ALBERT

Description: BRCA1 is essential for repair of DNA double-strand breaks by homologous recombination, and hence for survival. Complete loss of its function is lethal during early embryonic development. Patients who are compound heterozygous for BRCA1 truncating mutations and missense alleles that retain some DNA repair capacity may survive, albeit with very high risk of early onset breast or ovarian cancer and features of Fanconi anemia. However, a mechanism enabling survival of patients homozygous for BRCA1 truncating mutations has not been described. We studied two unrelated families in which four children presented with multiple congenital anomalies and severe chromosomal fragility. One child developed T cell acute lymphocytic leukemia (ALL), and a second child developed neuroblastoma. Each of the four children was homozygous for a nonsense mutation in BRCA1 exon 11. Homozygosity for the nonsense mutations was viable thanks to the presence of a naturally occurring alternative splice donor in BRCA1 exon 11 that lies 5′ of the mutations. The mutations did not affect the alternative splice site, but transcription from it produced an in-frame BRCA1 message with deletion of 3,309 bp. The translated BRCA1 protein was only 40% of normal length, but with intact N- and C-terminal sequences. These patients extend the range of BRCA1-related phenotypes and illustrate how naturally occurring alternative splicing can enable survival, albeit with severe consequences, of otherwise lethal genotypes of an essential gene.

Description: Patients with beta-thalassemia major (BTM) are prone to tissue iron overloading in case that not adequately chelated. Among the iron chelators, development of oral chelators have improved patients’adherence to treatment. Deferasirox is a tridentate iron chelator, used once daily with a half life of 8-16 hours. The negative chelation effect is achieved at doses above 30 mg/kg/day and the currently FDA-approved maximum dose for use in patients is 40 mg/kg/day. However, some of the deferasirox side effects are dose-dependent increasing the occurences of adverse events at high doses. Additionally, although there is data that non-transferrin bound iron is effectively decreased by once daily dosing, the iron chelation effects of the drug may increase since in some of the patients half-life of the drug has been reported to be even less than 7 hours. Herein, we compared the iron chelation effect and tolerability of deferasirox in the same patients who were using deferasirox at a dose of 40 mg/kg/day. The patients with BTM who were under iron chelation with deferasirox at a dose of median 40 mg/kg/day (38-41) once daily for at least 6 months were included (n=10). These patients were receiving deferasirox at a maximum dose in the enrollment to the study related to either serum ferritin levels above 1500 ng/ml or moderate to severe iron loading in cardiac or liver tissues. These patients were put on a twice daily regimen of the same dose and followed up at a median time period of 7 months (4-17). The serum ferritin, ALT, creatinine levels and T2* MRI of heart and liver were obtained at the beginning of the twice daily dosing and by the end of the follow-up time. Patients were given a questionairre to investigate the tolerability and satisfaction of once daily and twice daily use. The median age of the study group was 21 years (3-34), hal were males. The patients’ serum ferritin, cardiac and hepatic iron loading levels were summarized in Table 1. There was a statistically significant decrease in serum ferritin levels with twice daily use of deferasirox compared to once daily use of the same dose. The initial and follow-up ALT and serum creatinine levels did not differ significantly (p〉0.05). None of the patient required a dose reduction or cessation of the drug related to a toxicity. The major tolerability concern of the patients in once daily dosing was nausea in 2 of the patients (20%). After twice daily dosing the major concern of the patients was twice daily use of the drug itself in 2 patients (20%). None of the patients reported nausea in twice-dosing. The patients’ satisfaction survey in the end of the study for once or twice daily use was for once daily use revealed preference for twice-use in 5 (50%) of the patients for either no nausea (n=2) with twice-use or better decrease in serum ferritin levels (n=3). Three (30%) preferred once daily use as a better way, related to lesser drug use. Two patients reported that there were no difference in terms of satisfaction. In conclusion, twice daily use of deferasirox at higher doses is much better tolerated and causes a better decline in serum ferritin levels of already high iron burden. Further studies in larger sample groups may be more definitive. Table 1. The initial and follow-up iron overloading evaluations Mean ± SD (Range) p Initial SF (ng/ml) Follow-up SF 2020±983 (1016-4128) 1533±1026 (521-4003) 0.047 Initial cardiac T2* MRI (ms) Follow-up cardiac T2* MRI 25.4±8.6 (14.6-38) 21.5±7.8 (13.8-30) 0.068 Initial liver T2* MRI (ms) Follow-up liver T2* MRI 3.6±1.9 (1.3-8) 6.7±5.1 (2.1-13.6) 0.465 SF: serum ferritin; SD: standard deviation Disclosures No relevant conflicts of interest to declare.

Description: Introduction Juvenile myelomonocytic leukemia (JMML) is a unique, aggressive hematopoietic disorder of childhood caused by excessive proliferation of cells of monocytic and granulocytic lineages. Childhood JMML is classified as a bridging disorder between myelodysplastic syndrome (MDS) and myeloproliferative diseases.More than 95% of JMML patients are diagnosed under the age of six years. Children with JMML mostly present with hepatosplenomegaly, lymphadenopathy, bleeding, anemia, fever, recurrent infections, rash, failure to thrive and pulmonary disease. Approximately 90% of patients carry either somatic or germline mutations of PTPN-11,K-RAS,N-RAS,CBL or NF-1 in their leukemic cells. Aim We want to describe the clinical and laboratory features in 55 cases of JMML seen at the Hacettepe University Pediatric Hematology Department during a 18 year period (January 2000-June 2018). Patients & Methods There were 38 males and 17 females aged between 1 months and 168 months (median 36 months). On admission mean Hb, WBC and platelet was found to be 9.1±1.9 g/dl (range 5.7-14.6g/dl), 38.7±4.3 x10 3 µ/L (range 1.4 - 214 x10 3 µ/L) and 156 ± 7.8x 109 range (8-1598x109/L) , respectively.Results of cytogenetic analysis showed monosomy 7/7qdel in 16 cases.Somatic PTPN11 mutation was found in 23 children whereas somatic KRAS mutation in 7 and germline mutation in one case, somatic NRAS mutation in 3 cases and c-CBL mutation in 5 cases. On admission 49% of patients had no blast cells on the peripheral blood smear.But 3 of 55 patients had 100% blast cells in peripheral blood smear.Monosomy 7 mutation was positive in all of these 3 patients and one of these case had an history of familial MDS and a positive GATA mutation, one other had NF-1 mutation.All three patients were died despite hematopoietic stem cell transplantation(HSCT). On admission, 7 out of 55 patients had 〉30% blast cells in bone marrow aspiration and 3 of them had %100 blast cells on the peripheral smear. The rest of this group except one who had a positive KRAS mutation and diagnosed as AML-M4 were treated with HSCT and 4/6 were stil alive.On the other hand, 7 out of 55 patients had 20-30% blast cells in bone marrow aspiration on admission and none of these patients had neither monosomy 7/7qdel nor trisomy 8 mutation. c-CBL mutation was found to be positive in 5 case and all were still alive (two siblings with c-CBL and one other patient had a diagnosis of juvenile xanthogranulamatosis), and one patient with c-CBL mutation had a diagnosis of portal hypertension.On the other hand two siblings with monosomy 7 have a diagnosis of GATA mutation and both were died after HSCT.Almost 40% of this pediatric group (20/55) were died after a median follow up time 16 months (1-211 months). Discussion JMML is a clonal hematopoietic disorder of infancy and early childhood which results from oncogenic mutations in genes involved in the Ras pathway and allogeneic HSCT remains the only curative treatment more than 50% of patients.However, the timing of diagnosis and treatment is critical to outcome.Prompt HSCT is recommended for all children with NF1, somatic PTPN11 and KRAS mutations, and for most children with somatic NRAS mutations.'Watch and wait' strategy is usually for the group of patients with germline CBL mutations, specific somatic NRAS mutation, and in Noonan syndrome patients, cause spontaneous resolution has been reported in this group. Our results were compatible with the literature , however it seems that in our group despite allogeneic HSCT, relapse is the main treatment failure. Disclosures Niemeyer: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: In the present study, molecular pathologies of 8 patients from unrelated 4 consanguineous and one non-consanguineous families were characterized; 4 novel and 1 previously reported homozygous mutations were identified in the P5N-1 gene. 1) A frameshift 700–701InsA mutation in exon 9 was identified in a 40 years old patient who has been followed as hereditary spherocytosis (HS) for many years because of marked spherocytes and highly increased osmotic fragility. He underwent splenectomy (11yr) and cholecystectomy (26yr). Afterwards, Hb level stayed between 8.5–11g/dl. Co-existence of Gilbert syndrome 7/7 TATA repeat genotype accounts for very high serum bilirubin level (18–28mg/dl). 2) A splicing mutation (IVS8+1 G〉A) was found in a 30 years old patient who was followed as HS due to associated spherocytosis and slightly increased osmotic fragility. She received blood transfusions regularly, underwent splenectomy and cholecystectomy (14yr). Afterwards Hb stayed at 9.6–10.5 g/dl. Co-existence of Gilbert 7/7 genotype accounts for the high bilirubin (15mg/dl). 3) A splicing (IVS7+1 G〉A) and a silent (T275C in exon 6) mutations were detected in 2 siblings. Despite non-consanguinity, identification of two homozygous mutations indicated the presence of a common ancestor. The girl received transfusion several times, underwent splenectomy and cholecystectomy(18yr). The boy, diagnosed in family survey (2yr) and underwent splenectomy (10yr), currently has highly increased osmotic fragility. Both had few acanthocytic spherocytes. After splenectomy Hb stayed around 11 gr/dl, bilirubin around 5–6 gr/dl in both. 4) A missense T220C mutation in exon 5 (C74R) was identified in 24 and 21 years old sisters. The elder had iron deficiency anemia and the younger diagnosed in a family survey had retinitis pigmentosa. Hb were around 10g/dl, bilirubin around 2.5–6 mg/dl, few acontocytic spherocytes observed in both. 5) A nonsense T543G mutation (Y181X) in exon 8 was detected in 14 and 7 years old brothers. The mutation was reported previously in another Turkish family, however, haplotype analysis failed to indicate founder effect. The older required blood transfusions while hospitalized several times due to recurrent attacks of cerebral infarcts. Hb fluctuated between 6.5–10 g/dl, bilirubin was around 3 mg/dl. He underwent splenectomy recently (17yr). The younger had iron deficiency anemia, Hb was between 7.2–9.9 gr/dl. Few aconthocytic spherocytes were observed in both. All mutations were screened in at least 100 individuals from a healthy Turkish population and no mutation was detected. In conclusion: 1- The mutation spectrum of P5N-1 gene is quite heterogeneous in Turkish population (7 different mutations in 9 unrelated families so far). 2- P5N-1 deficiency is classified as non-spherocytic hemolytic anemia, however, all patients of this study more or less had spherocyte in the peripheral blood smear. In addition, some patients even had increased erythrocyte osmotic fragility emerging as confounding factor in diagnosis. Taken together, there is a reason to suggest that the possibility of P5N-1 deficiency should be considered in at least some of the patients diagnosed as HS. 3- The co-existence of Gilbert disease could modify the clinical presentation of the disease. This study was supported by Hacettepe University Research Fund (02G116).

Description: Pediatric myelodysplastic syndrome (MDS) is a group of heterogeneous clonal disorder with lesser frequency compared to adults. Additionally, there is much rare data in pediatric age group in relation to presentational findings and treatment. The clinical and laboratory data, in addition to therapeutic interventions and outcomes of 47 patients in a single centre who were diagnosed between January 2001-May 2014 were summarized. Median age of the study group was 2.8 years (0.1-16.8). The most common complaints at presentation included fever (27%), fatigue (17%), bleeding (15%), abdominal distention (13%), in addition to other rare presentational complaints including pallor, rash, vomiting, irritability, jaundice, stridor, abdominal pain and easy bruising. The underlying disorder was established as: neurofibromatosis in 5, Down syndrome in 3, secondary to prior chemotherapy in 2 (ALL and PNET), Fanconi anemia in 1, Jacobsen syndrome in 1, Klinefelter in 1. Final diagnosis was MDS in 22, JMML in 19, hypoplastic MDS in 4 and chemotherapy related MDS in 2. Median Hb, WBC, thrombocyte counts at presentation were 8.7 g/dl (4.1-12.7), 10.3x109/L (1.3-117) and 55x109/L (4-1515), respectively. Of the mutations studied related to MDS in 22 of the patients, k-ras positivity was the most common (23%). The most common cytogenetic abnormality was chromosome 7 related abnormalities (25%). Of the patients, 21 (45%) are alive and of these alive patients 62% are alive subsequent to hematopoietic stem cell transplantation. The patients with pediatric MDS may present with various complaints and they may have underlying genetic diseases causing propensity for MDS. The survival is better among patients who underwent hematopoietic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

Description: BRCA1 and BRCA2 play critical roles in maintenance of genomic integrity, DNA repair, and cell cycle checkpoint control. Deficiency in BRCA-associated DNA interstrand crosslink (ICL) repair is connected to breast cancer susceptibility and Fanconi anemia (FA) which is an autosomal recessive disorder that causes genomic instability along with abnormalities in major organ systems, early-onset bone marrow failure, and a high predisposition to cancer. Homozygous or compound heterozygous mutations in the BRCA2 gene are well associated to severe FA phenotype (FANCD1) and solid tumor development. However, biallelic mutations in BRCA1 are extremely rare suggesting that most combinations of deleterious BRCA1 mutations might be lethal. In this study using whole exome sequencing we report the first homozygote nonsense BRCA1 mutation associated with Fanconi anemia in a three generation Turkish consanguineous family. The afected individual is a five year-old girl who was diagnosed to have stage IV neuroblastoma at the age of two and treated uneventfully, in addition to a surgical intervention for ASD and VSD. During the follow-up she was diagnosed to have FA. Spontaneous chromosamal breakages were high, despite normal MMC and DEB. The physical examination revealed, microcephaly, short stature, microphtalmia, cafe-au-lait spots. Hb 11.2 g/dl, MCV 87.5 fl, WBC: 4.3x109/L and platelets 130 x109/L. She had received no transfusions except for the those during the chemotherapy for neuroblastoma treatment. Growth hormone deficiency was found. Cranial MRI exhibited widespread gliosis areas and prominences in the cerebral cortical sulci. Her elder brother was 13 years-old at the diagnosis. The physical examination revealed mental retardation, microcephaly, microphtalmia, cafe-au-lait spots and undescended testis. Small kidneys and periventricular and subcortical gliotic areas and central adrenal insufficiency were identified. Spontaneous chromosomal breakages were high and further increased with DEB. Both parents and two other siblings were healthy although uterus Ca, esophagus Ca and lung Ca were detected in family members in the history. A homozygous stop codon mutation in exon 10 of BRCA1 gene, c.1151T〉G/ p.Leu384Stop was identified in the whole exome study using Illumina HiSeq 2000 platform. Both afected individuals were homozygote and the mother was heterozygote fort his mutation DNA of the healthy father of affected siblings was not available. To the best of our knowledge there are only two reports that compound heterozygote mutations in BRCA1 causes FA-like subtype (FANCS) (Domchek et al., Cancer Discov. 2013; 3:399-405 and Sawyer et al., Cancer Discov 2015; 5:135-142). In these reports, the patients do not display bone marrow failure, mutation carriers have higher risk of breast and ovarian tumors and lower onset age. However, one of the siblings in our report had mild thrombocytopenia. Also compatible with the previous report, our patients had intellectual disability. Interestingly, one of the siblings in our series had neuroblastoma and family members had uterus, esophagus and lung cancers. Our findings clearly show that homozygous deletorious BRCA1 mutations are vital and molecular analysis expands the spectrum of phenotypes associated with BRCA1 mutations. Disclosures No relevant conflicts of interest to declare.

Description: In the absence of adequate chelation therapy, cardiomyopathy caused by iron overload is the leading cause of death in patients with β-thalassemia major (BTM). Additionally, more than half of the adult patients with BTM suffer from hypogonadism (HG), osteoporosis, diabetes mellitus (DM) or hypothyroidism. The use of iron chelators is the mainstay of treatment in patients with BTM to ameliorate these complications. In this study, we aimed to compare the chelation effects of deferasirox (DFX) and other iron chelators on iron in heart, liver, in addition to pituitary, pancreas and thyroid glands. The study included a total of 37 patients with BTM, who were on the same iron chelator for at least 1 year of duration and above 7 years of age. All of the patients were on iron chelation therapies with either monotherapy with DFX (n=29), desferrioxamine (n=4), deferiprone (n=1) or combination therapy of desferrioxamine and deferiprone (n=3). The mean dose of DFX was 30.8 ± 6.3 mg/kg/day (20-40), the mean dose of desferrioxamine 43.1 ± 5.3 mg/kg/day (39-50) and mean dose of deferiprone was 73.26 ± 9.45 mg/kg/day (70-90). All of the patients were compliant to chelation treatment. Cardiac T2*, hepatic T2*, thyroid T2 and R2, pituitary T2 and R2, pancreas T2* and R2* MRI were ordered twice to the patients in order to measure the accumulation of iron. The median time interval between the two MRI was 6 months (range 6-11 months). The effect of DFX (n=29) on iron measurements in different organs were compared to the effects of other chelators group (OCG) (n=8). The mean age of patients participating in the study was 20.8 ± 6.3 years (7.1-36.8). Of the study group, 7.1% of the patients had DM, 8.1% had hypothyroidism and 13.5% had HG at enrollment. According to our previous study for the cut-off value determinations for iron accumulation in BTM with comparison to healthy controls (data unpublished), all of the patients in both groups were found to have pituitary iron accumulation at initial MRI. The changes in iron measures in various organs were summarized in Table 1, indicating a decrease in cardiac, pituitary and pancreas iron loading in both drug groups in follow-up MRI’s (p〉0.05). On the other hand δ Liver T2* was negative direction indicating a decrease in hepatic iron loading in DFX group, wheras positive in OCG indicating an increase in follow-up, although insignificant (Table 1, p=0.9). In both groups iron loading in thyroid was found to increase in follow-up and there was no difference between drug groups (Table 1). In conclusion, DFX is as effective as other drugs in chelation of iron from cardiac, hepatic, pituitary, pancreas and thyroid. The increase in iron in thyroid gland during follow-up in both groups may indicate that iron chelation may not be as efficient in thyroid as it is in other organs. Although, all patients had pituitary iron accumulation, only 13.5% were found to have HG, indicating that patients become symptomatic only occur after a threshold of accumulation was achieved. Our study is initiative for the measurements of iron accumulation with MRI in thyroid. Table 1. δT2* and δR2 change values between first and second MRI assessments Chelation type Mean±SD Median Range p δ Liver T2 * a (ms) Deferasirox -0.06 -8.5-7.20 0.90 Other chelators 0.79 -0.98-4.40 δ Cardiac T2 * b (ms) Deferasirox -3.83±9.5 0.88 Other chelators -3.2±8.82 δ Pituitary T2 b (ms) Deferasirox -0.7±11.3 0.09 Other chelators -1.4±6.4 δ Pituitary R2 a (Hz) Deferasirox 0.10 -6,20-3,10 0.25 Other chelators 0.20 -4,60-1,30 δ Thyroid R2 a (Hz) Deferasirox -1.4 -6,1-12,7 0.06 Other chelators -0.1 -3,80-8,1 δ Thyroid T2 a (ms) Deferasirox 4.8 -59,8-14,6 0.08 Other chelators 0.4 -20,6-20,1 δ Pancreas T2* b (ms) Deferasirox -7.46±21.6 0.99 Other chelators -7.52±9.63 δ Pancreas R2 * b (Hz) Deferasirox 9.24±45.23 0.11 Other chelators 56.4±73.3 SD: Standard Deviation; aNon-parametric variable, median values were provided; bParametric variables, mean±SD were provided. Disclosures No relevant conflicts of interest to declare.

Description: Aim: In disorders with ineffective erythropoiesis like thalassemia major (TM) iron absorption from gastrointestinal tract increases regardless of iron store. Iron is taken into enterocytes at duodenum via apical divalent metallo protein 1 (DMT-1) protein. Besides iron DMT-1 also transports other divalent metals like cadmium (Cd) , cobalt (Co), copper (Cu), lead (Pb), manganese, nickel, and zinc (Zn). In our study we hypothesized that absorption of heavy metals via DMT-1 protein may be elevated in particularly TM and other disorders with ineffective erythropoiesis, and we aimed to investigate blood heavy metal levels in these patients and compare with control group. Methods: Study was performed between December 2014 and April 2015 in Hacettepe University Children Hospital Pediatric Hematology Clinic and Gulhane Military Medical Academy Pediatric Hematology Clinic. Blood samples were taken from patients with TM, thalassemia intermedia (TI), congenital dyserythropoietic anemia (CDA), and age and sex matched healthy control group. Heavy metal exposure was searched via questionnaire for each participant. Serum heavy metals including aluminium (Al), Cd, Co, crom (Cr), Cu, Pb, selenyum (Se), and Zn were measured. Results: Blood samples were obtained from 51 patients with TM, 8 with TI, 9 with CDA, and control group included 65 healthy volunteers. General characteristics of patients and control group were showed on Table 1. There wasn't statistical difference between patients and control group in terms of age and heavy metal exposure. Plasma or serum heavy metal levels of patients and control group were shown in Table 2. Patients with TM were found to have lower Al, Pb, Se, and Zn, and higher Cd levels compared to control group. In patients with TM, Cd level was higher than control group, however median value of Cd was still in normal range. In TI group only Se level was lower (p; 0.003), in CDA group both Se (p; 0.008) and Zn (p; 0.02) levels were lower compared to control group. When we looked for patients with TM, TI, and CDA who were treated with deferasirox; Al, Pb, Se, and Zn levels were lower, and Cd level was higher than control group. Discussion: Opposite to our hypothesis Al, Pb, Se. and Zn levels were lower in TM group and in group of patients who were on deferasirox. Se and Zn levels in patients with TM have been studied before but contradictory results have been reported. Generally they were reported to be lower in patients with TM. These results have been explained with micronutrient deficiency, and chelator treatment. Deferasirox has been reported to bind to Al and Zn, as well. The lower Al and Zn levels in our study may be explained with deferasirox therapy. Deferasirox may also have capacity to bind Pb, related to the lower levels found in our study in deferasirox group. In addition to iron chelation, chelator treatment with deferasirox might have protective effects in patients with ineffective erythtopoiesis against heavy metal toxicity. Table 1. General characteristics of patient and control groups TM TI CDA Control Number 51 8 9 65 Age* (year) 20.2 (4.5-39) 8.8 (3.4-37) 9 (2.5-14.6) 16.8 (3-39) Male/female 23/28 3/5 6/3 33/32 Heavy metal exposure YesNo 2130 62 81 3134 Chelator usage NoDeferasiroxDeferoxamineDeferoxamine+ deferriprone 04641 2600 4500 TM: thalassemia major, TI: thalassemia intermedia, CDA: congenital dyserythropoietic anemia *Median and range Table 2. Heavy metal levels in patient and control groups TM TI CDA Control pa Aluminium (mcg/L) (0-50) 27.,6 (10.1-82.1)* 50.1 (18.2-91.3) 41.1 (11.3-48.5) 43.8 (2.4-175.6) 0.01 Copper (mcg/dL) (70-150) 71.7 (22.3-175.5) 90.5 (27.9-165.2) 59.9 (35.1-110.3) 78.5 (20.5-189) 0.1 Zinc (mcg/dL) (70-120) 56 (21.8-126.2) 63.8 (34.4-130.2) 59.3 (23.6-76.9) 70.8 (36-166.8) 0.001 Cadmium (mcg/L) (0.3-1.2) 0.75 (0.34-2.82) 0.77 (0.36-1.37) 0.59 (0.38-0.78) 0.58 (0.23-3.54) 0.002 Cobalt (mcg/L) (0.5-3.9) 0 (0-6.73) 0.19 (0-1.94) 0.08 (0-2.49) 0.39 (0-6.59) 0.23 Crom (mcg/L) (2.8-45) 45.08 (39.4-63.8) 45.1 (41.7-47.5) 45.7 (35.5-53.7) 46.9 (28.5-96.1) 0.49 Lead (mcg/L) (0-150) 3.83 (0-22.7) 6.71 (0-11.1) 5.18 (0-16.3) 9.31 (0-45.2) 0.01 Selenium (mcg/L) (0-150) 24.6 (11.8-42.5) 21.7 (8.7-35.3) 25.6 (20.3-31.8) 33.,3 (15.1-71.5)