Improved genetic risk scoring algorithm for type 1 diabetes prediction.

BACKGROUND: Precise risk prediction of type 1 diabetes (T1D) facilitates early intervention and identification of risk factors prior to irreversible beta-islet cell destruction, and can significantly improve T1D prevention and clinical care. Sharp et al. developed a genetic risk scoring (GRS) system for T1D (T1D-GRS2) capable of predicting T1D risk in children of European ancestry. The T1D-GRS2 was developed on the basis of causal genetic variants, thus may be applicable to minor populations, while a trans-ethnic GRS for T1D may avoid the exacerbation of health disparities due to the lack of genomic information in minorities. METHODS: Here, we describe a T1D-GRS2 calculator validated in two independent cohorts, including African American children and European American children. Participants were recruited by the Center for Applied Genomics at the Children's Hospital of Philadelphia. RESULTS: It demonstrates that GRS2 is applicable to the T1D risk prediction in the AA cohort, while population-specific thresholds are needed for different populations. CONCLUSIONS: The study highlights the potential to further improve T1D-GRS2 performance with the inclusion of additional genetic markers.

Growth in Children with a Fontan Circulation.

OBJECTIVE: To evaluate growth in a population of patients with Fontan circulation. STUDY DESIGN: We performed a cross-sectional evaluation of patients followed in our multidisciplinary Fontan clinic from January 2011 through August 2015. We reviewed the historical data, anthropometry, clinical, and laboratory studies and performed bivariate and multivariate analysis of factors associated with height z score. RESULTS: Patients (n = 210) were included in the study at median age 11.07 years (8.3, 14.73 years) (43% female); 138 (65%) had a dominant right systemic ventricle and 92 (44%) hypoplastic left heart syndrome. Median age at completion of Fontan circulation was 31 months (7.6, 135.8 months). Median height z score was -0.58 (-1.75, 0.26). Twenty-five (12%) had current or past history of protein-losing enteropathy (PLE). Median height z score for those with current or past history of PLE was -2.1 (-2.46, 1.24). Multivariate analysis revealed positive associations between height z score and body mass index z score, time since Fontan, mid-parental height, dominant systemic ventricle type, and serum alkaline phosphatase. Height correlated negatively with known genetic syndrome, PLE, use of stimulant or oral steroid medication. CONCLUSIONS: Children with Fontan circulation have mild deficits in height, with greater deficits in those with PLE. Height z score improves with time postsurgery. Improving weight, leading to improved body mass index, may be a modifiable factor that improves growth in those who are underweight. Biochemical markers may be helpful screening tests for high-risk groups in whom to intensify interventions.

Progress in understanding type 1 diabetes through its genetic overlap with other autoimmune diseases.

Type 1 diabetes mellitus (T1DM) is the most common autoimmune disease in pediatrics with a prevalence of roughly 1 in 500 children in the USA. Genome-wide association studies have identified more than 50 variants associated with increased risk for type 1 diabetes. Comparison of these variants with those identified in other autoimmune diseases reveals three important findings: (1) there is a high degree of overlap in implicated variants in diseases with similar pathophysiology, (2) in diseases with differing pathophysiology the same variants are often implicated in opposite roles, (3) in diseases with differing pathophysiology that have many non-overlapping or oppositely implicated variants there are still several variants which are overlapping or shared. Thus, the genetic overlap between T1DM and other autoimmune diseases forms the basis for our understanding of druggable targets in type 1 diabetes.

Dietary vitamin D is a novel modulator of tumor engraftment through regulation of GC protein abundance.

The vitamin D binding protein, the GC protein, is a multifunctional protein that binds circulating vitamin D and also increases macrophage killing of tumor cells. Injecting exogenous GC protein concurrent with experimental tumor implant decreases tumor engraftment rate. Until now serum abundance of this protein was thought to be controlled by estrogen, glucocorticoids and inflammatory cytokines, but, not by vitamin D itself(1, 2). Nonetheless, increasing dietary vitamin D is thought to increase serum vitamin D, which is 98% bound by the GC protein. Based on the protection that excess GC protein offers we sought to determine if decreased GC protein abundance might decrease tumor immunity. Relatedly, we theorized, by contrast to the current model, that dietary vitamin D might affect serum abundance of GC protein. If exogenous vitamin D alters available GC levels, then this effect might indicate a novel pathway by which vitamin D enhances immunity. To examine these possibilities, we examined the effect of GC protein absence on tumor persistence or engraftment on two different and common tumor types (prostate cancer and breast cancer). We further examined the relationship between dietary vitamin D and serum GC abundance. We found that absence of GC protein allowed significantly more engraftment of breast tumor cells in female mice and of prostate tumor cells in male mice. Further, we found a U-shaped response of serum GC protein to dietary vitamin D dosage as well as to serum vitamin D, indicating the potential benefit of high exogenous doses to enhance immunity and reduce tumor burden.

High dose dietary vitamin D allocates surplus calories to muscle and growth instead of fat via modulation of myostatin and leptin signaling.

Obesity occurs because the body stores surplus calories as fat rather than as muscle. Fat secretes a hormone, leptin, that modulates energy balance at the brain. Changes in fat mass are mirrored by changes in serum leptin. Elevated leptin prompts the brain to decrease appetite and increase energy expenditure. In obesity, however, impaired leptin sensitivity mutes these leptin-mediated changes. We have limited understanding of what controls leptin production by fat or leptin sensitivity in the brain. Muscle produces a hormone, myostatin, that plays a role in muscle analogous to the one that leptin plays in fat. Absent myostatin leads to increased muscle mass and strength. As with leptin, we also do not know what controls myostatin production or sensitivity. Although fat mass and muscle mass are closely linked, the interplay between leptin and myostatin remains obscure. Here we describe an interplay linked thru vitamin D. Conventionally, it is thought that vitamin D improves strength via trophic effects at the muscle. However, we find here that high dose dietary vitamin D allocates excess calories to muscle and linear growth instead of storage as fat. Vitamin D mediates this allocation by decreasing myostatin production and increasing leptin production and sensitivity. That is, high dose vitamin D improves integration of organismal energy balance. Obesity, aging and other chronic inflammatory diseases are associated with increased fat mass and decreased muscle mass and function (e.g. sarcopenia). Our work provides a physiologic framework for how high-dose vitamin D would increase allocation of calories to muscle instead of fat in these pathologies. Additionally, our work reveals a novel link between the myostatin and leptin signaling whereby myostatin conveys energy needs to modulate leptin effects on calorie allocation. This result provides evidence to update the conventional model of energy stores sensing to a new model of energy balance sensing. In our proposed model, integration of leptin and myostatin signaling allows control of body composition independent of weight. Furthermore, our work reveals how physiologic seasonal variation in vitamin D may be important in controlling season-specific metabolism and calorie allocation to fat in winter and muscle and growth in summer.

A Pilot Randomized Clinical Trial of Intranasal Oxytocin to Promote Weight Loss in Individuals With Hypothalamic Obesity.

Context: Hypothalamic obesity is a rare, treatment-resistant form of obesity. In preliminary studies, the hypothalamic hormone oxytocin (OXT) has shown promise as a potential weight loss therapy. Objective: To determine whether 8 weeks of intranasal OXT (vs 8 weeks of placebo) promotes weight loss in children, adolescents, and young adults with hypothalamic obesity. Methods: This randomized, double-blind, placebo-controlled, crossover pilot trial (NCT02849743), conducted at an outpatient academic medical center, included patients aged 10 to 35 years with hypothalamic obesity from hypothalamic/pituitary tumors. Participants received intranasal OXT (Syntocinon, 40 USP units/mL, 4 IU/spray) vs excipient-matched placebo, 16 to 24 IU 3 times daily at mealtimes. Weight loss attributable to OXT vs placebo and safety (adverse events) were assessed. Results: Of 13 individuals randomized (54% female, 31% pre-pubertal, median age 15.3 years, IQR 13.3-20.6), 10 completed the entire study. We observed a nonsignificant within-subject weight change of -0.6 kg (95% CI: -2.7, 1.5) attributable to OXT vs placebo. A subset (2/18 screened, 5/13 randomized) had prolonged QTc interval on electrocardiography prior to screening and/or in both treatment conditions. Overall, OXT was well-tolerated, and adverse events (epistaxis and nasal irritation, headache, nausea/vomiting, and changes in heart rate, blood pressure, and QTc interval) were similar between OXT and placebo. In exploratory analyses, benefits of OXT for anxiety and impulsivity were observed. Conclusion: In this pilot study in hypothalamic obesity, we did not detect a significant impact of intranasal OXT on body weight. OXT was well-tolerated, so future larger studies could examine different dosing, combination therapies, and potential psychosocial benefits.

Insights into non-autoimmune type 1 diabetes with 13 novel loci in low polygenic risk score patients.

With polygenic risk score (PRS) for autoimmune type 1 diabetes (T1D), this study identified T1D cases with low T1D PRS and searched for susceptibility loci in these cases. Our hypothesis is that genetic effects (likely mediated by relatively rare genetic variants) of non-mainstream (or non-autoimmune) T1D might have been diluted in the previous studies on T1D cases in general. Two cohorts for the PRS modeling and testing respectively were included. The first cohort consisted of 3302 T1D cases and 6181 controls, and the independent second cohort consisted of 3297 T1D cases and 6169 controls. Cases with low T1D PRS were identified using PRSice-2 and compared to controls with low T1D PRS by genome-wide association (GWA) test. Thirteen novel genetic loci with high imputation quality (Quality Score r2 > 0.91) were identified of SNPs/SNVs associated with low PRS T1D at genome-wide significance (P ≤ 5.0 × E-08), in addition to 4 established T1D loci, 3 reported loci by our previous study, as well as 9 potential novel loci represented by rare SNVs, but with relatively low imputation quality (Quality Score r2 < 0.90). For the 13 novel loci, 9 regions have been reported of association with obesity related traits by previous GWA studies. Three loci encoding long intergenic non-protein coding RNAs (lncRNA), and 2 loci involved in N-linked glycosylation are also highlighted in this study.

Genetic architecture of type 1 diabetes with low genetic risk score informed by 41 unreported loci.

Type 1 diabetes (T1D) patients with low genetic risk scores (GRS) may be non-autoimmune or autoimmune mediated by other genetic loci. The T1D-GRS2 provides us an opportunity to look into the genetic architecture of these patients. A total of 18,949 European individuals were included in this study, including 6599 T1D cases and 12,323 controls. 957 (14.5%) T1D patients were identified with low GRS (GRS < 8.43). The genome-wide association study on these patients identified 41 unreported loci. Two loci with common variants and 39 loci with rare variants were identified in this study. This study identified common SNPs associated with both low GRS T1D and expression levels of the interferon-α-induced MNDA gene, indicating the role of viral infection in T1D. Interestingly, 16 of the 41 unreported loci have been linked to autism spectrum disorder (ASD) by previous studies, suggesting that genes residing at these loci may underlie both T1D and autism.

Association of DLL1 with type 1 diabetes in patients characterized by low polygenic risk score.

Type 1 diabetes (T1D) is a heterogeneous disease. This study identified T1D cases with low polygenic risk score (PRS) to better represent T1D cases with less prominent autoimmune response (T1bD), and performed a gene-based association study to identify novel susceptibility loci in two independent cohorts, characterized by low PRS. The Notch ligand Delta-like 1 gene (DLL1) was identified with genome-wide significance in both cohorts, highlighting the roles of DLL1 genetic variants in T1D patients with low PRS, supported by functional evidence from a recent study by Rubey et al.

Differential Frequency of CYP2R1 Variants Across Populations Reveals Pathway Selection for Vitamin D Homeostasis.

CONTEXT: Normal vitamin D homeostasis is necessary to ensure optimal mineral metabolism. Dietary insufficiency of vitamin D and the lack of sunlight each have well understood roles in vitamin D deficiency; however, the extent to which common genetic variations in vitamin D metabolizing enzymes contribute to alterations in vitamin D homeostasis remains uncertain. OBJECTIVE: To examine the possibility that common coding variation in vitamin D metabolizing enzymes alters vitamin D homeostasis we determined the effect of 44 nonsynonymous polymorphisms in CYP2R1, the vitamin D 25-hydroxylase, on enzyme function. RESULTS: Twenty-one of these polymorphisms decreased activity, while 2 variants increased activity. The frequency of CYP2R1 alleles with decreased 25-hydroxylase activity is 3 in every 1000 Caucasians and 7 in every 1000 African Americans. In populations where exposure to sunlight is high, alleles with decreased function occur at a frequency as high as 8%. The pattern of selected variation as compared to nonselected variation is consistent with it being the result of positive selection for nonfunctional alleles closer to the equator. To examine this possibility, we examined the variation pattern in another protein in the vitamin D pathway, the vitamin D binding protein (GC protein). The pattern of selected variation in the GC protein as compared to nonselected variation is also consistent with it being the result of positive selection for nonfunctional alleles closer to the equator. CONCLUSIONS: CYP2R1 polymorphisms have important effects on vitamin D homeostasis, and the geographic variability of CYP2R1 alleles represents an adaptation to differential exposures to UVB irradiation from sunlight.

Preterm birth without progesterone withdrawal in 15-hydroxyprostaglandin dehydrogenase hypomorphic mice.

Parturition is a complex mammalian physiological process whose fundamental determinants have remained elusive. The increasing incidence of human preterm birth, a leading cause of infant mortality, highlights the importance of further understanding mechanisms regulating the timing of birth. Parturition is initiated in most nonprimate mammals, including mice, through a decrease in circulating progesterone caused by elevated prostaglandins. In humans, other higher primates, and guinea pigs, no consistent decrease in circulating progesterone occurs before the onset of labor. The divergence in endocrine control of labor initiation between most mammals compared with the great apes and guinea pigs gives rise to the question: how could a mechanism for the initiation of labor not requiring the withdrawal of progesterone evolve? Here, we genetically modulate prostaglandin signaling to determine the role of prostaglandin catabolism in the timing of birth. We find spontaneous preterm labor in the absence of progesterone withdrawal in 15-hydroxyprostaglandin dehydrogenase hypomorphic mice. The onset of labor in these hypomorphic mice is preceded by prematurely increased concentrations of prostaglandin E(2) and F(2alpha). Moreover, genetic crosses demonstrate a role for fetal genotype in birth timing. Together, these findings demonstrate a 15-hydroxyprostaglandin dehydrogenase-dependent shift in the physiology of murine parturition to one resembling the physiology of higher primates. Thus, endocrine control of labor has the capacity to plastically adapt to changes in genetically determined prostaglandin signals.

Early pregnancy loss in 15-hydroxyprostaglandin dehydrogenase knockout (15-HPGD-/-) mice due to requirement for embryo 15-HPGD activity.

Prostaglandins (PGs) have critical signaling functions in a variety of processes including the establishment and maintenance of pregnancy, and the initiation of labor. Most PGs are non-enzymatically degraded, however, the two PGs most prominently implicated in the termination of pregnancy, including the initiation of labor, prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α), are enzymatically degraded by 15-hydroxyprostaglandin dehydrogenase (15-HPGD). The role of PG metabolism by 15-HPGD in the maintenance of pregnancy remains largely unknown, as direct functional studies are lacking. To test the hypothesis that 15-PGDH-mediated PG metabolism is essential for pregnancy maintenance and normal labor timing, we generated and analyzed pregnancy in 15-HPGD knockout mice (Hpgd-/-). We report here that pregnancies resulting from matings between 15-HPGD KO mice (Hpgd-/- X Hpgd-/-KO mating) are terminated at mid gestation due to a requirement for embryo derived 15-HPGD. Aside from altered implantation site spacing, pregnancies from KO matings look grossly and histologically normal at days post coitum (dpc) 6.5 and 7.5 of pregnancy. However, virtually all of these pregnancies are resorbed by dpc 8.5. This resorption is preceded by elevation of PGF2∝ but is not preceded by a decrease in circulating progesterone, suggesting that pregnancy loss is a local inflammatory phenomenon rather than a centrally mediated phenomena. This pregnancy loss can be temporarily deferred by indomethacin treatment, but treated pregnancies are not maintained to term and indomethacin treatment increases maternal mortality. We conclude that PG metabolism to inactive products by embryo derived 15-HPGD is essential for pregnancy maintenance in mice, and may serve a similar function during human pregnancy.

Obesity Decreases Hepatic 25-Hydroxylase Activity Causing Low Serum 25-Hydroxyvitamin D.

Normal vitamin D homeostasis is critical for optimal health; nevertheless, vitamin D deficiency is a worldwide public health problem. Vitamin D insufficiency is most commonly due to inadequate cutaneous synthesis of cholecalciferol and/or insufficient intake of vitamin D, but can also arise as a consequence of pathological states such as obesity. Serum concentrations of 25(OH)D (calcidiol) are low in obesity, and fail to increase appropriately after vitamin D supplementation. Although sequestration of vitamin D in adipose tissues or dilution of ingested or cutaneously synthesized vitamin D in the large fat mass of obese patients has been proposed to explain these findings, here we investigate the alternative mechanism that reduced capacity to convert parent vitamin D to 25(OH)D due to decreased expression of CYP2R1, the principal hepatic vitamin D 25-hydroxylase. To test this hypothesis, we isolated livers from female mice of 6 to 24 weeks of age, weaned onto either a normal chow diet or a high-fat diet, and determined the abundance of Cyp2r1 mRNA using digital droplet-quantitative PCR. We observed a significant (p < 0.001) decrease in Cyp2r1 mRNA in the liver of high-fat diet-fed mice relative to lean-chow-fed female mice. Moreover, there was a significant (p < 0.01) relationship between levels of Cyp2r1 mRNA and serum 25(OH)D concentrations as well as between Cyp2R1 mRNA and the ratio of circulating 25(OH)D3 to cholecalciferol (p < 0.0001). Using linear regression we determined a curve with 25(OH)D3/cholecalciferol versus normalized Cyp2R1 mRNA abundance with an R2 value of 0.85. Finally, we performed ex vivo activity assays of isolated livers and found that obese mice generated significantly less 25(OH)D3 than lean mice (p < 0.05). Our findings indicate that expression of CYP2R1 is reduced in obesity and accounts in part for the decreased circulating 25(OH)D. © 2019 American Society for Bone and Mineral Research.

Decreased Serum 25-Hydroxyvitamin D in Aging Male Mice Is Associated With Reduced Hepatic Cyp2r1 Abundance.

The prevalence of vitamin D deficiency, as determined by circulating levels of 25-hydroxycalciferol [25(OH)D], is greater in older individuals compared with the young. To examine the hypothesis that altered production or inactivation of 25(OH)D contributes to lower circulating levels of 25(OH)D, we measured the serum levels of parent vitamin D3 (cholecalciferol) and 25(OH)D. We also determined the relative abundance of transcripts encoding hepatic CYP2R1 and CYP27B1, the principal 25-hydroxylases, transcripts encoding enzymes that degrade 25(OH)D in the liver (Cyp3A11) and kidney (Cyp24A1) and transcripts encoding megalin and cubilin, proteins critical to vitamin D resorption in the kidney in mice at three different ages. We observed a significant decline in the relative abundance of Cyp2R1 in the liver with aging (one-way ANOVA, P = 0.0077). Concurrent with the decrease in mRNA, a significant decline in hepatic CYP2R1 protein (one-way ANOVA for trend, P = 0.007) and 25(OH)D (one-way ANOVA for trend, P = 0.002) and in the ratio of 25(OH)D3 to cholecalciferol (one-way ANOVA, P = 0.0003). By contrast, levels of the transcripts encoding Cyp3a11, Cyp24a1, and Cyp27b1 megalin and cubilin were unchanged with aging. A significant positive correlation was found between Cyp2r1 mRNA and 25(OH)D, and a stronger correlation was found between Cyp2r1 mRNA and the ratio of 25(OH)D3 to cholecalciferol. These results indicate that decreased expression of CYP2R1 contributes to the reduced serum levels of 25(OH)D in aging.

CLEC16A regulates splenocyte and NK cell function in part through MEK signaling.

CLEC16A is implicated in multiple autoimmune diseases. We generated Clec16a inducible knockout (KO) mice to examine the functional link between CLEC16A auto-inflammation and autoimmunity. Clec16a KO mice exhibited weight loss and thymic and splenic atrophy. Mitochondrial potential was lowered in KO mice splenocytes resulting in aggregation of unhealthy mitochondria in B, T, and NK cells. In Clec16a KO mice we detected disrupted mitophagy in splenic B and T cells. NK cells from Clec16a KO mice exhibited increased cytotoxicity. Incomplete mitophagy was attenuated with PI3K and/or MEK inhibition in Clec16a KO mice. Our results demonstrate a functional link between CLEC16A and disrupted mitophagy in immune cells and show that incomplete mitophagy predisposes the KO mice to inflammation. Taken together, loss of function variants in CLEC16A that are associated with decreased CLEC16A expression levels may contribute to inflammation in autoimmunity through disrupted mitophagy. Drugs modulating mitophagy reverse the process and may be effective in treating and preventing autoimmunity in individuals with risk associated CLEC16A variants.

CYP3A4 mutation causes vitamin D-dependent rickets type 3.

Genetic forms of vitamin D-dependent rickets (VDDRs) are due to mutations impairing activation of vitamin D or decreasing vitamin D receptor responsiveness. Here we describe two unrelated patients with early-onset rickets, reduced serum levels of the vitamin D metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, and deficient responsiveness to parent and activated forms of vitamin D. Neither patient had a mutation in any genes known to cause VDDR; however, using whole exome sequencing analysis, we identified a recurrent de novo missense mutation, c.902T>C (p.I301T), in CYP3A4 in both subjects that alters the conformation of substrate recognition site 4 (SRS-4). In vitro, the mutant CYP3A4 oxidized 1,25-dihydroxyvitamin D with 10-fold greater activity than WT CYP3A4 and 2-fold greater activity than CYP24A1, the principal inactivator of vitamin D metabolites. As CYP3A4 mutations have not previously been linked to rickets, these findings provide insight into vitamin D metabolism and demonstrate that accelerated inactivation of vitamin D metabolites represents a mechanism for vitamin D deficiency.

Ablation of Oxytocin Neurons Causes a Deficit in Cold Stress Response.

The paraventricular nucleus (PVN) is a critical locus of energy balance control. Three sets of neurons in the PVN are involved in regulating energy balance: oxytocin-expressing neurons (OXT-neurons), thyrotropin-releasing hormone-expressing neurons, and corticotrophin-releasing hormone-expressing neurons. To examine the role of OXT-neurons in energy balance, we ablated these neurons in mice by injecting diphtheria toxin into mice possessing both the oxytocin promoter driving cre expression and a cre-inducible diphtheria toxin receptor. Immunohistochemistry and real-time reverse transcriptase polymerase chain reaction confirmed that this injection caused a significant decrease in PVN OXT-neurons and OXT-mRNA abundance. OXT-neuron ablation did not alter food intake, weight, or energy expenditure at room temperature on either chow or a high-fat diet. To further characterize OXT-neuron-ablated mice, we examined their response to 1) intraperitoneal cholecystokinin (CCK) injection and 2) thermogenic stress. OXT-neuron-ablated mice had a blunted decrease in feeding response to CCK. When exposed to the extreme cold (4°C) for 3 hours, OXT-neuron-ablated mice had significant decreases in both rectal and brown adipose tissue temperature relative to controls, which was rescued by OXT treatment. Thermographic imaging revealed that OXT-neuron-ablated mice had increased body surface temperature. Thus, we report that OXT-neuron ablation shows no role for OXT-neurons in energy homeostasis at neutral temperature but reveals a heretofore unappreciated role for OXT-neurons and oxytocin specifically in regulating the thermogenic stress response.