Albert B. Seymour

The serotonin transporter polymorphism, 5HTTLPR, is associated with a faster response time to sertraline in an elderly population with major depressive disorder

RationaleA common polymorphism (5HTTLPR) within the promoter region of the serotonin transporter gene (LSC6A4) has been shown to influence response time as well as overall response to selective serotonin reuptake inhibitors (SSRIs) in subjects with major depressive disorder. We hypothesized that a similar effect in response time to sertraline would be observed and that no effect on response time would be seen in a placebo arm.ObjectivesWe tested the hypothesis that subjects homozygous for the long allele at the 5HTTLPR polymorphism would respond more rapidly to sertraline than subjects carrying one or two copies of the short allele.MethodsHAM-D and CGI-I responses to sertraline and placebo were measured weekly in the context of an 8-week, placebo-controlled study in elderly depressed subjects. Genotyping of the 5HTTLPR polymorphism was performed to test for correlations with response at each week in the sertraline and placebo groups (n=206).ResultsSubjects homozygous for the long allele of 5HTTLPR showed a significant increase in response at week 1 and week 2, as assessed by the CGI-I scale compared with subjects carrying one or two copies of the short allele (P=0.01 at both weeks). No significant difference was observed in the placebo group.ConclusionsThese results suggest that genetic variation in the serotonin transporter gene effects the response time to sertraline and provides complementing evidence to previous reports that this polymorphism affects response time to other SSRIs.

Power in the phenotypic extremes: a simulation study of power in discovery and replication of rare variants.

Next‐generation sequencing technologies are making it possible to study the role of rare variants in human disease. Many studies balance statistical power with cost‐effectiveness by (a) sampling from phenotypic extremes and (b) utilizing a two‐stage design. Two‐stage designs include a broad‐based discovery phase and selection of a subset of potential causal genes/variants to be further examined in independent samples. We evaluate three parameters: first, the gain in statistical power due to extreme sampling to discover causal variants; second, the informativeness of initial (Phase I) association statistics to select genes/variants for follow‐up; third, the impact of extreme and random sampling in (Phase 2) replication. We present a quantitative method to select individuals from the phenotypic extremes of a binary trait, and simulate disease association studies under a variety of sample sizes and sampling schemes. First, we find that while studies sampling from extremes have excellent power to discover rare variants, they have limited power to associate them to phenotype—suggesting high false‐negative rates for upcoming studies. Second, consistent with previous studies, we find that the effect sizes estimated in these studies are expected to be systematically larger compared with the overall population effect size; in a well‐cited lipids study, we estimate the reported effect to be twofold larger. Third, replication studies require large samples from the general population to have sufficient power; extreme sampling could reduce the required sample size as much as fourfold. Our observations offer practical guidance for the design and interpretation of studies that utilize extreme sampling. Genet. Epidemiol. 35: 236‐246, 2011.  © 2011 Wiley‐Liss, Inc.

Application of pooled genotyping to scan candidate regions for association with HDL cholesterol levels

Association studies are used to identify genetic determinants of complex human traits of medical interest. With the large number of validated single nucleotide polymorphisms (SNPs) currently available, two limiting factors in association studies are genotyping capability and costs. Pooled DNA genotyping has been proposed as an efficient means of screening SNPs for allele frequency differences in case-control studies and for prioritising them for subsequent individual genotyping analysis. Here, we apply quantitative pooled genotyping followed by individual genotyping and replication to identify associations with human serum high-density lipoprotein (HDL) cholesterol levels. The DNA from individuals with low and high HDL cholesterol levels was pooled separately, each pool was amplified by polymerase chain reaction in triplicate and each amplified product was separately hybridised to a high-density oligonucleotide array. Allele frequency differences between case and control groups with low and high HDL cholesterol levels were estimated for 7,283 SNPs distributed across 71 candidate gene regions spanning a total of 17.1 megabases. A novel method was developed to take advantage of independently derived haplotype map information to improve the pooled estimates of allele frequency differences. A subset of SNPs with the largest estimated allele frequency differences between low and high HDL cholesterol groups was chosen for individual genotyping in the study population, as well as in a separate replication population. Four SNPs in a single haplotype block within the cholesteryl ester transfer protein (CETP) gene interval were significantly associated with HDL cholesterol levels in both populations. Our study is among the first to demonstrate the application of pooled genotyping followed by confirmation with individual genotyping to identify genetic determinants of a complex trait.

Relation of Increased Prebeta-1 High-Density Lipoprotein Levels to Risk of Coronary Heart Disease

Preβ-1 high-density lipoprotein (HDL) plays a key role in reverse cholesterol transport by promoting cholesterol efflux. Our aims were (1) to test previous associations between preβ-1 HDL and coronary heart disease (CHD) and (2) to investigate whether preβ-1 HDL levels also are associated with risk of myocardial infarction (MI). Plasma preβ-1 HDL was measured by an ultrafiltration-isotope dilution technique in 1,255 subjects recruited from the University of California-San Francisco Lipid and Cardiovascular Clinics and collaborating cardiologists. Preβ-1 HDL was significantly and positively associated with CHD and MI even after adjustment for established risk factors. Inclusion of preβ-1 HDL in a multivariable model for CHD led to a modest improvement in reclassification of subjects (net reclassification index 0.15, p = 0.01; integrated discrimination improvement 0.003, p = 0.2). In contrast, incorporation of preβ-1 HDL into a risk model of MI alone significantly improved reclassification of subjects (net reclassification index 0.21, p = 0.008; integrated discrimination improvement 0.01, p = 0.02), suggesting that preβ-1 HDL has more discriminatory power for MI than for CHD in our study population. In conclusion, these results confirm previous associations between preβ-1 HDL and CHD in a large well-characterized clinical cohort. Also, this is the first study in which preβ-1 HDL was identified as a novel and independent predictor of MI above and beyond traditional CHD risk factors.

siRNA Off-Target Effects Can Be Reduced at Concentrations That Match Their Individual Potency

Small interfering RNAs (siRNAs) are routinely used to reduce mRNA levels for a specific gene with the goal of studying its function. Several studies have demonstrated that siRNAs are not always specific and can have many off-target effects. The 3′ UTRs of off-target mRNAs are often enriched in sequences that are complementary to the seed-region of the siRNA. We demonstrate that siRNA off-targets can be significantly reduced when cells are treated with a dose of siRNA that is relatively low (e.g. 1 nM), but sufficient to effectively silence the intended target. The reduction in off-targets was demonstrated for both modified and unmodified siRNAs that targeted either STAT3 or hexokinase II. Low concentrations reduced silencing of transcripts with complementarity to the seed region of the siRNA. Similarly, off-targets that were not complementary to the siRNA were reduced at lower doses, including up-regulated genes that are involved in immune response. Importantly, the unintended induction of caspase activity following treatment with a siRNA that targeted hexokinase II was also shown to be a concentration-dependent off-target effect. We conclude that off-targets and their related phenotypic effects can be reduced for certain siRNA that potently silence their intended target at low concentrations.

Association of matrilin-3 polymorphisms with spinal disc degeneration and osteoarthritis of the first carpometacarpal joint of the hand

Background: Seven polymorphisms in the matrilin-3(MATN3) gene were previously tested for genetic association with hand osteoarthritis in an Icelandic cohort. One of the variants, involving a conserved amino acid substitution (T303M; SNP5), was related to idiopathic hand osteoarthritis. Objectives: To investigate SNP5 and two other promising polymorphisms (rs2242190; SNP3, rs8176070; SNP6) for association with radiographic and symptomatic hand osteoarthritis phenotypes, as well as other heritable phenotypes. Methods: Polymorphisms were examined in two distinct cohorts of subjects: a population based sample from the Rotterdam study (n = 809), and affected siblings from the genetics, osteoarthrosis and progression (GARP) study (n = 382). Results: The originally described association of T303M with the hand osteoarthritis phenotype was not observed in the populations studied. In the Rotterdam sample, however, carrying the T allele of T303M conferred an odds ratio of 2.9 (95% confidence interval (CI), 1.2 to 7.3; p = 0.02) for spinal disc degeneration. In the GARP study, carriers of the A allele of SNP6 had an odds ratio of 2.0 (95% CI, 1.3 to 3.1, p = 0.004) for osteoarthritis of the first carpometacarpal joint (CMC1) as compared with the Rotterdam sample as a control group. Subsequent haplotype analysis showed that a common haplotype, containing the risk allele of SNP6, conferred a significant risk in sibling pairs with CMC1 osteoarthritis (odds ratio = 1.7 (95% CI, 1.1 to 2.7, p = 0.02)). Conclusions: These associations suggest that the MATN3 region also determines susceptibility to spinal disc degeneration and CMC1 osteoarthritis.

Linkage analysis of X-linked cone-rod dystrophy : Localization to Xp11.4 and definition of a locus distinct from RP2 and RP3

Progressive X-linked cone-rod dystrophy (COD1) is a retinal disease affecting primarily the cone photoreceptors. The COD1 locus originally was localized, by the study of three independent families, to a region between Xp11.3 and Xp21.1, encompassing the retinitis pigmentosa (RP) 3 locus. We have refined the COD1 locus to a limited region of Xp11.4, using two families reported elsewhere and a new extended family. Genotype analysis was performed by use of eight microsatellite markers (tel-M6CA, DXS1068, DXS1058, DXS993, DXS228, DXS1201, DXS1003, and DXS1055-cent), spanning a distance of 20 cM. Nine-point linkage analysis, by use of the VITESSE program for X-linked disorders, established a maximum LOD score (17.5) between markers DXS1058 and DXS993, spanning 4.0 cM. Two additional markers, DXS977 and DXS556, which map between DXS1058 and DXS993, were used to further narrow the critical region. The RP3 gene, RPGR, was excluded on the basis of two obligate recombinants, observed in two independent families. In a third family, linkage analysis did not exclude the RPGR locus. The entire coding region of the RPGR gene from two affected males from family 2 was sequenced and was found to be normal. Haplotype analysis of two family branches, containing three obligate recombinants, two affected and one unaffected, defined the COD1 locus as distal to DXS993 and proximal to DXS556, a distance of approximately 1.0 Mb. This study excludes COD1 as an allelic variant of RP3 and establishes a novel locus that is sufficiently defined for positional cloning.

Rapid identification of somatic mutations in colorectal and breast cancer tissues using mismatch repair detection (MRD).

Mismatch repair detection (MRD) was used to screen 93 matched tumor‐normal sample pairs and 22 cell lines for somatic mutations in 30 cancer relevant genes. Using a starting amount of only 150 ng of genomic DNA, we screened 102 kb of sequence for somatic mutations in colon and breast cancer. A total of 152 somatic mutations were discovered, encompassing previously reported mutations, such as BRAF V600E and KRAS G12S, G12V, and G13D, as well as novel mutations, including some in genes in which somatic mutations have not previously been reported, such as MAP2K1 and MAP2K2. The distribution of mutations ranged widely within and across tumor types. The functional significance of many of these mutations is not understood, with patterns of selection only evident in KRAS and BRAF in colon cancer. These results present a novel approach to high‐throughput mutation screening using small amounts of starting material and reveal a mutation spectrum across 30 genes in a large cohort of breast and colorectal cancers. Hum Mutat 29(3), 441–450, 2008.

Emerging strategies and applications of pharmacogenomics

The rapid pace of genomic science advancements, including the completion of the human genome sequence, the extensive cataloguing of genetic variation and the acceleration of technologies to assess such variation, combined with clinical programmes with rich phenotypic data, serve as the foundation for the design and execution of pharmacogenomic studies which have an impact on the pharmaceutical pipeline from early discovery through to the marketplace. The authors discuss the required infrastructure to support pharmacogenomic studies and provide insight into the strategies and practical application to influence decision making in the pharmaceutical setting. Further, the influence of pharmacogenomics is currently affecting patient care in the oncology area and is highlighted as evident impact in the marketplace.

Abstract B119: Insulin‐like growth factor 1 receptor mutants are inhibited by figitumumab (CP‐751, 871) in vitro

To test the hypothesis that mutant insulin‐like growth factor 1 receptor (IGF‐1R) protein may differentially respond to inhibition by figitumumab (CP‐751, 871), potentially necessitating patient screening, we studied the in vitro efficacy of figitumumab against seven identified non‐synonymous somatic IGF‐1R mutations. The IGF‐1R signaling pathway has been implicated in numerous aspects of tumor biology, and inhibition of this pathway has been shown to provide beneficial anti‐tumor effects. Figitumumab is a fully human monoclonal IgG2 antibody against IGF‐1R that has been shown to block ligand binding, inhibit IGF‐1R tyrosine kinase activity, and induce IGF‐1R down‐modulation. There have been reports in the literature of targeted cancer therapies being affected by somatic mutations within the target of the therapy itself as well as downstream in the signaling cascade. To identify somatic DNA mutations within the IGF1R gene, 46 colon and 46 lung matched normal/tumor sets were screened using a mismatch repair detection method. The screening process identified seven unique mutations in either colon or lung cancer samples that altered the IGFIR coding sequence; 5 in colon and 2 in lung. These mutations were distributed across various domains of the receptor with three located in the extracellular alpha chain and four in the intracellular beta chain. Of the four located in the intracellular beta chain, three were in the tyrosine kinase domain. To study the effects of these mutations on the kinase activity of the receptor and the ability to inhibit the mutant receptors with figitumumab, stable cell lines containing wild‐type and the mutant receptors were created and evaluated by ELISA for down‐regulation of the IGF‐1R protein and IGF‐1R phosphorylation. All seven mutant forms of the protein, as well as wild‐type IGF‐1R were inhibited by figitumumab. Six of the seven mutant proteins maintained dependency on ligand stimulation for kinase activity while one mutant, a glycine to arginine change at amino acid position 1199, lacked kinase activity all together. The results of our experiments suggest that figitumumab effectively inhibited the wild‐type and mutant IGF‐1R receptors in this study and therefore screening of figitumumab patients for these mutations should not be necessary. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B119.