摘要：

目的 探讨基于毛细管电泳的片段分析和克隆测序在脊髓小脑共济失调（SCA）动态突变检测中的准确性和稳定性。方法 采用基于毛细管电泳的片段分析和克隆测序对14 例脊髓小脑共济失调患者（包括3 例SCA2 型、2 例SCA7 型、7 例SCA8 型和2 例SCA17 型）致病基因三核苷酸重复序列进行检测。结果 3 例SCA2 基因样本基于毛细管电泳的片段分析显示，扩展片段胞嘧啶-腺嘌呤-鸟嘌呤（CAG）重复序列分别为31、30 和32 次，每例样本取3 个菌落进行克隆测序，CAG 重复序列分别为37/40/40、37/38/39 和38/39/40 次；2 例SCA7 基因样本基于毛细管电泳的片段分析显示，扩展片段CAG 重复序列分别为57 和34 次，1 例取3 个菌落进行克隆测序，CAG 重复序列为69、74 和75 次，1 例为45 次；7 例SCA8 基因样本基于毛细管电泳的片段分析显示，扩展片段胞嘧啶-胸腺嘧啶-腺嘌呤（CTA）/胞嘧啶-胸腺嘧啶-鸟嘌呤（CTG）重复序列分别为99、111、104、92、89、104 和75 次，克隆测序分别为97、116、104、90、90、102 和76 次；2 例SCA17 基因样本基于毛细管电泳的片段分析显示，短片段/扩展片段CAG 重复序列为37/50 和36/45 次，扩展片段分别取3 和2 个菌落进行克隆测序，CAG 重复序列为51/50/52 和45/44 次。结论 基于毛细管电泳的片段分析在判读重复序列时存在一定偏倚，但可以预估，可重复性佳，不影响基因检测结果的判定；而克隆测序具有明显不稳定性，尤其是SCA2 和SCA7 基因，可能与其重复序列组成较为单纯有关。克隆测序不适宜作为检测基因动态突变的方法，更不适宜作为判定动态突变序列组成的标准。

关键词: 脊髓小脑共济失调, 重复序列, 核酸, 核苷酸类, 基因, 突变, 电泳, 琼脂凝胶, 序列分析

Abstract:

Objective  To estimate the accuracy and stability of capillary electrophoresis fragment analysis and clone sequencing in detecting dynamic mutations of spinocerebellar ataxia (SCA).  Methods  Capillary electrophoresis fragment analysis and clone sequencing were used in detecting trinucleotide repeated sequence of 14 SCA patients (3 cases of SCA2, 2 cases of SCA7, 7 cases of SCA8 and 2 cases of SCA17).  Results  Capillary electrophoresis fragment analysis of 3 SCA2 cases showed the expanded cytosine-adenine-guanine (CAG) repeats were 31, 30 and 32, and the copy numbers of 3 clone sequencing for 3 colonies in each case were 37/40/40, 37/38/39 and 38/39/40 respectively. Capillary electrophoresis fragment analysis of 2 SCA7 cases showed the expanded CAG repeats were 57 and 34, and the copy numbers of repeats were 69, 74, 75 in 3 colonies of one case, and was 45 in the other case. For the 7 SCA8 cases with the expanded cytosine-thymine-adenine (CTA)/cytosine-thymine-guanine (CTG) repeats of 99, 111, 104, 92, 89, 104 and 75, the results of clone sequencing were 97, 116, 104, 90, 90, 102 and 76 respectively. For 2 SCA17 cases with the short/expanded CAG repeats of 37/50 and 36/45, the results of clone sequencing were 51/50/52 and 45/44 for 3 and 2 colonies.  Conclusions  Although the higher mobility of polymerase chain reaction (PCR) products containing dynamic mutation in the capillary electrophoresis fragment analysis might cause the deviation for analysis of copy numbers, the deviation was predictable and the results were repeatable. The clone sequencing results showed obvious instability, especially for SCA2 and SCA7 genes, which might owing to their simple CAG repeats. Consequently, clone sequencing is not suited for detection of dynamic mutation, not to mention the quantitative criteria of dynamic mutation sequencing.

Key words: Spinocerebellar ataxia, Repetitive sequences, nucleic acid, Nucleotides, Genes, Mutation, Electrophoresis, agar gel, Sequence analysis