How to facilitate NHS professionals to recognise and use skills gained from global health engagement when back in the UK workforce? A participatory action research project to design, pilot and evaluate a series of online leadership workshops.

OBJECTIVES: Leadership knowledge and skills are known to be developed by health professionals during global health experiences overseas. However, volunteers struggle to recognise and use these new skills on return to their workplace. A series of bespoke leadership workshops were designed, delivered and evaluated by leadership experts to help enhance the transferability of leadership skills back to the UK National Health Service. DESIGN: A mixed-methods participatory action research methodology was employed to explore the impact of the workshops. This approach lends itself to a complex, situated project involving multiple partners. Quantitative and qualitative descriptive data were collected via online survey (n=29 participants) and focus groups (n=18 focus groups) and thematically analysed. SETTING: The authors delivered the tailored leadership workshops online to globally engaged National Health Service (NHS) healthcare professionals based in England who had all worked overseas within the past 5 years. PARTICIPANTS: 29 participants attended: 11 medical doctors; 6 nurses/midwives; 10 allied health professionals; 1 NHS manager and 1 student nurse (who was also working as a healthcare assistant). RESULTS: Participants were able to network both during the large group discussions and while in smaller breakout groups. Data highlighted the substantial benefits obtained from this networking, with 91% of participants reporting it enriched their learning experience, particularly within a multi-disciplinary context, and by having the time and space for facilitated reflection on leadership. Furthermore, 78% agreed that they learned new skills for influencing change beyond their position and 76% reported they could maximise the impact of this change for themselves and their employer. Participants also reported the development of systems and ethical leadership knowledge that they felt they could transfer to their NHS roles. CONCLUSIONS: This study extends explorations of global health experiences by moving beyond the skills gained while working in low-income and middle-income countries. The innovative online leadership workshops gave agency to individuals to recognise and use the skills gained from global health placements on return to the NHS.

Multiplex Lateral Flow Assay and the Sample Preparation Method for the Simultaneous Detection of Three Marine Toxins.

A multiplex lateral flow immunoassay (LFA) has been developed to detect the primary marine biotoxin groups: amnesic shellfish poisoning toxins, paralytic shellfish poisoning toxins, and diarrhetic shellfish poisoning toxins. The performance characteristics of the multiplex LFA were evaluated for its suitability as a screening method for the detection of toxins in shellfish. The marine toxin-specific antibodies were class-specific, and there was no cross-reactivity between the three toxin groups. The test is capable of detecting all three marine toxin groups, with working ranges of 0.2-1.5, 2.5-65.0, and 8.2-140.3 ng/mL for okadaic acid, saxitoxin, and domoic acid, respectively. This allows the multiplex LFA to detect all three toxin groups at the EU regulatory limits, with a single sample extraction method and dilution volume. No matrix effects were observed on the performance of the LFA with mussel samples spiked with toxins. The developed LFA uses a simple and pocket-sized, portable Cube Reader to provide an accurate result. We also evaluated the use of this Cube Reader with commercially available monoplex lateral flow assays for marine toxins.

Progression of Mineral Ion Abnormalities in Patients With Jansen Metaphyseal Chondrodysplasia.

Context: Five different activating PTH/PTH-related peptide (PTHrP) receptor (PTHR1) mutations have been reported as causes of Jansen metaphyseal chondrodysplasia (JMC), a rare disorder characterized by severe growth plate abnormalities and PTH-independent hypercalcemia. Objectives: Assess the natural history of clinical and laboratory findings in 24 patients with JMC and characterize the disease-causing mutant receptors in vitro. Patients and Methods: The H223R mutation occurred in 18 patients. T410P, I458R and I458K each occurred in single cases; T410R was present in a father and his two sons. Laboratory records were analyzed individually and in aggregate. Results: Postnatal calcium levels were normal in most patients, but elevated between 0.15 and 10 years (11.8 ± 1.37 mg/dL) and tended to normalize in adults (10.0 ± 1.03 mg/dL). Mean phosphate levels were at the lower end of the age-specific normal ranges. Urinary calcium/creatinine (mg/mg) were consistently elevated (children, 0.80 ± 0.40; adults, 0.28 ± 0.19). Adult heights were well below the 3rd percentile for all patients, except for those with the T410R mutation. Most patients with JMC had undergone orthopedic surgical procedures, most had nephrocalcinosis, and two had advanced chronic kidney disease. The five PTHR1 mutants showed varying degrees of constitutive and PTH-stimulated cAMP signaling activity when expressed in HEK293 reporter cells. The inverse agonist [L11,dW12,W23,Y36]PTHrP(7-36) reduced basal cAMP signaling for each PTHR1 mutant. Conclusions: Except for T410R, the other PTHR1 mutations were associated with indistinguishable mineral ion abnormalities and cause similarly severe growth impairment. Hypercalciuria persisted into adulthood. An inverse agonist ligand effectively reduced in vitro PTH-independent cAMP formation at all five PTHR1 mutants, suggesting a potential path toward therapy.

Epigenomic Modifications Mediating Antibody Maturation.

Epigenetic modifications, such as histone modifications, DNA methylation status, and non-coding RNAs (ncRNA), all contribute to antibody maturation during somatic hypermutation (SHM) and class-switch recombination (CSR). Histone modifications alter the chromatin landscape and, together with DNA primary and tertiary structures, they help recruit Activation-Induced Cytidine Deaminase (AID) to the immunoglobulin (Ig) locus. AID is a potent DNA mutator, which catalyzes cytosine-to-uracil deamination on single-stranded DNA to create U:G mismatches. It has been shown that alternate chromatin modifications, in concert with ncRNAs and potentially DNA methylation, regulate AID recruitment and stabilize DNA repair factors. We, hereby, assess the combination of these distinct modifications and discuss how they contribute to initiating differential DNA repair pathways at the Ig locus, which ultimately leads to enhanced antibody-antigen binding affinity (SHM) or antibody isotype switching (CSR). We will also highlight how misregulation of epigenomic regulation during DNA repair can compromise antibody development and lead to a number of immunological syndromes and cancer.

Chytrid fungus infection in zebrafish demonstrates that the pathogen can parasitize non-amphibian vertebrate hosts.

Aquatic chytrid fungi threaten amphibian biodiversity worldwide owing to their ability to rapidly expand their geographical distributions and to infect a wide range of hosts. Combating this risk requires an understanding of chytrid host range to identify potential reservoirs of infection and to safeguard uninfected regions through enhanced biosecurity. Here we extend our knowledge on the host range of the chytrid Batrachochytrium dendrobatidis by demonstrating infection of a non-amphibian vertebrate host, the zebrafish. We observe dose-dependent mortality and show that chytrid can infect and proliferate on zebrafish tissue. We also show that infection phenotypes (fin erosion, cell apoptosis and muscle degeneration) are direct symptoms of infection. Successful infection is dependent on disrupting the zebrafish microbiome, highlighting that, as is widely found in amphibians, commensal bacteria confer protection against this pathogen. Collectively, our findings greatly expand the limited tool kit available to study pathogenesis and host response to chytrid infection.

Tracking the amphibian pathogens Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans using a highly specific monoclonal antibody and lateral-flow technology.

The fungus Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a lethal epizootic disease of amphibians. Rapid identification of the pathogen and biosecurity is essential to prevent its spread, but current laboratory-based tests are time-consuming and require specialist equipment. Here, we describe the generation of an IgM monoclonal antibody (mAb), 5C4, specific to Bd as well as the related salamander and newt pathogen Batrachochytrium salamandrivorans (Bsal). The mAb, which binds to a glycoprotein antigen present on the surface of zoospores, sporangia and zoosporangia, was used to develop a lateral-flow assay (LFA) for rapid (15 min) detection of the pathogens. The LFA detects known lineages of Bd and also Bsal, as well as the closely related fungus Homolaphlyctis polyrhiza, but does not detect a wide range of related and unrelated fungi and oomycetes likely to be present in amphibian habitats. When combined with a simple swabbing procedure, the LFA was 100% accurate in detecting the water-soluble 5C4 antigen present in skin, foot and pelvic samples from frogs, newts and salamanders naturally infected with Bd or Bsal. Our results demonstrate the potential of the portable LFA as a rapid qualitative assay for tracking these amphibian pathogens and as an adjunct test to nucleic acid-based detection methods.

Contribution of murine IgG Fc regions to antibody binding to the capsule of Burkholderia pseudomallei.

Immunoglobulin G3 (IgG3) is the predominant IgG subclass elicited in response to polysaccharide antigens in mice. This specific subclass has been shown to crosslink its fragment crystallizable (Fc) regions following binding to multivalent polysaccharides. Crosslinking leads to increased affinity through avidity, which theoretically should lead to more effective protection against bacteria and yeast displaying capsular polysaccharides on their surface. To investigate this further we have analyzed the binding characteristics of 2 IgG monoclonal antibody (mAb) subclass families that bind to the capsular polysaccharide (CPS) of Burkholderia pseudomallei. The first subclass family originated from an IgG3 hybridoma cell line (3C5); the second family was generated from an IgG1 cell line (2A5). When the Fc region of the 3C5 IgG3 is removed by proteolytic cleavage, the resulting F(ab')2 fragments exhibit decreased affinity compared to the full-length mAb. Similarly, when the parent IgG3 mAb is subclass-switched to IgG1, IgG2b, and IgG2a, all of these subclasses exhibit decreased affinity. This decrease in affinity is not seen when the 2A5 IgG1 mAb is switched to an IgG2b or IgG2a, strongly suggesting the drop in affinity is related to the IgG3 Fc region.

Burkholderia pseudomallei Capsular Polysaccharide Recognition by a Monoclonal Antibody Reveals Key Details toward a Biodefense Vaccine and Diagnostics against Melioidosis.

Burkholderia pseudomallei is the bacterium responsible for melioidosis, an infectious disease with high mortality rates. Since melioidosis is a significant public health concern in endemic regions and the organism is currently classified as a potential biothreat agent, the development of effective vaccines and rapid diagnostics is a priority. The capsular polysaccharide (CPS) expressed by B. pseudomallei is a highly conserved virulence factor and a protective antigen. Because of this, CPS is considered an attractive antigen for use in the development of both vaccines and diagnostics. In the present study, we describe the interactions of CPS with the murine monoclonal antibody (mAb) 4C4 using a multidisciplinary approach including organic synthesis, molecular biology techniques, surface plasmon resonance, and nuclear magnetic spectroscopy. Using these methods, we determined the mode of binding between mAb 4C4 and native CPS or ad hoc synthesized capsular polysaccharide fragments. Interestingly, we demonstrated that the O-acetyl moiety of CPS is essential for the interaction of the CPS epitope with mAb 4C4. Collectively, our results provide important insights into the structural features of B. pseudomallei CPS that enable antibody recognition that may help the rational design of CPS-based vaccine candidates. In addition, our findings confirm that the mAb 4C4 is suitable for use in an antibody-based detection assay for diagnosis of B. pseudomallei infections.

Angioplasty for renovascular hypertension in 78 children.

OBJECTIVES: To evaluate the outcome of percutaneous transluminal angioplasty (PTA) in children with renovascular hypertension (RVH) treated at a single centre over 29 years. METHODS: A retrospective study of the medical charts of all children with RVH who underwent PTA between 1984 and 2012. The primary outcome measurement was blood pressure (BP) achieved after the procedure. The BP before the procedure was compared with that at last available follow-up, 6 (range 0.6-16) years after the initial procedure. RESULTS: Seventy-eight children with median (range) age of 6.5 (0.5-17) years were studied. Twenty-three (29.5%) had an underlying syndrome, 35 (44.9%) children had bilateral renal artery stenosis (RAS), 18 (23%) intrarenal disease and 11(14%) showed bilateral RAS and intrarenal disease. Twenty (25.6%) children had mid-aortic syndrome and 14 (17.9%) cerebrovascular disease. One hundred and fourteen PTA procedures were carried out including 31 stent insertions. Following PTA, BP was improved in 49 (62.8%) children and of those 18 (23.1%) were cured. Children with involvement of only the main renal arteries showed improved BP control in 79.9% of the children with cure in 39.5%. BP was intentionally maintained above the 95th centile for age and height in four children with coexistent cerebrovascular disease. No change in BP was seen in 18 children despite observed technical success of the PTA, and in seven children due to technical failure of the procedure. CONCLUSIONS: PTA provided a clinical benefit in 62.8% of children with RVH.

Development of a prototype lateral flow immunoassay (LFI) for the rapid diagnosis of melioidosis.

Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.

Systemic polyarteritis nodosa in the young: a single-center experience over thirty-two years.

OBJECTIVE: Polyarteritis nodosa (PAN) is a rare disease of childhood. The aims of this study were to describe the clinical features, treatment, and outcome of systemic childhood PAN and to identify predictors of relapse. METHODS: A single-center retrospective medical records review of children with PAN fulfilling the European League Against Rheumatism (EULAR)/Paediatric Rheumatology European Society (PRES)/Paediatric Rheumatology International Trials Organisation (PRINTO) classification criteria who were seen over a 32-year period was performed. Data on demographic and clinical features, treatments, relapses (recurrence of clinical signs/symptoms or occurrence of new symptoms after initial remission requiring escalation or resumption of immunosuppressive therapy), and deaths were recorded. A disease activity score was retrospectively assigned using the Paediatric Vasculitis Activity Score (PVAS) instrument. Cox regression analysis was used to identify significant predictors of relapse. RESULTS: Sixty-nine children with PAN were identified; 55% were male, and their median age was 8.5 years (range 0.9-15.8 years). Their clinical features at presentation were fever (87%), myalgia (83%), skin (88%), renal (19%), severe gastrointestinal (GI) (10%), and neurologic (10%) involvement. The PVAS at presentation was 9 of 63 (range 4-24). Histopathologic analysis of the skin showed necrotizing vasculitis in biopsy samples from 40 of 50 children. Results of selective visceral arteriography suggested the presence of PAN in 96% of patients. Treatment included cyclophosphamide and corticosteroids (83%), plasma exchange (9%), and biologic agents (after 2002; 13%). The relapse rate was 35%, and the mortality rate was 4%. Severe GI involvement was associated with increased risk of relapse (P = 0.031), while longer time to induce remission (P = 0.022) and increased cumulative dose of cyclophosphamide (P = 0.005) were associated with lower relapse risk. CONCLUSION: Childhood PAN is a severe inflammatory disease of insidious onset and variable clinical presentation. Relapses occurred more frequently in those with severe GI involvement. A higher cumulative dose of cyclophosphamide was associated with a lower risk of relapse.

Hypertension with or without adrenal hyperplasia due to different inherited mutations in the potassium channel KCNJ5.

We recently implicated two recurrent somatic mutations in an adrenal potassium channel, KCNJ5, as a cause of aldosterone-producing adrenal adenomas (APAs) and one inherited KCNJ5 mutation in a Mendelian form of early severe hypertension with massive adrenal hyperplasia. The mutations identified all altered the channel selectivity filter, producing increased Na(+) conductance and membrane depolarization, the signal for aldosterone production and proliferation of adrenal glomerulosa cells. We report herein members of four kindreds with early onset primary aldosteronism of unknown cause. Sequencing of KCNJ5 revealed that affected members of two kindreds had KCNJ5(G151R) mutations, identical to one of the prevalent recurrent mutations in APAs. These individuals had severe progressive aldosteronism and hyperplasia requiring bilateral adrenalectomy in childhood for blood pressure control. Affected members of the other two kindreds had KCNJ5(G151E) mutations, which are not seen in APAs. These subjects had easily controlled hypertension and no evidence of hyperplasia. Surprisingly, electrophysiology of channels expressed in 293T cells demonstrated that KCNJ5(G151E) was the more extreme mutation, producing a much larger Na(+) conductance than KCNJ5(G151R), resulting in rapid Na(+)-dependent cell lethality. We infer that this increased lethality limits adrenocortical cell mass and the severity of aldosteronism in vivo, accounting for the milder phenotype among these patients. These findings demonstrate striking variations in phenotypes and clinical outcome resulting from different mutations of the same amino acid in KCNJ5 and have implications for the diagnosis and pathogenesis of primary aldosteronism with and without adrenal hyperplasia.

Medium-size-vessel vasculitis.

Medium-size-artery vasculitides do occur in childhood and manifest, in the main, as polyarteritis nodosa (PAN), cutaneous PAN and Kawasaki disease. Of these, PAN is the most serious, with high morbidity and not inconsequential mortality rates. New classification criteria for PAN have been validated that will have value in epidemiological studies and clinical trials. Renal involvement is common and recent therapeutic advances may result in improved treatment options. Cutaneous PAN is a milder disease characterised by periodic exacerbations and often associated with streptococcal infection. There is controversy as to whether this is a separate entity or part of the systemic PAN spectrum. Kawasaki disease is an acute self-limiting systemic vasculitis, the second commonest vasculitis in childhood and the commonest cause of childhood-acquired heart disease. Renal manifestations occur and include tubulointerstitial nephritis and renal failure. An infectious trigger and a genetic predisposition seem likely. Intravenous immunoglobulin (IV-Ig) and aspirin are effective therapeutically, but in resistant cases, either steroid or infliximab have a role. Greater understanding of the pathogenetic mechanisms involved in these three types of vasculitis and better long-term follow-up data will lead to improved therapy and prediction of prognosis.

Results of surgical treatment for renovascular hypertension in children: 30 year single centre experience.

BACKGROUND: We retrospectively reviewed the medical records of all patients who underwent surgery as part of the treatment of renovascular hypertension (RVH) at our centre between 1979 and 2008. Patients. Thirty-seven children (65% male) with a median age of 7.6 (0.4-17.9) years were identified with a median systolic blood pressure (SBP) of 140 (105-300) mm Hg prior to surgery. Bilateral renal artery stenosis and intra-renal disease were present in 19 (51%) patients, mid-aortic syndrome in 15 (40%), involvement of visceral arteries in eight out of 35 (23%) and coexisting cerebral disease in eight out of 30 (26%) investigated patients. RESULTS: Surgical procedures (n = 53) included (i) nephrectomy (18, of which two unplanned and two secondary due to technical failure), (ii) renovascular surgery on the renal arteries (28, of which 18 had autologous surgery and 10 synthetic grafts inserted for revascularisation) and (iii) aortic reconstruction with (6) and without (1) a synthetic graft. Post-operative complications were haemorrhage (5), septicaemia (5) and chylous ascites (1). There were no perioperative deaths; two children died during follow-up. The SBP post-surgery improved to a median value of 116 (range 90-160) mm Hg. Twelve months after surgery, 16 (43%) children had normal blood pressure without treatment, 15 (41%) normal or improved on one to four antihypertensive drugs and four (11%) unchanged; no data were available for two (5%) children. CONCLUSION: Surgery effectively treated the hypertension of 90% of our children, when performed in conjunction with medical therapy and interventional radiology. In spite of aggressive surgical treatment, RVH is sometimes a progressive disease.

Mid-aortic syndrome: long-term outcome of 36 children.

The clinical characteristics and outcomes of children with mid-aortic syndrome (MAS) and the effectiveness of different therapeutic approaches in reducing hypertension are still debated. We conducted a single-centre retrospective review of the records of children with MAS over 30 years. Children with angiographic evidence of a narrowed abdominal aorta were included. Therapeutic approaches included medical management, percutaneous transluminal angioplasty and/or surgical intervention. Thirty-six children had presented at a median age of 2.7 years (10 days-10 years). Thirteen (36%) patients had associated syndromes, and 44% had been diagnosed with cerebrovascular disease. All patients had involvement of multiple arteries. The mortality rate was 8% after a median follow-up period of 4.5 (range 1.1-19.7) years. Among the children who survived, 90% had obtained a reduction in their blood pressure (BP). Of the patients, 76% had had a normal estimated glomerular filtration rate (eGFR) at the last follow-up examination. Seventeen percent (six of 36) had renal dysfunction at presentation. Although MAS is a severe and widespread disease, in most cases it can be effectively treated with a combination of medical, angioplasty and surgical interventions.

Management of high blood pressure in children and adolescents: recommendations of the European Society of Hypertension.

Hypertension in children and adolescents has gained ground in cardiovascular medicine, thanks to the progress made in several areas of pathophysiological and clinical research. These guidelines represent a consensus among specialists involved in the detection and control of high blood pressure in children and adolescents. The guidelines synthesize a considerable amount of scientific data and clinical experience and represent best clinical wisdom upon which physicians, nurses and families should base their decisions. They call attention to the burden of hypertension in children and adolescents, and its contribution to the current epidemic of cardiovascular disease, these guidelines should encourage public policy makers, to develop a global effort to improve identification and treatment of high blood pressure among children and adolescents.

Biologic therapy in primary systemic vasculitis of the young.

OBJECTIVES: To describe the biologic treatment regimens and report the efficacy and safety of biologic therapies in a multicentre series of children with primary systemic vasculitis (PSV). METHODS: This was a retrospective descriptive case series of children with PSV treated with biologic therapy between February 2002 and November 2007. Primary retrospective outcome assessment measures were: daily corticosteroid dose; Birmingham Vasculitis Activity Score (BVAS); and adverse events (including infection rate). RESULTS: Twenty-five patients median age 8.8 (range 2.4-16) years; 11 male with active PSV (n = 6 with anti-neutrophil cytoplasmic antibody associated vasculitides, n = 11 with polyarteritis nodosa, n = 7 with unclassified vasculitis and n = 1 with Behçet's disease) were treated with biologic agents including infliximab (n = 7), rituximab (n = 6), etanercept (n = 4), adalimumab (n = 1) or multiple biologics sequentially (n = 7). Overall, there was a significant reduction in BVAS from a median of 8.5 (range 5-32) at start of therapy to 4 (range 0-19) at median 32 months follow-up (P = 0.003) accompanied by significant reduction in median daily prednisolone requirement from 1 (range 0.2-2) to 0.25 (range 0-1) mg/kg/day, P = 0.000. For those receiving multiple biologic agents sequentially, a similar clinical improvement was observed with corticosteroid sparing. Infections occurred in 24%, the most severe in those receiving infliximab. CONCLUSION: Our data provide retrospective evidence of efficacy of these agents, and highlight the associated infectious complications. Further multicentre standardization of treatment protocols and data collection to inform clinical trials of biologic therapy in systemic vasculitis of the young is required.

Mutational analysis of CLC-5, cofilin and CLC-4 in patients with Dent's disease.

BACKGROUND/AIMS: Dent's disease is caused by mutations in the chloride/proton antiporter, CLC-5, or oculo-cerebro-renal-syndrome-of-Lowe (OCRL1) genes. METHODS: Eighteen probands with Dent's disease were investigated for mutations in CLC-5 and two of its interacting proteins, CLC-4 and cofilin. Wild-type and mutant CLC-5s were assessed in kidney cells. Urinary calcium excretion following an oral calcium challenge was studied in one family. RESULTS: Seven different CLC-5 mutations consisting of two nonsense mutations (Arg347Stop and Arg718Stop), two missense mutations (Ser244Leu and Arg516Trp), one intron 3 donor splice site mutation, one deletion-insertion (nt930delTCinsA) and an in-frame deletion (523delVal) were identified in 8 patients. In the remaining 10 patients, DNA sequence abnormalities were not detected in the coding regions of CLC-4 or cofilin, and were independently excluded for OCRL1. Patients with CLC-5 mutations were phenotypically similar to those without. The donor splice site CLC-5 mutation resulted in exon 3 skipping. Electrophysiology demonstrated that the 523delVal CLC-5 mutation abolished CLC-5-mediated chloride conductance. Sixty percent of women with the CLC-5 deletion-insertion had nephrolithiasis, although calcium excretion before and after oral calcium challenge was similar to that in unaffected females. CONCLUSIONS: Three novel CLC-5 mutations were identified, and mutations in OCRL1, CLC-4 and cofilin excluded in causing Dent's disease in this patient cohort.

Epilepsy, ataxia, sensorineural deafness, tubulopathy, and KCNJ10 mutations.

BACKGROUND: Five children from two consanguineous families presented with epilepsy beginning in infancy and severe ataxia, moderate sensorineural deafness, and a renal salt-losing tubulopathy with normotensive hypokalemic metabolic alkalosis. We investigated the genetic basis of this autosomal recessive disease, which we call the EAST syndrome (the presence of epilepsy, ataxia, sensorineural deafness, and tubulopathy). METHODS: Whole-genome linkage analysis was performed in the four affected children in one of the families. Newly identified mutations in a potassium-channel gene were evaluated with the use of a heterologous expression system. Protein expression and function were further investigated in genetically modified mice. RESULTS: Linkage analysis identified a single significant locus on chromosome 1q23.2 with a lod score of 4.98. This region contained the KCNJ10 gene, which encodes a potassium channel expressed in the brain, inner ear, and kidney. Sequencing of this candidate gene revealed homozygous missense mutations in affected persons in both families. These mutations, when expressed heterologously in xenopus oocytes, caused significant and specific decreases in potassium currents. Mice with Kcnj10 deletions became dehydrated, with definitive evidence of renal salt wasting. CONCLUSIONS: Mutations in KCNJ10 cause a specific disorder, consisting of epilepsy, ataxia, sensorineural deafness, and tubulopathy. Our findings indicate that KCNJ10 plays a major role in renal salt handling and, hence, possibly also in blood-pressure maintenance and its regulation.