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1.  Exome sequencing: the expert view 
Genome Biology  2011;12(9):128.
To complement our special issue on exome sequencing, Genome Biology asked several leaders in the field for their views on this new approach. Leslie G Biesecker (LGB), Jim C Mullikin (JM) and Kevin V Shianna (KVS) discuss the reasons for the popularity of exome sequencing and its contribution to genomics.
doi:10.1186/gb-2011-12-9-128
PMCID: PMC3308041  PMID: 21920051
2.  A genome-wide association study of resistance to HIV infection in highly exposed uninfected individuals with hemophilia A 
Human Molecular Genetics  2013;22(9):1903-1910.
Human genetic variation contributes to differences in susceptibility to HIV-1 infection. To search for novel host resistance factors, we performed a genome-wide association study (GWAS) in hemophilia patients highly exposed to potentially contaminated factor VIII infusions.
Individuals with hemophilia A and a documented history of factor VIII infusions before the introduction of viral inactivation procedures (1979–1984) were recruited from 36 hemophilia treatment centers (HTCs), and their genome-wide genetic variants were compared with those from matched HIV-infected individuals. Homozygous carriers of known CCR5 resistance mutations were excluded. Single nucleotide polymorphisms (SNPs) and inferred copy number variants (CNVs) were tested using logistic regression. In addition, we performed a pathway enrichment analysis, a heritability analysis, and a search for epistatic interactions with CCR5 Δ32 heterozygosity.
A total of 560 HIV-uninfected cases were recruited: 36 (6.4%) were homozygous for CCR5 Δ32 or m303. After quality control and SNP imputation, we tested 1 081 435 SNPs and 3686 CNVs for association with HIV-1 serostatus in 431 cases and 765 HIV-infected controls. No SNP or CNV reached genome-wide significance. The additional analyses did not reveal any strong genetic effect.
Highly exposed, yet uninfected hemophiliacs form an ideal study group to investigate host resistance factors. Using a genome-wide approach, we did not detect any significant associations between SNPs and HIV-1 susceptibility, indicating that common genetic variants of major effect are unlikely to explain the observed resistance phenotype in this population.
doi:10.1093/hmg/ddt033
PMCID: PMC3613165  PMID: 23372042
3.  Single nucleotide polymorphism upstream of interleukin 28B associated with phase 1 and phase 2 of early viral kinetics in patients infected with HCV genotype 1 
Journal of hepatology  2011;56(3):10.1016/j.jhep.2011.10.004.
Background & Aims
We studied the relationship between IL28B gene-related SNP rs12979860 and early viral kinetics (day 0–28) during peginterferon and ribavirin treatment, in 173 African Americans (AA) and 188 Caucasian Americans (CA) with HCV genotype 1.
Methods
We studied the relationship between IL28B 16 gene-related SNP rs12979860 and early viral kinetics (day 0–28) 17 during peginterferon and ribavirin treatment, in 171 African 18 Americans (AA) and 188 Caucasian Americans (CA) with HCV 19 genotype 1.
Results
Compared to non-C/C genotypes, C/C was associated with greater declines in serum HCV RNA during phase 1 (day 0–2), phase 2 (day 7–28), and day 0–28 and higher response (undetected HCV RNA) rates at weeks 4 and 12 in AA and CA. A static phase and increases in HCV RNA from day 2 to 7 were more common in patients with non-C/C genotypes. C/C was also associated with higher week 24, 48, and 72 response rates in CA (p <0.01) but not in AA. At baseline, SNP genotype was the only independent predictor of phase 1; SNP genotype and phase 1 were independent predictors of phase 2 (p<0.001). There were no racial differences in HCV RNA declines during phase 1, day 2–7, phase 2, and day 0–28 with the same SNP genotype. AA with C/C and C/T genotypes had lower week 24, 48, and 72 (SVR) rates than did CA (p = 0.03). SNP C/C predicted higher SVR rates in AA and CA with high baseline HCV RNA (≥ 600,000 IU/ml), and in CA with ≥ 1 log10 IU/ml decrease in HCV RNA from day 0 to 28.
Conclusions
SNP rs12979860 is strongly associated with both phase 1 and phase 2 HCV RNA kinetics in AA and CA with HCV genotype 1.
doi:10.1016/j.jhep.2011.10.004
PMCID: PMC3884806  PMID: 22027585
IL28B gene; Hepatitis C virus; Viral kinetics; Single nucleotide polymorphism
4.  IL28B genotype is associated with differential expression of intrahepatic interferon-stimulated genes in chronic hepatitis C patients 
Hepatology (Baltimore, Md.)  2010;52(6):1888-1896.
Genetic variation in the IL28B region has been associated with sustained virological response (SVR) rates in chronic hepatitis C (CHC) patients treated with peginterferon-α and ribavirin. We hypothesized that IL28B polymorphism is associated with intrahepatic expression of interferon-stimulated genes (ISGs), known to influence treatment outcome. IL28B genotyping (rs12979860) and whole-genome RNA expression were performed using liver biopsies from 61 North American CHC patients. After correction for multiple testing (false discovery rate < 0.10), 164 transcripts were found to be differentially expressed by IL28B-type. The interferon signaling pathway was the most enriched canonical pathway differentially expressed by IL28B-type (p < 10−5), with most genes showing higher expression in livers of individuals carrying the poor-response IL28B-type. In 25 patients for which treatment response data were available, IL28B-type was associated with SVR (p = 0.0054). ISG expression was also associated with SVR; however, this was not independent of IL28B-type. Analysis of miR-122 expression in liver biopsies showed reduced miR-122 levels associated with poorer treatment outcome, independently of IL28B-type. No association was observed between IL28B-type and levels of liver IL28B or IL28A mRNA expression. IL28B protein sequence variants associated with rs12979860 were therefore investigated in vitro: no differences in ISG induction or inhibition of HCV replication were observed in Huh7.5 cells.
Conclusion
The good response IL28B variant was strongly associated with lower level ISG expression. The results suggest that IL28B genotype may explain the relationship between hepatic ISG expression and HCV treatment outcome, and this is independent of miR-122 expression. IL28B-type was not associated with intrahepatic IL28B mRNA expression in vivo. Further investigation of the precise molecular mechanism(s) by which IL28B genetic variation influences HCV outcomes is warranted.
doi:10.1002/hep.23912
PMCID: PMC3653303  PMID: 20931559
interferon lambda; hepatitis C virus; gene expression
5.  A polymorphism in the HCP5 gene associated with HLA-B*5701 does not restrict HIV-1 in vitro 
AIDS (London, England)  2010;24(1):155-157.
A single-nucleotide polymorphism (rs2395029) in the HCP5 gene associated with HLA-B*5701 is correlated with lower HIV-1 viral set point. The two allelic forms of coding region were ectopically expressed in TZM-bl cells for an effect on HIV-1 replication. No significant HIV-1 restriction was observed in the cells with infectivity assays throughout HIV-1 life cycle, suggesting that the association of HCP5 variant with viral control is likely due to HLA-B*5701-related effect or other functional variants in the haplotype or both.
doi:10.1097/QAD.0b013e32833202f5
PMCID: PMC3652654  PMID: 19935381
6.  Genome-wide association study of interferon-related cytopenia in chronic hepatitis C patients 
Journal of hepatology  2011;56(2):313-319.
Background & Aims
Interferon-alfa (IFN)-related cytopenias are common and may be dose-limiting. We performed a genome wide association study on a well-characterized genotype 1 HCV cohort to identify genetic determinants of peginterferon-α (peg-IFN)-related thrombocytopenia, neutropenia, and leukopenia.
Methods
1604/3070 patients in the IDEAL study consented to genetic testing. Trial inclusion criteria included a platelet (Pl) count ≥80 × 109/L and an absolute neutrophil count (ANC) ≥ 1500/mm3. Samples were genotyped using the Illumina Human610-quad BeadChip. The primary analyses focused on the genetic determinants of quantitative change in cell counts (Pl, ANC, lymphocytes, monocytes, eosinophils, and basophils) at week 4 in patients >80% adherent to therapy (n = 1294).
Results
6 SNPs on chromosome 20 were positively associated with Pl reduction (top SNP rs965469, p = 10−10). These tag SNPs are in high linkage disequilibrium with 2 functional variants in the ITPA gene, rs1127354 and rs7270101, that cause ITPase deficiency and protect against ribavirin (RBV)-induced hemolytic anemia (HA). rs1127354 and rs7270101 showed strong independent associations with Pl reduction (p = 10−12, p = 10−7) and entirely explained the genome-wide significant associations. We believe this is an example of an indirect genetic association due to a reactive thrombocytosis to RBV-induced anemia: Hb decline was inversely correlated with Pl reduction (r = −0.28, p = 10−17) and Hb change largely attenuated the association between the ITPA variants and Pl reduction in regression models. No common genetic variants were associated with pegIFN-induced neutropenia or leucopenia.
Conclusions
Two ITPA variants were associated with thrombocytopenia; this was largely explained by a thrombocytotic response to RBV-induced HA attenuating IFN-related thrombocytopenia. No genetic determinants of pegIFN-induced neutropenia were identified.
doi:10.1016/j.jhep.2011.04.021
PMCID: PMC3634361  PMID: 21703177
GWAS; ITPA; Thrombocytopenia; Hepatitis C; Neutropenia; IL28B
7.  De novo mutations in ATP1A3 cause alternating hemiplegia of childhood 
Heinzen, Erin L. | Swoboda, Kathryn J. | Hitomi, Yuki | Gurrieri, Fiorella | Nicole, Sophie | de Vries, Boukje | Tiziano, F. Danilo | Fontaine, Bertrand | Walley, Nicole M. | Heavin, Sinéad | Panagiotakaki, Eleni | Fiori, Stefania | Abiusi, Emanuela | Di Pietro, Lorena | Sweney, Matthew T. | Newcomb, Tara M. | Viollet, Louis | Huff, Chad | Jorde, Lynn B. | Reyna, Sandra P. | Murphy, Kelley J. | Shianna, Kevin V. | Gumbs, Curtis E. | Little, Latasha | Silver, Kenneth | Ptác̆ek, Louis J. | Haan, Joost | Ferrari, Michel D. | Bye, Ann M. | Herkes, Geoffrey K. | Whitelaw, Charlotte M. | Webb, David | Lynch, Bryan J. | Uldall, Peter | King, Mary D. | Scheffer, Ingrid E. | Neri, Giovanni | Arzimanoglou, Alexis | van den Maagdenberg, Arn M.J.M. | Sisodiya, Sanjay M. | Mikati, Mohamad A. | Goldstein, David B. | Nicole, Sophie | Gurrieri, Fiorella | Neri, Giovanni | de Vries, Boukje | Koelewijn, Stephany | Kamphorst, Jessica | Geilenkirchen, Marije | Pelzer, Nadine | Laan, Laura | Haan, Joost | Ferrari, Michel | van den Maagdenberg, Arn | Zucca, Claudio | Bassi, Maria Teresa | Franchini, Filippo | Vavassori, Rosaria | Giannotta, Melania | Gobbi, Giuseppe | Granata, Tiziana | Nardocci, Nardo | De Grandis, Elisa | Veneselli, Edvige | Stagnaro, Michela | Gurrieri, Fiorella | Neri, Giovanni | Vigevano, Federico | Panagiotakaki, Eleni | Oechsler, Claudia | Arzimanoglou, Alexis | Nicole, Sophie | Giannotta, Melania | Gobbi, Giuseppe | Ninan, Miriam | Neville, Brian | Ebinger, Friedrich | Fons, Carmen | Campistol, Jaume | Kemlink, David | Nevsimalova, Sona | Laan, Laura | Peeters-Scholte, Cacha | van den Maagdenberg, Arn | Casaer, Paul | Casari, Giorgio | Sange, Guenter | Spiel, Georg | Boneschi, Filippo Martinelli | Zucca, Claudio | Bassi, Maria Teresa | Schyns, Tsveta | Crawley, Francis | Poncelin, Dominique | Vavassori, Rosaria
Nature genetics  2012;44(9):1030-1034.
Alternating hemiplegia of childhood (AHC) is a rare, severe neurodevelopmental syndrome characterized by recurrent hemiplegic episodes and distinct neurologic manifestations. AHC is usually a sporadic disorder with unknown etiology. Using exome sequencing of seven patients with AHC, and their unaffected parents, we identified de novo nonsynonymous mutations in ATP1A3 in all seven AHC patients. Subsequent sequence analysis of ATP1A3 in 98 additional patients revealed that 78% of AHC cases have a likely causal ATP1A3 mutation, including one inherited mutation in a familial case of AHC. Remarkably, six ATP1A3 mutations explain the majority of patients, including one observed in 36 patients. Unlike ATP1A3 mutations that cause rapid-onset-dystonia-parkinsonism, AHC-causing mutations revealed consistent reductions in ATPase activity without effects on protein expression. This work identifies de novo ATP1A3 mutations as the primary cause of AHC, and offers insight into disease pathophysiology by expanding the spectrum of phenotypes associated with mutations in this gene.
doi:10.1038/ng.2358
PMCID: PMC3442240  PMID: 22842232
8.  Variants in the ITPA Gene Protect Against Ribavirin-Induced Hemolytic Anemia in HIV/HCV-Coinfected Patients With All HCV Genotypes 
The Journal of Infectious Diseases  2011;205(3):376-383.
Background. A recent genome-wide association study reported a strong association with a single-nucleotide polymorphism (SNP) in the inosine triphosphate (ITPA) gene and hemolytic anemia in patients infected with hepatitis C virus (HCV) receiving pegylated interferon and ribavirin. We investigate these polymorphisms in a cohort of human immunodeficiency virus (HIV)/HCV–coinfected patients.
Methods. DNA was available for 161 patients with validated outcomes. We analyzed the association between the variants and week 4 hemoglobin reduction. Anemia over the course of therapy, ribavirin (RBV) dose reduction, serum RBV level, and rapid virological response (RVR) and sustained virological response (SVR) were also investigated. Using a candidate gene approach, ITPA variants rs1127354 and rs7270101 were tested using the ABI TaqMan kit. Multivariable models were used to identify predictors of anemia.
Results. A significant minority (33%) of patients were predicted to have reduced ITPase activity. The minor allele of each variant was associated with protection against week 4 anemia. In multivariable models only the genetic variants, creatinine, and zidovudine exposure remained significant. ITPase deficiency was not associated with RBV-dose reduction, RVR, or SVR.
Conclusions. This study confirms that polymorphisms in the ITPA gene are associated with protection from RBV-induced anemia in HIV/HCV-coinfected patients but not improved clinical outcomes.
doi:10.1093/infdis/jir754
PMCID: PMC3283113  PMID: 22158703
9.  A whole-genome analysis of premature termination codons 
Genomics  2011;98(5):337-342.
We sequenced the genomes of ten unrelated individuals and identified heterozygous stop gain variants in protein-coding genes: we then sequenced their transcriptomes and assessed the expression levels of the stop gain alleles. An ANOVA showed statistically significant differences between their expression levels (p=4×10-16). This difference was almost entirely accounted for by whether the stop gain variant had a second, non-protein-truncating function in or near an alternate transcript: stop gains without alternate functions were generally not found in the cDNA (p=3×10-5). Additionally, stop gain variants in two intronless genes were not expressed, an unexpected outcome given previous studies. In this study, stop gain variants were either well expressed in all individuals or were never expressed. Our finding that stop gain variants were generally expressed only when they had an alternate function suggests that most naturally occurring stop gain variants in protein-coding genes are either not transcribed or have their transcripts destroyed.
doi:10.1016/j.ygeno.2011.07.001
PMCID: PMC3282586  PMID: 21803148
Nonsense-mediated decay; whole-genome sequencing; RNA-Seq; premature termination codons
10.  Genome-wide mapping for clinically relevant predictors of lamotrigine- and phenytoin-induced hypersensitivity reactions 
Pharmacogenomics  2012;13(4):399-405.
Aims
An association between carbamazepine-induced hypersensitivity and HLA-A*3101 has been reported in populations of both European and Asian descent. We aimed to investigate HLA-A*3101 and other common variants across the genome as markers for cutaneous adverse drug reactions (cADRs) attributed to lamotrigine and phenytoin.
Materials & methods
We recruited patients with lamotrigine-induced cADRs (n = 46) and patients with phenytoin-cADRs (n = 44) and the 1958 British birth cohort was used as a control (n = 1296). HLA-A*3101 was imputed from genome-wide association study data. We applied genome-wide association to study lamotrigine- and phenytoin-induced cADR, and total cADR cases combined.
Results
Neither HLA-A*3101 nor any other genetic marker significantly predicted lamotrigine- or phenytoin-induced cADRs.
Conclusion
HLA-A*3101 does not appear to be a predictor for lamotrigine- and phenytoin-induced cADRs in Europeans. Our genome-wide association study results do not support the existence of a clinically relevant common variant for the development of lamotrigine- or phenytoin-induced cADRs. As a predictive marker, HLA-A*3101 appears to be specific for carbamazepine-induced cADRs.
doi:10.2217/pgs.11.165
PMCID: PMC3428903  PMID: 22379998
epilepsy; GWAS; HLA-A*3101; hypersensitivity; lamotrigine; phenytoin
11.  Cold Urticaria, Immunodeficiency, and Autoimmunity Related to PLCG2 Deletions 
The New England Journal of Medicine  2012;366(4):330-338.
Background
Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance.
Methods
We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing.
Results
Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ2 (PLCγ2), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures.
Conclusions
Genomic deletions in PLCG2 cause gain of PLCγ2 function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.)
doi:10.1056/NEJMoa1102140
PMCID: PMC3298368  PMID: 22236196
12.  Clinical application of exome sequencing in undiagnosed genetic conditions 
Journal of Medical Genetics  2012;49(6):353-361.
Background
There is considerable interest in the use of next-generation sequencing to help diagnose unidentified genetic conditions, but it is difficult to predict the success rate in a clinical setting that includes patients with a broad range of phenotypic presentations.
Methods
The authors present a pilot programme of whole-exome sequencing on 12 patients with unexplained and apparent genetic conditions, along with their unaffected parents. Unlike many previous studies, the authors did not seek patients with similar phenotypes, but rather enrolled any undiagnosed proband with an apparent genetic condition when predetermined criteria were met.
Results
This undertaking resulted in a likely genetic diagnosis in 6 of the 12 probands, including the identification of apparently causal mutations in four genes known to cause Mendelian disease (TCF4, EFTUD2, SCN2A and SMAD4) and one gene related to known Mendelian disease genes (NGLY1). Of particular interest is that at the time of this study, EFTUD2 was not yet known as a Mendelian disease gene but was nominated as a likely cause based on the observation of de novo mutations in two unrelated probands. In a seventh case with multiple disparate clinical features, the authors were able to identify homozygous mutations in EFEMP1 as a likely cause for macular degeneration (though likely not for other features).
Conclusions
This study provides evidence that next-generation sequencing can have high success rates in a clinical setting, but also highlights key challenges. It further suggests that the presentation of known Mendelian conditions may be considerably broader than currently recognised.
doi:10.1136/jmedgenet-2012-100819
PMCID: PMC3375064  PMID: 22581936
Exome sequencing; unidentified genetic conditions; medical genetics; paediatrics; clinical genetics; complex traits; genetic screening/counselling; genetics; genome-wide; psychotic disorders (including schizophrenia); molecular genetics; gastroenterology; immunology (including allergy).
13.  Host Genetic Determinants of T Cell Responses to the MRKAd5 HIV-1 gag/pol/nef Vaccine in the Step Trial 
The Journal of Infectious Diseases  2011;203(6):773-779.
Understanding how human genetic variation impacts individual response to immunogens is fundamental for rational vaccine development. To explore host mechanisms involved in cellular immune responses to the MRKAd5 human immunodeficiency virus type 1 (HIV-1) gag/pol/nef vaccine tested in the Step trial, we performed a genome-wide association study of determinants of HIV-specific T cell responses, measured by interferon γ enzyme-linked immunospot assays. No human genetic variant reached genome-wide significance, but polymorphisms located in the major histocompatibility complex (MHC) region showed the strongest association with response to the HIV-1 Gag protein: HLA-B alleles known to be associated with differences in HIV-1 control were responsible for these associations. The implication of the same HLA alleles in vaccine-induced cellular immunity and in natural immune control is of relevance for vaccine design. Furthermore, our results demonstrate the importance of considering the host immunogenetic background in the analysis of immune responses to T cell vaccines.
doi:10.1093/infdis/jiq125
PMCID: PMC3071133  PMID: 21278214
14.  COMMON HUMAN GENETIC VARIANTS AND HIV-1 SUSCEPTIBILITY: A GENOME-WIDE SURVEY IN A HOMOGENEOUS AFRICAN POPULATION 
AIDS (London, England)  2011;25(4):513-518.
To date, CCR5 variants remain the only human genetic factors to be confirmed to impact HIV-1 acquisition. However, protective CCR5 variants are largely absent in African populations, in which sporadic resistance to HIV-1 infection is still unexplained. Here we perform a genome-wide association study (GWAS) in a population of 1,532 individuals from Malawi, a country with high prevalence of HIV-1 infection, to investigate whether common genetic variants associate with HIV-1 susceptibility in Africans. Using single nucleotide polymorphisms (SNPs) present on the genome-wide chip, we also investigated previously reported associations with HIV-1 susceptibility or acquisition. Recruitment was coordinated by the Center for HIV/AIDS Vaccine Immunology at two sexually transmitted infection clinics. HIV status was determined by HIV rapid tests and nucleic acid testing.
After quality control, the population consisted of 848 high-risk seronegative and 531 HIV-1 seropositive individuals. Logistic regression testing in an additive genetic model was performed for SNPs that passed quality control. No single SNP yielded a significant P-value after correction for multiple testing. The study was sufficiently powered to detect markers with genotype relative risk ≥ 2.0 and minor allele frequencies ≥12%. This is the first GWAS of host determinants of HIV-1 susceptibility, performed in an African population. The absence of any significant association can have many possible explanations: rarer genetic variants or common variants with weaker effect could be responsible for the resistance phenotype; alternatively, resistance to HIV-1 infection might be due to non-genetic parameters or to complex interactions between genes, immunity and environment.
doi:10.1097/QAD.0b013e328343817b
PMCID: PMC3150594  PMID: 21160409
Human immunodeficiency virus (HIV-1); acquisition; resistance; Genome Wide Association Study (GWAS); Africa
15.  Quantitation of Pretreatment Serum IP-10 Improves the Predictive Value of an IL28B Gene Polymorphism for Hepatitis C Treatment Response 
Hepatology (Baltimore, Md.)  2011;53(1):14-22.
Polymorphisms of IL28B gene are highly associated with sustained virological response (SVR) in patients with chronic hepatitis C treated with peginterferon and ribavirin. Quantitation of Interferon-γ Inducible Protein-10 (IP-10) may also differentiate antiviral response. We evaluated IP-10 levels in pretreatment serum from 115 non-responders and 157 sustained responders in the VIRAHEP-C cohort, including African Americans (AA) and Caucasian Americans (CA). Mean IP-10 was lower in sustained responders compared to non-responders (460 ± 37 pg/ml vs 697 ± 49 pg/ml, p<0.001), both in AA and CA. The positive predictive value of low IP-10 levels (<600 pg/ml) for SVR was 69% while the negative predictive value of high IP-10 levels (>600 pg/ml) was 67%. We assessed the combination of pretreatment IP-10 levels with IL28B genotype as predictors of treatment response. The IL28B polymorphism rs12979860 was tested in 210 participants. CC, CT, or TT genotypes were found in 30%, 49%, and 21%, respectively, with corresponding SVR rates of 87%, 50%, and 39% (p<0.0001). Serum IP-10 levels within the IL28B genotype groups provided additional information regarding the likelihood of SVR (p< 0.0001). CT carriers with low IP-10 had 64% SVR versus 24% with high IP-10. Similarly, a higher SVR rate was identified for TT and CC carriers with low versus high IP-10 (TT: 48% versus 20%, CC: 89% versus 79%). IL28B genotype and baseline IP-10 levels were additive but independent when predicting SVR in both AA and CA.
Conclusions
When IL28B genotype is combined with pretreatment serum IP-10 measurement, the predictive value for discrimination between SVR and non-response is significantly improved, especially in non-CC genotypes. This relationship warrants further investigation to elucidate mechanisms of antiviral response and prospective validation.
doi:10.1002/hep.24056
PMCID: PMC3083026  PMID: 21254158
Peginterferon; Ribavirin; Race; Biomarker; Genotype
17.  Copy Number Variation of KIR Genes Influences HIV-1 Control 
PLoS Biology  2011;9(11):e1001208.
The authors that the number of activating and inhibitory KIR genes varies between individuals and plays a role in the regulation of immune mechanisms that determine HIV-1 control.
A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection.
Author Summary
There is marked intrinsic variation in the extent to which individuals are able to control HIV-1. We have identified a genetic copy number variable region (CNV) in humans that plays a significant role in the control of HIV-1. This CNV is located in the genomic region that encodes the killer cell immunoglobulin-like receptors (KIRs) and specifically affects the KIR3DS1 and KIR3DL1 genes, encoding two KIRs that interact with human leukocyte antigen B (HLA-B) ligands. KIRs are expressed on the surface of natural killer (NK) cells, which serve as important players in the innate immune response, and are involved in the recognition of infected and malignant cells through a loss or alteration in “self” ligands. We use both genetic association and functional evidence to show a strong interaction between KIR3DL1 and KIR3DS1, indicating that increasing gene counts for KIR3DL1 confer increasing levels of protection against HIV-1, but only in the presence of at least one copy of KIR3DS1. This effect was associated with a dramatic increase in the abundance of KIR3DS1+ NK cells in the peripheral blood, and strongly associated with a more robust capacity of peripheral NK cells to suppress HIV-1 replication in vitro. This work provides one of the few examples of an association between a relatively common CNV and a human complex trait.
doi:10.1371/journal.pbio.1001208
PMCID: PMC3226550  PMID: 22140359
18.  HLA-A★3101 and Carbamazepine-Induced Hypersensitivity Reactions in Europeans 
The New England journal of medicine  2011;364(12):1134-1143.
BACKGROUND
Carbamazepine causes various forms of hypersensitivity reactions, ranging from maculopapular exanthema to severe blistering reactions. The HLA-B★1502 allele has been shown to be strongly correlated with carbamazepine-induced Stevens–Johnson syndrome and toxic epidermal necrolysis (SJS–TEN) in the Han Chinese and other Asian populations but not in European populations.
METHODS
We performed a genomewide association study of samples obtained from 22 subjects with carbamazepine-induced hypersensitivity syndrome, 43 subjects with carbamazepine-induced maculopapular exanthema, and 3987 control subjects, all of European descent. We tested for an association between disease and HLA alleles through proxy single-nucleotide polymorphisms and imputation, confirming associations by high-resolution sequence-based HLA typing. We replicated the associations in samples from 145 subjects with carbamazepine-induced hypersensitivity reactions.
RESULTS
The HLA-A★3101 allele, which has a prevalence of 2 to 5% in Northern European populations, was significantly associated with the hypersensitivity syndrome (P = 3.5×10−8). An independent genomewide association study of samples from subjects with maculopapular exanthema also showed an association with the HLA-A★3101 allele (P = 1.1×10−6). Follow-up genotyping confirmed the variant as a risk factor for the hypersensitivity syndrome (odds ratio, 12.41; 95% confidence interval [CI], 1.27 to 121.03), maculopapular exanthema (odds ratio, 8.33; 95% CI, 3.59 to 19.36), and SJS–TEN (odds ratio, 25.93; 95% CI, 4.93 to 116.18).
CONCLUSIONS
The presence of the HLA-A★3101 allele was associated with carbamazepine-induced hypersensitivity reactions among subjects of Northern European ancestry. The presence of the allele increased the risk from 5.0% to 26.0%, whereas its absence reduced the risk from 5.0% to 3.8%. (Funded by the U.K. Department of Health and others.)
doi:10.1056/NEJMoa1013297
PMCID: PMC3113609  PMID: 21428769
19.  SVA: software for annotating and visualizing sequenced human genomes 
Bioinformatics  2011;27(14):1998-2000.
Summary: Here we present Sequence Variant Analyzer (SVA), a software tool that assigns a predicted biological function to variants identified in next-generation sequencing studies and provides a browser to visualize the variants in their genomic contexts. SVA also provides for flexible interaction with software implementing variant association tests allowing users to consider both the bioinformatic annotation of identified variants and the strength of their associations with studied traits. We illustrate the annotation features of SVA using two simple examples of sequenced genomes that harbor Mendelian mutations.
Availability and implementation: Freely available on the web at http://www.svaproject.org.
Contact: d.ge@duke.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btr317
PMCID: PMC3129530  PMID: 21624899
20.  Variants in the ITPA Gene Protect Against Ribavirin-Induced Hemolytic Anemia and Decrease the Need for Ribavirin Dose Reduction 
Gastroenterology  2010;139(4):1181-1189.
BACKGROUND & AIMS
In a genome-wide association study of patients being treated for chronic hepatitis C, 2 functional variants in ITPA that cause inosine triphosphatase (ITPase) deficiency were shown to protect against ribavirin (RBV)-induced hemolytic anemia during early stages of treatment. We aimed to replicate this finding in an independent cohort from the Study of Viral Resistance to Antiviral Therapy of Chronic Hepatitis C and to investigate the effects of these variants beyond week 4.
METHODS
Genetic material was available from 318 patients. The ITPA variants, rs1127354 (exon 2, P32T) and rs7270101 (intron 2, splice altering), were genotyped and tested for association with hemoglobin (Hb) reduction at week 4. An ITPase deficiency variable was defined that combined both ITPA variants according to documented effect on ITPase activity. We investigated the impact of ITPA variants on Hb levels over the course of therapy and on the need for RBV dose reduction.
RESULTS
The final analysis included 304 patients with genotype 1 hepatitis C virus (167 white patients and 137 black patients). The polymorphisms rs1127354 and rs7270101 were associated with Hb reduction at week 4 (P = 3.1 × 10−13 and 1.3 × 10−3, respectively). The minor alleles of each variant protected against Hb reduction. Combining the variants into the ITPase deficiency variable strengthened the association (P = 2.4 × 10−18). The ITPase deficiency variable was associated with lower rates of anemia over the entire treatment period (48 weeks), as well as a lower rate of anemia-related RBV dose reduction (hazard ratio, 0.52; P = .0037). No association with sustained virological response was observed.
CONCLUSIONS
Two polymorphisms that cause ITPase deficiency are strongly associated with protection from RBV-induced hemolytic anemia and decrease the need for RBV dose reduction.
doi:10.1053/j.gastro.2010.06.016
PMCID: PMC3086671  PMID: 20547162
Pharmacogenomics; Genome-Wide Association Study; Polymorphism; Single Nucleotide Polymorphism; HCV; Adverse Event
21.  Host determinants of HIV-1 control in African Americans 
The Journal of infectious diseases  2010;201(8):1141-1149.
We performed a whole-genome association study on HIV-1 viral load setpoint in an African American cohort (n=515), and an intronic SNP in the HLA-B gene showed one of the strongest associations. Using a subset of patients, we show that this SNP reflects the effect of the HLA-B*5703 allele, which shows a genome-wide significant association with HIV-1 VL setpoint (p=5.6×10−10). These analyses therefore confirm a member of the HLA-B*57 group of alleles as the most important common variant influencing viral load variation in African Americans, consistent with what is observed in individuals of European ancestry in which the most important common variant is HLA-B*5701.
doi:10.1086/651382
PMCID: PMC2838940  PMID: 20205591
HIV; viral load setpoint; host genetics; association study; HLA
22.  Common variants conferring risk of schizophrenia 
Stefansson, Hreinn | Ophoff, Roel A. | Steinberg, Stacy | Andreassen, Ole A. | Cichon, Sven | Rujescu, Dan | Werge, Thomas | Pietiläinen, Olli P. H. | Mors, Ole | Mortensen, Preben B. | Sigurdsson, Engilbert | Gustafsson, Omar | Nyegaard, Mette | Tuulio-Henriksson, Annamari | Ingason, Andres | Hansen, Thomas | Suvisaari, Jaana | Lonnqvist, Jouko | Paunio, Tiina | Børglum, Anders D. | Hartmann, Annette | Fink-Jensen, Anders | Nordentoft, Merete | Hougaard, David | Norgaard-Pedersen, Bent | Böttcher, Yvonne | Olesen, Jes | Breuer, René | Möller, Hans-Jürgen | Giegling, Ina | Rasmussen, Henrik B. | Timm, Sally | Mattheisen, Manuel | Bitter, István | Réthelyi, János M. | Magnusdottir, Brynja B. | Sigmundsson, Thordur | Olason, Pall | Masson, Gisli | Gulcher, Jeffrey R. | Haraldsson, Magnus | Fossdal, Ragnheidur | Thorgeirsson, Thorgeir E. | Thorsteinsdottir, Unnur | Ruggeri, Mirella | Tosato, Sarah | Franke, Barbara | Strengman, Eric | Kiemeney, Lambertus A. | Melle, Ingrid | Djurovic, Srdjan | Abramova, Lilia | Kaleda, Vasily | Sanjuan, Julio | de Frutos, Rosa | Bramon, Elvira | Vassos, Evangelos | Fraser, Gillian | Ettinger, Ulrich | Picchioni, Marco | Walker, Nicholas | Toulopoulou, Timi | Need, Anna C. | Ge, Dongliang | Yoon, Joeng Lim | Shianna, Kevin V. | Freimer, Nelson B. | Cantor, Rita M. | Murray, Robin | Kong, Augustine | Golimbet, Vera | Carracedo, Angel | Arango, Celso | Costas, Javier | Jönsson, Erik G. | Terenius, Lars | Agartz, Ingrid | Petursson, Hannes | Nöthen, Markus M. | Rietschel, Marcella | Matthews, Paul M. | Muglia, Pierandrea | Peltonen, Leena | St Clair, David | Goldstein, David B. | Stefansson, Kari | Collier, David A.
Nature  2009;460(7256):744-747.
Schizophrenia is a complex disorder, caused by both genetic and environmental factors and their interactions. Research on pathogenesis has traditionally focused on neurotransmitter systems in the brain, particularly those involving dopamine. Schizophrenia has been considered a separate disease for over a century, but in the absence of clear biological markers, diagnosis has historically been based on signs and symptoms. A fundamental message emerging from genome-wide association studies of copy number variations (CNVs) associated with the disease is that its genetic basis does not necessarily conform to classical nosological disease boundaries. Certain CNVs confer not only high relative risk of schizophrenia but also of other psychiatric disorders1–3. The structural variations associated with schizophrenia can involve several genes and the phenotypic syndromes, or the ‘genomic disorders’, have not yet been characterized4. Single nucleotide polymorphism (SNP)-based genome-wide association studies with the potential to implicate individual genes in complex diseases may reveal underlying biological pathways. Here we combined SNP data from several large genome-wide scans and followed up the most significant association signals. We found significant association with several markers spanning the major histocompatibility complex (MHC) region on chromosome 6p21.3-22.1, a marker located upstream of the neurogranin gene (NRGN) on 11q24.2 and a marker in intron four of transcription factor 4 (TCF4) on 18q21.2. Our findings implicating the MHC region are consistent with an immune component to schizophrenia risk, whereas the association with NRGN and TCF4 points to perturbation of pathways involved in brain development, memory and cognition.
doi:10.1038/nature08186
PMCID: PMC3077530  PMID: 19571808
23.  A genome-wide study of common SNPs and CNVs in cognitive performance in the CANTAB 
Human Molecular Genetics  2009;18(23):4650-4661.
Psychiatric disorders such as schizophrenia are commonly accompanied by cognitive impairments that are treatment resistant and crucial to functional outcome. There has been great interest in studying cognitive measures as endophenotypes for psychiatric disorders, with the hope that their genetic basis will be clearer. To investigate this, we performed a genome-wide association study involving 11 cognitive phenotypes from the Cambridge Neuropsychological Test Automated Battery. We showed these measures to be heritable by comparing the correlation in 100 monozygotic and 100 dizygotic twin pairs. The full battery was tested in ∼750 subjects, and for spatial and verbal recognition memory, we investigated a further 500 individuals to search for smaller genetic effects. We were unable to find any genome-wide significant associations with either SNPs or common copy number variants. Nor could we formally replicate any polymorphism that has been previously associated with cognition, although we found a weak signal of lower than expected P-values for variants in a set of 10 candidate genes. We additionally investigated SNPs in genomic loci that have been shown to harbor rare variants that associate with neuropsychiatric disorders, to see if they showed any suggestion of association when considered as a separate set. Only NRXN1 showed evidence of significant association with cognition. These results suggest that common genetic variation does not strongly influence cognition in healthy subjects and that cognitive measures do not represent a more tractable genetic trait than clinical endpoints such as schizophrenia. We discuss a possible role for rare variation in cognitive genomics.
doi:10.1093/hmg/ddp413
PMCID: PMC2773267  PMID: 19734545
24.  Host Genetics and HIV-1: The Final Phase? 
PLoS Pathogens  2010;6(10):e1001033.
This is a crucial transition time for human genetics in general, and for HIV host genetics in particular. After years of equivocal results from candidate gene analyses, several genome-wide association studies have been published that looked at plasma viral load or disease progression. Results from other studies that used various large-scale approaches (siRNA screens, transcriptome or proteome analysis, comparative genomics) have also shed new light on retroviral pathogenesis. However, most of the inter-individual variability in response to HIV-1 infection remains to be explained: genome resequencing and systems biology approaches are now required to progress toward a better understanding of the complex interactions between HIV-1 and its human host.
doi:10.1371/journal.ppat.1001033
PMCID: PMC2954832  PMID: 20976252
25.  The Characterization of Twenty Sequenced Human Genomes 
PLoS Genetics  2010;6(9):e1001111.
We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten “case” genomes from individuals with severe hemophilia A and ten “control” genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs) discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways.
Author Summary
We report here the nearly complete genomic sequence of 20 different individuals, determined using “next-generation” sequencing technologies. We use these data to characterize the type of genetic variation carried by humans in a sample of this size, which is to our knowledge the largest set of unrelated genomic sequences that have been reported. We summarize different categories of variation in each genome, and in total across all 20 of the genomes, finding a surprising number of variants predicted to reduce or remove the proteins encoded by many different genes. This work provides important fundamental information about the scope of human genetic variation, and suggests ways to further explore the relationship between these genetic variants and human disease.
doi:10.1371/journal.pgen.1001111
PMCID: PMC2936541  PMID: 20838461

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