Đột Biến Gen α-Synuclein Được Xác Định Trong Cộng Đồng Gia Đình Bệnh Parkinson

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DNA samples were collected after informed consent. For mutation analysis genomic DNA was amplified with primer 3 (5′-GCTAATCAGCAATTTAAGGCTAG-3′) and primer 13 (5′-GATATGTTCTTAGATGCTCAG-3′) of the DNA sequence (GenBank ID U46898) under standard PCR conditions. Sequence analysis was performed with the Perkin-Elmer dye terminator cycle sequencing kit on an ABI 373 fluorescent sequencer (ABI Foster City CA). Restriction digestion was done after the PCR with Tsp45 I according to the manufacturer's protocol (New England Biolabs Beverly MA). The digested PCR products were separated by electrophoresis on a 6% Visigel (Stratagene La Jolla CA) and visualized by ethidium bromide staining. Pedigree structures in Fig. 2 have been slightly modified to protect patient confidentiality.

Total RNA was extracted from the lymphoblastoid cell line of an affected individual and first-strand synthesis was performed by oligo (dT) priming (Gibco-BRL Gaithersburg MD). Primer 1F: (5′-ACGACAGTGTGGTGTAAAGG-3′) and primer 13R 5′-AACATCTGTCAGCAGATCTC-3′ corresponding to nucleotides 21 to 40 and 520 to 501 of the published sequence were used to amplify a product of 500 bp containing the mutation nucleotide 209. PCR products were subjected to restriction digestion by Tsp45 I (5). The mutation at nucleotide 209 creates a novel Tsp45 I site (Fig. 1) so that the normal allele can be cut into four fragments of 249 218 24 and 9 bp whereas the mutant allele will have five fragments of 249 185 33 24 and 9 bp (Fig. 3). Size standards used were the 100-bp ladder as provided by Gibco-BRL (Gaithersburg MD).

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Abbreviations for the amino acid residues are: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Try.

The authors would like to thank V. V. McKoy and Y. Chen for technical assistance V. Bonavita for sample collection Z. Hall for his continuing encouragement and support the NINDS intramural research program for resources and F. Collins and H. Varmus for critical review of the manuscript.