联合应用PCR扩增和Sanger测序对脊髓小脑型共济失调进行基因诊断的临床意义

doi:10.3969/j.issn.1673-8640.2019.06.012

摘要/Abstract

摘要：

目的分析1个遗传性脊髓小脑型共济失调（SCA）家系的基因突变情况,对该家系进行分型,为遗传咨询提供依据。方法采集1个SCA家系5例个体（含3例患者）的外周血,提取基因组DNA。设计9对引物,采用聚合酶链反应（PCR）分别扩增SCA1、SCA2、SCA3、SCA6、SCA7、SCA8、SCA12、SCA17、齿状肌麻痹性萎缩（DRPLA）基因,筛选该家系的致病基因。对筛选出的SCA致病基因通过Sanger测序进行验证。结果 3例患者SCA3基因中CAG重复序列数分别为76、78和75,超出文献报道的12~44重复数的正常范围,确诊为SCA3家系。通过PCR扩增及Sanger测序,家系中1例携带CAG重复序列数为74的个体被确诊为SCA3致病基因携带者。结论联合应用PCR扩增和Sanger测序是一种简单易行的SCA基因诊断途径,可应用于携带者诊断和产前诊断。

关键词: 遗传性脊髓小脑型共济失调, 聚合酶链反应, Sanger测序, 基因诊断

Abstract:

Objective To investigate the causative mutations in a spinocerebellar ataxia（SCA） pedigree and the types of the SCA pedigree,and to provide a reference for genetic counseling. Methods Genomic DNA was extracted from peripheral blood of 5 subjects,including 3 SCA patients,in the SCA pedigree. Totally,9 sets of primers were designed for the polymerase chain reaction（PCR） amplification of 9 fragments,including SCA1,SCA2,SCA3,SCA6,SCA7,SCA8,SCA12,SCA17 and dentatorubral-pallidoluysian atrophy（DRPLA） gene,respectively. Causative mutations were screened and were validated by Sanger sequencing. Results Repeat lengths of 76,78 and 75,above that of 12-44 CAG triplets reported in healthy people from literatures,were observed in the 3 patients from the SCA3 pedigree. Another member carrying a repeat length of 74 in the pedigree was diagnosed as a causative mutation of the SCA3 gene carrier by Sanger sequencing. Conclusions PCR amplification combined with Sanger sequencing is a convenient and efficient method to identify causative mutations for SCA gene,which can be used for carrier screening and prenatal diagnosis.

Key words: Spinocerebellar ataxia, Polymerase chain reaction, Sanger sequencing, Genetic diagnosis

中图分类号:

R446.1

陈韭铭, 顾鸣敏, 孙顺昌. 联合应用PCR扩增和Sanger测序对脊髓小脑型共济失调进行基因诊断的临床意义[J]. 检验医学, 2019, 34(6): 534-538.

CHEN Jiuming, GU Mingmin, SUN Shunchang. Genetic diagnosis of spinocerebellar ataxia by PCR amplification and Sanger sequencing[J]. Laboratory Medicine, 2019, 34(6): 534-538.

图/表 7

参考文献 12

[1]	KANACK A J,NEWSOM O J,SCAGLIONE K M.Most mutations that cause spinocerebellar ataxia autosomal recessive type 16(SCAR16) destabilize the protein quality-control E3 ligase CHIP[J]. J Biol Chem,2018,293(8):2735-2743.
[2]	HIGUCHI Y,OKUNUSHI R,HARA T,et al.Mutations in COA7 cause spinocerebellar ataxia with axonal neuropathy[J]. Brain,2018,141(6):1622-1636.
[3]	RAMANI B,HARRIS G M,HUANG R,et al.A knockin mouse model of spinocerebellar ataxia type 3 exhibits prominent aggregate pathology and aberrant splicing of the disease gene transcript[J]. Hum Mol Genet,2017,26(16):3232-3233.
[4]	YOKOYAMA T,ISHIYAMA M,HASEGAWA K,et al.Novel neuronal cytoplasmic inclusions in a patient carrying SCA8 expansion mutation[J]. Neuropathology,2014,34(1):27-31.
[5]	MAGAÑA J J,TAPIA-GUERRERO Y S,VELÁZQUEZ-PÉREZ L,et al. Analysis of CAG repeats in five SCA loci in Mexican population:epidemiological evidence of a SCA7 founder effect[J]. Clin Genet,2014,85(2):159-165.
[6]	LI M,PANG S Y,SONG Y,et al.Whole exome sequencing identifies a novel mutation in the transglutaminase 6 gene for spinocerebellar ataxia in a Chinese family[J]. Clin Genet,2013,83(3):269-273.
[7]	KRYSA W,SULEK A,RAKOWICZ M,et al.High relative frequency of SCA1 in Poland reflecting a potential founder effect[J]. Neurol Sci,2016,37(8):1319-1325.
[8]	MONGELLI A,SARRO L,RIZZO E,et al.Multiple system atrophy and CAG repeat length:a genetic screening of polyglutamine disease genes in Italian patients[J]. Neurosci Lett,2018,678:37-42.
[9]	PULST S M.Degenerative ataxias,from genes to therapies:the 2015 cotzias lecture[J]. Neurology,2016,86(24):2284-2290.
[10]	SHANG X J,XU H L,YANG J S,et al.Homozygote of spinocerebellar Ataxia type 3 correlating with severe phenotype based on analyses of clinical features[J]. J Neurol Sci,2018,390:111-114.
[11]	DIALLO A,JACOBI H,COOK A,et al.Survival in patients with spinocerebellar ataxia types 1,2,3,and 6(EUROSCA):a longitudinal cohort study[J]. Lancet Neurol,2018,17(4):327-334.
[12]	MELO A R,RAMOS A,KAZACHKOVA N,et al.Triplet repeat primed PCR(TP-PCR) in molecular diagnostic testing for spinocerebellar ataxia type 3(SCA3)[J]. Mol Diagn Ther,2016,20(6):617-622.

引物名称	引物序列（5~3'）	退火温度（℃）	扩增片段长度（bp）
SCA1	F：5'-GGTCCTCCCAATACAGTGGA-3'	60	364~403
SCA1	R：5'-TTCTGCGGAGAACTGGAAAT-3'
SCA2	F：5'-CTCCGCCTCAGACTGTTTTG-3'	60	810~837
SCA2	R：5'-CTGACCATCGCCGCTACC-3'
SCA3	F：5'-TCTGTATCAGACTAACTGCTCTTGC-3'	60	369~438
SCA3	R：5'-GAGGGAATGAAGAATAATGTAAAGC-3'
SCA6	F：5'-TCCCGTGTCTCCTTTGATTT-3'	60	378~411
SCA6	R：5'-GACCCGCCTCTCCATCCT-3'
SCA7	F：5'-TAGGAGCGGAAAGAATGTCG-3'	57	280~310
SCA7	R：5'-CCCAGCATCACTTCAGGACT-3'
SCA8	F：5'-GCAGTATGAGGAAGTATGGAAAAA-3'	60	230~335
SCA8	R：5'-TTCTGACTCCCAGCTTCCAC-3'
SCA12	F：5'-CCACTGCAGCAAAGAGCA-3'	60	280~355
SCA12	R：5'-GGAATGAGGGTGCTGGTC-3'
SCA17	F：5'-GACC CCACAGCCTATTCAGA-3'	60	196~247
SCA17	R：5'-GCCTGAGGTTCCCTGTGTT-3'
DRPLA	F：5'-CCACCACCTCCTCCCTATG-3'	60	209~257
DRPLA	R：5'-AGTGGGTGGGGAAATGCT-3'

引物名称	引物序列（5~3'）	退火温度（℃）	扩增片段长度（bp）
SCA1	F：5'-GGTCCTCCCAATACAGTGGA-3'	60	364~403
SCA1	R：5'-TTCTGCGGAGAACTGGAAAT-3'
SCA2	F：5'-CTCCGCCTCAGACTGTTTTG-3'	60	810~837
SCA2	R：5'-CTGACCATCGCCGCTACC-3'
SCA3	F：5'-TCTGTATCAGACTAACTGCTCTTGC-3'	60	369~438
SCA3	R：5'-GAGGGAATGAAGAATAATGTAAAGC-3'
SCA6	F：5'-TCCCGTGTCTCCTTTGATTT-3'	60	378~411
SCA6	R：5'-GACCCGCCTCTCCATCCT-3'
SCA7	F：5'-TAGGAGCGGAAAGAATGTCG-3'	57	280~310
SCA7	R：5'-CCCAGCATCACTTCAGGACT-3'
SCA8	F：5'-GCAGTATGAGGAAGTATGGAAAAA-3'	60	230~335
SCA8	R：5'-TTCTGACTCCCAGCTTCCAC-3'
SCA12	F：5'-CCACTGCAGCAAAGAGCA-3'	60	280~355
SCA12	R：5'-GGAATGAGGGTGCTGGTC-3'
SCA17	F：5'-GACC CCACAGCCTATTCAGA-3'	60	196~247
SCA17	R：5'-GCCTGAGGTTCCCTGTGTT-3'
DRPLA	F：5'-CCACCACCTCCTCCCTATG-3'	60	209~257
DRPLA	R：5'-AGTGGGTGGGGAAATGCT-3'

反应组分	体积（µL）	终浓度
pMD20-T载体	1.0	5 ng/µL
纯化DNA产物	1.0
2×连接缓冲液	5.0	1×
H2O	3.0

反应组分	体积（µL）	终浓度
pMD20-T载体	1.0	5 ng/µL
纯化DNA产物	1.0
2×连接缓冲液	5.0	1×
H2O	3.0

个体	重复序列	重复序列数
Ⅲ-1	Q3KQ23/Q3KQ71	27/75
Ⅲ-4	Q3KQ22/Q3KQ72	26/76
Ⅳ-2	Q3KQ10/Q3KQ74	14/78
Ⅳ-1	Q3KQ10/Q3KQ70	14/74
Ⅲ-5	Q3KQ10/Q3KQ10	14/14