Therapeutic Phlebotomy Research Articles

A Novel Allogeneic Transplant Conditioning Regimen Designed for Tolerance Induction in Patients with Severe Sickle Cell Disease.

Allogeneic hematopoietic stem cell (HSC) transplantation remains the only curative approach for sickle cell disease (SCD) patients, yet high risk of procedural toxicities and graft-versus-host disease limits this approach. We chose a low-dose radiation approach utilizing rapamycin based upon its unique ability to promote T cell tolerance. We tested this approach in vivo in a murine bone marrow transplantation model comparing a short course of conventional immunosuppression with cyclosporine to that with rapamycin, with long-term, high-level chimerism attained only in mice treated with rapamycin. We have now begun accrual to an IRB approved clinical trial testing this approach in SCD adults and report the results in our first five subjects. Protocol entry criteria include irreversible complications from sickle cell disease (such as prior strokes or pulmonary hypertension defined as a tricuspid-regurgitant jet velocity [TRV] ≥2.5 m/s), or reversible complications (such as frequent vaso-occlusive crises or acute chest syndrome) not ameliorated by a 6-month course of hydroxyurea. Conditioning was achieved with a single radiation dose of 300cGy, alemtuzumab (1mg/kg total), and oral rapamycin targeting trough levels between 10–20 ng/ml. All patients received unmanipulated mobilized peripheral blood progenitors obtained from an HLA-matched sibling. Assessment of donor chimerism was measured by microsatellite PCR of CD3 and CD14/15 positive white cells. Conditioning was well tolerated in all patients. Two patients inadvertently received only 200 cGy. CD34+ cell doses ranged from 5.72–10 × 10e6/kg, and CD3+ cell doses ranged from 1.93–5.35 × 10e8/kg. In 4 of the 5 patients in whom follow-up is sufficient, an early peak in myeloid chimerism was seen at 95–100% at day 28, with stabilization at 48–100% long-term. Lower level lymphoid chimerism was seen early, ranging from 3–23%, with stabilization at 5–47% long-term. Hemoglobin electrophoresis revealed complete replacement by donor type hemoglobin in all 4 patients by day 120, with amelioration of the phenotype allowing for therapeutic phlebotomy. Unfortunately, two patients have experienced recurrent sickle cell disease, though T cell responses to donor assessed by the mixed lymphocyte reaction revealed continued tolerance to donor with preserved T cell responses to third party antigens. These results argue against rejection and suggest competition by surviving endogenous HSCs as a cause of graft loss. Additionally, retransplant of one of the patients in relapse thus far using 400 cGy, alemtuzumab, and rapamycin has re-established full donor erythroid chimerism. Stable mixed chimerism persists out to 23 months in one patient and 10 months in another. Finally, no patient to date has developed any symptoms or signs of acute or chronic GVHD. Our results demonstrate the ability of allogeneic HSC transplantation using a relatively simple conditioning regimen to achieve mixed hematopoietic chimerism without the development of GVHD, suggest the importance of adequate hematopoietic suppression, and may allow extension to alternative donor transplantation.

Screening for Hereditary Hemochromatosis: A Systematic Review for the U.S. Preventive Services Task Force

The U.S. Preventive Services Task Force (USPSTF) has not previously considered screening for hereditary hemochromatosis for a recommendation as a clinical preventive service for primary care clinicians. To conduct a focused systematic review of hereditary hemochromatosis screening relating to 2 USPSTF criteria, the burden of suffering and the potential effectiveness of a preventive intervention, to determine whether evidence is sufficient for a USPSTF recommendation. MEDLINE, CINAHL, and Cochrane Library databases from 1966 through February 2005. The authors supplemented literature searches with source materials from experts in the field and the bibliographies of key reviews and included studies. Studies were retrieved to answer 3 key questions: 1) What is the risk for developing clinical hemochromatosis among those with a homozygous C282Y genotype? 2) Does earlier therapeutic phlebotomy of individuals with primary iron overload due to hereditary hemochromatosis reduce morbidity and mortality compared with treatment after diagnosis in routine clinical care? 3) Are there groups at increased risk for developing hereditary hemochromatosis that can be readily identified before genetic screening? The authors critically appraised studies using quality criteria specific to their design. The authors abstracted all studies into evidence tables using condition definitions and diagnostic criteria. Data were insufficient to define a very precise estimate of penetrance. Available data suggest that up to 38% to 50% of C282Y homozygotes may develop iron overload, with up to 10% to 33% eventually developing hemochromatosis-associated morbidity. Prevalence of C282Y homozygosity is higher in family members of probands and other high-risk patient groups defined by signs, symptoms, and phenotypic screening. This review considered genetic screening for HFE-related hereditary hemochromatosis in C282Y homozygotes only. Available research is limited, is based solely on observational designs, and is plagued by poor or inconsistent reporting. Research addressing genetic screening for hereditary hemochromatosis remains insufficient to confidently project the impact of, or estimate the benefit from, widespread or high-risk genetic screening for hereditary hemochromatosis.

Iron overload and prolonged ingestion of iron supplements: Clinical features and mutation analysis of hemochromatosis‐associated genes in four cases

We evaluated and treated four white adults (one man, three women) who had iron overload associated with daily ingestion of iron supplements for 7, 15, 35, and 61 years, respectively. We performed HFE mutation analysis to detect C282Y, H63D, and S65C in each patient; in two patients, HFE exons were sequenced. In two patients, direct sequencing was performed to detect coding region mutations of TFR2, HAMP, FPN1, HJV, and ALAS2. Patients 1-4 ingested 153, 547, 1,341, and 4,898 g of inorganic iron as supplements. Patient 1 had hemochromatosis, HFE C282Y homozygosity, and beta-thalassemia minor. Patient 2 had spherocytosis and no HFE coding region mutations. Patient 3 had no anemia, a normal HFE genotype, and no coding region mutations in HAMP, FPN1, HJV, or ALAS2; she was heterozygous for the TFR2 coding region mutation V583I (nt 1,747 G-->A, exon 15). Patient 4 had no anemia and no coding region mutations in HFE, TFR2, HAMP, FPN1, HJV, or ALAS2. Iron removed by phlebotomy was 32.4, 10.4, 15.2, and 4.0 g, respectively. There was a positive correlation of log(10) serum ferritin and the quantity of iron removed by phlebotomy (P = 0.0371). Estimated absorption of iron from supplements in patients 1-4 was 20.9%, 1.9%, 1.1%, and 0.08%. We conclude that the clinical phenotypes and hemochromatosis genotypes of adults who develop iron overload after ingesting iron supplements over long periods are heterogeneous. Therapeutic phlebotomy is feasible and effective, and would prevent complications of iron overload.

Iron overload in chronic liver diseases and clinical usefulness of therapeutic phlebotomy: Our experience

Iron overload in chronic liver diseases and clinical usefulness of therapeutic phlebotomy: Our experience

High prevalence of abnormal glucose homeostasis secondary to decreased insulin secretion in individuals with hereditary haemochromatosis

The prevalence and mechanisms of diabetes in hereditary haemochromatosis are not known. We therefore measured glucose tolerance, insulin secretory capacity and insulin sensitivity in adults with haemochromatosis. Subjects recruited from referrals to a haemochromatosis clinic underwent OGTT and frequently sampled IVGTT. A chart review of former clinic patients was also performed. The prevalence of diabetes (23%) and IGT (30%) was increased in haemochromatosis compared with matched control subjects (0% diabetes and 14% IGT). Subjects with haemochromatosis and diabetes were overweight (14%) or obese (86%). The prevalence of diabetes, as determined by chart review of fasting glucose values, in subjects who had haemochromatosis and were in the 40-79 years age range was 26%. Overall, patients with haemochromatosis and control subjects had similar values for acute insulin response to glucose and insulin sensitivity. However, patients with haemochromatosis and IGT had a 68% decrease in acute insulin response to glucose (p<0.02) compared with those with NGT. They were not insulin-resistant, exhibiting instead a 62% increase in insulin sensitivity (NS). Haemochromatosis subjects with diabetes exhibited further declines in acute insulin response to glucose, insulin resistance, or both. Diabetes and IGT are common in haemochromatosis, justifying screening for diabetes and therapeutic phlebotomy. The major abnormality associated with IGT is decreased insulin secretory capacity. Diabetes is usually associated with obesity and concomitant insulin resistance.

Hereditary Hemochromatosis: A Review of the Genetics, Mechanism, Diagnosis, and Treatment of Iron Overload

Hereditary hemochromatosis is a relatively common genetic disorder characterized by excess dietary iron absorption and deposition in tissues with resulting end-organ damage. Early diagnosis and initiation of therapeutic phlebotomy can provide a normal life expectancy for affected individuals.

A New Perspective on the Natural History of Hereditary Hemochromatosis.

A few homozygotes for the C282Y mutation of HFE manifest severe and even fatal iron-storage disease; most have only a modest increase in iron stores, show no clinical manifestations, and have no shortening of lifespan. Screening for hemochromatosis has been advocated so that therapeutic phlebotomy could be offered to the mildly affected subjects, often termed "pre-cirrhotic", presumably protecting them against progression of the disease. The rationale of this approach depends upon the implicit assumption that the natural history of the disease is one of progressive accumulation of iron and consequently ever-increasing tissue damage. In 1998 we began screening for the C282Y homozygous genotype among patients in the Kaiser Permanente Health Appraisal Clinic in San Diego. We have now followed 144 patients from this cohort of 156 C282Y homozygotes with medical records spanning up to 10 years, including 35 patients who declined phlebotomy. Data from this analysis and other sources now suggest that the natural history of the disease might be quite different than has heretofore been assumed. Our revised perspective is based upon both cross-sectional and longitudinal observations. Cross-sectional analysis of levels of serum ferritin, a surrogate for total body iron, shows little or no change with age in C282Y homozygotes. Neither are abnormal liver function tests, which occur in a small minority of homozygotes, age-related. In Denmark, longitudinal analyses of serum ferritin levels in untreated patients show no increase over as much as 20 years. Our own longitudinal data on serum ferritin levels in homozygotes who were not phlebotomized confirm this observation. Longitudinal analyses of abnormal ALT levels in homozygotes show no upward trend over a four-year period. Accordingly, it seems likely that in hemochromatosis the maximum extent of disease has been achieved in most patients by the time they reach adulthood, and that patients who are diagnosed with mild disease are unlikely to show progression.

Hematocrit-Driven Referrals for Measurement of RBC Mass. Is the Natural History of Polycythemia Vera Changing?.

Red blood cell mass (RCM) elevation is the sine qua non for diagnosis of polycythemia vera (pvera). The Polycythemia Vera Study Group (PVSG) employed therapeutic phlebotomy to lower the HCT to <55% as initial therapy. In the current era of frequent blood testing, RCM is usually requested based only on a high HCT often <55%. Thus the natural history of pvera may be changing if it is being diagnosed at an earlier stage. We reviewed 101 consecutive patients referred in 2002–2003 for RCM testing to characterize RCM requests from a large community referral base. There were 61 men and 40 women with mean HCT<55% (Table 1). 20 had lung disease or were smokers, 3 presented with arterial or venous thrombosis, 7 had cancer, 2 had renal cysts. HCT of men was higher than HCT of women if RCM was normal, but HCT were equivalent between genders if RCM was elevated. HCT was higher in men (p=0.0010) and women (p<0.0001) if RCM was elevated versus normal. HCT elevation was similar in secondary polycythemia vs pvera in men (51.0±4.02 vs 54.3±6.23; p=0.3397) or women (50.5±4.80 vs 49.3±2.12; p=0.4762).Table 1HCT of patients referred for RCM measurementGenderWhole GroupNormal RCMHigh RCMnHCTnHCTnHCTMale6148.3 (47.8) ±4.894546.9 (47.2) ±4.01652.1 (51.3) ±5.16Female4045.0 (43.8)± 4.723243.3 (42.5) ±3.691050.0 (50.0) ±3.84p value0.0001<0.00010.4932HCT shown as Mean (Median) ±SDWe examined whether, despite a lower presenting HCT, those with normal RCM demonstrated PVSG criteria that justified referral to our Nuclear Medicine department. Only 7 of the 75 patients with normal RCM met at least 2 of the PVSG “B” criteria or had splenomegaly, thus might have been diagnosed with pvera had RCM been high (Table 2).Table 22 PVSG “B” criteria or splenomegaly and normal RCMGenderWBC ≥12KPLT ≥400KB12 ≥900≥ LAP 100SplenomegalyM14.4413640158“normal”M12.1585133653“normal”M9.4428113968“normal”M8.6132134315 cmM8.231249218617 cmF18.258271923“normal”F6.3226318“enlarged”F7.114681315.3 cmOverall, 10 of 40 women and 16 of 61 men had an elevated RCM: 4 women and 5 men with polycythemia vera; 6 women and 11 men with secondary polycythemia. One other man was diagnosed with polycythemia vera on the basis of a borderline elevated RCM (33.8 ml/kg), normal O2 Sat on room air, popliteal artery thrombosis, and 3 “B” criteria. Serum erythropoietin (EPO) was low at 2.6 mU/ml (ref range 4–16). He was receiving therapeutic phlebotomy for hereditary hemochromatosis when his RCM was measured. He was the only patient with pvera whose EPO was low (three others had normal EPO levels). The 11 patients with secondary polycythemia who had EPO measured had normal levels. In summary, the HCT was the primary criterion for RCM testing for 2/3 of these patients. Only 5 presented with HCT>55%; mean HCT was ~50% in patients with elevated RCM. The bottom quartile HCT of women with elevated RCM in our patient population was 48.7%. This is a sensitive “cut-off” for finding an elevated RCM (p<0.0001, Chi square with Yates correction). We conclude that patients are referred for RCM testing when a high HCT is found, but at HCT far below the original PVSG parameters. Therefore polycythemia vera is now diagnosed earlier and in mostly asymptomatic patients. A normal EPO level does not rule out a diagnosis of pvera. A HCT < 48.7% may not warrant the measurement of RBC mass.

Magnetic Ressonance Image T2* for the Evaluation of Iron Overload in Patients with Hereditary Hemochromatosis. A New Guide for the Indication of Therapeutic Phlebotomies.

Hereditary hemochromatosis is highly prevalent in those with northern European ancestry (1 in 8 individuals are heterozygous and 1 in 100 – 200 are homozygous). It is characterized by laboratory tests as ferritin > 150 ng/mL, transferrine saturation > 40% and HFE mutations (C282Y and H63D). Presently the indications for treatment (therapeutic phlebotomies, TP) are based solely on the ferritin levels. It is possible that patients with moderate or even high levels of ferritin do not have iron overload. Performing liver biopsies would be an option for the actual evaluation, though invasive and risky. A non-invasive method to evaluate this deposition would be helpful in order to determine which patients actually demand TP.To evaluate if the use of Magnetic Ressonance Image (MRI) is a method for measuring tissue iron and could be a new guide for the indication of therapeutic phlebotomies, nineteen patients (mean age 43,47 y.o, +/− 9,85, gender = 16 male, 3 female) with hereditary hemochromatosis were scanned with T2- star (T2*) (GE equipment, Milwaukee, USA). The median of ferritin level was 594 (21–9300) The MRI method was previously validated to chemical estimation of iron in thalassemic major patients undergoing liver biopsies. The evaluated organs were liver and heart.All patients were in normal range of myocardial T2*. The images of four patients (25%), showed liver iron deposition. Eleven patients who presented serum ferritin levels below 600 ng/ml showed no liver iron deposition. Just one among five patients (20%), who presented ferritin levels between 601 and 1000 ng/ml showed hepatic iron overload. The three patients with ferritin levels higher than 1000 ng/ml had liver iron deposition quantified using liver T2* MRI techique.MRI T2* showed that some patients who would have an indication for TP based on laboratory tests, might avoid these procedures based on image results of internal organs. These patients can have an image follow up in order to decide when would be the appropriate time to start TP. This method can also be used to evaluate the efficacy of TP in patients who have already received this treatment. A larger group of patients would have to be evaluated in order to validate these results.

A Novel Allogeneic Transplant Conditioning Regimen Designed for Tolerance Induction in Patients with Severe Sickle Cell Disease.

Allogeneic transplantation of hematopoietic stem cells remains the only curative approach for patients with sickle cell disease (SCD), yet procedural toxicities and graft-versus-host disease limit this approach to children. We chose a low-dose radiation approach utilizing rapamycin based upon its unique ability to promote T cell tolerance even when T cells are stimulated in the presence of costimulation (Powell, et al, J Immunol. 1999). We confirmed this approach in vivo in a murine bone marrow transplantation model comparing a short course of conventional immunosuppression with cyclosporine to that with rapamycin, with long-term, high-level chimerism attained only in mice treated with rapamycin (Powell et al., manuscript submitted). We have now begun accrual to an IRB approved clinical trial testing this approach in adults with SCD and herein report the results in our first two subjects. Protocol entry criteria include those previously reported (Walters, et al, NEJM, 1996) with the addition of pulmonary hypertension defined as a tricuspid-regurgitant jet velocity (TRV) > 2.5 m/s (Gladwin et al, NEJM, 2004). Additionally, patients must have failed a 6-month course of hydroxyurea. The conditioning regimen consists of a single radiation dose of 300cGy, alemtuzumab (1mg/kg total), and oral rapamycin targeting trough levels between 10–20 ng/ml. The graft consists of unmanipulated mobilized peripheral blood progenitors obtained from an HLA-matched sibling. The first patient is a 24-year-old female referred due to recurrent transient ischemic attacks and strokes despite chronic exchange transfusions. Her initial evaluation was notable for evidence of significant hemolysis with a total bilirubin of 9.8 mg/dl, an LDH 1,172 units/L (range 113–226), an absolute reticulocyte count of 318,000/uL and an undetectable haptoglobin. Cardiac doppler-echocardiogram revealed a TRV of 3.7 m/s, consistent with severe pulmonary hypertension. The conditioning was well tolerated and the patient did not require parenteral antibiotics or nutritional support. Assessment of donor chimerism, measured by microsatellite PCR and hemoglobin electrophoresis, revealed an early peak of myeloid chimerism which has stabilized at approximately 60%, with lower levels of lymphoid chimerism at approximately 5–10% now more than 300 days post-transplant. Hemoglobin levels stabilized at 11–12 g/dL, without detectable sickle hemoglobin, now allowing for therapeutic phlebotomy. Remarkably, the LDH and TRV fell concurrently toward the normal range. Patient 2 is a 26 year old male with frequent crises requiring hospitalization twice monthly. At one month post-transplant, myeloid chimerism is 97%, with lymphoid chimerism currently unmeasurable due to lymphopenia, and similar to that seen in patient 1 at the same time point. Patient 2 also required neither parenteral antibiotics nor nutritional support. Finally, neither patient to date has developed any symptoms or signs of either acute or chronic GVHD. Thus with two patients accrued to date, we have demonstrated the ability of allogeneic HSC transplantation to achieve mixed hematopoietic chimerism without the development of GVHD and for the first time, established the reversibility of the associated pulmonary hypertension.

Screening for Hereditary Hemochromatosis: A Clinical Practice Guideline from the American College of Physicians

Hereditary hemochromatosis is a genetic disorder of iron metabolism. Diagnosis of hereditary hemochromatosis is usually based on a combination of various genetic or phenotypic criteria. Decisions regarding screening are difficult because of the variable penetrance of mutations of the HFE gene and the absence of any definitive trials addressing the benefits and risks of therapeutic phlebotomy in asymptomatic patients or those with only laboratory abnormalities. The purpose of this guideline is to increase physician awareness of hereditary hemochromatosis, particularly the variable penetrance of genetic mutations; aid in case finding; and explain the role of genetic testing. This guideline provides recommendations based on a review of evidence in the accompanying background paper by Schmitt and colleagues. The target audience for this guideline is internists and other primary care physicians. The target patient population is all persons who have a probability or susceptibility of developing hereditary hemochromatosis, including the relatives of individuals who already have the disease.

Restless legs syndrome in patients with hereditary hemochromatosis

Iron deficiency is important in the pathogenesis of restless legs syndrome (RLS), and iron supplementation improves its symptoms.1,2 Hereditary hemochromatosis (HH) is an autosomal recessive condition caused by mutations of the HFE gene in which excessive intestinal iron absorption leads to iron deposition in multiple organs.3 Iron therapy for RLS precipitated symptoms of iron overload in one patient with previously unrecognized HH,4 suggesting that monitoring of iron indexes may be required during iron therapy in RLS. We investigated the frequency of RLS and the relationship between RLS and iron indexes in consecutive adult patients with HH attending a large teaching hospital for therapeutic phlebotomy over a 3-month period. All 61 HH patients seen agreed to participate; 48 (79%) were men, and the median (range) age was 53 (24 to 82) years. The median (range) duration of phlebotomy was 12 (2 to 108) months, and 35 (62%) had serum ferritin level of <50 ng/mL. …

Management of patients with polycythaemia vera: results of a survey among Swedish haematologists

The prevailing attitudes regarding diagnostic and therapeutic procedures in patients with polycythaemia vera (PV) among Swedish haematologists were surveyed by way of a mailed questionnaire in August 2002. Among diagnostic procedures frequent use is reported for arterial O(2) saturation, spleen size determination, bone marrow histology, serum erythropoietin, serum cobalamins and leukocyte alkaline phosphatase score, while direct determination of the red blood cell mass is used infrequently (seldom or never by 82%). Among therapeutic modalities hydroxyurea and phlebotomy alone were most frequently used. The (32)P therapy was used at least sometimes by 57% of the physicians, and more widely in the university clinics. Anagrelide and alfa-interferon was used in a minority of patients only. The use of prophylactic acetylsalicylic acid was very variable. The majority of the physicians had an aim for their phlebotomy treatment at a level of 0.45 or less, but 21% used a level of 0.46-0.49 and 8% a level of 0.55-0.60 (in younger patients). The platelet level, at which myelosuppressive therapy was initiated, also varied, from 400 x 10(9)/L to >1500 x 10(9)/L. It can be concluded that in practical clinical work in Sweden the diagnosis of PV is established by frequent use of serum erythropoietin, bone marrow examination and spleen size determination. The use of different therapeutic modalities is very variable. Many physicians carry out their phlebotomy treatment with less intensity compared with national and international recommendations.

Hereditary hemochromatosis

Hereditary hemochromatosis (HH) is a disorder of iron metabolism characterized by a progressive tissue iron overload which leads to an irreversible organ damage if it is not treated timely. The recent developments in the field of molecular medicine have radically changed the physiopathology and the diagnosis of this disease. However, transferrin saturation and serum ferritin are still the most reliable tests for the detection of subjects with HH. Therapeutic phlebotomy is the mainstay of the treatment of HH. If phlebotomy is started before the onset of irreversible organ damages, the life expectancy of these patients is similar to that of normal population.

Recent advances in hereditary hemochromatosis

Hereditary hemochromatosis, a very common genetic defect in the Caucasian population, is characterized by progressive tissue iron overload which leads to irreversible organ damage if it is not treated in a timely manner. Recent developments in the field of molecular medicine have radically improved the understanding of the physiopathology and diagnosis of this disease. However, transferrin saturation and serum ferritin are still the most reliable tests for identifying subjects with hereditary hemochromatosis. Therapeutic phlebotomy is the mainstay of the treatment of this disease and the life expectancy of these patients is similar to that of the normal population if phlebotomy is started before the onset of irreversible organ damage. In this review we discuss the genetics, pathophysiology, diagnosis, clinical features, and management of hereditary hemochromatosis.

Soluble transferrin receptor in hemochromatosis patients during phlebotomy therapy

Background The monitoring of phlebotomies in hemochromatosis patients depends on iron status measured by ferritin and transferrin saturation (TS). However, in the presence of inflammation or liver injury, soluble transferrin receptor (sTfR) determination was proposed to replace ferritin for diagnosing iron deficiency (ID). The present study evaluated performances of sTfR for the prediction of iron deficiency in a large number of hemochromatosis patients under phlebotomy therapy. Methods We studied 52 patients undergoing therapeutic phlebotomies and obtained 2 samples from 37 patients. Biological parameters were determined before each phlebotomy began. Performances of sTfR and TS in the diagnosis of iron deficiency were compared, according to ferritin levels under 12 μg/l. Results Ferritin and TS were correlated with removed iron ( r=0.473, p<0.005 and r=0.345, p<0.05, respectively) and sTfR was correlated with the decrease in hemoglobin levels induced by phlebotomies ( r=−0.678, p<0.0001). Areas under Receiver Operating Characteristics (ROC) curves for sTfR and TS were not statistically different for prediction of iron deficiency and sensitivity/specificity of sTfR at 1.64 mg/l were 67/86%. Conclusions sTfR determination could be used to predict iron depletion induced by phlebotomies when ferritin is of limited interest, to avoid the appearance of anemia.

HLA haplotype A*03-B*07 in hemochromatosis probands with HFE C282Y homozygosity: frequency disparity in men and women and lack of association with severity of iron overload

Before the discovery of HFE, reports suggested that hemochromatosis patients with the ancestral haplotype (or some element thereof) have more severe iron overload than those without the haplotype. We performed univariate and multivariate analyses of the relationships of human leukocyte antigen (HLA)-A*03 and HLA haplotype A*03-B*07 to iron measures (serum iron concentration, transferrin saturation, and serum ferritin concentration at diagnosis and units of phlebotomy to achieve iron depletion) in hemochromatosis probands homozygous for HFE C282Y diagnosed in medical care. Iron overload was defined by demonstration of hepatic iron index of ≥1.9 or removal of ≥2.0 g Fe by therapeutic phlebotomy. We tabulated the phenotype frequencies of HLA-A*03 and the frequencies of common HLA haplotypes A*01-B*08, A*02-B*44, A*03-B*07, and A*03-B*14 in three groups of white adults: (1) 141 hemochromatosis probands with C282Y homozygosity; (2) 195 index cases with IgG subclass deficiency (IgGSD) or common variable immunodeficiency (CVID), disorders typically linked to Ch6p, and (3) 750 control subjects. Among probands, 86 men and 42 women had iron overload. Frequencies of HLA-A and -B alleles in probands did not depart significantly from Hardy-Weinberg equilibrium. The phenotype frequency of A*03 did not differ significantly between men and women in the each of the respective three groups. The frequency of haplotype A*03-B*07 was greater in men than women with hemochromatosis (0.3081 vs. 0.1455; P = 0.0019). The frequency of A*03-B*014 was significantly greater in women than men with hemochromatosis (0.1182 vs. 0.0407, respectively; P = 0.0134). Mean values of most iron measures were not affected by numbers of copies of A*03 or by presence of A*03-B*07 in either men or women in univariate analysis. ANOVA models of sex, age at diagnosis, and all HLA alleles and haplotypes in probands were used to determine effects of these variables on iron measures. ANOVA models revealed that (1) there were no significant predictors for serum iron concentration; (2) B*14 is associated with higher transferrin saturation in women and lower transferrin saturation in men; (3) A*01-B*08 is associated with a trend of higher serum ferritin levels; and (4) A*03-B*14 is associated with exaggeration of the age-associated upward trend in units of phlebotomy to achieve iron depletion. In hemochromatosis probands with HFE C282Y homozygosity, we conclude that (1) disparate frequencies of HLA haplotypes A*03-B*07 and A*03-B*14 occur in men and women and (2) HLA-A*03 and HLA-A*03-B*07 are not independent variables associated with iron overload severity.

Molecular Diagnosis of the First Ferroportin Mutation (C326Y) in the Far East Causing a Dominant Form of Inherited Iron Overload.

Genetic hemochromatosis (HH) is a common inherited disorder in populations of European origin in which different types of genetic hemochromatosis (type 1–4) have been characterized. Most hemochromatosis-type 1 patients are homozygotes or compound heterozygotes for two HFE mutations C282Y and H63D. Studies of several non-HFE iron overload families led to identification of mutations in hemojuvelin and hepcidin (juvenile form-HFE2A and B), transferrin receptor 2 (HFE3) and ferroportin (HFE4) as a cause of different forms of hemochromatosis. In the Far East, inherited hemochromatosis has rarely been reported and may have been misdiagnosed due to the high prevalence of secondary iron loading from hemoglobin disorders. This report describes, for the first time, non-HFE iron overload in patients from Southeast Asia. The affected Thai family presented with a distinctive clinical phenotype including macrocytosis and elevated transferrin saturation (>95%), increased non-transferrin bound iron (NTBI) as well as raised serum ferritin and marked hepatic hemochromatosis. Our patients tolerated therapeutic phlebotomy well. DNAs from peripheral blood leukocytes were firstly analyzed for three common HFE mutations (C282Y, H63D and IVS5+1 G→A). Subsequently, we screened all coding sequences, promoters and exon/intron boundaries of the HFE, HAMP, TfR2, HJV and SLC40A1 genes using denaturing high performance liquid chromatography (DHPLC). The entire coding region and splice sites of these genes were amplified and directly sequenced. We identified a novel mutation (C326Y) in ferroportin (SLC40A1, IREG-1, MTP-1), a membrane iron transport protein due to a G→A substitution at nucleotide 1281 in exon 7. This mutation was confirmed by restriction fragment length polymorphism (RFLP) analysis using Sfa NI. Six hundred Thai and two hundred Vietnamese chromosomes were analyzed for the C326Y mutation by RFLP analysis and it was not detected in any of the healthy controls studied. This result suggested that the G→A substitution is not a common polymorphism and is likely to be the causative mutation for the phenotype in this family. Previous reported mutations of ferroportin, including A77D and V162del, which lead to type IV hemochromatosis, were characterized by increased serum ferritin despite normal transferrin saturation, in contrast to our patients' phenotype. These autosomal dominant mutants are postulated to lead to disease due to loss of iron exporting function. Preliminary in vivo assay using transient transfection of wild-type and ferroportin mutants in HeLa or 293T cells revealed, as expected, a loss of function and diminished surface membrane localisation in A77D and V162del mutants. Surprisingly, the C326Y mutant was indistinguishable from wt ferroportin in both iron status of the cell and protein localization suggesting different pathophysiology leading to iron overload in our patients.

Hemochromatosis,&lt;I&gt; HFE&lt;/I&gt; C282Y Homozygosity, and Bariatric Surgery: Report of Three Cases

Iron absorption is decreased in some individuals who have undergone bariatric surgery. We evaluated measures of iron metabolism and therapeutic phlebotomy in 3 adults with hemochromatosis and HFE C282Y homozygosity who underwent bariatric surgery. 1 male and 1 female had surgery before diagnosis of hemochromatosis (jejuno-ileal bypass and Roux-en-Y gastric bypass, respectively); neither had iron overload. Another man was treated with serial phlebotomy to induce iron depletion; later, he underwent Roux-en-Y gastric bypass. His maintenance phlebotomy requirement for hemochromatosis decreased substantially (on average approximately 1 unit each 71 days before surgery, and approximately 1 unit each 173 days after surgery). None of these patients developed iron deficiency, and none took supplemental iron. Iron absorption is decreased in some patients with hemochromatosis and HFE C282Y homozygosity after bariatric surgery, but their risk of developing iron deficiency may be diminished.

Iron out the details of therapeutic phlebotomy.

Iron out the details of therapeutic phlebotomy.