Patients and consent
This study was approved by and performed in accordance with the ethical standards of the QIMR Berghofer Human Research Ethics Committee and with the Helsinki Declaration of 1975, as revised in 1983. Informed and written consent was obtained for the studies described in this report.
Cases of clinically identified atypical iron overload of unknown cause were referred to our laboratory for sequencing by primary clinicians. Cases were required to demonstrate persistently (two or more occasions) abnormal iron indices (including significantly elevated serum ferritin > 1000 μg/L for males and > 700 for females) and to be negative for p.C282Y homozygosity. Referring clinicians were also requested to undertake a detailed iron metabolism-specific patient history for other possible attributing causes, such as high alcohol consumption. In total, 31 referred patients have been sequenced to date at our center .
Sequencing of genomic DNA using an iron-associated gene panel, covering 39 genes and 11 promoters associated with iron overload or anemia, was performed as previously described . Samples were sequenced to an average of 400-fold coverage using an Ion Torrent PGM, with post run QC/QA filtering performed using Torrent Suite (v3.6; Life Technologies). Sequences were mapped to the Human Genome (HG19) and variants identified using the Torrent Variant Caller (version 3.6.59049 with Germ Line - Low Stringency configuration). Identified variants were annotated using the wANNOVAR tool , and candidate mutations confirmed by Sanger sequencing.
BMP6 expression constructs
Constructs containing double-tagged human BMP6 coding sequence were synthesized and validated by BioMatik (Delaware, USA). Constructs were generated for both the wildtype and p.Q118dup sequence and contained a hemagglutinin (HA)—tag inserted between amino acids 23 and 24 (N-terminus of the pro-protein) and a V5-tag at the carboxyl-terminus of the mature protein.
Chinese hamster ovary (CHO) cells and the hepatic endothelial cell line, SK-HEP-1  (HTB-52, ATCC, Manassas, Virginia), were transiently transfected with the BMP6 expression constructs by reverse transfection using Viafect (Promega, Alexandria, NSW, Australia) according to the manufacturer’s instructions. Stably expressing cells were obtained by placing transfected cells under selection with puromycin at 10 μg/ml (CHO cells) or 1 μg/ml (SK-HEP-1 cell).
Transfected cells were plated on glass coverslips to a confluency of 50–70% for 24 h. Cells were washed twice in PBSCM (phosphate buffered saline with 1 mM CaCl2, 1 mM MgCl2) and then fixed (in methanol at − 20 °C for 5 min for CHO cells then at room temperature (RT) for 10 min and in 3% paraformaldehyde for SK-HEP Hep-1 cells which were then permeabilized using 0.1% saponin in PBSCM for 15 min). Primary antibodies were then applied: anti-HA at 1:10 (12CA5, tissue culture supernatant) and anti-V5 at 1:3000 (Merck-Millipore). After three PBSCM washes, cells were incubated in Alexa Fluor donkey secondary antibodies: anti-mouse 488/594 and anti-rabbit 594/488 (Invitrogen). Following three final PBSCM washes, coverslips were mounted in Prolong Gold anti-fade mounting media (Invitrogen) and imaged by microscopy at × 63 magnification using a Zeiss Axioscope NLO-780 or a Leica TCS SP5. Z stacks were imaged for each focal point, and the final images were processed using the in-built deconvolution algorithms in the Zen software. The immunofluorescence studies were performed at least three times for each cell line.
Conditioned media from transfected cells were precipitated with 10% trichloroacetic acid overnight at 4 °C, and the precipitate was washed with cold acetone and resuspended in SDS sample buffer (SB) (50 mm Tris pH 6.8, 2% SDS, 10% glycerol, 0.1% bromophenol blue, 5% β-mercaptoethanol). Cell lysates were also resuspended in SDS SB, and all samples were loaded onto a 12% SDS-PAG and electrophoresed. Following transfer onto 0.2 μm nitrocellulose (Bio-Rad), blots were blocked for 3 h at RT in Western blocking buffer WBB (10% skim milk powder in 0.5% Tween-20 in TBS (TBST)). Primary anti-V5 (2.5 ng/ml) or anti-HA (1:100) diluted in WBB was applied overnight at 4 °C. Following three washes in TBST, blots were incubated with secondary anti-mouse horseradish peroxidase (1:10,000 in WBB) for 1 h at RT. Blots were washed extensively with TBST, Luminata Forte Western Chemiluminescent HRP Substrate (Merck-Millipore) was applied for 5 min, and blots were then exposed to film. Western blotting was repeated three times for each cell line and quantified. Band volume and density were quantitated using ImageJ software (National Institutes of Health, Maryland, USA).