A comparative mRNA- and miRNA transcriptomics reveals novel molecular signatures associated with metastatic prostate cancers.

Background: Prostate cancer (PC) is a fatally aggressive urogenital cancer killing millions of men, globally. Thus, this study aims to identify key miRNAs, target genes, and drug targets associated with prostate cancer metastasis. Methods: The miRNA and mRNA expression datasets of 148 prostate tissue biopsies (39 tumours and 109 normal tissues), were analysed by differential gene expression analysis, protein interactome mapping, biological pathway analysis, miRNA-mRNA networking, drug target analysis, and survival curve analysis. Results: The dysregulated expression of 53 miRNAs and their 250 target genes involved in Hedgehog, ErbB, and cAMP signalling pathways connected to cell growth, migration, and proliferation of prostate cancer cells was detected. The subsequent miRNA-mRNA network and expression status analysis have helped us in narrowing down their number to 3 hub miRNAs (hsa-miR-455-3p, hsa-miR-548c-3p, and hsa-miR-582-5p) and 9 hub genes (NFIB, DICER1, GSK3B, DCAF7, FGFR1OP, ABHD2, NACC2, NR3C1, and FGF2). Further investigations with different systems biology methods have prioritized NR3C1, ABHD2, and GSK3B as potential genes involved in prostate cancer metastasis owing to their high mutation load and expression status. Interestingly, down regulation of NR3C1 seems to improve the prostate cancer patient survival rate beyond 150 months. The NR3C1, ABHD2, and GSK3B genes are predicted to be targeted by hsa-miR-582-5p, besides some antibodies, PROTACs and inhibitory molecules. Conclusion: This study identified key miRNAs (miR-548c-3p and miR-582-5p) and target genes (NR3C1, ABHD2, and GSK3B) as potential biomarkers for metastatic prostate cancers from large-scale gene expression data using systems biology approaches.

The association between different blood group systems and susceptibility to COVID-19: a single center cross-sectional study from Saudi Arabia.

Background: Since the beginning of COVID-19 pandemic, many associated factors have been investigated to clarify the susceptibility and severity among the affected individuals. Biological markers can play an important role in identification of individual susceptibility to such pandemic. Growing evidence suggest the influence of different blood group systems on susceptibility to COVID-19 virus, with a particular blood type conferring selection advantage. Objectives: The study aimed to determine the association of ABO, Rhesus (D) and P1 blood groups with COVID-19 susceptibility in Taif city, Western Saudi Arabia. Methods: ABO, D and P1 blood antigens were determined in 104 blood samples of COVID-19 patients versus 100 control samples using either automated immunohematology analyser or test tube method. Statistical differences between patients and control samples were calculated based on p-value where results of ≤ 0.05 were considered significant. Results: O+ve blood group constituted the predominant type among the studied samples. Determination of P1 antigen showed significant association where Anti-P1 was positive in 76.9% of patients compared to 61.0% of controls with a P value of 0.01 conferring the susceptibility of P1+ve patients to COVID-19. Conclusion: Although our study showed no significant association between ABO and D, and susceptibility to COVID-19, there was a significant association between P1+ve and COVID-19. P1+ve participants were 2.131 times more associated with the risk of COVID-19 infection than those with Anti P1-ve. Thus, P1 antigen can be used as a biological marker for identification of individuals susceptibility to COVID-19. It is strongly advised that such individuals should consider extra protective measures. Further studies on other contributing factors should also be considered for more scientific clarity.

Genotype-protein phenotype characterization of NOD2 and IL23R missense variants associated with inflammatory bowel disease: A paradigm from molecular modelling, dynamics, and docking simulations.

Inflammatory bowel disease (IBD) is a gastrointestinal disease with an underlying contribution of genetic, microbial, environment, immunity factors. The coding region risk markers identified by IBD genome wide association studies have not been well characterized at protein phenotype level. Therefore, this study is conducted to characterize the role of NOD2 (Arg675Trp and Gly908Arg) and IL23R (Gly149Arg and Arg381Gln) missense variants on the structural and functional features of corresponding proteins. Thus, we used different variant pathogenicity assays, molecular modelling, secondary structure, stability, molecular dynamics, and molecular docking analysis methods. Our findings suggest that SIFT, Polyphen, GREP++, PhyloP, SiPhy and REVEL methods are very sensitive in determining pathogenicity of NOD2 and IL23R missense variants. We have also noticed that all the tested missense variants could potentially alter secondary (α-helices, ß-strands, and coils) and tertiary (residue level deviations) structural features. Moreover, our molecular dynamics (MD) simulation findings have simulated that NOD2 (Arg675Trp and Gly908Arg) and IL23R (Gly149Arg and Arg381Gln) variants creates rigid local structures comprising the protein flexibility and conformations. These predictions are corroborated by molecular docking results, where we noticed that NOD2 and IL23R missense variants induce molecular interaction deformities with RIPK2 and JAK2 ligand molecules, respectively. These functional alterations could potentially alter the signal transduction pathway cascade involved in inflammation and autoimmunity. Drug library searches and findings from docking studies have identified the inhibitory effects of Tacrolimus and Celecoxib drugs on NOD2 and IL23R variant forms, underlining their potential to contribute to personalized medicine for IBD. The present study supports the utilization of computational methods as primary filters (pre-in vitro and in vivo) in studying the disease potential mutations in the context of genptype-protein phenotype characteristics.

Computational approaches for discovering significant microRNAs, microRNA-mRNA regulatory pathways, and therapeutic protein targets in endometrial cancer.

Endometrial cancer (EC) is a urogenital cancer affecting millions of post-menopausal women, globally. This study aims to identify key miRNAs, target genes, and drug targets associated with EC metastasis. The global miRNA and mRNA expression datasets of endometrial tissue biopsies (24 tumors +3 healthy tissues for mRNA and 18 tumor +4 healthy tissues for miRNAs), were extensively analyzed by mapping of DEGs, DEMi, biological pathway enrichment, miRNA-mRNA networking, drug target identification, and survival curve output for differentially expressed genes. Our results reveal the dysregulated expression of 26 miRNAs and their 66 target genes involved in focal adhesions, p53 signaling pathway, ECM-receptor interaction, Hedgehog signaling pathway, fat digestion and absorption, glioma as well as retinol metabolism involved in cell growth, migration, and proliferation of endometrial cancer cells. The subsequent miRNA-mRNA network and expression status analysis have narrowed down to 2 hub miRNAs (hsa-mir-200a, hsa-mir-429) and 6 hub genes (PTCH1, FOSB, PDGFRA, CCND2, ABL1, ALDH1A1). Further investigations with different systems biology methods have prioritized ALDH1A1, ABL1 and CCND2 as potential genes involved in endometrial cancer metastasis owing to their high mutation load and expression status. Interestingly, overexpression of PTCH1, ABL1 and FOSB genes are reported to be associated with a low survival rate among cancer patients. The upregulated hsa-mir-200a-b is associated with the decreased expression of the PTCH1, CCND2, PDGFRA, FOSB and ABL1 genes in endometrial cancer tissue while hsa-mir-429 is correlated with the decreased expression of the ALDH1A1 gene, besides some antibodies, PROTACs and inhibitory molecules. In conclusion, this study identified key miRNAs (hsa-mir-200a, hsa-mir-429) and target genes ALDH1A1, ABL1 and CCND2 as potential biomarkers for metastatic endometrial cancers from large-scale gene expression data using systems biology approaches.

Investigation of Isocitrate Dehydrogenase 1 and 2 Mutations in Acute Leukemia Patients in Saudi Arabia.

Different forms of human cancer show mutations for isocitrate dehydrogenases 1 and 2 (IDH1/2). Mutation of these genes can cause aberrant methylation of the genome CpG islands (CGIs), which leads to an increase of suppressed oncogenes transcription or repression of active tumor suppressor gene transcription. This study aimed to identify the prevalence of IDH1/2 mutations in acute leukemia patients. The study cohort included 43 AML patients and 30 childhood ALL patients, from whom DNA bone marrow samples were taken. The alteration hotspots in codons IDH1 (R132) and IDH2 (R172 and R140) were examined via direct sequencing. Mutations in IDH1 were detected in 7 out of 43 (16.2%) AML patients; 5 of them occurred at codon R132. The other two mutations included a single-nucleotide polymorphism, which affected codon G105 in one patient. However, no mutation was detected in the IDH2 in any of the patients. Moreover, no mutations were detected in either IDH1 or IDH2 in ALL patients. The dominance of IDH1 mutations in AML, which was 16%, emphasizes the existence of the mutation in our population. On the other hand, IDH2 mutation was observed to be less frequent in both illnesses. Due to the limitation of using a small sample size, larger cohort screening is recommended to determine their usefulness as prognostic indicators.

Novel MYO1D Missense Variant Identified Through Whole Exome Sequencing and Computational Biology Analysis Expands the Spectrum of Causal Genes of Laterality Defects.

Laterality defects (LDs) or asymmetrically positioned organs are a group of rare developmental disorders caused by environmental and/or genetic factors. However, the exact molecular pathophysiology of LD is not yet fully characterised. In this context, studying Arab population presents an ideal opportunity to discover the novel molecular basis of diseases owing to the high rate of consanguinity and genetic disorders. Therefore, in the present study, we studied the molecular basis of LD in Arab patients, using next-generation sequencing method. We discovered an extremely rare novel missense variant in MYO1D gene (Pro765Ser) presenting with visceral heterotaxy and left isomerism with polysplenia syndrome. The proband in this index family has inherited this homozygous variant from her heterozygous parents following the autosomal recessive pattern. This is the first report to show MYO1D genetic variant causing left-right axis defects in humans, besides previous known evidence from zebrafish, frog and Drosophila models. Moreover, our multilevel bioinformatics-based structural (protein variant structural modelling, divergence, and stability) analysis has suggested that Ser765 causes minor structural drifts and stability changes, potentially affecting the biophysical and functional properties of MYO1D protein like calmodulin binding and microfilament motor activities. Functional bioinformatics analysis has shown that MYO1D is ubiquitously expressed across several human tissues and is reported to induce severe phenotypes in knockout mouse models. In conclusion, our findings show the expanded genetic spectrum of LD, which could potentially pave way for the novel drug target identification and development of personalised medicine for high-risk families.

Molecular differential analysis of uterine leiomyomas and leiomyosarcomas through weighted gene network and pathway tracing approaches.

Uterine smooth muscular neoplastic growths like benign leiomyomas (UL) and metastatic leiomyosarcomas (ULMS) share similar clinical symptoms, radiological and histological appearances making their clinical distinction a difficult task. Therefore, the objective of this study is to identify key genes and pathways involved in transformation of UL to ULMS through molecular differential analysis. Global gene expression profiles of 25 ULMS, 25 UL, and 29 myometrium (Myo) tissues generated on Affymetrix U133A 2.0 human genome microarrays were analyzed by deploying robust statistical, molecular interaction network, and pathway enrichment methods. The comparison of expression signals across Myo vs UL, Myo vs ULMS, and UL vs ULMS groups identified 249, 1037, and 716 significantly expressed genes, respectively (p ≤ 0.05). The analysis of 249 DEGs from Myo vs UL confirms multistage dysregulation of various key pathways in extracellular matrix, collagen, cell contact inhibition, and cytokine receptors transform normal myometrial cells to benign leiomyomas (p value ≤ 0.01). The 716 DEGs between UL vs ULMS were found to affect cell cycle, cell division related Rho GTPases and PI3K signaling pathways triggering uncontrolled growth and metastasis of tumor cells (p value ≤ 0.01). Integration of gene networking data, with additional parameters like estimation of mutation burden of tumors and cancer driver gene identification, has led to the finding of 4 hubs (JUN, VCAN, TOP2A, and COL1A1) and 8 bottleneck genes (PIK3R1, MYH11, KDR, ESR1, WT1, CCND1, EZH2, and CDKN2A), which showed a clear distinction in their distribution pattern among leiomyomas and leiomyosarcomas. This study provides vital clues for molecular distinction of UL and ULMS which could further assist in identification of specific diagnostic markers and therapeutic targets.Abbreviations UL: Uterine Leiomyomas; ULMS: Uterine Leiomyosarcoma; Myo: Myometrium; DEGs: Differential Expressed Genes; RMA: Robust Multiarray Average; DC: Degree of Centrality; BC: Betweenness of Centrality; CGC: Cancer Gene Census; FDR: False Discovery Rate; TCGA: Cancer Genome Atlas; BP: Biological Process; CC: Cellular Components; MF: Molecular Function; PPI: Protein-Protein Interaction.

Molecular profiling of lamellar ichthyosis pathogenic missense mutations on the structural and stability aspects of TGM1 protein.

Lamellar ichthyosis (LI) is a rare inherited disease where affected infants present a extensive skin scaling characterized by hyperkeratosis. Inherited mutations in the Transglutaminase 1 (TGM1) protein is one of the known causative genetic factor for the LI. The main objective of this study is to explore the impact of LI causative missense mutations on the structural and stability aspects of TGM1 protein using structural modeling, molecular docking and molecular dynamics approaches. By testing all LI causative TMG1 mutations against multiple stability prediction methods, we found that L362R and L388P mutations positioned in the Transglut_core domain were most destabilizing to the stability of TGM1 protein. These 2 mutations were 3D protein modeled and further analyzed by molecular docking and dynamic simulation methods. Molecular docking of these TGM1 mutant structures with chitosan, a natural polyphenolic compound and known inducer for transglutaminase enzyme, has shown stable molecular interactions between the native TGM1-chitosan and TGM1(L388P)-chitosan complex, when compared to the TGM1(L362R)-chitosan complex. Interestingly, molecular dynamics analysis have also yielded similar findings, where L388P-chitosan complex is shown to develop B-sheets and attain better stability, whereas TGM1-L362R complex possessed coils over the simulation period, pointing its highly destabilizing behavior on the protein structure. This study concludes that missense mutations in Transglut_core domain of the TGM1 are deleterious to the stability and structural changes of TGM1 protein and also suggest that chitosan molecule could act as a natural activator against few pathogenic TGM1 mutations. Communicated by Ramaswamy H. Sarma.

The influence of polymorphisms in the drug transporter, ABCB1 on the toxicity of glucocorticoids in Saudi children with acute lymphoblastic leukaemia.

BACKGROUND: Glucocorticoids play essential roles in the treatment of childhood acute lymphoblastic leukaemia (ALL); however, treatment with these agents can result in severe side-effects. This study, the first of its kind in a Saudi population, investigates associations of ABCB1 gene polymorphisms (pharmacodynamics and pharmacokinetic) with the development of toxicity and side effects (glucose abnormality, liver toxicity and infection) in a small population of Saudi children with ALL. METHODS: Three single nucleotide polymorphisms (SNPs) of the ABCB1 gene (rs 3213619 T129C, rs 2032582 G2677T and rs1045642 C3435T) were analysed in 70 Saudi children with ALL and 60 control subjects. Participants were treated according to the ALL 2000 study protocol. Toxicities were assessed and associations with genotypes were evaluated according to Common Toxicity Criteria (NCI-CTC). RESULTS: Significant associations were observed among carriers and the mutated genotype C3435T (ABCB1), which had an incidence of infection (p = 0.05). Although no correlations were found between liver toxicity and glucose abnormalities for patients carrying ABCB1 SNPs, risk factors for liver toxicity were elevated by a factor of three for patients carrying the SNP G2677T, OR 3.00 (1.034-8.702). The risk factor of glucose abnormality toxicity for the patients carring T129C were increased three times OR 3.06 (0.486-19.198). CONCLUSIONS: In terms of infection incidence, polymorphism C3435T may contribute to potential life-threatening infections during paediatric ALL therapy, through glucocorticoid usage.

Association Between NR3C1 Gene Polymorphisms and Toxicity Induced by Glucocorticoids Therapy in Saudi Children with Acute Lymphoblastic Leukemia

Background: Glucocorticoids (GCs) are key hormones used for the treatment of acute lymphoblastic leukemia (ALL) in children, but their cytotoxic effects are not well defined. The aim of this study was to evaluate the association between polymorphisms in NR3C1 encoding for protein involved in the GCs metabolism and its role in the development of ALL and the toxicity outcome, in terms of liver toxicity, glucose abnormality and infections, in ALL Saudi children. Methods: The following polymorphisms BCII rs41423247, ER22/23 EK rs6189 and rs6190 and N363S rs6195 in NR3C1 were analyzed in 70 children with ALL treated according to the ALL 2000 study protocol in comparison to 60 control subjects. Treatment toxicities and their association with genotypes were evaluated according to Common Toxicity Criteria (NCI-CTC). Results: This study demonstrated that the NR3C1 did not contribute to the development of childhood ALL. Homozygous ER22/23EK polymorphism was not found in both ALL patients and in control group whereas the heterozygous polymorphism was only observed in the control group (6.66%). The toxicology data in this study showed a significant difference between ALL patients carrying N363S polymorphism and wild type (40% and 6.51% respectively, P= 0.009) and a high-risk factor in the toxicity of glucose abnormality (OR=10.167; 1.302-79.339). BCII shows increased risk factors towards the liver toxicity (OR=2.667; 0.526-7.330) as well as the glucose abnormality (OR=7.5; 1.039-54.116). Conclusion: This study suggested that the polymorphisms in NR3C1 were not associated with the development of ALL in children. N363S polymorphism was sensitive to glucocorticoids and it may contribute to the glucose abnormality for these patients.

The GALNT9, BNC1 and CCDC8 genes are frequently epigenetically dysregulated in breast tumours that metastasise to the brain.

BACKGROUND: Tumour metastasis to the brain is a common and deadly development in certain cancers; 18-30 % of breast tumours metastasise to the brain. The contribution that gene silencing through epigenetic mechanisms plays in these metastatic tumours is not well understood. RESULTS: We have carried out a bioinformatic screen of genome-wide breast tumour methylation data available at The Cancer Genome Atlas (TCGA) and a broad literature review to identify candidate genes that may contribute to breast to brain metastasis (BBM). This analysis identified 82 candidates. We investigated the methylation status of these genes using Combined Bisulfite and Restriction Analysis (CoBRA) and identified 21 genes frequently methylated in BBM. We have identified three genes, GALNT9, CCDC8 and BNC1, that were frequently methylated (55, 73 and 71 %, respectively) and silenced in BBM and infrequently methylated in primary breast tumours. CCDC8 was commonly methylated in brain metastases and their associated primary tumours whereas GALNT9 and BNC1 were methylated and silenced only in brain metastases, but not in the associated primary breast tumours from individual patients. This suggests differing roles for these genes in the evolution of metastatic tumours; CCDC8 methylation occurs at an early stage of metastatic evolution whereas methylation of GANLT9 and BNC1 occurs at a later stage of tumour evolution. Knockdown of these genes by RNAi resulted in a significant increase in the migratory and invasive potential of breast cancer cell lines. CONCLUSIONS: These findings indicate that GALNT9 (an initiator of O-glycosylation), CCDC8 (a regulator of microtubule dynamics) and BNC1 (a transcription factor with a broad range of targets) may play a role in the progression of primary breast tumours to brain metastases. These genes may be useful as prognostic markers and their products may provide novel therapeutic targets.

RASSF6 exhibits promoter hypermethylation in metastatic melanoma and inhibits invasion in melanoma cells.

Brain metastasis is a major contributor to cancer mortality, yet, the genetic changes underlying the development of this capacity remain poorly understood. RASSF proteins are a family of tumor suppressors that often suffer epigenetic inactivation during tumorigenesis. However, their epigenetic status in brain metastases has not been well characterized. We have examined the promoter methylation of the classical RASSF members (RASSF1A-RASSF6) in a panel of metastatic brain tumor samples. RASSF1A and RASSF2 have been shown to undergo promoter methylation at high frequency in primary lung and breast tumors and in brain metastases. Other members exhibited little or no methylation in these tumors. In examining melanoma metastases, however, we found that RASSF6 exhibits the highest frequency of inactivation in melanoma and in melanoma brain metastases. Most melanomas are driven by an activating mutation in B-Raf. Introduction of RASSF6 into a B-Raf(V600E)-containing metastatic melanoma cell line inhibited its ability to invade through collagen and suppressed MAPK pathway activation and AKT. RASSF6 also appears to increase the association of mutant B-Raf and MST1, providing a potential mechanism by which RASSF6 is able to suppress MAPK activation. Thus, we have identified a novel potential role for RASSF6 in melanoma development. Promoter methylation leading to reduced expression of RASSF6 may play an important role in melanoma development and may contribute to brain metastases.

Stability of the CpG island methylator phenotype during glioma progression and identification of methylated loci in secondary glioblastomas.

BACKGROUND: Grade IV glioblastomas exist in two forms, primary (de novo) glioblastomas (pGBM) that arise without precursor lesions, and the less common secondary glioblastomas (sGBM) which develop from earlier lower grade lesions. Genetic heterogeneity between pGBM and sGBM has been documented as have differences in the methylation of individual genes. A hypermethylator phenotype in grade IV GBMs is now well documented however there has been little comparison between global methylation profiles of pGBM and sGBM samples or of methylation profiles between paired early and late sGBM samples. METHODS: We performed genome-wide methylation profiling of 20 matched pairs of early and late gliomas using the Infinium HumanMethylation450 BeadChips to assess methylation at >485,000 cytosine positions within the human genome. RESULTS: Clustering of our data demonstrated a frequent hypermethylator phenotype that associated with IDH1 mutation in sGBM tumors. In 80% of cases, the hypermethylator status was retained in both the early and late tumor of the same patient, indicating limited alterations to genome-wide methylation during progression and that the CIMP phenotype is an early event. Analysis of hypermethylated loci identified 218 genes frequently methylated across grade II, III and IV tumors indicating a possible role in sGBM tumorigenesis. Comparison of our sGBM data with TCGA pGBM data indicate that IDH1 mutated GBM samples have very similar hypermethylator phenotypes, however the methylation profiles of the majority of samples with WT IDH1 that do not demonstrate a hypermethylator phenotype cluster separately from sGBM samples, indicating underlying differences in methylation profiles. We also identified 180 genes that were methylated only in sGBM. Further analysis of these genes may lead to a better understanding of the pathology of sGBM vs pGBM. CONCLUSION: This is the first study to have documented genome-wide methylation changes within paired early/late astrocytic gliomas on such a large CpG probe set, revealing a number of genes that maybe relevant to secondary gliomagenesis.

DNA methylation profiles of long- and short-term glioblastoma survivors.

Glioblastoma (GBM) is the most common and malignant type of primary brain tumor in adults and prognosis of most GBM patients is poor. However, a small percentage of patients show a long term survival of 36 mo or longer after diagnosis. Epigenetic profiles can provide molecular markers for patient prognosis: recently, a G-CIMP positive phenotype associated with IDH1 mutations has been described for GBMs with good prognosis. In the present analysis we performed genome-wide DNA methylation profiling of short-term survivors (STS; overall survival < 1 y) and long-term survivors (LTS; overall survival > 3 y) by utilizing the HumanMethylation450K BeadChips to assess quantitative methylation at > 480,000 CpG sites. Cluster analysis has shown that a subset of LTS showed a G-CIMP positive phenotype that was tightly associated with IDH1 mutation status and was confirmed by analysis of the G-CIMP signature genes. Using high stringency criteria for differential hypermethylation between non-cancer brain and tumor samples, we identified 2,638 hypermethylated CpG loci (890 genes) in STS GBMs, 3,101 hypermethylated CpG loci (1,062 genes) in LTS (wild type IDH1) and 11,293 hypermethylated CpG loci in LTS (mutated for IDH1), reflecting the CIMP positive phenotype. The location of differentially hypermethylated CpG loci with respect to CpG content, neighborhood context and functional genomic distribution was similar in our sample set, with the majority of CpG loci residing in CpG islands and in gene promoters. Our preliminary study also identified a set of CpG loci differentially hypermethylated between STS and LTS cases, including members of the homeobox gene family (HOXD8, HOXD13 and HOXC4), the transcription factors NR2F2 and TFAP2A, and Dickkopf 2, a negative regulator of the wnt/ß-catenin signaling pathway.

KIBRA gene methylation is associated with unfavorable biological prognostic parameters in chronic lymphocytic leukemia.

Ras-association domain family (RASSF) members are a family of genes containing an RA domain in either the C-terminus (RASSF1-RASSF6) or in the N-terminus (RASSF7-RASSF10). Members of this gene family are core members of the Salvador/Warts/Hippo (SWH) tumor suppressor network and have been shown to be involved in human tumorigenesis. Among the RASSF genes, RASSF1A is one of the most frequently methylated genes in a wide range of epithelial cancers, and we previously demonstrated that RASSF6 and RASSF10 genes are frequently epigenetically inactivated in acute leukemias, particularly in those of the B cell type. We here determined the methylation profiles of all members of the RASSF gene family as well as two recently identified (KIBRA, CRB3) upstream members of the SWH pathway in the leukemic B cells obtained from a well-characterized cohort of 95 patients with chronic lymphocytic leukemia (CLL). Among the RASSF genes, RASSF10 (50%) was the most frequently methylated gene, followed by RASSF6 (16%). The remaining RASSF genes were either unmethylated or showed a frequency of methylation < 10%. The upstream SWH member KIBRA was also frequently methylated in CLL (35%) in contrast to CRB3. Interestingly, the analysis of clinical-pathological parameters showed that KIBRA methylation was associated with unfavorable biological prognostic parameters, including unmutated IGHV genes (p = 0.007) and high CD38 expression (p < 0.05).