Molekularna osnova hronične limfocitne leukemije : korelacija između mutacionog statusa teških lanaca imunoglobulina i ekspresije apoptotskih gena

Abstract

Hronična limfocitna leukemija (HLL), najčešći tip leukemije u Evropi i Severnoj Americi, se manifestuje kao klonska ekspanzija zrelih CD5+ CD19+ CD23+ sIgM+/- B limfocita i karakteriše se izuzetno heterogenim kliničkim tokom. Leukemični, kao i normalni B limfociti, na svojoj površini eksprimiraju imunoglobulinski antigenski receptor. Struktura varijabilnog regiona njegovog teškog lanca (IGH) je determinisana specifičnim rearanžmanima između IGHV, IGHD i IGHJ gena, koji se odvijaju tokom diferencijacije B ćelija. Nakon susreta sa antigenom, B limfociti ulaze u proces afinitetnog sazrevanja u germinalnim centrima sekundarnih limfnih folikula, tokom koga dolazi do akumulacije somatskih hipermutacija u IGHV-IGHD-IGHJ rearanžmanima. Pokazano je da je mutacioni status rearanžiranih gena za varijabilni region teških lanaca imunoglobulina (IGHV) najpouzdaniji molekularni marker u HLL, koji definiše dva podtipa bolesti: M-HLL i N-HLL. Pacijenti sa malim procentom ili bez IGHV mutacija (N-HLL) obično imaju mnogo agresivniji tok bolesti i lošiju prognozu od pacijenata sa mutiranim IGH rearanžmanima (M-HLL). Razlike u IGHV genskom repertoaru između M-HLL i N-HLL klonova, populacione varijacije u učestalosti određenih IGHV gena u HLL rearanžmanima kao i ekspresija visoko homologih, ˝stereotipnih˝ rearanžmana, ukazuju na ulogu antigenske stimulacije u patogenezi HLL.HLL se smatra tipičnim primerom maligniteta uzrokovanog poremećajima u procesu apoptoze. U HLL su detektovane genetičke promene i aberantna ekspresija brojnih proteina regulatora apoptoze, koji su uključeni kako u spoljašnji tako i u unutrašnji put aktivacije ovog procesa. Smatra se da je smanjeni apoptotski potencijal HLL klonova uzrokovan, između ostalog, i poremećajima u ekspresiji proteina Bcl2 familije. U ovom radu je analiziran IGHV mutacioni status i genski repertoar IGHV-IGHD-IGHJ rearanžmana kod pacijenata obolelih od HLL. Pored toga, analizirana je ekspresija gena Bcl2 familije, kao osnovnih regulatora unutrašnjeg puta aktivacije apoptoze, u cilju utvrđivanja njihove uloge u apoptotskoj rezistenciji HLL B limfocita. qRT-PCR metodom je merena ekspresija Bcl2, Bax i Bcl2L12 gena, i ispitivana asocijacija nivoa njihove ekspresije sa odabranim kliničkim i molekularnim prognostičkim faktorima (IGHV mutacionim statusom, ekspresijom CD38 i lipoprotein lipaze). Bcl2L12 je novi član Bcl2 familije apoptotskih proteina čija pro- ili anti-apoptotska funkcija još uvek nije razjašnjena. Pored analize ekspresije Bcl2L12 gena u HLL, jedan od ciljeva ovog rada je bilo i definisanje promotorskog regiona i mesta starta transkripcije Bcl2L12, što je neophodno za proučavanje mehanizama njegove transkripcione regulacije. Određivanjem IGHV mutacionog statusa je pokazano da 55.3% analiziranih pacijenata pripada M-HLL, a 44.7% N-HLL podtipu, kao i da u N-HLL preovlađuju pacijenti sa progresivnim oblikom bolesti. U analiziranim rearanžmanima su sa najvećom frekvencom bili zastupljeni geni IGHV3 familije (55.7%), a zatim IGHV1 (27.3%), IGHV4 (12.5%), IGHV5 (2.3%), IGHV2 (1.1%) i IGHV6 (1.1%) geni. Pokazano je prisustvo tzv. ˝stereotipnih˝ rearanžmana kod 15.3% pacijenata, predominantno u N-HLL podtipu. Na osnovu određenih frekvenci IGHV, IGHD i IGHJ gena i genskih familija je zaključeno da je IGH genski repertoar leukemičnih klonova kod pacijenata iz Srbije veoma sličan repertoaru detektovanom kod pacijenata iz zemalja mediteranskog područja, sa izuzetkom gena IGHV4 familije koji su kod pacijenata u ovoj studiji zastupljeni sa manjom učestalošću.Analizom ekspresije Bcl2, Bax i Bcl2L12 su detektovani znatno viši nivoi ekspresije sva tri gena kod HLL pacijenata u odnosu na zdrave kontrole, pri čemu je povećanje ekspresije bilo najizraženije u slučaju Bcl2 gena. Povišena ekspresija Bcl2 je pokazala asocijaciju sa nepovoljnim prognostičkim parametrima: progresivnim tipom bolesti, visokim nivoom serumskog β2-mikroglobulina i povišenim nivoom ekspresije gena za lipoprotein lipazu (LPL). Ekspresija Bax je pokazala korelaciju samo sa ekspresijom LPL, dok je ekspresija Bcl2L12 bila relativno homogena među HLL pacijentima i, kao takva, nije ispoljila značajnu asocijaciju sa većinom kliničkih i molekularnih prognostičkih faktora. Ekspresija Bcl2, Bax i Bcl2L12 je bila viša u grupi N-HLL pacijenata u odnosu na M-HLL pacijente, ali ova razlika nije dostigla statističku značajnost.Kloniranjem fragmenata 5' kraja Bcl2L12 gena i funkcionalnom analizom reporterskih konstrukata, detektovan je region koji ispoljava jaku promotorsku aktivnost. Ovaj region se prostire od 338 nukleotida uzvodno, do 148 nizvodno od početka kodirajućeg dela egzona 1, a esejom usporene elektroforetske pokretljivosti (˝EMSA˝) je pokazano da se za njega vezuju Sp1 i GATA-1 transkripcioni faktori. Metodom elongacije reverznog prajmera (˝primer extension˝) je određen položaj starta transkripcije Bcl2L12 gena, 33 nukleotida uzvodno od translacionog start kodona

Chronic lymphocytic leukemia (CLL), the most common type of leukemia in Western countries, manifests as clonal expansion of mature CD5+ CD19+ CD23+ sIgMlow B lymphocytes and it is characterized by an extremely heterogeneous clinical course. Leukemic, as well as normal B lymphocytes, express immunoglobulin antigenic receptor at their surface. The structure of its heavy chain (IGH) variable region is being formed during B-cell differentiation, through rearrangements between IGHV, IGHD and IGHJ genes. After antigen encouner, B lymphocytes undergo the process of affinity maturation in germinal centers of secondary lymphoid follicles, durring which IGHV-IGHD-IGHJ rearrangements accumulate somatic hypermutations. It has been shown that the mutational status of rearranged immunoglobulin heavy variable genes (IGHV) represents the most reliable molecular marker in CLL, which defines two CLL subsets: M-CLL and U-CLL. The patients without or with a small percentage of IGHV mutations (UCLL) usually have more agressive disease and inferior prognosis in comparison to patients expressing mutated IGH rearrangements (M-CLL). Biased IGHV gene repertoire between MCLL and U-CLL clones, population differences in IGHV gene usage, as well as the expression of highly homologous, ˝stereotyped˝ rearrangements, strongly imply the role of antigenic stimulation in pathogenesis of CLL.CLL typifies the malignancy caused by defective apoptosis. Genetic alterations and aberrant expression of numerous proteins involved in extrinsic and intrinsic pathways of apoptosis regulation have been described in CLL. Reduced apoptotic capacity of CLL clones is, in part, caused by disturbances in the expression of Bcl2 family proteins. In this study, we analized IGHV mutational status and gene repertoire of IGHV-IGHD-IGHJ rearrangements in CLL patients. Furthermore, the expression of Bcl2 family genes, the key regulators of intrinsic apoptotic pathway, has been studied, in order to elucidate their role in resistance of CLL B lymphocytes to apoptosis. The expression levels of Bcl2, Bax and Bcl2L12 genes were measured by qRT-PCR, and association of their expression with clinical and molecular prognostic factors (IGHV mutational status, expression of CD38 and lipoprotein lipase) was analized. Bcl2L12 is a novel member of Bcl2 family of apoptotic proteins, whose pro- or anti-apoptotic function has not been elucidated yet. Besides the expression analysis of Bcl2L12 gene in CLL, one of the aims of this dissertation was defining the promoter region and transcription start site of Bcl2L12, which is essential for the study of mechanisms involved in its transcriptional regulation. The analysis of IGHV mutational status showed that 55.3% of patients enrolled in this study belonged to M-CLL, and 44.7% to U-CLL subset, and that in U-CLL predominated patients with the progressive form of the disease. The most frequently expressed genes were those belonging to IGHV3 family (55.7%), followed by IGHV1 (27.3%), IGHV4 (12.5%), IGHV5 (2.3%), IGHV2 (1.1%) i IGHV6 (1.1%) genes. The presence of ˝stereotyped˝ rearrangements was detected in 15.3% of patients, predominantly in U-CLL subset. Based on the determined frequencies of IGHV, IGHD and IGHJ genes and gene families, we concluded that the IGH gene repertoire of leukemic clones of Serbian CLL patients closely resembles the repertoire observed in patients from Mediterranean countries, with the exception of IGHV4 family, which was underrepresented in our cohort. The expression analysis of Bcl2, Bax and Bcl2L12 showed that those three genes were overexpressed in CLL patients compared to healthy controls, and that elevation of expression level was the most prominent in the case of Bcl2 gene. High expression levels of Bcl2 were associated with unfavorable prognostic parameters: progressive form of the disease, elevated β2- microglobulin and high expression of lipoprotein lipase gene (LPL). The expression of Bax was correlated only with the expression of LPL, while the expression of Bcl2L12 turned out to be relatively homogenous among CLL patients and, as such, failed to show association with the majority of clinical and molecular prognostic factors. Expression levels of Bcl2, Bax and Bcl2L12 were higher in U-CLL in comparison to M-CLL group of patients, but this difference did not reach statistical significance.Cloning of fragments corresponding to 5' end of Bcl2L12 gene and functional analysis of reporter constructs, led to identification of a region which exerts high promoter activity. This region extends from 338 nucleotides upstream, to 148 nucleotides downstream relative to the beginning of exon 1 coding sequence, and electromobility shift assay (˝EMSA˝) showed that it binds Sp1 and GATA-1 transcription factors. Transcription start site of Bcl2L12 gene was determined by primer extension assay, which showed that it is located 33 nucleotides upstream of translation start codon.