Huseyin Tokgoz

Spectrum of the Mutations in Bernard–Soulier Syndrome

Bernard–Soulier syndrome (BSS) is a rare autosomal recessive bleeding disorder characterized by defects of the GPIb‐IX‐V complex, a platelet receptor for von Willebrand factor (VWF). Most of the mutations identified in the genes encoding for the GP1BA (GPIbα), GP1BB (GPIbβ), and GP9 (GPIX) subunits prevent expression of the complex at the platelet membrane or more rarely its interaction with VWF. As a consequence, platelets are unable to adhere to the vascular subendothelium and agglutinate in response to ristocetin. In order to collect information on BSS patients, we established an International Consortium for the study of BSS, allowing us to enrol and genotype 132 families (56 previously unreported). With 79 additional families for which molecular data were gleaned from the literature, the 211 families characterized so far have mutations in the GP1BA (28%), GP1BB (28%), or GP9 (44%) genes. There is a wide spectrum of mutations with 112 different variants, including 22 novel alterations. Consistent with the rarity of the disease, 85% of the probands carry homozygous mutations with evidence of founder effects in some geographical areas. This overview provides the first global picture of the molecular basis of BSS and will lead to improve patient diagnosis and management.

Variable presentation of primary immune deficiency: Two cases with CD3 gamma deficiency presenting with only autoimmunity

CD3 chain expression defects including CD3 gamma, epsilon, delta, and zeta chain subunits, are autosomal recessive inherited severe combined immunodeficiencies (SCID). The phenotype is usually T‐B+NK+ SCID with lymphopenia where the clinical findings may be mild (CD3γ) or severe (CD3δ, ε, ζ) owing to the underlying molecular defect. There is limited information about the disease in literature.

CD3G Gene Defects in Familial Autoimmune Thyroiditis

The patients with CD3γ deficiency can present with different clinical findings despite having the same homozygous mutation. We report three new CD3gamma‐deficient siblings from a consanguineous family with a combined T−B+NK+ immunodeficiency and their variable clinical and cellular phenotypes despite the same homozygous mutation of the CD3G gene (c.80‐1G>C). We also re‐evaluate a previously reported non‐consanguineous family with two CD3gamma‐deficient siblings with the same mutation. The median age at diagnosis was 11 years (14 months–20 years). We found all five patients to display autoimmunity: autoimmune thyroiditis (n = 5), autoimmune haemolytic anaemia (n = 2), immune thrombocytopenia (n = 1), autoimmune hepatitis (n = 1), minimal change nephrotic syndrome (n = 1), vitiligo (n = 1) and positive antinuclear antibodies (n = 3) as well as high IgE (n = 2) and atopic eczema (n = 2). While CD3+TCRαβ+T cell percentages were low in all patients, only one had lymphopenia and 3 had CD3+T cell lymphopenia. Strikingly, we report frequent and multiple autoimmunity in tested heterozygous carriers in both families (n = 6; in 67%), and frequent autoimmunity in family members not available for testing (n = 5, in 80%). The results suggest that CD3G should be studied as a candidate gene for autoimmunity and that CD3gamma deficiency should be considered among other primary immunodeficiencies with predominantly autoimmune manifestations.

Ophthalmic Manifestations in Recently Diagnosed Childhood Leukemia

Purpose To determine the prevalence and the pattern of ocular involvement in children with leukemia at the time of diagnosis. Methods The data of patients with leukemia who underwent complete ophthalmic examination at the time of diagnosis between January 2005 and December 2014 were retrospectively reviewed. Demographic data, type of leukemia, ocular findings, blood parameters, and duration of follow-up were analyzed. Results A total of 185 patients (111 male and 74 female) were included in the study, with a median age of 6.0 years (range 0.5-18.0 years) and a median follow-up time of 36.0 months (range 0.5-108.0 months). Ocular signs were present in 24.3% of the patients at the time of diagnosis and 37.8% of them were symptomatic. The prevalence of ocular involvement was 20.4% in patients with acute lymphocytic leukemia (ALL) and 36.4% in patients with acute myelocytic leukemia (AML) (p = 0.051). Fatality rate was significantly higher in subjects with AML compared with ALL (p = 0.019), but was not significantly different between patients with and without ocular involvement (p = 0.166). There were no significant differences in hemoglobin levels, white blood cell counts, or platelet counts between patients with ALL and AML. Platelet counts were significantly lower in patients with ocular signs compared with subjects without ocular involvement (p = 0.012), while hemoglobin levels and white blood cell counts did not differ significantly. Conclusions Various ocular signs may be present at the time of diagnosis in childhood leukemia, even in patients without any symptoms. Routine ophthalmic examination should be performed in recently diagnosed children with leukemia.

Successful use of fondaparinux in a child with heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a well described side effect of heparin therapy. A 12-year-old boy developed deep-vein thrombosis. Risk factors for initial thrombosis are antiphospholipid syndrome and heterozygous mutation for prothrombin G20210A. Anticoagulant therapy with warfarin for 12 months was effective, but discontinuation of warfarin after 12 months resulted in recurrence of thrombosis. Unfractionated heparin (UFH) was initiated during the acute period, but heparin-induced thrombocytopenia developed. Transition from UFH to fondaparinux resulted in successful anticoagulation for a period of platelet recovery. We report a case of HIT developing with a background of prothrombotic genetic risk factors and antiphospholipid syndrome. This case study highlights several difficulties in pediatric HIT cases.

Persistent Nasal Bleeding Due to Nasal CPAP Application in 2 Premature Newborns Successfully Treated With Topical “Ankaferd Blood Stopper”

Respiratory distress syndrome (RDS) is one of the most common causes of hospitalization of premature babies in newborn intensive care units. Nasal continuous positive airway pressure (CPAP) has been used widely for a long time in the treatment of RDS. Nasal bleeding due to nasal mucosal injury is not rare in the course of nasal CPAP application. Nasal bleeding can usually be controlled uneventfully with simple applications. However, to bring these bleedings under control can sometimes be difficult and time consuming. Furthermore, continuous bleeding can occlude the respiratory route by forming a plug. Ankaferd Blood Stopper (ABS) is a herbal product that can stop cutaneous, dental, and postoperational external bleeding in a few minutes. Herein, we wanted to share our experience in using topical ABS to control epistaxis that had appeared in 2 premature babies related to CPAP application, and which could not be controlled using conventional methods. Nasal CPAP was applied on 2 premature babies born at 30 and 32 weeks of gestation and hospitalized for RDS treatment. However, unilateral nasal bleeding began on the first and the third day of treatment, respectively. Nasal bleeding was attempted to be controlled using conventional methods (pressure on the nose, irrigation with hot water, topical vasoconstrictor agent application, etc), but bleeding could not be stopped. Thrombocyte counts and routine hemostasis tests of both babies were normal. Intramuscular vitamin K (0.5 mg) was administered at birth. Ankaferd blood stopper (Ankaferd Health Products Ltd, _ Istanbul, Turkey) was applied onto the bleeding nostrils of both babies as a topical spray form. Bleeding stopped in a few minutes following ABS application and did not recur. No systemic or local adverse effects were observed. Ankaferd blood stopper is a hemostatic agent consisting of 5 distinct herbal extracts named Urtica dioica, Vitis vinifera, Glycrrhiza glabra, Alpinia officinarum, and Thymus vulgaris. This product, which is used in traditional Turkish medicine, has been licensed by the Ministry of Health. There are many clinical and experimental studies proving its effect on controlling digestive tract, oral, nasal, and cutaneous bleedings, which show whether there they accompany any coagulation disorders. In published studies, it has been shown that this product has no serious adverse effects. Ankaferd blood stopper achieves hemostasis independently from coagulation factors and the classical coagulation cascade. It constitutes a structural network via interference with blood proteins, mainly with fibrinogen, and thus, it provides vital aggregation of erythrocytes. It can be used both for individuals with normal hemostasis and primary or secondary hemostasis disorders as it does not interfere with the cascade working upon coagulation factors. Based on our experience in these 2 cases, we can conclude that topical ABS application is a practical, effective, and safe hemostatic treatment modality. We believe that topical ABS application will create a novel treatment modality in superficial cutaneous and mucosal bleedings in newborns including premature babies.

Novel mutations of integrin αIIb and β3 genes in Turkish children with Glanzmann’s thrombasthenia

Abstract Glanzmann’s thrombasthenia (GT) is an inherited disorder of platelet aggregation, characterized by qualitative and quantitative defect on platelet αIIbβ3 integrin (GpIIb/IIIa), resulting in lifelong bleeding tendency due to defective platelet plug formation. The αIIb gene (ITGA2B) and β3 gene (ITGB3) are closely located at chromosome 17q21.31-32. ITGA2B consist of 30 exons and encoding α chain, whereas ITGB3 has 15 exons and encoding β chain. Until now, according to the Human Gene Mutation Database (HGMD), 138 mutations at ITGA2B gene and 101 mutations at ITGB3 gene have been identified. We aimed to determine whether there was any mutation in the ITGA2B and ITGB3 genes, and a correlation between clinical phenotype and genotype in Turkish GT patients. We examined 20 patients with GT followed at the Department of Pediatric Hematology, Meram Faculty of Medicine, for Clinical and Laboratory Findings and Molecular Genetic Analysis. Peripheral blood was collected from patients, and a written informed consent for genetic analysis was obtained from parents. DNA was isolated from by proteinase K and phenol/chloroform extraction. ITGA2B and ITGB3 genes were screened by polymerase chain reaction. There were 12 females and 8 males with a median age of 15.25 years. Major clinical presentations of these patients were mucocutaneous bleedings. The most common bleeding type was epistaxis (85%). Life-threatening bleedings were seen in five patients. Seven (35%) patients showed various mutations in the ITGA2B or ITGB3 genes. We detected four novel mutations in three different regions and two mutations defined previously within the ITGA2B gene. These changes are at exon 4; c.570 T > G alteration, at exon 13 c.1277 T > A, c.1291 T > G alterations, at exon 19 c.1921A > G alterations. And from the start point of exon 14, behind 107 bases, we detected a heterozygous alteration at Thymine to Guanine. According to PolyPhen Database Program and NCBI Multiple Alignment Tool Database, four transitions are conserved at evolutionary process, so we can say that these transitions are novel mutations. c. 468T > G alteration at exon 4 and c. 1378 T > A alteration at exon 13 were reported to HGMD previously. Screening the exons of the ITGB3 gene from the same patient groups, we reported a novel missense mutation at exon 5, at nucleotide 680. No correlation was found between clinical phenotype and genotype. These mutations were described for the first time in Turkish population, and all novel mutations are not defined previously. Furthermore, collaborative studies are needed for the population point of view.

Novel plasminogen gene mutations in Turkish patients with type I plasminogen deficiency.

The plasminogen (Plg) protein is the inactive proenzyme form of plasmin that dissolves fibrin thrombi by a process called fibrinolysis. It has been shown that homozygous or compound-heterozygous deficiency of this protein is a major cause of a rare inflammatory disease affecting mainly mucous membranes found in different body sites. In this study, five individual Turkish patients and nine Turkish families with type 1 Plg deficiency were investigated for PLG gene mutations. All of the coding regions of the PLG gene mutations were screened for mutations using denaturing high-pressure liquid chromatography (DHPLC). Samples showing a different DHPLC profile were subjected to DNA sequencing analysis. Here, we described five novel mutations namely, Cys49Ter, +1 IVS6 G>A, Gly218Val, Tyr283Cys, and Gly703Asp. Previously identified five nonsynonymous (Lys38Glu, Glu180Lys, Gly420Asp, Asp453Asn, Pro763Ser), five synonymous (330 C>T, 582 C>T, 771 T>C, 1083 A>G, 2286 T>G), and a 3′ untranslated region (3′ UTR) mutation (c.*45 A>G) were also reported in this present study. In this study, we have identified a total of eight mutations, five of which are novel. The mutations/polymorphisms identified in eight of the patients do not explain the disease phenotype. These cases probably carry other pathological mutations (homozygous or compound heterozygous) that cannot be detected by DHPLC.

A national registry of thalassemia in Turkey; demographic and disease characteristics of patients, achievements and challenges in prevention

Objective: The Turkish Society of Pediatric Hematology set up a National Hemoglobinopathy Registry to demonstrate the demographic and disease characteristics of patients and assess the efficacy of a hemoglobinopathy control program (HCP) over 10 years in Turkey. Materials and Methods: A total of 2046 patients from 27 thalassemia centers were registered, of which 1988 were eligible for analysis. This cohort mainly comprised patients with β-thalassemia major (n=1658, 83.4%) and intermedia (n=215, 10.8%). Results: The majority of patients were from the coastal areas of Turkey. The high number of patients in Southeastern Anatolia was due to that area having the highest rates of consanguineous marriage and fertility. The most common 11 mutations represented 90% of all β-thalassemia alleles and 47% of those were IVS1-110(G->A) mutations. The probability of undergoing splenectomy within the first 10 years of life was 20%, a rate unchanged since the 1980s. Iron chelators were administered as monotherapy regimens in 95% of patients and deferasirox was prescribed in 81.3% of those cases. Deferasirox administration was the highest (93.6%) in patients aged <10 years. Of the thalassemia major patients, 5.8% had match-related hemopoietic stem cell transplantation with a success rate of 77%. Cardiac disease was detected as a major cause of death and did not show a decreasing trend in 5-year cohorts since 1999. Conclusion: While the HCP has been implemented since 2003, the affected births have shown a consistent decrease only after 2009, being at lowest 34 cases per year. This program failure resulted from a lack of premarital screening in the majority of cases. Additional problems were unawareness of the risk and misinformation of the at-risk couples. In addition, prenatal diagnosis was either not offered to or was not accepted by the at-risk families. This study indicated that a continuous effort is needed for optimizing the management of thalassemia and the development of strategies is essential for further achievements in the HCP in Turkey.

Differences in Granule Morphology yet Equally Impaired Exocytosis among Cytotoxic T Cells and NK Cells from Chediak–Higashi Syndrome Patients

Chediak–Higashi syndrome (CHS) is caused by autosomal recessive mutations in LYST, resulting in enlarged lysosomal compartments in multiple cell types. CHS patients display oculocutaneous albinism and may develop life-threatening hemophagocytic lymphohistiocytosis (HLH). While NK cell-mediated cytotoxicity has been reported to be uniformly defective, variable defects in T cell-mediated cytotoxicity has been observed. The latter has been linked to the degree of HLH susceptibility. Since the discrepancies in NK cell- and T cell-mediated cellular cytotoxicity might result from differences in regulation of cytotoxic granule release, we here evaluated perforin-containing secretory lysosome size and number in freshly isolated lymphocytes from CHS patients and furthermore compared their exocytic capacities. Whereas NK cells from CHS patients generally contained a single, gigantic perforin-containing granule, cytotoxic T cells predominantly contained several smaller granules. Nonetheless, in a cohort of 21 CHS patients, cytotoxic T cell and NK cell granule exocytosis were similarly impaired upon activating receptor stimulation. Mechanistically, polarization of cytotoxic granules was defective in cytotoxic lymphocytes from CHS patients, with EEA1, a marker of early endosomes, mislocalizing to lysosomal structures. The results leads to the conclusion that lysosome enlargement corresponds to loss of distinct organelle identity in the endocytic pathway, which on a subcellular level more adversely affects NK cells than T cells. Hence, vesicular size or numbers do not per se dictate the impairment of lysosomal exocytosis in the two cell types studied.