Pamela L. St. Jean

An Abundance of Rare Functional Variants in 202 Drug Target Genes Sequenced in 14,002 People

A Deep Look Into Our Genes Recent debates have focused on the degree of genetic variation and its impact upon health at the genomic level in humans (see the Perspective by Casals and Bertranpetit). Tennessen et al. (p. 64, published online 17 May), looking at all of the protein-coding genes in the human genome, and Nelson et al. (p. 100, published online 17 May), looking at genes that encode drug targets, address this question through deep sequencing efforts on samples from multiple individuals. The findings suggest that most human variation is rare, not shared between populations, and that rare variants are likely to play a role in human health. A pharmacogenomics analysis shows how challenging it will be to associate rare variants with phenotypes. Rare genetic variants contribute to complex disease risk; however, the abundance of rare variants in human populations remains unknown. We explored this spectrum of variation by sequencing 202 genes encoding drug targets in 14,002 individuals. We find rare variants are abundant (1 every 17 bases) and geographically localized, so that even with large sample sizes, rare variant catalogs will be largely incomplete. We used the observed patterns of variation to estimate population growth parameters, the proportion of variants in a given frequency class that are putatively deleterious, and mutation rates for each gene. We conclude that because of rapid population growth and weak purifying selection, human populations harbor an abundance of rare variants, many of which are deleterious and have relevance to understanding disease risk.

A Genome-Wide Investigation of SNPs and CNVs in Schizophrenia

We report a genome-wide assessment of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) in schizophrenia. We investigated SNPs using 871 patients and 863 controls, following up the top hits in four independent cohorts comprising 1,460 patients and 12,995 controls, all of European origin. We found no genome-wide significant associations, nor could we provide support for any previously reported candidate gene or genome-wide associations. We went on to examine CNVs using a subset of 1,013 cases and 1,084 controls of European ancestry, and a further set of 60 cases and 64 controls of African ancestry. We found that eight cases and zero controls carried deletions greater than 2 Mb, of which two, at 8p22 and 16p13.11-p12.4, are newly reported here. A further evaluation of 1,378 controls identified no deletions greater than 2 Mb, suggesting a high prior probability of disease involvement when such deletions are observed in cases. We also provide further evidence for some smaller, previously reported, schizophrenia-associated CNVs, such as those in NRXN1 and APBA2. We could not provide strong support for the hypothesis that schizophrenia patients have a significantly greater “load” of large (>100 kb), rare CNVs, nor could we find common CNVs that associate with schizophrenia. Finally, we did not provide support for the suggestion that schizophrenia-associated CNVs may preferentially disrupt genes in neurodevelopmental pathways. Collectively, these analyses provide the first integrated study of SNPs and CNVs in schizophrenia and support the emerging view that rare deleterious variants may be more important in schizophrenia predisposition than common polymorphisms. While our analyses do not suggest that implicated CNVs impinge on particular key pathways, we do support the contribution of specific genomic regions in schizophrenia, presumably due to recurrent mutation. On balance, these data suggest that very few schizophrenia patients share identical genomic causation, potentially complicating efforts to personalize treatment regimens.

Functional Polymorphism in the Matrix Metalloproteinase-9 Promoter as a Potential Risk Factor for Intracranial Aneurysm

BACKGROUND AND PURPOSE There is convincing evidence that susceptibility to intracranial aneurysms (ICAs) has a genetic component. However, few studies have sought to identify functional variation in specific candidate genes that may predispose individuals to develop an ICA. METHODS ICA cases and controls were genotyped for a simple length polymorphism in the promoter of matrix metalloproteinase-9 (MMP-9) to test for association between variation in the promoter and the occurrence of ICA. Alternative alleles were cloned into an in vitro reporter vector, transfected into human HT1080 fibroblasts, and assayed for promoter activity by beta-gal and luciferase assays. Electrophoretic gel shift assays were used to assess nuclear factor binding. RESULTS A length polymorphism in the promoter of MMP-9 was nonrandomly associated with the occurrence of ICA in a case-control study. This polymorphism was shown, by direct sequencing of 36 individuals, to be the only sequence variation within a 736-base pair region proximal to the transcriptional start site of the gene. Variation in the length of this repetitive element was shown to modulate promoter activity in an in vitro reporter assay, with the highest promoter activity being observed in constructs bearing the longest [(CA)23] element. Electrophoretic mobility shift assays were used to show that the (CA) element is bound by a sequence-specific DNA-binding protein. CONCLUSIONS Genetic variation in the promoter of the MMP-9 gene results in variation in its expression at the level of transcription. This may result in subtle differences in MMP-9 activity within the circle of Willis, leading to increased susceptibility to ICA formation.

Rare Variants in APP, PSEN1 and PSEN2 Increase Risk for AD in Late-Onset Alzheimer's Disease Families

Pathogenic mutations in APP, PSEN1, PSEN2, MAPT and GRN have previously been linked to familial early onset forms of dementia. Mutation screening in these genes has been performed in either very small series or in single families with late onset AD (LOAD). Similarly, studies in single families have reported mutations in MAPT and GRN associated with clinical AD but no systematic screen of a large dataset has been performed to determine how frequently this occurs. We report sequence data for 439 probands from late-onset AD families with a history of four or more affected individuals. Sixty sequenced individuals (13.7%) carried a novel or pathogenic mutation. Eight pathogenic variants, (one each in APP and MAPT, two in PSEN1 and four in GRN) three of which are novel, were found in 14 samples. Thirteen additional variants, present in 23 families, did not segregate with disease, but the frequency of these variants is higher in AD cases than controls, indicating that these variants may also modify risk for disease. The frequency of rare variants in these genes in this series is significantly higher than in the 1,000 genome project (p = 5.09×10−5; OR = 2.21; 95%CI = 1.49–3.28) or an unselected population of 12,481 samples (p = 6.82×10−5; OR = 2.19; 95%CI = 1.347–3.26). Rare coding variants in APP, PSEN1 and PSEN2, increase risk for or cause late onset AD. The presence of variants in these genes in LOAD and early-onset AD demonstrates that factors other than the mutation can impact the age at onset and penetrance of at least some variants associated with AD. MAPT and GRN mutations can be found in clinical series of AD most likely due to misdiagnosis. This study clearly demonstrates that rare variants in these genes could explain an important proportion of genetic heritability of AD, which is not detected by GWAS.

Disease-specific target selection: a critical first step down the right road.

Relevance of a drug target for a disease is often inferred with strong belief but fragile evidence. Here, a program for early identification of human disease-specific drug targets using high-throughput genetic associations is described. Large numbers of well-characterized patients (>1000) and matched controls are screened for genetic associations using several thousand (>7000) single nucleotide polymorphisms from more than 1500 genes. The genes were selected because they are members of target classes for which there are precedents for high-throughput chemical screening technology. This review summarizes the methods and intensive data analyses leading to target gene identification for type 2 diabetes mellitus, including the statistical permutation methodology used to correct for many variables.

The influence of genomic context on mutation patterns in the human genome inferred from rare variants

Understanding patterns of spontaneous mutations is of fundamental interest in studies of human genome evolution and genetic disease. Here, we used extremely rare variants in humans to model the molecular spectrum of single-nucleotide mutations. Compared to common variants in humans and human-chimpanzee fixed differences (substitutions), rare variants, on average, arose more recently in the human lineage and are less affected by the potentially confounding effects of natural selection, population demographic history, and biased gene conversion. We analyzed variants obtained from a population-based sequencing study of 202 genes in >14,000 individuals. We observed considerable variability in the per-gene mutation rate, which was correlated with local GC content, but not recombination rate. Using >20,000 variants with a derived allele frequency ≤ 10(-4), we examined the effect of local GC content and recombination rate on individual variant subtypes and performed comparisons with common variants and substitutions. The influence of local GC content on rare variants differed from that on common variants or substitutions, and the differences varied by variant subtype. Furthermore, recombination rate and recombination hotspots have little effect on rare variants of any subtype, yet both have a relatively strong impact on multiple variant subtypes in common variants and substitutions. This observation is consistent with the effect of biased gene conversion or selection-dependent processes. Our results highlight the distinct biases inherent in the initial mutation patterns and subsequent evolutionary processes that affect segregating variants.

Evaluation of the effect of UGT1A1 polymorphisms on dolutegravir pharmacokinetics

AIM To evaluate potential pharmacogenetic effects of UGT1A1 polymorphisms on the pharmacokinetics (PK) of dolutegravir (Tivicay®; ViiV Healthcare, NC, USA), an HIV-1 integrase inhibitor. PATIENTS & METHODS Analysis of pooled data from nine Phase I and II clinical studies was undertaken for 89 subjects receiving repeat dolutegravir 50 mg once daily (tablet formulation) who were genotyped for known UGT1A1 functional variants. RESULTS Geometric mean ratio (92% CI) for subjects carrying low (*28/*28 and *28/*37) and reduced activity (*1/*6, *1/*28, *1/*37, *28/*36 and *36/*37) polymorphisms compared with subjects with normal activity (*1/*1 and *1/*36) showed decreased oral clearance (CL/F; 0.765 [92% CI: 0.659-0.889]), increased area under the concentration-time curve (AUC(0-τ); 1.307 [1.125-1.518]) and C(max) (1.221 [1.063-1.402]), respectively. CONCLUSION Increased dolutegravir exposure in carriers of UGT1A1 reduced function polymorphisms is not clinically significant based on accumulated safety data so dose adjustment in these individuals is not required.

Deep sequencing of the LRRK2 gene in 14,002 individuals reveals evidence of purifying selection and independent origin of the p.Arg1628Pro mutation in Europe†

Genetic variation in LRRK2 predisposes to Parkinson disease (PD), which underpins its development as a therapeutic target. Here, we aimed to identify novel genotype–phenotype associations that might support developing LRRK2 therapies for other conditions. We sequenced the 51 exons of LRRK2 in cases comprising 12 common diseases (n = 9,582), and in 4,420 population controls. We identified 739 single‐nucleotide variants, 62% of which were observed in only one person, including 316 novel exonic variants. We found evidence of purifying selection for the LRRK2 gene and a trend suggesting that this is more pronounced in the central (ROC–COR–kinase) core protein domains of LRRK2 than the flanking domains. Population genetic analyses revealed that LRRK2 is not especially polymorphic or differentiated in comparison to 201 other drug target genes. Among Europeans, we identified 17 carriers (0.13%) of pathogenic LRRK2 mutations that were not significantly enriched within any disease or in those reporting a family history of PD. Analysis of pathogenic mutations within Europe reveals that the p.Arg1628Pro (c4883G>C) mutation arose independently in Europe and Asia. Taken together, these findings demonstrate how targeted deep sequencing can help to reveal fundamental characteristics of clinically important loci. Hum Mutat 33:1087–1098, 2012.

Analysis of rare variant population structure in Europeans explains differential stratification of gene-based tests

There is substantial interest in the role of rare genetic variants in the etiology of complex human diseases. Several gene-based tests have been developed to simultaneously analyze multiple rare variants for association with phenotypic traits. The tests can largely be partitioned into two classes – ‘burden’ tests and ‘joint’ tests – based on how they accumulate evidence of association across sites. We used the empirical joint site frequency spectra of rare, nonsynonymous variation from a large multi-population sequencing study to explore the effect of realistic rare variant population structure on gene-based tests. We observed an important difference between the two test classes: their susceptibility to population stratification. Focusing on European samples, we found that joint tests, which allow variants to have opposite directions of effect, consistently showed higher levels of P-value inflation than burden tests. We determined that the differential stratification was caused by two specific patterns in the interpopulation distribution of rare variants, each correlating with inflation in one of the test classes. The pattern that inflates joint tests is more prevalent in real data, explaining the higher levels of inflation in these tests. Furthermore, we show that the different sources of inflation between tests lead to heterogeneous responses to genomic control correction and the number of variants analyzed. Our results indicate that care must be taken when interpreting joint and burden analyses of the same set of rare variants, in particular, to avoid mistaking inflated P-values in joint tests for stronger signals of true associations.

Genetic Deficiency of Plasma Lipoprotein-Associated Phospholipase A2 (PLA2G7 V297F Null Mutation) and Risk of Alzheimer's Disease in Japan

High plasma lipoprotein phospholipase A2 activity (Lp-PLA2) is reported to be a risk factor for dementia. A loss of function polymorphism in the Lp-PLA2 gene - PLA2G7 V279F - is found almost exclusively in Asians. In 1,952 subjects with late-onset AD and 2,079 non-demented controls recruited from Japan, the PLA2G7 null allele was not associated with risk or age at onset of AD: logistic regression OR 0.98 (95% CI 0.86-1.12, p=0.81) per additional null allele, adjusted for age/age at onset, gender, and APOE ε4. Genetic deficiency of Lp-PLA2 activity is not associated with a reduced risk of AD in the Japanese population.