12例X连锁慢性肉芽肿病的基因诊断

Genetic diagnosis for twelve cases of X-linked chronic granulomatous disease

目的:通过分析12例X连锁慢性肉芽肿病(X-CGD)的CYBB基因变异,探讨基因诊断在确诊该病中的价值。方法:根据患儿临床特征及四唑氮蓝试验(NBT)初步筛查结果:,运用流式细胞术中性粒细胞呼吸爆发试验,检测11例X-CGD患者及家系成员的中性粒细胞氧化功能,采用PCR及RT-PCR直接测序法,分析12例患儿及其家系外周血细胞的CYBB基因,并为1例CYBB突变基因携带者的胎儿分析羊水细胞的CYBB基因。结果:患儿均有反复感染史,NBT显著降低(0～6%),11例患儿中性粒细胞氧化指数(NOI)为1～2,明显低于正常人(>100),初步诊断为X-CGD;12例患儿中,11例存在CYBB基因突变:缺失突变4例,拼接错误突变4例,无义突变2例及错义突变1例。其中7种突变(1341delT,1182delC,1091delG,IVS10-2A>C,IVS10+2dupT,497-498ins115bp,591T>C)为新型突变。除了例3患儿母亲不是携带者,例9患儿母亲是否为携带者尚不明确,其余9个患儿母亲均为携带者,例6母亲的胎儿正常。结论:CYBB基因分析有助于X-CGD患儿的进一步明确诊断,在检测携带者及产前诊断中起着重要作用,并为干细胞移植治疗患者提供依据。

To explore the value of genetic diagnosis for X-linked chronic granulomatous disease （X-CGD）, we analyzed the variation of CYBB gene in twelve cases of X-CGD. Clinical features and laboratory data of patients with X-CGD showed that all of patients suffered from recurrent infections. Nitroblue tetrazolium （NBT） test and flow cytometry-DHR assay were used for evaluating neutrophil function. The NBT results before and after PMA and LPS stimulation were markedly lower （0-6% ）; the neutrophil oxidative index in 11 X-CGD patients tested by Flow cytometry-DHR assay was 1-2, significantly lower than healthy controls （〉100）, diagnosed as XCGD. By direct PCR or RT-PCR sequencing, the CYBB gene from the patients and their family members were analyzed. The Genomic DNA from fetus amniotic fluid cells, whose mother was a carrier of mutant CYBB gene, was used to sequencing CYBB gene. By sequence analysis, 1 patient had normal CYBB gene and 11 patients were found with CYBB gene mutations, including four deletion mutations, four splicing errors, two nonsense mutations, and a missense mutation. And seven of those were novel mutations. In the mothers of the 11 mutated patients, 9 mothers are carriers of gene mutation, 1 mother is indefinite, and a mother is not a carrier of gene mutation. These results mean that genetic diagnosis is useful for identification of X-CGD patients and represents an important tool for carrier detection as well as prenatal diagnosis. Furthermore, genetic diagnosis also provides the evidences for hematopoietic stem cell transplantation treatment in XCGD patients.