Genome-Wide Association Studies of Hypertension: Light at the End of the Tunnel

Despite its significant genetic component, the study of hypertension by genome-wide association presents more challenges than other common complex diseases. Its high prevalence, heterogeneity, and somewhat unclear definition are the challenges that need to be overcome on one hand. On the other hand, there are issues of small effect sizes and pleiotropism that are not specific to hypertension alone but nonetheless magnify the problems of genetic dissection when coupled with phenotypic misclassification. We discuss issues of study design and summarise published genome-wide association studies (GWASs) of hypertension and blood pressure. With careful study design and analysis success is possible, as demonstrated by the recent large-scale studies. Following these, there is still further scope to advance the field through high fidelity phenotyping and deep sequencing.

Introduction
Hypertension is the major factor responsible for the most deaths worldwide (around 7 million or 12.8% per year) and this is 46% more than tobacco usage, the next major risk factor [1]. From an epidemiological and clinical perspective, blood pressure at the higher end of the normal population distribution is associated with an increased risk of cardiovascular mortality and morbidity. But clinical risk assessment is based on a predefined threshold at which the quantitative blood pressure phenotype in converted into a binary trait (hypertension) and management strategies are directed towards blood pressure reduction below this threshold at which the risk of excess cardiovascular events is abolished. Hence large-scale efforts to dissect the genetic underpinnings of hypertension are justified. While the causation of hypertension is multifactorial with both genetic and environmental components, the purpose of this paper is to discuss the genetic determinants of high blood pressure and GWAS study design.
For any genetic study, the key initial step is phenotypic specification. Here whether studying blood pressure as a quantitative trait or the qualitative hypertension phenotype depends on whether one follows the Pickering argument that blood pressure is inherited as a "graded character," and hence a complex non-Mendelian trait, or the Platt suggestion that hypertension is a qualitative abnormality and a simple Mendelian disease [2]. The observed normal unimodal distribution of blood pressure and its complex multifactorial aetiology support studies of blood pressure as a quantitative phenotype. According to the latter argument, if hypertension is a dichotomous risk trait, one would expect a bimodal distribution and this is not observed. However both approaches are useful and complementary in the genetic dissection of this trait which has so far been extremely resistant to the GWAS approach.

Evidence for a Genetic Component in Hypertension
There are multiple strands of evidence showing that genetic factors contribute to blood pressure and hypertension. Firstly, the normal distribution of blood pressure in the general population indicates the presence of multiple environmental and genetic factors and thus a polygenic aetiology. Secondly, rare monogenic forms of hypertension associated with major defects in renal salt handling prove that gene 2 International Journal of Hypertension mutations can cause hypertension leading to a hypothesis that minor variations in these genes may contribute to the common essential hypertension. Finally from a population perspective, there is considerable evidence from twins and family aggregation studies indicating the presence of a heritable component. It is estimated that around 30% of variation in blood pressure is due to genetic factors [3]. Hypertension is about twice as common in individuals who have one or two hypertensive parents, and blood pressure is more closely correlated in monozygotic than dizygotic twins [4,5]. In the Montreal Adoption Study investigators compared blood pressure correlation between biological sibling pairs and adoptive sibling pairs (as well as parent-child correlations). SBP correlation coefficients were 0.38 and 0.16 for biological and adopted siblings, respectively, and DBP coefficients 0.53 compared with 0.29 respectively [6].
Two measures that are commonly used to assess the genetic component of a trait are heritability (h 2 ) which is the fraction of variation in disease susceptibility due to genetic factors, and sibling recurrent risk (λs) which is the degree of elevated risk of disease for a sibling of an affected individual compared with a member of the general population. The heritability of clinic systolic blood pressure is around 15-40% and 15-30% for clinic diastolic blood pressure [7,8]; whereas for ambulatory night-time systolic and diastolic BP the heritabilities are 69% and 51% [7]. It is pertinent to point out that though the heritability estimates are considerable, this does not equate to magnitude of genetic effect. This is because the denominator in the estimate of heritability comprises measurement error and variances attributable to genes, shared environment, unshared environment and unmeasured determinants. This is illustrated by the example above where minimizing measurement errors by using ambulatory night-time values inflates the heritability estimates. Heritability is also a property of the population studied and low heritability estimates would suggest that genetic mapping would be difficult for that phenotype. The sibling recurrent risk of hypertension is around 1.2-1.5 [9], indicating a phenotype with modest genetic effect.

Strategies for Gene Mapping of Complex Diseases
The field of common complex disease genetics has in recent years moved from linkage to association study design because association analysis has far greater power to detect variants of modest effect and of lower frequency. The initial successes of hypertension gene mapping have come from studies of monogenic syndromes. The monogenic diseases are examples of diseases which are under very strong negative selection primarily because they affect fitness and are less likely to be transmitted to the next generation. The monogenic Mendelian forms of hypertension are thus rare, and responsible gene mutations are highly penetrant, and under very strong selection which keeps them at low frequencies with high levels of allelic heterogeneity. Thus these are highly amenable to linkage analysis. In contrast, susceptibility variants involved in essential hypertension are likely to have low or medium penetrance and are probably not subject to such strong selection resulting in lower allelic heterogeneity and greater prevalence of the trait. Thus linkage analysis as expected has not really provided robust validated loci for hypertension. To detect loci conferring a genotypic relative risk of 1.5 (minor allele frequency (MAF) = 0.1) by linkage analysis requires an estimated 67,816 affected sibling pairs (ASPs) whereas detection is possible through association with just 2218 singletons [10]. Moreover it is easier to recruit participants from the general population (than families as required for linkage), and there are fewer sampling restrictions in some disease categories such as late onset. Association studies are typically performed in unrelated population samples (although it is possible to conduct them on related individuals). For qualitative traits they measure statistical association between a disease (phenotype) and genetic marker (genotype) directly by comparing allele frequencies of cases and controls. The aim is to establish whether a particular allele occurs in cases, compared with controls, more frequently than expected by chance. Quantitative traits, for example, blood pressure, cholesterol, and glucose, are assessed for association using linear regression. GWAS is typically an indirect or map-based approach. This measures the association between a phenotype and a marker allele (or "tag" SNP), which is correlated with the true causal allele due to linkage disequilibrium (LD). Linkage disequilibrium is defined as "the statistical association, within gametes in a population, of the alleles at two loci" [11] (on the same chromosome). It is assumed that typically a causal variant will not have been typed in GWAS which take advantage of LD to genotype a set of tag SNPs as proxies for the entire set of SNPs. The amount of LD between two loci is summarised by the metric r 2 (squared correlation coefficient for each SNP) which varies between 0 and 1. The maximum r 2 can be used to translate coverage (measure of how well the SNPs that are part of a genotyping set capture all known variants) to the sample size that is required for an indirect association study. To cover unobserved loci well an r 2 value of ≥ 0.8 with typed loci is considered sufficient [12]. In general SNPs in LD are more likely to be inherited together because they are physically close to each other on the chromosome. But this is not necessarily the case; studies have shown that levels of local LD vary, with some adjacent SNPs being independent despite their proximity and others of ≥100 kb apart being in a high or a significant LD [13]. Patterns of LD are affected by many factors such as population growth, population structure, admixture, natural selection, genetic drift, rate of recombination and mutation, and gene conversion [14].
Traditionally association studies tested hypotheses based on candidate genes, for which there was prior evidence (of known physiological pathways that affect the phenotype in question) that a genetic variant influenced disease risk. Unsurprisingly many of the candidate genes for hypertension are involved in sodium balance. No candidate gene study has yet demonstrated a reproducible association with hypertension [15]. There are several potential reasons for this, which International Journal of Hypertension 3 highlight the drawbacks of candidate gene studies: (i) the wrong genes may have been selected; (ii) the causative genes may be upstream or downstream from the genes studied; (iii) discovery of genetic variants in novel pathways is not possible as candidate gene studies rely on a priori information regarding disease mechanisms.
In addition to the above there are the possibilities of population stratification, phenotypic and locus heterogeneity, and insufficient sample size: problems common to candidate gene studies and GWAS. Finally the SNPs studied might not provide complete coverage of the variants within the genes.

The GWAS Strategy
The chance of detecting genetic variants that influence common disease depends on the underlying genetic architecture. That is to say, the number of susceptibility alleles, whether they are common or rare, their frequency, and whether their action is neutral or deleterious. Allelic spectra vary greatly between disease genes. Common alleles and those of high frequency are of course easier to detect, as are deleterious alleles. The entire GWAS strategy is based on the Common Disease Common Variant (CDCV) hypothesis, that the causative genes for common diseases have relatively simple allelic spectra, that is, one or a few predisposing alleles of relatively high frequency. For GWAS it has been suggested that, as a rough guide, SNPs should meet a threshold of MAF ≥ 1% [16] or MAF ≥ 5% [11] to be considered common. As yet there is insufficient empirical evidence to determine the validity of the CDCV hypothesis, and arguments for and against have been put forward. These are crucial to research using SNP mapping to predict common disease risk, which assumes that the theory is by and large accurate (linkage studies of families or ASPs, by contrast, are robust to allelic heterogeneity).
A key part of the argument against the CDCV hypothesis rests on the fact that the risk of common disease depends on the interaction of many genes and environmental factors. In particular late-onset disorders of high prevalence in modern western society have been heavily influenced by changes in lifestyle factors such as diet and physical activity, and not by common disease-predisposing alleles. The risk conferred by any one factor, whether genetic or environmental, is weak and most cases are not predominantly determined by genetic variance. On the other hand, the relative risks observed in family members (a more rapid than linear decline in risk with increasing distance of relationship) support the conclusion that the majority of risk is due to a modest number of loci with common disease-predisposing alleles. The existing evidence suggests that alleles of both high and low frequency play a part in common disease [17][18][19][20][21][22].
Wang et al. have argued against making a distinction between rare and common disease-predisposing alleles [12]. Instead they propose that the allelic spectrum of disease associated variants be considered in the context of all variants in the human genome. In this framework the neutral model is that the allelic spectrum of disease variants and that of all variants are the same. Most susceptibility variants would be rare (MAF < 1%); however SNPs with MAF > 1% would still account for more than 90% of genetic differences between individuals and therefore make a significant contribution to phenotypic variation [23]. This lies somewhere between the two opposing views regarding the CDCV hypothesis. Common diseases vary in their allelic spectra depending on the evolutionary forces exerted upon them; nevertheless it is estimated that each will likely have hundreds of common and rare variants contributing to their familial clustering [12].
The International HapMap Project is a global consortium mapping all common SNPs in different populations across the world. The availability of SNP maps from HapMap led to a revolution in the dissection of common diseases and traits based on the common disease common variant hypothesis using the GWAS approach. This is a hypothesis-generating approach where no assumptions are made regarding the location or function of the causal variant. The dense genotyping chips that are now available contain hundreds of thousands of tag SNPs and offer increasingly better coverage of the human genome (whether within or outside genes). Adequate coverage requires ≥300 000 SNPs with more needed for African samples due to greater genetic diversity in those populations [11] and thus less LD [24,25]. Though there is some concern that a set of tag SNPs that were selected in one population may not perform well in another [11,14], the availability of dense SNP arrays will overcome this. There is also evidence of good tag SNP transference across populations [26,27]. This is especially true for different populations within the same continent; the greatest disparities are between African and non-African samples.
While GWASs scan the genome for association signals without selecting for gene regions, Jorgenson and Witte have argued for a gene-centric approach to GWAS [28]. The reasons they outline are the following: genic variants are more likely to be functionally important than nongenic; variants in many genes are in lower LD than those outside genes so may be difficult to capture through indirect association. By focusing solely on genes and not the whole genome there is potential to increase coverage of genes and decrease the genotyping burden. The genic approach has greater power to detect variants within genes but suffers from a loss of power for nongenic variants. Despite this Jorgenson and Witte demonstrated empirically using HapMap data that it is more efficient in detecting causal variants than the indirect whole-genome approach when related to genotyping burden. Their suggestion of the best overall GWAS approach is to combine indirect genotyping data with gene-based SNPs in high priority regions, or alternatively to use a more stringent LD threshold in genic regions to "over-capture" genic SNPs.
A limitation of GWAS is that they are very expensive, especially with the large sample sizes that are required for small effects. However, technological advancements are rapidly reducing the cost of genotyping. In a bid to further reduce costs, some researchers have adopted a two-stage 4 International Journal of Hypertension GWAS study approach. In stage 1 a proportion of the samples are genotyped on all markers, and then in stage 2 a proportion of these markers are genotyped in the remaining samples [29]. Another approach to make studies more economical is the use of common controls for several groups of different disease cases. This was recently demonstrated by the Wellcome Trust Case Control Consortium (WTCCC) [30]. In 2008 Donnelly summarised all GWAS recorded at that time in the National Human Genome Research Institute catalogue [31], which amounted to more than 300 replicated associations for more than 70 common diseases and quantitative traits [32]. As of September 2009, there were more than 500 published GWAS at P ≤ 5 × 10 −8 (see Figure 1).

Study Design Issues in GWAS
5.1. Significance Thresholds. By convention statistical significance using frequentist methods is determined using the P-value threshold of .05, with values below this considered significant (i.e., there is evidence to reject the null hypothesis of no effect). This is not appropriate for GWAS because the large number of tests performed increases the chance of type I error. An alternative threshold, proposed by Risch and Merikangas and now widely adopted, is P < 5 × 10 −8 , which corresponds to an equivalent false positive rate of 5% for 1 000 000 independent tests of association [10]. This is calculated using the simple Bonferroni correction for multiple testing, which calculates a new significance threshold by dividing .05 by the number of tests performed. In practice this is conservative as it does not take levels of LD into account; the use of tagging SNPs means the genome can be covered sufficiently with around half this number of SNPs (i.e., ∼500 000). This threshold is preferable to the traditional .05 but it has been argued that P value alone is not adequate for assessing significance. In addition to the possibility of false positives within a study, the issue of multiple testing can be viewed in the context of replication across studies. If several groups publish the same nominally significant association and these are combined, then the problem arises [11].

Statistical Power.
Statistical power to detect a phenotype-genotype association is dependent upon the magnitude of effect, the frequency of causal allele(s), and the sample size. Moreover, for indirect association it is not only the disease predisposing allele frequency that matters, but also the marker allele (or tag SNP) frequency, and the power to detect an association is the greatest when these frequencies match [33]. The extent of LD also influences the likelihood of observing an association. However if the effect size is large this is less important with power being high even at low to moderate LD. Effect size for case control studies is measured as an odds ratio (OR), which estimates the odds of an individual in a given exposure group (i.e., with a certain genetic variant) being a case versus being a control. If the OR is significantly greater than 1 then the variant confers susceptibility to the disease, if it is significantly less than 1 then its effect is protective. Unfortunately large effects are usually rare.
For common complex diseases most published genetic effects have to date been modest (OR ∼ 1.1-1.5) [17,34], so a reasonable level of LD is necessary as well as the disease allele being common and close to the marker allele frequency. These conditions translate to a feasible sample size of several thousands of cases and controls. An exception, which no doubt increased expectations of similar findings, is the association between APOE4 and late-onset Alzheimer disease [35] for which the allelic OR is 3.3 [33]. As yet only a small percentage of the human genome has been subject to well-designed association study so it is unknown whether the published effect sizes are representative of the genome overall [12]. The effect sizes observed are expected due to the multifactorial nature of the diseases concerned and individually translate to only a small increase in population absolute risk. Multiple common risk variants of small effect have been combined theoretically, however, to construct risk scores of greater practical significance. Studies of the distribution of genetic effect sizes in other species such as rodents, Drosophila melanogaster, crops, and livestock suggest that there will be few genetic loci of large effect and many loci of small effect [36][37][38][39][40][41][42]. This view is now widely accepted in the field of common disease genetics [43]. [44][45][46]. It occurs when there are two or more strata in a population, and both the risk of disease and the frequency of marker alleles differ between strata. It therefore may appear that the risk of disease is related to the marker alleles when in fact it is not. A similar concept is admixture, which refers to "the mixture of two or more genetically distinct populations" [12]. The International HapMap Project has demonstrated clear genetic differences between geographically separated populations [47].

Population Stratification. Population stratification acts as a confounder and can result in artefactual evidence of association
The effect of stratification on analysis increases with increasing sample size because even modest levels of underlying population structure are amplified [48,49]. This has particular relevance to GWAS as they are employing larger and larger samples. A final important point is that confounding by population stratification tends to actually decrease (counter intuitively) with increasing number of ethnic groups [50]. This is because the direction of bias may differ between groups so that the overall combined effect is diluted. Whether population stratification is a real concern or not, to avoid any possibility of bias it is now commonplace in studies of unrelated cases and controls to employ stratification detection and correction methods [46].
Aside from matching cases and controls for genetic background and relevant environmental factors as well as possible and straightforward adjustment for ethnic group, a number of possible solutions to population stratification have been proposed. One is genomic control (GC) which employs detection and correction methods, for example, by using a bank of randomly selected markers (preferably > 100 [11]) that are unrelated to the question of interest to assess   International Journal of Hypertension artefactual association [48,[51][52][53]. A scaling factor is then applied to the association results to adjust for the level of ethnic variation observed. A similar approach is structure assessment (SA), which too uses unlinked genetic markers for detection but then attempts to match homogeneous subgroups of the sample for association analysis within these subpopulations [54][55][56][57]. It is assumed that any significant association observed within a subpopulation cannot be due to population structure; there is an issue, however, about how many subpopulations to apply since they are a theoretical concept [11]. Explicit detection/correction methods by principal components analysis have also been employed [58,59]. Due to the large number of markers genotyped in GWAS it is possible to detect low levels of stratification. However, a caveat to all of these detection methods is that with a large enough sample size even small biases will be statistically significant and may lead to overcorrection.

Replication.
Many published association findings have failed to be replicated. This is partly due to the so-called "winner's curse," which is a bias whereby genetic effect size estimates are overestimated in initial discovery studies of disease-predisposing variants. The degree of effect size inflation can be reduced or eliminated by increasing sample size, and several correction methods have been proposed (e.g., [60,61]). Technical bias can occur if cases and controls are not genotyped and analysed together in the same way. It is also thought that poor choice of controls and population stratification affect data quality and therefore play a part in replication failure. Meta-analyses that have examined replication failure indicate that in the majority of instances false positives are to blame [17,62].
Replication of GWAS findings is as important as replication of candidate gene associations, if not more so. The large number of SNPs studied in GWAS and resulting volume of statistical tests performed increases the likelihood of observing type I errors, that is, false positives. In 2007 an NCI-NHGRI (National Cancer Institute and the National Human Genome Research Institute in the US) Working Group on Replication in Association Studies published an excellent summary of their recommendations on the reporting of initial association studies and criteria for replication [63]. There are certain situations in which there are insufficient participant numbers for replication, such as rare diseases or environmental exposures. These concerns do not affect most association studies of common diseases, though, so usually replication is advised. A strategy that increases power over that of individual studies and may be more cost effective than replication is meta-analysis of genome wide datasets [64,65]. This collaborative way of working is increasingly common as investigators attempt to detect loci with smaller effects.

GWAS of Hypertension/Blood Pressure
To date there have been few GWAS of hypertension and/or blood pressure, with varying degrees of success in detecting associations with genetic variants. The WTCCC, made up of over 50 British research groups, conducted a GWAS study of 2000 cases each for 7 complex diseases of major public health importance; bipolar disorder, coronary artery disease, Crohn's disease, hypertension, rheumatoid arthritis, type 1 diabetes, and type 2 diabetes. These were compared with 3000 shared common controls that came from two sources: 1500 from the 1958 British Birth Cohort and 1500 blood donors that were recruited for the project. The 2000 hypertension cases were unrelated participants from the British Genetics of Hypertension (BRIGHT) study [9]. Over the entire genome there were 21 SNPs identified with P values lower than the genome wide significance threshold of 5 × 10 −7 . Of these, 10 were known associations. Unfortunately, of the 7 diseases of interest, hypertension could be described as the loser in that it was not associated with any SNPs at P < 5 × 10 −7 . Moreover there was no evidence for any of the variants previously associated with hypertension (at least partly due to some not being well tagged by the Affymetrix chip, for example, promoter of the WNK1 gene). There were, however, a similar number and distribution of marginal results (with P-values between 10 −4 and 10 −7 ) to the other case groups.
It was speculated that the lack of a positive result for hypertension in the WTCCC study may have been due to poorly tagged variants or that hypertension may have few common risk alleles with larger effect sizes. Furthermore, misclassification bias may have reduced the power of detecting effects. The common controls were not specifically phenotyped for blood pressure. Due to the high prevalence of hypertension and its existence on the continuum of blood pressure some of the controls may have been misclassified cases. It was estimated that the misclassification of 5% of controls (i.e., if 5% of controls were in fact undiagnosed cases) would translate to a loss of power equivalent to a 10% reduction in sample size [30]. This is because of the dilution of any observable genetic difference, caused by the blurring of the distinction between cases and controls. Considering the expense of genome-wide association analysis and the anticipated relatively small effect sizes any reduction in power poses a serious problem. Moreover individuals with blood pressure in the mid-range of normotension (that is not considered to pose a risk clinically) may still be at increased risk in relation to individuals with low-blood pressure.
Two recent studies that have had considerably more success are the Global Blood Pressure Genetics (Global BPgen) Consortium study, conducted by Newton-Cheh et al. (2009) [71], and the Cohorts for Heart and Aging Research in Genome Epidemiology (CHARGE) Consortium study, conducted by Levy et al. (2009) [72]. But in contrast to the WTCCC, these were studies of blood pressure as a quantitative trait and on hypertension. Both had very large discovery samples, providing sufficient power to overcome variations in genotyping platform, participant ascertainment and method of blood pressure measurement between component studies. Each dealt with the confounding effect of antihypertensive medication by adding 15 mmHg to recorded SBP and 10 mmHg to DBP for those who were on such treatment.
In the Global BPgen discovery GWAS (N = 34, 433) two SNPs achieved P < 5 × 10 −8 (considered genome wide significance for this analysis). Once these and borderline significant findings were meta-analysed with validation data there were eight loci associated with either DBP or SBP genome-wide. Investigators also assessed whether these loci were associated with hypertension. The initial GWAS was not conducted for hypertension due to lack of power, so instead the significant loci were examined in planned secondary analysis (N range = 57, 410-99, 802). In the secondary samples all eight alleles showed association with hypertension in the same direction of effect as continuous blood pressure. It should be noted that all of the reported associations translate to a very small change in blood pressure, approximately 1 mmHg per allele SBP or 0.5 mmHg per allele DBP. However, the effects of multiple variants can be combined to produce a meaningful change in population cardiovascular risk. Taking a genome wide significance threshold of P < 4 × 10 −7 , the CHARGE discovery GWAS (N = 29, 136) identified 13 significant SNP associations for SBP, 20 for DBP, and ten for hypertension. There is quite a bit of overlap between phenotypes with many of the top hits attaining significance in the same direction of effect for more than one phenotype.
The top ten loci for SBP, DBP, and hypertension (30 in total) in the CHARGE cohort were checked for significance in the Global BPgen results. One SNP for SBP, four for DBP, and one for hypertension were assessed for independent replication in Global BPgen. Five SNPs out of this six attained P < .008, the threshold for external replication in Global BPgen. When the results for the same 30 SNPs in both studies were analysed together by meta-analysis, there were four associations of genome wide significance (P < 5 × 10 −8 ) for SBP, six for DBP, and one for hypertension. Again effect sizes were very small, approximately 1 mmHg change in SBP per allele or 0.5 mmHg change in DBP per allele.
The remaining published studies have demonstrated far less success in identifying genetic variants that are associated with either hypertension, SBP, or DBP. Many have not observed any SNPs that reached genome-wide significance [66,67,[73][74][75]. One possible reason for this failure in some studies is that hypertension and/or blood pressure were not the primary trait of interest [73], or not of a priori interest when the cohort was recruited [75] therefore phenotyping may not have been necessarily thorough. Another possibility is that there were not enough SNPs studied to provide sufficient coverage of the whole genome; in some studies fewer than 100 000 SNPs passed quality control measures [66,67,74]. Table 1 summarises the most significant hits from GWAS of hypertension and/or blood pressure published since 2007 (other than WTCCC, Global BPgen, and CHARGE), along with meta-analysed discovery and replication results if replication was attempted. One study conducted by Sabatti et al. [75] is omitted from the table because the authors did not publish any results for blood pressure, instead reporting that analysis of blood pressure did not produce any genomewide significant results. However it was not the primary trait of interest; the authors also studied triglycerides, HDL, LDL, CRP, glucose, insulin, and BMI.
Levy et al. [66] and Wang et al. [67] failed to find any associations of genome-wide significance but had limited genomic coverage, studying just 70,897 and 79,447 SNPs, respectively. Furthermore the discovery sample employed by Wang et al. was small at 542 participants. Org et al. [68] and Cho et al. [69] also did not find any associations of genomewide significance.
The vast majority of GWAS thus far have been conducted on samples of Caucasian individuals of European ancestry.
One of the few studies to examine African Americans was that of Adeyemo et al. [70]. Their initial findings were promising; however replication was either not attempted (in the case of SBP) or the replication findings were in the opposite direction of effect (DBP and hypertension).

Future Directions
Following an initial dearth of positive results from GWAS of hypertension and blood pressure, the methodological advancement and achievements of recent studies provide optimism and guidance for continued work in this area. Where to now in the search for further novel gene polymorphisms? One approach that has been proposed to increase the likelihood of detecting genetic effects is the recruitment of hypercontrols and individuals with severe hypertension [15]. This requires high fidelity phenotyping, with the overall study design comparing cases and controls at the extreme high and low ends, respectively, of the blood pressure distribution. This would markedly reduce the risk of misclassification bias and thus minimise any resultant loss of power.
Much of the unexplained variation in blood pressure may be due to rare variants that are undetectable through traditional GWAS case-control study design. However, intensively genotyped samples are becoming available from the 1000 Genomes Project, an open resource catalogue of human genetic variation [76]. The project is run by an international consortium and aims to describe over 90% of genetic variation down to 1% MAF. To date the genomes of more than 1000 individuals have been sequenced (with planned expansion to 2000 individuals in 2010) and around 10 million novel variants identified. The resource will enable the study of low frequency variants and aid fine mapping of regions of interest. Furthermore, next-generation deep sequencing technology is now available which will also increase the chances of rare variant detection [77]. All of this is carried out with the ambition to improve the diagnosis and treatment of this widespread condition.


s2fieldsofstudy: Biology Medicine

text:
A phase 2, randomized, double-blind safety and pharmacokinetic assessment of respiratory syncytial virus (RSV) prophylaxis with motavizumab and palivizumab administered in the same season

Background Respiratory syncytial virus (RSV) is an important pathogen causing annual epidemics of bronchiolitis and pneumonia among infants worldwide. High-risk infants currently receive RSV prophylaxis with palivizumab, a humanized RSV monoclonal antibody (MAb). In preclinical in vitro and in vivo (cotton-rat model) studies, motavizumab, a new RSV MAb, was shown to have greater anti-RSV activity than palivizumab. Motavizumab is currently under review for licensing approval. Since both MAbs may be available concurrently, this study evaluated their safety and tolerability when administered sequentially during the same RSV season. Methods Between April 2006 and May 2006, 260 high-risk infants were randomly assigned 1:1:1 to receive monthly intramuscular injections: 2 doses of motavizumab followed by 3 doses of palivizumab (M/P); 2 doses of palivizumab followed by 3 doses of motavizumab (P/M); or 5 doses of motavizumab (control). Adverse events (AEs, serious AEs [SAEs]), development of antidrug antibody (ADA), and serum drug trough concentrations were assessed. Results Most children received all 5 doses (246/260 [94.6%]) and completed the study (241/260 [92.7%]). While overall AE rates were similar (mostly level 1 or 2 in severity), SAEs and level 3 AEs occurred more frequently in the M/P group (SAEs: 22.9% M/P, 8.4% P/M, 11.8% motavizumab only; level 3 AEs: 15.7% M/P, 6.0% P/M, 6.5% motavizumab only). This trend in AE rates occurred before and after switching from motavizumab to palivizumab, suggesting a cause other than the combined regimen. Frequencies of AEs judged by the investigator to be related to study drug were similar among groups. Two deaths occurred on study (both in the M/P group, before palivizumab administration); neither was considered by the site investigator to be related to study drug. Mean serum drug trough concentrations were comparable among groups; ADA detection was infrequent (5.1% or less of any group). Conclusions The conclusions drawn from this study are limited by the small sample size per group. However, within this small study, overall AE rates, serum drug trough concentrations, and development of ADA associated with administering motavizumab and palivizumab sequentially to high-risk children appear comparable to administering motavizumab alone during the same RSV season. Trial Registration clinicaltrials.gov NCT00316264

children at high risk for severe RSV disease, including premature infants, infants with chronic lung disease (CLD), and infants with complicated congenital heart disease [4][5][6].
These high-risk infants currently receive prophylaxis for RSV with palivizumab (MedImmune, Gaithersburg, MD, USA), which is recommended and indicated for the prevention of severe RSV disease in high-risk children [7,8]. Palivizumab is a humanized monoclonal antibody that recognizes a highly conserved neutralizing epitope in the A antigenic site of the F glycoprotein of RSV [9]. Monthly palivizumab administration has been shown to reduce RSV hospitalizations by approximately 50% compared with placebo in high-risk children with prematurity, CLD of prematurity, and congenital heart disease [10][11][12]. In addition, in the IMpact study, preterm infants without CLD who received prophylaxis with palivizumab had an even greater reduction in RSV-related hospitalizations that approached 80% [12].
Motavizumab (MEDI-524, MedImmune) is an investigational monoclonal antibody developed by affinity maturation of palivizumab. Compared with palivizumab, motavizumab has an approximately 75-fold greater affinity for the RSV F protein [13,14], is approximately 20-fold more active in microneutralization studies, and, in the cotton rat model, reduces nasal and lung RSV titers 25and 100-fold, respectively [14,15]. In comparison with controls, in a mouse model of RSV, motavizumab was also found to be associated with significant reductions in RSV replication and concentrations of cytokines (interleukin-1 alpha, interleukin-12p70 and tumor necrosis factor alpha, interferon gamma) that are probably related to improvements observed in clinical markers of disease severity [16].
Early pediatric clinical studies of motavizumab in which children received monthly intramuscular dosing demonstrated no dose-limiting toxicities, and serum pharmacokinetics were found to be consistent with published data for palivizumab [17,18]. In addition, in a proof-of-concept phase 1 study, children hospitalized with RSV illness were given a single dose of intravenous motavizumab or placebo [18]. In that study, motavizumab significantly reduced viral load and culturable RSV was eliminated 1 day post-treatment in a greater proportion of treated children compared to those who received placebo. Significant anti-RSV effects were not seen in the upper respiratory tracts of infants in a similar study conducted with palivizumab [19]. Recently, a large clinical study of over 6600 infants [20] to evaluate monthly immunoprophylaxis during the RSV season with motavizumab compared to palivizumab was completed. In this study of premature infants with and without CLD, motavizumab was shown to be noninferior to palivizumab with a 26% relative reduction in the primary endpoint of RSV hospi-talizations (1.4% vs 1.9%, respectively; relative risk, 0.740; 95% confidence interval: 0.503, 1.083) [20]. In addition, in the outpatient setting, a secondary endpoint, motavizumab was superior to palivizumab, effecting a 50% relative reduction of RSV-specific, medically attended, lower respiratory tract infections (2.0% vs 3.9%, respectively; P = 0.005) [20].
Motavizumab is currently under review by the US Food and Drug Administration for the prevention of serious lower respiratory tract disease caused by RSV in children at high risk of RSV disease. Because it is likely that initially both products will be available concurrently for commercial use, there could be times when a child receives both agents sequentially within the same RSV season. Therefore, the primary objective of this study was to provide initial data on the safety of motavizumab and palivizumab when administered sequentially to high-risk, preterm infants during the same RSV season. Additional objectives of the study were to determine serum trough concentrations of motavizumab and palivizumab when administered during the same RSV season and to evaluate the development of antimotavizumab and antipalivizumab antidrug antibodies (ADA).

Study Design
This was a phase 2, randomized, double-blind study in which motavizumab and palivizumab were administered during the same RSV season to premature infants with and without CLD. The study was conducted between April 2006 and February 2007. During the 2006 RSV season, eligible infants were enrolled at 18 sites in the southern hemisphere, which included Chile (7 sites), New Zealand (5 sites), and Australia (6 sites). Eligible children were randomly assigned (1:1:1, stratified by site) to one of 3 treatment groups using an automated randomization system: 2 doses of motavizumab followed by 3 doses of palivizumab (M/P); 2 doses of palivizumab followed by 3 doses of motavizumab (P/M); or 5 doses of motavizumab only (control). Motavizumab and/or palivizumab were administered at 15 mg/kg by intramuscular injection every 30 days, for a total of 5 planned doses. These are the prescribed dose and regimen as currently used for palivizumab. Doses were administered on study days 0, 30, 60, 90, and 120. Drug administration of dose 2 (day 30) was allowed between day 25 and day 30. For all other doses, a prespecified ± 5-day window was allowed.
The study was conducted in accordance with the Declaration of Helsinki and in compliance with the ethical principles of the International Conference on Harmonization Guidelines for Good Clinical Practice. The study was approved by the institutional review board or independent ethics committee of each participating center.
Parents or legal guardians provided written informed consent for each subject before enrollment.

Study Endpoints
The primary endpoints were safety and tolerability of motavizumab and palivizumab administered sequentially during the same RSV season, as assessed by summarizing adverse events (AEs), serious AEs (SAEs), and laboratory evaluations. Secondary endpoints were ADA and serum trough concentrations of motavizumab and palivizumab. AEs were defined as any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease that occurred while the child was enrolled in the study from the time of randomization through study day 150, whether or not the event was considered to be related to the study treatment. SAEs were defined as AEs that resulted in death, risk of life, hospitalization or prolongation of existing hospitalization, persistent or significant disability or incapacity, or that required medical or surgical intervention to prevent any of these outcomes. AEs and SAEs were summarized by system organ class and preferred terms using the Medical Dictionary for Regulatory Activities (MedDRA), by severity (levels 1-4, which corresponded to mild, requiring no treatment; moderate, possibly requiring symptomatic therapy; severe, generally requiring a more immediate medical evaluation or treatment; and life-threatening, requiring immediate medical attention to support vital functions, respectively), and by relationship to study treatment (none, remote, possible, probable, definite). AEs were assessed by the investigator for severity, relationship to the study treatment, and whether the event met criteria as an SAE (ie, not all level 4 AEs, such as laboratory abnormalities, met criteria for SAE). All summaries of AEs include both nonserious AEs and SAEs.

Study Inclusion and Exclusion Criteria
Preterm children were eligible to participate if the gestational age was ≤35 weeks and the chronologic age was ≤6 months at the time of entry into the study, or if they were ≤24 months of age at the time of entry into the study and had a diagnosis of CLD of prematurity requiring medical management within 6 months before randomization. Eligible children had to be in good health at the time of study entry. They could not be hospitalized (unless discharge was expected within 10 days); be receiving chronic oxygen therapy or any ventilatory support; have significant congenital heart disease; have evidence of infection with hepatitis A, B, or C virus or HIV; have any acute illness, including acute RSV infection; known renal impairment, hepatic dysfunction, chronic seizure disorder, or immunodeficiency; have suspected serious allergic or immune-mediated events in association with prior receipt of immunoglobulins, blood products, or other foreign proteins; have received within the past 120 days or currently be receiving immunoglobulin products, palivizumab, or any investigational agent.

Study Assessments
Subjects were evaluated before each injection of study drug, at study day 150, which corresponded to 30 days after the fifth and final planned dose of study drug, and at the final follow-up visit between study days 270 and 300, which corresponded to 120 to 150 days after the fifth dose of study drug. Subjects were monitored for AEs and SAEs from the time of the first study drug administration through study day 150. Blood was collected to assess serum drug trough concentrations and development of ADA on day 0 (pre-drug baseline), day 60 (before dose 3), and day 150. Final assessments of antimotavizumab and antipalivizumab antibody and serum trough concentrations of motavizumab and palivizumab were performed between study days 270 and 300.
Enzyme linked immunosorbent assays to quantify ADAs and trough serum concentrations of motavizumab and palivizumab were developed by MedImmune and validated and performed by PPD Development, LP (Richmond, VA). These methods have been described previously [9,17]. No data on the prevention of RSV disease were collected.

Study Products
Study drugs were provided in sterile, preservative-free vials containing 100 mg motavizumab in 1 mL 25 mM histidine-HCL, pH 6.0, or 100 mg palivizumab in 1 mL 25 mM histidine, 1.6 mM glycine, pH 6.0. Both study products were stored at 2° to 8°C. Motavizumab was administered at the same prescribed dose and regimen as is currently used for palivizumab (15 mg/kg administered by intramuscular injection at monthly intervals for 5 months).

Statistical Methods and Analysis Populations
As no hypothesis testing was planned to compare treatments, no formal sample size calculations were performed. However, AE rates were compared between treatment groups using a 2-sided Fisher's exact test to note significant differences. Statistically significant differences were reported for P values < 0.05. Given the number of subjects enrolled and included in the safety analyses, the minimum statistical difference that could have been detected with 80% power between the control arm (N = 93) and either mixed-dose arm (N = 83) was 10 percentage points for event rates of 1.0% and 22 percentage points for event rates of 50%. For example, if one treatment group had an event rate of 1%, an event rate of at least 11% would have been necessary in the comparison treatment group to allow at least an 80% chance of detecting a statistically significant difference between the two treatment groups. Similarly, if one treatment group had an event rate of 50%, an event rate of at least 72% would have been necessary in the comparison treatment group. Due to the number of tests that were performed, the P values should be interpreted with caution. No adjustments for multiple comparisons were made.
Statistical analyses were performed using SAS/STAT ® Version 8.2. Categorical data are summarized by the number and percent of subjects in each category. Continuous variables are summarized by descriptive statistics including mean, standard deviation, minimum, and maximum. Study day 0 was defined as the day of randomization.
The intent-to-treat population included all randomized subjects. The safety population included all randomized subjects who received study drug and had any safety follow-up. The serum drug trough concentration and ADA population included all subjects in the safety population who did not receive commercial palivizumab during the 120 days before study day 0. Subjects were excluded from any individual time-point summary of serum drug trough concentration and ADA if they did not receive the correct number of study treatment doses before that time point.

Study Population
A total of 260 subjects were enrolled into the 3 treatment groups during April and May 2006: M/P, n = 83; P/M, n = 84; and motavizumab only (control), n = 93; 259 subjects received at least one dose of study drug. Demographic characteristics at study entry are presented in Table 1. Overall, the treatment groups were balanced with respect to chronologic age, weight, and gestational age. There were 2 minor imbalances noted that were not felt to affect the outcome of the study: 1) the baseline incidence of CLD was slightly lower in the M/P treatment group (13.3%) than in the P/M and motavizumab-only (control) treatment groups (16.7% and 17.2%, respectively); and 2) there were slightly more male subjects enrolled in the M/ P treatment group (61% in M/P compared with 51% and 52% in the P/M and the motavizumab-only [control] groups, respectively).

Subject Compliance, Disposition, and Discontinuations
Subjects who remained in the study for the final followup visit between study days 270 and 300 were deemed to have completed the study. The majority (246/260, 94.6%) of subjects received all 5 doses of study drug, and a total of 241/260 (92.7%) subjects completed the study ( Figure  1). Two children who had study drug discontinued due to SAEs continued with follow-up until the completion of the study; both were in the M/P group (one developed erythema multiforme after 2 doses; another developed staphylococcal scalded skin syndrome after 3 doses). Another child who discontinued study drug after an SAE of visual disturbance (also in the M/P group) after receiving 1 dose of study drug did not continue in the study. All 3 of these children were included in the safety population. One subject assigned to the P/M treatment group did not receive study drug and was therefore not included in the safety, ADA, or serum drug trough concentration analyses.

Safety: Adverse Events
A comparative overview of AEs for the 3 groups overall and by severity is presented in Table 2. Overall, 89% to 93% of subjects in all 3 groups experienced at least 1 AE.
In general, AE rates were similar among the treatment groups, with the exception of level 3 events reported as the highest severity and SAEs, for which the M/P treatment group had more subjects with these events than the other 2 groups. In the M/P group, 15.7% reported at least one level 3 AE, compared with 6.0% in the P/M group and 6.5% in the motavizumab-only (control) group. The incidence of level 3 and level 4 AEs in the M/P group was significantly higher compared with the P/M group (19.3% vs. 6.0%, respectively; P < 0.05) but did not differ significantly from the rate in the motavizumab-only (control) group (10.8%). More than half of level 3 or level 4 events in any treatment group (15/17, 5/7, and 6/10 events in the M/P, P/M, and motavizumab-only [control] groups, respectively) were classified in the MedDRA "infections and infestations" system organ class. A total of 24 SAEs occurred in 19 children in the M/P group, 9 SAEs occurred in 7 children in the P/M group, and 12 SAEs occurred in 11 children in the motavizumab-only (control) group (Table 2).
Overall, the types and numbers of specific AEs were similar among the 3 treatment groups; those occurring in at least 5% of subjects in any group are summarized in Table 3. The most frequent AEs were infectious in nature and were expected for this population of high-risk children. Due to the higher frequencies of level 3 AEs (as the highest severity) and SAEs in the M/P treatment group, a post hoc analysis was performed to determine the incidence of AEs before and after receipt of dose 3 (when the switch to the second study drug occurred). It is important to note that the reporting period for this analysis encompassed 60 days before dose 3 and 90 days after dose 3. An overview of safety data before and after dose 3 is presented in Table 4. The increases in level 3 or 4 AEs and SAEs were consistently observed both before and after dose 3 in the M/P group. After 2 doses of study drug (ie, prior to dose 3), all groups had comparable rates of AEs and AEs deemed by the site investigators to be related to Figure 1 Subject disposition through the end of the study period. a Intent-to-treat population. b Includes 1 subject with an SAE of visual disturbance who discontinued drug after 1 dose and did not complete the study; 2 additional subjects discontinued drug after receiving < 5 doses of study drug (1 with erythema multiforme after 2 doses and 1 with staphylococcal scalded skin syndrome after 3 doses, both in the M/P group), but since they were followed through the end of the study period are classified as having completed the study, thus they are not included in this total. c Includes 1 subject for whom consent was withdrawn on day 13 and who died of a bowel obstruction on day 153. d Completed the study through study days 270-300 (120-150 days after the final dose). study treatment. Before dose 3, the rates of level 3 AEs as the highest severity, level 4 AEs, SAEs, SAEs judged by the investigator to be related to study drug, and AEs resulting in discontinuation of study drug were all higher among children in the M/P group than in the motavizumab-only (control) treatment group. Some of these differences persisted after receipt of dose 3 of study drug, with the M/P group exhibiting higher rates of level 3 AEs (as the highest severity) and SAEs compared with either the motavizumab-only (control) or the P/M treatment group. The number of children who experienced SAEs before and after dose 3 was similar for both dosing periods in each group. In the M/P group, 14 of the 24 SAEs were level 3 or level 4 in severity; similarly, 14 of the 17 level 3 or level 4 AEs were considered SAEs. In the P/M group, 4 of the 9 SAEs were level 3 in severity; likewise, 4 of the 7 level 3 AEs were considered SAEs. No level 4 AEs were reported in this group. In the motavizumab-only (control) group, 7 of the 12 SAEs were level 3 or level 4 in severity; similarly, 7 of the 10 level 3 or level 4 AEs were considered SAEs. At least one-half of SAEs in any treatment group (19/24 in the M/P group, 7/9 in the P/M group, 6/12 in the motavizumab-only [control] group) were infection-related. In the M/P group, a total of 12 of 24 SAEs occurred after receipt of dose 3 (ie, when sub-jects had received both study drugs). All 12 were infectious in nature, and 11 of the 12 were respiratory infections. Ten of the 11 respiratory infections had local viral testing performed, with only 2 tests positive for Rsv.

Adverse Events Judged by the Site Investigators as Related to Study Treatment
Overall, the frequencies of AEs deemed by the investigator to be related to treatment with palivizumab or motavizumab were comparable among all 3 treatment groups (M/P, 16.9%; P/M, 19.3%; motavizumab only [control], 22.6%). The 2 most common AEs judged by the investigator to be related to study drug were injection-site erythema (M/P, 2.4%; P/M, 10.8%; motavizumab only, 8.6%) and irritability (4.8%, 7.2%, and 7.5%, respectively). The rate of injection-site erythema was significantly lower in the M/P group compared with the P/M group (P < 0.05), and the events were balanced before and after dose 3. These AEs were all level 1 in severity except in 2 subjects in the M/P group, each of whom experienced 1 level 2 event. All other drug-related AEs were level 1 or level 2 in severity, with the exception of 1 SAE of abnormal liver function tests in the motavizumab-only (control) group that was reported by the investigator at the day 150 study visit as level 4 in severity (reported as having returned to  normal as of 142 days after the child received the fifth and final dose of motavizumab).

SAEs Judged by the Site Investigators as Related to Study Treatment
Three SAEs were considered to be possibly or probably related to study treatment by the site investigators: visual disturbance, erythema multiforme (1 subject each; M/P group), and abnormal liver function tests (1 subject, motavizumab-only [control] group). Each subject had received only motavizumab before the onset of each SAE. The child with the reported event of visual disturbance was a 5-month-old male with a gestational age of 26 weeks, and a medical history of respiratory distress syndrome, bronchopulmonary dysplasia, necrotizing enterocolitis, gastroesophageal reflux, and intestinal obstruction. He had an initial retinopathy of prematurity screening at 6 weeks of age that was normal. He had received only 1 dose of motavizumab before the event. Two days after receiving motavizumab, the child was noted to have abnormal roving eye movements, which were random and not associated with any alteration in sensorium or vital signs. The event of visual disturbance  a The total number of subjects who received at least 3 doses of study drug. b None of the subjects who reported a level 4 AE had a level 3 AE. c Includes 1 subject with an SAE of visual disturbance who discontinued after 1 dose (followed to the end of the study period by an ophthalmologist, but did not complete the study follow-up) and 1 subject with an SAE of erythema multiforme who discontinued after 2 doses (followed to the end of the study period and completed the study follow-up). A third subject experienced staphylococcal scalded skin syndrome after receiving 3 doses and also completed the study follow-up. All 3 subjects are included in the safety population. d This subject withdrew consent on study day 13 and died on study day 153.
was reported by the investigator as an SAE of level 2 severity and was judged to be possibly related to study treatment because of the temporal relationship, and study drug was permanently discontinued. The child was withdrawn from the study but was monitored by an ophthalmologist through study day 150, with the visual disturbance still present. There was no formal diagnosis of his visual abnormality. The child with the event of erythema multiforme was a 2-month-old male with a gestational age of 34 weeks and a history of wet lung, hyperbilirubinemia, bilateral pyelectasis, anemia of prematurity, and seborrheic dermatitis. He had previously taken cefadroxil, but not for the preceding 10 days. Two days after the second dose of motavizumab he experienced an abdominal rash. Four days after receiving motavizumab he presented to the investigational site with a generalized rash that was felt to be consistent with erythema multiforme. There were no respiratory symptoms or mucosal involvement, and his general condition was good. A diagnosis of erythema multiforme minor was made. He was treated with chlorpheniramine with resolution of the rash 2 days later. This event was reported as an SAE of level 2 severity and judged by the investigator to be probably related to study treatment, and study drug was permanently discontinued after its occurrence. Study treatment was permanently discontinued because of an AE in a total of 3 patients (all in the M/P group). These included the events of visual disturbance and erythema multiforme, which are described above, and another child who experienced a level 3 SAE of staphylococcal scalded skin syndrome, which the investigator considered remotely related to study treatment. This event occurred 3 days after this child received the third dose of study drug (2 doses motavizumab and 1 dose palivizumab). The child's condition improved quickly and resolved after receiving antibiotics for bullous impetigo, and a dermatologist and infectious disease consultant felt that the clinical picture was consistent with staphylococcal scalded skin syndrome. This child and the child with erythema multiforme were monitored through the end of the study period.

Deaths
There were 2 deaths during the study (one case of pneumonia and one case of sepsis). Both subjects were in the M/P treatment group and both deaths occurred before receiving palivizumab. The child who died from pneumonia was born prematurely at 29 weeks' gestation with a medical history significant for CLD, apnea of prematurity, and previous infection. She received 2 doses of motavizumab, the last dose administered 13 days before her death. She was found not breathing on the morning of her death; autopsy findings were consistent with acute bronchopneumonia. The child who died from sepsis had a gestational age of approximately 35 weeks with a history of suspected sepsis at birth. She had received 1 dose of motavizumab 3 days before her death at 10 days of age. On the day of her death, she was found not breathing while sleeping in bed with her parents; autopsy findings were consistent with sepsis and included hepatitis, septic adrenalitis, and neonatal pneumonia. Neither child had a known diagnosis of RSV at the time of death, and both deaths were considered by the investigator to be unrelated to study treatment. A third child died on study day 153 of an intestinal obstruction. The child had received 1 dose of motavizumab at the time of withdrawal. This death occurred outside of the study period, 140 days after withdrawal of consent and beyond the AE follow-up period (day 150). It was not considered by the site investigator to be related to study drug and is presented here for completeness.

Antimotavizumab and Antipalivizumab Antibodies
Few subjects developed detectable antibodies to either study drug; ADA titers observed at different time points during the study are illustrated in Figure 2. A total of 13 subjects (M/P, n = 8; P/M, n = 4; motavizumab only, n = 1) had detectable antipalivizumab and/or antimotavizumab antibody at any time during the study. All antimotavizumab antibodies were detected at study day 150 and/ or 270-300 (except 1 subject in the P/M group who had antimotavizumab antibody detected at baseline prior to receiving study drug, and had no subsequent antibody detection after exposure to study drug). The subject in the M/P group with the SAE of erythema multiforme on study day 28 described above had antimotavizumab antibody of 1:50 detected on study day 150. Antimotavizumab antibody titers ranged from 1:40 to 1:1250 in the M/P treatment group and from 1:10 to 1:250 in the P/M treatment group. Antipalivizumab antibody titers were also low, ranging from 1:10 to 1:20 and were generally detected at study day 150 and/or 270-300. Cross-reactivity was low (1.1-1.3%). Only 1 subject each in the M/P and the motavizumab-only (control) treatment groups had detectable antipalivizumab antibodies without receiving palivizumab. The antipalivizumab titers of these subjects were low (≤1:20).

Serum Drug Trough Concentrations
Mean serum trough concentrations of motavizumab and palivizumab for each treatment group are illustrated in Figure 3. As expected, serum concentrations of each drug increased with treatment. On study day 60, mean serum trough concentrations of motavizumab in the M/P and motavizumab-only (control) groups were 74.74 μg/mL and 78.02 μg/mL, respectively. On study day 150, mean serum trough concentrations of motavizumab in the P/M and motavizumab-only (control) groups were 93.05 μg/ mL and 105.8 μg/mL, respectively. For palivizumab, the mean serum trough concentration in the P/M group was 87.37 μg/mL on day 60.

Discussion
In this small study evaluating the safety and tolerability of motavizumab and palivizumab administered sequentially during the same season for RSV prophylaxis, both study drugs were found to have an acceptable safety profile. Over 90% of subjects in the 3 treatment groups received all 5 doses of study drug and completed the study. Overall, the rates of AEs were similar among all 3 treatment groups. The most frequent AEs were infectious in nature, and were events common to and expected in this highrisk pediatric population. Of interest was the increased rate of level 3 AEs and SAEs in the M/P treatment group compared with the other treatment groups. This difference was present both before dose 3 (motavizumab for 2 doses) and after dose 3 (palivizumab for 3 doses), suggesting a cause other than receipt of both study drugs. In addition, before the switch from motavizumab to palivizumab, the rates of level 3 AEs as the highest severity, level 4 AEs, SAEs, SAEs judged by the site investigator to be related to study drug, and AEs that resulted in discontinuation of study drug were higher among children in the M/P group than in the motavizumab-only (control) group, again suggesting a difference that could not be explained by study treatment since both groups had received the same drug and number of doses.
When the increased incidence of SAEs in the M/P treatment group was further evaluated, it was noted that the increase in either time period was attributable to SAEs in the MedDRA "infections and infestations" system organ class. Since this was not an efficacy study, formal study data on RSV infections were not collected. However, some sites performed local RSV testing and reported the results. Of the 10 children in the M/P group with SAEs of respiratory infections who had local viral testing reported, 2 tests were positive for RSV. In addition, half of the SAEs in the M/P group occurred before dose 3 (ie, when subjects had received only motavizumab). For the 3 SAEs that were judged by the site investigators to be related to study treatment, all occurred after the receipt of motavizumab only.  Overall, antimotavizumab and antipalivizumab antibody detection was infrequent. A total of 13 subjects had antipalivizumab and/or antimotavizumab antibody at any time during the study, and no antimotavizumab antibody was detected in any subject in the motavizumab-only (control) treatment group. Mean serum trough concentrations of motavizumab and palivizumab in the mixeddose and motavizumab-only (control) treatment groups were comparable based on the number of doses received. Only one child had a reported event consistent with a hypersensitivity reaction (erythema multiforme minor) after 2 doses of motavizumab. This event subsequently resolved; further study dosing was discontinued by the site investigator. ADA was detected in this child on study day 150, 124 days after final dose on study day 26. The causal relation of ADA with the event cannot be determined. No other events that could be considered immune-mediated were noted in children with and without ADA.
The conclusions drawn from this study are limited by the small sample size per group. Yet, in this group of high-risk children, the rates of AEs, serum drug trough concentrations, and occurrence of ADA associated with the sequential administration of motavizumab and palivizumab appear comparable to those observed in subjects administered motavizumab alone during the same RSV season.
While it did not address effects of sequential dosing, a large clinical study by Carbonell-Estrany et al recently compared the safety and efficacy of motavizumab (n = 3315) to palivizumab (n = 3298) and identified no significant overall difference in subjects reporting at least 1 AE (motavizumab, 85.6%; palivizumab, 86.0%), nor in subjects reporting at least 1 SAE (motavizumab, 14.6%;  [20]. The overall rates of AEs and SAEs observed in the study by Carbonell-Estrany et al are similar to those in our study. Regarding AEs in the Med-DRA system organ class of "infections and infestations," the study by Carbonell-Estrany et al found rates that were comparable for motavizumab and palivizumab. The children who received motavizumab had an overall AE infection rate of 66.3% with an SAE infection rate of 8.2%. In the palivizumab group, the overall AE infection rate was 68.3% with an SAE infection rate of 9.3% (G.A. Losonsky, MedImmune, LLC, personal communication). It is likely that differences among groups in our study could be attributed to the small sample size and not to the study drug administered or the sequence of administration.

Conclusions
In this small study, motavizumab and palivizumab administered sequentially during the same RSV season exhibited an acceptable safety profile in high-risk preterm children. These results suggest that the safety, serum drug trough concentrations, and ADA profiles of palivizumab and motavizumab given in varying sequence are comparable to those observed in subjects receiving only motavizumab during the season.