M. Dawn Teare

Genetic linkage studies

Linkage analysis is used to map genetic loci by use of observations of related individuals. We provide an introduction to methods commonly used to map loci that predispose to disease. Linkage analysis methods can be applied to both major gene disorders (parametric linkage) and complex diseases (model-free or non-parametric linkage). Evidence for linkage is most commonly expressed as a logarithm of the odds score. We provide a framework for interpretation of these scores and discuss the role of simulation in assessment of statistical significance and estimation of power. Genetic and phenotypic heterogeneity can also affect the success of a study, and several methods exist to address such problems.

Replication of Lung Cancer Susceptibility Loci at Chromosomes 15q25, 5p15, and 6p21: A Pooled Analysis From the International Lung Cancer Consortium

BACKGROUND Genome-wide association studies have identified three chromosomal regions at 15q25, 5p15, and 6p21 as being associated with the risk of lung cancer. To confirm these associations in independent studies and investigate heterogeneity of these associations within specific subgroups, we conducted a coordinated genotyping study within the International Lung Cancer Consortium based on independent studies that were not included in previous genome-wide association studies. METHODS Genotype data for single-nucleotide polymorphisms at chromosomes 15q25 (rs16969968, rs8034191), 5p15 (rs2736100, rs402710), and 6p21 (rs2256543, rs4324798) from 21 case-control studies for 11 645 lung cancer case patients and 14 954 control subjects, of whom 85% were white and 15% were Asian, were pooled. Associations between the variants and the risk of lung cancer were estimated by logistic regression models. All statistical tests were two-sided. RESULTS Associations between 15q25 and the risk of lung cancer were replicated in white ever-smokers (rs16969968: odds ratio [OR] = 1.26, 95% confidence interval [CI] = 1.21 to 1.32, P(trend) = 2 x 10(-26)), and this association was stronger for those diagnosed at younger ages. There was no association in never-smokers or in Asians between either of the 15q25 variants and the risk of lung cancer. For the chromosome 5p15 region, we confirmed statistically significant associations in whites for both rs2736100 (OR = 1.15, 95% CI = 1.10 to 1.20, P(trend) = 1 x 10(-10)) and rs402710 (OR = 1.14, 95% CI = 1.09 to 1.19, P(trend) = 5 x 10(-8)) and identified similar associations in Asians (rs2736100: OR = 1.23, 95% CI = 1.12 to 1.35, P(trend) = 2 x 10(-5); rs402710: OR = 1.15, 95% CI = 1.04 to 1.27, P(trend) = .007). The associations between the 5p15 variants and lung cancer differed by histology; odds ratios for rs2736100 were highest in adenocarcinoma and for rs402710 were highest in adenocarcinoma and squamous cell carcinomas. This pattern was observed in both ethnic groups. Neither of the two variants on chromosome 6p21 was associated with the risk of lung cancer. CONCLUSIONS In this international genetic association study of lung cancer, previous associations found in white populations were replicated and new associations were identified in Asian populations. Future genetic studies of lung cancer should include detailed stratification by histology.

Mutations in the Gene Encoding Capillary Morphogenesis Protein 2 Cause Juvenile Hyaline Fibromatosis and Infantile Systemic Hyalinosis

Juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) are autosomal recessive conditions characterized by multiple subcutaneous skin nodules, gingival hypertrophy, joint contractures, and hyaline deposition. We previously mapped the gene for JHF to chromosome 4q21. We now report the identification of 15 different mutations in the gene encoding capillary morphogenesis protein 2 (CMG2) in 17 families with JHF or ISH. CMG2 is a transmembrane protein that is induced during capillary morphogenesis and that binds laminin and collagen IV via a von Willebrand factor type A (vWA) domain. Of interest, CMG2 also functions as a cellular receptor for anthrax toxin. Preliminary genotype-phenotype analyses suggest that abrogation of binding by the vWA domain results in severe disease typical of ISH, whereas in-frame mutations affecting a novel, highly conserved cytoplasmic domain result in a milder phenotype. These data (1) demonstrate that JHF and ISH are allelic conditions and (2) implicate perturbation of basement-membrane matrix assembly as the cause of the characteristic perivascular hyaline deposition seen in these conditions.

Obesity, metabolic factors and risk of different histological types of lung cancer: A Mendelian randomization study.

Background Assessing the relationship between lung cancer and metabolic conditions is challenging because of the confounding effect of tobacco. Mendelian randomization (MR), or the use of genetic instrumental variables to assess causality, may help to identify the metabolic drivers of lung cancer. Methods and findings We identified genetic instruments for potential metabolic risk factors and evaluated these in relation to risk using 29,266 lung cancer cases (including 11,273 adenocarcinomas, 7,426 squamous cell and 2,664 small cell cases) and 56,450 controls. The MR risk analysis suggested a causal effect of body mass index (BMI) on lung cancer risk for two of the three major histological subtypes, with evidence of a risk increase for squamous cell carcinoma (odds ratio (OR) [95% confidence interval (CI)] = 1.20 [1.01–1.43] and for small cell lung cancer (OR [95%CI] = 1.52 [1.15–2.00]) for each standard deviation (SD) increase in BMI [4.6 kg/m2]), but not for adenocarcinoma (OR [95%CI] = 0.93 [0.79–1.08]) (Pheterogeneity = 4.3x10-3). Additional analysis using a genetic instrument for BMI showed that each SD increase in BMI increased cigarette consumption by 1.27 cigarettes per day (P = 2.1x10-3), providing novel evidence that a genetic susceptibility to obesity influences smoking patterns. There was also evidence that low-density lipoprotein cholesterol was inversely associated with lung cancer overall risk (OR [95%CI] = 0.90 [0.84–0.97] per SD of 38 mg/dl), while fasting insulin was positively associated (OR [95%CI] = 1.63 [1.25–2.13] per SD of 44.4 pmol/l). Sensitivity analyses including a weighted-median approach and MR-Egger test did not detect other pleiotropic effects biasing the main results. Conclusions Our results are consistent with a causal role of fasting insulin and low-density lipoprotein cholesterol in lung cancer etiology, as well as for BMI in squamous cell and small cell carcinoma. The latter relation may be mediated by a previously unrecognized effect of obesity on smoking behavior.

Optimizing the yield and utility of circulating cell-free DNA from plasma and serum

BACKGROUND Cell-free DNA (CFDNA) in the plasma/serum of patients with cancer demonstrates tumour-associated genetic alterations, offering possibilities for diagnosis, prognostication and disease monitoring. There is wide variation in the reported levels of CFDNA, associated with different methods used to collect, process and analyze blood samples. We therefore evaluated different aspects of laboratory protocols for the processing and purification of CFDNA in clinical studies. METHODS We evaluated and compared the QIAamp kit and a Triton/Heat/Phenol protocol (THP) for CFDNA purification. Total CFDNA was quantified by PicoGreen assay and SYBR-Green real-time PCR assay was used to amplify specific genes to estimate the efficiency of different protocols. RESULTS The efficiency of DNA extraction was 18.6% using the standard QIAamp protocol and 38.7% using the THP method (p < 0.0001, unpaired t-test). A modified QIAamp protocol that included a proteinase incubation stage and elution volumes up to 300 microl increased DNA yields, but was not as good as the THP method. CONCLUSIONS Blood samples should be kept at/or below room temperature (18 degrees C-22 degrees C) for no more than 2 h before plasma separation by double-spin. Because of its higher efficiency, low-cost and good-quality products, the THP protocol is preferred for extraction of CFDNA.

Sample size requirements to estimate key design parameters from external pilot randomised controlled trials: a simulation study

BackgroundExternal pilot or feasibility studies can be used to estimate key unknown parameters to inform the design of the definitive randomised controlled trial (RCT). However, there is little consensus on how large pilot studies need to be, and some suggest inflating estimates to adjust for the lack of precision when planning the definitive RCT.MethodsWe use a simulation approach to illustrate the sampling distribution of the standard deviation for continuous outcomes and the event rate for binary outcomes. We present the impact of increasing the pilot sample size on the precision and bias of these estimates, and predicted power under three realistic scenarios. We also illustrate the consequences of using a confidence interval argument to inflate estimates so the required power is achieved with a pre-specified level of confidence. We limit our attention to external pilot and feasibility studies prior to a two-parallel-balanced-group superiority RCT.ResultsFor normally distributed outcomes, the relative gain in precision of the pooled standard deviation (SDp) is less than 10% (for each five subjects added per group) once the total sample size is 70. For true proportions between 0.1 and 0.5, we find the gain in precision for each five subjects added to the pilot sample is less than 5% once the sample size is 60. Adjusting the required sample sizes for the imprecision in the pilot study estimates can result in excessively large definitive RCTs and also requires a pilot sample size of 60 to 90 for the true effect sizes considered here.ConclusionsWe recommend that an external pilot study has at least 70 measured subjects (35 per group) when estimating the SDp for a continuous outcome. If the event rate in an intervention group needs to be estimated by the pilot then a total of 60 to 100 subjects is required. Hence if the primary outcome is binary a total of at least 120 subjects (60 in each group) may be required in the pilot trial. It is very much more efficient to use a larger pilot study, than to guard against the lack of precision by using inflated estimates.

Atrial fibrillation associated with ivabradine treatment: meta-analysis of randomised controlled trials

Objective To quantify any risk of atrial fibrillation (AF) associated with ivabradine treatment by meta-analysis of clinical trial data. Methods Medline, Embase, Web of Knowledge and the Cochrane central register of controlled trials were searched for double-blinded randomised controlled trials of ivabradine with a minimum follow-up period of 4 weeks. For studies where AF data were unpublished, safety data were obtained from the European Medicines Agency (EMeA) website and personal communications. Studies were appraised for risk of bias using components recommended by the Cochrane Collaboration. Meta-analyses were performed of relative risk of AF and absolute risk difference of AF per year of treatment. The main outcome measure was incident AF during the follow-up period. Results AF data were available from 11 studies: one from the published report, six from the EMeA and four from personal communications. Ivabradine treatment was associated with a relative risk of AF of 1.15 (95% CI 1.07 to 1.24, p=0.0027) among 21 571 patients in the meta-analysis. From this we estimated that the number needed to harm for ivabradine would be 208 (95% CI 122 to 667) per year of treatment. Conclusions AF is a substantially more common side effect of ivabradine treatment than one patient in 10 000, the risk presently reported in the product literature. The incidence of AF has not routinely been reported in clinical trials of ivabradine.

Increased risk of lung cancer in individuals with a family history of the disease: A pooled analysis from the International Lung Cancer Consortium

BACKGROUND AND METHODS Familial aggregation of lung cancer exists after accounting for cigarette smoking. However, the extent to which family history affects risk by smoking status, histology, relative type and ethnicity is not well described. This pooled analysis included 24 case-control studies in the International Lung Cancer Consortium. Each study collected age of onset/interview, gender, race/ethnicity, cigarette smoking, histology and first-degree family history of lung cancer. Data from 24,380 lung cancer cases and 23,305 healthy controls were analysed. Unconditional logistic regression models and generalised estimating equations were used to estimate odds ratios and 95% confidence intervals. RESULTS Individuals with a first-degree relative with lung cancer had a 1.51-fold increase in the risk of lung cancer, after adjustment for smoking and other potential confounders (95% CI: 1.39, 1.63). The association was strongest for those with a family history in a sibling, after adjustment (odds ratios (OR) = 1.82, 95% CI: 1.62, 2.05). No modifying effect by histologic type was found. Never smokers showed a lower association with positive familial history of lung cancer (OR = 1.25, 95% CI: 1.03, 1.52), slightly stronger for those with an affected sibling (OR = 1.44, 95% CI: 1.07, 1.93), after adjustment. CONCLUSIONS The occurrence of lung cancer among never smokers and similar magnitudes of the effect of family history on lung cancer risk across histological types suggests familial aggregation of lung cancer is independent of those risks associated with cigarette smoking. While the role of genetic variation in the aetiology of lung cancer remains to be fully characterised, family history assessment is immediately available and those with a positive history represent a higher risk group.

The Gene for Juvenile Hyaline Fibromatosis Maps to Chromosome 4q21

Juvenile hyaline fibromatosis (JHF) is an autosomal recessive condition characterized by multiple subcutaneous nodular tumors, gingival fibromatosis, flexion contractures of the joints, and an accumulation of hyaline in the dermis. We performed a genomewide linkage search in two families with JHF from the same region of the Indian state of Gujarat and identified a region of homozygosity on chromosome 4q21. Dense microsatellite analyses within this interval in five families with JHF who were from diverse origins demonstrate that all are compatible with linkage to chromosome 4q21 (multipoint LOD score 5.5). Meiotic recombinants place the gene for JHF within a 7-cM interval bounded by D4S2393 and D4S395.

Localization of the gene for distal hereditary motor neuronopathy VII (dHMN-VII) to chromosome 2q14

Distal hereditary motor neuronopathy type VII (dHMN-VII) is an autosomal dominant disorder characterized by distal muscular atrophy and vocal cord paralysis. We performed a genomewide linkage search in a large Welsh pedigree with dHMN-VII and established linkage to chromosome 2q14. Analyses of a second family with dHMN-VII confirmed the location of the gene and provided evidence for a founder mutation segregating in both pedigrees. The maximum three-point LOD score in the combined pedigree was 7.49 at D2S274. Expansion of a polyalanine tract in Engrailed-1, a transcription factor strongly expressed in the spinal cord, was excluded as the cause of dHMN-VII.