摘要目的 研究糖原累积病Ⅲ型(glycogen storage diseases type Ⅲ,GSD Ⅲ)患者糖原脱支酶AGL基因突变情况.方法 采用PeR、DNA直接测序法,对临床表现为肝大、低血糖、生长落后、运动耐力下降的10例GSDⅢ患者的糖原脱支酶基因进行突变检测.检出突变的片段再经DNA双向测序证实.采用限制性内切酶片段长度多态性和荧光PCR结合基因扫描方法证实5个新突变.结果 10例患者中共检出13种突变类型,包括4种剪切突变:IVS6+1G>A、IVS6-1G>A、IVS14+1G>T、IVS26-2A>C;5种无义突变:R469X、R864X、S929X、R977X、Y1428X;3种缺失突变:c.408-411delTTTG、c.2717-2721delAGATC、c.2823delT;1种插入突变:c.4234insT.除IVS14+1G>T、R864X和R977X,其余10种均未见报道过.限制性内切酶片段长度多态性分析证实了3个新剪切突变,荧光PCR结合基因扫描方法证实2个缺失突变的缺失碱基数.2例患者仪检出1个致病突变,其余患者均明确有2个致病突变,突变检出率为90%.IVS14+1G>T突变频率最高,占25%.结论 10例GSDⅢ型患者的糖原脱支酶基因突变呈高度异质性,发现13种突变,其中10种是新突变,IVS14+1G>T是较常见的突变类型.

abstractsObjective Glycogen debranching enzyme (AGL) plays an important role in complete degradation of the glycogen, and has two independent catalytic activities, i.e., those of α-1, 4-glucantransferase (EC 2.4.1.25) and amylo-1,6-glucosidase (EC 3.2.1.33). A deficiency in activities of AGL causes excessive accumulation of glycogen with short branched outer chains and results in glycogen storage disease type Ⅲ (GSD Ⅲ ; MIM #232 400), an autosomal recessive inborn disorder of glycogen metabolism. The present study aimed to investigate the mutation of AGL in 10 Chinese patients with GSD Ⅲ. Method Clinical and laboratory data of 10 patients with typical clinical manifestations of GSD Ⅲ suggesting hypoglycemia, hyperlipoidemia, increased creatine-phosphokinase and its isozyme were collected. The ceding regions and their flanking introns of AGL gene of the 10 patients were amplified by PCR and analyzed by direct DNA sequencing. All the mutated alleles were confirmed by bidirectional DNA sequencing. The 3 novel splicing mutations were analyzed by restriction fragment length polymorphism (RFLP) in 50 healthy children (control). The 2 small deletions (c. 408-411delTlTG, c. 2717-2721delAGATC) were analyzed by fluorescent polymerase chain reaction and gene scan analysis to confirm the number of deleted bases. Result Thirteen different mutations were identified, including 4 splicing mutations (IVS6+IG>A, IVS6-1G>A, IVS14+1G>T, IVS26-2A>C), 5 nonsense mutations (R469X, R864X, S929X, R977X, Y1428X), 3 small deletions (c. 408-411delTITG, c. 2717-2721delAGATC, c. 2823delT) and 1 insert mutation (c. 4234insT). Except for IVS14 + 1G > T, R864X, and R977X, the other 10 mutations are novel; 18 mutated alleles were identified in the 20 alleles (90%). IVS14 +IG >T was the most frequently seen mutation, accounting for 5 of 20(25%) alleles examined. None of homozygote and heterozygote of the 3 novel splicing mutations was found in the 50 healthy controls by RFLP analysis. With the fluorescent polymerase chain reaction and gene scan analysis, c. 408-411delTITG mutation and c. 2717-2721delAGATC mutation were confirmed to have 4 and 5 bases deletion respectively. Conclusion Thirteen mutations were identified in the 10 cases with GSD Ⅲ, with 10 novel mutations. IVS14 + 1G>T was a relatively common mutation. This study revealed the heterozygosity of AGL gene in Chinese patients with GSD Ⅲ.