One of the most common observation in Mediterranean areas is the migration of contractional deformation and associated slabs through time toward external orogenic areas, associated with lower plate ...crustal accretion. The Dinarides orogen of Central Europe is an optimal place to study such a sequence of contractional deformation. Compared with other areas, contraction in the Dinarides was less overprinted by subsequent extension, while a remnant of the subducted slab is observed in a far external orogenic position. Understanding the deformational evolution of the Dinarides is hampered by the reduced availability of kinematic studies. Therefore, we have performed a surface kinematic study in the external parts of the Dinarides. By correlating with available geophysical and evolutionary constraints, we constructed two large‐scale, kinematically controlled regional transects. The results demonstrate a long‐lived evolution of shortening that affected the Dinarides lower orogenic plate. While the Late Jurassic‐earliest Cretaceous deformation was associated with an earlier obduction moment, the latest Cretaceous onset of continental collision has gradually focused deformation at inherited rheological weakness zones. We show that shortening was interrupted by a period of Miocene extension that affected all orogenic areas and created the Dinarides Lake System. The extension was followed by renewed shortening, which started during the latest Miocene and remains presently active, whose kinematics in the central and SE part of the Dinarides is revealed for the first time by our study. These results indicate a lower plate crustal accretion mechanism that was spatially and temporally connected with gradual slab retreat in the Dinarides.
Key Points
The migration of contractional deformation in the Dinarides can be associated with lower plate crustal accretion during slab rollback
Along the Dinarides orogenic strike there is a lateral variability of contractional deformation
The entire Dinarides orogen was affected by Miocene extension and subsequent inversion
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Understanding the structural and kinematic effects of indentation is still debated due to the large number of competing mechanisms associated with the complex orogenic build-up. Among the many ...examples available worldwide, the evolution of the Adriatic continental microplate in the Mediterranean domain provides one of the best places to understand the mechanics of indentation. This understanding is hampered by the lack of structural and kinematic data in the Dinarides, an orogen situated at the critical transition between the Alps, Albanides and Hellenides, and across the Adriatic margin of the Apennines. We have studied the less known area of the central and south-eastern Dinarides by focussing on collecting a new kinematic dataset for structures formed during the Adriatic indentation, which postdates the main Late Jurassic – Paleogene orogenic structuration. Our results are in agreement with previous interpretations of an early-middle Miocene period of extension that affected the entire orogen across its strike and is incompatible with indentation effects in the studied parts of the Dinarides. More importantly, we demonstrate for the first time that the post- middle Miocene Dinarides deformation was characterized by a coherent regional system of large offset dextral strike-slip faults, which transfer gradually their offsets to thrusts and high-angle reverse faults. The overall deformation transfer mechanism can be described as a special class of continental restraining bends or stepovers, whose geometry is controlled by rheological distribution. The integration of our results in the larger geodynamic context shows that the post-middle Miocene Dinarides fault system accommodates the differential motion between the N- to NE-wards Adriatic indentation and the rapid S- to SW- ward movement of a Hellenides area situated SE of the Kefalonia Fault, driven by the Aegean slab-roll back.
•Along-strike transfer of deformation during indentation and subduction;•Across-strike transfer of deformation accommodated by the interplay between strike-slip and reverse faulting;•Continental restraining bends and stepovers controlled by rheological distribution;•Regional Dinarides fault system accommodating the differential motion between the Adriatic indentation and Aegean slab roll-back.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Neurofibromatosis type 1 (NF1) is caused by inactivating mutations in NF1. Due to the size, complexity, and high mutation rate at the NF1 locus, the identification of causative variants can be ...challenging. To obtain a molecular diagnosis in 15 individuals meeting diagnostic criteria for NF1, we performed transcriptome analysis (RNA‐seq) on RNA obtained from cultured skin fibroblasts. In each case, routine molecular DNA diagnostics had failed to identify a disease‐causing variant in NF1. A pathogenic variant or abnormal mRNA splicing was identified in 13 cases: 6 deep intronic variants and 2 transposon insertions causing noncanonical splicing, 3 postzygotic changes, 1 branch point mutation and, in 1 case, abnormal splicing for which the responsible DNA change remains to be identified. These findings helped resolve the molecular findings for an additional 17 individuals in multiple families with NF1, demonstrating the utility of skin‐fibroblast‐based transcriptome analysis for molecular diagnostics. RNA‐seq improves mutation detection in NF1 and provides a powerful complementary approach to DNA‐based methods. Importantly, our approach is applicable to other genetic disorders, particularly those caused by a wide variety of variants in a limited number of genes and specifically for individuals in whom routine molecular DNA diagnostics did not identify the causative variant.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. ...Transcriptome analysis (RNA sequencing RNA-seq) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals’ cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual’s specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics.
We present a user-friendly web application to detect pathogenic outlier gene expression and mRNA splicing in transcriptome data. Application of this pipeline in a cohort of 67 undiagnosed individuals with neurodevelopmental disorders increased the diagnostic yield by 13%. This approach will facilitate wider implementation of RNA-seq in routine genome diagnostics.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
See Uzquiano and Francis (doi:10.1093/brain/awz048) for a scientific commentary on this article.
Mutations in RTTN, which encodes Rotatin, give rise to various brain malformations. Vandervore et al. ...reveal mitotic failure, aneuploidy, apoptosis and defective ciliogenesis in patient cells. Rotatin binds to myosin subunits in the leading edge of human neurons, which may explain the proliferation and migration defects observed.
Abstract
Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects.
Neurofibromatosis type 1 (NF1) and Legius syndrome (LS) are caused by inactivating variants in NF1 and SPRED1. NF1 encodes neurofibromin (NF), a GTPase-activating protein (GAP) for RAS that interacts ...with the SPRED1 product, Sprouty-related protein with an EVH (Ena/Vasp homology) domain 1 (SPRED1). Obtaining a clinical and molecular diagnosis of NF1 or LS can be challenging due to the phenotypic diversity, the size and complexity of the NF1 and SPRED1 loci, and uncertainty over the effects of some NF1 and SPRED1 variants on pre-mRNA splicing and/or protein expression and function. To improve NF1 and SPRED1 variant classification and establish pathogenicity for NF1 and SPRED1 variants identified in individuals with NF1 or LS, we analyzed patient RNA by RT-PCR and performed in vitro exon trap experiments and estimated NF and SPRED1 protein expression, RAS GAP activity, and interaction. We obtained evidence to support pathogenicity according to American College of Medical Genetics guidelines for 73/114 variants tested, demonstrating the utility of functional approaches for NF1 and SPRED1 variant classification and NF and LS diagnostics.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Pediatric cardiomyopathies are a clinically and genetically heterogeneous group of heart muscle disorders associated with high morbidity and mortality. Although knowledge of the genetic basis of ...pediatric cardiomyopathy has improved considerably, the underlying cause remains elusive in a substantial proportion of cases.
Exome sequencing was used to screen for the causative genetic defect in a pair of siblings with rapidly progressive dilated cardiomyopathy and death in early infancy. Protein expression was assessed in patient samples, followed by an in vitro tail-anchored protein insertion assay and functional analyses in zebrafish.
We identified compound heterozygous variants in the highly conserved
gene (arsA arsenite transporter, ATP-binding, homolog), which encodes an ATPase required for post-translational membrane insertion of tail-anchored proteins. The c.913C>T variant on the paternal allele is predicted to result in a premature stop codon p.(Gln305*), and likely explains the decreased protein expression observed in myocardial tissue and skin fibroblasts. The c.488T>C variant on the maternal allele results in a valine to alanine substitution at residue 163 (p.Val163Ala). Functional studies showed that this variant leads to protein misfolding as well as less effective tail-anchored protein insertion. Loss of
in zebrafish resulted in reduced cardiac contractility and early lethality. In contrast to wild-type mRNA, injection of either mutant mRNA failed to rescue this phenotype.
Biallelic variants in
cause severe pediatric cardiomyopathy and early death. Our findings point toward a critical role of the tail-anchored membrane protein insertion pathway in vertebrate cardiac function and disease.
Recent studies have reported that alleles in the premutation range in the FMR1 gene in males result in increased FMR1 mRNA levels and at the same time mildly reduced FMR1 protein levels. Some elderly ...males with premutations exhibit an unique neurodegenerative syndrome characterized by progressive intention tremor and ataxia. We describe neurohistological, biochemical and molecular studies of the brains of mice with an expanded CGG repeat and report elevated Fmr1 mRNA levels and intranuclear inclusions with ubiquitin, Hsp40 and the 20S catalytic core complex of the proteasome as constituents. An increase was observed of both the number and the size of the inclusions during the course of life, which correlates with the progressive character of the cerebellar tremor/ataxia syndrome in humans. The observations in expanded-repeat mice support a direct role of the Fmr1 gene, by either CGG expansion per se or by mRNA level, in the formation of the inclusions and suggest a correlation between the presence of intranuclear inclusions in distinct regions of the brain and the clinical features in symptomatic premutation carriers. This mouse model will facilitate the possibilities to perform studies at the molecular level from onset of symptoms until the final stage of the disease.
Mutations to the TSC1 and TSC2 genes cause the disease tuberous sclerosis complex. The TSC1 and TSC2 gene products form a protein complex that integrates multiple metabolic signals to regulate the ...activity of the target of rapamycin (TOR) complex 1 (TORC1) and thereby control cell growth. Here we investigate the quaternary structure of the TSC1-TSC2 complex by gel filtration and coimmunoprecipitation.
TSC1 and TSC2 co-eluted in high molecular weight fractions by gel filtration. Coimmunoprecipitation of distinct tagged TSC1 and TSC2 isoforms demonstrated that TSC1-TSC2 complexes contain multiple TSC1 and TSC2 subunits.
TSC1 and TSC2 interact to form large complexes containing multiple TSC1 and TSC2 subunits.
Pediatric cardiomyopathies are a clinically and genetically heterogeneous group of heart muscle disorders associated with high morbidity and mortality. Although knowledge of the genetic basis of ...pediatric cardiomyopathy has improved considerably, the underlying cause remains elusive in a substantial proportion of cases. In this study, we sought to identify the underlying genetic defect in a pair of siblings with rapidly progressive dilated cardiomyopathy leading to death in early infancy.Whole exome sequencing revealed compound heterozygous variants in both siblings in the highly conserved ASNA1 gene. ASNA1, also known as TRC40 or GET3, is an ATPase required for post-translational insertion of tail-anchored proteins into the endoplasmic reticulum. Histological and immunohistochemical analysis in myocardial tissue from both patients revealed disruption of intercalated discs and decreased ASNA1 expression. ASNA1 protein levels were also decreased in fibroblasts of one patient. In an in vitro tail-anchored protein insertion assay, recombinant mutant ASNA1 was more aggregation prone and less functional than the wild-type protein. Loss of asna1 in zebrafish resulted in reduced cardiac contractility, decreased circulation, and early lethality. Overexpression of human wild-type ASNA1 mRNA rescued this phenotype, whereas injection of either paternal or maternal mutant mRNA showed no improvement.In conclusion, we propose ASNA1, encoding a cytosolic targeting factor for tail-anchored proteins, as a novel gene for pediatric cardiomyopathy with acute onset and rapid progression. We hypothesize that mislocalization of cardiac tail-anchored proteins (such as emerin and phospholamban) contributes to myocardial dysfunction. Our findings point toward a critical role of the tail-anchored membrane protein insertion pathway in vertebrate heart development and disease.
PDF
