Whole-Exome Sequencing Identifies Causative Mutations in Families with Congenital Anomalies of the Kidney and Urinary Tract.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) are the most prevalent cause of kidney disease in the first three decades of life. Previous gene panel studies showed monogenic causation in up to 12% of patients with CAKUT. METHODS: We applied whole-exome sequencing to analyze the genotypes of individuals from 232 families with CAKUT, evaluating for mutations in single genes known to cause human CAKUT and genes known to cause CAKUT in mice. In consanguineous or multiplex families, we additionally performed a search for novel monogenic causes of CAKUT. RESULTS: In 29 families (13%), we detected a causative mutation in a known gene for isolated or syndromic CAKUT that sufficiently explained the patient's CAKUT phenotype. In three families (1%), we detected a mutation in a gene reported to cause a phenocopy of CAKUT. In 15 of 155 families with isolated CAKUT, we detected deleterious mutations in syndromic CAKUT genes. Our additional search for novel monogenic causes of CAKUT in consanguineous and multiplex families revealed a potential single, novel monogenic CAKUT gene in 19 of 232 families (8%). CONCLUSIONS: We identified monogenic mutations in a known human CAKUT gene or CAKUT phenocopy gene as the cause of disease in 14% of the CAKUT families in this study. Whole-exome sequencing provides an etiologic diagnosis in a high fraction of patients with CAKUT and will provide a new basis for the mechanistic understanding of CAKUT.

Mutations in TBX18 Cause Dominant Urinary Tract Malformations via Transcriptional Dysregulation of Ureter Development.

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life. Identification of single-gene mutations that cause CAKUT permits the first insights into related disease mechanisms. However, for most cases the underlying defect remains elusive. We identified a kindred with an autosomal-dominant form of CAKUT with predominant ureteropelvic junction obstruction. By whole exome sequencing, we identified a heterozygous truncating mutation (c.1010delG) of T-Box transcription factor 18 (TBX18) in seven affected members of the large kindred. A screen of additional families with CAKUT identified three families harboring two heterozygous TBX18 mutations (c.1570C>T and c.487A>G). TBX18 is essential for developmental specification of the ureteric mesenchyme and ureteric smooth muscle cells. We found that all three TBX18 altered proteins still dimerized with the wild-type protein but had prolonged protein half life and exhibited reduced transcriptional repression activity compared to wild-type TBX18. The p.Lys163Glu substitution altered an amino acid residue critical for TBX18-DNA interaction, resulting in impaired TBX18-DNA binding. These data indicate that dominant-negative TBX18 mutations cause human CAKUT by interference with TBX18 transcriptional repression, thus implicating ureter smooth muscle cell development in the pathogenesis of human CAKUT.

Exome Sequencing Discerns Syndromes in Patients from Consanguineous Families with Congenital Anomalies of the Kidneys and Urinary Tract.

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the leading cause of CKD in children, featuring a broad variety of malformations. A monogenic cause can be detected in around 12% of patients. However, the morphologic clinical phenotype of CAKUT frequently does not indicate specific genes to be examined. To determine the likelihood of detecting causative recessive mutations by whole-exome sequencing (WES), we analyzed individuals with CAKUT from 33 different consanguineous families. Using homozygosity mapping and WES, we identified the causative mutations in nine of the 33 families studied (27%). We detected recessive mutations in nine known disease-causing genes: ZBTB24, WFS1, HPSE2, ATRX, ASPH, AGXT, AQP2, CTNS, and PKHD1 Notably, when mutated, these genes cause multiorgan syndromes that may include CAKUT as a feature (syndromic CAKUT) or cause renal diseases that may manifest as phenocopies of CAKUT. None of the above monogenic disease-causing genes were suspected on clinical grounds before this study. Follow-up clinical characterization of those patients allowed us to revise and detect relevant new clinical features in a more appropriate pathogenetic context. Thus, applying WES to the diagnostic approach in CAKUT provides opportunities for an accurate and early etiology-based diagnosis and improved clinical management.

Protein-protein interactions indicate composition of a 480 kDa SELMA complex in the second outermost membrane of diatom complex plastids.

Most secondary plastids of red algal origin are surrounded by four membranes and nucleus-encoded plastid proteins have to traverse these barriers. Translocation across the second outermost plastid membrane, the periplastidal membrane (PPM), is facilitated by a ERAD-(ER-associated degradation) derived machinery termed SELMA (symbiont-specific ERAD-like machinery). In the last years, important subunits of this translocator have been identified, which clearly imply compositional similarities between SELMA and ERAD. Here we investigated, via protein-protein interaction studies, if the composition of SELMA is comparable to the known ERAD complex. As a result, our data suggest that the membrane proteins of SELMA, the derlin proteins, are linked to the soluble sCdc48 complex via the UBX protein sUBX. This is similar to the ERAD machinery whereas the additional SELMA components, sPUB und a second Cdc48 copy might indicate the influence of functional constraints in developing a translocation machinery from ERAD-related factors. In addition, we show for the first time that a rhomboid protease is a central interaction partner of the membrane proteins of the SELMA system in complex plastids.

Mutations of the SLIT2-ROBO2 pathway genes SLIT2 and SRGAP1 confer risk for congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) account for 40-50% of chronic kidney disease that manifests in the first two decades of life. Thus far, 31 monogenic causes of isolated CAKUT have been described, explaining ~12% of cases. To identify additional CAKUT-causing genes, we performed whole-exome sequencing followed by a genetic burden analysis in 26 genetically unsolved families with CAKUT. We identified two heterozygous mutations in SRGAP1 in 2 unrelated families. SRGAP1 is a small GTPase-activating protein in the SLIT2-ROBO2 signaling pathway, which is essential for development of the metanephric kidney. We then examined the pathway-derived candidate gene SLIT2 for mutations in cohort of 749 individuals with CAKUT and we identified 3 unrelated individuals with heterozygous mutations. The clinical phenotypes of individuals with mutations in SLIT2 or SRGAP1 were cystic dysplastic kidneys, unilateral renal agenesis, and duplicated collecting system. We show that SRGAP1 is expressed in early mouse nephrogenic mesenchyme and that it is coexpressed with ROBO2 in SIX2-positive nephron progenitor cells of the cap mesenchyme in developing rat kidney. We demonstrate that the newly identified mutations in SRGAP1 lead to an augmented inhibition of RAC1 in cultured human embryonic kidney cells and that the SLIT2 mutations compromise the ability of the SLIT2 ligand to inhibit cell migration. Thus, we report on two novel candidate genes for causing monogenic isolated CAKUT in humans.

Photonic quadrupole topological insulator using orbital-induced synthetic flux.

The rich physical properties of multiatomic crystals are determined, to a significant extent, by the underlying geometry and connectivity of atomic orbitals. The mixing of orbitals with distinct parity representations, such as s and p orbitals, has been shown to be useful for generating systems that require alternating phase patterns, as with the sign of couplings within a lattice. Here we show that by breaking the symmetries of such mixed-orbital lattices, it is possible to generate synthetic magnetic flux threading the lattice. We use this insight to experimentally demonstrate quadrupole topological insulators in two-dimensional photonic lattices, leveraging both s and p orbital-type modes. We confirm the nontrivial quadrupole topology by observing the presence of protected zero-dimensional states, which are spatially confined to the corners, and by confirming that these states sit at mid-gap. Our approach is also applicable to a broader range of time-reversal-invariant synthetic materials that do not allow for tailored connectivity, and in which synthetic fluxes are essential.

Generalized laws of refraction and reflection at interfaces between different photonic artificial gauge fields.

Artificial gauge fields the control over the dynamics of uncharged particles by engineering the potential landscape such that the particles behave as if effective external fields are acting on them. Recent years have witnessed a growing interest in artificial gauge fields generated either by the geometry or by time-dependent modulation, as they have been enablers of topological phenomena and synthetic dimensions in many physical settings, e.g., photonics, cold atoms, and acoustic waves. Here, we formulate and experimentally demonstrate the generalized laws of refraction and reflection at an interface between two regions with different artificial gauge fields. We use the symmetries in the system to obtain the generalized Snell law for such a gauge interface and solve for reflection and transmission. We identify total internal reflection (TIR) and complete transmission and demonstrate the concept in experiments. In addition, we calculate the artificial magnetic flux at the interface of two regions with different artificial gauge fields and present a method to concatenate several gauge interfaces. As an example, we propose a scheme to make a gauge imaging system-a device that can reconstruct (image) the shape of an arbitrary wavepacket launched from a certain position to a predesigned location.

Artificial gauge field switching using orbital angular momentum modes in optical waveguides.

The discovery of artificial gauge fields controlling the dynamics of uncharged particles that otherwise elude the influence of standard electromagnetic fields has revolutionised the field of quantum simulation. Hence, developing new techniques to induce these fields is essential to boost quantum simulation of photonic structures. Here, we experimentally demonstrate the generation of an artificial gauge field in a photonic lattice by modifying the topological charge of a light beam, overcoming the need to modify the geometry along the evolution or impose external fields. In particular, we show that an effective magnetic flux naturally appears when a light beam carrying orbital angular momentum is injected into a waveguide lattice with a diamond chain configuration. To demonstrate the existence of this flux, we measure an effect that derives solely from the presence of a magnetic flux, the Aharonov-Bohm caging effect, which is a localisation phenomenon of wavepackets due to destructive interference. Therefore, we prove the possibility of switching on and off artificial gauge fields just by changing the topological charge of the input state, paving the way to accessing different topological regimes in a single structure, which represents an important step forward for optical quantum simulation.

Impact of different pretreatments and aging procedures on the flexural strength and phase structure of zirconia ceramics.

OBJECTIVE: To test the impact of zirconia pretreatment and aging on flexural strength and phase structure. METHODS: For flexural strength measurements, 180 3Y-TZP0.25 specimens were fabricated and pretreated: (i) air-abraded (105-µm alumina, 0.25MPa), (ii) air-abraded (50-µm alumina, 0.25MPa), (iii) air-abraded (30-µm silica-coated alumina, 0.28MPa) (iv) non-pretreated. Each pretreated group (n=15) was aged: (a) hydrothermal (134°C, 0.23MPa, 2h) (b) in a mastication simulator (1,200,000×, 5/55°C) and (c) not aged. The fractured specimens were stored dry for 5 years (23°C) for analysis of phase transformation. Additionally, specimens were fabricated from 3Y-TZP0.25 (n=12) and 3Y-TZP0.05 (n=8), pretreated (i, ii, iii, iv), and hydrothermally aged. Each air-abrasion method was alternated using 0.05, 0.25 and 0.4MPa pressure. The phase transformation was examined by Raman spectroscopy and surface topography by scanning electron microscope. Data were analyzed using univariate ANOVA with the Scheffé post hoc test and partial-eta-squared (ƞp²) (α=0.05). RESULTS: The highest impact on flexural strength was exerted by the pretreatment (ηP²=0.261, p<0.001), followed by interactions between pretreatment and aging (ηP²=0.077, p=0.033). Non-pretreated and non-aged specimens showed the lowest monoclinic percentage. Hydrothermal aging and 5 years of storage at room temperature increased the monolithic percentage of 3Y-TZP0.25. The highest phase transformation was observed in groups air-abraded with 105-µm alumina particles. Increasing pressure during the air-abrading process increased the content of the monoclinic phase in zirconia surfaces. SIGNIFICANCE: Air-abrasion with 30-µm silica-coated alumina powder can be recommended for pretreatment of 3Y-TZP0.25 and 3Y-TZP0.05. For air-abrasion using alumina powder lower pressure should be used.

The Central Vacuole of the Diatom Phaeodactylum tricornutum: Identification of New Vacuolar Membrane Proteins and of a Functional Di-leucine-based Targeting Motif.

Diatoms are unicellular organisms evolved by secondary endosymbiosis. Although studied in many aspects, the functions of vacuolar-like structures of these organisms are rarely investigated. One of these structures is a dominant central vacuole-like compartment with a marbled phenotype, which is supposed to represent a chrysolaminarin-storing and carbohydrate mobilization compartment. However, other functions as well as targeting of proteins to this compartment are not shown experimentally. In order to study trafficking of membrane proteins to the vacuolar membrane, we scanned the genome for intrinsic vacuolar membrane proteins and used one representative for targeting studies. Our work led to the identification of several proteins located in the vacuolar membrane as well as the sub-compartmentalized localization of one protein. In addition, we show that a di-leucine-based motif is an important signal for correct targeting to the central vacuole of diatoms, like it is in plants.

Compound analysis of gallstones using dual energy computed tomography--results in a phantom model.

PURPOSE: The potential of dual energy computed tomography (DECT) for the analysis of gallstone compounds was investigated. The main goal was to find parameters, that can reliably define high percentage (>70%) cholesterol stones without calcium components. MATERIALS AND METHODS: 35 gallstones were analyzed with DECT using a phantom model. Stone samples were put into specimen containers filled with formalin. Containers were put into a water-filled cylindrical acrylic glass phantom. DECT scans were performed using a tube voltage/current of 140 kV/83 mAs (tube A) and 80 kV/340 mAs (tube B). ROI-measurements to determine CT attenuation of each sector of the stones that had different appearance on the CT images were performed. Finally, semi-quantitative infrared spectroscopy (FTIR) of these sectors was performed for chemical analysis. RESULTS: ROI-measurements were performed in 45 different sectors in 35 gallstones. Sectors containing >70% of cholesterol and no calcium component (n=20) on FTIR could be identified with 95% sensitivity and 100% specificity on DECT. These sectors showed typical attenuation of -8+/-4 HU at 80 kV and +22+/-3 HU at 140 kV. Even the presence of a small calcium component (<10%) hindered the reliable identification of cholesterol components as such. CONCLUSION: Dual energy CT allows for reliable identification of gallstones containing a high percentage of cholesterol and no calcium component in this pre-clinical phantom model. Results from in vivo or anthropomorphic phantom trials will have to confirm these results. This may enable the identification of patients eligible for non-surgical treatment options in the future.