Definition of three minimal deleted regions by comprehensive allelotyping and mutational screening of FHIT,p16(INK4A), and p19(ARF) genes in nasopharyngeal carcinoma.
BACKGROUND
Recurrent deletion on a chromosomal location in tumor cells can be detected by frequent allelic loss and generally is considered to be an indication of the existence of a tumor suppressor gene (TSG) in the region. In the current study, using fluorescent-labeled, high-density microsatellite markers for allelotyping, the authors pinpointed three minimal deleted regions (MDRs) and screened mutations of putative TSGs on chromosomes 3, 9, and 11 in nasopharyngeal carcinoma (NPC) cases occurring in Taiwan.


METHODS
A total of 133 informative microsatellite markers were used on chromosomes 3, 9, and 11 with an average marker density of 4 centimorgans (cM) for the allelotyping of genomic DNAs isolated from NPC tissues and their corresponding lymphocytes in 48 patients. The correlation between allelic loss and the clinicopathologic parameters of NPC tissues was examined. In addition, putative TSGs including FHIT, p16(INK4a), and p19(ARF) were selected for mutation screening to investigate their potential participation in NPC tumorigenesis.


RESULTS
Of 3787 informative allelotyping data, 25 frequent allelic losses (or loss of heterozygosity [LOH]) in 13 cytogenetic loci were identified based on a deletion frequency that was greater than the average of allelic loss on that particular chromosome. Several significant associations were determined after statistical analysis of the correlation between allelic loss and clinicopathologic parameters. The allelic losses by D9S318 and D11S1304 were associated with N2/N3 (P = 0.035 and P = 0.005, respectively), and those by D9S905 and D11S1304 were associated with grouped American Joint Committee on Cancer (AJCC) Stage III/IV samples (P = 0.022 and P = 0.017, respectively) of NPC tissues. In addition, three MDRs were revealed on 3p25.3-24.1 (< 19 cM), 3p23-21.31 (< 9 cM), and 11q22.1-23.2 (< 8 cM). To examine somatic mutations in previously reported TSGs located near these frequent LOH loci, three candidate genes, p16(INK4a), p19(ARF), and FHIT, were analyzed. Point mutations in the coding region of FHIT and in the intron 1 splicing acceptor site of both p16(INK4a) and p19(ARF) were detected in NPC cell lines. Sequence analysis of both p16(INK4a) and p19(ARF) transcripts revealed that the point mutation resulted in skipping of exon 2 and the generation of shorter transcripts.


CONCLUSIONS
High-density allelotyping permitted the discovery of 3 MDRs on 3p25.3-24.1 (< 19 cM), 3p23-21.31 (< 9 cM), and 11q22.1-23.2 (< 8 cM) and a correlation was determined between allelic loss and clinicopathologic parameters of NPC tissues. More important, one somatic mutation in NPC cell lines on the intron 1/exon 2 splicing acceptor site of the INK4a/ARF locus was found to result in exon 2 skipping both p16(INK4a) and p19(ARF) transcripts, which presumably inactivates the functions of both the p16(INK4a) and p19(ARF) proteins.
Ko
Jenq-Yuh
JY
Department of Otolaryngology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Lee
Tso-Ching
TC
Hsiao
Chin-Fu
CF
Lin
Guan-Lu
GL
Yen
Sang-Hue
SH
Chen
Kuang Y
KY
Hsiung
Chao A
CA
Chen
Pei-Jer
PJ
Hsu
Mow-Ming
MM
Jou
Yuh-Shan
YS
D017766:Acid Anhydride Hydrolases; D000483:Alleles; D002869:Chromosome Aberrations; D002872:Chromosome Deletion; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics; D002893:Chromosomes, Human, Pair 3 / Q000235:genetics; D002899:Chromosomes, Human, Pair 9 / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000032:analysis; D016147:Genes, Tumor Suppressor; D006801:Humans; D019656:Loss of Heterozygosity; D018895:Microsatellite Repeats / Q000235:genetics; D009303:Nasopharyngeal Neoplasms / Q000235:genetics* / Q000473:pathology; D009363:Neoplasm Proteins / Q000235:genetics*; D009364:Neoplasm Recurrence, Local / Q000235:genetics / Q000473:pathology; D009367:Neoplasm Staging; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics*; D000099170:Bis(5'-adenosyl)triphosphatase
D017766:Acid Anhydride Hydrolases; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D025803:Tumor Suppressor Protein p14ARF; D000099170:Bis(5'-adenosyl)triphosphatase; C417743:Cdkn2a protein, mouse