Diagnostic value of targeted next‐generation sequencing in suspected hemochromatosis patients with a single copy of the <i>HFE</i> p.Cys282Tyr causative allele
Authors: Kevin Uguen, Virginie Scotet, Chandran Ka, Isabelle Gourlaouen, Carine L'hostis, Marie‐Christine Merour, Tania Cuppens, Claude Ferec, Gerald Le Gac
Journal: American Journal of Hematology (2017)

The best-known and most prevalent form of hemochromatosis is an adult-onset autosomal recessive condition usually associated with the HFE p.[Cys282Tyr];[Cys282Tyr] genotype. In Caucasians, this genotype is carried by approximately 1 person in 200–300. A second HFE variant, p.His63Asp, has proved to be enriched on the non-p.Cys282Tyr alleles of hemochromatosis patients, and it has been argued that the p.[Cys282Tyr];[His63Asp], but not the p.[His63Asp]+[His63Asp], compound heterozygous genotype increases the risk of disease.1 Apart from the widespread p.His63Asp allele, rare HFE mutations can be found in trans with the causative p.Cys282Tyr substitution. Complex alleles associating the p.His63Asp variant and a second amino-acid alteration have also been observed.2 Sequencing the entire HFE coding region in suspected hemochromatosis patients where the standard HFE genetic test reveals a heterozygote status for p.Cys282Tyr is, however, rarely performed. We used targeted next-generation sequencing to investigate all hemochromatosis genes (HFE, HFE2, HAMP, TFR2, and SLC40A1) and BMP6, which has recently been associated with mild to moderate late-onset iron overload,3 in 42 patients with unexplained hyperferritinemia (men ≥300 µg/L and women ≥200 µg/L). All had serum transferrin saturation of at least 60% (men) or at least 50% (women), consistent with the natural history of hemochromatosis. The patient group was composed of 7 p.Cys282Tyr heterozygotes and 35 p.Cys282Tyr/p.His63Asp compound heterozygotes. The splice site c.76 + 2T>C and nonsense p.Trp155* HFE mutations were separately detected in two females firstly identified as simple p.Cys282Tyr heterozygotes. They both had a severe phenotype, manifested by very high levels of serum ferritin and amounts of iron removed by phlebotomy to restore normal iron indices greater than 3 g in the absence of comorbid factors (Table 1). The c.76 + 2T>C splicing mutation is present in the GnomAD database but at a very low frequency (8.19 × 10−6) as it was only seen in two individuals (one Latino and one European). Here, we demonstrate its functional impact on HFE pre-mRNA splicing: it results in intron retention and may give rise to a truncated and inactive protein (Supporting Information Figure 1). To the best of our knowledge, the p.Trp155* nonsense mutation has never previously been reported. It was not detected in 1,460 control chromosomes from the western part of France and, thus, it is also expected to be very rare or private. AIR (g) We identified a rare HFE missense mutation, which changes glutamic acid at position 168 to glutamine (p.Glu168Gln; p.E168Q), in a 51-year-old man with 61% transferrin saturation and 487 μg/L serum ferritin at diagnosis (Table 1). On clinical interview, the patient declared moderate but daily alcohol consumption (approximately 16 U per week). Cloning genomic HFE sequences, from exon 2 to exon 3 and from exon 3 to exon 4, revealed that the p.Glu168Gln substitution was in cis with the p.His63Asp allele (Supporting Information Figure 2). This observation is consistent with the study of an Italian pedigree, where the p.Glu168Gln and p.His63Asp variants were inherited together (in cis) and independently of the p.Cys282Tyr-bearing chromosome.4 By sequencing the chromosomes of 730 healthy French adults, we found the frequency of the complex allele to be 0.00274. There is at present moderate evidence to support a causal role for the HFE p.Glu168Gln missense mutation. This consists in the c.502G>C transversion reported by the GnomAD Consortium at very low frequency in non-Finnish Europeans (<0.001; rs146519482). Apart from the Italian pedigree, it has been reported only twice in the literature,2 and genotype-phenotype correlation is impossible. In silico evaluation does not clarify the contribution of this rare genetic variant to disease. Indeed, substitution of glutamic acid by glutamine is a biochemically moderate change with a Grantham distance of 29 (0–215). This change at position 168 of the HFE protein is predicted to be neutral by Align GVGD (score: C0; GV: 54.02-GD: 0.00), Mutation Taster (polymorphism; P value: 0.707) and SIFT (tolerated; score: 0.06), whereas it is predicted to be probably damaging by PolyPhen-2 (score: 0.980; HumVar model). More importantly, however, is perhaps the finding that glutamic acid 168 is part of a region that may contribute considerably to the interaction between HFE and transferrin receptor 1 (TfR1).5 We have previously hypothesized that the p.Leu96Pro missense mutation is enriched in French patients with hyperferritinemia and no mutations in genes commonly associated with hemochromatosis.6 This mutation has been proven to decrease BMP6 secretion in opossum kidney (OK) cells.3 We report here, for the first time, the case of a 43-year-old woman bearing both the p.[Cys282Tyr];[His63Asp] compound heterozygous HFE genotype and the p.[Leu96Pro];[=] heterozygous BMP6 genotype (Table 1). The patient presented with a moderate degree of iron accumulation in the body as revealed by slightly elevated transferrin saturation (69%) and serum ferritin (402 µg/L) levels and also evaluated from the amount of iron removed by phlebotomy (1.1 g). No familial investigations were carried out to identify symptomatic or asymptomatic relatives with identical genotypes. Intrafamilial phenotypic heterogeneity has been observed in pedigrees with BMP6 propeptide missense mutations.3 On the other hand, subjects with the HFE p.[Cys282Tyr];[His63Asp] genotype and no comorbid factors dot not usually develop iron overload.1 Both HFE and BMP6 modulate hepcidin synthesis in response to body iron status7. An interesting question is whether mild cases of iron overload can involve the two disease loci. To conclude, our study highlights the benefit of investigating the whole HFE coding sequence in p.Cys282Tyr heterozygotes after exclusion of the most common causes of acquired hyperferritinemia. We report an unusual case of moderate iron overload associated with a complex HFE allele (p.[His63Asp;Glu168Gln]). This further indicates that screening p.His63Asp substitution in p.[Cys282Tyr];[His63Asp] heterozygotes with a well-defined iron overload phenotype has the potential to reveal rare HFE variants. If correctly interpreted, these uncommon variants may reduce diagnostic uncertainty and improve genetic counseling. Last but not least, we revealed disease-associated alleles in two different genes that interact within the same pathway. Further observations will help to determine whether mutations in BMP6 may partially explain the large phenotypic heterogeneity observed in HFE p.[Cys282Tyr];[His63Asp] individuals and confer higher risk of iron overload. The authors sincerely thank the physicians from the French Blood Agency who agreed to participate to the EMSAI study. This work has been funded by the French Blood Agency (grants APR-2010 and APR-2013). The authors declare that there is no conflict of interest. G.LG. designed the study. K.U., C.K., and I.G. performed molecular analyses. G.LG., V.S., C.H., T.C., MC.M., and C.F. analyzed the data. G.LG. wrote the paper. Additional Supporting Information may be found online in the supporting information tab for this article. Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.