A critical spotlight on the paradigms of FFPE-DNA sequencing.

In the late 19th century, formalin fixation with paraffin-embedding (FFPE) of tissues was developed as a fixation and conservation method and is still used to this day in routine clinical and pathological practice. The implementation of state-of-the-art nucleic acid sequencing technologies has sparked much interest for using historical FFPE samples stored in biobanks as they hold promise in extracting new information from these valuable samples. However, formalin fixation chemically modifies DNA, which potentially leads to incorrect sequences or misinterpretations in downstream processing and data analysis. Many publications have concentrated on one type of DNA damage, but few have addressed the complete spectrum of FFPE-DNA damage. Here, we review mitigation strategies in (I) pre-analytical sample quality control, (II) DNA repair treatments, (III) analytical sample preparation and (IV) bioinformatic analysis of FFPE-DNA. We then provide recommendations that are tested and illustrated with DNA from 13-year-old liver specimens, one FFPE preserved and one fresh frozen, applying target-enriched sequencing. Thus, we show how DNA damage can be compensated, even when using low quantities (50 ng) of fragmented FFPE-DNA (DNA integrity number 2.0) that cannot be amplified well (Q129 bp/Q41 bp = 5%). Finally, we provide a checklist called 'ERROR-FFPE-DNA' that summarises recommendations for the minimal information in publications required for assessing fitness-for-purpose and inter-study comparison when using FFPE samples.

Convert-Pheno: A software toolkit for the interconversion of standard data models for phenotypic data.

Efficient sharing and integration of phenotypic data is crucial for advancing biomedical research and enhancing patient outcomes in precision medicine and public health. To achieve this, the health data community has developed standards to promote the harmonization of variable names and values. However, the use of diverse standards across different research centers can hinder progress. Here we present Convert-Pheno, an open-source software toolkit that enables the interconversion of common data models for phenotypic data such as Beacon v2 Models, CDISC-ODM, OMOP-CDM, Phenopackets v2, and REDCap. Along with the software, we have created a detailed documentation that includes information on deployment and installation.

The RD-Connect Genome-Phenome Analysis Platform: Accelerating diagnosis, research, and gene discovery for rare diseases.

Rare disease patients are more likely to receive a rapid molecular diagnosis nowadays thanks to the wide adoption of next-generation sequencing. However, many cases remain undiagnosed even after exome or genome analysis, because the methods used missed the molecular cause in a known gene, or a novel causative gene could not be identified and/or confirmed. To address these challenges, the RD-Connect Genome-Phenome Analysis Platform (GPAP) facilitates the collation, discovery, sharing, and analysis of standardized genome-phenome data within a collaborative environment. Authorized clinicians and researchers submit pseudonymised phenotypic profiles encoded using the Human Phenotype Ontology, and raw genomic data which is processed through a standardized pipeline. After an optional embargo period, the data are shared with other platform users, with the objective that similar cases in the system and queries from peers may help diagnose the case. Additionally, the platform enables bidirectional discovery of similar cases in other databases from the Matchmaker Exchange network. To facilitate genome-phenome analysis and interpretation by clinical researchers, the RD-Connect GPAP provides a powerful user-friendly interface and leverages tens of information sources. As a result, the resource has already helped diagnose hundreds of rare disease patients and discover new disease causing genes.

Labelled regulatory elements are pervasive features of the macrophage genome and are dynamically utilized by classical and alternative polarization signals.

The concept of tissue-specific gene expression posits that lineage-determining transcription factors (LDTFs) determine the open chromatin profile of a cell via collaborative binding, providing molecular beacons to signal-dependent transcription factors (SDTFs). However, the guiding principles of LDTF binding, chromatin accessibility and enhancer activity have not yet been systematically evaluated. We sought to study these features of the macrophage genome by the combination of experimental (ChIP-seq, ATAC-seq and GRO-seq) and computational approaches. We show that Random Forest and Support Vector Regression machine learning methods can accurately predict chromatin accessibility using the binding patterns of the LDTF PU.1 and four other key TFs of macrophages (IRF8, JUNB, CEBPA and RUNX1). Any of these TFs alone were not sufficient to predict open chromatin, indicating that TF binding is widespread at closed or weakly opened chromatin regions. Analysis of the PU.1 cistrome revealed that two-thirds of PU.1 binding occurs at low accessible chromatin. We termed these sites labelled regulatory elements (LREs), which may represent a dormant state of a future enhancer and contribute to macrophage cellular plasticity. Collectively, our work demonstrates the existence of LREs occupied by various key TFs, regulating specific gene expression programs triggered by divergent macrophage polarizing stimuli.

Genomic evidence for recurrent genetic admixture during the domestication of Mediterranean olive trees (Olea europaea L.).

BACKGROUND: Olive tree (Olea europaea L. subsp. europaea, Oleaceae) has been the most emblematic perennial crop for Mediterranean countries since its domestication around 6000 years ago in the Levant. Two taxonomic varieties are currently recognized: cultivated (var. europaea) and wild (var. sylvestris) trees. However, it remains unclear whether olive cultivars derive from a single initial domestication event followed by secondary diversification, or whether cultivated lineages are the result of more than a single, independent primary domestication event. To shed light into the recent evolution and domestication of the olive tree, here we analyze a group of newly sequenced and available genomes using a phylogenomics and population genomics framework. RESULTS: We improved the assembly and annotation of the reference genome, newly sequenced the genomes of twelve individuals: ten var. europaea, one var. sylvestris, and one outgroup taxon (subsp. cuspidata)-and assembled a dataset comprising whole genome data from 46 var. europaea and 10 var. sylvestris. Phylogenomic and population structure analyses support a continuous process of olive tree domestication, involving a major domestication event, followed by recurrent independent genetic admixture events with wild populations across the Mediterranean Basin. Cultivated olives exhibit only slightly lower levels of genetic diversity than wild forms, which can be partially explained by the occurrence of a mild population bottleneck 3000-14,000 years ago during the primary domestication period, followed by recurrent introgression from wild populations. Genes associated with stress response and developmental processes were positively selected in cultivars, but we did not find evidence that genes involved in fruit size or oil content were under positive selection. This suggests that complex selective processes other than directional selection of a few genes are in place. CONCLUSIONS: Altogether, our results suggest that a primary domestication area in the eastern Mediterranean basin was followed by numerous secondary events across most countries of southern Europe and northern Africa, often involving genetic admixture with genetically rich wild populations, particularly from the western Mediterranean Basin.

gemBS: high throughput processing for DNA methylation data from bisulfite sequencing.

MOTIVATION: DNA methylation is essential for normal embryogenesis and development in mammals and can be captured at single base pair resolution by whole genome bisulfite sequencing (WGBS). Current available analysis tools are becoming rapidly outdated as they lack sensible functionality and efficiency to handle large amounts of data now commonly created. RESULTS: We developed gemBS, a fast high-throughput bioinformatics pipeline specifically designed for large scale BS-Seq analysis that combines a high performance BS-mapper (GEM3) and a variant caller specifically for BS-Seq data (BScall). gemBS provides genotype information and methylation estimates for all genomic cytosines in different contexts (CpG and non-CpG) and a set of quality reports for comprehensive and reproducible analysis. gemBS is highly modular and can be easily automated, while producing robust and accurate results. AVAILABILITY AND IMPLEMENTATION: gemBS is released under the GNU GPLv3+ license. Source code and documentation are freely available from www.statgen.cat/gemBS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Recurrent somatic mutations reveal new insights into consequences of mutagenic processes in cancer.

The sheer size of the human genome makes it improbable that identical somatic mutations at the exact same position are observed in multiple tumours solely by chance. The scarcity of cancer driver mutations also precludes positive selection as the sole explanation. Therefore, recurrent mutations may be highly informative of characteristics of mutational processes. To explore the potential, we use recurrence as a starting point to cluster >2,500 whole genomes of a pan-cancer cohort. We describe each genome with 13 recurrence-based and 29 general mutational features. Using principal component analysis we reduce the dimensionality and create independent features. We apply hierarchical clustering to the first 18 principal components followed by k-means clustering. We show that the resulting 16 clusters capture clinically relevant cancer phenotypes. High levels of recurrent substitutions separate the clusters that we link to UV-light exposure and deregulated activity of POLE from the one representing defective mismatch repair, which shows high levels of recurrent insertions/deletions. Recurrence of both mutation types characterizes cancer genomes with somatic hypermutation of immunoglobulin genes and the cluster of genomes exposed to gastric acid. Low levels of recurrence are observed for the cluster where tobacco-smoke exposure induces mutagenesis and the one linked to increased activity of cytidine deaminases. Notably, the majority of substitutions are recurrent in a single tumour type, while recurrent insertions/deletions point to shared processes between tumour types. Recurrence also reveals susceptible sequence motifs, including TT[C>A]TTT and AAC[T>G]T for the POLE and 'gastric-acid exposure' clusters, respectively. Moreover, we refine knowledge of mutagenesis, including increased C/G deletion levels in general for lung tumours and specifically in midsize homopolymer sequence contexts for microsatellite instable tumours. Our findings are an important step towards the development of a generic cancer diagnostic test for clinical practice based on whole-genome sequencing that could replace multiple diagnostics currently in use.

Whole-epigenome analysis in multiple myeloma reveals DNA hypermethylation of B cell-specific enhancers.

While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM.

Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification.

Many fungi restructured their proteomes through incorporation of serine (Ser) at thousands of protein sites coded by the leucine (Leu) CUG codon. How these fungi survived this potentially lethal genetic code alteration and its relevance for their biology are not understood. Interestingly, the human pathogen Candida albicans maintains variable Ser and Leu incorporation levels at CUG sites, suggesting that this atypical codon assignment flexibility provided an effective mechanism to alter the genetic code. To test this hypothesis, we have engineered C. albicans strains to misincorporate increasing levels of Leu at protein CUG sites. Tolerance to the misincorporations was very high, and one strain accommodated the complete reversion of CUG identity from Ser back to Leu. Increasing levels of Leu misincorporation decreased growth rate, but production of phenotypic diversity on a phenotypic array probing various metabolic networks, drug resistance, and host immune cell responses was impressive. Genome resequencing revealed an increasing number of genotype changes at polymorphic sites compared with the control strain, and 80% of Leu misincorporation resulted in complete loss of heterozygosity in a large region of chromosome V. The data unveil unanticipated links between gene translational fidelity, proteome instability and variability, genome diversification, and adaptive phenotypic diversity. They also explain the high heterozygosity of the C. albicans genome and open the door to produce microorganisms with genetic code alterations for basic and applied research.

Genomic characterization of mutant laboratory mouse strains by exome sequencing and annotation lift-over.

BACKGROUND: Exome sequencing has become a popular method to evaluate undirected mutagenesis experiments in mice. However, the most suitable mouse strain for the biological model may be relatively distant from the standard mouse reference genome. For pinpointing causative variants, a matching reference with gene annotations is essential, but not always readily available. RESULTS: We present an approach that allows to use murine Ensembl annotations on alternative mouse strain assemblies. We resolved ENU-induced mutation screening for 8 phenotypic mutant lines generated on C3HeB/FeJ background aligning the sequences against the closely related, but not annotated reference of C3H/HeJ. Variants occurring in all strains were filtered out as specific for the C3HeB/FeJ strain but unrelated to mutagenesis. Variants occurring exclusively in all individuals of one mutant line and matching the inheritance model were selected as mutagenesis-related. These variants were annotated with gene and exon names lifted over from the standard murine reference mm9 to C3H/HeJ using megablast. For each mutant line, we could restrict the results to exonic variants in between 1 and 23 genes. CONCLUSIONS: The presented method of exonic annotation lift-over proved to be a valuable tool in the search for mutagenesis-derived coding genomic variants and the assessment of genotype-phenotype relationships.

Involvement of a citrus meiotic recombination TTC-repeat motif in the formation of gross deletions generated by ionizing radiation and MULE activation.

BACKGROUND: Transposable-element mediated chromosomal rearrangements require the involvement of two transposons and two double-strand breaks (DSB) located in close proximity. In radiobiology, DSB proximity is also a major factor contributing to rearrangements. However, the whole issue of DSB proximity remains virtually unexplored. RESULTS: Based on DNA sequencing analysis we show that the genomes of 2 derived mutations, Arrufatina (sport) and Nero (irradiation), share a similar 2 Mb deletion of chromosome 3. A 7 kb Mutator-like element found in Clemenules was present in Arrufatina in inverted orientation flanking the 5' end of the deletion. The Arrufatina Mule displayed "dissimilar" 9-bp target site duplications separated by 2 Mb. Fine-scale single nucleotide variant analyses of the deleted fragments identified a TTC-repeat sequence motif located in the center of the deletion responsible of a meiotic crossover detected in the citrus reference genome. CONCLUSIONS: Taken together, this information is compatible with the proposal that in both mutants, the TTC-repeat motif formed a triplex DNA structure generating a loop that brought in close proximity the originally distinct reactive ends. In Arrufatina, the loop brought the Mule ends nearby the 2 distinct insertion target sites and the inverted insertion of the transposable element between these target sites provoked the release of the in-between fragment. This proposal requires the involvement of a unique transposon and sheds light on the unresolved question of how two distinct sites become located in close proximity. These observations confer a crucial role to the TTC-repeats in fundamental plant processes as meiotic recombination and chromosomal rearrangements.

Distinct DNA methylomes of newborns and centenarians.

Human aging cannot be fully understood in terms of the constrained genetic setting. Epigenetic drift is an alternative means of explaining age-associated alterations. To address this issue, we performed whole-genome bisulfite sequencing (WGBS) of newborn and centenarian genomes. The centenarian DNA had a lower DNA methylation content and a reduced correlation in the methylation status of neighboring cytosine--phosphate--guanine (CpGs) throughout the genome in comparison with the more homogeneously methylated newborn DNA. The more hypomethylated CpGs observed in the centenarian DNA compared with the neonate covered all genomic compartments, such as promoters, exonic, intronic, and intergenic regions. For regulatory regions, the most hypomethylated sequences in the centenarian DNA were present mainly at CpG-poor promoters and in tissue-specific genes, whereas a greater level of DNA methylation was observed in CpG island promoters. We extended the study to a larger cohort of newborn and nonagenarian samples using a 450,000 CpG-site DNA methylation microarray that reinforced the observation of more hypomethylated DNA sequences in the advanced age group. WGBS and 450,000 analyses of middle-age individuals demonstrated DNA methylomes in the crossroad between the newborn and the nonagenarian/centenarian groups. Our study constitutes a unique DNA methylation analysis of the extreme points of human life at a single-nucleotide resolution level.

RD-Connect: an integrated platform connecting databases, registries, biobanks and clinical bioinformatics for rare disease research.

Research into rare diseases is typically fragmented by data type and disease. Individual efforts often have poor interoperability and do not systematically connect data across clinical phenotype, genomic data, biomaterial availability, and research/trial data sets. Such data must be linked at both an individual-patient and whole-cohort level to enable researchers to gain a complete view of their disease and patient population of interest. Data access and authorization procedures are required to allow researchers in multiple institutions to securely compare results and gain new insights. Funded by the European Union's Seventh Framework Programme under the International Rare Diseases Research Consortium (IRDiRC), RD-Connect is a global infrastructure project initiated in November 2012 that links genomic data with registries, biobanks, and clinical bioinformatics tools to produce a central research resource for rare diseases.

A revisit of high collision energy effects on collision-induced dissociation spectra using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-LIFT-TOF/TOF): application to the sequencing of RNA/DNA chimeras.

RATIONALE: High-energy collision-induced dissociation (CID) spectra of isomeric RNA/DNA chimeras using matrix-assisted laser desorption/ionization time-of-flight LIFT mass spectrometry (MALDI-LIFT-TOF/TOF) can potentially be applied for an exhaustive fragment characterization in a nucleic acid sequencing scheme. These chimeras contain deoxynucleotides and at the 3'-end a ribonucleotide with a 3'-phosphate group. METHODS: Deprotonated RNA/DNA chimeras of 4-, 5-, 7- and 10-mers are analyzed by CID. This enhances consecutive dissociations from both the precursor and prompt product anions generated by MALDI and metastable fragmentations prior to entering the LIFT cell. RESULTS: Gas-phase fragmentations of 4- and 5-mers produced many fragment ions, from base release prior to consecutive cleavage of the nucleotide phosphate bond linkage phosphate. The unusual a4(-) product ion is a specific and diagnostic dissociation of the 4-mer if the ribonucleotide contains cytosine. As the size of RNA/DNA chimeras increase, several abundant product ions are generated mainly from zwitterionic forms (deprotonated phosphate ester and protonated base sites): [(M-H)-BiH](-), [ai-BiH](-), wj(-), [wj, (ai-BiH)](-) (if Bi ≠ T) as internal product ion, and more rarely [wj-BiH](-). The absence of the majority of the [ai-BiH](-) series although the wj (-) series suggested that the higher critical energy processes with a loose transition state are favored yielding the wj(-) series. A large number of abundant fragment ions are detected which enable each isomer to be sequenced. CONCLUSIONS: This sequencing method is high-throughput, accurate and could be used to sequence isomers of up to 10-mers and also oligonucleotides of unknown sequence. However, RNA/DNA chimeras without thymine must be sufficiently concentrated to reach desorption of deprotonated molecular species to be selected in LIFT to produce all fragment ions within measurable abundances.

Structural studies of the sBBI/trypsin non-covalent complex using covalent modification and mass spectrometry.

RATIONALE: The study of protein recognition sites is crucial for understanding the mechanisms of protein interaction. Mass spectrometry can be a method of choice for the investigation of the contact surface within the protein non-covalent complexes. METHODS: Probing the reactivity of essential amino acid residues of soybean Bowman-Birk inhibitor (sBBI) within the non-covalent sBBI/bovine trypsin complex was performed using covalent labeling by the BS3 cross-linker and charge tag with a quaternary ammonium group in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis. RESULTS: Significant modulation of the reactivity of essential K16 and S17 residues in the sBBI molecule upon binding to trypsin was established. The studies of sBBI proteolytic peptides with the same structure but carrying different labels using metastable dissociation in LIFT mode demonstrated that fragmentation pathways were oriented by used modification (BS3 cross-linker or charge tag). CONCLUSIONS: The effectiveness of the mass spectrometric approach including covalent modification for exploring protein-protein interaction sites has been demonstrated. The alteration of the reactivity of functionally important amino acid residues in the sBBI molecule is most likely related to changes in their microenvironment. It has been suggested that in the presence of charge tags fragmentation in LIFT mode proceeds through the formation of salt bridges between quaternary ammonium groups and acidic residues due to the occurrence of zwitterions (including basic and acidic residues). Despite the presence of one or several charge tags, fragmentation takes place yielding modulated bi /yj ion series depending on the positions of the tags.

Multi-organ single-cell transcriptomics of immune cells uncovered organ-specific gene expression and functions.

Despite the wealth of publicly available single-cell datasets, our understanding of distinct resident immune cells and their unique features in diverse human organs remains limited. To address this, we compiled a meta-analysis dataset of 114,275 CD45+ immune cells sourced from 14 organs in healthy donors. While the transcriptome of immune cells remains relatively consistent across organs, our analysis has unveiled organ-specific gene expression differences (GTPX3 in kidney, DNTT and ACVR2B in thymus). These alterations are linked to different transcriptional factor activities and pathways including metabolism. TNF-α signaling through the NFkB pathway was found in several organs and immune compartments. The presence of distinct expression profiles for NFkB family genes and their target genes, including cytokines, underscores their pivotal role in cell positioning. Taken together, immune cells serve a dual role: safeguarding the organs and dynamically adjusting to the intricacies of the host organ environment, thereby actively contributing to its functionality and overall homeostasis.

High-specificity single-tube multiplex genotyping using Ribo-PAP PCR, tag primers, alkali cleavage of RNA/DNA chimeras and MALDI-TOF MS.

Here, we describe a high-throughput, single-tube, allele-specific ribonucleotide analog pyrophosphorolysis-activated polymerization (ribo-PAP) PCR multiplex genotyping and resequencing method. An RNA/DNA chimeric PCR product is generated using genomic DNA as starting template, a panel of allele-selective 5'-tagged primers, a reverse primer, one nucleotide in the ribo-form (90-100%), the other nucleotides in the deoxy-form, a DNA polymerase capable of incorporating ribonucleotides, a suitable buffer and thermal cycling. The RNA/DNA chimeric PCR products are fragmented by treatment with alkali and analyzed by mass spectrometry. All allele-selective primers have a 5' repetitive motif where each repeat unit has a unique, distinct mass upon reverse copying and alkali fragmentation. The mass of the complement repeat fragment or flag identifies the primer or primers that were recruited in the ribo-PAP PCR. The method readily identifies homozygous and heterozygous positions in simplex or duplex ribo-PAP PCR. Many different tags can be analyzed simultaneously. The assay can genotype several SNPs in a single tube. It thus constitutes the simplest genotyping protocol with multiplex analysis. This novel genotyping and resequencing protocol was applied to different genomic loci: NOS1 and H19 in 30 individuals in simplex ribo-PAP PCR and at two SLCO1B1 loci in 95 individuals in duplex ribo-PAP PCR.

The genome sequencing of an albino Western lowland gorilla reveals inbreeding in the wild.

BACKGROUND: The only known albino gorilla, named Snowflake, was a male wild born individual from Equatorial Guinea who lived at the Barcelona Zoo for almost 40 years. He was diagnosed with non-syndromic oculocutaneous albinism, i.e. white hair, light eyes, pink skin, photophobia and reduced visual acuity. Despite previous efforts to explain the genetic cause, this is still unknown. Here, we study the genetic cause of his albinism and making use of whole genome sequencing data we find a higher inbreeding coefficient compared to other gorillas. RESULTS: We successfully identified the causal genetic variant for Snowflake's albinism, a non-synonymous single nucleotide variant located in a transmembrane region of SLC45A2. This transporter is known to be involved in oculocutaneous albinism type 4 (OCA4) in humans. We provide experimental evidence that shows that this amino acid replacement alters the membrane spanning capability of this transmembrane region. Finally, we provide a comprehensive study of genome-wide patterns of autozygogosity revealing that Snowflake's parents were related, being this the first report of inbreeding in a wild born Western lowland gorilla. CONCLUSIONS: In this study we demonstrate how the use of whole genome sequencing can be extended to link genotype and phenotype in non-model organisms and it can be a powerful tool in conservation genetics (e.g., inbreeding and genetic diversity) with the expected decrease in sequencing cost.

A large-scale, consortium-based genomewide association study of asthma.

BACKGROUND: Susceptibility to asthma is influenced by genes and environment; implicated genes may indicate pathways for therapeutic intervention. Genetic risk factors may be useful in identifying subtypes of asthma and determining whether intermediate phenotypes, such as elevation of the total serum IgE level, are causally linked to disease. METHODS: We carried out a genomewide association study by genotyping 10,365 persons with physician-diagnosed asthma and 16,110 unaffected persons, all of whom were matched for ancestry. We used random-effects pooled analysis to test for association in the overall study population and in subgroups of subjects with childhood-onset asthma (defined as asthma developing before 16 years of age), later-onset asthma, severe asthma, and occupational asthma. RESULTS: We observed associations of genomewide significance between asthma and the following single-nucleotide polymorphisms: rs3771166 on chromosome 2, implicating IL1RL1/IL18R1 (P=3×10(−9)); rs9273349 on chromosome 6, implicating HLA-DQ (P=7×10(−14)); rs1342326 on chromosome 9, flanking IL33 (P=9×10(−10)); rs744910 on chromosome 15 in SMAD3 (P=4×10(−9)); and rs2284033 on chromosome 22 in IL2RB (P=1.1×10(−8)). Association with the ORMDL3/GSDMB locus on chromosome 17q21 was specific to childhood-onset disease (rs2305480, P=6×10(−23)). Only HLA-DR showed a significant genomewide association with the total serum IgE concentration, and loci strongly associated with IgE levels were not associated with asthma. CONCLUSIONS: Asthma is genetically heterogeneous. A few common alleles are associated with disease risk at all ages. Implicated genes suggest a role for communication of epithelial damage to the adaptive immune system and activation of airway inflammation. Variants at the ORMDL3/GSDMB locus are associated only with childhood-onset disease. Elevation of total serum IgE levels has a minor role in the development of asthma. (Funded by the European Commission and others.)

Genetic variants regulating ORMDL3 expression contribute to the risk of childhood asthma.

Asthma is caused by a combination of poorly understood genetic and environmental factors. We have systematically mapped the effects of single nucleotide polymorphisms (SNPs) on the presence of childhood onset asthma by genome-wide association. We characterized more than 317,000 SNPs in DNA from 994 patients with childhood onset asthma and 1,243 non-asthmatics, using family and case-referent panels. Here we show multiple markers on chromosome 17q21 to be strongly and reproducibly associated with childhood onset asthma in family and case-referent panels with a combined P value of P < 10(-12). In independent replication studies the 17q21 locus showed strong association with diagnosis of childhood asthma in 2,320 subjects from a cohort of German children (P = 0.0003) and in 3,301 subjects from the British 1958 Birth Cohort (P = 0.0005). We systematically evaluated the relationships between markers of the 17q21 locus and transcript levels of genes in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines from children in the asthma family panel used in our association study. The SNPs associated with childhood asthma were consistently and strongly associated (P < 10(-22)) in cis with transcript levels of ORMDL3, a member of a gene family that encodes transmembrane proteins anchored in the endoplasmic reticulum. The results indicate that genetic variants regulating ORMDL3 expression are determinants of susceptibility to childhood asthma.