Analysis of association between components of the folate metabolic pathway and autism spectrum disorder in eastern Indian subjects.

BACKGROUND: Folate has a pivotal role in maintaining different cellular processes including DNA integrity and neurotransmitter levels. Further, folate deficiency was reported in subjects with neuropsychiatric disorders including autism spectrum disorder (ASD). METHODS AND RESULTS: We recruited ASD probands following the Diagnostic and Statistical Manual of Mental Disorder-IV/-5. Severity was assessed by the Childhood Autism Rating Scale2-Standard Test (CARS2-ST). Functional SNPs in reduced folate carrier1 (rs1051266), methylenetetrahydrofolate dehydrogenase (rs2236225), methylenetetrahydrofolate methyltransferase (rs1805087), methylenetetrahydrofolate reductase (rs1801133 and rs1801131), cystathionine-beta- synthase (rs5742905), and serine hydroxymethyltransferase (rs1979277) genes were analyzed in the ASD probands (N = 203), their parents and controls (N = 250) by PCR/TaqMan based methods. Plasma homocysteine and vitamin B12 levels were examined by Enzyme-Linked ImmunoSorbent Assay. Statistical analysis revealed higher frequencies of rs1051266 and rs1805087 "A" alleles (P = 8.233e-005 and P = 0.010 respectively) and rs1051266 "AA" genotype (P = 0.02) in the ASD probands. Gender based stratified analysis revealed higher frequency of rs1051266 "AA" in the male probands (P = 0.001) while frequencies of rs1805087 "A" (P = 0.001) and "AA" (P < 0.05), and rs2236225 "CC" (P = 0.03) were higher in the females. The case-control analysis also exhibited a significant difference in the occurrence of biallelic and triallelic haplotypes. rs1051266 "A", rs1979277 "T" and rs5742905 "C" alleles showed biased parental transmission (P = 0.02). CARS2-ST scores were higher in the presence of rs5742905 "T" while scores were lower in the presence of rs1979277 "T" and rs1051266 "A". ASD probands showed vitamin B12 deficiency. CONCLUSION: Based on these observations, we infer that components needed for proper folate metabolism may influence ASD severity in this population.

Retromer retrieves the Wilson disease protein ATP7B from endolysosomes in a copper-dependent manner.

The Wilson disease protein, ATP7B maintains copper (herein referring to the Cu+ ion) homeostasis in the liver. ATP7B traffics from trans-Golgi network to endolysosomes to export excess copper. Regulation of ATP7B trafficking to and from endolysosomes is not well understood. We investigated the fate of ATP7B after copper export. At high copper levels, ATP7B traffics primarily to acidic, active hydrolase (cathepsin-B)-positive endolysosomes and, upon subsequent copper chelation, returns to the trans-Golgi network (TGN). At high copper, ATP7B colocalizes with endolysosomal markers and with a core member of retromer complex, VPS35. Knocking down VPS35 did not abrogate the copper export function of ATP7B or its copper-responsive anterograde trafficking to vesicles; rather upon subsequent copper chelation, ATP7B failed to relocalize to the TGN, which was rescued by overexpressing wild-type VPS35. Overexpressing mutants of the retromer complex-associated proteins Rab7A and COMMD1 yielded a similar non-recycling phenotype of ATP7B. At high copper, VPS35 and ATP7B are juxtaposed on the same endolysosome and form a large complex that is stabilized by in vivo photoamino acid labeling and UV-crosslinking. We demonstrate that retromer regulates endolysosome to TGN trafficking of copper transporter ATP7B in a manner that is dependent upon intracellular copper.

Folate System Gene Variant rs1801394 66A>G may have a Causal Role in Down Syndrome in the Eastern Indian Population.

Down syndrome (DS) is associated with trisomy of the 21st chromosome in more than 95% cases. The extra chromosome mostly derives due to abnormal chromosomal segregation, i.e. non-disjunction, during meiosis. Earlier reports showed that abnormal folate metabolism can lead to DNA hypomethylation and abnormal chromosomal segregation. We analyzed three functional folate gene variants, namely 5-methyltetrahydrofolate-homocysteine methyltransferase rs1805087, 5-methyltetrahydrofolate-homocysteine methyltransferase reductase rs1801394, and reduced folate carrier 1 rs1051266, for contribution in the etiology of DS. Ethnically matched subjects including DS probands (N=183), their parents (N=273), and controls (N=286) were recruited after obtaining informed written consent for participation. Karyotype analysis confirmed trisomy 21 in DS patients recruited. Genomic DNA, purified from peripheral blood leukocytes was used for genotyping of the target sites by PCR based methods, and data obtained was subjected to population- as well as family-based association analysis. Frequency of rs1801394 'G' allele and 'GG' genotype was higher in DS probands (P < 0.0001). Statistically significant higher occurrence of the 'G' allele in parents of DS probands (P < 0.0001) and maternal bias in transmission of the "G" allele was also noticed (P < 0.0001). Genetic model analysis demonstrated rs1801394 "G" as a risk allele under both dominant and recessive models. DS probands also showed higher occurrence of rs1051266 "G" (P = 0.05). Quantitative trait analysis revealed significant negative influence of rs1805087 "A" on birth weight. Screening for rs1801394 "G" could be useful in monitoring the risk of DS, at least in the studied population.

Autistic traits and components of the folate metabolic system: an explorative analysis in the eastern Indian ASD subjects.

Objectives: Proper metabolism of the folate is crucial for maintaining DNA integrity, chromosome structure, methylation, as well as gene expression, and thus, folate is speculated to contribute to the etiology of different disorders. Since the etiology of autism spectrum disorder (ASD) is believed to be influenced by both genetic and environmental factors, we hypothesized that functional single nucleotide polymorphisms (SNPs) affecting folate metabolic pathway may have a causal role in the etiology of ASD. Methods: We analyzed three SNPs, rs2071010, rs2298444 and rs1801198 (in the folate receptor 1, folate receptor 2 and transcobalamin 2, respectively), in 867 ethnically matched subjects including 206 ASD probands and 286 controls. Plasma vitamin B6 and folate were measured in age-matched probands and controls. Results: ASD probands showed a higher frequency of rs2298444 'A' allele (P = 0.01) and genotypes with 'A' allele (P = 0.03) when compared with the controls. rs1801198 'C' allele and 'CG' genotype also showed higher occurrence in the probands (P = 0.009 and 0.005, respectively). Gender-based stratified analysis revealed a significant higher frequency of rs2298444 'A' allele (P = 0.003), genotypes with rs2298444 'A' allele (P = 0.003) and rs1801198 CG (P = 0.001) in the male probands. Studied variants also showed statistically significant associations with ASD-associated traits measured by the Childhood Autism Rating Scale. ASD subjects exhibited gross deficiency in vitamin B6 level when compared with age-matched controls (P < 0.001), which correlated with risk genetic variants. Discussion: We infer from this pioneering study on eastern Indian subjects that vitamin B6 deficiency, along with risk gene variants, may affect ASD-associated symptoms, warranting further investigation in large cohorts.

Genetic variants of the folate metabolic system and mild hyperhomocysteinemia may affect ADHD associated behavioral problems.

An etiologically complex disorder, Attention Deficit Hyperactivity Disorder (ADHD), is often associated with various levels of cognitive deficit. Folate/vitamin B9 is crucial for numerous biochemical pathways including neural stem cell proliferation and differentiation, regulation of gene expression, neurotransmitter synthesis, myelin synthesis and repair, etc. and a scarcity has often been linked to cognitive deficit. Our pilot study in the field revealed significant association of few genetic variants with ADHD. Mild hyperhomocysteinemia and vitamin B12 deficiency was also noticed in the probands. In the present study additional genetic variants, folate and vitamin B6, which may affect folate-homocysteine metabolic pathway, were investigated in 866 individuals including nuclear families with ADHD probands (N=221) and ethnically matched controls (N=286) to find out whether ADHD associated traits are affected by these factors. Population based analysis revealed significant over representation of MTRR rs1801394 "G" allele and "GG" genotype in all as well as male probands. Stratified analysis showed significantly higher frequency of RFC1 rs1051266 and BHMT rs3733890 "AG" genotypes in full term and prematurely delivered ADHD probands respectively. Probands with rs1801394 "GG" genotype and BHMT rs3733890 "G" allele showed association with hyperhomocysteinemia. MTHFR rs1801131, MTR rs1805087 and BHMT rs3733890 also showed association with ADHD index. While rs1051266, rs1801131, and rs1805087 showed association with behavioral problems, rs3733890 was associated with ODD score. Conduct problem exhibited association with RFC1 rs1051266, MTHFR rs1801133 and MTRR rs1801394. Gene-gene interaction analysis revealed positive synergistic interactions between rs1051266, rs1801131 and rs1801394 in the probands as compared to the controls. It can be inferred from the data obtained that folate system genetic variants and mild hyperhomocysteimenia may affect ADHD associated traits by attenuating folate metabolism.

Pilot study indicate role of preferentially transmitted monoamine oxidase gene variants in behavioral problems of male ADHD probands.

BACKGROUND: Attention deficit hyperactivity disorder (ADHD) is an etiologically complex childhood onset neurobehavioral disorder characterized by age-inappropriate inattention, hyperactivity, and impulsivity. Symptom severity varies widely and boys are diagnosed more frequently than girls. ADHD probands were reported to have abnormal transmissions of dopamine, serotonin, and/or noradrenaline. Monoamine oxidase A (MAOA) and B (MAOB), mitochondrial outer membrane bound two isoenzymes, mediate degradation of these neurotransmitters and thus regulating their circulating levels. Case-control analyses in different populations, including Indians, suggested involvement of MAOA and MAOB genes in the etiology of ADHD. Due to high heritability rate of ADHD, we tested familial transmission of MAOA and MAOB variants to ADHD probands in 190 nuclear families having ADHD probands from Indo-Caucasoid ethnicity. METHODS: Subjects were recruited following the Diagnostic and Statistical Manual of Mental Disorders-4th edition (DSM-IV). Appropriate scales were used for measuring the behavioral traits in probands. Genotyping was performed through PCR-based amplification of target sites followed by DNA-sequencing and/or gel-electrophoresis. Data obtained were analyzed by family based statistical methods. RESULTS: Out of 58 variants present in the analyzed sites only 15 were found to be polymorphic (30 bp-uVNTR, rs5906883, rs1465107, rs1465108, rs5905809, rs5906957, rs6323, rs1137070 from MAOA and rs4824562, rs56220155, rs2283728, rs2283727, rs3027441, rs6324, rs3027440 from MAOB). Statistically significant maternal transmission of alleles to male probands was observed for MAOA rs5905809 'G' (p = 0.04), rs5906957 'A' (p = 0.04), rs6323 'G' (p = 0.0001) and MAOB rs56220155 'A' (p = 0.002), rs2283728 'C' (p = 0.0008), rs2283727 'C' (p = 0.0008), rs3027441 'T' (p = 0.003), rs6324 'C' (p = 0.003), rs3027440 'T' (p = 0.0002). Significantly preferential maternal transmissions of different haplotype combinations to male probands were also noticed (p < 0.05), while female probands did not reveal such transmission bias. Behavioral traits of male probands exhibited significant association with gene variants. Age of the mother at pregnancy also revealed association with risk variants of male probands. CONCLUSIONS: It may be inferred that the MAOA and MAOB variants may contribute to the etiology of ADHD in the Indo-Caucasoid population and could be responsible for higher occurrence of ADHD in the boys.

Components of the folate metabolic pathway and ADHD core traits: an exploration in eastern Indian probands.

We investigated role of the folate-homocysteine metabolic pathway in the etiology of attention-deficit hyperactivity disorder (ADHD) due to its importance in maintaining DNA integrity as well as neurotransmission. Functional gene variants in MTR (rs1805087), CBS (rs5742905), MTHFR (rs1801133 &rs1801131), MTHFD (rs2236225), RFC1 (rs1051266), plasma vitamin B12, folate and homocysteine were analyzed. rs1805087 'A' showed strong association with ADHD. Vitamin B12 deficiency of ADHD probands (P=0.01) correlated with rs1801133 'T' and rs1805087'GG'. Mild hyperhomocysteinemia (P=0.05) in the probands was associated with rs1805087 'AA'. Probands having rs1805087 'GG' and rs1051266 'G' was more inattentive. Hyperactivity-impulsivity score revealed association with rs5742905 'TT' and rs2236225 'CC', while rs1801133 'CC' showed association with inattentiveness and hyperactivity-impulsivity. rs1801131 exhibited strong synergistic interaction with rs1051266 and rs2236225. This indicated that the folate-homocysteine pathway gene variants may affect ADHD etiology through mild hyperhomocysteinemia and vitamin B12 deficiency, factors known to be associated with cognitive deficit.