Instability of the Octarepeat Region of the Human Prion Protein Gene

Prion diseases are a family of unique fatal transmissible neurodegenerative diseases that affect humans and many animals. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common prion disease in humans, accounting for 85–90% of all human prion cases, and exhibits a high degree of diversity in phenotypes. The etiology of sCJD remains to be elucidated. The human prion protein gene has an octapeptide repeat region (octarepeats) that normally contains 5 repeats of 24–27 bp (1 nonapeptide and 4 octapeptide coding sequences). An increase of the octarepeat numbers to six or more or a decrease of the octarepeat number to three is linked to genetic prion diseases with heterogeneous phenotypes in humans. Here we report that the human octarepeat region is prone to either contraction or expansion when subjected to PCR amplification in vitro using Taq or Pwo polymerase and when replicated in wild type E. coli cells. Octarepeat insertion mutants were even less stable, and the mutation rate for the wild type octarepeats was much higher when replicated in DNA mismatch repair-deficient E.coli cells. All observed octarepeat mutants resulting from DNA replication in E.coli were contained in head-to-head plasmid dimers and DNA mfold analysis (http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form) indicates that both DNA strands of the octarepeat region would likely form multiple stable hairpin structures, suggesting that the octarepeat sequence may form stable hairpin structures during DNA replication or repair to cause octarepeat instability. These results provide the first evidence supporting a somatic octarepeat mutation-based model for human sCJD etiology: 1) the instability of the octarepeat region leads to accumulation of somatic octarepeat mutations in brain cells during development and aging, 2) this instability is augmented by compromised DNA mismatch repair in aged cells, and 3) eventually some of the octarepeat mutation-containing brain cells start spontaneous de novo prion formation and replication to initiate sCJD

Instability of octarepeats during DNA replication in DH5α cells.

<p>(A) Mutant clones from replication of pOct5 in DH5α cells. pOct5 was transformed into DH5α. Plasmid DNAs were prepared from the resulting colonies, digested with <i>Sac</i>II and <i>Spe</i>I and separated on a 2% agarose gel. Shown are plasmid DNAs from 3 colonies that produced two octarepeat bands of equal molar ratio. (B) Mutant clones from replication of pOct11b in DH5α cells. pOct11b was transformed into DH5α. Plasmid DNAs were prepared from the resulting colonies, digested with <i>Sac</i>II and <i>Spe</i>I and separated on a 2% agarose gel. Shown are plasmid DNAs from 4 colonies that produced two octarepeat bands of equal molar ratio. (C–D) Unusual mutant clones from replication of pOct11b in DH5α cells. Shown are plasmid DNAs from two pOct11b-transformed DH5α colonies that produced 2–3 octarepeat bands upon digestion with <i>Sac</i>II and <i>Spe</i>I, of which the template-sized band is much stronger than the mutant bands (C). The unequal molar ratio of the octarepeat bands suggests the presence in these colonies of mixed plasmid DNA species where each species produced one of the octarepeat bands. Re-transformation of these plasmid DNAs into DH5α cells resulted in separation of the mixed plasmid DNA species and produced colonies that each contained only one plasmid DNA species as confirmed by restriction analysis and sequencing (D). For all panels, the octarepeat sequence is indicated above each lane, the arrowhead points to the band whose sequence is shown above the lane, and the black box marks the template-sized Oct5 or Oct11 band from a non-mutant clone. Rep. No., number of repeats; M,100-bp DNA Ladder.</p

A hairpin structure-based model for mutation of octarepeats.

<p>(A) Diagram of hairpin structures of Oct5 and Oct11b as predicted by mfold. The likely secondary structures of Oct5 and Oct11b octarepeat sequences were examined with the online mfold program (<a href="http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form" target="_blank">http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form</a>). Only the most stable structure for each sequence as predicted by mfold at the PCR annealing condition (50 mM Na⁺, 1.5 mM Mg²⁺, 58.5°C) is depicted, but the <i>E.coli</i> condition (170 mM Na⁺, 37°C) yielded similar results. The three colored hairpins represent the common ones shared by Oct5 and Oct11b sequences; the insertion of 6 extra repeats in Oct11b led to 6 additional hairpins (in black) that are identical to the first hairpin (in orange). (B) Hairpin-based slippage model for contraction or expansion of the octarepeats. The parental DNA strands are in thick lines and the newly synthesized DNA strands (leading strand or lagging stand) are in thin lines. The red lines denote the octarepeat region on parental DNA strands while green lines denote the octarepeat region on the newly synthesized DNA strands; hairpin formation on the parental DNA strand leads to contraction whereas hairpin formation on the lagging strand being synthesized results in expansion. Adopted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026635#pone-0026635-g003" target="_blank">Figure 3</a> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026635#pone.0026635-Mirkin1" target="_blank">[29]</a>.</p

Instability of octarepeats during PCR amplification by Pwo polymerase.

<p>(A) PCR products from the PrP-Oct5 and PrP-Oct11a templates. The octarepeat regions PCR amplified by Pwo polymerase from PrP-Oct5 and PrP-Oct11a with primers HP20 and HP306r were cleaned up and separated on a 2% agarose gel. Bl, blank control. (B) Mutant octarepeat clones from PCR amplification of the PrP-Oct5 template: restriction analysis with <i>Sac</i>II and <i>Spe</i>I. Six mutant clones and one wild type clone are shown. The black box marks the template-sized Oct5 band from a non-mutant clone. (C) Mutant octarepeat clones from PCR amplification of the PrP-Oct11a template: restriction analysis with <i>Sac</i>II and <i>Spe</i>I. Same as in (B) except that PrPOct11a was the template DNA. Eighteen mutant clones and one wild type clone are shown. The black box marks the template-sized Oct11 band from a non-mutant clone. (D) A mutant octarepeat clone containing two octarepeat inserts from PCR amplification of PrP-Oct5. <i>Sac</i>II and <i>Spe</i>I digestion of this mutant plasmid clone produced two octarepeat inserts; one was the wild type Oct5 while the other was a 2-repeat deletion mutant (R1-R2). The arrowhead points to the band whose sequence is shown above the lane. The black box marks the template-sized Oct5 band from a non-mutant clone. (E) Mutant octarepeat clones containing two octarepeat inserts from PCR amplification of PrP-Oct11a. <i>Sac</i>II and <i>Spe</i>I digestion of the 3 mutant clones produced two octarepeat inserts; one was the 11-repeat parental Oct11a in all clones while the other was a mutant octarepeat sequence of varying sizes and sequences. The arrowhead points to the band whose sequence is shown above the lane. The black box marks the template-sized Oct11 band from a non-mutant clone. For all panels, the octarepeat sequence is indicated above each lane; Rep. No., number of repeats; M,100-bp DNA Ladder.</p

Schematic diagrams for analysis of octarepeat mutation rate during PCR or replication in <i>E.coli</i>.

<p>(A) Measurement of octarepeat mutants resulting from PCR amplification of octarepeats. Cloned PrP ORF sequences (PrP-Oct5 and PrP-Oct11a) were subjected to PCR with primers HP20 and HP306r. The PCR products (depicted in a shaded box), which contained input (green line) and PCR-mutant (red line) octarepeat sequences as well as “paired” molecules (two parallel lines), were cleaned up and ligated to the pGEM-T vector, producing 4 kinds of ligation products: mutant monomer, wild type monomer, wild type dimer, and mutant dimer. The ligation products were transformed into DH5α competent cells, and the resulting colonies were directly examined by PCR with primers HP50f and HP293r. Plasmid DNAs were extracted from colonies containing mutant octarepeats, subjected to restriction analysis with <i>Sac</i>II and <i>Spe</i>I, and sequenced. The number of mutant colonies over the total number of colonies screened was calculated to represent the octarepeat mutation rate during the PCR process. Small oval: individual <i>E.coli</i> cell; big dashed-line oval: <i>E.coli</i> colony. (B) Measurement of octarepeat mutants resulting from DNA replication in <i>E.coli</i>. pOct5 or pOct11b was used to transform competent DH5α or XL-1 Red <i>E.coli</i> cells, and plasmid DNA sample prepared from a single colony (depicted in a shaded box) was used to transform competent DH5α cells. The mutant colonies (containing only mutant plasmid, red dashed-line oval) and new mutant colonies [containing some cells with the NEW replication-mutant octarepeat insert (light purple), light blue dashed-line oval] were screened out as in (A). The number of mutant colonies (excluding the new mutant colonies) over the total number of colonies examined should reflect the octarepeat mutation rate during the 1^st round of plasmid replication and cell division in <i>E.coli</i> (DH5α or XL-1 Red).</p

Human <i>PRNP</i> octarepeat sequences and cloned octarepeats for instability analysis.

<p>(A) Wild type and mutant human octarepeat sequences. In the mutant octarepeats, the mutated bases are in bold case and underlined. R14 could be a chimera repeat between R1 and R4; R1a could be a chimera repeat between R1 and R3; R2a could be a chimera between R2 and R3. The repeats in pOct5, pOct11a and pOct11b are listed. (B) Diagram of cloned wild type human <i>PRNP</i> octarepeats used for instability analysis. PrP-Oct5: a region encompassing the wild type PrP ORF (762 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into pGEM-T after PCR amplification (template: wt human genomic DNA, primers: 42F and 45R). pOct5: the wild type octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct5, primers: HP20 and HP306r). Arrows denote the primers. (C) Diagram of cloned insertion mutant human <i>PRNP</i> octarepeats used for instability analysis. PrP-Oct11a or PrP-Oct11b: a region encompassing an 11-repeat mutant PrP ORF (906 bp), 232 bp upstream non-coding sequence and 271 bp downstream non-coding sequence subcloned into pGEM-T after PCR amplification (template: one of two human genomic DNA samples containing different 11-repeat octarepeats, primers: 42F and 45R). pOct11a or pOct11b: the 11-repeat octarepeat region subcloned into pGEM-T after PCR amplification (template: PrP-Oct11a or PrP-11b, primers: HP20 and HP306r). Arrows denote the primers.</p

Mutation rate of octarepeats during PCR or DNA replication in <i>E.coli.</i>

a<p>Number of colonies with mutant octarepeats/total colonies screened; percentage in the parenthesis.</p>b<p>Each of the three plasmid preparations was made from a single <i>E.coli</i> colony.</p

Instability of octarepeats during PCR amplification by Taq Polymerase.

<p>(A) PCR products from the PrP-Oct5 and PrP-Oct11a templates. The octarepeat regions PCR amplified by Taq polymerase from PrP-Oct5 and PrP-Oct11a with primers HP20 and HP306r were cleaned up and separated on a 2% agarose gel. (B) Mutant octarepeat clones from PCR amplification of the PrP-Oct5 template: restriction analysis with <i>Sac</i>II and <i>Spe</i>I. Six mutant clones and one wild type clone are shown. The black box marks the template-sized Oct5 band from a wild type clone. (C) Mutant octarepeat clones from PCR amplification of the PrP-Oct11a template: restriction analysis with <i>Sac</i>II and <i>Spe</i>I. Same as in (B) except that PrPOct11a was the template. Fifteen mutant clones and one wild type clone are shown. The black box marks the template-sized Oct11 band from a non-mutant clone. (D) A mutant octarepeat clone containing two octarepeat inserts from PCR amplification of PrP-Oct5. <i>Sac</i>II and <i>Spe</i>I digestion of this mutant clone produced two octarepeat inserts; one was the 5-repeat wild type Oct5 while the other was a 2-repeat deletion mutant (R1a-R4). The arrowhead points to the band whose sequence is shown above the lane. The black box marks the template-sized Oct5 band from a non-mutant clone. (E) Mutant octarepeat clones containing two octarepeat inserts from PCR amplification of PrP-Oct11a. <i>Sac</i>II and <i>Spe</i>I digestion of the 10 mutant clones produced two octarepeat inserts; one was the 11-repeat parental Oct11a in all clones while the other was a mutant octarepeat sequence of varying sizes and sequences. The arrowhead points to the band whose sequence is shown above the lane. The black box marks the template-sized Oct11 band from a non-mutant clone. For all panels, the octarepeat sequence is indicated above each lane; Rep. No., number of repeats; M,100-bp DNA Ladder.</p