Refinement of variant selection for the LDL cholesterol genetic risk score in the diagnosis of the polygenic form of clinical familial hypercholesterolemia and replication in samples from 6 countries.

BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused by mutations in 1 of 3 genes. In the 60% of patients who are mutation negative, we have recently shown that the clinical phenotype can be associated with an accumulation of common small-effect LDL cholesterol (LDL-C)-raising alleles by use of a 12-single nucleotide polymorphism (12-SNP) score. The aims of the study were to improve the selection of SNPs and replicate the results in additional samples. METHODS: We used ROC curves to determine the optimum number of LDL-C SNPs. For replication analysis, we genotyped patients with a clinical diagnosis of FH from 6 countries for 6 LDL-C-associated alleles. We compared the weighted SNP score among patients with no confirmed mutation (FH/M-), those with a mutation (FH/M+), and controls from a UK population sample (WHII). RESULTS: Increasing the number of SNPs to 33 did not improve the ability of the score to discriminate between FH/M- and controls, whereas sequential removal of SNPs with smaller effects/lower frequency showed that a weighted score of 6 SNPs performed as well as the 12-SNP score. Metaanalysis of the weighted 6-SNP score, on the basis of polymorphisms in CELSR2 (cadherin, EGF LAG 7-pass G-type receptor 2), APOB (apolipoprotein B), ABCG5/8 [ATP-binding cassette, sub-family G (WHITE), member 5/8], LDLR (low density lipoprotein receptor), and APOE (apolipoprotein E) loci, in the independent FH/M- cohorts showed a consistently higher score in comparison to the WHII population (P < 2.2 × 10(-16)). Modeling in individuals with a 6-SNP score in the top three-fourths of the score distribution indicated a >95% likelihood of a polygenic explanation of their increased LDL-C. CONCLUSIONS: A 6-SNP LDL-C score consistently distinguishes FH/M- patients from healthy individuals. The hypercholesterolemia in 88% of mutation-negative patients is likely to have a polygenic basis.

Prohibitin 2 represents a novel nuclear AKT substrate during all-trans retinoic acid-induced differentiation of acute promyelocytic leukemia cells.

The AKT/PKB kinase is essential for cell survival, proliferation, and differentiation; however, aberrant AKT activation leads to the aggressiveness and drug resistance of many human neoplasias. In the human acute promyelocytic leukemia cell line NB4, nuclear AKT activity increases during all-trans retinoic acid (ATRA)-mediated differentiation. As nuclear AKT activity is associated with differentiation, we sought to identify the nuclear substrates of AKT that were phosphorylated after ATRA treatment. A proteomics-based search for nuclear substrates of AKT in ATRA-treated NB4 cells was undertaken by using 2D-electrophoresis/mass spectrometry (MS) in combination with an anti-AKT phospho-substrate antibody. Western blot analysis, an in vitro kinase assay, and/or site-directed mutagenesis were performed to further characterize the MS findings. MS analysis revealed prohibitin (PHB)-2, a multifunctional protein involved in cell cycle progression and the suppression of oxidative stress, to be a putative nuclear substrate of AKT. Follow-up studies confirmed that AKT phosphorylates PHB2 on Ser-91 and that forced expression of the PHB2(S91A) mutant results in a rapid loss of viability and apoptotic cell death. Activation of nuclear AKT during ATRA-mediated differentiation results in the phosphorylation of several proteins, including PHB2, which may serve to coordinate nuclear-mitochondrial events during differentiation.

Association of USF1 and APOA5 polymorphisms with familial combined hyperlipidemia in an Italian population.

BACKGROUND: Familial combined hyperlipidemia (FCH) is a polygenic and multifactorial disease characterized by a variable phenotype showing increased levels of triglycerides and/or cholesterol. The aim of this study was to identify single nucleotides (SNPs) in lipid-related genes associated with FCH. METHODS AND RESULTS: Twenty SNPs in lipid-related genes were studied in 142 control subjects and 165 FCH patients after excluding patients with mutations in the LDLR gene and patients with the E2/E2 genotype of APOE. In particular, we studied the 9996G > A (rs2073658) and 11235C > T (rs3737787) variants in the Upstream Stimulatory Factor 1 gene (USF1), and the -1131T > C (rs662799) and S19W (rs3135506) variants in the Apolipoprotein A-V gene (APOA5). We found that the frequencies of these variants differed between patients and controls and that are associated with different lipid profiles. At multivariate logistic regression SNP S19W in APOA5 remained significantly associated with FCH independently of age, sex, BMI, cholesterol and triglycerides. CONCLUSIONS: Our results show that the USF1 and APOA5 polymorphisms are associated with FCH and that the S19W SNP in the APOA5 gene is associated to the disease independently of total cholesterol, triglycerides and BMI. However, more extensive studies including other SNPs such as rs2516839 in USF1, are required.

Phosphoinositide-specific phospholipase C ß 1b (PI-PLCß1b) interactome: affinity purification-mass spectrometry analysis of PI-PLCß1b with nuclear protein.

Two isoforms of inositide-dependent phospholipase C ß1 (PI-PLCß1) are generated by alternative splicing (PLCß1a and PLCß1b). Both isoforms are present within the nucleus, but in contrast to PLCß1a, the vast majority of PLCß1b is nuclear. In mouse erythroid leukemia cells, PI-PLCß1 is involved in the regulation of cell division and the balance between cell proliferation and differentiation. It has been demonstrated that nuclear localization is crucial for the enzymatic function of PI-PLCß1, although the mechanism by which this nuclear import occurs has never been fully characterized. The aim of this study was to characterize both the mechanism of nuclear localization and the molecular function of nuclear PI-PLCß1 by identifying its interactome in Friend's erythroleukemia isolated nuclei, utilizing a procedure that coupled immuno-affinity purification with tandem mass spectrometry analysis. Using this procedure, 160 proteins were demonstrated to be in association with PI-PLCß1b, some of which have been previously characterized, such as the splicing factor SRp20 (Srsf3) and Lamin B (Lmnb1). Co-immunoprecipitation analysis of selected proteins confirmed the data obtained via mass spectrometry. Of particular interest was the identification of the nuclear import proteins Kpna2, Kpna4, Kpnb1, Ran, and Rangap1, as well as factors involved in hematological malignancies and several anti-apoptotic proteins. These data give new insight into possible mechanisms of nuclear trafficking and functioning of this critical signaling molecule.

Identification of the PKR nuclear interactome reveals roles in ribosome biogenesis, mRNA processing and cell division.

The double-strand RNA-dependent protein kinase, PKR, plays a central role in inflammatory/chronic stress-mediated pathologies such as cancer, diabetes, and neuro/muscular degenerative diseases. Although a significant amount of research has been conducted to elucidate the role of PKR signaling in the cytosol, only recently has attention been paid to the role of PKR in the nuclear compartment. Previously our group reported that phosphorylated forms of PKR are present in the nucleus of acute leukemic cell lines, representing a reservoir of active kinase that responds to stress. Using the CCRF-CEM acute T-cell leukemia cell line, a PKR-specific inhibitor, co-immunoprecipitation and a proteomics approach, which included affinity purified mass spectrometry analysis (AP/MS), we identified the proteins present in active and inactive PKR nuclear complexes. Of the proteins identified in the PKR complexes, sixty-nine (69) were specific to the active complex, while thirty-eight (38) were specific to the inactive complex. An additional thirteen (13) proteins associated specifically with both complexes. The majority of the proteins identified are involved in, ribosome biogenesis, RNA splicing, mRNA stability, gene expression, cell cycle, or chromatin organization, including several with known significance to normal hematopoiesis and/or hematological disease. In agreement with the AP/MS data, basal- or over-expression of PKR under normal growth conditions favored cell proliferation in the tested cell lines, whereas pharmacological inhibition of PKR or shRNA-mediated knock-down did not. PKR was also found to influence the isoform and the level of expression of the proto-oncogene MYC.

A role for PLCß1 in myotonic dystrophies type 1 and 2.

Phosphoinositide-phospholipase C ß1 (PLCß1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation. We previously demonstrated that nuclear PLCß1 activates the cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLCß1 is essential for cyclin D3 promoter activation and gene transcription, through c-jun/AP1. Myotonic dystrophy (DM) is the most prevalent form of muscular dystrophy in adults. DM type 1 (DM1) and type 2 (DM2) are dominantly inherited multisystem disorders. DM1 is triggered by the pathological expansion of a (CTG)(n) triplet repeat in the gene coding for DMPK, the dystrophia myotonica-protein kinase, whereas a (CCTG)(n) tetranucleotide repeat expansion in the ZNF9 gene, encoding a CCHC-type zinc finger protein, causes DM2. We found that, unlike in normal myotubes, the level of expression of PLCß1 in DM1 and DM2 cells was already elevated in proliferating cells. Treatment with insulin induced a dramatic decrease in the amount of PLCß1. During differentiation, cyclin D3 and myogenin were elevated in normal myotubes, whereas differentiating DM1 and DM2 cells did not increase these proteins. Forced expression of PLCß1 in DM1 and DM2 cells increased the expression of differentiation markers, myogenin and cyclin D3, and enhanced fusion of DM myoblasts. These results highlight again that PLCß1 expression is a key player in myoblast differentiation, functioning as a positive regulator in the correction of delayed differentiation of skeletal muscle in DM human myoblasts.

Are overweight and obese patients who receive autologous free-flap breast reconstruction satisfied with their postoperative outcome? A single-centre study.

INTRODUCTION: Obese and overweight patients represent a challenging surgical group for autologous free-flap breast reconstruction. There is a paucity of information regarding post-operative patient-reported satisfaction in this increasingly prevalent cohort. This retrospective study aimed to determine using BREAST-Q patient-reported satisfaction amongst normal, overweight and obese patients receiving autologous free-flap breast reconstruction. METHODS: BREAST-Q (post-reconstruction) module was sent to 174 autologous breast free flap reconstruction patients between 2001 and 2012. Clinical data were collated for patients who returned questionnaires. Post-operative complications and satisfaction scores were compared between normal versus overweight and obese patients. RESULTS: A total of 101 patients (normal body mass index (BMI) = 27; overweight BMI = 48 and obese BMI = 25) completed BREAST-Q (response rate 66%). Obese and overweight patients are significantly more likely to develop major (44.8% and 31.1% vs. 29.6%) and minor (34.4% and 20% vs. 7.4%) complications in comparison to normal BMI patients (p < 0.02). Overweight and obese patients demonstrated similar levels of satisfaction compared with normal patients with the shape of their reconstructed breasts (73.2 and 72.1 vs. 71.2; p > 0.05) and overall surgical outcome (75.8 and 78.9 vs. 75.4; p > 0.05). CONCLUSIONS: Patient post-operative satisfaction is gaining increasing relevance in assessing the outcomes from autologous breast reconstruction. Overweight and obese women benefit from autologous breast reconstruction, despite being at increased risk of post-operative complications, and eventually achieve comparable levels of post-operative satisfaction. This should be reflected in pre-operative counselling.