Non-HFE mutations in haemochromatosis in China: combination of heterozygous mutations involving HJV signal peptide variants

Introduction Hereditary haemochromatosis (HH) caused by a homozygous p.C282Y mutation in haemochromatosis ( HFE ) gene has been well documented. However, less is known about the causative non- HFE mutation. We aimed to assess mutation patterns of haemochromatosis-related genes in Chinese patients with primary iron overload. Methods Patients were preanalysed for mutations in the classic HH-related genes: HFE , HJV , HAMP , TFR2 and SLC40A1 . Whole exome sequencing was conducted for cases with variants in HJV signal peptide region. Representative variants were analysed for biological function. Results None of the cases analysed harboured the HFE p.C282Y; however, 21 of 22 primary iron-overload cases harboured at least one non-synonymous variant in the non- HFE genes. Specifically, p.E3D or p.Q6H variants in the HJV signal peptide region were identified in nine cases (40.9%). In two of three probands with the HJV p.E3D, exome sequencing identified accompanying variants in BMP/SMAD pathway genes, including TMPRSS6 p.T331M and BMP4 p.R269Q, and interestingly, SUGP2 p.R639Q was identified in all the three cases. Pedigree analysis showed a similar pattern of combination of heterozygous mutations in cases with HJV p.E3D or p.Q6H, with SUGP2 p.R639Q or HJV p.C321X being common mutation. In vitro siRNA interference of SUGP2 showed a novel role of downregulating the BMP/SMAD pathway. Site-directed mutagenesis of HJV p.Q6H/p.C321X in cell lines resulted in loss of membrane localisation of mutant HJV, and downregulation of p-SMAD1/5 and HAMP . Conclusion Compound heterozygous mutations of HJV or combined heterozygous mutations of BMP/SMAD pathway genes, marked by HJV variants in the signal peptide region, may represent a novel aetiological factor for HH.