Abstract:

Objective To investigate the feasibility of molecular determination for Prader-Willi syndrome（PWS） through methylation-specific（MS）-polymerase chain reaction（PCR） and MS-multiplex ligation-dependent probe amplification（MLPA）,with remaining fixed cells from peripheral blood chromosome examination or fluorescence in situ hybridization（FISH） being treated as the source of DNA,so as to lay a foundation for carrying out chromosomal,FISH and molecular determination simultaneously with a little amount of whole blood sample. Methods A total of 13 samples were collected and classified into normal control group（3 cases）,confirmed group（2 cases） and suspected group（8 cases）. All frozen fixed cells were centrifuged,the stationary liquid was discarded,DNA extraction was conducted with genome DNA extraction kit,and MS-PCR combined with MS-MLPA was performed to determine PWS in the 13 samples. Results The average A₂₆₀/A₂₈₀ ratio of the 13 extracted DNA samples was 1.94±0.1,the average concentration was （64.0±10） ng/μL,and all the fragment sizes were >10 kb. The results of MS-PCR indicated that both paternal and maternal bands existed in normal control group and suspected group,while only maternal band existed in confirmed group. The results of MS-MLPA demonstrated that the results were normal except for 2 cases in confirmed group,which showed paternal deletion type PWS. Conclusions Genome DNA from fixed cells satisfies the experimental requirements of MS-PCR and MS-MLPA in the aspects of purity,concentration and integrity,and it could be used for clinical molecular determination for PWS as well as relevant experiments.

Key words: Prader-Willi syndrome, DNA extraction, Methylation-specific-polymerase chain reaction, Methylation-specific-multiplex ligation-dependent probe amplification, Fixed cell