Mycobacterium leprae, rarely affects children younger than 5 years. Here, we studied a multiplex leprosy family that included monozygotic twins aged 22 months suffering from paucibacillary leprosy. Whole genome sequencing identified three amino acid mutations previously associated with Crohn’s disease and Parkinson’s disease as candidate variants for early onset leprosy: LRRK2 N551K, R1398H and NOD2 R702W. In genome-edited macrophages, we demonstrated that cells expressing the LRRK2 mutations displayed reduced apoptosis activity following mycobacterial challenge independently of NOD2. However, employing co-immunoprecipitation and confocal microscopy we showed that LRRK2 and NOD2 proteins interacted in RAW cells and monocyte-derived macrophages, and that this interaction was substantially reduced for the NOD2 R702W mutation. Moreover, we observed a joint effect of LRRK2 and NOD2 variants on Bacillus Calmette-Guérin (BCG)-induced respiratory burst, NF-κB activation and cytokine/chemokine secretion with a strong impact for the genotypes found in the twins consistent with a role of the identified mutations in the development of early onset leprosy.Leprosy, caused by  Mycobacterium leprae. Here, we report a case of a family with three generation of leprosy cases including a rare pair of identical twins of less than two years of age. To investigate if genetic factors in the twins may explain the early disease onset, we conducted a whole genome sequence analysis of family members. We found three amino acid changes in LRRK2 and NOD2 as candidate susceptibility variants for early onset leprosy. In follow-up functional assays, we demonstrated that LRRK2 and NOD2 proteins physically interact in macrophages and that this interaction was strongly impacted by the NOD2 variant. Furthermore, we showed a joint effect of LRRK2 and NOD2 variants that results in a reduced antimycobacterial response in these cells. Interestingly, the variants in LRRK2 and NOD2 found in the twins had previously been identified as genetic modulators for risk of Parkinson’s and Crohn’s diseases with the two LRRK2 variants showing antagonistic pleiotropy. This suggests pleiotropic effects of the NOD2 and LRRK2 variants across neurodegenerative, inflammatory and infectious diseases.Children younger than 5-years old are rarely affected by leprosy, a chronic infectious disease of the skin and peripheral nerves caused by  Mycobacterium leprae or M. lepromatosis. The mode of transmission of leprosy is not completely understood. Evidence suggests zoonotic transmission from armadillos in isolated leprosy cases, and experimental models have presented Reduviidae bugs and ticks as potential vectors .,16.15,16 in ACP5 . The twiin silico  was a co-dominant risk factor for CD  and incubated with the primary antibodies diluted in blocking buffer–rabbit anti-LRRK2 1:500 and mouse anti-FLAG 1:250 (for NOD2)–overnight at 4°C. Cells were washed with PBS for 3 x 5 minutes and incubated with secondary antibodies prepared in a diluted (1:5) blocking buffer [Alexa Fluor 488 goat anti-mouse IgG (H+L) 1:1000 and Alexa Fluor 594 donkey anti-rabbit IgG (H+L) 1:1000] for 1 hour. Cells were washed with PBS for 3 x 5 minutes and nuclei were stained with DAPI. Images were obtained by confocal microscopy. Colocalization between LRRK2 and NOD2 was measured from 25–30 cells by Zeiss 2012 ZEN confocal software.6 PBMC (approximately 2x105 monocytes) were plated per well, in a 24-well plate containing coverslips, in a final volume of 500μl of RPMI medium supplemented by 2% FBS, 2mM of L-GLU and 100 μg/ml of PEES for 2h at 37°C. After 2 hrs, non-adherent cells were gently washed 2 times with warm PBS and then incubated with 1ml of RPMI medium supplemented by 10% FBS, 2mM of L-GLU, 100 μg/mL of PEES and 100ng of GM-CSF (PrepoTech). After 3 days, half of the medium volume was removed and completed with another 500 μl of RPMI medium supplemented by 10% FBS, 2mM of L-GLU, 100μg /ml of PEES and 100ng of GM-CSF. On day 6 the cells were treated with 10ug/ml of N-glycolyl-MDP for another 24 hrs.Primary monocytes were obtained from peripheral blood monocytes from a healthy volunteer and two members of the studied family: the father and one of the twin girls . The bloAfter that, coverslips with cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) solution for 24 hrs and then 2% paraformaldehyde solution for another 48h. Then the coverslips were submitted to the immunofluorescence labeling protocol, which consists of a step of 30 minutes in a blocking solution: PBS supplemented by 5% normal goat serum (NGS) (Life technologies ref: PCN5000), 2% BSA (Sigma ref. A9418) and 0.1% triton X-100 . This step was followed by a 1-hr incubation with the primary antibodies against LRRK2 (AB133474) at a dilution of 1:50 and against NOD2 (MA116611) at a dilution of 1:100. After the incubation time with the primary antibodies, the coverslips were washed 3 times in PBS. Then the coverslips were incubated with the secondary antibodies, Alexa 633 Goat anti-Rabbit (ThermoFisher Scientific) (1:500) and Alexa 546 Goat anti-Mouse (ThermoFisher Scientific) (1:400) for 30 minutes. After incubation with the secondary antibodies, coverslips were incubated with DAPI for 2 minutes and then washed 3 times in PBS. Coverslips were mounted on glass slides with ProLong Gold Antifade Mountant (ThermoFisher Scientific) and sealed with Permount Mounting Medium (Fisher Scientific). Images were obtained by confocal microscopy. Colocalization between LRRK2 and NOD2 was measured from 40 cells by LAS X Software (Leica).6 LRRK2 WT, DM or KO RAW 264.7 cells were suspended in 100μl of a solution (buffer R) containing 10μg of pcDNA3.1+/C-(K)DYK-NOD2, pcDNA3.1+/C-(K)DYK-NOD2 R702W or empty vector control, 3μg of NF-κB firefly-Luc plasmid, and 1μg of Renilla-Luc plasmid pRL-TK. Cells were transfected by electroporation using the Neon Transfection System (Thermo Fisher Scientific) at 1,680V for 20 ms and 1 pulse. Cells from each transfection reaction were plated in a 24-well plate with 1×105 cells per well. Twenty-four hours after electroporation, cells were treated with 10μg/ml of N-glycolyl MDP for another 24 hours or left untreated; following this, cell lysates were prepared by using Passive Lysis Buffer (Promega). Luciferase activity was determined from a 20-μL cell lysates and measured on the microplate reader. Firefly luciferase activity was normalized to Renilla luciferase activity.Luciferase reporter assays were performed using Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Briefly, 1×10For cytokine and chemokine measurements, twenty-four hours after electroporation cells were incubated with or without BCG-Russia (MOI 10:1) for another 24 hours. Cell culture supernatants were collected and centrifuged to remove debris. Cytokines and Chemokines were detected using Milliplex Map  multiplex magnetic bead-based antibody detection kits according to the manufacturer’s instructions. A 6-plex premixed kit was used, which included analytes IL-1β, IL-6, IL-10, IL-12 (p40), MCP-1 and TNF. Assay was read using xPONENT 3.1 acquisition software and MAGPIX instrument . Data was analyzed using Mulliplex Analyst software v4.2 (EMD Millipore) and presented as picogram of cytokine per milliliter of supernatants (pg/ml).www.graphpad.com). Kinetics of ROS production were analyzed by comparing the mutant cells with the wild-type cells at each timepoint using two-way ANOVA with Bonferroni correction. In apoptosis, NF-κB activation and cytokine secretion assays, one-way ANOVA and post hoc t test with Bonferroni correction was used to compare means between two groups. Co-localization analysis was done using one-way ANOVA, comparing each condition with the control using Kruskal-Wallis test. Adjusted P < 0.05 was used as significance threshold, which is represented by asterisks in the figures. Data visualization for bar plots and box plots was done using ggplot2 package in R version 3.6.3 (https://www.R-project.org/" https://www.R-project.org/).Statistical analysis was conducted using GraphPad Prism 5 . Then, seven different filtering approaches were applied (approaches #1 to #7). These filtering steps were based i) on the variant frequencies in public databases, ii) on the model of inheritance and iii) on the age-at-diagnosis of the leprosy-affected family members. Specifically, recessive (#1 to #4) and dominant (#5 to #7) models were tested based on the presence of the variant in all affected family members (#1 and #5), only in the cases younger than 30 years (#2 and #6) and only in the early-onset twins with less than 2 years . In the pedigree, men and women are represented by boxes and circles, respectively. Leprosy patients, regardless of the subtype, are indicated by filled symbols. Monozygocity is represented by a triangle. The number zero in blue represents the reference allele and the number one in red corresponds to the variant. The sample ID is the same as (TIF)Click here for additional data file.S2 FigLRRK2 missense variants found in the family were rs2256408 (R50H), rs7308720 (N551K), rs78365431 (Q1111H), rs7133914 (R1398H), rs11564148 (S1647T) and rs3761863 (M2397T). Four missense variants were detected in NOD2, which were rs2066842 (P268S), rs104895438 (A612T), rs2066844 (R702W) and rs5743278 (A725G). Among these variants, LRKK2 N551K and R1398H passed filtering approaches #2 and #3, respectively; while NOD2 R702W passed filtering approach #6 Click here for additional data file.S3 Fig(A) First and second components are plotted on the x and y axis, respectively. (B) First and third components are plotted on the x and y axis, respectively.Each dot represents an individual, including the six family members from the present study and 2,504 unrelated individuals from the 1000 Genomes Consortium representing the five super populations: African/African American (AFR), Admixed American/Latin (AMR), East Asian (EAS), European (EUR) and South Asian (SAS). (TIF)Click here for additional data file.S4 Fig(A) Kinetics of reactive oxygen species (ROS) production upon BCG challenge in RAW cells expressing wild-type (WT), 1398R/H heterozygous (HET) or 1398H/H homozygous (HOM) LRRK2 proteins. Box plots presents the results at each time point showing the ROS measurement on the y axis and the genotype groups on the x axis. (B) Effect of LRRK2 WT, R1398H HET and HOM on apoptosis in response to BCG. Percentage of total apoptotic cells, including cells with early and late apoptosis, was derived for non-infected (left box plot) and BCG-infected cells (right box plot). (A-B) LRRK2 R1398H HET and HOM cells were compared to WT using (A) two-way ANOVA  and (B) one-way ANOVA , followed by post-hoc t test with Bonferroni correction. Pairwise comparisons between WT and HET or HOM are represented by the blue and red lines on top of the box plots, respectively. A linear regression model was used to analyze the dose-dependent effect of LRRK2 R1398H minor allele on: (A) ROS production by time point and (B) apoptosis by infection status (WT→HET→HOM). Results from the trend tests are shown in black with the regression lines presented as dotted lines and the P-values shown below the box plots. **** P < 0.0001; ** 0.001 ≤ P < 0.01; ns: non-significant. BCG: Bacillus Calmette–Guérin.(TIF)Click here for additional data file.S5 FigLRRK2 Wild-type (WT), CRISPR/Cas-edited LRRK2 double-mutant  and LRRK2 knock-out (KO) RAW264.7 cell lines were transfected with plasmids expressing NOD2 WT, NOD2 mutant (R702W) or an empty vector [pcDNA3.1+/C-(K)DYK] as a control. Twenty-four hours post-transfection, cells were left uninfected or infected with live bacillus Calmette–Guérin (BCG)-Russia (MOI 10:1) for another 24 hours. Cells were then harvested, stained with Annexin V/ZA-FVD, and analyzed by flow cytometry for apoptosis. The illustrated result is a representative of two independent experiments with similar results (done in triplicates).(TIF)Click here for additional data file.S6 Fig(A) NOD2 WT and (B) NOD2 R702W. (C) LRRK2 KO cells (top panel) and untransfected LRRK2 WT cells (bottom panel) were used as a negative control for antibody specificity. (A-D) RAW264.7 cell lines with the three LRRK2 genotypes were transfected with NOD2 plasmid and a LRRK2 WT cell line was kept untransfected. Twenty-four hours after electroporation, the transfected and untransfected cells were treated with or without 10 μg/mL of N-glycolyl MDP for another 24 hours. Cells were fixed with 4% paraformaldehyde, permeabilized and double stained for LRRK2 and NOD2 with rabbit anti-LRRK2 (1:500) and mouse anti-FLAG (1:250) antibodies. Nuclei were stained with DAPI. Images were obtained by confocal microscopy. (D) Colocalization between LRRK2 and NOD2 was measured from 25–30 cells by Zeiss 2012 ZEN confocal software.Colocalization of LRRK2 wild-type (WT) and CRISPR/Cas-edited LRRK2 double-mutant  in cells transfected with plasmids expressing (TIF)Click here for additional data file.S7 FigLRRK2 Wild-type (WT), CRISPR/Cas-edited LRRK2 double-mutant  and LRRK2 knock-out (KO) RAW264.7 cell lines were transfected with plasmids expressing NOD2 WT, NOD2 mutant (R702W) or an empty vector [pcDNA3.1+/C-(K)DYK] as a control. Twenty-four hours post-transfection, cells were left untreated or treated with different concentrations of N-glycolyl MDP as indicated, for another 24 hours. Cell lysates were prepared and the phosphorylation of RIP2 (p-RIP2) in the transfected cell lines was analyzed by immunoblotting with a specific antibody against RIP2 when phosphorylated at Ser 176. GAPDH was used as a loading control.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Method(DOCX)Click here for additional data file.


Pathological cardiac hypertrophy involves multiple regulators and several signal transduction pathways. Currently, the mechanisms of it are not well understood. Differentially expressed in FDCP 6 homolog (DEF6) was reported to participate in immunity, bone remodeling, and cancers. The effects of DEF6 on pathological cardiac hypertrophy, however, have not yet been fully characterized. We initially determined the expression profile of DEF6 and found that DEF6 was upregulated in hypertrophic hearts and cardiomyocytes. Our in vivo results revealed that DEF6 deficiency in mice alleviated transverse aortic constriction (TAC)-induced cardiac hypertrophy, fibrosis, dilation and dysfunction of left ventricle. Conversely, cardiomyocyte-specific DEF6-overexpression aggravated the hypertrophic phenotype in mice under chronic pressure overload. Similar to the animal experiments, the in vitro data showed that adenovirus-mediated knockdown of DEF6 remarkably inhibited phenylephrine (PE)-induced cardiomyocyte hypertrophy, whereas DEF6 overexpression exerted the opposite effects. Mechanistically, exploration of the signal pathways showed that the mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2)-extracellular signal-regulated kinase 1/2 (ERK1/2) cascade might be involved in the prohypertrophic effect of DEF6. Coimmunoprecipitation and GST (glutathione S-transferase) pulldown analyses demonstrated that DEF6 can directly interact with small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), and the Rac1 activity assay revealed that the activity of Rac1 is altered with DEF6 expression in TAC-cardiac hypertrophy and PE-triggered cardiomyocyte hypertrophy. In the end, western blot and rescue experiments using Rac1 inhibitor NSC23766 and the constitutively active mutant Rac1(G12V) verified the requirement of Rac1 and MEK1/2-ERK1/2 activation for DEF6-mediated pathological cardiac hypertrophy. Our study substantiates that DEF6 acts as a deleterious regulator of cardiac hypertrophy by activating the Rac1 and MEK1/2-ERK1/2 signaling pathways, and suggests that DEF6 may be a potential treatment target for heart failure. Heart failure, the terminal stage of heart disease, limits the quality of life and imposes a major global health burden. Heart failure occurs since the heart is incapable of maintaining normal ventricular ejection to meet an individual’s needs even at normal filling pressures. Cardiac hypertrophy as a compensatory remodeling to pressure overload and other hypertrophic stimuli is a major risk factor of heart failure . Hallmar2+ signaling, and T-cell adhesion [Differentially expressed in FDCP 6 homolog (Def6), which contains 631 amino acids, is mapped to human chromosome 6p21.31 , 5. Becaadhesion –12. Manyadhesion –15. DEF6adhesion . Additioadhesion . Furtheradhesion –20. Plenadhesion , and conadhesion , we sougWe conducted a suite of experiments to explore this possibility. First, remarkably increased expression levels of DEF6 were observed in the hypertrophic hearts of mice processed with TAC and in cardiomyocytes administered with phenylephrine (PE). Then, DEF6 loss- and gain-of-function assays conducted both in vitro and in vivo revealed a deleterious regulatory role for DEF6 in pathological cardiac hypertrophy. In terms of mechanism, RAC1 and the MEK-ERK signaling cascade activation largely contributed to DEF6’s deleterious role in cardiac hypertrophy. In conclusion, activation of Rac1 and MEK-ERK signaling cascades are some of the mechanisms by which DEF6 accelerates the development of pathological cardiac hypertrophy.Abundant expression of DEF6, which plays a crucial role in adaptive and innate immunity, has been described in the immune system, especially in T cells , 16. TheThe above findings prompted the investigation into the impact of DEF6 on cardiac hypertrophy. To achieve this goal, we constructed a global DEF6-knockout (KO) mouse strain Fig. , and WesWe then subjected the KO and WT mice to TAC or sham. Four weeks later, the hypertrophic responses of the indicated groups were analyzed. At the basal level, DEF6 deficiency caused no apparent abnormalities; however, compared with the control condition, it exerted a remarkable ability to protect against pressure overload-induced increases in heart mass and cardiomyocyte size, as indicated by the decreased heart weight (HW), HW/body weight (BW), HW/tibia length (TL), gross heart sizes, and cardiomyocyte cross-sectional area values in KO mice Fig. C, D. TheWe explored the impact of DEF6 ablation on pressure overload-induced cardiac fibrosis using PSR staining. Four weeks after TAC, KO mice exhibited lower LV collagen volumes than WT mice, indicating reduced cardiac fibrosis Fig. . These cTo explore the overall auxo-action of DEF6 in pathological cardiac hypertrophy, cardiomyocyte-specific DEF6-overexpressing mice (AAV9-DEF6) were constructed via tail vein injection of an AAV9 vector containing the DEF6 gene controlled by the cardiomyocyte-specific promoter cTNT. Mice injected with an empty AAV9 vector (AAV9-vector) were used as controls. The cardiomyocyte-specific overexpression of DEF6 was confirmed by Western blot Fig. .Fig. 3OvDEF6-overexpressing (AAV9-DEF6) and control (AAV9-vector) mice underwent TAC or sham surgery and were analyzed 4 weeks later. Under basal conditions (sham), AAV9-DEF6 mice displayed no differences from AAV9-vector mice with regard to cardiac structure and contractile function. However, DEF6 overexpression significantly deteriorated pressure overload-induced cardiac hypertrophy and pulmonary congestion, as shown by increments in HW, HW/BW, LW/BW, HW/TL Fig. , gross hCoinciding with the progression of cardiac hypertrophy and fibrosis, DEF6 overexpression remarkably enhanced the mRNA levels of the cardiac hypertrophic and fibrotic markers Fig. D, I. ConThe above in vivo data prompted further exploration about whether the hypertrophic effect of DEF6 acts mainly on cardiomyocytes. Therefore, further assays were conducted on cultured NRCMs that were infected with adenoviral vector containing short hairpin RNA against DEF6 (AdshDEF6) to knock down DEF6 or adenoviral vector containing DEF6 (AdDEF6) to overexpress this gene. In addition, an adenovirus encoding empty short hairpin RNA (AdshRNA) and an adenovirus encoding the empty vector (Advector) were used as controls. The adenovirus-mediated expression modification of DEF6 was verified by Western blot Fig. A, D. AftMitogen-activated protein kinase (MAPK) signaling is crucial for cardiac hypertrophy provoked by pressure overload or other pathological stimulation , 27–29. As an upstream activator of MEK-ERK signaling, RAC1 has also been implicated in pathological cardiac hypertrophy and activated by DEF6 in the immune system and cell morphology modification , 30, 31.We initially explored whether DEF6 and RAC1 directly act upon each other. Flag-tagged DEF6 and HA-tagged RCA1 were co-translated in HEK293T cells for Co-IP experiment. As shown in Fig. Then, we assessed the influence of DEF6 expression alteration on the activities of Rac1 after hypertrophic stimulations. Both in vivo and in vitro data exhibited that DEF6 knockdown reduced the expression of activated Rac1 and DEF6 overexpression remarkably enhanced activities of Rac1 in response to TAC or PE treatment Fig. . Those fMany previous studies have confirmed the involvement of Rac1-MEK-ERK1/2 signaling cascade in cardiac hypertrophy , 32. TheOn the other hand, AdRac1(G12V) or Adcontrol was co-transfected with AdshDEF6 or AdshRNA into NRCMs before PE treatment (24 h). As expected, the Western blotting data also suggested that activation of MEK1/2 and ERK1/2 triggered by PE was inhibited by DEF6 knockdown but this ameliorative effect reversed by constitutively Rac1 activation Fig. . MeanwhiThe high incidence of heart failure necessitates illumination of the underlying mechanisms of pathological cardiac hypertrophy to identify useful therapeutic targets. In this context, the current study started from an observation of elevated expression levels of DEF6 in the hypertrophic myocardium and in hypertrophic cardiomyocytes. Our further experiments uncovered that DEF6 aggravated pathological hypertrophy, contractile dysfunction, fibrosis, and cardiomyocyte hypertrophy induced by pro-hypertrophic stimuli, but did not work under physiological conditions. A subsequent mechanistic study revealed that DEF6 execute its prohypertrophic effect by directly interacting with and activating RAC1, thereby activating downstream MEK-ERK1/2 signaling. Thus, we first demonstrated that the expression level of DEF6 is sensitive to the severity of pathological cardiac hypertrophy and DEF6 can remarkably exacerbates the pressure overload-induced cardiac hypertrophy.The significantly elevated expression levels of DEF6 were observed in hypertrophic heart and cardiomyocytes, however, the mechanism of this expression alteration induced by hypertrophic stimuli remains unknown. Plenty of evidence has demonstrated that a number of GEFs can be activated by hypertrophic stimuli  through GPCRs, receptor tyrosine kinases, and other membrane receptors to activate Rac1 and eventually lead to cardiac hypertrophy –35. SincMechanistically, our research reveals that MAPK signaling participates in the effects of DEF6 on cardiac hypertrophy. MAPK signaling pathway, which includes three members, ERKs, JNKs, and p38, plays an extraordinarily vital role in cardiac hypertrophy provoked by overload and other pathological stimuli , 27–29. Ras-related C3 botulinum toxin substrate GTPases  belongs to the Rho small GTPase family. There are three Rac isoforms: Rac1 is widely expressed; Rac2 is expressed specifically in hematopoietic cells; and Rac3 has been reported to be expressed in the brain, lungs, liver, and pancreas . NumerouPrevious study on DEF6 mainly focused on its function in immunity and cancers. However, our current research first reveals the deleterious regulatory effects of DEF6 in cardiac hypertrophy through the Rac1-MEK1/2-ERK1/2 pathways Fig. . It reveAnimals were applied to experiments with the approval of the Ethics Committee of Shengjing Hospital of China Medical University (NO. 2022PS458K). Animal experiments in this study were performed according to the Guide for the Care and Use of Laboratory Animals.http://chopchop.cbu.uib.no/) was utilized to design guiding sequences targeting the second exon of the mouse DEF6 gene (guideRNA1 target site: GGACACCGGGCCATCATCGTCATCC-CGG), and pUC57-sgRNA  was used to generate the DEF6-sgRNA expression vector. The in vitro transcription products of the DEF6-sgRNA expression vector and Cas9 expression vector pST1374-CAS9  were purified and mixed. In the next step, fertilized eggs collected from C57BL/6 mice were microinjected with this mixture using a FemtoJet 5247 microinjection system and subsequently transplanted into pseudopregnant foster mothers' oviducts. After 19-21 days, F0-generation mice were born. Two weeks later, genomic samples were obtained from the toes of F0 mice, and the genotypes were tested using the following pair of primers: F : 5′-CCGGATGCAGAAAAGCAACC-3′, R : 5′-CGCGGGAGCTAAGAGAGATG-3′.Production of DEF6-knockout (KO) mice: An online CRISPR design tool  of the AAV9-DEF6 or AAV9-vector. The relevant primers: DEF6 F: 5′-CACGCTTAACTAGCTAGCCACCATGGCCCTGCGCAAGGAG-3′,DEF6 R: 5′-AGCTCCGCTTCCACGCGTCTTATCGTCGTCATCCTTGTAATCATTCCCTGGTGCTGGATCCAG-3′.AAV9-DEF6 and AAV9 control vectors (AAV9-vector) were constructed as described previously . BrieflyIn this study, we performed TAC surgery to generate pressure overload-provoked model of cardiac hypertrophy as described previously . Nine- tFor echocardiographic analysis, animals were anesthetized using isoflurane (1.5–2%). Cardiac function evaluation and hemodynamic analysis were performed by transthoracic echocardiography with an Animal Ultrasound Imaging System  equipped with a 30 MHz probe (MS400). The left ventricular (LV) end-diastolic diameter (LVEDd), LV end-systolic diameter (LVESd), and LV posterior wall diastolic thickness (LVPWd) were measured for three beats from each projection and averaged. Cardiac function was determined by calculating the LV fractional shortening (FS) and LV ejection fraction (EF). Echocardiography was conducted by investigators blinded to the studyThe mouse hearts were excised and stopped in the diastolic period with a 10% KCl solution after 4 weeks of sham or AB surgery and then processed by 10% formalin and embedded in paraffin. For morphological analysis and collagen deposition detection, the paraffin blocks were cut into five- micrometer-thick slices, which were then stained with hematoxylin  & eosin  (H&E) or picrosirius red . Image-Pro Plus 6.0 (IPP 6.0) was used for image analysis.6 cells/well as previously described [The rat full-length DEF6 cDNA or the constitutively active mouse Rac1(G12V) mutant was cloned into a replication-deficient adenoviral vector that contained a cytomegalovirus promoter ((AdDEF6) or AdRac1(G12V)). The control was an empty adenoviral vector (Advector). Adenoviral constructs contains short hairpin RNA against rat DEF6 (AdshDEF6) was used to silence DEF6 gene expression, and AdshRNA was utilized as a nontargeting control. Then, we isolated NRCMs from the hearts of neonatal sprague-dawley (SD) rats, NRCMs were seeded in culture plates coated with gelatin at 1.8×10escribed . The carescribed . The relAdDEF6-Rat-F: GGCTAGCGATATCGGATCCGCCACCATGGCCCTGCGCAAGGAGAdDEF6-Rat -R: CGTCCTTGTAATCACTAGTATTTTCTGGCGCTGCATCCAGAdshDEF6-Rat-F: CCGGGGTCCTTCACATCCCTCATGACTCGAGTCATGAGGGATGTGAAGGACCTTTTTGAdshDEF6-Rat-R: AATTCAAAAAGGTCCTTCACATCCCTCATGACTCGAGTCATGAGGGATGTGAAGGACCAdRac1(G12V)-Mouse-F: GGCTAGCGATATCGGATCCGCCACCATGCAGGCCATCAAGTGTGTGGTGGTGGGAGACGTAGCTGTTGGAdRac1(G12V)-Mouse-R: CGTCCTTGTAATCACTAGTCAACAGCAGGCATTTTCTCTTCCTCAfter stimulation with PE, the NRCM surface area was measured by immunofluorescence. The NRCMs were processed in 4% formaldehyde for 30 min and then washed more than three times. After that, the cardiomyocytes were processed with 0.2% Triton X-100 in PBS for 5 min, incubated with α-actinin antibody  and then labeled with a fluorescent secondary antibody . The nucleus of the cells was stained with DAPI. Image analysis of NRCM surface area was conducted with IPP 6.0.Briefly, cultured HEK293T cells were co-transfected with plasmids expressing the fusion proteins and then lysed. The supernatant obtained from the lysates was incubated with Protein G-conjugated agarose beads  and corresponding antibodies against the label (anti-HA or anti-Flag) overnight at temperature of 4 °C. After washing by IP buffer, the proteins were eluted and then collected from the agarose beads. Finally, the proteins were tested and analyzed by Western blotting. The primers of the involved fusion proteins are presented in Supplementary Table Briefly, HEK293T cells were transfected with plasmids expressing the fusion proteins GST-HA-Rac1, GST-HA-DEF6, Flag-Rac1, and Flag-DEF6 and then lysed. After purification, the fusion proteins GST-HA-Rac1 and Flag-DEF6 or Flag-Rac1 and GST-HA-DEF6 were mixed and then incubated overnight at 4 °C. The immunocomplex was washed and collected with the corresponding buffers . Finally, the proteins were tested and analyzed by Western blotting.Rac1 activation “pull-down” assay (Upstate Biotechnology) was used to assess the activities of Rac1 . AccordiTM XRS+ system (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. Information on the antibodies is listed in Supplementary Table Total proteins extraction were described as a previous study . DetectiTRIzol reagent  was applied for total RNA extraction from cardiac tissue or NRCMs. Two micrograms of RNA were reverse-transcribed into first-strand cDNA using a Transcriptor First Strand cDNA Synthesis Kit , and PCR amplification was performed using SYBR Green PCR Master Mix . The target gene expression levels were normalized against GAPDH. The primers of the involved genes are presented in Supplementary Table n ≥ 6 were used for pathology studies or functional analysis. In the cell experiments, at least 3 independent replications were performed. All data were subjected to the normality test. The means of 2-group samples were compared using two-tailed Student’s t tests (n ≥ 5) or Mann–Whitney U test (n ≤ 4) . One-way ANOVA with the Bonferroni test  or the Tamhane T2 test  and Kruskal–Wallis test  were used to test differences among more than 2 groups. P < 0.05 was considered to indicate statistical significance.In our study, all data are described as the mean ± standard deviation (SD), and visualized using GraphPad Prism 8.0. Data analysis were performed using SPSS (version 26.0). In animal experiments, SUPPLEMENTARY FIGURE AND TABLE LEGENDSFigure Supplementary1Figure Supplementary2Supplementary table 1, 2, 3Original Data FileOriginal Data FileReproducibility checklist


Triple negative breast cancer (TNBC) is an aggressive clinical tumor, accounting for about 25% of breast cancer (BC) related deaths. Chemotherapy is the only therapeutic option to treat TNBC, hence a detailed understanding of the biology and its categorization is required. To investigate the clinical relevance of BCL11A in TNBC subtype, we focused on gene and protein expression and its mutational status in a large cohort of this molecular subtype.Gene expression profiling of BCL11A and its isoforms  has been determined in Luminal A, Luminal B, HER2-enriched and TNBC subtypes. BCL11A protein expression has been analyzed by immunohistochemistry (IHC) and its mutational status by Sanger sequencing.P = 0.03) and AR downregulation , as well as a higher proliferative index in TNBC cells. BCL11A-L expression is associated with more aggressive TNBC histological types, such as medullary and metaplastic carcinoma.In our study, BCL11A was significantly overexpressed in TNBC both at transcriptional and translational levels compared to other BC molecular subtypes. A total of 404 TNBCs were selected and examined showing a high prevalence of BCL11A-XL (37.3%) and BCL11A-L (31.4%) isoform expression in TNBC, associated with a 26% of BCL11A protein expression levels. BCL11A protein expression predicts scarce LIV (HR = 0.52; 95% CI, 0.29–0.92, Our finding showed that BCL11A protein expression acts as an unfavorable prognostic factor in TNBC patients, especially in non luminal TNBCs subgroups. These results may yield a better treatment strategy by providing a new parameter for TNBC classification.The online version contains supplementary material available at 10.1007/s00432-022-04301-w. Triple negative breast cancer, which accounts for 10–20% of all invasive breast cancer (BC) subtypes, is characterized by the lack of immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), and HER2 and/or HER2 gene amplification. TNBC is most prevalent in women aged < 50 years and shows aggressive clinical behavior   in human B-cell non-Hodgkin’s lymphomas  tumor blocks from surgical specimens. Three experienced pathologists independently reviewed all cases. Histologic subtyping was performed according to current WHO classification  recommendations for immunohistochemical testing of hormone receptors in BC ]; nine Luminal B ; eight HER2-enriched ; sixty-one TNBC . The ordinal Allred scoring system was used to assess BCL11A immunostaining quantity in tumor cells, based on intensity  and percentage of stained cells ; the combination of intensity + percentage gives an Allred score between 0 and 8. Tumor with Allred score > 2 was defined as positive for BCL11A expression . The RNA integrity was assessed by the RNA Integrity Number (RIN) using the Agilent RNA 6000 Nano Kit on the BioAnalyzer 2100 .Genomic DNA was obtained from neoplastic tissue, and total RNA was obtained from neoplastic and non-neoplastic specimens. Nucleic acids were extracted using the QIAmp DNA Mini Kit and miRNeasy Mini Kit . The quantity and the quality of nucleic acids were assessed using Nanodrop ND1000 . The RNA quantity was evaluated by QubitGene expression profiles of BCL11A were analyzed in all BC molecular intrinsic subtypes. Two µg of total RNA were reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit . BCL11A encodes three mRNA variants and each isoform of BCL11A has specific expression patterns. Primers for BCL11A , the isoforms BCL11A-S (Hs01093198_m1), BCL11A-L (Hs01093199-m1), BCL11A-XL (Hs00250581_s1) and 18S rRNA  human genes were chosen using Assays-on-Demand™-Products (Applied Biosystems). Neoplastic and non-neoplastic tissues were analyzed by quantitative real time PCR (qRT-PCR) using the ABI 7900HT Sequence Detection System (Applied Biosystems)  of the BCL11A-XL protein, where several most common missense mutations were identified in patients affected by autism, intelligence disabilities  frequencies. Chi-squared or Fisher exact tests were used to detect any statistical differences in the comparison of qualitative variables between down and up regulation of BCL11A gene or low and high protein expression. Logistic regression analysis was performed to assess the relationship between BCL11A upregulation or high protein expression and clinicopathological TNBC characteristics. Survival rate differences between down and upregulation or low and high protein expression were detected with Kaplan–Meier analysis. P-value less than 0.05 was considered statistically significant. Stata 17  statistical software was used for every statistical computation.P < 0.05)  significantly correlated with TNBC pathology . No significant differences were shown between HER2-enriched and other molecular intrinsic subtypes and NBT. BCL11A-XL was overexpressed in TNBC vs Luminal A and B , whereas BCL11A-L and BCL11A-S were overexpressed in TNBC vs Luminal B   and B (P: 0.002) while a significant BCL11A downregulation was present in Luminal A and B compared to NBT (P: 0.002 and Focusing on BCL11A protein expression profile we performed IHC on the 87 primary BC molecular subtypes used for gene expression profile. A BCL11A protein overexpression was found in 16 out of 61 26.2%) TNBCs, in 4 out of 12 (33.3%) NBT, whereas no protein expression was detected in Luminal A (nine cases), Luminal B (nine cases) and HER2-enriched (eight cases) tumors. The tumors immunostained positively showed high mRNA levels compared with those with negative immunostaining. Immunohistochemistry analysis of BCL11A in an independent validation cohort of 343 TNBC samples, confirmed that BCL11A protein expression agreed with the first cohort examined: 79 BCL11A-overexpressing TNBCs out of 343 (23.0%). Figure 6.2% TNBCTable P: 0.05). BCL11A-L mRNA overexpression was associated with some histological types such as medullary and metaplastic carcinomas (P: 0.04)  and lymphovascular invasion (LVI) were detected in 52.9 and 25.5%, respectively. AR expression was found in 30.9% cases. A total of 8 patients out of 61 (13.1%) died. The clinicopathological data of the validation cohort is reported in Table S1. TNBCs with BCL11A and BCL11A-L mRNA overexpression were more frequently associated with AR expression < 10% (BCL11A protein expression was associated with ki-67 > 35% (P: 0.004), and with absence of LIV and AR downregulation  (Table P < 0.001) are independent prognostic factors for TNBCs overexpressing mRNA BCL11A or its isoforms  and AR expression  are independent prognostic factors for overall survival (OS) in BCL11A-L transcripts overexpressing TNBCs. High protein expression levels of BCL11A  and AR  are independent prognostic factors for TNBCs showing high BCL11A protein expression levels , was detected in all BC molecular subtypes. CC genotype was prevalent in all BC molecular subtypes (60–62.5%). In TNBC subtype, no TT homozygous were present while 40% of them showed CT genotype.BCL11A is a proto-oncogene which maps on chromosome 2p16. Alternative splicing generates at least three most common BCL11A transcripts, BCL11A-XL, BCL11A-L and BCL11A-S containing differing numbers of C-terminal C2H2 finger motifs, and showing low expression in normal human tissue, except in fetal liver, hematopoietic tissue and brain  significantly correlated with TNBC pathology. Additionally, tumors positively immunostained showed high BCL11A mRNA levels compared with those with negative immunostaining. Our results confirmed recent data correlating BCL11A overexpression and TNBC subtype . Among TNBC histotypes, the medullary type of pattern is often associated with variable immunohistochemical expression of basal markers  gains of BCL11A had a higher rate of relapse and metastasis and a lower rate of survival Below is the link to the electronic supplementary material.


Tandem repeat expansions are enriched in autism spectrum disorder, including CTG expansion in the DMPK gene that underlines myotonic muscular dystrophy type 1. Although the clinical connection of autism to myotonic dystrophy is corroborated, the molecular links remained unknown. Here, we show a mechanistic path of autism via repeat expansion in myotonic dystrophy. We found that inhibition of muscleblind-like (MBNL) splicing factors by expanded CUG RNAs alerts the splicing of autism-risk genes during brain development especially a class of autism-relevant microexons. To provide in vivo evidence that the CTG expansion and MBNL inhibition axis leads to the presentation of autistic traits, we demonstrate that CTG expansion and MBNL-null mouse models recapitulate autism-relevant mis-splicing profiles and demonstrate social deficits. Our findings indicate that DMPK CTG expansion-associated autism arises from developmental mis-splicing. Understanding this pathomechanistic connection provides an opportunity for greater in-depth investigations of mechanistic threads in autism. ASD affects 1 in 36 children, and more than 95% of them have at least one additional physical or mental health condition3. Despite hundreds of genes known to confer risk for ASD, the molecular mechanisms explaining ASD and comorbid condition manifestations remain elusive4.Autism spectrum disorder (ASD) is a genetically and clinically heterogeneous neurodevelopmental condition that affects communication and social interactions with restricted interests and repetitive behaviorsexp) in the 3’ untranslated region (3’UTR) of the DMPK gene6. A recent large-scale newborn genetic screen estimated the prevalence of DMPK CTGexp mutations approximately 1:2,100 newborns, with a relatively high detection rate of pre-mutations that increase the risk of germline expansions in the subsequent generation8. The DMPK 3’UTR CTGexp mutation causes myotonic dystrophy type 1 (DM1), one of the most variable genetic disorders with onset times that span from in utero to late adulthood, with highly variable symptom severity and multisystem involvement10. Previous studies reported comorbidity of DM1 and ASD, and showed that the presence of ASD inversely correlates with DM1 age of onset15. However, a molecular mechanism explaining the manifestation of ASD in DM1 families is unknown.Recently, large-scale whole-genome sequencing studies identified tandem repeat mutations that contribute to ~4% of ASD risk. One the most recurrent mutations include a CTG expansion (CTGexp RNAs provide a large array of high-affinity binding sites for muscleblind-like (MBNL) RNA-binding proteins resulting in MBNL inhibition and formation of biomolecular condensates known as RNA foci22. MBNL proteins, including MBNL1 and MBNL2, are trans-acting factors regulating alternative splicing (AS) during organism development23. MBNL inhibition leads to adult-to-fetal reversion of the AS program resulting in specific DM1 clinical symptoms, including myotonia26.In DM1 neurons and muscles, DMPK 3’UTR CUG29. Neuronal microexons (miEs), which are 3 to ~30 nucleotides (nt) in length, are estimated to be mis-regulated in one-third of idiopathic ASD brains30. MiEs play an essential role in nervous system development and function by encoding post-translational modification sites and modulating protein-protein interaction networks32. Importantly, alterations that recapitulate neuronal miE mis-splicing can lead to ASD-like phenotypes in mice, including social avoidance34. Although numerous DM1 mouse models have been utilized to investigate typical DM1 manifestations, the ASD traits have not been elucidated36. A molecular mechanism explaining the manifestation of ASD in DM1 families could open both diagnostic and therapeutic avenues.Developmental mis-splicing is not only a feature of DM1 but also ASDDmpk 3’UTR CTGexp knock-in (KI), as well as a Mbnl knock-out (KO) mouse models. Our results provide insights into the molecular mechanism underlying DM1-associated ASD where developmental mis-splicing of ASD-linked genes arises by loss of MBNL activity due to CUG repeat expansions.Here, we performed an integrative transcriptomic and genomic analysis of RNA mis-splicing in human DM1 and ASD brains as well as multiple DM1 mouse models. For greater in-depth analysis, we focused on the mis-splicing of neuronal miEs in high-confidence ASD-risk genes directly regulated by MBNL proteins during human and mouse brain development. Finally, we assessed social interaction deficits in specific DM1 mouse models, a 37. To test the hypothesis that the DMPK 3’UTR CTGexp mutation leads to mis-splicing of ASD-risk genes, we analyzed human prefrontal cortex  RNA-seq data generated from DM1 (unknown ASD status) and unaffected control samples (Supplementary Table 1)38. For differential AS analysis, we computed the change of percent spliced in (DPSI) for skipped exons (SE), mutually exclusive exons (MXE), alternative 5′ and 3′ splice sites (A5SS and A3SS) and retained introns (RI). Of all identified AS events (100%) and genes (100%) in DM1 cortex splicing analysis, 1% of AS events met our mis-splicing criteria in the total pool of 7% of mis-spliced genes (−11) database (−6), ‘strong candidate’  and ‘suggestive evidence’  gene categories. Our analysis also revealed a significant enrichment of high-confidence ASD-risk genes identified in two large Autism Speaks MSSNG-based whole-genome sequencing studies: MSSNG-201739  and MSSNG-202240   since a previous study demonstrated the strongest positive correlation between those CTG sizes and general mis-splicing level in DM1 prefrontal cortexes38. This analysis revealed a significant positive correlation between CTGexp size and the number of mis-spliced events in ASD-risk genes from the SFARI  and MSSNG-2017 study   versus wild-type (WT) frontal cortex samples43. The Mbnl cDKO mice bypass the embryonic lethality of constitutive Mbnl1−/−; Mbnl2−/− DKO mice and provide a nervous system-specific model where Mbnl1 expression is absent in all tissues while Mbnl2 is lost only in neuronal and glial precursor cells. The Mbnl cDKO is characterized by RNA mis-splicing, altered cortical neuronal and synaptic structures and widespread brain anatomical changes46. In total, 5% of AS events in 13% of detected genes were mis-spliced , MSSNG-2017 and MSSNG-2022  studies  between mouse and human frontal cortex  (The strong correlation between CTG% to 58% . In totact test) .34. We noticed significant disproportional miE mis-spliced events in both Mbnl cDKO and DM1 cortex , as well as 15%, 23% and 35% when they are found within ASD-risk genes from the SFARI , MSSNG-2022 , and MSSNG-2017  studies, respectively. Similarly, the proportion of miE mis-splicing events increased from 2% to 8% in DM1 prefrontal cortex , and to 19%, 17%, and 44% in ASD-risk genes from the SFARI , MSSNG-2022 , and MSSNG-2017  studies, respectively. In total, we identified mis-spliced miE events in 33 genes that are present in both human DM1 and mouse Mbnl cDKO cortex, including evolutionary conserved miEs in high-confidence ASD-risk genes, such as ANK2, TANC2, and DMD  mis-splicing is a hallmark of ASD brains, which can lead to ASD-like behaviors in mice1 cortex . MiEs co and DMD –g.30, we performed comparative in silico modeling of peptides with/without miE-encoded amino acid (aa) sequences to test their potential for protein modulation. This analysis showed that some mis-spliced miEs might modulate internal  or C-terminal  protein structures (Ank2 miE (12 nt) along with the use of a proximal alternative 3′ splice sites (A3SS) results in protein isoform with a TIP aa sequence, whereas miE exclusion promotes distal A3SS usage (15 nt), and results in a protein isoform with a LRSF aa sequence containing a S901 phosphorylation site47 .Since previous studies have shown that miEs can locally modulate protein structure49. Our analysis showed an evolutionarily conserved increase of MBNL2 expression during neonate/P0 to middle childhood/P14 brain development , MSSNG-2017 , and MSSNG-2022  studies , MSSNG-2017 , and MSSNG-2022  studies , constitutive Mbnl2−/− KO (hereafter Mbnl2 KO) and WT mice 51. We performed differential splicing analysis followed by ASD-risk gene enrichment analysis. In agreement with the relatively low embryonic Mbnl1 and Mbnl2 expression levels , MSSNG-2017  and MSSNG-2022  studies in the DM1 brain organoid, including previously identified DMD miE , MSSNG-2017  and MSSNG-2022  studies  versus control51. In total, 5% of AS events in 15% of detected genes were mis-spliced , MSSNG-2017  and MSSNG-2022  studies , MSSNG-2017  and MSSNG-2022  studies were significantly enriched in Mbnl DKD CAD cells  neuronal cell line with siRNA-mediated AD cells . We iden regions .57, we determined YGCYGCY and YGCY(N)0–5YGCY as high affinity MBNL-binding sequences. We performed a genome-wide distribution analysis of intronic MBNL-binding motifs ±500 bp from alternative SE splice sites. We detected a significant enrichment of Mbnl binding sequences in mis-spliced ASD genes in DM1  and Mbnl cDKO  frontal cortex, Mbnl DKD CAD cells  as well as Mbnl2 KO hippocampus   .55. A cluster of CLIP-seq reads indicates an Mbnl2-RNA interaction region. In agreement with previous studies demonstrating that Mbnl binding within the downstream intron of an alternative SE promotes its inclusion58, we identified a Mbnl2-CLIP-seq cluster covering a conserved TGCT(N)3TGCT(N)13–18TGCT/C sequence ~55 bp downstream 5’ splice site in Ank2/ANK2 intron . Since splicing of the Ank2 miE to the A3SS results in mRNA isoforms differing by only 3 nt (as explained earlier), we took advantage of our previously developed Atp2a1 E22 inclusion minigene (Atp2a1-WT) assay for RNA-MBNL interactions56. We deleted an experimentally confirmed MBNL-binding motif within the downstream intron 22 of the mouse Atp2a1-WT minigene (Atp2a1-D) and inserted the 90 bp mouse Ank2 and human ANK2 conserved intronic sequence TGCT(N)3TGCT(N)13–18TGCT/C  and mutated GGCT(N)3TGAT(N)13–18TGTT/C sequences   expression vectors. Atp2a1-Ank2 E22 and Atp2a1-ANK2 E22 were significantly more included than Atp2a1-mutAnk2 and Atp2a1-mutANK2  samples from adult WT hippocampierlined) –g. The dproteins . To supp59. Our differential AS analysis revealed 0.3% of mis-spliced events in 2% of analyzed genes , including ANK2 miE  cell line with siRNA-mediated Srrm3; Srrm4 double knockdowns (hereafter Srrm DKD) versus control60. Our differential AS analysis revealed 2% of mis-spliced events in 7% of analyzed genes . As anticipated, Srrm DKD preferentially altered miE splicing , including miE in ASD-risk genes from the SFARI , MSSNG-2017  and MSSNG-2022  studies . In total, we identified a non-random overlap of 153 mis-spliced AS events between Mbnl DKD CAD and Srrm DKD N2a , including 34 altered miE events . 41% of overlapping mis-spliced miEs showed concordant DPSI changes in Mbnl DKD and Srrm DKD, all of which demonstrated exon exclusion in the transcripts (DPSI < 0)  samples from the PsychENCODE Consortium (Supplementary Table 1)ANK2 miE . PreviouKD cells  and 6e.61. In contrast, MBNL proteins bind to downstream intronic UGCY motifs to promote alternative exon inclusion62. To support that MBNL and SRRM4 regulate Ank2 miE inclusion binding to distinct sequences, we retrieved available CLIP-seq data from an N2a cell line expressing flagged SRRM4 protein61. As expected, we identified a SRRM4-CLIP-seq reads cluster covering a conserved UGC motif 9 nt upstream Ank2 miE, and there were no reads supporting SRRM4 interaction with the MBNL binding site and vice versa . As predicted, Srrm4 and Srrm3 gene expression levels were unchanged in Mbnl cDKO frontal cortex, Mbnl2 KO hippocampus, and Mbnl DKD CAD cells . Interestingly, we noticed the significant 28% reduction of SRRM4 RNA in DM1 brain, however this downregulation did not correlate with CTGexp  . These results indicate that the MBNL and SRRM proteins regulate splicing of ASD-relevant miEs, such as ANK2 miE 480/WT knockin (hereafter Dmpk-(CTG)480/WT KI) and homozygous Dmpk-(CTG)480/480 KI mouse models to study phenotypic outcomes. Both Dmpk-(CTG)480/WT KI and Dmpk-(CTG)480/480 KI reproduce characteristic DM1 pathological molecular signatures, including MBNL sequestration on Dmpk 3’UTR (CUG)480 RNAs, RNA mis-splicing in the vulnerable cell types, and DMPK protein loss65. Importantly, the molecular phenotypes are significantly more exaggerated in homozygous Dmpk-(CTG)480/480KIcompared to heterozygous Dmpk-(CTG)480/WT KI65. In the sociability phase of the three-chamber test, WT and heterozygous Dmpk-(CTG)480/WT KI mice spent significantly more time in the chamber with a novel animal (Stranger 1) than a novel object (Dmpk-(CTG)480/480 KI mice showed no significant preference for the chamber with novel animal over the novel object , immune system and vision pathology67, whereas the Mbnl2 KO exhibits central nervous system abnormalities, including neuronal morphology and synaptic changes68. Like homozygous Dmpk-(CTG)480/480KI, and in contrast to WT, Mbnl2 KO mice did not spend significantly more time in the chamber with a novel animal  and a novel animal (Stranger 2) in the social novelty phase . Since Mbnl1 is the dominant Mbnl paralog expressed in skeletal muscles, testing Mbnl1 KO mice in the social test failed to provide reliable results due to their profoundly limited mobility evident during the habituation phase 480/480 expression or MBNL2 protein loss led to social interaction deficits, a key diagnostic feature of DM1-associated ASD. The variability observed in the three-chamber test for both homozygous Dmpk-(CTG)480/480 and Mbnl2 KO mice suggests incomplete penetrance of this phenotype.These mouse behavioral results showed that either DMPK 3’UTR CTGexp and its subsequent inhibition of MBNL’s RNA splicing activity adversely impacts the developmental ASD-risk gene splicing program, which leads to social interaction deficits, as we demonstrated in the mouse models. Thus, we propose that ASD can arise from a gene-specific tandem repeat expansion through an RNA-mediated gain-of-function mechanism whereby symptoms are a consequence of altered RNA splicing of multiple ASD-risk genes during brain development.Here, we delineate the mechanisms underlying a specific ASD-linked tandem repeat expansion and its phenotypic consequences. We provide evidence that 30. Although miEs are regulated by multiple RNA-binding proteins, their abnormal exclusion in ASD brains has been linked to downregulated SRRM4 expression. For example, the ANK2 miE 12 nt analyzed in this study is commonly mis-spliced in both DM1 and ASD brains and is co-regulated by MBNL and SRRM4 proteins. Like MBNL inhibition, SRRM4 haploinsufficiency not only causes miE mis-splicing, but also a social deficit in mice33. Additionally, AS events in the DM1 brain can mimic ASD-associated variants. For example, SCN2A MXE mis-splicing results in a protein isoform differing by a single negatively charged amino acid  in the extracellular loop of the Nav1.2 channel voltage-sensing domain . Previous research has demonstrated that similar to the ‘fetal’ MXE inclusion, ASD-associated SCN2A variants reduce neuronal excitability71. The role of mis-splicing in DM1-associated ASD is additionally supported by recent clinical trial results for tideglusib (AMO-02). ASD symptoms were improved in some of the treated children with DM172. In preclinical studies tideglusib, a small-molecule inhibitor of glycogen synthase kinase 3 (GSK3), reduces CUGexp RNA levels and corrects aberrant splicing in DM1-derived cells and two DM1 repeat expansion mouse models73.Aberrant RNA splicing is a characteristic feature of the ASD brain, including neuronal miE mis-splicing shown in approximately one-third of ASD casesFMR1 5’ untranslated region (5’UTR). The FMR1 5’UTR CGGexp underlies Fragile X-Associated Disorders, including Fragile X Syndrome (FXS) which is the most common monogenic disorder comorbid with ASD74. Here, we provide a molecular mechanism for the DMPK 3’UTR CTGexp as a second example of a tandem repeat expansion leading to ASD traits.Studies on tandem repeat expansions provide a unique opportunity to investigate the mechanistic threads in ASD, as was successfully demonstrated for the prototypical example of the CGG expansion in the Mbnl1DE3/DE3 (Mbnl1 KO) and B6.129S1-Mbnl2DE2/DE2 (Mbnl2 KO) have been described66. B6-Dmpk-(CTG)480 (³N6) line was derived from the previously described FVB-Dmpk-(CTG)48065. We used Mbnl2 KO  and WT littermate mice  as well as heterozygous Dmpk-(CTG)480/WT , homozygous Dmpk-(CTG)480/480 , and WT littermate mice . All behavioral analyses were performed between 8 weeks and 6 months of age followed by brain harvesting. Mice were housed under specific pathogen-free conditions. Both the humidity (50%–70%) and temperature (70–75°F) were controlled, and the room was maintained on a 12:12 light:dark cycle (lights off at 8:00 pm). Mice were ear-notched, and tail-snipped for identification and genotyping. Same-sex littermates were group-caged (2–4 mice/cage) at weaning in cages with water and standard rodent chow available ad-lib. The mice remained in the same cage group throughout the behavioral experiments.All relevant ethical regulations for animal testing and research were observed, and this study received approval from the University of Florida Institutional Animal Care and Use Committee (IACUC). All animal procedures and endpoints were in accordance with IACUC guidelines and animals were sacrificed in accordance with IACUC-approved protocols. B6.129S1-Three chamber test was used to assess sociability in mouse models. The rectangular three-chambered apparatus consisted of three 20 cm × 40.5 cm × 22 cm chambers separated by clear Plexiglass walls. The walls had small doors that could be lifted or closed between phases to allow chamber access or prevent it. Throughout the test, the center chamber remained empty, and objects or target (Stranger) mice were placed in the left or right chambers. The test mouse was the mouse that had its behavior analyzed. The target mice (Strangers 1 and 2) were matched in both age and sex to the test mouse and were placed into the test to provide a social stimulus. The test mice did not undergo any other experiments prior to being placed in the three-chambered social test. Similarly, the target mice only were subject to being novel mice in the three-chambered test and were not involved in any other experiments. Wire cups were used to confine the target mice while allowing for social investigation by the test mouse. Before beginning, the two target mice were habituated for ten minutes in the inverted wire cups that they were subsequently placed in during the social test. During this habituation, we observed if target mice exhibited aggression or abnormal behaviors, such as excessive grooming, bar-biting, and jumping, that could interfere with the test and provided grounds for their exclusion. None of the target mice used in this study met these criteria for exclusion.The habituation phase for the test mouse followed the habituation of the target mice. All chambers were completely empty during this phase. This phase allowed the test mouse to acclimate to the chambers and allowed us to assess if they showed a preference for one side before any novel objects or animals had been placed in the chamber.For the sociability phase, an empty inverted wire cup was placed in one chamber while an inverted wire cup with one of the target animals (Stranger 1) was placed in the chamber on the opposite side. The chamber that contained the target animal alternated with each animal that was being tested. The test mouse was placed in the center chamber, and once the doors were lifted, left to explore all chambers for ten minutes. The test animal was allowed to interact with the cup with or without a social partner present for ten minutes.For the social novelty phase, the same target animal (Stranger 1) that was used in the sociability phase remained in its place and the previously empty wire cup became occupied by a novel target mouse (Stranger 2). The test mouse was placed in the center chamber to begin and allowed to explore all chambers for ten minutes once the doors were lifted. This phase assessed whether the animal displayed more investigative behavior towards the novel target mouse (Stranger 2) or displayed a preference for the familiar mouse (Stranger 1).After the three phases of the social test were completed and the animals were placed back in their home cages, the interior of the chambers and the wire cups were sanitized with ethanol before proceeding with another test mouse. Illumination was kept even on both sides of the apparatus. The test was conducted in a quiet room with minimal visual distractions and was recorded overhead using a video camera. Each video specified the date, test animal ID, and target mice used.76.The recorded videos were observationally coded by human raters using Behavioral Observation Research Interactive System (BORIS v 8.1.2) software77. Time in each chamber and the number of social/object interactions were coded during the sociability and social novelty phases, respectively. Social interactions were operationally defined as the test mouse sniffing the target mouse, which could include nose-to-nose interaction, the test mouse sniffing any other part of the body of the target mouse, or nose-to-cup interaction, and rearing on the wire cup with the target mouse64. Object interactions only applied to the sociability phase and were defined as sniffing or rearing on the wire cup that did not have a target mouse in it. During the social novelty phase, social interactions were coded for both chambers, differentiating which animal was the novel one and which was the familiar one. Twenty per cent of the coded observations were randomly selected and independently scored by another researcher to determine the agreement between raters. A criterion of 85% or greater inter-observer agreement was established. If the behavioral scores were recorded between 1 second of each other for point events and, for durations of behavior, were 2 seconds of the start and stop time, it was counted as a scoring agreement. These parameters were set to account for the reaction time of the scorers. All the data included in this study met the criteria for 85% inter-rater agreement.Mouse video tracking during habituation phase was performed using ToxTrac (v 2.98)Mice were acclimated to the procedure room for approximately two hours before the test. For the open field, test mice were then placed in the center of the darkened activity-monitoring 17” × 17” chamber (Med Associates), and mouse movement was traced for 30 min. Analysis was performed with Activity Monitor  software. For the final statistical analysis, only 5–30 min interval was taken.2.HeLa cells were cultured in were grown in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum and 1x antibiotic and antimycotic (All Thermo Fisher Scientific) at 37°C with 5% CO57. Mouse Ank2 and mutAnk2 as well as human ANK2 and mutANK2 splicing minigenes were generated by cloning DNA oligonucleotides between NotI and SalI restriction sites in Atp2a1-D minigene. 120 bp DNA oligonucleotides contained selected TGCT(N)3TGCT(N)13–18TGCT/C or mutated GGCT(N)3TGAT(N)13–18TGTC sequences (substitutions are underlined) as well as NotI and SalI restriction sites at 5’ and 3’ ends respectively. DNA oligonucleotide sequences are listed in the key resources table. The complementary single-stranded DNA oligonucleotides (100 μM) were annealed in Annealing Buffer  at 95°C for 5 minutes followed by cooling to 25°C for 45 minutes. Annealed oligonucleotides were digested with NotI and SalI restriction enzymes (New England Biolabs), purified using Clean-Up Concentrator Kit (A&A Biotechnology), and ligated. The design of the hybrid Atp2a1 minigenes preserves RNA structures within a thermodynamically stable region at the Atp2a1 insertion site57. Final splicing minigenes were tested by Sanger sequencing.EGFP-MBNL1-41, EGFP-MBNL2-38, Atp2a1-WT and Atp2a1-D minigenes were previously describedHeLa cells were seeded on 12-well plates filled with 1 mL of medium and allowed to grow up to 50–60% of confluence priori transfection. Cells were transfected with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. For exogenous splicing analysis, HeLa cells were co-transfected with 200 ng of the indicated minigene construct and 500 ng of the EGFP-MBNL1-41, EGFP-MBNL2-38 or EGFP expressing vector. The cells were harvested 48 hours after transfection.Mouse tissues were homogenized in TRIzol (Ambion) with 1.5 mm zirconium beads in a Bead Ruptor 12 . Total RNA from mouse tissues and HeLa cells were isolated by using TRIzol Reagent (Invitrogen)/TRI Reagent (Sigma-Aldrich) and the Direct-zol RNA MiniPrep Kit (Zymo Research)/Total RNA Zol-Out D Kit (A&A Biotechnology) with on-column DNase digestion according to the manufacturer’s protocol.Total RNA (1–2 μg) was reverse transcribed using the GoScript Reverse Transcription System (Promega)/High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) with Random Primers  according to the manufacturer’s protocol. PCR was conducted using GoTaq G2 Flexi DNA Polymerase (Promega). PCR products were resolved on 2% agarose gels stained with ethidium bromide and gels visualized on a Molecular Imager ChemiDoc XRS + (BioRad)/G:Box (Syngene) and analyzed using Image Lab (BioRad)/GeneTools software (Syngene). All primers and PCR product sizes are listed in the key resources table.78. Splicing analysis was performed using rMATS (v 4.1.0)79. Sashimi plots were generated using ggsashimi.py script80. Median coverage was used to generate the plot (-A median). The total numbers of junction reads are showed. The introns were compressed for better representation (--shrink). Transcript expression quantification was performed using Salmon (v 1.1)81, and differential gene expression analysis was performed using DESeq2 (v 1.32.C)82.All RNA-seq and CLIP-seq data accession numbers are listed in the key resources table. Reads were aligned to the human hg38 or mouse mm10 genomes using STAR (v 2.7.5c)See Supplementary Table 2.apps.kaessmannlab.org/evodevoapp/)49. dbGaP accession number phs000424.v8.p2 .Evo-devo mammalian organs (RNAfold (rna.tbi.univie.ac.at).84. Protein fragments used for structure modeling with miE-encoded residues are underlined.The modeled structures of mouse proteins up to 50 aa or 214 aa for SHANK3 were predicted using the UCSC ChimeraX AlphaFold tool with the use of ColabFold, an optimized version of AlphaFold2 with default parametersShank3 without miE-encoded sequence: GGLGSLLDPAKKSPIAAARLFSSLGELSTISAQRSPGGPGGGASYSVRPSGRYPVARRAPSPVKPASLERVEGLGAGVGGAGRPFGLTPPTILKSSSLSIPHEPKEVRFVVRSVSARSRSPSPSPLPSPSPGSGPSAGPRRPFQQKPLQLWSKFDVGDWLESIHLGEHRDRFEDHEIEGAHLPALTKEDFVELGVTRVGHRMNIERALRQLDGSCAVVPSAGCALQQPRShank3 with miE-encoded sequence: GGLGSLLDPAKKSPIAAARNrxn1 without miE-encoded sequence: RLPDLISDALFCNGQIERGCEGPSTTCQEDSCSNQGVCLQQVALMKADLQGPSTTCQEDSCSNQGVCLQQNrxn1 with miE-encoded sequence: RLPDLISDALFCNGQIERGCEDmd without miE-encoded sequence: TGLEEVMEQLNNSFPSSRGHNVGSLFHMADDLGRAMESLVSVMTDEEGAEDmd sith miE-encoded sequence: TGLEEVMEQLNNSFPSSRGRNAPGKPMREDTMwww.phosphosite.org).PhosphoSitePlus . Experiments were performed on P56d old male C57BL/6J mice.For a detailed description of in situ hybridization (ISH) procedure and informatics data processing see: help.brain-map.org/display/mousebrain/Documentation.Mouse Brain Atlas  software. RNA-seq maximum group sizes and sample characteristics were predetermined. We analyzed sex- and age-matched groups.79 and DESeq2 (v 1.32.C)82, respectively. The odds ratio (OR) was calculated using ‘epitools’ package in R, and the statistical significance was determined based on Fisher’s exact test followed by the multiple comparison correction using the FDR method. Other statistical analyses were performed using GraphPad Prism (v 9.5.1). The normal distribution was assessed by the Shapiro–Wilk test followed by parametric or nonparametric tests and the post hoc test for multiple comparisons. Graphs were generated in R using the ‘ggplot2’ package and GraphPad Prism (v 9.5.1) software. Details are specified in the figure legends.Whole transcriptome statistical analysis for splicing and gene expression was performed using rMATS (v 4.1.0)


H9N2 avian influenza A viruses (AIVs) cause economic losses in the poultry industry and provide internal genomic segments for the evolution of H5N1 and H7N9 AIVs into more detrimental strains for poultry and humans. In addition to the endemic Y439/Korea-lineage H9N2 viruses, the Y280-lineage spread to Korea since 2020. Conventional recombinant H9N2 vaccine strains, which bear mammalian pathogenic internal genomes of the PR8 strain, are pathogenic in BALB/c mice. To reduce the mammalian pathogenicity of the vaccine strains, the PR8 PB2 was replaced with the non-pathogenic and highly productive PB2 of the H9N2 vaccine strain 01310CE20. However, the 01310CE20 PB2 did not coordinate well with the hemagglutinin (HA) and neuraminidase (NA) of the Korean Y280-lineage strain, resulting in a 10-fold lower virus titer compared to the PR8 PB2. To increase the virus titer, the 01310CE20 PB2 was mutated (I66M-I109V-I133V) to enhance the polymerase trimer integrity with PB1 and PA, which restored the decreased virus titer without causing mouse pathogenicity. The reverse mutation (L226Q) of HA, which was believed to decrease mammalian pathogenicity by reducing mammalian receptor affinity, was verified to increase mouse pathogenicity and change antigenicity. The monovalent Y280-lineage oil emulsion vaccine produced high antibody titers for homologous antigens but undetectable titers for heterologous (Y439/Korea-lineage) antigens. However, this defect was corrected by the bivalent vaccine. Therefore, the balance of polymerase and HA/NA activities can be achieved by fine-tuning PB2 activity, and a bivalent vaccine may be more effective in controlling concurrent H9N2 viruses with different antigenicities. H9N2 avian influenza viruses (AIVs) pose a threat to the poultry industry, leading to decreased egg production and increased mortality when complicated with other pathogens. Since the first outbreak in 1994, these viruses have diverged into several lineages, including Y439/Korea in Korea, Y280/G9 in China, and G1 in Egypt . The Y43Wild aquatic birds serve as natural reservoirs for AIVs, and the transmission between different flocks and species may occur mainly through contaminated water that is shared by various wild birds. Chickens, which are highly susceptible and raised in close, densely populated spaces, play a role in amplifying the virus population and hastening the selection of viral quasi-species with better fitness. The Y280 and G1-lineages of H9N2 viruses have evolved into multiple sub-branches and clusters with high nucleotide and amino acid variability, and they are still being isolated from poultry . As a reTo date, various mammalian pathogenicity-related mutations (MPMs) have been reported in different genes of AIVs. However, only a few mutations have been proven to have been acquired during mammalian infections, such as E158G, G591R/K, E627K, and D701N in the PB2 gene ,15,16. TA/Puerto Rico/8/1934 (H1N1) (PR8)-derived recombinant vaccine strains for poultry have been developed, containing the HA and NA genes of contemporary field viruses and the six internal genomic segments of PR8. While these vaccine strains showed high replication efficiency in embryonated chicken eggs (ECEs), some grew poorly in the ECEs and showed high mammalian pathogenicity ,30. One SL20wt was found to have the highest nucleotide identity with the H9N2 strains of A/Korean native chicken/South Korea/N20-99/2020 (N20-99) and A/chicken/Shandong/1844/2019 (SD1844) among the available Korean and Chinese H9N2 strains . The nuc9.41±0.12 and 109.42±0.14 EID50/mL, respectively) were comparable to that of SL20wt (109.67±0.29 EID50/mL). The compatibility of SL20wt’s HA and NA genes with PR8’s six internal protein genes and the lack of effect of the L226Q mutation on virus replication in ECEs were demonstrated. However, the virus titer of rSL20(P)-310PB2 was significantly lower (108.57±0.46 EID50/mL). To resolve this issue, the MVV mutations were introduced into 01310 PB2 (MVV310PB2) to increase polymerase activity, resulting in the increased virus titer of rSL20(P)-MVV310PB2 (109.58±0.14 EID50/mL), comparable to SL20wt and others. Additionally, the MVV310PB2 gene was compatible with SL20wt’s internal protein genes, causing no significant decrease in the replication efficiency of rSL20-MVV310PB2 in ECEs (109.33±0.52 EID50/mL).Five recombinant viruses with different combinations of the wild-type or mutant HA (L226Q) and NA of SL20wt, and wild-type or mutant (MVV) PB2 of 01310 with six internal genomic segments of PR8, were generated. The replication efficiency in ECEs was compared with SL20wt . The higrSL20(P) and rSL20(P)-L226Q had a higher ability to replicate and produce higher viral titers compared to the PR8 virus in both MDCK and A549 cells during 48 hpi. On the other hand, the SL20wt virus only replicated in MDCK but not in A549 cells . The repThe pathogenicity of three virus strains, rSL20(P), rSL20(P)-L226Q, and rSL20(P)-MVV310PB2, was evaluated in BALB/c mice . The resThe susceptibility of rSL20(P) and rSL20(P)-L226Q to nonspecific inhibitors present in serum samples from SPF chickens and mice was compared using the hemagglutination inhibition (HI) test . The resThree inactivated oil emulsion vaccines, V-rSL20(P), V-rSL20(P)-L226Q, and V-rSL20(P)-MVV310PB2, were inoculated into five three-week-old SPF chickens and the serum collected at three and four weeks post vaccination (wpv) for evaluation of their immunogenicity. They elicited high HI titers with a minimum of 256 at 3 wpv . The vac8.75 (Exp. 1) and 109.25 EID50/mL of r310-NS28 (Exp. 2), induced 128 and 294 HI titers at 3 wpv and 294 and 294 HI titers at 4 wpv to r310-NS28, respectively. However, it induced lower HI titers to rSL20(P)-MVV310PB2. The monovalent V-rSL20(P)-MVV310PB2 vaccine, with an average of 109.00 and 109.00 EID50/mL of rSL20(P)-MVV310PB2 (Exp. 1 and 2), induced 2048 and 891 HI titers at 3 wpv and 3104 and 446 HI titers at 4 wpv in average to rSL20(P)-MVV310PB2, respectively, but did not induce any detectable HI titers to r310-NS28. The bivalent vaccines, composed of 108.75 EID50/mL of r310-NS28 and 109.00 EID50/mL of rSL20(P)-MVV310PB2 (Exp. 1), induced higher HI titers at 3 wpv (338 vs. 128 to r310-NS28 and 2353 vs. 2048 to rSL20(P)-MVV310PB2) and lower HI titers at 4 wpv (194 vs. 294 to r310-NS28 and 1783 vs. 3104 to rSL20(P)-MVV310PB2) compared to the corresponding HI titers of the monovalent vaccines. However, the bivalent vaccine composed of 109.25 r310-NS28 and 109.00 EID50/mL rSL20(P)-MVV310PB2 (Exp. 2) induced lower HI titers at 2 (11 vs. 74 to r310-NS28 and 169 vs. 194 to rSL20(P)-MVV310PB2), 3 (194 vs. 294 to r310-NS28 and 388 vs. 891 to rSL20(P)-MVV310PB2), and 4 wpv (147 vs. 294 to r310-NS28 and 223 vs. 446 to rSL20(P)-MVV310PB2) compared to the corresponding HI titers of monovalent vaccines. As the bivalent vaccine induced the antibody to both antigens, it is better than the monovalent vaccines. The bivalent vaccine composed of 108.75 of r310-NS28 and 109.00 of EID50/mL rSL20(P)-MVV310PB2 is more preferable than the bivalent vaccine composed of 109.25 r310-NS28 and 109.00 EID50/mL rSL20(P)-MVV310PB2 due to comparable antibody titers to the corresponding monovalent vaccines.The immunogenicity of monovalent and bivalent oil emulsion vaccines composed of r310-NS28 and rSL20(P)-MVV310PB2 of different antigenic structures was compared . The r31The NA activities of rSL20(P)-MVV310PB2 and r310-NS28 were similar when tested at the same HA titers A. HoweveNation-wide recurrent outbreaks of the Y439/Korea-lineage virus in poultry since 1999 might have started with the multi-focal introduction of similar but apparently different viruses from migratory wild birds. The outbreaks might have resulted from farm-to-farm secondary transmission ,51. The The distribution of Korean Y280-lineage viruses in infected chicken tissues differs from that of Y439/Korea-lineage viruses, indicating differences in pathogenicity and level of chicken adaptation . The preDespite similarities in growth kinetics between SL20wt and PR8 in MDCK cells, the public health risk of SL20wt may not be high as it does not replicate in A549 cells. However, the higher virus titers of rSL20(P) and rSL20(P)-L226Q than rPR8 in both cells raises strong public health concerns about the conventional reverse genetics system using the highly pathogenic PR8 PB2 gene. Our experiences in testing the growth kinetics of various PR8-derived clade 2.3.2.1c H5N1, clade 2.3.4.4 H5N6 and H5N8, Y439/Korea-lineage, and G1-lineage Egyptian H9N2 recombinant strains in MDCK and/or A549 cells showed that rSL20(P) and rSL20(P)-L226Q outgrow rPR8, similar to the G1-lineage Egyptian H9N2 strain ,30,35,36The L226Q mutation decreased the replication efficiency in A549 cells but increased its pathogenicity in BALB/c mice. This resulted in severe weight loss and a 10-fold higher virus titer of rSL20(P)-L226Q in the lungs compared to rSL20(P) . The conThe optimal vaccine, V-rSL20(P)-MVV310PB2, induced very high HI antibody titers to the homologous antigen, but not to the heterologous antigen (r310-NS28). In contrast, the V-r310-NS28 induced low but cross-reactive HI antibodies to rSL20(P)-MVV310PB2 . The resThe A/chicken/Korea/SL20/2020 (H9N2) (SL20) was isolated from oropharyngeal swab samples of Korean native chickens at a LPM of Korea and cultured in specific-pathogen-free (SPF) ECEs . The previously developed Y439/Korea-lineage H9N2 recombinant r310-NS28 vaccine strain was used for monovalent and bivalent vaccine preparation . Recombihttps://www.rcsb.org/3d-view (accessed on 12 January 2023).RNA was extracted from harvested allantoic fluid using a Viral Gene-spin™ Viral DNA/RNA Extraction Kit  and cDNA was synthesized by a TOPscript™ cDNA Synthesis kit . Full genomes were amplified using universal primer sets and sequenced as previously described ,65. The The L226Q mutation was introduced into the HA gene of SL20 using the Muta-direct site-directed mutagenesis kit  with the specific primer sets detailed in v/v) chicken red blood cells, and the full genomes of the virus were confirmed by RT-PCR and Sanger sequencing [The generation of various recombinant Y280-lineage H9N2 viruses was carried out to obtain different gene constellations and mutations as listed in quencing . The con50) by inoculating serially 10-fold diluted virus into 5 10-day-old SPF ECEs. After incubating the virus-inoculated eggs at 37 °C for 72 h, followed by chilling at 4 °C before harvest, the EID50 was calculated using the hemagglutination test in each dilution and the Spearman–Karber method [50 of harvested virus after inoculating 100 EID50/0.1 mL of each virus into 10-day-old SPF ECEs and incubating for 72 h.The titer of each virus was determined as the 50% egg infectious dose (EIDr method . The rep50) in MDCK cells. The supernatant was serially diluted and inoculated into MDCK cells in a 96-well plate, and the virus in each dilution was checked by hemagglutination assay after 72 h to calculate the TCID50 using the Spearman–Karber method.Virus replication in mammalian cells, specifically MDCK and A549 cells, and the pathogenicity of the viruses in mice were evaluated in order to select a safe recombinant Y280-lineage H9N2 vaccine strain that was free from potential human infection risks. The viruses were diluted in DMEM with 1 µg/mL TPCK-trypsin and 0.1 MOI of virus was infected with confluent MDCK and A549 cells in a 12-well plate. Following a 1-h incubation period, the inoculum was replaced with fresh media, and the supernatant was harvested at specific time points  to measure the viral titer using the 50% tissue culture infectious dose  was inoculated with the same volume of sterile phosphate-buffered saline (PBS). Three of eight mice in each group were euthanized through CO2 asphyxiation to test virus replication and virus titers in the lungs at three days-post-inoculation (dpi). The remaining mice were observed for weight loss and mortality for 7 dpi. The mice were euthanized by CO2 asphyxiation when the body weight of each mouse was reduced by more than 20%. The collected lungs were homogenized using a TissueLyzer 2  equipped with 5 mm diameter stainless steel beads and mixed with PBS (10 volumes of lung weight). Following centrifugation at 13,000 rpm for 10 min, 0.1 mL of each supernatant and 10-fold diluted pooled supernatants were inoculated into ECEs as described above. The presence of virus in harvested allantoic fluid was tested by the plate HA test with 1% chicken RBCs. The virus titer of pooled lung specimens was calculated as described above.The mouse infection experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (IACUC-SNU-210811-2) and conducted in an animal biosafety level 2 facility at the Animal Center for Pharmaceutical Research of Seoul National University  according to the national guidelines for the care and use of laboratory animals. Eight six-week-old female BALB/c mice  of each group were sedated by an intraperitoneal injection of Zoletil 50 , and 10v/v) chicken RBCs were added and incubated for another 40 min at 4 °C, and the HI result was recorded [Serum samples from SPF chickens and BALB/c mice were prepared to detect non-specific inhibitors of the viral hemagglutination. One group of serum was heat-treated at 56 °C for 30 min, while the other group was treated with three volumes of RDE  at 37 °C overnight before heat inactivation at 56 °C for 30 min. The hemagglutination inhibition (HI) test was performed as below. The serum was serially 2-fold diluted with PBS, and 25 µL of the diluted serum was mixed with the same volume of the antigen virus with 4 hemagglutination unit. They were incubated at 4 °C for 40 min to allow binding of any non-specific inhibitors present in the serum before the hemagglutination reaction of the virus. Then, 25 µL of 1% (recorded 50/100 µL) was inoculated into SPF ECEs for propagation and titrated as EID50/mL prior to inactivation. The recombinant virus was then inactivated using 0.2% formaldehyde (Sigma-Aldrich) and inoculated into 10-day-old SPF ECEs after a 24-h incubation period to confirm virus inactivation. The monovalent and bivalent vaccines were prepared by mixing one or two inactivated viruses with ISA70  in a ratio of 3:7 (virus-to-oil). A negative control vaccine was prepared by utilizing negative allantoic fluid of 10-day-old ECEs as above.Each virus  of Seoul National University (IACUC-SNU-220806-1) and conducted in an animal biosafety level 1 facility of Seoul National University. Five three-week-old SPF chickens per each vaccination group were subcutaneously vaccinated with 0.5 mL of the prepared inactivated oil emulsion vaccine. Serum samples were collected at 0, 3, and 4 weeks post vaccination (wpv), and serum antibody titers were determined using the HI test The neuraminidase activity of virus and neuraminidase inhibiting activity of serum samples were measured using the NA-star™ Influenza Neuraminidase Inhibitor Resistance Detection Kit.  according to the manufacturer’s manual. The NA activity of virus with varying HA titers ranging from 21 to 27 was measured and 8 HAU of virus was selected for NI test. The heat-treated (56 °C for 30 min) serum samples collected at 3 wpv of each group in Exp. 2 was utilized to compare the NI antibody titers. Briefly, serially 2-fold diluted serum sample was incubated with the same volume of virus (25 µL:25 µL) at room temperature for 20 min. A total of 10 µL of 1:1000 diluted NA substrate was added to every well and incubated at room temperature for 30 min. Then, 60 µL of NA-star accelerator was added, and luminescence was measured immediately using an Infinite 200 PRO .p < 0.05 was defined as statistically significant.The significance of the results between experimental groups was evaluated by one-way analysis of variance  , and The optimal recombinant vaccine strains for AIVs can be engineered by balancing the activity of HA/NA with polymerase activity, and the polymerase activity can be fine-tuned through PB2 mutations. To protect against antigenically distinct H9N2 viruses, a bivalent vaccine is better than a monovalent vaccine, but the ratio of antigens in the bivalent vaccine needs to be optimized for improved vaccine efficacy.Korea pending patent 10-2021-0191058 (MAMMALIAN AVIRULENT AND EMBRYONATED EGG HIGHLY REPLICATIVE RECOMBINANT INFLUENZA VIRUS).