Cytokinin is an important phytohormone that employs a multistep phosphorelay to transduce the signal from receptors to the nucleus, culminating in activation of type-B response regulators which function as transcription factors. Recent chromatin immunoprecipitation-sequencing (ChIP-seq) studies have identified targets of type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs) and integrated these into the cytokinin-activated transcriptional network. Primary targets of the type-B ARRs are enriched for genes involved in hormonal regulation, emphasizing the extensive crosstalk that can occur between cytokinin, auxin, abscisic acid, brassinosteroids, gibberellic acid, ethylene, jasmonic acid, and salicylic acid. Examination of hormone-related targets reveals multiple regulatory points including biosynthesis, degradation/inactivation, transport, and signal transduction. Here, we consider this early response to cytokinin in terms of the hormones involved, points of regulatory crosstalk, and physiological significance. ARRs which encode a second family of response regulators that lack a DNA-binding motif and negatively regulate cytokinin responses [Physcomitrella patens [Cytokinins are a class of phytohormones that regulate multiple aspects of plant growth and development including shoot and root growth, chloroplast development, seed fill, senescence, and nutrient uptake ,2,3,4,5.esponses ,11,12. Sa patens ,14.Chlamydomonas reinhardtii PSR1, and Arabidopsis PHR1 transcription factors [The type-B response regulators have a modular structure. They have a receiver domain, the diagnostic domain of a response regulator, at their N-terminus. The receiver domain contains a conserved Asp residue which serves as the site for regulatory phosphorylation when the multistep phosphorelay is activated ,18,19,20 factors . The Myb factors  but opti factors ,23,24. F factors ,26,27. Aarr1 arr10 arr12 abolishes the majority of cytokinin-mediated gene expression as well as rendering the plant largely insensitive to cytokinin based on a variety of physiological assays [ARRs, which are primary response genes that negatively regulate cytokinin responses, are among the most uniformly upregulated members of the Golden List [Although Arabidopsis contains 11 type-B ARRs, these do not all contribute equally in cytokinin signal transduction ,28,29,30l assays ,33. Not Recently, two complementary Chromatin Immunoprecipitation-sequencing (ChIP-seq) studies were carried out with type-B ARRs of Arabidopsis, these studies providing the first genome-wide views of the type-B ARR targets involved in the initiation of the cytokinin response ,34. Resuarr1 arr10 arr12 cytokinin-insensitive mutant line, the 35S:ARR10-GFP construct complementing a variety of arr1 arr10 arr12 mutant phenotypes and resulting in a hypersensitive cytokinin response [35S:ARR10-GFP rescuing the loss of not only the native ARR10 activity but that of all three type-B ARRs . The cytokinin-hypersensitivity, coupled to the lack of competition from ARR1 and ARR12, were predicted to increase the sensitivity of these lines for identifying targets of ARR10 by ChIP-seq. Cytokinin treatment was performed on ~3 week old green seedlings for 30 min with 5 µM of the cytokinin benzyladenine (BA) [Two complementary ChIP-seq studies were carried out by Zubo et al.  and Xie response . These dine (BA) , BA haviine (BA) ,41,42. Tine (BA) .ARR1, ARR10, and ARR12 and which mimics, as closely as possible, the native expression state of these genes both in terms of expression levels and organ-specificity [arr1 arr10 arr12 mutant line, the same line exploited by Zubo et al. [arr1, arr10, and arr12 alleles all being cytokinin hyposensitive to varying extents [ARR1, ARR10, and ARR12 lines, allowing them to identify candidate gene targets for each type-B ARR [The ChIP-seq approach taken by Xie et al.  differedcificity . The reco et al. , each tro et al. . The rec extents ,29,30. H extents , the red extents  performe extents ; thus, t extents  would ha extents ,34. Xie  extents   analysis reveals that the primary targets of the type-B ARRs are enriched genes involved in various hormone responses, emphasizing the extensive hormone crosstalk mediated through action of cytokinin and the type-B ARRs ,34. ExamWUSCHEL is a type-B ARR target transcriptionally induced by cytokinin, although it does not generally appear in the transcriptomic studies, this likely being because WUSCHEL expression occurs predominantly in a subset of meristem cells and also exhibits a long-term kinetic response to cytokinin [Of particular interest are those genes for which expression information is available for their response to cytokinin. These type-B ARR target genes, based on transcriptomic studies to identify cytokinin-responsive genes ,30,33, aytokinin ,34,43,44CKXs which encode cytokinin oxidases and the UGTs which encode glucosyl transferases for conjugation of glucose to cytokinin [CKX4 was identified as a primary response gene regulated by the type-B ARR and ARR1 [ARRs [Prior studies indicate that one transcriptional response to cytokinin is the induction of genes that negatively regulate cytokinin activity ,45,46,47ytokinin ,49. The R1 [ARRs ,40,45,46R1 [ARRs ,47. The R1 [ARRs ,50,51. HR1 [ARRs . AdditioR1 [ARRs ,53.CKX and UGT families for the degradation and inactivation of cytokinins as well as the biosynthetic gene IPT5, the expression of which is downregulated in response to cytokinin [ARRs, members of the type-A ARR family exhibiting some of the strongest binding by the type-B ARRs along with a pronounced cytokinin dependence for this binding [Based on expression analysis, cytokinin induces multiple negative feedback loops to regulate both cytokinin metabolism and signal transduction with such negative feedback loops being a common feature of hormone responses to allow for inactivation as well as adaptation so that the organism can respond to increasing hormone concentrations ,55. The ytokinin . In term binding ,34. Not LOG2 is a target that is upregulated in response to cytokinin, potentially serving to help replenish the cytokinin pool, although this is not a family member implicated in playing a major role in either the shoot apical meristem or the root [AHK4, which encodes a cytokinin receptor is also a primary target upregulated in response to cytokinin. However, AHK4, like many histidine kinases, possesses both histidine kinase and phosphatase activity and so has the potential to function as both a positive and a negative regulator of the pathway, dependent on cytokinin levels [Although the majority of type-B ARR primary targets are consistent with negative regulation of cytokinin activity, there are two potential exceptions. First, the cytokinin biosynthetic gene the root . Second,n levels .ARRs ARR7 and ARR15 to inhibit the cytokinin response during Arabidopsis root embryogenesis [AUX/IAA gene SHY2/IAA3 which functions as a negative regulator of auxin signaling to inhibit auxin responses and cell proliferation in the primary root [AUX1 auxin efflux carrier, affecting the shootward movement of auxin from the root tip and resulting in decreased root cell expansion [Auxin controls fundamental aspects of plant growth, organogenesis, and responses to the environment ,60,61,62ogenesis . Howeverogenesis . Relatinary root ,67,68. Cxpansion .IAMT1 and ILL6 and induces the expression of GH3.6 which encodes an acyl acid amido synthetase for the inactivation of auxin by conjugation to amino acids, consistent with their being primary targets and indicating that cytokinin would likely reduce the levels of available auxin. Interestingly, although the four GH3 genes involved in auxin inactivation are all conserved type-B ARR candidates, of the five auxin biosynthesis genes, only NIT1 is a conserved B-ARR candidate. However, in the study by Xie et al. [CYP79B3 was bound by ARR1 after 3 h cytokinin treatment, and ILL6, IAMT1, and CYP79B3 all bound by ARR1 after three days of treatment; additionally, IAMT1 was bound by ARR10 without cytokinin treatment [The ChIP-seq studies indicate that the type-B ARRs target genes are involved in auxin metabolism, transport, and signal transduction . Expresse et al. , CYP79B3reatment . This mareatment ,70,71,72AUX1 is downregulated in response to cytokinin, the cytokinin-dependent peak(s) for type-B ARR binding being found in the eighth intron of AUX1 [Genes related to auxin transport, including both efflux and influx carriers, are represented among the B-ARR targets and candidate targets . The PIN of AUX1 ,69. We n of AUX1 , this be of AUX1 .AUX/IAA genes, and four ARF genes. Cytokinin-dependent expression information is only available for a few of these, the most significant being for SHY2/IAA3, a negative regulator of auxin activity. The gene SHY2/IAA3 is induced in response to cytokinin and plays a pivotal role in regulating the balance of auxin to cytokinin activity in the root, its induction by cytokinin reducing auxin activity and resulting in a decrease in cell proliferation by the primary root [Type-B ARR binding sites are also found associated with genes encoding elements of the auxin signal transduction pathway, including two genes encoding auxin receptors, ten ary root ,64,67,68ahk2 ahk3 double mutant [AHK cytokinin receptors and the type-A and type-B ARRs involved in cytokinin signaling and the SnRK2 kinases involved in ABA signaling [ARRs, which are rapidly induced cytokinin primary response genes, have been identified as key players mediating this antagonistic interaction in several instances. For instance, the ability of ABA to inhibit seed germination and cotyledon greening requires the ABA-activated transcription factor ABI4 to suppress transcription of type-A ARRs [ABI5, as well as negatively regulate its expression, ABI5 being rapidly upregulated in response to ABA and playing a key role in mediating the ABA response [Abscisic acid (ABA) regulates plant responses to such abiotic stresses as drought and cold ,77,78. Ae mutant ,82. Antaignaling ,84. The e-A ARRs . Additioresponse .ABA1; (2) the induction of ABA degradation/inactivation genes such as CYP707A1, which encodes an hydroxylase involved in ABA catabolism, and the UGTs, which encode UDP glucosyl transferases, involved in conjugation of glucose to ABA; (3) the downregulation of NRT1.2 for ABA influx; and (4) the upregulation of HAI1 and HAI2, which encode PP2C phosphatases that negatively regulate ABA signaling. We note that although many of the candidate targets involved in metabolism are not among the “conserved” B-ARR dataset, the study by Xie et al. [BCH1 and ABA1, and ARR12 binding to ABA4 and NCED6.Type-B ARR binding sites are found associated with genes involved in biosynthesis, degradation/inactivation, transport, and signaling . Based oe et al.  does supCycD3 gene, and in some cases brassinosteroids can substitute for cytokinin to promote cell proliferation in Arabidopsis callus and suspension cells [Brassinosteroids are polyhydroxylated steroid hormones that regulate various aspects of plant growth and development, such as cell proliferation and expansion, vascular differentiation, reproduction, photomorphogenesis, and senescence ,86,87. Con cells . In anoton cells .BAK1, SERK1,4), signal transduction , and transcriptional regulation   . Howevergai mutant  under an anther- and pollen-specific promoter resulted in male sterility that was reverted by kinetin.Gibberellins are plant hormones that play key roles in developmental programs including seed germination, stem elongation, flowering, trichome development, and leaf expansion ,95,96. GGA3OX1, GA2, and GIM2), whereas expression of the GID1b receptor is repressed. Thus, cytokinin-gibberellic acid crosstalk points to a complex set of interactions between these two phytohormones; the type-B ARRs interact with DELLAs to facilitate the cytokinin transcriptional response and oppose gibberellin activity [Type-B ARR binding sites are found associated with genes involved in biosynthesis, inactivation, and signaling . Based oactivity , but theEthylene is a gaseous hormone that regulates cell proliferation and expansion, senescence, fruit ripening, and responses to biotic and abiotic stresses ,102. CytACS genes are identified, ACS2 expression being upregulated by cytokinin, consistent with cytokinin-activating ethylene biosynthesis in a type-B ARR-dependent manner [ARGOS and ARL from the ARGOS gene family which are transmembrane proteins that act at the level of the receptors and desensitize the plant to ethylene [TRP1 which functions as a positive regulator of ethylene signaling potentially by binding to receptors and interfering with their ability to interact with CTR1 [ETP and EBF genes which encode F-box proteins involved in the degradation of EIN2 and EIN3, respectively [Type-B ARR binding sites are found to be associated with genes involved in ethylene biosynthesis and signal transduction ; note tht manner ,106. Addethylene ,111,112;ith CTR1 ; and ETPectively ,115,116.Jasmonic acid is an important regulator of plant development and stress responses, influencing flower development and senescence, as well as responses to herbivorous insects, necrotrophic pathogens, and wounding ,118,119.Type-B ARR binding sites are found to be associated with genes involved in biosynthesis, degradation/inactivation, and signaling . ExpressSID2 for salicylic acid biosynthesis and PR1, a marker gene for salicylic acid responses [PR1 [Salicylic acid is a phytohormone that plays a central role in plant defense against biotrophic pathogens, contrasting with jasmonic acid and its central role in resistance to necrotrophic pathogens ,127,128.esponses ,130. Thises [PR1 ,129.EDS5, a “common” target that is induced by cytokinin. EDS5 is a MATE-transporter involved in the movement of salicylic acid from the chloroplasts, where salicylic acid biosynthesis occurs, to the cytosol and its induction by cytokinin could thus facilitate salicylic acid activity [HAC5, and the NPR1-suppressor NIMIN1 [Analysis of type-B ARR ChIP-seq data suggests that additional mechanisms may also play a role in mediating crosstalk between cytokinin and salicylic acid, because type-B ARR binding sites are found associated with genes involved in the metabolism, transport, and signaling of salicylic acid . Perhapsactivity ,131. Manr NIMIN1 ,127,128.WUSCHEL as a type-B ARR target transcriptionally induced by cytokinin, although it does not generally appear in transcriptomic studies [As described above, analysis of the type-B ARR ChIP-seq datasets reveals that one mechanism for cytokinin crosstalk with other phytohormones is the transcriptional regulation of elements involved in their metabolism, transport, and signal transduction ,34. That studies ,34,43,44 studies ,99,129.


Codonopsis tsinglingensis, belonging to the Campanulaceae family, is a perennial medicinal herb highly valued in Chinese traditional medicine. The complete chloroplast genome of C. tsinglingensis was sequenced using the Illumina Hiseq 4000 platform. The size of the C. tsinglingensis chloroplast genome is 170,253 bp, with an average GC content of 38.3%. This circular molecule has a typical quadripartite structure containing a large single copy (LSC) region of 85,408 bp, a small single copy (SSC) region of 8179 bp, and two inverted (IRs) repeat regions of 38,333 bp. The genome contains 138 genes, including 92 protein-coding genes, 38 transfer RNA genes (tRNA), 8 ribosomal RNA genes (rRNA). Phylogenetic analysis based on complete chloroplast genome sequences of 14 species indicates that C. tsinglingensisis closely related to Codonopsis minima in the Campanulaceae family. Codonopsisis a dicotyledonous genus containing about 42 species of perennial plants,mainly distributed in East, South and Central Asia , is a kind of famous traditional Chinese medicine, which have long been used to lower the blood pressure and treat watery stool with poor appetite . Total genomic DNA was extracted from leaf tissue using the Plant Genomic DNA kit . The whole-genome sequencing was conducted with 350 bp pair-end reads on the Illumina Hiseq 4000platform  by Novogene, Beijing, China. After trimming, the high-quality paired-end reads were assembled with the programme MITObim v1.7  regions of 38,333 bp, the large single-copy (LSC) region and small single-copy (SSC) region of 85,408and 8179 bp. A total of 138 genes were successfully annotated containing 92 protein-coding genes, 38 transfer RNA genes, 8ribosomal RNA genes. Among these genes, 12 genes  have one intron, and three genes  have two introns.The chloroplast genome of Brassica juncea as outgroup. The phylogenetic relationships analysis was inferred using the maximum likelihood (ML) method based on complete cp genomes, which was performed using RAxML (Stamatakis C. tsinglingensis was most closely related to C. minima with 100% bootstrap support (To conduct phylogenetic analysis, we downloaded complete chloroplast genome sequences of 13 Campanulaceae species from NCBI and  support .


Dense deposit disease (DDD), a subtype of complement component 3 (C3) glomerulopathy (C3G), results from alternative complement pathway hyperactivity leading to membrane attack complex formation. DDD treatment strategies are limited. We report a case of a 13-year-old girl diagnosed with DDD at 9 years of age, with nephritic and nephrotic syndrome and C3 nephritic factor-negative alternative complement pathway activation. Initial treatment with prednisolone, methylprednisolone pulses (MPs), and mizoribines was effective for 3 years, after which she relapsed. Despite MP treatment followed by prednisolone and mycophenolate mofetil (MMF), her kidney function and proteinuria deteriorated with a high soluble (s)C5b-9 level; she also developed dyspnea and pleural effusion (PE). Three days after the first eculizumab (ECZ) infusion, urine volume increased, respiratory condition improved, PE resolved, and proteinuria decreased in 1 month. Serum creatinine level decreased, and kidney function completely normalized within 7 weeks. The sC5b-9 level normalized, and although proteinuria decreased, nephrotic range proteinuria persisted during ECZ treatment with MMF for 53 weeks, even with increased treatment interval. Thus, complement activation pathway-targeted therapy may be useful for rapidly progressing DDD. Our data support the role of complement pathway abnormalities in C3G with DDD.  Dense deposit disease (DDD) is a rare subtype of complement component 3 (C3) glomerulopathy (C3G) defined by the glomerular deposition of C3, but not other immunoglobulins (Igs), and intramembranous electron-dense material in the glomerular basement membrane, caused by the dysregulation of the alternative complement pathway . It mostThe pathogenesis of DDD offers novel therapeutic opportunities to target complement pathways. Eculizumab (ECZ), a humanized monoclonal antibody against human C5, blocks the activation of the alternative complement pathway and prevents the generation of terminal complement complex C5b-9 , 20, 21.2 based on Uemura’s eGFR formula for Japanese children [A 9-year-old female patient presented with macrohematuria and persistent proteinuria (0.2 g/g creatinine) for several months, high serum creatinine level (0.58 mg/dL), low serum creatinine-based estimated glomerular filtration rate (Cr-eGFR) of 81 mL/min/1.73mchildren , hyperac2) and normalized urine for 3 years. During this time, C3 and CH50 were undetectable . However, when the patient was 13 years old, nephritic and nephrotic syndrome relapsed. A second biopsy confirmed DDD with severe diffuse endocapillary and mesangial proliferative glomerulonephritis with 1 cellular crescent. Treatment with MP, angiotensin-converting enzyme inhibitor (ACEI), and angiotensin II receptor blocker (ARB) was initiated, followed by treatment with prednisolone, dipyridamole, warfarin, and MMF (900 mg/m2) instead of mizoribine, for 1 month. However, her kidney function further deteriorated, with Cr 1.02 mg/dL (Cr-eGFR 57 mL/min/1.73m2), proteinuria increased (12.3 g/g creatinine), and dyspnea requiring oxygen occurred owing to pleural effusion (PE). Although FFP was administered, there was no change. Methylprednisolone pulses (MPs) were administered at 1 g/day, 3 times a week for 3 weeks, and were followed by prednisolone, mizoribine, dipyridamole, and warfarin therapies. These treatments resulted in complete renal function recovery   . In the 7 ng/mL) . No advest ECZ infusion, immunofluorescence indicated that isolated C3 deposits along the glomerular basement membrane and IgG deposition had decreased compared with those at the second biopsy. 49 weeks after the first ECZ treatment, the treatment interval was extended from 2 to 3 or 4 weeks. Under this regimen, the sC5b-9 level was normal , and kidney function (Cr-eGFR 107 mL/min/1.73m2), proteinuria (~ 2.5 g/g creatinine), and hematuria (10 – 19/HPF) were stable throughout the 53-week follow-up period; leukocyturia was not observed. After the 53-week follow-up period, proteinuria and leukocyturia started to increase at 6 weeks after the final ECZ infusion. At 17 weeks after the final ECZ infusion, nephritic and nephrotic syndromes reappeared, and we restarted ECZ treatment. Consequently, leukocyturia disappeared and proteinuria started to decrease at 1 week after ECZ retreatment without kidney injury. After discharge from the hospital, treatment was continued in an outpatient ward with MMF, ARB, ACEI, dipyridamole, and gradual prednisolone tapering. At the third biopsy after the 41We observed an impressive and significant response to ECZ in a pediatric patient with DDD, despite the lack of a notable response to other immunosuppressive therapies. Proteinuria decreased over the 53-week treatment period, even when the treatment interval was extended from 2 to 3 or 4 weeks . DDD treatment has historically focused on supportive measures (ARB and ACEI), plasmapheresis to remove autoantibodies, and antiproliferative agents such as prednisolone, MMF, and anti-CD20 antibody , 7, 26. In our patient, at the third biopsy after ECZ treatment accompanied by a substantial decrease in the sC5b-9 level, immunofluorescence of C3 deposits showed an improvement, indicating slowed disease progression . ECZ is Use of ECZ for pediatric DDD has been reported in 9 patients  9, 10, , 19. MosTo the best of our knowledge, this is the first report of a pediatric DDD patient with information on sC5b-9 level before and after ECZ treatment and treatment interval changes. After a 12-week washout, sC5b-9 should return to levels observed before the first ECZ infusion , but thiThe contribution of combined immunosuppressive therapies to our patient’s clinical response cannot be ruled out. However, these therapies had been applied for more than 1 month before ECZ treatment, with continued deterioration of kidney function and proteinuria. In contrast, an improvement was evident within days after the first ECZ treatment, with further biopsy and sC5b-9 measurement showing continued resolution. This response is similar to that reported in previous cases , 18, sugInterestingly, the ECZ dose used in this study was different from the recommended dose. For patients with a body weight over 40 kg, the normal practice is to administer ECZ at 900 mg/week for the first 4 weeks and then use 1,200 mg/week in the subsequent weeks , 23. HowIn conclusion, we report a case of a pediatric patient with proliferative DDD resistant to immunosuppressive therapies, who received ECZ for 53 weeks. Importantly, her condition and sC5b-9 level were stable even with prolonged treatment interval. Our findings contribute to the pool of evidence supporting the pivotal role of complement alternative pathway abnormalities in DDD. The authors thank Dr. Inoue, Dr. Hidaka, Dr. Fukumori, and the Japanese Association for Complement Research for genetic analysis. The Japanese Association for Complement Research for Genetic Analysis, which was supported by Alexion Pharma GK as a company-sponsored research grant. All authors have no conflict of interest to declare.


MYH7) and myosin-binding protein C (MYBPC3) explain 60%–70% of observed clinical cases. The heterogeneity of phenotypes observed in HCM patients, however, suggests that novel causative genes or genetic modifiers likely exist. Here, we systemically evaluated RNA-seq data from 28 HCM patients and 9 healthy controls with pathogenic variant identification, differential expression analysis, and gene co-expression and protein–protein interaction network analyses. We identified 43 potential pathogenic variants in 19 genes in 24 HCM patients. Genes with more than one variant included the following: MYBPC3, TTN, MYH7, PSEN2, and LDB3. A total of 2538 protein-coding genes, six microRNAs (miRNAs), and 1617 long noncoding RNAs (lncRNAs) were identified differentially expressed between the groups, including several well-characterized cardiomyopathy-related genes . Gene enrichment analysis revealed that those genes are significantly involved in heart development and physiology. Furthermore, we highlighted four subnetworks: mtDNA-subnetwork, DSP-subnetwork, MYH7-subnetwork, and MYBPC3-subnetwork, which could play significant roles in the progression of HCM. Our findings further illustrate that HCM is a complex disease, which results from mutations in multiple protein-coding genes, modulation by non-coding RNAs and perturbations in gene networks.Hypertrophic cardiomyopathy (HCM) is an inherited disorder of the myocardium, and pathogenic mutations in the sarcomere genes myosin heavy chain 7 ( Hypertrophic cardiomyopathy (HCM) is a global, inherited cardiovascular disease that can lead to arrhythmia, heart failure and even sudden cardiac death ,2. It isMYH7) and cardiac myosin-binding protein C (MYBPC3) account for 60%–70% of identified HCM cases [TNNT2, TNNI3, TPM1, MYL2, MYL3, ACTC1, and TNNC1) and some non-sarcomere protein genes  [Variants in sarcomere and sarcomere-associated protein genes represent the greatest genetic contributor to HCM cases. Previous studies have shown that pathogenic mutations in myosin heavy chain 7  in nine patients  was found in PLN. For the 10 patients with previously identified patients . HCM518 MYH7 variants were found in nine samples (MYH7 mutation in isolation and one (HCM437) possessing MYH7, MYBPC3, and COX15 variants. For the remaining MYH7 HCM samples, variants in the following other cardiomyopathy genes were also found: DSP, MHY6, NEBL, PSEN1, RBM20, and TTN. Seven missense  samples , with twABCC9 (c.T2935C:p.W979R) in HCM541, SGCD (c.A845G:p.Q282R) and TTN (c.C28730T:p.P9577L) in HCM493, CSRP3 (c.A585C:p.X195C) in HCM395, NEXN (c.C643T:p.R215C) in HCM282, DSP (c.G6119A:p.R2040Q) and PSEN2 (c.G640T:p.V214L) in HCM591, and NEBL (c.G561C:p.Q187H) in HCM552. However, we were unable to identify any variants in the remaining four genetically undiagnosed patients  < 0.05 and fold change > 1.5), a total of 4161 genes were identified as DEGs , of whicMYBPC3 and MYH7 between the groups. However, several genes that have been implicated in cardiomyopathy did demonstrate large differences between normal controls and HCM patients, including ANKRD1, FHL2, and TGFB3  candidate pairs ranged from 25.5% (miR-154) to 76.5% (miR-3671). There are only six miRNAs that showed significant differences, with patients A–F. Amonfibrosis ,14. NextTTN, ACTC1, TPM1, JPH2, ANKRD1, DTNA, TMPO, FHL2, CTNNA3, GATA4, LAMA2, PRDM16, POLG, etc. Further GO enrichment analysis illustrated that these genes are significantly enriched in cardiovascular-related categories  those lncRNAs , includitegories , includiMYH7 and MYBPC3, respectively. To gain insight into the functional organization of the transcriptomes from myocardial tissue, we conducted gene co-expression network analysis with WGCNA. By applying data transformation and filtration according to the WGCNA pipeline , 8417 protein-coding genes were used for constructing gene co-expression networks. With the soft-thresholding power β = 12 determined by scale-free topology A,B, we ip < 0.05) with the measured phenotypes (Supplementary Data 8), including NDUFS2, DSP, JUP, TNNI1, etc. These genes could be directly associated with the phenotypes, while the rest may modulate the HCM phenotypes by interacting with those 18 genes.Next, we explored the PPIs for the genes in the MHY7- and MYBPC3-involved modules (M14 and M16). By performing MCL clustering, we highlighted four subnetworks that could play significant roles in HCM, including one mtDNA-subnetwork and three cardiomyopathy-gene-centered networks: MYBPC3-, MYH7-, and DSP-subnetworks . Next, wTPM1, and ACTC1, which are critical for the maintenance of sarcomere thickness [DES are associated with inherited skeletal and cardiac myopathies [TCAP is required for sarcomerogenesis in striated muscles, and mutations in TCAP have been identified in patients with HCM and dilated cardiomyopathy (DCM) [JUP have been diagnosed with HCM [DSG2 levels appear to be specifically associated with arrhythmogenic right ventricular cardiomyopathy (ARVC) [MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-CO1, MT-CO2, MT-CO3, MT-ATP6, MT-ATP8, and MT-CYB have been identified in cardiomyopathy patients [NDUFV1, a component of complex I in the electron transport chain, are significantly decreased in the myocardium of DCM patients, and NDUFS2 gene mutations cause complex I deficiency with associated DCM [Besides the subnetwork hub genes, other genes were also found to be associated with cardiomyopathy. For instance, hickness ,16, havehickness ,18,19. Topathies . TCAP ishy (DCM) . Patientwith HCM ,23, and y (ARVC) . For thepatients ,26,27,28ated DCM ,30. MYH7 variants and nine with pathogenic MYBPC3 variants. One patient (HCM437) had both MYH7 and MYBPC3 variants, along with one in COX15, whose function appears to be essential for cytochrome c oxidase (COX) biogenesis in the electron transport chain (ETC). Mutations in this gene are associated with Leigh syndrome and COX deficiency with infantile HCM, and the presence of this variant is interesting in the context of the enriched mitochondrial PPI-subnetwork  have another cardiomyopathy gene identified. Desmoplakin, which is encoded by DSP, is a desmosomal plaque protein, and variants in this gene are implicated in ARVC [PSEN2 mutations, like the one in HCM591, have been reported in association with familial Alzheimer’s disease and DCM, but not HCM, and those associated with DCM impair calcium signaling and handling [DSG2), another component of the desmosome, and protein networks implicate gene–gene interactions between the presenilin components and a number of desmosomal proteins, including desmoplakin [DSP variant may be a pro-HCM modifier gene. Overall, the NEXN, NEBL, and DSP mutations may represent important modifier variants, as they are disproportionately represented in this sample population.Both of the patients with the  in ARVC . PSEN2 mhandling . The premoplakin . This prMYBPC3 or MYH7 variant had mutations in ABCC9, SGCD, TTN, CSRP3, and PLN. Variants in ABCC9, which encodes an ATP-sensitive K+ channel, have been implicated in familial atrial fibrillation, sporadic DCM, and hypertrichotic osteochondrodysplasia. DCM variants have been described in exon 38 and likely modify a nucleotide-binding domain in the SUR2A portion of the channel, altering overall channel gating [ABCC9 is found in exon 24, which corresponds to the SunT domain of the protein (not a transmembrane region), and may represent an HCM mutational hotspot.The remaining patients without an identified pathogenic SGCD encodes the δ component of the sarcoglycan complex, and mutations in this gene are associated with autosomal recessive limb-girdle muscular dystrophy type VI and a mild autosomal dominant DCM phenotype [TTN, titin maintains the structure of the sarcomeric unit in both skeletal and cardiac muscle during contraction and interacts with actin filaments and myosin [SGCD mutation may further disrupt titin networks that are already disorganized by the TTN variant itself and thus work synergistically to produce HCM.henotype ,40. The henotype . Encodedd myosin . While bCSRP3 encodes an LIM domain-containing scaffolding protein that maintains the structure of the Z-disk and the costamere and is implicated in actin remodeling and cardiomyocyte differentiation. Mutations in this gene are associated with both familial HCM and DCM [ and DCM ,44. HCM3PLN, phospholamban is a substrate of protein kinase A (PKA), a cAMP dependent kinase, in cardiomyocytes. The phosphorylation of phospholamban by PKA relieves its inhibition of ATP2A2, a sarcoplasmic reticulum Ca2+-ATPase, allowing for the sequestration of calcium ions and cardiac muscle relaxation in diastole [PLN variants, like the unreported one found in HCM486, have been identified in patients with HCM; the PLN inheritance pattern for DCM is less clear [Produced by diastole ,46. Hetess clear . InteresMYBPC3 and MYH7, there is often dramatic phenotypic variability, even within a single family, with some family members having other cardiomyopathy subtypes, including DCM and ARVC, or hybrid phenotypes. This variability is most likely attributable to the presence of multiple modifier variants, and hints at the concept that cardiomyopathy is a polygenic trait. While HCM has been attributed to monogenic perturbations in sarcomeric genes, like ANKRD1, FHL2, and TGFB3 between the HCM patients and normal controls demonstrated this idea. Mutations in FHL2 have been implicated in HCM, so the reduced expression of these genes in HCM hearts mirrors actual inherited modifications or deficiencies in these proteins [ANKRD1 expression in DCM heart samples correlates with the progression of heart failure, and this reflects the increased expression that we saw in the HCM patients [TGFB3 is an important driver of the fibrosing process [TGFB3 expression in our HCM samples; likewise, missense TGFB3 mutations have loosely been associated with HCM itself [Beyond the single gene and synergistic modifier gene perspectives, pathway perspectives illustrate the idea that even single gene mutations may in turn modify other genes in a specific pathway or network. The differences in expression levels of proteins ,49,50. Opatients . Finally process . This isM itself . miR-487a, miR-654, miR-30d, miR-154, miR-3193 and miR-3671) were observed to be significantly up- or down-regulated in HCM patients. Among them, Serum miR-30d has been reported to be associated with acute heart failure [miR-30d-5p was decreased 1.58-fold in patients with ST-elevated myocardial infarction [MiR-154 has been reported to promote myocardial fibrosis through β-catenin signaling pathways [FGF1, known as acidic fibroblast growth factor, shares regulation by miR-487a and miR-154. A pathophysiologic role for fibroblast growth factors in idiopathic cardiomyopathy has been implicated in the past [CACNA2D1, CACNA1B, CACNA1S, CACNG4, and CACNG8) are also targeted by those miRNAs. It has been reported that mutations in CACNA2D1 cause a variant of short QT syndrome, which is associated with sudden cardiac death in humans [As post-transcriptional regulators, both miRNAs and lncRNAs can interfere with gene expression ,55. We e failure  and cons failure . In addifarction . MiR-154pathways ,58. It ithe past . Addition humans . TTN, ACTC1, TPM1, JPH2, ANKRD1, DTNA, TMPO, and FHL2) or physically close to these genes. The lncRNA-annotated protein-coding genes are enriched in the GO terms of heart development and cardiomyocyte differentiation  in HCM. However, those genes are largely neglected on HCM genetic screening panels. Mutations in sarcomeric genes are associated with inefficient energy utilization . When thMYH7 and MYBPC3 pathogenic variants that have been identified in the original research, but also discovered eight candidate variants in six genetically undiagnosed patients. In addition, we also found most of the patients carry muti-genic variants, even for the MYH7 and MYBPC3 diagnosed patients, suggesting that a heterogeneous phenotype could exist among those patients. Second, we identified thousands of differentially expressed coding and noncoding genes. For the coding genes, we performed functional enrichment analysis, which highlighted that those genes significantly involved in regulating cardiovascular system development and cardiac physiology . This limited our genotype–phenotype correlations, especially for evaluating the clinical outcome differences between the patients carry muti-genic and single gene mutation. Third, the larger sample size would be beneficial for covering broader variants’ spectrum and increase the statistical power for differential expression analysis and gene co-expression network construction. There are several limitations for our studies. First, calling variants from RNA-seq is confronted with several issues, due to the complexity of the transcriptome. In order to avoid false positives/negatives, we applied SNP and Indel calling with the recommendation of the Broad Institute’s GATK best practices workflow. In addition, for the candidate variants, we also “eye” inspected the alignment to avoid false positive/negatives raised by too much complexity around the variant region. Further, our results confirmed the https://www.ncbi.nlm.nih.gov/sra/SRP186138). This data set contains 28 myocardial samples from HCM patients and 9 samples from healthy donors. The detailed clinical characteristics of the studied samples are listed in The RNA-seq data of SRP186138  were dowhttps://software.broadinstitute.org/gatk/documentation/article.php?id=3891). Briefly, paired-end reads (PE300) were mapped onto the human reference genome (GRCh38) using the STAR 2-pass alignment method [http://broadinstitute.github.io/picard/) was used to add read groups, sort, mark duplicates, and create index. Then, the SplitNCigarReads function, implemented in GATK, was used to split reads into exon segments. Finally, variant calling and filtering were done with HaplotypeCaller and VariantFiltration, respectively.The raw reads for each sample first underwent adaptor trimming and filtering with the fastp software , allowint method . The firThe variant call was first annotated with wANNOWAR . Then, tp-values were adjusted with the false discovery rate (FDR) based on Benjamini and Hochberg’s method [A comparison of HCM patients with normal controls was used to identify differentially expressed genes (DEGs). Gene-level read counts were obtained using featureCount version 0.6.1 with Ensembl annotation of the GRCh38 reference genome, which includes both coding and non-coding genes. Raw counts were analyzed with DESeq2 version 1.10.1  to idents method , and DEGMiRNAs are a class of non-coding small RNAs (~22 nucleotides), which are widely found in plant and animal cells. Cleavage of mRNA transcripts or the inhibition of translation initiation is achieved by inaccurate complementary base pairing with the target gene . To dateLncRNAs are a class of non-coding RNAs that are greater than 200 nucleotides in length . Althoughttps://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/). We first normalized the raw read count with the Transcripts Per Million (TPM) method and then removed genes whose expression was consistently low (TPM < 10 in more than 90% of the samples). The filtered data was log-transformed with log2(x+1), which was used as input for WGCNA analysis. Based on the scale-free topology, threshold power β = 5 was used for constructing the adjacency matrix and the Topological Overlap Matrix. Then, genes were aggregated into modules with hierarchical clustering and further refined using the dynamic tree cut algorithm. In order to identify the clinical-trait-associated modules, we performed Pearson’s correlation coefficient analysis between the traits and MEs, which are defined as the first principal component of a given module. Gene co-expression networks was constructed using the WGCNA package in R , accordihttps://string-db.org/) [The PPI network analysis of module genes was based on the protein interaction information retrieved from the online database STRING (db.org/) . To defihttp://www.webgestalt.org/) with default parameters [Gene overrepresentation analysis for GO  and KEGG pathway was analyzed with WebGestalt (rameters . The humrameters  was usedhttps://www.ncbi.nlm.nih.gov/sra/) under the accession number SRP186138 [Raw RNA-seq data had been deposited in the NCBI Sequence Read Archive database (SRA; RP186138 .In summary, by analyzing myocardial RNA-seq data from HCM and control patients, we provide evidence that HCM is a complex genetic disorder, with pathogenic and modifier protein-coding mutations, differential gene expression, and non-coding RNA modulation contributing to extensive phenotypic variability. In addition, this work highlights the importance of integrative analysis with gene co-expression and protein–protein interaction networks for the further identification of functional HCM genes within gene subnetworks. Further elucidation of HCM genes, whether at the level of the single protein-coding gene or the gene network, will lead to improvements in genetic screening for HCM patients without an identified genetic cause and to the development of new, personalized therapeutics.


Although vertebral osteomyelitis (VO) is commonly associated with high morbidity and high recurrence rate, effective diagnostic and prognostic biomarkers of VO are still lacking.Case 1: a 60-year-old male had had upper back pain for 3 days. Case 2: a 71-year-old female presented upper back pain for 2 days.Staphylococcus aureus VO.Based on physical examination and findings of magnetic resonance imaging and findings by matrix-assisted laser desorption ionization-time of flight mass spectrometry, they were diagnosed with TM Protein Array by analyzing autoantibodies in both VO patients, potential biomarkers of VO were explored.Using Sengenics ImmunomeA, and keratin type II cytoskeletal 8 were highly differentially expressed among VO and controls. Relevant auto-antibody profiles were discovered after intra-group and inter-group comparison, and based on functional rationality, an adapter protein 14-3-3 protein gamma, and pterin-4-alpha-carbinolamine dehydratase that involved in tetrahydrobiopterin biosynthesis, might serve as valuable diagnostic biomarkers.Four subjects with more than 1600 antigens screened while the results showed that 14-3-3 protein gamma, pterin-4-alpha-carbinolamine dehydratase, fructose-bisphosphate aldolase This pilot study on 4 subjects with more than 1600 antigens screened on the Sengenics Immunome protein array provided a general outlook on autoantibody biomarker profiles of VO subjects. Future large-scale trials with longer follow-up times are warranted. Delayed diagnosis leading to disabling morbidity is common, and functional disabilities occur in 25% to 50% of cases. Early identification of VO patients is critical to ensure accurate clinical decisions and improved prognosis. Currently, the gold standard of VO diagnosis is magnetic resonance imaging (MRI). In a preliminary study, we focused on the clinical presentation of VO in Taiwan. However, the clinical diagnosis of VO remains challenging since it lacks sensitive and accurate markers for detection, and does not have a convenient gold standard for investigation. Furthermore, it is still unclear what factors might determine the relatively high morbidity and mortality, and which of these is the key determinant. Therefore, diagnostic judgments require further studies to identify prognostic biomarkers that can predict potential complications in VO patients.Vertebral osteomyelitis (VO) is an infection of the vertebrae, contiguous intervertebral disc space, and the paravertebral space. C-reactive protein (CRP), and procalcitonin (PCT) are routinely used in the diagnosis of these infections, but no specific laboratory test exists, with the exception of the isolation of pathogenic organisms from the bone or synovial fluid. CRP and ESR are currently used in all patients suspected of having VO; however, they lack the accuracy to differentiate between patients with and without VO. Anti-glucosaminidase IgG, iron-regulated surface determinant protein B, CCL11/eotaxin, interleukin-6, S100 calcium binding protein A11, E-selectin, and matrix metalloproteinase-1 have been reported as potential biomarkers; yet, they have not been used for the clinical diagnosis of VO. A review of the literature, employing a data mining approach using Open Targets Platform , presented a list of VO-associated antigens showing elevated autoantibody responses; the results are shown in Supplemental Digital Content . Finding diagnostic biomarkers that could reduce the delayed diagnosis time from VO onset, allow early initiation of the correct treatment, and have the potential to reduce overall mortality would be beneficial. However, there is currently a deficiency in available diagnostic VO biomarkers.At present, there are no reliable biomarkers that could identify VO patients with bacteremia in the clinic. Recent studies have identified several VO biomarkers, which are listed in Supplemental Digital Content (Appendix 1). Total white cell count, erythrocyte sedimentation rate (ESR), The ImmunomeTM Protein Array utilizes an immunomics technique employing a biotin carboxyl carrier protein domain affinity tag to promote fusion of post-translational biotinylated proteins to an array surface. This surface includes 1,631 proteins that serve important functions in the human immune response, including kinases and transcription factors.Development of biomarkers with high predictive value for infectious diseases is 1 of the goals for precision medicine. Early diagnosis, patient stratification, and predictive treatment of infectious diseases using biomarkers can significantly improve treatment effectiveness and the survival rate of patients, especially in the case of acute and difficult infectious diseases.TM profiling. The ImmunomeTM Protein Array also allows for the detection of autoantibodies in patient serum samples, making it an effective tool for biomarker discovery. Therefore, this study aimed to determine the differential expression levels of immune-related proteins identified in patients with different VO classifications, in order to identify proteomic biomarkers in VO patients. Furthermore, it was designed to provide insights into potential pathways of VO progression and enable the development of novel treatment strategies and appropriate clinical management of VO patients.We described 2 VO cases. Both cases are difficult to diagnose VO at the critical moment because there is lack of accurate VO biomarkers. And we designed this pilot study to identify potential VO biomarkers, those immune-related proteins expressed in VO patients with different clinical condition by Immunome2Staphylococcus aureus in 2 subjects. The initial MRI images were compatible with osteomyelitis in 2 subjects. All subjects survived. Previous potential biomarkers of VO, including ESR, CRP, and PCT were found to be partially significant.In order to detecting biomarkers of VO, we designed the approach method as shown in Supplemental Digital Content (Appendix 3). In brief, 4 subjects, including 2 VO subjects, 1 negative control subject (bone degeneration disease patient without infection), and 1 healthy control subject, were enrolled between July 1, 2018 to December 31, 2018. Of the 4 subjects, including 2 patients were analyzed ) while 2 had diabetes mellitus, 2 were hypertensive, and 1 had end-stage renal disease. None of the subjects had a predisposing traumatic condition. As for the initial signs and symptoms before admission, 3 had a painful sensation over the lesion site, and 2 had fever. The initial laboratory data showed that 2 had leucocytosis, 2 had elevated ESR, 1 had elevated CRP level, and 1 had elevated PCT level. Initial blood culture showed the presence of 2.13; ESR, 32 mm/hr; CRP, 3.48 mg/L and PCT, 0.6 ng/mL. The magnetic resonance imaging (MRI) of the spine showed that abnormal collection with T1 and T2 high signal intensity at the C4 to T2 level, around the vertebrae and suspected connection with C7/T1 disk space and adjacent C7-T1 anterior epidural space involvement are found. The C7-T1 diskitis with VO was impressed. Staphylococcus aureus (2/2 sets) was identified by using matrix-assisted laser desorption ionization-time of flight mass spectrometry  on 3th admission day, and it was oxacillin resistant strain according to the susceptibility test. Oxacillin resistant S. aureus VO was suspected, and parenteral vancomycin 500 mg plus daptomycin 300 mg (6 mg/kg) every other day after dialysis was prescribed. He received the 42-days combination regimens without adverse and unanticipated events. The serial follow-up laboratory data normalized. He was discharged on 45th admission days and was follow-up at outpatient department where he recovered well.A 60-year-old male, who had had diabetes mellitus, hypertension and end stage renal disease, was sent to our emergency department because of upper back pain for 3 days, and he presented with episodic fever and general malaise. He was admitted for further evaluation and management. He denied any traumatic injury and acupuncture and invasive therapeutics. At admission Day, his vital sign showed a blood pressure of 120/80 mmHg, body temperature 37.6°C, respiratory rate 24/min and heart beat 88/min. Physical examination and important clinical findings showed the painful sensation over intra-scapular area and there were no significant neurological deficit. The initial laboratory data revealed white cell count, 36700/mm2.23; ESR, 86 mm/h; CRP, 0.28 mg/L and PCT, 0.03 ng/mL. The MRI of the spine showed that abnormal signal intensity in the T8–9 vertebrae. The T8–9 VO was impressed. S aureus (2/2 sets) was identified by using matrix-assisted laser desorption ionization-time of flight mass spectrometry  on 3th admission day, and it was oxacillin susceptible strain according to the susceptibility test. Oxacillin susceptible S. aureus VO was suspected, and parenteral oxacillin 2000 mg every 4 hours was prescribed. She received the 42-days combination regimens without adverse and unanticipated events. The serial follow-up laboratory data normalized. She was discharged on 44th admission days and was follow-up at outpatient department where she recovered well.A 71-year-old healthy female visited to our emergency department because of upper back pain for 2 days, and she presented with fever. She was admitted for further evaluation and management. She denied any traumatic injury and acupuncture and invasive therapeutics. At admission Day, her vital sign showed a blood pressure of 140/90 mm Hg, body temperature 38.2°C, respiratory rate 26/min and heart beat 110/min. Physical examination and important clinical findings showed the painful sensation over middle part of T spine area and there were no significant neurological deficit. The initial laboratory data revealed white cell count, 9300/mmThe penetrance fold change-based method was performed for the following 2 sets of analysis. First, analysis of VO plasma versus non-VO control (bone-disease patient without infection) plasma was conducted; Supplemental Digital Content (Appendix 5) shows the top 10 antigens with high autoantibody titers identified from the analysis. Penetrance fold changes between VO plasma and non-VO control were as follows: geranylgeranyl pyrophosphate synthase (GGPS1), 20.78; replication protein A 32 kDa subunit (RPA2), 18.884; Sjoegren syndrome nuclear autoantigen 1 (SSNA1), 17.281; ornithine decarboxylase (ODC1), 14.832; keratin, type II cytoskeletal 8 (KRT8), 14.481; alpha-crystallin B chain (CRYAB), 14.137; keratin, type I cytoskeletal 19 (KRT19), 14.026; fructose-bisphosphate aldolase A (ALDOA), 13.233; cysteine-rich secretory protein 2 (CRISP2), 12.781; and cAMP-dependent protein kinase type I-alpha regulatory subunit (PRKAR1A), 12.661. Second, analysis of VO plasma compared to healthy control  plasma was performed; Supplemental Digital Content (Appendix 6) shows the top 10 antigens with high autoantibody titers identified from this analysis. Penetrance fold changes between VO plasma and the healthy control were as follows: zinc finger C4H2 domain-containing protein (ZC4H2), 11.391; KRT8, 8.819; transforming acidic coiled-coil-containing protein 1 (TACC1), 8.61; ODC1, 6.949; MOB kinase activator 3A (MOB3A), 6.291; pterin-4-alpha-carbinolamine dehydratase (PCBD1), 6.286; ALDOA, 6.027; 14-3-3 protein gamma (YWHAG), 5.998; thymidine kinase, cytosolic (TK1), 5.752; and DnaJ homolog subfamily B member 1 (DNAJB1), 5.685.An unsupervised clustering was performed for the following biomarkers across patients, controls, and pooled normal samples using a hierarchical clustering method with distance calculated based on Euclidean distance. The heatmap shown in Figures A comparison of autoantibody profiles among VO, non-VO control, healthy control, and pooled normal samples for the most clinically significant common biomarkers identified based on penetrance fold change analysis is shown in Figure Figure Clustering was performed according to Ward's method, and the Euclidean distance was calculated. Unsupervised clustering of individual fold change profiles for the 50 biomarkers among VO, non-VO control, healthy control, and pooled normal samples is shown in Figure In the comparison between the VO patients and the non-VO control, KRT8 was selected because the penetrance fold change was 14.481, and the penetrance percentage frequency was 100%. ALDOA was selected because the penetrance fold change between VO patients and the non-VO control was 13.233 and the penetrance percentage frequency was 100%. In addition, YWHAG showed a penetrance fold change of 5.998 after comparing VO patients and the healthy control group, and a penetrance percentage frequency of 100%. PCBD1 showed a penetrance fold change of 6.286 between VO patients and healthy controls, and a penetrance percentage frequency of 100%.3TM Protein Array platform. We identified 2 distinct and novel biomarkers in this preliminary study conducted in Taiwan. There were also several other major findings. First, partial significance was found for known potential biomarkers of VO, including ESR, CRP, and PCT. Second, the following target antigens showed high penetrance fold change in a comparison between VO plasma and non-VO control plasma: GGPS1, RPA2, SSNA1, ODC1, KRT8, CRYAB, KRT19, ALDOA, CRISP2, and PRKAR1A. In addition, in an analysis comparing VO plasma and healthy control plasma, the following target antigens showed high penetrance fold change: ZC4H2 KRT8, TACC1, ODC1, MOB3A, PCBD1, ALDOA, YWHAG, TK1, and DNAJB1. Third, the top ten identified antigens obtained upon comparing the autoantibody profiles among VO, non-VO control, healthy control, and pooled normal samples were GGPS1, RPA2, SSNA1, KRT8, CRYAB, KRT19, ALDOA, CRISP2, PRKAR1A, ALDOA, KRT8, MIF, ODC1, PCBD1, TACC1, TGF, YWHAG, MOB3A, and cellular tumor antigen p53. Fourth, PCBD1, ILF, TFG, GFAP, ALDOA, ODC1, KRT8, TACC1, ZC4H2 and MOB3A were identified as the top ten antigens throughout the analysis. Finally, YWHAG and PCBD1 were identified as potentially clinically significant as diagnostic biomarkers of VO after intersecting at relevant auto-antibody profiles after intra-group and inter-group comparison and interpreting based on functional rationality. However, further study is needed in order to validate these novel biomarkers. Generally, ESR, CRP, or PCT cannot be used to diagnose VO patients. This study showed that previously reported potential biomarkers of VO, including ESR, CRP, and PCT were partially significant. Yet, these findings are important for identifying clinically significant common diagnostic biomarkers of VO, and the combination of biomarkers with diagnostic and prognostic potential in a defined VO condition can improve the overall diagnostic accuracy rate. This study aimed to discover the most significant autoantibody biomarkers of VO. The proposed functional annotations of the top 10 antigens from the Immunome protein microarray platform, with significant autoantibody responses in VO vs non-VO control samples, are listed in Supplemental Digital Content (Appendix 3). The comparison of their annotations for VO patient samples vs healthy control samples is listed in Supplemental Digital Content (Appendix 7). Out of all of the significant autoantibody biomarkers listed for VO, the following biomarkers may have clinical relevance.This research explored biomarkers of VO using the Sengenics Immunomehttps://www.uniprot.org/), YWHAG was discovered to be an adapter protein to regulate both general and specialized signaling pathways. YWHAG binds to many partners, often by recognizing a phosphoserine or phosphothreonine motif. Further, YWHAG binding usually creates the modulation of the activity of its binding partner. Chu et al. reported for the first time that the expression level of miR-222 was reduced in osteosarcoma (OS) tissues as well as in OS cell lines. Furthermore, miR-222 can inhibit cell proliferation and invasion via down-regulating YWHAG. These data suggest a potential target for the biological treatment of OS.YWHAG displayed a penetrance fold change of 5.998 between VO patients and the negative control and a penetrance percentage frequency of 100%. After employing a data mining functional approach using Universal Protein Resource (UniProt) Platform  and increases the cell count, effects which are blocked by addition of IGF-I receptor antibodies. Herzlieb et al. also hypothesized that IGF-I affects cells via 2 pathways: by promoting proliferative responses and by suppressing PCD. The differential responses of PCD and cell number to caspase 1 inhibitors suggest the existence of an independent control system for these processes.The second potential biomarkers with clinical relevance in VO is PCBD1, with a penetrance fold change of 6.286 between VO patients and healthy controls and a penetrance percentage frequency of 100%. PCBD1 was found to be involved in tetrahydrobiopterin biosynthesis, and it seems to both prevent the formation of 7-pterins and accelerate the formation of quinonoid-BH2. In addition, it may also function as a scaffolding protein. The biomarker points resistance to cisplatin for osteosarcoma and proposes for the early and accurate use of cisplatin to treat osteosarcoma. Zhang et al. described that ALDOA and PGK1 might become appropriate biomarkers for treating osteosarcoma with cisplatin.The third potential biomarker is ALDOA, with a penetrance fold change of 13.223 between VO patients and non-VO control and a penetrance percentage frequency of 100%. Between VO patients and the healthy control group the penetrance fold change was 6.027 and the penetrance percentage frequency was 100%. It was found that ALDOA plays a key role in glycolysis and gluconeogenesis. Le Henaff et al. described that cystic fibrosis patients show low bone mass and alterations of bone structure, and mice bringing the F508del genetic mutation in the cystic fibrosis conductance regulator (Cftr) gene present decreased bone formation and reduced bone mass. The KRT8 could cause to correct osteoblast dysfunctions, to alter bone formation. And osteopenia in F508del-Cftr mice provides the KRT8-F508del-CFTR reaction to reform the abnormal bone structure in cystic fibrosis. In addition, the potential proteins associated with VO disease showing elevated autoantibody responses were reviewed based on literature and a data mining approach using Open Targets Platform  as well as UniProt Platform (https://www.uniprot.org/). The results are shown in Supplemental Digital Content . Furthermore, Supplemental Digital Content (Appendix 9) lists annotations of biomarkers of bone diseases, including VO. Other biomarkers were observed which might play a significant role in immune responses related to VO. The autoantibody profiles of each case will gain unique significance when associated with the pathophysiology of the patients. This will help to identify potentially sustainable biomarkers that can assist with VO diagnosis. After discovering relevant autoantibody profiles and performing intra-group and inter-group comparisons, as well as interpreting them based on functional rationality, YWHAG and PCBD1 in particular showed potential as valuable diagnostic biomarkers of VO.The fourth potential biomarker is KRT8, with a penetrance fold change of 14.481 between VO patients and the non-VO control group and a penetrance percentage frequency of 100%. Between VO patients and the healthy control group the penetrance fold change was 8.819 and the penetrance percentage frequency was 100%. It has been reported that KRT8 can link the contract apparatus to dystrophin at the striated muscle.This pilot study on 4 subjects with more than 1600 antigens screened on the Sengenics Immunome protein array provided a general outlook on autoantibody biomarker profiles of VO subjects. YWHAG, and PCBD1 in particular might serve as valuable diagnostic biomarkers. Therefore, further research including a large-scale autoantibody profiling study to identify biomarkers with diagnostic and prognostic potential in a defined VO condition is warranted to validate these novel biomarkers.This research project would not have been possible without the support of many people. The authors wish to express their gratitude to the staffs of the Department of Neurosurgery, Orthopedic Surgery, Division of Critical Care Medicine, the Division of Infectious Diseases, the Department of Pharmacology, the Department of Computer Science and the Department of Healthcare Quality at Changhua Christian Hospital who were extremely helpful and provided invaluable assistance and support. The authors would also like to thank AllBio Co. Ltd.  and Sengenics Corporation Pte Ltd. (Singapore) for technical support. This manuscript was edited by Editage Editing.Acquisition of data: CHC, ILC, HCY, and SHWAnalysis and/or interpretation of data: CHC, SCL, and CCHApproval of the version of the manuscript to be published: CHC, ILC, HCY, SHW, SCL, and CCHConception and design of study: CHC, ILC, HCY, SHW, JSL, and CCHDrafting the manuscript: CHC, and CCHRevising the manuscript critically for important intellectual content: CHC, and CCH


KCNA2, encoding for the voltage-gated potassium channel Kv1.2, have been identified as the cause for an evolving spectrum of neurological disorders. Affected individuals show early-onset developmental and epileptic encephalopathy, intellectual disability, and movement disorders resulting from cerebellar dysfunction. In addition, individuals with a milder course of epilepsy, complicated hereditary spastic paraplegia, and episodic ataxia have been reported. By analyzing phenotypic, functional, and genetic data from published reports and novel cases, we refine and further delineate phenotypic as well as functional subgroups of KCNA2-associated disorders. Carriers of variants, leading to complex and mixed channel dysfunction that are associated with a gain- and loss-of-potassium conductance, more often show early developmental abnormalities and an earlier onset of epilepsy compared to individuals with variants resulting in loss- or gain-of-function. We describe seven additional individuals harboring three known and the novel KCNA2 variants p.(Pro407Ala) and p.(Tyr417Cys). The location of variants reported here highlights the importance of the proline(405)–valine(406)–proline(407) (PVP) motif in transmembrane domain S6 as a mutational hotspot. A novel case of self-limited infantile seizures suggests a continuous clinical spectrum of KCNA2-related disorders. Our study provides further insights into the clinical spectrum, genotype–phenotype correlation, variability, and predicted functional impact of KCNA2 variants.Pathogenic variants in  KCNA2, encoding for the voltage-gated potassium channel subfamily A member 2  is a member of the shaker-like delayed rectifier potassium channel family [v-subfamily members contribute to functional channel characteristics [v1.2 consists of six transmembrane segments (S1–S6) forming the voltage-sensing domain (S4) and the ion-conducting pore (S5-S6) with its selectivity filter localized in the pore loop [v1 channel components, such as leucine-rich glioma-inactivated 1, contactin-associated protein 2, and particularly Kv1 channel subunits [Potassium channels are involved in many vital processes such as neuronal excitability, neurotransmitter release, and cellular osmoregulation ,2. KCNA2l family . It is pl family ,5. Homo-ore loop . The C-tore loop ,10. Sevesubunits ,12.KCNA2 were first described in six individuals with early-onset severe epilepsy, cognitive impairment, and ataxia [v1.2 [KCNA2 variant c.881G > A, leading to an amino-acid exchange at position 294 within the voltage sensing domain, was identified in individuals with hereditary spastic paraplegia (HSP) as an additional phenotype [v1.2 dysfunction: GOF, LOF, and a mixed dysfunctional type (GOF/LOF) [In 2015, pathogenic de novo variants in d ataxia . Two pheia [v1.2 ,14. The GOF/LOF) .KCNA2. We reevaluate phenotypic, functional, and genetic data from all published cases to refine and further delineate the assumed phenotypic and functional subgroups. We analyze the genetic distribution of all known pathogenic and likely pathogenic variants in KCNA2 and predict the functional impact of all possible missense variations in KCNA2.In this study, we present additional individuals with known and novel variants in KCNA2 variants from 28 publications (78 cases) and ClinVar database entries (37 cases). Additionally, we report clinical data from seven novel cases, six individuals with P and LP KCNA2 variants, and one individual with a VUS. After the exclusion of all individuals without sufficient clinical information, we included 76 of 115 cases into further analyses  yses see .KCNA2 are on record to date, with 26 single-nucleotide variants (SNV), one in-frame deletion, and three different deletions  and novel variants (n = 2) in KCNA2 were ascertained. Genetic and phenotypic data are summarized in Data from seven previously unpublished cases with known pathogenic (n = 18).Mean age at inclusion and publication was 17 years . No individual had died until the last reported study visit. Nearly all (91%) reported cases developed seizures. Mean age at seizure onset was 13 months . In total, 41 male and 33 female individuals were included (three cases with missing information). No relevant differences in gender distribution on clinical manifestation or genotype were observed. Seven individuals were reported as not having seizures at all, of whom five harbor the variant p.(Arg294His) associated with hereditary spastic paraplegia and ataxia (HSP)  and two KCNA2, electrophysiological channel characterization has been available in 13/30 (43%) variants. Six different electrophysiological LOF variants in KCNA2 have been described [KCNA2 variants caused GOF in cellular expression systems [v1.2 for phenotypic comparison , later onset of epilepsy (p = 0.033), more favorable epilepsy outcome (seizure-free) (p = 0.014), and less severe intellectual disability (p = 0.029). Compared to individuals with LOF/GOF variants, epilepsy onset is significantly later in GOF individuals (p = 0.008), whereas development before seizures, epilepsy outcome, and intellectual disability did not differ significantly. Comparing individuals with LOF and GOF variants, development before seizures, epilepsy onset, seizure outcome, and intellectual disability was not significantly different. Compared to individuals with a mixed dysfunctional type (LOF/GOF), individuals with LOF variants showed better development before seizure onset , a continuous spectrum from mild to severe neurologic manifestations and paroxysmal movement disorders can be delineated [Recently, different neurological disorders, including epilepsy syndromes, ataxia, and hereditary spastic paraplegia were associated to Klineated ,45. By cKCNA2. The variant p.(Tyr417Cys) was identified in a three-year-old boy (#1) with benign epilepsy of infancy (BFIS) and normal development. No other variants in known genes for BFIS were identified. The variant was inherited from an unaffected father. It is classified as a variant of unknown significance (VUS) according to the American College of Medical Genetics and Genomics (ACMG)  [KCNA2-related epilepsy. Therefore, a role of the specific KCNA2 variant p.Tyr417Cys in benign infantile epilepsy (BFIS) appears possible, with a reduced penetrance, similar to other known genetic causes of infantile seizures such as PRRT2 [SCN2A [SCN8A [KCNA2- and recently in KCNA1-associated early-onset epileptic encephalopathy [v-channel suggested a non-conducting mutant channel as the underlying dysfunction [We here report seven children with three known pathogenic variants and two novel variants in P2, PP3) . This vaP2, PP3) ,48. The as PRRT2 , SCN2A [2 [SCN2A , and SCNA [SCN8A . The novA [SCN8A . The varalopathy ,36,39,52function . This noThe variant p.(Pro405Leu) affects the first proline of the PVP motif. We report two additional children with infantile-onset epilepsy and prolonged febrile seizures, harboring this recurrent variant. One of the two children had moderate intellectual disability, while the other one was described as normally developed. The mean age at seizure onset in all reported individuals with p.(Pro405Leu) was 9 months, and the main seizure types included focal and generalized seizures as well as febrile seizures. Continuous spikes and waves during sleep (CSWS) were reported in 8/13 individuals ,34,37,38We identified the variant p.Arg297Gln) in a 9-year-old male patient (#5) with infantile onset epileptic encephalopathy and ataxia, showing significant overlap to previously reported individuals with this GOF variant, who constantly developed ataxia ,27,28,297Gln in aCACNA1A [In twins, one boy and one girl (#6 and #7), we identified the recurrent variant c.881G>A; p.(Arg294His), which was described previously in families with hereditary spastic paraplegia ,20. SimiCACNA1A . SimilarKCNA2 variants with a genotype–phenotype correlation based on the electrophysiological channel properties were suggested [v1.2 both lead to epileptic encephalopathy as well. Recently, Shore et al. (2020) provided data, showing how GOF variants can lead to epilepsy [KCNT1-gain-of-function variants in excitatory and inhibitory neurons, they were able to demonstrate specific mutational effects affecting inhibitory neurons only, especially interneurons with fast-spiking activity, promoting network hyperexcitability and hypersynchronicity. Cell-type-specific functional changes from distinct variants might also explain the more severe epilepsy course in children carrying variants with mixed LOF and GOF effects. It can be hypothesized that specific effects dominate in different neuronal subpopulations, leading to an impaired GABA-ergic response from interneurons on one side and to impaired glutamatergic transmission in excitatory neurons on the other side, affecting different inhibitory and excitatory networks. Interestingly, the homologous shaker mutation of the LOF variant p.(Arg294His) was shown to lead to proton currents in addition to the pure loss-of-Kv1.2 function when expressed in Xenopus laevis oocytes [v1.2.Previously, three distinct subgroups of uggested . We hereepilepsy . By expr oocytes ,56. Thesn = 7) had neonatal refractory epilepsy. All showed severe intellectual disability, and in four of seven individuals, spastic cerebral palsy was reported  are more likely to have febrile seizures and CSWS than other individuals in the LOF subgroup. Individuals with the variant p.(Arg294His) developed spastic paraplegia and ataxia as prominent manifestations. Seizures were described only in two previously reported cases  and in tv1.2  and p.(Pro417Tyr) suggests effects on protein stability and protein structure of Kv1.2 . Importaalopathy .KCNA2 variants containing clinical and/or genetic data were identified using PubMed, Human Gene Mutation Database (HGMD), and ClinVar  and all individuals without sufficient clinical information , 76 individuals were included into further statistical analyses . Variants were standardized to the NM_004974.3 transcript of the GRCh37/hg19 human reference genome using the Mutalyzer Nomenclature Checker web tool [KCNA2 coding sequence were computed and translated to the corresponding genomic position. Subsequently, established variant effect prediction (VEP) scores  were annotated using the VEP web tool of Ensembl Release 101  in order to evaluate the likelihood of pathogenic variant effects [v1.2 protein using the geom_smooth and geom_density function, respectively, of the ggplot2 library in RStudio  [To visualize differences in variant distribution between affected individuals and unaffected controls, we compiled all currently known pathogenic and benign web tool . Additio effects ,63,64,65MA, USA) . The REVMA, USA) . TherefoKCNA2 missense variants p.(Pro405Leu), p.(Pro407Ala), and p.(Tyr417Cys) in the quaternary protein structure were analyzed and visualized using PyMol  installed through Anaconda  and the existing PDB template 3LUT (chain B: α-subunit) of rat Kv1.2.Localization and structural consequence of the identified Due to limited and heterogeneous quality of data in publications, phenotypic features might not have been completely accessible. Therefore, the number of patients with available information is specified with each clinical feature. Due to differing age at imaging, the detectability of cerebellar changes might vary between reports. Despite the reasonable ability of the software programs to predict pathogenicity, they still cannot discriminate among functional LOF, GOF, and LOF/GOF-type variants. This is a critical limitation of computed predictions of variant effects in clinical practice and for genetic counseling. Furthermore, some recurrent variants represent a significant proportion of the cohort and variant-specific effects may dominate the phenotypic presentation of the whole functional subgroup.KCNA2 variants. The location of variants reported here highlights the importance of the PVP motif in transmembrane domain S6 as a mutational hotspot. A novel case of self-limited infantile seizures suggests a continuous clinical spectrum of KCNA2-related disorders. The prediction of functional relevance of variants allowed us to identify hotspots of functional impact and recurrent mutations.Our study provides further insights into the clinical spectrum, genotype–phenotype correlations, variability, and predicted functional impact of


PKD1 or PKD2, which are causal genes for autosomal dominant polycystic kidney disease (ADPKD). To determine whether PKD gene overexpression is a universal mechanism driving cystogenesis or is merely restricted to rodents, other animal models are required. Previously, we failed to observe any renal cysts in a transgenic porcine model of PKD2 overexpression partially due to epigenetic silencing of the transgene. Thus, to explore the feasibility of porcine models and identify potential genes/pathways affected in ADPKD, LLC-PK1 cells with high PKD2 expression were generated. mRNA sequencing (RNA-seq) was performed, and MYC, IER3, and ADM were found to be upregulated genes common to the different PKD2 overexpression cell models. MYC is a well-characterized factor contributing to cystogenesis, and ADM is a biomarker for chronic kidney disease. Thus, these genes might be indicators of disease progression. Additionally, some ADPKD-associated pathways, e.g., the mitogen-activated protein kinase (MAPK) pathway, were enriched in the cells. Moreover, gene ontology (GO) analysis demonstrated that proliferation, apoptosis, and cell cycle regulation, which are hallmarks of ADPKD, were altered. Therefore, our experiment identified some biomarkers or indicators of ADPKD, indicating that high PKD2 expression would likely drive cystogenesis in future porcine models.Consistent with the gene dosage effect hypothesis, renal cysts can arise in transgenic murine models overexpressing either  PKD1 and PKD2 are two causal genes for ADPKD and account for approximately 85% and 15% of cases, respectively [2+ homeostasis, apoptosis, extracellular matrix deposition, planar cell polarity, and fluid secretion across the epithelium [Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic causes of end-stage renal disease, affecting millions of people worldwide . As a syectively ,5. The pithelium .PKD1/2 expression) [PKD1 or PKD2 gene with the inherited germline mutant allele drives cysts formation. However, exceptional cases were observed, in which persistent expression of polycystins could be noted in cyst lining epithelia of some ADPKD patients, making dosage effect of polycystin above/under certain threshold a plausible explanation of ADPKD [PKD1 and PKD2 transgenic rodent models exhibited renal cysts and other organ abnormalities [PKD genes could drive cystogenesis due to the overexpression dosage effect. However, to date, only murine models have been established, and other animal models are required to recapitulate the phenotype and validate the overexpression model to exclude the species-specific propensity of renal cystogenesis. As pigs, which are similar to human beings in physiology, anatomy, and metabolism, are ideal large animal models for human diseases, we attempted to construct a PKD2 overexpression porcine model [Several genetic hypotheses, e.g., the two-hit ,8, thirdression) ,13, haveof ADPKD ,15. In tmalities ,17. Thusne model . Unfortune model ,20. One ne model ,20. Therne model .PKD2 overexpression porcine models and identifying potential disrupted genes/pathways, LLC-PK1 cells were transfected with four PKD2 constructs of different genotypes. Via mRNA sequencing (RNA-seq) and enrichment analysis, three commonly upregulated genes , along with enrichment of MAPK pathways and several biological processes involved in proliferation, apoptosis, and cell cycle regulation, were identified in our cellular overexpression models, indicating that these cellular models likely recapitulate ADPKD. The results not only provide functional information, e.g., biomarkers and disrupted pathways, regarding the progression of ADPKD but also facilitate the future characterization of porcine models of ADPKD.In the current study, to explore the feasibility of constructing PKD2 gene sequence was customized and purchased from Cyagen . Then, a primer pair (PKD2-up and PKD2-down) was used to amplify the entire PKD2-3×FLAG sequence using Phusion high-fidelity polymerase . The amplified fragment was gel purified  and ligated into the NotI-digested pCAG-floxP-neo-pH11 plasmid using the In-fusion method  to construct the pCAG-WThPKD2-3×FLAG-floxP-neo-pH11 (WT) plasmid. Finally, the mutant PKD2 plasmids pCAG-muhPKD2 c.A1532T/p.D511V-3×FLAG-floxP-neo-pH11 (Tg1), pCAG-muhPKD2 c.T1967G/p.L656W-3×FLAG-floxP-neo-pH11 (Tg2), and pCAG-muhPKD2 c.C2224T/p.R742X-3×FLAG-floxP-neo-pH11 (Tg3) were constructed using a site-specific mutation kit . A diagram of the PKD2 overexpression plasmid is shown in The plasmid pCAG-hPKD2-3×FLAG containing the WT human PKD2-containing plasmid and empty plasmid  were transfected in quadruplicate using Lipofectamine 3000  according to the guidelines. Forty-eight hours later, one of the quadruplicate samples was used for protein extraction, while the other three replicates were used for RNA isolation.LLC-PK1 cells were obtained from the American Type Culture Collection  and cultured in 6-well plates in M199 medium  containing 3% fetal bovine serum . When the cells reached 80% confluency, three micrograms of each Protein extraction was performed using Western and IP Cell Lysis Buffer . Approximately 30 µg of protein extract was loaded for SDS-PAGE. Anti-FLAG  was used for immunoblotting at a dilution of 1:1000. Ponceau staining was used as loading control according to the manufacturer’s guideline . For detecting the expression of Polycystin-2 and MYC levels in transgenic pigs, kidney proteins from five years old transgenic pigs were prepared using the Western and IP Cell Lysis Buffer  according to previously published protocols ,22. Antiftp://ftp.ensembl.org/pub/release-98/fasta/sus_scrofa/cdna/). Identification of the differentially expressed gene (DEG) set  was then performed with the DESeq2 (version 1.26.0) package [Forty-eight hours after transfection, cells were harvested, and total RNA was extracted using a MiniBEST Universal RNA extraction kit . Then, RNA was subjected to library construction and sequencing on the Illumina HiSeq X Ten platform , and quality control was performed according to the Novogene  pipeline. Salmon software (version 0.14.1) , which c package . In addi package ,26. The https://david.ncifcrf.gov) to identify significantly enriched gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways [p-value of ≤ 0.05 were identified as functionally enriched.DEGs were submitted to the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool  according to a previously established protocol . The priPCR data .PKD2, pig PKD2, MYC, ADM, IER3, and PKD1. Data are presented as the means ± standard errors of the mean (SEMs). A p-value of less than 0.05 was considered to indicate significance.One-way ANOVA with Dunnett’s post hoc test was employed to compare the expression levels of human All the procedures were conducted according to the guidelines developed by the China Council on Animal Care and Protocol, and approved by the Animal Welfare Committee of Zhejiang University (no. 11834 issued on 26 February 2018). The experiments conducted on the transgenic pigs were also approved by the China Agricultural University , where these transgenic pigs were.PKD2 gene. This cell line was selected because it was isolated from the renal proximal tubule epithelium of a male Hampshire pig and is thus an ideal model for both kidney research and pig biomedical studies [Currently, no clinically approved drug can halt or reverse ADPKD. Tolvaptan, which is the only available chemical to slow disease progression, targets and inhibits arginine vasopressin V2 receptor mediated cyclic adenosine monophosphate (cAMP) signaling pathways in collecting duct epithelial cells . Since c studies .PKD2 was used to establish the pure overexpression model of ADPKD, and the Tg1, Tg2, and Tg3 mutations were selected based on the information in the ADPKD mutation database (http://pkdb.mayo.edu) [Tg1 and Tg3 are listed as definitely pathogenic; Tg1 is considered a dominant-negative mutant disrupting intracellular calcium release [Tg3 lacks the C-terminal tail and cannot interact with PC1 [Tg2 was identified in a patient with early-onset ADPKD who harbored a homozygous PKD2-L656W mutation due to uniparental disomy [Tg2 mutant is likely pathogenic owing to the disruption of the channel pore structure [WT ayo.edu) . Tg1 and release , while Twith PC1 . Tg2 wasl disomy . The restructure . The bactructure . Thus, tPKD2. As shown in PKD2 had a very high expression level compared to NC, while the endogenous pig PKD genes were not affected. As we assumed that overexpression of these PKD2 constructs affects several signaling pathways and biological processes, some commonly regulated genes and pathways might be identified as potential contributors to ADPKD. Thus, RNA-seq was carried out, and DEGs were identified by separately comparing the reads from the WT, Tg1, Tg2, and Tg3 models with those from the NC model  [IER3 was upregulated in a model of CKD [ADM and IER3 upregulation might be useful as indicators of disease progression.After transfection of LLC-PK1 cells with the five plasmids, Western blotting was performed to confirm the expression of these proteins except Tg3, which lacks the C-terminal region and the FLAG epitope a. qRT-PCNC model . Ten uprNC model . A totalNC model . In the NC model . The Tg3NC model . Among tenotypes a, and thenotypes . The proof ADPKD ,37. In apression ,39,40,41ogenesis . In our al years . Howeveral years ,46,47,48 studies ,51,52,53se (CKD) ,55. One l of CKD . Since APKD2, additional terms such as reactive oxygen species, morphogenesis of epithelium, nitric oxygen biosynthesis, MAPK regulation, and cytokine-mediated pathways were also enriched. The 10 DEGs overexpressed in the WT model were predicted to be involved in the MAPK, JNK, and p38MAPK pathways (+/-PKD2 kidneys with TNF-α led to aggravated cyst formation [Next, the DEGs identified in these cells were subjected to GO and KEGG enrichment analyses using the DAVID tool . The ovepathways b. Thus, ormation . Thus, bPKD2 overexpression strategy, we identified several disrupted pathways/processes related to ADPKD. Additionally, MYC, ADM, and IER3 were identified as potential disease contributors or indicators. This information not only provides the first identification of the shared genes and pathways affected based on the overexpression hypothesis in ADPKD but also lays a foundation for future characterization of porcine models of PKD2 overexpression.In the current study, by employing a cellular ADPKDAutosomal dominant polycystic kidney diseasePKD1/PKD2Polycystic kidney disease gene 1/2PC1/2Polycystin-1/2DEGDifferentially expressed geneGOGene ontology; KEGGKyotoEncyclopedia of Genes and GenomesWTWild typeTgTransgenicRNA-seqmRNA sequencingMAPKMitogen-activated protein kinasecDNAComplementary deoxyribonucleic acidcAMPCyclic adenosin monophosphate