Genetic heterogeneity of beta thalassemia mutations in Kahramanmaraş province in Southern Turkey: preliminary report

Background: Beta thalassemia is one of the most common autosomal single-gene disorders in the world. The prevalence of the disease is in the “thalassemia belt” which includes the Mediterranean region of Turkey; throughout the country the gene frequency is estimated to be 2.1%, but in certain regions, this figure increases to 10%. Aim: In this first study, we aimed to determine the frequency of β-thalassemia trait and distrubition of mutations in Kahramanmaraş province, which is located in the southern part of Turkey. Materials and Methods: In this study; 5 ml blood samples was taken from 14 thalassemic patients and their relatives who were taking care of Sutcu Imam University Hospital at Kahramanmaraş. Also, we collected blood samples from 245 adults for screening beta thalassemia trait. Haematological data were obtained by cell counter.  HbA2 was determined by HPLC. Ten common mutations were screened by ARMS  (Amplification Refractory Mutation System) method. These β-thalassemia mutations are -30 (T>A), Fsc8 (-AA), Fsc8/9 (+G), IVS1-1 (G>A), IVS1-5 (G>C), IVS1-6 (T>C), IVS1-110 (G>A ), Cd 39 ( C>T), IVS2-1 (G>A), IVS 2-745 (C>G). A rare mutation; Fsc44 (-C) was charecterized by DNA sequencing. Results: Ten patients were detected as homozygous for IVS1-110 (seven cases), Fsc 44 (two cases) and IVS1-5 (only one case). Rest of the 4 patients were double heterozygous (two: IVS1-110/IVS1-6, one: Fsc8/Fsc8-9, one: IVS2-1/IVS1-5). In 245 adult, five  β-thalassemia trait were detected by screening survey.  Conclusion: Sixteen alleles were detected as IVS1-110 in 57.1%. It was seen the most common mutation in Kahramanmaraş. Seven different β-thalassemia mutations were found in this study. Each of 10 families have only one thalassemic patient, other two families have double thalassemic patient in total 12 family.

SURGICAL MANAGEMENT OF TRAUMATIC VERTEBRAL BODY COMPRESSION FRACTURE WITH CORD COMPRESSION IN A THALASSEMIC PATIENT. CASE REPORT

Extramedullary hematopoiesis associated with thalassemia causing spinal cord compression is a rare event in the course of the disease. Management of these patients remains controversial. We present a case of traumatic vertebral body compression fracture with cord compression in association with thalassemia, which was treated successfully with spinal decompression and fusion, xation using implants and bone graft.

Role of Exosomes in Hepcidin Regulation in β-Thalassemia

β-thalassemia is characterized by ineffective erythropoiesis and iron overload. Ineffective erythropoiesis causes iron overload by suppressing hepcidin, the main negative regulator of iron absorption and recycling, and is mediated by secretion of erythroferrone from bone marrow cells. Targeted treatment for ineffective erythropoiesis is unavailable. Furthermore, molecular mechanisms involved in ineffective erythropoiesis and the details of how erythropoiesis regulates iron metabolism are incompletely understood. Lastly, while loss of erythroferrone in β-thalassemic mice leads to partial reversal of iron overload [Kautz Blood 2015], erythroferrone ablated mice are still able to suppress hepcidin after phlebotomy [Kautz Nat Med 2014]. These finding provide evidence of additional regulatory crosstalk between erythropoiesis and iron metabolism. We hypothesize that bone-marrow derived exosomes regulate iron metabolism by modulating hepcidin. Exosomes are small extracellular vesicles derived from multi-vesicular bodies forming intraluminal vesicles which fuse with the plasma membrane and are released by many different cell types [Thery Nat Rev Immun 2002]. In light of their capacity for cell-cell communication and modification of the microenvironment, exosomes have been widely studied in multiple diseases [Valadi Nat Cell Bio 2007] despite which, erythropoiesis-derived exosomes and their role in iron metabolism regulation remain unexplored. Our preliminary data demonstrate that phlebotomy in wild type mice results in increased exosome concentration in serum and that exosomes are increased in th3/+ mouse serum (Figure 1a). Furthermore, hepcidin induction by exosome depleted-FBS is decreased relative to FBS (Figure 1b), and exosomes isolated from FBS induce hepcidin in a dose response manner in vitro (Figure 1c). We thus propose to explore the mechanistic relationship between exosomes and hepcidin regulation in β-thalassemia. Serum samples from patients with β-thalassemia major and age / gender matched controls were collected; all patients were treated with iron chelation therapy and all samples were collected immediately prior to transfusion. Exosome fractions were purified and analyzed in patients relative to controls. Although there is no difference in the number of exosomes or mean particle size within the exosomal fraction, exosomal protein content per volume of serum is significantly decreased in patients relative to controls. In addition, the treatment of primary wild type mouse hepatocytes with sera from patients and controls reveals the expected relatively decreased hepcidin induction in β-thalassemic patient sera treated hepatocytes relative to control sera; a similar difference is seen in hepatocytes treated with exosome-depleted sera from patients and controls (Figure 2a). These findings suggest that hepcidin suppression is a consequence of the exosome-free portion of serum from control and β-thalassemic samples. Furthermore, only exosomes derived from β-thalassemic patient sera induces hepcidin expression in primary wild type mouse hepatocyte cultures (Figure 2b). Lastly, exosomes derived from β-thalassemic patient sera do not affect ERK1/2 and STAT3 signaling in primary hepatocytes but increase SMAD1/5/8 (Figure 2c) and decrease AKT signaling (Figure 2d). Taken together, these findings demonstrate that exosomes enhance hepcidin expression via increased SMAD1/5/8 signaling, that increased hepcidin may influence multiple signaling pathways by an autocrine mechanism in response to exosomes, and that exosomes counterbalance hepcidin suppressive substances in the exosome-depleted serum from β-thalassemic samples. Our studies provide novel insights into the important previously unexplored mechanism of hepcidin regulation by exosomes in both physiologic and pathologic states. Disclosures Coates: apo pharma: Consultancy, Honoraria, Speakers Bureau; vifor: Consultancy, Honoraria; celgene: Consultancy, Honoraria, Other: steering committee of clinical study; agios pharma: Consultancy, Honoraria. Ginzburg:La Jolla Pharma: Membership on an entity's Board of Directors or advisory committees.

Compressive Dorsal Myelopathy Secondary to Extramedullary Hematopoiesis in a Thalassemic Patient

Background. Extramedullary hematopoiesis (EMH) is one of the rare causes of spinal cord compression (SCC). It results from noncancerous proliferation of hematopoietic tissue outside the bone marrow as a compensatory mechanism for ineffective erythropoiesis. It occurs in the paraspinal area in 11–15% of thalassemic patients in intermediate and severe cases causing a paraspinal compressive mass. We present a rare case of spinal EMH with thoracic cord compression in a 22-year-old female with beta thalassemia who presented with paraparesis and we provide a review of literature. Case Report. A 22-year-old female patient with a known history of beta thalassemia presented with subacute onset of weakness and numbness of both lower limbs with a sensory level at T6. Magnetic resonance imaging (MRI) of the dorsal spine showed cord compression secondary to paraspinal EMH from T2 to T9 with most prominent compression over T5. She was managed with blood transfusion and low-dose radiotherapy. Conclusion. Although rare, EMH should be suspected in thalassemic patients presenting with paraplegia. Treatment with blood transfusions is usually effective. Other options include radiotherapy, surgery, hydroxyurea or a combination of these modalities.

Hepatitis C Virus Infection, but Not Hepatic Iron Overload Is the Dominant Risk Factor for the Manifestation of Hepatocellular Carcinoma Among Greek Thalassemic Patients

Background: Effective iron chelation and other supportive treatment have a substantial impact on survival prolongation of thalassemic patients, who may reach late adulthood, and therefore, may manifest various neoplastic disorders. However, no systemic analysis of the prevalence of neoplastic disorders in this patient population has been performed. Aims: We systematically analyzed all cases of malignant neoplastic disorders, occurred in a large cohort of Greek thalassemic patients and investigated for possible underlying predisposing factors. Patients and methods: Data of 3652 patients with hemoglobinopathies (Group I: beta-thalassemia homozygous N=1981, Group II: thalassemia Intermedia N=746, Group III: sickle-cell disease +/- beta-Thalassemia n=751, Group IV: hemoglobinopathy-H N=174) were retrieved, followed up at 24 specific Hospital Units, between 1985 and 2018. Totally, 165 cases of a malignant disorder were identified (overall prevalence 4.52%). The significance of the following predisposing factors was investigated: familial history of neoplasia, occupational exposure to known mutagens, previous autoimmunity, previous splenectomy, tobacco smoking, alcohol use, HBV, HCV or HIV infection, iron overload, hydroxyurea treatment, previous irradiation for extramedullary hematopoietic tumors and systemic use of androgens/estrogens. Results: Patients were 84 males and 81 females with a median age at diagnosis of the neoplastic disorder of 45 years (range 9-73 years). Higher prevalence of neoplasia was noted among patients of Groups I and II (4.99% vs 3.03% among patients of Groups III and IV, p<0.05). Table 1 shows histological diagnosis of the 165 neoplastic disorders, of which 139 (84.2%) were solid tumors and 26 (15.8%) hematological malignancies. The dominant malignancy was hepatocellular carcinoma, diagnosed in 63 patients, followed by thyroid cancer (17 cases), non-Hodgkin's lymphoma (13 cases), and renal cell carcinoma (10 cases). There was a strong positive association between hepatitis C virus infection and hepatocellular carcinoma, and a negative one between HCV infection and thyroid and renal cancer. Active HCV infection was found in 81 patients (49.1%) compared to an estimated prevalence of about 25% among the whole thalassemic patient population. Hepatocellular carcinoma was more frequently diagnosed in men (M/F ratio 1.86) of the fourth and fifth decade (median age 45 years) with thalassemia homozygous or intermedia (89% of the cases), with long-standing, untreated HCV infection (76%), irrespective of the burden of hepatic iron load, estimated with MRI T2*. Indeed, no difference in the occurrence of hepatocellular carcinoma, as well as of any other type of cancer was found, in relation to Liver Iron Concentration (LIC). Moreover, no preponderance of any HCV genotype was identified, but interestingly, all 1b HCV genotype-associated neoplasms were hepatocellular carcinoma (7 cases). Finally, no association between any of the remaining potential risk factors with the manifestation of any specific neoplastic disorder was observed. Discussion: In our large thalassemic patient cohort, representative of the whole country of Greece, we have identified increased prevalence of four types of cancer. Besides hepatocellular carcinoma, we have unexpectedly encountered high prevalence of thyroid and renal cancer, as well as of all types of lymphomas. These primary findings deserve further investigation, since, excluding hepatocellular carcinoma, no prominent or speculative causality can be currently attributed for the remaining malignancies. Disclosures Kattamis: Vifor Pharma: Consultancy; ApoPharma: Honoraria; CELGENE: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Unc 51–like autophagy-activating kinase (ULK1) mediates clearance of free α-globin in β-thalassemia

Erythroid maturation is coordinated to maximize the production of hemoglobin A heterotetramers (α2β2) and minimize the accumulation of potentially toxic free α- or β-globin subunits. In β-thalassemia, mutations in the β-globin gene cause a build-up of free α-globin, which forms intracellular precipitates that impair erythroid cell maturation and viability. Protein quality-control systems mitigate β-thalassemia pathophysiology by degrading toxic free α-globin. We show that loss of the Unc 51–like autophagy-activating kinase geneUlk1in β-thalassemic mice reduces autophagic clearance of α-globin in red cell precursors and exacerbates disease phenotypes, whereas inactivation of the canonical autophagy geneAtg5has minimal effects. Systemic treatment with rapamycin to inhibit the ULK1 inhibitor mTORC1 reduces α-globin precipitates and lessens pathologies in β-thalassemic mice, but not in those lackingUlk1. Similarly, rapamycin reduces free α-globin accumulation in erythroblasts derived from β-thalassemic patient CD34+hematopoietic progenitors. Our findings identify a new, drug-regulatable pathway for ameliorating β-thalassemia.One Sentence SummaryRapamycin alleviates β-thalassemia by stimulating ULK1-dependent autophagy of toxic free α-globin.

Thalassaemiás betegen észlelt anaemia perniciosa – a diagnózis nehézségei

The bone marrow aspiration, which was done in a leukopenic, hypochromic, microcytic, progressive anemic, thalassemic patient, revealed megaloblastic morphology. The low level of vitamin B12 and the reticulocytosis following the B12 supportation strenghtened the diagnosis of pernicious anemia. The set of the right diagnosis has been delayed by the fact that even in severe anemia one could not obtain the typical signs of B12 deficiency, having a hypochromic, microcytic erythrocyte morphology, due to the thalassemia minor disorder. Orv Hetil. 2018; 159(33): 1368–1371.

Spontaneous fertility in a male thalassemic patient after allogeneic hematopoietic cell transplantation

Patients with thalassemia major who received allogeneic hematopoietic cell transplantation are at increased risk of gonadal insufficiency and reduced fertility due to the toxicity of both the transfusional iron overload and the gonadotoxic effects of drugs used in the conditioning regimen. We present a case of an ex-thalassemic patient with spontaneous recovery of spermatogenesis that fathered a healthy, term male neonate. Maternal hemoglobin electrophoresis was within normal limits. At the age of 9.5 years the patient underwent hematopoietic cell transplantation. The conditioning therapy included busulfan (16 mg/kg) and cyclophosphamide (200 mg/kg). No irradiation was administered. Thirty-two days after the hematopoietic cell transplantation the patient developed acute graft-versus-host disease needing long-term treatment with methylprednisolone, cyclosporine and immunoglobulin. Although consecutive semen analyses after the hematopoietic cell transplantation revealed azoospermia, the last semen analysis before conception, at the age of 33 years, was improved and normal follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (Te) levels were detected. The current pregnancy was the result of physical conception. In this case, it seems that thalassemia major along with the respective treatment prior to- and posthematopoietic cell transplantation did not irreparably impair spermatogenesis, probably due to the pre-pubertal time frame they were implemented. 对于接受异基因造血细胞移植的重型地中海贫血患者，由于输注性铁过载的毒性和预处理方案中所用药物性腺毒性作用这两方面的原因，都使其面临更大的性腺功能不全风险和更低的生育力。本文报道一例精子发生出现自然恢复的原重型地中海贫血患者，他成功孕育出一个健康的足月男婴。母体血红蛋白电泳在正常范围内。患者在9岁半时接受了造血细胞移植。预处理治疗包括白消安（16 mg/kg）和环磷酰胺（200 mg/kg）。未给予照射。造血细胞移植32天后，患者出现急性移植物抗宿主病，需要长期使用甲基强的松龙、环孢素和免疫球蛋白治疗。虽然造血细胞移植后连续的精液分析显示无精子症，但在33岁时受精前的最后一次精液分析有所改善，经检测发现卵泡刺激素（FSH）、黄体生成素（LH）和睾酮（Te）水平正常。目前的怀孕是自然受孕的结果。在这个病例中，看来重型地中海贫血以及造血细胞移植前后相应的治疗并没有对精子发生造成不可恢复的破坏，这可能是由于移植时处于青春发育期前时间段的原因。