Dimitris Loukopoulos

Bone resorption is increased in young adults with thalassaemia major

Bone disease in patients with thalassaemia major is a multifactorial and still poorly understood process. The present study evaluated 45 thalassaemic patients using dual X‐ray absorptiometry at three sites (lumbar spine, head of femur and forearm) to assess bone mineral density, in parallel with a series of biochemical markers to measure bone formation and bone resorption. To identify possible interfering factors, our patients were grouped according to whether or not they needed transfusion therapy; the presence of hypogonadism was also considered. Our results showed that patients on regular transfusions had a markedly low bone mineral density in contrast to those not requiring blood support and that this finding was more pronounced in the hypogonadic group, irrespectively of sex. The decrease of bone mineral density values was more prominent in the forearm, thus making this site particularly interesting for such studies. Bone formation, as evidenced by the levels of serum alkaline phosphatase and osteocalcin, did not appear to be impaired, while bone resorption was grossly increased in all patient groups. The latter process was clearly evident using the recently introduced measurement of the urinary N‐terminal peptides of collagen type I, the sensitivity of which has already been established in other groups of osteoporotic patients. Our conclusion is that, in spite of the severe bone destruction that occurs in thalassaemia major, the fact that bone formation remains intact calls for a more intensive treatment comprising hormonal replacement, bisphosphonates and other agents.

Pamidronate is an effective treatment for osteoporosis in patients with beta‐thalassaemia

Summary.  Osteoporosis in beta‐thalassaemia is multifactorial; increased osteoclast function seems to play an important role in its pathogenesis. The aim of this study was to evaluate the effect of pamidronate on the osteoporosis of thalassaemia. To this effect we studied 26 patients who received this drug in doses of 30 or 60 mg i.v. once a month over 12 months. The effects were monitored by measuring bone mineral density (BMD) in association with markers of osteoclast function [soluble receptor activator of nuclear factor‐kappa B ligand (sRANKL), osteoprotegerin (OPG)] and of bone remodelling [N‐telopeptide of collagen type‐I (NTX), tartrate‐resistant acid phosphatase isoform‐5b (TRACP‐5b), bone‐alkaline phosphatase (bALP), and osteocalcin (OC)]. Thirty healthy individuals were also studied, as controls. NTX, TRACP‐5b, bALP and OC levels were significantly higher in thalassaemic patients compared with controls; in contrast, OPG levels were significantly lower, while the levels of sRANKL varied within normal limits. Administration of pamidronate was followed by a clear decrease of NTX, TRACP‐5b, OPG, and OC, and by a significant increase in the BMD of the lumbar spine, which was similar in patients of both treatment groups. These data suggest that pamidronate, at a monthly dose of 30 mg, is an effective treatment for thalassaemic osteoporosis.

βs haplotypes in various world populations

SummaryWe have determined the βs haplotypes in 709 patients with sickle cell anemia, 30 with SC disease, 91 with S-β-thalassemia, and in 322 Hb S heterozygotes from different countries. The methodology concerned the detection of mutations in the promoter sequences of the Gγ- and Aγ-globin genes through dot blot analysis of amplified DNA with 32P-labeled probes, and an analysis of isolated Hb F by reversed phase high performance liquid chromatography to detect the presence of the AγT chain [Aγ75 (E19) Ile→Thr] that is characteristic for haplotype 17 (Cameroon). The results support previously published data obtained with conventional methodology that indicates that the βs gene arose separately in different locations. The present methodology has the advantage of being relatively inexpensive and fast, allowing the collection of a vast body of data in a short period of time. It also offers the opportunity of identifying unusual βs haplotypes that may be associated with a milder expression of the disease. The numerous blood samples obtained from many SS patients living in different countries made it possible to compare their hematological data. Such information is included (as average values) for 395 SS patients with haplotype 19/19, for 2 with haplotype 17/17, for 50 with haplotype 20/20, for 2 with haplotype 3/3, and for 37 with haplotype 31/31. Some information on haplotype characteristics of normal βA chromosomes is also presented.

Arterial Calcifications in β-Thalassemia

The purpose of this study was to define the incidence of arterial calcifications in patients with β-Thalassemia. β-thalassemia patients have been shown to present a high preva lence of angioid streaks and skin lesions characteristic of pseudoxanthoma elasticum (PXE). Given the fact that vascular involvement in the form of arterial calcifications is also a common manifestation of PXE, the authors investigated radiographically the presence of arterial calcifications in β-thalassemia patients. They studied 40 patients with β-thalassemia over 30 years of age. Forty healthy, age- and sex-matched subjects were chosen as a control group. Radiographs of the tibias were performed in order to disclose arterial calcifications. The occurrence of PXE skin lesions and of angioid streaks (AS) was also investigated. Arterial calcifications were detected in the posterior tibial artery in 22 (55%) β-thalassemia patients and in six (15%) controls (P<0.01 for the comparison). PXE skin lesions and AS were found in eight (20%) and 21 (52%) patients respectively. A total of 34 patients (85%) had at least one of the three lesions, namely, arterial calcifications, angioid streaks, and/or PXE-like skin lesions. Stepwise logistic regression analysis did not reveal prognostic value in independent variables such as transfusions, chelation therapy, pseudoxanthoma elasticum skin lesions and/or angioid streaks, diabetes, hemoglobin, serum ferritin, and uric acid. It was concluded that arterial calcifications are common in older β-thalassemia patients. This finding could be a manifestation of an acquired PXE syndrome associated with β-thalassemia, and consequently, vascular events complicating PXE should be expected in these patients.

Clinical and laboratory effects of long‐term administration of hydroxyurea to patients with sickle‐cell/β‐thalassaemia

Hydroxyurea (HU), a widely used cytostatic, has been given over a long period of time to 14 adult Caucasian compound heterozygotes for βs and various β‐thalassaemia genes. All patients had severe pain crises and other complications prior to receiving the drug. After 4‐8 weeks on high ‘sub‐toxic’doses of HU all patients responded with a multifold increase of fetal haemoglobin (HbF) and a marked increase of MCV and MCH; they also felt significantly better and ceased having pains or other complaints. Haematological toxicity was minimal and rapidly reversible. Follow‐up of the patients has now exceeded 100 weeks and goes up to 180 weeks in two of them. Pain crises have never recurred. Maintenance of high levels of HBF requires continuous administration of high doses of HU; whenever the latter were decrease in various attempts to avoid potential long‐term toxicity, the observed changes gradually faded. The effect of HU in HbS/β‐thalassaemia may be better than that reported for homozygous HbS disease because the synthesized λ‐chains not only inhibit the sickling process but they also neutralize the noxious effects of the excess α‐chains and cut down the ineffective erythropoiesis of the patients.

Alpha-Chain of Human Hemoglobin: Occurrence in vivo

A minor hemoglobin component, apparently representing uncombined α-chains has been detected in the hemolysates of persons with inherited β-chain deficiency of moderate or severe degree. This finding supports the hypothesis that there is independent control of the synthesis of α-chains.

Analysis of 14 cystic fibrosis mutations in five South European populations

SummaryWe have analysed five Southern European populations (Albanian, Greek, Italian, Spanish and Yugoslavian) for 14 cystic fibrosis (CF) mutations. The most frequent mutations, apart from ΔF508, were G542X (6.04%), R1162X (3.61%) and N1303K (3.24%). Each of the other analysed mutations were present at a frequency of less than 1% (R347P, R334W, S549RA, S549I, G551D, R553X and W1282X), and four mutations (D110H, ΔI507, S549RT, and S1255X) were not found in this sample. The data presented here allows the use of mutation analysis in 69.5% of Spanish, 58% of Greek, and 56.5% of Italian CF cases.

Somatic hypermutation of immunoglobulin variable region genes: focus on follicular lymphoma and multiple myeloma

Summary: Analysis of the rearranged immunoglobulin variable region gene hypermutation has provided important information concerning the clonal history and ontogenetic origin of various B‐cell lymphoproliferative disorders. Under the selective pressure of antigen, mutational events in immunoglobulin genes will fine tune survival of B‐cell clones bearing immunoglobulin with high affinity for antigen. Our studies aimed at analyzing neoplastic disorders originating from germinal and post‐germinal center B‐cells: follicular lymphoma and multiple myeloma. respectively. Despite the already acknowledged evidence for a selectable distribution of mutations within the clonal immunoglobulin variable heavy chain genes, very little is known about the contribution of light chains in the process of antigen selection. In follicular lymphoma. a more limited pattern of somatic mutation with less evidence of antigen selection was observed in variable K light chain genes (40%) than in their partner heavy chain genes (80%). In myeloma, hypermutation of variable light chain genes, with a distribution suggestive of antigen selection, was frequently observed. Based on these data and recent reports it appears that the light chain expressed by the clonogenic myeloma B‐cells plays a pivotal role in the antigen selection process. Additionally, abortive K light chain variable region genes in X‐expressing myeloma carried a significant number of somatic mutations indicating that the cell of origin is open to the hypermutation machinery at that particular developmental stage irrespective of antigen selection.

Comparison of homozygous sickle cell disease in Northern Greece and Jamaica

The clinical and haematological features of homozygous sickle cell (SS) disease were compared in 30 Greek and 310 Jamacian patients. Deletional alpha-thalassaemia, which modifies SS disease, is rare among Greek patients, so only Jamacian patients with four alpha-globin genes were included in the control group. Greek patients had higher total haemoglobin concentration and red cell counts, and lower mean cell haemoglobin concentration (MCHC) and reticulocyte counts. They also had a more normal body build and more adults had persistent splenomegaly. Fewer had a history of leg ulceration or priapism but more reported acute chest syndrome. The comparatively mild disease in Greek patients is consistent with less haemolysis and sickling and therefore less bone marrow expansion. In the absence of amelioriating factors such as high HbF concentration or alpha-thalassaemia, these findings may be explained by the low MCHC.

The Cretan type of non-deletional hereditary persistence of fetal hemoglobin [Aγ–158C→T] results from two independent gene conversion events

Abstract We report a new type of non-deletional hereditary persistence of fetal hemoglobin that is due to a C→T transition at position –158, relative to the Cap site of the human Aγ-globin gene. This mutation was identified in three unrelated adult cases presenting slightly elevated levels of fetal hemoglobin (Hb F), i.e. 2.9–5.1%, and normal hematological indices. Our sequencing results, from both polymerase chain reaction-amplified and subcloned DNA fragments, indicate that the Aγ–158C→T mutation occurred by two independent gene conversion events in the three cases studied. In addition, hematological and molecular data, including restriction fragment length polymorphism haplotyping in the β-globin gene cluster, extended haplotype analysis inside the γ-globin gene region and routine analysis of three tandem repeat loci (D1S80, 3′-HVR/apoB and F8vWf), led us to conclude that the Aγ–158C→T mutation in one of the three cases occurred recently in the parental germ line (P=99.47%), representing the first example of a de novo gene conversion event identified in humans.