TRPC6 Single Nucleotide Polymorphisms and Progression of Idiopathic Membranous Nephropathy

Background

Activating mutations in the Transient Receptor Potential channel C6 (TRPC6) cause autosomal dominant focal segmental glomerular sclerosis (FSGS). TRPC6 expression is upregulated in renal biopsies of patients with idiopathic membranous glomerulopathy (iMN) and animal models thereof. In iMN, disease progression is characterized by glomerulosclerosis. In addition, a context-dependent TRPC6 overexpression was recently suggested in complement-mediated podocyte injury in e.g. iMN. Hence, we hypothesized that genetic variants in TRPC6 might affect susceptibility to development or progression of iMN.

Methods & Results

Genomic DNA was isolated from blood samples of 101 iMN patients and 292 controls. By direct sequencing of the entire TRPC6 gene, 13 single nucleotide polymorphisms (SNPs) were identified in the iMN cohort, two of which were causing an amino acid substitution (rs3802829; Pro15Ser and rs36111323, Ala404Val). No statistically significant differences in genotypes or allele frequencies between patients and controls were observed. Clinical outcome in patients was determined (remission n = 26, renal failure n = 46, persistent proteinuria n = 29, follow-up median 80 months {range 51–166}). The 13 identified SNPs showed no association with remission or renal failure. There were no differences in genotypes or allele frequencies between patients in remission and progressors.

Conclusions

Our data suggest that TRPC6 polymorphisms do not affect susceptibility to iMN, or clinical outcome in iMN.     

NOX4 Mediates BMP4-Induced Upregulation of TRPC1 and 6 Protein Expressions in Distal Pulmonary Arterial Smooth Muscle Cells

Rationale

Our previous studies demonstrated that bone morphogenetic protein 4 (BMP4) mediated, elevated expression of canonical transient receptor potential (TRPC) largely accounts for the enhanced proliferation in pulmonary arterial smooth muscle cells (PASMCs). In the present study, we sought to determine the signaling pathway through which BMP4 up-regulates TRPC expression.

Methods

We employed recombinant human BMP4 (rhBMP4) to determine the effects of BMP4 on NADPH oxidase 4 (NOX4) and reactive oxygen species (ROS) production in rat distal PASMCs. We also designed small interfering RNA targeting NOX4 (siNOX4) and detected whether NOX4 knockdown affects rhBMP4-induced ROS, TRPC1 and 6 expression, cell proliferation and intracellular Ca²⁺ determination in PASMCs.

Results

In rhBMP4 treated rat distal PASMCs, NOX4 expression was (226.73±11.13) %, and the mean ROS level was (123.65±1.62) % of that in untreated control cell. siNOX4 transfection significantly reduced rhBMP4-induced elevation of the mean ROS level in PASMCs. Moreover, siNOX4 transfection markedly reduced rhBMP4-induced elevation of TRPC1 and 6 proteins, basal [Ca²⁺]_i and SOCE. Furthermore, compared with control group (0.21±0.001), the proliferation of rhBMP4 treated cells was significantly enhanced (0.41±0.001) (P<0.01). However, such increase was attenuated by knockdown of NOX4. Moreover, external ROS (H₂O₂ 100 µM, 24 h) rescued the effects of NOX4 knockdown, which included the declining of TRPC1 and 6 expression, basal intracellular calcium concentration ([Ca²⁺]_i) and store-operated calcium entry (SOCE), suggesting that NOX4 plays as an important mediator in BMP4-induced proliferation and intracellular calcium homeostasis.

Conclusion

These results suggest that BMP4 may increase ROS level, enhance TRPC1 and 6 expression and proliferation by up-regulating NOX4 expression in PASMCs.     

Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2)

Gain-of-function mutations in the canonical transient receptor potential 6 (TRPC6) gene are a cause of autosomal dominant focal segmental glomerulosclerosis (FSGS). The mechanisms whereby abnormal TRPC6 activity results in proteinuria remain unknown. The ERK1/2 MAPKs are activated in glomeruli and podocytes in several proteinuric disease models. We therefore examined whether FSGS-associated mutations in TRPC6 result in activation of these kinases. In 293T cells and cultured podocytes, overexpression of gain-of-function TRPC6 mutants resulted in increased ERK1/2 phosphorylation, an effect dependent upon channel function. Pharmacologic inhibitor studies implicated several signaling mediators, including calmodulin and calcineurin, supporting the importance of TRPC6-mediated calcium influx in this process. Through medium transfer experiments, we uncovered two distinct mechanisms for ERK activation by mutant TRPC6, a cell-autonomous, EGF receptor-independent mechanism and a non-cell-autonomous mechanism involving metalloprotease-mediated release of a presumed EGF receptor ligand. The inhibitors KN-92 and H89 were able to block both pathways in mutant TRPC6 expressing cells as well as the prolonged elevation of intracellular calcium levels upon carbachol stimulation seen in these cells. However, these effects appear to be independent of their effects on calcium/calmodulin-dependent protein kinase II and PKA, respectively. Phosphorylation of Thr-70, Ser-282, and Tyr-31/285 were not necessary for ERK activation by mutant TRPC6, although a phosphomimetic TRPC6 S282E mutant was capable of ERK activation. Taken together, these results identify two pathways downstream of mutant TRPC6 leading to ERK activation that may play a role in the development of FSGS.     

Chronic Hypoxia Increases TRPC6 Expression and Basal Intracellular Ca2+ Concentration in Rat Distal Pulmonary Venous Smooth Muscle

Background

Hypoxia causes remodeling and contractile responses in both pulmonary artery (PA) and pulmonary vein (PV). Here we explore the effect of hypoxia on PV and pulmonary venous smooth muscle cells (PVSMCs).

Methods

Chronic hypoxic pulmonary hypertension (CHPH) model was established by exposing rats to 10% O₂ for 21 days. Rat distal PVSMCs were isolated and cultured for in vitro experiments. The fura-2 based fluorescence calcium imaging was used to measure the basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) and store-operated Ca²⁺ entry (SOCE). Quantitative RT-PCR and western blotting were performed to measure the expression of mRNA and levels of canonical transient receptor potential (TRPC) protein respectively.

Results

Hypoxia increased the basal [Ca²⁺]_i and SOCE in both freshly dissociated and serum cultured distal PVSMCs. Moreover, hypoxia increased TRPC6 expression at mRNA and protein levels in both cultured PVSMCs exposed to prolonged hypoxia (4% O₂, 60 h) and distal PV isolated from CHPH rats. Hypoxia also enhanced proliferation and migration of rat distal PVSMCs.

Conclusions

Hypoxia induces elevation of SOCE in distal PVSMCs, leading to enhancement of basal [Ca²⁺]_i in PVSMCs. This enhancement is potentially correlated with the increased expression of TRPC6. Hypoxia triggered intracellular calcium contributes to promoted proliferation and migration of PVSMCs.     

Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair

Background

Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K⁺ channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before.

Results

Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair.

Conclusion

Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.     

Urinary Peptide Profiling to Differentiate between Minimal Change Disease and Focal Segmental Glomerulosclerosis

Background

Minimal change disease (MCD) and primary focal segmental glomerulosclerosis (FSGS) are the main causes of primary idiopathic nephrotic syndrome in children and adults, with diagnosis being essential for the appropriate choice of therapy and requiring renal biopsy. However, the presence of only normal glomeruli on renal biopsy of FSGS patients may lead to the misclassification of these patients as having MCD. The aim of this study was to (i) compare the peptide profile of MCD and FSGS patients with that of a group of healthy subjects, (ii) generate and validate a class prediction model to classify MCD and FSGS patients and (ii) identify candidate biomarkers of these glomerular entities by analysis of the urinary peptidome.

Methods

The urinary peptide profile was analyzed by magnetic bead-based technology combined with MALDI-TOF mass spectrometry in 44 patients diagnosed of MCD (n = 22) and FSGS (n = 22). The resulting spectra were compiled and analyzed using ClinProTools software.

Results

A class prediction model was developed to differentiate MCD and FSGS patients. The validation of this model correctly classified 81.8% (9/11) of MCD patients and 72.7% (8/11) of FSGS patients. Moreover, the signal with m/z 1913.60, identified as a fragment of uromodulin, and the signal with m/z 2392.54, identified as a fragment of alpha-1-antitrypsin, showed higher and lower peak areas, respectively, in FSGS patients compared with MCD patients.

Conclusions

The simple, non-invasive technique described in the present study may be a useful tool to help clinicians by confirming diagnoses achieved by renal biopsy, thereby reducing misdiagnoses and avoiding the implementation of inappropriate therapies.     

Candida albicans possesses Sap7 as a pepstatin A-insensitive secreted aspartic protease

Background

Candida albicans, a commensal organism, is a part of the normal flora of healthy individuals. However, once the host immunity is compromised, C. albicans opportunistically causes recurrent superficial or fatal systemic candidiasis. Secreted aspartic proteases (Sap), encoded by 10 types of SAP genes, have been suggested to contribute to various virulence processes. Thus, it is important to elucidate their biochemical properties for better understanding of the molecular mechanisms that how Sap isozymes damage host tissues.

Methodology/Principal Findings

The SAP7 gene was cloned from C. albicans SC5314 and heterogeneously produced by Pichia pastoris. Measurement of Sap7 proteolytic activity using the FRETS-25Ala library showed that Sap7 was a pepstatin A-insensitive protease. To understand why Sap7 was insensitive to pepstatin A, alanine substitution mutants of Sap7 were constructed. We found that M242A and T467A mutants had normal proteolytic activity and sensitivity to pepstatin A. M242 and T467 were located in close proximity to the entrance to an active site, and alanine substitution at these positions widened the entrance. Our results suggest that this alteration might allow increased accessibility of pepstatin A to the active site. This inference was supported by the observation that the T467A mutant has stronger proteolytic activity than the wild type.

Conclusions/Significance

We found that Sap7 was a pepstatin A-insensitive protease, and that M242 and T467 restricted the accessibility of pepstatin A to the active site. This finding will lead to the development of a novel protease inhibitor beyond pepstatin A. Such a novel inhibitor will be an important research tool as well as pharmaceutical agent for patients suffering from candidiasis.     

Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block

Background

Understanding the interactions between ion channels and blockers remains an important goal that has implications for delineating the basic mechanisms of ion channel function and for the discovery and development of ion channel directed drugs.

Methodology/Principal Findings

We used genetic selection methods to probe the interaction of two ion channel blockers, barium and amantadine, with the miniature viral potassium channel Kcv. Selection for Kcv mutants that were resistant to either blocker identified a mutant bearing multiple changes that was resistant to both. Implementation of a PCR shuffling and backcrossing procedure uncovered that the blocker resistance could be attributed to a single change, T63S, at a position that is likely to form the binding site for the inner ion in the selectivity filter (site 4). A combination of electrophysiological and biochemical assays revealed a distinct difference in the ability of the mutant channel to interact with the blockers. Studies of the analogous mutation in the mammalian inward rectifier Kir2.1 show that the T→S mutation affects barium block as well as the stability of the conductive state. Comparison of the effects of similar barium resistant mutations in Kcv and Kir2.1 shows that neighboring amino acids in the Kcv selectivity filter affect blocker binding.

Conclusions/Significance

The data support the idea that permeant ions have an integral role in stabilizing potassium channel structure, suggest that both barium and amantadine act at a similar site, and demonstrate how genetic selections can be used to map blocker binding sites and reveal mechanistic features.     

Regulation of TRPC6 channels by non-steroidal anti-inflammatory drugs

Family focal segmental glomerulosclerosis (FSGS) is characterized by sclerosis and hyalinosis of particular loops of glomeruli and is one of the causes of the nephrotic syndrome. Certain mutations in the structure of TRPC6 channels are the genetic impetus for FSGS development resulting in podocytes functional abnormalities and various nephropathies. We have recently demonstrated that non-steroid antiinflammatory drugs (NSAID) ibuprofen and diclofenac decrease the activity of endogenous TRPC-like calcium channels in the podocytes of the freshly isolated rat glomeruli. It has also been shown that TRPC6 channels are expressed in the podocytes. In the current study we have functionally reconstituted TRPC6 channels in mammalian cells to investigate the effects of diclofenac on the activity of wild type TRPC6 channel and TRPC6^P112Q channel containing a mutation in the N-terminus that was described in FSGS patients. Intracellular calcium level measurements in transfected cells revealed a more intensive carbachol-induced increase of calcium concentration in HEK-293 cells expressing TRPC6^P112Q versus the cells expressing wild-type TRPC6. We also performed patch-clamp experiments to study TRPC6 channels reconstituted in Chinese hamster ovary (CHO) cell line and found that application of diclofenac (500 μM) acutely reduced single channel activity. Preincubation with diclofenac (100 μM) also decreased the whole-cell current in CHO cells overexpressing TRPC6^P112Q. Therefore, our previously published data on the effects of NSAID on TRPC-like channels in the isolated rat glomeruli, along with this current investigation on the cultured overexpressed mammalian cells, allows hypothesizing that TRPC6 channels may be a target for NSAID that can be important in the treatment of FSGS.     

Emergence of differentially regulated pathways associated with the development of regional specificity in chicken skin

Background

Regional specificity allows different skin regions to exhibit different characteristics, enabling complementary functions to make effective use of the integumentary surface. Chickens exhibit a high degree of regional specificity in the skin and can serve as a good model for when and how these regional differences begin to emerge.

Results

We used developing feather and scale regions in embryonic chickens as a model to gauge the differences in their molecular pathways. We employed cosine similarity analysis to identify the differentially regulated and co-regulated genes. We applied low cell techniques for expression validation and chromatin immunoprecipitation (ChIP)-based enhancer identification to overcome limited cell availabilities from embryonic chicken skin.We identified a specific set of genes demonstrating a high correlation as being differentially expressed during feather and scale development and maturation. Some members of the WNT, TGF-beta/BMP, and Notch family known to be involved in feathering skin differentiation were found to be differentially regulated. Interestingly, we also found genes along calcium channel pathways that are differentially regulated. From the analysis of differentially regulated pathways, we used calcium signaling pathways as an example for further verification. Some voltage-gated calcium channel subunits, particularly CACNA1D, are expressed spatio-temporally in the skin epithelium. These calcium signaling pathway members may be involved in developmental decisions, morphogenesis, or epithelial maturation. We further characterized enhancers associated with histone modifications, including H3K4me1, H3K27ac, and H3K27me3, near calcium channel-related genes and identified signature intensive hotspots that may be correlated with certain voltage-gated calcium channel genes.

Conclusion

We demonstrated the applicability of cosine similarity analysis for identifying novel regulatory pathways that are differentially regulated during development. Our study concerning the effects of signaling pathways and histone signatures on enhancers suggests that voltage-gated calcium signaling may be involved in early skin development. This work lays the foundation for studying the roles of these gene pathways and their genomic regulation during the establishment of skin regional specificity.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-014-1202-9) contains supplementary material, which is available to authorized users.     

Correlations in Ion Channel mRNA in Rhythmically Active Neurons

Background

To what extent do identified neurons from different animals vary in their expression of ion channel genes? In neurons of the same type, is ion channel expression highly variable and/or is there any relationship between ion channel expression that is conserved?

Methodology/Principal Findings

To address these questions we measured ion channel mRNA in large cells (LCs) of the crab cardiac ganglion. We cloned a calcium channel, caco, and a potassium channel, shaker. Using single-cell quantitative PCR, we measured levels of mRNA for these and 6 other different ion channels in cardiac ganglion LCs. Across the population of LCs we measured 3–9 fold ranges of mRNA levels, and we found correlations in the expression of many pairs of conductances

Conclusions/Significance

In previous measurements from the crab stomatogastric ganglion (STG), ion channel expression was variable, but many pairs of channels had correlated expression. However, each STG cell type had a unique combination of ion channel correlations. Our findings from the crab cardiac ganglion are similar, but the correlations in the LCs are different from those in STG neurons, supporting the idea that such correlations could be markers of cell identity or activity.     

Identification of a novel gene product that promotes survival of Mycobacterium smegmatis in macrophages

Background

Bacteria of the suborder Corynebacterineae include significant human pathogens such as Mycobacterium tuberculosis and M. leprae. Drug resistance in mycobacteria is increasingly common making identification of new antimicrobials a priority. Mycobacteria replicate intracellularly, most commonly within the phagosomes of macrophages, and bacterial proteins essential for intracellular survival and persistence are particularly attractive targets for intervention with new generations of anti-mycobacterial drugs.

Methodology/Principal Findings

We have identified a novel gene that, when inactivated, leads to accelerated death of M. smegmatis within a macrophage cell line in the first eight hours following infection. Complementation of the mutant with an intact copy of the gene restored survival to near wild type levels. Gene disruption did not affect growth compared to wild type M. smegmatis in axenic culture or in the presence of low pH or reactive oxygen intermediates, suggesting the growth defect is not related to increased susceptibility to these stresses. The disrupted gene, MSMEG_5817, is conserved in all mycobacteria for which genome sequence information is available, and designated Rv0807 in M. tuberculosis. Although homology searches suggest that MSMEG_5817 is similar to the serine:pyruvate aminotransferase of Brevibacterium linens suggesting a possible role in glyoxylate metabolism, enzymatic assays comparing activity in wild type and mutant strains demonstrated no differences in the capacity to metabolize glyoxylate.

Conclusions/Significance

MSMEG_5817 is a previously uncharacterized gene that facilitates intracellular survival of mycobacteria. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of mycobacterial pathogens by assisting the host immune system in clearance of persistent intracellular bacteria.     

An ENU-mutagenesis screen in the mouse: identification of novel developmental gene functions

Background

Mutagenesis screens in the mouse have been proven useful for the identification of novel gene functions and generation of interesting mutant alleles. Here we describe a phenotype-based screen for recessive mutations affecting embryonic development.

Methodology/Principal Findings

Mice were mutagenized with N-ethyl-N-nitrosurea (ENU) and following incrossing the offspring, embryos were analyzed at embryonic day 10.5. Mutant phenotypes that arose in our screen include cardiac and nuchal edema, neural tube defects, situs inversus of the heart, posterior truncation and the absence of limbs and lungs. We isolated amongst others novel mutant alleles for Dll1, Ptprb, Plexin-B2, Fgf10, Wnt3a, Ncx1, Scrib(Scrib, Scribbled homolog [Drosophila]) and Sec24b. We found both nonsense alleles leading to severe protein truncations and mutants with single-amino acid substitutions that are informative at a molecular level. Novel findings include an ectopic neural tube in our Dll1 mutant and lung defects in the planar cell polarity mutants for Sec24b and Scrib.

Conclusions/Significance

Using a forward genetics approach, we have generated a number of novel mutant alleles that are linked to disturbed morphogenesis during development.     

Mutants in Drosophila TRPC Channels Reduce Olfactory Sensitivity to Carbon Dioxide

Background

Members of the canonical Transient Receptor Potential (TRPC) class of cationic channels function downstream of Gαq and PLCβ in Drosophila photoreceptors for transducing visual stimuli. Gαq has recently been implicated in olfactory sensing of carbon dioxide (CO₂) and other odorants. Here we investigated the role of PLCβ and TRPC channels for sensing CO₂ in Drosophila.

Methodology/Principal Findings

Through behavioral assays it was demonstrated that Drosophila mutants for plc21c, trp and trpl have a reduced sensitivity for CO₂. Immuno-histochemical staining for TRP, TRPL and TRPγ indicates that all three channels are expressed in Drosophila antennae including the sensory neurons that express CO₂ receptors. Electrophysiological recordings obtained from the antennae of protein null alleles of TRP (trp³⁴³) and TRPL (trpl³⁰²), showed that the sensory response to multiple concentrations of CO₂ was reduced. However, trpl³⁰²; trp³⁴³ double mutants still have a residual response to CO₂. Down-regulation of TRPC channels specifically in CO₂ sensing olfactory neurons reduced the response to CO₂ and this reduction was obtained even upon down-regulation of the TRPCs in adult olfactory sensory neurons. Thus the reduced response to CO₂ obtained from the antennae of TRPC RNAi strains is not due to a developmental defect.

Conclusion

These observations show that reduction in TRPC channel function significantly reduces the sensitivity of the olfactory response to CO₂ concentrations of 5% or less in adult Drosophila. It is possible that the CO₂ receptors Gr63a and Gr21a activate the TRPC channels through Gαq and PLC21C.     

Characterization of novel StAR (steroidogenic acute regulatory protein) mutations causing non-classic lipoid adrenal hyperplasia

Context

Steroidogenic acute regulatory protein (StAR) is crucial for transport of cholesterol to mitochondria where biosynthesis of steroids is initiated. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH).

Objective

StAR gene mutations causing partial loss of function manifest atypical and may be mistaken as familial glucocorticoid deficiency. Only a few mutations have been reported.

Design

To report clinical, biochemical, genetic, protein structure and functional data on two novel StAR mutations, and to compare them with published literature.

Setting

Collaboration between the University Children''s Hospital Bern, Switzerland, and the CIBERER, Hospital Vall d''Hebron, Autonomous University, Barcelona, Spain.

Patients

Two subjects of a non-consanguineous Caucasian family were studied. The 46,XX phenotypic normal female was diagnosed with adrenal insufficiency at the age of 10 months, had normal pubertal development and still has no signs of hypergonodatropic hypogonadism at 32 years of age. Her 46,XY brother was born with normal male external genitalia and was diagnosed with adrenal insufficiency at 14 months. Puberty was normal and no signs of hypergonadotropic hypogonadism are present at 29 years of age.

Results

StAR gene analysis revealed two novel compound heterozygote mutations T44HfsX3 and G221S. T44HfsX3 is a loss-of-function StAR mutation. G221S retains partial activity (∼30%) and is therefore responsible for a milder, non-classic phenotype. G221S is located in the cholesterol binding pocket and seems to alter binding/release of cholesterol.

Conclusions

StAR mutations located in the cholesterol binding pocket (V187M, R188C, R192C, G221D/S) seem to cause non-classic lipoid CAH. Accuracy of genotype-phenotype prediction by in vitro testing may vary with the assays employed.     

Interactions of attenuated Mycobacterium tuberculosis phoP mutant with human macrophages

Background

Mycobacterium tuberculosis phoP mutant SO2 derived from a clinical isolate was shown to be attenuated in mouse bone marrow-derived macrophages and in vivo mouse infection model and has demonstrated a high potential as attenuated vaccine candidate against tuberculosis.

Methodology/Principal Findings

In this study, we analyze the adhesion and the intracellular growth and trafficking of SO2 in human macrophages. Our results indicate an enhanced adhesion to phagocitic cells and impaired intracellular replication of SO2 in both monocyte-derived macrophages and human cell line THP-1 in comparison with the wild type strain, consistent with murine model. Intracellular trafficking analysis in human THP-1 cells suggest that attenuation of SO2 within macrophages could be due to an impaired ability to block phagosome-lysosome fusion compared with the parental M. tuberculosis strain. No differences were found between SO2 and the wild-type strains in the release and mycobacterial susceptibility to nitric oxide (NO) produced by infected macrophages.

Conclusions/Significance

SO2 has enhanced ability to bind human macrophages and differs in intracellular trafficking as to wild-type M. tuberculosis. The altered lipid profile expression of the phoP mutant SO2 and its inability to secrete ESAT-6 is discussed.     

A novel and lethal de novo LQT-3 mutation in a newborn with distinct molecular pharmacology and therapeutic response

Background

SCN5A encodes the α-subunit (Na_v1.5) of the principle Na⁺ channel in the human heart. Genetic lesions in SCN5A can cause congenital long QT syndrome (LQTS) variant 3 (LQT-3) in adults by disrupting inactivation of the Na_v1.5 channel. Pharmacological targeting of mutation-altered Na⁺ channels has proven promising in developing a gene-specific therapeutic strategy to manage specifically this LQTS variant. SCN5A mutations that cause similar channel dysfunction may also contribute to sudden infant death syndrome (SIDS) and other arrhythmias in newborns, but the prevalence, impact, and therapeutic management of SCN5A mutations may be distinct in infants compared with adults.

Methods and Results

Here, in a multidisciplinary approach, we report a de novo SCN5A mutation (F1473C) discovered in a newborn presenting with extreme QT prolongation and differential responses to the Na⁺ channel blockers flecainide and mexiletine. Our goal was to determine the Na⁺ channel phenotype caused by this severe mutation and to determine whether distinct effects of different Na⁺ channel blockers on mutant channel activity provide a mechanistic understanding of the distinct therapeutic responsiveness of the mutation carrier. Sequence analysis of the proband revealed the novel missense SCN5A mutation (F1473C) and a common variant in KCNH2 (K897T). Patch clamp analysis of HEK 293 cells transiently transfected with wild-type or mutant Na⁺ channels revealed significant changes in channel biophysics, all contributing to the proband''s phenotype as predicted by in silico modeling. Furthermore, subtle differences in drug action were detected in correcting mutant channel activity that, together with both the known genetic background and age of the patient, contribute to the distinct therapeutic responses observed clinically.

Significance

The results of our study provide further evidence of the grave vulnerability of newborns to Na⁺ channel defects and suggest that both genetic background and age are particularly important in developing a mutation-specific therapeutic personalized approach to manage disorders in the young.