PDF(Pubmed)
Abstract：
BACKGROUND: Spinal muscular atrophy (SMA) is a rare autosomal recessive hereditary neuromuscular disease caused by survival motor neuron 1 (SMN1) gene deletion or mutation. Homozygous deletions of exon 7 in SMN1 result in 95% of SMA cases, while the remaining 5% are caused by other pathogenic variants of SMN1.
METHODS: We analyzed two SMA-suspected cases that were collected, with no SMN1 gene deletion and point mutation in whole-exome sequencing. Exon 1 deletion of the SMN gene was detected using Multiplex ligation-dependent probe amplification (MLPA) P021. We used long-range polymerase chain reaction (PCR) to isolate the SMN1 template, optimized-MLPA P021 for copy number variation (CNV) analysis within SMN1 only, and validated the findings via third-generation sequencing.
RESULTS: Two unrelated families shared a genotype with one copy of exon 7 and a novel variant, g.70919941_70927324del, in isolated exon 1 of the SMN1 gene. Case F1-II.1 demonstrated no exon 1 but retained other exons, whereas F2-II.1 had an exon 1 deletion in a single SMN1 gene. The read coverage in the third-generation sequencing results of both F1-II.1 and F2-II.1 revealed a deletion of approximately 7.3 kb in the 5\' region of SMN1. The first nucleotide in the sequence data aligned to the 7385 bp of NG_008691.1.
CONCLUSIONS: Remarkably, two proband families demonstrated identical SMN1 exon 1 breakpoint sites, hinting at a potential novel mutation hotspot in Chinese SMA, expanding the variation spectrum of the SMN1 gene and corroborating the specificity of isolated exon 1 deletion in SMA pathogenesis. The optimized-MLPA P021 determined a novel variant (g.70919941_70927324del) in isolated exon 1 of the SMN1 gene based on long-range PCR, enabling efficient and affordable detection of SMN gene variations in patients with SMA, providing new insight into SMA diagnosis to SMN1 deficiency and an optimized workflow for single exon CNV testing of the SMN gene.
METHODS: We analyzed two SMA-suspected cases that were collected, with no SMN1 gene deletion and point mutation in whole-exome sequencing. Exon 1 deletion of the SMN gene was detected using Multiplex ligation-dependent probe amplification (MLPA) P021. We used long-range polymerase chain reaction (PCR) to isolate the SMN1 template, optimized-MLPA P021 for copy number variation (CNV) analysis within SMN1 only, and validated the findings via third-generation sequencing.
RESULTS: Two unrelated families shared a genotype with one copy of exon 7 and a novel variant, g.70919941_70927324del, in isolated exon 1 of the SMN1 gene. Case F1-II.1 demonstrated no exon 1 but retained other exons, whereas F2-II.1 had an exon 1 deletion in a single SMN1 gene. The read coverage in the third-generation sequencing results of both F1-II.1 and F2-II.1 revealed a deletion of approximately 7.3 kb in the 5\' region of SMN1. The first nucleotide in the sequence data aligned to the 7385 bp of NG_008691.1.
CONCLUSIONS: Remarkably, two proband families demonstrated identical SMN1 exon 1 breakpoint sites, hinting at a potential novel mutation hotspot in Chinese SMA, expanding the variation spectrum of the SMN1 gene and corroborating the specificity of isolated exon 1 deletion in SMA pathogenesis. The optimized-MLPA P021 determined a novel variant (g.70919941_70927324del) in isolated exon 1 of the SMN1 gene based on long-range PCR, enabling efficient and affordable detection of SMN gene variations in patients with SMA, providing new insight into SMA diagnosis to SMN1 deficiency and an optimized workflow for single exon CNV testing of the SMN gene.
摘要：
背景：脊髓性肌萎缩症（SMA）是一种罕见的常染色体隐性遗传性神经肌肉疾病，由运动神经元存活1（SMN1）基因缺失或突变引起。SMN1中外显子7的纯合缺失导致95%的SMA病例，而其余5%是由SMN1的其他致病变种引起的。
方法：我们分析了收集的两个SMA疑似病例，全外显子组测序中无SMN1基因缺失和点突变。使用多重连接依赖性探针扩增(MLPA)P021检测SMN基因的外显子1缺失。我们使用远程聚合酶链反应（PCR）来分离SMN1模板，优化的MLPAP021，仅用于SMN1内的拷贝数变异（CNV）分析，并通过第三代测序验证了研究结果。
结果：两个不相关的家族共有一个基因型，一个外显子7拷贝和一个新的变异体，g.70919941_70927324del,在SMN1基因的分离外显子1中。病例F1-II.1显示没有外显子1，但保留了其他外显子，而F2-II.1在单个SMN1基因中有外显子1缺失。F1-II.1和F2-II.1的第三代测序结果中的读段覆盖显示SMN1的5'区缺失约7.3kb。序列数据中的第一个核苷酸与NG_008691.1的7385bp比对。
结论：值得注意的是，两个先证者家族显示相同的SMN1外显子1断点位点，暗示中国SMA潜在的新突变热点，扩大SMN1基因的变异谱，证实分离的外显子1缺失在SMA发病机制中的特异性。优化的MLPAP021基于远程PCR在SMN1基因的分离外显子1中确定了一个新的变体（g.70919941_70927324del），能够有效和负担得起的检测SMA患者的SMN基因变异，为SMN1缺陷的SMA诊断提供了新的见解，并为SMN基因的单外显子CNV测试提供了优化的工作流程。
方法：我们分析了收集的两个SMA疑似病例，全外显子组测序中无SMN1基因缺失和点突变。使用多重连接依赖性探针扩增(MLPA)P021检测SMN基因的外显子1缺失。我们使用远程聚合酶链反应（PCR）来分离SMN1模板，优化的MLPAP021，仅用于SMN1内的拷贝数变异（CNV）分析，并通过第三代测序验证了研究结果。
结果：两个不相关的家族共有一个基因型，一个外显子7拷贝和一个新的变异体，g.70919941_70927324del,在SMN1基因的分离外显子1中。病例F1-II.1显示没有外显子1，但保留了其他外显子，而F2-II.1在单个SMN1基因中有外显子1缺失。F1-II.1和F2-II.1的第三代测序结果中的读段覆盖显示SMN1的5'区缺失约7.3kb。序列数据中的第一个核苷酸与NG_008691.1的7385bp比对。
结论：值得注意的是，两个先证者家族显示相同的SMN1外显子1断点位点，暗示中国SMA潜在的新突变热点，扩大SMN1基因的变异谱，证实分离的外显子1缺失在SMA发病机制中的特异性。优化的MLPAP021基于远程PCR在SMN1基因的分离外显子1中确定了一个新的变体（g.70919941_70927324del），能够有效和负担得起的检测SMA患者的SMN基因变异，为SMN1缺陷的SMA诊断提供了新的见解，并为SMN基因的单外显子CNV测试提供了优化的工作流程。
