儿童遗传性球形红细胞增多症临床表型及分子遗传学分析

Study of clinical characteristics and related gene mutations in children with hereditary spherocytosis

目的分析儿童遗传性球形红细胞增多症(HS)的临床表型及分子遗传学特点。方法收集2014年6月至2018年6月郑州大学附属儿童医院HS患儿的临床病例资料,进行回顾性分析。采集20例患儿及其父母外周血样本,应用二代测序技术进行靶向捕获或全外显子组测序,对可疑突变位点应用Sanger测序进行验证。结果共43例患儿纳入研究(男23例,女20例),中位发病年龄1岁11个月(1个月~10岁)。面色苍白(27/43例,62.79%)是主要的发病症状,典型临床表现为贫血(36/43例,83.72%)、黄疸(35/43例,81.40%)、脾大(33/43例,76.74%)、肝大(27/43例,62.79%)。外周血及骨髓血涂片球形红细胞比例≥0.1的患儿分别为23例(23/43例,53.49%)、17例(17/43例,39.53%),红细胞渗透脆性试验阳性20例(20/43例,46.51%)。29例患儿输血前后比较:输血后血红蛋白(Hb)[(88.69±11.22) g/L比(78.24±14.47) g/L]、平均红细胞体积(MCV)[(89.37±7.15) fL比(84.08±7.49) fL]、平均红细胞血红蛋白含量(MCH)[(29.12±2.70) pg比(27.36±1.95) pg]、平均血红蛋白浓度(MCHC)[(361.79±32.27) g/L比(356.31±31.43) g/L]均升高,总胆红素(TB)水平较输血前下降[(33.27±16.42) μmol/L比(41.58±15.40) μmol/L],差异均有统计学意义(t=-3.538、-5.187、-5.412、-7.404、2.527,均P<0.05)。7例患儿行脾切除术:术后Hb[(116.00±5.54) g/L比(75.71±9.96) g/L]、MCH[(29.87±1.62) pg比(24.61±1.65) pg]、MCHC[(391.14±12.99) g/L比(315.14±51.99) g/L]均升高,差异均有统计学意义(t=-9.234、-4.330、-4.031,均P<0.05)。手术组患儿Hb较输血组升高明显,治疗效果更显著(t=-9.247,P<0.05)。13例患儿在3种基因(ANK1、SPTB及SPTA1)检出13种致病变异(11种是未报道的新变异),8例为ANK1变异,4例为SPTB变异,1例为SPTA1变异。13例变异中,除1例遗传自母亲外,余12例为新生变异(de novo)。结论儿童HS多以贫血、面色苍白为首发症状,输血可暂时改善贫血、黄疸症状,脾切除是有效的根本治疗手段。13例患儿获得基因诊断,为下一胎遗传咨询提供依据;在ANK1、SPTB、SPTA1基因中共发现11种新变异,扩展了HS的基因突变谱。

Objective To analyze the clinical phenotypes and molecular genetic characteristics of hereditary spherocytosis(HS)in children.Methods The clinical data of children with HS in the Affiliated Children′s Hospital of Zhengzhou University from June 2014 to June 2018 were collected and analyzed retrospectively.The peripheral blood samples of 20 children and their parents were collected and targeted capture or whole exon group sequencing was performed using the second generation sequencing technique,and the suspicious mutation sites were verified by Sanger sequencing.Results A total of 43 pediatric patients(23 males,20 female)were included in this study.The median age of onset of 1 year old and 11 months(1 month to 10 years old).Pallor(27/43 cases,62.79%)was the main symptom.Typical clinical manifestations were anemia(36/43 cases,83.72%),jaundice(35/43 cases,81.40%),splenomegaly(33/43 cases,76.74%),and hepatomegaly(27/43 cases,62.79%).The proportion of spherical erythrocytes in peripheral blood of 23 cases(23/43 cases,53.49%)≥0.1,and the proportion of spherical erythrocytes in bone marrow smears of 17 cases(39.53%)was≥0.1,and 20 cases were positive in erythrocyte osmotic fragility test(20/43 cases,46.51%).Comparison of 29 children before and after blood transfusion:post-transfusion hemoglobin(Hb)[(88.69±11.22)g/L vs.(78.24±14.47)g/L],mean corpuscular volume(MCV)[(89.37±7.15)fL vs.(84.08±7.49)fL],mean corpuscular hemoglobin(MCH)[(29.12±2.70)pg vs.(27.36±1.95)pg],mean corpuscular hemoglobin corpuscular(MCHC)[(361.79±32.27)g/L vs.(356.31±31.43)g/L]were increased,and the total bilirubin(TB)level was lower after blood transfusion than before blood transfusion[(33.27±16.42)μmol/L vs.(41.58±15.40)μmol/L],the differences were statistically significant(t=-3.538,-5.187,-5.412,-7.404,2.527,all P<0.05).Seven children underwent splenectomy:postoperative Hb[(116.00±5.54)g/L vs.(75.71±9.96)g/L],MCH[(29.87±1.62)pg vs.(24.61±1.65)pg],MCHC[(391.14±12.99)g/L vs.(315.14±51.99)g/L]all increased,the difference was statistically significant(t=-9.234,-4.330,-4.031,all P<0.05).The Hb in the operation group was significantly higher than that in the blood transfusion group,including the treatment effect of operation was more significant(t=-9.247,P<0.05).Thirteen pathogenic mutations(11 new mutations not reported)were detected in 3 genes(ANK1,SPTB and SPTA1)in 13 children,8 of who were mutations in ANK1,4 cases of which were mutations in SPTB and 1 case of which was mutation in SPTA1.Among the 13 mutations,12 cases were de novo except one inherited from the mother.Conclusions Anemia and pallor are the initial symptoms of HS in children.Blood transfusion can temporarily improve the symptoms of anemia and jaundice.Splenectomy is an effective and fundamental treatment.The gene diagnosis of 13 pediatric patients got gene diagnosis,which provided basis for genetic consultation of the next fetus.Eleven new mutations were found in ANK1,SPTB,SPTA1 genes,which exten-ded the gene mutation spectrum of HS.