Human Mutations Research Articles

Novel homozygous SPAG17 variants cause human male infertility through multiple morphological abnormalities of spermatozoal flagella related to axonemal microtubule doublets.

Male infertility can result from impaired sperm motility caused by multiple morphological abnormalities of the flagella (MMAF). Distinct projections encircling the central microtubules of the spermatozoal axoneme play pivotal roles in flagellar bending and spermatozoal movement. Mammalian sperm-associated antigen 17 (SPAG17) encodes a conserved axonemal protein of cilia and flagella, forming part of the C1a projection of the central apparatus, with functions related to ciliary/flagellar motility, skeletal growth, and male fertility. This study investigated two novel homozygous SPAG17 mutations (M1: NM_206996.2, c.829+1G>T, p.Asp212_Glu276del; and M2: c.2120del, p.Leu707*) identified in four infertile patients from two consanguineous Pakistani families. These patients displayed the MMAF phenotype confirmed by Papanicolaou staining and scanning electron microscopy assays of spermatozoa. Quantitative real-time polymerase chain reaction (PCR) of patients' spermatozoa also revealed a significant decrease in SPAG17 mRNA expression, and immunofluorescence staining showed the absence of SPAG17 protein signals along the flagella. However, no apparent ciliary-related symptoms or skeletal malformations were observed in the chest X-rays of any of the patients. Transmission electron microscopy of axoneme cross-sections from the patients showed incomplete C1a projection and a higher frequency of missing microtubule doublets 1 and 9 compared with those from fertile controls. Immunofluorescence staining and Western blot analyses of spermatogenesis-associated protein 17 (SPATA17), a component of the C1a projection, and sperm-associated antigen 6 (SPAG6), a marker of the spring layer, revealed disrupted expression of both proteins in the patients' spermatozoa. Altogether, these findings demonstrated that SPAG17 maintains the integrity of spermatozoal flagellar axoneme, expanding the phenotypic spectrum of SPAG17 mutations in humans.

Insights into RNA-mediated pathology in new mouse models of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative polyglutamine (polyQ) disease resulting from the expansion of CAG repeats located in the ORF of the huntingtin gene (HTT). The extent to which mutant mRNA-driven disruptions contribute to HD pathogenesis, particularly in comparison to the dominant mechanisms related to the gain-of-function effects of the mutant polyQ protein, is still debatable. To evaluate this contribution invivo, we generated two mouse models through a knock-in strategy at the Rosa26 locus. These models expressed distinct variants of human mutant HTT cDNA fragment: a translated variant (HD/100Q model, serving as a reference) and a nontranslated variant (HD/100CAG model). The cohorts of animals were subjected to a broad spectrum of molecular, behavioral, and cognitive analysis for 21 months. Behavioral testing revealed alterations in both models, with the HD/100Q model exhibiting late disease phenotype. The rotarod, static rod, and open-field tests showed some motor deficits in HD/100CAG and HD/100Q model mice during the light phase, while ActiMot indicated hyperkinesis during the dark phase. Both models also exhibited certain gene deregulations in the striatum that are related to disrupted pathways and phenotype alterations observed in HD. In conclusion, we provide invivo evidence for a minor contributory role of mutant RNA in HD pathogenesis. The separated effects resulting from the presence of mutant RNA in the HD/100CAG model led to less severe but, to some extent, similar types of impairments as in the HD/100Q model. Increased anxiety was one of the most substantial effects caused by mutant HTT RNA.

Identification of novel variants of XPA and POLH/XPV genes in xeroderma pigmentosum patients in Vietnam.

Xeroderma pigmentosum (XP) disorder is recognized as a genetic condition inherited by autosomal recessive fashion. XP results from a defective DNA repair mechanism that significantly increases skin cancer risk. Fifteen Vietnamese patients were investigated with typical clinical manifestations of XP. Eight XP genes (XPA to XPG and POLH/XPV) were sequenced using peripheral blood samples. Overall, three novel variants on the XPA and XPV genes were detected in members of two families. One novel missense variant c.388A>G (p.R130G) of XPA was found in three patients with XP group A, two novel variants: c.680G>A (p.C227Y) and c.1652dupC (p.Gln553Profs*8) of XPV in one patient with XP group F/G. Our study contributes to the recognition of new mutations in XP patients which have not been reported in Human Gene Mutation Database (HGMD).

Single-Nucleus RNA Sequencing Reveals the Spatiotemporal Dynamics of Disease-Associated Microglia in Amyotrophic Lateral Sclerosis.

Disease-associated microglia (DAM) are observed in neurodegenerative diseases, demyelinating disorders, and aging. However, the spatiotemporal dynamics and evolutionary trajectory of DAM during the progression of amyotrophic lateral sclerosis (ALS) remain unclear. Using a mouse model of ALS that expresses a human SOD1 gene mutation, we found that the microglia subtype DAM begins to appear following motor neuron degeneration, primarily in the brain stem and spinal cord. Using reverse transcription quantitative polymerase chain reaction, RNAscope insitu hybridization, and flow cytometry, we found that DAM increased in number as the disease progressed, reaching their peak in the late disease stage. DAM responded to disease progression in both SOD1G93A mice and sporadic ALS and C9orf72-mutated patients. Motor neuron loss in SOD1G93A mice exhibited 2 accelerated phases: P90 to P110 (early stage) and P130 to P150 (late stage). Some markers were synchronized with the accelerated phase of motor neuron loss, suggesting that these proteins may be particularly responsive to disease progression. Through pseudotime trajectory analysis, we tracked the dynamic transition of homeostatic microglia into DAM and cluster 6 microglia. Interestingly, we used the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia in SOD1G93A mice and observed that DAM survival is independent of CSF1R. An invitro phagocytosis assay directly confirmed that DAM could phagocytose more beads than other microglia subtypes. These findings reveal that the induction of the DAM phenotype is a shared cross-species and cross-subtype characteristic in ALS. Inducing the DAM phenotype and enhancing its function during the early phase of disease progression, or the time window between P130 and P150 where motor neuron loss slows, could serve as a neuroprotective strategy for ALS.

Parallel HIV-1 fitness landscapes shape viral dynamics in humans and macaques that develop broadly neutralizing antibodies.

Human immunodeficiency virus (HIV)-1 exhibits remarkable genetic diversity. For this reason, an effective HIV-1 vaccine must elicit antibodies that can neutralize many variants of the virus. While broadly neutralizing antibodies (bnAbs) have been isolated from HIV-1 infected individuals, a general understanding of the virus-antibody coevolutionary processes that lead to their development remains incomplete. We performed a quantitative study of HIV-1 evolution in two individuals who developed bnAbs. We observed strong selection early in infection for mutations affecting HIV-1 envelope glycosylation and escape from autologous strain-specific antibodies, followed by weaker selection for bnAb resistance later in infection. To confirm our findings, we analyzed data from rhesus macaques infected with viruses derived from the same two individuals. We inferred remarkably similar fitness effects of HIV-1 mutations in humans and macaques. Moreover, we observed a striking pattern of rapid HIV-1 evolution, consistent in both humans and macaques, that precedes the development of bnAbs. Our work highlights strong parallels between infection in rhesus macaques and humans, and it reveals a quantitative evolutionary signature of bnAb development.

A fully human IgG1 antibody targeting connexin 32 extracellular domain blocks CMTX1 hemichannel dysfunction in an in vitro model

Connexins (Cxs) are fundamental in cell–cell communication, functioning as gap junction channels (GJCs) that facilitate solute exchange between adjacent cells and as hemichannels (HCs) that mediate solute exchange between the cytoplasm and the extracellular environment. Mutations in the GJB1 gene, which encodes Cx32, lead to X-linked Charcot-Marie-Tooth type 1 (CMTX1), a rare hereditary demyelinating disorder of the peripheral nervous system (PNS) without an effective cure or treatment. In Schwann cells, Cx32 HCs are thought to play a role in myelination by enhancing intracellular and intercellular Ca2+ signaling, which is crucial for proper PNS myelination. Single-point mutations (p.S85C, p.D178Y, p.F235C) generate pathological Cx32 HCs characterized by increased permeability (“leaky”) or excessive activity (“hyperactive”).We investigated the effects of abEC1.1-hIgG1, a fully human immunoglobulin G1 (hIgG1) monoclonal antibody, on wild-type (WT) and mutant Cx32D178Y HCs. Using HeLa DH cells conditionally co-expressing Cx and a genetically encoded Ca2+ biosensor (GCaMP6s), we demonstrated that mutant HCs facilitated 58% greater Ca2+ uptake in response to elevated extracellular Ca2+ concentrations ([Ca2+]ex) compared to WT HCs. abEC1.1-hIgG1 dose-dependently inhibited Ca2+ uptake, achieving a 50% inhibitory concentration (EC50) of ~ 10 nM for WT HCs and ~ 80 nM for mutant HCs. Additionally, the antibody suppressed DAPI uptake and ATP release. An atomistic computational model revealed that serine 56 (S56) of the antibody interacts with aspartate 178 (D178) of WT Cx32 HCs, contributing to binding affinity. Despite the p.D178Y mutation weakening this interaction, the antibody maintained binding to the mutant HC epitope at sub-micromolar concentrations.In conclusion, our study shows that abEC1.1-hIgG1 effectively inhibits both WT and mutant Cx32 HCs, highlighting its potential as a therapeutic approach for CMTX1. These findings expand the antibody’s applicability for treating diseases associated with Cx HCs and inform the rational design of next-generation antibodies with enhanced affinity and efficacy against mutant HCs.

Lithium normalizes ASD-related neuronal, synaptic, and behavioral phenotypes in DYRK1A-knockin mice.

Dyrk1A deficiency is linked to various neurodevelopmental disorders, including developmental delays, intellectual disability (ID) and autism spectrum disorders (ASD). Haploinsufficiency of Dyrk1a in mice reportedly leads to ASD-related phenotypes. However, the key pathological mechanisms remain unclear and human DYRK1A mutations remain uncharacterized in mice. Here, we generated and studied Dyrk1a-knockin mice carrying a human ASD patient mutation (Ile48LysfsX2; Dyrk1a-I48K mice). These mice display severe microcephaly, social and cognitive deficits, dendritic shrinkage, excitatory synaptic deficits, and altered phospho-proteomic patterns enriched for multiple signaling pathways and synaptic proteins. Early chronic lithium treatment of newborn mutant mice rescues the brain volume, behavior, dendritic, synaptic, and signaling/synapse phospho-proteomic phenotypes at juvenile and adult stages. These results suggest that signaling/synaptic alterations contribute to the phenotypic alterations seen in Dyrk1a-I48K mice, and that early correction of these alterations by lithium treatment has long-lasting effects in preventing juvenile and adult-stage phenotypes.

The co-chaperone DNAJA2 buffers proteasomal degradation of cytosolic proteins with missense mutations

Mutations can disrupt a protein's native function by causing misfolding, which is generally handled by an intricate protein quality control network. To better understand the triaging mechanisms of misfolded cytosolic proteins, we screened a human mutation library to identify a panel of unstable mutations. The degradation of these mutated cytosolic proteins is largely dependent on the ubiquitin proteasome system. Using BioID proximity labelling, we found that the co-chaperones DNAJA1 and DNAJA2 are key interactors with one of the mutated proteins. Notably, the absence of DNAJA2 increases the turnover of the mutant but not the wild-type protein. Our work indicates that specific missense mutations in cytosolic proteins can promote enhanced interactions with molecular chaperones. Assessment of the broader panel of cytosolic mutant proteins shows that the co-chaperone DNAJA2 exhibits two distinct behaviours: acting to stabilize a wide array of cytosolic proteins including wild type variants, and specifically “buffer” some mutant proteins to reduce their turnover. Our work illustrates how distinct elements of the protein homeostasis network are utilized in the presence of a cytosolic misfolded protein.

Human DNA Mutations and their Impact on Genetic Disorders.

DNA is a remarkably precise medium for copying and storing biological information. It serves as a design for cellular machinery that permits cells, organs, and even whole organisms to work. The fidelity of DNA replication results from the action of hundreds of genes involved in proofreading and damage repair. All human cells can acquire genetic changes in their DNA all over life. Genetic mutations are changes to the DNA sequence that happen during cell division when the cells make copies of themselves. Mutations in the DNA can cause genetic illnesses such as cancer, or they could help humans better adapt to their environment over time. The endogenous reactive metabolites, therapeutic medicines, and an excess of environmental mutagens, such as UV rays all continuously damage DNA, compromising its integrity. One or more chromosomal alterations and point mutations at a single site (monogenic mutation) including deletions, duplications, and inversions illustrate such DNA mutations. Genetic conditions can occur when an altered gene is inherited from parents, which increases the risk of developing that particular condition, or some gene alterations can happen randomly. Moreover, symptoms of genetic conditions depend on which gene has a mutation. There are many different diseases and conditions caused by mutations. Some of the most common genetic conditions are Alzheimer's disease, some cancers, cystic fibrosis, Down syndrome, and sickle cell disease. Interestingly, scientists find that DNA mutations are more common than formerly thought. This review outlines the main DNA mutations that occur along the human genome and their influence on human health. The subject of patents pertaining to DNA mutations and genetic disorders has been brought up.

PD-1 antibody interactions with Fc gamma receptors enable PD-1 agonism to inhibit T cell activation – therapeutic implications for autoimmunity

PD-1 has emerged as a central inhibitory checkpoint receptor in maintaining immune homeostasis and as a target in cancer immunotherapies. However, targeting PD-1 for the treatment of autoimmune diseases has been more challenging. We recently showed in a phase 2a trial that PD-1 could be stimulated with the PD-1 agonist antibody peresolimab to treat rheumatoid arthritis. Here, we demonstrate that PD-1 antibodies can elicit agonism and inhibit T cell activation by co-localization of PD-1 with the T cell receptor via Fcγ receptor (FcγR) engagement. Three PD-1 agonist antibodies with different antigen binding domains, including the clinically validated PD-1 blocking antibody pembrolizumab, suppressed T cell activation to a similar degree; this finding suggests that a specific PD-1-binding epitope is not required for PD-1 agonism. We next explored whether antibody-mediated clustering was an important driver of inhibition of T cell activation; however, we found that a monovalent PD-1 antibody was not inferior to a conventional bivalent antibody in its ability to suppress T cell activation. Importantly, we found that affinity to PD-1 correlated positively with inhibition of T cell activation, with higher affinity antibodies exhibiting higher levels of inhibition. Using a series of human Fc mutants with altered affinities to various FcγRs, we dissected the contributions of FcγRs and found that multiple FcγRs rather than a single receptor contribute to agonist activity. Our work reveals an important role for FcγR binding in the activity of PD-1 antibodies, which has implications for optimizing both PD-1 agonist and antagonist antibodies.

Generation of Ext1 Gene-Edited Mice Model Via Dual sgRNAs/Cas9 System and Phenotypic Analyses.

Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disease. Genetic linkage analyses have identified that mutations in the exostosin glycosyltransferase (EXT)1 and EXT2 genes are linked to HME pathogenesis, with EXT1 mutation being the most frequent. The aim of this study was to generate a mice model with Ext1 gene editing to simulate human EXT1 mutation and investigate the genetic pathogenicity of Ext1 through phenotypic analyses. We designed a pair of dual sgRNAs targeting exon 1 of the mice Ext1 gene for precise deletion of a 46bp DNA fragment, resulting in frameshift mutation of the Ext1 gene. The designed dual sgRNAs and Cas9 proteins were injected into mice zygotes cytoplasm. A total of 14 mice were obtained via embryo transfer, among which two genotypic chimera mice had a deletion of the 46bp DNA fragment in exon 1 of the Ext1 gene. By hybridization and breeding, we successfully generated heterozygous mice with edited Ext1 gene (Ext+/-). Off-target effect analysis did not reveal off-target mutations in Ext+/- mice caused by the two sgRNAs used. Compared to wild-type mice, Ext+/- mice exhibited lower body weights. X-ray imaging showed hyperplastic bone near caudal vertebrae only in male Ext+/- mice, with computed tomography values approximately at 200 HU for hyperplastic bone between ribs and spine regions. Furthermore, immunohistochemical analysis revealed fewer articular chondrocytes expressing EXT1 in edited mice compared to wild-type ones. Pathological section analysis demonstrated no structural or morphological abnormalities in heart, liver, lung, or kidney tissues from Ext+/- mice. In conclusion, we successfully generated an accurate DNA deletion model for studying Ext1 using dual sgRNAs/Cas9 systems. In conclusion, we successfully generated precise DNA deletions in the Ext1 mice model using the dual sgRNAs/Cas9 system. In conclusion, we observed significant phenotypic changes in Ext+/- mice, particularly bone hyperplasia in male individuals; however, no exostosis was detected in the gene-edited mice. The introduction of a frameshift mutation into the Ext1 gene through CRISPR/Cas9 technology resulted in novel phenotypic alterations, highlighting the genetic pathogenicity of Ext1. Therefore, our Ext+/- mice serve as a valuable model for further biomedical investigations related to the Ext1 gene.

Distinguishing PEX gene variant severity for mild, severe, and atypical peroxisome biogenesis disorders in Drosophila.

Peroxisomal biogenesis disorders (PBD) are autosomal recessive disorders caused by loss-of-function mutations of one of the PEX genes responsible for peroxisomal formation. Impaired peroxisome assembly causes severe multisystemic failure with patient phenotypes ranging from epilepsy, liver disease, feeding issues, biochemical abnormalities, and neurodegeneration. Variants in the same PEX gene can produce wide differences in severity, ranging from individuals with death in the first year of life to adults with milder complications. To study this strong genotype-phenotype correlation, we selected specific human PEX gene mutations and utilized Drosophila as a model organism. We generated flies replacing the coding sequence of our Pex gene of interest with a KozakGAL4 (KZ) promoter trap sequence. These cassettes simultaneously knock-out of the Pex gene and knock-in a GAL4 driver, ideal for making "humanized" flies in which the human PEX gene can replace the fly loss. We assessed Pex2 KZ and Pex16 KZ lines in lifespan, bang sensitivity, and climbing assays and confirmed that these are strong loss-of-function alleles. In parallel, we generated human reference and variant UAS-cDNA lines of PEX2 and PEX16 variants in Drosophila . We observed nearly complete phenotypic rescue of Drosophila Pex2 and Pex16 loss when human PEX2 Ref or PEX16 Ref , respectively, were expressed. We also provide evidence for an allele severity spectrum in PEX2 and PEX16 in which some missense alleles, such as PEX2 C247R , are equally severe as early truncations, such as PEX2 R119* . We also observed that alleles associated with mild PBD, such as PEX2 E55K , show variability depending on the assay but do not fully rescue. Finally, alleles associated with atypical ataxia phenotypes, such as PEX16 F332Del , can perform as well as PEX16 Ref , depending on the assay. Altogether, these Drosophila lines effectively model the range of severity of peroxisomal biogenesis disorders.

Detailed phenotyping of Tbr1-2A-CreER knock-in mice demonstrates significant impacts on TBR1 protein levels and axon development.

Cre recombinase knock-in mouse lines have served as invaluable genetic tools for understanding key developmental processes altered in autism. However, insertion of exogenous DNA into the genome can have unintended effects on local gene regulation or protein function that must be carefully considered. Here, we analyze a recently generated Tbr1-2A-CreER knock-in mouse line, where a 2A-CreER cassette was inserted in-frame before the stop codon of the transcription factor gene Tbr1. Heterozygous TBR1 mutations in humans and mice are known to cause autism or autism-like behavioral phenotypes accompanied by structural brain malformations, most frequently a reduction of the anterior commissure (AC). Thus, it is critical for modified versions of Tbr1 to exhibit true wild-type-like activity. We evaluated the Tbr1-2A-CreER allele for its potential impact on Tbr1 function and complementation to Tbr1 loss-of-function alleles. In mice with one copy of the Tbr1-2A-CreER allele, we identified reduction of TBR1 protein in early postnatal cortex along with thinning of the AC, suggesting hypersensitivity of this structure to TBR1 dosage. Comparing Tbr1-2A-CreER and Tbr1-null mice to Tbr1-null complementation crosses showed reductions of TBR1 dosage ranging from 20% to 100%. Using six combinatorial genotypes, we found that moderate to severe TBR1 reductions (≥44%) were associated with cortical layer 5 expansion, while only the complete absence of TBR1 was associated with reeler-like "inverted" cortical layering. In total, these results strongly support the conclusion that Tbr1-2A-CreER is a hypomorphic allele. We advise caution when interpreting experiments using this allele, considering the sensitivity of various corticogenic processes to TBR1 dosage and the association of heterozygous TBR1 mutations with complex neurodevelopmental disorders.

Mechanism-based approach in designing patient-specific combination therapies for nonsense mutation diseases.

Premature termination codon (PTC) diseases, arising as a consequence of nonsense mutations in patient DNA, account for approximately 12% of all human disease mutations. Currently there are no FDA approved treatments for increasing PTC readthrough in nonsense mutation diseases, although one translational readthrough inducing drug, ataluren, has had conditional approval for treatment of Duchenne muscular dystrophy in Europe and elsewhere for 10 years. Ataluren displays consistent low toxicity in clinical trials for treatment of several different PTC diseases, but its therapeutic effects on such diseases are inconsistent. The identity of the stop codon and its sequence context are major determinants of PTC readthrough efficiency in both the absence and presence of nonsense suppressors. Previously we have shown that ataluren stimulates readthrough exclusively by competitively inhibiting release factor complex (RFC, eRF1.eRF3.GTP)-dependent catalysis of translation termination. Here, using an in vitro reconstituted system (PURE-LITE) and both ensemble and single molecule assays, we demonstrate that PTC identity and the immediately adjacent mRNA sequence contexts modulate the catalytic activity of RFC in terminating peptide elongation. Such modulation largely determines the effectiveness of ataluren in stimulating readthrough, whether added alone or in combination with either the aminoglycoside G418 or an anticodon edited aa-tRNA, each of which stimulate readthrough by mechanisms orthogonal to that of ataluren. Our results provide an attractive rationale for the variability of ataluren effectiveness in stimulating readthrough in clinical trials. Patients harboring a PTC mutation with a sequence context promoting strong interaction with RFC are predicted to be resistant to ataluren, whereas ataluren treatment should be more effective for patient sequences conferring weaker interaction with RFC.

A CADASIL NOTCH3 mutation leads to clonal hematopoiesis and expansion of Dnmt3a-R878H hematopoietic clones.

Clonal hematopoiesis (CH) is nearly universal in the elderly. The molecular and cellular mechanisms driving CH and the clinical consequences of carrying clonally derived mutant mature blood cells are poorly understood. We recently identified a C223Y mutation in the extracellular domain (ECD) of NOTCH3 as a putative CH driver in mice. Provocatively, germline NOTCH3 ECD mutations perturbing cysteine numbers cause Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), a type of vascular dementia, suggesting an unexpected link between CADASIL and CH. Here, we formally demonstrated that mouse hematopoietic stem and progenitor cells (HSPCs) expressing CADASIL-related NOTCH3C455R exhibit a proliferative advantage resulting in robust cellular expansion in vivo and in vitro. Co-expression of NOTCH3C455R and Dnmt3aR878H, homologous to a frequent human CH mutation, increased the fitness of NOTCH3C455R HSPCs, demonstrating their functional cooperation. Surprisingly, the presence of NOTCH3C455R hematopoietic cells supported the expansion of Dnmt3aR878H HSPCs in a non-cell autonomous fashion in vivo, strongly suggesting that CADASIL patients and asymptomatic carriers can be highly predisposed to DNMT3AR882H-driven CH. Considering that CADASIL-related NOTCH3 mutations are more frequent in the general population than anticipated (~1 carrier in 400 people), the effect of these NOTCH3 mutations on CH development should be considered.

BIOM-23. DEUTERIUM METABOLIC IMAGING OF RESPONSE TO PRECISION THERAPY IN BRAF-V600E MUTANT GLIOMAS

Abstract Oncogenic BRAF-V600E mutations are frequently observed in low- and high-grade gliomas in adults and children. BRAF transduces signals from receptor tyrosine kinases to downstream effectors such as MEK1/2 and ERK, which regulate both metabolism and proliferation. The combination of the BRAF-V600E inhibitor dabrafenib and the MEK1/2 inhibitor trametinib (Dab/Tra) improves patient survival. However, there is considerable variation in the durability of response, underscoring the need for early biomarkers of treatment response. Magnetic resonance imaging (MRI), which is the gold standard for patient management, does not reliably assess response to therapy. Since the BRAF/MEK pathway regulates metabolism, the goal of this study was to determine whether Dab/Tra induces alterations in glucose metabolism that can be leveraged for non-invasive imaging. Using gene expression profiling, in vivo metabolomics, and stable isotope tracing, we show that Dab/Tra downregulates glucose metabolism to lactate in human and murine BRAF-V600E mutant models (AM38, 2341). In vivo infusion with [U-13C]-glucose in mice bearing intracranial AM38 tumors confirmed that Dab/Tra downregulates glucose metabolism to lactate in the tumor in vivo. Mechanistically, Dab/Tra destabilizes hypoxia inducible factor-1a and downregulates glycolytic genes, including SLC2A1, HK2, PFKFB3, and LDHA. Deuterium metabolic imaging (DMI) is a novel, clinical stage method of tracing glucose metabolism in vivo. Our studies show that lactate production from [6,6-2H]-glucose is reduced in AM38 and 2341 cells treated with Dab/Tra. Importantly, lactate production is reduced within 48 h of treatment with Dab/Tra in mice bearing intracranial AM38 tumors at the clinical magnetic field strength of 3T. Furthermore, this drop in 2H-lactate production is observed in the absence of MRI-detectable volumetric alterations and is predictive of extended survival. Collectively, our studies mechanistically link BRAF/MEK inhibition with downregulation of glycolysis and identify [6,6’-2H]-glucose as a novel contrast agent for imaging early response to therapy. Clinical translation of our studies will enable precision imaging of response to therapy for patients with BRAF-mutant gliomas.

Hyperphosphorylated tau targeting human serum albumin Fusion protein as therapeutics for Alzheimer’s diseases

IntroductionNeurofibrillary tangles (NFTs) are composed of hyperphosphorylated forms of microtubule-associated protein tau (Tau), which is responsible for neurodegeneration in Alzheimer’s disease (AD). The hippocampal region has been a major focus of AD research because the deposits of phosphorylated tau protein in these regions are correlated with early memory deficits. Despite extensive studies, therapeutic strategies to reduce tau hyperphosphorylation and NFTs deposition remain unclear. AL04, a recently developed recombinant fusion protein comprising Cystatin C, human serum albumin, and a novel blood brain barrier (BBB) penetrating peptide, is currently under investigation. Previous studies have demonstrated its effectiveness in reducing amyloid beta plaques in AD mouse model. MethodsIn this study, we investigated the effects of AL04 on lowering hyperphosphorylated tau and NFTs in JNPL3 mouse model harboring human tau-P301L mutation. 3-month-old female mice intraperitoneally received AL04 (5 mg/kg) or PBS treatment every other week for 24 weeks. We used confocal microscopy and western blot to visualize and analyze changes in hyperphosphorylated tau Ser202/Thr205 labeled with AT8 antibody in the brain. ResultsWe found that the AL04 treatment decreases hyperphosphorylated tau at PP2A-sensitive epitope Ser202/Thr205 in the hippocampus of the brain. In the brain lysates of AL04 treated mice, we observed the reduction of I2PP2A, inhibitor of PP2A, and the induction of autophagy receptor proteins such as SQSTM-1/p62 and OPTN. ConclusionOur data suggests that AL04 can be used as an AD prophylactic/therapeutic agent as it lowers the hyperphosphorylated tau by downregulating I2PP2A. We also propose that AL04 can induce the degradation of hyperphosphorylated tau aggregates through the upregulation of the autophagy pathway.

R391 human dominant mutation does not affect TubB4b localization and sensory hair cells structure in zebrafish inner ear and lateral line

Heterozygous R391 TUBB4B pathogenic variations are responsible for an association of hearing loss and retinal dystrophy in human. With the goal of understanding the functions of TuBB4b and the pathogenic role of R391 variations, we characterized tubB4B in zebrafish and identified the gene regulatory elements necessary and sufficient for expression of TubB4b as in endogenous tissues. Using knock-out and transgenic approaches, we determined that R391 mutations impair neither localization of TubB4B within sensory hair cells (SHC) nor their structure, but induced to a small decrease in SHC number from anterior crista. Expression of R391 mutations in sensory hair cells has no effect on zebrafish audition, suggesting a different equilibrium between various tubulin isotypes in zebrafish possibly due to compensatory mechanisms. The careful expression analysis and transgenic tools generated in this study could help understand how recently described pathogenic variants lead to more severe clinical forms of TUBB4B-related diseases.

Mutational signature analyses in multi-child families reveal sources of age-related increases in human germline mutations.

Whole-genome sequencing studies of parent-offspring trios have provided valuable insights into the potential impact of de novo mutations (DNMs) on human health and disease. However, the molecular mechanisms that drive DNMs are unclear. Studies with multi-child families can provide important insight into the causes of inter-family variability in DNM rates but they are highly limited. We characterized 2479 de novo single nucleotide variants (SNVs) in 13 multi-child families of Mexican-American ethnicity. We observed a strong paternal age effect on validated de novo SNVs with extensive inter-family variability in the yearly rate of increase. Children of older fathers showed more C > T transitions at CpG sites than children from younger fathers. Validated SNVs were examined against one cancer (COSMIC) and two non-cancer (human germline and CRISPR-Cas 9 knockout of human DNA repair genes) mutational signature databases. These analyses suggest that inaccurate DNA mismatch repair during repair initiation and excision processes, along with DNA damage and replication errors, are major sources of human germline de novo SNVs. Our findings provide important information for understanding the potential sources of human germline de novo SNVs and the critical role of DNA mismatch repair in their genesis.

Cytosine base editors with increased PAM and deaminase motif flexibility for gene editing in zebrafish

Cytosine base editing is a powerful tool for making precise single nucleotide changes in cells and model organisms like zebrafish, which are valuable for studying human diseases. However, current base editors struggle to edit cytosines in certain DNA contexts, particularly those with GC and CC pairs, limiting their use in modelling disease-related mutations. Here we show the development of zevoCDA1, an optimized cytosine base editor for zebrafish that improves editing efficiency across various DNA contexts and reduces restrictions imposed by the protospacer adjacent motif. We also create zevoCDA1-198, a more precise editor with a narrower editing window of five nucleotides, minimizing off-target effects. Using these advanced tools, we successfully generate zebrafish models of diseases that were previously challenging to create due to sequence limitations. This work enhances the ability to introduce human pathogenic mutations in zebrafish, broadening the scope for genomic research with improved precision and efficiency.