Juvenile onset central nervous system folate deficiency and rheumatoid arthritis.

Isolated cerebral folate deficiency was detected in a 13-year-old girl with cognitive and motor difficulties and juvenile rheumatoid arthritis. Her serum contains autoantibodies that block membrane-bound folate receptors that are on the choroid plexus and diminish the uptake of folate into the spinal fluid. Whereas her serum folate exceeded 21 ng/mL, her spinal fluid contained 3.2 ng/mL of 5-methyltetrahydrofolate as a consequence of the autoantibodies diminishing the uptake of this folate.

Autoantibodies to folate receptors in the cerebral folate deficiency syndrome.

In infantile-onset cerebral folate deficiency, 5-methyltetrahydrofolate (5MTHF) levels in the cerebrospinal fluid are low, but folate levels in the serum and erythrocytes are normal. We examined serum specimens from 28 children with cerebral folate deficiency, 5 of their mothers, 28 age-matched control subjects, and 41 patients with an unrelated neurologic disorder. Serum from 25 of the 28 patients and 0 of 28 control subjects contained high-affinity blocking autoantibodies against membrane-bound folate receptors that are present on the choroid plexus. Oral folinic acid normalized 5MTHF levels in the cerebrospinal fluid and led to clinical improvement. Cerebral folate deficiency is a disorder in which autoantibodies can prevent the transfer of folate from the plasma to the cerebrospinal fluid.

Autoantibodies against folate receptors in women with a pregnancy complicated by a neural-tube defect.

BACKGROUND: In the absence of clinical folate deficiency, periconceptional supplementation with folic acid reduces a woman's risk of having an infant with a neural-tube defect. Since antiserum to folate receptors induces embryo resorption and malformations in rats, we hypothesized that autoantibodies against folate receptors in women may be associated with pregnancy complicated by a neural-tube defect. METHODS: Serum from 12 women who were or had been pregnant with a fetus with a neural-tube defect and from 24 control women (20 with current or prior normal pregnancies and 4 who were nulligravid) was analyzed for autoantibodies by incubation with human placental folate receptors radiolabeled with [3H]folic acid. The properties of these autoantibodies were characterized by incubating serum and the autoantibodies isolated from serum with placental membranes, ED27 cells, and KB cells, which express the folate receptors. RESULTS: Serum from 9 of 12 women with a current or previous affected pregnancy (index subjects) and 2 of 20 control subjects contained autoantibodies against folate receptors (P<0.001). The autoantibodies blocked the binding of [3H]folic acid to folate receptors on placental membranes and on ED27 and KB cells incubated at 4 degrees C and blocked the uptake of [3H]folic acid by KB cells when incubated at 37 degrees C. CONCLUSIONS: Serum from women with a pregnancy complicated by a neural-tube defect contains autoantibodies that bind to folate receptors and can block the cellular uptake of folate. Further study is warranted to assess whether the observed association between maternal autoantibodies against folate receptors and neural-tube defects reflects a causal relation.

A genetic polymorphism in the coding region of the gastric intrinsic factor gene (GIF) is associated with congenital intrinsic factor deficiency.

Congenital intrinsic factor (IF) deficiency is a disorder characterized by megaloblastic anemia due to the absence of gastric IF (GIF, GenBank NM_005142) and GIF antibodies, with probable autosomal recessive inheritance. Most of the reported patients are isolated cases without genetic studies of the parents or siblings. Complete exonic sequences were determined from the PCR products generated from genomic DNA of five affected individuals. All probands had the identical variant (g.68A>G) in the second position of the fifth codon in the coding sequence of the gene that introduces a restriction enzyme site for Msp I and predicts a change in the mature protein from glutamine(5) (CAG) to arginine(5) (CGG). Three subjects were homozygous for this base exchange and two subjects were heterozygous, one of which was apparently a compound heterozygote at positions 1 and 2 of the fifth codon ([g.67C>G] + [g.68A>G]). The other patient, heterozygous for position 2, had one heterozygous unaffected parent. Most parents were heterozygous for this base exchange, confirming the pattern of autosomal recessive inheritance for congenital IF deficiency. cDNA encoding GIF was mutated at base pair g.68 (A>G) and expressed in COS-7 cells. The apparent size, secretion rate, and sensitivity to pepsin hydrolysis of the expressed IF were similar to native IF. The allelic frequency of g.68A>G was 0.067 and 0.038 in two control populations. This sequence aberration is not the cause of the phenotype, but is associated with the genotype of congenital IF deficiency and could serve as a marker for inheritance of this disorder.

Identification of a 4-base deletion in the gene in inherited intrinsic factor deficiency.

A 4-base deletion has been identified in the coding region of the gene for gastric intrinsic factor (IF) in an 11-year-old girl with severe anemia and cobalamin (Cbl) deficiency. The bone marrow showed frank megaloblastic morphology, and the Schilling test indicated a failure to absorb Cbl that was corrected by coadministration of IF. Pentagastrin administration induced acid secretion, but the gastric juice lacked IF as determined by CbI binding, by fractionation of protein-bound CbI, and by immunoprecipitation with anti-IF antiserum. Individual exons were amplified by the polymerase chain reaction by using primers to the flanking intronic regions, and the nucleotide sequence analysis identified a 4-base deletion (c183_186delGAAT) spanning positions 104 to 107 in exon 2, resulting in premature termination of translation. This mutation also eliminates a site for Bst XI endonuclease and introduces a site for BsaBI for identifying this deletion in hereditary IF deficiency.

Antibodies to folate receptors impair embryogenesis and fetal development in the rat.

BACKGROUND: Folic acid (FA) supplementation reduces neural tube defects (NTDs) by 70%. However, the cause of most NTDs cannot be attributed to folate deficiency, to mutations of genes that encode folate pathway enzymes, and folate receptors (FRs) that mediate cellular folate uptake. Mouse embryos nullizygous for the ortholog of the FRalpha gene have lethal congenital abnormalities that are preventable by administration of folinic acid to the dams. To determine whether antibodies to FRs are similarly teratogenic, we studied a rat model. METHODS: Immunohistochemistry with an antiserum to rat FRs was used to identify the receptors on reproductive tissues and embryos. Gestation day (GD) 8 rats received intraperitoneal injections of antiserum to the FRs, and their embryos were examined 2-9 days later. Some rats received pharmacologic doses of folinic acid or dexamethasone before the antiserum was administered. RESULTS: The FRs are present on oocytes, the oviduct, and uterine epithelial cells, and in the embryo at all stages examined between GD4 and GD15. The antiserum has a dose-related effect on embryo viability and organogenesis. Folinic acid prevented teratogenicity resulting from smaller doses of antiserum, but not that caused by larger doses. Resorption of embryos with the larger doses of the antiserum was prevented by dexamethasone. CONCLUSIONS: FRs are expressed on oocytes, epithelial cells of reproductive organs, and embryonic and extraembryonic tissues. Antiserum to FRs administered to pregnant rats causes embryonic damage. Embryo lethality with smaller doses of antiserum is preventable by administration of folinic acid, while larger doses cause embryo damage by immune-mediated cell lysis, which can be prevented by dexamethasone.

The half-life of the transcript encoding the folate receptor alpha in KB cells is reduced by cytosolic proteins expressed in folate-replete and not in folate-depleted cells.

The KB cell, a transformed human cell line, constitutively expresses a high level of the glycosylphosphatidylinositol (GPI) anchored folate receptor protein alpha (FR alpha) and thereby can grow in medium containing <1 nM folate. When transferred from a folate-replete (FR) medium to one folate-deficient (FD), intracellular folate diminishes about 50-fold and expression of the FR alpha increases 6-fold. This up-regulation is mediated by a 4.5-fold increase in the initial transcription rate and a 2.4-fold prolongation of the mRNA half-life that together provide a higher level of the steady-state mRNA abundance. An RNA gel -shift assay of a 5' region of the mRNA that includes all of the non-coding and 24 nt of coding sequence, and a 3' region comprised only of coding sequence, identified unique complexes with cytosolic proteins from the FR-KB cells that were not observed with the cytosol from FD-KB cells. It appears, therefore, that expression of these folate-dependent cytosolic trans-active proteins function to maintain a shorter half-life of the mRNA in the FR-KB cells by binding to 5' and 3' cis elements, reducing the stability of this transcript.

Congenital transcobalamin II deficiency due to errors in RNA editing.

Transcobalamin II (TCII) is a plasma protein essential for the transport and cellular uptake of vitamin B12 (B12; cobalamin, Cbl). Congenital deficiency of functional TCII is an autosomal recessive genetic disorder that results in clinical B12 deficiency usually within several months following birth. In this report, we describe the molecular basis for TCII deficiency in two patients who developed a megaloblastic anemia in early infancy. The serum of both patients contained immunoreactive TCII that did not bind [57Co]Cbl. The fibroblasts from each patient secreted a similarly nonfunctional TCII, yet full-length TCII transcripts were identified by Northern blot. Overlapping cDNA fragments were generated by reverse transcription-polymerase chain reaction and several mutations were identified in the coding region of the cDNA, one of which was common to both patients. However, amplification of the corresponding regions of the gene from genomic DNA failed to identify these mutations. These findings were confirmed by replicate analyses and support the proposal that a variance in RNA editing is the likely mechanism for the mutations that resulted in the expression of a nonfunctional TCII protein in these patients.

Direct assay for cobalamin bound to transcobalamin (holo-transcobalamin) in serum.

BACKGROUND: Only cobalamin carried by transcobalamin (holo-transcobalamin) is available for cellular uptake and hence is physiologically relevant. However, no reliable or accurate methods for quantifying holo-transcobalamin are available. We report a novel holo-transcobalamin assay based on solid-phase capture of transcobalamin. METHODS: A monoclonal antibody specific for human transcobalamin with an affinity constant >10(10) L/mol was immobilized on magnetic microspheres to capture and concentrate transcobalamin. The cobalamin bound to transcobalamin was then released and assayed by a competitive binding radioassay. The quantification of holo-transcobalamin was accomplished using calibrators composed of recombinant, human holo-transcobalamin. RESULTS: The assay was specific for holo-transcobalamin and had a detection limit of 5 pmol/L. Within-run and total imprecision (CV) was 5% and 8-9%, respectively. The working range (CV <20%) was 5-370 pmol/L. Dilutions of serum were linear in the assay range. The recovery of recombinant, human holo-transcobalamin added to serum was 93-108%. A 95% reference interval of 24-157 pmol/L was established for holo-transcobalamin in 105 healthy volunteers 20-80 years of age. For 72 of these sera, holo-haptocorrin and total cobalamin were also determined. Whereas holo-haptocorrin correlated well (r(2) = 0.87) with total cobalamin, holo-transcobalamin correlated poorly (r(2) = 0.23) with total cobalamin or holo-haptocorrin. CONCLUSIONS: The solid-phase capture assay provides a simple, reliable method for quantitative determination of holo-transcobalamin in serum.