Genetic Epidemiologic Survey Of AIS In Chongqing Of China And Comparative Analysis Of Sequence Alignment Of SH3GL1

Background and Objects of the StudyAdolescent Idiopathic Scoliosis (AIS) is a three-dimensional deformation of dorsal spine structural perversion with vertebral body rotation. The incidence of children and teenagers is 2%~5% from reports abroad while incidence is 1.5%~3% from domestic reports. The incidence of severe deformation is 0.2~0.5% with female patients obviously more than male ones. Though ALS ususlly does not cause severe pathologic state except deformation, severe thoracocyrtosis may cause heart and lung hypofunction, leading to conspicuous somatotype deformation and even life. At present, the available treatments focus on correcting and curing on deformation including clinical observation, orthosis treatment, and operation on patients with lateral curvature above 45o. However, the treatment process was complicated with spinal deformity and dorsal spine activity obstacle left, producing no ideal effect. The ideal effect is to eliminate deformity. If diagnosis and cure on etiological factor can be made before symptoms and birth, or even gene diagnosis and cure can be made for pregnant women with family medical history before 6 days of ferrtilization and embryo nidation, spinal deformity will be cut off and AIS eliminated clinically. But etiological factor and invasive gene mechanism need to be identified first.So far, The definite etiological factor of AIS has not been detected. Past researches concentrated on pathogenic mechanism of histology factors such as cartilage, ossature, nerves and muscle, but no definite conclusions were drawn. With unceasing deepening and progress of researches on medical genetics and medical molecular biology, clinically pathogenesy of many agnogenic common diseases has been proved relevant with heridity and virulence gene. At present, medical molecular biology holds that generation of various kinds of diseases is related with one or many genes. Thus people are easy to think that whether AIS is concerned with heredity? Recently many scholars deem AIS correlates with heredity. If it is related with heredity, what is the heredity mode? monogenic inheritance mode or polygenic inheritance mode? How is the genic location and molecule mechanism of disease? The approach to influence of genetic factors on disease and possible hereditary modes is the task of genetic epidemiology, while the approach to disease genic location and mechanism evoking disease, the research task of medical molecular biology.There are few research reports about AIS epidemiology and genetic epidemiology, but these reports show familial aggregation. Genetic factor was deemed to be involved with AIS, which has several inheritance modes including autosomal dominant inheritance mode, X-linked dominant inheritance mode, and polygenic inheritance mode. That is to say, no final conclusion is drawn about whether AIS is genetic disorder and what kind of hereditary mode it is. No definit hereditary mode bring obstacle for chromosomal assignment study on AIS pathological gene The screening and identification of pathological gene were also affected consequently. Different analytical methods were applied in accordance with corresponding inheritance mode during the process of Chromosomal assignment analysis on pathological gene. Linkage analytical method was applied for monogenic disorder while correlation analysis for polygenic disease. The analytical results were indefinite with indefinite inheritance mode, which gave rise to different research results of Chromosomal assignment study. The decisive role was played by discussions on the influence of genetic factor on AIS and the possible inheritance mode in Chromosomal assignment study on AIS pathological gene as well as screening and identification of pathological gene. Only by determining AIS genetic factor and inheritance mode can Chromosomal assignment of pathological gene be conducted by employing linkage analysis (analytical method aiming at monogenic inheritance disorder) or correlation analysis (analytical method aiming at polygenic inheritance disorder) under the guidance of the inheritance mode.Some studies on AIS molecular genetics mechanism have been reported. Two strategies (Norientation or uorientation ) were employed to the study. However the study is still at the stage of early chromosomal assignment study of the disease gene locus, and up to now a consensus gene locus has not been determined. Norientation strategy (classical genetics), namely the cloning methods based on gene function, was oriented to function of proteic substance or differential gene expression of mRNA. Multiple molecular biology methods oriented to differential hybridization were applied. Initially specific expression and the lacked gene fragment were acquired by subtraction, and then chromosomal assignment ending with obtaining whole gene was conducted. Studies on mRNA or differential expression of proteic substance of different tissues of AIS diseased region have been reported. They focused onⅠ,Ⅱ,X type collagen, TGF-?1,bFGF and osteoblast protein expression differences inside intervertebral cartilages in lateral curvature dorsal spine protruding and curved side andⅠ,Ⅱ,Ⅸtype collagen mRNA expression difference. Although it has been unanimously reported that differential expression existed, yet it has not been determined whether the differential expression was etiological factor or the result of disease. That is to say, the genuine etiological factor has not been detected. The reason of this status quo is that research and design use contrast of protruding and curved side. If differences of genuine tissue, protein or mRNA are needed to be found, contrast research of proband before and after invasion or research between proband and normal people should be made. But it is hard to make clinically, so the result is hard to break through with the only hope for"reversing"research.Positional cloning genetic analysis was applied to the chromosomal assignment, screening, and identification of disease gene in the strategy of reverse genetics (molecular genetics). The method was premised on the genetics principles that any gene has its gene locus in chromatin body, and genetics characters determined by genes were closely correlated with these gene loci. position information of disease gene in chromatin body was obtained through genetics analysis, then according to the position information, the physical marking of"physical map"was applied to the transformation from genetics information to definite chromatin region represented by the physical marking, then the expressible gene was screened in accordance with linked gene fragment grouping of the correlated regions, and transcription map of the region could be made. Finally two methods of positioning candidate cloning and function candidate cloning were employed to compare the differences of architectural gene between the patients and the healthy one, thus changes correlated with the disease were defined, and disease gene was screened and identificated. 7 reports about positional cloning study on AIS disease gene has been retrived, yet a consensus conclusion has not been drawn due to different study groups, genetics statistical analytical method, and gene loci of chromosomal assignment. No received positioning results were got respectively for chromosome 6,9,10q,16,17,17p11,19p13.3,19 p 13 and Xq21. It should be noticed that two scholars positioned the disease gene locus in 19p13 in their positional cloning studies. Their trail objects were Americans and Cantonese respectively. The key-gene locus of the latter was 19p13.3, and minor gene locus was 2p, which were approved to be located in the vicinity of the mark of D19S216 , ranging in the breadth of 5.2cM between D19S894～D19S1034 with the approximate length of 1.9Mb through further accurate positioning and recombination. Research objects that receive experiment are Cantonese. More than 400 fluorescently-labeled microsatellite signals were applied to make the whole genome screening, and two correlation analysis statistical methods of parameter and nonparameter were adopted. Influence of hereditary mode on results was basically eliminated. Besides, results from one family constellation were checked in other 6 family constellations with large sample size. Affectoi of article is as high as over 10.0, producing a convincing result. Although the disease gene locus was accurately positioned in the vicinity of the mark of D19S216 in the study, the research was still in the province of chromosomal assignment study of disease gene. The following screening and identification of pathological gene were not reported, which adequately approved that the gene locus was highly likely to correlate with AIS. Methods of positioning candidate cloning were worthy applying to the study on screening and identification of 198 genes in the position area.To make sure the number of intragenes 19p13.3, 198 known genes inside 3 subband are retrived, among which only 17 are genes of known diseases. Gene reports related with AIS has not been attained.If 198 genes inside 3 subband are to be screened, which gene to be start with were worthy studie. Certainly, gene that is the most related with AIS is chosen. Accurate positioning of Chan is in the vicinity of the mark of D19S216. From physical map, SH3GL1 is just located in this area. In report, Chan also pointed that SH3GL1 is concerned with osteoclast metabolism, and may be the important virulence gene of AIS.SH3GL1 is the first choice.There are not many research reports about function of gene SH3GL1. People like Wise reported that gene SH3GL1 is revelant with cytophagocytesis, and participated in adjustment of Ca ion channel of cell internalization and excretion couplant. Some researches also think that SH3GL1 is related with leukemia in human beings.Therefore the study was designed to make genetic epidemiologic survey and analysis of AIS in Chongqing and discuss the effect of inheritance factors on the AIS and the possible inheritance mode. Moreover, the method of positioning candidate cloning was applied to the analytical study on the Exon Sequence Alignment of the AIS Disease Candidate Gene SH3GL1, making technology preparation for the screening and identification of the disease gene.Research Method1.Genetic epidemiologic survey: The genetic epidemiology questionnaire for AIS was designed, and the data of index case who received medical treatment in Southwest Hospital were collected. Diagnostic code: affected individual: std. A-P position X-ray Cobb Ang > 10o; susc.: Cobb Ang <10o; the undetermined : normal phaenotype and age under 12. Initially trained investigators inquired index cases and their family members, and then filled the questionnaires. Investigating methods include out-patient clinic, hospital room inquiry, and telephone inquiry. Index cases and their fathers and mothers were inquired respectively to avoid memory errors. The suspected affected individual and susc. were received clinical inspections, Adam waist-bending test, and X-ray check when necessary.2. Statistical analysis of genetic epidemiology: SPSS10.0 statistics software was applied to the analysis of the genetic epidemiology survey data. The influence of genetic factor was determined by analyses on disease age incidence, familial aggregation, and calculation of degree of heritability. Inheritance mode was determined according to analysis of features of the disease of monogenic or multifactorial inheritance.3. Positioning candidate cloning:"case-sibling control design"research scheme based on family constellation was designed. A sib pair was choosed to collect blood preparation, and genome DNA was extracted. According to nucleotide sequence of gene SH3GL1, primer pair for PCR amplification, cloning, and sequencing with 10 exons as emphasis was designed.4. Sequence comparative analysis: sequence comparative analysis for exon sequencing result between sib pairs, and that between sib pair and NCBI were conducted through GeneTool software to judge whether basic group mutation occurred or not. amino acid sequence comparative analysis for predict was made.Research Results1. Genetic epidemiologic survey: The genetic epidemiology questionnaire for AIS was designed and genetic epidemiology survey on 73 family constellations was conducted successfully according to the questionnaire.2. Statistical analysis of genetic epidemiology: analysis of disease age incidence: morbility of the disease of 75% occurred between 11and 14. The mean morbility age was 10.84. Analysis of familial aggregation: family total disease incidence of the case was 4.86%. Disease incidence of first degree relative was 16.93%, and disease incidence of second degree relative was7.21%, and disease incidence of third degree relative was 4.36%, and disease incidence of the reference family was 2.25%. The incidences were significant differences by statistics analysis. (u=6.451,P﹤0.01 )Calculation of degree of heritability (Falconer Method): degree of heritability of first degree relative was 84%, degree of heritability of second degree relative was 88%, and degree of heritability of third degree relativ was above 85%, which had statistical significance through significance test (P<0.01)3. Positioning candidate cloning: 10 exons of the candidate gene SH3GL1 were successfully amplified and cloned in genome DNA of an AIS sib pair, and the sequencings obtained positive results.4. Sequence comparative analysis: 12 basic group mutations were found in 10 exons of the candidate gene SH3GL1 of patients with AIS. These mutations were located in the second exon (2 mutations), the fourth exon, the fifth exon (3 mutations), the sixth exon, the eighth exon, and the tenth exon (2 mutations, noncoding region). If basic group in 515 of mRNA was mutationed to T, stop codon(TAG) was made and open reading frame was altered , the sequence of proteinum was predicted and show brachytmema proteinum was encoded which could cause changes of primary structure.Conclusions:1. AIS was genetic disorder. Morbility of the disease usually occurred between 11 and 14 according to the analysis of disease age incidence. The family disease incidence of the case, disease incidence of first degree relative and the second degree relative and third degree relative were found obviously higher than the average disease incidence of the reference group. AIS was found to have obvious familial aggregation. The degree of heritability of first degree relative and second degree relative and third degree relativ was above 84%, which approved AIS was significantly correlated with heredity. AIS belonged to genetic disorder through comprehensive analysis. 2.AIS was a disease of multifactorial inheritance. On the whole, AIS was a genetic disorder; and the disease incidences of the index case's all degrees of relatives were found to be higher than that of the reference crowd. Sibling disease incidence was found far lower than 1/2 or 1. The closer blood relationship with the index case means higher disease incidence. The disease has obvious familial aggregation. Among 73 family constellations, only 3 of them were found to have the family history of the disease. Index cases in other family constellations were all single cases. AIS belonged to the category of a disease of multifactorial inheritance through comprehensive analysis.3. Gene SH3GL1 was one of the virulence genes of AIS. sequence comparative analysis show that Basic group mutations were found in exons of the candidate gene SH3GL1, and 10 of the mutations were located in the coding region and 2 of the mutations were located in the noncoding region. If basic group in 515 of mRNA was mutationed from C to T, stop codon(TAG) was made and open reading frame was altered , the sequence of proteinum predict show brachytmema proteinum was encoded which could cause changes of primary structure. It was demonstrated that Gene SH3GL1 was one of the virulence genes of AIS.