晚发型糖原贮积症Ⅱ型患儿1例的溶酶体酶活性分析及遗传学分析

Analysis of lysosomal enzyme activity and genetic variants in a child with late-onset Pompe disease

目的探讨1例晚发型糖原贮积症Ⅱ型(LOPD)型患儿的临床特征、GAA基因变异特点及溶酶体酶α-葡萄糖苷酶(GAA)活性改变情况。方法收集2020年8月在四川大学华西第二医院临床确诊的1例GSDⅡ患儿的病历资料,采集患儿及其父母的外周血样,分别用于外周血白细胞、淋巴细胞分离及基因组DNA提取,检测2种细胞中加入GAA同工酶抑制剂阿卡波糖和不加抑制剂时溶酶体酶GAA的活性。同时收集20例健康受试者的外周血样,混合后分别分离白细胞与淋巴细胞,与患儿家系同批次进行GAA活性测定,作为正常参考值。基因组DNA用于神经肌肉病基因panel检测,并对候选变异进行Sanger测序验证和保守性分析。结果患儿为9岁女性,2岁11个月时发现语言运动发育落后,就诊时走路不稳、上台阶困难及明显脊柱侧弯,患儿血清肌酸激酶显著升高、肌电图异常,心脏彩色多普勒超声未见异常。基因检测发现其携带GAA基因c.1996dupG(p.A666Gfs*71)和c.701C>T(p.T234M)复合杂合变异。根据美国医学遗传学与基因组学学会相关指南,分别评级为致病变异(PVS1+PM2_Supporting+PM3)和可能致病变异(PM1+PM2_Supporting+PM3+PM5+PP3)。患儿、患儿父亲、母亲白细胞中GAA活性在不加抑制剂时分别为同批次正常值的76.1%、91.3%、95.6%,加入抑制剂时分别为同批次正常值的70.8%、112.9%、128.2%,白细胞中GAA活性在加入抑制剂后降低约6~9倍;患儿、患儿父亲、母亲淋巴细胞中GAA活性在不加抑制剂时分别为同批次正常值的68.3%、59.0%、59.5%,在加抑制剂时分别为同批次正常值的41.0%、89.5%、57.7%,淋巴细胞中GAA活性在加入抑制剂后降低约2~5倍。结论患儿被诊断为LOPD,其GAA基因的c.1996dupG和c.701C>T复合杂合变异可能是其遗传学病因。LOPD患儿GAA残余酶活性范围较宽,酶活性改变不典型,在诊断时不能仅依据酶活性检测,而需要结合临床表现、基因检测和酶活性检测进行综合诊断。

Objective To explore the clinical features,lysosomal enzymatic[acidα-glucosidase(GAA)]activities and genetic variants in a child with late-onset Pompe disease(LOPD).Methods Clinical data of a child who had presented at the Genetic Counseling Clinic of West China Second University Hospital in August 2020 was retrospectively analyzed.Blood samples were collected from the patient and her parents for the isolation of leukocytes and lymphocytes as well as DNA extraction.The activity of lysosomal enzyme GAA in leukocytes and lymphocytes was analyzed with or without addition of inhibitor of GAA isozyme.Potential variants in genes associated with neuromuscular disorders were analyzed,in addition with conservation of the variant sites and protein structure.The remaining samples from 20 individuals undergoing peripheral blood lymphocyte chromosomal karyotyping were mixed and used as the normal reference for the enzymatic activities.Results The child,a 9-year-old female,had featured delayed language and motor development from 2 years and 11 months.Physical examination revealed unstable walking,difficulty in going upstairs and obvious scoliosis.Her serum creatine kinase was significantly increased,along with abnormal electromyography,whilst no abnormality was found by cardiac ultrasound.Genetic testing revealed that she has harbored compound heterozygous variants of the GAA gene,namely c.1996dupG(p.A666Gfs*71)(maternal)and c.701C>T(p.T234M)(paternal).Based on the guidelines from the American College of Medical Genetics and Genomics,the c.1996dupG(p.A666Gfs*71)was rated as pathogenic(PVS1+PM2_Supporting+PM3),whilst the c.701C>T(p.T234M)was rated as likely pathogenic(PM1+PM2_Supporting+PM3+PM5+PP3).The GAA in the leukocytes from the patient,her father and mother were respectively 76.1%,91.3%and 95.6%of the normal value without the inhibitor,and 70.8%,112.9%and 128.2%of the normal value with the inhibitor,whilst the activity of GAA in their leukocytes had decreased by 6~9 times after adding the inhibitor.GAA in lymphocytes of the patient,her father and mother were 68.3%,59.0%and 59.5%of the normal value without the inhibitor,and 41.0%,89.5%and 57.7%of the normal value with the inhibitor,the activity of GAA in lymphocytes has decreased by 2~5 times after adding the inhibitor.Conclusion The child was diagnosed with LOPD due to the c.1996dupG and c.701C>T compound heterozygous variants of the GAA gene.The residual activity of GAA among LOPD patients can range widely and the changes may be atypical.The diagnosis of LOPD should not be based solely on the results of enzymatic activity but combined clinical manifestation,genetic testing and measurement of enzymatic activity.