Synonymous Polymorphisms at Splicing Regulatory Sites are Associated with CpGs in Neurodegenerative Disease-Related Genes

Neuronal plasticity is associated with alternative splicing and epigenetic modulation. Recent evidence reveals the association of cytosine methylation with alternative splicing and splicing regulatory mechanisms. Single nucleotide polymorphisms (SNPs) are generally less frequent in conserved coding regions and probably in splice sites, compared to non-coding regions. CpG polymorphisms in coding regions and splice sites and their association with splicing regulatory elements have not been investigated till presently. We currently analyzed the CpG variability in 28 genes (361 constitutive and 105 alternative exons and the corresponding splice sites) associated with neurodegenerative diseases (ND). CpG polymorphisms in the splice sites of these genes are particularly frequent when compared to those at AG sequences. Moreover, in both constitutive and alternative exons, polymorphisms in CpGs are more frequent than in AG, GT sequences. On the contrary, in the polypyrimidine acceptor sequence C/T conservation is prominent indicating that in this locus the sequence of cytosines and thymines is preserved. Bioinformatic analysis of the splicing-associated regulatory elements in these exons and splice sites reveals that 18 out of a total of 39 SNPs which could strongly affect splicing (>1.5 score difference) contain CpG sequences. Cytosines are considerably more frequent and variable than expected at the position preceding the GT splice donors, while sites of epigenetic modification are absent from acceptors. The high CpG frequency in polymorphic splicing-associated sites implicates the involvement of epigenetic mechanisms in splicing selection decisions regulated by these sites, and indicates the complexity of genetic studies involving these, tentatively critical, polymorphisms in ND.     

G:C > A:T mutations and potential epigenetic regulation of <Emphasis Type="Italic">p53</Emphasis> in breast cancer

Analysis of germline p53 mutations in breast cancer reveals that the Li-Fraumeni and Li-Fraumeni-like syndromes are mostly related to the loss of initiation codon 133 of regulatory TP53 isoforms (Delta133p53). In eight codons of exons 5-8 (including 133), mutations are frequent in Li-Fraumeni-related, but scarce in sporadic breast cancer, while in six more codons they are frequent both in familial and sporadic breast cancers. At the proximity of these codons, we observed in somatic mutation databases, 16 codons (minihotspots mostly in exons 7, 8) which undergo frequent G:C > A:T transitions (non-CpG) in all sporadic cancers. In addition, in sporadic breast cancer we observed 35 adjacent codons in which the following types of mutation are observed: frequent G:C > A:T transitions at CCs/GGs, frequent silent mutations in exons 5,6 and suppressed nonsense mutations (5 codons, few records). Non-CpG G:C > A:T transitions in the 35 codons are rare in familial cancers (p53, BRCA1, or BRCA2-related), but frequent in sporadic cancers in organs where Li-Fraumeni-related carcinogenesis is common e.g. adrenal cortex, soft tissues. These data are in support of the following tissue-specific processes: in sporadic breast cancer (sarcomas etc.), loss of methylation sites (in 35 codons mostly next to codon 133), might lead to loss of silencing of TP53 isoforms which are suppressed in these tissues. On the contrary, "spreading" of cytosine methylation (asymmetric) in a G:C-rich region next to common hotspots (codons 238-252 in minihotspots) and mutagenesis probably destabilizes all tissues. Frequent C > T activation at non-CpG is also observed in prostate sporadic cancer, which similarly to breast, undergoes age-related crisis. The above data reveal that tissue-specific epigenetic regulatory mechanisms might be involved in p53 instability.     

Integrated epigenomic and transcriptomic analysis reveals TP63 as a novel player in clinically aggressive chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) stereotyped subsets #6 and #8 include cases expressing unmutated B cell receptor immunoglobulin (BcR IG) (U-CLL). Subset #6 (IGHV1-69/IGKV3-20) is less aggressive compared to subset #8 (IGHV4-39/IGKV1(D)-39) which has the highest risk for Richter's transformation among all CLL. The underlying reasons for this divergent clinical behavior are not fully elucidated. To gain insight into this issue, here we focused on epigenomic signatures and their links with gene expression, particularly investigating genome-wide DNA methylation profiles in subsets #6 and #8 as well as other U-CLL cases not expressing stereotyped BcR IG. We found that subset #8 showed a distinctive DNA methylation profile compared to all other U-CLL cases, including subset #6. Integrated analysis of DNA methylation and gene expression revealed significant correlation for several genes, particularly highlighting a relevant role for the TP63 gene which was hypomethylated and overexpressed in subset #8. This observation was validated by quantitative PCR, which also revealed TP63 mRNA overexpression in additional nonsubset U-CLL cases. BcR stimulation had distinct effects on p63 protein expression, particularly leading to induction in subset #8, accompanied by increased CLL cell survival. This pro-survival effect was also supported by siRNA-mediated downregulation of p63 expression resulting in increased apoptosis. In conclusion, we report that DNA methylation profiles may vary even among CLL patients with similar somatic hypermutation status, supporting a compartmentalized approach to dissecting CLL biology. Furthermore, we highlight p63 as a novel prosurvival factor in CLL, thus identifying another piece of the complex puzzle of clinical aggressiveness.     

Li–Fraumeni and Li–Fraumeni‐like syndrome mutations in p53 are associated with exonic methylation and splicing regulatory elements

Mutations in codon 133 of p53, which cause the loss of the Δ133 isoform(s) expression, are very frequent in the Li–Fraumeni (LF) and Li–Fraumeni‐like (LFL) syndromes. In sporadic cancers, silent p53 mutations are correlated with exonic splicing enhancers (ESEs) and exonic methylated sites. The present study shows that mutations in splice sites are also very frequent in LF/LFL syndromes, while missense mutations are less common compared to other familial or sporadic cancers (P = 0 in both cases). Furthermore, it is shown that the codons at which LF/LFL germline missense mutations occur, correlate with CpG‐containing ESEs (r = 0.181, P = 0.014) which are all methylated in p53. While both silent and LF/LFL missense mutations correlate with SC35 motifs, only the latter are associated with SRp55. On the contrary, only silent mutations in sporadic cancers correlate with SF2/ASF motifs in p53. Moreover, 12.1% of LF/LFL missense mutations involve the formation of potential splice sites of considerable splicing scores. Finally, mutations that are not at, or adjacent to CpGs (±1 codon, 34% of all LF/LFL mutated sites), introduce considerable changes of the ESE scores (>1.3 score change). The above data verify that LF/LFL missense mutations probably result also in splicing deregulation, in addition to any changes of the protein function and are mostly associated with alterations of the exonic methylation landscape. Some of the ESEs affected in LF/LFL syndromes are also genetically unstable in sporadic cancers but non‐CpG cytosine instability, which is predominantly associated with specific ESEs, is only common in sporadic cancers. Mol. Carcinog. © 2009 Wiley‐Liss, Inc.     

SpliceIT: A hybrid method for splice signal identification based on probabilistic and biological inference

Splice sites define the boundaries of exonic regions and dictate protein synthesis and function. The splicing mechanism involves complex interactions among positional and compositional features of different lengths. Computational modeling of the underlying constructive information is especially challenging, in order to decipher splicing-inducing elements and alternative splicing factors. SpliceIT (Splice Identification Technique) introduces a hybrid method for splice site prediction that couples probabilistic modeling with discriminative computational or experimental features inferred from published studies in two subsequent classification steps. The first step is undertaken by a Gaussian support vector machine (SVM) trained on the probabilistic profile that is extracted using two alternative position-dependent feature selection methods. In the second step, the extracted predictions are combined with known species-specific regulatory elements, in order to induce a tree-based modeling. The performance evaluation on human and Arabidopsis thaliana splice site datasets shows that SpliceIT is highly accurate compared to current state-of-the-art predictors in terms of the maximum sensitivity, specificity tradeoff without compromising space complexity and in a time-effective way. The source code and supplementary material are available at: http://www.med.auth.gr/research/spliceit/.     

Nursing personnel in inpatient rehabilitation--work satisfaction,motivation for interdisciplinary cooperation and for achieving psychosocial competence

106 nurses from eleven rehabilitation clinics were questioned by means of an anonymous clinical questionnaire on the issues of job satisfaction, interdisciplinary cooperation, and expectations towards future psychosocial staff training (main fields of the participants: orthopaedics, cardiology, dermatology and neurology). Two thirds were altogether satisfied with their jobs, primarily with regard to their relationships with the patients and the colleague nurses (76%), to some extent less so, however, with the doctors (63%). More than 90% expected psychosocial service to deliver feedback for the team and close interdisciplinary communication. Main expectations of psychosocial staff focused on the improvement of communication skills with strained patients, on improved psychological and especially psychodiagnostic knowledge as well as more functional coping with conflicts with patients and team-members (60% expressed marked expectations with regard to each issue). More than half of the staff indicated that they would prefer regular training sessions of one to two hours' duration, and more than 20% favoured each one-day- or weekend-workshops. The most popular ways of working in staff training were "discussion of team conflicts", "exchange of experience", "seeking emotional release", "discussing cases" and "training of communication skills" (approx. 60% each). Nearly 90% found "coping with aggressive patients" as well as "depression and suicide" to be the most important issues, followed by "ways of self-protection", "dealing with anxious patients", "coping with illness", "improvement of team-atmosphere" and "coping with death and dying". The findings of the study recommend an integration of psychosocial issues into education--and training--curricula for the various professions in rehabilitation.     

Staphylococcus aureus Isolated from Ruminants with Mastitis in Northern Greece Dairy Herds: Genetic Relatedness and Phenotypic and Genotypic Characterization

Staphylococcus aureus is the most common mastitis-related pathogen in dairy cattle, goats, and sheep worldwide. However, the population structure and genomic characteristics of mastitis-associated S. aureus in small ruminants are limited. Furthermore, the genotypic and phenotypic characteristics involved in the pathogenicity of S. aureus have been thoroughly defined, yet their association with the severity of mastitis is not fully established. Here, we performed genotyping by pulsed-field gel electrophoresis (PFGE) and spa analyses to assess the genetic diversity and relatedness of 162 S. aureus strains recovered from clinical mastitis (CM) and subclinical mastitis (SCM) cases from goats, sheep, and bovines. PFGE analysis revealed 108 distinguishable pulsotypes and 3 main clusters that comprised isolates from the three host species, while according to spa typing, 32 different spa types were identified. Genotypic analysis revealed a spreading of genetically related or indistinguishable S. aureus strains among ovine, caprine, and bovine farms of distant geographical regions. In total, 28 different staphylococcal enterotoxin (SE) gene profiles were observed, revealing a diverse range of SE genes among isolates. By evaluating the antimicrobial resistance, we found low phenotypic antimicrobial resistance among all ruminant isolates. We also performed multiple correspondence analysis, which indicated that the presence of the sec gene, biofilm production, and high autoaggregation ability are associated with CM cases.