Methylation-specific Polymerase Chain Reaction Method Research Articles

Analysis of allelic imbalances at multiple cancer-related chromosomal loci and microsatellite instability within the same tumor using a single tumor gland from colorectal carcinomas

Genetic changes related to colorectal carcinomas are accumulated in individual tumor glands during disease progression. Microsatellite allelic analysis of individual tumor glands from 30 colorectal carcinomas using a polymerase chain reaction (PCR) assay coupled with crypt isolation was used to detect intratumoral genetic heterogeneity, the sequence of allelic imbalances (AIs) and the microsatellite instability status of single tumor glands during neoplastic progression. In addition, the CpG islands methylated phenotype (CIMP) status was examined using a methylation-specific PCR method. The specimens were divided into 2 groups: a pooled gland sample, which was composed of more than 50 tumor glands, and a single tumor gland sample. The latter consisted of 10 single tumor glands, which were obtained from the same tumor separately. Most colorectal carcinomas (27 of 30 tumors) examined were heterogeneous for at least one genetic alteration, with from 2 to 7 genotypically different subclones detected per tumor. In 12 of the 27 heterogeneous tumors, it was possible to define the order of genetic alterations during the tumor progression. By analyzing multiple single tumor glands within the same tumor, we found that various subclonal expansions were seen within the same tumors. Finally, the AI pattern of single tumor glands was not correlated with CIMP status. Most carcinomas appeared to have a heterogeneous composition. This may have resulted from the successful progression of one clone that had different AIs in many chromosomal regions. This suggests that knowledge of the different genotypes of multiple single tumor glands may help clarify the process of tumor progression.

Correlations of partial and extensive methylation at the p14(ARF) locus with reduced mRNA expression in colorectal cancer cell lines and clinicopathological features in primary tumors.

p14(ARF) is a putative tumor suppressor gene thought to modify the levels of p53. CpG sites within the 5'-flanking region and exon 1beta of p14(ARF) are targets of aberrant methylation and transcriptional silencing in human colorectal cancer (CRC). Here we have developed methylation-specific polymerase chain reaction (MSPCR) methods to detect methylation of CpG sites in p14(ARF) in CRC cell lines and primary CRC tumors, and correlated p14(ARF) mRNA expression with methylation in CRC cell lines using competitive quantitative reverse transcription-polymerase chain reaction methods. Ten CRC cell lines were studied; three (DLD-1, HCT15 and SW48) showed extensive methylation and six (Colo320, SW480, HT29, Caco2, SW837 and WiDr) were unmethylated; the other cell line, LoVo, showed partial methylation that affected exon 1beta but not the immediate upstream CpG sites. p14(ARF) mRNA was expressed at extremely low levels in fully methylated cell lines and at 10(4)- to 10(5)-fold higher levels in unmethylated cell lines. p14(ARF) expression in the partially methylated LoVo cell line was intermediate. Treatment of LoVo cells with 2 microM 5-aza-2'-deoxycytidine for 72 h was associated with marked (100-fold) induction of mRNA levels. Of 119 primary CRCs, 18% contained p14(ARF) methylation, although partial methylation was the most common pattern observed (in 67% of methylated tumors). Methylation of p14(ARF) was often accompanied by p16(INK4a) methylation; however, 50% of p14(ARF) methylated tumors contained unmethylated p16(INK4a). Methylation at p14(ARF) was associated with female gender, greater age, proximal anatomic location and poor differentiation, but not stage at diagnosis. A two-step MSPCR method for assaying p14(ARF) methylation in human tumors is described.

Prader-Willi and Angelman syndromes: Diagnosis with a bisulfite-treated methylation-specific PCR method

The putative promoter region of the SNRPN gene contains a CpG island which is heavily methylated in the maternally derived allele and unmethylated in the paternally derived allele. In patients with Prader-Willi syndrome (PWS) only the methylated allele is present, while in those with Angelman syndrome (AS) only the unmethylated allele is present. The purpose of this paper is to report a polymerase chain reaction (PCR)-based assay to evaluate methylation status of the CpG island of the SNRPN gene and to show that this assay allows rapid diagnosis of PWS and AS. Methylated cytosines in the CpG dinucleotide are resistant to chemical modification by sodium bisulfite. In contrast, bisulfite treatment converts all unmethylated cytosines to uracil. Based on this differential effect, the bisulfite-modified DNA sequence of a methylated allele was successfully distinguished from that of an unmethylated allele using 2 sets of allele-specific primer pairs: a methylated allele-specific primer pair (MET) and an unmethylated allele-specific primer pair (UNMET). Bisulfite-modified DNA from 10 patients with PWS amplified only with the MET pair while modified DNA from 5 patients with AS amplified only with the UNMET pair. Modified DNA from 50 normal unrelated individuals amplified with both primer pairs. In that methylation-specific PCR (MSPCR) can detect all presently testable causes of PWS and AS in a rapid and cost-effective fashion, serious consideration should be given to the use of this test in the initial evaluation of all patients in which PWS or AS is being considered.