Genome-wide association study indicates two novel resistance loci for severe malaria.

Malaria causes approximately one million fatalities per year, mostly among African children. Although highlighted by the strong protective effect of the sickle-cell trait, the full impact of human genetics on resistance to the disease remains largely unexplored. 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 successful. 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 erythrocytes, 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 cells 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 O. Our findings underline the potential of the GWA approach to provide candidates for the development of control measures against infectious diseases in humans.

The Prevalence of Sickle Cell Disease and Its Implication for Newborn Screening in Germany (Hamburg Metropolitan Area).

Sickle cell disease is among hereditary diseases with evidence that early diagnoses and treatment improves the clinical outcome. So far sickle cell disease has not been included in the German newborn screening program despite immigration from countries with populations at risk. To determine the birth prevalence we tested 17,018 newborns. High pressure liquid chromatography and subsequent molecular-genetic testing were used for the detection and confirmation of hemoglobin variants. The frequency of sickle cell disease-consistent genotypes was one in 2,385 newborns. Duffy-blood group typing showed evidence that affected children were likely of Sub-Saharan ancestry. An inclusion of sickle cell disease into the German newborn screening seems reasonable.

CCN1 mutation is associated with atrial septal defect.

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.

A -436C>A polymorphism in the human FAS gene promoter associated with severe childhood malaria.

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, p(empirical) = 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.

The spectrum of ß-thalassemia mutations in Gaza Strip, Palestine.

BACKGROUND: ß-Thalassemia is a disorder caused by mutations at the hemoglobin ß-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 ß-thalassemia major. DESIGN AND METHODS: An assessment of mutations was performed in 49 transfusion dependent patients with ß-thalassemia major and in 176 ß-thalassemia carriers diagnosed with a mean erythrocyte cell volume (MCV) <80fl and a proportion of HbA2>3.5%. In addition 39 individuals suspicious for ß-thalassemia carrier status due to a reduced MCV (<80fl) 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) ß-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 ß-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 ß-thalassemia in Gaza Strip, which will improve the screening protocol and genetic counseling in the future.

The new Powerplex® ESX17 and ESI17 kits in paternity and maternity analyses involving people from Africa--including allele frequencies for three African populations.

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.

FCGR2A functional genetic variant associated with susceptibility to severe malarial anaemia in Ghanaian children.

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 (FcgammaRIIa, CD32). The presence of an arginine instead of a histidine residue at amino acid position 131 (H131R) in the extracellular domain of FcgammaRIIa 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 FcgammaRIIa(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, FcgammaRIIa(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 FcgammaRIIa supports evidence for a role of CRP mediated defence mechanisms in the pathogenesis of severe malarial anaemia.

Malaria, mummies, mutations: Tutankhamun's archaeological autopsy.

The cause of death of the Egyptian pharoah Tutankhamun has now for decades been matter of speculation and various hypotheses. A recent article in the Journal of the American Medical Association (JAMA) provided new evidence and suggested malaria, together with Köhler's disease, as the most probable cause of death of the boy king. We are sceptical towards this elucidation of the cause of death of King Tut and discuss alternative and differential diagnoses, among them, in particular, sickle cell disease and Gauche's disease.

Genome-wide linkage analysis of malaria infection intensity and mild disease.

Although balancing selection with the sickle-cell trait and other red blood cell disorders has emphasized the interaction between malaria and human genetics, no systematic approach has so far been undertaken towards a comprehensive search for human genome variants influencing malaria. By screening 2,551 families in rural Ghana, West Africa, 108 nuclear families were identified who were exposed to hyperendemic malaria transmission and were homozygous wild-type for the established malaria resistance factors of hemoglobin (Hb)S, HbC, alpha(+) thalassemia, and glucose-6-phosphate-dehydrogenase deficiency. Of these families, 392 siblings aged 0.5-11 y were characterized for malaria susceptibility by closely monitoring parasite counts, malaria fever episodes, and anemia over 8 mo. An autosome-wide linkage analysis based on 10,000 single-nucleotide polymorphisms was conducted in 68 selected families including 241 siblings forming 330 sib pairs. Several regions were identified which showed evidence for linkage to the parasitological and clinical phenotypes studied, among them a prominent signal on Chromosome 10p15 obtained with malaria fever episodes (asymptotic z score = 4.37, empirical p-value = 4.0 x 10(-5), locus-specific heritability of 37.7%; 95% confidence interval, 15.7%-59.7%). The identification of genetic variants underlying the linkage signals may reveal as yet unrecognized pathways influencing human resistance to malaria.

Incidence of TNFRSF1A mutations in German children: epidemiological, clinical and genetic characteristics.

OBJECTIVE: TNF receptor 1-associated periodic syndrome (TRAPS) is a rare disease belonging to the heterogeneous group of hereditary periodic fever (HPF) syndromes. By their monogenic origins, the HPF syndromes are clearly differentiated from other periodic inflammatory episodes occurring in autoimmune, neoplastic and infectious diseases. We aim to determine the incidence of TRAPS and the spectrum of mutations in the TNFRSF1A gene, and to give a brief survey of clinical signs. METHODS: A prospective surveillance of children with TRAPS was conducted in Germany during a time period of 3 years (2003-06). Monthly inquiries were sent to 370 children's hospitals by the German Pediatric Surveillance Unit (Clinic-ESPED, n1) and to 23 laboratories (Laboratory-ESPED, n2). Inclusion criteria were TNFRSF1A mutation-positive patients < or =16 years of age, more than three self-limiting episodes of fever >38.5 degrees C, and increased inflammation markers. Clinical, epidemiological and genetic data were evaluated via questionnaires. RESULTS: Of the 23 cases included, 19 were identical in 20 clinical and 22 laboratory reports. The incidence of TRAPS in German children was estimated to be approximately 5.6 per 10(7) person-years. In 20 TRAPS patients of the Clinic-ESPED, median age of onset and duration of fever periods were 6 (range 1-16) years and 6.3 (range 2-24) days, respectively. Main symptoms were arthralgia, abdominal pain, lymphadenopathy, headache and skin involvement. The R92Q substitution was found in 19 (83%) of 23 cases. CONCLUSION: The incidence of TRAPS is low and corresponds to 6-10 newly diagnosed patients < or =16 years per year in Germany.

Allele frequencies of 11 X-chromosomal loci in a population sample from Ghana.

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.

Hemoglobin variants and disease manifestations in severe falciparum malaria.

CONTEXT: The geographical distributions of hemoglobin S (HbS), hemoglobin C (HbC), and alpha+-thalassemia (-alpha) strongly suggest balancing selection with malaria. However, whereas several studies indicate that the HbS carrier state protects against all major forms of clinical malaria, malaria protection on clinical grounds has been more difficult to confirm for HbC and -alpha, and questions remain as to whether it applies to all forms of the disease. OBJECTIVE: To assess the association between major clinical forms of severe falciparum malaria and HbS, HbC, and -alpha. DESIGN, SETTING, AND PARTICIPANTS: Case-control study of 2591 children with severe falciparum malaria enrolled at a tertiary referral center in Ghana, West Africa, and 2048 age-, sex-, and ethnicity-matched control participants recruited by community surveys. MAIN OUTCOME MEASURES: Frequencies of HbS, HbC, and -alpha in patients and controls, including stratifications of patients for signs of disease. RESULTS: Patients presented with partly overlapping signs of disease, including severe anemia (64%), cerebral malaria (22%), respiratory distress (30%), hyperparasitemia (32%), prostration (52%), acidosis (59%), and hyperlactatemia (56%). Carrier states of HbS, HbC, and -alpha were found in 1.4%, 9.4%, and 25.2% of the patients, respectively, and 14.8%, 8.7%, and 27.3% of controls. The HbS carrier state was negatively associated with all forms of the disease studied (overall odds ratio [OR], 0.08; 95% confidence interval [CI], 0.06-0.12). The HbC carrier state showed a negative association selectively with cerebral malaria (OR, 0.64; 95% CI, 0.45-0.91), and the -alpha carrier state showed a negative association selectively with severe anemia (OR, 0.82; 95% CI, 0.69-0.96). CONCLUSION: Whereas the HbS carrier state was found to be negatively associated with all major forms of severe falciparum malaria, the negative associations of the carrier states of HbC and -alpha appeared to be limited to cerebral malaria and severe anemia, respectively.

Lack of Association of CD55 Receptor Genetic Variants and Severe Malaria in Ghanaian Children.

In a recent report, the cellular receptor CD55 was identified as a molecule essential for the invasion of human erythrocytes by Plasmodium falciparum, the causal agent of the most severe form of malaria. As this invasion process represents a critical step during infection with the parasite, it was hypothesized that genetic variants in the gene could affect severe malaria (SM) susceptibility. We performed high-resolution variant discovery of rare and common genetic variants in the human CD55 gene. Association testing of these variants in over 1700 SM cases and unaffected control individuals from the malaria-endemic Ashanti Region in Ghana, West Africa, were performed on the basis of single variants, combined rare variant analyses, and reconstructed haplotypes. A total of 26 genetic variants were detected in coding and regulatory regions of CD55 Five variants were previously unknown. None of the single variants, rare variants, or haplotypes showed evidence for association with SM or P. falciparum density. Here, we present the first comprehensive analysis of variation in the CD55 gene in the context of SM and show that genetic variants present in a Ghanaian study group appear not to influence susceptibility to the disease.

Hereditary periodic fever with systemic amyloidosis: is hyper-IgD syndrome really a benign disease?

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.

Malaria transmission intensity and dynamics of clinical malaria incidence in a mountainous forest region of Ghana.

Background: Malaria transmission is heterogeneous. Villages close to each other may have very different transmission characteristics. The presence and abundance of malaria vectors is governed by local ecology and microclimate. Knowledge of the dynamics of transmission is important for planning and evaluation of malaria control strategies. This study investigated the heterogeneity of malaria transmission in preparation for a vaccine trial and offers insights into dynamics of malaria incidence in the forest zone of Ghana. Methods: Malaria transmission was assessed in four villages with different micro-ecological features in the forest zone of the Akwapim-Mampong Range in Ghana, water shed with rivers flowing north to Lake Volta in the south. Human landing catches (HLC) of mosquitoes were conducted and Plasmodium falciparum circumsporozoite rates were assessed by ELISA. Sporozoite prevalence, annual biting rates (ABR) and entomological inoculation rates (EIR) from the four study sites were compared with climatological and ecological data. Regression analysis was used to compare transmission data and blood parasite prevalence, parasite density (PD) and malaria episodes from children in the study area. Additionally we examined trends in confirmed clinical malaria incidence from 2005 -2012. Results: In total 1307 Anopheles gambiae s.l. and 54 An. funestus females were caught by HLC from November 2003 to August 2005. Sporozoites in Anopheles vectors in four villages ranged from 4.0 to 10.2%, ABR from 371 to 1890 and EIR from 40 to 158. Linear regression on parasitological and clinical data of children from the villages revealed that the ABR significantly influenced the parasite density (PD) of P. falciparum. Conclusion: Malaria transmission was intense and heterogeneous and corresponded to the micro-ecological differences. Malaria transmission in the early evening hours before people went to sleep was enough to sustain stable malaria. Scaling up preventive measures to reduce exposure to vectors will be effective in reducing parasitemia in children. Variations in transmission intensity must be considered when evaluating impact of control strategies and interventions such as the vaccine trials.

Human genetic resistance to Onchocerca volvulus: evidence for linkage to chromosome 2p from an autosome-wide scan.

BACKGROUND: Human infections with the tissue nematode Onchocerca volvulus show strong interindividual variation in intensity, which cannot be explained by differences in exposure alone. Several lines of evidence suggest a relevant influence of human genetics. METHODS: In a genome-wide search for genetic determinants of resistance, we studied 196 siblings from 51 families exposed to endemic O. volvulus transmission in the forest zone of Ghana, West Africa. The numbers of worm larvae in the skin (i.e., microfilariae), which are the established measure of O. volvulus infection intensity, were counted in 4 small skin biopsy specimens (i.e., skin snips), and the numbers of palpable subcutaneous worm nodules (i.e., onchocercomata) were assessed. Numbers were corrected for age and exposure and were analyzed for linkage to 377 autosomal microsatellite markers and additional markers in genomic regions of interest. RESULTS: Linkage was detected between the numbers of microfilariae and chromosome 2p21-p14 (maximum multipoint log(10) of odds (LOD) score of 3.80 at marker position D2S2378; empirical P=2.9 x 10(-5)). CONCLUSIONS: This finding provides strong evidence that a human genetic factor influences the intensity of O. volvulus infection. The strength of the linkage signal may facilitate the identification of the decisive genetic variants.

Colchicine use in children and adolescents with familial Mediterranean fever: literature review and consensus statement.

The daily application of colchicine is the standard therapy for prophylaxis of attacks and amyloid deposition in familial Mediterranean fever. However, because of many issues (eg, dosage, time of introduction, etc), no standardized treatment recommendations have been established. In this work we review the available literature on colchicine use with respect to its indication, efficacy, mode of application, and safety in children and adolescents with familial Mediterranean fever. On the basis of this analysis, a consensus statement on the application of colchicine in children and adolescents with familial Mediterranean fever was developed by caregivers from Germany, Austria, and Turkey.