IMPDH2 genetic polymorphism: a promoter single-nucleotide polymorphism disrupts a cyclic adenosine monophosphate responsive element.

Inosine 5'-monophosphate dehydrogenase (IMPDH), which catalyzes a key step in the de novo biosynthesis of guanine nucleotide, is mediated by two highly conserved isoforms, IMPDH1 and IMPDH2. In this study, IMPDH2 genetic polymorphism was investigated in 96 individuals of Caucasian origin. Four single-nucleotide polymorphisms were identified, comprising one previously described single base-pair substitution in the close vicinity of the consensus donor splice site of intron 7 (IVS7+10T>C), and three novel polymorphisms, one silent substitution in exon 9 (c.915C>G), one single base-pair insertion (g.6971_6972insT) within the 3'-untranslated region of the gene, and one substitution located in the promoter region (c.-95T>G) in a transcription factor binding site CRE(A) (cyclic adenosine monophosphate [cAMP] response element). Considering the nature and location of this latter polymorphism, its functional relevance was examined by transfecting HEK293 and Jurkat cell lines with constructs of the related region of IMPDH2/luciferase reporter gene. The c.-95T>G mutation leads to a significant decrease of luciferase activity (HEK293: 55% decrease, p < 0.05; Jurkat: 65% decrease, p < 0.05) compared with the wild-type promoter sequence and, therefore, is likely to determine interindividual differences in IMPDH2 transcriptional regulation. These results might contribute to a better understanding of the variability in clinical outcome and dose adjustments of certain immunosuppressors that are metabolized through the IMPDH pathway or that are IMPDH inhibitors.

Xenogenic bone matrix extracts induce osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells.

Colloss and Colloss-E are sterile acellular lyophilizates extracted from bovine and equine bone matrix, respectively. Animal and clinical studies have shown that these xenogenic bone matrix extracts (BMEs) are effective as bone graft substitutes. In this report, we investigated the effect of Colloss and Colloss-E on human adult in vitro-expanded bone marrow-derived mesenchymal stem cells (BMMSCs). Specifically, we assessed whether these xenogenic BMEs induced osteoblastic differentiation of cultured BMMSC. We show that BMMSCs treated with either Colloss or Colloss-E exhibited characteristic osteoblastic morphological changes accompanied by the expression of osteoblast-specific markers, such as alkaline phosphatase activity, osteopontin secretion and calcium deposits, explicitly demonstrating that these bone matrix extracts induce osteoblastic differentiation of BMMSCs in vitro. Hence, xenogenic BMEs induce bone-specific differentiation of BMMSCs, presumably through providing stem cells with structural and soluble mediators that mimic the in vivo microenvironment. These results may explain the in vivo mode of action of these medical devices, and potentially provide a novel tissue engineering-based treatment of bone defect, using autologous BMMSCs pretreated with BMEs.

[Crohn's disease associated with interferon and ribavirin treatment for chronic hepatitis C]. / Maladie de Crohn au cours du traitement de l'hépatite chronique C par interféron et ribavirine.

Digestive side effects associated with interferon and ribavirin in the treatment of chronic hepatitis C seem to be non specific. So far, inflammatory bowel diseases have only been rarely described in this context. We report two cases of Crohn's disease which occurred a few months after interferon and ribavirin treatment for chronic hepatitis C. The first case was a 43 year old man treated for 7 months with a interferon and ribavirin combination for chronic hepatitis C who experienced colitis related to Crohn's disease, with the occurrence of articular and cutaneous manifestations. The second case was a 43 year old women treated for 8 months with a pegylated interferon and ribavirin combination for chronic hepatitis C who developed Crohn's disease with right ileo-colic topography and severe evolution requiring an ileo-coecal resection because of the failure of medical treatment. The alpha interferon used for its antiviral action against hepatitis C virus, has complex immuno-modulation effects which could reveal latent Crohn's disease.

Identification and functional analysis of two rare allelic variants of the thiopurine S-methyltransferase gene, TPMT*16 and TPMT*19.

Human thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. TPMT is genetically polymorphic and is associated with large interindividual variations in thiopurine drug toxicity and therapeutic efficacy. During routine genotyping of patients with Crohn's disease, one novel missense mutation, 365A>C (TPMT*19, Lys(122)Thr), and a recently described missense mutation, 488G>A (TPMT*16, Arg(163)His), were identified in a Caucasian and a Moroccan patient, respectively. Using a heterologous yeast expression system, kinetic parameters (K(m) and V(max)) of the two variants with respect to 6-thioguanine S-methylation were determined and compared with those obtained with the wild-type enzyme. The Lys(122)Thr exchange did not significantly decrease the intrinsic clearance value (V(max)/K(m)) of the variant enzyme. In contrast, the Arg(163)His substitution significantly decreased the intrinsic clearance value by three-fold. The Arg(163) is located in a highly conserved region of the human TPMT protein and, as such, the Arg(163)His substitution is expected to result in a marked reduction of enzyme activity, as confirmed by the in vitro data. Phenotyping by measurement of red blood cell TPMT activity indicated that the patient heterozygous for the Lys(122)Thr mutation had normal TPMT activity, whereas the patient heterozygous for the Arg(163)His mutation was an intermediate methylator, which demonstrated a positive correlation between TPMT phenotyping and the in vitro data. The identification of a novel non-functional allele of the TPMT gene improves our knowledge of the genetic basis of interindividual variability in TPMT activity. These data further enhance the efficiency of genotyping methods to predict patients at risk of an inadequate response to thiopurine therapy.

In vitro characterization of four novel non-functional variants of the thiopurine S-methyltransferase.

Human thiopurine S-methyltransferase (TPMT) is an enzyme responsible for the detoxification of widely used thiopurine drugs such as azathioprine (Aza). Its activity is inversely related to the risk of developing severe hematopoietic toxicity in certain patients treated with standard doses of thiopurines. DNA samples from four leucopenic patients treated with Aza were screened by PCR-SSCP analysis for mutations in the 10 exons of the TPMT gene. Four missense mutations comprising two novel mutations, A83T (TPMT*13, Glu(28)Val) and C374T (TPMT*12, Ser(125)Leu), and two previously described mutations, G430C (TPMT*10, Gly(144)Arg) and T681G (TPMT*7, His(227)Gln) were identified. Using a recombinant yeast expression system, kinetic parameters (K(m) and V(max)) of 6-thioguanine S-methylation of the four TPMT variants were determined and compared to those obtained with wild-type TPMT. This functional analysis suggests that these rare allelic variants are defective TPMT alleles. The His(227)Gln variant retained only 10% of the intrinsic clearance value (V(max)/K(m) ratio) of the wild-type enzyme. The Ser(125)Leu and Gly(144)Arg variants were associated with a significant decrease in intrinsic clearance values, retaining about 30% of the wild-type enzyme, whereas the Glu(28)Val variant produced a more modest decrease (57% of the wild-type enzyme). The data suggest that the sporadic contribution of the rare Glu(28)Val, Ser(125)Leu, Gly(144)Arg, and His(227)Gln variants may account for the occurrence of altered metabolism of TPMT substrates. These findings improve our knowledge of the genetic basis of interindividual variability in TPMT activity and would enhance the efficiency of genotyping methods to predict patients at risk of inadequate responses to thiopurine therapy.