Identification of novel non-HFE mutations in Chinese patients with hereditary hemochromatosis

Abstract

Backgrounds: Hereditary hemochromatosis (HH) is mainly caused by homozygous p.C282Y mutations in HFE in the Caucasians. We recently reported non-HFE mutations constitute the major cause of HH in Chinese. However, there is still a relatively high proportion of cases with primary iron overload from unexplained causes. We aimed to explore novel non-HFE mutations in Chinese patients with primary iron overload.

Methods: Whole exome sequence was conducted to screen mutations in novel HH-related genes in the 9 cases with unexplained primary iron overload. Then the representative candidate genes were screened for mutations in another cohort of 18 HH cases. The biological function of the selected genes and variants were analyzed in vitro.

Results: Whole exome sequencing of 9 cases with unexplained primary iron overload identified 42 missense variants in 40 genes associated with iron metabolism pathway genes such as UBE2O p.K689R and PCSK7 p.R711W. Subsequent Sanger sequencing of the UBE2O and PCSK7 genes in the 27 cases with primary iron overload identified p.K689R in UBE2O, p.R711W and p.V143F in PCSK7 at frequency of 2/27,1/27 and 2/27 respectively. In vitro siRNA interference of UBE2O and PCSK7 resulted in down-regulated HAMP mRNA expression. Adenovirus generation of UBE2O p.K689R in cell lines resulted in increased expression of SMAD6 and SMAD7 and downregulation of p-SMAD1/5 and HAMP expression, and the reduction of hepcidin level.

Conclusions: Our study identified a series of novel candidate non-HFE mutations in Chinese patients with HH. These may provide insights into the genetic basis of unexplained primary iron overload.

Trial registration: ClinicalTrials.gov NCT03131427.

Keywords: Gene mutation; Hereditary hemochromatosis; Iron overload; Non-HFE; PCSK7; UBE2O.

Conflict of interest statement

All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

© 2022. The Author(s).

Figures

Fig. 1

Representative sequencing of the novel variants in UBE2O, and PCSK7 in cases with primary iron overload. Sequencing of the heterozygous missense mutations UBE2O p.K689R (A) and PCSK7 p.R711W (B)

Fig. 2

Analysis of HAMP mRNA expression in UBE2O and PCSK7 knockdown HCC cells. AHAMP mRNA levels in Huh-7 and HepG2 cells transfected with UBE2O siRNA or control siRNA. BHAMP mRNA levels in Huh-7 and HepG2 cells transfected with PCSK7 siRNA or control siRNA

Fig. 3

Analysis of SMAD6 and SMAD7 expression in UBE2O knockdown HCC cells. Western blot analysis of SMAD6, SMAD7, and pSmad1/5 expression in UBE2O knockdown Huh-7 and HepG2 cells showed that higher expression of Smad6 and Smad7 in UBE2O-knockdown HCC cells, and higher ratio of pSmad1/5/tSmad1 in Huh-7 cells, but lower ratio of pSmad1/5/tSmad1 in HepG2 cells

Fig. 4

Analysis of HAMP expression in Huh7 and HepG2 cells infected with UBE2O or UBE2O p.K689R adenovirus. A. HAMP mRNA levels in Huh-7 and HepG2 cells were analyzed by real-time PCR assays. B and C Hepcidin was analyzed in Huh-7 and HepG2 cells by immunofluorescence and ELISA

Fig. 5

Analysis of SMAD6 and SMAD7 expression in Huh7 and HepG2 cells infected with UBE2O or UBE2O p.K689R adenovirus. Western blot analysis of SMAD6, SMAD7, and pSmad1/5 expression in Huh-7 and HepG2 cells showed higher expression of Smad6 and Smad7 and lower ratio of pSmad1/5/tSmad1 in UBE2O p.K689R HCC cells than wild-type HCC cells