Hereditary hemochromatosis: Mutations in genes involved in iron homeostasis in Brazilian patients

Blood Cells, Molecules, and Diseases 46 (2011) 302–307

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Hereditary hemochromatosis: Mutations in genes involved in iron homeostasis in Brazilian patients Paulo C.J.L. Santos a,⁎, Rodolfo D. Cançado b, Alexandre C. Pereira c, Isolmar T. Schettert c,d, Renata A.G. Soares c, Regina A. Pagliusi e, Rosario D.C. Hirata a, Mario H. Hirata a, Ana C. Teixeira a, Maria Stella Figueiredo f, Carlos S. Chiattone b, Jose E. Krieger c, Elvira M. Guerra-Shinohara a a

Department of Clinical Chemistry and Toxicology, School of Pharmaceutical Sciences, University of Sao Paulo, SP, Brazil Santa Casa Medical School, Sao Paulo, SP, Brazil c Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of Sao Paulo Medical School, SP, Brazil d Novo Atibaia Hospital, SP, Brazil e Adolfo Lutz Institute, Sao Jose do Rio Preto, Brazil f Hematology and Hemotherapy, EPM/UNIFESP, SP, Brazil b

a r t i c l e

i n f o

Article history: Submitted 6 January 2011 Revised 11 February 2011 (Communicated by Sir D. Weatherall, F.R.S., 17 February 2011) Keywords: Hemochromatosis HFE Primary iron overload Sequencing

a b s t r a c t Background: p.C282Y mutation and rare variants in the HFE gene have been associated with hereditary hemochromatosis (HH). HH is also caused by mutations in other genes, such as the hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1). The low rate homozygous p.C282Y mutation in Brazil is suggestive that mutations in non-HFE genes may be linked to HH phenotype. Aim: To screen exon-by-exon DNA sequences of HFE, HJV, HAMP, TFR2 and SLC40A1 genes to characterize the molecular basis of HH in a sample of the Brazilian population. Materials and methods: Fifty-one patients with primary iron overload (transferrin saturation ≥50% in females and ≥ 60% in males) were selected. Subsequent bidirectional DNA sequencing of HFE, HJV, HAMP, TFR2 and SLC40A1 exons was performed. Results: Thirty-seven (72.5%) out of the 51 patients presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n = 11, 21.6%). In addition, heterozygous HFE p.S65C mutation was found in combination with p.H63D in two patients and homozygous HFE p.H63D was found in two patients as well. Sequencing in the HJV and HAMP genes revealed HJV p.E302K, HJV p.A310G, HJV p.G320V and HAMP p.R59G alterations. Molecular and clinical diagnosis of juvenile hemochromatosis (homozygous form for the HJV p.G320V) was described for the first time in Brazil. Three TFR2 polymorphisms (p.A75V, p.A617A and p.R752H) and six SLC40A1 polymorphisms (rs13008848, rs11568351, rs11568345, rs11568344, rs2304704, rs11568346) and the novel mutation SLC40A1 p.G204S were also found. Conclusions: The HFE p.C282Y in homozygosity or in heterozygosity with p.H63D was the most frequent mutation associated with HH in this sample. The HJV p.E302K and HAMP p.R59G variants, and the novel SLC40A1 p.G204S mutation may also be linked to primary iron overload but their role in the pathophysiology of HH remain to be elucidated. © 2011 Elsevier Inc. All rights reserved.

Introduction Hereditary hemochromatosis (HH) is the most common autosomal recessive disorder in populations of Northern European origin. It is characterized by enhanced intestinal absorption of dietary iron leading to multiple organ dysfunctions as cirrhosis, hepatoma, cardiomyopathy, diabetes mellitus, arthritis and hypogonadism [1]. p.C282Y and p.H63D mutations in the HFE (OMIM ID: 235200) have

⁎ Corresponding author. E-mail address: [email protected] (P.C.J.L. Santos). 1079-9796/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2011.02.008

been implicated in the pathogenesis of type 1 HH [2]. Most HH patients carry homozygous p.C282Y or heterozygous p.C282Y/p.H63D [1,3]. Recent reports have suggested that rare HFE variants, such as p.G43A, p.L46W, p.D129N, are also linked to HH thus contributing to genetic and phenotypic heterogeneity of the disease [4–7]. The HFE p.C282Y homozygous genotype is particularly common in Northern European populations (1 in 200–300 healthy subjects) and the HFE 282Y allele frequency is high (5.1% to 8.2%) [8]. In the Brazilian population, the prevalence of HH is low and reduced frequency of the HFE 282Y allele (2.3%) was observed in blood donors [9,10]. Thus, rare functional mutations may be present in Brazilian HH patients.

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Mutations in the genes that encode hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1) have been associated with non-HFE forms of HH [5,11]. The juvenile hemochromatosis (JH) is a rare form of iron overload that leads to organ damage before the age of 30, and frequently causes cardiomyopathy, hypogonadotrophic hypogonadism and endocrine dysfunctions [11]. JH types 2A and 2B are caused by mutations in HJV (OMIM ID: 602390) and HAMP (OMIM ID: 606464), respectively [12,13]. Hepcidin is a peptide that plays a role in the iron absorption related to ferroportin of the enterocyte and it is inappropriately decreased in HFE-HH patients [13]. Patients with JH type 2A patients have low urinary hepcidin concentration implying that HJV is hepcidin-related [12]. The HJV p.G320V is the most frequent JHrelated mutation [12,14], whereas mutations in HAMP are a rare cause of JH [13,15,16]. Type 3 HH is a rare disease caused by mutations in TFR2 (OMIM ID: 604720) and patients had iron overload similar to HFE-HH phenotype [17]. The function of TFR2 protein has been indicated as a regulator of hepcidin because, in both animal and patient TFR2-HH models, hepcidin concentrations are low [18,19]. Some TFR2 mutations, such as p.R105X, p.M172K, p.Y250X, p.Q317X, p.R455Q and p.Q690P, have been shown to be associated with hemochromatosis [17,20–23]. Type 4 HH is related to mutations in SLC40A1 (OMIM ID: 604353) and it had an autosomal dominant pattern. Ferroportin protein is a membrane transporter that modulates iron efflux [24]. SLC40A1 mutations, such as p.A77D, p.N144H, p.V162X, p.Q182H, p.Q248H, p.G267D and p.G323V, were associated with HH [24–27]. The information on mutations in genes involved in primary iron overload in our population is still limited. In this scenario, HFE, HJV, HAMP, TFR2 and SLC40A1exons were sequenced to identify variants that may contribute to the molecular basis of the HH in Brazil.

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HCV® kit (Murex Biotech S.A, South Africa), and hepatitis B was detected by Hepanostika anti-HBc Uni-Form® and Hepanostika HbsAg Uni-FormII® kits (BioMérieux, Boxtel, Netherlands). DNA extraction, sequencing and high-resolution melting analysis Genomic DNA was isolated from peripheral blood leukocytes by a salting-out method [29]. Coding sequences of the HFE (6 exons), HJV (4 exons), HAMP (3 exons), TFR2 (18 exons) and SLC40A1 (8 exons) were amplified by a polymerase chain reaction (PCR) using the previously described primer sequences [14,21,30–32]. PCR products were purified using ExoSAP-IT® reagent (GE Healthcare, NJ, USA) and were bidirectionally sequenced using the ABI Terminator Sequencing Kit according to the manufacturer's instructions and an ABI 3500XL Sequencer® (Applied Biosystems, Foster City, CA, USA). High-resolution melting analysis (HRM) was used to analyze the novel mutation SLC40A1 p.G204S, found by DNA sequencing, in HHaffected and in control samples (n = 305). The PCR was performed in a Rotor Gene 6000® (Qiagen, Courtaboeuf, France) using the following primers: sense 5′-TGAATGCCACAATACGAAGG-3′ and antisense 5′CCAAGTTCCATCCCGAAATA-3′ (fragment size 124 base pairs), with addition of fluorescent DNA-intercalating SYTO9® (Invitrogen, Carlsbad, CA, USA). In the HRM phase, the fluorescence was measured at a 0.1 °C increase in the range of 78–84 °C [33]. Melting curves reveal nucleotide changes of p.G204S (c.G610A). Samples of the heterozygous and homozygous genotypes were added in the HRM analysis. In silico studies ClustalX v.2.0 software (www.clustal.org/) was used for analysis of multiple sequence alignment of the HFE and ferroportin proteins from different species (Homo sapiens, Pan troglodytes, Macaca mulatta, Diceros bicornis, Bos taurus, Rattus norvegicus, Mus musculus).

Materials and methods Subjects Fifty-one Brazilian patients with primary iron overload were selected at the Santa Casa Medical School of Sao Paulo, SP, Brazil, and Novo Atibaia Hospital, SP, Brazil. Three hundred and five blood donors [10] were also included for evaluating the frequencies of novel mutations. The study protocol was approved by the Institutional Ethics Committees (Santa Casa Medical School; Novo Atibaia Hospital; School of Pharmaceutical Sciences), and written informed consent was obtained from all participants prior to entering the study. General characteristics of patients were obtained through an interview. The diagnosis of primary iron overload was based on transferrin saturation values over 50% and 60% for female and male, respectively. Patients with secondary causes of iron overload, such as parenteral or dietary iron overload, alcohol abuse, chronic hemolytic anemia, thalassemias and hepatitis [28], and/or low levels of transferrin saturation were not included in this study. Blood sampling and laboratory determinations Blood was drawn using the BD Vacutainers System® (Becton Dickinson, NJ, USA) for blood cell count and genetic analysis (K2EDTA) and for measurement of biochemical and immunological data (without anticoagulant). The concentrations of serum iron (SI) and total iron binding capacity (TIBC) and the activities of alanine transaminase (ALT) and aspartate transaminase (AST) were measured using an automation system Advia 1650® (Bayer Diagnostics, Tarrytown, NY, USA). The value of transferrin saturation (TS) was obtained by the ratio between SI and TIBC values. Serum ferritin (SF) was determined by the Axsym System® (Abbott Laboratories, Abbott Park, IL, USA). Determination of hepatitis C was performed by Murex anti-

Statistical analysis Categorical variables were presented as frequencies and continuous variables as medians and 25–75% percentiles. The Chi-squared test was performed to compare the frequencies of the homozygous HFE p.C282Y genotype between this study and other studies. Kruskal– Wallis and Mann–Whitney tests were used for comparing the SF and TS medians according to gender. All statistical analyses were carried out using SPSS software (v. 16.0), with the level of significance set at P b 0.05. Results Of the fifty-one subjects with primary iron overload included, thirteen (25.5%) were female and thirty-eight (74.5%) male. The mean age of studied patients was 58.9 (±13.6) years in the females and 53.2 (±10.9) years in the males. The group of patients was separated in self-identified ethnic sub-groups as White (n = 34, 66.6%), Intermediate (n = 11, 21.6%), Black (n = 3, 5.9%), and Yellow (n = 3, 5.9%) [10]. One out of the 51 patients had clinical characteristics compatible to JH: 22 years old, severe iron overload, hypogonadotrophic hypogonadism and glucose intolerance. Thirty-seven (72.5%) out of the 51 patients with primary iron overload presented at least one HFE mutation located at the exon 2 (p.H63D and p.S65C) or exon 4 (p.C282Y and p.V256I), as previously described [30]. HFE 282Y (31.4%) and 63D (23.5%) were the most common alleles in this sample. The homozygous genotype for the p.C282Y mutation (p.C282Y/p.C282Y) was found in 11 patients (21.6%). The p.C282Y was presented in heterozygosity with the wild-type (p.C282Y/WT) in 4 patients (7.8%) and it was also found in combination with p.H63D (p.C282Y/p.H63D; n = 6, 11.7%). The p.H63D mutation was presented in heterozygosity (p.H63D/WT) in 11 patients (21.6%) and in the homozygosis form (p.H63D/p.H63D) in 2 patients (3.8%). HFE p.S65C mutation was found in heterozygosity

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with p.H63D mutation (p.H63D/p.S65C) in two patients (3.8%). An isoleucine at amino acid position 256 (p.V256I) was detected in exon 4 [30]. Three mutations (p.E302K, p.A310G and p.G320V) were found in exon 4 of HJV, but none was detected in exons 1, 2 and 3. HJV p.E302K mutation was found in heterozygosity (c.G904A, Fig. 1A) in two males (61 and 65 years old) carrying HFE p.H63D in heterozygosity. HJV p.A310G variant was detected in heterozygosity (c.C929G, rs7540883) in one 58-year-old female. HJV p.G320V mutation was observed in homozygosis (c.G1284T, rs74315323, Fig. 1C) in a 22year-old male. This patient presented transferrin saturation value of 99%, serum ferritin concentration of 3500 μg/L, hypogonadotrophic hypogonadism and glucose intolerance. His patterns presented normal iron status and no clinical event [34]. In HAMP exon 3, the p.R59G mutation in heterozygosity was identified in one 63-year-old male, who presented no mutation in HFE (c.A175G, Fig. 1B). Screening of HAMP exons 1 and 2 did not indicate any alterations. Three previously described polymorphisms were observed in the TFR2 (p.A75V, p.A617A e p.R752H). TFR2 p.A75V polymorphism was identified in heterozygosity (c.C224T, exon 2) in one patient. TFR2 p.A617A synonymous–polymorphism was observed in 7 patients (6 in heterozygosity and 1 in homozygosity; c.C1878T, exon 16). TFR2 p.R752H polymorphism was detected in heterozygosity (c.G2296A, rs41295942, exon 18) in 3 patients. SLC40A1 sequencing revealed 6 polymorphisms (rs13008848, rs11568351, rs11568345, rs11568344, rs2304704, and rs11568346) and a guanine-to-adenine substitution (c.G610A) that corresponds to the novel mutation p.G204S. Two polymorphisms located in exon 1, rs13008848, UTR c.C98G and rs11568351, UTR c.G8C, presented variant allele frequencies of 22.5% and 20.6%, respectively. In exon 4, SLC40A1 p.I109I and p.L129L synonymous–polymorphisms were identified in heterozygosity (c.C678T, rs11568345 and c.C738T, rs11568344) in 2 patients and in 1 patient, respectively. And SLC40A1 p.V221V synonymous–polymorphism (c.T1014C, rs2304704, exon 6) was detected with variant allele frequency of 39.2%. SLC40A1 p.R561G polymorphism was identified in homozygous (c.A1681G, rs11568346, exon 8) in one 59-year-old male. Novel SLC40A1 p.G204S mutation was observed in homozygosis (c.G610A, exon 6, Fig. 2A) in one 52-year-old female patient carrying no HFE gene mutation. To her knowledge, her family has not

developed iron overload. Two daughters of the patient (31 and 33 years old) were investigated for p.G204S (Fig. 2B), which was found in heterozygosity. They had normal results of iron status tests and reported no clinical symptoms and signs. In multiple sequence alignment, the p.G204 is a conserved residue in all organisms studied (Fig. 2C). The evaluation of p.G204S variant by HRM analysis presented different pattern curves for the mutant homozygous (Fig. 3C), for the heterozygous (Fig. 3B), and for the wild-type genotypes (Fig. 3A, found in 305 blood donors). Allele frequencies of variants in HFE, HJV, HAMP, TFR2 and SLC40A1 genes were shown in Table 1. Gender, age, and iron status in patients with iron overload carrying hemochromatosis-related variants were demonstrated in Table 2. No relationship was found between HFE, HJV, HAMP, SLC40A1 and TFR2 mutations or polymorphisms and SF and TS values in this sample population (data not shown). Discussion The frequency of the homozygous genotype for the p.C282Y mutation (p.C282Y/p.C282Y) was significantly lower (21.6%) in this sample than in predominantly Caucasian populations (64.0% to 96.3%; P b 0.05) [35–38]. Similar to our results, in some countries of Asia, Africa and South America, an increased number of patients with primary iron overload did not carry p.C282Y/p.C282Y or p.C282Y/ p.H63D genotypes [39–42]. In this sample, heterozygous p.H63D and p.S65C mutations were found in two patients (3.8%) and homozygous p.H63D mutation was detected in another two patients (3.8%), in the absence of other functional mutations. It is likely that these alterations isolated or in combination modify the iron homeostasis [43]. However, the functional role of these mutations on hemochromatosis phenotype still remains unclear. Homozygous genotype for the HJV p.G320V mutation was identified in a young man with clinical data indicative of JH. It is likely this mutation plays a major role in JH pathophysiology as it has been suggested in other populations [44]. In addition, in this first Brazilian JH case, the normalization of iron parameters was achieved by administration of deferasirox in combination with venesections during the initial treatment phase, followed by normalization of the endocrine and liver dysfunction, as previously reported [34].

Fig. 1. HJV and HAMP sequencing results. A: Heterozygous genotype for the HJV p.E302K (c.G904A) mutation. B: Heterozygous genotype for the HAMP p.R59G (c.A175G) mutation. C: Homozygosity for the HJV p.G320V (c. G1284T) mutation. The box shows the difference between sequence patient (GTG-Val) and sequence control (GGG-Gly).

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Fig. 2. A: SLC40A1 p.G204S (c.G610A, exon 6) in homozygosis. B: SLC40A1 p.G204S in heterozygosity. C: Multiple sequence alignment of SLC40A1 protein from different species (Homo sapiens, Pan troglodytes, Macaca mulatta, Diceros bicornis, Bos taurus, Rattus norvegicus, Mus musculus, respectively). Similar residues are indicated by an asterisk (*); and semiconserved substitutions by a dot (.).

In our study, two patients carrying HJV p.E302K and one carrying HAMP p.R59G in heterozygosity did not present higher iron overload compared with other Brazilian patients. However, the implication of causality of these mutations found should be considered in interaction with mutations in intronic or regulatory regions of genes related to HH. The HJV p.E302K and HAMP p.R59G mutations in combination with HFE mutations have been suggested to modify HH phenotype [45–47]. While the HJV p.A310G polymorphism has frequencies of 7% and 2% in controls and iron-overload patients from North America, respectively, its lack of association with HH was thus suggested [5,14]. Three previously described TFR2 polymorphisms (p.A75V, p.A617A, and p.R752H) were identified in our study. TFR2 p.R752H polymorphism was found in 2 of 23 HH patients, but this alteration was also found in 9 of 65 healthy individuals [22,32,48]. Thus, TFR2 p.R752H presented similar allelic frequencies between patients and controls and it was classified as frequent polymorphism [22,32,48]. Studies reported that TFR2 p.A75V and p.A617A polymorphisms are not likely to be linked to iron overload [22,32,48]. Six SLC40A1 polymorphisms were found in our study. Two (c.C98G and c.G8C) were identified in the promoter region and one previous study reported that these polymorphisms were not associated with ferritin or transferrin saturation levels in normal subjects [49]. Three synonymous–polymorphisms (p.I109I, p.L129L and p.V221V) were found and these were described as non-pathogenic [49,50]. In a previous study, similar frequencies for the p.R561G polymorphism were observed in case and control subjects suggesting a lack of linkage to HH [50].

Fig. 3. Graphs of the nucleotide changes result in different curve patterns using highresolution melting analysis (SLC40A1 p.G204S genotypes). A: Wild-type genotype (blood donors; N = 305); B: heterozygous genotype; C: homozygous genotype for the SLC40A1 p.G204S.

The novel SLC40A1 p.G204S mutation may not be associated with the patient's iron overload. Because HH related to SLC40A1 had an autosomal dominant pattern [25–27] and the daughters of the affected patient did not have iron overload, they presented heterozygous genotype for the p.G204S mutation. However, the role of the p.G204S mutation on HH pathophysiology could not be excluded in this case, considering that the daughters are young adults and HH phenotype usually is manifested in the fifth or sixth decade of life. It is conceivable that individuals without mutation identified in our study may be carrying mutations located in intronic or regulatory regions of the studied genes and/or even in other hemochromatosisrelated very rare genes, such as DMT1, ALAS2, TF, and FTH [51]. Furthermore, environmental factors as well may contribute to the iron overload expression in the studied patients [1,7]. In conclusion, the HFE p.C282Y in homozygosity or in heterozygosity with p.H63D was the most frequent mutation associated with HH in this sample. The HJV p.E302K and HAMP p.R59G variants, and the novel SLC40A1 p.G204S mutation may also be linked to primary

Table. 1 Allele frequencies of variants in HFE, HJV, HAMP, TFR2 and SLC40A1 genes in patients with iron overload. Variants

Number of individuals Genotype (HM or HT) Allele frequency (%)

HFE gene p.C282Y p.H63D p.S65C p.V256I

21 22 2 1

HJV gene p.E302K p.A310G p.G320V

2 1 1

2 HT 1HT 1HM

2.0 1.0 2.0

HAMP gene p.R59G

1

1HT

1.0

TFR2 gene p.A75V p.A617A p.R752H

1 7 3

1HT 1 HM, 6 HT 3HT

1.0 7.8 2.9

2 HM, 17 HT 3 HM, 17 HT 2 HT 1HT 4 HM, 32 HT 1HM 1HM

20.6 22.5 2.0 1.0 39.2 2.0 2.0

SLC40A1 gene UTR c.G8C 19 UTR c.C98G 19 p.I109I 2 p.L129L 1 p.V221V 36 p.R561G 1 p.G204S 1

11 HM, 10 HT 2 HM, 20 HT 2 HT 1HT

HM: homozygosity; HT: heterozygosity.

31.4 23.5 2.0 1.0

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Table 2 Gender, age, and iron status in patients with iron overload carrying hemochormatosis-related variants. Genes and variants

Gender

Age (years)

Transferrin saturation (%)

Serum ferritin (μg/L)

HFE p.C282Y/p.C282Y (N = 17) HFE p.C282Y/p.H63D (N = 6) HFE p.H63D/p.H63D (N = 2) HFE p.H63D/p.S65C (N = 2) HFE p.V256I/p.H63D (N = 1) HJV p.E302K (N = 2) HJV p.G320V (N = 1) HAMP p.R59G (N = 1) SLC40A1 p.G204S (N = 1)

4 F–13 M 6M 2M 2M M 2M M M F

56 (44–63) 55 (42–62) 59; 56 43; 75 44 61; 65 22 63 52

84.2 (67.2–89.2) 67.0 (64.6–75.4) 72.0; 66.4 62.4; 97.5 62.0 68.0; 82.0 99.0 69.6 100.0

1,051 (560–2085) 569 (439–1144) 1662; 1500 829; 697 114 356; 1623 3500 1310 5236

N: number of individuals; F: female; M: male. Age, transferrin saturation and serum ferritin are expressed as median (25–75% percentiles). No statistical relationship was found between HFE, HJV, HAMP, SLC40A1 and TFR2 mutations and serum ferritin and transferrin saturation values.

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