ORIGINAL ARTICLE
Primary haemochromatosis: a missed cause
of chronic fatigue syndrome?
D.W. Swinkels1*, N. Aalbers4, L.D. Elving2, G. Bleijenberg3, C.M.A. Swanink5,
J.W.M. van der Meer2**
Departments of 1Clinical Chemistry 564, 2Internal Medicine and 3Medical Psychology,
University Medical Centre St Radboud, PO Box 9101, 6500 HB, Nijmegen, the Netherlands,
tel.: +31 (0)24-361 47 77, fax: +31 (0)24-24354 17 43, e-mail: [email protected],
4
Department of Internal Medicine, 5Laboratory of Medical Microbiology and Medical Immunology,
Rijnstate Hospital, PO Box 9025, 6800 EG Arnhem, the Netherlands, * corresponding author
ABSTRACT
INTRODUCTION
Objective: To determine whether patients previously
diagnosed as chronic fatigue syndrome (CFS) actually
have primary haemochomatosis (PH).
Primary haemochomatosis (PH) is one of the most common
genetic disorders known in subjects of northern European
descent.1,2 The identification of the C282Y mutation in
the HFE gene in most patients with PH has been a major
advance, resulting in a diagnostic genotypic test for this
form of iron overload.3 As much as 10% of the population is
heterozygous for the C282Y mutation, and the homozygous
state is believed to affect as much as 0.5% of the population,
or more than 80,000 people in the Netherlands.2 In some
patients another mutation, the H63D mutation in the
HFE gene on one chromosome, has been associated with
PH in combination with the C282Y mutation on the
other chromosome.4
In affected individuals, inappropriately increased absorption
of iron may result in its progressive accumulation in the
liver, heart, pancreas and other organs, eventually producing
hepatic cirrhosis, cardiac failure, diabetes mellitus, arthritis,
gonadal dysfunction and other disorders.1,5-8 Suggestive
laboratory findings of PH are persistently increased transferrin saturation and ferritin values, reflecting circulating
and body iron levels, respectively.6,9 In the past, iron overload was confirmed by the detection of increased hepatic
iron in a liver biopsy specimen.6,8 With the availability of
genetic testing, haemochomatosis can be confirmed by
genotyping.6,8 Population studies suggest that the disorder
is greatly underdiagnosed.10 If patients are identified early
in the course of the disease, removal of the excess iron
by phlebotomy can prevent the subsequent development
of irreversible tissue damage and restore normal life
expectancy.7
Although PH varies in clinical severity, its most commonly
Methods: The setting was a Dutch referral centre.
Transferrin saturation (TS) was retrospectively evaluated
in banked blood samples of 88 patients diagnosed as CFS.
Patients with elevated TS values were asked to provide a new
overnight fasting blood sample for a second determination
of TS and measurement of serum ferritin. The DNA was
investigated for mutations in the HFE gene when one of
these iron parameters was elevated.
Results: For 19 out of 88 patients with CFS an elevated TS
was found. A new blood sample was obtained from 11 of
these 19: six had increased TS and two had elevated serum
ferritin values. These eight patients were neither C282Y
homozygotes nor compound C282Y-H63D heterozygotes.
In the eight cases where no new blood samples could be
obtained, the TS was >50% for two of the five men and
<45% for the three female patients.
Conclusion: In a group of 88 CFS patients we could exclude
PH in all but two of them (prevalence 2.3%; 95% confidence
interval 0-5.5%). In our population of CFS patients PH is not
more common than in a control population of northern
European descent (prevalence 0.25-0.50%).
** J.W.M. van der Meer was not involved in the handling and review process of this paper.
© 2002 Van Zuiden Communications B.V. All rights reserved.
DECEMBER 2002, VOL. 60, NO. 11
429
presenting feature is fatigue.5 Therefore, the question arises
whether patients previously diagnosed as suffering from
chronic fatigue syndrome (CFS) actually have PH. CFS is
characterised by severe disabling fatigue of at least six
month’s duration,11 which has led to considerable impairment
in daily functioning, and for which no explanation has been
found. To date, only anecdotal information is available on
patients with PH originally being misdiagnosed as CFS.12
In 1992 a cohort of 88 CFS patients was studied in our
outpatient clinic for aetiological factors.13 As at that time
the iron status of the of CFS patients was not tested, PH
was not excluded as a cause of their fatigue. In this study
we therefore aimed to determine the prevalence of primary
haemochomatosis among 88 patients who had previously
been diagnosed as having CFS.
studied at the Department of General Internal Medicine
of the University Medical Centre St Radboud, Nijmegen,
a Dutch tertiary CFS referral centre.13,15 The medical
ethics committee approved the study. All 88 patients were
self-referred and gave permission to store serum for future
CFS studies. In 1992 the mean age of the 88 patients was
40 (SD 10.7 years, range 20-66 years); the male to female
ratio was 1:3 (23 males and 65 females).13 All had normal
serum chemistry (minerals, kidney and liver function
tests) and haematological tests. The C-reactive protein
was low (<10 mg/l) in all patients. Of the patients, 65%
were taking medication (mainly analgesics, vitamins and
homeopathic drugs).13 Samples were stored at -80°C.
In 1999, the preserved non-fasting samples of these patients
were studied for transferrin saturation (TS) and, the quantity
of material allowing, serum ferritin concentration. All of
the patients with elevated TS values (female (f) >40% and
male (m) >45%) that we could locate (n=15) received a
questionnaire and were asked to provide a new overnight
fasting blood sample for a second determination of TS
and the measurement of serum ferritin (table 1, figure 1).
METHODS
Study design
In 1992, 88 patients fulfilling the criteria for CFS14 were
Table 1
Demographic, laboratory and clinical characteristics of 19 CFS patients with elevated transferrin saturation (TS) in
banked serum samples from 1992 and fresh collected samples in 1999
PATIENT
NO.
ALAT
U/L
1999
GT
U/L
1999
TS1
%
1992
2
12
5
60
46
49
_
_
_
43
_
_
_
_
44
_
_
18
15
45
35
16
24
49
32
HB
CRP
MMOL/L MG/L
1999
1999
TS1
%
1999
SEX
AGE
(YEARS)
1999
FERRITIN
G/L
1992
1
F
40
7.8
24
F
53
_
34
F
40
_
_
4
M
50
8.9
1
5
F
49
8.9
14
6
F
60
8.4
<5
25
14
46
41
75
M
43
10.6
9
28
41
45
34
84
F
50
_
_
_
_
40
_
_
9
F
44
7.8
<5
14
10
51
66
40
10
F
59
8.7
5
20
11
50
52
61
11
F
46
7.4
<5
17
13
47
30
31
124
M
50
_
_
_
_
98
_
13
F
55
9.2
2
39
16
40
32
FERRITIN
G/L
1999
HFE
MUTATION
282/63
19992
SYMPTOMS
199219993
42
+-/—
≈
_
_
?
_
_
?
_
128
_
≈
158
249
—/—
↓
97
114
+-/—
↓
294
426
+-/—
↓
_
_
?
30
—/+-
↓
60
+-/—
↓
16
_
↓
246
_
_
?
_
39
_
≈
14
F
74
8.2
1
23
17
42
40
_
101
—/—
↑
156
M
52
_
_
_
_
55
_
153
_
_
↑
166
M
48
_
_
_
_
46
_
35
_
_
↓
17
F
62
7.9
2
42
33
42
43
_
167
—/—
≈
187
M
28
_
_
_
_
46
_
159
_
_
↓↓
197
M
72
_
_
_
_
46
_
100
_
_
↓↓
1
TS = transferrin saturation, criteria used for increased levels: m >45%, f >40%, 2 +/- heterozygote, — wildtype, 3 subjective change in severity of fatigue in 1999
in comparison with 1992, ≈ unchanged, F = female, M = male, 4,6,7 patients who did not provide a new blood sample in 1999, 4,7 patients who did not fill in the
questionnaire, 4 patients who could not be located in 1999, 5 consumption of three to five alcoholic units per day in 1999, 6 patients did not donate blood because
they were too busy, but they did fill in the questionnaire, 7 patients neither donated blood nor filled in questionnaire because they had no symptoms of CFS.
Swinkels, et al. Primary haemochromatosis: a missed cause of chronic fatigue syndrome?
DECEMBER 2002, VOL. 60, NO. 11
430
m <50 and f <35 U/l) serum iron (SI) and total iron binding
capacity (TIBC) were measured on a Hitachi 474 analyser
(Roche Diagnostics). TS is expressed as the ratio of serum
iron concentration and total iron binding capacity. Serum
ferritin levels were determined on the Immulite 1 of DPC
(Diagnostic Product Corporation) using a two-sided
immunometric assay (references values: m 15-280 g/l,
pre- and postmenopausal f 6-80 g/l and 15-190 g/l,
respectively).
88 Banked serum samples of CFS patients
Transferrin saturation (TS)
Possible PH: 19 patients TS ↑1,2
Request for blood
donation in 1999
8 Non-responders
11 Responders
3 f TS 40-45%1
3 m TS 45-50%1
2 m TS >50%1,3,4
TS and ferritin
DNA tests
DNA was isolated from EDTA blood samples using the
QIAmp Kit (Qiagen Ltd.). Genotyping of the C282Y and
H63D mutation was based on two separate polymerase chain
reaction (PCR) amplifications of the DNA spanning the
C282Y locus using the PCR-primers reverse (5’-TACCTCCTCAGGCACTCCT-3’) and forward (5’-TGGCAAGGGTAAACAGATCC-3’), and the primers spanning the H63D locus
reverse (5’-GCCTCAGAGCAGGACCTTGG-3’) and forward
(5’-CAGCTGTTTCCTTCAAGATGC-3’), respectively.3,16
Amplification was carried out on the thermocycler GeneAmp
PCR system 9700 (Applied Biosystems). Amplification
products were digested with RsaI for the C282Y mutation
and BsphI for the H63D mutation and visualised by
agarose gel electrophoresis.17
6 x TS ↑
2 x ferritin ↑5,6
0 x TS ↑ and ferritin ↑
DNA tests (n=8)
R E S U LT S
C282Y/C282Y none
C282Y/H63D none
From the original 1992 cohort, 19 out of 88 CFS patients
had increased serum TS levels upon retrospective evaluation
of stored blood samples (figure 1, table 1). In 1999, 13 of these
19 patients filled in questionnaires. Two additional patients
refused to return the questionnaires because they had no
symptoms of CFS. Nine, four and two patients reported
diminished, unchanged and deteriorated symptoms,
respectively, of CFS since 1992 (table 1). One out of 13
patients consumed three to five alcoholic units per day
(patient 7), while the other 12 patients drank less than that.
This patient had the highest ferritin level with a normal
TS.
Eleven of the 19 patients with a retrospectively detected
elevated TS provided a new blood sample in 1999, of
whom six had increased TS and two had elevated serum
ferritin values (figure 1, table 1). None of the patients had
both. The eight patients with either increased fasting TS
or ferritin levels were neither C282Y homozygotes nor
compound C282Y-H63D heterozygotes (figure 1, table 1).
All patients had normal Hb, CRP, ALAT and GT levels,
except for a slightly increased CRP (14 mg/l) for patient 5
and a relatively high and low Hb for patients 7 and 11,
respectively (table 1). Remarkably, both the patients with
increased ferritin levels of 249 g/l (patients 5, CRP 14 mg/l)
and 426 g/l (patient 7, CRP 9 mg/l), respectively, reported
Figure 1
Results of diagnostic work-up of CFS patients for primary
haemochomatosis
CFS = chronic fatigue syndrome, PH = primary haemochomatosis,
TS = transferrin saturation, m = male, f = female, 1 values obtained in
1999 using stored blood samples from 1992, 2 m >45%, f >40%, 3,4,5,6
patients 12, 15, 5 and 7, respectively (table 1).
The questionnaire contained questions on changes in
the severity of their fatigue since 1992 and alcohol
consumption. Patients were asked to abstain from
multivitamins, vitamin C and iron supplements
24 hours before the blood was taken. The DNA was
investigated for mutations in the HFE gene if either
TS (m >45%, f >40%) or ferritin levels were elevated
(figure 1).
Laboratory tests
C-reactive protein (CRP, normal value <10 mg/l), liver
function (ALAT, normal value <45 U/l; GT, normal value
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DECEMBER 2002, VOL. 60, NO. 11
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