The postprandial secretion of peptide YY1-36 and 3-36 in obesity is differentially increased after gastric bypass versus sleeve gastrectomy.

OBJECTIVES: Peptide tyrosine tyrosine (PYY) exists as two species, PYY1-36 and PYY3-36 , with distinct effects on insulin secretion and appetite regulation. The detailed effects of bariatric surgery on PYY1-36 and PYY3-36 secretion are not known as previous studies have used nonspecific immunoassays to measure total PYY. Our objective was to characterize the effect of sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB) on fasting and postprandial PYY1-36 and PYY3-36 secretion using a newly developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. DESIGN AND SUBJECTS: Observational study in 10 healthy nonobese volunteers and 30 participants with obesity who underwent RYGB (n = 24) or SG (n = 6) at the Imperial Weight Centre [NCT01945840]. Participants were studied using a standardized mixed meal test (MMT) before and 1 year after surgery. The outcome measures were PYY1-36 and PYY3-36 concentrations. RESULTS: Presurgery, the fasting and postprandial levels of PYY1-36 and PYY3-36 were low, with minimal responses to the MMT, and these did not differ from healthy nonobese volunteers. The postprandial secretion of both PYY1-36 and PYY3-36 at 1 year was amplified after RYGB, but not SG, with the response being significantly higher in RYGB compared with SG. CONCLUSIONS: There appears to be no difference in PYY secretion between nonobese and obese volunteers at baseline. At 1 year after surgery, RYGB, but not SG, is associated with increased postprandial secretion of PYY1-36 and PYY3-36 , which may account for long-term differences in efficacy and adverse effects between the two types of surgery.

Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2 - Part 1 liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays - impact of 3-epi-25-hydroxyvitamin D3 on assay performance.

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays used for the determination of serum total 25-hydroxyvitamin D (25(OH)D), which is the sum of 25-hydroxyvitamin D2 (25(OH)D2) and 25-hydroxyvitamin D3 (25(OH)D3). A set of 50 single-donor samples was assigned target values for concentrations of 25(OH)D2, 25(OH)D3, 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3), and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) using isotope dilution liquid chromatography - tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 1 includes results from 14 laboratories using 14 custom LC-MS/MS assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 53% of the LC-MS/MS assays met the VDSP criterion of mean % bias ≤ |±5%|. For the LC-MS/MS assays not meeting the ≤ |±5%| criterion, four assays had mean % bias of between 12 and 21%. Based on multivariable regression analysis using the concentrations of the four individual vitamin D metabolites in the 50 single-donor samples, the performance of several LC-MS/MS assays was found to be influenced by the presence of 3-epi-25(OH)D3. The results of this interlaboratory study represent the most comprehensive comparison of LC-MS/MS assay performance for serum total 25(OH)D and document the significant impact of the lack of separation of 3-epi-25(OH)D3 and 25(OH)D3 on assay performance, particularly with regard to mean % bias.

Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2 - Part 2 ligand binding assays - impact of 25-hydroxyvitamin D2 and 24R,25-dihydroxyvitamin D3 on assay performance.

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of ligand binding assays (Part 2) for the determination of serum total 25-hydroxyvitamin D [25(OH)D]. Fifty single-donor samples were assigned target values for concentrations of 25-hydroxyvitamin D2 [25(OH)D2], 25-hydroxyvitamin D3 [25(OH)D3], 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3], and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] using isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 2 includes results from 17 laboratories using 32 ligand binding assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 50% of the ligand binding assays achieved the VDSP criterion of mean % bias ≤ |± 5%|. For the 13 unique ligand binding assays evaluated in this study, only 4 assays were consistently within ± 5% mean bias and 4 assays were consistently outside ± 5% mean bias regardless of the laboratory performing the assay. Based on multivariable regression analysis using the concentrations of individual vitamin D metabolites in the 50 single-donor samples, most assays underestimate 25(OH)D2 and several assays (Abbott, bioMérieux, DiaSorin, IDS-EIA, and IDS-iSYS) may have cross-reactivity from 24R,25(OH)2D3. The results of this interlaboratory study represent the most comprehensive comparison of 25(OH)D ligand binding assays published to date and is the only study to assess the impact of 24R,25(OH)2D3 content using results from a reference measurement procedure.

Assessment of serum total 25-hydroxyvitamin D assays for Vitamin D External Quality Assessment Scheme (DEQAS) materials distributed at ambient and frozen conditions.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at -40 °C prior to distribution and the participants are instructed to store the samples frozen at -20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p < 0.05 for four assays (DiaSorin, DIAsource, Siemens, and SNIBE prototype). For all 14 LC-MS/MS assays, the differences in the results for the ambient- and frozen-shipped samples were not found to be significant at p < 0.05 indicating that these analytes were stable during shipment at ambient conditions. Even though assay results have been shown to vary considerably among different 25(OH)D assays in other studies, the results of this study also indicate that sample handling/transport conditions may influence 25(OH)D assay response for several assays.

Vitamin D Supplementation and Sunlight Exposure on Serum Vitamin D Concentrations in 2 Parallel, Double-Blind, Randomized, Placebo-Controlled Trials.

BACKGROUND: Vitamin D concentrations are a function of sunlight exposure and dietary intake. However, current dietary vitamin D recommendations do not consider differences in country-specific sunlight availability or spontaneous individual exposure. OBJECTIVES: We aimed to investigate the effects of vitamin D supplementation and sunlight exposure on vitamin D concentrations in Brazilian women living in high compared with low latitudes. METHODS: In 2 parallel, double-blind, randomized placebo-controlled trials, Brazilian women living in England (51°N) composed "without ultraviolet B (UVB) exposure" groups and those living in Brazil (16°S) composed the "with UVB exposure" groups (mean age, 31.39 ± 8.7 years). Participants received 15 µg cholecalciferol or placebo daily for 12 weeks during wintertime. Serum 25-hydroxyvitamin D [25(OH)D] concentrations, the primary outcome, were assessed by HPLC-MS/MS, vitamin D intakes were assessed by 4-day diet diaries, and sunlight exposure was assessed by UVB dosimeters. The effects of supplementation and UVB exposure were tested by the intention to treat with a linear mixed model. RESULTS: The 25(OH)D concentrations increased in both supplemented groups [from 75.1 ± 22.0 to 84.8 ± 21.0 nmol/L (P = 0.004) in the group with UVB exposure; from 38.1 ± 15.9 to 55.1 ± 12.2 nmol/L (P < 0.001) in the group without UVB exposure], with no significant changes in either placebo group. Concentrations in both supplemented groups were higher than those in the placebo group without UVB exposure (P = 0.0002 in the group with UVB exposure; P = 0.0035 in the group without UVB exposure). Postintervention 25(OH)D concentrations were significantly affected by serum 25(OH)D concentrations at baseline (P < 0.0001) and by intervention (placebo or supplement; P > 0.0001), with a large effect size (Cohen's D = 0.768), but were not affected by UVB exposure (with or without; P = 0.1386), nor by the interaction between the intervention (placebo or supplement) and UVB exposure (with or without; P = 0.9845). CONCLUSIONS: Moderate supplementation of 15 ug/d cholecalciferol, in accordance with current recommendations, supports an adequate vitamin D status in adult women, irrespective of latitude, and might concomitantly prevent an increase in parathyroid hormone. The Interaction Between Vitamin D Supplementation and Sunlight Exposure in Women Living in Opposite Latitudes (D-SOL) study was registered at clinicaltrials.gov as NCT03318029.

Assessment of serum total 25-hydroxyvitamin D assay commutability of Standard Reference Materials and College of American Pathologists Accuracy-Based Vitamin D (ABVD) Scheme and Vitamin D External Quality Assessment Scheme (DEQAS) materials: Vitamin D Standardization Program (VDSP) Commutability Study 2.

An interlaboratory study was conducted through the Vitamin D Standardization Program (VDSP) to assess commutability of Standard Reference Materials® (SRMs) and proficiency testing/external quality assessment (PT/EQA) samples for determination of serum total 25-hydroxyvitamin D [25(OH)D] using ligand binding assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A set of 50 single-donor serum samples were assigned target values for 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] using reference measurement procedures (RMPs). SRM and PT/EQA samples evaluated included SRM 972a (four levels), SRM 2973, six College of American Pathologists (CAP) Accuracy-Based Vitamin D (ABVD) samples, and nine Vitamin D External Quality Assessment Scheme (DEQAS) samples. Results were received from 28 different laboratories using 20 ligand binding assays and 14 LC-MS/MS methods. Using the test assay results for total serum 25(OH)D (i.e., the sum of 25(OH)D2 and 25(OH)D3) determined for the single-donor samples and the RMP target values, the linear regression and 95% prediction intervals (PIs) were calculated. Using a subset of 42 samples that had concentrations of 25(OH)D2 below 30 nmol/L, one or more of the SRM and PT/EQA samples with high concentrations of 25(OH)D2 were deemed non-commutable using 5 of 11 unique ligand binding assays. SRM 972a (level 4), which has high exogenous concentration of 3-epi-25(OH)D3, was deemed non-commutable for 50% of the LC-MS/MS assays.

Redefining the stress cortisol response to surgery.

BACKGROUND: Cortisol levels rise with the physiological stress of surgery. Previous studies have used older, less-specific assays, have not differentiated by severity or only studied procedures of a defined type. The aim of this study was to examine this phenomenon in surgeries of varying severity using a widely used cortisol immunoassay. METHODS: Euadrenal patients undergoing elective surgery were enrolled prospectively. Serum samples were taken at 8 am on surgical day, induction and 1 hour, 2 hour, 4 hour and 8 hour after. Subsequent samples were taken daily at 8 am until postoperative day 5 or hospital discharge. Total cortisol was measured using an Abbott Architect immunoassay, and cortisol-binding globulin (CBG) using a radioimmunoassay. Surgical severity was classified by POSSUM operative severity score. RESULTS: Ninety-three patients underwent surgery: Major/Major+ (n = 37), Moderate (n = 33) and Minor (n = 23). Peak cortisol positively correlated to severity: Major/Major+ median 680 [range 375-1452], Moderate 581 [270-1009] and Minor 574 [272-1066] nmol/L (Kruskal-Wallis test, P = .0031). CBG fell by 23%; the magnitude of the drop positively correlated to severity. CONCLUSIONS: The range in baseline and peak cortisol response to surgery is wide, and peak cortisol levels are lower than previously appreciated. Improvements in surgery, anaesthetic techniques and cortisol assays might explain our observed lower peak cortisols. The criteria for the dynamic testing of cortisol response may need to be reduced to take account of these factors. Our data also support a lower-dose, stratified approach to dosing of steroid replacement in hypoadrenal patients, to minimize the deleterious effects of over-replacement.

Reversible inhibitors of LSD1 as therapeutic agents in acute myeloid leukemia: clinical significance and progress to date.

In the 10 years since the discovery of lysine-specific demethylase 1 (LSD1), this epigenetic eraser has emerged as an important target of interest in oncology. More specifically, research has demonstrated that it plays an essential role in the self-renewal of leukemic stem cells in acute myeloid leukemia (AML). This review will cover clinical aspects of AML, the role of epigenetics in the disease, and discuss the research that led to the first irreversible inhibitors of LSD1 entering clinical trials for the treatment of AML in 2014. We also review recent achievements and progress in the development of potent and selective reversible inhibitors of LSD1. These compounds differ in their mode of action from tranylcypromine derivatives and could facilitate novel biochemical studies to probe the pathways mediated by LSD1. In this review, we will critically evaluate the strengths and weaknesses of published series of reversible LSD1 inhibitors. Overall, while the development of reversible inhibitors to date has been less fruitful than that of irreversible inhibitors, there is still the possibility for their use to facilitate further research into the roles and functions of LSD1 and to expand the therapeutic applications of LSD1 inhibitors in the clinic.

Determination of 24,25-dihydroxyvitamin D3 in Vitamin D External Quality Assessment Scheme samples using a reference measurement procedure.

Ninety archived human serum samples from the Vitamin D External Quality Assessment Scheme (DEQAS) were analyzed using a reference measurement procedure (RMP) based on isotope dilution liquid chromatography - tandem mass spectrometry (ID LC-MS/MS) for the determination of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3]. These 24,25(OH)2D3 results, in conjunction with concentration values assigned using RMPs for 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3], provide a valuable resource for assessing the accuracy of measurements for 24,25(OH)2D3 and for investigating the relationship between 24,25(OH)2D3 and 25(OH)D3. Results for 24,25(OH)2D3 using the RMP were compared to DEQAS consensus values demonstrating that the consensus values were not sufficient to assess the accuracy of measurements among different laboratories and methods. A multivariable regression analysis approach using historical DEQAS consensus values for various total 25(OH)D assays was used to assess the contribution of 24,25(OH)2D3 concentration on the assay response. The response of several ligand binding assays for total 25(OH)D was shown to be impacted by the presence of 24,25(OH)2D3.

The enzyme 4-hydroxy-2-oxoglutarate aldolase is deficient in primary hyperoxaluria type 3.

BACKGROUND: Mutations in the 4-hydroxy-2-oxoglutarate aldolase (HOGA1) gene have been recently identified in patients with atypical primary hyperoxaluria (PH). However, it was not clearly established whether these mutations caused disease via loss of function or activation of the gene product. METHODS: Whole-gene sequencing of HOGA1 was conducted in 28 unrelated patients with a high clinical suspicion of PH and in whom Types 1 and 2 had been excluded. RESULTS: Fifteen patients were homozygous or compound heterozygous for mutations in HOGA1. In total, seven different mutations were identified including three novel changes: a missense mutation, c.107C > T (p.Ala36Val), and two nonsense mutations c.117C > A (p.Tyr39X) and c.208C > T (p.Arg70X) as well as the previously documented c.860G > T (p.Gly297Val), c.907C > T (p.Arg303Cys) and in-frame c.944_946delAGG (p.Glu315del) mutations. The recurrent c.700 + 5G > T splice site mutation in intron 5 was most common with a frequency of 67%. Expression studies on hepatic messenger RNA demonstrated the pathogenicity of this mutation. CONCLUSIONS: The detection of a patient with two novel nonsense mutations within exon 1 of the gene, c.117C > A (p.Tyr39X) and c.208C > T (p.Arg70X), provides definitive proof that PH Type 3 is due to deficiency of the 4-hydroxy-2-oxoglutarate aldolase enzyme.

Late-night salivary cortisol and cortisone should be the initial screening test for Cushing's syndrome.

Endogenous Cushing's syndrome (CS) poses considerable diagnostic challenges. Although late-night salivary cortisol (LNSC) is recommended as a first-line screening investigation, it remains the least widely used test in many countries. The combined measurement of LNSC and late-night salivary cortisone (LNS cortisone) has shown to further improve diagnostic accuracy. We present a retrospective study in a tertiary referral centre comparing LNSC, LNS cortisone, overnight dexamethasone suppression test, low-dose dexamethasone suppression test and 24-h urinary free cortisol results of patients investigated for CS. Patients were categorised into those who had CS (21 patients) and those who did not (33 patients). LNSC had a sensitivity of 95% and a specificity of 91%. LNS cortisone had a specificity of 100% and a sensitivity of 86%. With an optimal cut-off for LNS cortisone of >14.5 nmol/L the sensitivity was 95.2%, and the specificity was 100% with an area under the curve of 0.997, for diagnosing CS. Saliva collection is non-invasive and can be carried out at home. We therefore advocate simultaneous measurement of LNSC and LNS cortisone as the first-line screening test to evaluate patients with suspected CS.

Clinical Evaluation of Assays for Plasma Renin Activity and Aldosterone Measurement by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Aldosterone and renin are pivotal hormones in the regulation of salt and water homeostasis and blood pressure. Measurement of renin and aldosterone in serum/plasma is essential for the investigation of primary hyperaldosteronism (PA) and monitoring of glucocorticoid replacement therapy. METHODS: We report 2 LC-MS/MS methods developed to measure aldosterone and plasma renin activity (PRA). PRA was determined by endogenous enzymatic generation of angiotensin I using 150 µL of sample. Generated angiotensin I was purified by solid phase extraction prior to chromatographic separation and mass spectrometry. Aldosterone measurement required 300 µL of sample extracted with MTBE prior to LC-MS/MS analysis. RESULTS: The PRA method was linear (1.2-193 nmol/L), sensitive (LLOQ = 1.2 nmol/L), precise (CV = 4.1%), and specific (no cross reactivity for a number of structurally similar steroids). Dilutional linearity and recovery (84%) were acceptable. Accuracy was confirmed by comparison against our current RIA method. The aldosterone method had equally acceptable performance characteristics. Reference ranges in 110 healthy normotensive subjects were: PRA 0.2-3.7 nmol/L/h and aldosterone 50-950 pmol/L. Consecutive patients (n = 62) with adrenal incidentalomas shown to have no functional adrenal disease; their post overnight 1 mg dexamethasone test values were: PRA 0.2-2.6 nmol/L/h and aldosterone 55-480 pmol/L. Serum aldosterone values after 2 liter saline suppression were-normal subjects (n = 17): 78-238 pmol/L and confirmed primary hyperaldosteronism (n = 25): 131-1080 pmol/L. CONCLUSIONS: We have developed robust assays for PRA and aldosterone with appropriate clinical evaluation. These assays are now in routine practice in the UK.

The relationship between vitamin D status, intake and exercise performance in UK University-level athletes and healthy inactive controls.

The potential ergogenic effects of vitamin D (vitD) in high performing athletes has received considerable attention in the literature and media. However, little is known about non-supplemented university athletes and students residing at a higher latitude. This study aimed to investigate the effects of vitD (biochemical status and dietary intake) on exercise performance in UK university athletes and sedentary students. A total of 34 athletes and 16 sedentary controls were studied during the spring and summer months. Serum vitD status and sunlight exposure were assessed using LC-MS/MS and dosimetry, respectively. Muscular strength of the upper and lower body was assessed using handgrip and knee extensor dynamometry (KE). Countermovement jump (CMJ) and aerobic fitness were measured using an Optojump and VO2max test, respectively. Statistical analysis was performed using paired/ independent t-tests, ANCOVA and Pearson/ Spearman correlations, depending on normality. VitD status increased significantly over the seasons, with athletes measuring higher status both in spring (51.7±20.5 vs. 37.2±18.9 nmol/L, p = 0.03) and summer (66.7±15.8 vs 55.6±18.8 nmol/L, p = 0.04) when compared to controls, respectively. Notably, 22% of the subjects recruited were vitD deficient during the spring term only (<25nmol/L, n 9). Subjects with 'insufficient' vitD status (<50nmol/L) elicited significantly lower CMJ when contrasted to the vitD 'sufficient' (>50nmol/l) group (p = 0.055) and a lower VO2 max (p = 0.05) in the spring and summer term (p = 0.05 and p = 0.01, respectively). However, an ANCOVA test showed no significant difference detected for either CMJ or VO2max following adjustments for co-variates. In conclusion, we provide novel information on the vitD status, dietary intake, physical fitness and sunlight exposure of UK young adults across two separate seasons, for which there is limited data at present.

Seasonal variation in vitamin D status, bone health and athletic performance in competitive university student athletes: a longitudinal study.

Vitamin D deficiency has been commonly reported in elite athletes, but the vitamin D status of UK university athletes in different training environments remains unknown. The present study aimed to determine any seasonal changes in vitamin D status among indoor and outdoor athletes, and whether there was any relationship between vitamin D status and indices of physical performance and bone health. A group of forty-seven university athletes (indoor n 22, outdoor n 25) were tested during autumn and spring for serum vitamin D status, bone health and physical performance parameters. Blood samples were analysed for serum 25-hydroxyvitamin D (s-25(OH)D) status. Peak isometric knee extensor torque using an isokinetic dynamometer and jump height was assessed using an Optojump. Aerobic capacity was estimated using the Yo-Yo intermittent recovery test. Peripheral quantitative computed tomography scans measured radial bone mineral density. Statistical analyses were performed using appropriate parametric/non-parametric testing depending on the normality of the data. s-25(OH)D significantly fell between autumn (52·8 (sd 22·0) nmol/l) and spring (31·0 (sd 16·5) nmol/l; P < 0·001). In spring, 34 % of participants were considered to be vitamin D deficient (<25 nmol/l) according to the revised 2016 UK guidelines. These data suggest that UK university athletes are at risk of vitamin D deficiency. Thus, further research is warranted to investigate the concomitant effects of low vitamin D status on health and performance outcomes in university athletes residing at northern latitudes.

Pre-clinical activity of combined LSD1 and mTORC1 inhibition in MLL-translocated acute myeloid leukaemia.

The histone demethylase lysine-specific demethylase 1 (LSD1 or KDM1A) has emerged as a candidate therapeutic target in acute myeloid leukaemia (AML); tranylcypromine-derivative inhibitors induce loss of clonogenic activity and promote differentiation, in particular in the MLL-translocated molecular subtype of AML. In AML, the use of drugs in combination often delivers superior clinical activity. To identify genes and cellular pathways that collaborate with LSD1 to maintain the leukaemic phenotype, and which could be targeted by combination therapies, we performed a genome-wide CRISPR-Cas9 dropout screen. We identified multiple components of the amino acid sensing arm of mTORC1 signalling-RRAGA, MLST8, WDR24 and LAMTOR2-as cellular sensitizers to LSD1 inhibition. Knockdown of mTORC1 components, or mTORC1 pharmacologic inhibition, in combination with LSD1 inhibition enhanced differentiation in both cell line and primary cell settings, in vitro and in vivo, and substantially reduced the frequency of clonogenic primary human AML cells in a modelled minimal residual disease setting. Synergistic upregulation of a set of transcription factor genes associated with terminal monocytic lineage differentiation was observed. Thus, dual mTORC1 and LSD1 inhibition represents a candidate combination approach for enhanced differentiation in MLL-translocated AML which could be evaluated in early phase clinical trials.

Primary hyperoxaluria type 1: update and additional mutation analysis of the AGXT gene.

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive, inherited disorder of glyoxylate metabolism arising from a deficiency of the alanine:glyoxylate aminotransferase (AGT) enzyme, encoded by the AGXT gene. The disease is manifested by excessive endogenous oxalate production, which leads to impaired renal function and associated morbidity. At least 146 mutations have now been described, 50 of which are newly reported here. The mutations, which occur along the length of the AGXT gene, are predominantly single-nucleotide substitutions (75%), 73 are missense, 19 nonsense, and 18 splice mutations; but 36 major and minor deletions and insertions are also included. There is little association of mutation with ethnicity, the most obvious exception being the p.Ile244Thr mutation, which appears to have North African/Spanish origins. A common, polymorphic variant encoding leucine at codon 11, the so-called minor allele, has significantly lower catalytic activity in vitro, and has a higher frequency in PH1 compared to the rest of the population. This polymorphism influences enzyme targeting in the presence of the most common Gly170Arg mutation and potentiates the effect of several other pathological sequence variants. This review discusses the spectrum of AGXT mutations and polymorphisms, their clinical significance, and their diagnostic relevance.

Anti-TNF-induced lupus.

The use of protein-based anti-TNF-alpha therapies such as antibodies and soluble TNF-alpha receptors is commonly associated with the induction of autoantibodies, whereas anti-TNF-induced lupus (ATIL) is rare. ATIL can occur with any of the available TNF inhibitors, but the frequency and clinical characteristics of ATIL vary between different drugs. Cutaneous, renal and cerebral involvement as well as dsDNA antibodies are more common in ATIL compared to classical drug-induced lupus (DIL), suggesting different pathogenic mechanisms of ATIL and DIL. True ATIL must be clinically differentiated from mixed CTD, SLE or overlap syndromes unmasked, but not induced, by anti-TNF-alpha treatment of unclassified polyarthritis. The pathogenesis of ATIL is still unknown. Concomitant immunosuppression can reduce autoantibody formation in ATIL, and withdrawal of anti-TNF-alpha therapy usually leads to resolution of symptoms. Steroids and/or immunosuppressive therapy may be required in severe cases.

Derepression of the Iroquois Homeodomain Transcription Factor Gene IRX3 Confers Differentiation Block in Acute Leukemia.

The Iroquois homeodomain transcription factor gene IRX3 is expressed in the developing nervous system, limb buds, and heart, and transcript levels specify obesity risk in humans. We now report a functional role for IRX3 in human acute leukemia. Although transcript levels are very low in normal human bone marrow cells, high IRX3 expression is found in ∼30% of patients with acute myeloid leukemia (AML), ∼50% with T-acute lymphoblastic leukemia, and ∼20% with B-acute lymphoblastic leukemia, frequently in association with high-level HOXA gene expression. Expression of IRX3 alone was sufficient to immortalize hematopoietic stem and progenitor cells (HSPCs) in myeloid culture and induce lymphoid leukemias in vivo. IRX3 knockdown induced terminal differentiation of AML cells. Combined IRX3 and Hoxa9 expression in murine HSPCs impeded normal T-progenitor differentiation in lymphoid culture and substantially enhanced the morphologic and phenotypic differentiation block of AML in myeloid leukemia transplantation experiments through suppression of a terminal myelomonocytic program. Likewise, in cases of primary human AML, high IRX3 expression is strongly associated with reduced myelomonocytic differentiation. Thus, tissue-inappropriate derepression of IRX3 contributes significantly to the block in differentiation, which is the pathognomonic feature of human acute leukemias.

Enhancer Activation by Pharmacologic Displacement of LSD1 from GFI1 Induces Differentiation in Acute Myeloid Leukemia.

Pharmacologic inhibition of LSD1 promotes blast cell differentiation in acute myeloid leukemia (AML) with MLL translocations. The assumption has been that differentiation is induced through blockade of LSD1's histone demethylase activity. However, we observed that rapid, extensive, drug-induced changes in transcription occurred without genome-wide accumulation of the histone modifications targeted for demethylation by LSD1 at sites of LSD1 binding and that a demethylase-defective mutant rescued LSD1 knockdown AML cells as efficiently as wild-type protein. Rather, LSD1 inhibitors disrupt the interaction of LSD1 and RCOR1 with the SNAG-domain transcription repressor GFI1, which is bound to a discrete set of enhancers located close to transcription factor genes that regulate myeloid differentiation. Physical separation of LSD1/RCOR1 from GFI1 is required for drug-induced differentiation. The consequent inactivation of GFI1 leads to increased enhancer histone acetylation within hours, which directly correlates with the upregulation of nearby subordinate genes.