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目的对498例地中海贫血产前基因诊断结果和方法进行回顾性分析。方法对498例地贫产前诊断样本,包括羊水样本445例、绒毛样本50例和脐血样本3例,提取基因组DNA。缺失型α地贫的产前诊断采用跨越断裂点PCR(Gap polymerase chain reaction,Gap-PCR)技术和多重连接依赖式探针扩增(multiplex ligation-dependent probe amplificatio,MLPA)技术同时进行检测;非缺失型α地贫的产前诊断采用反向斑点杂交(reverse dot blot,RDB)技术进行检测。β地贫的产前诊断采用RDB技术和多色探针熔解曲线技术(multicolor melting curve analysis,MMCA)同时进行检测。对RDB方法检测不出的IVS-Ⅰ-1(G→T)纯合突变,改用DNA测序和MMCA的方法同时检测。对少见的缺失型β地贫采用MLPA方法进行检测。婴儿出生后半年电话随访婴儿表型。结果 498例产前诊断样本孕妇来自包括河北在内的17个省市,其中中重型α地贫高风险胎儿样本295例,中重型β地贫高风险胎儿样本134例,其他情况69例。α地贫基因检测共446例,其中产前诊断严重类型地贫87例(19.51%),包括巴氏水肿胎66例(14.80%)和Hb H病21例(4.71%),轻型186例(41.7%),正常胎儿173例(38.79%);β地贫基因检测共389例,其中严重类型地贫31例(7.97%),轻型98例(25.19%),正常胎儿260例(66.84%)。经DNA测序和MMCA检测到1例胎儿为IVS-Ⅰ-1(G→T)纯合突变。MLPA检测到1例胎儿父亲为β地贫东南亚型缺失(HPFH of SEA type),胎儿未检测到此缺失。1例样本结果为轻型α地贫同时合并18三体。87例严重类型α地贫胎儿、31例严重类型β地贫胎儿和1例轻型α地贫伴18三体胎儿均在围产期前后接受了终止妊娠的处理。结论地贫的人群分布已逐渐从南方地区向北方地区扩展。对中重型地贫高风险孕妇进行产前基因诊断,检出严重类型地贫胎儿,是降低高危地域严重类型地贫患儿出生的有效手段。对于RDB方法检测不出的纯合突变类型,可改用DNA测序和MMCA的方法进行检测;同时,对于β地贫的少见缺失类型应进行进一步检测,防止漏诊。另外,在进行地贫基因产前诊断的同时,要注意排除胎儿是否患有染色体病。
Objective To retrospectively analyze the diagnostic results and methods of 498 cases of thalassemia prenatal gene. Methods 498 cases of prenatal diagnosis of thalassemia were collected, including 445 cases of amniotic fluid samples, 50 cases of villus samples and 3 cases of umbilical cord blood samples. Genomic DNA was extracted. Prenatal diagnosis of deletional alpha thalassemia was performed simultaneously by Gap-PCR and multiplex ligation-dependent probe amplifica- tion (MLPA) techniques; Prenatal diagnosis of deletional alpha thalassemia was performed by reverse dot blot (RDB) technique. Prenatal diagnosis of beta thalassemia was performed simultaneously using RDB technique and multicolor melting curve analysis (MMCA). The IVS-I-1 (G → T) homozygous mutation that could not be detected by RDB method was detected simultaneously by DNA sequencing and MMCA. The MLPA method was used to detect the rare deletion type β thalassemia. Six months after birth, the baby was followed up by telephone for the baby’s phenotype. Results A total of 498 prenatal diagnostic samples from 17 provinces and cities including Hebei were included. Among them, 295 were high risk fetuses of medium and heavy α-thalassemia, 134 were high risk β-thalassemia fetuses and 69 were other cases. There were 446 cases of alpha thalassemia gene, including 87 (19.51%) cases of prenatal diagnosis of thalassemia major, including 66 cases of Pap smear (14.80%) and 21 cases (4.71%) of Hb disease, 186 cases of mild 41.7%), and 173 normal fetuses (38.79%). A total of 389 cases of β thalassemia gene were detected, of which 31 cases were severe thalassemia (7.97%), 98 (25.19%) were mild cases and 260 (66.84% . A fetus was homozygous for IVS-I-1 (G → T) detected by DNA sequencing and MMCA. One MLF paired MLPA detected a loss of beta-thalassemia subtype (HPFH of SEA type), which was undetectable in the fetus. One case of the sample was light α-thalassemia combined with trisomy 18. 87 cases of severe type α thalassemia, 31 cases of severe type β-thalassemia and 1 case of light α-thalassemia with trisomy 18 were given termination of pregnancy before and after perinatal period. Conclusion The distribution of thalassemia population has gradually expanded from the southern region to the northern region. Prenatal genetic diagnosis of high-risk women with mid-heavy type of thalassemia major and detection of severe type of thalamic fetus is an effective means of reducing the risk of birth in children with thalassemia major in high-risk areas. For homozygous mutation types that can not be detected by RDB method, DNA sequencing and MMCA can be used instead. For the rare type of β-thalassemia, further detection should be performed to prevent misdiagnosis. In addition, in the prenatal diagnosis of thalassemia gene, pay attention to rule out whether the fetus has a chromosome disease.