Detection of Homozygous Deletions and Mutations in the CDKN2A Gene in Hydatidiform Moles

Discussion

The human CDKN2A gene which is located on chromosome 9p21 is a cyclin-dependent kinase inhibitor gene that encodes 2 different tumor suppressor proteins. One is designated as p16^INK4a which is encoded within exon 1α, exon 2 and 3. The other is designated as p14ARF and is encoded within exon 1β, exon 2 and 3. The p16^INK4a and p14^ARF genes share exon 2 and 3, while they have different open-reading frames and their amino sequences are quite disparate. The CDKN2A gene regulates cell proliferation by 2 different pathways^[2]: p16^INK4a-CDK4/CDK6-pRb and p14ARF-mdm2-p53. Variation of the gene or inactivation of the protein will result in loss of control of regulatory pathways, excessive cell proliferation and induction of tumorigenesis^[3-5].

DHPLC is a sensitive technique for mutation detection^[6,7]. The technique separates heteroduplex from homoduplex molecules by ion-pair reverse-phase liquid chromatography and, since it is automated, renders screening for mutations in a less expensive and timely manner. The power of the approach is especially realized in those instances when sequence variation is low, and consequently only homoduplex patterns are typically observed^[8,9]. In our study, all spectra of PCR products of the p14^ARF exon 1 and p16^INK4a exon 1 and 2 were single peak curves, indicating that sequences of the amplified products were coincident with the reference (wild-type) sequences and no mutations existed.

Homozygous deletions in the p16^INK4a exon 1 were detected in 5 hydatidiform mole cases in our study. These genetic variations may result in disordered regulation, abnormal cell division and excessive proliferation, suggesting that the genetic variation of the CDKN2A gene may be related to proliferation of the placental hydatidi-form moles. Wang et al.^[10] reported that deletion rates of the p16 gene and p15 gene and their proteins were significantly higher in hydatidiform moles compared to in villi samples. In 2004, Xue et al.^[11] detected hypermethylation of genes (p16, HIC-1, TIMP-3, GSTP1, DAPK, E-cadberin) in hydatidiform mole and chorioepithelioma samples using methylation specific PCR (MSP), and found that hypermethylation of the p16 gene showed a highly correlated relationship with the progression of hydatidiform moles.

Variations in the p16^INK4a/p14^ARF genes, including homozygous deletion, heterozygous deletion, mutation and promoter hypermethylation, have been found in many kinds of tumor malignancies. Pineau et al.^[12] detected homozygous deletions in 246 gene sites in lung cancer, ovarian cancer and squamous cell carcinoma of the head and neck, and found that homozygous deletions of p16^INK4a/p14^ARF were the most common event.

In 2005, Grafstrom et al.^[13] studied the relationship between homozygous deletions of the INK4 gene in 86 malignant melanoma patients and their prognosis, and found that these deletions were the most important genetic change which correlated to poor prognosis. Using Southern blots, PCR-single-strand conformation polymorphism (PCR-SSCP), MSP and reverse transcription PCR (RT-PCR), Chang et al.^[14] detected variations in the p16^INK4a/p14^ARF gene, and discovered that most deletions occurred in exon 2, and that promoter hypermethylation took place in 60% of the cases. All the above studies indicated that variations in the p16^INK4a/p14^ARF gene existed in numerable tumors and related to their progression.

In conclusion, our results indicated that homozygous deletions in the CDKN2A gene were highly related to the occurrence of hydatidiform moles, while mutation may be not the major cause for its genetic variation. Combined with previous studies, we suggest that homozygous deletions and promoter hypermethylation of the CDKN2A gene may be the major cause for its variations in hydatidiform moles. Further studies with larger number of cases should be conducted to explain the occurrence of these tumors, and to elucidate their development.