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PubMed2024

Development of SacB-based counterselection for efficient allelic exchange in Fusobacterium nucleatum.

Fusobacterium nucleatum, prevalent in the oral cavity, is significantly linked to overall human health. Our molecular comprehension of its role in oral biofilm formation and its interactions with the host under various pathological circumstances has seen considerable advancements in recent years, primarily due to the development of various genetic tools for DNA manipulation in this bacterium. Of these, counterselection-based unmarked in-frame mutation methods have proved notably effective. Under suitable growth conditions, cells carrying a counterselectable gene die, enabling efficient selection of rare, defined allelic exchange mutants. The sacB gene from Bacillus subtilis, encoding levansucrase, is a widely used counterselective marker partly due to the easy availability of sucrose. Yet, its potential application in F. nucleatum genetic study remains untested. We demonstrated that F. nucleatum cells expressing sacB in either a shuttle or suicide plasmid exhibit a lethal sensitivity to supplemental sucrose. Utilizing sucrose counterselection, we created an in-frame deletion of the F. nucleatum tonB gene, a critical gene for energy-dependent transport processes in Gram-negative bacteria, and a precise knock-in of the luciferase gene immediately following the stop codon of the hslO gene, the last gene of a five-gene operon possibly related to the natural competence of F. nucleatum. Post-counterselection with 5% sucrose, chromosomal plasmid loss occurred in all colonies, leading to gene alternations in half of the screened isolates. This sacB-based counterselection technique provides a reliable method for isolating unmarked gene mutations in wild-type F. nucleatum, enriching the toolkit for fusobacterial research.IMPORTANCEInvestigations into Fusobacterium nucleatum's role in related diseases significantly benefit from the strategies of creating unmarked gene mutations, which hinge on using a counterselective marker. Previously, the galk-based allelic exchange method, although effective, faced an inherent limitation-the need for a modified host. This study aims to surmount this limitation by substituting galK with sacB for gene modification in F. nucleatum. Our application of the sacB-based methodology successfully yielded a tonB in-frame deletion mutant and a luciferase gene knock-in at the precise chromosomal location in the wild-type background. The new method augments the existing toolkit for F. nucleatum research and has far-reaching implications due to the easy accessibility to the counterselection compound sucrose. We anticipate its broader adoption in further exploration, thereby reinforcing its critical role in propelling our understanding of F. nucleatum.

Fusobacterium nucleatum↗
PubMed1992

Cystic fibrosis patients with mutation 1949del84 in exon 13 of the CFTR gene have a similar clinical severity as delta F508 homozygotes.

The majority of the identified cystic fibrosis (CF) mutations are very uncommon in the total patient population, making the correlation between the clinical presentation and the molecular alterations difficult. The largest deletion that has been described so far in CF is of 84 bp in exon 13, which corresponds to the regulatory (R) domain of the CF transmembrane conductance regulator (CFTR) protein. We have analysed 340 Spanish CF patients for this deletion, named 1949del84, and found three further compound heterozygous patients for mutations 1949del84 and delta F508, and one for 1949del84 and an unknown mutation. Evaluation of the clinical data in these patients suggests that this in-frame deletion, when associated with delta F508, has a similar disease severity to that of delta F508 homozygous patients.

Adolescent↗
PubMed2025

Pathogenic XPO1 variants cause a dominant neurodevelopmental disorder.

PURPOSE: XPO1 functions in key cellular processes, including nucleo-cytoplasmic export and mitosis. The gene is deleted in a subset of patients with the 2p15p16.1 microdeletion syndrome; however, no monogenic XPO1-related disorder has been described to date. METHODS: We collected clinical data of individuals with de novo XPO1 variants through online matchmaking. We used Drosophila to study XPO1 function in development and habituation learning. RESULTS: A total of 22 individuals met the criteria to be included in the main study cohort. Of these, half have putative loss-of-function variants, and half have coding variants (10 missense and 1 in-frame deletion variant). We found an overlapping phenotype, consistent with a monogenic neurodevelopmental disorder. We demonstrate XPO1 functions in development by ubiquitous and neuron-specific knockdown in Drosophila. GABAergic neuron specific knockdown flies demonstrated impaired habituation. CONCLUSION: Our results establish XPO1 as a novel dominant monogenic neurodevelopmental disorder gene and demonstrate a central role for XPO1 in development.

Exportin 1 Protein↗
PubMed2026

A Novel SLC25A4 Variant Causing Mitochondrial Dysfunction, Myopathy and Cardiomyopathy: A Functional and Molecular Characterization.

SLC25A4, solute carrier family 25 member 4, gene is a member of the mitochondrial carrier subfamily within the solute carrier protein family. Pathogenic variants in SLC25A4 are associated with a spectrum of mitochondrial disorders that exhibit variable inheritance patterns and clinical manifestations. Specifically, dominantly inherited variants are typically associated with progressive external ophthalmoplegia with mitochondrial DNA deletions, recessively inherited variants are linked to myopathy and cardiomyopathy, and de novo variants can result in early-onset fatal disease presentations. In this study, we aimed to identify and characterize the disease-causing mutation(s) in a nine-year-old female patient from a consanguineous Saudi family. The patient was asymptomatic until the age of 3 years, when she presented with cardiomyopathy and myopathy. Comprehensive genetic analysis inclusive of whole exome sequencing and segregation analysis using Sanger sequencing identified an SLC25A4 variant (NM_001151.4: exon 2: c.112-1G>C) as the most likely cause of the disease. To assess transcript-level effects, we performed RT-PCR on RNA extracted from the patient's cultured lymphoblast cell lines (LCLs) and fibroblast cell lines (FCLs). RT-PCR analysis demonstrated that the variant causes aberrant splicing, resulting in a 6 bp in-frame deletion (p.Gln37_Val38del) in the ANT1 protein. Quantitative RT-PCR demonstrated reduced SLC25A4 transcript levels in both FCLs and LCLs. Quantitative PCR analysis of mitochondrial DNA demonstrated a trend toward increased mtDNA copy number in patient-derived FCLs compared with controls, suggesting a possible compensatory response to mitochondrial dysfunction. Furthermore, Seahorse assays revealed marked reductions in both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in patient-derived FCLs compared with controls. These findings expand the molecular and functional spectrum of SLC25A4-associated disease and may inform clinical practice, including genetic interventions such as preimplantation genetic diagnosis, premarital genetic screening, targeted genetic counseling, and cascade testing of at-risk family members.

Humans↗
PubMed1992

Screening for mutations by expressing patient cDNA segments in E. coli: homocystinuria due to cystathionine beta-synthase deficiency.

Deficiency of cystathionine beta-synthase (CBS) causes the most common form of inherited homocystinuria. We developed a simple CBS expression system in E. coli to screen for pathogenic mutations in affected individuals. Portions of patient cDNAs were amplified by PCR and used to replace the corresponding segments of normal human CBS cDNA in the bacterial expression plasmid pHCS3. Hybrid CBS was expressed in E. coli and the segments of patient's cDNA which extinguished CBS activity were sequenced to identify the mutation. The first study of a pyridoxine-responsive patient using this screen revealed that of the clones which contained either the middle or the 3'-portion of his cDNA, about half were devoid of catalytic activity. Subsequent sequencing of the affected segments confirmed a compound heterozygosity for a maternal T833-->C transition (I278T) and for a paternal A-->C transversion in the intron 11 splice acceptor. The latter mutation leads to an in-frame deletion of exon 12 (nt 1224-1358, amino acids W408 to G453). This bacterial expression system proved to be a rapid screening method for localizing pathogenic mutations in CBS, allowing us to sequence the affected portions of mutant cDNA within 7-10 days of harvesting cultured fibroblasts.

Base Sequence↗
PubMed2026

The pseudokinase domain PK1 of UNC-89/obscurin is required for mitochondrial morphology and function in C. elegans.

UNC-89 is a giant modular protein located at the sarcomeric M-line of C. elegans striated muscle and is required for sarcomere organization and function. UNC-89 contains two protein kinase domains, PK1 and PK2, separated by 850 residues, that includes a 645-residue long intrinsically disordered sequence that acts like an elastic spring. Bioinformatic analysis suggests that PK2 is an active kinase whereas PK1 is a pseudokinase. We recently reported that a genome-edited worm, unc-89(sf22), that expresses UNC-89 carrying a kinase-inactivating point mutation in PK2 has an unusual phenotype with normally organized sarcomeres and SR, normal muscle function and yet fragmented mitochondria, increased ATP levels, increased glycolysis and alterations in electron transport chain complexes and respiration. Here, we show that a genome-edited worm unc-89(sf23), that expresses UNC-89 with an in-frame deletion of the C-lobe of PK1 has approximately the same phenotype as the PK2 catalytically dead mutant. The fact that mutations in two different regions of UNC-89 result in a mitochondrial phenotype is further evidence of communication between the sarcomere and mitochondria. We further demonstrate that in vitro PK2 interacts with full length PK1 and the C-lobe of PK1. The protein kinase domains of giant sarcomeric proteins are autoinhibited by parts of their own sequence, and this is also likely for PK2, but the mechanism by which PK2 would be activated is unknown. Our data is compatible with a model in which PK1 interacts with PK2 and thereby stimulates PK2 kinase activity.

Animals↗
PubMed2025

ELFN1 deficiency: The mechanistic basis and phenotypic spectrum of a neurodevelopmental disorder with epilepsy.

PURPOSE: Synaptic communication deficits are central to many neurodevelopmental disorders. However, for rare monogenic conditions, these disorders remain poorly defined, with limited understanding of their molecular etiology. A homozygous frameshift variant in the synaptic cell adhesion molecule ELFN1 was reported in a family with 3 affected siblings with epileptic encephalopathy, alongside a missense variant of uncertain significance in a cohort study involving a family with intellectual disability. Therefore, we sought to evaluate the role and mechanism of biallelic ELFN1 variants in disease pathogenesis. METHODS: We describe 8 newly identified individuals from 5 unrelated families, all carrying homozygous ELFN1 variants, including frameshift and in-frame deletions. By integrating data from these cases with clinical details from 6 previously reported individuals, we delineate the phenotypic spectrum associated with ELFN1 variants. RESULTS: Clinical features include varying degrees of developmental delay/intellectual disability, epilepsy, and movement disorders. Molecular investigations reveal that these variants disrupt ELFN1 protein trafficking to the cell surface, resulting in loss of function. Functional modeling in mice and zebrafish demonstrates the role of Elfn1 loss in motor activity abnormalities and seizures. CONCLUSION: Our findings establish ELFN1 deficiency as the cause of a distinct, rare neurodevelopmental disorder, providing a foundation for future investigations into its pathophysiology and therapeutic strategies.

Humans↗
PubMed1992

Mutations in the V2 vasopressin receptor gene are associated with X-linked nephrogenic diabetes insipidus.

X-linked nephrogenic diabetes insipidus (NDI) is a rare disorder in which the kidney is insensitive to the antidiuretic hormone, vasopressin. It has been proposed that the kidney-specific V2 vasopressin receptor, a G protein-coupled receptor, is defective in this disorder as both the disease and the receptor map to Xq28. We report six unique mutations in the V2 receptor gene of five unrelated NDI patients, with one patient having two mutations. The most severely affected patient has a nonsense mutation which would terminate the protein in transmembrane domain III. Other mutations include three missense mutations, a frameshift and one small in-frame deletion. These results represent one of the first examples of recessive mutations affecting a G protein-coupled receptor.

Amino Acid Sequence↗
PubMed2025

Pharmacogenomic Assessment of Genes Implicated in Thiopurine Metabolism and Toxicity in a UK Cohort of Pediatric Patients With Inflammatory Bowel Disease.

BACKGROUND: Thiopurine drugs are effective treatment options in inflammatory bowel disease and other conditions but discontinued in some patients due to toxicity. METHODS: We investigated thiopurine-induced toxicity in a pediatric inflammatory bowel disease cohort by utilizing exome sequencing data across a panel of 46 genes, including TPMT and NUDT15. RESULTS: The cohort included 487 patients with a median age of 13.1 years. Of the 396 patients exposed to thiopurines, myelosuppression was observed in 11%, gastroenterological intolerance in 11%, hepatotoxicity in 4.5%, pancreatitis in 1.8%, and "other" adverse effects in 2.8%. TPMT (thiopurine S-methyltransferase) enzyme activity was normal in 87.4%, intermediate 12.3%, and deficient in 0.2%; 26% of patients with intermediate activity developed toxicity to thiopurines. Routinely genotyped TPMT alleles associated with defective enzyme activity were identified in 28 (7%) patients: TPMT*3A in 4.5%, *3B in 1%, and *3C in 1.5%. Of these, only 6 (21%) patients developed toxic responses. Three rare TPMT alleles (*3D, *39, and *40) not assessed on routine genotyping were identified in 3 patients, who all developed toxic responses. The missense variant p.R139C (NUDT15*3 allele) was identified in 4 patients (azathioprine 1.6 mg/kg/d), but only 1 developed toxicity. One patient with an in-frame deletion variant p.G13del in NUDT15 developed myelosuppression at low doses. Per-gene deleteriousness score GenePy identified a significant association for toxicity in the AOX1 and DHFR genes. CONCLUSIONS: A significant association for toxicity was observed in the AOX1 and DHFR genes in individuals negative for the TPMT and NUDT15 variants. Patients harboring the NUDT15*3 allele, which is associated with myelosuppression, did not show an increased risk of toxicity.

Humans↗
PubMed2025

Distinct phenotypic consequences of cholangiocarcinoma-associated FGFR2 alterations depend on biliary epithelial cell state.

Epithelial cancers disrupt tissue architecture and are often driven by mutations in genes that play important roles in normal epithelial morphogenesis. The intrahepatic biliary system is an epithelial tubular network that forms within the developing liver via the de novo initiation and expansion of apical lumens. Intrahepatic biliary tumors (intrahepatic cholangiocarcinoma) commonly harbor activating genomic alterations in the FGFR2 receptor tyrosine kinase, which plays important roles in epithelial morphogenesis in other developmental settings. Using a physiologic and quantitative 3D model we demonstrate that FGFR signaling is important for biliary morphogenesis and that oncogenic FGFR2 fusions and in-frame deletions disrupt biliary architecture. Importantly, we show that the trafficking of and signaling from the FGFR2 mutants, as well as their phenotypic impacts, are governed by the epithelial state of the cell. Unexpectedly, we also found that distinct tumor-driving FGFR2 mutants disrupt biliary morphogenesis in completely different and clinically relevant ways, informing our understanding of morphogenesis and tumorigenesis and highlighting the importance of convergent studies of both.

Journal Article↗
PubMed2024

Consider CUX1 variants in children with a variation of sex development: a case report and review of the literature.

BACKGROUND: The Cut Homeobox 1 (CUX1) gene has been implicated in a number of developmental processes and has recently emerged as an important cause of developmental delay and impaired intellectual development. Individuals with variants in CUX1 have been described with a variety of co-morbidities including variations in sex development (VSD) although these features have not been closely documented. CASE PRESENTATION: The proband is a 14-year-old male who presented with congenital complex hypospadias, neurodevelopmental differences, and subtle dysmorphism. A family history of neurodevelopmental differences and VSD was noted. Microarray testing and whole exome sequencing found the 46,XY proband had a large heterozygous in-frame deletion of exons 4-10 of the CUX1 gene. CONCLUSIONS: Our review of the literature has revealed that variants in CUX1 are associated with a range of VSD and suggest this gene should be considered in cases where a VSD is noted at birth, especially if there is a familial history of VSD and/or neurodevelopmental differences. Further work is required to fully investigate the role and regulation of CUX1 in sex development.

Humans↗
PubMed2026

Transposon insertion sequencing of Pseudomonas aeruginosa identifies multiple intersecting pathways essential for extreme colistin resistance.

Colistin is used to treat antibiotic resistant gram-negative infections, including those caused by Pseudomonas aeruginosa (Pa). Using a diverse collection of clinical isolates, we identified BWH047, a colistin-resistant isolate with an extremely high minimum inhibitory concentration (MIC, 1280 µg/mL). To characterize the genes conditionally essential for colistin resistance in BWH047, we employed transposon insertion sequencing and identified 20 gene candidates. In-frame deletion validated 75% of the candidates and identified genes in several new pathways that contribute to colistin resistance in Pa, including algU and wapH. We also identified several candidate genes from previously reported colistin resistance pathways (e.g., arn, pmrAB). We further investigated the impact of a colistin resistance-associated inner membrane DedA-family undecaprenyl phosphate flippase, which we named DpcA (DedA of Pseudomonas necessary for colistin resistance A). Deletion of dpcA in BWH047 restored sensitivity to colistin (MIC = 0.5 µg/mL) and resulted in several unique changes to the structure of lipopolysaccharide (LPS), including production of decreased amounts of the colistin resistance-conferring 4-amino-4-deoxy-L-arabinose (L-Ara4N) modification on lipid A. This work represents a robust analysis of colistin resistance in Pa and identifies intersecting pathways that contribute to extreme phenotypic resistance.

Pseudomonas aeruginosa↗
PubMed2021

Detection of PKD1 and PKD2 Somatic Variants in Autosomal Dominant Polycystic Kidney Cyst Epithelial Cells by Whole-Genome Sequencing.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by the development of multiple cysts in the kidneys. It is often caused by pathogenic mutations in PKD1 and PKD2 genes that encode polycystin proteins. Although the molecular mechanisms for cystogenesis are not established, concurrent inactivating germline and somatic mutations in PKD1 and PKD2 have been previously observed in renal tubular epithelium (RTE). METHODS: To further investigate the cellular recessive mechanism of cystogenesis in RTE, we conducted whole-genome DNA sequencing analysis to identify germline variants and somatic alterations in RTE of 90 unique kidney cysts obtained during nephrectomy from 24 unrelated participants. RESULTS: Kidney cysts were overall genomically stable, with low burdens of somatic short mutations or large-scale structural alterations. Pathogenic somatic "second hit" alterations disrupting PKD1 or PKD2 were identified in 93% of the cysts. Of these, 77% of cysts acquired short mutations in PKD1 or PKD2 ; specifically, 60% resulted in protein truncations (nonsense, frameshift, or splice site) and 17% caused non-truncating mutations (missense, in-frame insertions, or deletions). Another 18% of cysts acquired somatic chromosomal loss of heterozygosity (LOH) events encompassing PKD1 or PKD2 ranging from 2.6 to 81.3 Mb. 14% of these cysts harbored copy number neutral LOH events, while the other 3% had hemizygous chromosomal deletions. LOH events frequently occurred at chromosomal fragile sites, or in regions comprising chromosome microdeletion diseases/syndromes. Almost all somatic "second hit" alterations occurred at the same germline mutated PKD1/2 gene. CONCLUSIONS: These findings further support a cellular recessive mechanism for cystogenesis in ADPKD primarily caused by inactivating germline and somatic variants of PKD1 or PKD2 genes in kidney cyst epithelium.

Humans↗
PubMed1992

Molecular basis of phenylketonuria and related hyperphenylalaninemias: mutations and polymorphisms in the human phenylalanine hydroxylase gene.

Mutations in the human phenylalanine hydroxylase gene producing phenylketonuria or hyperphenylalaninemia have now been identified in many patients from various ethnic groups. These mutations all exhibit a high degree of association with specific restriction fragment-length polymorphism haplotypes at the PAH locus. About 50 of these mutations are single-base substitutions, including six nonsense mutations and eight splicing mutations, with the remainder being missense mutations. One splicing mutation results in a 3 amino acid in-frame insertion. Two or 3 large deletions, 2 single codon deletions, and 2 single base deletions have been found. Twelve of the missense mutations apparently result from the methylation and subsequent deamination of highly mutagenic CpG dinucleotides. Recurrent mutation has been observed at several of these sites, producing associations with different haplotypes in different populations. About half of all missense mutations have been examined by in vitro expression analysis, and a significant correlation has been observed between residual PAH activity and disease phenotype. Since continuing advances in molecular methodologies have dramatically accelerated the rate in which new mutations are being identified and characterized, this register of mutations will be updated periodically.

Amino Acid Metabolism, Inborn Errors↗
PubMed1992

Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination.

The human dystrophin gene, mutations of which cause Duchenne and Becker muscular dystrophy, measures 2.4 Mb. This size seriously limits its cloning as a single DNA fragment and subsequent in-vitro expression studies. We have used stepwise in-vivo recombination between overlapping yeast artificial chromosomes (YACs) to reconstruct the dystrophin gene. The recombinant YACs are mitotically stable upon propagation in haploid yeast cells. In contrast, specific combinations of YACs display a remarkable mitotic and meiotic instability in diploid cells. Non-disjunction is rare for overlapping YACs, but increases upon sporulation of diploid cells containing non-overlapping molecules. We have exploited this feature in a three-point recombination to bridge a 280 kb gap between two non-overlapping YACs for which no YAC of proper polarity existed. Our largest recombinant YAC measures 2.3 Mb and contains the entire muscle specific DMD-gene with the exception of a 100 kb region containing the in-frame exon 60. The latter segment has a high tendency to undergo deletions in multi-molecular interactions, probably due to the presence of as yet unidentified instability-enhancing sequences. Fluorescent in situ hybridizations confirmed that the 2.3 Mb DMD YAC contained Xp21-sequences only and indicated a compact tertiary structure of the DMD-gene in interphase lymphocyte nuclei. We conclude that the yeast system is a flexible, efficient and generally applicable tool to reconstruct or build genomic regions from overlapping YAC constituents. Its application to the human dystrophin gene has provided many possibilities for future studies.

Chromosomes, Fungal↗
PubMed2026

Genetic Mutation and Epigenetic Silencing Drive Antigen-Negative Relapse in CD7 CAR T-Treated T-cell Lymphoid Malignancies.

UNLABELLED: CD7 is a promising target for chimeric antigen receptor (CAR) T-cell therapy in T-cell lymphoid malignancies; however, antigen loss-mediated relapse has emerged as a major challenge. In this study, we systematically analyzed the genetic and epigenetic alterations of paired specimens (pretreatment and relapsed) from 10 patients with T-cell lymphoma/leukemia receiving CD7 CAR T cells. Overall, we identified three distinct mechanisms underlying CD7 loss: first, frameshift insertion (patient 4; c.164dupG:p.R55fs) or deletion (patient 7; c.122delG:p.G41Efs*19) resulting in truncation of the CD7 transmembrane domain in two of 10 patients; second, hypermethylation of the CD7 promoter in seven of 10 patients without CD7 mutation; third, simultaneous occurrence of promoter region hypermethylation and multiple in-frame mutations with predicted functional interference in one of 10 patients (patient 2). Collectively, these findings demonstrate that both clonal heterogeneity and epigenetic plasticity drive antigen-negative relapse in T-cell lymphoid malignancies under the selective pressure of CD7 CAR T-cell therapy. SIGNIFICANCE: Understanding mechanisms of antigen-negative relapse is critical for developing effective CD7-targeting CAR-T therapies against T-cell lymphoid malignancies. Our study identifies both genetic truncation mutations and epigenetic silencing as contributors to CD7-negative relapse. Monitoring and preventing these events is warranted to improve treatment outcomes.

Humans↗
PubMed2024

Progressive cardiomyopathy with intercalated disc disorganization in a rat model of Becker dystrophy.

Becker muscular dystrophy (BMD) is an X-linked disorder due to in-frame mutations in the DMD gene, leading to a less abundant and truncated dystrophin. BMD is less common and severe than Duchenne muscular dystrophy (DMD) as well as less investigated. To accelerate the search for innovative treatments, we developed a rat model of BMD by deleting the exons 45-47 of the Dmd gene. Here, we report a functional and histopathological evaluation of these rats during their first year of life, compared to DMD and control littermates. BMD rats exhibit moderate damage to locomotor and diaphragmatic muscles but suffer from a progressive cardiomyopathy. Single nuclei RNA-seq analysis of cardiac samples revealed shared transcriptomic abnormalities in BMD and DMD rats and highlighted an altered end-addressing of TMEM65 and Connexin-43 at the intercalated disc, along with electrocardiographic abnormalities. Our study documents the natural history of a translational preclinical model of BMD and reports a cellular mechanism for the cardiac dysfunction in BMD and DMD offering opportunities to further investigate the organization role of dystrophin in intercellular communication.

Animals↗