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.

A mutation in the SUV39H2 gene in Labrador Retrievers with hereditary nasal parakeratosis (HNPK) provides insights into the epigenetics of keratinocyte differentiation.

Hereditary nasal parakeratosis (HNPK), an inherited monogenic autosomal recessive skin disorder, leads to crusts and fissures on the nasal planum of Labrador Retrievers. We performed a genome-wide association study (GWAS) using 13 HNPK cases and 23 controls. We obtained a single strong association signal on chromosome 2 (p(raw) = 4.4×10⁻¹⁴). The analysis of shared haplotypes among the 13 cases defined a critical interval of 1.6 Mb with 25 predicted genes. We re-sequenced the genome of one case at 38× coverage and detected 3 non-synonymous variants in the critical interval with respect to the reference genome assembly. We genotyped these variants in larger cohorts of dogs and only one was perfectly associated with the HNPK phenotype in a cohort of more than 500 dogs. This candidate causative variant is a missense variant in the SUV39H2 gene encoding a histone 3 lysine 9 (H3K9) methyltransferase, which mediates chromatin silencing. The variant c.972T>G is predicted to change an evolutionary conserved asparagine into a lysine in the catalytically active domain of the enzyme (p.N324K). We further studied the histopathological alterations in the epidermis in vivo. Our data suggest that the HNPK phenotype is not caused by hyperproliferation, but rather delayed terminal differentiation of keratinocytes. Thus, our data provide evidence that SUV39H2 is involved in the epigenetic regulation of keratinocyte differentiation ensuring proper stratification and tight sealing of the mammalian epidermis.

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.

N-terminal aromatic residues closely impact the cytolytic activity of cupiennin 1a, a major spider venom peptide

Cupiennins are small cationic a-helical peptides from the venom of the ctenid spider Cupiennius salei which are characterized by high bactericidal as well as hemolytic activities. To gain insight into the determinants responsible for the broad cytolytic activities, two analogues of cupiennin 1a with different N-terminal hydrophobicities were designed. The insecticidal, bactericidal and hemolytic activities of these analogues were assayed and compared to the native peptide. Specifically, substitution of two N-terminal Phe residues by Ala results in less pronounced insecticidal and cytolytic activity, whereas a substitution by Lys reduces strongly its bactericidal activity and completely diminishes its hemolytic activity up to very high tested concentrations. Biophysical analyses of peptide/bilayer membrane interactions point to distinct interactions of the analogues with lipid bilayers, and dependence upon membrane surface charge. Indeed, we find that lower hemolytic activity was correlated with less surface association of the analogues. In contrast, our data indicate that the reduced bactericidal activity of the two cupiennin 1a analogues likely correspond to greater bilayer-surface localization of the peptides. Overall, ultimate insertion and destruction of the host cell membrane is highly dependent on the presence of Phe-2 and Phe-6 (Cu 1a) or Leu-6 (Cu 2a) in the N-terminal sequences of native cupiennins.

Mutation analysis of the PVRL1 gene in caucasians with nonsyndromic cleft lip/palate.

Nonsyndromic cleft lip with or without cleft palate (nsCL/P, MIM 119530) is perhaps the most common major birth defect. Homozygous PVRL1 loss-of-function mutations result in an autosomal recessive CL/P syndrome, CLPED1, and a PVRL1 nonsense mutation is associated with sporadic nsCL/P in Northern Venezuela. To address the more general role of PVRL1 variation in risk of nsCL/P, we carried out mutation analysis of PVRL1 in North American and Australian nsCL/P cases and population-matched controls. We identified a total of 15 variants, 5 of which were seen in both populations and 1 of which, an in-frame insertion at Glu442, was more frequent in patients than in controls in both populations, though the difference was not statistically significant. Another variant, which is specific to the PVRL1 beta (HIgR) isoform, S447L, was marginally associated with nsCL/P in North American Caucasian patients, but not in Australian patients, and overall variants that affect the beta-isoform were significantly more frequent among North American patients. One Australian patient had a splice junction mutation of PVRL1. Our results suggest that PVRL1 may play a minor role in susceptibility to the occurrence of nsCL/P in some Caucasian populations, and that variation involving the beta (HIgR) isoform might have particular importance for risk of orofacial clefts. Nevertheless, these results underscore the need for studies that involve very large numbers when assessing the possible role of rare variants in risk of complex traits such as nsCL/P.

The C-terminal domain of MinC inhibits assembly of the Z ring in Escherichia coli.

In Escherichia coli, the Min system, consisting of three proteins, MinC, MinD, and MinE, negatively regulates FtsZ assembly at the cell poles, helping to ensure that the Z ring will assemble only at midcell. Of the three Min proteins, MinC is sufficient to inhibit Z-ring assembly. By binding to MinD, which is mostly localized at the membrane near the cell poles, MinC is sequestered away from the cell midpoint, increasing the probability of Z-ring assembly there. Previously, it has been shown that the two halves of MinC have two distinct functions. The N-terminal half is sufficient for inhibition of FtsZ assembly, whereas the C-terminal half of the protein is required for binding to MinD as well as to a component of the division septum. In this study, we discovered that overproduction of the C-terminal half of MinC (MinC(122-231)) could also inhibit cell division and that this inhibition was at the level of Z-ring disassembly and dependent on MinD. We also found that fusing green fluorescent protein to either the N-terminal end of MinC(122-231), the C terminus of full-length MinC, or the C terminus of MinC(122-231) perturbed MinC function, which may explain why cell division inhibition by MinC(122-231) was not detected previously. These results suggest that the C-terminal half of MinC has an additional function in the regulation of Z-ring assembly.

Syntaxin 3b is a t-SNARE specific for ribbon synapses of the retina.

Previous studies have demonstrated that ribbon synapses in the retina do not contain the t-SNARE (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 1A that is found in conventional synapses of the nervous system. In contrast, ribbon synapses of the retina contain the related isoform syntaxin 3. In addition to its localization in ribbon synapses, syntaxin 3 is also found in nonneuronal cells, where it has been implicated in the trafficking of transport vesicles to the apical plasma membrane of polarized cells. The syntaxin 3 gene codes for four different splice forms, syntaxins 3A, 3B, 3C, and 3D. We demonstrate here by using analysis of EST databases, RT-PCR, in situ hybridization, and Northern blot analysis that cells in the mouse retina express only syntaxin 3B. In contrast, nonneuronal tissues, such as kidney, express only syntaxin 3A. The two major syntaxin isoforms (3A and 3B) have an identical N-terminal domain but differ in the C-terminal half of the SNARE domain and the C-terminal transmembrane domain. These two domains are thought to be directly involved in synaptic vesicle fusion. The interaction of syntaxin 1A and syntaxin 3B with other synaptic proteins was examined. We found that both proteins bind Munc18/N-sec1 with similar affinity. In contrast, syntaxin 3B had a much lower binding affinity for the t-SNARE SNAP25 compared with syntaxin 1A. By using an in vitro fusion assay, we could demonstrate that vesicles containing syntaxin 3B and SNAP25 could fuse with vesicles containing synaptobrevin2/VAMP2, demonstrating that syntaxin 3B can function as a t-SNARE.

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.

Higher frequency of genetic variants conferring increased risk for ADRs for commonly used drugs treating cancer, AIDS and tuberculosis in persons of African descent

There is established clinical evidence for differences in drug response, cure rates and survival outcomes between different ethnic populations, but the causes are poorly understood. Differences in frequencies of functional genetic variants in key drug response and metabolism genes may significantly influence drug response differences in different populations. To assess this, we genotyped 1330 individuals of African (n=372) and European (n=958) descent for 4535 single-nucleotide polymorphisms in 350 key drug absorption, distribution, metabolism, elimination and toxicity genes. Important and remarkable differences in the distribution of genetic variants were observed between Africans and Europeans and among the African populations. These could translate into significant differences in drug efficacy and safety profiles, and also in the required dose to achieve the desired therapeutic effect in different populations. Our data points to the need for population-specific genetic variation in personalizing medicine and care.

Generation and characterization of antigenic variants induced by exposure of tumor cells to UV radiation in vitro

Antigenic changes present in nonantigenic tumor cells exposed to UV radiation (UV) in vitro were investigated by addressing the following questions: (1) Are antigenic variants (AV) produced that are rejected in normal but not immunosuppressed mice? (2) Does generation of AV depend upon intrinsic properties of the cells exposed or result from the action of UV? (3) Is antigenic modification induced by UV due to increased histocompatibility antigen expression? (4) Do AV crossreact immunologically with parental tumor or with other AV? and (5) Is the UV-associated common antigen expressed on UV-induced tumors present on UV-irradiated tumor cells? AV were generated at different frequencies following in vitro UV irradiation of a spontaneous murine fibrosarcoma (51% of cell lines tested), a murine melanoma (56%), and two melanoma clones (100% and 11%). This indicated that the percentage of AV produced is an intrinsic property of the cell line exposed. The increased antigenicity did not correlate with an increased expression of class I histocompatibility antigens. Immunological experiments demonstrated that the AV and parental cells shared a determinant that was susceptible to immune recognition, but incapable of inducing immunity. In contrast, the AV were noncrossreactive, suggesting that variant-specific antigens were also expressed. Finally, the AV were recognized by UV-induced suppressor cells, indicating that the UV-associated common antigen expressed by UV-induced tumors was also present. This investigation provides new information on the susceptibility of tumors to antigenic modification by UV and on the relationship between tumor antigens and neoplastic transformation. Furthermore, it suggests an immunological approach for cancer therapy. ^

Immune response to regressor and progressor tumor variants

Most skin cancers induced in mice by Ultraviolet (UV) radiation express highly immunogenic Tumor specific transplantation antigens (TSTAs) and thus exhibit a regressor phenotype. In this study, I have used cloned genes encoding tumor antigens and oncogenes in conjunction with DNA transfection technique to isolate and characterize regressor variants from progressor tumors and vice versa. The purpose of this study was (1) to determine whether the product of a cloned gene (216) from UV-1591 tumor, which encodes a novel MHC class I antigen can function as a tumor rejection antigen when expressed on unrelated, nonantigenic, murine tumor cells or whether its function is restricted to UV-induced tumors, and (2) to determine the processes by which progressor variants derived from a regressor UV-2240 cell line by transfection with an activated Ha-ras oncogene escape the immune defenses of the normal immunocompetent host.^ To answer the first question, a spontaneously transformed, nonimmunogenic cell line (10T-1) was cotransfected with DNA from p216 and pSV2-neo plasmids. Results demonstrate that the product of a cloned TSTA gene from a UV-induced murine tumor is capable of functioning as a tumor rejection antigen when expressed on unrelated, nonantigenic tumor cells. In addition, these results indicate that this approach could be used to augment the immune response against poorly antigenic tumors.^ To answer the second question, progressor variants were isolated from a highly antigenic UV radiation-induced C3H mouse regressor fibrosarcoma cell line, UV-2240, by transfection with an activated Ha-ras oncogene. Subcutaneous injection of Ha-ras-transfected UV-2240 cells into immunocompetent C3H mice produced tumors in 4 of 36 animals. In addition, the Ha-ras-induced progressor variants produced experimental lung metastasis in both normal C3H and nude mice, although they induced more lung nodules in nude mice than in normal C3H mice. Results indicate that the progressor phenotype of the Ha-ras-induced tumor variants is not due to loss of TSTAs or MHC class I antigens. This implies that some tumors can escape the immune defenses of the normal immunocompetent host by mechanisms other than the loss of TSTAs and MHC class I antigens. (Abstract shortened with permission of author.) ^

A molecular study of the rhodopsin locus in patients with retinitis pigmentosa

DNA for this study was collected from a sample of 133 retinitis pigmentosa (RP) patients and the rhodopsin locus molecularly analyzed by linkage and for disease specific mutations. The cohort of patients consisted of 85 individuals diagnosed with autosomal dominant RP (adRP), and 48 patients representing other forms of retinitis pigmentosa or retinal dystrophy related disease. In three large families with adRP rhodopsin was excluded from linkage to the disease locus. A search for subtle mutations in the rhodopsin coding region using single strand conformational polymorphisms (SSCP) and sequencing detected a total of 14 unique sequence variants in 24 unrelated patients. These variants included one splicing variant, 5168 -1G-A, one deletion variant of 17 base pairs causing a frame shift at codon 332, and 12 misense variants: Pro23His, Leu46Arg, Gly106Trp, Arg135Pro, Pro171Glu, Pro180Ala, Glu181Lys, Asp190Asn, His211Arg, Ser270Arg, Leu328Pro and Pro347Thr. All but three of the missense variants change amino acids that are evolutionarily conserved. The Pro23His mutation was found in 10 unrelated individuals with family histories of adRP and not in any normal controls (over 80 chromosomes tested). The Pro180Ala mutation was present in a patient with simplex RP and probably represents a new mutation. Three normal polymorphic nucleotide substitutions, A-269-G, T-3982-C, and G-5145-A, were also identified. We conclude, based on this study, that 25% of adRP cases are attributable to rhodopsin mutations.^ Clinical data, including ERG results and visual field testing, was available for patients with eleven different mutations. The eleven patients were all diagnosed with RP, however the severity of the disease varied with five patients mildly affected and diagnosed with type II adRP and 5 patients severely affected and diagnosed with type I adRP. The patient with simplex RP was mildly affected. The location of the mutations within the rhodopsin protein was randomly associated with the severity of the disease in those patients evaluated. However, four mutations, Pro23His, Leu46Arg, Pro347Thr, and 5168 -1G-A, are particularly interesting. The Pro23His mutation appears to have radiated from a recent common ancestor of the affected patients as all of them share a common haplotype at the rhodopsin locus. The Leu46Arg mutation causes an unusually severe form of RP. Hydropathy analysis of the mutated sequence revealed a marked change in the hydrophobicity of this first transmembrane spanning region. Codon 347 has been the target of multiple mutations with at least six documented changes at the position, significantly more than expected by a random distribution of mutations. Finally the splice-site variant is extremely variable in its expression in the family studied. Similar mutations have been reported in other cases of adRP and postulated to be involved in autosomal recessive RP (arRP). Mechanisms to account for the variable expression of rhodopsin mutations in relation to RP heterogeneity are discussed. (Abstract shortened by UMI.) ^

Transcriptional regulation and functional analysis of the Wilms' tumor gene WT1

The Wilms' tumor gene, WT1, encodes a zinc finger transcription factor which functions as a tumor suppressor. Defects in the WT1 gene can result in the development of nephroblastoma. WT1 is expressed during development, primarily in the metanephric kidney, the mesothelial lining of the abdomen and thorax, and the developing gonads. WT1 expression is tightly regulated and is essential for renal development. The WT1 gene encodes a protein with a proline-rich N-terminus which functions as a transcriptional repressor and C-terminus contains 4 zinc fingers that mediate DNA binding. WT1 represses transcription from a number of growth factors and growth factor receptors. WT1 mRNA undergoes alternative splicing at two sites, resulting in 4 mRNA species and polypeptide products. Exon 5, encoding 17 amino acids is alternatively spliced, and is located between the transcriptional repression domain and the DNA binding domain. The second alternative splice is the terminal 9 nucleotides of zinc finger 3, encoding the tripeptide Lys-Thr-Ser (KTS). The presence or absence of KTS within the zinc fingers of WT1 alters DNA binding.^ I have investigated transcriptional regulation of WT1, characterizing two means of repressing WT1 transcription. I have cloned a transcriptional silencer of the WT1 promoter which is located in the third intron of the WT1 gene. The silencer is 460 bp in length and contains an Alu repeat. The silencer functions in cells of non-renal origin.^ I have found that WT1 protein can autoregulate the WT1 promoter. Using the autoregulation of the WT1 promoter as a functional assay, I have defined differential consensus DNA binding motifs of WT1 isoforms lacking and containing the KTS tripeptide insertion. With these refined consensus DNA binding motifs, I have identified two additional targets of WT1 transcriptional repression, the proto-oncogenes bcl-2 and c-myc.^ I have investigated the ability of the alternatively spliced exon 5 to influence cell growth. In cell proliferation assays, isoforms of WT1 lacking exon 5 repress cell growth. WT1 isoforms containing exon 5 fail to repress cell growth to the same extent, but alter the morphology of the cells. These experiments demonstrate that the alternative splice isoforms of WT1 have differential effects on the function of WT1. These findings suggest a role for the alternative splicing of WT1 in metanephric development. ^