Genetic abnormalities and juvenile hemochromatosis: mutations of the HJV gene encoding hemojuvelin

The first description of what is now designated as juvenile hemochromatosis was published in 1932 by Bezançon et al.¹  Their patient, age 20 years, was described as having pigmentary cirrhosis with an enlarged liver, infantilism, and multiple endocrine insufficiencies. He died of cardiac failure.

Those findings correspond closely to the syndrome as described more recently.²  Hypogonadotrophic hypogonadism and cardiac failure, not only an early age of onset of iron overload, distinguish this disorder clinically from the much more common type of hemochromatosis that results from mutations of the HFE gene. Most cases of juvenile hemochromatosis were found to be genetically linked to chromosome 1q.^3-6  However, a few patients with the same syndrome did not have this linkage and were shown to have mutations of the HAMP gene encoding hepcidin.⁷ 

Because no gene known to regulate iron homeostasis was known to exist on chromosome 1q, this putative juvenile hemochromatosis gene became a prime target for positional cloning. Very recently, the putative gene responsible for the Ch1q-linked form of the disorder was cloned. It encodes a transcription unit of previously unknown function (LOC148738) that has been designated hemojuvelin.⁸  The suggested designation of the gene encoding hemojuvelin, namely HFE2, is inappropriate because it is contrary to the accepted guidelines for gene nomenclature.⁹  The use of an Arabic numeral is recommended for designation of a gene family, but the gene encoding hemojuvelin is not a part of the HFE family. Thus, a more appropriate designation for this gene is HJV, which has been adopted by Online Mendelian Inheritance in Man (OMIM).

We previously described 7 cases of juvenile hemochromatosis in 2 kinships, including one that had originally been reported in 1962.⁴  We have now sequenced the HJV gene of these patients with early-onset iron overload. We confirm that, indeed, mutations of this gene are responsible for the syndrome of juvenile hemochromatosis in most patients. We report the existence of 2 mutations of the HJV gene that have not been previously described and define the haplotypes in which they occur.

We evaluated persons in each of 2 unrelated kinships from the southeast United States; all were white. In kinship A, we previously reported 4 siblings with a juvenile hemochromatosis clinical phenotype⁴  and demonstrated that they were homozygous for the same Ch1q haplotype. The third of the 4 siblings, an 18-year-old woman who had amenorrhea since age 12, was selected for the present genetic analyses. At diagnosis, she had transferrin saturation 92%, serum ferritin 2003 μg/L, severe hypogonadotrophic hypogonadism, hepatomegaly, and hyperpigmentation. We also evaluated her father's cousin, one of the original 1967 probands who presented at age 23 years with amenorrhea, severe hypogonadism, and transferrin saturation 96% and had phlebotomy-proven iron overload.^4,10  This woman had one Ch1q haplotype apparently identical with that of her younger first cousin once removed and a second, different Ch1q haplotype, and we had presumed her to be a compound heterozygote for the Ch1q-linked gene. In kinship B, we evaluated the parents of a woman who died at age 23 years of cardiomyopathy and was discovered at autopsy to have severe, multiorgan iron overload and hepatic cirrhosis. At age 17 years, she experienced cessation of menses and later developed other signs of severe hypogonadism and hyperpigmentation.⁴  The clinical characteristics of these patients are summarized in Table 1. Approval was obtained from the Scripps Research Institutional Review Board and from the Brookwood Medical Center Institutional Review Board for these studies. Informed consent was provided according to the Declaration of Helsinki.

The primers used to amplify the HJV gene are shown in Table 2. Amplification was performed in a 50-μL reaction mix containing 50 to 200 ng genomic DNA, 150 ng of each primer, 33.5 mM Tris (tris(hydroxymethyl)aminomethane) HCl pH 8.8, 8.3 mM (NH₃)₂SO₄, 3.35 mM MgCl₂, 85 μg/mL bovine serum albumin, and 1 U Taq polymerase. After initial denaturation at 95°C for 4 minutes, amplification was performed for 30 cycles at 95°C for 1 minute, 60°C for 30 seconds, and 72°C for 30 to 50 seconds.

The primers and conditions for the amplification of the HFE gene¹¹  and the hepcidin gene¹²  were as described previously. The region around the HJV gene contained single nucleotide polymorphisms and microsatellites that were useful for haplotyping the subjects. The primers and annealing temperatures for haplotype analysis are described in the supplementary material available at the Blood website (see the Supplemental Tables link at the top of the online article). Amplification was performed using the polymerase chain reaction (PCR) conditions described earlier.

Amplified DNA products were purified by using Qiaquick PCR purification kits (Qiagen, Valencia, CA). Genotypes were determined by direct sequencing using the ABI 3100 DNA sequencer (Applied Biosystems, Foster City, CA).

The pedigrees and the result of mutation analysis of the 2 kinships are shown in Figures 1 and 2. Subject II-5 from kinship A was a compound heterozygote with c.238T>C (C80R) and c.302T>C (L101P) mutations in the hemojuvelin gene. Subject III-2 in kinship A was, as expected from the chromosome 1q haplotype that had been determined earlier, homozygous for a hemojuvelin mutation, L101P. The C80R and L101P mutations have not been reported previously. The haplotypes in which they were found are defined as a series of single nucleotide polymorphisms (SNPs) in the region surrounding the HJV gene and are summarized in the Supplemental Tables. A large number of genes in the region of the HJV gene were sequenced in subject II-5 and/or subject III-3. No mutations were found. The genes that were examined are listed in the Supplemental Tables.

Subjects I-1 and I-2 from kinship B were the parents of a woman who died at age 23 of putative juvenile hemochromatosis.⁴  Subject I-1 was a simple heterozygote carrying the c.959G>T (G320V) mutation in the HJV gene. Subject I-2 was a simple heterozygote carrying the c.665T>A (I222N) mutation in the HJV gene. Both the G320V and the I222N mutations have been described previously.⁸  The deceased subject II-1 from kinship B is, therefore, presumed to be a compound heterozygote carrying both mutations in the HJV gene.

The results of these investigations confirm the assignment of the gene causing chromosome 1q-linked juvenile hemochromatosis. In 2 families manifesting the typical clinical picture of this disorder, mutations were found in the HJV gene. The heterogeneity of the HJV mutations and Ch1q haplotypes demonstrated in the present kinships and in those previously reported^4,5,8  are typical of rare heritable disorders. Most patients with juvenile hemochromatosis are diagnosed younger than age 30 years and have hypogonadism, hepatic fibrosis, or cirrhosis, or cardiomyopathy.^8,13-15  However, some patients, their siblings, or other affected members with juvenile hemochromatosis have fewer complications of iron overload at diagnosis. This observation suggests that, although age of onset may influence the phenotype, juvenile hemochromatosis appears to have unique features, especially endocrinopathies (hypogonadotrophic hypogonadism) and cardiomyopathy. These complications may be the result of age-dependent organ susceptibility to iron, unusually high levels of non-transferrin-bound iron, tissue-specific iron deposition, transport or avidity, or other peculiarities of the mutations themselves. It is clear from kinship A, in which 4 siblings are affected, that the penetrance of HJV mutations is very high, in contrast with hemochromatosis associated with HFE mutations.

Although the appearance of the disease phenotype in 2 successive generations might have suggested dominant inheritance with low penetrance, genetic analysis makes it clear that the inheritance is autosomal recessive, and that the pattern observed represents pseudodominance. Such pseudodominant inheritance is common when the disease gene frequencies are high. For example, it was once believed that Gaucher disease was a dominant disorder,¹⁶  but it is now clear that the apparent dominance was due to the mating of heterozygotes with a homozygotes in a population in which about 7% were heterozygous. Pseudodominance must be much less common in the case of rare mutations such as those of hemojuvelin.

The technical assistance of Mrs Terri Gelbart and Mrs Carol Halloran is gratefully acknowledged.