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To try to further define the mechanisms that increase the levels of hemoglobin F (HbF) in the blood of patients with severe forms of ß thalassemia, we have studied two comparable populations of hemoglobin E (HbE)/ß thalassemics, one regularly transfused and one receiving only occasional blood transfusions. Regular transfusion was associated with a significant decrease in soluble transferrin receptor and erythropoietin levels. Globin chain synthesis studies also show a highly significant decrease in HbF synthesis relative to HbE in the transfused patients. This effect was confirmed by sequential data on one patient, studied before and after the commencement of regular blood transfusion; blood transfusion was followed by a marked increase in the ?/?, ßE/?, and HbE/HbF ratios. These data suggest that the high HbF levels in HbE/ß thalassemia, and other ß thalassemia syndromes, result from increased erythropoietin levels leading to bone marrow expansion, and possibly increased F-cell production, combined with ineffective erythropoiesis giving a survival advantage to F cells. This study also suggests that alteration in blood transfusion regimes must be taken into account when interpreting changes in HbF levels seen in trials of HbF-promoting drugs.

To try to further define the mechanisms that increase the levels of hemoglobin F (HbF) in the blood of patients with severe forms of β thalassemia, we have studied two comparable populations of hemoglobin E (HbE)/β thalassemics, one regularly transfused and one receiving only occasional blood transfusions. Regular transfusion was associated with a significant decrease in soluble transferrin receptor and erythropoietin levels. Globin chain synthesis studies also show a highly significant decrease in HbF synthesis relative to HbE in the transfused patients. This effect was confirmed by sequential data on one patient, studied before and after the commencement of regular blood transfusion; blood transfusion was followed by a marked increase in the ∝/γ, βE/γ, and HbE/HbF ratios. These data suggest that the high HbF levels in HbE/β thalassemia, and other β thalassemia syndromes, result from increased erythropoietin levels leading to bone marrow expansion, and possibly increased F-cell production, combined with ineffective erythropoiesis giving a survival advantage to F cells. This study also suggests that alteration in blood transfusion regimes must be taken into account when interpreting changes in HbF levels seen in trials of HbF-promoting drugs.

We have characterised an unusual, highly polymorphic region of DNA located 8‐kb downstream of the human alpha‐globin gene complex. This hypervariable region (alpha‐globin 3′ HVR) is composed of an array of 17‐bp tandem repeats, the number of which differs considerably (70‐450) from one allele to another. The sequence of the 17‐bp repeats is highly conserved within and between alleles. Furthermore, this sequence identifies a core oligonucleotide [5′‐GNGGGG(N)ACAG‐3′] that is common to three previously characterised hypervariable regions. At reduced stringency, a probe to the 3′ HVR detects a new family of multiallelic loci that will be of value in the study of human genetics.

The molecular basis of alpha‐thalassaemia has been established in 48 Thai subjects with Hb H disease and 15 with the Hb Bart's hydrops fetalis syndrome. This study has shown that in this population there are at least 18 different types of chromosome carrying seven independent alpha‐thalassaemia mutations one of which is a novel deletion removing the entire alpha‐globin gene complex. Although there are a limited number of alpha‐thalassaemia determinants in the Thai population, there is a remarkable degree of variation in the genetic markers which flank them. These markers may be of value in establishing the evolutionary history of the alpha‐thalassaemias.

Hemoglobin E (HbE; ?2ß226glu-lys), globally the commonest hemoglobin variant, is synthesized at a slightly reduced rate and has a homozygous phenotype similar to heterozygous ß thalassemia. Yet, when it is inherited together with a ß thalassemia allele, the resulting condition, HbE/ß thalassemia, is sometimes characterized by a severe, transfusion-dependent thalassemia major. The severity of this interaction has not been explained. We have explored the possibility that it may reflect the instability of HbE consequent upon globin chain imbalance imposed by the ß thalassemia allele. Time-course and pulse-chase globin chain synthesis studies at 37°C on peripheral blood and bone marrow suggest that hemoglobin instability is not significant in steady-state HbE/ß thalassemia; this is confirmed by density-gradient centrifugation studies that show no decrease in HbE levels relative to HbA as HbE/ß+ thalassemia red blood cells age. Globin binding to membranes was assessed and only ? globin chains were found, in contrast to other unstable hemoglobins in which both ? and ß chains were present. However, in experiments performed on blood from HbE/ß thalassemics in the temperature range 39°C to 41°C, there was evidence of instability of HbE, a finding that was also observed in homozygous HbE. These findings suggest that the phenotype of HbE/ß thalassemia is primarily the result of the interaction of two ß thalassemia alleles; however, hemoglobin instability may be important during febrile episodes, contributing to worsening anemia. © 1998 by The American Society of Hematology.

The molecular basis of alpha‐thalassaemia has been established in 48 Thai subjects with Hb H disease and 15 with the Hb Bart's hydrops fetalis syndrome. This study has shown that in this population there are at least 18 different types of chromosome carrying seven independent alpha‐thalassaemia mutations one of which is a novel deletion removing the entire alpha‐globin gene complex. Although there are a limited number of alpha‐thalassaemia determinants in the Thai population, there is a remarkable degree of variation in the genetic markers which flank them. These markers may be of value in establishing the evolutionary history of the alpha‐thalassaemias.

We have characterised an unusual, highly polymorphic region of DNA located 8‐kb downstream of the human alpha‐globin gene complex. This hypervariable region (alpha‐globin 3′ HVR) is composed of an array of 17‐bp tandem repeats, the number of which differs considerably (70‐450) from one allele to another. The sequence of the 17‐bp repeats is highly conserved within and between alleles. Furthermore, this sequence identifies a core oligonucleotide [5′‐GNGGGG(N)ACAG‐3′] that is common to three previously characterised hypervariable regions. At reduced stringency, a probe to the 3′ HVR detects a new family of multiallelic loci that will be of value in the study of human genetics.

The Gγand Aγgenes of an individual homozygous for the British form of Aγnondeletion hereditary persistence of fetal hemoglobin have been cloned and partially sequenced. The Gγgene was normal, but the Aγgene was found to have a single base change (T →* C) at –198 bp relative to the cap site. Supercoiled plasmids containing normal γ-genes or the mutant Aγ-gene displayed an S1-hypersensitive site immediately 5′ to the base change.

We conducted a prospective community-based malaria surveillance study on a cohort of children <10 years old living in an area of hyperendemic malaria (spleen rates >50% in children aged 2–9 years) in Vanuatu, Melanesia, supported by a concurrent prospective descriptive study of malaria admissions to the local hospital. The incidence of clinical malaria in children <10 years old was 1·9 episodes/year. The annual incidence of severe malaria (severe malarial anaemia and cerebral malaria) was only in children aged <5 years. The only manifestation of severe malaria seen in indigenous children was anaemia. No death could be attributed to malaria. While the incidence of uncomplicated clinical malaria in this population was comparable to that in many parts of Africa, the incidence of severe forms of the disease was significantly lower. This could not be attributed to differing rates of malaria transmission, chloroquine resistance, or to host protective or behavioural factors. These findings suggest that studies which compare disease patterns in geographically disparate populations may be instrumental in developing a better understanding of the determinants of clinical outcome in Plasmodium falciparummalaria and that such regional differences must be considered when planning or interpreting the effects of malaria interventions.

To further explore the cause for variation in hemoglobin F (Hb F) levels in sickle cell disease, the β globin restriction-fragment length polymorphism haplotypes were determined in a total of 303 (126 SS, 141 AS, 17 Sβ°,7 Aβ,° and 12 AA) Indians from the state of Orissa. The βsglobin gene was found to be linked almost exclusively to a βshaplotype (+ ++ - + +—), which is also common in Saudi Arabian patients from the Eastern Province (referred to as the Asian β*haplotype). By contrast, the majority of βAand β°thalassemia globin genes are linked to haplotypes common in all European and Asian populations (+—— [+ /- ]; -+ + + +). Family studies showed that there is a genetic factor elevating Hb F levels dominantly in homozy-gotes (SS). This factor appears to be related to the Asian βs globin haplotype, and a mechanism for its action is discussed. There is also a high prevalence of an independent Swiss type hereditary persistence of fetal hemoglobin (HPFH) determinant active in both the sickle cell trait and in sickle cell disease.

aThalassemia modifies the hematologic expression of homozygous sickle cell (SS) disease, resulting in increased total hemoglobin and HbA2and decreased HbF, mean cell volume, reticulocytes, irreversibly sickled cells, and bilirubin levels. The age at which these changes develop in children with SS disease is unknown. Ascertainment of globin gene status in a large representative sample of children with SS disease has afforded an opportunity to study the hematologic indices in nine children homozygous for athalassemia 2 (two-gene group), 90 children heterozygous for athalassemia 2 (three-gene group), and 167 children with a normal aglobin gene complement (four-gene group). The two-gene group had significantly lower mean cell volumes from birth, higher red cell counts from one month, lower reticulocytes from three months, and higher HbA2 levels from one year, as compared with the four-gene group. Children with three genes had intermediate indices but resembled more closely the four-gene group. Differences in total hemoglobin or in fetal hemoglobin between the groups were not apparent by eight years of age. The most characteristic differences of the two-gene group were the raised proportional HbA2 level and low mean cell volume, the latter having some predictive value for a thalassemia status at birth. © 1986 by Grune & Stratton, Inc.

The application of the amplification refractory mutation system (ARMS) to the detection of individual β-thalassaemia mutations in heterozygous parents and "at risk" fetuses has been assessed in Indian and Cypriot immigrant populations in the UK. 100 first trimester prenatal diagnoses have been done, entailing the detection of 17 different mutations. The method, which allows the determination of the mutations in both parental and fetal DNA on the same day, should have wide application to the carrier detection and prenatal diagnosis of monogenic diseases with heterogeneous molecular defects.

SummaryHuman bone marrow was cultured with alpha medium, 10−6m hydrocortisone and a mixture of horse and fetal calf serum in a long‐term liquid culture system. An adherent layer was formed which contained fat cells, macrophages, fibroblast‐like and epithelioid cells. The layer harboured myeloid cells and their presumptive precursors whilst the nonadherent cells were composed of immature myeloid elements plus mature macrophages and granulocytes whose numbers were maintained for periods of up to 12 weeks. Experiments showed that the use of serum mixtures was superior to horse or fetal calf serum alone and successful culture was accompanied by early growth of the hydrocortisone‐dependent fat cells in the adherent layer.

A form of α°-thalassemia found in subjects of Mediterranean origin has been analyzed by gene mapping and DNA sequencing. Homozygotes have the hemoglobin Bart's hydrops fetalis syndrome, while compound heterozygotes for this defect and α‘ ‘-thalassemia have hemoglobin H disease. It results from a deletion that removes 20.5 kilobases of DNA from within the a-globin gene cluster. Sequence data from the regions adjacent to the breakpoint indicate that the recombination event that caused this deletion occurred between the α1-gene and an unusual AluI sequence located between the embryonic ζ genes. © 1985 by Grune & Stratton, Inc.

Studies of the prevalence and incidence of malaria were conducted in children <10 years old living in 10 rural villages on the island of Espiritu Santo, Vanuatu, south-west Pacific. Malaria prevalence remained stable at 30% throughout the year but the relative contributions of the 2 major species were highly dependent on season. Plasmodium falciparumpredominated in the long wet season (November–May) and P. vivaxin the dry season (June–October). Case definitions for malaria, derived using a multiple logistic regression method, showed that parasite densities associated with clinical disease were low; case definitions for P. falciparum(>1000 parasites/µL in children > 1 year old and >500/µL in infants) and P. vivax(>500 parasites/µL at all ages) were both associated with a specificity and sensitivity of >90%. Like prevalence data, malaria morbidity was highly seasonal; 80% of clinical P. falciparuminfections occurred in the wet season and 66% of clinical P. vivaxin the dry season. Mixed infections were rare. Malaria was an important cause of morbidity with children <5 years old experiencing 1.3–2.0 episodes of clinical malaria per year and 23% of fevers being attributable to malaria in this age group. Children aged 5–9 years continued to suffer one episode of clinical malaria per year. The peak incidence of P. vivaxmalaria occurred earlier in life than the peak incidence of P. falciparummalaria. The possible interactions between these 2 parasite species are discussed.

Analysis of DNA from 852 Island Melanesians has revealed a high frequency of single- and triple-7-globin genes in this population. Homozygotes for triple- and single-7 genes have normal hematologic findings, normal hemoglobin F (HbF) levels, and when there is coexisting athalassemia, appropriate levels of Bart's Hb (74) at birth. In addition, we have identified an individual with a quadruple-7 gene chromosome who also has a normal HbF level. All single-7 genes were A7, all triple-7 genes GγGγAγ.and the quadru- ple-7 gene GγGγGγGγ. Analysis of Gγ:Aγ ratios in cord bloods and HbF levels in adults showed that these additional γ genes are expressed and are down regulated appropriately by the fetal to adult Hb switch. Analysis of the restriction enzyme haplotypes of these various chromosomes indicates that intrachromosomal cross-overs are more likely to have produced these variants than interchro-mo soreal recombination events.

A 12-yr-old Malay boy who was studied because his youngest brother and both his parents had the slow-moving Hb X components (earlier reported to lead to Hb H disease when combined with α-thalassemia) was found to be homozygous for the same slow-moving components. He had splenomegaly and a just palpable liver, mild anemia with microcytosis, hypochromia, slight morphologic changes of the red blood cells, and slight reticulocytosis. Of eight children in the family, six had the trait for the abnormality, one was normal, and one, the propositus, was homozygous. Structural studies of the isolated abnormal hemoglobin showed it to be identical to Hb Constant Spring (Hb CoSp), an α-chain variant with 172 residues instead of the usual 141, the additional 31 being attached to the C-terminal end. In addition to the abnormal α variant for which the propositus was homozygous, he also had normal Hb A and normal Hb A2with normal α-chains. If the theory that Hb CoSp is due to a structural mutation affecting the terminator codon is correct, this case provides evidence for a duplication of the gene for α-chain production. Results of study of several erythrocyte enzymes are also reported.

In a previous study, we described a form of nondeletion a-thalassemia (αTSaudiα) found in subjects of Saudi Arabian origin. In the current study, using synthetic oligoprobe hybridization and restriction enzyme analysis, we have demonstrated that the molecular basis of αT Saudiα is due solely to a single base mutation (AATAAA → AATAAG) in the polyadenylation signal of the α2 gene and that the frameshift mutation in codon 14 of the linked α1 gene is the result of a cloning artefact. The α2polyadenylation signal mutation occurs in other Middle Eastern and the Mediterranean populations and is responsible for the clinical phenotype of Hb H disease in some Saudi Arabian individuals with five α genes (αT Saudiα/(ααα)T Saudi). Evidence suggests that the (ααα)T Saudi haplotype has arisen as a result of a recombination between two misaligned chromosomes bearing the α T Saudi α defect.

Hb F levels were determined on samples from 750 normal blood donors. Six individuals (0.8 %) had Hb F levels in excess of 1.1 %, the upper end of the continuous distribution. Eight individuals at the upper end and 7 individuals at the lower end of the range were selected for family studies. These studies revealed that the control of Hb F levels in adults, as judged by the more sensitive F-cell technique, has a major genetic component. Structural analysis of the Hb F in several cases demonstrated that both Gγ and Aγ chains were present, but in variable proportions. These results are discussed in light of current concepts of adult F-cell production.

Data on the distribution of β thalassemia among over 6,000 Melanesians reveals a major difference in the carrier rates between populations in the malarious coastal regions of New Guinea and those living in the historically malaria-free Highlands. The island of Maewo in Vanuatu has a particularly high incidence of β+thalassemia associated with a single restriction enzyme haplotype. Direct cloning into a plasmid vector and sequence analysis demonstrate that the mutation is a G to C transversion at position 5 of intron 1 of the β-globin gene. Oligonucleotide probe surveys indicate that this variant accounted for all cases of βthalassemia studied from Maewo. It is also common in coastal Papua New Guinea where haplotype and oligonucleotide probe data suggest that the molecular basis of βthalassmia is more heterogeneous.

Haemoglobin Bart's was detected in cord blood samples from 81% of 217 infants born in Madang on the north coast of Papua New Guinea. Analysis of the α globin genes of 30 infants and adults from the same region showed that all but 3 were heterozygous or homozygous for the deletion form of α+thalassaemia. None of 18 cord blood samples from infants born in Goroka in the Eastern Highlands Province had haemoglobin Bart's, and in each case the α globin genes were normal. Preliminary geographical and linguistic analyses of both groups suggest that the prevalence of a thalassaemia may be related to altitude rather than to linguistic grouping and hence that resistance to malaria may be at least one reason why α thalassaemia is so common in some populations.

J Pediatr 1998;132:765-7

Studies on the biosynthesis of the Summaryminor adult hæmoglobin A2may be relevant to an understanding of some of the β thalassæmias.

Iron absorption and rates of iron accumulation were analysed in a group of patients with β-thalassæmia intermedia. Iron absorption was strikingly increased and there was a progressive iron loading with increasing age. Balance studies indicated that by the time many of these patients reach the third or fourth decades their total iron loads may be of a similar magnitude to those of transfusion-dependent β-thalassæmia homozygotes. If these patients are to be protected from cardiac, hepatic, and endocrine complications of iron overload in middle life it will be necessary to reduce gastrointestinal iron absorption, starting from early childhood.

Absorption of 59Fe ferrous sulphate was measured in patients with a variety of iron-loading anæmias both before and during subcutaneous infusions of desferrioxamine. The infusions were associated with a reduction in absorption (P<0·025). This effect was not altered by saturation with ascorbic acid.

A highly polymorphic DNA probe genetically linked to the locus of autosomal dominant polycystic kidney disease was used in linkage studies for prenatal diagnosis in a nine-week fetus at risk for the disease. The fetus was judged to have inherited the polycystic kidney disease mutation, and this was confirmed by microscopic examination of the fetal kidneys at necropsy.

Slow-moving hæmoglobin components have been found in small amounts in some patients with hœmoglobin H disease from Greece, Thailand, Malaysia, and Hong Kong. Chemical analysis indicates that in each case the abnormal hæmoglobins are identical to hæmoglobin Constant Spring (Hb CS), a variant with an elongated α-chain.

Although considered a mild disorder, beta thalassemia trait (uncomplicated by any other hematologic abnormality) has been reportedly associated with symptoms of anemia, while in other series hepatosplenomegaly has been reported as associated with this diagnosis. No controlled study has examined the symptoms and signs of individuals with beta thalassemia trait and compared these with age- and sex-matched normal individuals. We administered a questionnaire to 397 parents (67% females) of children with beta thalassemia major attending the National Thalassemia Centre in Kurunagala, Sri Lanka, and to 87 normal volunteers (90% females). All individuals underwent physical examination by the same investigator, as well as testing by automated cell counter (Coulter), and HPLC (Bio Rad). The mean age of the parents (33 years) and controls (29.5 years) was not significantly different. In parents with thalassemia trait, 20% of males and 42% of females complained of reduced exercise tolerance compared to 12.5% of males and 38% of females in the controls (P= NS). The questionnaire revealed that 41% of parents, and 36% of controls, complained of recurrent headache; 18% of parents, and 15% of controls, complained of lethargy (P=NS). A history of malaria infection was reported in 30% of parents with thalassemia trait, and in 24% of controls. Mean quality-of-life score, recorded formally by a linear analog scale, from 0 (poorest) to 10 (greatest) was 7.1 in parents and 7.2 in controls. Splenomegaly was detected in 6% of parents with beta thalassemia trait (mean spleen size 1.5 cm below the costal margin), and 5% of normal controls (mean spleen size 1 cm below the costal margin). These findings suggest that, by contrast to many reports in the older literature, in uncomplicated beta thalassemia trait there appears to be no symptoms or signs greater than those observed in a normal control population and that fatigue, lethargy, exercise intolerance, or splenomegaly in a patient with beta thalassemia trait should not be attributed to the underlying hemoglobinopathy.

A series of restriction fragment length polymorphisms which are due to DNA rearrangements have been identified within two highly variable regions flanking the human α globin genes. The existence of such highly polymorphic areas provides a large number of individual genetic markers for the α globin gene cluster on chromosome 16. If, as seems likely, such regions occur frequently throughout the human genome they should be of considerable value in the antenatal diagnosis of genetic disease.

DNA from individuals heterozygous for Gγ(δβ)° thalassaemia has been studied by restriction endonuclease analysis. The results reveal a new molecular defect associated with this condition. A total of three defects is now responsible for the one single phenotype, thereby emphasising the complex relationship between genotype and phenotype among the disorders of β-like globin synthesis in man.

The first example of a duplication involving the human β-like globin genes has been characterised in DNA from a native of Vanuatu. Restriction endonuclease mapping has shown that a 5 kb insert of DNA in the γ-δ-β gene cluster is due to duplication of the Gγ-globin gene and results in a new rearrangement ‘5′-ɛ-Gγ-Gγ-Aγ-δ-β-3’

We have examined the molecular structure of the human α globin gene complex from individuals with a common form of a thalassaemia in which one of the duplicated pair of α genes (αα) has been deleted (−α3·7). Restriction mapping and DNA sequence analysis of the mutants indicate that different −α3·7 chromosomes are the result of at least three independent events. In each case the genetic crossover has occurred within a region of complete homology between the α1 and α2 genes. Since the −α chromosomes may reflect the processes of crossover fixation and gene conversion between the two genes, their structures may provide some insight into the mechanism by which the concerted evolution of the human α globin genes occurs.

We studied the aetiology of malnutrition in a cohort of 1511 children <10 years old in Espiritu Santo, Vanuatu. Malnutrition was categorized using standard anthropometrie criteria as: underweight [weight-for-age (WA) Z score <–2], wasting [weight-for-height (WH) Z <–2], or stunting [height-for-age (HA) Z <–2]. On multiple logistic regression analysis, the only factors significantly associated with wasting were age <5 years [OR (95% CI) 1.8 (1.2–2.9), p = 0.01] and having suffered one or more episodes of clinical P. vivax malaria in the 6 months preceding nutritional assessment [OR 2.4 (1.3–4.4), p = 0.006]. The incidence of P. vivax infection was significantly higher during the 6 months preceding assessment in underweight vs. non-underweight children [incidence rate ratio (IRR) 2.6 (1.5–4.4), p≤0.0001). These groups had similar incidences of clinical P. falciparum infection during the same period [IRR 1.1 (0.57–2.1) p = 0.8] and of either species during the 6 months following assessment [IRR P. vivax 1.3 (0.9–2.0) p = 0.2; IRR P. falciparum 1.3 (0.9–1.9) p = 0.2]. In these children, P. vivax malaria was a major predictor of acute malnutrition; P. faldparum was not. Wasting neither predisposed to nor protected against malaria of either species. Although P. vivax malaria is generally regarded as benign, it may produce considerable global mortality through malnutrition.