Contribution of genome-wide significant single-nucleotide polymorphisms and antiretroviral therapy to dyslipidemia in HIV-infected individuals: a longitudinal study

BACKGROUND: HIV-infected individuals have an increased risk of myocardial infarction. Antiretroviral therapy (ART) is regarded as a major determinant of dyslipidemia in HIV-infected individuals. Previous genetic studies have been limited by the validity of the single-nucleotide polymorphisms (SNPs) interrogated and by cross-sectional design. Recent genome-wide association studies have reliably associated common SNPs to dyslipidemia in the general population. METHODS AND RESULTS: We validated the contribution of 42 SNPs (33 identified in genome-wide association studies and 9 previously reported SNPs not included in genome-wide association study chips) and of longitudinally measured key nongenetic variables (ART, underlying conditions, sex, age, ethnicity, and HIV disease parameters) to dyslipidemia in 745 HIV-infected study participants (n=34 565 lipid measurements; median follow-up, 7.6 years). The relative impact of SNPs and ART to lipid variation in the study population and their cumulative influence on sustained dyslipidemia at the level of the individual were calculated. SNPs were associated with lipid changes consistent with genome-wide association study estimates. SNPs explained up to 7.6% (non-high-density lipoprotein cholesterol), 6.2% (high-density lipoprotein cholesterol), and 6.8% (triglycerides) of lipid variation; ART explained 3.9% (non-high-density lipoprotein cholesterol), 1.5% (high-density lipoprotein cholesterol), and 6.2% (triglycerides). An individual with the most dyslipidemic antiretroviral and genetic background had an approximately 3- to 5-fold increased risk of sustained dyslipidemia compared with an individual with the least dyslipidemic therapy and genetic background. CONCLUSIONS: In the HIV-infected population treated with ART, the weight of the contribution of common SNPs and ART to dyslipidemia was similar. When selecting an ART regimen, genetic information should be considered in addition to the dyslipidemic effects of ART agents.

Host genetic determinants of spontaneous hepatitis C clearance

Acute infection with the hepatitis C virus (HCV) induces a wide range of innate and adaptive immune responses. A total of 20-50% of acutely HCV-infected individuals permanently control the virus, referred to as 'spontaneous hepatitis C clearance', while the infection progresses to chronic hepatitis C in the majority of cases. Numerous studies have examined host genetic determinants of hepatitis C infection outcome and revealed the influence of genetic polymorphisms of human leukocyte antigens, killer immunoglobulin-like receptors, chemokines, interleukins and interferon-stimulated genes on spontaneous hepatitis C clearance. However, most genetic associations were not confirmed in independent cohorts, revealed opposing results in diverse populations or were limited by varying definitions of hepatitis C outcomes or small sample size. Coordinated efforts are needed in the search for key genetic determinants of spontaneous hepatitis C clearance that include well-conducted candidate genetic and genome-wide association studies, direct sequencing and follow-up functional studies.

The EmsB tandemly repeated multilocus microsatellite: a new tool to investigate genetic diversity of Echinococcus granulosus sensu lato

Cystic echinococcosis (CE) is a widespread and severe zoonotic disease caused by infection with the larval stage of the eucestode Echinococcus granulosus sensu lato. The polymorphism exhibited by nuclear and mitochondrial markers conventionally used for the genotyping of different parasite species and strains does not reach the level necessary for the identification of genetic variants linked to restricted geographical areas. EmsB is a tandemly repeated multilocus microsatellite that proved its usefulness for the study of genetic polymorphisms within the species E. multilocularis, the causative agent of alveolar echinococcosis. In the present study, EmsB was used to characterize E. granulosus sensu lato samples collected from different host species (sheep, cattle, dromedaries, dogs, and human patients) originating from six different countries (Algeria, Mauritania, Romania, Serbia, Brazil, and the People's Republic of China). The conventional mitochondrial cox1 and nad1 markers identified genotypes G1, G3, G5, G6, and G7, which are clustered into three groups corresponding to the species E. granulosus sensu stricto, E. ortleppi, and E. canadensis. With the same samples, EmsB provided a higher degree of genetic discrimination and identified variations that correlated with the relatively small-scale geographic origins of the samples. In addition, one of the Brazilian single hydatid cysts presented a hybrid genotypic profile that suggested genetic exchanges between E. granulosus sensu stricto and E. ortleppi. In summary, the EmsB microsatellite exhibits an interesting potential for the elaboration of a detailed map of the distribution of genetic variants and therefore for the determination and tracking of the source of CE.

Clinical and genetic characteristics of long QT syndrome

Long QT syndrome (LQTS) is an arrhythmogenic ion channel disorder characterized by severely abnormal ventricular repolarization, which results in prolongation of the electrocardiographic QT interval. The condition is associated with sudden cardiac death due to malignant ventricular arrhythmias similar in form to the hallmark torsade de pointes. Eleven years after the identification of the principle cardiac channels involved in the condition, hundreds of mutations in, to date, 10 genes have been associated with the syndrome. Genetic investigations carried out up until the present have shown that, although the severe form of the disease is sporadic, there are a number of common polymorphisms in genes associated with the condition that may confer susceptibility to the development of torsade de pointes in some individuals, particularly when specific drugs are being administered. Moreover, some polymorphisms have been shown to have regulatory properties that either enhance or counteract a particular mutation's impact. Understanding of the molecular processes underlying the syndrome has enabled treatment to be optimized and has led to better survival among sufferers, thereby demonstrating a key correspondence between genotype, phenotype and therapy. Despite these developments, a quarter of patients do not have mutations in the genes identified to date. Consequently, LQTS continues to be an area of active research. This article contains a summary of the main clinical and genetic developments concerning the syndrome that have taken place during the last decade.

A genome-to-genome analysis of associations between human genetic variation, HIV-1 sequence diversity, and viral control

HIV-1 sequence diversity is affected by selection pressures arising from host genomic factors. Using paired human and viral data from 1071 individuals, we ran >3000 genome-wide scans, testing for associations between host DNA polymorphisms, HIV-1 sequence variation and plasma viral load (VL), while considering human and viral population structure. We observed significant human SNP associations to a total of 48 HIV-1 amino acid variants (p<2.4 × 10−12). All associated SNPs mapped to the HLA class I region. Clinical relevance of host and pathogen variation was assessed using VL results. We identified two critical advantages to the use of viral variation for identifying host factors: (1) association signals are much stronger for HIV-1 sequence variants than VL, reflecting the ‘intermediate phenotype’ nature of viral variation; (2) association testing can be run without any clinical data. The proposed genome-to-genome approach highlights sites of genomic conflict and is a strategy generally applicable to studies of host–pathogen interaction.

Contribution of Genetic Background, Traditional Risk Factors, and HIV-Related Factors to Coronary Artery Disease Events in HIV-Positive Persons

Background Persons infected with human immunodeficiency virus (HIV) have increased rates of coronary artery disease (CAD). The relative contribution of genetic background, HIV-related factors, antiretroviral medications, and traditional risk factors to CAD has not been fully evaluated in the setting of HIV infection. Methods In the general population, 23 common single-nucleotide polymorphisms (SNPs) were shown to be associated with CAD through genome-wide association analysis. Using the Metabochip, we genotyped 1875 HIV-positive, white individuals enrolled in 24 HIV observational studies, including 571 participants with a first CAD event during the 9-year study period and 1304 controls matched on sex and cohort. Results A genetic risk score built from 23 CAD-associated SNPs contributed significantly to CAD (P = 2.9×10−4). In the final multivariable model, participants with an unfavorable genetic background (top genetic score quartile) had a CAD odds ratio (OR) of 1.47 (95% confidence interval [CI], 1.05–2.04). This effect was similar to hypertension (OR = 1.36; 95% CI, 1.06–1.73), hypercholesterolemia (OR = 1.51; 95% CI, 1.16–1.96), diabetes (OR = 1.66; 95% CI, 1.10–2.49), ≥1 year lopinavir exposure (OR = 1.36; 95% CI, 1.06–1.73), and current abacavir treatment (OR = 1.56; 95% CI, 1.17–2.07). The effect of the genetic risk score was additive to the effect of nongenetic CAD risk factors, and did not change after adjustment for family history of CAD. Conclusions In the setting of HIV infection, the effect of an unfavorable genetic background was similar to traditional CAD risk factors and certain adverse antiretroviral exposures. Genetic testing may provide prognostic information complementary to family history of CAD.

Origins of Shared Genetic Variation in African Cichlids

Cichlid fishes have evolved tremendous morphological and behavioral diversity in the waters of East Africa. Within each of the Great Lakes Tanganyika, Malawi, and Victoria, the phenomena of hybridization and retention of ancestral polymorphism explain allele sharing across species. Here, we explore the sharing of single nucleotide polymorphisms (SNPs) between the major East African cichlid assemblages. A set of approximately 200 genic and nongenic SNPs was ascertained in five Lake Malawi species and genotyped in a diverse collection of 160 species from across Africa. We observed segregating polymorphism outside of the Malawi lineage for more than 50% of these loci; this holds similarly for genic versus nongenic SNPs, as well as for SNPs at putative CpG versus non-CpG sites. Bayesian and principal component analyses of genetic structure in the data demonstrate that the Lake Malawi endemic flock is not monophyletic and that river species have likely contributed significantly to Malawi genomes. Coalescent simulations support the hypothesis that river cichlids have transported polymorphism between lake assemblages. We observed strong genetic differentiation between Malawi lineages for approximately 8% of loci, with contributions from both genic and nongenic SNPs. Notably, more than half of these outlier loci between Malawi groups are polymorphic outside of the lake. Cichlid fishes have evolved diversity in Lake Malawi as new mutations combined with standing genetic variation shared across East Africa.

Genetic testing for TMEM154 mutations associated with lentivirus susceptibility in sheep.

In sheep, small ruminant lentiviruses cause an incurable, progressive, lymphoproliferative disease that affects millions of animals worldwide. Known as ovine progressive pneumonia virus (OPPV) in the U.S., and Visna/Maedi virus (VMV) elsewhere, these viruses reduce an animal's health, productivity, and lifespan. Genetic variation in the ovine transmembrane protein 154 gene (TMEM154) has been previously associated with OPPV infection in U.S. sheep. Sheep with the ancestral TMEM154 haplotype encoding glutamate (E) at position 35, and either form of an N70I variant, were highly-susceptible compared to sheep homozygous for the K35 missense mutation. Our current overall aim was to characterize TMEM154 in sheep from around the world to develop an efficient genetic test for reduced susceptibility. The average frequency of TMEM154 E35 among 74 breeds was 0.51 and indicated that highly-susceptible alleles were present in most breeds around the world. Analysis of whole genome sequences from an international panel of 75 sheep revealed more than 1,300 previously unreported polymorphisms in a 62 kb region containing TMEM154 and confirmed that the most susceptible haplotypes were distributed worldwide. Novel missense mutations were discovered in the signal peptide (A13V) and the extracellular domains (E31Q, I74F, and I102T) of TMEM154. A matrix-assisted laser desorption/ionization-time-of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect these and six previously reported missense and two deletion mutations in TMEM154. In blinded trials, the call rate for the eight most common coding polymorphisms was 99.4% for 499 sheep tested and 96.0% of the animals were assigned paired TMEM154 haplotypes (i.e., diplotypes). The widespread distribution of highly-susceptible TMEM154 alleles suggests that genetic testing and selection may improve the health and productivity of infected flocks.

Higher frequency of genetic variants conferring increased risk for ADRs for commonly used drugs treating cancer, AIDS and tuberculosis in persons of African descent

There is established clinical evidence for differences in drug response, cure rates and survival outcomes between different ethnic populations, but the causes are poorly understood. Differences in frequencies of functional genetic variants in key drug response and metabolism genes may significantly influence drug response differences in different populations. To assess this, we genotyped 1330 individuals of African (n=372) and European (n=958) descent for 4535 single-nucleotide polymorphisms in 350 key drug absorption, distribution, metabolism, elimination and toxicity genes. Important and remarkable differences in the distribution of genetic variants were observed between Africans and Europeans and among the African populations. These could translate into significant differences in drug efficacy and safety profiles, and also in the required dose to achieve the desired therapeutic effect in different populations. Our data points to the need for population-specific genetic variation in personalizing medicine and care.

Nuclear markers reveal unexpected genetic variation and a Congolese-Nilotic origin of the Lake Victoria cichlid species flock

Phylogenetic analyses based on mitochondrial (mt) DNA have indicated that the cichlid species flock of the Lake Victoria region is derived from a single ancestral species found in East African rivers, closely related to the ancestor of the Lake Malawi cichlid species flock. The Lake Victoria flock contains ten times less mtDNA variation than the Lake Malawi radiation, consistent with current estimates of the ages of the lakes. We present results of a phylogenetic investigation using nuclear (amplified fragment length polymorphism) markers and a wider coverage of riverine haplochromines. We demonstrate that the Lake Victoria–Edward flock is derived from the morphologically and ecologically diverse cichlid genus Thoracochromis from the Congo and Nile, rather than from the phenotypically conservative East African Astatotilapia. This implies that the ability to express much of the morphological diversity found in the species flock may by far pre–date the origin of the flock. Our data indicate that the nuclear diversity of the Lake Victoria–Edward species flock is similar to that of the Lake Malawi flock, indicating that the genetic diversity is considerably older than the 15 000 years that have passed since the lake began to refill. Most of this variation is manifested in trans–species polymorphisms, indicating very recent cladogenesis from a genetically very diverse founder stock. Our data do not confirm strict monophyly of either of the species flocks, but raise the possibility that these flocks have arisen from hybrid swarms.

Genetic and microbial factors modulating the ubiquitin proteasome system in inflammatory bowel disease

OBJECTIVE: Altered microbiota composition, changes in immune responses and impaired intestinal barrier functions are observed in IBD. Most of these features are controlled by proteases and their inhibitors to maintain gut homeostasis. Unrestrained or excessive proteolysis can lead to pathological gastrointestinal conditions. The aim was to validate the identified protease IBD candidates from a previously performed systematic review through a genetic association study and functional follow-up. DESIGN: We performed a genetic association study in a large multicentre cohort of patients with Crohn's disease (CD) and UC from five European IBD referral centres in a total of 2320 CD patients, 2112 UC patients and 1796 healthy controls. Subsequently, we did an extensive functional assessment of the candidate genes to explore their causality in IBD pathogenesis. RESULTS: Ten single nucleotide polymorphisms (SNPs) in four genes were significantly associated with CD: CYLD, USP40, APEH and USP3. CYLD was the most significant gene with the intronically located rs12324931 the strongest associated SNP (pFDR=1.74e-17, OR=2.24 (1.83 to 2.74)). Five SNPs in four genes were significantly associated with UC: USP40, APEH, DAG1 and USP3. CYLD, as well as some of the other associated genes, is part of the ubiquitin proteasome system (UPS). We therefore determined if the IBD-associated adherent-invasive Escherichia coli (AIEC) can modulate the UPS functioning. Infection of intestinal epithelial cells with the AIEC LF82 reference strain modulated the UPS turnover by reducing poly-ubiquitin conjugate accumulation, increasing 26S proteasome activities and decreasing protein levels of the NF-κB regulator CYLD. This resulted in IκB-α degradation and NF-κB activation. This activity was very important for the pathogenicity of AIEC since decreased CYLD resulted in increased ability of AIEC LF82 to replicate intracellularly. CONCLUSIONS: Our results reveal the UPS, and CYLD specifically, as an important contributor to IBD pathogenesis, which is favoured by both genetic and microbial factors.

Genetic Analyses Reveal a Role for Vitamin D Insufficiency in HCV-Associated Hepatocellular Carcinoma Development

Background Vitamin D insufficiency has been associated with the occurrence of various types of cancer, but causal relationships remain elusive. We therefore aimed to determine the relationship between genetic determinants of vitamin D serum levels and the risk of developing hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). Methodology/Principal Findings Associations between CYP2R1, GC, and DHCR7 genotypes that are determinants of reduced 25-hydroxyvitamin D (25[OH]D3) serum levels and the risk of HCV-related HCC development were investigated for 1279 chronic hepatitis C patients with HCC and 4325 without HCC, respectively. The well-known associations between CYP2R1 (rs1993116, rs10741657), GC (rs2282679), and DHCR7 (rs7944926, rs12785878) genotypes and 25(OH)D3 serum levels were also apparent in patients with chronic hepatitis C. The same genotypes of these single nucleotide polymorphisms (SNPs) that are associated with reduced 25(OH)D3 serum levels were found to be associated with HCV-related HCC (P = 0.07 [OR = 1.13, 95% CI = 0.99–1.28] for CYP2R1, P = 0.007 [OR = 1.56, 95% CI = 1.12–2.15] for GC, P = 0.003 [OR = 1.42, 95% CI = 1.13–1.78] for DHCR7; ORs for risk genotypes). In contrast, no association between these genetic variations and liver fibrosis progression rate (P>0.2 for each SNP) or outcome of standard therapy with pegylated interferon-α and ribavirin (P>0.2 for each SNP) was observed, suggesting a specific influence of the genetic determinants of 25(OH)D3 serum levels on hepatocarcinogenesis. Conclusions/Significance Our data suggest a relatively weak but functionally relevant role for vitamin D in the prevention of HCV-related hepatocarcinogenesis.

CYP3A5*3 and POR*28 genetic variants influence the required dose of tacrolimus in heart transplant recipients

BACKGROUND After heart transplantation (HTx), the interindividual pharmacokinetic variability of immunosuppressive drugs represents a major therapeutic challenge due to the narrow therapeutic window between over-immunosuppression causing toxicity and under-immunosuppression leading to graft rejection. Although genetic polymorphisms have been shown to influence pharmacokinetics of immunosuppressants, data in the context of HTx are scarce. We thus assessed the role of genetic variation in CYP3A4, CYP3A5, POR, NR1I2, and ABCB1 acting jointly in immunosuppressive drug pathways in tacrolimus (TAC) and ciclosporin (CSA) dose requirement in HTx recipients. METHODS Associations between 7 functional genetic variants and blood dose-adjusted trough (C0) concentrations of TAC and CSA at 1, 3, 6, and 12 months after HTx were evaluated in cohorts of 52 and 45 patients, respectively. RESULTS Compared with CYP3A5 nonexpressors (*3/*3 genotype), CYP3A5 expressors (*1/*3 or *1/*1 genotype) required around 2.2- to 2.6-fold higher daily TAC doses to reach the targeted C0 concentration at all studied time points (P ≤ 0.003). Additionally, the POR*28 variant carriers showed higher dose-adjusted TAC-C0 concentrations at all time points resulting in significant differences at 3 (P = 0.025) and 6 months (P = 0.047) after HTx. No significant associations were observed between the genetic variants and the CSA dose requirement. CONCLUSIONS The CYP3A5*3 variant has a major influence on the required TAC dose in HTx recipients, whereas the POR*28 may additionally contribute to the observed variability. These results support the importance of genetic markers in TAC dose optimization after HTx.

Bovine prion protein gene (PRNP) promoter polymorphisms modulate PRNP expression and may be responsible for differences in bovine spongiform encephalopathy susceptibility

The susceptibility of humans to the variant Creutzfeldt-Jakob disease is greatly influenced by polymorphisms within the human prion protein gene (PRNP). Similar genetic differences exist in sheep, in which PRNP polymorphisms modify the susceptibility to scrapie. However, the known coding polymorphisms within the bovine PRNP gene have little or no effect on bovine spongiform encephalopathy (BSE) susceptibility in cattle. We have recently found a tentative association between PRNP promoter polymorphisms and BSE susceptibility in German cattle (Sander, P., Hamann, H., Pfeiffer, I., Wemheuer, W., Brenig, B., Groschup, M., Ziegler, U., Distl, O., and Leeb, T. (2004) Neurogenetics 5, 19-25). A plausible hypothesis explaining this observation could be that the bovine PRNP promoter polymorphisms cause changes in PRNP expression that might be responsible for differences in BSE incubation time and/or BSE susceptibility. To test this hypothesis, we performed a functional promoter analysis of the different bovine PRNP promoter alleles by reporter gene assays in vitro and by measuring PRNP mRNA levels in calves with different PRNP genotypes in vivo. Two variable sites, a 23-bp insertion/deletion (indel) polymorphism containing a RP58-binding site and a 12-bp indel polymorphism containing an SP1-binding site, were investigated. Band shift assays indicated differences in transcription factor binding to the different alleles at the two polymorphisms. Reporter gene assays demonstrated an interaction between the two postulated transcription factors and lower expression levels of the ins/ins allele compared with the del/del allele. The in vivo data revealed substantial individual variation of PRNP expression in different tissues. In intestinal lymph nodes, expression levels differed between the different PRNP genotypes.

Polymorphisms within the canine MLPH gene are associated with dilute coat color in dogs

BACKGROUND Pinschers and other dogs with coat color dilution show a characteristic pigmentation phenotype. The fur colors are a lighter shade, e.g. silvery grey (blue) instead of black and a sandy color (Isabella fawn) instead of red or brown. In some dogs the coat color dilution is sometimes accompanied by hair loss and recurrent skin inflammation, the so called color dilution alopecia (CDA) or black hair follicular dysplasia (BHFD). In humans and mice a comparable pigmentation phenotype without any documented hair loss is caused by mutations within the melanophilin gene (MLPH). RESULTS We sequenced the canine MLPH gene and performed a mutation analysis of the MLPH exons in 6 Doberman Pinschers and 5 German Pinschers. A total of 48 sequence variations was identified within and between the breeds. Three families of dogs showed co-segregation for at least one polymorphism in an MLPH exon and the dilute phenotype. No single polymorphism was identified in the coding sequences or at splice sites that is likely to be causative for the dilute phenotype of all dogs examined. In 18 German Pinschers a mutation in exon 7 (R199H) was consistently associated with the dilute phenotype. However, as this mutation was present in homozygous state in four dogs of other breeds with wildtype pigmentation, it seems unlikely that this mutation is truly causative for coat color dilution. In Doberman Pinschers as well as in Large Munsterlanders with BHFD, a set of single nucleotide polymorphisms (SNPs) around exon 2 was identified that show a highly significant association to the dilute phenotype. CONCLUSION This study provides evidence that coat color dilution is caused by one or more mutations within or near the MLPH gene in several dog breeds. The data on polymorphisms that are strongly associated with the dilute phenotype will allow the genetic testing of Pinschers to facilitate the breeding of dogs with defined coat colors and to select against Large Munsterlanders carrying BHFD.