Mutations in Danish patients with long QT syndrome and the identification of a large founder family with p.F29L in KCNH2.

BACKGROUND: Long QT syndrome (LQTS) is a cardiac ion channelopathy which presents clinically with palpitations, syncope or sudden death. More than 700 LQTS-causing mutations have been identified in 13 genes, all of which encode proteins involved in the execution of the cardiac action potential. The most frequently affected genes, covering > 90% of cases, are KCNQ1, KCNH2 and SCN5A. METHODS: We describe 64 different mutations in 70 unrelated Danish families using a routine five-gene screen, comprising KCNQ1, KCNH2 and SCN5A as well as KCNE1 and KCNE2. RESULTS: Twenty-two mutations were found in KCNQ1, 28 in KCNH2, 9 in SCN5A, 3 in KCNE1 and 2 in KCNE2. Twenty-six of these have only been described in the Danish population and 18 are novel. One double heterozygote (1.4% of families) was found. A founder mutation, p.F29L in KCNH2, was identified in 5 "unrelated" families. Disease association, in 31.2% of cases, was based on the type of mutation identified (nonsense, insertion/deletion, frameshift or splice-site). Functional data was available for 22.7% of the missense mutations. None of the mutations were found in 364 Danish alleles and only three, all functionally characterised, were recorded in the Exome Variation Server, albeit at a frequency of < 1:1000. CONCLUSION: The genetic etiology of LQTS in Denmark is similar to that found in other populations. A large founder family with p.F29L in KCNH2 was identified. In 48.4% of the mutations disease causation was based on mutation type or functional analysis.

Localization of A11-reactive oligomeric species in prion diseases.

AIMS: To investigate in prion diseases the in-situ localization of prion protein oligomers sharing a common epitope with amyloid oligomers involved in a range of neurodegenerative diseases. METHODS AND RESULTS: We performed immunohistochemistry on sporadic Creutzfeldt-Jakob disease (sCJD) (n = 9) and hereditary Gerstmann-Sträussler-Scheinker disease (GSS) (n = 1) specimens with the anti-oligomer antibody A11 to determine the localization of reactive species. We found that A11 reactivity in the sCJD specimens was localized to the cerebral and cerebellar cortices both in spongiform and adjacent, non-spongiform areas, reminiscent of multicentric or diffuse plaques. In the GSS specimens, we found that staining was closely associated with kuru-like plaques, and that A11-reactive species colocalized with protease-resistant prion protein (Prp(Sc)). We also observed sporadic neuronal cytosolic staining in both types of specimen. CONCLUSIONS: We confirm that intracellular and extracellular A11-reactive species are present in situ in sCJD cases and GSS, and that immunoreactivity for A11 and Prp(Sc) overlaps. We argue that the A11-reactive species are indeed composed of oligomeric Prp(Sc), and suggest that the toxic effects of Prp(Sc) oligomers could be related to the generic oligomeric conformation recognized by A11.

The KCNE genes in hypertrophic cardiomyopathy: a candidate gene study.

BACKGROUND: The gene family KCNE1-5, which encode modulating ß-subunits of several repolarising K+-ion channels, has been associated with genetic cardiac diseases such as long QT syndrome, atrial fibrillation and Brugada syndrome. The minK peptide, encoded by KCNE1, is attached to the Z-disc of the sarcomere as well as the T-tubules of the sarcolemma. It has been suggested that minK forms part of an "electro-mechanical feed-back" which links cardiomyocyte stretching to changes in ion channel function. We examined whether mutations in KCNE genes were associated with hypertrophic cardiomyopathy (HCM), a genetic disease associated with an improper hypertrophic response. RESULTS: The coding regions of KCNE1, KCNE2, KCNE3, KCNE4, and KCNE5 were examined, by direct DNA sequencing, in a cohort of 93 unrelated HCM probands and 188 blood donor controls.Fifteen genetic variants, four previously unknown, were identified in the HCM probands. Eight variants were non-synonymous and one was located in the 3'UTR-region of KCNE4. No disease-causing mutations were found and no significant difference in the frequency of genetic variants was found between HCM probands and controls. Two variants of likely functional significance were found in controls only. CONCLUSIONS: Mutations in KCNE genes are not a common cause of HCM and polymorphisms in these genes do not seem to be associated with a propensity to develop arrhythmia.

Evolutionary dissection of mtDNA hg H: a susceptibility factor for hypertrophic cardiomyopathy.

Mitochondrial DNA (mtDNA) haplogroup (hg) H has been reported as a susceptibility factor for hypertrophic cardiomyopathy (HCM). This was established in genetic association studies, however, the SNP or SNP's that are associated with the increased risk have not been identified. Hg H is the most frequent European mtDNA hg with greater than 80 subhaplogroups (subhgs) each defined by specific SNPs. We tested the hypothesis that the distribution of H subhgs might differ between HCM patients and controls. The subhg H distribution in 55 HCM index cases was compared to that of two Danish mtDNA hg H control groups (n = 170 and n = 908, respectively). In the HCM group, H and 12 different H subhgs were found. All these, except subhgs H73, were also found in both control groups. The HCM group was also characterized by a higher proportion of H3 compared to H2. In the HCM group the H3/H2 proportion was 1.7, whereas it was 0.45 and 0.54 in the control groups. This tendency was replicated in an independent group of Hg H HCM index cases (n = 39) from Queensland, Australia, where the H3/H2 ratio was 1.5. In conclusion, the H subhgs distribution differs between HCM cases and controls, but the difference is subtle, and the understanding of the pathogenic significance is hampered by the lack of functional studies on the subhgs of H.

Private mitochondrial DNA variants in danish patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetic cardiac disease primarily caused by mutations in genes coding for sarcomeric proteins. A molecular-genetic etiology can be established in ~60% of cases. Evolutionarily conserved mitochondrial DNA (mtDNA) haplogroups are susceptibility factors for HCM. Several polymorphic mtDNA variants are associated with a variety of late-onset degenerative diseases and affect mitochondrial function. We examined the role of private, non-haplogroup associated, mitochondrial variants in the etiology of HCM. In 87 Danish HCM patients, full mtDNA sequencing revealed 446 variants. After elimination of 312 (69.9%) non-coding and synonymous variants, a further 109 (24.4%) with a global prevalence > 0.1%, three (0.7%) haplogroup associated and 19 (2.0%) variants with a low predicted in silico likelihood of pathogenicity, three variants: MT-TC: m.5772G>A, MT-TF: m.644A>G, and MT-CYB: m.15024G>A, p.C93Y remained. A detailed analysis of these variants indicated that none of them are likely to cause HCM. In conclusion, private mtDNA mutations are frequent, but they are rarely, if ever, associated with HCM.

The role of CAV3 in long-QT syndrome: clinical and functional assessment of a caveolin-3/Kv11.1 double heterozygote versus caveolin-3 single heterozygote.

BACKGROUND: Mutations in CAV3, coding for caveolin-3, the major constituent scaffolding protein of cardiac caveolae, have been associated with skeletal muscle disease, cardiomyopathy, and most recently long-QT syndrome (LQTS) and sudden infant death syndrome. We examined the occurrence of CAV3 mutations in a large cohort of patients with LQTS. METHODS AND RESULTS: Probands with LQTS (n=167) were screened for mutations in CAV3 using direct DNA sequencing. A single proband (0.6%) was found to be a heterozygous carrier of a previously described missense mutation, caveolin-3:p.T78M. The proband was also a heterozygous carrier of the trafficking-deficient Kv11.1:p.I400N mutation. The caveolin-3:p.T78M mutation was found isolated in 3 family members, none of whom had a prolonged QTc interval. Coimmunoprecipitations of caveolin-3 and the voltage-gated potassium channel subunit (Kv11.1) were performed, and the electrophysiological classification of the Kv11.1 mutant was carried out by patch-clamp technique in human embryonic kidney 293 cells. Furthermore, the T-wave morphology was assessed in mutation carriers, double mutation carriers, and nonmutation carriers by applying a morphology combination score. The morphology combination score was normal for isolated caveolin-3:p.T78M carriers and of LQT2 type in double heterozygotes. CONCLUSIONS: Mutations in CAV3 are rare in LQTS. Furthermore, caveolin-3:p.T78M did not exhibit a LQTS phenotype. Because no association has ever been found between LQTS and isolated CAV3 mutations, we suggest that LQTS9 is considered a provisional entity.

Mitochondrial haplogroups modify the risk of developing hypertrophic cardiomyopathy in a Danish population.

Hypertrophic cardiomyopathy (HCM) is a genetic disorder caused by mutations in genes coding for proteins involved in sarcomere function. The disease is associated with mitochondrial dysfunction. Evolutionarily developed variation in mitochondrial DNA (mtDNA), defining mtDNA haplogroups and haplogroup clusters, is associated with functional differences in mitochondrial function and susceptibility to various diseases, including ischemic cardiomyopathy. We hypothesized that mtDNA haplogroups, in particular H, J and K, might modify disease susceptibility to HCM. Mitochondrial DNA, isolated from blood, was sequenced and haplogroups identified in 91 probands with HCM. The association with HCM was ascertained using two Danish control populations. Haplogroup H was more prevalent in HCM patients, 60% versus 46% (p = 0.006) and 41% (p = 0.003), in the two control populations. Haplogroup J was less prevalent, 3% vs. 12.4% (p = 0.017) and 9.1%, (p = 0.06). Likewise, the UK haplogroup cluster was less prevalent in HCM, 11% vs. 22.1% (p = 0.02) and 22.8% (p = 0.04). These results indicate that haplogroup H constitutes a susceptibility factor and that haplogroup J and haplogroup cluster UK are protective factors in the development of HCM. Thus, constitutive differences in mitochondrial function may influence the occurrence and clinical presentation of HCM. This could explain some of the phenotypic variability in HCM. The fact that haplogroup H and J are also modifying factors in ischemic cardiomyopathy suggests that mtDNA haplotypes may be of significance in determining whether a physiological hypertrophy develops into myopathy. mtDNA haplotypes may have the potential of becoming significant biomarkers in cardiomyopathy.

MT-CYB mutations in hypertrophic cardiomyopathy.

Mitochondrial dysfunction is a characteristic of heart failure. Mutations in mitochondrial DNA, particularly in MT-CYB coding for cytochrome B in complex III (CIII), have been associated with isolated hypertrophic cardiomyopathy (HCM). We hypothesized that MT-CYB mutations might play an important causal or modifying role in HCM. The MT-CYB gene was sequenced from DNA isolated from blood from 91 Danish HCM probands. Nonsynonymous variants were analyzed by bioinformatics, molecular modeling and simulation. Two germline-inherited, putative disease-causing, nonsynonymous variants: m.15024G>A; p.C93Y and m.15482T>C; p.S246P were identified. Modeling showed that the p.C93Y mutation leads to disruption of the tertiary structure of Cytb by helix displacement, interfering with protein-heme interaction. The p.S246P mutation induces a diproline structure, which alters local secondary structure and induces a kink in the protein backbone, interfering with macromolecular interactions. These molecular effects are compatible with a leaky phenotype, that is, limited but progressive mitochondrial dysfunction. In conclusion, we find that rare, putative leaky mtDNA variants in MT-CYB can be identified in a cohort of HCM patients. We propose that further patients with HCM should be examined for mutations in MT-CYB in order to clarify the role of these variants.