Genetic link between renal birth defects and congenital heart disease

Structural birth defects in the kidney and urinary tract are observed in 0.5% of live births and are a major cause of end-stage renal disease, but their genetic aetiology is not well understood. Here we analyse 135 lines of mice identified in large-scale mouse mutagenesis screen and show that 29% of mutations causing congenital heart disease (CHD) also cause renal anomalies. The renal anomalies included duplex and multiplex kidneys, renal agenesis, hydronephrosis and cystic kidney disease. To assess the clinical relevance of these findings, we examined patients with CHD and observed a 30% co-occurrence of renal anomalies of a similar spectrum. Together, these findings demonstrate a common shared genetic aetiology for CHD and renal anomalies, indicating that CHD patients are at increased risk for complications from renal anomalies. This collection of mutant mouse models provides a resource for further studies to elucidate the developmental link between renal anomalies and CHD

Discovery of four recessive developmental disorders using probabilistic genotype and phenotype matching among 4,125 families.

Discovery of most autosomal recessive disease-associated genes has involved analysis of large, often consanguineous multiplex families or small cohorts of unrelated individuals with a well-defined clinical condition. Discovery of new dominant causes of rare, genetically heterogeneous developmental disorders has been revolutionized by exome analysis of large cohorts of phenotypically diverse parent-offspring trios. Here we analyzed 4,125 families with diverse, rare and genetically heterogeneous developmental disorders and identified four new autosomal recessive disorders. These four disorders were identified by integrating Mendelian filtering (selecting probands with rare, biallelic and putatively damaging variants in the same gene) with statistical assessments of (i) the likelihood of sampling the observed genotypes from the general population and (ii) the phenotypic similarity of patients with recessive variants in the same candidate gene. This new paradigm promises to catalyze the discovery of novel recessive disorders, especially those with less consistent or nonspecific clinical presentations and those caused predominantly by compound heterozygous genotypes

Exome-chip meta-analysis identifies novel loci associated with cardiac conduction, including ADAMTS6.

BACKGROUND: Genome-wide association studies conducted on QRS duration, an electrocardiographic measurement associated with heart failure and sudden cardiac death, have led to novel biological insights into cardiac function. However, the variants identified fall predominantly in non-coding regions and their underlying mechanisms remain unclear. RESULTS: Here, we identify putative functional coding variation associated with changes in the QRS interval duration by combining Illumina HumanExome BeadChip genotype data from 77,898 participants of European ancestry and 7695 of African descent in our discovery cohort, followed by replication in 111,874 individuals of European ancestry from the UK Biobank and deCODE cohorts. We identify ten novel loci, seven within coding regions, including ADAMTS6, significantly associated with QRS duration in gene-based analyses. ADAMTS6 encodes a secreted metalloprotease of currently unknown function. In vitro validation analysis shows that the QRS-associated variants lead to impaired ADAMTS6 secretion and loss-of function analysis in mice demonstrates a previously unappreciated role for ADAMTS6 in connexin 43 gap junction expression, which is essential for myocardial conduction. CONCLUSIONS: Our approach identifies novel coding and non-coding variants underlying ventricular depolarization and provides a possible mechanism for the ADAMTS6-associated conduction changes.BH

A structural analysis of the architectural diversity in the centriolar cartwheel-containing region

Centrioles are evolutionary conserved, supramolecular organelles, characterized by a blade-like assembly of nine microtubule triplets. They are critical for essential cellular functions of centrosome assembly and cilium templating. Polarized with distinct microtubule-binding structures along the proximal-distal axis, the proximal region mediates centriole assembly. Seminal studies of centriole architecture in species such as Trichonympha and Chlamydomonas have provided insights into the structural organization of the proximal region, but the question remains: is this organization ubiquitous through evolution? Using cryo-electron tomography to visualize the native proximal architecture of Naegleria gruberi, Chlamydomonas reinhardii, Paramecium tetraurelia, and human centrioles, we unveiled that the cartwheel structure is a stack of ring pairs with a 4 nm periodicity, the cartwheel inner density is a conserved structural entity, and that the cartwheel is connected to the A-C linker through the triplet base, rooted in the pinhead. This work provides unprecedented insights into centriole biogenesis. Furthermore, the tomograms generated in these 4 species allowed us to explore other centriolar regions. We identified that at the centriolar core region, a helical inner scaffold interconnects the microtubule triplets, and helps to main centriole cohesion and integrity. This work has unveiled sub-structural connections previously undescribed and defined the boundaries of the proximal and central core regions. Our work has generated the most structurally comprehensive model of the centriole to date

Visualizing the native cellular organization by coupling cryofixation with expansion microscopy (Cryo-ExM)

Cryofixation has proven to be the gold standard for efficient preservation of native cell ultrastructure compared to chemical fixation, but this approach is not widely used in fluorescence microscopy owing to implementation challenges. Here, we develop Cryo-ExM, a method that preserves native cellular organization by coupling cryofixation with expansion microscopy. This method bypasses artifacts associated with chemical fixation and its simplicity will contribute to its widespread use in super-resolution microscopy.</p

Isolation and Fluorescence Imaging for Single-particle Reconstruction of <em>Chlamydomonas</em> Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies

An In-depth Guide to the Ultrastructural Expansion Microscopy (U-ExM) of <em>Chlamydomonas reinhardtii</em>

Expansion microscopy is an innovative method that enables super-resolution imaging of biological materials using a simple confocal microscope. The principle of this method relies on the physical isotropic expansion of a biological specimen cross-linked to a swellable polymer, stained with antibodies, and imaged. Since its first development, several improved versions of expansion microscopy and adaptations for different types of samples have been produced. Here, we show the application of ultrastructure expansion microscopy (U-ExM) to investigate the 3D organization of the green algae Chlamydomonas reinhardtii cellular ultrastructure, with a particular emphasis on the different types of sample fixation that can be used, as well as compatible staining procedures including membrane.</p

Kinesin-1 activity recorded in living cells with a precipitating dye

Kinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. The ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate (QPD-OTf) for kinesin-1 that yields a precipitating dye along its walking path on microtubules (MTs). QPD-OTf enables to monitor native kinesin-1 transport activity in cellulo without external modifications. In vitro assays show that kinesin-1 and MTs are sufficient to yield fluorescent crystals; in cells, kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time. These findings are further supported by docking studies, which suggest the binding of the activity-based substrate in the nucleotide binding site of kinesin-1