TBX5 haploinsufficiency causes congenital conduction disorders, whereas increased expression levels of TBX5 in human heart samples has been associated with atrial fibrillation (AF). We deleted the conserved mouse orthologues of two independent AF-associated genomic regions in the Tbx5 locus, one intronic (RE(int)) and one downstream (RE(down)) of Tbx5. In both lines, we observed a modest (30%) increase of Tbx5 in the postnatal atria. To gain insight into the effects of slight dosage increase in vivo, we investigated the atrial transcriptional, epigenetic and electrophysiological properties of both lines. Increased atrial Tbx5 expression was associated with induction of genes involved in development, ion transport and conduction, with increased susceptibility to atrial arrhythmias, and increased action potential duration of atrial cardiomyocytes. We identified an AF-associated variant in the human RE(int) that increases its transcriptional activity. Expression of the AF-associated transcription factor Prrx1 was induced in RE(int)KOTbx5 cardiomyocytes. We found that some of the transcriptional and functional changes in the atria caused by increased Tbx5 expression were normalized when reducing cardiac Prrx1 expression in RE(int)KOTbx5 mice, indicating an interaction between these two AF genes. We conclude that modest increases in expression of dose-dependent transcription factors, caused by common regulatory variants, significantly impact on the cardiac gene regulatory network and disease susceptibility.Heart development and rhythm control are highly Tbx5 dosage-sensitive.  The lifetime risk of developing a common disease, such as cardiovascular or neurodegenerative conditions, is influenced by genetic predisposition resulting from large numbers of inherited common genetic variants . Disease-associated variants are typically found in noncoding genomic regions and are thought to affect the functionality of regulatory elements (REs) such as enhancers or elements involved in chromatin conformation . These rPITX2, TBX5, and PRRX1, suggesting that altered expression levels of such factors cause imbalances in gene regulatory networks that control heart rhythm and function  have identified many common variants in over 100 genetic loci associated with atrial fibrillation (AF) risk, the most prevalent arrhythmia associated with high comorbidity and increased mortality risk . The idefunction . Indeed,function . Heterozfunction . Interesfunction . Furtherysmal AF . Moreove with AF . The effTBX5 locus to investigate how variant regions associated with a common disease modulate phenotype in a tissue- and developmental stage-specific manner. Each deletion caused a modest 30% increase in Tbx5 expression in different heart compartments and at different stages of life. We report the relatively large effect of this modest increase in Tbx5 expression on atrial function including arrhythmia susceptibility, and on the gene regulatory network. Decreased expression of Prrx1 has been associated with AF in human and mouse models  harbors accessible chromatin sites in the left atria and ventricles, and EMERGE enhancer prediction signal KOTbx5 adult mice expressed approximately 30% more Tbx5, whereas only the left atrium and lungs of RE(down)KOTbx5 adult mice expressed more Tbx5 (RE(int)KOTbx5 mice (RE(int)KOTbx5 KO mice . We confE(int)KO . Interesc tissue . This su–6) (Arabidopsis SOL1/2 (TCX/TCX2) (sine oculis (SIX) homeodomain transcription factor binding motif   using lu–6) . Of the –6) . ComparaCX/TCX2) , which aCX/TCX2) , a DNA-bCX/TCX2) . We alsong motif . Lin54 ang motif .Tbx5. Both RE(int)KOTbx5 and RE(down)KOTbx5 mice had slower and more variable heart rates ; standard deviation of normal to normal R-R intervals (SDNN)) (RE(int)KOTbx5 mice, but remained unaffected in RE(down)KOTbx5 mice (RE(int)KOTbx5, but not RE(down)KOTbx5 mice compared to controls  to determine the functional consequences of such a modest increase in  (SDNN)) . Additio (SDNN)) . PR inte)KO mice . Other E)KO mice . Heart r)KO mice  were inc)KO mice , suggestRE(int)KOTbx5 mice we analyzed the properties of single isolated left atrial cardiomyocytes using patch-clamp. At 6 Hz stimulation, we observed APD prolongation in mutants at all measured repolarization stages, with no changes in AP upstroke velocity, resting membrane potential and maximal AP amplitude (Tbx5 has important roles in intracellular Ca2+ handling (2+ concentration ([Ca2+]i) in isolated atrial CMs using fluorescent calcium indicator Indo-1. We did not observe any changes in systolic or diastolic [Ca2+]i concentration KOTbx5 and RE(down)KOTbx5, respectively, of which 816 were significantly up- and 1235 were downregulated in RE(int)KOTbx5 (RE(down)KOTbx5 (RE(down)KOTbx5 compared to RE(int)KOTbx5 (RE(int)KOTbx5 atria and in RE(down)KOTbx5 atria are comparably affected qualitatively, but not quantitatively, consistent with the slightly higher increase in Tbx5 expression in RE(int)KOTbx5 compared to RE(down)KOTbx5. Gene Ontology analysis KO . These danalysis  revealedanalysis . Accordichanges,  were sigchanges, .Tbx5 in left atria (RE(int)KOTbx5) to that of a gain-of-function missense mutation (G125R/+Tbx5) (Tbx5 deletion (Tbx5iKO) KOTbx5 mice  , and notG125R/+) . In contTbx5iKO)  revealedTbx5iKO) , thus in control , and was control . However)KO mice .Tbx5 expression in cardiomyocytes results in changes in chromatin accessibility, we performed ATAC-sequencing profiling (ATACseq) of RE(int)KOTbx5 left atrial cardiomyocytes (Tbx5 (one of which caused by the deletion of the intronic genomic fragment in RE(int)KOTbx5 mice), suggestive for increased direct transcriptional autoregulation in RE(int)KOTbx5 mice (Tbx5 expression does not significantly change transcription factor occupancy or epigenetic state.To determine whether a slight increase in myocytes . After pmyocytes . Two of )KO mice . The verPRRX1, encoding the transcription factor Paired Related Homeobox 1, has been linked to AF-predisposition (PRRX1 expression in human cardiac tissues has been associated with AF (PRRX1 (Prrx1(enh)) to investigate its role in gene regulation and rhythm control ((enh)KOPrrx1 mice express less Prrx1 specifically in cardiomyocytes compared to controls, and show atrial conduction slowing, lower AP upstroke velocity KOTbx5 with (enh)KOPrrx1 mice (decreased Prrx1 in cardiomyocytes), and investigated cardiac transcriptomes and phenotypes across genotypes. We isolated cardiomyocyte and non-cardiomyocyte nuclei from whole hearts using a PCM-1 antibody and interrogated Tbx5 and Prrx1 expression across all genotypes. Tbx5 was upregulated in RE(int)KOTbx5 cardiomyocytes, as expected, but not deregulated in (enh)KOPrrx1 or double mutant cardiomyocytes (Prrx1 levels were increased in RE(int)KOTbx5 cardiomyocytes and decreased in (enh)KOPrrx1, as expected, and also decreased in double mutant cardiomyocytes  deletion.We considered whether the presence of two AF-risk alleles would exacerbate the phenotype(s) consistent with AF. position . Reduced with AF . We prev control . Prrx1(enduction . To explmyocytes . Prrx1 lmyocytes . There wmyocytes . These dRE(int)KO;Prrx1(enh)KOTbx5 double mutants were more similar to the transcript changes found in RE(int)KOTbx5 than the ones found in (enh)KOPrrx1, when comparing all lines to WT, suggesting a dominant contribution of RE(int)KOTbx5 to the transcriptional changes in double mutants (RE(int)KOTbx5 mice, those of RE(int)KO;Prrx1(enh)KOTbx5 double homozygous showed less variance with WT atrial transcriptomes KOTbx5 would also be rescued by the presence of this second AF-risk allele. In vivo ECGs and burst pacing experiments revealed that RR, PR interval, and WBCL were normalized in double homozygous mice, whereas SDNN, cSNRT, and AA inducibility remained unchanged KOPrrx1 mice, and increased Tbx5 imposes changes in expression and electrophysiological properties, in part through increasing Prrx1 expression in cardiomyocytes.Taken together, our findings suggest that an interaction between TBX5 locus modulate Tbx5 expression in tissue- and stage-specific manners, causing distinct specific phenotypes. While the impact of 50–100% dose reduction of Tbx5 in humans and model systems has been well-investigated  on phenotype. Our models provide insight into the mechanisms underlying the pleiotropic effects and interactions of disease-associated regulatory variants, which typically cause small changes in target gene expression in particular cell-types, conditions or stages of development  may increase Tbx5 expression in both the left atrium and pulmonary vein myocardium. The pulmonary veins have been strongly implicated in AF as they are the most common source of triggered activity KOTbx5 mice were slower and more variable than of controls. In addition, PR interval was prolonged in RE(int)KOTbx5 mice combination with increased Wenckebach cycle lengths. The latter is in line with the important role of Tbx5 in sinus node and AV node development and function (RE(int)KOTbx5 mice suggesting electrophysiological remodeling of the atria. It has been shown that mice haploinsufficient for Tbx5 show atrial downregulation of genes encoding proteins involved in cardiac conduction (e.g. Gja1/Cx43 and Scn5a/Nav1.5) and Ca2+ handling (e.g Atp2a2/Serca2a and Ryr2/Ryr2) KOTbx5 mice, where expression of Tbx5 was slightly elevated, transcriptomic analysis indicated upregulation of genes involved in cardiac conduction including Scn5a and Cx43. The Ca2+ handling genes Atp2a2/Serca2a and Ryr2/Ryr2 were not differently expressed between RE(int)KOTbx5 mice and controls. Accordingly, in our mice, we did not observe any changes in Ca2+ transients and long decay constants as seen in Tbx5 haploinsufficient mice KOTbx5 mice. This is consistent with the observed longer atrial APD in RE(int)KOTbx5 mice compared to controls, which may predispose to increased arrhythmia inducibility, as seen in mice harboring the pathogenic TBX5-G125R variant or Prrx1 enhancer deletion (Heart rates of the function . Moreover2/Ryr2) . This leopensity . In the ent mice . Expressdeletion . Althougdeletion , prolongdeletion .Tbx5 iKO (Tbx5 deletion induced in adult mice), of Tbx5+/G125R (heterozygous for a gain of function missense mutation) and of RE(int)KOTbx5 mice show APD prolongation (RE(int)KOTbx5 and Tbx5+/G125R models, whereas significantly differentially expressed genes common to both RE(int)KOTbx5 and Tbx5 iKO models were changed in opposite direction  do not necessarily predict changes in APD. Furthermore, Pitx2 was downregulated in left atria of Tbx5 iKO mice and upregulated in right atria of Tbx5+/G125R mice (Pitx2 expression in the atria of RE(int)KOTbx5 mice. Together, these findings indicate that decreasing or increasing Tbx5 dose or changing Tbx5 function induces a large number of divergent transcriptional responses that disturb the balance in the genetic networks underlying functional  properties of the atria, such as APD, leading to similar phenotypic outcomes.The molecular mechanisms underlying electrophysiological remodeling have remained unclear. For example, atrial cardiomyocytes of ongation . When coirection . This su25R mice , and Tbx25R mice . Yet, wePrrx1 expression, and that Tbx5 levels are Prrx1-dependent (or Prrx1 target-dependent) in specific contexts. We then asked whether combining two alleles modeling the changes in gene expression conferred by AF-associated variant regions would cause a greater effect on heart phenotype, and by extension, arrhythmia predisposition, than each allele alone. Here, we found that introduction of this AF-susceptibility allele RE(int)KOTbx5 mice rescued heart rate, PR interval, and atrioventricular node phenotypes. This example shows that two variant regions that are both independently associated with increased risk for a particular disease, do not necessarily cumulatively increase disease predisposing phenotype, but may neutralize each other’s effect. Previously, the atrial electrophysiological phenotype of Tbx5 haploinsufficient mice was observed to be rescued by haploinsufficiency of either Pitx2 or Gata4, both of which have been associated with AF as well , guide 2 (GGACTGTTGGGTCACCTTGT), and RE(down) guide 1 (GGCTCCTTCGTCAGTAAATA), guide 2 (AAACAAGGGCTCTCTGGCGTTT). The deleted coordinates are the following in mm10: RE(int) chr5:119,874,013–119,880,148, and RE(down) chr5:119,891,017–119,915,744. Founders were backcrossed with wildtype FVB/NJ mice to obtain stable lines. Downstream experiments were performed on F3-F7 mice, backcrossed with wild-type FVB mice. All transgenic mice were maintained on a FVB/NJ background commercially obtained from Jackson laboratory (stock number 100800).Mutant mice were generated using CRISPR/Cas9. Guide RNA (sgRNA) constructs were designed with ZiFiT Targeter . The sgR2 inhalation followed by cervical dislocation.For tissue harvest, animals were euthanized by 20% COTimed pregnant female mice received intraperitoneal injection of 100 mg/kg EdU one hour before sacrifice by isoflurane and cervical dislocation. E14.5 fetuses were isolated in 1xPBS on ice, the head was removed and the body fixed in 4% PFA for 24 hr. 7 μm sections were stained with mouse-anti-actin , goat-anti-nkx2.5 , DAPI (1:1000) and EdU Click-IT  before imaging and counting. Nkx2.5 positive nuclei were designated cardiomyocytes, Nkx2.5 and EdU double positive nuclei were designated proliferating cardiomyocytes. Ratios were normalized within each litter.TGTTGGATATGCCCTTGACT, GATTCAACTTGCGCTCATCT; Ppia: GGGTGGTGACTTTACACGCC, CTTGCCATCCAGCCATTCAG; Rpl32: GCCTCTGGTGAAGCCCAAG, TTGTTGCTCCCATAACCGATGT; Tbx5: CCCGGAGACAGCTTTTATCG, TGGTTGGAGGTGACTTTGTG; Prrx1: CACAAGCAGACGAAAGTGTGG, GTTGTCCTGTTTCTCCGCTG; Tbx3: CGCCGTTACTGCCTATCAGAA; GCCATTGCCAGTGTCTCGAA.Total RNA was isolated from atria, ventricle and lungs from P21 and left and right atria from adult male and female mice using ReliaPrep RNA Tissue Miniprep System  according to the manufacturer’s protocol. cDNA was reverse transcribed with oligo dT primers from 500 ng of total RNA, or random hexamers from 500 pg of CM nuclear RNA, according to the manufacturer’s protocol of the Superscript II Reverse Transcriptase system . Expression levels of candidate target genes were determined by quantitative real-time PCR using a LightCycler 480 Instrument II . Expression levels were measured using LightCycler 480 SYBR Green I Master  and the primers had a concentration of 1 pmol/L. The amplification protocol consisted of 5 min 95 °C followed by 45 cycles of 10 s 95 °C, 20 s 60 °C and 20 s 72 °C. Relative start concentration (N0) was calculated using LinRegPCR . Values CVCL_0303) were grown in 24-well plates in Claycomb medium  supplemented with chemically defined HL-1 FBS substitute , Glutamax  and Pen/Strep . Cells were transfected using polyethylenimine 25 kDa  at a 1:3 ratio (DNA:PEI). Standard transfections were carried out using 200 ng of reporter construct per well. 24 hr after transfection, cells were lysed using Renilla luciferase assay lysis buffer  and luciferase activity measured. Luciferase measurements were performed using a GloMax Explorer . During the measurement, 100 µL D-Luciferin  was injected (150 µL/s) followed by a 1-s delay and 5 s of measurement. Transfections were carried out at least three times and measured in duplicates. HL-1 cell cultures were routinely tested negative for mycoplasma contamination. HL-1 cell lines were easily distinguished based on cellular morphology and contractility. HL-1 is not found in the database of commonly misidentified cell lines that is maintained by the International Cell Line Authentication Committee.RE sub fragments were cloned into a modified pGL2-Basic plasmid containing an SV40 minimal promoter and an adjusted multiple cloning site for in vitro analysis by a transfection luciferase assay. HL1 cells  and placed on thermostated mat (36 °C) with a steady flow of 1.5% isoflurane during all experiments. Electrodes were inserted subcutaneously in the limbs and connected to an ECG amplifier . The electrocardiogram (ECG) was measured for 5 min. ECG parameters were determined in Lead II (L-R) based on the last 60 s of the recording. For atrial stimulation, an octapolar CIB’er electrode (NuMED) was advanced through the esophagus to achieve atrial capture. Atrial capture thresholds were determined for each mouse, and all pacing protocols were performed at 2 x threshold. For sinus node recovery time (SNRT) measurements, a 4-s pacing train with a cycle length of 120 or 100ms was used. SNRT was defined as the interval between the last pacing stimulus and onset of the first P wave. To control for differences in sinus rate, SNRT was normalized to resting heart rate . To determine the Wenckebach cycle length (WBCL) we applied a 4s pacing train starting at a cycle length of 100ms, and decreasing by 2ms until atrioventricular (AV) block was first observed. Atrial arrhythmia (AA) was induced by 1 or 2 s bursts starting with a cycle length of 60ms, decreasing successively with a 2 ms decrement, down to a cycle length of 10ms. AA duration was the sum of time each mouse spent under and AA episode after completion of the two passes. Atrial arrhythmia (AA) inducibility was scored as the number of mice in which at least one episode lasting >1 s of arrhythmia was induced after pacing. Mice of both sexes were used for atrial arrhythmia induction experiments.2 1.8, MgCl2 1.0, glucose 5.5, HEPES 5.0; pH 7.4 (set with NaOH). Subsequently, the hearts were perfused with Tyrode’s solution containing a low Ca2+-concentration (10 μmol/L) for 10 min, after which Liberase TM research grade  and Elastase from porcine pancreas  were added for 12 min at a concentration of 0.038 mg/mL and 0.01 mg/mL, respectively. All solutions were saturated with 100% O2 and the temperature was maintained at 37 °C. To obtain single cells, the digested left atria was cut into small pieces which were triturated for 4 min through a pipette (tip diameter: 0.8 mm) in the low Ca2+ Tyrode’s solution, supplemented with 10 mg/ml Bovine Serum Albumin . Single cells were stored at room temperature for at least 45 min before they were used. Quiescent single rod-shaped cells with smooth surfaces were selected for electrophysiological measurements.Single cells were isolated from left atria of adult male mice by enzymatic dissociation. Therefore, excised hearts were perfused for 5 min in a Langendorff system with a modified Tyrode’s solution containing (in mmol/L): NaCl 140, KCl 5.4, CaClAction potentials (APs) were recorded with the amphotericin-B perforated patch-clamp technique using an Axopatch 200B amplifier . Voltage control and data acquisition were performed as described previously , and APs20, APD50, and APD90, respectively). Parameters from 10 consecutive APs were averaged.APs were recorded at 36 ± 0.2°C using the modified Tyrode’s solution. Pipettes ) were filled with solution containing (in mmol/l): K-gluc 125, KCl 20, NaCl 5, amphotericin-B 0.44, HEPES 10, pH 7.2 (KOH). APs were elicited at 2–8 Hz by 3ms,~1.2 × threshold current pulses through the patch pipette. We analyzed resting membrane potential (RMP), maximal AP amplitude (APA), maximum AP upstroke velocity, and AP duration at 20, 50, and 90% repolarization (APD2+]i were measured at 25 °C in HEPES solution ((mmol/l): Na+ 156, K+ 4.7, Ca2+ 1.3, Mg2+ 2.0, Cl− 150.6, HCO3 4.3, HPO2− 1.4, HEPES 17, Glucose 11 and 1% fatty acid free albumin, pH 7.3) using the fluorescent probe Indo-1 as described previously . After a 10 min incubation in the lysis buffer, an additional 10 strokes were performed with a tight pestle. The lysis procedure was monitored by light microscopy to ensure complete tissue and cell lysis and efficient nuclear extraction. The crude lysate was successively passed through 100  and 30 µm mesh filters. The final lysate was spun at 1000 × g for 5  min and the resulting pellet was resuspended in 500  µl staining buffer (5% BSA in PBS) supplemented with RNAse inhibitor. Isolated nuclei were incubated with rabbit polyclonal antibodies specific for pericentriolar material 1 (PCM1)  at a dilution of 1:400 for 1  hr rotating at 4  °C. Next, Alexa Fluor 647-conjugated donkey-anti-rabbit 647 antibodies , and DAPI (1:1000 dilution) were added and the incubation was continued for an additional hour. Samples were spun at 1000 × g for 10  min and washed with 500  µl staining buffer before resuspension in 500  µl staining buffer supplemented with RNAse inhibitor. Intact CM nuclei were sorted on a BD Influx FACS on the basis of DAPI and Alexa Fluor 647 positivity into cold BL +TG buffer from the ReliaPrep RNA Tissue Miniprep System  for RNA isolation, and into resuspension buffer for ATACseq . RNA wasRE(int)KOTbx5, 3 RE(down)KOTbx5, 4 (enh)KOPrrx1, and 3 Double homozygous.For RNA isolated from left atria, 500 ng was used for library generation with the KAPA mRNA HyperPrep kit (Roche) and sequenced on the HiSeq4000 system (Illumina) with 50 bp single-end reads. RNAseq sample sizes are as follows: Whole adult left atria: 4 WT, 3 http://cran.rproject.org/web/packages/pheatmap/index.html). PANTHER KOTbx5, all with random genome as background in HOMER.200 bp summits were determined of ATAC-seq performed in atrial cells . In totahttp://cran.r-project.org/web/packages/pheatmap/index.html).Unsupervised hierarchical clustering was performed on differentially detected peaks and genes in RNA-seq and ATAC-seq using the R package pheatmap, version 1.0.8.  duration was determined with Mann Whitney Wilcoxon test and differences in AA inducibility were tested using Fisher’s exact test. Conduction velocity was analyzed with unpaired t-tests with Welch’s correction. For single cell and whole tissue action potential (AP) duration (APD) normality and equal variance assumptions were tested with the Kolmogorov-Smirnov and the Levene median test, respectively. Two groups were compared with unpaired t-test or repeated measures ANOVA followed by pairwise comparison using the Student-Newman-Keuls test. Differences in Ca2+ transient amplitude as well as changes in diastolic and systolic Ca2+ concentrations in atria were tested using two-way ANOVA. Multiple testing corrections were performed independently within each hypothesis. Data are presented as individual data points and mean or mean ± standard error of the mean (SEM) or standard deviation (SD), as indicated, and <0.05 defines statistical significance. Statistical analysis was performed using GraphPad Prism 9.The experimenters were blind to mouse genotype during all measurements and outcome assessment. Datasets were tested for normality using Shapiro-Wilk test unless specified otherwise. Whole tissue RT-qPCR fold change vs WT in fetal ventricles and adult whole left atria corrected expression changes were analyzed with unpaired t-tests and reference gene-corrected  Molecular mechanisms of atrial fibrillation, a highly prevalent arrhythmia, have been challenging to contextualize. The investigators, in a series of experiments using the deletion of two loci in TBX5 (one intronic and one downstream), achieved a modest increase in Tbx5 in the atria. In a series of elegant experiments they show that this increase in TbX5 in turn impacted the cardiac gene regulatory network that resulted in a higher susceptibility to AF. These results will provide useful insights into the mechanisms of disease especially the variable phenotypes observed with functional variants of a gene. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:Tbx5 levels and arrhythmia susceptibility" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Kalyanam Shivkumar as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Benoit Bruneau (Reviewer #2).Thank you for submitting your article "An atrial fibrillation-associated regulatory region modulates cardiac The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:Please address the discussion points raised by reviewers # 1 and 2.1. Please provide data for this comment by reviewer # 2-Does the increase in Tbx5 gene expression result in an increase in protein expression?2. Please address the extra data sought by reviewer # 3 in the detailed comments.Reviewer #1 (Recommendations for the authors):1. Why is the assumption that the regulatory elements "modulate the expression of TBX5 only" as opposed to have trans effects on distant gene encoding regions? For example, in reference 41 on the topic of promoter capture Hi-C maps, "Remarkably, more than 90% of SNP-target gene interactions did not involve the nearest gene, while 40% of SNPs interacted with at least two genes, demonstrating the importance of considering long-range chromatin interactions when interpreting functional targets of disease loci." Aside from the two regions for selected to study, were additional regions identified?2. As "13790 and 13564 different transcripts in RE(int)-/- and RE (down)-/-" were identified, how is it known that the transcriptional changes are from increased TBX5 levels as opposed to a direct, trans effects due to the deletion of the two loci at TBX5? This seems at odds with the claim that "[d]eletion of either of the two RE-containing regions only affected Tbx5 expression".3. As the SNP of interest rs7312625 has been identified as a variant associated with the electrocardiographic P wave (https://pubmed.ncbi.nlm.nih.gov/28794112/) and the transgenic mice in this study had slower heart rates, sinus pauses, inverted P waves , PR prolongation, corrected sinus node recovery time, and Wenckebach cycle length, could the findings be more explanatory for nodal (sinus and atrioventricular) function and the cardiac conduction system more generally rather than atrial fibrillation in particular? The study seems as though it could have been re-framed as an evaluation of nodal function rather than atrial fibrillation. Of note, these findings were of note in the RE(down)-/- mice, which did not have increased atrial arrhythmia inducibility.4. The authors cite reference 19 as showing a "slight increase…in cardiac TBX5 expression in human heart tissues [as being] associated with AF". What was the magnitude of the increase? Challenging to gather this information on this reviewer's review of reference 19.5. Of all the genes that are modulated by changes in TBX5 expression, why was Prrx1 chosen? How do the authors explain that lack of change noted for Pitx2?Reviewer #2 (Recommendations for the authors):The rationale for focusing on RE(down) is not clearly appreciated based on Figure 1. This is in contrast to the apparent evidence in support of RE(int). Promoter Hi-C data shows DNA interactions in human that appears to span beyond the presumptive syntenic region of RE(down) in mouse, as well as a lack of chromatin accessibility, histone marks or EMERGE prediction for RE(down). Perhaps highlighting relevant features for RE(down) more explicitly would address this minor concern.For supplemental figure 1, please define LD as "linkage disequilibrium" prior to abbreviation, as appropriate, and list the abbreviation at the beginning of the manuscript. As well, a scale bar is not apparent.Does the increase in Tbx5 gene expression result in an increase in protein expression?It would be interesting to compare these mouse models to the recently published TBX5 gain of function model.For Figure 3, please define RR as "R-R interval" in legend and SDNN as "standard deviation of normal to normal R-R intervals", as appropriate, and add to abbreviation list.In Figure 5, where is Prrx1 highlighted in the transcriptomic analysis?For Figure 6C, how does Prrx1(enh)-/- compare in the PCA analysis? Please display it.Reviewer #3 (Recommendations for the authors):1. TBX5 mutations in humans and animal models are known to generate congenital heart defects and upper limb/radial malformations. Perhaps of relevance to this manuscript, human patients with TBX5 duplications also show many of these same phenotypes . Do RE(int) and RE(down) homozygous mutations result in congenital heart or limb malformations? Structural heart defects can certainly impact cardiac electrophysiology and reporting structural defects, or lack of them can help put the phenotype into context.2. The RE(int) deletion appears to be more severe than the RE(down) deletion. Does the RE(int) deletion interfere with the splicing of the last two exons? Depending on library construction, perhaps the RNA-seq can address this.3. In Figure 2E, the authors report that rs7312625 A>G appears to be the functional SNP as it alone results in increased Tbx5 expression in vitro. As this is a predicted regulatory element, what transcription factor binding motif(s) is this SNP predicted to interfere or generate? Does this motif change make sense with gained Tbx5 expression in cardiomyocytes/HL1 cells?4. In Figure 3C, the authors report RE(int) mutants have an increased PR interval and in Figure 4C, the authors report RE(int) have an increased APD20, 50, and 90. Single-copy loss of Tbx5 in mice has been reported to cause these same phenotypes . Would you not expect the opposite phenotype, i.e., decreased PR interval and APD20/50/90? What is the explanation for these paradoxical results?5. Several transcriptional profiling datasets have been generated for various Tbx5 mutations in mouse and human including a few by the authors (by cursory search of GEO for "TBX5"). How do the gene expression changes seen in the current datasets compare with previously generated datasets? Are the changes largely consistent with expectations of increased Tbx5 levels in RE(int) and RE(down)? It appears most public datasets are in Tbx5/TBX5 loss-of-function contexts, so we are expecting the opposite results. Does this data support that hypothesis or is there something else going on?6. Figures 2A and 2C report that RE(down) demonstrated increased expression of Tbx5 in the adult left atria and P21 lungs, specifically, but there is little to no mention of this in the text or discussion. Given the importance of the pulmonary veins in the development of atrial fibrillation and prior publication history from the authors and colleagues on the topic of left atrial Tbx5, can the authors expand their discussion on the RE(down) related SNPs? The SNPs associated with RE(down) are independently segregating and the differences observed in this manuscript may hint at an independent, tissue-dependent function for that regulatory element. Essential revisions:Please address the discussion points raised by reviewers # 1 and 2.1. Please provide data for this comment by reviewer # 2-Does the increase in Tbx5 gene expression result in an increase in protein expression?2. Please address the extra data sought by reviewer # 3 in the detailed comments.We would like to thank the reviewers for their constructive criticism and suggestions. In our revised manuscript, we have addressed all comments and questions raised as detailed below. We believe that the incorporated changes have greatly improved the manuscript.Reviewer #1 (Recommendations for the authors):1. Why is the assumption that the regulatory elements "modulate the expression of TBX5 only" as opposed to have trans effects on distant gene encoding regions? For example, in reference 41 on the topic of promoter capture Hi-C maps, "Remarkably, more than 90% of SNP-target gene interactions did not involve the nearest gene, while 40% of SNPs interacted with at least two genes, demonstrating the importance of considering long-range chromatin interactions when interpreting functional targets of disease loci." Aside from the two regions for selected to study, were additional regions identified?Tbx5 regulatory region is confined to its own TAD . Additionally, we examined the expression of genes adjacent to the Tbx5 TAD, Rbm19, Tbx3 and Med13l, in atria and ventricles of mice with deletions by RT-qPCR, and found no changes in their expression . These data confirm that the deleted regulatory regions target Tbx5 and not genes in adjacent TADs.Although a regulatory element, potentially containing SNP(s), usually shares the topologically associating domain (TAD) with its target genes, it does not necessarily interact with the nearest gene. In the referred study, promoter capture Hi-C identified only a fraction of the interactions, and showed a bias against detecting proximal interactions because of the high background signal associated with genomic locations close to each other. Moreover, the interactions reflect conformation (3D structure) of the chromatin and do not necessarily imply functional interactions. Nevertheless, our group has previously shown that the TBX5/Tbx5 locus, namely, in the second to last intron, which can be seen in Figure 1. However, we chose not to study this region because of the decreased accessibility in atrial tissue (ATACseq) and the comparably lower degree of conservation across species (GERP).We identified at least one additional AF-associated variant region with regulatory potential in the 2. As "13790 and 13564 different transcripts in RE(int)-/- and RE (down)-/-" were identified, how is it known that the transcriptional changes are from increased TBX5 levels as opposed to a direct, trans effects due to the deletion of the two loci at TBX5? This seems at odds with the claim that "[d]eletion of either of the two RE-containing regions only affected Tbx5 expression".Tbx5, we anticipate that the transcriptional changes are a result of increased Tbx5 levels rather than a consequence of the deletion of the variant regions. As mentioned above in response to comment 1 of the reviewer, previous research indicated that the regulatory elements of Tbx5 are restricted to the Tbx5 TAD , and we confirmed that nearby genes in adjacent TADs were not influenced by the deletions . Regulatory element-target promoter interactions are generally assumed to be confined to the same TAD , and it is considered very unlikely that these interactions span multiple TADs or chromosomes. Furthermore, we observed the upregulation of several well established TBX5 target genes , suggesting that increased Tbx5 dose is causal. Finally, we compared the transcriptomes of left atria of RE(int)-/- mice  to the reported transcriptome of left atria of mice in which Tbx5 was inactivated . We observed a strikingly opposite transcriptional response , indicating that the Tbx5-dependent regulatory network responds in opposite direction to increased and decreased Tbx5 dose, respectively.Although we cannot fully rule out that the deleted regulatory regions target other genes in addition to 3. As the SNP of interest rs7312625 has been identified as a variant associated with the electrocardiographic P wave (https://pubmed.ncbi.nlm.nih.gov/28794112/) and the transgenic mice in this study had slower heart rates, sinus pauses, inverted P waves , PR prolongation, corrected sinus node recovery time, and Wenckebach cycle length, could the findings be more explanatory for nodal (sinus and atrioventricular) function and the cardiac conduction system more generally rather than atrial fibrillation in particular? The study seems as though it could have been re-framed as an evaluation of nodal function rather than atrial fibrillation. Of note, these findings were of note in the RE(down)-/- mice, which did not have increased atrial arrhythmia inducibility.TBX5 and SCN5A, involved in atrial cardiomyocyte electrophysiology. We addressed this aspect here, and found that atrial cardiomyocytes showed altered electrophysiological properties facilitating arrhythmia. The observed inverted P waves, changed SAN recovery times and changed WCL indeed suggest that SAN and AVN function may be affected in addition. Therefore, it is possible that the atrial arrhythmia is a culmination of multiple triggers, including changes in nodal and working cardiomyocytes. While we certainly want to further investigate a role of Tbx5 in SAN function, we feel that further characterization of nodal function remains outside the scope of this study.We thank the reviewer for this comment. The study the reviewer refers to states: “The P wave on an ECG is a measure of atrial electric function, and its characteristics may serve as predictors for atrial arrhythmias.” Therefore, the SNPs identified in this study may impact on expression of genes, including 4. The authors cite reference 19 as showing a "slight increase…in cardiac TBX5 expression in human heart tissues [as being] associated with AF". What was the magnitude of the increase? Challenging to gather this information on this reviewer's review of reference 19.TBX5 expression in lines 96-98 and 144-145.We thank the reviewer for pointing this out. We have amended the text in the revised manuscript to clarify a 30% increase in cardiac 5. Of all the genes that are modulated by changes in TBX5 expression, why was Prrx1 chosen?PRRX1 locus has been strongly associated with AF. PRRX1 encodes a transcription factor that we and others have shown to be causally linked to atrial fibrillation susceptibility. Therefore, we chose to intercross RE(int)-/- and Prrx1(enh)-/- mice to test whether the two established AF-risk alleles would aggravated the arrhythmogenic phenotype. Strikingly, we found a partial rescue of the phenotypes instead. We later determined that Prrx1 was upregulated in RE(int)-/- specifically in cardiomyocytes, but not in bulk RNAseq, likely because Prrx1 is most highly expressed in non-cardiomyocyte cell populations.In GWAS studies, the We have changed the revised manuscript to clarify this in 229-2243, as follows:PRRX1, encoding the transcription factor Paired Related Homeobox 1, has been linked to AF-predisposition . Reduced PRRX1 expression in human cardiac tissues has been associated with AF (19). We previously deleted the mouse orthologous variant region near PRRX1 (Prrx1(enh)) to investigate its role in gene regulation and rhythm control (25). -/-Prrx1(enh) mice express less Prrx1 specifically in cardiomyocytes compared to controls, and show atrial conduction slowing, lower AP upstroke velocity (indicative for lower Na+ current densities) (58), as well as increased systolic and diastolic [ca2+]i concentration that culminate in increased susceptibility to atrial arrhythmia induction (25). To explore whether and how these two AF-risk genes may interact, we intercrossed -/RE(int)- with -/-Prrx1(enh) mice (decreased Prrx1 in cardiomyocytes), and investigated cardiac transcriptomes and phenotypes across genotypes.”“We considered whether the presence of two AF-risk alleles would exacerbate the phenotype(s) consistent with AF. How do the authors explain that lack of change noted for Pitx2?Pitx2 expression in the left atria of our mutant mice. We surmise that this is because a 30-35% increase of Tbx5 expression is not sufficient to induce a proportional increase in Pitx2. We recently characterized a Tbx5 gain-of-function mouse model  in which Pitx2 was increased in the right atrium, while the left atrium remained unaffected. We therefore examined whether Pitx2 expression was changed in the right atria of RE(int)-/- by RT-qPCR and found that it is indeed not affected, thus suggesting that the modest increase in Tbx5 level does not influence Pitx2 expression.We did not observe a change in We included this result in lines 215-216, new Figure 5—figure supplement 3, and discussion in lines 348-356.Reviewer #2 (Recommendations for the authors):The rationale for focusing on RE(down) is not clearly appreciated based on Figure 1. This is in contrast to the apparent evidence in support of RE(int). Promoter Hi-C data shows DNA interactions in human that appears to span beyond the presumptive syntenic region of RE(down) in mouse, as well as a lack of chromatin accessibility, histone marks or EMERGE prediction for RE(down). Perhaps highlighting relevant features for RE(down) more explicitly would address this minor concern.We would like to thank the reviewer for this suggestion. While the absence of interactions as assayed in Promoter Capture Hi-C does not necessarily indicate a lack of interaction, the interactions beyond the RE(down) region indeed indicate possible distal downstream regulatory elements. Consistently, Smemo et al.  identified multiple enhancer candidates downstream of the REdown region, one of which was found to harbor a SNP associated with CHD (non-HOS). We chose to include the RE(down) region in our study because it harbors AF-associated variants, and H3K4me1 modifications in cardiomyocytes. We have clarified this in lines 125-126 of the revised manuscript.For supplemental figure 1, please define LD as "linkage disequilibrium" prior to abbreviation, as appropriate, and list the abbreviation at the beginning of the manuscript. As well, a scale bar is not apparent.We thank the reviewer for pointing out these oversights. We have changed the text in the supplement as indicated.Does the increase in Tbx5 gene expression result in an increase in protein expression?-/- and WT mice by Western Blot. However, we were unable to detect Tbx5 protein. We performed immunofluorescence staining on adult atria to show that indeed Tbx5 protein is detected in the atria of mutant and control mice, even though a 30% increase is not quantifiable using this method. However, the analysis shows that Tbx5 remains selectively expressed in PCM-1+ cardiomyocyte nuclei in RE(int)-/- atria .We attempted to quantify protein expression in adult atria of RE(int)It would be interesting to compare these mouse models to the recently published TBX5 gain of function model.-/- and Tbx5G125R/+ as Supplementary file 4 and in Figure 5—figure supplement 2, respectively. Surprisingly, we found very few differentially expressed genes (DEGs) common to both models, while their electrophysiological phenotypes were more alike- inverted P-waves, slower and more variable heart rates, increased atrial arrhythmia induction, and prolonged APD. Conspicuously, an inducible loss-of-function Tbx5 mouse model  also displays irregular RR, prolonged APD and arrhythmia. We have also compared transcriptomes of left atria of our RE(int) model to those of this induced adult Tbx5 deletion model  and found opposite response of many of the same DEGs, as expected. Together, this suggests that perturbation of a Tbx5–dependent network independently of direction of change of many of the network genes results in overlapping phenotypic changes culminating in atrial dysfunction.We thank the reviewer for this valuable suggestion. We now include an additional table and a scatter plot showing a comparison between adult left atria from RE(int)We have included this comparison in lines 204-208.For Figure 3, please define RR as "R-R interval" in legend and SDNN as "standard deviation of normal to normal R-R intervals", as appropriate, and add to abbreviation list.We apologize for the oversight. The definitions have been added to the revised manuscript as indicated.In Figure 5, where is Prrx1 highlighted in the transcriptomic analysis?Prrx1 was, in fact, not found to be differentially expressed in left atrial RNAseq data. We found that the induction of Prrx1 occurs only in atrial cardiomyocytes of RE(int)-/- mice, consistent with cardiomyocyte-specific expression-, and thereby induction in RE(int)-/-, of Tbx5 . We surmise that this change was not detectable in bulk RNAseq because Prrx1 is mostly expressed in non-cardiomyocytes, which is likely not affected in our mutants.Tbx5 and Prrx1 is muddled without this observation. Therefore, we have changed the text in lines 229-238, as specified in reviewer 1 point 5.We understand that the rationale for studying the interaction between For Figure 6C, how does Prrx1(enh)-/- compare in the PCA analysis? Please display it.-/- RNAseq dataset in the PCA because it was obtained, along with its own WT control group, in a different session . It appeared that this difference in session greatly influences the PCs. However, we compared the L2FC (and p(adj)) values in each transcriptomic comparison (WT vs RE(int)-/-, WT vs Prrx1(enh)-/-, WT vs RE(int)-/-;Prrx1(enh)-/- (Double mutant)), as these parameters are relatively unaffected by inter-session variation. We observed that (1) the number of genes differentially expressed between mutant and WT decreased going from RE(int)-/-, Double mutant, Prrx1(enh)-/-, (2) the direction of change of DEGs was similar between RE(int)-/- and Double mutant, but different from Prrx1(enh)-/-, (3) the distribution of the L2FCs was slightly smaller in Double mutant than RE(int)-/-. This is shown in the revised Supplemental figure 10. Together, these analyses suggest that RE(int)-/- has a much larger effect on the atrial transcriptome than Prrx1(enh)-/-, and that the effect of RE(int)-/- is slightly normalized by the inclusion of Prrx1(enh)-/- in RE(int)-/-.Unfortunately, we are unable to directly compare the Prrx1(enh)Reviewer #3 (Recommendations for the authors):1. TBX5 mutations in humans and animal models are known to generate congenital heart defects and upper limb/radial malformations. Perhaps of relevance to this manuscript, human patients with TBX5 duplications also show many of these same phenotypes . Do RE(int) and RE(down) homozygous mutations result in congenital heart or limb malformations? Structural heart defects can certainly impact cardiac electrophysiology and reporting structural defects, or lack of them can help put the phenotype into context.Tbx5 expression in RE(down)-/- mice is selectively induced in atrial and lung tissue after birth, and therefore limb defects are not expected. Similarly, atrial induction in RE(int)-/- mice is observed after birth. However, we noted a mild induction of Tbx5 expression in prenatal ventricles of RE(int)-/- mice (not in RE(down)-/- mice). While this does not lead to structural defects, the ventricles may be slightly larger. We are currently investigating this possible phenotype.We thank the reviewer for this suggestion. We have included the notion of CHD in patients with duplications and these references in the Introduction section of the revised manuscript in lines 85-86. We did not observe any structural defects of limbs or heart. 2. The RE(int) deletion appears to be more severe than the RE(down) deletion. Does the RE(int) deletion interfere with the splicing of the last two exons? Depending on library construction, perhaps the RNA-seq can address this.-/- . Moreover, when zooming in on the intronic region, we clearly see absence of reads mapped to the deleted region, validating the absence of this genomic sequence. Furthermore, when we performed RT-qPCR of WT and RE(int)-/- atrial samples using intron/exon-spanning primers in exons 6-8, 7-9 and 8-9, respectively, we obtained the same result for both groups, and the obtained amplicons did not include fragments in which exon 8 was skipped .This is a very insightful suggestion. Inspection of the RNAseq data sets revealed that the exon read coverage of exons relative to each other, including the ones flanking the intra-intronic deletion, is highly similar between WT and RE(int)3. In Figure 2E, the authors report that rs7312625 A>G appears to be the functional SNP as it alone results in increased Tbx5 expression in vitro. As this is a predicted regulatory element, what transcription factor binding motif(s) is this SNP predicted to interfere or generate? Does this motif change make sense with gained Tbx5 expression in cardiomyocytes/HL1 cells?sine oculis (SIX) homeodomain transcription factor binding motif with the risk allele (new Supplementary file 1). SIX transcription factors can act as transcriptional activators or repressors depending on interactions with other highly conserved regulators including Paired-box (Pax), Eyes absent (Eya), Dachshund (Dach), and Groucho (Grg) proteins . Both Six4 and Six5 are expressed in atrial tissue of mice (see Supplementary table 1) and human ). We speculate the SNP could create a SIX binding site, causing loss of repressive activity of the RE. We have added this information to the revised manuscript in lines 152-159 of the Results section and 295-298 in the discussion.We analyzed whether the rs7312625 substitution interfered/changed predicted binding motifs and found that the risk allele disrupts motifs for SOL1/2 (TCX/TCX2/CHC), which are homologues of the animal CHC protein LIN54 that binds DNA in a sequence-specific manner and is a component of DREAM complexes regulating cell cycle-dependent transcription. Furthermore, we observed gain of a 4. In Figure 3C, the authors report RE(int) mutants have an increased PR interval and in Figure 4C, the authors report RE(int) have an increased APD20, 50, and 90. Single-copy loss of Tbx5 in mice has been reported to cause these same phenotypes . Would you not expect the opposite phenotype, i.e., decreased PR interval and APD20/50/90? What is the explanation for these paradoxical results?Tbx5. When comparing the transcriptional response of increased Tbx5 in left atria (RE(int)-/-) to that of Tbx5G125R/+  ) we observed that it was unexpectedly divergent. When comparing the published left atrial transcriptomes of Tbx5 iKO ; Tbx5 deletion induced in adult mice) with that of RE(int)-/-, we found, as expected, that most significantly differentially expressed genes (DEGs) common to both datasets were changed in opposite direction . Remarkably, all three Tbx5 mouse models display APD prolongation. This suggests that a (probably) large number of transcriptional changes occur in RE(int)-/- that disturb the balance between AP duration-increasing and -decreasing gene expression, leading to phenotypic outcome similar to that of both Tbx5 loss of function and Tbx5 G125R missense variant. This further indicates that the response to Tbx5 dose is complex. Moreover, as indicated by the reviewer, loss- and gain of TBX5 gene copy results in CHD phenotypes, consistent with the notion that both reduced and increased Tbx5 levels can result in similar phenotypic outcomes.This observation is puzzling to us as well, and may reflect the complexity of responses to either decreased or increased levels of 5. Several transcriptional profiling datasets have been generated for various Tbx5 mutations in mouse and human including a few by the authors (by cursory search of GEO for "TBX5"). How do the gene expression changes seen in the current datasets compare with previously generated datasets? Are the changes largely consistent with expectations of increased Tbx5 levels in RE(int) and RE(down)? It appears most public datasets are in Tbx5/TBX5 loss-of-function contexts, so we are expecting the opposite results. Does this data support that hypothesis or is there something else going on?-/- mice to those of a Tbx5 gain-of-function variant mouse model  and an adult-specific knockout mouse model . Please see reviewer 2 point 4, and the response this reviewer above.We have included a new supplemental figure and accompanying tables in which we compare transcriptional profiles of left atria of RE(int)6. Figures 2A and 2C report that RE(down) demonstrated increased expression of Tbx5 in the adult left atria and P21 lungs, specifically, but there is little to no mention of this in the text or discussion. Given the importance of the pulmonary veins in the development of atrial fibrillation and prior publication history from the authors and colleagues on the topic of left atrial Tbx5, can the authors expand their discussion on the RE(down) related SNPs? The SNPs associated with RE(down) are independently segregating and the differences observed in this manuscript may hint at an independent, tissue-dependent function for that regulatory element.This is an excellent suggestion. We have addressed this point in the Discussion lines 302-313 as follows:RE(down) may increase Tbx5 expression in both the left atrium and pulmonary vein myocardium. The pulmonary veins have been strongly implicated in AF as they are the most common source of triggered activity . TBX5 levels in pulmonary vein myocardium may influence gene regulation independently from the atrial TBX5 levels (66). The variants in RE(down) segregate independently from those in RE(int), indicating these variant RE regions act through distinct tissue-specific transcriptional mechanisms that are influenced by AF-predisposing common variants. A further implication is they may cumulatively increase AF predisposition in a manner dependent on risk variant dose, in which homozygous carriers of risk haplotypes in RE(int) and RE(down) have the largest relative predisposition. These relations could be addressed in future genotype-phenotype analyses of human atrial and pulmonary vein samples.”“…the AF-associated variants in ReferencesDixon, J.R., Selvaraj, S., Yue, F., Kim, A., Li, Y., Shen, Y., Hu, M., Liu, J.S., Ren, B., 2012. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–80. https://doi.org/10.1038/nature11082Meurer, L., Ferdman, L., Belcher, B., Camarata, T., 2021. The SIX Family of Transcription Factors: Common Themes Integrating Developmental and Cancer Biology. Front. Cell Dev. Biol. 9.Nadadur, R.D., Broman, M.T., Boukens, B., Mazurek, S.R., Yang, X., Van Den Boogaard, M., Bekeny, J., Gadek, M., Ward, T., Zhang, M., Qiao, Y., Martin, J.F., Seidman, C.E., Seidman, J., Christoffels, V., Efimov, I.R., McNally, E.M., Weber, C.R., Moskowitz, I.P., 2016. Pitx2 modulates a Tbx5-dependent gene regulatory network to maintain atrial rhythm. Sci. Transl. Med. 8. https://doi.org/10.1126/scitranslmed.aaf4891Nora, E.P., Lajoie, B.R., Schulz, E.G., Giorgetti, L., Okamoto, I., Servant, N., Piolot, T., Van Berkum, N.L., Meisig, J., Sedat, J., Gribnau, J., Barillot, E., Blüthgen, N., Dekker, J., Heard, E., 2012. Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485, 381–385. https://doi.org/10.1038/nature11049Drosophila genome. Cell 148, 458–472. https://doi.org/10.1016/j.cell.2012.01.010Sexton, T., Yaffe, E., Kenigsberg, E., Bantignies, F., Leblanc, B., Hoichman, M., Parrinello, H., Tanay, A., Cavalli, G., 2012. Three-dimensional folding and functional organization principles of the Smemo, S., Campos, L.C., Moskowitz, I.P., Krieger, J.E., Pereira, A.C., Nobrega, M.A., 2012. Regulatory variation in a TBX5 enhancer leads to isolated congenital heart disease. Hum. Mol. Genet. 21, 3255–63. https://doi.org/10.1093/hmg/dds165van Ouwerkerk, A.F., Bosada, F.M., van Duijvenboden, K., Houweling, A.C., Scholman, K.T., Wakker, V., Allaart, C.P., Uhm, J.-S., Mathijssen, I.B., Baartscheer, T., Postma, A.V., Barnett, P., Verkerk, A.O., Boukens, B.J., Christoffels, V.M., 2022. Patient-Specific TBX5-G125R Variant Induces Profound Transcriptional Deregulation and Atrial Dysfunction. Circulation 145, 606–619. https://doi.org/10.1161/CIRCULATIONAHA.121.054347Weerd, J.H. van, Badi, I., Boogaard, M. van den, Stefanovic, S., Werken, H.J.G. van de, Gomez-Velazquez, M., Badia-Careaga, C., Manzanares, M., Laat, W. de, Barnett, P., Christoffels, V.M., 2014. 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The intrinsic n-type of epitaxial graphene on SiC substrate limits its applications in microelectronic devices, and it is thus vital to modulate and achieve p-type and charge-neutral graphene. The main groups of metal intercalations, such as Ge and Sn, are found to be excellent candidates to achieve this goal based on the first-principle calculation results. They can modulate the conduction type of graphene via intercalation coverages and bring out interesting magnetic properties to the entire intercalation structures without inducing magnetism to graphene, which is superior to the transition metal intercalations, such as Fe and Mn. It is found that the Ge intercalation leads to ambipolar doping of graphene, and the p-type graphene can only be obtained when forming the Ge adatom between Ge layer and graphene. Charge-neutral graphene can be achieved under high Sn intercalation coverage (7/8 bilayer) owing to the significantly increased distance between graphene and deformed Sn intercalation. These findings would open up an avenue for developing novel graphene-based spintronic and electric devices on SiC substrate. The linear dispersion of π-bands endows graphene with novel physical properties and vast potential for applications in electric, optoelectronic, and photo-electrochemical fields ,2,3,4,5.xCy, as reported in our previous work [Generally, the intercalation between the buffer layer and SiC substrate is beneficial for turning the buffer carbon layer into freestanding graphene by saturating the Si dangling bonds. Graphene may be doped with electrons or holes depending on the type and properties of intercalations. The most widely used H intercalation ,15,16,17ous work ,34. Howeous work ,35,36,37ous work ,31; Fe ious work . Overall1-xGex alloy intercalations with different Sn:Ge ratios [To achieve the stable p-type and charge-neutral graphene, the intercalation materials need to meet the following basic screening conditions. Firstly, the intercalated atomic orbitals should have strong electron providing and holding capacity at the same time. Secondly, the intercalation layer could stably exist between the SiC substrate and graphene. Finally, the intercalation could only modulate the electronic structure without introducing any magnetism to graphene. The intercalation of the main group metals without d orbitals may overcome the negative influence on the insurmountable electron doping and magnetic interactions of graphene. The Ge ,40,41,42e ratios  could bee ratios . Furthere ratios . Althoug1-xGex intercalated systems. The effect of the coverage of intercalations and the location of the intercalated atoms on the electronic structure of graphene has been highlighted. The calculation results verified that the p-type and charge-neutral graphene could be achieved by high-coverage Ge and Sn intercalations, which is attributed to the charge transfer mechanism. Both the Ge and Sn intercalations could effectively inhibit the induced magnetism in graphene, which was different from the cases of TM intercalations [To answer these questions, we performed first-principles calculations to investigate the structures and electronic structure of the Ge, Sn, and Snalations ,30,31,32The band structures of (2 × 2)Gr − (√3 × √3)SiC are compared to the (2 × 4)Gr − (√3 × 2√3)SiC structure to evaluate the effect of the computational cell size on the electronic structure of graphene c,d. It iEf), and we will not discuss these cases anymore. The intercalation energies indicated that all the intercalation structures are stable, and the relative stability of the intercalation structures depends on the coverage—high coverages show better thermodynamic stability. Then, their thermal stabilities were tested using AIMD simulations. It was reported that graphene was experimentally synthesized by the thermal decomposition of SiC at 900 °C, and Ge (Sn) intercalation was generated at 600–800 °C [The Ge intercalations can decouple the interactions between the 0LG and SiC substrates, as shown in 0–800 °C . The AIMHere, the effect of the intercalation coverage and atom location on the electronic structure modulation of graphene has been explored. The experiments showed that some Ge atoms deintercalated from the system with increasing temperature, resulting in a decrease in the Ge coverage gradually . Angle-rThe Ge-intercalated structures show a coverage-dependent electronic ambipolar doping of graphene. As shown in 1) and other Ge atoms  in the second Ge layer were ~0.8 Å smaller than those between the Ge atoms in the same layer  calculated using the following Equation (2).When the Ge coverages are as high as 7/8 and 4/8 BL, the graphene becomes p-type with the Dirac point at 0.18 eV above the Fermi level a,d. The me layer . In orde1-Ge2/3/4 are an order of magnitude smaller than the others, demonstrating that Ge1 and Ge2/3/4 have strong chemical bonding interactions. Such strong chemical bonding of the Ge adatom to the Ge layer induces the charge transfer from graphene to the Ge intercalation. Bader charge calculation results show that the graphene loses 0.14 |e|, while the Ge intercalation gains 0.18 |e|, demonstrating the formation of p-type graphene in the presence of the Ge adatom. Meanwhile, the presence of the Ge adatom for the 4/8 and 7/8 BL coverages results in a significant change in the distance between the layers. Specifically, the distances between the Ge layers are largely decreased by 0.3–0.6 Å, and the distances between graphene and the Ge layer are slightly increased by 0.11–0.14 Å.It is usually treated as a descriptor of the bond strength in compounds, and the negative value of ICOHP indicates strong interaction between atoms. The calculated ICOHP values of GeFor the low Ge coverages , the effect of intercalation atom location on the band structures is also investigated. We found that the atomic location only slightly influences the position of the Dirac point, and the electronic states near the Fermi level as well as the conduction type of graphene remain almost the same. This observation is consistent with the experimental conclusion that the graphene doping type is not sensitive to the order of the intercalation layer structure . Since tB. However, the magnetism has not been extended to graphene owing to the complete graphene π-bonds and little interaction with metal intercalations. When the Ge coverage reduces to 1/16 ML, the Ge-intercalated structure becomes half-metal, which arises from the electronic and magnetic couplings of the Ge layer with the SiC surface rather than the graphene. It indicates that such a structure may fabricate spin batteries and ideal magnetic tunnel junctions for spintronic applications.Since Ge has metal characteristics that may introduce magnetism into the graphene analogously to the Fe intercalation, the spin DOS and the electron density of Ge-intercalated structures were calculated and showed in 2 planarity of the graphene is higher than the energy gain from forming the covalent Sn-C bonds. Meanwhile, the Ef and EI of Sn intercalation system are also used to reflect the possible and preferable coverage ranges of the intercalations in terms of energy, as shown in Similarly, the Sn intercalation can also transform 0LG into free-state graphene, as shown in We also found the coverage-dependent regulation of electronic structure, but the effect and mechanism are different from Ge intercalation. Firstly, the distances between graphene and Sn intercalations are very large at high coverages. Normally, the total energy of the whole structure is contributed to two components—elastic contribution (positive effect) and electronic effect (negative effect) . The intOn the other hand, the electron doping of graphene, owing to the Sn intercalation, is overall stronger than that for Ge intercalation. This difference can be attributed to the more metallic character of Sn than Ge, resulting in the more electron transfer to graphene. As a result, the Sn intercalation can be regulated to produce charge-neutral graphene experimentally . Our calB, which is the same as the maximum magnetic moment of Ge intercalated structure. Notably, when the Sn coverage reduces to 1/16 ML, the spin-up channel is metallic and the spin-down channel exhibits an electronic structure similar to the Dirac point of graphene. This change provides the capability to modulate the magnetism and ultra-fast electronic mobility in the same structure. The spin polarization ratio is defined as Equation (3).↑ and ρ↓ are the absolute values of DOS at the Fermi level for spin-up and spin-down channels, respectively. For the 1/16 ML coverage, both the 1LG/Ge/SiC and 1LG/Sn/SiC is half-metals with 100% spin–polarization ratios at the Fermi level, which can be advantageous for nanoscale spintronic-electric applications.The spin DOS and the electron density of Sn-intercalated structures were calculated and showed in According to our theoretical research results, experimental achievement of the p-type and charge-neutral graphene on SiC substrate is based on the precise regulation of Ge and Sn intercalation coverages. It should be noticed that the precise control of intercalation coverage has been achieved experimentally for many years. For example, Gierz et.al successfully traced the intercalation atom deposition process by using angle-resolved photoemission spectroscopy (ARPES) and precisely control the Bi intercalation coverages of 0→0.28→0.38→0.46 atoms/u.c. (the amount of Bi atoms per graphene unit cell) . Moreove1-xGex alloy layers between graphene and the SiC substrate at 600–800 °C. The Sn:Ge ratio was modulated by changing the preparation temperature, and it was inferred that the doping characteristics of graphene could thus be affected. It was also experimentally found that the Ge atoms prefer to locate as the bottom layer and bind to the Si dangling bonds of the SiC substrate, while the Sn atoms are thus the top layer of the alloy intercalation. This phenomenon is confirmed by theoretical calculations. The calculated binding energy between Ge and interfacial Si atoms is −4.19 eV, while that of Sn is −3.81 eV, indicating that Ge atoms are more preferable to bind with the Si-terminated surface than Sn. The calculated ICOHP values of Ge-Si and Sn-Si are −4.86 and −4.13 respectively  in the Perdew–Burke–Ernzertaly (PBE) form [−4 eV/atom for the residual force and total energy, respectively. To provide a good approximation of the experimentally reported (6√3 × 6√3) R30° reconstruction [First-principles calculations were performed based on the Vienna ab initio simulation package (VASP) ,52 in coBE) form  was adoptruction , an intetruction . The ab 1-xGex metal intercalations on the structure and electronic structure of epitaxial graphene on SiC substrate by performing DFT calculations. It is found that the buffer carbon layer could turn into monolayer quasi-free-standing graphene by intercalating these metal layers between graphene and SiC substrate, and the doping properties of graphene depend on the type of metal intercalations and the intercalation coverages. The Ge intercalation induces graphene ambipolar doping, and the p-type graphene could be obtained with 4/8 and 7/8 BL coverages. The p-type graphene originates from the strong interaction between the adatom Ge and its nearby Ge layer. On the other hand, the charge-neutral graphene is tailored by the Sn intercalation with as high coverage as 7/8 BL since the charge transfer between graphene and the Sn layer can be eliminated by the massive deformation of the Sn layer and the resulting enlarged distance between them. Further studies show that the group-IV metal intercalations induce interesting magnetic properties to the entire structure while keeping graphene free of magnetism. These results help understand the effect and mechanism of main-group metal intercalation on the electronic and magnetic property regulation of graphene. It also provides a new path to develop novel spintronic-electric devices based on these composite layered structures.In summary, we investigated the effect of group-IV Ge, Sn, and Sn


Sarcoidosis is a granulomatous inflammatory disease that could potentially involve multiple organ systems. It causes noncaseating granulomas in tissues, and at least two organs must be involved to make a diagnosis. In sarcoidosis patients, if there is a mass in the testicles, a testicular biopsy should be performed to exclude malignancies because of infrequent testicular involvement. We present a 23-year-old male diagnosed with sarcoidosis who had a bilateral testicular mass. A testicular biopsy was performed because of bilateral involvement. The biopsy revealed a diagnosis of sarcoidosis. After high-dose steroid treatment, the lesions regressed. This paper presents a sarcoidosis case with testicular involvement that imitates testicular tumors. Testicular tumors and testicular involvement of sarcoidosis are two different pathologies that may mimic each other, confuse clinicians, and/or lead to misdiagnosis and mistreatment. Sarcoidosis is a granulomatous inflammatory disease that could potentially involve multiple organ systems and causes non-caseating granulomas in tissues . AlthougA 23-year-old male patient with known asthma and a sarcoidosis history came to our outpatient urology clinic with a complaint of left testicular pain. He had been previously diagnosed with sarcoidosis by transbronchial biopsy and mediastinoscopy and had received steroid treatment six months before coming to our clinic. Upon physical examination, left testicular tenderness was detected. Scrotal Doppler ultrasonography (USG) exposed multiple hypoechoic lesions with irregular borders in the left testicle, with the largest being 6 × 6 mm. The USG also showed a regularly bordered hypoechoic 2 mm lesion in the right testicle. The laboratory analysis showed that the patient’s ß-human chorionic gonadotropin (β-HCG) and alpha-fetoprotein (AFP) levels were normal, and his lactate dehydrogenase (LDH) level was measured as 274 U/L. Contrast-enhanced thoracoabdominal computed tomography (CT) documented multiple conglomerated lymphadenopathies (LAPs) in every lymphatic station in the mediastinum, with the largest lymph node’s size being 5 × 4 cm Figure . MultiplBecause the patient had a diagnosis of sarcoidosis and bilateral testicular involvement, we evaluated the patient via contrast-enhanced abdominopelvic magnetic resonance imaging (MRI), which showed multiple nodular lesions smaller than 6 mm in size with weak heterogeneous contrast uptake Figure .Afterward, we recommended radical orchiectomy or testicular biopsy because of his sarcoidosis diagnosis, bilateral testicular involvement, and desire to preserve fertility. We also recommended a spermiogram to evaluate the patient’s fertility state, given that sarcoidosis and testicular tumors might lead to infertility, which the patient refused. He decided to undergo a testicular biopsy. Under spinal anesthesia, the left testicle was exposed through a left scrotal horizontal incision. A USG-assisted needle biopsy was performed on the lesion. Frozen section analysis of the biopsy specimen showed non-necrotizing granulomatous findings in the testicle. No testicular tumor cells were seen. Therefore, we referred the patient to the pulmonology department for treatment. In accordance with the recommendations of the pulmonologists, he started to receive high-dose steroid treatment by getting 32 milligrams of methylprednisolone per day, and the therapy terminated in a dose diminishing way within six months. After receiving high-dose steroid treatment for six months, the patient visited our outpatient clinic for control, as we suggested. Afterward, we checked the testicular tumor marker levels again, and his LDH, AFP, and β-HCG levels were all normal. In the control USG, testicular parenchyma was homogenous and isoechoic, and there were no solid lesions on either side. In non-contrast thoracal CT, the parenchymal nodules were dramatically regressed, and mediastinal LAPs were smaller in size Figures -3B.There were no abnormal findings in the MRI, including LAPs and solid lesions Figure .Sarcoidosis is a multisystemic disease characterized by non-caseating granulomas. Urogenital sarcoidosis is quite rare, and the urogenital system is affected in only 0.2% of all cases . In a puFor differential diagnosis, tuberculosis, fungal infections, and granulomatous diseases such as berylliosis must be excluded, and testicular cancer should be kept in mind . TesticuThe effects of sarcoidosis on fertility have not yet been illuminated. The development of obstructive granuloma and fibrosis secondary to epididymal involvement may lead to azoospermia. Infertility has been reported due to sarcoidosis in some cases . OligospThere is no consensus on the management of testicular sarcoidosis in the literature. The general opinion among the authors is radical orchiectomy, especially in patients with unilateral masses due to the increased risk of malignancy. However, if sarcoidosis is suspected and tumor markers are negative in patients with bilateral involvement, the biopsy is preferred . ExcisioTesticular sarcoidosis should be considered in patients with known sarcoidosis histories applying with testicular mass. Testicular biopsy or radical orchiectomy options may be offered to the patient. Moreover, because there is an increased risk of testicular cancer in patients with sarcoidosis, urologists should regularly examine them for testicular tumors.


While most forms of sarcoidosis of the skin do not require treatment, 40% of patients initially diagnosed with cutaneous sarcoidosis are found to have an asymptomatic disease involving other organ systems. It is the involvement of the lungs, heart, eyes, and nervous system which most often contributes to morbidity/mortality. An early and accurate diagnosis of sarcoidosis is difficult because patients may be asymptomatic, initial presentations may vary, and there is no single reliable diagnostic test except biopsy. We present a case of scar sarcoidosis which led to the diagnosis of stage II pulmonary sarcoidosis in a woman in her 50s. Her scar sarcoidosis presented as well-circumscribed, reddish-brown macules surrounding an atrophic scar from a prior skin graft on the right leg. Biopsy revealed scattered, well-formed, non-necrotizing granulomas of the dermis composed of epithelioid histiocytes and multinucleated giant cells, surrounded by a sparse infiltrate of lymphocytes and histiocytes. A CT chest demonstrated extensive hilar lymphadenopathy, leading to a diagnosis of stage II pulmonary sarcoidosis with cutaneous involvement. This case illustrates the interesting presentation of scar sarcoidosis and underscores the importance of a broad differential including sarcoidosis for skin changes around scars and underscores the need for early biopsy. Prompt cutaneous diagnosis leads to earlier systemic evaluation, therapeutics, and better outcomes. While most forms of sarcoidosis of the skin are mild and do not require treatment, approximately 40% of patients initially diagnosed with cutaneous sarcoidosis are found to have asymptomatic disease involving other organ systems , 2. FurtA woman in her 50s with a history of hand dermatitis, gastroparesis, and achalasia was hospitalized for feeding tube dysfunction. Dermatology was consulted for an itchy and painful rash on her chest and right leg. The patient had been seen for a similar rash on the chest one year prior and was diagnosed with contact dermatitis for which she was treated with triamcinolone 0.1% ointment twice a day with no improvement. A biopsy of the chest had not been performed. She denied any fevers, chills, or recent new lotions or washes. A physical examination revealed an erythematous, scaly patch on her anterior chest surrounding a previous surgical scar  and wellSarcoidosis is a multisystem granulomatous disease; although cutaneous symptoms are seen in only 25% of patients, the diagnosis is more commonly made based on skin findings rather than pulmonary symptoms . HoweverIn the case of our patient, her rash was initially diagnosed as contact dermatitis, and she did not follow-up with dermatology as an outpatient even though her rash did not resolve. Once she was readmitted to the hospital a year later, a prompt skin biopsy ultimately led to her pulmonary sarcoidosis diagnosis. This illustrates the importance of having a strong clinical suspicion for sarcoidosis, as this is often necessary to uncover underlying systemic diseases.For asymptomatic and stable patients, treatment is not always necessary. For those with a skin-limited disease, topical or intralesional corticosteroids are the first line . PatientIn summary, we report a rare case of scar sarcoidosis along with periportal and perigastric lymph node involvement that developed on old scar areas. The skin is the second most common organ affected in sarcoidosis but often the first organ system involved in initial presentation. Prompt cutaneous diagnosis leads to earlier systemic evaluation and potential treatment.


A novel functionalized multi-walled carbon nanotube (FMWCNT)/hydroxyethyl cellulose (HEC) composite-based humidity sensor was successfully developed for humidity monitoring applications. FMWCNTs were synthesized by covalently functionalizing multi-walled carbon nanotubes (MWCNTs) in a mixture of sulfuric and nitric acid to enhance their hydrophilicity. The FMWCNTs were characterized using transmission electron microscopy, Raman spectroscopy, Fourier transform infrared spectroscopy and dispersion analysis to verify the presence of functional hydroxyl and carboxyl groups. A FMWCNT/HEC (1 : 6 w/w) composite ink was formulated using the solution blending technique with 2.5 wt% FMWCNTs. A multi-layered humidity sensor was fabricated using additive print manufacturing processes on a flexible polyethylene terephthalate (PET) substrate. Screen printing and gravure printing processes were used to deposit the bottom silver (Ag) electrode and FWMCNT/HEC sensing layers, respectively. The capability of the fabricated humidity sensor was investigated by measuring its resistive response towards relative humidity (RH) varying from 20% RH to 80% RH. As the RH was increased from 20% RH to 80% RH in steps of 10% RH at 25 °C, it was observed that the resistance of the printed sensor increased linearly. The printed sensor demonstrated resistance changes as high as ≈290% at 80% RH, when compared to its base resistance at 20% RH. A sensitivity and a response time of 0.048/%RH and ≈20 s were obtained for the printed sensor, respectively. The results thus demonstrated the feasibility of employing additive print manufacturing processes to develop a highly sensitive sensor for humidity monitoring applications. Fully printed, functionalized multi-walled carbon nanotube (FMWCNT)/hydroxyethyl cellulose (HEC) composite-based humidity sensor. Owing to their large surface area to volume ratio and hollow cylindrical nanostructure,16 multiwalled carbon nanotubes (MWCNTs) have been extensively employed for sensing various molecules adsorbed on their surface.17,18 Research has been reported on MWCNT based resistive type humidity sensors. Liu et al. reported a resistive type humidity sensor based on pristine MWCNT networks and demonstrated its capability to sense a broad range of relative humidities (25% RH to 85% RH) with good linearity and an excellent response time of 3 s.19 However, this device suffered from a low sensitivity of 0.5%/%RH towards humidity sensing.19 To improve the sensitivity of the MWCNT based humidity sensor, Cao et al.20 chemically treated the surface of MWCNTs and compared the humidity sensing characteristics of both chemically treated and untreated MWCNTs. It was observed that the former had greater sensitivity than the latter due to the presence of hydrophilic functional groups on the surface of the chemically treated MWCNTs.20 Thus, MWCNTs are promising as humidity sensitive materials. However, more efforts are needed towards the development of novel MWCNT based, highly sensitive, resistive type humidity sensors.Resistive type RH humidity sensors are the most commonly implemented type in the industry, as they are much easier to integrate and use relatively simpler electronics to monitor and control ambient humidity.21,22 These techniques are relatively expensive and time consuming as they require high vacuum, large power density, and a high temperature environment. Moreover, the rigid nature of the substrates prevents their use in applications which require mechanical flexibility and conformal form factors. To overcome these limitations, additive print manufacturing processes such as screen, inkjet, gravure and flexography can be used for the development of cost efficient, flexible and conformal humidity sensors. These additive print manufacturing processes have already enabled an emerging field called flexible hybrid electronics to develop novel, cost-efficient electronic devices for applications that demand flexibility and conformability. Accordingly, many researchers have actively employed these processes for the development of RFID tags,23,24 sensors,25–34 solar cells,35,36 antennas37 and circuits.38,39 In a recent study, a CNT based humidity sensor fabricated using two printing processes, screen and gravure printing, was reported.40 In addition, Xie et al. also reported an inkjet printed MWCNT based humidity sensor.41 Although the printed sensors in both the cases responded towards ambient humidity, there is still a need to improve their sensitivity. Therefore, the use of additive print manufacturing processes to develop highly sensitive humidity sensors employing MWCNTs and/or MWCNTs/polymer composites is envisioned to advance the field of humidity sensing.Conventionally, humidity sensors have been manufactured on rigid structures like glass or ceramics using techniques such as photolithography and sputtering.In this work, a multi-layered functionalized multi-walled carbon nanotube (FMWCNT)/hydroxyethyl cellulose (HEC) composite-based humidity sensor was developed using additive print manufacturing processes on a flexible polyethylene terephthalate (PET) substrate. MWCNTs were subjected to acid functionalization in a mixture of sulfuric and nitric acid. Transmission electron microscopy, Raman spectroscopy and Fourier transform infrared spectroscopy were performed to verify the functionalization of MWCNTs. Screen printing, a push through process was employed to deposit the electrodes in an interdigitated structure. The FMWCNT/HEC composite ink was formulated and deposited on the electrodes as the humidity sensing layer using gravure printing. The performance of the printed humidity sensor was investigated by measuring its resistive response towards relative humidity (RH) varying from 20% RH to 80% RH at a constant temperature of 25 °C.2.2.12SO4) and 70% nitric acid (HNO3) from Sigma-Aldrich Chemical Company were used for the functionalization of the MWCNTs via acid treatment. Hygroscopic HEC  and polyvinylpolypyrrolidone (PVPP) polymer  were used as the polymer matrix and binder in the FMWCNT/HEC ink, respectively. Acetone, from Sigma-Aldrich Chemical Company, and de-ionized (DI) water were used as the cleaning solvents for the Ag and FMWCNT/HEC inks, respectively. The electrical connections for the printed sensor were made using contact flat flex interconnects (Model no. 1-88997-2) from TE Connectivity AMP Connectors. The headers of these interconnects were connected using male to female jumper wires (Model no. 1568-1511-ND) from SparkFun Electronics.A flexible PET film (MELINEX® ST730) from DuPont Teijin Films was used as the substrate. Conductive Ag ink (AG-800) from Applied Ink Solutions was used for the electrodes. The conductive filler for the sensing layer was MWCNTs, with diameters and lengths of 20–30 nm and 10–30 nm, respectively . Analytical grade 95% sulfuric acid  mixture of H2SO4 and HNO3, respectively and the mixture was refluxed at 140 °C using continuous magnetic stirring for 1 hour.11 The mixture was allowed to cool down to room temperature after the reflux. Then, the obtained mixture was titrated against NH3OH until a pH of 5.5 was attained.44 The neutralized solution was then vacuum filtered to separate the FMWCNTs using 0.2 μm pore size polytetrafluoroethylene membrane filters. Finally, the FMWCNTs were washed with DI water several times and dried for 12 hours at 140 °C.The MWCNTs were subjected to a covalent functionalization process which involves surface modification of the MWCNTs by incorporating hydrophilic substituents onto the exterior MWCNT sidewalls2 Fig. S1. The aim2.3Transmission electron microscopy (TEM) and Raman spectroscopy were used to obtain morphological information and to determine the degree of functionalization, respectively, of the MWCNTs before and after acid treatment. A JEOL 100 CXII TEM with an accelerated voltage of 200 kV was used to record the TEM micrographs and analyze the morphology of MWCNTs. Two samples were prepared by dispersing the MWCNTs in ethanol and the FMWCNTs in DI water. The prepared samples were dropped on a carbon coated 200-mesh copper grid for imaging.44 A spectrometer (QE 6500) from Ocean Optics Inc. was used to obtain the Raman spectra. The Raman spectra were then analyzed using Spectra Suite Software from Ocean Optics Inc.The experimental setup for Raman spectroscopy is shown in Fig. S2.Fourier transform infrared spectroscopy (FT-IR) was performed to verify the presence of hydrophilic constituents on the surface of the FMWCNTs. Two samples, one with MWCNTs and another with the FMWCNTs, were prepared by mechanically mixing them with potassium bromide powder. The mixtures were pressed into discs and were placed in a ThermoFisher Nicolet iS5 spectrometer. The FT-IR spectra were analyzed using OriginPro software.44 0.125 g (0.5 wt%) of MWCNTs and FMWCNTs were dispersed in 25 mL of DI water. The solutions were stirred vigorously for 60 minutes and held for 5 days, which is a sufficient duration required to formulate the ink and fabricate the sensor.The dispersion analysis was performed on both MWCNTs and FMWCNTs to compare their suspension stability in DI water, thereby demonstrating the efficiency of functionalization.2.445 The FMWCNT/HEC composite for the ink was prepared using the solution blending technique.11 FMWCNT/HEC composites with varying ratios (1 : 4 w/w and 1 : 6 w/w) were prepared for studying the humidity response. In addition to being a hygroscopic polymer, HEC is also an efficient dispersant in the ink system as it is completely water soluble and swells in the presence of water, thereby preventing agglomeration between the FMWCNTs.11,46 The mixture was stirred for 16 hours to obtain homogeneity in the composite. Finally, PVPP was added and magnetically stirred for 3 hours to form the FMWCNT/HEC ink (The FMWCNT/HEC ink was formulated using DI water as the solvent. /HEC ink  shows th2.5−1) and 56.2 (degrees) with a goniometer (First Ten Angstroms FTA-200) using the pendant drop method49 and sessile drop method,50 respectively (−1) with the goniometer using the Owens Wendt method.48 It was observed that the ST of the ink was 18.7 units greater than the SE of the substrate. However, a good print compatibility between the printed layer and the substrate is obtained when the SE of the substrate is greater than the ST of the ink.47 This is because the SE of the substrate, which is the energy due to the intermolecular forces, should be able to break the resistance of the ink and to deform it into a new surface.47 The resistance offered by the ink, due to attractive intermolecular forces formed on its interfacial surface, is the ST of the ink.47 Therefore, 1 wt% Ecosurf™  surfactant was added to the prepared FMWCNT/HEC ink and was magnetically stirred for 3 hours for reducing its ST. It was observed that the ST decreased to 23.8 (dynes cm−1), which is 21.6 units less than the SE of the substrate (The surface tension (ST) and contact angle (CA) of the formulated FMWCNT/HEC ink were measured to be 64.1  of 1.49 ± 0.05 μm and 0.94 ± 0.03 μm and an average surface roughness (Sa) of 0.52 ± 0.04 μm and 0.28 ± 0.05 μm were obtained for the Ag electrodes and FMWCNT/HEC sensing layer, respectively.A Bruker Contour GT-K vertical scanning interferometer was used to characterize the thickness and roughness of the printed layers . An aver2.8The experimental setup for investigating the response of the printed humidity sensor towards varying relative humidity (%RH) is shown in 3.3.120,42 The diameter and length of the MWCNTs were measured to be in the range of ≈20 nm to 30 nm  . This ph1 Fig. S3.3.2−1 and ≈1613 cm−1, respectively.44,52 The relative intensity (I(D)/(G)), which is the ratio of the D-band intensity (I(D)) to the G-band intensity (I(D)), provides information on the degree of functionalization. I(D)/(G) values of 1.05 and 1.33 were calculated for the MWCNTs and FMWCNTs, respectively, indicating that there was a 27% increase in the number of defective sites on the surface of the FMWCNTs. The increase in the number of defects can be attributed to the acid functionalization process that breaks the bond between the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C atoms and inserts functional groups.44,533.354 These dipoles can be detected as the IR spectra during the FT-IR analysis.44,54 In MWCNTs, the symmetrical structure results in a relatively silent spectrum as weak infrared signals are detected due to the weak difference of charge states between the symmetrical carbon atoms.44 However, in the FMWCNTs, multiple characteristic peaks were observed in the FT-IR spectra indicating the presence of functional groups that are introduced due to the acid treatment. Broad peaks between 3000 cm−1 and 3500 cm−1 were obtained indicating the characteristic stretching vibrations of hydroxyl (O–H) bonds.44 Further, the FT-IR spectrum of the FMWCNTs showed the peaks of carboxyl (CO) and carbonyl (O–CO) groups at ≈1650 cm−1 and ≈1450 cm−1, respectively.44,55,56 Thus, these peaks from the FT-IR indicated the presence of functional groups on the surface of FMWCNTs after covalent acid functionalization.3.444 On the other hand, the FMWCNTs showed better suspension stability for up to 5 days, as no sedimentation was observed. This stability is due to the presence of the equally charged functional hydroxyl and carboxyl groups which enable the MWCNTs to repel each other and, thus, keep the solution dispersed.44The results of the dispersion analysis for the MWCNTs and FMWCNTs are shown in Fig. S4.3.5Before subjecting the humidity sensors to varying humidity levels, the sensors were pre-heated on a VWR 810 ceramic hot plate at 90 °C for 1 hour to remove the water molecules trapped around the FMWCNTs and in between the electrodes. 57 When exposed to humidity in the environment, the resistance changes in the MWCNTs depend on their interaction with water molecules. These water molecules are adsorbed onto the surface of the MWCNTs through physisorption, and tend to transfer electrons to MWCNTs due to the difference in electrical potential between them.19,58 This charge transfer mechanism decreases the number of holes and consequently increases the electrical resistance of MWCNTs. The RH level of the environment directly dictates the amount of water molecules to be adsorbed on the MWCNTs. As the RH level increases, more water molecules are adsorbed, and more electrons will be transferred, leading to a further increase in the resistance value.11 In addition, the hydroxyl and carboxyl groups present on the defective sites of the FMWCNTs show increased affinity towards water molecules (hydrophilicity). This improved hydrophilicity not only enhances the interaction between FMWCNTs and water molecules, but also aids in increasing the number of electrons transferred, contributing to a much higher resistance change.11 Further, the HEC consists of abundant hydroxyl groups and exhibits physical swelling upon exposure to humidity due to the adsorption of a large number of water molecules.59 This swelling property of HEC can effectively increase the contact gap of the FMWCNT intertube junction in the composite, thereby contributing to the increase in the resistance with humidification.11 However, during de-humidification the number of effective charge carriers (holes) in the FMWCNTs increases as the water molecules desorb from their surface.64 This increase in the number of effective charge carriers results in a decrease in the resistance values. In addition, HEC starts to physically shrink as the water molecules desorb from its surface.65 This phenomenon decreases the contact gap of the FMWCNT intertube junction in the composite, hence resulting in a decrease in the resistance during dehumidification.S) of 0.0485/%RH was mathematically calculated for the fabricated humidity sensor using RiT and RfT are the resistance values obtained at initial relative humidity RHi and final temperature RHf. The results obtained for the printed humidity sensor demonstrated a sensitivity which is an order of magnitude greater than the sensitivity of a MWCNT network-based humidity sensor (0.005/%RH) reported by Liu et al.19 In addition, the sensitivity of the printed FMWCNT/HEC based humidity sensor was better than those of several other MWCNT and MWCNT/polymer-based humidity sensors reported15,19,20,60–62 and a comparison is shown in 66 The trapped charge contributes to a comparatively higher resistance, during de-humidification when compared to the resistance, during humidification.The reproducibility of the sensor performance was investigated by analyzing the resistive response of three different sensors printed using the FMWCNT/HEC ink, formulated separately for each sensor . It was 6,63 The recovery curve of the printed humidity sensor when the relative humidity of the chamber was varied from 60 %RH to 50 %RH is shown in 6The response curve of the printed humidity sensor is shown in 4.In this work, a fully printed, highly sensitive, humidity sensor based on a FMWCNT and HEC composite was successfully developed for humidity monitoring applications. To enhance the hydrophilicity, MWCNTs were treated in a mixture of sulfuric and nitric acid. Transmission electron microscopy, Raman spectroscopy, Fourier transform infrared spectroscopy and dispersion analysis were performed to confirm the functionalization and the presence of functional groups on the surface of the MWCNTs. A FMWCNT/HEC composite ink was formulated with 2.5 wt% MWCNTs and the ratio between FMWCNTs and HEC was maintained at 1 : 6. A pair of electrodes with 24 fingers in an interdigitated geometry were deposited using the screen printing process. The formulated FMWCNT/HEC ink was deposited on the electrodes as the sensing layer using the gravure printing process. The performance of the printed humidity sensor was investigated by measuring its resistive response towards relative humidity varying from 20% RH to 80% RH, in steps of 10% RH, at a constant temperature of 25 °C. The printed sensor exhibited resistive changes as high as 290% at 80% RH, when compared to its base resistance at 20% RH with a sensitivity of 0.048/%RH. The obtained sensitivity of the sensor was greater than those of several other MWCNT and MWCNT/polymer based humidity sensors reported. The reproducibility in terms of ink formulation and fabrication of sensors was also investigated. In addition, the printed humidity sensor demonstrated a good response time of ≈20 s when measured against a commercial humidity sensor equipped in the environmental chamber.These results demonstrate that humidity sensors fabricated using additive print manufacturing processes on flexible substrates, with the FMWCNT/HEC composite as the sensing material, have a significant potential in humidity sensing applications. Moreover, the printed humidity sensor can potentially be implemented in applications where its lightweight and conformal features along with large scale manufacturing capabilities are paramount for improved user–device interactions. Further research is underway to investigate the performance of the printed humidity sensor for varying temperatures along with the compensation strategies. Research is also being focused on the monolithic integration of a printed micro-heater with the humidity sensor and, thereby, the improvement of the reliability of the sensor by reducing the hysteresis. Finally, the effect of mechanical stresses such as bending, twisting, and stretching on the performance of the sensor is also under investigation to determine appropriate output compensation required, before implementing the printed humidity sensor into a field deployable sensing system.There are no conflicts to declare.NA-001-C9NA00179D-s001