The splicing defect at IVS-I-110 is by far (43.15%) the most common beta-thalassaemia mutation in Greece. The - 117 (G-->A) Agamma hereditary persistence of fetal hemoglobin (Greek HPFH) is also the most frequent nondeletional HPFH in Greece. We report a case in which these two defects co-segregates. She is a healthy female where the total Hb is 12.3 g/dl with 51% HbF and normal HbA2. Her Ggamma/Agamma ratio is 35:65 differing from that of 10 simple heterozygotes for the Greek HPFH who have ratio of 8:92. Molecular analysis of the beta-globin genotype revealed the presence of the IVS-I-110 beta+ mutation in trans to the -117 G-->A Greek HPFH. Both mutations are linked to Ia. Her father has Greek HPFH in trans to the -158 C-->T on the Ggamma promoter, which is linked with haplotype IIIalpha. He has 13% HbF with a Ggamma/Agamma ratio 32:68. Her sister is a compound heterozygote for the IVS-I-110 mutation in trans to the - 158 C-->T, with HbF levels of 3% and a Ggamma/Agamma ratio 72:28.

Hydroxycarbamide (hydroxyurea; HU) is now considered as the main pharmacological agent capable to prevent the painful crises, to reduce the frequency and length of hospital admissions, and to improve the quality of life of patients with sickle-cell disease (SCD); whether HU can prevent the severe chronic complications or modify the mortality of these patients remains still an interesting unresolved question. The present study aims to evaluate the above effects of HU in a large number of SCD patients who received HU over long period of time and were followed in a single Center. To this effect, we evaluated the records of 330 patients with SCD who have been followed in the Thalassemia Center of Laikon Hospital over the last twenty years (136M/194F; median age: 42 years, range: 20- 76 years); 34 with homozygous HbS (SS) and 296 compound heterozygotes for HbS and beta-thalassemia. Of the latter, 131 patients had HbS/beta0-thal and 165 HbS/beta+-thal (107 with the IVSI-110 and 58 with the IVSI-6 thalassemic mutation). Administration of HU was started as early as 1991; progressively, the total number of patients who received HU reached 131 (“group A”); the remaining 199 were treated conventionally (“group B”). The mean age of the patients in the two groups was similar (mean ±SD: 43.3±9.9 years for group A and 44.1±12.6 years for group B) as well as the levels of HbF. The usual dosage of HU was 20 mg/kg/day, except for some patients, where toxicity or lack of effectiveness imposed modifications of the dosage in the range of 15 to 35 mg/kg/day. The median follow-up period was 8 years (range: 0.1–17 years) for the patients who received HU and 5 years (range: 0.1–18 years) for those who did not receive HU. Apart rare exceptions, patients reported every 4–8 weeks to the Outpatient Clinic, where they had a thorough clinical and laboratory evaluation, including FBC and reticulocytes, LDH, bilirubin, other basic biochemistries and HbF using conventional techniques. The percentage of HbS/HbS, HbS/beta0-thal, and HbS/IVSI-110 patients who were given HU was 70%, 49% and 40% respectively; in contrast, very few HbS/IVSI-6 compound heterozygotes had symptoms justifying use of the drug (3.6%). Patients receiving HU showed a dramatic reduction of the frequency of severe painful crises (from 7.4±6.5 episodes per year pre-HU to 0.2±0.4 episodes post-HU; p<0.001) along with a significant reduction of transfusion requirements (mean number of administered packed red cell units: 1.5±5.9 per year pre-HU diminishing to almost zero during HU treatment; p<0.001). These results led to a significant reduction of hospital admissions from 2.1±2.9 per year pre-HU to 0.06±0.02 per year post-HU (p<0.001). Moreover, we noticed a significant reduction of the frequency of chest syndrome from 6.1% pre-HU to 0.8% during the HU period (p=0.013). In contrast, we failed to observe any significant differences in the frequency of aseptic avascular necrosis (p=0.219) or cerebrovascular episodes among the patients who received and those who did not receive HU (p=0.65). Importantly, the death rate in the group of patients receiving HU was significantly lower than that observed among patients who were conventionally treated (12 deaths in group A vs. 47 in group B; 9.1% vs. 23.6%, respectively; p=0.001). The probability of 10-year survival was 91% for the patients treated with HU, while that of the patients conventionally treated was only 70% (Figure). This difference is further magnified if one considers that prior to starting HU the patients of group A had significantly higher transfusion requirements, frequency of painful crises, yearly number of admissions to hospital and intensity of hemolysis in comparison to the group of patients who were not given HU (p=0.004, <0.001, <0.001, and 0.01, respectively). Factors that predicted for a superior survival in the HU group include the increased level of total hemoglobin, a reduced platelet and leukocyte count, increased HbF levels, low reticulocyte counts and low bilirubin and serum LDH levels at 6 months post-HU administration. Side-effects of HU therapy were minimal, predictable and easily manageable. Our study provides evidence that, in addition to reducing the number of painful crises, the frequency of hospital admissions and the transfusion requirements, the long-term administration of HU in adult patients with sickle cell syndromes can improve survival. These results highlight the beneficial effect of HU in SCD and raise the issue of expanding the use of HU in all patients with these conditions. Figure Figure

Osteoporosis represents an important cause of morbidity in adult patients with thalassemia. Its pathogenesis is multifactorial, and includes mainly bone marrow expansion, endocrine dysfunction and iron overload. Bone metabolism is altered in thalassemia. Osteoclast function is elevated, while osteoblast activity seems to be reduced and thus the balance of bone remodeling is in favor of bone loss. The exact mechanisms of osteoblast dysfunction have not been fully clarified to-date. Wingless-type (Wnt) signaling is an important pathway for osteoblast differentiation. Dickkopf-1 (Dkk-1) protein is an inhibitor of Wnt pathway and is implicated in the pathogenesis of several bone disorders. Collagen type-I is the main structural protein of the bone. The collagen type-I alpha (COLIA)-1 specific protein (Sp)-1 polymorphism has been related to osteoporosis in thalassemia. The aim of this study was to evaluate the serum levels of Dkk-1 in patients with thalassemia-induced osteoporosis who receive therapy with zoledronic acid (ZOL) and evaluate possible correlations with clinical and laboratory data, including the COLIA-1 Sp1 polymorphism. Sixty-six patients (21M/45F; median age 35.5 years) with thalassemia and osteoporosis were studied. Patients were blindly randomized to receive ZOL at a dose of 4 mg, iv, in 15 min infusion, every 6 months (group A, n=23) or every 3 months (group B, n=21), or to receive placebo every 3 months (group C, n=22) for a period of one year. All patients received oral calcium (500 mg) during the treatment period. Dkk-1 was measured at baseline and after 12 months of therapy using ELISA methodology (Biomedica Medizinprodukte, Wien, Austria) along with a series of serum bone remodeling indices: bone resorption markers [C-telopeptide of type-I collagen (CTX), tartrate-resistant acid phosphatase isoform-5b (TRACP-5b)], bone formation markers [bone-specific alkaline phosphatase (bALP), osteocalcin, and C-terminal propeptide of collagen type-I (CICP)], and osteoclast regulators [receptor activator of nuclear factor-kappa B ligand (RANKL), osteoprotegerin (OPG), and osteopontin]. The above bone markers were also evaluated in 30, age- and gender-matched, healthy controls. The G-->T mutation at base 1 of intron 1 at the binding site of the Sp1 transcription factor of the COLIA-1 gene was detected by polymerase chain reaction using mutagenesis primers followed by restriction enzyme analysis in all patients. BMD of the lumbar spine (L1-L4), femoral neck (FN) and wrist (W) was determined using DEXA, before and 12 months after treatment. At baseline, all patients had increased serum levels of Dkk-1 (mean±SD: 39±17.1 pmol/L) compared to controls (27.4±9.7 pmol/L; p<0.0001). Furthermore, thalassemia patients had increased values of CTX (p<0.0001), bALP (p<0.001), CICP (p=0.003), sRANKL (p=0.02), and OPG (p=0.001) compared to controls. Results for the COLIA-1 Sp1 polymorphism were available for 53 patients. Seventeen patients (32%) were G/T heterozygotes at the polymorphic Sp1 site (Ss), while 3 (5.6%) were T/T homozygotes (ss). Dkk-1 serum levels correlated with L1-L4 BMD (r=−0.290, p=0.022) and W-BMD (r=−0.415. p=0.001), but also with TRACP-5b (r=0.310, p=0.011) and bALP levels (r=−0.289, p=0.018). Ss and ss patients tended to have lower L1-L4 BMD compared with SS patients (p=0.09). No significant correlations were observed between Ss and ss patients with the measured bone markers or the response to ZOL. As reported previously, patients of group B experienced an increase of L1-L4 BMD, while no other alterations in BMD were observed in the 3 studied groups after 12 months of ZOL administration. Interestingly, patients of groups A+B showed a strong reduction of Dkk-1 after 12 months of ZOL (from 39.6±16.6 to 28.9±16.3 pmol/L; p=0.004); indeed they almost normalized Dkk-1 levels (no difference from control values). In contrast, patients of group C showed a borderline increase of Dkk-1 (from 33.1±16.8 to 40.1±23.2 pmol/L, p=0.08). These results show for the first time in the literature that Dkk-1 is increased in the serum of patients with thalassemia and osteoporosis, correlates with their BMD and is reduced post-ZOL therapy. This Dkk-1 elevation may be at least partially responsible for osteoblast dysfunction in thalassemia and reveal a novel possible target for the development of new agents for the management of bone loss in thalassemia patients.