Molecular analyses of an acidic transthyretin Asn 90 variant

A mutation in transthyretin (TTR Asn 90) has been identified in the Portuguese and German populations. This variant has a lower pI and was found by screening analyses in 2/4,000 German subjects and in 4/1,200 Portuguese by using either double one-dimensional (D1-D) electrophoresis with isoelectric focusing (IEF) or hybrid isoelectric focusing in immobilized pH gradient (HIEF) as the final separation step. The Portuguese population sample was from the area where TTR Met 30-associated familial amyloidotic polyneuropathy (FAP) prevails, and it was divided into (a) a group of 500 individuals belonging to FAP kindreds and (b) a group of 700 collected at random. HIEF showed two particular situations: (1) one case, from an FAP kindred, was simultaneously carrier of the Met 30 substitution and the acidic variant, and (2) one individual, from the randomly selected Portuguese sample, had only the acidic monomer. Comparative peptide mapping, by HPLC, of the acidic variant carriers and of normal TTR showed the presence of an abnormal tryptic peptide, not present in the normal TTR digests, with an asparagine-for-histidine substitution at position 90 explained by a single base change of adenine for cytosine in the histidine codon. This was confirmed at the DNA level by RFLP analyses of PCR-amplified material after digestion with SphI and BsmI. In all carriers of the Asn 90 substitution, no indicators were found for an association with traits characteristic for FAP.