Analysis Of Four T2DM Associated Genes And Their Phenotypes And The Establishment Of A Detection Method With Multicolor Encoded Fluorescent Microspheres

Type2diabetes mellitus (T2DM) is a chronic metabolic diseases, contributedsynergistically by multiple genes and environmental factors, and its chroniccomplications cause severe mental and financial burden to patients with a longduration. Therefore, the pathogenesis of and targeting interference against T2DM areof great importance and are focued on by T2DM researchers. Recently, with thedevelopment of single nucleotide polymorphism (SNP) technology, identification ofT2DM susceptibility genes has been extensively focused on, and there have beenmore than100SNP sites of T2DM susceptibility genes being reported. However,little is known about the correlation of these genetypes with their phenotypes, thoughthe identification of the correlation can facilitate the early diagnosis and clinicalprevention and treatment of T2DM. Nowadays, there have been multiple methods toidentify the SNPs, however, most of these methods are still on the way to clinicalassay, being limited by inadequate reliability, simplicity of operation, instrumentprecision, throughput of screening. Therefore, a novel method of low cost, simpleoperation and high throughput is in need to identify the SNPs of T2DMsusceptibility genes.Ligase detection reaction (LDR）is based on the specific binding of thermostableligase to SNP sites under high temperature. If there were a completecomplementation between the upstream, downstream probes and the SNP site,without gap between the two probes, the ligation completes; While the lagation fails,if the probe sequences mismatched the mutated SNP site. The lenghth of probe sequences varies with SNPs of genptypes, as can be identified by the fluorescencescanning of the fluorescence labeled at the end of various probes, basing on theemission peak of various SNP genotypes of various fragment length. Basing on theprinciple of LDR technology, multiple LDR developes, in which, there are multipleprobes targeting multiple SNPs, capable of multiple SNPs identificationsimultaneously. However, the LDR detection technology is restricted to large-scaleclinical application by its high cost, complicated procedures, including DNAsequencing.The development of Multicolor encoded Fluoresent Microspheres (MFMs) hasopened a novel area for high-throughput detection technology. Compared withconventional organic dyes, fluorescent nano-materials emission is stronger, morestable, therefore, more suitable for the fluorescent biomarker research. Moreover,because of their good biocompatibility, excellent fluorescence properties, MFMs areparticularly suitable for simultaneous high throughput analysis of multiple biologicalmacromolecules, by being coupled to a variety of biological molecules, Especially,the mature of surface modification of nano-materials and bio-link technologiespromote the further application of MFMs in the biomedical field. MFMs can giveoligonucleotide probes unique color, let along their length, thereby facilitating thesimutanuos detection of multiple SNPs in same reaction system.Magnetic nanoparticles (MNPs) have been widely applicated in the biomedicalfield, the magnetic separation technology can achieve rapid separation andpurification of biomolecules. And the molecular modification technique can couplethe MNPs to various biomolecules via multiple functional groups on MNPs, withoutchanging the characteristics of coupled molecules. Basing the functionalized specificligand-receptor interaction of magnetic nanoparticles, DNA products can be rapidlyand efficiently purified without procedures, such as gel electrophoresis recycling,and the DNA purification can be carried out in any subsequent experiments rapidly.In present study, we constructed a novel detection method for molecularetiology and for clinical prevention and treatment, basing on LDR, MNPs and MNPs, and then identified SNPs of four susceptibility genes to T2DM via the method.There are foure parts in this study:1. We determined the SNPs of SLC30A8, CDKN2A/2B, HHEX, TCF7L2gene, and analyzed the correlation of these SNPs with T2DM. LDR method wasadopted to identify the SNPs of four T2DM susceptability genes of T2DM patients,i.e. the site of rs13266634in SLC30A8, the site of rs10811661in CDKN2A/2B, thesite of rs1111875in HHEX and the site of rs7903146in TCF7L2. Then thedistribution frequency of each site was analyzed in T2DM patients of Han innortheast China. And the acuracy of LDR results were reconfirmed with DNAsequencing. In addition, we also determined the association of the polymorphisms inSLC30A8, CDKN2A/2B, HHEX and TCF7L2gene with the T2DM morbidity, aswell as with the abnormal glucose metabolism, abnormal lipid metabolism andchronic complications.2. Synthesis, modification and DNA-conjugation of MNPs and MFMs.SiO2-coated Fe3O4nanoparticles were prepared by coprecipitation method, theSiO2-based magnetic nanoparticles were obtained under strict control of the particlesize and magnetic density by regulating the size and thickness of the silica shell to ofthe aggregates. By covalent coupling method, electrostatic adsorption andpolyelectrolyte bridges and other methods, we prepared four kinds of SiO2fluorescence coded nanoparticles with multi-color fluorescence, i.e. MFMs. Then themagnetic and fluorescent nanospheres surface was modified with carboxyl group andcoupled to streptavidin-biotin, and thus these particles were capable to bind tobiological macromolecules to develop SiO2-based magnetic or fluorescentnanoparticles-streptavidin-biotin complex. Thus, these particles were utilized toconjunct to the biotin modified5’ end of LDR upstream probe, or the biotin modified3’ end of LDR downstream probe.3. Optimization of multiple PCR-LDR assay for multiple SNPs. According tothe principles of multiple PCR and multiple LDR, PCR primers and LDR probeswere designed targeting the site of rs13266634in SLC30A8, the site of rs10811661 in CDKN2A/2B, the site of rs1111875in HHEX and the site of rs7903146inTCF7L2. The reaction conditions of the reaction system, with whole genomic DNAas template, were optimezed to establish the optimal reaction system for T2DMsusceptable SNPs identification, by multiple LDR.4. Establishment of the detection system for T2DM susceptable gene SNPs,basing on MFMs.The optimized multiple LDR technique was adopted, with wholeblood genomic DNA as template, magnetic and with nanoparticles coupled upstreamLDR probes, the amplificated product of multiple LDR was rapidly and specificallyisolated from the irrelevant downstream probe. The fluorescent nanospheres coupledLDR downstream probe effectively promoted the detection specificity basing on theabsorbance of the fluorescence with different color, and promoted the detectioncapbility of mutiple SNPs simultaneously in the same reaction system. Thus, weestablished a multi-LDR detection system for T2DM susceptibility gene SNPs,basing on nanomaterials technology.Research conclusions are as following:1We have successfully identified the SNP of rs13266634in SLC30A8,rs10811661in CDKN2A/2B, rs1111875in HHEX and rs7903146in TCF7L2, of113cases of T2DM patients and107healthy subjects. And we found that there was anassociation of C allele at rs13266634of SLC30A8gene with fasting insulinsecretion deficiency, Islet function failure, increased cholesterol and increasedincidence of cardiovascular complications. And we also found that there was anassociation of T allele at rs10811661of CDKN2A/2B gene with islet β celldysfunction, Islet function failure, insulin secretion reduction, increased incidence ofmicrovascular complications.2We have successfully synthesized the streptavidin-biotin-modifiedSiO2-composed magnetic nanospheres and multicolor fluorescence codedmicrospheres. And then we respectively coupled the magnetic nanospheres with theupstream LDR probe, coupled the multicolor fluorescence coded microspheres withthe downstream LDR probe. This is the first successful preparation of the oligonucleotide probes coupled with multicolor fluorescence coded microspheres forthe high throughput detection of T2DM susceptibility genes.3We have optimized the reaction conditions of multiple PCR and multipleLDR, and we found that non-specific PCR products could be significantly reduced,and the target PCR products could be elevated by appropriately increasing theconcentration of MgCl2and dNTP, adjusting the ratio of the primers pairs of eachgene, by reducing the amount of probe, or decreasing the annealing temperature ofthe reaction system to improving the resolution of multiple LDR. These findingslayed the technical foundation for the simultaneous detection of clinical T2DMsusceptibility gene SNPs.4We have identified the T2DM susceptability gene SNPs via the combinedusage of the multiple PCR, multiple LDR technology, the high efficient purificationtechnology with MNPs, and the oligonucleotide probes with four kinds of MFMs.We confirmed for the first time, MFMs could be utilezed to label the oligonucleotideprobes for LDR detection, and we established a detection method, simutaneouslyidentifying SNPs of four T2DM susceptability genes, laying the foundation forhigh-throughput detection of T2DM susceptibility gene SNPs.In present study, we established a cheap, simple, rapid, and high-throughputdetection method for the identification of T2DM susceptability gene SNPs, with thecombination of the simplicity, high-throughput of muliple LDR, the simplicity andrepeatability of MNPs, and the high-throughput of MFMs. It could effectively reducetesting costs, was suitable for clinical application, with an effective combination ofthe biological research and clinical prevention etiology.