| Objective: Multiple Myeloma(MM)is a disease caused by malignant proliferation of plasma cells in bone marrow which is the second most common hematological malignancy.Proteasome inhibitors(PIs),particularly bortezomib(BTZ),have drawn much attention due to their unprecedented efficacy in significantly improving overall survival in patients with MM,but they are still not able to cure the disease.Meanwhile,PIs treatment of MM may produce a series of side effects,especially the Peripheral Neuropathy(PN),which seriously affects the subsequent treatment and quality of life of MM patients.We observed that there is a difference in the PN occurence when the equivalent dose of bortezomib,that was converted based on body surface area,was administered.The variation and susceptibility between individuals were observed to be associated with the occurrence of drug-related adverse events.The unpredictability of Bortezomib-induced Peripheral Neuropathy(BIPN)was reported to be partly due to genetic differences.However,further study is needed to substrantiate the association.Currently,the genetic factors and possible mechanism of BIPN have not been reported in China.In this study,we analyzed the association between single nucleotide polymorphism(SNP)and gene expression of related genes,and BIPN.If appropriate genetic risk profiles could be identified for patients at risk of BIPN,it will help to guide the clinical treatment and ultimately benefit patients.Therefore,we examined the drug response related SNPs of bortezomib in Chinese population that could provide valuable insights in the experimental analysis and also facilitate the construction of pharmacogenomic data based on Chinese population.Methods:The study was designed for the detection of bortezomib metabolism and treatment-related genes based on the known SNPs and important genes related to the therapeutic response of bortezomib as first-line drug in MM treatment,and known SNPs and important genes related to peripheral neuropathy.We collaborated with Mesirui(Beijing)Biotechnology Company and designed a second-generation sequencing panel based on multiple PCR amplification technology.The panel contains 25100 SNPs,involving 550 genes that were used for detecting bortezomib drug metabolism,treatment and peripheral neuropathy related genes.A total of 204 patients diagnosed with MM and treated with bortezomib were selected from the hematology department of our hospital from 10 May 2019 to 10 November 2021.Clinical information and DNA sample was collected from all patients enrolled.RT-PCR was used to detect the changes of MTHFR and ALDH1A1 gene expression in peripheral blood of patients with MM before and after bortezomib treatment.We included the SNPs of MTHFR and ALDH1A1 that were reported to be expressed in peripheral blood.Statistical analysis was performed to compare the frequency differences of SNPs in different groups.The RNA expression of the above 2 genes were detected in peripheral blood of 62 samples,including 26 patients with untreated MM,19 patients after treatment with bortezomib,and 17 healthy people with physical examination at the same time as the control group.Statistical analysis was performed to compare the differences in clinical information,variation sites in DNA sequencing data and RT-PCR detection dates of peripheral blood RNA expression among different groups.Results:First part:In this study,204 eligible patients with MM were enrolled,including 115 patients who developed PN side effects after bortezomib treatment and 89 patients without PN.There were 77 mild patients treated as usual(G1 and G2 without pain)and 38 severe patients changed treatment plans(G2 with pain,G3 and G4)with peripheral neuritis.The incidence of PN was 56.4% and that of severe PN was 18.6%.There was no statistical difference in gender,age,blood routine and other baseline characteristics and clinical information between patients with and without PN(various indicators with P>0.05).There was also no statistical difference in gender,age,blood routine and other baseline characteristics and clinical information between severe patients with PN and patients without PN(various indicators with P>0.05).Second part:Peripheral neuritis after bortezomib treatment may be associated with the following SNP i.e.rs1801131(T>G,variable frequency(AF): 0.217 vs 0.096,OR=2.631,P=0.001;T/T>T/G,variable frequency(AF): 0.383 vs 0.191,OR=2.743,P=0.002),rs180113(A>G,AF: 0.678 vs 0.466,OR=2.413,P=0.000;A/A>G/G,AF: 0.530 vs 0.393,OR=5.364,P=0.001),rs17421511(G>A,AF: 0.152 vs 0.039,OR=4.385,P=0.000;G/G>G/A,AF: 0.304 vs 0.079,OR=5.132,P=0.000)of MTHFR gene;rs1051740(C>T,AF:0.700 vs 0.472,OR=2.611,P=0.000;T/C>T/T,AF: 0.496 vs 0.191,OR=3.566,P=0.000)of EPHX1 gene;rs2016848(G>A,AF: 0.348 vs 0.124,OR=3.782,P=0.001;G/G>A/G,AF: 0.330 vs 0.112,OR=3.960,P=0.000)of MEE gene;rs6151031(GCTG>G,AF: 0.061 vs 0.011,OR=5.704,P=0.010;GCTG/ GCTG>G/GCTG,AF: 0.122 vs 0.022,OR=6.027,P=0.009)of ALDH1A1 gene;rs1935349(C>T,AF: 0.343 vs 0.208,OR=1.994,P=0.003;C/C>T/T,AF: 0.139 vs 0.022,OR=8.313,P=0.002)of HTR7 gene;rs8192720(G/A>A/A,AF: 0.130 vs 0,OR=infinite,P=0.001;G/G>A/A,AF: 0.130 vs 0,OR=infinite,P=0.000)of CYP2A6 gene.All the genotypes mentioned above were risk karyotypes of BIPN.Multivariate model analysis showed that G/A genotype at rs17421511 had 9.578 times higher risk of peripheral neuropathy than G/G genotype(P=0.002,OR=9.578(95%CI 2.296-39.955)),the risk of peripheral neuropathy in rs1051740 T/T genotype was 6.030 times higher than that in T/C genotype(P=0.001,OR=6.030(95%CI 2.000-18.181)),the risk of peripheral neuropathy was 4.925 times higher in A/A genotype of rs2016848 than in G/G genotype(P=0.026,OR=4.925(95%CI 1.213-19.996)),the risk of peripheral neuropathy was 7.377 times higher in GCTG/G genotype at rs6151031 than in GCTG/GCTG genotype(P=0.046,OR=7.377(95%CI 1.778-39.978)).Third part: The results of RT-PCR showed that :(1)MTHFR m RNA expression in the newly diagnosed and without treatment group(group C)was higher than that in the control group(2.55±1.02 vs 1.95±0.92,P=0.050),but there was no statistical difference;MTHFR m RNA expression in treated group(group F)was higher than that in control group(3.02±1.89 vs 1.95±0.92,P=0.041),and the difference was statistically significant.There was no difference in MTHFR m RNA expression between the newly diagnosed and without treatment group(C group)and the treated group(F group)(2.55±1.02 vs3.02±1.89,P=0.264).MTHFR m RNA expression in C group patients without PN after treatment(CN group)was higher than that in C group patients with PN after treatment(CP group)(2.81±0.97 vs 1.70±0.77,P=0.009),and the difference was statistically significant.There was no difference in MTHFR m RNA expression between patients without PN after treatment(FN group)and patients with PN after treatment(FP group)(2.56±0.78 vs 3.30±2.31,P=0.426).(2)Compared with the control group,there was no difference in the m RNA expression level of ALDH1A1 in group C(6.74±4.76 vs 4.63±5.67,P=0.182);the m RNA expression level of ALDH1A1 in F group was higher than that in normal control group(11.15±11.92 vs 4.63±5.67,P=0.048),and the difference was statistically significant.Meanwhile,there was no difference in ALDH1A1 m RNA expression between group C and group F(6.74±4.76 vs 11.15±11.92,P=0.080),in ALDH1A1 m RNA expression between CN group and CP group(6.92±5.27 vs 5.15±2.26,P=0.375)and in ALDH1A1 m RNA expression between FN group and FP group(16.24±17.23 vs 8.15±6.67,P=0.159).Conclusion : rs1801131,rs1801133 and rs17421511 of MTHFR,rs6151031 of ALDH1A1,rs1051740 of EPHX1,rs2016848 of MME,rs1935349 of HTR7 and rs8192720 of CYP2A6 were correlated with BIPN.The m RNA expression level of MTHFR gene in peripheral blood was different between patients with and without BIPN in the newly diagnosed group.The genes of SNPs identified in this study were involved in the development and maintenance of nerve cells(ALDH1A1,HTR7,MME),in the transformation and metabolism of neurotoxic substances into amino acids(MTHFR),in the metabolism of boron tezomib(CYP2A6),and in neuroinflammatory responses(EPHX1).These SNPs will provide theoretical and experimental evidence for clinical use as genetic markers of PN after bortezomib treatment. |
