Ronald A. Booth

Assessment of thiopurine S-methyltransferase activity in patients prescribed thiopurines: a systematic review.

BACKGROUND The evidence for testing thiopurine S-methyltransferase (TPMT) enzymatic activity or genotype before starting therapy with thiopurine-based drugs is unclear. PURPOSE To examine the sensitivity and specificity of TPMT genotyping for TPMT enzymatic activity, reducing harm from thiopurine by pretesting, and the association of thiopurine toxicity with TPMT status in adults and children with chronic inflammatory diseases. DATA SOURCES MEDLINE, EMBASE, the Cochrane Library, and Ovid HealthSTAR (from inception to December 2010) and BIOSIS and Genetics Abstracts (to May 2009). STUDY SELECTION Two reviewers screened records and identified relevant studies in English. DATA EXTRACTION Data on patient characteristics, outcomes, and risk for bias were extracted by one reviewer and independently identified by another. DATA SYNTHESIS 54 observational studies and 1 randomized, controlled trial were included. Insufficient evidence addressed the effectiveness of pretesting. Genotyping sensitivity to identify patients with low and intermediate TPMT enzymatic activity ranged from 70.33% to 86.15% (lower-bound 95% CI, 54.52% to 70.88%; upper-bound CI, 78.50% to 96.33%). Sparse data precluded estimation of genotype sensitivity to identify patients with low to absent enzymatic activity. Genotyping specificity approached 100%. Compared with noncarriers, heterozygous and homozygous genotypes were both associated with leukopenia (odds ratios, 4.29 [CI, 2.67 to 6.89] and 20.84 [CI, 3.42 to 126.89], respectively). Compared with intermediate or normal activity, low TPMT enzymatic activity was significantly associated with myelotoxicity and leukopenia. LIMITATION Available evidence was not rigorous and was underpowered to detect a difference in outcomes. CONCLUSION Insufficient evidence addresses the effectiveness of TPMT pretesting in patients with chronic inflammatory diseases. Estimates of the sensitivity of genotyping are imprecise. Evidence confirms the known associations of leukopenia or myelotoxicity with reduced TPMT activity or variant genotype. PRIMARY FUNDING SOURCE Agency for Healthcare Research and Quality.

Regulation of Xenopus oocyte meiosis arrest by G protein βγ subunits

Abstract Background: Progesterone induces the resumption of meiosis (maturation) in Xenopus oocytes through a nongenomic mechanism involving inhibition of an oocyte adenylyl cyclase and reduction of intracellular cAMP. However, progesterone action in Xenopus oocytes is not blocked by pertussis toxin, and this finding indicates that the inhibition of the oocyte adenylyl cyclase is not mediated by the α subunits of classical G i -type G proteins. Results: To investigate the possibility that G protein βγ subunits, rather than α subunits, play a key role in regulating oocyte maturation, we have employed two structurally distinct G protein βγ scavengers (G t α and βARK-C CAAX ) to sequester free Gβγ dimers. We demonstrated that the injection of mRNA encoding either of these Gβγ scavengers induced oocyte maturation. The Gβγ scavengers bound an endogenous, membrane-associated Gβ subunit, indistinguishable from Xenopus Gβ1 derived from mRNA injection. The injection of Xenopus Gβ1 mRNA, together with bovine Gγ2 mRNA, elevated oocyte cAMP levels and inhibited progesterone-induced oocyte maturation. Conclusion: An endogenous G protein βγ dimer, likely including Xenopus Gβ1, is responsible for maintaining oocyte meiosis arrest. Resumption of meiosis is induced by Gβγ scavengers in vitro or, naturally, by progesterone via a mechanism that suppresses the release of Gβγ.

Correlation of a multi-cytokine panel with clinical disease activity in patients with rheumatoid arthritis

OBJECTIVE Explore the potential use of a cytokine panel as biochemical markers of disease activity in rheumatoid arthritis (RA) patients. DESIGN AND METHODS 57 adult RA patients were assessed using five validated clinical disease activity tools: Health Assessment Questionnaire (HAQ), standard 28-joint Disease Activity Score (DAS28), DAS28 using C-reactive protein (DAS28-CRP), Clinical Disease Activity Index (CDAI), and Simple Disease Activity Index (SDAI). Plasma cytokine levels (IL-2, IL-4, IL-6, IL-8, IL-10, VEGF, IFN-γ, TNF-α, IL1α, IL1β, MCP1, and EGF) were measured in 47 of the 57 patients and correlated with clinical indicators. RESULTS We found significant correlations between plasma levels of IL-6 and all clinical measures of disease activity; Spearman coefficients (p values) were: HAQ: 0.347(0.017); DAS28: 0.409(0.005); DAS-CRP: 0.378(0.011); CDAI: 0.312(0.033); SDAI: 0.310(0.039); ESR: 0.448(0.002); and CRP: 0.513(0.001). IFN-γ also correlated with DAS-CRP: 0.309(0.039) and SDAI: 0.301(0.044). Furthermore, the levels of IL-6 and IFN-γ increased significantly with worsening disease, as defined by the European League Against Rheumatism (EULAR) classification of disease activity. CONCLUSION A significant correlation between plasma levels of IL-6 and clinical disease activity in patients with RA suggests a future role of IL6 as a disease activity marker.

A systematic review of BNP and NT-proBNP in the management of heart failure: overview and methods

B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) levels are increased in persons with heart failure (HF); low levels of these peptides rule out HF. We systematically reviewed the literature to assess the use of BNP and NT-proBNP in the diagnosis, prognosis, and treatment for HF. We also examined the biological variation of these peptides in persons with and without HF. We searched Medline, Embase, AMED, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and CINAHL for English-language studies published between January 1989 and June 2012. Supplemental searches involved the gray literature and the reference lists of included studies. Trained reviewers used standardized forms to screen articles for inclusion in the review and to extract data from included papers. We examined the risk of bias with QUADAS-2 for diagnosis studies, the Hayden criteria for prognosis studies, and the Jadad scale for treatment studies. We assessed the strength of evidence in four domains (risk of bias, consistency, directness, and precision) for the diagnosis and treatment studies. Results were reported as narrative syntheses. Additional meta-analyses were conducted for the diagnosis studies. Three hundred ten articles passed through screening and were included in the review. One hundred four articles applied to diagnostic accuracy, 190 papers pertained to prognosis, and nine articles addressed BNP- or NT-proBNP-guided treatment. Each individual paper in this series reports, summarizes, and discusses the evidence regarding diagnosis, prognosis, or treatment.

Performance of BNP and NT-proBNP for diagnosis of heart failure in primary care patients: a systematic review

National and international guidelines have been published recommending the use of natriuretic peptides as an aid to the diagnosis of heart failure (HF) in acute settings; however, few specific recommendations exist for governing the use of these peptides in primary care populations. To summarize the available data relevant to the diagnosis of HF in primary care patient population, we systematically reviewed the literature to identify original articles that investigated the diagnostic accuracy of B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) in primary care settings. The search yielded 25,864 articles in total: 12 investigating BNP and 20 investigating NT-proBNP were relevant to our objective and included in the review. QUADAS-2 and GRADE were used to assess the quality of the included articles. Diagnostic data were pooled based on three cutpoints: lowest and optimal, as chosen by study authors, and manufacturers’ suggested. The effect of various determinants (e.g., age, gender, BMI, and renal function) on diagnostic performance was also investigated. Pooled sensitivity and specificity of BNP and NT-proBNP using the lowest [0.85 (sensitivity) and 0.54 (specificity)], optimal (0.80 and 0.61), and manufacturers’ (0.74 and 0.67) cutpoints showed good performance for diagnosing HF. Similar performance was seen for NT-proBNP: lowest (0.90 and 0.50), optimal (0.86 and 0.58), and manufacturers’ (0.82 and 0.58) cutpoints. Overall, we rated the strength of evidence as high because further studies will be unlikely to change the estimates diagnostic performance.

Pre-analytic and analytic sources of variations in thiopurine methyltransferase activity measurement in patients prescribed thiopurine-based drugs: A systematic review☆

OBJECTIVES Low thiopurine S-methyltransferase (TPMT) enzyme activity is associated with increased thiopurine drug toxicity, particularly myelotoxicity. Pre-analytic and analytic variables for TPMT genotype and phenotype (enzyme activity) testing were reviewed. DESIGN AND METHODS A systematic literature review was performed, and diagnostic laboratories were surveyed. RESULTS Thirty-five studies reported relevant data for pre-analytic variables (patient age, gender, race, hematocrit, co-morbidity, co-administered drugs and specimen stability) and thirty-three for analytic variables (accuracy, reproducibility). TPMT is stable in blood when stored for up to 7 days at room temperature, and 3 months at -30°C. Pre-analytic patient variables do not affect TPMT activity. Fifteen drugs studied to date exerted no clinically significant effects in vivo. Enzymatic assay is the preferred technique. Radiochemical and HPLC techniques had intra- and inter-assay coefficients of variation (CVs) below 10%. CONCLUSION TPMT is a stable enzyme, and its assay is not affected by age, gender, race or co-morbidity.

Spot urine protein measurements: are these accurate in kidney transplant recipients?

Background Proteinuria and albuminuria are important markers of allograft pathology and are associated with graft loss and cardiovascular disease. Traditionally, these have been quantified using a 24-hr urine collection, but spot urine measurements (albumin-creatinine and protein-creatinine ratios) have become popular because of convenience. Aside from tests of correlation, there has been little evaluation of these measurements in kidney transplantation. Methods To further assess the value of albumin-creatinine and protein-creatinine ratios, we measured protein-creatinine ratio and 24-hr urine protein excretion (n=192) and albumin-creatinine ratio and 24-hr urine albumin excretion (n=189) in stable renal transplant patients. Bias (measured minus estimated value), precision, and accuracy was calculated. Results For the protein-creatinine ratio, percent bias ranged from 12% to 21%, and the accuracy (within 30% of 24-hr collection) was only 47% to 56% depending on the degree of proteinuria. For the albumin-creatinine ratio, percent bias ranged from 9% to 21%, and the accuracy (within 30%) ranged from 38% to 80% depending on the degree of albuminuria. There was no statistical difference between accuracy of protein-creatinine and albumin-creatinine ratios. Conclusions The ability of the albumin-creatinine and protein-creatinine ratios to accurately predict 24-hr albumin and protein excretion is modest. Given the similar accuracy of both measurements, either protein-creatinine ratio or albumin-creatinine ratio can be used for monitoring protein excretion. However, given the limitations of both the albumin-creatinine ratio and protein-creatinine ratio in this population, a 24-hr urine collection should be considered before making major clinical decisions (e.g., biopsy) based on the presence of proteinuria.

Xenopus laevis TRK-fused gene (TFG) is an SH3 domain binding protein highly expressed in the cement gland

TRK‐fused gene (TFG) was originally identified in humans as the N‐terminus of an oncogenic fusion protein TRK‐T3, associated with papillary thyroid carcinoma. An amino‐terminal coiled coil domain of TFG is responsible for mediating oligomerization of the TRK‐T3 oncoprotein, resulting in constitutive activation of the TRK protein tyrosine kinase and oncogenesis. We have cloned the Xenopus laevis homologue of TFG and demonstrated that xTFG was highly expressed in the cement gland of tailbud embryos. Overexpression of xTFG2–136 (including the coiled coil domain) in early embryos, via mRNA microinjection as well as transgenic expression using the recently described restriction enzyme mediated integration (REMI) transgenesis, did not alter embryonic development or development of a functional cement gland, despite clear evidence that xTFG2–136 strongly interacted with endogenous xTFG. Finally, we have identified a potential SH3 binding motif in xTFG (and in TFG) and have demonstrated that xTFG selectively interacted with SH3 domains of Src, PLCγ, and the p85 phosphatidylinositol 3‐kinase subunit. Mol. Reprod. Dev. 56:336–344, 2000.

The Risk of Acute Rejection Following Kidney Transplant by 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D Status: A Prospective Cohort Study:

Background: Prediction of acute kidney transplant rejection remains imperfect despite several known risk factors. There is an increasing appreciation of the potential importance of the vitamin D pathway in immunological disease and transplantation. Objective: The purpose of this study was to determine the association of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D with acute rejection. Design: This was a prospective cohort study. Setting: Three academic adult kidney transplant programs in Ontario, Canada, were chosen. Patients: All consecutive adult patients at the 3 institutions who received a solitary kidney transplant, were able to provide written informed consent, and planned to be followed at the same center post-operatively were included. Measurements: Serum concentration of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were measured at baseline, 3, and 6 months post-transplantation. Acute rejection was classified using Banff criteria. Methods: The co-primary outcome was the association between 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D and time to first occurrence of biopsy-proven acute rejection (BPAR) within the first year after kidney transplantation. Cox proportional hazards models were fitted taking into account the time-varying nature of serum concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. Results: From 556 screened patients, data on 327 kidney transplant recipients are included. First BPAR occurred in 54 (16.5%) patients. In adjusted Cox proportional hazards models, the serum concentration of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D was not associated with acute renal transplant rejection (hazard ratio 1.00; 95% [confidence interval] CI, 0.87-1.14, per 10 nmol/L increase, and hazard ratio 0.97; 95% CI, 0.84-1.12, per 10 pmol/L increase, respectively). Limitations: Given the observational design, we cannot rule out the possibility of residual confounding that limited our ability to detect a clinically significant effect of vitamin D metabolites on acute rejection. Conclusions: A low serum concentration of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D is not associated with an increased risk of acute kidney transplant rejection following kidney transplantation.

Candidate recommendations for protein electrophoresis reporting from the Canadian Society of Clinical Chemists Monoclonal Gammopathy Working Group

Protein electrophoresis is commonly used as an aid in the diagnosis of monoclonal gammopathies and is performed in many laboratories in Canada and throughout the world. However, unlike many other diagnostic tests, there is limited guidance for standardization and neither guidance nor specific recommendations for clinical reporting of serum (SPE) or urine (UPE) protein electrophoresis and immunotyping available in the literature. Therefore, a Canadian effort was undertaken to recommend standards that cover all aspects of clinical reporting with an ultimate goal towards reporting standardization. The Canadian Society of Clinical Chemists (CSCC) Monoclonal Gammopathy Interest Group (MGIG), which is composed of CSCC members with an interest in protein electrophoresis, has formed a Monoclonal Gammopathy Working Group (MGWG) to take initial steps towards standardization of SPE, UPE and immunotyping. Candidate standardization recommendations were developed, discussed and voted upon by the MGWG. Candidate recommendations that achieved 90% agreement are presented as consensus recommendations. Recommendations that did not achieve 90% consensus remain candidate recommendations and are presented with accompanying MGWG discussion. Eleven consensus recommendations along with candidate recommendations for nomenclature, protein fraction reporting, test utilization, interference handling and interpretive reporting options are presented.