Danish expanded newborn screening is a successful preventive public health programme.

INTRODUCTION: Newborn screening is a public health programme for early diagnosis of treatable diseases. METHODS: The subjects included were newborns born 2002-2019. Expanded newborn screening (eNBS) for metabolic diseases was introduced as a pilot project from 2002 to 2009, followed by routine screening with informed dissent. A total of 967,780 newborns were screened; 82,930 were unscreened. Furthermore, a historic cohort of clinically diagnosed children born in the 1992-2001 period was included. Children in the unscreened and historic cohorts were evaluated for the same diseases as were the screened children. Dried blood spot samples were collected locally and sent for screening analyses. We recorded newborns with true and false positive results as well as false negative results and their clinical signs at screening and at the last follow-up. RESULTS: A total of 603 samples were screen positive: 354 false positives and 249 true positives (222 newborns and 27 mothers). The positive predictive value (PPV) was 41% for the entire screening period; 62% for 2018. The false positive rate (FPR) was 0.036% overall; 0.024% for 2018. The overall prevalence of diseases was 1:3,900; in the historic cohort, the prevalence of the same diseases was 1:8,300; 7.3% had symptoms at the time of screening. At follow-up, 93% of the children had no clinically significant sequelae. Among 82,930 unscreened newborns, 27 (1:3,000) had eNBS panel diseases, some with severe manifestations. CONCLUSIONS: This update of eNBS in Denmark confirms that eNBS is a successful preventive public health programme. Early treatment in a latent phase of disease is effective and screening should be extended to other diseases not currently in the programme. FUNDING: The work was supported by grants from The Ronald McDonald Børnefond, Danmarks Sundhedsfond, Direktør Ib Henriksens Fond, Ragnhild Ibsens Legat til Medicinsk Forskning, Gerda og Aage Haenschs Fond, Dronning Louises Børnehospitals Forskningsfond, Læge Sofus Carl Emil Friis og Hustru Olga Doris Friis's Legat, Aase and Ejnar Danielsens Fond, Oda og Hans Svenningsens Fond, Fonden af 1870, Vanførefonden, Fonden til Lægevidenskabens Fremme and Danish Medical Research Council. TRIAL REGISTRATION: not relevant.

Foetal oestrogens and autism.

Elevated latent prenatal steroidogenic activity has been found in the amniotic fluid of autistic boys, based on measuring prenatal androgens and other steroid hormones. To date, it is unclear if other prenatal steroids also contribute to autism likelihood. Prenatal oestrogens need to be investigated, as they play a key role in synaptogenesis and corticogenesis during prenatal development, in both males and females. Here we test whether levels of prenatal oestriol, oestradiol, oestrone and oestrone sulphate in amniotic fluid are associated with autism, in the same Danish Historic Birth Cohort, in which prenatal androgens were measured, using univariate logistic regression (n = 98 cases, n = 177 controls). We also make a like-to-like comparison between the prenatal oestrogens and androgens. Oestradiol, oestrone, oestriol and progesterone each related to autism in univariate analyses after correction with false discovery rate. A comparison of standardised odds ratios showed that oestradiol, oestrone and progesterone had the largest effects on autism likelihood. These results for the first time show that prenatal oestrogens contribute to autism likelihood, extending the finding of elevated prenatal steroidogenic activity in autism. This likely affects sexual differentiation, brain development and function.

Autism spectrum disorders, endocrine disrupting compounds, and heavy metals in amniotic fluid: a case-control study.

Background: Evidence has indicated that some non-inherited factors such as exposure to environmental pollutants are associated with neurodevelopment disorders like autism spectrum disorder (ASD). Studies report that endocrine disrupting compounds (EDCs), including polychlorinated biphenyls, organochlorine pesticides, perfluoroalkyl substances (PFAS), and some metals, have adverse effects on the fetal neurodevelopment. The aim of this study was to measure the amniotic fluid (AF) levels of EDCs and metals as well as the receptor transactivities induced by AF and investigate the possible link between prenatal exposure to EDCs and heavy metals and ASD risk. Methods: In this case-control study, we included AF samples of 75 ASD cases and 135 frequency-matched controls and measured the levels of the endogenous sex hormones, PFAS, and elements including heavy metals. The combined effect of endogenous hormones and EDCs on the receptor of estrogen (ER), androgen (AR), aryl hydrocarbon (AhR), and thyroid hormone-like activity were also determined and expressed as receptor ligand equivalents. We assessed the associations of AF levels of chemicals, sex hormones, and receptor activities with ASD risk using unconditional logistical regression analyses. To control for multiple comparisons, the false discovery rate (FDR) was used and q values less than 0.25 were designated as statistical significance. Results: PFAS and metals were detectable in AF samples. The ASD cases had significantly lower AF levels of PFAS than controls, and the adjusted odds ratio (OR) was 0.410 (95% CI 0.174, 0.967; p = 0.042; FDR qvalue = 0.437) for perfluorooctane sulfonate (PFOS). The principal component, including PFAS congeners, copper, iron, and estrogenic activity, was significantly inversely associated with ASD risk (adjusted OR = 0.100; 95% CI 0.016, 0.630; p = 0.014; FDR qvalue = 0.098).Testosterone level in AF weakly associated with ASD risk (adjusted OR = 1.002; 95% CI 1.000, 1.004; p = 0.05). However, after multiple comparison correction, the association was not significant (FDR qvalue = 0.437). No significant associations between AF-induced receptor transactivities and ASD risk were observed. The adjusted OR was 2.176 (95%CI 0.115, 41.153) for the ratio of the combined androgenic activity to combined estrogenic activity. Conclusions: The presence of PFAS and heavy metals in AF indicates that they can cross the placenta. The inverse association between levels of PFAS congeners in AF and ASD risk might relate to the weak estrogenic activities and anti-androgenic activities of PFAS.The observed tendency of positive association between the ratio of combined androgenic effect to the combined estrogenic effect and ASD risk needs further studies to explore whether EDCs together with endogenous hormones play a role in the development of ASD.

The association between neonatal vitamin D status and risk of schizophrenia.

Clues from the epidemiology of schizophrenia, such as the increased risk in those born in winter/spring, have led to the hypothesis that prenatal vitamin D deficiency may increase the risk of later schizophrenia. We wish to explore this hypothesis in a large Danish case-control study (n = 2602). The concentration of 25 hydroxyvitamin D (25OHD) was assessed from neonatal dried blood samples. Incidence rate ratios (IRR) were calculated when examined for quintiles of 25OHD concentration. In addition, we examined statistical models that combined 25OHD concentration and the schizophrenia polygenic risk score (PRS) in a sample that combined the new sample with a previous study (total n = 3464; samples assayed and genotyped between 2008-2013). Compared to the reference (fourth) quintile, those in the lowest quintile (<20.4 nmol/L) had a significantly increased risk of schizophrenia (IRR = 1.44, 95%CI: 1.12-1.85). None of the other quintile comparisons were significantly different. There was no significant interaction between 25OHD and the PRS. Neonatal vitamin D deficiency was associated with an increased risk for schizophrenia in later life. These findings could have important public health implications related to the primary prevention of schizophrenia.

Amniotic Fluid INSL3 Measured During the Critical Time Window in Human Pregnancy Relates to Cryptorchidism, Hypospadias, and Phthalate Load: A Large Case-Control Study.

The period of the first to second trimester transition in human pregnancy represents a sensitive window for fetal organogenesis, particularly in regard to the development of the male reproductive system. This is a time of relative analytical inaccessibility. We have used a large national biobank of amniotic fluid samples collected at routine amniocentesis to determine the impacts of exogenous endocrine disruptor load on specific fetal biomarkers at this critical time. While adrenal and testicular steroids are highly correlated, they are also mostly positively influenced by increasing phthalate load, represented by the metabolites 7cx-MMeHP and 5cx-MEPP, by perfluorooctane sulfonate (PFOS) exposure, and by smoking, suggesting an adrenal stress response. In contrast, the testis specific biomarkers insulin-like peptide 3 (INSL3) and androstenedione are negatively impacted by the phthalate endocrine disruptors. Using a case-control design, we show that cryptorchidism and hypospadias are both significantly associated with increased amniotic concentration of INSL3 during gestational weeks 13-16, and some, though not all steroid biomarkers. Cases are also linked to a specifically increased variance in the Leydig cell biomarker INSL3 compared to controls, an effect exacerbated by maternal smoking. No influence of phthalate metabolites or PFOS was evident on the distribution of cases and controls. Considering that several animal and human studies have shown a negative impact of phthalate load on fetal and cord blood INSL3, respectively, the present results suggest that such endocrine disruptors may rather be altering the relative dynamics of testicular development and consequent hormone production, leading to a desynchronization of tissue organization during fetal development. Being born small for gestational age appears not to impact on the testicular biomarker INSL3 in second trimester amniotic fluid.

CASPR2 autoantibodies are raised during pregnancy in mothers of children with mental retardation and disorders of psychological development but not autism.

BACKGROUND, METHODS AND OBJECTIVES: Maternal autoantibodies to neuronal proteins may be one cause of neurodevelopmental disorders. This exploratory study used the Danish archived midgestational sera and their nationwide registers to search for antibodies to the N-methyl-D-aspartate receptor (NMDAR) and contactin-associated protein-like 2 (CASPR2) in maternal sera, and to relate them to subsequent psychiatric diagnoses in the woman or her child. RESULTS: In a sample of 192 women, there was no association between antibody status and subsequent psychosis in the mothers. However, NMDAR antibodies (n=4) or CASPR2 antibodies (n=1) were identified in 5/11 (45.5%) women whose children were given a diagnosis of mild or unspecified mental retardation or disorders of psychological and motor development (collectively abbreviated as mental retardation and/or disorders of psychological development (MR/DPD)) compared with 9/176 (5.1%) of the remaining mother (p<0.001). These findings were followed up in a specifically selected cohort, in which CASPR2 antibodies were detected in 7/171 (4.1%) mothers of MR/DPD progeny, compared with only 1/171 (0.6%) control mother (p=0.067). The combined sample showed a significantly higher frequency of CASPR2 antibodies in mothers of MD/DPD children (p=0.01). These autoantibodies were not increased in mothers of children with autistic spectrum disorder. CONCLUSIONS: These findings complement the known roles of CASPR2 in brain development, and warrant further epidemiological and experimental studies to clarify the role of CASPR2 and possibly other antibodies in neurodevelopmental disorders.

Influence of Polygenic Risk Scores on the Association Between Infections and Schizophrenia.

BACKGROUND: Several studies have suggested an important role of infections in the etiology of schizophrenia; however, shared genetic liability toward infections and schizophrenia could influence the association. We therefore investigated the possible effect of polygenic risk scores (PRSs) for schizophrenia on the association between infections and the risk of schizophrenia. METHODS: We conducted a nested case-control study on a Danish population-based sample born after 1981 comprising of 1692 cases diagnosed with schizophrenia between 1994 and 2008 and 1724 matched controls. All individuals were linked utilizing nationwide population-based registers with virtually complete registration of all hospital contacts for infections. PRSs were calculated using discovery effect size estimates weights from an independent meta-analysis (34,600 cases and 45,968 control individuals). RESULTS: A prior hospital contact with infection had occurred in 41% of the individuals with schizophrenia and increased the incidence rate ratio (IRR) of schizophrenia by 1.43 (95% confidence interval [CI] = 1.22-1.67). Adding PRS, which was robustly associated with schizophrenia (by an IRR of 1.46 [95% CI = 1.34-1.60] per standard deviation of the score), did not alter the association with infections and the increased risk of schizophrenia remained (IRR = 1.41; 95% CI = 1.20-1.66). Furthermore, there were no interactions between PRS and infections on the risk of developing schizophrenia (p = .554). Neither did PRS affect the risk of acquiring infections among patients with schizophrenia (odds ratio = 1.00; 95% CI = 0.89-1.12) nor among controls (odds ratio = 1.09; 95% CI: 0.96-1.24). CONCLUSIONS: PRS and a history of infections have independent effects on the risk for schizophrenia, and the common genetic risk measured by PRS did not account for the association with infection in this sample.

Perfluorooctane Sulfonate Concentrations in Amniotic Fluid, Biomarkers of Fetal Leydig Cell Function, and Cryptorchidism and Hypospadias in Danish Boys (1980-1996).

BACKGROUND: Exposure to perfluorooctane sulfonate (PFOS) may potentially disturb fetal Leydig cell hormone production and male genital development. OBJECTIVES: We aimed to study the associations between levels of amniotic fluid PFOS, fetal steroid hormone, and insulin-like factor 3 (INSL3) and the prevalence of cryptorchidism and hypospadias. METHODS: Using the Danish National Patient Registry, we selected 270 cryptorchidism cases, 75 hypospadias cases, and 300 controls with stored maternal amniotic fluid samples available in a Danish pregnancy-screening biobank (1980-1996). We used mass spectrometry to measure PFOS in amniotic fluid from 645 persons and steroid hormones in samples from 545 persons. INSL3 was measured by immunoassay from 475 persons. Associations between PFOS concentration in amniotic fluid, hormone levels, and genital malformations were assessed by confounder-adjusted linear and logistic regression. RESULTS: The highest tertile of PFOS exposure (> 1.4 ng/mL) in amniotic fluid was associated with a 40% (95% CI: -69, -11%) lower INSL3 level and an 18% (95% CI: 7, 29%) higher testosterone level compared with the lowest tertile (< 0.8 ng/mL). Amniotic fluid PFOS concentration was not associated with cryptorchidism or hypospadias. CONCLUSIONS: Environmental PFOS exposure was associated with steroid hormone and INSL3 concentrations in amniotic fluid, but was not associated with cryptorchidism or hypospadias in our study population. Additional studies are needed to determine whether associations with fetal hormone levels may have long-term implications for reproductive health. CITATION: Toft G, Jönsson BA, Bonde JP, Nørgaard-Pedersen B, Hougaard DM, Cohen A, Lindh CH, Ivell R, Anand-Ivell R, Lindhard MS. 2016. Perfluorooctane sulfonate concentrations in amniotic fluid, biomarkers of fetal Leydig cell function, and cryptorchidism and hypospadias in Danish boys (1980-1996). Environ Health Perspect 124:151-156; http://dx.doi.org/10.1289/ehp.1409288.

Amniotic fluid phthalate levels and male fetal gonad function.

BACKGROUND: Prenatal exposure to phthalates may pose a threat to human male reproduction. However, additional knowledge about the in vivo effect in humans is needed, and reported associations with genital abnormalities are inconclusive. We aimed to study prenatal di(2-ethylhexyl) phthalate (DEHP) and diisononyl phthalate (DiNP) exposure in relation to cryptorchidism, hypospadias, and human fetal Leydig cell function. METHODS: We studied 270 cryptorchidism cases, 75 hypospadias cases, and 300 controls. Second-trimester amniotic fluid samples were available from a Danish pregnancy-screening biobank (n = 25,105) covering 1980-1996. We assayed metabolites of DEHP and DiNP (n = 645) and steroid hormones (n = 545) by mass spectrometry. We assayed insulin-like factor 3 by immunoassay (n = 475) and analyzed data using linear or logistic regression. RESULTS: Mono(2-ethyl-5-carboxypentyl) phthalate (5cx-MEPP, DEHP metabolite) was not consistently associated with cryptorchidism or hypospadias. However, we observed an 18% higher (95% confidence interval [CI] = 5%-33%) testosterone level, and a 41% lower (-56% to -21%) insulin-like factor 3 level in the highest 5cx-MEPP tertile compared with the lowest. Mono(4-methyl-7-carboxyheptyl) phthalate (7cx-MMeHP, DiNP metabolite) showed elevated odds ratio point estimates for having cryptorchidism (odds ratio = 1.28 [95% CI = 0.80 to 2.01]) and hypospadias (1.69 [0.78 to 3.67]), but was not consistently associated with the steroid hormones or insulin-like factor 3. CONCLUSIONS: Data on the DEHP metabolite indicate possible interference with human male fetal gonadal function. Considering the DiNP metabolite, we cannot exclude (nor statistically confirm) an association with hypospadias and, less strongly, with cryptorchidism.

DNA methylome profiling using neonatal dried blood spot samples: a proof-of-principle study.

DNA methylation is the most common DNA modification and perhaps the best described epigenetic modification. It is believed to be important for genomic imprinting and gene regulation and has been associated with the development of diseases such as schizophrenia and some types of cancer. Neonatal dried blood spot samples, commonly known as Guthrie cards, are routinely collected worldwide to screen newborns for diseases. Some countries, including Denmark, have been storing the excess neonatal dried blood spot samples in biobanks for decades. Representing a high percentage of the population under a certain age, the neonatal dried blood spot samples are a potential alternative to collecting new samples to study diseases. As such, neonatal dried blood spot samples have previously been used for DNA genotyping studies with excellent results. However, the amount of material available for research is often limited, challenging researchers to generate the most data from a limited quantity of material. In this proof-of-principle study, we address whether two 3.2mm disks punched from a neonatal dried blood spot sample contain enough DNA for genome-wide methylome profiling, measuring 27,578 loci at the same time. We selected two subjects and carried out the following with each: 1) collected an adult whole-blood sample as reference, 2) spotted a fraction of the whole-blood sample onto a similar type of filter paper as used in the newborn screening and stored it for 3years to serve as a dried blood spot reference, and 3) identified the archived neonatal dried blood spot samples, stored for 26-28years, in the Danish Newborn Screening Biobank as a representative of the archived samples. For comparison, we used two different kits for DNA extraction. The DNA, extracted using the Extract-N-Amp Blood PCR kit, was analyzed, and no statistically significant differences were observed (P<0.001) when we compared the methylation profile of the reference whole-blood samples to the dried blood spot references. This indicates that two 3.2mm disks contain enough material for reliable methylome profiling and that storing the whole-blood sample on neonatal dried blood spot filter paper for 3years does not interfere with the outcome of the analysis. Furthermore, we compared the adult DNA methylation profile to the neonatal dried blood spot sample profile. Approximately 50 sites in the subjects were significantly (P<0.001) different in the newborn sample compared with the adult sample. Both being healthy adults and the high quality of the DNA methylation array led to the conclusion that the archived neonatal dried blood spot samples can be used for methylome profiling, despite decades of storage and DNA degradation. In conclusion, we show that reliable methylome data can be obtained from old neonatal dried blood spot samples, by using a reasonable amount of the limited resource. This further adds to the use of neonatal dried blood spot samples in genetic research and screening and paves the way for unique population-based studies of epigenetic modifications after birth.

Neonatal chemokine levels and risk of autism spectrum disorders: findings from a Danish historic birth cohort follow-up study.

A potential role of chemokines in the pathophysiology of Autism Spectrum Disorders (ASDs) has been previously suggested. In a recent study we examined levels of three inflammatory chemokines (MCP-1, MIP-1α and RANTES) in samples of amniotic fluid of children diagnosed later in life with ASD and controls frequency-matched to cases on gender and year of birth. In this follow-up study, levels of the same chemokines were analyzed postnatally in dried blood spot samples from the same subjects utilizing the Danish Newborn Screening Biobank. Crude estimates showed decreased levels of RANTES. In the adjusted estimates, no differences were found in levels of the three examined chemokines in ASD cases compared to controls. Our findings may cautiously suggest an altered cell-mediated immunity during the early neonatal period in ASD. Further research is needed to examine the relationship between maternal/fetal and neonatal chemokine levels and their role in ASD.

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

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-ß compared to controls. Analyzing individuals diagnosed only with ICD-10 codes yielded significantly elevated levels of IL-4, IL-10, TNF-α and TNF-ß in ASD patients. Restricting analysis to infantile autism cases showed significantly elevated levels of IL-4, TNF-α and TNF-ß 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.

Joint analysis of SNPs and proteins identifies regulatory IL18 gene variations decreasing the chance of spastic cerebral palsy.

Cerebral palsy (CP) is a permanent disorder, affecting 2-3 per 1,000 live born children, disturbing movement and posture. Spastic limbs affects about 70-80% of the CP children, and this group is the target of our study. CP is considered a multifactorial condition believed to be provoked by, for example, preterm birth, infection during pregnancy, neural disorders, and genetics, to mention some. Interestingly, the cytokine network is believed to be involved in many of these disorders. In this study, including 203 spastic CP cases and 167 controls, we measured the levels of 25 cytokine proteins, and genotyped 159 SNPs in their gene loci. Using logistic regression, we estimated the genetic association of SNP genotypes to spastic CP. In addition, fitting a Tobit regression model for each protein and each SNP in the respective gene loci, we estimated three regression coefficients corresponding three different effects of the genetic variation on the protein level. Intriguingly, two IL18 loci SNPs (rs549908:A>C and rs1290349:C>A) showed a protective effect against spastic CP, and interestingly both were associated to a decreased epidemiological expression of IL-18 protein. By joining protein data to genetic information, we have provided new data suggesting IL18's involvement in the pathogenesis of spastic CP.

Infections during pregnancy and after birth, and the risk of autism spectrum disorders: a register-based study utilizing a Danish historic birth cohort.

OBJECTIVE: Mounting evidence suggests that immune dysfunction may play a crucial role in the pathophysiology of autism spectrum disorders (ASD). In addition, several studies have reported that congenital and postnatal infections may contribute to the neurobiological basis of ASD. This study aimed to investigate the relationship between infections during pregnancy and after birth, and ASD. METHODS: A case-control study design was adopted. Both cases and controls were retrieved from a historic birth cohort (HBC) maintained at Statens Serum Institute in Copenhagen/Denmark and were followed up retrospectively during pregnancy and after birth over four pre-defined periods. Study subjects were followed-up utilizing Danish nation-wide health registers for outpatient and hospital admissions due to infections. Associations between infections and ASD were analyzed using Mantel-Haenszel estimate of the odds ratio (OR) and logistic regression models. RESULTS: In total, 414 ASD cases and 820 controls were followed-up during pregnancy and a mean 16.3 years after birth. Crude, but not adjusted estimates showed that ASD cases had an increased risk of hospital admission due to infection at the end of the first year of life (OR = 1.48 [range: 1.07-2.05], P = 0.02) and at the end of the follow-up period (OR = 1.30 [range: 1.02-1.64], P = 0.03). CONCLUSION: The present findings indicate that infections have a potential role in the pathophysiology of ASD; however, further studies are necessary to determine if infections etiologically contribute to ASD or if they act as an epiphenomenon due to distorted immunity in children with ASD.

Parvovirus B19 infection in the first trimester of pregnancy and risk of fetal loss: a population-based case-control study.

Because parvovirus B19 infection during pregnancy has been associated with increased risk of fetal loss in small or selected study populations, the authors evaluated the risk in a population-based study. A nested case-control study was conducted by using a population-based screening for syphilis in 3 regions in Denmark from 1992 to 1994. Cases of women with fetal loss were identified in the National Patient Register (n = 2,918), and control women with live-born children were identified in the Medical Birth Register (n = 8,429) by matching on age and sampling week. First-trimester serum samples were tested for parvovirus B19 immunoglobulin M positivity. Parvovirus B19 immunoglobulin M positivity was associated with a 71% increased risk of fetal loss (odds ratio = 1.71, 95% confidence interval: 1.02, 2.86). Adjustment for number of children or stratifying for gestational age at loss did not change the risk estimate. Assuming causality, only 0.1% of fetal losses were attributable to parvovirus B19 positivity, a proportion which could increase to approximately 1% during epidemic periods. In conclusion, acute parvovirus B19 infection during the first trimester of pregnancy was associated with an increased risk of fetal loss. However, the impact on the overall burden of fetal losses appeared small even during epidemics.

Amniotic fluid MMP-9 and neurotrophins in autism spectrum disorders: an exploratory study.

Evidence suggests that some developmental disorders, such as autism spectrum disorders (ASDs), are caused by errors in brain plasticity. Given the important role of matrix metalloproteinases (MMPs) and neurotrophins (NTs) in neuroplasticity, amniotic fluid samples for 331 ASD cases and 698 frequency-matched controls were analyzed for levels of MMP-9, brain-derived neurotrophic factor, NT-4 and transforming growth factor-ß utilizing a Danish historic birth cohort and Danish nationwide health registers. Laboratory measurements were performed using an in-house multiplex sandwich immunoassay Luminex xMAP method, and measurements were analyzed using tobit and logistic regression. Results showed elevated levels of MMP-9 in ASD cases compared with controls (crude and adjusted tobit regression P-values: 0.01 and 0.06). Our results highlight the importance of exploring the biologic impact of MMP-9 and potential therapeutic roles of its inhibitors in ASD and may indicate that neuroplastic impairments in ASD may present during pregnancy.

Neonatal levels of cytokines and risk of autism spectrum disorders: an exploratory register-based historic birth cohort study utilizing the Danish Newborn Screening Biobank.

The aim of the study was to analyze cytokine profiles in neonatal dried blood samples (n-DBSS) retrieved from The Danish Newborn Screening Biobank of children developing Autism Spectrum Disorders (ASD) later in life and controls. Samples of 359 ASD cases and 741 controls were analyzed using Luminex xMAP technology and clinical data were retrieved from nationwide registers. Findings showed that children developing ASD were more likely to have decreased levels of both T helper-1(Th-1)-like cytokines (i.e. IFN-γ) and Th-2like cytokines (i.e. IL-4, IL-10) which may suggest a depressed or hypoactive immune cell activity during neonatal period in ASD.

Toxoplasma gondii infection and self-directed violence in mothers.

CONTEXT: Two studies based on clinical samples have found an association between Toxoplasma gondii infection and history of suicide attempt. To our knowledge, these findings have never been replicated in a prospective cohort study. OBJECTIVE: To examine whether T gondii­infected mothers have an increased risk of self-directed violence, violent suicide attempts, and suicide and whether the risk depends on the level of T gondii IgG antibodies. DESIGN: Register-based prospective cohort study. Women were followed up from the date of delivery, 1992 to 1995 until 2006. SETTING: Denmark. PARTICIPANTS: A cohort of 45 788 women born in Denmark whose level of Toxoplasma-specific IgG antibodies was measured in connection with child birth between 1992 and 1995. MAIN OUTCOME MEASURES: Incidence rates of self-directed violence, violent suicide attempts, and suicide in relation to T gondii seropositivity and serointensity. RESULTS: T gondii­infected mothers had a relative risk of self-directed violence of 1.53 (95% CI, 1.27-1.85) compared with noninfected mothers, and the risk seemed to increase with increasing IgG antibody level. For violent suicide attempts, the relative risk was 1.81 (95% CI, 1.13-2.84) and for suicide, 2.05 (95% CI, 0.78-5.20). A similar association was found for repetition of self-directed violence, with a relative risk of 1.54 (95% CI, 0.98-2.39). CONCLUSION: Women with a T gondii infection have an increased risk of self-directed violence.

Biochemical screening of 504,049 newborns in Denmark, the Faroe Islands and Greenland--experience and development of a routine program for expanded newborn screening.

Expanded newborn screening for selected inborn errors of metabolism (IEM) in Denmark, the Faroe Islands and Greenland was introduced in 2002. We now present clinical, biochemical, and statistical results of expanded screening (excluding PKU) of 504,049 newborns during nine years as well as diagnoses and clinical findings in 82,930 unscreened newborns born in the same period. The frequencies of diagnoses made within the panel of disorders screened for are compared with the frequencies of the disorders in the decade preceding expanded newborn screening. The expanded screening was performed as a pilot study during the first seven years, and the experience obtained during these years was used in the development of the routine neonatal screening program introduced in 2009. Methods for screening included tandem mass spectrometry and an assay for determination of biotinidase activity. A total of 310 samples from 504,049 newborns gave positive screening results. Of the 310 results, 114 were true positive, including results from 12 newborns in which the disease in question was subsequently diagnosed in their mothers. Thus, the overall frequency of an IEM in the screening panel was 1:4942 (mothers excluded) or 1:4421 (mothers included). The false positive rate was 0.038% and positive predictive value 37%. Overall specificity was 99.99%. All patients with true positive results were followed in The Center for Inherited Metabolic Disorders in Copenhagen, and the mean follow-up period was 45 months (range 2109 months). There were no deaths among the 102 children, and 94% had no clinically significant sequelae at last follow-up. Our study confirms the higher frequency of selected IEM after implementation of expanded newborn screening and suggests an improved outcome for several disorders. We argue that newborn screening for these disorders should be standard of care, though unresolved issues remain, e.g. about newborns with a potential for remaining asymptomatic throughout life. Well organized logistics of the screening program from screening laboratory to centralized, clinical management is important.