Mapping a 50-spin-qubit network through correlated sensing.

Spins associated to optically accessible solid-state defects have emerged as a versatile platform for exploring quantum simulation, quantum sensing and quantum communication. Pioneering experiments have shown the sensing, imaging, and control of multiple nuclear spins surrounding a single electron spin defect. However, the accessible size of these spin networks has been constrained by the spectral resolution of current methods. Here, we map a network of 50 coupled spins through high-resolution correlated sensing schemes, using a single nitrogen-vacancy center in diamond. We develop concatenated double-resonance sequences that identify spin-chains through the network. These chains reveal the characteristic spin frequencies and their interconnections with high spectral resolution, and can be fused together to map out the network. Our results provide new opportunities for quantum simulations by increasing the number of available spin qubits. Additionally, our methods might find applications in nano-scale imaging of complex spin systems external to the host crystal.

Variation in human genes encoding adhesion and proinflammatory molecules are associated with severe malaria in the Vietnamese.

The genetic basis for susceptibility to malaria has been studied widely in African populations but less is known of the contribution of specific genetic variants in Asian populations. We genotyped 67 single-nucleotide polymorphisms (SNPs) in 1030 severe malaria cases and 2840 controls from Vietnam. After data quality control, genotyping data of 956 cases and 2350 controls were analysed for 65 SNPs (3 gender confirmation, 62 positioned in/near 42 malarial candidate genes). A total of 14 SNPs were monomorphic and 2 (rs8078340 and rs33950507) were not in Hardy-Weinberg equilibrium in controls (P<0.01). In all, 7/46 SNPs in 6 genes (ICAM1, IL1A, IL17RC, IL13, LTA and TNF) were associated with severe malaria, with 3/7 SNPs in the TNF/LTA region. Genotype-phenotype correlations between SNPs and clinical parameters revealed that genotypes of rs708567 (IL17RC) correlate with parasitemia (P=0.028, r(2)=0.0086), with GG homozygotes having the lowest parasite burden. Additionally, rs708567 GG homozygotes had a decreased risk of severe malaria (P=0.007, OR=0.78 (95% CI; 0.65-0.93)) and death (P=0.028, OR=0.58 (95% CI; 0.37-0.93)) than those with AA and AG genotypes. In summary, variants in six genes encoding adhesion and proinflammatory molecules are associated with severe malaria in the Vietnamese. Further replicative studies in independent populations will be necessary to confirm these findings.

Genetic association study for RSV bronchiolitis in infancy at the 5q31 cytokine cluster.

BACKGROUND: The pathophysiological basis of severe respiratory syncytial virus (RSV) bronchiolitis in infancy is poorly understood and has hindered vaccine development. Studies implicate the cell-mediated immune response in the pathogenesis of the disease. A recent twin study estimated a heritable contribution of 22% to RSV bronchiolitis. Genetic epidemiology provides a new approach to identifying important immune determinants of disease severity. METHODS: A comprehensive high-density gene-region association study for severe RSV bronchiolitis in infancy at 5q31 across 11 genes including the Th2-cytokine cluster was performed. A haplotype tagging approach was used to analyse genetic variation at 113 single nucleotide polymorphisms (SNPs) in 780 independent cases and 1045 controls. The study had sufficient power to detect small effects, perform extensive haplotype analysis and analyse both a principal phenotype and a refined age-limited phenotype enriched for first-exposure RSV infection. RESULTS: SNP associations were found at IL4 and a highly significant risk haplotype was identified across IL13 CNS-1 and IL4 (odds ratio 1.69, p<0.0001), present in both case-control and family-based analyses. All associations were strongest for a phenotype limited to <6 months of age, implicating this locus in primary RSV disease. The same risk haplotype has previously been shown to be associated with increased IL13 expression. CONCLUSIONS: A haplotype at IL13-1L4, which is associated with increased IL13 production, confers an increased risk of severe primary RSV bronchiolitis in early infancy. This study, together with previous studies implicating the same locus in atopic sensitisation, suggests that primary RSV bronchiolitis and atopy share a genetic contribution at the IL13-IL4 locus.

Variation in the ICAM1 gene is not associated with severe malaria phenotypes.

Evidence from autopsy and in vitro binding studies suggests that adhesion of erythrocytes infected with Plasmodium falciparum to the human host intercellular adhesion molecule (ICAM)-1 receptor is important in the pathogenesis of severe malaria. Previous association studies between polymorphisms in the ICAM1 gene and susceptibility to severe malarial phenotypes have been inconclusive and often contradictory. We performed genetic association studies with 15 single nucleotide polymorphisms (SNPs) around the ICAM1 locus. All SNPs were screened in a family study of 1071 trios from The Gambia, Malawi and Kenya. Two key non-synonymous SNPs with previously reported associations, rs5491 (K56M or 'ICAM-1(Kilifi)') and rs5498 (K469E), were tested in an additional 708 Gambian trios and a case-control study of 4058 individuals. None of the polymorphisms were associated with severe malaria phenotypes. Pooled results across our studies for ICAM-1(Kilifi) were, in severe malaria, odds ratio (OR) 1.02, 95% confidence interval (CI) 0.96-1.09, P=0.54, and cerebral malaria OR 1.07, CI 0.97-1.17, P=0.17. We assess the available epidemiological, population genetic and functional evidence that links ICAM-1(Kilifi) to severe malaria susceptibility.

Perturbation analysis: a simple method for filtering SNPs with erroneous genotyping in genome-wide association studies.

We introduce a simple and yet scientifically objective criterion for identifying SNPs with genotyping errors due to poor clustering. This yields a metric for assessing the stability of the assigned genotypes by evaluating the extent of discordance between the calls made with the unperturbed and perturbed intensities. The efficacy of the metric is evaluated by: (1) estimating the extent of over-dispersion of the Hardy-Weinberg equilibrium chi-square test statistics; (2) an interim case-control study, where we investigated the efficacy of the introduced metric and standard quality control filters in reducing the number of SNPs with evidence of phenotypic association which are attributed to genotyping errors; (3) investigating the call and concordance rates of SNPs identified by perturbation analysis which have been genotyped on both Affymetrix and Illumina platforms. Removing SNPs identified by the extent of discordance can reduce the degree of over-dispersion of the HWE test statistic. Sensible use of perturbation analysis in an association study can correctly identify SNPs with problematic genotyping, reducing the number required for visual inspection. SNPs identified by perturbation analysis had lower call and concordance rates, and removal of these SNPs significantly improved the performance for the remaining SNPs.

Interferon regulatory factor-1 polymorphisms are associated with the control of Plasmodium falciparum infection.

We describe the haplotypic structure of the interferon regulatory factor-1 (IRF-1) locus in two West African ethnic groups, Fulani and Mossi, that differ in their susceptibility and immune response to Plasmodium falciparum malaria. Both populations showed significant associations between IRF-1 polymorphisms and carriage of P. falciparum infection, with different patterns of association that may reflect their different haplotypic architecture. Genetic variation at this locus does not therefore account for the Fulani-specific resistance to malaria while it could contribute to parasite clearance's ability in populations living in endemic areas. We then conducted a case-control study of three haplotype-tagging single nucleotide polymorphisms (htSNPs) in 370 hospitalised malaria patients (160 severe and 210 uncomplicated) and 410 healthy population controls, all from the Mossi ethnic group. All three htSNPs showed correlation with blood infection levels in malaria patients, and the rs10065633 polymorphism was associated with severe disease (P=0.02). These findings provide the first evidence of the involvement in malaria susceptibility of a specific locus within the 5q31 region, previously shown to be linked with P. falciparum infection levels.

Haplotypic diversity in human CEACAM genes: effects on susceptibility to meningococcal disease.

Adhesion between the opacity-associated adhesin (Opa) proteins of Neisseria meningitidis and human carcino-embryonic antigen cell adhesion molecule (CEACAM) proteins is an important stage in the pathogenesis of meningococcal disease, a globally important bacterial infection. Most disease is caused by a small number of meningococcal genotypes known as hyperinvasive lineages. As these are also carried asymptomatically, acquisition of them alone cannot explain why only some hosts develop meningococcal disease. Our aim was to determine whether genetic diversity in CEACAM is associated with susceptibility to meningococcal disease. Frequency distributions of alleles, genotypes and haplotypes were compared in four CEACAM genes in 384 case samples and 190 controls. Linkage disequilibrium among polymorphic sites, haplotype structures and relationships were also analysed. A number of polymorphisms were observed in CEACAM genes but the diversity of CEACAM1, to which most Opa proteins bind, was lower, and a small number of high-frequency haplotypes were detected. Dose-dependent associations of three CEACAM haplotypes with meningococcal disease were observed, with the effect of carrying these haplotypes amplified in homozygous individuals. Two haplotypes were protective while one haplotype in CEACAM6 was associated with a twofold increase in disease susceptibility. These data imply that human CEACAM may be one determinant of human susceptibility to meningococcal disease.

Genetic variation at the TNF locus and the risk of severe sequelae of ocular Chlamydia trachomatis infection in Gambians.

Tumor necrosis factor (TNF) is thought to be a key mediator of the inflammatory and fibrotic response to Chlamydia trachomatis (Ct) infection. A large matched-pair case-control study investigated putative functional single nucleotide polymorphisms (SNPs) across the major histocompatibility complex (MHC) class III region, including TNF and its immediate neighbors nuclear factor of kappa light polypeptide gene enhancer in B cells (IkappaBL), inhibitor like 1 and lymphotoxin alpha (LTA) in relation to the risk of scarring sequelae of ocular Ct infection. Haplotype and linkage disequilibrium analysis demonstrated two haplotypes, differing at position TNF-308, conferring an increased risk of trichiasis. The TNF-308A allele, and its bearing haplotype, correlated with increased TNF production in lymphocyte cultures stimulated with chlamydial elementary body antigen. Thus TNF-308A may determine directly, or be a marker of a high TNF producer phenotype associated with increased risk of sequelae of chlamydial infection. Multivariate analysis provided evidence for the presence of additional risk-associated variants near the TNF locus.

A comparison of case-control and family-based association methods: the example of sickle-cell and malaria.

There has been much debate about the relative merits of population- and family-based strategies for testing genetic association, yet there is little empirical data that directly compare the two approaches. Here we compare case-control and transmission/disequilibrium test (TDT) study designs using a well-established genetic association, the protective effect of the sickle-cell trait against severe malaria. We find that the two methods give similar estimates of the level of protection (case-control odds ratio = 0.10, 95% confidence interval 0.03-0.23; family-based estimate of the odds ratio = 0.11, 95% confidence interval 0.04-0.25) and similar statistical significance of the result (case-control: chi2= 41.26, p= 10(-10), TDT: chi2= 39.06, p= 10(-10)) when 315 TDT cases are compared to 583 controls. We propose a family plus population control study design, which allows both case-control and TDT analysis of the cases. This combination is robust against the respective weaknesses of the case-control and TDT study designs, namely population structure and segregation distortion. The combined study design is especially cost-effective when cases are difficult to ascertain and, when the case-control and TDT results agree, offers greater confidence in the result.

Risk of trachomatous scarring and trichiasis in Gambians varies with SNP haplotypes at the interferon-gamma and interleukin-10 loci.

Experimental evidence implicates interferon gamma (IFNgamma) in protection from and resolution of chlamydial infection. Conversely, interleukin 10 (IL10) is associated with susceptibility and persistence of infection and pathology. We studied genetic variation within the IL10 and IFNgamma loci in relation to the risk of developing severe complications of human ocular Chlamydia trachomatis infection. A total of 651 Gambian subjects with scarring trachoma, of whom 307 also had potentially blinding trichiasis and pair-matched controls with normal eyelids, were screened for associations between single-nucleotide polymorphisms (SNPs), SNP haplotypes and the risk of disease. MassEXTEND (Sequenom) and MALDI-TOF mass spectrometry were used for detection and analysis of SNPs and the programs PHASE and SNPHAP used to infer haplotypes from population genetic data. Multivariate conditional logistic regression analysis identified IL10 and IFNgamma SNP haplotypes associated with increased risk of both trachomatous scarring and trichiasis. SNPs in putative IFNgamma and IL10 regulatory regions lay within the disease-associated haplotypes. The IFNgamma +874A allele, previously linked to lower IFNgamma production, lies in the IFNgamma risk haplotype and was more common among cases than controls, but not significantly so. The promoter IL10-1082G allele, previously associated with high IL10 expression, is in both susceptibility and resistance haplotypes.

Increased in vivo transcription of an IL-8 haplotype associated with respiratory syncytial virus disease-susceptibility.

Interleukin-8 (IL-8) has been implicated in the pathogenesis of RSV-induced bronchiolitis. Previously, we have described an association between bronchiolitis disease severity and a specific IL-8 haplotype comprising six single-nucleotide polymorphisms (SNPs) (-251A/+396G/+781T/+1238delA/+1633T/+2767T, haplotype 2). Here we investigated the functional basis for this association by measuring haplotype-specific transcription in vivo in human primary cells. We found a significant increase in transcript level derived from the IL-8 haplotype 2 relative to the mirror haplotype 1 (-251T/+396T/+781C/+1238insA/+1633C/+2767A) in respiratory epithelial cells but not in lymphocytes. A promoter polymorphism, -251A, present on the high producer haplotype, had no significant affect on the allele-specific level of transcription when analyzed in reporter gene experiments in human respiratory epithelial A549 cells. We proceeded to systematically screen for allele-specific protein-DNA binding in this functional haplotype, which revealed significant differential binding at the +781T/C polymorphism. C/EBP beta was identified as being part of a transcription factor binding complex that preferentially bound in the presence of the +781 T allele. These results suggest that the mechanism for disease susceptibility to RSV-induced bronchiolitis may occur through a haplotype-specific increase in IL-8 transcription, which may be mediated by functional polymorphisms within that haplotype.

Complex haplotypic structure of the central MHC region flanking TNF in a West African population.

TNF polymorphisms have been associated with susceptibility to malaria and other infectious and inflammatory conditions. We investigated a sample of 150 West African chromosomes to determine linkage disequilibrium (LD) between 25 SNP markers located in an 80 kb segment of the MHC Class III region encompassing TNF and eight neighbouring genes. We observed 45 haplotypes, and 22 of them comprise 80% of the sample. The pattern of LD is remarkably patchy, such that many markers show no LD with adjacent markers but high LD with markers that are much further away. We introduce a method of examining the implications of LD data for disease association studies based on sample size considerations: this shows that certain TNF polymorphisms would be likely to yield positive associations if the true disease allele resided in LTA or BAT1. We conclude that detailed marker maps are needed to resolve the causal origin of disease associations observed at the TNF locus.

Haplotypic relationship between SNP and microsatellite markers at the NOS2A locus in two populations.

The density of genetic markers required for successful association mapping of complex diseases depends on linkage disequilibrium (LD) between non-functional markers and functional variants. The haplotypic relationship between stable markers and potentially unstable but highly informative markers (e.g. microsatellites) indicates that LD might be maintained over considerable genetic distance in non-African populations, supporting the use of such 'mixed marker haplotypes' in LD-based mapping, and allowing inferences to be drawn about human origins. We investigated sequence variation in the proximal 2.6 kb of the inducible nitric oxide synthase (NOS2A) promoter and the relationship between SNP haplotypes and a pentanucleotide microsatellite (the 'NOS2A(-2.6) microsatellite') in Gambians and UK Caucasians. UK Caucasians exhibited a subset of sequence diversity observed in Gambians, sharing four of 11 SNPs and a similar haplotypic structure. Five SNPs were found in the sequence of interspersed repetitive DNA elements. In both populations, there was dramatic loss of LD between SNP haplotypes and microsatellite alleles across a very short physical distance, suggesting a high intrinsic mutation rate of the NOS2A(-2.6) microsatellite, the SNP haplotypes are relatively ancient, or that this was a region of frequent recombination. Understanding locus- and population-specific LD is essential when designing and interpreting genetic association studies.

Stimulators of tumour necrosis factor production released by damaged erythrocytes.

We sought to characterize factors released by sonicated human erythrocytes that stimulate peripheral blood mononuclear cells (PBMC) to release tumour necrosis factor-alpha (TNF). This response is not inhibited by polymyxin B, indicating that contaminating lipopolysaccharide (LPS) is not responsible. When erythrocyte lysates are fractionated by reverse-phase chromatography using a gradient of n-propanol on Sep-Pak C18 cartridges, the TNF-inducing activity elutes as a single peak. The erythrocyte-derived TNF-inducing activity is unaffected by digestion with proteases but is destroyed by mild base hydrolysis or digestion by lipases, indicating that compounds containing ester-linked acyl chains may be essential. These properties are similar to those of TNF stimulators that we have previously identified in erythrocytes infected with malaria parasites, except that the TNF-inducing activity per cell is about 200 times higher in parasitized erythrocytes than in uninfected erythrocytes. Lipase-digested erythrocyte lysates inhibit the TNF-inducing factors of both normal and malaria-infected erythrocytes, suggesting that lipase digestion creates partial structures which compete with active components for macrophage receptors. Such receptors may recognize a common structure that contains an inositol monophosphate (IMP)-like component, as IMP also inhibits the TNF response to erythrocyte-derived factors and to parasite lysates whereas it does not affect the response to LPS. We conclude that lysed erythrocytes release specific cytokine-inducing factors that may contribute to the fever response to non-infectious tissue injury.

Severe malaria in Gambian children is not due to lack of previous exposure to malaria.

The reasons why only a small proportion of African children infected with Plasmodium falciparum develop severe or fatal malaria are not known. One possible reason is that children who develop severe disease have had less previous exposure to malaria infection, and hence have less acquired immunity, than children who develop a mild clinical attack. To investigate this possibility we have measured titres of a wide range of anti-P. falciparum antibodies in plasma samples obtained from children with severe malaria, children with mild malaria and from children with other illnesses. Mean antibody levels in patients with malaria were higher than those in patients with other conditions but, with only one exception, there were no significant differences in antibody titres between cases of severe or mild malaria. A parasitized-erythrocyte agglutination assay was used to estimate the diversity of parasite isolates to which children had been exposed; plasma samples obtained from children with cerebral malaria recognized as many isolates as did samples obtained from children with mild disease. Our findings do not provide any support for the view that the development of severe malaria in a small proportion of African children infected with P. falciparum is due to lack of previous exposure to the infection.