Function of liver activation-regulated chemokine/CC chemokine ligand 20 is differently affected by cathepsin B and cathepsin D processing

Chemokine processing by proteases is emerging as an important regulatory mechanism of leukocyte functions and possibly also of cancer progression. We screened a large panel of chemokines for degradation by cathepsins B and D, two proteases involved in tumor progression. Among the few substrates processed by both proteases, we focused on CCL20, the unique chemokine ligand of CCR6 that is expressed on immature dendritic cells and subtypes of memory lymphocytes. Analysis of the cleavage sites demonstrate that cathepsin B specifically cleaves off four C-terminally located amino acids and generates a CCL20(1-66) isoform with full functional activity. By contrast, cathepsin D totally inactivates the chemotactic potency of CCL20 by generating CCL20(1-55), CCL20(1-52), and a 12-aa C-terminal peptide CCL20(59-70). Proteolytic cleavage of CCL20 occurs also with chemokine bound to glycosaminoglycans. In addition, we characterized human melanoma cells as a novel CCL20 source and as cathepsin producers. CCL20 production was up-regulated by IL-1alpha and TNF-alpha in all cell lines tested, and in human metastatic melanoma cells. Whereas cathepsin D is secreted in the extracellular milieu, cathepsin B activity is confined to cytosol and cellular membranes. Our studies suggest that CCL20 processing in the extracellular environment of melanoma cells is exclusively mediated by cathepsin D. Thus, we propose a model where cathepsin D inactivates CCL20 and possibly prevents the establishment of an effective antitumoral immune response in melanomas.

Basistests Suisse Sport Test Konzept: Validierung einer sportmotorischen Basistestbatterie für den Schul- und Nachwuchssport

Physical fitness can be evaluated in competitive and school sports with different field tests under different conditions and goals. To produce valid results, a field test must be practical and reach high standards of test criteria (objectivity, reliability, validity). The purpose of this study was to investigate the test criteria and the practicability of a group of field tests called «SUISSE Sport Test Konzept Basis Feldtestbatterie». For 20-m sprint, ventral trunk muscle test, standing long jump, 2-kg medicine ball shot, obstacle course and cooper-test, test quality and practicability were evaluated. 221 children and adolescents from competitive sports and different school levels took part in the study. According to school level, they were divided into 3 groups (P: 7–11.5 y, S1: 11.6–15.5 y, S2: 15.6–21.8 y). Objectivity was tested for time or distance measurement in all tests as well as for error rating in obstacle test. For reliability measurement, 162 subjects performed the field tests twice within a few weeks. For validity results of standing long jump were compared with counter movement jump performance on a force plate. Correlation analysis was performed and level of significance was set for p < 0.05. For accuracy standard error was calculated. All tests achieved sufficient to excellent objectiv - ity with correlation-coefficient (r) lying between 0.85 and 0.99. Reliability was very good (r = 0.84–0.97). In cooper- and trunk test, reliability was higher for athletes than for pupils (trunk test: r = 0.95 vs. r = 0.62, cooper-test: r = 0.90 vs. r = 0.78). In those tests the reliability decreases with increasing age (cooper-test: P: r = 0.84, S1: r = 0.69, S2: r = 0.52; trunk-test: P: r = 0.69, S1: r = 0.71; S2: r = 0.39). Validity for standing long jump was good (r = 0.75–0.86). The standard error of the mean was between 4–8%, with the exception for cooper-test (athletes: 6%, pupils: 11%) and trunk test (athletes: 14%, pupils: 46%). The results show that the evaluated group of field tests is a practicable, objective and reliable tool to determine physical skills in young athletes as well as in a scholar setting over a broad age range. Most of the tests achieved the test criteria with the grades good to excellent. The lower coefficient of reliability for cooper- and trunk test by the pupils could be explained by motivational problems in this setting. For up to 20 subjects, a tester can accomplish the tests within 3 h. Finally, age-dependent grades were elaborated

Biochemical typing of pathological prion protein in aging cattle with BSE

BACKGROUND: The broad enforcement of active surveillance for bovine spongiform encephalopathy (BSE) in 2000 led to the discovery of previously unnoticed, atypical BSE phenotypes in aged cattle that differed from classical BSE (C-type) in biochemical properties of the pathological prion protein. Depending on the molecular mass and the degree of glycosylation of its proteinase K resistant core fragment (PrPres), mainly determined in samples derived from the medulla oblongata, these atypical cases are currently classified into low (L)-type or high (H)-type BSE. In the present study we address the question to what extent such atypical BSE cases are part of the BSE epidemic in Switzerland. RESULTS: To this end we analyzed the biochemical PrPres type by Western blot in a total of 33 BSE cases in cattle with a minimum age of eight years, targeting up to ten different brain regions. Our work confirmed H-type BSE in a zebu but classified all other cases as C-type BSE; indicating a very low incidence of H- and L-type BSE in Switzerland. It was documented for the first time that the biochemical PrPres type was consistent across different brain regions of aging animals with C-type and H-type BSE, i.e. independent of the neuroanatomical structure investigated. CONCLUSION: Taken together this study provides further characteristics of the BSE epidemic in Switzerland and generates new baseline data for the definition of C- and H-type BSE phenotypes, thereby underpinning the notion that they indeed represent distinct prion disease entities.

Spectrum and prevalence of mutations involving BrS1- through BrS12-susceptibility genes in a cohort of unrelated patients referred for Brugada syndrome genetic testing: implications for genetic testing

OBJECTIVES The aim of this study was to provide the spectrum and prevalence of mutations in the 12 Brugada syndrome (BrS)-susceptibility genes discovered to date in a single large cohort of unrelated BrS patients. BACKGROUND BrS is a potentially lethal heritable arrhythmia syndrome diagnosed electrocardiographically by coved-type ST-segment elevation in the right precordial leads (V1 to V3; type 1 Brugada electrocardiographic [ECG] pattern) and the presence of a personal/family history of cardiac events. METHODS Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, comprehensive mutational analysis of BrS1- through BrS12-susceptibility genes was performed in 129 unrelated patients with possible/probable BrS (46 with clinically diagnosed BrS [ECG pattern plus personal/family history of a cardiac event] and 83 with a type 1 BrS ECG pattern only). RESULTS Overall, 27 patients (21%) had a putative pathogenic mutation, absent in 1,400 Caucasian reference alleles, including 21 patients with an SCN5A mutation, 2 with a CACNB2B mutation, and 1 each with a KCNJ8 mutation, a KCND3 mutation, an SCN1Bb mutation, and an HCN4 mutation. The overall mutation yield was 23% in the type 1 BrS ECG pattern-only patients versus 17% in the clinically diagnosed BrS patients and was significantly greater among young men<20 years of age with clinically diagnosed BrS and among patients who had a prolonged PQ interval. CONCLUSIONS We identified putative pathogenic mutations in ∼20% of our BrS cohort, with BrS genes 2 through 12 accounting for <5%. Importantly, the yield was similar between patients with only a type 1 BrS ECG pattern and those with clinically established BrS. The yield approaches 40% for SCN5A-mediated BrS (BrS1) when the PQ interval exceeds 200 ms. Calcium channel-mediated BrS is extremely unlikely in the absence of a short QT interval.

Cardiac channel molecular autopsy: insights from 173 consecutive cases of autopsy-negative sudden unexplained death referred for postmortem genetic testing

OBJECTIVE To perform long QT syndrome and catecholaminergic polymorphic ventricular tachycardia cardiac channel postmortem genetic testing (molecular autopsy) for a large cohort of cases of autopsy-negative sudden unexplained death (SUD). METHODS From September 1, 1998, through October 31, 2010, 173 cases of SUD (106 males; mean ± SD age, 18.4 ± 12.9 years; age range, 1-69 years; 89% white) were referred by medical examiners or coroners for a cardiac channel molecular autopsy. Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, a comprehensive mutational analysis of the long QT syndrome susceptibility genes (KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2) and a targeted analysis of the catecholaminergic polymorphic ventricular tachycardia type 1-associated gene (RYR2) were conducted. RESULTS Overall, 45 putative pathogenic mutations absent in 400 to 700 controls were identified in 45 autopsy-negative SUD cases (26.0%). Females had a higher yield (26/67 [38.8%]) than males (19/106 [17.9%]; P<.005). Among SUD cases with exercise-induced death, the yield trended higher among the 1- to 10-year-olds (8/12 [66.7%]) compared with the 11- to 20-year-olds (4/27 [14.8%]; P=.002). In contrast, for those who died during a period of sleep, the 11- to 20-year-olds had a higher yield (9/25 [36.0%]) than the 1- to 10-year-olds (1/24 [4.2%]; P=.01). CONCLUSION Cardiac channel molecular autopsy should be considered in the evaluation of autopsy-negative SUD. Several interesting genotype-phenotype observations may provide insight into the expected yields of postmortem genetic testing for SUD and assist in selecting cases with the greatest potential for mutation discovery and directing genetic testing efforts.

A novel rare variant in SCN1Bb linked to Brugada syndrome and SIDS by combined modulation of Na(v)1.5 and K(v)4.3 channel currents.

BACKGROUND Cardiac sodium channel β-subunit mutations have been associated with several inherited cardiac arrhythmia syndromes. OBJECTIVE To identify and characterize variations in SCN1Bb associated with Brugada syndrome (BrS) and sudden infant death syndrome (SIDS). METHODS All known exons and intron borders of the BrS-susceptibility genes were amplified and sequenced in both directions. Wild type (WT) and mutant genes were expressed in TSA201 cells and studied using co-immunoprecipitation and whole-cell patch-clamp techniques. RESULTS Patient 1 was a 44-year-old man with an ajmaline-induced type 1 ST-segment elevation in V1 and V2 supporting the diagnosis of BrS. Patient 2 was a 62-year-old woman displaying a coved-type BrS electrocardiogram who developed cardiac arrest during fever. Patient 3 was a 4-month-old female SIDS case. A R214Q variant was detected in exon 3A of SCN1Bb (Na(v)1B) in all three probands, but not in any other gene previously associated with BrS or SIDS. R214Q was identified in 4 of 807 ethnically-matched healthy controls (0.50%). Co-expression of SCN5A/WT + SCN1Bb/R214Q resulted in peak sodium channel current (I(Na)) 56.5% smaller compared to SCN5A/WT + SCN1Bb/WT (n = 11-12, P<0.05). Co-expression of KCND3/WT + SCN1Bb/R214Q induced a Kv4.3 current (transient outward potassium current, I(to)) 70.6% greater compared with KCND3/WT + SCN1Bb/WT (n = 10-11, P<0.01). Co-immunoprecipitation indicated structural association between Na(v)β1B and Na(v)1.5 and K(v)4.3. CONCLUSION Our results suggest that R214Q variation in SCN1Bb is a functional polymorphism that may serve as a modifier of the substrate responsible for BrS or SIDS phenotypes via a combined loss of function of sodium channel current and gain of function of transient outward potassium current.

LQTS-associated mutation A257G in α1-syntrophin interacts with the intragenic variant P74L to modify its biophysical phenotype.

The SNTA1-encoded α1-syntrophin (SNTA1) missense mutation, p.A257G, causes long QT syndrome (LQTS) by pathogenic accentuation of Nav1.5's sodium current (I Na). Subsequently, we found p.A257G in combination with the SNTA1 polymorphism, p.P74L in 4 victims of sudden infant death syndrome (SIDS) as well as in 3 adult controls. We hypothesized that p.P74L-SNTA1 could functionally modify the pathogenic phenotype of p.A257G-SNTA1, thus explaining its occurrence in non-LQTS populations. The SNTA1 variants p.P74L, p.A257G, and the combination variant p.P74L/p.A257G were engineered using PCR-based overlap-extension and were co-expressed heterologously with SCN5A in HEK293 cells. I Na was recorded using the whole-cell method. Compared to wild-type (WT), the significant increase in peak I Na and window current found with p.A257G was reversed by the intragenic variant p.P74L (p.P74L/p.A257G). These results report for the first time the intragenic rescue of an LQT-associated SNTA1 mutation when found in combination with the SNTA1 polymorphism p.P74L, suggesting an ever-increasing picture of complexity in terms of genetic risk stratification for arrhythmia.

Unexplained drownings and the cardiac channelopathies: a molecular autopsy series.

OBJECTIVE To determine the prevalence and spectrum of mutations associated with long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT) in a seemingly unexplained drowning cohort. PATIENTS AND METHODS From September 1, 1998, through October 31, 2010, 35 unexplained drowning victims (23 male and 12 female; mean ± SD age, 17±12 years [range, 4-69 years]) were referred for a cardiac channel molecular autopsy. Of these, 28 (20 male and 8 female) drowned while swimming, and 7 (3 male and 4 female) were bathtub submersions. Polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing were used for a comprehensive mutational analysis of the 3 major LQTS-susceptibility genes (KCNQ1, KCNH2, and SCN5A), and a targeted analysis of the CPVT1-associated, RYR2-encoded cardiac ryanodine receptor was conducted. RESULTS Of the 28 victims of swimming-related drowning, 8 (28.6%) were mutation positive, including 2 with KCNQ1 mutations (L273F, AAPdel71-73 plus V524G) and 6 with RYR2 mutations (R414C, I419F, R1013Q, V2321A, R2401H, and V2475F). None of the bathtub victims were mutation positive. Of the 28 victims who drowned while swimming, women were more likely to be mutation positive than men (5/8 [62.5%] vs 3/20 [15%]; P=.02). Although none of the mutation-positive, swimming-related drowning victims had a premortem diagnosis of LQTS or CPVT, a family history of cardiac arrest, family history of prior drowning, or QT prolongation was present in 50%. CONCLUSION Nearly 30% of the victims of swimming-related drowning hosted a cardiac channel mutation. Genetic testing should be considered in the postmortem evaluation of an unexplained drowning, especially if a positive personal or family history is elicited.

Loss-of-function mutations in the KCNJ8-encoded Kir6.1 K(ATP) channel and sudden infant death syndrome.

BACKGROUND Approximately 10% of sudden infant death syndrome (SIDS) may stem from cardiac channelopathies. The KCNJ8-encoded Kir6.1 (K(ATP)) channel critically regulates vascular tone and cardiac adaptive response to systemic metabolic stressors, including sepsis. KCNJ8-deficient mice are prone to premature sudden death, particularly with infection. We determined the spectrum, prevalence, and function of KCNJ8 mutations in a large SIDS cohort. METHODS AND RESULTS Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, comprehensive open reading frame/splice-site mutational analysis of KCNJ8 was performed on genomic DNA isolated from necropsy tissue on 292 unrelated SIDS cases (178 males, 204 white; age, 2.9±1.9 months). KCNJ8 mutations were coexpressed heterologously with SUR2A in COS-1 cells and characterized using whole-cell patch-clamp. Two novel KCNJ8 mutations were identified. A 5-month-old white male had an in-frame deletion (E332del) and a 2-month-old black female had a missense mutation (V346I). Both mutations localized to Kir6.1's C-terminus, involved conserved residues and were absent in 400 and 200 ethnic-matched reference alleles respectively. Both cases were negative for mutations in established channelopathic genes. Compared with WT, the pinacidil-activated K(ATP) current was decreased 45% to 68% for Kir6.1-E332del and 40% to 57% for V346I between -20 mV and 40 mV. CONCLUSIONS Molecular and functional evidence implicated loss-of-function KCNJ8 mutations as a novel pathogenic mechanism in SIDS, possibly by predisposition of a maladaptive cardiac response to systemic metabolic stressors akin to the mouse models of KCNJ8 deficiency.

Gain-of-function mutation S422L in the KCNJ8-encoded cardiac K(ATP) channel Kir6.1 as a pathogenic substrate for J-wave syndromes.

BACKGROUND J-wave syndromes have emerged conceptually to encompass the pleiotropic expression of J-point abnormalities including Brugada syndrome (BrS) and early repolarization syndrome (ERS). KCNJ8, which encodes the cardiac K(ATP) Kir6.1 channel, recently has been implicated in ERS following identification of the functionally uncharacterized missense mutation S422L. OBJECTIVE The purpose of this study was to further explore KCNJ8 as a novel susceptibility gene for J-wave syndromes. METHODS Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, comprehensive open reading frame/splice site mutational analysis of KCNJ8 was performed in 101 unrelated patients with J-wave syndromes, including 87 with BrS and 14 with ERS. Six hundred healthy individuals were examined to assess the allelic frequency for all variants detected. KCNJ8 mutation(s) was engineered by site-directed mutagenesis and coexpressed heterologously with SUR2A in COS-1 cells. Ion currents were recorded using whole-cell configuration of the patch-clamp technique. RESULTS One BrS case and one ERS case hosted the identical missense mutation S422L, which was reported previously. KCNJ8-S422L involves a highly conserved residue and was absent in 1,200 reference alleles. Both cases were negative for mutations in all known BrS and ERS susceptibility genes. K(ATP) current of the Kir6.1-S422L mutation was increased significantly over the voltage range from 0 to 40 mV compared to Kir6.1-WT channels (n = 16-21; P <.05). CONCLUSION These findings further implicate KCNJ8 as a novel J-wave syndrome susceptibility gene and a marked gain of function in the cardiac K(ATP) Kir6.1 channel secondary to KCNJ8-S422L as a novel pathogenic mechanism for the phenotypic expression of both BrS and ERS.

Sudden infant death syndrome-associated mutations in the sodium channel beta subunits.

BACKGROUND Approximately 10% of sudden infant death syndrome (SIDS) cases may stem from potentially lethal cardiac channelopathies, with approximately half of channelopathic SIDS involving the Na(V)1.5 cardiac sodium channel. Recently, Na(V) beta subunits have been implicated in various cardiac arrhythmias. Thus, the 4 genes encoding Na(V) beta subunits represent plausible candidate genes for SIDS. OBJECTIVE This study sought to determine the spectrum, prevalence, and functional consequences of sodium channel beta-subunit mutations in a SIDS cohort. METHODS In this institutional review board-approved study, mutational analysis of the 4 beta-subunit genes, SCN1B to 4B, was performed using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing of DNA derived from 292 SIDS cases. Engineered mutations were coexpressed with SCN5A in HEK 293 cells and were whole-cell patch clamped. One of the putative SIDS-associated mutations was similarly studied in adenovirally transduced adult rat ventricular myocytes. RESULTS Three rare (absent in 200 to 800 reference alleles) missense mutations (beta3-V36M, beta3-V54G, and beta4-S206L) were identified in 3 of 292 SIDS cases. Compared with SCN5A+beta3-WT, beta3-V36M significantly decreased peak I(Na) and increased late I(Na), whereas beta3-V54G resulted in a marked loss of function. beta4-S206L accentuated late I(Na) and positively shifted the midpoint of inactivation compared with SCN5A+beta4-WT. In native cardiomyocytes, beta4-S206L accentuated late I(Na) and increased the ventricular action potential duration compared with beta4-WT. CONCLUSION This study provides the first molecular and functional evidence to implicate the Na(V) beta subunits in SIDS pathogenesis. Altered Na(V)1.5 sodium channel function due to beta-subunit mutations may account for the molecular pathogenic mechanism underlying approximately 1% of SIDS cases.

Alpha1-syntrophin mutations identified in sudden infant death syndrome cause an increase in late cardiac sodium current.

BACKGROUND Sudden infant death syndrome (SIDS) is a leading cause of death during the first 6 months after birth. About 5% to 10% of SIDS may stem from cardiac channelopathies such as long-QT syndrome. We recently implicated mutations in alpha1-syntrophin (SNTA1) as a novel cause of long-QT syndrome, whereby mutant SNTA1 released inhibition of associated neuronal nitric oxide synthase by the plasma membrane Ca-ATPase PMCA4b, causing increased peak and late sodium current (I(Na)) via S-nitrosylation of the cardiac sodium channel. This study determined the prevalence and functional properties of SIDS-associated SNTA1 mutations. METHODS AND RESULTS Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing of SNTA1's open reading frame, 6 rare (absent in 800 reference alleles) missense mutations (G54R, P56S, T262P, S287R, T372M, and G460S) were identified in 8 (approximately 3%) of 292 SIDS cases. These mutations were engineered using polymerase chain reaction-based overlap extension and were coexpressed heterologously with SCN5A, neuronal nitric oxide synthase, and PMCA4b in HEK293 cells. I(Na) was recorded using the whole-cell method. A significant 1.4- to 1.5-fold increase in peak I(Na) and 2.3- to 2.7-fold increase in late I(Na) compared with controls was evident for S287R-, T372M-, and G460S-SNTA1 and was reversed by a neuronal nitric oxide synthase inhibitor. These 3 mutations also caused a significant depolarizing shift in channel inactivation, thereby increasing the overlap of the activation and inactivation curves to increase window current. CONCLUSIONS Abnormal biophysical phenotypes implicate mutations in SNTA1 as a novel pathogenic mechanism for the subset of channelopathic SIDS. Functional studies are essential to distinguish pathogenic perturbations in channel interacting proteins such as alpha1-syntrophin from similarly rare but innocuous ones.

The RYR2-encoded ryanodine receptor/calcium release channel in patients diagnosed previously with either catecholaminergic polymorphic ventricular tachycardia or genotype negative, exercise-induced long QT syndrome: a comprehensive open reading frame mutational analysis.

OBJECTIVES This study was undertaken to determine the spectrum and prevalence of mutations in the RYR2-encoded cardiac ryanodine receptor in cases with exertional syncope and normal corrected QT interval (QTc). BACKGROUND Mutations in RYR2 cause type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a cardiac channelopathy with increased propensity for lethal ventricular dysrhythmias. Most RYR2 mutational analyses target 3 canonical domains encoded by <40% of the translated exons. The extent of CPVT1-associated mutations localizing outside of these domains remains unknown as RYR2 has not been examined comprehensively in most patient cohorts. METHODS Mutational analysis of all RYR2 exons was performed using polymerase chain reaction, high-performance liquid chromatography, and deoxyribonucleic acid sequencing on 155 unrelated patients (49% females, 96% Caucasian, age at diagnosis 20 +/- 15 years, mean QTc 428 +/- 29 ms), with either clinical diagnosis of CPVT (n = 110) or an initial diagnosis of exercise-induced long QT syndrome but with QTc <480 ms and a subsequent negative long QT syndrome genetic test (n = 45). RESULTS Sixty-three (34 novel) possible CPVT1-associated mutations, absent in 400 reference alleles, were detected in 73 unrelated patients (47%). Thirteen new mutation-containing exons were identified. Two-thirds of the CPVT1-positive patients had mutations that localized to 1 of 16 exons. CONCLUSIONS Possible CPVT1 mutations in RYR2 were identified in nearly one-half of this cohort; 45 of the 105 translated exons are now known to host possible mutations. Considering that approximately 65% of CPVT1-positive cases would be discovered by selective analysis of 16 exons, a tiered targeting strategy for CPVT genetic testing should be considered.