Investigation of the molecular defect causing the phenotype of familial hypercholesterolemia in the greek population: genes LDL-R, APOB-100, PCSK9 and ARH

Atherosclerosis constitutes the fundamental cause of cardiovascular diseases, such as infarction, angina pectoris and vasal cerebral episodes. Hypercholesterolemia is the main cause for the 30-50% cardiovascular diseases, which constitute the main cause of death of young in most developed countries and are accountable for up to 50% of deaths. The illness begins from childhood and develops during adulthood. Many studies have shown that the risk factors that contribute to the development of atherosclerosis are distinguished in lipidaimic and not lipidaimic factors. Lipidaemic risk factors that contribute to the development of atherosclerosis are: increased total cholesterol (TC), increased levels of triglycerides (TG), low density cholesterol (HDL-C), low density lipoprotein (LDL-C) Familial hypercholesterolemia is a common disease that is presented in 1% of adults and is considered to be responsible for 10% of early events of illness. Children with inherited defects in lipid metabolism and particularly in the gene encoding the LDL receptor are characterised as heterozygotes with a frequency of 1:500. They present levels of total cholesterol TC > 200 mg/dl and develop cardiovascular diseases in the adult life. Homozygous children are presented with a frequency of 1:1.000.000. The levels of total cholesterol range from 500 to 2000 mg/dl while clinically present tendon xanthomas up to the age of 5 years, corneal arcus as well as severe damage of coronary arteries up to the age of 20 years. Aim of my work is to study of Greek patients expressing the phenotype of familial hypercholesterolemia, with positive family history. Exclusion of any mutation in the LDL-R gene will lead to the analysis of the ApoB-100 gene for the 3 most frequent mutations, R3500Q, R3500W, and R3531C In the case that the study of ApoB100 mutations is negative then the third phase of the study was performed on the new gene, PCSK9 follows. The patients with phenotypical ADH and with a positive family history and no mutations in the genes of LDL-R and ApoB-100, will constitute our inquiring material for likely mutations in PCSK9 gene, for the first time in Greece. The detection of possible mutations in this gene will be done by PCR and sequencing. Firstly, we will focus on the four most frequent mutations in the gene and afterwards, if no mutations are found, we will amplify all the exons by PCR and after sequencing of the whole gene our interest will be focused on new mutations In addition, studies have shown that from the 311 polymorphisms of PCSK9 detected up to date some are associated with high and others with low levels of lipids, a fact that can characterize these genetic indicators as cardioprotective or not. So we will study 9 polymorphisms of the PCSK9 gene. For the first time in Greece the R3500Q mutation apoB100 gene was detected in a child while mutations in PCSK9 do not constitute a fundamental cause of familial hypercholesterolemia in Greece. The identification of polymorphisms showed that indeed the A53V, rs 615563, rs 644000 are associated with levels of lipids, A53V is associated with high levels of HDLc and can be considered as cardioprotective.Η αθηρωμάτωση, αποτελεί βασική αιτία εμφάνισης καρδιαγγειακών νοσημάτων, όπως έμφραγμα, στηθάγχη και αγγειακά εγκεφαλικά επεισόδια. Η υπερχοληστερολαιμία ενοχοποιείται για το 30-50% των καρδιαγγειακών νοσημάτων, τα οποία αποτελούν την κύρια αιτία θανάτου των ενηλίκων στις περισσότερες ανεπτυγμένες χώρες και ευθύνονται για ποσοστό έως και 50% περίπου του συνόλου των θανάτων. Η νόσος αρχίζει από την παιδική ηλικία και εξελίσσεται κατά την ενήλικο ζωή. Μεγάλες μελέτες έχουν δείξει ότι οι επιβαρυντικοί παράγοντες που συμβάλλουν στη διεργασία της αθηροσκλήρωσης διακρίνονται σε λιπιδαιμικούς και μη λιπιδαιμικούς. Επιβαρυντικοί παράγοντες που συμβάλλουν στη διεργασία της αθηροσκλήρωσης: αυξημένη ολική χοληστερόλη (TC), αυξημένα τριγλυκερίδια (TG), χαμηλή υψηλής πυκνότητας χοληστερόλη (HDL-C), υψηλή χαμηλής πυκνότητας χοληστερόλη (LDL-C) Η οικογενής υπερχοληστερολαιμία είναι μία κοινή διαταραχή που συναντάται σε ποσοστό 1% των ενηλίκων και θεωρείται υπεύθυνη για το 10% των πρώιμων εκδηλώσεων της νόσου. Παιδιά με κληρονομούμενη βλάβη στον μεταβολισμό των λιπιδίων και ιδιαίτερα στο γονίδιο του LDL υποδοχέα εμφανίζονται ως ετεροζυγώτες με συχνότητα 1:500. Παρουσιάζουν τιμές ολικής χοληστερόλης (ΤC) >200 mg/dl και εκδηλώνουν καρδιαγγειακά νοσήματα στην ενήλικο ζωή. Παιδιά ομοζυγώτες εμφανίζονται με συχνότητα 1:1.000.000. Τα επίπεδα ολικής χοληστερόλης (ΤC) κυμαίνονται από 400 έως 1000 mg/dl ενώ κλινικά εμφανίζουν τενόντια ξανθώματα μέχρι την ηλικία των 5 ετών, καθώς και σοβαρές βλάβες των στεφανιαίων αρτηριών μέχρι την ηλικία των 20 ετών. Σκοπός της εργασίας είναι η μελέτη Ελλήνων ασθενών με τον φαινότυπο της οικογενούς υπερχοληστερολαιμίας και με θετικό οικογενειακό ιστορικό. Απόκλειση οποιασδήποτε μετάλλαξης του LDL-R γονιδίου θα οδηγήσει στην ανάλυση του γονιδίου της ApoB-100 για τις 3 πιο συχνές μεταλλάξεις, R3500Q, R3500W, και R3531C. Εφόσον, η παραπάνω μελέτη αποβεί αρνητική, τότε θα ακολουθήσει η τρίτη φάση της μελέτης για το νέο γονίδιο, PCSK9. Άρα οι ασθενείς με φαινότυπο ADH και με θετικό οικογενειακό ιστορικό στους οποίους δεν υπάρχει μετάλλαξη στα γονίδια του LDL-R και της ApoB-100, θα αποτελέσουν το ερευνητικό μας υλικό για πιθανή μετάλλαξη στο γονίδιο PCSK9, που επίσης θα γίνει για πρώτη φορά στην Ελλάδα. Η ανίχνευση κάποιας μετάλλαξης αυτού του γονιδίου θα γίνει με την PCR και τον άμεσο προσδιορισμό αλληλουχίας. Σε πρώτη φάση θα ανιχνευτούν οι τέσσερις πιο συχνές μεταλλάξεις του νέου γονιδίου και στη συνέχεια, αν δεν εντοπιστούν, θα πολλαπλασιάστούν όλα τα εξόνια του γονιδίου με PCR και θα εντοπιστούν κάποιες νέες μετάλλαξεις με τον άμεσο προσδιορισμό αλληλουχίας. Τέλος, θα ταυτοποιηθούν 9 πολυμορφισμοί του PCSK9 αφού μελέτες έχουν δείξει ότι από τους 311 ανιχνευθέντες μέχρι σήμερα κάποιοι συσχετίζονται με υψηλά και άλλοι με χαμηλά επίπεδα λιπιδίων, γεγονός που μπορεί να χαρακτηρίσει τους γενετικούς αυτούς δέικτες καρδιοπροστατευτικούς και μη. Για πρώτη φορά στην Ελλάδα ανιχνεύτηκε μετάλλαξη ApoB100, η R3500Q, σε παιδί ενώ οι μεταλλάξεις του PCSK9 δεν αποτελούν βασική αιτία της οικογενούς υπερχοληστερολαιμίας για την Ελλάδα. Η ταυτοποίηση των πολυμορφισμών έδειξε ότι πράγματι οι A53V, rs 615563, rs 644000 συσχετίζονται με επίπεδα λιπιδίων από τους οποίους ο A53V συσχετίζεται με υψηλά επίπεδα HDLc και μπορεί να θεωρηθεί καρδιοπροστατευτικός

A Genetic study in assisted reproduction and the risk of congenital anomalies

In vitro fertilization is one of the most common and effective procedure for thousands of couples worldwide who want to have a child and are unable to do so for various reasons. Diverse studies show that couples who conceive naturally after one year of trying had newborns with an increased risk of prematurity and low birth weight, compared with couples who conceived before completing one year of trying. Children from assisted reproduction (AR), have a 30% increased risk of prematurity and low birth weight, compared with children from infertile fathers. Regarding the conflicting results the present study aimed to record the frequency of genetic, congenital anomalies in children and adolescents who had examined in the last decade to the Clinical Genetics Clinic of the National and Kapodistrian University of Athens whose mothers had undergone assisted reproduction. The research process was conducted at the "Aghia Sofia" Children's Hospital based in Athens. However, the cases that were studied came from all over Greece. Initially, the researcher recorded the cases that came to the clinic of Clinical Genetics and whose conception occurred after technical assisted reproduction. After telephone communication and the consent of the parents, a live appointment was scheduled. In this meeting-interview all the provisions of the investigation and the protocol were asked and some elements of the medical history of the cases were confirmed. The total sample included 230 children and adolescents. The resulting data were recorded on a printed form/questionnaire. Then, they were registered electronically in the program SPSS 25.0 (Statistical Package for Social Sciences) with a specific unit code for each case/patient, followed by the processing and statistical analysis of the data as well as the recording of the results. The gender of the participants was male for 118 participants (51.3%) and 112 females (48.7%). Mean and standard deviation (SD) of maternal, paternal (at the time of delivery) age was equal to 36.38 (5.94) and 39.94 (6.58) respectively. The observed abdormalities were 35.53% psychomotor retardation, 23.68% facial abnormalities, 23.68% spinal cord abnormalities, 21.05% morphological abnormalities, 20.61% short stature, 19.74% developmental disorders, 19.30% heart disease, 16.67% neurological diseases, 14.47% genetic syndromes, 11.40% genital abnormalities, 8.33% limb abnormalities, 7.46% dermatological abnormalities, 6.14% eye abnormalities, 6.14% hypothyroidism, 5.70% endocrine disorders, 5.26%otolaryngology abnormalities, 2.63% disease of kidney, intestine, 2.19% vascular malformations. Regarding the karyotype chromosome analysis by G-banding technique, from the 230 children in: 24 (10.43%) a pathological result was found, in 158 children (68.70%) it was found normal (46, XX or 46, XY by case) without other findings, while in 48 children (20.87%) the test was not performed for various reasons. Regarding the results of molecular analysis (DNA) from the 230 children, in 50 (21.74%) a pathological finding was found, in 56 children (24.35%) no abnormalities were found and in 124 children (53.91%) no molecular analysis was performed for various reasons. In conclusion, the sample of this descriptive study is characterized as uniform in terms of the method of assisted reproduction since 96.24% had followed the classic IVF. Full-term pregnancy was associated with the appearance of malignancy and head morphological abnormalities (64.6%), normal pregnancy was associated with genetic syndromes (18.2%) and facial abnormalities (11.1%). It is recommended the screening oocyte and sperm donors in order to help protect the safety and health of donors, recipients, and future offspring. The present study confirms the association of the presence of congenital anomalies after in vitro fertilization (IVF). However, the absolute risk of developing severe dysplasias after an IVF procedure is limited

Serum Spexin is Correlated with Lipoprotein(a) and Androgens in Female Adolescents

The Spexin gene is considered the most dysregulated in obese human fat. Limited data suggest that the novel peptide spexin may potentially impact food intake, weight regulation and body adiposity. The aim of this case-control study was to compare fasting serum spexin concentrations between normal weight (NW) and overweight/obese (OB/OW) adolescent females and explore the relationship between circulating spexin and anthropometric, bone and fat mass, metabolic and hormonal parameters. Eighty post-menarcheal females (mean age ± SD 16.23 ± 2.26 years); 55 NW (mean BMI ± SD 19.72 ± 2.52 kg/m2) and 25 OB/OW (mean BMI ± SD 29.35 ± 3.89 kg/m2) participated in the study. Circulating spexin levels did not differ significantly (p = 0.378) between NW (median (interquartile range), 0.26 (0.17) ng/mL) and OB/OW (median (interquartile range), 0.28 (0.06) ng/mL) adolescents and did not correlate with BMI (rs = −0.090, p = 0.438), % body fat (rs = −0.173, p = 0.409), glucose or insulin resistance indices derived from fasting and oral glucose tolerance states. In the total study sample, spexin concentrations correlated positively with lipoprotein(a) (rs = 0.402, p = 0.046). In the OB/OW adolescents spexin levels correlated positively with testosterone (rs = 0.727, p = 0.011) and free androgen index (rs = 0.755, p = 0.007). In the NW adolescents, spexin concentrations correlated negatively with dehydroepiandrosterone sulphate (rs = −0.445, p = 0.038). Results may suggest potential involvement of spexin in the regulation of lipoprotein(a) and of the reproductive/adrenal axis in post-menarcheal adolescent females

Untargeted Plasma Metabolomics Unravels a Metabolic Signature for Tissue Sensitivity to Glucocorticoids in Healthy Subjects: Its Implications in Dietary Planning for a Healthy Lifestyle

In clinical practice, differences in glucocorticoid sensitivity among healthy subjects may influence the outcome and any adverse effects of glucocorticoid therapy. Thus, a fast and accurate methodology that could enable the classification of individuals based on their tissue glucocorticoid sensitivity would be of value. We investigated the usefulness of untargeted plasma metabolomics in identifying a panel of metabolites to distinguish glucocorticoid-resistant from glucocorticoid-sensitive healthy subjects who do not carry mutations in the human glucocorticoid receptor (NR3C1) gene. Applying a published methodology designed for the study of glucocorticoid sensitivity in healthy adults, 101 healthy subjects were ranked according to their tissue glucocorticoid sensitivity based on 8:00 a.m. serum cortisol concentrations following a very low-dose dexamethasone suppression test. Ten percent of the cohort, i.e., 11 participants, on each side of the ranking, with no NR3C1 mutations or polymorphisms, were selected, respectively, as the most glucocorticoid-sensitive and most glucocorticoid-resistant of the cohort to be analyzed and compared with untargeted blood plasma metabolomics using gas chromatography–mass spectrometry (GC–MS). The acquired metabolic profiles were evaluated using multivariate statistical analysis methods. Nineteen metabolites were identified with significantly lower abundance in the most sensitive compared to the most resistant group of the cohort, including fatty acids, sugar alcohols, and serine/threonine metabolism intermediates. These results, combined with a higher glucose, sorbitol, and lactate abundance, suggest a higher Cori cycle, polyol pathway, and inter-tissue one-carbon metabolism rate and a lower fat mobilization rate at the fasting state in the most sensitive compared to the most resistant group. In fact, this was the first study correlating tissue glucocorticoid sensitivity with serine/threonine metabolism. Overall, the observed metabolic signature in this cohort implies a worse cardiometabolic profile in the most glucocorticoid-sensitive compared to the most glucocorticoid-resistant healthy subjects. These findings offer a metabolic signature that distinguishes most glucocorticoid-sensitive from most glucocorticoid-resistant healthy subjects to be further validated in larger cohorts. Moreover, they support the correlation of tissue glucocorticoid sensitivity with insulin resistance and metabolic syndrome-associated pathways, further emphasizing the need for nutritionists and doctors to consider the tissue glucocorticoid sensitivity in dietary and exercise planning, particularly when these subjects are to be treated with glucocorticoids

Transcriptomics in tissue glucocorticoid sensitivity

Background Synthetic glucocorticoids are widely used in the treatment of several inflammatory, autoimmune and lymphoproliferative disorders. However, considerable variation in response to therapeutic doses of glucocorticoids has been documented among individuals. The aim of our study was to identify novel glucocorticoid sensitivity-determining genes using genome-wide expression profiling in healthy subjects. Methods One hundred one healthy subjects [mean age +/- standard error of the mean (SEM); 26.52 +/- 0.50 years] were given 0.25 mg dexamethasone at midnight, and serum cortisol concentrations were determined at 08:00 hours the following morning. Subjects were stratified into the 10% most glucocorticoid-sensitive and 10% most glucocorticoid-resistant according to the serum cortisol concentrations. Genomic DNA, RNA and plasma samples were obtained in the 22 subjects one month later. Results Transcriptomic analysis showed variability between glucocorticoid-resistant and glucocorticoid-sensitive subjects. One hundred thirty-three genes were upregulated and 49 downregulated in the glucocorticoid-resistant compared to the glucocorticoid-sensitive group. Further analysis revealed differences between 3 glucocorticoid-resistant and 3 glucocorticoid-sensitive subjects. The majority of the 1058 upregulated genes and 1139 downregulated genes were found to participate in telomere maintenance, systemic lupus erythematosus and Alzheimer’s disease. Interestingly, Synuclein A, a key molecule in Parkinson’s disease, was upregulated in the subgroup of glucocorticoid-sensitive subjects. Conclusions We have identified differences in tissue sensitivity to glucocorticoids among healthy subjects at the transcriptomic level. These differences are associated with differential expression of genes related to autoimmune and neurological disorders

Atrial fibrillation in heart failure patients: An update on renin–angiotensin–aldosterone system pathway blockade as a therapeutic and prevention target

Heart failure (HF) and atrial fibrillation (AF) are two cardiovascular (CV) entities that affect millions of individuals worldwide and their prevalence is translated into a significant impact on health care systems. The common pathophysiological pathways that these two share have created an important clinical interrelation, as the coexistence of HF and AF is associated with worse prognosis and treatment challenges. Renin–angiotensin–aldosterone system (RAAS), a critical mechanism in blood pressure (BP) control, was proved to be involved in the pathogenesis of both conditions contributing to their further coexistence. Successful control of BP is of great importance to the management of HF, crucial for the prevention of arrhythmiogenic substrates, while RAAS antagonists may possibly affect the development of new-onset AF as well. There are numerous studies that evaluated the effectiveness of RAAS blockade in AF/HF population and despite comparable or modest results, there is a well-established suggestion that RAAS blockers may contribute to a reduction of HF, CV events and recurrence of AF, along with their potential effective role in the new-onset AF prophylaxis. Angiotensin receptor blockers, according to the evidence, are more effective in that direction, followed by angiotensin converting enzyme inhibitors, whereas the data on aldosterone antagonists are not encouraging, yet do have the potential of significant CV disease modificators regardless of their effects on BP

Anti-Diabetic Therapy, Heart Failure and Oxidative Stress: An Update

Diabetes mellitus (DM) and heart failure (HF) are two chronic disorders that affect millions worldwide. Hyperglycemia can induce excessive generation of highly reactive free radicals that promote oxidative stress and further exacerbate diabetes progression and its complications. Vascular dysfunction and damage to cellular proteins, membrane lipids and nucleic acids can stem from overproduction and/or insufficient removal of free radicals. The aim of this article is to review the literature regarding the use of antidiabetic drugs and their role in glycemic control in patients with heart failure and oxidative stress. Metformin exerts a minor benefit to these patients. Thiazolidinediones are not recommended in diabetic patients, as they increase the risk of HF. There is a lack of robust evidence on the use of meglinitides and acarbose. Insulin and dipeptidyl peptidase-4 (DPP-4) inhibitors may have a neutral cardiovascular effect on diabetic patients. The majority of current research focuses on sodium glucose cotransporter 2 (SGLT2) inhibitors and glucagon-like peptide 1 (GLP-1) receptor agonists. SGLT2 inhibitors induce positive cardiovascular effects in diabetic patients, leading to a reduction in cardiovascular mortality and HF hospitalization. GLP-1 receptor agonists may also be used in HF patients, but in the case of chronic kidney disease, SLGT2 inhibitors should be preferred

Plasma Proteomics in Healthy Subjects with Differences in Tissue Glucocorticoid Sensitivity Identifies A Novel Proteomic Signature

Significant inter-individual variation in terms of susceptibility to several stress-related disorders, such as myocardial infarction and Alzheimer’s disease, and therapeutic response has been observed among healthy subjects. The molecular features responsible for this phenomenon have not been fully elucidated. Proteomics, in association with bioinformatics analysis, offer a comprehensive description of molecular phenotypes with clear links to human disease pathophysiology. The aim of this study was to conduct a comparative plasma proteomics analysis of glucocorticoid resistant and glucocorticoid sensitive healthy subjects and provide clues of the underlying physiological differences. For this purpose, 101 healthy volunteers were given a very low dose (0.25 mg) of dexamethasone at midnight, and were stratified into the 10% most glucocorticoid sensitive (S) (n = 11) and 10% most glucocorticoid resistant (R) (n = 11) according to the 08:00 h serum cortisol concentrations determined the following morning. One month following the very-low dose dexamethasone suppression test, DNA and plasma samples were collected from the 22 selected individuals. Sequencing analysis did not reveal any genetic defects in the human glucocorticoid receptor (NR3C1) gene. To investigate the proteomic profile of plasma samples, we used Liquid Chromatography-Mass Spectrometry (LC-MS/MS) and found 110 up-regulated and 66 down-regulated proteins in the S compared to the R group. The majority of the up-regulated proteins in the S group were implicated in platelet activation. To predict response to cortisol prior to administration, a random forest classifier was developed by using the proteomics data in order to distinguish S from R individuals. Apolipoprotein A4 (APOA4) and gelsolin (GSN) were the most important variables in the classification, and warrant further investigation. Our results indicate that a proteomics signature may differentiate the S from the R healthy subjects, and may be useful in clinical practice. In addition, it may provide clues of the underlying molecular mechanisms of the chronic stress-related diseases, including myocardial infarction and Alzheimer’s disease

Biophysical Studies and In Vitro Effects of Tumor Cell Lines of Cannabidiol and Its Cyclodextrin Inclusion Complexes

Phytocannabinoids possess anticancer properties, as established in vitro and in vivo. However, they are characterized by high lipophilicity. To improve the properties of cannabidiol (CBD), such as solubility, stability, and bioavailability, CBD inclusion complexes with cyclodextrins (CDs) might be employed, offering targeted, faster, and prolonged CBD release. The aim of the present study is to investigate the in vitro effects of CBD and its inclusion complexes in randomly methylated β-CD (RM-β-CD) and 2-hyroxypropyl-β-CD (HP-β-CD). The enhanced solubility of CBD upon complexation with CDs was examined by phase solubility study, and the structure of the inclusion complexes of CBD in 2,6-di-O-methyl-β-CD (DM-β-CD) and 2,3,6-tri-O-methyl-β-CD (TM-β-CD) was determined by X-ray crystallography. The structural investigation was complemented by molecular dynamics simulations. The cytotoxicity of CBD and its complexes with RM-β-CD and HP-β-CD was tested on two cell lines, the A172 glioblastoma and TE671 rhabdomyosarcoma cell lines. Methylated β-CDs exhibited the best inclusion ability for CBD. A dose-dependent effect of CBD on both cancer cell lines and improved efficacy of the CBD–CDs complexes were verified. Thus, cannabinoids may be considered in future clinical trials beyond their palliative use as possible inhibitors of cancer growth

Biophysical Studies and In Vitro Effects of Tumor Cell Lines of Cannabidiol and Its Cyclodextrin Inclusion Complexes

Phytocannabinoids possess anticancer properties, as established in vitro and in vivo. However, they are characterized by high lipophilicity. To improve the properties of cannabidiol (CBD), such as solubility, stability, and bioavailability, CBD inclusion complexes with cyclodextrins (CDs) might be employed, offering targeted, faster, and prolonged CBD release. The aim of the present study is to investigate the in vitro effects of CBD and its inclusion complexes in randomly methylated β-CD (RM-β-CD) and 2-hyroxypropyl-β-CD (HP-β-CD). The enhanced solubility of CBD upon complexation with CDs was examined by phase solubility study, and the structure of the inclusion complexes of CBD in 2,6-di-O-methyl-β-CD (DM-β-CD) and 2,3,6-tri-O-methyl-β-CD (TM-β-CD) was determined by X-ray crystallography. The structural investigation was complemented by molecular dynamics simulations. The cytotoxicity of CBD and its complexes with RM-β-CD and HP-β-CD was tested on two cell lines, the A172 glioblastoma and TE671 rhabdomyosarcoma cell lines. Methylated β-CDs exhibited the best inclusion ability for CBD. A dose-dependent effect of CBD on both cancer cell lines and improved efficacy of the CBD–CDs complexes were verified. Thus, cannabinoids may be considered in future clinical trials beyond their palliative use as possible inhibitors of cancer growth