The psychiatric risk gene BRD1 modulates mitochondrial bioenergetics by transcriptional regulation.

Bromodomain containing 1 (BRD1) encodes an epigenetic regulator that controls the expression of genetic networks linked to mental illness. BRD1 is essential for normal brain development and its role in psychopathology has been demonstrated in genetic and preclinical studies. However, the neurobiology that bridges its molecular and neuropathological effects remains poorly explored. Here, using publicly available datasets, we find that BRD1 targets nuclear genes encoding mitochondrial proteins in cell lines and that modulation of BRD1 expression, irrespective of whether it is downregulation or upregulation of one or the other existing BRD1 isoforms (BRD1-L and BRD1-S), leads to distinct shifts in the expression profile of these genes. We further show that the expression of nuclear genes encoding mitochondrial proteins is negatively correlated with the expression of BRD1 mRNA during human brain development. In accordance, we identify the key gate-keeper of mitochondrial metabolism, Peroxisome proliferator-activated receptor (PPAR) among BRD1's co-transcription factors and provide evidence that BRD1 acts as a co-repressor of PPAR-mediated transcription. Lastly, when using quantitative PCR, mitochondria-targeted fluorescent probes, and the Seahorse XFe96 Analyzer, we demonstrate that modulation of BRD1 expression in cell lines alters mitochondrial physiology (mtDNA content and mitochondrial mass), metabolism (reducing power), and bioenergetics (among others, basal, maximal, and spare respiration) in an expression level- and isoform-dependent manner. Collectively, our data suggest that BRD1 is a transcriptional regulator of nuclear-encoded mitochondrial proteins and that disruption of BRD1's genomic actions alters mitochondrial functions. This may be the mechanism underlying the cellular and atrophic changes of neurons previously associated with BRD1 deficiency and suggests that mitochondrial dysfunction may be a possible link between genetic variation in BRD1 and psychopathology in humans.

Inactivation of the Schizophrenia-associated BRD1 gene in Brain Causes Failure-to-thrive, Seizure Susceptibility and Abnormal Histone H3 Acetylation and N-tail Clipping.

Genetic studies have repeatedly shown that the Bromodomain containing 1 gene, BRD1, is involved in determining mental health, and the importance of the BRD1 protein for normal brain function has been studied in both cell models and constitutive haploinsufficient Brd1+/- mice. Homozygosity for inactivated Brd1 alleles is lethal during embryonic development in mice. In order to further characterize the molecular functions of BRD1 in the brain, we have developed a novel Brd1 knockout mouse model (Brd1-/-) with bi-allelic conditional inactivation of Brd1 in the central nervous system. Brd1-/- mice were viable but smaller and with reduced muscle strength. They showed reduced exploratory behavior and increased sensitivity to pentylenetetrazole-induced seizures supporting the previously described GABAergic dysfunction in constitutive Brd1+/- mice. Because BRD1 takes part in protein complexes with histone binding and modifying functions, we investigated the effect of BRD1 depletion on the global histone modification pattern in mouse brain by mass spectrometry. We found decreased levels of histone H3 acetylation (H3K9ac, H3K14ac, and H3K18ac) and increased N-tail clipping in consequence of BRD1 depletion. Collectively, the presented results support that BRD1 controls gene expression at the epigenetic level by regulating histone H3 proteoforms in the brain.

Co-occurring hydrocephalus in autism spectrum disorder: a Danish population-based cohort study.

BACKGROUND: The association between autism spectrum disorder and hydrocephalus is not well understood, despite demonstrated links between autism spectrum disorder and cerebrospinal fluid abnormalities. Based on the hypothesis that autism spectrum disorder and hydrocephalus may, at least in some cases, be two manifestations of a shared congenital brain pathology, we investigated the potential association between autism spectrum disorder and hydrocephalus in a large Danish population-based cohort. METHODS: Patients and controls were obtained from the Lundbeck Foundation Initiative for Integrative Psychiatric Research iPSYCH2012 case-cohort, which includes all patients with selected psychiatric disorders born in Denmark 1981-2005 along with randomly selected population controls (end of follow-up, December 31, 2016). The associations between individual psychiatric disorders and hydrocephalus were estimated using binary logistic regression with adjustment for age and sex. RESULTS: The cohort consisted of 86,571 individuals, of which 14,654 were diagnosed with autism spectrum disorder, 28,606 were population controls, and the remaining were diagnosed with other psychiatric disorders. We identified 201 hydrocephalus cases; 68 among autism spectrum disorder patients and 40 among controls (OR 3.77, 95% CI 2.48-5.78), which corresponds to an absolute risk of 0.46 % (i.e. approximately one in 217 children with autism spectrum disorder had co-occurring hydrocephalus). The autism spectrum disorder-hydrocephalus association was significant over the entire subgroup spectrum of autism spectrum disorder. CONCLUSIONS: Given the considerable risk of hydrocephalus among patients with autism spectrum disorder, we suggest that patients with autism spectrum disorder should be evaluated for co-occurring hydrocephalus on a routine basis as timely neurosurgical intervention is important. Likewise, attention must be paid to traits of autism spectrum disorder in children with hydrocephalus. The results of this study call for future investigations on a potential shared aetiology between hydrocephalus and autism spectrum disorder, including the role abnormal CSF dynamics in the pathogenesis of autism spectrum disorder.

Association Between Childhood Green Space, Genetic Liability, and the Incidence of Schizophrenia.

Childhood exposure to green space has previously been associated with lower risk of developing schizophrenia later in life. It is unclear whether this association is mediated by genetic liability or whether the 2 risk factors work additively. Here, we investigate possible gene-environment associations with the hazard ratio (HR) of schizophrenia by combining (1) an estimate of childhood exposure to residential-level green space based on the normalized difference vegetation index (NDVI) from Landsat satellite images, with (2) genetic liability estimates based on polygenic risk scores for 19 746 genotyped individuals from the Danish iPSYCH sample. We used information from the Danish registers of health, residential address, and socioeconomic status to adjust HR estimates for established confounders, ie, parents' socioeconomic status, and family history of mental illness. The adjusted HRs show that growing up surrounded by the highest compared to the lowest decile of NDVI was associated with a 0.52-fold (95% confidence interval [CI]: 0.40 to 0.66) lower schizophrenia risk, and children with the highest polygenic risk score had a 1.24-fold (95% CI: 1.18 to 1.30) higher schizophrenia risk. We found that NDVI explained 1.45% (95% CI: 1.07 to 1.90) of the variance on the liability scale, while polygenic risk score for schizophrenia explained 1.01% (95% CI: 0.77 to 1.46). Together they explained 2.40% (95% CI: 1.99 to 3.07) with no indication of a gene-environment interaction (P = .29). Our results suggest that risk of schizophrenia is associated additively with green space exposure and genetic liability, and provide no support for an environment-gene interaction between NDVI and schizophrenia.

Association of Childhood Exposure to Nitrogen Dioxide and Polygenic Risk Score for Schizophrenia With the Risk of Developing Schizophrenia.

Importance: Schizophrenia is a highly heritable psychiatric disorder, and recent studies have suggested that exposure to nitrogen dioxide (NO2) during childhood is associated with an elevated risk of subsequently developing schizophrenia. However, it is not known whether the increased risk associated with NO2 exposure is owing to a greater genetic liability among those exposed to highest NO2 levels. Objective: To examine the associations between childhood NO2 exposure and genetic liability for schizophrenia (as measured by a polygenic risk score), and risk of developing schizophrenia. Design, Setting, and Participants: Population-based cohort study including individuals with schizophrenia (International Statistical Classification of Diseases and Related Health Problems, Tenth Revision code F20) and a randomly selected subcohort. Using national registry data, all individuals born in Denmark between May 1, 1981, and December 31, 2002, were followed up from their 10th birthday until the first occurrence of schizophrenia, emigration, death, or December 31, 2012, whichever came first. Statistical analyses were conducted between October 24, 2018, and June 17, 2019. Exposures: Individual exposure to NO2 during childhood estimated as mean daily exposure to NO2 at residential addresses from birth to the 10th birthday. Polygenic risk scores were calculated as the weighted sum of risk alleles at selected single-nucleotide polymorphisms based on genetic material obtained from dried blood spot samples from the Danish Newborn Screening Biobank and on the Psychiatric Genomics Consortium genome-wide association study summary statistics file. Main Outcomes and Measures: The main outcome was schizophrenia. Weighted Cox proportional hazards regression models were fitted to estimate adjusted hazard ratios (AHRs) for schizophrenia with 95% CIs according to the exposures. Results: Of a total of 23 355 individuals, 11 976 (51.3%) were male and all had Danish-born parents. During the period of the study, 3531 were diagnosed with schizophrenia. Higher polygenic risk scores were correlated with higher childhood NO2 exposure (ρ = 0.0782; 95% CI, 0.065-0.091; P < .001). A 10-µg/m3 increase in childhood daily NO2 exposure (AHR, 1.23; 95% CI, 1.15-1.32) and a 1-SD increase in polygenic risk score (AHR, 1.29; 95% CI, 1.23-1.35) were independently associated with increased schizophrenia risk. Conclusions and Relevance: These findings suggest that the apparent association between NO2 exposure and schizophrenia is only slightly confounded by a higher polygenic risk score for schizophrenia among individuals living in areas with greater NO2. The findings demonstrate the utility of including polygenic risk scores in epidemiologic studies.

Differential activity of transcribed enhancers in the prefrontal cortex of 537 cases with schizophrenia and controls.

Transcription at enhancers is a widespread phenomenon which produces so-called enhancer RNA (eRNA) and occurs in an activity-dependent manner. However, the role of eRNA and its utility in exploring disease-associated changes in enhancer function, and the downstream coding transcripts that they regulate, is not well established. We used transcriptomic and epigenomic data to interrogate the relationship of eRNA transcription to disease status and how genetic variants alter enhancer transcriptional activity in the human brain. We combined RNA-seq data from 537 postmortem brain samples from the CommonMind Consortium with cap analysis of gene expression and enhancer identification, using the assay for transposase-accessible chromatin followed by sequencing (ATACseq). We find 118 differentially transcribed eRNAs in schizophrenia and identify schizophrenia-associated gene/eRNA co-expression modules. Perturbations of a key module are associated with the polygenic risk scores. Furthermore, we identify genetic variants affecting expression of 927 enhancers, which we refer to as enhancer expression quantitative loci or eeQTLs. Enhancer expression patterns are consistent across studies, including differentially expressed eRNAs and eeQTLs. Combining eeQTLs with a genome-wide association study of schizophrenia identifies a genetic variant that alters enhancer function and expression of its target gene, GOLPH3L. Our novel approach to analyzing enhancer transcription is adaptable to other large-scale, non-poly-A depleted, RNA-seq studies.

Brain proteome changes in female Brd1+/- mice unmask dendritic spine pathology and show enrichment for schizophrenia risk.

Genetic and molecular studies have implicated the Bromodomain containing 1 (BRD1) gene in the pathogenesis of schizophrenia and bipolar disorder. Accordingly, mice heterozygous for a targeted deletion of Brd1 (Brd1+/- mice) show behavioral phenotypes with broad translational relevance to psychiatric disorders. BRD1 encodes a scaffold protein that affects the expression of many genes through modulation of histone acetylation. BRD1 target genes have been identified in cell lines; however the impact of reduced Brd1 levels on the brain proteome is largely unknown. In this study, we applied label-based quantitative mass spectrometry to profile the frontal cortex, hippocampus and striatum proteome and synaptosomal proteome of female Brd1+/- mice. We successfully quantified between 1537 and 2196 proteins and show widespread changes in protein abundancies and compartmentalization. By integrative analysis of human genetic data, we find that the differentially abundant proteins in frontal cortex and hippocampus are enriched for schizophrenia risk further linking the actions of BRD1 to psychiatric disorders. Affected proteins were further enriched for proteins involved in processes known to influence neuronal and dendritic spine morphology e.g. regulation of cytoskeleton dynamics and mitochondrial function. Directly prompted in these findings, we investigated dendritic spine morphology of pyramidal neurons in anterior cingulate cortex and found them significantly altered, including reduced size of small dendritic spines and decreased number of the mature mushroom type. Collectively, our study describes known as well as new mechanisms related to BRD1 dysfunction and its role in psychiatric disorders, and provides evidence for the molecular and cellular dysfunctions underlying altered neurosignalling and cognition in Brd1+/- mice.

The importance of data structure in statistical analysis of dendritic spine morphology.

BACKGROUND: Dendritic spine morphology is heterogeneous and highly dynamic. To study the changing or aberrant morphology in test setups, often spines from several neurons from a few experimental units e.g. mice or primary neuronal cultures are measured. This strategy results in a multilevel data structure, which, when not properly addressed, has a high risk of producing false positive and false negative findings. METHODS: We used mixed-effects models to deal with data with a multilevel data structure and compared this method to analyses at each level. We apply these statistical tests to a dataset of dendritic spine morphology parameters to illustrate advantages of multilevel mixed-effects model, and disadvantages of other models. RESULTS: We present an application of mixed-effects models for analyzing dendritic spine morphology datasets while correcting for the data structure. COMPARISON WITH EXISTING METHODS: We further show that analyses at spine level and aggregated levels do not adequately account for the data structure, and that they may lead to erroneous results. CONCLUSION: We highlight the importance of data structure in dendritic spine morphology analyses and highly recommend the use of mixed-effects models or other appropriate statistical methods to deal with multilevel datasets. Mixed-effects models are easy to use and superior to commonly used methods by including the data structure and the addition of other explanatory variables, for example sex, and age, etc., as well as interactions between variables or between variables and level identifiers.

The Schizophrenia-Associated BRD1 Gene Regulates Behavior, Neurotransmission, and Expression of Schizophrenia Risk Enriched Gene Sets in Mice.

BACKGROUND: The schizophrenia-associated BRD1 gene encodes a transcriptional regulator whose comprehensive chromatin interactome is enriched with schizophrenia risk genes. However, the biology underlying the disease association of BRD1 remains speculative. METHODS: This study assessed the transcriptional drive of a schizophrenia-associated BRD1 risk variant in vitro. Accordingly, to examine the effects of reduced Brd1 expression, we generated a genetically modified Brd1+/- mouse and subjected it to behavioral, electrophysiological, molecular, and integrative genomic analyses with focus on schizophrenia-relevant parameters. RESULTS: Brd1+/- mice displayed cerebral histone H3K14 hypoacetylation and a broad range of behavioral changes with translational relevance to schizophrenia. These behaviors were accompanied by striatal dopamine/serotonin abnormalities and cortical excitation-inhibition imbalances involving loss of parvalbumin immunoreactive interneurons. RNA-sequencing analyses of cortical and striatal micropunches from Brd1+/- and wild-type mice revealed differential expression of genes enriched for schizophrenia risk, including several schizophrenia genome-wide association study risk genes (e.g., calcium channel subunits [Cacna1c and Cacnb2], cholinergic muscarinic receptor 4 [Chrm4)], dopamine receptor D2 [Drd2], and transcription factor 4 [Tcf4]). Integrative analyses further found differentially expressed genes to cluster in functional networks and canonical pathways associated with mental illness and molecular signaling processes (e.g., glutamatergic, monoaminergic, calcium, cyclic adenosine monophosphate [cAMP], dopamine- and cAMP-regulated neuronal phosphoprotein 32 kDa [DARPP-32], and cAMP responsive element binding protein signaling [CREB]). CONCLUSIONS: Our study bridges the gap between genetic association and pathogenic effects and yields novel insights into the unfolding molecular changes in the brain of a new schizophrenia model that incorporates genetic risk at three levels: allelic, chromatin interactomic, and brain transcriptomic.

Schizophrenia risk variants affecting microRNA function and site-specific regulation of NT5C2 by miR-206.

Despite the identification of numerous schizophrenia-associated genetic variants, few have been examined functionally to identify and characterize the causal variants. To mitigate this, we aimed at identifying functional variants affecting miRNA function. Using data from a large-scale genome-wide association study of schizophrenia, we looked for schizophrenia risk variants altering either miRNA binding sites, miRNA genes, promoters for miRNA genes, or variants that were expression quantitative trait loci (eQTLs) for miRNA genes. We hereby identified several potentially functional variants relating to miRNA function with our top finding being a schizophrenia protective allele that disrupts miR-206׳s binding to NT5C2 thus leading to increased expression of this gene. A subsequent experimental follow-up of the variant using a luciferase-based reporter assay confirmed that the allele disrupts the binding. Our study therefore suggests that miR-206 may contribute to schizophrenia risk through allele-dependent regulation of the genome-wide significant gene NT5C2.

DNA Methylation at the Neonatal State and at the Time of Diagnosis: Preliminary Support for an Association with the Estrogen Receptor 1, Gamma-Aminobutyric Acid B Receptor 1, and Myelin Oligodendrocyte Glycoprotein in Female Adolescent Patients with OCD.

Obsessive-compulsive disorder (OCD) is a neuropsychiatric disorder. Non-genetic factors and their interaction with genes have attracted increasing attention. Epigenetics is regarded an important interface between environmental signals and activation/repression of genomic responses. Epigenetic mechanisms have not previously been examined in OCD in children and adolescents. The aim of the present study was to examine the DNA methylation profile of selected genes in blood spots from neonates later diagnosed with OCD and in the same children/adolescents at the time of diagnosis compared with age- and sex-matched controls. Furthermore, we wanted to characterize the association of the differential methylation profiles with the severity of OCD and treatment outcome. Dried and new blood spot samples were obtained from 21 female children/adolescents with verified OCD and 12 female controls. The differential methylation was analyzed using a linear model and the correlation with the severity of OCD and treatment outcome was analyzed using the Pearson correlation. We evaluated selected Illumina Infinium HumanMethylation450 BeadChip probes within and up to 100,000 bp up- and downstream of 14 genes previously associated with OCD (SLC1A1, SLC25A12, GABBR1, GAD1, DLGAP1, MOG, BDNF, OLIG2, NTRK2 and 3, ESR1, SL6A4, TPH2, and COMT). The study found no significantly differential methylation. However, preliminary support for a difference was found for the gamma-aminobutyric acid (GABA) B receptor 1 (cg10234998, cg17099072) in blood samples at birth and for the estrogen receptor 1 (ESR1) (cg10939667), the myelin oligodendrocyte glycoprotein (MOG) (cg16650906), and the brain-derived neurotrophic factor (BDNF) (cg14080521) in blood samples at the time of diagnosis. Preliminary support for an association was observed between the methylation profiles of GABBR1 and MOG and baseline severity, treatment effect, and responder status; and between the methylation profile of ESR1 and baseline severity. To our knowledge, this is the first study to examine the DNA methylation profiles in OCD. The study points towards possible differences in the methylation profiles and suggests a correlation with the severity of OCD. However, the results warrant further studies in larger sample sets.

Quantitative assessment of methyl-esterification and other side reactions in a standard propionylation protocol for detection of histone modifications.

Histone modifications play an important role in regulating chromatin stability and gene expression, but to date, investigating them remains challenging. In order to obtain peptides suitable for MS-based analysis, chemical derivatization of N-terminus and lysine residues by propionic anhydride is commonly performed. Several side reactions (methyl-esterification, amidation, solvolysis, overpropionylation, and missed propionylation) during propionylation protocols have been described, yet their relative abundances remain vague. Because methyl-esterification could interfere with correct interpretation of the modification pattern, it is essential to take measures to avoid it. Here we present in-depth quantitative analyses of methyl-esterification and the other side reactions in a standard propionylation protocol containing methanol, and when replacing methanol with isopropanol or acetonitrile. We show that the use of alternative solvents can eliminate methyl-esterification and that even though other side reactions are not prevented, their contribution can be kept relatively small. We also show that replacing methanol can be of importance also in other proteomics methods, such as mixed cation exchange, using methanol under acidic conditions.

High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA.

Stored neonatal dried blood spot (DBS) samples from neonatal screening programmes are a valuable diagnostic and research resource. Combined with information from national health registries they can be used in population-based studies of genetic diseases. DNA extracted from neonatal DBSs can be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject we analysed a neonatal DBS sample and corresponding adult whole-blood (WB) reference sample. Different DNA sample types were prepared for each of the subjects. Pilot 1: wgaDNA of 2x3.2mm neonatal DBSs (DBS_2x3.2) and raw DNA extract of the WB reference sample (WB_ref). Pilot 2: DBS_2x3.2, WB_ref and a WB_ref replica sharing DNA extract with the WB_ref sample. Pilot 3: DBS_2x3.2, WB_ref, wgaDNA of 2x1.6 mm neonatal DBSs and wgaDNA of the WB reference sample. Following sequencing and data analysis, we compared pairwise variant calls to obtain a measure of similarity--the concordance rate. Concordance rates were slightly lower when comparing DBS vs WB sample types than for any two WB sample types of the same subject before filtering of the variant calls. The overall concordance rates were dependent on the variant type, with SNPs performing best. Post-filtering, the comparisons of DBS vs WB and WB vs WB sample types yielded similar concordance rates, with values close to 100%. WgaDNA of neonatal DBS samples performs with great accuracy and efficiency in exome sequencing. The wgaDNA performed similarly to matched high-quality reference--whole-blood DNA--based on concordance rates calculated from variant calls. No differences were observed substituting 2x3.2 with 2x1.6 mm discs, allowing for additional reduction of sample material in future projects.

Analyzing the Role of MicroRNAs in Schizophrenia in the Context of Common Genetic Risk Variants.

IMPORTANCE: The recent implication of 108 genomic loci in schizophrenia marked a great advancement in our understanding of the disease. Against the background of its polygenic nature there is a necessity to identify how schizophrenia risk genes interplay. As regulators of gene expression, microRNAs (miRNAs) have repeatedly been implicated in schizophrenia etiology. It is therefore of interest to establish their role in the regulation of schizophrenia risk genes in disease-relevant biological processes. OBJECTIVE: To examine the role of miRNAs in schizophrenia in the context of disease-associated genetic variation. DESIGN, SETTING, AND PARTICIPANTS: The basis of this study was summary statistics from the largest schizophrenia genome-wide association study meta-analysis to date (83 550 individuals in a meta-analysis of 52 genome-wide association studies) completed in 2014 along with publicly available data for predicted miRNA targets. We examined whether schizophrenia risk genes were more likely to be regulated by miRNA. Further, we used gene set analyses to identify miRNAs that are regulators of schizophrenia risk genes. MAIN OUTCOMES AND MEASURES: Results from association tests for miRNA targetomes and related analyses. RESULTS: In line with previous studies, we found that similar to other complex traits, schizophrenia risk genes were more likely to be regulated by miRNAs (P < 2 × 10-16). Further, the gene set analyses revealed several miRNAs regulating schizophrenia risk genes, with the strongest enrichment for targets of miR-9-5p (P = .0056 for enrichment among the top 1% most-associated single-nucleotide polymorphisms, corrected for multiple testing). It is further of note that MIR9-2 is located in a genomic region showing strong evidence for association with schizophrenia (P = 7.1 × 10-8). The second and third strongest gene set signals were seen for the targets of miR-485-5p and miR-137, respectively. CONCLUSIONS AND RELEVANCE: This study provides evidence for a role of miR-9-5p in the etiology of schizophrenia. Its implication is of particular interest as the functions of this neurodevelopmental miRNA tie in with established disease biology: it has a regulatory loop with the fragile X mental retardation homologue FXR1 and regulates dopamine D2 receptor density.

[Genomics of mental disorders].

The heritability of mental disorders is high, but it has been very difficult to unravel the genetic architecture. In the past five years, unprecedented progress has been made on the insight of the genetic background of several mental disorders, and the present article describes the current results. The disorders are extremely polygenic with thousands of common as well as rare risk variants, some of which are shared across disorders. Epigenetic modifications and potentially gene-environment interactions are adding further complexity to the picture.

A genome-wide search for linkage to allergic rhinitis in Danish sib-pair families.

Allergic rhinitis (AR) is a complex disorder with a polygenic, multifactorial aetiology. Twin studies have found the genetic contribution to be substantial. We collected and clinically characterised a sample consisting of 127 Danish nuclear families with at least two siblings suffering from AR or allergic conjunctivitis including 540 individuals (286 children and 254 parents). A whole-genome linkage scan, using 424 microsatellite markers, was performed on both this sample and an earlier collected sample consisting of 130 families with atopic dermatitis and other atopic disorders. A third sib-pair family sample, which was previously collected and genotyped, was added to the analysis increasing the total sample size to 357 families consisting of 1508 individuals. In total, 190 families with AR was included. The linkage analysis software Genehunter NPL, Genehunter MOD, and Genehunter Imprinting were used to obtain nonparametric and parametric linkage results. Family-based association analysis of positional candidate SNPs was carried out using the FBAT program. We obtained genome-wide significant linkage to a novel AR locus at 1p13 and suggestive linkage to two novel regions at 1q31-q32 and 20p12, respectively. Family-based association analysis of SNPs in the candidate locus DNND1B/CRB1 at 1q31 showed no significant association and could not explain the linkage signal observed. Suggestive evidence of linkage was also obtained at three AR loci previously reported (2q14-q23, 2q23, and 12p13) and indication of linkage was observed at a number of additional loci. Likely maternal imprinting was observed at 2q23, and possible maternal imprinting at 3q28.

A functional CD86 polymorphism associated with asthma and related allergic disorders.

BACKGROUND: Several studies have documented a substantial genetic component in the aetiology of allergic diseases and a number of atopy susceptibility loci have been suggested. One of these loci is 3q21, at which linkage to multiple atopy phenotypes has been reported. This region harbours the CD86 gene encoding the costimulatory B7.2 protein. The costimulatory system, consisting of receptor proteins, cytokines and associated factors, activates T cells and regulates the immune response upon allergen challenge. METHODS: We sequenced the CD86 gene in patients with atopy from 10 families that showed evidence of linkage to 3q21. Identified polymorphisms were analysed in a subsequent family-based association study of two independent Danish samples, respectively comprising 135 and 100 trios of children with atopy and their parents. Functional analysis of the costimulatory effect on cytokine production was performed in an autologous cell-based system based on cells expressing CD86 variants. RESULTS: Two polymorphisms were identified, encoding the amino acid changes Ile179Val and Ala304Thr, respectively. Significant associations were observed between the Ile179Val polymorphism and allergy phenotypes in both samples (eg, asthma, p = 4 x 10(-3) in the two samples combined). The undertransmitted (protective) Val179 allele was found to induce higher production of both Th1 and Th2 cytokines than the overtransmitted (risk) Ile179 allele, suggesting a functional impact of the polymorphism. CONCLUSION: The CD86 gene, and specifically the Ile179Val polymorphism, may be a novel aetiological factor in the development of asthma and related allergic disorders.