St Lukes Hospital

Aicardi-Goutières syndrome is an inflammatory disease occurring due to mutations in any of TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR or IFIH1. We report on 374 patients from 299 families with mutations in these seven genes. Most patients conformed to one of two fairly stereotyped clinical profiles; either exhibiting an in utero disease-onset (74 patients; 22.8% of all patients where data were available), or a post-natal presentation, usually within the first year of life (223 patients; 68.6%), characterized by a sub-acute encephalopathy and a loss of previously acquired skills. Other clinically distinct phenotypes were also observed; particularly, bilateral striatal necrosis (13 patients; 3.6%) and non-syndromic spastic paraparesis (12 patients; 3.4%). We recorded 69 deaths (19.3% of patients with follow-up data). Of 285 patients for whom data were available, 210 (73.7%) were profoundly disabled, with no useful motor, speech and intellectual function. Chilblains, glaucoma, hypothyroidism, cardiomyopathy, intracerebral vasculitis, peripheral neuropathy, bowel inflammation and systemic lupus erythematosus were seen frequently enough to be confirmed as real associations with the Aicardi-Goutieres syndrome phenotype. We observed a robust relationship between mutations in all seven genes with increased type I interferon activity in cerebrospinal fluid and serum, and the increased expression of interferon-stimulated gene transcripts in peripheral blood. We recorded a positive correlation between the level of cerebrospinal fluid interferon activity assayed within one year of disease presentation and the degree of subsequent disability. Interferon-stimulated gene transcripts remained high in most patients, indicating an ongoing disease process. On the basis of substantial morbidity and mortality, our data highlight the urgent need to define coherent treatment strategies for the phenotypes associated with mutations in the Aicardi-Goutières syndrome-related genes. Our findings also make it clear that a window of therapeutic opportunity exists relevant to the majority of affected patients and indicate that the assessment of type I interferon activity might serve as a useful biomarker in future clinical trials.

In a sample of 60 schizophrenic patients encompassing all grades of severity and chronicity memory impairment was found to be prevalent, often substantial, and disproportionate to the overall level of intellectual impairment. The deficits were not easily attributable to poor cooperation, attention or motivation; nor were they related to neuroleptic or anticholinergic medication. Memory impairment was significantly associated with severity and chronicity of illness and also with negative symptoms and formal thought disorder. There was evidence from the sample as a whole, and from a more detailed examination of five patients with relatively isolated deficits, that schizophrenic memory impairment conformed to the pattern seen in the classical amnesic syndrome. Additionally, there was preliminary evidence for a marked deficit in semantic memory.

Memory impairment is not usually considered to form part of the clinical picture of schizophrenia, except perhaps in severely deteriorated patients. In a survey of 60 patients encompassing all grades of severity and chronicity poor memory performance was found to be common, sometimes substantial, and disproportionately pronounced compared to the degree of general intellectual impairment. Although associated with severity and chronicity of illness, impaired memory was by no means confined to old, institutionalized, or markedly deteriorated patients. The pattern of deficit appeared to resemble that of the classic amnesic syndrome rather than that seen in Alzheimer-type dementia.

BACKGROUND: Bipolar disorder (BD) is a leading cause of disability. Systematic reviews of randomized trials for the treatment of the maintenance phase of BD are lacking. OBJECTIVES: To determine the efficacy and tolerability of mood stabilizers and antipsychotics in the maintenance treatment of BD. METHODS: We systematically reviewed randomized controlled trials of licensed medications for the treatment of any phase of BD. We included randomized controlled trials comparing a medication to placebo or another medication. Comprehensive searches of electronic databases were conducted to March 2005. Outcomes investigated were relapse due to mania, depression or any mood episode, and withdrawal due to any reason or due to an adverse event. Data were combined through meta-analysis. RESULTS: Fourteen studies (n = 2,526) met the inclusion criteria. Lithium, lamotrigine, olanzapine and valproate semisodium each demonstrated evidence to support long-term use. Compared with placebo, all medications were more effective at preventing relapse because of any mood episode. Hazard ratios (HR) were 0.68 [95% confidence interval (CI) = 0.53-0.86] for lithium, 0.68 (95% CI = 0.55-0.85) for lamotrigine, and 0.82 (95% CI = 0.57-1.20) for valproate semisodium; for olanzapine, the risk ratio (RR) was 0.58 (95% CI = 0.49-0.69). Lithium and olanzapine significantly reduced manic relapses (HR = 0.53; 95% CI = 0.35-0.79 and RR = 0.37; 95% CI = 0.24-0.57, respectively). Lamotrigine and valproate semisodium significantly reduced depressive relapses (HR = 0.65; 95% CI = 0.46-0.91 and RR = 0.40; 95% CI = 0.20-0.82, respectively). Lithium significantly reduced manic relapses compared with lamotrigine (HR = 0.56; 95% CI = 0.34-0.92) and olanzapine significantly reduced manic relapses compared with lithium (RR = 1.69; 95% CI = 1.12-2.55). Withdrawal due to an adverse event was approximately twice as likely with lithium compared with valproate semisodium (RR = 1.81; 95% CI = 1.08-3.03) and lamotrigine (RR = 2.20; 95% CI = 1.31-3.70). There were few data for carbamazepine or medications given as adjunct therapy. CONCLUSIONS: Mood stabilizers have differing profiles of efficacy and tolerability, suggesting complementary roles in long-term maintenance treatment.

OBJECTIVE: Psoriatic arthritis (PsA) is characterized by inflammatory arthritis in the presence of psoriasis. Certain clinical features help characterize this disorder, one of which is dactylitis. Hitherto an instrument for quantifying dactylitis has not been developed. METHODS: A dactylitis score sheet was developed. The score is a function of finger circumference and tenderness, assessed and summed across all dactylitic digits. Initial results were obtained on a small sample of patients attending clinics. Inter and intraobserver agreement on the presence of dactylitis using kappa agreement statistics, and the validity and reliability of the instrument, using intraclass correlation coefficients (ICC), were assessed in a further group of 7 patients with PsA. RESULTS: Tender dactylitis was deemed present in 74 digits out of a total of 280 (140 digits on each occasion). Kappa agreement scores for the presence of tender dactylitis were poor to good, both within and between observers (0.25 to 0.89 between observers and 0.29 to 0.91 within observers). Agreement scores for non-tender dactylitis were poor (0.01 to 0.66 between observers and 0.01 to 0.59 within-observer agreement). The new dactylitis instrument was simple and easy to administer and was found to measure appropriate scores in patients with different severity of dactylitis. Inter and intraobserver agreement was good (interobserver ICC 0.90, 95% CI 0.74-0.98; intraobserver ICC 0.84, 95% CI 0.71-0.92). Intraobserver ICC improved but interobserver ICC deteriorated by rating simply presence or absence, rather than a 4 point grade, of tenderness. CONCLUSION: A new method for quantifying dactylitis based on digital circumference and tenderness has been described. This instrument has shown good inter and intraobserver reliability. Further studies of responsiveness are now required.

BACKGROUND: Coronary angiography may not reliably predict whether a stenosis causes exercise-induced ischemia. Intracoronary Doppler ultrasound may enhance diagnostic accuracy by providing a physiological assessment of stenosis severity. The goal of this study was to compare intracoronary Doppler ultrasound with both 201Tl imaging and coronary angiography. METHODS AND RESULTS: Fifty-five patients with 67 stenotic coronary arteries underwent coronary angiography with intracoronary Doppler ultrasound and had exercise 201Tl testing within a 1-week period. Coronary flow reserve was measured, and analyses were performed by independent core laboratories. The mean stenosis was 59+/-12%; 51 of 67 stenoses were intermediate in severity (40% to 70%). A coronary flow reserve < 1.7 predicted the presence of a stress 201Tl defect in 56 of 67 stenoses (agreement=84%; kappa=0.67; 95% CI=0.48 to 0.86). In the patients who achieved 75% of their predicted maximum heart rate, the Doppler and 201Tl imaging data agreed in 46 of 52 stenoses (agreement=88%; kappa=0.77; 95%CI=0.57 to 0.97). Scatter was evident when angiography was compared with coronary flow reserve (r=.43), and the angiogram did not reliably predict the results of the 201Tl stress test (kappa=0.21; agreement=57% to 63%). CONCLUSIONS: Doppler-derived coronary flow reserve accurately predicts the presence of exercise-induced ischemia on stress 201Tl imaging, and coronary angiography does not reliably assess the physiological significance of an intermediate coronary stenosis.

The relations between estradiol, testosterone, insulin, lipids, and prevalent ischemic heart disease were examined using the cross-sectional data from the Caerphilly Heart Disease Study, a cohort of 2,512 men (aged 45-59 years) surveyed between 1978 and 1982. Endogenous levels of estradiol were associated directly with high density lipoprotein (HDL) cholesterol (r = 0.106, p less than 0.001), but this relation was removed after adjustment for testosterone and insulin levels. Estradiol was not associated with prevalent ischemic heart disease. Endogenous levels of testosterone were associated directly with HDL cholesterol (r = 0.148, p less than 0.001) and inversely with triglyceride (r = -0.217, p less than 0.001). Persons with prevalent ischemic heart disease had significantly lower testosterone levels than persons without ischemic heart disease (mean levels 20.9 vs. 22.0 nmol/liter, p less than 0.01). These relations were confounded by associations with insulin. The associations between testosterone and the lipids persist after adjusting for body mass index, age, and insulin. The association between testosterone and prevalent ischemic heart disease was reduced after adjusting for insulin and/or triglyceride levels. The results suggest that insulin and testosterone may have an interdependent regulatory effect on lipid metabolism. The effect of testosterone on ischemic heart disease appears to be primarily mediated through its association with insulin. Future work on sex hormones and ischemic heart disease will need to account for the effects of insulin.

The diagnosis of non-islet cell tumour (hypoinsulinaemic) hypoglycamia has been complicated by contradictory biochemical evidence. Although insulin-like growth factor II (IGF-II) has been identified as the hypoglycaemic agent, plasma levels are often not elevated. In this study specific radioimmunoassay procedures for the measurement of IGF-I and IGF-II are described. Reference data on plasma IGF-II concentrations in relation to a wide range of IGF-I levels have been accumulated using plasma samples from acromegalic, hypopituitary and insulinoma (i.e. hyperinsulinaemic hypoglycaemia) patients as well as normal subjects from all age groups. The reference data indicate that a low plasma IGF-I value is normally associated with a relatively low plasma IGF-II level. Within a group of hypoinsulinaemic hypoglycaemia patients, a small number, invariably with evidence of a neoplasm, had low plasma IGF-I concentrations but apparently normal IGF-II levels. We propose that, in such cases, an apparently normal plasma IGF-II value is inappropriately high for the low plasma IGF-I level and, in association with non-ketotic hypoinsulinaemia and suppressed plasma growth hormone (GH), is diagnostic of a non-islet cell tumour as the cause of hypoglycaemia.

INTRODUCTION: Cardiovascular disease is more prevalent in patients with severe mental illness (SMI) than in the general population. METHOD: Seven geographically diverse centres were assigned a nurse to monitor the physical health of SMI patients in secondary care over a 2-year period in the "Well-being Support Programme" (WSP). A physical health screen was performed and patients were given individual weight and lifestyle advice including smoking cessation to reduce cardiovascular risk. RESULTS: Nine hundred and sixty-six outpatients with SMI >2 years were enrolled. The completion rate at 2 years was 80%. Significant improvements were observed in levels of physical activity (p<0.0001), smoking (p<0.05) and diet (p<0.0001). There were no changes in mean BMI although 42% lost weight over 2 years. Self-esteem improved significantly. Low self-esteem decreased from 43% at baseline to 15% at 2 years (p<0.0001). At the end of the programme significant cardiovascular risk factors remained, 46% of subjects smoked, 26% had hypertension and 81% had BMI >25. CONCLUSION: Physical health problems are common in SMI subjects. Many patients completed 2 years follow up suggesting that this format of programme is an acceptable option for SMI patients. Cardiovascular risk factors were significantly improved. Interventions such as the Well-being Support Programme should be made widely available to people with SMI.

Two studies are reported. In the first, of 62 schizophrenic patients, no correlation between negative symptom scores (rated blindly) and any measure of positive symptoms was found. This independence was confirmed by factor and cluster analyses, which left the question of a third 'disorganisation' class of schizophrenic symptoms open. In the second study, of 80 patients, formal thought disorder separated unequivocally into 'positive formal thought disorder' and 'alogia' syndromes on the basis of correlations with positive and negative symptoms. Catatonic motor disorder also showed evidence of a corresponding positive: negative division, although this only emerged when severity or chronicity of illness was controlled for. Cognitive impairment showed a broad range of affiliations and its particular correlation with negative symptoms was perhaps artefactual.

The incidence of argentaffin and Paneth cells in epithelial tumours of the large intestine was investigated. Argentaffin cells were found in adenomatous polyps, villus adenomas, polyposis coli, Peutz-Jehgers' polyps, juvenile polyposis, and adenocarcinomas. Paneth cells were not found in metaplastic or juvenile polyps. The crypt unit was destroyed in neoplasia and argentaffin and Paneth cells occurred either as a result of sequestration or were taking part in the neoplastic process. The crypt unit was retained in the disorders of epithelial growth. The identification of argentaffin and Paneth cells enabled the crypt to be defined and thus provided a useful, practical aid in the differentiation between neoplasms and disorders of epithelial growth.

The present randomized, double-blind, placebo and active-drug controlled, crossover study assessed residual sedation after zaleplon 10 mg, flurazepam 30 mg (as an active control), and placebo, taken during a nocturnal awakening in patients with sleep maintenance insomnia. Twenty-two healthy sleep maintenance insomniacs (11 men; mean age, 42 y) received zaleplon, flurazepam, or placebo after an experimental awakening 3.5 hours after bedtime on two consecutive nights in each of three conditions. Residual sedation was measured with sleep latency testing (5 and 6.5 h postdrug), digit symbol substitution, symbol copying, and subjective sleepiness by visual analog scale, each twice each morning. Zaleplon did not differ from placebo on any measure of residual sedation; flurazepam showed significant sedation on all measures. No residual sedative effects were detected 5 or 6.5 hours after ingestion of zaleplon during the middle of the night by sleep maintenance insomniacs.

SUMMARY A study of influenza in residential schools provided the opportunity to assess the significance of antibody as a predictor of immunity. Five hundred and fifty-six pupils from 8 schools were included in the investigations, and the outcome for these children in 27 naturally occurring outbreaks of influenza was analysed. The outbreaks comprised 5 caused by strains of influenza A H3N2, 10 caused by strains of influenza A H1N1, and 12 caused by strains of influenza B. On 8 occasions a second outbreak of the same serotype occurred in a school. There was a general correlation between the presence of antibody to the outbreak strain and protection from infection. For each of the three influenza virus serotypes the infection rate in those with no detectable antibody was approximately 80%. Those with past experience of the virus but no antibody to the outbreak strain experienced lower infection rates (62% overall) but the infection rates were lowest in those with intermediate and high level antibody to the challenge strain (18% overall). Vaccine was used by three of the schools. The effect of antibody derived from recent experience, either natural or vaccine-induced, on subsequent challenge with a drifted strain i.e. one showing antigenic drift away from the previous strain, was compared. Intermediate or high level antibody to the challenge strain in those who had experienced a recent natural infection was associated with a low infection rate (9%). A similar level of antibody produced in response to vaccination was associated with a significantly higher infection rate (23%:P 0-025). Among the vaccinees who had produced such antibody the infection rate was highest (32%) in those who had responded to vaccine in the presence of antibody to the vaccine strain. The evidence from this study indicates that whilst antibody surveys of populations may provide some information about susceptibility to challenge with new strains of influenza viruses, the cirucumstances of the induction of the antibody affect its value as a predictor of immunity.

Abstract Context. —Accurate specimen identification is critical for quality patient care. Improperly identified specimens can result in delayed diagnosis, additional laboratory testing, treatment of the wrong patient for the wrong disease, and severe transfusion reactions. Specimen identification errors have been reported to occur at rates of 0.1% to 5%. Objective. —To determine the frequency of labeling errors in a multi-institutional survey. Design. —Labeling errors were categorized as: (1) mislabeled, (2) unlabeled, (3) partially labeled, (4) incompletely labeled, and (5) illegible label. Blood specimens for routine or stat chemistry, hematology, and coagulation testing were included. Labeling error rates were calculated for each participant and tested for associations with institutional demographic and practice variable information. Results. —More than 3.3 million specimen labels were reviewed by 147 laboratories. Labeling errors were identified at a rate of 0.92 per 1000 labels. Two variables were statistically associated with lower labeling error rates: (1) laboratories with current, ongoing quality monitors for specimen identification ( P = .008) and (2) institutions with 24/7 phlebotomy services for inpatients ( P = .02). Most institutions had written policies for specimen labeling at the bedside or in outpatient phlebotomy areas (96% and 98%, respectively). Allowance of relabeling of blood specimens by primary collecting personnel was reported by 42% of institutions. Conclusions. —Laboratories actively engaged in ongoing specimen labeling quality monitors had fewer specimen labeling errors. Also, 24/7 phlebotomy services were associated with lower specimen error rates. Establishing quality metrics for specimen labeling and deploying 24/7 phlebotomy operations may contribute to improving the accuracy of specimen labeling for the clinical laboratory.

OBJECTIVES: Image-guided musculoskeletal interventional procedures around the hip are widely used in daily clinical practice. The need for clarity concerning the actual added value of imaging guidance and types of medications to be offered led the Ultrasound and the Interventional Subcommittees of the European Society of Musculoskeletal Radiology (ESSR) to promote, with the support of its Research Committee, a collaborative project to review the published literature on image-guided musculoskeletal interventional procedures in the lower limb in order to derive a list of clinical indications. METHODS: In this article, we report the results of a Delphi-based consensus of 53 experts who reviewed the published literature for evidence on image-guided interventional procedures offered in the joint and soft tissues around the hip in order of their clinical indications. RESULTS: Ten statements concerning image-guided treatment procedures around the hip have been collected by the panel of ESSR experts. CONCLUSIONS: This work highlighted that there is still low evidence in the existing literature on some of these interventional procedures. Further large prospective randomized trials are essential to better confirm the benefits and objectively clarify the role of imaging to guide musculoskeletal interventional procedures around the hip. KEY POINTS: • Expert consensus produced a list of 10 evidence-based statements on clinical indications of image-guided interventional procedures around the hip. • The highest level of evidence was only reached for one statement. • Strong consensus was obtained for all statements.

Peripheral venous plasma insulin and C-peptide concentrations were measured in 10 healthy volunteers, given either 100 g glucose orally or sufficient intravenous (i.v.) glucose to produce similar glucose concentrations when measured in arterialized blood. The incremental areas under both the insulin and C-peptide curves were significantly increased after oral as compared with i.v. glucose administration by 229% and 138%, respectively. Arteriovenous plasma glucose differences were higher after oral glucose administration and were positively correlated with plasma insulin concentrations. Plasma gastric inhibitory polypeptide (GIP) and insulin concentrations were measured in seven healthy volunteers given oral glucose loads ranging from 25 to 200 g. Both the magnitude and duration of the GIP and insulin responses after oral glucose ingestion were dose dependent. These results suggest that the main cause of the increase in peripheral insulin levels after large oral carbohydrate loads is augmented insulin secretion rather than reduced hepatic extraction, indicating the possibility that an enteroinsular factor does exist, in accordance with the "incretin" concept. They also emphasize the need to document both arterial and venous glucose concentrations for the correct interpretation of experiments investigating glucose homeostasis.

The aim of this article is to explain the basic principles of digital radiography, and to discuss the intra- and extra-oral imaging systems currently available. There are two main types of digital sensors available. One is based on charge coupled device technology and the other consists of phosphor storage plates. The advantages and disadvantages of each are highlighted with particular attention to orthodontics.

BACKGROUND: There is currently conflicting evidence surrounding the effects of obesity on postoperative outcomes. Previous studies have found obesity to be associated with adverse events, but others have found no association. The aim of this study was to determine whether increasing body mass index (BMI) is an independent risk factor for development of major postoperative complications. METHODS: This was a multicentre prospective cohort study across the UK and Republic of Ireland. Consecutive patients undergoing elective or emergency gastrointestinal surgery over a 4-month interval (October-December 2014) were eligible for inclusion. The primary outcome was the 30-day major complication rate (Clavien-Dindo grade III-V). BMI was grouped according to the World Health Organization classification. Multilevel logistic regression models were used to adjust for patient, operative and hospital-level effects, creating odds ratios (ORs) and 95 per cent confidence intervals (c.i.). RESULTS: Of 7965 patients, 2545 (32·0 per cent) were of normal weight, 2673 (33·6 per cent) were overweight and 2747 (34·5 per cent) were obese. Overall, 4925 (61·8 per cent) underwent elective and 3038 (38·1 per cent) emergency operations. The 30-day major complication rate was 11·4 per cent (908 of 7965). In adjusted models, a significant interaction was found between BMI and diagnosis, with an association seen between BMI and major complications for patients with malignancy (overweight: OR 1·59, 95 per cent c.i. 1·12 to 2·29, P = 0·008; obese: OR 1·91, 1·31 to 2·83, P = 0·002; compared with normal weight) but not benign disease (overweight: OR 0·89, 0·71 to 1·12, P = 0·329; obese: OR 0·84, 0·66 to 1·06, P = 0·147). CONCLUSION: Overweight and obese patients undergoing surgery for gastrointestinal malignancy are at increased risk of major postoperative complications compared with those of normal weight.

BACKGROUND: Singapore was affected by an outbreak of severe acute respiratory syndrome (SARS) from 25 February to 31 May 2003, with 238 probable cases and 33 deaths. AIMS: To study usage of personal protective equipment (PPE) among three groups of healthcare workers (HCWs: doctors, nurses, and administrative staff), to determine if the appropriate PPE were used by the different groups and to examine the factors that may determine inappropriate use. METHODS: A self-administered questionnaire survey of 14,554 HCWs in nine healthcare settings, which included tertiary care hospitals, community hospitals, and polyclinics, was carried out in May-July 2003. Only doctors, nurses, and clerical staff were selected for subsequent analysis. RESULTS: A total of 10 236 valid questionnaires were returned (70.3% response); 873 doctors, 4404 nurses, and 921 clerical staff were studied. A total of 32.5% of doctors, 48.7% of nurses, and 77.1% of the administrative staff agreed that paper and/or surgical masks were "useful in protecting from contracting SARS". Among this group, 23.6% of doctors and 42.3% of nurses reported working with SARS patients. The view that a paper and/or surgical mask was adequate protection against SARS was held by 33.3% of doctors and 55.9% of nurses working at the A&E unit, 30.5% of doctors and 49.4% of nurses from medical wards, and 27.5% of doctors and 37.1% of nurses from intensive care units. Factors which predicted for agreement that paper and/or surgical masks were protective against SARS, included HCW's job title, reported contact with SARS patients, area of work, and Impact Events Scale scores. CONCLUSION: A variety of factors determine appropriate use of personal protective equipment by HCWs in the face of a major SARS outbreak.

The identification of disease alleles underlying human autoinflammatory diseases can provide important insights into the mechanisms that maintain neutrophil homeostasis. Here, we focused our attention on generalized pustular psoriasis (GPP), a potentially life-threatening disorder presenting with cutaneous and systemic neutrophilia. Following the whole-exome sequencing of 19 unrelated affected individuals, we identified a subject harboring a homozygous splice-site mutation (c.2031−2A>C) in MPO. This encodes myeloperoxidase, an essential component of neutrophil azurophil granules. MPO screening in conditions phenotypically related to GPP uncovered further disease alleles in one subject with acral pustular psoriasis (c.2031−2A>C;c.2031−2A>C) and in two individuals with acute generalized exanthematous pustulosis (c.1705C>T;c.2031−2A>C and c.1552_1565del;c.1552_1565del). A subsequent analysis of UK Biobank data demonstrated that the c.2031−2A>C and c.1705C>T (p.Arg569Trp) disease alleles were also associated with increased neutrophil abundance in the general population (p = 5.1 × 10−6 and p = 3.6 × 10−5, respectively). The same applied to three further deleterious variants that had been genotyped in the cohort, with two alleles (c.995C>T [p.Ala332Val] and c.752T>C [p.Met251Thr]) yielding p values < 10−10. Finally, treatment of healthy neutrophils with an MPO inhibitor (4-Aminobenzoic acid hydrazide) increased cell viability and delayed apoptosis, highlighting a mechanism whereby MPO mutations affect granulocyte numbers. These findings identify MPO as a genetic determinant of pustular skin disease and neutrophil abundance. Given the recent interest in the development of MPO antagonists for the treatment of neurodegenerative disease, our results also suggest that the pro-inflammatory effects of these agents should be closely monitored. The identification of disease alleles underlying human autoinflammatory diseases can provide important insights into the mechanisms that maintain neutrophil homeostasis. Here, we focused our attention on generalized pustular psoriasis (GPP), a potentially life-threatening disorder presenting with cutaneous and systemic neutrophilia. Following the whole-exome sequencing of 19 unrelated affected individuals, we identified a subject harboring a homozygous splice-site mutation (c.2031−2A>C) in MPO. This encodes myeloperoxidase, an essential component of neutrophil azurophil granules. MPO screening in conditions phenotypically related to GPP uncovered further disease alleles in one subject with acral pustular psoriasis (c.2031−2A>C;c.2031−2A>C) and in two individuals with acute generalized exanthematous pustulosis (c.1705C>T;c.2031−2A>C and c.1552_1565del;c.1552_1565del). A subsequent analysis of UK Biobank data demonstrated that the c.2031−2A>C and c.1705C>T (p.Arg569Trp) disease alleles were also associated with increased neutrophil abundance in the general population (p = 5.1 × 10−6 and p = 3.6 × 10−5, respectively). The same applied to three further deleterious variants that had been genotyped in the cohort, with two alleles (c.995C>T [p.Ala332Val] and c.752T>C [p.Met251Thr]) yielding p values < 10−10. Finally, treatment of healthy neutrophils with an MPO inhibitor (4-Aminobenzoic acid hydrazide) increased cell viability and delayed apoptosis, highlighting a mechanism whereby MPO mutations affect granulocyte numbers. These findings identify MPO as a genetic determinant of pustular skin disease and neutrophil abundance. Given the recent interest in the development of MPO antagonists for the treatment of neurodegenerative disease, our results also suggest that the pro-inflammatory effects of these agents should be closely monitored. A tight regulation of neutrophil numbers is crucial to innate immune homeostasis. As mature granulocytes do not divide, their accumulation depends on the balance between progenitor proliferation, release of differentiated cells into the bloodstream, and clearance of aging cells.1Nicolás-Ávila J.A. Adrover J.M. Hidalgo A. Neutrophils in Homeostasis, Immunity, and Cancer.Immunity. 2017; 46: 15-28Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar Given the difficulty of manipulating primary neutrophils, the mechanisms that regulate these processes have mostly been investigated in animal models. In this context, the genetic characterization of human autoinflammatory diseases can provide crucial insights into the pathways that maintain neutrophil homeostasis. Here we focused our attention on generalized pustular psoriasis (GPP [MIM: 614204]), a potentially life-threatening condition presenting with flares of neutrophilic skin inflammation (pustular eruptions), fever, increased production of acute phase reactants, and neutrophilia. While disease alleles have been described in IL36RN, AP1S3, and CARD14, the majority of affected individuals do not carry deleterious changes at these loci.2Twelves S. Mostafa A. Dand N. Burri E. Farkas K. Wilson R. Cooper H.L. Irvine A.D. Oon H.H. Kingo K. et al.Clinical and genetic differences between pustular psoriasis subtypes.J. Allergy Clin. Immunol. 2019; 143: 1021-1026Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar To identify genetic determinants for GPP, we undertook whole-exome sequencing in 19 unrelated affected individuals of varying ancestry (Table S1, Figure 1A). Given the severity of the condition and the lack of parent-offspring transmissions, we hypothesized the presence of recessive loss-of-function alleles. We therefore filtered the variant profiles to retain rare homozygous changes predicted to cause premature protein truncation. This identified six candidate mutations, each affecting a single individual (Table S2). The c.2031−2A>C substitution in MPO (MIM: 606989; GenBank: NM_000250) (Figure 1B) was selected for follow-up, as the gene encodes myeloperoxidase, a major component of neutrophil azurophilic granules. Of note, the c.2031−2A>C change has been previously observed in individuals presenting with myeloperoxidase deficiency (MPOD [MIM: 254600]) (Table S3), an inherited defect of neutrophil microbicidal activity.3Marchetti C. Patriarca P. Solero G.P. Baralle F.E. Romano M. Genetic characterization of myeloperoxidase deficiency in Italy.Hum. Mutat. 2004; 23: 496-505Crossref PubMed Scopus (27) Google Scholar Specifically, Marchetti et al. demonstrated that the substitution affects splicing and leads to the production of a truncated protein lacking enzymatic activity.3Marchetti C. Patriarca P. Solero G.P. Baralle F.E. Romano M. Genetic characterization of myeloperoxidase deficiency in Italy.Hum. Mutat. 2004; 23: 496-505Crossref PubMed Scopus (27) Google Scholar Here, the frequency of the c.2031−2C;c.2031−2C genotype among European GPP case subjects was much higher than that observed in the non-Finnish European exomes sequenced by the gnomAD consortium (5.3% versus 0.003%; p = 0.001) (Table 1). Given that frequency estimates obtained in small datasets are not always robust, the MPO coding region was next examined in a validation sample, including 14 GPP subjects and 109 individuals with acral variants of pustular psoriasis (APP). This uncovered a further study participant harboring a homozygous c.2031−2A>C substitution (Figure 1B, Table 2, and Supplemental Note).Table 1Frequency of the c.2031−2C;c.2031−2C Genotypes in Case Subjects versus Control SubjectsGenotype Counts (%)p ValueCasesControlsDiscovery cohort1/19 (5.3%)2/56,7466 (0.003%)0.001Replication cohort1/123 (0.8%)0/32,264 (0%)0.004Combined study cohort2/142 (1.4%)2/89,010 (0.002%)1.5 × 10−5c.2031−2A>C was the only truncating change observed in the homozygous state in the control subjects, obviating the need for a burden association test. Open table in a new tab Table 2Disease Features Observed in Individuals with Bi-allelic MPO MutationsSubjectsSexAge of OnsetDiagnosisaThe most likely culprit drug for each AGEP subject is reported in parentheses. AGEP, acute generalized exanthematous pustulosis; APP, acral pustular psoriasis; GPP, generalized pustular psoriasis.Systemic InvolvementGenotypeGYFAP0014F36GPPfever and neutrophiliac.2031−2A>C;c.2031−2A>CDDPLM0001F24APP–c.2031−2A>C;c.2031−2A>CSCAR2124F80AGEP (methotrexate)fever and neutrophiliac.1552_1565del;c.1552_1565delSCAR2567F69AGEP (hydroxychloroquine)feverc.2031−2A>C;c.1705C>TNone of the affected individuals reported a history of recurrent infections.a The most likely culprit drug for each AGEP subject is reported in parentheses. AGEP, acute generalized exanthematous pustulosis; APP, acral pustular psoriasis; GPP, generalized pustular psoriasis. Open table in a new tab c.2031−2A>C was the only truncating change observed in the homozygous state in the control subjects, obviating the need for a burden association test. None of the affected individuals reported a history of recurrent infections. While no other bi-allelic or truncating MPO changes were detected (Table S4), a comparison of the replication cohort against a second gnomAD dataset (32,264 Non-Finnish European genomes) confirmed the elevated frequency of the c.2031−2C;c.2031−2C genotype in case subjects versus control subjects (0.8% versus 0%; p = 0.004) (Table 1). Finally, the analysis of the combined study resource (142 case subjects versus 89,010 control subjects) yielded a p value of 1.5 × 10−5 (Table 1). Importantly, the c.2031−2C;c.2031−2C genotype was also absent from 590 British exomes processed with our in-house pipeline. Thus, the association with GPP/-APP is unlikely to be a technical artifact or to reflect population stratification between case and control subjects. To further investigate the impact of MPO recessive alleles, we queried exome-sequencing data available for 96 unrelated individuals affected by acute generalized exanthematous pustulosis (AGEP). This is a severe cutaneous adverse reaction, which can be triggered by drugs (mostly antibiotics and antifungals), leading to flares of skin pustulation, fever, and systemic neutrophilia.4Sidoroff A. Halevy S. Bavinck J.N. Vaillant L. Roujeau J.C. Acute generalized exanthematous pustulosis (AGEP)--a clinical reaction pattern.J. Cutan. Pathol. 2001; 28: 113-119Crossref PubMed Scopus (615) Google Scholar Our analysis identified an affected individual, who was homozygous for a 14 bp MPO deletion (c.1552_1565del [p.Met519Profs∗21]). A second study participant had inherited the c.2031−2A>C substitution previously observed in GPP/APP, in conjunction with a damaging c.1705C>T (p.Arg569Trp) change (CADD score: 35.0) (Table 2, Supplemental Note). Of note, homozygous c.1705C>T (p.Arg569Trp) mutations have been repeatedly observed in individuals affected by MPOD.5Kizaki M. Miller C.W. Selsted M.E. Koeffler H.P. Myeloperoxidase (MPO) gene mutation in hereditary MPO deficiency.Blood. 1994; 83: 1935-1940Crossref PubMed Google Scholar,6Nauseef W.M. Brigham S. Cogley M. Hereditary myeloperoxidase deficiency due to a missense mutation of arginine 569 to tryptophan.J. Biol. Chem. 1994; 269: 1212-1216Abstract Full Text PDF PubMed Google Scholar A c.1555_1568del variant, which overlaps the c.1552_1565del variant described here, has also been documented in an affected-relative pair, where it triggered nonsense-mediated decay in at least one individual7Romano M. Dri P. Da Dalt L. Patriarca P. Baralle F.E. Biochemical and molecular characterization of hereditary myeloperoxidase deficiency.Blood. 1997; 90: 4126-4134Crossref PubMed Google Scholar (Table S3). Thus, all the MPO alleles observed in our dataset have a well-established impact on protein function. MPOD is a mild immune deficiency that is clinically well characterized. We therefore undertook a systematic literature review, to better understand the connection between MPO mutations, MPOD, and skin pustulation. We examined 28 articles describing the presentation of MPOD in 217 individuals. This uncovered four case reports where the disease manifested with pustular eruptions and a fifth where it was associated with the severe neutrophilic dermatosis known as pyoderma gangrenosum (Table S5). Given the very low prevalence of the above conditions (<1:100,000), these observations strengthen the link between MPO dysfunction and neutrophilic inflammation. To investigate the mechanisms whereby MPO mutations contribute to disease, we explored the phenotypic effects of the c.2031−2A>C variant though a Phenome-Wide Association study (PheWAS). We queried the UK Biobank dataset, which includes genotype information and health data for a well-characterized population cohort (>450,000 individuals).8Sudlow C. Gallacher J. Allen N. Beral V. Burton P. Danesh J. Downey P. Elliott P. Green J. Landray M. et al.UK biobank: an open access resource for identifying the causes of a wide range of complex diseases of middle and old age.PLoS Med. 2015; 12: e1001779Crossref PubMed Scopus (3405) Google Scholar While the fraction of c.2031−2C homozygotes (0.003%) (Table S3) present in the biobank was very small, c.2031−2A>C heterozygotes accounted for approximately 1% of study participants. Thus, we were able to analyze the c.2031−2A>C genotypes against 778 available phenotypes. This revealed that the traits showing the most significant associations with c.2031−2A>C were related to leukocyte counts. In this context, the largest effect size (beta) was observed for the association with neutrophil abundance (beta = 0.45; p = 5.1 × 10−6; Figure 2A). To validate these findings, we examined the c.1705C>T (p.Arg569Trp) change and three additional MPOD alleles (c.518A>G [p.Tyr173Cys], c.752T>C [p.Met251Thr], c.995C>T [p.Ala332Val]) for which genotype data were available in UK Biobank (Table S3). We found that all were associated with increased neutrophil accumulation, with p values ranging from 0.008 to 3.9 × 10−28 (Figure 2B). To explore the pathways underlying the effects of MPO alleles on granulocyte numbers, we examined RNA-sequencing profiles generated in pure neutrophil populations (see Supplemental Subjects and Methods). Specifically, we compared gene expression in the c.2031−2A>C homozygous GPP individual versus 11 healthy control subjects. We observed that MPO was expressed at comparable levels in the affected subject and the unaffected control subjects (FDR > 0.5). As c.2031−2A>C affects the splicing of the last exon, this is in keeping with the expectation of an escape from nonsense-mediated decay. Conversely, we found that 95 genes were upregulated in the affected individual (FDR < 0.05) (Table S6). The majority of these loci (85/95) were not overexpressed in 7 unrelated individuals with GPP (all MPO wild type) examined in parallel, indicating that the changes are unlikely to be a secondary effect of inflammation. While the experiment was limited by the modest sample size and the number of differentially expressed genes was too small for pathway enrichment analyses, we noted that two of the five most upregulated loci (PBK and GUCYA2) encode proteins (PDZ binding kinase and soluble guanylate cyclase alpha-2 subunit) that can inhibit apoptosis.9Liu Y. Liu H. Cao H. Song B. Zhang W. Zhang W. PBK/TOPK mediates promyelocyte proliferation via Nrf2-regulated cell cycle progression and apoptosis.Oncol. Rep. 2015; 34: 3288-3296Crossref PubMed Scopus (26) Google Scholar,10Weissmann N. Lobo B. Pichl A. Parajuli N. Seimetz M. Puig-Pey R. Ferrer E. Peinado V.I. Domínguez-Fandos D. Fysikopoulos A. et al.Stimulation of soluble guanylate cyclase prevents cigarette smoke-induced pulmonary hypertension and emphysema.Am. J. Respir. Crit. Care Med. 2014; 189: 1359-1373Crossref PubMed Scopus (67) Google Scholar This suggests that MPO mutations may affect neutrophil survival. We investigated this possibility by using a myeloperoxidase inhibitor (4-Aminobenzoic acid hydrazide, ABAH) to mimic the effects of MPO disease alleles in cell culture experiments. We induced neutrophil apoptosis through Phorbol 12-myristate 13-acetate (PMA) stimulation and assessed the effects of ABAH pre-treatment on this process. While PMA caused substantial neutrophil death, we found that ABAH supplementation caused an increase in cell viability (Figure 2C) and a reduction in the number of apoptotic cells (Figure 2D). Thus, neutrophil apoptosis is downregulated in the absence of MPO activity. Our findings (and those independently reported by Haskamp et al.11Haskamp S. Bruns H. Hahn M. Hoffmann M. Gregor A. Löhr S. Hahn J. Schauer C. Ringer M. Flamann C. et al.Myeloperoxidase modulates inflammation in generalized pustular psoriasis and additional rare pustular skin diseases.Am. J. Hum. Genet. 2020; 107 (Published online August 5, 2020)https://doi.org/10.1016/j.ajhg.2020.06.020Abstract Full Text Full Text PDF Scopus (0) Google Scholar) demonstrate a significant association between MPO mutations and pustular skin disease. While the disease alleles described here have also been implicated in MPOD, we did not observe any evidence of immune deficiency in the individuals examined in this study. Likewise, pustular skin disease is present in only a fraction of people affected by MPOD. Thus, it is tempting to speculate that the manifestations of MPO mutations may be influenced by background polygenic variation, especially as a similar phenomenon has been documented in rare hematological phenotypes.12Vuckovic D. Bao E.L. Akbari P. Lareau C. Moussas A. Jiang T. Chen M.H. Raffield L.M. Tardaguila M. Huffman J.E. et al.The Polygenic and Monogenic Basis of Blood Traits and Diseases.medRxiv. 2020; https://doi.org/10.1101/2020.02.02.20020065Crossref Scopus (0) Google Scholar Of note, the c.2031−2C;c.2031−2C GPP individual described here also harbored a deleterious AP1S3 allele (GenBank: NM_001039569.2; c.97C>T [p.Arg33Trp]),13Setta-Kaffetzi N. Simpson M.A. Navarini A.A. Patel V.M. Lu H.C. Allen M.H. Duckworth M. Bachelez H. Burden A.D. Choon S.E. et al.AP1S3 mutations are associated with pustular psoriasis and impaired Toll-like receptor 3 trafficking.Am. J. Hum. Genet. 2014; 94: 790-797Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar which further supports the involvement of genetic modifiers. An increased prevalence of spondyloarthropathy has also been reported among individuals with MPO deficiency.14Kutter D. Devaquet P. Vanderstocken G. Paulus J.M. Marchal V. Gothot A. Consequences of total and subtotal myeloperoxidase deficiency: risk or benefit?.Acta Haematol. 2000; 104: 10-15Crossref PubMed Scopus (213) Google Scholar This suggests that the disruption of neutrophil apoptosis may affect immune homeostasis in multiple organs. Given that MPO inhibitors are being developed for the treatment of neurodegenerative disease,15Galijasevic S. The development of myeloperoxidase inhibitors.Bioorg. Med. Chem. Lett. 2019; 29: 1-7Crossref PubMed Scopus (33) Google Scholar our data suggest that the inflammatory side effects of these agents should be closely monitored during clinical trials. The results of our PheWAS indicate that the effects of MPO alleles are likely to be mediated by a systemic upregulation of neutrophil numbers. Of note, a significant association between granulocyte abundance and a common MPO variant has previously been documented,16Astle W.J. Elding H. Jiang T. Allen D. Ruklisa D. Mann A.L. Mead D. Bouman H. Riveros-Mckay F. Kostadima M.A. et al.The Allelic Landscape of Human Blood Cell Trait Variation and Links to Common Complex Disease.Cell. 2016; 167: 1415-1429.e19Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar further supporting the role of the gene in neutrophil homeostasis. Further studies will be required to dissect the molecular mechanisms whereby MPO deficiency downregulates cell death. Given that PBK (one of the most upregulated genes in the c.2031−2A>C homozygous individual) is an inhibitor of myeloid cell apoptosis,9Liu Y. Liu H. Cao H. Song B. Zhang W. Zhang W. PBK/TOPK mediates promyelocyte proliferation via Nrf2-regulated cell cycle progression and apoptosis.Oncol. Rep. 2015; 34: 3288-3296Crossref PubMed Scopus (26) Google Scholar its role is worthy of further examination. A proposed link between MPO-related oxidative stress, NF-κB activation, and apoptotic signaling17Kanayama A. Miyamoto Y. Apoptosis triggered by phagocytosis-related oxidative stress through FLIPS down-regulation and JNK activation.J. Leukoc. Biol. 2007; 82: 1344-1352Crossref PubMed Scopus (43) Google Scholar should also be investigated. While experimentally demanding, these studies have the potential to illuminate key regulators of innate immune homeostasis and uncover new candidate genes for neutrophilic conditions. The neutrophil RNA-sequencing dataset described in this study may be obtained from the Gene Expression Omnibus using has from has been a on clinical and studies by and has from and from has and from and has for clinical from through and are has to from and This has been using the UK Biobank We are to and Dand for their technical and We from the of via a of to and in with and We also from the The is by the and This has been by a European of and to and is by the is by a by a and by an is by the is a also from the and the and In and are of The of by from the European and in and by a consortium of with Supplemental Supplemental Subjects and and Expression myeloperoxidase mutations are associated with increased neutrophil and pustular skin et al.The of Human of Human PDF Open