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1.  Novel regional age-associated DNA methylation changes within human common disease-associated loci 
Genome Biology  2016;17:193.
Advancing age progressively impacts on risk and severity of chronic disease. It also modifies the epigenome, with changes in DNA methylation, due to both random drift and variation within specific functional loci.
In a discovery set of 2238 peripheral-blood genome-wide DNA methylomes aged 19–82 years, we identify 71 age-associated differentially methylated regions within the linkage disequilibrium blocks of the single nucleotide polymorphisms from the NIH genome-wide association study catalogue. This included 52 novel regions, 29 within loci not covered by 450 k or 27 k Illumina array, and with enrichment for DNase-I Hypersensitivity sites across the full range of tissues. These age-associated differentially methylated regions also show marked enrichment for enhancers and poised promoters across multiple cell types. In a replication set of 2084 DNA methylomes, 95.7 % of the age-associated differentially methylated regions showed the same direction of ageing effect, with 80.3 % and 53.5 % replicated to p < 0.05 and p < 1.85 × 10–8, respectively.
By analysing the functionally enriched disease and trait-associated regions of the human genome, we identify novel epigenetic ageing changes, which could be useful biomarkers or provide mechanistic insights into age-related common diseases.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-016-1051-8) contains supplementary material, which is available to authorized users.
PMCID: PMC5034469  PMID: 27663977
Epigenetics; DNA methylation; Ageing; Common Disease; Human
2.  The stress regulator FKBP51 drives chronic pain by modulating spinal glucocorticoid signaling 
Science translational medicine  2016;8(325):325ra19.
Polymorphisms in FKBP51 are associated with stress-related psychiatric disorders and influence the severity of pain symptoms experienced after trauma. Here, we report that FKBP51 (FK506 binding protein 51) is crucial for the full development and maintenance of long-term pain states and that this is independent from its effect on mood. Indeed, FKBP51 knock out mice but also mice with silencing of FKBP51 restricted to the spinal cord showed reduced hypersensitivity in a number of persistent pain models. FKBP51 deletion did not compromise the detection of acute painful stimuli, a critical protective mechanism. Moreover, the specific FKBP51 inhibitor SAFit2 intrathecally administered reduced the severity of an established pain state, confirming the crucial role of spinal FKBP51 in nociceptive processing. Finally, glucocorticoid signaling, which is known to modulate persistent pain states in rodents, was impaired in FKBP51 knock out mice. This suggested that FKBP51 regulates chronic pain by modulation of glucocorticoid signaling. In conclusion, FKBP51 is a central mediator of chronic pain, likely in humans as well as rodents, and is a new pharmacologically tractable target for the treatment of long term pain states.
PMCID: PMC4880036  PMID: 26865567
3.  Contribution of Heritability and Epigenetic Factors to Skeletal Muscle Mass Variation in United Kingdom Twins 
Skeletal muscle mass (SMM) is one of the major components of human body composition, with deviations from normal values often leading to sarcopenia.
Our major aim was to conduct a genome-wide DNA methylation study in an attempt to identify potential genomic regions associated with SMM.
This was a mixed cross-sectional and longitudinal study.
Community-based study.
A total of 1550 middle-aged United Kingdom twins (monozygotic [MZ] and dizygotic [DZ]), 297 of which were repeatedly measured participated in the study.
Main Outcome Measure:
Appendicular lean mass assessed using dual-energy X-ray absorptiometry technology, and methylated DNA immunoprecipitation sequencing DNA methylation profiling genome-wide were obtained from each individual.
Heritability estimate of SMM, with simultaneous adjustment for covariates obtained using variance decomposition analysis, was h2 = 0.809 ± 0.050. After quality control and analysis of longitudinal stability, the DNA methylation data comprised of 723 029 genomic sites, with positive correlations between repeated measurements (Rrepeated = 0.114–0.905). Correlations between MZ and DZ twins were 0.51 and 0.38 at a genome-wide average, respectively, and clearly increased with Rrepeated. Testing for DNA methylation association with SMM in 50 discordant MZ twins revealed 36 081 nominally significant results, of which the top-ranked 134 signals (P < .01 and Rrepeated > 0.40) were subjected to replication in the sample of 1196 individuals. Seven SMM methylation association signals replicated at a false discovery rate less than 0.1, and these were located in or near genes DNAH12, CAND1, CYP4F29P, and ZFP64, which have previously been highlighted in muscle-related studies. Adjusting for age, smoking, and blood cell heterogeneity did not alter significance of these associations.
This epigenome-wide study, testing longitudinally stable methylation sites, discovered and replicated a number of associations between DNA methylation at CpG loci and SMM. Four replicated signals were related to genes with potential muscle functions, suggesting that the methylome of whole blood may be informative of SMM variation.
“We conducted a genome-wide DNA methylation study in an attempt to identify potential genomic regions associated with skeletal muscle mass variation. Four replicated signals were found close to genes with potential muscle functions.”
PMCID: PMC4891794  PMID: 27144936
4.  Integrative DNA methylome analysis of pan-cancer biomarkers in cancer discordant monozygotic twin-pairs 
A key focus in cancer research is the discovery of biomarkers that accurately diagnose early lesions in non-invasive tissues. Several studies have identified malignancy-associated DNA methylation changes in blood, yet no general cancer biomarker has been identified to date. Here, we explore the potential of blood DNA methylation as a biomarker of pan-cancer (cancer of multiple different origins) in 41 female cancer discordant monozygotic (MZ) twin-pairs sampled before or after diagnosis using the Illumina HumanMethylation450 BeadChip.
We analysed epigenome-wide DNA methylation profiles in 41 cancer discordant MZ twin-pairs with affected individuals diagnosed with tumours at different single primary sites: the breast, cervix, colon, endometrium, thyroid gland, skin (melanoma), ovary, and pancreas. No significant global differences in whole blood DNA methylation profiles were observed. Epigenome-wide analyses identified one novel pan-cancer differentially methylated position at false discovery rate (FDR) threshold of 10 % (cg02444695, P = 1.8 × 10−7) in an intergenic region 70 kb upstream of the SASH1 tumour suppressor gene, and three suggestive signals in COL11A2, AXL, and LINC00340. Replication of the four top-ranked signals in an independent sample of nine cancer-discordant MZ twin-pairs showed a similar direction of association at COL11A2, AXL, and LINC00340, and significantly greater methylation discordance at AXL compared to 480 healthy concordant MZ twin-pairs. The effects at cg02444695 (near SASH1), COL11A2, and LINC00340 were the most promising in biomarker potential because the DNA methylation differences were found to pre-exist in samples obtained prior to diagnosis and were limited to a 5-year period before diagnosis. Gene expression follow-up at the top-ranked signals in 283 healthy individuals showed correlation between blood methylation and gene expression in lymphoblastoid cell lines at PRL, and in the skin tissue at AXL. A significant enrichment of differential DNA methylation was observed in enhancer regions (P = 0.03).
We identified DNA methylation signatures in blood associated with pan-cancer, at or near SASH1, COL11A2, AXL, and LINC00340. Three of these signals were present up to 5 years prior to cancer diagnosis, highlighting the potential clinical utility of whole blood DNA methylation analysis in cancer surveillance.
Electronic supplementary material
The online version of this article (doi:10.1186/s13148-016-0172-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4721070  PMID: 26798410
DNA methylation; Cancer; Discordant monozygotic twins; Epigenetics; Biomarker; Twin study
5.  The epigenomic interface between genome and environment in common complex diseases 
Briefings in Functional Genomics  2010;9(5-6):477-485.
The epigenome plays the pivotal role as interface between genome and environment. True genome-wide assessments of epigenetic marks, such as DNA methylation (methylomes) or chromatin modifications (chromatinomes), are now possible, either through high-throughput arrays or increasingly by second-generation DNA sequencing methods. The ability to collect these data at this level of resolution enables us to begin to be able to propose detailed questions, and interrogate this information, with regards to changes that occur due to development, lineage and tissue-specificity, and significantly those caused by environmental influence, such as ageing, stress, diet, hormones or toxins. Common complex traits are under variable levels of genetic influence and additionally epigenetic effect. The detection of pathological epigenetic alterations will reveal additional insights into their aetiology and how possible environmental modulation of this mechanism may occur. Due to the reversibility of these marks, the potential for sequence-specific targeted therapeutics exists. This review surveys recent epigenomic advances and their current and prospective application to the study of common diseases.
PMCID: PMC3080746  PMID: 21062751
Genomics; epigenetics; epigenomics; common disease; complex traits; gene environment interaction
6.  Human-specific epigenetic variation in the immunological Leukotriene B4 Receptor (LTB4R/BLT1) implicated in common inflammatory diseases 
Genome Medicine  2014;6(3):19.
Common human diseases are caused by the complex interplay of genetic susceptibility as well as environmental factors. Due to the environment’s influence on the epigenome, and therefore genome function, as well as conversely the genome’s facilitative effect on the epigenome, analysis of this level of regulation may increase our knowledge of disease pathogenesis.
In order to identify human-specific epigenetic influences, we have performed a novel genome-wide DNA methylation analysis comparing human, chimpanzee and rhesus macaque.
We have identified that the immunological Leukotriene B4 receptor (LTB4R, BLT1 receptor) is the most epigenetically divergent human gene in peripheral blood in comparison with other primates. This difference is due to the co-ordinated active state of human-specific hypomethylation in the promoter and human-specific increased gene body methylation. This gene is significant in innate immunity and the LTB4/LTB4R pathway is involved in the pathogenesis of the spectrum of human inflammatory diseases. This finding was confirmed by additional neutrophil-only DNA methylome and lymphoblastoid H3K4me3 chromatin comparative data. Additionally we show through functional analysis that this receptor has increased expression and a higher response to the LTB4 ligand in human versus rhesus macaque peripheral blood mononuclear cells. Genome-wide we also find human species-specific differentially methylated regions (human s-DMRs) are more prevalent in CpG island shores than within the islands themselves, and within the latter are associated with the CTCF motif.
This result further emphasises the exclusive nature of the human immunological system, its divergent adaptation even from very closely related primates, and the power of comparative epigenomics to identify and understand human uniqueness.
PMCID: PMC4062055  PMID: 24598577
7.  Human-specific CpG “beacons” identify loci associated with human-specific traits and disease 
Epigenetics  2012;7(10):1188-1199.
Regulatory change has long been hypothesized to drive the delineation of the human phenotype from other closely related primates. Here we provide evidence that CpG dinucleotides play a special role in this process. CpGs enable epigenome variability via DNA methylation, and this epigenetic mark functions as a regulatory mechanism. Therefore, species-specific CpGs may influence species-specific regulation. We report non-polymorphic species-specific CpG dinucleotides (termed “CpG beacons”) as a distinct genomic feature associated with CpG island (CGI) evolution, human traits and disease. Using an inter-primate comparison, we identified 21 extreme CpG beacon clusters (≥ 20/kb peaks, empirical p < 1.0 × 10−3) in humans, which include associations with four monogenic developmental and neurological disease related genes (Benjamini-Hochberg corrected p = 6.03 × 10−3). We also demonstrate that beacon-mediated CpG density gain in CGIs correlates with reduced methylation in these species in orthologous CGIs over time, via human, chimpanzee and macaque MeDIP-seq. Therefore mapping into both the genomic and epigenomic space the identified CpG beacon clusters define points of intersection where a substantial two-way interaction between genetic sequence and epigenetic state has occurred. Taken together, our data support a model for CpG beacons to contribute to CGI evolution from genesis to tissue-specific to constitutively active CGIs.
PMCID: PMC3469460  PMID: 22968434
epigenetics; epigenomics; CpG islands; gene regulation; evolution; human disease
8.  An integrated epigenomic analysis for type 2 diabetes susceptibility loci in monozygotic twins 
Nature Communications  2014;5:5719.
DNA methylation has a great potential for understanding the aetiology of common complex traits such as Type 2 diabetes (T2D). Here we perform genome-wide methylated DNA immunoprecipitation sequencing (MeDIP-seq) in whole-blood-derived DNA from 27 monozygotic twin pairs and follow up results with replication and integrated omics analyses. We identify predominately hypermethylated T2D-related differentially methylated regions (DMRs) and replicate the top signals in 42 unrelated T2D cases and 221 controls. The strongest signal is in the promoter of the MALT1 gene, involved in insulin and glycaemic pathways, and related to taurocholate levels in blood. Integrating the DNA methylome findings with T2D GWAS meta-analysis results reveals a strong enrichment for DMRs in T2D-susceptibility loci. We also detect signals specific to T2D-discordant twins in the GPR61 and PRKCB genes. These replicated T2D associations reflect both likely causal and consequential pathways of the disease. The analysis indicates how an integrated genomics and epigenomics approach, utilizing an MZ twin design, can provide pathogenic insights as well as potential drug targets and biomarkers for T2D and other complex traits.
Type 2 diabetes (T2D) is a highly heterogeneous disease with a strong genetic component. Here the authors examine genome-wide methylation patterns in T2D-discordant, T2D-concordant and healthy concordant monozygotic twin pairs, and identify DNA methylation signals that may represent new biomarkers or drug targets for T2D.
PMCID: PMC4284644  PMID: 25502755
9.  Measurement of the clinical utility of a combined mutation detection protocol in carriers of Duchenne and Becker muscular dystrophy 
Journal of Medical Genetics  2007;44(6):368-372.
Recent methodological advances have improved the detection rate for dystrophin mutations, but there are no published studies that have measured the clinical utility of these protocols for carrier detection compared with conventional carrier testing protocols that use pedigree, serum creatine kinase levels and linkage analysis.
Methods and subjects
The clinical utility of a combined mutation detection protocol was measured. It involved quantitative PCR procedures followed by DNA sequence analysis for the identification of dystrophin mutation carriers in 2101 women at risk of being carriers from 348 mutation‐known Duchenne or Becker muscular dystrophy pedigrees.
The combined mutation detection protocol identified a mutation in 96% and 82% of index cases of Duchenne muscular dystrophy and Becker muscular dystrophy, respectively. An additional 692 (33%) potential carriers were correctly classified by the combined mutation detection protocol compared with pedigree, serum creatine kinase levels and linkage analysis. Significantly lower mutation carrier rates were identified in the mothers of isolated cases with deletion mutations than predicted from theoretical considerations, but these findings were not confirmed for duplication and DNA sequence mutations.
There are significant clinical benefits to be gained from a combined mutation detection protocol for carrier detection. It is recommended that mutation‐specific carrier frequencies for the different classes of dystrophin mutations should be taken into account in genetic counselling practice.
PMCID: PMC2740880  PMID: 17259292
10.  The Long-Time Chronoamperometric Current at an Inlaid Microband (or Laminar) Electrode 
Sensors (Basel, Switzerland)  2013;13(1):626-647.
In this article, we derive an approximate asymptotic analytical expression for the long-time chronoamperometric current response at an inlaid microband (or laminar) electrode. The expression is applicable when the length of the microband is much greater than the width, so that the diffusion of the electrochemical species can be regarded as two-dimensional. We extend the previously known result for the diffusion-limited current response (Aoki, K. et al. J. Electroanal. Chem. 1987, 225, 19–32 and Phillips, C.G. J. Electroanal. Chem. 1992, 333, 11–32) to accommodate quasi-reversible reactions and unequal diffusion coefficients of the oxidant and the reductant. Comparison with numerical calculations validates the analytical expression, and we demonstrate that unequal diffusion coefficients can substantially change the current response. Finally, we discuss the form of the long-time current response for a one-step, one-electron redox reaction if the rate constants are modelled in the Butler–Volmer framework, and indicate the importance of choosing the width of the microband appropriately to allow accurate experimental determination of the standard kinetic rate constant and the electron transfer coefficient.
PMCID: PMC3574695  PMID: 23291578
chronoamperometry; two-dimensional; electrode; band; microband; lamina; voltammetry
11.  Identification of Type 1 Diabetes–Associated DNA Methylation Variable Positions That Precede Disease Diagnosis 
PLoS Genetics  2011;7(9):e1002300.
Monozygotic (MZ) twin pair discordance for childhood-onset Type 1 Diabetes (T1D) is ∼50%, implicating roles for genetic and non-genetic factors in the aetiology of this complex autoimmune disease. Although significant progress has been made in elucidating the genetics of T1D in recent years, the non-genetic component has remained poorly defined. We hypothesized that epigenetic variation could underlie some of the non-genetic component of T1D aetiology and, thus, performed an epigenome-wide association study (EWAS) for this disease. We generated genome-wide DNA methylation profiles of purified CD14+ monocytes (an immune effector cell type relevant to T1D pathogenesis) from 15 T1D–discordant MZ twin pairs. This identified 132 different CpG sites at which the direction of the intra-MZ pair DNA methylation difference significantly correlated with the diabetic state, i.e. T1D–associated methylation variable positions (T1D–MVPs). We confirmed these T1D–MVPs display statistically significant intra-MZ pair DNA methylation differences in the expected direction in an independent set of T1D–discordant MZ pairs (P = 0.035). Then, to establish the temporal origins of the T1D–MVPs, we generated two further genome-wide datasets and established that, when compared with controls, T1D–MVPs are enriched in singletons both before (P = 0.001) and at (P = 0.015) disease diagnosis, and also in singletons positive for diabetes-associated autoantibodies but disease-free even after 12 years follow-up (P = 0.0023). Combined, these results suggest that T1D–MVPs arise very early in the etiological process that leads to overt T1D. Our EWAS of T1D represents an important contribution toward understanding the etiological role of epigenetic variation in type 1 diabetes, and it is also the first systematic analysis of the temporal origins of disease-associated epigenetic variation for any human complex disease.
Author Summary
Type 1 diabetes (T1D) is a complex autoimmune disease affecting >30 million people worldwide. It is caused by a combination of genetic and non-genetic factors, leading to destruction of insulin-secreting cells. Although significant progress has recently been made in elucidating the genetics of T1D, the non-genetic component has remained poorly defined. Epigenetic modifications, such as methylation of DNA, are indispensable for genomic processes such as transcriptional regulation and are frequently perturbed in human disease. We therefore hypothesized that epigenetic variation could underlie some of the non-genetic component of T1D aetiology, and we performed a genome-wide DNA methylation analysis of a specific subset of immune cells (monocytes) from monozygotic twins discordant for T1D. This revealed the presence of T1D–specific methylation variable positions (T1D–MVPs) in the T1D–affected co-twins. Since these T1D–MVPs were found in MZ twins, they cannot be due to genetic differences. Additional experiments revealed that some of these T1D–MVPs are found in individuals before T1D diagnosis, suggesting they arise very early in the process that leads to overt T1D and are not simply due to post-disease associated factors (e.g. medication or long-term metabolic changes). T1D–MVPs may thus potentially represent a previously unappreciated, and important, component of type 1 diabetes risk.
PMCID: PMC3183089  PMID: 21980303
12.  Integrated Genetic and Epigenetic Analysis Identifies Haplotype-Specific Methylation in the FTO Type 2 Diabetes and Obesity Susceptibility Locus 
PLoS ONE  2010;5(11):e14040.
Recent multi-dimensional approaches to the study of complex disease have revealed powerful insights into how genetic and epigenetic factors may underlie their aetiopathogenesis. We examined genotype-epigenotype interactions in the context of Type 2 Diabetes (T2D), focussing on known regions of genomic susceptibility. We assayed DNA methylation in 60 females, stratified according to disease susceptibility haplotype using previously identified association loci. CpG methylation was assessed using methylated DNA immunoprecipitation on a targeted array (MeDIP-chip) and absolute methylation values were estimated using a Bayesian algorithm (BATMAN). Absolute methylation levels were quantified across LD blocks, and we identified increased DNA methylation on the FTO obesity susceptibility haplotype, tagged by the rs8050136 risk allele A (p = 9.40×10−4, permutation p = 1.0×10−3). Further analysis across the 46 kb LD block using sliding windows localised the most significant difference to be within a 7.7 kb region (p = 1.13×10−7). Sequence level analysis, followed by pyrosequencing validation, revealed that the methylation difference was driven by the co-ordinated phase of CpG-creating SNPs across the risk haplotype. This 7.7 kb region of haplotype-specific methylation (HSM), encapsulates a Highly Conserved Non-Coding Element (HCNE) that has previously been validated as a long-range enhancer, supported by the histone H3K4me1 enhancer signature. This study demonstrates that integration of Genome-Wide Association (GWA) SNP and epigenomic DNA methylation data can identify potential novel genotype-epigenotype interactions within disease-associated loci, thus providing a novel route to aid unravelling common complex diseases.
PMCID: PMC2987816  PMID: 21124985
13.  Genome-wide DNA methylation analysis for diabetic nephropathy in type 1 diabetes mellitus 
BMC Medical Genomics  2010;3:33.
Diabetic nephropathy is a serious complication of diabetes mellitus and is associated with considerable morbidity and high mortality. There is increasing evidence to suggest that dysregulation of the epigenome is involved in diabetic nephropathy. We assessed whether epigenetic modification of DNA methylation is associated with diabetic nephropathy in a case-control study of 192 Irish patients with type 1 diabetes mellitus (T1D). Cases had T1D and nephropathy whereas controls had T1D but no evidence of renal disease.
We performed DNA methylation profiling in bisulphite converted DNA from cases and controls using the recently developed Illumina Infinium® HumanMethylation27 BeadChip, that enables the direct investigation of 27,578 individual cytosines at CpG loci throughout the genome, which are focused on the promoter regions of 14,495 genes.
Singular Value Decomposition (SVD) analysis indicated that significant components of DNA methylation variation correlated with patient age, time to onset of diabetic nephropathy, and sex. Adjusting for confounding factors using multivariate Cox-regression analyses, and with a false discovery rate (FDR) of 0.05, we observed 19 CpG sites that demonstrated correlations with time to development of diabetic nephropathy. Of note, this included one CpG site located 18 bp upstream of the transcription start site of UNC13B, a gene in which the first intronic SNP rs13293564 has recently been reported to be associated with diabetic nephropathy.
This high throughput platform was able to successfully interrogate the methylation state of individual cytosines and identified 19 prospective CpG sites associated with risk of diabetic nephropathy. These differences in DNA methylation are worthy of further follow-up in replication studies using larger cohorts of diabetic patients with and without nephropathy.
PMCID: PMC2924253  PMID: 20687937
14.  Advances in the identification and analysis of allele-specific expression 
Genome Medicine  2009;1(5):56.
Allele-specific expression (ASE) is essential for normal development and many cellular processes but, if impaired, can result in disease. ASE is a feature of organisms with genomes consisting of more than one set of homologous chromosomes. The higher the number of chromosome sets (ploidy) per cell, the higher the potential complexity of ASE. Humans, for instance, are diploid (except germ cells, which are haploid), resulting in multiple possible expression states in time and space for each set of alleles. ASE is invoked and modulated by both genetic and epigenetic changes, affecting the underlying DNA sequence or chromatin of each allele, respectively. Although numerous methods have been developed to assay ASE, they usually require RNA to be available and are dependent upon genetic polymorphisms (such as single nucleotide polymorphisms (SNPs)) to differentiate between allelic transcripts. The rapid convergence to second-generation sequencing as the method of choice to examine genomic, epigenomic and transcriptomic data enables an integrated and more general approach to define and predict ASE, independent of SNPs. This 'Omni-Seq' approach has the potential to advance our understanding of the biology and pathophysiology of ASE-mediated processes by elucidating subtle combinatorial effects, leading to the accurate delineation of sub-phenotypes with consequential benefit for improved insight into disease etiology.
PMCID: PMC2689448  PMID: 19490587
15.  Analysis of sequence variability in the CART gene in relation to obesity in a Caucasian population 
BMC Genetics  2005;6:19.
Cocaine and amphetamine regulated transcript (CART) is an anorectic neuropeptide located principally in hypothalamus. CART has been shown to be involved in control of feeding behavior, but a direct relationship with obesity has not been established. The aim of this study was to evaluate the effect of polymorphisms within the CART gene with regards to a possible association with obesity in a Caucasian population.
Screening of the entire gene as well as a 3.7 kb region of 5' upstream sequence revealed 31 SNPs and 3 rare variants ; 14 of which were subsequently genotyped in 292 French morbidly obese subjects and 368 controls. Haplotype analysis suggested an association with obesity which was found to be mainly due to SNP-3608T>C (rs7379701) (p = 0.009). Genotyping additional cases and controls also of European Caucasian origin supported further this possible association between the CART SNP -3608T>C T allele and obesity (global p-value = 0.0005). Functional studies also suggested that the SNP -3608T>C could modulate nuclear protein binding.
CART SNP -3608T>C may possibly contribute to the genetic risk for obesity in the Caucasian population. However confirmation of the importance of the role of the CART gene in energy homeostasis and obesity will require investigation and replication in further populations.
PMCID: PMC1087839  PMID: 15823203

Results 1-15 (15)