Abstract

A transthyretin (TTR) mutation is described in a 44 year old French woman from Caen who presented at the age of 40 with neuropathy in all four extremities, diarrhoea, and orthostatic hypotension. Her father died with a similar syndrome including vitreous opacities. A nerve biopsy from the proband showed amyloid deposits which stained with anti-transthyretin. Direct genomic DNA sequencing of TTR exon 3 showed both thymine and cytosine in the position corresponding to the second base of codon 71. This codes for a variant alanine (GCG) as well as the normal valine (GTG), indicating that the proband is heterozygous for the substitution. Since this substitution does not result in the creation or abolition of a restriction endonuclease recognition site, a new technique (PCR-IMRA) was used to create an RFLP. Using a 24 bp nucleotide mutagenesis primer in the PCR reaction, a new NspBII site is created on amplification of the variant allele. With this method a 170 bp TTR exon 3 PCR product was generated for both the normal and the variant allele. On digestion of the PCR product with NspBII, DNA from a heterozygous subject showed both the 170 bp undigested product from the normal allele and a 146 bp digestion product from the variant allele. By PCR-IMRA, two of five children of the proband were positive for the variant allele. This non-radioactive technique gives a rapid method for testing subjects at risk for this mutation.