A MULTILEVEL MODEL TO ADDRESS BATCH EFFECTS IN COPY NUMBER ESTIMATION USING SNP ARRAYS

Submicroscopic changes in chromosomal DNA copy number dosage are common and have been implicated in many heritable diseases and cancers. Recent high-throughput technologies have a resolution that permits the detection of segmental changes in DNA copy number that span thousands of basepairs across the genome. Genome-wide association studies (GWAS) may simultaneously screen for copy number-phenotype and SNP-phenotype associations as part of the analytic strategy. However, genome-wide array analyses are particularly susceptible to batch effects as the logistics of preparing DNA and processing thousands of arrays often involves multiple laboratories and technicians, or changes over calendar time to the reagents and laboratory equipment. Failure to adjust for batch effects can lead to incorrect inference and requires inefficient post-hoc quality control procedures that exclude regions that are associated with batch. Our work extends previous model-based approaches for copy number estimation by explicitly modeling batch effects and using shrinkage to improve locus-specific estimates of copy number uncertainty. Key features of this approach include the use of diallelic genotype calls from experimental data to estimate batch- and locus-specific parameters of background and signal without the requirement of training data. We illustrate these ideas using a study of bipolar disease and a study of chromosome 21 trisomy. The former has batch effects that dominate much of the observed variation in quantile-normalized intensities, while the latter illustrates the robustness of our approach to datasets where as many as 25% of the samples have altered copy number. Locus-specific estimates of copy number can be plotted on the copy-number scale to investigate mosaicism and guide the choice of appropriate downstream approaches for smoothing the copy number as a function of physical position. The software is open source and implemented in the R package CRLMM available at Bioconductor (http:www.bioconductor.org).

Acetaldehyde as an underestimated risk factor for cancer development: role of genetics in ethanol metabolism

Chronic ethanol consumption is a strong risk factor for the development of certain types of cancer including those of the upper aerodigestive tract, the liver, the large intestine and the female breast. Multiple mechanisms are involved in alcohol-mediated carcinogenesis. Among those the action of acetaldehyde (AA), the first metabolite of ethanol oxidation is of particular interest. AA is toxic, mutagenic and carcinogenic in animal experiments. AA binds to DNA and forms carcinogenic adducts. Direct evidence of the role of AA in alcohol-associated carcinogenesis derived from genetic linkage studies in alcoholics. Polymorphisms or mutations of genes coding for AA generation or detoxifying enzymes resulting in elevated AA concentrations are associated with increased cancer risk. Approximately 40% of Japanese, Koreans or Chinese carry the AA dehydrogenase 2*2 (ALDH2*2) allele in its heterozygous form. This allele codes for an ALDH2 enzyme with little activity leading to high AA concentrations after the consumption of even small amounts of alcohol. When individuals with this allele consume ethanol chronically, a significant increased risk for upper alimentary tract and colorectal cancer is noted. In Caucasians, alcohol dehydrogenase 1C*1 (ADH1C*1) allele encodes for an ADH isoenzyme which produces 2.5 times more AA than the corresponding allele ADH1C*2. In studies with moderate to high alcohol intake, ADH1C*1 allele frequency and rate of homozygosity was found to be significantly associated with an increased risk for cancer of the upper aerodigestive tract, the liver, the colon and the female breast. These studies underline the important role of acetaldehyde in ethanol-mediated carcinogenesis.

MEP1A allele for meprin A metalloprotease is a susceptibility gene for inflammatory bowel disease

The MEP1A gene, located on human chromosome 6p (mouse chromosome 17) in a susceptibility region for inflammatory bowel disease (IBD), encodes the alpha-subunit of metalloproteinase meprin A, which is expressed in the intestinal epithelium. This study shows a genetic association of MEP1A with IBD in a cohort of ulcerative colitis (UC) patients. There were four single-nucleotide polymorphisms in the coding region (P=0.0012-0.04), and one in the 3'-untranslated region (P=2 x 10(-7)) that displayed associations with UC. Moreover, meprin-alpha mRNA was decreased in inflamed mucosa of IBD patients. Meprin-alpha knockout mice exhibited a more severe intestinal injury and inflammation than their wild-type counterparts following oral administration of dextran sulfate sodium. Collectively, the data implicate MEP1A as a UC susceptibility gene and indicate that decreased meprin-alpha expression is associated with intestinal inflammation in IBD patients and in a mouse experimental model of IBD.

Neurogranin controls the spatiotemporal pattern of postsynaptic Ca2+/CaM signaling.

Neurogranin (Ng) is a postsynaptic IQ-motif containing protein that accelerates Ca(2+) dissociation from calmodulin (CaM), a key regulator of long-term potentiation and long-term depression in CA1 pyramidal neurons. The exact physiological role of Ng, however, remains controversial. Two genetic knockout studies of Ng showed opposite outcomes in terms of the induction of synaptic plasticity. To understand its function, we test the hypothesis that Ng could regulate the spatial range of action of Ca(2+)/CaM based on its ability to accelerate the dissociation of Ca(2+) from CaM. Using a mathematical model constructed on the known biochemistry of Ng, we calculate the cycle time that CaM molecules alternate between the fully Ca(2+) saturated state and the Ca(2+) unbound state. We then use these results and include diffusion of CaM to illustrate the impact that Ng has on modulating the spatial profile of Ca(2+)-saturated CaM within a model spine compartment. Finally, the first-passage time of CaM to transition from the Ca(2+)-free state to the Ca(2+)-saturated state was calculated with or without Ng present. These analyses suggest that Ng regulates the encounter rate between Ca(2+) saturated CaM and its downstream targets during postsynaptic Ca(2+) transients.

A mutation in the FAM83G gene in dogs with hereditary footpad hyperkeratosis (HFH)

Hereditary footpad hyperkeratosis (HFH) represents a palmoplantar hyperkeratosis, which is inherited as a monogenic autosomal recessive trait in several dog breeds, such as e.g. Kromfohrländer and Irish Terriers. We performed genome-wide association studies (GWAS) in both breeds. In Kromfohrländer we obtained a single strong association signal on chromosome 5 (p(raw) = 1.0×10(-13)) using 13 HFH cases and 29 controls. The association signal replicated in an independent cohort of Irish Terriers with 10 cases and 21 controls (p(raw) = 6.9×10(-10)). The analysis of shared haplotypes among the combined Kromfohrländer and Irish Terrier cases defined a critical interval of 611 kb with 13 predicted genes. We re-sequenced the genome of one affected Kromfohrländer at 23.5× coverage. The comparison of the sequence data with 46 genomes of non-affected dogs from other breeds revealed a single private non-synonymous variant in the critical interval with respect to the reference genome assembly. The variant is a missense variant (c.155G>C) in the FAM83G gene encoding a protein with largely unknown function. It is predicted to change an evolutionary conserved arginine into a proline residue (p.R52P). We genotyped this variant in a larger cohort of dogs and found perfect association with the HFH phenotype. We further studied the clinical and histopathological alterations in the epidermis in vivo. Affected dogs show a moderate to severe orthokeratotic hyperplasia of the palmoplantar epidermis. Thus, our data provide the first evidence that FAM83G has an essential role for maintaining the integrity of the palmoplantar epidermis.

Impact of common risk factors of fibrosis progression in chronic hepatitis C.

OBJECTIVE The natural course of chronic hepatitis C varies widely. To improve the profiling of patients at risk of developing advanced liver disease, we assessed the relative contribution of factors for liver fibrosis progression in hepatitis C. DESIGN We analysed 1461 patients with chronic hepatitis C with an estimated date of infection and at least one liver biopsy. Risk factors for accelerated fibrosis progression rate (FPR), defined as ≥0.13 Metavir fibrosis units per year, were identified by logistic regression. Examined factors included age at infection, sex, route of infection, HCV genotype, body mass index (BMI), significant alcohol drinking (≥20 g/day for ≥5 years), HIV coinfection and diabetes. In a subgroup of 575 patients, we assessed the impact of single nucleotide polymorphisms previously associated with fibrosis progression in genome-wide association studies. Results were expressed as attributable fraction (AF) of risk for accelerated FPR. RESULTS Age at infection (AF 28.7%), sex (AF 8.2%), route of infection (AF 16.5%) and HCV genotype (AF 7.9%) contributed to accelerated FPR in the Swiss Hepatitis C Cohort Study, whereas significant alcohol drinking, anti-HIV, diabetes and BMI did not. In genotyped patients, variants at rs9380516 (TULP1), rs738409 (PNPLA3), rs4374383 (MERTK) (AF 19.2%) and rs910049 (major histocompatibility complex region) significantly added to the risk of accelerated FPR. Results were replicated in three additional independent cohorts, and a meta-analysis confirmed the role of age at infection, sex, route of infection, HCV genotype, rs738409, rs4374383 and rs910049 in accelerating FPR. CONCLUSIONS Most factors accelerating liver fibrosis progression in chronic hepatitis C are unmodifiable.

8p23 beta-defensin copy number determination by single-locus pseudogene-based paralog ratio tests risk bias due to low-frequency sequence variations.

BACKGROUND The copy number variation (CNV) in beta-defensin genes (DEFB) on human chromosome 8p23 has been proposed to contribute to the phenotypic differences in inflammatory diseases. However, determination of exact DEFB CN is a major challenge in association studies. Quantitative real-time PCR (qPCR), paralog ratio tests (PRT) and multiplex ligation-dependent probe amplification (MLPA) have been extensively used to determine DEFB CN in different laboratories, but inter-method inconsistencies were observed frequently. In this study we asked which one is superior among the three methods for DEFB CN determination. RESULTS We developed a clustering approach for MLPA and PRT to statistically correlate data from a single experiment. Then we compared qPCR, a newly designed PRT and MLPA for DEFB CN determination in 285 DNA samples. We found MLPA had the best convergence and clustering results of the raw data and the highest call rate. In addition, the concordance rates between MLPA or PRT and qPCR (32.12% and 37.99%, respectively) were unacceptably low with underestimated CN by qPCR. Concordance rate between MLPA and PRT (90.52%) was high but PRT systematically underestimated CN by one in a subset of samples. In these samples a sequence variant which caused complete PCR dropout of the respective DEFB cluster copies was found in one primer binding site of one of the targeted paralogous pseudogenes. CONCLUSION MLPA is superior to PRT and even more to qPCR for DEFB CN determination. Although the applied PRT provides in most cases reliable results, such a test is particularly sensitive to low-frequency sequence variations preferably accumulating in loci like pseudogenes which are most likely not under selective pressure. In the light of the superior performance of multiplex assays, the drawbacks of such single PRTs could be overcome by combining more test markers.

Variants in the Mannose-binding Lectin Gene MBL2 do not Associate With Sepsis Susceptibility or Survival in a Large European Cohort.

BACKGROUND  Sepsis is an increasingly common condition, which continues to be associated with unacceptably high mortality. A large number of association studies have investigated susceptibility to, or mortality from, sepsis for variants in the functionally important immune-related gene MBL2. These studies have largely been underpowered and contradictory. METHODS  We genotyped and analyzed 4 important MBL2 single nucleotide polymorphisms (SNPs; rs5030737, rs1800450, rs1800451, and rs7096206) in 1839 European community-acquired pneumonia (CAP) and peritonitis sepsis cases, and 477 controls from the United Kingdom. We analyzed the following predefined subgroups and outcomes: 28-day and 6 month mortality from sepsis due to CAP or peritonitis combined, 28-day mortality from CAP sepsis, peritonitis sepsis, pneumococcal sepsis or sepsis in younger patients, and susceptibility to CAP sepsis or pneumococcal sepsis in the United Kingdom. RESULTS  There were no significant associations (all P-values were greater than .05 after correction for multiple testing) between MBL2 genotypes and any of our predefined analyses. CONCLUSIONS  In this large, well-defined cohort of immune competent adult patients, no associations between MBL2 genotype and sepsis susceptibility or outcome were identified.

Genome-Wide Analyses Suggest Mechanisms Involving Early B-Cell Development in Canine IgA Deficiency.

Immunoglobulin A deficiency (IgAD) is the most common primary immune deficiency disorder in both humans and dogs, characterized by recurrent mucosal tract infections and a predisposition for allergic and other immune mediated diseases. In several dog breeds, low IgA levels have been observed at a high frequency and with a clinical resemblance to human IgAD. In this study, we used genome-wide association studies (GWAS) to identify genomic regions associated with low IgA levels in dogs as a comparative model for human IgAD. We used a novel percentile groups-approach to establish breed-specific cut-offs and to perform analyses in a close to continuous manner. GWAS performed in four breeds prone to low IgA levels (German shepherd, Golden retriever, Labrador retriever and Shar-Pei) identified 35 genomic loci suggestively associated (p <0.0005) to IgA levels. In German shepherd, three genomic regions (candidate genes include KIRREL3 and SERPINA9) were genome-wide significantly associated (p <0.0002) with IgA levels. A ~20kb long haplotype on CFA28, significantly associated (p = 0.0005) to IgA levels in Shar-Pei, was positioned within the first intron of the gene SLIT1. Both KIRREL3 and SLIT1 are highly expressed in the central nervous system and in bone marrow and are potentially important during B-cell development. SERPINA9 expression is restricted to B-cells and peaks at the time-point when B-cells proliferate into antibody-producing plasma cells. The suggestively associated regions were enriched for genes in Gene Ontology gene sets involving inflammation and early immune cell development.

Imputation of sequence level genotypes in the Franches-Montagnes horse breed

BACKGROUND A cost-effective strategy to increase the density of available markers within a population is to sequence a small proportion of the population and impute whole-genome sequence data for the remaining population. Increased densities of typed markers are advantageous for genome-wide association studies (GWAS) and genomic predictions. METHODS We obtained genotypes for 54 602 SNPs (single nucleotide polymorphisms) in 1077 Franches-Montagnes (FM) horses and Illumina paired-end whole-genome sequencing data for 30 FM horses and 14 Warmblood horses. After variant calling, the sequence-derived SNP genotypes (~13 million SNPs) were used for genotype imputation with the software programs Beagle, Impute2 and FImpute. RESULTS The mean imputation accuracy of FM horses using Impute2 was 92.0%. Imputation accuracy using Beagle and FImpute was 74.3% and 77.2%, respectively. In addition, for Impute2 we determined the imputation accuracy of all individual horses in the validation population, which ranged from 85.7% to 99.8%. The subsequent inclusion of Warmblood sequence data further increased the correlation between true and imputed genotypes for most horses, especially for horses with a high level of admixture. The final imputation accuracy of the horses ranged from 91.2% to 99.5%. CONCLUSIONS Using Impute2, the imputation accuracy was higher than 91% for all horses in the validation population, which indicates that direct imputation of 50k SNP-chip data to sequence level genotypes is feasible in the FM population. The individual imputation accuracy depended mainly on the applied software and the level of admixture.

Proteomic analysis of Plasmodium in the mosquito: progress and pitfalls

Here we discuss proteomic analyses of whole cell preparations of the mosquito stages of malaria parasite development (i.e. gametocytes, microgamete, ookinete, oocyst and sporozoite) of Plasmodium berghei. We also include critiques of the proteomes of two cell fractions from the purified ookinete, namely the micronemes and cell surface. Whereas we summarise key biological interpretations of the data, we also try to identify key methodological constraints we have met, only some of which we were able to resolve. Recognising the need to translate the potential of current genome sequencing into functional understanding, we report our efforts to develop more powerful combinations of methods for the in silico prediction of protein function and location. We have applied this analysis to the proteome of the male gamete, a cell whose very simple structural organisation facilitated interpretation of data. Some of the in silico predictions made have now been supported by ongoing protein tagging and genetic knockout studies. We hope this discussion may assist future studies.

Candidate gene identification following linkage: Search for hypertension susceptibility genes

Following up genetic linkage studies to identify the underlying susceptibility gene(s) for complex disease traits is an arduous yet biologically and clinically important task. Complex traits, such as hypertension, are considered polygenic with many genes influencing risk, each with small effects. Chromosome 2 has been consistently identified as a genomic region with genetic linkage evidence suggesting that one or more loci contribute to blood pressure levels and hypertension status. Using combined positional candidate gene methods, the Family Blood Pressure Program has concentrated efforts in investigating this region of chromosome 2 in an effort to identify underlying candidate hypertension susceptibility gene(s). Initial informatics efforts identified the boundaries of the region and the known genes within it. A total of 82 polymorphic sites in eight positional candidate genes were genotyped in a large hypothesis-generating sample consisting of 1640 African Americans, 1339 whites, and 1616 Mexican Americans. To adjust for multiple comparisons, resampling-based false discovery adjustment was applied, extending traditional resampling methods to sibship samples. Following this adjustment for multiple comparisons, SLC4A5, a sodium bicarbonate transporter, was identified as a primary candidate gene for hypertension. Polymorphisms in SLC4A5 were subsequently genotyped and analyzed for validation in two populations of African Americans (N = 461; N = 778) and two of whites (N = 550; N = 967). Again, SNPs within SLC4A5 were significantly associated with blood pressure levels and hypertension status. While not identifying a single causal DNA sequence variation that is significantly associated with blood pressure levels and hypertension status across all samples, the results further implicate SLC4A5 as a candidate hypertension susceptibility gene, validating previous evidence for one or more genes on chromosome 2 that influence hypertension related phenotypes in the population-at-large. The methodology and results reported provide a case study of one approach for following up the results of genetic linkage analyses to identify genes influencing complex traits. ^

Instrumental variable method for the causal relationship between soluble receptor for advanced glycation end-products (sRAGE) and risk of pancreatic cancer

This thesis project is motivated by the potential problem of using observational data to draw inferences about a causal relationship in observational epidemiology research when controlled randomization is not applicable. Instrumental variable (IV) method is one of the statistical tools to overcome this problem. Mendelian randomization study uses genetic variants as IVs in genetic association study. In this thesis, the IV method, as well as standard logistic and linear regression models, is used to investigate the causal association between risk of pancreatic cancer and the circulating levels of soluble receptor for advanced glycation end-products (sRAGE). Higher levels of serum sRAGE were found to be associated with a lower risk of pancreatic cancer in a previous observational study (255 cases and 485 controls). However, such a novel association may be biased by unknown confounding factors. In a case-control study, we aimed to use the IV approach to confirm or refute this observation in a subset of study subjects for whom the genotyping data were available (178 cases and 177 controls). Two-stage IV method using generalized method of moments-structural mean models (GMM-SMM) was conducted and the relative risk (RR) was calculated. In the first stage analysis, we found that the single nucleotide polymorphism (SNP) rs2070600 of the receptor for advanced glycation end-products (AGER) gene meets all three general assumptions for a genetic IV in examining the causal association between sRAGE and risk of pancreatic cancer. The variant allele of SNP rs2070600 of the AGER gene was associated with lower levels of sRAGE, and it was neither associated with risk of pancreatic cancer, nor with the confounding factors. It was a potential strong IV (F statistic = 29.2). However, in the second stage analysis, the GMM-SMM model failed to converge due to non- concaveness probably because of the small sample size. Therefore, the IV analysis could not support the causality of the association between serum sRAGE levels and risk of pancreatic cancer. Nevertheless, these analyses suggest that rs2070600 was a potentially good genetic IV for testing the causality between the risk of pancreatic cancer and sRAGE levels. A larger sample size is required to conduct a credible IV analysis.^

Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA

Genetic analysis of limiting quantities of genomic DNA play an important role in DNA forensics, paleoarcheology, genetic disease diagnosis, genetic linkage analysis, and genetic diversity studies. We have tested the ability of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to amplify picogram quantities of human genomic DNA for the purpose of increasing the amount of template for genotyping with microsatellite repeat markers. DNA was uniformly amplified at a large number of typable loci throughout the human genome with starting template DNAs from as little as 15 pg to as much as 400 ng. A much greater-fold enrichment was seen for the smaller genomic DOP-PCRs. All markers tested were amplified from starting genomic DNAs in the range of 0.6–40 ng with amplifications of 200- to 600-fold. The DOP-PCR-amplified genomic DNA was an excellent and reliable template for genotyping with microsatellites, which give distinct bands with no increase in stutter artifact on di-, tri-, and tetranucleotide repeats. There appears to be equal amplification of genomic DNA from 55 of 55 tested discrete microsatellites implying near complete coverage of the human genome. Thus, DOP-PCR appears to allow unbiased, hundreds-fold whole genome amplification of human genomic DNA for genotypic analysis.

Targeted development of informative microsatellite (SSR) markers

We describe a novel approach, selectively amplified microsatellite (SAM) analysis, for the targeted development of informative simple sequence repeat (SSR) markers. A modified selectively amplified microsatellite polymorphic loci assay is used to generate multi-locus SSR fingerprints that provide a source of polymorphic DNA markers (SAMs) for use in genetic studies. These polymorphisms capture the repeat length variation associated with SSRs and allow their chromosomal location to be determined prior to the expense of isolating and characterising individual loci. SAMs can then be converted to locus-specific SSR markers with the design and synthesis of a single primer specific to the conserved region flanking the repeat. This approach offers a cost-efficient and rapid method for developing SSR markers for predetermined chromosomal locations and of potential informativeness. The high recovery rate of useful SSR markers makes this strategy a valuable tool for population and genetic mapping studies. The utility of SAM analysis was demonstrated by the development of SSR markers in bread wheat.