Diagnosis of late-infantile neuronal ceroid lipofuscinosis using dried blood spot-based assay for TPPI enzyme activity: TPPI diagnostic assay from DBS.

BACKGROUND: The neuronal ceroid lipofuscinosis 2 (NCL2) or classic late-infantile neuronal ceroid lipofuscinosis (LINCL) is a neurogenetic disorder caused by mutations in the TPPI gene, which codes for the lysosomal tripeptidyl peptidase 1 (TPPI) EC 3.4.14.9. Loss of functional TPPI activity results in progressive visual and neurological symptoms starting at around 1-2 years of age causing early death. METHODS: We report a DBS-based TPPI assay that cleaves a synthetic tetrapeptide substrate generating a product that is detected by HPLC. Probands and carriers were identified with 100% accuracy (7 probands, 30 carriers, 13 controls). RESULTS: The assay detected a single TPPI activity at a lower pH towards the substrate tested. TPPI activity measurable when extracted at lower pH while inactive at neutral pH showed steady increase for at least 8 h incubation. No loss in TPPI activity was observed when DBS were stored for at least 2 weeks either in freezer, refrigerator, room temperature or 42 °C. CONCLUSION: A sequence variant causing Arg339Gln substitution in a proband had 12% TPPI. TPPI activity can be reliably measured in DBS, giving an opportunity to diagnose NCL2 at birth and refer patients for enzyme replacement or other therapies for earliest intervention, or alternatively offers a second-tier confirmatory test.

DNA Methylation Profiling at Single-Base Resolution Reveals Gestational Folic Acid Supplementation Influences the Epigenome of Mouse Offspring Cerebellum.

It is becoming increasingly more evident that lifestyle, environmental factors, and maternal nutrition during gestation can influence the epigenome of the developing fetus and thus modulate the physiological outcome. Variations in the intake of maternal nutrients affecting one-carbon metabolism may influence brain development and exert long-term effects on the health of the progeny. In this study, we investigated whether supplementation with high maternal folic acid during gestation alters DNA methylation and gene expression in the cerebellum of mouse offspring. We used reduced representation bisulfite sequencing to analyze the DNA methylation profile at the single-base resolution level. The genome-wide DNA methylation analysis revealed that supplementation with higher maternal folic acid resulted in distinct methylation patterns (P < 0.05) of CpG and non-CpG sites in the cerebellum of offspring. Such variations of methylation and gene expression in the cerebellum of offspring were highly sex-specific, including several genes of the neuronal pathways. These findings demonstrate that alterations in the level of maternal folic acid during gestation can influence methylation and gene expression in the cerebellum of offspring. Such changes in the offspring epigenome may alter neurodevelopment and influence the functional outcome of neurologic and psychiatric diseases.

High Gestational Folic Acid Supplementation Alters Expression of Imprinted and Candidate Autism Susceptibility Genes in a sex-Specific Manner in Mouse Offspring.

Maternal nutrients play critical roles in modulating epigenetic events and exert long-term influences on the progeny's health. Folic acid (FA) supplementation during pregnancy has decreased the incidence of neural tube defects in newborns, but the influence of high doses of maternal FA supplementation on infants' brain development is unclear. The present study was aimed at investigating the effects of a high dose of gestational FA on the expression of genes in the cerebral hemispheres (CHs) of 1-day-old pups. One week prior to mating and throughout the entire period of gestation, female C57BL/6J mice were fed a diet, containing FA at either 2 mg/kg (control diet (CD)) or 20 mg/kg (high maternal folic acid (HMFA)). At postnatal day 1, pups from different dams were sacrificed and CH tissues were collected. Quantitative RT-PCR and Western blot analysis confirmed sex-specific alterations in the expression of several genes that modulate various cellular functions (P < 0.05) in pups from the HMFA group. Genomic DNA methylation analysis showed no difference in the level of overall methylation in pups from the HMFA group. These findings demonstrate that HMFA supplementation alters offsprings' CH gene expression in a sex-specific manner. These changes may influence infants' brain development.

Lifestyle, pregnancy and epigenetic effects.

Rapidly growing evidences link maternal lifestyle and prenatal factors with serious health consequences and diseases later in life. Extensive epidemiological studies have identified a number of factors such as diet, stress, gestational diabetes, exposure to tobacco and alcohol during gestation as influencing normal fetal development. In light of recent discoveries, epigenetic mechanisms such as alteration of DNA methylation, chromatin modifications and modulation of gene expression during gestation are believed to possibly account for various types of plasticity such as neural tube defects, autism spectrum disorder, congenital heart defects, oral clefts, allergies and cancer. The purpose of this article is to review a number of published studies to fill the gap in our understanding of how maternal lifestyle and intrauterine environment influence molecular modifications in the offspring, with an emphasis on epigenetic alterations. To support these associations, we highlighted laboratory studies of rodents and epidemiological studies of human based on sampling population cohorts.

Microarray analysis reveals higher gestational folic Acid alters expression of genes in the cerebellum of mice offspring-a pilot study.

Folate is a water-soluble vitamin that is critical for nucleotide synthesis and can modulate methylation of DNA by altering one-carbon metabolism. Previous studies have shown that folate status during pregnancy is associated with various congenital defects including the risk of aberrant neural tube closure. Maternal exposure to a methyl supplemented diet also can alter DNA methylation and gene expression, which may influence the phenotype of offspring. We investigated if higher gestational folic acid (FA) in the diet dysregulates the expression of genes in the cerebellum of offspring in C57BL/6 J mice. One week before gestation and throughout the pregnancy, groups of dams were supplemented with FA either at 2 mg/kg or 20 mg/kg of diet. Microarray analysis was used to investigate the genome wide gene expression profile in the cerebellum from day old pups. Our results revealed that exposure to the higher dose FA diet during gestation dysregulated expression of several genes in the cerebellum of both male and female pups. Several transcription factors, imprinted genes, neuro-developmental genes and genes associated with autism spectrum disorder exhibited altered expression levels. These findings suggest that higher gestational FA potentially dysregulates gene expression in the offspring brain and such changes may adversely alter fetal programming and overall brain development.

Folic acid supplementation in pregnancy and implications in health and disease.

Maternal exposure to dietary factors during pregnancy can influence embryonic development and may modulate the phenotype of offspring through epigenetic programming. Folate is critical for nucleotide synthesis, and preconceptional intake of dietary folic acid (FA) is credited with reduced incidences of neural tube defects in infants. While fortification of grains with FA resulted in a positive public-health outcome, concern has been raised for the need for further investigation of unintended consequences and potential health hazards arising from excessive FA intakes, especially following reports that FA may exert epigenetic effects. The objective of this article is to discuss the role of FA in human health and to review the benefits, concerns and epigenetic effects of maternal FA on the basis of recent findings that are important to design future studies.

Increasing maternal or post-weaning folic acid alters gene expression and moderately changes behavior in the offspring.

BACKGROUND: Studies have indicated that altered maternal micronutrients and vitamins influence the development of newborns and altered nutrient exposure throughout the lifetime may have potential health effects and increased susceptibility to chronic diseases. In recent years, folic acid (FA) exposure has significantly increased as a result of mandatory FA fortification and supplementation during pregnancy. Since FA modulates DNA methylation and affects gene expression, we investigated whether the amount of FA ingested during gestation alters gene expression in the newborn cerebral hemisphere, and if the increased exposure to FA during gestation and throughout the lifetime alters behavior in C57BL/6J mice. METHODS: Dams were fed FA either at 0.4 mg or 4 mg/kg diet throughout the pregnancy and the resulting pups were maintained on the diet throughout experimentation. Newborn pups brain cerebral hemispheres were used for microarray analysis. To confirm alteration of several genes, quantitative RT-PCR (qRT-PCR) and Western blot analyses were performed. In addition, various behavior assessments were conducted on neonatal and adult offspring. RESULTS: Results from microarray analysis suggest that the higher dose of FA supplementation during gestation alters the expression of a number of genes in the newborns' cerebral hemispheres, including many involved in development. QRT-PCR confirmed alterations of nine genes including down-regulation of Cpn2, Htr4, Zfp353, Vgll2 and up-regulation of Xist, Nkx6-3, Leprel1, Nfix, Slc17a7. The alterations in the expression of Slc17a7 and Vgll2 were confirmed at the protein level. Pups exposed to the higher dose of FA exhibited increased ultrasonic vocalizations, greater anxiety-like behavior and hyperactivity. These findings suggest that although FA plays a significant role in mammalian cellular machinery, there may be a loss of benefit from higher amounts of FA. Unregulated high FA supplementation during pregnancy and throughout the life course may have lasting effects, with alterations in brain development resulting in changes in behavior.

Single-base resolution of mouse offspring brain methylome reveals epigenome modifications caused by gestational folic acid.

BACKGROUND: Epigenetic modifications, such as cytosine methylation in CpG-rich regions, regulate multiple functions in mammalian development. Maternal nutrients affecting one-carbon metabolism during gestation can exert long-term effects on the health of the progeny. Using C57BL/6 J mice, we investigated whether the amount of ingested maternal folic acid (FA) during gestation impacted DNA methylation in the offspring's cerebral hemispheres. Reduced representation bisulfite sequencing at single-base resolution was performed to analyze genome-wide DNA methylation profiles. RESULTS: We identified widespread differences in the methylation patterns of CpG and non-CpG sites of key developmental genes, including imprinted and candidate autism susceptibility genes (P <0.05). Such differential methylation of the CpG and non-CpG sites may use different mechanisms to alter gene expressions. Quantitative real time reverse transcription-polymerase chain reaction confirmed altered expression of several genes. CONCLUSIONS: These finding demonstrate that high maternal FA during gestation induces substantial alteration in methylation pattern and gene expression of several genes in the cerebral hemispheres of the offspring, and such changes may influence the overall development. Our findings provide a foundation for future studies to explore the influence of gestational FA on genetic/epigenetic susceptibility to altered development and disease in offspring.

Folic acid supplementation dysregulates gene expression in lymphoblastoid cells--implications in nutrition.

For over a decade, folic acid (FA) supplementation has been widely prescribed to pregnant women to prevent neural tube closure defects in newborns. Although neural tube closure occurs within the first trimester, high doses of FA are given throughout pregnancy, the physiological consequences of which are unknown. FA can cause epigenetic modification of the cytosine residues in the CpG dinucleotide, thereby affecting gene expression. Dysregulation of crucial gene expression during gestational development may have lifelong adverse effects or lead to neurodevelopmental defects, such as autism. We have investigated the effect of FA supplementation on gene expression in lymphoblastoid cells by whole-genome expression microarrays. The results showed that high FA caused dysregulation by ≥ four-fold up or down to more than 1000 genes, including many imprinted genes. The aberrant expression of three genes (FMR1, GPR37L1, TSSK3) was confirmed by Western blot analyses. The level of altered gene expression changed in an FA concentration-dependent manner. We found significant dysregulation in gene expression at concentrations as low as 15 ng/ml, a level that is lower than what has been achieved in the blood through FA fortification guidelines. We found evidence of aberrant promoter methylation in the CpG island of the TSSK3 gene. Excessive FA supplementation may require careful monitoring in women who are planning for, or are in the early stages of pregnancy. Aberrant expression of genes during early brain development may have an impact on behavioural characteristics.

Glyoxalase I polymorphism rs2736654 causing the Ala111Glu substitution modulates enzyme activity--implications for autism.

Autism is a pervasive, heterogeneous, neurodevelopmental disability characterized by impairments in verbal communications, reciprocal social interactions, and restricted repetitive stereotyped behaviors. Evidence suggests the involvement of multiple genetic factors in the etiology of autism, and extensive genome-wide association studies have revealed several candidate genes that bear single nucleotide polymorphisms (SNPs) in non-coding and coding regions. We have shown that a non-conservative, non-synonymous SNP in the glyoxalase I gene, GLOI, may be an autism susceptibility factor. The GLOI rs2736654 SNP is a C→A change that causes an Ala111Glu change in the Glo1 enzyme. To identify the significance of the SNP, we have conducted functional assays for Glo1. We now present evidence that the presence of the A-allele at rs2736654 results in reduced enzyme activity. Glo1 activity is decreased in lymphoblastoid cells that are homozygous for the A allele. The Glu-isoform of Glo1 in these cells is hyperphosphorylated. Direct HPLC measurements of the glyoxalase I substrate, methylglyoxal (MG), show an increase in MG in these cells. Western blot analysis revealed elevated levels of the receptor for advanced glycation end products (RAGEs). We also show that MG is toxic to the developing neuronal cells. We suggest that accumulation of MG results in the formation of AGEs, which induce expression of the RAGE that during crucial neuronal development may be a factor in the pathology of autism.

A critical tryptophan and Ca2+ in activation and catalysis of TPPI, the enzyme deficient in classic late-infantile neuronal ceroid lipofuscinosis.

BACKGROUND: Tripeptidyl aminopeptidase I (TPPI) is a crucial lysosomal enzyme that is deficient in the fatal neurodegenerative disorder called classic late-infantile neuronal ceroid lipofuscinosis (LINCL). It is involved in the catabolism of proteins in the lysosomes. Recent X-ray crystallographic studies have provided insights into the structural/functional aspects of TPPI catalysis, and indicated presence of an octahedrally coordinated Ca(2+). METHODOLOGY: Purified precursor and mature TPPI were used to study inhibition by NBS and EDTA using biochemical and immunological approaches. Site-directed mutagenesis with confocal imaging technique identified a critical W residue in TPPI activity, and the processing of precursor into mature enzyme. PRINCIPAL FINDINGS: NBS is a potent inhibitor of the purified TPPI. In mammalian TPPI, W542 is critical for tripeptidyl peptidase activity as well as autocatalysis. Transfection studies have indicated that mutants of the TPPI that harbor residues other than W at position 542 have delayed processing, and are retained in the ER rather than transported to lysosomes. EDTA inhibits the autocatalytic processing of the precursor TPPI. CONCLUSIONS/SIGNIFICANCE: We propose that W542 and Ca(2+) are critical for maintaining the proper tertiary structure of the precursor proprotein as well as the mature TPPI. Additionally, Ca(2+) is necessary for the autocatalytic processing of the precursor protein into the mature TPPI. We have identified NBS as a potent TPPI inhibitor, which led in delineating a critical role for W542 residue. Studies with such compounds will prove valuable in identifying the critical residues in the TPPI catalysis and its structure-function analysis.

Differential expression of small heat shock protein 27 (Hsp27) in Ataxia telangiectasia brains.

Ataxia telangiectasia (A-T) is a progressive neurodegenerative disorder caused by disruption of the gene, ataxia telangiectasia mutated (ATM). Present study was aimed at identifying proteins that are present in abnormal levels in A-T brain that may identify alternative targets for therapeutic interventions. Proteomic and Western blot analysis have shown massive expression of the small heat shock protein 27 (Hsp27) in frontal cortices of A-T brains compared to negligible levels in controls. The expression of other stress proteins, Hsp70, alphaB-crystallin, and prohibitin remained unchanged in the A-T and control brains. Significant decreases in reactive oxygen species, protein carbonyl groups and lipid peroxidation products were observed in the A-T brains. There is no evidence of caspase 3 activation or DAXX mediated apoptosis. We propose that neurons in the frontal lobe are protected by the expression of Hsp27, which scavenges the oxidative stress molecules formed consequent to the primary loss of ATM function.

Proteomic studies identified a single nucleotide polymorphism in glyoxalase I as autism susceptibility factor.

Autism is a neurodevelopmental disability characterized by deficits in verbal communications, impairments in social interactions, and repetitive behaviors. Several studies have indicated strong involvement of multigenic components in the etiology of autism. Linkage analyses and candidate gene search approaches so far have not identified any reliable susceptibility genes. We are using a proteomic approach to identify protein abnormalities due to aberrant gene expression in autopsied autism brains. In four of eight autism brains, we have found an increase in polarity (more acidic) of glyoxalase I (Glo1) by two-dimensional gel electrophoresis. To identify the molecular change resulting in the shift of Glo1 polarity, we undertook sequencing of GLO1 gene. Direct sequencing of GLO1 gene/mRNA in these brains, has identified a single nucleotide polymorphism (SNP), C419A. The SNP causes an Ala111Glu change in the protein sequence. Population genetics of GLO1 C419A SNP studied in autism (71 samples) and normal and neurological controls (49 samples) showed significantly higher frequency for the A419 (allele frequency 0.6 in autism and 0.4 in controls, one-tailed Fisher's test P < 0.0079). Biochemical measurements have revealed a 38% decrease in Glo1 enzyme activity in autism brains (one-tailed t-test P < 0.026). Western blot analysis has also shown accumulation of advanced glycation end products (AGE's) in autism brains. These data suggest that homozygosity for A419 GLO1 resulting in Glu111 is a predisposing factor in the etiology of autism.