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Application of Fluorescence-PCR on CAG Repeats of


    Application of Fluorescence-PCR on CAG Repeats of Spinocerebellar Ataxias TypeI, Type II and Type III Gene Studying*

    Miao Jiang1, 2 **, Yunqing Li3, Chunlian Jing1, Weitian Han2, Changkun Lin1, Guangrong Qin1,Zonglan Liu4, Xuefang Yang2, Kailai Sun1

    Abstract: To investigate the normal range of (CAG)n in SCA1, SCA2 and SCA3/MJD gene of Han population of Northeastern of China, we assessed the genotypes that clinically were diagnosed SCA individuals including 25 patients from 8 families and 6 sporadics, so as to make presymptomatic diagnosis. DNA fragments from healthy people were detected by fluorescence-PCR, homozygosity were selected for DNA sequencing. The results are following, the normal range of (CAG)n of SCA1, SCA2 and SCA3/MJD were 20-39, 15-29 and 14-38 repeats respectively, SCA1 was mostly 26 and 27 repeats, allele frequence was 34.09% and 20.91%, heterozygosity was 84.55%, SCA2 was mostly 22 (49.55%) and 23 (25.91%), heterozygosity was 20.00%, SCA3/MJD was mostly 14 repeats, allele frequence was 39.55%, heterozygosity was 78.18%. (CAG)68 of SCA3/MJD gene of one affected individual had been found in a family, but no CAG mutative expansion in related members was observed. So we have some conclusions, the variation of CAG repeats is different in areas and races; SCAs genotyping is the first choice in presymptomatic and prenatal diagnosis; assessing the number of repeats exactly is the key to research spinocerebellar ataxia.

    Key words: spinocerebellar ataxias type I (SCA1); spinocerebellar ataxias typeII(SCA2); spinocerebellar ataxias typeIII (SCA3/MJD); fluorescence-PCR; presymptomatic diagnosis

    The autosomal dominant spinocerebellar ataxias (SCAs) are a clinically and genetically heterogeneous complex group of neurodegenerative disorders. They are charactered by neuronal loss affecting to varying extent, the cerebellum, and brain stem nuclei and spinocerebellar. These are severe diseases that lead to uncoordinated gait (ataxia) and dysarthria (such as opthalmopl-moplegia, decreased vibration sense, sphincter disturbances), difficulities in swallowing, and other clinical features may be presented in some patients. SCA1, SCA2 and SCA3/MJD are three high frequency subtypes that are caused by a CAG expansion mutation encoded polyglucamines; patients are delayed to adults with progressive aggravation. Repeats of CAG mutative expansion have negative correlation with age of onset, but positive with severity of the disease. Similar symptoms may appear when the repeats exceed certain critical value in any subtype. We applied Fluorescence-PCR to assess the repeats of CAG in SCA1, SCA2 and SCA3/MJD genes of Han population in northeastern of China.


    25 affectted members of 8 families with hereditary ataxia and 6 sporadics were Han population from northeast China, they were clinically examined and collected by Neural Department of First Affiliated Hospital of China Medical University, 110 control subjects were collected by Liaoning Research Institute for Family Planning, they are Han population from northeast China, too.

    The genomic DNA samples were extracted by a standard proteinase K/phenol-chloroform. The Fluorescence polymerase china reaction (F-PCR) was carried out in a 25l reaction volume using 100ng DNA (2l), 1buffer (MgCl2 1.5mM), 0.1mM dNTP, 0.5M primer, 2 unites Taq polymerase, 10% DMSO. Primers were as describered [1-3]. Initial DNA denaturation at 94 for 3 min was followed by 30 cycles of 30 seconds each at 94, 57 and 72, respectively, and a final elongation step of 10 min at 72. Products of PCR were tested by POP-4 gel in electrophoresis under 15kV, 24 min, capillaries 47cm50M (caliber).

    Fragment length and genotype analysis: The shortest amplified fragment was respectively selected for DNA sequencing, CAG repeats of SCA1, SCA2 and SCA3/MJD for every sample was calculated by comparing fragment length with the shortest DNA sequence mensuration [4].


    The number of CAG repeats in normal allele for SCA1 ranged from 20~39, with 84.55% heterozygosity, 26 and 27 repeats being the allele peaks (34.09% and 20.91%), see Fig. 1-a. For SCA2 there were 15 to 29 CAG repeats, with 20.00% heterozygosity, and two different allele peaks of 22 (49.55%) and 23 (25.91%), see Fig. 1-b. For SCA3/MJD there were 14~38 CAG repeats, with 39.55% heterozygosity, 14 repeats being in the allele peaks (39.55%), see Fig. 1-c. Only one patient among all cases was found 68 CAG repeats in SCA3/MJD gene, mutation repeats in other member of this family were not tested (Fig. 2).

    a. SCA1

    Fig. 2 Fluorescence-PCR test result of SCA3/MJD family

    a: SCA3/MJD pedigree. b, c and d were II1, II3 and III1 fragment length of Fluorescence-PCR test respectively, derived CAG repeats through calculation and analysis are 21/68, 21/23 and 21/28


    Late onest cerebellar ataxias are a group of severe neurodegenerative disorders with an estimated prevalence in our population of 1 in 1,000,000[5]. Spinocerebellar ataxia is one of autosomal dominant cerebellar ataxias (ADCA). So far, the locus of 19 subtypes of SCAs, including SCA1~8, SCA10~17, SCA19, SCA21, DRPLA were found, SCA1~3, SCA6~7, SCA12, SCA17, DRPLA are caused by CAG mutative expansion, while SCA10 is caused by pentanucleotide mutative expansion [6]. SCA1, SCA2 and SCA3/MJD are the most familiar ones, whose genes are separately locus on 6p23, 12p24 and 14p24.3-q31. SCA1 is constituted by 9 exons and 8 introns. Its gene contains 450kb and mRNA forms a 2448bp opening reading frame. (CAG)n locates in 588bp of coding sequence [7]. The SCA2 gene encodes a basic 145 kD protein and the CAG repeats locats in the 5-prime end of the coding region [8]. SCA3/MJD is constituted by 11 exons and 10 introns, its gene contains 48,240bp and mRNA is expressed in all tissues of human, CAG repeats locates in the third exon [9].

    We studied the CAG repeat distribution at the SCA1, SCA2 and SCA3/MJD loci in Han population from northeast China from which the patients originated. This would help us to determine whether the prevalence varies between different ethnic groups. We had found that the normal range of (CAG)n of SCA1 is 20~39, mostly 26 or 27 with allele frequency 34.09% and 20.91% and 13 alleles, heterozygosity frequency 84.55%. This didn't accord with other relevant reports such as CAG repeats in Spanish were 6~39, mostly 31 with heterozygosity frequency 72% [10], while among 9 minorities of India the range was 23~36, mostly 27~31 with heterozygosity frequency 72%~85% [11]. At SCA2 locus, we had also found that the normal range of CAG repeats 15 to 29 with 20.00% heterozygosity, and two different allele peaks of 22 (49.55%) and 23 (25.91%), while they in Spanish were 17~29, mostly 22 with heterozygosity frequency 89%, and among 9 minorities of India the range was 14~37, mostly 22 with heterozygosity frequency 0%~36%. Meanwhile we have found CAG repeats in SCA3/MJD locus were 14~38 with 39.55% heterozygosity, 14 repeats being the most frequenct allele (39.55%), but they in Spanish were 17~29, mostly 22 with heterozygosity frequency 89%, while among 9 minorities of India the range was 14~37, mostly 22 with heterozygosity frequency 0%~36%. Therefore it is important for us to detect the number of repeats exactly, especially for population up limit of CAG repeats is close to the mutation level limit of patients. Fluorescence-PCR was high sensitivity and can show segment length of PCR product directly precisely, especially for heterozygosity that is difficult to test DNA sequence.

    The relative frequencies of the hereditary ataxias vary worldwide and also among different ethnic groups. SCA1 has been reported to be far more common in Russia than any other SCAs subtypes, whereas the general prevalence of SCA1 and SCA2 is significantly higher among white SCA pedigrees (15% and 14%, respectively) than that in the Japanese [12]. SCA1, SCA2 and SCA3/MJD were first reported of 4.70%, 5.88%, and 48.23% in China in 1999, but still about 41% of ADCA cases remained genetically unclassified. From the study of 8 cases of unrelated Chinese Han SCAs families, we only detected 1 (CAG)68 (12.5%) mutation at SCA3/MID locus, we have detected three presymptomatic subjects for SCA3/MJD in this family with CAG repeats expansions of 21/23, 21/28, so they were normal.

    Finally, the analysis of 6 sporadic cases of SCAs has indeciated the probable presence of a different genetic mutation mechanism in these patients.


    We are grateful to all of the families for their collaboration. We wish to thank many doctors of neurology department of First Affiliated Hospital of China Medical University for referring families and helping in obtaining clinical information.


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    (Edited by Tiebo Zhou, Xia Gao and Yingqi Zhao)

    * Supported by Research Foundation from Department of Science and Technology of Liaoning Provience, Project No. 99225003

    ** Corresponding to Miao Jiang, Tel: 024-86800662, 13019351149; E-mail: Jiangmiaocpt@yahoo.com.cn

    1 Department of Medical Genetics, China Medical University, Shenyang, Liaoning, China, Postcode: 110001;

    2 Liaoning Family-planning Institute, Shenyang, Liaoning, China, Postcode: 110001

    3 Departiment of 87th English Clincal Medicine, China Medical University, Shenyang, Liaoning, China, Postcode: 110001

    4 First Affiliated Hospital of China Medical University, Shenyang, Liaoning, China, Postcode: 110001





    Application of Fluorescence-PCR on CAG Repeats of Spinocerebellar Ataxias TypeI, Type II and Type III Gene Studying



    Jan.2005,Volume 2, No.1 (Serial No.2) Journal of US-China Medical Science, ISSN 1548-6648,USA