Jeffrey M. Sequeira

Cerebral folate receptor autoantibodies in autism spectrum disorder

Cerebral folate deficiency (CFD) syndrome is a neurodevelopmental disorder typically caused by folate receptor autoantibodies (FRAs) that interfere with folate transport across the blood–brain barrier. Autism spectrum disorders (ASDs) and improvements in ASD symptoms with leucovorin (folinic acid) treatment have been reported in some children with CFD. In children with ASD, the prevalence of FRAs and the response to leucovorin in FRA-positive children has not been systematically investigated. In this study, serum FRA concentrations were measured in 93 children with ASD and a high prevalence (75.3%) of FRAs was found. In 16 children, the concentration of blocking FRA significantly correlated with cerebrospinal fluid 5-methyltetrahydrofolate concentrations, which were below the normative mean in every case. Children with FRAs were treated with oral leucovorin calcium (2 mg kg−1 per day; maximum 50 mg per day). Treatment response was measured and compared with a wait-list control group. Compared with controls, significantly higher improvement ratings were observed in treated children over a mean period of 4 months in verbal communication, receptive and expressive language, attention and stereotypical behavior. Approximately one-third of treated children demonstrated moderate to much improvement. The incidence of adverse effects was low. This study suggests that FRAs may be important in ASD and that FRA-positive children with ASD may benefit from leucovorin calcium treatment. Given these results, empirical treatment with leucovorin calcium may be a reasonable and non-invasive approach in FRA-positive children with ASD. Additional studies of folate receptor autoimmunity and leucovorin calcium treatment in children with ASD are warranted.

Folate Receptor Autoimmunity and Cerebral Folate Deficiency in Low-Functioning Autism with Neurological Deficits

Reduced folate transport to the CNS was identified in two autism spectrum disorders, i.e., Rett syndrome and infantile low-functioning autism with neurological abnormalities. Twenty-five patients with early-onset low-functioning autism with or without neurological deficits, were evaluated for serum folate, cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5MTHF), and serum FR autoantibodies of the blocking type to determine the significance of folate receptor (FR) autoantibodies with respect to folate transport across the blood-CSF barrier. In spite of normal serum folate, CSF 5MTHF was low in 23 of 25 patients. The reduced CSF folate in 19 of these 23 patients could be explained by serum FR autoantibodies blocking the folate binding site of the membrane-attached FR on the choroid epithelial cells. Oral folinic acid supplements led to normal CSF 5MTHF and partial or complete clinical recovery after 12 months. Serum FR autoimmunity appears to represent an important factor in the pathogenesis of reduced folate transport to the nervous system among children with early-onset low-functioning autism associated with or without neurological deficits. Early detection of FR autoantibodies may be a key factor in the prevention and therapeutic intervention among this subgroup of patients with autism.

The protein and the gene encoding the receptor for the cellular uptake of transcobalamin bound cobalamin

The transcobalamin (TC, TCII) receptor (TCblR) on the plasma membrane binds TC- cobalamin (Cbl) and internalizes the complex by endocytosis. This receptor was purified from human placental membranes by affinity chromatography. Tryptic digest of the protein extracted from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and subjected to liquid chromatography/mass spectrometry identified 4 peptides that matched with a membrane protein in the data bank. TCblR belongs to the low-density lipoprotein receptor family, with 2 low-density lipoprotein receptor type A domains separated by a complement-like cysteine-rich region. The 282-amino acid sequence includes a signal peptide of 31 residues, extracellular domain of 198 residues, a transmembrane region of 21 residues, and a cytoplasmic domain of 32 residues. The binding of TC-Cbl does not require the cytoplasmic domain or its orientation in the plasma membrane because the recombinant extracellular domain binds TC-Cbl with high affinity and specificity. The protein is heavily glycosylated and accounts for the 58-kDa size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The human gene first identified as 8D6A and more recently as CD 320 encoding TCblR is located at p13.2 on the short arm of chromosome 19, spans a length of 6.224 kb, and is composed of 5 exons and 4 introns.

A milk-free diet downregulates folate receptor autoimmunity in cerebral folate deficiency syndrome.

In cerebral folate deficiency syndrome, the presence of autoantibodies against the folate receptor (FR) explains decreased folate transport to the central nervous system and the clinical response to folinic acid. Autoantibody crossreactivity with milk FR from different species prompted us to test the effect of a milk‐free diet. Intervention with a milkfree diet in 12 children (nine males, three females; mean age 6y [SD 4y 11mo], range 1‐19y), decreased autoantibody titer significantly from 2.08pmol of FR blocked per ml of serum (SD 2.1; range 0.24‐8.35) to 0.35pmol (SD 0.49; range 0‐1.32; p=0.012) over 3 to 13 months, whereas FR autoantibody titer increased significantly to 6.53 (SD 6.08; range 0.54‐14.07; p=0.013) in nine children who were reexposed to milk for 6 to 14 weeks. In 12 children on a normal diet (eight males, four females; mean age 5y 5mo [SD 4y 1mo], range 1y 6mo‐16y 4mo), the antibody titer increased significantly from 0.84pmol of FR blocked per ml (SD 0.39; range 0.24‐1.44) to 3.04pmol (SD 1.42; range 0.84‐6.01; p=0.001) over 10 to 24 months. Decreasing the autoantibody titer with a milk‐free diet in conjunction with folinic acid therapy may be advocated for these patients.

Mitochondrial diseases associated with cerebral folate deficiency

Cerebral folate deficiency (CFD) has been defined as any neuropsychiatric condition associated with isolated lowering of 5-methyltetrahydrofolate (5-MTHF) levels in CSF and normal systemic folate metabolism.1 CFD has been detected in the infantile-onset CFD syndrome (mediated by serum folate receptor [FR] autoantibodies of the blocking type1) and Aicardi-Goutieres and Rett syndromes. In Kearns-Sayre syndrome (KSS), systemic folate deficiency or low CSF 5-MTHF have long been recognized.2,3 Because active transport of 5-MTHF across the choroid plexus epithelial cells is mediated by ATP-dependent processes, we conducted a study to determine CSF 5-MTHF in a series of patients with mitochondrial disorders. ### Methods. Twenty-eight patients with different mitochondrial disorders and fulfilling the previously defined diagnostic criteria4 were recruited from the Hospital Sant Joan de Deu, Barcelona, Spain (21 patients), the University Clinic Aachen, Germany (5 patients), and Hospital 12 de Octubre, Madrid, Spain (2 patients). Diagnosis included KSS (5 patients), neuropathy, ataxia, and retinitis pigmentosa (1 patient), mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke (1 patient), and various clinically heterogeneous respiratory chain enzyme deficiencies (RCD) (21 patients). Brain MRI was performed for each patient. Serum folate and CSF 5-MTHF, glucose, protein, and cells were determined as per established protocol.5 CSF 5-MTHF …

Cerebral folate deficiency and CNS inflammatory markers in Alpers disease

We describe a 3.5-year-old female with Alpers disease with a POLG genotype of p.A467T/p.G848S and with a lethal outcome. Laboratory investigation revealed elevated CSF neopterin, IL-6, IL-8, IFN-gamma, reduced CSF 5-methyltetrahydrofolate (5MTHF), and increased serum as well as CSF folate receptor blocking autoantibodies. Treatment with oral Leucovorine (5-formyl-tetrahydrofolate) was initiated at 0.25mg/kg bid, and later increased to 4mg/kg bid. Under treatment CSF levels of 5MTHF, seizure frequency and communicative abilities improved. Over a time span of 17months, CSF levels of IL-6 and IFN-gamma decreased, levels of folate receptor blocking autoantibodies continued to raise, whereas CSF IL-8 remained elevated 1500-fold above normal. The child died without apparent stress at the age of 5.5years. Alpers disease, a neurodegenerative disease usually presents in the first years of life as a progressive encephalopathy with multifocal myoclonic seizures, developmental regression, cortical blindness and early death. The underlying genetic defect has been attributed to mutations of the catalytic subunit of the mitochondrial DNA polymerase-gamma leading to an organ-specific mitochondrial DNA depletion syndrome with reduced activity of respiratory chain enzyme complexes in the brain and the liver. A curative therapy is not available. This case report of Alpers disease provides new insights into the pathophysiology of Alpers disease, where mitochondrial dysfunction in conjunction with inflammatory cytokines and blocking folate receptor autoantibodies may lead to a secondary cerebral folate deficiency syndrome. The treatment of the latter provides relief to the patient without stopping the underlying disease.

Acetoacetate reduces growth and ATP concentration in cancer cell lines which over-express uncoupling protein 2

BackgroundRecent evidence suggests that several human cancers are capable of uncoupling of mitochondrial ATP generation in the presence of intact tricarboxylic acid (TCA) enzymes. The goal of the current study was to test the hypothesis that ketone bodies can inhibit cell growth in aggressive cancers and that expression of uncoupling protein 2 is a contributing factor. The proposed mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration.MethodsSeven aggressive human cancer cell lines, and three control fibroblast lines were grown in vitro in either 10 mM glucose medium (GM), or in glucose plus 10 mM acetoacetate [G+AcA]. The cells were assayed for cell growth, ATP production and expression of UCP2.ResultsThere was a high correlation of cell growth with ATP concentration (r = 0.948) in a continuum across all cell lines. Controls demonstrated normal cell growth and ATP with the lowest density of mitochondrial UCP2 staining while all cancer lines demonstrated proportionally inhibited growth and ATP, and over-expression of UCP2 (p < 0.05).ConclusionSeven human cancer cell lines grown in glucose plus acetoacetate medium showed tightly coupled reduction of growth and ATP concentration. The findings were not observed in control fibroblasts. The observed over-expression of UCP2 in cancer lines, but not in controls, provides a plausible molecular mechanism by which acetoacetate spares normal cells but suppresses growth in cancer lines. The results bear on the hypothesized potential for ketogenic diets as therapeutic strategies.

Positive newborn screen for methylmalonic aciduria identifies the first mutation in TCblR/CD320, the gene for cellular uptake of transcobalamin‐bound vitamin B12

Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin‐bound cobalamin (holo‐TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo‐TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low‐density lipoprotein receptor type A‐like domain. Inserting the codon by site‐directed mutagenesis fully restored TCblR function. Hum Mutat 31:1–6, 2010.

Role of folate receptor autoantibodies in infantile autism

During pregnancy, maternal folate receptor alpha (FRα) autoantibodies could block folate transfer to the fetus and increase the risk of neural tube defects.1 The prevalence of blocking FRα antibodies in healthy adult women was estimated at 4–7% in Spain,2 9–13% in Ireland3 and 10–15% in the US population.1 A low titer of this antibody in a fraction of the adult population appears to be non-pathologic. Postnatally acquired FR autoantibodies blocking folate transport to the brain have been associated with the infantile-onset cerebral folate deficiency syndrome,4 which in a number of patients manifests as low-functioning autism with neurological deficits.5 In a US population with autism spectrum disorder (ASD), either the blocking- or the binding-type autoantibody was detected in 75% of the children and high-dose folinic-acid supplementation improved the core symptoms of autism in these children.6 We studied a population of infantile autism, non-autistic controls with developmental deficits and their parents in Belgium (research project supported by FNRS Belgium No: 3.4.540.09.F). Serum was tested for FRα-blocking autoantibodies as described previously.4 Plasma homocysteine and serum folate were also measured. The samples were coded and blinded to all analysis.

Transcobalamin II receptor polymorphisms are associated with increased risk for neural tube defects

Objective Women who have low cobalamin (vitamin B12) levels are at increased risk for having children with neural tube defects (NTDs). The transcobalamin II receptor (TCblR) mediates uptake of cobalamin into cells. Inherited variants in the TCblR gene as NTD risk factors were evaluated. Methods Case–control and family-based tests of association were used to screen common variation in TCblR as genetic risk factors for NTDs in a large Irish group. A confirmatory group of NTD triads was used to test positive findings. Results 2 tightly linked variants associated with NTDs in a recessive model were found: TCblR rs2336573 (G220R; pcorr=0.0080, corrected for multiple hypothesis testing) and TCblR rs9426 (pcorr=0.0279). These variants were also associated with NTDs in a family-based test before multiple test correction (log-linear analysis of a recessive model: rs2336573 (G220R; RR=6.59, p=0.0037) and rs9426 (RR=6.71, p=0.0035)). A copy number variant distal to TCblR and two previously unreported exonic insertion–deletion polymorphisms were described. Conclusions TCblR rs2336573 (G220R) and TCblR rs9426 represent a significant risk factor in NTD cases in the Irish population. The homozygous risk genotype was not detected in nearly 1000 controls, indicating that this NTD risk factor may be of low frequency and high penetrance. 9 other variants are in perfect linkage disequilibrium with the associated single nucleotide polymorphisms. Additional work is required to identify the disease-causing variant. Our data suggest that variation in TCblR plays a role in NTD risk and that these variants may modulate cobalamin metabolism.