Prof. Marc De Braekeleer, Laboratoire d'Anthropologie et de Démographie Génétiques, Université Victor Segalen Bordeaux 2, 3ter, Place de la Victoire, 33076 Bordeaux cedex, France. E-mail: firstname.lastname@example.org.
A mutation analysis of the HFE gene followed, when applicable, by sequencing was performed on 47 patients with hereditary haemochromatosis (HH) living in Saguenay–Lac-Saint-Jean. The C282Y and H63D mutations were present on 50% and 20·3% of the HH chromosomes respectively. These frequencies were very different from those found in other populations and could be, at least partially, the result of a founder effect. No new mutation was identified among the remaining 28·1% of the HH chromosomes. Five of the eight probands with no mutation in the HFE gene had a severe and early onset suggestive of juvenile haemochromatosis.
Hereditary haemochromatosis (HH) is the most common autosomal recessive disorder in individuals of north European descent, with an estimated homozygote frequency of 1/300 and a carrier rate of 1/7 ( Bothwell et al, 1989 ; De Braekeleer, 1993).
It is caused by an inborn error of iron metabolism, leading to increased intestinal absorption of iron and progressive overload. HH patients accumulate iron in parenchymal organs, which is responsible for damage such as liver cirrhosis, diabetes, heart failure and hypogonadism. Early diagnosis and treatment can restore a normal life expectancy ( Bothwell et al, 1989 ).
Several hereditary disorders have been shown to have a high frequency in Saguenay–Lac-Saint-Jean (SLSJ), a geographically isolated region located 200 km north-east of Quebec City and surrounded by tracts of unsettled land and forests ( De Braekeleer, 1991, 1995). At the beginning of the 1990s, we conducted a clinical, biochemical and genetic study on 30 families with HH in the region ( De Braekeleer et al, 1991 , 1992a,b, 1993; De Braekeleer & Simard, 1995).
Based on HLA typing, the gene frequency was estimated at 0·12 (homozygote frequency at 0·014, heterozygote frequency at 0·21). The values of the mean inbreeding and kinship coefficients were much higher than those found in other HH populations or in subpopulations with autosomal recessive disorders in SLSJ ( De Braekeleer, 1995; De Braekeleer & Gauthier, 1996). Seven consanguineous marriages were identified; they included one marriage between first-degree cousins, one between first-degree cousins once removed and three between second-degree cousins.
Although the clinical manifestations of HH were similar to those described previously, the mean age at diagnosis of the HH probands was much lower than usually reported (36 vs. 50 years) and was associated with a greater severity at a young age. Our results suggested that the high variability in the phenotype was the result not only of mutation variability, but also of the action of modifying genes and/or other genes involved in iron metabolism ( De Braekeleer & Simard, 1995).
Given our previous results, we decided to analyse the mutations in the HFE gene among 47 individuals with HH identified by strict diagnostic criteria and regularly followed in SLSJ.
PATIENTS and METHODS
All the haemochromatosis patients followed on a regular basis for phlebotomies at the Department of Haematology of the Complexe Hospitalier de la Sagamie were invited to participate in the present study. The diagnosis was made on the basis of clinical history, physical examination and persistently raised iron indices (% transferrin saturation and serum ferritin). Liver biopsy was performed in at least one affected member of each sibship.
Forty-seven patients participated; they represented 97·9% of those currently followed. They included 32 probands and 15 affected siblings. We also obtained blood samples from four parents.
DNA was extracted using standard methods from the blood of these 51 individuals. All three substitutions (C282Y, H63D and S65C) were analysed as described previously ( Mura et al, 1997 , 1999). All seven coding exons and exon–intron junctions of the HFE gene were sequenced after specific amplification of the relevant region by polymerase chain reaction (PCR). The PCR reactions were carried out under standard conditions, and the amplified products were sequenced by the termination dideoxynucleotide chain termination method, as described previously ( Mura et al, 1997 , 1999).
Thirty-two probands were genotyped ( Table I). The frequency of the C282Y mutation was 50%, the H63D mutation being present on 20·3% of the HFE chromosomes. Homozygosity for the C282Y mutation was seen in 14 of the 32 probands (43·8%) Four probands (12·5%) were homozygous for the H63D mutation, whereas three (9·4%) were compound heterozygous for both mutations. The S65C mutation was identified in a heterozygous state in one proband.
A high proportion (28·1%) of haemochromatosis chromosomes carried an unidentified mutation. The coding exons and exon–intron junctions of the HFE gene of the patients carrying at least one unidentified mutation were sequenced. No new mutation was identified.
More than one sibling had haemochromatosis in 11 families. Identical genotypes were found among affected members in seven sibships, but not in the remaining four sibships (nine HH patients). All nine patients agreed to give a second blood sample to check their results; they were identical to those found previously. We also obtained blood from the four living parents in these sibships for mutation analysis. The results are presented in Fig 1.
Although both sibs in family 7 shared the same HLA haplotypes, the genotypes they carried were discordant, in that one brother was homozygous for the C282Y mutation and the second compound heterozygous for the C282Y and H63D mutations. In two sibships (families 15 and 27), one affected individual carried two unidentified mutations in the HFE gene, whereas their respective siblings were heterozygotes for the C282Y mutation. The proband in family 15 was diagnosed at 22 years old after a blood check-up for unspecified symptoms and skin hyperpigmentation. His brother, who was 25 years old at the time of family screening (with HLA), had no clinical signs, but had a transferrin saturation of 100%. Both had identical HLA haplotypes. The proband in family 27 was diagnosed at 28 years old; she had had amenorrhoea for the past 2 years and had liver cirrhosis at the time of the diagnosis. Her younger sister had amenorrhoea since she was 19 years-old; she also had hypothyroidism and liver cirrhosis.
Two siblings in family 36 were homozygous for the C282Y mutation, while a third was a compound heterozygote for the C282Y and H63D mutations. Their mother was also found to be a compound heterozygote. She had 13 children and is now 76 years-old. Presently, she has no clinical signs suggestive of haemochromatosis, and her iron parameters are normal.
Our results confirmed that the C282Y mutation in the HFE gene is strongly associated with haemochromatosis. It is notable that its frequency in the HH population of SLSJ is much lower than those observed thus far in other populations (Table I). The frequency of probands who were homozygous for C282Y was also much lower in SLSJ than elsewhere. In contrast, the frequency of the H63D mutation was much higher than reported previously in other studies (Table I), as was also the case for the frequency of patients homozygous for the H63D mutation.
Our results were consistent with our observations in cystic fibrosis, another highly prevalent autosomal recessive disorder in Caucasian populations. Although the mutations found in the SLSJ cystic fibrosis population are the same as those identified elsewhere, the frequencies of three of them are very different and probably due to a founder effect ( Rozen et al, 1990 , 1992). The same conclusions apply to haemochromatosis.
HH patients had discordant HFE genotypes in four sibships. A non-expressing HH parent is a likely explanation for the discrepancy observed in two sibships. Indeed, in one family (7), both parents died in their seventies and had no signs of haemochromatosis. The mother had 14 children, eight of them with transferrin saturation levels higher than 60% ( De Braekeleer et al, 1992a ). In another family (36), the mother was found to be a compound heterozygote. She had 13 children, five of whom had HH. Non-expressing homozygotes for the C282Y and compound heterozygotes for the C282Y and H63D mutations have already been described by others ( Crawford et al, 1998 ; Moirand et al, 1999 ).
In families 15 and 27, we cannot exclude the possibility that haemochromatosis was the result of mutations in another gene, the presence of the C282Y mutation in one sibling in each family being a result of the high frequency of the mutation in the general population. Both sisters in family 27 had a very severe and early onset consistent with juvenile haemochromatosis, the locus of which was recently mapped to chromosome 1 ( Cazzola et al, 1983 ; Roetto et al, 1999 ).
Eleven of the 32 HH probands had at least one unidentified mutation in the HFE gene. This was much higher than reported elsewhere (Table I). Several workers have attempted, without success, to identify new mutations in the HFE gene ( Feder et al, 1996 ; Beutler et al, 1997; Carella et al, 1997 ; Mura et al, 1997 ). The presence of patients without the C282Y or H63D mutations suggests that mutations in the non-coding sequences of the HFE gene or other loci may be implicated in the disease.
Most of the probands who had two unidentified mutations in the HFE gene had a milder disorder characterized by lower values of serum iron and transferrin saturation ( Datz et al, 1997; Sham et al, 1997 ; Mura et al, 1999 ). The situation might be different in SLSJ. Indeed, five of the eight probands with two unidentified mutations in the HFE gene had a severe and early onset suggestive of juvenile haemochromatosis. At the present time, we cannot exclude the presence of a cluster in SLSJ. Juvenile haemochromatosis has also seldom been identified in other studies ( The UK Haemochromatosis Consortium, 1997; Ryan et al, 1998 ).
In conclusion, our results differ from those relating to other populations in north America and Europe. The C282Y mutation has a lower frequency and the H63D mutation a higher frequency than reported previously. The differences could be result not only from a founder effect but also from the presence of a high number of families in which a gene for juvenile haemochromatosis may be isolated.
The authors thank Mrs Marielle Gagnon and Mr Jocelyn Tremblay for their invaluable help. This research was supported by INSERM grants from CRI9607, the Association de Transfusion Sanguine et de Biogénétique Gaëtan Saleun and a grant from the Décanat des Etudes de Cycles Supérieurs et de la Recherche at the Université du Québec à Chicoutimi.