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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 falciparum predominated in the long wet season (November-May) and P. vivax in 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/microL in children > 1 year old and > 500 microL in infants) and P. vivax (> 500 parasites/microL 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. falciparum infections occurred in the wet season and 66% of clinical P. vivax in the dry season. Mixed infections were rare. Malaria was important cause of morbidity with children < 5 years old experiencing 1.3-3.0 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. vivax malaria occurred earlier in life than the peak incidence of P. falciparum malaria. The possible interactions between these 2 parasite species are discussed.

Most eukaryotic messenger RNAs have the sequence AAUAAA 11-30 nucleotides from the 3'-terminal poly(A) tract. Since this is the only significant sequence homology in the 3' non-coding region it has been suggested that it may be a recognition site for enzymes involved in polyadenylation and/or termination of polymerase II transcription. This idea is strengthened by observations on the effect of deletion mutations in or around the AATAAA sequence on polyadenylation of late simian virus 40 (SV40) mRNA; removal of this sequence prevents poly(A) addition. Naturally occurring variants of this hexanucleotide are rare and hitherto their functional significance has not been assessed. We have now identified a human alpha 2-globin gene which contains a single point mutation in this hexanucleotide (AATAAA leads to AATAAG). The paired alpha 1 gene on the same chromosome is completely inactivated by a frame-shift mutation. This unique combination has enabled the expression of the mutant alpha 2 gene to be studied in vivo where it has been found that the accumulated level of alpha 2-specific mRNA in erythroid cells is reduced. Furthermore, readthrough transcripts extending beyond the normal poly(A) addition site are detected in mRNA obtained from HeLa cells transfected with cloned DNA from the mutant alpha 2 gene, suggesting that the single nucleotide change in the AATAAA sequence is the cause of its abnormal expression.

The alpha+-thalassaemias are the commonest known human genetic disorders, affecting up to 80 per cent of some populations. Although there is good evidence from both epidemiological and clinical studies that these gene frequencies reflect selection by, and protection from, malaria, the mechanism is unknown. We have studied the epidemiology of malaria in childhood on the southwestern Pacific island of Espiritu Santo in Vanuatu and here we report that, paradoxically, both the incidence of uncomplicated malaria and the prevalence of splenomegaly, an index of malaria infection, are significantly higher in young children with alpha+-thalassaemia than in normal children. Furthermore, this effect is most marked in the youngest children and for the non-lethal parasite Plasmodium vivax. The alpha+-thalassaemias may have been selected for their ability beneficially to increase susceptibility to P. vivax, which, by acting as a natural vaccine in this community, induces limited cross-species protection against subsequent severe P. falciparum malaria.

Soluble transferrin receptor (sTfR) concentration is a sensitive index of iron deficiency when used in conjunction with ferritin measurements in adults. One advantage of this assay is that unlike ferritin it does not appear to be affected by a range of infectious and inflammatory conditions or by pregnancy, rendering it a promising adjunct to the diagnosis of iron deficiency in tropical populations. We have measured plasma sTfR concentrations in a group of malaria patients (n = 21) and asymptomatic (18) and aparasitemic (76) controls in Vanuatu. Plasma sTfR concentration was significantly reduced in individuals with acute malaria (P = 0.003). While this observation provides evidence that erythropoeitic suppression may be an important etiologic component in malarial anemia, it also suggests that malaria may be a confounding factor when interpreting sTfR concentrations in such populations. The role of sTfR in the diagnosis of iron deficiency in tropical populations remains to be established.

Extensive allelic diversity in variable numbers of tandem repeats (VNTRs) has been discovered in the human genome. For population genetic studies of VNTRs, such as forensic applications, it is important to know whether a neutral mutation-drift balance of VNTR polymorphism can be represented by the infinite alleles model. The assumption of the infinite alleles model that each new mutant is unique is very likely to be violated by unequal sister chromatid exchange (USCE), the primary process believed to generate VNTR mutants. We show that increasing both mutation rates and misalignment constraint for intrachromosomal recombination in a computer simulation model reduces simulated VNTR diversity below the expectations of the infinite alleles model. Maximal constraint, represented as slippage of single repeats, reduces simulated VNTR diversity to levels expected from the stepwise mutation model. Although misalignment rule is the more important variable, mutation rate also has an effect. At moderate rates of USCE, simulated VNTR diversity fluctuates around infinite alleles expectation. However, if rates of USCE are high, as for hypervariable VNTRs, simulated VNTR diversity is consistently lower than predicted by the infinite alleles model. This has been observed for many VNTRs and accounted for by technical problems in distinguishing alleles of neighboring size classes. We use sampling theory to confirm the intrinsically poor fit to the infinite alleles model of both simulated VNTR diversity and observed VNTR polymorphisms sampled from two Papua New Guinean populations.

Data on the distribution of beta 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 beta + 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 beta- globin gene. Oligonucleotide probe surveys indicate that this variant accounted for all cases of beta thalassemia studied from Maewo. It is also common in coastal Papua New Guinea where haplotype and oligonucleotide probe data suggest that the molecular basis of beta thalassmia is more heterogeneous.

We describe a simple method to directly clone any DNA fragment for which a flanking restriction enzyme map is known. Genomic DNA is digested with multiple enzymes cutting outside the fragment to be cloned, selected by electroelution from an agarose gel, and cloned directly into a plasmid vector. It is only necessary to screen 10-1000 colonies and recombinant DNA is ready for immediate molecular analysis without further subcloning. The use of this technique is demonstrated for the cloning of a sequence from within the human alpha-globin complex that was previously shown to be "unclonable" in bacteriophage and cosmid vectors and which is a multiallelic general genetic marker, as well as both beta-globin alleles from an individual with beta-thalassaemia.

Four minor haplotypes that produce abnormal haemoglobin phenotypes in horses have been characterized. Two of them, AIIb and V, are copy number variants with, respectively, one and three alpha genes instead of the normal complement of two. The AIIa and C haplotypes, on the other hand, each have two alpha genes but, as a result of probable gene conversions, they now encode identical, though haplotype specific, globins. Two out of 60 unrelated and phenotypically normal horses studied had an unusual triplicated rearrangement in the embryonic zeta-gene locus. Each of these variants appears to have been produced by aberrant recombination events.

Objectives: This investigation was performed to examine genetic variation at the β-globin locus in a sample of 30 healthy individuals from native populations in South America. The patterns of haplotypic variation were compared with those of previous studies including samples for various worldwide populations in an attempt to make inferences about the occupation of the Americas from a deeper temporal perspective than is typically available with haploid markers. Methods: A 2.67-kb segment containing the β-globin gene and its flanking regions was examined for genetic variation in a sample of 60 chromosomes from native populations in South America. The fragment was PCR-amplified and directly sequenced. To determine linkage relationships in compound heterozygotes, we used the amplification refractory mutation system. In addition, we assessed genetic variability and differentiation among populations, and we performed tests of selective neutrality. These analyses were performed for Brazilian Amerindian group and other worldwide populations previously studied. Results: Eleven polymorphic sites were found in the studied fragment, which distinguished eight different haplotypes, three recombinants haplotypes (present as single copies) and five previously described haplotypes, including some of those most highly differentiated. Genetic variation found in the pooled sample is substantial. Conclusions: Although only five known haplotypes are observed in Amazonia, some of these are highly divergent, resulting in patterns of molecular polymorphism equal to or higher than those from other world regions. Am. J. Hum. Biol. 2012. © 2012 Wiley Periodicals, Inc.

We report here restriction endonuclease maps of the β-like globin gene cluster for the British form of HPFH and for a case of Gγβ+ HPFH, and also confirm and extend previous reports of the map for the Greek form of HPFH. These results show that all these conditions belong to a group lacking any substantial deletion or rearrangement of DNA sequence in this gene cluster. The absence of any gross disruption of the structure of this region of the genome, together with evidence that the HPFH genotype is either allelic with, or closely linked to, the β-like globin gene cluster, suggests that the responsible lesions are nearer to being purely regulatory in nature than in forms of HPFH due to substantial deletions. Thus these conditions promise to provide less equivocal evidence about the regulation of β-like globin gene expression than has so far been available.

The promoter region of the UDP glucuronosyltransferase 1 gene (UGT1A1) contains a run of thymine-adenine (TA) repeats, usually six (TA)(6). As well as its relationship to Gilbert's syndrome, homozygosity for the extended sequence, (TA)(7) (TA)(7), has been found to be an important risk factor for hyperbilirubinemia and gallstones in patients with hemoglobin E-beta-thalassemia and other intermediate forms of beta thalassemia. To assess the importance of this polymorphism in these common disorders a wide-scale population study of the relative frequency of the size alleles of the UGT1A1 promoter has been carried out. Homozygosity for the (TA)(7) allele occurs in 10-25% of the populations of Africa and the Indian subcontinent, with a variable frequency in Europe. It occurs at a much lower frequency in Southeast Asia, Melanesia, and the Pacific Islands, ranging from 0 to 5%. African populations show a much greater diversity of length alleles than other populations. These findings define those populations with a high frequency of hemoglobin E-beta-thalassemia and related disorders that are at increased risk for hyperbilirubinemia and gall bladder disease and provide evolutionary insights into how these polymorphisms have arisen and are so unequally distributed among human populations.

Polymerase chain reaction protocols were designed specifically to amplify regions of the alpha globin complex that contain the nine common polymorphic haplotyping sites. These reactions provided a quicker and more sensitive approach to determining alpha globin haplotypes than Southern blotting methods.

Summary Background Thalassaemias pose an increasing problem for the Indian subcontinent and many Asian countries. We analysed the different types of thalassaemia in the Sri Lankan population, surveyed gene frequencies in schoolchildren, and estimated the burden of disease and requirements for its control. Methods We analysed blood samples from patients attending clinics in nine hospitals and defined the different types of β thalassaemia by high-performance liquid chromatography (HPLC) and DNA analysis. The range of mutations was obtained by analysis of β-globin genes. Capillary blood was obtained from schoolchildren from different parts of the island and analysed by HPLC to provide an approximate assessment of the carrier frequency of β thalassaemia and haemoglobin E (HbE). To estimate the frequency of α thalassaemia the α-globin genotypes were also analysed when it was possible. Findings Blood samples were obtained from 703 patients with β thalassaemia and from 1600 schoolchildren. The thalassaemia mutations were unevenly spread. Although 23 different β-thalassaemia mutations were found, three accounted for the thalassaemia phenotype in about 70% of the patients, most whom are homozygotes or compound heterozygotes for IVS1–5 (G→C) or IVS1–1 (G→A). The third common mutation, codon 26 (G→A), which produces HbE, interacts with one or other of these mutations to produce HbE/β thalassaemia; this comprises 13·0–30·9% of cases in the main centres. Samples from 472 patients were analysed to determine the α-globin genotype. Overall, 15·5% patients were carriers for deletion forms of α + thalassaemia. Average gene frequencies showed that there will be more than 2000 patients requiring treatment at any one time, in the future, of whom those with HbE/β thalassaemia will account for about 40%. Interpretation In Sri Lanka, interactions of the two common β-thalassaemia alleles will nearly always result in a transfusion-dependent disorder. However, about 40% of patients will have HbE/β thalassaemia, which has a variable course. The management of these disorders could require about 5% of the total health budget. We need to learn more about the natural history and appropriate management of HbE/β thalassaemia if resources are to be used effectively.

We describe a sensitive, reliable and reproducible method, based on three multiplex PCR assays, for the rapid detection of seven common alpha-thalassaemia deletions and one alpha-globin gene triplication. The new assay detects the alpha0 deletions - -SEA, - (alpha)20.5, - -MED, - -FIL and - -THAI in the first multiplex PCR, the second multiplex detects the -alpha3.7 deletion and alphaalphaalphaanti3.7 variant, the third multiplex detects the -alpha4.2 deletion. This simple multiplex method should greatly facilitate the genetic screening and molecular diagnosis of these determinants in populations where alpha-thalassaemias are prevalent.

DNA sequence polymorphism and divergence was examined in the vicinity of the human beta-globin gene cluster origin of replication initiation region (IR), a 1.3-kb genomic region located immediately 5' of the adult-expressed beta-globin gene. DNA sequence variation in the replication origin IR and 5 kb of flanking DNA was surveyed in samples drawn from two populations, one African (from the Gambia, West Africa) and the other European (from Oxford, England). In these samples, levels of nucleotide and length polymorphism in the IR were found to be more than two times as high as adjacent non-IR-associated regions (estimates of per-nucleotide heterozygosity were 0.30% and 0.12%, respectively). Most polymorphic positions identified in the origin IR fall within or just adjacent to a 52-bp alternating purine-pyrimidine ((RY)n) sequence repeat. Within- and between-populations divergence is highest in this portion of the IR, and interspecific divergence in the same region, determined by comparison with an orthologous sequence from the chimpanzee, is also pronounced. Higher levels of diversity in this subregion are not, however, primarily attributable to slippage-mediated repeat unit changes, as nucleotide substitution contributes disproportionately to allelic heterogeneity. An estimate of helical stability in the sequenced region suggests that the hypervariable (RY)n constitutes the major DNA unwinding element (DUE) of the replication origin IR, the location at which the DNA duplex first unwinds and new strand synthesis begins. These findings suggest that the beta-globin IR experiences a higher underlying rate of neutral mutation than do adjacent genomic regions and that enzyme fidelity associated with the initiation of DNA replication at this origin may be compromised. The significance of these findings for our understanding of eukaryotic replication origin biology is discussed.

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.

The hemoglobinopathies are probably the world's most common genetic diseases: The World Health Organization has estimated that at least 5% of the population are carriers for one or other of the most serious forms, the alpha- and beta-thalassemias and the structural variant hemoglobins S, C, and E, which are found at polymorphic frequencies in many countries. All these hemoglobinopathies are believed to provide protection against malaria, and it is thought that, in malarial regions of the world, natural selection has been responsible for elevating and maintaining their gene frequencies, an idea first proposed 50 years ago by J.B.S. Haldane. Epidemiological studies undertaken in the 1950s on hemoglobin S in Africa provided support for the "malaria hypothesis," but until recently it has proved extremely difficult to verify it for the thalassemias. The application of molecular methods has, however, provided new opportunities to address this old question. Population and molecular genetic analysis of thalassemia variants, and microepidemiological studies of the relationship between alpha-thalassemia and malaria in the southwest Pacific, have provided unequivocal evidence for protection. Surprisingly, some of this protection appears to derive from enhanced susceptibility in very young thalassemic children to both Plasmodium falciparum and, especially, P. vivax, and this early exposure appears to provide the basis for better protection in later life.

Southeast Asian ovalocytosis (SAO) occurs at high frequency in malarious regions of the western Pacific and may afford a survival advantage against malaria. It is caused by a deletion of the erythrocyte membrane band 3 gene and the band 3 protein mediates the cytoadherence of parasitized erythrocytes in vitro. The SAO band 3 variant may prevent cerebral malaria but it exacerbates malaria anemia and may also increase acidosis, a major determinant of mortality in malaria. We undertook a case-control study of children admitted to hospital in a malarious region of Papua New Guinea. The SAO band 3, detected by the polymerase chain reaction, was present in 0 of 68 children with cerebral malaria compared with six (8.8%) of 68 matched community controls (odds ratio = 0, 95% confidence interval = 0-0.85). Median hemoglobin levels were 1.2 g/dl lower in malaria cases with SAO than in controls (P = 0.035) but acidosis was not affected. The remarkable protection that SAO band 3 affords against cerebral malaria may offer a valuable approach to a better understanding of the mechanisms of adherence of parasitized erythrocytes to vascular endothelium, and thus of the pathogenesis of cerebral malaria.

Mitochondrial DNA (mtDNA) analysis has proved useful in studies of recent human evolution and the genetic affinities of human groups of different geographical regions. As part of an extensive survey of mtDNA diversity in present-day Pacific populations, we obtained sequence information of the hypervariable mtDNA control region of 452 individuals from various localities in the western Pacific. The mtDNA types fell into three major groups which reflect the settlement history of the area. Interestingly, we detected an extremely rare point mutation at high frequency in the small island of Nguna in the Melanesian archipelago of Vanuatu. Phylogenetic analysis of the mtDNA data indicated that the mutation was present in individuals of separate mtDNA lineages. We propose that the multiple occurrence of a rare mutation event in one isolated locality is highly improbable, and that recombination between different mtDNA types is a more likely explanation for our observation. If correct, this conclusion has important implications for the use of mtDNA in phylogenetic and evolutionary studies.

The human β-globin gene complex spans a region of 70 kb and contains numerous sequence variants. These variant sites form a 5′ cluster (5′ β-haplotype) and a 3′ cluster (3′ β-haplotype) with strong linkage disequilibrium among the sites within each cluster, but not between the two clusters. The 9-kb region between the 5′ and 3′ clusters has been estimated to have rates of recombination that are 3 to 30 times normal, and the region has therefore been proposed as a ‘hotspot’ of recombination. We describe three families with evidence of meiotic recombination within this ‘hotspot’ of the β-globin gene cluster and in which the cross-over breakpoints have been defined at the sequence level. In one family, the recombination has occurred in the maternal chromosome within a region of 361 bp between positions −911 and −550 5′ to the β-globin gene. In the other two families, the recombination has occurred in the paternal chromosome within a region of approximately 1,100 bp between positions −542 and +568 relative to the β-globin gene cap site. Both regions occur within the 2-kb region of replication initiation (IR) in the β-globin gene domain with no overlap. The IR region contains a consensus sequence for a protein (Pur), which binds preferentially to single-stranded DNA, a role implicated in recombination events.

The human β-globin gene complex spans a region of 70 kb and contains numerous sequence variants. These variant sites form a 5′ cluster (5′ β-haplotype) and a 3′ cluster (3′ β-haplotype) with strong linkage disequilibrium among the sites within each cluster, but not between the two clusters. The 9-kb region between the 5′ and 3′ clusters has been estimated to have rates of recombination that are 3 to 30 times normal, and the region has therefore been proposed as a ‘hotspot’ of recombination. We describe three families with evidence of meiotic recombination within this ‘hotspot’ of the β-globin gene cluster and in which the cross-over breakpoints have been defined at the sequence level. In one family, the recombination has occurred in the maternal chromosome within a region of 361 bp between positions −911 and −550 5′ to the β-globin gene. In the other two families, the recombination has occurred in the paternal chromosome within a region of approximately 1,100 bp between positions −542 and +568 relative to the β-globin gene cap site. Both regions occur within the 2-kb region of replication initiation (IR) in the β-globin gene domain with no overlap. The IR region contains a consensus sequence for a protein (Pur), which binds preferentially to single-stranded DNA, a role implicated in recombination events.

Although alpha+ thalassaemia is the commonest haemoglobinopathy in the world, it is not known if it is associated with significant ineffective erythropoiesis, a fact of importance in interpreting its complex interaction with malaria. To study this problem, we have measured the concentrations of soluble transferrin receptor (sTIR) and ferritin in 181 children from Vanuatu with heterozygous (68) and homozygous (46) alpha+ thalassaemia, and normal controls (67). sTfR concentrations were significantly higher in both homozygotes (mean 3.1 mg/l, range 2.8-3.4) and heterozygotes (2.86 mg/l, 2.6-3.2) compared to the normal controls (2.48 mg/l, 2.3-2.7), suggesting that although globin chain imbalance is minimal, there is ineffective erythropoiesis in both these conditions. Age was also shown to significantly affect sTfR, with peak levels occurring in the 5-9 years age group. Ferritin concentrations showed a similar trend, being higher in the thalassaemic groups, although this did not reach statistical significance. No individuals had low ferritin concentrations, although two had significantly elevated sTfR levels. These observations suggest that the alpha+ thalassaemia phenotype includes an expansion of the erythron, and may suggest possible mechanisms for the increased susceptibility in babies with alpha thalassaemia to both P. falciparum and P. vivax malaria.

Hemoglobin E (HbE; 2β226glu-lys), globally the commonest hemoglobin variant, is synthesized at a slightly reduced rate and has a homozygous phenotype similar to heterozygous β thalassemia. Yet, when it is inherited together with a β thalassemia allele, the resulting condition, HbE/β 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 β 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/β thalassemia; this is confirmed by density-gradient centrifugation studies that show no decrease in HbE levels relative to HbA as HbE/β+ 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 β chains were present. However, in experiments performed on blood from HbE/β 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/β thalassemia is primarily the result of the interaction of two β thalassemia alleles; however, hemoglobin instability may be important during febrile episodes, contributing to worsening anemia. © 1998 by The American Society of Hematology.

The notion that selection during epidemics or longer periods of exposure to infectious diseases may have had a major effect in modifying the constitution of the human genome is not new. It was proposed, at least in outline, by A.E. Garrod in 1931. In his remarkable book, The Inborn Factors in Disease, he suggested that infectious diseases may have been a major selective force in human evolution and in shaping our biochemical individuality. In 1948, J.B.S. Haldane, unimpressed with the idea that the extremely high frequency of thalassemia in certain racial groups from the Mediterranean region might reflect an unusually high mutation rate in these populations, proposed that these diseases might have come under intense selection because of heterozygote advantage against malaria. It was, in effect, Haldane’s remarkable insight that opened up the field of the investigation of genetic susceptibility to infection. Although considerable progress was made in relating the frequency and distribution of different protein polymorphisms to past or present infection, until recently the field was bedeviled by difficulties relating to population homogeneity, founder effects, and gene drift. However, with an increasing ability to analyze human variability at the DNA level, progress has been much more rapid. Comparing the sequences of genes common to rodents and humans, Murphy (1993), found that host defense genes are much more diverse than those for other families of proteins, an observation suggesting that selection in many species has resulted from exposure to different infectious agents. The way in which DNA analysis is transforming our understanding of the reasons for the distribution and high frequency of the human hemoglobin variants, the problem first posed by Haldane, is reviewed by Flint et al. (1993). Clearly, an analysis of the human genome with respect to variable susceptibility to infection is already beginning to provide important new insights into the mechanisms of human diversity. In considering genetic susceptibility to infectious disease it is important not only to consider the genetic makeup of the host but also that of the infectious agent. Here, we focus mainly on recent studies of the genetic factors that may modify susceptibility to infection in humans and touch only briefly on some recent developments in microbial genetics that may play an important role in this interaction. Mouse models for studying genetic susceptibility to infection have been reviewed recently elsewhere (McLeod et al. 1995).

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 2/1000 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 falciparum malaria and that such regional differences must be considered when planning or interpreting the effects of malaria interventions.

We have analyzed allelic sequence variation in sixty-one 3-kb beta-globin sequences from the Melanesian population of Vanuatu to demonstrate the value of (1) turning to the autosomal nuclear genome for studies on the evolution of modern humans and (2) using new analytical methods based on a coalescent model. After excluding recombination events, beta-globin sequence variants were connected in a unique gene tree. A gene tree provides more information for inferences on the population genealogy than simple summary statistics such as the average pairwise sequence difference. Estimates of the time to the most recent common ancestor (MRCA) and of the ages of each mutation, conditional on the gene tree, were made using new maximum likelihood methods assuming a coalescent model. We found that allelic beta-globin variation coalesces to a single shared ancestral haplotype over a time scale of approximately 900,000 years. Three major haplotypes (A1, B1, C3) that are older than 200,000 years identify ancestral diversity contemporaneous with the single MRCA for mitochondrial variation.

The alpha+ thalassaemias are the most common single gene disorders of humans, yet little is known about their haematological characteristics in childhood. Blood samples have been collected randomly from more than 2000 individuals in village communities in Vanuatu in the South West Pacific and analysed for alpha thalassaemia and associated haematological changes. Here we describe the haematological effects of the alpha+ thalassaemias from early childhood through to maturity in this population. Mean cell volume (MCV) and mean cell haemoglobin (MCH) levels in individuals of normal, heterozygous and homozygous genotype differed significantly from one another throughout the entire age range (2P < 0.05). In contrast, haemoglobin levels in heterozygous and homozygous individuals were well maintained throughout development. Adults of normal genotype attain Hb levels which are indistinguishable from Caucasian reference values, a finding made all the more remarkable given the high frequency of clinical malaria in this population. It is clear from these findings that haematological data are valuable in screening for carriers of alpha+ thalassaemia in this population. MCH is clearly the most sensitive discriminator. None of the homozygous adults tested had an MCH of > 27 pg, whereas < 10% of normals had a value of < 27 pg. These data provide reference values for areas in which the alpha+ thalassaemias are common and often confused with iron-deficiency anaemia.

A Bangladeshi family is described in which the genes for both hemoglobin E (Hb E) and pyrimidine 5′ nucleotidase deficiency are segregating. An individual homozygous for both these conditions has a severe hemolytic anemia, whereas family members who are homozygous for Hb E are asymptomatic and those homozygous for pyrimidine 5′ nucleotidase deficiency have the mild hemolytic anemia that is characteristic of this disorder. Globin-chain synthesis experiments have shown that the mechanism underlying the interaction between these two genotypes is a marked decrease in the stability of Hb E in pyrimidine 5′ nucleotidase-deficient red blood cells (RBCs). It has also been found that in the enzyme-deficient RBCs in which Hb E is highly unstable, free alpha-chains, though not beta E-chains, acoumulate on the membrane. In view of the increasing evidence that the hemolysis associated with pyrimidine 5′ nucleotidase deficiency results not only from an increase in the level of erythrocyte pyrimidines, but also from inhibition of the hexose monophosphate shunt activity in young erythrocytes, it is likely that the marked instability of Hb E in the enzyme-deficient cells results from oxidant damage acting on a mildly unstable Hb variant. These observations may have important implications for the better understanding of the pathophysiology of Hb E/beta-thalassemia, globally the commonest important form of thalassemia.

The introduction of molecular genetic techniques to survey DNA variation directly has greatly advanced the study of inter- and intra-population variability. One of the first DNA polymorphisms to be surveyed for population variation was the α-globin copy number variants in Melanesia, and this study indicated a new role for malarial selection. Subsequently, DNA sequence polymorphism was investigated by the definition of haplotypes, which result from linkage disequilibrium among site polymorphisms. Distributions of α-globin haplotypes clearly demonstrate the effects of migration on population affinities in Melanesia and Polynesia. Analysis of neutral diversity in the nuclear genome has been enriched by the discovery of variable numbers of tandem repeats (VNTRs), particularly the hypervariable mini-satellites. Highly allelic VNTRs are sensitive to the effects of genetic drift caused by bottlenecks or population subdivision, and in Oceania they reflect the demography of colonization. Because VNTRs are subject to high and variable rates of mutation, they are informative within a range of recent evolutionary time scales and, together with other DNA polymorphisms, contribute to a more complete interpretation of a population's history. © 1996 Wiley-Liss, Inc.

Allelic sequence polymorphism at the beta-globin locus was investigated in a group of 36 Melanesians. A 3-kilobase fragment containing the gene and its flanking regions was sequenced in 60 normal (beta A) and 12 thalassemic (intron 1, position 5, G-->C) chromosomes. Haplotype relationships between linked polymorphisms were derived by allele-specific PCR amplification and sequencing. Seventeen nucleotide polymorphisms and 2 length variants were identified, and these sites segregated as 17 sequence haplotypes in the normal chromosomes. This haplotype diversity is higher than that expected on the basis of the nucleotide polymorphism observed and is probably due to recombination and gene conversion. Nucleotide diversity at synonymous sites in the sample is 0.14%, suggesting an average age of sequence divergence of approximately 450,000 years, consistent with that expected for a neutrally evolving human nuclear locus.

The haemoglobinopathies are the commonest single-gene disorders known, almost certainly because of the protection they provide against malaria, as attested by a number of observations. The geographical distributions of malaria and haemoglobinopathies largely overlap, and microepidemiological surveys confirm the close relationship between them. For two of the commonest disorders, haemoglobin S and alpha(+)-thalassaemia, there is also good clinical evidence for protection against malaria morbidity. However, not all the evidence appears to support this view. In some parts of the world malaria and haemoglobinopathies are not, and never have been, coexistent. It is also difficult to explain why the majority of haemoglobinopathies appear to be recent mutations and are regionally specific. Here we argue that these apparent inconsistencies in the malaria hypothesis are the result of processes such as genetic drift and migration and of demographic changes that have occurred during the past 10,000 years. When these factors are taken into account, selection by malaria remains the force responsible for the prevalence of the haemoglobinopathies.

Venous thrombosis is a common problem, predominantly afflicting people of European origin. This European predisposition has been explained to some extent by the recent characterization of factor V Leiden, and the G20210A prothrombin variant. Although it is clear that factor V Leiden is largely confined to Europeans, the world distribution of the prothrombin variant is not known. We have analysed samples from 22 different non-European countries and shown that this prothrombin variant is very rare outside Europe: one case occurring in India. The reason for the confined distribution of these two mutations is unclear.

The genes which direct the structure of human fetal and adult haemoglobin consist of a linked pair of alpha-chain loci on chromosome 16 and the G gamma-A gamma-delta-beta loci complex on chromosome 11. The delta-and beta-chain genes contain inserts similar to those of the mouse and rabbit globin genes. The structure of the various messenger RNAs transcribed from these loci is now worked out although the function of the non-coding regions is not known. The abnormal haemoglobin disorders and thalassaemias result from a variety of lesions at these loci which include single base substitutions, deletions of one or more bases or entire loci, insertions, frame-shifts, fusion genes caused by abnormal crossing over, chain termination mutations and ill-defined defects which lead to a reduced rate of transcription or abnormal structure of messenger RNA. Some progress has been made towards an understanding of the cellular mechanisms whereby the haemoglobin polymorphisms have been maintained. Very little is known about the regulatory mechanisms involved in the switch from fetal to adult haemoglobin production, although it is likely that certain specific areas of the gamma-delta-beta gene complex are involved in its control.

The effect of maternal alpha+ -thalassaemia on pregnancy was assessed in the north coastal region of Papua New Guinea (PNG), where malaria is hyperendemic and alpha+ -thalassaemia is extremely common. In a prospective study of 987 singleton hospital deliveries, we correlated maternal alpha-globin genotype with markers of reproductive fitness (age in primigravidae, gravidity, pregnancy interval and the number of miscarriages and stillbirths), Plasmodium falciparum(P. falciparum) infection of the mother and placenta, maternal haemoglobin, preterm delivery and birthweight. The frequency of the -alpha genotype in mothers was 0.61. Markers of reproductive fitness were similar in women with and without alpha+ -thalassaemia. Median haemoglobin concentration during pregnancy and after delivery was about 1.0 g/dl lower in homozygous alpha+ -thalassaemia than in women with a normal alpha- globin genotype (P < or = 0.001). The frequency of placental P. falciparum infection and systemic malaria infection after delivery showed no consistent relationship to alpha-globin genotype. The frequency of preterm delivery and low birthweight did not vary significantly according to maternal alpha-globin genotype. Maternal alpha+ -thalassaemia does not affect reproductive fitness or susceptibility to malaria during pregnancy. Although median haemoglobin concentration was significantly lower in mothers homozygous for alpha+ -thalassaemia than those with a normal alpha-globin genotype, this did not result in an adverse outcome of pregnancy.

Blocks of linkage disequilibrium (LD) in the human genome represent segments of ancestral chromosomes. To investigate the relationship between LD and genealogy, we analysed diversity associated with restriction fragment length polymorphism (RFLP) haplotypes of the 5' beta-globin gene complex. Genealogical analyses were based on sequence alleles that spanned a 12.2-kb interval, covering 3.1 kb around the psibeta gene and 6.2 kb of the delta-globin gene and its 5' flanking sequence known as the R/T region. Diversity was sampled from a Kenyan Luo population where recent malarial selection has contributed to substantial LD. A single common sequence allele spanning the 12.2-kb interval exclusively identified the ancestral chromosome bearing the "Bantu" beta(s) (sickle-cell) RFLP haplotype. Other common 5' RFLP haplotypes comprised interspersed segments from multiple ancestral chromosomes. Nucleotide diversity was similar between psibeta and R/T-delta-globin but was non-uniformly distributed within the R/T-delta-globin region. High diversity associated with the 5' R/T identified two ancestral lineages that probably date back more than 2 million years. Within this genealogy, variation has been introduced into the 3' R/T by gene conversion from other ancestral chromosomes. Diversity in delta-globin was found to lead through parts of the main genealogy but to coalesce in a more recent ancestor. The well-known recombination hotspot is clearly restricted to the region 3' of delta-globin. Our analyses show that, whereas one common haplotype in a block of high LD represents a long segment from a single ancestral chromosome, others are mosaics of short segments from multiple ancestors related in genealogies of unsuspected complexity.

We have implemented a technique combining allele-specific PCR (AS-PCR) and denaturing high-performance liquid chromatography (DHPLC) to identify new polymorphic variants within an intergenic region in the beta-globin cluster. This technique is applicable to the detection of new variants in genomic regions where variation is apportioned into distinct classes of haplotype. Duplexes for DHPLC analysis were created by denaturation and re-annealing of a mixture of two AS-PCR products of known and unknown sequence from the same haplotypic class, permitting detection of new haplotypes in each class. A 454bp fragment 3.5kb 5' to the human delta-globin gene, which may have a gene regulatory function, was analysed in 840 chromosomes from a global sampling of human populations using this method. Two divergent haplotypes were found to predominate in all populations studied, possibly as a result of balancing selection.

Recombination was measured across nine intervals in the human beta-globin gene cluster by single-sperm analysis. A recombination fraction of approximately 0.9% was calculated across an approximately 11 kb region using a new method to estimate recombination fractions from single-sperm typing data. No recombination was detected in an adjacent approximately 90 kb region that extends upstream of the beta-globin cluster. These data are consistent with previous estimates based on population genetic analysis, and suggest a recombination rate of nearly two orders of magnitude greater than the genome average of approximately 1 cM/Mb. Because recombination hot spots will destroy linkage disequilibrium across small physical regions, knowledge about the location and strength of such hot spots could be extremely valuable for genetic association studies.

A large and ethnically well-defined Mandenka sample from eastern Senegal was analyzed for the polymorphism of the beta-globin gene cluster on chromosome 11. Five RFLP sites of the 5' region were investigated in 193 individuals revealing the presence of 10 different haplotypes. The frequency of the sickle-cell anemia causing mutation (beta(S)) in the Mandenka estimated from this sample is 11.7%. This mutation was found strictly associated with the single Senegal haplotype. Approximately 600 bp of the upstream region of the beta-globin gene were sequenced for a subset of 94 chromosomes, showing the presence of four transversions, five transitions, and a composite microsatellite polymorphism. The sequence of 22 beta(S) chromosomes was also identical to the previously defined Senegal haplotype, suggesting that this mutation is very recent. Monte Carlo simulations (allowing for a specific balancing selection model, a logistic growth of the population, and variable initial frequencies of the Senegal haplotype) were used to estimate the age of the beta(S) mutation. Resulting maximum-likelihood estimates are 45-70 generations (1,350-2,100 years) for very different demographic scenarios. Smallest confidence intervals (25-690 generations) are obtained under the hypothesis that the Mandenka population is large (N(e) >5,000) and stationary or that it has undergone a rapid demographic expansion to a current size of >5,000 reproducing individuals, which is quite likely in view of the great diversity found on beta(A) chromosomes.

We have used a new method for binning minisatellite alleles (semi-automated allele aggregation) and report the extent of population diversity detectable by eleven minisatellite loci in 2,689 individuals from 19 human populations distributed widely throughout the world. Whereas population relationships are consistent with those found in other studies, our estimate of genetic differentiation (F(st)) between populations is less than 8%, which is lower than comparative estimates of between 10%-15% obtained by using other sources of polymorphism data. We infer that mutational processes are involved in reducing F(st) estimates from minisatellite data because, first, the lowest F(st) estimates are found at loci showing autocorrelated frequencies among alleles of similar size and, second, F(st) declines with heterozygosity but by more than predicted assuming simple models of mutation. These conclusions are consistent with the view that minisatellites are subject to selective or mutational constraints in addition to those expected under simple step-wise mutation models.

Sequences of the alpha1, alpha2 and theta globin genes from six equid species have been determined to investigate relationships within the genus Equus. Analyses using standard phylogenetic methods, or an approach designed to account for the effects of gene conversion between the alpha genes, gave broadly similar results and show that the horses diverged from the zebra/ass ancestor approximately 2.4 million years ago and that the zebra and ass species arose in a rapid radiation approximately 0.9 million years ago. These results from the alpha genes are corroborated by theta gene data and are in contrast to mitochondrial DNA studies of the phylogeny of this genus, which suggest a more gradual set of speciation events.

Aim—To identify causes of preterm delivery and intrauterine growth retardation (IUGR) in a malaria endemic region of Papua New Guinea. Methods—Independent predictors of preterm delivery and birthweight in term infants were identified using multiple regression analysis in a prospective study of 987 singleton live births delivered in Madang Hospital. Results—Overall, Plasmodium falciparum infection of the placenta was associated with a reduction in birthweight of 130 g. Malaria was significantly more common in primigravidae than multigravidae and probably contributed to both preterm delivery and IUGR. Maternal haemoglobin concentrations were significantly lower in malaria infected than noninfected women and reduced haemoglobin was the main determinant of preterm delivery. Poorer maternal nutritional status and smoking were associated with both prematurity and IUGR. Greater antenatal clinic attendance predicted increased birthweight in term infants. Conclusions—Protection against malaria during pregnancy, especially in primigravidae, improved nutrition in women and discouragement of smoking would probably reduce both preterm delivery and IUGR. Greater use of existing antenatal clinics might increase birthweight in term infants. (Arch Dis Child Fetal Neonatal Ed 1998;79:F135‐F140)

Southeast-Asian ovalocytosis (SAO) was diagnosed in children from Madang, Papua New Guinea, by detection of the SAO band 3 gene variant using the polymerase chain reaction. SAO band 3 was present in 16/241 (6.6%) children living in the community and 32/389 (8.2%) children with acute Plasmodium falciparum malaria (P=0.42). SAO band 3 was detected in 8.2% (23/281) of alpha+-thalassaemia homozygotes, 9.4% (20/214) of heterozygotes and 2.4% (2/85) of children with a normal alpha-globin genotype (P=0.12). The most consistent feature of SAO band 3 on microscopy of thin blood films was red cells with two or more linear or irregularly-shaped pale regions. In children living in the community, these were present in 15 with SAO band 3 (sensitivity 93.8%) and only two normals (specificity 99.1%). The presence ofor = 20% ovalocytosis was a poorer indicator of SAO band 3 (sensitivity 68.8% and specificity 100%). Haematological data were similar in SAO band 3 and normal children. However, in children with acute malaria, haemoglobin levels and red cell counts were significantly lower in SAO band 3 than normal children. The degree of ovalocytosis was lower in children with SAO band 3 during acute malaria, suggesting that a selective loss of ovalocytes may contribute to malaria anaemia in Southeast-Asian ovalocytosis.

In the South West Pacific region, the striking geographical correlation between the frequency of alpha+-thalassemia and the endemicity of Plasmodium falciparum suggests that this hemoglobinopathy provides a selective advantage against malaria. In Vanuatu, paradoxically, alpha+-thalassemia increases the incidence of contracting mild malaria in the first 2 years of life, but severe disease was too uncommon to assess adequately. Therefore, we undertook a prospective case-control study of children with severe malaria on the north coast of Papua New Guinea, where malaria transmission is intense and alpha+-thalassemia affects more than 90% of the population. Compared with normal children, the risk of having severe malaria was 0.40 (95% confidence interval 0.22-0.74) in alpha+-thalassemia homozygotes and 0.66 (0.37-1.20) in heterozygotes. Unexpectedly, the risk of hospital admission with infections other than malaria also was reduced to a similar degree in homozygous (0. 36; 95% confidence interval 0.22-0.60) and heterozygous (0.63; 0. 38-1.07) children. This clinical study demonstrates that a malaria resistance gene protects against disease caused by infections other than malaria. The mechanism of the remarkable protective effect of alpha+-thalassemia against severe childhood disease remains unclear but must encompass the clear interaction between this hemoglobinopathy and both malarial and nonmalarial infections.A prospective case-control study conducted on the north coast of Papua New Guinea in 1993-96 investigated the hypothesis that alpha(+)-thalassemia provides a selective advantage against endemic Plasmodium falciparum. In this geographic area, alpha(+)-thalassemia affects more than 90% of the population. 249 children admitted to Madang Hospital with one or more symptoms of severe malaria (severe anemia, coma, hypoglycemia, acidosis, and hyperlactatemia) were paired with 249 randomly selected healthy age- and sex-matched controls. Also enrolled were 233 children with infections other than malaria, primarily respiratory infections, gastroenteritis, and meningitis. Compared with healthy community controls, the risk of severe malaria was 0.40 (95% confidence interval (CI), 0.22-0.74) in alpha(+)-thalassemia homozygotes and 0.66 (95% CI, 0.37-1.20) in heterozygotes. The lowest odds ratios for alpha(+)-thalassemia homozygotes were recorded in the acidosis and hyperlactatemia subgroups--the two complications most predictive of malaria mortality. Unexpectedly, the risk of hospital admission with infections other than malaria was reduced to a similar degree in homozygous (0.36; 95% CI, 0.22-0.60) and heterozygous (0.63; 95% CI, 0.38-1.07) children. Although the relevant mechanism is unknown, these findings confirm a clear interaction between thalassemia hemoglobinopathy and both malarial and other infections. Given its high frequency and protective effect against malaria and some other infections, alpha(+)-thalassemia is likely to have a major impact on child survival in coastal Papua New Guinea.

We studied the aetiology of malnutrition in a cohort of 1511 children10 years old in Espiritu Santo, Vanuatu. Malnutrition was categorized using standard anthropometric 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 age5 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), por = 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. falciparum 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.The etiology of malnutrition was investigated in a cohort of all 1511 children under 10 years of age living in 13 villages in the island of Espiritu Santo, Vanuatu, where malaria is endemic. 18% of children under 5 years were underweight, 5% were wasted, and 20% were stunted. The mean weight-for-age Z score for the 1114 children resident in hyperendemic villages was significantly lower (-0.99) than that of the 397 children living in the mesoendemic area (-0.77). According to multiple logistic regression analysis, the only factors significantly associated with wasting in the hyperendemic area were age under 5 years (odds ratio (OR), 1.8; 95% confidence interval (CI), 1.2-2.9) and 1 or more episodes of clinical Plasmodium vivax malaria in the 6 months preceding nutritional assessment (OR, 2.4; 95% CI, 1.3-4.4). Only male sex and low birth weight were significantly associated with stunting. The incidence of P. vivax infection in the 6 months preceding the survey was significantly higher in underweight compared to non-underweight children (relative risk (RR), 2.6; 95% CI, 1.5-4.4). The incidence of P. falciparum malaria was not significantly different between groups, suggesting that this is not a major cause of malnutrition on the island. Wasting neither predisposed to nor protected against malaria of either species. Although P. vivax malaria is generally regarded as benign, it may produce substantial global mortality through malnutrition.

The clinical features of severe falciparum malaria and risk factors for mortality were studied in 489 children admitted with malaria to Madang Hospital, Papua New Guinea. The most common severe manifestations of malaria were severe anaemia (22%) and coma (16%). Children with severe anaemia were younger than those with coma (median age 2.2 vs. 3.7 years) and had been ill for longer before admission (median 7 vs. 4 days, respectively). Although the clinical features of coma in Madang children with malaria resembled closely those reported in African children, mortality was lower (8% vs. 17-25%, respectively). Overall, 17 (3.5%) children died, most within 12 h of admission. A high level of plasma lactate (> or = 5 mmol/l) was common (20%) and was the major predictor of death in multiple regression analysis. Raised plasma creatinine and decreased plasma bicarbonate were also independent predictors of mortality. Coma was not predictive of death, although a high proportion of children with profound coma died. Investigation of the causes of acidosis in children with malaria is a high research priority. In view of the short time interval between admission and death in many children, emphasis must be placed on the prevention or early recognition and treatment of acidosis in the district health clinic as well as the central hospital.