Mutations In Different Genes Research Articles

Abstract 4138690: A Case of Hypertrophic Cardimyopathy: Digenic Variants of Uncertain Significance Mutations in MHY7 and RYR2 Genes

Introduction: Hypertrophic Cardiomyopathy (HCM) is primarily a disease caused by mutations in single genes, but around 5% of HCM patients, exhibit 2 (digenic) or more (oligogenic) causal mutations in the same gene or in different genes. We present a case of familial HCM with variants of uncertain significance (VUS) mutations in the MYH7 and RYR2 genes, highlighting the complexity of genetic inheritance and its implications on disease severity. Case presentation: A 40-year-old male presented with a history of hypertension, HCM, and two prior myocardial infarctions. He had syncope, non-sustained ventricular tachycardia, and a family history of sudden cardiac death (SCD) in his maternal grandfather. A dual-chamber ICD was placed for primary prevention of SCD because of his high-risk profile. Echocardiography demonstrated normal LV size and function with moderate asymmetric septal hypertrophy. Cardiac MRI indicated diffuse asymmetric hypertrophy and a 10.9% LV myocardial scar burden. Left heart catheterization revealed normal coronary arteries with LVOT gradient up to 60 mmHg, consistent with HCM. Genetic testing revealed VUS in the MYH7 (c.1305G>A, p.Met435Ile) and RYR2 (c.10528C>A, p.Arg3510Ser) genes (Figure 1). Further family genetic testing confirmed these VUS mutations in multiple relatives (Figure 2). Discussion: The identification of VUS mutations in MYH7 and RYR2 genes in multiple family members highlights the complexity of genetic inheritance in HCM. These findings underscore the challenges in clinical interpretation and risk stratification, as the co-occurrence of mutations in different genes may have synergistic effects on disease severity and phenotype expression. Comprehensive genetic evaluation, including family studies, is crucial for understanding the clinical significance of these mutations and guiding personalized management strategies. Further research is needed to elucidate the impact of VUS mutations on the clinical outcomes of HCM patients.

Amyotrophic Lateral Sclerosis (ALS): An Overview of Genetic and Metabolic Signaling Mechanisms.

Amyotrophic Lateral Sclerosis (ALS) is a rare, progressive, and incurable disease. Sporadic (sALS) accounts for ninety percent of ALS cases, while familial ALS (fALS) accounts for around fifteen percent. Reports have identified over 30 different forms of familial ALS. Multiple types of fALS exhibit comparable symptoms with mutations in different genes and possibly with different predominant metabolic signals. Clinical diagnosis takes into account patient history but not genetic mutations, misfolded proteins, or metabolic signaling. As research on genetics and metabolic pathways advances, it is expected that the intricate complexity of ALS will compound further. Clinicians discuss whether a gene's presence is a cause of the disease or just an association or consequence. They believe that a mutant gene alone is insufficient to diagnose ALS. ALS, often perceived as a single disease, appears to be a complex collection of diseases with similar symptoms. This review highlights gene mutations, metabolic pathways, and muscle-neuron interactions.

Clinical trials of new therapies for mucopolysaccharidoses

Background: Mucopolysaccharidoses (MPS) are a group of rare genetic diseases with a metabolic background. Currently, there are no effective methods of treating MPS, and new ways of treating patients are constantly being sought.Aim of the study: The purpose of this article is to review the available literature on clinical trials of new treatments for mucopolysaccharidoses.Material and methods: The review of research literature published between 1999 and 2023 was conducted, with a specific focus on the last ten years. The literature for this article was selected from publications available online in databases such as Google Scholar or PubMed. Research was based on keywords like: mucopolysaccharidoses, clinical trials, gene therapy, MPS. Results: A total of 104 publications were considered in the study, including 95 scientific articles and 9 clinical trial reports (consisting of 1 FDA approval). Out of those 104 articles, 82 discussed potential future therapies for patients with MPS. Among them, as many as 51 focused on gene therapy.Conclusions: The only currently approved treatments for mucopolysaccharidoses are enzyme replacement therapy and hematopoietic stem cell transplantation, which are not suitable for all types of MPS and have their limitations. In addition, there is no single therapy for all types of MPS, as they are the result of mutations in different genes and result from the deficiency of different enzymes. Numerous preclinical and clinical studies are being conducted on therapies for mucopolysaccharidoses. These include therapies that allow manipulation of cellular pathways, substrate reduction, or gene therapy, which is the most promising form of treatment. Some of the studies have successfully passed the first and second research phases.

The Roles of Drift and Selection on Short Stamen Loss in Arabidopsis thaliana along an Elevational Gradient in the Spanish Pyrenees.

Traits that have lost function sometimes persist through evolutionary time. These traits may be maintained by a lack of standing genetic variation for the trait, if selection against the trait is weak relative to drift, or if they have a residual function. To determine the evolutionary processes shaping whether nonfunctional traits are retained or lost, we investigated short stamens in 16 populations of Arabidopsis thaliana along an elevational cline in the Spanish Pyrenees. We found a cline in short stamen number from retention of short stamens in high elevation populations to incomplete loss in low elevation populations. We did not find evidence that limited genetic variation constrains the loss of short stamens at high elevations nor evidence for divergent selection on short stamens between high and low elevations. Finally, we identified loci associated with short stamens in the Spanish Pyrenees that are different from loci associated with variation in short stamen number across latitudes from a previous study. Overall, we did not identify the evolutionary mechanisms maintaining an elevational cline in short stamen number but did identify different genetic loci underlying the variation in short stamen along similar phenotypic clines.

Innovative Therapeutic Approaches in Congenital Myasthenic Syndromes.

To provide real-word clinical follow-up data on patients carrying variations of congenital myasthenic syndromes (CMS) and who respond to some innovative drugs. Patients recruited from the Neurology Department of the Mustapha Bacha university hospital in Algiers. Treated with innovative drugs, they were monitored and their clinical progress was evaluated on the basis of clinical arguments suggestive of CMSs, but also para clinical arguments (electromyography and genetic study). Six patients carrying different mutations in different genes of CMSs were studied. They had different pathophysiologic profiles (slow or fast channel syndromes, low expressor of receptor). Their therapeutic management was based on innovative drugs, normally indicated in other, non-neurological pathologies. Their outcome was toward a clear clinical improvement. This work relates the interest of proposing treatments (outside of Pyridostigmine) in the management of CMSs. These therapies can greatly modify the prognosis of patients suffering from this orphan disease. This study provides Class IV evidence that for patients with congenital myasthenic syndromes, some innovative treatments are effective.

Convergence and molecular evolution of floral fragrance after independent transitions to self-fertilization

Studying the independent evolution of similar traits provides valuable insights into the ecological and genetic factors driving phenotypic evolution.1 The transition from outcrossing to self-fertilization is common in plant evolution2 and is often associated with a reduction in floral attractive features such as display size, chemical signals, and pollinator rewards.3 These changes are believed to result from the reallocation of the resources used for building attractive flowers, as the need to attract pollinators decreases.2,3 We investigated the similarities in the evolution of flower fragrance following independent transitions to self-fertilization in Capsella.4,5,6,7,8,9 We identified several compounds that exhibited similar changes in different selfer lineages, such that the flower scent composition reflects mating systems rather than evolutionary history within this genus. We further demonstrate that the repeated loss of β-ocimene emission, one of the compounds most strongly affected by these transitions, was caused by mutations in different genes. In one of the Capsella selfing lineages, the loss of its emission was associated with a mutation altering subcellular localization of the ortholog of TERPENE SYNTHASE 2. This mutation appears to have been fixed early after the transition to selfing through the capture of variants segregating in the ancestral outcrossing population. The large extent of convergence in the independent evolution of flower scent, together with the evolutionary history and molecular consequences of a causal mutation, suggests that the emission of specific volatiles evolved as a response to changes in ecological pressures rather than resource limitation.

Assessment of structural and activity-related contributions of various PIM-1 kinase inhibitors in the treatment of leukemia and prostate cancer.

One of the most perilous illnesses in the world is cancer. The cancer may be associated with the mutation of different genes inside the body. The PIM kinase, also known as the serine/threonine kinase, plays a critical role in the biology of different kinds of cancer. They are widely distributed and associated with several biological processes, including cell division, proliferation, and death. Aberration of PIM-1 kinase is found in varieties of cancer. Prostate cancer and leukemia can both be effectively treated with PIM-1 kinase inhibitors. There are several potent compounds that have been explored in this review based on heterocyclic compounds for the treatment of prostate cancer and leukemia that have strong effects on the suppression of PIM-1 kinase. The present review summarizes the PIM-1 kinase pathway, their inhibitors under clinical trial, related patents, and SAR studies of several monocyclic, bicyclic, and polycyclic compounds. The study related to their molecular interactions with receptors is also included in the present manuscript. The study may be beneficial to scientists for the development of novel compounds as PIM-1 inhibitors in the treatment of prostate cancer and leukemia.

The role of long noncoding RNAs in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with a poor prognosis leading to death. The diagnosis and treatment of ALS are inherently challenging due to its complex pathomechanism. Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides involved in different cellular processes, incisively gene expression. In recent years, more studies have been conducted on lncRNA classes and interference in different disease pathologies, showing their promising contribution to diagnosing and treating neurodegenerative diseases. In this review, we discussed the role of lncRNAs like NEAT1 and C9orf72-as in ALS pathogenesis mechanisms caused by mutations in different genes, including TAR DNA-binding protein-43 (TDP-43), fused in sarcoma (FUS), superoxide dismutase type 1 (SOD1). NEAT1 is a well-established lncRNA in ALS pathogenesis; hence, we elaborate on its involvement in forming paraspeckles, stress response, inflammatory response, and apoptosis. Furthermore, antisense lncRNAs (as-lncRNAs), a key group of transcripts from the opposite strand of genes, including ZEB1-AS1 and ATXN2-AS, are discussed as newly identified components in the pathology of ALS. Ultimately, we review the current standing of using lncRNAs as biomarkers and therapeutic agents and the future vision of further studies on lncRNA applications.

Special Breeding Techniques for Use in Mouse Mutation Analysis.

Certain specialized breeding techniques may come in handy during the analysis of a mutation in order to further understanding of the mutation and its interactions with other genes. Different mutant alleles of the gene in question might be available from other sources or mutations with similar phenotypes could potentially be alleles. This could be determined by complementation testing. In the production of a conditional allele, retention of exogenous DNA in the allele could fortuitously disrupt a regulatory element and thus result in a hypomorphic allele, which can be simply tested by breeding. Mutations in different genes frequently affect the same organ, tissue, or cell type through genetic interactions. Common approaches to investigate and interpret genetic interactions are detailed here for gene families, in which there may be redundancy or genetic compensation of different genes, for genes that constitute different components of a biochemical pathway, for genes with overlapping expression patterns, and for unrelated genes that produce similar mutant phenotypes.

Genetic epilepsy and role of mutation variants in 27 epileptic children: results from a "single tertiary centre" and literature review

<sec><title>Aim and scope</title><p>The wide use of next-generation sequencing has allowed professionals to reach relevant progress in the medical field including diagnoses, prognoses, and genetic counselling and to help in recognizing the pathogenic mechanisms which underlie several epileptic disorders. Genetic epilepsy refers to a disorder in which genetic mutations are recognized as the primary cause of epileptic seizures in the patients. Various types of epilepsy have been highlighted alongside a clear etiologic relationship with genetic mutations; however, for a select number of patients, some doubts remain on the role played by the variant mutations, as well as the adverse pathologic events which may interfere with the clinical manifestations. In this study, twenty-seven children affected by epileptic seizures are reported, with the aim to describe the clinical and neurological involvement presented by the children according to the genes and variant mutations, and to compare the neurological signs observed in these children with those reported in children with similar genetic mutations. Additionally, the roles played by single or associated variants on the clinical expression of the affected children are discussed.</p></sec><sec><title>Material and methods</title><p>A retrospective observational study was conducted on the gene mutations observed in 27 children affected by a clinical history and diagnosis of epilepsy, alongside an electroencephalographic analysis performed with the Pediatric Department of the University-Hospital S. Marco during the years ranging from January 2020 to January 2022. A genetic analysis was performed using an array comparative genomic hybridization (array CGH), a gene panel for epilepsy, Whole Exome Sequencing (WES), and a single nucleotide polymorphism (SNP) array.</p></sec><sec><title>Results</title><p>Nineteen children showed a single nucleotide variant, eight children carried two or more gene mutations in different genes, and one child was not reported. In this study, we identified a total of thirty-six gene mutations, where seventeen were de novo mutations, and twenty-two had either a maternal or paternal inheritance. The genes in which one or more allelic variants were found included <italic>GRIN2A</italic>, <italic>GABRA1</italic>, <italic>SCN2A1,</italic> <italic>KCNT1</italic>, <italic>PCDH19</italic>, <italic>SCN8A</italic>, <italic>KCNK4</italic>, <italic>SLC2A1</italic>, <italic>DNM1</italic>, <italic>ARID1B</italic>, <italic>WWOX</italic>, <italic>GABRG2,</italic> and others.</p></sec><sec><title>Conclusions</title><p>Genetic analyses are recognized to have a central role in the diagnostic process of children with epileptic seizures, alongside their family history, and clinical, neuroradiologic, and neurophysiologic examinations. The gene mutation anomalies reported in this study may be useful to be included in the wide field of factors related to epileptic seizures in children. The roles played by single or associated variant mutations in the clinical expression of the affected epileptic children remain difficult to be defined. Moreover, other factors such as pre-, peri-, and post-natal adverse events may negatively interfere with the severity of the clinical manifestations.</p></sec>

Unique coexistence of SHOX and PTHLH gene mutations in a 12-year-old boy with syndromic short stature. Case report with literature review

Despite the availability of advanced genetic testing that enables accurate and reliable genotype assessment, clinicians continue to face diagnostic problems, especially in patients with potentially pathogenic mutations in different genes. SHOX gene mutations are the most common cause of proportional monogenic short stature, whereas the PTHLH gene encodes a parathormone-related protein that plays a crucial role in the regulation of cell growth, calcium ion transport, and bone development. The authors present the case of a 12-year-old boy with a short stature and a mosaic of developmental malformations; nephrocalcinosis; and calcium and magnesium metabolism disorders; with a unique coexistence of mutations in the SHOX and PTHLH genes. The presented case extends the clinical spectrum associated with these rare mutations and shows the usefulness of whole exome sequencing in diagnosing patients with atypical phenotypes.

Mice Overexpressing Wild-Type RRAS2 Are a Novel Model for Preclinical Testing of Anti-Chronic Lymphocytic Leukemia Therapies.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common type of leukemia in the Western world. Mutation in different genes, such as TP53 and ATM, and deletions at specific chromosomic regions, among which are 11q or 17p, have been described to be associated to worse disease prognosis. Recent research from our group has demonstrated that, contrary to what is the usual cancer development process through missense mutations, B-CLL is driven by the overexpression of the small GTPase RRAS2 in its wild-type form without activating mutations. Some mouse models of this disease have been developed to date and are commonly used in B-CLL research, but they present different disadvantages such as the long waiting period until the leukemia fully develops, the need to do cell engraftment or, in some cases, the fact that the model does not recapitulate the alterations found in human patients. We have recently described Rosa26-RRAS2fl/flxmb1-Cre as a new mouse model of B-CLL with a full penetrance of the disease. In this work, we have validated this mouse model as a novel tool for the development of new therapies for B-CLL, by testing two of the most broadly applied targeted agents: ibrutinib and venetoclax. This also opens the door to new targeted agents against R-RAS2 itself, an approach not yet explored in the clinic.

Assessment of Plasmodium falciparum drug resistance associated molecular markers in Mandla, Madhya Pradesh, India

BackgroundResistance against artemisinin-based combination therapy is one of the challenges to malaria control and elimination globally. Mutations in different genes (Pfdhfr, Pfdhps, Pfk-13 and Pfmdr1) confer resistance to artesunate and sulfadoxine–pyrimethamine (AS + SP) were analysed from Mandla district, Madhya Pradesh, to assess the effectiveness of the current treatment regimen against uncomplicated Plasmodium falciparum.MethodsDried blood spots were collected during the active fever survey and mass screening and treatment activities as part of the Malaria Elimination Demonstration Project (MEDP) from 2019 to 2020. Isolated DNA samples were used to amplify the Pfdhfr, Pfdhps, Pfk13 and Pfmdr1 genes using nested PCR and sequenced for mutation analysis using the Sanger sequencing method.ResultsA total of 393 samples were subjected to PCR amplification, sequencing and sequence analysis; 199, 215, 235, and 141 samples were successfully sequenced for Pfdhfr, Pfdhps, Pfk13, Pfmdr1, respectively. Analysis revealed that the 53.3% double mutation (C59R, S108N) in Pfdhfr, 89.3% single mutation (G437A) in Pfdhps, 13.5% single mutants (N86Y), and 51.1% synonymous mutations in Pfmdr1 in the study area. Five different non-synonymous and two synonymous point mutations found in Pfk13, which were not associated to artemisinin resistance.ConclusionThe study has found that mutations linked to SP resistance are increasing in frequency, which may reduce the effectiveness of this drug as a future partner in artemisinin-based combinations. No evidence of mutations linked to artemisinin resistance in Pfk13 was found, suggesting that parasites are sensitive to artemisinin derivatives in the study area. These findings are a baseline for routine molecular surveillance to proactively identify the emergence and spread of artemisinin-resistant parasites.

A Comprehensive Genomic Profiling of Myeloid Malignancies Demonstrates Mutational Spectrum of DNA Variants Including FLT3-ITDs, and Gene Fusions

Introduction Myeloid malignancies are a group of clonal hematopoietic disorders including acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML). These diseases are heterogenous and genetically complex and the number of genetic alterations associated with them continues to grow. To address the need for comprehensive genetics research we developed the Oncomine TM Myeloid Assay and the Oncomine TM Myeloid Assay GX v2, which both detect genomic alterations associated with myeloid malignancies including 45 DNA genes and >30 fusion driver genes with the ability to detect >700 fusion isoforms. Our panel includes genetic alterations in AML, MPN, and MDS such as FLT3-Internal Tandem Duplications (ITDs), IDH1/2, CEBPA, CALR, and TP53. Methods Here we describe the genomic profiles of 8503 clinical research samples (including AML, MPN, MDS, CML, CMML, or JMML). A total of 4723 samples were run on the Ion GeneStudio TM S5 System and analyzed using the Oncomine TM Myeloid Research workflow on Ion Reporter TM 5.18. A total of 3780 samples were run on the Ion Torrent TM Genexus Software 6.6 and analyzed using the Oncomine TM Myeloid Assay GX v2. Results The turnaround time (the time between starting the run and NGS data report) was 23-25 hours and the hands-on time was around 1 hour, which enabled fast and accurate profiling of myeloid malignancies, and facilitated the timely availability of results. The success rate of the samples was 100%. Frequency of relevant mutations by gene Some genes had a higher rate of genetic mutations. TET2 has the highest percentage of detection (12.6%, 1068 positive sample/8503 total number of samples). Other important genes with the highest percentage of detection included ASXL1 9.3% (787/8503), DNMT3A 7.8% (665/8503), TP53 7.5%, (640/8503), SRSF2 6.9% (587/8503), JAK2 6.6%, (562/8503), RUNX1 5.2% (446/8503), SF3B1 5.0% (423/8503), IDH2 4.0% (337/8503), NRAS 3.8% (318/8503), FLT3 3.4% (292/8503), ABL1 3.2% (273/8503), BCOR 2.7% (229/8503), NPM1 2.4% (201/8503), and IDH1 1.0% (170/8503). Overall, 4797 (56.4%) of samples had a mutation in at least one gene in the panels, and the average number of mutations per samples was 2.3 within these samples. The most co-occurring gene pairs include ASXL1 and TET2, as well as SRSF2 and TET2, both are detected in more than 2% co-occurring gene pairs. Variant allele frequency by gene We observed a substantial difference between the variant allele frequency (VAF) of relevant DNA mutations (SNP + Insertions + Deletions) in different genes. ANKRD26 has a median VAF of 66%, whereas MYD88 has a median VAF of only 7%. (Fig.1) Mutation spectrum of FLT3ITD variants We observed FLT3-ITD in 3% of samples. We observed the VAF of FLT3-ITD variants averaged around 0.32. Interestingly, the length of the FLT3-ITD duplication is multi-modal, with the highest peak at around 34 bp. The maximum duplication length detected is 151 bp. Mutation spectrums of fusions Of the 8480 samples with RNA fusion profiling, 710 were fusion positive (8.4%). ABL1 is the most common fusion driver (432 samples, 5.1%), with 88% of these being BCR- ABL1. Other common fusion drivers include KMT2A (171 samples, 2.0%; 29% are KMT2A-MLLT3 and 26% are KMT2A-MLLT1), MYH11 (126 samples, 1.5%; all are CBFB-MYH11), RARA (117 samples, 1.4%; all are PML-RARA), RUNX1 (95 samples, 1.1%; 56% are RUNX1-RUNX1T1). A total of 17 distinct driver genes, 49 distinct gene pairs and 110 distinct gene isoforms were observed, including rare fusions (for example, ZMYM2-FGFR1, KAT6A-CREBBP, etc) that are not detectable by traditional methods like qPCR (quantitative polymerase chain reaction) or FISH (fluorescence in-situ hybridization). The read count of the fusion positive samples ranges from 21 to 113,000. (Fig.2) Conclusions The Oncomine Myeloid Assay is a fast, accurate, robust, and reproducible solution for comprehensive genomic profiling of myeloid malignancies. We describe the mutational spectrum of DNA variants and RNA fusions in a range of clinical research samples, exploring the VAF, the abundance of mutations in different genes, and the properties of observed gene fusions. (For research use only. Not for use in diagnostic procedures.)

Genetic diagnosis of skeletal dysplasias causing short stature in children

Short stature may often be due to skeletal dysplasias affecting the limbs, spine, or both. A careful clinical evaluation will help in identifying the rhizomelic and mesomelic shortening of the limbs and scoliosis. The mutations in different genes involved in various pathways in skeletal development lead to phenotypes that present from infancy to childhood or adulthood. A systematic clinical evaluation with identification of the limb bowing or deformity, dysmorphic features, radiological findings from skull to toes, and a single gene or multi-gene panel testing will help in making an appropriate diagnosis. The clinical clues to skeletal dysplasia include skeletal disproportion, unexplained limb bowing, recurrent fractures, facial dysmorphism including flat facies and blue sclera in severe cases, and sometimes typical digital or cardiac abnormalities. The following review focuses on the postnatal presentation of skeletal dysplasias mostly referred for evaluation of short stature.

#5114 THE GENOTYPE DISTRIBUTION IN A SINGLE-CENTRE COHORT OF PATIENTS WITH POLYCYSTIC KIDNEY DISEASE

Abstract Background and Aims Polycystic kidney disease (PKD) is one of the most common causes of chronic kidney disease (CKD) and could lead to the end-stage kidney disease (ESKD) both in adulthood and in childhood. In the last few years many genes were discovered to be associated with PKD beyond the classical genes PKD1 and PKD2, that account for the majority of patients with ADPKD (Autosomal Dominant Polycystic Kidney Disease), and PKHD1 and DZIPIL that are responsible of ARPKD (Autosomal Recessive Polycystic Kidney Disease). The new genes are associated with different type of extra-renal involvement and with variable chance to develop CKD or ESKD. Thus, it is important for clinicians to define what mutation is involved in each patient to improve care in a context of precision medicine. We here describe a cohort of 65 consecutive patients followed at our PKD outpatient clinic who underwent the genetic analysis. Method We conducted a retrospective study of patients followed at our PKD outpatient clinic who underwent a genetic test for clinical reasons. We recorded why test was performed, with which modality and what was the result. If the analysis was positive we analyzed if only one or more genes were found mutated and if they belong to new genes described as being involved in PKD or if they are usually described in association with different diseases. Then, we collected demographic and clinical information (age, sex, renal function/presence of absence of ESKD) and the radiological description of kidneys in order to distinguish between typical and atypical PKDs. Results Among the 65 patients of our cohort 28 (43%) were males and 37 (57%) were females, the mean age was 56±18.6 years old; 20% had ESKD. They underwent genetic analysis for different reasons: 31 because of atypical PKD forms, 6 to define a suspected de novo mutation, 14 as a familiar screening and 14 for a better prognosis definition or when they approached the kidney transplant. The genetic analysis was conducted in two main ways: sequencing of PKD1 and PKD2 genes according to modern standards or exome sequencing. The first type of analysis was used for typical ADPKD forms whereas the second type was used when patients displayed an atypical presentation for both cysts distribution and kidney diameter (usually not enlarged kidneys with multiple bilateral cysts) and in two patients in which PKD1 and PKD2 sequencing was negative despite the PKD phenotype. We found that 25% of patients were negative, and the others are almost equally distributed between typical gene mutations and mutations in other genes (40% and 35% respectively) (Figure 1A). Of those with typical mutation, 81% was PKD1 positive and 19% PKD2 positive, a distribution that reflects that of literature (Figure 1B). Of those with other genes positivity: 52% had a DNAJB11 mutation, 17% a mutations in other PKD genes (ALG8, LRP5, PKHD1, COL4A1, IFT140), 32% had a mutation in genes usually associated with CAKUT (congenital anomalies of kidney and urinary tract) or with syndromic or extra-renal diseases (Figure 1C). 18 patients presented with multiple mutations, usually 2, in one case three and in another 4. Of them, the 22% had a double PKD1 mutation, while the others showed mutations in different genes associated with syndromic or extra-renal diseases (Figure 1D). Conclusion Our cohort revealed a wide variety of genetic mutations and also an almost equal distribution between typical genes and atypical/other genes. This could be the result of the small number of patients involved and of the monocentric design of the study (we have a cluster of patients with DNAJB11 belonging to the same small geographical area), but also of the larger inclusion of patients presenting with atypical PKDs. Unfortunately a 25% of patients remained genetically unresolved despite the use of exome sequencing.

Misrouting to mitochondria of renin carrying dominant mutations in the leader peptide or pro-segment.

Autosomal Dominant Tubulointerstitial Kidney Disease, a rare genetic disorder characterised by progressive chronic kidney disease, is caused by mutations in different genes including REN, encoding renin. Renin is a secreted protease composed of 3 domains: the leader peptide allowing insertion in the endoplasmic reticulum (ER), a pro-segment regulating its activity, and the mature part. Mutations in mature renin lead to ER retention of mutant protein and to late onset disease, while mutations in the leader peptide, associated with defective ER translocation, and mutations in the pro-segment, accumulating in the ER-to-Golgi compartment, lead to a more severe, early-onset disease. In this study we demonstrate a common, unprecedented effect of mutations in the leader peptide and pro-segment as they lead to full or partial mistargeting of mutated protein to mitochondria. The mutated pre-pro sequence of renin is necessary and sufficient to drive mitochondrial rerouting, mitochondrial import defect and fragmentation. Mitochondrial localisation and fragmentation are also observed for wild type renin when affecting ER translocation. These results expand the spectrum of cellular phenotypes associated with ADTKD-REN mutations providing new insight into the disease molecular pathogenesis.

Ceftazidime resistance in Pseudomonas aeruginosa is multigenic and complex.

Pseudomonas aeruginosa causes a wide range of severe infections. Ceftazidime, a cephalosporin, is a key antibiotic for treating infections but a significant proportion of isolates are ceftazidime-resistant. The aim of this research was to identify mutations that contribute to resistance, and to quantify the impacts of individual mutations and mutation combinations. Thirty-five mutants with reduced susceptibility to ceftazidime were evolved from two antibiotic-sensitive P. aeruginosa reference strains PAO1 and PA14. Mutations were identified by whole genome sequencing. The evolved mutants tolerated ceftazidime at concentrations between 4 and 1000 times that of the parental bacteria, with most mutants being ceftazidime resistant (minimum inhibitory concentration [MIC] ≥ 32 mg/L). Many mutants were also resistant to meropenem, a carbapenem antibiotic. Twenty-eight genes were mutated in multiple mutants, with dacB and mpl being the most frequently mutated. Mutations in six key genes were engineered into the genome of strain PAO1 individually and in combinations. A dacB mutation by itself increased the ceftazidime MIC by 16-fold although the mutant bacteria remained ceftazidime sensitive (MIC < 32 mg/L). Mutations in ampC, mexR, nalC or nalD increased the MIC by 2- to 4-fold. The MIC of a dacB mutant was increased when combined with a mutation in ampC, rendering the bacteria resistant, whereas other mutation combinations did not increase the MIC above those of single mutants. To determine the clinical relevance of mutations identified through experimental evolution, 173 ceftazidime-resistant and 166 sensitive clinical isolates were analysed for the presence of sequence variants that likely alter function of resistance-associated genes. dacB and ampC sequence variants occur most frequently in both resistant and sensitive clinical isolates. Our findings quantify the individual and combinatorial effects of mutations in different genes on ceftazidime susceptibility and demonstrate that the genetic basis of ceftazidime resistance is complex and multifactorial.

Molecular Characterization of katG and inhA Mutations by Genotype MTBDRplus Line Probe Assay To Guide Isoniazid and Ethionamide Use for Drug-Resistant Tuberculosis.

Introduction Drug-resistant tuberculosis (TB) continues to be a global health threat in all its forms. Significant resistance has been observed against isoniazid (INH), one of the most important therapeutic options for treating TB. Molecular testing methods such as line probe assay (LPA) provide rapid diagnosis and early management. Mutations in different genes can be detected, which indicate INH and ethionamide (ETH) drug resistance. We aimed to determine the frequency of these mutations in katG and inhA genes by LPA to guide INH and ETH use for drug-resistant TB. Materials and methods Two consecutive sputum samples were collected from each patient, followed by decontamination by N‑acetyl‑L‑cysteine and sodium hydroxide method. LPA was performed on the decontaminated samples by GenoType MTBDRplus, and the strips were analyzed. Results Out of the 3,398 smear-positive samples tested by LPA, valid results were found in 3,085 (90.79%) samples. Of the 3,085 samples, INH resistance was seen in 295 samples (9.56%), of which mono INH resistance was in 204 samples, and 91 were multidrug resistant. katG S315T was the most common mutation responsible for high-level INH resistance. At the same time, inhA c15t was the most common mutation associated with low-level INH resistance and ETH cross-resistance. The average turnaround time for the processing and reporting of samples was five days. Conclusions The high burden of INH resistance is alarming and can be a major obstacle to TB elimination. Although molecular methods have reduced the reporting time leading to early management of the patients still, a large knowledge gap persists.

Abstract 4648: How inherited mutations affect single cells within the tumor microenvironment in breast tumors stratified by receptor status

Abstract The tumor microenvironment (TME) plays important roles in tumor progression, therapy response and patient survival. In this comprehensive study, we performed a detailed multiomic single cell analysis of breast cancers with inherited mutations in different genes (BRCA1, BRCA2, PALB2, ATM, CHEK2), and wild type mutation negative (WT) patients, stratified by hormone receptor status, to understand the relationship between inherited mutation and immunogenicity. Using spatial tissue multiplexing (PhenoCycler), single cell RNAseq (10X Genomics) and bulk RNAseq, we evaluated cellular markers within the TME associated with DNA damage as well as phenotypes with tumor suppressive (M1, CD8 T cells, NK cells) or tumor promotive (M2, Treg, CAF) properties. We developed a 43-plex antibody panel and characterized eight tissue microarrays containing 443 patient samples from mutation carriers (BRCA1: n=142, BRCA2: n=118, CHEK2: n=2, TP53: n=3, PALB2: n=7) and WT tumors (n=171). We addressed how the TME differs in mutation carriers versus WT tumors stratified by receptor status. To further investigate immune cell function (cytolytic function, exhaustion, immune checkpoint) as well as pathways involved in immune suppression and/or promotion, we performed scRNA sequencing using isolated single cells from fresh collected BC samples (WT ER: n=3, BRCA2 ER: n=1, CHEK2 ER: n=1, VUS ER: n=1) and bulk RNA (BRCA1: n=17, BRCA2: n=9). To determine immune cell (IC) frequency, we analyzed CD45+ cells detectable in each cohort and their co-expression of markers to determine specific phenotypes. Our data suggest that within BRCA mutation-associated BC, there are two groups: 1) with increased tumor infiltrating lymphocytes (TILs) including CD8+ cytotoxic T cells with high cytolytic function; and 2) with decreased TILs and cytolytic function. The frequency of CD8+ T cells is significantly decreased in breast cancers with BRCA2 mutations compared to BRCA1. Characterizing ER+ CHEK2 BC reveals T cell exhaustion compared to WT ER+ BC. Furthermore, preliminary scRNA results reveal increased fibroblast markers in ER+ CHEK2 and ER+ WT compared to ER+ BRCA2 tumors. Interestingly, cancer associated fibroblast (CAFs) markers (ACTA2, COL1A1, FAP, PDGFRA, PDGFRB, PDPN, THY1) as well as TAMs (CCL2, CD163, CD206, CD68, IL10, LOX, PLOD2, SIGLEC1) are increased in ER+ CHEK2 tumors compared to ER+ WT. These data could provide insights into how the TME associated with ER+ CHEK2 mutations might be driven by CAFs and TAMs with a frequency much higher than in ER+ WT. The detection of immune suppressive (Treg, M2) and immune promotive (M1) phenotypes differs in BRCA1 compared to BRCA2 mutated and WT TNBC. Our findings could decipher the role of DNA repair gene mutations and their effect on cells within the TME that might influence the design of treatment options in patients with inherited mutations based on their receptor status. Citation Format: Dana Pueschl, Derek A. Oldridge, Jonathan Belman, William Chandler, Anupma Nayak, Bradley Wubbenhorst, John Pluta, Michael Feldman, E. John Wherry, Heather Thorne, Georgia Chenevix-Trench, Kathleen Cuningham Foundation Consortium for Resea kConFab, Susan M. Domchek, Katherine L. Nathanson. How inherited mutations affect single cells within the tumor microenvironment in breast tumors stratified by receptor status. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4648.