Circulating Acylcarnitines Associated with Hypertrophic Cardiomyopathy Severity: an Exploratory Cross-Sectional Study in MYBPC3 Founder Variant Carriers.

Hypertrophic cardiomyopathy (HCM) is a relatively common genetic heart disease characterised by myocardial hypertrophy. HCM can cause outflow tract obstruction, sudden cardiac death and heart failure, but severity is highly variable. In this exploratory cross-sectional study, circulating acylcarnitines were assessed as potential biomarkers in 124 MYBPC3 founder variant carriers (59 with severe HCM, 26 with mild HCM and 39 phenotype-negative [G + P-]). Elastic net logistic regression identified eight acylcarnitines associated with HCM severity. C3, C4, C6-DC, C8:1, C16, C18 and C18:2 were significantly increased in severe HCM compared to G + P-, and C3, C6-DC, C8:1 and C18 in mild HCM compared to G + P-. In multivariable linear regression, C6-DC and C8:1 correlated to log-transformed maximum wall thickness (coefficient 5.01, p = 0.005 and coefficient 0.803, p = 0.007, respectively), and C6-DC to log-transformed ejection fraction (coefficient -2.50, p = 0.004). Acylcarnitines seem promising biomarkers for HCM severity, however prospective studies are required to determine their prognostic value.

BIO FOr CARE: biomarkers of hypertrophic cardiomyopathy development and progression in carriers of Dutch founder truncating MYBPC3 variants-design and status.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disease, commonly caused by truncating variants in the MYBPC3 gene. HCM is an important cause of sudden cardiac death; however, overall prognosis is good and penetrance in genotype-positive individuals is incomplete. The underlying mechanisms are poorly understood and risk stratification remains limited. AIM: To create a nationwide cohort of carriers of truncating MYBPC3 variants for identification of predictive biomarkers for HCM development and progression. METHODS: In the multicentre, observational BIO FOr CARe (Identification of BIOmarkers of hypertrophic cardiomyopathy development and progression in Dutch MYBPC3 FOunder variant CARriers) cohort, carriers of the c.2373dupG, c.2827C > T, c.2864_2865delCT and c.3776delA MYBPC3 variants are included and prospectively undergo longitudinal blood collection. Clinical data are collected from first presentation onwards. The primary outcome constitutes a composite endpoint of HCM progression (maximum wall thickness ≥ 20 mm, septal reduction therapy, heart failure occurrence, sustained ventricular arrhythmia and sudden cardiac death). RESULTS: So far, 250 subjects (median age 54.9 years (interquartile range 43.3, 66.6), 54.8% male) have been included. HCM was diagnosed in 169 subjects and dilated cardiomyopathy in 4. The primary outcome was met in 115 subjects. Blood samples were collected from 131 subjects. CONCLUSION: BIO FOr CARe is a genetically homogeneous, phenotypically heterogeneous cohort incorporating a clinical data registry and longitudinal blood collection. This provides a unique opportunity to study biomarkers for HCM development and prognosis. The established infrastructure can be extended to study other genetic variants. Other centres are invited to join our consortium.

A Dutch MYH7 founder mutation, p.(Asn1918Lys), is associated with early onset cardiomyopathy and congenital heart defects.

BACKGROUND: Mutations in the myosin heavy chain 7 (MYH7) gene commonly cause cardiomyopathy but are less frequently associated with congenital heart defects. METHODS: In this study, we describe a mutation in the MYH7 gene, c. 5754C > G; p. (Asn1918Lys), present in 15 probands and 65 family members. RESULTS: Of the 80 carriers (age range 0-88 years), 46 (57.5%) had cardiomyopathy (mainly dilated cardiomyopathy (DCM)) and seven (8.8%) had a congenital heart defect. Childhood onset of cardiomyopathy was present in almost 10% of carriers. However, in only a slight majority (53.7%) was the left ventricular ejection fraction reduced and almost no arrhythmias or conduction disorders were noted. Moreover, only one carrier required heart transplantation and nine (11.3%) an implantable cardioverter defibrillator. In addition, the standardised mortality ratio for MYH7 carriers was not significantly increased. Whole exome sequencing in several cases with paediatric onset of DCM and one with isolated congenital heart defects did not reveal additional known disease-causing variants. Haplotype analysis suggests that the MYH7 variant is a founder mutation, and is therefore the first Dutch founder mutation identified in the MYH7 gene. The mutation appears to have originated in the western region of the province of South Holland between 500 and 900 years ago. CONCLUSION: Clinically, the p. (Asn1918Lys) mutation is associated with congenital heart defects and/or cardiomyopathy at young age but with a relatively benign course.

Recurrent and founder mutations in the Netherlands: the cardiac phenotype of DES founder mutations p.S13F and p.N342D.

BACKGROUND: Desmin-related myopathy (DRM) is an autosomally inherited skeletal and cardiac myopathy, mainly caused by dominant mutations in the desmin gene (DES). We describe new families carrying the p.S13F or p.N342D DES mutations, the cardiac phenotype of all carriers, and the founder effects. METHODS: We collected the clinical details of all carriers of p.S13F or p.N342D. The founder effects were studied using genealogy and haplotype analysis. RESULTS: We identified three new index patients carrying the p.S13F mutation and two new families carrying the p.N342D mutation. In total, we summarised the clinical details of 39 p.S13F carriers (eight index patients) and of 21 p.N342D carriers (three index patients). The cardiac phenotype of p.S13F carriers is fully penetrant and severe, characterised by cardiac conduction disease and cardiomyopathy, often with right ventricular involvement. Although muscle weakness is a prominent and presenting symptom in p.N342D carriers, their cardiac phenotype is similar to that of p.S13F carriers. The founder effects of p.S13F and p.N342D were demonstrated by genealogy and haplotype analysis. CONCLUSION: DRM may occur as an apparently isolated cardiological disorder. The cardiac phenotypes of the DES founder mutations p.S13F and p.N342D are characterised by cardiac conduction disease and cardiomyopathy, often with right ventricular involvement.

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.

Desmin-related myopathy.

Desmin-related myopathy (DRM) is an autosomally inherited skeletal and cardiac myopathy, mainly caused by dominant mutations in the desmin gene (DES). We provide (i) a literature review on DRM, including clinical manifestations, inheritance, molecular genetics, myopathology and management and (ii) a meta-analysis of reported DES mutation carriers, focusing on their clinical characteristics and potential genotype-phenotype correlations. Meta-analysis: DES mutation carriers (n = 159) with 40 different mutations were included. Neurological signs were present in 74% and cardiological signs in 74% of carriers (both neurological and cardiological signs in 49%, isolated neurological signs in 22%, and isolated cardiological signs in 22%). More than 70% of carriers exhibited myopathy or muscular weakness, with normal creatine kinase levels present in one third of them. Up to 50% of carriers had cardiomyopathy and around 60% had cardiac conduction disease or arrhythmias, with atrioventricular block as an important hallmark. Symptoms generally started during the 30s; a quarter of carriers died at a mean age of 49 years. Sudden cardiac death occurred in two patients with a pacemaker, suggesting a ventricular tachyarrhythmia as cause of death. The majority of DES mutations were missense mutations, mostly located in the 2B domain. Mutations in the 2B domain were predominant in patients with an isolated neurological phenotype, whereas head and tail domain mutations were predominant in patients with an isolated cardiological phenotype.

Haplotype sharing test maps genes for familial cardiomyopathies.

Identifying a mutation in a heterogeneous disease such as inherited cardiomyopathy is a challenge because classical methods, like linkage analysis, can often not be applied as there are too few meioses between affected individuals. However, if affected individuals share the same causal mutation, they will also share a genomic region surrounding it. High-density genotyping arrays are able to identify such regions shared among affected individuals. We hypothesize that the longest shared haplotype is most likely to contain the disease-causing mutation. We applied this method to two pedigrees: one with arrhythmogenic right ventricular cardiomyopathy (ARVC) and one with dilated cardiomyopathy (DCM), using high-density genome-wide SNP arrays. In the ARVC pedigree, the largest haplotype was on chromosome 12 and contained a causative PKP2 mutation. In the DCM pedigree, a causative MYH7 mutation was present on a large shared haplotype on chromosome 14. We calculated that a pedigree containing at least seven meioses has a high chance of correctly detecting the mutation-containing haplotype as the largest. Our data show that haplotype sharing analysis can assist in identifying causative genes in families with low penetrance Mendelian diseases, in which standard tools cannot be used due to lack of sufficient pedigree information.

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.).

Distinction between major and minor Bacillus signal peptidases based on phylogenetic and structural criteria.

The processing of secretory preproteins by signal peptidases (SPases) is essential for cell viability. As previously shown for Bacillus subtilis, only certain SPases of organisms containing multiple paralogous SPases are essential. This allows a distinction between SPases that are of major and minor importance for cell viability. Notably, the functional difference between major and minor SPases is not reflected clearly in sequence alignments. Here, we have successfully used molecular phylogeny to predict major and minor SPases. The results were verified with SPases from various bacilli. As predicted, the latter enzymes behaved as major or minor SPases when expressed in B. subtilis. Strikingly, molecular modeling indicated that the active site geometry is not a critical parameter for the classification of major and minor Bacillus SPases. Even though the substrate binding site of the minor SPase SipV is smaller than that of other known SPases, SipV could be converted into a major SPase without changing this site. Instead, replacement of amino-terminal residues of SipV with corresponding residues of the major SPase SipS was sufficient for conversion of SipV into a major SPase. This suggests that differences between major and minor SPases are based on activities other than substrate cleavage site selection.

A truncated soluble Bacillus signal peptidase produced in Escherichia coli is subject to self-cleavage at its active site.

Soluble forms of Bacillus signal peptidases which lack their unique amino-terminal membrane anchor are prone to degradation, which precludes their high-level production in the cytoplasm of Escherichia coli. Here, we show that the degradation of soluble forms of the Bacillus signal peptidase SipS is largely due to self-cleavage. First, catalytically inactive soluble forms of this signal peptidase were not prone to degradation; in fact, these mutant proteins were produced at very high levels in E. coli. Second, the purified active soluble form of SipS displayed self-cleavage in vitro. Third, as determined by N-terminal sequencing, at least one of the sites of self-cleavage (between Ser15 and Met16 of the truncated enzyme) strongly resembles a typical signal peptidase cleavage site. Self-cleavage at the latter position results in complete inactivation of the enzyme, as Ser15 forms a catalytic dyad with Lys55. Ironically, self-cleavage between Ser15 and Met16 cannot be prevented by mutagenesis of Gly13 and Ser15, which conform to the -1, -3 rule for signal peptidase recognition, because these residues are critical for signal peptidase activity.

TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway.

The recent discovery of a ubiquitous translocation pathway, specifically required for proteins with a twin-arginine motif in their signal peptide, has focused interest on its membrane-bound components, one of which is known as TatC. Unlike most organisms of which the genome has been sequenced completely, the Gram-positive eubacterium Bacillus subtilis contains two tatC-like genes denoted tatCd and tatCy. The corresponding TatCd and TatCy proteins have the potential to be involved in the translocation of 27 proteins with putative twin-arginine signal peptides of which approximately 6-14 are likely to be secreted into the growth medium. Using a proteomic approach, we show that PhoD of B. subtilis, a phosphodiesterase belonging to a novel protein family of which all known members are synthesized with typical twin-arginine signal peptides, is secreted via the twin-arginine translocation pathway. Strikingly, TatCd is of major importance for the secretion of PhoD, whereas TatCy is not required for this process. Thus, TatC appears to be a specificity determinant for protein secretion via the Tat pathway. Based on our observations, we hypothesize that the TatC-determined pathway specificity is based on specific interactions between TatC-like proteins and other pathway components, such as TatA, of which three paralogues are present in B. subtilis.

Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome.

One of the most salient features of Bacillus subtilis and related bacilli is their natural capacity to secrete a variety of proteins into their environment, frequently to high concentrations. This has led to the commercial exploitation of bacilli as major "cell factories" for secreted enzymes. The recent sequencing of the genome of B. subtilis has provided major new impulse for analysis of the molecular mechanisms underlying protein secretion by this organism. Most importantly, the genome sequence has allowed predictions about the composition of the secretome, which includes both the pathways for protein transport and the secreted proteins. The present survey of the secretome describes four distinct pathways for protein export from the cytoplasm and approximately 300 proteins with the potential to be exported. By far the largest number of exported proteins are predicted to follow the major "Sec" pathway for protein secretion. In contrast, the twin-arginine translocation "Tat" pathway, a type IV prepilin-like export pathway for competence development, and ATP-binding cassette transporters can be regarded as "special-purpose" pathways, through which only a few proteins are transported. The properties of distinct classes of amino-terminal signal peptides, directing proteins into the various protein transport pathways, as well as the major components of each pathway are discussed. The predictions and comparisons in this review pinpoint important differences as well as similarities between protein transport systems in B. subtilis and other well-studied organisms, such as Escherichia coli and the yeast Saccharomyces cerevisiae. Thus, they may serve as a lead for future research and applications.

A bacterial signal peptidase enhances processing of a recombinant single chain antibody fragment in insect cells.

The production of an antibody single chain fragment (scFv) in insect cells was accompanied by the formation of an insoluble intracellular precursor even with the inclusion of the bee melittin signal peptide. The presence of the precursor polypeptide suggests a limitation in the processing of the signal peptide so a baculovirus containing a signal peptidase from Bacillus subtilis (SipS) was constructed for expression studies. When the wild type SipS was coexpressed with scFv, preprocessed scFv fragments were no longer detected in insect cell lysates. Conversely, coexpression of scFv alone or with an inactive mutant SipS resulted in at least 30% of the intracellular polypeptide in an unprocessed form at 3 days post infection. Production of scFv in the medium was also enhanced in the presence of SipS; however, low secretion levels indicate the presence of a post-processing bottleneck.