Cigdem Altay

Application of endonuclease mapping to the analysis and prenatal diagnosis of thalassemias caused by globin-gene deletion.

We applied a recently developed and more direct technic to diagnose thalassemia syndromes associated with deletion of particular globin structural genes and to assess a fetus at risk for one of those conditions, deltabeta-thalassemia. The method allows assessment of the globin genes present in total cellular DNA and is applicable to amniotic-fluid cell DNA. Cellular DNA fragments produced by cleavage using two specific restriction endonucleases are separated on the basis of size by agarose-gel electrophoresis, and the distribution of specific sequences among the DNA fragments determined by molecular hybridization. We observed the total deletion of alpha-globin genes in homozygous alpha-thalassemia (hydrops fetalis with hemoglobin Barts) and the deletion of particular beta and beta-like sequences in cases homozygous for hereditary persistence of fetal hemoglobin and deltabeta-thalassemia. Analysis of amniotic-fluid cell DNA from a fetus at risk for deltabeta-thalassemia demonstrated the feasibility of these improved methods for antenatal diagnosis. The molecular studies confirmed the diagnosis predicted by analysis of fetal blood and established at birth.

The molecular basis of α-thalassemias: Frequent occurrence of dysfunctional α loci among non-Asians with Hb H disease

Abstract Study of Asians has previously indicated that deletion of α-globin structural genes is the predominant lesion in α-thalassemias and that Hb H disease occurs when three of four normal α loci per cell are deleted. To test the generality of this model, Hb H disease DNAs of both Asian and non-Asian origin were analyzed by restriction endonuclease mapping using the technique of Southern (1975). Whereas in normal DNA, α sequences are present in a single Eco RI fragment of cellular DNA approximately 22.5 kb long, fragments of 22.5, 20 and 2.6 kb were found in various Hb H disease DNAs. The 20 kb Eco RI fragment alone, in which a single α-globin structural locus resides, was found in Asian Hb H disease DNA. This finding is consistent with the deletion model of α-thalassemia. In contrast, seven of eight non-Asian Hb H disease DNAs displayed a more complex molecular composition. The fragment patterns observed were 22.5 kb alone, 22.5 plus 2.6 kb, 20 plus 2.6 kb and 20 kb alone. Non-Asian Hb H disease DNAs contained one, two or three α loci per cell in contrast to the one locus predicted by the simple deletion model of α-thalassemia. The data are best explained by the existence of defective α loci in certain individuals with α-thalassemia, particularly outside the Asian population. Restriction mapping of the 20 kb Eco RI fragment found in Asian and some non-Asian Hb H disease DNAs demonstrated a striking similarity in the placement of restriction sites about the single α gene compared with sites about the two genes in the 22.5 kb Eco RI fragment seen in normal DNA. These data are consistent with origin of the 20 kb fragment from the 22.5 kb normal Eco RI fragment by either unequal crossing-over or a deletion event. The molecular heterogeneity and frequent occurrence of defective α loci in non-Asian Hb H disease DNAs described here may explain, in part, the clinical heterogeneity of α-thalassemias and the absence of the homozygous deletion state (hydrops fetalis) in non-Asians. Further study of cellular DNA fragments containing the defective α loci identified in this work may indicate the types of specific mutations responsible for abnormal globin gene expression and complement similar studies on abnormal β genes in β-thalassemias.

Deletion of the A gamma-globin gene in G gamma-delta beta-thalassemia.

In an individual homozygous for G gamma-delta beta-thalassemia, a physical alteration in gamma-globin gene organization was detected by restriction enzyme mapping. The data indicated that the absence of A gamma-globin chains resulted from extension of the DNA deletion from the delta beta-globin gene region into the gamma-globin gene region rather than a functional disturbance of gamma-gene expression.

Variable pathogenicity of exon 43del (FAA) in four Fanconi anaemia patients within a consanguineous family

Four Fanconi anaemia group A (FAA) patients within two related consanguineous families are presented: the propositus (male, 13 years, transplanted at age 10), and his three cousins (one male, 8 years, and two female newborns). Assignment of the patients to FAA was based on the functional complementation analysis by somatic cell hybridization and confirmed by mutation screening showing a homozygous deletion of exon 43 (4267–4404del) in the FAA gene to be present in all four patients. The newborn patients had been diagnosed prenatally by DNA analysis. In spite of identical molecular pathology and close familial relationship the clinical phenotypes of the four patients were not concordant. Discordant symptoms included birthweight, pigmentation abnormalities, skeletal, renal and genital abnormalities, whereas microcephaly and possibly the haematological course were concordant. Differences in environmental conditions and/or genetic make‐up along with chance effects during development may explain discordant phenotypes despite identical molecular pathology in these patients. However, our results do not rule out the possibility that the exon 43del mutation may have prognostic value for the haematological course of the disease.

Analysis of some clinical and laboratory aspects of adolescent patients with thrombosis.

A total of 360 pediatric patients aged 1 month to 18 years were diagnosed as having thrombosis between January 1998 and April 2003. Of these patients, those aged 11–18 years (n = 131) were regarded as adolescents and the rest as children. The proportion of adolescents in the whole group excluding the neonates was 36%. The peak age of diagnosis in adolescents was 11–14 years, comprising 58% of all thrombotic events in adolescents. In 73% of the adolescents, there was at least one risk factor. The four most common underlying disorders were infection, malignancy, connective tissue and cardiac disorders, in decreasing order of frequency. In children, on the other hand, infection was followed by congenital heart disease, malignancy and liver disease. Three common types of thrombosis in adolescents were deep venous thrombosis, cerebro-vascular events and portal venous thrombosis, while cerebro-vascular events were the most common in children. The frequency of factor V G1691A mutation in the adolescents (22.1%) was significantly higher than that found in a group of healthy controls (7.4%) and this mutation was associated with a 3.6-fold increase in the risk of developing thrombosis (95% confidence interval, 1.4–9.0). The carrier frequency of prothrombin G20210A mutation (3.1%) in adolescents did not differ significantly from that of the healthy population (2.3%) and no association was observed between this mutation and a risk of developing thrombosis in this group (odds ratio, 1.3; 95% confidence interval, 0.2–7.5). The rate of recurrent thrombosis was 6%.

Incidence of high erythrocyte count in infants and young children with iron deficiency anemia: Re-evaluation of an old parameter

Purpose To investigate the frequency of high erythrocyte count (red blood cell count ≥5.0 × 106/&mgr;L) in infants and young children with iron deficiency anemia and to document the differences in hematologic parameters at diagnosis and during iron therapy in IDA patients with and without a high erythrocyte count. Patients and Methods A total of 140 infants and young children aged 6 to 48 months with nutritional IDA without a history of any bleeding disorder were the subjects of this study. The patients were divided into three groups according to the severity of anemia. Group A1 children had Hb values 8.0 g/dL or less (severe anemia); group A2, 8.1 to 10.0 g/dL (moderate anemia); and group A3, 10.1–11.0 g/dL (mild anemia). All children received oral iron (3–5 mg/kg per day) for 12 weeks. Complete blood counts were done weekly during treatment. Results A total of 36 of the 140 patients (26%) had a high erythrocyte count. Of the 140 patients, 37 were in group A1, 80 in A2, and 23 in A3. The frequency of high erythrocyte count was 11%, 23%, and 61% in groups A1, A2, and A3, respectively. The patients with a high erythrocyte count had significantly higher Hb and Hct but significantly lower mean corpuscular volume and mean corpuscular hemoglobin (MCH) values than those with a low erythrocyte count (n = 104). A continuous elevation in the erythrocyte count has been observed in patients with a high red cell count, as in those with a low red cell count, after the institution of iron therapy. Conclusions A high erythrocyte count is a common feature of iron deficiency anemia in infants and young children, with an increasing frequency from severe to moderate to mild anemia. High erythrocyte count cannot be regarded as a reliable preliminary parameter in differentiating iron deficiency from thalassemias in infants and children aged up to 48 months.

Haematological findings in children with inborn errors of metabolism

SummaryEarly detection and therapy of haematological abnormalities and/or diseases may improve the prognosis of metabolic disorders. Accordingly, we aimed to evaluate the frequency and types of haematological abnormalities in children[-31pc] with various inherited metabolic disorders. The study group comprised 46 children with metabolic disorders who were followed at the Pediatric Metabolism Unit and were referred to the Pediatric Hematology Unit for evaluation of anaemia between June 2000 and 2005. The mean age of the children was 55.2 ± 64.8 months at haematological evaluation (range 1 month–18 years, median 22.0 months); 16 were female and 30 were male. Of these 46 patients with anaemia, 25 of (54.3%) had anaemia of chronic disease (ACD), 9 (19.6%) had iron-deficiency anaemia (IDA), 7 (15.2%) had megaloblastic anaemia due to vitamin B12 deficiency, 3 (6.5%) had chronic haemolytic anaemia, 2 (4.3%) had autoimmune haemolytic anaemia, 1 had β-thalassaemia major, and 1 had hereditary spherocytosis. In addition to the anaemia, bicytopenia or pancytopenia was found in 8 of 46 children (17.4%). The study indicated that in organic acidaemias including methylmalonic acidaemia, propionic acidaemia, isovaleric acidaemia, and argininosuccinic acidaemia, the majority of patients had ACD (75%), which was followed by vitamin B12 deficiency anaemia and IDA (p < 0.001). In PKU, both nutritional anaemias and ACD were present at about same frequency: 46.7% and 40%, respectively (p > 0.05). This study suggested that congenital anaemias such as hereditary spherocytosis or thalassaemias should be kept in mind as a coexisting haematological diseases in young patients with inborn errors of metabolism. In conclusion, ACD and nutritional anaemias are the most prevalent anaemias seen in patients with inborn errors of metabolism. Early detection of the disease, early administration of specific diet, and close monitoring of the patients are very important factors to prevent the development of haematological diseases in patients with inborn errors of metabolism.

Poor Prognosis in Children with Acute Nonlymphoblastic Leukemia in Turkey

Two hundred fourteen children with acute nonlymphoblastic leukemia (ANLL) were diagnosed between July 1975 to January 1987 in our center. Only 106 (50%) of the children could be treated. An initial hematologic remission was attained in 61 (58%) with a median duration of hematologic remission of 7.5 months and median survival of 12 months. Five patients who completed 36 months of therapy are still in initial remission, <84 months. Exophthalmos at presentation, which is clearly related to shorter duration of remission, was observed in 11% of these children. In two cases, it was the first sign of leukemia, 6 and 7 months prior to obvious bone marrow involvement. Twenty percent of the 61 patients who achieved remission discontinued their chemotherapy during the course of treatment while they were in remission, which indicates a different problem in the treatment of these patients in Turkey. Despite administration of similar treatment protocols, the prognosis of our patients was worse than the results observed in developed countries. We conclude that poor socioeconomic and nutritional conditions should also be considered among the high-risk factors for children with ANLL, as experienced in our patients with acute lymphoblastic leukemia (ALL).

Long term biweekly 1 mg oral vitamin B12 ensures normal hematological parameters, but does not correct all other markers of vitamin B12 deficiency. A study in patients with inherited vitamin B12 deficiency

Parenteral vitamin B12 is considered the treatment of choice for vitamin B12 deficiency, but also treatment with 1–2 mg daily oral vitamin B12 is recommended.[1][1],[2][2] Recently, alternative regimes have been proposed, such as an oral dose of 1 mg vitamin B12 daily for ten days, weekly for four

Clinical and laboratory evaluation of Turkish children with thrombosis for homozygous factor V G1691A mutation

Factor V (FV) G1691A mutation, in a heterozygous state, is one of the most common inherited risk factors for development of thrombosis. However, the clinical manifestations of homozygosity for the FV G1691A mutation in children is largely unknown because of the limited number of studies reported. The purpose of this study was to evaluate the clinical manifestations and laboratory findings of children with thrombosis who were homozygous for this mutation. Ten patients (four male/six female; mean age, 4.5 years; age range, 1–13 years) who were found to be homozygous for the FV G1691A mutation among 360 consecutive children with thrombosis (2.8%) were the subjects of this study. Six of the 10 patients had venous thrombosis, two had purpura fulminans, one had diffuse skin ecchymosis and one had arterial thrombosis. No history of thrombosis was present in their family members. Seven of the 10 children were under the age of 5 years. One or more additional risk factors (infection, protein S and protein C deficiencies, elevated factor VIII, etc.) were also present in nine of these patients. None of these patients had prothrombin G20210A mutation but one patient had risk-associated plasminogen activator inhibitor-1 gene 4G/4G genotype. These findings suggest that, in the presence of other underlying risk factors, homozygosity for FV G1691A mutation may lead to development of thrombosis at a very young age.