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Recently, a common genetic variant E756del in the human gene PIEZO1 was associated with protection from severe malaria. Here, we performed a genetic association study of this gain-of-function variant in a large case-control study including 4149 children from the Ashanti Region in Ghana, West Africa. The statistical analysis did not indicate an association with protection from severe malaria and, thus, providing evidence against a strong protective effect of the PIEZO1 E756del variant on severe malaria susceptibility.

The concept of interferon-γ (IFN-γ) having a central role in cell-mediated immune defence to Mycobacterium tuberculosis has long been proposed. Observations made through early candidate gene studies of constituents of the IFN-γ pathway have identified moderately associated variants associated with resistance or susceptibility to tuberculosis (TB). By analysing 20 major genes whose proteins contribute to IFN-γ signalling we have assessed a large fraction of the variability in genes that might contribute to susceptibility to TB. Genetic variants were identified by sequencing the promoter regions and all exons of IFNG, IFNGR1, IFNGR2, IRF1, IL12A, IL12B, IL12RB1, IL12RB2, IL23A, IL23R, IL27, EBI3, IL27RA, IL6ST, SOCS1, STAT1, STAT4, JAK2, TYK2 and TBX21 in 69 DNA samples from Ghana. In addition, we screened all exons of IFNGR1 in a Ghanaian study group comprising 1999 TB cases and 2589 controls by high-resolution melting point analysis. The fine-mapping approach allows for a detailed screening of all variants, common and rare. Statistical comparisons of cases and controls, however, did not yield significant results after correction for multiple testing with any of the 246 variants selected for genotyping in this investigation. Gene-wise haplotype tests and analysis of rare variants did not reveal any significant association with susceptibility to TB in our investigation as well. Although this analysis was applied on a plausible set of IFN-γ pathway genes in the largest African TB cohort available so far, the lack of significant results challenges the view that genetic marker of the IFN-γ pathway have an important impact on susceptibility to TB.

Human genetic factors predispose to tuberculosis (TB). We studied 7.6 million genetic variants in 5,530 people with pulmonary TB and in 5,607 healthy controls. In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10(-11) for rs4733781; P = 1.0 × 10(-10) for rs10956514). Dendritic cells (DCs) showed high ASAP1 expression that was reduced after Mycobacterium tuberculosis infection, and rs10956514 was associated with the level of reduction of ASAP1 expression. The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes. The ASAP1-depleted DCs showed impaired matrix degradation and migration. Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis-infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB.

The genetic basis of congenital heart disease remains unknown in most of the cases. Recently, a novel mouse model shed new light on the role of CCN1/CYR61, a matricellular regulatory factor, in cardiac morphogenesis. In a candidate gene approach, we analyzed a cohort of 143 patients with atrial septal defects (ASD) by sequencing the coding exons of CCN1. In addition to three frequent polymorphisms, we identified an extremely rare novel heterozygous missense mutation (c.139C > T; p.R47W) in one patient with severe ASD. The mutation leads to an exchange of residues with quite different properties in a highly conserved position of the N-terminal insulin-like growth factor binding protein module. Further bioinformatic analysis, exclusion of known ASD disease genes as well as the exclusion of the mutation in a very high number of ethnically matched controls (more than 1,000 individuals) and in public genetic databases, indicates that the p.R47W variant is a probable disease-associated mutation. The report about ASD in mice in heterozygous Ccn 1 +/- animals strongly supports this notion. Our study is the first to suggest a relationship between a probable CCN1 mutation and ASD. Our purpose here was to draw attention to CCN1, a gene that we believe may be important for genetic analysis in patients with congenital heart disease.

The molecular mechanisms determining the transmissibility and prevalence of drug-resistant tuberculosis in a population were investigated through whole-genome sequencing of 1,000 prospectively obtained patient isolates from Russia. Two-thirds belonged to the Beijing lineage, which was dominated by two homogeneous clades. Multidrug-resistant (MDR) genotypes were found in 48% of isolates overall and in 87% of the major clades. The most common rpoB mutation was associated with fitness-compensatory mutations in rpoA or rpoC, and a new intragenic compensatory substitution was identified. The proportion of MDR cases with extensively drug-resistant (XDR) tuberculosis was 16% overall, with 65% of MDR isolates harboring eis mutations, selected by kanamycin therapy, which may drive the expansion of strains with enhanced virulence. The combination of drug resistance and compensatory mutations displayed by the major clades confers clinical resistance without compromising fitness and transmissibility, showing that, in addition to weaknesses in the tuberculosis control program, biological factors drive the persistence and spread of MDR and XDR tuberculosis in Russia and beyond.

Background: β-Thalassemia is a disorder caused by mutations at the hemoglobin β-gene (HBB) locus. Its most important manifestation, the major form, is characterized by severe hypochromic and hemolytic anemia and is inherited in an autosomal recessive mode. In Gaza Strip, Palestine 0.02% of the population has been identified as β-thalassemia major. Design and methods: An assessment of mutations was performed in 49 transfusion dependent patients with β-thalassemia major nd in 176 β-thalassemia carriers diagnosed with a mean erythrocyte cell volume (MCV) b80 fl and a proportion of HbA2>3.5%. In addition 39 individuals suspicious for β-thalassemia carrier status due to a reduced MCV (b80 fl) but a normal HBA2 were screened. Results: By screening with three hybridization assays a proportion of 80% of the thalassemic chromosomes from patients and carriers was identified to carry five different mutations of the hemoglobin (Hb) β-gene. Subsequent DNA sequencing confirmed these and revealed further 9% of the chromosomes to be affected by other mutations. In addition six chromosomes from suspicious carriers were detected to carry β-thalassemia mutations. Of the 15 different HBB mutations identified the variant IVS-I-110 G>A was the most frequent mutation identified in 34% of the thalassemic chromosomes, followed by IVS-I-1 G>A, IVS-I-6 T>C, Codon 39 C>T, and Codon 37 G>A. Three novel HBB variants were discovered by direct sequencing of the gene: 5′ UTR-50 (−/G), 5′ UTR-43 C>T, and IVS-II-26 T>G. Conclusions: The spectrum of HBB mutations described is of the Mediterranean type whereby the allele frequencies of the most common mutations differ from those, which were previously described for the population of the Gaza Strip and other Palestinian populations. The data presented may promote the introduction of molecular testing to the Palestinian premarital screening program for β-thalassemia in Gaza Strip, which will improve the screening protocol and genetic counseling in the future.

We combined two tuberculosis genome-wide association studies from Ghana and The Gambia with subsequent replication in a combined 11,425 individuals. rs4331426, located in a gene-poor region on chromosome 18q11.2, was associated with disease (combined P = 6.8 x 10(-9), odds ratio = 1.19, 95% CI = 1.13-1.27). Our study demonstrates that genome-wide association studies can identify new susceptibility loci for infectious diseases, even in African populations, in which levels of linkage disequilibrium are particularly low.

A genome-wide association study in Ghana, West Africa, to identify genetic variants associated with malaria pathogenesis reveals two previously unknown loci on chromosomes 1 and 16. This genome-wide association study in 2,645 cases and 3,050 controls from Ghana in West Africa identifies genetic variants that might affect any one of the many steps in malaria pathogenesis. Two novel loci are described. The first is on chromosome 1, in the vicinity of the ATP2B4 gene, which encodes the main calcium pump of erythrocytes, the host cells of the pathogenic stage of the malaria parasite. The second is on chromosome 16, close to MARVELD3, which encodes a tight-junction protein that is expressed on endothelial cells. These genetic variants may confer resistance by affecting key steps in disease development that could generate possible antimalarial targets. Malaria causes approximately one million fatalities per year, mostly among African children1. Although highlighted by the strong protective effect of the sickle-cell trait2,3, the full impact of human genetics on resistance to the disease remains largely unexplored4. Genome-wide association (GWA) studies are designed to unravel relevant genetic variants comprehensively; however, in malaria, as in other infectious diseases, these studies have been only partly successful5. Here we identify two previously unknown loci associated with severe falciparum malaria in patients and controls from Ghana, West Africa. We applied the GWA approach to the diverse clinical syndromes of severe falciparum malaria, thereby targeting human genetic variants influencing any step in the complex pathogenesis of the disease. One of the loci was identified on chromosome 1q32 within the ATP2B4 gene, which encodes the main calcium pump of erythrocytes6, the host cells of the pathogenic stage of malaria parasites. The second was indicated by an intergenic single nucleotide polymorphism on chromosome 16q22.2, possibly linked to a neighbouring gene encoding the tight-junction protein MARVELD3. The protein is expressed on endothelial cells7 and might therefore have a role in microvascular damage caused by endothelial adherence of parasitized erythrocytes. We also confirmed previous reports on protective effects of the sickle-cell trait and blood group O5,8,9. Our findings underline the potential of the GWA approach to provide candidates for the development of control measures against infectious diseases in humans.

Isoniazid (INH) and rifampicin (RMP) resistance in Mycobacterium tuberculosis complex (MTC) isolates are mainly based on mutations in a limited number of genes. However, mutation frequencies vary in different mycobacterial populations. In this work, we analyzed the distribution of resistance-associated mutations in M. tuberculosis and M. africanum strains from Ghana, West Africa. The distribution of mutations in katG, fabG1-inhA, ahpC, and rpoB was determined by DNA sequencing in 217 INH-resistant (INH(r)) and 45 multidrug-resistant (MDR) MTC strains isolated in Ghana from 2001 to 2004. A total of 247 out of 262 strains investigated (94.3%) carried a mutation in katG (72.5%), fabG1-inhA (25.1%), or ahpC (6.5%), respectively. M. tuberculosis strains mainly had katG 315 mutations (80.1%), whereas this proportion was significantly lower in M. africanum West-African 1 (WA1) strains (43.1%; p

Paternity and maternity investigations in immigration procedures are frequently done in Germany. Since mostly only one parent and one or more children are investigated, the occurrence of possible mutational events has to be interpreted with great care and the analysis of as many STRs as possible is recommended. The new Powerplex® ESX17 and Powerplex® ESI17 kits from Promega comprising both eleven established STRs and additionally the loci D1S1656, D2S441, D10S1248, D12S391, and D22S1045 (in different order) are potential tools in such paternity or maternity analyses, but only few allele frequency data for the five new loci exist. Here, we provide allele frequencies for the five additional STRs from three different populations from Africa. In addition, we present two maternity cases and one paternity case in which a clear inclusion or exclusion of the alleged parent could only be achieved by the additional application of the new Powerplex® ESX17 kit.

Although Mycobacterium africanum is being isolated in a significant proportion of cases of pulmonary tuberculosis in West Africa, its pathogenic potential remains a matter of discussion. Recent reports leave the question of whether M. africanum causes more severe pathology than M. tuberculosis or resembles opportunistic pathogens and might gain importance in the course of the HIV pandemic. Patients with pulmonary tuberculosis associated with M. africanum (n=556) and M. tuberculosis (n=1350) were studied in Ghana, West Africa, and compared regarding self-reported signs and symptoms, chest radiography, HIV status, mycobacterial drug resistance and mycobacterial clustering as determined by spoligotyping and IS6110 fingerprints. The rate of M. africanum infections was similar in HIV-positive (27%) and HIV-negative (30%) patients. M. africanum clustered less than M. tuberculosis (21% vs 79%; OR, 0.38; 95% CI, 0.3-0.5; p

The contribution of interleukin- (IL-) 4 receptor-alpha- (Rα-) dependent events in the pathogenesis of tuberculosis (TB) is controversial. We have recently shown IL-13 overexpression in mice to cause recrudescentMtbreplication and centrally necrotizing granulomas strongly resembling pathology of human TB. A deletion of IL-4Rαcompletely abrogates TB tissue pathology in these mice. To validate our results in human TB patients, we here determined the association of distinct variants of theIL4,IL13,IL4RA,IL13RA1, andIL13RA2genes with cavity formation in a large Ghanaian cohort of HIV-negative individuals with newly diagnosed pulmonary TB. In fact, the structural variant of theIL4RAI50V, previously shown to result in enhanced signal transduction, was significantly associated with greater cavity size, and a variant ofIL13RA2was associated with disease in females. To evaluate whether the human-like TB pathology in IL-13-overexpressing mice is specifically mediated through the IL-4Rαsubunit, we analyzed IL-13 transgenic mice with a genetic ablation of the IL-4Rα. In these mice, the IL-13-mediated increased susceptibility, human-like pathology of collagen deposition around centrally necrotizing granulomas, and alternative macrophage activation were abolished. Together, our genetic association study in human TB patients further supports the assumption that IL-13/IL-4Rα-dependent mechanisms are involved in mediating tissue pathology of human TB.

Toll like receptors (TLR) are key elements of the innate immune response and involved in the recognition of pathogens. To test common and rare TLR variants involved in susceptibility or resistance to infection with Mycobacterium tuberculosis we screened the exons of the genes encoding TLR 1, 2, 4, and the adaptor molecule TIRAP in more than 4500 tuberculosis (TB) cases and controls from Ghana. The analysis yielded 109 variants with possible functional impact, including 101 non-synonymous variants, three stop-variants, and five indels. Association analyses yielded a significant result for the TLR1 variant rs3923647, conferring strong protection against TB (Odds ratio [OR] 0.21, CI confidence interval [CI] 0.05–0.6, Pnominal 1 x 10−3) when applying a recessive model of inheritance. Replication analyses with an additional 3370 Ghanaian cases and control samples, and with data from a recent TB study of 533 African-Americans confirmed the protective effect and resulted in a combined OR of 0.19, with a nominal P value of 2.2 x 10−5, and a corrected P value of 4.1 x 10−4. The SNP is located near the binding pocket of TLR1 and causes an amino acid exchange from histidine to leucine at position 305. The observed effect may, therefore, be attributable to structural changes in the recognition site of the TLR1 molecule, allowing to bind those mycobacterial ligands which preferentially may induce a protective immune response. This is supported by the analysis of BCG-stimulated peripheral blood mononuclear cells, showing increased induction of the proinflammatory cytokine IFN-γ in carriers of the mutant TLR1 rs3923647 TT genotype, compared to the IFN-γ levels of individuals with the AT and AA genotypes.

We report a case of amyloidosis in association with hyperimmunoglobulinemia D syndrome (HIDS). The patient showed typical clinical features of HIDS. He had crescentic glomerulonephritis progressing to end-stage renal disease at age 13 years. Eight years later, he developed an AA-type amyloidosis with extensive involvement of the intestine, respiratory tract, and thyroid gland. These unusual complications of HIDS seriously challenge the assumption that the disease is associated with a good prognosis.

SummaryDeterminations of in vitro proliferative and secretory activities of peripheral blood cells are used widely for research in clinical immunology but, to our knowledge, have not been evaluated as to their power to reflect in vivo activities quantitatively. Here, we addressed this question by quantitatively correlating the in vitro secretion of interleukin (IL)-5 by peripheral blood cells to the in vivo activity of IL-5 as reflected by peripheral-blood eosinophil counts. Studying 458 humans exposed to transmission of the nematode Onchocerca volvulus, IL-5 was measured in the supernatants of 0·02-ml whole-blood cells cultured in the presence of O. volvulus extract or mitogen. O. volvulus-reactive IL-5 secretion was correlated significantly to blood eosinophilia in a quantitative manner explaining 15·1% (95% CI 8·3–19·9%) of the variability of eosinophil counts. Interestingly, correlations were obtained only if parasite counts were included in the calculation using multiple regression analysis. The results show that in vitro assays of minute amounts of blood lymphocytes may quantitatively reflect activities of the entire lymphocyte population in vivo.

Increasing resistance, recrudescences, and treatment failure have led to the replacement of chloroquine with the combination of pyrimethamine (PYR) and sulfadoxine (SDX) as the first-line antimalarial drugs for treatment of uncomplicated Plasmodium falciparum malaria in several areas where this disease is endemic. The development of resistance to PYR-SDX is favored by incomplete treatment courses or by subtherapeutic levels in plasma. PYR-SDX resistance has been associated with several single-nucleotide polymorphisms (SNPs) in the P. falciparum dihydrofolate reductase ( pfdhfr ) and the P. falciparum dihydropteroate synthetase ( pfdhps ) genes. We have established assays based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that conveniently allow the identification of SNPs associated with PYR resistance. Variants occurring at codon positions 16, 51, 59, and 108 of the pfdhfr gene were analyzed by MALDI-TOF MS in synthetic oligonucleotides to determine the detection threshold. In addition, 63 blood samples from subjects with P. falciparum parasitemia of various degrees were analyzed. The results were compared to those obtained by DNA sequencing of the respective gene fragment. The results of MALDI-TOF MS and DNA sequencing were consistent in 40 samples. In 23 samples two or three pfdhfr variants were detected by MALDI-TOF assays, whereas DNA-sequencing revealed one variant only. Simultaneous detection of two different mutations by biplex assays was, in principle, feasible. As demonstrated by the example of PYR resistance, MALDI-TOF MS allows for rapid and automated high-throughput assessment of drug sensitivity in P. falciparum malaria.

The cattle chromosome region BTA5q21-q25 is associated with immune defense and is linked with several economically important traits for growth, ovulation, twinning and milk yield. Chromosome microdissection of the region was performed to generate fragment-specific DNA libraries and to provide new bovine-specific DNA sequences as starting material for physical fine-mapping approaches or linkage studies. A first library screening resulted in the identification of 16 region-specific sequence-tagged sites (STS). Fifteen STS represent new unique cattle DNA sequences, of which one identifies a new microsatellite. Homology with the gene CDK2 was found for the remaining STS.

Trypanosomosis, or sleeping sickness, is a major disease constraint on livestock productivity in sub-Saharan Africa. To identify quantitative trait loci (QTL) controlling resistance to trypanosomosis in cattle, an experimental cross was made between trypanotolerant African N'Dama ( Bos taurus ) and trypanosusceptible improved Kenya Boran ( Bos indicus ) cattle. Sixteen phenotypic traits were defined describing anemia, body weight, and parasitemia. One hundred seventy-seven F 2 animals and their parents and grandparents were genotyped at 477 molecular marker loci covering all 29 cattle autosomes. Total genome coverage was 82%. Putative QTL were mapped to 18 autosomes at a genomewise false discovery rate of 2 cross between N'Dama and Boran cattle could produce a synthetic breed with higher trypanotolerance levels than currently exist in the parental breeds.

Studying 12 selected individuals from a malaria-endemic area in West Africa, 24 variants of the CD36 gene were found, 21 of them novel ones. These included three single-nucleotide substitutions causing non-conservative amino acid exchanges E123K, T174A, and I271T as well as a three base pair (bp) insertion resulting in the addition of an asparagine residue (N232-233ins). The E123K variant was located within the putative ligand-binding domain for oxidized low density lipoprotein, while the other substitutions resided outside any of the binding sites for reaction partners mapped on CD36 so far. Twelve single-nucleotide polymorphisms (SNPs) were identified in untranslated parts of the exons and in introns. Five additional SNPs were located in the promoter region whereby -144G-->T, -53G-->T, and -2A-->G alter putative binding sites for the transcription factors purine factor (PuF), phorbol ester-responsive element AP-2, and CCAAT/enhancer-binding protein. A G-->T exchange at position -50 appears to introduce a new recognition site for PuF. Calculations of nucleotide diversity revealed extraordinarily high numbers for all parts of the gene, which may, however, to some extent be due to the selection of individuals studied.

The epidemiological features of human infection with Plasmodium were studied in a community-based survey of 35 villages in the Ashanti region of Ghana. The overall prevalences of malarial parasitaemia in subjects aged > or = 2 years were 50.72% in forest areas and 49.72% in savanna. Plasmodium falciparum was the predominant species everywhere, followed by P. malariae in the savanna and P. ovale in the forest. The highest prevalence of asexual parasitaemia (of any species) occurred in the youngest age-group (2-9 years). The geometric mean intensities of parasitaemia among the parasitaemic (i.e. the parasite density indices) were 557, 640 and 452 parasites/microliter for P. falciparum, P. ovale and P. malariae, respectively. For each Plasmodium species encountered, the mean intensity of parasitaemia decreased with age. Mixed infections were observed in 24% and 30% of the parasitaemic subjects from the forest and savanna, respectively. Those infected with P. falciparum were more likely to carry P. ovale (odds ratio = 2.02) or P. malariae (odds ratio = 2.63) than those who were not infected with P. falciparum. Mean intensities of the parasitaemias in mixed infections were substantially higher than the sums of those in the corresponding single infections. When comparing villages, parasite density indices were found to be correlated with the prevalences of parasitaemia (r = 0.56).

Studying the genetic polymorphism of the major histocompatibility complex class II genes in cattle, we identified an allele (BNI13) which encodes a typical class II alpha chain. Its transcription was confirmed by RNA analysis. Sequence comparisons, Southern blot, and phylogenetic analyses indicate that (1) BNI13 represents a distinct DQA locus which we propose to designate BoLA-DQA5, (2) BoLA-DQA1 and BoLA-DQA5 separated after the divergence of BoLA-DQA1 and BoLA-DQA2, but prior to the separation of sheep DQA1 and cattle DQA1, and (3) DQA5 is distributed among various cattle breeds but is confined to certain haplotypes.

Eleven X-chromosomal short tandem repeats (STRs) from two multiplex PCR approaches (DXS6807, DXS8378, DXS7132, DXS6800, DXS9898, DXS7424, DXS101, DXS7133, HPRTB, DXS8377, and DXS7423), located in four different X-chromosomal linkage groups, were typed in a population sample from Ghana, Africa. After genotyping unrelated men (129) and women (114) from the Ashanti population, forensic efficiency parameters such as polymorphism information content and mean exclusion chance were calculated. A deviation from the Hardy-Weinberg equilibrium could not be found. The investigation of 11 father-daughter and seven mother-son meioses revealed no mutations in any STR analyzed. Our data were compared with European, African-American, and Asian populations from the literature.

Parkinson's disease (PD) is a common degenerative neurologic disorder, which is pathologically characterized by a selective degeneration of dopaminergic neurons of the substantia nigra pars compacta, and the presence of characteristic eosinophilic inclusions, known as Lewy-bodies in affected brain areas. The cause of PD is unknown but, in recent years, genetic factors have been implicated in the aetiology of the disease. Firstly, clinico-genetic, epidemiologic and twin studies revealed inheritable effects and questioned earlier studies which had denied such influences. Secondly, several family studies suggested autosomal-dominant inheritance of syndromes which, to variable degrees, resembled sporadic PD clinically and in some cases also neuropathologically. Recently, a disease locus has been mapped to chromosome 4q21-22 in a large Mediterranean pedigree, in which disease expression is clinically and pathologically within the spectrum of sporadic PD; being atypical only for a relatively young mean age at onset of 46 years and rapid course of 10 years from onset to death. In affected individuals of this family and of three unrelated Greek kindreds, a putative disease-causing mutation has been identified in the gene encoding alpha-synuclein. With the first variant being defined, genetic heterogeneity has become apparent, as in other families parkinsonism was not linked to the 4q-locus and was not associated with the alpha-synuclein mutation (unpublished data). We describe a different genetic locus that appears to be involved in the development of parkinsonism closely resembling sporadic PD including a similar mean age of onset (59 years in the families, 59.7 years in sporadic PD; ref. 12). This locus was detected in a group of families of European origin. In two of these families, there is genetic evidence for a common founder. The penetrance of the mutation appears to be low, most likely below 40%. This is compatible with a possible role of this locus not only in familial, but also in typical (sporadic) PD.

To the Editor: The directly observed treatment strategy (DOTS) for tuberculosis (TB) treatment has been implemented in Ghana since 1994. Before then, TB was treated without adherence to any concerted guidelines. The 2003 report of the Ghanaian National Tuberculosis Programme (NTP) stated a TB incidence of 281/100,000 (1). NTP ensures treatment of all patients with an 8-month course of streptomycin, isoniazid, rifampin, and pyrazinamide (for 2 months), followed by thiacetazone and isoniazid (6 months). The cure rate for 2003 was >50% (1), and >75% is anticipated for 2005. To determine the extent of drug resistance and to make suggestions for future Ghanaian NTP strategies, we assessed resistance against anti-TB drugs used in Ghana. A total of 2,064 patients with new cases of pulmonary TB were recruited at Korle Bu Teaching Hospital, Accra; Komfo Anokye Teaching Hospital, Kumasi; 15 periurban hospitals; and hospitals in the Ashanti, Eastern, and Central Regions of Ghana. These patients were consecutively enrolled in a cross-sectional study from September 2001 to December 2004. On all patients’ clinical examinations, chest radiographs, sputum smears for staining of acid-fast bacteria, HIV testing, and culturing of Mycobacterium tuberculosis complex strains were performed. Samples were taken only after informed consent was given. The study was approved by the appropriate ethics committees. A total of 2,064 Mycobacterium isolates were cultured at the Kumasi Centre for Collaborative Research. After decontamination of sputum samples (N-acetyl-L-cysteine/NaOH) and centrifugation, sediments were transferred onto Lowenstein-Jensen (LJ) media, incubated (37°C), and read weekly for 10 weeks for mycobacterial growth. Subsequently, cultures were sent to the German National Reference Centre for Mycobacteria in Borstel, Germany, a reference laboratory of the World Health Organization, for drug sensitivity testing (DST; proportion method on LJ media). Sensitivity to isoniazid, rifampin, pyrazinamide, ethambutol, and streptomycin was determined for 2,064 isolates and to thiacetazone for 1,288 isolates. For ambiguous results and DST of thiacetazone, the modified proportion method (Bactec 460TB; Becton Dickinson, Cockeysville, MD, USA) was performed. Data were analyzed with EpiInfo (Centers for Disease Control and Prevention, Atlanta, GA, USA) and Fourth Dimension (ACI Group, San Jose, CA, USA) software programs. Of the isolates, 32.8% were from female patients, and 67.8% were from male patients. The mean age of participants (33 years, range 10–60) did not differ by sex. HIV prevalence was 14.3% (males, n = 179, females, n = 117). A total of 1,578 (76.5%) isolates were susceptible to all drugs tested, whereas 304 (14.7%) were monodrug resistant, and 177 (8.7%) were multi- or polydrug resistant to combinations (multidrug resistance meant resistance to at least isoniazid and rifampin (2.2%); polydrug resistance meant resistance to several drugs, excluding combined resistance to isoniazid and rifampin (6.5%). The overall prevalence of any drug resistance was 23.5% (486 isolates) (Table). No differences were observed between HIV-negative and HIV-positive patients. The highest level of resistance was against streptomycin, followed by isoniazid. Resistance to rifampin, pyrazinamide, and thiacetazone was lower. Monoresistance to ethambutol was not observed; resistance to ethambutol combined with other drugs occurred in 0.9% of isolates. Table Resistance to first-line antituberculosis drugs, Ghana* In all, 6.5% of isolates were polydrug resistant and virtually always included resistance to isoniazid. Among isolates with double- and triple drug resistance, combinations of resistance to isoniazid and streptomycin and to isoniazid-thiacetazone-streptomycin occurred most frequently. Other combinations were relatively rare. In 1989, an initial drug resistance rate of 54.5% in pulmonary TB was observed in Ghana (2); 27% were resistant to isoniazid, 23% to streptomycin, 29% to thiacetazone, 16% to streptomycin-isoniazid, and 5% to thiacetazone-streptomycin-isoniazid. A later study reported a high prevalence of primary drug resistance to isoniazid (23%), while sensitivity to rifampicin, pyrazinamide, ethambutol, streptomycin, and ciprofloxacin was maintained (3). However, the number of isolates tested was fewer in both studies (n = 99 and 25, respectively) than in ours. This report supplements data from patients in Ghana whose conditions were newly diagnosed as HIV-negative and HIV-positive. Samples were collected in 2 large regions of Ghana, the Greater Accra and the Ashanti Regions, and were supplemented by samples from additional regions. Thus, these results are likely representative of the entire country. The overall primary drug resistance rate of 23.5% in Ghanaian TB patients ranks Ghana among those African countries with a high prevalence of drug-resistant TB. The high degree of mono-, multi- and polyresistance to streptomycin may be the result of selective pressure exerted by treatment of other infections with streptomycin and to incomplete treatment courses. Drug resistance to streptomycin and isoniazid are of concern, since these drugs are core components of the NTP. The relative ineffectiveness of streptomycin and the low level of resistance to ethambutol justify the most recent replacement of streptomycin by ethambutol by the Ghanaian NTP. Low rates of initial drug resistance have been reported in countries in which the DOTS strategy has been successfully implemented. Adequate use of standardized treatment regimens under DOTS will limit further emergence of drug resistance but not substantially reduce the current degree of resistance (4). Although the levels of drug resistance in Africa are lower than in several other countries (5), measures to provide controlled application of second-line drugs, supervision of drug distribution and compliance, enforcement of DOTS protocols, and sustained training of all personnel involved in TB management are crucial.

Summary The adhesion of infected red blood cells (iRBCs) to human endothelium is considered a key event in the pathogenesis of cerebral malaria and other life‐threatening complications caused by the most prevalent malaria parasite Plasmodium falciparum. In the past 30 years, 14 endothelial receptors for iRBCs have been identified. Exposing 10 additional surface proteins of endothelial cells to a mixture of P. falciparum isolates from three Ghanaian malaria patients, we identified seven new iRBC receptors, all expressed in brain vessels. This finding strongly suggests that endothelial binding of P. falciparum iRBCs is promiscuous and may use a combination of endothelial surface moieties.

Most cases of α-thalassemia result from large deletions at the α-globin locus (1). The α-globin gene cluster contains a tandem array of 2 nearly identical α-globin genes ( HBA ; Fig. 1A⇓ ) (2). The α-thalassemias are characterized by deletions that inactivate both α-globin genes of a given chromosome, whereas in α+-thalassemias, one gene remains functional. The most widespread α+-thalassemias are those designated −α3.7 and −α4.2, according to the lengths of the deleted fragments (3). The α+-thalassemias cannot be diagnosed on clinical grounds, and routine laboratory tests fail, except in the perinatal period, when transient expression of hemoglobin (Hb) Barts may be found (4). Diagnostic assays are also important for genetic counseling because, when occurring in compound heterozygosity with α-thalassemia, α+-thalassemia causes Hb H disease with a thalassemia intermedia phenotype (5). The classic method to diagnose α-thalassemias is the laborious and expensive Southern blot hybridization. Alternative assays include qualitative gap-PCRs (3)(6), which depend on the generation of long PCR products, which is not consistently reproducible (3) in the GC-rich sequences of the α-globin gene cluster. In addition, gap-PCRs require the use of deletion-type–specific primers, which limits their sensitivities to distinct variants. Moreover, as for any PCR-based, allele-specific method, they carry the risk that alleles may be amplified from minimal contaminants, such as maternal DNA in prenatal diagnostics or fetal DNA circulating in maternal blood (7)(8)(9). Quantitative PCR has been proposed to avoid these problems (9). Because of limited sensitivity, however, real-time PCR approaches have been successful only for the diagnosis of α-thalassemias (9)(10)(11). We studied a group of 122 individuals from the Ashanti Region of Ghana, West Africa, which has a moderate prevalence of −α3.7-thalassemia. …

SUMMARYInvasive properties of pathogenicEntamoeba histolyticahave been postulated to depend on the secretion or release of cysteine proteinases and pore-forming peptides (amoebapores) by trophozoites. To establish whether such toxic molecules are released by viable trophozoites or upon cellular disintegration, amoebae were maintained in various culture media, and activities in supernatants were monitored over time in correlation to cellular integrity. By measuring the release of the cytoplasmic marker enzyme NADP+-alcohol dehydrogenase, it became apparent that release of amoebapore was accompanied by cellular disintegration. In contrast, considerable quantities of cysteine proteinases were found to be present in culture supernatants also when amoebae remained intact. Treatment of amoebae with concanavalin A, bacterial lipo-polysaccharides or the calcium ionophore A23187 did not result in amoebapore secretion suggesting that here target cell contact is required as an essential stimulus.

Extensively drug-resistant (XDR) tuberculosis (TB), which is resistant to both first- and second-line antibiotics, is an escalating problem, particularly in the Russian Federation. Molecular fingerprinting of 2348 Mycobacterium tuberculosis isolates collected in Samara Oblast, Russia, revealed that 72% belonged to the Beijing lineage, a genotype associated with enhanced acquisition of drug resistance and increased virulence. Whole-genome sequencing of 34 Samaran isolates, plus 25 isolates representing global M. tuberculosis complex diversity, revealed that Beijing isolates originating in Eastern Europe formed a monophyletic group. Homoplasic polymorphisms within this clade were almost invariably associated with antibiotic resistance, indicating that the evolution of this population is primarily driven by drug therapy. Resistance genotypes showed a strong correlation with drug susceptibility phenotypes. A novel homoplasic mutation in rpoC, found only in isolates carrying a common rpoB rifampicin-resistance mutation, may play a role in fitness compensation. Most multidrug-resistant (MDR) isolates also had mutations in the promoter of a virulence gene, eis, which increase its expression and confer kanamycin resistance. Kanamycin therapy may thus select for mutants with increased virulence, helping preserve bacterial fitness and promoting transmission of drug-resistant TB strains. The East European clade was dominated by two MDR clusters, each disseminated across Samara. Polymorphisms conferring fluoroquinolone resistance were independently acquired multiple times within each cluster, indicating that XDR TB is currently not widely transmitted.

After imputation of data from the 1000 Genomes Project into a genome-wide dataset of Ghanaian individuals with tuberculosis and controls, we identified a resistance locus on chromosome 11p13 downstream of the WT1 gene (encoding Wilms tumor 1). The strongest signal was obtained at the rs2057178 SNP (P = 2.63 × 10 -9). Replication in Gambian, Indonesian and Russian tuberculosis case-control study cohorts increased the significance level for the association with this SNP to P = 2.57 × 10 -11. © 2012 Nature America, Inc. All rights reserved.

Human infections with the tissue nematode Onchocerca volvulus result in a variety of clinical conditions that possibly include protective immunity. In a West African area hyperendemic for human onchocerciasis, 120 residents were classified according to clinical and laboratory findings as presenting with generalized onchocerciasis, localized onchocerciasis, or as being putatively immune. The three groups differed in the distribution of HLA-D variants as determined by DNA typing. The most pronounced differences were found among alleles of the DQ loci. The haplotype DQA1*0501-DQB1*0301 was significantly more frequent among putatively immune individuals than among patients with generalized or localized disease. Conversely, DQA1*0101-DQB1*0501 and, independently, the allele DQB1*0201 were more frequent in generalized disease than in localized disease or putative immunity. In these correlations, the frequencies of allelic variants were in localized disease intermediate to those of the two other groups. The only distinct association found with localized disease was that of the DP allele DPB1*0402. The findings indicate that HLA-D variants influence the course of O. volvulus infection and help to define a state that may reflect protective immunity.

Background The monocyte chemoattractant protein 1 (MCP-1) is involved in the recruitment of lymphocytes and monocytes and their migration to sites of injury and cellular immune reactions. In a Ghanaian tuberculosis (TB) case-control study group, associations of the MCP1 -362C and the MCP1 -2581G alleles with resistance to TB were recently described. The latter association was in contrast to genetic effects previously described in study groups originating from Mexico, Korea, Peru and Zambia. This inconsistency prompted us to further investigate the MCP1 gene in order to determine causal variants or haplotypes genetically and functionally. Results A 14 base-pair deletion in the first MCP1 intron, int1del554-567, was strongly associated with protection against pulmonary TB (OR = 0.84, CI 0.77-0.92, Pcorrected = 0.00098). Compared to the wildtype combination, a haplotype comprising the -2581G and -362C promoter variants and the intronic deletion conferred an even stronger protection than did the -362C variant alone (OR = 0.78, CI 0.69-0.87, Pnominal = 0.00002; adjusted Pglobal = 0.0028). In a luciferase reporter gene assay, a significant reduction of luciferase gene expression was observed in the two constructs carrying the MCP1 mutations -2581 A or G plus the combination -362C and int1del554-567 compared to the wildtype haplotype (P = 0.02 and P = 0.006). The associated variants, in particular the haplotypes composed of these latter variants, result in decreased MCP-1 expression and a decreased risk of pulmonary TB. Conclusions In addition to the results of the previous study of the Ghanaian TB case-control sample, we have now identified the haplotype combination -2581G/-362C/int1del554-567 that mediates considerably stronger protection than does the MCP1 -362C allele alone (OR = 0.78, CI 0.69-0.87 vs OR = 0.83, CI 0.76-0.91). Our findings in both the genetic analysis and the reporter gene study further indicate a largely negligible role of the variant at position -2581 in the Ghanaian population studied.

Using segregation analyses, control of malaria parasites has previously been linked to a major gene within the chromosomal region 5q31-33, but also to complex genetic factors in which effects are under substantial age-dependent influence. However, the responsible gene variants have not yet been identified for this chromosomal region. In order to perform association analyses of 5q31-33 locus candidate single nucleotide polymorphisms (SNPs), 1015 children were recruited at the age of 3 months and followed monthly until the age of 2 years in an area holoendemic for Plasmodium falciparum malaria in Ghana. Quantitative (incidence rates of malaria episodes) and qualitative phenotypes (i.e. 'more than one malaria episode' or 'not more than one malaria episode') were used in population- and family-based analyses. The strongest signal was observed for the interleukin 3 gene (IL3) SNP rs40401 (P = 3.4 × 10(-7), P(c)= 1.4 × 10(-4)). The IL3 genotypes rs40401(CT) and rs40401(TT) were found to exert a protective effect of 25% [incidence rate ratio (IRR) 0.75, P = 4.1 × 10(-5)] and 33% (IRR 0.67, P = 3.2 × 10(-8)), respectively, against malaria attacks. The association was confirmed in transmission disequilibrium tests (TDT, qTDT). The results could argue for a role of IL3 in the pathophysiology of falciparum malaria.

Summary Immunoregulatory eicosanoids have been implicated in protection from mycobacterial infection in cell and animal models. Recently, a study of the zebrafish embryo demonstrated that mutants of the lta4h gene, which encodes the leukotriene A4 hydrolase (LTA4H) enzyme of the eicosanoid pathway, have hypersusceptibility to Mycobacterium marinum infection. It also reported that heterozygosity at the two single nucleotide polymorphisms rs1978331 and rs2660898 located in introns of the LTA4H gene, a human homologue of lta4h, is associated with protection from pulmonary tuberculosis. To replicate this association we genotyped six LTA4H gene polymorphisms in samples from 3703 pulmonary tuberculosis patients and 5412 healthy controls collected in Russia. We found no evidence of the protective effect of heterozygosity at the polymorphisms rs1978331 and rs2660898 (P = 0.29 and 0.49) and no association of the alleles of any of the six polymorphisms (P = 0.13–0.81). These results suggest that common polymorphisms in the LTA4H gene do not play any major role in susceptibility to clinical pulmonary tuberculosis.

Structural variants of the Mannose Binding Lectin (MBL) cause quantitative and qualitative functional deficiencies, which are associated with various patterns of susceptibility to infectious diseases and other disorders. We determined genetic MBL variants in 2010 Ghanaian patients with pulmonary tuberculosis (TB) and 2346 controls and characterized the mycobacterial isolates of the patients. Assuming a recessive mode of inheritance, we found a protective association between TB and the MBL2 G57E variant (odds ratio 0.60, confidence interval 0.4-0.9, P 0.008) and the corresponding LYQC haplotype (P(corrected) 0.007) which applied, however, only to TB caused by M. africanum but not to TB caused by M. tuberculosis. In vitro, M. africanum isolates bound recombinant human MBL more efficiently than did isolates of M. tuberculosis. We conclude that MBL binding may facilitate the uptake of M. africanum by macrophages, thereby promoting infection and that selection by TB may have favoured the spread of functional MBL deficiencies in regions endemic for M. africanum.

Summary The lytic effect of complement activated through the alternative pathway (AP) was studied on pathogenic and nonpathogenic Entamoeba histolytica recently isolated from stool samples. Recent nonpathogenic isolates were nearly unaffected by exposure to AP whereas recent pathogenic stool isolates were highly susceptible to AP dependent complement-mediated lysis. Complement susceptible pathogenic stool isolates developed complement resistance in vivo during hamster liver passage and in vitro during cultivation in the presence of increasing concentrations of normal human serum (NHS). Since a clone of pathogenic HM-l.IMSS which initially was highly susceptible also acquired complement resistance during cultivation in the presence of NHS, it is concluded that complement resistance was caused by induction rather than by selection alone. Because cultivation in the presence of heat-inactivated NHS did not affect complement susceptibility of the cloned HM-l.IMSS, complement activation itself might induce complement resistance in pathogenic E. histolytica.

Human genetics and immune responses are considered to critically influence the outcome of malaria infections including life-threatening syndromes caused by Plasmodium falciparum. An important role in immune regulation is assigned to the apoptosis-signaling cell surface receptor CD95 (Fas, APO-1), encoded by the gene FAS. Here, a candidate-gene association study including variant discovery at the FAS gene locus was carried out in a case-control group comprising 1,195 pediatric cases of severe falciparum malaria and 769 unaffected controls from a region highly endemic for malaria in Ghana, West Africa. We found the A allele of c.−436C>A (rs9658676) located in the promoter region of FAS to be significantly associated with protection from severe childhood malaria (odds ratio 0.71, 95% confidence interval 0.58–0.88, pempirical = 0.02) and confirmed this finding in a replication group of 1,412 additional severe malaria cases and 2,659 community controls from the same geographic area. The combined analysis resulted in an odds ratio of 0.71 (95% confidence interval 0.62–0.80, p = 1.8×10−7, n = 6035). The association applied to c.−436AA homozygotes (odds ratio 0.47, 95% confidence interval 0.36–0.60) and to a lesser extent to c.−436AC heterozygotes (odds ratio 0.73, 95% confidence interval 0.63–0.84), and also to all phenotypic subgroups studied, including severe malaria anemia, cerebral malaria, and other malaria complications. Quantitative FACS analyses assessing CD95 surface expression of peripheral blood mononuclear cells of naïve donors showed a significantly higher proportion of CD69+CD95+ cells among persons homozygous for the protective A allele compared to AC heterozygotes and CC homozygotes, indicating a functional role of the associated CD95 variant, possibly in supporting lymphocyte apoptosis., Author Summary Severe malaria caused by infection with the protozoan parasite Plasmodium falciparum is a major health burden, causing approximately one million fatalities annually, predominantly among young children in Sub-Saharan Africa. The occurrence of severe malaria may depend on a complex interplay of transmission dynamics and the development of a protective immune response but also on heritable differences in the susceptibility to the disease. In two large studies including a total of 2,607 affected children and 3,428 apparently healthy individuals from Ghana, West Africa, we investigated genetic variants of the FAS gene, which encodes CD95, a molecule critically involved in the programmed cell death of lymphocytes. We found that a single nucleotide variant in the FAS promoter was associated with a 29%–reduced risk of developing severe malaria. In individuals carrying two copies of the protective allele, a higher proportion of activated lymphocytes was found to express CD95. These findings indicate that a predisposition to an increased expression of CD95 may help to protect from severe malaria, possibly by rendering activated T-lymphocytes more susceptible to programmed cell death.

M protein, the major virulence factor of group A streptococci, has antiopsonic activity in that it inhibits activation of the alternative complement pathway on the streptococcal surface. Two properties of M protein have been claimed to account for the inhibitory activity, namely, (i) its binding affinity for complement factor H, which is an inhibitor of alternative pathway activation, and (ii) its high binding affinity for fibrinogen. We have recently shown that fibrinogen, like M protein, inhibits alternative pathway activation by possessing binding affinity for factor H. Here we report that fibrinogen effectively competes with factor H for binding to M protein but retains its own binding affinity for factor H. The presence of fibrinogen did not significantly affect alternative pathway inhibition on the streptococcal surface.

After the identification of the human interferon-gamma (IFNG) variant G54D (c.287GA, ss105106770) in DNA samples from Ghana, West Africa, systematic mutation screening of IFNG by the LightCycler((R))-based procedure of high-resolution melting (HRM) revealed additional rare mutations. All variants occurred heterozygously only and were confirmed either by their detection in other individuals and/or by repeated DNA sequencing of independent PCR products.

Rationale: Susceptibility to tuberculosis is not only determined by Mycobacterium tuberculosis infection, but also by the genetic component of the host. The pleiotropic cytokine tumor necrosis factor-α is essential to control tuberculosis infection, and various tumor necrosis factor family members and their respective receptors may contribute to tuberculosis risk. Objectives: To investigate four functionally relevant polymorphisms in the tumor necrosis factor receptor 2–encoding gene, tumor necrosis factor receptor superfamily member 1B, for association with tuberculosis susceptibility. Methods: Genotyping of four polymorphisms was performed in independent populations from South Africa (429 cases and 482 control subjects) and Ghana (640 cases and 1,158 control subjects), and the association of the variants with tuberculosis was tested using two case-control association studies. Measurements and Main Results: Single-point and haplotype analysis in South Africans revealed an association in the 3′untranslated region of the investigated gene. The T allele of rs3397 alone and/or the 3′ untranslated region haplotype GTT may confer protection against tuberculosis insofar as both allele and haplotype frequencies were significantly lower in case subjects than in controls. The GTT genotype had previously been shown to increase the decay of tumor necrosis factor receptor 2 messenger ribonucleic acid, and messenger ribonucleic acid destabilization may represent a key molecular mechanism for disease susceptibility. Interestingly, the association signal appeared to be restricted to women. The genetic finding was validated in female participants from Ghana. The combined P value in the haplotype analysis was P = 0.00011. Conclusions: Our finding emphasizes the importance of tumor necrosis factor/tumor necrosis factor receptor–mediated immune responses in the pathogenesis of tuberculosis.

Background Severe malarial anaemia is a major cause of mortality from malaria. Although of enormous relevance, its pathogenesis is largely unknown. Interestingly, the extent of anaemia greatly exceeds the loss of erythrocytes due to direct destruction by the pathogen Plasmodium falciparum . Immune response against the parasite is partially mediated through the Fc receptor for immunoglobulin (Ig) G IIa (FcγRIIa, CD32). The presence of an arginine instead of a histidine residue at amino acid position 131 (H131R) in the extracellular domain of FcγRIIa reduces the affinity of the receptor for IgG 2 and IgG 3 isotypes but increases the binding activity for C reactive protein (CRP). Methods In Ghana, West Africa, 2504 children with severe malaria and 2027 matched healthy controls were studied for the FcγRIIa H131R polymorphism in order to ascertain its influence on major manifestations of the disease. The study group included patients with partly overlapping symptoms of severe malaria, among them 1591 cases with severe anaemia, 562 cases with cerebral malaria, and 497 cases with other malaria complications. Results Analyses of the genotype distributions indicated that, under a recessive model, FcγRIIa 131RR was positively associated with severe malaria collectively (OR 1.20, 95% CI 1.05 to 1.38; p=0.007, p corrected =0.021) and, after stratification for phenotypes, with severe anaemia (OR 1.33, 95% CI 1.13 to 1.57; p=0.001, p corrected =0.009), but not with cerebral malaria (OR 1.04, 95% CI 0.82 to 1.33; p=0.733) or other malaria complications (OR 1.03, 95% CI 0.78 to 1.37; p=0.827). No association was found with levels of parasitaemia. Conclusion The positive association with a CRP binding variant of FcγRIIa supports evidence for a role of CRP mediated defence mechanisms in the pathogenesis of severe malarial anaemia.

Evidence from genetic association and twin studies indicates that susceptibility to tuberculosis (TB) is under genetic control. One gene implicated in susceptibility to TB is that encoding interleukin-10 (IL10). In a group of 2010 Ghanaian patients with pulmonary TB and 2346 healthy controls exposed to Mycobacterium tuberculosis, among them 129 individuals lacking a tuberculin skin test (PPD) response, we genotyped four IL10 promoter variants at positions 22849 , 21082 , 2819 , and 2592 and reconstructed the haplotypes. The IL10 low-producer haplotype 22849A/21082A/2819C/2592C, compared to the high-producer haplotype 22849G/21082G/2819C/2592C, occurred less frequent among PPD-negative controls than among cases (OR 2.15, CI 1.3–3.6) and PPD-positive controls (OR 2.09, CI 1.2–3.5). Lower IL-10 plasma levels in homozygous 22849A/21082A/2819C/2592C carriers, compared to homozygous 22849G/21082G/2819C/2592C carriers, were confirmed by a IL-10 ELISA (p = 0.016). Although we did not observe differences between the TB patients and all controls, our results provide evidence that a group of individuals exposed to M. tuberculosis transmission is genetically distinct from healthy PPD positives and TB cases. In these PPD-negative individuals, higher IL-10 production appears to reflect IL-10-dependent suppression of adaptive immune responses and sustained long-term specific anergy.

The gene of Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA4), a negative regulator of T lymphocytes, contains a single-nucleotide polymorphism (SNP) at position +6230A->G (ct60A->G), which has been found associated with several autoimmune diseases and appears to reduce T-cell inhibitory activity. In Ghana, West Africa, we compared the frequencies of CTLA4 +6230 A/G and 6 haplotype-tagging SNPs in 2010 smear-positive, HIV-negative patients with pulmonary tuberculosis (TB) and 2346 controls matched for age, gender and ethnicity. We found no difference in allele frequencies between cases and controls. However, +6230A and a distinct CTLA4 haplotype and a diplotype comprising the +6230A allele were significantly less frequent among cases with large opacities in chest radiographs compared to those with small ones (P(corrected [cor]) = 0.002, P(cor) = 0.00045, P = 0.0005, respectively). This finding suggests that an increased T-cell activity associated with the CTLA4 +6230G allele contributes to pathology rather than to protection in pulmonary TB.

Previously unrecognized variants of human leukocyte antigens (HLA) are currently being analyzed by in vitro amplification and sequencing of the variable gene segments. In heterozygous individuals, molecular cloning is required to separate the two concomitantly amplified haplotypic gene segments. A method is presented which facilitates the procedure of separating the two haplotypic gene segments by using a temperature-gradient gel electrophoresis (TGGE). The procedure comprises PCR amplification of the variable HLA gene segments, allele separation by TGGE, re-amplification of each of the separated allelic segments, and direct DNA sequencing using the PCR primers.