Effect of the new HJV-L165X mutation on penetrance of HFE

To the editor:

We present a new homozygous truncating mutation, L165X, of the hemojuvelin gene (HJV), observed in one male patient with severe juvenile hemochromatosis (JH). Because the C282Y-variant in the hemochromatosis gene (HFE) was also common in this family, we investigated whether the inactivating mutation HJV-L165X influenced penetrance of HFE-C282Y homozygosity.

The proband, born in 1956 and diagnosed in 1972 by family screening (B III-54),¹  presented with increased serum iron values and heavy iron accumulation in the hepatocytes.^1,2  There was no consanguinity between his parents or grandparents,¹  and linkage to HLA was excluded in his familybranch, providing the first clue of genetic heterogeneity in hemochromatosis.³  In late 2005, he provided contact information on relatives. The institutional review board approved this study and informed consent was obtained from all participants (n = 20). Non-fasting blood and urine samples from the proband and his relatives were collected between 7 and 9pmon the same day. Information on the number of phlebotomies and the time between the last phlebotomy and sample collection was provided. Urinary hepcidin was measured by mass spectometry.⁴  For the proband, we sequenced the hepcidin (HAMP) and HJV genes. Mutations in the HAMP gene were absent. The HJV gene was sequenced using previously reported primers.⁵  For exon 3 we designed new primers: Ex3a1935F 5′-GCAAACTACACTCCGATAGAG-3′ and Ex3a2253R 5′-GCTGGATCATCAGGTCTTCG-3′, resulting in a 319 bp product, and Ex3b2202F 5′-GACCTCGCCTTCCATTCG-3′ and Ex3b2603R 5′-GAATCTCATGAGGTGGATCGG-3′, leading to a 402 bp product (GenBank NT_004 434/gi:88 943 080). We observed a novel homozygous mutation in exon 3 of the HJV gene. The 494T→A transversion leads to a premature stop codon at position 165 of the HJV protein: L165X. This probably leads to nonsense-mediated decay of the corresponding messenger RNA. If the aberrant message is translated, however, it would code for a protein that lacks the GPI anchor signal, such that it remains in the endoplasmic reticulum.⁶  In both cases, it can be anticipated that upstream regulation of hepcidin is impaired.⁷ 

Relatives of the proband were investigated for the HFE-C282Y and the HJV-L165X mutation by a restriction fragment length polymorphism analysis using the Ex3b2202F and Ex3b2603R primers and the restriction enzyme HpyCH4V (New England Biolabs, Ipswich, MA).

HJV-L165X homozygosity was only present in the proband, while heterozygosity was common among relatives (allele frequency: 14/40 = 35.0%). Furthermore, the HFE-C282Y mutation was observed frequently (allele frequency: 27/40 = 67.5%) (Table 1). Phlebotomies were only reported in individuals later found to be homozygous for either HJV-L165X (n = 1; proband) or HFE-C282Y (n = 8). Iron indices (current and from the early seventies^1,2 ; J. P. G., unpublished data, early 1970s) are copied into Table 1.

Current serum iron parameters are not appropriate as a measure of iron burden, as most relatives have been adequately phlebotomized. We found an alternative in the following parameters: quantity of iron removed by phlebotomies (iron removed/age),⁸  a rough estimate hampered by the probability that intestinal iron uptake may increase upon phlebotomy; transferrin saturation (TS) values and desferrioxamine (DFO) test results from the early seventies, before treatment; and urinary hepcidin levels, measured with our improved MS assay.⁴  The iron removed/age, urinary hepcidin levels, and iron indices from the early seventies were similar for HJV-heterozygotes (L165X) and the HJV-wildtypes, also when stratified by HFE genotype, indicating the absence of a clinically relevant modifying effect. Others reported HJV a modifier,^9,10  although not consistently.¹¹  Furthermore, our data agree with recent findings that HFE and HJV participate, at least partially, in distinct regulatory pathways.¹²  Finally, against a background of multiple small variations, we cannot exclude a minor effect of the heterozygous HJV-L165X mutation on iron homeostasis.