| BackgroundMultiple myeloma（MM）is a malignant hematological tumor which originating from plasma cells.Its incidence has increased every year and it has become the second most common hematological tumor.At present,the pathogenesis of multiple myeloma is unknown.A number of studies have shown that MM may be a genetic disease and multi-gene alteration play an important role in the pathogenesis of MM.In polygenic diseases,the influence of each gene on the disease is limited,and the participation of environmental factors and other random factors makes the pathogenesis of the disease complicated and difficult to explain.Genome-wide association studies（GWAS）are considered to be effective methods for discovering susceptibility genes involved in complex diseases.GWAS is based on the linkage disequilibrium（LD）of single nucleotide polymorphisms that the most common type of human heritable variation for searching the sites or areas that are closely related to the disease.However,the SNP detected by GWAS was common SNPs that be defined as the tag SNP of the genome rather than covering the whole genome.It is helpful to discover new disease-associated mutations by further sequencing the susceptibility gene region identified by GWAS.Low-density lipoprotein receptor-related protein 1B（LRP1B）is a potential tumor suppressor gene that encodes a cell surface receptor with multiple functions.It belongs to the low-density lipoprotein receptor family.The GWAS of MM patients in the European population showed that the rs61070260 locus of the LRP1B gene is associated with MM survival(P=8.05×10-6).At present,the susceptibility of LRP1B gene polymorphism to MM patients in Han Chinese population has not been reported.PurposeOur study aimed to investigate the association between the susceptibility and clinical phenotype of MM patients in Han Chinese population and LRP1B polymorphism through case-control study.In addition,we look for possible pathogenic mutations in MM patients by sequencing the designated location that in linkage disequilibrium with rs61070260 locus.Methods1.Blood samples of patients with multiple myeloma and healthy individuals were collected and the genomic DNA was extracted.Genotyping was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.chi-square test and logistic regression analysis were used to analyze the difference of the allele frequency,genotype frequency and three genetic models of rs61070260locus in order to evaluate the association of rs61070260 locus and the risk of MM.2.The association of immunoglobulin typing,light chain typing and the evaluation indicators of the clinical manifestations of MM patients,such as serum calcium,creatinine,β2-microglobulin and hemoglobin,and rs61070260 locus were analysed by logistic regression analysis.3.Haploview software was used to analyze the linkage disequilibrium around100kb of rs61070260 locus using the genotyping data of Han Chinese population in1000 Genomes Project.4.The linkage disequilibrium region of rs61070260 locus was sequenced by Sanger sequencing in 178 MM patients to discover the potential causal variants in linkage disequilibrium with rs61070260 in MM.Results1.A total of 739 patients with multiple myeloma and 592 healthy controls were collected for the association analysis of multiple myeloma susceptibility.The genotyping results showed no significant deviation from Hardy-Weinberg equilibrium was observed for rs61070260 in the controls（P>0.05）which indicate that the sample is representative.The allele frequencies and genotype frequency of the rs61070260 locus on the LRP1B gene were significantly different(P=3.937×10-37;P=1.347×10-150)in the case group and the control group.And the results of the dominant model,recessive model and additive model also showed significant difference(P=1.551×10-134;P=3.261×10-9;P=9.622×10-74).2.There were no correlation between immunoglobulin typing,light chain typing and the clinical indicators（serum creatinine,β2-microglobulin and hemoglobin）and rs61070260 locus（P>0.05）.3.The linkage disequilibrium region around 100kb of rs61070260 locus was analysed and the heat map of the linkage disequilibrium and was constructed by the Haploview software.The physical location of the linkage disequilibrium region of rs61070260 locus is 2:141601530-141611630.4.We detected three SNP loci by sequencing the linkage disequilibrium region of rs61070260 locus and the mutation hotspots between the haplotype block of LRP1B gene in 178 multiple myeloma patients.One patient was detected the rs762074421（T>C）locus,one patient was detected rs756168629（C>T）locus,and four patients were detected rs113600691（A>G）locus.The SNP rs756168629 is a missense mutation located in the coding region of LRP1B gene,which results in a substitution from arginine to histidine at position 1661 of the LRP1B protein.SIFT and Poly Phen2 software predict that the mutation is deleterious.We searched the gnomAD database for the frequency of mutations in the Asian population,rs756168629 mutation has not been reported.Conclusions1.The rs61070260 locus of LRP1B gene is associated with MM susceptibility in Han Chinese population.2.There was no correlation between immunoglobulin typing,light chain typing and clinical phenotype of MM patients and rs61070260 locus in MM patients（P>0.05）.3.The rs756168629 locus detected in MM patients is a nonsynonymous mutation,which was predicted as deleterious that has not been reported in the Asian population.The correlation between rs756168629 locus and MM pathogenesis needs further study. |
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