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

Introduction: Hereditary haemochromatosis (HH) caused by a homozygous p.C282Y mutation in haemochromatosis () gene has been well documented. However, less is known about the causative non- 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: , , , and . Whole exome sequencing was conducted for cases with variants in signal peptide region. Representative variants were analysed for biological function.

Results: None of the cases analysed harboured the p.C282Y; however, 21 of 22 primary iron-overload cases harboured at least one non-synonymous variant in the non- genes. Specifically, p.E3D or p.Q6H variants in the signal peptide region were identified in nine cases (40.9%). In two of three probands with the p.E3D, exome sequencing identified accompanying variants in BMP/SMAD pathway genes, including p.T331M and p.R269Q, and interestingly, p.R639Q was identified in all the three cases. Pedigree analysis showed a similar pattern of combination of heterozygous mutations in cases with p.E3D or p.Q6H, with p.R639Q or p.C321X being common mutation. In vitro siRNA interference of showed a novel role of downregulating the BMP/SMAD pathway. Site-directed mutagenesis of p.Q6H/p.C321X in cell lines resulted in loss of membrane localisation of mutant HJV, and downregulation of p-SMAD1/5 and .

Conclusion: Compound heterozygous mutations of or combined heterozygous mutations of BMP/SMAD pathway genes, marked by variants in the signal peptide region, may represent a novel aetiological factor for HH.