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

IntroductionHereditary 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.MethodsPatients 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.ResultsNone 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.ConclusionCompound 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.

Variant <i>GDF9</i> mRNA is likely not the main cause of larger litter size in Iranian Lori-Bakhtyari, Shal, Ghezel, and Afshari sheep breeds

This study was carried out to screen the GDF9 gene and evaluate the polymorphism effect on litter size of four Iranian sheep breeds using the PCR-RFLP and PCR-SSCP methods. First, sequencing of the GDF9 gene in 16 twin-birth, 4 triplet-birth, and 2 infertile ewes showed that, in addition to G2, G3, G4, G5, and G6 mutations that have been previously reported in other breeds, a new G0 mutation, called C25T, exists in the GDF9 sequence of 1 out of 22 ewes and causes L9F substitution in the signal peptide region. None of the triplet-birth or infertile ewes carried G1, G4, G7, FecGE, G8, or FecGT mutations. In the second experiment, a large dataset was used: 605 individuals including 496 ewes (145 Afshari, 54 Shal, 126 Ghezel, and 171 Lori-Bakhtyari sheep), and 109 rams (26 Afshari, 23 Shal, 10 Ghezel, and 50 Lori-Bakhtyari sheep. There were no sheep carrying the G7, G8, or Thoka mutations. Among all 109 rams that were used in this study, none of them were homozygous for the G1 mutation. Moreover, abundance of heterozygote rams (G1/G+) varied from 0.0 (Afshari) to 28.6 % (Lori-Bakhtyari and Ghezel). The highest and the lowest frequencies of the G4 mutation were 30.6 and 3.0 % in Shal and Afshari breeds, respectively. Moreover, G4 abundance varied from 0.0 to 42.3 %, from 3.0 to 26.9, and from 3.0 to 30.6 % in rams, ewes, and overall, respectively. There was a significant difference in the abundance of G1 and G4 mutations between breeds. However, neither the G1 nor the G4 mutation was associated with litter size in Afshari, Ghezel, Lori-Bakhtyari, or Shal sheep breeds. In conclusion, the results of this study showed that GDF9 G1 and G4 mutations are not the reason for higher litter size in Iranian sheep. Moreover, the GDF9 G0 and G6 mutations do not cause triplet births or infertility in Iranian ewes. Therefore, it is unlikely that variant GDF9 mRNA induces larger litter size or infertility in Iranian ewes.