Moderate Hereditary Spherocytosis Research Articles

A clinical and experimental study of adult hereditary spherocytosis in the Chinese population.

Hereditary spherocytosis (HS) is often misdiagnosed due to lack of specific diagnostic methods. Our study summarized clinical characteristics and described the diagnostic workflow for mild and moderate HS in Chinese individuals, using data from 20 adults, 8 of whom presented a familial history for HS. We used scanning electron microscopy (SEM) to diagnose HS. We observed reduced eosin maleimide fluorescence activity (5.50 mean channel fluorescence (MCF) units) in the 10 cases of HS, which differed significantly when compared with 10 normal adults (15.50 units), iron deficiency anemia (15.50 MCF units), and megaloblastic anemia (12.00 MCF units) values (P < .05). Next generation sequencing results revealed that 9 out of 10 patients were found to have mutations in the spectrin alpha chain (SPTB), anchor protein (ANK1), and SLC4A1 genes. These mutations were not reported in the Human Gene Mutation Database (HGMD), 1000 human genome, ExAC, and dbSNP147 databases. Splenectomy proved to be beneficial in alleviating HS symptoms in 10 cases. It was found that for the diagnosis of HS, SEM and next generation gene sequencing method proved to be more ideal than red blood cell membrane protein analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting.

The Complexity of Genotype-Phenotype Correlations in Hereditary Spherocytosis: A Cohort of 95 Patients: Genotype-Phenotype Correlation in Hereditary Spherocytosis.

Hereditary spherocytosis (HS) is a phenotypically and genetically heterogeneous disease. With the increased use of Next Generation Sequencing (NGS) techniques in the diagnosis of red blood cell disorders, the list of unique pathogenic mutations underlying HS is growing rapidly. In this study, we aimed to explore genotype-phenotype correlation in 95 HS patients genotyped by targeted NGS as part of routine diagnostics (UMC Utrecht, Utrecht, The Netherlands). In 85/95 (89%) of patients a pathogenic mutation was identified, including 56 novel mutations. SPTA1 mutations were most frequently encountered (36%, 31/85 patients), primarily in patients with autosomal recessive forms of HS. Three SPTA1 (α-spectrin) mutations showed autosomal dominant inheritance. ANK1 (ankyrin1) mutations accounted for 27% (23/85 patients) and SPTB (β-spectrin) mutations for 20% (17/85 patients). Moderate or severe HS was more frequent in patients with SPTB or ANK1 mutations, reflected by lower hemoglobin concentrations and higher reticulocyte counts. Interestingly, mutations affecting spectrin association domains of ANK1, SPTA1 and SPTB resulted in more severe phenotypes. Additionally, we observed a clear association between phenotype and aspects of red cell deformability as determined by the Laser assisted Optical Rotational Cell Analyzer (LoRRca MaxSis). Both maximal deformability and area under the curve were negatively associated with disease severity (respectively r = -0.46, p < 0.01, and r = -0.39, p = 0.01). Genotype-phenotype prediction in HS facilitates insight in consequences of pathogenic mutations for the assembly and dynamic interactions of the red cell cytoskeleton. In addition, we show that measurements of red blood cell deformability are clearly correlated with HS severity.

S902 THE COMPLEXITY OF GENOTYPE‐PHENOTYPE CORRELATIONS IN HEREDITARY SPHEROCYTOSIS: A COHORT OF 95 PATIENTS

Background:Hereditary spherocytosis (HS) presents as a highly heterogeneous disease, both phenotypically and genetically. Disease severity is directly related to the extent of loss of surface area of the red cell. The diagnosis is multifaceted and includes functional testing of red cell deformability. Until recently, establishing or confirming the diagnosis at the DNA level was only rarely done. With increased use of Next‐Generation‐Sequencing (NGS) techniques in diagnosis of red blood cell disorders, the list of unique pathogenic mutations underlying HS is growing rapidly. However, insight into the impact of genetic defects on the function of membrane proteins, and consequently on phenotypic presentation, is still limited.Aims:Explore genotype‐phenotype correlation in a large cohort of patients with HS.Methods:Retrospective analysis on a cohort of 95 HS patients genotyped by targeted‐NGS (SPTA1, SPTB, ANK1, SLC4A1, EPB41, EPB42, RHAG) as part of routine diagnostics (UMC Utrecht, Utrecht, The Netherlands).Results:Our cohort included 95 patients suspected to have HS. In 85/95 (89%) of the patients a pathogenic mutation was identified, including 56 novel mutations. Mutations in SPTA1 were most frequently encountered (36%, 31/85 patients), primarily in patients with autosomal recessive forms of HS. Three SPTA1 mutations showed autosomal dominant inheritance. ANK1 mutations accounted for 27% (23/85 patients) and SPTB mutations for 20% (17/85 patients) of the cases, all showing autosomal dominant inheritance. Moderate or severe HS was more frequently observed in patients with SPTB or ANK1 mutations, reflected by significantly lower hemoglobin concentrations (SPTB 10.5 g/dL, ANK1 10.8 g/dL compared to SLC4A1 13.2 g/dL, both p &lt; 0.05) and higher absolute reticulocyte counts (SPTB 580x10^9/L, ANK1 506x10^9/L compared to SPTA1 298x10^9/L and SLC4A1 259x10^9/L, all p &lt; 0.01) in non‐splenectomized patients.We investigated the correlation between phenotype and genetic mutations using protein structure information (Figure). Mutations positioned in the spectrin‐association domains of ANK1, SPTA1 and SPTB resulted in a more severe phenotype. Interestingly, deletion of the whole SPTB gene resulted in a mild phenotype, while the majority of other pathogenic mutations in SPTB were associated with a more severe disease phenotype, suggesting that incorporation of truncated or dysfunctional protein occurs, which might be more harmful than an absolute decrease of otherwise normal protein.Our cohort included nine families and broad phenotypic heterogeneity was observed in four of them. Phenotypic heterogeneity could not be directly explained by the underlying genotype, suggesting that other (genetic) yet‐unknown modifiers contribute to disrupted interactions in the red cell cytoskeleton. To identify alternative predictors of clinical severity we analyzed red cell Osmoscan profiles from the Laser‐assisted Optical Rotational Cell Analyzer. Data were available in a subset of our cohort (44 not splenectomized patients). We observed a clear association between clinical disease severity and the maximal deformability, and the area under the curve (respectively r = −0.48, p &lt; 0.01, and r = −0.38, p = 0.01).Summary/Conclusion:Genotype‐phenotype prediction in HS requires additional insight in the consequences of pathogenic mutations for assembly of and dynamic interactions in the red blood cell cytoskeleton. Currently, in conjunction to the molecular deficit, functional analysis of red cell deformability adds to the prediction of phenotypic variability.image

PS1205 META‐ANALYSIS USING DATA OF RED BLOOD CELL ENZYME ASSAY AND PROTEOME IN PATIENTS WITH DIAMOND‐BLACKFAN ANEMIA

Background:Diamond‐Blackfan anemia (DBA) is a form of congenital bone marrow failure syndrome, which is characterized by RBC aplasia with varied malformation in infants. Elevated enzymatic activity of adenosine deaminase (ADA) is known to be a valuable biomarker of DBA. The ADA assay has been shown to be more sensitive than the gene test of ribosome subunit genes. We recently reported that reduced glutathione (GSH) in red blood cells (RBCs) is another biomarker of DBA, and simultaneous assessment of ADA and GSH enabled us to discriminate DBA patients from normal subjects as well as unaffected family members (Blood Cells Mol Dis 59:31–36, 2016).Aims:This study was performed to investigate the detailed changes in enzymatic activity and enzyme protein mass in DBA erythrocytes. We reanalyzed the enzymatic activity and GSH in 14 DBA patients and compared the results with those obtained from simultaneously assayed 14 normal subjects. Furthermore, we performed a meta‐analysis using proteome data as recently reported by Pesciotta, et al. (PLoS ONE 10:e0140036, 2015).Methods:The gene mutations of the enrolled patients were previously confirmed. We estimated the activity of RBC enzymes and GSH content by a standard method using a spectrophotometer. We calculated the relative expression of both enzymatic activity and protein content. The statistical analyses were performed by Wilcoxon rank‐sum test and a P value of &lt;0.001 was considered statistically significant.Results:The combined analyses of proteomic and enzymatic data revealed that ADA, PGD, and HK‐1 (proteome)/HK (activity) were elevated, while PFKL (proteome)/PFK (activity) and PKLR (proteome)/PK (activity) were decreased. The G6PD enzyme activity, but not its protein level, was characteristically increased.Summary/Conclusion:We speculate that the discrepancy between the activity and protein level of G6PD was attributable to the decreased inhibition of G6PD activity by NADPH. In other words, G6PD activity was supposedly activated by a lower NADPH level in DBA. To maintain the affordable level of GSH in RBCs, reduction of GSSG was necessarily maximized through the reaction catalyzed by glutathione reductase, which used NADPH as a cofactor. As a result, NADPH content was presumably exhausted, resulting in decreased inhibition of G6PD. In this study, we found that HK activity and HK‐1 content were elevated significantly. On the other hand, the activity and protein content of two important rate‐limiting enzymes (PFK/PK and PFKL/PKLR) involved in glycolysis were found to be decreased. Higher demand for NADPH production through the pentose phosphate pathway leads to a reduction in glucose consumption in the glycolytic pathway, and/or activation of p53 leads to inhibition of glycolysis, as previously reported. The present study shows the inhibition of glycolysis in the RBCs in DBA. It needs to be elucidated whether impaired glycolysis partly accounts for premature cell death of erythroblasts in DBA.Abbreviations: DBA: Diamond‐Blackfan anemia; RBC: red blood cell; ADA: adenosine deaminase; GSH: reduced glutathione; PGD: 6‐phosphogluconate dehydrogenase; HK: hexokinase; PFK (L): 6‐phosphofructokinase (liver type); PK (LR): pyruvate kinase (liver and erythrocytes type); G6PD: glucose‐6‐phosphate dehydrogenase; NADPH: reduced nicotinamide adenine dinucleotide phosphate; GSSG: glutathione disulfide.

Hereditary spherocytosis: Retrospective evaluation of 65 children.

Güngör A, Yaralı N, Fettah A, Ok-Bozkaya İ, Özbek N, Kara A. Hereditary spherocytosis: Retrospective evaluation of 65 children. Turk J Pediatr 2018; 60: 264-269. Hereditary spherocytosis (HS) is a common cause of congenital hemolytic anemia in Caucasians and it could be diagnosed at any age. The aim of this study is to examine the demographic characteristics, clinical features and laboratory findings of children with HS and their complications observed during follow up. Sixty-five patients, with hereditary spherocytosis between January 2008 and September 2013, were enrolled into this retrospective study. The age of patients at the time of diagnosis varied between 15 days and 17 years. The median age of patients at diagnosis was 48 months (IQR 2-78). The female/male ratio was 1.1. Forty-seven patients (72.3%) had a family history of HS. The patients were classified according to laboratory findings: 13 of them (20%) were diagnosed as mild HS, 36 (55.4%) as moderate HS and of 16 (24.6%) as severe HS. During follow-up, nine patients (13.8%) experienced an aplastic crisis. Megaloblastic crisis was not observed in any patient. Twenty patients (30.8%) had cholelithiasis. Splenectomy was performed in 20% of patients and the mean age for splenectomy was 8.3 years. Complications such as sepsis or thrombosis were not detected after splenectomy. Hereditary spherocytosis should be kept in mind in patients with anemia, jaundice and splenomegaly and the family history must be questioned. The most common complication was gallstone; even patients without severe hemolysis should be followed intermittently by abdominal ultrasonography in order to control the development of gallstone.

Misdiagnosis of two cases of hereditary spherocytosis in a family and review of published reports

BackgroundHereditary spherocytosis is usually diagnosed based on a combination of clinical and family histories, physical examination, and laboratory data. Milder or atypical cases can be difficult to diagnose. MethodsWe report one case each of mild and moderate hereditary spherocytosis whose diagnoses were made by the First Affiliated Hospital, Guangxi Medical University in China. ResultsIn case 1, laboratory test results were as follows: hemoglobin 81.40g/l, mean corpuscular hemoglobin concentration 339.00g/l, mean corpuscular volume 82.88fl, reticulocyte count 19%, mean sphered corpuscular volume 71.66fl, total bilirubin 57.60μmol/l, and blood smears showed increased spherocytosis. In case 2, laboratory test results were as follows: hemoglobin 133.10g/l, mean corpuscular hemoglobin concentration 358.60g/l, mean corpuscular volume 96.75fl, reticulocyte count 17%, mean sphered corpuscular volume 77.78fl, total bilirubin 62.50μmol/l, and blood smears showed increased spherocytosis. ConclusionsMild and moderate hereditary spherocytosis can be easily misdiagnosed. Assessment of total, direct, and indirect serum bilirubin, erythrocyte morphology and red cell characteristics (particularly mean corpuscular volume and mean sphered corpuscular volume) clearly distinguishes hereditary spherocytosis from autoimmune hemolytic anemia, glucose-6-phosphate dehydrogenase deficiency, thalassemia, and autoimmune hepatitis.

PS-090 Hereditary Spherocytosis And Red Cell Indices Mchc, Mcv, Rdw

Background Hereditary spherocytosis (HS) is a common inherited disorder that is characterised by anaemia, jaundice, and splenomegaly. Clinical severity is variable with most patients having a well-compensated hemolytic anaemia. The primary lesion in HS is loss of membrane surface area, leading to reduced deformability due to defects in the membrane proteins ankyrin, band 3, beta spectrin, alpha spectrin, or protein 4.2. Many isolated mutations have been identified in the genes encoding these membrane proteins; common hereditary spherocytosis-associated mutations have not been identified. The classic laboratory features of HS include minimal or no anaemia, reticulocytosis, an increased mean corpuscular haemoglobin concentration (MCHC), spherocytes on the peripheral blood smear, hyperbilirubinemia, and abnormal results on the osmotic fragility test. Aim Of the study is to evaluate the role of MCV, MCHC as a screen test to diagnose spherocytosis Methods In our study are included 60 subjects, 30 children with HS and 30 children-control groups. Our patients with anaemia, jaundice, and splenomegaly are diagnose with HS by incubated osmotic fragility test, performed after incubating RBCs for 18–24 h under sterile conditions at 37°C. Results We found that 25% of pts. have mild HS, 20% moderate HS, 30% moderate to severe HS and 25% severe HS. In peripheral blood smear 7% of pts. had 0–5 spherocites for field, 30% had 5–10 spherocites for field and 63% had 10–15 spherocites for field. 70% of pts. With HS have MCHC > 38%. There are a positive correlation between MCHC and spherocites in peripheral blood smear (r = 0,898, p Conclusion The dedication of hiperdense erythrociyte today is used as a new tool in diagnosing HS. The determination of MCHC constantly growing with other red cell index, MCV 15 obtained from an electronic cell counter usually is enough to suggest for HS. Key Words Spherocytosis, MCHC, anaemia, children.

Comments on 'clinical course of 63 children with hereditary spherocytosis: a retrospective study'

Comments on 'clinical course of 63 children with hereditary spherocytosis: a retrospective study'

Steroid therapy may be effective in augmenting hemoglobin levels during hemolytic crises in children with hereditary spherocytosis

The course of hereditary spherocytosis (HS) may be subject to hemolytic episodes, sometimes requiring blood transfusion. The aim of this study was to evaluate the efficacy of a short course of steroid therapy in elevating hemoglobin levels during hemolytic crisis. The files of all patients followed for HS from 1968 to 2009 at our hospital were reviewed. Outcome of hemolytic crises was compared between steroid-treated and untreated patients; patients given packed red blood cell transfusion(s) or erythropoietin treatment were excluded. A good outcome was defined as an increase of at least 20% in hemoglobin level from the nadir within 1 week. Of the 118 patients with HS who attended our hospital during the study period, 20 were treated with steroids and 9 received no treatment. Mean nadir hemoglobin level in both groups was 6.9 g/dl. The study group had a total of 50 steroid-treated hemolytic crises of which 37 (74%) responded favorably to treatment. Treatment failure was significantly associated with a low dose (<1 mg/kg/day) or short duration (<1 week) of treatment. The nine untreated patients had 16 hemolytic crises, of which 25% had a good outcome. Steroid therapy was significantly more effective than no therapy in increasing hemoglobin level (P = 0.015) in these hemolytic crises. Steroid therapy may be effective in augmenting hemoglobin levels during hemolytic crises in patients with moderate HS and eventually will result in a reduced need for RBC transfusion.

Hereditary Spherocytosis with Leg Ulcers Healing After Splenectomy

Hereditary spherocytosis (HS) is a familial hemolytic disorder with marked heterogeneity. A refractory chronic leg ulcer is an uncommon complication of HS, reported in fewer than two percent of patients. We present the case of a 28-year-old man who was suffering from a leg ulcer and was unresponsive to treatment of two years' duration with various conservative modalities. He had been suffering from repeated attacks of jaundice, pallor, and splenomegaly since he was 10 years old. Upon admission, physical and laboratory findings revealed moderate HS. However, complete clearance of the leg ulcer was not achieved until two months after a splenectomy.

Additional erythrocytic and reticulocytic parameters helpful for diagnosis of hereditary spherocytosis: results of a multicentre study

Hereditary spherocytosis (HS) is characterised by weakened vertical linkages between the membrane skeleton and the red blood cell's lipid bilayer, leading to the release of microparticles. All the reference tests suffer from specific limitations. The aim of this study was to develop easy to use diagnostic tool for screening of hereditary spherocytosis based on routinely acquired haematological parameters like percentage of microcytes, percentage of hypochromic cells, reticulocyte counts, and percentage of immature reticulocytes. The levels of haemoglobin, mean cell volume, mean corpuscular haemoglobin concentration, reticulocytes (Ret), immature reticulocytes fraction (IRF), hypochromic erythrocytes (Hypo-He) and microcytic erythrocytes (MicroR) were determined on EDTA samples on Sysmex instruments from a cohort of 45 confirmed SH. The HS group was then compared with haemolytical disorders, microcytic anaemia, healthy individuals and routine samples (n = 1,488). HS is characterised by a high Ret count without an equally elevated IRF. All 45 HS have Ret >80,000/μl and Ret(10(9)/L)/IRF (%) greater than 7.7 (rule 1). Trait and mild HS had a Ret/IRF ratio greater than 19. Moderate and severe HS had increased MicroR and MicroR/Hypo-He (rule 2). Combination of both rules gave predictive positive value and negative predictive value of respectively 75% and 100% (n=1,488), which is much greater than single parameters or existing rules. This simple and fast diagnostic method could be used as an excellent screening tool for HS. It is also valid for mild HS, neonates and ABO incompatibilities and overcomes the lack of sensitivity of electrophoresis in ankyrin deficiencies.

Complementary markers for the clinical severity classification of hereditary spherocytosis in unsplenectomized patients

Hereditary spherocytosis (HS) is usually classified as mild, moderate or severe using conventional features, namely, hemoglobin (Hb) concentration, reticulocyte count and bilirubin levels, which do not always contribute to an adequate clinical classification. The aim of our study was to establish the importance of some laboratory routine parameters, as markers of HS clinical outcome, by studying a control group ( n = 26) and unsplenectomized HS patients ( n = 82) presenting mild, moderate or severe HS. We performed a basic hematologic study and evaluated the reticulocyte count, bilirubin, erythropoietin (EPO) and soluble transferrin receptor (sTfR) levels; the osmotic fragility (OFT) and criohemolysis tests (CHT); the ratios Hb/MCHC (mean cell hemoglobin concentration), Hb/RDW (red cell distribution width) and MCHC/RDW, were calculated. Hb changed significantly in accordance with HS severity, but not reticulocytes or bilirubin. We found that MCHC, RDW, EPO, sTfR, OFT, CHT and the calculated ratios were significantly changed in patients, and, therefore, were valuable as complementary diagnostic tools for HS. Moreover, RDW, Hb/MCHC, Hb/RDW and MCHC/RDW changed significantly with worsening of HS; thus, they are also good markers for the clinical outcome of HS. In conclusion, we propose the use of these routine parameters as useful to complement the analysis of HS severity.

Medical and Quality of Life Indications for Splenectomy in Children with Hereditary Spherocytosis.

Abstract 5082 BackgroundSplenectomy has been considered the treatment of choice for patients with “symptomatic” hereditary spherocytosis (HS). However, given the long-term risks associated with asplenia, including sepsis and thrombosis, the risks and benefits of splenectomy in HS require ongoing analysis. To our knowledge, no comprehensive institutional review summarizing the medical and quality of life indications for splenectomy in children with HS has been reported to date. MethodsA retrospective record review was performed of 64 children, age ' 18 yrs, having splenectomy for HS at Children's Medical Center Dallas from January 1973 through March 2009. The following patient characteristics were abstracted: age at splenectomy, family history of HS and/or splenectomy, baseline hemoglobin concentration and reticulocyte count prior to splenectomy, degree of splenomegaly, total number of prior transfusions, hyperhemolytic events, and aplastic crisis. Patients were classified based on mean hemoglobin concentration as having mild (≥11.0 g/dL), moderate (8.0-10.9 g/dL), or severe HS ('7.9 g/dL) (Bolton-Maggs PH, et al. Br J Haematol 2004;126:455-74). The primary indication for splenectomy was characterized in each case as either a “quality of life (QOL) indication” or a “medical indication.” QOL indicators included jaundice, pallor, maxillary hyperplasia, poor growth, fatigue, exercise intolerance, and family history of splenectomy for HS which, having been deemed successful, fostered parental preference for splenectomy in their child. Medical indicators included baseline hemoglobin approaching transfusion-dependent threshold, multiple or severe hyperhemolytic events, severe aplastic crisis, symptomatic gallbladder disease, marked splenomegaly, and the need for numerous transfusions. Some patients had both medical and QOL indications but only one was designated as the primary indication for splenectomy based on documentation in the medical record. ResultsWhen classifying HS patients by hemoglobin concentration, 16 patients (25%) were mild, 38 (59%) moderate, and 10 (16%) severe. The following table summarizes the patient characteristics.Patient CharacteristicsMild HS (n=16)Moderate HS (n=38)Severe HS (n=10)Median age at splenectomy, yr11.410.34.7Family history of HS, n (%)16 (100%)25 (65.8%)6 (60%)Family history of splenectomy, n (%)14 (87.5%)18 (47.4%)5 (50%)Pre-splenectomy: Hemoglobin level (g/dL), median (range)11.6 (11.0-13.7)9.7 (8.2-10.9)7.4 (5.0-7.9)Reticulocyte count (%), median (range)9.8 (3.9-28.7)11.7 (1.1-21.1)12.4 (6.5-25.3)Splenomegaly (cm), median (range)3.5 (0-7.0)4.0 (0-12.0)4.7 (2.0-8.0)Despite having just mild HS, 16 patients had splenectomy, 6 for QOL indications. Moreover, 11 of 38 moderate HS patients had splenectomy for a primary QOL indication. Medical indications predominated in all 10 patients with severe HS. Of the 17 patients with QOL indications who had splenectomy, 8 (47%) also had documentation of medical indications. Among 45 patients with primary medical indications for splenectomy, 18 (40%) also had QOL issues contributing to the decision. Two patients' indications for splenectomy were unclear. ConclusionsAlthough splenectomy is generally felt to be unnecessary in patients with mild HS and many with moderate HS as well, our experience demonstrates that splenectomy is often performed primarily for QOL indications in both risk categories. A validated assessment tool that incorporates QOL indicators in addition to the traditional medical reasons for splenectomy in patients with HS would be of great value in an evidence-based assessment of the patient's need for splenectomy. We recommend inclusion of QOL indicators when considering splenectomy in patients with mild and moderate HS. DisclosuresNo relevant conflicts of interest to declare.

Erythropoietic Stimuli and Response in Hereditary Spherocytosis

Hereditary Spherocytosis (HS) is the most common non-immune hemolytic anemia in Europe. HS ranges from assymptomatic to transfusion-dependent hemolytic anemia and is clinically classified as mild, moderate or severe. Clinical, biochemical and molecular heterogeneous patterns are HS trademarks. In a previous work, we reported that in HS patients the erythropoietic response appears to be impaired in the more severe cases of the disease, since erythropoietin (EPO) levels did not correlate with reticulocyte count or with reticulocyte production index (RPI) [Rocha S. et al. (2005) Br J Haematol. 131(4):534]. It is known that spherocytes are removed by splenic macrophages. The activation of these cells is associated with cytokine release, namely, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), both known as EPO inhibitors. The aim of our work was to evaluate the erythropoietic stimuli and response according to HS severity and to clarify the role of these cytokines in HS. We studied 31 HS patients and 25 healthy individuals as the control group. The HS group included only unsplenectomised patients classified as presenting mild (n=19), moderate (n=9) or severe HS (n=3) according to the Guidelines for the diagnosis and management of hereditary spherocytosis [Bolton-Maggs et al. (2004) Br J Haematol. 126(4):455]. The basic hematological study was performed in all samples, as well as the reticulocyte count and RPI calculation; the plasma levels of EPO and soluble transferrin receptor (sTfR) (as markers of erythropoietic stimuli), of bilirubin (as a marker of hemolysis) and of TNF-α and IFN-γ (known as EPO inhibitors) were also determined. Data was analysed according to the severity of HS and versus the control group. Erythrocyte count, hemoglobin concentration, hematocrit and mean cell volume were found to be significantly lower in patients than in controls and their values decreased with HS severity; for MCHC, RDW, reticulocytes, RPI, bilirubin, EPO and sTfR patients presented significantly higher values than controls, and their values increased with HS severity. However, the increment in EPO and sTfR levels was more marked than for reticulocytes and RPI. As for TNF-α and IFN-γ no differences were found between patients and controls or between HS severities; no correlations with the other studied variables were observed. In mild HS patients, sTfR was found to be statistically and positively correlated with reticulocytes (r=0.783; p<0.001), RPI (r=0.748; p<0.001) and bilirubin (r=0.870; p<0.001); in patients with more severe forms (moderate and severe HS) these correlations were lower and only a statistically and positively correlation between sTfR and reticulocytes (r=0.657; p<0.05) was found. Our results confirm the inadequate erythropoietic response in the more severe cases of HS, given that, the erythropoietic stimuli (shown by higher EPO, sTfR and indirectly, by bilirubin levels) was not able to trigger an adequate erythropoietic response as shown by the reticulocyte number and RPI, as occured in mild HS patients. Our hypothesis, that this disturbance could be due to an increase in TNF-α and IFN-γ, was not confirmed, as we did not found any significant increase in these cytokines that could explain the non adequate erythropoietic response in moderate/severe HS patients. In conclusion, the erythropoietic stimuli seem to increase with HS severity; however, the response to that stimulus appears to be impaired in the more severe cases of HS. Further studies are needed to clarify this erythropoietic disturbance in HS patients, which may contribute to the severity of the clinical presentation of the disease.

Coinheritance of Gilbert Syndrome Increases the Risk for Developing Gallstones in Patients With Hereditary Spherocytosis: Presented in part at the 40th Annual Meeting of the American Society of Hematology, Miami Beach, FL, December 4-8, 1998 and published in Blood 92:470a, 1998 (abstr, suppl 1) .

The precocious formation of bilirubinate gallstones is the most common complication of hereditary spherocytosis (HS), and the prevention of this problem represents a major impetus for splenectomy in many patients with compensated hemolysis. Because Gilbert syndrome has been considered a risk factor for gallstone formation, there are reasons for postulating that the association of this common inherited disorder of hepatic bilirubin metabolism with HS could increase cholelithiasis. To test this hypothesis, 103 children with mild to moderate HS who, from age 1, have undergone a liver and biliary tree ultrasonography every year, were retrospectively examined. The 2-bp (TA) insertion within the promoter of the uridine diphosphate-glucuronosyltransferase gene (UGT1A1), associated with Gilbert syndrome, was screened. The risk of developing gallstones was statistically different among the 3 groups of patients: homozygotes for the normal UGT1A1 allele, heterozygotes, and homozygotes for the allele with the TA insertion. Fitting a Cox regression model, in fact, a statistically significant hazard ratio of 2.19 (95% confidence interval: 1.31 to 3.66) was estimated from one to the next of these genetic classes. The individual proneness to form gallstones from TA insertion in the TATA-box of the UGT1A1 promoter should be considered during the follow-up of patients with HS. Although patients with HS were the only ones studied, extrapolating these data to patients who have different forms of inherited (eg, thalassemia, intraerythrocytic enzymatic deficiency) or acquired (eg, autoimmune hemolytic anemia, hemolysis from mechanical heart valve replacement) chronic hemolysis can be warranted.

Red blood cell membrane disorders.

The recent discovery of the specific molecular defects in many patients with hereditary spherocytosis and hereditary elliptocytosis/pyropoikilocytosis partially clarifies the molecular pathology of these diseases. HE and HPP are caused by defects in the horizontal interactions that hold the membrane skeleton together, particularly the critical spectrin self-association reaction. Single gene defects cause red cells to elongate as they circulate, by a unknown mechanism, and are clinically harmless. The combination of two defective genes or one severe alpha spectrin defect and a thalassaemia-like defect in the opposite allele (alphaLELY) results in fragile cells that fragment into bizarre shapes in the circulation, with haemolysis and sometimes life-threatening anaemia. A few of the alpha spectrin defects are common, suggesting they provide an advantage against malaria or some other threat. HS, in contrast, is nearly always caused by family-specific private mutations. These involve the five proteins that link the membrane skeleton to the overlying lipid bilayer: alpha and beta spectrin, ankyrin, band 3 and protein 4.2. Somehow, perhaps through loss of the anchorage band 3 provides its lipid neighbours (Peters et al, 1996), microvesiculation of the membrane surface ensues, leading to spherocytosis, splenic sequestration and haemolysis. Future research will need to focus on how each type of defect causes its associated disease, how the spleen aggravates membrane skeleton defects (a process termed 'conditioning'), how defective red, cells are recognized and removed in the spleen, and why patients with similar or even identical defects can have different clinical severity. Emphasis also needs to be given to improving diagnostic tests, particularly for HS, and exploring new options for therapy, like partial splenectomy, which can ameliorate symptoms while better protecting patients from bacterial sepsis and red cell parasites, and perhaps from atherosclerosis (Robinette & Franmeni, 1977) and venous thrombosis (Stewart et al, 1996).

Hereditary spherocytosis with band 3 deficiency. Association with a nonsense mutation of the band 3 gene (allele Lyon), and aggravation by a low-expression allele occurring in trans (allele Genas)

We describe an 18-year-old with moderate hereditary spherocytosis. The condition was associated with a 35% decrease in band 3. The underlying mutation was Arg to stop at codon 150 (CGA--&gt;TGA) and was designated R150X, which defined allele Lyon of the EPB3 gene. The inheritance pattern was dominant. However, the mother, who also carried the allele Lyon, had a milder clinical presentation and only a 16% decrease of band 3. We suggested that the father had transmitted a modifying mutation that remained silent in the heterozygous state. Nucleotide sequencing after single strand conformation polymorphism analysis of the band 3 cDNA and promoter region revealed a G--&gt;A substitution at position 89 from the cap site in the 5′-untranslated region, designated 89G--&gt;A, which defined allele Genas. A ribonuclease protection assay showed that (1) the allele Genas (father) resulted in a 33% decrease in the amount of band 3 mRNA, (2) the reduction caused by the allele Lyon (mother) was 42%, and (3) the compound heterozygous state for both alleles (proband) resulted in a 58% decrease. These results suggest that some mildly deleterious alleles of the EPB3 gene are compensated for by the normal allele in the heterozygous state. They are shown through the aggravation of the clinical picture, based on more obvious molecular alterations when they occur in trans to an allele causing a manifest reduction of band 3 membrane protein concentration.

Hereditary Spherocytosis With Band 3 Deficiency. Association With a Nonsense Mutation of the Band 3 Gene (Allele Lyon), and Aggravation by a Low-Expression Allele Occurring in trans (Allele Genas)

We describe an 18-year-old with moderate hereditary spherocytosis. The condition was associated with a 35% decrease in band 3. The underlying mutation was Arg to stop at codon 150 (CGA-->TGA) and was designated R150X, which defined allele Lyon of the EPB3 gene. The inheritance pattern was dominant. However, the mother, who also carried the allele Lyon, had a milder clinical presentation and only a 16% decrease of band 3. We suggested that the father had transmitted a modifying mutation that remained silent in the heterozygous state. Nucleotide sequencing after single strand conformation polymorphism analysis of the band 3 cDNA and promoter region revealed a G-->A substitution at position 89 from the cap site in the 5'-untranslated region, designated 89G-->A, which defined allele Genas. A ribonuclease protection assay showed that (1) the allele Genas (father) resulted in a 33% decrease in the amount of band 3 mRNA, (2) the reduction caused by the allele Lyon (mother) was 42%, and (3) the compound heterozygous state for both alleles (proband) resulted in a 58% decrease. These results suggest that some mildly deleterious alleles of the EPB3 gene are compensated for by the normal allele in the heterozygous state. They are shown through the aggravation of the clinical picture, based on more obvious molecular alterations when they occur in trans to an allele causing a manifest reduction of band 3 membrane protein concentration.