The safety of peri-articular local anaesthetic injection for patients undergoing total knee replacement with autologous blood transfusion: a randomised trial.

Intra-operative, peri-articular injection of local anaesthesia is an increasingly popular way of controlling pain following total knee replacement. At the same time, the problems associated with allogenic blood transfusion have led to interest in alternative methods for managing blood loss after total knee replacement, including the use of auto-transfusion of fluid from the patient's surgical drain. It is safe to combine peri-articular infiltration with auto-transfusion from the drain. We performed a randomised clinical trial to compare the concentration of local anaesthetic in the blood and in the fluid collected in the knee drain in patients having either a peri-articular injection or a femoral nerve block. Clinically relevant concentrations of local anaesthetic were found in the fluid from the drains of patients having peri-articular injections (4.92 µg/ml (sd 3.151)). However, none of the patients having femoral nerve blockade had detectable levels. None of the patients in either group had clinically relevant concentrations of local anaesthetic in their blood after re-transfusion. The evidence from this study suggests that it is safe to use peri-articular injection in combination with auto-transfusion of blood from peri-articular drains during knee replacement surgery.

First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin.

BACKGROUND: Transferrin receptor 2 (TfR2) is a key molecule involved in the regulation of iron homeostasis. Mutations in humans cause type 3 haemochromatosis and a targeted mutation in mice leads to iron overload with a similar phenotype. We have previously described the generation of a complete TfR2-knockout (KO) mouse. AIMS: The aims of this study were to determine the phenotype and analyse expression of iron related molecules in the liver, duodenum, and spleen of homozygous TfR2-KO, heterozygous, and wild-type mice. METHODS: Serum and tissue iron levels were determined in 10 week old male mice. Expression of iron related mRNA transcripts were analysed in the liver, duodenum, and spleen using real time polymerase chain reaction. Expression of iron related proteins in the liver were analysed by immunoblotting and immunohistochemistry. RESULTS: Homozygous TfR2-KO mice had no TfR2 protein expression and developed significant iron overload typical of TfR2 associated haemochromatosis. In the liver of TfR2-KO mice there was no upregulation of hepcidin mRNA or prohepcidin protein in response to iron loading. CONCLUSIONS: Our results suggest that TfR2 is required for iron regulated expression of hepcidin and is involved in a pathway related to Hfe and hemojuvelin.

A novel mutation in ferroportin1 is associated with haemochromatosis in a Solomon Islands patient.

BACKGROUND: A severe form of iron overload with the clinicopathological features of haemochromatosis inherited in an autosomal dominant manner has been described in the Solomon Islands. The genetic basis of the disorder has not been identified. The disorder has similarities to type 4 haemochromatosis, which is caused by mutations in ferroportin1. AIMS: The aims of this study were to identify the genetic basis of iron overload in a patient from the Solomon Islands. PATIENT AND METHODS: Genomic DNA was isolated from peripheral blood leucocytes of a Solomon Islands man with severe iron overload. The entire coding region and splice sites of the ferroportin1 gene was sequenced. RESULTS AND CONCLUSIONS: A novel missense mutation (431A>C; N144T) was identified in exon 5 of the ferroportin1 gene. A novel restriction endonuclease based assay which identifies both the N144T and N144H mutations was developed which will simplify the diagnosis and screening of patients for iron overload in the Solomon Islands and other populations. This is the first identified mutation associated with haemochromatosis in the Solomon Islands population.

Atypical haemochromatosis: phenotypic spectrum and beta2-microglobulin candidate gene analysis.

Beta2-microglobulin was investigated in atypical haemochromatosis patients not homozygous for the C282Y mutation of HFE (OMIM *235200), because the HFE protein binds beta2-microglobulin, and in mice beta2-microglobulin gene knockout causes hepatic iron overload. Six unrelated patients with atypical haemochromatosis were studied. Five patients had normal HFE coding sequence and the sixth was heterozygous for C282Y. We show that the spectrum of atypical haemochromatosis includes two distinct familial forms: juvenile haemochromatosis (OMIM *602390) and a novel form of familial iron overload, with apparently autosomal dominant inheritance, predominant Kupffer cell siderosis, and possible minimal dyserythropoiesis on bone marrow examination. Serial serum beta2-microglobulin estimation showed normal levels in all patients. Southern blot analysis showed normal beta2-microglobulin gene structure, excluding major gene rearrangement. Several corrections to the published beta2-microglobulin sequence were identified, but all six patients had normal beta2-microglobulin sequence. Western blot analysis of serum showed beta2-microglobulin protein of normal size. In conclusion, we found no evidence to implicate beta2-microglobulin mutation in atypical haemochromatosis. Two forms of familial iron overload appear unrelated to either HFE or beta2-microglobulin. Linkage studies are required to identify the genes involved, which may encode novel proteins crucial to the regulation of iron metabolism. Identification of these loci will aid the diagnosis, counselling, and treatment of iron overload disorders.

A novel mutation of HFE explains the classical phenotype of genetic hemochromatosis in a C282Y heterozygote.

BACKGROUND & AIMS: Most patients with genetic hemochromatosis are homozygous for a single mutation of the HFE gene (C282Y). There is a second mutation, H63D, but its role in iron overload is less conclusive. The aim of this study was to investigate the basis of iron overload in a patient with classical hemochromatosis who was only heterozygous for C282Y and negative for H63D. METHODS: Genotype for the C282Y, H63D, and S65C mutations of HFE was determined in patient RFH, his family members, and 365 controls. The HFE gene was sequenced in patient RFH. Allele-specific reverse-transcription polymerase chain reaction was performed to investigate RNA splicing. Allele frequency was determined by allele-specific oligonucleotide hybridization. RESULTS: The patient is compound heterozygous for C282Y and a novel splice site mutation (IVS3 + 1G --> T). His sister has an identical genotype and elevated serum ferritin and transferrin saturation. The novel mutation functionally alters messenger RNA splicing, causing obligate skipping of exon 3. However, the IVS3 + 1G --> T mutation was found to be rare and was not detected in 630 control European chromosomes. CONCLUSIONS: IVS3 + 1G --> T in the compound heterozygous state with C282Y results in iron overload that can progress to a severe phenotype of classical hemochromatosis. The demonstration of IVS3 +1G --> T highlights the possibility of other rare HFE mutations, particularly in C282Y heterozygotes with iron overload.

Monocyte-macrophage ferric reductase activity is inhibited by iron and stimulated by cellular differentiation.

The enzyme ferric reductase catalyses the reduction of Fe(III) as a prerequisite to its transportation across the cell membrane. Duodenal mucosal biopsies from iron overloaded patients with genetic haemochromatosis (GH) have increased ferric reductase activity and iron absorption compared with controls, yet the GH mucosa is iron deficient. A similar GH-related iron deficiency is also seen in macrophages. The aim of this study was to investigate whether macrophage ferric reductase activity is altered in GH, and to determine ferric reductase activity in monocytes and differentiated macrophages. The erythroleukaemic K562 cell line was studied as a clonal reference cell line. The basal K562 ferric reductase activity is characteristic of a membrane bound enzyme, being both temperature and protease sensitive. Ferric reductase activity was also demonstrated in human leucocyte, monocyte and macrophage preparations. Assays of K562 and macrophage cell supernatants confirmed that the ferric reductase activity was not due to a secreted factor. Assay of ferric reductase in normalized-iron and iron-enriched (100 microM ferric citrate) conditions showed no significant difference between Cys282Tyr (Cys282-->Tyr) homozygous GH macrophages and Cys282-Tyr negative control activities (P>0.05). However, a 900% increase in ferric reductase activity was observed during monocyte to macrophage differentiation (P<0.05), possibly reflecting the co-ordinate up-regulation of iron metabolism in these cells. The demonstration of approx. 25% activity after macrophage differentiation at high free-iron concentrations compared with 'normalized' iron is consistent with repression of human ferric reductase activity by iron. The identification of the human ferric reductase gene and its protein will ultimately provide insight into its regulation and role in mammalian iron metabolism.

A 6p22 reference map of leukocyte DNA: exclusion of rearrangement in four cases of atypical haemochromatosis.

We describe a 4 Mb reference map of the haemochromatosis gene region in leukocyte DNA from seven controls and four atypical haemochromatosis patients. Three patients had normal coding sequence for HFE, the candidate gene for genetic haemochromatosis (GH). The fourth patient had classical GH but was heterozygous for Cys282Tyr with otherwise normal coding sequence. The genomic DNA was mapped by pulsed-field gel electrophoresis (PFGE) using five rare-cutting enzymes. Seventeen probes including HFE were positioned on the map. Despite proximity to the highly polymorphic major histocompatibility complex (MHC), no polymorphism was observed in the control group with these telomeric probes. Furthermore, major rearrangement of the HFE region was excluded as a mutation contributing to iron overload in these atypical patients. Maps of cloned DNA are linked through genes and other probes to this reference map of the HFE region in uncloned genomic DNA.