Differential HFE allele expression in hemochromatosis heterozygotes Olivier Rosmorduc, Raoul Poupon, Isabelle Nion, Dominique Wendum, John Feder, Gilbert Béréziat, Brigitte Hermelin  Gastroenterology  Volume 119, Issue 4, Pages 1075-1086 (October 2000) DOI: 10.1053/gast.2000.18146 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Detection of HFE mRNAs by RT-PCR analysis in PBMCs of normal subjects and C282Y and H63D homozygotes. After amplification of the regions encompassing nucleotide 187 and nucleotide 845 (B), the RT-PCR products were digested with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) the restriction enzymes DpnII and RsaI and identified by electrophoresis on (A) 2.0% and (B) 2.5% agarose gels. M, markers. Gastroenterology 2000 119, 1075-1086DOI: (10.1053/gast.2000.18146) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 HFE mRNAs encoded by one allele are preferentially detected in PBMCs. Three families (Bi, Le, and Be) were studied; mRNAs coded by mutated and wild-type HFE genes were studied in PBMCs from compound heterozygotes (Bi3, Le3, Be2, and Be3), patients heterozygous for H63D (Bi1, Le2, and Be4), and patients heterozygous for C282Y (Bi2 and Be1) and homozygous for C282Y (Le1). (A) Pedigrees of 3 families (Bi, Le, and Be). (B) After amplification of the region surrounding nucleotide 187 and digestion by DpnII, a major band at 170 bp corresponding to the (187C > G; H63D) mutated transcripts was detected in all patients heterozygous for H63D (Bi1, Le2, and Be4) and compound heterozygotes (Bi3, Le3, Be2, and Be3). The C282Y homozygote (Le1) showed 100% nonmutated transcripts (84 + 86 pb). M, markers (123-bp DNA ladder; GIBCO BRL); lane 1, PCR products before digestion (170 pb); lane 2, negative control without cDNA. (C) After amplification of the region surrounding nucleotide 845 and restriction enzyme cleavage by RsaI, a major band at 217 bp corresponding to the nonmutated transcripts was detected in patients heterozygous for C282Y (Bi2 and Be1) and in compound heterozygotes (Bi3, Le3, Be2, and Be3); a single band at 217 bp was exclusively detected in patients heterozygous for H63D (Bi1, Le2, and Be4). A single band at 188 bp corresponding to the (845G > A; C282Y) mutated transcripts was detected in the patient homozygous for C282Y (Le1). The expected 76-bp band was present in all cases; M, markers (123-bp DNA ladder; GIBCO BRL); lane 1, PCR products before digestion (293 pb); lane 2, negative control without cDNA. Gastroenterology 2000 119, 1075-1086DOI: (10.1053/gast.2000.18146) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Relative-quantitative analysis of mutated and nonmutated transcripts. After amplification, products of the regions encompassing nucleotides 187 and 845, obtained using primers 3 and 8 coupled to a fluorophore (6 FAM), and the size of PCR products and the intensity of fluorescence were automatically measured using the Genescan software (version 1.2) of an ABI model 373 sequencer (Perkin Elmer). (A) Signals obtained after amplification of the region surrounding nucleotide 187 and digestion of the RT-PCR products with DpnII in a homozygous normal subject (panel 1) showing 100% nonmutated transcripts; in a patient homozygous for H63D (panel 2) showing 100% (187C > G; H63D) transcripts; and in a typical compound heterozygote (panel 3) showing 74% (187C > G; H63D) transcripts and 26% nonmutated transcripts. (B) Signals obtained after amplification of the region surrounding nucleotide 845 and digestion of the RT-PCR products with RsaI in a homozygous normal subject (panel 1) showing 100% nonmutated transcripts; in a patient homozygous for C282Y (panel 2) showing 100% (845G > A; C282Y) transcripts; and in a typical compound heterozygote (panel 3) showing 73% nonmutated transcripts and 27% (845G > A; C282Y) transcripts. (C) The percentages (mean ± SEM) of wild-type and (187C > G; H63D) and (845G > A; C282Y) transcripts were determined for 9 C282Y heterozygotes (wild-type transcripts, 68.5% ± 2.7%); 14 compound heterozygotes [(187C > G; H63D) transcripts, 71.9% ± 1.2%; (845G > A; C282Y) transcripts, 30.7% ± 1.4%]; and 11 H63D heterozygotes [(187C > G; H63D) transcripts, 70.6% ± 1.5%]. Gastroenterology 2000 119, 1075-1086DOI: (10.1053/gast.2000.18146) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Western blot analysis of the HFE protein extracted from EBV-transformed lymphocytes. (A) Proteins were extracted from 50 × 106 EBV-transformed lymphocytes. Equal amounts of proteins were fractionated by 10% SDS-PAGE. After transfer to a nitrocellulose membrane, HFE was detected using a rabbit antibody for human HFE at a dilution of 1/500 (see Patients and Methods). A band at 45 kilodaltons corresponding to the mature form of HFE and a band at 43 kilodaltons corresponding to the unglycosylated form of the protein were detected in 2 C282Y homozygotes, 2 H63D homozygotes, and a compound heterozygote. (B) HFE protein amount was quantified by optical density of the blot and represented on a histogram. Gastroenterology 2000 119, 1075-1086DOI: (10.1053/gast.2000.18146) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Distribution between the different transcripts is the same in PBMCs and liver and duodenal samples. (A) After amplification of the region surrounding nucleotide 187 and restriction enzyme cleavage with DpnII, PBMCs and liver and duodenal samples from H63D heterozygotes (Gue) and compound heterozygote (Le3 and Be2) show a major band at 170 bp corresponding to (187C > G; H63D) transcripts; mRNAs from a C282Y homozygote (Chi) show 100% of nonmutated transcripts (84 + 86 bp) in PBMCs (a), liver (b), and duodenum (c). M, markers; lane 1, PCR products before digestion (170 pb); lane 2, negative control. (B) After amplification of the region surrounding nucleotide 845 and restriction enzyme cleavage with RsaI, PBMCs and liver and duodenal samples from heterozygotes (Le3, Gu, and Be2) show a major band at 217 bp corresponding to nonmutated transcripts; mRNAs from a patient homozygous for C282Y (Chi) show 100% (845G > A; C282Y) transcripts (188 bp) in PBMCs (a), liver (b), and duodenum (c). A 76-bp band was present in all cases. M, markers; lane 1, PCR products before digestion (293 bp); lane 2, negative control. Gastroenterology 2000 119, 1075-1086DOI: (10.1053/gast.2000.18146) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Identification of a new mutation in the HFE gene: a G-to-C transversion resulting in a cys > ser missense mutation at amino acid 282 in the family Be. (A) Sequence analysis of genomic DNA, with an antisense primer, reveals the presence of a double peak (A + C) caused by the presence of 2 missense mutations at nucleotide 845, and a G-to-A transition (845G > A; C282Y) on one allele and a G-to-C transversion (845G > C; C282S) on the other allele in an affected compound heterozygote (Be2). (B) Sequencing of cDNA with an antisense primer reveals the presence of a major peak of cytosine caused by preferential detection of mRNAs encoded by one allele bearing (845G > C; C282S) and (187C > G; H63D) mutations. Gastroenterology 2000 119, 1075-1086DOI: (10.1053/gast.2000.18146) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Detection of HFE protein and iron overload in the liver from a C282Y/C282S heterozygote. Liver biopsy samples were obtained from (A and B) a C282Y homozygote and (C and D) one of the C282Y/C282S heterozygotes (Be2). (A and C) HFE protein was detected by immunohistochemistry; (B and D) iron overload was detected using Perls' Prussian blue staining on liver tissue sections. Similar iron overload and intracellular HFE staining were observed in the hepatocytes from both patients. Gastroenterology 2000 119, 1075-1086DOI: (10.1053/gast.2000.18146) Copyright © 2000 American Gastroenterological Association Terms and Conditions