Early detection of individuals with hereditary hemochromatosis (HH) is
important to manage iron levels and prevent future organ damage. Although theHFE mutations that cause most cases of HH
have been identified, their geographic distribution is highly variable, and their
contribution to iron overload is not fully understood. All new registered blood
donors at the Sahlgrenska University hospital between 1998 and 2015 were included in
the study. Donors with signs of iron overload at baseline and subsequent follow-up
testing were recommended genotyping of the HFE
gene. Of the 50,493 donors that were included in the study, 950 (1.9%) had signs of
iron overload on both test occasions. Of the 840 donors with iron overload that
performed HFE genotyping, 117 were homozygous for
C282Y, and 97 were compound heterozygotes. The prevalence of C282Y homozygosity was
0.23%. Iron overload screening effectively detects individuals at risk of carrying
the C282Y mutation of the HFE gene and enables
early treatment to prevent HH complications.
Hinweise
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Introduction
Hereditary hemochromatosis (HH) is caused by mutations in the HFE gene, leading to a low production of hepcidin
resulting in high uptake of iron from the intestine [1]. The subsequent iron-overload is often asymptomatic but may,
left untreated, lead to liver cirrhosis, diabetes mellitus, hypothyroidism, cardiac
arrhythmia and arthropathy [1]. The risk
of developing sequelae is further increased by environmental factors such as
excessive alcohol consumption and obesity [2].
Individuals homozygous for C282Y make up only 0.4% of the population
[3], but many of them will gradually
accumulate iron and eventually develop symptoms of the disease. The overwhelming
majority of patients with HH are either C282Y homozygotes or C282Y/H63D compound
heterozygotes. Around 70% of C282Y homozygotes have biochemical signs of iron
overload, with levels between 73 and 94% reported in males and 55 and 69% in females
[4‐7]. However, it
should be noted that these studies have used different cutoff levels for the
definition of iron overload.
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Early identification of individuals with HH is important, allowing for
monitoring of iron levels and the application of therapeutic phlebotomy when needed
to avoid further complications of the disease [8]. Presently, population screening for HFE mutation is not recommended due to unfavourable cost-benefit
ratio [3]. Evaluations of screening
approaches where risk groups with iron-overload are identified for subsequentHFE genotyping shows promising results
[9, 10], but the variability in both prevalence and penetrance of
C282Y mutations together with the relative scarcity of large iron-overload screening
studies highlights the need of further studies to assess the cost-benefit of
iron-overload screening for detection of individuals at risk of hereditary
hemochromatosis.
Therefore, the aim of the study was to investigate the feasibility and
usefulness of an iron-overload screening program to identify previously unknownHFE C282Y and H63y mutations in newly
registered blood donors. We will also evaluate how using different cutoff levels
will affect the ability of the screening program to identify HFE mutations.
Materials and methods
The Sahlgrenska Iron Overload Study (SIOS) was started in 1998 with the
aim of investigating causes and outcome of iron overload in blood donors. The study
was approved by the local ethics committee in Gothenburg (approval number: 593-17;
170930).
All new registered blood donors between 1998 and 2015 that fulfilled
criteria for blood donation and were not previously diagnosed with hereditary
hemochromatosis or had known HFE mutations were
included in the study. Eligibility for blood donation was established during the
first visit using structured interview, checklists and blood sampling with
subsequent analysis of s-Fe, s-total iron–binding capacity (TIBC) and s-ferritin. No
blood was donated during the first visit. All donors that fulfilled our criteria for
iron overload (transferrin saturation (TS) > 50%) were selected for subsequent
control measurement of TS% and s-ferritin (μg/L). Based on the results from the
control measurement, donors having TS > 50% or elevated s-ferritin (s-ferritin
> 130 for men/s-ferritin > 100 for women) were recommended HFE genotyping. In all, 50,493 blood donors were
screened, and 2864 were found to have TS > 50%. Of the donors with baseline TS
> 50%, 74% (2131 donors) returned for control measurement with a mean time
between baseline and control visit of 154 days. Control measurements were performed
prior to blood donation. Of the 950 donors with elevated levels of TS or s-ferritin,
840 (88%) were tested for the HFE C282Y and H63D
mutations. Levels of s-Fe, s-TIBC and s-ferritin were determined using standard
laboratory methods. Figure 1 illustrates the
inclusion and testing procedure.
×
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Genetic analyses
HFE C282Y and H63D were detected from EDTA whole-blood samples using
ABI 7500 Real-Time PCR system (Applied Biosystems). Allele discrimination was
performed using the ABI 7500 SDS software. Participants negative for C282Y and
H63D were designated wild type.
Statistical analyses
Patients were grouped according to HFE status. Demographic differences were analysed using the
unpaired t test (age) and χ2 (sex). Levels of iron
overload markers were compared between the groups with C282Y or H63D alleles and
the wild type group using the unpaired t
test. To evaluate diagnostic value for the identification of C282Y homozygotes,
we calculated sensitivity/specificity, positive/negative likelihood ratio and
positive/negative predictive value of different levels of TS% and s-ferritin
using cross-tabulation. All analyses were performed using IBM SPSS software
(version 19.0).
Results
As can be seen in Table 1, the
largest group of donors with iron-overload did not have either the C282Y or the H63D
mutations. The majority were male irrespectively of HFE status, but the male dominance was least pronounced in the C282Y
homozygous and C282Y/H63D compound heterozygous groups. These groups also have the
most pronounced iron-overload compared with the wild type group.
Table
1
Study
participants
C282/C282
C282/H63
H63/H63
C282/WT
H63/WT
WT/WT
N
117
97
31
125
131
339
Age
31.1 ± 10.6
29.1 ± 10.6
28.7 ± 11.3
28.6 ± 9.8
30.8 ± 11.2
29.1 ± 9.0
Age range
18–57
18–56
18–58
18–60
18–59
18–62
Male sex %
62**
75*
84
88
82
85
TS baseline
72.5 ± 13.6**
60.6 ± 10.7**
58.4 ± 8.8
57.1 ± 7.4
57.3 ± 7.6
57.1 ± 7.4
TS F-U
67.8 ± 16.2**
54.8 ± 16.3**
51.7 ± 14.6*
48.7 ± 14.5*
46.8 ± 14.5
44.5 ± 15.1
S-ferritin
383 ± 334**
204 ± 186**
170 ± 119
140 ± 89
161 ± 106
147 ± 89
Groups
carrying at least one allele of C282Y (C282) or H63D (H63) were compared
with wild type donors. Values are given as mean value ±
SD
TS
Transferrin saturation %. F-U
follow-up
*P
value < 0.05 vs wild type. **P
value < 0.001 vs wild type
Tables 2 and 3 shows participant characteristics divided by sex.
Again, the C282Y homozygotic group have the highest levels of iron deposits, but
only males in the C282Y/H63D group have elevated iron levels compared with the
wild-type group. The difference between the iron levels of the C282Y homozygotic and
C282Y/H63D compound heterozygotic groups and the wild type group is generally more
pronounced in the follow-up testing.
Table
2
Iron status andHFE mutations for male
participants
C282/C282
C282/H63
H63/H63
C282/WT
H63/WT
WT/WT
N
73
73
26
101
108
287
Age
30.9 ± 10.5
28.9 ± 10.3
27.5 ± 10.1
27.7 ± 8.6
29.9 ± 11.1
28.7 ± 8.8
Age range
18–57
18–53
18–57
18–54
18–59
18–62
Baseline
S-Fe
35.8 ± 7.3**
33.9 ± 6.1*
33.3 ± 6.7
32.0 ± 4.9
33.1 ± 5.4
32.5 ± 5.3
S-TIBC
47.1 ± 5.5**
55.6 ± 6.2
56.0 ± 5.2
55.8 ± 6.4
57.5 ± 6.9
57.0 ± 6.7
TS
76.0 ± 12.2**
61.3 ± 11.3**
59.4 ± 9.2
57.4 ± 7.8
57.6 ± 8.0
56.9 ± 7.7
Follow-up
S-Fe
32.5 ± 8.3**
30.3 ± 9.8**
29.7 ± 7.9*
26.9 ± 8.1
26.7 ± 8.5
25.1 ± 9.1
S-TIBC
47.4 ± 6.8**
55.5 ± 6.4*
57.1 ± 5.8
55.8 ± 6.5
58.0 ± 7.0
57.4 ± 7.3
TS
69.1 ± 16.5**
55.0 ± 17.2**
51.7 ± 14.6*
48.4 ± 13.8*
46.1 ± 13.9
43.9 ± 15.0
S-ferritin
478 ± 324**
231 ± 147**
179 ± 127
150 ± 88
174 ± 110
159 ± 90
Groups
carrying at least one allele of C282Y (C282) or H63D (H63) were compared
with wild type donors. Values are given as mean value ±
SD
S-Fe
Serum iron. S-TIBC serum total iron
binding capacity. TS transferrin
saturation %. F-U follow-up. S-Fe and
S-TIBC are reported as μmol/L, S-ferritin is reported as
μg/L
*P value
< 0.05 vs wild type. **P value
< 0.001 vs wild type
Table
3
Iron status andHFE mutations for female
participants
C282/C282
C282/H63
H63/H63
C282/WT
H63/WT
WT/WT
N
44
24
5
24
23
52
Age
31.4 ± 11.0
30.1 ± 11.5
35.0 ± 16.2
32.0 ± 13.3
35.5 ± 10.8
31.1 ± 9.5
Age range
18–53
18–56
21–58
18–60
21–58
19–51
Baseline
S-Fe
32.9 ± 7.5
32.2 ± 5.5
31.2 ± 3.9
32.0 ± 5.4
32.8 ± 6.4
32.8 ± 5.0
S-TIBC
49.6 ± 8.6**
55.3 ± 6.0
58.8 ± 7.0
57.2 ± 7.8
60.4 ± 10.4
57.0 ± 8.6
TS
66.8 ± 13.9**
58.3 ± 8.3
53.0 ± 1.6
55.9 ± 5.7
56.3 ± 5.5
57.7 ± 5.9
Follow-up
S-Fe
31.4 ± 7.5*
28.9 ± 7.3
31.6 ± 15.1
28.2 ± 9.9
29.4 ± 10.6
26.9 ± 9.8
S-TIBC
48.2 ± 6.9**
52.3 ± 7.9*
61.4 ± 6.1
57.0 ± 7.0
58.5 ± 7.7
56.8 ± 9.1
TS
65.7 ± 15.7**
54.2 ± 13.5
52.0 ± 14.6
49.8 ± 17.6
49.9 ± 16.6
47.6 ± 15.4
S-ferritin
230 ± 294**
125 ± 260
124 ± 55*
97 ± 81
102 ± 57.3
79 ± 48
Groups
carrying at least one allele of C282Y (C282) or H63D (H63) were compared
with wild type donors. Values are given as mean value ±
SD
S-Fe
Serum iron. S-TIBC serum total
iron-binding capacity. TS transferrin
saturation %. F-U follow-up. S-Fe and
S-TIBC are reported as μmol/L, S-ferritin is reported as
μg/L
*P value
< 0.05 vs wild type. **P value
< 0.001 vs wild type
A comparison between the SIOS participants that fulfilled the criteria
for HFE genotyping and the general population is
displayed in Fig. 2. All groups carrying a
mutation, with the exception for H63D/WT, were more prevalent in the SIOS group. The
C282Y homozygous and C282Y/H63D compound heterozygous groups showed the highest
overrepresentation compared with expected prevalence.
×
Table 4 displays a comparison
of different TS% cutoff values for the discovery of C282 homozygotes. Positive
likelihood ratio increased with increasing cutoff levels in both men and women with
the highest levels seen for s-ferritin > 350 μg/L in men and s-ferritin
> 150 μg/L in women. Defining iron overload as TS > 50% and assuming 71%
penetrance of iron overload in C282 homozygotes, we performed a cross tabulation on
the entire cohort resulting in high specificity and positive likelihood
ratios.
Table
4
Comparison of different TS%
cutoff values for the identification of C282
homozygotes
Sens.
Spec.
+ LR
− LR
PPV
NPV
Men
F-U TS% > 50
84
56
1.9
(1.7–2.2)
0.3 (0.2–0.5)
18 (16–20)
97
(95–98)
F-U TS% > 55
76
70
2.5
(2.1–3.0)
0.3 (0.2–0.5)
22 (19–26)
96
(94–98)
F-U TS% > 60
71
80
3.5
(2.8–4.4)
0.4 (0.3–0.5)
29 (24–34)
96
(94–97)
F-U s-ferritin
> 130
93
36
1.5 (1.3–1.6)
0.2
(0.1–0.5)
15
(14–16)
98
(95–99)
F-U s-ferritin
> 350
63
94
10 (7.2–14)
0.4
(0.3–0.5)
56
(47–64)
96
(94–97)
Women
F-U TS%
> 50
81
38
1.3 (1.1–1.6)
0.5
(0.3–0.9)
31
(27–35)
86
(76–92)
F-U TS% > 55
77
61
2.0
(1.5–2.6)
0.4 (0.2–0.7)
40 (33–46)
89
(81–93)
F-U TS% > 60
60
76
2.5
(1.7–3.7)
0.5 (0.4–0.8)
46 (37–56)
85
(79–89)
F-U s-Ferritin
>100
64
59
1.5 (1.1–2.1)
0.6
(0.4–0.9)
35
(28–42)
82
(75–88)
F-U s-Ferritin
>150
41
88
3.4 (1.9–6.2)
0.7
(0.5–0.9)
55
(40–68)
81
(40–68)
Whole cohort assuming 71% penetrance of
iron-overload in C282
homozygotes
The Sahlgrenska iron-overload study successfully screened 50,493 blood
donors for iron-overload and was able to identify 117 donors that were homozygous
for C282Y. The screening process considerably reduced the number of donors
fulfilling the criteria for HFE genotyping,
resulting in 840 (1.7%) donors ultimately genotyped. C282Y homozygotes and
C282Y/H63D compound heterozygotes were highly overrepresented in the group that was
genotyped compared with previous reports on the prevalence of C282Y and H63D alleles
in the general population [11].
C282Y homozygotes made up 14% of the 1.7% of the cohort that performedHFE typing, indicating that the screening
procedure produced a group with a high number of mutation carriers. The 117 C282Y
homozygotes identified correspond to a prevalence of 0.23% in the screened cohort.
Although the prevalence of C282Y and H63D alleles is highly variable across
geographic regions in the world [12],
studies on subjects with similar ancestry as ours have reported prevalence of C282Y
homozygotes between 0.30 and 0.75% [4‐6, 11, 13, 14]. Applying an iron-overload penetrance of 71% in homozygotes,
[4‐7], results in an
estimation of 165 homozygotes in the cohort corresponding to a prevalence of 0.33%
which is at the lower end of previously reported values. The estimated prevalence is
likely too low, possibly reflecting a lower iron-overload penetrance in our young
and healthy study population.
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The levels of TS% and s-ferritin differed between the groups that
fulfilled the screening criteria. The highest levels were seen in the C282Y
homozygotes and C282Y/H63D groups. Previous studies that have investigated iron
levels and HFE status in the population without
applying screening criteria have found similar levels of TS% and s-ferritin among
C282Y homozygotes as we found in our screened group [4, 5, 11]. The other groups, however, have lower
levels of TS% and s-ferritin in studies without screening criteria leading us to
conclude that the applied conditions for eligibility for HFE genotyping in the SIOS mainly discriminate donors with HFE mutations less strongly linked to hemochromatosis.
Further support for this conclusion can be found when comparing the composition of
the genotyped group in the SIOS compared with what has been reported in the general
population (Fig. 2). The C282Y homozygous
group (11.5%) and the C282Y/H63D compound heterozygotic group (13.9%) were highly
prevalent in our iron-overload group compared to reported prevalence in the
population [4, 5, 11]. Thus we conclude that the screening process was an efficient
tool to select a group of individuals where relevant HFE mutations can be expected to be highly overrepresented.
Finding the correct cutoff value for inclusion into a screening program
is fundamental. Similar screening studies have employed varying cutoff levels,
ranging between TS > 45 and TS > 55%. In retrospect, it would have been useful
to have had a lower cutoff of TS > 45% in the SIOS to better evaluate the varying
cutoff levels that have been used in previous studies, and also because recent
findings have shown that TS > 45% may be the best cutoff point for identifying
C282Y homozygotes [15], which is also
reflected in recent recommendations [16]. Nonetheless, the screening method employed in the SIOS using TS
> 50% yielded a group with high prevalence of C282Y homozygotes. When applying a
71% penetrance of iron-overload in C282Y homozygotes in the whole cohort, we found
that the screening process resulted in high sensitivity and positive predictive
values, especially so when applying a two-step approach with control measurements.
However, there may be situations, such as in large population screening or where a
low overall cost for the program is necessary, where a high positive predictive
value is more important than a high sensitivity. In those situations, based on our
findings, it may be advantageous to increase the follow-up TS cutoff to 55% in both
men and women resulting in a substantial increase in specificity at the cost of a
small reduction in sensitivity. It should be noted, however, that it may not be
necessary to identify all C282Y homozygotes as C282Y homozygotes without signs of
iron overload seem to be at low risk of developing HH complications [17]. The trend towards increased HFE typing in individuals without biochemical signs of
iron overload [18] highlights the need
for the establishment of iron overload prior to genotyping for a more favourable
cost-benefit ratio.
The contribution of mutations other than C282Y homozygotes to iron
overload is not fully understood. Our findings that blood donors with iron overload
are more likely to be C282Y/H63D compound heterozygotes, C282Y heterozygotes or H63D
homozygotes is in line with previous studies [19]. The relative low penetrance of these mutations on iron
overload has not yet been determined, and the probable cause is genetic and
environmental factors. Additionally, C282Y heterozygotes may carry rare mutations
contributing to iron overload [20].
Limitations
Blood donors may not be representative for the general population.
Although the majority of HFE mutation
carriers are asymptomatic and that the SIOS cohort is young (mean age 29.6) and
may not have had time to develop symptoms, it is possible that signs of the
disease may have discouraged some individuals from blood donation resulting in
an underestimation of the prevalence of HFE
mutation carriers. Additionally, although blood donors are required to have a
good command of Swedish and minorities are believed to be underrepresented as
donors, we do not record ethnic origin of blood donors which could have affected
the mutation frequencies. A limitation of the present study is also the lack of
data for other mutations than C282 and H63. Although the C282 HFE mutation is the principal cause of HH, other
mutations may also give rise to iron overload [21]. Another issue with the study design is the lack of
standardization of test setting. The blood for laboratory analyses were drawn
when the participant registered to become a blood donor, which could have
happened any time during the day. As circadian rhythms may potentially affect
the results [22], it would have
been preferable to standardize the blood collection reflecting this.
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Conclusions
Iron overload screening using TS% effectively identifies a population
with high prevalence of C282Y and H63D mutation carriers, enabling monitoring and
early treatment to prevent HH complications.
Acknowledgements
We would like to thank blood donors, staff and laboratory personnel at the
department of immunology and transfusion medicine at Sahlgrenska University
Hospital.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Disclaimer
The funding sources were not involved in the drafting of this
manuscript.
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