ABC | Volume 110, Nº2, February 2018

Original Article Lorenzo et al Clinical characterization of patients with HF Arq Bras Cardiol. 2018; 110(2):119-123 Institute of Cardiology in Rio de Janeiro, Brazil. Subjects with LDL-C > 190 mg/dl and in use of lipid-lowering drug were selected after review of the lipid panel results over 6 months. These patients were invited by phone call to take part in the study, and those with acute coronary syndromes or myocardial revascularization in the previous 30 days, autoimmune diseases, thyroid disorders, chronic renal failure, liver diseases, malignancy, using steroids, or pregnant or breastfeeding were excluded. For this study, a convenience sample was used, including all patients who had been genetically screened to date. Once considered eligible, all participants read and signed an informed consent document approved by the institutional Ethics Committee. The study was undertaken in accordance with the Helsinki Declaration of 1975, revised in 2000. The study patients underwent clinical evaluation and peripheral blood collection. The FH phenotype was classified according to the Dutch Lipid Clinic Network criteria. 3 Prior cardiovascular disease was defined as a history of myocardial infarction, evidence of obstructive coronary artery disease at coronary angiography (> 50% stenosis of any epicardial coronary artery), myocardial revascularization (either percutaneous or coronary artery bypass surgery) or stroke. Hypertension was defined as blood pressure ≥ 140/90 mmHg and/or antihypertensive drug use. Diabetes mellitus was defined by history and use of insulin or oral hypoglycemic medications, or fasting glucose levels > 126 mg/dl. Anthropometric measurement All patients underwent assessment of body composition. Body mass index (BMI) was calculated as weight in Kg/ (height) 2 . Body composition (body fat percentage [%], visceral fat area [cm²] and phase angle [degrees]) was estimated by bioelectrical impedance, using the multifrequency analyzer octopolar (In‑Body 720; Biospace). The measurements were made with the patient in the supine position, with the arms lying parallel and separated from the trunk and with the legs separated, so that the thighs were not touching. Two electrodes were placed on the hand and wrist and another two were positioned on the foot and ankle of the non-dominant side of the body. An electrical current measured at six different frequencies (1, 5, 50, 250, 500 and 1000 KHz) was introduced into the subject, and resistance and reactance were measured. The phase angle was calculated according to the following equation: Phase Angle = arctangent (inductance / resistance) × 180º/ π. 9 Laboratory measurements For biochemical testing, venous blood samples were obtained in the morning after 12 h of fasting. The patients took their usual medications on the morning of the tests. Laboratory evaluations were performed by an automated method (ARCHITECT ci8200, Abbott ARCHITECT®, Abbott Park, IL, USA) using commercial kits (Abbott ARCHITECT c8000®, Abbott Park, IL, USA). Serum triglyceride levels, total cholesterol, LDL cholesterol (LDL-C), HDL-cholesterol (HDL-C), apolipoproteins A (apo A) and B (apo B) and C-reactive protein (CRP) were evaluated. Genomic DNA was extracted from peripheral blood following a standard salting-out procedure. All DNA stock samples were quantified with Qubit dsDNA BR Assay Kit (Thermo Fisher) and diluted to 10 ng/ul for enrichment with Ion AmpliSeq Custom Kit (Thermo Fisher). Samples were enriched for six FH-related genes: LDLR, APOB, PCSK9, LDLRAP1, LIPA and APOE. Patients who tested positive for any of these mutations were considered to have genetically confirmed FH. Target regions were considered as coding exons plus 10bp of introns up and downstream. Templates were prepared on Ion One Touch System and sequenced in Ion Torrent PGM ® platform, with 32 samples per run in a 316v2 Ion Chip. All FASTQ files were imported to CLC Genomics Workbench 9.5 (QIAGEN) and analyzed in a custom pipeline. Minimum quality requirements for variant call were: Base quality of PhredQ ≥ 20; Target-region coverage ≥ 10x; Frequency of variant allele ≥ 20% and bidirectional presence of variant allele. After polymorphism filtering with control populations (NHLBI-ESP6500, ExAC and 1000Genomes), all potential mutations were consulted for previous description in ClinVar, Human Genome Mutation Database (HGMD), British Heart Foundation and Jojo Genetics databases. Functional impact prediction was performed with SIFT, PROVEAN and PolyPhen-2 and mutations without previous description should be pointed as damaging in at least two algorithms to be considered as potentially pathogenic. Individuals with negative results were also screened for large insertions and deletions via MLPA (MRC-Holland). Statistical analysis Continuous data were analyzed using two-tailed unpaired Student’s t test or Mann-Whitney´s test, and categorical variables with chi -squared test. Kolmogorov - Smirnov test was performed t o determine whether sample data was normally distributed. Continuous variables are reported as means ± standard deviations, and categorical variables are presented as percentages. Correlations between continuous variables were analyzed with Pearson`s test. Analyses were performed with SPSS software, version 21.0, and p values < 0.05 were considered statistically significant. Statistical review of the study was performed by a biomedical statistician. Results Forty-five patients with LDL-C> 190 mg/dl were studied, of which fifteenhadpositive testing for familial hypercholesterolemia and thirty had negative. Comparing patients with genetically confirmed FH to those without it (Table 1), the former had a higher clinical score for FH, were more frequently considered to have definite FH, andmore often had xanthelasma. Of note, the prevalence of prior coronary artery disease or stroke were not significantly different between patients with or without the genetic diagnosis of FH. Mean LDL-C and apo B levels were higher in patients with a molecular diagnosis of FH (Table 2). When the correlations between LDL-C levels and other clinical, demographic and anthropometric variables were examined, there was a weak, although significant correlation between LDL-C and the clinical point score (R = 0.382, p = 0.037) and a moderate and significant correlation between LDL-C and body fat (R = 0.461, p = 0.01). 120