microglial homeostasis, enhance phagocytosis, and reduce neuroinflammation. While these approaches show promise in in vivo/in vitro studies, their clinical translation may be challenged by disease heterogeneity and mutation-specific responses. Additionally, determining the appropriate timing and precise dosing will be essential. Full article

Recombinantly produced monoclonal antibodies (mabs) belong to the fastest growing class of biotherapeutics. In humans, antibodies are classified into five different classes: IgA, IgD, IgE, IgG and IgM. Most of the therapeutic mabs used in the clinic belong to the IgG class, albeit other antibody classes, e.g., IgM, have been evaluated in clinical stages. Antibodies are composed of heavy chains paired with a light chain. In IgM and IgA, an additional chain, the J-chain, is present. Two types of light chains exist in humans: the κ-light chain and the λ-light chain. The κ-light chain predominates in humans and is used in the vast majority of therapeutic IgG. The reason for the preference of the κ-light chain in humans is not known. Our study investigates whether light-chain selection influences the productivity of the clinically validated mabs adalimumab and trastuzumab. Both mabs were expressed as IgG and IgM with a κ- or a λ-light chain in HEK293 cells. Besides comparing the expression levels of the different mabs, we also evaluated whether the passage number of the cell line has an impact on product yield. In addition, the expressions of adalimumab, trastuzumab, an anti-CD38 and an anti-PD-L1-antibody were analyzed in HEK293 and CHO cells when both the κ- and λ-light chains are present. In summary, IgG outperformed IgM variants in expression efficacy, while light-chain selection had minimal impact on the overall expression levels. The yields of all mab variants were higher in fresh cells, despite cell cultures with a high cell passage number having higher cell densities and cell numbers at the time of harvest. The incorporation of a particular light chain occurred at similar rates in HEK293 and CHO cells. Full article

Background: Parkinson’s disease (PD) is a genetically complex neurodegenerative disorder. Up to 15% of cases are considered monogenic. However, research on monogenic PD has largely focused on populations of European ancestry, leaving gaps in our understanding of genetic variability in other populations. This study addresses this gap by analysing the allele frequencies of pathogenic and likely pathogenic variants in known monogenic PD genes across eight global populations, using data from the gnomAD database. Methods: We compiled a list of 27 genes associated with Mendelian PD from the Online Mendelian Inheritance in Man (OMIM) database, and identified pathogenic and likely pathogenic variants using ClinVar. We then performed pairwise comparisons of allele frequencies across populations included in the gnomAD database. Variants with significant frequency differences were further assessed using in silico pathogenicity predictions. Results: We identified 81 variants across 17 genes with statistically significant allele frequency differences between at least two populations. Variants in GBA1 were the most prevalent among monogenic PD-related genes, followed by PLA2G6, ATP13A2, VPS13C, and PRKN. GBA1 exhibited the greatest variability in allele frequencies, particularly the NM_000157.4:c.1226A>G (p.Asn409Ser) variant. Additionally, we observed significant population-specific differences in PD-related variants, such as the NM_032409.3:c.1040T>C (p.Leu347Pro) variant in PINK1, which was most prevalent in East Asian populations. Conclusions: Our findings reveal substantial population-specific differences in the allele frequencies of pathogenic variants linked to monogenic PD, emphasising the need for broader genetic studies beyond European populations. These insights have important implications for PD research, genetic screening, and understanding the pathogenesis of PD in diverse populations. Full article

Breast cancer is the most common and deadly female-specific malignancy in the world. Four immunohistochemical subtypes are distinguished: luminal A, luminal B, HER2-positive, and triple-negative. In turn, the HER2-positive subtype presents two variants depending on the status of the hormone receptors. The variant that expresses them can benefit from both anti-HER2 and anti-hormonal therapy. Today, MCTP finds application in maintenance therapy after standard of care and in advanced breast cancer when the patient’s clinical condition is already seriously compromised by metastatic disease; in this context, it is used as a first-line treatment, in pre-treated subjects, or as a rescue treatment. Here, the use of adjuvant oral MCTP after surgery at an early stage in HER-2 and hormone-positive local breast cancer is proposed, where effective treatment options are available, such as anti-HER2 therapy (e.g., trastuzumab, pertuzumab), anti-hormonal therapy (e.g., tamoxifen, letrozole), radiotherapy, and, in case of strong PD-1 positivity, immunotherapy. Full article

Upfront Next-Generation Sequencing (NGS) is increasingly recommended in advanced NSCLC to guide targeted therapy. This prospective single-center study in Romania evaluated routine, upfront NGS in advanced NSCLC at baseline (tissue and/or liquid) and progression (liquid). Baseline FoundationOne NGS (tissue/liquid) was performed in 119 consecutive stage IV NSCLC patients, along with PD-L1 immunohistochemistry (IHC, SP263). Liquid biopsy was repeated at progression. Turnaround time (TAT), the prevalence of actionable targets, and clinical utility were assessed. Patients were predominantly male (68.1%) with a median age of 62 years (range 30–86). Most had ECOG PS 0–1 (79%) and non-squamous histology (67.2%). Never-smokers accounted for 25.2%. The median TAT for the NGS results was 9 days (range 5–21). Overall, 671 genetic alterations were detected in 149 genes. The mean number of distinct mutations per patient dropped from 5.6 at baseline to 4.3 at progression. Tissue samples yielded more alterations (6 per patient) than baseline liquid biopsies (4.6). Squamous tumors had more alterations (7.1 vs. 4.8 in non-squamous), and the number of smokers exceeded that of never-smokers (6 vs. 4.5). TP53 was the most frequent (70.59%). Actionable variants were found in 74.8% of patients, though only 35.3% received personalized therapy, largely due to performance status deterioration, reimbursement, or trial availability barriers. Common targets in non-squamous tumors included EGFR (21%), KRAS G12C (11%), NF1 (11%), and ERBB2 (6%); in squamous tumors, common targets included NF1 (24%), PIK3CA (18%), and ERBB2 (8%). Among smokers, driver mutations were often NF1 (15%), PIK3CA (11%), KRAS G12C (9%), and ERBB2 (8%); never-smokers were dominated by EGFR (45%), NF1 (15%), and KRAS G12C (8%). TMB ≥ 10 mut/Mb was seen in 26.9%; no patients were MSI-H. PD-L1 TPS was <1% in 33% of patients, 1–49% in 20%, ≥50% in 18%, and unknown in 29%. Upfront NGS offers rapid, comprehensive genomic data, guiding tailored therapies and trials in advanced NSCLC. Liquid rebiopsy at progression further refines treatment decisions. Full article

Pennsylvania started newborn screening for Pompe disease (PD) in 2016. As a result, the prevalence of PD has increased with early detection, primarily of late-onset Pompe disease (LOPD). No clear guidelines exist regarding if and when to initiate enzyme replacement therapy (ERT) in patients identified through a newborn screen (NBS). To help define the natural history and indications for starting ERT, we present the long-term follow-up data of 45 patients identified through NBS from 2016 to 2021. These patients were evaluated at regular intervals through our multi-disciplinary clinic at the Children’s Hospital of Philadelphia (CHOP) with physical examinations, physical therapy evaluations, muscle biomarkers including creatine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and hexosaminidase 4 levels (Hex4), as well as cardiac evaluation at certain points in time. We found that newborn screening of acid alpha-glucosidase (GAA) enzyme detected primarily LOPD. One case of infantile-onset PD (IOPD) was detected. Muscle biomarkers in LOPD were elevated at birth and showed a general downward trend over time. NBS GAA levels and initial CK levels helped to differentiate LOPD cases from unaffected infants (carriers, pseudodeficiency alleles), while Hex4 was not a meaningful discriminator. On repeat NBS, there was a significant difference between mean GAA levels for the unaffected vs. compound heterozygote groups and unaffected vs. homozygote groups for the common splice site pathogenic variant (c.-32-13T>G). Echocardiogram and electrocardiogram (EKG) are essentially normal at the first evaluation in LOPD. One LOPD patient was started on ERT at age 4.5 months. Continued data collection on these patients is critical for developing management guidelines, including timing of ERT and improved genotype–phenotype correlation. Full article

Periodontitis (PD), an oral inflammatory disease, is primarily caused by P. gingivalis. Peptidylarginine deiminase (PPAD) is considered an attractive virulence factor because, due to protein citrullination, it may have deleterious effects on host tissues. In this study, the ppad gene sequences from P. gingivalis were analyzed in the context of its impact on bacterial virulence and potential targets for PD therapy. Analyses of ppad sequences from 58 patients with various clinical stages of PD, 20 controls, and 60 sequences from public databases were conducted. Overall, 55 substitutions assigned as polymorphic variants (4), missense mutations (10), or synonymous variants (35) were identified in PD, and 22 synonymous variants were identified in controls. Among them, the G231N, E232T, N235D variant was found in ~25% of P. gingivalis strains from PD samples. It was located close to the catalytic triad and had two-fold higher activity in comparison with reference P. gingivalis, upregulated expression of key inflammatory mediators, and contributed to worsening periodontium conditions in advanced PD, suggesting their unambiguous impact on P. gingivalis virulence. Our results indicate the G231N, E232T, N235D variant of the ppad gene as a potential candidate, opening a path to searching for novel targets for supportive therapy of PD. Further validation of the identified mutations is needed in future studies. Full article

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency (HMGCS2D) is a rare metabolic disorder that impairs the body’s ability to produce ketone bodies and regulate energy metabolism. Diagnosing HMGCS2D is challenging because patients typically remain asymptomatic unless they experience fasting or illness. Due to the absence of reliable biochemical markers, genetic testing has become the definitive method for diagnosing HMGCS2D. This study included 19 patients from 14 unrelated families diagnosed with HMGCS2D in our department between October 2018 and October 2024. The clinical presentations, biochemical findings, molecular characteristics, and management strategies were systematically summarized and analyzed. Of the 19 cases studied, 16 were symptomatic, and 3 were asymptomatic. The onset of the first acute episode occurred between 10 days and 28 months of age. Triggers for the initial crisis in the symptomatic cases included poor feeding (93.8%), vomiting (56.3%), diarrhea (25.0%), and fever (18.8%). Clinical manifestations during the first episode were lethargy/coma (81.3%), rapid breathing (68.8%), hepatomegaly (56.3%), shock (37.5%), and seizures (18.8%). The biochemical abnormalities observed included elevated plasma transaminases (100%), metabolic acidosis (75%), hypoglycemia (56.3%), and elevated plasma ammonia levels (31.3%). Additionally, low free carnitine levels were found in seven cases, elevated C2 levels were found in one case, dicarboxylic aciduria was found in two cases, and ketonuria was found in two cases. Abnormal brain MRI findings were detected in three patients. Genetic analysis revealed seven HMGCS2 gene variants across the 19 cases. Notably, a novel variant, c.407A>T (p.D136V), was identified and has not been reported in any existing databases. Two common variants, c.559+1G>A and c.1090T>A (p.F364I), were present in 11 out of 19 cases (57.9%) and 10 out of 19 cases (55.5%), respectively. The implementation of a high glucose infusion and proactive management strategies—such as preventing prolonged fasting and providing enteral carbohydrate/glucose infusion during illness—effectively reduced the rate of acute relapses following accurate diagnosis. Currently, all 19 patients are alive, with ages ranging from 5 months to 14 years, and exhibit normal physical development. To the best of our knowledge, this study represents the first reported cases of HMGCS2D in Vietnamese patients. Our findings contribute to a broader understanding of the clinical phenotype and expand the known spectrum of HMGCS2 gene variants, enhancing current knowledge of this rare metabolic disorder. Full article

Preimplantation genetic testing (PGT) is practiced worldwide, allowing the prevention of the transmission and expression of various genetic conditions. Socio-ethical considerations of justified applications for PGT are part of an ongoing debate. Pathogenic variants in the glucocerebrosidase (GBA1) gene, causing Gaucher disease (GD), have emerged as a risk factor for Parkinson’s disease (PD) in both patients and carriers. Genotype–phenotype correlations exist between different GBA1 pathogenic variants and the risk to develop PD: mild pathogenic variants increase the risk of developing PD by ~3-fold, while severe pathogenic variants increase this risk by ~15-fold, occurring at a younger age. A woman with GD, a compound heterozygote of N370S (now commonly described as c.1226A>G (N409S)—mild pathogenic variant) and 84insG (severe pathogenic variant), had PGT consulting before planned in vitro-fertilization. Her mother, an 84insG carrier, had early-onset PD. GBA1 sequencing of her spouse was negative. We discussed the selection for N370S carrier embryos to reduce PD risk. This case report demonstrates the expansion of PGT for late-onset conditions. These novel indications will increase the number of subjects who would be candidates for PGT. The medical and bioethical considerations of these cases should be acknowledged by the professional community and discussed with couples during genetic counseling. Full article

Ticks are important vectors of pathogenic bacteria that cause diseases in both humans and animals. Analysis of tick microbiota via massive sequencing allows rapid and comprehensive identification of almost all bacteria inhabiting ticks. This has improved the detection of emerging pathogens and has helped define their relationship with public health. In Mexico, the brown dog tick (Rhipicephalus sanguineus sensu lato) is a public health problem, especially in northeast Durango. In the present study, the bacterial microbiota of this tick was determined using third-generation massive sequencing (PacBio, V1–V9 region of the 16S rRNA gene); bacteria with pathogenic potential that are transmitted by salivation and those that can be transmitted by accidental regurgitation of the parasite were also identified. In 2024, 60 dogs were searched for unfed ticks; then, 15 groups of female ticks and 15 groups of male ticks were formed, with each group consisting of 30 individuals. DNA was extracted from each tick pool, and the complete 16S rRNA gene was amplified (PacBio). Bioinformatics analysis was performed in QIIME2 (Quantitative Insights into Microbial Ecology) to obtain amplicon sequence variants (ASVs). Alpha and beta diversity metrics, as well as statistical analyses, were performed to test for differences between the microbiota of females and males. The bacterial taxa were classified into 21 phyla, 24 classes, 81 orders, 137 families, 339 genera, and 565 species. The male microbiota presented a significantly greater number of ASVs and a greater phylogenetic diversity index (FaithPD). Additionally, the unweighted UniFrac metric was significantly different between the sexes. The endosymbiont Coxiella mudrowiae was significantly more abundant in females, and Ehrlichia canis was more abundant in males. The pathogens E. canis and Anaplasma platys (transmitted by salivation) were detected, as well as 75 species of potential pathogens recorded in this tick that could enter the host in case of accidental regurgitation of the parasite (e.g., Staphylococcus, Streptococcus, Acinetobacter, Corynebacterium, Proteus, Clostridum, Klebsiella, Bacillus, Escherichia, Fusobacterium, and Pasteurella). It is necessary to continue analyzing the microbiota of ticks through massive sequencing for the benefit of public health and to establish new alternatives for controlling these parasites. Full article

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an enzymopathy that affects approximately 500 million people worldwide. A great number of mutations in the G6PD gene have been described. However, three class A G6PD variants known as G6PD Tomah (C385R), G6PD Kangnam (C385G), and G6PD Madrid (C385W) have been reported to be clinically important due to their associations with severe clinical manifestations such as hemolytic anemia. Therefore, this work aimed to perform, for the first time, biochemical and functional characterizations of these variants. The G6PD variants were cloned and purified for this purpose, followed by analyses of their kinetic parameters and thermal stability, as well as in silico studies. The results showed that the mutations induced changes in the proteins. Regarding the kinetic parameters, it was observed that the three variants showed lower affinities for G6P and NADP⁺, as well as lower thermal stability compared to WT-G6PD. Molecular dynamics simulations showed that C385 mutations induced changes around neighboring amino acids. Metadynamics simulations showed that most remarkable changes account for the binding pocket volumes, particularly in the structural NADP⁺ binding site, with a concomitant loss of affinity for catalytic processes. Full article

Background: Glucose 6 phosphate dehydrogenase (G6PD) is a rate-limiting enzyme of the pentose phosphate pathway. The loss of G6PD activity in red blood cells increases the risk of acute haemolytic anaemia under oxidative stress induced by infections, some medications, or fava beans. More than 200 single missense mutations are known in the G6PD gene. A 41-year-old woman with a family history of favism coming from the Basilicata region (Italy) was evaluated at our hospital for G6PD abnormalities. Methods: DNA was extracted from a peripheral blood sample and genotyped for the most common G6PD pathogenic variants (PVs). Positive results obtained by Restriction Fragment Length Polymorphism (RFLP), as per practice in our laboratory, were then reconfirmed in Sanger sequencing. Results: RFLP analysis highlighted a variant compatible with the G6PD Cassano variant. Confirmatory testing by Sanger unexpectedly identified a novel variant: c.1357G>A, p.(Val453Met) (NM_001360016.2); the same variant was found in the patient’s mother. In silico models predicted a deleterious effect of this variant at the protein level. The novel G6PD variant was named “G6PD Potenza” on the basis of the patient’s regional origin. Conclusions: This case describes a novel G6PD variant. It also highlights how the Sanger sequencing technique still represents an indispensable confirmatory standard method for variants that could be misinterpreted by only using a “first-level” approach, such as the RFLP. We stress that the evaluation of clinical manifestations in G6PD-deficient patients is of primary importance for the classification of each new G6PD mutation, in agreement with the new WHO guidelines. Full article