| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
Spectrum of mutations in the HFE gene implicated in haemochromatosis and porphyria
Introduction
Results
Mutation analysis
Analysis of patients referred for a molecular diagnosis of HH
Family study
Analysis of patients referred for a molecular diagnosis of VP
Discussion
Materials And Methods
Patients and controls
DNA analysis
Statistical analysis
Abbreviations
Acknowledgements
References
Spectrum of mutations in the HFE gene implicated in haemochromatosis and porphyria
Received March 29, 1999; Revised and Accepted April 30, 1999
Mutation analysis was performed on DNA samples of 965 individuals from four different ethnic groups in South Africa, in an attempt to determine the spectrum of sequence variants in the haemochromatosis (HFE) gene. This population screening approach, utilizing a combined heteroduplex and single-strand conformation polymorphism (HEX-SSCP) method, revealed three previously described and four novel missense mutations. Novel variants V53M and V59M were identified in exon 2, Q127H in exon 3 and R330M in exon 5. The exon 5 variant was identified in one of 13 patients referred for a molecular diagnosis of hereditary haemochromatosis (HH), who tested negative for the known C282Y and H63D mutations. Mutation Q127H was detected in exon 3 of the HFE gene together with mutation H63D in an apparently severely affected patient previously shown to carry the protoporphyrinogen oxidase (PPOX) gene mutation R59W, which accounts for dominantly inherited variegate porphyria (VP) in >80% of affected South Africans. The mutant allele frequency of the C282Y mutation was found to be significantly lower in 73 apparently unrelated VP patients with the R59W mutation than in 102 controls drawn from the same population (P = 0.005). The population screening approach used in this study revealed considerable genotypic variation in the HFE gene and supports previous data on the involvement of this gene in the porphyria phenotype.
INTRODUCTION
Hereditary haemochromatosis (HH) is considered to be the most common autosomal recessive disorder in the Caucasian population of Northern European descent. The incidence of HH is estimated to be ~1/300 in this population, with a carrier frequency of 1/8-1/10 (1,2). The disorder leads to excess iron storage in the parenchymal cells of major organs, primarily the liver, pancreas and heart, and can lead to severe tissue damage and premature death if left untreated (3).
Two human haemochromatosis (HFE) gene mutations, C282Y and H63D, have been identified in 60-100% of clinically diagnosed HH patients (4-6). Mutation H63D is associated with iron overload in the compound heterozygous state with the C282Y mutation (7,8). Both these mutations have also been linked to porphyria cutanea tarda (PCT), one of the cutaneous porphyrias presenting with hepatic siderosis and iron overload (9-11). This relationship recently has been confirmed in South African patients with PCT (12,13). Mutations H63D and C282Y have not been detected in a small cohort of Southern African subjects diagnosed with African iron overload (14).
A worldwide frequency of ~1.9% was reported for the mutant C282Y allele compared with 8.1% for the H63D mutation (15). Mutation C282Y appears to be absent in Africans, while mutation H63D was detected at a very low frequency in some non-Caucasoid populations. DNA screening data of individuals from the general South African population were in accordance with this finding and demonstrated a carrier frequency of ~17% (mutant allele frequency 9%) for the C282Y mutation among Caucasians, implying that up to 1/115 South Africans of European descent may be homozygous for this founder-type mutation (6). Since the initiation of a molecular diagnostic service for HH in South Africa in 1997, 244 subjects from 215 apparently unrelated families have been referred for DNA analysis. Seventy-seven per cent of unrelated patients clinically diagnosed with HH were homozygous for the C282Y mutation, 14% were compound heterozygotes for mutations C282Y and H63D, and 9% of HH referrals did not exhibit either mutation (6). C282Y- and H63D-negative HH patients may either indicate the presence of unknown disease-causing mutations in the HFE gene or represent unrecognized cases of secondary iron overload and, hence, an alternative diagnosis.
In this study, we included a group of 13 mutation-negative South African patients referred for H63D and C282Y mutation screening, in order to determine whether additional HFE gene mutations may contribute to their abnormal iron profiles. In light of the fact that the two known HFE mutations have also been associated with sporadic PCT, we included 236 patients referred for a molecular diagnosis of variegate porphyria (VP), the most common form of porphyria in South Africa (16). The rationale for including the VP referrals was to assess whether variation in the HFE gene might possibly contribute to the disease phenotype in these patients. VP is very common (3/1000) in the Afrikaner population of South Africa due to a founder effect (16), and the protoporphyrinogen oxidase (PPOX) gene mutation R59W has been shown to be responsible for the disease in >80% of affected patients (17,18). Screening for mutation R59W in clinically defined VP families demonstrated that this mutation has variable penetrance (L. Warnich and J.N.P. de Villiers, unpublished data), confirming previous reports that VP may be unexpressed in up to 80% of cases (19). A number of probands with mutation R59W, however, appeared to be more severely affected than could be ascribed to a single copy of the mutation, implying that other genetic and/or environmental factors may contribute to the disease phenotype.
RESULTS
Mutation analysis
Twelve sequence changes, including eight exonic and four intronic variants, were detected in the study population (Table 1). Aberrations identified in exon 2 by combined heteroduplex and single-strand conformation polymorphism (HEX-SSCP) analysis are demonstrated in Figure 1. A restriction enzyme recognition site was either created or abolished as a consequence of the sequence change in two of the newly described missense mutations and all of the intronic variants. Where possible, restriction enzyme analysis was performed to verify the sequence changes in polymerase chain reaction (PCR)-amplified genomic DNA (data not shown). Mutations not involving restriction enzyme recognition sites were confirmed by bi-directional sequencing.
Figure 1. A 10% polyacrylamide gel (1%C, supplemented with 7.5% urea) stained with ethidium bromide displaying the various SSCP mobility shifts representative of mutations/genotypes V59M (lane 1), H63D/V53M (lane 2), S65C/S65C (lane 3), S65C (lane 4), H63H (lane 5), H63D (lane 7), H63D/H63D (lane 8) and V53M (lane 9). Lane 6, PCR-amplified DNA of a normal control.
Table 1. HFE sequence variants identified in the South African population
| Location | Nucleotide change | Amino acid change | Mutation designation | Restriction enzyme | Reference | |
| Created | Abolished | |||||
| Exon 2 | G->A at 157 | Val53->Met | V53M | NlaIII | MaeII | a |
| Exon 2 | G->A at 175 | Val59->Met | V59M | NlaIII | a | |
| Exon 2 | C->G at 187 | His63->Asp | H63D | MboI | 4 | |
| Exon 2 | T->C at 189 | His63->His | H63H | NlaIII | 20 | |
| Exon 2 | A->T at 193 | Ser65->Cys | S65C | HinfI | 21 | |
| Intron 2 | IVS2+4T->C | RsaI | 22 | |||
| Exon 3 | A->C at 381 | Gln127->His | Q127H | a | ||
| Exon 4 | G->A at 845 | Cys282->Tyr | C282Y | RsaI | 4 | |
| Intron 4 | IVS4+37A->G | MseI | a | |||
| Intron 4 | IVS4+109A->G | FokI | a | |||
| Intron 4 | IVS4+115T->C | HaeIII | DdeI | a | ||
| Exon 5 | G->T at 989 | Arg330->Met | R330M | a |
Utilization of the combined HEX-SSCP method to screen the study population initially for the documented H63D and C282Y mutations in exons 2 and 4, respectively, of the HFE gene resulted in the identification of two novel missense mutations V53M and V59M in exon 2, in addition to the recently described base changes at nucleotide positions 189 (T->C) (20) and 193 (A->T) (21). In contrast, application of this sensitive mutation detection method did not reveal additional mutations in exon 4 of the gene. Of the mutations detected in exon 2, V53M was identified only in the South African black and bushman (Khoisan) populations, whereas mutations V59M and S65C were confined to the Caucasian population (Table 2). The presence of mutations C282Y and H63D in the subjects analysed was confirmed by restriction enzyme analysis using RsaI and MboI, respectively. Gene frequencies in the South African Caucasian, coloured (mixed ancestry) and black populations have been described elsewhere (6). Mutation H63D was absent in the 340 Xhosa, Zulu, Venda, Pedi and Sotho individuals analysed, while only one individual (Xhosa) carried the C282Y mutation. This is in apparent contrast to the higher frequency of these mutations (0.03) reported among African-Americans, which can probably be ascribed to Caucasoid admixture (23). Mutation H63D was detected in a single Khoisan individual, while mutation C282Y was absent in this population.
Table 2. Prevalence of missense mutations in the HFE gene in South African patient and control populations
| Population | Total | V53M | V59M | S65C | Q127H | R330M | |
| Patients | |||||||
| HH mutation- | Caucasian | 13 | 0 | 0 | 0 | 0 | 1 |
| VP R59W+ | Caucasian | 73 | 0 | 0 | 1 | 1 | 0 |
| VP R59W- | Caucasian | 163 | 0 | 0 | 5 | 1 | 0 |
| Controls | |||||||
| Black | Sotho/Pedi | 31 | 1 | 0 | 0 | 0 | 0 |
| Venda | 101 | 1 | 0 | 0 | 0 | 0 | |
| Xhosa | 112 | 0 | 0 | 0 | 0 | 0 | |
| Zulu | 96 | 1 | 0 | 0 | 0 | 0 | |
| Bushman | Khoisan | 118 | 5 | 0 | 0 | 0 | 0 |
| Coloured | Western Cape | 156 | 0 | 0 | 0 | 0 | 0 |
| White | Caucasian | 102 | 0 | 1 | 0 | 0 | 0 |
| Total | 965 | 8 | 1 | 6 | 2 | 1 |
+, presence of a mutation; -, absence of a mutation.
Analysis of patients referred for a molecular diagnosis of HH
None of the 13 HH patient referrals subjected to extensive mutation screening of the promoter and coding region of the HFE gene was homozygous or compound heterozygous for the C282Y and/or H63D mutations. The novel mutation R330M, identified in exon 5 in one of the patients, was absent in 40 control individuals screened by HEX-SSCP analysis. The intronic sequence variants (IVS4+37A->G, IVS4+109A->G and IVS4+115T->C) identified in intron 4 were not pursued further, since these base changes were not expected to affect gene splicing. The genotype distribution and allele frequency of the previously described (22) RsaI polymorphism (IVS2+4T->C) were compared in the HH patient group and Caucasian controls, in order to assess possible allelic effects/associations. The frequency of the mutant C-allele was determined to be 0.25 in 24 control individuals, compared with 0.38 in the small cohort of 13 HH patient referrals. The apparent excess of the mutated allele in the patient group may be due to chance, but this aspect should be investigated further in order to determine the likelihood of a mild phenotypic effect or linkage disequilibrium with another disease-related mutation in the patient cohort.
Family study
Mutation Q127H was identified (Fig. 2) together with mutation H63D (compound heterozygote) in an apparently `severely affected' VP patient with the founder-related PPOX gene defect, R59W. This 11-year-old proband often suffers from headaches, fatigue, respiratory problems, photosensitivity and severe abdominal distress, the latter symptoms usually presenting at an older age in VP patients. Further testing is necessary to determine whether these symptoms are related to the PPOX and/or HFE gene mutations, but biochemical porphyrin measurements are usually relatively uninformative in children and it is also unlikely that the patient's iron levels would be elevated at this young age. Segregation analysis of the R59W, H63D and Q127H mutations in 20 additional family members revealed that only the proband demonstrated compound heterozygosity for the two HFE gene mutations, in combination with the VP mutation R59W (data not shown). Mutation Q127H was paternally transmitted (father R59W-positive), and mutation H63D was maternal in origin. Mutation C282Y was not detected in any of the family members.
Figure 2. Identification of mutation Q127H in exon 3 of the HFE gene. (A) A 10% polyacrylamide gel (1%C, supplemented with 7.5% urea) stained with ethidium bromide displaying the mobility shift characteristic of mutation Q127H in the proband (lane 1) and her father (lane 4). Lanes 2 (brother) and 3 (mother), PCR-amplified DNA of family members without the mutation. (B) Direct DNA sequencing (sense strand) of PCR products from a control individual (wild-type) and the proband (A, lane 1). Mutation Q127H is indicated by an asterisk.
Analysis of patients referred for a molecular diagnosis of VP
Analysis of the C282Y and H63D mutations in patients referred for a molecular diagnosis of VP demonstrated that seven of 163 R59W mutation-negative cases (4%) carried two HFE gene mutations (four individuals C282Y/C282Y and three individuals C282Y/H63D), while these `iron overload' genotypes were not detected in any of 73 genetically proven VP patients. Furthermore, the mutant allele frequency of the C282Y mutation was found to be significantly lower in R59W-positive VP patients compared with controls drawn from the same population ([chi]2 = 7.61, df = 1, P < 0.05) (Table 3). The carrier frequency of the C282Y mutation was also significantly lower in the porphyria patient referrals (4% in R59W-positive and 9% in R59W-negative subjects) than in control individuals (16.9%). A reduced C282Y mutation frequency in VP patients has been confirmed independently in another laboratory, with none of 32 additional R59W-positive cases carrying the C282Y mutation (L. Warnich, unpublished data). The carrier frequency of the H63D mutation was similar in the VP patient (26%) and control (21%) populations of European descent (data not shown).
Table 3. Comparison of genotype distribution and allele frequencies of the C282Y HFE gene mutation in VP patients with the R59W PPOX gene mutation and controls from the same population
| VP patients (n = 73) | Controlsa (n = 102) | |
| Genotype | ||
| C282Y+/+ | 0 | 2 (2%) |
| C282Y+/- | 3 (4%) | 15 (15%) |
| C282Y-/- | 70 (96%) | 85 (83%) |
| Allele | ||
| 282-G (normal) | 0.98 | 0.91 |
| 282-A (mutant) | 0.02b | 0.09 |
| Carrier frequency | 4% | 16.9% |
bVP versus control: P = 0.005, [chi]2 = 7.61.
DISCUSSION
We report the identification of four novel missense mutations and provide evidence that sequence variation in the HFE gene may explain the porphyria-like symptoms in a number of patient referrals without VP-related mutations. This investigation was prompted by a recent study (24), which demonstrated that many individuals in South Africa have been misdiagnosed with VP, based on biochemical determinations and clinical features suggestive of porphyria. It has been reported previously that either an excess or a deficiency of iron may affect the haem biosynthetic pathway and precipitate porphyria (9,11,25). High serum iron levels were reported in 75% of VP patients studied by Bothwell et al. (26), the highest levels being detected during presentation of the severest symptoms of the acute attack, whilst normal levels were measured in remission. Iron serves as a cofactor to many non-haem and haemoproteins, and is a regulator of haem synthesis (27). Whilst iron overload accelerates the inactivation of hepatic uroporphyrinogen decarboxylase in animal models of sporadic PCT (28,29), the effect of iron deficiency on haem biosynthesis is unclear at this time.
DNA screening for the H63D and C282Y mutations in the study population comprising 965 subjects revealed a relatively high mutation frequency in exon 2 of the HFE gene. Two novel missense mutations, V53M and V59M, were detected in this exon, in addition to a silent T->C base change at nucleotide position 189 (20) and mutation S65C, recently implicated in a mild form of haemochromatosis (21). Detection of mutation S65C in 3% of molecularly uncharacterized porphyria patient referrals (5/163), and in none of the control individuals (including 102 Caucasians), provides preliminary evidence that this mutation may also be associated with the porphyria phenotype in a subset of South African patients. Mutations V53M and S65C occur at CpG dinucleotides, but the frequency of these potential mutational hotspots is not higher in exon 2 than in exon 4. Notably, all the exon 2 missense mutations identified in the study population involve amino acids which have remained evolutionarily conserved in human, mouse and rat. Further studies are needed to investigate the likelihood that the newly described exon 2 missense mutations V53M and V59M, and possibly also mutation S65C implicated in a mild form of HH (21), may alter the affinity of HFE for the transferrin receptor in a manner similar to that of mutation H63D (30). Predominance of mutation C282Y in HH patients worldwide complicates assessment of the potential role of other HFE gene mutations in disease expression, in particular since subtle phenotypic effects would probably not be detected by conventional biochemical assays of iron status.
Identification of the HFE gene mutation Q127H in a VP patient found to carry the founder-type PPOX gene mutation R59W raises the possibility that the apparently severe disease expression in this 11-year-old girl is a consequence of compound heterozygosity with mutation H63D. Her clinical features were not compatible with the usual VP phenotype, but were also not as severe as those described earlier in a proposed clinical VP homozygote (31), who was shown to be a compound heterozygote for PPOX gene mutations R59W and R168C (17). Segregation analysis in 20 additional family members demonstrated that although several individuals had inherited both the R59W (PPOX, chromosome 1) and Q127H (HFE, chromosome 6) mutations, co-existence of H63D with this genotype was not detected. Mutation Q127H occurs in the [alpha]2 chain in a disulfide-binding area, and may resemble the situation reported for the C282Y mutation, which disrupts a critical disulfide bond in the [alpha]3 extracellular loop of the HFE protein (32). Mutation R330M, identified in the heterozygous form (implying the presence of a second unidentified mutation elsewhere) in one of the patients analysed for HH, is located in the transmembrane region of the HFE protein. Further studies are needed to determine whether an alteration at position 330, a conserved tryptophan in the protein used by Feder et al. (4) to generate the HFE sequence alignment, may affect the manner in which HFE behaves in a membrane.
In an attempt to determine the possible impact of HFE gene mutations in the South African VP population, we also screened patients referred for a molecular diagnosis of VP for HFE mutations Q127H, C282Y and H63D. The novel exon 3 Q127H mutation was detected in a single VP patient referral without the R59W mutation, but was absent in the molecularly characterized VP patients and 40 control individuals from the Caucasian population. Screening for the two common mutations revealed four homozygotes and three compound heterozygotes among the 163 referrals without the PPOX gene mutation. However, none of 73 VP patients who tested positive for the founder mutation R59W demonstrated these `iron overload' genotypes.
It has been estimated previously, based on a carrier frequency of 16.9% for the C282Y mutation, that ~1/115 Caucasians in South Africa are homozygous for this mutation (6). Detection of four C282Y homozygotes among 163 R59W-negative porphyria patients clearly represents an excess of this highly penetrant disease genotype. Since mutations C282Y and H63D are also associated with PCT (9-11), it is highly likely that the raised iron levels associated with these mutations explain, at least in part, the porphyria phenotype in a number of biochemically and molecularly uncharacterized patients referred for a molecular diagnosis of VP. The cutaneous features of VP, although distinctive, are not limited to VP and are also present in patients with PCT, hereditary co-proporphyria and congenital erythropoietic porphyria. Due to the sensitivity of the convenient molecular test used to largely exclude or confirm VP, many patient referrals are not being screened at the biochemical level, and, therefore, several patients shown to be homozygous or compound heterozygous for mutations C282Y and/or H63D may indeed suffer from PCT.
Another intriguing observation was the low carrier frequency of the C282Y mutation in patients presenting with porphyria symptoms compared with that previously reported (6) in the general South African Caucasian population. This finding cannot be ascribed to a dilution effect as a consequence of possible absence of mutation C282Y in the original founder couple who introduced the R59W mutation into South Africa (16), since the HFE and PPOX genes are located on different chromosomes and the R59W mutation has been expanded over at least 12 generations after its introduction into the South African population >300 years ago. It is therefore possible that not only homozygosity for mutation C282Y, but also the absence of this mutation, most likely together with other genetic and/or environmental challenges causing iron deficiency, could precipitate porphyria or porphyria-like symptoms. This may be the case in many South African patients previously misdiagnosed with VP, since none of the probands studied by Kotze et al. (24) who subsequently were subjected to HFE mutation screening were homozygous for the C282Y mutation (data not shown). Although the low C282Y mutation frequency in R59W-positive VP patients may be a reflection of the founder mutation being the major cause of their disease (17,18), as opposed to possible involvement of gene-gene interaction in phenotypic expression of VP, this apparent `selection by phenotype' can nevertheless be accepted as genetic confirmation of the importance of iron in the haem biosynthesis pathway (27). Subtle genetic abnormalities of iron metabolism, which might escape detection by standard parameters of iron status (11), may thus underlie the disease symptoms in some patients with unexplained porphyria-like disease(s).
Our data appear to emphasize the significance of iron levels and iron-related mutations in haem biosynthesis and may eventually contribute to a better understanding of the nature of porphyria-related diseases. It seems plausible that both the presence and absence of specific HFE gene mutations, possibly in combination with other genetic and/or environmental factors, are important inducers of a porphyria phenotype. Since previous studies have shown that not only homozygosity, but also heterozygosity for disease-related mutations in the HFE gene are important determinants of iron status (33), further clinical, biochemical and genetic studies in well-defined patient and control groups are warranted to investigate the role of specific HFE gene mutations in haem and iron-related diseases.
MATERIALS AND METHODS
Patients and controls
Blood samples were obtained from 965 individuals from four different ethnic groups in South Africa, including two patient cohorts comprising 13 Caucasians referred for molecular diagnosis of HH and 236 Caucasians referred for a molecular diagnosis of VP (Table 1). In this study, `white' or `Afrikaner' refers to an individual of European descent, mainly Dutch, French, German and British stock; `coloured' refers to an individual of mixed ancestry, including San, Khoi, African Negro, Madagascar, Javanese and Western European origin; and `black' refers to South Africans of central African descent.
All the study participants were screened for common mutations H63D and C282Y in exons 2 and 4, respectively, of the HFE gene (4). DNA samples of the 13 patients likely to have HH, but who tested negative for mutations C282Y and H63D, were selected for analysis of the promoter region and entire coding region of the HFE gene. They were referred for a molecular diagnosis of HH based on the presence of elevated serum iron and ferritin levels, liver biopsy findings and/or response to phlebotomy (34).
Criteria for referral of patients for a molecular diagnosis of VP included: (i) a positive clinical picture of VP; (ii) a positive family history of VP/porphyria symptoms; and/or (iii) a positive biochemical laboratory test on stool, urine and blood specimens, demonstrating excretion of specific metabolites typical of VP. The founder-related PPOX gene mutation R59W was present in 73 of the VP referrals, as determined by restriction enzyme analysis of amplified products using AvaII or StyI (17,18). The DNA sample of one of the mutation-positive cases, an 11-year-old girl with an apparently severe clinical expression of the disease, was subjected to extensive exon-by-exon screening of the HFE gene. The reported porphyria-like symptoms in this patient could not be ascribed to any other disease/condition, despite extensive medical examination. Her father had also been diagnosed with VP based on both biochemical and DNA testing. In this family, blood samples were obtained with informed consent from 21 subjects. The study protocol was approved by the Ethics Review Committee of the University of Stellenbosch.
DNA analysis
Genomic DNA was extracted from 5 ml of whole blood preserved in EDTA, using a standard lysis method (35). Screening of PCR-amplified DNA for mutations C282Y and H63D was performed using a non-radioactive HEX-SSCP mutation detection method (36). This method was selected as opposed to restriction enzyme analysis alone, since it is cost-effective and allows the concurrent identification of additional aberrations within the amplified DNA fragment. PCR products corresponding to aberrant bands were sequenced using an automated system (ABI 373A; Perkin Elmer, Foster City, CA). Primer sequences for amplification of the promoter and coding region of the HFE gene were kindly supplied by Dr Clara Camaschella (University of Torino, Italy).
Statistical analysis
Allele frequencies were determined by allele counting. Testing for significance of heterogeneity in mutation frequencies among patient and control groups was based on the [chi]2 test.
ABBREVIATIONS
HEX-SSCP, heteroduplex-single-strand conformation polymorphism; HFE, human haemochromatosis gene; HH, hereditary haemochromatosis; PPOX, protoporphyrinogen oxidase gene.
ACKNOWLEDGEMENTS
The authors thank Prof. E.P.G. Mansvelt, Dr E.J. Cawood, Dr R. Delport, C.F. Hoogendijk, C.L. Scholtz and E. Langenhoven for provision of blood or DNA samples. Prof. L. Warnich, Prof. P. Meissner, Dr R. Hift, R.N. Rooney and M. Viviers are thanked for helpful discussions. Pathologists and clinicians are thanked for patient referrals and clinical data. The University of Stellenbosch, the Harry and Doris Crossley Foundation and the Freda and David Becker Trust are acknowledged for financial support, and the South African Medical Research Council for a bursary awarded to J.N.P.d.V.
REFERENCES
*To whom correspondence should be addressed. Tel: +27 21 9389441; Fax: +27 21 9317810; Email: mjk{at}gerga.sun.ac.za
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