| Background Multiple sclerosis?MS?is a central nervous system's white matter inflammatory demyelinating autoimmune disease that due to the attack of the immune system.The onset age of disease is mostly 20?40 years old,and women account for about 65%?70%.The white matter surrounding of ventricular,cerebral stem,cerebellum,optic nerve and spinal cord are frequently affected by multiple sclerosis.The primary clinical manifestations are repeated episodes of extremities weakness,paresthesia and visual impairment,ect.Neuromyelitis optica?NMO?is also an autoimmune disease that principally involving the optic nerve and spinal cord,and it is distinguished by the acute or subacute demyelinating lesion.Young adults are in the majority,but women have significantly higher incidence that females outnumber males by five?ten to one.Fatigue,frigidity,diarrhea and other incentives are contributing to NMO,and the disease is customarily accompanied by other autoimmune diseases.The dominating clinical manifestations are attacks of eyesight,weakness of extremities,sore eyes,nausea and vomiting,urination and defecation function disturbance.The clinical diagnosis can be made according to the simultaneously or successively occurrence of optic neuritis and myelitis,what's more,the auxiliary inspection results of cerebral and spinal cord MRI and NMO-Ig G serological tests.MS and NMO are the most common types of demyelinating disease of the central nervous system,and NMO was invariably regarded as a subtype of MS.There are plenty of similarities between them,for instance,demyelinating is a mutual characteristic manifestation in their's pathological process.The pathogenesis of two diseases is still a brainteaser.However,the mainstream standpoint of contemporary society considers that MS and NMO might be the consequence of the comprehensive effects of genetic susceptibility,environmental elements and infectious elements.Owing to the absence of specific therapeutic drugs,diseases bring about diverse extent of disability frequently and reduce the living standards of patients seriously.Both MS and NMO have a tendency of attacking on females and simultaneously have a distinct gender distribution.Abundant of clinical observations and experiments have manifested that pregnancy can significantly affect the occurrence and development,the severity and recurrence of MS and NMO.Symptoms can be significantly reduced during pregnancy and rarely recur in remission.Although the specific mechanism of the above-mentioned phenomena is ill-defined,it is speculated that the pathogenesis of MS and NMO might be associated with estrogen.Estrogen can be divided into ER? and ER?,and it plays a vital physiological role in the human body by integrating with its specific receptor.The expression and function of estrogen receptor influence the biological effect of estrogen.The effects of estrogen on our prevenient researches have draw a conclusion that icariin?ICA?can play its therapeutical effects for autoimmune encephalomyelitis experimental autoimmune encephalomyelitis?EAE?by ER?.Besides,a plentiful of researches has indicated that estrogen is bound up with the pathogenesis and treatment of MS and NMO,and the mechanism is associated with estrogen receptor gene polymorphism.ER? gene frequent polymorphic loci are Pvu? and Xba?,and the ER? gene frequent polymorphic loci are Rsa? and Alu?.The endonuclease enzyme of Pvu? and Xba?were used for the cleavage of ER? gene,so that it can distinguish three genotypes respectively,PP,Pp,pp and XX,Xx,xx.The endonuclease enzyme of Rsa? and Alu? were used for the cleavage of ER? gene,so that it also can distinguish three genotypes respectively,RR,Rr,rr and AA,Aa,aa.However,the relationship between the above-mentioned ER gene polymorphism and MS and NMO is ambiguous.Therefore,study on the correlation between ER gene polymorphism and MS and NMO have turned into a trending topics for the past few years.The diagnosed MS and NMO patients were incorporated into this research as a case group.Peripheral venous blood was collected,and the genotypes of single nucleotide polymorphism?SNP?sites were detected by polymerase chain reaction?PCR?and restriction fragment length polymorphism?RFLP?.To investigate the correlation between ER gene polymorphism and MS and NMO ultimately.Objective To study the polymorphism distribution of ER gene and the correlation between different types of polymorphism in MS and NMO patients:by means of Pvu? and Xba? restriction fragment length polymorphism enzyme recognition sites of ER? gene,and Rsa? and Alu? enzyme recognition sites of ER? gene,polymerase chain reactionrestriction fragment length polymorphism?PCR-RFLP?analysis were conducted.To comprehend the ER? and ER? gene polymorphism distribution in MS and NMO patients and in healthy volunteers as well as to probe into the correlation between them and MS and NMO.Consequently,objective biological experimental basis can be provided for the researches on the susceptibility of MS and NMO and the influencing factors of curative effect and the formulation of comprehensive treatment scheme.Methods Part I The selection of patients with multiple sclerosis and optic neuromyelitis as well as clinical information.Blood samples were gathered from all MS and NMO patients who came to the hospital for treatment.Peripheral venous blood samples?3 ml?were collected,and EDTA was used for anticoagulation.Samples were numbered and recorded for backup.All MS patients conformed to?Mc Donald diagnostic criteria for multiple sclerosis,2017 revision?,and all NMO patients conformed to?Guidelines for diagnosis and treatment of optic neuromyelitis spectrum diseases in China ?.The patients who accompanied by other autoimmune diseases,tumors and craniocerebral infectious diseases were excluded.Healthy volunteers of identical age and gender were chosen for the control group,and 3ml of peripheral blood was collected for backup.There were 46 cases in MS and NMO patient group,including 5males and 41 females,that aged from 17 to 67 years,with an average age of?43.63±13.76?years.Disease duration ranged from 5 days to 12 years,with an average of?2.61±3.98?years.There were 58 cases in the healthy control group,including 8 males and 50 females,that aged from 17 to 72 years,with an average age of?46.20±14.44?years.Part II PCR-RFLP was applied for detecting the genotypes of each SNP locus.First of all,genomic DNA was extracted from whole blood and PCR amplification.And then,PCR products were inspected by two percent agarose gel electrophoresis.Last but not least,restriction enzyme digestion was performed on the amplification products and the restriction enzyme genotype was analyzed according to the electrophoresis stripes.Part III The analysis of the correlation between different types of ER gene polymorphism in MS and NMO patients According to the polymorphism types of ER gene that are screened in each patient,correlation analysis was carried out by clinical information of patient,for instance,gender,onset age,disease duration and initial symptom,then to further study the correlation between gene polymorphism type and clinical phenotype.Part IV Statistical approach The software SPSS version 20 was used for analysis.Firstly,the measurement data was described by mean±standard deviation.Secondly,t-test was applied for two groups,one-way analysis of variance was used to contrast the parameters between groups,and LSD-t test was applied for the pairwise comparison of multigroups.Thirdly,Pearson correlation analysis was applied for the variables with correlative trends.Fourthly,the chi-square goodness of fit test was applied for whether each genotype distribution of two sets abides by Hardy-Weinberg equilibrium,and employing chi-square test or Fisher's Exact Test to contrast difference that shown by the genotype frequency and allele frequency distribution of two groups respectively.Finally,by virtue of the Logistic regression's odds ratio and 95% confidence interval to analyze the relative allele risk of age and gender.Results Part I The distribution of ER gene polymorphism in MS and NMO patients as well as healthy volunteers.There was no significant difference in the frequency distribution of ER? gene's PP,Pp and pp genotype between MS and NMO case group and control group?P=0.598?.The distribution of P and p allele frequency between two groups was not statistically significant?P=0.456?.The difference between case group and control group was statistically significant in men?P=0.043?.To be specific,the frequency of P and p allele were uniform in case group of MS and NMO.The majority were Pp genotype,and the minority were PP and pp genotype.In control group,the frequency of P allele were higher than p allele.The majority were PP genotype,and the minority were pp genotype,and PP genotype couldn't be detected.However,the difference between case group and control group was not statistically significant in women?P=0.414?.Frequency distribution of ER? gene's XX,Xx and xx was statistically significant between MS and NMO case group and control group?P=0.021?.Further pairwise comparison showed that the frequency distribution of Xx and Xx genotype was statistically significant between case group and control group?P=0.006?.By means of Logistic regression analysis,suggesting Xba? genes were independent factors affecting MS and NMO morbidity.Among them,distribution of Xx and Xx gene frequency between patient group and control group was statistically significant?P=0.001,OR=4.622,95%CI:1.803?11.852?.It is suggested that the prevalence rate of Xx genotype is higher than that of xx genotype,and the odds ratio is 4.622.There was no statistically significant difference between case group and control group in men?P=0.598?,but there was a statistically significant difference in women?P=0.005?.That is to say,the frequency of X and x allele were analogous in MS and NMO case group.The majority were Xx genotype,and the minority were XX and xx genotype.In control group,the frequency of x allele were higher than X allele obviously.The majority were Xx and xx genotype,and the minority were XX genotype.There was no significant difference in the frequency distribution of ER? gene's RR,Rr and rr between MS and NMO case group and control group?P=0.797?.The distribution of R and r allele frequency between two groups was not statistically significant?P=0.797?.There was no significant difference between case and control group in both male and female(Pmale=0.715;Pfemale=0.891).There was no significant difference in the frequency distribution of ER? gene's AA,Aa and aa between MS and NMO case group and control group?P=0.620?.The distribution of A and a allele frequency between two groups was not statistically significant?P=0.843?.There was no significant difference between case and control group in both male and female(Pmale=0.188;Pfemale=0.347).Part II The correlation between ER genes and MS and NMO.1.Correlation between genotypes and onset age in patient group:the results showed that there was no significant correlation between ER? and ER? genotypes and the onset age in patient group.?1?Comparison of onset age of Pvu? each genotype:PP and Pp?P=0.317?;PP and pp?P=0.622?;Pp and pp?P=0.590?.?2?Comparison of onset age of Xba? each genotype:XX and Xx?P=0.885?;XX and xx?P=0.761?;Xx and xx?P=0.717?.?3?Comparison of onset age of RR and rr genotype:RR and rr?P=0.782?.?4?Comparison of onset age of Alu? each genotype:AA and Aa?P=0.903?;AA and aa?P=0.430?;Aa and aa?P=0.340?.2.Correlation between genotypes and disease duration in patient group:?1?The difference was not statistically significant in disease duration of Pvu? each genotype:PP and Pp?P=0.406?;PP and pp?P=0.625?;Pp and pp?P=0.583?.?2?Comparison of disease duration of Xba? each genotype:The difference was statistically significant in disease duration of XX and Xx genotype,and the disease duration of Xx genotype is longer than that of XX genotype?P=0.006?.Whereas,there was no significant difference in disease duration of XX and xx genotype as well as Xx and xx genotype?P value respectively:P=0.282;P=0.412?.?3?The difference was not statistically significant in disease duration of RR and rr genotype:RR and rr?P=0.263?.?4?The difference was not statistically significant in disease duration of Alu? each genotype:AA and Aa?P=0.227?;AA and aa?P=0.983?;Aa and aa?P=0.306?.3.Correlation between genotypes and gender:Logistic regression analysis showed the comparison of Xx and xx genotype frequency distribution in gender exists difference?P=0.047,OR=7.500,95%CI:1.023?54.996?.It is suggested that patients with Xx genotype are more likely to be female than those with xx genotype,and the odds ratio is 7.500.4.Correlation between genotypes and first-episode symptom:the majority of homozygous genotypes of PP,pp,XX,xx,RR,rr,AA and aa had extremities weakness as the first-episode symptom,whereas the majority of heterozygous genotypes of Pp,Xx and Aa had paresthesia as the first-episode symptom.Conclusions1.Xba? gene polymorphisms in ER? gene have correlation with MS and NMO.Xba? gene could be a risk factor of MS and NMO pathogenesis,especially the women with Xx genotype are more vulnerable.2.Xba? gene polymorphisms in ER? gene may impact the disease duration of MS and NMO,or rather,the disease duration of Xx genotype persists longer than Xx genotype.3.Neither Pvu? gene polymorphisms in ER? gene nor Rsa? and Alu?gene polymorphisms in ER? gene has correlation with MS and NMO.4.The majority of homozygous genotypes had extremities weakness as the first-episode symptom,whereas the majority of heterozygous genotypes had paresthesia as the first-episode symptom. |
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