A Streamlined PCR-based Fragment Analysis Assay that Resolves...
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Figure 1. Genotyping APOE and TOMM40 in a streamlined workflow from sample-to answer. A) APOE is found on human Chromosome 19 (figure adapted from Liu et. al. 5 ). Three common alleles designated ε2, ε3, and ε4 corresponding to 2 SNP variants in Exon 4 were associated with AD in 1993 6 . Unique primers were designed to specifically amplify each allele differentiated based by amplicon size. B) The variable poly-T tract of TOMM40 is located in intron 6 of the gene (blue star). This length polymorphism was binned into the following genotypes: Short (≤19 Ts), Long (20-29 Ts), and Very Long (≥30 T). C) For the prototype assay, PCR and thermocycling conditions were independent but similar for the two targets. Dye-labelled amplicons were combined in a single CE injection after PCR. The unified assay has a turnaround time of ~4.5 hrs for 24 samples and a total hands-on time of ~1.5 hrs. APOE SNP Cys (T) Cys (T) Cys (T) Arg (C) Arg (C) Arg (C) 136 150 167 206 244 272 299 COOH NH2 1 rs:429358 (aa:112) rs:7412 (aa:158) Receptor Binding Lipid Binding Hinge Helix 1 Helix 2 Helix 3 aa ...CGTG [T/C] GCGG...GAAG [T/C] GCCT... ...GCAC [A/G] CGCC...CTTC [A/G] CGGA... + 5' - 3' 5' 3' ApoE 2 ApoE 3 ApoE 4 Summary • Alzheimer’s disease (AD) is an irreversible neurodegenerative disorder that progresses slowly over time, affecting 5.4M Americans in 2016, or 1 in 10 Americans age 65 and older. • Sequence variants in TOMM40 and APOE are two genetic risk predictors of AD. However, these sequences are difficult to genotype using conventional PCR due to their AT- and GC-rich character. • We unified a proof-of-concept APOE assay with our AmplideX ® PCR/CE TOMM40 Kit (RUO) to simultaneously genotype both genetic markers. • Here we report the first single-assay workflow that achieves reproducible and accurate resolution of TOMM40 poly-T length polymorphisms and all 6 APOE SNP genotypes. Introduction Alzheimer’s disease (AD) accounts for 60-80% of all dementias in the US 1 . Previous studies have identified an AD susceptibility locus in a region that includes Apolipoprotein E (APOE) and Translocase Of Outer Mitochondrial Membrane 40 (TOMM40). Of the three common APOE alleles, ε4 is the most informative but in only ~25% of Caucasians 2 . Independently, poly-T polymorphisms within the TOMM40 gene have been reported to influence both age of onset in late-onset AD (LOAD) and measures of cognitive decline elevating informative risk data for LOAD to ~97% of individuals 2,3,4 . The AmplideX ® PCR/CE TOMM40 Kit (RUO) is a commercially available option for this marker. Here we describe the first PCR-based workflow that unifies APOE and TOMM40 genotyping. Materials and Methods Cell-line gDNA samples (14 AD, 104 other) were acquired from the Coriell Institute. Whole blood (WB; 97) and matched WB/buccal gDNA samples (77) were isolated from presumed healthy donors (Asuragen, Equitech Enterprises). Sample gDNA was PCR amplified using prototype AmplideX reagents (Asuragen) by different operators on different days. Amplicons tagged with FAM (TOMM40) and HEX (APOE) were resolved on a 3500xL Genetic Analyzer (Thermo Fisher Scientific) CE instrument using POP-7™ polymer with 2.5kV, 20 sec injection and 20 min run time. Genotype analysis utilized a ROX 400HD size ladder (Thermo Fisher Scientific) in separate color channels from target amplicon patterns (APOE) or from the mobility of target amplicon (TOMM40) relative to a three-point cell-line DNA calibrator (16T, 29T, 36T poly-T lengths). References 1.https://www.alz.org/documents_custom/2017-facts-and-figures.pdf, (Sourced Jun 30 2017). 2.Crenshaw DG, et al. (2013) Clin Pharmacol Ther. 93(2), 177–85. 3.Saunders, A. M., et al. (2000) Biochim Biophys Acta - Mol Basis Dis. 1502, 85–94. 4.Yu, L., et al. (2017) Neurology. 88(7), 661–668. 5.Liu, Chia-Chen, et al. (2013) Nat Rev Neuro. 9(2), 106–118. 6.Corder, E. H., et al. (1993) Science. 261 (5123), 921–923. 7.Lutz, M. W., et al. (2016) Alz & Dem. 2(1), 30–44. 8.Roses, A. D., et al. (2010) Pharmacogenomics J. 10, 375–84. Amplidex PCR/CE TOMM40 Kit - Research Use Only – Not for Use in Diagnostic Procedures Prototype APOE reagents used to gather preliminary research data. The performance characteristics of this assay have not yet been established. Presented at AAIC 2017 A Streamlined PCR-based Fragment Analysis Assay that Resolves Both Single Nucleotide and Poly-T Length Polymorphisms at APOE and TOMM40 Susceptibility Loci in Alzheimer’s Disease Bradley Hall, Sarah Statt, Julie R Thibert, Jacob Wisotsky, Jon Kemppainen, and Gary J Latham Asuragen, Inc., Austin, TX Results Conclusions • A single PCR/CE technology resolved SNP and length polymorphisms in APOE and TOMM40, respectively, and produced concordant genotypes from 292 samples across three gDNA sources, multiple operators and different days. • This streamlined workflow analyzes purified DNA with a turn-around time of 4.5 hrs. • This multimodal approach has the potential to advance clinical research using a standardized assay that can harmonize results across laboratories. P1-270 #17750 Figure 2. All six possible APOE and TOMM40 genotypes were resolved using PCR methods. A) Five of six APOE genotypes were identified within Coriell cell-line and WB samples. ε2-ε2 was positively identified in PC3 cell line (ATCC). GS is a positive gene specific control. B) The TOMM40 poly-T repeat number was determined within a single nucleotide for all sample genotypes assayed between 15 and 48Ts. C) Genotype distribution for 174 WB samples recapitulated the prevalence of APOE and TOMM40 variants that were previously reported. Although 21.5% of samples had TOMM40 genotypes that fell near allele length boundaries, all calls were repeatable. Figure 3. APOE genotypes resolved by the prototype assay agreed with reference calls for all 292 cell- line, blood and buccal cell samples. A subset of samples and controls were duplicated across different operators and days. All results were repeatable. Input gDNA concentration was measured by A 260 and ranged from 5.5ng to 176.8ng. A) The APOE TaqMan ® SNP Genotyping qPCR Assay (Thermo Fisher Scientific) was used as a reference method to determine agreement. Matched WB (43) and each AD Coriell cell- line genotype (4/6) and NTC (1) are shown. B) Representative WB and buccal PCR electropherograms demonstrate 4 of the 6 possible APOE genotypes. Figure 4. Assayed APOE and TOMM40 genotypes reflect known allele-variant associations. A) Distribution of APOE and TOMM40 genotypes matched for all independent samples from WB (160) and B) Coriell cell-line non- AD (104). Colors represent samples containing one or both alleles with a poly-T length of 30±2 (L/VL boundary) for all but one WB sample with a 20 T allele (S/L boundary). 3A 3B 4A 5A 4B 2A 2B 2C 1A Figure 5. APOE and TOMM40 PCR amplicons could be resolved from a single-assay PCR/CE workflow to assess AD risk. A) Risk algorithm and prevalence were previously published 5,7 . TOMM40 poly-T length was found to have a strong relationship with age of onset, further refining the APOE association with AD with L showing the highest risk 8 . B) Co-injection on CE simplified both the workflow and data analysis, and stratified the largest subset of APOE genotypes (ε3-ε3 and ε4-ε4) into high and low risk groups. Start Yes At least 1 APOE 2 APOE 2- 4 APOE 4- 4 Yes 1-2% Population 3.6% AD incidence No 16-17% Population 3.6% AD incidence '523 L/L or L/VL No No APOE 3- 3 or 3- 4 Yes '523 VL/VL No Yes '523 S/S 77yo S/L 74yo S/VL 76yo No Yes No S 1-19 Ts L 20-29 Ts VL 30-60+ Ts 80% Population 68.3% AD incidence Yes 1-2% Population 24.4% AD incidence Low Risk APOE 2- 2 or 2- 3 or '523 VL/VL High Risk 5B 1B TOMM40 Poly-T 12.39 kb Forward strand Short (S): ≤19 Long (L): 20-29 Very Long (VL): ≥ 30 P 1C PCR CE DATA ANALYSIS TOTAL 30 min ea 15 min 20 min 95 min 120 min 60 min 0 min 3.0 hr Hands-on Time Instrument Time
Transcript of A Streamlined PCR-based Fragment Analysis Assay that Resolves...
Figure 1. Genotyping APOE and TOMM40 in a streamlined workflow from sample-to answer. A) APOE is found on human Chromosome 19 (figure adapted from Liu et. al.5). Three common alleles designated ε2, ε3, and ε4 corresponding to 2 SNP variants in Exon 4 were associated with AD in 19936. Unique primers were designed to specifically amplify each allele differentiated based by amplicon size. B) The variable poly-T tract of TOMM40 is located in intron 6 of the gene (blue star). This length polymorphism was binned into the following genotypes: Short (≤19 Ts), Long (20-29 Ts), and Very Long (≥30 T). C) For the prototype assay, PCR and thermocycling conditions were independent but similar for the two targets. Dye-labelled amplicons were combined in a single CE injection after PCR. The unified assay has a turnaround time of ~4.5 hrs for 24 samples and a total hands-on time of ~1.5 hrs.
APOE SNP
Cys (T) Cys (T)Cys (T) Arg (C)
Arg (C)Arg (C)
136 150 167 206 244 272 299
COOHNH2
1
rs:429358 (aa:112) rs:7412 (aa:158)
ReceptorBinding
LipidBinding
HingeHelix 1 Helix 2 Helix 3
aa
...CGTG[T/C]GCGG...GAAG[T/C]GCCT...
...GCAC[A/G]CGCC...CTTC[A/G]CGGA...+ 5'
- 3' 5'
3'
ApoE 2ApoE 3ApoE 4
Summary• Alzheimer’s disease (AD) is an irreversible neurodegenerative disorder that
progresses slowly over time, affecting 5.4M Americans in 2016, or 1 in 10 Americans age 65 and older.
• Sequence variants in TOMM40 and APOE are two genetic risk predictors of AD. However, these sequences are difficult to genotype using conventional PCR due to their AT- and GC-rich character.
• We unified a proof-of-concept APOE assay with our AmplideX® PCR/CE TOMM40 Kit (RUO) to simultaneously genotype both genetic markers.
• Here we report the first single-assay workflow that achieves reproducible and accurate resolution of TOMM40 poly-T length polymorphisms and all 6 APOE SNP genotypes.
IntroductionAlzheimer’s disease (AD) accounts for 60-80% of all dementias in the US1. Previous studies have identified an AD susceptibility locus in a region that includes Apolipoprotein E (APOE) and Translocase Of Outer Mitochondrial Membrane 40 (TOMM40). Of the three common APOE alleles, ε4 is the most informative but in only ~25% of Caucasians2. Independently, poly-T polymorphisms within the TOMM40 gene have been reported to influence both age of onset in late-onset AD (LOAD) and measures of cognitive decline elevating informative risk data for LOAD to ~97% of individuals2,3,4. The AmplideX® PCR/CE TOMM40 Kit (RUO) is a commercially available option for this marker. Here we describe the first PCR-based workflow that unifies APOE and TOMM40 genotyping.
Materials and MethodsCell-line gDNA samples (14 AD, 104 other) were acquired from the Coriell Institute. Whole blood (WB; 97) and matched WB/buccal gDNA samples (77) were isolated from presumed healthy donors (Asuragen, Equitech Enterprises). Sample gDNA was PCR amplified using prototype AmplideX reagents (Asuragen) by different operators on different days. Amplicons tagged with FAM (TOMM40) and HEX (APOE) were resolved on a 3500xL Genetic Analyzer (Thermo Fisher Scientific) CE instrument using POP-7™ polymer with 2.5kV, 20 sec injection and 20 min run time. Genotype analysis utilized a ROX 400HD size ladder (Thermo Fisher Scientific) in separate color channels from target amplicon patterns (APOE) or from the mobility of target amplicon (TOMM40) relative to a three-point cell-line DNA calibrator (16T, 29T, 36T poly-T lengths).
References1. https://www.alz.org/documents_custom/2017-facts-and-figures.pdf,
(Sourced Jun 30 2017).2. Crenshaw DG, et al. (2013) Clin Pharmacol Ther. 93(2), 177–85. 3. Saunders, A. M., et al. (2000) Biochim Biophys Acta - Mol Basis Dis.
1502, 85–94.4. Yu, L., et al. (2017) Neurology. 88(7), 661–668.5. Liu, Chia-Chen, et al. (2013) Nat Rev Neuro. 9(2), 106–118. 6. Corder, E. H., et al. (1993) Science. 261 (5123), 921–923. 7. Lutz, M. W., et al. (2016) Alz & Dem. 2(1), 30–44.8. Roses, A. D., et al. (2010) Pharmacogenomics J. 10, 375–84.
Amplidex PCR/CE TOMM40 Kit - Research Use Only – Not for Use in Diagnostic ProceduresPrototype APOE reagents used to gather preliminary research data. The performance characteristics of this assay have not yet been established. Presented at AAIC 2017
A Streamlined PCR-based Fragment Analysis Assay that Resolves Both Single Nucleotide and Poly-T Length Polymorphisms at APOE and TOMM40 Susceptibility Loci in Alzheimer’s DiseaseBradley Hall, Sarah Statt, Julie R Thibert, Jacob Wisotsky, Jon Kemppainen, and Gary J LathamAsuragen, Inc., Austin, TX
Results
Conclusions• A single PCR/CE technology resolved SNP and length polymorphisms in APOE and
TOMM40, respectively, and produced concordant genotypes from 292 samples across three gDNA sources, multiple operators and different days.
• This streamlined workflow analyzes purified DNA with a turn-around time of 4.5 hrs.• This multimodal approach has the potential to advance clinical research using a
standardized assay that can harmonize results across laboratories.
P1-270#17750
Figure 2. All six possible APOE and TOMM40 genotypes were resolved using PCR methods. A) Five of six APOE genotypes were identified within Coriell cell-line and WB samples. ε2-ε2 was positively identified in PC3 cell line (ATCC). GS is a positive gene specific control. B) The TOMM40 poly-T repeat number was determined within a single nucleotide for all sample genotypes assayed between 15 and 48Ts. C) Genotype distribution for 174 WB samples recapitulated the prevalence of APOE and TOMM40 variants that were previously reported. Although 21.5% of samples had TOMM40 genotypes that fell near allele length boundaries, all calls were repeatable.
Figure 3. APOE genotypes resolved by the prototype assay agreed with reference calls for all 292 cell-line, blood and buccal cell samples. A subset of samples and controls were duplicated across different operators and days. All results were repeatable. Input gDNA concentration was measured by A260 and ranged from 5.5ng to 176.8ng. A) The APOE TaqMan® SNP Genotyping qPCR Assay (Thermo Fisher Scientific) was used as a reference method to determine agreement. Matched WB (43) and each AD Coriell cell-line genotype (4/6) and NTC (1) are shown. B) Representative WB and buccal PCR electropherograms demonstrate 4 of the 6 possible APOE genotypes.
Figure 4. Assayed APOE and TOMM40 genotypes reflect known allele-variant associations. A) Distribution of APOE and TOMM40 genotypes matched for all independent samples from WB (160) and B) Coriell cell-line non-AD (104). Colors represent samples containing one or both alleles with a poly-T length of 30±2 (L/VL boundary) for all but one WB sample with a 20 T allele (S/L boundary).
3A 3B
4A
5A
4B
2A 2B 2C
1A
Figure 5. APOE and TOMM40 PCR amplicons could be resolved from a single-assay PCR/CE workflow to assess AD risk. A) Risk algorithm and prevalence were previously published5,7. TOMM40 poly-T length was found to have a strong relationship with age of onset, further refining the APOE association with AD with L showing the highest risk8. B) Co-injection on CE simplified both the workflow and data analysis, and stratified the largest subset of APOE genotypes (ε3-ε3 and ε4-ε4) into high and low risk groups.
Start
YesAt least 1 APOE 2
APOE2- 4
APOE4- 4
Yes1-2% Population3.6% AD incidence
No16-17% Population3.6% AD incidence
'523 L/Lor L/VL
No
No
APOE3- 3
or 3- 4
Yes
'523 VL/VL
No
Yes
'523 S/S 77yo S/L 74yo
S/VL 76yo
No
YesNo
S 1-19 TsL 20-29 Ts
VL 30-60+ Ts
80% Population68.3% AD incidence
Yes1-2% Population24.4% AD incidence
Low RiskAPOE 2- 2
or 2- 3or '523 VL/VL
High Risk
5B
1B TOMM40 Poly-T12.39 kb Forward strand
Short (S): ≤19
Long (L): 20-29
Very Long (VL): ≥ 30
P
1C PCR CE DATAANALYSIS
TOTAL
30min ea
15min
20min
95min
120min
60min
0min
3.0hr
Hands-on Time
Instrument Time
