Susanna Barella

A mutation in the TMPRSS6 gene, encoding a transmembrane serine protease that suppresses hepcidin production, in familial iron deficiency anemia refractory to oral iron

Previous studies have described a familial syndrome characterized by iron malabsorption, hypoferremia, and microcytic anemia that did not respond to oral iron and responded only partly to parenteral iron. In this work, Melis and coworkers studied a Sardinian family with this inherited condition. They found a homozygous causal mutation in the TMPRSS6 gene in affected members, who had inappropriately elevated levels of serum and urinary hepcidin. See related perspective article on page 1441. Background Hepcidin plays a key role in body iron metabolism by preventing the release of iron from macrophages and intestinal cells. Defective hepcidin synthesis causes iron loading, while overproduction results in defective reticuloendothelial iron release and iron absorption. Design and Methods We studied a Sardinian family in which microcytic anemia due to defective iron absorption and utilization is inherited as a recessive character. Five members showed iron deficiency anemia that was not responsive to oral iron and only partially responsive to parenteral iron administration. To investigate the involvement of known genes implicated in iron metabolism we carried out linkage analysis with microsatellite markers mapping close to these genes. Afterwards, a genome-wide search was performed. Results No linkage was found between the phenotype of the patients and several known human genes involved in iron metabolism (DMT1, TF, TFRC, ZIRTL, HAMP, HJV). Genome-wide scanning by microsatellites and single nucleotide polymorphisms showed a multipoint LOD score of 5.6 on chromosome 22q12.3–13.1, where the matriptase-2 (also known as transmembrane protease, serine 6 or TMPRSS6) gene is located. Its murine counterpart (Tmprss6) has recently been found to be an essential component of a pathway that detects iron deficiency and suppresses hepcidin production. Sequencing analysis of TMPRSS6 revealed a homozygous causal mutation, predicting a splicing error and a truncated TMPRSS6 protein in affected members. Homozygous subjects had inappropriately elevated levels of serum and urinary hepcidin. Conclusions The findings of this study suggest that the observed TMPRSS6 mutation leads to overproduction of hepcidin and, in turn, to defective iron absorption and utilization. More generally, they confirm in humans the inhibitory effect of matriptase-2 on hepcidin synthesis already demonstrated in mice.

Amelioration of Sardinian β0 thalassemia by genetic modifiers

Sardinian beta-thalassemia patients all are homozygotes for the same null allele in the beta-globin gene, but the clinical manifestations are extremely variable in severity. Previous studies have shown that the coinheritance of alpha-thalassemia or the presence of genetic variants that sustain fetal hemoglobin production has a strong impact on ameliorating the clinical phenotype. Here we evaluate the contribution of variants in the BCL11A, and HBS1L-MYB genes, implicated in the regulation of fetal hemoglobin, and of alpha-thalassemia coinheritance in 50 thalassemia intermedia and 75 thalassemia major patients. We confirm that alpha-thalassemia and allele C of single nucleotide polymorphism rs-11886868 in BCL11A were selectively represented in thalassemia intermedia patients. Moreover, allele G at single nucleotide polymorphism rs9389268 in the HBS1L-MYB locus was significantly more frequent in the thalassemia intermedia patients. This trio of genetic factors can account for 75% of the variation differences in phenotype severity.

Relationship between transfusion regimen and suppression of erythropoiesis in β-thalassaemia major

In the management of β‐thalassaemia major, different transfusion schemes are employed with baseline haemoglobin levels ranging from 8 to over 12 g/dl. We studied the relationship between transfusion regimen and suppression of erythropoiesis in 52 patients with β‐thalassaemia major whose mean pretransfusion haemoglobin levels ranged from 8.6 to 10.9 g/dl. Multiple, regression analysis showed that serum transferrin receptor was the parameter more closely related to mean pretransfusion haemoglobin (r = ‐0.77, P < 0.001). As measured through serum transferrin receptor, erythroid activity was 1‐2 times normal for pretransfusion haemoglobin levels between 10 and 11 g/dl, 1‐4 times normal for levels from 9 to 10 g/dl, and 2‐6 times normal for levels from 8.6 to 9 g/dl. Mean pretransfusion haemoglobin was also inversely related to serum erythropoietin (r = ‐0.72, P < 0.001), whereas it showed no or a weak relationship with Hb F, reticulocyte count, or circulating nucleated red cell count. This study suggests that serum transferrin receptor is a reliable indicator of suppression of erythropoiesis in β‐thalassaemia major. On the basis of our findings, pretransfusion haemoglobin values of ≦ 9 g/dl should be adopted with caution, because these levels can be associated with an insufficient inhibition of erythroid marrow expansion. However, a transfusion programme, with a baseline haemoglobin of 9‐10 g/dl, may provide enough suppression of erythropoiesis and allow a reduction in blood consumption as compared with the classic hyper‐ or supertransfusion schemes. Since fixed haemoglobin levels may not be the best target for transfusion treatment in all thalassaemic patients, assay of serum transferrin receptor may be helpful for individualizing the transfusion regimens.

A pilot trial of deferiprone for neurodegeneration with brain iron accumulation

Deferiprone was shown to reverse iron deposition in Friedreich’s ataxia. This multi-center, unblinded, single-arm pilot study evaluated safety and efficacy of deferiprone for reducing cerebral iron accumulation in neurodegeneration with brain iron accumulation. Four patients with genetically-confirmed pantothenate kinase-associated neurodegeneration, and 2 with parkinsonism and focal dystonia, but inconclusive genetic tests, received 15 mg/kg deferiprone bid. Magnetic resonance imaging and neurological examinations were conducted at baseline, six and 12 months. Chelation treatment caused no apparent hematologic or neurological side effects. Magnetic resonance imaging revealed decreased iron accumulation in the globus pallidus of 2 patients (one with pantothenate kinase-associated neurodegeneration). Clinical rating scales and blinded video rating evaluations documented mild-to-moderate motor improvement in 3 patients (2 with pantothenate kinase-associated neurodegeneration). These results underline the safety and tolerability of deferiprone, and suggest that chelating treatment might be effective in improving neurological manifestations associated with iron accumulation.

Cholelithiasis and Gilbert's syndrome in homozygous β-thalassaemia

Cholelithiasis has been reported with a variable incidence in homozygous β‐thalassaemia, the reasons for which have only partially been defined. Disease‐associated factors or specific modifier genes may be implicated. We assessed the prevalence of cholelithiasis and the effect of co‐inherited Gilberts syndrome genotype on its development in 261 thalassaemia major (TM) and 35 thalassaemia intermedia (TI) patients. Cholelithiasis was found in 20·3% of TM and in 57·1% of TI patients. Its incidence was higher (Pu2003<u20030·05) in patients homozygous for the (TA7) motif in the promoter of the UGT1‐A1 gene, the genotype associated with Gilberts syndrome, which seems to be a risk factor for the development of gallstones in TM and TI patients.

Soluble transferrin receptor as a potential determinant of iron loading in congenital anaemias due to ineffective erythropoiesis

Congenital anaemias due to ineffective erythropoiesis may be associated with excessive iron absorption and progressive iron loading. We investigated whether the soluble transferrin receptor (TfR) level was related to the degree of iron overload in 20 patients with thalassaemia intermedia, six patients with congenital dyserythropoietic anaemia type II (CDA II) and four patients with X‐linked congenital sideroblastic anaemia (XLSA). All but two patients had increased serum ferritin levels (median 601u2003μg/l, range 105–2855u2003μg/l). Multiple regression analysis showed that 62% (Pu2003<u20030.0001) of the variation in serum ferritin was explained by age and by changes in soluble TfR.

Hyperbilirubinaemia in heterozygous β‐thalassaemia is related to co‐inherited Gilbert's syndrome

The reasons why heterozygotes for β‐thalassaemia have considerable variation in serum bilirubin levels are unkown. High levels of bilirubin could be related to the co‐inherited Gilberts syndrome, determined either by mutations of the coding region or by variation in the A(TA)nTAA motif of the promoter of the bilirubin UDP‐glucuronosyltransferase gene (UGT‐1). We sequenced the coding and the promoter region of UGT‐1A or characterized the A(TA)nTAA motif of the promoter by denaturing gel electrophoresis of radioactive amplified products. The results were correlated with bilirubin levels in 49 β‐thalassaemia heterozygotes for codon 39 (CAGu2003→u2003TAG) nonsense mutation. 21 normal individuals and 32 unrelated patients with Gilberts syndrome served as controls. The coding sequence region of the UGT‐1A was normal. Five β‐thalassaemia heterozygotes, who were homozygous for the extra (TA) bases in the A(TA)nTAA element of the promoter of UGT‐1A, the configuration present in homozygosity in Gilberts syndrome, had higher bilirubin levels compared to those with the (TA)6/(TA)7 or (TA)6/(TA)6 configurations.

Efficacy and safety of deferiprone for the treatment of pantothenate kinase-associated neurodegeneration (PKAN) and neurodegeneration with brain iron accumulation (NBIA): Results from a four years follow-up

OBJECTIVEnTo evaluate the long-term effect of Deferiprone (DFP) in reducing brain iron overload and improving neurological manifestations in patients with NBIA.nnnMETHODSn6 NBIA patients (5 with genetically confirmed PKAN), received DFP solution at 15xa0mg/kg po bid. They were assessed by UPDRS/III and UDRS scales and blinded video rating, performed at baseline and every six months. All patients underwent brain MRI at baseline and during follow up. Quantitative assessment of brain iron was performed with T2* relaxometry, using a gradient multi-echo T2* sequence.nnnRESULTSnAfter 48 months of treatment clinical rating scales and blinded video rating indicated a stabilization in motor symptoms in 5/6 Pts. In the same subjects MRI evaluation showed reduced hypointensity in the globus pallidus (GP); quantitative assessment confirmed a significant increment in the T2* value, and hence reduction of the iron content of the GP.nnnCONCLUSIONnThe data from our 4-years follow-up study confirm the safety of DFP as a chelator agent for iron accumulation. The clinical stabilization observed in 5/6 of our patients suggests that DFP may be a reasonable therapeutic option for the treatment of the neurological manifestations linked with iron accumulation and neurodegeneration, especially in adult patients at early stage of the disease. (Clinicaltrials.gov identifier: NTC00907283).

No evidence of cardiac iron in 20 never- or minimally-transfused patients with thalassemia intermedia

Iron loading in non-transfused thalassemia intermedia (TI) patients is mainly due to an increased intestinal absorption secondary to chronic anemia and varies from 2 to 5 grams per year.[1][1] In thalassemia major (TM) iron derived from red blood cell breakdown accumulates first in the

HbH Disease in Sardinia: Molecular, Hematological and Clinical Aspects

In this study we have defined the molecular basis and correlated the clinical phenotype with the alpha-globin genotype in a large series of patients of Sardinian descent with HbH disease. The most prevalent molecular defect was the deletion of 3 alpha-globin structural genes most commonly the (--/-alpha 3.7) genotype (83.6%) and rarely the (--/-alpha 4.2) genotype (1.4%), followed in decreasing order of incidence by the combination of deletion alpha zero-thalassemia and initiation codon mutation of the alpha 2-gene (--/alpha NcoI alpha = 9.8%), deletion alpha zero-thalassemia and pentanucleotide deletion of IVS-I of the alpha 2-globin gene, (--/alpha HphI alpha = 3.3%) deletion alpha zero-thalassemia and initiation codon mutation of the alpha 1-gene (--/alpha alpha NcoI = 1.3%), a homozygous state for initiation codon mutation of the alpha 2-gene (alpha Nco alpha/alpha NcoI alpha = 0.7%). Patients with the (--/alpha thal alpha) genotypes showed severer clinical and hematological features as compared to those with the (--/-alpha) or those with the (--/alpha alpha thal) genotypes. The single patient with the (alpha Nco alpha/alpha Nco alpha) genotype had a clinical phenotype intermediate between HbH disease and the alpha-thalassemia carrier status. This heterogeneity depends on the fact that the alpha 2-globin gene produces 2-3 times alpha-globin chains than the alpha 1-gene and the single remaining alpha 1-like globin gene in the -alpha 3.7 chromosome has a compensatory increase in the alpha-globin chain output. alpha-Globin gene mapping of HbH disease patients may be useful for predicting the clinical outcome and to improve genetic counseling.