Balkan Journal of Medical Genetics

IGHV MUTATIONAL STATUS IN A COHORT OF BULGARIAN CLL PATIENTS: HIGH UNMUTATED CLL PREVALENCE IN NORTH-EAST BULGARIA
Yosifova A, Micheva I, Donchev M, Tincheva S, Ormandjiev S, Genova J, Pavlova Z, Todorova A
*Corresponding Author: Angelina Yosifova, Genetic Medico-Diagnostic Laboratory “Genica”, Sofia, Bulgaria. Email: andjim91@gmail.com; Phone: +359888979313
page: 15

MATERIALS AND METHODS

The present prospective study includes a total of 105 newly diagnosed CLL patients (72 males and 33 females) at the ages of 38-80 years from different Bulgarian regions. To the best of our knowledge, all of them have previously tested negative by FISH for 17p13 deletion, and none of them have received treatment prior to IGHV testing. All patient samples were obtained after signing an informed consent form. Samples collection and cells extraction: IGHV muta- tional status was determined by PCR/Sanger sequencing on genomic DNA (gDNA) or complementary DNA (cDNA) extracted from venous blood mononuclear cells collected in EDTA tubes. During the whole process of testing, ERIC Recommendations were strictly followed [6]. Mononuclear cells were obtained after density gradi- ent separation by FiColl-Paque PLUS with some in-house modifications: after gradient centrifugation, the mononu- clear cell pellet formed between the upper plasma layer and FiColl fraction was dissolved in Dulbecco’s Phosphate- Buffered Saline (D-PBS 1x – calcium and magnesium free), instead of Roswell Park Memorial Institute (RPMI 1640 Medium 1x). Total DNA/RNA was extracted from collected mono- nuclear cells. The preferred target for us is gDNA be- cause there is no need for a reverse transcription step. High molecular weight gDNA was extracted by QIAGEN - QIAmp ® DNA Blood kit following the manufacture’s instructions. Total RNA (extracted by ZYMO Research - Quick-RNA Viral kit), followed by cDNA synthesis (Sensi- FAST cDNA Synthesis Kit) was used only in 9 problematic cases, in which gDNA amplification showed unproductive rearrangements or poor quality of the sequencing profile. Following ERIC recommendations, the PCR ampli- fication was performed with leader primers in a multiplex reaction. In rare cases (n=3 patients), the amplification of the clonotypic IG rearrangement was successful only by utilization of internal IGHV FR1 (framework) prim- ers (Figure 2). In all 3 cases the amplification with the leader primers failed. Consensus primers targeting the IGHJ genes were used in reverse direction. These results were interpreted with caution, because FR1 primers do not amplify the entire V region [7]. The PCR amplification was performed with primers and protocols, according to ERIC recommendations and BIOMED2 [8,9]. The analysis of the rearranged IG sequences in FAS- TA format was performed with the IMGT/V-QUEST tool [10]. Cases with ≥98% identity were considered unmu- tated, while those with a homology less than 98% - mu- tant type, and cases were considered borderline when the homology is between 97-97.99% [11]. BCR stereotyped subsets were determined by AR- ResT/AssignSubsets online tool [12, 13]. Statistical analysis for correlation was performed by using Fisher’s Exact Test.

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