A Rapid and Reliable Test for BRCA1 Promoter Hypermethylation in Paraffin Tissue Using Pyrosequencing
Abstract
:1. Introduction
2. Materials and Methods
3. Results
3.1. Design and Interpretation of the Pyrosequencing Assay
3.2. Accuracy
3.3. Analytical Sensitivity
3.4. Intra- and Inter-Run Reproducibility
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Pos. 1 | Pos. 2 | Pos. 3 | Pos. 4 | Pos. 5 | Pos. 6 | Pos. 7 | Pos. 8 | Pos. 9 | Pos. 10 | Pos. 11 | |||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
ID | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Mean | Pyro Result | TCGA Result | Real-Time PCR Result |
29 1694 | 55.2 | 71.3 | 53.6 | 49.9 | 54.1 | 53.1 | 58.6 | 53.9 | 48.5 | 43.8 | 43.3 | 53.2 | PRESENT | METH | |
29 1707 | 50.6 | 65.9 | 48.3 | 45.6 | 49.2 | 50.3 | 53.3 | 47.2 | 42.4 | 41.1 | 39.4 | 48.5 | PRESENT | METH | |
29 1711 | 61.9 | 71.8 | 58.7 | 55.9 | 58.7 | 58.5 | 65.4 | 61.2 | 58.8 | 52.5 | 53.6 | 59.7 | PRESENT | METH | |
JB 2335 | 44.1 | 58.0 | 44.5 | 40.9 | 39.5 | 47.6 | 50.1 | 44.4 | 37.6 | 42.1 | 38.5 | 44.3 | PRESENT | METH | Positive |
JB 2595 | 28.0 | 51.1 | 36.7 | 31.3 | 28.9 | 34.7 | 42.7 | 43.1 | 30.1 | 29.6 | 25.4 | 34.7 | PRESENT | METH | Positive |
JB 2670 | 62.3 | 85.6 | 62.4 | 61.1 | 67.0 | 63.8 | 69.5 | 65.6 | 60.2 | 58.4 | 56.3 | 64.7 | PRESENT | METH | Positive |
JB 1412 | 36.3 | 59.6 | 41.2 | 40.4 | 38.9 | 40.5 | 47.4 | 42.2 | 34.9 | 38.2 | 36.0 | 41.4 | PRESENT | Positive | |
JB 1468 | 44.1 | 61.3 | 41.2 | 40.8 | 42.4 | 42.8 | 48.1 | 42.4 | 35.0 | 37.5 | 29.0 | 42.2 | PRESENT | Positive | |
JB 3081 | 41.1 | 57.7 | 43.8 | 39.4 | 45.0 | 44.5 | 48.0 | 42.3 | 38.1 | 36.5 | 35.5 | 42.9 | PRESENT | Positive | |
JB 3393 | 32.8 | 49.3 | 34.5 | 32.0 | 31.9 | 34.9 | 37.1 | 31.8 | 27.9 | 22.4 | 25.1 | 32.7 | PRESENT | Positive | |
29 1696 | 2.8 | 8.8 | 2.4 | 2.3 | 1.8 | 4.9 | 3.6 | 2.2 | 1.8 | 3.9 | 6.2 | 3.7 | Not Present | Not Meth | |
29 1710 | 2.5 | 9.8 | 2.5 | 3.1 | 2.4 | 5.8 | 3.9 | 2.1 | 3.1 | 3.9 | 6.2 | 4.1 | Not Present | Not Meth | |
29 1762 | 1.8 | 11.4 | 2.8 | 2.1 | 2.1 | 6.9 | 4.1 | 1.9 | 1.5 | 3.0 | 7.2 | 4.1 | Not Present | Not Meth | |
29 1763 | 3.8 | 24.6 | 7.3 | 6.4 | 3.7 | 11.8 | 8.8 | 5.2 | 4.6 | 8.9 | 11.5 | 8.8 | Not Present | Not Meth | |
29 1776 | 3.0 | 13.1 | 3.4 | 2.8 | 2.8 | 8.0 | 5.2 | 3.3 | 3.9 | 6.6 | 9.5 | 5.6 | Not Present | Not Meth | |
29 1784 | 2.2 | 9.7 | 3.8 | 2.9 | 2.5 | 6.5 | 4.6 | 2.4 | 2.0 | 4.0 | 7.3 | 4.4 | Not Present | Not Meth | |
BRME 23 | 0.7 | 12.5 | 2.4 | 1.9 | 1.6 | 7.0 | 3.1 | 2.2 | 1.0 | 3.6 | 7.3 | 3.9 | Not Present | Negative | |
JB 2206 | 1.8 | 10.8 | 1.7 | 1.5 | 1.5 | 4.5 | 2.9 | 1.2 | 1.4 | 2.9 | 5.3 | 3.2 | Not Present | Negative | |
JB 3717 | 1.5 | 10.0 | 1.6 | 2.0 | 1.5 | 4.2 | 2.5 | 1.1 | 1.5 | 2.9 | 4.8 | 3.1 | Not Present | Negative | |
JB 2388 | 2.1 | 9.5 | 3.0 | 3.0 | 2.2 | 5.4 | 4.2 | 2.5 | 1.9 | 4.5 | 6.5 | 4.1 | Not Present | Negative |
(A) | |||||||||||||||
Pos. 1 | Pos. 2 | Pos. 3 | Pos. 4 | Pos. 5 | Pos. 6 | Pos. 7 | Pos. 8 | Pos. 9 | Pos. 10 | Pos. 11 | |||||
Dilution | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Mean | |||
Neg. | Run#1 | 1.4 | 14.5 | 3.3 | 3.2 | 2.1 | 7.0 | 5.1 | 2.4 | 2.2 | 5.5 | 8.9 | 5.1 | ||
Neg. | Run#2 | 5.1 | 17.8 | 5.8 | 4.0 | 3.4 | 8.6 | 6.7 | 3.0 | 3.4 | 7.3 | 11.4 | 7.0 | ||
100% | Run#1 | 95.0 | 100.0 | 90.1 | 79.7 | 91.4 | 90.1 | 95.1 | 91.3 | 92.0 | 79.4 | 81.2 | 89.6 | ||
100% | Run#2 | 94.9 | 100.0 | 91.3 | 83.1 | 93.7 | 90.6 | 93.9 | 91.3 | 88.5 | 86.7 | 74.4 | 89.9 | ||
50% | Run#1 | 55.6 | 74.2 | 55.2 | 49.3 | 54.2 | 57.3 | 62.4 | 57.7 | 52.4 | 49.1 | 52.1 | 56.3 | ||
50% | Run#2 | 55.5 | 67.1 | 53.0 | 49.2 | 52.2 | 53.9 | 59.1 | 55.0 | 50.8 | 47.1 | 48.8 | 53.8 | ||
25% | Run#1 | 29.3 | 41.9 | 28.2 | 26.5 | 27.5 | 30.9 | 34.5 | 30.3 | 27.0 | 25.9 | 28.7 | 30.0 | ||
25% | Run#2 | 30.6 | 44.3 | 30.0 | 28.3 | 28.5 | 30.5 | 34.0 | 26.6 | 26.6 | 27.8 | 29.1 | 30.6 | ||
12.50% | Run#1 | 18.0 | 32.4 | 18.8 | 18.7 | 17.9 | 22.5 | 23.4 | 20.0 | 16.7 | 18.3 | 21.2 | 20.7 | ||
12.50% | Run#2 | 16.1 | 30.9 | 17.2 | 18.8 | 17.6 | 21.3 | 22.1 | 18.6 | 14.5 | 17.1 | 21.4 | 19.6 | ||
6.25% | Run#1 | 13.0 | 24.1 | 12.3 | 12.5 | 11.2 | 15.9 | 16.1 | 12.2 | 11.3 | 12.9 | 15.9 | 14.3 | ||
6.25% | Run#2 | 13.6 | 23.3 | 12.5 | 12.1 | 10.0 | 15.5 | 14.8 | 11.6 | 9.9 | 11.8 | 16.4 | 13.8 | ||
3.13% | Run#1 | 8.2 | 18.5 | 8.6 | 7.8 | 7.5 | 12.0 | 11.1 | 8.1 | 6.6 | 10.1 | 13.1 | 10.1 | ||
3.13% | Run#2 | 7.5 | 19.8 | 8.2 | 7.9 | 7.4 | 11.2 | 10.5 | 8.2 | 6.3 | 8.9 | 12.6 | 9.9 | ||
1.56% | Run#1 | 5.5 | 20.3 | 5.7 | 6.0 | 4.3 | 10.3 | 8.9 | 5.5 | 4.5 | 7.2 | 12.0 | 8.2 | ||
1.56% | Run#2 | 6.0 | 20.4 | 7.4 | 6.2 | 4.9 | 10.2 | 8.4 | 5.5 | 4.7 | 7.6 | 11.6 | 8.4 | ||
(B) | |||||||||||||||
Pos. 1 | Pos. 2 | Pos. 3 | Pos. 4 | Pos. 5 | Pos. 6 | Pos. 7 | Pos. 8 | Pos. 9 | Pos. 10 | Pos. 11 | |||||
Dilution | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Mean (measured) | Expected | Pyro Result | |
100% | Run#1 | 59.5 | 82.9 | 57.4 | 55.6 | 62.5 | 59.3 | 69.6 | 61.5 | 54.0 | 54.7 | 56.1 | 61.2 | DETECTED | |
100% | Run#2 | 72.0 | 87.1 | 70.2 | 61.0 | 71.8 | 71.0 | 80.5 | 67.8 | 65.3 | 54.2 | 61.4 | 69.3 | DETECTED | |
50% | Run#1 | 18.9 | 25.6 | 18.0 | 17.3 | 18.0 | 18.2 | 21.4 | 19.5 | 14.2 | 16.2 | 13.2 | 18.2 | 32.5 | DETECTED |
50% | Run#2 | 19.7 | 31.2 | 21.4 | 19.0 | 16.8 | 21.7 | 21.7 | 16.7 | 12.5 | 13.2 | 18.5 | 19.3 | 32.5 | DETECTED |
25% | Run#1 | 13.0 | 19.1 | 12.9 | 13.6 | 12.3 | 15.1 | 18.2 | 14.0 | 10.7 | 11.4 | 13.7 | 14.0 | 16.2 | DETECTED |
25% | Run#2 | 9.6 | 22.9 | 10.6 | 11.0 | 10.1 | 14.9 | 14.5 | 12.3 | 7.5 | 9.1 | 13.4 | 12.4 | 16.2 | DETECTED |
12.50% | Run#1 | 6.4 | 14.7 | 5.4 | 4.7 | 4.9 | 8.8 | 7.2 | 5.4 | 2.2 | 6.8 | 9.3 | 6.9 | 8.1 | NOT DETECTED |
12.50% | Run#2 | 7.7 | 16.9 | 8.6 | 7.5 | 6.7 | 11.2 | 12.1 | 7.9 | 5.0 | 8.9 | 12.9 | 9.6 | 8.1 | NOT DETECTED |
6.25% | Run#1 | 8.1 | 14.9 | 7.8 | 8.1 | 7.0 | 10.5 | 7.6 | 3.8 | 3.4 | 5.2 | 9.0 | 7.8 | 4.0 | NOT DETECTED |
6.25% | Run#2 | 6.2 | 16.9 | 7.3 | 8.7 | 4.9 | 11.6 | 9.9 | 5.9 | 4.4 | 8.2 | 10.9 | 8.6 | 4.0 | NOT DETECTED |
3.13% | Run#1 | 4.1 | 12.8 | 3.2 | 3.8 | 4.6 | 5.9 | 5.6 | 3.2 | 2.2 | 3.3 | 8.7 | 5.2 | 2.0 | NOT DETECTED |
3.13% | Run#2 | 3.3 | 11.4 | 5.4 | 3.6 | 3.5 | 6.2 | 4.1 | 2.6 | 2.6 | 4.4 | 6.0 | 4.8 | 2.0 | NOT DETECTED |
(A) | |||||||||||||||
Pos. 1 | Pos. 2 | Pos. 3 | Pos. 4 | Pos. 5 | Pos. 6 | Pos. 7 | Pos. 8 | Pos. 9 | Pos. 10 | Pos. 11 | |||||
Case # | ID | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Mean | Result | |
21 | JB 1412 | 50.6 | 84.4 | 49.9 | 48.3 | 49.7 | 54.4 | 53.9 | 51.8 | 40.8 | 44.1 | 45.8 | 52.2 | PRESENT | |
21 | JB 1412 | 36.3 | 59.6 | 41.2 | 40.4 | 38.9 | 40.5 | 47.4 | 42.2 | 34.9 | 38.2 | 36.0 | 41.4 | PRESENT | |
21 | JB1412 | 50.9 | 76.8 | 47.5 | 44.3 | 46.1 | 49.1 | 55.4 | 49.8 | 45.6 | 44.7 | 45.6 | 50.5 | PRESENT | |
22 | JB 1468 | 41.9 | 63.4 | 39.6 | 39.4 | 40.6 | 41.3 | 47.4 | 41.7 | 35.7 | 37.4 | 28.5 | 41.5 | PRESENT | |
22 | JB 1468 | 42.0 | 62.0 | 41.0 | 38.5 | 39.5 | 40.7 | 46.7 | 41.7 | 36.2 | 36.2 | 29.4 | 41.3 | PRESENT | |
22 | JB 1468 | 43.0 | 60.4 | 41.6 | 40.9 | 42.5 | 41.4 | 47.8 | 42.2 | 35.6 | 39.0 | 29.9 | 42.2 | PRESENT | |
23 | JB 2400 | 3.0 | 13.1 | 3.3 | 2.5 | 1.6 | 6.5 | 4.3 | 1.8 | 2.4 | 4.2 | 7.3 | 4.5 | Not Present | |
23 | JB 2400 | 3.4 | 12.0 | 4.2 | 3.2 | 2.5 | 7.6 | 4.9 | 2.6 | 1.7 | 4.9 | 6.7 | 4.9 | Not Present | |
23 | JB 2400 | 2.7 | 14.0 | 4.0 | 3.5 | 1.7 | 6.8 | 4.5 | 2.3 | 1.9 | 4.5 | 7.2 | 4.8 | Not Present | |
24 | JB 2505 | 61.1 | 97.5 | 66.3 | 63.1 | 67.4 | 68.2 | 75.0 | 67.2 | 62.4 | 61.9 | 61.0 | 68.3 | PRESENT | |
24 | JB 2505 | 65.4 | 90.3 | 65.3 | 60.8 | 66.3 | 67.1 | 72.4 | 67.7 | 61.5 | 60.8 | 60.0 | 67.0 | PRESENT | |
24 | JB 2505 | 62.6 | 89.7 | 66.0 | 58.2 | 63.6 | 66.3 | 71.2 | 64.3 | 63.6 | 60.3 | 59.0 | 65.9 | PRESENT | |
25 | JB 2520 | 4.0 | 13.7 | 5.0 | 3.6 | 2.7 | 7.0 | 5.5 | 3.0 | 2.5 | 5.9 | 8.7 | 5.6 | Not Present | |
25 | JB 2520 | 2.2 | 14.5 | 3.6 | 2.9 | 2.4 | 6.9 | 4.4 | 3.1 | 2.8 | 4.6 | 9.0 | 5.1 | Not Present | |
25 | JB 2520 | 2.7 | 18.0 | 3.4 | 2.2 | 2.2 | 6.0 | 7.3 | 2.9 | 2.4 | 5.1 | 8.6 | 5.5 | Not Present | |
26 | JB 2540 | 2.7 | 12.8 | 4.0 | 4.2 | 3.9 | 7.5 | 5.9 | 3.2 | 2.1 | 4.7 | 7.0 | 5.3 | Not Present | |
26 | JB 2540 | 5.8 | 12.3 | 5.1 | 4.9 | 3.2 | 8.5 | 5.6 | 2.7 | 2.3 | 5.6 | 9.6 | 6.0 | Not Present | |
26 | JB 2540 | 3.7 | 15.0 | 5.4 | 5.1 | 4.6 | 7.7 | 5.7 | 3.5 | 3.2 | 5.9 | 6.5 | 6.0 | Not Present | |
(B) | |||||||||||||||
Pos. 1 | Pos. 2 | Pos. 3 | Pos. 4 | Pos. 5 | Pos. 6 | Pos. 7 | Pos. 8 | Pos. 9 | Pos. 10 | Pos. 11 | |||||
Case # | ID | Run# | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Meth. (%) | Mean | Result |
16 | BRME 23 | Run#1 | 0.7 | 12.5 | 2.4 | 1.9 | 1.6 | 7.0 | 3.1 | 2.2 | 1.0 | 3.6 | 7.3 | 3.9 | Not Present |
16 | BRME 23 | Run#2 | 2.7 | 11.0 | 2.9 | 3.1 | 0.8 | 8.3 | 2.0 | 0.0 | 1.5 | 9.6 | 9.6 | 4.7 | Not Present |
16 | BRME 23 | Run#3 | 3.4 | 17.3 | 3.9 | 4.5 | 5.0 | 9.1 | 7.3 | 4.1 | 3.4 | 6.9 | 8.0 | 6.6 | Not Present |
17 | JB 1468 | Run#1 | 42.0 | 62.0 | 41.0 | 38.5 | 39.5 | 40.7 | 46.7 | 41.7 | 36.2 | 36.2 | 29.4 | 41.3 | PRESENT |
17 | JB 1468 | Run#2 | 28.7 | 56.0 | 36.9 | 35.4 | 36.1 | 36.7 | 37.1 | 38.5 | 34.2 | 33.9 | 27.6 | 36.5 | PRESENT |
17 | JB 1468 | Run#3 | 34.3 | 62.1 | 40.2 | 38.7 | 40.3 | 42.0 | 46.4 | 41.3 | 33.2 | 36.7 | 30.5 | 40.5 | PRESENT |
13 | JB 2335 | Run#1 | 48.3 | 63.6 | 46.7 | 44.6 | 44.6 | 41.6 | 55.5 | 41.3 | 31.6 | 35.5 | 36.2 | 44.5 | PRESENT |
13 | JB 2335 | Run#2 | 44.1 | 58.0 | 44.5 | 40.9 | 39.5 | 47.6 | 50.1 | 44.4 | 37.6 | 42.1 | 38.5 | 44.3 | PRESENT |
13 | JB 2335 | Run#3 | 38.4 | 73.0 | 47.7 | 41.2 | 40.8 | 48.3 | 52.8 | 44.5 | 35.4 | 34.6 | 42.0 | 45.3 | PRESENT |
18 | JB 2400 | Run#1 | 3.4 | 12.0 | 4.2 | 3.2 | 2.5 | 7.6 | 4.9 | 2.6 | 1.7 | 4.9 | 6.7 | 4.9 | Not Present |
18 | JB 2400 | Run#2 | 2.5 | 12.1 | 3.8 | 4.1 | 3.9 | 6.4 | 0.0 | 0.0 | 0.0 | 8.0 | 4.8 | 4.1 | Not Present |
18 | JB 2400 | Run#3 | 3.3 | 8.8 | 3.5 | 2.9 | 3.1 | 6.0 | 5.0 | 4.9 | 3.1 | 4.2 | 4.8 | 4.5 | Not Present |
19 | JB 2540 | Run#1 | 4.9 | 16.9 | 7.0 | 6.9 | 3.7 | 8.2 | 8.5 | 4.2 | 4.8 | 6.6 | 11.8 | 7.6 | Not Present |
19 | JB 2540 | Run#2 | 2.3 | 10.6 | 2.8 | 2.8 | 3.2 | 5.3 | 7.4 | 3.2 | 2.8 | 3.7 | 7.5 | 4.7 | Not Present |
19 | JB 2540 | Run#3 | 4.3 | 28.5 | 8.2 | 7.6 | 5.3 | 12.5 | 13.0 | 6.2 | 3.9 | 7.3 | 11.1 | 9.8 | Not Present |
20 | JB 3393 | Run#1 | 32.8 | 49.3 | 34.5 | 32.0 | 31.9 | 34.9 | 37.1 | 31.8 | 27.9 | 22.4 | 25.1 | 32.7 | PRESENT |
20 | JB 3393 | Run#2 | 32.3 | 44.7 | 33.0 | 33.3 | 33.1 | 33.0 | 39.1 | 33.9 | 28.9 | 24.6 | 22.1 | 32.5 | PRESENT |
20 | JB 3393 | Run#3 | 23.6 | 59.9 | 32.2 | 31.1 | 29.6 | 35.3 | 37.9 | 31.6 | 24.9 | 24.8 | 26.4 | 32.5 | PRESENT |
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Bacares, R.; Soslow, R.; Olvera, N.; Levine, D.A.; Zhang, L. A Rapid and Reliable Test for BRCA1 Promoter Hypermethylation in Paraffin Tissue Using Pyrosequencing. Diagnostics 2025, 15, 601. https://doi.org/10.3390/diagnostics15050601
Bacares R, Soslow R, Olvera N, Levine DA, Zhang L. A Rapid and Reliable Test for BRCA1 Promoter Hypermethylation in Paraffin Tissue Using Pyrosequencing. Diagnostics. 2025; 15(5):601. https://doi.org/10.3390/diagnostics15050601
Chicago/Turabian StyleBacares, Ruben, Robert Soslow, Narciso Olvera, Douglas A. Levine, and Liying Zhang. 2025. "A Rapid and Reliable Test for BRCA1 Promoter Hypermethylation in Paraffin Tissue Using Pyrosequencing" Diagnostics 15, no. 5: 601. https://doi.org/10.3390/diagnostics15050601
APA StyleBacares, R., Soslow, R., Olvera, N., Levine, D. A., & Zhang, L. (2025). A Rapid and Reliable Test for BRCA1 Promoter Hypermethylation in Paraffin Tissue Using Pyrosequencing. Diagnostics, 15(5), 601. https://doi.org/10.3390/diagnostics15050601