背景:急性淋巴细胞白血病(ALL)的特征是高度遗传异质性,由于融合基因复发,基因突变,基因内缺失,和基因过度表达,这对临床检测提出了重大挑战。RNA测序(RNA-seq)是检测多种遗传异常的强大工具,尤其是隐秘的基因重排,在一个单一的测试。
方法:60个样本(B-ALL,n=49;T-ALL,n=9;混合表型急性白血病(MPAL),n=2)和20个对照通过我们实验室开发的507个基因的靶向RNA-seq小组进行分析。其中,使用51个基因的下一代测序小组在DNA水平上同时分析了16名患者的基因突变。融合基因,CRLF2表达,和IKZF1基因内缺失也通过逆转录聚合酶链反应(RT-PCR)检测到。在培养24小时后,使用R-显带和G-显带技术对骨髓细胞进行核型分析。使用荧光原位杂交(FISH)分析部分融合基因。
结果:与核型分析结果相比,FISH,和RT-PCR,靶向RNA-seq对融合基因的检出率从48.3%提高到58.3%,发现了六个意想不到的融合基因,以及一种罕见的IKZF1基因内缺失同种型(IK10)。对16例ALL患者的DNA测序分析显示,在DNA水平上鉴定出的96.2%(25/26)的基因突变也可以在RNA水平上检测到。除了一个具有低变异等位基因分数的突变。CRLF2过表达的检测在RT-PCR和RNA-seq之间显示出完全一致。
结论:利用RNA-seq能够鉴定可能通过常规检测方法检测不到的临床上显著的遗传异常。其稳健的分析性能可能为临床诊断带来巨大的应用价值,预后,在所有的治疗。
BACKGROUND: Acute lymphoblastic leukemia (
ALL) is characterized by highly genetic heterogeneity, owing to recurrent fusion genes, gene mutations, intragenic deletion, and gene overexpression, which poses significant challenges in clinical detection. RNA sequencing (RNA-seq) is a powerful tool for detecting multiple genetic abnormalities, especially cryptic gene rearrangements, in a single test.
METHODS: Sixty samples (B-
ALL, n = 49; T-
ALL, n = 9; mixed phenotype acute leukemia (MPAL), n = 2) and 20 controls were analyzed by targeted RNA-seq panel of 507 genes developed by our lab. Of these, 16 patients were simultaneously analyzed for gene mutations at the DNA level using a next-generation sequencing panel of 51 genes. Fusion genes, CRLF2 expression, and IKZF1 intragenic deletion were also detected by reverse transcription-polymerase chain reaction (RT-PCR). Karyotype analysis was performed using the R-banding and G-banding technique on bone marrow cells after 24 hours of culture. Partial fusion genes were analyzed using fluorescence in situ hybridization (FISH).
RESULTS: Compared with the results of Karyotype analysis, FISH, and RT-PCR, the detection rate of fusion genes by targeted RNA-seq increased from 48.3% to 58.3%, and six unexpected fusion genes were discovered, along with one rare isoform of IKZF1 intragenic deletion (IK10). The DNA sequencing analysis of 16
ALL patients revealed that 96.2% (25/26) of gene mutations identified at the DNA level were also detectable at the RNA level, except for one mutation with a low variant allele fraction. The detection of CRLF2 overexpression exhibited complete concordance between RT-PCR and RNA-seq.
CONCLUSIONS: The utilization of RNA-seq enables the identification of clinically significant genetic abnormalities that may go undetected through conventional detection methods. Its robust analytical performance might bring great application value for clinical diagnosis, prognosis, and therapy in
ALL.