RAG1 Gene Research Articles

An unexpected new red-bellied Stumpffia (Microhylidae) from forest fragments in central Madagascar highlights remaining cryptic diversity.

The Madagascan endemic subfamily Cophylinae in the family Microhylidae, is an example of a taxonomic group for which much is still to be discovered. Indeed, the cophyline frogs present a large portion of Madagascar's cryptic and microendemic amphibian diversity, yet they remain understudied. A new red-bellied species of the microhylid frog genus Stumpffia is described from the central plateau of Madagascar. Visual encounter surveys in Ambohitantely and Anjozorobe in 2019 and 2020 identified this previously unknown Stumpffia species, which closely resembles Stumpffiakibomena known from Andasibe in the east. Stumpffialynnae sp. nov. adds another species to the red-bellied species complex, differing from S.kibomena by genetic differentiation in the mitochondrial 16S rRNA gene (3.6-3.9%) and distinct nuclear RAG1 haplotypes, as well as strongly by its advertisement call. The new species is known from across Ambohitantely Special Reserve and Anjozorobe Angavo protected area, but is known only from one complete specimen and eight individual tissue samples. Based on the rarity of the species, the small number of locations in which it has been found, and its disappearing forest habitat, its IUCN Red List classification is suggested as "Endangered". This species is the first Stumpffia described from Madagascar's central plateau, highlighting the importance of conserving the remnant forest fragments in this area and the ongoing need to survey and protect this threatened habitat type.

Genome‐wide Association Study of Oxidative Phosphorylation in Cocks Semen after Freezing

The biochemical processes associated with the generation of energy (ATP) ‐ oxidative phosphorylation (OXPHOS) and glycolysis ‐ occupy a central place in the vital activity of cells. OXPHOS is the terminal process of cellular respiration (CR). Aim of the study was Genome‐wide Association Study of OXPHOS in cocks sperm after freezing. Semen from 96 cocks was frozen. CR was assessed by the polarographic method. OXPHOS can be evaluated by the reaction CR on adding 2,4‐dinitrophenol (2,4 ‐ DNP) to semen sample. 2,4 ‐ DNP disrupt the proton gradient by carrying protons across a membrane and uncouples proton pumping from ATP synthesis because it carries protons across the inner mitochondrial membrane. As a result it increases respiration rate. If the respiration rate increases – there is good OXPHOS. Sperm motility was evaluated by computer‐assisted semen analysis (CASA) (ArgusSoft, Russia) before and after freezing. The evaluation was carried out using an Axio Imager microscope (Carl Zeiss Microscopy GmbH, Germany). DNA was isolated from sperm samples for genotyping on Gallus Gallus – Affymetrix Axiom®600k Array. Calculations were performed using the PLINK and EMMAX programs. The Genome‐Wide Association Studies (GWAS) was used for the analysis of genomic associations. Results are depicted as ranges, percentages of totals and averages ± SEs. The volume ejaculate varied from 0.4 ml to 1.4 ml, motility before freezing was from 20 to 95% and motility after freezing was from 5 to 80%. The 2.4‐DNP (FSRA) stimulation of respiration after freezing ranged from 1 to 8.09, averaging 1.66±0.114. The high repeatability of the FSRA score across samples suggests the presence of genetic factors, the identification of which can serve as a test for selecting animals with sperm whose quality remains fertile after freezing. 28 potential SNPs associated with FSRA the were found. На 5 хромосоме было найден также блок SNPs (большинство из них significant, 4 последних ‐ suggestive: AX‐76791596 (P‐value=6,20E‐09), AX‐76791535 (P‐value=1,83E‐08), AX‐76791611 (P‐value=1,83E‐08), AX‐76791454 (P‐value=1,87E‐08), AX‐76791629 P‐value=2,11E‐08), AX‐76791570 (P‐value=4,04E‐08), AX‐76791651 (P‐value=5,43E‐08), AX‐76791640 (P‐value=8,84E‐08), AX‐76791553 (P‐value=9,94E‐08), AX‐76791503 (P‐value=1,18E‐07), AX‐76791443 (P‐value=5,49E‐07), AX‐76791439 (P‐value=1,41E‐06), AX‐76791501 (P‐value=1,63E‐06). The significant SNPs found were located in the RAG2 gene. We suggest that the polymorphism in this gene is associated with the selectivity of fertilization, and the efficiency the work is associated with ATP‐dependent remodeling of ATPase subunits. Studies have opened up the possibility of studying the changes in the cells after freezing. Found SNPs markers will be used to select males whose semen is suitable for cryopreservation.

Risky business blockade: RAG2 basic region blocks V(D)J recombinase function upon genotoxic stress in DNA damage response

Abstract The V(D)J recombinase, composed of the recombination activating gene (RAG) proteins, RAG1 and RAG2, generate functional antigen receptor genes through the repeated generation of DNA double strand breaks and DNA repair. However, this process can lead to increased genomic instability particularly in the presence of excess DNA breaks. Previously, we showed in pre-B cells that full length RAG2, a nuclear protein, will export to the cytoplasm with DNA damage in an ATM-dependent manner with eventual recovery back into the nucleus following DNA repair. We propose that re-localization of RAG2 prevents RAG-mediated DNA cleavage in the presence of excess DNA ends. Here, by using fluorescently tagged RAG proteins, we show that DNA damage-induced export of the V(D)J recombinase can be recapitulated in non-lymphoid cells. Further, we determined that mutations in the intrinsically-disordered region of RAG2 have distinct effects on this process. Specifically, a basic region mutant of RAG2 (K/R 518–524 A) alters the localization pattern of the V(D)J recombinase, and yet its pre-DNA damage localization pattern is re-established upon DNA repair. Conversely, a T490A mutant blocks relocalization of RAG2 following genotoxic stress for both the single T490A mutant and the combined T490A/basic region mutant. Together, these results demonstrate the role of the intrinsically-disordered region in RAG2 in regulating the mobility and subcellular localization of the V(D)J recombinase upon cellular conditions that may be deemed too risky for continued DNA rearrangements. Supported by NIA T32AG052363, NIH AI128137, OCAST

Ezrin promotes antigen receptor diversity during B cell development by supporting immunoglobulin heavy chain variable gene recombination

Abstract Genome-level rearrangements of immunoglobulin genes during B cell development are critical for generation of a diverse repertoire of B cell antigen receptors (BCRs) that bind to a multitude of foreign antigens and some self-antigens. Bone marrow B cell development involves a variety of cell-cell interactions, cell migration and receptor signaling that likely benefit from the activity of membrane-cytoskeletal reorganizing proteins. However, the specific contribution of such proteins towards BCR repertoire diversification is poorly understood. Ezrin is a membrane-cytoskeletal linker protein that regulates mature B cell activation through spatial organization of the BCR. We employed next generation sequencing to investigate if Ezrin plays a role in immunoglobulin heavy chain rearrangements and generation of BCR diversity in developing bone marrow B cells. BCR repertoire development occurred stochastically in B cell progenitors from both control and B cell conditional Ezrin-deficient mice. However, the loss of Ezrin resulted in fewer unique CDR3s in the BCRs and reduced Shannon entropy. Ezrin-deficient pro-, pre- and immature B cells utilized similar number of joining (J) genes but significantly fewer variable (V) genes, thereby decreasing V-J combinatorial diversity. V-J junctional diversity, measured by CDR3 length, nucleotide additions and deletions, was also altered in Ezrin-deficient pro-, pre- and immature B cells. Mechanistically, Ezrin-deficient bone marrow B cells showed a marked decrease in RAG1 gene expression, indicating a less efficient DNA recombination machinery. Overall, our results demonstrate a novel role for Ezrin in shaping the BCR repertoire through combinatorial and junctional diversification. Supported by grants from NIH (R01 AR067705) to N.G.

MiRAGDB: A Knowledgebase of RAG Regulators.

RAG1 and RAG2 genes generate diversity in immunoglobulin and TCR genes by initiating the process of V-D-J recombination. RAGs recognize specific sequences (heptamer-nonamer) to generate a diversity of immunoglobulins. RAG expression is limited to early B and T cell developmental stages. Aberrant expression of RAG can lead to double strand breaks and translocations as observed in leukemia and lymphoma. The expression of RAG is tightly regulated at the transcriptional and posttranscriptional levels. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in the post-transcriptional regulation of gene expression. This study aimed to identify and catalog RAG regulation by miRNA during normal development and cancer. NGS data from normal B-cell and T-cell developmental stages and blood cancer samples have been analyzed for the expression of miRNAs against RAG1 (1,173 against human RAG1 and 749 against mouse RAG1). The analyzed data has been organized to retrieve the miRNA and mRNA expression of various RAG regulators (10 transcription factors and interacting partners) in normal and diseased states. The database allows users to navigate through the human and mouse RAG regulators, visualize and plot expression. miRAGDB is freely available and can be accessed at http://52.4.112.252/shiny/miragdb/.

Severe combined immunodeficiencies: Expanding the mutation spectrum in Turkey and identification of 12 novel variants.

Human Inborn Errors of Immunity (IEIs) are clinically and genetically heterogeneous group of diseases, with relatively mild clinical course or severe types that can be life-threatening. Severe combined immunodeficiency (SCID) is the most severe form of IEIs, which is caused by monogenic defects that impair the proliferation and function of T, B, and NK cells. According to the most recent report by the International Union of Immunological Societies (IUIS), SCID is caused by mutations in IL2RG, JAK3, FOXN1, CORO1A, PTPRC, CD3D, CD3E, CD247, ADA, AK2, NHEJ1, LIG4, PRKDC, DCLRE1C, RAG1 and RAG2 genes. The targeted next-generation sequencing (TNGS) workflow based on Ion AmpliSeq™ Primary Immune Deficiency Research Panel was designed for sequencing 264 IEI-related genes on Ion S5™ Sequencer. Herein, we present 21 disease-causing variants (12 novel) which were identified in 22 patients in eight different SCID genes. Next-generation sequencing allowed a rapid and an accurate diagnosis SCID patients.

Strategies for enhanced adaptive immune responses of Pterophyllum scalare larvae against Aeromonas hydrophila

The present study examines host pathogen response in developmental stage of Pterophyllum scalare (angelfish) infected with Aeromonas hydrophila. The study conducted had two different experiments in which the fish were subjected to formalin-killed Aeromonas hydrophila (FKA) and immunostimulant (β-glucan) by immersion method. The first experiment had three treatment groups viz., immunized, non-immunized and control groups and the exposure time ranged between 12 and 32 dph (days posthatch) of larval rearing. The immunized group exhibited a higher fold expression of RAG-1, RAG-2 and IgM genes compared with non-immunized group. The results demonstrated that the vaccination of 12 dph developmental stage prior to bacterial challenge of angelfish makes it more immunocompetent against the pathogens. Moreover, another experiment using β-glucan was conducted to further examine the development of immune responses in angelfish larvae. The study consisted four treatments containing graded levels of β-glucan dosages such as 100, 200 and 300 mg/L including a control (0 mg/L) for 20 days from 12 to 32 dph. The higher fold change in mRNA expression of RAG-1, RAG-2 and IgM genes was observed in β-glucan (200 mg/L) treated group as compared to non-treated and control group. The highest survivability was observed in the immunized group and immunostimulant treated group in the survival analysis. Hence, the present study suggested that either immunization with FKA or β-glucan (200 mg/L) could be used as a strategy to mitigate the mortality rate and combat infection during the early larval rearing of P. scalare.

Discovery of Kerivoula kachinensis and a validity of K. titania (Chiroptera: Vespertilionidae) in China

Abstract In April 2019, 15 (10♂, 5♀) Kerivoula bats were collected by harp traps from Xishuangbanna, Yunnan Province, China. External and craniodental examination, multivariate statistical analyses and molecular phylogenetic inference (CoI, Cytb and Rag2 gene markers) indicated they are Kerivoula kachinensis and Kerivoula titania, respectively. Former represents a new chiropteran record from China, while the latter is a valid occurrence of K. titania in this region because recent study indicate a misidentification of “K. titania” in Guangdong, Guangxi and Hainan, China. All specimens are presently preserved at Key Laboratory of Conservation and Application in Biodiversity of South China in Guangzhou University, Guangzhou, China. Nowadays, four woolly bats occur in China including, Kerivoula furva, K. kachinensis, Kerivoula picta and K. titania, whilst there is a risk of underestimation the actual species diversity in China region when comparing those of neighboring region such as Vietnam. Supports for field survey need to be continued in future.

Hidden diversity within a polytypic species: The enigmatic Sceloporus torquatus Wiegmann, 1828 (Reptilia, Squamata, Phrynosomatidae)

The spiny lizard genus Sceloporus was described by Wiegmann in 1828, with S. torquatus posteriorly designated as the type species. The taxonomic history of S. torquatus is complicated, as it has been confused with other taxa by numerous authors. Many modern systematics works have been published on Sceloporus, but none have included all five recognized S. torquatus subspecies: S. t. torquatus, S. t. melanogaster, S. t. binocularis, S. t. mikeprestoni, and S. t. madrensis. Additionally, there is previous evidence for at least one unnamed taxon. The present study is the first taxonomic revision of the enigmatic S. torquatus based on molecular phylogenies using combined molecular data from 12S, ND4 and RAG1 genes, and Maximum Likelihood and Bayesian inference phylogenetic methods. This work includes the most extensive sampling across the entire distribution, as well as divergence time estimates and environmental niche modelling, which combined offer a spatio-temporal framework for understanding the evolution of the species. Additionally, a series of morphological characters are analyzed to identify significant differences between lineages consistently recovered in the molecular phylogenies. Using this integrative approach, evidence is presented for eight lineages within the S. torquatus complex, five of which correspond to previously recognized subspecies and three represent unnamed taxa masked by morphological conservatism. Finally, to maintain taxonomic stability a lectotype and paralectoype are designated for S. torquatus, and certain taxonomic changes are suggested in order to reflect the phylogenetic relationships within the S. torquatus complex.

Aberrant Dntt Expression, TdT-Aided Gene Length Mutations, and Prognosis Relevance in ATG/Alg Based Regimen Allo-HSCT in Acute Myeloid Leukemias

DNTT encodes the most template-independent DNA polymerases TdT. The canonical function of TdT is to boost the diversity of immunoglobulin and T cell receptors by incorporating non-templated nucleotides (NTN) to their variable regions via RAG1/2 mediated DNA breaks and non-homologous end joining (NHEJ) rearrangement process. This study aimed to investigate the relationship between aberrant DNTT expression and illegitimately TdT-aided microhomology-mediated replication-dependent recombination (MMRDR) with the mutagenesis of gene length mutations (LMs) in acute myeloid leukemia (AML), and their prognosis relevance.A cohort of 578 AML cases was enrolled. Fifty healthy donors for allogeneic hematopoietic stem cell transplantation (allo-HSCT), 393 B cell acute lymphoblastic leukemia (B-ALL) cases, 78 T-ALL cases, and 25 mixed-phenotype acute leukemia (MPAL) cases were used as control. Next-generation sequencing was performed for mutation analysis of 86 leukemia driver genes. RNA-seq was used to analyze the expression of DNTT and other non-homologous end joining (NHEJ) associated genes. Prognostic was investigated in a subset of 239 AML cases who underwent anti-thymocyte globulin (ATG) or anti-lymphocyte globulin (ALG) based regimen allo-HSCT.Based on sequence anatomy that considers the MMRDR mechanism and nucleotides characteristics of TdT mediated NTN incorporation, we formulate a classification algorithm for LMs and divide them into four subtypes (type I-IV). Type-I indicates pure duplicated/triplicated germline sequences with identifiable ≥2bp canonical triple microhomology (MH) sequences; type-II indicates pure duplicated/triplicated germline sequences without ≥2bp canonical MH sequences; type-III indicates any LMs with NTN insertions; type-IV indicates any other LMs, mainly deletions.FLT3-LMs has the highest overall incidence and occur across multiple lineage leukemias. We observed a significant FLT3-LMs subtypes distribution bias among acute leukemia subtypes (Figure 1A). Type-I FLT3 LMs are only observed in AML; there are mainly type-III FLT3 LMs in T-ALL and MPAL; type-II, III, IV FLT3 LMs are predominant in B-ALL. The overall DNTT expression was significantly lower in AML than in other leukemia subtypes and control groups (P < 0.001). This supports that FLT3-LMs subtypes distribution bias might be attributed to the difference in the overall DNTT expression among leukemia lineages.A total of 458 LMs events were observed in 295 cases (51.0%) within 25 genes (FLT3, NPM1, CEBPA, RUNX1, KIT, etc.) in our AML cohort. The incidence of type-II and type-III LMs, both of which the mutagenesis relies on TdT-aided MMRDR in theoretically speculate, were 31.2 % and 47.8 %, respectively. Type-I and type-II, which manifested as pure germline sequence duplications, account for 43.6% of the FLT3 LMs; type-III LMs, which with additional inserted NTN sequences, account for as high as 50.4% of the FLT3 LMs.We analyzed the G/C nucleotide contents adjacent to LMs junctions. A significantly high G/C bias was observed at +1 nucleotide position in type-II and type-III subsets (Figure 1B), suggesting that the TdT-aided MMRDR mechanism plays a role in the mutagenesis in these cases. We also observed a strong positive correlation between fragment length and G/C content of the inserted NTN sequences (P < 0.001) within the type-III subset of the 25 LMs genes (Figure 1C), suggesting a higher TdT activity mediates longer inserted sequences.DNTT expressions level of type-III LMs cases were significantly higher than that of type-I, II, IV LMs cases and cases without LMs in the total 25 LMs genes (Figure 1D) and the FLT3 LMs subset. Similar expression signatures of other NHEJ associated genes RAG2, XRCC4, and XRCC6 were also observed. For the survival analysis in the ATG/ALG based regimen allo-HSCT AML subset, we observed a significantly better overall survival (P = 0.024) in cases positive for type-III FLT3-LMs than that of type-I, II (Figure 1E).In this study, we proposed a subclassification algorithm for LMs (type I-IV) in AML. Both DNTT gene expression and sequence character suggesting that TdT-aided MMRDR plays a role in the mutagenesis of type-III and type-II LMs. We also observed AML cases with type-III FLT3 LMs benefit more from ATG/ALG based regimen allo-HSCT than cases with other FLT3 LMs types, which may be attributed to the aberrant lymphoid lineage antigen expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical, Immunological, and Molecular Variability of RAG Deficiency: A Retrospective Analysis of 22 RAG Patients

PurposeWe described clinical, immunological, and molecular characterization within a cohort of 22 RAG patients focused on the possible correlation between clinical and genetic data.MethodsImmunological and genetic features were investigated by multiparametric flow cytometry and by Sanger or next generation sequencing (NGS) as appropriate.ResultsPatients represented a broad spectrum of RAG deficiencies: SCID, OS, LS/AS, and CID. Three novel mutations in RAG1 gene and one in RAG2 were reported. The primary symptom at presentation was infections (81.8%). Infections and autoimmunity occurred together in the majority of cases (63.6%). Fifteen out of 22 (68.2%) patients presented autoimmune or inflammatory manifestations. Five patients experienced severe autoimmune cytopenia refractory to different lines of therapy. Total lymphocytes count was reduced or almost lacking in SCID group and higher in OS patients. B lymphocytes were variably detected in LS/AS and CID groups. Eighteen patients underwent HSCT permitting definitive control of autoimmune/hyperinflammatory manifestations in twelve of them (80%).ConclusionWe reinforce the notion that different clinical phenotype can be found in patients with identical mutations even within the same family. Infections may influence genotype–phenotype correlation and function as trigger for immune dysregulation or autoimmune manifestations. Severe and early autoimmune refractory cytopenia is frequent and could be the first symptom of onset. Prompt recognition of RAG deficiency in patients with early onset of autoimmune/hyperinflammatory manifestations could contribute to the choice of a timely and specific treatment preventing the onset of other complications.

ETV6-RUNX1 and RUNX1 directly regulate RAG1 expression: one more step in the understanding of childhood B-cell\xa0acute lymphoblastic leukemia leukemogenesis

HighlightsETV6-RUNX1 and RUNX1 directly promote RAG1 expression.ETV6-RUNX1 and RUNX1 preferentially bind to the −1200 bp enhancer of RAG1 and the −80 bp promoter of RAG1 gene respectively, and compete for these bindings.ETV6-RUNX1 and RUNX1 induce an excessive RAG recombinase activity.ETV6-RUNX1 participates directly in two events of the multi-hit ALL leukemogenesis: as an initiating event and as an activator of RAG1 expression.

New Mitogenome of the Hainan Mole Mogera hainana and Taxonomic Implications Based on Molecular Data

The Hainan mole Mogera hainana was described as a full species by Thomas (1910), but its taxonomic status of this animal has been debated since then and remains controversial. In this study, we determined the mitochondrial genome of M. hainana. We also estimated its phylogenetic relationships using mitochondrial Cytb and nuclear Rag1 genes and conducted molecular species delimitation analyses using Bayesian Poisson tree processes, Automatic Barcode Gap Discovery, and Assemble Species by Automatic Partitioning to determine the evolutionary position and putative taxonomic status of M. hainana. The mitogenome of M. hainana is 16 845 base pairs, composed of 13 protein-coding genes, 22 tRNAs, two rRNAs, and the control region. We further compared this sequence with those of other Mogera species. The phylogenetic trees support a sister relationship between M. hainana and M. kanoana and close relationships among M. hainana, M. kanoana, M. insularis, and M. latouchei. Species delimitation analyses suggest that M. hainana is distinct from other recognized species and thus is likely a distinct species.

Guardian of the Genome: An Alternative RAG/Transib Co-Evolution Hypothesis for the Origin of V(D)J Recombination.

The appearance of adaptive immunity in jawed vertebrates is termed the immunological ‘Big Bang’ because of the short evolutionary time over which it developed. Underlying it is the recombination activating gene (RAG)-based V(D)J recombination system, which initiates the sequence diversification of the immunoglobulins and lymphocyte antigen receptors. It was convincingly argued that the RAG1 and RAG2 genes originated from a single transposon. The current dogma postulates that the V(D)J recombination system was established by the split of a primordial vertebrate immune receptor gene into V and J segments by a RAG1/2 transposon, in parallel with the domestication of the same transposable element in a separate genomic locus as the RAG recombinase. Here, based on a new interpretation of previously published data, we propose an alternative evolutionary hypothesis suggesting that two different elements, a RAG1/2 transposase and a Transib transposon invader with RSS-like terminal inverted repeats, co-evolved to work together, resulting in a functional recombination process. This hypothesis offers an alternative understanding of the acquisition of recombinase function by RAGs and the origin of the V(D)J system.

Immunodeficiency Caused Byheterozygous RAG1 Gene Mutation in a Boy Manifested As Severe Granulomatous Diseases and Eosinophilia: A Case Report

Severe combined immunodeficiency (SCID) is an inherited diseases caused by the defective development of T- and/or B-lymphocytes refractory to common therapy. We report a 13-year old boy with combined immunodeficiency caused by a heterozygous mutation at cDNA position 1435 (c.1435C>T) in RAG1 gene. His father and grand-father carried the same heterozygous RAG1 gene mutation, and experienced anaphylactic rhinitis. His mother had mutations in 6 immunodeficiency-related heterozygous genes but no RAG1 mutation, and experienced thrombocytopenia. Both his parents had normal NK and T cell numbers, and serum immunoglobulin (Ig) levels. After birth, this patient had severe allergies to many food and drugs, and suffered from recurrent upper and lower respiratory tract infections every year. After admission, the patient complained of a generalized body rash with itches lasting about several years. We found severe granuloma lesions in the face, mouth and nose, lymphadenectasis and splenomegaly by physical examination (Figure 1a). Laboratory examinations demonstrated that he had a high eosinophilic granulocyte count as 2.19×109/L, but decreased NK cell numbers and CD4+/CD8+ T cell ratios, and IgG, IgM and IgA serum levels. This patient has been previously treated with immune-modulatory drugs including anti-histamine drugs, prednisone, γ-globulin, and chemotherapy including Cyclophosphamide, but lacking clinical improvement. In our center, after Methotrexate (1g/m2 q.w.)+L-asparaginase (10000 unit q.d.×10 days) + Dexamethasone (10mg/m2q.d.×5 days) chemotherapy, and immune enhancement therapy with γ-globulin(400mg/kg×5 days per month), the patient got his symptoms ameliorated (Figure 1b). Haploidentical hematopoietic stem cell transplantation (HCT) from his HLA 7/10 matched mother was subsequently applied, and the patient remained remission without HCT-related complications for at least 8 months after transplantation (Figure 1c). Our results thus showed the immune disorder in a boy caused by a novel heterozygous RAG1 gene c.1435C>T mutation which has been efficiently treated by our specific chemotherapy combined with HCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Neonatal Lymphopenia Screening Is Important for Early Diagnosis of Severe Combined Immunodeficiency.

T-cell receptor excision circles are expensive for neonatal severe combined immunodeficiency screening in developing countries. We aimed to detect immunodeficiencies presenting with lymphopenia to enable screening in the general population and to improve awareness regarding lymphopenia among clinicians. This study was conducted prospectively. In all newborns included, complete blood count from umbilical cord blood samples was recorded. Absolute lymphopenia was defined as absolute lymphocyte count <3,000/mm3 in umbilical cord blood sample. Complete blood count was repeated at month 1 in cases found to have lymphopenia. Overall, 2,000 newborns were included in the study. Absolute lymphopenia was detected in 42 newborns (2.1%), while lymphocyte count was >3,000/mm3 in 1,958 newborns (97.9%). Two infants with persisted lymphopenia at the end of the first month; therefore, further evaluations such as lymphocyte subsets for severe combined immunodeficiency (SCID) were done. In the first infant, the lymphocyte subgroups were detected as compatible with T (-), B (-), natural killer cells (NK) (+) SCID phenotype RAG defect. Sanger sequencing revealed that NM_000448 c.2209C > T (p.R737C) homozygous mutation of RAG1 gene. In the other infant, the lymphocyte subgroups were found as considered with T (-), B (+) NK (-) SCID phenotype JAK3 defect. Both patients underwent hematopoietic stem cell transplantation from human leukocyte antigen-matched family member. Absolute lymphopenia by complete blood count is a more simpler, relatively noninvasive and inexpensive screening methodfor detection of SCID in newborns compared with T-cell receptor excision circles technique. · Our study was conducted with a much smaller number of study groups compared with the previous ones.. · However, SCID was found at a higher rate compared with other studies.. · Our study for this disease that is common in our country where consanguineous marriages are common.

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity is required for V(D)J recombination.

A whole-genome CRISPR/Cas9 screen identified ATP2A2, the gene encoding the Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2 protein, as being important for V(D)J recombination. SERCAs are ER transmembrane proteins that pump Ca2+ from the cytosol into the ER lumen to maintain the ER Ca2+ reservoir and regulate cytosolic Ca2+-dependent processes. In preB cells, loss of SERCA2 leads to reduced V(D)J recombination kinetics due to diminished RAG-mediated DNA cleavage. SERCA2 deficiency in B cells leads to increased expression of SERCA3, and combined loss of SERCA2 and SERCA3 results in decreased ER Ca2+ levels, increased cytosolic Ca2+ levels, reduction in RAG1 and RAG2 gene expression, and a profound block in V(D)J recombination. Mice with B cells deficient in SERCA2 and humans with Darier disease, caused by heterozygous ATP2A2 mutations, have reduced numbers of mature B cells. We conclude that SERCA proteins modulate intracellular Ca2+ levels to regulate RAG1 and RAG2 gene expression and V(D)J recombination and that defects in SERCA functions cause lymphopenia.

RAG enhances BCR-ABL1-positive leukemic cell growth through its endonuclease activity in vitro and in vivo.

BCR‐ABL1 gene fusion associated with additional DNA lesions involves the pathogenesis of chronic myelogenous leukemia (CML) from a chronic phase (CP) to a blast crisis of B lymphoid (CML‐LBC) lineage and BCR‐ABL1 + acute lymphoblastic leukemia (BCR‐ABL1 + ALL). The recombination‐activating gene RAG1 and RAG2 (collectively, RAG) proteins that assemble a diverse set of antigen receptor genes during lymphocyte development are abnormally expressed in CML‐LBC and BCR‐ABL1 + ALL. However, the direct involvement of dysregulated RAG in disease progression remains unclear. Here, we generate human wild‐type (WT) RAG and catalytically inactive RAG‐expressing BCR‐ABL1 + and BCR‐ABL1 − cell lines, respectively, and demonstrate that BCR‐ABL1 specifically collaborates with RAG recombinase to promote cell survival in vitro and in xenograft mice models. WT RAG‐expressing BCR‐ABL1 + cell lines and primary CD34+ bone marrow cells from CML‐LBC samples maintain more double‐strand breaks (DSB) compared to catalytically inactive RAG‐expressing BCR‐ABL1 + cell lines and RAG‐deficient CML‐CP samples, which are measured by γ‐H2AX. WT RAG‐expressing BCR‐ABL1 + cells are biased to repair RAG‐mediated DSB by the alternative non–homologous end joining pathway (a‐NHEJ), which could contribute genomic instability through increasing the expression of a‐NHEJ‐related MRE11 and RAD50 proteins. As a result, RAG‐expressing BCR‐ABL1 + cells decrease sensitivity to tyrosine kinase inhibitors (TKI) by activating BCR‐ABL1 signaling but independent of the levels of BCR‐ABL1 expression and mutations in the BCR‐ABL1 tyrosine kinase domain. These findings identify a surprising and novel role of RAG in the functional specialization of disease progression in BCR‐ABL1 + leukemia through its endonuclease activity.

Cutting Edge: TCR-β Selection Is Required at the CD4+CD8+ Stage of Human T Cell Development.

T cell development is predicated on the successful rearrangement of the TCR gene loci, which encode for Ag-specific receptors. Recombination-activating gene (RAG) 2 is required for TCR gene rearrangements, which occur during specific stages of T cell development. In this study, we differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) to elucidate the requirement for the TCR β-chain in mediating β-selection during human T cell development. In stark contrast to mice, human RAG2-KO T lineage progenitors progressed to the CD4+CD8+ double-positive (DP) stage in the absence of TCRβ rearrangements. Nonetheless, RAG2-KO DPs retrovirally transduced to express a rearranged TCR β-chain showed increased survival and proliferation as compared with control-transduced RAG2-KO DPs. Furthermore, transcriptomic analysis showed that TCRβ- and control-transduced RAG2-KO DPs differed in gene pathways related to survival and proliferation. Our results provide important insights as to the distinct requirement for the TCR β-chain during human T cell development.

The effect of CRISPR constructs microinjection on the expression of developmental genes in Rag1 knocked-out mice embryo.

Despite all the advances in the production of transgenic mice, the production efficiency of these animal models is still low. Given that the expression of developmental genes has a critical role in growth and development of embryo, we determined the expression pattern of pluripotency, trophectoderm and imprinting genes in the Rag1 (recombination‐activating gene 1) knocked‐out blastocysts resulting from microinjection of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9) constructs into the zygote cytoplasm of C57bl6 mice. Following microinjection, the embryos were cultured and the gene expression of developed blastocysts and natural blastocysts (Sham and control groups) were evaluated using real‐time PCR. The agarose gel to confirm the deletion in the Rag1 gene in Rag1 knocked‐out blastocyst. Our results showed that the expression of trophectoderm genes (‐TEAD‐4 and Cdx2), pluripotency genes (Nanog and Oct‐4) and imprinting gene (H19) in the Rag1 knocked‐out group was significantly lower compared with the embryos obtained from Natural fertilization. According to these findings, manipulation, embryo culture and microinjection of CRISPR constructs into the zygote cytoplasm of mice led to reduced expression of imprinting, pluripotency and trophectoderm genes. Therefore, the Rag1 knocked‐out embryos produced by the CRISPR/Cas9 system are of low quality, which reduces the chances of live birth in these animals and may cause various abnormalities in fetuses.