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Χαρακτηρισμός ανοσογενετικού προφίλ με στοχευμένη αλληλούχηση μεγάλης κλίμακας Β λεμφοκυττάρων σε διαχρονικά δείγματα ασθενών με χρόνια λεμφοκυτταρική λευχαιμία (Bachelor thesis)

Μουράτη, Σοφία

Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia worldwide. It is characterized by a diverse clinical course. Although the genetic and molecular mechanisms that lead to the disease pathogenesis remain at large unknown, what is clear is that there is a major malignant B cell clone, carrying a unique antigenic specificity through its B-cell receptor immunoglobulin (BcR IG); its cells represent approximately 90% of the peripheral blood (PB) cells. The molecular characteristics of the BcR IG define the affinity with the selecting antigen (Ag) and the type of signaling transmitted through the BcR, ultimately affecting clonal behavior and disease outcome. Indeed, CLL is divided in two prognostic categories, based on the extent of somatic hypermutation in the IGHV gene, namely: (i) mutated (<98% germline identity, GI), characterized by anergic BcRs and indolent course, and (ii) unmutated (>98% GI), characterized by agressive disease course and adverse clinical outcomes. Generally, the disease is considered incurable and many patients relapse after treatment. The purpose of this thesis was the characterization of B-cell receptor repertoire of 5 patients in the above timepoints, in order to investigate possible (sub)clonal evolution, associated with clinical relapse. The first step of the workflow consisted of PBMCs isolation from patients’ blood samples. Then, DNA was extracted from the isolated cells and was used as template for the library preparation. Library preparation was accomplished by a single PCR reaction, using primers which were both properly adapted and indexed with probes compatible with Illumina’s MiSeq platform. When the libraries were evaluated for their quantity and quality, the targeted PCR products were sequenced. The raw data underwent a multistage analysis, using purpose-built bioinformatic tools. The main message arised was that the major clonotype of each patient was exactly the same at diagnosis and relapse. A number of different, less frequent clonotypes were identified in each sample and they were compared with the major one, based on their unique molecular features. In particular, clonotypes which had the same CDR3 length, fewer than two or three amino acid substitutions within CDR3 and used the same gene IGHV in their productive rearrangement were clustered together and formed a new cluster, because they may come from the major one as a result of mutagenesis. Last but not least, it is of great importance to keep in mind that Next Generation Sequencing (NGS) offers new insights in the field of Immunogenetics, as it can provide information for many different sequences which are present in the sample in really low frequencies.