Gene expression profiling of yeasts overexpressing wild type or misfolded Pma1 variants reveals activation of the Hog1 MAPK pathway

Dominant negative PMA1 mutants render misfolded proteins that are retained in the endoplasmic reticulum (ER) and slowly degraded by ER-associated degradation. Accumulation of misfolded proteins in the ER activates an ER-to-nucleus signalling pathway termed the unfolded protein response (UPR). We have used a PMA1-D378T dominant negative mutant to analyse its impact on UPR induction. Our results show that overexpression of the misfolded mutant Pma1 does not lead to activation of the UPR. In addition, in mutants with a constitutively activated UPR the turnover rate of the mutant ATPase is not altered. To determine if the expression of the misfolded mutant protein induces some other kind of response we performed global gene expression analysis experiments in yeasts overexpressing either wild type or dominant lethal PMA1 alleles. The results suggest that the high osmolarity glycerol (Hog1) mitogen-activated protein kinase (MAPK) pathway is activated by both wild type and mutant ATPases. We show that expression of the PMA1 alleles induces phosphorylation of Hog1 and activation of the Hog1 MAPK cascade. This activation is mediated by the Sln1 branch of the stress-dependent Hog1 MAPK network. Finally, we show that at least two other plasma membrane proteins are also able to activate the Hog1 MAPK system.     

Efficient degradation of misfolded mutant Pma1 by endoplasmic reticulum-associated degradation requires Atg19 and the Cvt/autophagy pathway

Misfolded proteins are usually arrested in the endoplasmic reticulum (ER) and degraded by the ER-associated degradation (ERAD) machinery. Several mutant alleles of PMA1, the gene coding for the plasma membrane H(+)-ATPase, render misfolded proteins that are retained in the ER and degraded by ERAD. A subset of misfolded PMA1 mutants exhibit a dominant negative effect on yeast growth since, when coexpressed with the wild-type allele, both proteins are retained in the ER. We have used a pma1-D378T dominant negative mutant to identify new genes involved in ERAD. A genetic screen was performed for isolation of multicopy suppressors of a GAL1-pma1-D378T allele. ATG19, a member of the cytoplasm to vacuole targeting (Cvt) pathway, was found to suppress the growth arrest phenotype caused by the expression of pma1-D378T. ATG19 accelerates the degradation of pma1-D378T thus allowing the co-retained wild-type Pma1 to reach the plasma membrane. ATG19 was also able to suppress other dominant lethal PMA1 mutations. The degradation of the mutant ATPase occurs in the proteasome and requires intact both ERAD and Cvt/autophagy pathways. We propose the cooperation of both pathways for an efficient degradation of misfolded Pma1.     

Loss of Vacuolar H+-ATPase Activity in Organelles Signals Ubiquitination and Endocytosis of the Yeast Plasma Membrane Proton pump Pma1p

Yeast mutants lacking the intracellular V-ATPase proton pump (vma mutants) have reduced levels of the Pma1p proton pump at the plasma membrane and increased levels in organelles including the vacuolar lumen. We examined the mechanism and physiological consequences of Pma1p mislocalization. Pma1p is ubiquitinated in vma mutants, and ubiquitination depends on the ubiquitin ligase Rsp5p and the arrestin-related adaptor protein Rim8p. vma mutant strains containing rsp5 or rim8 mutations maintain Pma1p at the plasma membrane, suggesting that ubiquitination is required for Pma1p internalization. Acute inhibition of V-ATPase activity with concanamycin A triggers Pma1p ubiquitination and internalization. In an endocytosis-deficient mutant (end4Δ) Pma1p is ubiquitinated but retained at the plasma membrane during concanamycin A treatment. Consistent with specificity in signaling loss of V-ATPase activity to Pma1p, another plasma membrane transporter, Mup1p, is not internalized in a vma mutant, and loss of the Mup1p adaptor Art1p does not prevent Pma1p internalization in a vma mutant. Very poor growth of vma2 rsp5-1 and vma2 rim8Δ double mutants suggests that Pma1p internalization benefits the vma mutants. We hypothesize that loss of V-ATPase-mediated organelle acidification signals ubiquitination, internalization, and degradation of a portion of Pma1p as a means of balancing overall pH homeostasis.     

Novel Genes Involved in Endosomal Traffic in Yeast Revealed by Suppression of a Targeting-defective Plasma Membrane ATPase Mutant

A novel genetic selection was used to identify genes regulating traffic in the yeast endosomal system. We took advantage of a temperature-sensitive mutant in PMA1, encoding the plasma membrane ATPase, in which newly synthesized Pma1 is mislocalized to the vacuole via the endosome. Diversion of mutant Pma1 from vacuolar delivery and rerouting to the plasma membrane is a major mechanism of suppression of pma1^ts. 16 independent suppressor of pma1 (sop) mutants were isolated. Identification of the corresponding genes reveals eight that are identical with VPS genes required for delivery of newly synthesized vacuolar proteins. A second group of SOP genes participates in vacuolar delivery of mutant Pma1 but is not essential for delivery of the vacuolar protease carboxypeptidase Y. Because the biosynthetic pathway to the vacuole intersects with the endocytic pathway, internalization of a bulk membrane endocytic marker FM 4-64 was assayed in the sop mutants. By this means, defective endosome-to-vacuole trafficking was revealed in a subset of sop mutants. Another subset of sop mutants displays perturbed trafficking between endosome and Golgi: impaired pro-α factor processing in these strains was found to be due to defective recycling of the trans-Golgi protease Kex2. One of these strains defective in Kex2 trafficking carries a mutation in SOP2, encoding a homologue of mammalian synaptojanin (implicated in synaptic vesicle endocytosis and recycling). Thus, cell surface delivery of mutant Pma1 can occur as a consequence of disturbances at several different sites in the endosomal system.     

Characterization of an allele-nonspecific intragenic suppressor in the yeast plasma membrane H+-ATPase gene (Pma1).

We have analyzed the ability of A165V, V169I/D170N, and P536L mutations to suppress pma1 dominant lethal alleles and found that the P536L mutation is able to suppress the dominant lethality of the pma1-R271T, -D378N, -D378E, and -K474R mutant alleles. Genetic and biochemical analyses of site-directed mutants at Pro-536 suggest that this amino acid may not be essential for function but is important for biogenesis of the ATPase. Proteins encoded by dominant lethal pma1 alleles are retained in the endoplasmic reticulum, thus interfering with transport of wild-type Pma1. Immunofluorescence studies of yeast conditionally expressing revertant alleles show that the mutant enzymes are correctly located at the plasma membrane and do not disturb targeting of the wild-type enzyme. We propose that changes in Pro-536 may influence the folding of the protein encoded by a dominant negative allele so that it is no longer recognized and retained as a misfolded protein by the endoplasmic reticulum.     

A Dominant Negative Mutant of Pma1 Interferes with the Folding of the Wild Type Enzyme

Misfolded proteins are usually arrested in the endoplasmic reticulum (ER) and degraded by the ER-associated degradation (ERAD) machinery. Several mutant alleles of PMA1 , the gene coding for the plasma membrane H ⁺-ATPase, render misfolded proteins that are subjected to ERAD. A subset of misfolded PMA1 mutants exhibits a dominant negative effect on yeast growth since, when co-expressed with the wild type allele, both proteins are retained in the ER and degraded. We have used a PMA1 -D378T dominant lethal allele to analyse the mechanism underlying the retention of the wild type enzyme by the dominant negative mutant. A genetic screen was performed for isolation of intragenic suppressors of PMA1 -D378T allele. This analysis pointed to transmembrane helix 10 (TM10) as an important element in the establishment of the dominant lethality. Deletion of the TM10 was able to suppress not only the PMA1 -D378T but all the dominant lethal alleles tested. Biochemical analyses suggest that dominant lethal proteins obstruct, through TM10, the correct folding of the wild type enzyme leading to its retention and degradation by ERAD.     

A Mutant Plasma Membrane Protein Is Stabilized Upon Loss of Yvh1, a Novel Ribosome Assembly Factor

Pma1-10 is a mutant plasma membrane ATPase defective at the restrictive temperature in stability at the cell surface. At 37°, Pma1-10 is ubiquitinated and internalized from the plasma membrane for degradation in the vacuole. YVH1, encoding a tyrosine phosphatase, is a mutant suppressor of pma1-10; in the absence of Yvh1, Pma1-10 remains stable at the plasma membrane, thereby permitting cells to grow. The RING finger domain of Yvh1, but not its phosphatase domain, is required for removal of mutant Pma1-10 from the plasma membrane. Yvh1 is a novel ribosome assembly factor: in yvh1Δ cells, free 60S and 80S ribosomal subunits are decreased, free 40S subunits are increased, and half-mer polysomes are accumulated. Pma1-10 is also stabilized by deletion of 60S ribosomal proteins Rpl19a and Rpl35a. We propose that changes in ribosome biogenesis caused by loss of Yvh1 or specific ribosomal proteins have effects on the plasma membrane, perhaps by producing specific translational changes.     

Impaired Iron Transport Activity of Ferroportin 1 in Hereditary Iron Overload

To investigate the functional significance of mutations in Ferroportin that cause hereditary iron overload, we directly measured the iron efflux activity of the proteins expressed in Xenopus oocytes. We found that wild type and mutant Ferroportin molecules (A77D, N144H, Q248H and V162Δ) were all expressed at the plasma membrane at similar levels. All mutations caused significant reductions in ⁵⁹Fe efflux compared to wild type but all retained some residual transport activity. A77D had the strongest effect on ⁵⁹Fe efflux (remaining activity 9% of wild-type control), whereas the N144H mutation retained the highest efflux activity (42% of control). The Q248H and V162Δ mutations were intermediate between these values. Co-injection of mutant and wild-type mRNAs revealed that the A77D and N144H mutations had a dominant negative effect on the function of the WT protein.     

Eps1, a novel PDI-related protein involved in ER quality control in yeast.

PMA1 is an essential gene encoding the yeast plasma membrane [H(+)]ATPase. A pma1-D378N mutant has a dominant-negative effect on cell growth because both newly synthesized mutant and wild-type Pma1 molecules are retained and degraded in the endoplasmic reticulum (ER). Like other substrates for ER-associated degradation, Pma1-D378N is stabilized in mutants defective in components of the ubiquitination machinery. A genetic selection was performed for eps (ER-retained pma1 suppressing) mutants in which the growth defect caused by the D378N allele is suppressed. In an eps1 mutant, both mutant and wild-type Pma1 molecules are allowed to travel to the plasma membrane; however, normal retention of resident ER proteins Shr3 and Kar2 is not perturbed. Eps1 is a novel membrane protein belonging to the protein disulfide isomerase (PDI) family, and Eps1 co-localizes with Pma1-D378N in the ER. In the absence of Pma1-D378N, ER export of wild-type Pma1 is not affected by eps1 deletion, but export of the plasma membrane protein Gas1 is delayed. Because Eps1 is required for retention and degradation of Pma1-D378N, we propose a model in which Eps1 acts as a novel membrane-bound chaperone in ER quality control.     

An endosome-to-plasma membrane pathway involved in trafficking of a mutant plasma membrane ATPase in yeast

The plasma membrane ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway. Previously, we characterized a temperature-sensitive pma1 mutant in which newly synthesized Pma1-7 is not delivered to the plasma membrane but is mislocalized instead to the vacuole at 37 degrees C. Several vps mutants, which are defective in vacuolar protein sorting, suppress targeting-defective pma1 by allowing mutant Pma1 to move once again to the plasma membrane. In this study, we have analyzed trafficking in the endosomal system by monitoring the movement of Pma1-7 in vps36, vps1, and vps8 mutants. Upon induction of expression, mutant Pma1 accumulates in the prevacuolar compartment in vps36 cells. After chase, a fraction of newly synthesized Pma1-7 is delivered to the plasma membrane. In both vps1 and vps8 cells, newly synthesized mutant Pma1 appears in small punctate structures before arrival at the cell surface. Nevertheless, biosynthetic membrane traffic appears to follow different routes in vps8 and vps1: the vacuolar protein-sorting receptor Vps10p is stable in vps8 but not in vps1. Furthermore, a defect in endocytic delivery to the vacuole was revealed in vps8 (and vps36) but not vps1 by endocytosis of the bulk membrane marker FM 4-64. Moreover, in vps8 cells, there is defective down-regulation from the cell surface of the mating receptor Ste3, consistent with persistent receptor recycling from an endosomal compartment to the plasma membrane. These data support a model in which mutant Pma1 is diverted from the Golgi to the surface in vps1 cells. We hypothesize that in vps8 and vps36, in contrast to vps1, mutant Pma1 moves to the surface via endosomal intermediates, implicating an endosome-to-surface traffic pathway.     

Evaluation of the antimicrobial activity of ebselen: role of the yeast plasma membrane H+-ATPase

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is a selenium-containing antioxidant demonstrating anti-inflammatory and cytoprotective properties in mammalian cells and cytotoxicity in lower organisms. The mechanism underlying the antimicrobial activity of ebselen remains unclear. It has recently been proposed that, in lower organisms like yeast, the plasma membrane H+-ATPase (Pma1p) could serve as a potential target for this synthetic organoselenium compound. Using yeast and bacteria, the present study found ebselen to inhibit microbial growth in a concentration- and time-dependent manner, and yeast and Gram-positive bacteria to be more sensitive to this action (IC50 approximately 2-5 microM) than Gram-negative bacteria (IC50 < 80 microM). Washout experiments and scanning electron microscopic analysis revealed ebselen to possess fungicidal activity. In addition, ebselen was found to inhibit medium acidification by PMA1-proficient haploid yeast in a concentration-dependent manner. Additional studies comparing PMA1 (+/-) and PMA1 (+/+) diploid yeast cells revealed the mutant to be more sensitive to treatment with ebselen than the wild type. Ebselen also inhibited the ATPase activity of Pma1p from S. cerevisiae in a concentration-dependent manner. The interaction of ebselen with the sulfhydryl-containing compounds L-cysteine and reduced glutathione resulted in the complete and partial prevention, respectively, of the inhibition of Pma1p ATPase activity by ebselen. Taken together, these results suggest that the fungicidal action of ebselen is due, at least in part, to interference with both the proton-translocating function and the ATPase activity of the plasma membrane H+-ATPase.     

Use of Targeted Exome Sequencing in Genetic Diagnosis of Chinese Familial Hypercholesterolemia

Familial hypercholesterolemia is an autosomal dominant inherited disease characterized by elevated plasma low-density lipoprotein cholesterol (LDL-C). It is mainly caused by mutations of the low-density lipoprotein receptor (LDLR) gene. Currently, the methods of whole genome sequencing or whole exome sequencing for screening mutations in familial hypercholesterolemia are not applicable in China due to high cost. We performed targeted exome sequencing of 167 genes implicated in the homozygous phenotype of a proband pedigree to identify candidate mutations, validated them in the family of the proband, studied the functions of the mutant protein, and followed up serum lipid levels after treatment. We discovered that exon 9 c.1268 T>C and exon 8 c.1129 T>G compound heterozygous mutations in the LDLR gene in the proband derived from the mother and father, respectively, in which the mutation of c.1129 T>G has not been reported previously. The mutant LDL-R protein had 57% and 52% binding and internalization functions, respectively, compared with that of the wild type. After 6 months of therapy, the LDL-C level of the proband decreased by more than 50% and the LDL-C of the other family members with heterozygous mutation also reduced to normal. Targeted exome sequencing is an effective method for screening mutation genes in familial hypercholesterolemia. The exon 8 and 9 mutations of the LDLR gene were pedigree mutations. The functions of the mutant LDL-R protein were decreased significantly compared with that of the wild type. Simvastatin plus ezetimibe was proven safe and effective in this preschool-age child.     

Quality control in the yeast secretory pathway: a misfolded PMA1 H+-ATPase reveals two checkpoints

The yeast plasma-membrane H(+)-ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway and has recently emerged as an excellent system for identifying quality control mechanisms along the pathway. In the present study, we have tracked the biogenesis of Pma1-G381A, a misfolded mutant form of the H(+)-ATPase. Although this mutant ATPase is arrested transiently in the peripheral endoplasmic reticulum, it does not become a substrate for endoplasmic reticulum-associated degradation nor does it appear to stimulate an unfolded protein response. Instead, Pma1-G381A accumulates in Kar2p-containing vesicular-tubular clusters that resemble those previously described in mammalian cells. Like their mammalian counterparts, the yeast vesicular-tubular clusters may correspond to specific exit ports from the endoplasmic reticulum, since Pma1-G381A eventually escapes from them (still in a misfolded, trypsin-sensitive form) to reach the plasma membrane. By comparison with wild-type ATPase, Pma1-G381A spends a short half-life at the plasma membrane before being removed and sent to the vacuole for degradation in a process that requires both End4p and Pep4p. Finally, in a separate set of experiments, Pma1-G381A was found to impose its phenotype on co-expressed wild-type ATPase, transiently retarding the wild-type protein in the ER and later stimulating its degradation in the vacuole. Both effects serve to lower the steady-state amount of wild-type ATPase in the plasma membrane and, thus, can explain the co-dominant genetic behavior of the G381A mutation. Taken together, the results of this study establish Pma1-G381A as a useful new probe for the yeast secretory system.     

Altered distribution of the yeast plasma membrane H+-ATPase as a feature of vacuolar H+-ATPase null mutants

The effect of vacuolar H(+)-ATPase (V-ATPase) null mutations on the targeting of the plasma membrane H(+)-ATPase (Pma1p) through the secretory pathway was analyzed. Gas1p, which is another plasma membrane component, was used as a control for the experiments with Pma1p. Contrary to Gas1p, which is not affected by the deletion of the V-ATPase complex in the V-ATPase null mutants, the amount of Pma1p in the plasma membrane is markedly reduced, and there is a large accumulation of the protein in the endoplasmic reticulum. Kex2p and Gef1p, which are considered to reside in the post-Golgi vesicles, were suggested as required for the V-ATPase function; hence, their null mutant phenotype should have been similar to the V-ATPase null mutants. We show that, in addition to the known differences between those yeast phenotypes, deletions of KEX2 or GEF1 in yeast do not affect the distribution of Pma1p as the V-ATPase null mutant does. The possible location of the vital site of acidification by V-ATPase along the secretory pathway is discussed.     

Proline 146 is critical to the structure, function and targeting of sod2, the Na/H exchanger of Schizosaccharomyces pombe

Sod2 is the Na⁺/H⁺ exchanger of the fission yeast Schizosaccharomyces pombe that is principally responsible for salt tolerance. We examined the role of nine polar, membrane associated amino acids in the ability of the protein to confer salt tolerance in S. pombe. Wild type sod2 protein with a C-terminal GFP tag effectively rescued salt tolerance in S. pombe with deleted endogenous sod2. Sod2 protein with the mutations P163A, P183A, D298N, D389N, E390Q, E392Q and E397Q also conveyed salt tolerance as effectively as the wild type sod2 protein. In contrast, the mutation P146A resulted in a protein that did not convey salt tolerance nearly as effectively as the wild type and did not extrude Na⁺ as well as the wild type. Mutation of Pro¹⁴⁶ to Ser, Asp or Lys had an intermediate effect. Mutation of Thr¹⁴² to Ser resulted in a slightly defective protein. Western blot analysis showed that all mutant proteins were expressed at similar levels as wild type sod2 protein. Examination of the localization of the proteins showed that wild type and most sod2 mutants were present in the plasma membrane while the P146A mutant had an intracellular localization. Limited tryptic digestion suggested that the P146A sod2 protein had a change in conformation in comparison to the wild type protein. The results suggest that Pro¹⁴⁶ is an amino acid critical to sod2 structure, function and localization.     

Novel UMOD mutations in familial juvenile hyperuricemic nephropathy lead to abnormal uromodulin intracellular trafficking

Background

Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal dominant disorder characterized by hyperuricemia and progressive chronic kidney disease. Uromodulin gene (UMOD) mutations, leading to abnormalities of uromodulin intracellular trafficking contribute to the progress of the disease.

Methods

We did UMOD screening in three Chinese FJHN families. We thus constructed mutant uromodulin express plasmids by site-mutagenesis from wild type uromodulin vector and transfected them into HEK293 (human embryonic kidney) cells. And then we detected uromodulin expression by western blot and observed intracellular distribution by immunofluorescence.

Results

We found three heterozygous mutations. Mutation Val109Glu (c.326T/A; p.Val109Glu) and mutation Pro236Gln (c.707C/A; p.Pro236Gln) were newly indentified mutations in two distinct families (family F1 and family F3). Another previously reported UMOD mutation Cys248Trp (c.744C/G; p.Cys248Trp) was detected in family F2. Phenotypes varied both within the same family and between different families. Uromodulin expression is abnormal in the patient biopsy. Functional analysis of mutation showed that mutant types of uromodulin were secreted into the supernatant medium much less when compared with wild type. In mutant type uromodulin transfected cells, intracellular uromodulin localized less in the Golgi apparatus and more in endoplasmic reticulum(ER).

Conclusions

Our results suggested that the novel uromodulin mutations found in the Chinese families lead to misfolded protein, which was retained in the endoplasmic reticulum, finally contributed to the phenotype of FJHN.     

An ER membrane protein,Sop4, facilitates ER export of the yeast plasma membrane [H+]ATPase,Pma1

We have analyzed the mechanism by which Sop4, a novel ER membrane protein, regulates quality control and intracellular transport of Pma1–7, a mutant plasma membrane ATPase. At the restrictive temperature, newly synthesized Pma1–7 is targeted for vacuolar degradation instead of being correctly delivered to the cell surface. Loss of Sop4 at least partially corrects vacuolar mislocalization, allowing Pma1–7 routing to the plasma membrane. Ste2–3 is a mutant pheromone receptor which, like Pma1–7, is defective in targeting to the cell surface, resulting in a mating defect. sop4Δ suppresses the mating defect of ste2–3 cells as well as the growth defect of pma1–7 . Visualization of newly synthesized Pma1–7 in sop4Δ cells by indirect immunofluorescence reveals delayed export from the ER. Similarly, ER export of wild-type Pma1 is delayed in the absence of Sop4 although intracellular transport of Gas1 and CPY is unaffected. These observations suggest a model in which a selective increase in ER residence time for Pma1–7 may allow it to achieve a more favorable conformation for subsequent delivery to the plasma membrane. In support of this model, newly synthesized Pma1–7 is also routed to the plasma membrane upon release from a general block of ER-to-Golgi transport in sec13–1 cells.     

A Mutation Associated with CMT2A Neuropathy Causes Defects in Fzo1 GTP Hydrolysis,Ubiquitylation, and Protein Turnover

Charcot-Marie-Tooth disease type 2A (CMT2A) is caused by mutations in the gene MFN2 and is one of the most common inherited peripheral neuropathies. Mfn2 is one of two mammalian mitofusin GTPases that promote mitochondrial fusion and maintain organelle integrity. It is not known how mitofusin mutations cause axonal degeneration and CMT2A disease. We used the conserved yeast mitofusin FZO1 to study the molecular consequences of CMT2A mutations on Fzo1 function in vivo and in vitro. One mutation (analogous to the CMT2A I213T substitution in the GTPase domain of Mfn2) not only abolishes GTP hydrolysis and mitochondrial membrane fusion but also reduces Mdm30-mediated ubiquitylation and degradation of the mutant protein. Importantly, complexes of wild type and the mutant Fzo1 protein are GTPase active and restore ubiquitylation and degradation of the latter. These studies identify diverse and unexpected effects of CMT2A mutations, including a possible role for mitofusin ubiquitylation and degradation in CMT2A pathogenesis, and provide evidence for a novel link between Fzo1 GTP hydrolysis, ubiquitylation, and mitochondrial fusion.     

DISSECT Method Using PNA-LNA Clamp Improves Detection of EGFR T790m Mutation

Non-small cell lung cancer (NSCLC) patients treated with small molecule EGFR inhibitors, such as gefitinib, frequently develop drug resistance due to the presence of secondary mutations like the T790M mutation on EGFR exon 20. These mutations may originate from small subclonal populations in the primary tumor that become dominant later on during treatment. In order to detect these low-level DNA variations in the primary tumor or to monitor their progress in plasma, it is important to apply reliable and sensitive mutation detection methods. Here, we combine two recently developed methodologies, Differential Strand Separation at Critical Temperature (DISSECT), with peptide nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) for the detection of T790M EGFR mutation. DISSECT pre-enriches low-abundance T790M EGFR mutations from target DNA prior to implementing PNA-LNA PCR, a method that can detect 1 mutant allele in a background of 100–1000 wild type alleles. The combination of DISSECT and PNA-LNA PCR enables the detection of 1 mutant allele in a background of 10,000 wild type alleles. The combined DISSECT-PNA-LNA PCR methodology is amenable to adaptation for the sensitive detection of additional emerging resistance mutations in cancer.     

Functional dominant-negative mutation of sodium channel subunit gene SCN3B associated with atrial fibrillation in a Chinese GeneID population

Atrial fibrillation (AF) is the most common cardiac arrhythmia in the clinic, and accounts for more than 15% of strokes. Mutations in cardiac sodium channel α, β1 and β2 subunit genes (SCN5A, SCN1B, and SCN2B) have been identified in AF patients. We hypothesize that mutations in the sodium channel β3 subunit gene SCN3B are also associated with AF. To test this hypothesis, we carried out a large scale sequencing analysis of all coding exons and exon-intron boundaries of SCN3B in 477 AF patients (28.5% lone AF) from the GeneID Chinese Han population. A novel A130V mutation was identified in a 46-year-old patient with lone AF, and the mutation was absent in 500 controls. Mutation A130V dramatically decreased the cardiac sodium current density when expressed in HEK293/Na_v1.5 stable cell line, but did not have significant effect on kinetics of activation, inactivation, and channel recovery from inactivation. When co-expressed with wild type SCN3B, the A130V mutant SCN3B negated the function of wild type SCN3B, suggesting that A130V acts by a dominant negative mechanism. Western blot analysis with biotinylated plasma membrane protein extracts revealed that A130V did not affect cell surface expression of Na_v1.5 or SCN3B, suggesting that mutant A130V SCN3B may not inhibit sodium channel trafficking, instead may affect conduction of sodium ions due to its malfunction as an integral component of the channel complex. This study identifies the first AF-associated mutation in SCN3B, and suggests that mutations in SCN3B may be a new pathogenic cause of AF.