24 de julio de 2012

Cerebral folate receptor autoantibodies in autism spectrum disorder

Fuente original: Molecular Psychiatry advance online publication 10 January 2012; doi: 10.1038/mp.2011.175
R E Frye1, J M Sequeira2, E V Quadros2, S J James1 and D A Rossignol3

1Department of Pediatrics, Arkansas Children's Hospital Research Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA
2Department of Medicine, State University of New York—Downstate Medical Center, Brooklyn, NY, USA
3International Child Development Resource Center, Melbourne, FL, USA
Correspondence: Dr RE Frye, MD, PhD, Department of Pediatrics, Arkansas Children's Hospital Research Institute, University of Arkansas for Medical Sciences, Little Rock, AR 72202, USA. E-mail: REFrye@uams.edu

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 (2mgkg−1 per day; maximum 50mg 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.

 Keywords: autism spectrum disorders; cerebral folate deficiency; folate receptor autoantibody; folinic acid; leucovorin calcium

Cerebral folate deficiency (CFD) syndrome is a recently described neurometabolic disorder characterized by low concentrations of 5-methyltetrahydrofolate (5MTHF) in the cerebrospinal fluid (CSF), despite normal systemic folate levels. CFD is believed to be caused by the impaired transport of folates across the blood–brain barrier. The first reported cases of CFD manifested severe neurodevelopmental symptoms including spastic paraplegia, cerebellar ataxia, dyskinesia, seizures, acquired microcephaly and developmental regression, which occurred as early as 4 months of age.1, 2 Central visual disturbances (optic atrophy and blindness) and hearing loss occurred after age 3 and 6 years, respectively, in some cases.2
Since its original description, the phenotype of CFD has been expanded. Six studies have reported autism spectrum disorder (ASD) in a subset of children with CFD.2, 3, 4, 5, 6, 7 Most of these children with ASD were low functioning and had significant neurological abnormalities.4, 6 Although the biological mechanisms linking CFD to ASD are not known, deficits in folate within the central nervous system (CNS) could explain several findings documented in ASD. For example, deficits in folate one-carbon metabolism have been shown to promote methylation deficits and oxidative stress in some children with ASD,8, 9, 10 as well as alter DNA methylation in the brain of an animal model.11 Examination of some postmortem ASD brains has verified alterations in DNA methylation in the frontal cortex12 and increased oxidative stress in several cortical regions, including those associated with speech processing and emotional and social behavior.13 Ramaekers et al.14 suggested that expression of normally silenced genes could be caused by either alterations in the methyl CpG-binding protein 2 gene, as occurs in Rett's syndrome or alterations in DNA methylation resulting from CFD, thus explaining the similarity between the clinical symptoms of the original description of CFD and Rett's syndrome. Moreover, as chronic oxidative stress can result in mitochondrial dysfunction,15 the increased oxidative stress noted in postmortem ASD brain samples13 could account for the mitochondrial dysfunction observed in similar cortical regions in postmortem ASD brain samples.16 As folate is essential for production of purines and pyrimidines, the nucleotide precursors of RNA and DNA, low folate levels can result in abnormalities in cellular proliferation, as well as transcription and translation, and therefore contribute to DNA instability17 and chromosome breakage.18 Finally, animal models demonstrate that the folate receptor protein alpha (FRα) is important in the repair and regeneration of the CNS after injury.19

Methylfolate is transported across the blood–brain barrier by FRα, which has a high affinity for both folic acid and 5MTHF and is located on both sides of the epithelial surface of the choroid plexus.20 FRα is part of an ATP-dependent receptor-mediated system that transports folate derivatives across the blood–brain barrier through an endocytosis mechanism. FRα is essential for folate transport across the blood–brain barrier when extracellular folate concentrations are low.20 FRα is also located on thyroid cells,6 the microvillus plasma membrane of the placenta,21 as well as in the epithelium of the fallopian tubes, uterus and epididymis, acinar cells of the breast, submandibular salivary and bronchial glands and the alveolar lining including pneumocytes.22 Around 2005, autoantibodies were described that bind to the FRα, greatly impairing its function.6 FRα autoantibodies (FRAs) have been linked to CFD in cases with4 and without6 ASD and also in gestations with neural tube defects23 and subfertile women.24 In a 2007 case report, mitochondrial complex I deficiency was linked to CFD in a FRA-negative child,25 and in 2008, a wider array of mitochondrial diseases was associated with CFD.26

Folate can also be transported across cellular membranes using the folate receptor-β,27 the reduced folate carrier (RFC)28 and the proton-coupled folate transporter.29 Although folate receptor-β seems important in the placental transport of folate,27 the proton-coupled folate transporter is critical in folate transport in the gastrointestinal tract especially as impairment of this transporter function is responsible for hereditary folate malabsorption.29 The RFC is a transmembrane protein that is expressed in a wide range of tissues, including the placenta, kidney, intestine and both the basolateral and apical surfaces of the choroid plexus.30 Unlike the folate transport involving FRα, the RFC allows bidirectional transport of folate across the cellular membrane.30 The RFC is responsible for folate transport across the blood–brain barrier when extracellular folate concentrations are high20 and has a higher affinity for reduced forms of folate such as 5MTHF and folinic acid (leucovorin) as compared with folic acid.28 As leucovorin can enter the CNS through the RFC, it can normalize CSF 5MTHF levels in individuals with CFD.6 In some cases, clinical response is dramatic, especially if treatment is started early in life.1, 31 In children with concomitant ASD and CFD, treatment with oral leucovorin (0.5 to 2mgkg−1 per day) has resulted in improvements ranging from partial improvements in communication, social interaction, attention and stereotypical behavior3, 5, 6, 7 to complete recovery of both neurological and ASD symptoms.
The aforementioned reports provide a compelling reason to study the FRA and CFD in more depth in children with ASD. However, a systematic study of FRA or CFD prevalence in children with ASD has not been performed. Unfortunately, the diagnosis of CFD requires a lumbar puncture (LP), a rather invasive procedure, to measure CSF 5MTHF levels. However, FRA concentrations can be measured using a blood test, thereby providing a minimally invasive method for identifying children who might be at increased risk for CFD. Starting in 2010, two of the authors (REF and DAR) offered FRA testing to patients evaluated in their clinics. Parents of children who were FRA positive were offered two treatment options for their children: either a diagnostic LP to measure the CSF 5MTHF level or an empirical treatment with oral leucovorin if they did not wish their child to undergo an invasive procedure. Response to oral leucovorin treatment in the child was rated by parents using a standardized scale. Surprisingly, FRA prevalence was very high in children with ASD, and the response to leucovorin in children was rated by parents as favorable on several cognitive dimensions.