Innovative and New Approaches to Laboratory Diagnosis of Zika and Dengue: A Meeting Report

Epidemics of dengue, Zika, and other arboviral diseases are increasing in frequency and severity. Current efforts to rapidly identify and manage these epidemics are limited by the short diagnostic window in acute infection, the extensive serologic cross-reactivity among flaviviruses, and the lack of point-of-care diagnostic tools to detect these viral species in primary care settings. The Partnership for Dengue Control organized a workshop to review the current landscape of Flavivirus diagnostic tools, identified current gaps, and developed strategies to accelerate the adoption of promising novel technologies into national programs. The rate-limiting step to bringing new diagnostic tools to the market is access to reference materials and well-characterized clinical samples to facilitate performance evaluation. We suggest the creation of an international laboratory-response consortium for flaviviruses with a decentralized biobank of well-characterized samples to facilitate assay validation. Access to proficiency panels are needed to ensure quality control, in additional to in-country capacity building

Loss-of-Function Mutations in WDR73 Are Responsible for Microcephaly and Steroid-Resistant Nephrotic Syndrome: Galloway-Mowat Syndrome

Galloway-Mowat syndrome is a rare autosomal-recessive condition characterized by nephrotic syndrome associated with microcephaly and neurological impairment. Through a combination of autozygosity mapping and whole-exome sequencing, we identified WDR73 as a gene in which mutations cause Galloway-Mowat syndrome in two unrelated families. WDR73 encodes a WD40-repeat-containing protein of unknown function. Here, we show that WDR73 was present in the brain and kidney and was located diffusely in the cytoplasm during interphase but relocalized to spindle poles and astral microtubules during mitosis. Fibroblasts from one affected child and WDR73-depleted podocytes displayed abnormal nuclear morphology, low cell viability, and alterations of the microtubule network. These data suggest that WDR73 plays a crucial role in the maintenance of cell architecture and cell survival. Altogether, WDR73 mutations cause Galloway-Mowat syndrome in a particular subset of individuals presenting with late-onset nephrotic syndrome, postnatal microcephaly, severe intellectual disability, and homogenous brain MRI features. WDR73 is another example of a gene involved in a disease affecting both the kidney glomerulus and the CNS

Nail-patella syndrome. Overview on clinical and molecular findings.

Item does not contain fulltextNail-patella syndrome (NPS) is a rare autosomal dominant pleiotropic disorder characterized by dysplasia of the nails, patellar aplasia or hypoplasia, iliac horns, dysplasia of the elbows, and frequently glaucoma and progressive nephropathy. The recent identification of the causative gene for this syndrome has initiated further studies of the phenotype and molecular pathogenesis of kidney disease in NPS. The gene underlying NPS, LMX1B, is a LIM-homeodomain transcription factor involved in normal patterning of the dorsoventral axis of the limb during development and early morphogenesis of the glomerular basement membrane. Molecular studies of Lmx1b, combined with genetic and immunohistochemical investigation of different alpha chains of type IV collagen in the Lmx1b null mice kidney, a mouse model for NPS, have provided evidence that Lmx1b is involved in the pathogenesis of NPS glomerulopathy. At present evidence for a correlation between the presence and severity of the renal and extrarenal anomalies and LMX1B genotype is lacking. This review focuses on the recent advances in clinical and molecular genetic studies of NPS

An update on Zika virus infection.

The epidemic history of Zika virus began in 2007, with its emergence in Yap Island in the western Pacific, followed in 2013-14 by a larger epidemic in French Polynesia, south Pacific, where the first severe complications and non-vector-borne transmission of the virus were reported. Zika virus emerged in Brazil in 2015 and was declared a national public health emergency after local researchers and physicians reported an increase in microcephaly cases. In 2016, WHO declared the recent cluster of microcephaly cases and other neurological disorders reported in Brazil a global public health emergency. Similar clusters of microcephaly cases were also observed retrospectively in French Polynesia in 2014. In 2015-16, Zika virus continued its spread to cause outbreaks in the Americas and the Pacific, and the first outbreaks were reported in continental USA, Africa, and southeast Asia. Non-vector-borne transmission was confirmed and Zika virus was established as a cause of severe neurological complications in fetuses, neonates, and adults. This Review focuses on important updates and gaps in the knowledge of Zika virus as of early 2017

Interpreting the autobiographical archive

In this study we have investigated the presence of apoptotic cells in renal biopsy material of seven patients with hemolytic uremic syndrome (HUS) by using an improved and stringent terminal deoxynucleotidyl nick-end labeling (TUNEL) technique. Renal biopsy material was taken in the second or third week after onset of the disease. Renal biopsy material of patients with minimal lesions nephrotic syndrome or thin basement syndrome were used as control. It has been reported that nonapoptotic cells can be labeled nonspecifically due to proteinase K pretreatment or a delay in fixation when only TUNEL technique is used. In post mortem material this delay in fixation is seen. Moreover, it has been described that mainly nonapoptotic cells that shows signs of active gene transcription can be labeled in this nonspecific way. For this reason we used the TUNEL technique in combination with a label for RNA synthesis and splicing factor (SC-35). Indeed, we found nonspecific labeling of nonapoptotic nuclei in biopsy material of HUS patients, but not in control biopsy material. By using co-labeling with RNA synthesis factor SC-35, we were able to identify true apoptotic cells. There was a significant increase (p < 0.05) in the presence of apoptotic cells in biopsy material of HUS patients compared with material of controls. About 80 % of apoptotic cells were detected in tubuli and only 20 % in glomeruli of the renal biopsies of HUS patients. Furthermore, most apoptotic cells were detected in those patients that had received peritoneal dialysis suggesting that there is a relationship between severity of the disease and amount of apoptotic cells. The finding of apoptotic cells suggest that apoptosis plays a role in HUS

Glomerular extracellular matrix and growth factors in diffuse mesangial sclerosis.

Item does not contain fulltextDiffuse mesangial sclerosis, isolated (IDMS) or observed in the context of Denys-Drash syndrome (DDS) due to WT1 mutation, is characterized by early onset nephrotic syndrome progressing to renal failure. A striking morphological feature is the rapid development of glomerulosclerosis. The aims of our study were: (1) to analyze the glomerular distribution of extracellular matrix (ECM) antigens at the early stage of DMS, (2) to determine the composition of the ECM accumulated within the mesangial areas and leading to glomerular sclerosis, and (3) to analyze the expression of growth factors, transforming growth factor-beta 1 (TGF beta 1) and platelet-derived growth factor A (PDGFA). In glomeruli of patients with IDMS and DDS, the glomerular basement membrane (GBM) expression of the heparan sulfate chain of heparan sulfate proteoglycan (HSPG) was decreased from the early stage of DMS, at a time when ECM proteins retained a normal distribution. In fully developed lesions, mesangial and subendothelial accumulation of collagenous and noncollagenous glycoproteins normally expressed in the mesangial area (types IV [alpha 1(IV)2 alpha 2(IV)] and VI collagen, beta 1 laminin, fibronectin, tenascin, and perlecan) increased with progression of mesangial sclerosis. This was associated with mesangial expression of proteins normally restricted to the GBM (agrin, alpha 1/alpha 5, beta 2, and gamma 1 laminin chains) and with accumulation of chondroitin sulfate proteoglycan. The distribution of the alpha 3-alpha 5 chains of type IV collagen was normal. Focal accumulation of types I, III, and V collagen was seen only in severely sclerotic glomeruli. Expression of growth factors TGF beta 1 and PDGFA was increased in 9 of 10 and 5 of 10 patients, respectively. Early decreased GBM expression of the heparan sulfate chain of HSPG could play a role in the proteinuria of DMS patients. Changes in the composition of the ECM accumulated within the mesangial areas are not specific. We speculate that deregulation of the expression of growth factors normally downregulated by WT1, is one of the factors responsible for the rapid and massive mesangial deposition of basement membrane material in DDS

Glomerular extracellular matrix and growth factors in diffuse mesangial sclerosis.

Item does not contain fulltextDiffuse mesangial sclerosis, isolated (IDMS) or observed in the context of Denys-Drash syndrome (DDS) due to WT1 mutation, is characterized by early onset nephrotic syndrome progressing to renal failure. A striking morphological feature is the rapid development of glomerulosclerosis. The aims of our study were: (1) to analyze the glomerular distribution of extracellular matrix (ECM) antigens at the early stage of DMS, (2) to determine the composition of the ECM accumulated within the mesangial areas and leading to glomerular sclerosis, and (3) to analyze the expression of growth factors, transforming growth factor-beta 1 (TGF beta 1) and platelet-derived growth factor A (PDGFA). In glomeruli of patients with IDMS and DDS, the glomerular basement membrane (GBM) expression of the heparan sulfate chain of heparan sulfate proteoglycan (HSPG) was decreased from the early stage of DMS, at a time when ECM proteins retained a normal distribution. In fully developed lesions, mesangial and subendothelial accumulation of collagenous and noncollagenous glycoproteins normally expressed in the mesangial area (types IV [alpha 1(IV)2 alpha 2(IV)] and VI collagen, beta 1 laminin, fibronectin, tenascin, and perlecan) increased with progression of mesangial sclerosis. This was associated with mesangial expression of proteins normally restricted to the GBM (agrin, alpha 1/alpha 5, beta 2, and gamma 1 laminin chains) and with accumulation of chondroitin sulfate proteoglycan. The distribution of the alpha 3-alpha 5 chains of type IV collagen was normal. Focal accumulation of types I, III, and V collagen was seen only in severely sclerotic glomeruli. Expression of growth factors TGF beta 1 and PDGFA was increased in 9 of 10 and 5 of 10 patients, respectively. Early decreased GBM expression of the heparan sulfate chain of HSPG could play a role in the proteinuria of DMS patients. Changes in the composition of the ECM accumulated within the mesangial areas are not specific. We speculate that deregulation of the expression of growth factors normally downregulated by WT1, is one of the factors responsible for the rapid and massive mesangial deposition of basement membrane material in DDS

Two splice variants of the Wilms' tumor 1 gene have distinct functions during sex determination and nephron formation

Alternative splicing of Wt1 results in the insertion or omission of the three amino acids KTS between zinc fingers 3 and 4. In vitro experiments suggest distinct molecular functions for + and -KTS isoforms. We have generated mouse strains in which specific isoforms have been removed. Heterozygous mice with a reduction of +KTS levels develop glomerulosclerosis and represent a model for Frasier syndrome. Homozygous mutants of both strains die after birth due to kidney defects. Strikingly, mice lacking +KTS isoforms show a complete XY sex reversal due to a dramatic reduction of Sry expression levels. Our data demonstrate distinct functions for the two splice variants and place the +KTS variants as important regulators for Sry in the sex determination pathway

A murine model of Denys-Drash syndrome reveals novel transcriptional targets of WT1 in podocytes

The Wilms tumor-suppressor gene WT1, a key player in renal development, also has a crucial role in maintenance of the glomerulus in the mature kidney. However, molecular pathways orchestrated by WT1 in podocytes, where it is highly expressed, remain unknown. Their defects are thought to modify the cross-talk between podocytes and other glomerular cells and ultimately lead to glomerular sclerosis, as observed in diffuse mesangial sclerosis (DMS) a nephropathy associated with WT1 mutations. To identify podocyte WT1 targets, we generated a novel DMS mouse line, performed gene expression profiling in isolated glomeruli and identified excellent candidates that may modify podocyte differentiation and growth factor signaling in glomeruli. Scel, encoding sciellin, a protein of the cornified envelope in the skin, and Sulf1, encoding a 6-O endosulfatase, are shown to be expressed in wild-type podocytes and to be strongly down-regulated in mutants. Co-expression of Wt1, Scel and Sulf1 was also found in a mesonephric cell line, and siRNA-mediated knockdown of WT1 decreased Scel and Sulf1 mRNAs and proteins. By ChIP we show that Scel and Sulf1 are direct WT1 targets. Cyp26a1, encoding an enzyme involved in the degradation of retinoic acid, is shown to be up-regulated in mutant podocytes. Cyp26a1 may play a role in the development of glomerular lesions but does not seem to be regulated by WT1. These results provide novel clues in our understanding of normal glomerular function and early events involved in glomerulosclerosis

In vivo expression of putative LMX1B targets in nail-patella syndrome kidneys.

The nail-patella syndrome (NPS) is characterized by nail and bone abnormalities, associated with glomerular involvement in approximately 40% of patients. Typical glomerular changes consist of fibrillar material in the irregularly thickened glomerular basement membrane. NPS is inherited as an autosomal dominant trait and caused by heterozygous loss of function mutations in LMX1B, a member of the LIM homeodomain protein family. Mice with homozygous inactivation of the gene exhibit nail and skeletal defects, similar to those observed in patients, associated with glomerular abnormalities. Strong reduction in the glomerular expression of the alpha3 and alpha4 chains of type IV collagen, and of podocin and CD2AP, two podocyte proteins critical for glomerular function, has been observed in Lmx1b null mice. The expression of these proteins appeared to be regulated by Lmx1b. To determine whether these changes in podocyte gene expression are involved in the development of NPS nephropathy, using immunohistological techniques, we analyzed the podocyte phenotype and the renal distribution of type IV collagen chains in the kidneys of seven NPS patients with severe glomerular disease. We also examined the nature of the fibrillar material present within the glomerular extracellular matrix. The glomerular basement membrane fibrillar material was specifically labeled with anti-type III collagen antibodies, suggesting a possible regulation of type III collagen expression by LMX1B. The expression of the alpha3 and alpha4 chains of type IV collagen, and of podocin and CD2AP, was found to be normal in the seven patients. These findings indicate that heterozygous mutations of LMX1B do not appear to dramatically affect the expression of type IV collagen chains, podocin, or CD2AP in NPS patients