Recurrent and founder mutations in the Netherlands: cardiac Troponin I (TNNI3) gene mutations as a cause of severe forms of hypertrophic and restrictive cardiomyopathy.

BACKGROUND: About 2-7% of familial cardiomyopathy cases are caused by a mutation in the gene encoding cardiac troponin I (TNNI3). The related clinical phenotype is usually severe with early onset. Here we report on all currently known mutations in the Dutch population and compared these with those described in literature. METHODS: TheTNNI3 gene was screened for mutations in all coding exons and flanking intronic sequences in a large cohort of cardiomyopathy patients. All Dutch index cases carrying a TNNI3 mutation that are described in this study underwent extensive cardiological evaluation and were listed by their postal codes. RESULTS: In 30 families, 14 different mutations were identified. Three TNNI3 mutations were found relatively frequently in both familial and non-familial cases of hypertrophic cardiomyopathy (HCM) or restrictive cardiomyopathy (RCM). Haplotype analysis showed that p.Arg145Trp and p.Ser166Phe are founder mutations in the Netherlands, while p.Glu209Ala is not. The majority of Dutch TNNI3 mutations were associated with a HCM phenotype. Mean age at diagnosis was 36.5 years. Mutations causing RCM occurred less frequently, but were identified in very young children with a poor prognosis. CONCLUSION: In line with previously published data, we found TNNI3 mutations to be rare and associated with early onset and severe clinical presentation.

Recurrent and Founder Mutations in the Netherlands: the Long-QT Syndrome.

BACKGROUND AND OBJECTIVE: The long-QT syndrome (LQTS) is associated with premature sudden cardiac deaths affecting whole families and is caused by mutations in genes encoding for cardiac proteins. When the same mutation is found in different families (recurrent mutations), this may imply either a common ancestor (founder) or multiple de novo mutations. We aimed to review recurrent mutations in patients with LQTS. METHODS: By use of our databases, we investigated the number of mutations that were found recurrently (at least three times) in LQT type 1-3 patients in the Netherlands. We studied familial links in the apparently unrelated probands, and we visualised the geographical distribution of these probands. Our results were compared with published literature of founder effects in LQTS outside the Netherlands. RESULTS: We counted 14 recurrent LQT mutations in the Netherlands. There are 326 identified carriers of one of these mutations. For three of these mutations, familial links were found between apparently unrelated probands. CONCLUSION: Whereas true LQT founder mutations are described elsewhere in the world, we cannot yet demonstrate a real founder effect of these recurrent mutations in the Netherlands. Further studies on the prevalence of these mutations are indicated, and haplotype-sharing of the mutation carriers is pertinent to provide more evidence for founder mutation-based LQTS pathology in our country.

Founder mutations in hypertrophic cardiomyopathy patients in the Netherlands.

In this part of a series on cardiogenetic founder mutations in the Netherlands, we review the Dutch founder mutations in hypertrophic cardiomyopathy (HCM) patients. HCM is a common autosomal dominant genetic disease affecting at least one in 500 persons in the general population. Worldwide, most mutations in HCM patients are identified in genes encoding sarcomeric proteins, mainly in the myosin-binding protein C gene (MYBPC3, OMIM #600958) and the beta myosin heavy chain gene (MYH7, OMIM #160760). In the Netherlands, the great majority of mutations occur in the MYBPC3, involving mainly three Dutch founder mutations in the MYBPC3 gene, the c.2373_2374insG, the c.2864_2865delCT and the c.2827C>T mutation. In this review, we describe the genetics of HCM, the genotype-phenotype relation of Dutch founder MYBPC3 gene mutations, the prevalence and the geographic distribution of the Dutch founder mutations, and the consequences for genetic counselling and testing. (Neth Heart J 2010;18:248-54.).

Recurrent and founder mutations in the Netherlands : Plakophilin-2 p.Arg79X mutation causing arrhythmogenic right ventricular cardiomyopathy/dysplasia.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is an inherited cardiac disease with reduced penetrance and a highly variable expression. Mutations in the gene encoding the plakophilin-2 gene (PKP2) are detected in about 50% of ARVC/D patients. The p.Arg79X mutation in PKP2 has been identified in Europe and North America and has been functionally characterised. We evaluated the prevalence of the p.Arg79X mutation in PKP2 in the Dutch population. METHODS: Twelve index patients and 41 family members were evaluated in three university hospitals in the Netherlands. The diagnosis of ARVC/D was established according to the recently revised Task Force Criteria. Segregation of the p.Arg79X mutation was studied and haplotypes were reconstructed to determine whether the p.Arg79X mutation was a recurrent or a founder mutation. RESULTS: The p.Arg79X mutation in PKP2 was identified in 12 index patients. Haplotype analysis revealed a shared haplotype among Dutch p.Arg79X mutation carriers, indicating a common founder. Six index patients (50%) had a first- or second-degree relative who had died of sudden cardiac death below 40 years of age. At age 60, only 60% of the mutation carriers had experienced any symptoms. There was no significant difference in symptom-free survival and event-free survival between men and women. CONCLUSION: We have identified the largest series of patients with the same desmosome gene mutation in ARVC/D reported to date. This p.Arg79X mutation in PKP2 is a founder mutation in the Dutch population. The phenotypes of PKP2 p.Arg79X mutation carriers illustrate the clinical variability and reduced penetrance often seen in ARVC/D. (Neth Heart J 2010;18:583-91.).

Defective complex I assembly due to C20orf7 mutations as a new cause of Leigh syndrome.

BACKGROUND: Leigh syndrome is an early onset, progressive, neurodegenerative disorder with developmental and motor skills regression. Characteristic magnetic resonance imaging abnormalities consist of focal bilateral lesions in the basal ganglia and/or the brainstem. The main cause is a deficiency in oxidative phosphorylation due to mutations in an mtDNA or nuclear oxidative phosphorylation gene. METHODS AND RESULTS: A consanguineous Moroccan family with Leigh syndrome comprise 11 children, three of which are affected. Marker analysis revealed a homozygous region of 11.5 Mb on chromosome 20, containing 111 genes. Eight possible mitochondrial candidate genes were sequenced. Patients were homozygous for an unclassified variant (p.P193L) in the cardiolipin synthase gene (CRLS1). As this variant was present in 20% of a Moroccan control population and enzyme activity was only reduced to 50%, this could not explain the rare clinical phenotype in our family. Patients were also homozygous for an amino acid substitution (p.L159F) in C20orf7, a new complex I assembly factor. Parents were heterozygous and unaffected sibs heterozygous or homozygous wild type. The mutation affects the predicted S-adenosylmethionine (SAM) dependent methyltransferase domain of C20orf7, possibly involved in methylation of NDUFB3 during the assembly process. Blue native gel electrophoresis showed an altered complex I assembly with only 30-40% of mature complex I present in patients and 70-90% in carriers. CONCLUSIONS: A new cause of Leigh syndrome can be a defect in early complex I assembly due to C20orf7 mutations.

A single hERG mutation underlying a spectrum of acquired and congenital long QT syndrome phenotypes.

The long QT syndrome (LQTS) is a multi-factorial disorder that predisposes to life-threatening arrhythmias. Both hereditary and acquired subforms have been identified. Here, we present clinical and biophysical evidence that the hERG mutation c.1039 C>T (p.Pro347Ser or P347S) is responsible for both the acquired and the congenital phenotype. In one case the genotype remained silent for years until the administration of several QT-prolonging drugs resulted into a full-blown phenotype, that was reversible upon cessation of these compounds. On the other hand the mutation was responsible for a symptomatic congenital LQTS in a Dutch family, displaying a substantial heterogeneity of the clinical symptoms. Biophysical characterization of the p.Pro347Ser potassium channels using whole-cell patch clamp experiments revealed a novel pathogenic mechanism of reciprocal changes in the inactivation kinetics combined with a dominant-negative reduction of the functional expression in the heterozygous situation, yielding a modest genetic predisposition for LQTS. Our data show that in the context of the multi-factorial aetiology underlying LQTS a modest reduction of the repolarizing power can give rise to a spectrum of phenotypes originating from one mutation. This observation increases the complexity of genotype-phenotype correlations in more lenient manifestations of the disease and underscores the difficulty of predicting the expressivity of the LQTS especially for mutations with a more subtle impact such as p.Pro347Ser.

[Genetic identification of patients and families with a long-QT syndrome: large regional differences in the result of 10 years]. / Genetische identificatie van patiënten en families met lange-QT-syndroom: grote regionale verschillen in de resultaten van 10 jaar.

OBJECTIVE: To determine the pattern of referral of Dutch patients with a long-QT syndrome (LQTS) on the basis of the postal codes of the LQTS probands from whom blood samples were submitted for DNA diagnostics. DESIGN: . Retrospective cohort study. METHOD: From the databases that are coupled to DNA diagnostics, all index patients were included for whom LQTS diagnostics had been requested during the period 1996-2005 at two clinical genetics centres (the University Medical Centre in Amsterdam and Maastricht University Hospital). The results were related to the postal code of the referred patient and corrected for the number of inhabitants of the region concerned. RESULTS: A total of 421 potential LQTS probands were included. Corrected for the numbers of inhabitants in the various postal codes, the number of referrals varied from 3 per million to 110 per million inhabitants. In view of the most recent estimated prevalence of LQTS (1:2000), this means that only 15% ofthe carriers of the LQTS mutation have so far been detected. CONCLUSION: There were large regional differences in the Netherlands in the requests for DNA diagnostics in patients with clinical LQTS. The overwhelming majority of the LQTS patients in the Netherlands have not yet been referred or identified. Expanding the available courses for general practitioners and cardiologists that are given by the staff of the cardiogenetic centres would seem to be indicated.

Methods in molecular cardiology: DHPLC mutation detection analysis.

An increasing number of mutations have been identified in genes involved in cardiac disorders which has led to novel insights in the pathophysiology of inherited cardiac diseases. As a result of these findings, techniques specialised in automated high-throughput analysis are implemented to handle the increasing number of diagnostic genetic requests. Denaturing high-performance liquid chromatography (DHPLC) is one such novel technique that fulfils the criteria of speed, sensitivity and accuracy. This issue focuses on the basic principle of the technique and illustrates how genetic alterations can be identified.

Contribution of agricultural and non-agricultural use of pesticides to the environmental impact on aquatic life in regional surface water systems.

By means of a modelling tool an analysis was made of the local variation in the use of pesticides in the province of Utrecht in The Netherlands, and the potential environmental impact of pesticide emissions on the aquatic ecosystems. The aim of this study was to identify and quantify the major sources of pesticide use and environmental impact, taking the regional variation of pesticide use into account. The analysis was targeted at different levels: detailed (individual active substances, individual agricultural crops, civil land-use types, hydrological catchment basins) and globally covering agricultural use, non-agricultural use (some civil sectors) and recreational shipping. The results can be used for the (re)design of environmental monitoring programmes of pesticides in surface waters and for the development of region based policies towards sustainable pesticide use. The analysis tool that was developed is considered to be applicable for other regions as well.

Diffuse pollution of surface water by pharmaceutical products.

Pharmaceutical products for humans or animals, as well as their related metabolites (degradation products) end up in the aquatic environment after use. Recent investigations from abroad show that low concentrations of pharmaceuticals are detectable in municipal waste water, surface water, groundwater and even drinking water. Little is known about the effects, and with that the risk, of long term exposure to low concentrations of pharmaceuticals for aquatic organisms. On the basis of the current knowledge, further attention to map the presence and effects of pharmaceutical residues on aquatic organisms is justified. To map the Dutch situation, recently a monitoring program has started.

Pearson syndrome and the role of deletion dimers and duplications in the mtDNA.

Pearson syndrome is an often fatal multisystem disease associated with mitochondrial DNA rearrangements. Here we report a patient with a novel mtDNA deletion of 3.4 kb ranging from nucleotides 6097 to 9541 in combination with deletion dimers. The mutation percentage in different tissues (blood, muscle and liver) varied between 64% and 95%. After a remission period of about a year, the patient suddenly died at the age of 3 years owing to a severe lactic acidosis. A second patient with a previously reported deletion of 8 kb and a milder phenotype was found to have mitochondrial duplications and died at the age of 10 years. From these data and data from previous reports, we hypothesize that duplications might be beneficial in the clinical course of the disease and in life expectancy.

Novel KCNQ1 and HERG missense mutations in Dutch long-QT families.

Congenital long QT syndrome (cLQTS) is electrocardiographically characterized by a prolonged QT interval and polymorphic ventricular arrhythmias (torsade de pointes). These cardiac arrhythmias may result in recurrent syncopes, seizure, or sudden death. LQTS can occur either as an autosomal dominant (Romano Ward) or as an autosomal recessive disorder (Jervell and Lange-Nielsen syndrome). Mutations in at least five genes have been associated with the LQTS. Four genes, encoding cardiac ion channels, have been identified. The most common forms of LQTS are due to mutations in the potassium-channel genes KCNQ1 and HERG. We have screened 24 Dutch LQTS families for mutations in KCNQ1 and HERG. Fourteen missense mutations were identified. Eight of these missense mutations were novel: three in KCNQ1 and five in HERG. Novel missense mutations in KCNQ1 were Y184S, S373P, and W392R and novel missense mutations in HERG were A558P, R582C, G604S, T613M, and F640L. The KCNQ1 mutation G189R and the HERG mutation R582C were detected in two families. The pathogenicity of the mutations was based on segregation in families, absence in control individuals, the nature of the amino acid substitution, and localization in the protein. Genotype-phenotype studies indicated that auditory stimuli as trigger of cardiac events differentiate LQTS2 and LQTS1. In LQTS1, exercise was the predominant trigger. In addition, a number of asymptomatic gene defect carriers were identified. Asymptomatic carriers are still at risk of the development of life-threatening arrhythmias, underlining the importance of DNA analyses for unequivocal diagnosis of patients with LQTS.

Auditory stimuli as a trigger for arrhythmic events differentiate HERG-related (LQTS2) patients from KVLQT1-related patients (LQTS1).

OBJECTIVE: This study was performed to identify a possible relationship between genotype and phenotype in the congenital familial long QT syndrome (cLQTS). BACKGROUND: The cLQTS, which occurs as an autosomal dominant or recessive trait, is characterized by QT-interval prolongation on the electrocardiogram and torsade de pointes arrhythmias, which may give rise to recurrent syncope or sudden cardiac death. Precipitators for cardiac events are exercise or emotion and occasionally acoustic stimuli. METHODS: The trigger for cardiac events (syncope, documented cardiac arrhythmias, sudden cardiac death) was analyzed in 11 families with a familial LQTS and a determined genotype. RESULTS: The families were subdivided in KVLQT1-related families (LQTS1, n = 5) and HERG (human ether-a-gogo-related gene)-related families (LQTS2, n = 6) based on single-strand conformation polymorphism analysis and sequencing. Whereas exercise-related cardiac events dominate the clinical picture of LQTS1 patients, auditory stimuli as a trigger for arrhythmic events were only seen in LQTS2 patients. CONCLUSIONS: Arrhythmic events triggered by auditory stimuli may differentiate LQTS2 from LQTS1 patients.

Non-starch polysaccharides in pig feeding.

In pigs and humans, the nutrients starch, protein, fat and some minerals need to be digested prior to the terminal ileum for optimal use of these nutrients. In contrast, the non-starch polysaccharides (NSP) are mainly fermented by microbes in the hindgut. Results of experiments in pigs showed that NSP negatively affected apparent digestion of protein, fat and some minerals. In addition, large amounts of fermented NSP increased the empty weight of the hindgut. Because tissue of organs like the intestinal tract are metabolically very active, it may have required more energy for maintenance, hence leaving less energy for growth. Despite all the negative effects as mentioned above, including NSP-rich ingredients in pig diets also has quite a lot of advantages. Their energy supply can cover the energy requirements for maintenance. In addition, positive effects on the well-being and health of pigs, and on the excretion of ammonia are claimed. In conclusion, in future pig diet formulation not only the nutritional aspects of NSP-rich ingredients should be taken into account, but also their non-nutritional aspects. This might be realized by developing nutrient based feed evaluation systems, rather than the energy based systems which are presently used.

The long QT syndrome: a novel missense mutation in the S6 region of the KVLQT1 gene.

The Romano Ward long QT syndrome (LQTS) has an autosomal dominant mode of inheritance. Patients suffer from syncopal attacks often resulting in sudden cardiac death. The main diagnostic parameter is a prolonged QT(c) interval as judged by electro-cardiographic investigation. LQTS is a genetically heterogeneous disease with four loci having been identified to date: chromosome 11p15.5 (LQT1), 7q35-36 (LQT2), 3p21-24 (LQT3) and 4q25-26 (LQT4). The corresponding genes code for potassium channels KVLQT1 (LQT1) and HERG (LQT2) and the sodium channel SCN5A (LQT3). The KVLQT1 gene is characterized by six transmembrane domains (S1-S6), a pore region situated between the S5 and S6 domains and a C-terminal domain accounting for approximately 60% of the channel. This domain is thought to be co-associated with another protein, viz. minK (minimal potassium channel). We have studied a Romano Ward family with several affected individuals showing a severe LQTS phenotype (syncopes and occurrence of sudden death). Most affected individuals had considerable prolongations of QT(c). By using haplotyping with a set of markers covering the four LQT loci, strong linkage was established to the LQT1 locus, whereas the other loci (LQT2, LQT3 and LQT4) could be excluded. Single-strand conformation polymorphism analysis and direct sequencing were used to screen the KVLQT1 gene for mutations in the S1-S6 region, including the pore domain. We identified a Gly-216-Arg substitution in the S6 transmembrane domain of KVLQT1. The mutation was present in all affected family members but absent in normal control individuals, providing evidence that the mutated KVLQT1-gene product indeed caused LQTS in this family. The mutated KVLQT1-gene product thus probably results in a dominant negative suppression of channel activity.

A probabilistic model for deriving soil quality criteria based on secondary poisoning of top predators. I. Model description and uncertainty analysis.

In previous studies, the risk of toxicant accumulation in food chains was used to calculate quality criteria for surface water and soil. A simple algorithm was used to calculate maximum permissable concentrations [MPC = no-observed-effect concentration/bioconcentration factor(NOEC/BCF)]. These studies were limited to simple food chains. This study presents a method to calculate MPCs for more complex food webs of predators. The previous method is expanded. First, toxicity data (NOECs) for several compounds were corrected for differences between laboratory animals and animals in the wild. Second, for each compound, it was assumed these NOECs were a sample of a log-logistic distribution of mammalian and avian NOECs. Third, bioaccumulation factors (BAFs) for major food items of predators were collected and were assumed to derive from different log-logistic distributions of BAFs. Fourth, MPCs for each compound were calculated using Monte Carlo sampling from NOEC and BAF distributions. An uncertainty analysis for cadmium was performed to identify the most uncertain parameters of the model. Model analysis indicated that most of the prediction uncertainty of the model can be ascribed to uncertainty of species sensitivity as expressed by NOECs. A very small proportion of model uncertainty is contributed by BAFs from food webs. Correction factors for the conversion of NOECs from laboratory conditions to the field have some influence on the final value of MPC5, but the total prediction uncertainty of the MPC is quite large. It is concluded that the uncertainty in species sensitivity is quite large. To avoid unethical toxicity testing with mammalian or avian predators, it cannot be avoided to use this uncertainty in the method proposed to calculate MPC distributions. The fifth percentile of the MPC is suggested as a safe value for top predators.

A probabilistic model for deriving soil quality criteria based on secondary poisoning of top predators. II. Calculations for dichlorodiphenyltrichloroethane (DDT) and cadmium.

A simplified food web with three trophic levels is designed: plants and invertebrates at the first, small birds and mammals at the second, and birds and beasts of prey at the third trophic level. Exposure of top predators via separate food chains is analyzed. However, most top predator species are exposed via more than one food chain (food web). Therefore, a species-specific approach is followed too, for which four bird of prey species and two beast of prey species with different food choices are selected: sparrow hawk, kestrel, barn owl, little owl, badger, and weasel. The most critical food chains for secondary poisoning of top predators are soil --> worm/insect --> bird --> bird of prey for dichlorodiphenyltrichloroethane (DDT), and soil --> worm --> bird/mammal --> bird of prey for cadmium (Cd). The risk for the selected top predator species is much lower than the risk based on these critical food chains because the critical food chains constitute a minor part of their food webs. Species feeding on birds (sparrow hawk) and small carnivorous mammals (barn owl) are exposed to DDT and Cd to a much higher extent than species mainly feeding on small herbivorous mammals (kestrel and weasel). It is recommended to include exposure via the pathways soil --> worm/insect --> bird/mammal --> top predator in procedures for derivation of environmental quality objectives for persistent and highly lipophilic compounds.

Apparent digestibility of nutrients in diets with different energy density, as estimated by direct and marker methods for pigs with or without ileo-cecal cannulas.

The objective of this study were a) to compare the apparent total tract digestibility (TD) between non-cannulated (intact) and cannulated (steered ileo-cecal valve technique, SICV) pigs fed diets differing in energy density (Exp. 1) and b) to compare the direct vs marker (Cr2O3) methods for estimation of the TD and apparent ileal digestibility (ID) in SICV-cannulated pigs (Exp. 2). In Exp. 1, 24 intact and 18 SICV-cannulated castrates of approximately 40 kg initial BW were randomly assigned to six treatments in a 2 x 3 x 2 factorial arrangement (two pig types, three carbohydrate sources, and two fat levels). In Exp. 2, the same SICV-cannulated pigs from Exp. 1 were given those treatments in a 2 x 3 x 2 factorial arrangement (two methods of digestibility estimation, three carbohydrates sources, and two fat levels). In both experiments either cornstarch, soybean hulls, or pure cellulose, without or with fat, were incorporated into a barely-soybean meal based diet to alter energy density. Daily diets were isoenergetic (based on NEf), and water supply was .33 L/MJ of NEf. In Exp. 1, the pig type effect on the TD of DM, OM, CP, and the pig type x carbohydrate interactions for the TD of DM, OM, and crude fiber (CF) were significant (P < .05), merely due to a larger difference found for the diet enriched with cellulose. In Exp. 2, the TD and ID evaluated with the marker method were significantly lower (except for the TD of CF) than with the direct method, mainly because Cr recovery was below 100%. Overall, the marker method seems to be superior because the TD means obtained from Cr ratios were closer to the TD obtained from intact pigs. In general, the SICV technique seems to be suitable for long-term digestibility studies to measure the TD and ID in the same pig fed low-or high-fiber diets.