Reduced editing of low-affinity kainate receptor subunits in optic nerve glial cells.

We have analyzed the degree of editing of adult optic nerve mRNAs encoding the low-affinity kainate receptor subunits, GluR5 and GluR6, the two major constituents of native receptors in this family. To this end, we used reverse transcription (RT) followed by polymerase chain reaction (PCR), and subsequent cloning and sequencing of the amplified fragments. Our results revealed that the GluR5 subunit is unedited at the Q/R site of cismembrane domain 2 (M2), whereas the GluR6 subunit is edited to a low extent at this site. These findings are in contrast to those reported by others using mRNAs from the adult brain in which GluR5 and GluR6 are edited at the Q/R site of M2 to a larger extent. In addition, we found that the adenosine deaminases, DRADA, RED1 and RED2, which edit ionotropic glutamate receptors in the brain, are expressed in the adult optic nerve and in oligodendrocytes, the major cell type in this structure. It thus appears that editing of kainate receptors in optic nerve cells is not limited by the availability of editing enzymes but rather by other, as yet unidentified factors. Overall, reduced editing of kainate receptor subunits in glial cells may result in different functional responses of the native receptors present in these cells with respect to those in neurons.

Identification of a new apolipoprotein E variant (E2 Arg142-->Leu) in type III hyperlipidemia.

A new rare apolipoprotein E mutant was identified as we were investigating the apolipoprotein E genotype of patients with type III hyperlipidemia (HLP III). The unusual DNA restriction fragment length polymorphism profile and then the sequence analysis of a PCR amplified fragment of the proband's apo E gene revealed a simple base substitution (G-->T) at nucleotide 3836. This mutation leads to the replacement of arginine by leucine at position 142 of the mature protein. The proband carried the mutant allele at the heterozygous status with an epsilon 3 allele. Subsequently, analysis of the proband's father's apo E gene showed that same mutated allele associated with an epsilon 2 allele. The two subjects presented a dysbetalipoproteinemia in which this new apo E variant could be implicated.

Genotyping of a patient homozygous for a rare apolipoprotein E1 [Gly127-->Asp; Arg158-->Cys] (Weisgraber allele).

We examined the apolipoprotein E polymorphism of an obese patient presenting non-insulin-dependent diabetes, hypertension and moderate lipid disturbances. The apolipoprotein E genotyping carried out from leukocyte DNA using PCR amplification and restriction enzyme digestion demonstrated homozygosity for the rare apoE1[Gly127-->Asp; Arg158--> Cys] (Weisgraber allele). The nucleotide change results in a glycine to aspartic acid substitution at amino acid 127 in the apolipoprotein E2.

Application of PCR site-directed mutagenesis for a rapid and accurate detection of mutation 3500 (Arg-->Gln) of human apolipoprotein B-100.

A rapid detection of the Arg3500-->Gln mutation of human apolipoprotein B-100 is of particular interest because of its prevalence in familial forms of hypercholesterolemia. A simple procedure, based on amplification by polymerase chain reaction (PCR) and NlaIII endonuclease restriction cleavage, allows this diagnosis without ambiguity. By using two oligonucleotide primers carrying one mismatch each, two permanent restriction sites were generated in the normal allele, while one of them disappeared in the mutant allele. Thus, the two alleles can be differentiated by their specific N/aIII restriction profile.

Common and rare genotypes of human apolipoprotein E determined by specific restriction profiles of polymerase chain reaction-amplified DNA.

The three common isoforms of human apolipoprotein E (apo E) differ at positions 112 and 158 and are named E3, E4, and E2 according to phenotyping by isoelectric focusing (IEF). The polymerase chain reaction (PCR) method allows the detection of common and several rare allelic apo E variants not detected by IEF. We propose a genotyping procedure for apo E that characterizes a given allele on the basis of amplification of specific sequences of the gene followed by the action of restriction endonucleases. When the nucleotide change does not lead to a restriction site, PCR-directed mutagenesis creates the discriminant site, and the differentiation of the three common alleles and five rare variants is possible. We present here profiles of common alleles and of three rare alleles, Weisgraber [Cys112/Asp127/Cys158], Christchurch [Cys112/Ser136/Arg158], and a new rare variant [Cys112/Leu142/Cys158].