Haemochromatosis in patients with b-thalassaemia trait

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1 British Journal of Haematology, 2000, 111, 908±914 Haemochromatosis in patients with b-thalassaemia trait Alberto Piperno, 1 Raffaella Mariani, 1 Cristina Arosio, 2 Anna Vergani, 1 Sandra Bosio, 3 Silvia Fargion, 4 Maurizio Sampietro, 4 Domenico Girelli, 5 Mirella Fraquelli, 6 Dario Conte, 6 Gemino Fiorelli 4 and Clara Camaschella 3 1 Clinica Medica, UniversitaÁ degli Studi di Milano-Bicocca, Azienda Ospedaliera San Gerardo, Monza, 2 Istituto Auxologico Italiano, Ospedale San Luca, Milano, 3 Dipartimento di Scienze Cliniche e Biologiche, UniversitaÁ di Torino, Azienda Ospedaliera San Luigi, Orbassano, Torino, 4 Istituto di Medicina Interna e Fisiologia Medica, UniversitaÁ di Milano, IRCCS, Ospedale Maggiore, Milano, 5 Istituto di Clinica Medica, UniversitaÁ di Verona, Verona, and 6 Cattedra di Gastroenterologia, UniversitaÁ di Milano, IRCCS, Ospedale Maggiore, Milano, Italy Received 23 March 2000; accepted for publication 17 July 2000 Summary. Severe iron overload has been reported in patients with the b-thalassaemia trait. Studies performed before the discovery of the haemochromatosis gene (HFE) have yielded conflicting results: some suggest that iron overload might arise from the interaction of the b- thalassaemia trait with heterozygosity for haemochromatosis, some with homozygosity for haemochromatosis and others that it was unrelated to haemochromatosis. We have studied the clinical phenotype, iron indices and HFE genotypes of 22 unrelated patients with the b-thalassaemia trait and haemochromatosis, the inheritance of chromosome 6p and 1q haplotypes in families of nonhomozygous C282Y probands and serum measures of iron status in relatives heterozygous for C282Y with or without the b-thalassaemia trait. We demonstrate that the b-thalassaemia trait aggravates the clinical picture of C282Y homozygotes, favouring higher rates of iron accumulation and the development of severe iron-related complications. We suggest that the coexistence of the b- thalassaemia trait might also increase the risk of iron overload in patients with HFE genotypes at a mild risk of haemochromatosis. Our findings do not support the hypothesis that the association of the b-thalassaemia trait with a single C282Y or H63D allele might lead to iron overload and suggest that other non-hfe-related inherited factors are present in haemochromatosis patients with incomplete HFE genotypes. Keywords: haemochromatosis, b-thalassaemia, HFE, iron overload, ineffective erythropoiesis. The b-thalassaemia trait is characterized by mild, ineffective erythropoiesis and erythroid hyperplasia that could induce excess iron absorption and ultimately lead to iron overload (Williams & Siemsen, 1968; Parfrey et al, 1981; Pippard & Wainscoat, 1987). However, only a minority of patients with the b-thalassaemia trait develop iron overload, indicating that other factors are involved in these cases (Bowdler & Huens, 1963; Fargion et al, 2000). Some authors have suggested that iron overload is the result of a synergistic effect of the b-thalassaemia trait and heterozygosity for haemochromatosis (HH) (Fargion et al, 1985), but the data are controversial (Edwards et al, 1981) and date back to the era before the identification of the haemochromatosis gene (HFE). The isolation of HFE and the identification of causal mutations (Feder et al, 1996) enables direct testing of this hypothesis. The majority of Correspondence: Dr Alberto Piperno, Clinica Medica, Azienda Ospedaliera San Gerardo via Donizetti 106, 20052, Monza, Italy. Alberto.Piperno@unimi.it patients with HH are homozygous for the C282Y mutation, a few are compound heterozygotes C282Y/H63D or H63D homozygotes (Feder et al, 1996; Pietrangelo & Camaschella, 1998). A minority of cases presenting with a classical HH phenotype are C282Y or H63D heterozygotes, or carry the wild-type genotype. Compared with Northern Europe, these genotypes appear to be more common in Italy, with the highest prevalence in the South of Italy where they account for more than 50% of the HFE genotypes associated with HH phenotype (Piperno et al, 1998). Further studies have demonstrated the existence of non-hfe genetic determinants in adult (Camaschella et al, 1999; Pietrangelo et al, 1999) and juvenile forms of HH (JH) (Roetto et al, 1999). In the present study, we have studied the clinical phenotype, iron indices and HFE genotypes of 22 unrelated patients with the b-thalassaemia trait who met the phenotypical criteria for HH. In addition, we compared serum iron indices in relatives heterozygous for the C282Y mutation with and without the b-thalassaemia trait. Finally, in order to understand whether genetic components other than HFE 908 q 2000 Blackwell Science Ltd

2 Haemochromatosis and b-thalassaemia Trait 909 Table I. Frequency of C282Y and H63D genotypes and alleles of HFE gene in haemochromatosis (HH) patients with and without the b- thalassaemia trait (b-thal) (allelic frequency is calculated on the number of chromosomes). C282Y genotype H63D genotype HH patients with b-thal n ˆ 22 (%) P-value HH patients without b-thal n ˆ 156 (%) 1/1 ±/± 7 (31 8) (71 8) 1/± ±/± 2* (9 1) NS 9 (5 8) 1/± 1/± 2 (9 1) NS 9 (5 8) ±/± 1/± 4 (18 2) (5 1) ±/± 1/1 1 (4 5) NS 4 (2 5) ±/± ±/± 6 (27 3) (9) Alleles C282Y 18 (40 9), (77 6) H63D² 8 (18 2) (8) S65C 1 (2 3) ± Wild type 17 (38 6), (14 4) *One patient was C282Y/S65C compound heterozygote (see also Table II). ²The frequency of H63D corrected for the number of chromosomes at risk is 30 7% in patients with b-thalassaemia and 35 7% in those without b-thalassaemia. were present in our patients, we sequenced the HFE gene and analysed the inheritance of chromosome 6p and chromosome 1q (where the JH gene has been mapped) in families with multiple-affected siblings non-homozygous for the C282Y mutation. PATIENTS AND METHODS Patients. We studied 22 unrelated Italian patients (20 men and two women) with the b-thalassaemia trait and a classical HH phenotype. Family members numbering 62 individuals were also analysed. Italian unrelated probands (n ˆ 156; 129 men and 27 women) with the HH phenotype without the b-thalassaemia trait were studied to compare clinical and iron data, frequency of HFE genotype and geographical origin. HH phenotype was defined according to classical criteria (Piperno et al, 1998). Diagnosis of the b- thalassaemia trait was defined by low mean corpuscular volume (MCV) and haemoglobin (Hb) concentration, increased amounts of HbA2 and normal HbF (assessed by high performance liquid chromatography), and family analysis. Patients with Hb, 9 g/dl and evidence of other chronic haemolytic anaemias, chronic viral hepatitis, a history of blood transfusions and parenteral iron therapy, or heavy alcohol intake, were excluded. All patients and relatives gave informed consent to the study. Clinical studies. The presence of fibrosis or cirrhosis was determined at liver biopsy. The presence of clinical complications related to HH and total iron removed was determined as previously described (Piperno et al, 1996). Transferrin saturation and serum ferritin were measured by standard methods. Hepatocellular iron overload was graded according to Scheuer et al (1962). The hepatic iron concentration (HIC) was measured by atomic absorption spectrophotometry (Perkin-Elmer S2380, Norwalk, CT, USA) and the hepatic iron index (HII) was calculated as the ratio of HIC (mmol/g dry weight) to age (years). The geographical origin of the patients was ascertained up to the third generation. Molecular studies. Genomic DNA was extracted from peripheral leucocytes. The C282Y, H63D and S65C mutations of the HFE gene were detected as previously described (Feder et al, 1996; Mura et al, 1999). Microsatellites D6S265, D6S105 and D6S1281 were analysed as described (Camaschella et al, 1996). Linkage to chromosome 1q was assessed by analysis of microsatellites D1S2344, D1S442 and D1S498 (Roetto et al, 1999). Haplotypes were constructed manually on the basis of intrafamilial segregation of the marker alleles. The entire sequence of the HFE gene was amplified by polymerase chain reaction (PCR) using previously described primers (Carella et al, 1997) in six unrelated probands with an incomplete HFE genotype and in three affected siblings (see below). The nucleotide sequence of both single strands was independently determined using the dideoxy chain-termination reaction with sequenase version 2 0 (USB Amersham, Cleveland, OH, USA) and [ 35 S-a]-dATP according to the protocol recommended by the manufacturer (USB Amersham). Differences between groups of patients were evaluated by the non-parametric Mann±Whitney test and Fischer's exact test. They were calculated using the statistical package In Stat 2 01 (GraphPad Software, San Diego, CA, USA). All parameters are expressed as means ^ standard deviation. RESULTS Patients The two groups of patients with or without the b- thalassaemia trait did not differ between age (47 ^ 12 years vs. 46 ^ 16 years), transferrin saturation (82 ^ 17% vs. 85 ^ 14%), serum ferritin [2847 ^ 1952 mg/l (range 620±6757) vs ^ 2124 mg/l

3 910 A. Piperno et al Table II. Individual data of patients with haemochromatosis and b-thalassaemia trait according to their HFE genotypes. Number HFE genotype Sex Age (years) Hb (g/ dl) TS (%) SF mg/l Hepatic iron grade HIC* mmol/g HII² IR³ (g) Organ damage 1 C282Y1/1 Male C, D, CA, H 2 C282Y1/1 Male C, D, H 3 C282Y1/1 Male ± C, CA 4 C282Y1/1 Male F 5 C282Y1/1 Male C, H 6 C282Y1/1 Male ± C, CA, H 7 C282Y1/1 Female C 8 C282Y 1/± Male F 9 C282Y 1/± Female none S65C 1/± 10 C282Y 1/± Male F H63D 1/± 11 C282Y 1/± Male C, D H63D 1/± 12 H63D 1/± Male F 13 H63D 1/± Male none 14 H63D 1/± Male ± C 15 H63D 1/± Male ± ± ± C 16 H63D1/1 Male F 17 Wild type Male ± ± 8 4 F 18 Wild type Male ± ± 9 C 19 Wild type Male C 20 Wild type Male C, CA, A, H 21 Wild type Male none 22 Wild type Male F *Hepatic iron concentration. ²Hepatic iron index [HIC (mmol/g dry weight)/age (years)]. ³Iron removed. TS, transferrin saturation; SF, serum ferritin; F, fibrosis; C, cirrhosis; CA, cardiopathy; A, arthropathy; H, hypogonadism; D, diabetes. (range 144±11389)], HIC (327 ^ 138 mmol/g vs. 359 ^ 180 mmol/g dry weight), HII (7 6 ^ 4 3 vs. 8 2 ^ 4 8), and male to female ratio. By contrast, the geographical origin of the patients significantly differed between the groups: 10 out of 22 (45 5%) patients with the b- thalassaemia trait originated from Southern Italy compared with 15 out of 156 (11 5%) patients without the b- thalassaemia trait (P ˆ ). Table I shows the frequency of the C282Y and H63D genotypes and alleles in patients with and without the b-thalassaemia trait. The C282Y and the wild-type alleles were, respectively, significantly lower and higher in the former than in the latter group. The frequency of HFE genotypes in subgroups of similar geographical origin did not differ significantly in patients with and without b-thalassaemia, although the limited number of patients does not allow a proper analysis (data not shown). Table II shows the individual data of the patients with the b-thalassaemia trait grouped according to their HFE genotypes. All C282Y homozygotes had a severe HH phenotype, as shown by the high amount of iron overload and the presence of HH-related clinical complications. Compared with 93 C282Y homozygote men without b-thalassaemia, the six men with the b-thalassaemia trait were younger (36 2 ^ 5 2 years vs ^ 11 years, P ˆ 0 041), and had higher levels of serum ferritin (4951 ^ 1707 mg/l vs ^ 2239 mg/l, P ˆ 0 012) and HII (12 5 ^ 4 1 vs. 8 6 ^ 4 3, P ˆ 0 041). Moreover, they also showed higher prevalences of heart failure (50% vs. 6 5%, P, 0 01), clinically manifested pituitary hypogonadism (66 6% vs. 10 7%, P, 0 01) and hepatic pre-cirrhosis or cirrhosis (100% vs. 50 6%, P ˆ 0 029). Among male patients with the b-thalassaemia trait, C282Y homozygotes were younger, had higher serum ferritin and HII levels than wild-type homozygotes and H63D heterozygotes in the order, P ˆ 0 005, P ˆ 0 04 and P, respectively (non-parametric Kruskal±Wallis test). However, as shown in Table II, patients with the wild-type genotype had a heterogenous expression from moderate to very severe. Family studies Family studies were performed in five C282Y homozygotes and seven C282Y non-homozygous probands. Five affected siblings were detected: one was homozygous for C282Y and four were not. In the families of the C282Y homozygous probands, we identified 25 individuals (11 with and 14 without the b-thalassaemia trait) who were heterozygous for C282Y and aged 48 7 ^ 13 4 years

4 Haemochromatosis and b-thalassaemia Trait 911 Fig 1. Inheritance of chromosome 6p haplotype in four pedigrees in which non-homozygous C282Y haemochromatosis (black square) and the b-thalassaemia trait (asterisk) co-exist. The arrows indicate the probands. Numbers above the circles and squares are pedigree numbers; haplotypes are reported below and are defined by D6S265, D6S105, D6S1281 alleles and HFE mutations. (range 28±73 years). Serum iron indices did not differ according to the presence or absence of the b-thalassaemia trait [transferrin saturation: 38 ^ 16% (range 11±58) vs. 36 ^ 19% (range 14±88), serum ferritin: 183 ^ 104 mg/ l (range 38±343) vs. 149 ^ 138 mg/l (range 17±483) respectively], even when data were corrected for sex and age (data not shown). Figure 1 shows the pedigrees of the four families with affected siblings non-homozygous for the C282Y mutation and Table III shows haematological and iron data of the individuals from the respective families. In family 1, the two siblings showed the classical HH phenotype; they were both heterozygous for b-thalassaemia and C282Y and carried the same chromosome 6p haplotypes. The father was heterozygous for b-thalassaemia and C282Y in addition, but had only slightly increased transferrin saturation. Sequence analysis detected the presence of three already described polymorphisms only [IVS.2 (15) T!C, IVS.4 (244) T!C and IVS.5 (247) G!A respectively) (Beutler & West, 1997) trans to the C282Y allele. In family 2, the two affected siblings shared the same chromosome 6p haplotypes, a single H63D allele and the b-thalassaemia trait. The third brother, heterozygous for both b-thalassaemia and H63D, shared only one haplotype with the proband and had only slightly increased transferrin saturation. Sequence analysis did not demonstrate causal mutations, but only the IVS.2 (15) and IVS.5 (247) polymorphic changes in cis to H63D. In family 3, the two affected siblings were both heterozygous for b-thalassaemia and H63D. A third brother without the b-thalassaemia trait, but with a haplotype identical to the proband, had a history of high alcohol intake (. 80 g/d) and slightly increased levels of serum ferritin, alanine aminotransferase and g-glutamyltransferase. The woman refused a liver biopsy, but her iron data are consistent with iron overload that has developed despite the presence of several protective factors (abundant menses, four pregnancies, peptic ulcer and late menopause). Her sons were all H63D heterozygotes, two of them without the b-thalassaemia trait, and had increased serum iron indices; the third son, with the b-thalassaemia trait, had normal iron indices. In family 4, parents and siblings were all heterozygous for b-thalassaemia and carried the wild-type genotype, confirmed by sequencing the HFE gene. The two siblings were 6p haploidentical and the phenotype expression was more severe in the younger sibling. Although the phenotype of the probands was not

5 912 A. Piperno et al Table III. Haematological data and iron status of the individuals of the four families with affected siblings with incomplete HFE genotypes (see pedigrees in Fig 1). Family Individual Age (years) Hb (g/dl) TS (%) SF (mg/l) Hepatic iron grade HIC² (mmol/g) HII³ 1 I-1* ± ± ± I ± ± ± II-1* II-2* II-1* II-2* ±175 ± ± ± II-3* I-1* ± ± ± I ± ± ± I-3* ± ± II ± ± ± II ± ± ± II-3* ±199 ± ± ± 4 I-1* ± ± ± I-2* ± ± ± II-1* II-2* *b-thalassaemia trait. ²Hepatic iron concentration. ³Hepatic iron index [HIC (mmol/g dry weight)/age (years)]. Magnetic resonance imaging positive for the presence of hepatic iron. compatible with JH, linkage to 1q was assessed and excluded in families 3 and 4. DISCUSSION The association of the b-thalassaemia trait with C282Y homozygosity is rather rare; this is not unexpected, given the different ethnic background of the two molecular defects. However, as shown in this study, the b-thalassaemia trait aggravates the clinical picture of C282Y homozygotes, favouring the development of marked iron overload and severe iron-related complications. C282Y homozygotes lack the function of HFE that normally regulates iron uptake, by reducing the affinity of transferrin receptor for transferrin (Feder et al, 1998). It has been suggested that HFE in cryptic duodenal cells might respond to body iron stores and demands, and that a failure in this regulatory loop might cause a dissociation between iron absorption and body iron stores in C282Y homozygotes (Pietrangelo & Camaschella, 1998; Waheed et al, 1999). We speculate that the coexistence of the b-thalassaemia trait, probably through a mild ineffective erythropoiesis, increases the rate of intestinal iron absorption beyond that induced by the lack of HFE in C282Y homozygous patients, possibly through an alternative regulatory mechanism that modulates intestinal iron absorption in response to the needs of the erythron (Levy et al, 2000). Accordingly, it is possible that coinheritance of the b-thalassaemia trait increases the risk of developing iron overload in patients with mild HFE genotypes, such as C282Y/H63D or S65C compound heterozygotes and H63D homozygotes. By contrast, our results in patients and families indicate that the coexistence of the b-thalassaemia trait with a single C282Y allele is unable to produce a HH phenotype. Other family studies are in agreement with our own results and suggest that an associated increase in erythroid activity does not appreciably enhance iron absorption in C282Y heterozygotes with haemolytic anaemia (Dykes et al, 1998) and thalassaemia intermedia (Cappellini et al, 1998). It is also improbable that the association of the b-thalassaemia trait with a single H63D defect could lead to the development of HH because of the very mild effect of H63D on HFE function (Feder et al, 1998). It is probable that other genetic determinants exist in HH patients with incomplete HFE genotypes. The presence of rare mutations other than C282Y, H63D and S65C has recently been reported in HH patients (Barton et al, 1999; Wallace et al, 1999; Piperno et al, 2000), but no causal mutations were found in our patients by sequencing the HFE gene. In order to investigate the presence of non-hferelated determinants, we analysed the inheritance of chromosome 6p haplotypes in the four families of patients non-homozygous for C282Y. In families 1 and 2, the two affected brothers shared C282Y or H63D in the heterozygous state and the same 6p haplotypes, suggesting the existence of genetic factors related to chromosome 6. Other defects might exist in the promoter region of the HFE gene that we did not analyse. The presence of other chromosome 6p gene(s) modulating the function of HFE has been recently suggested (Pratiwi et al, 1999; Levy et al, 2000),

6 but it is still to be formally proved. It is also possible that the identity of chromosome 6p haplotypes in the two affected sibling pairs is coincidental and that other defects not linked to HFE exist in those families. In families 1, 2 and 3, a synergistic effect of the b-thalassaemia trait and heterozygosity for C282Y or H63D seems improbable considering the normal iron indices in subjects I-1 in family 1, II-2 in family 2 and II-3 in family 3. In the latter family, the nonidentity of the two affected brothers at 6p and 1q suggests a disorder not linked to chromosomes 6 and 1. In family 4, data are consistent with a recessive non-hfe, non-jh disorder in which the association of the b-thalassaemia trait with a single affected allele is not able to produce a HH phenotype. 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