Use of Array Comparative Genomic Hybridization for the Diagnosis of DiGeorge Syndrome in Saudi Arabian Population.

DiGeorge syndrome (DGS) is a genetic disorder known as a clinically variable syndrome with over 180 associated phenotypic features. It is caused by a common human deletion in the 22q11.2 chromosomal region and currently is affecting approximately 1 in 4,000 individuals. Despite the prevalence of inherited diseases mainly due to consanguineous marriages, the current diagnosis of DGS in Saudi Arabia is mainly based on conventional high-resolution chromosome banding (karyotyping) and FISH techniques. However, advanced genome-wide studies for detecting microdeletions or duplications across the whole genome are needed. The aim of this study is to implement and use aCGH technology in clinical diagnosis of the 22q11.2 deletion in Saudi Arabian DGS patients and to confirm its effectiveness compared to conventional FISH and chromosome banding techniques. Thirty suspected DGS patients were assessed for chromosome 22q11.2 deletion using high-resolution G-banding, FISH, and aCGH. The aCGH results were compared with those obtained by the other 2 cytogenetic techniques. G-banding detected the 22q11.2 deletion in only 1 patient in the cohort. Moreover, it detected additional chromosomal aberrations in 3 other patients. Using FISH, allowed for detection of the 22q11.2 deletion in 2 out of 30 patients. Interestingly, the use of aCGH technique showed deletions in the chromosome 22q11.2 region in 8 patients, indicating a 4-fold increase in diagnostic detection capacity compared to FISH. Our results show the effectiveness of aCGH to overcome the limitations of FISH and G-banding in terms of diagnostic yield and allow whole genome screening and detection of a larger number of deletions and/or duplications in Saudi Arabian DGS patients. Except for balanced translocations and inversions, our data demonstrate the suitability of aCGH in the diagnostics of submicroscopic deletion syndromes such as DGS and most chromosomal aberrations or complex abnormalities scattered throughout the human genome. Our results recommend the implementation of aCGH in clinical genomic testing in Saudi Arabia to improve the diagnostic capabilities of health services while maintaining the use of conventional cytogenetic techniques for subsequent validation or for specific and known aberrations whenever required.

CD44 regulates the apoptotic response and promotes disease development in chronic lymphocytic leukemia.

The cell-surface glycoprotein CD44 is expressed in chronic lymphocytic leukemia (CLL), but its functional role in this disease is poorly characterized. We therefore investigated the contribution of CD44 to CLL in a murine disease model, the Eµ-TCL1 transgenic mouse, and in CLL patients. Surface CD44 increased during murine CLL development. CD44 expression in human CLL was induced by stimulation with interleukin 4/soluble CD40 ligand and by stroma cell contact. Engagement of CD44 by its natural ligands, hyaluronic acid or chondroitin sulfate, protected CLL cells from apoptosis, while anti-CD44 small interfering RNAs impaired tumor cell viability. Deletion of CD44 during TCL1-driven murine leukemogenesis reduced the tumor burden in peripheral blood and spleen and led to a prolonged overall survival. The leukemic cells from these CD44 knockout animals revealed lower levels of antiapoptotic MCL1, a higher propensity to apoptosis, and a diminished B-cell receptor kinase response. The inhibitory anti-CD44 antibodies IM7 and A3D8 impaired the viability of CLL cells in suspension cultures, in stroma contact models, and in vivo via MCL1 reduction and by effector caspase activation. Taken together, CD44 expression in CLL is mediated by the tumor microenvironment. As a coreceptor, CD44 promotes leukemogenesis by regulating stimuli of MCL1 expression. Moreover, CD44 can be addressed therapeutically in CLL by specific antibodies.

Cell-cycle regulator E2F1 and microRNA-223 comprise an autoregulatory negative feedback loop in acute myeloid leukemia.

Transcription factor CCAAT enhancer binding protein alpha (C/EBPalpha) is essential for granulopoiesis and its function is deregulated in leukemia. Inhibition of E2F1, the master regulator of cell-cycle progression, by C/EBPalpha is pivotal for granulopoiesis. Recent studies show microRNA-223 (miR-223), a transcriptional target of C/EBPalpha, as a critical player during granulopoiesis. In this report, we demonstrate that during granulopoiesis microRNA-223 targets E2F1. E2F1 protein was up-regulated in miR-223 null mice. We show that miR-223 blocks cell-cycle progression in myeloid cells. miR-223 is down-regulated in different subtypes of acute myeloid leukemia (AML). We further show that E2F1 binds to the miR-223 promoter in AML blast cells and inhibits miR-223 transcription, suggesting that E2F1 is a transcriptional repressor of the miR-223 gene in AML. Our study supports a molecular network involving miR-223, C/EBPalpha, and E2F1 as major components of the granulocyte differentiation program, which is deregulated in AML.

Elevated c-Jun expression in acute myeloid leukemias inhibits C/EBPalpha DNA binding via leucine zipper domain interaction.

Transcription factor C/EBPalpha induces normal myeloid differentiation, inactivation of C/EBPalpha leads to a differentiation block in acute myeloid leukemias (AML), and overexpression of C/EBPalpha results in AML growth arrest and differentiation. Recent reports suggest that C/EBPalpha is activated or inactivated via protein-protein interactions. We previously reported that C/EBPalpha needs to inactivate the proto-oncogene c-Jun via leucine zipper domain interaction in order to induce granulocytic differentiation. We, therefore, hypothesized that c-Jun expression might be elevated in AML and subsequently inactivate C/EBPalpha. In fact, compared to normal bone marrow mononuclear cells, c-Jun expression is increased in AML patient samples (Affymetrix expression microarray analysis, n=166). c-Jun binds to C/EBPalpha via the leucine zipper domains and prevents C/EBPalpha from DNA binding. Inactivation of C/EBPalpha by c-Jun is necessary for c-Jun to induce proliferation because c-Jun-induced proliferation can be prevented by ectopic overexpression of C/EBPalpha. The dominant-negative 30-kDa C/EBPalpha protein, found in AML, fails to downregulate c-Jun mRNA expression in AML patient samples. Thus, our data suggest a model for AML in which c-Jun promotes proliferation and prevents differentiation by inhibiting C/EBPalpha DNA binding via leucine zipper domain interaction. It might depend on the expression levels of C/EBPalpha and c-Jun, if inhibition of C/EBPalpha by c-Jun or if inhibition of c-Jun by C/EBPalpha is more predominant: proliferation versus differentiation; AML versus normal myeloid development.

Advancing mass spectrometry-based clinical proteomics in Saudi Arabia. Establishing a Saudi Proteomics Society.

Rapid developments in the field of mass spectrometry-based quantitative proteomic technologies holds great promise in the search for clinically useful protein biomarkers for early detection, diagnosis, and prognosis of disease in general, and for monitoring response to therapy. Proteomics may contribute to finding novel drug targets and unravel molecular pathways associated with disease. Despite the application of clinical proteomics to a wide spectrum of disease research globally, however, there is lack of data with regard to quantitative proteomics in the Kingdom of Saudi Arabia (KSA) for the diseases common to the Saudi population. In this review therefore, we will discuss some aspects of clinical proteomics with regard to the occurrence of common diseases, and outline our perspectives and vision in the context of KSA. Furthermore, we aim to introduce the concept of a national Saudi Proteomics Society in the Kingdom as part of an advanced research and development program.

Targeting the disturbed redox equilibrium in chronic lymphocytic leukemia by novel reactive oxygen species-catalytic 'sensor/effector' compounds.

Precursor transformation, clonal sustenance, and therapeutic resistance in cancer are significantly mediated by deregulated reactive oxygen species (ROS), which primarily act as DNA-stressors. Here, we demonstrate that elevated ROS in chronic lymphocytic leukemia (CLL) may represent a promising therapeutic target. We designed organochalcogens, which, based on a 'sensor/effector' principle, would confer selective cytotoxicity through the generation of intolerably high ROS levels preferentially in CLL cells, as these carry a high-level redox burden. Our novel compounds show an encouraging profile of efficient induction of apoptosis, low normal cell toxicity, and promising chemotherapy synergism. These findings warrant further mechanistic and preclinical studies of this therapeutic principle in CLL.

Target proteins of C/EBPalphap30 in AML: C/EBPalphap30 enhances sumoylation of C/EBPalphap42 via up-regulation of Ubc9.

CCAAT/enhancer-binding protein alpha (C/EBPalpha) is a critical regulator for early myeloid differentiation. Mutations in C/EBPalpha occur in 10% of patients with acute myeloid leukemia (AML), leading to the expression of a 30-kDa dominant-negative isoform (C/EBPalphap30). In the present study, using a global proteomics approach to identify the target proteins of C/EBPalphap30, we show that Ubc9, an E2-conjugating enzyme essential for sumoylation, is increased in its expression when C/EBPalphap30 is induced. We confirmed the increased expression of Ubc9 in patients with AML with C/EBPalphap30 mutations compared with other subtypes. We further confirmed that the increase of Ubc9 expression was mediated through increased transcription. Furthermore, we show that Ubc9-mediated enhanced sumoylation of C/EBPalphap42 decreases the transactivation capacity on a minimal C/EBPalpha promoter. Importantly, overexpression of C/EBPalphap30 in granulocyte colony-stimulating factor (G-CSF)-stimulated human CD34(+) cells leads to a differentiation block, which was overcome by the siRNA-mediated silencing of Ubc9. In summary, our data indicate that Ubc9 is an important C/EBPalphap30 target through which C/EBPalphap30 enhances the sumoylation of C/EBPalphap42 to inhibit granulocytic differentiation.