Establishing Of STR Analysis Model For Risk Assessment Of Polygenic Genetic Diseases And Its Experimental Basis

Background and Objectives:Short tandem repeat(STR)is heritable short nucleotide repeat with the advantages of wide distribution in the genome,high polymorphism,high heterozygosity,low mutation rate,easy and fast detection.Since its development and utilization,it has been widely used in many fields such as forensic identification,disease gene localization,diagnosis of genetic diseases,biochemistry and molecular biology of tumors.It is currently the most widely used genetic marker.According to the theory of programmed onset of disease for an individual,to study the association with the occurrence of cancer and STR polymorphism,to establish the genetic risk of death from major cancers;to establish the genetic risk assessment method of polygenic genetic diseases based on STR model;to identify the types and patterns of STR mutation in gastric cancer tissues and to identify microsatellite instability in tumor tissues;to study the correlation between microsatellite instability and HLA gene polymorphism in gastric cancer patients and its potential mechanism.Methods:1.Fifty patients(27 males and 23 females)with lung cancer and fifty patients(33males and 17 females)with liver cancer diagnosed by postoperative pathology in the Third Hospital of Dalian from September 2016 to June 2017,were included in the cancer group and 200 healthy blood donors(100 males and 100 females)were selected as the control group.After blood collection,DNA was extracted and 15 specific STR gene loci were detected by the STR typing technique.Cox regression was used to assess the difference in the average age of onset of lung cancer and liver cancer patients with or without STR alleles and to determine the genes with the different average ages of onset.Logistic binary regression was then used for cross-validation to identify alleles that were significantly different from the cancer group and the control group,and to find STR alleles associated with cancer.The results of the case-control study were translated into those of the cohort study.And the mortality rates of lung and liver cancer in individuals carrying two or one cancer-related STR alleles or without carrying cancer-related STR alleles were calculated based on the proportional mortality rate.2.A total of 396 groups of children and their biological parents that received paternity testing in Dalian Blood Center during the period of January to December in2018 were selected.The genomic DNA was extracted after blood collection and 19 specific STR loci were detected by the STR typing technique.The number of tandem repeats of two alleles in an STR locus was assumed to be the quantitative genetic intensity of the incidence of disease,and the sum of the number of tandem repeats of 19 STR loci in an individual was the quantitative genetic intensity of the individual.Thresholds of the STR between paternal,maternal and childhood data were recorded.As an exemplar,with the first quarter threshold range assigned a value of "1",while the other samples were assigned a value of "0".Genetic concordance rates were calculated based on the difference in child incidence of parents in the group with or without the disease.The ratio of the observed genetic concordance rate for the expected genetic concordance rate was defined as the genetic index(HI)to build the STR model.Another121 children and their biological parents were selected for a finger-crossover test to validate the STR model,with data obtained from self-reports or telephone interviews.3.From September 2018 to August 2019,35 patients(23 males and 12 females)with pathologically confirmed gastric cancer were collected from the First Affiliated Hospital of Dalian Medical University.The gastric cancer tumor paraffin-embedded tissues were obtained from the Department of Pathology,and the corresponding blood samples were obtained from the Department of Clinical Laboratory.After blood collection,DNA was extracted and 21 STR loci were detected by STR typing technique in all samples.The STR typing results of tumor tissues and blood samples of the same individual were compared,and the STR mutation sites and types were recorded and the corresponding statistics were performed to determine the MSI and LOH phenotypes.4.DNA from tumour tissue and blood samples from MSI gastric cancer patients were amplified for HLA-A,-B and-DR loci,and HLA-A and-B locus alleles were sequenced and gene frequencies were compared using PCR-SBT technology.The National Library of Medicine literature search system NCBI,Protein Structure Database PDB and PROSITE databases were searched online and combined with Discovery Studio(DS)software to analyse and predict the interaction between two HLA molecules,HLA-B *44,HLA-A*02 and TCR.Results:1.After Cox regression and Logistic regression cross-validation,D18S51-20 was confirmed to be a lung cancer-related allele,D21S11-30.2 and D6S1043-18 were confirmed to be liver cancer-related alleles.The probability of death from cancer in individuals with or without the D18S51-20,D21S11-30.2,and D6S1043-18 alleles ranged from 0.115 to 0.395.2.The individual quantitative genetic intensities of the 396 children,when expressed as a bar chart,formed a curve that approximated a normal distribution,indicating that the individual quantitative genetic intensities were consistent with a polygenic inheritance phenomenon and followed a normal distribution by the Kolmogorov-Smirnov test(p>0.05).The data from the finger-crossover test is substituted for the STR model,as CHe was 0.221,implying that the HI for the mode of holding the fingers was 0.950.3.Compared to the STR typing results of blood samples of gastric cancer patients,four types of STR variants were observed in the same individual tumour tissue:additional alleles,new alleles,complete loss of heterozygosity and partial loss of heterozygosity,the first three could cause genotype alteration.The variation rate of the21 STR loci ranged from 1.43% to 10.00%,with the highest variation rate(10.00%)in the D19S433 locus.23 of the 35 gastric cancer patients had mutations in the STR locus of the tumour tissue,a variation rate of 65.71%(23/35).Only additional alleles were detected in 6 cases,both additional alleles and new alleles variants were detected in 2cases,both types of variants representing microsatellite instability.Loss of heterozygosity alone was detected in 9 cases,and variants with both microsatellite instability and complete loss of heterozygosity were detected in 6 cases.The overall incidence of microsatellite instability was 40.00%(14/35).Microsatellite instability high was defined as variants occurring in greater than or equal to two loci(6 cases).4.PCR amplification and electrophoresis of HLA-A,-B and-DR loci were performed on DNA from tumour tissues and blood samples of 14 gastric cancer patients with MSI,showing negative results for A and B loci and positive results for DR loci in tumour tissues;while positive results were obtained for A,B and DR loci in blood samples of the same individuals.Allele frequencies were calculated after sequence the HLA-A and-B loci in blood samples of 14 gastric cancer patients,and the gene frequency of HLA-B*44(0.1071)was higher than that reported in the literature(0.0205),which was statistically different by analysis of the binomial test in the non-parametric test(p=0.019,p<0.05).Bioinformatic analysis revealed that the affinity ranking of HLA for TCR was HLA-B*44>HLA-A*02.The major loci at which HLA-B*44 and TCR formed non-bonded interactions were 82 GLU,86ARG and89 GLN,whereas HLA-A*02 did not form major non-bonded interactions with TCR at these three loci.However,there were also some common sites(93,96,100,103)of driving force for both molecules to stabilize binding to the TCR.Conclusions:1.According to the theory of programmed onset of disease and STR locus genetic marker analysis can create an effective analysis method to assess individual genetic risk and can be used to assess the genetic risk of mortality of cancers.2.The STR model was successfully developed to analyse polygenic genetic phenomena and the accuracy of the STR model was confirmed with data from actual family surveys(finger crossing tests).The STR model can be used to assess the risk of polygenic inheritance in first-degree relatives or to assess the incidence of polygenic inheritance in unrelated populations.3.STR genotype alterations may present in tumour tissues;the MSI phenotype of gastric cancer may be determined by comparing the variants of STR loci in tumor tissues and blood samples.STR genetic markers have potential as diagnostic markers and immunotherapy sensitivity markers for patients with gastric cancer.4.HLA-B*44 may correlate with the MSI phenotype of gastric cancer and deletion of highly polymorphic HLA class I molecules may be associated with tumourigenesis,progression and prediction of immunotherapy efficacy.5.The sites that cause the binding affinity of HLA-B*44-TCR complex to be stronger than that of HLA-A*02-TCR complex may be at positions 82,86 and 89,while the four sites at positions 93,96,100 and 103 are more important for HLA-TCR binding,and if mutations occur here,they may easily lead to weakening or loss of driving force and increase the risk of tumor immune escape.