Unstable TTTTA/TTTCA expansions in MARCH6 are associated with Familial Adult Myoclonic Epilepsy type 3

Familial Adult Myoclonic Epilepsy (FAME) is a genetically heterogeneous disorder characterized by cortical tremor and seizures. Intronic TTTTA/TTTCA repeat expansions in SAMD12 (FAME1) are the main cause of FAME in Asia. Using genome sequencing and repeat-primed PCR, we identify another site of this repeat expansion, in MARCH6 (FAME3) in four European families. Analysis of single DNA molecules with nanopore sequencing and molecular combing show that expansions range from 3.3 to 14 kb on average. However, we observe considerable variability in expansion length and structure, supporting the existence of multiple expansion configurations in blood cells and fibroblasts of the same individual. Moreover, the largest expansions are associated with micro-rearrangements occurring near the expansion in 20% of cells. This study provides further evidence that FAME is caused by intronic TTTTA/TTTCA expansions in distinct genes and reveals that expansions exhibit an unexpectedly high somatic instability that can ultimately result in genomic rearrangements

Comprehensive characterization of RB1 mutant and MYCN amplified retinoblastoma cell lines

In retinoblastoma research tumor-derived cell lines remain an important model to investigate tumorigenesis and new therapy options, due to limited tumor material and lack of adequate animal models. A panel of 10 retinoblastoma cell lines was characterized with respect to mutation, methylation and expression of RB1 and MYCN. These established retinoblastoma cell lines represent the most frequent types of RB1 inactivation and together with the MYCN amplification status, three classes can be distinguished: RB1(mut)/MYCNnonA,RB1(mut)/MYCNA and RB1(wt)/MYCNA. MYCN amplification was identified in five cell lines, whereby two of them, RB522 and RB3823, harbor no aberration in RB1. Targeted sequencing of 160 genes often mutated in cancer identified only few variants in tumor-associated genes other than in RB1. None of these variants was recurrent. mRNA expression analyses of retinal markers, cell cycle regulators and members of the TP53 signaling pathway revealed a high variability between cell lines but no class-specific differences. The here presented thorough validation of retinoblastoma cell lines, including microsatellite analysis for cell line authentication, provides the basis for further in vitro studies on retinoblastoma

Translational investigation of electrophysiology in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) patients are at increased risk of ventricular arrhythmias and sudden cardiac death, which can occur even in the absence of structural changes of the heart. HCM mouse models suggest mutations in myofilament components to affect Ca2+ homeostasis and thereby favor arrhythmia development. Additionally, some of them show indications of pro-arrhythmic changes in cardiac electrophysiology. In this study, we explored arrhythmia mechanisms in mice carrying a HCM mutation in Mybpc3 (Mybpc3-KI) and tested the translatability of our findings in human engineered heart tissues (EHTs) derived from CRISPR/Cas9-generated homozygous MYBPC3 mutant (MYBPC3hom) in induced pluripotent stem cells (iPSC) and to left ventricular septum samples obtained from HCM patients. We observed higher arrhythmia susceptibility in contractility measurements of field-stimulated intact cardiomyocytes and ventricular muscle strips as well as in electromyogram recordings of Langendorff-perfused hearts from adult Mybpc3-KI mice than in wild-type (WT) controls. The latter only occurred in homozygous (Hom-KI) but not in heterozygous (Het-KI) mouse hearts. Both Het- and Hom-KI are known to display pro-arrhythmic increased Ca2+ myofilament sensitivity as a direct consequence of the mutation. In the electrophysiological characterization of the model, we observed smaller repolarizing K+ currents in single cell patch clamp, longer ventricular action potentials in sharp microelectrode recordings and longer ventricular refractory periods in Langendorff-perfused hearts in Hom-KI, but not Het-KI. Interestingly, reduced K+ channel subunit transcript levels and prolonged action potentials were already detectable in newborn, pre-hypertrophic Hom-KI mice. Human iPSC-derived MYBPC3hom EHTs, which genetically mimicked the Hom-KI mice, did exhibit lower mutant mRNA and protein levels, lower force, beating frequency and relaxation time, but no significant alteration of the force-Ca2+ relation in skinned EHTs. Furthermore, MYBPC3hom EHTs did show higher spontaneous arrhythmic behavior, whereas action potentials measured by sharp microelectrode did not differ to isogenic controls. Action potentials measured in septal myectomy samples did not differ between patients with HCM and patients with aortic stenosis, except for the only sample with a MYBPC3 mutation. The data demonstrate that increased myofilament Ca2+ sensitivity is not sufficient to induce arrhythmias in the Mybpc3-KI mouse model and suggest that reduced K+ currents can be a pro-arrhythmic trigger in Hom-KI mice, probably already in early disease stages. However, neither data from EHTs nor from left ventricular samples indicate relevant reduction of K+ currents in human HCM. Therefore, our study highlights the species difference between mouse and human and emphasizes the importance of research in human samples and human-like models.publishedVersionPeer reviewe

Unstable TTTTA/TTTCA expansions in MARCH6 are associated with Familial Adult Myoclonic Epilepsy type 3

Familial Adult Myoclonic Epilepsy (FAME) is a genetically heterogeneous disorder characterized by cortical tremor and seizures. Intronic TTTTA/TTTCA repeat expansions in SAMD12 (FAME1) are the main cause of FAME in Asia. Using genome sequencing and repeat-primed PCR, we identify another site of this repeat expansion, in MARCH6 (FAME3) in four European families. Analysis of single DNA molecules with nanopore sequencing and molecular combing show that expansions range from 3.3 to 14 kb on average. However, we observe considerable variability in expansion length and structure, supporting the existence of multiple expansion configurations in blood cells and fibroblasts of the same individual. Moreover, the largest expansions are associated with micro-rearrangements occurring near the expansion in 20% of cells. This study provides further evidence that FAME is caused by intronic TTTTA/TTTCA expansions in distinct genes and reveals that expansions exhibit an unexpectedly high somatic instability that can ultimately result in genomic rearrangements

Unstable TTTTA/TTTCA expansions in MARCH6 are associated with Familial Adult Myoclonic Epilepsy type 3

Familial Adult Myoclonic Epilepsy (FAME) is a genetically heterogeneous disorder characterized by cortical tremor and seizures. Intronic TTTTA/TTTCA repeat expansions in SAMD12 (FAME1) are the main cause of FAME in Asia. Using genome sequencing and repeat-primed PCR, we identify another site of this repeat expansion, in MARCH6 (FAME3) in four European families. Analysis of single DNA molecules with nanopore sequencing and molecular combing show that expansions range from 3.3 to 14 kb on average. However, we observe considerable variability in expansion length and structure, supporting the existence of multiple expansion configurations in blood cells and fibroblasts of the same individual. Moreover, the largest expansions are associated with micro-rearrangements occurring near the expansion in 20% of cells. This study provides further evidence that FAME is caused by intronic TTTTA/TTTCA expansions in distinct genes and reveals that expansions exhibit an unexpectedly high somatic instability that can ultimately result in genomic rearrangements