| Part one SCN9 A missense mutations contribute to congenital insensitivity to pain ——An unexpected genotype-phenotype relationshipBackground:congenital insensitivity to pain?CIP?is an extremely rare autosomal recessive disorder caused by loss-of-function mutations of Nav1.7 encoded by SCN9 A.So far,only about 40 CIP cases have been reported,and the complex relationship between genotype and phenotype remains unclear.This article emphasized the genotype-phenotype relationship in three CIP patients of China.Purpose:To identify the SCN9 A mutations in three Chinese patients with CIP,and to explore the genotype-phenotype relationship and pathogenic mechanisms.Methods:1.The written informed consents were obtained from patients and their family before study.The phenotype of the patient was assessed by a questionnaire and physical examinations.The genomic DNA was extracted and analyzed for gene mutations using the Gene Chip technology.The detected genes included sixty pain-related genes,such as SCN9 A,SCN10A,NGF,NTRK1,and PRDM12.2.Bioinformatics analysis was performed according to different mutation types: to analysis the effect of frameshift mutations on the structure of Nav1.7 according to the position and reading frame,to analysis the effect of intron mutations on the splicing function of SCN9 A,to analysia the conservation of Nav1.7 amino acids and pathogenicity of Nav1.7 by software Clustal Omega and Polyphen 2/SIFT respectively.3.The wild-type and mutant vectors was constructed,and to transfect the HEK293 cells,and to obtain the HEK293 cells express the wild-type or mutant Nav1.7.Then to analysis the effect of intron mutations of patient 2 on the splicing of Nav1.7 m RNA,to determine the effect of missense mutations of patient 3 on the electrophysiological function of Nav1.7 by the whole cell patch clamp,and to assess the effect of missense mutations of patient 3 on the Nav1.7 expression and subcellular localization by RT-PCR?Reverse transcription polymerase chain reaction,RT-PCR,western blotting and immunofluorescence.Results:1.The main clinical phenotypes of three CIP patients were as follows: 1?congenital insensitivity to pain: the patients had no pain and no unpleasant expression to noxious stimulation?such as blood injection,injury,boiled water?,and often had self-mutilation,such as biting injury to the tongue;2)Anosmia,the patients were unable to distinguish the smell of water,alcohol,and white vinegar.2.All the patients were carried compound heterozygous mutations and identified six novel SCN9 A mutations in three patients,and did not detect the other gene mutations.CIP patient 1 carried compound heterozygous frameshift mutations in SCN9 A,c.850 del G,p.Glu284Lysfs*3)and c.129141del TGAAGAAGCCCCA,p.Asp43Glufs*43;the CIP patient 2 carried compound heterozygous intron mutations in SCN9 A,c.4174-1G>C and c.4228-10A>G;and the CIP patient 3 had compound heterozygous missense mutations in SCN9 A,c.296G>A?p.Arg99His?and c.2749T>G?p.Trp917Gly?.3.The results of bioinformatics analysis showed that 1)in CIP patient 1,two frameshift mutations resulting in truncated Nav1.7 protein;2)in CIP patient 2,two splice mutations,may resul in splicing abnormalities of Nav1.7 m RNA;3)in CIP patient 3,residues Arg99 and Trp917 were mapped to the Nav1.7 N-terminus and domain II selectivity filter?P-loop?,respectively,were highly conserved from chicken to human and among sodium channel subtypes in human.The mutations in patient 3,p.Arg99 His and p.Trp917 Gly are highly pathogenic and Poly Phen-2 predicts: 0.953 and 1.000.4.The results of the effect of intron mutations on the splicing of Nav1.7 m RNA showed that the mutations c.4174-1G>C and c.4228-10A>G resulted the splicing abnormalities of Nav1.7 m RNA and truncated Nav1.7.5.The whole-cell patch clamp detection of missense mutations in patient 3 revealed that compared to the wild-type Nav1.7 channel,the p.Arg99 His mutant Nav1.7 channel current was significantly reduced and approximately 50% that of the wild-type;The p.Trp917 Gly mutant Nav1.7 channel current almost disappeared.The detection of Nav1.7 expression level showed that both p.Arg99 His mutation and p.Trp917 Gly mutation had no effect on the expression of Nav1.7 m RNA;p.Arg99 His mutation resulted in a significant decrease of Nav1.7 channel protein expression,which was about 48% of the wild type?P<0.001?,the expression level of Nav1.7 in the cell membrane also decreased significantly,about 43% of the wild type?P<0.001?,while the p.Trp917 Gly mutation did not affect the expression level of Nav1.7 channel protein?P>0.05?.Conclusions:1.The main clinical phenotypes of three Chinese CIP patients were complete painlessness and anosmia.We identified six novel SCN9 A mutations in three CIP patient who carried compound heterozygous SCN9 A mutations.2.The frameshift mutation in SCN9 A gene of patient 1 and the intron mutation in SCN9 A gene of patient 2 all resulted in truncated Nav1.7.3.CIP patient 3 carries compound heterozygous SCN9 A missense mutations,c.296G>A?p.Arg99His?and c.2749T>G?p.Trp917Gly?.The whole-cell patch-clamp revealed that mutations in Nav1.7 in CIP patient 3 still retained partial channel function,but the patient showed complete painlessness,which was inconsistent with previous findings "Nav1.7 total loss-of-function leads to painlessness",this unexpected genotypic-phenotypic relationship should be emphasized.4.Detection of electrophysiological and expression levels of missense mutations carried in patient 3 revealed that the amino acid residue p.Arg99 located at the N-terminal of Nav1.7 plays an important role in maintaining Nav1.7 expression and membrane localization,and the amino acid residue p.Trp917 mapped to the site of pore selectivity filterformation plays an important role in maintaining the normal electrophysiological function of Nav1.7,which will be helpful to the development of Nav1.7-targeted analgesic drugs.Part two The role of Nav1.7 in acute postoperative painBackground:Postoperative pain remains a complex problem that is difficult to manage in the clinical context,seriously affecting rehabilitation and the quality of life of patients after surgery.However,the mechanism of postopetative pain is unclear.At present,it is believed that the increasing of excitability of peripheral nociceptors is an important mechanism of postoperative pain.Therefore,preventing the transmission of pain signals from the source to the central axis is the key to prevent and treat postoperative pain.During the generation and transmission of pain signals,the DRG?Dorsal root ganglion?is the bond between the body's pain signal and the spinal cord.The sodium channel Nav1.7 encoded by the SCN9 A is most important in mediating action potential of DRG.Nav1.7 regulates neuronal excitability by determining the initial threshold of action potential,which in turn plays an important role in the generation and transmission of pain signals.Hyperactivity of Nav1.7 can causes hyperexcitability of nociceptors,leading to erythromelalgia?PE?,paroxysmal extreme pain disorder?PEPD?,and Nav1.7 loss-of-function mutations cause hypoexcitability of nociceptor,leading to congenital insensitivity to pain?CIP?.The studies on animal confirmed that Nav1.7 knockout mice no longer have acute inflammatory pain triggered by nerve growth factor,complete Freund's adjuvant,etc.,and Nav1.7 inhibitors can inhibit acute inflammatory pain in rats.The previous study of our research group found that the polymorphism of SCN9 A encoding Nav1.7 affects the basal pain sensitivity of the general population and postoperative pain sensitivity of surgical patients;in addition,CIP patients not only showed no pain to acute noxious stimulation?such as injections,fractures,etc.?,but also showed no pain after surgery.Based on this,we speculate that the enhancing Nav1.7 in DRG may play an important role in postoperative pain,and it is necessary to study the expression and the effect of DRG Nav1.7 in postoperative pain.Purpose:To observe the dynamic expression of DRG Nav1.7 in the process of postoperative pain after the plantar incision in rat,and investigate the role of Nav1.7 channel protein in the development of postoperative pain.Methods:1.A rat model of plantar incision was established,before and at 2 h,24 h,48 h,72 h and 120 h after surgery,then the mechanical withdrawal threshold?MWT?,thermal withdrawal latency?TWL?and cumulative pain scores was measured,and the Nav1.7 expression was detected by Quantitative PCR?Q-PCR?and Western blotting.2.The intrathcal catheters were constructed,and lidocaine test was used to determine the success or not.The rat that intrathcal catheters constructed successfully were used for subsequent experiments.The rats were randomly divided into four groups: control group,plantar incision group,Nav1.7 interference lentivirus?SCN9A-RNAi-LV?+incision group,negative control lentivirus?NC-LV?+incision group.After 3 days,a rat model of plantar incision was established and SCN9A-RNAi-LV or NC-LV was administered intrathecally.Pain behavior were test before and 2 hours,24 hours,and 48 hours after surgery,including MWT,TWL,and cumulative pain score.Because the pain threshold was lowest and Nav1.7 expression was lowest in 2 hours after surgery in the previous experiment,we detected the expression of Nav1.7 m RNA in rat DRG by Q-PCR at 2 hours after surgery.At the same time,the expression of Nav1.7 protein in rat DRG was detected by Western Blotting and immunohistochemistry.Results:1.After plantar incision,the MWT and TWL of rats decreased significantly,and the pain cumulative score increased significantly?P<0.05?.At the same time,the expression of Nav1.7 m RNA and DRG Nav1.7 protein in the rat increased significantly?P<0.05?.At 2 hours after surgery,MWT and TWL decreased most significantly,Nav1.7 m RNA and Nav1.7 channel protein were highest,and gradually recovered after that.2.After SCN9A-RNAi-LV or NC-LV was administered intrathecally,the expression of Nav1.7 m RNA and protein in rat DRG was significantly decreased?P<0.05?but without affecting the expression of Nav1.8 and Nav1.9?P>0.05?.3.Compared with incision group,Nav1.7 in DRG of SCN9A-RNAi-LV+incision group was significantly decreased after plantar plantar incision?P<0.05?,MWT,TWL were significantly increased,and pain cumulative score was significantly reduced?P<0.05?.Conclusion:The study find that after plantar incision in rats,the MWT,TWL decreased significantly,and cumulative pain scores increased significantly,while the Nav1.7 m RNA and Nav1.7 protein expression in rat DRG increased significantly.While,specifically down-regulated the expression of DRG Nav1.7 in rat by administering SCN9A-RNAi-LV.Intrathecally,then after surgery,the MWT and TWL were increased significantly,and cumulative pain scores were reduced significantly.This indicates that increased expression of Nav1.7 in DRG is involved in the development of postoperative pain. |
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