University Children's Hospital, Belgrade

Skin tests are of paramount importance for the evaluation of drug hypersensitivity reactions. Drug skin tests are often not carried out because of lack of concise information on specific test concentrations. The diagnosis of drug allergy is often based on history alone, which is an unreliable indicator of true hypersensitivity.To promote and standardize reproducible skin testing with safe and nonirritant drug concentrations in the clinical practice, the European Network and European Academy of Allergy and Clinical Immunology (EAACI) Interest Group on Drug Allergy has performed a literature search on skin test drug concentration in MEDLINE and EMBASE, reviewed and evaluated the literature in five languages using the GRADE system for quality of evidence and strength of recommendation. Where the literature is poor, we have taken into consideration the collective experience of the group.We recommend drug concentration for skin testing aiming to achieve a specificity of at least 95%. It has been possible to recommend specific drug concentration for betalactam antibiotics, perioperative drugs, heparins, platinum salts and radiocontrast media. For many other drugs, there is insufficient evidence to recommend appropriate drug concentration. There is urgent need for multicentre studies designed to establish and validate drug skin test concentration using standard protocols. For most drugs, sensitivity of skin testing is higher in immediate hypersensitivity compared to nonimmediate hypersensitivity.

Analyzing the type and frequency of patient-specific mutations that give rise to Duchenne muscular dystrophy (DMD) is an invaluable tool for diagnostics, basic scientific research, trial planning, and improved clinical care. Locus-specific databases allow for the collection, organization, storage, and analysis of genetic variants of disease. Here, we describe the development and analysis of the TREAT-NMD DMD Global database (http://umd.be/TREAT_DMD/). We analyzed genetic data for 7,149 DMD mutations held within the database. A total of 5,682 large mutations were observed (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions and 199 (14%) affected the splice sites. Point mutations totalled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations).

Evidence-based guidelines, or recommendations, for the management of infants with seizures are lacking. A Task Force of the Commission of Pediatrics developed a consensus document addressing diagnostic markers, management interventions, and outcome measures for infants with seizures. Levels of evidence to support recommendations and statements were assessed using the American Academy of Neurology Guidelines and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system. The report contains recommendations for different levels of care, noting which would be regarded as standard care, compared to optimal care, or "state of the art" interventions. The incidence of epilepsy in the infantile period is the highest of all age groups (strong evidence), with epileptic spasms the largest single subgroup and, in the first 2 years of life, febrile seizures are the most commonly occurring seizures. Acute intervention at the time of a febrile seizure does not alter the risk for subsequent epilepsy (class 1 evidence). The use of antipyretic agents does not alter the recurrence rate (class 1 evidence), and there is no evidence to support initiation of regular antiepileptic drugs for simple febrile seizures (class 1 evidence). Infants with abnormal movements whose routine electroencephalography (EEG) study is not diagnostic, would benefit from video-EEG analysis, or home video to capture events (expert opinion, level U recommendation). Neuroimaging is recommended at all levels of care for infants presenting with epilepsy, with magnetic resonance imaging (MRI) recommended as the standard investigation at tertiary level (level A recommendation). Genetic screening should not be undertaken at primary or secondary level care (expert opinion). Standard care should permit genetic counseling by trained personal at all levels of care (expert opinion). Genetic evaluation for Dravet syndrome, and other infantile-onset epileptic encephalopathies, should be available in tertiary care (weak evidence, level C recommendation). Patients should be referred from primary or secondary to tertiary level care after failure of one antiepileptic drug (standard care) and optimal care equates to referral of all infants after presentation with a seizure (expert opinion, level U evidence). Infants with recurrent seizures warrant urgent assessment for initiation of antiepileptic drugs (expert opinion, level U recommendation). Infantile encephalopathies should have rapid introduction and increment of antiepileptic drug dosage (expert opinion, level U recommendation). There is no high level evidence to support any particular current agents for use in infants with seizures. For focal seizures, levetiracetam is effective (strong evidence); for generalized seizures, weak evidence supports levetiracetam, valproate, lamotrigine, topiramate, and clobazam; for Dravet syndrome, strong evidence supports that stiripentol is effective (in combination with valproate and clobazam), whereas weak evidence supports that topiramate, zonisamide, valproate, bromide, and the ketogenic diet are possibly effective; and for Ohtahara syndrome, there is weak evidence that most antiepileptic drugs are poorly effective. For epileptic spasms, clinical suspicion remains central to the diagnosis and is supported by EEG, which ideally is prolonged (level C recommendation). Adrenocorticotropic hormone (ACTH) is preferred for short-term control of epileptic spasms (level B recommendation), oral steroids are probably effective in short-term control of spasms (level C recommendation), and a shorter interval from the onset of spasms to treatment initiation may improve long-term neurodevelopmental outcome (level C recommendation). The ketogenic diet is the treatment of choice for epilepsy related to glucose transporter 1 deficiency syndrome and pyruvate dehydrogenase deficiency (expert opinion, level U recommendation). The identification of patients as potential candidates for epilepsy surgery should be part of standard practice at primary and secondary level care. Tertiary care facilities with experience in epilepsy surgery should undertake the screening for epilepsy surgical candidates (level U recommendation). There is insufficient evidence to conclude if there is benefit from vagus nerve stimulation (level U recommendation). The key recommendations are summarized into an executive summary. The full report is available as Supporting Information. This report provides a comprehensive foundation of an approach to infants with seizures, while identifying where there are inadequate data to support recommended practice, and where further data collection is needed to address these deficits.

Hypersensitivity reactions to betalactams (BLs) are classified as immediate or nonimmediate. The former usually appear within 1 h of drug‐intake and are mediated by specific IgE‐antibodies. Nonimmediate reactions are those occurring more than 1 h after drug‐intake, and they can be T‐cell mediated. The diagnostic evaluation of allergic reactions to BLs has changed over the last 5 years, for several reasons. Major and minor determinants are no longer commercially available for skin testing in many countries. In immediate allergic reactions, the sensitivity of skin testing and immunoassays is decreasing and new in vitro methods, such as the basophil activation test, are gaining importance for diagnosis. For nonimmediate reactions, skin testing appears to be less sensitive than previous results, although more studies need to be carried out in this direction. Nevertheless, the drug provocation test is still necessary for diagnosis.

Hypersensitivity reactions to betalactams (BLs) are classified as immediate or nonimmediate. The former usually appear within 1 h of drug-intake and are mediated by specific IgE-antibodies. Nonimmediate reactions are those occurring more than 1 h after drug-intake, and they can be T-cell mediated. The diagnostic evaluation of allergic reactions to BLs has changed over the last 5 years, for several reasons. Major and minor determinants are no longer commercially available for skin testing in many countries. In immediate allergic reactions, the sensitivity of skin testing and immunoassays is decreasing and new in vitro methods, such as the basophil activation test, are gaining importance for diagnosis. For nonimmediate reactions, skin testing appears to be less sensitive than previous results, although more studies need to be carried out in this direction. Nevertheless, the drug provocation test is still necessary for diagnosis.

A recent survey of the European Academy of Allergy and Clinical Immunology (EAACI) Drug Allergy Interest Group (DAIG) on how European allergy specialists deal with beta-lactam (BL) hypersensitivity demonstrated a significant heterogeneity in current practice, suggesting the need to review and update existing EAACI guidelines in order to make the diagnostic procedures as safe and accurate, but also as cost-effective, as possible. For this purpose, a bibliographic search on large studies regarding BL hypersensitivity diagnosis was performed by an EAACI task force, which reviewed and evaluated the literature data using the GRADE system for quality of evidence and strength of recommendation. The updated guidelines provide a risk stratification in BL hypersensitivity according to index reaction(s), as well as an algorithmic approach, based on cross-reactivity studies, in patients with a suspicion of BL hypersensitivity and an immediate need for antibiotic therapy, when referral to an allergist is not feasible. Furthermore, the update addresses availability and concentrations of skin test (ST) reagents, ST and drug provocation test (DPT) protocols, and diagnostic algorithms and administration of alternative BL in allergic subjects. Specifically, distinct diagnostic algorithms are suggested depending on risk stratification of the patient into high and low risk based on the morphology and chronology of the reaction, immediate (ie, occurring within 1-6 hours after the last administered dose) or nonimmediate (ie, occurring more than 1 hour after the initial drug administration), and the reaction severity. Regarding the allergy workup, the main novelty of this document is the fact that in some low-risk nonimmediate reactions ST are not mandatory, especially in children. For DPT, further studies are necessary to provide data supporting the standardization of protocols, especially of those regarding nonimmediate reactions, for which there is currently no consensus.

When questioned, about 10% of the parents report suspected hypersensitivity to at least one drug in their children. However, only a few of these reactions can be confirmed as allergic after a diagnostic workup. There is still a lack of knowledge on drug hypersensitivity (DH) epidemiology, clinical spectrum, and appropriate diagnostic methods particularly in children. Meanwhile, the tools used for DH management in adults are applied also for children. Whereas this appears generally acceptable, some aspects of DH and management differ with age. Most reactions in children are still attributed to betalactams. Some manifestations, such as nonsteroidal anti-inflammatory drug-associated angioedema and serum sickness-like reactions, are more frequent among young patients as compared to adults. Risk factors such as viral infections are particularly frequent in children, making the diagnosis challenging. The practicability and validity of skin test and other diagnostic procedures need further assessment in children. This study presents an up-to-date review on epidemiology, clinical spectrum, diagnostic tools, and current management of DH in children. A new general algorithm for the study of these reactions in children is proposed. Data are presented focusing on reported differences between pediatric and adult patients, also identifying unmet needs to be addressed in further research.

We investigated the value of genetic, histopathologic, and early treatment response information in prognosing long-term renal outcome in children with primary steroid-resistant nephrotic syndrome. From the PodoNet Registry, we obtained longitudinal clinical information for 1354 patients (disease onset at >3 months and <20 years of age): 612 had documented responsiveness to intensified immunosuppression (IIS), 1155 had kidney biopsy results, and 212 had an established genetic diagnosis. We assessed risk factors for ESRD using multivariate Cox regression models. Complete and partial remission of proteinuria within 12 months of disease onset occurred in 24.5% and 16.5% of children, respectively, with the highest remission rates achieved with calcineurin inhibitor-based protocols. Ten-year ESRD-free survival rates were 43%, 94%, and 72% in children with IIS resistance, complete remission, and partial remission, respectively; 27% in children with a genetic diagnosis; and 79% and 52% in children with histopathologic findings of minimal change glomerulopathy and FSGS, respectively. Five-year ESRD-free survival rate was 21% for diffuse mesangial sclerosis. IIS responsiveness, presence of a genetic diagnosis, and FSGS or diffuse mesangial sclerosis on initial biopsy as well as age, serum albumin concentration, and CKD stage at onset affected ESRD risk. Our findings suggest that responsiveness to initial IIS and detection of a hereditary podocytopathy are prognostic indicators of favorable and poor long-term outcome, respectively, in children with steroid-resistant nephrotic syndrome. Children with multidrug-resistant sporadic disease show better renal survival than those with genetic disease. Furthermore, histopathologic findings may retain prognostic relevance when a genetic diagnosis is established.

BACKGROUND: Recent short-term clinical trials in patients with Duchenne Muscular Dystrophy (DMD) have indicated greater disease variability in terms of progression than expected. In addition, as average life-expectancy increases, reliable data is required on clinical progression in the older DMD population. OBJECTIVE: To determine the effects of corticosteroids on major clinical outcomes of DMD in a large multinational cohort of genetically confirmed DMD patients. METHODS: In this cross-sectional study we analysed clinical data from 5345 genetically confirmed DMD patients from 31 countries held within the TREAT-NMD global DMD database. For analysis patients were categorised by corticosteroid background and further stratified by age. RESULTS: Loss of ambulation in non-steroid treated patients was 10 years and in corticosteroid treated patients 13 years old (p = 0.0001). Corticosteroid treated patients were less likely to need scoliosis surgery (p < 0.001) or ventilatory support (p < 0.001) and there was a mild cardioprotective effect of corticosteroids in the patient population aged 20 years and older (p = 0.0035). Patients with a single deletion of exon 45 showed an increased survival in contrast to other single exon deletions. CONCLUSIONS: This study provides data on clinical outcomes of DMD across many healthcare settings and including a sizeable cohort of older patients. Our data confirm the benefits of corticosteroid treatment on ambulation, need for scoliosis surgery, ventilation and, to a lesser extent, cardiomyopathy. This study underlines the importance of data collection via patient registries and the critical role of multi-centre collaboration in the rare disease field.

Mobile health (mHealth) uses mobile communication devices such as smartphones and tablet computers to support and improve health-related services, data and information flow, patient self-management, surveillance, and disease management from the moment of first diagnosis to an optimized treatment. The European Academy of Allergy and Clinical Immunology created a task force to assess the state of the art and future potential of mHealth in allergology. The task force endorsed the "Be He@lthy, Be Mobile" WHO initiative and debated the quality, usability, efficiency, advantages, limitations, and risks of mobile solutions for allergic diseases. The results are summarized in this position paper, analyzing also the regulatory background with regard to the "General Data Protection Regulation" and Medical Directives of the European Community. The task force assessed the design, user engagement, content, potential of inducing behavioral change, credibility/accountability, and privacy policies of mHealth products. The perspectives of healthcare professionals and allergic patients are discussed, underlining the need of thorough investigation for an effective design of mHealth technologies as auxiliary tools to improve quality of care. Within the context of precision medicine, these could facilitate the change in perspective from clinician- to patient-centered care. The current and future potential of mHealth is then examined for specific areas of allergology, including allergic rhinitis, aerobiology, allergen immunotherapy, asthma, dermatological diseases, food allergies, anaphylaxis, insect venom, and drug allergy. The impact of mobile technologies and associated big data sets are outlined. Facts and recommendations for future mHealth initiatives within EAACI are listed.

Gold nanoparticles (GNPs) are claimed as outstanding biomedical tools for cancer diagnostics and photo-thermal therapy, but without enough evidence on their potentially adverse immunological effects. Using a model of human dendritic cells (DCs), we showed that 10 nm- and 50 nm-sized GNPs (GNP10 and GNP50, respectively) were internalized predominantly via dynamin-dependent mechanisms, and they both impaired LPS-induced maturation and allostimulatory capacity of DCs, although the effect of GNP10 was more prominent. However, GNP10 inhibited LPS-induced production of IL-12p70 by DCs, and potentiated their Th2 polarization capacity, while GNP50 promoted Th17 polarization. Such effects of GNP10 correlated with a stronger inhibition of LPS-induced changes in Ca2+ oscillations, their higher number per DC, and more frequent extra-endosomal localization, as judged by live-cell imaging, proton, and electron microscopy, respectively. Even when released from heat-killed necrotic HEp-2 cells, GNP10 inhibited the necrotic tumor cell-induced maturation and functions of DCs, potentiated their Th2/Th17 polarization capacity, and thus, impaired the DCs' capacity to induce T cell-mediated anti-tumor cytotoxicity in vitro. Therefore, GNP10 could potentially induce more adverse DC-mediated immunological effects, compared to GNP50.

INTRODUCTION: Because many aspects of the management of esophageal atresia (EA) are still controversial, we evaluated the practice patterns of this condition across Europe. METHODS: A survey was completed by 178 delegates (from 45 [27 European] countries; 88% senior respondents) at the EUPSA-BAPS 2012. RESULTS: Approximately 66% of respondents work in centers where more than five EA repairs are performed per year. Preoperatively, 81% of respondents request an echocardiogram, and only 43% of respondents routinely perform preoperative bronchoscopy. Approximately 94% of respondents prefer an open approach, which is extrapleural in 71% of respondents. There were no differences in use of thoracoscopy between Europeans (10%) and non-Europeans (11%, p = nonsignificant). Approximately 60% of respondents measure the gap intraoperatively. A transanastomotic tube (90%) and chest drain (69%) are left in situ. Elective paralysis is adopted by 56% of respondents mainly for anastomosis tension (65%). About 72% of respondents routinely request a contrast study on postoperative day 7 (2-14). Approximately 54% of respondents use parenteral nutrition, 40% of respondents start transanastomotic feeds on postoperative day 1, and 89% of respondents start oral feeds after postoperative day 5. Pure EA: 46% of respondents work in centers that repair two or more than two pure EA a year. About 60% of respondents opt for delayed primary anastomosis at 3 months (1-12 months) with gastrostomy formation without esophagostomy. Anastomosis is achieved with open approach by 85% of respondents. About 47% of respondents attempt elongation of esophageal ends via Foker technique (43%) or with serial dilations with bougies (41%). Approximately 67% of respondents always attempt an anastomosis. Gastric interposition is the commonest esophageal substitution. CONCLUSION: Many aspects of EA management are lacking consensus. Minimally invasive repair is still sporadic. We recommend establishment of an EA registry.

INTRODUCTION: Peyronie's disease is a sexual medicine condition that may adversely affect male sexual function. AIM: To provide expert opinions/recommendations concerning state-of-the-art knowledge for the pathophysiology, diagnosis and treatment of Peyronie's disease. METHODS: An International Consultation in collaboration with the major urology and sexual medicine associations assembled over 200 multidisciplinary experts from 60 countries into 17 committees. Committee members established specific objectives and scopes for various male and female sexual medicine topics. The recommendations concerning state-of-the-art knowledge in the respective sexual medicine topic represent the opinion of experts from five continents developed in a scientific and debate process. Concerning the Peyronnie's disease committee, there were 10 experts from six countries. MAIN OUTCOME MEASURE: Expert opinions/recommendations are based on grading of evidence-based medical literature, extensive internal committee discussion over 2 years, public presentation and deliberation. RESULTS: Peyronie's disease is characterized by an inflammatory response beneath the tunica albuginea with fibroblast proliferation forming a thickened fibrous plaque that may cause penile pain, penile curvature and erectile dysfunction. Medical treatments have been described but few prospective controlled trials have revealed significant clinical benefits. Surgical treatments (penile plication, Nesbit excision, plaque incision and grafting and penile prosthesis insertion) should be considered after Peyronie's disease has stabilized. Surgical outcome studies reveal that a stable deformity is best corrected with the least postoperative ED by a Nesbit procedure. Plaque incision and grafting should be reserved for men with good erectile function and marked penile shortening although there is a higher prevalence of postoperative ED. Implantation of a penile prosthesis is an excellent option for men with an accompanying erectile deficit. CONCLUSIONS: Safe and effective treatments for Peyronie's disease examined by prospective, placebo-controlled, multi-institutional clinical trials are needed.

The main remit of the European Society for Paediatric Anaesthesiology (ESPA) Pain Committee is to improve the quality of pain management in children. The ESPA Pain Management Ladder is a clinical practice advisory based upon expert consensus to help to ensure a basic standard of perioperative pain management for all children. Further steps are suggested to improve pain management once a basic standard has been achieved. The guidance is grouped by the type of surgical procedure and layered to suggest basic, intermediate, and advanced pain management methods. The committee members are aware that there are marked differences in financial and personal resources in different institutions and countries and also considerable variations in the availability of analgesic drugs across Europe. We recommend that the guidance should be used as a framework to guide best practice.

Dysfunction of motile monocilia, altering the leftward flow at the embryonic node essential for determination of left-right body asymmetry, is a major cause of laterality defects. Laterality defects are also often associated with reduced mucociliary clearance caused by defective multiple motile cilia of the airway and are responsible for destructive airway disease. Outer dynein arms (ODAs) are essential for ciliary beat generation, and human respiratory cilia contain different ODA heavy chains (HCs): the panaxonemally distributed γ-HC DNAH5, proximally located β-HC DNAH11 (defining ODA type 1), and the distally localized β-HC DNAH9 (defining ODA type 2). Here we report loss-of-function mutations in DNAH9 in five independent families causing situs abnormalities associated with subtle respiratory ciliary dysfunction. Consistent with the observed subtle respiratory phenotype, high-speed video microscopy demonstrates distally impaired ciliary bending in DNAH9 mutant respiratory cilia. DNAH9-deficient cilia also lack other ODA components such as DNAH5, DNAI1, and DNAI2 from the distal axonemal compartment, demonstrating an essential role of DNAH9 for distal axonemal assembly of ODAs type 2. Yeast two-hybrid and co-immunoprecipitation analyses indicate interaction of DNAH9 with the ODA components DNAH5 and DNAI2 as well as the ODA-docking complex component CCDC114. We further show that during ciliogenesis of respiratory cilia, first proximally located DNAH11 and then distally located DNAH9 is assembled in the axoneme. We propose that the β-HC paralogs DNAH9 and DNAH11 achieved specific functional roles for the distinct axonemal compartments during evolution with human DNAH9 function matching that of ancient β-HCs such as that of the unicellular Chlamydomonas reinhardtii.

Ciliopathies are genetically heterogeneous disorders characterized by variable expressivity and overlaps between different disease entities. This is exemplified by the short rib-polydactyly syndromes, Jeune, Sensenbrenner, and Mainzer-Saldino chondrodysplasia syndromes. These three syndromes are frequently caused by mutations in intraflagellar transport (IFT) genes affecting the primary cilia, which play a crucial role in skeletal and chondral development. Here, we identified mutations in IFT140, an IFT complex A gene, in five Jeune asphyxiating thoracic dystrophy (JATD) and two Mainzer-Saldino syndrome (MSS) families, by screening a cohort of 66 JATD/MSS patients using whole exome sequencing and targeted resequencing of a customized ciliopathy gene panel. We also found an enrichment of rare IFT140 alleles in JATD compared with nonciliopathy diseases, implying putative modifier effects for certain alleles. IFT140 patients presented with mild chest narrowing, but all had end-stage renal failure under 13 years of age and retinal dystrophy when examined for ocular dysfunction. This is consistent with the severe cystic phenotype of Ift140 conditional knockout mice, and the higher level of Ift140 expression in kidney and retina compared with the skeleton at E15.5 in the mouse. IFT140 is therefore a major cause of cono-renal syndromes (JATD and MSS). The present study strengthens the rationale for IFT140 screening in skeletal ciliopathy spectrum patients that have kidney disease and/or retinal dystrophy.

INTRODUCTION: There are three different types of priapism: low-flow, ischemic, anoxic or veno-occlusive priapism; high-flow, arterial or nonischemic priapism; and recurrent or stuttering priapism. AIM: To provide recommendations/guidelines concerning state-of-the-art knowledge for the diagnosis and treatment of priapism. METHODS: An International Consultation in collaboration with the major urology and sexual medicine associations assembled over 200 multidisciplinary experts from 60 countries into 17 committees. Committee members established specific objectives and scopes for various male and female sexual medicine topics. The recommendations concerning state-of-the-art knowledge in the respective sexual medicine topic represent the opinion of experts from five continents developed in a process over a 2-year period. Concerning the Priapism Committee, there were 10 experts from six countries. MAIN OUTCOME MEASURE: Expert opinion was based on grading of evidence-based medical literature, widespread internal committee discussion, public presentation and debate. RESULTS: Concerning ischemic priapism, persistent cavernous smooth muscle relaxation and failure of contraction is a compartment syndrome with increasing intracavernosal anoxia, rising pCO2 and acidosis. Urgent medical attention should be sought for an erection lasting >4 hours; 90% with priapism >24 hours develop complete erectile dysfunction. After diagnosis and counselling, intracavernosal aspiration and alpha-blockers should precede surgical shunting. Concerning high-flow priapism (congenital, traumatic or iatrogenic), intervention is not urgent and often unnecessary. Definitive management is by selective embolization with autologous blood clot. Concerning recurrent/stuttering priapism, the pathophysiology may be central or local (sickle cell disease). Management needs to be individualized; androgen deprivation has proved useful but has adverse effects. CONCLUSIONS: There is need for prospective, clinical trials to define safe and effective management strategies for patients with low-flow, high-flow or recurrent priapism.

Congenital sodium diarrhea (CSD) refers to an intractable diarrhea of intrauterine onset with high fecal sodium loss. CSD is clinically and genetically heterogeneous. Syndromic CSD is caused by SPINT2 mutations. While we recently described four cases of the non-syndromic form of CSD that were caused by dominant activating mutations in intestinal receptor guanylate cyclase C (GC-C), the genetic cause for the majority of CSD is still unknown. Therefore, we aimed to determine the genetic cause for non-GC-C non-syndromic CSD in 18 patients from 16 unrelated families applying whole-exome sequencing and/or chromosomal microarray analyses and/or direct Sanger sequencing. SLC9A3 missense, splicing and truncation mutations, including an instance of uniparental disomy, and whole-gene deletion were identified in nine patients from eight families with CSD. Two of these nine patients developed inflammatory bowel disease (IBD) at 4 and 16 years of age. SLC9A3 encodes Na(+)/H(+) antiporter 3 (NHE3), which is the major intestinal brush-border Na(+)/H(+) exchanger. All mutations were in the NHE3 N-terminal transport domain, and all missense mutations were in the putative membrane-spanning domains. Identified SLC9A3 missense mutations were functionally characterized in plasma membrane NHE null fibroblasts. SLC9A3 missense mutations compromised NHE3 activity by reducing basal surface expression and/or loss of basal transport function of NHE3 molecules, whereas acute regulation was normal. This study identifies recessive mutations in NHE3, a downstream target of GC-C, as a cause of CSD and implies primary basal NHE3 malfunction as a predisposition for IBD in a subset of patients.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive tubular disorder caused by CLDN16 mutations. CLDN16 encodes the renal tight junction protein claudin-16, which is important for the paracellular reabsorption of calcium and magnesium in the thick ascending limb of Henle's loop. That FHHNC is frequently associated with progressive renal failure suggests additional roles for claudin-16 in the maintenance of tight junction integrity. An investigation of 32 patients with FHHNC and 17 different mutations was previously reported; here, the analysis is expanded to 39 additional patients and 12 new mutations. Expression studies revealed that five of the 12 new mutations led to partial loss of claudin-16 function and the remaining seven led to complete loss of function. The 23 patients who had mutations resulting in complete loss of function of both alleles were significantly younger at the onset of symptoms than the 46 patients who had at least one mutant allele providing partial function (2.2 versus 5.6 years; P < 0.01). In addition, those with complete loss of function had a more rapid decline in GFR (7.3 versus 2.9 ml/min per 1.72 m2/y; P < 0.01), leading to 54% requiring renal replacement therapy by age 15 compared with 20% of those with residual function (P < 0.05). These data suggest that residual function of claudin-16 may delay the progression of renal failure in FHHNC. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC; OMIM *248250) is a rare autosomal recessive tubular disorder. It is characterized by massive urinary losses of magnesium (Mg) and calcium (Ca) leading to hypomagnesemia and bilateral nephrocalcinosis. The disease usually presents with recurrent urinary tract infections and polyuria/polydipsia. Additional symptoms include rickets, nephrolithiasis, hematuria, muscular tetanies, seizures, failure to thrive, vomiting, and abdominal pain.1,2 Ocular abnormalities and hearing impairment have been described in a subset of patients with FHHNC.3,4 Additional biochemical abnormalities include signs of incomplete distal renal tubular acidosis, hypocitraturia, increased parathyroid hormone (PTH) levels (independent of GFR), and hyperuricemia.3,5–8 Unlike most other inherited tubular diseases affecting electrolyte homeostasis, FHHNC is generally complicated by progressive renal failure during childhood or adolescence, but the pathogenesis of chronic renal failure remains a matter of debate. Clearance studies in patients with FHHNC localized the site of disturbed handling of Mg and Ca to the thick ascending limb (TAL) of Henle's loop.6 The TAL plays an important role in the tubular reabsorption of Mg and Ca, which occurs by paracellular flux, a process driven by the lumen-positive transepithelial potential in this nephron segment. In 1999, Simon et al.9 identified a new gene (CLDN16, formerly PCLN1) and characterized mutations in this gene as the underlying molecular defect in FHHNC. Since then, approximately 30 additional families with FHHNC as a result of CLDN16 defects have been described.2,10–13 Hypercalciuria and nephrolithiasis have also been observed in heterozygous FHHNC mutation carriers.2,3 FHHNC is a genetically heterogeneous disease because recently CLDN19 mutations have been identified in a cohort of patients mainly originating from Spain.14 The renal phenotype of these patients is very similar to patients exhibiting CLDN16 mutations; however, patients with CLDN19 mutations also have severe ocular abnormalities in most cases. CLDN16 and CLDN19 encode the tight junction (TJ) proteins claudin-16 (paracellin-1) and claudin-19, both being members of the claudin multigene family. Claudins are important components of the TJ strands in various tissues.15 They are part of a complex protein network built up by a variety of different proteins, and there is clear evidence that claudins confer ion selectivity to the paracellular pathway.16–18 In the kidney, claudin-16 expression is restricted to the TAL of Henle's loop. It was speculated that claudin-16 constitutes the core of an intercellular pore, allowing paracellular reabsorption of Mg and Ca ions.9,19 Following the observation that a naturally occurring knockout model in Japanese black cattle shows early-onset renal failure with diffuse interstitial nephritis,20 it was recently speculated that claudin-16, like other claudins, may also be involved in the regulation of cell growth, proliferation, differentiation, and dedifferentiation.21 Hou et al.22 described the functional analysis of claudin-16 in polarized cell lines. They demonstrated that in LLC-PK1 cells, claudin-16 modulated the ion selectivity of the TJ by selectively increasing the permeability of Na+ with no effects on Cl−, resulting in a high permeability ratio of Na+ to Cl−. Instead, Mg flux across cell monolayers showed a far less pronounced change after claudin-16 expression. From these data, it might be concluded that claudin-16 defects lead to a loss of cation selectivity with a subsequent decrease in lumen-positive potential that is the driving force for the paracellular flux of cations. This hypothesis of a nonselective paracellular cation channel is supported by a mouse model using transgenic RNAi depletion of claudin-16. Loss of CLDN16 in this model caused TJ in TAL to lose the cation selectivity.23 Hou et al.22 also analyzed the consequences of most of the reported human CLDN16 mutations by heterologous expression in vitro. Whereas most mutations resulted in a complete loss of function, some mutations retained a substantial residual function. One of these mutations (L151F) is by far the most frequent FHHNC mutation, occurring in almost 50% of the patients described so far.2 Combining this information with additional functional analysis of new mutations using the same expression system, we present a genotype/phenotype correlation with a special focus on the progression of renal failure in a large cohort of patients with FHHNC. We provide clinical data indicating that homozygous or compound heterozygous patients who carry at least one CLDN16 mutation with residual function have a much more benign course of the disease than patients with a complete loss of function. RESULTS Mutation Analysis of CLDN16 In our initial study,2CLDN16 mutations were identified in 32 patients with FHHNC, 67% of the mutant alleles exhibited a missense mutation affecting the first extracellular loop of claudin-16, and 48% were affected by an L151F exchange. This mutational hot spot is due to a widespread founder effect (Germany and Eastern European countries). Mutation analysis in families F29 to F88 (with 39 affected individuals) revealed 17 different mutations (Table 1). In addition to the ones already described, 12 novel CLDN16 mutations were identified, including seven missense mutations, one nonsense mutation truncating the protein in the fourth transmembrane domain; one small internal deletion, one splice-site mutation in exon four, one frameshift mutation truncating the protein before the first extracellular domain, and one mutation resulting in the loss of the translation initiation start site (Figure 1A). Figure 1B depicts the amino acids affected by missense mutations in the claudin-16 protein. All mutations co-segregated with the FHHNC phenotype, and none of the mutations was observed in at least 100 control chromosomes. Both mutant alleles were detected in 34 affected individuals; however, in the remaining five patients from three families (F37, F61, and F73), only one heterozygous mutation could be identified. Families F37 and F61 both have two affected children. Therefore, we performed haplotype analysis to demonstrate identical haplotypes also for the second mutant allele. In both families, the affected siblings share the same haplotypes compatible with linkage to the CLDN16 locus. Thus, it is most likely that the second mutation was missed because it is not located in the coding sequence. Family F73 could not be studied by haplotype analysis because there is only one affected child. Functional Analysis of the New Mutations The functional consequences of the new CLDN16 missense mutations were analyzed after heterologous expression in MDCK and LLC-PK1 cells. In addition, we studied H141D and L151W, which have been described previously2 but not functionally characterized by Hou et al.22 The profile of expression and localization of the mutant proteins are summarized in Table 2. Most of the mutant proteins display a normal trafficking to the cell membrane. Only two mutations (M71T and C131R) were retained inside the cell; the M71T mutation is localized to lysosomes, whereas C131R is retained in the endoplasmic reticulum (Figure 2). The mutants that were properly targeted to TJ showed a significant loss of function compared with wild-type claudin-16 when expressed in LLC-PK1 cells. We observed a significant decrease of the dilution potential and the ratio of permeability of Na+ over Cl−. This indicates a disturbance of the cation selectivity of LLC-PK1 cells expressing wild-type claudin-16. As in the previous study, not all mutations showed a complete loss of function (CL; Figure 3). Several mutants retain a significant residual claudin-16 function. To compare the results obtained in this study with the data from the initial expression study,22 we calculated the residual function as percentage relative to wild-type claudin-16 function. Mutants with a residual function >40% were considered “partial” loss of function (PL); the remaining mutants were considered CL. Theses categories were also used for the genotype/phenotype analysis (see next section). Genotype/Phenotype Correlation Assignment of Mutation Category To analyze a possible genotype/phenotype correlation in FHHNC, we assigned CLDN16 mutations to three different categories: (1) Complete loss of function (CL) mutations including missense mutations resulting in a complete loss of function after heterologous expression (Figure 3). In addition, nonsense, truncating and splice-site mutations were attributed to this category. Bioinformatic analysis of the three splice site mutations clearly indicated significant disturbance of CLDN16 mRNA splicing. (2) Missense mutations that displayed substantial residual function >40% as compared with wild-type claudin-16 function were considered as mutations with partial loss of function (PL) (Figure 3). (3) Missense mutations that could not be assigned in categories CL or PL are given as X. This assignment was done separately for both mutant alleles. Following these criteria, 23 patients had a CL/CL genotype, 29 patients had PL/PL, 12 patients had PL/CL, five patients had PL/X, one patient had X/X, and one patient had CL/X. Because of the lack of information in X/X and CL/X patients, these two patients were completely excluded from the genotype/phenotype analysis (Table 3). Confirmation of the Recessive Nature of FHHNC In a first step of the genotype/phenotype analysis, only the groups with classified mutations on both mutant alleles were analyzed (CL/CL, PL/CL, and PL/PL). PL/X patients were excluded from this first statistical analysis because no information is available with respect to the severity of the second allele (for comparison, the data are shown in Figure 4). In theory, given the recessive nature of the disease, one might expect that the effect of a PL mutation can also be observed if this mutation occurs in compound heterozygosity with a severe mutation (CL). This has been previously shown for other recessive diseases, for example, with respect to the preservation of pancreatic sufficiency in cystic fibrosis24: as long as a loss of function mutant was in compound heterozygosity with a mutant with residual function, no severe pancreatic phenotype was observed. Analysis of the three groups with categorized mutations on both alleles (CL/CL, PL/CL, and PL/PL) revealed a significant correlation between the genotype and age at onset (P < 0.05) and decline of renal function (P < 0.01). CL/CL patients were significantly younger at presentation of first symptoms (2.2 yr; 95% confidence interval [CI] 1.1 to 3.3 yr) compared with PL/PL patients (5.7 yr; 95% CI 3.3 to 8.2 yr), whereas age at onset in PL/CL patients (4.9 yr; 95% CI 2.3 to 7.6 yr) was similar to that of PL/PL patients, nevertheless not reaching statistical significance (Table 4, Figure 4A). The progression of renal failure expressed as loss of GFR/yr was significantly faster in the CL/CL patients (7.3 ml/min per 1.73 m2/yr; 95% CI 5.0 to 9.6) as compared with either PL/PL (3.3 ml/yr; 95% CI 1.6 to 4.9) or PL/CL patients (3.0 ml/yr; 95% CI 1.1 to 4.9), whereas no difference between PL/PL and PL/CL patients could be observed (Table 4, Figure 4B). The decline of GFR in the various groups during the study period is shown in Figure 5. The similar clinical courses of the PL/PL and PL/CL which is in to the CL/CL indicates that in FHHNC, the nature of the second allele in with a PL mutation is not important for the clinical The PL/X which was excluded from this first analysis, these (Table 4, and all criteria, the PL/X is very similar to either the PL/PL or the PL/CL We PL/PL, PL/CL, but also PL/X patients one large This was compared with the of CL/CL at and Loss of CL and PL at onset in the CL loss of function of both was significantly than in the PL loss of function of at least one with CI 1.1 to 3.3 yr) versus 5.6 CI to yr; P < Table 4). CL patients showed a faster decline of GFR than PL patients, as expressed by a loss of GFR of versus 2.9 ml/min per 1.73 (P < 0.01). As a at the of the the renal function significantly between both groups (P < CL patients had a GFR ml/min per 1.73 compared with PL patients versus and more CL than PL patients had a GFR ml/min per 1.73 versus or renal replacement therapy versus Table CL patients also in than PL the age of 15 of CL patients renal replacement therapy versus 20% of PL patients (P < these increased to in CL and in PL by the age of We also analyzed the Mg and Ca but it has to be in that of with Mg and Ca at different of chronic renal failure may these The Mg and Ca levels were in both groups but not significantly between the CL and PL Mg was high in both groups but a significant In the urinary Ca was significantly in the CL versus the PL both in and in spot levels were high in both groups when compared with control patients with chronic renal failure of other (Figure In to most other tubular affected by FHHNC are at high to progressive renal one of the patients already during adolescence, requiring renal replacement The pathogenesis of chronic renal failure in FHHNC remains a matter of debate. The progressive in FHHNC has been attributed to the hypercalciuria and however, other have this because other disease associated with early-onset nephrocalcinosis are not by a severe of renal All patients with FHHNC are affected by but of renal is to a The for this has been genotype/phenotype correlation could be in the patient that had first been analyzed for CLDN16 however, the of et in two different large mutations of CLDN16 in Japanese cattle already to a function of claudin-16 for tubular These present with chronic interstitial diffuse and with renal studies in these cattle characterized an nephron with and This knockout model suggests that the complete loss of claudin-16 is associated with a severe of renal function, whereas the heterozygous remains In no large CLDN16 gene have been however, the recently of Hou et demonstrated for the first functional in the human CLDN16 missense mutations identified so human missense mutations were in a system, and TJ localization and ion were of the mutants showed a CL TJ mutants showed a partial loss of function localization but ion and one mutant displayed a normal function to the wild-type protein not identified in our patient Mutants with residual function were expressed and targeted to TJ but showed a significant not loss of function compared with wild-type claudin-16. In this study, we performed mutational analysis of CLDN16 in 39 new patients with FHHNC and analyzed the course of renal in this cohort with the 32 patients of our initial the expression studies by functional data for the new mutations, all patients were to mutation for genotype/phenotype The clinical course in patients two mutations with CL also and splice-site is significantly compared with patients with at least one mutation with residual function with CL present symptoms in and a faster decline of As a significantly more patients with two CL mutations had a GFR ml/min per 1.73 at the of the study or renal replacement and was in than in the patients with PL of at least one allele. Whereas in the CL most of the families a CLDN16 mutation, in the PL the of patients carry the L151F founder mutation on at least one mutant allele. Only families have other mutations with PL on both mutant alleles. This the that the less severe phenotype in the PL on the L151F if this be it because the expression studies not functional for the courses of loss of GFR in the families L151F in the PL are to the patients with L151F mutations with the two patients a normal GFR at age and The previously observation of a with respect to the clinical course in the of the families the important role of CLDN16 for the progression of renal The handling of Mg and Ca in FHHNC was also analyzed in this As we hypomagnesemia and a a that is in FHHNC. We also could previous results increased levels already before the onset of chronic renal however, we were to between patients with CL and levels before chronic renal failure may be by the loss of Ca with subsequent from In addition, hypomagnesemia is to in of very severe in which it to have an This is observed in patients with hypomagnesemia with In these patients, is frequently very but the Mg levels in hypomagnesemia with are far the levels in FHHNC. In to Ca, and urinary Ca in FHHNC to on the CLDN16 CL patients more Ca in the than PL this is a effect of the residual claudin-16 function is a residual claudin-16 function, as shown in the expression system, also in affected by these PL mutations, one expect reabsorption for both Ca and however, Mg not between the but we have to that either Mg or in the of of renal failure are we could not for The increased Ca in CL patients also may be to which has been demonstrated to Ca in patients with It might be possible that patients with FHHNC and PL mutations have renal more than patients with renal which is more frequent in the CL The of tubular handling of Ca and Mg to be in much more in progression of renal could be between patients two mutations with residual function and patients affected by one mutation with residual function and one mutation with CL on the second allele. These results are in with the recessive nature of the One mutation with residual function is to a clinical we completely that mutations may different effects on the genotype of the second allele a can we in patients affected by mutations in characterized by interstitial and tubular is the to for patients with FHHNC of our initial demonstrated Ca tubular and interstitial to a In interstitial tubular cells were reported as with loss of both and to the membrane. between and was and the tubular was used to the of the cells of the renal These are with a process of tubular cells new that are considered to a role in the progression of renal is characterized by the of junction and the loss of cell cells cells. These suggest that TJ could be an in et demonstrated that of in is associated with of claudins and proteins of the TJ Therefore, one might that claudin-16 function could be an in of the tubular TJ by of cell and an of tubular who have FHHNC and are affected by two mutations have an onset of chronic renal failure as demonstrated in this patients with at least one mutation with residual function localization of the protein to the TJ are from rapid loss of renal function. a second study to the consequences of human CLDN16 mutations was That study on and the with the results of Hou et al.22 in however, with respect to there were some Most two of the mutations with residual function and could not be detected at the cell by et one mutation which was characterized as a trafficking mutant by Hou et was to be targeted to TJ by et respect to the two studies are not because different cell were It that the residual function described by Hou et as an phenotype between control and wild-type claudin-16 with respect to the permeability ratio of Na+ over and potential can be only in LLC-PK1 cells and not in cells, which were used by et These are to different transepithelial In LLC-PK1 cells, paracellular as a result of the transepithelial We also analyzed the of this study at onset and loss of in our patients with respect to the various of mutations by et attributed the patients either to expression at the cell as Mg by et or to at onset and loss of GFR were identical in both groups versus yr; loss of GFR versus ml/min per 1.73 that the on trafficking evidence of residual claudin-16 function is not for a genotype/phenotype This is supported by the observation of additional mutations that are targeted to TJ but display a CL (Table 2). our in data suggest that residual function of claudin-16 may delay the progression of renal failure in in patients with FHHNC the of the clinical course to some and of affected and Families patients who had FHHNC from families were in this study and and molecular results of families to families with affected individuals) were reported The remaining patients to 39 affected from were the same as in our initial hypercalciuria in the and bilateral and the of data of and were reported was in 15 17 families were with two or more affected The study was by the and was obtained from the patients and levels of Ca, and were analyzed using Additional urinary of Ca and Ca of per was considered for who were than The for and in spot in younger and were on the by et The control for levels with respect to GFR of to yr) with chronic renal failure tubular disorder. The GFR was calculated using the The loss of GFR was calculated from first and loss of only patients with a were Mutation Analysis CLDN16 mutation analysis was performed in families F29 by analysis and as described In all affected in analysis not both mutant CLDN16 was completely (F37, F61, and all new mutations, at least 100 control were In families F37 and F61, haplotype analysis was performed as described Expression The human claudin-16 was the The was performed with a for mutant were by were by of the cell with as described The were used for of MDCK and LLC-PK1 cells. The expression and localization of claudin-16 mutant proteins were by and with studies were performed on cell monolayers on as described The significance of difference between groups was by with and by as of different groups was compared by and not results are expressed as with 95% were used of Mutations are the presentation of CLDN16 mutations in protein model from affected by novel missense mutations are CL mutations are in PL mutations are in CLDN16 mutations resulting in trafficking localization of claudin-16 mutants in LLC-PK1 and MDCK cells. M71T is in the with a of the C131R shows a and indicating in the endoplasmic potential of CLDN16 missense mutations after heterologous expression in LLC-PK1 cells. are given as percentage relative to wild-type claudin-16 function. New mutations are the remaining mutations were from Hou et at of first clinical of renal failure expressed as loss of GFR per 1.73 are was performed only between the groups with categorized mutations on both alleles. < < of renal failure over in FHHNC to the mutation category. are levels with GFR in patients with FHHNC and control is indicated in analysis of the new patients with Functional analysis of CLDN16 mutations in LLC-PK1 Functional consequences of the CLDN16 correlation in FHHNC, correlation in are to the patients and families for We and for We also the and

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used in the pediatric population as antipyretics/analgesics and anti-inflammatory medications. Hypersensitivity (HS) reactions to NSAID in this age group, while similar to adults, have unique diagnostic and management issues. Although slowly accumulating, published data in this age group are still relatively rare and lacking a unifying consensus. This work is a summary of current knowledge and consensus recommendations utilizing both published data and expert opinion from the European Network of Drug Allergy (ENDA) and the Drug Hypersensitivity interest group in the European Academy of Allergy and Clinical Immunology (EAACI). This position paper summarizes diagnostic and management guidelines for children and adolescents with NSAIDs hypersensitivity.