Spectrum of LDLR gene mutations, including a novel mutation causing familial hypercholesterolaemia, in North-western Greece

Background

Familial Hypercholesterolaemia (FH) is a clinical syndrome characterised by elevated serum low-density lipoprotein (LDL) cholesterol, by tendon xanthomata and clinical manifestations of ischaemic heart disease in early life. Typically, it results from mutations in the low-density lipoprotein receptor (LDLR) gene. Furthermore, there are 3 additional genetic disorders that cause clinical syndromes that mimic FH. These are: 1) familial ligand-defective apolipoprotein (apo)-B (FLDH), 2) familial hypercholesterolaemia type 3 (FH3) and 3) autosomal recessive hypercholesterolaemia (ARH). The aim of this study was to elaborate the impact of the above genetic disorders in Greek patients with a clinical diagnosis of FH.

Methods

In this study, we assessed the contribution of the LDLR, Apo B, ARH and PCSK9 genes in the expression of FH in North-western Greece. Two hundred and fifty-four (254) probands with a clinical diagnosis of FH were included in the study.

Results

One hundred and sixty-nine (169) patients had one of the following LDLR gene mutations: 81T>G, 1775G>A, 517T>C, 858C>A, 1352T>C, 1285G>A, 761A>C, 1195G>A, 1646G>A and a deletion mutation g.387-410del24 in exon 4. We sequenced the Apo B, ARH and PCSK9 genes in 40, randomly selected patients, from the 85 patients with no identified LDLR gene defects. In these 40, randomly selected patients, with the exception of benign single nucleotide polymorphisms, no functional mutations were identified for all the above mentioned sequenced genes.

Conclusion

Our results reveal substantial genetic heterogeneity for FH in North-western Greece with at least ten LDLR gene mutations present in the study population. One of these mutations although quite rare is reported here for the first time in the scientific literature. The detection of these mutations is important as they may be used to design multiplex detection assays for large scale population screening programmes to facilitate primary and secondary prevention of cardiovascular disease in the region. Finally, ARH, Apo B and PCSK9 gene defects were excluded from causing FH in a subgroup of the study population indicating that other yet unrecognized genes may be involved in causing the clinical feature of FH, and/or that large scale deletions/duplications evaded the applied mutation detection techniques of this study.     

<Emphasis Type="Italic">Anisakis</Emphasis> spp. burden in <Emphasis Type="Italic">Trachurus trachurus</Emphasis>

Anisakis is a parasite of marine mammals that uses a great number of fish species as intermediate or paratenic hosts. It is common in commercially important marine fishes and its presence is of great concern for both human health and economic reasons. Horse mackerels (Trachurus trachurus) originated from the Northern Aegean Sea were examined for the presence of Anisakis spp. larvae. The prevalence of Anisakis spp. was found 98.8 %. The number of parasites was significantly related to the host’s length but was not related to the fish gender. The month of sampling affected the size of the fishes and consequently the number of parasites. The length of larvae was not related to the host’s length. The present study resulted in the design of a prediction model for the number of existing parasites in the fish by measuring only its Fixed Length.     

First report of Troglostrongylus brevior in a kitten in Greece

The first case of a natural infestation with Troglostrongylus brevior in a kitten in Greece is described here. A ～40-day-old stray cat was referred to a private veterinary clinic with signs of respiratory distress. First stage larvae of a metastrongyloid nematode were observed in the wet mount faecal preparation. Despite an anthelmintic treatment, the respiratory signs worsened and the kitten died 2 days later. The larvae in the faeces were identified morphologically and genetically as T. brevior. The present evidence suggests a vertical or direct infestation of the kitten and a severe pathogenic role of T. brevior in young cats. This report expands the recent published cases of troglostrongylosis in domestic cats to a wider geographical distribution and opens new questions on the apparent spreading of T. brevior from wild to domestic hosts.     

Stabilization of human urine doping control samples: III. Recombinant human erythropoietin

BackgroundThe tampering of athlete's urine samples by the addition of proteolytic enzymes during the doping control sampling procedure was reported recently. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in urine samples to chemically inactivate proteolytic enzymes and improve the electrophoteric signal of erythropoietin (EPO) in human urine.MethodsThe stabilization mixture applied was a combination of antibiotics, antimycotic substances and protease inhibitors. A series of incubation experiments were conducted under controlled conditions in the presence and absence of the stabilization mixture in urine aliquots spiked with six proteases. Two different analytical techniques were applied for the qualitative and quantitative EPO measurement: isoelectric focusing (IEF) and chemiluminescent immunoassay respectively.ResultsThe addition of the chemical stabilization mixture into urine aliquots substantially improved EPO detection in the presence of proteolytic enzymes following incubation at 37 °C or storage at ? 20 °C.ConclusionsThe results of this study indicated that the stabilization of urine prior to the sample collection procedure with the proposed chemical mixture might prove to be a useful tool for the preservation of anti-doping samples.     

Pharmacological treatment of non-alcoholic steatohepatitis: The current evidence

¹²⁵I-protein-radioiodinated pure pancreatic juice samples from patients with adenocarcinoma of the pancreas, chronic pancreatitis, or intact pancreas were analysed by high-resolution SDS-polyacrylamide gel electrophoresis and subsequent autoradiography. Experiments resulted in the detection of a 180K protein, probably a glycoprotein, in the pure pancreatic juice from pancreatic carcinoma (93%) and chronic pancreatitis (73%) patients, which was completely absent from pancreatic juice from intact pancreas. Sephadex G-200-isolated 18UK protein was found to be different from carcinoembryonic antigen (CEA) when traced by a commercial CEA radioimmunoassay, but it seemed identical in pancreatic juice samples from patients with pancreatic carcinoma and chronic pancreatitis, at least with regard to isoelectric point. In brief, the present results suggest that 180K protein identification in pancreatic juice permits adenocarcinoma of the pancreas and chronic pancreatitis to be differentiated from normal conditions but that distinction between pancreatic carcinoma and chronic pancreatitis is unlikely.     

Plasma high sensitivity C-reactive protein and its relationship with cytokine levels in children with newly diagnosed type 1 diabetes and ketoacidosis

BackgroundHigh-sensitivity C-reactive protein (hs-CRP) and pro-inflammatory cytokines have been suggested as sensitive markers of endothelial dysfunction. Our aim was to monitor plasma hs-CRP levels at different time-points and in different degrees of ketoacidosis severity, its association with cytokine levels and its role as a marker of severe ketoacidosis complications.Patients and methodsWe studied in 38 newly diagnosed children with type 1 diabetes and ketoacidosis, aged 7.7 ± 3.1 years, hs-CRP, white blood cell count (WBC), and plasma levels of cytokines IL-1β (interleukin-1β), IL-2, IL-6, IL-8, IL-10, TNF-α (tumor necrosis factor-α) prior to and during DKA management.ResultsOn admission, the levels of WBC, PMN, IL-6 and IL-10 were elevated, but were all reduced within 120 h after ketoacidosis management. In the group with moderate/severe ketoacidosis, but not in mild ketoacidosis, hs-CRP levels were significantly reduced at 24 h (p = 0.021), WBC and IL-6 at 120 h (p = 0.003), while IL-10 was prematurely reduced at 6–8 h (p = 0.008). Moreover hs-CRP was significantly associated with WBC (p = 0.023) and IL-6 (p = 0.028) on admission, with IL-6 (p = 0.002) and IL-8 (p = 0.014) at 24 h and with IL-10 (p = 0.027) at 120 h. The above were not observed in the group with mild ketoacidosis.ConclusionsIn the children with moderate/severe diabetic ketoacidosis of our study, increased levels of hs-CRP and IL-6 were observed, together with leukocytosis and neutrophilia, without the presence of infection. As hs-CRP was found to be strongly associated with the inflammatory IL-6, the prolonged elevation of hs-CRP levels in children with severe ketoacidosis could serve as a marker for the development of its severe complications.     

Restoring methicillin-resistant Staphylococcus aureus susceptibility to β-lactam antibiotics

Despite the need for new antibiotics to treat drug-resistant bacteria, current clinical combinations are largely restricted to β-lactam antibiotics paired with β-lactamase inhibitors. We have adapted a Staphylococcus aureus antisense knockdown strategy to genetically identify the cell division Z ring components-FtsA, FtsZ, and FtsW-as β-lactam susceptibility determinants of methicillin-resistant S. aureus (MRSA). We demonstrate that the FtsZ-specific inhibitor PC190723 acts synergistically with β-lactam antibiotics in vitro and in vivo and that this combination is efficacious in a murine model of MRSA infection. Fluorescence microscopy localization studies reveal that synergy between these agents is likely to be elicited by the concomitant delocalization of their cognate drug targets (FtsZ and PBP2) in MRSA treated with PC190723. A 2.0 ? crystal structure of S. aureus FtsZ in complex with PC190723 identifies the compound binding site, which corresponds to the predominant location of mutations conferring resistance to PC190723 (PC190723(R)). Although structural studies suggested that these drug resistance mutations may be difficult to combat through chemical modification of PC190723, combining PC190723 with the β-lactam antibiotic imipenem markedly reduced the spontaneous frequency of PC190723(R) mutants. Multiple MRSA PC190723(R) FtsZ mutants also displayed attenuated virulence and restored susceptibility to β-lactam antibiotics in vitro and in a mouse model of imipenem efficacy. Collectively, these data support a target-based approach to rationally develop synergistic combination agents that mitigate drug resistance and effectively treat MRSA infections.