Gestational vitamin D deficiency and autism-related traits: the Generation R Study.

There is intense interest in identifying modifiable risk factors associated with autism-spectrum disorders (ASD). Autism-related traits, which can be assessed in a continuous fashion, share risk factors with ASD, and thus can serve as informative phenotypes in population-based cohort studies. Based on the growing body of research linking gestational vitamin D deficiency with altered brain development, this common exposure is a candidate modifiable risk factor for ASD and autism-related traits. The association between gestational vitamin D deficiency and a continuous measure of autism-related traits at ~6 years (Social Responsiveness Scale; SRS) was determined in a large population-based cohort of mothers and their children (n=4229). 25-hydroxyvitamin D (25OHD) was assessed from maternal mid-gestation sera and from neonatal sera (collected from cord blood). Vitamin D deficiency was defined as 25OHD concentrations less than 25 nmol l-1. Compared with the 25OHD sufficient group (25OHD>50 nmol l-1), those who were 25OHD deficient had significantly higher (more abnormal) SRS scores (mid-gestation n=2866, ß=0.06, P<0.001; cord blood n=1712, ß=0.03, P=0.01). The findings persisted (a) when we restricted the models to offspring with European ancestry, (b) when we adjusted for sample structure using genetic data, (c) when 25OHD was entered as a continuous measure in the models and (d) when we corrected for the effect of season of blood sampling. Gestational vitamin D deficiency was associated with autism-related traits in a large population-based sample. Because gestational vitamin D deficiency is readily preventable with safe, cheap and accessible supplements, this candidate risk factor warrants closer scrutiny.

Associations of maternal and fetal 25-hydroxyvitamin D levels with childhood lung function and asthma: the Generation R Study.

BACKGROUND: Exposure to low levels of vitamin D in fetal life might be a risk factor for childhood asthma. OBJECTIVE: We examined whether 25-hydroxyvitamin D levels in mid-gestation and at birth were associated with higher airway resistance and inflammation, and increased risks of wheezing and asthma in school-age children. METHODS: We performed a population-based prospective cohort study among 3130 mothers and their children. Maternal blood samples in mid-gestation and umbilical cord blood samples at birth were used to determine 25-hydroxyvitamin D levels. At age of 6, airway resistance (Rint) was measured by interrupter technique and airway inflammation by fractional exhaled nitric oxide (FENO) using NIOX chemiluminescence analyser. Wheezing and asthma were prospectively assessed by annual questionnaires until age 6. RESULTS: Maternal levels of 25-hydroxyvitamin D in mid-gestation were not associated with Rint, FeNO, wheezing patterns, or asthma. Children in the lowest tertile of 25-hydroxyvitamin D levels at birth had a higher Rint (Z-score (95% confidence interval [95% CI]): -0.42 (-0.84, -0.01), P-value for trend< 0.05), compared to those in the highest tertile group. The effect estimate attenuated when child's current 25-hydroxyvitamin D level was taken into account [Z-score (95% CI): -0.55 (-1.08, 0.01)]. CONCLUSION AND CLINICAL RELEVANCE: Low levels of 25-hydroxyvitamin D at birth were associated with a higher airway resistance in childhood. Additional adjustment for child's current 25-hydroxyvitamin D level reduced the effect size of the association. Further studies are needed to replicate these findings and to examine mechanisms underlying the observed association and the long-term consequences.

Dopamine, psychosis and schizophrenia: the widening gap between basic and clinical neuroscience.

The stagnation in drug development for schizophrenia highlights the need for better translation between basic and clinical research. Understanding the neurobiology of schizophrenia presents substantial challenges but a key feature continues to be the involvement of subcortical dopaminergic dysfunction in those with psychotic symptoms. Our contemporary knowledge regarding dopamine dysfunction has clarified where and when dopaminergic alterations may present in schizophrenia. For example, clinical studies have shown patients with schizophrenia show increased presynaptic dopamine function in the associative striatum, rather than the limbic striatum as previously presumed. Furthermore, subjects deemed at high risk of developing schizophrenia show similar presynaptic dopamine abnormalities in the associative striatum. Thus, our view of subcortical dopamine function in schizophrenia continues to evolve as we accommodate this newly acquired information. However, basic research in animal models has been slow to incorporate these clinical findings. For example, psychostimulant-induced locomotion, the commonly utilised phenotype for positive symptoms in rodents, is heavily associated with dopaminergic activation in the limbic striatum. This anatomical misalignment has brought into question how we assess positive symptoms in animal models and represents an opportunity for improved translation between basic and clinical research. The current review focuses on the role of subcortical dopamine dysfunction in psychosis and schizophrenia. We present and discuss alternative phenotypes that may provide a more translational approach to assess the neurobiology of positive symptoms in schizophrenia. Incorporation of recent clinical findings is essential if we are to develop meaningful translational animal models.

Vitamin D status during fetal life and childhood kidney outcomes.

BACKGROUND/OBJECTIVES: Maternal vitamin D deficiency during pregnancy may influence offspring kidney health. We aimed to examine the associations of 25-hydroxyvitamin D (25(OH)D) blood levels during fetal life with kidney outcomes at school age. SUBJECTS/METHODS: This study was embedded in a population-based prospective cohort study among 4212 mother-child pairs. We measured maternal second trimester (18-25 weeks) and fetal cord blood (at birth) 25(OH)D levels. At a median age of 6.0 years, we measured children's combined kidney volume, glomerular filtration rate (eGFR) from creatinine and cystatin C serum levels, and microalbuminuria from albumin and creatinine urine levels. RESULTS: Of all mothers, 21.9% had severely deficient levels (25(OH)D <25.0 nmol/l), 25.7% had deficient levels (25.0-49.9 nmol/l), 25% had sufficient levels (50.0-74.9 nmol/l) and 27.4% had optimal levels (⩾75.0 nmol/l). Maternal 25(OH)D levels were not consistently associated with childhood combined kidney volume. Higher maternal 25(OH)D levels were associated with lower childhood eGFR (difference -0.94 ml/min per 1.73 m(2) (95% confidence interval, -1.73; -0.15) per 1 standard deviation (s.d.) increase in 25(OH)D). Maternal 25(OH)D levels were not associated with microalbuminuria. Cord blood 25(OH)D levels were not associated with childhood kidney outcomes. The associations of maternal 25(OH)D levels with childhood eGFR were partly explained by childhood vitamin D status. CONCLUSIONS: Our findings suggest that maternal 25(OH)D levels during pregnancy may influence childhood kidney outcomes. These results should be considered hypothesis generating. Further studies are needed to replicate the observations, to examine the underlying mechanisms and to identify the long-term clinical consequences.

Developmental Vitamin D3 deficiency alters the adult rat brain.

There is growing evidence that Vitamin D(3) (1,25-dihydroxyvitamin D(3)) is involved in brain development. We have recently shown that the brains of newborn rats from Vitamin D(3) deficient dams were larger than controls, had increased cell proliferation, larger lateral ventricles, and reduced cortical thickness. Brains from these animals also had reduced expression of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor. The aim of the current study was to examine if there were any permanent outcomes into adulthood when the offspring of Vitamin D(3) deficient dams were restored to a normal diet. The brains of adult rats were examined at 10 weeks of age after Vitamin D(3) deficiency until birth or weaning. Compared to controls animals that were exposed to transient early Vitamin D(3) deficiency had larger lateral ventricles, reduced NGF protein content, and reduced expression of a number genes involved in neuronal structure, i.e. neurofilament or MAP-2 or neurotransmission, i.e. GABA-A(alpha4). We conclude that transient early life hypovitaminosis D(3) not only disrupts brain development but leads to persistent changes in the adult brain. In light of the high incidence of hypovitaminosis D(3) in women of child-bearing age, the public health implications of these findings warrant attention.

Vitamin D regulates tyrosine hydroxylase expression: N-cadherin a possible mediator.

Vitamin D is a neuroactive steroid. Its genomic actions are mediated via the active form of vitamin D, 1,25(OH)2D3, binding to the vitamin D receptor (VDR). The VDR emerges in the rat mesencephalon at embryonic day 12, representing the peak period of dopaminergic cell birth. Our prior studies reveal that developmental vitamin D (DVD)-deficiency alters the ontogeny of dopaminergic neurons in the developing mesencephalon. There is also consistent evidence from others that 1,25(OH)2D3 promotes the survival of dopaminergic neurons in models of dopaminergic toxicity. In both developmental and toxicological studies it has been proposed that 1,25(OH)2D3 may modulate the differentiation and maturation of dopaminergic neurons; however, to date there is lack of direct evidence. The aim of the current study is to investigate this both in vitro using a human SH-SY5Y cell line transfected with rodent VDR and in vivo using a DVD-deficient model. Here we show that in VDR-expressing SH-SY5Y cells, 1,25(OH)2D3 significantly increased production of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. This effect was dose- and time-dependent, but was not due to an increase in TH-positive cell number, nor was it due to the production of trophic survival factors for dopamine neurons such as glial-derived neurotrophic factor (GDNF). In accordance with 1,25(OH)2D3's anti-proliferative actions in the brain, 1,25(OH)2D3 reduced the percentage of dividing cells from approximately 15-10%. Given the recently reported role of N-cadherin in the direct differentiation of dopaminergic neurons, we examined here whether it may be elevated by 1,25(OH)2D3. We confirmed this in vitro and more importantly, we showed DVD-deficiency decreases N-cadherin expression in the embryonic mesencephalon. In summary, in our in vitro model we have shown 1,25(OH)2D3 increases TH expression, decreases proliferation and elevates N-cadherin, a potential factor that mediates these processes. Accordingly all of these findings are reversed in the developing brain in our DVD-deficiency model. Remarkably our findings in the DVD-deficiency model phenocopy those found in a recent model where N-cadherin was regionally ablated from the mesencephalon. This study has, for the first time, shown that vitamin D directly modulates TH expression and strongly suggests N-cadherin may be a plausible mediator of this process both in vitro and in vivo. Our findings may help to explain epidemiological data linking DVD deficiency with schizophrenia.

Quantitative analysis of two pyridinium metabolites of haloperidol in patients with schizophrenia.

OBJECTIVE: A pyridinium metabolite (HPP+) of the neuroleptic drug haloperidol has been identified in rats and in the urine of patients. The purpose of this study was to measure the steady-state blood and plasma concentrations and daily urinary excretion of HPP+ in patients treated with haloperidol. METHODS: HPP+ was measured by HPLC with fluorescence detection. The chromatograms also revealed the presence of a previously unknown pyridinium species, which was identified in urine by liquid chromatography/mass spectrometry/mass spectrometry as 4-(4-chlorophenyl)-1-4-(4-fluorophenyl)-4-hydroxybutylpyridinium (RHPP+). Concentrations of RHPP+ were then measured by HPLC. RESULTS: The steady-state concentrations of HPP+ or RHPP+ in blood and plasma from 34 patients were virtually identical. The plasma concentrations of each metabolite were related to the daily dose of haloperidol and to its plasma concentrations. Nonlinearity in the elimination of RHPP+ was suggested by the increase in the ratio between RHPP+ and HPP+ plasma concentrations with dose or steady-state concentrations of haloperidol. The concentrations of RHPP+ in plasma and urine generally exceeded those of HPP+; the ratio between them in plasma ranged from 0.9 to 14.1. The daily urinary excretion of HPP+ and RHPP+ accounted for 0.40% +/- 0.18% and 2.3% +/- 1.4% of the haloperidol dose, respectively. The renal clearance of each species was 4.5 +/- 2.5 and 11.3 +/- 5.3 L/hr, respectively. CONCLUSIONS: The presence of these pyridinium species in humans raises the concern that they may be neurotoxic in a manner similar to the dopaminergic pro-neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Stereospecific reduction of haloperidol in human tissues.

In the current study, we have examined the catalytic activity and stereospecificity of haloperidol (HP) reductase activity in the cytosolic fractions of human brain and liver and in whole blood. The reductase activity was NADPH-dependent and inhibited by menadione, features typical of the ketone reductases (EC 1.2.1). The Vmax in the brain was about 4-fold higher than in the liver. Moreover, the reaction was stereospecific in that only the S(-) enantiomer was detected in brain and blood and 99.2 +/- 0.1% of the reduced HP (RHP) produced in the liver was S(-). The potential clinical implications of our results are unknown because until now all binding and pharmacodynamic studies with RHP have been performed with the racemate.

Formation of pyridinium species of haloperidol in human liver and brain.

Recent interest in the neurotoxicity of haloperidol is based on its oxidation in rodents to the pyridinium derivative, HPP+, a structural analog of the neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). Recently, we reported that HPP+ and a newly identified reduced pyridinium, RHPP+, were present in blood and urine of haloperidol-treated schizophrenics and that the concentrations of RHPP+ exceeded those of HPP+. In this study, we examined pathways for formation of RHPP+ in subcellular fractions of human liver (n = 5) and brain (basal ganglia; n = 5). The major pathway was reduction of HPP+ (20 microM) to RHPP+ in cytosol (0.17-0.39 and 0.03-0.07 microM RHPP+/g cytosolic protein per h in liver and brain, respectively). The reactions were inhibited significantly by menadione and in brain also by daunorubicin. The inhibition profile, cytosolic location and strict NADPH dependence suggest that the enzymes involved are ketone reductases. A second pathway was oxidation of reduced haloperidol (50 microM), a major metabolite of haloperidol in blood and brain, to RHPP+. In liver microsomes, 0.17-0.63 mumol RHPP+ was formed /g microsomal protein per h. A potent inhibitor of the pathway was ketoconazole (IC50, 0.8 microM), which suggests that P-450 3A isozymes could be involved. In brain mitochondria but not microsomes, reduced haloperidol (120 microM) was oxidised to RHPP+ at a small but significant rate (0.005-0.020 mumol RHPP+/g mitochondrial protein per h) which was not attenuated by SKF 525A, quinidine, ketoconazole, or monoamine oxidase inhibitors. Further studies are warranted to establish the biological importance of these metabolites in vivo.

Nonlinear relationship between circulating concentrations of reduced haloperidol and haloperidol: evaluation of possible mechanisms.

In patients taking haloperidol (HP), circulating concentrations of reduced haloperidol (RHP increase disproportionately to the dose or concentration of the parent drug. In the current study, we tested the hypothesis that the nonlinearity is due to preferential saturation of the reoxidation of RHP to HP, and two factors that could amplify the nonlinearity-concentration-dependent binding of RHP by plasma proteins, or by red blood cells. In 25 patients with schizophrenia who were taking HP, the unbound fraction of HP (0.085 +/- 0.016) and RHP (0.244 +/- 0.026) in plasma, and the blood:plasma ratio for each compound were independent of their concentration. Thus, saturable binding of RHP to plasma proteins or red blood cells can be excluded. HP reductase and RHP oxidase activity were measured in human liver cytosol and microsomal fractions, respectively. Because ketone reductase-catalysed formation of RHP is stereospecific, we examined each enantiomer of RHP separately. The Vmax for the oxidation of the S(-) and R(+) RHP enantiomers in four livers was 0.23 +/- 0.15 and 0.60 +/- 0.32 mumol/g protein per min (mean +/- SD), respectively. The Km was 110 +/- 40 and 70 +/- 10 microM, respectively. In contrast, HP reductase activity displayed greater capacity and was not saturable. The rate of production of RHP at a HP concentration of 122 microM (the limit of HP solubility) in the same livers was 2.6 +/- 0.7 mumol/g protein per min. Despite the observed nonlinearity between the enzymatic pathways in vitro, RHP concentrations in vivo are 2-3 orders of magnitude lower than the Km for oxidation of each enantiomer of RHP.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of haloperidol and reduced haloperidol in the plasma and blood of patients on depot haloperidol.

We developed a sensitive HPLC assay to measure haloperidol (HA) and its metabolite, reduced haloperidol (RH), in plasma and whole blood. The conditions under which HA might be converted to RH during collection and analysis of blood were examined. Provided the blood was kept at 0 degrees C, erythrocyte ketone reductase activity was insignificant. The solid phase extraction method did not generate RH. We studied ten patients taking 25-400 mg/month of HA decanoate and one patient for 4 weeks after the daily oral dose of 120 mg HA was ceased. In the patients on depot HA, the plasma and blood concentrations of HA were not significantly different (P greater than 0.1). For the first time, RH was detected in plasma patients on depot drug, but only in three cases. In contrast, RH was present in the blood of eight of these patients. The accumulation of RH in red blood cells was also evident in the patient on oral HA, in whom the mean ratio of RH concentrations in whole blood to plasma was 3.6 +/- 1.1. Plasma concentrations of HA correlated highly with total neuroleptic activity measured by a radioreceptor assay. Compared to plasma, analysis of concentrations of HA and RH in blood has the advantages of greater sensitivity, of using smaller volumes of blood and of avoiding the efflux of HA and RH during separation of plasma and red cells.

Neuronal calcium-binding proteins and schizophrenia.

Calcium-binding proteins (CBPs) such as calbindin, parvalbumin and calretinin are used as immunohistochemical markers for discrete neuronal subpopulations. They are particularly useful in identifying the various subpopulations of GABAergic interneurons that control output from prefrontal and cingulate cortices as well as from the hippocampus. The strategic role these interneurons play in regulating output from these three crucial brain regions has made them a focus for neuropathological investigation in schizophrenia. The number of pathological reports detailing subtle changes in these CBP-containing interneurons in patients with schizophrenia is rapidly growing. These proteins however are more than convenient neuronal markers. They confer survival advantages to neurons and can increase the neuron's ability to sustain firing. These properties may be important in the subtle pathophysiology of nondegenerative phenomena such as schizophrenia. The aim of this review is to introduce the reader to the functional properties of CBPs and to examine the emerging literature reporting alterations in these proteins in schizophrenia as well as draw some conclusions about the significance of these findings.

Chirality of reduced haloperidol in humans.

In vitro, cytosolic human ketone reductases catalyse the stereospecific (i.e. >99%) formation of S(-) reduced haloperidol (RHP) from haloperidol (HP). Whether this situation is reflected in patients taking the drug is unknown. In this study in nine patients taking HP, only 73.2+/-18.2% of the RHP excreted in urine was the S(-) enantiomer. Thus, enzymes other than cytosolic ketone reductases must be responsible for the formation of the minor enantiomer.

Two pyridinium metabolites of haloperidol are present in the brain of patients at post-mortem.

We have shown in patients taking the antipsychotic drug haloperidol (HP) that two pyridinium metabolites (HPP+ and RHPP+) are present in blood and urine in nM concentrations. These metabolites are structurally analogous to MPP+, the neurotoxic metabolite of the well-known parkinsonian-producing protoxin, MPTP. In this study we measured the concentrations of HPP+ and RHPP+ in seven regions of the brain (putamen, substantia nigra, globus pallidus, caudate, hippocampus, cerebellum and occipital cortex) obtained at post-mortem from three patients who were taking HP before death. Blood, urine, and bile from one patient were analysed as well. HPP+ was present in all regions (except for substantia nigra in one patient and globus pallidus in another); the amount/g ranged from 1.6-8.3 pMol but there was no preferential sequestration of the metabolite in dopaminergic regions. Similarly, RHPP+ was present relatively uniformly in all regions; the amount/g ranged from 1.1-7.6 pMol. The concentrations of HPP+ and RHPP+ in one patient were 24 and 13 nM in blood, 660 and 230 nM in urine, and 13.0 and 1.4 microM in bile, respectively. The presence of these pyridinums in brain adds another important piece of information to the case that, at least for HP, metabolite-induced neurotoxicity could contribute to the extrapyramidal side-effects in patients receiving long-term therapy.

Mitochondrial ultrastructure and density in a primate model of persistent tardive dyskinesia.

The use of neuroleptic drugs to treat schizophrenia is almost invariably associated with extrapyramidal movement disorders. One of these disorders, tardive dyskinesia (TD), can persist long after neuroleptic withdrawal suggesting that permanent neurological damage is produced. However, there appears to be no convincing pathology of TD and its pathogenesis remains unknown. Findings that neuroleptics interfere with normal mitochondrial function and produce mitochondrial ultrastructural changes in the basal ganglia of patients and animals suggest that mitochondrial dysfunction plays a role in TD. We have established a model for persistent TD in baboons that appears to involve compromised mitochondrial function. In this study, we evaluated two animals treated for 41 weeks with a derivative of haloperidol and two treated with vehicle only. Treatment was then withdrawn and the animals observed for a further 17-18 weeks. Treated animals developed abnormal orofacial signs that were consistent with TD. These symptoms persisted during the drug-free period. The animals were euthanased, the brains perfused-fixed then post-fixed in 4% paraformaldehyde and the caudate and putamen prepared for electron microscopy. Regardless of whether mitochondria were located in neural soma, excitatory terminals, glia or in non-somal neuropil there was no consistent difference either in size or number between treated and control animals. Thus, even if mitochondria in striatal neurons undergo ultrastructural alterations during neuroleptic therapy, these changes do not persist after drug withdrawal.

Haloperidol and its tetrahydropyridine derivative (HPTP) are metabolized to potentially neurotoxic pyridinium species in the baboon.

The in vivo metabolic fate of haloperidol (HP) and its tetrahydropyridine analog HPTP have been examined in the baboon to investigate the formation of potentially neurotoxic pyridinium metabolites that have been observed previously in humans. Urine samples collected from baboons treated with HPTP were shown to contain, in addition to the parent drug, the corresponding reduced HPTP (RHPTP), generated by reduction of the butyrophenone carbonyl group. RHPTP was characterized by comparison with a synthetic standard using HPLC with electrochemical detection and HPLC/MS/MS. Another compound identified by LC/MS/MS was a glucuronide metabolite of RHPTP. The HP pyridinium (HPP+) and reduced pyridinium (RHPP+) metabolites were shown to be present in urine from both HP and HPTP treated baboons by HPLC using fluorescence detection. The urinary excretion profile of HPP+ and RHPP+ in both groups was essentially identical and, in contrast to that observed in rodents, closely paralleled the profile found in humans treated with HP. These data in the baboon suggest that the metabolic processes involved in the production of the pyridinium metabolites of HP are similar to those in humans. Furthermore, the HPTP-treated baboon may be an appropriate model in which to study the role of pyridinium metabolites in the induction of tardive dyskinesia.

Intracellular distribution of the vitamin D receptor in the brain: comparison with classic target tissues and redistribution with development.

Apart from its role in regulating calcium there is growing evidence that vitamin D is a neuroactive steroid capable of regulating multiple pathways important for both brain development and mature brain function. Vitamin D induces its genomic effects through its nuclear receptor the vitamin D receptor (VDR). Although there is abundant evidence for this receptor's presence in the mammalian brain from studies employing immunohistochemistry, Western blot or quantitative RNA studies there remains some dispute regarding the validity of these studies. In this study we provide unambiguous confirmation for the VDR in adult rodent brain using proteomic techniques. However Western blot experiments show that compared to more classic target organs such as the gut and kidney, VDR expression is quantitatively lower in the brain. In addition we have examined VDR subcellular distribution in the gut, kidney and brain from both embryonic and adult tissues. We show that in all embryonic tissues VDR distribution is mostly nuclear, however by adulthood it appears that at least in the gut and kidney, VDR presence in the plasma membrane is more prominent perhaps reflecting some change in VDR function with the maturation of these tissues. Finally the subcellular distribution of VDR in the embryo did not appear to be altered by vitamin D deficiency indicating that perhaps there are other mechanisms at play in vivo to stabilize this receptor in the absence of its ligand.

The vitamin D receptor in dopamine neurons; its presence in human substantia nigra and its ontogenesis in rat midbrain.

There is growing evidence that vitamin D is a neuroactive steroid capable of regulating multiple pathways important for both brain development and mature brain function. In particular, there is evidence from rodent models that prenatal vitamin D deficiency alters the development of dopaminergic pathways and this disruption is associated with altered behavior and neurochemistry in the adult brain. Although the presence of the vitamin D receptor (VDR) has been noted in the human substantia nigra, there is a lack of direct evidence showing that VDR is present in dopaminergic cells. Here we confirm that the VDR is present in the nucleus of tyrosine hydroxylase (TH)-positive neurons in both the human and rat substantia nigra, and it emerges early in development in the rat, between embryonic day 12 (E12) and E15. Consistent evidence based on immunohistochemistry, real-time PCR and western blot confirmed a pattern of increasing VDR expression in the rat midbrain until weaning. The nuclear expression of VDR in TH-positive neurons during critical periods of brain development suggests that alterations in early life vitamin D status may influence the orderly development of dopaminergic neurons.

Effect of vitamin D deficiency during pregnancy on offspring bone structure, composition and quality in later life.

During foetal development, calcium requirements are met as a consequence of maternal adaptations independent of vitamin D status. In contrast, after birth, dependency on vitamin D appears necessary for calcium metabolism and skeletal health. We used a rodent model (Sprague-Dawley rats), to determine if maternal vitamin D deficiency during pregnancy had a deleterious effect on bone structure at birth. Vitamin D deplete females were maintained under deplete conditions until birth of the pups, whereupon all dams were fed a vitamin D replete diet. Offspring were harvested at birth, and 140 days of age. Bones were analyzed using micro-computed tomography and strength tested to study differences in bone structure, density and strength and subjected to elemental analysis using plasma mass spectrometry to determine strontium, barium and calcium contents. Offspring from deplete mothers displayed altered trabecular parameters in the femur at birth and 140 days of age. In addition, at 140 days of age there was evidence of premature mineralization of the secondary ossification centre of the femoral head. Elemental analysis showed increased strontium uptake in the femur of the developmentally vitamin D-deficient offspring. Vitamin D depletion during development in the offspring may have a long-lasting effect, despite repletion of vitamin D from birth. This may have consequences for human health given the low vitamin D levels seen during pregnancy and current lifestyle of sun avoidance due to the risk of skin cancer.

Increased de novo copy number variants in the offspring of older males.

The offspring of older fathers have an increased risk of neurodevelopmental disorders, such as schizophrenia and autism. In light of the evidence implicating copy number variants (CNVs) with schizophrenia and autism, we used a mouse model to explore the hypothesis that the offspring of older males have an increased risk of de novo CNVs. C57BL/6J sires that were 3- and 12-16-months old were mated with 3-month-old dams to create control offspring and offspring of old sires, respectively. Applying genome-wide microarray screening technology, 7 distinct CNVs were identified in a set of 12 offspring and their parents. Competitive quantitative PCR confirmed these CNVs in the original set and also established their frequency in an independent set of 77 offspring and their parents. On the basis of the combined samples, six de novo CNVs were detected in the offspring of older sires, whereas none were detected in the control group. Two of the CNVs were associated with behavioral and/or neuroanatomical phenotypic features. One of the de novo CNVs involved Auts2 (autism susceptibility candidate 2), and other CNVs included genes linked to schizophrenia, autism and brain development. This is the first experimental demonstration that the offspring of older males have an increased risk of de novo CNVs. Our results support the hypothesis that the offspring of older fathers have an increased risk of neurodevelopmental disorders such as schizophrenia and autism by generation of de novo CNVs in the male germline.