Pompe disease is a lysosomal storage disorder caused by the deficiency of acid alpha-glucosidase (EC. 3.2.1.20) due to mutations in human GAA gene. The objective of the present study was to examine clinical and molecular characteristics of infantile-onset Pompe disease (IOPD) in Thailand. Twelve patients with infantile-onset Pompe disease (IOPD) including 10 Thai and two other Asian ethnicities were enrolled. To examine the molecular characteristics of Pompe patients, GAA gene was analyzed by PCR amplification and direct Sanger-sequencing of 20 exons coding region. The novel mutations were transiently transfected in COS-7 cells for functional verification. The severity of the mutation was rated by study of the GAA enzyme activity detected in transfected cells and culture media, as well as the quantity and quality of the proper sized GAA protein demonstrated by western blot analysis. The GAA three dimensional structures were visualized by PyMol software tool. All patients had hypertrophic cardiomyopathy, generalized muscle weakness, and undetectable or < 1% of GAA normal activity. Three patients received enzyme replacement therapy with variable outcome depending on the age of the start of enzyme replacement therapy (ERT). Seventeen pathogenic mutations including four novel variants: c.876C > G (p.Tyr292X), c.1226insG (p.Asp409GlyfsX95), c.1538G > A (p.Asp513Gly), c.1895 T > G (p.Leu632Arg), and a previously reported rare allele of unknown significance: c.781G > A (p.Ala261Thr) were identified. The rating system ranked p.Tyr292X, p. Asp513Gly and p. Leu632Arg as class “B” and p. Ala261Thr as class “D” or “E”. These novel mutations were located in the N-terminal beta-sheet domain and the catalytic domain. The present study provides useful information on the mutations of GAA gene in the underrepresented population of Asia which are more diverse than previously described and showing the hotspots in exons 14 and 5, accounting for 62% of mutant alleles. Almost all mutations identified are in class A/B. These data can benefit rapid molecular diagnosis of IOPD and severity rating of the mutations can serve as a partial substitute for cross reactive immunological material (CRIM) study.

中文翻译：

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泰国婴儿发作性庞贝病的临床病程，突变及其功能特征

庞贝病是一种溶酶体贮积病，由人GAA基因突变引起的酸性α-葡萄糖苷酶缺乏症（EC.3.2.1.20）引起。本研究的目的是检查泰国婴儿型庞贝病（IOPD）的临床和分子特征。招募了12名婴儿期庞贝病（IOPD）患者，其中包括10个泰国人和另外两个亚洲人。为了检查庞贝患者的分子特征，通过PCR扩增和20个外显子编码区的直接Sanger测序分析了GAA基因。在COS-7细胞中瞬时转染了新的突变，以进行功能验证。通过研究在转染的细胞和培养基中检测到的GAA酶活性来评估突变的严重程度，以及通过蛋白质印迹分析证明合适大小的GAA蛋白的数量和质量。GAA三维结构通过PyMol软件工具可视化。所有患者均患有肥厚型心肌病，全身性肌无力，且GAA正常活动未检出或<1％。3名患者接受酶替代疗法的结果不同，具体取决于酶替代疗法（ERT）开始的年龄。十七种致病突变，包括四个新变体：c.876C> G（p.Tyr292X），c.1226insG（p.Asp409GlyfsX95），c.1538G> A（p.Asp513Gly），c.1895 T> G（p.Leu632Arg） ，以及先前报道的罕见等位基因，其重要性不明：鉴定出c.781G> A（p.Ala261Thr）。评级系统对p.Tyr292X，p。进行了排名。Asp513Gly和p。Leu632Arg归类为“ B”类和p。Ala261Thr为“ D”或“ E”类。这些新颖的突变位于N端β-sheet域和催化域。本研究提供了有关GAA基因突变的有用信息，其在代表性不足的亚洲人群中比以前描述的更加多样化，并显示了外显子14和5的热点，占突变等位基因的62％。几乎所有鉴定出的突变都在A / B类中。这些数据可以使IOPD的快速分子诊断受益，并且突变的严重性等级可以部分替代交叉反应免疫材料（CRIM）研究。本研究提供了有关GAA基因突变的有用信息，其在代表性不足的亚洲人群中比以前描述的更加多样化，并显示了外显子14和5的热点，占突变等位基因的62％。几乎所有鉴定出的突变都在A / B类中。这些数据可以使IOPD的快速分子诊断受益，并且突变的严重性等级可以部分替代交叉反应免疫材料（CRIM）研究。本研究提供了有关GAA基因突变的有用信息，该突变在亚洲代表性不足的人群中比以前描述的更加多样化，并显示了外显子14和5的热点，占突变等位基因的62％。几乎所有鉴定出的突变都在A / B类中。这些数据可以使IOPD的快速分子诊断受益，并且突变的严重性等级可以部分替代交叉反应免疫材料（CRIM）研究。