Aus der Medizinischen Klinik mit Schwerpunkt Hämatologie und Onkologie der Medizinischen Fakultät Charité – Universitätsmedizin Berlin DISSERTATION Sequence Analysis of the NUMB Gene in Chronic Myeloid Leukaemia Patients

Better understanding of the transformation of chronic myeloid leukemia (CML) from chronic phase (CP) to the invariably fatal blast phase (BP) is of critical importance for the clinical management of patients with CML. However, the mechanisms responsible for triggering disease progression have eluded investigators’ efforts. Recently, our group verified reported data showing increased levels of Musashi-2 (MSI2) transcripts in patients with CML in BP compared to those in CP, implying a role for MSI2 in CML transformation. The Musashi gene family is reported to control critical cell fate decisions by binding to target mRNAs, including the NUMB mRNA, thereby inhibiting translation. Unregulated increased expression of MSI2 results in the dysfunction of NUMB-Notch signalling, leading to haematopoietic stem cell (HSC) proliferation, impaired myeloid differentiation and worse clinical prognosis in CML. Therefore, we hypothesized that mutations mapping to the NUMB gene may perturb this signalling pathway and thereby influence CML transformation. I tested this notion by directly sequencing the entire NUMB transcript in 22 patients with CML of whom 10 were in CP and 12 were in BP. Archived RNA extracted from peripheral blood from subjects with CML was reverse transcribed to cDNA and the entire NUMB gene transcript was amplified. The amplified products were subjected to Sanger sequencing. For the 22 patients with CML, the NUMB gene transcript sequence was determined to be identical to the published wild type sequence, apart from two previously reported single nucleotide polymorphisms (SNP) mapping to the 3’-UTR: rs11625196 (C/G) and rs7202 (C/T) . I observed no significant difference in the distribution of the genotypes of the two SNP between that reported for normal healthy individuals and the CML patients, nor between the different disease phases. However, rs7202 genotype had significant influence on the mortality rate of patients in BP – an observation which was fortuitously biased by different treatment modalities. The software tools Mfold and SNPfold predicted a negligible effect of the two SNP on the secondary structure of NUMB mRNA. In a summary, these observations suggest that NUMB, which regulates Notch, Hedgehog and p53 signalling, is not the primary cause of CML evolution. However, it would be prudent to confirm this finding in a study with greater number of CML CP and BP patients and/or using a sequencing method with higher sensitivity, such as deep-gene sequencing.