Study On Mutations Spectrum And Relationships Analysis On Genetypes And TCM Syndrome Types Of Chinese WD Patients

BackgroundWilson’s disease, also known as hepatolenticular degeneration(HLD),is an autosomalrecessively inherited disorder of copper metabolism,ATP7B gene is the pathogenicgenes. The mutations of ATP7B gene lead to reduce copper excretion in the bile,andthen the copper ion is deposited in the tissues or organs of liver,brain,corneal,and so on.The main clinical manifestations are hepatic cirrhosis, serious extraparamidalneurological symptoms, Kayser-Fleischer ring (K-F ring) and psychiatric symptoms.Most studies have shown that WD has obvious genetic heterogeneity with too manytypes of mutations and polymorphisms of ATP7B gene, and so far more than600kindsof mutations or polymorphisms distributed in the21exons have been found.Themutations mostly are compound heterozygous mutation. The common mutation typesare rare, more rare mutations. The differences of mutation characteristics are obviousbetween different races, groups and regions.Therefore, the mutation spectrum resultof different races, groups and regions is a prerequisite to carry out practical clinicaldiagnosis of WD gene.Although currently there are too many studies about WD gene mutations of Chinesehuman, but the number of cases about the studies is less.There have no studies withlarge-scale WD population more than100cases are reported,and so the systemicresearch data about WD gene mutation spectrum of Chinese people is absent.In recentyears, with the emergence of high throughput and relatively low cost of genetic testingtechnology such as MassArray, making a systematic study of a large sample of WDgene mutation spectrum becomes possible.In our study, based on the research status of WD gene mutations, under theauspices of the Ministry of Health national clinical key discipline construction projects,we applicate the high-throughput PCR-MassArray detection platform to launch thelarge sample studies of Chinese human WD gene mutation spectrum. Collection the datas of genotype and phenotype of the WD patients, to research the characteristics ofgene mutations in different areas and ethnic, and try to explore the relationshipbetween the types of gene mutations in different TCM syndrome types and clinicalsyndrome types.Objective1. By using PCR-MassArray technique to detect the ATP7B gene mutations of ChineseWD patients,and clear the mutation spectrum.2. To study the characteristics of ATP7B gene mutation of different regions based on alarge sample of WD gene mutation spectrum of Chinese people.3.To explore the relationship between the common genotype of ATP7B gene and theTCM syndrome types and clinical phenotype.MethodThe name, gender, age, disease duration, initial symptoms and datas of TCMsyndrome type of the WD patients in our hospital between2008~2013years arecollected.Extract of genomic DNA, and then make use of PCR-MassArray analysismethod to detect the ATP7B gene mutation sites, to clear the WD gene muetationspectrum of Chinese WD patients, analyse the patterns and characteristics of themutation of ATP7B gene. Then explore the relationship between the common genotypeof ATP7B gene and TCM syndrome types in patients.Result1.General clinical data of WD patients(1)In this study we collected from years2008to2013confirmed cases in the AffiliatedHospital of Anhui University of Chinese Medical, Institute of Neurology total of1216cases of hospitalization, they came from800different families, in some cases from thesame family,756cases of male and female460. Age of onset1to57years, with an average age of14.30±7.26, duration of0.1to36years, with an average6.99±5.86years.1208cases of Han, Hui two cases, Manchu four cases, one case of Mongolian,Uygur one case. From Anhui (290cases), Jiangsu (111cases), Zhejiang (107cases),Henan (105cases), Shandong (101cases), Jiangxi (80cases) and other provinces.(2)With the first symptom of jaundice, ascites and other symptoms of liver patients are409cases, with the first symptom of extrapyramidal and psychiatric symptoms inpatients are609cases. Patients with blood system, digestive system, as well as boneand joint symptoms as the first symptom are198cases.(3)There are588patients of cerebral type with the extrapyramidal symptoms onset, orthe main clinical manifestations is extrapyramidal symptoms in the duration, and haveno obvious symptoms of liver;447cases with the symptoms of liver damage ofappetite lossing, jaundice, ascites or hypersplenism are liver-type; The WD patientswhose symptoms of liver disease and neurological symptoms are the main feature,concomitant damage to other organs of the brain-visceral are150patients; There arepatients,31cases of non-obvious clinical symptoms, attributed to subclinical disease.(4) Based on symptoms, signs, tongue, pulse, four diagnostic parameters,139typicalcases are classified into five types:106were liver kidney yin insufficiency type,62were Qixue insufficiency type,102patients belonged to the phlegmatic heat internalresistance type,6were phlegm accumulating with stagnation type and2is Phlegm-Firedisturbance heart.2.The results of ATP7B gene mutations in WD patients(1)There are59different mutations of1216WD patients. The mutations ofp.Arg778Leu in exon8and p.Pro992Leu in exon13are the highest frequency, the ratesare32.32%and18.54%respectively; The frequencies of mutations of p.Ile1148Thr inexon16,p.Arg919Gly in exon12, p.Asn1270Ser in exon18, p.Val1216Met in exon17,p.Met769Hisfs*26in exon8, p.Ser975Tyr in exon13, p.Thr935Met in exon12, arerespectively1.81%,1.77%,1.60%,1.60%,1.52%,1.44%,1.07%. The other mutationsfrequencies are less than1%. One patient was detected p.His1069Gln in exon14(the mutation hotspot in Europe and America).(2)The frequencies of exons of8,13,12,16,18, respectively are34.58%,21.30%,3.42%,3.20%and3.12%; The mutation rate located in the transmembrane domain (exons of7,8,12,13,19,20) is60.49%, ATP enzyme function domain (exons of10~11,14~18)is11.19%, copper ion binding domain (exons of2~3,5) is2.43%.(3)In1216cases of patients, the Han population is1,208cases, their highestfrequency of mutation are p.Arg778Leu (32.07%) and p.Pro992Leu (18.50%);minority has eight, including three cases of p.Arg778Leu and one ofp.Pro992Leu,frequencies respectively are of37.50%and12.50%, suggesting thatthese two mutations are also the hot spots of ethnic minorities.(4)There are724cases in East China, followed by the South China (236cases), North(94cases), the Northeast (84cases), Southwest (40cases), Northwestern (38cases),the main mutations are mainly p.Arg778Leu and p.Pro992Leu, the frequencies of thetwo respectively are33.08%,19.27%in East China,28.81%,16.74%inSouth,32.45%,11.17%in North,35.12%,15.48%in Northeast,27.50%,21.25%inSouthwest,35.53%,17.11%in Northwestern.these two mutations is no pointthroughout the range significant difference (P>0.05).3.p.Arg778Leu mutation group compared with non-mutation group has obviousdifference(P <0.05),and no significant difference in the clinical classification (P>0.05);p.Arg919Gly mutation group compared with non-mutation has obvious difference inthe clinical classification and first syndrome(P <0.05), and no significant difference inthe gender(P>0.05);The mutation groups of p.Pro992Leu, p.Ile1148Thr, p.Asn1270Serand p.Val1216Met compared with non-mutation group have no significant difference inthe clinical classification, the first symptom and gender (P>0.05).4.p.Arg778Leu mutation group with liver kidney yin insufficiency type is significantdifference (P <0.05), p.Pro992Leu、p.Ile1148Thr and p.Arg919Gly have no significantdifference between mutations and TCM syndrome types(P>0.05). Conclusion1.The exons8and13are the first mutation hotspots, the exons12,16and18are thesecond hotspots; mutation hotspots of ATP7B gene are located transmembrane domain,The functional domain and copper ion binding domain are not hotspots of Chinese WDpatients.2.p.Arg778Leu in exon8and p.Pro992Leu in13exons are high-frequency mutationsof ATP7B gene of Chinese WD patients (mutation rate were32.32%and18.54%,respectively). The frequencies of p.Ile1148Thr in exon16,p.Arg919Gly in exon12,p.Asn1270Ser in exon18, p.Val1216Met in exon17, p.Met769HisfsX26in exon8,p.Ser975Tyr in exon13, p.Thr935Met in exon12, are respectively1.81%,1.77%,1.60%,1.60%,1.52%,1.44%,1.07%.3.There was no differencies between the genotype and regions(P>0.05).4. p.Arg778Leu mutation group, p.Arg919Gly mutation group compared withnon-mutation group has significant difference in the clinical classification;p.Arg919Gly mutation group compared with non-mutation group has significantdifference in the first symptom; p.Arg778Leu, p.Pro992Leu, p.Ile1148Thr,p.Arg919Gly, p.Asn1270Ser and p.Ser975Tyr mutation groups has no significantdifference in the gender.5. p.Arg778Leu in WD maybe correlated with the type of liver kidney yininsufficiency type(P<0.05).