Critical Reviews in Clinical Laboratory Sciences

ISSN: 1040-8363 (Print) 1549-781X (Online) Journal homepage: http://www.tandfonline.com/loi/ilab20

Telomere length measurement as a clinical

biomarker of aging and disease

Clare L. Fasching

To cite this article: Clare L. Fasching (2018): Telomere length measurement as a clinical
biomarker of aging and disease, Critical Reviews in Clinical Laboratory Sciences, DOI:
10.1080/10408363.2018.1504274

To link to this article: https://doi.org/10.1080/10408363.2018.1504274

Published online: 28 Sep 2018.

Submit your article to this journal

View Crossmark data

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=ilab20
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES
https://doi.org/10.1080/10408363.2018.1504274

REVIEW ARTICLE

Telomere length measurement as a clinical biomarker of aging and disease

Clare L. Fasching
Telomere Diagnostics, Inc., Menlo Park, CA, USA

ABSTRACT ARTICLE HISTORY

Telomere length measurement is increasingly recognized as a clinical gauge for age-related dis- Received 30 March 2018
ease risk. There are several methods for studying blood telomere length (BTL) as a clinical bio- Revised 11 July 2018
marker. The first is an observational study approach, which directly measures telomere lengths Accepted 22 July 2018
using either cross-sectional or longitudinal patient cohorts and compares them to a population Published online 2 2 2
of age- and sex-matched individuals. These direct traceable measurements can be considered KEYWORDS
reflective of an individual’s current health or disease state. Escalating interest in personalized Leukocyte; telomere length;
medicine, access to high-throughput genotyping and resulting acquisition of large volumes of cardiovascular disease; all-
genetic data corroborates the second method, Mendelian randomization (MR). MR employs telo- cause mortality; age-related
mere length-associated genetic variants to indicate predisposition to disease risk based on the disease; observational
genomic composition of the individual. When assessed from cells in the bloodstream, telomeres studies; Mendelian
can show variation from their genetically predisposed lengths due to environmental-induced randomization
changes. These alterations in telomere length act as an indicator of cellular health, which, in
turn, can provide disease risk status. Overall, BTL measurement is a dynamic marker of biological
health and well-being that together with genetically defined telomere lengths can provide
insights into improved healthcare for the individual.

Abbreviations: apoAI: apolipoprotein AI; BMI: body mass index; BP: blood pressure; BTL: blood
telomere length; CAC: coronary artery calcification; CAD: coronary artery disease; CHD: coronary
heart disease; CLL: chronic lymphocytic leukemia; CMV: cytomegalovirus; CRP: C-reactive protein;
CST: CTC1-STN1-TEN1; CT: computed tomography; CVD: cardiovascular disease; DNA: deoxyribo-
nucleic acid; DC: Dyskeratosis congenita; FISH: fluorescence in-situ hybridization; GWAS: genome-
wide association study; HbA1c: glycated hemoglobin; HDL-C: high-density lipoprotein cholesterol;
HH: Hoyeraal–Hreidarsson syndrome; HSC: hematopoietic stem cell; IGF1: insulin-like growth fac-
tor-1; IHD: ischemic heart disease; IL-6: interleukin-6; LDL-C: low-density lipoprotein cholesterol;
NT-proBNP: N-terminal pro B-type natriuretic peptide; MI: myocardial infarction; MR: Mendelian
randomization; PBMC: peripheral blood mononuclear cell; PC: progenitor cell; PCR: polymerase
chain reaction; qPCR: quantitative polymerase chain reaction; ROS: reactive oxygen species; SCD:
sudden cardiac death; SNP: single nucleotide polymorphism; ssDNA: single-stranded deoxyribo-
nucleic acid; STELA: single telomere length analysis; T2DM: type 2 diabetes mellitus; TC: total
cholesterol; TRF: terminal restriction fragment; TRF1: Terminal repeat Factor 1; TRF2: Terminal
repeat Factor 2; UCP2: uncoupling protein 2; US: United States; WBCs: white blood cells

1. Introduction predisposition. The number and breadth of studies

Telomeres are nucleoprotein structures found at the
ends of linear chromosomes, comprised of repetitive
hexameric deoxyribonucleic acid (DNA) sequence (50 -
TTAGGG-30 ) and specific associated proteins [1]. In add-
ition to providing a mechanism for the aging cell to
determine its replicative lifespan [2], telomeres serve as
molecular beacons for DNA damage that signal the cell
to activate repair mechanisms [3] or, in the case of
extreme damage, to signal senescence or cell death
pathways [4]. Considerable interest has developed
around the effectiveness and utility of using telomere
length as a clinical biomarker for disease and/or disease
associating blood telomere length (BTL) with lifetime
disease risks continues to increase, including reported
associations with autoimmune disorders [5–7] and, in
particular, age-related conditions that include cardio-
vascular disease (CVD, e.g. coronary heart disease
(CHD), atherosclerosis), type 2 diabetes mellitus (T2DM),
stroke, and cancer (Table 1). In a similar fashion, gen-
ome-wide association studies (GWAS) have found telo-
mere length-associated genetic variants that associate
with predisposition to disease risk (Table 1). At face
value, these studies suggest that telomere length meas-
urement and/or specific genetic variants could have

CONTACT Clare L. Fasching cfasching@telomeredx.com Telomere Diagnostics, Inc., 3603 Haven Ave., Suite A Menlo Park, CA 94025, USA
ß 2018 Informa UK Limited, trading as Taylor & Francis Group
2 C. L. FASCHING

Table 1. Summary of studies and cohorts by indication.

TL measurement
Indication All-cause mortality cohorts Sample size Study type method Reference
All-cause mortality Leiden Longevity Study 3359 Observational/MR qPCR [64]
All-cause mortality Copenhagen City Heart Study and 64367 Observational/MR qPCR [66]
Copenhagen General Population Study
All-cause mortality Epidemiological Study on the Chances of 12199 Observational qPCR [68]
Prevention, Early Recognition, and
Optimized Treatment of Chronic Diseases
in the Older Population, Nurse’s
Health Study
All-cause mortality Copenhagen City Heart Study 4576 Observational qPCR [67]
All-cause mortality National Health and Nutrition Examination 3091 Observational qPCR [69]
Survey, 1999–2002
All-cause mortality Zutphen Elderly Study 203 Observational qPCR [72]
All-cause mortality Steno Diabetes Center patients 273 Observational Southern blot [101]
All-cause mortality Italian National Research Center on Aging 568 Observational qPCR [100]
recruitment
All-cause mortality Osteoporotic Fractures in Men Study 2744 Observational qPCR [73]
All-cause mortality Tokyo Oldest Old Survey on Total Health, 1554 Observational Southern blot [15]
Tokyo Centenarians Study, Japanese
Semi-Supercentenarians Study
General Health Genetic Epidemiology Research on Adult 110266 Observational qPCR [65]
Health and Aging
All-cause mortality Lothian Birth cohort (1921) 550 Observational qPCR [74]
All-cause mortality Heart and Soul Study 780 Observational qPCR [71]
All-cause mortality Heart and Soul Study 608 Observational qPCR [70]
TL measurement
Indication Cardiovascular Disease Cohorts Sample Size Study Type method Reference
CVD Copenhagen General Populations Study, 64367 Observational/MR qPCR [66]
Copenhagen City Heart Study
MI INTERHEART study 3972 cases/ Observational qPCR [86]
4321 controls
CHD Mental Stress Ischemia Prognosis Study 566 Observational qPCR [84]
CAD West of Scotland Primary Prevention Study 484 Observational Southern Blot [82]
IHD Copenhagen General Populations Study, 290022 Observational/MR qPCR [87]
Copenhagen City Heart Study,
Copenhagen Ischemic Heart Disease
Study, CARDIoGRAM consortium,
CARDIoGRAMplusC4D consortium
MI/stroke Cardiovascular Health Study 419 Observational Southern Blot [85]
CVD Cardiovascular Disease in Intermittent 241 cases/ Observational qPCR [90]
Claudication study 249 controls
CHD Recruited Healthy people 203 Observational Southern Blot [81]
CVD Bruneck Study 88 Observational Southern Blot [88]
CAC Healthy individuals recruited in 325 Observational qPCR [91]
South Carolina
CAC National Heart, Lung and Blood Institute 3169 Observational Southern Blot [92]
Family Heart Study
CHD Hypercoagulability and Impaired Fibrinolytic 1517 cases/ MR N/A [111]
function MECHanisms, Coronary Artery 1601 controls
Bypass Graft, Simon Broome Familial
Hypercholesterolemia study, University
College London Diabetes and
Cardiovascular Disease Study, European
Atherosclerosis Research Study II
CAD CARDIoGRAM consortium 22233 cases/ MR N/A [105]
64762 controls
CVD UK BioBank 134773 MR N/A [133]
CHD ENGAGE Telomere Consortium, 22233 cases/ MR N/A [134]
CARDIoGRAM consortium, MAGIC consor- 64762 controls
tium, DIAGRAM consortium, GIANT
Consortium, Global Lipids Genetics con-
sortium, ENGAGE 1000 Genome
Consortium, 1000 Genome Consortium
for CHD
TL measurement
Indication Metabolic Disorder Cohorts Sample Size Study Type method Reference
T2DM Recruited urban Chinese population 930 cases/ Observational/MR qPCR [98]
867 controls
T2DM Strong Heart Family Study 2328 Observational qPCR [97]
(continued)
 CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 3

T2DM Bruneck Study 606 Observational qPCR [96]

Insulin resistance Framingham Heart Study-men 174 cases/ Observational Southern Blot [48]
156 controls
T2DM National Health and Nutrition Examination 3921 Observational qPCR [99]
Survey, 1999-2002
Oxidative stress Recruited rural Australian of 950 MR N/A [137]
European descent
T2DM University College London Diabetes and 569 MR N/A [136]
Cardiovascular Disease Study
TL measurement
Indication Cancer Cohorts Sample Size Study Type method Reference
Cancer Copenhagen City Heart Study, Copenhagen 95568 Observational/MR qPCR [140]
General Population Study
Cancer Copenhagen City Heart Study, Copenhagen 47102 Observational qPCR [139]
General Population Study
Lung Cancer Recruited from University of Texas MD 1385 cases/ Observational qPCR [142]
Anderson Cancer Center 1385 controls
Cancer Bruneck Study 787 Observational qPCR [141]
Pancreatic Cancer European Prospective Investigation into 331 cases/ Observational qPCR [143]
Cancer and Nutrition 331 controls
Chronic myelogenous UK Medical Research Council CMLIII Trial 59 cases/ Observational Southern Blot [145]
Leukemia 75 controls
Chronic myelogenous Recruited from Terry Fox Laboratory 174 cases/ Observational qPCR, Southern [144]
Leukemia 301 controls Blot, Flow FISH
Chronic Lymphocytic Recruited from University Hospital, 152 cases Observational qPCR [146]
Leukemia Ulm, Germany
Chronic Lymphocytic Recruited from University of Turin, Amedeo 401 cases/ Observational Southern Blot [147]
Leukemia Avogadro University of Eastern Piedmont 30 controls
Chronic Lymphocytic Recruited from Department of Clinical and 173 cases Observational qPCR [148]
Leukemia Experimental Medicine, Hematology
Section, Padova and Department of
Hematology, Vicenza
Chronic Lymphocytic UK Lloyd’s Register Foundation CLL4 trial 384 cases Observational qPCR, STELA [149]
Leukemia
Chronic Lymphocytic Recruited from Cardiff University 41 cases Observational STELA [150]
Leukemia
Chronic Lymphocytic Recruited from University Hospitals Umeå, 310 cases Observational qPCR, Southern [151]
Leukemia Uppsala and Link€ oping Blot, Flow FISH
Breast and Ovarian SEARCH study, Sisters in Breast Screening 103991 breast MR N/A [40]
Cancer (SIBS) study, EPIC-Norfolk study, cases, 39774 ovar-
Copenhagen City Heart Study, ian cases, 11705
Copenhagen General Population study. BRCA1 mutation
carrier cases, 53724
leukocyte telomere
length analysis
Colorectal Cancer Wellcome Trust case-control consortium 2157 cases/ MR N/A [155]
National Blood Service cohort, Scottish 3912 controls
population for the COGS, UK CORGI study
controls, VICTOR study, Scottish cases
from COGS, colorectal cancer cases
from CORGI
Glioma UCSF Adult Glioma Study, Wellcome Trust 1644 cases/ MR N/A [41]
Case-Control Consortium, The Mayo 7736 controls
Clinic, Illumina iControls, The Cancer
Genome Atlas
Cancer Genetic Associations and Mechanisms in 51725 cases/ MR N/A [152]
Oncology consortium 62035 controls
Chronic lymphocytic Eastern Cooperative Oncology Group (Mayo 273 cases/ MR N/A [153]
leukemia Clinic), Recruited at the Dana-Farber 5725 controls
Cancer Institute
Non-Hodgkin Meta-analysis of 9 prospective cohort stud- 10102 cases/ MR N/A [154]
lymphoma ies, 8 population based case/control stud- 9562 controls
ies, 5 clinic based case/control studies
Cancer and Non- Meta-analysis compiled by The Telomere 420081 cases MR N/A [157]
Neoplastic Disease Mendelian Randomization Collaboration (median 2526/dis-
ease/ 1093105 con-
trols (median
6789/disease)
COGS: consortium on the genetics of schizophrenia; CORGI: colorectal tumor gene identification; ENGAGE: European network for genetic and genomic epi-
demiology; FISH: fluorescence in-situ hybridization; CARDIoGRAM: coronary artery disease genome-wide replication and meta-analysis;
CARDIoGRAMplusC4D: coronary artery disease genome-wide replication and meta-analysis plus coronary artery disease; DIAGRAM: diabetes genetic repli-
cation and meta-analysis; GIANT: genetic investigation of anthropometric traits; MAGIC: meta-analysis of glucose and insulin-related traits consortium; MR:
Mendelian randomization; TL: telomere length; UCSF: University of California, San Francisco; UK: United Kingdom.
4 C. L. FASCHING
clinical utility in evaluating increased disease risk and/
or as sentinels to monitor health status and disease
progression.
The aim of this review is to assess the mounting evi-
dence for a relationship between telomere length meas-
urement and disease risk and to discuss limitations
surrounding its use as a biomarker for age-related dis-
ease risk. As background to genetic predisposition of an
individual’s telomere length and factors that can directly
influence measured telomere length, an overview of
studies on telomere length inheritance as well as factors
that modulate telomere length during the course of a
lifetime are presented. Two primary classes of telomere
length studies, observational and Mendelian randomiza-
tion (MR), provide the data under review. Using disease-
specific clinical cohorts, observational studies directly
monitor telomere length from the case population and
compare them to those from a control population.
Repeat sampling can detect changes in the BTLs attrib-
uted to fluctuations in the microenvironment from life-
style, health, or disease status of an individual. These
changes present as a dynamic, measurable feature that
can provide insight into the current disease risk status
of an individual. In MR studies, genomic DNA polymor-
phisms, single nucleotide polymorphisms (SNPs) associ-
ated with short or long telomere lengths, highlight
genetic predisposition to disease risk in case-control
clinical studies. The genetic nature of the MR studies
avoids the dynamic aspect of direct telomere length
measurement and focuses primarily on personalized
telomere length with regard to its disease risk predis-
position. Benefits and caveats of these two potentially
useful approaches will be discussed in the context of
age-related diseases.
2. Determinants of telomere length: genetics,
environmental factors, and
molecular pathways
2.1. Telomere length inheritance
Innate telomere lengths, defined as average basal telo-
mere length distributions characteristic of an individual’s
stem cells, vary modestly across populations and are
determined by inherited genetic components. High her-
itability has been shown in twin and sibling studies [8],
and is dramatically illustrated in a telomere-associated
genetic syndrome with polygenic X-linked, autosomal-
recessive and autosomal-dominant variations,
Dyskeratosis congenita (DC). DC is a genetic disorder
with most known mutations residing within genes
involved in telomere biology, including DKC1, TERC,
TERT, TIN2, NOLA2, and NOLA3 [9]. A common molecular
feature of all DC patients is abnormally short telomeres
[9], which fall below the first percentile, that is, telomere
lengths 99% of the population [10]. Phenotypes associ-
ated with DC, include idiopathic pulmonary fibrosis,
aplastic anemia, and cancer [11]. Lineages with the auto-
somal-dominant form of DC display genetic anticipation,
in which telomere lengths shorten across generations
and the disease presents earlier and with more severity
in offspring relative to the previous generation [11].
Studies of DC families strongly support telomere length
heritability. Studies assessing telomere lengths in healthy
individuals and their offspring also support high herit-
ability, with some identifying maternal [8] or paternal
paths [12–15]. In one study of DC families with individu-
als haplo-insufficient for TERT [16], affected individuals
(n ¼ 15) had telomere lengths below the 10th percentile,
with 13/15 below the first percentile [16]. Seven of nine
of their genotypically normal offspring fell below the
10th percentile in telomere lengths, suggesting a genetic
inheritance of the parental telomere length [16]. The
remaining two genotypically normal offspring had lon-
ger telomere lengths than their parents, illustrating the
genetic complexity of telomere length inheritance [16].
A large meta-analysis of six cohorts (n ¼ 19,713) illus-
trated a strong association between parental and off-
spring telomere lengths, with a slightly greater maternal
association [8]. This study also found telomere lengths in
monozygotic twins were more similar than in dizygotic
twins [8]. The telomere lengths of the dizygotic twins
and their non-twin siblings were similar [8]. Heritable fac-
tors are not the only drivers of telomere length. An asso-
ciation between the telomere lengths of spouses,
especially among older couples, suggests there may also
be environmental components influencing length [8].
Evaluation of parental age and offspring telomere
lengths reveals an association between increased pater-
nal age at conception and longer telomere lengths in
their offspring [17–20]. Inquiry into telomere length
inheritance and associated diseases reveals the genetic
complexity of the telomere length set-point. Extrinsic
conditions such as socioeconomic status and psycho-
logical stress can affect maternal telomere lengths [21]
and may also affect the newborn [22]. Given that an indi-
vidual’s telomere length set-point is genetically inherited
and is also influenced by age, sex, environment, stress,
and health status, what mechanisms control and desta-
bilize telomere length?
2.2. Telomere length homeostasis:
molecular mechanisms
An individual’s telomere length set-point derives from
the actions of a combination of heritable pathways,
 CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 5

including factors such as telomerase [1] (i.e. the ribonu-
cleoprotein complex that actively lengthens telomeres
during development), a telomere trimming mechanism
that prevents over-lengthening [23], and the protein
complexes that protect the telomeric DNA, shelterin
[24,25], and CTC1-STN1-TEN1(CST) [26,27]. Regardless of
the initial set-point, telomere lengths decrease with
every cell division in an individual due to the “end-repli-
cation problem” [1]. Both oxidative and replication
stress can induce rapid telomere shortening due to
DNA damage [28]. Any one or all of these mechanisms
could be active on blood telomeres at a given time,
influencing its length distribution. In addition to these
intrinsic factors, viral infections including human
immunodeficiency virus showing a viral load [29,30],
active hepatitis C virus [30], and persistent cytomegalo-
virus [31] have been shown to associate with
short BTLs.
Telomerase and telomere trimming. Telomerase
extends telomeres using an RNA moiety, hTR. This moi-
ety partially anneals to a single-stranded DNA 30 over-
hang structure of the telomere providing a template for
hTERT, the catalytic subunit of telomerase (Figure 1(A))
[32–34]. Telomerase is active during embryonic devel-
opment to lengthen telomeres during cellular expan-
sion and remains active postnatally in tissues that self-
renew, such as stem cells [1]. This complex can be epi-
sodically activated in tissues that require further cell
expansion, for instance the hematopoietic system [1].
Excessive telomere lengths are prone to replication fork

(A) (B)

hTERT
Nuclease

TTAGGGTTAGGG ECTR
AAUCCC

Shelterin

hTR
Dyskerin NHP2 NOP10 GAR1 NAF1 RAP1 TRF2 TRF1 TIN2 TPP1 POT1

(C) (D)
5’ 3’
3’ 5’ Reactive Oxygen Species
(H2O2)

5’ 3’
3’
Telomere length

5’
Cell divisions

Replication stress

5’ 3’
3’ 5’

Base modification
5’ 3’
3’ 5’ (MNNG)

5’ 3’
3’ 5’

Figure 1. Processes involved in telomere length homeostasis. (A) Telomerase is a ribonucleoprotein that uses an RNA moiety,
hTR, as a template for adding sequence to the leading strand of the telomere. Several proteins, Dyskerin, NHP2, NOP10, and
NAF1 are associated with hTR during its maturation. GAR1 replaces NAF1 in the mature holoenzyme. (B) Telomere trimming, his-
torically known as telomere rapid deletion, involves cleavage of the terminal portion of the telomere, which is thought to involve
the t-loop, and results in the release of linear and circular extra-chromosomal telomeric DNA (ECTR). (C) Telomere attrition, also
known as the “end replication problem”, occurs during every cell division in the absence of any telomere lengthening mechan-
ism. This process results in the gradual shortening of telomere length and provides the means for the characteristic “cellular life-
span.” (D) DNA damage can affect telomere length by introducing single-strand breaks and gaps (H2O2), stalled replication forks
(replication stress), or bulky adducts on the DNA (MNNG), all of which require dissolution or resolution and often result in dra-
matic shortening of the affected telomere.
6 C. L. FASCHING
stalling or arrest due to slippage, or secondary structure
formations (e.g. G-quadruplex), both of which require
resolution [35]. Telomere trimming is a mechanism that
rapidly reduces telomere length by removing part of
the end structure (Figure 1(B)) [23]. This mechanism has
been demonstrated in cancer cells [36] that have been
modified to have very long telomeres, in leukocytes
that have been stimulated with a mitogen [37], and in
human stem cells [38]. Together, telomerase and telo-
mere trimming provide cells with a homeostasis path-
way for telomere length regulation [23].
Telomere length, DNA replication, and replicative cap-
acity. Recently, Gadalla et al. [39] showed improved sur-
vival among recipients of bone marrow transplants for
treatment of aplastic anemia when donor tissues had
longer telomere lengths [39]. This increase in survival
was attributed to an increase in telomere-mediated rep-
licative capacity that helped reestablish a productive
hematopoietic system. Because post-replicative removal
of the Okazaki fragment generates a gap and a single-
stranded 30 DNA overhang, telomeres shorten after
every cell division in a process known as “the end-repli-
cation problem” [1] (Figure 1(C)). Eventually, telomeres
reach a point of critical shortness, after which cells
enter a senescent state of permanent growth arrest [1].
Telomere attrition has long been associated with cellu-
lar aging and provides proliferating cells with a means
to determine cellular lifespan [2] or replicative capacity.
Genetic predisposition for longer telomere lengths has
been associated with increased cancer risk [34,40,41].
Simply, longer telomere lengths promote more cell divi-
sions prior to senescence or cell death [1]. As bone mar-
row transplant recipients exemplify [39], repopulating a
cellular niche necessitates sufficient telomere-mediated
replicative capacity for success.
Telomere length, DNA damage, and oxidative stress. In
addition to gradual telomere length attrition, DNA dam-
age or resolution of replication stress at the telomere
can cause rapid shortening (Figure 1(D)). A major
source of DNA damage is oxidative stress resulting from
reactive oxygen species (ROS), due to inefficient
removal or depletion of antioxidants [28]. ROS directly
damages DNA in the form of single-stranded DNA
breaks or indirectly through generation of aldehyde
metabolites that induce formation of DNA adducts,
leading to damage during the replication process [42].
The formation of single-stranded DNA gaps and breaks
within the telomere can collapse into double-stranded
breaks during replication, shortening the telomere
[43,44]. Oxidative stress has also been proposed to con-
tribute to telomere shortening by altering the binding
of telomere-specific proteins, terminal repeat factor 1
(TRF1) and 2 (TRF2), to the telomeric DNA [45]. These
genetically distinct pathways contribute to setting an
individual’s general telomere length and provide the
complexity that contributes to changes in telomere
length depending on age and health status.
3. Telomere lengths in the clinic: sampling a
potential biomarker
3.1. White blood cells: a DNA source for dynamic
and genetic telomere length measurement
Complete blood counts and leukocyte counts/analysis
have been recognized for years as useful and viable
markers of an individual’s health [46] and white blood
cells (WBCs) are the sample of choice for telomere
length studies. This is due, in part, to the critical role
they perform in immunity, inflammation, and cellular
clearance as well as the fact that WBCs are exposed to
the metabolic by-products of other tissues, including
ROS [47]. Chronic inflammation and ROS, indicative of
adverse health conditions, are observed to shorten BTLs
[48] and therefore can provide an indicator of disease
or disease risk. In itself, blood is a minimally invasive
source for genomic biomarker analysis and is amenable
to repeated sampling over time. Telomere length-asso-
ciated genetic variants inform an individual as to their
predisposition to disease risk. WBC progenitor cells
(PCs), hematopoietic stem cells (HSCs), are capable of
self-renewal [49], using telomerase to delay telomere
shortening [1]. It is estimated that approximately 1012
new WBCs enter the blood every day in healthy adults
[50]. The telomere lengths of these newly circulating
cells are reflective of the unadulterated stem cell popu-
lation, as the fresh cohort has genetically defined HSC
telomere lengths and thus are subject to telomere
shortening influenced by lifestyle and disease status.
Similar to measures of blood pressure (BP), heart rate,
and glucose levels, these changes create a dynamic
indicator of health status.
3.2. Molecular methods to measure
telomere length
Several broadly established methods are available to
measure telomere length, each with application-
dependent benefits and caveats. The most frequently
reported method relies on quantitative PCR (qPCR) to
calculate relative average telomere length. qPCR pro-
vides rapid, high-throughput analysis for large-scale
clinical studies [51,52]. Fluorescence-in situ-hybridiza-
tion (FISH) [53] and Flow-FISH [54] provide the means
to estimate telomere length within a single cell or

across a specific population of cells when combined
with specific antibody staining. Terminal restriction
fragment (TRF) analysis provides the ability to estimate
absolute telomere length in DNA base pairs [55]. Single
telomere length analysis (STELA) allows measurement
of a near-exact length of chromosome-specific telo-
meres [56]. When executed with standard reference
materials, controls and validated methodologies, any of
these methods can provide useful information about
telomere length. This reviewer recommends to the
interested reader four in-depth reviews that discuss
these methodologies, their benefits, and cav-
eats [57–60].
Although each of these methodologies provides
valuable data, it is important to note that their assay-
specific definitions of telomere length typically do not
allow telomere length comparisons across methods. In
addition, similar techniques performed in different labo-
ratories may utilize different quality control compo-
nents, resulting in different absolute values [61]. For
example, relative average telomere length values as
defined by qPCR are based on the source DNA used for
the standard curve, which is not standardized across
laboratories. TRF protocols vary and are not standar-
dized to one enzyme cocktail. Absolute or relative aver-
age telomere length data produced across separate,
non-standardized studies cannot be easily compared or
aggregated, as variations in methodologies may con-
found analyses. To bypass these limitations, this review
will focus on study data that have been categorized as
percentile divisions of the study population, allowing
comparison of population data independent of telo-
mere measurement method.
4. Telomere length and disease: is telomere
length a reliable clinical marker for non-
neoplastic age-related disease risk?
4.1. Observational studies: BTLs as indicators of
health or disease
The prevalence of high-powered clinical studies pro-
vides strong evidence of an association between telo-
mere length measurement and several age-related
diseases. From a clinical perspective, telomere lengths
that fall within the lowest age- and sex-matched per-
centiles (shorter telomeres) increase that individual’s
disease risk for all-cause mortality, CVD and age-related
metabolic disorders (Table 1). The observational studies
that link disease and/or mortality risk to short telomeres
are agnostic to the mechanism by which these telo-
mere lengths are acquired.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 7
Although telomere length predisposition is heritable,
dynamic variation of telomere length in an individual’s
WBCs is influenced by lifestyle, health, and disease state
[21]. Genetic telomere length has been shown to con-
tribute a relatively low percentage to the overall vari-
ability of telomere length [62], potentially justifying
direct measurement as a biomarker for non-neoplastic
age-related diseases, especially in individuals between
the ages of 50 and 75 years (Table 2). As aging popula-
tions adopt habits to improve health and are living lon-
ger, definitions of age groups tend to evolve. For the
2010 census, the US Census Bureau divided age groups
into: <18 years, 18–44 years, 45–64 years, and 65þ years
[63]. For the purpose of this review, middle-age is
defined as 45–64 years, older adults as 65–74 years, and
75þ as elderly. For studies that explored telomere
lengths in nonagenarians and above, those age groups
will be identified as ultra-elderly. As this review dis-
cusses the studies, it will start with those of the ultra-
elderly, as this population is quite informative when
compared to control groups with shorter life spans.
All-cause mortality and telomere length. Several
observational studies have associated direct BTL meas-
urement with all-cause mortality risk (Table 1), a classifi-
cation that encompasses death from any cause. Several
studies evaluated the relationship between BTL and all-
cause mortality in the ultra-elderly and their offspring.
The inverse association between all-cause mortality and
BTL in both middle-aged and ultra-elderly individuals
supports the monitoring of telomere lengths to collect
additional information for healthcare providers and
their long-lived patients. The Leiden Longevity Study
(LLS) cohort was used to evaluate the association of
telomere length to aging and age-related diseases in a
population of long-lived people, in which nonagenar-
ians (i.e. 90 years), represented the long-lived
(n ¼ 944; mean age: 93; age range: 89–104 years), their
offspring represented the genetically predisposed to be
long-lived, (n ¼ 1,671; mean age: 61; age range:
39–81 years), and their offspring’s spouses represented
the control population, (n ¼ 744; mean age: 60; age
range: 36–79 years) [64]. These studies show an inverse
association with BTL and all-cause mortality in the mid-
dle-aged groups, a combination of the LLS offspring
and their spouses, as well as in the nonagenarians,
quantified as a 25% decrease in mortality risk/unit BTL
[64]. After adjusting for covariates including immune-
related markers (i.e. insulin-like growth factor-1 (IGF-1),
C-reactive protein (CRP), interleukin-6 (IL-6), cyto-
megalovirus (CMV) infection and lymphocyte counts),
using a Cox proportional hazard model, the association
of short BTL with all-cause mortality was found to be
8 C. L. FASCHING

Table 2. Association of direct telomere length measurement and age-related diseases.

Middle-aged Older adult Ultra-elderly
Disease Associated BTL Adult 18–44 (years) 45–64 (years) 65–74 (years) Elderly 75–89 (years) 90 þ (years)
All-cause mortality Short Weischer et al. [67], Deelen et al. [64], Farzaneh-Far et al. Deelen et al. [64] Deelen et al. [64]
Astrup et al. [101] Rode et al. [66], [71], Bonfigli
Weischer et al. [67], et al. [100]
Mons et al. [68],
Goglin et al. [70],
Astrup et al. [101]
None N/A N/A N/A Houben et al. [72], N/A
Svensson et al. [73],
Marioni et al. [74]
CVD Short N/A Brouilette et al. [81], Fitzpatrick et al. [85], N/A N/A
Brouilette et al. [82], D’Mello et al. [86],
Hammadah et al. Willeit et al. [88]
[84], D’Mello et al.
[86], Scheller Madrid
et al. [87], Willeit
et al. [88],
Raschenberger et al.
[90], Mainous et al.
[91], Hunt et al. [92]
None N/A N/A Svensson et al. [73], N/A
Willeit et al. [88]
T2DM Short N/A Demissie et al. [48], N/A N/A N/A
Willeit et al. [96],
Zhao et al. [97],
Xiao et al. [98]
None Menke et al. [99] Menke et al. [99] N/A N/A N/A
Cancer Long N/A Sanchez-Espiridion N/A N/A N/A
et al. [142]
Short N/A Willeit et al. [141], Willeit et al. [141] N/A N/A
Sanchez-Espiridion
et al. [142]
None Campa et al. [143] Weischer et al. [139], Weischer et al. [139], Svensson et al. [73] N/A
Rode et al. [140], Rode et al. [140],
Campa et al. [143] Campa et al. [143]

independent of these covariates in the offspring/
spouses and the nonagenarians [64]. No difference in
BTL or in rate of telomere attrition was found between
the long-lived offspring and the controls, suggesting
that BTL in the middle-aged does not necessarily
explain the long-lived characteristic of these families.
This similarity in telomere characteristics between the
long-lived offspring and their spouses implicates con-
tribution from other (i.e. non-genetic) factors affecting
telomere lengths. In a similar study, Arai et al. [15]
examined the telomere lengths in centenarians and
their offspring and found that telomere lengths
decreased up to the age of 100 years, after which
they began lengthening [15]. These long-lived popu-
lations and their offspring had longer telomeres than
expected for their age group [15]. Similarly, a large
study using the Genetic Epidemiology Research on
Adult Health and Aging (GERA) cohort (n ¼ 110,266)
confirmed the age-related decline in telomere lengths
up to 75 years of age while the population older
than 75 years showed increasing telomere length [65].
This association of longer telomere lengths and
increased survival in these elderly populations is con-
sistent with a survivor effect.
The following studies, when taken together, illustrate
an inverse association between telomere length and all-
cause mortality for middle-aged individuals (Table 2),
suggesting that telomere length measurement could
provide additional information to healthcare manage-
ment when used as a biomarker for mortality risk. A
study using the Copenhagen City Heart Study (CCHS)
cohort and the Copenhagen General Population Study
(CGPS) cohort (n ¼ 64,367) evaluated the association
between telomere lengths and all-cause mortality in
the middle-aged [66]. At the time of follow-up (median:
7 years; range: 0–22 years), 12% of individuals had died
(n ¼ 7,607). The telomere length data was subdivided
into deciles, highlighting an inverse association
between the shortest telomere length decile compared
to the longest and all-cause mortality [66]. Increased
age, sex, and lifestyle factors including current smoking,
increased body mass index (BMI), physical inactivity,
and increased alcohol intake were the focus of a smaller
study using the CCHS cohort (n ¼ 4576), in which the
cross-sectional data showed a modest association
between short telomere length and all-cause mortality
at both baseline and the 10-year follow-up [67]. After
adjusting for age and sex, if necessary, smoking, BMI,

alcohol consumption, and physical activity, Mons et al.
[68] found the lowest quintile BTL associated with all-
cause mortality in the Epidemiological Study on the
Chances of Prevention, Early Recognition, and
Optimized Treatment of Chronic Diseases in the Older
Population (ESTHER; n ¼ 3,566), and the Nurse’s Health
Study (NHS; (n ¼ 8,633), cohorts [68]. Similarly, in the
National Health and Nutrition Examination Survey
(NHANES), 1999–2002 cohort, the two shortest telomere
length quartiles were modestly associated with all-
cause mortality, when compared to the longest [69]. In
one of two studies in individuals with stable CHD using
the Hearts and Soul cohort [70,71], Goglin et al. [70]
found telomere shortening was associated with
increased mortality in individuals with stable coronary
artery disease (CAD) over a 5-year period (n ¼ 608) [70].
In the second study, Farzaneh-Far et al. [71] sought an
association between telomere length and other clinical,
inflammatory and echocardiographic variables that may
be used as prognostic determinants in individuals with
stable CAD (n ¼ 780) [71]. After adjusting for clinical fac-
tors, those in the shortest quartile compared to those in
the longest were found to have an increased risk of all-
cause mortality [71]. Collectively, these studies promote
monitoring of BTL as a clinical biomarker for all-cause
mortality risk in the middle-aged.
In contrast to studies of middle-aged and long-
lived populations, several independent studies focus-
ing on the elderly age group failed to show an asso-
ciation between telomere length and all-cause
mortality (Tables 1 and 2). Several explanations could
play a role, including the advanced mean age of the
cohort participants, the relatively narrow age ranges
of the participants in these studies, and the tendency
for an increase in telomere lengths in this age
group. A group of men from the Zutphen Elderly
Study (n ¼ 203) were evaluated for association of BTL
and all-cause and cause-specific mortality. The subse-
quent analysis showed no association between all-
cause mortality and telomere length, socioeconomic,
or lifestyle factors [72]. Paired samples from the sur-
vivors (n ¼ 75), collected in 1993 (mean age: 78 years)
and 2000 (mean age: 83 years), were analyzed for
BTL; no association with telomere length and all-
cause mortality, CVD mortality or cancer mortality in
this longitudinal study was found [72]. A larger study
in the MrOS-Sweden study cohort (n ¼ 2,744), of eld-
erly men (mean age: 75.5; age range: 68–81 years;
average follow-up: 6 years) where approximately one-
fifth of the participants had died (n ¼ 556), showed
no association between baseline BTL and all-cause
mortality, CVD mortality, or cancer mortality after
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 9
multivariate adjusted analysis [73]. Longitudinal data
from the Lothian Birth cohorts in 1921 (n ¼ 550) and
1936 (n ¼ 1091) showed a modest association of
increased BTL with decreased mortality risk in the
1921 cohort. However, when evaluating the 1936
cohort, independently or combined with the 1921
cohort, no significant association between telomere
length and all-cause mortality was identified [74].
These studies fail to signify an association between
telomere length and all-cause mortality in elderly
men. The propensity of the telomeres of some eld-
erly to increase in length [15,65] could diminish the
usefulness of BTL measurement as a biomarker for
this age group.
CVD and telomere length. CVD in the United
States is comprised of 45.1% CHD, 16.5% stroke,
8.5% heart failure, 9.1% high BP, 3.2% artery disease,
and 17.6% other [75]. Underlying CAD can precipi-
tate into sudden cardiac death (SCD), which is
defined as death from an unexpected cardiac event
[76,77]. SCD primarily occurs in adults 35þ years
and accounts for approximately 20% of all deaths
due, in part, to undiagnosed CHD [78,79]. SCD
occurs in 44–52% of men and 59–69% of women
without previous history of CVD [79]. Current meth-
ods of identifying individuals at risk for SCD include
family history of CVD or sudden cardiac arrest, bio-
chemical and genetic markers for atherosclerosis,
coagulation, endothelial function, inflammation, oxi-
dative stress, renal function, euro-humoral status,
and cardiac pump functionality [78]. When account-
ing for age, sex, and ethnicity, a CVD risk score such
as the Framingham or cardiovascular mortality score
can be generated based on BP, total cholesterol
(TC), low-density lipoprotein cholesterol (LDL-C), and
high-density lipoprotein cholesterol (HDL-C) values
[78,80]. According to the American Heart Association,
several lifestyle factors (e.g. smoking, physical activ-
ity, diet, and weight) and health factors (e.g. TC, BP,
and glycemic control) are indicative of CHD risk [75].
Together, these risk factors and risk scores are the
current measure by which CVD is identified
and treated.
As early as 2003, short BTLs have been associated
with increased risk of myocardial infarction (MI) and
CHD (Table 1). Numerous subsequent studies uphold
the utility of BTL as a clinical biomarker for CVD/CHD
risk in individuals not otherwise identified by standard
cardiac risk factors, including BMI, smoking, and TC
(Table 1). Included in the studies showing an inverse
relationship between telomere length and CVD/CHD
risk are as follows: CHD/CAD/ischemic heart disease
10 C. L. FASCHING
(IHD), MI, coronary artery calcification (CAC), and ath-
erosclerosis (Tables 1 and 2). Brouilette and colleagues
[81] evaluated 203 individuals who had experienced an
MI by 50 years of age and compared their BTLs to an
age-, sex-, and smoking status-matched control group
(n ¼ 180) with no history of CHD. The two shortest telo-
mere length quartiles showed an inverse association
with CHD [81]. In a study of the West of Scotland
Primary Prevention Study (WOSCOPS) cohort (n ¼ 484),
Brouilette and colleagues [82] again assessed the asso-
ciation of telomere length with prediction of CAD [82].
A higher risk of CAD was associated with telomere
lengths in the middle and shortest tertiles [82]. When
these groups were given statins, their CAD risk lowered,
while those in the middle and shortest tertiles of the
placebo group remained at risk for CAD [82]. As statin
treatment is the standard of care to reduce CVD risk
[83], the association with longer telomere length may
provide useful data on the use of telomere length as a
biomarker to monitor statin efficacy and CVD risk.
Hammadah et al. [84] hypothesized that since reduced
numbers of circulating PC had been linked to CAD, and
telomere length could directly impact the replicative
capacity of PCs, PCs would mitigate the association
between telomere length and adverse CVD outcomes.
However, in a population of stable CAD patients using
the Mental Stress Ischemia Prognosis study (MIPS)
cohort (n ¼ 566; average age 63 ± 9 years), they found
history of MI, but not CAD severity associated with
shorter telomere lengths after multivariate analysis [84].
The lowest quartile of BTL and PCs were each associ-
ated with increased risk of CAD; however, no significant
interaction was found between PCs and telomere
lengths [84]. These data indicate an independent asso-
ciation of short telomere length and decreased number
of PCs with future CVD events. Similar to data for all-
cause mortality, individuals under the age of 73 years
from the Cardiovascular Health Study (CHS) cohort
(n ¼ 419) showed an association between short telo-
mere length and MI, as well as stroke [85]. After adjust-
ing for diabetes status, physical activity, hypertensive
status, smoking status, age or ethnicity, an inverse asso-
ciation of telomere lengths with MI risk was also seen
in the INTERHEART cohort (n ¼ 3972 cases; n ¼ 4321
controls) [86]. An inverse association of telomere
lengths with IHD was found in each of the three short-
est telomere length quartiles when compared with the
longest, after multifactorial adjustment in two Danish
cohorts (CCHS and CGPS; n ¼ 62996) [87]. Willeit et al.
[88] studied the association between telomere length
and CVD in the Bruneck cohort. Individuals that devel-
oped CVD at follow-up (n ¼ 88) were positive for
shorter telomere lengths; after adjusting for age and
sex, BTL was associated with HDL-C and apolipoprotein
AI (apoAI) and inversely associated with diabetes, CRP
and CVD in multivariable adjusted models [88]. Analysis
of the CVD group based on age showed an inverse rela-
tionship between baseline telomere lengths and dis-
ease presentation of MI or stroke, in the first three age
groups, 45–54, 55–64, and 65–74 years [88]. Similar
to studies of all-cause mortality, individuals in the
75–84 year age range did not show an association
between telomere length and CVD [88]. These data fur-
ther strengthen the evidence that telomere length
measurement could be an additional predictive bio-
marker for the development of CVD between the ages
of 45–74 years (Table 2).
Atherosclerosis, a chronic disease in which choles-
terol-rich deposits, scar tissue, inflammatory factors,
and cells build up on the artery walls, contributes to
underlying CVD [75,89]. Different methods of measuring
atherosclerosis-mediated CVD risk include ultrasound to
determine brachial artery flow mediated dilution, intima
media thickness, and carotid plaques, as well as CT
scans to evaluate CAC [89]. Atherosclerosis is another
facet of CVD that may use BTL as a biomarker for dis-
ease risk. Willeit et al. [88] evaluated telomere lengths
in the development of atherosclerosis in the Bruneck
cohort (n ¼ 623), a third of which had developed ath-
erosclerosis (n ¼ 294) [88]. Short telomere lengths asso-
ciated with advanced, but not early atherosclerosis in
this cohort [88], suggesting their diagnostic, but not
prognostic potential for this indication. Peripheral artery
disease is caused by atherosclerosis and increases the
risk of CVD or stroke [90]. An inverse association
between telomere length and peripheral artery disease
risk was identified in Caucasian males, which remained
after multivariate adjustment of age, log-CRP, HDL-C,
current smoking, and log-N-terminal B-type natriuretic
peptide (NT-proBNP) [91]. Growing evidence suggests
that assessing progression of CAC may provide a posi-
tive cost–benefit ratio and treatment strategies for CHD
[75]. The following studies explored the association of
CAC with BTL. Mainous and colleagues [91] hypothe-
sized that based on the association of atherosclerosis
with increasing age and telomere length with cellular
age [2] and CAD, telomere length may be associated
with CAC. They evaluated the association between BTL
and CAC in a group of healthy individuals (n ¼ 235; age
range: 40–64 years), with no history of CVD and found
telomere lengths in the shortest tertile were associated
with greater atherosclerosis in a multivariate adjusted
model of age, sex, race, metabolic syndrome, and smok-
ing, when compared to the longest telomere length

tertile, even after including Framingham Risk Score [91].
Using CAC as a metric of CAD risk and atherosclerosis
burden, a study using the National Heart, Lung and
Blood Institute Family Heart Study cohort (n ¼ 3169)
found the shortest telomere length tertile was associ-
ated with increased CAC category [92]. These associa-
tions support measuring BTL as a disease indicator for
atherosclerosis as well as CAD risk (Tables 1 and 2). The
scale of studies associating telomere length with CVD
risk heightens the utility of telomere length measure-
ment as a clinical biomarker for disease risk.
Age-related metabolic disorders. Metabolic syndrome
is characterized by central obesity, impaired glucose
tolerance, hypertension, dyslipidemia, high triglyceride
concentration, and low HDL-C concentration, which
collectively increase risk of developing T2DM [93].
T2DM, a prominent metabolic disease, occurs primar-
ily in middle-aged individuals between 60 and
74 years [94]. T2DM is associated with an increase in
insulin resistance and is a risk factor for other age-
related diseases, such as atherosclerosis and CVD [94].
Insulin resistance is induced by a sustained inflamma-
tory response and leads with T2DM, hypertension,
atherosclerosis, and metabolic syndrome [95]. Studies
on the relationship of telomere length with T2DM
and related metabolic disorders are few (Tables 1 and
2), but present a compelling picture.
Diabetes has been associated with oxidative stress,
which in turn affects BTLs. In a prospective study using
the Bruneck cohort (n ¼ 606) to determine the associ-
ation of BTL with risk of new-onset T2DM, Willeit et al.
[96] measured BTL prior to the clinical detection of
T2DM [96]. Although no association between BTL and
oxidative stress, inflammation or hyperglycemia was
detected, the lowest quartile of BTL was inversely asso-
ciated with T2DM risk [96]. Another prospective study
used the Strong Heart Family Study (n ¼ 2328) to asso-
ciate BTL and T2DM risk [97]. Patient samples with
prevalent T2DM or CHD at baseline were excluded and
followed up after 5.5 years [97]. The 292 individuals that
developed T2DM had shorter baseline BTL. After multi-
variate adjustment for sex, age, BMI, fasting glucose,
and total triglycerides, the shortest quartile of BTL
showed a higher risk of developing T2DM compared to
the longest [97]. Insulin resistance and oxidative stress
are associated with hypertension leading to CVD [48].
Based on its association with oxidative stress, BTL was
assessed for an association with hypertension using
Framingham Heart Study men (n ¼ 156 controls;
n ¼ 174 hypertensive) [48]. Demissie et al. [48] found
BTL was negatively associated with insulin resistance
and systemic oxidative stress. After adjusting for age,
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 11
the hypertensive group had shorter BTL that could be
attributed to its association with insulin resistance [48].
In another study, Xiao and colleagues [98] explored the
association of BTL and T2DM in a Chinese population
(n ¼ 930 cases; n ¼ 867 controls). They found shorter
BTL in the T2DM population compared to controls even
after multivariate adjustment [98]. BTL was inversely
associated with age and sex (male) in the control popu-
lation, but not in the T2DM population [98]. In contrast
a study using the NHANES (1999–2002) cohort
(n ¼ 3,921), found no association between telomere
length and T2DM status or duration after adjusting for
age, ethnicity, sex, education, income, smoking, pack-
years smoked, alcohol consumption, BMI, and CRP [99].
Bonfigli et al. [100] explored the predictive value of
BTL measurement for all-cause mortality risk in a
T2DM population [100]. BTL was predictive for all-
cause mortality, even when adjusted for T2DM com-
plications, age, sex, and disease duration [100]. In a
Kaplan–Meier comparison of T2DM duration and BTL,
the first 76 months showed the highest mortality risk
was conferred by the group that had longer T2DM
duration and shorter telomere lengths at baseline
[100]. A study in type I diabetic patients (n ¼ 273)
also showed shorter telomere lengths associated with
all-cause mortality, when adjusted for age, sex, smok-
ing, systolic BP, TC, glycated hemoglobin (HbAlc), log
urinary albumin excretion rate, previous MI, or apo-
plexia [101]. Collectively, these studies show a modest
association of BTL, insulin resistance, and T2DM.
Together, these studies present compelling data asso-
ciating telomere lengths, diabetes disease status, and
all-cause mortality risk. More studies are needed to
elucidate the association of metabolic disorders and
telomere lengths.
4.2. MR: genetic variants as predictors of disease
risk attributable to telomere length
Many studies, including those described above in
Observational Studies, report on the association
between disease risk and telomere length that have
been directly measured for each subject; one of the
core strengths of the observational data. Direct obser-
vation of telomere length as a phenotype, however,
can be influenced by confounding factors including
lifestyle, socioeconomic status, health status, reverse
causation, and selection bias [102]. Increasingly, MR
methods are being used to model the contribution of
telomere length to disease risk to evaluate its potential
as a biomarker for predisposition to disease risk
or state, and in some cases, to assign a causal agent.
12 C. L. FASCHING

Table 3. ENGAGE Telomere consortium single nucleotide polymorphisms.

SNP Tel-related gene Pathway Locus Chromosome Reference
rs10936599 TERC Telomerase MYNN 3 [105]
rs2736100 TERT Telomerase TERT 5 [105]
rs7675998 NAF1 Telomerase NAF1 4 [105]
rs9420907 OBFC1/STN1 Telomere end protection OBFC1 10 [105]
rs755017 RTEL1 Telomere replication ZBTB46 20 [105]
rs8105767 Unknown Unknown ZNF208 19 [105]
rs11125529 Unknown Muscle differentiation ACYP2 2 [105]

This method is used extensively in epidemiological Table 4. Complete list of IVs used in Mendelian randomiza-
studies where a genotype closely associated with a tion studies.
phenotype is used as a proxy for the phenotype, most Association with No association
often in GWAS analyses for disease risk [102–104]. leukocyte telomere with telomere
Gene Chromosome length lengths
The rationale for MR methods is rooted in Mendel’s CXCR4 2 [108] Unknown
law of independent assortment, where alleles inherited ACYP2 2 [105,106] Unknown
TERC 3 [62,106–110,155,159] Unknown
from both parents are randomly distributed to off- MNNY 3 [105,110] Unknown
spring. SNPs associated with a particular telomere PXK 3 [106] Unknown
length are used as a proxy for telomere length in stud- NAF1 4 [105,106] Unknown
TERT 5 [40,105,106,159] Unknown
ies for disease risk and should be randomly distributed ZNF311 6 [106] Unknown
throughout an unbiased general population. These TNKS 8 [156] Unknown
OBFC1 10 [62,106,108,111] Unknown
SNPs are assumed to have no direct association with MEN1 11 [156] Unknown
the disease of interest, but can, however, potentially Mre11 11 [156] Unknown
UCP2 11 [136,137] [98]
provide assignment of a causal influence by the pheno- DDX11 12 [160] Unknown
type on the disease risk [104]. Telomere length-associ- BICD1 12 [161] Unknown
CEP95 17 [110] Unknown
ated SNPs have been identified using GWAS by SMURF2 17 [110] Unknown
analyzing the allelic association to specific telomere CTC1 17 [62] Unknown
RECQL5 17 [156] Unknown
lengths (Tables 3 and 4). In addition to determining the ZNF676 19 [62,108] Unknown
telomere length associated with a particular SNP, there ZNF208 19 [105] Unknown
are several assumptions that must hold true for the SNP BCL2L1 20 [106] Unknown
DHX35 20 [62] Unknown
to be considered appropriate for use in MR studies. The RTEL1 20 [105,106] [156]
first assumption is the SNP must only affect the disease ZBTB46 20 [105] Unknown
through its effect on telomere length. To this end, there
should be some understanding of the functional or bio- associate with telomere length. Replication/validation
logical effect of the SNP on the telomere length. In studies in six additional cohorts supported the associ-
other words, the genotype must be strongly associated ation of seven SNPs with mean BTL even after adjust-
with the phenotype it represents and not be associated ment for age, sex, study-specific covariates, genomic
with confounding factors commonly seen in observa- inflation, and other control factors (Table 3) [105]. Five
tional studies. The SNP must be related to the disease of the genetic variants were near genes associated with
risk only through the phenotype it represents [102]. telomere biology: TERC, TERT, OBFC1, NAF1, and RTEL1
Finally, the SNPs should be well-established genotypes [105]. The connection of the two remaining loci,
that have been confirmed across several studies and ZNF208 and ACYP2, to telomere biology remains unclear
laboratories. Several limitations complicate the use of [105]. Several additional screens have confirmed TERT
MR, including linkage disequilibrium, pleiotropy, gen- [106], TERC [62,106–110], OBFC1 [62,106,108,111], NAF1
etic heterogeneity, and population stratification [102]. It [106], and ACYP2 [106] are associated with telomere
may be advantageous to use a set of SNPs instead of length. Many other loci have been associated with telo-
one or a few to assess the genetic relationship to dis- mere length (Table 3); however, most have not been
ease risk. A number of alleles have been identified and replicated across laboratories (Table 4). The most con-
are being used in MR studies to assess the nature of sistent associations between telomere length and dis-
telomere length involvement in age-related disease risk ease risk are found when using a combination of alleles
(Tables 3 and 4). rather than an individual SNP. This corroborates the
Genetic variants associated with telomere length. In requisite use of only those variants that have been
the most extensive GWAS to date, 15 cohorts replicated across laboratories and have a strong associ-
(n ¼ 37,684) were used to identify genetic variants that ation with telomere biology.

Shelterin
RAP1 TRF2 TRF1 TIN2 TPP1
Figure 2. Telomere-associated protein complexes. Shelterin is a six subunit protein complex, which includes TRF1, TRF2, RAP1,
TIN2, TPP1, and POT1, all of which serve to protect the telomere end. CST is a trimeric protein complex, which includes CTC1,
STN1 (OBFC1), and TEN1, that participates in lagging strand replication and has been implicated in telomerase regulation at
the telomere.
Two protein complexes involved in telomere length
protection and regulation at each chromosome end are
shelterin and CST. Shelterin is composed of six proteins
including TRF1, TRF2, TIN2, TPP1, POT1, and RAP1
(Figure 2), which together preserve the telomere struc-
ture and protect the telomere ends in part by prevent-
ing DNA repair mechanisms from identifying them as
double-strand DNA breaks [24,25]. The double-strand
telomere repeat sequence-binding proteins, TRF1 and
TRF2, act as a scaffold for the shelterin complex mem-
bers (Figure 2) [24]. While depletion of TRF1 results in
cellular senescence [25], overexpression of TRF1 [112],
or TRF2 [113] causes a decrease in telomere length.
TIN2 interacts with both TRF1 and TRF2 (Figure 2) and
has been shown to destabilize the shelterin complex
when depleted [114]. It provides the bridge to TPP1,
which binds and recruits POT1, the single-strand telo-
mere binding protein (Figure 2) [25,115]. In addition,
TPP1 interacts with telomerase [24] and is thought to
recruit and regulate it [116,117]. Decreased expression
of POT1 or TPP1 induces telomere lengthening, sug-
gesting that POT1 is involved in telomere length regula-
tion [25]. RAP1 relies on binding to TRF2 for its
telomeric localization [24] and is thought to participate
in repression of rapid telomere deletion and repression
of telomere fusion [118]. Given the intimate association
of the shelterin complex to telomere length, how have
no genetic variants been associated with variation in
telomere length? Two shelterin members, TIN2
[119,120] and TPP1 [121,122], are implicated in clinical
syndromes characterized by extremely short telomere
length, DC and the more severe form,
Hoyeraal–Hreidarsson syndrome (HH), respectively.
Deficiency of several shelterin members in mice includ-
ing Trf1 [123], Trf2 [124], Tin2 [125], Tpp1 [126], and
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 13
TPP1
POT1
CST
STN1/
POT1 TEN1 CTC1
OBFC1
Pot1a [127] result in embryonic lethality. These studies
illustrate the importance of shelterin complex members
in vivo, which may in part explain their absence as use-
ful proxies for genetic telomere length in MR studies.
There are reports of genetic variants in POT1 associat-
ing with cancer, but not telomere length [128,129]. The
fact that so few SNPs associated with shelterin have
been identified as candidate genetic variants for MR
studies suggests that an additional level of regulation
on genetic telomere length outside the known path-
ways may play an important role.
While shelterin has not been associated with genetic
telomere length, two members of CST have. CST, a tri-
meric protein complex comprised of CTC1, STN1, and
TEN1, was identified and is thought to play a role in
telomere length homeostasis [26,27]. STN1, also known
as OBFC1, binds nonspecifically to single-stranded DNA
[130,131]. Genetic variants in OBFC1 (STN1) associated
with telomere length have been identified by multiple
laboratories [62,106,108,111], making them useful for
MR studies. Driven by CTC1, the CST complex localizes
to the single-stranded portion of the telomere (Figure
2) [26,131]. Although TEN1 is necessary for the telomere
capping function of the CST complex, its exact telo-
meric function remains a mystery [131]. Its depletion,
however, results in telomere dysfunction [132].
Decreasing the expression of any of the three CST com-
plex members results in an increase in telomere length
in telomerase-positive cells, suggesting a role together
with telomerase in telomere length homeostasis [27]. It
has been proposed that the TPP1/POT1 complex
recruits and promotes telomerase activity at the telo-
meres (Figure 2) and the CST complex reduces recruit-
ment and subsequent telomerase activity and
promotes lagging strand synthesis to complete
14 C. L. FASCHING

telomere elongation (Figure 2) [27,34,131]. In contrast
to the shelterin members, genetic variants from OBFC1,
and less frequently CTC1, have been associated with
short/long telomere length, substantiating their use in
MR studies. The number of proteins and complexity of
the mechanisms associated with telomere length
homeostasis and telomere protection illustrate a ration-
ale for the broad range of telomere lengths at a given
age and sex. Further research will help resolve the con-
undrum of genetic variants with unknown biological
relationships to telomere length and pathways within
telomere biology that do not seem to significantly asso-
ciate with genetic telomere length.
Non-neoplastic disease associated with genetic telo-
mere lengths. Identification of telomere length-associ-
ated predisposition to disease risk using genetic
methodologies is ever more popular. The studies below
explore this avenue within several non-neoplastic dis-
ease categories (Tables 2 and 5). In a first glance at the
association of genetic telomere length and disease risk,
a common theme appears: the power of numbers.
Using a small population (n ¼ 3118; 1601 controls; 1517
cases) [111], or a general healthy population [64,66],
several studies were unable to find an association
between genetic telomere lengths and CHD [111],
T2DM [111], all-cause mortality [64,66], and CVD [64,66].
Many studies have the means to use cohorts with
greatly increased overall population numbers, some
using populations of general health and others includ-
ing case-control populations (Tables 1 and 6). Studies
using sufficient numbers of cases yield some associ-
ation between disease risk and genetic telomere length
(Table 6). As an example, using the most vetted SNPs
for genetic telomere lengths (i.e. OBFC1, TERC, and
TERT), two of the strongest studies found different
results [66,87]. The first study used the CCHS cohort
(n ¼ 64,367) and found no association of genetic telo-
mere length and all-cause or cardiovascular mortality
[66]. Using the combined CCHS data and the CGPS
data, the second study began by characterizing genetic
telomere length and found a 67 bp decrease in telo-
mere length/allele, which accounted for 0.7% of the
telomere length variation [87]. A modest association
between genetic telomere length and IHD risk was
seen when the SNPs were applied as an allele sum on
the combined Danish cohorts, Copenhagen Ischemic
Heart Disease Study (CIHDS), CGPS, and CCHS
(n ¼ 105,000 total; n ¼ 17,000 IHD), which was improved
by including data from the CARDIoGRAMplusC4D con-
sortium [87]. These differences in association between
genetic telomere length and disease risk, when includ-
ing the CIHDS and CARDIoGRAMplusC4D cohorts, sug-
gest the association may be masked in the general
population and becomes more apparent with inclusion
of a sufficient disease population. This illustrates the
importance of not only increased study power in

Table 5. Association of direct telomere length measurement in cancer types and subtypes.
Observational studies
Cancer subtypes Direct association Inverse association No association
Lung cancer N/A Rode et al. [140] N/A
Squamous cell carcinoma of the lung N/A Sanchez-Espiridion et al. [142] N/A
Adenocarcinoma of the lung Sanchez-Espiridion N/A N/A
et al. [142]
Gastric cancer N/A Willeit et al. [141] N/A
Ovarian cancer N/A Willeit et al. [141] N/A
CLL N/A Roos et al. [146], Rossi et al. [147], Rampazzo et al. [148], N/A
Strefford et al. [149], Grabowski et al. [151]
CML N/A Brummendorf et al. [144], Boultwood et al. [145] N/A
Melanoma N/A Rode et al. [140] N/A
Leukemia N/A Rode et al. [140] N/A
Pancreatic cancer N/A N/A Campa et al. [143]

Table 6. Association of genetic telomere length and age-related diseases.

Disease Genetic telomere length Associated studies
All-cause mortality Longer allele N/A
Shorter allele Deelen et al. [64], Rode et al. [66], Mauberet et al. [111]
No association Said et al. [133]
CVD Longer allele N/A
Shorter allele Deelen et al. [64], Rode et al. [66], Scheller Madrid et al. [87], Codd et al.
[105], Mauberet et al. [111], Said et al. [133], Zhan et al. [134]
Cancer Longer allele Bojesen et al. [40], Walsh et al. [41], Rode et al. [66], Rode et al. [140],
Zhang et al. [152], Ojha et al. [153], Machiela et al. [154], Jones et al.
[155], Haycock et al. [157]
Shorter allele Said et al. [133]

general, but especially of the cases. The value of
increased “case” study power is further illustrated by
Codd et al. [105], who, using ENGAGE SNPs (Table 3),
explored the association of genetic telomere length
with CAD in the CARDIoGRAM cohort (n ¼ 22,233 cases;
n ¼ 64,762 controls) [105]. The genetic risk score of the
set of all seven SNPs showed an association between
genetically shorter telomere length and higher risk of
CAD [105]. These results were similar to a study using
the Danish cohorts combined with the CARDIoGRAM
cohort, which evaluated three of the telomere associ-
ated SNPs, suggesting that OBFC1, TERC, and TERT are
sufficient to determine CHD disease risk. The first study,
using a UK Biobank population (n ¼ 134,773), reported
an association between genetically short telomeres and
increased hypertension, CVD, and cancer, but not dia-
betes or all-cause mortality after adjusting for age, sex,
and genotyping arrays [133]. These data illustrate the
potential of using significantly powered studies, specif-
ically demonstrating the number of cases with hyper-
tension (n ¼ 41.847), CVD (n ¼ 46,979), and cancer
(n ¼ 22,448), compared to T2DM (n ¼ 7,969). Similar to
previous studies [87,105], analyses of the CARDIoGRAM
consortium data and the ENGAGE Telomere consortium
SNPs (Table 3) showed genetically longer telomere
lengths associated with decreased risk of CHD [134].
Zhan et al. [134] also examined the association of gen-
etic telomere lengths to several metabolic markers and
found an association with fasting insulin after replica-
tion in an independent cohort [134]. Increased fasting
insulin was also associated with higher CHD risk, sug-
gesting that it acts as a mediator between genetic telo-
mere length and CHD [134]. Insulin resistance and
T2DM are associated with uncoupling protein 2 (UCP2),
an inner mitochondrial membrane protein [135].
Genetic variants in UCP2 have been found to associate
with T2DM [135] and oxidative stress levels [98]. Several
studies have explored the association of telomere
length to two UCP2 SNPs. For example, in a population
of urban Chinese, Xiao et al. [98] found the UCP2–886A
allele to be a risk factor for T2DM, but neither the A nor
G allele was associated with BTL [98]. In a population of
Caucasians with T2DM, the UCP-866A allele is associated
with shorter BTL than the UCP-866G allele [136]; neither
of the UCP-A55V alleles were associated with BTL [136].
These first two studies show conflicting results with
regard to BTL and UCP2 variants, which could be attrib-
uted to underlying population genomic differences.
Based on the association of UCP2 and oxidative stress,
Zhou et al. [137] explored the association of UCP2 with
telomere length in a control population lacking T2DM.
They found the UCP2–866A allele confers an increase in
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 15
BTL/allele and the UCP2-A55V-CC haplotype is associ-
ated with shorter telomere lengths than the TT and CT
haplotypes [137]. These data highlight the complexity
of using genetic variants as proxies for telomere length
with regard to disease risk and illustrate the rationale
for replication across several studies, populations, and
laboratories. There are a number of messages that can
be taken from these studies. First, association of genetic
telomere length and non-neoplastic disease risk may
not be empowered using all of the SNPs. Second, a suf-
ficiently high number of cases may be required for
identification of an association between genetic telo-
mere length and disease risk. This may be due to subtle
differences in genomic backgrounds of study partici-
pants that could confer compensatory or enhancing
functional effects. Finally, the majority of these studies
used the CARDIoGRAM consortium or
CARDIoGRAMplusC4D consortium for CHD, resulting in
similar outcomes. Additional studies in a variety of pop-
ulations and laboratories will strengthen or challenge
the validity of these associations.
5. Telomere length and disease: is telomere
length a reliable clinical marker for assessing
cancer risk?
The investigations exploring a link between cancer and
telomeres have been long and multifaceted. The pro-
gression of non-neoplastic cells to a neoplastic state
includes activation of a telomere maintenance mechan-
ism [138]. Eighty-five to 90% of cancers activate tel-
omerase; 10%–15% use a recombination-based
mechanism, alternative lengthening of telomeres
[1,138]. While directly measuring telomere length in
tumors may provide useful information, it is unclear
how measurement of BTL strengthens the understand-
ing of cancer or cancer risk (Tables 2 and 5). There may
be systemic changes, such as activation of cancer asso-
ciated with inflammatory pathways that can affect BTL,
but the impact of these changes has not proven to be
as promising as compared to other diseases, rendering
the clinical utility of BTL as a biomarker for cancer risk
unsubstantiated. The following observational studies
show that BTL is directly, inversely, or not associated
with cancer risk, illustrating this challenge (Tables 2
and 5).
BTL measurement for cancer risk analysis. A large
population (n ¼ 47,102) of the CCHS and the CGPS
cohorts was evaluated for years of survival after cancer
and cancer risk [139]. Compared to individuals in the
first quartile, representing the longest telomere lengths,
those in the fourth quartile had reduced survival time
16 C. L. FASCHING

Table 7. Association of genetic telomere length in cancer the accelerated phase more quickly [145]. Shorter telo-
types and subtypes. mere length was also associated with Philadelphia
Mendelian randomization studies chromosome-positive cells [144]. In chronic lymphocytic
Cancer subtypes Longer allele Shorter allele leukemia (CLL), BTL is a predictor of treatment-free sur-
Lung cancer Rode et al. [40] N/A vival and overall survival [146–149]. B cells purified
Adenocarcinoma of the lung Zhang et al. [152], N/A
Haycock et al. [157] from CLL patients had higher genomic rearrangements
Ovarian cancer Bojesen et al. [40], N/A in the population with the shortest telomeres [150].
Haycock et al. [157]
Breast cancer Bojesen et al. [40] N/A Roos et al. [146] found similar genomic aberrations
Chronic Lymphocytic Leukemia Ojha et al. [153], N/A independent of telomere length, but short telomere
Machiela et al. [154]
Melanoma Rode et al. [140], N/A length was associated with an unfavorable clinical out-
Haycock et al. [157] come, thus, could serve as an independent prognostic
Endometrial cancer Haycock et al. [157] N/A
Kidney cancer Haycock et al. [157] N/A
indicator for treatment-free survival [146]. B-CLL
Colorectal carcinoma Jones et al. [155] N/A patients with mutated VH genes were able to be further
Colorectal adenoma Haycock et al. [157] N/A
Bladder cancer Haycock et al. [157] N/A
subcategorized based on telomere lengths with shorter
Testicular germ-cell cancer Haycock et al. [157] N/A telomere length lending a worse prognosis, similar to
Glioma Walsh et al. [4], N/A the unmutated VH genes [151]. There is a high clinical
Haycock et al. [157]
Neuroblastoma Haycock et al. [157] N/A utility for telomere length measurement in hemato-
logical cancers, which in addition to disease phase and
disease survival, in CLL may provide useful independent
after cancer. Stratification of the cohort based on length prognostic information [151].
of survival/early death and for cancer type, including Genetic telomere length association with cancer risk
lung cancer, melanoma, leukemia, and esophageal can- analysis. Similar to CVD, the utilization of genetic telo-
cer, showed an association between telomere length mere length studies using MR is becoming common for
and early death, but no significant association with can- surveying cancer risk (Tables 6 and 7). The ENGAGE
cer risk [139]. These results were corroborated in an Telomere consortium SNPs (Table 3) were originally
additional study using these same cohorts [140]. Willeit characterized with regard to their association to telo-
and colleagues evaluated the Bruneck Study cohort mere length [105]. One study assessing genetic telo-
(n ¼ 787; age range: 40–79 years) and found an associ- mere length and cancer risk or cancer mortality found
ation between short telomere length and gastric, lung lower cancer mortality significantly associated with the
and ovarian cancer prognosis as well as cancer mortal- shortest genetic telomere length from the allele sum of
ity, when the longest telomere lengths were compared TERC, TERT, and OBFC1 [66]. In a subsequent study, gen-
with the middle and the shortest [141]. In a case-control etically increased telomere length was associated with
study, Sanchez-Espiridion and colleagues [142] eval- increased cancer risk individually and as an allele sum
uated histological subtypes of lung cancer and found of TERT, TERC, and OBFC1 alleles [140]. When Rode et al.
adenocarcinomas were associated with longer BTL, [140] evaluated specific cancer types, longer per allele
independent of sex, age, and smoking. In contrast, genetic telomere length was associated with increased
squamous cell carcinomas of the lung were associated risk in melanomas and lung cancer [140]. Using the
with short telomere length [142]. A study on pancreatic GAME-ON consortium data, another MR study eval-
cancer using the European Prospective Investigation uated the association between cancer risk from five
into Cancer and Nutrition (EPIC) cohort, (n ¼ 331 cases; common cancer types (i.e. breast, lung, colorectal, ovar-
n ¼ 331 controls), found no association, indicating that ian, and prostate) and telomere-associated genetic var-
BTL is not a strong predictor of pancreatic cancer risk iants corresponding to the TERC, TERT, NAF1, OBFC1,
[143]. These observational studies indicate that BTL PXK, ZNF208, RTEL1, ZNF676, CTC1, and ACPY2 loci [152].
measurement as a global biomarker for cancer risk Zhang et al. [152] found NAF1, OBFC1, TERT, and ZNF208
remains inconclusive (Table 2) and the clinical utility as associated with adenocarcinoma of the lung. Ojha and
a biomarker for specific cancer subtypes (Table 5) is at colleagues [153] studied the association of genetic telo-
best limited. Hematological cancers seem to be the mere length with risk of a hematopoietic malignancy,
exception. Compared to a normal population, average CLL, using the ENGAGE SNPs (Table 3) plus CTC1. The
BTL in chronic myelogenous leukemia patients was TERC, TERT, and OBFC1 alleles corresponding to longer
shorter [144] and associated with time from diagnosis telomere length were associated with increased CLL
to entering the accelerated and blast phase [144,145]; risk [153]. A study on non-Hodgkin lymphoma found
those with shorter BTL at the time of diagnosis entered that in 3104 patients, genetically longer telomere
 CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 17

length was associated with increased CLL and small
lymphocytic lymphoma risk [154]. SNPs showing the
strongest association were TERC, TERT, and OBFC1 [154].
Five SNPs in the region of TERC were identified after
GWAS analyses of telomere biology associated gene
pathway SNPs by BTL [155]. Jones et al. [155] went on
to assess these SNPs for colorectal carcinoma risk using
a GWAS approach and found one SNP, rs10936599, pre-
viously associated with longer telomeres [156], to be
associated with colorectal carcinoma and adenomas
[155]. This study highlights the association of cancer
risk with telomere maintenance as an alternative cause
for the association with genetic telomere length. A
meta-analysis identified an additional SNP near TERC
that is associated with glioma risk [41]. Previously iden-
tified alleles near TERC and TERT [105], representing lon-
ger BTL, were also associated with increased glioma risk
[41]. On the other hand, RTEL1 SNPs associated with
increased glioma risk showed no significant association
with regard to telomere length [41]. This study indicates
two loci, TERC and TERT may provide helpful informa-
tion in glioma-specific studies. In an extensive analysis
of the TERT locus for SNPs associated with mean telo-
mere length, breast, and ovarian cancer, Bojesen and
colleagues [40] found telomere length-dependent as
well as telomere length-independent SNPs associated
with increased cancer risk across three regions. The
minor alleles of two BTL- and cancer-associated SNPs in
the first two regions were associated with long telo-
mere length. A common theme seems to be emerging,
in which two of the most common SNPs associated
with cancer risk are found at loci, TERC and TERT, which
encode macromolecules essential for telomerase func-
tion [1]. This relationship between telomerase and can-
cer may present a more relevant association with these
SNPs than telomere length in the neoplastic studies.
The Telomeres Mendelian Randomization Collaboration
undertook a large meta-analysis of 22 different cancer
types [157]. Using 16 different SNPs that represented 10
different regions, longer genetic telomere length was
associated with glioma, endometrial cancer, kidney can-
cer, testicular germ-cell cancer, melanoma, bladder can-
cer, neuroblastoma, lung adenocarcinoma, and serous
low malignancy potential ovarian cancer [157].
Collectively, these observational and MR studies under-
score the early nature of BTL associations in the elucida-
tion of cancer risk and the need for more research.
6. Summary and perspectives
The importance of telomere length measurement as a
clinical biomarker is gaining acceptance. There is
increasing evidence from large cohort studies demon-
strating that direct measurement of telomere length is
relevant for all-cause mortality and CVD risk assessment
in individuals under the age of 75 years. Some age-
related metabolic disorders also indicate the potential
clinical usefulness of BTL measurement that will likely
become clearer with additional studies including more
diverse populations. The relationship between

Disease
Disease risk
states
(e.g. CVD)
(e.g. T2DM)

Leukocyte Telomere
Lengths
(Dynamic)

Genetic
Other
Telomere
Genetic
Length
(Set point)
Variants

Environmental
Factors
(e.g. inflammation,
chronic infection)

Figure 3. Contributions to telomere length as a clinical biomarker. Many factors play a role in the dynamic nature of measured
telomere length. Leukocyte telomere length can be affected by disease risk, as well as disease state driven by environmental fac-
tors, such as inflammation and insulin resistance. Genetic telomere length provides an inherited telomere length set point predis-
position to disease risk. Environmental factors, such as chronic viral infection or other causes of inflammation as well as
additional contributing factors such as disease state (e.g. type 2 diabetes mellitus (T2DM), atherosclerosis), can create shortened
telomere length in adulthood that increases disease risk (e.g. cardiovascular disease (CVD)). Genetic variants (e.g. UCP2) associ-
ated with environmental factors, such as oxidative stress, may affect telomere length and disease risk directly.
18 C. L. FASCHING
neoplastic diseases and telomeres, however, is compli-
cated, overshadowing BTLs as a clinical indication of
cancer. Studies evaluating predisposition to disease risk
based on genetic telomere length are also gaining
momentum. Direct and genetic telomere length meas-
urement approaches each provide interrelated comple-
mentary data. One provides a glimpse into an
individual’s genetic disease risk and the other a glimpse
into the effects of current health and lifestyle choices.
Knowledge from each provides valuable information for
downstream health and lifestyle decisions.
A number of different methodologies are currently
being explored to measure telomere lengths in a clin-
ical setting, as well as being used in the basic and clin-
ical research environment. A study comparing
methodologies resulted in large coefficients of variation
that would be unacceptable in a clinical laboratory
[158]. Challenges for implementation of direct telomere
length measurement in a clinical setting include devel-
oping standard methods, reagents, and protocols for
proficiency testing across methodologies, as well as
standardizing the tissue analyzed across laboratories.
Studies have used whole blood, isolated peripheral
blood mononuclear cells (PBMCs), fractionated subsets
of WBCs or saliva as the tissue for telomere length ana-
lysis, each of which may provide subtle, but measurable
differences. To minimize laboratory-induced variation in
telomere length, storage and handling of the samples,
both prior to and after DNA extraction, should also be
standardized. Regardless of these challenges, many
studies report acceptable variation and present their
data in percentile format, which allows the data to
be reconciled.
Genomic analysis to determine genetic telomere
length is not subject to the same challenges that direct
telomere length measurement incurs. Interpretation of
genetic telomere length is, however, not without a few
challenges of its own. The number of individuals for
which genetic telomere length confers increased dis-
ease risk is representative of only a small percentage of
the population. Caution in choosing genetic variants
that have demonstrably been associated with telomere
length seems paramount to the success of this strategy.
A larger challenge toward implementation of genetic
telomere length analysis as a personalized screen in the
clinic is to increase the diversity in the genomic back-
grounds necessary to apply these data broadly across
populations. Currently, the majority of studies have
been performed using northern European populations.
Understanding common risk alleles across genomic
backgrounds seems key to clinical implementation of
this methodology, given the potential different genetic
clades in an individual.
The collection of studies presented in this review
provides an interwoven picture of BTL measurement,
disease risk, and genetic predisposition (Figure 3). BTLs
are dynamic, reflecting the body’s current physiological
state. As new WBCs emerge into circulation, their telo-
meres are subjected to environmental conditions such
as chronic viral infection, to which lifestyle and disease
states, such as inflammation and T2DM, may contribute
(Figure 3). These environmental conditions and molecu-
lar disruptions can exacerbate disease risk, which are
reflected in the BTLs (Figure 3). Observational studies
monitoring dynamic telomere length provide strong
evidence for telomere length measurement as a viable
clinical biomarker in non-neoplastic diseases, especially
for the middle-aged. Genetic telomere length analysis
contributes an additional level of understanding to per-
sonalized disease risk predisposition (Figure 3).
Acknowledgements
I would like to thank Dr. Lyndal Hesterberg, Dr. Kirk Ehmsen,
Dr. Janet Warrington, Dr. Sara Cole, and Dr. Jessica Lao for
critical review and comments on this manuscript.
Disclosure statement
This author alone is responsible for the content and writing
of this article. The author is an employee and has received
stock options from Telomere Diagnostics, Inc.
References
[1] Reddel RR. Telomere maintenance mechanisms in
cancer: clinical implications. CPD. 2014;20:6361–6374.
[2] Allsopp RC, Vaziri H, Patterson C, et al. Telomere
length predicts replicative capacity of human fibro-
blasts. Proc Natl Acad Sci USA. 1992;89:10114–10118.
[3] Verdun RE, Crabbe L, Haggblom C, et al. Functional
human telomeres are recognized as DNA damage in
G2 of the cell cycle. Mol Cell. 2005;20:551–561.
[4] Ramirez R, Carracedo J, Jimenez R, et al. Massive
telomere loss is an early event of DNA damage-
induced apoptosis. J Biol Chem. 2003;278:836–842.
[5] Andrews NP, Fujii H, Goronzy JJ, et al. Telomeres and
immunological diseases of aging. Gerontology.
2010;56:390–403.
[6] Costenbader KH, Prescott J, Zee RY, et al.
Immunosenescence and rheumatoid arthritis: does
telomere shortening predict impending disease?
Autoimmun Rev. 2011;10:569–573.
[7] Kordinas V, Ioannidis A, Chatzipanagiotou S. The
telomere/telomerase system in chronic inflammatory
diseases. Cause or effect? Genes (Basel). 2016;7:60.
[8] Broer L, Codd V, Nyholt DR, et al. Meta-analysis of
telomere length in 19,713 subjects reveals high

heritability, stronger maternal inheritance and a
paternal age effect. Eur J Hum Genet.
2013;21:1163–1168.
[9] Savage SA, Alter BP. Dyskeratosis congenita. Hematol
Oncol Clin North Am. 2009;23:215–231.
[10] Alter BP, Baerlocher GM, Savage SA, et al. Very short
telomere length by flow fluorescence in situ hybrid-
ization identifies patients with dyskeratosis conge-
nita. Blood. 2007;110:1439–1447.
[11] Armanios M, Blackburn EH. The telomere syndromes.
Nat Rev Genet. 2012;13:693–704.
[12] Nordfjall K, Larefalk A, Lindgren P, et al. Telomere
length and heredity: indications of paternal inherit-
ance. Proc Natl Acad Sci USA. 2005;102:16374–16378.
[13] Nordfjall K, Svenson U, Norrback KF, et al. Large-scale
parent-child comparison confirms a strong paternal
influence on telomere length. Eur J Hum Genet.
2010;18:385–389.
[14] Njajou OT, Cawthon RM, Damcott CM, et al.
Telomere length is paternally inherited and is associ-
ated with parental lifespan. Proc Natl Acad Sci USA.
2007;104:12135–12139.
[15] Arai Y, Martin-Ruiz CM, Takayama M, et al.
Inflammation, but not telomere length, predicts suc-
cessful ageing at extreme old age: a longitudinal
study of semi-supercentenarians. EBioMedicine.
2015;2:1549–1558.
[16] Chiang YJ, Calado RT, Hathcock KS, et al. Telomere
length is inherited with resetting of the telomere
set-point. Proc Natl Acad Sci USA. 2010;107:
10148–10153.
[17] De Meyer T, Rietzschel ER, De Buyzere ML, et al.
Paternal age at birth is an important determinant of
offspring telomere length. Hum Mol Genet.
2007;16:3097–3102.
[18] Kimura M, Cherkas LF, Kato BS, et al. Offspring’s
leukocyte telomere length, paternal age, and telo-
mere elongation in sperm. PLoS Genet. 2008;4:e37
[19] Hjelmborg JB, Dalgard C, Mangino M, et al. Paternal
age and telomere length in twins: the germ stem
cell selection paradigm. Aging Cell. 2015;14:701–703.
[20] Factor-Litvak P, Susser E, Kezios K, et al. Leukocyte
telomere length in newborns: implications for the
role of telomeres in human disease. Pediatrics.
2016;137:e20153927.
[21] Lin J, Epel E, Blackburn E. Telomeres and lifestyle
factors: roles in cellular aging. Mutat Res. 2012;
730:85–89.
[22] Entringer S, Epel ES, Kumsta R, et al. Stress exposure
in intrauterine life is associated with shorter telomere
length in young adulthood. Proc Natl Acad Sci USA.
2011;108:E513–E518.
[23] Pickett HA, Reddel RR. The role of telomere trimming
in normal telomere length dynamics. Cell Cycle.
2012;11:1309–1315.
[24] Palm W, de Lange T. How shelterin protects mamma-
lian telomeres. Annu Rev Genet. 2008;42:301–334.
[25] Chan SS, Chang S. Defending the end zone: studying
the players involved in protecting chromosome
ends. FEBS Lett. 2010;584:3773–3778.
[26] Miyake Y, Nakamura M, Nabetani A, et al. RPA-like
mammalian Ctc1-Stn1-Ten1 complex binds to single-
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 19
stranded DNA and protects telomeres independently
of the Pot1 pathway. Mol Cell. 2009;36:193–206.
[27] Chen LY, Redon S, Lingner J. The human CST com-
plex is a terminator of telomerase activity. Nature.
2012;488:540–544.
[28] Halliwell B, Aruoma OI. DNA damage by oxygen-
derived species. Its mechanism and measurement in
mammalian systems. FEBS Lett. 1991;281:9–19.
[29] Cote HC, Soudeyns H, Thorne A, et al. Leukocyte
telomere length in HIV-infected and HIV-exposed
uninfected children: shorter telomeres with uncon-
trolled HIV viremia. PLoS One. 2012;7:e39266.
[30] Zanet DL, Thorne A, Singer J, et al. Association
between short leukocyte telomere length and HIV
infection in a cohort study: no evidence of a relation-
ship with antiretroviral therapy. Clin Infect Dis.
2014;58:1322–1332.
[31] van de Berg PJ, Griffiths SJ, Yong SL, et al.
Cytomegalovirus infection reduces telomere length
of the circulating T cell pool. J Immunol. 2010;184:
3417–3423.
[32] Greider CW, Blackburn EH. A telomeric sequence in
the RNA of Tetrahymena telomerase required for
telomere repeat synthesis. Nature. 1989;337:331–337.
[33] Schmidt JC, Cech TR. Human telomerase: biogenesis,
trafficking, recruitment, and activation. Genes Dev.
2015;29:1095–1105.
[34] Blackburn EH, Epel ES, Lin J. Human telomere biol-
ogy: a contributory and interactive factor in aging,
disease risks, and protection. Science. 2015;350:
1193–1198.
[35] Vannier JB, Pavicic-Kaltenbrunner V, Petalcorin MI,
et al. RTEL1 dismantles T loops and counteracts telo-
meric G4-DNA to maintain telomere integrity. Cell.
2012;149:795–806.
[36] Pickett HA, Cesare AJ, Johnston RL, et al. Control of
telomere length by a trimming mechanism that
involves generation of t-circles. Embo J.
2009;28:799–809.
[37] Pickett HA, Henson JD, Au AY, et al. Normal mamma-
lian cells negatively regulate telomere length by
telomere trimming. Hum Mol Genet. 2011;20:
4684–4692.
[38] Rivera T, Haggblom C, Cosconati S, et al. A balance
between elongation and trimming regulates telo-
mere stability in stem cells. Nat Struct Mol Biol.
2017;24:30–39.
[39] Gadalla SM, Wang T, Haagenson M, et al. Association
between donor leukocyte telomere length and sur-
vival after unrelated allogeneic hematopoietic cell
transplantation for severe aplastic anemia. JAMA.
2015;313:594–602.
[40] Bojesen SE, Pooley KA, Johnatty SE, et al. Multiple
independent variants at the TERT locus are associ-
ated with telomere length and risks of breast and
ovarian cancer. Nat Genet. 2013;45:371–384.
384e371-372.
[41] Walsh KM, Codd V, Smirnov IV, et al. Variants near
TERT and TERC influencing telomere length are asso-
ciated with high-grade glioma risk. Nat Genet.
2014;46:731–735.
20 C. L. FASCHING
[42] Marnett LJ. Oxyradicals and DNA damage.
Carcinogenesis. 2000;21:361–370.
[43] Petersen S, Saretzki G, von Zglinicki T. Preferential
accumulation of single-stranded regions in telomeres
of human fibroblasts. Exp Cell Res. 1998;239:
152–160.
[44] Oikawa S, Kawanishi S. Site-specific DNA damage at
GGG sequence by oxidative stress may accelerate
telomere shortening. FEBS Lett. 1999;453:365–368.
[45] Opresko PL, Fan J, Danzy S, et al. 3rd, Bohr VA.
Oxidative damage in telomeric DNA disrupts recogni-
tion by TRF1 and TRF2. Nucleic Acids Res.
2005;33:1230–1239.,
[46] Madjid M, Fatemi O. Components of the complete
blood count as risk predictors for coronary heart dis-
ease: in-depth review and update. Tex Heart Inst J.
2013;40:17–29.
[47] Mittal M, Siddiqui MR, Tran K, et al. Reactive oxygen
species in inflammation and tissue injury. Antioxid
Redox Signal. 2014;20:1126–1167.
[48] Demissie S, Levy D, Benjamin EJ, et al. Insulin resist-
ance, oxidative stress, hypertension, and leukocyte
telomere length in men from the Framingham Heart
Study. Aging Cell. 2006;5:325–330.
[49] Doulatov S, Notta F, Laurenti E, et al. Hematopoiesis:
a human perspective. Cell Stem Cell. 2012;10:
120–136.
[50] Rufer N, Brummendorf TH, Kolvraa S, et al. Telomere
fluorescence measurements in granulocytes and T
lymphocyte subsets point to a high turnover of hem-
atopoietic stem cells and memory T cells in early
childhood. J Exp Med. 1999;190:157–167.
[51] Cawthon RM. Telomere measurement by quantitative
PCR. Nucleic Acids Res. 2002;30:e47
[52] Cawthon RM. Telomere length measurement by a
novel monochrome multiplex quantitative PCR
method. Nucleic Acids Res. 2009;37:e21
[53] Canela A, Vera E, Klatt P, et al. High-throughput telo-
mere length quantification by FISH and its applica-
tion to human population studies. Proc Natl Acad Sci
USA. 2007;104:5300–5305.
[54] Poon SS, Martens UM, Ward RK, et al. Telomere
length measurements using digital fluorescence
microscopy. Cytometry. 1999;36:267–278.
[55] Kimura M, Stone RC, Hunt SC, et al. Measurement of
telomere length by the Southern blot analysis of ter-
minal restriction fragment lengths. Nat Protoc.
2010;5:1596–1607.
[56] Baird DM, Rowson J, Wynford-Thomas D, et al.
Extensive allelic variation and ultrashort telomeres in
senescent human cells. Nat Genet. 2003;33:203–207.
[57] Aubert G, Hills M, Lansdorp PM. Telomere length
measurement-caveats and a critical assessment of
the available technologies and tools. Mutat Res.
2012;730:59–67.
[58] Lai TP, Wright WE, Shay JW. Comparison of telomere
length measurement methods. Phil Trans R Soc B.
2018;373:20160451.
[59] Sanders JL, Newman AB. Telomere length in epi-
demiology: a biomarker of aging, age-related dis-
ease, both, or neither? Epidemiol Rev. 2013;35:
112–131.
[60] Bojesen SE. Telomeres and human health. J Intern
Med. 2013;274:399–413.
[61] Martin-Ruiz CM, Baird D, Roger L, et al.
Reproducibility of Telomere Length Assessment–An
International Collaborative Study. Int J Epidemiol.
2015;44:1749–1754.
[62] Mangino M, Hwang SJ, Spector TD, et al. Genome-
wide meta-analysis points to CTC1 and ZNF676 as
genes regulating telomere homeostasis in humans.
Hum Mol Genet. 2012;21:5385–5394.
[63] Howden LMaJAM. Age and Sex Composition: 2010.
In: Bureau USC, ed. Washington, D.C.: U. S. Census
Bureau; 2011.
[64] Deelen J, Beekman M, Codd V, et al. Leukocyte telo-
mere length associates with prospective mortality
independent of immune-related parameters and
known genetic markers. Int J Epidemiol. 2014;43:
878–886.
[65] Lapham K, Kvale MN, Lin J, et al. Automated assay of
telomere length measurement and informatics for
100,000 subjects in the genetic epidemiology
research on adult health and aging (GERA) cohort.
Genetics. 2015;200:1061–1072.
[66] Rode L, Nordestgaard BG, Bojesen SE. Peripheral
blood leukocyte telomere length and mortality
among 64,637 individuals from the general popula-
tion. J Natl Cancer Inst. 2015;107:djv074
[67] Weischer M, Bojesen SE, Nordestgaard BG. Telomere
shortening unrelated to smoking, body weight, phys-
ical activity, and alcohol intake: 4,576 general popu-
lation individuals with repeat measurements 10
years apart. PLoS Genet. 2014;10:e1004191.
[68] Mons U, Muezzinler A, Schottker B, et al. Leukocyte
telomere length and all-cause, cardiovascular disease,
and cancer mortality: results from individual-partici-
pant-data meta-analysis of 2 large prospective cohort
studies. Am J Epidemiol. 2017;185:1317–1326.
[69] Needham BL, Rehkopf D, Adler N, et al. Leukocyte
telomere length and mortality in the National Health
and Nutrition Examination Survey, 1999-2002.
Epidemiology. 2015;26:528–535.
[70] Goglin SE, Farzaneh-Far R, Epel ES, et al. Change in
leukocyte telomere length predicts mortality in
patients with stable coronary heart disease from the
Heart and Soul Study. PLoS One. 2016;11:e0168868.
[71] Farzaneh-Far R, Cawthon RM, Na B, et al. Prognostic
value of leukocyte telomere length in patients with
stable coronary artery disease: data from the Heart
and Soul Study. Arterioscler Thromb Vasc Biol.
2008;28:1379–1384.
[72] Houben JM, Giltay EJ, Rius-Ottenheim N, et al.
Telomere length and mortality in elderly men: the
Zutphen Elderly Study. J Gerontol A Biol Sci Med Sci.
2011;66:38–44.
[73] Svensson J, Karlsson MK, Ljunggren O, et al.
Leukocyte telomere length is not associated with
mortality in older men. Exp Gerontol. 2014;57:6–12.
[74] Marioni RE, Harris SE, Shah S, et al. The epigenetic
clock and telomere length are independently associ-
ated with chronological age and mortality. Int J
Epidemiol. 2016;45:424–432.

[75] Benjamin EJ, Blaha MJ, Chiuve SE, et al. Heart
Disease and Stroke Statistics-2017 Update: A Report
From the American Heart Association. Circulation.
2017;135:e146–e603.
[76] Bezzina CR, Lahrouchi N, Priori SG. Genetics of sud-
den cardiac death. Circ Res. 2015;116:1919–1936.
[77] Yang KC, Kyle JW, Makielski JC, et al. Mechanisms of
sudden cardiac death: oxidants and metabolism. Circ
Res. 2015;116:1937–1955.
[78] Wellens HJ, Schwartz PJ, Lindemans FW, et al. Risk
stratification for sudden cardiac death: current status
and challenges for the future. Eur Heart J.
2014;35:1642–1651.
[79] Hayashi M, Shimizu W, Albert CM. The spectrum of
epidemiology underlying sudden cardiac death. Circ
Res. 2015;116:1887–1906.
[80] Wilson PW, D’Agostino RB, Levy D, et al. Prediction
of coronary heart disease using risk factor categories.
Circulation. 1998;97:1837–1847.
[81] Brouilette S, Singh RK, Thompson JR, et al. White cell
telomere length and risk of premature myocardial
infarction. Arterioscler Thromb Vasc Biol. 2003;23:
842–846.
[82] Brouilette SW, Moore JS, McMahon AD, et al.
Telomere length, risk of coronary heart disease, and
statin treatment in the West of Scotland Primary
Prevention Study: a nested case-control study.
Lancet. 2007;369:107–114.
[83] Stone NJ, Robinson JG, Lichtenstein AH, et al. ACC/
AHA guideline on the treatment of blood cholesterol
to reduce atherosclerotic cardiovascular risk in
adults: a report of the American College of
Cardiology/American Heart Association Task Force on
Practice Guidelines. Circulation. 2013;2014:S1–S45.
[84] Hammadah M, Al Mheid I, Wilmot K, et al. Telomere
shortening, regenerative capacity, and cardiovascular
outcomes. Circ Res. 2017;120:1130–1138.
[85] Fitzpatrick AL, Kronmal RA, Gardner JP, et al.
Leukocyte telomere length and cardiovascular dis-
ease in the cardiovascular health study. Am J
Epidemiol. 2007;165:14–21.
[86] D’Mello MJJ, Ross SA, Anand SS, et al. Telomere
Length and Risk of Myocardial Infarction in a
MultiEthnic Population: The INTERHEART Study. J Am
Coll Cardiol. 2016;67:1863–1865.
[87] Scheller Madrid A, Rode L, Nordestgaard BG, et al.
Short Telomere Length and Ischemic Heart Disease:
Observational and Genetic Studies in 290 022
Individuals. Clin Chem. 2016;62:1140–1149.
[88] Willeit P, Willeit J, Mayr A, et al. Telomere length and
risk of incident cancer and cancer mortality. JAMA.
2010;304:69–75.
[89] Peters SA, den Ruijter HM, Bots ML, et al.
Improvements in risk stratification for the occurrence
of cardiovascular disease by imaging subclinical ath-
erosclerosis: a systematic review. Heart. 2012;98:
177–184.
[90] Raschenberger J, Kollerits B, Hammerer-Lercher A,
et al. The association of relative telomere length with
symptomatic peripheral arterial disease: results from
the CAVASIC study. Atherosclerosis. 2013;229:469–474.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 21
[91] Mainous AG, 3rd, Codd V, Diaz VA, et al. Leukocyte
telomere length and coronary artery calcification.
Atherosclerosis. 2010;210:262–267.
[92] Hunt SC, Kimura M, Hopkins PN, et al. Leukocyte
telomere length and coronary artery calcium. Am J
Cardiol. 2015;116:214–218.
[93] Rask-Madsen C, Kahn CR. Tissue-specific insulin
signaling, metabolic syndrome, and cardiovascular
disease. Arterioscler Thromb Vasc Biol. 2012;32:
2052–2059.
[94] Goldsworthy ME, Potter PK. Modelling age-related
metabolic disorders in the mouse. Mamm Genome.
2014;25:487–496.
[95] Abel ED, O’Shea KM, Ramasamy R. Insulin resistance:
metabolic mechanisms and consequences in the
heart. Arterioscler Thromb Vasc Biol. 2012;32:
2068–2076.
[96] Willeit P, Raschenberger J, Heydon EE, et al.
Leucocyte telomere length and risk of type 2 dia-
betes mellitus: new prospective cohort study and lit-
erature-based meta-analysis. PLoS One. 2014;9:
e112483.
[97] Zhao J, Zhu Y, Lin J, et al. Short leukocyte telomere
length predicts risk of diabetes in American Indians:
the strong heart family study. Diabetes. 2014;63:
354–362.
[98] Xiao F, Zheng X, Cui M, et al. Telomere dys-
function-related serological markers are associated
with type 2 diabetes. Diabetes Care. 2011;34:
2273–2278.
[99] Menke A, Casagrande S, Cowie CC. Leukocyte telo-
mere length and diabetes status, duration, and
control: the 1999-2002 National Health and
Nutrition Examination Survey. BMC Endocr Disord.
2015;15:52.
[100] Bonfigli AR, Spazzafumo L, Prattichizzo F, et al.
Leukocyte telomere length and mortality risk in
patients with type 2 diabetes. Oncotarget.
2016;7:50835–50844.
[101] Astrup AS, Tarnow L, Jorsal A, et al. Telomere length
predicts all-cause mortality in patients with type 1
diabetes. Diabetologia. 2010;53:45–48.
[102] Lawlor DA, Harbord RM, Sterne JA, et al. Mendelian
randomization: using genes as instruments for mak-
ing causal inferences in epidemiology. Statist Med.
2008;27:1133–1163.
[103] Thomas DC, Conti DV. Commentary: the concept of
’Mendelian Randomization’. Int J Epidemiol. 2004;33:
21–25.
[104] Didelez V, Sheehan N. Mendelian randomization as
an instrumental variable approach to causal infer-
ence. Stat Methods Med Res. 2007;16:309–330.
[105] Codd V, Nelson CP, Albrecht E, et al. Identification of
seven loci affecting mean telomere length and their
association with disease. Nat Genet. 2013;45:
422–427.
[106] Pooley KA, Bojesen SE, Weischer M, et al. A genome-
wide association scan (GWAS) for mean telomere
length within the COGS project: identified loci show
little association with hormone-related cancer risk.
Hum Mol Genet. 2013;22:5056–5064.
22 C. L. FASCHING
[107] Codd V, Mangino M, van der Harst P, et al. Common
variants near TERC are associated with mean telo-
mere length. Nat Genet. 2010;42:197–199.
[108] Levy D, Neuhausen SL, Hunt SC, et al. Genome-wide
association identifies OBFC1 as a locus involved in
human leukocyte telomere biology. Proc Natl Acad
Sci USA. 2010;107:9293–9298.
[109] Prescott J, Kraft P, Chasman DI, et al. Genome-wide
association study of relative telomere length. PLoS
One. 2011;6:e19635
[110] Lee JH, Cheng R, Honig LS, et al. Genome wide asso-
ciation and linkage analyses identified three loci-
4q25, 17q23.2, and 10q11.21-associated with vari-
ation in leukocyte telomere length: the Long Life
Family Study. Front Genet. 2014;4:310.
[111] Maubaret CG, Salpea KD, Romanoski CE, et al.
Association of TERC and OBFC1 haplotypes with
mean leukocyte telomere length and risk for coron-
ary heart disease. PLoS One. 2013;8:e83122
[112] van Steensel B, de Lange T. Control of telomere
length by the human telomeric protein TRF1. Nature.
1997;385:740–743.
[113] Smogorzewska A, van Steensel B, Bianchi A, et al.
Control of human telomere length by TRF1 and
TRF2. Mol Cell Biol. 2000;20:1659–1668.
[114] Ye JZ, Donigian JR, van Overbeek M, et al. TIN2
binds TRF1 and TRF2 simultaneously and stabilizes
the TRF2 complex on telomeres. J Biol Chem.
2004;279:47264–47271.
[115] Takai KK, Kibe T, Donigian JR, et al. Telomere protec-
tion by TPP1/POT1 requires tethering to TIN2. Mol
Cell. 2011;44:647–659.
[116] Nandakumar J, Cech TR. Finding the end: recruit-
ment of telomerase to telomeres. Nat Rev Mol Cell
Biol. 2013;14:69–82.
[117] Sexton AN, Regalado SG, Lai CS, et al. Genetic and
molecular identification of three human TPP1 func-
tions in telomerase action: recruitment, activation,
and homeostasis set point regulation. Genes Dev.
2014;28:1885–1899.
[118] Rai R, Chen Y, Lei M, et al. TRF2-RAP1 is required to
protect telomeres from engaging in homologous
recombination-mediated deletions and fusions. Nat
Comms. 2016;7:10881.
[119] Savage SA, Giri N, Baerlocher GM, et al. TINF2, a
component of the shelterin telomere protection
complex, is mutated in dyskeratosis congenita. Am J
Hum Genet. 2008;82:501–509.
[120] Walne AJ, Vulliamy T, Beswick R, et al. TINF2 muta-
tions result in very short telomeres: analysis of a
large cohort of patients with dyskeratosis congenita
and related bone marrow failure syndromes. Blood.
2008;112:3594–3600.
[121] Kocak H, Ballew BJ, Bisht K, et al. Hoyeraal-
Hreidarsson syndrome caused by a germline muta-
tion in the TEL patch of the telomere protein TPP1.
Genes Dev. 2014;28:2090–2102.
[122] Guo Y, Kartawinata M, Li J, et al. Inherited bone mar-
row failure associated with germline mutation of
ACD, the gene encoding telomere protein TPP1.
Blood. 2014;124:2767–2774.
[123] Karlseder J, Kachatrian L, Takai H, et al. Targeted
deletion reveals an essential function for the telo-
mere length regulator Trf1. Mol Cell Biol.
2003;23:6533–6541.
[124] Celli GB, de Lange T. DNA processing is not required
for ATM-mediated telomere damage response after
TRF2 deletion. Nat Cell Biol. 2005;7:712–718.
[125] Chiang YJ, Kim SH, Tessarollo L, et al. Telomere-asso-
ciated protein TIN2 is essential for early embryonic
development through a telomerase-independent
pathway. Mol Cell Biol. 2004;24:6631–6634.
[126] Kibe T, Osawa GA, Keegan CE, et al. Telomere protec-
tion by TPP1 is mediated by POT1a and POT1b. Mol
Cell Biol. 2010;30:1059–1066.
[127] Hockemeyer D, Daniels JP, Takai H, et al. Recent
expansion of the telomeric complex in rodents: two
distinct POT1 proteins protect mouse telomeres. Cell.
2006;126:63–77.
[128] Robles-Espinoza CD, Harland M, Ramsay AJ, et al.
POT1 loss-of-function variants predispose to familial
melanoma. Nat Genet. 2014;46:478–481.
[129] Speedy HE, Kinnersley B, Chubb D, et al. Germ line
mutations in shelterin complex genes are associated
with familial chronic lymphocytic leukemia. Blood.
2016;128:2319–2326.
[130] Wan M, Qin J, Songyang Z, et al. OB fold-containing
protein 1 (OBFC1), a human homolog of yeast Stn1,
associates with TPP1 and is implicated in telomere
length regulation. J Biol Chem. 2009;284:
26725–26731.
[131] Rice C, Skordalakes E. Structure and function of the
telomeric CST complex. Comput Struct Biotechnol J.
2016;14:161–167.
[132] Kasbek C, Wang F, Price CM. Human TEN1 maintains
telomere integrity and functions in genome-wide
replication restart. J Biol Chem. 2013;288:
30139–30150.
[133] Said MA, Eppinga RN, Hagemeijer Y, et al. Telomere
length and risk of cardiovascular disease and cancer.
J Am Coll Cardiol. 2017;70:506–507.
[134] Zhan Y, Karlsson IK, Karlsson R, et al. Exploring the
Causal Pathway From Telomere Length to Coronary
Heart Disease: A Network Mendelian Randomization
Study. Circ Res. 2017;121:214–219.
[135] Wang H, Chu WS, Lu T, et al. Uncoupling protein-2
polymorphisms in type 2 diabetes, obesity, and insu-
lin secretion. Am J Physiol Endocrinol Metab.
2004;286:E1–E7.
[136] Salpea KD, Talmud PJ, Cooper JA, et al. Association
of telomere length with type 2 diabetes, oxidative
stress and UCP2 gene variation. Atherosclerosis.
2010;209:42–50.
[137] Zhou Y, Simmons D, Hambly BD, et al. Interactions
between UCP2 SNPs and telomere length exist in
the absence of diabetes or pre-diabetes. Sci Rep.
2016;6:33147
[138] Reddel RR. The role of senescence and immortaliza-
tion in carcinogenesis. Carcinogenesis. 2000;21:
477–484.
[139] Weischer M, Nordestgaard BG, Cawthon RM, et al.
Short telomere length, cancer survival, and cancer

risk in 47102 individuals. J Natl Cancer Inst.
2013;105:459–468.
[140] Rode L, Nordestgaard BG, Bojesen SE. Long telo-
meres and cancer risk among 95 568 individuals
from the general population. Int J Epidemiol.
2016;45:1634–1643.
[141] Willeit P, Willeit J, Kloss-Brandstatter A, et al. Fifteen-
year follow-up of association between telomere
length and incident cancer and cancer mortality.
JAMA. 2011;306:42–44.
[142] Sanchez-Espiridion B, Chen M, Chang JY, et al.
Telomere length in peripheral blood leukocytes and
lung cancer risk: a large case-control study in
Caucasians. Cancer Res. 2014;74:2476–2486.
[143] Campa D, Mergarten B, De Vivo I, et al. Leukocyte
telomere length in relation to pancreatic cancer risk:
a prospective study. Cancer Epidemiol Biomarkers
Prev. 2014;23:2447–2454.
[144] Brummendorf TH, Holyoake TL, Rufer N, et al.
Prognostic implications of differences in telomere
length between normal and malignant cells from
patients with chronic myeloid leukemia measured by
flow cytometry. Blood. 2000;95:1883–1890.
[145] Boultwood J, Peniket A, Watkins F, et al. Telomere
length shortening in chronic myelogenous leukemia
is associated with reduced time to accelerated
phase. Blood. 2000;96:358–361.
[146] Roos G, Krober A, Grabowski P, et al. Short telomeres
are associated with genetic complexity, high-risk
genomic aberrations, and short survival in chronic
lymphocytic leukemia. Blood. 2008;111:2246–2252.
[147] Rossi D, Lobetti Bodoni C, Genuardi E, et al.
Telomere length is an independent predictor of sur-
vival, treatment requirement and Richter’s syndrome
transformation in chronic lymphocytic leukemia.
Leukemia. 2009;23:1062–1072.
[148] Rampazzo E, Bonaldi L, Trentin L, et al. Telomere
length and telomerase levels delineate subgroups of
B-cell chronic lymphocytic leukemia with different
biological characteristics and clinical outcomes.
Haematologica. 2012;97:56–63.
[149] Strefford JC, Kadalayil L, Forster J, et al. Telomere
length predicts progression and overall survival in
chronic lymphocytic leukemia: data from the UK LRF
CLL4 trial. Leukemia. 2015;29:2411–2414.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 23
[150] Lin TT, Letsolo BT, Jones RE, et al. Telomere dysfunc-
tion and fusion during the progression of chronic
lymphocytic leukemia: evidence for a telomere crisis.
Blood. 2010;116:1899–1907.
[151] Grabowski P, Hultdin M, Karlsson K, et al. Telomere
length as a prognostic parameter in chronic lympho-
cytic leukemia with special reference to VH gene
mutation status. Blood. 2005;105:4807–4812.
[152] Zhang C, Doherty JA, Burgess S, et al. Genetic deter-
minants of telomere length and risk of common can-
cers: a Mendelian randomization study. Hum Mol
Genet. 2015;24:5356–5366.
[153] Ojha J, Codd V, Nelson CP, et al. Genetic variation
associated with longer telomere length increases risk
of chronic lymphocytic leukemia. Cancer Epidemiol
Biomarkers Prev. 2016;25:1043–1049.
[154] Machiela MJ, Lan Q, Slager SL, et al. Genetically pre-
dicted longer telomere length is associated with
increased risk of B-cell lymphoma subtypes. Hum
Mol Genet. 2016;25:1663–1676.
[155] Jones AM, Beggs AD, Carvajal-Carmona L, et al. TERC
polymorphisms are associated both with susceptibil-
ity to colorectal cancer and with longer telomeres.
Gut. 2012;61:248–254.
[156] Mirabello L, Yu K, Kraft P, et al. The association of
telomere length and genetic variation in telomere
biology genes. Hum Mutat. 2010;31:1050–1058.
[157] Haycock PC, Burgess S, Nounu A, et al. Association
Between Telomere Length and Risk of Cancer and
Non-Neoplastic Diseases: A Mendelian
Randomization Study. JAMA Oncol. 2017;3:636–651.
[158] Martin-Ruiz CM, Baird D, Roger L, et al.
Reproducibility of telomere length assessment: an
international collaborative study. Int J Epidemiol.
2015;44:1673–1683.
[159] Melin BS, Nordfjall K, Andersson U, et al. hTERT can-
cer risk genotypes are associated with telomere
length. Genet Epidemiol. 2012;36:368–372.
[160] Vasa-Nicotera M, Brouilette S, Mangino M, et al.
Mapping of a major locus that determines telomere
length in humans. Am J Hum Genet.
2005;76:147–151.
[161] Mangino M, Brouilette S, Braund P, et al. A regula-
tory SNP of the BICD1 gene contributes to telomere
length variation in humans. Hum Mol Genet.
2008;17:2518–2523.