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Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of chronic kidney disease. Here, we identified recessive mutations in the gene encoding the actin-binding protein advillin (AVIL) in 3 unrelated families with SRNS. While all AVIL mutations resulted in a marked loss of its actin-bundling ability, truncation of AVIL also disrupted colocalization with F-actin, thereby leading to impaired actin binding and severing. Additionally, AVIL colocalized and interacted with the phospholipase enzyme PLCE1 and with the ARP2/3 actin-modulating complex. Knockdown of AVIL in human podocytes reduced actin stress fibers at the cell periphery, prevented recruitment of PLCE1 to the ARP3-rich lamellipodia, blocked EGF-induced generation of diacylglycerol (DAG) by PLCE1, and attenuated the podocyte migration rate (PMR). These effects were reversed by overexpression of WT AVIL but not by overexpression of any of the 3 patient-derived AVIL mutants. The PMR was increased by overexpression of WT Avilor PLCE1, or by EGF stimulation; however, this increased PMR was ameliorated by inhibition of the ARP2/3 complex, indicating that ARP-dependent lamellipodia formation occurs downstream of AVIL and PLCE1 function. Together, these results delineate a comprehensive pathogenic axis of SRNS that integrates loss of AVIL function with alterations in the action of PLCE1, an established SRNS protein., Introduction Nephrotic syndrome (NS) is characterized by proteinuria caused by disruption of the glomerular filtration barrier (1). It is the second most frequent cause of chronic kidney disease before the [...]

Aldosterone-producing adenomas (APAs) are benign tumors of the adrenal gland that constitutively produce the saltretaining steroid hormone aldosterone and cause millions of cases of severe hypertension worldwide. Either of 2 somatic mutations in the potassium channel KCNJ5 (G151R and L168R, hereafter referred to as [KCNJ5.sup.MUT]) in adrenocortical cellsaccount for half of APAs worldwide. These mutations alter channel selectivity to allow abnormal [Na.sup.+] conductance, resulting in membrane depolarization, calcium influx, aldosterone production, and cell proliferation. Because APA diagnosis requires a difficult invasive procedure, patients often remain undiagnosed and inadequately treated. Inhibitors of [KCNJ5.sup.MUT] could allow noninvasive diagnosis and therapy of APAs carrying KCNJ5 mutations. Here, we developed a high-throughput screen for rescue of [KCNJ5.sup.MUT]-induced lethality and identified a series of macrolide antibiotics, including roxithromycin, that potently inhibit [KCNJ5.sup.MUT], but not [KCNJ5.sup.WT]. Electrophysiology demonstrated direct [KCNJ5.sup.MUT] inhibition. In human aldosteroneproducing adrenocortical cancer cell lines, roxithromycin inhibited [KCNJ5.sup.MUT]-induced induction of CYP11B2 (encoding aldosterone synthase) expression and aldosterone production. Further exploration of macrolides showed that [KCNJ5.sup.MUT] was similarly selectively inhibited by idremcinal, a macrolide motilin receptor agonist, and by synthesized macrolide derivatives lacking antibiotic or motilide activity. Macrolide-derived selective [KCNJ5.sup.MUT] inhibitors thus have the potential to advance the diagnosis and treatment of APAs harboring [KCNJ5.sup.MUT]., Introduction Hypertension affects more than 1.1 billion people (1) and is a major risk factor for heart attack, stroke, and congestive heart failure, contributing to more than nine million deaths [...]

Dominantly inherited isolated polycystic liver disease (PCLD) consists of liver cysts that are radiologically and pathologically identical to those seen in autosomal dominant polycystic kidney disease, but without clinically relevant kidney cysts. The causative genes are known for fewer than 40% of PCLD index cases. Here, we have used whole exome sequencing in a discovery cohort of 102 unrelated patients who were excluded for mutations in the 2 most common PCLD genes, PRKCSH and SEC63, to identify heterozygous loss-of-function mutations in 3 additional genes, ALC8, CANAB, and SEC61B. Similarly to PRKCSH and SEC63, these genes encode proteins that are integral to the protein biogenesis pathway in the endoplasmic reticulum. We inactivated these candidate genes in cell line models to show that loss of function of each results in defective maturation and trafficking of polycystin-1, the central determinant of cyst pathogenesis. Despite acting in a common pathway, each PCLD gene product demonstrated distinct effects on polycystin-1 biogenesis. We also found enrichment on a genome-wide basis of heterozygous mutations in the autosomal recessive polycystic kidney disease gene PKHD1, indicating that adult PKHD1 carriers can present with clinical PCLD. These findings define genetic and biochemical modulators of polycystin-1 function and provide a more complete definition of the spectrum of dominant human polycystic diseases., Introduction Isolated polycystic liver disease (PCLD; also called ADPLD) presents in adult life with radiologically and pathologically identical liver cysts to those seen in autosomal dominant polycystic kidney disease (ADPKD), [...]

BACKGROUND. Recurrence and/or metastasis occurs in more than half of patients with head and neck squamous cell carcinoma (HNSCC), and these events pose the greatest threats to long-term survival. We set out to identify genetic alterations that underlie recurrent/metastatic HNSCC. METHODS. Whole-exome sequencing (WES) was performed on genomic DNA extracted from fresh-frozen whole blood and patient-matched tumor pairs from 13 HNSCC patients with synchronous lymph node metastases and 10 patients with metachronous recurrent tumors. Mutational concordance within and between tumor pairs was used to analyze the spatiotemporal evolution of HNSCC in individual patients and to identify potential therapeutic targets for functional evaluation. RESULTS. Approximately 86% and 60% of single somatic nucleotide variants (SSNVs) identified in synchronous nodal metastases and metachronous recurrent tumors, respectively, were transmitted from the primary index tumor. Genes that were mutated in more than one metastatic or recurrent tumor, but not in the respective primary tumors, include C17orf104, inositol 1,4,5-trisphosphate receptor, type 3 (ITPR3), and discoidin domain receptor tyrosine kinase 2 (DDR2). Select DDR2 mutations have been shown to confer enhanced sensitivity to SRC-family kinase (SFK) inhibitors in other malignancies. Similarly, HNSCC cell lines harboring endogenous and engineered DDR2 mutations were more sensitive to the SFK inhibitor dasatinib than those with WT DDR2. CONCLUSION. In this WES study of patient-matched tumor pairs in HNSCC, we found synchronous lymph node metastases to be genetically more similar to their paired index primary tumors than metachronous recurrent tumors. This study outlines a compendium of somatic mutations in primary, metastatic, and/or recurrent HNSCC cancers, with potential implications for precision medicine approaches. FUNDING. National Cancer Institute, American Cancer Society, Agency for Science, Technology and Research of Singapore, and Gilead Sciences Inc., Introduction Head and neck squamous cell carcinoma (HNSCC) is the seventh most common incident cancer worldwide, with more than 600,000 new cases each year (1). The major risk factors for [...]

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of progressive renal function decline and affects millions of people. In a recent study, 30% of SRNS cases evaluated were the result of monogenic mutations in 1 of 27 different genes. Here, using homozygosity mapping and whole-exome sequencing, we identified recessive mutations in kidney ankyrin repeat-containing protein 1 (KANK1), KANK2, and KANK4 in individuals with nephrotic syndrome. In an independent functional genetic screen of Drosophila cardiac nephrocytes, which are equivalents of mammalian podocytes, we determined that the Drosophila KANK homolog (dKank) is essential for nephrocyte function. RNAi-mediated knockdown of dKank in nephrocytes disrupted slit diaphragm filtration structures and lacuna channel structures. In rats, KANK1, KANK2, and KANK4 all localized to podocytes in glomeruli, and KANK1 partially colocalized with synaptopodin. Knockdown of kank2 in zebrafish recapitulated a nephrotic syndrome phenotype, resulting in proteinuria and podocyte foot process effacement. In rat glomeruli and cultured human podocytes, KANK2 interacted with ARHGDIA, a known regulator of RHO GTPases in podocytes that is dysfunctional in some types of nephrotic syndrome. Knockdown of KANK2 in cultured podocytes increased active GTP-bound RHOA and decreased migration. Together, these data suggest that KANK family genes play evolutionarily conserved roles in podocyte function, likely through regulating RHO GTPase signaling., Introduction Podocytes are polarized epithelial cells that are critical for the renal glomerular filtration barrier through their interdigitated foot processes. The filtration barrier is responsible for ultrafiltration of the blood [...]

Widespread reversion of genetic disease is rare; however, such events are particularly evident in some skin disorders in which normal clones develop on a background of affected skin. We previously demonstrated that mutations in keratin 10 (KRT10) cause ichthyosis with confetti (IWC), a severe dominant disorder that is characterized by progressive development of hundreds of normal skin spots via revertant mosaicism. Here, we report on a clinical and histological IWC subtype in which affected subjects have red, scaly skin at birth, experience worsening palmoplantar keratoderma in childhood, and develop hundreds of normal skin spots, beginning at around 20 years of age, that increase in size and number over time. We identified a causal de novo mutation in keratin 1 (KRT1). Similar to IWC-causing KRT10 mutations, this mutation in KRT1 resulted in a C-terminal frameshift, replacing 22 C-terminal amino acids with an alternate 30-residue peptide. Mutant KRT1 caused partial collapse of the cytoplasmic intermediate filament network and mislocalized to the nucleus. As with KRT10 mutations causing IWC, reversion of KRT1 mutations occurred via mitotic recombination. Because reversion is not observed with other diseasecausing keratin mutations, the results of this study implicate KRT1 and KRT10 C-terminal frameshift mutations in the high frequency of revertant mosaicism in IWC., Introduction While somatic loss-of-heterozygosity (LOH) events affecting tumor suppressor alleles are common in carcinogenesis (1), similar events leading to reversion of dominantly inherited disease traits are much less frequent. In [...]

Multiple studies have linked podocyte gene variants to diverse sporadic nephropathies, including HIV-1-associated nephropathy (HIVAN). We previously used linkage analysis to identify a major HIVAN susceptibility locus in mouse, HIVAN1. We performed expression quantitative trait locus (eQTL) analysis of podocyte genes in HIV-1 transgenic mice to gain further insight into genetic susceptibility to HIVAN. In 2 independent crosses, we found that transcript levels of the podocyte gene nephrosis 2 homolog (Nphs2), were heritable and controlled by an ancestral cis-eQTL that conferred a 3-fold variation in expression and produced reactive changes in other podocyte genes. In addition, Nphs2 expression was controlled by 2 trans-eQTLs that localized to the nephropathy susceptibility intervals HIVAN1 and HIVAN2. Transregulation of podocyte genes was observed in the absence of HIV-1 or glomerulosclerosis, indicating that nephropathy susceptibility alleles induce latent perturbations in the podocyte expression network. Presence of the HIV-1 transgene interfered with transregulation, demonstrating effects of gene-environment interactions on disease. These data demonstrate that transcript levels of Nphs2 and related podocyte-expressed genes are networked and suggest that the genetic lesions introduced by HIVAN susceptibility alleles perturb this regulatory pathway and transcriptional responses to HIV-1, increasing susceptibility to nephropathy., Introduction In the past decade, studies in humans and murine models have implicated defects in the glomerular visceral epithelial cells, the podocytes, as a principal factor in the development of [...]

To define the factors that control the tissue effects of IL-4, we compared the effects of Tg IL-4 in Balb/c and C57BL/6 mice. In the former, IL-4 caused modest eosinophilic [...]

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of progressive renal function decline and affects millions of people. In a recent study, 30% of SRNS cases evaluated were the result of monogenic mutations in 1 of 27 different genes. Here, using homozygosity mapping and whole-exome sequencing, we identified recessive mutations in kidney ankyrin repeat-containing protein 1 (KANK1), KANK2, and KANK4 in individuals with nephrotic syndrome. In an independent functional genetic screen of Drosophila cardiac nephrocytes, which are equivalents of mammalian podocytes, we determined that the Drosophila KANK homolog (dKank) is essential for nephrocyte function. RNAi-mediated knockdown of dKank in nephrocytes disrupted slit diaphragm filtration structures and lacuna channel structures. In rats, KANK1, KANK2, and KANK4 all localized to podocytes in glomeruli, and KANK1 partially colocalized with synaptopodin. Knockdown of kank2 in zebrafish recapitulated a nephrotic syndrome phenotype, resulting in proteinuria and podocyte foot process effacement. In rat glomeruli and cultured human podocytes, KANK2 interacted with ARHGDIA, a known regulator of RHO GTPases in podocytes that is dysfunctional in some types of nephrotic syndrome. Knockdown of KANK2 in cultured podocytes increased active GTP-bound RHOA and decreased migration. Together, these data suggest that KANK family genes play evolutionarily conserved roles in podocyte function, likely through regulating RHO GTPase signaling. [ABSTRACT FROM AUTHOR]

To define the factors that control the tissue effects of IL-4, we compared the effects of Tg IL-4 in Balb/c and C57BL/6 mice. In the former, IL-4 caused modest eosinophilic inflammation and mild airway fibrosis and did not shorten survival. In C57BL/6 mice, IL-4 caused profound eosinophilic inflammation, airway fibrosis, emphysematous alveolar destruction, and premature death. These differences could not be accounted for by changes in Th2 or Th1 cytokines, receptor components, STAT6 activation, MMPs, or cathepsins. In contrast, in C57BL/6 mice, alveolar remodeling was associated with decreased levels of tissue inhibitors of metalloproteinase 2, -3, and -4 and 1-antitrypsin, and fibrosis was associated with increased levels of total and bioactive TGF-ß1. Impressive differences in adenosine metabolism were also appreciated, with increased tissue adenosine levels and A1, A2B, and A3 adenosine receptor expression and decreased adenosine deaminase (ADA) activity in C57BL/6 animals. Treatment with ADA also reduced the inflammation, fibrosis, and emphysematous destruction and improved the survival of C57BL/6 Tg animals. These studies demonstrate that genetic influences control IL-4 effector pathways in the murine lung. They also demonstrate that IL-4 has different effects on adenosine metabolism in Balb/c and C57BL/6 mice and that these differences contribute to the different responses that IL-4 induces in these inbred animals. [ABSTRACT FROM AUTHOR]