[General Data Protection Regulation and medical research: friend or foe?] / Algemene Verordening Gegevensbescherming.

As of May 2018, the use of personal data in medical research is regulated under the General Data Protection Regulation (GDPR). While, as before, in principle patients' consent for the use of their personal data is still required, exemptions for medical research still exist. When all of the criteria for the exemptions are met, and the other requirements of the GDPR are adhered to, personal data can be used in medical research without consent. In this paper we present a brief outline of a number of GDPR-related requirements for use of personal data in medical research. Furthermore, we discuss how GDPR interlinks with the Medical Research Involving Human Subjects Act (WMO) and in which areas GDPR remains subject to interpretation. Medical researchers using personal data need to be aware when consent is required and on which grounds personal data can be used without consent in the Netherlands.

The human Cx40 promoter polymorphism -44G-->A differentially affects transcriptional regulation by Sp1 and GATA4.

Expression of the tissue-specific gap junction protein connexin(Cx)40 is regulated by the interaction of ubiquitous and tissue-specific factors such as Sp1 and GATA4. Cardiac Cx40 expression is altered under pathological conditions such as atrial fibrillation. A human promoter polymorphism, a G-->A change at position -44 that has been associated with atrial-specific arrhythmias, is located between the TBE-NKE-Sp and GATA consensus transcription factor binding sites important for the regulation of the mouse Cx40 gene. The presence of the A-allele at position -44 in promoter-reporter constructs significantly reduces promoter activity. Using electrophoretic mobility shift assays and luciferase reporter assays in various cell types, we show that Sp1 and GATA4 are important regulators of human Cx40 gene transcription and that the -44 G-->A polymorphism negatively affects the promoter regulation by the transcription factors Sp1 and GATA4.

Polymorphisms in human connexin40 gene promoter are associated with increased risk of hypertension in men.

OBJECTIVE: Gap junctions, formed by connexins (Cx), are important in the regulation of vascular tone. Previously, we reported two closely linked polymorphisms (-44G --> A and +71A --> G) within regulatory regions of the gene for Cx40, a major connexin in the vascular wall and the kidney. In the present study, we examined the hypothesis that these polymorphic variants are associated with hypertension and that they interact with blood pressure in healthy individuals. METHODS: Cx40 genotypes were determined in 191 subjects with essential hypertension, 198 normotensive individuals, and a healthy control population (178 twin pairs, 108 monozygotic, 70 dizygotic). RESULTS: We found a significant contribution of the minor Cx40 allele or genotype (-44AA/+71GG) to the risk of hypertension in men (P = 0.013 or P = 0.035; odds ratio, 1.87 or 2.10, respectively), but not in women. Moreover, in the healthy control population a significant effect of Cx40 genotype and sex on systolic blood pressure was found (P < 0.05 and P < 0.0001, respectively). Women carrying the minor Cx40 genotype had significantly higher systolic blood pressure compared with non-carriers (P < 0.05). In men, systolic blood pressure in carriers of the minor Cx40 genotype was not significantly different from the other two genotypes, possibly because of the small number of men in this group. However, men carrying the -44GA/+71AG genotype had higher standing systolic blood pressure compared with the more common Cx40 genotype (-44GG; P = 0.033). CONCLUSION: These findings suggest that the Cx40 polymorphisms may form a genetic susceptibility factor for essential hypertension in men.

Association of human connexin40 gene polymorphisms with atrial vulnerability as a risk factor for idiopathic atrial fibrillation.

Alterations in distribution, density, and properties of cardiac gap junctions, which mediate electrical coupling of cardiomyocytes, are considered potentially arrhythmogenic. We recently reported 2 linked polymorphisms within regulatory regions of the gene for the atrial gap junction protein connexin40 (Cx40) at nucleotides -44 (G-->A) and +71 (A-->G), which were associated with familial atrial standstill. The present study examined whether these Cx40 polymorphisms were associated with increased atrial vulnerability in vivo and arrhythmia susceptibility. In 30 subjects without structural heart disease, of whom 14 had documented sporadic paroxysmal atrial fibrillation (AF) and 16 had no AF history, inducibility of AF was assessed using an increasingly aggressive atrial stimulation protocol. Coefficient of spatial dispersion of refractoriness (CD) was calculated. CD was defined as the SD of 12 local mean fibrillatory intervals recorded at right atrial sites, expressed as a percentage of the overall mean fibrillatory interval. Cx40 genotypes were determined by direct DNA sequencing. Subjects were stratified according to normal or increased CD with a cutoff value of 3.0, because CD >3.0 was previously shown to be strongly associated with enhanced atrial vulnerability. The prevalence of the minor Cx40 allele (-44A) and -44AA genotype was significantly higher in subjects with increased dispersion (n=13) compared with those with CD < or =3.0 (n=17; P=0.00046 and P=0.025; odds ratios of 6.7 and 7.4) and a control population (n=253; P=0.00002 and P=3.90x10(-7)). Carriers of -44AA genotype had a significantly higher CD compared with those with -44GG genotype (6.37+/-1.21 versus 2.38+/-0.39, P=0.018), whereas heterozygotes had intermediate values (3.95+/-1.38, NS). All subjects with increased CD had a history of idiopathic AF compared with only 1 subject with normal CD. The -44A allele and -44AA genotype were significantly more frequent in subjects with prior AF than in those without (P=0.0019 and P=0.031; odds ratios 5.3 and 6.2). This study provides strong evidence linking Cx40 polymorphisms to enhanced atrial vulnerability and increased risk of AF. The full text of this article is available online at http://circres.ahajournals.org.

Compound heterozygosity for mutations (W156X and R225W) in SCN5A associated with severe cardiac conduction disturbances and degenerative changes in the conduction system.

Cardiac conduction defects associate with mutations in SCN5A, the gene encoding the cardiac Na+ channel. In the present study, we characterized a family in which the proband was born in severe distress with irregular wide complex tachycardia. His older sister died at 1 year of age from severe conduction disease with similarly widened QRS-complexes. Mutational analysis of SCN5A in the proband demonstrated compound heterozygosity for a nonsense mutation (W156X), inherited from the father, and a missense mutation (R225W), inherited from the mother. Genotyping on DNA extracted from tissue from the deceased sibling revealed the same SCN5A genotype. Injection of cRNA encoding the W156X mutation in Xenopus oocytes did not produce any current. The R225W substitution neutralizes the third Arg residue within the voltage-sensing segment of domain I. Expression studies showed that this mutation leads to a severe reduction in I(Na) and is also associated with gating changes. Histological examination of the heart from the deceased sibling revealed changes consistent with a dilated type of cardiomyopathy and severe degenerative abnormalities of the specialized conduction system. The occurrence of compound heterozygosity for these two mutations implies that the proband carries solely severely dysfunctional cardiac Na+ channels. This explains his severe phenotype and that of his deceased sister who had been a carrier of the same genotype. The morphological changes within the heart of the deceased sibling may have occurred secondary to the Na+ channel abnormality and contributed to the severity of the disorder in this individual.

A cardiac sodium channel mutation cosegregates with a rare connexin40 genotype in familial atrial standstill.

Atrial standstill (AS) is a rare arrhythmia that occasionally appears to be genetically determined. This study investigates the genetic background of this arrhythmogenic disorder in a large family. Forty-four family members were clinically evaluated. One deceased and three living relatives were unambiguously affected by AS. All other relatives appeared unaffected. Candidate gene screening revealed a novel mutation in the cardiac sodium channel gene SCN5A (D1275N) in all three affected living relatives and in five unaffected relatives, and the deceased relative was an obligate carrier. In addition, two closely linked polymorphisms were detected within regulatory regions of the gene for the atrial-specific gap junction protein connexin40 (Cx40) at nucleotides -44 (G-->A) and +71 (A-->G). Eight relatives were homozygous for both polymorphisms, which occurred in only approximately 7% of control subjects, and three of these relatives were affected by AS. The three living AS patients exclusively coinherited both the rare Cx40 genotype and the SCN5A-D1275N mutation. SCN5A-D1275N channels showed a small depolarizing shift in activation compared with wild-type channels. Rare Cx40 genotype reporter gene analysis showed a reduction in reporter gene expression compared with the more common Cx40 genotype. In this study, familial AS was associated with the concurrence of a cardiac sodium channel mutation and rare polymorphisms in the atrial-specific Cx40 gene. We propose that, although the functional effect of each genetic change is relatively benign, the combined effect of genetic changes eventually progresses to total AS.

Cx40 polymorphism in human atrial fibrillation.

UNLABELLED: Previous studies have shown that two linked polymorphisms within regulatory regions of the gene for connexin40 (Cx40), at nucleotides -44 (G --> A) and +71 (A --> G) occur in about 7% of the general population. Cx40 is abundant in the atrium, and homozygosity for the linked polymorphisms combined with an SCN5A mutation appeared to be responsible for familial atrial standstill. We hypothesized that these polymorphisms are associated with the atrial electrophysiologic substrate favoring reentrant mechanisms for initiation of atrial fibrillation (AF). Reentry is promoted by spatial dispersion of refractoriness that can be expressed as a coefficient of dispersion (CD). METHODS: CD was calculated from the standard deviation of 12 local mean fibrillatory intervals recorded at right atrial sites during induced AF in 30 patients without structural heart disease (14 sporadic AF episodes, 16 no AF history). CD

Congenital atrial standstill associated with coinheritance of a novel SCN5A mutation and connexin 40 polymorphisms.

BACKGROUND: Congenital atrial standstill has been linked to SCN5A. Incomplete penetrance observed in atrial standstill has been attributed in part to the digenic inheritance of polymorphisms in the atrial-specific gap junction connexin 40 (Cx40) in conjunction with an SCN5A mutation. OBJECTIVES: The purpose of this study was to determine the clinical and biophysical characteristics of a novel SCN5A mutation identified in a family with atrial standstill. METHODS: Family members of an apparently sporadic case of atrial standstill underwent genetic screening of SCN5A and atrial-specific genes including Cx40. Biophysical properties of the wild-type (WT) and mutant SCN5A channels in a heterologous expression system were studied using the whole-cell patch clamp technique. RESULTS: The novel SCN5A mutation L212P was identified in the proband (age 11 years) and his father. The father was in normal sinus rhythm. The proband had no P waves on surface ECG, and his right atrium could not be captured by pacing. The recombinant L212P Na channel showed a large hyperpolarizing shift in both the voltage dependence of activation (WT: -48.1 +/- 0.9 mV; L212P: -63.5 +/- 1.5 mV; P < .001) and inactivation (WT: -86.6 +/- 0.9 mV; L212P: -95.6 +/- 0.8 mV; P < .001) and delayed recovery from inactivation. Further screenings for genetic variations that might mitigate L212P dysfunction revealed that the proband, but not his father, carries Cx40 polymorphisms inherited from his asymptomatic mother. CONCLUSION: These results suggest that genetic defects in SCN5A most likely underlie atrial standstill. Coinheritance of Cx40 polymorphisms is a possible genetic factor that modifies the clinical manifestation of this inherited arrhythmia.

A novel LQT3 mutation implicates the human cardiac sodium channel domain IVS6 in inactivation kinetics.

UNLABELLED: The Long QT3 syndrome is associated with mutations in the cardiac sodium channel gene SCN5A. OBJECTIVE: The aim of the present study was the identification and functional characterization of a mutation in a family with the long QT3 syndrome. METHODS: The human cardiac sodium channel gene SCN5A was screened for mutations by single-stranded conformation polymorphism. The functional consequences of mutant sodium channels were characterized after expressing mutant and wild-type cRNAs in Xenopus oocytes by two-electrode voltage clamp measurements. RESULTS: SCN5A screening revealed an A-->G substitution at codon 1768, close to the C-terminal end of domain IVS6, which changes an isoleucine to a valine. Functional expression of mutant I1768V-channels in Xenopus oocytes showed that the voltage-dependence and slope factors of activation and inactivation were unchanged compared to wild-type channels. No difference in persistent TTX-sensitive current could be detected between wild-type and I1768V channels, a channel feature often increased in LQT3 mutants. However, I1768V mutant channels recovered faster from inactivation (2.4 times) than wild-type channels and displayed less slow inactivation. CONCLUSIONS: We postulate that severe destabilization of the inactivated state leads to increased arrhythmogenesis and QT prolongation in I1768V mutation carriers in the absence of a persistent inward sodium current.

Heterogeneous Connexin43 distribution in heart failure is associated with dispersed conduction and enhanced susceptibility to ventricular arrhythmias.

AIMS: Sudden arrhythmogenic cardiac death is a major cause of mortality in patients with congestive heart failure (CHF). To investigate determinants of the increased arrhythmogenic susceptibility, we studied cardiac remodelling and arrhythmogenicity in CHF patients and in a mouse model of chronic pressure overload. METHODS AND RESULTS: Clinical and (immuno)histological data of myocardial biopsies from CHF patients with (VT+) and without (VT-) documented ventricular arrhythmia were compared with controls. In CHF patients, ejection fraction was decreased and QRS duration was increased. Cell size and interstitial fibrosis were increased, but Connexin43 (Cx43) levels, the most abundant gap junction in ventricular myocardium, were unchanged. No differences were found between VT+ and VT- patients, except for the distribution pattern of Cx43, which was significantly more heterogeneous in VT+. Mice were subjected to transverse aortic constriction (TAC) or sham operated. At 16 weeks, cardiac function was determined by echocardiography and epicardial ventricular activation mapping was performed. Transverse aortic constriction mice had decreased fractional shortening and prolonged QRS duration. Right ventricular conduction velocity was reduced, and polymorphic VTs were induced in 44% TAC and 0% sham mice. Interstitial fibrosis was increased and Cx43 quantity was unchanged in TAC mice with and without arrhythmias. Similar to CHF patients, heterogeneous Cx43 distribution was significantly associated with arrhythmias in TAC mice and with spatial heterogeneity of impulse conduction. CONCLUSION: Heterogeneous Cx43 expression during CHF is associated with dispersed impulse conduction and may underlie enhanced susceptibility to ventricular tachyarrhythmias.

Functional analysis of novel KCNQ2 mutations found in patients with Benign Familial Neonatal Convulsions.

Benign Familial Neonatal Convulsions (BFNC) are a rare epilepsy disorder with an autosomal-dominant inheritance. It is linked to mutations in the potassium channel genes KCNQ2 and KCNQ3. These encode for Kv7.2 and Kv7.3 potassium ion channels, which produce an M-current that regulates the potential firing action in neurons through modulation of the membrane potential. We report on the biophysical and biochemical properties of V589X, T359K and P410fs12X mutant-KCNQ2 ion channels that were detected in three BFNC families. Mutant KCNQ2 cDNAs were co-expressed with WT-KCNQ2 and KCNQ3 cDNAs in HEK293 cells to mimic heterozygous expression of the KCNQ2 mutations in BFNC patients. The resulting potassium currents were measured using patch-clamp techniques and showed an approximately 75% reduction in current and a depolarized shift in the voltage dependence of activation. Furthermore, the time-constant of activation of M-currents in cells expressing T359K and P410fs12X was slower compared to cells expressing only wild-type proteins. Immunofluorescent labeling of HEK293 cells stably expressing GFP-tagged KCNQ2-WT or mutant alpha-subunits indicated cell surface expression of WT, V589X and T359K mutants, suggesting a loss-of-function, while P410fs12X was predominantly retained in the ER and sub-cellular compartments outside the ER suggesting an effectively haplo-insufficient effect.

Na+ channel mutation leading to loss of function and non-progressive cardiac conduction defects.

BACKGROUND: We previously described a Dutch family in which congenital cardiac conduction disorder has clinically been identified. The ECG of the index patient showed a first-degree AV block associated with extensive ventricular conduction delay. Sequencing of the SCN5A locus coding for the human cardiac Na+ channel revealed a single nucleotide deletion at position 5280, resulting in a frame-shift in the sequence coding for the pore region of domain IV and a premature stop codon at the C-terminus. METHODS AND RESULTS: Wild type and mutant Na+ channel proteins were expressed in Xenopus laevis oocytes and in mammalian cells. Voltage clamp experiments demonstrated the presence of fast activating and inactivating inward currents in cells expressing the wild type channel alone or in combination with the beta1 subinut (SCN1B). In contrast, cells expressing the mutant channels did not show any activation of inward current with or without the beta1 subunit. Culturing transfected cells at 25 degrees C did not restore the Na+ channel activity of the mutant protein. Transient expression of WT and mutant Na+ channels in the form of GFP fusion proteins in COS-7 cells indicated protein expression in the cytosol. But in contrast to WT channels were not associated with the plasma membrane. CONCLUSIONS: The SCN5A/5280delG mutation results in the translation into non-function channel proteins that do not reach the plasma membrane. This could explain the cardiac conduction defects in patients carrying the mutation.