Safety and Efficacy of the New Combination Iron Chelation Regimens in Patients with Transfusion-Dependent Thalassemia and Severe Iron Overload.

The aim of this study is the evaluation of the safety and the efficacy of long-term combination therapy deferasirox plus desferrioxamine and deferasirox plus deferiprone in a large group of transfusion-dependent thalassemia patients with high values of serum ferritin and/or magnetic resonance, indicative of severe liver and cardiac iron accumulation. Sixteen adults with transfusion-dependent thalassemia were treated simultaneously with deferasirox plus desferrioxamine, while another 42 patients (seven children) were treated with deferasirox plus deferiprone. The hepatic and cardiac iron overload was assessed prior to treatment and then annually with magnetic resonance imaging, and the serum ferritin was measured monthly. Adverse events were checked at each transfusion visit. The safety of both the combinations was consistent with established monotherapies. Both treatments were able to decrease the serum ferritin and liver iron concentration over time, depending on the level of compliance with therapy. Cardiac iron measured as R2* did not significantly change in patients treated with deferasirox plus desferrioxamine. Most patients with MRI indicative of myocardial siderosis at the beginning of treatment reached normal values of cardiac iron at the last determination if treated with deferasirox plus desferrioxamine. The greatest limitation of these therapies was low patient adherence to the two drugs, which is not surprising considering that the need for an intensive chelation is generally linked to previous issues of compliance.

Impact of the direct-acting antiviral agents (DAAs) on chronic hepatitis C in Sardinian patients with transfusion-dependent Thalassemia major.

BACKGROUND AND AIMS: Direct antiviral agents (DAAs) have revolutionised the standard of care for the treatment of hepatitis even in patients with hemoglobinopathies. The aim of this study is to show how, thanks to DAAs, HCV infection has been substantially eradicated in one of the biggest Centres for the management of Thalassemia in Europe. METHODS: Thalassemia major patients regularly transfused and iron chelated in Cagliari (Italy) who were HCV-RNA positive were evaluated for the potential prescription of antiviral therapy. RESULTS: A total of 99 patients, 26 of whom had been diagnosed with cirrhosis, were treated with at least one dose of DAAs, which proved to be safe and well tolerated. Two of the patients died during the treatment after becoming HCV-RNA negative while another voluntarily interrupted the therapy. The final SVR in the patients who completed the treatment was 100%, while measuring 97% (96/99) in the Intention-to-Treat analysis. After DAAs, no new cases of hepatocellular carcinoma have been reported. CONCLUSIONS: The use of DAAs in patients suffering from beta-Thalassemia major with chronic hepatitis C or cirrhosis can be considered safe and effective. Close monitoring for hepatocellular carcinoma development is, in any case, recommended indefinitely post-SVR.

Thalassemia major between liver and heart: Where we are now.

The aim of the study was to assess the current state in terms of liver and heart iron overload as well as of liver and heart related morbidity and mortality in a large cohort of thalassemia patients. Myocardial iron loading was present in 28.9% patients, which was severe in 3.2%. Liver iron was normal in 9.3% and severe in 15%. The rate of cardiac deaths started to decrease between 2000 and 2003 and dropped significantly afterwards. The prescription of combination therapy soon after the hospital admission for decompensated heart failure was associated with a decrease in the short-term mortality. In 111 adult patients who underwent liver elastometry, 14 HCVRNA positive subjects and 2 HCVRNA negative, had stiffness values suggestive of cirrhosis. No cases of hepatocarcinoma were reported. Liver "iron free foci" occurred in a HCV negative patient and the occurrence of a malignant epithelioid hemangioendothelioma led to liver transplantation in another. The study suggests that a subset of patients continues to develop progressive hemosiderosis that may lead to cardiac disease and death. Beyond its key role in preventing myocardial iron overload, liver iron chelation is essential for hampering the onset of hepatic tumors, which may not be limited to hepatocarcinoma.

The homozygosity index (HI) approach reveals high allele frequency for Wilson disease in the Sardinian population.

Wilson disease (WD) is an autosomal recessive disorder resulting in pathological progressive copper accumulation in liver and other tissues. The worldwide prevalence (P) is about 30/million, while in Sardinia it is in the order of 1/10,000. However, all of these estimates are likely to suffer from an underdiagnosis bias. Indeed, a recent molecular neonatal screening in Sardinia reported a WD prevalence of 1:2707. In this study, we used a new approach that makes it possible to estimate the allelic frequency (q) of an autosomal recessive disorder if one knows the proportion between homozygous and compound heterozygous patients (the homozygosity index or HI) and the inbreeding coefficient (F) in a sample of affected individuals. We applied the method to a set of 178 Sardinian individuals (3 of whom born to consanguineous parents), each with a clinical and molecular diagnosis of WD. Taking into account the geographical provenance of the parents of every patient within Sardinia (to make F computation more precise), we obtained a q=0.0191 (F=7.8 × 10(-4), HI=0.476) and a corresponding prevalence P=1:2732. This result confirms that the prevalence of WD is largely underestimated in Sardinia. On the other hand, the general reliability and applicability of the HI approach to other autosomal recessive disorders is confirmed, especially if one is interested in the genetic epidemiology of populations with high frequency of consanguineous marriages.

Wilson's disease in two consecutive generations: the detection of three mutated alleles in the ATP7B gene in two Sardinian families.

BACKGROUND: Wilson's disease diagnosis is still a challenge for clinicians. AIM: To underline the importance of genetic testing in carrier detection and diagnosis of atypical Wilson's disease cases. METHODS: Two families with Wilson's disease in two consecutive generations were analysed with clinical, biochemical and genetic testing. RESULTS: In one family with triplet siblings, two of whom monozygotic, molecular screening of ATP7B, the gene responsible for Wilson's disease phenotype, allowed detection of 3 disease alleles, the discrimination between carrier and disease state and the postmortem diagnosis of Wilson's disease in the siblings' father. In the second family, molecular analysis detected 3 disease alleles and confirmed the diagnosis of Wilson's disease in two asymptomatic monozygotic twins. CONCLUSION: These results demonstrate that mutational analysis is determinant for carrier identification and diagnosis of atypical Wilson's disease patients.

Mutation analysis of the ATP7B gene in a new group of Wilson's disease patients: contribution to diagnosis.

Wilson's disease (WD), an autosomal recessive disorder of copper transport with a broad range of genotypic and phenotypic characteristics, results from mutations in the ATP7B gene. Herein we report the results of mutation analysis of the ATP7B gene in a group of 118 Wilson disease families (236 chromosomes) prevalently of Italian origin. Using DNA sequencing we identified 83 disease-causing mutations. Eleven were novel, while twenty one already described mutations were identified in new populations in this study. In particular, mutation analysis of 13 families of Romanian origin showed a high prevalence of the p.H1069Q mutation (50%). Detection of new mutations in the ATP7B gene in new populations increases our capability of molecular analysis that is essential for early diagnosis and treatment of WD.

Feasibility of RNA studies on illegitimate transcription for molecular characterization of splicing mutations in the ATP7B gene: a case report.

Approximately 520 Wilson disease-causing mutations in the ATP7B gene have been described to date. In this study we report DNA and RNA analyses carried out for molecular characterization of a consensus sequence splicing mutation found in homozygosity in a Swiss Wilson disease patient. RNA analysis of 1946 +6 T→C in both the peripheral lymphoblasts and liver resulted in the production in the propositus of only an alternative transcript lacking exons 6, 7, and 8 resulting most likely in alterations of cell biochemistry and disease. The patient presents an early form of severe hepatic disease characterized by hepatosplenomegaly, reduced hepatic function, anemia and thrombocytopenia indicating that 1946 +6 T→C is a severe mutation. Since identical results were obtained from both peripheral lymphoblasts and liver they also suggest that RNA studies of illegitimate transcripts can be safely used for molecular characterization of ATP7B splicing mutations, thus improving genetic counseling and diagnosis of Wilson disease. Moreover these studies, contribute to reveal the exact molecular mechanisms producing Wilson disease.

DNA and RNA studies for molecular characterization of a gross deletion detected in homozygosity in the NH2-terminal region of the ATP7B gene in a Wilson disease patient.

Wilson disease is an autosomal recessive disorder caused by defective function of the copper transporting protein ATP7B. Approximately 520 Wilson disease-causing mutations have been described to date. In this study we report the use of DNA and RNA analysis for molecular characterization of a gross deletion of the ATP7B gene detected in homozygosity in a Wilson disease patient. The c.51+384_1708-953del mutation spans an 8798 bp region of the ATP7B gene from exon 2 to intron 4. The results obtained suggest that the combination of DNA and RNA analyses can be used for molecular characterization of gross ATP7B deletions, thus improving genetic counselling and diagnosis of Wilson disease. Moreover these studies, help to better establish the molecular mechanisms producing Wilson disease.

Molecular analysis of glycogen storage disease type Ib in Sardinian population: evidence for a founder effect.

We describe epidemiological, genetic, and clinical data of the 1124-2del mutation in the G6PT gene, detected in homozygosity in three glycogen storage disease type Ib patients of Sardinian origin. This mutation was found to be associated with four sequence variations: c.593 A>T (p.N198I), c.625+19 C>T, c.1062 C>T (N354N), and c.1224 G>A (p.T408T) in the G6PT gene. RNA studies were performed for c.1124-2del and c.625+19 C>T. The c.1124-1del2 acceptor splicing mutation showed skipping of 31 nucleotides of exon 9 due to the activation of a downstream cryptic acceptor splice site in 1154-1155 nucleotide positions, resulting in a downstream stop codon at aa position 402. RNA analysis of c.625+19 C>T variation showed a small amount of alternative splicing with skipping of exon 4, resulting in a stop codon at aa position 211. Our cases present most of features of the severe form of disease, including early onset with chronic neutropenia, frequent infections, and inflammatory bowel disease. Our results suggest a founder effect for glycogen storage disease type Ib that facilitates diagnosis using mutation analysis, sparing patients from liver biopsy. DNA-based diagnosis will enable us to make accurate determination of carrier status and prenatal diagnosis, thus improving genetic counseling.

Twenty-four novel mutations in Wilson disease patients of predominantly Italian origin.

Herein we report the results of mutation analysis of the ATP7B gene in a group of 134 Wilson disease (WD) families (268 chromosomes) prevalently of Italian origin. Using the SSCP and sequencing methods we identified 71 disease-causing mutations. Twenty-four were novel, while 19 more mutations already described, were identified in new populations in this study. A known mutation G591D showed a regional distribution, since it was only detected in 38.5% of the analyzed chromosomes in WD patients originating from Apulia, a region of South Italy. Detection of new mutations in the ATP7B gene increases our capability of molecular analysis that is essential for early diagnosis and treatment of WD.

Randomized phase II trial of deferasirox (Exjade, ICL670), a once-daily, orally-administered iron chelator, in comparison to deferoxamine in thalassemia patients with transfusional iron overload.

BACKGROUND AND OBJECTIVES: Iron accumulation is an inevitable consequence of chronic blood transfusions and results in serious complications in the absence of chelation treatment to remove excess iron. Deferoxamine (Desferal, DFO) reduces morbidity and mortality although the administration schedule of slow, parenteral infusions several days each week limits compliance and negatively affects long-term outcome. Deferasirox (Exjade, ICL670) is an oral chelator with high iron-binding potency and selectivity. In a phase II study, the tolerability and efficacy of deferasirox were compared with those of DFO in 71 adults with transfusional hemosiderosis. DESIGN AND METHODS: Patients were randomized to receive once-daily deferasirox (10 or 20 mg/kg; n=24 in both groups) or DFO (40 mg/kg, 5 days/week; n=23) for 48 weeks. Results. Both treatments were well tolerated and no patient discontinued deferasirox due to drug-related adverse events. The reported frequency of transient, mild to moderate gastrointestinal disturbances was higher in the deferasirox group than in the DFO group, but these disturbances settled spontaneously without dose interruption in all patients. Decreases in liver iron concentration (LIC) were comparable in the deferasirox 20 mg/kg/day and DFO groups; baseline values of 8.5 and 7.9 mg Fe/g dw fell to 6.6 and 5.9 mg Fe/g dw, respectively, by week 48. Deferasirox showed a plasma elimination half-life of 8-16 hours, supporting its once-daily administration. INTERPRETATION AND CONCLUSIONS: Deferasirox at daily doses of 10 or 20 mg/kg was well tolerated and, at 20 mg/kg, showed similar efficacy to DFO 40 mg/kg in terms of decreases in LIC.

The canine copper toxicosis gene MURR1 is not implicated in the pathogenesis of Wilson disease.

BACKGROUND: It has recently been demonstrated that the Wilson disease (WD) protein directly interacts with the human homolog of the MURR1 protein in vitro and in vivo, and that this interaction is specific for the copper transporter. The aim of the present study was to clarify the role of MURR1 in the pathogenesis of WD as well as in other WD-like disorders of hepatic copper metabolism of unknown origin. METHODS: Using the single-strand conformation polymorphism (SSCP) method followed by sequencing, we analyzed the 5' untranslated region (UTR) and three exons of the MURR1 gene in three groups of patients: 19 WD: patients in whom no mutations were detected in the ATP7B gene, 53 WD: patients in whom only one mutation in the ATP7B gene was found, and 34 patients in whom clinical and laboratory data suggested a WD-like disorder of hepatic copper metabolism of unknown origin. RESULTS: We detected in these patients six rare nucleotide substitutions, namely one splice-site consensus sequence and one missense and four silent nucleotide substitutions. All substitutions except one were found in the heterozygous state. No difference in the frequencies of the various substitutions was observed between patients and controls. CONCLUSIONS: These data suggest that the MURR1 gene and its protein product are unlikely to play a primary role in the pathogenesis of Wilson disease. More extensive studies with larger numbers of clinically homogeneous patients should be carried out to establish whether nucleotide alterations in the MURR1 gene may have a role in causing WD or WD-like disorders or act as modifying factors in the phenotype variability in WD.