Identification of new mutations of the HFE, hepcidin, and transferrin receptor 2 genes by denaturing HPLC analysis of individuals with biochemical indications of iron overload

BACKGROUND: Hereditary hemochromatosis is a recessive disorder characterized by iron accumulation in parenchymal cells, followed by organ damage and failure. The disorder is mainly attributable to the C282Y and H63D mutations in the HFE gene, but additional mutations in the HFE, transferrin receptor 2 (TfR2), and hepcidin genes have been reported. The copresence of mutations in different genes may explain the phenotypic heterogeneity of the disorder and its variable penetrance. METHODS: We used denaturing HPLC (DHPLC) for rapid DNA scanning of the HFE (exons 2, 3, and 4), hepcidin, and TfR2 (exons 2, 4 and 6) genes in a cohort of 657 individuals with altered indicators of iron status. RESULTS: DHPLC identification of C282Y and H63D HFE alleles was in perfect agreement with the restriction endonuclease assay. Fourteen DNA samples were heterozygous for the HFE S65C mutation. In addition, we found novel mutations: two in HFE (R66C in exon 2 and R224G in exon 4), one in the hepcidin gene (G71D), and one in TfR2 (V22I), plus several intronic or silent substitutions. Six of the seven individuals with hepcidin or TfR2 coding mutations carried also HFE C282Y or S65C mutations. CONCLUSION: DHPLC is an efficient method for mutational screening for the genes involved in hereditary hemochromatosis and for the study of their copresence.     

X-PERT: weight reduction with orlistat in obese subjects receiving a mildly or moderately reduced-energy diet: early response to treatment predicts weight maintenance

AIM: To determine the effect of two different levels of energy deficit on weight loss in obese patients treated with orlistat. METHODS: Patients (n=430) were randomized in a 1-year, multicentre, open-label, parallel group study conducted at 23 hospital centres and university medical departments worldwide. Obese outpatients (body mass index 30--43 kg/m(2)) aged 18--70 years with a body weight of >or=90 kg and a waist circumference of >or=88 cm (women) or >or=102 cm (men) were treated with orlistat 120 mg three times daily plus a diet that provided an energy deficit of either 500 or 1,000 kcal/day for 1 year. Orlistat treatment was discontinued in patients who did not achieve >or=5% weight loss after assessment at 3 and 6 months. The primary outcome measure was change in body weight from baseline at week 52. RESULTS: Reported mean difference in energy intake between the two groups (500-1,000 kcal/day deficit) at weeks 24 and 52 was actually 111 and 95 kcal/day respectively. Of the 430 patients involved in the study, 295 achieved >or=5% weight loss at both 3 and 6 months. In this population, at week 52, weight loss from baseline was similar for patients randomized to either the 500 or the 1,000 kcal/day deficit diet (-11.4 kg vs. -11.8 kg, respectively; p=0.778). After 12 months of treatment with orlistat, 84% (n=118/141) and 85% (n=131/154) of patients in the 500 and 1,000 kcal/day deficit groups, respectively, achieved >or=5% weight loss, and 50% (n=70/141) and 53% (n=82/154) of patients, respectively, achieved >or=10% weight loss. Patients in both the diet treatment groups showed similar significant improvements in blood pressure, lipid levels and waist circumference at week 52. CONCLUSIONS: Treatment with orlistat was associated with a clinically beneficial weight loss, irrespective of the prescribed dietary energy restriction (-500 or -1000 kcal/day). Patients who achieved >or=5% weight loss at 3 months achieved long-term, clinically beneficial weight loss with orlistat plus either diet. Therefore, identifying patients who lose at least 5% weight after 3 months and who maintain this weight loss up to 6 months is a valuable treatment algorithm to select patients who will benefit most from orlistat treatment in combination with diet.     

Splenectomy prolongs in vivo survival of erythrocytes differently in spectrin/ankyrin- and band 3-deficient hereditary spherocytosis

Red cell (RBC) deformability and membrane-bound immunoglobulin G (IgG) were studied to better understand premature clearance of erythrocytes in hereditary spherocytosis. Averaged deformability profiles from cells having comparable cell age revealed that splenectomy was more beneficial for spectrin/ankyrin-deficient than for band 3-deficient RBCs. Splenectomy prevented an early loss of young cells in both types of deficiencies. It had an additional beneficial effect on spectrin/ankyrin-deficient but not band 3-deficient RBCs. It prolonged the survival of mature spectrin/ankyrin-deficient RBCs such that they lost their deformability more slowly than RBCs from patients who had not undergone splenectomy. Band 3-deficient RBCs lost their deformability at the same rate before and after splenectomy. In HS patients with band 3 deficiency who underwent splenectomy, RBC deformability inversely correlated with the number of RBC-bound IgG (up to 140 molecules per cell). In spectrin/ankyrin deficiency, RBC-bound IgG remained at control levels (60 IgG or less per cell). It appears that spectrin/ankyrin-deficient RBCs escaped opsonization by releasing band 3-containing vesicles because their band 3 content and deformability dropped in parallel with increasing cell age. Band 3-deficient RBCs did not lose band 3 with increasing cell age. Hence, it is possible that band 3 clusters required for bivalent binding of low-affinity-IgG, naturally occurring antibodies were retained in band 3-deficient RBCs with a relative excess of skeletal proteins but were released from spectrin/ankyrin-deficient RBCs, in which vesicle budding was facilitated by an impaired skeleton.     

Lean and Fat Mass Loss in Obese Patients Before and After Roux-en-Y Gastric Bypass: A New Application for Ultrasound Technique

Objective  

This study aims to evaluate the thickness of the femoral quadriceps and biceps brachii and brachialis muscles bilaterally and the adjacent subcutaneous fat in patients undergoing gastric bypass Roux-en-Y before and after surgery, using ultrasound as the diagnostic method of choice.     

Impaired Abdominal Skin Sensory Function in Morbid Obesity and After Bariatric Surgery

Background  

Bariatric surgery reduces body weight, but creates the need for surgical correction of the patient’s body shape, especially of the abdomen. The abdominal skin of the ex-obese has a lower quantity of fibrous and non-fibrous components; however, its functional status has not yet been studied. This study quantified, at different depths, the neurological function of the abdominal skin of the obese and morbidly ex-obese.     

Functional study of lipids of PNH red cell membranes: susceptibility of liposomes to reactive lysis

Lipids extracted with chloroform-methanol from red blood cell membranes of 7 PNH and 13 control subjects were used for the preparation of liposomes, which were then examined with the reactive lysis test. PNH liposomes lysed to a higher extent than control liposomes as indicated by the higher dilution of the limiting complement reagent that was necessary to lyse 50% of the PNH liposomes. A similar finding was also observed with liposomes made of lipids from AET-treated red cells. The enhanced reactive lysis can be attributed to the polar lipid fraction, as indicated by the increased lysis of hybrid liposomes prepared from this polar lipids extracted from PNH erythrocyte membrane and lipids extracted from normal erythrocyte membrane. The increased susceptibility to reactive lysis does not seem to be specific of PNH liposomes, since it was also observed with liposomes prepared from lipids of red cells from beta-thalassemia major and autoimmune hemolytic disease.