The authors have declared that no competing interests exist.
Conceived and designed the experiments: VS GLG CF. Performed the experiments: MCM IG SA. Analyzed the data: VS PS GLG. Contributed reagents/materials/analysis tools: PG CLM KR. Wrote the paper: VS GLG CF. Patient inclusion and data collection: AYM BC JBN.
Despite type I haemochromatosis (HC) is mainly associated with the
Haemochromatosis (HC; EASL consensus conference 2000
The best-known and predominant form of HC is an adult-onset autosomal recessive condition mostly associated with the
A second
It is therefore probable that co-morbid factors are more frequent in C282Y/H63D heterozygotes diagnosed than in C282Y/C282Y homozygotes because of an hyperferritinemia, and that hyperferritinemia observed in most of C282Y/H63D compound heterozygotes does not totally match with the classical iron overload hallmarks of HC. If checked, this assumption will emphasize the importance of acting on co-morbid factors during care.
The aim of our study was to assess the profile of the C282Y/H63D patients treated by phlebotomies and to investigate the role of the co-morbid factors in the phenotypic expression. For that, we analysed a cohort of patients cared in a blood centre of western Brittany (France), where HC is particularly common
The study protocol was approved by our local ethical committee (University Hospital of Brest). Written informed consent was obtained from each patient included in the study.
The present study was conducted from the cohort of patients enrolled in a phlebotomy program in a blood centre of western Brittany (Brest, France) between January 1st 2004 and December 31st 2007 (year from which the H63D was not systematically searched for in our laboratory) (n = 325). These patients were referred to the blood centre by general practitioners or gastroenterologists, for the achievement of phlebotomies because they had elevated serum ferritin (≥300 µg/L for males or ≥200 µg/L for females).
Our attention was mainly focused on patients carrying the
The study relies on the data collected in a clinical questionnaire, which is completed by a referral physician at entrance in the phlebotomy program. As previously described
Data related to the treatment (number and volume of phlebotomies) are recorded thereafter, when depletion is reached. These data enable to assess the amount of iron removed (AIR; in grams) needed for normalising iron stores (
Regarding lifestyle factors, daily consumption of alcohol was estimated by asking patients to state the number of glasses of different kinds of alcohol (beer, wine, fortified wine, hard liquor) they drank each day. In our area, people mainly drink red wine (especially during meals) and, in second line, beer. Daily consumption corresponded to the sum of the numbers of glasses declared. Whatever the liquor, a standard drink (representing one unit of alcohol) contains the same amount of pure alcohol (ethanol), namely 10 grams in our country. Thereby, in France, a glass of 25 cl of beer (at 5°), of 10 cl of wine (12°), of 7 cl of fortified wine (18°) or of 3 cl of spirits (40°) contains about 10 grams of pure alcohol. In accordance with the WHO definition, alcohol intake was considered abusive when consumption exceeded 3 glasses per day for men (
Iron markers were measured by standard biochemical methods. The C282Y and H63D variants were studied using the 5′- nuclease allelic discrimination method or TaqMan method (Applied Biosystems, Foster City, CA).
Statistical analysis was carried out using the SAS software (version 9.02; SAS Institute Inc, Cary, NC). All tests were performed two-sided, and a p-value less than 5% was considered significant.
First, we first described the socio-demographic characteristics of the population under study. Then, we assessed the impact of the C282Y/H63D genotype and proceeded, for that, in three steps:
We quantified the deficit of C282Y/H63D compound heterozygotes observed among patients who come to medical attention. For that, we determined the prevalence of the C282Y/H63D and C282Y/C282Y genotypes in the study population (
We compared the nature and severity of iron overload (assessed by TS, SF and AIR) in C282Y/H63D and C282Y/C282Y patients who came to medical attention. We performed linear regression analysis to investigate the influence of the genotype on the level of iron overload. We performed logarithmic transformation (log10) of the dependant variables (
We examined whether the C282Y/H63D patients had more commonly co-morbid factors known to cause hyperferritinemia (alcohol abuse and overweight). We therefore compared the frequency of these factors in the two genotypic groups.
Over the study period, 325 patients were enrolled in a phlebotomy program at the blood centre of Brest.
One way to confirm the milder effect of the C282Y/H63D genotype was to compare the prevalence of the various
Study population | General population | |||
n | % | n | % | |
C282Y/C282Y | 172 | 56.2% | 7 | 0.9% |
C282Y/H63D | 58 | 18.9% | 34 | 4.3% |
H63D/H63D | 18 | 5.9% | 15 | 1.9% |
C282Y/wt* | 11 | 3.6% | 105 | 13.1% |
H63D/wt* | 25 | 8.2% | 164 | 20.6% |
wt*/wt* | 22 | 7.2% | 472 | 59.2% |
Total | 306 | 100.0% | 797 | 100.0% |
*wt: wild type.
The C282Y/H63D genotype appears relatively common in our population. It is carried by 1 individual in 23 (
By focusing on the C282Y/C282Y and C282Y/H63D genotypes, we observed that, in the general population, the C282Y/H63D genotype was 4.9 times more frequent than the C282Y/C282Y genotype (4.3%
We then compared the severity of iron overload in C282Y/H63D and C282Y/C282Y patients treated by phlebotomies. As illustrated in
C282Y/H63D | C282Y/C282Y | p | |
Median (Q1–Q3)* | Median (Q1–Q3)* | ||
Males | 57.0 (47.0–69.0) | 87.5 (75.5–94.5) | <0.0001 |
Females | 42.0 (37.0–51.0) | 73.5 (64.0–89.0) | <0.0001 |
Males | 2.3 (1.7–2.9) | 5.2 (3.2–7.0) | <0.0001 |
Females | 1.3 (1.0–2.2) | 2.1 (1.5–3.5) | 0.0132 |
Males | 659.0 (448.0–1072.0) | 927.0 (649.0–1700.0) | 0.0032 |
Females | 422.0 (300.0–665.0) | 414.0 (272.0–733.0) | 0.7913 |
*Q1: first quartile; Q3: third quartile.
Similar findings were obtained with the AIR by phlebotomies, which is a good marker of the iron burden. This marker was indeed significantly lower in the C282Y/H63D patients cared by phlebotomies than in the C282Y/C282Y ones (median: 2.1
The milder role of the C282Y/H63D genotype on the level of iron overload was confirmed by linear regression analysis (
Variables | 10β | (95% CI) | p | |
Genotype (ref: C282Y/C282Y) | ||||
Gender (ref: women) | 1.15 | (1.07–1.25) | 0.0005 | |
Age at diagnosis (ref: <40 y.) | ||||
[40 y. –50 y.] | 1.07 | (0.95–1.20) | 0.2731 | |
[50 y. –60 y.] | 1.08 | (0.96–1.21) | 0.1790 | |
≥60 y. | 1.09 | (0.95–1.24) | 0.2235 | |
Heavy drinking | 1.05 | (0.95–1.16) | 0.3273 | |
Overweight | 0.92 | (0.85–0.99) | 0.0353 | |
Genotype (ref: C282Y/C282Y) | ||||
Gender (ref: women) | 1.11 | (1.03–1.20) | 0.0082 | |
Age at diagnosis (ref: <40 y.) | ||||
[40 y. –50 y.] | 0.96 | (0.85–1.07) | 0.4472 | |
[50 y. –60 y.] | 1.00 | (0.89–1.12) | 0.9730 | |
≥60 y. | 0.96 | (0.84–1.09) | 0.5005 | |
Heavy drinking | 1.11 | (1.00–1.22) | 0.0441 | |
Overweight | 0.96 | (0.89–1.04) | 0.3459 | |
Genotype (ref: C282Y/C282Y) | ||||
Gender (ref: women) | 1.89 | (1.58–2.27) | <0.0001 | |
Age at diagnosis (ref: <40 y.) | ||||
[40 y. –50 y.] | 1.16 | (0.89–1.53) | 0.2768 | |
[50 y. –60 y.] | 1.36 | (1.04–1.77) | 0.0250 | |
≥60 y. | 1.30 | (0.97–1.75) | 0.0799 | |
Heavy drinking | 1.76 | (1.39–2.22) | <0.0001 | |
Overweight | 1.12 | (0.94–1.34) | 0.2130 |
In contrast, the level of hyperferritinaemia appeared similar between the two genotypic groups (median, 529.0
Finally, we checked whether the C282Y/H63D patients who came to medical attention had more commonly co-morbid factors that could explain their hyperferritinemia. Data on alcohol intake and on body mass index were not documented for 15 (6.5%) and 16 (7.0%) patients respectively, but the frequency of these missing values did not differ according to genotype (p = 0.0917).
We observed that the co-morbid factors were significantly more frequent in the group of C282Y/H63D patients (
In summary, three quarters of the C282Y/H63D patients had at least one of these co-morbid factors (75.0%
The present study confirms that the C282Y/H63D genotype is clearly associated with a milder iron overload. Patients carrying this genotype that are cared by phlebotomies have both a milder amount of plasma iron (reflected by lower TS) and of tissue iron (reflected by lower AIR required to reach depletion). These patients also exhibit more frequently co-morbid factors (as heavy drinking and/or overweight) that may alone explain their hyperferritinemia (whose level did not seem different from that of the C282Y/C282Y patients). This study confirms therefore the modest weight of the C282Y/H63D genotype and the major role of co-morbid factors in the occurrence of biological expression of HC.
The lack of C282Y/H63D genotypes among patients enrolled in a phlebotomy program is an additional argument reflecting the modest role of this genotype in the occurrence of iron overload. Indeed, we have shown here that the C282Y/H63D genotype was 3.0 times less frequent than the C282Y/C282Y genotype among patients enrolled in a phlebotomy program whereas it was 4.9 times more frequent in the general population. As expected, most of the patients carrying this genotype do not come to medical attention because of their mild, if any, iron overload.
Beyond these results, it is also important to keep in mind that this C282Y/H63D genotype has only rarely been reported in a family context. In our experience, we have never seen a family with two or more sibling carrier of the genotype C282Y/H63D that present an iron overload.
It should be noted that, in our cohort, less than one third of patients had a SF ≥1000 µg/L (30.9%
The originality of our study was to investigate the role of both
One limitation of our study was our inability to determine the true prevalence of the metabolic syndrome, which combines the presence of several metabolic abnormalities (abdominal obesity, low HDL cholesterol, high triglycerides, glucose intolerance, hypertension)
Another limitation was that the possible existence of modifier genes. We re-sequenced the five genes associated with haemochromatosis (
Our findings are consistent with data reported in the literature illustrating that the perception of the H63D variant has moved, in recent years, toward the notion of susceptibility factor (
Regarding penetrance, a recent study, based on a cohort of 6020 Danish men aged 30 to 53 years has estimated the biological penetrance at 87.5% for the C282Y homozygotes and at only 9.1% for the C282Y/H63D compound heterozygotes (based on TS ≥50% and SF ≥300 µg/L)
Beyond this, we should also keep in mind that, at protein level, the H63D does not alter the HFE protein structure, in contrast to the C282Y that prevents interaction with beta2-microglobulin and presentation on cell surface
In conclusion, our study provides evidence for the milder effect of the C282Y/H63D genotype in comparison to the C282Y/C282Y genotype by three ways: 1) through the lower frequency of this genotype among patients cared by phlebotomies than expected, 2) through the higher prevalence of associated co-morbid factors in these patients 3) and finally through the milder degree of iron overload in these patients.
Our results combined with data from the literature underline that the H63D variant, which is common in the general population, is a discrete genetic susceptibility factor whose expression is most visible in association with other co-factors such as alcohol abuse and/or overweight. Our study clearly highlights the importance of searching for co-morbidities in these diagnostic situations and of acting on these factors during care. In most cases, lifestyle and dietary advices should be sufficient to reduce the number of phlebotomies requested in those patients. This work should therefore contribute to improve the management of haemochromatosis patients.
The authors are grateful to Emmanuelle GENIN from Inserm (France) for her assistance on population genetic issues.