| The Human Genome Project (HGP), a marvelous science approach to human biology,is attempting to elucidate the structure and constituent of human genome ,and to decipher thesequence of DNA. These achievements facilitate the human being to understand themselveswell and unravel the mystery of human life.The human genome is the whole set of DNA arrayed in 24 distinct chromosomes, whichconsists of 3.2 billions of base pairs. The genomic DNA is 99.9%identical to that of otherhumans and 0.1% of variable .One of the fruits of the Human Genome Project is the discoveryof millions of DNA sequence variants in the human genome. The use of these genetic markershas been playing an increasing part in genetics studies.Autism is a developmental disorder characterized by significant disturbances in social,communicative, and behavioral functioning. About 3/4 patients have also obvious mentalretardation,part of them have good ability in some factors under the background of generalintellectual ability lag. The incidence of autism is 1/1000-1/2500 among childhood, and morein boys than in girls. Developmental abnormalities are apparent in the first 3 years of life,having chronic course and bad prognosis, therefore, the illness not only causes subjectssuffering in person, but also brings heavy burdens to families and societies. While the exactetiological factor and mechanism for autism remains unknown. So it is important to establisha procedure of treating and preventing autism.Recently, family and twin studies indicate that autism is one of the most stronglygenetic neuropsychiatric disorders, while the autism is not simple Mendelian disease but lookslike a complex disease involving several genes. The completion of the HGP has provided agood opportunity for mapping all the genes involved in human diseases, including autism.Basically. there are two steps for mapping a disease-related gene in the human genome, thelinkage-based genome-wide scan and the regional mapping with linkage disequilibrium (LD)analysis. At present, genome-wide scan has suggested that the following chromosomal regionsmay contain several genes contributing to autism, including 1p,2q, 3p, 6q16-21,7q31-33,13q,15q,16,17 and X. But the positioning study of predisposing genes is still disputing.In this study ,we selected FMR1 gene on X chromosome region, SLC25A12 gene andSCN2A2 gene on 2q24-33 region ,and NOTCH4 gene on 6q21 region. The study screened thegene locuses associated with autism by using a family-based LD and case-control analysis.The details of methodology and major results obtained in this study is as follows:1. Association between CGG repeated sequence polymorphism of FMR1 gene andautismThis research was performed in 321 Japanese autism family trios and 850 healthypersons and detected CGG repeated sequence polymorphism of FMR1 gene by using the7-deaza-dGTP polymerase chain reaction (7-deaza-dGTP PCR) and ALFred DNA sequencedetect method. The data was analysised by SSPS12.0. The results are as follows: 33 variousalleles were detected , the variation range of CGG repeated frequency were 8-50 in 1158 Xchromosomes among 832 normal human-beings;13 various alleles were detected , thevariation range of CGG repeated frequency were 16-36 in 121 X chromosomes among 109autism children;19 various alleles were detected , the variation range of CGG repeatedfrequency were 17-49 in 318 X chromosomes among 212 autism children's parents. CGGrepeated regions in FMR1 gene displayed polymorphism,moreover, the more being detectedpersons were, the more being detected genotypes were. We detected no premutation or fullmutation in three groups, all of them were in normal extent (n=7-60). Althoughheterozygosis rate was a litter higher in autism children group (41.67%),there was nosignificant difference between autism children's parent group (25.47%)and normal personsgroup(34.97%).Seeing the distribution of FMR1 gene CGG repeated sequence polymorphismsitus, the prominent peaks were all made of 24, 25, 26, 27, 28, 29 alleles in three groups, andtheir ratio was respectively 76.6% in normal persons group, 80.9% in autism children's parentgroup, 83.4% in autism children group. Although we could find the tendency of increase bydegrees, it was not significant in statistics (χ2=2.853, Р=0.358).The total distribution of FMR1gene CGG repeated sequence polymorphism situs in autism children group was ahead ofnormal persons group, which indicated that the opportunity of FMR1 gene CGG repeatedsequence mutation was not more increasing in autism children group than normal personsgroup. FMR1 gene CGG repeated sequence polymorphism situs had no significant differencebetween autism children group and their parents group(χ2=23.194,df=20, P =0.279). Thisstudy concluded that the abnormal expand of FMR1 gene CGG repeated sequence had norelation with childhood autism.2. Association between SLC25A12, SCN2A2, NOTCH4 gene single nucleotidepolymorphism and childhood autismThis research investigated 315 members in 105 autistic disorder cores familyconstellations, selected rs997508, rs3769955, rs3770448 and rs3765166 as genetic markersin SCN2A2 and SLC25A12 genes, which were familiar SNPs in Japnese (yusukereported) ,by looking through the known genes and SNPs of each gene on the 2q24-33region using bioinformatics method and http://www.ncbi.nlm.nih.gov. Genotypes weredetected by DHPLC, PCR-SSCP and direct sequencing (DS).On account of genealogy ,LDwas analyzed by SSCP and UNPHASED statistics programs. At the same time, to investigatethe association between rs3770448 in SLC25A12 locus and autism, the case-control studywas designed. The subjects were 323 autism cores family members and 328 normal controlpersons.In addition, we did a research among 24 autism children and 30 normal children atnorthern China, which selected two SNP locus i.e. rs367398 and rs520688 in NOTCH4 geneas genetic determinant and used PCR-RFLP to detect genotype in order to analysis therelevance with autism.2.1 The H-W equilibriumThe goodness of fit test showed that genotype frequency distributions of all subjectswere not deviated from the H-W equilibrium, thus these samples were suitable for the geneticanalysis.2.2 LD between paired SNPsThe estimated LD showed that rs3770448-rs3765166 were in the same LD block withUNPHASED program.2.3 Association between SNPs and autism2.3.1The TDT analysis and the HRR analysisThe TDT analysis and the HRR analysis revealed that neither rs99750 and rs3769955allele polymorphism in SCN2A2 gene,nor rs3770448 and rs3765166 allele polymorphismin SLC25A12 gene related with autism predisposing genes(P >0.05).2.3.2 Analysis for multi-site SNPs haplotype transmissionHaplotype transmission analysis indicated that the observed number of rs3770448(C)-rs3765166(G)monoploid transmitted by parents was significantly lower than expectednumber (χ2=9.995,P=0.0016)among autism children, the same was rs3769955(T)-rs3770448(C)-rs3765166(G)(χ2=10.660,P=0.0011). Simultaneously, the observednumber of rs997508(C)-rs3769955(T)-rs3770448(C)-rs3765166(G)monoploid transmittedby parents was significantly lower than expected number (χ2=6.306,P=0.0120)among autismchildren, and the same was rs997508(T)-rs3769955(T)-rs3770448(C)-rs3765166(G)(χ2=5.041,P=0.0248). The same character among these haplotypes was that they all hadrs3770448(C)-rs3765166(G)and they all associated with autism. Global χ2 test showed thatrs3770448-rs3765166 and rs3769955-rs3770448-rs3765166 haplotype systems were relatedwith autism ( P = 0.0133 , P = 0.0110 ) . Above all ,we can conclude thatrs3770448-rs3765166 and rs3769955-rs3770448-rs3765166 haplotype systems probably closelink with either autism predispoding genes or autism resistance gene .2.3.3 Analysis for case-control study about rs3770448 locus in SLC25A12 geneAnalysis for case-control study about rs3770448 locus in SLC25A12 gene discoveredthat genotype and allele distribution had significant difference between case group and normalcontrol group(χ2=7.336 Р=0.026;χ2=7.290 Р=0.007),so the study showed that rs3770448locus in SLC25A12 gene correlated with autism. At the same time, allele T frequency washigher in parents group and father group than that in normal control group(Р<0.05), whichmanifested that rs3770448 locus in SLC25A12 gene associated with autism familyconstellation.2.4 Association between NOTCH4 gene and autismAnalysis for case-control study about 2 SNPs in NOTCH4 gene found that allele andgenotype frequency had no significant difference between two groups(P>0.05). NOTCH4allele and genotype distribute in mild or moderate childhood autism group compared to heavychildhood autism group indicated that rs367398 和 rs520688 locus had no correlation withpathogenetic condition of autism. We should farther study through expand sample size.Summarily, the present study demonstrated that there may be many autism susceptibilitydeterminate genes located on 2q24-33 region;CGG repeated enlarge in FMR1 gene had nocorrelation with autism;and the association between NOTCH4 gene and autism need fartherresearch.These findings are advancing front topics of disease genomics in postgenome period.The consequences not only are benefit for profoundly illuminating molecular geneticsmachine of autism, but also are very important for establishing experiment foundation topredict autism in genetic level, to improve health care level, to develop new method todiagnosis, prevention and cure, to exploit new drugs.
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