A novel approach to simultaneously scan genes at fragile sites.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Willem
Pascale
P
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. pascale.willem@nhls.co.za
Brown
Jacqueline
J
Schouten
Jan
J
D017766:Acid Anhydride Hydrolases / Q000235:genetics; D002277:Carcinoma / Q000235:genetics; D002872:Chromosome Deletion; D043283:Chromosome Fragile Sites; D002885:Chromosomes, Human, Pair 16; D002893:Chromosomes, Human, Pair 3; D015342:DNA Probes; D004938:Esophageal Neoplasms / Q000235:genetics; D005260:Female; D018628:Gene Dosage; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D009363:Neoplasm Proteins / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010088:Oxidoreductases / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D000074584:WW Domain-Containing Oxidoreductase; D000099170:Bis(5'-adenosyl)triphosphatase
D017766:Acid Anhydride Hydrolases; D015342:DNA Probes; D009363:Neoplasm Proteins; D010088:Oxidoreductases; D025521:Tumor Suppressor Proteins; D000074584:WW Domain-Containing Oxidoreductase; D000099170:Bis(5'-adenosyl)triphosphatase; C486071:WWOX protein, human