Massimiliano Ammirabile

Hypocholesterolemia in adult patients with thalassemia: a link with the severity of genotype in thalassemia intermedia patients.

Objectives:  Hypocholesterolemia has been previously described in patients affected by thalassemia. In this study we retrospectively evaluated the cholesterol level in two groups of patients affected by either thalassemia major (TM) or thalassemia intermedia (TI), with the aim of establishing factors correlated to hypocholesterolemia within both populations.

Combined chelation therapy in thalassemia major with deferiprone and desferrioxamine: a retrospective study

Objectives: The benefits of combined chelation therapy with daily deferiprone (DFP) and subcutaneous desferrioxamine (DFO) have been widely reported in literature. We retrospectively evaluated the efficacy of different schedules of combined chelation therapy and the incidence of adverse events. Methods: We evaluated 36 patients affected by thalassemia major treated with combined chelation therapy. Patients were subdivided into four treatment arms according to severity of iron overload and previous onset of adverse events to DFP therapy: Group 1 (13 pts) DFP 75 mg/kg per d plus DFO (25–35 mg/kg per d for 5 d); Group 2 (6 pts) DFP 50 mg/kg per d plus DFO (25–35 mg/kg for 5 d), Group 3 (10 pts) DFP 75 mg/kg per d plus DFO (25–35 mg/kg for 3 d), and Group 4 (7 pts) DFP 50 mg/kg per d plus DFO (25–35 mg/kg for 3 d). Change in serum ferritin level was evaluated in all patients. Results: Overall, ferritin decreased from 2592 ± 1701 to 899 ± 833 ng/mL (P < 0.001). All treatments were able to reduce ferritin levels, but in patients of group 1 and group 2 the highest mean decrease in serum ferritin level and the greatest improvement in liver iron concentration (LIC) and in T2* values were observed. Conclusions: This study showed that the administration of DFO for 5 d a wk in combination with daily administration of DFP at 75 mg/Kg seemed to be the most efficacy and rapid method for reducing iron overload at liver and heart level. Furthermore, the use of different schedules of combined DFO and DFP administration was not associated with different incidence of adverse effects between the groups.

Splenectomy is a risk factor for developing hyperuricemia and nephrolithiasis in patients with thalassemia intermedia: a retrospective study.

Few data are available on the prevalence and the risk factors for the presence of kidney stones and hyperuricemia in patients with thalassemia intermedia. We retrospectively reviewed the charts and radiological studies of 89 patients with thalassemia intermedia followed at our clinic with routine biochemical examination and radiological imaging of the urinary tract. Renal calculi were identified in 11 patients (12%) and 22 patients (25%) were under uricosuric treatment for hyperucemia. The prevalence of nephrolithiasis increased with age but not in a statistically significant manner. Major risk factors for renal stone formation were splenectomy (in 91% of the cases) and higher number of erythroblasts. Patients with renal stones had higher mean creatinine level and lower GFR value with respect to those observed in patients not affected. Our data suggest that splenectomy, by further increasing erythrocyte turnover and number, may be directly involved in the pathogenesis of hyperuricemia and nephrolithiasis observed in thalassemia intermedia patients.

The impact of previous or concomitant IFN therapy on deferiprone-induced agranulocytosis and neutropenia: a retrospective study.

Objective: Although IFN therapy is known to cause neutropenia, data on the risk of deferiprone (DFP)-induced haematological complications in patients receiving IFN are lacking. Research design and methods: This was a retrospective single-centre study to assess the association between exposure to IFN for hepatitis C virus treatment and haematological side effects of DFP therapy in patients with thalassemia major and intermedia using a large database spanning 2001 – 2008. During observation time, a total of 66 patients, including 63 affected by thalassemia major and 3 by thalassemia intermedia, were treated with chelation DFP-based regimens. A subset of 25 patients was treated at least for 3 months also with IFN (6 were cotreated and 19 were pretreated). Results: Overall, the incidence of neutropenia and agranulocytosis was 9.83 and 1.14/100 patient/year, respectively. Receipt of IFN was significantly associated with increased risk of haematological complications of DFP therapy: among patients receiving IFN, 48 and 12% experienced at least one episode of neutropenia and agranulocytosis, respectively. Conclusions: These results suggest that IFN therapy may increase the risk of complications of DFP-based iron chelation therapy in patients with thalassemia. Further research is needed to assess whether the association observed in this retrospective single-centre observational study is due to IFN or other factors.

Identification and molecular characterization of the ‐‐CAMPANIA deletion, a novel α°‐thalassemic defect, in two unrelated Italian families

To the Editor: Alpha-thalassemia is characterized by decreased or complete lack of synthesis of a-globin chains. The a-globin gene cluster is located on the short arm of chromosome 16 and includes, in the 50-30 order, an embrionic globin gene (f2), three pseudogenes (pseudo-f, pseudo-a2, and pseudo-a1), two a-globin genes (a2 and a1), and a gene of undetermined function (y1). Most common defects result from large deletions involving one (-a) or both (--a) of the a-globin genes, although increasing number of point mutations have also been described [1–3]. Loss of function of three of four a-globin genes gives rise to HbH disease, an intermediate clinical form of athalassemia [4]. Mutations are population specific, and their distribution has been extensively determined, providing support for prevention programs [5,6]. During a study on the molecular basis for a-thalassemia in our region, we identified two patients with a novel type of a-thalassemia determinant resulting in the deletion of the entire f-a gene cluster. Clinical and hematological data (Supporting Information, Fig. S1A) were suggestive of HbH disease. Molecular analysis of the most common Mediterranean deletion forms of a-thalassemia indicated an apparent homozygosis for the -a deletion. As in both families only one parent was heterozygous for this mutation (Supporting information, Fig. S1B), the two patients were actually compound heterozygotes for the -a deletion and an uncommon a8 rearrangement. In both cases, hybridization with a f-globin probe on genomic DNA digested with Xba I failed to reveal any abnormal fragment (data not shown), whereas a L1 probe located 50 to the f-globin gene [7] showed an abnormal 6.3-kb fragment along with a normal 6.6-kb fragment suggestive of a deletion encompassing the entire f-a gene cluster (Supporting Information, Fig. S1C). This pattern was similar to that detected in heterozygotes for the --a deletion ( 6.0 kb/6.6 kb), first described in an Italian patient [8] and subsequently in a Spanish family [9]. An allele-specific PCR reaction previously described for the --a deletion [6] produced in both cases an amplicon of 2.1 kb instead of the 491bp size expected for this type of deletion (Supporting Information, Fig. S2A). Sequencing analysis of the 2.1-kb fragment allowed to identify the 31-kb region removed by this novel deletion and its resulting 50and 30-breakpoints located within the Alu Jb (coordinates 8635-8924) and Alu Sp (coordinates 39835-40133) repeats (Fig. 1A), respectively. As both patients were from Campania, a southern Italy region, the novel deletion was designated as CAMPANIA. Our results indicate that this rearrangement was produced by a mechanism of Alu-mediated recombination, which is rather common in this chromosomal region [10]. Definition of the specific Alu elements also demonstrates that the CAMPANIA and CAL deletions arose from independent mutational events, although their breakpoints lie very close to each other (Fig. 1B). Linkage analysis of polymorphic sites located upstream and downstream the deletion breakpoints (50HVR, XbaI RFLP, yPstI RFLP, and 30HVR) [11,12] showed for both patients the same junctional sequence and polymorphic genotypes in the regions surrounding the deletion: 50HVR (allele size of 830 bp), XbaI (2), yPstI (2), 30HVR (allele size of 2,100 bp), therefore supporting the hypothesis of a unique origin for this deletion (Fig. 1A and Supporting Information, Fig. S2D). Definition of the deletion breakpoints allowed to design an allele-specific amplification reaction for this novel deletion, which was found at the heterozygous condition in the obligate carriers (subjects I-2 from family a and I-2 from family b) and in another sibling from family b (II-2) (Supporting information, Fig. S2E). Our study contributes to define the wide spectrum of mutations that underlie the thalassemia syndromes in the Mediterranean area. Furthermore, involvement of Alu repeats remarks the high susceptibility of the a-globin gene cluster region to unequal crossing-over events. Therefore, given the high frequency with which these sequences occur within this region, the overall incidence of unusual deletional forms of a-thalassemia in our population is to be expected much higher than what was supposed so far [13]. Our study also indicates that exhaustive molecular studies are necessary to avoid potential pitfalls in genetic counseling. This requires specific, reliable, and easily accessible genetic tests to allow accurate large-scale screening programs and prenatal diagnosis services in regions where such uncommon a-thalassemic determinants could occur.

The long-term and extensive efficacy of low dose thalidomide in a case of an untransfusable patient with Non-Transfusion-Dependent Thalassemia

Patients with Non-Transfusion-Dependent Thalassemia may require regular transfusion therapy. However, these patients are at risk of developing irregular antibodies, making them untransfusable. Second line treatment usually includes hydroxyurea, which however is not effective in all patients. Other treatment options include thalidomide, which has been reported to be safe and effective in selected patients. We report the case of a patient who experienced improvement of hemoglobin levels and of a part of NTDT related complications, following 36months of continuous therapy with low doses of thalidomide.

Nephrolithiasis in patients exposed to deferasirox and desferioxamine: probably an age-linked event with different effects on some renal parameters

Dear Editor, We refer this letter to the manuscript by Efthimia et al. [1] in which they recently reported that in thalassemia major (TM) patients, the start of deferasirox (DFX) has been associated with increased risk of developing nephrolithiasis. However, in that series, several patients were also cotreated with calcium and vitamin D supplements, and the authors correctly hypothesized that it could have facilitated the renal calculi formation. Nevertheless, that clinical observation was not easy to evaluate by the fact that it was not a population-based case– control study and the definition of the population and the duration of patient exposure to DFX treatment were not reported; furthermore, the assessment of new cases was made only through the detection of renal colic. We reviewed the charts and radiological studies of our patients with TM to retrospectively evaluate the incidence of nephrolithiasis in those treated with DFX and in a matched control population treated with desferioxamine (DFO). The presence or the absence of nephrolithiasis had to be assessed by abdominal ultrasonography. To further analyze data, total therapy exposure and the incidence of nephrolithiasis adjusted to 100 years of patient exposure were calculated for each treatment. To make series comparable, data from patients under DFO treatment were collected since January 2010. The study was approved by the Ethics Committee of the Cardarelli Hospital, Napoli. Table 1 shows demographics and clinical characteristics of patients divided in those exposed to DFX and to DFO at baseline and at the end of drug exposure (October 2012). At baseline, both populations were comparable for previous chelation history, for the percentage of patients splenectomized and using calcium and vitaminD, for the mean creatinine level and GFR value, and for the prevalence of renal calculi [2], but patients treated with DFX tended to be younger and to have lower level of uric acid. Patients already affected by renal calculi were in average 40 years old and frequently splenectomized, but they did not use vitamin D and calcium supplementation more frequently than those without stones and were not always symptomatic; however, in those under DFO treatment, a decrease in GFR was observed following drug exposure. Comparing basal and final values in a population without stones, an increase in creatinine level and a decrease in GFR were observed in the DFX group. We observed eight (5.26/100 patients/year ) and five (4.76/100 patients/year) new cases of renal stones in patients treated with DFX and DFO, respectively, and the O.R. for developing nephrolithiasis under DFX was 1.17 , p=0.79 (data not shown). Interestingly, among stone formers following DFX treatment, a significant decrease in uric acid level without a decrease in renal function parameters was observed. Estimates of the incidence of renal stones in TM patients are lacking [3]. In spite of this, a sustained rate of renal stone formation under DFX treatment was confirmed; these data highlight the presence of an asymptomatic disease that may have contributed to its underestimation in the DFO group in Efthimia’s series [1]. In our series, renal stone formation appears to involve mainly patients older than 30 while the P. Ricchi :M. Ammirabile : S. Costantini :A. Spasiano : P. Cinque :M. Casale :A. Filosa : L. Prossomariti UOSD Centro delle Microcitemie “A. Mastrobuoni”, AORN A. Cardarelli, Naples, Italy

Hepatitis C virus distribution and clearance following interferon-monotherapy among thalassaemia major and intermedia patients.

Additional Supporting Information may be found in the online version of this article: Table SI. Complete Steady state plasma amino acid concentrations in SCA patients who subsequently died compared to age and sex matched SCA patients who were alive as of 1st July 2009. Table SII. Available laboratory markers of hemolysis and liver and kidney function in patients at enrollment into the cohort (steady-state) and at the time of amino acid steady-state sample collection. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

Hb Southern Italy: coexistence of two missence mutations (the Hb Sun Prairie α2 130 Ala Pro and Hb Caserta α2 26 Ala Thr) in a single HBA2 gene

This study describes a new molecular condition in the α2‐globin gene (HBA2) found in six unrelated families from Southern Italy (Campania and Sicily). This new double mutant form of haemoglobin is called Hb Southern Italy and originated from the coexistence of two known mutations occurring in the same globin gene, HBA2 26 G→A (Hb Caserta) and HBA2 130 G→C (Hb Sun Prairie). Hb Sun Prairie was originally observed in Indian patients in either the homozygous state, with severe hemolytic anemia, and in the heterozygous state with microcytosis, or in asymptomatic cases as an α‐thalassemia carrier phenotype. Hb Caserta was observed for the first time in a Casertian family (South Italy) that displayed a slowmigrating haemoglobin upon investigation. We report the clinical phenotype and molecular study of this new double mutant form of haemoglobin in heterozygous and homozygous subjects, as well as in association with α° delectional thalassemia.

Hepatitis B virus reactivation during combined therapy with deferiprone and desferioxamine in a hepatitis B surface antigen thalassemic carrier

In this report we firstly describe a case of reactivation of hepatitis B virus (HBV) replication occurred in a patient affected by Thalassemia major which underwent a combined chelation therapy with desferioxamine (DFO) and deferiprone (DFP). Clinical symptom and increase in alanine aminotransferase (ALT) level were detected when the prescription of DFO (30 mg/kg) was increased from 3 to 5 days/week; a raise in HBV-DNA levels of greater than or equal to tenfold compared with baseline was thereafter detected. Diagnosis was troublesome because increasing ALT levels, first suggested toxicity to DFP administration. However, HBV reactivation in our patient cannot be definitively attributed to combined regimen administration: the patient was on regular transfusion therapy and either coincidental further infection or spontaneous reactivation of HBV could not be completely ruled out. Furthermore, a background of immunologic abnormalities had been previously reported in thalassemia and postulated to be secondary to iron overload or DFO therapy itself.