Metabolomic disorders: confirmed presence of potentially treatable abnormalities in patients with treatment refractory depression and suicidal behavior.

BACKGROUND: Refractory depression is a devastating condition with significant morbidity, mortality, and societal cost. Approximately 15% of patients with major depressive disorder are refractory to currently available treatments. We hypothesized metabolic abnormalities contributing to treatment refractory depression are associated with distinct findings identifiable in the cerebrospinal fluid (CSF). Our hypothesis was confirmed by a previous small case-controlled study. Here we present a second, larger replication study. METHODS: We conducted a case-controlled, targeted, metabolomic evaluation of 141 adolescent and adult patients with well-characterized history of depression refractory to three maximum-dose, adequate-duration medication treatments, and 36 healthy controls. Plasma, urine, and CSF metabolic profiling were performed by coupled gas chromatography/mass spectrometry, and high-performance liquid chromatography, electrospray ionization, tandem mass spectrometry. RESULTS: Abnormalities were identified in 67 of 141 treatment refractory depression participants. The CSF abnormalities included: low cerebral folate (n = 20), low tetrahydrobiopterin intermediates (n = 11), and borderline low-tetrahydrobiopterin intermediates (n = 20). Serum abnormalities included abnormal acylcarnitine profile (n = 12) and abnormal serum amino acids (n = 20). Eighteen patients presented with two or more abnormal metabolic findings. Sixteen patients with cerebral folate deficiency and seven with low tetrahydrobiopterin intermediates in CSF showed improvement in depression symptom inventories after treatment with folinic acid and sapropterin, respectively. No healthy controls had a metabolite abnormality. CONCLUSIONS: Examination of metabolic disorders in treatment refractory depression identified an unexpectedly large proportion of patients with potentially treatable abnormalities. The etiology of these abnormalities and their potential roles in pathogenesis remain to be determined.

Problem-solving, Adherence to Lifestyle Goals, and Weight Loss Among Individuals Participating in a Weight Loss Study.

BACKGROUND: The role of problem-solving is not well understood in behavioral weight loss interventions. In a 12-month behavioral weight loss study, we examined whether problem-solving changed over time and the relationships between problem-solving and changes in adherence to calorie, fat, and physical activity (PA) goals and percent weight change. METHODS: One of the 24 intervention sessions (15th) was devoted to problem-solving. Participants received individualized calorie and fat goals and were given a 150 min/week moderate-to-vigorous PA goal. Adherence to calorie/fat goals and PA goals was calculated at 1, 6, and 12 months using self-reported food intake in a mobile-based weight loss app and accelerometer data, respectively. Weight was measured via a digital scale at baseline, and 6 and 12 months. A general linear model was used to compare problem-solving across time points; post hoc linear mixed modeling was used to examine the relationships between problem-solving and changes in adherence to lifestyle goals and percent weight change. RESULTS: The sample (N = 150) was mostly female (90.7%), white (80.70%), with a mean age of 51.1 ± 10.2 years, and a mean body mass index of 34.1 + 4.6 kg/m2. The mean total score of problem-solving at baseline was 81.2 ± 12.3. Problem-solving total and subscale scores did not significantly change over time. Baseline problem-solving was not significantly associated with changes in adherence to lifestyle goals and percent weight change (P > 0.05). CONCLUSION: A behavioral weight loss study did not impact problem-solving, and problem-solving may not influence lifestyle adherence and weight changes. Future work needs to examine problem-solving in larger and more diverse samples.

Non-invasive epigenomic molecular phenotyping of the human brain via liquid biopsy of cerebrospinal fluid and next generation sequencing.

Our goal was to undertake a genome-wide epigenomic liquid biopsy of cerebrospinal fluid (CSF) for the comprehensive analysis of cell-free DNA (cfDNA) methylation signatures in the human central nervous system (CNS). Solution-phase hybridization and massively parallel sequencing of bisulfite converted human DNA was employed to compare methylation signatures of cfDNA obtained from CSF with plasma. Recovery of cfDNA from CSF was relatively low (68-840 pg/mL) compared to plasma (2720-8390 pg/mL) and cfDNA fragments from CSF were approximately 20 bp shorter than their plasma-derived counterparts. Distributions of CpG methylation signatures were significantly altered between CSF and plasma, both globally and at the level of functional elements including exons, introns, CpG islands, and shores. Sliding window analysis was used to identify differentially methylated regions. We found numerous gene/locus-specific differences in CpG methylation between cfDNA from CSF and plasma. These loci were more frequently hypomethylated in CSF compared to plasma. Differentially methylated CpGs in CSF were identified in genes related to branching of neurites and neuronal development. Using the GTEx RNA expression database, we found clear association between tissue-specific gene expression in the CNS and cfDNA methylation patterns in CSF. Ingenuity pathway analysis of differentially methylated regions identified an enrichment of functional pathways related to neurobiology. In conclusion, we present a genome-wide analysis of DNA methylation in human CSF. Our methods and the resulting data demonstrate the potential of epigenomic liquid biopsy of the human CNS for molecular phenotyping of brain-derived DNA methylation signatures.

Rare variants and biological pathways identified in treatment-refractory depression.

Individuals diagnosed with major depressive disorder not responding to at least two adequate treatments are defined as treatment-refractory major depressive disorder (TR-MDD). Some TR-MDD patients have altered metabolic phenotypes that may be pharmacologically reversed. The characterization of these phenotypes and their underlying etiologies is paramount, particularly their genetic components. In this study, TR-MDD patients (n = 124) were recruited and metabolites were quantified in their cerebrospinal fluid (CSF) and peripheral blood. Three sub-categories of deficiencies were examined, namely 5-methyltetrahydrofolte (in CSF; n = 13), tetrahydrobiopterin (in CSF; n = 11), and abnormal acylcarnitine profiles (in peripheral blood; n = 8). Whole exome sequencing was performed on genomic DNA from the entire TR-MDD cohort and exonic variant allele frequencies for cases were compared to a control cohort (1:5 matching on ancestry). Low frequency, damaging alleles were identified and used for in silico pathway analyses. Three association signals for TR-MDD approached genome-wide significance on chromosomes 22, 7, and 3. Three risk-associated variants from a prior depression study were replicated. Relevant biological pathways were identified that contained an enrichment of rare, damaging variants in central nervous system (CNS)-specific pathways, including neurotransmitter receptors, potassium channels, and synapse transmission. Some TR-MDD patients had rare variants in genes that were previously associated with other psychiatric disorders, psychiatric endophenotypes, CNS structural defects, and CNS-related cellular and molecular functions. Exome analysis of metabolically phenotyped TR-MDD patients has identified potentially functional gene pathways and low frequency, deleterious gene variants for further investigation. Further studies in larger cohorts of biochemically phenotyped TR-MDD patients are desirable to extend and confirm these findings.

Genetic association and differential expression of PITX2 with acute appendicitis.

Appendicitis affects 9% of Americans and is the most common diagnosis requiring hospitalization of both children and adults. We performed a genome-wide association study of self-reported appendectomy with 18,773 affected adults and 114,907 unaffected adults of European American ancestry. A significant association with appendectomy was observed at 4q25 near the gene PITX2 (rs2129979, p value = 8.82 × 10-14) and was replicated in an independent sample of Caucasians (59 affected, 607 unaffected; p value = 0.005). Meta-analysis of the associated variant across our two cohorts and cohorts from Iceland and the Netherlands (in which this association had previously been reported) showed strong cumulative evidence of association (OR = 1.12; 95% CI 1.09-1.14; p value = 1.81 × 10-23) and some evidence for effect heterogeneity (p value = 0.03). Eight other loci were identified at suggestive significance in the discovery GWAS. Associations were followed up by measuring gene expression across resected appendices with varying levels of inflammation (N = 75). We measured expression of 27 genes based on physical proximity to the GWAS signals, evidence of being targeted by eQTLs near the signals according to RegulomeDB (score = 1), or both. Four of the 27 genes (including PITX2) showed significant evidence (p values < 0.0033) of differential expression across categories of appendix inflammation. An additional ten genes showed nominal evidence (p value < 0.05) of differential expression, which, together with the significant genes, is more than expected by chance (p value = 6.6 × 10-12). PITX2 impacts morphological development of intestinal tissue, promotes an anti-oxidant response, and its expression correlates with levels of intestinal bacteria and colonic inflammation. Further studies of the role of PITX2 in appendicitis are warranted.

Treatment of Fabry's Disease with the Pharmacologic Chaperone Migalastat.

BACKGROUND: Fabry's disease, an X-linked disorder of lysosomal α-galactosidase deficiency, leads to substrate accumulation in multiple organs. Migalastat, an oral pharmacologic chaperone, stabilizes specific mutant forms of α-galactosidase, increasing enzyme trafficking to lysosomes. METHODS: The initial assay of mutant α-galactosidase forms that we used to categorize 67 patients with Fabry's disease for randomization to 6 months of double-blind migalastat or placebo (stage 1), followed by open-label migalastat from 6 to 12 months (stage 2) plus an additional year, had certain limitations. Before unblinding, a new, validated assay showed that 50 of the 67 participants had mutant α-galactosidase forms suitable for targeting by migalastat. The primary end point was the percentage of patients who had a response (≥50% reduction in the number of globotriaosylceramide inclusions per kidney interstitial capillary) at 6 months. We assessed safety along with disease substrates and renal, cardiovascular, and patient-reported outcomes. RESULTS: The primary end-point analysis, involving patients with mutant α-galactosidase forms that were suitable or not suitable for migalastat therapy, did not show a significant treatment effect: 13 of 32 patients (41%) who received migalastat and 9 of 32 patients (28%) who received placebo had a response at 6 months (P=0.30). Among patients with suitable mutant α-galactosidase who received migalastat for up to 24 months, the annualized changes from baseline in the estimated glomerular filtration rate (GFR) and measured GFR were -0.30±0.66 and -1.51±1.33 ml per minute per 1.73 m(2) of body-surface area, respectively. The left-ventricular-mass index decreased significantly from baseline (-7.7 g per square meter; 95% confidence interval [CI], -15.4 to -0.01), particularly when left ventricular hypertrophy was present (-18.6 g per square meter; 95% CI, -38.2 to 1.0). The severity of diarrhea, reflux, and indigestion decreased. CONCLUSIONS: Among all randomly assigned patients (with mutant α-galactosidase forms that were suitable or not suitable for migalastat therapy), the percentage of patients who had a response at 6 months did not differ significantly between the migalastat group and the placebo group. (Funded by Amicus Therapeutics; ClinicalTrials.gov numbers, NCT00925301 [study AT1001-011] and NCT01458119 [study AT1001-041].).

The transcriptome of the Didelphis virginiana opossum kidney OK proximal tubule cell line.

The OK cell line derived from the kidney of a female opossum Didelphis virginiana has proven to be a useful model in which to investigate the unique regulation of ion transport and membrane trafficking mechanisms in the proximal tubule (PT). Sequence data and comparison of the transcriptome of this cell line to eutherian mammal PTs would further broaden the utility of this culture model. However, the genomic sequence for D. virginiana is not available and although a draft genome sequence for the opossum Monodelphis domestica (sequenced in 2012 by the Broad Institute) exists, transcripts sequenced from both species show significant divergence. The M. domestica sequence is not highly annotated, and the majority of transcripts are predicted rather than experimentally validated. Using deep RNA sequencing of the D. virginiana OK cell line, we characterized its transcriptome via de novo transcriptome assembly and alignment to the M. domestica genome. The quality of the de novo assembled transcriptome was assessed by the extent of homology to sequences in nucleotide and protein databases. Gene expression levels in the OK cell line, from both the de novo transcriptome and genes aligned to the M. domestica genome, were compared with publicly available rat kidney nephron segment expression data. Our studies demonstrate the expression in OK cells of numerous PT-specific ion transporters and other key proteins relevant for rodent and human PT function. Additionally, the sequence and expression data reported here provide an important resource for genetic manipulation and other studies on PT cell function using these cells.

Rapid progression and mortality of lysosomal acid lipase deficiency presenting in infants.

PURPOSE: The purpose of this study was to enhance understanding of lysosomal acid lipase deficiency (LALD) in infancy. METHODS: Investigators reviewed medical records of infants with LALD and summarized data for the overall population and for patients with and without early growth failure (GF). Kaplan-Meier survival analyses were conducted for the overall population and for treated and untreated patients. RESULTS: Records for 35 patients, 26 with early GF, were analyzed. Prominent symptom manifestations included vomiting, diarrhea, and steatorrhea. Median age at death was 3.7 months; estimated probability of survival past age 12 months was 0.114 (95% confidence interval (CI): 0.009-0.220). Among patients with early GF, median age at death was 3.5 months; estimated probability of survival past age 12 months was 0.038 (95% CI: 0.000-0.112). Treated patients (hematopoietic stem cell transplant (HSCT), n = 9; HSCT and liver transplant, n = 1) in the overall population and the early GF subset survived longer than untreated patients, but survival was still poor (median age at death, 8.6 months). CONCLUSIONS: These data confirm and expand earlier insights on the progression and course of LALD presenting in infancy. Despite variations in the nature, onset, and severity of clinical manifestations, and treatment attempts, clinical outcome was poor.Genet Med 18 5, 452-458.

Altered dynamics of a lipid raft associated protein in a kidney model of Fabry disease.

Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin-Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed.

Evaluation of catatonia in autism and severe depression revealing Phelan-McDermid syndrome and tetrahydrobiopterin deficiency.

The authors describe a female in her late twenties, presenting with catatonia and diagnosed with epilepsy, autism spectrum disorder, mild intellectual disability, psychosis, dysthymia, anxiety and bipolar disorder, receiving weekly electroconvulsive therapy (ECT). After testing, findings indicated an interstitial deletion in the 22q13.33 region associated with Phelan-McDermid syndrome. In addition, the patient had low cerebral spinal fluid tetrahydrobiopterin (BH4) levels, suggesting dysfunction in the pterin biosynthetic pathway. As a result, the patient started on sapropterin, a BH4 replacement small molecule. After sapropterin treatment, catatonia improved, and the need for ECT decreased. There was an improvement in her cognitive ability, attention and independence. However, there has been no improvement in seizure frequency.

GJC2 missense mutations cause human lymphedema.

Lymphedema is the clinical manifestation of defects in lymphatic structure or function. Mutations identified in genes regulating lymphatic development result in inherited lymphedema. No mutations have yet been identified in genes mediating lymphatic function that result in inherited lymphedema. Survey microarray studies comparing lymphatic and blood endothelial cells identified expression of several connexins in lymphatic endothelial cells. Additionally, gap junctions are implicated in maintaining lymphatic flow. By sequencing GJA1, GJA4, and GJC2 in a group of families with dominantly inherited lymphedema, we identified six probands with unique missense mutations in GJC2 (encoding connexin [Cx] 47). Two larger families cosegregate lymphedema and GJC2 mutation (LOD score = 6.5). We hypothesize that missense mutations in GJC2 alter gap junction function and disrupt lymphatic flow. Until now, GJC2 mutations were only thought to cause dysmyelination, with primary expression of Cx47 limited to the central nervous system. The identification of GJC2 mutations as a cause of primary lymphedema raises the possibility of novel gap-junction-modifying agents as potential therapy for some forms of lymphedema.

Metabolic features of treatment-refractory major depressive disorder with suicidal ideation.

Peripheral blood metabolomics was used to gain chemical insight into the biology of treatment-refractory Major Depressive Disorder with suicidal ideation, and to identify individualized differences for personalized care. The study cohort consisted of 99 patients with treatment-refractory major depressive disorder and suicidal ideation (trMDD-SI n = 52 females and 47 males) and 94 age- and sex-matched healthy controls (n = 48 females and 46 males). The median age was 29 years (IQR 22-42). Targeted, broad-spectrum metabolomics measured 448 metabolites. Fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) were measured as biomarkers of mitochondrial dysfunction. The diagnostic accuracy of plasma metabolomics was over 90% (95%CI: 0.80-1.0) by area under the receiver operator characteristic (AUROC) curve analysis. Over 55% of the metabolic impact in males and 75% in females came from abnormalities in lipids. Modified purines and pyrimidines from tRNA, rRNA, and mRNA turnover were increased in the trMDD-SI group. FGF21 was increased in both males and females. Increased lactate, glutamate, and saccharopine, and decreased cystine provided evidence of reductive stress. Seventy-five percent of the metabolomic abnormalities found were individualized. Personalized deficiencies in CoQ10, flavin adenine dinucleotide (FAD), citrulline, lutein, carnitine, or folate were found. Pathways regulated by mitochondrial function dominated the metabolic signature. Peripheral blood metabolomics identified mitochondrial dysfunction and reductive stress as common denominators in suicidal ideation associated with treatment-refractory major depressive disorder. Individualized metabolic differences were found that may help with personalized management.

Selective hypermethylation is evident in small intestine samples from infants with necrotizing enterocolitis.

OBJECTIVE: Necrotizing enterocolitis (NEC) is the most common and lethal gastrointestinal disease affecting preterm infants. NEC develops suddenly and is characterized by gut barrier destruction, an inflammatory response, intestinal necrosis and multi-system organ failure. There is currently no method for early NEC detection, and the pathogenesis of NEC remains unclear. DESIGN: To further understand the molecular mechanisms that support NEC, we used solution phase hybridization and next-generation DNA sequencing of bisulfite converted DNA to perform targeted genome-wide analysis of DNA methylation at high read depth. RESULTS: We found that ileal samples from surgical NEC infants (n = 5) exist in a broadly hypermethylated state relative to their non-NEC counterparts (n = 9). These trends were not uniform, with hypermethylation being most consistently observed outside CpG islands and promoters. We further identified several biologically interesting gene promoters that displayed differential methylation in NEC and a number of biological pathways that appear dysregulated in NEC. We also found that DNA methylation patterns identified in ileal NEC tissue were correlated with those found and published previously in stool samples from NEC-affected infants. CONCLUSION: We confirmed that surgical NEC is associated with broad DNA hypermethylation in the ileum, and this may be detectable in stool samples of affected individuals. Thus, an epigenomic liquid biopsy of stool may have significant potential as a biomarker with respect to the diagnostic/predictive detection of NEC. Our findings, along with recent similar observations in colon, suggest that epigenomic dysregulation is a significant feature of surgical NEC. These findings motivate future studies which will involve the longitudinal screening of samples obtained prior to the onset of NEC. Our long-term goal is the development of novel screening, diagnostic and phenotyping methods for NEC.

Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis.

Lymphatic vessels are essential for the removal of interstitial fluid and prevention of tissue edema. Lymphatic capillaries lack associated mural cells, and collecting lymphatic vessels have valves, which prevent lymph backflow. In lymphedema-distichiasis (LD), lymphatic vessel function fails because of mutations affecting the forkhead transcription factor FOXC2. We report that Foxc2(-/-) mice show abnormal lymphatic vascular patterning, increased pericyte investment of lymphatic vessels, agenesis of valves and lymphatic dysfunction. In addition, an abnormally large proportion of skin lymphatic vessels was covered with smooth muscle cells in individuals with LD and in mice heterozygous for Foxc2 and for the gene encoding lymphatic endothelial receptor, Vegfr3 (also known as Flt4). Our data show that Foxc2 is essential for the morphogenesis of lymphatic valves and the establishment of a pericyte-free lymphatic capillary network and that it cooperates with Vegfr3 in the latter process. Our results indicate that an abnormal interaction between the lymphatic endothelial cells and pericytes, as well as valve defects, underlie the pathogenesis of LD.

Neonatal necrotizing enterocolitis-associated DNA methylation signatures in the colon are evident in stool samples of affected individuals.

Aim: Neonatal necrotizing enterocolitis (NEC) is a deadly and unpredictable gastrointestinal disease, for which no biomarker exists. We aimed to describe the methylation patterns in stool and colon from infants with NEC. Methods: We performed a high-resolution genome-wide epigenomic analysis using solution-phase hybridization and next-generation sequencing of bisulfite-converted DNA. Results: Our data reveal significant genomic hypermethylation in NEC tissues compared with non-NEC controls. These changes were more pronounced in regions outside CpG islands and gene regulatory elements, suggesting that NEC-specific hypermethylation is not a nonspecific global phenomenon. Conclusions: This study provides evidence of a methylomic signature associated with NEC that is detectable noninvasively and provides a new opportunity for the development of a novel diagnostic method for NEC.

Breath Acetone Sensing Based on Single-Walled Carbon Nanotube-Titanium Dioxide Hybrids Enabled by a Custom-Built Dehumidifier.

Acetone is a metabolic byproduct found in the exhaled breath and can be measured to monitor the metabolic degree of ketosis. In this state, the body uses free fatty acids as its main source of fuel because there is limited access to glucose. Monitoring ketosis is important for type I diabetes patients to prevent ketoacidosis, a potentially fatal condition, and individuals adjusting to a low-carbohydrate diet. Here, we demonstrate that a chemiresistor fabricated from oxidized single-walled carbon nanotubes functionalized with titanium dioxide (SWCNT@TiO2) can be used to detect acetone in dried breath samples. Initially, due to the high cross sensitivity of the acetone sensor to water vapor, the acetone sensor was unable to detect acetone in humid gas samples. To resolve this cross-sensitivity issue, a dehumidifier was designed and fabricated to dehydrate the breath samples. Sensor response to the acetone in dried breath samples from three volunteers was shown to be linearly correlated with the two other ketone bodies, acetoacetic acid in urine and ß-hydroxybutyric acid in the blood. The breath sampling and analysis methodology had a calculated acetone detection limit of 1.6 ppm and capable of detecting up to at least 100 ppm of acetone, which is the dynamic range of breath acetone for someone with ketosis. Finally, the application of the sensor as a breath acetone detector was studied by incorporating the sensor into a handheld prototype breathalyzer.

Newborn screening for galactosemia: a review of 5 years of data and audit of a revised reporting approach.

BACKGROUND: Availability of the galactose-1-phosphate uridyltransferase (GALT) assay for newborn (NB) screening has improved identification of classic galactosemia. Previously defined critical cutoffs for total galactose (Gal), typically 1.110 mmol/L (20 mg/dL), are still in use in laboratories measuring total Gal for the diagnosis of nonclassic galactosemias. Urgent notification/referral to a treatment center follows, although few of the NBs will need treatment. METHODS: We reviewed all NB galactosemia-screening results and their corresponding clinical outcomes over a 5-year period (first phase, 1.32 x 10(6) NBs) and then over a 2-year period (second phase, 274 960 NBs). Each NB was screened for Gal and GALT. When Gal was increased and/or GALT was deficient, testing for percentage galactose-1-phosphate and/or DNA testing for common GALT mutations were performed. RESULTS: Of 209 reported positive results, 89% did not indicate GALT deficiency. These non-GALT-deficient results represented mostly clinically benign cases with a Gal threshold of > or = 1.110 mmol/L (> or = 20 mg/dL). The positive predictive value of a GALT cutoff of < or = 40 micromol/L was 83%. After a protocol change that redefined a critical result as a GALT value < or = 40 micromol/L and/or a Gal value > or = 1.665 mmol/L (> or = 30 mg/dL), results were monitored for an additional 2 years. The new protocol dramatically reduced the number of urgent calls/referrals and reduced the total number of referrals by nearly half. CONCLUSIONS: Use of a GALT cutoff of < or = 40 micromol/L/L and a Gal cutoff of > or = 1.665 mmol/L (> or = 30 mg/dL) for urgent notification/referral dramatically reduces false positives and unnecessary follow-up, thereby reducing the stress on healthcare resources.

Sudden death in medium chain acyl-coenzyme a dehydrogenase deficiency (MCADD) despite newborn screening.

INTRODUCTION: Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is the most frequent of the fatty acid oxidation disorders (FAOD), a group caused by defects in the mitochondrial B-oxidation of fatty acids. Fatty acid oxidation is critical in supplying energy during periods when glucose is limited or when energy needs are increased beyond the availability of glucose. In MCADD, this energy shortage can result in acute metabolic episodes or sudden death. The prevention of sudden death from MCADD served as the primary impetus to expand newborn screening. However, we have experienced sudden death in four children with MCADD despite their detection by newborn screening. The purpose of this report is to alert others to the danger of sudden death in MCADD even when it is detected by newborn screening, to identify the clinical symptoms that precede sudden death, and to examine the relationship between the newborn screening result and the risk for sudden death. METHODS: We describe these children and their metabolic findings with emphasis on their newborn screening octanoylcarnitine (C8) level, the primary marker for newborn detection of MCADD. We also performed a literature search of cases of sudden death in MCADD in which the clinical status preceding death is described. RESULTS: The newborn screening C8 levels in our four cases were markedly elevated, ranging from 8.4 to 24.8micromol/L (cut off<0.8micromol/L). Only two of the children were homozygous for the common c.985A>G MCAD mutation; the other two were heterozygous for this mutation. Similarly, among the eight reported cases which included MCAD genotypes, five were homozygous for the c.985A>G mutation, while two were heterozygous and one was homozygous for a splice site mutation. Vomiting 12-24h before sudden death was present in all four of our cases, and the review of reported cases of sudden death in MCADD disclosed vomiting as a frequent symptom. CONCLUSION: We suggest that in MCADD (1) a newborn screening C8 level of 6micromol/L or greater represents particular risk of sudden death; (2) that MCAD genotypes other than homozygosity for the c.985A>G mutation are also associated with sudden death; (3) that vomiting is a frequent symptom preceding sudden death; and (4) social support and medical follow-up of these families are crucial in reducing the occurrence of sudden death.

The influence of macrophage migration inhibitory factor gene polymorphisms on outcome from community-acquired pneumonia.

The cytokine, macrophage migration inhibitory factor (MIF), is encoded in a functionally polymorphic locus and subjects with high-expression MIF alleles are at an increased risk of inflammatory disease. Severe sepsis is the leading cause of death in intensive care units, and the prevailing hypothesis is that an excessive innate response contributes to its pathogenesis. To assess if MIF alleles influence the clinical course of infection, we conducted a case-control study to assess susceptibility and a parallel inception cohort study of community-acquired pneumonia (CAP) to assess risk of severe sepsis and 90-d mortality. Two distinct polymorphisms in the MIF promoter were analyzed: a G/C transition at -173 and a CATT repeat at -794. The frequency of both polymorphisms was similar in the CAP cohort (n=1739) and controls (n=639); however, the 90-d mortality was lower for the high-expression C allele (P=0.003). This association remained significant after adjusting for demographics, comorbid conditions, and disease severity score [hazard ratio=0.64 (0.44-0.91), P=0.01]. The hazard ratio was similar in different geographic subcohorts, and the association remained significant after adjusting for false discovery. These data indicate that polymorphisms associated with higher MIF expression may have a beneficial effect in community-acquired pneumonia.

Effects of flutamide therapy on craniofacial growth and development in a model of craniosynostosis.

Research has implicated the faulty regulation of transforming growth factor beta signaling as one mechanism for premature calvaria suture fusion. Androgens have been shown to increase the expression and activity of the transforming growth factor beta, resulting in increased osteoblast proliferation and differentiation and possibly premature suture fusion. The present study was designed to test the hypothesis that flutamide, an androgen receptor-blocking agent, would "rescue" a coronal suture destined to fuse and improve craniofacial growth in a familial rabbit model of craniosynostosis. Thirty rabbits with delayed-onset, coronal suture synostosis were examined via longitudinal cephalometry. The rabbits were divided into 4 groups: (1) sham surgical controls (n = 10), (2) bovine serum albumin (500 ng) protein controls (n = 6), (3) flutamide diluent controls (n = 6), and (4) flutamide (15 mg dissolved in ethanol) experimental group (n = 8). At 10 days of age, radiopaque amalgam markers were implanted in all rabbits on either side of the coronal suture to monitor sutural growth. At 25 days of age, the bovine serum albumin, ethanol, and flutamide were combined with a slow-resorbing collagen vehicle and injected subperiosteally above the coronal suture into the respective groups. Although results revealed a slight but significant increase in coronal suture marker separation in flutamide-treated rabbits compared with controls at 42 days of age, few significant differences were noted for craniofacial growth and intracranial volume among groups. Results suggest that androgen receptor-blocking using flutamide may only provide a transient rescue to suture fusion in this model. Further research is needed to investigate the effects of hormones on suture development and maintenance.