Nontransfusional Iron Overload in Thalassemia: Association With Hereditary Hemochromatosis

To the Editor:

Hemochromatosis is inherited as an autosomal recessive trait and is manifested by excessive absorption of dietary iron in homozygotes leading to complications of iron overload and premature death.1 In 1996, a candidate gene for hemochromatosis, HLA-H, was isolated.2 A missense mutation that led to the substitution of a tyrosine for cysteine at codon 282 (Cys282Tyr) was identified in the vast majority of patients with hemochromatosis, with 80% to 100% of patients with hemochromatosis being homozygous for the HLA-H Cys282Tyr allele.2-5 A second mutation resulting in the substitution of aspartic acid for histidine at codon 63 (His63Asp)2 was also identified, but its role is uncertain. Recent surveys have shown that the Cys282Tyr allele is frequent in Northern Europeans, with a frequency of 10% in some populations, but rare in Africans and Asians.6 Although the levels of serum ferritin, serum iron, and transferrin saturation are higher than normal in heterozygotes for hemochromatosis, complications due to iron overload are extremely rare in these individuals.7 

Apart from hereditary hemochromatosis, there are a number of other conditions in which nontransfusional iron loading occurs; these include sideroblastic anemia, porphyria cutanea tarda, the thalassemia syndromes, pyruvate kinase deficiency, and hereditary atransferrinemia. Among these, thalassemia is the only one that occurs commonly and is prevalent in the tropical and subtropical regions.8 Carriers for β thalassemia may have a very mild anemia but rarely iron overload; heterozygosity for β thalassemia also does not appear to accentuate iron loading in individuals homozygous or heterozygous for hemochromatosis.9 Unlike heterozygous β thalassemia, which is clinically asymptomatic, thalassemia intermedia is manifested by a symptomatic anemia associated with increased iron absorption, but regular transfusions are not necessary.8 

We have screened 81 thalassaemia intermedia patients (46 with 2 β thalassemia alleles, 6 β thalassemia heterozygotes with ααα/ααα or α α/ααα genotypes, 6 with dominantly inherited β thalassemia, and 23 hemoglobin [Hb] E/β thalassemia) for the Cys282Try and His63Asp mutations in the HLA-H gene. Their ages ranged from 10 to 65 years, with the following ethnic mix: Asian Indian (58%), Middle Eastern (17%), Mediterranean (15%), English (7%), and Chinese (3%). None of the patients is on a regular transfusion regime.

The mutations were detected by restriction enzyme analysis of polymerase chain reaction (PCR)-amplified DNA⁶; the Cys282Tyr mutation creates a new Rsa I site, whereas the His63Asp mutation removes an Mbo I site. Deletional α and β thalassemia and rearrangement of the α and β globin gene complex were screened for by Southern blot hybridization. Point mutations causing β thalassemia were detected by sequence analysis of PCR-amplified β globin genes.10 

The Cys282Tyr mutation was detected in only 1 of the 81 individuals with thalassemia intermedia. The proband presented in 1975 at the age of 42 years with joint pains. Hepatomegaly was noted with a Hb level of 9.8 g/dL and a serum iron level of 44.1 mmol/L with 71% transferrin saturation. A diagnosis of thalassemia and gout was made. He was lost to follow-up, but re-presented in 1981 with diabetes mellitus and was started on oral hypoglycemic agents, later needing insulin. Over the next decade, although his Hb level remained fairly stable, his ferritin level gradually increased with clear evidence of overload (Table 1). In 1994, his condition had worsened, with a Hb level of 6.5 g/dL and 9% reticulocytes; deep skin pigmentation and marked hepatosplenomegaly were noted. He received a single blood transfusion for the first time. In 1995, aged 62 years, he had a splenectomy and liver biopsy. Liver histology showed markedly increased iron (hepatic iron was 1.86% dry weight; normal <0.13%) and the hemosiderin granules were distributed mainly in hepatocytes with relative sparing of the Kupffer cells, features typical of primary hemosiderosis. There was background hepatitis, but autoantibody screen was negative and there was no evidence of hepatitis A, B, or C. After splenectomy, his Hb level increased to 7 to 9 g/dL. He was started on subcutaneous desferrioxamine. In 1996, he suffered a myocardial infarction and received a second blood transfusion in an attempt to improve his myocardial oxygenation.

There is no family history of anemia, diabetes, hemochromatosis, or arthritis. The proband was born in the Punjab, India, and had never been transfused until 1994, at the age of 61 years. He is married with four children, one of whom (daughter) was accessible for investigation.

Results of the investigation are summarized in Table 2. The proband has a marked globin chain imbalance with an α/β globin chain synthesis ratio of 4.1, which is much more severe than that encountered in β thalassemia trait. However, DNA analysis could identify only a single β thalassemia mutation (codon 41/42 −TCTT), but compound heterozygosity for a mild β thalassemia mutant is implicated from clinical, hematologic, and family studies. His daughter has mild but significant hypochromic microcytic red blood cells, with an α/β globin chain synthesis ratio of 2.1 that is typical of β thalassemia trait. However, extensive sequence analysis of the β globin genes (from position −630 upstream of the mRNA cap site of the β globin gene to 290 bp downstream of the termination site including the whole β gene) showed only a single β thalassemia allele in the father and no mutation in the daughter. DNA mapping of the α and β globin cluster in both father and daughter showed no deletions or rearrangements; in particular, there was no extra α globin gene. Serum transferrin receptor (sTfR) level was markedly elevated in the proband, suggesting significant expansion of the erythron consistent with thalassemia intermedia, whereas normal levels were found in the daughter and wife. Electron microscopy of the father's bone marrow showed intraerythroblastic inclusions indistinguishable from the precipitated α-globin chains seen in β thalassemia. The proportion of erythroblast sections containing such inclusions (17%) was considerably higher than that in β thalassemia trait and similar to that in the thalassemia intermedia syndromes caused by Hb E/β thalassemia.11 

Both father and daughter were heterozygous for the Cys282Tyr mutation in the HLA-H gene; neither had the His63Asp minor mutation. The mother was normal for both HLA-H alleles.

It seems likely that the phenotype of thalassemia intermedia in the proband is produced by compound heterozygosity for the FS 41/42 (−4 bp), a common β thalassemia mutation in Asian Indians,8 and a mild β⁺ thalassemia allele that has not been identified. Such uncharacterized β thalassemia mutations have been described, some of which may not be linked to the β globin complex.10 Other possible diagnoses for the unusually severe anemia in the proband, such as congenital dyserythropoietic anemia and sideroblastic anemia, are excluded by the electron microscopy findings.

Although excessive iron absorption is well described in untransfused thalassemia intermedia,12 there are few reports of such individuals developing the complications of severe iron overload as seen in our patient. Certainly the majority of thalassemia intermedia patients present with symptoms of anemia, unlike the proband, whose manner and age of presentation is typical of homozygous hereditary hemochromatosis. This is one of the first examples of the heterozygous state for hemochromatosis expressing a disease phenotype and appears to result from the interacting effects of the chronic anemia from thalassemia intermedia and the Cys282Tyr HLA-H mutation. The nature of the interaction between these alleles is unclear, because the mechanisms for excessive iron absorption in both conditions are not established.

The interaction between thalassemia intermedia and hereditary hemochromatosis must also occur in many other individuals, even if the populations in which these two traits are common do not coincide. A recent report has suggested that the HLA-H Cys282Tyr mutation is an important predisposing factor in spontaneous porphyria cutanea tarda.13 Our study supports the emerging importance of genetic screening for hereditary hemochromatosis in other iron-loading conditions.