Current screening methods, such as single strand conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (dHPLC) that are used for detecting mutations in familial hypercholesterolaemia (FH) subjects are time consuming, costly and only 80–90% sensitive. Here we have tested high-resolution melt (HRM) analysis for mutation detection using the Rotor-Gene6000 realtime rotary analyser.
Polymerase chain reaction and melt conditions (HRM) for 23 fragments of the LDL-receptor gene, a region of exon 26 in the APOB gene (including p.R3527Q) and exon 7 of the PCSK9 gene (including p.D374Y) were optimized. Two double stranded DNA saturating dyes, LC-Green and Syto9, were compared for sensitivity. Eighty-two samples with known mutations were used as positive controls. Twenty-eight Greek FH heterozygous patients and two homozygous patients from the UK and Croatia were screened.
HRM was able to identify all the positive control mutations tested, with similar results with either dye. Eight different variations were found in 17 of the 28 Greek FH patients for an overall detection rate of 61%: c.41delT (1), p.W165X (1), p.C173R (3), p.S286R (2), p.V429M (4), p.G549D (4), p.V613I (1), and a previously unreported mutation p.F694V (1) which is predicted to be FH-causing by functional algorithms. Mutations were found in both the homozygous patients; p.Q92X (Croatia) and p.Y489C (UK); both patients were homozygous for their respective mutations.
HRM is a sensitive, robust technique that could significantly reduce the time and cost of screening for mutations in a clinical setting.
Current gold standard markers for myocardial damage are troponins I and T, which are both sensitive and specific for the detection of myocardial infarction, but require up to 6 h to become reliably elevated in serum. Investigation into markers with potential to identify patients with early ischaemic changes is therefore intense. Choline is reported to be prognostic in patients presenting with acute coronary syndromes via its release from ischaemic cell membranes.
Liquid chromatography tandem mass spectrometry was used to develop a method to quantitate choline in plasma and blood. The method involves addition of a deuterated internal standard to an aliquot of plasma or blood followed by organic solvent addition, which precipitates the proteins in the sample. Preparation was carried out directly into a 96-deep-well plate. Chromatography of choline used a strong cation exchange column and separation used a Waters Atlantis dC18 analytical column positioned directly before the mass spectrometer source, allowing on-line preanalytical clean up of the sample.
The lower limit of quantitation was 0.38 µmol/L, linearity was observed up to 754 µmol/L, with a working concentration range of 0.38–224 µmol/L, inter- and intra-assay coefficients of variation were <6% and <4%, respectively. Samples were stable throughout five freeze–thaw cycles and recovery was between 94% and 114%.
The assay was successfully validated in accordance with FDA guidelines and is suitable for quantitation of choline in research and clinical settings.
Measurement of the faecal markers calprotectin, lactoferrin and tumour M2-PK has been reported to be useful in the diagnosis and management of a range of gastrointestinal disorders in both children and adults. The aim of this study was to investigate the requirement for age-related reference ranges.
Faecal samples were obtained from 132 healthy subjects and analysis of calprotectin, lactoferrin and tumour M2-PK was performed using commercially available enzyme-linked immunosorbent assay.
In healthy subjects median concentrations were as follows: for calprotectin 2–9 y 34 µg/g, 10–59 y 22 µg/g and ≥60 y 27 µg/g; for lactoferrin 2–9 y 2.2 µg/g, ≥10 y 0.5 µg/g; for tumour M2-PK all subjects <1 mU/L. Significant differences between age groups for different markers resulted in the following age-related reference ranges: calprotectin 2–9 y <79 µg/g, 10–59 y <22 µg/g, ≥60 y <44 µg/g; lactoferrin 2–9 y <18 µg/g, ≥10 y <2.4 µg/g; tumour M2-PK ≥2 y >3.9 mU/L.
In healthy individuals, we found variation in the faecal inflammatory markers calprotectin and lactoferrin with age. For both calprotectin and lactoferrin, children aged 2–9 y had significantly higher concentrations than subjects aged ≥10 y. For calprotectin but not lactoferrin, adults ≥60 y had a higher concentration than those aged 10–59 y. There was no change with age in the metabolomic marker faecal tumour M2-PK in healthy subjects. Knowledge of age-related reference ranges in healthy subjects is important to fully interpret changes in gastrointestinal disease.