Molecular Causes Research Articles

Targeting lipid peroxidation-associated ferroptosis suppresses lung carcinoma progression by regulating cell cycle arrest

Lung carcinoma is a frequently encountered cancerous growth that affects the respiratory tract and has a high occurrence rate globally. In light of the ongoing worldwide health emergency, the significance of efficient therapeutic agents and strategies is of utmost importance. A meticulous control of the cell cycle is crucial for comprehending the pathophysiology and molecular causes of lung cancer, as well as for the formulation of efficacious therapeutic medicines. The mechanism by which cells synchronize cell cycle with cell survival and death is still not fully understood. In this study, we demonstrate that the halting of the cell cycle has a strong inhibitory impact on ferroptosis, a specific type of controlled cell death triggered by excessive lipid peroxidation at the membranes of cells. Ferroptosis is halted through the mechanism of cell cycle arrest, which involves the deposition of intracellular lipids mediated by diacylglycerol acyltransferase (DGAT). Excessive amounts of polyunsaturated fatty acids (PUFAs) are stored as triacylglycerols (TAGs) within inactive cells. As a result, inhibiting DGAT causes a rearrangement of PUFAs from TAGs to phospholipids and makes arrested cells more susceptible to ferroptosis. We demonstrate that certain lung cancer cells that are resistant to antimitotic drugs and have a slow-cycling behavior exhibit an increase in lipid droplets. Furthermore, we find that the growth of tumors resistant to 5-fluorouracil, lorlatinib, and docetaxel can be effectively suppressed by a combination treatment involving the use of ferroptosis inducers and DGAT inhibitors, which induces ferroptosis. Collectively, these findings demonstrate the involvement of cell cycle arrest in conferring resistance to ferroptosis and propose a potential therapeutic approach for addressing the challenge of slow-cycling malignancies that exhibit resistance to ferroptosis.

Tubulin-binding region alters tau-lipid interactions and changes toxicity of tau fibrils formed in the presence of phosphatidylserine lipids.

Alzheimer's disease is the fastest-growing neurodegenerative disease that affects over six million Americans. The abnormal aggregation of amyloid β peptide and Tau protein is the expected molecular cause of the loss of neurons in brains of AD patients. A growing body of evidence indicates that lipids can alter the aggregation rate of amyloid β peptide and modify the toxicity of amyloid β aggregates. However, the role of lipids in Tau aggregation remains unclear. In this study, we utilized a set of biophysical methods to determine the extent to which phospatidylserine (PS) altered the aggregation properties of Tau isoforms with one (1N4R) and two (2N4R) N terminal inserts that enhance the binding of Tau to tubulin. We found that the length and saturation of fatty acids (FAs) in PS altered the aggregation rate of 2N4R isoform, while no changes in the aggregation rate of 1N4R were observed. These results indicate that N terminal inserts play an important role in protein-lipid interactions. We also found that PS could change the toxicity of 1N4R and 2N4R Tau fibrils, as well as alter molecular mechanisms by which these aggregates exert cytotoxicity to neurons. Finally, we found that although Tau fibrils formed in the presence and absence of PS endocytosed by cells, only fibril species that were formed in the presence of PS exert strong impairment of the cell mitochondria.

Approach to the Patient With Suspected Silver-Russell Syndrome.

Silver-Russell syndrome (SRS) is a clinical diagnosis requiring the fulfillment of ≥ 4/6 Netchine-Harbison Clinical Scoring System (NH-CSS) criteria. A score of ≥ 4/6 NH-CSS (or ≥ 3/6 with strong clinical suspicion) warrants (epi)genetic confirmation, identifiable in ∼60% patients. The approach to the investigation and diagnosis of SRS is detailed in the only international consensus guidance, published in 2016. In the intervening years, the clinical, biochemical, and (epi)genetic characteristics of SRS have rapidly expanded, largely attributable to advancing molecular genetic techniques and a greater awareness of related disorders. The most common etiologies of SRS remain loss of methylation of chromosome 11p15 (11p15LOM) and maternal uniparental disomy of chromosome 7 (upd(7)mat). Rarer causes of SRS include monogenic pathogenic variants in imprinted (CDKN1C and IGF2) and non-imprinted (PLAG1 and HMGA2) genes. Although the age-specific NH-CSS can identify more common molecular causes of SRS, its use in identifying monogenic causes is unclear. Preliminary data suggest that NH-CSS is poor at identifying many of these cases. Additionally, there has been increased recognition of conditions with phenotypes overlapping with SRS that may fulfill NH-CSS criteria but have distinct genetic etiologies and disease trajectories. This group of conditions is frequently overlooked and under-investigated, leading to no or delayed diagnosis. Like SRS, these conditions are multisystemic disorders requiring multidisciplinary care and tailored management strategies. Early identification is crucial to improve outcomes and reduce the major burden of the diagnostic odyssey for patients and families. This article aims to enable clinicians to identify key features of rarer causes of SRS and conditions with overlapping phenotypes, show a logical approach to the molecular investigation, and highlight the differences in clinical management strategies.

The N-glycosylation defect in Lec5 and Lec9 CHO cells is caused by absence of the DHRSX gene.

Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.

Enhancing probiotic impact: engineering Saccharomyces boulardii for optimal acetic acid production and gastric passage tolerance.

Saccharomyces boulardii has been a subject of growing interest due to its potential as a probiotic microorganism with applications in gastrointestinal health, but the molecular cause for its probiotic potency has remained elusive. The recent discovery that S. boulardii contains unique mutations causing high acetic acid accumulation and inhibition of bacterial growth provides a possible clue. The natural S. boulardii isolates Sb.P and Sb.A are homozygous for the recessive mutation whi2S270* and accumulate unusually high amounts of acetic acid, which strongly inhibit bacterial growth. However, the homozygous whi2S270* mutation also leads to acetic acid sensitivity and acid sensitivity in general. In the present study, we have constructed a new S. boulardii strain, derived from the widely therapeutically used CMCN I-745 strain (isolated from the pharmaceutical product Enterol), producing even higher levels of acetic acid while keeping the same tolerance toward low pH as the parent Enterol (ENT) strain. This newly engineered strain, named ENT3, has a homozygous deletion of ACH1 and strong overexpression of ALD4. It is also able to accumulate much higher acetic acid concentrations when growing on low glucose levels, in contrast to the ENT wild-type and Sb.P strains. Moreover, we show the antimicrobial capacity of ENT3 against gut pathogens in vitro and observed that higher acetic acid production might correlate with better persistence in the gut in healthy mice. These findings underscore the possible role of the unique acetic acid production and its potential for improvement of the probiotic action of S. boulardii.IMPORTANCESuperior variants of the probiotic yeast Saccharomyces boulardii produce high levels of acetic acid, which inhibit the growth of bacterial pathogens. However, these strains also show increased acid sensitivity, which can compromise the viability of the cells during their passage through the stomach. In this work, we have developed by genetic engineering a variant of Saccharomyces boulardii that produces even higher levels of acetic acid and does not show enhanced acid sensitivity. We also show that the S. boulardii yeasts with higher acetic acid production persist longer in the gut, in agreement with a previous work indicating competition between probiotic yeast and bacteria for residence in the gut.

Abstract A013: NF1 loss is syntetic lethal with Trastuzumab emtansine

Abstract There is great interest in the identification of biomarkers to guide development of antibody-drug conjugates (ADC). Most research has focused on target expression, but key predictors of payload efficacy have not been indeitifed. NF1 is a tumor suppressor classically considered as an inhibitor of RAS signaling, and often mutated in metastatic HER2+ breast cancer (BC). We screened multiple approved drugs for differential sensitivityin CRISPR-engineeredf NF1 KO cells. HER2-targeted agents (small molecules or antibodies) were found to be less effective upon NF1 loss; surprisingly, we identified increased sensitivity to the approved ADC T-DM1, but not to Trastuzumab Deruxtecan (T-Dxd). We then elucidated the underlying molecular cause employing in vivo, in vitro and in vitro reductionist systems. To measure in vivo MT dynamics, we transiently transfected the MT end-binding protein EB3- GFP and reconstructed MT trajectories by live-cell imaging. Upon DM1 treatment, KO cells showed a highly significant reduction in MT speed, demonstrating a direct role for NF1 on MT dynamics in cells. In tubulin polymerization assays, recombinant NF1 greatly accelerated polymerization and completely rescued DM1-induced inhibition. NF1 induced significant MT bundling, a defining feature of many MT-associated proteins, which generates signal indistinguishable from true MT polymerization in turbidity assays. To follow the dynamics of individual microtubules, we applied Total Internal Reflection (TIRF) microscopy on glass-immobilized MTs. As expected, polymerization in the presence of NF1 led to a dose-dependent significant increase in MT dynamics (fraction of elongating MTs, elongation speed, catastrophe rate). Expectedly, DM1 led to significant reduction in the fraction of elongating MTs and speed, but these defects were completely or partially rescued by NF1. Importantly, DM1 did not only lead to MT shortening (as proposed by the current model), but also to clear and frequent MT fracturing, indicating that the drug is not only engaging MT ends but also intra-tubular binding sites. This is consistent with recent models of MT formation which incorporate the frequent presence of areas of discontinuity or damage induced by mechanical stress, exposing intratubular DM1 binding sites. Interestingly, adding NF1 to DM1-treated MTs generated areas of de novo intra-tubular tubulin insertion, coincident with damaged sites, suggesting an entirely novel role for NF1 in MT repair. In conclusion, we provide evidence for a model in which maytansinoids bind not only to soluble tubulin dimers and MT ends, but also to intratubular damaged sites. Thus, the number of binding sites in cells would be proportional to MT damage, suggesting a mechanism for differential efficacy across tumor types and a potential avenue for combinatorial drug development. These results prompt the use of NF1 as a biomarker to select patients for ADC treatment. Funding: FIEO fellowship 2023, AIRC (n25791), Italian MoH-Ricerca Corrente di Rete (ACCORD) 2022, Next Generation EU – PNRR M6C2 – PNRR-MAD-2022-12376934 Citation Format: Eleonora Messuti, Bruno Achutti Duso, Alessia Castiglioni, Giulia Tini, Emanuele Bonetti, Giuseppe Ciossani, Silvia Monzani, Daria Khuntsariya, Marcus Braun, Zdenek Lansky, Luigi Scietti, Luca Mazzarella. NF1 loss is syntetic lethal with Trastuzumab emtansine [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A013.

Molecular mechanisms of human overgrowth and use of omics in its diagnostics: chances and challenges.

Overgrowth disorders comprise a group of entities with a variable phenotypic spectrum ranging from tall stature to isolated or lateralized overgrowth of body parts and or organs. Depending on the underlying physiological pathway affected by pathogenic genetic alterations, overgrowth syndromes are associated with a broad spectrum of neoplasia predisposition, (cardio) vascular and neurodevelopmental anomalies, and dysmorphisms. Pathologic overgrowth may be of prenatal or postnatal onset. It either results from an increased number of cells (intrinsic cellular hyperplasia), hypertrophy of the normal number of cells, an increase in interstitial spaces, or from a combination of all of these. The underlying molecular causes comprise a growing number of genetic alterations affecting skeletal growth and Growth-relevant signaling cascades as major effectors, and they can affect the whole body or parts of it (mosaicism). Furthermore, epigenetic modifications play a critical role in the manifestation of some overgrowth diseases. The diagnosis of overgrowth syndromes as the prerequisite of a personalized clinical management can be challenging, due to their clinical and molecular heterogeneity. Physicians should consider molecular genetic testing as a first diagnostic step in overgrowth syndromes. In particular, the urgent need for a precise diagnosis in tumor predisposition syndromes has to be taken into account as the basis for an early monitoring and therapy. With the (future) implementation of next-generation sequencing approaches and further omic technologies, clinical diagnoses can not only be verified, but they also confirm the clinical and molecular spectrum of overgrowth disorders, including unexpected findings and identification of atypical cases. However, the limitations of the applied assays have to be considered, for each of the disorders of interest, the spectrum of possible types of genomic variants has to be considered as they might require different methodological strategies. Additionally, the integration of artificial intelligence (AI) in diagnostic workflows significantly contribute to the phenotype-driven selection and interpretation of molecular and physiological data.

Elucidating the mechanisms of α-Synuclein-lipid interactions using site-directed mutagenesis

α-Synuclein (α-syn) is a small protein that is involved in cell vesicle trafficking in neuronal synapses. A progressive aggregation of this protein is the expected molecular cause of Parkinson's disease, a disease that affects millions of people around the world. A growing body of evidence indicates that phospholipids can strongly accelerate α-syn aggregation and alter the toxicity of α-syn oligomers and fibrils formed in the presence of lipid vesicles. This effect is attributed to the presence of high copies of lysines in the N-terminus of the protein. In this study, we performed site-directed mutagenesis and replaced one out of two lysines at each of the five sites located in the α-syn N-terminus. Using several biophysical and cellular approaches, we investigated the extent to which six negatively charged fatty acids (FAs) could alter the aggregation properties of K10A, K23A, K32A, K43A, and K58A α-syn. We found that FAs uniquely modified the aggregation properties of K43A, K58A, and WT α-syn, as well as changed morphology of amyloid fibrils formed by these mutants. At the same time, FAs failed to cause substantial changes in the aggregation rates of K10A, K23A, and K32A α-syn, as well as alter the morphology and toxicity of the corresponding amyloid fibrils. Based on these results, we can conclude that K10, K23, and K32 amino acid residues play a critical role in protein-lipid interactions since their replacement on non-polar alanines strongly suppressed α-syn-lipid interactions.

Current and future therapeutic approaches of CFTR and airway dysbiosis in an era of personalized medicine

ABSTRACT Cystic fibrosis (CF) is a life-threatening genetic disorder caused by mutations in the CFTR gene. This leads to a defective protein that impairs chloride transport, resulting in thick mucus buildup and chronic inflammation in the airways. The review discusses current and future therapeutic approaches for CFTR dysfunction and airway dysbiosis in the era of personalized medicine. Personalized medicine has revolutionized CF treatment with the advent of CFTR modulator therapies that target specific genetic mutations. These therapies have significantly improved patient outcomes, slowing disease progression, and enhancing quality of life. It also highlights the growing recognition of the airway microbiome’s role in CF pathogenesis and discusses strategies to modulate the microbiome to further improve patient outcomes. This review discusses various therapeutic approaches for cystic fibrosis (CFTR) mutations, including adenovirus gene treatments, nonviral vectors, CRISPR/cas9 methods, RNA replacement, antisense-oligonucleotide-mediated DNA-based therapies, and cell-based therapies. It also introduces airway dysbiosis with CF and how microbes influence the lungs. The review highlights the importance of understanding the cellular and molecular causes of CF and the development of personalized medicine to improve quality of life and health outcomes.

Characterization of cellular heterogeneity in recurrent pediatric glioblastoma: Machine learning-enhanced single-cell RNA-Seq unveils regulatory signatures

The most common oncologic cause of mortality in children is pediatric glioblastoma, an extremely dangerous brain tumor. The tumor progress is almost inevitable and recurs after first-line standard care. Because surgical resection is often more effective when tumors are localized and smaller, early identification and action may be essential to assure favourable outcomes for the recurring disease. This study aims to employ single-cell RNA-Sequencing data (scRNA-Seq data) for clustering and explainable Artificial intelligence framework to find gene biomarkers and signature cell types for the diagnosis and prognosis of reoccurring pediatric glioblastoma. Distinct cell types and statistically significant DEGs were found using scRNA-Seq data retrieved from the Gene Expression Omnibus database. Random forest (RF) and extreme gradient boosting (XGBoost) machine learning (ML) classifiers were constructed to select genes significantly contributing to the disease using Shapley (SHAP) values, an explainable artificial intelligence (EAI) framework. Potential biomarkers were chosen based on the shared genes among statistically discovered DEGs and SHAP-based relevance. B cells, macrophages, CD8+ T cells, T cells, and NK cells were identified as distinct cell types, which played an essential role in disease recurrence. Also, five significant genes, namely HMGB2, H2AFZ, HIST1H4C, KIAA0101, and DUT, were screened and in silico validated through survival analysis and feature plot, hence, proposed as biomarkers for recurring pediatric glioblastoma. Utilising these five genes may improve disease prognosis and provide a crucial understanding of the molecular causes of recurrent pediatric glioblastoma.

Transcranial sonography of subcortical structures in tic/tourette disorder

Functional neuroimaging studies demonstrate disinhibition of the cortico-striatal-thalamo-cortical circuit. However, structural imaging studies revealed conflicting results, some suggesting smaller volumes of the caudate nucleus (CN) in children with Gilles de la Tourette syndrome (TS). Here we wanted to find out whether transcranial sonography (TCS) detects alterations of raphe nuclei, substantia nigra, lenticular nucleus (LN), or CN in children with Tic disorder or TS (TIC/TS).The study included 25 treatment-naive children (age: 12.2 ± 2.5 years) with a DSM-V based diagnosis of Tic disorder or TS (10 subjects), without other psychiatric or neurologic diagnosis, and 25 healthy controls (age: 12.17 ± 2.57 years), matched for age and sex. Parental rating of behavioral, emotional abnormalities, somatic complaints and social competencies of the participants were assessed using the Child Behavior Check List (CBCL/4-18R). TCS of deep brain structures was conducted through the preauricular acoustic bone windows using a 2.5-MHz phased-array ultrasound system.Fisher's exact test and Mann-Whitney-U test were used for comparisons between TIC/TS patients and healthy volunteers. The number of participants with hyperechogenic area of left CN in the TIC/TS sample was increased, compared to the healthy control group. TIC/TS patients with hyperechogenic CN showed an increased occurrence of thought- and obsessive-compulsive problems. This TCS study revealed pathologic structural changes in CN, its higher occurrence in TIC/TS compared to healthy controls and the relation to comorbidity of thought problems. Further research should focus on the molecular cause of these alterations, probably the disturbed iron metabolism.

Post-stroke cognitive impairment: exploring molecular mechanisms and omics biomarkers for early identification and intervention

Post-stroke cognitive impairment (PSCI) is a major stroke consequence that has a severe impact on patients’ quality of life and survival rate. For this reason, it is especially crucial to identify and intervene early in high-risk groups during the acute phase of stroke. Currently, there are no reliable and efficient techniques for the early diagnosis, appropriate evaluation, or prognostication of PSCI. Instead, plenty of biomarkers in stroke patients have progressively been linked to cognitive impairment in recent years. High-throughput omics techniques that generate large amounts of data and process it to a high quality have been used to screen and identify biomarkers of PSCI in order to investigate the molecular mechanisms of the disease. These techniques include metabolomics, which explores dynamic changes in the organism, gut microbiomics, which studies host–microbe interactions, genomics, which elucidates deeper disease mechanisms, transcriptomics and proteomics, which describe gene expression and regulation. We looked through electronic databases like PubMed, the Cochrane Library, Embase, Web of Science, and common databases for each omics to find biomarkers that might be connected to the pathophysiology of PSCI. As all, we found 34 studies: 14 in the field of metabolomics, 5 in the field of gut microbiomics, 5 in the field of genomics, 4 in the field of transcriptomics, and 7 in the field of proteomics. We discovered that neuroinflammation, oxidative stress, and atherosclerosis may be the primary causes of PSCI development, and that metabolomics may play a role in the molecular mechanisms of PSCI. In this study, we summarized the existing issues across omics technologies and discuss the latest discoveries of PSCI biomarkers in the context of omics, with the goal of investigating the molecular causes of post-stroke cognitive impairment. We also discuss the potential therapeutic utility of omics platforms for PSCI mechanisms, diagnosis, and intervention in order to promote the area’s advancement towards precision PSCI treatment.

Comprehensive Molecular Analysis of Disease-Related Genes as First-Tier Test for Early Diagnosis, Classification, and Management of Patients Affected by Nonsyndromic Ichthyosis.

Inherited ichthyoses are a group of clinically and genetically heterogeneous rare disorders of skin keratinization with overlapping phenotypes. The clinical picture and family history are crucial to formulating the diagnostic hypothesis, but only the identification of the genetic defect allows the correct classification. In the attempt to molecularly classify 17 unrelated Italian patients referred with congenital nonsyndromic ichthyosis, we performed massively parallel sequencing of over 50 ichthyosis-related genes. Genetic data of 300 Italian unaffected subjects were also analyzed to evaluate frequencies of putative disease-causing alleles in our population. For all patients, we identified the molecular cause of the disease. Eight patients were affected by autosomal recessive congenital ichthyosis associated with ALOX12B, NIPAL4, and TGM1 mutations. Three patients had biallelic loss-of-function variants in FLG, whereas 6/11 males were affected by X-linked ichthyosis. Among the 24 different disease-causing alleles we identified, 8 carried novel variants, including a synonymous TGM1 variant that resulted in a splicing defect. Moreover, we generated a priority list of the ichthyosis-related genes that showed a significant number of rare and novel variants in our population. In conclusion, our comprehensive molecular analysis resulted in an effective first-tier test for the early classification of ichthyosis patients. It also expands the genetic, mutational, and phenotypic spectra of inherited ichthyosis and provides new insight into the current understanding of etiologies and epidemiology of this group of rare disorders.

Diagnosis and management of non-CAH 46,XX disorders/differences in sex development.

Prenatal-onset androgen excess leads to abnormal sexual development in 46,XX individuals. This androgen excess can be caused endogenously by the adrenals or gonads or by exposure to exogenous androgens. The most common cause of 46,XX disorders/differences in sex development (DSD) is congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, comprising >90% of 46,XX DSD cases. Deficiencies of 11β-hydroxylase, 3β-hydroxysteroid dehydrogenase, and P450-oxidoreductase (POR) are rare types of CAH, resulting in 46,XX DSD. In all CAH forms, patients have normal ovarian development. The molecular genetic causes of 46,XX DSD, besides CAH, are uncommon. These etiologies include primary glucocorticoid resistance (PGCR) and aromatase deficiency with normal ovarian development. Additionally, 46,XX gonads can differentiate into testes, causing 46,XX testicular (T) DSD or a coexistence of ovarian and testicular tissue, defined as 46,XX ovotesticular (OT)-DSD. PGCR is caused by inactivating variants in NR3C1, resulting in glucocorticoid insensitivity and the signs of mineralocorticoid and androgen excess. Pathogenic variants in the CYP19A1 gene lead to aromatase deficiency, causing androgen excess. Many genes are involved in the mechanisms of gonadal development, and genes associated with 46,XX T/OT-DSD include translocations of the SRY; copy number variants in NR2F2, NR0B1, SOX3, SOX9, SOX10, and FGF9, and sequence variants in NR5A1, NR2F2, RSPO1, SOX9, WNT2B, WNT4, and WT1. Progress in cytogenetic and molecular genetic techniques has significantly improved our understanding of the etiology of non-CAH 46,XX DSD. Nonetheless, uncertainties about gonadal function and gender outcomes may make the management of these conditions challenging. This review explores the intricate landscape of diagnosing and managing these conditions, shedding light on the unique aspects that distinguish them from other types of DSD.

A new Finnish flavor of feline coat coloration, "salmiak," is associated with a 95-kb deletion downstream of the KIT gene.

Cats with a distinctive white hair pattern of unknown molecular cause have been discovered in the Finnish domestic cat population. Based on the unique appearance of these cats, we have named this phenotype salmiak ("salty licorice"). The use of a commercially available panel test to genotype four salmiak-colored cats revealed the absence of all known variants associated with white-haired phenotypic loci: full White (W), Spotting (Ws) and the Birman white Gloves associated (wg) allele of the KIT proto-oncogene (KIT) gene. Whole-genome sequencing on two salmiak-colored cats was conducted to search for candidate causal variants in the KIT gene. Despite a lack of coding variants, visual inspection of the short read alignments revealed a large ~95 kb deletion located ~65 kb downstream of the KIT gene in the salmiak cats. Additional PCR genotyping of 180 domestic cats and three salmiak-colored cats confirmed the homozygous derived variant genotype fully concordant with the salmiak phenotype. We suggest the newly identified variant be designated as wsal for "w salmiak".

Clinician-Driven Reanalysis of Exome Sequencing Data From Patients With Inherited Retinal Diseases

Despite advances in next-generation sequencing (NGS), a significant proportion of patients with inherited retinal disease (IRD) remain undiagnosed after initial genetic testing. Exome sequencing (ES) reanalysis in the clinical setting has been suggested as one method for improving diagnosis of IRD. To investigate the association of clinician-led reanalysis of ES data, which incorporates updated clinical information and comprehensive bioinformatic analysis, with the diagnostic yield in a cohort of patients with IRDs in Korea. This was a multicenter prospective cohort study involving 264 unrelated patients with IRDs, conducted in Korea between March 2018 and February 2020. Comprehensive ophthalmologic examinations and ES analyses were performed, and ES data were reanalyzed by an IRD specialist for single nucleotide variants, copy number variants, mobile element insertions, and mitochondrial variants. Data were analyzed from March to July 2023. Diagnostic rate of conventional bioinformatic analysis and clinician-driven ES reanalysis. A total of 264 participants (151 [57.2%] male; mean [SD] age at genetic testing, 33.6 [18.9] years) were enrolled, including 129 patients (48.9%) with retinitis pigmentosa and 26 patients (9.8%) with Stargardt disease or macular dystrophy. Initial bioinformatic analysis diagnosed 166 patients (62.9%). Clinician-driven reanalysis identified the molecular cause of diseases in an additional 22 patients, corresponding to an 8.3-percentage point increase in diagnostic rate. Key factors associated with new molecular diagnoses included clinical phenotype updates (4 patients) and detection of previously overlooked variation, such as structural variants (9 patients), mitochondrial variants (3 patients), filtered or not captured variants (4 patients), and noncanonical splicing variants (2 patients). Among the 22 patients, variants in 7 patients (31.8%) were observed in the initial analysis but not reported to patients, while those in the remaining 15 patients (68.2%) were newly detected by the ES reanalysis. In this cohort study, clinician-centered reanalysis of ES data was associated with improved molecular diagnostic yields in patients with IRD. This approach is important for uncovering missed genetic causes of retinal disease.

Biallelic Variants of MRPS36 Cause a New Form of Leigh Syndrome.

The MRPS36 gene encodes a recently identified component of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme of the Krebs cycle catalyzing the oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. Defective OGDHC activity causes a clinically variable metabolic disorder characterized by global developmental delay, severe neurological impairment, liver failure, and early-onset lactic acidosis. We investigated the molecular cause underlying Leigh syndrome with bilateral striatal necrosis in two siblings through exome sequencing. Functional studies included measurement of the OGDHC enzymatic activity and MRPS36 mRNA levels in fibroblasts, assessment of protein stability in transfected cells, and structural analysis. A literature review was performed to define the etiological and phenotypic spectrum of OGDHC deficiency. In the two affected brothers, exome sequencing identified a homozygous nonsense variant (c.283G>T, p.Glu95*) of MRPS36. The variant did not affect transcript processing and stability, nor protein levels, but resulted in a shorter protein lacking nine residues that contribute to the structural and functional organization of the OGDHC complex. OGDHC enzymatic activity was significantly reduced. The review of previously reported cases of OGDHC deficiency supports the association of this enzymatic defect with Leigh phenotypic spectrum and early-onset movement disorder. Slightly elevated plasma levels of glutamate and glutamine were observed in our and literature patients with OGDHC defect. Our findings point to MRPS36 as a new disease gene implicated in Leigh syndrome. The slight elevation of plasma levels of glutamate and glutamine observed in patients with OGDHC deficiency represents a candidate metabolic signature of this neurometabolic disorder. © 2024 International Parkinson and Movement Disorder Society.

α2δ1-mediated maladaptive sensory plasticity disrupts adipose tissue homeostasis following spinal cord injury

Spinal cord injury (SCI) increases the risk of cardiometabolic disorders, including hypertension, dyslipidemia, and insulin resistance. Not only does SCI lead to pathological expansion of adipose tissue, but it also leads to ectopic lipid accumulation in organs integral to glucose and insulin metabolism. The pathophysiological changes that underlie adipose tissue dysfunction after SCI are unknown. Here, we find that SCI exacerbates lipolysis in epididymal white adipose tissue (eWAT). Whereas expression of the α2δ1 subunit of voltage-gated calcium channels increases in calcitonin gene-related peptide-positive dorsal root ganglia neurons that project to eWAT, conditional deletion of the gene encoding α2δ1 in these neurons normalizes eWAT lipolysis after SCI. Furthermore, α2δ1 pharmacological blockade through systemic administration of gabapentin also normalizes eWAT lipolysis after SCI, preventing ectopic lipid accumulation in the liver. Thus, our study provides insight into molecular causes of maladaptive sensory processing in eWAT, facilitating the development of strategies to reduce metabolic and cardiovascular complications after SCI.

Redox Regulation of LAT Enhances T Cell-Mediated Inflammation.

The positional cloning of single nucleotide polymorphisms (SNPs) of the neutrophil cytosolic factor 1 (Ncf1) gene, advocating that a low oxidative burst drives autoimmune disease, demands an understanding of the underlying molecular causes. A cellular target could be T cells, which have been shown to be regulated by reactive oxygen species (ROS). However, the pathways by which ROS mediate T cell signaling remain unclear. The adaptor molecule linker for activation of T cells (LAT) is essential for coupling T cell receptor-mediated antigen recognition to downstream responses, and it contains several cysteine residues that have previously been suggested to be involved in redox regulation. To address the possibility that ROS regulate T cell-dependent inflammation through LAT, we established a mouse strain with cysteine-to-serine mutations at positions 120 and 172 (LATSS). We found that redox regulation of LAT through C120 and C172 mediate its localization and phosphorylation. LATSS mice had reduced numbers of double-positive thymocytes and naïve peripheral T cells. Importantly, redox insensitivity of LAT enhanced T cell-dependent autoimmune inflammation in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). This effect was reversed on an NCF1-mutated (NCF1m1j), ROS-deficient, background. Overall, our data show that LAT is redox-regulated, acts to repress T cell activation, and is targeted by ROS induced by NCF1 in antigen-presenting cells (APCs).

Optic Nerve Neuroprotection in Glaucoma: A Narrative Review.

In recent years, researchers have been interested in neuroprotective therapies as a cutting-edge therapeutic strategy to treat neurodegenerative disorders by shielding the brain system from harmful events. Millions of individuals worldwide suffer from glaucoma, an ocular neurodegenerative disease characterized by gradual excavation of the optic nerve head, retinal axonal damage, and consequent visual loss. The pathology's molecular cause is still mostly unknown, and the current treatments are not able to alter the disease's natural progression. Thus, the modern approach to treating glaucoma consists of prescribing medications with neuroprotective properties, in line with the treatment strategy suggested for other neurodegenerative diseases. For this reason, several naturally derived compounds, including nicotinamide and citicoline, have been studied throughout time to try to improve glaucoma management by exploiting their neuroprotective properties. The purpose of this review is to examine the naturally derived compounds that are currently utilized in clinical practice for neuroprotection in glaucomatous patients based on scientific data, emphasizing these compounds' pivotal mechanism of action as well as their proven therapeutic and neuroprotective benefits.