cag repeats  (Addgene inc)

Journal: eLife

Article Title: Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (p97-PROTAC)

doi: 10.7554/eLife.101496

Figure Lengend Snippet: HeLa cells were transiently co-transfected with HTT GFP-tagged plasmids containing either 23 CAG repeats (EGFP-HTT Q23 -wild-type HTT), 74 CAG repeats (EGFP-HTT Q74 : mutant HTT), or 24 CAG repeats (EGFP-HTT Q24 ). ( A ) Cells were co-transfected with GFP-HTT Q23 and increasing amounts of the p97 PROTAC UBX-Nb (GFP) , degradation was determined by western blot analysis. ( B ) Quantification of A, 2 μg ‘p-value’ 0.0041 (**), 4 μg ‘p-value’ 0.0014 (** ). ( C ) Immunofluorescence showing the recruitment of p97 PROTAC UBX-Nb (GFP) to GFP-HTT Q23 . ( D ) HeLa cells were co-transfected with GFP-HTT Q74 and increasing amounts of the p97 PROTAC UBX-Nb (GFP) , degradation was determined by western blot analysis. ( E ) Quantification of D, 2 μg ‘p-value’ 0.0016 (**), 4 μg ‘p-value’ 0.0003 (*** ). ( F ) Immunofluorescence to demonstrate the recruitment of p97 PROTAC UBX-Nb (GFP) to GFP-HTT Q74 . Western blots were quantified and statistically analyzed using a Student’s t-test. p<0.05 compared to controls. n=3. Figure 5—source data 1. Raw Western blots showing p97-PROTAC–mediated degradation of GFP-HTTQ23 and GFP-HTTQ74. Figure 5—source data 2. Annotated Western blots indicating protein bands for p97-PROTAC–mediated degradation of GFP-HTTQ23 and GFP-HTTQ74.

Article Snippet: Cells were transiently transfected with the following vectors: pcDNA5 FRT/TO GFP-ETV1, pcDNA5 FRT/TO GFP-Emerin, pEYFP-Coilin, pcDNA FRT/TO GFP, VCP(wt)-EGFP (Addgene, #23971), pEGFP-C1-tagged plasmids containing the exon 1 of HTT with 23 CAG repeats (pEGFP-Q23: wild-type HTT, Addgene, #40261) or 74 CAG repeats (pEGFP-Q74: mutant HTT, Addgene, #40262) and pEGFP-Q24 (generously provided by Dr. Maite Castro, Universidad Austral de Chile).

Techniques: Transfection, Mutagenesis, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Genetic modifiers of somatic expansion and clinical phenotypes in Huntington’s disease reveal shared and tissue-specific effects

doi: 10.1101/2024.06.10.597797

Figure Lengend Snippet: A. The somatic expansion ratio of the HTT exon 1 CAG repeat in blood DNA is allele length- and age-dependent. The scatterplot shows the somatic expansion ratio (SER = in blood DNA plotted against age at sampling in 3,999 Enroll-HD participants. Points are color-coded based on the length of the inherited uninterrupted CAG length determined by MiSeq. Lines on the scatterplot are the SER values predicted by the multiple linear regression ln(SER) ∼ β 0 + β 1 .CAG + β 2 .age + β 3 .(CAG x age) + β 4 .CAG 2 + β 5 .age 2 + β 6 .(CAG 2 x age) + β 7 .(CAG x age 2 ) + ε 0 . Boxplot (25th and 75th percentiles (box), median (line), and range (whiskers, capped at 1.5x the interquartile range)) of the relationship between canonical and non-canonical HTT repeat sequences with the somatic expansion ratio in blood DNA adjusted for CAG length, age, CCG length and PCR batch ( i.e., the residuals of the multiple linear regression ln(SER) ∼ β 0 + β 1 .CAG + β 2 .age + β 3 .(CAG x age) + β 4 .CAG 2 + β 5 .age 2 + β 6 .(CAG 2 x age) + β 7 .(CAG x age 2 ) + β 8 .CCG + β 9 .PCRbatch). B. Boxplot of average number of CAG repeats gained over four weeks for HTT exon 1 variants knocked into the AAVS1 locus in RPE1 cells. Clonal knock-in RPE1 lines with canonical (n = 7), CAACAG-dup (n = 8), or CAA/CCA-loss (n = 11) HTT exon 1 variants are each represented by a single dot, positioned based on the average repeat gain of triplicate cultures. The average repeat gain represents the difference between mean CAG repeat (weighted on the fragment analysis peak height) at four-weeks and day-zero, which was adjusted for the effect size of day zero mean repeat length that ranged between 112-120 CAGs. Both CAACAG-dup and CAA/CCA-loss alleles showed significantly reduced CAG repeat expansion compared to canonical (p < 0.001 and = 0.015, respectively). C. Somatic expansion ratio length in post-mortem frontal cortex from 488 HD individuals carrying canonical expanded repeat sequences (open circles) and 5 carrying CAA/CCA-loss HD alleles (filled red circles) is plotted versus modal HTT CAG repeat D. Somatic expansion ratio in post-mortem frontal cortex from 488 HD individuals carrying canonical repeat sequences (open circles) and 5 carrying CAA/CCA-loss HD chromosomes (filled red circles) is plotted versus their ages at death. E. Boxplot of the relationship between canonical and non-canonical HTT repeat sequences with the somatic expansion ratio in HD frontal cortex DNA, adjusted for CAG length, age, CCG length and PCR batch as described in B. The CAA/CCA-loss allele carriers were not significantly different from those with canonical alleles (p = 0.96)

Article Snippet: The resulting plasmids with matching uninterrupted CAG repeat sequences were nanopore sequenced (Plasmidsaurus, SNPsaurus LLC) to confirm sequence identity and the repeat size was quantified by fragment analysis as described in the HTT CAG repeat genotyping section.

Techniques: Sampling, Knock-In

Journal: bioRxiv

Article Title: Genetic modifiers of somatic expansion and clinical phenotypes in Huntington’s disease reveal shared and tissue-specific effects

doi: 10.1101/2024.06.10.597797

Figure Lengend Snippet: A . GWAS of somatic CAG expansion measured by the PPS method applied to ABI blood CAG sizing traces is shown for SNVs with MAF > 1%. The dashed black line represents the threshold for genome-wide significance (p = 5.0E-08). B . An example of ABI traces from a CAACAG-dup allele (top) and a canonical allele (bottom), both carrying uninterrupted repeat lengths of 44 CAGs. The top panel reveals a mispriming artefact that scores as CAG expansion in the PPS method, leading to spurious signal at HTT in the GWAS of panel A. C . Association analysis of the PPS CAG expansion phenotype conditioned on rs183415333, which tags the non-canonical CAACAG-dup allele on HD chromosomes, is shown for SNVs with MAF > 1% in the HTT region. Conditioning removes the spurious signal and leaves rs146151652 as the top SNV, the same HTT 5’-UTR SNV detected in the MiSeq somatic CAG expansion GWAS ( , ).

Article Snippet: The resulting plasmids with matching uninterrupted CAG repeat sequences were nanopore sequenced (Plasmidsaurus, SNPsaurus LLC) to confirm sequence identity and the repeat size was quantified by fragment analysis as described in the HTT CAG repeat genotyping section.

Techniques: Genome Wide

Journal: bioRxiv

Article Title: Genetic modifiers of somatic expansion and clinical phenotypes in Huntington’s disease reveal shared and tissue-specific effects

doi: 10.1101/2024.06.10.597797

Figure Lengend Snippet: A. The somatic expansion ratio of the HTT exon 1 CAG repeat in blood DNA is allele length- and age-dependent. The scatterplot shows the somatic expansion ratio (SER = in blood DNA plotted against age at sampling in 3,999 Enroll-HD participants. Points are color-coded based on the length of the inherited uninterrupted CAG length determined by MiSeq. Lines on the scatterplot are the SER values predicted by the multiple linear regression ln(SER) ∼ β 0 + β 1 .CAG + β 2 .age + β 3 .(CAG x age) + β 4 .CAG 2 + β 5 .age 2 + β 6 .(CAG 2 x age) + β 7 .(CAG x age 2 ) + ε 0 . Boxplot (25th and 75th percentiles (box), median (line), and range (whiskers, capped at 1.5x the interquartile range)) of the relationship between canonical and non-canonical HTT repeat sequences with the somatic expansion ratio in blood DNA adjusted for CAG length, age, CCG length and PCR batch ( i.e., the residuals of the multiple linear regression ln(SER) ∼ β 0 + β 1 .CAG + β 2 .age + β 3 .(CAG x age) + β 4 .CAG 2 + β 5 .age 2 + β 6 .(CAG 2 x age) + β 7 .(CAG x age 2 ) + β 8 .CCG + β 9 .PCRbatch). B. Boxplot of average number of CAG repeats gained over four weeks for HTT exon 1 variants knocked into the AAVS1 locus in RPE1 cells. Clonal knock-in RPE1 lines with canonical (n = 7), CAACAG-dup (n = 8), or CAA/CCA-loss (n = 11) HTT exon 1 variants are each represented by a single dot, positioned based on the average repeat gain of triplicate cultures. The average repeat gain represents the difference between mean CAG repeat (weighted on the fragment analysis peak height) at four-weeks and day-zero, which was adjusted for the effect size of day zero mean repeat length that ranged between 112-120 CAGs. Both CAACAG-dup and CAA/CCA-loss alleles showed significantly reduced CAG repeat expansion compared to canonical (p < 0.001 and = 0.015, respectively). C. Somatic expansion ratio length in post-mortem frontal cortex from 488 HD individuals carrying canonical expanded repeat sequences (open circles) and 5 carrying CAA/CCA-loss HD alleles (filled red circles) is plotted versus modal HTT CAG repeat D. Somatic expansion ratio in post-mortem frontal cortex from 488 HD individuals carrying canonical repeat sequences (open circles) and 5 carrying CAA/CCA-loss HD chromosomes (filled red circles) is plotted versus their ages at death. E. Boxplot of the relationship between canonical and non-canonical HTT repeat sequences with the somatic expansion ratio in HD frontal cortex DNA, adjusted for CAG length, age, CCG length and PCR batch as described in B. The CAA/CCA-loss allele carriers were not significantly different from those with canonical alleles (p = 0.96)

Article Snippet: The resulting plasmids with matching uninterrupted CAG repeat sequences were nanopore sequenced (Plasmidsaurus, SNPsaurus LLC) to confirm sequence identity and the repeat size was quantified by fragment analysis as described in the HTT CAG repeat genotyping section.

Techniques: Sampling, Knock-In

Journal: bioRxiv

Article Title: Genetic modifiers of somatic expansion and clinical phenotypes in Huntington’s disease reveal shared and tissue-specific effects

doi: 10.1101/2024.06.10.597797

Figure Lengend Snippet: A . GWAS of somatic CAG expansion measured by the PPS method applied to ABI blood CAG sizing traces is shown for SNVs with MAF > 1%. The dashed black line represents the threshold for genome-wide significance (p = 5.0E-08). B . An example of ABI traces from a CAACAG-dup allele (top) and a canonical allele (bottom), both carrying uninterrupted repeat lengths of 44 CAGs. The top panel reveals a mispriming artefact that scores as CAG expansion in the PPS method, leading to spurious signal at HTT in the GWAS of panel A. C . Association analysis of the PPS CAG expansion phenotype conditioned on rs183415333, which tags the non-canonical CAACAG-dup allele on HD chromosomes, is shown for SNVs with MAF > 1% in the HTT region. Conditioning removes the spurious signal and leaves rs146151652 as the top SNV, the same HTT 5’-UTR SNV detected in the MiSeq somatic CAG expansion GWAS ( , ).

Article Snippet: The resulting plasmids with matching uninterrupted CAG repeat sequences were nanopore sequenced (Plasmidsaurus, SNPsaurus LLC) to confirm sequence identity and the repeat size was quantified by fragment analysis as described in the HTT CAG repeat genotyping section.

Techniques: Genome Wide