Loss of sodium current caused by a Brugada syndrome-associated variant is determined by patient-specific genetic background.

BACKGROUND: Brugada syndrome (BrS) is an inherited cardiac arrhythmogenic disease that predisposes patients to sudden cardiac death. It is associated with mutations in SCN5A, which encodes the cardiac sodium channel alpha subunit (NaV1.5). BrS-related mutations have incomplete penetrance and variable expressivity within families. OBJECTIVE: The purpose of this study was to determine the role of patient-specific genetic background on the cellular and clinical phenotype among carriers of NaV1.5_p.V1525M. METHODS: We studied sodium currents from patient-specific human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and heterologously transfected human embryonic kidney (HEK) tsA201 cells using the whole-cell patch-clamp technique. We determined gene and protein expression by quantitative polymerase chain reaction, RNA sequencing, and western blot and performed a genetic panel for arrhythmogenic diseases. RESULTS: Our results showed a large reduction in INa density in hiPSC-CM derived from 2 V1525M single nucleotide variant (SNV) carriers compared with hiPSC-CM derived from a noncarrier, suggesting a dominant-negative effect of the NaV1.5_p.V1525M channel. INa was not affected in hiPSC-CMs derived from a V1525M SNV carrier who also carries the NaV1.5_p.H558R polymorphism. Heterozygous expression of V1525M in HEK-293T cells produced a loss of INa function, not observed when this variant was expressed together with H558R. In addition, the antiarrhythmic drug mexiletine rescued INa function in hiPSC-CM. SCN5A expression was increased in the V1525M carrier who also expresses NaV1.5_p.H558R. CONCLUSION: Our results in patient-specific hiPSC-CM point to a dominant-negative effect of NaV1.5_p.V1525M, which can be reverted by the presence of NaV1.5_p.H558R. Overall, our data points to a role of patient-specific genetic background as a determinant for incomplete penetrance in BrS.

Generation of the induced pluripotent stem cell line ESi108-A from a familial atrial fibrillation patient.

Tissue-specific cells differentiated from patient-derived human induced pluripotent stem cells (hiPSC) are a relevant cellular model to study several diseases. We obtained a hiPSC line from skin fibroblasts of a patient affected by familial atrial fibrillation by nucleofection of non-integrating episomal vectors. The resulting hiPSC line displays a normal karyotype, expresses pluripotency surface markers and pluripotency genes, and differentiates into cells of the 3 germ layers. Therefore, it represents a reliable model to study the disease in a physiologically relevant cellular environment.

Generation of four induced pluripotent stem cell lines from a family harboring a single nucleotide variant in SCN5A.

Patient-derived induced pluripotent stem cells (iPSC) are a valuable approach to model cardiovascular diseases. We nucleofected non-integrating episomal vectors in skin fibroblasts of three family members carrying a single nucleotide variant (SNV) in SCN5A, which encodes the cardiac-type sodium channel, and of a related healthy control. The SNV SCN5A_c.4573G > A had been previously identified in a Brugada Syndrome patient. The resulting iPS cell lines differentiate into cells of the 3 germ layers, display normal karyotypes and express pluripotency surface markers and genes. Thus, they are a reliable source to study the effect of the identified mutation in a physiologically relevant environment.

Generation of an induced pluripotent stem cell line from a healthy Caucasian male.

The effects of genetic mutations on protein function can be studied in a physiologically relevant environment using tissue-specific cells differentiated from patient-derived induced pluripotent stem cells (iPSC). However, it is crucial to use iPSC derived from healthy individuals as control. We generated an iPS cell line from skin fibroblasts of a healthy Caucasian male by nucleofection of non-integrating episomal vectors. This cell line has normal karyotype, expresses pluripotency surface markers and pluripotency genes, and successfully differentiates into cells of the 3 germ layers. Therefore, it can be used as control for any disease of interest that is modelled using iPSC.

An SCN1B Variant Affects Both Cardiac-Type (NaV1.5) and Brain-Type (NaV1.1) Sodium Currents and Contributes to Complex Concomitant Brain and Cardiac Disorders.

Voltage-gated sodium (NaV) channels are transmembrane proteins that initiate and propagate neuronal and cardiac action potentials. NaV channel ß subunits have been widely studied due to their modulatory role. Mice null for Scn1b, which encodes NaV ß1 and ß1b subunits, have defects in neuronal development and excitability, spontaneous generalized seizures, cardiac arrhythmias, and early mortality. A mutation in exon 3 of SCN1B, c.308A>T leading to ß1_p.D103V and ß1b_p.D103V, was previously found in a patient with a history of proarrhythmic conditions with progressive atrial standstill as well as cognitive and motor deficits accompanying structural brain abnormalities. We investigated whether ß1 or ß1b subunits carrying this mutation affect NaV1.5 and/or NaV1.1 currents using a whole cell patch-clamp technique in tsA201 cells. We observed a decrease in sodium current density in cells co-expressing NaV1.5 or NaV1.1 and ß1D103V compared to ß1WT. Interestingly, ß1bD103V did not affect NaV1.1 sodium current density but induced a positive shift in the voltage dependence of inactivation and a faster recovery from inactivation compared to ß1bWT. The ß1bD103V isoform did not affect NaV1.5 current properties. Although the SCN1B_c.308A>T mutation may not be the sole cause of the patient's symptoms, we observed a clear loss of function in both cardiac and brain sodium channels. Our results suggest that the mutant ß1 and ß1b subunits play a fundamental role in the observed electrical dysfunction.

Extra Virgin Olive Oil Contains a Phenolic Inhibitor of the Histone Demethylase LSD1/KDM1A.

The lysine-specific histone demethylase 1A (LSD1) also known as lysine (K)-specific demethylase 1A (KDM1A) is a central epigenetic regulator of metabolic reprogramming in obesity-associated diseases, neurological disorders, and cancer. Here, we evaluated the ability of oleacein, a biophenol secoiridoid naturally present in extra virgin olive oil (EVOO), to target LSD1. Molecular docking and dynamic simulation approaches revealed that oleacein could target the binding site of the LSD1 cofactor flavin adenosine dinucleotide with high affinity and at low concentrations. At higher concentrations, oleacein was predicted to target the interaction of LSD1 with histone H3 and the LSD1 co-repressor (RCOR1/CoREST), likely disturbing the anchorage of LSD1 to chromatin. AlphaScreen-based in vitro assays confirmed the ability of oleacein to act as a direct inhibitor of recombinant LSD1, with an IC50 as low as 2.5 µmol/L. Further, oleacein fully suppressed the expression of the transcription factor SOX2 (SEX determining Region Y-box 2) in cancer stem-like and induced pluripotent stem (iPS) cells, which specifically occurs under the control of an LSD1-targeted distal enhancer. Conversely, oleacein failed to modify ectopic SOX2 overexpression driven by a constitutive promoter. Overall, our findings provide the first evidence that EVOO contains a naturally occurring phenolic inhibitor of LSD1, and support the use of oleacein as a template to design new secoiridoid-based LSD1 inhibitors.

Experimental Models of Brugada syndrome.

Brugada syndrome is an inherited, rare cardiac arrhythmogenic disease, associated with sudden cardiac death. It accounts for up to 20% of sudden deaths in patients without structural cardiac abnormalities. The majority of mutations involve the cardiac sodium channel gene SCN5A and give rise to classical abnormal electrocardiogram with ST segment elevation in the right precordial leads V1 to V3 and a predisposition to ventricular fibrillation. The pathophysiological mechanisms of Brugada syndrome have been investigated using model systems including transgenic mice, canine heart preparations, and expression systems to study different SCN5A mutations. These models have a number of limitations. The recent development of pluripotent stem cell technology creates an opportunity to study cardiomyocytes derived from patients and healthy individuals. To date, only a few studies have been done using Brugada syndrome patient-specific iPS-CM, which have provided novel insights into the mechanisms and pathophysiology of Brugada syndrome. This review provides an evaluation of the strengths and limitations of each of these model systems and summarizes the key mechanisms that have been identified to date.

Sodium channel current loss of function in induced pluripotent stem cell-derived cardiomyocytes from a Brugada syndrome patient.

Brugada syndrome predisposes to sudden death due to disruption of normal cardiac ion channel function, yet our understanding of the underlying cellular mechanisms is incomplete. Commonly used heterologous expression models lack many characteristics of native cardiomyocytes and, in particular, the individual genetic background of a patient. Patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CM) may uncover cellular phenotypical characteristics not observed in heterologous models. Our objective was to determine the properties of the sodium current in iPS-CM with a mutation in SCN5A associated with Brugada syndrome. Dermal fibroblasts from a Brugada syndrome patient with a mutation in SCN5A (c.1100G>A, leading to Nav1.5_p.R367H) were reprogrammed to iPS cells. Clones were characterized and differentiated to form beating clusters and sheets. Patient and control iPS-CM were structurally indistinguishable. Sodium current properties of patient and control iPS-CM were compared. These results were contrasted with those obtained in tsA201 cells heterologously expressing sodium channels with the same mutation. Patient-derived iPS-CM showed a 33.1-45.5% reduction in INa density, a shift in both activation and inactivation voltage-dependence curves, and faster recovery from inactivation. Co-expression of wild-type and mutant channels in tsA201 cells did not compromise channel trafficking to the membrane, but resulted in a reduction of 49.8% in sodium current density without affecting any other parameters. Cardiomyocytes derived from iPS cells from a Brugada syndrome patient with a mutation in SCN5A recapitulate the loss of function of sodium channel current associated with this syndrome; including pro-arrhythmic changes in channel function not detected using conventional heterologous expression systems.

Large Genomic Imbalances in Brugada Syndrome.

PURPOSE: Brugada syndrome (BrS) is a form of cardiac arrhythmia which may lead to sudden cardiac death. The recommended genetic testing (direct sequencing of SCN5A) uncovers disease-causing SNVs and/or indels in ~20% of cases. Limited information exists about the frequency of copy number variants (CNVs) in SCN5A in BrS patients, and the role of CNVs in BrS-minor genes is a completely unexplored field. METHODS: 220 BrS patients with negative genetic results were studied to detect CNVs in SCN5A. 63 cases were also screened for CNVs in BrS-minor genes. Studies were performed by Multiplex ligation-dependent probe amplification or Next-Generation Sequencing (NGS). RESULTS: The detection rate for CNVs in SCN5A was 0.45% (1/220). The detected imbalance consisted of a duplication from exon 15 to exon 28, and could potentially explain the BrS phenotype. No CNVs were found in BrS-minor genes. CONCLUSION: CNVs in current BrS-related genes are uncommon among BrS patients. However, as these rearrangements may underlie a portion of cases and they undergo unnoticed by traditional sequencing, an appealing alternative to conventional studies in these patients could be targeted NGS, including in a single experiment the study of SNVs, indels and CNVs in all the known BrS-related genes.

Comprehensive Genetic Characterization of a Spanish Brugada Syndrome Cohort.

BACKGROUND: Brugada syndrome (BrS) is a rare genetic cardiac arrhythmia that can lead to sudden cardiac death in patients with a structurally normal heart. Genetic variations in SCN5A can be identified in approximately 20-25% of BrS cases. The aim of our work was to determine the spectrum and prevalence of genetic variations in a Spanish cohort diagnosed with BrS. METHODOLOGY/PRINCIPAL FINDINGS: We directly sequenced fourteen genes reported to be associated with BrS in 55 unrelated patients clinically diagnosed. Our genetic screening allowed the identification of 61 genetic variants. Of them, 20 potentially pathogenic variations were found in 18 of the 55 patients (32.7% of the patients, 83.3% males). Nineteen of them were located in SCN5A, and had either been previously reported as pathogenic variations or had a potentially pathogenic effect. Regarding the sequencing of the minority genes, we discovered a potentially pathogenic variation in SCN2B that was described to alter sodium current, and one nonsense variant of unknown significance in RANGRF. In addition, we also identified 40 single nucleotide variations which were either synonymous variants (four of them had not been reported yet) or common genetic variants. We next performed MLPA analysis of SCN5A for the 37 patients without an identified genetic variation, and no major rearrangements were detected. Additionally, we show that being at the 30-50 years range or exhibiting symptoms are factors for an increased potentially pathogenic variation discovery yield. CONCLUSIONS: In summary, the present study is the first comprehensive genetic evaluation of 14 BrS-susceptibility genes and MLPA of SCN5A in a Spanish BrS cohort. The mean pathogenic variation discovery yield is higher than that described for other European BrS cohorts (32.7% vs 20-25%, respectively), and is even higher for patients in the 30-50 years age range.

Post-mortem genetic analysis in juvenile cases of sudden cardiac death.

BACKGROUND: The reason behind a sudden death of a young individual remains unknown in up to 50% of postmortem cases. Pathogenic mutations in genes encoding heart proteins are known to cause sudden cardiac death. OBJECTIVE: The aim of our study was to ascertain whether genetic alterations could provide an explanation for sudden cardiac death in a juvenile cohort with no-conclusive cause of death after comprehensive autopsy. METHODS: Twenty-nine cases <15 years showing no-conclusive cause of death after a complete autopsy were studied. Genetic analysis of 7 main genes associated with sudden cardiac death was performed using Sanger technology in low quality DNA cases, while in good quality cases the analysis of 55 genes associated with sudden cardiac death was performed using Next Generation Sequencing technology. RESULTS: Thirty-five genetic variants were identified in 12 cases (41.37%). Ten genetic/variants in genes encoding cardiac ion channels were identified in 8 cases (27.58%). We also identified 9 cases (31.03%) carrying 25 genetic variants in genes encoding structural cardiac proteins. Nine cases carried more than one genetic variation, 5 of them combining structural and non-structural genes. CONCLUSIONS: Our study supports the inclusion of molecular autopsy in forensic routine protocols when no conclusive cause of death is identified. Around 40% of sudden cardiac death young cases carry a genetic variant that could provide an explanation for the cause of death. Because relatives could be at risk of sudden cardiac death, our data reinforce their need of clinical assessment and, if indicated, of genetic analysis.

The smooth muscle-type ß1 subunit potentiates activation by DiBAC4(3) in recombinant BK channels.

Large-conductance Ca(2+)-activated (BK) channels, expressed in a variety of tissues, play a fundamental role in regulating and maintaining arterial tone. We recently demonstrated that the slow voltage indicator DiBAC4(3) does not depend, as initially proposed, on the ß 1 or ß 4 subunits to activate native arterial smooth muscle BK channels. Using recombinant mslo BK channels, we now show that the ß 1 subunit is not essential to this activation but exerts a large potentiating effect. DiBAC4(3) promotes concentration-dependent activation of BK channels and slows deactivation kinetics, changes that are independent of Ca(2+). Kd values for BK channel activation by DiBAC4(3) in 0 mM Ca(2+) are approximately 20 µM (α) and 5 µM (α+ß 1), and G-V curves shift up to -40 mV and -110 mV, respectively. ß1 to ß2 mutations R11A and C18E do not interfere with the potentiating effect of the subunit. Our findings should help refine the role of the ß 1 subunit in cardiovascular pharmacology.

Molecular heterogeneity of large-conductance calcium-activated potassium channels in canine intracardiac ganglia.

Large conductance calcium-activated potassium (BK) channels are widely expressed in the nervous system. We have recently shown that principal neurons from canine intracardiac ganglia (ICG) express a paxilline- and TEA-sensitive BK current, which increases neuronal excitability. In the present work, we further explore the molecular constituents of the BK current in canine ICG. We found that the ß1 and ß4 regulatory subunits are expressed in ICG. Single channel voltage-dependence at different calcium concentrations suggested that association of the BKα with a particular ß subunit was not enough to explain the channel activity in this tissue. Indeed, we detected the presence of several splice variants of the BKα subunit. In conclusion, BK channels in canine ICG may result from the arrangement of different BKα splice variants, plus accessory ß subunits. The particular combinations expressed in canine IC neurons likely rule the excitatory role of BK current in this tissue.

A missense mutation in the sodium channel ß2 subunit reveals SCN2B as a new candidate gene for Brugada syndrome.

Brugada Syndrome (BrS) is a familial disease associated with sudden cardiac death. A 20%-25% of BrS patients carry genetic defects that cause loss-of-function of the voltage-gated cardiac sodium channel. Thus, 70%-75% of patients remain without a genetic diagnosis. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium ß2 subunit encoded by SCN2B, in a woman diagnosed with BrS. We studied the sodium current (INa ) from cells coexpressing Nav 1.5 and wild-type (ß2WT) or mutant (ß2D211G) ß2 subunits. Our electrophysiological analysis showed a 39.4% reduction in INa density when Nav 1.5 was coexpressed with the ß2D211G. Single channel analysis showed that the mutation did not affect the Nav 1.5 unitary channel conductance. Instead, protein membrane detection experiments suggested that ß2D211G decreases Nav 1.5 cell surface expression. The effect of the mutant ß2 subunit on the INa strongly suggests that SCN2B is a new candidate gene associated with BrS.

DiBAC4(3) hits a "sweet spot" for the activation of arterial large-conductance Ca²âº-activated potassium channels independently of the ß1-subunit.

The voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)] has been reported as a novel large-conductance Ca²âº-activated K⁺ (BK) channel activator with selectivity for its ß1- or ß4-subunits. In arterial smooth muscle, BK channels are formed by a pore-forming α-subunit and a smooth muscle-abundant regulatory ß1-subunit. This tissue specificity has driven extensive pharmacological research aimed at regulating arterial tone. Using animals with a disruption of the gene for the ß1-subunit, we explored the effects of DiBAC4(3) in native channels from arterial smooth muscle. We tested the hypothesis that, in native BK channels, activation by DiBAC4(3) relies mostly on its α-subunit. We studied BK channels from wild-type and transgenic ß1-knockout mice in excised patches. BK channels from brain arteries, with or without the ß1-subunit, were similarly activated by DiBAC4(3). In addition, we found that saturating concentrations of DiBAC4(3) (~30 µM) promote an unprecedented persistent activation of the channel that negatively shifts its voltage dependence by as much as -300 mV. This "sweet spot" for persistent activation is independent of Ca²âº and/or the ß1₋4-subunits and is fully achieved when DiBAC4(3) is applied to the intracellular side of the channel. Arterial BK channel response to DiBAC4(3) varies across species and/or vascular beds. DiBAC4(3) unique effects can reveal details of BK channel gating mechanisms and help in the rational design of BK channel activators.

A novel missense mutation, I890T, in the pore region of cardiac sodium channel causes Brugada syndrome.

Brugada syndrome (BrS) is a life-threatening, inherited arrhythmogenic syndrome associated with autosomal dominant mutations in SCN5A, the gene encoding the cardiac Na(+) channel alpha subunit (Na(v)1.5). The aim of this work was to characterize the functional alterations caused by a novel SCN5A mutation, I890T, and thus establish whether this mutation is associated with BrS. The mutation was identified by direct sequencing of SCN5A from the proband's DNA. Wild-type (WT) or I890T Na(v)1.5 channels were heterologously expressed in human embryonic kidney cells. Sodium currents were studied using standard whole cell patch-clamp protocols and immunodetection experiments were performed using an antibody against human Na(v)1.5 channel. A marked decrease in current density was observed in cells expressing the I890T channel (from -52.0 ± 6.5 pA/pF, n = 15 to -35.9 ± 3.4 pA/pF, n = 22, at -20 mV, WT and I890T, respectively). Moreover, a positive shift of the activation curve was identified (V(1/2) = -32.0 ± 0.3 mV, n = 18, and -27.3 ± 0.3 mV, n = 22, WT and I890T, respectively). No changes between WT and I890T currents were observed in steady-state inactivation, time course of inactivation, slow inactivation or recovery from inactivation parameters. Cell surface protein biotinylation analyses confirmed that Na(v)1.5 channel membrane expression levels were similar in WT and I890T cells. In summary, our data reveal that the I890T mutation, located within the pore of Na(v)1.5, causes an evident loss-of-function of the channel. Thus, the BrS phenotype observed in the proband is most likely due to this mutation.

Identification of novel Sp1 targets involved in proliferation and cancer by functional genomics.

Sp1 is a transcription factor regulating many genes through its DNA binding domain, containing three zinc fingers. We were interested in identifying target genes regulated by Sp1, particularly those involved in proliferation and cancer. Our approach was to treat HeLa cells with a siRNA directed against Sp1 mRNA to decrease the expression of Sp1 and, in turn, the genes activated by this transcription factor. Sp1-siRNA treatment led to a great number of differentially expressed genes as determined by whole genome cDNA microarray analysis. Underexpressed genes were selected since they represent putative genes activated by Sp1 and classified in six Gene Onthology categories, namely proliferation and cancer, mRNA processing, lipid metabolism, glucidic metabolism, transcription and translation. Putative Sp1 binding sites were found in the promoters of the selected genes using the Match™ software. After literature mining, 11 genes were selected for further validation. Underexpression by qRT-PCR was confirmed for the 11 genes plus Sp1 in HeLa cells after Sp1-siRNA treatment. EMSA and ChIP assays were performed to test for binding of Sp1 to the promoters of these genes. We observed binding of Sp1 to the promoters of RAB20, FGF21, IHPK2, ARHGAP18, NPM3, SRSF7, CALM3, PGD and Sp1 itself. Furthermore, the mRNA levels of RAB20, FGF21 and IHPK2 and luciferase activity for these three genes related to proliferation and cancer, were determined after overexpression of Sp1 in HeLa cells, to confirm their regulation by Sp1. Involvement of these three genes in proliferation was validated by gene silencing using polypurine reverse hoogsteen hairpins.

Transcriptional profiling of striatal neurons in response to single or concurrent activation of dopamine D2, adenosine A(2A) and metabotropic glutamate type 5 receptors: focus on beta-synuclein expression.

G protein-coupled receptor oligomerization is a concept which is changing the understanding of classical pharmacology. Both, oligomerization and functional interaction between adenosine A(2A,) dopamine D(2) and metabotropic glutamate type 5 receptors have been demonstrated in the striatum. However, the transcriptional consequences of receptors co-activation are still unexplored. We aim here to determine the changes in gene expression of striatal primary cultured neurons upon isolated or simultaneous receptor activation. Interestingly, we found that 95 genes of the total analyzed (15,866 transcripts and variants) changed their expression in response to simultaneous stimulation of all three receptors. Among these genes, we focused on the ß-synuclein (ß-Syn) gene (SCNB). Quantitative PCR verified the magnitude and direction of change in expression of SCNB. Since ß-Syn belongs to the homologous synuclein family and may be considered a natural regulator of α-synuclein (α-Syn), it has been proposed that ß-Syn might act protectively against α-Syn neuropathology.

Underexpression of miR-224 in methotrexate resistant human colon cancer cells.

MicroRNAs (miRNAs) are small non-coding RNAs involved in RNA silencing that play a role in many biological processes. They are involved in the development of many diseases, including cancer. Extensive experimental data show that they play a role in the pathogenesis of cancer as well as the development of drug resistance during treatments. The aim of this work was to detect differentially expressed miRNAs in MTX-resistant cells. Thus, miRNA microarrays of sensitive and MTX-resistant HT29 colon cancer cells were performed. The results were analyzed using the GeneSpring GX11.5 software. Differentially expressed miRNAs in resistant cells were identified and miR-224, which was one of the most differentially expressed miRNAs and with high raw signal values, was selected for further studies. The underexpression of miR-224 was also observed in CaCo-2 and K562 cells resistant to MTX. Putative targets were predicted using TargetScan 5.1 software and integrated with the data from expression microarrays previously performed. This approach allowed us to identify miR-224 targets that were differentially expressed more than 2-fold in resistant cells. Among them CDS2, DCP2, HSPC159, MYST3 and SLC4A4 were validated at the mRNA level by qRT-PCR. Functional assays using an anti-miR against miR-224 desensitized the cells towards MTX, mimicking the resistant phenotype. On the other hand, siRNA treatment against SLC4A4 or incubation of Poly Purine Reverse Hoogsteen (PPRH) hairpins against CDS2 or HSPC159 increased sensitivity to MTX. These results revealed a role for miR-224 and its targets in MTX resistance in HT29 colon cancer cells.

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with conjugated linoleic acid during gestation and suckling.

BACKGROUND: Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. RESULTS: The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip(®) Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. CONCLUSIONS: Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.