Edna Efrati

Influence of glutathione S-transferase A1, P1, M1, T1 polymorphisms on oral busulfan pharmacokinetics in children with congenital hemoglobinopathies undergoing hematopoietic stem cell transplantation†

Busulfan (BU), often used in high dose for myeloablation before hematopoietic stem cell transplantation (HSCT), has been implicated in certain HSCT toxicities, including the occurrence of hepatic veno‐occlusive disease (HVOD). In addition to weight and age, gene polymorphisms in specific members of the glutathione‐transferase (GST) gene family (A1, P1, M1, and T1), involved in BU metabolism, may play a role in the wide inter‐patient variability in systemic BU concentrations.

Risk Factors for Serious Adverse Effects of Thiopurines in Patients with Crohn’s Disease

PURPOSE Thiopurines are effective in attaining and maintaining remission in patients with inflammatory bowel diseases (IBD). The major drawback of these drugs are their serious adverse effects (SAE), highlighting the importance of preemptive identification of patients at risk. We aimed to examine whether gene polymorphisms in GSTM1, GSTT1 and TPMT, combined with various clinical parameters, can predict thiopurine induced SAE. METHODS A retrospective cohort of 176 Crohns Disease (CD) patients treated with thiopurines (131 with 6MP and 45 with azathioprine) was genotyped for common polymorphisms in GSTM1, GSTT1 and TPMT. Clinical data including SAE, age, ethnicity, gender and smoking status were extracted from patient charts. SAEs evaluated were myelosuppression, hepatotoxicity and pancreatitis. Associations between demographic, clinical, and genetic variables and thiopurine induced SAE were assessed. RESULTS Twenty-four patients (14%) developed SAE, revealing a significant association between thiopurine induced SAE and GSTM1-null genotype (P=0.05), older age (P=0.016) and active smoking status (P=0.043) and SAE. On multi-variant analysis, past or current smokers were at increased risk for developing thiopurine related SAE (OR 2.915, CI 95%: 1.199- 7.084), specifically pancreatitis (p<0.001). No association was found between TPMT or GSTT1 polymorphisms and the development of SAE. CONCLUSIONS Active smoking and GSTM1-null genotype appear to be risk factors for thiopurine induced SAEs (i.e. myelosuppression, hepatotoxicity and pancreatitis) in patients with CD. Corroboration of these associations in larger cohorts is warranted.

Distribution of TPMT risk alleles for thioupurine toxicity in the Israeli population

Background and objectiveIndividuals with intermediate or no thiopurine S-methyltransferase (TPMT) activity are at risk of hematotoxicity when treated with standard doses of thiopurines, thus, pretreatment identification of these individuals is of major importance. The purpose of this study was to determine the frequency and distribution of TPMT polymorphic variants, known to functionally impair TPMT activity, in the highly heterogeneous Israeli population.MethodsTPMT genotyping of individuals representing three major demographic groups in Israel was carried out by PCR restriction fragment length polymorphism and high-resolution melting.ResultsFrequencies of TPMT risk alleles differed significantly among the screened Israeli subpopulations: Druze showed fivefold and twofold higher frequencies than Jews and Moslems, respectively. Specifically, allelic frequencies of TPMT*3A were 0.73% (95% CI 0.34-1.45%), 0.79% (95% CI 0.16-2.39%), and 3.19% (95% CI 1.78-5.58%) in Jews, Moslems, and Druze, respectively. Although not found in Jews, TPMT*3C was found at an allelic frequency of 1.05% (95% CI 0.31-2.76%) and 0.75% (95% CI 0.02-2.84%) in Moslems and Druze. TPMT*2 and TPMT*3B were not detected in any of the Israeli subpopulations studied.ConclusionThese data indicate that the Israeli population displays a distinct TPMT genetic variability that is comprised of a mix of three major genetically diverse subpopulations, each with its unique TPMT allelic frequency distribution pattern and likelihood of developing an adverse reaction to thiopurine drugs.

Transcriptional Regulation of the Pendrin Gene

Pendrin (SLC26A4), a Cl-/anion exchanger encoded by the gene PDS, is highly expressed in the kidney, thyroid and inner ear epithelia and is essential for bicarbonate secretion /chloride reabsorption, iodide accumulation and endolymph ion balance, respectively. The molecular mechanisms controlling pendrin activity in renal, thyroid and inner ear epithelia have been the subject of recent studies. The effects of ambient pH, the hormone aldosterone and the peptide uroguanylin (UGN; the “intestinal natriuretic hormone”), known modulators of electrolyte balance, on transcription of the pendrin gene, have been investigated. Luciferase reporter plasmids containing different length fragments of the human PDS (hPDS) promoter were transfected into renal HEK293, thyroid LA2, and inner ear VOT36 epithelial cells. Acidic pH decreased and alkaline pH increased hPDS promoter activity in transfected HEK293 and VOT36, but not in LA2 cells. Aldosterone reduced hPDS promoter activity in HEK293 but had no effect in LA2 and VOT36 cells. These pH and aldosterone-induced effects on the hPDS promoter occurred within 96-bp and 89-bp regions, respectively, which likely contain distinct response elements to these modulators. Injection of UGN into mice resulted in decreased pendrin mRNA and protein expression in the kidney. Exposure of transfected HEK293 to UGN decreased hPDS promoter activity. The findings provided evidence for the presence of a UGN response element within the 96-bp region overlapping with the pH response element on the hPDSpromoter. Pendrin is also expressed in airway epithelium. The cytokins interleukin 4 (IL-4) and interleukin-13 (IL-13), known regulators of airway surface function, have been shown to increase hPDS promoter activity by a STAT6-dependent mechanism. In conclusion, systemic pH, the hormone aldosterone, and the peptide UGN influence renal tubular pendrin gene expression and, perhaps, pendrin-mediated Cl-/HCO3- exchange at the transcriptional level. Pendrin-driven anion transport in the endolymph and at the airway surface may be regulated transcriptionally by systemic pH and IL-3/IL-4, respectively. The distinct response elements and the corresponding transcription factors mediating the effect of these modulators on the PDS promoter remain to be identified and characterized.

Pharmacokinetic and Pharmacogenetic Analysis of Oral Busulfan in Stem Cell Transplantation: Prediction of Poor Drug Metabolism to Prevent Drug Toxicity

High dose busulfan (BU) has become a mainstay in conditioning regimens for hematopoietic stem cell transplantation (HSCT), despite its unpredictable response, narrow therapeutic index and severe toxicity. The present study provides an integration of pharmacokinetic and genetic data of 63 adults with acute myeloid leukemia (AML) preconditioned for HSCT with high dose oral BU, with the aim of defining biomarkers predictive of poor BU metabolism. BU area under the concentration time curve (AUC) demonstrated that 76% of the patients achieved target AUC; 24% required dose modification. The main findings of this study were: (1) AML patients carrying the GSTP1 rs1695 variant allele were at risk of developing supra-therapeutic BU-AUC due to reduced BU clearance. (2) Combined polymorphisms in GSTM1 and ABCB1 were associated with BU clearance and AUC rates. In conclusion, GST and ABCB1 genotyping may assist care-givers in personalizing BU dosage with less trial-and-error and may enable preemptive identification of patients at risk for BU toxicity.

Thiopurine effectiveness in patients with Crohn's disease: a study of genetic and clinical predictive factors.

Background: Thiopurines are efficacious in the treatment of Crohns disease and were recently shown to induce T-cell apoptosis by modulation of Rac1 activation. To assess whether polymorphisms in Rac1 and other apoptosis-related genes, combined with clinical parameters, can predict response to thiopurines. Methods: A retrospective cohort of 156 thiopurine-treated patients with Crohns disease was genotyped for 11 single-nucleotide polymorphisms (SNPs): 9 SNPs in Rac1, 1 SNP in the Fas ligand −843 T>C, and 1 SNP in the Caspase-9 93 C>T. Clinical data were extracted from the medical charts. Odds ratios (ORs) and 95% confidence intervals (CIs) of the association between demographic, clinical, and genetic variables and thiopurine response rates were calculated. Results: The overall response rate to thiopurines was 74% (115/156). The Rac1 SNP rs34932801 heterozygote genotype GC was associated with a lower response rate compared with the wild-type GG genotype (46% versus 76%; OR = 0.26; 95% CI, 0.08–0.91; P = 0.036). Only wild-type homozygotes were found for 5 Rac1 SNPs. None of the other 3 Rac1 SNPs were associated with response to thiopurines. Patients with Montreal B3 behavior pattern responded worse than those with a B1 behavior pattern (59%, versus 80%; OR = 0.37; 95% CI, 0.17–0.83; P = 0.016). Sephardic Jews had a lower response rate to thiopurines compared with Jews of Ashkenazi or mixed ancestry (60% versus 82%; OR = 0.32; 95% CI, 0.15–0.69, P = 0.003). Conclusions: Rac1 SNP rs34932801carriage, Montreal B3 disease behavior, and a Sephardic Jewish origin were associated with unfavorable response to thiopurines. Corroboration of these associations in larger cohorts is warranted.

LNA-based PCR clamping enrichment assay for the identification of KRAS mutations.

INTRODUCTION KRAS mutations in colon carcinomas are associated with lack of response to anti-EGFR monoclonal antibody treatment. Therefore, patients must undergo genetic testing to be eligible for treatment. Several methods for KRAS mutation analysis exist, but many are not sensitive enough to detect a mutation in samples with low fraction of malignant cells. In the present study, we developed a KRAS mutations detection method that is both simple and sensitive. METHODS Using a locked nucleic acid (LNA) containing oligonucleotide, we developed a PCR clamping method that preferentially amplifies the mutated over wild type KRAS. We evaluated the sensitivity of this method using serial dilutions of plasmids containing wild-type and mutated KRAS fragments. Additionally, KRAS mutation status was evaluated on 60 archived tissue samples of colon carcinoma, and compared to direct sequencing and high resolution melting (HRM) methods. RESULTS The PCR clamping method could detect as little as 1% mutated DNA in the sample analyzed. Of the 29 KRAS mutations identified by the PCR clamping method, only 23 (79%) were identified by standard direct sequencing. The results of PCR clamping correlated with HRM results. CONCLUSIONS LNA based PCR clamping method is a simple and highly sensitive method for the detection of KRAS mutations.

Transcriptional regulation of the claudin-16 gene by Mg2+ availability.

Background/Aims: Renal tubular Mg²⁺ reabsorption is mediated predominantly by the tight junction channel protein claudin-16 which is encoded by the gene CLDN16. Hypermagnesemia decreases, whereas hypomagnesemia increases Mg²⁺ reabsorption. This study examines the role of claudin-16 in the adaptive response of the kidney to Mg²⁺ availability. Methods/Results: Mice received a low-, normal- or high Mg²⁺ diet for up to 3 days. Mg²⁺-loaded animals displayed hypermagnesemia with increasing urine Mg²⁺/Ca²⁺ levels paralleled by a decrease in claudin-16 protein and mRNA in the kidney. Mg²⁺- deprived animals developed hypomagnesemia with decreasing urine Mg²⁺/Ca²⁺ levels associated with an increase in claudin-16 protein and mRNA abundance. Mg²⁺ depletion markedly increased and Mg²⁺ load decreased endogenous claudin-16 mRNA levels in calcium-sensing receptor-transfected HEK293 cells compared with native HEK293 cells. The effect of Mg²⁺ availability on the human CLDN16 (hCLDN16) gene promoter was examined. Using a 2.5kb hCLDN16 5′-flanking DNA sequence, we show that magnesium depletion increases and Mg²⁺ load decreases hCLDN16 promoter activity in transfected HEK293 cells. Conclusions: Changes in Mg²⁺ availability may influence claudin-16 mediated Mg²⁺ transport at the transcriptional level. The possible involvement of the cell membrane bound Ca²⁺/Mg²⁺ sensing receptor or the potential role of a hypothetical Mg²⁺ response element on the CLDN16 promoter in the Mg²⁺-induced response remains to be explored.

Pendrin, a Novel Transcriptional Target of the Uroguanylin System

Guanylin (GN) and uroguanylin (UGN) are low-molecular-weight peptide hormones produced mainly in the intestinal mucosa in response to oral salt load. GN and UGN (guanylin peptides) induce secretion of electrolytes and water in both intestine and kidney. Thought to act as “intestinal natriuretic factors”, GN and UGN modulate renal salt secretion by both endocrine mechanisms (linking the digestive system and kidney) and paracrine/autocrine (intrarenal) mechanisms. The cellular function of GN and UGN in intestine and proximal tubule is mediated by guanylyl cyclase C (GC-C)-, cGMP-, and G protein-dependent pathways, whereas, in principal cells of the cortical collecting duct (CCD), these peptide hormones act via GC-C-independent signaling through phospholipase A2 (PLA2). The Cl-/HCO-3 exchanger pendrin (SLC26A4), encoded by the PDS gene, is expressed in non-α intercalated cells of the CCD. Pendrin is essential for CCD bicarbonate secretion and is also involved in NaCl balance and blood pressure regulation. Our recent studies have provided evidence that pendrin-mediated anion exchange in the CCD is regulated at the transcriptional level by UGN. UGN exerts an inhibitory effect on the pendrin gene promoter likely via heat shock factor 1 (HSF1) action at a defined heat shock element (HSE) site. Recent studies have unraveled novel roles for guanylin peptides in several organ systems including involvement in appetite regulation, olfactory function, cell proliferation and differentiation, inflammation, and reproductive function. Both the guanylin system and pendrin have also been implicated in airway function. Future molecular research into the receptors and signal transduction pathways involved in the action of guanylin peptides and the pendrin anion exchanger in the kidney and other organs, and into the links between them, may facilitate discovery of new therapies for hypertension, heart failure, hepatic failure and other fluid retention syndromes, as well as for diverse diseases such as obesity, asthma, and cancer.

The claudin-16 channel gene is transcriptionally inhibited by 1,25-dihydroxyvitamin D.

What is the central question of this study? In the kidney, the bulk of the filtered Mg2+ is reabsorbed in the thick ascending limb by paracellular conductance, mediated by the tight junction protein, claudin‐16, which is encoded by the gene CLDN16. The role of 1,25‐dihydroxyvitamin D [1,25(OH)2VitD] in renal Mg2+ handling is unclear. We aimed to explore the molecular mechanisms underlying the effect of 1,25(OH)2VitD on claudin‐16‐mediated Mg2+ transport. What is the main finding and its importance? Paracellular, claudin‐16‐mediated Mg2+ transport is transcriptionally repressed by 1,25(OH)2VitD, probably via a Ca2+‐sensing receptor‐dependent mechanism. This renal effect of 1,25(OH)2VitD may serve as an adaptive mechanism to the 1,25(OH)2VitD‐induced enteric hyperabsorption of dietary Mg2+.