Danish Neonatal Screening Biobank Research Articles

Mothers’ Views on the Storage and Usage of Their Children’s Biological Material Under the Danish Biobanking Model: A Narrative Approach Using Epistemic Injustice

ObjectivesTo investigate the knowledge and attitudes of mothers living in Denmark on the storage and usage of their children’s biological material. The Danish Neonatal Screening Biobank contains blood from the Phenylketonuria-screening test. Legal, ethical, and moral concerns have been raised in several countries of how consent is obtained best in pediatric biobank governance. Research on knowledge and attitudes of Danish parents on the usage of their children’s biological material is scarce. MethodsA coproduced study between a mother and 2 researchers. We analyzed 5 online focus group interviews using Ricoeur’s hermeneutical narrative analysis. ResultsMothers have very little knowledge on the storage and usage of their children’s biological material. They consider the Phenylketonuria-screening test to be part of a birth package, which leaves very little option of choice. They accept donating the material as a token of appreciation in an act of altruism toward the wider society but are only comfortable supporting Danish research. ConclusionsAn exploration of the communal narrative build in the interviews reveal an overall feeling of duty to help benefit society, an overwhelming trust toward the health system, and epistemic unjust storage information practices.

The impact of the COVID-19 lockdown on birthweight among singleton term birth in Denmark.

In Denmark, a nationwide COVID-19 lockdown was implemented on March 12, 2020 and eased on April 14, 2020. The COVID-19 lockdown featured reduced prevalence of extremely preterm or extremely low birthweight births. This study aims to explore the impact of this COVID-19 lockdown on term birthweights in Denmark. We conducted a nationwide register-based cohort study on 27,870 live singleton infants, born at term (weeks 37-41), between March 12 and April 14, 2015-2020, using data from the Danish Neonatal Screening Biobank. Primary outcomes, corrected for confounders, were birthweight, small-for-gestational-age (SGA), and large-for-gestational-age (LGA), comparing the COVID-19 lockdown to the previous five years. Data were analysed using linear regression to assess associations with birthweight. Multinomial logistic regression was used to assess associations with relative-size-for-gestational-age (xGA) categories. Adjusted mean birthweight was significantly increased by 16.9 g (95% CI = 4.1-31.3) during the lockdown period. A dip in mean birthweight was found in gestational weeks 37 and 38 balanced by an increase in weeks 40 and 41. The 2020 lockdown period was associated with an increased LGA prevalence (aOR 1.13, 95% CI = 1.05-1.21). No significant changes in proportions of xGA groups were found between 2015 and 2019. The nationwide COVID-19 lockdown resulted in a small but significant increase in birthweight and proportion of LGA infants, driven by an increase in birthweight in gestational weeks 40 and 41.

Gestational age-dependent development of the neonatal metabolome.

Prematurity is a severe pathophysiological condition, however, little is known about the gestational age-dependent development of the neonatal metabolome. Using an untargeted liquid chromatography-tandem mass spectrometry metabolomics protocol, we measured over 9000 metabolites in 298 neonatal residual heel prick dried blood spots retrieved from the Danish Neonatal Screening Biobank. By combining multiple state-of-the-art metabolome mining tools, we retrieved chemical structural information at a broad level for over 5000 (60%) metabolites and assessed their relation to gestational age. A total of 1459 (~16%) metabolites were significantly correlated with gestational age (false discovery rate-adjusted P < 0.05), whereas 83 metabolites explained on average 48% of the variance in gestational age. Using a custom algorithm based on hypergeometric testing, we identified compound classes (617 metabolites) overrepresented with metabolites correlating with gestational age (P < 0.05). Metabolites significantly related to gestational age included bile acids, carnitines, polyamines, amino acid-derived compounds, nucleotides, phosphatidylcholines and dipeptides, as well as treatment-related metabolites, such as antibiotics and caffeine. Our findings elucidate the gestational age-dependent development of the neonatal blood metabolome and suggest that the application of metabolomics tools has great potential to reveal novel biochemical underpinnings of disease and improve our understanding of complex pathophysiological mechanisms underlying prematurity-associated disorders. A large variation in the neonatal dried blood spot metabolome from residual heel pricks stored at the Danish Neonatal Screening Biobank can be explained by gestational age. While previous studies have assessed the relation of selected metabolic markers to gestational age, this study assesses metabolome-wide changes related to prematurity. Using a combination of recently developed metabolome mining tools, we assess the relation of over 9000 metabolic features to gestational age. The ability to assess metabolome-wide changes related to prematurity in neonates could pave the way to finding novel biochemical underpinnings of health complications related to preterm birth.

Adolescent residential mobility, genetic liability and risk of schizophrenia, bipolar disorder and major depression.

Residential mobility during upbringing, and especially adolescence, is associated with multiple negative mental health outcomes. However, whether associations are confounded by unmeasured familial factors, including genetic liability, is unclear. We used a population-based case-cohort study to assess whether polygenic risk scores (PRSs) for schizophrenia, bipolar disorder and major depression were associated with mobility from ages 10-14 years, and whether PRS and parental history of mental disorder together explained associations between mobility and each disorder. Information on cases (n = 4207 schizophrenia, n = 1402 bipolar disorder, n = 18 215 major depression) and a random population sample (n = 17 582), born 1981-1997, was linked between Danish civil and psychiatric registries. Genome-wide data were obtained from the Danish Neonatal Screening Biobank and PRSs were calculated based on results of separate, large meta-analyses. PRSs for schizophrenia and major depression were weakly associated with moving once (odds ratio 1.07, 95% CI 1.00-1.16; and odds ratio 1.10, 95% CI 1.04-1.17, respectively), but not twice or three or more times. Mobility was positively associated with each disorder, with more moves associated with greater risk. Adjustment for PRS produced slight reductions in the magnitude of associations. Adjustment for PRS and parental history of mental disorder together reduced estimates by 5-11%. In fully adjusted models mobility was associated with all three disorders; hazard ratios ranged from 1.33 (95% CI 1.08-1.62; one move and bipolar disorder) to 3.05 (95% CI 1.92-4.86; three or more moves and bipolar disorder). Associations of mobility with schizophrenia, bipolar disorder and depression do not appear to be attributable to genetic liability as measured here. Potential familial confounding of mobility associations may be predominantly environmental in nature.

Including Classical Galactosaemia in the Expanded Newborn Screening Panel Using Tandem Mass Spectrometry for Galactose-1-Phosphate

Galactosaemia has been included in various newborn screening programs since 1963. Several methods are used for screening; however, the predominant methods used today are based on the determination of either galactose-1-phosphate uridyltransferase (GALT) activity or the concentration of total galactose. These methods cannot be multiplexed and therefore require one full punch per sample. Since the introduction of mass spectrometry in newborn screening, many diseases have been included in newborn screening programs. Here, we present a method for including classical galactosaemia in an expanded newborn screening panel based on the specific determination of galactose-1-phosphate by tandem mass spectrometry. The existing workflow only needs minor adjustments, and it can be run on the tandem mass spectrometers in routine use. Furthermore, compared to the currently used methods, this novel method has a superior screening performance, producing significantly fewer false positive results. We present data from 5500 routine newborn screening samples from the Danish Neonatal Screening Biobank. The cohort was enriched by including 14 confirmed galactosaemia positive samples and 10 samples positive for other metabolic disorders diagnosed through the Danish newborn screening program. All galactosaemia positive samples were identified by the method with no false positives. Furthermore, the screening performance for other metabolic disorders was unaffected.

8 - COMMON RISK VARIANTS AND POLYGENIC HETEROGENEITY IN AUTISM

Background Autism Spectrum Disorder (ASD) is a psychiatric disorder with a World Wide prevalence of about 1%. ASD is characterized by impairments in social interaction, communication and repetitive stereotypic behavior. The etiology of ASD is largely unknown, but it is highly heritable and common variation is estimated to explain half of the genetic risk. Shared genetics between different psychiatric disorders have been widely reported, as have overlaps with phenotypes such as educational attainment and IQ. Over the past couple of years, iPSYCH and Broad/MGH have conducted the largest GWAS of ASD to date. Altogether more than 47k subjects from the Danish Neonatal Screening Biobank were included in the ASD GWAS and subsequently meta-analyzed together with additional 11k samples ASD GWAS from the Psychiatric Genomics Consortium (PGC). Methods The iPSYCH sample is a population sample with cases from the Danish Psychiatric Central Research Register and a random sample from the same birth cohorts as control group. All subjects were identified in the Danish Neonatal Screening Biobank, their DNA extracted, whole-genome amplified and genotyped on the PsychChip. We conducted the analyses in the Ricopili pipeline of PGC, and using LDSC, GCTA and R. Results We report here on the final freeze of the ASD GWAS. Almost 36k were analyzed as part of the CEU sample that was subsequently meta-analyzed together with the additional 11k samples ASD GWAS from the Psychiatric Genomics Consortium (PGC). While we will describe the 3 genome-wide significant the single locus findings as well as 2 more genome-wide significant loci in the meta-analysis with the replication sample, the main focus will be on polygenic features. We confirm previously reported genetic correlations, eg. for education (rg= 0:20, p=2.6E-9 for SSGAC and rg= 0.27, p=2.1E-11 for UKB) and follow up with results for newly analyzed phenotypes. Moreover, we will show an emerging polygenic structure that relates polygenic risk scores (PRS) to ASD subtypes (Asperger, childhood autism, atypical autism, other pervasive developmental disorder and pervasive developmental disorder, unspecified). E.g. the PRS for educational attainment loads differently on disjoint subtype groups, p=2.6E-11 (p=5.3E-8 when excluding intellectual disability) while for schizophrenia there is no such heterogeneity, p=0.98. Discussion Despite being the largest GWAS of autism yet conducted and despite showing a handful of genome-wide significant loci, it is clear that it will take a significant increase in sample size before ASD GWASes will be powered well enough for full-fledged gene-discovery. There is however already sufficient power that we can begin to dissect the polygenic structures and here new and interesting contours are developing with PRSs for other phenotypes loading differential on ASD subtypes.

13 - MITOCHONDRIAL DNA HAPLOGROUPS ARE ASSOCIATED WITH PSYCHIATRIC DISEASE: A NATION-WIDE STUDY OF 74,763 DANES

Background Disturbed mitochondrial function has been implicated in psychiatric disease. All mitochondria contain a small maternally inherited DNA (mtDNA) of 16.6 kb. Through evolution and genetic drift, the mtDNA sequence has become fixed into haplogroups (hgs) with a characteristic population and geographical distribution. Different hgs exhibit variable functional capacity and have been demonstrated to be susceptibility factors for various, predominantly degenerative, diseases. Associations with Parkinsons and Alzheimers diseases, as well as cardiac, metabolic and psychiatric disorders have been reported. These findings have prompted the suggestion that variations in mitochondrial function might be susceptibility factors for disease. However, reports on association between mtDNA hgs and disease have frequently been conflicting. This is partly due to population stratification in examined populations and low-powered study designs. We analysed the association between mtDNA hgs and psychiatric disease using a very large geographically unbiased cohort, iPSYCH, comprising 2% of Danes as controls and 50,000+ psychiatric patients. Methods Using DNA extracted from dried blood spots from 50,567 Danish psychiatric patients (ADHD: 13,103; ASD: 12,891; Anorexia: 2,124; BD: 1,242; SCZ: 2,438, and AD: 16,225) and 24,196 controls we examined the association between psychiatric disease and mtDNA haplogroup in the Danish population. Psychiatric patients were identified from the Danish Psychiatric Central Registry. DNA was obtained from the Danish Neonatal Screening Biobank. Haplotyping was performed using the PGC developed PsychChip v.1.0. containing 418 SNPs in mtDNA. The SNP data were manually analysed and haplotyped according to haplogroup defining SNPs in Phylotree. Results Hg M was associated with affective disorder (n = 17260) with an OR of 0.47 (cfi.95%: 0.39 – 0.58) (p = 1*10–14), ADHD (n = 13395) with an OR of 0.50 (0.40 – 0.62)(p = 2*10–11). Among patients belonging to the macro-hg N, patients with schizophrenia (n = 2589), had a high proportion of hg A (n = 45), OR: 4.52 (cfi.95%: 3.0 – 6.7)(p = 1.2*10–12). No significant associations between mtDNA hgs and BD or ASD were found. In a mitoGWAS for all six conditions, three mitoSNPs were highly associated with affective disorder (p-values ranging from 2*10E-25 – 2*10E-21), ADHD (p-values: 2*10E-11 – 1*10E-10), and anorexia (p-values: 6*10E -8 - 6*10E-7) and another mitoSNP with schizophrenia (p = 2*10–11). Discussion Psychiatric disease seems to be bigenomic diseases with contributions from both the mitochondrial and nuclear genome. Haplogroup A is a risk factor for schizophrenia and haplogroup M is a protective factor for ADHD and affective disorder. However, both hgs A and M are infrequent in the Danish population and markers of Greenlandic and Asian origin, respectively. So it will be important through studies of interaction between mtDNA and the genomic DNA to precisely define the role of each of these factors in the etiology of psychiatric disease.

26 - ESTIMATED DNA METHYLATION GESTATIONAL AGE IN NEWBORN MONOZYGOTIC TWINS ASSOCIATE WITH LATER PSYCHIATRIC DISORDERS BETWEEN CON/DISCORDANT PAIRS

Background Neonate developmental state at birth has previously been associated with later occurring psychiatric disease. As a novel possible measure of neonate epigenetic developmental state, we investigate the association between Accelerated Gestational Age (AccGA) and later occurring psychiatric disorder in six neuropsychiatric illnesses: Schizophrenia (SZ), Bipolar Disorder (BD), Major Depressive Disorder (MDD), Autism Spectrum Disorder (ASD), Attention Deficit Hyperactivity Disorder (ADHD) and Anorexia (ANX), in a cohort of dis/concordant Monozygotic (MZ) twins. Methods Neonatal dried blood spots, of 548 MZ twins (274 pairs) born between 1981 and 2005 were obtained from the Danish Neonatal Screening Biobank. The samples were analysed on the Illumina Infinium HumanMethylation 450k BeadChip. Estimated DNA methylation gestational age (EstGA) was calculated from 148 CpG probes across the array (described by Knight et al. 2016). Residual values of the linear fit between EstGA and actual gestational age, were used as measures of the in-cohort AccGA. We applied a linear mixed model for the twin pedigree structure - regressing AccGA against the six neuropsychiatric disorders and sex, while Twin Pair ID served as random effect in the twin model. Statistical significance was reached at p-value Results The EstGA correlated well with the actual gestational age (pearson r=0.86), and the AccGA correlated between twin-cotwin (Pearson r=0.67). Within the full cohort BD (N=7) and ANX (N=44) patients had significantly (p-val=0.007 and p-val=0.024) elevated AccGAs over healthy cotwins and cohort (0.967+/-0.356 and 0.310+/-0.137 weeks, respectively). For SZ (N=18) the opposite was observed (pval=0.005) with lowered AccGA (-0.585+/-0.206 weeks). Neither MDD, ASD nor ADHD were significantly associated with AccGA. Within Twin pair analysis of BD (N=6 pairs) and SZ (N=17 pairs), revealed the same significant (p-val=0.016, p-val=0.022, respectively) difference as observed above (1.185+/-0.341 and -0.697+/-0.275, respectively), while ANX (N=40 pairs) showed the same tendency although not significantly (p-val=0.053, 0.235+/-0.118). This association was independent of twin-cotwin birth weight percentile differences (Pearson r=0.0002, Not significant). Discussion We found significant AccGA differences within con/discordant MZ twin pairs and cohort of at least three main psychiatric disorders - detectable already at birth. AccGA has previously been correlated with birth weight percentile. Twin-cotwin differences of AccGA was however not explained by birth weight percentile differences, suggesting that the within pair differences caught by the estimator describes other aspects of neonate development, independent of weight. Our findings suggest that gestational age estimation, as assessed by the 148 CpG probes, in MZ twin pairs, can indicate a not previously reported epigenetic link to later psychiatric illness, this should be further investigated.

55 - LARGE META-ANALYSIS OF SCANDINAVIAN EXOME SEQUENCING STUDIES OF SCHIZOPHRENIA

Background The current data indicated that the allelic architecture of complex traits is caused by many common and rare variants. Large GWAS meta-analyses have been successfully applied in the search for common variants affecting the risk of developing psychiatric disorders, in particular schizophrenia and Major Depressive Disorder. However, these studies are designed to examine only the common variant proportion of the genomic landscape whereas high-throughput sequencing allows direct examination of both common and rare alleles. Schizophrenia is one of the most disabling mental disorders; it has a lifetime prevalence of around 1% and a heritability estimated as high as 80%. Although around one third of the heritability can be attributed to common variants, prior studies indicate that rare variants also contribute to its etiology. Methods A collaboration between iPSYCH (The Lundbeck Initiative for Integrative Psychiatric Research, Denmark) and the Broad Institute (in Cambridge, MA, USA) is conducting large scale exome sequencing of major psychiatric disorders, including schizophrenia. DNA is extracted from dried blood spots (DBS) stored at the Danish Neonatal Screening Biobank. The samples can be matched to a wide range of phenotypic information available in Danish registries. Results We present here the analysis of a large collection of schizophrenia individuals recruited in clinical centers (1,866) and from DBS samples (3,710): a total of 5,576 cases and 7,468 controls from Denmark. We follow up in a similarly sized sample from Sweden with 4,969 cases and 6,245 controls. The combined analysis represents the largest exome sequencing study in schizophrenia to date. We have found evidence showing a higher per-person rate of disruptive ultra-rare loss-of-function variants located in constrained genes among schizophrenia cases compared to controls (combined p-value 1.6e-07). Meta-analysis SKAT of rare and ultra-rare variants at gene level has been performed. Discussion The results from burden and enrichment analysis, as well as ranking of mutational load, will be discussed in the larger perspective of findings from both GWAS and other exome analyses in schizophrenia.

Fetal human leukocyte antigen-C and maternal killer-cell immunoglobulin-like receptors in cases of severe preeclampsia

IntroductionThe pathogenesis of preeclampsia may involve inadequate trophoblast invasion caused by excessive inhibition of uterine natural killer cells (uNK) by extravillous trophoblast cells (EVT). This may be the result of a combination of maternal killer-cell immunoglobin-like receptor (KIR) AA genotype and fetal human leukocyte antigen-C2 (HLA-C2) genotype. A few studies have reported a significantly increased frequency of the maternal KIR AA/fetal HLA-C2 combination in cases of preeclampsia compared to controls. MethodsStudy subjects were 259 cases of severe preeclampsia/eclampsia and 259 matched pregnant women without preeclampsia or eclampsia. All pregnancies were singleton pregnancies, and mothers were preferentially primigravidae. Blood samples from women and their newborns were obtained from the Danish National Birth Cohort (DNBC) and the Danish Neonatal Screening Biobank. Significant differences in the frequencies of KIR AA and HLA-C2 between cases and controls were investigated. ResultsNo significant difference was observed between cases and controls in the frequency of maternal KIR AA (OR = 0.86, 95%CI = 0.60–1.23, P = 0.41), neither when the fetus carried an HLA-C2 allele (OR = 0.85, 95%CI = 0.52–1.38, P = 0.51), nor when the fetus carried an HLA-C2 allele more than its mother (OR = 0.75, 95%CI = 0.34–1.64, P = 0.47). ConclusionThe Results show no influence of HLA-C/KIR genetic variation on the risk of severe preeclampsia, contrary to what some previous studies have observed. An explanation could be that severe preeclampsia represents a separate pathological entity compared to mild preeclampsia.

The role of genetic liability in the association of urbanicity at birth and during upbringing with schizophrenia in Denmark.

Studies have indicated that the association of urbanicity at birth and during upbringing with schizophrenia may be driven by familial factors such as genetic liability. We used a population-based nested case-control study to assess whether polygenic risk score (PRS) for schizophrenia was associated with urbanicity at birth and at age 15, and to assess whether PRS and parental history of mental disorder together explained the association between urbanicity and schizophrenia. Data were drawn from Danish population registries. Cases born since 1981 and diagnosed with schizophrenia between 1994 and 2009 were matched to controls with the same sex and birthdate (1549 pairs). Genome-wide data were obtained from the Danish Neonatal Screening Biobank and PRSs were calculated based on results of a separate, large meta-analysis. Those with higher PRS were more likely reside in the capital compared with rural areas at age 15 [odds ratio (OR) 1.19, 95% confidence interval (CI) 1.01-1.40], but not at birth (OR 1.09, 95% CI 0.95-1.26). Adjustment for PRS produced almost no change in relative risks of schizophrenia associated with urbanicity at birth, but slightly attenuated those for urban residence at age 15. Additional adjustment for parental history led to slight attenuation of relative risks for urbanicity at birth [incidence rate ratio (IRR) for birth in capital = 1.54, 95% CI 1.18-2.02; overall p = 0.016] and further attenuation of relative risks for urbanicity at age 15 (IRR for residence in capital = 1.32, 95% CI 0.97-1.78; overall p = 0.148). While results regarding urbanicity during upbringing were somewhat equivocal, genetic liability as measured here does not appear to explain the association between urbanicity at birth and schizophrenia.

35. The Role of Genetic Liability in the Association Between Urbanicity and Schizophrenia

Abstract Background: The association of urbanicity at birth and during upbringing with schizophrenia remains unexplained. Some studies have indicated that it may be driven by familial selective factors such as genetic liability. However, no studies have used measures of genetic risk other than family history to test this hypothesis with respect to urbanicity at birth. We assessed whether genetic liability to schizophrenia, measured by polygenic risk score (PRS), was associated with being born and raised in urban areas, and whether adjustment for PRS and parental history of mental disorder together explained the effect of urbanicity. Methods: We conducted a nested case–control study using Danish population registry data. Cases in the Danish Psychiatric Central Register (n = 1692) who were born since 1981 and diagnosed with schizophrenia between 1994 and 2009 were matched to controls (n = 1724) with the same sex and birthdate. Genome-wide data were obtained from the Danish Neonatal Screening Biobank, and PRSs were calculated based on a Psychiatric Genomics Consortium meta-analysis that excluded Danish samples. PRSs were adjusted for ancestry using the first 10 principal components. Results: PRS generally increased with increasing level of urbanicity. Those with higher PRSs were significantly more likely to reside in the capital compared to rural areas at age 15 (odds ratio [OR] = 1.19, 95% confidence interval [CI] =1.00–1.42) but not at birth (OR = 1.09, 95%CI = 0.94–1.26). Adjustment for PRS produced almost no change in the excess risk of schizophrenia associated with urbanicity at birth (relative risk [RR] = 1.67, 95%CI = 1.29–2.17). However, the RR associated with urbanicity at age 15 decreased from 1.58 (95%CI = 1.20–2.09) to 1.47 (95%CI = 1.10–1.97) after adjustment for PRS. After adjustment for PRS and parental history together, the association between urbanicity at birth and schizophrenia remained (P = .016) but that for urbanicity at age 15 was fully attenuated (P = .148). Conclusion: We found evidence consistent with a role of selective migration in the association between urbanicity and schizophrenia, especially with respect to urbanicity during upbringing. However, our results failed to support the notion that genetic liability fully explains the excess risk of schizophrenia associated with urbanicity at birth. Additional research is needed to identify other familial mechanisms or constituent exposures that explain the robust association between urbanicity at birth and schizophrenia.

RNA sequencing of archived neonatal dried blood spots

Neonatal dried blood spots (DBS) are routinely collected on standard Guthrie cards for all-comprising national newborn screening programs for inborn errors of metabolism, hypothyroidism and other diseases. In Denmark, the Guthrie cards are stored at −20°C in the Danish Neonatal Screening Biobank and each sample is linked to elaborate social and medical registries. This provides a unique biospecimen repository to enable large population research at a perinatal level. Here, we demonstrate the feasibility to obtain gene expression data from DBS using next-generation RNA sequencing (RNA-seq). RNA-seq was performed on five males and five females. Sequencing results have an average of >30 million reads per sample. 26,799 annotated features can be identified with 64% features detectable without fragments per kilobase of transcript per million mapped reads (FPKM) cutoff; number of detectable features dropped to 18% when FPKM≥1. Sex can be discriminated using blood-based sex-specific gene set identified by the Genotype-Tissue Expression consortium. Here, we demonstrate the feasibility to acquire biologically-relevant gene expression from DBS using RNA-seq which provide a new avenue to investigate perinatal diseases in a high throughput manner.

UGT1A1*28 Genotypes and Respiratory Disease in Very Preterm Infants: A Cohort Study

Background: Respiratory disease in the very preterm infant is frequent and often severe. Bilirubin is both a potent neurotoxin and antioxidant, and may have a clinical impact on preterm respiratory disease. The Gilbert genotype, the UGT1A1*28 allele, is the major known genetic cause of variation in bilirubin. Objectives: To study the association between respiratory disease in the very preterm infant and the UGT1A1*28 allele. Methods: This is a cohort study of 1,354 very preterm infants (gestational age <32 weeks) born in Jutland, Denmark in 1997-2011. Genotypes were obtained from the Danish Neonatal Screening Biobank, and clinical information was obtained from the databases of two tertiary neonatal intensive care units. Outcomes were the need for surfactant therapy, any need for and duration of supplementary oxygen and bronchopulmonary dysplasia (BPD). Results: Per UGT1A1*28 allele, odds were increased for any need of supplementary oxygen (odds ratio 1.26; 1.05-1.50) and for BPD (odds ratio 1.71; 1.23-2.39), the need of supplementary oxygen increased by 6.38 days (1.87-10.89), and chance per day of no longer needing supplementary oxygen was reduced (hazard rate 0.84; 0.76-0.93). No effect was observed for need of surfactant treatment (odds ratio 1.08; 0.91-1.28). Hardy-Weinberg equilibrium was unlikely for the cohort (p < 0.012). This could be explained by death prior to genotype sampling. In tests of robustness this failed to explain the primary results. Conclusions: Compared to the common genotype, UGT1A1*28 genotypes were associated with an increased need of oxygen supplementation and risk of BPD in very preterm newborns.

Genetic Variation in NFKBIE Is Associated With Increased Risk of Pneumococcal Meningitis in Children

BackgroundStreptococcus pneumoniae and Neisseria meningitidis are frequent pathogens in life-threatening infections. Genetic variation in the immune system may predispose to these infections. Nuclear factor-κB is a key component of the TLR-pathway, controlled by inhibitors, encoded by the genes NFKBIA, NFKBIE and NFKBIZ. We aimed to replicate previous findings of genetic variation associated with invasive pneumococcal disease (IPD), and to assess whether similar associations could be found in invasive meningococcal disease (IMD). MethodsCases with IPD and IMD and controls were identified by linking Danish national registries. DNA was obtained from the Danish Neonatal Screening Biobank. The association between SNPs and susceptibility to IPD and IMD, mortality and pneumococcal serotypes was investigated. Results372 children with pneumococcal meningitis, 907 with pneumococcal bacteremia and 1273 controls were included. We included 406 cases with meningococcal meningitis, 272 with meningococcal bacteremia, and 672 controls.The NFKBIE SNP was associated with increased risk of pneumococcal meningitis (aOR 1.68; 95% CI: 1.20–2.36), but not bacteremia (aOR 1.08; 95% CI: 0.86–1.35). The remaining SNPs were not associated with susceptibility to invasive disease. None of the SNPs were associated with risk of IMD or mortality. ConclusionsA NFKBIE polymorphism was associated with increased risk of pneumococcal meningitis.

Mannose-Binding Lectin Gene, MBL2, Polymorphisms Do Not Increase Susceptibility to Invasive Meningococcal Disease in a Population of Danish Children.

Background. Neisseria meningitidis is the cause of meningococcal bacteremia and meningitis, and nasopharyngeal colonization with this pathogen is common. The incidence of invasive disease is highest in infants, whereas adolescents more often are carriers. Altered regulation or dysfunction of the innate immune system may predispose to invasive meningococcal disease (IMD). In this study, we investigated the effect of genetic variation in the mannose-binding lectin gene, MBL2, and its promoter on susceptibility to IMD and IMD-associated mortality among children. Methods. Children (<5 years) diagnosed during 1982-2007 with IMD and controls were identified through Danish national registries. DNA was obtained from the Danish Neonatal Screening Biobank. The associations between MBL2 diplotypes and IMD susceptibility and 30- and 90-day mortality were investigated using logistic regression analysis. Results. We included 1351 children: 406 with meningitis, 272 with bacteremia, and 673 age- and sex-matched controls. Of the children studied, 1292 (96%) were successfully genotyped and assigned MBL2 diplotypes. The median age in IMD cases was 19.1 months (interquartile range [IQR], 8.8-32.2 months). Children with defective MBL2 diplotypes were not at higher risk for meningococcal meningitis than children with intermediate and normal diplotypes (odds ratio [OR] = 0.69; 95% confidence interval [CI], .47-1.02). Similar results were found for children with bacteremia and defective diplotypes (OR = 0.84; 95% CI, .53-1.32) as well as for all cases (OR = 0.75; 95% CI, .56-1.01). There was no association between MBL2 diplotypes and mortality. Conclusions. Defective MBL2 diplotypes did not predict either an increased IMD susceptibility or mortality in a Danish population of children.

Gene expression profiling of archived dried blood spot samples from the Danish Neonatal Screening Biobank.

A large part of the human genome is transcribed into various forms of RNA, and the global gene expression profile (GEP) has been studied for several years using technology such as RNA-microarrays. In this study, we evaluate whether neonatal dried blood spot (DBS) samples stored in the Danish Neonatal Screening Biobank (DNSB) can be used for GEP. This paper is divided into sub-studies examining the effects of: 1) different whole transcriptome amplification kits (WTA); 2) years of storage and storage in room temperature (RT) versus freezers (-20°C) on DNSB DBS samples; 3) effects of RT storage vs freezer storage on DBS samples from the USA and DNSB, and 4) using smaller disc sizes, thereby decreasing DBS use. We present evidence that reliable and reproducible GEPs can be obtained using neonatal DBS samples. The main source of variation is the storage condition. When samples are stored at -20°C, the dynamic range is increased, and Pearson correlations are higher. Differential analysis reveals no statistically significant differences between samples collected a decade apart and stored at -20°C. However, samples stored at RT show differential expression for a third of the gene-specific probes. Our data also suggests that using alternate WTA kits significantly changes the GEP. Finally, the amount of input material, i.e., the size and number of DBS discs used, can be reduced to preserve this valuable and limited material. We conclude that DNSB DBS samples provide a reproducible resource for GEP. Results are improved if the cards are stored at -20°C. Furthermore, it is important to use a single type of kit for analysis because using alternate kits introduces differential expression.

The role of the sodium current complex in a nonreferred nationwide cohort of sudden infant death syndrome.

Sudden infant death syndrome (SIDS) is the most common cause of death in infants between the age of 1 month and 1 year. Rare variants in Nav1.5 encoded by SCN5A are known to play a role in SIDS; however, the combined role of the sodium current complex is unknown. The purpose of this study was to investigate the role of the sodium current complex in a nonreferred nationwide cohort of SIDS cases. DNA was extracted from dried blood spot samples from the Danish Neonatal Screening Biobank. In total, 66 non-referred SIDS cases born in Denmark in the period of 2000-2006 were screened for genetic variants in the 8 major genes involved in the regulation of the Nav1.5 channel complex: SCN5A, SCN1B, SCN2B, SCN3B, SCN4B, GPD1L, SNTA1, and CAV3. Patch-clamp analyses were performed on variants not previously characterized. In total, 8 patients (12%) had nonsynonymous rare variants in the sodium current genes. SCN5A harbored 6 rare variants (R458C, R535*, S1103Y, R1193Q, S1609L, and Q1909R); CAV3, 1 rare variant (T78M); GPD1L, 1 rare variant (R220H); and SCN3B, 1 rare variant (L10P). Four variants were considered likely pathogenic and 5 variants of unknown significance. SCN5A R1193Q and GPD1L R220H (both considered variants of unknown significance) were present in the same infant. Functional analysis of variants not previously characterized (R458C, S1609L, and Q1909R in SCN5A) predominantly revealed increased transient and sustained sodium current. In a nonreferred nationwide Danish cohort of SIDS cases, up to 5/66 (7.5%) of SIDS cases can be explained by genetic variants in the sodium channel complex genes.

Delay in blood sampling for routine newborn screening is associated with increased risk of schizophrenia

BackgroundThe Danish Neonatal Screening Biobank, containing dried blood spot samples from all newborn in Denmark, is a unique source of data that can be utilized for analyses of genetic and environmental exposures related to schizophrenia and other mental disorders. In previous analyses, we have found that early and late blood sampling, compared to sampling at day 5, was associated with increased risk of schizophrenia. As delay in sampling of blood for neonatal screening cannot in itself influence the risk of schizophrenia, it must be seen as a proxy for unknown underlying causes responsible for this association. Therefore, we investigated whether the increased risk can be explained by other risk factors for schizophrenia. MethodsA case–control design was applied. A total of 846 cases with schizophrenia were selected from the Danish Psychiatric Case Register. One control was selected for each case, matched on sex and exact date of birth. ResultsBoth early and late blood sampling was associated with increased risk for schizophrenia. Compared to blood sampling at day 5, sampling at days 0 to 4 after birth was associated with an incidence rate ratio (IRR) of 1.46 (95% CI 1.15–1.87) for development of schizophrenia, and sampling at days 6 to 9 and at days 10 to 53 was associated with an IRR of 1.5 (95% CI 1.13–1.98) and 3.00 (95% CI 1.59–5.67), respectively. After adjusting the estimates for place of birth, both parents' psychiatric illness, maternal and paternal age, parents' country of origin, child admission, and parental education and income, the estimates were slightly different. Thus, blood collection at 0–4days was associated with an IRR of 1.27 (95% CI 0.94–1.71), 6–9days 1.31 (95% CI 0.94–1.84) and 10+days 3.52 (95% CI 1.50 to 8.24). DiscussionAfter adjusting risk estimates for well-known risk factors, delay in sampling of blood for neonatal screening was associated with unexplained increased risk of schizophrenia. Thus, a key finding is that age at test is a proxy for unobserved risk factors for schizophrenia due to unexplained reasons for late blood sampling. Date of sampling will be included in future analyses of genetic and environmental risk factors.

Extreme neonatal hyperbilirubinemia and a specific genotype: a population-based case-control study.

Extreme hyperbilirubinemia (plasma bilirubin ≥ 24.5 mg/dL) is an important risk factor for severe bilirubin encephalopathy. Several risk factors for hyperbilirubinemia are known, but in a large number of patients, a causal factor is never established. UGT1A1 is the rate-limiting enzyme in bilirubin's metabolism. The genotype of Gilbert syndrome, the UGT1A1*28 allele, causes markedly reduced activity of this enzyme, but its association with neonatal hyperbilirubinemia is uncertain and its relationship with extreme hyperbilirubinemia has not been studied. We examined whether the UGT1A1*28 allele is associated with extreme hyperbilirubinemia. The UGT1A1*28 allele was assessed in a case-control study of 231 white infants who had extreme hyperbilirubinemia in Denmark from 2000 to 2007 and 432 white controls. Cases were identified in the Danish Extreme Hyperbilirubinemia Database that covers the entire population. Genotypes were obtained through the Danish Neonatal Screening Biobank. Subgroup analysis was done for AB0 incompatible cases. No association was found between the UGT1A1*28 allele and extreme hyperbilirubinemia. With the common genotype as reference, the odds ratio of extreme hyperbilirubinemia was 0.87 (range, 0.68-1.13) for UGT1A1*28 heterozygotes and 0.77 (range, 0.46-1.27) for homozygotes. Also, no association was found for AB0 incompatible cases. The UGT1A1*28 allele was not associated with risk for extreme hyperbilirubinemia in this study.