आणविक और आनुवंशिक चिकित्सा

The increasing prevalence of type 2 diabetes (T2D) underlines the urgent need for proactive strategies to prevent and control T2D. A fairly large number of studies show that T2D is a complex metabolic disease caused by lifestyle, environment and genetic factors. Thus, the study of a genetic predisposition to diabetes is of great importance. At the same time, one of the most effective methods, besides genome-wide researches, is the use of polymorphic markers of various candidate genes, i.e., those genes, protein products of which (enzymes, regulatory proteins and peptides, structural proteins) can be potentially involved in the development of this disease. The study of genetic markers of type 2 diabetes association is relevant worldwide. The aim of this study was to search for genetic markers associated with the development of T2D in individuals of the Kazakh population.

Introduction: Chromosomal mosaicism is characterized by the presence of more than one chromosomally different cell line in an individual. Preimplantation chromosomal mosaicism is characterized by the presence of a mixture of chromosomally different cell lines in an embryo. Studies show that mosaicism for whole chromosomes (aneuploidies) in one or more cells (blastomeres) occurred in more than 75% of cleavage stage embryos, whilst 3%-24% of blastocyst stage embryos are chromosomally mosaic.

Aim: The purpose of this study was to standardize and validate a Next Generation Sequencing (NGS) method for comprehensive chromosome testing for aneuploidies and to study the level of mosaicism in cleavages stage vs. blastocyst stage embryos.

Methods: The validation involved a retrospective blind assessment of whole genome amplification (WGA) products from 14 cleavages stage embryo biopsies (blastomeres), 6 blastocyst stage embryo biopsies (TE), in addition to their 20 discarded blastocyst stage whole embryos. 42.8% of the cleavages stage embryos showed mosaicism, whilst results between the trophectoderm (TE) biopsies (TEB) and their whole embryos at blastocyst stage showed total concordance as no mosaicism was observed. NGS sensitivity and specificity for calling aneuploidy was found to be 100%.

Conclusion: This is the first study reporting preclinical validation and accuracy assessment of the Ion semiconductor sequencing technology in studying the level of mosaicism in cleavage stage and TE biopsies blastocyst stage embryos vs. their whole embryos.

The high level of mosaicism in cleavages stage embryos compared to blastocyst stage embryos does not recommend the PGT-A to be performed on cleavage stage embryos. The NGS proved to be a robust methodology in clinical application of PGT-A.

Background: Glucocorticoid-induced diabetes mellitus (GIDM) is a serious side effect of glucocorticoid (GC) treatment that is associated with both increased mortality and morbidity, but not all patients develop GIDM when treated with GC. The reason is not known, and clinical risk factors predictive of type 2 diabetes do not predict GIDM. Previous metabolomics studies have found specific metabolic disturbances prior to clinical type 2 diabetes. This could also be true for GIDM. The primary aim of this study was to investigate whether distinct metabolic patterns in patients treated with high dose GC can predict development of GIDM.
Material and Methods: Serum from 116 patients about to be treated with or in the first days of treatment with high-dose GC (>100 mg prednisolone equivalent) was analyzed with liquid chromatography-mass spectrometry (LC-MS) based nontargeted metabolomics. Clinical data were collected at baseline and through a 3-week follow-up period. 52 patients developed GIDM and 64 did not (control group). A logistic regression model and a predictive model was build and differences in the metabolome due to treatment with GC was tested in serum from patients without GC treatment (n=6) and patients with GC treatment (n=107).
Results and Discussion: At univariate analysis three metabolites were associated with the development of GIDM. These metabolites could not be annotated to specific metabolites. A multi-metabolite approach could not predict GIDM, and this is different from previous findings in T2DM. This supports the hypothesis that the etiology of T2DM and GIDM is different. The biological significance of our finding remains unknown, but with the rapid development in the field of metabolomics and databases with increasing numbers of characterized metabolites, these metabolites may be identified. Conclusion: Our data indicate that the typical metabolic shifts in T2DM are not the same in GIDM. This supports the hypothesis that GIDM may have a pathophysiology different from T2DM. Furthermore, our data suggest that there is potential for identifying patients at risk of GIDM before clinical manifestation.

Purpose: The objective of this study was to investigate the factors involved in mycophenolate mofetil (MMF) disposition on the risk of diarrhoea in renal transplanted children.

Methods: Patients’ characteristics, immunosuppression and polymorphisms of UGT1A8, UGT1A9, UGT2B7, ABCC2, IMPDH1, and IMPDH2 genes were explored. Statistical analyses were performed using logistic regression.

Results: Eighty three renal transplanted patients were included and 28/83 (33%) developed diarrhoea requiring MMF discontinuation or switched to the enteric-coated formulation EC-MPS during follow-up. In the multivariate analysis, the risk of diarrhoea was significantly higher in ABCC2 -24CC wild-type patients carrying IMPDH2 IVS7 + 10 T>C variant.

Conclusion: IMPDH2 (IVS7+10T>C) and ABCC2 (c.-24C>T) are biomarkers associated with diarrhoea in children treated with MMF.

Bacillus subtilis (B. subtilis) is an ideal host system in production of homologous proteins. However, the production of heterologous proteins in B. subtilis is rare due to low expression levels encountered in most cases. Inteins, also known as ‘protein intron’, which is capable of excised itself from its fusion partners, have been employed for the expression of recombinant proteins in various host systems especially in Escherichia coli (E. coli) but yet, only few paucity of employment of inteins for protein expression in B. subtilis has been found. In this communication, we demonstrated that B. subtilis was able to facilitate auto-cleavages between intein and C-extein. The construct, pECBS1-H6-DnaE-bFGF, in which a 6x His-tag (H6) and basic fibroblast growth factor (bFGF) were fused at the N- and C-terminus of Asp DnaE (intein) respectively, was shown to be capable of processing intracellular expression and auto-cleavages of bFGF with same primary sequence as Homo Sapiens. Moreover, switching shake of flask cultivation to small fermentative scale yielded 113 mg L^-1 of biologically active bFGF. This approach of using intein Asp DnaE for the production of heterologous proteins is highly productive and should be explored further for industrial application.