Absence of DKC1 exon 3 mutation in common human cancers

To the Editor

Dyskeratosis congenita is a genetic disease caused by point muations in the DKC1 gene [1]. Patients with dyskeratosis congenita show bone marrow failure syndrome characterized by abnormal skin pigmentation and mucosal leukoplakia [2]. Of note, a subset of patients with dyskeratosis congenita developed malignant tumors of various histologic origins [2]. Also, DKC1 mutant mice were highly susceptible to tumor development, and the most common tumors were lung and breast tumors [3]. These observations suggested that DKC1 may be a tumor suppressor gene. DKC1 protein normally binds with small nucleolar RNAs and the RNA components of telomerase, and these functions are impaired in the cells of the DKC1 mutant mice [3].

In the individuals with dyskeratosis congenita, DKC1 gene mutation is detected usually in the exon 3, strongly suggesting that these mutations may related with the disease phenotypes [1], [4], [5]. It could be hypothesized that increased incidence of malignant tumors in the dyskeratosis congenita patients might be related to the DKC1 mutations. It is of interest to see whether the DKC1 gene is somatically mutated in human cancers. However, to date, the data on the somatic mutations of DKC1 in human sporadic cancers is lacking.

The aim of this study was to see whether common human sporadic cancers harbor DKC1 mutation. We have analyzed methacarn-fixed tissues of 140 gastric carcinomas, 104 colorectal carcinomas, 94 breast ductal carcinomas, 100 non-small cell caners and 69 hepatocellular carcinomas. All of the patients of the cancers were Asians (Koreans). The gastric carcinomas consisted of 60 diffuse-type, 49 intestinal-type and 31 mixed-type gastric adenocarcinomas by Lauren's classification, and 25 early and 115 advanced gastric carcinomas according to the depth of invasion. The colorectal carcinomas originated from cecum (n = 2), ascending colon (n = 19), transverse colon (n = 6), descending colon (n = 4), sigmoid colon (n = 28) and rectum (n = 45). The breast carcinomas consisted of 15 intraductal and 79 invasive ductal carcinomas. The non-small cell lung cancer samples consisted of 50 adenocarcinomas, 47 squamous cell carcinomas, 1 adenosquamous carcinomas and 2 large cell carcinomas. The hepatocellular carcinomas consisted of Edmondson grade I (n = 8), grade II (n = 30) and grade III (n = 31) according to Edmondson and Steiner's criteria [6].

Malignant cells and normal cells from the same patients were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle (Becton Dickinson, Franklin Lakes, NJ) affixed to a micromanipulator, as described previously [7].

DNA extraction was performed by a modified single-step DNA extraction method [7]. Because the most common site of the germline mutations of DKC1 was reported to be the exon 3 [1], [4], [5], we analyzed the exon 3 in this study. Genomic DNA each from tumor cells and corresponding normal cells were amplified with a primer pair covering the DNA sequences in the exon 3. For the detection of DKC1 mutations, we analyzed the exon by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP). Radioisotope ([³²P]dCTP) was incorporated into the PCR products for detection by SSCP autoradiogram. Other procedures of PCR and SSCP analysis were performed as described previously [8]. However, the SSCP from the tumors did not reveal any aberrantly migrating band compared to the wild-type bands from the normal tissues. To confirm the SSCP results, we also analyzed the PCR products by direct DNA sequencing, but we could not detect any evidence of DNA sequence alteration both in the normal tissues and in the tumor samples. We repeated the experiments twice, including tissue microdissection, PCR, SSCP and direct DNA sequencing analysis to ensure the specificity of the results, and found that the data were consistent.

Because the previous studies strongly suggested the association of germline DKC1 mutations with tumor development both in human and mice [1], [3–5], we expected to detect some DKC1 mutations in the sporadic tumor samples. However, we detected no DKC1 exon 3 mutation in the 507 samples. Our data could be interpreted by several ways. One possibility is that DKC1 gene may not be mutated in human cancers, which is a contrast to the association of germline DKC1 mutation with tumor development. The other possibility is that the DKC1 mutation might be present in the other areas besides the exon 3. To confirm this, studies are needed that attempt to find DKC1 mutation in other exons of DKC1 gene. In conclusion, our data demonstrated that in disagreement with the germline mutation data of DKC1 gene, somatic mutations in the DKC1 exon 3 is uncommon in common human cancers. Also, the data suggested that somatic mutations in the DKC1 exon 3 may not contribute the development of human cancers.

This work was supported by the funds from KOSEF (R01-2004-000-10463-0).