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Background Bipolar disorder is the fourth most common mental health condition, affecting ~ 1% of UK adults. Lithium is an effective treatment for prevention of relapse and hospital admission, and is widely recommended as a first-line treatment. We previously showed in other areas that laboratory testing patterns are variable with sub-optimal conformity to guidance. We therefore examined lithium results and requesting patterns relative to monitoring recommendations. Methods Data on serum lithium levels and intervals between requests were extracted from Clinical Biochemistry laboratory information systems at the University Hospitals of North Midlands, Salford Royal Foundation Trust and Pennine Acute Hospitals from 2012 to 2018 (46,555 requests; 3371 individuals). Data were examined with respect to region/source of request, age and sex. Results Across all sites, lithium levels on many requests were outside the recommended UK therapeutic range (0.4–0.99 mmol/L); 19.2% below the range and 6.1% above the range (median [Li]: 0.60 mmol/L). A small percentage were found at the extremes (3.2% at Conclusions Our data showed: (a) lithium levels are often maintained at the lower end of the recommended therapeutic range, (b) patterns of lithium results and testing frequency were comparable across three UK sites with differing models of care and, (c) re-test intervals demonstrate a noticeable peak at the recommended 3-monthly, but not at 6-monthly intervals. Many tests were repeated outside expected frequencies, indicating the need for measures to minimise inappropriate testing.

BackgroundLithium is viewed as the first-line long-term treatment for prevention of relapse in people with bipolar disorder.AimsThis study examined factors associated with the likelihood of maintaining serum lithium levels within the recommended range and explored whether the monitoring interval could be extended in some cases.MethodWe included 46 555 lithium rest requests in 3371 individuals over 7 years from three UK centres. Using lithium results in four categories (ResultsFor tests within the recommended range (0.40–0.99 mmol/L categories), 84.5% of subsequent tests remained within this range. Overall, 3 monthly testing was associated with 90% of lithium results remaining within range, compared with 85% at 6 monthly intervals. In cases where the lithium level in the previous 12 months was on target (0.40–0.79 mmol/L; British National Formulary/National Institute for Health and Care Excellence criteria), 90% remained within the target range at 6 months. Neither age nor duration of lithium therapy had any significant effect on lithium level stability. Levels within the 0.80–0.99 mmol/L category were linked to a higher probability of moving to the ≥1.0 mmol/L category (10%) compared with those in the 0.4–0.79 mmol/L group (2%), irrespective of testing frequency.ConclusionWe propose that for those who achieve 12 months of lithium tests within the 0.40–0.79 mmol/L range, the interval between tests could increase to 6 months, irrespective of age. Where lithium levels are 0.80–0.99 mmol/L, the test interval should remain at 3 months. This could reduce lithium test numbers by 15% and costs by ~$0.4 m p.a.

Context Adrenal incidentalomas (AIs) are increasingly being identified during unrelated imaging. Unlike AI clinical management, data on referral patterns in routine practice are lacking. Objective This work aimed to identify factors associated with AI referral. Methods We linked data from imaging reports and outpatient bookings from a large UK teaching hospital. We examined (i) AI prevalence and (ii) pattern of referral to endocrinology, stratified by age, imaging modality, scan anatomical site, requesting clinical specialty, and temporal trends. Using key radiology phrases to identify scans reporting potential AI, we identified 4097 individuals from 479 945 scan reports (2015-2019). Main outcome measures included prevalence of AI and referral rates. Results Overall, AI lesions were identified in 1.2% of scans. They were more prevalent in abdomen computed tomography and magnetic resonance imaging scans (3.0% and 0.6%, respectively). Scans performed increased 7.7% year-on-year from 2015 to 2019, with a more pronounced increase in the number with AI lesions (14.7% per year). Only 394 of 4097 patients (9.6%) had a documented endocrinology referral code within 90 days, with medical (11.8%) more likely to refer than surgical (7.2%) specialties (P Conclusion While overall AI prevalence appeared low, scan numbers are large and rising; the number with identified AI are increasing still further. The poor AI referral rates, even in centers such as ours where dedicated AI multidisciplinary team meetings and digital management systems are used, highlights the need for new streamlined, clinically effective systems and processes to appropriately manage the AI workload.

Recently, the UK’s national Targeted Lung Health Checks programme produced recommendations for the management of incidental findings identified during the scans performed as part of the lung cancer screening programme. We identified significant discrepancies between the recommendations for adrenal incidentaloma management and those currently implemented into UK practice (2016 European Society of Endocrinology guidelines). This may create conflict and confusion between referrers (respiratory clinicians) and receivers (endocrinologists), with potential negative impact on patients, delay and inefficient use of resources. We also address the potential cost implications of adopting a more vigilant approach as advised by the European Society of Endocrinology. Urgent multidisciplinary and unified guidelines should be established in the interest of clinical- and cost-effectiveness.

ObjectivesOther than age, diabetes is the largest contributor to overall healthcare costs and reduced life expectancy in Europe. This paper aims to more exactly quantify the net impact of diabetes on different aspects of healthcare provision in hospitals in England, building on previous work that looked at the determinants of outcome in type 1 diabetes (T1DM) and type 2 diabetes (T2DM).SettingNHS Digital Hospital Episode Statistics (HES) in England was combined with the National Diabetes Audit (NDA) to provide the total number in practice of people with T1DM/T2DM.Outcome measuresWe compared differences between T1DM/T2DM and non-diabetes individuals in relation to hospital activity and associated cost.ResultsThe study captured 90% of hospital activity and £36 billion/year of hospital spend. The NDA Register showed that out of a total reported population of 58 million, 2.9 million (6.5%) had T2DM and 240 000 (0.6%) had T1DM. Bed-day analysis showed 17% of beds are occupied by T2DM and 3% by T1DM. The overall cost of hospital care for people with diabetes is £5.5 billion/year. Once the normally expected costs including the older age of T2DM hospital attenders are allowed for this fell to £3.0 billion/year or 8% of the total captured secondary care costs. This equates to £560/non-diabetes person compared with £3280/person with T1DM and £1686/person with T2DM. For people with diabetes, the net excess impact on non-elective/emergency work is £1.2 billion with additional estimated diabetes-related accident & emergency attendances at 440 000 costing the NHS £70 million/year. T1DM individuals required five times more secondary care support than non-diabetes individuals. T2DM individuals, even allowing for the age, require twice as much support as non-diabetes individuals.ConclusionsThis analysis shows that additional cost of provision of hospital services due to their diabetes comorbidities is £3 billion above that for non-diabetes, and that within this, T1DM has three times as much cost impact as T2DM. We suggest that supporting patients in diabetes management may significantly reduce hospital activity.

Whilst the estimated prevalence of adrenal incidentaloma is 0.5-2% following abdominal CT scan (1), little is known about subsequent endocrine referral patterns according to clinical specialty. We therefore examined the range of specialties that were responsible for requesting CT and MRI scans that led to reporting potential adrenal lesions. We also explored the referral pattern to endocrinology following identification. We extracted data from all radiology reports for all CT and MRI scans from Jan 2018-Oct 2019. We utilised a key phrase search strategy (eg adrenal adenoma/lesion/mass/nodule/incidentaloma, incidental adrenal, indeterminate adrenal). Where possible we excluded false hits (eg no adrenal lesion). These were lined to the referral patterns as identified by a referral logged or an attendance (new or follow-up) to endocrine clinic 3 months post index scan. These data were stratified by requesting specialty. Preliminary data showed that, out of 127878 scans, 2021 patients were identified to have a potential adrenal incidentaloma. These requests came from a total of 45 different clinical specialities (medical and surgical). The top 12 specialties accounted from 82.8% of these referrals. Medical specialties (renal, gastroenterology, respiratory, general medicine, acute medicine and geriatric medicine) accounted for 50.2%. The remaining 32.6% were via surgical specialities. The overall referral pattern in these cases was 8.5% with no difference between medical (8.6%) and surgical (8.4%) specialties. In conclusion, adrenal incidentalomas are potentially identifiable across a wide range of specialties. Currently, despite a dedicated adrenal multidisciplinary team and nationally-acknowledged quality improvement programme for the management of adrenal incidentalomas (2), the majority of cases are overlooked and not referred for endocrine review as suggested in current guidelines. We expect the findings in our centre to be reproducible elsewhere. We are considering innovative approaches to improve the process and cope with the additional workload cost-effectively. 1. Barzon L, Sonino N, Fallo F, Palu G, Boscaro M. Prevalence and natural history of adrenal incidentalomas. Eur J Endocrinol. 2003;149:273-285. Hanna FWF, Issa BG, Lea SC, George C, Golash A, Firn M, Ogunmekan S, Maddock E, Sim J, Xydopoulos G, Fordham R, Fryer AA. Adrenal lesions found incidentally: how to improve clinical and cost-effectiveness. BMJ Open Quality. 2019;In press.

Background: The National Health Service spends £170 million on blood glucose monitoring (BGM) strips each year and there are pressures to use cheaper less accurate strips. Technology is also being used to increase test frequency with less focus on accuracy. Previous modeling/real-world data analysis highlighted that actual blood glucose variability can be more than twice blood glucose meter reported variability (BGMV). We applied those results to the Parkes error grid to highlight potential clinical impact. Method: BGMV is defined as the percent of deviation from reference that contains 95% of results. Four categories were modeled: laboratory (Results: Applying to single readings, almost all strip accuracy ranges derived in a controlled setting fell within the category: clinically accurate/no effect on outcomes areas. However modeling the possible blood glucose distribution in more detail, 30.6% of longer term results of the strips with current ISO accuracy would fall into the “alter clinical action” category. For previous ISO strips, this rose to 44.1%, and for the latest higher accuracy strips, this fell to 12.8%. Conclusion: There is a minimum standard of accuracy needed to ensure that clinical outcomes are not put at risk. This study highlights the potential for amplification of imprecision with less accurate BGM strips.

IntroductionAdrenal incidentalomas are lesions that are incidentally identified while scanning for other conditions. While most are benign and hormonally non-functional, around 20% are malignant and/or hormonally active, requiring prompt intervention. Malignant lesions can be aggressive and life-threatening, while hormonally active tumours cause various endocrine disorders, with significant morbidity and mortality. Despite this, management of patients with adrenal incidentalomas is variable, with no robust evidence base. This project aimed to establish more effective and timely management of these patients.MethodsWe developed a web-based, electronic Adrenal Incidentaloma Management System (eAIMS), which incorporated the evidence-based and National Health Service–aligned 2016 European guidelines. The system captures key clinical, biochemical and radiological information necessary for adrenal incidentaloma patient management and generates a pre-populated outcome letter, saving clinical and administrative time while ensuring timely management plans with enhanced safety. Furthermore, we developed a prioritisation strategy, with members of the multidisciplinary team, which prioritised high-risk individuals for detailed discussion and management. Patient focus groups informed process-mapping and multidisciplinary team process re-design and patient information leaflet development. The project was partnered by University Hospital of South Manchester to maximise generalisability.ResultsImplementation of eAIMS, along with improvements in the prioritisation strategy, resulted in a 49% reduction in staff hands-on time, as well as a 78% reduction in the time from adrenal incidentaloma identification to multidisciplinary team decision. A health economic analysis identified a 28% reduction in costs.ConclusionsThe system’s in-built data validation and the automatic generation of the multidisciplinary team outcome letter improved patient safety through a reduction in transcription errors. We are currently developing the next stage of the programme to proactively identify all new adrenal incidentaloma cases.

A monomeric form of C-reactive protein (CRP) which precipitates with cell wall pneumococcal C polysaccharide (CWPS) and retains the ability to reversibly bind to its ligand phosphocholine has been produced through urea-induced dissociation at an optimized concentration of 3 M urea over a 10 weeks period. Dissociated samples were purified via size exclusion chromatography and characterized by western blot, phosphocholine affinity chromatography and CWPS precipitation. Human serum samples from patients with raised CRP levels (>100 mg/L as determined by the clinical laboratory assay) were purified by affinity and size exclusion chromatography and analyzed (n = 40) to determine whether circulating monomeric CRP could be detected ex vivo. All 40 samples tested positive for pentameric CRP via western blot and enzyme linked immunosorbent assay (ELISA) analysis. Monomeric C-reactive protein was also identified in all 40 patient samples tested, with an average level recorded of 1.03 mg/L (SE = ±0.11). Both the in vitro monomeric C-reactive protein and the human serum monomeric protein displayed a molecular weight of approximately 23 kDa, both were recognized by the same anti-CRP monoclonal antibody and both reversibly bound to phosphocholine in a calcium-dependent manner. In common with native pentameric CRP, the in vitro mCRP precipitated with CWPS. These overlapping characteristics suggest that a physiologically relevant, near-native monomeric CRP, which retains the structure and binding properties of native CRP subunits, has been produced through in vitro dissociation of pentameric CRP and also isolated from serum with markedly elevated CRP levels. This provides a clear route toward the in-depth study of the structure and function of physiological monomeric CRP.

What you need to know A 67 year old woman with no significant medical history required urgent orthopaedic surgery after a fall. In preparation, her bloods were collected and a random cortisol was requested, which came back at 763 nmol/L. She did not have diabetes or hypertension. The anaesthetist reviewed and commented: “High cortisol, not safe for surgery until further evaluation.” A 24 year old man with previously well controlled type 1 diabetes presented with repeated hypoglycaemic episodes. Serum cortisol in clinic at 9 am came back as 143 nmol/L. Cortisol, secreted by the adrenal cortex, regulates blood pressure, glucose metabolism, and physiological responses to stress. Both cortisol over-secretion (hypercortisolism, Cushing’s syndrome) and under-secretion (hypocortisolism such as in Addison’s disease) are uncommon: the prevalence of Addison’s disease is 6-9 per 100 000,1 while that of Cushing’s syndrome is 4/100 000.2 However, given the potential for life threatening consequences (such as in acute adrenal crisis) and the range of associated non-specific symptoms, it is critical for clinicians to understand how to interpret and manage cortisol status. Cortisol excess could be due to an adrenal cortisol-secreting tumour or to a functional pituitary tumour (Cushing’s disease), or, less commonly, malignant tumours can …

ObjectiveEvaluate relative clinical effectiveness of treatment options for type 2 diabetes mellitus (T2DM) using a statistical model of real-world evidence within UK general practitioner practices (GPP), to quantify the opportunities for diabetes care performance improvement.MethodFrom the National Diabetes Audit in 2015–2016 and 2016–2017, GPP target glycaemic control (TGC—%HbA1c ≤58 mmol/mol) and higher glycaemic risk (HGR —%HbA1c results >86 mmol/mol) outcomes were linked using multivariate linear regression to prescribing, demographics and practice service indicators. This was carried out both cross-sectionally (XS) (within year) and longitudinally (Lo) (across years) on 35 indicators. Standardised β coefficients were used to show relative level of impact of each factor. Improvement opportunity was calculated as impact on TGC & HGR numbers.ResultsValues from 6525 GPP with 2.7 million T2DM individuals were included. The cross-sectional model accounted for up to 28% TGC variance and 35% HGR variance, and the longitudinal model accounted for up to 9% TGC and 17% HGR variance. Practice service indicators including % achieving routine checks/blood pressure/cholesterol control targets were positively correlated, while demographic indicators including % younger age/social deprivation/white ethnicity were negatively correlated. The β values for selected molecules are shown as (increased TGC; decreased HGR), canagliflozin (XS 0.07;0.145/Lo 0.04;0.07), metformin (XS 0.12;0.04/Lo –;–), sitagliptin (XS 0.06;0.02/Lo 0.10;0.06), empagliflozin (XS–;0.07/Lo 0.09;0.07), dapagliflozin (XS –;0.04/Lo –;0.4), sulphonylurea (XS −0.18;−0.12/Lo–;–) and insulin (XS−0.14;0.02/ Lo−0.09;–). Moving all GPP prescribing and interventions to the equivalent of the top performing decile of GPP could result in total patients in TGC increasing from 1.90 million to 2.14 million, and total HGR falling from 191 000 to 123 000.ConclusionsGPP using more legacy therapies such as sulphonylurea/insulin demonstrate poorer outcomes, while those applying holistic patient management/use of newer molecules demonstrate improved glycaemic outcomes. If all GPP moved service levels/prescribing to those of the top decile, both TGC/HGR could be substantially improved.

Background: The most common functional abnormality in adrenal incidentalomas (AI) is autonomous cortisol secretion. Most guidelines recommend a 1 mg overnight dexamethasone suppression test (ONDST) as a screening test for this abnormality. There is some evidence that simultaneous measurement of post dexamethasone serum dexamethasone (SD) with serum cortisol (SC) improves the accuracy of the ONDST. We have therefore been routinely measuring SD in our unit to ensure adequate concentrations when interpreting the results of this test. Aim: To measure the correlation between SD and SC concentrations and SD with the decrement in serum cortisol levels from baseline (9 am the day before the test) to the post dexamethasone level (DSC) in the ONDST. Method: We examined the results of 57 patients with unilateral or bilateral adrenal incidentalomas who underwent a 1 mg ONDST on our unit. Using linear regression analysis, we correlated SC and SD concentrations and serum SD concentration with the DSC. We also examined whether lower levels of SD were associated with adequate suppression of SC following ONDST. Results: SD levels range 16, median 7.656 nmol/L. SC range

BackgroundWe previously showed, in patients with diabetes, that >50% of monitoring tests for glycated haemoglobin (HbA1c) are outside recommended intervals and that this is linked to diabetes control. Here, we examined the effect of tests/year on achievement of commonly utilised HbA1ctargets and on HbA1cchanges over time.MethodsData on 20,690 adults with diabetes with a baseline HbA1cof >53 mmol/mol (7%) were extracted from Clinical Biochemistry Laboratory records at three UK hospitals. We examined the effect of HbA1ctests/year on (i) the probability of achieving targets of ≤53 mmol/mol (7%) and ≤48 mmol/mol (6.5%) in a year using multi-state modelling and (ii) the changes in mean HbA1cusing a linear mixed-effects model.ResultsThe probabilities of achieving ≤53 mmol/mol (7%) and ≤48 mmol/mol (6.5%) targets within 1 year were 0.20 (95% confidence interval: 0.19–0.21) and 0.10 (0.09–0.10), respectively. Compared with four tests/year, having one test or more than four tests/year were associated with lower likelihoods of achieving either target; two to three tests/year gave similar likelihoods to four tests/year. Mean HbA1clevels were higher in patients who had one test/year compared to those with four tests/year (mean difference: 2.64 mmol/mol [0.24%], pConclusionsWe showed that ≥80% of patients with suboptimal control are not achieving commonly recommended HbA1ctargets within 1 year, highlighting the major challenge facing healthcare services. We also demonstrated that, although appropriate monitoring frequency is important, testing every 6 months is as effective as quarterly testing, supporting international recommendations. We suggest that the importance HbA1cmonitoring frequency is being insufficiently recognised in diabetes management.

What you need to know ### Sources and selection criteria References were selected from Medline search, focusing on published guidelines. Sub-references were selected to ensure full coverage of the areas identified within the initial search. An incidental adrenal tumour, described in this article as an “adrenal incidentaloma,” is an adrenal mass discovered during imaging that was not performed for suspected adrenal disease. These are being detected more frequently in an ageing population1 through the increased use of computed tomography (eg, computed tomography urogram, or colonoscopy) and magnetic resonance imaging (MRI) scans with enhanced resolution.2 This article highlights the management of adrenal incidentalomas, collating recommendations from international guidelines, and is aimed at non-specialists. Adrenal incidentalomas are defined as adrenal masses incidentally discovered during imaging that was not performed for suspected adrenal disease. Hence, an adrenal lesion discovered while investigating raised catecholamines would not be classified as an adrenal incidentaloma. Most definitions have restricted the diagnosis to lesions ≥10 mm34 unless there are clinical stigmas of adrenal hormone over-production identified after the detection of the lesion that warrant further investigation. In view of variation in diagnostic criteria, referral patterns, and surgical rates, it is challenging to precisely classify the underlying adrenal incidentaloma pathology from published literature.2Figure 1 provides a simplified description of the adrenal gland components, their role, and the consequences of excess hormone production. Fig 1 The components of the adrenal gland and consequences of excess hormone production Prevalence is estimated between ~2% (range 1.0%-8.7%) from autopsy studies3 …

Aims/hypothesis Pre-eclampsia is a pregnancy-specific multisystem disorder and a state of physiological insulin resistance. Our aim was to systematically evaluate and quantify the evidence on the relationship between pre-eclampsia and the future risk of diabetes. Methods We conducted a systematic review and meta-analysis of studies that evaluated diabetes in women with and without pre-eclampsia. We performed a systematic search of MEDLINE and EMBASE to identify relevant studies. Independent double data extractions were conducted by four reviewers. Random-effects meta-analysis was used to estimate the risk of future diabetes following pre-eclampsia. Results A total of 21 studies were identified with more than 2.8 million women, including more than 72,500 women with pre-eclampsia. Meta-analysis of studies that adjusted for potential confounders demonstrated that pre-eclampsia was independently associated with an increased risk of future diabetes (RR 2.37 [95% CI 1.89, 2.97]). This risk appeared in studies that followed women from less than 1 year postpartum (RR 1.97 [95% CI 1.35, 2.87]) and persisted to more than 10 years postpartum (RR 1.95 [95% CI 1.28, 2.97]). After adjusting for BMI or gestational diabetes, pre-eclampsia remained linked with an increased risk of future diabetes (RR 2.38 [95% CI 1.74, 3.24] and RR 2.36 [95% CI 1.94, 2.88], respectively). Conclusions/interpretation Pre-eclampsia is independently associated with a twofold increase in future diabetes. Our study highlights the importance of clinical risk assessment for the future development of diabetes in women with pre-eclampsia. We recommend detailed evaluation of a screening programme for diabetes in this high-risk population. Electronic supplementary material The online version of this article (doi:10.1007/s00125-016-4098-x) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Introduction:\ud Inappropriate DNA methylation is frequently associated with human tumour development, and in specific cases, is associated with clinical outcomes. Previous reports of DNA methylation in low/intermediate grade non-muscle invasive bladder cancer (NMIBC) have suggested that specific patterns of DNA methylation may have a role as diagnostic or prognostic biomarkers. In view of the aggressive and clinically unpredictable nature of high-grade (HG) NMIBC, and the current shortage of the preferred treatment option (Bacillus:Calmette-Guerin), novel methylation analyses may similarly reveal biomarkers of disease outcome that could risk-stratify patients and guide clinical management at initial diagnosis.\ud \ud Methods:\ud Promoter-associated CpG island methylation was determined in primary tumour tissue of 36 initial presentation high-grade NMIBCs, 12 low/intermediate-grade NMIBCs and 3 normal bladder controls. The genes HOXA9, ISL1, NKX6-2, SPAG6, ZIC1 and ZNF154 were selected for investigation on the basis of previous reports and/or prognostic utility in low/intermediate-grade NMIBC. Methylation was determined by Pyrosequencing of sodium-bisulphite converted DNA, and then correlated with gene expression using RT-qPCR. Methylation was additionally correlated with tumour behaviour, including tumour recurrence and progression to muscle invasive bladder cancer or metastases.\ud \ud Results:\ud The ISL1 genes' promoter-associated island was more frequently methylated in recurrent and progressive high-grade tumours than their non-recurrent counterparts (60.0% vs. 18.2%, p = 0.008). ISL1 and HOXA9 showed significantly higher mean methylation in recurrent and progressive tumours compared to non-recurrent tumours (43.3% vs. 20.9%, p = 0.016 and 34.5% vs 17.6%, p = 0.017, respectively). Concurrent ISL1/HOXA9 methylation in HG-NMIBC reliably predicted tumour recurrence and progression within one year (Positive Predictive Value 91.7%), and was associated with disease-specific mortality (DSM).\ud \ud Conclusions:\ud In this study we report methylation differences and similarities between clinical sub-types of high-grade NMIBC. We report the potential ability of methylation biomarkers, at initial diagnosis, to predict tumour recurrence and progression within one year of diagnosis. We found that specific biomarkers reliably predict disease outcome and therefore may help guide patient treatment despite the unpredictable clinical course and heterogeneity of high-grade NMIBC. Further investigation is required, including validation in a larger patient cohort, to confirm the clinical utility of methylation biomarkers in high-grade NMIBC.

Aims: Alterations in DNA methylation contribute to the abnormal phenotype of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). Herein, we performed genome-wide DNA methylation profiling of RA FLS derived from synovial fluid, a more readily accessible source of disease-associated cells. Patients & Methods: Synovial fluid-derived FLS were isolated and expanded in vitro from 12 patients with RA and osteoarthritis (OA). DNA methylation was interrogated using HumanMethylation450 BeadChips and bisulfite pyrosequencing. Results: Array analysis identified 328 CpGs, representing 195 genes, that were differentially methylated between RA and OA fluid-derived FLS. Comparison with the genes identified in two independent studies of tissue-derived FLS revealed 73 genes in common (~40%), of which 22 shared identity with both studies. Pyrosequencing confirmed altered methylation of these genes. Conclusions: Synovial fluid-derived RA FLS show methylation changes across multiple genes. A subset of these are common with tissue-derived FLS, supporting the use of fluid-derived FLS for future investigations.

Multiple reports now describe changes to the DNA methylome in rheumatoid arthritis and in many cases have analyzed methylation in mixed cell populations from whole blood. However, these approaches may preclude the identification of cell type-specific methylation, which may subsequently bias identification of disease-specific changes. To address this possibility, we conducted genome-wide DNA methylation profiling using HumanMethylation450 BeadChips to identify differences within matched pairs of T-lymphocytes and B-lymphocytes isolated from the peripheral blood of 10 healthy females. Array data were processed and differential methylation identified using NIMBL software. Validation of array data was performed by bisulfite pyrosequencing. Genome-wide DNA methylation was initially determined by analysis of LINE-1 sequences and was higher in B-lymphocytes than matched T-lymphocytes (69.8% vs. 65.2%, P ≤ 0.01). Pairwise analysis identified 679 CpGs, representing 250 genes, which were differentially methylated between T-lymphocytes and B-lymphocytes. The majority of sites (76.6%) were hypermethylated in B-lymphocytes. Pyrosequencing of selected candidates confirmed the array data in all cases. Hierarchical clustering revealed perfect segregation of samples into two distinct clusters based on cell type. Differentially methylated genes showed enrichment for biological functions/pathways associated with leukocytes and T-lymphocytes. Our work for the first time shows that T-lymphocytes and B-lymphocytes possess intrinsic differences in DNA methylation within a restricted set of functionally related genes. These data provide a foundation for investigating DNA methylation in diseases in which these cell types play important and distinct roles.

We investigated the association of developmental dysplasia of the hip (DDH) and primary protrusion acetabuli (PPA) with Vitamin D receptor polymorphisms Taq I and Fok I and oestrogen receptor polymorphisms Pvu II and Xba I. 45 patients with DDH and 20 patients with PPA were included in the study. Healthy controls (n = 101) aged 18–60 years were recruited from the same geographical area. The control subjects had a normal acetabular morphology based on a recent pelvic radiograph performed for an unrelated cause. DNA was obtained from all the subjects from peripheral blood. Genotype frequencies were compared in the three groups. The relationship between the genotype and morphology of the hip joint, severity of the disease, age at onset of disease and gender were examined. The oestrogen receptor Xba I wild-type genotype (XX, compared with Xx and xx combined) was more common in the DDH group (55.8%) than controls (37.9%), though this just failed to achieve statistical significance (p = 0.053, odds ratio = 2.1, 95% CI = 0.9–4.6). In the DDH group, homozygosity for the mutant Taq I Vitamin D receptor t allele was associated with higher acetabular index (Mann-Whitney U-test, p = 0.03). Pvu II pp oestrogen receptor genotype was associated with low centre edge angle (p = 0.07). This study suggests a possible correlation between gene polymorphism in the oestrogen and vitamin D receptors and susceptibility to, and severity of DDH. The Taq I vitamin D receptor polymorphisms may be associated with abnormal acetabular morphology leading to DDH while the Xba I oestrogen receptor XX genotype may be associated with increased risk of developing DDH. No such correlations were found in the group with PPA.

Background: The relationship between asthma severity and atopy is complex. Many studies have failed to show significant relationships between clinical severity or lung function and markers of atopic sensitisation. Aim: To determine whether increasing asthma severity is related to atopic sensitisation in a population of children with asthma. Methods: A total of 400 children (7–18 years) with asthma were recruited as part of a multicentre study of the genetics of asthma. Detailed phenotypic data were collected on all participants. Associations between measures of asthma severity and atopic sensitisation were sought using multilevel models allowing variation at the individual and family level. Results: Children recruited to the study had a range of asthma severities, with just over a third having mild persistent asthma. The logarithm of total serum IgE was associated with increased asthma severity score, decreased FEV 1 , increased airways obstruction, risk of hospital admission, and inhaled steroid use. Increasing skin prick test reactivity to a panel of seven aeroallergens was associated with increased risk of hospital admission, use of an inhaled steroid, and airways obstruction. The results remained highly significant after corrections for age, gender, and birth order. Conclusions: In children with asthma, increasing atopy is associated with increasing asthma severity. However, the relationships between asthma severity and skin prick tests, and asthma severity and total serum IgE values, appear subtly different.

Recent reports, primarily in patients with secondary progressive and relapsing-remitting multiple sclerosis have suggested that the apolipoprotein (APOE) e4 allele may be associated with a worse prognosis.1-3 The e class of APOE is involved in lipid transport and plays a part in brain development, synapse repair, and response to neuronal injury. It has been postulated that patients possessing the e4 allele might have a reduced capacity for neuronal remodelling after multiple sclerosis relapses.3 There is general agreement that patients with primary progressive multiple sclerosis have a worse outcome both in relation to development of disability over time and in mortality. Such patients do not have a relapsing course. Moreover, there are pathological and imaging differences between primary progressive and secondary progressive multiple sclerosis suggesting that primary progressive disease may be a less inflammatory form of the disease.4 The relation between APOE and outcome has not been specifically considered in patients with primary progressive multiple sclerosis, although 11 patients with …

OBJECTIVE—To determine whether glutathione S-transferase GSTM1, GSTM3, GSTT1, and GSTP1 genotypes influence susceptibility or outcome in rheumatoid arthritis (RA). METHODS—277 RA patients were compared with 577 controls to examine any associations between GST genotypes and susceptibility to RA. The effect of genotypes on outcome (Larsen and functional scores) and time integrated acute phase responses (erythrocyte sedimentation rate and C reactive protein) was assessed in 122 patients with disease duration of 5-10 years. GST and HLA-DRB1 genotypes were determined using polymerase chain reaction based assays. Data were analysed using multiple regression analysis with correction for age, sex, disease duration, and the DRB1 associated shared epitope (SE) and rheumatoid factor (RF) positivity where appropriate. RESULTS—The GSTM1*A/*B genotype was less common in RA cases (3 of 276) than in controls (22 of 591) (exact p=0.047), though significance was lost when adjustment was made for multiple comparisons. The Larsen score was higher (p=0.039) in the GSTM1 null patients (89.9) than those with other GSTM1 genotypes (74.7), and this was independent of the SE. Again, correction for multiple testing resulted in loss of significance. The difference in Larsen scores between patients homozygous or negative for the SE (87.9 v 74.3) was similar to that between GSTM1 null and non-null patients. No associations between GSTM3 or GSTT1 genotypes and disease markers were identified although the association between GSTP1*B/*B and Larsen score approached significance (p=0.096). CONCLUSION—It is proposed that certain GSTs may influence susceptibility and radiological progression in RA and that this is independent of the effect of the HLA-DRB1 associated SE. The mechanism for this effect is presumed to be because of differences in the ability of various GST enzymes to utilise the cytotoxic products of oxidant stress. Although significance was lost after correction for multiple testing, the data indicate that further studies may be of value in RA to determine the influence of the GST and other genes involved in cellular protection against oxidative stress. Keywords: rheumatoid arthritis; glutathione S-transferase; polymorphism; shared epitope

Allelism in the glutathione S-transferase, GSTM3 gene has been identified using PCR with specific primers to exon 6/exon 7. Sequencing showed the mutant GSTM3*B allele to have a three-base deletion in intron 6 with a frequency of 0.158. The mutation generates a recognition sequence, 5′-AAGATA-3′, for the negative transcription factor YY1. GSTM3*B was significantly associated with GSTM1*A.

We describe the identification of the GSTM1 null, GSTM1 A, GSTM1 B and GSTM1 A,B polymorphisms at the glutathione S-transferase GSTM1 locus using a single-step PCR method. Target DNA was amplified using primers to intron 6 and exon 7 with site-directed mutagenesis being used to introduce a restriction site in DNA amplified from GSTM1 *A, thereby allowing differentiation of this allele and GSTM1 *B. The accuracy of this approach in identifying the GSTM1 A, GSTM1 B, GSTM1 A,B and GSTM1 null polymorphisms was confirmed by comparison with, firstly, an established PCR method that distinguishes GSTM1 *0 homozygotes from individuals with the other GSTM1 genotypes and, secondly, GSTM1 phenotypes determined using chromatofocusing.

Studies were carried out to test the hypothesis that the GSTM1 null phenotype at the mu (mu) class glutathione S-transferase 1 locus is associated with an increased predisposition to primary biliary cirrhosis. Starch gel electrophoresis was used to compare the prevalence of GSTM1 null phenotype 0 in patients with end stage primary biliary cirrhosis and a group of controls without evidence of liver disease. The prevalence of GSTM1 null phenotype in the primary biliary cirrhosis and control groups was similar; 39% and 45% respectively. In the primary biliary cirrhosis group all subjects were of the common GSTM1 0, GSTM1 A, GSTM1 B or GSTM1 A, B phenotypes while in the controls, one subject showed an isoform with an anodal mobility compatible with it being a product of the putative GSTM1*3 allele. As the GSTM1 phenotype might be changed by the disease process, the polymerase chain reaction was used to amplify the exon 4-exon 5 region of GSTM1 and show that in 13 control subjects and 11 patients with primary biliary cirrhosis, GSTM1 positive and negative genotypes were associated with corresponding GSTM1 expressing and non-expressing phenotypes respectively. The control subject with GSTM1 3 phenotype showed a positive genotype.

Maternal factors including atopy and smoking during pregnancy are associated with asthma risk during childhood. Suggested mechanisms include transmission of specific maternal alleles and maternal influences on the intrauterine environment. We have previously shown that polymorphism in glutathione S -transferase, GSTP1 is associated with airway hyperresponsiveness (AHR) and atopy in adults. We now hypothesise that GSTP1 genotypes in the mother and child, but not the father, mediate asthma phenotypes in the child. One hundred and forty-five Caucasian families were recruited via an asthmatic proband aged 7–18 years. Atopy and asthma were assessed using a questionnaire, skin prick testing, serum IgE, spirometry and methacholine challenge (PC 20 , dose–response slope—DRS). GSTP1 genotyping was determined using PCR. GSTP1 Val 105 /Val 105 genotype in the child was associated with a reduced risk of atopy ( P =0.038) and AHR (PC 20 , P =0.046; DRS, P =0.032). In mothers ( P =0.014) but not fathers ( P =0.623), Val 105 /Val 105 was associated with a reduced risk of AHR in the child. We have identified, for the first time, an association between maternal genotype and the child's asthma phenotype that appears not to be due to transmission of specific maternal alleles. This preliminary data supports the view of in utero effects of maternal genotype and adds new insights into the possible mechanisms by which maternal factors may influence development of childhood asthma.

Allelic variation at the melanocyte stimulating hormone receptor (MC1R) gene has been linked with sun-sensitive skin types, suggesting it is a susceptibility candidate for melanoma. We determined the frequency of the val92met, asp294his, and asp84glu MC1R alleles in 190 Caucasian controls and 306 melanoma cases and studied their association with skin type and hair color. The percentage of controls with at least one val92met, asp294his, or asp84glu allele was 17.3%, 6.8%, and 3.5%, respectively. Individually, frequencies of the val92met, asp294his, or asp84glu alleles in the controls with skin types 3 and 4 were similar to those with skin types 1 and 2. Trend analysis, however, did identify an association (exact p = 0.048, two-sided test) between skin type and MC1R variants in the group comprising all controls with any one or more of these alleles. There was no association between MC1R alleles and hair color. Allele frequencies were not different in melanoma cases and controls. There were no associations between skin types and the proportion of cases with the asp294his or asp84glu alleles, though the association between skin type and the val92met allele approached significance (exact p = 0.09, two-sided test). Unexpectedly, in the group comprising all cases with one or more variant alleles, the proportion of subjects with variant alleles increased with skin types associated with tanning rather than burning, although trend analysis showed that this association did not quite reach statistical significance (exact p = 0.08, two-sided test). Asp84glu (but not val92met or asp294his) variant alleles were more common in subjects with blonde hair, although the relationship between the asp84glu allele and hair color did not achieve statistical significance (chi(2)3 = 6.16, exact p = 0.10). We interpret the data presented as indicating that polymorphism at MC1R does not appear a major determinant of skin type, at least in terms of these allelic variants. Furthermore, considered alone, these alleles are not susceptibility candidates for malignant melanoma.