Sheffield Children's Hospital

OBJECTIVE: Twin concordance data for rheumatoid arthritis (RA) on their own provide only limited insight into the relative genetic and environmental contribution to the disease. We applied quantitative genetic methods to assess the heritability of RA and to examine for evidence of differences in the genetic contribution according to sex, age, and clinical disease characteristics. METHODS: Data were analyzed from 2 previously published nationwide studies of twins with RA conducted in Finland and the United Kingdom. Heritability was assessed by variance components analysis. Differences in the genetic contribution by sex, age, age at disease onset, and clinical characteristics were examined by stratification. The power of the twin study design to detect these differences was examined through simulation. RESULTS: The heritability of RA was 65% (95% confidence interval [95% CI] 50-77) in the Finnish data and 53% (95% CI 40-65) in the UK data. There was no significant difference in the strength of the genetic contribution according to sex, age, age at onset, or disease severity subgroup. Both study designs had power to detect a contribution of at least 40% from the common family environment, and a difference in the genetic contribution of at least 50% between subgroups. CONCLUSION: Genetic factors have a substantial contribution to RA in the population, accounting for approximately 60% of the variation in liability to disease. Although tempered by power considerations, there is no evidence in these twin data that the overall genetic contribution to RA differs by sex, age, age at disease onset, and disease severity.

BACKGROUND: Patients with permanent neonatal diabetes usually present within the first three months of life and require insulin treatment. In most, the cause is unknown. Because ATP-sensitive potassium (K(ATP)) channels mediate glucose-stimulated insulin secretion from the pancreatic beta cells, we hypothesized that activating mutations in the gene encoding the Kir6.2 subunit of this channel (KCNJ11) cause neonatal diabetes. METHODS: We sequenced the KCNJ11 gene in 29 patients with permanent neonatal diabetes. The insulin secretory response to intravenous glucagon, glucose, and the sulfonylurea tolbutamide was assessed in patients who had mutations in the gene. RESULTS: Six novel, heterozygous missense mutations were identified in 10 of the 29 patients. In two patients the diabetes was familial, and in eight it arose from a spontaneous mutation. Their neonatal diabetes was characterized by ketoacidosis or marked hyperglycemia and was treated with insulin. Patients did not secrete insulin in response to glucose or glucagon but did secrete insulin in response to tolbutamide. Four of the patients also had severe developmental delay and muscle weakness; three of them also had epilepsy and mild dysmorphic features. When the most common mutation in Kir6.2 was coexpressed with sulfonylurea receptor 1 in Xenopus laevis oocytes, the ability of ATP to block mutant K(ATP) channels was greatly reduced. CONCLUSIONS: Heterozygous activating mutations in the gene encoding Kir6.2 cause permanent neonatal diabetes and may also be associated with developmental delay, muscle weakness, and epilepsy. Identification of the genetic cause of permanent neonatal diabetes may facilitate the treatment of this disease with sulfonylureas.

Abstract The genetic make-up of an individual contributes to the susceptibility and response to viral infection. Although environmental, clinical and social factors have a role in the chance of exposure to SARS-CoV-2 and the severity of COVID-19 1,2 , host genetics may also be important. Identifying host-specific genetic factors may reveal biological mechanisms of therapeutic relevance and clarify causal relationships of modifiable environmental risk factors for SARS-CoV-2 infection and outcomes. We formed a global network of researchers to investigate the role of human genetics in SARS-CoV-2 infection and COVID-19 severity. Here we describe the results of three genome-wide association meta-analyses that consist of up to 49,562 patients with COVID-19 from 46 studies across 19 countries. We report 13 genome-wide significant loci that are associated with SARS-CoV-2 infection or severe manifestations of COVID-19. Several of these loci correspond to previously documented associations to lung or autoimmune and inflammatory diseases 3–7 . They also represent potentially actionable mechanisms in response to infection. Mendelian randomization analyses support a causal role for smoking and body-mass index for severe COVID-19 although not for type II diabetes. The identification of novel host genetic factors associated with COVID-19 was made possible by the community of human genetics researchers coming together to prioritize the sharing of data, results, resources and analytical frameworks. This working model of international collaboration underscores what is possible for future genetic discoveries in emerging pandemics, or indeed for any complex human disease.

Wilson disease is an inherited autosomal recessive disorder of hepatic copper metabolism leading to copper accumulation in hepatocytes and in extrahepatic organs such as the brain and the cornea. Originally Wilson disease was described as a neurodegerative disorder associated with cirrhosis of the liver. Later, Wilson disease was observed in children and adolescents presenting with acute or chronic liver disease without any neurologic symptoms. While diagnosis of neurologic Wilson disease is straightforward, it may be quite difficult in non-neurologic cases. Up to now, no single diagnostic test can exclude or confirm Wilson disease with 100% certainty. In 1993, the gene responsible for Wilson disease was cloned and localized on chromosome 13q14.3 (MIM277900) (1, 2). The Wilson disease gene ATP7B encodes a P-type ATPase. More than 200 disease causing mutations of this gene have been described so far (3). Most of these mutations occur in single families, only a few are more frequent (like H1069Q, 3400delC and 2299insC in Caucasian (4-6) or R778L in Japanese (7), Chinese and Korean patients). Studies of phenotype-genotype relations are hampered by the lack of standard diagnostic criteria and phenotypic classifications. To overcome this problem, a working party discussed these problems in depth at the 8th International Meeting on Wilson disease and Menkes disease in Leipzig/Germany (April 16-18, 2001). After the meeting, a preliminary draft of a consensus report was mailed to all active participants and their comments were incorporated in the final text.

B cell acute lymphoblastic leukemia received lymphodepleting chemotherapy and anti-CD52 serotherapy, followed by a single-dose infusion of UCART19 cells. Molecular remissions were achieved within 28 days in both infants, and UCART19 cells persisted until conditioning ahead of successful allogeneic stem cell transplantation. This bridge-to-transplantation strategy demonstrates the therapeutic potential of gene-editing technology.

The nuclear-encoded Krebs cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDHB, -C and -D), act as tumour suppressors. Germline mutations in FH predispose individuals to leiomyomas and renal cell cancer (HLRCC), whereas mutations in SDH cause paragangliomas and phaeochromocytomas (HPGL). In this study, we have shown that FH-deficient cells and tumours accumulate fumarate and, to a lesser extent, succinate. SDH-deficient tumours principally accumulate succinate. In situ analyses showed that these tumours also have over-expression of hypoxia-inducible factor 1alpha (HIF1alpha), activation of HIF1alphatargets (such as vascular endothelial growth factor) and high microvessel density. We found no evidence of increased reactive oxygen species in our cells. Our data provide in vivo evidence to support the hypothesis that increased succinate and/or fumarate causes stabilization of HIF1alpha a plausible mechanism, inhibition of HIF prolyl hydroxylases, has previously been suggested by in vitro studies. The basic mechanism of tumorigenesis in HPGL and HLRCC is likely to be pseudo-hypoxic drive, just as it is in von Hippel-Lindau syndrome.

Chemokines are small peptides that are potent activators and chemoattractants for leukocyte subpopulations and some nonhemopoietic cells. Their actions are mediated by a family of 7-transmembrane G-protein-coupled receptors, the size of which has grown considerably in recent years and now includes 18 members. Chemokine receptor expression on different cell types and their binding and response to specific chemokines are highly variable. Significant advances have been made in understanding the regulation of chemokine receptor expression and the intracellular signaling mechanisms used in bringing about cell activation. Chemokine receptors have also recently been implicated in several disease states including allergy, psoriasis, atherosclerosis, and malaria. However, most fascinating has been the observation that some of these receptors are used by human immunodeficiency virus type 1 in gaining entry into permissive cells. This review will discuss structural and functional aspects of chemokine receptor biology and will consider the roles these receptors play in inflammation and in infectious diseases.

A collaboration of multidisciplinary experts on the delivery of pharmaceutical aerosols was facilitated by the European Respiratory Society (ERS) and the International Society for Aerosols in Medicine (ISAM), in order to draw up a consensus statement with clear, up-to-date recommendations that enable the pulmonary physician to choose the type of aerosol delivery device that is most suitable for their patient. The focus of the consensus statement is the patient-use aspect of the aerosol delivery devices that are currently available. The subject was divided into different topics, which were in turn assigned to at least two experts. The authors searched the literature according to their own strategies, with no central literature review being performed. To achieve consensus, draft reports and recommendations were reviewed and voted on by the entire panel. Specific recommendations for use of the devices can be found throughout the statement. Healthcare providers should ensure that their patients can and will use these devices correctly. This requires that the clinician: is aware of the devices that are currently available to deliver the prescribed drugs; knows the various techniques that are appropriate for each device; is able to evaluate the patient's inhalation technique to be sure they are using the devices properly; and ensures that the inhalation method is appropriate for each patient.

OBJECTIVE: To develop a North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition (NASPGHAN) and European Society for Pediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) international consensus on the diagnosis and management of gastroesophageal reflux and gastroesophageal reflux disease in the pediatric population. METHODS: An international panel of 9 pediatric gastroenterologists and 2 epidemiologists were selected by both societies, which developed these guidelines based on the Delphi principle. Statements were based on systematic literature searches using the best-available evidence from PubMed, Cumulative Index to Nursing and Allied Health Literature, and bibliographies. The committee convened in face-to-face meetings 3 times. Consensus was achieved for all recommendations through nominal group technique, a structured, quantitative method. Articles were evaluated using the Oxford Centre for Evidence-based Medicine Levels of Evidence. Using the Oxford Grades of Recommendation, the quality of evidence of each of the recommendations made by the committee was determined and is summarized in appendices. RESULTS: More than 600 articles were reviewed for this work. The document provides evidence-based guidelines for the diagnosis and management of gastroesophageal reflux and gastroesophageal reflux disease in the pediatric population. CONCLUSIONS: This document is intended to be used in daily practice for the development of future clinical practice guidelines and as a basis for clinical trials.

BACKGROUND: In depressed patients, low blood levels of eicosapentaenoic acid are seen. We tested the antidepressive effect of ethyl-eicosapentaenoate in these patients. METHODS: We included 70 patients with persistant depression despite ongoing treatment with an adequate dose of a standard antidepressant. Patients were randomized on a double-blind basis to placebo or ethyl-eicosapentaenoate at dosages of 1, 2, or 4 g/d for 12 weeks in addition to unchanged background medication. Patients underwent assessment using the 17-item Hamilton Depression Rating Scale, the Montgomery-Asberg Depression Rating Scale, and the Beck Depression Inventory. RESULTS: Forty-six (88%) of 52 patients receiving ethyl-eicosapentaenoate and 14 (78%) of 18 patients receiving placebo completed the 12-week study with no serious adverse events. The 1-g/d group showed a significantly better outcome than the placebo group on all 3 rating scales. In the intention-to-treat group, 5 (29%) of 17 patients receiving placebo and 9 (53%) of 17 patients receiving 1 g/d of ethyl-eicosapentaenoate achieved a 50% reduction on the Hamilton Depression Rating Scale score. In the per-protocol group, the corresponding figures were 3 (25%) of 12 patients for placebo and 9 (69%) of 13 patients for the 1-g/d group. The 2-g/d group showed little evidence of efficacy, whereas the 4-g/d group showed nonsignificant trends toward improvement. All of the individual items on all 3 rating scales improved with the 1-g/d dosage of ethyl-eicosapentaenoate vs placebo, with strong beneficial effects on items rating depression, anxiety, sleep, lassitude, libido, and suicidality. CONCLUSION: Treatment with ethyl-eicosapentaenoate at a dosage of 1 g/d was effective in treating depression in patients who remained depressed despite adequate standard therapy.

BACKGROUND: Hypophosphatasia results from mutations in the gene for the tissue-nonspecific isozyme of alkaline phosphatase (TNSALP). Inorganic pyrophosphate accumulates extracellularly, leading to rickets or osteomalacia. Severely affected babies often die from respiratory insufficiency due to progressive chest deformity or have persistent bone disease. There is no approved medical therapy. ENB-0040 is a bone-targeted, recombinant human TNSALP that prevents the manifestations of hypophosphatasia in Tnsalp knockout mice. METHODS: We enrolled infants and young children with life-threatening or debilitating perinatal or infantile hypophosphatasia in a multinational, open-label study of treatment with ENB-0040. The primary objective was the healing of rickets, as assessed by means of radiographic scales. Motor and cognitive development, respiratory function, and safety were evaluated, as well as the pharmacokinetics and pharmacodynamics of ENB-0040. RESULTS: Of the 11 patients recruited, 10 completed 6 months of therapy; 9 completed 1 year. Healing of rickets at 6 months in 9 patients was accompanied by improvement in developmental milestones and pulmonary function. Elevated plasma levels of the TNSALP substrates inorganic pyrophosphate and pyridoxal 5'-phosphate diminished. Increases in serum parathyroid hormone accompanied skeletal healing, often necessitating dietary calcium supplementation. There was no evidence of hypocalcemia, ectopic calcification, or definite drug-related serious adverse events. Low titers of anti-ENB-0040 antibodies developed in four patients, with no evident clinical, biochemical, or autoimmune abnormalities at 48 weeks of treatment. CONCLUSIONS: ENB-0040, an enzyme-replacement therapy, was associated with improved findings on skeletal radiographs and improved pulmonary and physical function in infants and young children with life-threatening hypophosphatasia. (Funded by Enobia Pharma and Shriners Hospitals for Children; ClinicalTrials.gov number, NCT00744042.).

IMPORTANCE: Limited information about the relationship between specific mutations in BRCA1 or BRCA2 (BRCA1/2) and cancer risk exists. OBJECTIVE: To identify mutation-specific cancer risks for carriers of BRCA1/2. DESIGN, SETTING, AND PARTICIPANTS: Observational study of women who were ascertained between 1937 and 2011 (median, 1999) and found to carry disease-associated BRCA1 or BRCA2 mutations. The international sample comprised 19,581 carriers of BRCA1 mutations and 11,900 carriers of BRCA2 mutations from 55 centers in 33 countries on 6 continents. We estimated hazard ratios for breast and ovarian cancer based on mutation type, function, and nucleotide position. We also estimated RHR, the ratio of breast vs ovarian cancer hazard ratios. A value of RHR greater than 1 indicated elevated breast cancer risk; a value of RHR less than 1 indicated elevated ovarian cancer risk. EXPOSURES: Mutations of BRCA1 or BRCA2. MAIN OUTCOMES AND MEASURES: Breast and ovarian cancer risks. RESULTS: Among BRCA1 mutation carriers, 9052 women (46%) were diagnosed with breast cancer, 2317 (12%) with ovarian cancer, 1041 (5%) with breast and ovarian cancer, and 7171 (37%) without cancer. Among BRCA2 mutation carriers, 6180 women (52%) were diagnosed with breast cancer, 682 (6%) with ovarian cancer, 272 (2%) with breast and ovarian cancer, and 4766 (40%) without cancer. In BRCA1, we identified 3 breast cancer cluster regions (BCCRs) located at c.179 to c.505 (BCCR1; RHR = 1.46; 95% CI, 1.22-1.74; P = 2 × 10(-6)), c.4328 to c.4945 (BCCR2; RHR = 1.34; 95% CI, 1.01-1.78; P = .04), and c. 5261 to c.5563 (BCCR2', RHR = 1.38; 95% CI, 1.22-1.55; P = 6 × 10(-9)). We also identified an ovarian cancer cluster region (OCCR) from c.1380 to c.4062 (approximately exon 11) with RHR = 0.62 (95% CI, 0.56-0.70; P = 9 × 10(-17)). In BRCA2, we observed multiple BCCRs spanning c.1 to c.596 (BCCR1; RHR = 1.71; 95% CI, 1.06-2.78; P = .03), c.772 to c.1806 (BCCR1'; RHR = 1.63; 95% CI, 1.10-2.40; P = .01), and c.7394 to c.8904 (BCCR2; RHR = 2.31; 95% CI, 1.69-3.16; P = .00002). We also identified 3 OCCRs: the first (OCCR1) spanned c.3249 to c.5681 that was adjacent to c.5946delT (6174delT; RHR = 0.51; 95% CI, 0.44-0.60; P = 6 × 10(-17)). The second OCCR spanned c.6645 to c.7471 (OCCR2; RHR = 0.57; 95% CI, 0.41-0.80; P = .001). Mutations conferring nonsense-mediated decay were associated with differential breast or ovarian cancer risks and an earlier age of breast cancer diagnosis for both BRCA1 and BRCA2 mutation carriers. CONCLUSIONS AND RELEVANCE: Breast and ovarian cancer risks varied by type and location of BRCA1/2 mutations. With appropriate validation, these data may have implications for risk assessment and cancer prevention decision making for carriers of BRCA1 and BRCA2 mutations.

BACKGROUND: Minimal residual disease (MRD) is the most sensitive and specific predictor of relapse risk in children with acute lymphoblastic leukaemia (ALL) during remission. We assessed whether treatment intensity could be adjusted for children and young adults according to MRD risk stratification. METHODS: Between Oct 1, 2003 and June 30, 2011, consecutive children and young adults (aged 1-25 years) with ALL from the UK and Ireland were recruited. Eligible patients were categorised into clinical standard, intermediate, and high risk groups on the basis of a combination of National Cancer Institute (NCI) criteria, cytogenetics, and early response to induction therapy, which was assessed by bone marrow blast counts taken at days 8 (NCI high-risk patients) and 15 (NCI standard-risk patients) after induction began. Clinical standard-risk and intermediate-risk patients were assessed for MRD. Those classified as MRD low risk (undetectable MRD at the end of induction [day 29] or detectable MRD at day 29 that became undetectable by week 11) were randomly assigned to receive one or two delayed intensification courses. Patients had received induction, consolidation, and interim maintenance therapy before they began delayed intensification. Delayed intensification consisted of pegylated asparaginase on day 4; vincristine, dexamethasone (alternate weeks), and doxorubicin for 3 weeks; and 4 weeks of cyclophosphamide and cytarabine. Computer randomisation was done with stratification by MRD result and balancing for sex, age, and white blood cell count at diagnosis by method of minimisation. Patients, clinicians, and data analysts were not masked to treatment allocation. The primary outcome was event-free survival (EFS), which was defined as time to relapse, secondary tumour, or death. Our aim was to rule out a 7% reduction in EFS in the group given one delayed intensification course relative to that given two delayed intensification courses. Analyses were by intention to treat. This trial is registered, number ISRCTN07355119. FINDINGS: Of 3207 patients registered in the trial overall, 521 MRD low-risk patients were randomly assigned to receive one (n=260) or two (n=261) delayed intensification courses. Median follow-up of these patients was 57 months (IQR 42-72). We recorded no significant difference in EFS between the group given one delayed intensification (94·4% at 5 years, 95% CI 91·1-97·7) and that given two delayed intensifications (95·5%, 92·8-98·2; unadjusted odds ratio 1·00, 95% CI 0·43-2·31; two-sided p=0·99). The difference in 5-year EFS between the two groups was 1·1% (95% CI -5·6 to 2·5). 11 patients (actuarial relapse at 5 years 5·6%, 95% CI 2·3-8·9) given one delayed intensification and six (2·4%, 0·2-4·6) given two delayed intensifications relapsed (p=0·23). Three patients (1·2%, 0-2·6) given two delayed intensifications died of treatment-related causes compared with none in the group given one delayed intensification (p=0·08). We recorded no significant difference between groups for serious adverse events and grade 3 or 4 toxic effects; however, the second delayed intensification course was associated with one (<1%) treatment-related death, and 74 episodes of grade 3 or 4 toxic effects in 45 patients (17%). INTERPRETATION: Treatment reduction is feasible for children and young adults with ALL who are predicted to have a low risk of relapse on the basis of rapid clearance of MRD by the end of induction therapy. FUNDING: Medical Research Council and Leukaemia and Lymphoma Research.

Abstract Objective : To investigate the role of sleeping arrangements as risk factors for the sudden infant death syndrome after a national risk reduction campaign. Design : Two year population based case-control study. Parental interviews were conducted for each infant who died and for four controls matched for age and date of interview. Setting : Three regions in England with a total population of 17 million people. Subjects : 195 babies who died and 780 matched controls. Results : Prone and side sleeping positions both carried increased risks of death compared with supine when adjusted for maternal age, parity, gestation, birth weight, exposure to smoke, and other relevant factors in the sleeping environment (multivariate odds ratio = 9.00 (95% confidence interval 2.84 to 28.47) and 1.84 (1.02 to 3.31), respectively). The higher incidence of side rather than prone sleeping led to a higher population attributable risk (side 18.4%, prone 14.2%). More of the infants who died were found with bed covers over their heads (21.58; 6.21 to 74.99). The use of a dummy had an apparent protective effect (0.38; 0.21 to 0.70). Bed sharing for the whole night was a significant risk factor for infants whose mothers smoked (9.25; 2.31 to 34.02). No protective effect of breast feeding could be identified on multivariate analysis. Conclusions : This study confirms the importance of certain risk factors for the sudden infant death syndrome and identifies others—for example, covers over the head, side sleeping position—which may be amenable to change by educating and informing parents and health care professionals. Key messages This large case-control study is the first after the national campaign to reduce the risk of the syndrome The risk of sudden infant death is increased by prone or side sleeping position; loose bedding (particularly duvets), which can slip over the baby's head; and bed sharing by mothers who smoke The risk may be reduced by supine sleeping position; placing the baby with feet at the foot of the cot (“feet to foot”); ensuring that bedding is securely tucked in; and avoiding the use of duvets

Asthma is the most common chronic lower respiratory disease in childhood throughout the world. Several guidelines and/or consensus documents are available to support medical decisions on pediatric asthma. Although there is no doubt that the use of common systematic approaches for management can considerably improve outcomes, dissemination and implementation of these are still major challenges. Consequently, the International Collaboration in Asthma, Allergy and Immunology (iCAALL), recently formed by the EAACI, AAAAI, ACAAI, and WAO, has decided to propose an International Consensus on (ICON) Pediatric Asthma. The purpose of this document is to highlight the key messages that are common to many of the existing guidelines, while critically reviewing and commenting on any differences, thus providing a concise reference. The principles of pediatric asthma management are generally accepted. Overall, the treatment goal is disease control. To achieve this, patients and their parents should be educated to optimally manage the disease, in collaboration with healthcare professionals. Identification and avoidance of triggers is also of significant importance. Assessment and monitoring should be performed regularly to re-evaluate and fine-tune treatment. Pharmacotherapy is the cornerstone of treatment. The optimal use of medication can, in most cases, help patients control symptoms and reduce the risk for future morbidity. The management of exacerbations is a major consideration, independent of chronic treatment. There is a trend toward considering phenotype-specific treatment choices; however, this goal has not yet been achieved.

A previous study (LMB89) of the French Society of Pediatric Oncology for childhood mature B-cell lymphoma (B-NHL) demonstrated a 92% 3-year event-free survival (EFS) for intermediate-risk group B defined as "non-resected" stage II/I and CNS-negative advanced-stage IIV/IV (70% of cases). We performed the FAB/LMB96 trial to assess the possibility of reducing treatment in children/adolescents with intermediate-risk B-NHL without jeopardizing survival. "Early responding" patients (tumor response > 20% at day 7) were randomized in a factorial design between 4 arms, 2 receiving half-dose of cyclophosphamide in the second induction course with cyclophosphamide, Oncovin (vincristine), prednisone, Adriamycin (doxorubicin), methotrexate (COPADM) and 2 not receiving the maintenance course M1. A total of 657 patients were randomized (May 1996 to June 2001) and 637 were analyzed. The analysis showed no significant effect of any of the treatment reductions on EFS and survival. The 4-year EFS was 93.4% and 90.9% in the groups with full-dose and half-dose of cyclophosphamide (RR = 1.3, P = .40) and 91.9% and 92.5% in the groups with and without M1 (RR = 1.01, P = .98). There was no interaction between the 2 treatment reductions or between each treatment reduction and LDH level or histologic subtypes (Burkitt/Burkitt-like or large B-cell). Children/adolescents with intermediate-risk B-NHL who have an early response and achieve a complete remission after the first consolidation course can be cured with a 4-course treatment with a total dose of only 3.3 g/m2 cyclophosphamide and 120 mg/m2 doxorubicin.

Functional iron deficiency (FID) is a state in which there is insufficient iron incorporation into erythroid precursors in the face of apparently adequate body iron stores, as defined by the presence of stainable iron in the bone marrow together with a serum ferritin value within normal limits (Macdougall et al, 1989). In its broadest sense this definition encompasses the partial block in iron transport to the erythroid marrow seen in subjects with infectious, inflammatory and malignant diseases, and is a major component of the anaemia of chronic disease (ACD). One form of FID, found in some subjects treated with erythropoiesis-stimulating agents (ESAs), has been the subject of numerous studies following the widespread use of these agents, especially in subjects with chronic kidney disease (CKD). The clinical assessment of iron status has largely been focussed on the level of iron stores, as reflected in the serum ferritin concentration. However iron in stores is metabolically inactive and is not only unavailable for immediate use but may be difficult to bring into use at all. The real clinical issue lies in active iron metabolism, the movement of iron from effete red cells and into further generations of developing red cells. It is nevertheless true that replenishment of iron lost from the red cell pool will be compromised and iron supply to the erythroid marrow will be suboptimal as iron stores become depleted. Irrespective of cause, inadequate iron supply leads to impaired haemoglobin production and a reduction in the mean cell haemoglobin (MCH) value that becomes apparent after several weeks of impairment. In contrast, it has long been evident that the adequacy of iron supply might be estimated from the haemoglobin content of the reticulocyte within a time span of a few days. With the widespread introduction of automated cell counters capable of measuring the numbers, volume and haemoglobin content of reticulocytes, many laboratories are now in a position to detect the early indications of a failure of iron supply in this way. In 2006 the National Institute for Health and Clinical Excellence (NICE) published a guideline entitled, ‘Anaemia management in people with chronic kidney disease (CKD).’ (NICE, 2006). Among tests recommended for the assessment of iron status was the percentage of hypochromic red cells (%HRC). This variable, which continues to be recommended in the updated guideline (guideline 114, NICE, 2011), has limited availability, whilst the reticulocyte measures mentioned above have become more widely available. It is thus timely to review the use of these newer variables, together with more established measures of iron status, in the management of patients with FID. The guideline group was selected to be representative of UK-based experts in the clinical and laboratory fields of iron metabolism, CKD, quality control and method evaluation. MEDLINE was searched systematically for publications in English from 1966-2011 using key words: functional iron deficiency and each of the parameters discussed. The writing group produced the draft guideline, which was subsequently revised by consensus by members of the Task Force of the British Committee for Standards in Haematology (BCSH). The guideline was then reviewed by a sounding board of UK haematologists and members of both the BCSH and the British Society for Haematology. Comments were incorporated where appropriate. The ‘GRADE’ system was used to quote levels and grades of evidence, details of which can be found in Appendix 1. The object of this guideline is to provide healthcare professionals with clear guidance on the management of FID, in particular with respect to patients with CKD but also to other disease states in which ESAs have been used. The guidance may not be appropriate to patients with inflammatory diseases and in all cases individual patient circumstances may dictate an alternative approach. This guideline is only applicable to adults, not children. The laboratory classification of iron status breaks down in the presence of inflammatory disease, as most of the variables used to define iron deficiency become abnormal despite the presence of adequate body iron reserves. ACD typically develops, a consistent feature of which is the retention of iron within body stores. As a result the supply of iron to the erythroid marrow becomes inadequate. This is the major form of FID; a second type often occurs when the erythroid marrow is stimulated by ESAs. Since the discovery of the iron regulatory peptide, hepcidin, a 25-amino acid peptide synthesized in the liver, our understanding of the biology of ACD has greatly improved (Goodnough et al, 2011). Hepcidin is upregulated in the setting of chronic inflammation and cancer, resulting in its increased synthesis in the liver stimulated by cytokines of which interleukin (IL) 6 is the most important. By degrading ferroportin, hepcidin decreases iron absorption from the gastrointestinal tract and decreases the accessibility of stored iron from macrophages. Where FID and inflammatory illness coexist it is likely that increased hepcidin synthesis will restrict the absorption of oral iron. Intravenous iron preparations might overcome this block. In patients treated with ESAs for the anaemia associated with CKD, the response rate improves when intravenous rather than oral iron supplements are given, and often allows a reduction in the ESA dose (Nemeth et al, 2004; van Wyck et al, 2004; Henry, 2010; Qunibi et al, 2010). Intravenous iron is routinely used in ESA-treated patients with CKD on dialysis and this practice is endorsed in national and international guidelines (NICE, 2011; National Kidney Foundation, 2006; Kidney Disease: Improving Global Outcomes (KDIGO) Anemia Work Group 2012). There are now a considerable number of red cell indices available for the assessment of iron status. Used in isolation as a diagnostic test, none is capable of differentiating between iron deficiency and FID. All are confounded by α°- and β-thalassaemia heterozygosity and in homozygotes and some heterozygotes for α+ thalassaemia. Patients with combined iron and vitamin B12/folate deficiencies or sideroblastic anaemia may also prove problematic. Once a diagnosis has been made, however, some of these variables may be used to monitor the response to ESA therapy and the requirement for iron. These indices can be divided into five groups; the traditional measures of MCV, MCH and mean cell haemoglobin concentration (MCHC): measures based on increased hypochromia: indices of reticulocyte volume and Hb content: red cell zinc protoporphyrin (ZPP) concentration: and recently introduced indices, such as red cell size factor (Rsf). The MCH is derived from the red blood cell (RBC) count and haemoglobin concentration (Hb), both of which are measured with considerable accuracy and precision by modern analysers. Values obtained from differing types of analyser are therefore largely interchangeable whereas MCV values, which are derived using differing analytical principles, are much less so. Unlike MCV, MCH is unaffected by several days of storage. As both MCV and MCH are derived from the entire circulating red cell mass, they are slow to change and have no value in detecting either the acute development of iron lack or the early response to iron therapy in patients treated with ESAs. The MCHC is of limited or no use in assessing changes in iron availability associated with ESA therapy. As originally identified using Siemens technology, hypochromic red cells are those with Hb <280 g/l. The clinical utility of this variable in detecting FID in patients with chronic renal failure treated with ESAs has long been recognized (Macdougall et al, 1992). A value of ≥6% was found to be superior to measurements of soluble transferrin receptor (sTfR), ZPP, ferritin and total iron-binding capacity (TIBC) in differentiating between iron-deficient and iron-sufficient patients with chronic renal failure receiving maintenance doses of ESAs (Tessitore et al, 2001). This value was incorporated into UK guidelines on anaemia management in this group (NICE, 2006). The measure is effective in the detection and monitoring of FID secondary to ESA therapy in anaemic subjects with advanced acquired immunodeficiency syndrome (Matzkies et al, 1999) and rheumatoid arthritis (Arndt et al, 2005). An increase in %HRC in subjects with low-risk myelodysplastic syndromes treated with ESAs may however reflect improved survival of a pre-existing population of abnormal hypochromic red cells, rather than ESA-induced FID (Ljung et al, 2004). Two other variables are now available for the assessment of hypochromia. One, %Hypo-He, is produced by some Sysmex blood counter analysers (XE 5000 and XN), and defines those red cells having MCH <17 pg. The measure has clinical utility in the differential diagnosis of anaemia (Urrechaga et al, 2009). The second, Low Haemoglobin Density (LHD%), is a variable based on a mathematical transformation of the MCHC value and is available on some Beckman Coulter instruments. Values correlate highly with those of %HRC (Urrechaga, 2010). The great increase in precision of the automated reticulocyte count and the provision of measures of immature reticulocyte fraction and the reticulocyte-specific indices of volume and haemoglobin content provide an opportunity to assess the effects of changing iron status on this transient population. The reticulocyte count itself cannot provide information about a patient's iron status. However a reticulocyte increase of ≥40 × 109/l from baseline by week four of ESA therapy in cancer patients has been shown to predict an adequate response, defined by a ≥ 6% rise in haematocrit above baseline, and it implies adequate iron stores (Henry et al, 1995). Automated blood counters capable of producing reticulocyte data generally group cells depending on RNA content. The immature reticulocyte fraction (IRF) is the component with highest RNA content. Immature reticulocytes are released during periods of intense erythropoietic stimulation, such as following haemorrhage or haemolysis, or in response to therapy with iron or ESAs. The IRF increases several days before the reticulocyte count (Davies, 1996) and is thus an early indicator of response to therapy. Nevertheless, the test is little used, probably because of a lack of standardization of methods and instrument- or method-specific reference ranges. CHr, the term used to describe the reticulocyte MCH as derived by Siemens analysers, was the first automated reticulocyte measure available for routine use. Among patients undergoing bone marrow examination for diagnostic purposes CHr had a better predictive value for iron depletion than MCV, serum ferritin or transferrin saturation values (Mast et al, 2002). CHr has been used as the standard against which other emerging variables have been assessed (Brugnara et al, 2006). Among subjects with presumed FID, CHr compared favourably with other measures of iron status in predicting a response to intravenous iron (Mittman et al, 1997; Chuang et al, 2003). The variable received US Federal Drug Administration approval in 1997 and was incorporated into the revised European Best Practice Guidelines for the management of patients with chronic renal failure (Locatelli et al, 2004). A target CHr of 29 pg was recommended (evidence level B). This value is indicative of the adequacy of iron incorporation into the developing erythron, although some patients with CHr values >29 pg responded to intravenous iron therapy, leading to a suggested cut-off value of 32 pg (Fishbane et al, 2001). In a study of sample stability, a small but statistically insignificant fall in CHr values over 24 h was demonstrated (Lippi et al, 2005). An alternative measure of reticulocyte haemoglobin content, Ret-He, is available on some analysers manufactured by the Sysmex Corporation. Although expressed in the standard unit of cellular haemoglobin content, (pg), Ret-He is a natural log transformation of Ret-Y, a measure of volume obtained from measurement of forward light scatter of reticulocytes and itself expressed in arbitrary units. A number of studies (Canals et al, 2005; Thomas et al, 2005; Brugnara et al, 2006; Garzia et al, 2007; Maconi et al, 2009; Miwa et al, 2010) have found excellent concordance between Ret-He and CHr in subjects with both iron deficiency and chronic renal failure. However Brugnara (2003) has reported that it is less clear that either low Ret-He or CHr values are predictive of response to intravenous iron or whether ESA usage can thus be reduced to the minimum required (Mast et al, 2008). Canals et al (2005) studied 504 patients with ACD or other iron-restricted states. Ret-He alone was able to distinguish iron-deficient and iron-sufficient subjects using a cut-off of 25 pg with reasonable sensitivity (0·76). However the groups that included ACD, mild iron deficiency anaemia and reduced iron stores showed significant overlap. The interquartile range for the ACD group in this study was below that of the reference range and the values of the ACD group were significantly different from those of the iron deficiency group. Although less sensitive (80% agreement with sTfR and sTfR/log ferritin values), a Ret-He cut-off of 25 pg may also to distinguish iron deficiency from ACD A Ret-He cut-off value of pg is a better of response to intravenous iron than baseline serum ferritin or transferrin saturation values in CKD patients undergoing et al, 2010). This variable is derived from the of the of the of and It with CHr, with better sensitivity and for the detection of iron-restricted Patients with values were more likely to have ACD, those with values to have iron deficiency (Urrechaga, 2009). protoporphyrin (ZPP) is a of synthesis and that limits iron supply to the erythroid marrow or synthesis leads to an increased concentration of in circulating red cells. The incorporation of iron into protoporphyrin is the of and an increase in is an indicator of at this The measure is therefore and values in iron FID, and in many iron-sufficient subjects with α°- and β-thalassaemia et al, There are of the measurement of by the used method of to the of the with found in the presence of et al, and in chronic renal failure a and increase in values is seen as Hb below about and many subjects with or anaemia have values of iron status. These can be overcome by to or the Hb concentration to a standard but sample is to lack of not be used in isolation as a diagnostic test, but a diagnosis is it may be used to monitor response to therapy. The concentration the entire circulating red cell population and is thus less sensitive than %HRC or CHr to acute changes in iron availability (Fishbane of body iron stores is both as a diagnostic and to monitor the effects of therapy with iron ESAs. This may be by of the iron content in bone or by use of the serum ferritin studies using iron have been from as they are these days and can be An assessment of iron stores in the bone marrow can be by use of Although a test, assessment can be insufficient is or more be available for review et al, and few haematologists can they this number on was a major factor in a study that that over of of of stainable iron were et al, 2001). the presence of stainable iron not define much can be and incorporated into the developing bone marrow examination is and not such as and The serum ferritin has become the standard test for the assessment of iron stores in serum from from or and in there is a between the such that each of ferritin in serum is to of iron in stores variables may this and the level of stored iron in some the it is derived are of the of serum ferritin but the by which this variable from the true measure of the iron stores is less often the ferritin value cannot in CKD, is to when stores to supply the term implies the patient will not to iron have it may be to an of serum ferritin concentration to provide a et al, such that it may be to intravenous and some CKD patients may to this therapy despite ferritin There is also to that CKD patients with low serum ferritin have a compared to patients with values in the range et al, 2005). some patients with CKD may have iron in the liver and within the bone marrow available for et al, the setting of an of serum ferritin concentration above which intravenous iron is not is not guidelines use a value of (NICE, or range National Kidney Foundation, for CKD patients on ESA therapy, that above these values there is a of iron with further therapy. However anaemic CKD patients with ferritin of showed an increase in Hb when treated with intravenous iron et al, These patients all had transferrin saturation levels to the presence of FID. The indicator of in this group was the response to intravenous iron. The serum ferritin concentration was in predicting response to ESA therapy in anaemia et al, 2003). The transferrin receptor is highly expressed on erythroid levels are found in associated with an erythroid marrow et al, et al, and also in iron The clinical utility of sTfR measurement has been by the lack of agreement the both of and of used to of the recently first Health for sTfR this et al, 2010). The major clinical of the is in differentiating the anaemia of iron lack from that by which has little or no on sTfR values, and in detecting the presence of iron lack when the coexist et al, et al Although in some studies of sTfR not prove superior to the serum ferritin in the detection of iron deficiency in patient groups of those seen in clinical practice (Mast et al, et al, et al, a review that its use improves the diagnosis of iron especially in the presence of chronic disease or gastrointestinal et al, 2009). In patients with chronic renal failure and kidney disease not receiving iron or the sTfR concentration alone to that of serum ferritin in detecting those with iron deficiency et al, However in a group of patients receiving maintenance doses of and with the measure superior to ZPP, transferrin saturation and serum but to %HRC and CHr, in differentiating between iron-deficient and subjects (Tessitore et al, 2001). The a of the ferritin concentration to that of sTfR et al, was found to be superior to ferritin alone in predicting response to intravenous iron in renal patients on ESA therapy et al, 2006). This to overcome to the use of sTfR as a test in monitoring iron status during ESA therapy, that of the therapy itself leading to increased of the erythroid marrow et al, 2002). The has been used, with a measure of iron availability to the erythroid such as %HRC or CHr, in the production of a diagnostic 2002). can be used to monitor the effects or with iron or ESAs et al, 2006). The value is derived from serum iron and values and is the most widely used of the The serum iron concentration within of the of inflammatory illness and also but to a This in reduced values that for the of the Although a measure of iron in transport and not of iron in stores, values of the for iron in the setting of anaemia treated with ESAs (Macdougall et al, Used in has sensitivity and in detecting those to intravenous iron et al, 1997; et al, although with variable improved accuracy and may be a alternative where and reticulocyte variables are not available. levels are not routinely in the setting of CKD and are not recommended in patients with anaemia and CKD Kidney Foundation, 2006). is clear is that for patients with the of CKD with anaemia there is a lack of serum However low levels have limited clinical value that some and are associated with of levels et al, et al, In a study by et al patients with and baseline levels were most likely to to ESA therapy. It to be seen whether such to ESA therapy to FID in cancer but with iron to response 2010). hepcidin has as the of iron availability to the bone marrow for its measurement in serum or have improved and this has that hepcidin might be a superior alternative to traditional of iron status. the that have been and (Macdougall et al, 2010). The of is that they are to are and to is that the used with both the inactive and thus the true level (Macdougall et al, 2010). This is when the is used to measure changes over with or but is less hepcidin values are are more detecting only but they are and levels of hepcidin are in acute and chronic inflammatory such as rheumatoid inflammatory disease and CKD, and are in iron such as however, may hepcidin and it is not clear whether this has in iron status or in the for iron. in a study of hepcidin measurement was to %HRC in predicting the response to intravenous iron (Tessitore et al, 2010). The hepcidin level in CKD patients may not be of diagnostic value than the ferritin but further studies are 2011; et al, 2012). there are no UK for hepcidin There is also a lack of of reference values the different The failure of adequate iron incorporation into the developing is component of ACD and With these the of and factor a of the erythron, early erythroid cell and reduced levels of both hepcidin and have the of iron to developing It is therefore difficult to predict the response to intravenous iron therapy in these this however there is to that ACD patients with of FID from iron et al, 2005). The development of FID in anaemic cancer patients the response to ESA therapy iron is et al, 1992). iron therapy has been the of for patients with iron Intravenous iron is and effective and in patients with ACD is superior to oral iron when used in with ESAs. patients with chronic inflammatory diseases or cancer with ESAs be of the development of FID. the variables above to therapy appropriate is less clear than for an of an for use in CKD quality control and for all reported variables be available. This is the for traditional variables such as MCV, MCH and reticulocyte count for newer variables used to detect or monitor FID and is not available and in some is the and information in these guidelines is to be true and at the time of to the the British Society for Haematology the for the content of these are when there is or not and can be to most as Where the of or not is less a is to individual as

OBJECTIVE: Age at onset of diagnostic motor manifestations in Huntington disease (HD) is strongly correlated with an expanded CAG trinucleotide repeat. The length of the normal CAG repeat allele has been reported also to influence age at onset, in interaction with the expanded allele. Due to profound implications for disease mechanism and modification, we tested whether the normal allele, interaction between the expanded and normal alleles, or presence of a second expanded allele affects age at onset of HD motor signs. METHODS: We modeled natural log-transformed age at onset as a function of CAG repeat lengths of expanded and normal alleles and their interaction by linear regression. RESULTS: An apparently significant effect of interaction on age at motor onset among 4,068 subjects was dependent on a single outlier data point. A rigorous statistical analysis with a well-behaved dataset that conformed to the fundamental assumptions of linear regression (e.g., constant variance and normally distributed error) revealed significance only for the expanded CAG repeat, with no effect of the normal CAG repeat. Ten subjects with 2 expanded alleles showed an age at motor onset consistent with the length of the larger expanded allele. CONCLUSIONS: Normal allele CAG length, interaction between expanded and normal alleles, and presence of a second expanded allele do not influence age at onset of motor manifestations, indicating that the rate of HD pathogenesis leading to motor diagnosis is determined by a completely dominant action of the longest expanded allele and as yet unidentified genetic or environmental factors.

BRCA1-associated breast and ovarian cancer risks can be modified by common genetic variants. To identify further cancer risk-modifying loci, we performed a multi-stage GWAS of 11,705 BRCA1 carriers (of whom 5,920 were diagnosed with breast and 1,839 were diagnosed with ovarian cancer), with a further replication in an additional sample of 2,646 BRCA1 carriers. We identified a novel breast cancer risk modifier locus at 1q32 for BRCA1 carriers (rs2290854, P = 2.7 × 10(-8), HR = 1.14, 95% CI: 1.09-1.20). In addition, we identified two novel ovarian cancer risk modifier loci: 17q21.31 (rs17631303, P = 1.4 × 10(-8), HR = 1.27, 95% CI: 1.17-1.38) and 4q32.3 (rs4691139, P = 3.4 × 10(-8), HR = 1.20, 95% CI: 1.17-1.38). The 4q32.3 locus was not associated with ovarian cancer risk in the general population or BRCA2 carriers, suggesting a BRCA1-specific association. The 17q21.31 locus was also associated with ovarian cancer risk in 8,211 BRCA2 carriers (P = 2×10(-4)). These loci may lead to an improved understanding of the etiology of breast and ovarian tumors in BRCA1 carriers. Based on the joint distribution of the known BRCA1 breast cancer risk-modifying loci, we estimated that the breast cancer lifetime risks for the 5% of BRCA1 carriers at lowest risk are 28%-50% compared to 81%-100% for the 5% at highest risk. Similarly, based on the known ovarian cancer risk-modifying loci, the 5% of BRCA1 carriers at lowest risk have an estimated lifetime risk of developing ovarian cancer of 28% or lower, whereas the 5% at highest risk will have a risk of 63% or higher. Such differences in risk may have important implications for risk prediction and clinical management for BRCA1 carriers.

BACKGROUND: Measurement of lung volumes across the life course is critical to the diagnosis and management of lung disease. The aim of the study was to use the Global Lung Function Initiative methodology to develop all-age multi-ethnic reference equations for lung volume indices determined using body plethysmography and gas dilution techniques. METHODS: Static lung volume data from body plethysmography and gas dilution techniques from individual, healthy participants were collated. Reference equations were derived using the LMS (lambda-mu-sigma) method and the generalised additive models of location shape and scale programme in R. The impact of measurement technique, equipment type and being overweight or obese on the derived lung volume reference ranges was assessed. RESULTS: Data from 17 centres were submitted and reference equations were derived from 7190 observations from participants of European ancestry between the ages of 5 and 80 years. Data from non-European ancestry populations were insufficient to develop multi-ethnic equations. Measurements of functional residual capacity (FRC) collected using plethysmography and dilution techniques showed physiologically insignificant differences and were combined. Sex-specific reference equations including height and age were developed for total lung capacity (TLC), FRC, residual volume (RV), inspiratory capacity, vital capacity, expiratory reserve volume and RV/TLC. The derived equations were similar to previously published equations for FRC and TLC, with closer agreement during childhood and adolescence than in adulthood. CONCLUSIONS: Global Lung Function Initiative reference equations for lung volumes provide a generalisable standard for reporting and interpretation of lung volumes measurements in individuals of European ancestry.