Willem Pascale, Brown Jacqueline, Schouten Jan
Department of Hematology and Molecular Medicine University of the Witwatersrand and the National Health Laboratory Services, WITS Medical School, 8 York road, 2193 Parktown, South Africa.
BMC Cancer. 2006 Aug 8;6:205. doi: 10.1186/1471-2407-6-205.
Fragile sites are regions of the genome sensitive to replication stress and to exposure to environmental carcinogens. The two most commonly expressed fragile sites FRA3B and FRA16D host the histidine triad (FHIT) and WW domain containing oxidoreductase (WWOX) genes respectively. There is growing evidence that both genes contribute to cancer development and they are frequently altered by allelic and homozygous deletions in a variety of tumors. Their status is linked to prognosis in several malignancies and they are thought to be involved in early tumorigenesis. The loci for FHIT and WWOX both span over a megabase but the genes encode for small transcripts. Thus the screening of intragenic deletion can be difficult and has relied on loss of heterozygosity LOH assays, or genomic arrays.
Multiplex ligation dependent probe amplification MLPA, allows for the detection of deletions/duplications and relative quantification of up to 40 specific probes in a single assay. A FHIT/WWOX MLPA assay was designed, applied and validated in five esophageal squamous cell carcinoma ESCC, cell lines established in South Africa where this cancer is of high prevalence. Sixteen probes covered all FHIT exons and 7 probes covered WWOX.
Both homozygous and hemizygous deletions were detected in FHIT, in four of the cell lines with a preferential deletion of exons 5 and 4. Chromosome 3 short arm was present in normal copy number indicating that deletions were site specific. In contrast WWOX was not altered in any cell lines. RT-PCR expression pattern paralleled the pattern of deletions. Ten primary ESCC tumor specimens were subsequently screened with this assay. FHIT exon deletions were found in four of them.
This method offers an alternative to loss of heterozygosity studies. Simultaneous scanning of FHIT and WWOX exons in the context of early tumorigenesis and tumor progression, may help clarify the mechanistic events related to cancer development which are not revealed by immuno histochemistry assays. The presence of site specific deletions of FHIT in these cell lines and primary tumors support its possible role in South African ESCC and justifies a wider screening.
脆性位点是基因组中对复制应激和环境致癌物暴露敏感的区域。两个最常表达的脆性位点FRA3B和FRA16D分别包含组氨酸三联体（FHIT）基因和含WW结构域的氧化还原酶（WWOX）基因。越来越多的证据表明，这两个基因都与癌症发展有关，并且在多种肿瘤中它们经常因等位基因和纯合缺失而发生改变。它们的状态与几种恶性肿瘤的预后相关，并且被认为参与早期肿瘤发生。FHIT和WWOX的基因座都跨越超过1兆碱基，但这些基因编码小转录本。因此，基因内缺失的筛查可能很困难，并且一直依赖于杂合性缺失（LOH）检测或基因组阵列。
多重连接依赖探针扩增（MLPA）允许在单一检测中检测缺失/重复以及多达40种特定探针的相对定量。设计了一种FHIT/WWOX MLPA检测方法，并在南非建立的5种食管鳞状细胞癌（ESCC）细胞系中应用和验证，南非这种癌症的患病率很高。16个探针覆盖了所有FHIT外显子，7个探针覆盖了WWOX。
在4个细胞系中检测到FHIT的纯合和半合子缺失，优先缺失外显子5和4。3号染色体短臂的拷贝数正常，表明缺失是位点特异性的。相比之下，任何细胞系中的WWOX均未改变。RT-PCR表达模式与缺失模式平行。随后用该检测方法筛查了10个原发性ESCC肿瘤标本。其中4个发现有FHIT外显子缺失。
该方法为杂合性缺失研究提供了一种替代方法。在早期肿瘤发生和肿瘤进展的背景下同时扫描FHIT和WWOX外显子，可能有助于阐明与癌症发展相关的机制事件，而免疫组织化学检测并未揭示这些事件。这些细胞系和原发性肿瘤中FHIT位点特异性缺失的存在支持了其在南非ESCC中的可能作用，并证明了更广泛筛查的合理性。
