Unraveling the metabolomic architecture of autism in a large Danish population-based cohort.

BACKGROUND: The prevalence of autism in Denmark has been increasing, reaching 1.65% among 10-year-old children, and similar trends are seen elsewhere. Although there are several factors associated with autism, including genetic, environmental, and prenatal factors, the molecular etiology of autism is largely unknown. Here, we use untargeted metabolomics to characterize the neonatal metabolome from dried blood spots collected shortly after birth. METHODS: We analyze the metabolomic profiles of a subset of a large Danish population-based cohort (iPSYCH2015) consisting of over 1400 newborns, who later are diagnosed with autism and matching controls and in two Swedish population-based cohorts comprising over 7000 adult participants. Mass spectrometry analysis was performed by a timsTOF Pro operated in QTOF mode, using data-dependent acquisition. By applying an untargeted metabolomics approach, we could reproducibly measure over 800 metabolite features. RESULTS: We detected underlying molecular perturbations across several metabolite classes that precede autism. In particular, the cyclic dipeptide cyclo-leucine-proline (FDR-adjusted p = 0.003) and the carnitine-related 5-aminovaleric acid betaine (5-AVAB) (FDR-adjusted p = 0.03), were associated with an increased probability for autism, independently of known prenatal and genetic risk factors. Analysis of genetic and dietary data in adults revealed that 5-AVAB was associated with increased habitual dietary intake of dairy (FDR-adjusted p < 0.05) and with variants near SLC22A4 and SLC22A5 (p < 5.0e - 8), coding for a transmembrane carnitine transporter protein involved in controlling intracellular carnitine levels. CONCLUSIONS: Cyclo-leucine-proline and 5-AVAB are associated with future diagnosis of autism in Danish neonates, both representing novel early biomarkers for autism. 5-AVAB is potentially modifiable and may influence carnitine homeostasis.

Vitamin D3 among neonates born after in vitro fertilization compared with neonates from the general population.

INTRODUCTION: Sufficient levels of vitamin D have been associated with higher chances for both clinical pregnancy and live birth among women undergoing assisted reproductive techniques, whereas low levels of maternal vitamin D have been associated with preeclampsia and late miscarriage. In Denmark, subgroups at risk for low vitamin D levels, including neonates and toddlers, are recommended to use supplementation. The aim was to study the level of vitamin D3 among neonates born after in vitro fertilization compared with neonates from the general population. MATERIAL AND METHODS: In this cohort study a random sample of 1326 neonates representing the general population and 1200 neonates conceived by in vitro fertilization born in Denmark from 1995 to 2002 were identified from registries covering the whole Danish population. Information on use of assisted reproduction was collected from the Danish In Vitro Fertilization register, ICD-10 code: DZ358F. 25-Hydroxyvitamin D was measured from dried blood spots routinely collected by heel prick 48-72 h after birth and corrected according to the hematocrit fraction for capillary blood of neonates. Linear regression analysis was performed, both crude and adjusted, for predefined putative confounders, identified through directed acyclic graphs. RESULTS: Vitamin D3 analysis could be performed from a total of 1105 neonates from the general population and 1072 neonates conceived by in vitro fertilization that were subsequently included in the study. The median vitamin D3 was 24.0 nmol/L (interquartile range [IQR] 14.1-39.3) and 33.0 nmol/L (IQR 21.3-48.8) among neonates from the general population and neonates conceived by in vitro fertilization, respectively. The adjusted mean difference between neonates from the general population and those conceived by in vitro fertilization was 6.1 nmol/L (95% confidence interval 4.1-8.1). CONCLUSIONS: In this study, children born after in vitro fertilization have a higher vitamin D3 than a random sample of neonates in Denmark.

Metabolic signature of the pathogenic 22q11.2 deletion identifies carriers and provides insight into systemic dysregulation.

Large deletions at chromosome 22q11.2 are known to cause severe clinical conditions collectively known as 22q11.2 deletion syndrome. Notwithstanding the pathogenicity of these deletions, affected individuals are typically diagnosed in late childhood or early adolescence, and little is known of the molecular signaling cascades and biological consequences immediately downstream of the deleted genes. Here, we used targeted metabolomics to compare neonatal dried blood spot samples from 203 individuals clinically identified as carriers of a deletion at chromosome 22q11.2 with 203 unaffected individuals. A total of 173 metabolites were successfully identified and used to inform on systemic dysregulation caused by the genomic lesion and to discriminate carriers from non-carriers. We found 84 metabolites to be differentially abundant between carriers and non-carriers of the 22q11.2 deletion. A predictive model based on all 173 metabolites achieved high Accuracy (89%), Area Under the Curve (93%), F1 (88%), Positive Predictive Value (94%), and Negative Predictive Value (84%) with tyrosine and proline having the highest individual contributions to the model as well as the highest interaction strength. Targeted metabolomics provides insight into the molecular consequences possibly contributing to the pathology underlying the clinical manifestations of the 22q11 deletion and is an easily applicable approach to first-pass screening for carrier status of the 22q11 to prompt subsequent verification of the genomic diagnosis.

First cases of SARS-CoV-2 BA.2.86 in Denmark, 2023.

We describe 10 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant BA.2.86 detected in Denmark, including molecular characteristics and results from wastewater surveillance that indicate that the variant is circulating in the country at a low level. This new variant with many spike gene mutations was classified as a variant under monitoring by the World Health Organization on 17 August 2023. Further global monitoring of COVID-19, BA.2.86 and other SARS-CoV-2 variants is highly warranted.

Patterns of testing in the extensive Danish national SARS-CoV-2 test set-up.

BACKGROUND: The Danish national SARS-CoV-2 mass test system was among the most ambitious worldwide. We describe its set-up and analyse differences in patterns of testing per demography and time period in relation to the three waves of SARS-CoV-2 transmission in Denmark. METHODS: We included all reported PCR- and rapid antigen-tests performed between 27 February 2020 and 10 March 2022 among all residents aged 2 years or above. Descriptive statistics and Poisson regression models were used to analyse characteristics of individuals tested for SARS-CoV-2 using a national cohort study design. RESULTS: A total of 63.7 million PCR-tests and 60.0 million rapid antigen-tests were performed in the study period, testing 90.9% and 78.8% of the Danish population at least once by PCR or antigen, respectively. Female sex, younger age, Danish heritage and living in the capital area were all factors positively associated with the frequency of PCR-testing. The association between COVID-19 vaccination and PCR-testing changed from negative to positive over time. CONCLUSION: We provide details of the widely available, free-of-charge, national SARS-CoV-2 test system, which served to identify infected individuals, assist isolation of infectious individuals and contact tracing, and thereby mitigating the spread of SARS-CoV-2 in the Danish population. The test system was utilized by nearly the entire population at least once, and widely accepted across different demographic groups. However, demographic differences in the test uptake did exist and should be considered in order not to cause biases in studies related to SARS-CoV-2, e.g., studies of transmission and vaccine effectiveness.

Multi-omic analyses of triptan-treated migraine attacks gives insight into molecular mechanisms.

Migraine is a common, polygenic disorder that is characterized by moderate to severe headache attacks. Migraine attacks are commonly treated with triptans, i.e. serotonin receptor agonists. However, triptans are effective in ~ 60% of the population, and the mechanisms of triptans are debated. Here, we aim to expose the mechanisms of triptan using metabolomics and transcriptomics in spontaneous migraine attacks. We collected temporal multi-omics profiles on 24 migraine patients, using samples collected at a migraine attack, 2 h after treatment with a triptan, when headache-free, and after a cold-pressor test. Differential metabolomic analysis was performed to find metabolites associated with treatment. Their effect was further investigated using correlation analysis and a machine learning approach. We found three differential metabolites: cortisol, sumatriptan and glutamine. The change in sumatriptan levels correlated with a change in GNAI1 and VIPR2 gene expression, both known to regulate cAMP levels. Furthermore, we found fatty acid oxidation to be affected, a mechanism known to be involved in migraine but not previously found in relation to triptans. In conclusion, using an integrative approach we find evidence for a role of glutamine, cAMP regulation, and fatty acid oxidation in the molecular mechanisms of migraine and/or the effect of triptans.

Evaluation of four laboratory-based high-throughput SARS-CoV-2 automated antigen tests compared to RT-PCR on nasal and oropharyngeal samples.

BACKGROUND: The demand for RT-PCR testing has been unprecedented during the SARS-CoV-2 pandemic. Fully automated antigen tests (AAT) are less cumbersome than RT-PCR, but data on performance compared to RT-PCR are scarce. METHODS: The study consists of two parts. A retrospective analytical part, comparing the performance of four different AAT on 100 negative and 204 RT-PCR positive deep oropharyngeal samples divided into four groups based on RT-PCR cycle of quantification levels. In the prospective clinical part, 206 individuals positive for and 199 individuals negative for SARS-CoV-2 were sampled from either the anterior nasal cavity (mid-turbinate) or by deep oropharyngeal swabs or both. The performance of AATs was compared to RT-PCR. RESULTS: The overall analytical sensitivity of the AATs differed significantly from 42% (95% CI 35-49) to 60% (95% CI 53-67) with 100% analytical specificity. Clinical sensitivity of the AATs differed significantly from 26% (95% CI 20-32) to 88% (95% CI 84-93) with significant higher sensitivity for mid-turbinate nasal swabs compared to deep oropharyngeal swabs. Clinical specificity varied from 97% to 100%. CONCLUSION: All AATs were highly specific for detection of SARS-CoV-2. Three of the four AATs were significantly more sensitive than the fourth AAT both in terms of analytical and clinical sensitivity. Anatomical test location significantly influenced the clinical sensitivity of AATs.

Effects of Long-Term Storage on the Biobanked Neonatal Dried Blood Spot Metabolome.

Over 2.5 million neonatal dried blood spots (DBS) are stored at the Danish National Biobank. These samples offer extraordinary possibilities for metabolomics research, including prediction of disease and understanding of underlying molecular mechanisms of disease development. Nevertheless, Danish neonatal DBS have been little explored in metabolomics studies. One question that remains underinvestigated is the long-term stability of the large number of metabolites typically assessed in untargeted metabolomics over long time periods of storage. Here, we investigate temporal trends of metabolites measured in 200 neonatal DBS collected over a time course of 10 years, using an untargeted liquid chromatography tandem mass spectrometry (LC-MS/MS) based metabolomics protocol. We found that a majority (71%) of the metabolome was stable during 10 years of storage at -20 °C. However, we found decreasing trends for lipid-related metabolites, such as glycerophosphocholines and acylcarnitines. A few metabolites, including glutathione and methionine, may be strongly influenced by storage, with changes in metabolite levels up to 0.1-0.2 standard deviation units per year. Our findings indicate that untargeted metabolomics of DBS samples, with long-term storage in biobanks, is suitable for retrospective epidemiological studies. We identify metabolites whose stability in DBS should be closely monitored in future studies of DBS samples with long-term storage.

Genetic correlates of vitamin D-binding protein and 25-hydroxyvitamin D in neonatal dried blood spots.

The vitamin D binding protein (DBP), encoded by the group-specific component (GC) gene, is a component of the vitamin D system. In a genome-wide association study of DBP concentration in 65,589 neonates we identify 26 independent loci, 17 of which are in or close to the GC gene, with fine-mapping identifying 2 missense variants on chromosomes 12 and 17 (within SH2B3 and GSDMA, respectively). When adjusted for GC haplotypes, we find 15 independent loci distributed over 10 chromosomes. Mendelian randomization analyses identify a unidirectional effect of higher DBP concentration and (a) higher 25-hydroxyvitamin D concentration, and (b) a reduced risk of multiple sclerosis and rheumatoid arthritis. A phenome-wide association study confirms that higher DBP concentration is associated with a reduced risk of vitamin D deficiency. Our findings provide valuable insights into the influence of DBP on vitamin D status and a range of health outcomes.

Diet-associated vertically transferred metabolites and risk of asthma, allergy, eczema, and infections in early childhood.

BACKGROUND: Evidence suggests maternal pregnancy dietary intake and nutrition in the early postnatal period to be of importance for the newborn child's health. However, studies investigating diet-related metabolites transferred from mother to child on disease risk in childhood are lacking. We sought to investigate the influence of vertically transferred metabolites on risk of atopic diseases and infections during preschool age. METHODS: In the Danish population-based COPSAC2010 mother-child cohort, information on 10 diet-related vertically transferred metabolites from metabolomics profiles of dried blood spots (DBS) at age 2-3 days was analyzed in relation to the risk of childhood asthma, allergy, eczema, and infections using principal component and single metabolite analyses. RESULTS: In 678 children with DBS measurements, a coffee-related metabolite profile reflected by principal component 1 was inversely associated with risk of asthma (odds ratio (95% CI) 0.78 (0.64; 0.95), p = .014) and eczema at age 6 years (0.79 (0.65; 0.97), p = .022). Furthermore, increasing stachydrine (fruit-related), 3-carboxy-4-methyl-5-propyl-2-furanpropanoate (fish-related), and ergothioneine (fruit-, green vegetables-, and fish-related) levels were all significantly associated with reduced risks of infections at age 0-3 years (p < .05). CONCLUSION: This study demonstrates associations between pregnancy diet-related vertically transferred metabolites measured in children in early life and risk of atopic diseases and infections in childhood. The specific metabolites associated with a reduced disease risk in children may contribute to the characterization of a healthy nutritional profile in pregnancy using a metabolomics-based unbiased tool for predicting childhood health.

Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations.

Multiple mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) increase transmission, disease severity, and immune evasion and facilitate zoonotic or anthropozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K, and N501Y, occurred in the SARS-CoV-2 spike glycoprotein in combinations that allow the simultaneous detection of VOCs. Here, we present two flexible reverse transcription-quantitative PCR (RT-qPCR) platforms for small- and large-scale screening (also known as variant PCR) to detect these mutations and schemes for adapting the platforms to future mutations. The large-scale RT-qPCR platform was validated by pairwise matching of RT-qPCR results with whole-genome sequencing (WGS) consensus genomes, showing high specificity and sensitivity. Both platforms are valuable examples of complementing WGS to support the rapid detection of VOCs. Our mutational signature approach served as an important intervention measure for the Danish public health system to detect and delay the emergence of new VOCs. IMPORTANCE Denmark weathered the SARS-CoV-2 crisis with relatively low rates of infection and death. Intensive testing strategies with the aim of detecting SARS-CoV-2 in symptomatic and nonsymptomatic individuals were available by establishing a national test system called TestCenter Denmark. This testing regime included the detection of SARS-CoV-2 signature mutations, with referral to the national health system, thereby delaying outbreaks of variants of concern. Our study describes the design of the large-scale RT-qPCR platform established at TestCenter Denmark in conjunction with whole-genome sequencing to report mutations of concern to the national health system. Validation of the large-scale RT-qPCR platform using paired WGS consensus genomes showed high sensitivity and specificity. For smaller laboratories with limited infrastructure, we developed a flexible small-scale RT-qPCR platform to detect three signature mutations in a single run. The RT-qPCR platforms are important tools to support the control of the SARS-CoV-2 endemic in Denmark.

Furan fatty acid metabolite in newborns predicts risk of asthma.

BACKGROUND: Intake of fish-oil and fatty fish during pregnancy has been shown to reduce the risk of childhood asthma but biomarkers of such intake are lacking. OBJECTIVE: To establish biomarkers of prenatal fish-oil exposure from newborn dry blood spot metabolomics profiles and assess their relevance for childhood asthma risk stratification. METHODS: The Danish COPSAC2010 mother-child cohort was utilized to investigate the effect of a double-blinded randomized controlled trial of fish-oil supplementation during pregnancy on dry blood spot liquid-chromatography mass spectrometry-based metabolomics profiles of 677 newborns. We thereafter investigated the association between fish-oil associated biomarkers in the newborn and development of asthma-related outcomes. Replication was sought in the independent observational COPSAC2000 cohort with 387 newborn metabolomics profiles. RESULTS: The newborn metabolomics profiles differed between children in the fish-oil vs. placebo group in COPSAC2010 (area under the receiver operator curve = 0.94 ± 0.03, p < .001). The fish-oil metabolomics profile and the top biomarker, 3-carboxy-4-methyl-5-propyl-2-furan propanoic acid (CMPF) were both associated with a decreased risk of asthma by age 6 years (HR = 0.89, p = .002 and HR = 0.67, p = .005, respectively). In COPSAC2000 , newborn CMPF level was also inversely associated with asthma risk by age 6 years (HR = 0.69, p = .01). Troublesome lung symptoms and common infections in the first 3 years were also inversely associated with newborn CMPF levels in both cohorts. CONCLUSIONS: Newborn children's blood levels of the furan fatty acid metabolite CMPF reflect fish-oil and fatty fish intake during pregnancy and are associated with a lower risk of asthma across two cohorts, which could aid newborn screening for childhood asthma.

Combined nasal- and oropharyngeal self-swab provides equivalent performance compared to professionally collected oropharyngeal swabs in detecting SARS-CoV-2 in a real-life setting.

PURPOSE: To investigate the performance of a combined nasal midturbinate- and oropharyngeal (NAOP) self-swab compared to a deep oropharyngeal (OP) swab by health care workers (HCW) in detecting SARS-CoV-2 in a real-life setting. METHODS: Paired swabs from 1119 participants were included. RT-PCR were used to detect SARS-CoV-2 in both swab samples. RESULTS: 330 participants tested positive. The sensitivity of the combined self-swab and OP swab was 96.9 % and 95.4 % respectively, whereas the Ct-values for self-swabs were significantly lower compared to OP swabs. CONCLUSION: The combined NAOP self-swab outperformed the OP swab and thus, the NAOP self-swab may be an alternative sampling method under the given circumstances.

Vitamin D status and severity of COVID-19.

We explored the association between COVID-19 severity and vitamin D status using information from Danish nation-wide health registers, the COVID-19 surveillance database and stored blood samples from the national biobank. 25-hydroxyvitamin D (25(OH)D) was measured using tandem mass spectroscopy. The association between 25(OH)D levels and COVID-19 severity, classified hierarchical as non-hospitalized, hospitalized but not admitted to an intensive care unit (ICU), admitted to ICU, and death, was evaluated by proportional odds ratios (POR) assuming proportionality between the four degrees of severity. Among 447 adults tested SARS-CoV-2 positive in the spring of 2020, low levels of 25(OH)D were associated with a higher risk of severe COVID-19. Thus, odds of experiencing more severe COVID-19 among individuals with insufficient (25 to < 50 nmol/L) and sufficient (≥ 50 nmol/L) 25(OH)D levels were approximately 50% of that among individuals with deficient levels (< 25 nmol/L) (POR = 0.49 (95% CI 0.25-0.94), POR = 0.51 (95% CI 0.27-0.96), respectively). Dividing sufficient vitamin D levels into 50 to < 75 nmol/L and ≥ 75 nmol/L revealed no additional beneficial effect of higher 25(OH)D levels. In this observational study, low levels of 25(OH)D were associated with a higher risk of severe COVID-19. A possible therapeutic role of vitamin D should be evaluated in well-designed interventional studies.

Multi-omics to predict changes during cold pressor test.

BACKGROUND: The cold pressor test (CPT) is a widely used pain provocation test to investigate both pain tolerance and cardiovascular responses. We hypothesize, that performing multi-omic analyses during CPT gives the opportunity to home in on molecular mechanisms involved. Twenty-two females were phenotypically assessed before and after a CPT, and blood samples were taken. RNA-Sequencing, steroid profiling and untargeted metabolomics were performed. Each 'omic level was analyzed separately at both single-feature and systems-level (principal component [PCA] and partial least squares [PLS] regression analysis) and all 'omic levels were combined using an integrative multi-omics approach, all using the paired-sample design. RESULTS: We showed that PCA was not able to discriminate time points, while PLS did significantly distinguish time points using metabolomics and/or transcriptomic data, but not using conventional physiological measures. Transcriptomic and metabolomic data revealed at feature-, systems- and integrative- level biologically relevant processes involved during CPT, e.g. lipid metabolism and stress response. CONCLUSION: Multi-omics strategies have a great potential in pain research, both at feature- and systems- level. Therefore, they should be exploited in intervention studies, such as pain provocation tests, to gain knowledge on the biological mechanisms involved in complex traits.

A RT-qPCR system using a degenerate probe for specific identification and differentiation of SARS-CoV-2 Omicron (B.1.1.529) variants of concern.

Fast surveillance strategies are needed to control the spread of new emerging SARS-CoV-2 variants and gain time for evaluation of their pathogenic potential. This was essential for the Omicron variant (B.1.1.529) that replaced the Delta variant (B.1.617.2) and is currently the dominant SARS-CoV-2 variant circulating worldwide. RT-qPCR strategies complement whole genome sequencing, especially in resource lean countries, but mutations in the targeting primer and probe sequences of new emerging variants can lead to a failure of the existing RT-qPCRs. Here, we introduced an RT-qPCR platform for detecting the Delta- and the Omicron variant simultaneously using a degenerate probe targeting the key ΔH69/V70 mutation in the spike protein. By inclusion of the L452R mutation into the RT-qPCR platform, we could detect not only the Delta and the Omicron variants, but also the Omicron sub-lineages BA.1, BA.2 and BA.4/BA.5. The RT-qPCR platform was validated in small- and large-scale. It can easily be incorporated for continued monitoring of Omicron sub-lineages, and offers a fast adaption strategy of existing RT-qPCRs to detect new emerging SARS-CoV-2 variants using degenerate probes.

Household transmission of the SARS-CoV-2 Omicron variant in Denmark.

In late 2021, the Omicron SARS-CoV-2 variant overtook the previously dominant Delta variant, but the extent to which this transition was driven by immune evasion or a change in the inherent transmissibility is currently unclear. We estimate SARS-CoV-2 transmission within Danish households during December 2021. Among 26,675 households (8,568 with the Omicron VOC), we identified 14,140 secondary infections within a 1-7-day follow-up period. The secondary attack rate was 29% and 21% in households infected with Omicron and Delta, respectively. For Omicron, the odds of infection were 1.10 (95%-CI: 1.00-1.21) times higher for unvaccinated, 2.38 (95%-CI: 2.23-2.54) times higher for fully vaccinated and 3.20 (95%-CI: 2.67-3.83) times higher for booster-vaccinated contacts compared to Delta. We conclude that the transition from Delta to Omicron VOC was primarily driven by immune evasiveness and to a lesser extent an inherent increase in the basic transmissibility of the Omicron variant.

A method to correct for the influence of bovine serum albumin-associated vitamin D metabolites in protein extracts from neonatal dried blood spots.

OBJECTIVE: We developed an assay to measure the concentration of 25 hydroxyvitamin D2 and D3 in protein extracts derived from stored neonatal dried blood spots. During this study, we postulated that these samples had been contaminated with exogenous vitamin D metabolites because of the addition of bovine serum albumin (BSA) as part of an extraction step undertaken 7 years earlier. The aim of the current study was to develop methods in order to adjust for this contamination. RESULTS: We identified between-plate variations in 25 hydroxyvitamin D2 and D3 concentrations which suggested the presence of three different BSA batches. Based on repeat extraction (without the addition of BSA) and testing of 395 samples, we developed models to correct for the exogenous 25 hydroxyvitamin D2 and D3. The regression models were Diff25OHD3 = - 8.2 + 1.8* Diff25OHD2 for low contamination, Diff25OHD3 = 23.8 + 1.7* Diff25OHD2 for middle contamination, and Diff25OHD3 = 14.3 + 3.0* Diff25OHD2 for high contamination. After these corrections, the three subsamples had comparable distributions within the expected range for both 25 hydroxyvitamin D2 and D3.