In Australia, Olynyk et al. (1) analyzed in 2009 the LIC by MRI of 52 consecutive patients who were referred for HF to a tertiary hospital for the evaluation of liver IO, either in the presence or absence of increased TSI. They described three different groups according to HFE mutations and TSI: Group A: no predisposing mutations (PM) for HH and TSI > 45%; Group B: PM for HH: C282Y/C282Y; C282Y/H63D, and TSI > 45 %; Group C: no PM for HH and normal TSI.
In the Basque country, PM differ, with prevalence of the H63D/H63D PM (2, 6) and a less important role of the C282Y/C282Y mutation than in Australia (1).
This was a prospective study including patients from January 2010 to December 2010 and was conducted in Mendaro hospital, a secondary hospital in the Basque health service, with a total catchment of 70,000 people. Other objectives of the study have been recently published (4, 9). The study protocol conforms to the 1975 ethical guidelines of the declaration of Helsinki, with the understanding and the consent of the patients. This study was approved by the ethical committee of the Mendaro hospital.
Inclusion criteria: consecutive outpatients referred for HF (serum ferritin (SF) > 200 µmol/g in women; >300 µmol/g in men, in accordance with the WHO criteria (10).
Exclusion criteria: systemic inflammation, infections, and renal or neoplastics diseases (excluded by clinical, laboratory and radiologic methods); history of transfusion or iron supplements; patients younger than 18 years. Patients were studied and treated according to the best practice.
We have studied 132 consecutive patients referred to a secondary hospital in Gipuzkoa, Basque country, Spain, with HF (4) for evaluation of possible IO, either in the presence or absence of a raised TSI.
In our study, we obtained 132 HF patients, with 120 HFE gene mutation studies, and 79 LIC determinations by MRI (SIR method) (5). In 71 patients, we had HF and the TS, HFE and LIC by MRI values.
To form the three groups, the predisposing genotypes for HH in group B were recorded according to the results obtained from epidemiological studies performed in Basque country (2, 6) and from a recent publication (7), including H63D homozygosity as a PM (C282Y/C282Y; C282Y/H63D; H63D/H63D).
3.1. LIC by MRI
The MRI technique used (SIR method) was that proposed by Alustiza et al. (5). This method has a high correlation with biochemical measurements (r = 0.937). MR images were obtained on a 1.5-Tesla imaging unit (Philips Intera, Osatek, Donostia) (5). The images were studied, blinded, by the same radiologist (JMA). Liver steatosis was discarded by assessing systematically T1-weighted in-phase and opposed-phase images (11). Fat may interfere in signal intensity measurements. To avoid this problem, we performed all the LIC sequences as in-phase sequences (11).
The LIC was considered normal if it was lower than 36 µmol/g (0-36 µmol/g). IO was diagnosed when the LIC was between 36 and 80 µmol/g. High IO was defined as a LIC ≥ 80 µmol/g.
3.2. Laboratory Analysis
DNA was extracted from the blood samples and HFE gene analysis was carried out by multiplex real-time PCR using lightcycler technology (LC 1.0). Simultaneous study of the HFE C282Y, H63D and S65C mutations was performed in a single capillary using LC-Red 640, LC-Red 705 and fluorescein-labelled hybridization probes (Tibmolbiol, Berlin, Germany). Melting curve analysis was performed to distinguish wild type and mutant alleles in each case (2, 4).
SF and the transferrin saturation index (TSI) were obtained from blood samples obtained during fasting from all patients included in the study (4, 9). Normal values from our laboratory were: SF in women (15 - 200 µg/L); SF in men (30 - 300 µg/L); TSI (15% - 45%) (4, 9).
3.3. Statistics
SPSS 15.0 software (SSPS Inc., Chicago, IL, USA) was used to perform the appropriate statistical analyses. Mean values with range and standard deviation were calculated for continuous variables and frequencies and percentages for categorical variables.
We compared the LIC mean values of the three groups using ANOVA. A P value < 0.05 was considered statistically significant.
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