Methylation profiling of SOCS1 , SOCS2 , SOCS3 , CISH and SHP1 in Philadelphia‐negative myeloproliferative neoplasm

Janus kinase‐signal transducer and activator of transcription ( JAK / STAT ) signalling, pivotal in Philadelphia‐negative (Ph‐ve) myeloproliferative neoplasm ( MPN ), is negatively regulated by molecules including SOCS s, CISH and SHP 1. SOCS 1 , SOCS 2 and SOCS 3 methylation have been studied in MPN with discordant results. Herein, we studied the methylation status of SOCS 1 , SOCS 2 and SOCS 3 , CISH and SHP 1 by methylation‐specific polymerase chain reaction ( MSP ) in cell lines and 45 diagnostic marrow samples of Ph‐ve MPN . Moreover, we attempted to explain the discordance of methylation frequency by mapping the studied MSP primers to the respective genes. Methylation was detected in normal controls using SOCS 2 MSP primers in the 3′translated exonic sequence, but not primers around the transcription start site in the 5′ untranslated regions (5′ UTR ). SOCS 1 , SOCS 2 , SOCS 3 and CISH were completely unmethylated in primary MPN samples and cell lines. In contrast, methylation of SHP 1 was detected in 8.9% primary marrow samples. Moreover, SHP 1 was completely methylated in K562 cell line, leading to reversible SHP 1 silencing. A review of methylation studies of SOCS 1 and SOCS 3 showed that spuriously high rates of SOCS methylation had been reported using MSP primers targeting CpG sites in the 3′translated exonic sequence, which is also methylated in normal controls. However, using MSP primers localized to the 5′ UTR , methylation of SOCS 1, SOCS 2 and SOCS 3 is infrequent across all studies. In summary, methylation of SOCS 1 , SOCS 2 , SOCS 3 and CISH is infrequent in Ph‐ve MPN . Appropriate MSP primers are important for accurate estimation of the methylation frequency. The role of SHP 1 methylation in the pathogenesis of MPN warrants further investigation.