Rabphillin-3A-Like gene is a candidate tumor suppressor in colorectal adenocarcinoma Free

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Background: The recently identified human ortholog of the Rabphillin-3A-Likegene (RPH3AL), located at the 17p13.3 locus, has been assessedfor molecular status and for its clinical significance in colorectal adenocarcinoma (CRC).

Methods: To determine the role of RPH3AL in CRC we screened 95 prospectively collected frozen CRCs andtheir matching control (benign epithelial) tissues for the mutational status of RPH3AL using the polymerase chain reaction (PCR) and direct sequencing methods. We assessed the loss of heterozygosity (LOH) at D17S643, DS17S849, D17S926 and D17S1866 loci in genomic DNA samples isolated from 53 CRCs and their matching controls. Also, the mRNA levels of this gene were measured by quantitative real-time-polymerase chain reaction (qRT-PCR) in 95 paired tissues. The LOH status of the RPH3AL gene was correlated with different clinicopathologic features and patient survival.

Results: The mutational analysis of RPH3AL has detected five novel missense mutations: at codons 67 (GTG > ATG; Val > Met) in 2 CRCs, at codon 175 (CCC > TCC; Pro > Ser) in 1 CRC, at codon 268 (GCC > GGC; Ala > Gly) in 1 CRC and at codon 290 (AGG > AAG; Arg > Lys) in 1 CRC, and a novel silent mutation at codon 98 (TGC> TGT; Cys > Cys) in 1 CRC. Additionally, 14 single nucleotide polymorphisms were identified: three at codon 49, nine at codon 62, and two at codon 303, in the coding region of the RPH3AL gene. Loss of heterozygosity was found in 26 of 44 (59%) informative cases demonstrating allelic loss in at least one of the four loci analyzed. The levels of mRNA expression were significantly reduced in CRCs (average copy number = 0.42, range 0.038-1.70) compared to their matching controls (average copy number=2.36, range 0.15-10.32) (χ², P<0.0001). The presence of LOH at the genomic region of RPH3AL was closely associated with down regulation of its mRNA expression. The incidence of LOH was found to be significantly associated with depth of the tumor (χ², P<0.05), nodal metastasis (χ², P=0.006), distant metastasis (χ², P<0.05), and high grade tumor differentiation (χ², P=0.038). Univariate Kaplan-Meier survival analysis of the LOH analyzed cohort demonstrated that patients who exhibited LOH had shorter survival than those patients without LOH (logrank, P=0.026).

Conclusions These preliminary findings suggest that the RPH3AL gene is a candidate tumor suppressor whose inactivation is involved in aggressiveness of colorectal adenocarcinoma.This work is supported partially by funds from the Departmentof Pathology, University of Alabama at Birmingham and by a grantfrom the National Institute of Health/National Cancer Institute(RO1-CA98932-01).