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Mutations in WNK1 and WNK4 lead to familial hyperkalemic hypertension (FHHt). Because FHHt associates net positive [Na.sup.+] balance together with [K.sup.+] and [H.sup.+] renal retention, the identification of WNK1 and WNK4 led to a new paradigm to explain how aldosterone can promote either [Na.sup.+] reabsorption or [K.sup.+] secretion in a hypovolemic or hyperkalemic state, respectively. WNK1 gives rise to L-WNK1, an ubiquitous kinase, and KS-WNK1, a kinase-defective isoform expressed in the distal convoluted tubule. By inactivating KS-WNK1 in mice, we show here that this isoform is an important regulator of sodium transport. KS-[WNK1.sup.-/-] mice display an increased activity of the Na-Cl cotransporter NCC, expressed specifically in the distal convoluted tubule, where it participates in the fine tuning of sodium reabsorption. Moreover, the expression of the ROMK and BKCa potassium channels was modified in KS-[WNK1.sup.-/-] mice, indicating that KS-WNK1 is also a regulator of potassium transport in the distal nephron. Finally, we provide an alternative model for FHHt. Previous studies suggested that the activation of NCC plays a central role in the development of hypertension and hyperkalemia. Even though the increase in NCC activity in KS-[WNK1.sup.-/-] mice was less pronounced than in mice overexpressing a mutant form of WNK4, our study suggests that the activation of Na--Cl cotransporter is not sufficient by itself to induce a hyperkalemic hypertension and that the deregulation of other channels, such as the Epithelial [Na.sup.+] channel (ENaC), is probably required. transgenic animals | water-electrolyte balance doi/ 10.1073/pnas.1006128107

Protein coding genes constitute only approximately 1% of the human genome but harbor 85% of the mutations with large effects on disease-related traits. Therefore, efficient strategies for selectively sequencing complete coding regions (i.e., 'whole exome') have the potential to contribute to the understanding of rare and common human diseases. Here we report a method for whole-exome sequencing coupling Roche/NimbleGen whole exome arrays to the Illumina DNA sequencing platform. We demonstrate the ability to capture approximately 95% of the targeted coding sequences with high sensitivity and specificity for detection of homozygous and heterozygous variants. We illustrate the utility of this approach by making an unanticipated genetic diagnosis of congenital chloride diarrhea in a patient referred with a suspected diagnosis of Bartter syndrome, a renal salt-wasting disease. The molecular diagnosis was based on the finding of a homozygous missense D652N mutation at a position in SLC26A3 (the known congenital chloride diarrhea locus) that is virtually completely conserved in orthologues and paralogues from invertebrates to humans, and clinical follow-up confirmed the diagnosis. To our knowledge, whole-exome (or genome) sequencing has not previously been used to make a genetic diagnosis. Five additional patients suspected to have Bartter syndrome but who did not have mutations in known genes for this disease had homozygous deleterious mutations in SLC26A3. These results demonstrate the clinical utility of whole-exome sequencing and have implications for disease gene discovery and clinical diagnosis. Bartter syndrome | congenital chloride diarrhea | next-generation sequencing | whole-exome sequencing | personal genomes www.pnas.org/cgi/doi/10.1073/pnas.0910672106

WNK4 (with no lysine kinase 4) inhibits ROMK channel activity in the distal nephron by stimulating clathrin-dependent endocytosis, an effect attenuated by SGK1 (serum-glucocorticoids-induced kinase)mediated phosphorylation. It has been suggested that increased ROMK activity because of SGK1-mediated inhibition of WNK4 plays a role in promoting renal K secretion in response to elevated serum K or high K (HK) intake. In contrast, intravascular volume depletion also increases SGK1 activity but fails to stimulate ROMK channels and K secretion. Because HK intake decreases Src family protein tyrosine kinase (PTK) activity an inhibitor of ROMK channels, it is possible that Src family PTK may modulate the effects of SGK1 on WNK4. Here, we show that c-Src prevents SGK1 from attenuating WNK4's inhibition of ROMK activity. This effect of c-Src was WNK4-dependent because c-Src had no effect on ROMK harboring mutation at the site of c-Src phosphorylation (R1Y337A) in the absence of WNK4. Moreover, expression c-Src diminished the SGK1-mediated increase in serine phosphorylation of WNK4, suggesting that c-Src enhances WNK4-mediated inhibition of ROMK channels by suppressing the SGK1-induced phosphorylation. This notion is also supported by the observation that c-Src was not able to modulate the interaction between SGK1 and WNK4 mutants ([WNK4.sub.S1169A] or [WNK4.sub.S1169D]) in which an SGK1-phosphorylation site (serine 1169) was mutated by alanine or aspartate. We conclude that c-Src inhibits SGK1-mediated phosphorylation hereby restoring the WNK4-mediated inhibition of ROMK channels thus suppressing K secretion. collecting duct | hyperkalemia | K secretion | Kir1.1

We describe members of 4 kindreds with a previously unrecognized syndrome characterized by seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (hypokalemia, metabolic alkalosis, and hypomagnesemia). By analysis of linkage we localize the putative causative gene to a 2.5-Mb segment of chromosome 1q23.2-23.3. Direct DNA sequencing of KCNJ10, which encodes an inwardly rectifying [K.sup.+] channel, identifies previously unidentified missense or nonsense mutations on both alleles in all affected subjects. These mutations alter highly conserved amino acids and are absent among control chromosomes. Many of these mutations have been shown to cause loss of function in related [K.sup.+] channels. These findings demonstrate that loss-of-function mutations in KCNJ10 cause this syndrome, which we name SESAME. KCNJ10 is expressed in gila in the brain and spinal cord, where it is believed to take up [K.sup.+] released by neuronal repolarization, in cochlea, where it is involved in the generation of endolymph, and on the basolateral membrane in the distal nephron. We propose that KCNJ10 is required in the kidney for normal salt reabsorption in the distal convoluted tubule because of the need for [K.sup.+] recycling across the basolateral membrane to enable normal activity of the [Na.sup.+]-[K.sup.+]-ATPase; loss of this function accounts for the observed electrolyte defects. Mice deficient for KCNJ10 show a related phenotype with seizures, ataxia, and hearing loss, further supporting KCNJ10's role in this syndrome. These findings define a unique human syndrome, and establish the essential role of basolateral [K.sup.+] channels in renal electrolyte homeostasis. Gitelman syndrome | hypokalemia | hypomagnesemia | inwardly rectifying [K.sup.+] channel | renal salt wasting

Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and high serum [K.sup.+] levels (hyperkalemia). WNK4 has distinct functional states that regulate the balance between renal salt reabsorption and [K.sup.+] secretion by modulating the activities of renal transporters and channels, including the Na-Cl cotransporter NCC and the [K.sup.+] channel ROMK. WNK4's functions could enable differential responses to intravascular volume depletion (hypovolemia) and hyperkalemia. Because hypovolemia is uniquely associated with high angiotensin II (Angll) levels, Angll signaling might modulate WNK4 activity. We show that Angll signaling in Xenopus oocytes increases NCC activity by abrogating WNK4's inhibition of NCC but does not alter WNK4's inhibition of ROMK. This effect requires Angll, its receptor AT1R, and WNK4, and is prevented by the AT1R inhibitor losartan. NCC activity is also increased by WNK4 harboring mutations found in PHAII, and this activity cannot be further augmented by Angll signaling, consistent with PHAII mutations providing constitutive activation of the signaling pathway between AT1R and NCC. Angll's effect on NCC is also dependent on the kinase SPAK because dominant-negative SPAK or elimination of the SPAK binding motif in NCC prevent activation of NCC by Angll signaling. These effects extend to mammalian cells. Angll increases phosphorylation of specific sites on SPAK and NCC that are necessary for activation of each in mpkDCT cells. These findings place WNK4 in the signaling pathway between Angll and NCC, and provide a mechanism by which hypovolemia maximizes renal salt reabsoprtion without concomitantly increasing [K.sup.+] secretion. angiotensin II receptor | hypertension | distal convoluted tubule | salt reabsorption | thiazide

The [Na.sup.+]:[K.sup.+]:[2Cl.sup.-] cotransporter (NKCC2) is the target of loop diuretics and is mutated in Bartter's syndrome, a heterogeneous autosomal recessive disease that impairs salt reabsorption in the kidney's thick ascending limb (TAL). Despite the importance of this cation/chloride cotransporter (CCC), the mechanisms that underlie its regulation are largely unknown. Here, we show that intracellular chloride depletion in Xenopus laevis oocytes, achieved by either coexpression of the K-CI cotransporter KCC2 or low-chloride hypotonic stress, activates NKCC2 by promoting the phosphorylation of three highly conserved threonines (96, 101, and 111) in the amino terminus. Elimination of these residues renders NKCC2 unresponsive to reductions of [[Cl-].sub.i]. The chloride-sensitive activation of NKCC2 requires the interaction of two serine-threonine kinases, WNK3 (related to WNK1 and WNK4, genes mutated in a Mendelian form of hypertension) and SPAK (a Ste20-type kinase known to interact with and phosphorylate other CCCs). WNK3 is positioned upstream of SPAK and appears to be the chloride-sensitive kinase. Elimination of WNK3's unique SPAK-binding motif prevents its activation of NKCC2, as does the mutation of threonines 96, 101, and 111. A catalytically inactive WNK3 mutant also completely prevents NKCC2 activation by intracellular chloride depletion. Together these data reveal a chloride-sensing mechanism that regulates NKCC2 and provide insight into how increases in the level of intracellular chloride in TAL cells, as seen in certain pathological states, could drastically impair renal salt reabsorption. ion transport | loop of Henle | protein serine-threonine kinases | hypertension | diuretics

Phosphate homeostasis is central to diverse physiologic processes including energy homeostasis, formation of lipid bilayers, and bone formation. Reduced phosphate levels due to excessive renal loss cause hypophosphatemic rickets, a disease characterized by prominent bone defects; conversely, hyperphosphatemia, a major complication of renal failure, is accompanied by parathyroid hyperplasia, hyperparathyroidism, and osteodystrophy. Here, we define a syndrome featuring both hypophosphatemic rickets and hyperparathyroidism due to parathyroid hyperplasia as well as other skeletal abnormalities. We show that this disease is due to a de novo translocation with a breakpoint adjacent to [alpha]-Klotho, which encodes a [beta]-glucuronidase, and is implicated in aging and regulation of FGF signaling. Plasma [alpha]-Klotho levels and [beta]glucuronidase activity are markedly increased in the affected patient; unexpectedly, the circulating FGF23 level is also markedly elevated. These findings suggest that the elevated [alpha]-Klotho level mimics aspects of the normal response to hyperphosphatemia and implicate [alpha]-Klotho in the selective regulation of phosphate levels and in the regulation of parathyroid mass and function; they also have implications for the pathogenesis and treatment of renal osteodystrophy in patients with kidney failure. bone | endocrinology | genetics | phosphorus

Homeostasis of intravascular volume, [Na.sup.+], [Cl.sup.-], and [K.sup.+] is interdependent and determined by the coordinated activities of structurally diverse mediators in the distal nephron and the distal colon. The behavior of these flux pathways is regulated by the rennin-angiotensin-aldosterone system; however, the mechanisms that allow independent modulation of individual elements have been obscure. Previous work has shown that mutations in WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension with hyperkalemia, due to altered activity of specific Na-Cl cotransporters, [K.sup.+] channels, and paracellular [Cl.sup.-] flux mediators of the distal nephron, By coexpression studies in Xenopus oocytes, we now demonstrate that WNK4 also inhibits the epithelial [Na.sup.+] channel (ENaC), the major mediator of aldosterone-sensitive [Na.sup.+] (re)absorption, via a mechanism that is independent of WNK4's kinase activity. This inhibition requires intact C termini in ENaC [beta]- and [gamma]-subunits, which contain PY motifs used to target ENaC for clearance from the plasma membrane. Importantly, PHAII-causing mutations eliminate WNK4's inhibition of ENaC, thereby paralleling other effects of PHAII to increase sodium balance. The relevance of these findings in vivo was studied in mice harboring PHAII-mutant WNK4. The colonic epithelium of these mice demonstrates markedly increased amiloride-sensitive [Na.sup.+] flux compared with wild-type littermates. These studies identify ENaC as a previously unrecognized downstream target of WNK4 and demonstrate a functional role for WNK4 in the regulation of colonic [Na.sup.+] absorption. These findings support a key role for WNK4 in coordinating the activities of diverse flux pathways to achieve integrated fluid and electrolyte homeostasis. aldosterone | hyperkalemia | hypertension | pseudohypoaldosteronism | renin-angiotensin system

The steroid hormone aldosterone is secreted both in the setting of intravascular volume depletion and hyperkalemia, raising the question of how the kidney maximizes NaCl reabsorption in the former state while maximizing [K.sup.+] secretion in the latter. Mutations in WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring increased renal NaCl reabsorption and impaired [K.sup.+] secretion. PHAII-mutant WNK4 achieves these effects by increasing activity of the Na-Cl cotransporter (NCC) and the [Na.sup.+] channel ENaC while concurrently inhibiting the renal outer medullary [K.sup.+] channel (ROMK). We now describe a functional state for WNK4 that promotes increased, rather than decreased, [K.sup.+] secretion. We show that WNK4 is phosphorylated by SGK1, a mediator of aldosterone signaling. Whereas wild-type WNK4 inhibits the activity of both ENaC and ROMK, a WNK4 mutation that mimics phosphorylation at the SGK1 site (WNK[4.sub.S1169D]) alleviates inhibition of both channels. The net result of these effects in the kidney would be increased [K.sup.+] secretion, because of both increased electrogenic [Na.sup.+] reabsorption and increased apical membrane [K.sup.+] permeability. Thus, modification at the PHAII and SGK1 sites in WNK4 impart opposite effects on [K.sup.+] secretion, decreasing or increasing ROMK activity and net [K.sup.+] secretion, respectively. This functional state for WNK4 would thus promote the desired physiologic response to hyperkalemia, and the fact that it is induced downstream of aldosterone signaling implicates WNK4 in the physiologic response to aldosterone with hyperkalemia. Together, the different states of WNK4 allow the kidney to provide distinct and appropriate integrated responses to intravascular volume depletion and hyperkalemia. hypertension | pseudohypoaldosteronism type II | epithelial [Na.sup.+] channel | renal outer medullary [K.sup.+] channel

SLC12A cation/[Cl.sup.-] cotransporters are mutated in human disease, are targets of diuretics, and are collectively involved in the regulation of cell volume, neuronal excitability, and blood pressure. This gene family has two major branches with different physiological functions and inverse regulation: K-CI cotransporters (KCC1-KCC4) mediate cellular [Cl.sup.-] efflux, are inhibited by phosphorylation, and are activated by dephosphorylation; Na-(K)-CI cotransporters (NCC and NKCC1/2) mediate cellular [Cl.sup.-] influx and are activated by phosphorylation. A single kinase/phosphatase pathway is thought to coordinate the activities of these cotransporters in a given cell; however, the mechanisms involved are as yet unknown. We previously demonstrated that WNK3, a paralog of serine-threonine kinases mutated in hereditary hypertension, is coexpressed with several cation/[Cl.sup.-] cotransporters and regulates their activity. Here, we show that WNK3 completely prevents the cell swelling-induced activation of KCC1-KCC4 in Xenopus oocytes. In contrast, catalytically inactive WNK3 abolishes the cell shrinkage- induced inhibition of KCC1-KCC4, resulting in a >100-fold stimulation of K-CI cotransport during conditions in which transport is normally inactive. This activation is completely abolished by calyculin A and cyclosporine A, inhibitors of protein phosphatase 1 and 2B, respectively. Wild-type WNK3 activates Na-(K)-CI cotransporters by increasing their phosphorylation, and catalytically inactive kinase inhibits Na-(K)-CI cotransporters by decreasing their phosphorylation, such that our data suggest that WNK3 is a crucial component of the kinase/phosphatase signaling pathway that coordinately regulates the [Cl.sup.-] influx and efflux branches of the SLC12A cotransporter family. ion transport | protein serine-threonine kinases | hypertension | cell volume regulation

The regulation of [CI.sup.-] transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion cotransporters: [Cl.sup.-] influx is mediated by the Na-K-2Cl cotransporter NKCC1 and [Cl.sup.-] efflux via K-Cl cotransporters, KCC1 or KCC2. A [Cl.sup.-]/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse [CI.sup.-] -transporting epithelia and in neurons expressing ionotropic GAB[A.sub.A] receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases [Cl.sup.-] influx via NKCC1, and that it inhibits [Cl.sup.-] exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular [Cl.sup.-] via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the [Cl.sup.-]/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission. cell volume | GABA | ion transport | protein serine--threonine kinases

WNK1 and WNK4 [WNK, with no lysine (K)] are serine--threonine kinases that function as molecular switches, eliciting coordinated effects on diverse ion transport pathways to maintain homeostasis during physiological perturbation. Gain-of-function mutations in either of these genes cause an inherited syndrome featuring hypertension and hyperkalemia due to increased renal NaCl reabsorption and decreased [K.sup.+] secretion. Here, we reveal unique biochemical and functional properties of WNK3, a related member of the WNK kinase family. Unlike WNK1 and WNK4, WNK3 is expressed throughout the nephron, predominantly at intercellular junctions. Because WNK4 is a potent inhibitor of members of the cation-cotransporter SLC12A family, we used coexpression studies in Xenopus oocytes to investigate the effect of WNK3 on NCC and NKCC2, related kidney-specific transporters that mediate apical NaCl reabsorption in the thick ascending limb and distal convoluted tubule, respectively. In contrast to WNK4's inhibitory activity, kinase-active WNK3 is a potent activator of both NKCC2 and NCC-mediated transport. Conversely, in its kinase-inactive state, WNK3 is a potent inhibitor of NKCC2 and NCC activity. WNK3 regulates the activity of these transporters by altering their expression at the plasma membrane. Wild-type WNK3 increases and kinase-inactive WNK3 decreases NKCC2 phosphorylation at Thr-184 and Thr-189, sites required for the vasopressin-mediated plasmalemmal translocation and activation of NKCC2 in vivo. The effects of WNK3 on these transporters and their coexpression in renal epithelia implicate WNK3 in NaCl, water, and blood pressure homeostasis, perhaps via signaling downstream of vasopressin. hypertension | ion transport | protein serine--threonine kinases

The multisubunit vacuolar-type [H.sup.+]ATPases mediate acidification of various intracellular organelles and in some tissues mediate [H.sup.+] secretion across the plasma membrane. Mutations in the B1-subunit of the apical [H.sup.+]ATPase that secretes protons in the distal nephron cause distal renal tubular acidosis in humans, a condition characterized by metabolic acidosis with an inappropriately alkaline urine. To examine the detailed cellular and organismal physiology resulting from this mutation, we have generated mice deficient in the B1-subunit (Atp6v1b[1.sup.-/-] mice). Urine pH is more alkaline and metabolic acidosis is more severe in Atp6v1b[1.sup.-/-] mice after oral acid challenge, demonstrating a failure of normal urinary acidification. In Atp6v1b[1.sup.-/-] mice, the normal urinary acidification induced by a lumen-negative potential in response to furosemide infusion is abolished. After an acute intracellular acidification, [Na.sup.+]-independent pH recovery rates of individual Atp6v1b[1.sup.-/-] intercalated cells of the cortical collecting duct are markedly reduced and show no further decrease after treatment with the selective [H.sup.+]ATPase inhibitor concanamycin. Apical expression of the alternative B-subunit isoform, B2, is increased in Atp6v1b[1.sup.-/-] medulla and colocalizes with the [H.sup.+]ATPase E-subunit; however, the greater severity of metabolic acidosis in Atp6v1b[1.sup.-/-] mice after oral acid challenge indicates that the B2-subunit cannot fully functionally compensate for the loss of B1. Our results indicate that the B1 isoform is the major B-subunit isoform that incorporates into functional, plasma membrane [H.sup.+]ATPases in intercalated cells of the cortical collecting duct and is required for maximal urinary acidification. collecting duct | intercalated cell | pH homeostasis | renal tubular acidosis | vacuolar [H.sup.+] ATPase

Patent ductus arteriosus (PDA) is a common congenital heart disease that results when the ductus arteriosus, a muscular artery, fails to remodel and close after birth. A syndromic form of this disorder, Char syndrome, is caused by mutation in TFAP2B, the gene encoding a neural crest-derived transcription factor. Established features of the syndrome are PDA, facial dysmorphology, and fifth-finger clinodactyly. Disease-causing mutations are missense and are proposed to be dominant negative. Because only a small number of families have been reported, there is limited information on the spectrum of mutations and resulting phenotypes. We report the characterization of two kindreds (K144 and K145) with Char syndrome containing 22 and 5 affected members, respectively. Genotyping revealed linkage to TFAP2B in both families. Sequencing of TFAP2B demonstrated mutations in both kindreds that were not found among control chromosomes. Both mutations altered highly conserved bases in introns required for normal splicing as demonstrated by biochemical studies in mammalian cells. The abnormal splicing results in mRNAs containing frameshift mutations that are expected to be degraded by nonsense-mediated mRNA decay, resulting in haploinsufficiency; even if produced, the protein in K144 would lack DNA binding and dimerization motifs and would likely result in haploinsufficiency. Examination of these two kindreds for phenotypes that segregate with TFAP2B mutations identified several phenotypes not previously linked to Char syndrome. These include parasomnia and dental and occipital-bone abnormalities. The striking sleep disorder in these kindreds implicates TFAP2B-dependent functions in the normal regulation of sleep. human genetics | parasomnia | congenital heart defects | Char

Paracellular ion flux across epithelia occurs through selective and regulated pores in tight junctions; this process is poorly understood. Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension and hyperkalemia. Whereas WNK4 is known to regulate several transcellular transporters and channels involved in NaCl and [K.sup.+] homeostasis, its localization to tight junctions suggests it might also regulate paracellular flux. We performed electrophysiology on mammalian kidney epithelia with inducible expression of various WNK4 constructs. Induction of wild-type WNK4 reduced transepithelial resistance by increasing absolute chloride permeability. PHAII-mutant WNK4 produced markedly larger effects, whereas kinase-mutant WNK4 had no effect. The electrochemical and pharmacologic properties of these effects indicate they are attributable to the paracellular pathway. The effects of WNK4 persist when induction is delayed until after tight-junction formation, demonstrating a dynamic effect. WNK4 did not alter the flux of uncharged solutes, or the expression or localization of selected tight-junction proteins. Transmission and freeze-fracture electron microscopy showed no effect of WNK4 on tight-junction structure. These findings implicate WNK signaling in the coordination of transcellular and paracellular flux to achieve NaCl and [K.sup.+] homeostasis, explain PHAII pathophysiology, and suggest that modifiers of WNK signaling may be potent antihypertensive agents.

HIV-1-associated nephropathy (HIVAN) is a major complication of HIV-1 infection with distinct pathologic features. Introduction of the HIV-1 genome into mice results in a renal disease with all of the histologic and clinical hallmarks of HIVAN on the FVB/N genetic background (TgFVB). We assessed the influence of genetic background on the development or progression of HIVAN by making [F.sub.1] hybrids of TgFVB with five other inbred strains (CBA, DBA/2, CAST/Ei, C3H/He, BALB/c) and determining phenotypes relevant to renal failure among transgenic offspring (histology, blood urea nitrogen, proteinuria, serum albumin, and serum cholesterol). We found striking variation in phenotypes among [F.sub.1]s, ranging from severe renal disease to no renal disease whatsoever (P < 0.001 for ANOVA across all groups). To map genes responsible for this variation, we produced a backcross of TgFVB/CAST [F.sub.1] x TgFVB. By genome-wide analysis of linkage in 185 heterozygous transgenic backcross mice, we identified a locus on chromosome 3A1-3, HIVAN1, that showed highly significant linkage to renal disease [logarithm of odds (lod) score 4.9 at D3Mit203, accounting for 15% of the variance in renal disease]. Other loci on chromosomes 11, 14, and 16 were suggestive of linkage to renal disease, and a locus on chromosome 9 influenced serum cholesterol but not nephropathy. Interestingly, HIVAN1 is syntenic to human chromosome 3q25-27, an interval showing suggestive evidence of linkage to various nephropathies. These findings demonstrate a strong genetic influence on HIVAN and demonstrate a major renal disease susceptibility locus on mouse chromosome 3A1-3.

Mutations in the serine-threonine kinase WNK4 [with no lysine (K) 4] cause pseudohypoaldosteronism type II, a Mendelian disease featuring hypertension with hyperkalemia. In the kidney, WNK4 regulates the balance between NaCl reabsorption and K+ secretion via variable inhibition of the thiazide-sensistive NaCl cotransporter and the K+ channel ROMK. We now demonstrate expression of WNK4 mRNA and protein outside the kidney. In extrarenal tissues, WNK4 is found almost exclusively in polarized epithelia, variably associating with tight junctions, lateral membranes, and cytoplasm. Epithelia expressing WNK4 include sweat ducts, colonic crypts, pancreatic ducts, bile ducts, and epididymis. WNK4 is also expressed in the specialized endothelium of the blood-brain barrier. These epithelia and endothelium all play important roles in Cl- transport. Because WNK4 is known to regulate renal Cl- handling, we tested WNK4's effect on the activity of mediators of epithelial Cl- flux whose extrarenal expression overlaps with WNK4. WNK4 proved to be a potent inhibitor of the activity of both the Na+ - K+ - 2Cl- cotransporter (NKCC1) and the Cl-/base exchanger SLC26A6 (CFEX) (> 95% inhibition of NKCC1-mediated [sup.86]Rb influx. P < 0.001; > 80% inhibition of CFEX-mediated [14C] formate uptake, P < 0.001), mediators of Cl- flux across basolateral and apical membranes, respectively. In contrast, WNK4 showed no inhibition of pendrin, a related Cl- /base exchanger. These findings indicate a general role for WNK4 in the regulation of electrolyte flux in diverse epithelia. Moreover, they reveal that WNK4 regulates the activities of a diverse group of structurally unrelated ion channels, cotransporters, and exchangers.

Gilles de la Tourette syndrome (GTS) is a potentially debilitating neuropsychiatric disorder defined by the presence of both vocal and motor tics. Despite evidence that this and a related phenotypic spectrum, including chronic tics (CT) and Obsessive Compulsive Disorder (OCD), are genetically mediated, no gene involved in disease etiology has been identified. Chromosomal abnormalities have long been proposed to play a causative role in isolated cases of GTS spectrum phenomena, but confirmation of this hypothesis has yet to be forthcoming. We describe an i(18q21.1-q22.2) inversion in a patient with CT and OCD. We have fine mapped the telomeric aspect of the rearrangement to within 1 Mb of a previously reported 18q22 breakpoint that cosegregated in a family with GTS and related phenotypes. A comprehensive characterization of this genomic interval led to the identification of two transcripts, neither of which was found to be structurally disrupted. Analysis of the epigenetic characteristics of the region demonstrated a significant increase in replication asynchrony in the patient compared to controls, with the inverted chromosome showing delayed replication timing across at least a 500-kb interval. These findings are consistent with long-range functional dysregulation of one or more genes in the region. Our data support a link between chromosomal aberrations and epigenetic mechanisms in GTS and suggest that the study of the functional consequences of balanced chromosomal rearrangements is warranted in patients with phenotypes of interest, irrespective of the findings regarding structurally disrupted transcripts.

Mutations in the serine-threonine kinases WNK1 and WNK4 [with no lysine (K) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The [Cl.sup.-] dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a 'mirror image' of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na-Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCT-mediated [Na.sup.+] influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in Xenopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter ([sup.22]Na-influx reduced 50%, P < 1 x [10.sup.-9], surface expression reduced 75%, P < 1 X [10.sup.-14] in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension. protein serine-threonine kinases | hypertension | thiazide-sensitive Na-Cl cotransporter | ion transport | medical genetics

Mutations in WNK1 and WNK4, genes encoding members of a novel family of serine--threonine kinases, have recently been shown to cause pseudohypoaldosteronism type II (PHAII), an autosomal dominant disorder featuring hypertension, hyperkalemia, and renal tubular acidosis. The localization of these kinases in the distal nephron and the [Cl.sup.-] dependence of these phenotypes suggest that these mutations increase renal [Cl.sup.-] reabsorption. Although WNK4 expression is limited to the kidney, WNK1 is expressed in many tissues. We have examined the distribution of WNK1 in these extrarenal tissues. Immunostaining using WNK1-specific antibodies demonstrated that WNK1 is not present in all cell types; rather, it is predominantly localized in polarized epithelia, including those lining the lumen of the hepatic biliary ducts, pancreatic ducts, epididymis, sweat ducts, colonic crypts, and gallbladder. WNK1 is also found in the basal layers of epidermis and throughout the esophageal epithelium. The subcellular localization of WNK1 varies among these epithelia. WNK1 is cytoplasmic in kidney, colon, gallbladder, sweat duct, skin, and esophagus; in contrast, it localizes to the lateral membrane in bile ducts, pancreatic ducts, and epididymis. These epithelia are all notable for their prominent role in [Cl.sup.-] flux. Moreover, these sites largely coincide with those involved in the pathology of cystic fibrosis, a disease characterized by deranged epithelial [Cl.sup.-] flux. Together with the known pathophysiology of PHAII, these findings suggest that WNK1 plays a general role in the regulation of epithelial [Cl.sup.-] flux, a finding that suggests the potential of new approaches to the selective modulation of these processes. protein-serine-threonine kinases | ion transport | cystic fibrosis | medical genetics

The causes of many sporadic diseases are unexplained; the contribution of recessive loci with reduced penetrance is one possibility that has been difficult to explore. We describe an approach to this problem by first searching for diseases with higher prevalence in populations with high rates of consanguinity, then determining whether disease cases are more commonly the product of consanguinous union than controls in such populations, followed by analysis of genetic linkage in consanguinous cases. We demonstrate the utility of this approach by investigation of congenital heart disease in Iran. We found that patent ductus arteriosus (PDA), a common congenital heart disease, accounts for a higher fraction of congenital heart disease in Iran (15%) than in the United States (2-7%). Moreover, Iranian PDA cases demonstrated a marked increase of parental consanguinity (63%), compared with the general Iranian population (25%) or control cases with tetralogy of Fallot (30%). The recurrence of PDA among siblings was 5%. A genomewide analysis of linkage in 21 unrelated consanguinous PDA cases demonstrated a multipoint logarithm of odds score of 6.27 in favor of linkage of PDA to a 3-centimorgan interval of chromosome 12q24, with 53% of kindreds linked. These findings together establish a recessive component to PDA and implicate a single locus, PDA1, in one third or more of all PDA cases in Iran; they further suggest a role for this locus in PDA worldwide. Finally, these results suggest a general approach to the identification of recessive contributions to sporadic diseases. linkage (genetics) | patent ductus arteriosus | consanguinity | human chromosomes | pair 12

Mutations in Krev1 interaction trapped gene 1 (KRIT1) cause cerebral cavernous malformation, an autosomal dominant disease featuring malformation of cerebral capillaries resulting in cerebral hemorrhage, strokes, and seizures. The biological functions of KRIT1 are unknown. We have investigated KRIT1 expression in endothelial cells by using specific anti-KRIT1 antibodies. By both microscopy and coimmunoprecipitation, we show that KRIT1 colocalizes with microtubules. In interphase cells, KRIT1 is found along the length of microtubules. During metaphase, KRIT1 is located on spindle pole bodies and the mitotic spindle. During late phases of mitosis, KRIT1 localizes in a pattern indicative of association with microtubule plus ends. In anaphase, the plus ends of the interpolar microtubules show strong KRIT1 staining and, in late telophase, KRIT1 stains the midbody remnant most strongly; this is the site of cytokinesis where plus ends of microtubules from dividing cells overlap. These results establish that KRIT1 is a microtubule-associated protein; its location at plus ends in mitosis suggests a possible role in microtubule targeting. These findings, coupled with evidence of interaction of KRIT1 with Krev1 and integrin cytoplasmic domain-associated protein-1 alpha (ICAP1 [alpha]), suggest that KRIT1 may help determine endothelial cell shape and function in response to cell-cell and cell-matrix interactions by guiding cytoskeletal structure. We propose that the loss of this targeting function leads to abnormal endothelial tube formation, thereby explaining the mechanism of formation of cerebral cavernous malformation (CCM) lesions.

Liddle syndrome is a mendelian form of hypertension characterized by constitutively elevated renal Na reabsorption that can result from activating mutations in the [beta] or [gamma] subunit of the epithelial Na channel. All reported mutations have deleted the last 45-76 normal amino acids from the cytoplasmic C terminus of one of these channel subunits. While these findings implicate these terminal segments in the normal negative regulation of channel activity, they do not identify the amino acid residues that are critical targets for these mutations. Potential targets include the short highly conserved Pro-rich segments present in the C terminus of [beta] and [gamma] subunits; these segments are similar to SH3-binding domains that mediate protein-protein interaction. We now report a kindred with Liddle syndrome in which affected patients have a mutation in codon 616 of the [beta] subunit resulting in substitution of a Leu for one of these highly conserved Pro residues. The functional significance of this mutation is demonstrated both by the finding that this is a de novo mutation appearing concordantly with the appearance of Liddle syndrome in the kindred and also by the marked activation of amiloride-sensitive Na channel activity seen in Xenopus oocytes expressing channels containing this mutant subunit (8.8-fold increase compared with control oocytes expressing normal channel subunits; P = 0.003). These findings demonstrate a de novo missense mutation causing Liddle syndrome and identify a critical channel residue important for the normal regulation of Na reabsorption in humans.

A Mendelian form of hypertension called glucocorticoid-remediable aldosteronism occurs due to mutations causing ectopic expression of aldosterone synthase enzymatic activity. Liddle syndrome, the Mendelian form of hypertension is activated due to the mutations in the beta subunit of the amiloride-sensitive epithelial sodium channel. The general variants at the angiotensinogen locus are responsible for the pathogenesis of the required hypertension in Caucasian subjects.

Cerebral cavernous malformation (CCM), a common disease of brain vasculature, is an autosomal dominant disorder linked to 7q11.2-q21 chromosome. It shows dilated thin-walled sinusoidal vessels and includes symptoms like headache, seizure and hemorrhagic stroke. Multipoint linkage of CCM to D7S669 gives a peak load score of 6.88 at a recombination fraction of zero and localizes gene to a 7-cM region in the interval between loci D7S802 and ELN.

A study has been conducted to examine how mutation of the channel beta subunit in the C terminus of the encoded protein of ENaC results to Liddle disease. Coexpression of the mutant beta subunit with the alpha and gamma subunits of the rat ENaC in Xenopus laevis oocytes resulted to an increase in channel activity. A deletion in the gamma subunit also resulted to an abnormal increase channel activity. These results suggest that mutations in subunits affecting channel activity in the cell membrane causes the Liddle disease.

Gain-of-function mutations in the CACNA1H gene (encoding the T-type calcium channel CaV3.2) cause autosomal-dominant familial hyperaldosteronism type IV (FH-IV) and early-onset hypertension in humans. We used CRISPR/Cas9 to generate Cacna1hM1560V/+ knockin mice as a model of the most common FH-IV mutation, along with corresponding knockout mice (Cacna1h-/-). Adrenal morphology of both Cacna1hM1560V/+ and Cacna1h-/- mice was normal. Cacna1hM1560V/+ mice had elevated aldosterone:renin ratios (a screening parameter for primary aldosteronism). Their adrenal Cyp11b2 (aldosterone synthase) expression was increased and remained elevated on a high-salt diet (relative autonomy, characteristic of primary aldosteronism), but plasma aldosterone was only elevated in male animals. The systolic blood pressure of Cacna1hM1560V/+ mice was 8 mmHg higher than in wild-type littermates and remained elevated on a high-salt diet. Cacna1h-/- mice had elevated renal Ren1 (renin-1) expression but normal adrenal Cyp11b2 levels, suggesting that in the absence of CaV3.2, stimulation of the renin-angiotensin system activates alternative calcium entry pathways to maintain normal aldosterone production. On a cellular level, Cacna1hM1560V/+ adrenal slices showed increased baseline and peak intracellular calcium concentrations in the zona glomerulosa compared to controls, but the frequency of calcium spikes did not rise. We conclude that FH-IV, on a molecular level, is caused by elevated intracellular Ca2+ concentrations as a signal for aldosterone production in adrenal glomerulosa cells. We demonstrate that a germline Cacna1h gain-of-function mutation is sufficient to cause mild primary aldosteronism, whereas loss of CaV3.2 channel function can be compensated for in a chronic setting. [ABSTRACT FROM AUTHOR]

Whole-exome sequencing (WES) has facilitated the discovery of genetic lesions underlying monogenic disorders. Incomplete penetrance and variable expressivity suggest a contribution of additional genetic lesions to clinical manifestations and outcome. Some monogenic disorders may therefore actually be digenic. However, only a few digenic disorders have been reported, all discovered by candidate gene approaches applied to at least one locus. We propose here a two-locus genome-wide test for detecting digenic inheritance in WES data. This approach uses the gene as the unit of analysis and tests all pairs of genes to detect pairwise gene × gene interactions underlying disease. It is a case-only method, which has several advantages over classic case-control tests, in particular by avoiding recruitment of controls. Our simulation studies based on real WES data identified two major sources of type I error inflation in this case-only test: linkage disequilibrium and population stratification. Both were corrected by specific procedures. Moreover, our case-only approach is more powerful than the corresponding case-control test for detecting digenic interactions in various population stratification scenarios. Finally, we confirmed the potential of our unbiased, genome-wide approach by successfully identifying a previously reported digenic lesion in patients with craniosynostosis. Our case-only test is a powerful and timely tool for detecting digenic inheritance in WES data from patients. [ABSTRACT FROM AUTHOR]

Craniosynostosis (CS) is a frequent congenital anomaly featuring the premature fusion of 1 or more sutures of the cranial vault. Syndromic cases, featuring additional congenital anomalies, make up 15% of CS. While many genes underlying syndromic CS have been identified, the cause of many syndromic cases remains unknown. We performed exome sequencing of 12 syndromic CS cases and their parents, in whom previous genetic evaluations were unrevealing. Damaging de novo or transmitted loss of function (LOF) mutations were found in 8 genes that are highly intolerant to LOF mutation (P = 4.0 x 10-8); additionally, a rare damaging mutation in SOX11, which has a lower level of intolerance, was identified. Four probands had rare damaging mutations (2 de novo) in TFAP2B, a transcription factor that orchestrates neural crest cell migration and differentiation; this mutation burden is highly significant (P = 8.2 x 10-12). Three probands had rare damaging mutations in GLI2, SOX11, or GPC4, which function in the Hedgehog, BMP, and Wnt signaling pathways; other genes in these pathways have previously been implicated in syndromic CS. Similarly, damaging de novo mutations were identified in genes encoding the chromatin modifier KAT6A, and CTNNA1, encoding catenin α-1. These findings establish TFAP2B as a CS gene, have implications for assessing risk to subsequent children in these families, and provide evidence implicating other genes in syndromic CS. This high yield indicates the value of performing exome sequencing of syndromic CS patients when sequencing of known disease loci is unrevealing. [ABSTRACT FROM AUTHOR]

Genomic analyses of patients with congenital heart disease (CHD) have identified significant contribution frommutations affecting cilia genes and chromatin remodeling genes; however, the mechanism(s) connecting chromatin remodeling to CHD is unknown. Histone H2B monoubiquitination (H2Bub1) is catalyzed by the RNF20 complex consisting of RNF20, RNF40, and UBE2B. Here, we show significant enrichment of loss-of-function mutations affecting H2Bub1 in CHD patients (enrichment 6.01, P = 1.67 × 10−03), some of whom had abnormal laterality associated with ciliary dysfunction. In Xenopus, knockdown of rnf20 and rnf40 results in abnormal heart looping, defective development of left-right (LR) asymmetry, and impaired cilia motility. Rnf20, Rnf40, and Ube2b affect LR patterning and cilia synergistically. Examination of global H2Bub1 level in Xenopus embryos shows that H2Bub1 is developmentally regulated and requires Rnf20. To examine gene-specific H2Bub1, we performed ChIP-seq of mouse ciliated and nonciliated tissues and showed tissue-specific H2Bub1 marks significantly enriched at cilia genes including the transcription factor Rfx3. Rnf20 knockdown results in decreased levels of rfx3 mRNA in Xenopus, and exogenous rfx3 can rescue the Rnf20 depletion phenotype. These data suggest that Rnf20 functions at the Rfx3 locus regulating cilia motility and cardiac situs and identify H2Bub1 as an upstream transcriptional regulator controlling tissuespecific expression of cilia genes. Our findings mechanistically link the two functional gene ontologies that have been implicated in human CHD: chromatin remodeling and cilia function. [ABSTRACT FROM AUTHOR]

Calcineurin is a calcium/calmodulin-regulated phosphatase known for its role in activation of T cells following engagement of the T cell receptor. Calcineurin inhibitors (CNIs) are widely used as immunosuppressive agents; common adverse effects of CNIs are hypertension and hyperkalemia. While previous studies have implicated activation of the Na-Cl cotransporter (NCC) in the renal distal convoluted tubule (DCT) in this toxicity, the molecular mechanism of this effect is unknown. The renal effects of CNIs mimic the hypertension and hyperkalemia that result from germ-line mutations in with-no-lysine (WNK) kinases and the Kelch-like 3 (KLHL3)- CUL3 ubiquitin ligase complex. WNK4 is an activator of NCC and is degraded by binding to KLHL3 followed by WNK4's ubiquitylation and proteasomal degradation. This binding is prevented by phosphorylation of KLHL3 at serine 433 (KLHL3S433-P) via protein kinase C, resulting in increased WNK4 levels and increased NCC activity. Mechanisms mediating KLHL3S433-P dephosphorylation have heretofore been unknown. We now demonstrate that calcineurin expressed in DCT is a potent KLHL3S433-P phosphatase. In mammalian cells, the calcium ionophore ionomycin, a calcineurin activator, reduces KLHL3S433-P levels, and this effect is reversed by the calcineurin inhibitor tacrolimus and by siRNA-mediated knockdown of calcineurin. In vivo, tacrolimus increases levels of KLHL3S433-P, resulting in increased levels of WNK4, phosphorylated SPAK, and NCC. Moreover, tacrolimus attenuates KLHL3- mediated WNK4 ubiquitylation and degradation, while this effect is absent in KLHL3 with S433A substitution. Additionally, increased extracellular K+ induced calcineurin-dependent dephosphorylation of KLHL3S433-P. These findings demonstrate that KLHL3S433-P is a calcineurin substrate and implicate increased KLHL3 phosphorylation in tacrolimus-induced pathologies. [ABSTRACT FROM AUTHOR]

Non-syndromic craniosynostosis (NSC) is a frequent congenital malformation in which one or more cranial sutures fuse prematurely. Mutations causing rare syndromic craniosynostoses in humans and engineered mouse models commonly increase signaling of the Wnt, bone morphogenetic protein (BMP), or Ras/ERK pathways, converging on shared nuclear targets that promote bone formation. In contrast, the genetics of NSC is largely unexplored. More than 95% of NSC is sporadic, suggesting a role for de novo mutations. Exome sequencing of 291 parent-offspring trios with midline NSC revealed 15 probands with heterozygous damaging de novo mutations in 12 negative regulators of Wnt, BMP, and Ras/ERK signaling (10.9-fold enrichment, P = 2.4 x 10-11). SMAD6 had 4 de novo and 14 transmitted mutations; no other gene had more than 1. Four familial NSC kindreds had mutations in genes previously implicated in syndromic disease. Collectively, these mutations contribute to 10% of probands. Mutations are predominantly loss-of-function, implicating haploinsufficiency as a frequent mechanism. A common risk variant near BMP2 increased the penetrance of SMAD6 mutations and was overtransmitted to patients with de novo mutations in other genes in these pathways, supporting a frequent two-locus pathogenesis. These findings implicate new genes in NSC and demonstrate related pathophysiology of common non-syndromic and rare syndromic craniosynostoses. These findings have implications for diagnosis, risk of recurrence, and risk of adverse neurodevelopmental outcomes. Finally, the use of pathways identified in rare syndromic disease to find genes accounting for non-syndromic cases may prove broadly relevant to understanding other congenital disorders featuring high locus heterogeneity. [ABSTRACT FROM AUTHOR]

With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphosite-specific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain. [ABSTRACT FROM AUTHOR]

Carcinosarcomas (CSs) of the uterus and ovary are highly aggressive neoplasms containing both carcinomatous and sarcomatous elements. We analyzed the mutational landscape of 68 uterine and ovarian CSs by whole-exome sequencing. We also performed multiregion whole-exome sequencing comprising two carcinoma and sarcoma samples from six tumors to resolve their evolutionary histories. The results demonstrated that carcinomatous and sarcomatous elements derive from a common precursor having mutations typical of carcinomas. In addition to mutations in cancer genes previously identified in uterine and ovarian carcinomas such as TP53, PIK3CA, PPP2R1A, KRAS, PTEN, CHD4, and BCOR, we found an excess of mutations in genes encoding histone H2A and H2B, as well as significant amplification of the segment of chromosome 6p harboring the histone gene cluster containing these genes. We also found frequent deletions of the genes TP53 and MBD3 (a member with CHD4 of the nucleosome remodeling deacetylase complex) and frequent amplification of chromosome segments containing the genes PIK3CA, TERT, and MYC. Stable transgenic expression of H2A and H2B in a uterine serous carcinoma cell line demonstrated that mutant, but not wild-type, histones increased expression of markers of epithelial-mesenchymal transition (EMT) as well as tumor migratory and invasive properties, suggesting a role in sarcomatous transformation. Comparison of the phylogenetic relationships of carcinomatous and sarcomatous elements of the same tumors demonstrated separate lineages leading to these two components. These findings define the genetic landscape of CSs and suggest therapeutic targets for these highly aggressive neoplasms. [ABSTRACT FROM AUTHOR]

Acyl CoA Oxidase 2 (ACOX2) encodes branched-chain acyl-CoA oxidase, a peroxisomal enzyme believed to be involved in the metabolism of branched-chain fatty acids and bile acid intermediates. Deficiency of this enzyme has not been described previously. We report an 8-y-old male with intermittently elevated transaminase levels, liver fibrosis, mild ataxia, and cognitive impairment. Exome sequencing revealed a previously unidentified homozygous premature termination mutation (p.Y69*) in ACOX2. Immunohistochemistry confirmed the absence of ACOX2 expression in the patient's liver, and biochemical analysis showed marked elevation of intermediate bile acids upstream of ACOX2. These findings define a potentially treatable inborn error of bile acid biosynthesis caused by ACOX2 deficiency. [ABSTRACT FROM AUTHOR]

Many aspects of the evolutionary process of tumorigenesis that are fundamental to cancer biology and targeted treatment have been challenging to reveal, such as the divergence times and genetic clonality of metastatic lineages. To address these challenges, we performed tumor phylogenetics using molecular evolutionary models, reconstructed ancestral states of somatic mutations, and inferred cancer chronograms to yield three conclusions. First, in contrast to a linear model of cancer progression, metastases can originate from divergent lineages within primary tumors. Evolved genetic changes in cancer lineages likely affect only the proclivity toward metastasis. Single genetic changes are unlikely to be necessary or sufficient for metastasis. Second, metastatic lineages can arise early in tumor development, sometimes long before diagnosis. The early genetic divergence of some metastatic lineages directs attention toward research on driver genes that are mutated early in cancer evolution. Last, the temporal order of occurrence of driver mutations can be inferred from phylogenetic analysis of cancer chronograms, guiding development of targeted therapeutics effective against primary tumors and metastases. [ABSTRACT FROM AUTHOR]

The presence of sarcomatoid features in clear cell renal cell carcinoma (ccRCC) confers a poor prognosis and is of unknown pathogenesis. We performed exome sequencing of matched normal-carcinomatous- sarcomatoid specimens from 21 subjects. Two tumors had hypermutation consistent with mismatch repair deficiency. In the remainder, sarcomatoid and carcinomatous elements shared 42% of somatic single-nucleotide variants (SSNVs). Sarcomatoid elements had a higher overall SSNV burden (mean 90 vs. 63 SSNVs, P = 4.0 × 10-4), increased frequency of nonsynonymous SSNVs in Pan- Cancer genes (mean 1.4 vs. 0.26, P = 0.002), and increased frequency of loss of heterozygosity (LOH) across the genome (median 913 vs. 460 Mb in LOH, P < 0.05), with significant recurrent LOH on chromosomes 1p, 9, 10, 14, 17p, 18, and 22. The most frequent SSNVs shared by carcinomatous and sarcomatoid elements were in known ccRCC genes including von Hippel-Lindau tumor suppressor (VHL), polybromo 1 (PBRM1), SET domain containing 2 (SETD2), phosphatase and tensin homolog (PTEN). Most interestingly, sarcomatoid elements acquired biallelic tumor protein p53 (TP53) mutations in 32% of tumors (P = 5.47 × 10-17); TP53 mutations were absent in carcinomatous elements in nonhypermutated tumors and rare in previously studied ccRCCs. Mutations in known cancer drivers ATrich interaction domain 1A (ARID1A) and BRCA1 associated protein 1 (BAP1) were significantly mutated in sarcomatoid elements and were mutually exclusive with TP53 and each other. These findings provide evidence that sarcomatoid elements arise from dedifferentiation of carcinomatous ccRCCs and implicate specific genes in this process. These findings have implications for the treatment of patients with these poor-prognosis cancers. [ABSTRACT FROM AUTHOR]

Hypertension contributes to the global burden of cardiovascular disease. Increased dietary K+ reduces blood pressure; however, the mechanism has been obscure. Human genetic studies have suggested that the mechanism is an obligatory inverse relationship between renal salt reabsorption and K+ secretion. Mutations in the kinases with-no-lysine 4 (WNK4) or WNK1, or in either Cullin 3 (CUL3) or Kelch-like 3 (KLHL3)—components of an E3 ubiquitin ligase complex that targets WNKs for degradation—cause constitutively increased renal salt reabsorption and impaired K+ secretion, resulting in hypertension and hyperkalemia. The normal mechanisms that regulate the activity of this ubiquitin ligase and levels of WNKs have been unknown. We posited that missense mutations in KLHL3 that impair binding of WNK4 might represent a phenocopy of the normal physiologic response to volume depletion in which salt reabsorption is maximized. We show that KLHL3 is phosphorylated at serine 433 in the Kelch domain (a site frequently mutated in hypertension with hyperkalemia) by protein kinase C in cultured cells and that this phosphorylation prevents WNK4 binding and degradation. This phosphorylation can be induced by angiotensin II (AII) signaling. Consistent with these in vitro observations, AII administration to mice, even in the absence of volume depletion, induces renal KLHL3S433 phosphorylation and increased levels of both WNK4 and the NaCl cotransporter. Thus, AII, which is selectively induced in volume depletion, provides the signal that prevents CUL3/KLHL3-mediated degradation of WNK4, directing the kidney to maximize renal salt reabsorption while inhibiting K+ secretion in the setting of volume depletion. [ABSTRACT FROM AUTHOR]

The renal phenotype induced by loss-of-function mutations of inwardly rectifying potassium channel (Kir), Kcnj10 (Kir4.1), includes salt wasting, hypomagnesemia, metabolic alkalosis and hypokalemia. However, the mechanism by which Kir.4.1 mutations cause the tubulopathy is not completely understood. Here we demonstrate that Kcnj10 is a main contributor to the basolateral K conductance in the early distal convoluted tubule (DCT1) and determines the expression of the apical Na-CI cotransporter (NCC) in the DCT. Immu-nostaining demonstrated Kcnj10 and Kcnj16 were expressed in the basolateral membrane of DCT, and patch-clamp studies detected a 40-pS K channel in the basolateral membrane of the DCT1 of p8/p10 wild-type Kcnj1(t+/+ mice (WT). This 40-pS K channel is absent in homozygous Kcnj10-/- (knockout) mice. The disruption of Kcnj10 almost completely eliminated the basolateral K conductance and decreased the negativity of the cell membrane potential in DCT1. Moreover, the lack of Kcnj10 decreased the basolateral Cl conductance, inhibited the expression of Ste20-related proline-alanine-rich kinase and diminished the apical NCC expression in DCT. We conclude that Kcnj10 plays a dominant role in determining the basolateral K conductance and membrane potential of DCT1 and that the basolateral K channel activity in the DCT determines the apical NCC expression possibly through a Ste20-related proline-alanine-rich kinase-dependent mechanism. [ABSTRACT FROM AUTHOR]

Pseudohypoaldosteronism type II (PHAII) is a rare Mendelian syndrome featuring hypertension and hyperkalemia resulting from constitutive renal salt reabsorption and impaired K+ secretion. Recently, mutations in Kelch-like 3 (KLHL3) and Cullin 3 (CUL3), components of an E3 ubiquitin ligase complex, were found to cause PHAII, suggesting that loss of this complex's ability to target specific substrates for ubiquitination leads to PHAII. By MS and coimmunoprecipitation, we show that KLHL3 normally binds to WNK1 and WNK4, members of WNK (with no lysine) kinase family that have previously been found mutated in PHAII. We show that this binding leads to ubiquitination, including polyubiquitination, of at least 15 specific sites in WNK4, resulting in reduced WNK4 levels. Dominant disease-causing mutations in KLHL3 and WNK4 both impair WNK4 binding, ubiquitination, and degradation. WNK4 normally induces clearance of the renal outer medullary K+ channel (ROMK) from the cell surface. We show that WT but not mutant KLHL3 inhibits WNK4-induced reduction of ROMK level. We show that PHAII-causing mutations in WNK4 lead to a marked increase in WNK4 protein levels in the kidney in vivo. These findings demonstrate that CUL3- RING (really interesting new gene) ligases that contain KLHL3 target ubiquitination of WNK4 and thereby regulate WNK4 levels, which in turn regulate levels of ROMK. These findings reveal a specific role of CUL3 and KLHL3 in electrolyte homeostasis and provide a molecular explanation for the effects of disease-causing mutations in both KLHL3 and WNK4. [ABSTRACT FROM AUTHOR]

Ubiquitin C-terminal hydrolase-L1 (UCHL1), a neuron-specific de-ubiquitinating enzyme, is one of the most abundant proteins in the brain. We describe three siblings from a consanguineous union with a previously unreported early-onset progressive neurodegenerative syndrome featuring childhood onset blindness, cerebellar ataxia, nystagmus, dorsal column dysfuction, and spasticity with upper motor neuron dysfunction. Through homozygosity mapping of the affected individuals followed by whole-exome sequencing of the index case, we identified a previously undescribed homozygous missense mutation within the ubiquitin binding domain of UCHL1 (UCHL1GLU7ALA), shared by all affected subjects. As demonstrated by isothermal titration calorimetry, purified UCHL1GLU7ALA, compared with WT, exhibited at least sevenfold reduced affinity for ubiquitin. In vitro, the mutation led to a near complete loss of UCHL1 hydrolase activity. The GLU7ALA variant is predicted to interfere with the substrate binding by restricting the proper positioning of the substrate for tunneling underneath the cross-over loop spanning the catalytic cleft of UCHL1. This interference with substrate binding, combined with near complete loss of hydrolase activity, resulted in a >100-fold reduction in the efficiency of UCHL1GLU7ALA relative to WT. These findings demonstrate a broad requirement of UCHL1 in the maintenance of the nervous system. [ABSTRACT FROM AUTHOR]

αβ T-cell repertoire selection is mediated by peptide-MHC complexes presented by thymic epithelial or myeloid cells, and by lipid-CD1 complexes expressed by thymocytes. γd T-cell repertoire selection, by contrast, is largely unresolved. Mice mutant for Skint-1, a unique Ig superfamily gene, do not develop canonical Vγ5Vd1+ dendritic epidermal T cells. This study shows that transgenic Skint-1, across a broad range of expression levels, precisely and selectively determines the Vγ5Vd1+ dendritic epidermal T-cell compartment. Skint-1 is expressed by medullary thymic epithelial cells, and unlike lipid-CD1 complexes, must be expressed by stromal cells to function efficiently. Its unusual transmembrane-cytoplasmic regions severely limit cell surface expression, yet increasing this or, conversely, retaining Skint1 intracellularly markedly compromises function. Each Skint1 domain appears nonredundant, including a unique decamer specifying IgV-domain processing. This investigation of Skint-1 biology points to complex events underpinning the positive selection of an intraepithelial γd repertoire. [ABSTRACT FROM AUTHOR]

Dominant human genetic diseases that impair reproductive fitness and have high locus heterogeneity constitute a problem for gene discovery because the usual criterion of finding more mutations in specific genes than expected by chance may require extremely large populations. Heterotaxy (Htx), a congenital heart disease resulting from abnormalities in left-right (LR) body patterning, has features suggesting that many cases fall into this category. In this setting, appropriate model systems may provide a means to support implication of specific genes. By high-resolution genotyping of 262 Htx subjects and 991 controls, we identify a twofold excess of subjects with rare genic copy number variations in Htx (14.5% vs. 7.4%, P = 1.5 × 10-4). Although 7 of 45 Htx copy number variations were large chromosomal abnormalities, 38 smaller copy number variations altered a total of 61 genes, 22 of which had Xenopus orthologs. In situ hybridization identified 7 of these 22 genes with expression in the ciliated LR organizer (gastrocoel roof plate), a marked enrichment compared with 40 of 845 previously studied genes (sevenfold enrichment, P < 10-6). Morpholino knockdown in Xenopus of Htx candidates demonstrated that five (NEK2, ROCK2, TGFBR2, GALNT11, and NUP188) strongly disrupted both morphological LR development and expression of pitx2, a molecular marker of LR patterning. These effects were specific, because 0 of 13 control genes from rare Htx or control copy number variations produced significant LR abnormalities (P = 0.001). These findings identify genes not previously implicated in LR patterning. [ABSTRACT FROM AUTHOR]

Vascular smooth muscle cell (VSMC) proliferation is an important event in atherosclerosis and other vasculopathies. PDGF signaling is a key mediator of SMC proliferation, but the mechanisms that control its activity remain unclear. We previously identified a mutation in LDL receptor-related protein 6 (LRP6), LRP6R611C, that causes early atherosclerosis. Examination of human atherosclerotic coronary arteries showed markedly increased expression of LRP6 and colocalization with PDGF receptor β (PDGFR-β). Further investigation showed that wild-type LRP6 inhibits but LRP6R611C promotes VSMC proliferation in response to PDGF. We found that wild-type LRP6 forms a complex with PDGFR-β and enhances its lysosomal degradation, functions that are severely impaired in LRP6R611C. Further, we observed that wild-type and mutant LRP6 regulate cell-cycle activity by triggering differential effects on PDGF-dependent pathways. These findings implicate LRP6 as a critical modulator of PDGF-dependent regulation of cell cycle in smooth muscle and indicate that loss of this function contributes to development of early atherosclerosis in humans. [ABSTRACT FROM AUTHOR]

Protein coding genes constitute only approximately 1% of the human genome but harbor 85% of the mutations with large effects on disease-related traits. Therefore, efficient strategies for selectively sequencing complete coding regions (i.e., "whole exome") have the potential to contribute to the understanding of rare and common human diseases. Here we report a method for whole-exome sequencing coupling Roche/NimbleGen whole exome arrays to the Illumina DNA sequencing platform. We demonstrate the ability to capture approximately 95% of the targeted coding sequences with high sensitivity and specificity for detection of homozygous and heterozygous variants. We illustrate the utility of this approach by making an unanticipated genetic diagnosis of congenital chloride diarrhea in a patient referred with a suspected diagnosis of Bartter syndrome, a renal salt-wasting disease. The molecular diagnosis was based on the finding of a homozygous missense D652N mutation at a position in 5LC26A3 (the known congenital chloride diarrhea locus) that is virtually completely conserved in orthologues and paralogues from invertebrates to humans, and clinical follow-up confirmed the diagnosis. To our knowledge, whole-exome (or genome) sequencing has not previously been used to make a genetic diagnosis. Five additional patients suspected to have Bartter syndrome but who did not have mutations in known genes for this disease had homozygous deleterious mutations in SLC26A3. These results demonstrate the clinical utility of whole-exome sequencing and have implications for disease gene discovery and clinical diagnosis. [ABSTRACT FROM AUTHOR]

WNK4 (with no lysine kinase 4) inhibits ROMK channel activity in the distal nephron by stimulating clathrin-dependent endocytosis, an effect attenuated by SGK1 (serum-glucocorticoids-induced kinase)-mediated phosphorylation. It has been suggested that increased ROMK activity because of SGK1-mediated inhibition of WNK4 plays a role in promoting renal K secretion in response to elevated serum K or high K (HK) intake. In contrast, intravascular volume depletion also increases SGK1 activity but fails to stimulate ROMK channels and K secretion. Because HK intake decreases Src family protein tyrosine kinase (PTK) activity an inhibitor of ROMK channels, it is possible that Src family PTK may modulate the effects of SGK1 on WNK4. Here, we show that c-Src prevents SGK1 from attenuating WNK4's inhibition of ROMK activity. This effect of c-Src was WNK4-dependent because c-Src had no effect on ROMK harboring mutation at the site of c-Src phosphorylation (R1Y337A) in the absence of WNK4. Moreover, expression c-Src diminished the SGK1-mediated increase in serine phosphorylation of WNK4, suggesting that c-Src enhances WNK4-mediated inhibition of ROMK channels by suppressing the SGK1-induced phosphorylation. This notion is also supported by the observation that c-Src was not able to modulate the interaction between SGK1 and WNK4 mutants (WNK4S1169A or WNK4S1169D) in which an SGK1-phosphorylation site (serine 1169) was mutated by alanine or aspartate. We conclude that c-Src inhibits SGK1-mediated phosphorylation hereby restoring the WNK4-mediated inhibition of ROMK channels thus suppressing K secretion. [ABSTRACT FROM AUTHOR]

Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and high serum K[sup+] levels (hyperkalemia). WNK4 has distinct functional states that regulate the balance between renal salt reabsorption and K[sup+] secretion by modulating the activities of renal transporters and channels, including the Na-Cl cotransporter NCC and the K[sup+] chan- nel ROMK. WNK4's functions could enable differential responses to intravascular volume depletion (hypovolemia) and hyperkalemia. Because hypovolemia is uniquely associated with high angiotensin II (Angli) levels. AnglI signaling might modulate WNK4 activity. We show that AnglI signaling in Xenopus oocytes increases NCC activity by abrogating WNK4's inhibition of NCC but does not alter WNK4's inhibition of ROMK. This effect requires AnglI, its receptor AT1R, and WNK4, and is prevented by the AT1R inhibitor losartan. NCC activity is also increased by WNK4 harboring mutations found in PHAII, and this activity cannot be further augmented by Angil signaling, consistent with PHAII mutations providing constitutive activation of the signaling pathway between AT1R and NCC. Angil's effect on NCC is also dependent on the kinase SPAK because dominant-negative SPAK or elimination of the SPAK binding motif in NCC prevent activation of NCC by Angll signaling. These effects extend to mammalian cells. AnglI increases phosphorylation of specific sites on SPAK and NCC that are necessary for activation of each in mpkDCT cells. These findings place WNK4 in the signaling pathway between AnglI and NCC. and provide a mechanism by which hypovolemia maximizes renal salt reabsoprtion without concomitantly increasing K secretion. [ABSTRACT FROM AUTHOR]

Phosphate homeostasis is central to diverse physiologic processes including energy homeostasis. formation of lipid bilayers, and bone formation. Reduced phosphate levels due to excessive renal loss cause hypophosphatemic rickets, a disease characterized by prominent bone defects; conversely, hyperphosphatemia, a major complication of renal failure, is accompanied by parathyroid hyperplasia, hyperparathyroidism, and osteodystrophy. Here, we define a syndrome featuring both hypophosphatemic rickets and hyperparathyroidism due to parathyroid hyperplasia as well as other skeletal abnormalities. We show that this disease is due to a de novo translocation with a breakpoint adjacent to a-K!otho, which encodes a f3-glucuronidase, and is implicated in aging and regulation of FGF signaling. Plasma a-Klotho levels and 13-glucuronidase activity are markedly increased in the affected patient; unexpectedly, the circulating FGF23 level is also markedly elevated. These findings suggest that the elevated a-Klotho level mimics aspects of the normal response to hyperphosphatemia and implicate a-Klotho in the selective regulation of phosphate levels and in the regulation of parathyroid mass and function; they also have implications for the pathogenesis and treatment of renal osteodystrophy in patients with kidney failure. [ABSTRACT FROM AUTHOR]

The steroid hormone aldosterone is secreted both in the setting of intravascular volume depletion and hyperkalemia, raising the question of how the kidney maximizes NaCl reabsorption in the former state while maximizing K+ secretion in the latter. Mutations in WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring increased renal NaCl reabsorption and impaired K+ secretion. PHAII-mutant WNK4 achieves these effects by increasing activity of the Na-Cl cotransporter (NCC) and the Na+ channel ENaC while concurrently inhibiting the renal outer medullary K+ channel (ROMK). We now describe a functional state for WNK4 that promotes increased, rather than decreased, K+ secretion. We show that WNK4 is phosphorylated by SGK1, a mediator of aldosterone signaling. Whereas wild-type WNK4 inhibits the activity of both ENaC and ROMK, a WNK4 mutation that mimics phosphorylation at the SGK1 site (WNK4S1169D) alleviates inhibition of both channels. The net result of these effects in the kidney would be increased K+ secretion, because of both increased electrogenic Na+ reabsorption and increased apical membrane K+ permeability. Thus, modification at the PHAII and SGK1 sites in WNK4 impart opposite effects on K+ secretion, decreasing or increasing ROMK activity and net K+ secretion, respectively. This functional state for WNK4 would thus promote the desired physiologic response to hyperkalemia, and the fact that it is induced downstream of aldosterone signaling implicates WNK4 in the physiologic response to aldosterone with hyperkalemia. Together, the different states of WNK4 allow the kidney to provide distinct and appropriate integrated responses to intravascular volume depletion and hyperkalemia. [ABSTRACT FROM AUTHOR]

Homeostasis of intravascular volume, Na+, Cl-, and K+ is interdependent and determined by the coordinated activities of structurally diverse mediators in the distal nephron and the distal colon. The behavior of these flux pathways is regulated by the reninangiotensin-aldosterone system; however, the mechanisms that allow independent modulation of individual elements have been obscure. Previous work has shown that mutations in WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension with hyperkalemia, due to altered activity of specific Na-Cl cotransporters, K+ channels, and paracellular Cl- flux mediators of the distal nephron. By coexpression studies in Xenopus oocytes, we now demonstrate that WNK4 also inhibits the epithelial Na+ channel (ENaC), the major mediator of aldosterone-sensitive Na+ (re)absorption, via a mechanism that is independent of WNK4's kinase activity. This inhibition requires intact C termini in ENaC β- and γ-subunits, which contain PY motifs used to target ENaC for clearance from the plasma membrane. Importantly, PHAII-causing mutations eliminate WNK4's inhibition of ENaC, thereby paralleling other effects of PHAII to increase sodium balance. The relevance of these findings in vivo was studied in mice harboring PHAII-mutant WNK4. The colonic epithelium of these mice demonstrates markedly increased amiloride-sensitive Na+ flux compared with wild-type littermates. These studies identify ENaC as a previously unrecognized downstream target of WNK4 and demonstrate a functional role for WNK4 in the regulation of colonic Na+ absorption. These findings support a key role for WNK4 in coordinating the activities of diverse flux pathways to achieve integrated fluid and electrolyte homeostasis. [ABSTRACT FROM AUTHOR]