Analysis of clonality in human endometriotic cysts based on evaluation of X chromosome inactivation in archival formalin-fixed, paraffin-embedded tissue.
The clonality of epithelial cells in ovarian endometriotic cysts was evaluated on the basis of a polymorphism of the X-linked phosphoglycerate kinase gene (PGK). The problems associated with clonality analysis of DNA samples extracted from archival formalin-fixed, paraffin-embedded tissue were addressed with the use of newly designed primers and a modified stepdown program for PCR amplification. The method relies on digestion of DNA with the methylation-sensitive restriction enzyme SnaBI, PCR amplification of PGK, and detection of a BstXI polymorphism. With this approach, only the inactive (methylated) PGK allele is selectively amplified by PCR. A total of 25 epithelial cells in endometriotic cysts and 25 matched normal ovarian stromal tissue specimens were analyzed. All of the control tissue samples yielded PCR products, with 11 of the 25 samples found to be heterozygous for the BstXI polymorphism. Ten of the 25 epithelial cells in endometriosis specimens were informative at this locus, and all of these 10 were shown to be monoclonal on the basis of PGK methylation. The results indicate that this new method of clonal analysis with archival formalin-fixed tissue is reliable and confirm that endometriotic cysts are monoclonal in origin.
Tamura
M
M
Department of Obstetrics and Gynecology, School of Medicine, Tohoku University, Sendai, Japan.
Fukaya
T
T
Murakami
T
T
Uehara
S
S
Yajima
A
A
D001483:Base Sequence; D002999:Clone Cells / Q000473:pathology; D003560:Cysts / Q000235:genetics* / Q000473:pathology*; D004715:Endometriosis / Q000235:genetics* / Q000473:pathology*; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011110:Polymorphism, Genetic / Q000235:genetics; D014960:X Chromosome / Q000502:physiology*
D005404:Fixatives; D005557:Formaldehyde; D010735:Phosphoglycerate Kinase