Intracellular Ca2+ signals are generated by Ca2+ release from intracellular Ca2+ stores and by Ca2+ entry through a multitude of Ca2+-permeable ion channels in the plasma membrane. Over the past decade, the transient receptor potential (TRP) channels have emerged as one of the most important families of ion channels mediating Ca2+ signaling in cardiac fibroblasts. TRP channels are a superfamily of non-voltage-gated, Ca2+-permeable non-selective cation channels. Their ability to respond to various stimulating cues makes TRP channels effective sensors of the many different pathophysiological events that stimulate cardiac fibrogenesis. This review focuses on the mechanisms of Ca2+ signaling in fibroblast differentiation and fibrosis-associated heart diseases and will highlight recent advances in the understanding of the roles that TRP and other Ca2+-permeable channels play in cardiac fibrosis.Cardiac fibrosis is the excessive deposition of extracellular matrix proteins by cardiac fibroblasts and myofibroblasts, and is a hallmark feature of most heart diseases, including arrhythmia, hypertrophy, and heart failure. This maladaptive process occurs in response to a variety of stimuli, including myocardial injury, inflammation, and mechanical overload. There are multiple signaling pathways and various cell types that influence the fibrogenesis cascade. Fibroblasts and myofibroblasts are central effectors. Although it is clear that Ca Cardiac fibrosis is involved in pathological remodeling of the heart, causing abnormalities in cardiac conduction, stiffness of the ventricular walls, reduced contractility, and impaired overall heart performance . Thus, c2+ has been found to play a particularly critical role .,46.36,46d in rat ,48, canid in rat ,50, and d in rat  fibroblad in rat , canine d in rat , and humd in rat  fibroblaeriments . Upregul failure . Anotherel, KATP ,52,54. Aecretion . Functiomyocytes . Severale (BKCa) ,57,58 ane (BKCa) , have bee (BKCa) .+ channels have been reported to be expressed in cardiac fibroblasts i) influence the activation of many TRP channels. A rise of intracellular Ca2+ ([Ca2+]i) activates several TRP channels, including TRPM4 [2+ ([Ca2+]i) can activate TRPV5 and TRPV6. TRPM2 can also be activated by multiple stimuli, including [Ca2+]i, ADP-ribose (ADPR), NAD+, and oxidative stress [TRP channels are not gated by voltage, but rather, these channels are responsive to a wide range of stimuli in a polymodal activation manner, including thermal, mechanical, oxidative, chemical, and nociceptive stresses, and local autocrine or paracrine environmental cues ,139. TRPntration ,149,150,ng TRPM4 , TRPM5 [ng TRPM4 ,153, TRPng TRPM4 , and TRPng TRPM4 ; in conte stress ,157,158.Many TRP channels have been detected at the RNA level by qPCR or RT-PCR in the cardiac fibroblasts of various species ,46,159. 2O2 treatment, suggesting hypoxia up-regulates TRPM2 in rat cardiac fibroblasts [2+ signaling in cardiac fibroblasts, and their potential roles in cardiac fibrosis and fibrosis-associated heart diseases [The first TRP channel current recorded in cardiac fibroblasts was the TRPM7 current recorded in rat cardiac fibroblasts . TRPM7 croblasts . The TRProblasts . Thus, mroblasts ,163. In diseases ,164,165 The role of TRPC channels in hypertrophy and heart failure has been extensively studied by the systematic or myocardial knockdown, overexpression, and knockout of TRPC channels, as previously reviewed ,165,201.2+ entry through TRPC3 [2+ release and subsequent Ca2+ entry via TRPC channel activation, which leads to fibroblast differentiation [Despite knowledge of the role of TRPC channels in fibroblasts in hypertrophic remodeling being limited, it has been shown that the pathological stimuli AngII and ET1 can indeed induce Cagh TRPC3  and TRPCgh TRPC3 , respectntiation . Nonethentiation ,166.TRPC3 and Fibrosis-Associated Arrhythmia and Heart Failure2+ signaling activates PKC, which then phosphorylates p47phox and activates NADPH oxidase 2 (Nox2) to generate ROS, thereby initiating the RhoA signaling pathway and ECM production [TRPC3 channels have been shown to play a role in cardiac fibrosis and fibrosis-associated heart diseases, such as atrial fibrillation (AF)  and hearoduction ,168,169.2+ signaling in fibroblasts is also involved in arrhythmogenesis [2+ influx, which activates extracellular signal-regulated kinase (ERK1/2) signaling [2+ signaling appears to reduce the susceptibility to AF, perhaps by reducing fibroblast cell proliferation. Overall, the data suggest that TRPC3 is likely a potential therapeutic target for fibrosis-associated atrial fibrillation [TRPC3-mediated Caogenesis . TRPC3 iogenesis . The mecogenesis . Enhanceignaling . TRPC3-mignaling . In vivoignaling . Thus, rillation .TRPC6-Mediated Fibroblast Differentiation and Heart Failure2+ influx by ET1 in neonatal rat ventricular fibroblasts activates NFAT, which acts as a negative regulator of ET1-induced fibroblast differentiation [2+ signaling is required for human cardiac fibroblast proliferation induced by AngII and OAG (1-oleoyl-2-acetyl-sn-glycerol); the latter is a diacylglycerol (DAG) analogue [-/-) were not able to differentiate into myofibroblasts in response to TGF-β1 stimulation [TRPC6 has been reported to regulate fibroblast differentiation induced by ET1 in rat ventricular fibroblasts . TRPC6-mntiation . In contntiation . It has analogue . Consistanalogue . It has analogue . Activatanalogue . Fibroblmulation . Also, mmulation . Consistmulation . Moreovemulation , suggestSeveral TRPV channels have been implicated in cardiac fibrosis, including TRPV1, TRPV2, TRPV3, and TRPV4. We summarize their putative roles below.TRPV1 and Cardiac FibrosisTRPV1 is predominately expressed in peripheral sensory neurons and is widespread in the cardiovascular system . The effTRPV2 and Cardiac Fibrosis2+-induced myocyte degeneration in dilated cardiomyopathy [2+ signaling in cardiac fibroblasts regulates fibroblast differentiation and cardiac fibrosis. Further studies are required to dissect the different roles of TRPV2 during heart injury.TRPV2 has been shown to play a role in Camyopathy . A knockmyopathy . A knockmyopathy . Applicamyopathy . Althougmyopathy . TRPV2 imyopathy . TRPV2 imyopathy . Thus, iTRPV3 and Cardiac FibrosisTRPV3 is expressed in the peripheral neurons and various other cell types. It has recently been found that TRPV3 is also expressed in cardiac myocytes  and fibrTRPV4 and Cardiac Fibrosis2+ influx. The application of a TRPV4 antagonist and an shRNA knockdown of TRPV4 inhibits TGF-β1-induced myofibroblast differentiation, whereas TGF-β1-treated fibroblasts exhibit enhanced TRPV4 expression and increased Ca2+ influx [TRPV4 has been shown to regulate fibroblasts’ differentiation into myofibroblasts by integrating TGF-β1 signals and mechanical stimulation ,184. In + influx . In a lu+ influx . Similar+ influx . These s2O2 [Several TRPM channels have been shown to play important physiological or pathological roles in the heart. TRPM2 has been found to be involved in ischemic cardiomyopathy, with protective roles reported by some studies ,187, and2O2 . However2O2 ,215,216.Physiological Function of TRPM7 in the Heart+/Δkinase) exhibit cardiac hypertrophy, fibrosis, and inflammation [+/Δkinase mice [+/Δkinase mice [TRPM7 is essential for embryogenesis , organogammation . It was ammation . Howeverase mice , it is pase mice .Expression of TRPM7 in Cardiac Fibroblasts2, but they have distinct single-channel conductances and pharmacological sensitivities to low and high concentrations of 2-APB [TRPM7 is also highly expressed in cardiac fibroblasts. Endogenous TRPM7-like currents were initially recorded in rat cardiac fibroblasts , and werof 2-APB ,161,224.TRPM7 and Fibroblast Differentiation2+ entry is significantly enhanced in fibroblasts from AF patients. Knockdown of TRPM7 inhibits TGF-β1-induced fibroblast proliferation, differentiation, and collagen production [2+ signaling is essential for TGF-β1-induced fibroblast proliferation, differentiation, and the fibrogenesis cascade [TRPM7-mediated Caoduction , whereasoduction . It appe cascade . A role  cascade ,195,196. cascade , and in 2+ influx in primary cultured human ventricular cardiac fibroblasts [2+/calmodulin-dependent protein kinase II (CaMKII) and calcineurin signaling pathways [TRPA1 is expressed in cardiac myocytes ,198 and roblasts . TRPA1 spathways . The knopathways . Moreovepathways . 2+-ATPase (SERCA) and the plasma membrane Na+/Ca2+ exchanger (NCX), the two major components that extrude cytosolic Ca2+ to the SR/ER and outside of the cell, the plasma membrane Ca2+-ATPase  was considered to be relatively insignificant in maintaining Ca2+ homeostasis in the heart [In contrast to the sarcoplasmic reticulum (SR) Cahe heart ,226. In 2+ level in PMCA4-KO fibroblasts is 25% higher than that in control fibroblasts. PMCA4 ablation also increases secreted, frizzled-related protein 2 (sFRP2) expression, which inhibits the hypertrophic response in myocytes [2+ homeostasis in fibroblasts can also mitigate fibrosis-associated heart diseases.Among the four PMCAs, the expression of PMCAs 1, 3, and 4 has been detected at the mRNA level in human cardiac fibroblasts . Recentlmyocytes . Moreovemyocytes . These r2+ homeostasis in many cell types, such as cardiac myocytes. In cardiac fibroblasts, the mRNA expression of NCX and SERCA has also been detected in human ventricular fibroblasts [2+ homeostasis in fibroblasts [2+, and extracellular Na+ replacement, on Ca2+ oscillations in human ventricular fibroblasts. However, the detailed characterization of NCX and SERCA in cardiac fibroblasts, and their roles in fibroblast function and in cardiac fibrosis, still remain largely unknown.NCX and SERCA are essential to maintaining Caroblasts . It was roblasts , based o2+ signaling mechanisms controlling cardiac fibroblast functions are not fully understood. Nevertheless, the advances in recent years have helped to shape our understanding of the complex nature of Ca2+ signaling mechanisms in fibroblasts, while TRP channels with their unique features have emerged as the most important ion channels that mediate Ca2+ signals in cardiac fibroblasts. Evidence suggests that many TRP channels have the potential to be therapeutic targets for drug intervention. However, more work is necessary to understand TRP channels’ functions in the heart. As TRP channels are widely expressed in different cell types, future investigations using cell type-specific deletions of TRP channel genes will provide more precise information regarding their specific roles in cardiac fibroblasts. Moreover, as in vitro fibroblast experiments are an important part of cardiac fibroblast research, finding the experimental conditions that closely mimic the in vivo properties of fibroblasts and myofibroblasts will be critical. Finally, since fibrogenesis involves multiple cell types in the heart, gaining a better understanding of the fibroblast differentiation process, and how Ca2+ signaling influences the interactions of fibroblasts and myofibroblasts with myocytes and other cell types, is an important challenge whose outcomes will provide new insights into the therapeutic potential of TRP channels in fibrosis-associated heart diseases. Fibrosis is a hallmark feature of most heart diseases. There are a multitude of signaling pathways, bioactive molecules, and various cell types that are involved in the cardiac fibrogenesis cascade. As the major cell type in the fibrogenesis cascade, fibroblasts are the receivers of various pathological stimuli and the producers of extracellular matrix proteins. However, the Ca


Cardiomyopathies are a heterogeneous group of inherited cardiac diseases characterized by progressive myocardium abnormalities associated with mechanical and/or electrical dysfunction. Massive genetic sequencing technologies allow a comprehensive genetic analysis to unravel the cause of disease. However, most identified genetic variants remain of unknown clinical significance due to incomplete penetrance and variable expressivity. Therefore, genetic interpretation of variants and translation into clinical practice remain a current challenge. We performed retrospective comprehensive clinical assessment and genetic analysis in six families, four diagnosed with arrhythmogenic cardiomyopathy, and two diagnosed with hypertrophic cardiomyopathy (HCM). Genetic testing identified three rare variants , each present in two families. Although each variant is currently classified as pathogenic and the cause of the diagnosed cardiomyopathy, the onset and/or clinical course differed in each patient. New genetic technology allows comprehensive yet cost-effective genetic analysis, although genetic interpretation, and clinical translation of identified variants should be carefully done in each family in a personalized manner. Sudden cardiac death (SCD) is a major cause of death with an incidence ranging from approximately 1:1000 to 1:3000 individuals per year in the general population . In popuIn post-mortem cases, current guidelines recommend molecular autopsy in all cases without a conclusive cause of death and without any cardiac alteration, but also if a cardiac alteration suspicious of cardiomyopathy is identified . In addiMassive sequencing technologies such as next generation sequencing (NGS) now allow comprehensive genetic analysis of families in a cost-effective way – genetic data can be obtained in reduced time and at low cost in comparison to traditional Sanger sequencing . The maiInformation of family history, segregation of genetic variants, and genotype-phenotype correlations are crucial to elucidate the role of each genetic variant . FurtherThis study was approved by the Ethics Committee of the Hospital Josep Trueta , following the Helsinki II declaration. Written informed consent was obtained from all relatives included in the study. Alive individuals were clinically evaluated at Hospital Josep Trueta , Hospital Clinic of Barcelona , or Hospital Sant Joan de Deu . All patients were Caucasian and native from Spain.We analyzed six families diagnosed with cardiomyopathies caused by 3 variants in 6 families. Each variant in two families. A multidisciplinary group including cardiologists, pediatricians, genetic counselors, molecular geneticists, and pathologists performed personalized genetic interpretation and clinical translation in each family. Families diagnosed with the same cardiomyopathy due to the same mutation were included in our study. Clinical evaluation of index cases and all available relatives included a complete physical examination, 12-lead electrocardiogram (ECG), 2-dimentional echocardiography (ECHO), cardiac magnetic resonance imaging (MRI), exercise stress test, 24-h Holter monitoring, and comprehensive genetic testing. Regarding post-mortem samples, a complete autopsy was performed according to current international regulations, including macroscopic, microscopic, and toxicological analyses . When maABCC9, ACTC1, ACTN2, AKAP9, ANK2, ANKRD1, BAG3, CACNA1C, CACNA2D1, CACNA1G, CACNA1H, CACNA1I, CACNB2, CALM1, CALM2, CALM3, CALR3, CASQ2, CAV3, CRYAB, CSRP3, CTNNA3, GJA1, CTF1, DES, DMD, DMPK, DPP6, DSC2, DSG2, DSP, DTNA, ECE1, EMD, EN1, EYA4, FHL2, FKTN, FLNA, FLNC, GAA, GJA5, GLA, GPD1L, HCN1, HCN2, HCN4, JPH2, JUP, KCNA5, KCND3, KCNE1, KCNE2, KCNE3, KCNE4, KCNE5, KCNH2, KCNJ2, KCNJ5, KCNJ8, KCNQ1, LAMA4, LAMP2, LDB3, LMNA, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYLK2, MYOZ2, MYPN, NEBL, NEXN, NOS1AP, NOTCH1, NPPA, NUP155, PDLIM3, PHOX2A, PHOX2B, PITX2, PKP2, PLN, PRKAG2, RANGRF, RBM20, RYR2, SCN1B, SCN2B, SCN3B, SCN4B, SCN5A, SCN10A, SDHA, SGCD, SLC22A5, SLC6A4, SLC8A1, SLMAP, SLN, SNTA1, TAZ, TCAP, TGFB3, TLX3, TMEM43, TMPO, TNNC1, TNNI3, TNNT2, TP63, TPM1, TRDN, TRIM63, TRPM4, TTN, TTR, and VCL) . We screened the most prevalent genes involved in cardiomyopathies and other pathologies associated with SCD (and VCL) . All genad hoc pipeline to generate raw VCF files. The final annotation steps provided information included in public databases. Identified variations were compared with DNA sequences from 500 healthy Spanish individuals  as control cases in order to identify potential founder mutations. In addition, in order to determinate the frequency of variants identified, they were contrasted with the Human Gene Mutation Database (HGMD4), HapMap5, 1000 Genomes Project6, Exome Variant Server (EVS7), Genome Aggregation Database (gnomAD8), and Exome Aggregation Consortium (ExAC9). Non-common genetic variants [minor allele frequency (MAF) <1%] identified in NGS analysis were confirmed by Sanger sequencing. Exons and exon-intron boundaries of each gene were amplified . PCR products were purified , and then were directly sequenced in both directions  with posterior SeqScape Software v2.5 analysis , comparing obtained results with the hg19 reference sequence. Sequence variants were described following rules outlined by the Human Genome Variation Society (HGVS10), and checked in Mutalyzer11. In silico prediction of pathogenicity of genetic variations was assessed in Functional Annotations for Non Synonymous SNVs (FannsDB12), Mutation Taster13, Protein Variation Effect Analyzer (PROVEAN14), and Polymorphism Phenotyping v2 (PPH215). Alignment of DNA sequences for different species was also performed for novel variations using Uniprot database16. Finally, protein structure and domains were consulted at STRING17 and SMART databases18.Coordinates of sequence data were based on UCSC human genome version hg19 (NCBI GRCh37 built). Biotinylated cRNA probe solution was used as a capture probe . Bioinformatic processing of fastq files consisted of low-quality base-trimming followed by a mapping step with BWA-MEM. After removal of duplicates and low-quality reads, variant call was performed with SAMtools v.1.2, and an Regarding copy number variation (CNV), our approach focused on capturing significant differences between expected normalized coverage and obtained normalized coverage for a given sample in a region of interest. Several samples were analyzed to corroborate similar levels of coverage between samples, as already published by our group . All CNV19   . This re19 . In addietation) . Finallyetation) . All invPKP2_p.(Leu92∗), families C and D carried the variant PKP2_p.(Arg413∗), and families E and F carried the variant MYBPC3_p.(Arg891Ala_fr∗160). Variants PKP2_p.(Leu92∗) and PKP2_p.(Arg413∗) are previously reported non-sense variants that are widely accepted as pathogenic. Both genetic abnormalities radically change the protein, resulting in a final protein that is shorter than wildtype PKP2  variant was reported first as a cause of ACM in a cohort of cases  variant was reported for the first time in 2006 in a cohort of cases with ACM , the frequency is very low . The third variant, MYBPC3_p.(Arg891Ala_fr∗160), is novel. Current global population databases do not include this variant, and in silico analyses predict a deleterious role. It is a small indel that induces a frameshift, resulting in a longer than wildtype protein. Therefore, all parameters analyzed indicate a pathogenic role   . Our NGSPKP2_p.(Leu92∗)- in three cases, II.1, III.1, and III.3. In addition, a rare variant in DSG2  was also identified in the index case (III.1), with the amino acid threonine  changed to isoleucine . This variant is reported in global databases with a low frequency . However, in silico analysis predicts an ambiguous role, and alignment between species showed no highly conserved domain. The variant in DSG2 was present in the index case , her sister , and her mother . One relative clinically diagnosed with ACM (III.4) did not carry this variant. Therefore, we disregarded DSG2_p.(Thr176Ile) as pathogenic, at least as the cause of ACM in this family.Family A included two members clinically diagnosed with ACM, III.1 -48 years old- and III.4 -42 years old- . Individnon-sense variant, PKP2_p.(Leu92∗). Relative III.1-39 years old- was definitely diagnosed with ACM , associated with ACM   and carried the same genetic variant, Arg413∗) . ConcernPKP2_p.(Arg413∗), MYH7_p.(Leu1591Gln) and TTN_p.(Ala20252Pro)-. All clinically affected relatives  carried the PKP2_p.(Arg413∗) variant. Three additional relatives  carried the same genetic variant, but all remain asymptomatic so far. Individual III.4 showed some cardiac abnormalities, although they were not diagnostic of ACM following current guidelines (MYH7_p.(Leu1591Gln) and TTN_p.(Ala20252Pro)-, previous studies published by our group disregard a pathogenic role  . Inverteidelines . Neithernic role .MYBPC3. This variant is a duplication of G (c.2670dupG), inducing a frameshift (p.Arg891Ala_fr∗160) (Families E and F were diagnosed with HCM . The dis_fr∗160) . This geMYBPC3_p.(Arg891Ala_fr∗160). Individual III.6 was diagnosed post-mortem –died at 38 years old-, during an extreme effort at work. Molecular autopsy identified the same variant (p.Arg891Ala_fr∗160) and forensic report described an asymmetric septal hypertrophy and microscopic analysis identified myocardial disarray with interstitial fibrosis. In addition, five relatives  carried the same genetic variant, but all remained asymptomatic and without any cardiac alteration, so far. Both grandparents (I.1 and I.2) died at old age (both >75 years old) due to oncologic and ischemic episodes, respectively. Neither had documented episodes of arrhythmia or syncope.Family E included five living family members  diagnosed with HCM . Only IIMYBPC3_p.(Arg891Ala_fr∗160). Neither grandparent (I.1 nor I.2) died suddenly or at a young age (both >75 years old).Family F included three living family members  diagnosed with HCM . IndividThe “five P” characteristics in current medicine – personalized, predictive, preventive, participative, and precision – are an innovative approach to the patient using available technologies to interpret individually all patient data. Recently updated guidance, first published in 2009 for the Heart Failure Society of America and ACMG, supports the use of NGS in genetic testing for cardiomyopathy to assist patient care and management of at-risk family members . In our PKP2_p.(Leu92∗) (CM102825). Our group previously published the index case of Family A (DSG2 (p.T176I) was not present in a relative clinically diagnosed with ACM (III.4). Therefore, we disregarded DSG2_p.(Thr176Ile) as pathogenic, at least in Family A, remaining the variant PKP2_p.(Leu92∗) is the most plausible cause of disease in Family A.Families A and B were diagnosed with ACM due to a pathogenic variant, Family A , and herPKP2_p.(Leu92∗) may be considered the cause of ACM, although other genetic abnormalities may modify onset, evolution, and outcome of the disease.Family B, despite only two genetic carriers showed incomplete penetrance and variable expressivity. The younger relative (III.1) showed an aggressive phenotype, with often-malignant arrhythmias, and had an ICD implanted. The older relative (II.1) showed a slow evolution and benevolent phenotype due to minimal symptoms throughout her life. Therefore, considering the clinical data in both Families A and B, PKP2 . Our group published on Family C in 2014  as cause of disease. Genotype-phenotype correlation suggests that PKP2_p.(Arg413∗) is the cause of disease in these families, whereas additional variants could play a role as phenotype modulators. We observed incomplete penetrance and variable expressivity, as occurs in other reported families affected by ACM . In Family C, relatives showed evident heart abnormalities at an early age, suggesting a highly aggressive role of the variant; however, in Family D, the abnormalities seemed to occur progressively, suggesting a pathogenic role of the variant but not with the same temporal onset. Therefore, despite PKP2_p.(Arg413∗) being the cause of ACM, other genetic abnormalities  may modify the onset, evolution, and outcome of ACM.Families C and D were also diagnosed with ACM and both carried a pathogenic variant (CM060431) in  in 2014 . All cliFamily D , and herd by ACM . Asymptod by ACM . TherefoMYBPC3 that results in a different protein than wildtype MYBPC3. All analyses suggest a pathogenic role. Family E showed incomplete penetrance and variable expressivity, with clinically diagnosed relatives carrying the MYBPC3_p.(Arg891Alafs∗160) variant and showing different phenotypic evolution of heart abnormalities. This agrees with several reports showing different phenotypes from one family diagnosed with HCM due to a single pathogenic variant (MYBPC3_p.(Arg891Alafs∗160) variant had a clinical diagnosis of HCM, including young family members. Therefore, MYBPC3_p.(Arg891Alafs∗160) is the likely cause of HCM in both Families E and F, and other genetic abnormalities  may modify onset, evolution, and outcome of the disease in each family.Families E and F were diagnosed with HCM due to a novel frameshift alteration in  variant . In Fami variant , 2012. FAutopsy in young individuals who die suddenly may be not conclusive and may not identify cardiac abnormalities. However, potential cardiomyopathy should not be disregarded, as ultramicroscopic changes that are not evident at autopsy may nonetheless induce arrhythmia responsible for SCD . MoleculIn conclusion, genetic testing has been progressively incorporated into clinical diagnosis, mainly due to improved NGS technology. Although genetic interpretation may classify rare variants as pathogenic, translation into clinical practice sometimes shows both interfamilial and intrafamilial phenotypic differences. We report here families carrying the same pathogenic variant associated with cardiomyopathies but with differential onset and/or clinical progression. Therefore, additional factors may modify the disease phenotype in each individual. Nonetheless, all individuals carrying a pathogenic variant, despite being asymptomatic, are at risk of disease therefore, clinical follow-up should be performed. Clinical translation remains a main challenge for cardiovascular genetic units and should be carefully performed in a personalized manner.in vivo and/or in vitro studies of each genetic variant may help clarify pathophysiological mechanisms associated with the pathology.A main limitation of this study is the lack of understanding of the reasons implicated in high expression variability even for the same variant and family. Additional analysis of the assessed genes as well as in other genes not included in our NGS custom-panel is necessary to identify additional genetic variants that could modify the phenotype. In addition, other genetic analyses focused on transcriptional/proteomic modifications are necessary to clarify the role of each genetic variant in each individual. It is important to remark the absence of data on exercise, an important contributor to phenotypic manifestation. Finally, This study was approved by the Ethics Committee of the Hospital Josep Trueta , following the Helsinki II declaration. Written informed consent was obtained from all relatives included in the study. Alive individuals were clinically evaluated at Hospital Josep Trueta , Hospital Clinic of Barcelona , or Hospital Sant Joan de Deu .OC, AF-F, GS-B, JB, and RB developed the concept. SC, MC, JM, EA, AG-Á, PJ, AP-S, BdO, CF, MA, VF, AI, MP, LL, FP, IM-S, and CT acquired, pre-processed, and analyzed the data. OC, AF-F, and GS-B prepared the manuscript. OC, JB, and RB supervised the study. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fragaria × ananassa) fruit development using an in vitro ripening system for 48 h. Also, we observed the effects of MeJA and the application of jarin-1 to MeJA-treated fruits (MeJA + jarin-1 treatment). We assessed changes of expression levels for the JA-Ile and MeJA biosynthetic (FaJAR1.2 and FaJMT), JA signaling-related (FaMYC2 and FaJAZ1), MYB-bHLH-WD40 (MBW) complex-related , and anthocyanin and PA biosynthetic  genes. In addition, the promoter region of MBW complex-related MYB genes was isolated and sequenced. We found a higher redness of strawberry fruit skin and anthocyanin content in MeJA-treated fruits with respect to jarin-1-treated ones concomitant with an upregulation of FaANS and FaUFGT genes. Inversely, the PA content was higher in jarin-1- and MeJA + jarin-1-treated than in MeJA-treated fruits. MeJA + jarin-1 treatment resulted in an upregulation of FaANR and associated transcription factors such as FabHLH33 and FaMYB9/11 along with FaJMT and FaJAR1.2. Finally, we found JA-responsive elements in the promoter regions of FaMYB1/9/10/11 genes. It is proposed that PA biosynthesis-related genes can be upregulated by the application of jarin-1 to MeJA-treated fruit, thus increasing PA accumulation in strawberry.Fleshy fruits are an important source of anthocyanins and proanthocyanidins (PAs), which protect plants against stress, and their consumption provides beneficial effects for human health. In strawberry fruit, the application of exogenous methyl jasmonate (MeJA) upregulates anthocyanin accumulation, although the relationship between the jasmonate pathway and anthocyanin and PA biosynthesis in fruits remains to be understood. Anthocyanin and PA accumulation is mainly regulated at the transcriptional level through R2R3-MYB and bHLH transcription factors in different plant species and organs. Here, the effect of jarin-1, a specific inhibitor of bioactive JA  biosynthesis, on anthocyanin and PA accumulation was evaluated during strawberry ( Fragaria × ananassa) consumption has a positive impact on human health, since this fruit is a relevant source of antioxidant compounds [Plant polyphenols play a central role in plant fitness, since these compounds are important in plant environment crosstalk, playing a role in plant responses to biotic and abiotic stress, and in flowers and fruits they are important for pollen fertility and animal attraction for pollination and seed dispersion ,2,3,4. Mompounds ,10. Straompounds . Strawbeompounds ,14,15,16ompounds .O malonyl-glucoside make up 80% and 14% of the total anthocyanin content, respectively [O-glucosyl transferase (UFGT) [Anthocyanins are widely distributed pigments in the plant kingdom and are responsible for blue, purple, violet, and red coloration in most plants, with their presence more obvious in flowers and fruits . On straectively , accumule (UFGT) .Proanthocyanidins, also called condensed tannins, are polymeric flavan-3-ols . StrawbeAtTT2 [AtTT8 [AtTTG1 [FaMYB9/FaMYB11, FabHLH3, and FaTTG1, respectively [F. × ananassa fruit, respectively [Many highly conserved transcription factors (TFs) regulate the expression of genes involved in late flavonoid/phenylpropanoid metabolism, including anthocyanin and PA biosynthesis. They are mainly R2R3-MYB TFs, interacting or not with basic helix-loop-helix (bHLH) TFs and/or with proteins containing the conserved WD40 repeats to form the so-called ternary MYB-bHLH-WD40 (MBW) complexes . In straAtTT2 , AtTT8 [2 [AtTT8 , and AtT [AtTTG1  were chaectively . Moreoveectively . Finallyectively ,23,28,29On the other hand, plant hormones regulate fruit development and ripening, and they could be related to the accumulation of interesting bioactive compounds in fruit . Unlike Fragaria chiloensis) fruit is related to an upregulation of the corresponding biosynthetic genes, among them DFR, ANS, and UFGT [In Arabidopsis seedlings, anthocyanin accumulation induced by JAs has been reported . The autand UFGT . Finallyand UFGT .Additionally, jarin-1 (from jasmonic acid:amino acid synthetase (JAR1) inhibitor; Fragaria × ananassa) fruits, and on transcriptional levels of genes encoding for MBW complex-associated TF and JA biosynthesis-related enzymes. We show that jarin-1 application to MeJA-treated fruit upregulates genes encoding for key MYB and bHLH components and increases PA level.Recently, our group identified and characterized the key JA metabolism- and signaling-related molecular components in strawberry at the genetic and transcriptional levels ,33. SpecFirst of all, as a main research objective, we proposed to analyze the effects of a JA-Ile biosynthesis inhibitor, jarin-1, on anthocyanin and PA accumulation during strawberry fruit ripening. Thus, we planned an experiment in which the expected MeJA-induced effects could be counteracted by jarin-1 in an in vitro fruit ripening system ,42. TherGain or loss in fruit color values could reflect changes in anthocyanin and PA contents. Accordingly, to record changes in fruit skin color, we measured L*, a*, and b* along with color dimensions chroma and hue angle (h°) . Jarin-1Regarding changes in fruit firmness and weight, jarin-1-treated fruit exhibited significant firmness reduction compared with its respective control at 12 and 24 h . A signiThe changes observed in the fruit skin color parameters  suggest Opposite to the observed results in TAC, an increase and decrease in total proanthocyanidin content (TPC), respectively, were observed in jarin-1- and MeJA-treated fruit at 48 h . Jarin-1Later, for the purpose of performing a qualitative analysis of PAs, we determined the mean degree of polymerization (mDP) of total PAs and found lower values in jarin-1- and MeJA + jarin-1-treated fruit at 48 h in mDP3 (%) , which mFaMYB9, FaMYB11, and FabHLH33 expression patterns, with the highest expression level in MeJA + jarin-1 treatment at 48 h compared to its respective control and other treatments , FaMYB9 , FaMYB10 , and FaMYB11 . Several phytohormone-related cis-acting elements were identified in a 1500 bp region of the promoters -responsive elements were observed in FaMYB9 and FaMYB11 promoter regions. Specifically, the putative JA-responsive element G-box and its variants were identified in the promoter regions of FaMYB1 (5′-CACGTGT-3′), FaMYB9 (5′-AACGTG-3′ and 5′-CACGTT-3′), FaMYB10 (three 5′-CACGTT-3′), and FaMYB11 (5′-CACATG-3′).Additionally, with the objective of finding some clues to the expression regulation of the romoters . By usinO-glucosyl transferase (UFGT), and leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), respectively. The expression patterns for the encoding genes for FaANS, FaUFGT, FaLAR, and FaANR were analyzed with the aim of verifying a possible coordination between the observed accumulation pattern of anthocyanin and PAs  and UDP glucose:flavonoid 3- and PAs  and the  and PAs . We note and PAs . The antand 48 h A,B. Thesand 48 h A. Treatmreatment A,B.FaLAR and FaANR, their expression patterns showed differences mainly at 24 and 48 h under different treatments  are among the most ubiquitous groups of plant phenolics distributed in plant-derived foods, such as fruits, and have several potential beneficial effects on human health . AlthougIn the present research, we report alterations on fruit firmness, weight, and skin color, PA and anthocyanin accumulation, and associated gene expression patterns when jarin-1, a JA-Ile biosynthesis inhibitor , was appF. × ananassa cv. Camarosa) fruit treated with MeJA in an in vitro ripening system, which had weight gain with respect to control fruits. Inversely, Concha et al. [Fragaria chiloensis fruit compared to controls, and a weight decrease in all treatments relative to the initial weight at 0 d was also shown [F. chiloensis fruit was significantly higher from 48 to 120 h of treatment. In turn, Pérez et al. [F. × ananassa (cv. Camarosa) fruits decreased following a sigmoidal curve, without differences between MeJA and control treatments. The differences in the reported weight and firmness gains or declines could reflect specific differences between species or cultivars and their behavior in in vitro ripening assays.In the case of fruit weight, we report significant increases in both MeJA and jarin-1 treatments at different times . Howevera et al.  reportedso shown . In termso shown  previousz et al.  reportedF. chiloensis and F. × ananassa (cv. Aromas) [F. × ananassa (cv. Albion)  ,38, the  Albion) B. On the Albion) A,B. We aultivars ,37,38, wultivars . In thisultivars , blockin1 mutant . Moreovethaliana . Until nFragaria vesca genome  between PA accumulation and FaMYB9, FaMYB11, FaANR, and FaLAR expression levels  in those treatments at 48 h compared to MeJA-treated fruit  in the ACGTG-3′ ,57,58,60ACGTG-3′  in the FACATG-3′  in the pFaMYC2 in MeJA treatments at 24 and 48 h, concomitant with the anthocyanin accumulation (MYC2 mutant (atmyc2-2) and wild-type plants showed anthocyanin accumulation induced by JA, which suggests that MYC2 would not have a significant effect on anthocyanin accumulation induced by JA. Nevertheless, Niu et al. [MYC2, MYC3 or MYC4 Arabidopsis seedlings accumulated higher anthocyanin content than wild-type plants. In strawberry fruit, more evidence is needed to assign a role for FaMYC2 in JA-induced anthocyanin accumulation. In the case of FaJAZ1, upregulation was observed in MeJA + jarin-1 treatment at 48 h that could be related to the PA accumulation with this treatment at this time  fruits at the large green stage, of similar size and without external damage, were harvested according to Garrido-Bigotes et al. [s et al.  from plas et al.  and Concs et al. . Fruits Briefly, the in vitro fruit ripening system consisted of the growth and development of picked fruits in a nutritious solution under controlled environmental conditions . In a grThe experiment was arranged in a randomized design to account for spatial variability. Each individual fruit was considered as a biological replicate. Six fruits were collected at 12, 24, and 48 h for each treatment and control and assessed for fruit quality. Then, 3 fruits were analyzed for anthocyanin and proanthocyanidin contents and 3 for gene expression. Values for MeJA-, jarin-1–, and MeJA + jarin-1-treated fruits under the parameters for each treatment time were calculated with respect to Control 1, Control 2, and the average of Control 1 and Control 2 values, respectively.2 + b*2)1/2) and hue angle (h° = arctan (b*/a*)) were calculated from numerical values of a* and b* as previously reported [Fruits from each treatment were weighed, and their firmness and color were estimated. Firmness of treated fruits was measured using a texture analyzer  with a TA-39 probe, a 3 mm target value, and a 1 mm/s test speed. Color changes on treated fruit skin were measured using a colorimeter  and expressed according to the CIELAB scale  along with the dimensions of color chroma and hue angle (h°). L*, a*, and b* indicate lightness, chromaticity on a green (−) to red (+) axis, and on a blue (−) to yellow (+) axis, respectively. Chroma (chroma = (a*reported . For firv/v), incubated overnight at 4 °C, and centrifuged for 10 min at 12,000 rpm at 4 °C. After that, 2 aliquots from the aqueous phase of each sample were diluted (1:4) with 2 different buffers: a pH 1 buffer (0.025 M KCl) and a pH 4.5 buffer (0.4 M sodium acetate). Finally, the sample absorbances were quantified at 516 and 700 nm. Buffers for each pH were used as blanks. The anthocyanin content of each sample was calculated based on the Lambert–Beer law, using the coefficient of molar extinction for the pelargonidin-3-glucoside  reported by Swain [Total anthocyanin content (TAC) was quantified by the pH differential method performed according to Lee et al.  and Debnby Swain . Results−1). Results are expressed as difference  between TPC  values of treatments and their respective controls at each time point. Mean degree of polymerization (mDP) of total PA was characterized by hydrophilic interaction chromatography (HILIC-HPLC) according to Pastene et al. [For each treatment at each sampling time point, a bulk of 3 g of strawberry fruits without achenes was ground with liquid nitrogen, homogenized in 9 mL of 80% acetone, and sonicated at room temperature for 30 min. Total proanthocyanidin content (TPC) was quantified according to Prior et al. . The reae et al. . F. × ananassa (cv. Albion) was achieved by combining the RNeasy Plus Mini Kit  and the cetyltrimethylammonium bromide (CTAB) method described by Liao et al. [Total RNA isolation from treated fruits of o et al.  with somFaJMT and FaJAR1.2 [FaMYC2 and FaJAZ1 [FaMYB9, FaMYB10, FaMYB11, FabHLH3, and FabHLH33 and repressor FaMYB1 [FaANS, FaUFGT, FaANR, and FaLAR [FaGAPDH [FaMYB10 and FabHLH33 were designed in the present research and the other primer sequences were as previously reported [We analyzed the expression level of genes previously reported as related to the following processes or pathways: JAs biosynthesis , JA d FaJAZ1 ), MBW cor FaMYB1 ), and annd FaLAR ,71,72, Sreported ,33,70,71®FAST qPCR Kit , according to the manufacturer’s instructions, and the PikoReal Real-Time PCR System . The PCR conditions were: 94 °C for 10 min; 40 cycles at 94 °C for 15 s, 60 °C for 15 s, and 72 °C for 15 s. Each reaction was repeated 3 times, using water as negative control. The relative gene expression levels correspond to the mean of 3 biological replicates normalized against the expression level of the housekeeping gene using untreated fruits (0 h)   as the ct method . RelativF. × ananassa MYB genes (FaMYB9 (GenBank JQ989281), FaMYB10 (GenBank MG456859), FaMYB11 (GenBank JQ989282), and the repressor FaMYB1 (GenBank AF401220)) were isolated by a PCR-based method using the Fragaria vesca genome as reference. mRNA sequences of MYB genes were used to search orthologous genes in the F. vesca genome , https://www.ncbi.nlm.nih.gov/genome/3314) by means of the BLAST genome search tool  (Promoter regions of ast.cgi) . Primersast.cgi) .F. × ananassa (cv. Aromas) was performed by the CTAB method [w/v) agarose gel and sequenced on both strands by primer walking . Sequences were assembled using SeqMan Pro software  and their identity was confirmed by BLAST Genome.Genomic DNA extraction from leaves of B method  with somhttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [Identification of putative cis elements in the sequenced promoters was performed using the PlantCARE database (e/html/) . We alsoe/html/) ,59,60,61p ≤ 0.05 were considered statistically significant.Data were analyzed by one-way analysis of variance (ANOVA) using Infostat software (version 2015). LDS test and Tukey post hoc test were used to evaluate significance for physiological data  and RT-qPCR gene expression analysis, respectively. Values of The regulation of PA and anthocyanin biosynthesis in fleshy fruits is a complex process due to the participation of several molecular components. In the present research, we found a connection between the JA pathway and PA and anthocyanin biosynthesis in strawberry fruit, although further investigation is needed to decipher how the MeJA and jarin-1 interaction works at the molecular level and what the specific roles of the JA signaling pathway-associated components are in anthocyanin and PA biosynthesis. This work provides evidence for potential uses of hormone inhibitors for the study of several biochemical pathways during fruit ripening and for chemical manipulation of the flavonoid pathway, especially in strawberry, which could enrich the nutritional quality of this fruit.


Arabidopsis. However, little is known about this process in androdioecious plants. Tapiscia sinensis Oliv. is a functionally androdioecious tree, with both male and hermaphroditic individuals. Hermaphroditic flowers (HFs) are female-fertile flowers that can produce functional pollen and set fruits. However, compared with male flowers (MFs), the pollen viability and number of pollen grains per flower are markedly reduced in HFs. MFs are female-sterile flowers that fail to set fruit and that eventually drop.Gametogenesis is a key step in the production of ovules or pollen in higher plants. The sex-determination aspects of gametogenesis have been well characterized in the model plant T. sinensis is when the early gynoecium meristem is disrupted. During the early stage of HF development (stage 6), the ring meristem begins to form as a ridge around the center of the flower. At this stage, the internal fourth-whorl organ is stem-like rather than carpelloid in MF.Compared with HF, a notable cause of MF female sterility in A total of 52,945 unigenes were identified as transcribed in MF and HF. A number of differentially expressed genes (DEGs) and metabolic pathways were detected as involved in the development of the gynoecium, especially the ovule, carpel and style. At the early gynoecium development stage, DEGs were shown to function in the metabolic pathways regulating ethylene biosynthesis and signal transduction (upstream regulator), auxin, cytokinin transport and signalling, and sex determination (or flower meristem identity).T. sinensis.Pathways for the female sterility model were initially proposed to shed light on the molecular mechanisms of gynoecium development at early stages in  Angiosperm plants exhibit a wide variety of breeding systems , 2. AmonCucumis sativus (cucumber) and Cucumis melo (melon) is regulated by the interaction of environmental cues, plant hormones and genetic factors that differentiate gender phenotypes [Shy Girl, arose via lineage-specific duplication of a cytokinin response regulator [Arabidopsis [Silene latifolia [Zea mays [Diospyros lotus [ANT promotes ovule primordium growth; the CUP-SHAPED COTYLEDON genes (CUCs) play a role in the establishment of the ovule primordium boundaries; when the functions of stk, shp1, and shp2 were lost in a triple mutant, fewer ovules were initiated, and ovule development was severely disrupted. Even more remarkably, the ABCDE model of flower determination and development has indicated that a specific class of MADS-box genes are key regulators of pistil development [AGAMOUS (AG), APETALA 2 (AP2), BELL 1, INNER NO OUTER (INO), AINTEGUMENTA (ANT), SPOROCYTELESS (SPL/NZZ), and SUPERMAN (SUP) [Separate sexes have evolved on numerous independent occasions from hermaphroditic ancestors in flowering plants . Mechanienotypes , 25, 30.egulator . At presbidopsis , 32, cotbidopsis , rice [3bidopsis , tomato bidopsis , Silene atifolia , 37, ZeaZea mays –40, Diosos lotus , and cucos lotus –26. Moleos lotus . For exaelopment , such asAN (SUP) , 44.Tapiscia sinensis Oliv. (Tapisciaceae) [, the male individuals have pistillode, some of which have a shorter column and abortive ovule, while the vast majority of pistils lack ovaries  –54, the es Figs.  and 2a. Tapiscia.To determine the molecular mechanism of female sterility in male individuals, we compared the developmental anatomy and performed transcript profiling using the RNA-Seq technique of the gynoecia of the HF and MF. A number of candidate genes and related pathways were revealed, which provided new insights into the genetic and biochemical controls for early stages of pistil development, and our results will be helpful in shedding light on the mechanism of sex differentiation in the functionally androdioecious trees of p < 0.05)  and the PL  were clearly shorter in MF than in HF. Remarkably, in MFs, the PL and OTD remain unchanged when the bud vertical diameter grew to 0.75 ± 0.05 mm   Fig. i, while 05) Fig. j. Corres05) Fig. g and an 05) Fig. h. In con05) Fig. j and an 05) Fig. i. The OTth) Fig. j.Fig. 3The SEM results showed that there were no obvious differences observed between MF and HF before stage 5 Fig. a. HoweveIn most MFs, during stage 6, the gynoecium primordium began to form Fig. b, but thT. sinensis, the course of individual flower development can be divided into five phases: early differentiation, calyx initiation, petal initiation, stamen initiation, and carpel initiation. The perianth was the first organ to be formed on the floral apex . The average GC percentage was 44.69%. The clean reads were de novo assembled using Trinity  into 63,p-values (p < 0.05) marked with coloured boxes, including profile 3, profile 4, profile 5, profile 6, and profile 7  marked with coloured boxes, including profile 1, profile 6, and profile 7  , a totalTo determine whether the DEGs between MF and HF are significantly related to specific pathways or biological function, GO and KEGG databases were used for enrichment analysis , AGAMOUS (Unigene0020941), SUP (Uniene0047137), SPL (Unigene0006964), INO (Unigene0025624), STM (Unigene16944), ANT1 (Unigene0002499), STK (AGL11) (Unigene0007536), SHP1 (Unigene0013614), AP2 (Unigene0015664), TSO1 (Ungene0002282), SPT (Unigene0034455), LUG (Unigene0041412), PI (Unigene0020391), SEUSS (Unigene0033961) and BEL1 (Unigene0039261)  and light microscopy (LM) show that most of the vestigial pistils of MF of ers Fig. a. In Ara Fig. AG , 61, 62, AG [SPL , INO [64and BEL1 , 65. Carbidopsis , and in To confirm that the unique-match genes from the Illumina sequencing and bioinformatics analysis were indeed differentially expressed, a total of 16 genes were selected from the DEGs related to flower development  for quantitative RT-PCR assays. For 16 genes, the qRT-PCR analysis revealed the same expression trends as the RNA-Seq data, despite some quantitative differences. All genes had different expression levels between MF and HF at stage 6 Fig. d. For exACO (Unigene0029908), ACO1 (Unigene0037768), ACS (Unigene0021193), ERS1 (Unigene0024060), CTR1 (Unigene0036282), TIR1 (Unigene0037222), ETR1/2 , EIN2/3/4   had different results in qRT-PCR vs RNA-Seq, confirming the reliability of the RNA-Seq data. 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is the rate-limiting enzyme in ethylene synthesis, and it is important in regulating ethylene biosynthesis. Unigene0021193 was downregulated at stage 6 in the M-H comparison. Two ACC oxidase homologous genes showed different expression patterns, but ACO and ACO1 were both upregulated in stage 6 in the MF-HF comparison.Ethylene regulates many aspects of plant development and responses to biotic and abiotic stress. We identified ten DEGs involved in the ethylene biosynthesis and signal transduction process Fig. a: ACO , ETR2 (Unigene0027393), ETR1 (Unigene0020715), ERS1 (Unigene0024060). At stage 6, the relative expression of Unigene0020715 displayed significant differences, but Unigene0027393 and Unigene0024060 were indistinctive. Unigene0020715, the repressor of ethylene signal transduction, was up-regulated in MF relative to HF, but Unigene0027393 displayed indistinctive expression. Similarly, Unigene0003076 displayed almost the same trend as ETR1 during stage 6, while Unigene0024060 was indistinctive. ERF1/2 , the downstream gene in the ethylene signal transduction pathway, displayed differential expression in both the M1-M2 and H1-H2 comparisons, being up-regulated in MF relative to HF at the key stage. Obviously, at stage 6, ACO (Unigene0029908), ACO1 (Unigene0037768), CTR1 (Unigene0036282), and EIN2 (Unigene0029458) were up-regulated in the MF-HF comparison, while ACS and EIN3 were down-regulated in MF relative to HF.In activity . From mRT. sisensis, we sprayed ethephon at concentrations of 150 mg/L. We found that ethephon had a significant effect on MF transformed into HF.To verify the role of ethylene in sex differentiation of IAA31 (Unigene0018133), IAA26 (Unigene0035359), IAA15 (Unigene0001116), IAA19 (Unigene0025767), GH3.1 (Unigene0015426), SAUR2 (Unigene0017779), SAUR14 (Unigene0050945), SAUR72 (Unigene0005319), SAUR50 (Unigene0010084), SAUR12 (Unigene0018267), SAUR31 (Unigene0049968), SAUR53 (Unigene0019490), KUP3 (Unigene0005158), MIR397B (Unigene0025946), ARR10 (Unigene0049560), AHP4 (Unigene0004154), AHP6 (Unigene0011561), AHP4 (Unigene0004325), IAA1 (Unigene0001116), ARF17 (Unigene0029174), CALS5 (Unigene0019058), PIN6 (Unigene0020253), PIN2 (Unigene0033811), PIN1 (Unigene0000533). To confirm that the unique-match genes from the Illumina sequencing and bioinformatics analysis were indeed differentially expressed, a total of 9 genes were selected from the DEGs related to flower development for quantitative RT-PCR assays. Among 9 of the 24 genes with different expression levels between MF and HF  show that dioecy has three separate origins; in one instance, dioecy evolved from hermaphroditism via androdioecy following the transition from insect to wind pollination [T. sinensis is a wind-pollinated and insect-pollinated species [T. sinensis, the tectum sculpture is reticulate in MF pollen grains, while that of HF is perforate. Additionally, globose pollen grains have the largest volume-to-surface area ratio, but with minimal regulatory function, which is a relatively primitive character [The breeding system and other traits mapped on the phylogenetic tree of lination , 86. Diolination , 88. T.  species , 53, and species . A trend species , 67, 89.haracter . Olive pharacter .T. sinensis. Transcriptome analyses showed that sixteen genes related to carpel and ovule development are expressed differentially at the key stage of MF pistil abortion and were verified by qRT-PCR. The molecular mechanisms of ovule development in Arabidopsis, cotton, and rice are relatively well known [AG (AGAMOUS) belongs to the MADS-box family, which determines stamen and carpel identity and plays a role in ovule development [SHP1 (SHATTERPROOF 1), SHP2 (SHATTERPROOF 2), and STK (SEEDSTICK) genes together with the SEP (SEPALLATA) genes have overlapping expression patterns with AG in an AG-independent manner [ANT (AINTEGUMENTA), HLL (HUELLENLOS), SIN2 (SHORT INTEGUMENT 2), INNER NO OUTER (INO), and SUPERMAN (SUP) as regulators of ovule outgrowth [AG is necessary for the determination and termination of floral meristem activity. In the ag mutant, delayed floral meristem activity results in the stamens and carpels being replaced by flowers [WUSCHEL (WUS), which specifies stem cell identity, and AG play crucial roles in regulating the initiation or termination of floral meristems [Admittedly, ovule primordia arise from the placenta; however, both ovules and placenta are completely absent in most MFs of ll known –26, 90. elopment , 44. Thet manner . ANT  and controls KNOX gene expression independently of the transcriptional repressor AS1 [STM plays a crucial role in preventing meristem cell differentiation by inducing the production of cytokinins (CK) and the ARR transduction pathway [STM causes floral phenotypes ranging from reduced formation of placental tissues and inhibited carpel fusion to complete loss of carpel development [T. sinensis, STM also showed differential expression, which indicated the role of STM in promoting ovule development.In addition, the ssor AS1 , 98. Mor pathway . The proelopment . In T. sArabidopsis, the ovule primordia arise from the placentas flanking the medial ridges, while leunig (lug) aintegumenta (ant) double mutants lack medial tissues, and the double mutant seu-3 ant-1 results in a complete loss of ovule initiation, caused by severe defects in early gynoecium development [CRABS CLAW (CRC) is a member of the YABBY gene family, whose expression is largely limited to carpels and nectaries, and plants expressing 35SCaMV::CRC produce variable flowers with partial transformations of sepals into carpels and reductions in flower organ number. Carpels are short and solid (ovule is completely absent) and composed primarily of stylar tissues. The morphology of the pistillode of MF in T. sinensis was similar to that of the Arabidopsis 35SCaMV::CRC pistil [In elopment . CRABS CC pistil .Ethylene, as an upstream factor for pistil or ovule formation, has been verified in many species; for instance, tobacco, pomegranate, and cucumber , 102.T. sinensis, ETR1 (Unigene0020715) was up-regulated in MF relative to HF. ERF1 and ERF2, as ethylene response signal factors, were differentially expressed between MF and HF. The down-regulation of ERF1/2 and up-regulation of ETR1 may be related to the flower development. ACO and ACS (ACS and ACO are rate-limiting enzyme) are a pivotal enzyme in ethylene biosynthesis, they are showed significant expression levels in pistil formation ant stage 6. Jointly, our findings indicated that ethylene may act as one of the upstream regulators influencing the development of pistil. Furthermore, ANT, an APETALAP2-like Gene, plays fundamental roles in ovule formation and megasporogenesis [ANT may be responded to ethylene. However, the expression of ANT and INO (INNER NO OUTER) may be regulated by AG and SPL.The ETR1 protein acts as negative regulator of ethylene responses , has theogenesis , 105, thArabidopsis, there are eight PIN proteins (PIN1–8), from which only PIN1 and PIN3 are expressed in the pistil and ovules. For example, polar auxin transport is mostly mediated by the PINFORMED1 (PIN1) efflux carriers [pin mutant, gynoecia display a range of phenotypes from almost normal structures with two valves, style and stigma, to stalk-like gynoecia with an elongated gynophore topped with a style and stigma [PINOID (PID) positively regulates auxin efflux, which can control an early general step in meristem development. In the pid-l flower, the internal fourth whorl organ is stem-like rather than carpelloid, although it is capped with a ring of stigmatic papillae. Furthermore, the morphology of the vestigial pistil of MF T. sinensis was similar to that of the Arabidopsis pid-l pistil [PINOID is required for formation of the stigma and style in rice [Arabidopsis Histidine Phosphotransfer Protein 6 (AHP6), an inhibitor, mediates an auxin-cytokinin crosstalk that regulates the timing of organ initiation at the shoot apical meristem. In this study, genes that were expressed differently in MF relative to HF also supported this point.Auxin maxima are fundamental for the formation of primordia, and auxin action has been well described for lateral roots and primordial flowers , 107. Thcarriers . Based od stigma . The prol pistil . Similar in rice . In addi in rice . SimultaT. sinensis. First, ethylene is an upstream factor in T. sinensis gynoecium development, and signal transduction of auxin and cytokinin is fundamental to the initiation of pistil primordia; MADS-box genes were also shown to be involved in the determination of the fourth-whorl flower organs. In addition, auxin signalling pathways were thought to be involved in pollen abortion  and hermaphroditic individuals  with winter fruits. Two types of flowers of T. sinensis were sampled from the natural geographical distribution in Shennongjia National Nature Reserve  and the voucher specimens were deposited archived in the herbarium of Structural Botany Laboratory (SBL), Northwest University. Prof. Liu Wenzhe undertook the formal identification of the samples. Different growth stages of flowers were collected for our study. The flowers used for microscopic observation were fixed immediately in 2.5% glutaraldehyde, and those used for transcriptome analysis were stored in liquid nitrogen (LN) at − 80 °C.T. sinensis. Similarly, H1, H2, and H3 represented the HF buds when their development was at stage 5, stage 6, and stage 8, respectively. Photographs are included showing the general developmental stages of the flowers . To describe the samples clearly, we used M1, M2, and M3 to represent MF buds when their development was at stage 5, stage 6, and stage 8, respectively Fig. ; these sTotal RNA (40 μg) was isolated from the M1, M2, M3 and H1, H2, and H3 samples using the TRIzol Kit  and treated with RNase-free DNase . RNA quantity and purity were assessed using a Nanodrop 1000 spectrophotometer . RNA integrity (> 8.0) was determined using a Bioanalyzer 2100 (Agilent) with an RNA 6000 Nanochip (Agilent). Methods of enrich Mrna, remove rRNA, RNA fragment, random haxamer primed cDNA synthesis, size selection and PCR amplification were performed as previously described , 113, anhttp://trinotate.sourceforge.net/) to identify open reading frames and assign best hits to UniProtKB (1e-03), PFAM-A (1e-03), Gene Ontology (GO) and eggNOG categories.Raw data were processed with Perl scripts to ensure the quality of the data used in further analyses. The adopted filtering criteria are as Zhang et al. (2018) , 114. Thhttp://www.r-project.org/) was used. We identified genes with greater than a two-fold change and a false discovery rate (FDR) < 0.05 in a comparison as significant DEGs. DEGs were then subjected to an enrichment analysis of GO functions and KEGG pathways. Read count for each gene in each sample was determined by HTSeq , and RPKM (reads per kilobase per million mapped reads) was then calculated to estimate the expression level of the genes in each sample [To identify DEGs across samples or groups, the edgeR package  enrichment of DEGs was implemented by the hypergeometric test, in which a p-value is calculated and adjusted as the q-value, and the data background is genes in the whole genome. GO terms with q < 0.05 were considered to be significantly enriched. GO enrichment analysis could reveal the biological functions of the DEGs [The GO  related to ethylene biosynthesis and signal transduction, nine genes involved in auxin and cytokinin signal transduction, and sixteen genes related to carpel and ovule formation. Primer sequences were designed using Primer premier 5.0, and Primer sequences and β - actin  sequence of internal reference gene are listed in Additional file T. sinensis of three developmental stages, and RNA extraction, cDNA synthesis, and Real-time PCR method as described in related references [qRT-PCR was performed not only to analyze the expression levels of DEGs but also to verify the RNA-Seq analysis, which included ten genes  Longitudinal section of HF. (B) Mature embryo sac in HF. (C) In Type III MF, embryo sac development stopped at the triad stage. (D) Longitudinal section of Type III MF. Sy, synergid; sn, secondary nucleus; hy, hypostase; ii, inner integument; oi, uter integument; mi, micropyle. Figure S2. Eight model profoles in TERM1 and TERM2, respectively. In TERM1, the expression patterns of 13,761 genes were analysed, eight model profiles were used to summarize, and five expression patterns of genes showed significant p-values (p < 0.05) (coloured boxes). In TERM 2, the expression patterns of 16,130 genes were analysed, and eight model profiles were used to summarize. Three expression patterns of genes showed significant p-values (p < 0.05) (coloured boxes). Each box represents a model expression profile with the model profile number and p-value. Colored boxes indicate that there are significant differences between floral stages. No color box means no difference. M1, M2 and M3 represent MF at stages 5, 6 and 10, respectively, while H1, H2 and H3 represent bisexual flowers at stages 5, 6 and 10, respectively. The meaning of the ‘significant p-value’ was a significant difference between floral stages. Figure S3. Numbers of differentially expressed genes. (A) TERM1 – trend all by gene number, trend all by P-value. In profile 3: 2364 gene  had stable expression in floral stages 5 and 6, but decreased in expression in stage 10; in profile 4, 1664 genes  had stable expression in floral stages 5 and 6, but increased in expression in stage 10; in profile 5, 2320 genes  increased in expression in floral stages 5 and 6, but decreased in stage 10; in profile 6, 2170 genes  had an increased expression in floral stages 5 and 6, but had stable expression in stage 10; in profile 7: 1471 genes  had an increased expression in floral stages 5 to 10. (B) TERM2 – trend all by gene number, trend all by P-value. In profile 1, the expression of 2308 genes  present the trend of first decline and then maintain stable from stage 5 to 10; profile 6: 3179 genes  had an increased expression in floral stages 5 and 6, but had stable expression in stage 10; profile 7: 1839 genes , the trend was always upward. (C) GO and KEGG pathway enrichment analysis in TERM1 and TERM2. The meaning of the ‘significant p-value’ was a significant difference between floral stages.


Myeloid immune cells, such as dendritic cells, monocytes, and macrophages, play a central role in the generation of immune responses and thus are often either disabled or even hijacked by tumors. These new tolerogenic activities of tumor-associated myeloid cells are controlled by an oncogenic transcription factor, signal transducer and activator of transcription 3 (STAT3). STAT3 multitasks to ensure tumors escape immune detection by impairing antigen presentation and reducing production of immunostimulatory molecules while augmenting the release of tolerogenic mediators, thereby reducing innate and adaptive antitumor immunity. Tumor-associated myeloid cells and STAT3 signaling in this compartment are now commonly recognized as an attractive cellular target for improving efficacy of standard therapies and immunotherapies. Hereby, we review the importance and functional complexity of STAT3 signaling in this immune cell compartment as well as potential strategies for cancer therapy. Src, K-ras and Bcr-Abl, growth factors and cytokine receptors can persistently activate STAT3, thereby leading to cellular transformation or promoting cancer cell survival [Pten phosphatase-deficient cancer cells, STAT3 can acquire an unexpected role as a tumor suppressor [In recent years, a number of clinical studies have demonstrated the efficacy of emerging immunotherapies against treatment refractory cancers, thereby gaining the attention of the medical community as well as patients ,2,3. In survival . Howeverppressor ,14. Therppressor ,15,16.One of the hallmarks of the tumor microenvironment is the accumulation of heterogeneous and undifferentiated MDSCs, or partly differentiated but dysfunctional, immature DCs and macrophages ,18,19. AStat3 deletion found that STAT3 is required primarily for differentiation of plasmacytoid DCs, specialized in type I interferon production, but not the conventional or tissue-resident conventional DCs, at least not at the later stages of their development [PRKCB), strongly inhibiting its expression and decreasing expression of DC differentiation markers, such as CD11c, MHC class I and II complexes, and costimulatory molecules. Interestingly, forced expression of PKCβII was able to decrease STAT3 activation by reducing the expression of several STAT3-inducing receptors, such as IL-6Rα, granulocyte colony-stimulating factor (G-CSF) receptor, and VEGF receptor 2 [+ T cells to regulatory T cells (Tregs) [DCs are highly specialized myeloid immune cells that control the activation of adaptive immunity by presenting antigens on major histocompatibility complex (MHC) class I or II molecules to cytotoxic CD8 or helper CD4 T cells, respectively . STAT3 helopment ,24. In celopment ,26. Tumoelopment . While telopment . Tumor-dceptor 2 . Thus, cceptor 2 . Beyond ceptor 2 . More receptor 2 . Despiteceptor 2 ,32,33,34ceptor 2 ,36. As s (Tregs) ,33,37,38 (Tregs) ,12,39. S (Tregs) ,38. At tMacrophages are key phagocytes in the organism, and show remarkable adaptability to changing conditions of the microenvironment. In response to stimulation by pathogens, products of dying cells, or cytokines, macrophages can differentiate functionally into M1 or M2 phenotypes, as characterized by the release of cytokines promoting Th1 or Th2 responses such as IL-4/IL-10/IL-13 . EndosomLIF is expressed more commonly than IL6 in human prostate cancers. Importantly, PMN-MDSCs and, to a lesser extent, M-MDSCs isolated from the blood of prostate cancer patients show high surface levels of LIF receptor and respond to LIF stimulation with STAT3 activation and increased T-cell inhibition. Tumor-induced STAT3 plays a central role in regulating both the differentiation and tolerogenic effects of MDSCs. First, STAT3 promotes both expansion and survival of MDSCs through upregulation of Bcl-XL, c-Myc, and Cyclin D1 [Tumors comprise a heterogeneous population of immature MDSCs, which have been associated with poor patient prognosis and tumor progression ,49,50. Byclin D1 . In addiyclin D1 ,54. The yclin D1 . Downstryclin D1 . Finallyyclin D1 ,56. As syclin D1 ,56. Simiyclin D1 . Conversyclin D1 . An elegyclin D1 . MDSCs uyclin D1 . These iyclin D1 . Many ofyclin D1 . TargetiDeregulated metabolism in the tumor microenvironment, and more specifically in tumor-associated myeloid cells, has been suggested as an essential element of cancer-related inflammation ,61. RapiDietary conditions, specifically the availability of lipids and their accumulation, can have profound effects on the immunogenic or tolerogenic functions of DCs in cancer ,64. In tDuring the differentiation of monocytes into tissue macrophages, cells undergo significant epigenetic remodeling of their genome and already at this stage can indicate metabolic differences related to the tolerant or “trained” innate immunity phenotype . MacrophARG1 was shown to be directly regulated by STAT3 binding to multiple sites in the ARG1 promoter [ARG1 expression [ARG1 expression [−) results in the nitration of molecules on the surface of T cells when in contact with MDSCs. Importantly, both T cell receptors (TCR) and CD8 molecules can undergo ROS-induced nitration and lose their ability to trigger T cell activation after the formation of the TCR–MHC complex with APCs [Although normal neutrophils primarily rely on glycolysis, tumor-associated MDSCs, including PMN-MDSCs, were recently shown to depend on FAO for their energy supply ,75. Suchpromoter . While ipression ,76. It cpression ,76. MDSCpression . The pripression . Generatith APCs .+ myeloid cells in vitro and in vivo. STAT3 inhibition and TLR9 immunostimulation disrupt the tolerogenic effects of the tumor microenvironment and thereby lead to potent antitumor immune responses in a variety of preclinical tumor models in mice [STAT3 activation in a variety of tumor-associated myeloid cells generates a powerful and multilayered backing for cancer progression and immune evasion. Therefore, STAT3 provides an exceptional molecular target at the key node of the immune system network, based on the antigen-presenting potential of DCs/macrophages or complex tumor-promoting activity of TAMs and MDSCs. Blocking STAT3 in tumor-associated myeloid cells can permit development of antitumor immune responses, at the same time eliminating tumor resistance to therapeutic assaults. Importantly, inhibition of STAT3 is not cytotoxic to non-malignant cells. The dominant-negative mutations of STAT3 found in patients with autosomal dominant HIES result in the increased susceptibility of patients to infections but are not lethal per se ,80. Desp in mice ,90,91,92 in mice ,53,93. A in mice . With th


In 1817, the London surgeon and pharmacist James Parkinson published a 66-page-long booklet entitled “An Essay on the Shaking Palsy”, which contains the first detailed, clinical description of the shaking palsy, or “paralysis agitans”, which is currently known as Parkinson’s disease (PD) . In factPD affects millions of people globally, but there is no cure, and its prevalence will double by 2030 -8. AlthoThe neuroscientific community has recently recognized that an increasing number of PD patients has died suddenly and unexpectedly, referred to as “sudden unexpected death in Parkinson’s disease” (SUDPAR) ,. SUDPARConsidering that SUDPAR is a rare phenomenon, difficult to diagnose, and only rarely reported, it is a phenomenon that has attracted the interest of the neuroscientific community since the late 1970s . More re2+ concentration ([Ca2+]c) and energy production in cardiomyocytes, such as Ca2+ channels, Ca2+-ATPases, the sarcoplasmic reticulum (SR), and mitochondria (MIT) (2+ homeostasis (PD-related cardiac dysfunction may manifest as ventricular arrhythmias due to the collapse of cardiac excitation-contraction coupling (CECC), primarily caused by persistent ionic deregulation in cardiomyocytes ,17,22,24ia (MIT) ,28. In mia (MIT) ,28. MIT eostasis ,28.2+ influx from the extracellular medium to the cytosol through L-type voltage-dependent Ca2+ channels (VDCCs) (2+ influx stimulates Ca2+-release (CICR) from the SR via ryanodine-sensitive Ca2+ channels (RyRs), generating a transient elevation in the [Ca2+]c and consecutive activation of the myosin-actin contractile myofilaments. This transient elevation in [Ca2+]c simultaneously increases the Ca2+ uptake by MIT and the Ca2+ concentration in the mitochondrial matrix ([Ca2+]m), which stimulates ATP production by the activation of the dehydrogenases in the tricarboxylic acid (TCA) cycle. To generate contractility for the ejection of blood from the heart, the activation of myosin by energy-stored ATP molecules is required to shift the head pulling on the actin filament and to shorten the sarcomere. The strength of myocardial contraction is directly related to the local Ca2+ concentration surrounding the myosin-actin myofilaments. Thus, the synchronization of [Ca2+]c transients throughout the myocardium is crucial for synchronous cardiac contraction  ,28. Thistraction ,28. Howehythmias ,28. In s2+ uptake by MIT is mainly mediated by the mitochondrial uniporter of Ca2+ (MUC), while its efflux is mainly mediated by the mitochondrial Na+/Ca2+-exchange channel (mNCE)  . Thus, t [Ca2+]m -30. Howeoduction ,27-29. TIt has been shown that mutations in genes causing PD, such as PINK1, parkin, DJ-1, alpha-synuclein, and LRRK2, cause mitochondrial dysfunctions, which is one of the reasons why they are called mitochondrial nigropathies . Mitocho2+ phosphate crystals that severely compromise the functional integrity of MIT . Hmyocytes  32). Si. Si2+ ov2+ channel, thereby reducing the influx of Ca2+ into MIT  also prevented cardiac arrhythmias and hemodynamic dysfunctions in laboratory animals exposed to cardiac IR injury . It has into MIT ,28,32,33myocytes ,28,32,33myocytes ,28,32,33Several studies suggested that myocardial dysfunctions similar to those induced by cardiac IR can be involved in SUDPAR ,17,22,24Scorza FA and Caricati-Neto A contributed to the design, and manuscript writing and editing. Menezes-Rodrigues FS, Errante PR and Tavares JGP contributed to the acquisition, analysis and interpretation of experimental data. Scorza CA, Ferraz HB, Finsterer J and Olszewer E contributed to the critical review of the manuscript.


MDM2 promoter are thought to have opposite effects on the binding of transcription factor SP1 (specificity protein 1), and consequently on MDM2 expression, p53 levels, cancer risk, age at onset, and prognosis. Here, we genotyped SNP309 and SNP285 in 406 Austrian breast cancer patients and 254 female controls. The SNP309GG genotype was associated with an increased breast cancer risk in p53 negative , but not p53 positive or unselected patients. In contrast, the SNP309TT genotype was associated with an earlier age at onset . 31% of SNP309TT, 26% of TG, and 13% of GG tumors were p53 positive (p = 0.034), indicating a lower selective pressure to mutate TP53 in the presence of the G-allele. Moreover, SNP309TT patients exhibited a shortened metastasis-free survival in multivariable analysis. Censoring carriers of the SNP285C-allele hardly altered the strength of these associations of SNP309, thus challenging the proposed antagonistic function of SNP285C towards SNP309G. The minor SNP285C-allele tended to be non-significantly associated with an increased breast cancer risk and a poor disease-free and metastasis-free survival, which may be bystander effects of its complete linkage disequilibrium with SNP309G. We conclude that the SNP309G-allele attenuates the p53-response and leads to a higher breast cancer risk, but also to a later onset of breast cancer and a trend towards a good prognosis.SNP309T>G (rs2279744) and SNP285G>C (rs117039649) in the  TP53 is the most commonly mutated gene in human cancer [TP53 occurs at a frequency of 25–35% and is often associated with an earlier age at onset, a poor prognosis and a triple-negative molecular subtype [TP53 is also a major mechanism of functional inactivation of p53 [Mdm2 knockout in mice leads to early embryonic lethality, whereas mice with a deletion of both Mdm2 and p53 are viable and develop normally, demonstrating that the negative regulation of p53 is a critical role of MDM2 [MDM2 amplification and/or overexpression may be an important mechanism of aberrant p53 inactivation, and has been observed in many tumors, often mutually exclusive with TP53 mutation [The tumor suppressor gene n cancer . In brea subtype ,5. p53 i subtype ,7. In adn of p53 . MDM2  is a sequence variant in the pression . In the pression . Howeverpression ,17,18,19MDM2 P2 promoter, SNP285G>C (rs117039649) [A potential mechanistic explanation for these discordant results was provided by the more recent discovery of another SNP in the 7039649) ,21. SNP27039649) . Like SN7039649) . In addi7039649) . Only a 7039649) ,27,28,297039649) ,25,29,30MDM2 SNP309 and SNP285 in a Central European (Austrian) study population. This closes an important geographical gap between the many Northern and several Southern European populations studied thus far, particularly in light of their divergent SNP285 allele frequencies. We further sought to analyze the association of SNP285 and SNP309 with breast cancer risk, with the age at breast cancer onset, with key clinical and histopathological parameters of breast cancer, in particular the p53 status, and with breast cancer survival and prognosis. By studying the potential mutual interference between SNP309 and SNP285 with respect to these associations, we aimed to address the hypothesis that SNP285 antagonizes the effects of SNP309, thus potentially confounding the many studies of SNP309 in Caucasians that have not taken SNP285 into account.The aim of our study was to determine the distribution and allele frequencies of MDM2 gene  were genotyped in an Austrian hospital-based case-control study of 406 breast cancer patients and 254 control subjects. p = 0.34) and SNP309 (p = 0.39). Likewise, the patient population was in Hardy-Weinberg equilibrium for both SNPs . The frequency of the minor G-allele of SNP309 (MAF) was 0.365 in patients and 0.347 in controls, close to the 0.367 MAF reported by the 1000 Genomes Project population database [p = 1.1 × 10−15), indicating that the C-allele of SNP285 is present exclusively in a distinct SNP285C/SNP309G haplotype. 9.6% (17/178) of SNP309TG patients (8.2% of controls), and 10.2% (6/59) of SNP309GG patients (7.4% of controls) exhibited the SNP285GC genotype. The promoter SNPs rs117039649 (SNP285G>C) and rs2279744 (SNP309T>G) in intron 1 of the database . SNP285 database ,22,25,31database ,22,25. Ap-values were determined. This analysis revealed moderately, but non-significantly increased odds ratios associated with the GG genotype, but not the TG genotype, indicating a recessive inheritance mode , 95% confidence intervals (CI), and nce mode . Censorince mode . We next analyzed the association of SNP285 with breast cancer risk. SNP285CC patients or controls were not observed in our study population, so we compared genotype GC vs. GG, which revealed no significant association . Excludip = 0.02; p = 0.04). A trend at borderline significance for an elevated odds ratio in the recessive model was also observed in patients older than 55 years, in patients with large tumors , and in stage II–IV patients  status and p53 status have been reported ,33,34,35patients . Analysepatients . The asspatients .p < 0.05, log-rank test; p < 0.04; Associations of SNP309 with the onset age of hereditary breast cancer have been reported in several studies ,38,39,40p = 0.03; Fisher’s exact test), indicating a higher rate of p53 mutation with increasing number of SNP309 T-alleles  as part of the routine clinical breast cancer diagnostics in Austria. p53 IHC positivity indicates an inactivating mutation of p53, since it leads to a defective p53-MDM2 negative feedback loop and hence an accumulation of stabilized p53 protein ,43,44,45-alleles . The rat-alleles . These dTP53. Detailed follow-up records for this survival analyses were available for 132 of the genotyped patients. For 115 of these 132 patients, TP53 status was determined by DNA sequencing in addition to IHC [p < 0.04; The overall survival (OS), disease-free survival (DFS) and metastasis-free survival (MFS) of patients with the three SNP309 genotypes were compared in Kaplan-Meier analyses of unselected patients, SNP285GG patients, ER-positive patients, and patients with wildtype n to IHC , and theMDM2 promoter P2 has led to an elegant hypothesis potentially solving a long-standing conundrum: Why is the SNP309G-allele associated with an increased cancer risk in Asian, but not in Caucasian populations [The discovery of SNP285 in ulations ,21,27,29ulations . Howeverulations ,22. On tulations ,18,19. Hulations ,32. We cBRCA1 mutation carriers [MDM2 and hence to attenuate the p53-pathway. We hypothesize that this seemingly paradoxical association may be due to the differential rate of p53 positivity found by us and others, which is low in SNP309GG, but high in TT-tumors (see discussion below). It has been convincingly established that TP53 mutant patients have a much younger age at onset [TP53 and hence to a much higher rate of these mutations, which then causes the earlier mean age at onset associated with that genotype. This effect of differential somatic TP53 mutation rate obviously does not exist in Li-Fraumeni patients who have a germline mutation in TP53, which may explain why they differ from sporadic cancer cases with respect to the association of SNP309 with the age at cancer onset. Consistently, we found an association of the age at breast cancer onset with MDM2 mRNA expression. Significantly elevated mean MDM2 mRNA levels were observed in patients with an age ≥55 years, as well as in post-menopausal patients .In the initial study of SNP309, the GG genotype was found to be associated with a significantly younger age at cancer onset in Li-Fraumeni patients . This ficarriers ,27,48,49carriers ,51,52,53carriers . Like oucarriers . This isp = 0.02), but not in p53 positive or unselected patients  in TP53 itself [We found that the SNP309GG genotype is significantly associated with an increased breast cancer risk in p53 negative patients, who harbour a functional p53 protein . Only women of Central European descent from the same geographical area were included as patients or controls. The response rates for both patients and controls were >80%. Two patients were excluded from further analyses due to failure to genotype SNP309, one additional patient and one control due to failure to genotype SNP285, and one additional patient since genotyping of both SNPs was unsuccessful. Accordingly, all analyses reported here are based on the 406 breast cancer patients and 254 controls for whom both SNP309 and SNP285 were successfully genotyped.This study was approved and is annually reviewed by the Institutional Review Board (“Ethikkommission”) of the Medical University of Vienna, Austria (MUV) . Written informed consent was obtained from all participants. Clinical and histopathological characteristics of the study population are shown in MDM2 gene was performed by qPCR with TaqMan Genotyping Mastermix and the following primers and allele-specific, fluorescently labeled probes : SNP285, forward primer, 5′-CGCGGGAGTTCAGGGTAAAG-3′; reverse primer: 5′-ACAGGCACCTGCGATCATC-3′; G-allele reporter probe: 5′-CCGGCCCCCGTGAC-3′; C-allele reporter probe: 5′-CGGCCCGCGTGAC-3′; SNP309, forward primer, 5′-CGGGAGTTCAGGGTAAAGGT-3′; reverse primer: 5′-ACAGGCACCTGCGATCATC-3′; T-allele reporter probe: 5′-CCGCGCCGAAGCG-3′; G-allele reporter probe: 5′-CCGCGCCGCAGCG-3′. PCR reactions were carried out on a CFX96 instrument for SNP285 , and a QS3 instrument for SNP309  with 20 ng of genomic DNA in a reaction volume of 20 µL following the manufacturer’s instructions. As a quality control, ~100 samples were genotyped in duplicate, 5 of them from separate DNA isolations, with 100% concordance. Laboratory staff was blinded with respect to the identity of these duplicates, and with respect to patient or control status of all samples. For technical reasons, the SNP285 genotype could not be determined for two patients and one control subject, and the SNP309 genotype for three patients. Genomic DNA has been extracted previously from peripheral lymphocytes with the QIAamp DNA Blood Midi kit , and from fresh-frozen primary tumour samples with the High Pure PCR Template Preparation Kit  as described ,69. Genop-values associated with odds ratios were calculated by the mid-P exact method. As advocated [t-tests, and between three or more groups by ANOVA. Statistical methods of survival analyses, as well as follow-up details of our study population have been described in References [p-Values to Kaplan-Meier analyses and analyses of the cumulative breast cancer incidence were calculated by log-rank tests, as described in Reference [TP53 status, and lymph node status. The subcategories of these variables were coded as follows: SNP309 genotype, TT = 0, TG = 1, GG = 2; SNP285 genotype, GG = 0, GC = 1; ER status, positive = 0, negative = 1; TP53 status, wildtype = 0, mutated = 1 ; lymph TP53 is reduced. An attenuated p53-response is presumably still functional to some extent as opposed to one that is completely inactivated by mutation, and consequently leads to a later onset of breast cancer and a trend towards a good prognosis. None of the observed associations of SNP285 were significant in our study, which may in part be due to the very low frequency of the variant C-allele. However, the observed trends towards an increased breast cancer risk and a poor survival are at odds with the proposed molecular function of the SNP285C-allele to reduce binding of SP1 and expression of MDM2. Accordingly, we conclude that these associations of SNP285C are bystander effects of its complete linkage disequilibrium with SNP309G. The odds ratios in subgroups according to SNP309 genotype (We found that the SNP309GG genotype was associated with an older age at breast cancer onset, a good prognosis in multivariable analysis of the metastasis-free survival and a trend in the same direction in related survival analyses, an increased breast cancer risk in specific subgroups, and a rate of p53 positivity less than half compared to the SNP309TT genotype. These findings are consistent with a model in which the p53-response is attenuated in the presence of the SNP309GG genotype. A direct consequence of this attenuation may be that the risk of breast cancer and other cancers is increased, but that the selective pressure to mutate genotype , and in genotype  support genotype . This isgenotype . In conc


The genetic predisposition to chronic kidney disease (CKD) has been widely evaluated especially using the genome-wide association studies, which highlighted some novel genetic susceptibility variants in many genes, and estimated glomerular filtration rate to diagnose and stage CKD. Of these variants, rs7805747 in PRKAG2 was identified to be significantly associated with both serum creatinine and CKD with genome wide significance level. Until now, the potential mechanism by which rs7805747 affects CKD risk is still unclear. Here, we performed a functional analysis of rs7805747 variant using multiple bioinformatics software and databases. Using RegulomeDB and HaploReg (version 4.1), rs7805747 was predicated to locate in enhancer histone marks . Using GWAS analysis in PhenoScanner, we showed that rs7805747 is not only associated with CKD, but also is significantly associated with other diseases or phenotypes. Using metabolite analysis in PhenoScanner, rs7805747 is identified to be significantly associated with not only the serum creatinine, but also with other 16 metabolites. Using eQTL analysis in PhenoScanner, rs7805747 is identified to be significantly associated with gene expression in multiple human tissues and multiple genes including PRKAG2. The gene expression analysis of PRKAG2 using 53 tissues from GTEx RNA-Seq of 8555 samples (570 donors) in GTEx showed that PRKAG2 had the highest median expression in Heart-Atrial Appendage. Using the gene expression profiles in human CKD, we further identified different expression of PRKAG2 gene in CKD cases compared with control samples. In summary, our findings provide new insight into the underlying susceptibility of PRKAG2 gene to CKD. Chronic kidney disease (CKD) is a major global problem caused by the permanent loss of kidney function, and is also associated with an increased risk for cardiovascular disease . The oveIn these CKD risk genes, a genetic variant rs7805747 in PRKAG2 was identified to be significantly associated with both serum creatinine and CKD with genome wide significance level . The rs7RegulomeDB database could annotate genetic variants with known and predicted regulatory elements in the intergenic regions of the human genome . In brieHaploReg is a tool for exploring annotations of the non-coding variants , 2016. HPhenoScanner included publicly available large-scale GWAS summary results, about 3 billion associations and over 10 million unique single nucleotide polymorphisms (SNPs) and a broad range of phenotypes . The resWe evaluated the expression of PRKAG2 using the RNA-Seq datasets from the NIH Genotype-Tissue Expression (GTEx) project, which was created to establish a sample and data resource for studies on the relationship between genetic variation and gene expression in multiple human tissues . The GTEP < 0.01.We analyzed a whole genome case-control expression profiles in human CKD . In the Using RegulomeDB, the predicted score is 5, which suggested that rs7805747 is likely to affect binding (TF binding) or DNase peak. The predicted binding protein is HNF4A (chr7:151407767-151408030 by ChIP-seq in Caco2 cell type) . The hisUsing HaploReg v4, rs7805747 is predicated to locate in enhancer histone marks , DNase hypersensitivity , and motifs changed  , 2016.P < 0.01. In addition to the CKD, we found that rs7805747 is also significantly associated with other diseases or phenotypes including Hemoglobin Hb, Hematocrit Hct, Red blood cell count RBC, systolic blood pressure (SBP), Breast cancer, Gout, Hypertension, and Extraversion, as described in Table Using PhenoScanner in GWAS option, we identified 43 significant association results with P < 0.01. These findings show that rs7805747 is significantly associated with gene expression in multiple human tissues including brain, cells transformed fibroblasts, colon sigmoid, colon transverse, heart left ventricle, liver, lung, skin, small intestine terminal ileum, stomach, and whole blood, as described in Table P = 3.81E-03 and 8.15E-04.Using PhenoScanner in eQTL option, we identified 23 significant associations between rs7805747 and gene expression with P < 0.01. These metabolites included Creatinine, Indolelactate, Phenol sulfate, Pseudouridine, Propionylcarnitine, C-glycosyltryptophan, Kynurenine, Myo-inositol, 3-carboxy-4-methyl-5-propyl-2-furanpropanoate (CMPF), 1-palmitoylglycerophosphoethanolamine, Phenyllactate (PLA), Erythronate, N-acetylthreonine, Citrulline, 3-methoxytyrosine, Urate, 2-methylbutyroylcarnitine, as described in Table Using PhenoScanner in metabolites option, we identified significant association between rs7805747 and 17 metabolites with Using the UCSC Genome Browser, the results showed that PRKAG2 had the highest median expression: 34.90 RPKM in Heart – Atrial Appendage . PRKAG2 had the total median expression 412.37 RPKM in all these 53 tissues. Figure P = 1.23E-07 and log2(fold change) = -2.07], and A_23_P44366 [P = 4.39E-03 and log2(fold change) = 0.74], and A_23_P314760 [P = 1.54E-02 and log2(fold change) = 0.62]. In Table P < 0.01. Table There are three probes to evaluate the expression of PRKAG2 gene in the gene expression profiles in human CKD including A_24_P384779, A_23_P44366, and A_23_P314760. Each of these three probes represents different regions of PRKAG2 gene. These three probes may have the same or different transcript or isoforms, or extrons. The results showed that all these probes about the PRKAG2 gene is significantly dysregulated in CKD cases compared with control samples including A_24_P384779 [It is reported that PRKAG2 could encode the gamma2-subunit isoform of 5′-AMP-activated protein kinase (AMPK) , 2017. AOver the past decade, GWAS have considerably improved our understanding of the genetic basis of kidney function and disease . A SNP rUsing PhenoScanner in GWAS option, we showed that rs7805747 is not only associated with CKD, but also is significantly associated with other diseases or phenotypes including Hemoglobin Hb, Hematocrit Hct, Red blood cell count RBC, SBP, Breast cancer, Gout, Hypertension, and Extraversion.Using PhenoScanner in eQTL option, rs7805747 is identified to be significantly associated with gene expression in multiple human tissues and multiple genes including PRKAG2. Previous study has reported rs7805747 to be associated with serum creatinine and CKD . Using PThe gene expression analysis of PRKAG2 using 53 tissues from GTEx RNA-Seq of 8555 samples (570 donors) in GTEx showed that PRKAG2 had the highest median expression in Heart – Atrial Appendage. Using the gene expression profiles in human CKD, we further identified different expression of PRKAG2 gene in CKD cases compared with control samples. All these findings indicate that rs7805747 is associated with CKD risk, PRKAG2 gene expression, and 17 metabolites. Meanwhile, gene expression analysis further showed that CKD cases had different expression of PRKAG2 gene. In summary, our findings provide new insight into the underlying susceptibility of PRKAG2 gene to CKD.EW conceived and initiated the project and performed the functional analysis. EW, HZ, DZ, LL, and LD wrote the manuscript. All authors reviewed the manuscript and contributed to the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.