Compound heterozygous C282Y/Q283P and Q283P/H63D mutations in haemochromatosis

The discovery of the haemochromatosis gene (HFE) (Feder et al, 1996) and its most common mutations, C282Y and H63D, in patients with iron overload has improved the diagnosis of hereditary haemochromatosis. A homozygous C282Y mutation or compound heterozygous C282Y/H63D or C282Y/S65C is present in most patients (Hanson et al, 2001; Adams et al, 2005). Other mutations in HFE are very rare, but can be of importance to explain iron overload in patients (Pointon et al, 2000; Swinkels et al, 2008). We found a very rare Q283P missense mutation, previously described in only one case in compound heterozygosity with C282Y (Le Gac et al, 2003). To the best of our knowledge, no other reports on this mutation have been published. The proband was a 45-year-old man, with an elevated transferrin saturation of 89% (reference value: <50%) and serum ferritin of 1747 μg/l (reference range: 20–250 μg/l) at diagnosis of hereditary haemochromatosis. A magnetic resonance imaging scan showed moderate iron overload. Liver biopsy showed diffuse haemochromatosis, multifocal macrovesicular steatosis and periportal fibrosis. No signs of cirrhosis were observed. Two brothers and two daughters were also investigated for the presence of HFE mutations. The proband's parents are deceased. The Q283P mutation is located in a β-strand position adjacent to the cysteine residue at position 282, which is involved with cysteine 225 in the key disulphide bridge of the HFE domain α3. Modelling studies (Ka et al, 2005) reveal strong arguments for a dramatic effect of the Q283P variant on the 3D structure of the wild-type folding. The Q283P mutation hampers the formation of the important Cys-225-Cys282 disulphide bond. Further biochemical experiments (Ka et al, 2005) confirmed that the Q283P mutation has structural consequences, affecting the protein function, similar to those described for the C282Y-mutated HFE protein. DNA was extracted from 100 μl of blood anti-coagulated with EDTA, using the automatic NucliSENS EasyMAG DNA isolation (BioMérieux, Zaltbommel, the Netherlands). The method of Mangasser-Stephan et al (1999) was used for simultaneous analysis of the two most common HFE mutations, C282Y and H63D. The primer design used in this method results in the amplification of polymerase chain reaction (PCR) fragments of 390 and 310 base pairs spanning the C282Y and H63D mutation sites. Detection of the mutation is based on hybridization probes using the fluorescent resonance transfer (FRET) technique and melting curve analysis. The melting curves showed specific melting temperatures for the HFE wild-type (C282C and H63H) and the variants (C282Y and H63D), respectively. Other mutations lying within the hybridization probe that spans the area of the mutation site mostly result in different melting curves. Sanger sequencing of the PCR products from the different melting curve revealed the Q283P mutation. Figure 1 shows the melting curves from the proband (C282Y/Q283P), his 14-year-old daughter (C282Y/H63D) and his 16-year-old daughter (Q283P/H63D). Both brothers of the proband were heterozygous for C282Y and did not have iron overload. The absorbance with a Tm at 50·5°C observed for the proband was different from the Tm of the wild-types C282C and H63H and the C282Y and H63D mutations. The absorbance with a Tm at 50·5°C could be attributed to the Q283P mutation. Figure 2 shows Sanger sequencing of the PCR fragment from the proband and his 16-year-old daughter (Q283P/H63D). The analysis shows both the 845 G>A (C282Y) and 848 A>C (Q283P) mutations in the proband and a single 848 A>C mutation (Q283P) in his daughter, indicating a C282Y/Q283P mutation in trans, i.e., on the two different DNA-strands in the proband. Our results indicate the association between the C282Y/Q283P compound heterozygous genotype in trans and the development of iron overload. A C282Y/Q283P compound heterozygous mutation in cis will not result in haemochromatosis, because a wild-type HFE gene can still be expressed. Because the impact of Q283P mutation is comparable to the C282Y, it is expected that the clinical implications of Q283P/H63D and C282Y/H63D are also comparable. The two daughters with compound heterozygous C282Y/H63D and Q283P/H63D genotype, respectively, showed no iron overload. However, it should be noted that they are only 14 and 16 years of age, respectively. Both are susceptible to iron overload in adult life. This is the second case report on the HFE Q283P mutation. As in the first report of the Q283P mutation in an individual from Brittany (Le Gac et al, 2003), our proband developed iron overload with the co-inheritance of a C282Y mutation. Also in this family the rare Q283P mutation is observed together with the C282Y. A heterozygous Q283P mutation has not been reported yet. Firstly, the prevalence of heterozygous Q283P is very low. Secondly, a heterozygous Q283P mutation apparently does not lead to iron overload and so the Q283Y mutation would not be subjected to DNA analysis. Finally, the DNA method used should be able to detect the Q283P mutation, while screening for the more common C282Y mutation. The method used in the present report could simultaneously detect the relevant HFE mutations H63D, S65C, C282Y and Q283P in one single PCR reaction. AvG wrote the manuscript and critically revised the manuscript. EdB and LS performed DNA analysis. PvW performed research and wrote or critically revised the manuscript. All authors approved the submitted version of the manuscript. The authors have no competing interests.