Differential expression profile of H/ACA ribonucleoprotein components in chronic lymphocytic leukemia patients

CARMEN STANGANELLI; IRMA SLAVUTSKY; JULIETA PANERO; RAIMUNDO BEZARES; PATRICIA DOS SANTOS; VIRGINIA PALAU NAGORE

Conpenhagen

Congreso; 21st Congress of the European Hematology Association; 2016

European Hematology Association

Background: Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. Telomere dysfunction has been proposed as an emerging prognostic factor in this pathology. Telomeres are essential to maintain chromosomal integrity and genome stability. They progressively shorten with repeated cell division, leading to telomere dysfunction and contributing to tumorigenesis. Telomerase is a ribonucleoprotein complex containing an internal RNA template (TERC) and a catalytic protein with telomere-specific reverse transcriptase activity (TERT). The human TERC gene consists of three major domains, among them the H/ACA domain is essential for telomere biogenesis. H/ACA ribonucleoprotein (RNP) complex is composed of four evolutionary conserved proteins, including dyskerin (DKC1), NOP10, NHP2 and GAR1. The first three proteins form a core trimer that directly binds to H/ACA RNAs. They are interdependent upon each other for stability and also regulate stability of the bound RNAs. GAR1 binds only to DKC1 and is needed for proper functioning of the H/ACA RNPs, but its absence does not reduce stability of the RNA. Aim: In this study, we have evaluated the expression profile of the H/ACA RNP complex: DKC1, NOP10, NHP2 and GAR1, as well as TERT and TERC mRNA levels, in patients with CLL. Results were correlated with telomere length (TL), genetic alterations, mutational status of IGHV (immunoglobulin heavy chain variable region) genes, and clinico-pathological characteristics of the disease. Methods: The study was performed on mononuclear cells isolated on a Ficoll-Paque Plus density gradient from peripheral blood samples of 60 patients with CLL at diagnosis (34 males; mean age: 66.8 years; range: 44-88 years) and 14 normal controls. All individuals gave their informed consent and the study was approved by the local Ethics Committee. Gene expression and absolute TL measurement was carried out by real-time quantitative PCR. IGHV gene rearrangements and mutational status were analyzed by RT-PCR and bi-directional sequencing. Cytogenetic and FISH analysis were also performed. Results: Gene expression analysis showed increased mRNA levels of TERT, NHP2 and GAR1, as well as decreased expression of TERC, DKC1 and NOP10 in CLL patients compared to controls (p=0.043). A significant correlation between GAR1-NHP2, GAR1-NOP10 and NOP10-NHP2 transcription levels (p=0.0001) was detected, supporting a strong interaction among them. Patients with short TL had higher expression of TERT (p=0.033) and DKC1 (p=0.02) than those with long telomeres. The analysis taking into account cytogenetic and FISH alterations showed higher mRNA levels of GAR1 and NHP2 in patients with two or more anomalies (p=0.02) compared to those with no/one alteration. An upregulation of TERT, TERC, GAR1 and NOP10 expression in unmutated IGHV CLL patients was also observed. No association between gene expression and clinical parameters was found. However, a tendency to a short treatment free survival in patients with increased TERT expression was detected. Summary/Conclusions: Our findings show a global modification in the expression of telomere associated genes in CLL, being, to our knowledge, the first analysis of TERC, NOP10, NHP2 and GAR1 in this pathology. Their association with higher number of genetic alterations and unmutated IGHV mutational status suggests a role for these telomere-associated genes in genomic instability and telomere dysfunction in CLL.