David M. Hougaard

Common variants conferring risk of schizophrenia

Schizophrenia is a complex disorder, caused by both genetic and environmental factors and their interactions. Research on pathogenesis has traditionally focused on neurotransmitter systems in the brain, particularly those involving dopamine. Schizophrenia has been considered a separate disease for over a century, but in the absence of clear biological markers, diagnosis has historically been based on signs and symptoms. A fundamental message emerging from genome-wide association studies of copy number variations (CNVs) associated with the disease is that its genetic basis does not necessarily conform to classical nosological disease boundaries. Certain CNVs confer not only high relative risk of schizophrenia but also of other psychiatric disorders. The structural variations associated with schizophrenia can involve several genes and the phenotypic syndromes, or the ‘genomic disorders’, have not yet been characterized. Single nucleotide polymorphism (SNP)-based genome-wide association studies with the potential to implicate individual genes in complex diseases may reveal underlying biological pathways. Here we combined SNP data from several large genome-wide scans and followed up the most significant association signals. We found significant association with several markers spanning the major histocompatibility complex (MHC) region on chromosome 6p21.3-22.1, a marker located upstream of the neurogranin gene (NRGN) on 11q24.2 and a marker in intron four of transcription factor 4 (TCF4) on 18q21.2. Our findings implicating the MHC region are consistent with an immune component to schizophrenia risk, whereas the association with NRGN and TCF4 points to perturbation of pathways involved in brain development, memory and cognition.

NEONATAL VITAMIN D STATUS AND RISK OF SCHIZOPHRENIA: A POPULATION-BASED CASE-CONTROL STUDY

CONTEXT Clues from the epidemiology of schizophrenia suggest that low levels of developmental vitamin D may be associated with increased risk of schizophrenia. OBJECTIVE To directly examine the association between neonatal vitamin D status and risk of schizophrenia. DESIGN Individually matched case-control study drawn from a population-based cohort. SETTING Danish national health registers and neonatal biobank. PARTICIPANTS A total of 424 individuals with schizophrenia and 424 controls matched for sex and date of birth. MAIN OUTCOME MEASURES The concentration of 25 hydroxyvitamin D(3) (25[OH]D3) was assessed from neonatal dried blood samples using a highly sensitive liquid chromatography tandem mass spectroscopy method. Relative risks were calculated for the matched pairs when examined for quintiles of 25(OH)D3. RESULTS Compared with neonates in the fourth quintile (with 25[OH]D3 concentrations between 40.5 and 50.9 nmol/L), those in each of the lower 3 quintiles had a significantly increased risk of schizophrenia (2-fold elevated risk). Unexpectedly, those in the highest quintile also had a significantly increased risk of schizophrenia. Based on this analysis, the population-attributable fraction associated with neonatal vitamin D status was 44%. The relationship was not explained by a wide range of potential confounding or interacting variables. CONCLUSIONS Both low and high concentrations of neonatal vitamin D are associated with increased risk of schizophrenia, and it is feasible that this exposure could contribute to a sizeable proportion of cases in Denmark. In light of the substantial public health implications of this finding, there is an urgent need to further explore the effect of vitamin D status on brain development and later mental health.

A genome-wide association study identifies CDHR3 as a susceptibility locus for early childhood asthma with severe exacerbations

Asthma exacerbations are among the most frequent causes of hospitalization during childhood, but the underlying mechanisms are poorly understood. We performed a genome-wide association study of a specific asthma phenotype characterized by recurrent, severe exacerbations occurring between 2 and 6 years of age in a total of 1,173 cases and 2,522 controls. Cases were identified from national health registries of hospitalization, and DNA was obtained from the Danish Neonatal Screening Biobank. We identified five loci with genome-wide significant association. Four of these, GSDMB, IL33, RAD50 and IL1RL1, were previously reported as asthma susceptibility loci, but the effect sizes for these loci in our cohort were considerably larger than in the previous genome-wide association studies of asthma. We also obtained strong evidence for a new susceptibility gene, CDHR3 (encoding cadherin-related family member 3), which is highly expressed in airway epithelium. These results demonstrate the strength of applying specific phenotyping in the search for asthma susceptibility genes.

Toxoplasma gondii as a risk factor for early-onset schizophrenia : Analysis of filter paper blood samples obtained at birth

BACKGROUND Infections during fetal life or neonatal period, including infections with Toxoplasma gondii, may be associated with a risk for schizophrenia and other mental disorders. The objectives of this study were to study the association between serological markers for maternal and neonatal infection and the risk for schizophrenia, related psychoses, and affective disorders in a national cohort of newborns. METHODS This study was a cohort-based, case-control study combining data from national population registers and patient registers and a national neonatal screening biobank in Denmark. Patients included persons born in Denmark in 1981 or later followed up through 1999 with respect to inpatient or outpatient treatment for schizophrenia or related disorders (ICD-10 F2) or affective disorders (ICD-10 F3). RESULTS Toxoplasma gondii immunoglobulin G (IgG) levels corresponding to the upper quartile among control subjects were significantly associated with schizophrenia risk (odds ratio [OR] = 1.79, p = .045) after adjustment for urbanicity of place of birth, year of birth, gender, and psychiatric diagnoses among first-degree relatives. There was no significant association between any marker of infection and other schizophrenia-like disorders or affective disorders. CONCLUSIONS Our study supports an association between Toxoplasma gondii and early-onset schizophrenia. Further studies are needed to establish if the association is causal and if it generalizes to cases with onset after age 18.

A sensitive LC/MS/MS assay of 25OH vitamin D3 and 25OH vitamin D2 in dried blood spots.

BACKGROUND Low levels of 25 hydroxyvitamin D (25OHD) during early development is associated with a range of adverse health outcomes. While a number of methods exist to measure 25OHD in sera, none have been specifically developed to examine dried blood spots (DBS). METHODS We describe an assay where 25 hydroxyvitamin D(3) (25OHD3) and 25 hydroxyvitamin D(2) (25OHD2) are extracted from 3.2 mm DBS punches, derivatised with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) prior to analysis with LC/MS/MS. We assessed assay precision, relative accuracy and examined the impact of storage conditions in samples stored for up to 22 years. RESULTS The new assay had good accuracy and precision, and was highly sensitive, being capable of detecting <1 nmol/l 25OHD3 and 2 nmol/l 25OHD2. CDER sensitivity criteria were slightly higher at 7.7 nmol/l for 25OHD3 and 10.7 nmol/l for 25OHD2. The mean 25OHD3 concentration in 118 archived DBS was 20.8+/-11.4, (4.8 to 67.8 nmol/l). 25OHD2 was detected in only two of these samples. 25OHD3 concentrations were significantly higher in DBS collected in summer compared to winter (p<0.0001). CONCLUSION Both 25OHD3 and 25OHD2 can be reliably quantified in archived 3.2 mm dried blood spots. We can not be certain that the levels we measure in archived samples are exactly the same as when they were collected. However, the fact that the DBS levels reflect the well-known seasonal variation in this vitamin and when corrected for sera, values fall within the normal range for 25OHD3, means that DBS are a useful tissue repository for testing a range of hypotheses linking developmental hypovitaminosis D and adverse health outcomes.

Common Variants at VRK2 and TCF4 Conferring Risk of Schizophrenia

Common sequence variants have recently joined rare structural polymorphisms as genetic factors with strong evidence for association with schizophrenia. Here we extend our previous genome-wide association study and meta-analysis (totalling 7 946 cases and 19 036 controls) by examining an expanded set of variants using an enlarged follow-up sample (up to 10 260 cases and 23 500 controls). In addition to previously reported alleles in the major histocompatibility complex region, near neurogranin (NRGN) and in an intron of transcription factor 4 (TCF4), we find two novel variants showing genome-wide significant association: rs2312147[C], upstream of vaccinia-related kinase 2 (VRK2) [odds ratio (OR) = 1.09, P = 1.9 × 10(-9)] and rs4309482[A], between coiled-coiled domain containing 68 (CCDC68) and TCF4, about 400 kb from the previously described risk allele, but not accounted for by its association (OR = 1.09, P = 7.8 × 10(-9)).

Weight loss larger than 10% is needed for general improvement of levels of circulating adiponectin and markers of inflammation in obese subjects: a 3-year weight loss study

OBJECTIVE To investigate the effects of: I) short- (8 weeks), II) long-term (3 years) weight loss, and III) the degree of weight loss on circulating levels of adiponectin, high sensitive-C reactive protein (hs-CRP), and fibrinogen in obese subjects. Moreover, to evaluate the effect of the lipase inhibitor, orlistat, on these parameters. DESIGN Weight loss induced in 93 obese subjects (mean weight: 108.9+/-15.8 kg) through 8-week very-low-energy diet (VLED, 800 kcal/day) followed by randomization to orlistat or placebo together with lifestyle intervention for further 3 years. Adiponectin and hs-CRP were measured at baseline, after 8 weeks of VLED and 6, 12, and 36 months after the VLED by flowmetric xMAP technology (Luminex Multi-Analyte Profiling System, Luminex Corp., Austin, TX, USA). Fibrinogen was measured in a coagulation assay. RESULTS Weight loss after VLED treatment was 14.3+/-4.5 kg and after 3 years 7.7+/-8.7 kg. Orlistat-treated subjects regained 3.9 kg less than placebo-treated from the end of the VLED to 3 years (P=0.01). No differences were detected between the two groups regarding changes in adiponectin, hs-CRP, or fibrinogen. Accordingly, the groups were combined for further analyses. Serum adiponectin increased by 22% (P<0.05) after the VLED but returned to baseline after 3 years. Both short- and long-term weight losses needed to be in excess of 10% (approximately 12 kg) in order to increase adiponectin levels significantly. Weight loss was associated with a significant decrease in hs-CRP. Fibrinogen decreased by 12% (P<0.05) after 3 years. CONCLUSIONS In obese subjects, weight loss was associated with an increase in serum adiponectin and a decrease in hs-CRP and plasma fibrinogen. Long-term weight loss (3 years) must exceed 10% to induce a combined significant improvement in these inflammatory markers.

CACNA1C (rs1006737) is associated with schizophrenia

molecules are not altered in all neuropsychiatric disorders. Taken together, these findings show that hyperinsulinemia may have a role in the onset of schizophrenia. This has important implications, as elevated insulin levels can have deleterious effects on brain function. In addition, this suggests the possibility that drugs that improve insulin signaling may represent a novel treatment strategy. In this regard, the insulin-related molecules identified here, and potentially other co-secreted insulin-secretory granule proteins, may have utility as biomarkers for patient stratification and for monitoring the responses to existing and novel therapeutic treatment strategies.

Expanding the range of ZNF804A variants conferring risk of psychosis

A trio of genome-wide association studies recently reported sequence variants at three loci to be significantly associated with schizophrenia. No sequence polymorphism had been unequivocally (P<5 × 10−8) associated with schizophrenia earlier. However, one variant, rs1344706[T], had come very close. This polymorphism, located in an intron of ZNF804A, was reported to associate with schizophrenia with a P-value of 1.6 × 10−7, and with psychosis (schizophrenia plus bipolar disorder) with a P-value of 1.0 × 10−8. In this study, using 5164 schizophrenia cases and 20 709 controls, we replicated the association with schizophrenia (odds ratio OR=1.08, P=0.0029) and, by adding bipolar disorder patients, we also confirmed the association with psychosis (added N=609, OR=1.09, P=0.00065). Furthermore, as it has been proposed that variants such as rs1344706[T]—common and with low relative risk—may also serve to identify regions harboring less common, higher-risk susceptibility alleles, we searched ZNF804A for large copy number variants (CNVs) in 4235 psychosis patients, 1173 patients with other psychiatric disorders and 39 481 controls. We identified two CNVs including at least part of ZNF804A in psychosis patients and no ZNF804A CNVs in controls (P=0.013 for association with psychosis). In addition, we found a ZNF804A CNV in an anxiety patient (P=0.0016 for association with the larger set of psychiatric disorders).

Genetic risk for autism spectrum disorders and neuropsychiatric variation in the general population

Almost all genetic risk factors for autism spectrum disorders (ASDs) can be found in the general population, but the effects of this risk are unclear in people not ascertained for neuropsychiatric symptoms. Using several large ASD consortium and population-based resources (total n > 38,000), we find genome-wide genetic links between ASDs and typical variation in social behavior and adaptive functioning. This finding is evidenced through both LD score correlation and de novo variant analysis, indicating that multiple types of genetic risk for ASDs influence a continuum of behavioral and developmental traits, the severe tail of which can result in diagnosis with an ASD or other neuropsychiatric disorder. A continuum model should inform the design and interpretation of studies of neuropsychiatric disease biology.