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Variation of iron loading expression in C282Y homozygous haemochromatosis probands and sib pairs
  1. Catherine Mura,
  2. Gérald Le Gac,
  3. Virginie Scotet,
  4. Odile Raguenes,
  5. Anne-Yvonne Mercier,
  6. Claude Férec
  1. Centre de Biogénétique, ETSBO, CHU, UBO, BP454, 46 rue Félix Le Dantec, F-29275 Brest, France
  1. Dr Mura, Catherine.Mura{at}

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Editor—Hereditary haemochromatosis (HH), a common autosomal recessive disease of iron metabolism, is more prevalent among populations of northern Europe with an affected rate of 1 in 200 to 400 and a carrier frequency of around 1 in 10.1 2 The disease is characterised by progressive iron overload, and the clinical onset usually appears after middle age. The phenotypic manifestations of HH are variable, and the severity of the disease is related to the iron loading; the most common symptoms of iron overload are fatigue, lethargy, arthropathy, and skin pigmentation, often along with more serious organ damage including cirrhosis, diabetes mellitus, myocardiopathy, and endocrine dysfunction. The assessment of iron loading is currently based on the levels of biochemical iron markers such as transferrin saturation percentage, serum ferritin, and serum iron concentrations. However, the diagnosis of haemochromatosis can now be confirmed using direct HFE mutation testing. The prognosis depends on early diagnosis and therapeutic venesections. Thus, population screening would allow early diagnosis during the asymptomatic phase and prophylactic treatment by repeated venesection to prevent the irreversible damage of iron overload,3 but predictive diagnosis requires a well established phenotype-genotype correlation.

The identification of the haemochromatosis gene,4 now referred to as HFE,5 enables the performance of direct genetic testing for diagnosis. The role of the HFE protein in iron metabolism has not yet been clearly established, but it seems that the complex of HFE with β2-microglobulin interacts with the transferrin receptor (TfR) on the cell surface, which decreases the affinity of TfR for transferrin.6-9 Some mutations characterised in theHFE gene and leading to a functional defect have been correlated with HH. Two missense mutations, 845G→A (C282Y) accounting for 80-90% of HH chromosomes,4 10-13 and 187C→G, (H63D) representing 40-70% of non-C282Y HH chromosomes,4 12-15 leading to the absence and decrease of HFE activity, respectively, have been described.16 17Another variant, 193A→T, leading to the missense substitution S65C, has been reported to be increased in HH chromosomes, accounting for 7.2% of HH chromosomes, neither 845A (C282Y) nor 187G (H63D).18

The considerable heterogeneity of iron loading observed in HH patients has been correlated with their genotype. Several studies have confirmed that HH patients homozygous for the C282Y mutation are associated with a more severe form of the disease than those carrying other genotypes (H63D/H63D, C282Y/H63D, C282Y/S65C).12 13 19 Phenotype-genotype correlation studies have shown discrepancies. Some reports have mentioned patients diagnosed with haemochromatosis who did not carry known HFE mutations on both chromosomes, accounting for up to 21% of the HH population.4 12 13 Thus, the aetiology of the iron loading in these patients remains unclear. Non-HFE related patient cases may have been included in these HH subjects because of misdiagnosis owing to secondary iron overload or atypical juvenile haemochromatosis linked to chromosome 1q20-21; this point still needs to be clarified. In addition, despite the prominent role of the C282Y mutation in HH, population screening indicates that 17.6% of homozygotes for C282Y were asymptomatic patients.22 Thus, C282Y penetrance confronts one with a problem and requires more investigation.

In the present study, we assessed the biochemical expression of iron loading in HFE C282Y homozygotes. We thus examined the parameters indicative of iron loading in a series of probands homozygous for the C282Y mutation. Then we conducted a family case study of HFE identical sibs enrolled because one of them had HH. The whole study showed a variable biochemical expression of iron overload related to the patients' age and sex, which was not correlated in subjects with an identical inherited genotype at the HFE locus.

Patients and methods


A series of 545 unrelated probands, all homozygous for C282Y, showing various symptoms of clinical haemochromatosis and referred from clinicians to our blood centre for treatment by venesection, was included in this study. Before treatment the diagnosis was confirmed by their iron status markers, and all of them showed at least two of the following criteria: (1) transferrin saturation higher than 60% in males and 50% in females, (2) serum ferritin concentration exceeding 400 μg/l in males and 300 μg/l in females, and (3) serum iron above 20 μmol/l. These iron status markers were measured by standard techniques.


As part of genetic counselling a family study was conducted. Partners and sibs of C282Y HH probands were screened forHFE mutations and biochemical iron markers. The study group consisted of 53 subjects from 24 unrelated HH families in whom at least one sib had exhibited symptoms and an increased total body iron loading, indicative of HH. This allowed the examination of 18 same sex sib pairs, homozygous for C282Y, and composed of 13 male pairs and five female pairs; eight others were opposite sex pairs. At the time of the biochemical diagnosis, all these sibs were 32 to 64 years old. The mean difference in age within a set of sibs was 5.6 years.


DNA was extracted from peripheral blood leucocytes. C282Y, H63Ds, and S65C substitutions were analysed as previously described.18


Measurements of transferrin saturation, serum ferritin, and serum iron are expressed as means (SD) and range is indicated. Comparisons between groups of subjects were made with Student'st test and correlation between iron parameters was assessed. The relationship between the iron parameters and the age of patients was studied separately in males and females using linear regression analysis.


A total of 545 unrelated subjects including 197 females and 348 males, all diagnosed with HH and homozygous for the C282Y mutation, were studied. Thus, the male to female sex ratio was 1.7:1 showing a reduced penetrance of the C282Y mutation in females compared to males. The difference in age at onset was recorded according to the sex of probands. The mean age was 44.1 (SD 10.9) and 49.8 (SD 12.5) in males and females, respectively. The difference in mean age at onset calculated between the males and the females using Student'st test was significant (t=4.57, p=3.3 × 10-6) showing that, at onset, females were significantly older than males. Approximately 70% of the males were diagnosed with HH before the age of 50, whereas only 48.6% of females were; 90% of the males and 76.5% of the females were diagnosed with HH before the age of 60 (table 1). These results show that the biochemical expression of haemochromatosis strongly depended on both sex and age in C282Y homozygotes. Iron marker values ranged between normal and significantly increased compared with control values and regardless of the proband's sex and age. However, transferrin saturation, serum ferritin, and serum iron values as a whole were significantly higher in males than in females (p=1.2 × 10-3, 6.7 × 10-8, 9 × 10-3, respectively).

Table 1

Iron status in 545 probands homozygous for the C282Y mutation

According to the age range, above 30 years old serum ferritin and serum iron concentrations were significantly higher in males than in females; transferrin saturation was significantly increased with age only after 40 years in males compared with females (table 1). A correlation analysis showed that, whereas transferrin saturation and serum iron remained stable with age in both sexes, there was a progressive increase of serum ferritin concentration with age in males (r=0.29) and females (r=0.23) (fig 1); it increased from 262 to 1355 μg/l in females and from 598 to 2349 μg/l in males aged from 30 to over 60 years of age. The linear regression analysis indicated a significant mean annual progression of serum ferritin of 39.9 μg/l (p<10-3) in males and 21.1 μg/l (p=10-2) in females.

Figure 1

Distribution and correlation of serum ferritin concentration in 348 males (r=0.29) and 197 females (r=0.23) according to age. The equation of the fitted regression line was Y=39.9X-43.4 in males (light line) and Y=21.1X-217.2 in females (dark line).

The biochemical data of families were reviewed following the discovery of one sib pair HFE identical by descent, in which one sib exhibited total body iron overload and was clinically diagnosed as HH. Sex matched sibs, homozygous for the C282Y mutation, were reviewed to determine the degree of iron loading in the other sib through transferrin saturation percentage and serum ferritin and serum iron concentrations. Opposite sex sibs were not compared because iron overload is known to be higher in male subjects compared to females. Therefore, 18 C282Y homozygous same sex sib pairs were examined; this showed that transferrin saturation ranged between 39 and 98%, serum ferritin between 159 and 4900 μg/l, and serum iron from 12.5 to 48 μmol/l. Transferrin saturation, serum ferritin, and serum iron means in probands were 79.4% (SD 13), 1382 μg/l (SD 1348), 39.5 μmol/l (SD 6.4), respectively, and in sib cases 74.2% (SD 19), 1069 μg/l (SD 1166), and 38.1 μmol/l (SD 8.1), respectively; these results were not significantly different. The concordance of these parameters between sib pairs was also assessed. There was no correlation of serum ferritin or serum iron levels between the HH diagnosed sibs and other sibs, while transferrin saturation level tended to be correlated, but remained non-significant (p=0.07); the mean differences in transferrin saturation, serum ferritin, and serum iron values in sib pairs were all significant (table 2). In addition, no significant correlation was found between the oldest and the youngest sibs for the three iron markers when all same sex pairs were considered, indicating that, in this case, the lack of correlation was not related to the age of the subjects. This intrafamilial study showed a variable level of iron overload for subjects with HFE genotype identical by descent. In addition, among the 18 same sex sibs, two, six, and one sibs were in the normal range of values for transferrin saturation percentage (<43%), serum ferritin concentration (<300 μg/l), and serum iron concentration (<20 μmol/l), respectively.

Table 2

Comparison of 18 same sex sib pairs homozygous for the C282Y mutation

Moreover, in a family of five HFE identical sibs homozygous for the C282Y mutation, ranging in age from 53 to 61 years old, the sibs displayed variable biochemical expression of iron loading (fig 2). One 60 year old female had no significant increase of any of the iron parameters. Her two sisters (mother of four and two children, respectively) and two brothers, who were C282Y homozygotes, were affected with HH and showed iron overload according to their iron status parameters.

Figure 2

A familial case of a female (II.2) homozygous for the C282Y mutation without haemochromatosis. The genotypes are given in order C282Y, H63D, and S65C. + indicates the presence of the mutant allele and − the presence of the wild type allele.

Another case of discrepancy between genotype and phenotype was discovered in a female homozygous for the C282Y mutation; she had five children. Her husband was genotyped to evaluate the potential risk for the children of having HH. He was found to be homozygous for C282Y and his biochemical iron status at 61 years of age did not show any sign of iron loading (fig 3). Their son, 35 years old and C282Y homozygous, showed signs of iron overloading whereas of their four daughters, aged 30 to 40 years old, only the oldest showed raised transferrin saturation and serum iron concentration.

Figure 3

A male case (I.1) homozygous for the C282Y mutation without reaching iron status level for haemochromatosis. The genotypes are given in order C282Y, H63D, and S65C. +indicates the presence of the mutant allele and − the presence of the wild type allele.


The present study reports on the relationship between the biochemical expression of iron loading and the homozygous genotype for the C282Y mutation. Iron loading was first examined in a series of 545 probands homozygous for the C282Y mutation. The iron loading was significantly lower in females than in males whatever the parameter investigated; moreover the study confirmed the reduced penetrance of C282Y in females with a male to female sex ratio of 1.7:1 in probands with clinical HH. The biochemical expression of HH, lower in females than in males, indicated that some C282Y homozygous females may not develop signs of iron overload. In a family study, one case of a female identical by descent to four sibs homozygous for C282Y and diagnosed with HH did not reach the threshold values for iron overload expression defined for haemochromatosis, which indicated that the C282Y mutation did not show complete penetrance in females.

In this series, iron marker values ranged between normal and significantly increased; transferrin saturation percentage seemed to be the best parameter to predict haemochromatosis in young C282Y homozygous subjects whereas serum ferritin, the only value to increase progressively, was proved better to show overloading extent. This study thus confirmed that the extent of iron loading in haemochromatosis C282Y homozygotes is directly related to the age and sex of probands.23-25 However, when considering age range, large variations in iron status values were observed in subjects homozygous for C282Y mutation; serum ferritin showed the largest variation. We thus checked for intrafamilial variation of iron markers in sib pairs homozygous for the C282Y mutation. The lack of correlation between sibs and the significant differences of the iron marker values between sib pairs clearly showed a variable biochemical expression of iron overload in sibs with genotype identical by descent at theHFE locus. Although the expression of the disease is strongly influenced by the C282Y mutation, an intrafamilial study of subjects also confirmed that an identical genotype for theHFE gene can show variable iron loading; thus, intrafamilial variations in iron loading do not only represent a variation in the genetic expression of theHFE gene. The cases of non-expressing C282Y homozygous males aged over 60 years old, and displaying iron parameters in the normal range, as reported here and by others,26showed that the biochemical expression of HH is under the influence of other factor(s). In the families studied, transferrin saturation, serum ferritin, and serum iron variations could result from environmental and non-genetic factors or other genetic factors than those examined. Recent population studies have shown that, compared with non-carriers, any genotype group defined as carriers of aHFE mutation had higher mean serum iron concentration and mean transferrin saturation, whereas only C282Y homozygotes showed higher serum ferritin concentration23; they also highlighted that some C282Y heterozygotes could be diagnosed as HH.22 Thus, despite a good correlation between HFE defined genotypes and phenotype in population studies,HFE genotyping does not show a clear level of iron loading in subjects and may have implications in genetic counselling.

  • Hereditary haemochromatosis (HH) is a common autosomal recessive disease characterised by progressive iron overload. The identification of the HFE gene and mutations involved in haemochromatosis allows direct genetic testing for diagnosis. However, correlations between phenotype and HFE genotypes showed discrepancies and mutation penetrance raises questions.

  • We examined the iron loading status in 545 probands and 18 same sex sibs, all homozygous for the C282Y mutation.

  • Our data support transferrin saturation percentage and serum ferritin concentration as the best biochemical iron marker for HH phenotype in young subjects and extent of overload in these patients, respectively. The results also confirm a clear correlation of the iron loading level with age and sex of the patients. However, the lack of correlation of the iron marker status between pairs of sibs, homozygous for C282Y identical by descent, indicated a variable phenotypic expression of iron loading independent of HFE genotype.


This work was supported by INSERM grants from CRI 9607 and Association de Transfusion Sanguine et de Biogénétique Gaetan Saleun.