Development Of A New Method For Detecting YMDD Mutations Of P Gene Of Hepatitis B Virus And Study Of Clinical Practical

Aims LAM has been developed as a therapeutic drug for anti-HBV in recent years which can inhibit hepatitis B virus replication thereby to reduce inflammatory activity,stabilize patients' condition and even improves liver histology. However,the effected period is short in inhibiting virus replication and liable to recurrence.With prolonged use of LAM, drug resistance emerges under action of drug and immunity pressure of body. It has been observed that YMDD mutations often emerge in YMDD motif of P gene of HBV during LAM treatment.Once YMDD mutation strains emerge, it can result in changes of liver function and HBV DNA breakthrough even exacerbation of patients' condition.It is very important to detect YMDD mutations for promptly adjusting treatment , rationally using drug and enhancing therapeutic effect. YMDD mutations of P gene of HBV were detected by mismatch amplify mutation assay in this study. Clinical apply of this method was evaluated and its clinical significance of YMDD mutations also was explored.Methods For amplification of fragment of DNA including different mutated sites, we designed different primers according to the specific nucleotide in YMDD motif. YMDD mutations were detected by mismatch amplification mutation assay,in this method the second base of 3' end in one primer was incomplementary with the corresponding base in template DNA.1.Establishing positive control system by molecular cloning method, this system including plasmids of YMDD, YVDD ,YIDD.2. Plasmids of YMDD, YVDD , YIDD were identified by PCR,restriction endonulease digestion and direct sequence analysis and related assays about sensitivity and specificity were also done.3.YMDD mutations were analyzed in 65 patitents who did not receive antivirual treatment.4. YMDD mutations were analyzed in 30 patitents with chronic hepatitis B who had received oral LAM and their HBV DNA levels were continueously positive for one year.Results1. Plasmids of YMDD, YVDD, YIDD were identified by PCR method, restriction endonulease method and direct sequence analysis, which indicated construction was successful.2. Plasmids of YMDD, YVDD ,YIDD had high sensitivity and specificity. Minimal concentration of plasmids of YMDD,YVDD,YIDD were detected in 1.87 copies/u 1,1.53 copies/u 1 and 1.53 copies/u 1 respectively. False positive results had not appeared in the concentration of plasmids of YMDD,YVDD,YIDD below 1.87X109 copies/u 1.1.53X1O8 copies/u 1 and 1.53 X 108 copies/U 1 respectively.3.YMDD mutations existed in 6 cases of 65 patitents of HBV without antivirual therapy. The rate of YMDD mutations was 9.07% including 1 YIDD mutation strain, 1 YI/YMDD mixed mutation strain,3 YVDD mutation strains and 1 YV/YMDD mixed mutation strain in 6 patitents. Compared with the patients without mutations, liver function and HBV DNA levels of those with mutations had no statistically significance.4. The rate of YMDD mutations was 16.67% in 30 patients of HBV who received oral LAM and their HBV DNA were persistently positive for half a year. There were 2 YVDD mutation strains, 1 YI/YMDD mixed mutation strain, 1 YIDD mutation strain in 5 patitents. The rate of YMDD mutations was 13.33% in 30 patients of HBV who received oral LAM and their HBV DNA were persistently positive for one year. There were 1 YIDD mutation strain and 3 YI/YMDD mixed mutation strains in 4 patitents. Liver function and HBV DNA levels of mutations' compared with those without mutations' had no statistically significance. YMDD mutations of over the age of 35 years was higher than under the age of 35 years from the age' s distribution of YMDD mutations.Conclusions1. YMDD mutations were found in the patients of HBV without antivirualtherapy. Pressure of antivirual drug is not unique factor that leads to YMDD mutations.2.YMDD mutations usually occurred in the patients who received oral LAM for long term but had not got therapeutic effects. It suggests that pressure of antivirual drug is still an important factor that results in YMDDmutations.3.YMDD mutations were found in the patients of HBV who had received oral LAM but had not got therapeutic effects for half a year. With time prolonged, YMDD mutations could change. Liver function and HBV DNA levels had not worsened obviously after YMDD mutations.4.This method has high sensitivity, specificity, which is easy, costless and practical in detecting YMDD mutations of P gene of HBV, available to detect YMDD mutations of P gene of HBV in clinic.