Brain autopsy of the proband was performed shortly after death with informed consent. Genomic DNA was extracted from the brain using Qiagen's DNA purification kit according to the manufacturer's instructions. The
PRNP open reading frame was amplified by polymerase chain reaction (PCR) using a protocol and primers described elsewhere [
4]. The genotype at codon 129 of
PRNP was determined by digestion with the restriction endonuclease Nsp I. Analysis of
PRNP sequences was performed by direct sequencing in a MacBAC sequencer (Pharmacia, USA). A missense mutation (T to G) was identified at the position of nt 341 in one
PRNP allele, leading to change from glycine (Gly) to valine (Val) at codon 114 (Figure
2B). No other nucleotide exchange was found in the rest of the
PRNP sequence. Nsp I digestion and direct sequencing of the amplified product revealed a methionine homozygous genotype at codon 129 of
PRNP. To identify the distribution of this point-mutation in the family, blood samples of five other family members, including the son of her first cousin (IV 2), were collected and the
PRNP genes were sequenced. As suspected, the same G114V mutation was observed in the
PRNP gene of IV 2. In addition, two other health family members, the proband's daughter (IV 10, age of 22) and the mother of the second case (III 1, age of 61), were found to have the same missense mutation. The son of the proband case (IV 9, age of 24) and the son of IV 2 (V 3, age of 9) were confirmed to have a wild-type
PRNP sequence without such mutation. All tested family members were homozygous for methionine (M/M) at codon 129 of
PRNP as in the profile of Han Chinese [
5].