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Multiple studies have confirmed the contribution of rare de novo copy number variations to the risk for autism spectrum disorders (1-3). But whereas denovo single nucleotide variants have been identified in affected individuals (4), their contribution to risk has yet to be clarified. Specifically, the frequency and distribution of these mutations have not been well characterized in matched unaffected controls, and such data are vital to the interpretation of de novo coding mutations observed in probands. Here we show, using whole-exome sequencing of 928 individuals, including 200 phenotypically discordant sibling pairs, that highly disruptive (nonsense and splice-site) de novo mutations in brain-expressed genes are associated with autism spectrum disorders and carry large effects. On the basis of mutation rates in unaffected individuals, we demonstrate that multiple independent denovosingle nucleotide variants in the same gene among unrelated probands reliably identifies risk alleles, providing a clear path forward for gene discovery. Among a total of 279 identified de novo coding mutations, there is a single instance in probands, and none in siblings, in which two independent nonsense variants disrupt the same gene, SCN2A (sodium channel, voltage-gated, type II, a subunit), a result that is highly unlikely by chance., We completed whole-exome sequencing in 238 families from the Simons Simplex Collection (SSC), a comprehensively phenotyped autism spectrum disorders (ASD) cohort consisting of pedigrees with two unaffected parents, an affected [...]

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

Somatic loss of wild-type alleles can produce disease traits such as neoplasia. Conversely, somatic loss of disease-causing mutations can revert phenotypes; however, these events are infrequently observed. Here we show that ichthyosis with confetti, a severe, sporadic skin disease in humans, is associated with thousands of revertant clones of normal skin that arise from loss of heterozygosity on chromosome 17q via mitotic recombination. This allowed us to map and identify disease-causing mutations in the gene encoding keratin 10 (KRT10); all result in frameshifts into the same alternative reading frame, producing an arginine-rich C-terminal peptide that redirects keratin 10 from the cytokeratin filament network to the nucleolus. The high frequency of somatic reversion in ichthyosis with confetti suggests that revertant stem cell clones are under strong positive selection and/or that the rate of mitotic recombination is elevated in individuals with this disorder. 10.1126/science.1192280

The development of the human cerebral cortex is an orchestrated process involving the generation of neural progenitors in the periventricular germinal zones, cell proliferation characterized by symmetric and asymmetric mitoses, followed by migration of post-mitotic neurons to their final destinations in six highly ordered, functionally specialized layers (1,2). An understanding of the molecular mechanisms guiding these intricate processes is in its infancy, substantially driven by the discovery of rare mutations that cause malformations of cortical development (3-6). Mapping of disease loci in putative Mendelian forms of malformations of cortical development has been hindered by marked locus heterogeneity, small kindred sizes and diagnostic classifications that may not reflect molecular pathogenesis. Here we demonstrate the use of whole-exome sequencing to overcome these obstacles by identifying recessive mutations in WD repeat domain 62 (WDR62)as the cause of a wide spectrum of severe cerebral cortical malformations including microcephaly, pachygyria with cortical thickening as well as hypoplasia of the corpus callosum. Some patients with mutations in WDR62 had evidence of additional abnormalities including lissencephaly, schizencephaly, polymicrogyria and, in one instance, cerebellar hypoplasia, all traits traditionally regarded as distinct entities. In mice and humans, WDR62 transcripts and protein are enriched in neural progenitors within the ventricular and subventricular zones. Expression of WDR62 in the neocortex is transient, spanning the period of embryonic neurogenesis. Unlike other known microcephaly genes, WDR62 does not apparently associate with centrosomes and is predominantly nuclear in localization. These findings unify previously disparate aspects of cerebral cortical development and highlight the use of whole-exome sequencing to identify disease loci in settings in which traditional methods have proved challenging., Malformations of cortical development are a diverse group of often devastating structural brain disorders reflecting deranged neuronal proliferation, migration or organization. Application of traditional mapping approaches has proved to be [...]

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

Tourette's syndrome (TS) is a genetically influenced developmental neuropsychiatric disorder characterized by chronic vocal and motor tics. We studied Slit and Trk-like 1 (SLITRK1) as a candidate gene on chromosome 13q31.1 because of its proximity to a de novo chromosomal inversion in a child with TS. Among 174 unrelated probands, we identified a frameshift mutation and two independent occurrences of the identical variant in the binding site for microRNA hsa-miR-189. These variants were absent from 3600 control chromosomes. SLITRK1 mRNA and hsa-miR-189 showed an overlapping expression pattern in brain regions previously implicated in TS. Wild-type SLITRK1, but not the frame-shift mutant, enhanced dendritic growth in primary neuronal cultures. Collectively, these findings support the association of rare SLITRK1 sequence variants with TS., TS is a potentially debilitating developmental neuropsychiatric disorder, characterized by the combination of persistent vocal and motor tics, that affects as many as 1 in 100 individuals (1, 2). A [...]

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

Hypertension and dyslipidemia are risk factors for atherosderosis and occur together more often than expected by chance. Although this clustering suggests shared causation, unifying factors remain unknown. We describe a large kindred with a syndrome including hypertension, hypercbolesterolemia, and hypomagnesemia. Each phenotype is transmitted on the maternal lineage with a pattern indicating mitochondrial inheritance. Analysis of the mitochondria[ genome of the maternal lineage identified a homoplasmic mutation substituting cytidine for uridine immediately 5' to the mitochondria[ transfer RN[A.sup.Ile] anticodon. Uridine at this position is nearly invariate among transfer RNAs because of its role in stabilizing the anticodon loop. Given the known loss of mitochondrial function with aging, these findings may have implications for the common clustering of these metabolic disorders., Hypertension and dyslipidemia are important risk factors for many common cardiovascular diseases, including myocardial infarction, stroke, and congestive heart failure (l, 2). These traits are concordant in individual patients more [...]

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.

Hypertension and pregnancy-related hypertension are major public health problems of largely unknown causes. We describe a mutation in the mineralocorticoid receptor (MR), S810L, that causes early-onset hypertension that is markedly exacerbated in pregnancy. This mutation results in constitutive MR activity and alters receptor specificity, with progesterone and other steroids lacking 21-hydroxyl groups, normally MR antagonists, becoming potent agonists. Structural and biochemical studies indicate that the mutation results in the gain of a van der Waals interaction between helix 5 and helix 3 that substitutes for interaction of the steroid 21-hydroxyl group with helix 3 in the wild-type receptor. This helix 5-helix 3 interaction is highly conserved among diverse nuclear hormone receptors, suggesting its general role in receptor activation., Although blood pressure is normally reduced throughout gestation, about 6% of pregnancies are complicated by the development of hypertension, raising the risk of pre-eclampsia, a hypertensive disorder of pregnancy that [...]

Epithelia permit selective and regulated flux from apical to basolateral surfaces by transcellular passage through cells or paracellular flux between cells. Tight junctions constitute the barrier to paracellular conductance; however, little is known about the specific molecules that mediate paracellular permeabilities. Renal magnesium ion ([Mg.sup.2+]) resorption occurs predominantly through a paracellular conductance in the thick ascending limb of Henle (TAL). Here, positional cloning has identified a human gene, paracellin-1 (PCLN-1), mutations in which cause renal [Mg.sup.2+] wasting. PCLN-1 is located in tight junctions of the TAL and is related to the claudin family of tight junction proteins. These findings provide insight into [Mg.sup.2+] homeostasis, demonstrate the role of a tight junction protein in human disease, and identify an essential component of a selective paracellular conductance., The composition of fluids on opposite sides of epithelial cell layers is often different, and is maintained by barriers to passage of fluid, electrolytes, nutrients, pathogens, and cells. Cellular membranes [...]