International network on endocrine complications in thalassaemia (I-CET): an opportunity to grow.

Most of the endocrine complications in thalassaemia are attributable to iron overload which may be the result of economic circumstances (expense of the chelation therapy), late onset of chelation therapy or poor compliance with the iron chelation therapy. The major difficulties reported by hematologists or pediatric endocrinologists experienced in thalassaemias or thalassaemia syndromes in following growth disorders and endocrine complications were: lack of familiarity with medical treatment of endocrine complications (40%), interpretation of endocrine tests (30%), costs (65%), absence of paediatric endocrinologist for consultation on growth disorders and endocrine complications (27%), facilities (27%), other (e.g. lack of collaboration and on-time consultation between thalassaemic Centers supervised by hematologists and endocrinologists) (17%). Because any progress we make in research into growth disorders and endocrine complications in thalassaemia should be passed on to all those suffering from it, guaranteeing them the same therapeutic benefits and the same quality of life, on the 8th of May, 2009 in Ferrara (Italy), the International Network on Endocrine Complications in Thalassemia (I-CET) was founded. The I-CET group is planning to conduct, in Ferrara in May 2012, a workshop, "MRI and Endocrine Complications in Thalassaemia", and in Doha (Qatar) in September 2012, a 3-day intensive course entitled, "Growth disorders and Endocrine Complications in Thalassaemia", to provide interested pediatricians, physicians and hematologists from all over the world with an in-depth approach to the diagnosis and management of growth and endocrine disorders in thalassaemic patients.

Quality of life in thalassemia.

Morbidity and mortality related to thalassemia have been reduced significantly with modern medical treatment, and quality of life (QOL) should now be considered an important index of effective health care. An assessment of QOL differs from other forms of medical assessment in that it focuses on the individuals' own views of their well-being and assesses other aspects of life, giving a more holistic view of well-being. There is very little published work on evaluation of QOL in thalassemia. A suitable tool should be reproducible, sensitive to the major features of the condition that affect patients' lives, and applicable in the range of different cultural, age, and social settings. Such an instrument would be valuable in evaluating new forms of treatment and in comparing health outcomes between different clinics. Two instruments have been assessed, one derived from the WHOQOL-100 questionnaire, and one designed specifically for thalassemia, which assesses psychosocial and clinical burden, as they affect adult patients, parents, and children. Further studies are required to develop and assess such tools for use in thalassemia. Another approach is to seek patients' own views of their routine treatment and the extent to which medical treatment affects QOL. Results from patient questionnaires in the United Kingdom and Cyprus are consistent in finding problems with organization of transfusions, insufficient options with chelation therapy, and poor communication. Practical measures could be taken to address these issues.

Global epidemiology of hemoglobin disorders.

Thalassemias and the hemoglobinopathies such as Hemoglobins S, C and E, are now a global problem. They have spread through migration from their native areas in the Mediterranean, Africa and Asia and are now endemic throughout Europe, the Americas and Australia. Comprehensive control programs in recent years have succeeded in limiting the numbers of new births and prolonging life in affected individuals. Such programs have been successful in a minority of countries and have little global impact. Over 300,000 infants with major syndromes are born every year and the majority die undiagnosed, untreated or under-treated. Countries may be divided into three general categories according to the services available: A. Endemic Mediterranean countries. In these long-established prevention programs have succeeded in achieving 80%-100% prevention. Specialized clinics able to provide optimum treatment. B. Areas of the developed, industrialized world where prevalence is increasing because of migration. These countries have the means to provide adequate control but have problems in reaching immigrant groups with different cultural background. C. Countries of the developing world where the provision of services is hampered by economic difficulties, other health priorities due to high infant mortality from infectious diseases, and religious/cultural constraints.

Fertility in female patients with thalassemia.

With recent therapeutic advances, thalassemic patients can now reach adulthood and attain reproductive capacity. Endocrine complications due to hemosiderosis and especially hypogonatotropic hypogonadism, which present either with sexual infantilism and primary amenorrhea or with secondary amenorrhea, are common in thalassemic women. The aim of this study was to estimate the frequency of fertility among our female thalassemic patients. Our population included 50 married women with thalassemia major (TM) and 12 with thalassemia intermedia (TI) who are regularly followed in our thalassemic centers. Of the 50 patients with TM, 7 had primary amenorrhea (PA), 9 had secondary amenorrhea (SA), and 34 had normal menstrual function (NM), as did all the patients with TI. Overall we had 62 women who were able to achieve 90 pregnancies and give birth to 87 healthy babies. Most of our patients became pregnant around the age of 25 years. Associated endocrine complications were rare except in the group of patients with PA, as expected. In all patients with PA and SA, the 17 pregnancies were induced (intercourse 10, insemination 3, IVF 4). In the patients with NM and TI, 66 pregnancies were achieved spontaneously and 7 following induction (insemination 3, IVF 4). There were four twin and one triple pregnancies, which all resulted in premature deliveries. Among the seven couples in which both partners had thalassemia major, sperm donation was used in 5 cases, ovum donation in one case, and one pregnancy was achieved spontaneously. These 90 pregnancies resulted in 69 full-term, 12 pre-term, 7 abortions and 2 stillbirths. No severe obstetric complication was observed except for two patients with preeclampsia. One patient with PA who carried the triple pregnancy developed severe cardiac failure, which was successfully treated. Transfusion requirements were increased during pregnancy. Discontinuation of desferrioxamine resulted in elevation of ferritin levels during the second and third trimesters of pregnancy and after delivery. Nine patients who were examined with cardiac echo had a transient increase of ESD and EDD during pregnancy, with return to normal after delivery. Labor was performed by Caesarian section in 26 births (26%) out of the 81 successful pregnancies. These collected data represent the largest number of pregnancies in thalassemic females reported so far and are clearly encouraging for the ultimate improvement of the quality of life in thalassemic patients.

Bone pain in thalassaemia: assessment of DEXA and MRI findings.

An increasing number of adult thalassaemics have been complaining of aches and pains of varying degrees of severity. In a minority the pains are debilitating and there is stiffness in movement. This study is an attempt to understand the osteoporosis of thalassaemia using DEXA and MRI as the main investigative tools. 122 patients with homozygous beta-thalassaemia were examined by DEXA. It was found that almost half had BMD below two standard deviations from the mean for the normal population, especially in the lumbar spine. There was no marked worsening with age. However the proportion of patients who had their first transfusion after the 3rd year (especially after the 6th) was significantly greater in those with the low BMD. There is also an excess of hypogonadic thalassaemics amongst those with low BMD. 72 thalassaemics were examined by MRI of marrow. Hypercellular, dark marrow on T1 weighted images found in young patients (20-30 yr) was replaced by fatty marrow in later life (30-40 yr). In a group of 21 older thalassaemics (33-62 yr) extreme bone marrow expansion was expressed by the reappearance of hypercellular areas, giving the impression of patchiness which affects not only the diaphyses but also the metaphyses. These patients mostly (66%) had thalassaemia intermedia and had started irregular transfusion after the 6th year of life. About 75% had a BMD below 2 SD. The conclusion is that patients who were late in receiving blood and especially those with thalassaemia intermedia had a more expanded bone marrow with pressure on cortical bone which caused pain in several cases. An attempt was made in 10 patients to reduce marrow hyperplasia by using hydroxyurea. Results showed a relief of pain and modification of magnetic signal intensity.

ICSH recommendations for the measurement of haemoglobin A2.

Although DNA analysis is needed for characterization of the mutations that cause ß-thalassaemia, measurement of the Hb A(2) is essential for the routine identification of people who are carriers of ß-thalassaemia. The methods of quantitating Hb A(2) are described together with pitfalls in undertaking these laboratory tests with particular emphasis on automated high-performance liquid chromatography and capillary electrophoresis.

ICSH recommendations for the measurement of haemoglobin F.

Measurement of the Haemoglobin F in red cell haemolysates is important in the diagnosis of 뫧 thalassaemia, hereditary persistence of fetal haemoglobin (HPFH) and in the diagnosis and management of sickle cell disease. The distribution of Hb F in red cells is useful in the diagnosis of HPFH and in the assessment of feto-maternal haemorrhage. The methods of quantifying Hb F are described together with pitfalls in undertaking these laboratory tests with particular emphasis on automated high-performance liquid chromatography and capillary electrophoresis.

Preimplantation diagnosis and HLA typing for haemoglobin disorders.

Haemoglobin disorders are among the most frequent indications for preimplantation genetic diagnosis (PGD), introduced as an important option to couples at risk for producing offspring with thalassaemia and sickle cell disease. Previous experience mainly included PGD for beta-thalassaemia, while PGD for alpha-thalassaemia resulting in an unaffected pregnancy has not been reported. This study presents the results of the world's largest experience of 197 PGD cycles for haemoglobin disorders, which includes PGD for alpha-thalassaemia, resulting in 53 clinical pregnancies and birth of 45 healthy children, with five still ongoing. Fifty-four of these cycles were performed in combination with HLA typing, allowing the birth of thalassaemia-free children who were also HLA identical to the affected sibling, with successful stem cell transplantation in one case. As an increasing proportion of patients requesting PGD with HLA typing are of advanced reproductive age, aneuploidy testing was performed simultaneously with PGD. The results show that PGD has now become a practical approach for prevention of haemoglobin disorders, and is gradually being used also for improving access to HLA compatible stem cell transplantation for this group of diseases.

The adolescent thalassemic. The complicant rebel.

Chronic hereditary conditions are expected to have a strong influence on the psychosocial development of the adolescent. Thalassaemia major is the commonest of these disorders in Mediterranean countries. In the past it was rarely seen in adolescence because the majority of patients died in childhood. The availability of treatment has allowed survival but the optimum treatment is difficult, especially chelation which consists of daily subcutaneous infusions of 10 hours duration. The adolescent patient must achieve social independence while he/she has a dependency on family, doctors and nurses and a need to strictly adhere to a difficult regime. Non-adherence will result in complications and possible fatality. Family and caretakers may become overprotective. Short stature, bone deformities and poor sexual development influence self image and self confidence. The purpose of this paper is to examine how various published studies have viewed the adolescent with Thalasaemia and to draw conclusions about the necessity for supportive services for the patients and their families. Earlier studies gave emphasis to negative psychosocial trends such as dysphoric moods, dependency, low self esteem, fears, anxiety etc. These were attributed to family stresses and over-protectiveness as well as the condition. Other studies from centers in Italy mainly, have demonstrated normal psychosocial development with better social adjustment than normal peers. Recently published data suggest that Italian patients are more compliant to treatment compared to Greek and Cypriot patients. This may be due to better psychosocial support. The need for such support is emphasized as is the need for training of doctors and nurses dealing with Thalassaemia major patients.

Prevention of thalassaemia in Cyprus.

A programme for the prevention of beta-homozygous thalassaemia has been operating in Cyprus from 1973. From 1976 there has been an increasing gap between the number of homozygotes born and the number expected, calculated as 1 in 135 of the total births. In 1978 23 homozygotes are known to have been born compared with 71 expected, and in 1979, 18 compared with 77 expected. All cases may not have yet to come to notice. the programme consists of public education, population screening, genetic counseling, and antenatal diagnosis.

Prevention and control of haemoglobinopathies.

In many developing countries the haemoglobinopathies (thalassaemias and sickle-cell disorder) are so common that they provide a convenient model for working out a genetic approach to control of chronic childhood diseases. At present, about 250 million people (4.5% of the world population) carry a potentially pathological haemoglobinopathy gene. Each year about 300,000 infants are born with major haemoglobinopathies. Haemoglobinopathy control programmes, based on WHO approaches and recommendations, have been established in different countries in all WHO Regions and have been successful in management of the problem. Following WHO recommendations the health burden of hereditary anaemias could be significantly reduced. This article summarizes the presentations and discussion made at a joint WHO/TIF (Thalassaemia International Federation) meeting, held in Cyprus in April 1993, and reviews the experience of programmes in several countries for the control of haemoglobinopathies in the world.

Novel cystic fibrosis mutation associated with mild disease in Cypriot patients.

Cyprus is an island in the eastern Mediterranean basin inhabited by people of Caucasian extraction, mostly Greek-Cypriots. The most common inherited disease among Caucasians is cystic fibrosis (CF). Although no careful scientific study had ever been done the impression was that CF was extremely rare among the Greek-Cypriots, with an incidence estimated at around 1:30,000. About 2 years ago, we introduced molecular diagnostic methodology in an effort to assist clinicians in safer diagnosis of patients presenting with atypical CF symptomatology, and also for testing the hypothesis that mutations that cause milder phenotypes might be responsible for misdiagnosis or for missing entirely some cases of CF. Initial screening for delta F508 revealed that it is indeed rare in the general population. Further screening of suspected CF patients revealed a novel mutation that converted leucine at position 346 to proline (L346P) in two unrelated families. The second CF mutation was delta F508 and 1677delTA in the two families respectively, both reportedly associated with severe phenotypes. Yet our patients did not present with typical CF pictures possibly because of the dominant nature of this novel mild mutation in exon 7. Symptoms included failure to thrive, chest infections and electrolyte disturbances. These findings raise the possibility that Cyprus might have been spared very severe CF phenotypes but not cystic fibrosis transmembrane conductance regulator (CFTR) mutations.

alpha-Thalassaemia in the population of Cyprus.

We have determined the alpha-thalassaemia (alpha-thal) determinants in 78 patients with Hb H disease from Cyprus; 25 were Turkish Cypriots and 53 were Greek Cypriots. Four deletional and three non-deletional alpha-thal alleles were present; the -alpha(3.7 kb) alpha-thal-2 and the --MED-I alpha-thal-1 were most frequently seen; --MED-II and -(alpha)20.5 deletions occurred at considerably lower frequencies. About 15% of all chromosomes carried a non-deletional alpha-thal-2 allele; of these the 5 nucleotide (nt) deletion at the first intervening sequence (IVS-I) donor splice site was present in approximately 8% of all chromosomes. Two types of polyadenylation signal (poly A) mutations were observed. No striking frequency differences were seen between Greek and Turkish Cypriot patients. Combinations of the various types of alpha-thal resulted in eight different forms of Hb H disease. The phenotypes were comparable except for great variations in the level of Hb H which was highest (average approximately 22%) in the 12 patients with the alpha 5nt alpha/--MED-I combination. One patient with the same form of Hb H disease but with an additional beta-thal (IVS-I-110,G-->A) heterozygosity had a most severe microcytosis and hypochromia with < 1% Hb H. Variations in the level of Hb H might correlate with the severity of the disease, although this was not evident from the haematological data.

Alpha-thalassaemia prenatal diagnosis by two PCR-based methods.

In Cyprus all couples carrying alpha0-thalassaemia mutations are detected in the course of the thalassaemia carrier screening program and prenatal diagnosis is offered to all of them. Prenatal diagnosis for alpha-thalassaemia is routinely done by two independent molecular methods. With the first method, the mutations of the parents are directly determined by gap-PCR and then the chorionic villus sample (CVS) is examined for the presence of these mutations. With the other method, a (CA)n repeat polymorphic site located between the psialpha1- and alpha2-globin genes is used for determining the presence or absence of the normal and mutant alleles. In the period from 1995 to 1999, molecular analysis of 46 couples in which haematological data were consistent with deletion of two alpha-globin genes in both partners indicated that only 13 of them were actually at risk for haemoglobin (Hb) Bart's hydrops fetalis and prenatal diagnosis was provided in 16 pregnancies. The molecular diagnosis was possible in all cases with the use of both gap-PCR and (CA)n repeat polymorphisms analysis. No misdiagnosed cases for alpha-thalassaemia have been reported to date.

Hb Bart's levels in cord blood and alpha-thalassemia mutations in Cyprus.

The purpose of this study was to examine the frequency of alpha-thalassemia in the population of Cyprus using cord blood samples. The levels of Hb Bart's were compared with the hematological indices and the results correlated with the presence of alpha-thalassemia mutations. The protocols for the polymerase chain reaction detection of the six most common alpha-globin mutations encountered in Cyprus were optimized, and the frequency of each mutation was determined through the screening of 495 random cord blood samples. The total allele frequency for the mutations examined was 10.6%, of which 1% is due to the triplication of the alpha-globin genes. The -alpha(3.7 kb) deletion accounts for 72.8% of all detectable mutations, while the--MED-I and -(alpha)-20.5 kb mutations account for 7.8%. The level of Hb Bart's and the MCV and MCH values in cord blood samples were found to correlate closely with the severity of alpha-thalassemia, although the -alpha(3.7 kb) deletion and perhaps other mild alpha-thalassemia mutations may not give detectable Hb Bart's levels. A reasonably accurate estimate of the alpha-thalassemia carrier frequency may be obtained from cord blood studies if Hb Bart's estimates are combined with hematological indices. When molecular methods are added, these give the best way to use cord bloods to survey populations for alpha-thalassemia.

Haematological aspects of antenatal diagnosis for thalassaemia in Britain.

The results are described of 200 antenatal diagnostic tests for haemoglobinopathies performed on samples of fetal blood obtained during the second trimester of pregnancy. Haemoglobin A synthesis in the fetus was measured by incorporation of tritiated leucine in vitro and separation of the globin chains on CM23 columns. The range of HbA synthesis detected was 3.5-8.0% in normal fetuses, 2.0-5.0% in fetuses with thalassaemia trait, and less than 1.6% in fetuses with thalassaemia major. There were eight cases in which other haemoglobinopathies were diagnosed. 29% of the pregnancies were terminated because thalassaemia major was diagnosed, and 9.5% of the remaining healthy fetuses were lost for obstetric reasons. Follow up has been possible for 96% of the 124 surviving babies and three misdiagnoses have come to light; one false positive (0.5%) and two false negatives (1%). These figures represent a first effort at antenatal diagnosis for haemoglobinopathies and it is likely that they will improve with the passage of time.

Comparative in vivo expression of beta(+)-thalassemia alleles.

Double heterozygotes who inherit one abnormal though stable beta-globin variant in association with a molecularly identified beta(+)-thalassaemia allele provide unique opportunities to quantify the in vivo expression of particular beta(+)-thalassemia alleles. The globin products of the two alleles can be separated, quantified and the output of the beta(+)-thalassaemia allele expressed as the MCH-beta(A) in pg beta(A)-globin/beta(+)-thalassemia allele/RBC = 0.5 MCH x Hb A%. In this communication we provide new quantitative data on the expression of five mutations as follows: the beta(+)-87 (C-->G) = 3.8 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 1); the beta(+) IVS-I-1 (G-->A) = 0.2 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 1); the beta(+) IVS-I-6 (T-->C) = 2.9 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 7); the beta(+) IVS-I-110 (G-->A) = 1.1 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 13), and the beta(+) IVS-II-745 (C-->G) = 1.74 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 2). The values obtained are compared with those of other beta(+)-thalassemia alleles from the literature. It can be seen that the MCH-beta(A) value may be a correct index of thalassemia severity useful for the correlation of genotype with phenotype, and for understanding the effects of mutations in beta-globin genes on pathophysiologically meaningful beta-globin gene expression.

Birth of healthy children after preimplantation diagnosis of thalassemias.

BACKGROUND: Preimplantation genetic diagnosis (PGD) allows couples at risk of having children with thalassemia to ensure the healthy outcome of their pregnancy. METHODS: Seventeen PGD clinical cycles were initiated for Cypriot couples at risk of having children with different thalassemia mutations, including IVSI-110, IVSI-6, and IVS II-745. Unaffected embryos for transfer were selected by testing oocytes, using first and second polar body (PB) removal and nested polymerase chain reaction analysis followed by restriction digestion. RESULTS: Unaffected embryos were selected in 16 of 17 PGD cycles. Of 166 oocytes studied from these cycles, 110 were analyzed by sequential analysis of both the first and the second PB, resulting in preselection and transfer of 45 unaffected embryos. This resulted in seven pregnancies and in the birth of five healthy thalassemia-free children. The embryos predicted to have inherited the affected allele were not transferred. Analysis of these embryos confirmed the PB diagnosis. CONCLUSIONS: Sequential first and second PB testing of oocytes is reliable for PGD of thalassemia and is a feasible alternative to prenatal diagnosis in high-risk populations.

The beta-thalassaemia mutations in the population of Cyprus.

We have identified the beta-thalassaemia alleles in nearly all known Turkish Cypriot beta-thalassaemia homozygotes and in over 700 Greek Cypriot beta-thalassaemia heterozygotes living on the island of Cyprus. The data confirmed earlier observations that the IVS-I-100 (G-->A) mutation is present for about 74-80%, while three other alleles [IVS-II-745 (C-->G), IVS-I-6 (T-->C), IVS-I-1 (G-->A)] occur at frequencies of 5-8%. Nearly identical percentages were observed for the two Cypriot groups, quite different from those for beta-thalassaemia patients from Greece and Turkey. This suggests close contacts between the two Cypriot communities during many centuries without a major recent influence from Greek or Turkish beta-thalassaemia carriers.