Zhang L P, Wang Y F
Department of Neurology, Heping Hospital Affiliated to Changzhi Medical College, Changzhi 046000,China.
Zhonghua Nei Ke Za Zhi. 2022 Dec 1;61(12):1343-1350. doi: 10.3760/cma.j.cn112138-20220912-00675.
To clarify the pathogenicity and further explore the association between genotype and clinical phenotype of this variant, analyzing a novel variation of SPAST gene in hereditary spastic paraplegia (HSP) family from Changzhi city, Shanxi Province. A family with HSP was tracked and collected in Neurology Department of Heping Hospital Affiliated to Changzhi Medical College in October 2019. Peripheral venous blood of 2 ml was extracted from the proband and 8 other members of the family, genomic DNA was extracted from the blood samples, and the genes of spastic paraplegia were screened by next-generation sequencing (NGS). HGMD, 1000G, OMIM databases and PolyPhen2, SIFT and other software were used for bioinformatics analysis of suspected mutations. Multiplex ligation-dependent probe amplification (MLPA) was used to further screen for total deletions/duplications in patients who remained negative after targeting NGS, and Sanger sequencing was performed to verify the suspected pathogenic mutation sites in the family to determine co-isolation of the mutation sites in the family members. Finally, it is necessary to refer to the latest version of The American College of Medical Genetics and Genomics (ACMG) sequence variation interpretation guidelines to interpret the mutation sites to determine pathogenicity. The HSP family consist 47 members of 4 generations and 10 patients, with onset ages ranging from 2 to 44 years. The proband's daughter only showed positive bilateral Babbitt signs on physical examination, and the rest of the patients showed spasticity and weakness of lower limbs with varying severity on this basis. Preliminary screening by next-generation sequencing technology showed that the proband had frame-shift variation of SPAST gene c.1057_1058insCC (p.Leu354HisfsTer11) and missense variation of DCTN1 gene c.2213A>G (p.Gln738Arg). Then, Sanger sequencing was used for in-family verification, which showed SPAST gene c.1057_1058insCC (p.Leu354HisfsTer11) was detected in the affected members include father, brother, son and daughter, and not detected in the unaffected normal members, the proband's wife, mother, sister and sister-in-law. However, the unaffected of mother detected missense variation of DCTN1 gene c.2213A>G (p.Gln738Arg), while the remaining members did not detect this variation. The results of MLPA showed that no large fragment variation was found. The genetic pattern of the HSP family was autosomal dominant, and the clinical characteristics were consistent with hereditary spastic paraplegia type 4 (SPG4). Co-segregation of SPAST gene c.1057_1058insCC (p.Leu354HisfsTer11) was found in the HSP family and was the pathogenicity cause of this SPG4 family, and it was a newly discovered mutation locus.
为阐明该变异的致病性并进一步探索其基因型与临床表型之间的关联，对山西省长治市一个遗传性痉挛性截瘫（HSP）家系中的SPAST基因新变异进行分析。2019年10月，长治医学院附属和平医院神经内科对一个HSP家系进行追踪并采集样本。从先证者及该家系的其他8名成员中抽取2ml外周静脉血，从血样中提取基因组DNA，采用二代测序（NGS）技术对痉挛性截瘫相关基因进行筛查。利用人类基因突变数据库（HGMD）、千人基因组计划（1000G）、在线孟德尔人类遗传数据库（OMIM）以及PolyPhen2、SIFT等软件对疑似突变进行生物信息学分析。对NGS靶向检测后仍为阴性的患者，采用多重连接依赖探针扩增技术（MLPA）进一步筛查大片段缺失/重复，并通过桑格测序验证家系中疑似致病突变位点，以确定家系成员中突变位点的共分离情况。最后，参照美国医学遗传学与基因组学学会（ACMG）序列变异解读最新指南对突变位点进行解读，以确定其致病性。该HSP家系共4代47人，其中患者10人，发病年龄为2至44岁。先证者女儿体格检查仅双侧巴宾斯基征阳性，其余患者在此基础上均有不同程度的下肢痉挛和无力。二代测序技术初步筛查显示，先证者存在SPAST基因c.1057_1058insCC（p.Leu354HisfsTer11）移码变异以及DCTN1基因c.2213A>G（p.Gln738Arg）错义变异。随后，采用桑格测序进行家系验证，结果显示，在患病成员（父亲、哥哥、儿子和女儿）中检测到SPAST基因c.1057_1058insCC（p.Leu354HisfsTer11），在先证者妻子、母亲、妹妹和嫂子等未患病的正常成员中未检测到。然而，未患病的母亲检测到DCTN1基因c.2213A>G（p.Gln738Arg）错义变异，其余成员未检测到该变异。MLPA结果显示未发现大片段变异。该HSP家系遗传模式为常染色体显性遗传，临床特征符合遗传性痉挛性截瘫4型（SPG4）。在该HSP家系中发现SPAST基因c.1057_1058insCC（p.Leu354HisfsTer11）共分离，是该SPG4家系的致病原因，且为新发现的突变位点。
