Gene Defects Research Articles

A large cohort from an immunology reference center and an algorithm for the follow-up of chronic neutropenia.

Chronic neutropenia causes involve nutritional deficiencies and inborn errors of immunity(IEI), such as severe congenital neutropenia. To classify common chronic neutropenia causes in a pediatric immunology unit. We enrolled 109 chronic neutropenia patients admitted to a pediatric immunology department between 2002-2022. We recorded clinical/laboratory features and genetic characteristics. The male/female ratio was 63/46. Fifty-eight patients had parental consanguinity(57.4%). 26.6% (n = 29) patients had at least one individual in their family with neutropenia. Common symtpoms at presentation were upper respiratory tract infections(URTI)(31.1%), oral aphthae(23.6%), skin infections(23.6%), pneumonia(20.8%), and recurrent abscesses(12.3%). Common infections during follow-up were URTI(56.8%), pneumonia(33%), skin infections(25.6%), gastroenteritis(18.3%), and recurrent abscesses(14,6%). Common long-term complications were dental problems(n = 51), osteoporosis(n = 22), growth retardation(n = 14), malignancy(n = 16)[myelodysplastic syndrome(n = 10), large granulocytic leukemia(n = 1), acute lymphoblastic leukemia(n = 1), Hodgkin lymphoma(n = 1), EBV-related lymphoma(n = 1), leiomyosarcoma(n = 1), and thyroid neoplasm(n = 1)]. We performed a genetic study in 86 patients, and 69(71%) got a genetic diagnosis. Common gene defects were HAX-1(n = 26), ELA-2 (ELANE)(n = 10), AP3B1(n = 4), and ADA-2(n = 4) gene defects. The IEI ratio(70.6%) was high. GCSF treatment(93.4%), immunoglobulin replacement therapy(18.7%), and HSCT(15.9%) were the treatment options. The mortality rate was 12.9%(n = 14). The most common long term complications were dental problems that is three times more common in patients with known genetic mutations. We prepared an algorithm for chronic neutropenia depending on the present cohort. An important rate of inborn errors of immunity, especially combined immunodeficiency(11.9%) was presented in addition to congenital phagocytic cell defects. Early diagnosis will allow us tailor the disease-specific treatment options sooner, preventing irreversible consequences.

Molecular analysis of inherited disorders of cornification in polish patients show novel variants and functional data and provokes questions on the significance of secondary findings.

The Mendelian Disorders of Cornification (MeDOC) comprise a large number of disorders that present with either localised (palmoplantar keratoderma, PPK) or generalised (ichthyoses) signs. The MeDOC are highly heterogenic in terms of genetics and phenotype. Consequently, diagnostic process is challenging and before implementation of the next generation sequencing, was mostly symptomatic, not causal, which limited research on those diseases. The aim of the study was to genetically characterise a cohort of 265 Polish patients with MeDOC and to get insight into the skin lesions using transcriptome and lipid profile analyses. We detected causal variants in 85% (226/265) patients. In addition to the primary gene defect, a pathogenic variant in another gene involved in MeDOC pathology was identified in 23 cases. We found 150 distinct variants in 33 genes, including 32 novel and 16 recurrent (present in > 5 alleles). In 43 alleles large rearrangements were detected, including deletions in the STS, SPINK5, CERS3 and recurrent duplication of exons 10-14 in TGM1. The RNA analysis using samples collected from 18 MeDOC patients and 22 controls identified 1377 differentially expressed genes - DEG. The gene ontology analysis revealed that 114 biological processes were upregulated in the MeDOC group, including i.e. epithelial cell differentiation, lipid metabolic process; homeostasis; regulation of water loss via skin; peptide cross-linking. The DEG between TGM1 and ALOX12B patients, showed that RNA profile is highly similar, though fatty acid profile in epidermal scrapings of those patients showed differences e.g. for the very long chain fatty acids (VLCFAs; FAs ≥ C20), the very long-chain monounsaturated fatty acids (VLC-MUFAs, FAs ≥ C20:1) and the n6 polyunsaturated fatty acids (n6 PUFAs). Our results show that NGS-based analysis is an effective MeDOC diagnostic tool. The Polish MeDOC patients are heterogenic, however recurrent variants are present. The novel variants and high number of TGM1 and SPINK5 copy number variations give further insight into molecular pathology of MeDOC. We show that secondary variants in MeDOC-related genes are present in a significant group of patients, which should be further investigated in the context of phenotype modifiers. Finally, we provide novel RNA and lipid data that characterise molecularly MeDOC epidermis.

Germline Variants in DNA Interstrand-Cross Link Repair Genes May Contribute to Increased Susceptibility for Serrated Polyposis Syndrome

Serrated polyposis syndrome (SPS) is characterized by the development of multiple colorectal serrated polyps and increased predisposition to colorectal cancer (CRC). However, the molecular basis of SPS, especially in cases presenting family history of SPS and/or polyps and/or CRC in first-degree relatives (SPS-FHP/CRC), is still poorly understood. In a previous study, we proposed the existence of two molecular entities amongst SPS-FHP/CRC families, proximal/whole-colon and distal SPS-FHP/CRC, according to the preferential location of lesions and somatic events involved in tumor initiation. In the present study, we aimed to investigate these distinct subgroups of SPS patients in a larger cohort at the germline level and to identify the genetic defects underlying an inherited susceptibility for these two entities. Next-generation sequencing was performed using multigene analysis with a custom-designed panel in a Miseq platform in 60 SPS patients (with and without/unknown FHP/CRC). We found germline pathogenic variants in 6/60 patients (ATM, FANCM, MITF, RAD50, RAD51C, and RNF43). We also found variants of unknown significance (VUS), with prediction of probable damaging effect in 23/60 patients (ATM, BLM, BRCA1, FAN1, ERCC2, ERCC3, FANCA, FANCD2, FANCL, MSH2, MSH6, NTHL1, PALB2, PDGFRA, PMS2, PTCH1, RAD51C, RAD51D, RECQL4, TSC2, WRN, and XRCC5 genes). Most variants were detected in gene coding for proteins of the Fanconi Anemia (FA) pathway involved in the DNA Interstrand-Cross Link repair (ICLR). Notably, variants in ICLR genes were significantly more frequent in the proximal/whole-colon than in the distal subgroup [15/44 (34%) vs 1/16 (6%), p = 0.025], as opposed to the non-ICLR genes that were slightly more frequent in the distal group [8/44 (18%) vs. 5/16 (31%), p > 0.05]. Germline defects in the DNA-ICLR genes may contribute to increased serrated colorectal polyps/carcinoma risk in SPS patients, particularly in proximal/whole-colon SPS. The inclusion of DNA-ICLR genes in the genetic diagnosis of SPS patients, mainly in those with proximal/whole-colon lesions, should be considered and validated by other studies. In addition, patients with germline defects in the DNA-ICLR genes may be more sensitive to treatment with platinum-based therapeutics, which can have implications in the clinical management of these patients.

The effect of hereditary thrombophilia on recurrent pregnancy loss: a retrospective cohort study

ObjectiveThrombophilia screening has been performed in patients with conditions such as previous fetal death, (fetal growth restriction) FGR, preeclampsia, (hemolysis. elevated liver enzyme, low platelet count) HELLP Syndrome, previous abruptio placentae, previous thrombosis in pregnancy, and abnormal placental histology. The actual role of hereditary thrombophilia in recurrent pregnancy loss (RPL) is still debated. This study was intended to determine the incidence of specific gene defects for hereditary thrombophilia and to ascertain their impact on RPL in central Anatolia in Turkey.MethodsThis retrospective cohort study was performed between January 2012 and December 2022. All pregnant women with a complete hereditary screening profile were included. The investigated gene polymorphisms were methylenetetrahydrofolate reductase (MTHFR) C677T, MTHFR A1298C, Factor V Leiden G1691A, and Factor II prothrombin G20210A. Cases of pregnant women at least two or more consecutive pregnancy losses before 22 weeks of gestation were defined as RPL. The rates of genetic screening and their association with RPL were analyzed.ResultsRPL was identified in 224 (27.58%) of the 812 pregnant women with complete genetic screening. Although there was no difference in terms of age, body mass index, numbers of ectopic pregnancies, molar pregnancies, or dilatation & curettage (p > 0.05), gravity (2.0 [2.0–3.0] vs. 4.0 [3.0–5.0]), parity (1.0 [1.0–2.0] vs. 1.0 [0–1.0]), live birth (1.0 [1.0–2.0] vs. 1.0 [0–1.0]), anembryonic pregnancy (0 [0–0] vs. 0 [0–0]), miscarriage (0 [0–1.0] vs. 3.0 [2.0–3.0]), and stillbirth (0 [0–0] vs. 0 [0–0]) numbers differed significantly between the groups (p < 0.05). While no significant differences were determined in MTHFR A1298C, Factor V Leiden, factor II prothrombin G20210A, or homocysteine levels (p > 0.05), the homozygous MTHFR C677T positivity rates differed significantly (6.3% in the non-RPL group vs. 11.6% in the RPL group, p = 0.027) .ConclusionThe homozygous MTHFR C677T polymorphisms was found to be more frequent in women with RPL. Further studies with larger cohorts are needed to confirm our results.

Laboratory and Molecular Diagnosis of Factor XI Deficiency.

The prevalence of factor XI (FXI) deficiency is 1 per 10 to 20,000 in the general population, much higher than that reported in most texts. The prevalence is higher in Ashkenazi Jews where it is about 1:20. Clinically, FXI deficiency presents as a mild bleeding disorder mostly associated with posttraumatic or postsurgical hemorrhages or unexplained minor bleeding. It is often discovered due to incidental finding of a prolonged activated partial thromboplastin time (aPTT) on routine laboratory screening. FXI deficiency is an autosomal recessive bleeding disorder with many causative F11 gene defects. Diagnosis is based on FXI activity, antigen levels, and molecular diagnostics. As FXI levels do not correlate with bleeding symptoms, identification of pathogenic genetic variants may be a more accurate predictor of bleeding risk and therefore aid in the clinical management of the patient. Two variants in the F11 gene account for most cases found in the Jewish and Arab populations. Patients with FXI deficiency can develop inhibitors to FXI although spontaneously acquired inhibitors are extremely rare. We will discuss laboratory and molecular assays used to diagnose FXI deficiency as well as interferences that can complicate diagnosis including new anticoagulants and acquired FXI inhibitors.

Deciphering the mystery of CHNG3

Congenital hypothyroidism (CH), characterized by insufficient thyroid hormone production due to abnormalities in the hypothalamic-pituitary-thyroid axis, is the most common congenital endocrine disorder. We previously conducted comprehensive genetic screening of 102 patients with permanent CH born in Kanagawa Prefecture, Japan and identified mutations in several genes in 19 CH patients, including defects in genes encoding dual oxidase 2, thyroglobulin, thyrotropin receptor, thyroid peroxidase, and paired-box 8. Despite these findings, approximately 80% of cases remain unexplained. CH pedigrees unexplained by known genetic forms of CH have been reported in the literature and registered as congenital hypothyroidism, nongoitrous, 3 (CHNG3; %609893) in Online Mendelian Inheritance in Man. We also identified a Japanese pedigree of CH that was compatible with CHNG3. However, the exact genetic cause of CHNG3 was not revealed by standard analysis methods such as exome sequencing and array comparative genomic hybridization. We therefore took a combined approach and analyzed a total of 11 undiagnosed CH pedigrees by whole genome sequencing to analyze a 3-Mb linkage region, and found a disease-causing variant affecting a TTTG microsatellite in a noncoding region on chromosome 15. Further analysis revealed that 13.9% of 989 Japanese CH patients had abnormalities involving the TTTG microsatellite, with a substantial proportion (41.5%) of familial CH cases carrying these mutations. Identification of the genetic cause of CHNG3 provides new insights into the pathogenesis of CH, and highlights the need for continued exploration of noncoding genomic regions in Mendelian disorders of unknown etiology.

CRISPR-Cas9: Revolutionizing Gene Therapy for Genetic Disorders

Faulty or mutated genes cause multiple human disorders such as cancer, neurogenerative disorders, and cardiovascular diseases. These mutations in genes are usually inherited. Many treatments have been established to cure such diseases, but gene therapy is the most promising strategy. It’s an application of biotechnology that is based on correcting and replacing the mutated gene with the healthy gene, providing the genes for the proper expression of desired proteins required for curing the disease. In clinical settings, gene therapy has proved itself as a very promising treatment but has some drawbacks. Traditionally, these methods involved viral vectors that are used to deliver correct genes and replace them with the mutated genes, in patients. These approaches have somehow potential to cure diseases but often off-target effects, limitations in the editing process, and immune responses caused by patients’ immune systems are the main challenges that are the main shortcomings of this method. However, the discovery of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease protein 9 (Cas9) genome editing system (CRISPR/Cas9) in 2012 and its development have increased the value of gene therapy in the therapeutic world [1]. CRISPR/Cas9 is a revolutionizing tool that has been in gene therapy for knocking in and out gene to correct the mutation associated with many genetic diseases. This system has evolved greatly and has many isomers using different strategies to improve both applied, basic research and its clinical application. The ability of CRISPR-Cas9 to target various genetic diseases is one of its greatest advantages. Mutation in single gene results in monogenic diseases such as cystic fibrosis, muscular dystrophy, and sickle cell anemia, and this system has shown the potential to edit or fix the defective genes, causing these diseases. Even in the case of complex disorders caused by the mutation in multiple genes such as several types of cancer, and cardiovascular diseases, CRISPR has shown very positive results. The application of CRISPR-Cas9 in gene therapy is very promising and has tremendous potential to give cures for many complex disorders. The ethical issue regarding the editing of human DNA and the inheritance of modified DNA into the next generation is still under discussion and causing hurdles in uncovering the full potential of this system. Researchers are working to increase its efficiency and specificity to reduce the off-target sequences so that only targeted modification can be achieved. Moreover, they are trying to give possible results to reduce ethical concerns. CRISPR has evolved greatly and has many isomers using different strategies to improve both applied, basic research and its clinical application in the future.

Myocardial injury and heart failure biomarkers in acute intermittent porphyria

Abstract Background Acute intermittent porphyria (AIP) is a rare metabolic disorder caused by the defect in the hydroxymethylbilane synthase gene, encoding the third enzyme in heme biosynthesis pathway. The main symptoms include abdominal pain, vomiting, paralysis, confusion. Exacerbation of AIP may manifest with elevation of blood pressure and tachycardia due to hyperactivity of sympathetic nervous system. Data regarding cardiac function in AIP are scarce. Purpose The aim of this study was to assess the myocardial injury and heart failure biomarkers during AIP exacerbations and remission. Methods This cohort prospective study encompassed patients at the age 18-65 years who had at least one exacerbation of AIP in their lifetime. Patients examined during the AIP exacerbations were reassessed in the remission phase. The concentrations of sodium, troponin T, CK-MB mass, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and metoxycatecholamines in blood were assessed. Blood pressure was measured with 24-hour ABPM. Echocardiography was performed according to the current recommendations. Results The total number of 70 patients with AIP were enrolled to the study (April 2019 - March 2023). We assessed 36 patients in both phases of disease, mean age 37 years (21-63), 75% females. The median of prior AIP exacerbations was 8. During the exacerbations a significant increase in the concentrations of NT-proBNP (250 [97 ; 501] vs. 89 [48 ; 140] pg/mL, p&amp;lt;0.001), metanephrine (49.0 [37.8 ; 61.2] vs. 41.4 [36.8 ; 53.3] pg/mL, p=0.009) and normetanephrine (169.8 [118.2 ; 327.2] vs. 93.2 [78.1 ; 120.9] pg/mL, p&amp;lt;0.001) was observed. Conversely, sodium concentrations decreased (136 [133 ; 140] vs. 140 [139 ; 142] mmol/L, p&amp;lt;0.001). There were no significant differences in the values of troponin (5.4 [4.0 ; 11.3] vs. 5.6 [3.7 ; 9.5] ng/L, p=0.445), CK-MB mass (1.88 [0.84 ; 2.64] vs. 1.21 [0.83 ; 2.80] ng/mL, p=0.80). In the acute phases of AIP the levels of NT-proBNP were correlated negatively with the values of sodium concentrations (r=-0.36, p=0.032) and positively with average systolic blood pressure (r=0.32, p=0.06). Echocardiography showed an increase in left ventricular (LV) mass indices in some patients hospitalized due to AIP symptoms. Sodium concentrations correlated negatively with these changes (r=-0.36, p=0.032). LV ejection fraction was slightly higher during AIP exacerbations. There were no significant differences in other parameters of LV systolic and diastolic function. Conclusions During acute intermittent porphyria (AIP) exacerbations there is an increase in the concentrations of NT-proBNP and catecholamines in blood. In most patients the levels of markers of myocardial injury, such as troponin and CK-MB mass, do not differ significantly between phases of disease. During AIP exacerbations some patients experience an increase in LV mass which may be associated with hyponatremia. In most cases cardiac function do not change significantly.Frequency of abnormal laboratory resultsResults of echocardiography

Emerging Bioengineering Approaches for Cancer and Tumor Therapy

Cancer has become a significant socioeconomic burden globally, with millions of new cases and deaths each year. The promising field of bioengineering has recently undergone significant advancements, providing new methods for combating cancer. Increasing attention has been directed toward understanding the molecular mechanisms of human diseases, supported by the availability of various genetic tools and rapid technological advancements. These developments have enabled the use of the latest gene therapy techniques for cancer treatment, including gene editing, gene deletion, and correcting defective genes through methods such as TALENs, Zinc fingers, RNAi, CRISPR, site-directed mutagenesis (SDM), and enzyme therapy to modulate catalytic activity. In addition, bioengineering vaccines like mRNA vaccines, bioinformatics, computational tools, artificial intelligence (AI), nanotechnology, and chemotherapy are emerging as significant cancer treatment strategies. Among these, gene editing and gene therapy have gained particular attention in recent years and are often used in combination with other therapeutic approaches. The engineering of enzymes and advancements in nanotechnology have also significantly progressed. AI and bioinformatics have contributed to more precise diagnosis, prediction, and prognosis, enabling tailored treatment of cancer and tumors. Imaging and radiotherapy, enhanced by AI, have improved surgical outcomes, even from remote locations. Precision oncology has emerged, using bacteria and viruses to target tumors directly. In this review, we discuss recent advancements and challenges in various cancer therapies.

Exploring RNA therapeutics for urea cycle disorders.

RNA has triggered a significant shift in modern medicine, providing a promising way to revolutionize disease treatment methods. Different therapeutic RNA modalities have shown promise to replace, supplement, correct, suppress, or eliminate the expression of a targeted gene. Currently, there are 22 RNA-based drugs approved for clinical use, including the COVID-19 mRNA vaccines, whose unprecedented worldwide success has meant a definitive boost in the RNA research field. Urea cycle disorders (UCD), liver diseases with high mortality and morbidity, may benefit from the progress achieved, as different genetic payloads have been successfully targeted to liver using viral vectors, N-acetylgalactosamine (GalNAc) conjugations or lipid nanoparticles (LNP). This review explores the potential of RNA-based medicines for UCD and the ongoing development of applications targeting specific gene defects, enzymes, or transporters taking part in the urea cycle. Notably, LNP-formulated mRNA therapy has been assayed preclinically for citrullinemia type I (CTLN1), adolescent and adult citrin deficiency, argininosuccinic aciduria, arginase deficiency and ornithine transcarbamylase deficiency, in the latter case has progressed to the clinical trials phase.

Nutrition in phenylketonuria

Phenylketonuria (PKU) is a genetic metabolic disease resulting from a deficiency in the enzyme phenylalanine hydroxylase. This defect prevents the conversion of phenylalanine to tyrosine, and as a result, the level of phenylalanine in the body increases abnormally. The accumulation of this amino acid may cause brain damage and grows development in these patients.The primary approach to treating the disease involves dietary management that restricts protein and phenylalanine intake, often alongside Tetrahydrobiopterin (BH4) supplementation. Medical foods specifically formulated for these patients are utilized to supply the necessary energy, protein, and tyrosine. Other treatment options, including the use of large neutral amino acids (LNAAs) and the enzyme Phenylalanine ammonia lyase (PAL), can be utilized to manage symptoms in adult patients. Advancements in gene therapy aimed at correcting defective genes in patients may help restore phenylalanine metabolism, though further research in this area is necessary. Effective personal management of phenylketonuria necessitates enhanced knowledge and collaboration among healthcare professionals to achieve optimal treatment outcomes for patients. This article emphasis the nutritional management of individuals with phenylketonuria across various stages of their lives.

In Search of Spinal Muscular Atrophy Disease Modifiers

The 5q Spinal Muscular Atrophy (SMA) is a hereditary autosomal recessive disease caused by defects in the survival motor neuron (SMN1) gene encoding survival motor neuron (SMN) protein. Currently, it is the leading cause of infantile mortality worldwide. SMA is a progressive neurodegenerative disease with “continuum of clinical severity”, which can be modulated by genetic and epigenetic factors known as disease modifiers (DMs). Individuals (even siblings) with the same defects in SMN1 gene might have strikingly different types of SMA, supposedly due to the impact of DMs. There are several therapeutic options for SMA, all of them focusing on the restoration of the SMN protein levels to normal. Determining DMs and the pathways in which they are involved might aid in enhancing existing curative approaches. Furthermore, DMs might become novel therapeutic targets or prognostic biomarkers of the disease. This narrative review provides a brief overview of the genetics and pathobiology of SMA, and its bona fide modifiers. We describe novel, emerging DMs, approaches and tools used to identify them, as well as their potential mechanisms of action and impact on disease severity. We also propose several disease-modifying molecular mechanisms which could provide a partial explanation of the staggering variability of SMA phenotypes.

Multiple outcomes of the germline p16INK4a mutation affecting senescence and immunity in human skin.

The integrated behaviour of multiple senescent cell types within a single human tissue leading to the development of malignancy is unclear. Patients with Familial Melanoma Syndrome (FMS) have heterozygous germline defects in the CDKN2A gene coding for the cyclin inhibitor p16INK4a. Melanocytes within skin biopsies from FMS patients express significantly less p16INK4a but express higher levels of the DNA-damage protein 𝛾H2AX a than fibroblastic cells. However, patient fibroblasts also exhibit defects since senescent cells do not increase in the skin during ageing and fibroblasts isolated from the skin of patients have increased replicative capacity compared to control fibroblasts invitro, culminating in abnormal nuclear morphology. Patient derived fibroblasts also secreted less SASP than control cells. Predisposition of FMS patients to melanoma may therefore result from integrated dysregulation of senescence in multiple cell types invivo. The inherently greater levels of DNA damage and the overdependence of melanocytes on p16 for cell cycle inhibition after DNA damage makes them exquisitely susceptible to malignant transformation. This may be accentuated by senescence-related defects in fibroblasts, in particular reduced SASP secretion that hinders recruitment of T cells in the steady state and thus reduces cutaneous immunosurveillance invivo.

An update on autophagy disorders.

Macroautophagy is a highly conserved cellular pathway for the degradation and recycling of defective cargo including proteins, organelles, and macromolecular complexes. As autophagy is particularly relevant for cellular homeostasis in post-mitotic tissues, congenital disorders of autophagy, due to monogenic defects in key autophagy genes, share a common "clinical signature" including neurodevelopmental, neurodegenerative, and neuromuscular features, as well as variable abnormalities of the eyes, skin, heart, bones, immune cells, and other organ systems, depending on the expression pattern and the specific function of the defective proteins. Since the clinical and genetic resolution of EPG5-related Vici syndrome, the paradigmatic congenital disorder of autophagy, the widespread use of massively parallel sequencing has resulted in the identification of a growing number of autophagy-associated disease genes, encoding members of the core autophagy machinery as well as related proteins. Recently identified monogenic disorders linking selective autophagy, vesicular trafficking, and other pathways have further expanded the molecular and phenotypical spectrum of congenital disorders of autophagy as a clinical disease spectrum. Moreover, significant advances in basic research have enhanced the understanding of the underlying pathophysiology as a basis for therapy development. Here, we review (i) autophagy in the context of other intracellular trafficking pathways; (ii) the main congenital disorders of autophagy and their typical clinico-pathological signatures; and (iii) the recommended primary health surveillance in monogenic disorders of autophagy based on available evidence. We further discuss recently identified molecular mechanisms that inform the current understanding of autophagy in health and disease, as well as perspectives on future therapeutic approaches.

Mycobacterium smegmatis NucS-promoted DNA mismatch repair involves limited resection by a 5'-3' exonuclease and is independent of homologous recombination and NHEJ.

The MutSL mismatch repair (MMR) systems in bacteria and eukaryotes correct mismatches made at the replication fork to maintain genome stability. A novel MMR system is represented by the EndoMS/NucS endonuclease from Actinobacterium Corynebacterium glutamicum, which recognizes mismatched substrates in vitro and creates dsDNA breaks at the mismatch. In this report, a genetic analysis shows that an M. smegmatis ΔnucS strain could be complemented by expression of wild type NucS protein, but not by one deleted of its last five amino acids, a region predicted to be critical for binding to the β-clamp at the replication fork. Oligo-recombineering was then leveraged to deliver defined mismatches to a defective hygromycin resistance gene on the M. smegmatis chromosome. We find that NucS recognizes and repairs G-G, G-T, and T-T mismatches in vivo, consistent with the recognition of these same mismatches in C. glutamicum in vitro, as well as mutation accumulation studies done in M. smegmatis. Finally, an assay that employs an oligo that promotes the generation of two mismatches in close proximity on the chromosome shows that a NucS-promoted cut is processed by a 5'-3' exonuclease (or 5'-Flap endonuclease)and that NucS-promoted MMR is independent of both homologous recombination and non-homologous end-joining.

Role of DOCK8 in Cytokine Storm Syndromes

Cytokine storm syndromes (CSS), including hemophagocytic lymphohistiocytosis (HLH), are increasingly recognized as hyperinflammatory states leading to multi-organ failure and death. Familial HLH (FHL) in infancy results from homozygous genetic defects in perforin-mediated cytolysis by CD8 T-lymphocytes and natural killer (NK) cells. Later onset CSS are frequently associated with heterozygous defects in FHL genes, but genetic etiologies for most are unknown. We identified rare DOCK8 variants in CSS patients. We explore the role of CSS patient-derived DOCK8 mutations on cytolytic activity in NK cells. We further study effects of DOCK8 deficiency in murine models of CSS. DOCK8 cDNA from 2 unrelated CSS patients with different missense mutations were introduced into human NK-92 NK cells by foamy virus transduction. NK cell degranulation (CD107a), cytolytic activity against K562 target cells, and interferon-gamma (IFNγ) production were explored by flow cytometry. A third CSS patient DOCK8 mRNA splice acceptor site variant was explored by exon trapping. Dock8-/- mice were assessed for features of CSS (weight loss, splenomegaly, hepatic inflammation, cytopenias, and IFNγ levels) upon challenge with lymphocytic choriomeningitis virus (LCMV) and excess IL-18. Both patient DOCK8 missense mutations decreased cytolytic function in NK cells in a partial dominant-negative fashion in vitro. The patient DOCK8 splice variant disrupted mRNA splicing in vitro. LCMV infection promoted CSS in Dock8-/- mice and interacted with excess IL-18 limiting T-cell numbers while promoting CD8 T-cell hyperactivation. Mutations in DOCK8 may contribute to CSS-like hyperinflammatory states by altering cytolytic function in a threshold model of disease.

The Aspergillus flavus hacA Gene in the Unfolded Protein Response Pathway Is a Candidate Target for Host-Induced Gene Silencing.

Fungal HacA/Hac1 transcription factors play a crucial role in regulating the unfolded protein response (UPR). The UPR helps cells to maintain endoplasmic reticulum (ER) protein homeostasis, which is critical for growth, development, and virulence. The Aspergillus flavus hacA gene encodes a domain rich in basic and acidic amino acids (Bsc) and a basic leucine zipper (bZip) domain, and features a non-conventional intron (Nt20). In this study, CRISPR/Cas9 was utilized to dissect the Bsc-coding, bZip-coding, and Nt20 sequences to elucidate the relationship between genotype and phenotype. In the Bsc and bZip experimental sets, all observed mutations in both coding sequences were in frame, suggesting that out-of-frame mutations are lethal. The survival rate of transformants in the Nt20 experiment set was low, at approximately 7%. Mutations in the intron primarily consisted of out-of-frame insertions and deletions. In addition to the wild-type-like conidial morphology, the mutants exhibited varied colony morphologies, including sclerotial, mixed (conidial and sclerotial), and mycelial morphologies. An ER stress test using dithiothreitol revealed that the sclerotial and mycelial mutants were much more sensitive than the conidial mutants. Additionally, the mycelial mutants were unable to produce aflatoxin but still produced aspergillic acid and kojic acid. RNAi experiments targeting the region encompassing Bsc and bZip indicated that transformant survival rates generally decreased, with a small number of transformants displaying phenotypic changes. Defects in the hacA gene at the DNA and transcript levels affected the survival, growth, and development of A. flavus. Thus, this gene may serve as a promising target for future host-induced gene-silencing strategies aimed at controlling infection and reducing aflatoxin contamination in crops.

Ultrarare Variants in DNA Damage Repair Genes in Pediatric Acute-Onset Neuropsychiatric Syndrome or Acute Behavioral Regression in Neurodevelopmental Disorders.

Acute onset of severe psychiatric symptoms or regression may occur in children with premorbid neurodevelopmental disorders, although typically developing children can also be affected. Infections or other stressors are likely triggers. The underlying causes are unclear, but a current hypothesis suggests the convergence of genes that influence neuronal and immunological function. We previously identified 11 genes in Pediatric Acute-Onset Neuropsychiatry Syndrome (PANS), in which two classes of genes related to either synaptic function or the immune system were found. Among the latter, three affect the DNA damage response (DDR): PPM1D, CHK2, and RAG1. We now report an additional 17 cases with mutations in PPM1D and other DDR genes in patients with acute onset of psychiatric symptoms and/or regression that their clinicians classified as PANS or another inflammatory brain condition. The genes include clusters affecting p53 DNA repair (PPM1D, ATM, ATR, 53BP1, and RMRP), and the Fanconi Anemia Complex (FANCE, SLX4/FANCP, FANCA, FANCI, and FANCC). We hypothesize that defects in DNA repair genes, in the context of infection or other stressors, could contribute to decompensated states through an increase in genomic instability with a concomitant accumulation of cytosolic DNA in immune cells triggering DNA sensors, such as cGAS-STING and AIM2 inflammasomes, as well as central deficits on neuroplasticity. In addition, increased senescence and defective apoptosis affecting immunological responses could be playing a role. These compelling preliminary findings motivate further genetic and functional characterization as the downstream impact of DDR deficits may point to novel treatment strategies.

Genotype-Phenotype Correlation of GNAS Gene: Review and Disease Management of a Hotspot Mutation.

Defects of the GNAS gene have been mainly associated with pseudohypoparathyroidism Ia. To date, pathogenic missense, frameshift, non-sense and splicing variants have been described in all the 13 exons of the GNAS gene. Of them, a specific mutation, namely the 4 bp deletion c.565_568delGACT, is currently considered a mutation hotspot. Recent articles performed genotype-phenotype correlations in patients with GNAS-related pseudohypoparathyroidism Ia (PHP1a) but a specific focus on this hotspot is still lacking. We reported two cases, from our department, of PHP1a associated with c.565_568delGACT deletion and performed a literature review of all the previously reported cases of the 4 bp deletion hotspot. We found a higher prevalence of brachydactyly, round face, intellectual disability and subcutaneous/heterotopic ossifications in patients with the c.565_568delGACT as compared to the other variants in the GNAS gene. The present study highlights the different prevalence of some clinical features in patients with the c.565_568delGACT variant in the GNAS gene, suggesting the possibility of a personalized diagnostic follow-up and surveillance for these patients.

Novel Compound Heterozygous Variants in the FAS Gene Lead to Fetal Onset of Autoimmune Lymphoproliferative Syndrome (ALPS).

FAS gene defects lead to autoimmune lymphoproliferative syndrome (ALPS), which is often inherited in an autosomal dominant and rarely in an autosomal recessive manner. We report a case of a newborn girl with novel compound heterozygous variants in FAS and reveal the underlying mechanism. Whole-exome sequencing (WES) was used to identify pathogenic variants. Multiparametric flow cytometry analysis, phosflow analysis, and FAS-induced apoptosis assays were used to explore the effects of the variants on FAS expression, apoptosis, and immunophenotype. The HEK293T cells were used to assess the impact of the variants on protein expression and FAS-induced apoptosis. The patient was born with hepatosplenomegaly, anemia, and thrombocytopenia. She also experienced COVID-19, rotavirus infection, herpes simplex virus infection, and severe pneumonia. The proportion of double-negative T cells (DNTs) was significantly elevated. Novel FAS compound heterozygous variants c.310T > A (p.C104S) and c.702_704del (p.T235del) were identified. The apoptotic ability of T cells was defective, and FAS expression on the surface of T cells was deficient. The T235del variant decreased FAS expression, and the C104S protein remained in the endoplasmic reticulum (ER) and could not translocate to the cell surface. Both mutations resulted in loss-of-function in terms of FAS-induced apoptosis in HEK293T cells. The DNTs were mainly terminally differentiated T (TEMRA) and CD45RA+HLA-DR+, with high expression of CD85j, PD-1, and CD57. The percentage of Th1, Tfh, and autoreactive B cells were significantly increased in the patient. The abnormal immunophenotyping was partially attenuated by sirolimus treatment. We identified two variants that significantly affect FAS expression or localization, leading to early disease onset of in the fetus. Abnormalities in the mTOR pathway are associated with a favorable response to sirolimus.