Bent Nørgaard-Pedersen

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.

Feasibility of neonatal screening for toxoplasma infection in the absence of prenatal treatment

BACKGROUND The best method for prevention and control of congenital toxoplasma infection is uncertain. Prenatal screening is done in Austria and France, but the effect of treatment during pregnancy is not well documented. The aim of our study was to find out the maternofetal transmission rate and outcome in infants born to mothers who were not treated during pregnancy. METHODS We analysed 89873 eluates from phenylketonuria (PKU) cards from neonates and paired first-trimester serum samples from the mothers for specific IgG antibodies to Toxoplasma gondii. Children born to mothers who seroconverted during pregnancy were followed-up clinically and serologically to 12 months of age. In addition, 21144 PKU cards were analysed for toxoplasma-specific IgM antibodies during the last 12 months of the study. FINDINGS In 24989 (27.8%) cases both the PKU eluate and the first-trimester samples were IgG positive, which indicates previous maternal infection. 139 of the 64884 seronegative women acquired toxoplasma infection during pregnancy and gave birth to 141 infants (two sets of twins). 27 of these children were diagnosed with congenital toxoplasma infection. The transmission rate was 19.4% (95% CI 13.2-27.0). Clinical signs and symptoms were found in four (15%) of the 27 children. The additional analysis for toxoplasma-specific IgM antibodies from the PKU card identified seven of nine children with congenital toxoplasma infection. The false-positive rate for the IgM test was 0.19 per 1000, and no false-negatives were found. INTERPRETATION The risks of transmission of infection and of disease in the infant are low in an area with a low risk of toxoplasma infection. A neonatal screening programme based on detection of toxoplasma-specific IgM antibodies alone will identify between 70% and 80% of cases of congenital toxoplasmosis.

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.

Reduction of the disintegrin and metalloprotease ADAM12 in preeclampsia.

Objectives: The secreted form of ADAM12 is a metalloprotease that may be involved in placental and fetal growth. We examined whether the concentration of ADAM12 in first-trimester maternal serum could be used as a marker for preeclampsia. Methods: We developed a semiautomated, time-resolved, immunofluorometric assay for the quantification of ADAM12 in serum. The assay detected ADAM12 in a range of 78–1248 &mgr;g/L. Serum samples derived from women in the first trimester of a normal pregnancy (n = 324) and from women who later developed preeclampsia during pregnancy (n = 160) were obtained from the First Trimester Copenhagen Study. ADAM12 levels were assayed in these serum samples. Serum levels of ADAM12 were converted to multiples of the median (MoM) after log-linear regression of concentration versus gestational age. Results: Serum ADAM12 levels in women who developed preeclampsia during pregnancy had a mean log MoM of –0.066, which was significantly lower than the mean log MoM of –0.001 for ADAM12 levels observed in serum samples from women with normal pregnancy (P = .008). The mean log MoM was even lower in serum derived from preeclamptic women whose infants weight at birth was less than 2,500 g (n = 27, mean log MoM of –0.120, P = .053). Conclusion: The maternal serum levels of ADAM12 are significantly lower during the first trimester in women who later develop preeclampsia during pregnancy when compared with levels in women with normal pregnancies. Because the secreted form of ADAM12 cleaves insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5, the IGF axis may play a role in preeclampsia. ADAM12 may be a useful early marker for preeclampsia. Level of Evidence: II-2

Storage policies and use of the Danish Newborn Screening Biobank

SummaryAfter routine newborn screening, residual dried blood spot samples (DBSS) are stored at −20°C in the Danish Newborn Screening Biobank (NBS-Biobank), which contains DBSS from virtually all newborns in Denmark since 1982—about 1.8 million samples. The purpose of the storage is: (1) diagnosis and treatment of congenital disorders including documentation, repeat testing, quality assurance, statistics and improvement of screening methods; (2) diagnostic use later in infancy after informed consent; (3) legal use after court order; (4) the possibility of research projects after approval by the Scientific Ethical Committee System in Denmark, The Danish Data Protection Agency and the NBS-Biobank Steering Committee. The operation and use of the NBS-Biobank has until recently been regulated by an executive order of 1993 from the Danish Ministry of Health. The Ethical Council, the Central Scientific Ethical Committee and the National Board of Health were also involved in the regulations. These regulations have now been replaced by detailed general operational guidelines for biobanks in Denmark according to Acts on Processing of Personal Data, Patient’s Rights, Health 546/2005 and the Biomedical Research Ethics Committee System. No specific Act on biobanks per se has been made in Denmark, but the new regulations and guidelines make the operations of the Danish NBS-Biobank even more clear-cut and safe. The Danish NBS-Biobank has been used in several research projects for aetiological studies of a number of disorders, recently employing new sensitive multiplex technologies and genetic analyses utilizing whole-genome amplified DNA.

Maternal serum markers in screening for Down syndrome.

The addition of two new markers in maternal serum, estriol and HCG, to those already known, namely the level of maternal serum alfa‐fetoprotein and maternal age, considerably improves the expected results of a screening strategy for Down syndrome. The detection rate is slightly increased from 53.0% to 57.6%, but, more importantly, the false‐positive rate decreases from 9.4% to 7.3%. It is our belief that, at least in women aged less than 35 years, a screening strategy based on a combination of maternal age and biochemical markers should be incorporated into antenatal care. For older women, the results of such a maternal serum test may refine counseling for genetic amniocentesis, as a much more explicit risk calculation can be performed than that based on age alone.

Effects of blood sample handling procedures on measurable inflammatory markers in plasma, serum and dried blood spot samples

The interests in monitoring inflammation by immunoassay determination of blood inflammatory markers call for information on the stability of these markers in relation to the handling of blood samples. The increasing use of stored biobank samples for such ventures that may have been collected and stored for other purposes, justifies the study hereof. Blood samples were stored for 0, 4, 24, and 48 h at 4 degrees C, room temperature (RT), and at 35 degrees C, respectively, before they were separated into serum or plasma and frozen. Dried blood spot samples (DBSS) were stored for 0, 1, 2, 3, 7, and 30 days at the same temperatures. 27 inflammatory markers in serum and plasma and 25 markers in DBSS were measured by a previously validated multiplex sandwich immunoassay using Luminex xMAP technology. The measurable concentrations of several cytokines in serum and plasma were significantly increased when blood samples were stored for a period of time before the centrifugation, for certain cytokines more than 1000 fold compared to serum and plasma isolated and frozen immediately after venepuncture. The concentrations in serum generally increased more than in plasma. The measurable concentrations of inflammatory markers also changed in DBSS stored under various conditions compared to controls frozen immediately after preparation, but to a much lesser degree than in plasma or serum. The study demonstrates that trustworthy measurement of several inflammatory markers relies on handling of whole blood samples at low temperatures and rapid isolation of plasma and serum. Effects of different handling procedures for all markers studied are given. DBSS proved to be a robust and convenient way to handle samples for immunoassay analysis of inflammatory markers in whole blood.

Amniotic fluid inflammatory cytokines: Potential markers of immunologic dysfunction in autism spectrum disorders

Abstract Objectives. The aim of the study was to analyze cytokine profiles in amniotic fluid (AF) samples of children developing autism spectrum disorders (ASD) and controls, adjusting for maternal autoimmune disorders and maternal infections during pregnancy. Methods. AF samples of 331 ASD cases and 698 controls were analyzed for inflammatory cytokines using Luminex xMAP technology utilizing a historic birth cohort. Clinical data were retrieved from nationwide registers, and case-control differences in AF cytokine levels were assessed using chi-square tests, logistic and tobit regression models. Results. Overall, individuals with ASD had significantly elevated AF levels of TNF-α and TNF-β compared to controls. Analyzing individuals diagnosed only with ICD-10 codes yielded significantly elevated levels of IL-4, IL-10, TNF-α and TNF-β in ASD patients. Restricting analysis to infantile autism cases showed significantly elevated levels of IL-4, TNF-α and TNF-β compared to controls with no psychiatric comorbidities. Elevated levels of IL-6 and IL-5 were found in individuals with other childhood psychiatric disorders (OCPD) when compared to controls with no psychiatric comorbidities. Conclusions. AF samples of individuals with ASD or OCPD showed differential cytokine profiles compared to frequency-matched controls. Further studies to examine the specificity of the reported cytokine profiles in ASD and OCPD are required.