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BACKGROUND Busulfan (Bu) conditioning used in hematopoietic stem cell transplantation (HSCT) may induce seizures, and prophylactic antiepileptic treatment is recommended. Following updated guidelines, in August 2019, the adult HSCT department of the Rambam Health Care Campus (Haifa, Israel) switched the antiepileptic prophylaxis protocol from phenytoin to oral levetiracetam during oral Bu conditioning. The aim of this study was to compare the pharmacokinetic parameters of Bu after oral dosing between patients receiving phenytoin and those receiving levetiracetam prophylaxis. METHODS This study was a retrospective cohort study in adults undergoing myoablative conditioning with oral busulfan between August 2018 and August 2020. Bu pharmacokinetic parameters (AUC0-6, C0, Cmax, Tmax) were compared in patients treated with phenytoin comedication (during the year before the change in policy) and levetiracetam comedication (during the year after the change). Potential confounders were accounted for, including age, azole co-medication, and body weight. RESULTS There were no significant differences in demographic and clinical parameters or weight-corrected busulfan dose between the phenytoin group (n = 28) and the levetiracetam group (n = 25). There was no difference in the rate of voriconazole comedication, but fluconazole was more common in the phenytoin group (P = 0.026). The median AUC0-6 was significantly lower in the levetiracetam group (949 micM*min; IQR=806 to 1101 micM*min) than in the phenytoin group (1208 micM*min; IQR=1087 to 1389 micM*min; P < 0.001). This is a clinically significant difference of 258 micM*min (21%). Azole use was not associated with Bu exposure. CONCLUSION The findings suggest that, after treatment with oral busulfan, oral levetiracetam comedication is associated with reduced systemic exposure compared to phenytoin comedication, possibly due to decreased bioavailability.

Meropenem is a broad-spectrum carbapenem antibiotic with mostly renal excretion. Conflicting data are available regarding meropenem pharmacokinetics in critically ill neonates on concomitant continuous renal replacement therapy (CRRT) and/or extracorporeal membrane oxygenation (ECMO). Our objectives were to assess meropenem clearance in a neonate on CRRT and ECMO, compare it to previously published data and assess whether dose recommendations can be generalized in this population.A 2.5 kg male infant with a large diaphragmatic hernia was delivered by cesarean section at week 35 and immediately mechanically ventilated due to shock and respiratory insufficiency. He underwent surgical correction of the hernia, but developed recurrent sepsis, multiorgan failure and pulmonary hypertension. He remained mechanically ventilated and required ECMO and continuous venovenous hemodiafiltration. He was started on meropenem 40 mg/kg/dose, every 8 hs for Enterobacter cloacae bacteremia and sepsis, but due to lack of clinical and microbiologic response despite in vitro susceptibility, he was started on a continuous meropenem infusion of 240 mg/kg/d, based on dose recommendations in a similar case. We measured steady state meropenem plasma concentrations on 2 occasions, during ECMO and continuous venovenous hemodiafiltration (CVVHDF) and then on CVVHDF only.Meropenem serum concentrations were 90 and 64 mg/L on the first and second occasion (therapeutic target concentration, 10 mg/L) corresponding to a clearance of 1.9 and 2.6 mL/kg/min. This clearance estimate was substantially lower than that reported in toddlers on CRRT and ECMO in some studies.In neonates and infants, meropenem clearance is difficult to predict because of dynamic ontogenetic changes in renal function. This problem is further aggravated in acutely ill infants with decreased renal function, renal replacement therapy and/or ECMO. Therefore, Target Concentration Intervention based on meropenem plasma concentrations is indispensable to ensure therapeutic exposure in this population.

Introduction Acute kidney injury (AKI) after high dose methotrexate (HD-MTX) is associated with delayed MTX-excretion and life-threatening toxicity. Glucapridase, the recommended therapy, is expensive and not always available. Case series We describe 3 cases (69, 67, 73 years) with diffuse large B-cell lymphoma who developed AKI and early-onset severely delayed MTX elimination after HD-MTX. MTX serum concentrations were 101 and 69 μmol/L at 24 h after administration in two patients and 34 μmol/L at 32 h in the third. Management and outcome Since glucarpidase was unavailable, we performed daily high-flux hemodialysis (HF-HD) or online hemodiafiltration (HDF) sessions (median duration, 6 h). The median serum MTX elimination half-life during HDF/HF-HD sessions was similar in all patients (median, 4.4 h; IQR, 3.8–5.3 h), but serum MTX concentrations rebounded after each dialysis by a median of 40% of the trough concentrations. The three patients underwent multiple dialysis sessions, until MTX serum concentrations remained sufficiently low to be neutralized by leucovorin. Only 1 patient developed severe pancytopenia, and renal function normalized in all patients after 3–6 weeks. Discussion In conclusion, when glucarpidase is unavailable or delayed, early, repeated and prolonged HDF/HF-HD effectively enhance MTX elimination and prevent toxicity in patients with AKI and severely delayed MTX elimination after HD-MTX.

BACKGROUND Therapeutic drug monitoring (TDM) for busulfan supports dose adjustment during conditioning for stem cell transplantation. The authors aimed to develop and validate limited sampling strategies (LSS) of 4-5 samples for a precise estimation of the area under concentration (AUC)-time curve of busulfan, in plasma as an alternative to an intensive sampling strategy (ISS) requiring 9-10 samples. METHODS ISS TDM data from 297 patients (≤18 years of age) were used. AUCLSS was calculated using the trapezoidal rule and multiple linear regression (MLR). Unlike more complex modeling methods, MLR does not require sophisticated software or advanced training of personnel. MLR coefficients were estimated in the development subset containing randomly selected 50% of the records and were then used to calculate the AUCLSS of the remaining records (the validation subset). The agreement between dose adjustment recommendations (DAR) based on ISS and LSS, in the validation subset, was evaluated by a Bland-Altman analysis. A DAR deviating from an ISS-based reference by

Liquid chromatography with mass spectrometry (LC-MS/MS) is the method of choice for the determination of everolimus whole blood concentrations but is not always available. Therefore, immunoassays have been developed for clinical monitoring of everolimus. In previous studies, the Quantitative Microsphere System (QMS) immunoassay had a positive bias compared with LC-MS/MS, but was judged acceptable, although clinical agreement (eg, 95% limits of agreement) was not reported. The objective of this study was to assess whether the agreement between the QMS assay and an LC-MS/MS method was clinically acceptable for use interchangeably in therapeutic everolimus monitoring.Whole blood samples from organ-transplanted patients on everolimus therapy were analyzed by both QMS (on Architect ci4100 analyzer) and LC-MS/MS. Paired results were compared using paired Student t test, Bland-Altman plots, and Deming regression analysis. The proportions of falsely supratherapeutic and subtherapeutic results on the QMS assay compared with the LC-MS/MS were calculated.Among 250 samples (169 patients), mean everolimus concentrations determined by LC-MS/MS and QMS assays were 4.8 ± 2.1 ng/mL and 6.3 ± 2.1 ng/mL, respectively (P0.001), with 95% lines of agreement between -2.1 and 5.2 ng/mL, a range corresponding to 152% of the mean concentration. When stratified by the type of transplant, a similar positive bias was found in each subgroup (all P0.014). Sixty-nine percent of the samples yielding supratherapeutic concentrations (8 ng/mL) on the QMS assay were within the therapeutic range on the LC-MS/MS.The everolimus QMS immunoassay, using the Architect ci4100 analyzer, had a significant positive bias compared with LC-MS/MS, with a wide range between the limits of agreement. The lack of agreement may result in inadequate everolimus dose adjustments, suggesting that the QMS assay cannot be used interchangeably with the LC-MS/MS method for therapeutic everolimus monitoring in organ-transplanted patients.

Infants exclusively breastfed with zinc-deficient milk, develop transient neonatal zinc deficiency (TNZD) due to inactivating mutations in the maternal zinc transporter SLC30A2/ZnT2. In the current study, DNA sequencing of SLC30A2/ ZnT2 in a mother exclusively breastfeeding her infant which presented with TNZD symptoms, identified a single nucleotide missense mutation c.839G>T. This mutation was initially predicted to result in a single amino acid substitution p.(Gly280Val). However, c.838G was also predicted to be located close to a splice site. Hence, using sequencing of mRNA isolated from the mother’s breast milk cells, we discovered that the c.839G>T mutation creates an alternative 3’-splice sites, introducing a 27 bp exon skipping. This mutant protein lacking 9 amino acids (Δ9AA) failed to dimerize with the wild type (WT) ZnT2 protein and displayed enhanced degradation. Furthermore, the zinc transport ability of this mutant protein was completely lost as shown by intracellular zinc accumulation using the zinc fluorescent probes Zinquin and Fluozin 3 AM. These findings reveal that the Δ9AA-ZnT2 variant did not exert a dominant negative effect over the WT ZnT2 protein. Hence, we provide direct evidence that a haploinsufficiency state occurs in women with heterozygous SLC30A2/ZnT2 mutations. Importantly, we herein developed a novel genetic test of expressed genes using maternal breast milk cells, which is well suited for the early diagnosis and prevention of TNZD and possibly other micronutrient deficiencies.

New Findings 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+. Magnesium is reabsorbed in the thick ascending limb by paracellular conductance, mediated by the CLDN16-encoded tight junction protein, claudin-16. However, the role of 1,25-dihydroxyvitamin D [1,25(OH)2VitD] in renal Mg2+ handling is unclear. We have shown that Mg2+ depletion increases and 1,25(OH)2VitD inhibits CLDN16 transcription. We have now explored further the molecular mechanisms underlying the effect of 1,25(OH)2VitD on claudin-16-mediated Mg2+ transport. Adult mice received parenteral 1,25(OH)2VitD or 1,25(OH)2VitD combined with either high-Mg2+ or low-Mg2+ diets. Administration of 1,25(OH)2VitD enhanced urinary excretion of Mg2+ and Ca2+. The 1,25(OH)2VitD also increased renal Ca2+-sensing receptor (CaSR) mRNA and decreased renal claudin-16 and claudin-19 mRNA and claudin-16 protein, but did not affect renal claudin-2 mRNA. The 1,25(OH)2VitD reversed the expected increase in claudin-16 mRNA in Mg2+-depleted animals. Comparably treated HEK 293 cells showed similar changes in claudin-16 mRNA, but 1,25(OH)2VitD did not alter mRNA of the TRPM6 Mg2+ channel. A luciferase reporter vector containing 2.5 kb of 5′-flanking DNA sequence from human CLDN16 (hCLDN16) was transfected into HEK 293 and OK cells. The hCLDN16 promoter activity was modestly decreased by 1,25(OH)2VitD, but markedly inhibited in HEK 293 cells coexpressing CaSR. Coexpression in OK cells of dominant-negative CaSR completely abolished inhibition of hCLDN16 promoter activity by 1,25(OH)2VitD. The 1,25(OH)2VitD-induced decrease in hCLDN16 promoter activity was attenuated in Mg2+-depleted HEK 293 cells. In conclusion, 1,25(OH)2VitD transcriptionally inhibits claudin-16 expression by a mechanism sensitive to CaSR and Mg2+. This renal effect of 1,25(OH)2VitD may serve as an adaptive response to the 1,25(OH)2VitD-induced increase in intestinal Mg2+ absorption.

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 Crohn's 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 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.

BACKGROUND Thiopurines are efficacious in the treatment of Crohn's 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 Crohn's 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.

The pendrin/SLC26A4 Cl−/HCO3− exchanger, encoded by the PDS gene, is expressed in cortical collecting duct (CCD) non-A intercalated cells. Pendrin is essential for CCD bicarbonate secretion and is also involved in NaCl balance and blood pressure regulation. The intestinal peptide uroguanylin (UGN) is produced in response to oral salt load and can function as an “intestinal natriuretic hormone.” We aimed to investigate whether UGN modulates pendrin activity and to explore the molecular mechanisms responsible for this modulation. Injection of UGN into mice resulted in decreased pendrin mRNA and protein expression in the kidney. UGN decreased endogenous pendrin mRNA levels in HEK293 cells. A 4.2-kb human PDS (h PDS) promoter sequence and consecutive 5′ deletion products were cloned into luciferase reporter vectors and transiently transfected into HEK293 cells. Exposure of transfected cells to UGN decreased h PDS promoter activity. This UGN-induced effect on the h PDS promoter occurred within a 52-bp region encompassing a single heat shock element (HSE). The effect of UGN on the promoter was abolished when the HSE located between nt −1119 and −1115 was absent or was mutated. Furthermore, treatment of HEK293 cells with heat shock factor 1 (HSF1) small interfering RNA (siRNA) reversed the UGN-induced decrease in endogenous PDS mRNA level. In conclusion, pendrin-mediated Cl−/HCO3− exchange in the renal tubule may be regulated transcriptionally by the peptide hormone UGN. UGN exerts its inhibitory activity on the h PDS promoter likely via HSF1 action at a defined HSE site. These data define a novel signaling pathway involved in the enterorenal axis controlling electrolyte and water homeostasis.

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 hPDS promoter. 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(-)/HCO(3)(-) 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.

Background 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. Procedure The present study integrated clinical data regarding the occurrence of HVOD, graft versus host disease (GVHD), BU pharmacokinetics and GSTA1, GSTP1, GSTM1, and GSTT1 genotypes of 18 children who received BU in their pre-HSCT conditioning regimen. The children were all treated for congenital hemoglobinopathies and were all of Arab Moslem descent. Results The data demonstrate an association between GSTA1 and GSTP1 genotypes and BU-maximal concentration (Cmax) (P = 0.01, P = 0.02, respectively), area under the concentration-time curve (AUC) (P = 0.02, P = 0.01, respectively) and oral BU clearance/kg body weight (P

Hematopoietic SCT (HSCT) from HLA-matched donors is sometimes complicated by GVHD or graft rejection, because of mismatched mHA. This study presents data suggesting the involvement of glutathione S-transferase theta-1 (GSTT1), a phase II detoxifying enzyme encoded by GSTT1, in Ab-mediated rejection of HSCT in children with congenital hemoglobinopathies (CHs). Mismatch of GSTT1, which often features a deletion polymorphism variant, can have major consequences in solid organ transplantation outcome. In liver transplantation, it has been shown to lead to de novo hepatitis, whereas in kidney transplantation, chronic allograft rejection has been documented. In this study on 18 children with CH who underwent HSCT, five cases of graft rejection occurred, all in GSTT1-null patients, four of which featured anti-GSTT1 antibodies. The data suggest that when GSTT1-null patients are transplanted with a GSTT1-positive graft, rejection due to an Ab-mediated immune response against GSTT1 displayed on transplanted stem cells may take place. Thus, it seems that detection of anti-GSTT1 antibodies in patients with a GSTT1-null genotype before transplantation may be predictive of graft rejection in the event of a GSTT1-positive donor.

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, w

Individuals 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. TPMT genotyping of individuals representing three major demographic groups in Israel was carried out by PCR restriction fragment length polymorphism and high-resolution melting. Frequencies 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. These 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.

Thiopurines are effective for maintenance of remission in inflammatory bowel disease (IBD) in only about half of patients. Predictors of response may assist in selecting the most appropriate patients for thiopurine therapy. Thiopurines inhibit Rac1, a GTPase that exerts an antiapoptotic effect on T-lymphocytes. A genetic association was recently demonstrated between a Rac1 single nucleotide polymorphism (SNP) and poorer response to thiopurines in adult patients with Crohn disease. We aimed to determine whether Rac1 SNPs are associated with response to thiopurines in children with IBD.Children with IBD treated with thiopurines were prospectively followed for 1 year and were genotyped for 3 Rac1 SNPs previously found to be relevant to IBD: rs10951982, rs4720672, and rs34932801. The rate of sustained steroid-free remission (SSFR) without treatment escalation by 12 months was compared between wild types (WTs) and heterozygotes.A total of 59 patients were studied (63% boys, 80% having Crohn disease, mean age 13 ± 4.1). Nineteen of the 41 WT (46%) and 9 of the 15 (60%) heterozygotes for rs10951982 were in SSFR (P = 0.55). Similarly, 21 of the 45 (47%) WT and 8 of the 12 (67%) heterozygotes for rs4720672 were in remission (P = 0.33). Finally, 21 of the 45 (47%) WT and 3 of the 5 (60%) heterozygotes for rs34932801 were in remission (P = 0.66). All of the 3 comparisons remained nonsignificant in a sensitivity analysis of only the patients with Crohn disease.We did not find an association between 3 Rac1 SNPs and thiopurine effectiveness by 12 months in a prospective study of children with IBD. Other predictors of response should be sought to optimize patient selection for thiopurine therapy.

The D1790G mutation was found in all 24 patients of an extended long QT family but not in 200 chromosomes carried by healthy individuals. We describe a 37-year-old man presenting with a typical spontaneous type 1 Brugada pattern who in electrophysiological testing had easily inducible ventricular fibrillation. At the age of 47 years he had an atrial ventricular type 2 block documented by an exercise test and a Holter monitor. Genetic analysis revealed a known D1790G mutation in the gene encoding of the sodium channel (SCN5A) that until now has been associated only with the long QT phenotype. Although this mutation has not been associated with a reduction of sodium channel expression, we hypothesize that sodium currents are further diminished due to the 20-mV shift of the steady-state inactivation curve, and this could contribute to the Brugada phenotype. This case is important as it allows a better understanding of the underlying molecular mechanisms of Brugada syndrome. Moreover, this observation raises concern about the safety of class IC drug therapy in long QT type 3 patients and quinidine therapy in Brugada patients, and emphasizes the importance of a thorough clinical and genetic evaluation.

Tubular reabsorption of Mg2+is mediated by the tight junction protein paracellin-1, which is encoded by the gene PCLN-1 (CLDN16) and exclusively expressed in the kidney. Tubular Mg2+reclamation is modulated by many hormones and factors. The aim of this study was to define regulatory elements essential for renal tubular cell-specific expression of human PCLN-1 ( hPCLN-1) and to explore the effect of Mg2+transport modulators on the paracellin-1 gene promoter. Endogenous paracellin-1 mRNA and protein were detected in renal cell lines opossom kidney (OK), HEK293, and MDCT, but not in the fibroblast cell line NIH3T3. A 7.5-kb hPCLN-1 5′-flanking DNA sequence along with seven 5′-deletion products were cloned into luciferase reporter vectors and transiently transfected into the renal and nonrenal cells. The highest levels of luciferase activity resulted from transfection of a 5′-flanking 2.5-kb fragment (pJ2M). This activity was maximal in OK cells, was orientation dependent, and was absent in NIH3T3 cells. Mg2+deprivation significantly increased pJ2M-driven activity in transfected OK cells, whereas Mg2+load decreased it compared with conditions of normal Mg2+. Deletion analysis along with electrophoretic mobility-shift assay demonstrated that OK cells contain nuclear proteins, which bind a 70-bp region between −1633 and −1703 of major functional significance. Deleting this 70-bp segment, which contains a single peroxisome proliferator-response element (PPRE), or mutating the PPRE, caused a 60% reduction in luciferase activity. Stimulating the 70-bp sequence with 1,25(OH)2vitamin D decreased luciferase activity by 52%. This effect of 1,25(OH)2vitamin D was abolished in the absence of PPRE or in the presence of mutated PPRE. We conclude that the PPRE within this 70-bp DNA region may play a key role in the cell-specific and regulatory activity of the hPCLN-1 promoter. Ambient Mg2+concentration and 1,25(OH)2vitamin D may modulate paracellular, paracellin-1-mediated, Mg2+transport at the transcriptional level. 1,25(OH)2vitamin D exerts its activity on the hPCLN-1 promoter likely via the PPRE site.

What is the central question of this study? In the kidney, the bulk of the filtered Mg(2+) 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)2 VitD] in renal Mg(2+) handling is unclear. We aimed to explore the molecular mechanisms underlying the effect of 1,25(OH)2 VitD on claudin-16-mediated Mg(2+) transport. What is the main finding and its importance? Paracellular, claudin-16-mediated Mg(2+) transport is transcriptionally repressed by 1,25(OH)2 VitD, probably via a Ca(2+)-sensing receptor-dependent mechanism. This renal effect of 1,25(OH)2 VitD may serve as an adaptive mechanism to the 1,25(OH)2 VitD-induced enteric hyperabsorption of dietary Mg(2+). Magnesium is reabsorbed in the thick ascending limb by paracellular conductance, mediated by the CLDN16-encoded tight junction protein, claudin-16. However, the role of 1,25-dihydroxyvitamin D [1,25(OH)2 VitD] in renal Mg(2+) handling is unclear. We have shown that Mg(2+) depletion increases and 1,25(OH)2 VitD inhibits CLDN16 transcription. We have now explored further the molecular mechanisms underlying the effect of 1,25(OH)2 VitD on claudin-16-mediated Mg(2+) transport. Adult mice received parenteral 1,25(OH)2 VitD or 1,25(OH)2 VitD combined with either high-Mg(2+) or low-Mg(2+) diets. Administration of 1,25(OH)2 VitD enhanced urinary excretion of Mg(2+) and Ca(2+). The 1,25(OH)2 VitD also increased renal Ca(2+)-sensing receptor (CaSR) mRNA and decreased renal claudin-16 and claudin-19 mRNA and claudin-16 protein, but did not affect renal claudin-2 mRNA. The 1,25(OH)2 VitD reversed the expected increase in claudin-16 mRNA in Mg(2+)-depleted animals. Comparably treated HEK 293 cells showed similar changes in claudin-16 mRNA, but 1,25(OH)2 VitD did not alter mRNA of the TRPM6 Mg(2+) channel. A luciferase reporter vector containing 2.5 kb of 5'-flanking DNA sequence from human CLDN16 (hCLDN16) was transfected into HEK 293 and OK cells. The hCLDN16 promoter activity was modestly decreased by 1,25(OH)2 VitD, but markedly inhibited in HEK 293 cells coexpressing CaSR. Coexpression in OK cells of dominant-negative CaSR completely abolished inhibition of hCLDN16 promoter activity by 1,25(OH)2 VitD. The 1,25(OH)2 VitD-induced decrease in hCLDN16 promoter activity was attenuated in Mg(2+)-depleted HEK 293 cells. In conclusion, 1,25(OH)2 VitD transcriptionally inhibits claudin-16 expression by a mechanism sensitive to CaSR and Mg(2+). This renal effect of 1,25(OH)2 VitD may serve as an adaptive response to the 1,25(OH)2 VitD-induced increase in intestinal Mg(2+) absorption.

8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG), a common oxidative DNA lesion, favors a syn-conformation in DNA, enabling formation of stable 8-oxo-dG·A base mispairs resulting in G·C → T·A transversion mutations. When human DNA polymerase (pol) β was used to copy a short single-stranded gap containing a site-directed 8-oxo-dG lesion, incorporation of dAMP opposite 8-oxo-dG was slightly favored over dCMP depending on “downstream” sequence context. Unexpectedly, however, a significant increase in dCMP·A and dGMP·A mispairs was also observed at the “upstream” 3′-template site adjacent to the lesion. Errors at these undamaged template sites occurred in four sequence contexts with both gapped and primed single-stranded DNA templates, but not when pol α replaced pol β. Error rates at sites adjacent to 8-oxo-dG were roughly 1% of the values opposite 8-oxo-dG, potentially generating tandem mutations during in vivo short-gap repair synthesis by pol β. When 8-oxo-dG was replaced with 8-bromo-2′-deoxyguanosine, incorporation of dCMP was strongly favored by both enzymes, with no detectable misincorporation occurring at neighboring template sites.

The attenuation in the capacity to respond to stress is one of the major characteristics of ageing organisms. There are several cellular pathways of response to various types of stress. We briefly discuss the NFkB pathway that is altered with age as was primarily shown by Papaconstantinou. We suggest that another important transcription factor system that should be investigated is that involved in the response to hypoxia. Our own work shows that the capacity to respond to elevated temperatures is considerably diminished in ageing organisms. Our studies demonstrate that this is due to faults in the control of the activation of the transcription factor, HSF, which is a major protein that activates many stress response genes. The nature of the alteration of the control of HSF with age is described and discussed.

Methotrexate (MTX), a folate antagonist employed for treating a wide range of malignancies, has an extensive interpatient variability, resulting in unpredictable toxicity. The present study evaluated the impact of single gene polymorphisms (SNPs: rs1801133 and rs1801131 in the MTHFR gene; rs4149056 and rs11045879 in the SLC01B1 gene; and rs2032582 and rs1045642 in the ABCB1 transporter gene) on MTX blood levels and toxicity in samples from 69 patients with diffuse large-B-cell lymphoma (DLBCL) treated with high dose intravenous (HD IV) MTX,2 g/m(2). None of the studied genotypes was found to be associated with a statistically significant risk for elevated MTX levels at 24-48 h after completing therapy with MTX. Ancestral alleles (T) for SLC01B1 rs4149056 (T521C) and SLC01B1 rs11045879 (intron C21273886T) were found to be associated with an increased risk for MTX-related toxicity (p0.05 and p = 0.07, respectively), emphasizing the potential importance of employing pharmacogenetic assessment for personalized medicine.

MTHFR C677T is a common gene polymorphism that has been shown to be associated with hyperhomocysteinemia. Studies on the role of MTHFR in inflammatory bowel diseases (IBD) have yielded conflicting results, perhaps due in part to genetic heterogeneity. The prevalence of the MTHFR C677T variant allele varies according to Jewish subpopulations: Ashkenazi vs non-Ashkenazi. The aim of this study was to examine the association between MTHFR C677T genotype and IBD in the different Jewish populations. DNA samples were assessed for the presence of the MTHFR C677T variant allele in 445 Jewish Israeli IBD patients: 338 with Crohn's disease [CD] (214 Ashkenazi and 124 non-Ashkenazi Jews) and 107 with ulcerative colitis [UC] (73 Ashkenazi and 34 non-Ashkenazi Jews), and in 347 healthy controls: 173 Ashkenazi and 174 Non-Ashkenazi Jews. Possible genotype–phenotype associations were investigated. We showed a significantly higher frequency of MTHFR 677T variant genotypes in non-Ashkenazi CD patients: Odds ratio of 1.86 for heterozygotes (CT) and 2.89 for homozygotes (TT) compared to non-Ashkenazi healthy controls. No significant association was found for UC in non-Ashkenazi patients or for CD or UC in Ashkenazi patients. Our findings suggest that the MTHFR 677T variant may contribute to the risk of CD in non-Ashkenazi but not Ashkenazi Jews. This may result from genetic heterogeneity and highlights the complexity of the genetic etiology of IBD.

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.

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: Usingalockednucleicacid(LNA)containingoligonucleotide, wedevelopedaPCRclampingmethodthatpreferentially 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 29KRAS 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.

KRAS mutation status has a significant role determining anti-epidermal growth factor receptor (anti-EGFR) treatment response in colon carcinoma patients. Malignant transformation is a dynamic process and therefore, it is conceivable that, at a certain point, the tumor cells’ mass might be heterogeneous for particular mutations. Therefore, the fraction of tumor cells carrying a particular mutation may be more relevant for treatment than the simple determination of presence or absence of mutation. The purpose of this study is to assess whether or not KRAS mutation status is heterogeneous and, if so, to what extent in colon carcinoma samples. Deoxyribonucleic acid was extracted from formalin-fixed paraffin-embedded samples of colon carcinoma and analyzed for the presence of KRAS mutation. The relative fraction of mutated versus wild-type KRAS alleles was evaluated by real-time polymerase chain reaction. Additionally, the relative fraction of cancer cells in the tissue sample was evaluated using computer assisted morphometric analysis. Using this data, we calculated the fraction of mutation containing cells in the samples. Colon carcinoma (169 cases) were analyzed, and a KRAS mutation was found in 75 cases (44%), of which 42 were available for morphometric analysis. In 41 (97.6%) of these cases, the fraction of mutation containing tumor cells was 50% or higher, indicating the absence of significant KRAS mutation status heterogeneity. There was a strong positive correlation (R = 0.66, P < 0.0001) between the fraction of mutated KRAS alleles and the fraction of cancer cells in the samples. The strong positive correlation between the fraction of mutated KRAS alleles and the fraction of cancer cells in the samples indicate homogeneity of KRAS mutation status in colorectal carcinoma.

The involvement of oxidant/antioxidant imbalance in the development of inflammatory bowel disease (IBD) is well documented. Two members of the glutathione S-transferase (GST) family of enzymes, GSTM1 and GSTT1, known to take part in cellular protection against electrophiles, demonstrate common deletion variants (termed null) associated with impaired enzyme function. To evaluate the effect of GSTM1/GSTT1 genotype on IBD susceptibility in a Israeli cohort and to study the correlation between GSTM1/GSTT1 genotype, smoking status, and a variety of clinical characteristics of IBD. A cohort of 606 Israeli IBD patients (453 with Crohn’s disease [CD] and 153 with ulcerative colitis [UC]) and 528 ethnically matched healthy controls were genotyped for the null variants of GSTM1 and GSTT1. In patients, phenotype–genotype correlations were examined. Ethnic stratification of healthy controls revealed a higher frequency of GSTT1-null in Jewish and Arab Moslem individuals compared to Druze individuals (P

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.The present study integrated clinical data regarding the occurrence of HVOD, graft versus host disease (GVHD), BU pharmacokinetics and GSTA1, GSTP1, GSTM1, and GSTT1 genotypes of 18 children who received BU in their pre-HSCT conditioning regimen. The children were all treated for congenital hemoglobinopathies and were all of Arab Moslem descent.The data demonstrate an association between GSTA1 and GSTP1 genotypes and BU-maximal concentration (C(max)) (P = 0.01, P = 0.02, respectively), area under the concentration-time curve (AUC) (P = 0.02, P = 0.01, respectively) and oral BU clearance/kg body weight (P0.02, P = 0.08, respectively). GSTM1-null individuals demonstrated lower BU-AUC/Kg compared to GSTM1-positive individuals. In addition, an association between GVHD and GSTM1-null genotype was found.GSTA1, GSTP1, and GSTM1 genotyping prior to HSCT in children with congenital hemoglobinopathies may allow better prediction of oral BU kinetics and the need for BU dose adjustment, as well as prediction of transplant related toxicity such as GVHD, thereby improving clinical outcome.

Renal tubular Mg(2+) reabsorption is mediated predominantly by the tight junction channel protein claudin-16 which is encoded by the gene CLDN16. Hypermagnesemia decreases, whereas hypomagnesemia increases Mg(2+) reabsorption. This study examines the role of claudin-16 in the adaptive response of the kidney to Mg(2+) availability.Mice received a low-, normal- or high Mg(2+) diet for up to 3 days. Mg(2+)-loaded animals displayed hypermagnesemia with increasing urine Mg(2+)/Ca(2+) levels paralleled by a decrease in claudin-16 protein and mRNA in the kidney. Mg(2+)- deprived animals developed hypomagnesemia with decreasing urine Mg(2+)/Ca(2+) levels associated with an increase in claudin-16 protein and mRNA abundance. Mg(2+) depletion markedly increased and Mg(2+) load decreased endogenous claudin-16 mRNA levels in calcium-sensing receptor-transfected HEK293 cells compared with native HEK293 cells. The effect of Mg(2+) 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 Mg(2+) load decreases hCLDN16 promoter activity in transfected HEK293 cells.Changes in Mg(2+) availability may influence claudin-16 mediated Mg(2+) transport at the transcriptional level. The possible involvement of the cell membrane bound Ca(2+)/Mg(2+) sensing receptor or the potential role of a hypothetical Mg(2+) response element on the CLDN16 promoter in the Mg(2+)-induced response remains to be explored.

Purpose: This study was designed to delineate the relative frequency of CYP2C9 and VKORC1 polymorphisms known to affect warfarin response in the highly heterogeneous Israeli population. Methods: Frequencies of CYP2C9 allelic variants CYP2C9*2, CYP2C9*3 and of VKORC1 single nucleotide polymorphisms (snps)- 1639G > A and D36Y were determined in genomic DNA of 438 healthy unrelated Israeli volunteers of Jewish, Druze and Arab Moslem descent, using allele specific PCR-RFLP. Genotyping results obtained were confirmed by probe free High Resolution Melt (HRM) Technology. Results: Arab Moslems had a higher frequency of warfarin “sensitive” CYP2C9*2, CYP2C9*3 and VKROC1-1639G > A alleles (0.21, 0.07 and 0.58, respectively) than both Jews (0.13, 0.11 and 0.57, respectively) and Druze (0.12, 0.06 and 0.53, respectively). Statistically significant differences were found in CYP2C9*2 between Druze and Moslems (p=0.01) and between Jews and Moslems (p=0.016) and in CYP2C9*3 between Druze and Jews (p=0.0086). VKORC1(-1639G > A) was the major gene polymorphism associated with warfarin sensitivity in all 3 subpopulations. In contrast, the warfarin “resistant” VKORC1 D36Y allele was very rare in the Israeli population (0- 0.015). The results presented demonstrate that allelic variants in CYP2C9 and VKORC1 are very common in Israel with ∼95% of Jews, ∼84% of Druze and ∼91% of Arab Moslems manifesting at least one known warfarin “sensitive” or “resistant” allele. Conclusions: Individualized genotype based warfarin therapy is highly relevant in the Israeli population due to the high incidence of genetic variations associated with warfarin sensitivity in all 3 non-mixing subpopulations tested.

Individuals 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.TPMT genotyping of individuals representing three major demographic groups in Israel was carried out by PCR restriction fragment length polymorphism and high-resolution melting.Frequencies 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.These 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.

Pendrin, 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, iodide accumulation, and endolymph ion balance, respectively. This study aimed to define promoter regulatory elements essential for renal, thyroid, and inner ear epithelial cell-specific expression of human PDS (hPDS) and to explore the effect of ambient pH and aldosterone on hPDS promoter activity. Endogenous pendrin mRNA and protein were detected in renal HEK293, thyroid LA2, and inner ear VOT36 epithelial cell lines, but not in the fibroblast cell line, NIH3T3. A 4.2-kb hPDS 5′-flanking DNA sequence and consecutive 5′-deletion products were cloned into luciferase reporter vectors and transiently transfected into the above cell lines. Distinct differences in expression/activity of deduced positive/negative regulatory elements within the hPDS promoter between HEK293, LA2, and VOT36 cells were demonstrated, with only basal activity in NIH3T3 cells. Acidic pH (7.0–7.1) decreased and alkaline pH (7.6–7.7) increased hPDS promoter activity in transfected HEK293 and VOT36, but not in LA2 cells. Aldosterone (10−8 M) 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. Acidic pH and aldosterone decreased, and alkaline pH increased, endogenous pendrin mRNA level in HEK293 cells. In conclusion, pendrin-mediated HCO3− secretion in the renal tubule and anion transport in the endolymph may be regulated transcriptionally by systemic pH and aldosterone.

Peripheral T-cell lymphomas (PTCLs) constitute a rare and very heterogeneous group of non-Hodgkin lymphoma (NHL), with various compositions in different subtypes, depending on geographic location a...

Background: The best described primary inherited proximal tubulopathies include X-linked hypercalciuric nephrolithiasis (XLHN), caused by a mutation in the chloride channel gene CLCN5, and classic Fanconi's syndrome, the genetic basis of which is unknown. The aim of this study is to examine the clinical, biochemical, and genetic characteristics of a highly consanguineous Druze family with autosomal recessive proximal tubulopathy and hypercalciuria (ARPTH), a syndrome not reported previously. Methods: Three children (2 girls, 1 boy) of the family referred for evaluation of renal glycosuria and hypercalciuria and 10 of their close relatives were evaluated clinically and biochemically. All study participants underwent genetic analysis to exclude involvement of the CLCN5 gene. Results: Evaluation of the 3 affected children showed glycosuria, generalized aminoaciduria, hypouricemia, uricosuria, low molecular weight (LMW) proteinuria, and hypercalciuria in all 3 children and phosphaturia in 2 children. They had no metabolic acidosis or renal insufficiency. One affected girl had nephrocalcinosis. Two children had a history of growth retardation and radiological findings of metabolic bone disease. Parathyroid hormone and 1,25-dihydroxyvitamin D [1,25(OH) 2 Vit D] blood levels in affected children were normal. Unaffected family members examined had no renal tubular defects or LMW proteinuria. Genetic linkage analysis excluded cosegregation of the ARPTH phenotype with the CLCN5 locus. Conclusion: ARPTH is a new syndrome characterized by nonacidotic proximal tubulopathy, hypercalciuria, metabolic bone disease, and growth retardation. It can be distinguished from XLHN by its autosomal recessive mode of inheritance and normal serum levels of calciotropic hormones, as well as the absence of LMW proteinuria in obligate carriers. The gene mutated in ARPTH remains to be identified.

The effects of abasic lesions on both primase activity and DNA polymerase alpha- (pol alpha) catalyzed elongation of primase-synthesized primers were examined. Abasic lesions were strong chain terminators during primer synthesis by primase. However, extension of primase-synthesized primers by pol alpha resulted in 60-93% bypass of abasic lesions. Sequencing of bypass products generated during this primase-coupled pol alpha activity showed that dAMP was preferentially incorporated opposite the abasic lesion, indicating that pol alpha was responsible for bypass. In contrast, previous analyses of pol alpha-catalyzed elongation of exogenously supplied DNA primer-templates showed that abasic lesions strongly terminated DNA synthesis. Thus, elongation of primase-synthesized primers by pol alpha-primase is fundamentally different than elongation of exogenously added primer-templates with respect to interaction with abasic lesions. Furthermore, this high level of abasic lesion bypass during primase-coupled pol alpha activity provides an additional mechanism for how translesional synthesis may occur in vivo, an event hypothesized to be mutagenic.

DNA synthesis and postreplication mismatch repair were measuredin vitrousing cell-free extracts from cultured human SY5Y neuroblastoma and WI38 fibroblast cells in different growth states. All extracts, including differentiated SY5Y and quiescent WI38 fibroblasts, catalyzed SV40 origin-dependent DNA synthesis, totally dependent on SV40 T-antigen. Thus, although differentiated neuroblastoma and quiescent fibroblasts cells were essentially nondividing, their extracts were competent for DNA replication using DNA polymerases δ, α, and possibly ε, with proliferating cell nuclear antigen. Nonreplicative DNA synthesis and lesion bypass by either α- or β-polymerases were detected independently in extracts using primed or gapped single-stranded DNA templates. Long-patch postreplication mismatch repair was measured for the first time in neuroblastoma cell-free extracts. Extracts from subconfluent and high-density SY5Y cells catalyzed postreplication mismatch repair with efficiencies comparable to those of HeLa cell extracts. No significant differences were observed in repair between SY5Y differentiated and undifferentiated cell extracts. Mismatch repair efficiencies were threefold lower in extracts from subconfluent WI38 cells, and repair in WI38 quiescent cells was fourfold less than in subconfluent cells, suggesting that mismatch repair may be regulated. The spectrum of mismatch repair in SY5Y extracts closely resembled the mismatch removal specificities of HeLa extracts: T · G and G · G mismatches were repaired most efficiently; C · A, A · A, A · G and a five-base loop were repaired with intermediate efficiency; repair of G · A, C · C, and T · T mismatches was extremely inefficient.