. 2018 Oct 24;7(11):183.

doi: 10.3390/cells7110183.

Telomere Length Calibration from qPCR Measurement: Limitations of Current Method

Youjin Wang¹, Sharon A Savage², Rotana Alsaggaf³, Geraldine Aubert^{4

5}, Casey L Dagnall^{6

7}, Stephen R Spellman⁸, Stephanie J Lee^{9

10}, Belynda Hicks^{11

12}, Kristine Jones^{13

14}, Hormuzd A Katki¹⁵, Shahinaz M Gadalla¹⁶

Affiliations

¹ Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. youjin.wang@nih.gov.
² Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. savagesh@mail.nih.gov.
³ Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. rotana.alsaggaf@nih.gov.
⁴ Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada. gaubert@bccrc.ca.
⁵ Repeat Diagnostics Inc., North Vancouver, BC V7M 1A5, Canada. gaubert@bccrc.ca.
⁶ Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. dagnallc@mail.nih.gov.
⁷ Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA. dagnallc@mail.nih.gov.
⁸ Center for International Blood and Marrow Transplant Research, Minneapolis, MN 55401, USA. sspellma@nmdp.org.
⁹ Center for International Blood and Marrow Transplant Research, Medical College of Wisconsin, Milwaukee, WI 53226, USA. sjlee@fredhutch.org.
¹⁰ Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. sjlee@fredhutch.org.
¹¹ Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. hicksbel@mail.nih.gov.
¹² Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA. hicksbel@mail.nih.gov.
¹³ Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. kristine.jones@nih.gov.
¹⁴ Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA. kristine.jones@nih.gov.
¹⁵ Biostatistics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. katkih@mail.nih.gov.
¹⁶ Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. shahinaz.gadalla@nih.gov.

PMID: 30352968
PMCID: PMC6262465
DOI: 10.3390/cells7110183

Telomere Length Calibration from qPCR Measurement: Limitations of Current Method

Youjin Wang et al. Cells. 2018.

. 2018 Oct 24;7(11):183.

doi: 10.3390/cells7110183.

Authors

Affiliations

¹ Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. youjin.wang@nih.gov.
² Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. savagesh@mail.nih.gov.
³ Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. rotana.alsaggaf@nih.gov.
⁴ Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada. gaubert@bccrc.ca.
⁵ Repeat Diagnostics Inc., North Vancouver, BC V7M 1A5, Canada. gaubert@bccrc.ca.
⁶ Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. dagnallc@mail.nih.gov.
⁷ Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA. dagnallc@mail.nih.gov.
⁸ Center for International Blood and Marrow Transplant Research, Minneapolis, MN 55401, USA. sspellma@nmdp.org.
⁹ Center for International Blood and Marrow Transplant Research, Medical College of Wisconsin, Milwaukee, WI 53226, USA. sjlee@fredhutch.org.
¹⁰ Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. sjlee@fredhutch.org.
¹¹ Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. hicksbel@mail.nih.gov.
¹² Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA. hicksbel@mail.nih.gov.
¹³ Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. kristine.jones@nih.gov.
¹⁴ Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA. kristine.jones@nih.gov.
¹⁵ Biostatistics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. katkih@mail.nih.gov.
¹⁶ Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. shahinaz.gadalla@nih.gov.

PMID: 30352968
PMCID: PMC6262465
DOI: 10.3390/cells7110183

Abstract

Telomere length (TL) comparisons from different methods are challenging due to differences in laboratory techniques and data configuration. This study aimed to assess the validity of converting the quantitative polymerase chain reaction (qPCR) telomere/single copy gene (T/S) ratio to TL in kilobases (kb). We developed a linear regression equation to predict TL from qPCR T/S using flow cytometry with fluorescence in situ hybridization (flow FISH) TL data from 181 healthy donors (age range = 19⁻53) from the National Marrow Donor Program (NMDP) biorepository. TL measurements by qPCR and flow FISH were modestly correlated (R² = 0.56, p < 0.0001). In Bland-Altman analyses, individuals with the shortest (≤10th percentile) or longest (≥90th) flow FISH TL had an over- or under-estimated qPCR TL (bias = 0.89 and -0.77 kb, respectively). Comparisons of calculated TL from the NMDP samples and 1810 age- and sex-matched individuals from the National Health and Nutrition Examination Survey showed significant differences (median = 7.1 versus 5.8 kb, respectively, p < 0.0001). Differences in annual TL attrition were also noted (31 versus 13 bp/year, respectively, p = 0.02). Our results demonstrate that TL calculated in kb from qPCR T/S may yield biased estimates for individuals with the shortest or longest TL, those often of high clinical interest. We also showed that calculated TL in kb from qPCR data are not comparable across populations and therefore are not necessarily useful.

Keywords: agreement; flow FISH; qPCR; telomere length.

PubMed Disclaimer

Conflict of interest statement

G.A. is a part-time employee of Repeat Diagnostics Inc., a company specializing in clinical telomere length measurements. There are no other conflicts of interest to disclose.

Figures

**Figure 1**
Correlation between telomere length (TL) and age (A) qPCR TL (T/S ratio); (B) Flow cytometry with fluorescence in situ hybridization (flow FISH) TL (kb).

**Figure 2**
Correlation and agreement between calculated telomere length (TL) from conversion equation and flow cytometry with fluorescence in situ hybridization (flow FISH) TL in the National Marrow Donor Program^® (NMDP) cohort. (A) Correlation between calculated qPCR TL (kb) and flow FISH TL; (B) Bland-Altman plot of agreement between calculated TL from conversion equation and flow FISH TL.

**Figure 3**
Flow cytometry with fluorescence in situ hybridization (Flow FISH) telomere length (TL) quintile stratified analysis of agreement between calculated TL from conversion equation and flow FISH TL in the National Marrow Donor Program^® (NMDP) cohort. (A) Above 10th and below 90th percentile (TL 5.8–8.4); (B) Flow FISH TL ≤ 10th percentile (TL 3.7–5.7); (C) ≥90th percentile (TL 8.5–11.2).

**Figure 4**
Distribution of calculated telomere length (TL) in the National Marrow Donor Program^® (NMDP) and National Health and Nutrition Examination Survey (NHANES) cohorts.

**Figure 5**
Annual telomere length (TL) attrition. (A) National Marrow Donor Program^® (NMDP) cohort: flow cytometry with fluorescence in situ hybridization (flow FISH) TL; (B) NMDP cohort: calculated TL using conversion equation generated from the qPCR T/S ratio; (C) National Health and Nutrition Examination Survey (NHANES): calculated TL using the published conversion equation.

See this image and copyright information in PMC

Cited by

C-peptide is a predictor of telomere shortening: A five-year longitudinal study.
Ghoussaini R, Tamim H, Elbejjani M, Makki M, Nasreddine L, Ismaeel H, Nasrallah MP, Zgheib NK. Ghoussaini R, et al. Front Endocrinol (Lausanne). 2022 Aug 18;13:978747. doi: 10.3389/fendo.2022.978747. eCollection 2022. Front Endocrinol (Lausanne). 2022. PMID: 36060975 Free PMC article.
Short telomere length predicts nonrelapse mortality after stem cell transplantation for myelodysplastic syndrome.
Myllymäki M, Redd R, Reilly CR, Saber W, Spellman SR, Gibson CJ, Hu ZH, Wang T, Orr EH, Grenier JG, Chen MM, Steensma DP, Cutler C, De Vivo I, Antin JH, Neuberg D, Agarwal S, Lindsley RC. Myllymäki M, et al. Blood. 2020 Dec 24;136(26):3070-3081. doi: 10.1182/blood.2020005397. Blood. 2020. PMID: 33367544 Free PMC article. Clinical Trial.
Telomere length in hematopoietic cell transplant.
Gadalla SM. Gadalla SM. Blood. 2020 Dec 24;136(26):2972-2973. doi: 10.1182/blood.2020008547. Blood. 2020. PMID: 33367550 Free PMC article.
Telomere Dynamics in Livestock.
Zhang N, Baker EC, Welsh TH Jr, Riley DG. Zhang N, et al. Biology (Basel). 2023 Oct 31;12(11):1389. doi: 10.3390/biology12111389. Biology (Basel). 2023. PMID: 37997988 Free PMC article. Review.
T cell memory revisited using single telomere length analysis.
Roger L, Miners KL, Leonard L, Grimstead JW, Price DA, Baird DM, Ladell K. Roger L, et al. Front Immunol. 2023 Sep 14;14:1100535. doi: 10.3389/fimmu.2023.1100535. eCollection 2023. Front Immunol. 2023. PMID: 37781376 Free PMC article.

See all "Cited by" articles

References

1. O’Sullivan R.J., Karlseder J. Telomeres: Protecting chromosomes against genome instability. Nat. Rev. Mol. Cell Biol. 2010;11:171–181. doi: 10.1038/nrm2848. - DOI - PMC - PubMed
1. Aubert G., Lansdorp P.M. Telomeres and aging. Physiol. Rev. 2008;88:557–579. doi: 10.1152/physrev.00026.2007. - DOI - PubMed
1. Counter C.M. The roles of telomeres and telomerase in cell life span. Mutat. Res./Rev. Genet. Toxicol. 1996;366:45–63. doi: 10.1016/S0165-1110(96)90006-8. - DOI - PubMed
1. Feldser D.M., Hackett J.A., Greider C.W. Telomere dysfunction and the initiation of genome instability. Nat. Rev. Cancer. 2003;3:623–627. doi: 10.1038/nrc1142. - DOI - PubMed
1. Savage S.A., Bertuch A.A. The genetics and clinical manifestations of telomere biology disorders. Genet. Med. 2010;12:753–764. doi: 10.1097/GIM.0b013e3181f415b5. - DOI - PMC - PubMed

Grants and funding

N0014-17-1-2850/The Office of Naval Research

LinkOut - more resources

Full Text Sources
Molecular Biology Databases
- NIAID Data Ecosystem - Find datasets on Infectious and Immune-mediated Diseases

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Telomere Length Calibration from qPCR Measurement: Limitations of Current Method

Affiliations

Telomere Length Calibration from qPCR Measurement: Limitations of Current Method

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Grants and funding

LinkOut - more resources

Full Text Sources

Molecular Biology Databases