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Cerebral cavernous malformations (CCMs) are prevalent slow-flow vascular lesions which harbour the risk to develop intracranial haemorrhages, focal neurological deficits, and epileptic seizures. Autosomal dominantly inherited CCMs were found to be associated with heterozygous inactivating mutations in 3 genes, CCM1(KRIT1), CCM2(MGC4607), and CCM3(PDCD10) in 1999, 2003 and 2005, respectively. Despite the availability of high-throughput sequencing techniques, no further CCM gene has been published since. Here, we report on the identification of an autosomal dominantly inherited frameshift mutation in a gene of thus far unknown function, FAM222B(C17orf63), through exome sequencing of CCM patients mutation-negative for CCM1-3. A yeast 2-hybrid screen revealed interactions of FAM222B with the tubulin cytoskeleton and STAMBP which is known to be associated with microcephaly-capillary malformation syndrome. However, a phenotype similar to existing models was not found, neither in fam222bb/fam222ba double mutant zebrafish generated by transcription activator-like effector nucleases nor in an in vitro sprouting assay using human umbilical vein endothelial cells transfected with siRNA against FAM222B. These observations led to the assumption that aberrant FAM222B is not involved in the formation of CCMs.
In classical models of tumorigenesis, the accumulation of tumor promoting chromosomal aberrations is described as a gradual process. Next-generation sequencing-based methods have recently revealed complex patterns of chromosomal aberrations, which are beyond explanation by these classical models of karyotypic evolution of tumor genomes. Thus, the term chromothripsis has been introduced to describe a phenomenon, where temporarily and spatially confined genomic instability results in dramatic chromosomal rearrangements limited to segments of one or a few chromosomes. Simultaneously arising and misrepaired DNA double-strand breaks are also the cause of another phenomenon called chromoplexy, which is characterized by the presence of chained translocations and interlinking deletion bridges involving several chromosomes. In this study, we demonstrate the genome-wide identification of chromosomal translocations based on the analysis of translocation-associated changes in spatial proximities of chromosome territories on the example of the cutaneous T-cell lymphoma cell line Se-Ax. We have used alterations of intra- and interchromosomal interaction probabilities as detected by genome-wide chromosome conformation capture (Hi-C) to infer the presence of translocations and to fine-map their breakpoints. The outcome of this analysis was subsequently compared to datasets on DNA copy number alterations and gene expression. The presence of chained translocations within the Se-Ax genome, partly connected by intervening deletion bridges, indicates a role of chromoplexy in the etiology of this cutaneous T-cell lymphoma. Notably, translocation breakpoints were significantly overrepresented in genes, which highlight gene-associated biological processes like transcription or other gene characteristics as a possible cause of the observed complex rearrangements. Given the relevance of chromosomal aberrations for basic and translational research, genome-wide high-resolution analysis of structural chromosomal aberrations will gain increasing importance.
The transcriptome of non-coding RNA (ncRNA) species is increasingly focused in Alzheimer’s disease (AD) research. NcRNAs comprise, among others, transfer RNAs, long non-coding RNAs and microRNAs (miRs), each with their own specific biological function. We used smallRNASeq to assess miR expression in the hippocampus of young (3 month old) and aged (8 month old) Tg4-42 mice, a model system for sporadic AD, as well as age-matched wildtype controls. Tg4-42 mice express N-truncated Aβ4–42, develop age-related neuron loss, reduced neurogenesis and behavioral deficits. Our results do not only confirm known miR-AD associations in Tg4-42 mice, but more importantly pinpoint 22 additional miRs associated to the disease. Twenty-five miRs were differentially expressed in both aged Tg4-42 and aged wildtype mice while eight miRs were differentially expressed only in aged wildtype mice, and 33 only in aged Tg4-42 mice. No significant alteration in the miRNome was detected in young mice, which indicates that the changes observed in aged mice are down-stream effects of Aβ-induced pathology in the Tg4-42 mouse model for AD. Targets of those miRs were predicted using miRWalk. For miRs that were differentially expressed only in the Tg4-42 model, 128 targets could be identified, whereas 18 genes were targeted by miRs only differentially expressed in wildtype mice and 85 genes were targeted by miRs differentially expressed in both mouse models. Genes targeted by differentially expressed miRs in the Tg4-42 model were enriched for negative regulation of long-term synaptic potentiation, learning or memory, regulation of trans-synaptic signaling and modulation of chemical synaptic transmission obtained. This untargeted miR sequencing approach supports previous reports on the Tg4-42 mice as a valuable model for AD. Furthermore, it revealed miRs involved in AD, which can serve as biomarkers or therapeutic targets.
Chronic kidney disease (CKD) is a major public health burden affecting more than 500 million people worldwide. Podocytopathies are the main cause for the majority of CKD cases due to pathogenic morphological as well as molecular biological alterations of postmitotic podocytes. Podocyte de-differentiation is associated with foot process effacement subsequently leading to proteinuria. Since currently no curative drugs are available, high throughput screening methods using a small number of animals are a promising and essential tool to identify potential drugs against CKD in the near future. Our study presents the implementation of the already established mouse GlomAssay as a semi-automated high-throughput screening method—shGlomAssay—allowing the analysis of several hundreds of FDA-verified compounds in combination with downstream pathway analysis like transcriptomic and proteomic analyses from the same samples, using a small number of animals. In an initial prescreening we have identified vitamin D3 and its analog calcipotriol to be protective on podocytes. Furthermore, by using RT-qPCR, Western blot, and RNA sequencing, we found that mRNA and protein expression of nephrin, the vitamin D receptor and specific podocyte markers were significantly up-regulated due to vitamin D3- and calcipotriol-treatment. In contrast, kidney injury markers were significantly down-regulated. Additionally, we found that vitamin D3 and calcipotriol have had neither influence on the expression of the miR-21 and miR-30a nor on miR-125a/b, a miRNA described to regulate the vitamin D receptor. In summary, we advanced the established mouse GlomAssay to a semi-automated high-throughput assay and combined it with downstream analysis techniques by using only a minimum number of animals. Hereby, we identified the vitamin D signaling pathway as podocyte protective and to be counteracting their de-differentiation.