Interferon-gamma improves splicing efficiency of CYBB gene transcripts in an interferon-responsive variant of chronic granulomatous disease due to a splice site consensus region mutation
Interferon-gamma improves splicing efficiency of CYBB gene transcripts in an interferon-responsive variant of chronic granulomatous disease due to a splice site consensus region mutation
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DOI：
10.1182/blood.v95.11.3548.011k36_3548_3554
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发表时间：
2000-06-01
期刊：
影响因子：
20.3
通讯作者：
Newburger, PE
中科院分区：
文献类型：
--
作者：
Condino-Neto, A;Newburger, PE
X连锁慢性肉芽肿性疾病(CGD)源于编码NADPH氧化酶gp91Phox成分的细胞色素B亚基基因的缺陷。我们研究了一个变异的CGD家系的分子基础，该变异是由于CYBB第一内含子第六位的单碱基替换所致。先前已经证明，患者的吞噬细胞在体外和体内都能极大地增加对干扰素-伽马的反应，释放超氧化物。在这一家系中，我们检测了1例EB病毒(EBV)转化的B细胞系中的CYBB基因的表达。这些细胞的总RNA(正常的5%)和核RNA(正常的1.4%)中的CYBB转录水平显著降低，尽管CGD和对照细胞的CYBB转录水平相同。与干扰素-γ孵育后，患者细胞中的细胞色素B合成酶总信使RNA(MRNA)水平增加了3倍，核转录产物减少到无法检测到的水平。RNA剪接的逆转录-聚合酶链式反应分析显示，在患者的核RNA中，未剪接的CYBB转录本占优势。在体外与干扰素-γ孵育后，CGD-B细胞系细胞核中的剪接相对非剪接的CYBB mRNA的比例增加了40%。从同一患者的单核细胞中提取的总RNA，在接受和不接受干扰素-γ治疗的情况下，在剪接方面显示出类似的改善。我们的结论是，干扰素-γ部分纠正了这种类型的由于CYBB基因内含子突变引起的核加工缺陷，最可能是通过增加正常转录本的核输出，以及提高第一内含子剪接的保真度。(血。2000；95：3548-3554)(C)2000美国血液病学会。
X-linked chronic granulomatous disease (CGD) derives from defects in the CYBB gene, which encodes the gp91-phox component of NADPH oxidase. We studied the molecular basis of the disease in a kindred with variant CGD, due to a single base substitution at the sixth position of CYBB first intron. The patients' phagocytes have been shown previously to greatly increase superoxide release in response to interferon-gamma (IFN-gamma) in vitro and in vivo. We examined CYBB gene expression in an Epstein-Barr virus (EBV)-transformed B-cell line from 1 patient in this kindred. These cells showed markedly decreased levels of CYBB transcripts in total RNA (5% of normal) and nuclear RNA (1.4% of normal), despite equal CYBB transcription rates in the CGD and control cells. Incubation with IFN-gamma produced a 3-fold increase in CYBB total messenger RNA (mRNA) levels in the patient's cells, and decreased nuclear transcripts to undetectable levels. Reverse transcriptase-polymerase chain reaction analysis of RNA splicing revealed a preponderance of unspliced CYBB transcripts in the patient's nuclear RNA. In vitro incubation with IFN-gamma increased by 40% the ratio of spliced relative to unspliced CYBB mRNA in nuclei from the CGD B-cell line. Total RNA harvested from the same patient's monocytes, on and off therapy with IFN-gamma, showed a similar improvement in splicing. We conclude that IFN-gamma partially corrects a nuclear processing defect due to the intronic mutation in the CYBB gene in this kindred, most likely by augmentation of nuclear export of normal transcripts, and improvement in the fidelity of splicing at the first intron. (Blood. 2000;95:3548-3554) (C) 2000 by The American Society of Hematology.