Coupling of mutated Met variants to DNA repair via Abl and Rad51

Abnormal activation of DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems is a compelling likelihood with significant implications in both cancer biology and treatment. Here, we show that due to a potential substrate switch, mutated variants of the receptor for hepatocyte growth factor Met, but not the wild-type form of the receptor, directly couple to the Abl tyrosine kinase and the Rad51 recombinase, two key signaling elements of homologous recombination-based DNA repair. Treatment of cells that express the mutated receptor variants with the Met inhibitor SU11274 leads, in a mutant-dependent manner, to a reduction of tyrosine phosphorylated levels of Abl and Rad51, impairs radiation-induced nuclear translocation of Rad51, and acts as a radiosensitizer together with the p53 inhibitor pifithrin-alpha by increasing cellular double-strand DNA break levels following exposure to ionizing radiation. Finally, we propose that in order to overcome a mutation-dependent resistance to SU11274, this aberrant molecular axis may alternatively be targeted with the Abl inhibitor, nilotinib.

Do common genetic variants in endotoxin signaling pathway contribute to predisposition to alcoholic liver cirrhosis?

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), produced by endotoxin-activated Kupffer cells, play a key role in the pathogenesis of alcoholic liver cirrhosis (ALC). Alleles TNFA -238A, IL1B -31T and variant IL1RN*2 of repeat polymorphism in the gene encoding the IL-1 receptor antagonist increase production of TNF-alpha and IL-1beta, respectively. Alleles CD14 -159T, TLR4 c.896G and TLR4 c.1196T modify activation of Kupffer cells by endotoxin. We confirmed the published associations between these common variants and genetic predisposition to ALC by means of a large case-control association study conducted on two Central European populations. METHODS: The study population comprised a Czech sample of 198 ALC patients and 370 controls (MONICA project), and a German sample of 173 ALC patients and 331 controls (KORA-Augsburg), and 109 heavy drinkers without liver disease. RESULTS: Single locus analysis revealed no significant difference between patients and controls in all tested loci. Diplotype [IL1RN 2/ 2; IL1B -31T+] was associated with increased risk of ALC in the pilot study, but not in the validation samples. CONCLUSIONS: Although cytokine mediated immune reactions play a role in the pathogenesis of ALC, hereditary susceptibility caused by variants in the corresponding genes is low in Central European populations.

High aldosterone-to-renin variants of CYP11B2 and pregnancy outcome

BACKGROUND: Increased aldosterone concentrations and volume expansion of normal pregnancies are hallmarks of normal pregnancies and blunted in pre-eclampsia. Accordingly, we hypothesized an active mineralocorticoid system to protect from pre-eclampsia. METHODS: In pregnant women (normotensive n = 44; pre-eclamptic n = 48), blood pressure, urinary tetrahydro-aldosterone excretion and activating polymorphisms (SF-1 site and intron 2) of the aldosterone synthase gene (CYP11B2) were determined; 185 non-pregnant normotensive individuals served as control. Amino acid-changing polymorphisms of the DNA- and agonist-binding regions of the mineralocorticoid receptor were evaluated by RT-PCR, SSCP and sequencing. RESULTS: Urinary tetrahydro-aldosterone excretion was reduced in pre-eclampsia as compared to normal pregnancy (P < 0.05). It inversely correlated with blood pressure (r = 0.99, P < 0.04). Homozygosity for activating CYP11B2 polymorphisms was preferably present in normotensive as compared to pre-eclamptic pregnancies, identified (intron 2, P = 0.005; SF-1 site, P = 0.016). Two mutant haplotypes decreased the risk of developing pre-eclampsia (RR 0.16; CI 0.05-0.54; P < 0.001). In contrast, intron 2 wild type predisposed to pre-eclampsia (P < 0.0015). No functional mineralocorticoid receptor mutant has been observed. CONCLUSIONS: High aldosterone availability is associated with lower maternal blood pressure. In line with this observation, gain-of-function variants of the CYP11B2 reduce the risk of developing pre-eclampsia. Mutants of the mineralocorticoid receptor cannot explain the frequent syndrome of pre-eclampsia.

Bacterial phenotype variants in group B streptococcal toxic shock syndrome

We conducted genetic and functional analyses of isolates from a patient with group B streptococcal (GBS) necrotizing fasciitis and toxic shock syndrome. Tissue cultures simultaneously showed colonies with high hemolysis (HH) and low hemolysis (LH). Conversely, the HH and LH variants exhibited low capsule (LC) and high capsule (HC) expression, respectively. Molecular analysis demonstrated that the 2 GBS variants were of the same clonal origin. Genetic analysis found a 3-bp deletion in the covR gene of the HH/LC variant. Functionally, this isolate was associated with an increased growth rate in vitro and with higher interleukin-8 induction. However, in whole blood, opsonophagocytic and intracellular killing assays, the LH/HC phenotype demonstrated higher resistance to host phagocytic killing. In a murine model, LH/HC resulted in higher levels of bacteremia and increased host mortality rate. These findings demonstrate differences in GBS isolates of the same clonal origin but varying phenotypes.

Staphylococcus aureus as an intracellular pathogen: the role of small colony variants

Increasing evidence indicates that Staphylococcus aureus might be a facultative intracellular pathogen. In particular, certain subpopulations, called small colony variants (SCVs), seem to be well adapted to the intracellular milieu. When compared to 'normal' staphylococcal strains, SCVs show increased uptake by host cells, resistance to intracellular defenses and reduced stimulation of host defenses. We propose that the ability to form two subpopulations with different phenotypes might allow S. aureus the option for both extra- cellular and intra-cellular survival in the host.

The novel ATP-competitive inhibitor of the MET hepatocyte growth factor receptor EMD1214063 displays inhibitory activity against selected MET-mutated variants

The receptor tyrosine kinase MET is a prime target in clinical oncology due to its aberrant activation and involvement in the pathogenesis of a broad spectrum of malignancies. Similar to other targeted kinases, primary and secondary mutations seem to represent an important resistance mechanism to MET inhibitors. Here, we report the biologic activity of a novel MET inhibitor, EMD1214063, on cells that ectopically express the mutated MET variants M1268T, Y1248H, H1112Y, L1213V, H1112L, V1110I, V1206L, and V1238I. Our results demonstrate a dose-dependent decrease in MET autophosphorylation in response to EMD1214063 in five out of the eight cell lines (IC50 2-43nM). Blockade of MET by EMD1214063 was accompanied by a reduced activation of downstream effectors in cells expressing EMD1214063-sensitive mutants. In all sensitive mutant-expressing lines, EMD1214063 altered cell cycle distribution, primarily with an increase in G1 phase. EMD1214063 strongly influenced MET-driven biological functions, such as cellular morphology, MET-dependent cell motility and anchorage-independent growth. To assess the in vivo efficacy of EMD1214063, we used a xenograft tumor model in immunocompromised mice bearing NIH3T3 cells expressing sensitive and resistant MET mutated variants. Animals were randomized for the treatment with EMD1214063 (50mg/kg/day) or vehicle only. Remarkably, five days of EMD1214063 treatment resulted in a complete regression of the sensitive H1112L-derived tumors, while tumor growth remained unaffected in mice with L1213V tumors and in vehicle-treated animals. Collectively, the current data identifies EMD1214063 as a potent MET small molecule inhibitor with selective activity towards mutated MET variants.

Understanding the role of argininosuccinate lyase transcript variants in the clinical and biochemical variability of the urea cycle disorder argininosuccinic aciduria

Argininosuccinic aciduria (ASA) is an autosomal recessive urea cycle disorder caused by deficiency of argininosuccinate lyase (ASL) with a wide clinical spectrum from asymptomatic to severe hyperammonemic neonatal onset life-threatening courses. We investigated the role of ASL transcript variants in the clinical and biochemical variability of ASA. Recombinant proteins for ASL wild type, mutant p.E189G, and the frequently occurring transcript variants with exon 2 or 7 deletions were (co-)expressed in human embryonic kidney 293T cells. We found that exon 2-deleted ASL forms a stable truncated protein with no relevant activity but a dose-dependent dominant negative effect on enzymatic activity after co-expression with wild type or mutant ASL, whereas exon 7-deleted ASL is unstable but seems to have, nevertheless, a dominant negative effect on mutant ASL. These findings were supported by structural modeling predictions for ASL heterotetramer/homotetramer formation. Illustrating the physiological relevance, the predominant occurrence of exon 7-deleted ASL was found in two patients who were both heterozygous for the ASL mutant p.E189G. Our results suggest that ASL transcripts can contribute to the highly variable phenotype in ASA patients if expressed at high levels. Especially, the exon 2-deleted ASL variant may form a heterotetramer with wild type or mutant ASL, causing markedly reduced ASL activity.

Characterization of 2 genetic variants of Na(v) 1.5-arginine 689 found in patients with cardiac arrhythmias

Hundreds of genetic variants in SCN5A, the gene coding for the pore-forming subunit of the cardiac sodium channel, Na(v) 1.5, have been described in patients with cardiac channelopathies as well as in individuals from control cohorts. The aim of this study was to characterize the biophysical properties of 2 naturally occurring Na(v) 1.5 variants, p.R689H and p.R689C, found in patients with cardiac arrhythmias and in control individuals. In addition, this study was motivated by the finding of the variant p.R689H in a family with sudden cardiac death (SCD) in children. When expressed in HEK293 cells, most of the sodium current (I(Na)) biophysical properties of both variants were indistinguishable from the wild-type (WT) channels. In both cases, however, an ∼2-fold increase of the tetrodotoxin-sensitive late I(Na) was observed. Action potential simulations and reconstruction of pseudo-ECGs demonstrated that such a subtle increase in the late I(Na) may prolong the QT interval in a nonlinear fashion. In conclusion, despite the fact that the causality link between p.R689H and the phenotype of the studied family cannot be demonstrated, this study supports the notion that subtle alterations of Na(v) 1.5 variants may increase the risk for cardiac arrhythmias.

Novel variants in the KIT and PAX3 genes in horses with white-spotted coat colour phenotypes.

Variants in the EDNRB, KIT, MITF, PAX3 and TRPM1 genes are known to cause white spotting phenotypes in horses, which can range from the common white markings up to completely white horses. In this study, we investigated these candidate genes in 169 horses with white spotting phenotypes not explained by the previously described variants. We identified a novel missense variant, PAX3:p.Pro32Arg, in Appaloosa horses with a splashed white phenotype in addition to their leopard complex spotting patterns. We also found three novel variants in the KIT gene. The splice site variant c.1346+1G>A occurred in a Swiss Warmblood horse with a pronounced depigmentation phenotype. The missense variant p.Tyr441Cys was present in several part-bred Arabians with sabino-like depigmentation phenotypes. Finally, we provide evidence suggesting that the common and widely distributed KIT:p.Arg682His variant has a very subtle white-increasing effect, which is much less pronounced than the effect of the other described KIT variants. We termed the new KIT variants W18-W20 to provide a simple and unambiguous nomenclature for future genetic testing applications.

DNA-based identification of novel bovine casein gene variants

In cattle, at least 39 variants of the 4 casein proteins (α(S1)-, β-, α(S2)- and κ-casein) have been described to date. Many of these variants are known to affect milk-production traits, cheese-processing properties, and the nutritive value of milk. They also provide valuable information for phylogenetic studies. So far, the majority of studies exploring the genetic variability of bovine caseins considered European taurine cattle breeds and were carried out at the protein level by electrophoretic techniques. This only allows the identification of variants that, due to amino acid exchanges, differ in their electric charge, molecular weight, or isoelectric point. In this study, the open reading frames of the casein genes CSN1S1, CSN2, CSN1S2, and CSN3 of 356 animals belonging to 14 taurine and 3 indicine cattle breeds were sequenced. With this approach, we identified 23 alleles, including 5 new DNA sequence variants, with a predicted effect on the protein sequence. The new variants were only found in indicine breeds and in one local Iranian breed, which has been phenotypically classified as a taurine breed. A multidimensional scaling approach based on available SNP chip data, however, revealed an admixture of taurine and indicine populations in this breed as well as in the local Iranian breed Golpayegani. Specific indicine casein alleles were also identified in a few European taurine breeds, indicating the introgression of indicine breeds into these populations. This study shows the existence of substantial undiscovered genetic variability of bovine casein loci, especially in indicine cattle breeds. The identification of new variants is a valuable tool for phylogenetic studies and investigations into the evolution of the milk protein genes.

Origin of Minority Drug-Resistant HIV-1 Variants in Primary HIV-1 Infection

Background. Drug-resistant human immunodeficiency virus type 1 (HIV-1) minority variants (MVs) are present in some antiretroviral therapy (ART)–naive patients. They may result from de novo mutagenesis or transmission. To date, the latter has not been proven. Methods. MVs were quantified by allele-specific polymerase chain reaction in 204 acute or recent seroconverters from the Zurich Primary HIV Infection study and 382 ART-naive, chronically infected patients. Phylogenetic analyses identified transmission clusters. Results. Three lines of evidence were observed in support of transmission of MVs. First, potential transmitters were identified for 12 of 16 acute or recent seroconverters harboring M184V MVs. These variants were also detected in plasma and/or peripheral blood mononuclear cells at the estimated time of transmission in 3 of 4 potential transmitters who experienced virological failure accompanied by the selection of the M184V mutation before transmission. Second, prevalence between MVs harboring the frequent mutation M184V and the particularly uncommon integrase mutation N155H differed highly significantly in acute or recent seroconverters (8.2% vs 0.5%; P < .001). Third, the prevalence of less-fit M184V MVs is significantly higher in acutely or recently than in chronically HIV-1–infected patients (8.2% vs 2.5%; P = .004). Conclusions. Drug-resistant HIV-1 MVs can be transmitted. To what extent the origin—transmission vs sporadic appearance—of these variants determines their impact on ART needs to be further explored.

Common noncoding UMOD gene variants induce salt-sensitive hypertension and kidney damage by increasing uromodulin expression

Hypertension and chronic kidney disease (CKD) are complex traits representing major global health problems1,2. Multiple genome-wide association studies have identified common variants in the promoter of the UMOD gene3–9, which encodes uromodulin, the major protein secreted in normal urine, that cause independent susceptibility to CKD and hypertension. Despite compelling genetic evidence for the association between UMOD risk variants and disease susceptibility in the general population, the underlying biological mechanism is not understood. Here, we demonstrate that UMOD risk variants increased UMOD expression in vitro and in vivo. Uromodulin overexpression in transgenic mice led to salt-sensitive hypertension and to the presence of age-dependent renal lesions similar to those observed in elderly individuals homozygous for UMOD promoter risk variants. The link between uromodulin and hypertension is due to activation of the renal sodium cotransporter NKCC2. We demonstrated the relevance of this mechanism in humans by showing that pharmacological inhibition of NKCC2 was more effective in lowering blood pressure in hypertensive patients who are homozygous for UMOD promoter risk variants than in other hypertensive patients. Our findings link genetic susceptibility to hypertension and CKD to the level of uromodulin expression and uromodulin’s effect on salt reabsorption in the kidney. These findings point to uromodulin as a therapeutic target for lowering blood pressure and preserving renal function.