WO2006119589A2 - Prevention and therapy of cerebral folate deficiency - Google Patents

Abstract

The present invention relates to methods and means to prevent CFD and/or to treat CFD at a very early stage, when CFD has not yet fully developed. It was found that circulating and blocking autoantibodies to FR represent one of the major causes of CFD and that prognosis improves the younger a child can be treated. The invention as such in particular relates to a method of screening infants and their mothers for the presence of circulating autoantibodies in their serum and/or for low 5MTHF CSF levels, followed by a prompt treatment of a subject in need thereof with a folate supplement in case the testing procedure is positive. Such screening should also be performed for all children or any other subjects as soon as at least 3 of the major criteria of CFD manifest. It was further found that the addition of antioxidants to a folate supplement maintains stability of 5MTHF and can help restore an impaired 5MTHF uptake in the nervous system due to the circulation of blocking autoantibodies. Avoidance of foods and products, containing proteins with similar amino acid sequences as compared to human FRs, is strongly preferred in the preparation of compounds or food products for the prevention and/or treatment of CFD. The methods and means of the invention have a major impact on the health of the population and can help to reduce the incidence of for instance autism and schizophrenia related to CFD.

Description

PREVENTION AND THERAPY OF CEREBRAL FOLATE DEFICIENCY

Field of the invention

[0001] The present invention is related to methods and means to prevent the occurrence of CFD during infancy, childhood, adolescence and adulthood; and provides guidelines for effective treatment after diagnosis of the disease and its causes.

Background

[0002] The physiological function of the various reduced folate forms that exist subserves the de novo synthesis of purines and thymidine, methylation of DNA, the conversion of homocysteine to methionine and the formation of the active methyl-group donor S- adenosylmethionine (SAM) , which is used for the transfer of methyl-groups in over 100 reactions.

[0003] Folates are required by all cell types and tissues including the brain. Normal brain development and function depends on the active transport of folates across choroid epithelial cells at the blood-CSF Barrier.

[0004] Folate transport across cell membranes is achieved by at least 3 different mechanisms: the folate receptor (FR) , the reduced folate carrier (RFC) , and an ATP-dependent folate exporter.

[0005] Yet, active folate transport across the blood- brain and blood-cerebrospinal fluid barriers is mediated primarily by membrane-associated FR. These FR have a high affinity constant (10⁹ to 10¹⁰ liters per mole) for several folate derivatives, including 5- methyltetrahydrofolate (5MTHF) and folic acid. [0006] Due to this active transport process CSF folate levels are at least 1.5 to 2 times higher than serum folate levels.

[0007] Cerebral folate deficiency (CFD) is defined by low cerebrospinal fluid (CSF) folates. The predominant and biologically active folate form 5MTHF is the single folate form known to pass the blood-CSF barriers and is significantly reduced in the CSF. Folate metabolism outside the nervous system is normal in CFD patients. [0008] CFD is a collective name for any neurological or neuropsychiatric syndrome associated with low CSF 5MTHF. The term "CFD" is meant to include any disease associated with CFD (also referred to as a CFD-related disease) and any secondary form of CFD. [0009] CFD can result either from a disturbed folate transport or from an increased folate turnover (utilization and/or breakdown) in the central nervous system (CNS) . [0010] CFD is a highly debilitating disease. The estimated incidence for CFD is 1:1000 to 1:1500. The real incidence is probably much higher.

[0011] CFD can affect people of all ages with a variable clinical phenotype including slowly progressive neurologic dysfunction affecting infants, children and even adults.

[0012] Infantile-onset CFD is a neurological syndrome that develops about 4-6 months after birth and can result in e.g. psychomotor retardation and autistic behavior. Neurodevelop-mental progress was mostly normal before the onset of CFD.

[0013] Secondary CFD is known to develop during chronic use of antifolate agents like methotrexate, anticonvulsant drugs and carbidopa, and in various known conditions such as Rett syndrome, Aicardi-Goutieres syndrome, 3-phosphoglycerate dehydrogenase deficiency, dihydropteridine reductase deficiency, aromatic amino acid decarboxylase deficiency, mitochondrial encephalopathies and Kearns-Sayre syndrome.

[0014] Some CFD patients have shown an improvement of their clinical symptoms after supplementation with folinic acid or L-5MTHF, but the causes of brain folate deficiencies remained unknown.

[0015] Baby formulas contain folic acid to ensure folate uptake via food. Supplements with folates have been prescribed to pregnant women, as a medical food for the dietary management of endothelial dysfunction, hyperhomocysteinemia, nutrient malabsorption or inadequate dietary intake by for instance Alzheimer patients .

[0016] The patents PCT/US97/01870 and US2001/6, 254, 904 disclose essential nutrient/vitamin supplements and food preparations for human consumption that comprise natural isomers of a reduced folate.

[0017] There is an ever increasing demand for improved methods and means for the treatment of CFD. Appropriate methods and means for the prevention of CFD have not been proposed yet.

[0018] There is a further need for an adequate and early diagnosis of CFD and of diseases associated with CFD, for an identification of its causes, and for an appropriate treatment following said diagnosis. Description of the figures

[0019] Figure 1 displays the age of onset of the main clinical features of Infantile-Onset CFD.

[0020] Figure 2 displays amounts of blocking auto- antibodies against the folate receptor (FR) present in the serum of children with CFD and age-matched control subjects. Mean ± SD of 28 patients was 0.87 ± 0.08 pmol of folate receptor blocked per ml of serum. No autoantibodies were present in the serum of controls. [0021] Figure 3 shows that reactive oxygen species

(ROS) exposure leads to significantly decreased 5MTHF uptake by KB cells ( human nasopharyngeal epidermoid carcinoma cells), which highly express the FRl. ROS: hatched bars. Control group: white bars. Results of a one-hour incubation at extracellular 5MTHF concentrations of 10 nM and 50 nM respectively. [0022] Figure 4 shows that preincubation with phosphatidyl-inositol-specific phospholipase C (PIPLC) or ROS decreases ¹⁴C-5MTHF uptake and membrane binding by KB cells. Coexposure with ROS and PIPLC leads to an additional decrease of ¹⁴C-5MTHF binding and uptake, indicating an FRl unrelated effect of ROS on 5MTHF uptake. Results of a one-hour incubation at a physiological 10 nM concentration. Open bars: controls and after PIPLC pretreatment ; Hatched bars: influence after ROS and after ROS + PIPLC.

[0023] Figure 5 shows 5MTHF uptake and binding after incubation with ROS and after combined incubation with ROS in the presence of vitamin C. [0024] Figure 6 shows 5MTHF stability after ROS exposure with more than 70 % loss of the 5MTHF concentration after 1 hour. Pretreatment with vitamin C could prevent 5MTHF catabolism by ROS. [0025] Figure 7 demonstrates the influence of a cows milk free diet (mostly soya products) on the levels of FR autoantibodies in 8 CFD patients, already treated with reduced folates and antioxidants. For each individual, the measured FR autoantibody levels in serum before and after at least four months on the cows milk free diet, have been represented by two points connected by a line. In 5 patients the autoantibodies became negative, while in the other 3 the FR autoantibodies decreased. The results are statistically significant.

[0026] Figure 8 represents the structural formula of folic acid. This is composed of an unreduced 2-amino-4- hydroxypteridine molecule linked through methylene (C9- position) to p-aminobenzoylmonoglutamate, while R represents the carried one-carbon group at different oxidation states in the form of e.g. methyl, methenyl or formyl, attached to the N-5 and/or N-IO positions as shown in the drawing. The metabolically active form tetrahydrofolate is reduced at the 5,6,7,8 positions of the pteridine group.

[0027] figure 9 compares the complete amino acid sequences for human FRl, bovine FR and chicken erythroied FR with 62% identical amino acids; for all three folate receptor forms the sequence homology is indicated by bold letters.

Summary of the invention

[0028] A first aspect of the invention concerns compositions or kits-in-parts comprising a pharmacologically effective amount of at least one folate, and possibly a pharmacologically effective amount of at least one antioxidant . [0029] The term " kit-in-parts" as used herein includes a medicament or other entity comprising at least one folate, and possibly at least one antioxidant, wherein the ingredients (the different folates and/or the different antioxidants) are not necessarily mixed from the start but may be contained in different compartments to be consumed preferably together, yet possibly consecutively. Before consumption the different ingredients may be mixed. The composition or "kit-in-parts" can be prepared for the administration through different routes, i.e. the oral, intravenous or intramuscular route or through any other route of administration which allows to obtain an optimal concentration of the pharmaceutical active compounds in blood, CSF and nervous tissues.

[0030] By a "pharmacologic effective amount" is meant an amount much greater than normal to overcome the disease in question, a folate deficiency, more in particular CFD. [0031] As used herein the term "folate" refers to folic acid and to derivatives thereof (Fig.8 and definitions below) . In compositions or kits-in-parts intended for the prevention and/or the treatment of CFD, reduced folates are preferred such as e.g. folinic acid. Most preferred, however, is 5-methyltetra-hydrofolate

(5MTHF) , the physiological and biologically active folate form, which passes the blood-brain barriers.

[0032] Examples of reduced folates include tetrahydrofolic acid, 5-formyltetrahydrofolic acid, 5- methyltetrahydrofolic acid, 10-formyltetrahydrofolic acid, 5, 10-methenyltetrahydrofolic acid, 5,10- methylenetetrahydrofolic acid, 5- formiminotetrahydrofolic acid, a Ca or a Na₂ salt of any of these, monoglutamyl-, and/or polyglutamyl derivatives of any of these. The synthetic and natural isomers and polyglutamyl forms thereof as described in patents PCT/US97/01870 and US2001/6, 254 , 904 (incorporated herein by reference) can be used, but also other synthetic forms may be used. The (reduced) folate may be present under the form of an L and/or a LD isomer. Natural isomers can be present in a chirally pure form, yet (some amounts) of unnatural isomers of reduced folates may also be present. See also http://www.merckeprova.ch

(incorporated by reference herein) and possible compounds listed there. The compound can be present as the Monoglutamate form; Polyglutamyl derivatives include di-, tri-, tetra-, penta- and hexaglutamyl derivatives, and compounds with more than six polyglutamyl- derivatives .

[0033] The present invention relates amongst others to a composition or kit-in-parts comprising a pharmacologically effective amount of at least one (reduced) folate and a pharmacologically effective amount of at least one antioxidant.

[0034] Advantageously, antioxidants are present herein in an amount of about at least 0.1 RDA, preferably of about at least 0.5 RDA, more preferably of about at least 1, 2, 2.5, 3,5 to up to 4 times the RDA (Recommended Dietary Allowance) , or possibly higher. [0035] The antioxidant may be any antioxidant that is suited for use in a mammal, in particular a human. Examples hereof include vitamin C, vitamin E, β carotene, Ubichinone 10 (QlO) and/or vitamin A. Vitamin C and/or vitamin E are preferred. Preferably both vitamin E (e.g. α-RRR-tocopherol) and vitamin C are present. In addition, known substances which directly or indirectly augment antioxidant defenses in a human can be utilized and regarded as antioxidants.

[0036] Below examples are given of when to use low and when to use high amounts of antioxidants. [0037] Antioxidant amounts >0.1 times the RDA up to about 1 time the RDA of the antioxidant in question are considered "low amounts" or "low doses". Amounts >1 up to 4 times the RDA are considered "high amounts" or "high doses" . [0038] By "low doses of vitamin C" is meant an amount from about 0.1 times the RDA to about 1 time the RDA. By "high doses of vitamin C" is meant a amount from about 1 to about 3.5 times the RDA of vitamin C (equivalent to about 50-200 mg/day for adults) . In the case of vitamin C, daily doses up to 20 mg/kg are tolerated by infants (0-1 year old) . Adults (>18 year old) would tolerate 100-400 mg/day.

[0039] By "low doses of vitamin E" is meant an amount from about 0.5 to about 5 mg/kg/day of α-RRR-tocopherol, or an amount equivalent thereto. By "high doses of vitamin E" is meant an amount from about 10 to about 125mg/kg/day of α-RRR-tocopherol, in particular from about 25 to 50 mg/kg/day of α-RRR-tocopherol, or an amount equivalent thereto (in the case that a vitamin E compound with antioxidant properties other than α-RRR- tocopherol would be used) . In some cases higher amounts may be tolerated. Vitamin E is not toxic at higher doses, yet in children at the age below 1 year, doses not higher than 2-5 mg/kg/day are recommended. [0040] Optimal amounts of antioxidants may vary from one individual to another and will depend on amongst others body weight, age, health, nutritional status, sex and the like. [0041] Preferably the composition or kit-in-parts according to the invention comprises at least 2 times the RDA, preferably at least 2.5 times, at least 3 times the RDA of a (reduced) folate. For children, doses corresponding to at least 2.5 to at least 3 times the RDA may be recommended. Of Leucovorin® (folinic acid; DL-5-Formyl-tetrahydrofolate) maximal doses up to 4 mg/kg/day or higher can be given to children. [0042] Advantageously, the total amount of the (reduced) folates in said composition is above 200%, more in particular above 210% of a human daily- requirement for folates per customarily consumed quantity of said composition [0043] The invention further relates to a (reduced) folate supplement, an "essential nutrient preparation", an "essential vitamin preparation", or any type of food preparation in general that comprises a composition or kit-in-parts as described above. [0044] An "essential nutrient preparation" is a material which contains one or more essential nutrients. As used herein, "essential nutrients" are those nutrients, which are required to sustain health but which can not be effectively produced by a human. An "essential vitamin" is a vitamin required to sustain health but which can not be effectively produced by a human .

[0045] These "essential nutrient preparations" or "essential vitamin preparations" in the context of the present invention are also referred to as a " (reduced) folate" supplement. By a "supplement" is meant products in e.g. capsule, tablet, powder or liquid form that provide essential nutrients, such as a vitamin, an essential mineral, a protein, an herb, or similar nutritional substance.

[0046] Food preparations are materials which contain one or more amino acids, peptides, proteins, carbohydrates, or fats, which are suitable for enteral consumption by a human. "Enteral consumption" includes oral, intragastric or transpyloric consumption or administration . [0047] The "food preparation" may be selected from the group consisting of a formula, a beverage or any type of food product for babies or infants, children, adolescents or adults. It may for instance be a milk formula for babies or infants, or may be maternal milk that is supplemented with a composition or a kit-in- parts according to the invention.

The nutrient preparation, essential vitamin preparation and/or food preparation advantageously is for human consumption . [0048] A composition or kit-in-parts of the invention (any of those mentioned thus far) may be used as fortification or as supplement in maternal milk or in any formula, beverage or food product for infants, children, adolescents or adults. [0049] The present invention in particular relates to a composition or kit-in-parts comprising at least one reduced folate (preferably 5MTHF) , wherein the total amount of the (reduced) folates in said composition is above 200%, more in particular above 210% of a human daily requirement for folates per customarily consumed quantity of said composition. Advantageously said composition is for human consumption. Even more preferred are compositions for human consumption wherein the total amount of (reduced) folates is above 250%, 300%, 350%, 400%, 450%, 500%, or even higher of a human daily requirement for folates per customarily consumed quantity of said composition. The amount preferably does not exceed the tolerable level of reduced folates . Preferably the total amount of (reduced) folates in said composition for human consumption is below 2000%, preferably below 1500%, below 1000%, below 900% of a human daily requirement for folates per customarily consumed quantity. [0050] Advantageously said composition or kit-in- parts for human consumption further comprises at least one antioxidant. Preferred antioxidants and preferred amounts are as indicated above. [0051] The composition, kit-in-parts or folate supplement for human consumption can be (integrated in) an essential nutrient preparation, an essential vitamin preparation, or any type of food preparation in general, such as a formula, a beverage or a food product for babies or infants, children, adolescents or adults. Preferred for infants is a milk formula for babies or infants or a supplemented maternal milk.

[0052] The present invention further relates to a composition or kit-in-parts according to the invention (any of those mentioned thus far) for use as a medicament.

[0053] The present invention also relates to the use of a composition or kit-in-parts of the invention (any of those mentioned thus far) for the preparation of a medicament to prevent and/or treat a folate deficiency, in particular CFD, in a subject.

[0054] By "subject" is meant any mammalian individual in general, but more in particular a human. It may be an individual suffering from a folate deficiency, such as CFD, and/or a disease associated therewith (a CFD patient); or it may be a control individual. A "control" may be a healthy individual (not suffering from any disease) , or it may be an individual not suffering from a folate deficiency, such as CFD.

[0055] A preferred subject to be treated is a child younger than 6, 5, 4, 3, 2 years old. Most preferably it is an infant (0-1 year) . [0056] By "prevention" or "preventing" is meant that the risk of developing a disease such as CFD can be determined or predicted in sufficient time so as to keep that disease, such as CFD or a disease associated therewith, as much as possible from occurring or from manifesting irreversible symptoms. [0057] Advantageously the subject to treat is at risk of developing CFD or a disease associated therewith due to blocking and circulating antibodies. By "at risk" is meant having a high chance to develop CFD or disease associated therewith (also referred to as a CFD-related disease) in the absence of appropriate measures. An early and appropriate treatment may prevent CFD from occurring in such subjects and/or may significantly reduce its impact if the disease would develop. [0058] The term "antibody" refers to an immunoglobulin of any class or subclass, a portion thereof or an active fragment thereof, wherein an active fragment of an antibody retains its specific binding capability. As used herein, an "autoantibody" refers to an antibody, e. g. an IgG antibody, in a subject that is directed against components of the subject's own body. An "autoimmune disorder" refers to a disorder or condition that a subject's immune system mistakenly attacks and leads to the destruction of the subject's own body cells and/or tissues.

[0059] An "autoantibody to the (cell membrane) folate receptors (FRs)" refers to any autoantibody that is directed against any isoform or peptide sequence of the FRs, including the isoforms of the FRs. A "blocking autoantibody" in the context of the invention refers to an autoantibody that binds with a cell membrane FR and thereby blocks folate binding to the cell membrane FRs and the subsequent folate uptake by the cells.

[0060] By "diagnosing", "diagnosis", "detecting" or "screening" is meant an act or process of identifying and/or determining the presence, nature and cause of CFD, through evaluation of patient history, examination, analytical procedures (serum and CSF analysis e.g.) etc. A patient may be diagnosed to have CFD on the basis of manifesting clinical symptoms (via classical exam) , the finding of low CSF 5MTHF levels compared to age-matched control subjects and/or the finding of blocking AuAb against a cell membrane FR in a subjects biological sample .

[0061] By "biological sample" is meant a clinical sample for testing, taken from any tissue or compartment of a mammal. Preferably the sample is a body fluid from a mammal, more preferably it is (human) serum and/or (human) cerebrospinal fluid.

[0062] "Treating" or "treatment" as used herein means to ameliorate, suppress, mitigate or eliminate the clinical symptoms after the onset (i.e. clinical manifestation) of a disease state. An effective or successful treatment provides a clinically observable improvement . [0063] The person, patient or individual suffering from CFD or from a disease associated therewith is a patient in which CSF 5MTHF levels are low. Such person may in addition possess blocking autoantibodies (AuAb) to cell membrane folate receptors (FR) .

[0064] Advantageously, the subject treated may be an individual with circulating (blocking) AuAb to FR, in which no clinical symptoms of CFD have manifested yet, or in which the clinical picture of CFD is not yet complete (for instance in which less than three of the major symptoms of CFD have manifested) . Such person is at risk of developing CFD due to blocking AuAb against FR. [0065] Alternatively, the subject treated may be a subject found to have CFD, in which typical clinical symptoms (preferably more than 3 of the major criteria) are manifesting or have manifested.

[0066] Many CFD patients suffer from an eating and/or from a nutritional disorder. A subpopulation of CFD patients suffers from an oxidative and/or nitrosative stress. For these subpopulations adequate treatments are provided infra.

[0067] The invention also concerns the use of a pharmacologically effective amount of reduced folates, or of a composition or kit-in-parts comprising a pharmacologically effective amount of at least one reduced folate, for the preparation of a medicament to prevent and/or treat CFD. [0068] In particular the invention concerns a method to prevent CFD or a CFD-related disease thereby. As indicated above, advantageously, the subject treated is an individual with circulating (blocking) AuAb to FR in which no clinical symptoms of CFD have manifested yet, or in which the clinical picture of CFD is not yet complete (for instance in which less than three of the major symptoms of CFD have manifested) .

[0069] The invention also concerns the use of a pharmacologically effective amount of reduced folates, or of a composition or kit-in-parts comprising a pharmacologically effective amount of at least one reduced folate, for the preparation of a medicament to treat CFD. [0070] Once again, 5MTHF and/or folinic acid are the preferred compounds. Preferred amounts of reduced folates have been given above.

[0071] Examples of diseases associated with CFD include autism spectrum disorders (e.g. Rett syndrome, Kanner autism or early infantile autism, Asperger syndrome, Heller syndrome or dementia infantilis, atypical autism or PDD-NOS, so-called double syndromes associated with autism, autistiform syndromes with neurological deficits etc) , depression and postnatal depression, schizophrenia, the Aicardi-Goutieres syndrome and the Aicardi-Goutieres variant, Attention Deficit Disorders and Hyperkinetic Disorders, neurological disorders in association with a disturbed blood platelets adhesion, and/or cases of acute and chronic inflammatory diseases of the central nervous system with secondary CFD as a result. Known conditions where in a number of cases secondary CFD is present, are mitochondrial diseases, Kearns-Sayre syndrome, Friedreich's ataxia, developmental disorders with intractable epilepsy and dementias.

[0072] Another aspect of the invention concerns a method for the early diagnosis of CFD, comprising the step of assaying a biological sample of a subject for the presence of (blocking) autoantibodies (AuAb) against a cell membrane folate receptor.

[0073] Blocking AuAb have been linked to folate deficiencies such as neural tube defects but have not been linked hitherto to CFD.

[0074] It may be derived from the presence and/or the amount of these (blocking) AuAb to FR that the subject is at risk to CFD (for instance in the case of a predisposition in the family, is developing CFD (for instance when the clinical picture of CFD is not yet complete), and/or that the subject is suffering from CFD, id est has CFD. AuAb levels are hereby compared to levels found in age-matched controls. [0075] The method for the early diagnosis of CFD according to the invention may further comprise the step of measuring CSF 5MTHF levels in the subject to confirm a CFD diagnosis on the basis of blocking autoantibodies

(AuAb) . Advantageously this method is an in vitro method. A confirmation of CFD may, however, also include a physical exam of a patient for CFD symptoms.

[0076] The method for the early diagnosis of CFD according to the invention may further comprise the step of assaying (in vitro) a subject's sample for indications of an oxidative and/or nitrosative stress. Aim thereof is to further identify the origin or cause for CFD before starting a (reduced) folate therapy (preferably with 5MTHF) . Another aim is to include correction of the causes for CFD, related to oxidative and/or nitrosative stress during therapy of CFD, for example supplementation with the lowered antioxidant enzyme cofactors selenium, zinc, copper or other factors . [0077] Another aspect of the invention concerns a test kit or assay for detecting (blocking) autoantibodies (AuAb) to FRs in a biological sample from a subject, comprising purified FRs from a human or homologous species, reagents for treating said biological sample, labeled folic acid or a labeled folate, at least one indicator which detects a complex of said purified FRs and said AuAb, and further means to identify an oxidative and/or nitrosative stress in the subject. Advantageously the test kit or assay can also determine the titer of said (blocking) AuAb and/or the apparent association constant (Ka) of said blocking AuAb to said FRs . The kit may be a kit-in-parts . [0078] Another aspect of the invention concerns a method for the prevention and/or the early treatment of a folate deficiency such as CFD via screening. [0079] It is recommended in the framework of CFD prevention to screen all infants or young children (preferably before reaching the age of 3 year) and/or the infants' mother for the presence of (blocking) AuAb in e.g. their serum and/or for a low 5MTHF CSF level. [0080] Accordingly, a further aspect of the invention concerns a method for the prevention and/or the early treatment of CFD, said method comprising the steps of — Testing a biological sample of an infant or a child for the presence of blocking AuAb to the cell membrane folate receptor; and/or

- Measuring the 5MTHF level in a CSF sample from said infant or said child; and — Starting a treatment of the infant or the child with a pharmacologically effective amount of folates promptly upon the detection of AuAb (or abnormal levels thereof) and/or promptly upon the measuring of low CSF 5MTHF levels in the infant or the child compared to age- matched control subjects.

[0081] The invention in addition relates to a method for the prevention and/or the early treatment of CFD, said method comprising the steps of

- Testing a biological sample of the mother of an infant on the presence of blocking AuAb to the cell membrane folate receptor; - Optionally, measuring the 5MTHF level in a CSF sample from the infant; and

- Starting a treatment of the infant with a pharmacologically effective amount of folates promptly upon the detection of AuAb (or abnormal levels thereof) in the mother and/or promptly upon the measuring of low CSF 5MTHF levels in the infant compared to age-matched control subjects.

[0082] The biological sample tested advantageously is cord blood or serum. [0083] Advantageously, an infant or a child is tested for the presence of (blocking) AuAb and/or for a low CSF 5MTHF level in the period between about 0 and about 3 year, more preferably in the period between about 0 and 1 year. The infant can be assayed at birth, but more preferably is tested or screened at the age of about 4 to about 6 months, the testing procedure possibly being repeated at least once, for instance at the age of about 1 year, 1.5, 2, 2.5 or 3 year, preferably at the age of about 1 year. When testing the mother of an infant this may be done before birth, at birth and/or shortly after birth of the infant. [0084] The early treatment of infants advantageously prevents CFD and/or prevents irreversibility of CFD symptoms and/or significantly improves CFD prognosis.

[0085] It is further recommended to screen each and every subject at the manifestation of one or more major criteria of CFD, preferably promptly after the onset or manifestation of at least three or more of the major criteria (see Fig. 1, and see below).

[0086] Accordingly, the invention further relates to a method for the early treatment of CFD, said method comprising the steps of, as soon as CFD symptoms manifest or break out:

— Testing a biological sample of a subject for the presence of blocking AuAb to a cell membrane receptor and/or

— Measuring the CSF 5MTHF level in said subject, and

— Starting a treatment of the subject with a pharmacologically effective amount of folates promptly upon the detection of AuAb and/or promptly upon the measuring of low CSF 5MTHF levels in the subject compared to age-matched control subjects.

[0087] Such testing is highly recommended for brothers or sisters of and children from a subject known to have CFD. Such testing further is recommended whenever the presence of CFD is suspected and/or when at least three of the following symptoms manifest in a subject: 1. Unrest, irritability, insomnia; 2. Decelerating head growth; 3. Neurodevelopmental delay, standstill, regression; 4. Hypotonia and ataxia; 5. Ascending pyramidal tract signs in legs; 6. Dyskinesias (choreoathetosis, ballismus); and 7. Epilepsy or seizures. These symptoms indicate or point to the possible presence of CFD.

[0088] The method for the prevention and/or the early treatment of CFD according to the invention may further comprise the step of assaying a subject's sample or a subject for a possible oxidative and/or nitrosative stress .

[0089] The following disorders or deficiencies may indicate or point to an oxidative and/or nitrosative stress: a mitochondrial disease such as a Kearns Sayre syndrome, Complex I-V deficiencies or mitochondrial depletion syndromes; a Glutathione Peroxidase deficiency associated with selenium deficiency; an extracellular and intracellular Superoxid Dismutase deficiency secondary to an intracellular and/or extracellular manganese, copper or zinc deficiency; Ubichinone-10 (QlO) deficiency states; or conditions associated with vitamin C and vitamin E deficiencies.

[0090] In a method for prevention and/or early treatment according to the invention, a subject in need thereof (e.g. one diagnosed to have CFD or at risk of developing CFD) is treated with a pharmacologically effective amount of (reduced) folates.

[0091] Advantageously the subject in need thereof is treated with any of the compositions or kits-in-parts according to the invention (any of those that have been mentioned thus far) .

[0092] Chances of success were found to be highest when the subject can be treated before reaching the age of 6, 5, 4 years, more preferably before reaching the age of 3, 2 years, most preferably before reaching the age of 1 year, for instance at the age of about 4 to 6 months . [0093] "CFD" as herein used also refers to a disease associated with CFD and/or a secondary form of CFD (see infra for an overview) . The CFD form may be caused by- circulating autoantibodies against a cell membrane folate receptor and/or may be caused by something else. [0094] The subject treated may have and/or may be at risk of developing autism or an autism spectrum disorder, depression, schizophrenia, the Aicardi- Goutieres syndrome and the Aicardi-Goutieres variant, Attention Deficit Disorders and Hyperkinetic Disorders in the absence of a proper and sufficiently early treatment. Also the earlier mentioned conditions can be considered. [0095] Yet another aspect of the invention relates to a composition to which a mammal (in casu a human) is exposed that in normal circumstances contains soluble or membrane-derived FR receptors, but from which these soluble or membrane-derived FR receptors have now been removed. Soluble or membrane-derived FR are amongst other present in human and cow milk.

[0096] Provided in the present invention is a milk formula for babies or a milk-based product for babies, infants, children, adolescents or adults that is deprived from soluble FR receptors or significantly reduced in these inducing soluble FR receptors. Id est, products are proposed in which these soluble FR receptors are no longer present, or are no longer present in an amount or form that can lead to the induction of AuAb to cell membrane FRs. [0097] The invention with respect to the prevention and/or treatment of CFD further relates to the use of special baby formulas or food products with elimination of soluble or membrane-derived FRs, as contained in cows milk, or the use of alternative products such as hydrolyzed milks, amino acid formulas or soya products. Subjects at risk of developing CFD or having CFD may be put on a diet with these products. [0098] The invention with respect to the prevention and/or treatment of CFD further relates to baby formulas or food products, characterized by the elimination of FRs or by the absence of FRs (i.e. soya or hydrolysed cow milk formulas) , which posses similarity to the human FR. A composition or kits-in-parts of at least one reduced folate and/or at least one antioxidant may be processed or added to these special formulas or products .

Detailed description of the invention

[0099] Folates are a family of structurally related compounds {Rosenblatt D, 1995. Inherited disorders of folate transport and metabolism. In: Scrlver CR, Beaudet AL, Sly WS, Valle D (eds) . The Metabolic and Molecular Basis of Inherited Disease . Sixth ed. New York: McGraw-Hill: pp 3111-3128) . The basic structure consists of a 2-amino-4-hydroxypteridine molecule linked through a methylene carbon to para- aminobenzoylmono- or polyglutamate (figure 8). [0100] Only the reduced folate forms can function as the active cofactors in cellular metabolism. These carry one-carbon units at different oxidation levels in the form of e.g. methyl, methenyl or formyl groups, attached to the N-5 and/or N-IO position of tetrahydrofolate, which can enzymatically be interconverted .

[0101] The disabilities of all those behavioral disorders and conditions associated with and linked to CFD (see infra) can be prevented, or at least be significantly improved, when a treatment with folates, in particular reduced folates can be started sufficiently early, as early as possible. [0102] The shorter the period between the onset or manifestation of CFD symptoms and start of therapy, the better its prognosis will be. Preferably therapy is started before the onset of clinical symptoms with the aim of preventing CFD. [0103] Neonatal, infantile-onset and late-onset CFD are major causes of psychomotor retardation, autism, epilepsy, movement disorders and neuropsychiatric conditions, which are irreversible and only respond partially in the event that these disorders are diagnosed after signs and symptoms have become manifest. [0104] A complete recovery and also prevention of CFD are possible upon an early and proper diagnosis, followed by an effective treatment with (reduced) folates promptly after said diagnosis. [0105] With "immediately after" or "promptly after" is meant that therapy is started as soon as possible, preferably from the moment that AuAb to FR and/or a low level of CSF 5MTHF are detected. Preferably treatment is started before the manifestation or onset of symptoms . If not possible before the onset of symptoms, therapy should be started promptly after the first CFD symptoms manifest to avoid irreversibility of the condition and only partial responses. [0106] After clear manifestation of CFD symptoms, a therapy with (reduced) folates can still be effective but may only partially or incompletely cure mental deficits, autistic signs and dyskinesias (Example 4) . [0107] The presence (e.g. in serum) of blocking AuAb against FR at the blood-brain barrier, which impairs 5MTHF transport to the nervous system, appears to be the major cause of CFD. [0108] Up to 90% of CFD patients tested positive for these AuAb. These AuAb are further believed to be present in a biological sample e.g. serum, before the real development or onset of CFD clinical symptoms . [0109] As such, a screening for the presence of these AuAb provides a useful tool for the early detection of CFD.

[0110] The testing of FR AuAb provides a good alternative to a lumbar puncture for the determination of 5MTHF levels in CFS, up to now the gold standard for the diagnosis of CFD. Testing of FR AuAb in e.g. serum is a simple non-invasive method (compared to a lumbar puncture). Such testing can be performed for subjects or patients of all ages, including infants and elderly people . [0111] Most CFD patients suffer from eating and/or nutritional disorders and as such benefit from a supplement of a low amount of antioxidants in addition to a pharmacologically effective amount of (reduced) folates . [0112] Administration of low amounts of antioxidants

(see infra), in addition to (reduced) folates, advantageously prevent that an oxidative stress and/or nitrosative stress will evolve or manifest. See supra for "low" and "high" amounts of an antioxidant. [0113] Patients with an oxidative stress and/or a nitrosative stress did not respond to standard treatments with a pharmacologically effective amount of

(reduced) folates, unless in addition to the folates a high amount of antioxidants was administered (see infra) .

[0114] The presence of elevated reactive oxygen radicals or other radical molecules appears to further prevent 5MTHF transfer to the nervous system, possibly due to damage of the blood-brain barrier membrane structures (Example 7) . Within or outside the nervous system, oxidative stress and/or nitrosative stress may also lead to increased catabolism of folates. In the presence of reactive oxygen species 5-MTHF appeared to be very unstable (fig.6). Vitamin C was able to protect against ROS, maintained 5MTHF stability and normalized 5MTHF binding plus uptake into KB cells (fig. 5,6) [0115] In other words it is advisable to exclude in CFD patients or in patients suspected to develop CFD (for instance those which have AuAb to FR and/or a low 5MTHF CSF level but do not show any symptoms yet) , with the most modern techniques in the art, the existence of an oxidative/nitrosative stress. If such stress is present, the treatment has to be adopted accordingly.

[0116] Conditions associated with oxidative and/or nitrosative stress include (1) an increased production of reactive oxygen species (ROS) : e.g., in the case of mitochondrial disorders or inflammatory processes; (2) a failure of antioxidant enzymes and radical scavenging deficiencies: e.g. a Glutathione Peroxidase deficiency associated with selenium deficiency; an extracellular and intracellular Superoxid Dismutase deficiency secondary to deficiencies of their cofactors manganese, copper and zinc; Ubichinone-10 (QlO) deficiency states; or all conditions associated with vitamin C and E deficiencies . [0117] It has been found that young children (below the age of β years) and older CFD patients with early diagnosis , can recover completely from CFD and lead a normal life. Beyond the age of 5 to 6 years, partial recovery with poorer response after treatment in infantile-onset CFD was found.

[0118] A first aspect of the invention therefore concerns a method for the early diagnosis of CFD and the identification of its causes, comprising the step of assaying a biological sample of a subject for the presence of (blocking) autoantibodies against a cell membrane folate receptor. Methods for the detection of anti-folate receptor AuAb in a biological sample are described in WO 2004/043233 A2 (which is incorporated by reference herein) and are given below (Example 1) .

[0119] One may further also screen for the presence of an oxidative and/or a nitrosative stress to improve the insight in the origin and cause of CFD. The frequently encountered antioxidant enzyme cofactor deficiencies for selenium, manganese, copper and zinc should be measured.

[0120] Children may be screened at birth or shortly after birth, but the optimal time for screening would be at the biological age between 4 and 6 months (range from birth to one year) , when the autoantibodies are believed to develop.

[0121] It is further recommended to screen before birth, at birth, or shortly after birth, the mother for the presence of the AuAb, certainly if she is breast- feeding, and especially in the case of a predisposition in the family.

[0122] It is further recommended to screen all other individuals who manifest one or preferably at least three of the following neurological signs before or after the age of 4-6 months:

1. Insomnia, agitation, crying, colics; 2. Deceleration of head growth from the age of 4-6 months; 3. Psychomotor retardation, sometimes with developmental standstill and regression; 4. Hypotonia and ataxia; 5. Ascending spasticity with pyramidal deficits starting in the lower limbs; 6. Dyskinesias (choreoathetosis, ballistic or other involuntary movement disorders); 7. Convulsions and epilepsy.

Screening is also recommended in autism spectrum disorders or suggestive features for autism with neurological deficits or in any behavioural and neuropsychiatric condition compatible with CFD or reported in association with CFD.

[0123] The screening for AuAb to FR may be combined with the analysis of 5MTHF CSF levels. 5MTHF CSF measurements are recommended in the case of a negative test for AuAb, especially when one or more of the symptoms listed in the previous paragraph manifest.

[0124] The selected group of babies, children, patients who possess AuAb against the FR and/or in whom low 5MTHF levels are present in CSF, should be supplemented with high amounts of (reduced) folates. [0125] Reduced folates (e.g. 5-formyltetrahydrofolate or folinic acid) and in particular 5MTHF (5-methyltetra- hydrofolate) are preferred to folic acid.

[0126] A child was found to recover from CFD upon a supplementation with folic acid. However, in a few cases supplementation with folic acid has led to the further depletion of the metabolically active pool of reduced folates in the central nervous system. This may have been due to the fact that the activity of the dihydrofolate reductase in the brain is (very) low and/or because the FR have a higher affinity for folic acid than for the reduced folate 5MTHF.

[0127] Accordingly, a composition or kit-in-parts of the invention may comprise folic acid in addition to reduced folates (at least one reduced folate) , yet the amount of reduced folates should be much higher than the amount of folic acid to avoid the problems indicated in the previous paragraph. [0128] As mentioned before a subpopulation of CFD patients benefits from the addition of a low or a high amount of antioxidants such as vitamin C and/or vitamin E. [0129] Especially a combination of the water-soluble vitamin C and the fat-soluble vitamin E proved effective. Vitamin C may be added under the form of ascorbic acid. Vitamin E may be added under the form of α-RRR-tocopherol or any equivalent Vitamin E form with antioxidative properties. [0130] CFD patients with an oxidative and/or nitrosative stress require the addition of a high amount (see supra) of antioxidants like vitamin C and E. These high doses are in that case needed to restore an impaired 5MTHF uptake and transport across the blood-CSF barrier due to the presence of reactive oxidative species (ROS) . Moreover, the antioxidants protect the reduced folate pool against degradation by ROS. [0131] High amounts of antioxidants help to maintain 5MTHF stability and improve the passage of 5MTHF across a barrier, for instance the blood-CSF barrier, when said barrier and 5MTHF transport across said barrier via a cell membrane FR are affected by (reactive) oxygen and/or nitrogen species. [0132] Another aspect of the invention therefore concerns the use of antioxidants in a composition or kit-in-part comprising at least one folate, to help restore transport of the folate across a barrier. These barriers comprise intestinal, placental and blood-brain or blood-CSF barriers. In addition high amounts of antioxidants maintain stability of 5MTHF ( reduced folate pool) and can ameliorate 5MTHF binding to and transport across cell membranes to the cell interior and into cell organelles.

[0133] Any type of administration known in the art is possible, but an oral or enteral form of administration is preferred. Tablets, powders, pastes, liquid formulations are preferred yet any other suitable type of formulation known in the art may be envisaged.

[0134] The folate compositions or kit-in-parts according to the invention can advantageously be combined with maternal milk, baby formulas, beverages for babies or children, or any other type of food product, nutrient or vitamin supplement.

[0135] Compositions or kit-in-parts comprising (L-) 5- methyltetrahydrofolate (5MTHF) are preferred for the following reasons: 5MTHF is the naturally circulating and metabolically active folate compound in humans and was demonstrated to have high bio-availability. Administration of 5MTHF may circumvent many genetic polymorphisms or deficiencies of enzymes, which are necessary to convert other pharmacologically available folate compounds (folic acid, 5-formyltetrahydrofolate) to the metabolically active 5MTHF form. Such deficiencies or polymorphisms may include methylenetetrahydrofolate reductase deficiency and polymorphisms, dihydrofolate reductase deficiencies etc. 5MTHF is further the only folate form known to cross the blood-brain and blood-CSF barriers and 5MTHF toxicity studies have been found to have no adverse effects. [0136] Hitherto, it was believed that for 5MTHF the same dosage would be needed as for the other (reduced) folates like folinic acid. Yet much lower concentrations, id est 1:6 to up to 1:10 times the amount needed of folinic acid suffice. [0137] The following doses are in particular recommended for an infant in the period between 0-1 year, for children at the age of 1-10 years, and males and females aged between 11-18 years:

[0138] For DL-5-methyltetrahydrofolate : Between about 30 and about 400 μg/kg/day, preferably between about 50 and about 170 μg/kg/day, more preferably between about 66 and about 133 μg/kg/day.

[0139] For L-5-methyltetrahydrofolate : Between about 15 and about 200 μg/kg/day, preferably between about 25 and 85 μg/kg/day, more preferably between about 33 and about 66 μg/kg/day.

[0140] For DL-calciumfolinate : Between about 0.2 and about 3.0 mg/kg/day, preferably between about 0.4 and about 1.2 mg/kg/day, more preferably between about 0.5 and about 1.0 mg/kg/day. [0141] For L-calciumfolinate: Between about 0.1 and about 1.5 mg/kg/day, preferably between about 0.2 and about 0.65 mg/kg/day, more preferably between about 0.25 and about 0.5 mg/kg/day. For each compound even higher doses may be required. [0142] For male and female adults above 18 years the following doses are recommended:

[0143] For DL-5-methyltetrahydrofolate: Between about 2 and about 10 mg/day, preferably between about 3 and about 9 mg/day, more preferably between about 4 and about 8 mg/day.

[0144] For L-5-methyltetrahydrofolate : Between 1 and about 5 mg/day, preferably between about 1.5 and 4.5 mg/day, more preferably between about 2 and 4 mg/day.

[0145] For DL-calciumfolinate: Between about 10 and about 80 mg/day, preferably between about 20 and about 70 mg/day, more preferably between about 30 and about 60 mg/day. [0146] For L-calciumfolinate : Between about 5 and about 40 mg/day, preferably between about 10 and about 35 mg/day, more preferably between about 15 and about 30 mg/day. [0147] The above doses are recommended for patients with autoantibodies to FR only and/or with low 5MTHF CSF levels, which manifest CFD symptoms irrespective of the presence or not of an oxidative/nitrosative stress. Yet, in some cases higher doses will be necessary to normalize CSF 5MTHF levels. Maximum amounts of a (reduced) folate that are tolerated vary from patient to patient and depend on body weight, age, sex, health, the amount of circulating AuAb in the serum, and the like. It is advisable to regularly monitor a patient and check if CSF 5MTHF has normalized, and if not (levels too high or too low) to adapt the therapy.

[0148] Below some examples are given of combinations that proved very efficient.

[0149] For an infant in the period 0.0-1.0 year in which FR AuAb have been detected: a daily supplementation with a combination of 33-66 μg/kg L-5- methyltetrahydrofolate, 2 mg/kg vitamin C and 0.4 mg/kg α-RRR-tocopherol . Instead of L-5-methyltetrahydrofolate, 0.5-1.0 mg/kg/day DL-calciumfolinate or 0.25-0.5 mg/kg/day L-calciumfolinate may be used.

[0150] The same would be recommended for children of 1-10 years old with CFD in which FR autoantibodies in serum and/or in which a lowered 5MTHF in cerebrospinal fluid have been detected.

[0151] For an infant in the period 0.0-1.0 year in which FR autoantibodies and oxidative stress have been detected: a daily supplementation with a combination of 33-66 μg/kg L-5-methyltetrahydrofolate, 10 mg/kg vitamin C and 5 mg/kg α-RRR-tocopherol. Instead of L-5- methyltetrahydrofolate, 0.5-1.0 mg/kg/day DL- calciumfolinate or 0.25-0.5 mg/kg/day L-calciumfolinate may be used. [0152] The same would be recommended for children of 1-10 years old with CFD in which FR autoantibodies in serum and/or a lowered 5MTHF in cerebrospinal fluid have been detected as well as an oxidative/nitrosative stress. The α-RRR-tocopherol dose for this age group with oxidative/nitrosative stress can be increased to 25 mg/kg/day.

[0153] For males and females aged between 11-18 years with CFD in which FR autoantibodies in serum and/or in which a lowered 5MTHF in cerebrospinal fluid have been detected: a daily supplementation with a combination of 33-66 μg/kg L-5-methyltetrahydrofolate, 2 mg/kg vitamin C and 4-5 mg/kg α-RRR-tocopherol. Instead of L-5- methyltetrahydrofolate, 0.5-1.0 mg/kg/day DL- calciumfolinate or 0.25-0.5 mg/kg/day L-calciumfolinate may be used.

[0154] For males and females aged between 11-18 years with CFD in which FR autoantibodies in their serum and/or in which a lowered 5MTHF in cerebrospinal fluid have been detected, as well as an oxidative/nitrosative stress: a daily supplementation with a combination of 33-66 μg/kg L-5-methyltetrahydrofolate, 200 mg/day vitamin C, and 25 mg/kg α-RRR-tocopherol . Instead of L- 5-methyltetrahydrofolate, 0.5-1.0 mg/kg/day DL- calciumfolinate or 0.25-0.5 mg/kg/day L-calciumfolinate may be used.

[0155] For males and females aged above 18 years with CFD in which FR autoantibodies in serum and/or in which a lowered 5MTHF in cerebrospinal fluid have been detected: a daily supplementation with a combination of 2-4 mg/day L-5-methyltetrahydrofolate, 200 mg/day vitamin C and 4-5 mg/kg α-RRR-tocopherol. Instead of L- 5-methyltetrahydrofolate, 30-60 mg/day DL- calciumfolinate or 15-30 mg/day L-calciumfolinate may be used.

[0156] The aim of these high-amount supplements of reduced folates, preferably 5MTHF, during the first year of life is to deliver from the time of detection of FR AuAb (and/or as soon as low CSF 5MTHF is detected) , higher amounts of 5MTHF to babies, so that their plasma L-5-methyltetrahydrofolate levels will be increased. [0157] This extra supply of reduced folates (e.g. 5MTHF) alone, or in combination with antioxidants, will overcome the occurrence of low levels of 5MTHF in CSF attributed to impaired folate transport across the blood-brain and blood-CSF barriers, or attributed to other causes. [0158] This fortification by reduced folates (e.g. 5MTHF) , alone or combined with antioxidants, during early infancy is designed to prevent many of the neuropsychiatric and behavioral disorders associated with CFD and their serious life-long disabilities. [0159] The compositions or kit-in-parts of the invention comprising a high amount of reduced folates , preferably 5MTHF, alone or in combination with antioxidants may be provided under the form of powders, tablets, liquid formulas, a paste or any other formulation known in the art that would be suitable. [0160] They may be provided either in the form of extra supplements or as fortification of baby milk formulas and maternal milk. In general, they may be added to any type of food product or beverage. Preferably, those baby formulas and food products are used where FRs with similarity to the human FRs have been removed or are not present. [0161] It needs no mentioning that when antioxidants are needed to restore an impaired 5MTHF uptake and passing across the blood-CSF barrier, preferably no or only very low levels of oxidants and/or radical forming species should be present. [0162] Further provided in the invention are special baby formulas to which reduced folates, alone or in combination with antioxidants are added, or wherein these replace folic acid present in the baby formula. A possible baby formula and its use is discussed in Example 8. [0163] Other possible procedures include a facultative use of baby formulas and food products with elimination of soluble FRs contained in cows milk, or the use of alternative products such as hydrolyzed milks, amino acid formulas or soya products. It may be advantageous to feed babies and infants thereon in order to prevent CFD and/or to reduce its incidence with the aforementioned procedures. [0164] It is believed that the AuAb can be induced by soluble folate binding proteins contained in human and bovine milk, which share more than 90 % amino acid sequence homology compared to human FRs ( Svendsen I et al, 1982, Carlsberg Res Commun 47:371-376). FR AuAb may also result from exposure of human individuals to and sensitization by known or unknown antigens with similar epitopes compared to human FRs ( for example exposure to chicken erythroid FR with 79 % amino acid sequence homology) . These soluble folate binding proteins in bovine milk share amino acid sequence homologies (90% similar) with the cell membrane folate receptors alpha and beta that are expressed on human choroid plexus epithelium (Pearson and Lipman, 1988, Proc Natl Acad Sci USA 85(8): 2444-2448). In addition, the folate receptors on the choroid plexus were shown to cross-react with rabbit antibodies against the human milk folate binding protein (Holm et al, 1991, Biochem J 280(Pt 1):267-271).

Autoantibodies against these epitopes could result in reduced folate transport into the CSF. In addition, serum AuAb against the human FR, isolated from CFD patients, were found to cross react with soluble FRs isolated from cows and human milk.

[0165] The term "CFD" as used herein refers to any known or unknown neuropsychiatric or systemic disorder associated with low 5-methyltetrahydrofolate in cerebrospinal fluid (CSF) in the presence of normal folate, vitamin B12 and homocystein outside the nervous system. [0166] There are four major underlying causes of CFD, as indicated below.

[0167] First, there may be a reduced transport across the blood-brain and blood-CSF barriers due to (1) disorders at the Folate Receptor (FR) ; (2) disorders leading to failure of energy metabolism and ATP production; (3) conditions damaging the transport functions of brain-endothelial vessel walls and choroid epithelial cells.

[0168] A reduced transport across the blood-brain and blood-CSF barriers may be due to (Ia) the presence in serum of blocking autoantibodies against the folate receptor (FR) : e.g. in the case of infantile-onset CFD, intermediate CFD, juvenile and adult CFD, Rett syndrome, autism spectrum disorders, autism with neurological deficits, part of attention deficit-hyperkinetic syndrome, depression, postnatal depression, schizophrenia, Aicardi-Goutieres syndrome, Friedreich's ataxia, postnatal depression, developmental disorders with intractable epilepsy; (Ib) genetic defects leading to loss of function of the FR, dysregulation of its expression or attachment processes to the external cellular membrane; (Ic) signal transduction disorders affecting the regulation of FR-mediated endocytosis; or (Id) disorders affecting membrane fluidity and composition: e.g., in the case of Smith-Lemli-Opitz syndrome . [0169] Disorders leading to failure of energy metabolism and ATP production may be due to (2a) mitochondrial disorders: e.g. in the case of Kearns Sayre syndrome, Complex I-V deficiencies, mitochondrial depletion syndromes; (2b) glucose transporter deficiency: e.g., a GLUT 1-deficiency; or (2c) any disorder or condition with intracellular energy failure. [0170] Conditions damaging the transport functions of brain- endothelial vessel walls and choroid epithelial cells may be due to (3a) intracranial choroid plexus bleeding: e.g., in premature infants; (3b) Xanthogranulomatous lesion of the choroid plexus; (3c) infectious, immunologic agents and traumatic lesions; or (3d) elevated reactive oxygen species or peroxynitrite with consequent damage to membranes and folate transporting proteins, e.g. FR, Reduced Folate Carrier (RFC) .

[0171] Second, there may be an increased utilization and consumption of folates within the nervous system due to (1) hereditary conditions; (2) iatrogenic conditions; or (3) infectious, parainfectious and immune-mediated conditions .

[0172] Hereditary conditions may be due to (Ia) an aromatic amino acid decarboxylase deficiency; or (Ib) a Dihydropteridine Reductase deficiency.

[0173] Iatrogenic conditions may be caused by inhibitors of the enzyme aromatic amino acid decarboxylase: e.g., use of carbidopa combined with L- Dopa in Parkinson disease. Other folate antagonists are known from literature to reduce the folate pool within the nervous system.

[0174] Infectious, parainfectious and immune-mediated conditions causing increased utilization and consumption of folates within the nervous system include Subacute Sclerosing Pan Encephalitis (SSPE) , Reactivated herpes infections and Rasmussen encephalitis.

[0175] Third, there may be an increased catabolism of reduced folates within the nervous system due to Conditions associated with oxidative and/or nitrosative stress or inflammatory processes. Conditions associated with oxidative and/or nitrosative stress include an (1) increased production of reactive oxygen species: e.g., in the case of mitochondrial disorders or inflammatory processes; (2) a failure of antioxidant enzymes and radical scavenging deficiencies: e.g. a Glutathione Peroxidase deficiency associated with selenium deficiency; an extracellular and intracellular Superoxid Dismutase deficiency secondary to intracellular and/or extracellular manganese, copper or zinc deficiency; Ubichinone-10 (QlO) deficiency states; or all conditions associated with vitamin C and E deficiencies. [0176] Fourth, there may be metabolic conditions affecting folate metabolism within the nervous system.

[0177] The above illustrates that many more individuals than ever believed may be affected by CFD or a CFD-related disease, and that several diseases/disorders appear to be linked with CFD and with the presence of circulating autoantibodies. A few examples are given below.

[0178] Evidence is accumulating that a considerable number of autism spectrum disorders, such as Rett syndrome, Kanner autism, Asperger syndrome, Heller syndrome, atypical autism or PPD-NOS (Pervasive Developmental Disorder-Not Otherwise Specified) , so- called double syndromes associated with autism, and autistiform syndromes associated with neurological deficits are caused or associated with CFD. A significant number of children with autism were found to have low CSF levels. A high percentage of these children were found to have AuAb against FR in their serum (see infra) . [0179] What has been called idiopathic CFD before, now appears to be an auto-immune disease of children with infantile-onset CFD. [0180] Also the Aicardi-Goutieres syndrome and the Aicardi-Goutieres variant appear to be linked with CFD (Blau et al, 2003, Neurology 61:642-7).

[0181] There are further indications that Attention Deficit Disorder and Hyperkinetic Disorder, 26% of the patients testing positive to AuAb, would be linked to CFD. There would further be a strong association with CFD in children with neurological disorders and a disturbed blood platelet adhesion or aggregation, indicative for disturbed intracellular signal transduction .

[0182] There is also evidence that in patients with schizophrenia, which do not respond or show a poor response to the usual medication, the underlying aetiology can be attributed to CFD with circulating and blocking FR AuAb. A 16-year old female, with schizophrenia, was found to have low CSF 5MTHF levels and tested positive for AuAb. Her schizophrenia was cured by high folinic acid therapy. That girl has a younger brother with infantile-onset CFD.

In adults with depression and a mother with postnatal depression, CFD due to blocking AuAb can also been found.

[0183] A few cases of acute and chronic inflammatory diseases of the central nervous system with secondary CFD as a result of damage to the blood-brain barrier and/or an increased folate consumption have been observed. [0184] There are several cases where an oxidative and/or nitrosative stress would be at the basis of a disturbed intracellular methylfolate transport and a disturbed transport across the blood-brain barrier and lead to CFD (see infra, Example 7) . Vitamin C was able to normalize 5MTHF membrane binding and uptake by KB cells exposed to ROS. In addition, in vitro experiments showed that 5MTHF stability during exposure to reactive oxygen species (ROS) became significantly reduced. The catabolism of 5MTHF after exposure to ROS could be prevented through addition of the radical scavenger vitamine C.

[0185] Chronic diseases such as multiple sclerosis, infections of the central nervous system and immunologic disorders (such as lupus) would also be linked with an increased folate consumption and/or catabolism of folates in the nervous system.

[0186] A treatment with inhibitors of aromatic amino acid decarboxylases (e.g. carbidopa in combination with L-dopa, or carbidopa in combination with 5- Hydroxytryptophane) would lead to an increased methylation of the accumulated L-dopa to 3-0-methyl-dopa and consequently an increased consumption of SAM and its precursor 5MTHF. Hereby the 5MTHF concentration in CSF is decreasing.

[0187] In the context of the present invention, the terms "CFD" and "a CFD related disease" cover all of the above. The term "CFD" includes secondary CFD. [0188] The invention will be described in further details in the following examples by reference to the enclosed drawings, which are not in any way intended to limit the scope of the invention as claimed Examples Example 1 : Autoantibodies against FR and Infantile-Onset CFD

[0189] It was postulated that the low 5MTHF concentration in the CSF of CFD patients is a consequence of impaired transport across these blood- brain and blood-CSF barriers. No abnormalities were found in genes encoding for these receptors among the CFD patients. Yet autoantibodies (AuAb) against folate receptors (FR) on the choroid plexus were found in the serum of these patients .

[0190] These AuAb appear to provide the mechanism that prevents the transfer of folate from the plasma into the CSF. CFD would as such be an autoimmune disease . [0191] Twenty-eight children with CFD and 28 age- matched controls were studied. The serum samples were blinded and assayed for AuAb that block the binding of [³H] folic acid to the purified folate receptors isolated from human placenta. All the parents agreed and signed an informed consent form after approval for this study by an Ethical Committee.

[0192] The pregnancies, births and neonatal history, were normal except for one child born prematurely (patient 18) at 28 weeks of gestation. All the parents were healthy and unrelated except for the parents of patient 12 who were first-line cousins.

[0193] Among these CFD patients 5MTHF, the major folate form in CSF, was measured by HPLC using electrochemical detection and compared to values derived in the laboratory from 99 normal controls, as previously described (mean 82.01 and range 44-181 nmol/L) (Blau N, Bonafe L and Blaskovics ME. Disorders of phenylalanine and tetrahydrobiopterine metabolism. In: Blau N, Duran M, Blascoviks ME, Gibson KM editors. Physicians Guide to the Laboratory Diagnosis of Metabolic Diseases. Berlin Springer Verlag 2003, page 96; Blau N et al, 2003, Neurology 61:642-647). [0194] In addition to the reduced concentration of 5MTHF in the CSF, the inclusion criteria, were that each child had at least three or more of the major clinical findings characteristic of the CFD syndrome (Ramaekers VT et al, 2002, Neuropediatrics 33:301-308; Ramaekers VT and Blau N, 2004, Dev Med Child Neurol 46:843-851) (Figure 1) .

[0195] Serum from 25 out of 28 children (20 males and 8 females; median age at the time of study: 7.1 and range: 2.5 -19.3 years) with idiopathic CFD was found to contain blocking AuAb with high affinity (mean Affinity constant Ka = 5.54 x 10¹⁰ liters per mole) to the cell membrane folate receptor, whereas all the control serums lacked the AuAb (chi-square test p<0.001) (Figure 2). [0196] A comparison between the group of patients with CFD and the normal controls was equal for age and gender distribution (mean age difference: 3.3; range 0-9 months). Serum folate concentrations were normal and there was no difference between both groups with respect to serum folate concentrations.

[0197] The history and neurologic examination of the 28 control subjects (17 males and 11 females; median age: 7.6 and range 1.9-19 years) lacked any of the symptoms of the CFD syndrome. In addition, the serum from 41 subjects with central nervous disease unrelated to CFD was tested for the presence of AuAb to FRs and found negative.

[0024198] The procedure for identifying in serum autoantibodies against the cell membrane folate receptors has been recently described (Rothenberg et al, 2004, N Engl J Med 350 (2) : 134-142) . A modification has now been introduced for these analyses whereby the sera to be tested for the AuAb are first incubated with solubilized purified folate receptors followed by the addition of [³H] folic acid. The presence of these blocking AuAb in the serum prevents the binding of [³H] folic acid to the folate receptors. [0199] For this assay, 100 μl of the serum(s) to be tested is first added to a button of dextran coated charcoal to remove the free folate. The serum (30 μl and 60 μl) is then incubated in a total volume of 500 μl of 0.01 M sodium phosphate buffer, pH 7.4 containing 0.5% Triton X-100, overnight at 4⁰C with 0.18 pmole of the solubilized apo-folate receptors purified from human placental membranes (Rothenberg et al, 2004). [³H] folic acid is then added and the mixture incubated for 30 minutes at room temperature. The free [³H] folic acid is removed by adsorption to the dextran coated charcoal and the receptor-bound radioactivity in the supernatant fraction is determined. The [³H] folic acid binds to the receptors^' on a 1:1 molar ratio and the radioactivity bound to the receptors in the assay is inversely related to the titer of the blocking AuAb and is expressed as pmoles of receptor blocked from binding the [³H] folic acid, normalized to one milliliter of the serum assayed. [0200] Endogenous folate binding protein in each serum sample was determined by the binding of [³H] folic acid and this value added to the 0.18 pmole of purified apo-folate receptor to determine the total amount of folate receptors blocked by the AuAb.

[0201] To establish that the AuAb are indeed immunoglobulins, 16 positive serum samples were analyzed by adding sufficient Protein A-Trisacryl to bind four times the average concentration of IgG in serum. Following a 1-hour incubation, the Protein A-Trisacryl was pelleted and the supernatant fraction lacked immunoglobulins and contained no blocking activity. Dissociation of the immunoglobulins from the Protein A- Trisacryl was obtained by acidification, and this fraction, following neutralization, contained the AuAb to the folate receptors.

[0202] The mean titer of the blocking AuAb in the serum of the CFD subjects was 0.87 pmoles of FR blocked per ml of serum. The mean apparent K_a for the binding of these AuAb to the folate receptor was 5.54 x 10¹⁰ liters per mole. The high affinity of these AuAb can prevent folate from binding to these receptors on the epithelial cells of the choroid plexus. Since AuAb with a mean K_a of 2.2 x 10¹⁰ liters per mole were shown to block the binding and cellular uptake of [³H] folic acid by KB cells (Rothenberg et al, 2004), the AuAb with a higher K_a in the serum from subjects with CFD would have a similar effect .

[0203] Circulating AuAb against the GPI-anchored folate receptors will preferentially bind to the epithelial cells on the plasma-side of the choroid plexus. The folate receptors expressed in the lungs and thyroid gland may also be affected by these blocking AuAb. The folate receptors on the luminal side of the proximal renal tubules will not be affected by the AuAb because immunoglobulins do not pass into the renal tubules of normal kidneys.

[0204] The effect of high-dose folinic acid treatment among CFD patients was subsequently evaluated. After the diagnosis of the CFD syndrome was established, treatment was started with folinic acid, 0.5-1 mg/kg daily in two divided doses. Patients were then examined at regular intervals after 1, 3 and 6 months, and thereafter every 6 months. Six months following treatment, a lumbar puncture was repeated to determine the CSF 5MTHF concentration whereupon the folinic acid dose was adjusted to maintain a normal CSF folate concentration. [0205] When compared to 99 normal controls, the pre- treatment CSF folate concentration for CFD patients was significantly lower (mean 20.6 and range 0-46.3 nmol/L; two-tailed t-test p < 0.001).

[0206] Following the administration of folinic acid, the CSF folate values normalized (mean 73.3 and range 45.4-120.7 nmol/L; t-test non-significant for post- treatment values versus normal controls) .

[0207] Serum from three children out of the 28 with CFD (patients 7, 9 and 21) lacked AuAb to FR. Patient 9, who had 4 of the 7 clinical criteria for the CFD syndrome, had frank autistic behavior and recovered completely after receiving a multivitamin preparation containing 400 μg of folic acid daily and currently attends a regular school. This patient was the first child identified to have the CFD syndrome . Patients 7 and 21 also exhibited remarkable improvement with folinic acid although not as dramatic as in patient 9.

Example 2 : Autoantibodies against FR and autism with neurological deficits [0208] Among the 25 children with blocking autoantibodies (AuAb, see Example 1), four children with CFD (patients 4, 16, 19, and 26) also fulfilled the criteria of late-infantile autism using the Autism Diagnostic Observation Schedule (ADOS) criteria (Lord C et al, 1989, J Autism Dev Disord 19:185-212).

[0209] These four children with mental retardation associated with autism had a very high titer of blocking AuAb (i.e. 1.27, 1.20, 0.65 and 1.27 pmol FR blocked/ml serum) .

[0210] Treatment with folinic acid improved communication skills and neurologic abnormalities among the two youngest autistic children diagnosed with CFD at age 2 and 3 years. The older children with this diagnosis, who were treated from the age of 5 and 12 years, had a poorer outcome and remained autistic. [0211] A screening test for AuAb to FR may thus be very helpful in the early detection of autism spectrum disorders associated with CFD. The earlier a treatment with folates can be started, id est the younger the children and the shorter the time period between disease onset, diagnosis and treatment, the better the prognosis it appears.

[0212] In a later study, it was confirmed that autism with neurological deficits may be linked to CFD. Again, a very high number of autistic patients with low CSF folate levels tested positive for FR AuAb in their serum. It concerns children with low-functioning (low IQ) autism (Kanner syndrome) , high-functioning (high IQ) autism (Asperger syndrome) , Rett syndrome, autistiform syndromes with neurological deficits, childhood disintegrative disorder and autism as part of a double syndrome (tuberose sclerosis and CFD with AuAb) .

[0213] Among twenty patients with low IQ (Kanner) autism associated with neurological deficits ( i.e. 3 or more of the 7 major CFD neurological criteria positive) , low CSF levels of 5MTHF were found in 19 out of the 20 subjects studied (95% of the subjects) . Values for serum homocysteine, folate, vitamin B12 and platelet function analysis were normal among all patients (age range 2 to 14 years; males to female range 14:6) . Results from patients with autism were hereby compared with age- matched controls.

[0214] The one patient with normal CSF folate levels tested negative for blocking AuAb. 16 out of the 19 subjects with low CSF levels of 5MTHF tested positive for blocking AuAb in their serum (84% of the CFD positive subjects and 80% of all 20 subjects with low-IQ autism and neurological deficits included in this study) . In addition, intermittent low levels of the antioxidant enzyme cofactors selenium, manganese and zinc were found in about 50% of all patients [0025215] The above indicates that at least the majority of individuals with low-IQ autism associated with neurological deficts appear to be suffering from CFD, and that the most important cause is the presence of circulating AuAb to FR. The latter prevent folate transfer into the CSF and nervous system which leads to folate deficiencies in the CNS during critical phases of its development. [0026216] These data support the hypothesis of an autoimmune basis for autism with neurological deficits where unidentified hereditary and environmental factors were believed to interact and/or to cause autism.

Example 3j Autoantibodies against FR and epilepsy/seizures

[0217] Eight children out of 28 (see Example 1) developed epilepsy with occasional seizures present in two patients (patients 20 and 25), and intractable epilepsy with absences, myoclonic astatic attacks and grand mal seizures requiring anticonvulsant therapy for six children. The response to classical anitiepileptic drugs (AEDs) was often poor in these children. [0218] After the diagnosis of low CSF folate was established, folinic acid was added to the treatment with the anticonvulsant drug combination following which these seizures were fully controlled.

Example 4 : Better prognosis when a therapy with folates can be started early [0219] The early detection and diagnosis of this apparent autoimmune disorder that results in CFD is important because a pharmacologic dose of folinic acid (and the 5MTHF derivative) seems to be able to by-pass the AuAb-blocked folate receptors, and enter the CSF via the RFCl (Reduced Folate Carrier 1) . Another possibility is that 5MTHF enters the CSF by displacing blocking AuAb to the FRs if the concentration of this folate is sufficiently high (~ 2 μM or greater) . A third mechanism for the folate to enter the CSF is by diffusion when the plasma concentration of 5MTHF is very high.

[0220] Fact is that a pharmacologic dose of (reduced) folates can restore the folate concentration within the CNS and can ameliorate the neuropsychiatric disorders . There appeared to be a better outcome among the 28 children (see Example 1) diagnosed with CFD and treated with folinic acid at a younger age .

[0221] The age span of onset of CFD clinical symptoms is shown in Figure 1. From this figure, it can be derived that the clinical picture of CFD below the age of 1 year is not fully developed.

[0222] Table 1 gives the percentage improvement after folate treatment (folinic acid) among the 28 patients with infantile-onset cerebral folate deficiency syndrome of Example 1 and two other children manifesting first features of CFD at the age of 2 and 3 months respectively. All children were found to have CFD symptoms before they were treated.

[0223] This table clearly shows that psychomotor retardation and dyskinesias are the symptoms for which the rate of improvement in symptoms was lowest. Overall 39% of psychomotor retardation and 33% of dyskinesias for all patients diagnosed between 1 and 16 years improved. Yet, as treatment after diagnosis was started earlier, the condition for the younger children appeared reversible .

Table 1 : Percentage improvement after folate treatment (folinic acid) among 30 patients with infantile-onset Cerebral Folate Deficiency syndrome . The indicated age in years represents the age of diagnosis immediately followed by therapy

[0224] When children are treated before they reach the age of 3, total % improvement was enormous: 100% <1 year, 90% before the age of 2 and 86% before the age of 3. [0225] It was found that if patients have a delayed diagnosis with late-onset treatment, lower success rates were obtained. A progressive visual loss (>3 years of age) or hearing loss (>6 years of age) will then develop (Fig. 1) . [0226] One child that since birth (normal pregnancy, normal birth) suffered from erratic myoclonic seizures and generalized tonic-clonic seizures, and that did not respond to Pyridoxine and several anticonvulsants, was subsequently treated at the age of 2 months with folinic acid. Another child developed hypotonia and refractory seizures from the age of three months.

[0227] Both children responded completely within 24 hours to a daily amount of 2-2.5 mg/kg folinic acid. Normal development was obtained with supplements of 2.0- 3.5 mg/kg/day folinic acid. Id est, these children, diagnosed before the age of 1 year, recovered completely and have no psychomotor retardation or any other problem whatsoever. In their serum AuAb to FR were found: 1.47 and 0.29 pmol FRs/ml serum. [0228] The above data indicate that there is a critical timeframe in which CFD symptoms have not yet fully developed, within which prognosis improves and irreversible psychomotoric retardation can be prevented. [0229] The early detection of AuAbs to FR by way of a screening and/or on indication ( in autistic syndromes or in case of neurological features, as shown by major criteria in Fig. 1) makes it possible to start a therapy as soon as possible, and preferably within this critical timeframe. The shorter the period between the outbreak or onset of manifestations and treatment, the better the prognosis will be.

Example 5; A very high percentage of CFD patients test positive for FR AuAb [0230] AuAb appear to be present in a very high number of individuals diagnosed to have CFD. Table 2 gives an overview of the percentage of CFD patients that tested positive.

[0231] AuAb to FR were found in the serum of at least 55% of the CFD patients screened and were found among all CFD groups tested: neonatal, infantile, intermediate, late-onset and Rett syndrome. Percentages as high as 90% were reached in the case of Infantile- Onset CFD.

Table 2 : Per group of CFD patients the percentage of individuals testing positive for FR AuAb

a Kanner autism b Asperger syndrome

[0232] These data demonstrate once more that circulating blocking AuAb may be the major cause of CFD. [0233] These data further demonstrate that AuAb are found in Neonatal (> 55%) and Infantile-Onset types of CFD (90%) .

[0234] CFD incidence is estimated at 1:1000 to 1:1500. The incidence of the Rett syndrome is estimated at 1:10,000 to 1:15,000.

[0235] A screening of infants followed by treatment below the age of 1 year would thus be very meaningful and can have serious consequences for the health and well-being of the human population if the incidence of CFD can thereby be drastically reduced and/or its disabilitating symptoms drastically improved.

Example 6 : Blocking AuAb against human FR in serum may be induced by soluble FR proteins of human and bovine milk. Effective treatment with a cow milk free diet, combined with reduced folates with or without antioxidants [0236] Based upon the observation of first clinical manifestations occurring after the age of 4 to 6 months, and the absence of AuAb in 5 mothers of children with CFD, the generation of AuAb probably occurs during the first 4 to 6 months of life in these children. [0237] These AuAb might be induced by soluble folate receptor proteins ( FR; synonym folate binding proteins) contained in human and bovine milk, or may result from exposure and/or sensitization by other unknown antigens with similar epitopes (Svendsen I et al, 1982, Carlsberg Res Commun 47:371-376). Soluble FR proteins in bovine milk share amino acid sequence homologies (90% similarity and 84% identity; Fig.9) with the cell membrane folate receptors alpha and beta that are expressed on human choroid plexus epithelium (Pearson WR and Lipman DJ, 1988, Proc Natl Acad Sci USA 85(8):2444- 2448) . In addition, the folate receptors on the choroid plexus were shown to cross-react with rabbit antibodies against the human milk folate binding protein (Holm et al, 1991, Biochem J 280(Pt 1):267-271). AuAb against these epitopes could result in reduced folate transport into the CSF. It was confirmed that serum FR AuAb against human FR from 5 CFD patients cross-reacted with both the soluble FRs contained in bovine and human milk. [0238] This seems to indicate that blocking AuAb can develop early in the infant's life. It can not be excluded, however, that AuAb are passed on from mother to child. Five mothers screened in the study of 28 children with infantile-onset CFD, were healthy and tested negative (see Example 1) . Yet, one mother of a child with infantile-onset CFD and with AuAb to FR, tested positive. She suffered from postnatal depression. [0239] Based upon these observations, the following may be concluded: [0240] First, if the AuAb would be induced by soluble folate receptor proteins contained in human and bovine milk, the preventive use of baby formulas, foods, beverages after elimination of soluble FRs contained in cows milk may be advisable, or the use of alternative products such as hydrolyzed milks, amino acid formulas or soya products.

[0241] Second, the screening of the mother of an infant at birth may be advisable since the AuAb might be passed on from the mother to her child. If the mother tests positive, the child should be followed closely. A screening of the mother is certainly recommended in the case of a predisposition in the family. [242] Third, additional treatment with a cow-milk free diet (mostly soya-based products) among 8 CFD patients showed disappearance of FR AuAb in 5 and reduction of titers in 3 patients after introduction of this soya- based dietary treatment to the combined supplements of folinic acid with or without antioxidants (Fig.7). For each subject the measurements before and after dietary treatment are represented by two points which are connected by a line.

Example 7 : ROS are capable to further diminish cellular uptake of 5MTHF and ROS lead to unstability of 5MTHF [0243] The trace element Manganese (Mn) in mammals is cofactor of many enzymes among which the extracellularly secreted and mitochondrial enzyme Mn-superoxide Dismutase (Mn-SOD) , which inactivates formed superoxide radicals to hydrogen peroxide. Mn deficiency in rats manifests with a phenotype of ataxia and seizures. [0244] Children with unexplained psychomotor retardation and ataxia since infancy were screened for disturbances of Mn, Selenium (Se) metabolism and/or mitochondrial diseases.

[0245] Eight patients were found to have a lowered serum Mn associated with low activity of plasma Mn-SOD (Oyanagui Y, 1984, Analytical Chemistry 142:290-296). In five out of eight patients the Mn content of red blood cells was also decreased. Mn content in red blood cells and serum were measured via standard procedures well known in the art . [0246] Two patients were found to suffer from an oxidative stress due to a Se deficiency (via methods well known in the art, e.g. atomic absorption spectrometry in serum and red blood cells) and 5 patients suffered from a mitochondrial disease (as well known in the art) .

[0247] CSF analysis in the Mn-deficient patients identified low 5MTHF values (mean 18.25, range 1.2-43.8 nmol/liter compared to a mean value in 99 healthy controls at 82, range 44-181 nmol/liter) . Serum and erythrocyte folate concentrations were normal. In the patients with selenium deficiency and the patients with mitochondrial encephalopathies CSF folate was also lowered.

[0248] Single oral Mn supplementation did not increase the serum and erythrocyte Mn values . Oral folinic acid supplements did not change the CSF 5MTHF levels . [0249] However, a combined administration of folinic acid with the radical scavengers vitamin C and E improved ataxia, raised the Mn values in serum and red blood cells and normalized CSF 5MTHF (mean 92.34, range 61-133 nmol/liter) . [0250] Especially a treatment with 100 mg/day vitamin C, 25 mg/kg/day vitamin E and a reduced folate (in the present case folinic acid) proved very efficient. [0251] CFD patients with a Se-deficiency of course are further treated with Se supplements in the form of 2-5 μg/kg/day selenite (anorganic form) or selenocysteine, often combined at the start of therapy with folinic acid. CFD patients with mitochondrial diseases were treated with a combination of a reduced folate (folinic acid in this case) and high doses of antioxidants (vitamin C and E in this case) , separate from a specific additional therapy with cofactors of the complexes with low enzymatic activity (e.g. extra doses QlO and riboflavin in the case of a complex I deficiency) .

[0252] Deficiencies of the cofactors selenium, manganese, copper and zinc associated with low activities of their antioxidant key enzymes (Gluthation Peroxidases, superoxide dismutases), and/or the presence of mitochondrial diseases were screened for or diagnosed with the most current techniques available and well known in the art. [0253] The combined treatment (high amount of a

(reduced) folates and a high amount of antioxidants) was based on the assumption that lowered activity of antioxidant enzymes or mitochondrial complex I-V leads to increased levels of superoxide and peroxynitrite radicals.

[0254] Consequently, oxidative and nitrosative stress may impair folate transport to the nervous system due to radical-mediated damage to brain endothelial vessels and/or to choroid epithelial cells and membrane proteins (FR, RFC: reduced folate carrier) subserving folate transport across the blood-brain barrier and choroid plexus. Reactive oxygen species (ROS) can further diminish 5MTHF binding to membrane-attached FR and RFC leading to disturbed 5MTHF uptake into cells. This results in impaired transport into the CSF and nervous system, which could be demonstrated in vitro { Fig.3-5) . In addition, ROS are capable to catabolise 5MTHF in vitro and thus lead to 5MTHF unstability (Fig.6). [0255] To study 5MTHF uptake by the folate receptor 1 (FRl) intracellular ¹⁴C 5MTHF was measured in KB cells at different time points in the presence and absence of ROS (Fig.3-5) . [0256] KB cells (human nasopharyngeal epidermoid carcinoma cells) and Minimum Essential Medium Eagle were obtained from the American Type Culture Collection

(ATCC) . In general KB-Cells were cultured in minimal essential medium, containing 10 % Foetal Calf Serum

(FCS) and 5000 IU penicillin and streptomycin (all from

BioConcept's AMIMED, Switzerland). Subcultivation was performed every 4-5 days at a ratio 1:3.

[0257] 24 h before each experiment 70-80 % confluent cell dishes (60.1 cm²) were incubated over 5 minutes with 0.15 M hydrogen chloride/sodium chloride solution (pH 3.5) to strip membrane-bound folates. After washing with PBS (Phosphate Buffered Saline from GIBCO, Invitrogen- Cooperation) , cells were divided in equal shares in new cell culture dishes and incubated overnight in folic acid-free medium (GIBCO, Invitrogen-Cooperation) , containing 10 % FCS and 5000 IU penicillin/streptomycine . [0258] Before starting an experiment, medium was exchanged and fresh folic acid-free medium was added (10 ml). Depending on the particular experimental setting, ¹⁴C 5MTHF (Amersham Biosciences) was added after preincubation of KB cells with PIPLC (phophatidyl- inositol-specific phospholipase C) , unlabelled 5MTHF (gift from Eprova, Switerland) , or ROS.

[0259] After incubation, cells were washed three times with PBS and harvested with 1:10 diluted Trypsin (Trypsin-EDTA (0.5%/0.2%), BioConcept's AMIMED). Viable cells were determined by tryphan-blue (Sigma Aldrich) and counted. Dishes containing more than 1.5 x 10⁶ cells were discarded.

[0260] Subsequently cells were lysed by three cycles of freezing and thawing. After centrifugation (3 minutes at 1090 U/m Hettich Rotina 46) supernatant was separated, 7.5ml Ultima Gold solution (Perkin Elmer) was added and radioactivity was counted (Packard liquid Scintillation Counter 1900 CA) . From the resulting counts per minute the background counts, achieved from one millilitre washing solution was subtracted. All experiments were done as triplicates and were expressed as pmol 5MTHF/10⁶ cells. [0261] To analyse the influence of ROS, incubation with ¹⁴C 5MTHF was performed in 10 ml Hanks balanced salt solution (HBSS) containing 25 mg bovine serum albumin (Fluka, Switzerland) 100 μg transferrin and 20 μg insulin. Three mM xanthine and 0.2 ϋ/1 xanthineoxidase (Sigma Aldrich) were added. Xanthine is metabolized by xanthineoxidase to uric acid under production of the oxygen radicals superoxide anion and hydrogen peroxide. Previous testing had shown that this mixture is able to produce ROS over 60 minutes, without obvious harm to the cells . [0262] Before and one hour after adding 10 nM or 50 nM ¹⁴C 5MTHF the amount of produced ROS was measured by luminometer counting (Turner Luminometer TD20/20) after addition of 10 μl Coelenterazine (Molecular probes Inc; Eugene, Oregon, USA). To exclude the possible oxidation of the added 5MTHF itself by free radicals, in a second experiment cells were exposed to ROS before adding 5MTHF. Only after oxygen radicals were not detectable, ¹⁴C 5MTHF was added. [0263] ROS (enzymatically produced) significantly reduced ¹⁴C 5MTHF uptake by KB cells (Fig. 3) both at low concentrations of 10 nM (45% decrease from 0.51 pmol/10⁶ cells to 0.28 pmol/10⁶ cells; p = 0.014) and at higher extracellular 5MTHF concentrations of 50 nM to an equal extent ( 44% decrease from 1.59 pmol/10⁶ cells to 0.89 pmol/10⁶ cells; p = 0.002).

[0264] It should be stressed that the exposure to ROS did not have any impact on cell count, viability or the binding of monoclonal antibodies (Movl8, Alexis Biochemicals) against FRl as determined by FACS analysis .

[0265] Further KB cell studies on 5MTHF membrane binding and uptake were done following the generation of ROS by xanthine/xanthineoxidase, quantified by coelenterazine

( chemiluminescent fluorescing probe) . KB cell cultures in HI-TESA medium ( Minimal essential medium with a balanced salt solution containing insulin, transferrin and albumin) could be incubated with ¹⁴C 5MTHF during one hour without affecting cell viability and integrity. After ROS exposure 5MTHF uptake by KB cells was reduced, but 5MTHF binding to cellular membranes was significantly decreased (Fig. 5). Both the slightly decreased uptake and the clearly decreased membrane binding were reversible after adding vitamin C in physiological concentration. This indicates that ROS are responsible for the changes of 5MTHF binding and uptake. Experiments have been repeated after preincubation with either PIPLC, ROS, or PIPLC combined with ROS (Fig. 4) . Compared to controls, PIPLC and ROS reduce 5MTHF binding and uptake, while coexposure with ROS on KB cells, pretreated with PIPLC, led to an additional decrease. Instead of FR removal by PIPLC, FR expression by KB cells was downregulated by 80% using small-interference RNA probes, leading to a 60% reduction of 5MTHF uptake. ROS led to a similar reduction of 5MTHF uptake in transfected and non-transfected KB cells, which could be reversed to normal by addition of vitamin C. These findings indicated that ROS diminish 5MTHF membrane binding and uptake through functional loss of both FR and RFC, and possibly other folate transporters. Vitamin C can neutralize these ROS-induced effects and protect intracellular folate transport.

The direct influence of ROS on ¹⁴C 5MTHF was analysed in vitro using a HPLC method with electrochemical detection, measuring 5MTHF concentration before and after incubation with ROS. It was found that 5MTHF concentration reduced by more than 70% after 1 hour incubation with ROS, which can be prevented by addition of vitamin C (Fig. 6) .

Example 8 : Example of an adapted baby milk formula and its use in the prevention and/or early treatment of CFD [0266] Below an example is given for the definition of a special baby milk formula without FR antigen, fortified with 40 μg L-methyltetrahydrofolate, a low amount (0.5-1 RDA) of 3 mg vitamin C and 0.4 mg vitamin E/ 100 ml solution Babyfood, that can be used for the prevention and/or early treatment of CFD. [0267] When testing at birth is positive for AuAb to FR, a child from birth onwards until the age of 4-6 months would be fed with a special baby formula to which 40 μg L-methyltetrahydrofolate, 3 mg vitamin C and 0.4 mg vitamin E have been added per 100 ml solution Babyfood. Soluble FRs derived from cow milk have to be removed by special processing or alternatively soya- based, partially or completely hydrolysed cows milk formulas can be used. Caution should be taken to the fact that the required amounts selenium, manganese, zinc and copper are present.

This would come down to administering to a breast-fed child during the first 6 months 1 drop of L- methyltetrahydrofolate solution per kg body weight ( 1 ml solution contains 20 drops; composition of the solution: 1200 μg 5MTHF/ml; 90 mg vitamin C/ml and 12 mg vitamin E/ml solution) . [0268] After repeated positive testing between the age of 4 and 6 months, the child would then be put from the age of 6-8 months on the high 5MTHF containing special baby formula with 80 μg L- methyltetrahydrofolate, 6 mg vitamin C and 0.8 mg vitamin E/100 ml solution Babyfood. The higher concentrations are required since the daily consumed amount will decrease to 500-600 ml from the age of 6-8 months. This would come down to giving to a breast-fed child 1 drop of a L-methyltetrahydrofolate solution per kg body weight (1 ml solution containing 1200 μg 5MTHF, 90 mg vitamin C and 12 mg vitamin E) .

[0269] Alternatively, a child testing positive from birth onwards can be fed with a baby formula containing more than 80 μg up to 210 μg L-methyltetrahydrofolate with low amounts vitamin C and E /100 ml solution Babyfood. For instance in the case of a very high titer of autoantibodies or a predisposition within the family. [0270] It is recommended that folate supplementation in the sense of the invention takes place under the supervision of and/or on the prescription of a clinician.

[0271] It is further recommended to start with lower doses of (reduced) folates to check a patients susceptibility, to then increase the concentration (possibly step-wise) , to regularly check patients and to use CSF 5MTHF levels for monitoring the effect of the treatment .

[0272] It is also recommended not to use too high concentrations of (reduced) folates. Hunter et al .

(1970, Lancet 1:61-3) have previously described in a study on healthy volunteers that daily administration of e.g. 15 mg folic acid to healthy volunteers for more than one month caused mental changes, sleep disturbances and irritability. Some became hyperactive, whereas others became depressed and confused and had increasing difficulties with concentration.

Claims

Claims

1. A composition or kit-in-parts comprising a pharmacologically effective amount of at least one reduced folate and a pharmacologically effective amount of at least one antioxidant, said antioxidant being in an amount of about at least 0.1 RDA, preferably at least 0.5 RDA, more preferably at least 1 RDA, most preferably at least 2 RDA.

2. A composition or kit-in-parts for human consumption comprising at least one reduced folate, wherein the total amount of reduced folates in said composition is above about 200%, in particular above about 210%, more in particular above about 250% of a human daily requirement for folates per customarily consumed quantity of said composition.

3. The composition of claim 2 wherein said composition or kit-in-parts further comprises at least one antioxidant, said antioxidant preferably being in an amount of about at least 0.1 RDA, preferably at least 0.5 RDA, more preferably at least 1 RDA, most preferably at least 2 RDA.

4. The composition or kit-in-parts of any of the preceding claims wherein the antioxidant is selected from the group consisting of vitamin C, vitamin E, β carotene, Ubichinone 10 and vitamin A.

5. The composition or kit-in-parts of claim 4 wherein the antioxidant is vitamin C and/or vitamin E.

6. The composition or kit-in-parts of any of the preceding claims wherein the reduced folate is present in an amount of about at least 2 times the RDA.

7. The composition or kit-in-parts of any of the preceding claims wherein the reduced folate is selected from the group consisting of tetrahydrofolic acid, 5- formyltetra-hydrofolic acid, 5-methyltetrahydrofolic acid, 10-formyltetrahydrofolic acid, 5,10- methenyltetrahydrofolic acid, 5,10- methylenetetrahydrofolic acid, 5- formiminotetrahydrofolic acid, a Ca or a Na₂ salt of any of these, a monoglutamate and polyglutamyl derivatives of any of these.

8. The composition or kit-in-parts of claim 7 wherein the reduced folate is L-5-methyltetrahydrofolic acid, said L- 5-methyltetrahydrofolic acid being present in an amount between about 15 and about 200 μg/kg/day, preferably between about 25 and about 85 μg/kg/day, more preferably between about 33 and about 66 μg/kg/day.

9. An essential nutrient preparation, an essential vitamin preparation, or a food preparation comprising a composition or kit-in-parts of any of the preceding claims .

10. The food preparation of claim 9 selected from the group consisting of a formula, a beverage or a food product for babies or infants, children, adolescents or adults .

11. The food product of claim 10 which is a milk formula for babies or infants, or is maternal milk.

12. The food product of claims 10 or 11, that is deprived of soluble FR receptors or significantly reduced in these soluble FR receptors .

13. The food product of claim 12 in that it is a hydrolyzed milk, an amino acid formula or a soya product.

14. The composition or kit-of-parts of any of the preceding claims for use as a medicament.

15. Use of a composition or kit-of-parts of any of the claims 1 to 14 for the preparation of a medicament to prevent CFD in a subject.

16. Use of a composition or kit-of-parts of any of the claims 1 to 14 for the preparation of a medicament to treat CFD in a subject.

17. Use of a composition or kit-in-parts comprising a pharmacologically effective amount of at least one reduced folate, preferably 5- methyltetrahydrofolic acid, for the preparation of a medicament to prevent CFD.

18. Use of a composition or kit-in-parts comprising a pharmacologically effective amount of at least one reduced folate, preferably 5- methyltetrahydrofolic acid, for the preparation of a medicament to treat CFD.

19. Use of claim 17 and 18 wherein the reduced folate is L-5-methyltetrahydrofolic acid, said L-5- methyltetrahydrofolic acid being present in an amount between about 15 and about 200 μg/kg/day, preferably between about 25 and about 85 μg/kg/day, more preferably between about 33 and about 66 μg/kg/day.

20. The use of any of the claim 15 to 19 wherein the subject is at risk of developing CFD.

21. The use of claim 20 wherein the subject possesses autoantibodies against a cell membrane folate receptor, but has not yet developed any symptoms of CFD.

22. The use of claim 20 wherein the subject possesses autoantibodies against a cell membrane folate receptor, the clinical picture of CFD not being complete though in said subject.

23. The use of any of the claims 15 to 22 to prevent and/or treat a disease selected from the group consisting of autism spectrum disorders, schizophrenia, depression, dementias, the Aicardi-Goutieres syndrome and the Aicardi-Goutieres variant, Attention Deficit Disorders and Hyperkinetic Disorders, neurological disorders in association with a disturbed blood platelets adhesion, and/or cases of acute and chronic inflammatory diseases of the central nervous system associated with a low level of 5MTHF acid in the CSF.

24. A method for the prevention and/or the early treatment of CFD, said method comprising the steps of

- Testing a biological sample of a subject for the presence of blocking AuAb to the cell membrane folate receptor; and/or

- Measuring the 5MTHF level in a CSF sample from said subject; and

- Starting a treatment of the subject with a pharmacologically effective amount of folates promptly upon the detection of AuAb and/or promptly upon the measuring of low CSF 5MTHF levels in the subject compared to age-matched control subjects.

25. A method for the prevention and/or the early treatment of CFD, said method comprising the steps of

- Testing a biological sample of the mother of an infant or child on the presence of blocking AuAb to the cell membrane folate receptor;

- Optionally, measuring the 5MTHF level in a CSF sample from the infant or child; and

- Starting a treatment of the infant or child with a pharmacologically effective amount of folates promptly upon the detection of AuAb in the mother and/or promptly upon the measuring of low CSF 5MTHF levels in the infant or child compared to age-matched control subjects.

26. The method of the claims 24 or 25 wherein the biological sample is cord blood or serum.

27. The method of any of the claims 24 to 26 wherein the subject is tested for the presence of autoantibodies and/or for a low CSF 5MTHF level in the period between about 0 and about 3 years, preferably in the period between about 0 and about 1 year.

28. The method of claim 27 wherein the subject is assayed at birth, but more preferably at the age of about 4 to about 6 months, the test possibly being repeated at least once, preferably at the age of about 1 year.

29. The method of claim 25 wherein the mother is tested before birth, at birth and/or shortly after birth of the infant.

30. The method of claim 24 or 25 wherein at least three of the following symptoms manifest in the subject tested: unrest, irritability, insomnia; decelerating head growth; neurodevelopmental delay, standstill, regression; hypotonia and ataxia; ascending pyramidal tract signs in legs; dyskinesias; epilepsy and seizures.

31. The method of any of the claims 24 to 30 further comprising the step of assaying a subject's sample or a subject for a possible oxidative and/or nitrosative stress.

32. The method of any of the claims 24 to 31 wherein the treatment consists of administering a composition or kit-in-parts of any of the claims 1 to 12.

33. A milk formula for babies or a milk-based product for babies, infants, children, adolescents or adults that is deprived of soluble FR receptors or significantly reduced in these inducing soluble FR receptors .

34. The use of at least one of the following: a milk formula or milk-based product of claim 33, a hydrolyzed milk, an amino acid formula or soya product for the prevention and/or treatment of CFD in a subject.

35. A test kit or assay for detecting blocking autoantibodies (AuAb) to FRs in a biological sample from a subject, said kit or assay comprising purified FRs from a human or homologous species, reagents for treating said biological sample, labeled folic acid or a labeled folate, at least one indicator which detects a complex of said purified FRs and said AuAb, and further means to identify an oxidative and/or nitrosative stress in the subject.