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Splenectomized patients are exposed to an increased risk of septicemia caused by encapsulated bacteria. Defense against infection is ensured by preformed serum antibodies produced by long-lived plasma cells and by memory B cells that secrete immunoglobulin in response to specific antigenic stimuli. Studying a group of asplenic individuals (57 adults and 21 children) without additional immunologic defects, we found that spleen removal does not alter serum anti-pneumococcal polysaccharide (PnPS) IgG concentration, but reduces the number of PnPS-specific memory B cells, of both IgM and IgG isotypes. The number of specific memory B cells was low in splenectomized adults and children that had received the PnPS vaccine either before or after splenectomy. Seven children were given the 13-valent pneumococcal conjugated vaccine after splenectomy. In this group, the number of PnPS-specific IgG memory B cells was similar to that of eusplenic children, suggesting that pneumococcal conjugated vaccine administered after splenectomy is able to restore the pool of anti-PnPS IgG memory B cells. Our data further elucidate the crucial role of the spleen in the immunological response to infections caused by encapsulated bacteria and suggest that glycoconjugated vaccines may be the most suitable choice to generate IgG-mediated protection in these patients.

Congenital dyserythropoietic anaemia type II (CDA II) is the most common congenital dyserythropoietic anaemia. CDA II is frequently misdiagnosed as Hereditary Spherocytosis (HS) due to the presence of mild chronic haemolytic anaemia with splenomegaly, increased osmotic fragility, and presence of microspherocytes. Accurate diagnosis of CDA II is important to prevent severe iron overload. Erythrocyte and reticulocyte indices were assessed in 10 patients from six families with CDA II, 18 patients from eight families with HS, and 50 normal controls. Characteristic increases in distribution width were present in CDA II for cell volume (RDW, anisocytosis) and in HS for cell haemoglobin concentration (HDW, anisochromia), resulting in an RDW/HDW ratio which was significantly greater in CDA than HS (P < 0.0002). A cut-off value for RDW/HDW of 5.34 resulted in 89% sensitivity and 70% specificity in distinguishing CDA II from HS. Distribution width for cell haemoglobin content of reticulocytes (CHDWr) was characteristically increased in CDA II, resulting in a CHDW/CHDWr ratio significantly lower in CDA II than HS (P < 0.0002). A cut-off value of 0.98 provided 89% sensitivity and 80% specificity in distinguishing CDA II from HS. These differences in distribution widths of flow-cytometric parameters of reticulocytes and mature erythrocytes reflect the different pathogeneses of the two diseases and are helpful for the differential diagnosis of these two conditions.

Diamond-Blackfan anemia (DBA) is a congenital pure red blood cell aplasia that often requires lifelong transfusional therapy. Autosomal dominant and recessive inheritance have both been reported, suggesting genetic heterogeneity, but most cases occur sporadically. The origin of impaired erythropoiesis is unknown. Several erythroid growth factors have been thought to have a role in the pathogenesis of DBA. However, there is neither molecular nor clinical evidence for the involvement of erythropoietin (EPO), its receptor, stem cell factor (SCF), or interleukin (IL)-3, even if the addition of SCF to IL-3 and EPO does significantly increase the growth of erythroid progenitors in in vitro cultures in most patients. In this work we evaluated the possible role of another early-acting erythroid growth factor, IL-9. We found that the addition of IL-9 to SCF, IL-3, and EPO further increases burst-forming unit-erythroid growth in in vitro cultures of those DBA patients who responded to SCF. To investigate the role of the IL-9 gene, we evaluated its segregation in 22 families with members who have DBA by using a polymorphic microsatellite located within its intron 4. Lod score analysis ruled out any statistically significant involvement of the IL-9 gene in the pathogenesis of DBA. Moreover, linkage analysis with 11 highly polymorphic markers spanning 5q31.1-q33.2 excluded this region, which is included in the major cluster of genes active in hematopoiesis of the human genome.

We sought to correlate hepcidin levels in inflammatory bowel disease [IBD] children with disease activity, inflammatory markers, and iron load test [ILT] and to compare IBD patients with coeliac and healthy patients.Between December 2012 and June 2013, 145 subjects [50 IBD patients, 45 coeliac patients and 50 healthy controls] were included in the study. All patients underwent the following examinations: blood count, iron status, erythropoiesis parameters, serum hepcidin, C-reactive protein [CRP], and erythrocyte sedimentation rate [ESR]. In order to evaluate the efficacy of iron absorption, ILT was performed in IBD patients. Disease activity indexes and IBD duration, localisation, and therapy were also evaluated, and a faecal sample for calprotectin collected.Serum hepcidin was significantly higher in IBD patients with active disease compared with both coeliac and healthy patients [p = 0.005, p = 0.003 respectively]. In a multivariate logistic regression model, having a Paediatric Crohn's Disease Activity Index [PCDAI] / Paediatric Ulcerative Colitis Activity Index [PUCAI] ≥ 30 resulted in the only variable independently associated with a positive serum hepcidin (odds ratio [OR] = 6.87; 95% confidence interval [CI] 1.4-33, p = 0.01]]. Patients with iron malabsorption [IM] showed higher values of ESR, CRP, and hepcidin [p = 0.02, p = 0.001, and p = 0.06, respectively]. Eight out of 12 [66.7%] children with IM showed an active disease compared with 6/31 [19.3%] children with normal ILT [p = 0.01]. Hepcidin levels correlated negatively with ILT [r = -0.451, p = 0.002], and positively with ferritin and CRP [r = 0.442, p = 0.0001; r = 0.243, p = 0.009, respectively]Our study demonstrates that serum hepcidin is increased in IBD children with active disease and it is responsible for IM.

Background Iron deficiency anemia in celiac disease is related to impaired duodenal mucosal uptake, due to villous atrophy. Iron enters the enterocytes through an apical divalent metal transporter, DMT1. Different DMT1 transcripts have been identified, depending on the presence of an iron-responsive element that allows DMT1 up-regulation during iron starvation. An intronic DMT1 polymorphism, IVS4+44C>A, has been associated with metal toxicity, and the CC-carriers show high iron levels. Aims This study investigates the association between DMT1 IVS4+44C>A and anemia in a cohort of 387 Italian celiac children, and the functional role of the polymorphism. Methods and results By association analysis, we found that DMT1 IVS4+44-AA genotype confers a four-fold risk of developing anemia, despite of atrophy degree. By analysis of mRNA from gastroesophageal biopsies, we found that total DMT1 is significantly upregulated in presence of mild, but not severe, atrophy, independently from IVS4+44C>A variant, and in normal but not in atrophic CC-biopsies. Moreover, we found that A-allele is associated to preferential expression of the DMT1 transcripts lacking the iron-responsive element, thus limiting the DMT1 overexpression that normally occurs to respond to iron starvation. Discussion Possibly, the IVS4+44-AA-related dysregulation of the iron-induced changes in DMT1 expression is not able to impair iron absorption in physiological condition. However, if exacerbated by the concomitant massive loss of functional absorbing tissue paralleling worsened stages of villus atrophy, it might be ineffective in counteracting iron deficiency, despite of DMT1 overexpression. Conclusion We suggest, for the first time, that celiac disease may unmask the contribution of the DMT1 IVS4+44C>A polymorphism to the risk of anemia.

Moyamoya syndrome (MMS) is the most common cerebral vasculopathy among children with neurofibromatosis type 1 (NF1). In this study, we clinically, radiologically, and genetically examined a cohort that was not previously described, comprising European children with NF1 and MMS. The NF1 genotyping had been registered. This study included 18 children. The mean age was 2.93 ± 3.03 years at the NF1 diagnosis and 7.43 ± 4.27 years at the MMS diagnosis. In seven patients, MMS was diagnosed before or at the same time as NF1. Neuroimaging was performed in 10 patients due to clinical symptoms, including headache (n = 6), cerebral infarction (n = 2), and complex partial seizures (n = 2). The remaining eight children (47%) had MMS diagnosed incidentally. Sixteen children were characterized molecularly. The features of MMS were similar between patients with and without NF1. Additionally, the NF1 phenotype and genotype were similar between children with and without MMS. Interestingly, three children experienced tumors with malignant histology or behavior. The presence of two first cousins in our cohort suggested that there may be potential genetic factors, not linked to NF1, with an additional role respect of NF1 might play a role in MMS pathogenesis. The incidental diagnosis of MMS, and the observation that, among children with NF1, those with MMS were clinically indistinguishable from those without MMS, suggested that it might be worthwhile to add an angiographic sequence to brain MRIs requested for children with NF1. A MMS diagnosis may assist in properly addressing an NF1 diagnosis in very young children who do not fulfill diagnostic criteria.

This retrospective study was conducted to determine whether the response to splenectomy is related to the response to previous treatments. We examined the records of 90 children splenectomized for chronic ITP. Platelet counts were constantly>50x10(9)/L in 68 patients (75%). An improvement in the quality of life was observed in 79 (85%). The success of splenectomy was strongly correlated with a good response to previous treatment. A negative response to any of the prior treatments had no predictive value. This finding is relevant when elective splenectomy is considered as a treatment option.

Background The ovary is an important site where gene variants modulate pubertal timing. The cannabinoid receptor 2 (CB2) is expressed in the ovary, plays a role in folliculogenesis and ovulation, and can be modulated by estrogens. Obesity is strictly associated with early menarche and is characterized by sex hormone and endocannabinoid derangement. Aim In this study, we investigated the role of the CB2 receptor in determining the age at menarche in obese girls. Methods We studied a cohort of 240 obese girls (age 11.9±3 years; BMI z-score 2.8±0.8). The age at menarche (if it had already occurred) was recorded at the time of the visit or via phonecall. The CNR2 rs35761398 polymorphism, which leads to the CB2 Q63R variant, was detected by the TaqMan assay. Results In total, 105 patients were homozygous for the R63-coding allele (RR), 113 were QR and 22 were QQ. Variance analysis revealed a significantly earlier age of menarche in subjects carrying the Q63 allele, which was also found after adjusting for BMI z-score (11±1.2 vs. 11.6±1.2 years, p = 0.0003). Logistic regression analysis demonstrated that patients homozygous for the Q allele had a 2.2-fold higher risk (odds ratio = 2.2; CI1.1-3.4; p = 0.02) of presenting with an early menarche (age at menarche

Objective: To determine the efficacy of high-dose dexamethasone in chronic idiopathic thrombocytopenic purpura of childhood. Methods: Seventeen patients entered the protocol. Dexamethasone was to be given orally in two divided doses at a dosage of 20 mg/m 2 for 4 consecutive days every 28 days for six courses. Results: One month after the end of the sixth course, six patients (35%) had platelet values within the normal range. One year later, five patients (29%) still have normal platelet values. Five patients discontinued treatment before completion because of lack of response and in one case for important side effects. Duration of the disease before treatment was inversely correlated with response to dexamethasone: 5 of 10 patients who had had thrombocytopenia for 30 months or less went into remission, as opposed to none of the seven who had been sick for a longer period ( p = 0.04). Side effects included fatigue or irritability, anxiety, abdominal pain, striae, hirsutism, acne, and weight gain. Conclusions: Contrary to what is observed in adults, in our patients pulsed dexamethasone therapy did not prove to be uniformly effective. However, in view of its effectiveness in a third of the patients, acceptable side effects, and low cost, we believe that this treatment could be considered in patients with chronic idiopathic thrombocytopenic purpura who do not tolerate the disease well, especially if no more than 3 years have elapsed since diagnosis. Larger studies will be necessary to define which patients will respond to this type of therapy.

Hypoxia-inducible factor 2α (HIF-2α) plays a pivotal role in the balancing of oxygen requirements throughout the body. The protein is a transcription factor that modulates the expression of a wide array of genes and, in turn, controls several key processes including energy metabolism, erythropoiesis and angiogenesis. We describe here the identification of two cases of familial erythrocytosis associated with heterozygous HIF2A missense mutations, namely Ile533Val and Gly537Arg. Ile533Val is a novel mutation and represents the genetic HIF2A change nearest to Pro-531, the primary hydroxyl acceptor residue, so far identified. The Gly537Arg missense mutation has already been described in familial erythrocytosis. However, our patient is the only described case of a de novo HIF2A mutation associated with the development of congenital polycythemia. Functional in vivo studies, based on exogenous expression of hybrid HIF-2α transcription factors, indicated that these genetic alterations lead to the stabilization of HIF-2α protein. All the identified polycythemic subjects with HIF2A mutations show serum erythropoietin in the normal range, independently of the hematocrit values and phlebotomy frequency. The erythroid precursors obtained from the peripheral blood of patients showed an altered phenotype, including an increased rate of growth and a modified expression of some HIF-2α target genes. These results suggest the novel proposal that polycythemia observed in subjects with HIF2A mutations might also be due to primary changes in hematopoietic cells and not only secondary to increased erythropoietin levels. © 2013 Ferrata Storti Foundation.

Celiac disease (CD) is a chronic inflammatory disease of the small bowel that occurs with the ingestion of gluten, found in several grains products. Although HLA-DQ2 variant is required for the gluten-derived peptide gliadin presentation by antigen-presenting cells to T-cells, non-HLA genetic factors account for the majority of heritable risk. Several genome-wide association studies have identified susceptibility loci for CD on chromosome 1. Cells of the immune system express the Cannabinoid Receptor type 2 (CB2), a plasma-membrane receptor activated by both endogenous and exogenous cannabinoids. Consistent data evidence that CB2 is linked to a variety of immune functional events and that, in the course of an inflammatory process, an increased number of receptors becomes available for activation. The Cannabinoid Receptor type 2 gene (CNR2; GeneID1269) maps on 1p36.11. In order to investigate the possible involvement of CB2 in CD establishment, immunohistochemistry toward CB2 receptor and CD4+ cells in small bowel biopsies from celiac children and association analysis, through TaqMan assay, of a CNR2 common missense variant, rs35761398 (CAA/CGG), resulting in the aminoacidic substitution of Glutamine at codon 63 with Arginine (Q63R), in a cohort of 327 South Italian children have been performed. We observed in this study that CB2 is up-regulated in CD small bowel biopsies and CNR2 rs35761398 is significantly associated with CD (χ 2 = 37.064; d.f. 1; p = 1.14 × 10 -9). Our findings suggest a role of CB2 in CD. The Q63R variant, increasing more than six-fold the risk for CD susceptibility, might eventually represent a novel molecular biomarker for CD risk stratification. Indeed, we provide here further evidence that CB2 receptor plays a critical role in autoimmunity susceptibility and indicates that it represents a molecular target to pharmacologically modulate the immune components in CD. © 2012 Elsevier Ltd.

Background/Objective: The management of chronic childhood idiopathic thrombocytopenic purpura (ITP) is distinct from acute ITP. Similar to the publication on acute ITP guidelines, the AIEOP (Associazione Italiana di Ematologia e Oncologia Pediatrica) considered it appropriate to develop consensus guidelines for chronic childhood ITP to provide useful and shared information for physicians, healthcare professionals, parents and patients. Design/Methods: A preliminary, evidence-based document issued by a select group of AIEOP pediatric hematologists was discussed, modified and approved during a Consensus Conference according to procedures previously validated by the AIEOP Board. Results: The guidelines give prominence to the periodical reevaluation of all the etiological hypotheses of thrombocytopenia in relation to its clinical condition. The majority of chronic ITP children do not require treatment, especially if bleeding is absent or minimal. The treatment decision depends on several factors other than the platelet count, and treatment options are suggested in relation to the therapeutic scenarios. Recommendations are given regarding support for surgery, particular hemorrhagic conditions, daily activities/sports, as well as for vaccines and drugs. Experimental treatments are also discussed.

We report the long-term follow-up (median 39.5 months) of 49 paediatric patients (33 females and 16 males) with refractory symptomatic immune thrombocytopenic purpura (ITP) treated with rituximab. The overall response rate was 69% (34/49 patients). Twenty-one responders had a platelet count >50 x 10(9)/l at a median 20.2 months from treatment. Kaplan-Meier analysis showed a probability of relapse-free survival (RFS) of 60% at 36 months from the first rituximab infusion. The number of infusions and a previous splenectomy did not influence overall response rate. Patients who achieved complete response were significantly older at diagnosis and first rituximab infusion than partial responders (P = 0.027). Older children displayed a significantly greater probability of sustained response (RFS) at 36 months than younger children (88.9% vs. 56.7%, P = 0.037). Earlier responses (within 20 d from treatment) were significantly associated with both complete (P = 0.004) and sustained response (P = 0.002). Only mild and transient side-effects were observed in 9/49 children; no major infections nor delayed toxicities were recorded during the follow-up.

The SLIT2 receptor ROBO2 plays a key role in the formation of the ureteric bud, and its inactivation in mice leads to supernumerary ureteric bud development, lack of ureter remodeling, and improper insertion of the ureters into the bladder. Recently, two heterozygous ROBO2 missense mutations were identified in two families with primary vesicoureteral reflux occurring in combination with congenital anomalies of the kidney and urinary tract (VUR/CAKUT). This study investigated a possible causal role of ROBO2 gene variants in 95 unrelated patients with primary VUR (n = 78) or VUR/CAKUT. Eighty-two percent of all patients had a family history of genitourinary anomalies. Twenty-four ROBO2 gene variants were identified by direct sequencing of all 26 exons and the exon-intron boundaries. Of these, four led to amino acid substitutions: Gly328Ser, Asn515Ile, Asp766Gly, and Arg797Gln. When the families were examined, the missense variants co-segregated with VUR (three families) or VUR/CAKUT (one family). These variants were not found in 190 control subjects, and the affected amino acids have been conserved through evolution. In conclusion, a relatively high frequency of ROBO2 variants (5.1%) was found in familial cases; however, functional studies and validation in other cohorts are warranted.

Background and Objectives: Hematopoietic stem cell transplantation (HSCT) still represents the only treatment potentially able to prevent/rescue the development of marrow failure and myeloid malignancies in patients with Fanconi anemia (FA). While in the past HSCT from an HLA-identical sibling was proven to cure many patients, a higher incidence of treatment failure has been reported in recipients of an unrelated donor (UD) or HLA-partially matched related allograft. Design and Methods: We analyzed the outcome of 64 FA patients (age range, 2-20 years) who underwent HSCT between January 1989 and December 2005. Patients were transplanted from either an HLA-identical sibling (n=31), an UD (n=26), or an HLA-partially matched relative (n=7). T-cell depletion of the graft was performed in patients transplanted from an HLA-disparate relative. Results: The 8-year estimate of overall survival (OS) for the whole cohort was 67%; it was 87%, 40% and 69% when the donor was an HLA-identical sibling, an UD and a mismatched relative, respectively (p

This retrospective study investigated the effects of rituximab in 19 pediatric patients (15 girls and 4 boys) with chronic refractory symptomatic immune thrombocytopenic purpura (ITP). Patients received from 2 to 5 weekly infusions of rituximab (375 mg/m(2)); 15 patients were younger than 12 years when treated. The median follow-up time was 30 months (range, 9-43 months). The overall response rate was 68% (13/19 patients). Six responders relapsed at a median of 4.5 months (range, 3-8 months). Seven patients still displayed a platelet count >150,000/microL at a median of 33 months (range, 14-43 months) after rituximab treatment. Six of 15 patients treated with 4 or 5 weekly infusions and 1 of 4 patients treated with 2 or 3 infusions are still in remission. No difference was detected between splenectomized and nonsplenectomized patients. The duration of ITP disease at the time of treatment did not influence the response rate. Patients still in remission showed significantly lower levels of CD19+ cells after 4 and 6 months than nonresponding or relapsed patients (P < .05). No major infections were reported during follow-up. Our data show the efficacy and tolerability of rituximab in young children with refractory symptomatic ITP. Nonrelapsed patients showed a more prolonged B-cell depletion.

Some selected oxidative stress parameters were measured in 56 Fanconi anaemia (FA) patients (42 untransplanted and 14 transplanted), 54 FA heterozygotes (parents) and 173 controls. Untransplanted FA patients showed a highly significant increase in leukocyte 8-hydroxy-2’-deoxyguanosine (8-OHdG) (p = 0.00003) and a borderline increase (p = 0.076) in urinary levels of 8-OHdG vs. child controls. These increases were more pronounced in female FA patients (p = 0.00005 for leukocyte 8-OHdG, and p = 0.021 for urinary 8-OHdG). Female FA patients also displayed a highly significant excess of spontaneous chromosomal breaks vs. male patients (p = 0.00026), in the same female:male ratio (≅ 1.4) as detected both for leukocyte and for urine 8-OHdG levels. Plasma methylglyoxal (MGlx) levels were increased in untransplanted FA patients vs. child controls (p = 0.032). The increases in leukocyte and urinary 8-OHdG, and in MGlx levels were detected in young FA patients (≤15 yrs), whereas patients aged 16 to 29 yrs failed to display any differences vs. controls in the same age group. A significant increase in oxidised:reduced glutathione (GSSG:GSH) ratio was observed (p = 0.046) in the FA patients aged ≤15 yrs, whereas those aged 16 to 29 yrs, both untransplanted and transplanted, displayed a decrease (p = 0.06) in the GSSG:GSH ratio vs. the controls of the respective age groups. No significant changes were detected in plasma levels of Vitamin C, Vitamin E, or uric acid. Transplanted FA patients showed lesser alterations in leukocyte 8-OHdG and in GSSG:GSH ratio vs. untransplanted patients. The parents of FA patients displayed a significant increase in plasma MGlx levels (p = 0.0014) vs. adult controls. The results suggest a gender- and age-related modulation of oxidative stress in FA patients. The observed increase in urinary 8-OHdG in untransplanted FA patients suggests a proficient removal of oxidised DNA bases. Key words: Fanconi anaemia; 8-hydroxy-2’-deoxyguanosine; methylglyoxal; glutathione; chromosomal instability

Vitamin A is a very intriguing natural compound. The molecule not only has a complex array of physiological functions, but also represents the precursor of promising and powerful new pharmacological agents. Although several aspects of human retinol metabolism, including absorption and tissue delivery, have been clarified, the type and amounts of vitamin A derivatives that are intracellularly produced remain quite elusive. In addition, their precise function and targets still need to be identified. Retinoic acids, undoubtedly, play a major role in explaining activities of retinol, but, recently, a large number of physiological functions have been attributed to different retinoids and to vitamin A itself. One of the primary roles this vitamin plays is in embryogenesis. Almost all steps in organogenesis are controlled by retinoic acids, thus suggesting that retinol is necessary for proper development of embryonic tissues. These considerations point to the dramatic importance of a sufficient intake of vitamin A and explain the consequences if intake of retinol is deficient. However, hypervitaminosis A also has a number of remarkable negative consequences, which, in same cases, could be fatal. Thus, the use of large doses of retinol in the treatment of some human diseases and the use of megavitamin therapy for certain chronic disorders as well as the growing tendency toward vitamin faddism should alert physicians to the possibility of vitamin overdose.

Autoimmune hemolytic anemia (AIHA) in children is sometimes characterized by a severe course, requiring prolonged administration of immunosuppressive therapy. Rituximab is able to cause selective in vivo destruction of B lymphocytes, with abrogation of antibody production. In a prospective study, we have evaluated the use of rituximab for the treatment of AIHA resistant to conventional treatment. Fifteen children with AIHA were given rituximab, 375 mg/m(2)/dose for a median of 3 weekly doses. All patients had previously received 2 or more courses of immunosuppressive therapy; 2 patients had undergone splenectomy. After completing treatment, all children received intravenous immunoglobulin for 6 months. Treatment was well tolerated. With a median follow-up of 13 months, 13 patients (87%) responded, whereas 2 patients did not show any improvement. Median hemoglobin levels increased from 7.7 g/dL to a 2-month posttreatment level of 11.8 g/dL (P

UNLABELLED: Obesity is a typical example of a complex multifactorial disease arising from behavioural, environmental and genetic factors that may affect individual responses to dietary intake and physical activity. Observational, longitudinal dietary interventional studies in obese patients present contrasting reports on the predictive value of baseline leptin levels. We report on the effect of a weight reduction programme in three different groups of obese children (82 patients in all) assembled on the basis of their baseline leptin levels adjusted for body mass index (BMI), gender and pubertal development. The effectiveness of this programme was decreased in patients with relative hyperleptinaemia or hypoleptinaemia compared to children with baseline leptin levels appropriate to BMI gender and pubertal development. CONCLUSION: Information gained from leptin assays could provide predictive insight into an individual's ability to lose body fat and may therefore have important implications for our approach to the treatment and prevention of childhood obesity.

Innovative approaches are needed for patients with systemic lupus erythematosus (SLE) who develop autoimmune haemolytic anaemia (AIHA) that does not respond to conventional treatment. Rituximab, a chimaeric anti-CD20 monoclonal antibody, has been demonstrated to be highly effective for in vivo B-cell depletion. We report an 18-year-old-girl with SLE and life-threatening AIHA that did not respond to steroids, intravenous immunoglobulin and cyclosporin A. Rituximab was given weekly at 375 mg/m2 for two doses. The drug was well tolerated and the patient had no adverse effects. Her haemolytic disorder markedly ameliorated, with a progressive increase of haemoglobin levels, starting a few days after therapy. The patient remains disease-free 7 months later.

About 75% of hereditary spherocytosis (HS) patients have the autosomal dominant form of the disease, whereas both parents of the remaining HS patients are clinically and haematologically normal. These patients could have either the autosomal recessive form of the disease or a de novo mutation. We studied 80 randomly chosen, Italian HS children with normal parents. They had different clinical phenotypes (16 mild, 40 moderate, 16 moderately severe and eight severe). These patients were screened for the occurrence of ankyrin or beta-spectrin de novo mutations. To search for ankyrin de novo mutations affecting mRNA accumulation, we studied a (AC)(n) microsatellite located in the non-coding sequence of the last exon of the ankyrin gene, and four different exonic polymorphisms in the beta-spectrin gene were utilized for the detection of de novo mutations influencing beta-spectrin mRNA stability. They were also screened for the presence of alpha-spectrin(LEPRA) as well as for the mutation -108T-->C in the ankyrin promoter, two variants previously found in some cases of genuinely recessive HS. Twenty-five patients showed ankyrin de novo mutations and 10 HS subjects had beta-spectrin de novo mutations. Furthermore, we found five patients to be heterozygous for alpha-spectrin(LEPRA) and one heterozygous for the mutation in the ankyrin promoter. Therefore, a molecular diagnosis was achieved in about 50% of the cases. Our data demonstrate that, among HS patients with normal parents, de novo dominant mutants are six times more common than recessive mutations. These results should be considered in view of the genetic counselling of a normal couple with a HS child.

The erythrocyte skeleton plays an essential role in determining the shape and deformability of the red cell. Disruption of the interaction between components of the red cell membrane skeleton may cause loss of structural and functional integrity of the membrane. Several observations based on studies in vitro strongly suggest that phosphorylation may modify interactions between proteins, leading to a reduced affinity. In particular, increased phosphorylation of beta-spectrin decreases membrane mechanical stability. In order to investigate the presence of membrane protein defects we investigated the erythrocyte membrane protein composition and phosphorylation in 22 children with leukemia at diagnosis and during the remission phase. Sixteen children had acute lymphoblastic leukemia (ALL), three had chronic myeloid leukemia (CML) and three had acute myeloid leukemia (AML). Ten patients (eight ALL and two CML) displayed elliptocytosis and poikilocytosis, an increase of spectrin dimers (41.8 +/- 15.6) and an enhanced phosphorylation of beta-spectrin (108 +/- 15%) at diagnosis. These alterations disappeared during the remission phase. This is the first demonstration of a reversible erythrocyte membrane alteration in leukemia. Since the beta-spectrin phosphate sites are located near the C-terminal region and close to the head of the beta-chain that is involved in dimer-dimer interaction, we supposed that the beta-chain phosphorylation has an effect upon the interactions between spectrin dimers, ie the tetramerization process. The weakening of this process should be responsible for the presence of elliptocytes and poikilocytes as reported in hereditary elliptocytosis and pyropoikilocytosis.

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.

Objective To evaluate the frequency of de novo monoallelic expression of the ANK1 gene in hereditary spherocytosis individuals appearing as recessive. Study design We studied 40 unrelated children with spherocytosis and their normal parents. The genomic distribution of the ankyrin (AC)n dinucleotide repeats was evaluated in the patients showing combined ankyrin and spectrin deficiency. To search for the absence of mRNA derived from one of the two ANK1 genes, cDNA from the heterozygous patients was amplified using polymerase chain reaction. This was analyzed for the (AC)n dinucleotide repeats. Results Thirty-three hereditary spherocytosis subjects had variable degrees of combined ankyrin and spectrin reduction; 19 were found to be heterozygous for the AC repeat lengths and were further studied. In 12, we found a cDNA polymerase chain reaction product from one ankyrin gene alone. These findings strongly suggested the nonexpression of one of the two ANK1 genes because of the de novo mutational events. Conclusion The de novo loss of an ankyrin allele expression is a frequent cause of hereditary spherocytosis in children with normal parents. Therefore the category of genuinely recessive hereditary spherocytosis cases is further reduced compared with spherocytosis cases because of de novo mutations. The determination of the (AC)n microsatellite polymorphisms appears as a helpful and reliable tool for the discrimination between these two categories.

Hereditary spherocytosis (HS) is a very heterogenous condition both at clinical and biochemical level. To establish the relationship between these aspects we performed a clinical and biochemical study in 87 Italian HS subjects. Patients were divided into three groups based on clinical severity (mild, typical and severe) and into five subgroups based on specific membrane abnormalities identified by polyacrylamide gel electrophoresis (isolated spectrin deficiency, spectrin deficiency combined with mild ankyrin reduction, spectrin deficiency combined with severe ankyrin reduction, band 3 reduction and isolated protein 4.2 reduction). We were not able to assess any alteration in six HS patients. A good correlation between clinical HS forms and membrane protein defects is shown. We conclude that erythrocyte membrane analysis should be carried out after diagnosis of HS in order to predict the clinical course of the disease.

Common HE is the most prevalent clinical form of hereditary elliptocytosis; its clinical findings vary considerably, ranging from an asymptomatic carrier state to a severe, even life-threatening hemolytic disorder. Structural modification and reduction of 4.1 protein, or abnormalities at the spectrin self-association site could lead to elliptocytes.Sixty-one Italian HE patients belonging to 28 families were studied. Analysis of red blood cell cytoskeleton was performed by means of SDS-PAGE, and spectrin dimer percentage was assessed by non denaturing polyacrylamide gel electrophoresis. Limited tryptic digestion of spectrin was employed in patients showing an abnormal dimer increase, and the amount of abnormal alpha I peptide was estimated. Molecular defects were detected by means of PCR of alpha and beta spectrin genes and direct sequencing of genomic DNA.We found a very heterogeneous spectrum of cytoskeletal alterations: 18 (29%) subjects showed partial protein 4.1 deficiency, whereas 31 (51%) displayed an increased amount of spectrin dimers; we were not able to detect any alteration in 12 (20%) HE patients. Patients enrolled in this study were widely distributed throughout Italy.The subgroup of HE patients related to 4.1 deficiency is homogeneously asymptomatic, whereas forms due to disruption of the spectrin tetramerization site are very heterogeneous, and clinical severity appears to be related to spectrin dimers and especially to spectrin content. These two parameters in turn are related to the presence of a low expression alpha allele in trans and to the degree of disruption of head-to-head contact between alpha and beta chains.

BackgroundGain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined.Methodology/principal findingsWe describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G-->T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34(+) cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway.Conclusions/significanceOur data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis.

Recently it has been clearly established that partial deficiency of spectrin (SP) evaluated by radioimmunoassay is a common feature of many different forms of hereditary spherocytosis (HS). In this paper the determination of SP density (spectrin/band 3 ratio) in 46 HS patients and in 50 normal controls is presented. The comparison between the membrane SP density of HS subjects and controls showed a statistically significant difference (P less than 0.0005). Moreover no overlap between normal and HS subjects was observed. Membrane spectrin/band 3 ratio has been found related to some clinical features: indeed patients with severe HS showed a smaller SP density than those with milder HS. Our results show that the evaluation of membrane SP density permits a prompt diagnosis of HS and avoids extensive and unnecessary studies for other anaemias.

We describe three Italian subjects from two unrelated families affected with isolated hereditary spherocytosis (HS) without other clinical abnormalities, associated with partial spectrin and ankyrin deficiency. In both families the propositus has normal biological parents, and thus appears to be the result of a new mutation; in one of them the disease is further transmitted in an autosomal dominant fashion. Cytogenetic analysis of the latter family excluded abnormalities of the short arm of chromosome 8. We speculate that in both kindreds ankyrin deficiency is the primary defect related to ankyrin gene mutation. Several pieces of evidence suggest that ankyrin deficiency is probably the most common molecular defect in HS. It is inherited in a, dominant manner and its clinical and biochemical expression is heterogenous.