Borkhardt A, Repp R, Haas O A, Leis T, Harbott J, Kreuder J, Hammermann J, Henn T, Lampert F
Department of Pediatrics, University of Giessen, Germany.
Oncogene. 1997 Jan 16;14(2):195-202. doi: 10.1038/sj.onc.1200814.
We report the cloning and characterization of the entire AFX gene which fuses to MLL in acute leukemias with a t(X;ll)(q13;q23). AFX consists of two exons and encodes for a protein of 501 amino acids. We found that normal B- and T-cells contain similar levels of AFX mRNA and that both the MLL/AFX as well as the AFX/MLL fusion transcripts are present in the cell line and the ANLL sample with a t(X;11)(q13;q23). The single intron of the AFX gene consists of 3706 nucleotides. It contains five simple sequence repeats with lengths of at least 12 bps, a chi-like octamer sequence (GCA/TGGA/TGG) and several immunoglobulin heptamer-like sequences (GATAGTG) that are distributed throughout the entire AFX intron sequence. In the KARPAS 45 cell line the breakpoints occur at nucleotides 2913/2914 of the AFX intron and at nucleotides 4900/4901 of the breakpoint cluster region of the MLL gene. The AFX protein belongs to the forkhead protein family. It is highly homologous to the human FKHR protein, the gene of which is disrupted by the t(2;13)(q35;q14), a chromosome rearrangement characteristic of alveolar rhabdomyosarcomas. It is noteworthy that the t(X;11)(q13;q23) in the KARPAS 45 cell line and in one acute nonlymphoblastic leukemia (ANLL) disrupts the forkhead domain of the AFX protein exactly at the same amino acids as does the t(2;13)(q35;q14) in case of the FKHR protein. In addition, the 5'-part of the AFX protein contains a conserved hexapeptide motif (QIYEWM) that is homologous to the functionally important conserved hexapeptide QIYPWM upstream of the homeobox domain in Hox proteins. This motif mediates the co-operative DNA binding of Pbx family members and Hox proteins and, therefore, plays an important role in physiologic and oncogenic processes. In acute leukemias with a t(X;11)(q13;q23), this hexapeptide motif is separated from the remaining forkhead domain within the AFX protein. The predicted amino acid sequence of AFX differs significantly from the partial AFX protein sequence published previously (Genes, Chromosomes and Cancer, 1994, 11, 79-84). This discrepancy can be explained by the occurrence of two sequencing errors in the earlier work at nucleotide number 783 and 844 (loss of a cytosine residue or guanosine residue, respectively) that lead to two reading frame shifts.
我们报告了整个AFX基因的克隆和特征分析，该基因在伴有t(X;11)(q13;q23)的急性白血病中与MLL融合。AFX由两个外显子组成，编码一个含501个氨基酸的蛋白质。我们发现正常B细胞和T细胞中AFX mRNA水平相似，并且在细胞系以及伴有t(X;11)(q13;q23)的急性非淋巴细胞白血病（ANLL）样本中均存在MLL/AFX以及AFX/MLL融合转录本。AFX基因的单个内含子由3706个核苷酸组成。它包含五个长度至少为12个碱基对的简单序列重复、一个类chi八聚体序列（GCA/TGGA/TGG）以及几个免疫球蛋白七聚体样序列（GATAGTG），这些序列分布于整个AFX内含子序列中。在KARPAS 45细胞系中，断点位于AFX内含子的第2913/2914个核苷酸处以及MLL基因断点簇区域的第4900/4901个核苷酸处。AFX蛋白属于叉头蛋白家族。它与人FKHR蛋白高度同源，FKHR基因在t(2;13)(q35;q14)时被破坏，这是肺泡横纹肌肉瘤的一种特征性染色体重排。值得注意的是，KARPAS 45细胞系以及一例急性非淋巴细胞白血病（ANLL）中的t(X;11)(q13;q23)与FKHR蛋白中的t(2;13)(q35;q14)一样，恰好破坏了AFX蛋白的叉头结构域中相同的氨基酸。此外，AFX蛋白的5'端包含一个保守的六肽基序（QIYEWM），它与Hox蛋白中同源异型框结构域上游功能重要的保守六肽QIYPWM同源。该基序介导Pbx家族成员与Hox蛋白的协同DNA结合，因此在生理和致癌过程中起重要作用。在伴有t(X;11)(q13;q23)的急性白血病中，这个六肽基序与AFX蛋白中其余的叉头结构域分离。AFX的预测氨基酸序列与先前发表的部分AFX蛋白序列（《基因、染色体与癌症》，1994年，第11卷，第79 - 84页）有显著差异。这种差异可以通过早期工作中在核苷酸783和844处出现的两个测序错误（分别丢失一个胞嘧啶残基或鸟苷残基）来解释，这导致了两个阅读框移位。
