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Regular Article
HEMATOPOIESIS AND STEM CELLS
A homozygous telomerase T-motif variant resulting in markedly reduced
repeat addition processivity in siblings with Hoyeraal
Hreidarsson syndrome
Maria M. Gramatges,1 Xiaodong Qi,2 Ghadir S. Sasa,1 Julian J.-L. Chen,2 and Alison A. Bertuch1
1
Department of Pediatrics, Baylor College of Medicine, Houston, TX; and 2Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ
Hoyeraal Hreidarsson syndrome (HHS) is a form of dyskeratosis congenita (DC) characterized by bone marrow failure, intrauterine growth retardation, developmental delay,
microcephaly, cerebellar hypoplasia, immunodeficiency, and extremely short telomeres. As
• Biallelic inheritance of a
with DC, mutations in genes encoding factors required for telomere maintenance, such as
telomerase T-motif mutation
telomerase reverse transcriptase (TERT), have been found in patients with HHS. We
selectively impairs repeat
describe 2 sibling HHS cases caused by a homozygous mutation (p.T567M) within the TERT
addition processivity and
T motif. This mutation resulted in a marked reduction in the capacity of telomerase to
results in severe disease.
processively synthesize telomeric repeats, indicating a role for the T motif in this unique
• Computational algorithms
aspect of telomerase function. We support this finding by demonstrating defective
commonly used to predict the
processivity in the previously reported p.K570N T-motif mutation. The consanguineous,
impact of variants on protein
heterozygous p.T567M parents exhibited telomere lengths around the first percentile and
function have limited sensitivity no evidence of a DC phenotype. Although heterozygous processivity defects have been
with regard to hTERT.
associated with familial, adult-onset pulmonary fibrosis, these cases demonstrate the
severe clinical and functional impact of biallelic processivity mutations. Thus, despite
retaining the capacity to add short stretches of telomeric repeats onto the shortest telomeres, sole expression of telomerase processivity
mutants can lead to a profound failure of telomere maintenance and early-onset multisystem disease. (Blood. 2013;121(18):3586-3593)
Key Points
Introduction
Telomeres, composed of repetitive TTAGGG hexamers and associated factors localized to chromosome ends, serve to maintain
chromosome integrity by preventing end-to-end fusions, potentially
deleterious recombination events, and loss of terminal coding regions
during DNA replication.1 Maintenance of telomeric DNA is crucial
to cell survival, as critically short telomeres are recognized as DNA
damage and provoke cellular senescence or apoptosis. In the absence
of this response, such dysfunctional telomeres give rise to genomic
instability and have the potential to promote malignant transformation. The hematopoietic consequences of defective telomere maintenance may manifest as bone marrow failure (BMF), myelodysplastic
syndrome (MDS), or acute myeloid leukemia (AML).
Telomerase, a specialized reverse transcriptase, maintains the
length of the telomeric repeats in select cell populations in which it
is expressed (eg, stem cells and germ cells). The catalytic protein
subunit of telomerase, hTERT, is encoded by telomerase reverse
transcriptase (TERT), and the integral RNA subunit, hTR, is encoded by telomerase RNA component (TERC).2,3 At present, 34
disease-associated sequence variants have been described within
TERT, the majority of which are missense changes.4 Markedly
short germ-line telomeres that result from such variants characterize disorders of telomere biology, the prototype of which is
dyskeratosis congenita (DC). DC includes a 90% lifetime risk for
BMF; predisposition to MDS, AML, and other malignancies; skin
and mucosal abnormalities; pulmonary fibrosis; liver disease; and
additional clinical features.5,6 Cells are very sensitive to a reduction
in levels of telomerase such that even heterozygous mutations in
TERT or TERC may result in short telomeres and a clinical
phenotype via haploinsufficiency.7,8 Variants in DKC1, TINF2,
NHP2, NOP10, WRAP53, CTC1, and RTEL1 have also been
associated with reduced telomere length and a clinical phenotype
consistent with DC.9-11
The telomere biology disorders represent a clinical spectrum.12
Mutations in DKC1, TERT, TERC, and TINF2 have been reported
not only in patients with classic DC but also in individuals with
a single predominant phenotype, such as BMF, MDS, liver disease,
or idiopathic pulmonary fibrosis.13,14 On the severe end of the
spectrum is Hoyeraal Hreidarsson syndrome (HHS), a multisystem
disorder characterized by intrauterine growth retardation and cerebellar hypoplasia in addition to features of DC.15,16 Correlating
Submitted August 17, 2012; accepted March 19, 2013. Prepublished online as
Blood First Edition paper, March 28, 2013; DOI 10.1182/blood-2012-08447755.
The online version of this article contains a data supplement.
M.M.G. and X.Q. contributed equally to this study, and J.J.-L.C. and A.A.B.
contributed equally to this study.
Presented in abstract form at the 54th annual meeting of the American Society
of Hematology, Atlanta, GA, December 8-11, 2012.
3586
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
© 2013 by The American Society of Hematology
BLOOD, 2 MAY 2013 x VOLUME 121, NUMBER 18
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BLOOD, 2 MAY 2013 x VOLUME 121, NUMBER 18
with severe clinical features, patients with HHS are noted to have
the most extremely shortened telomeres among the telomere
biology disorder spectrum of patients. HHS has been associated
with mutations in DKC1, TERT, and TINF2,17-19 as well as a splice
variant of DCLRE1B.20 As with classic DC, a substantial proportion of patients with HHS remain genetically uncharacterized.
In the HHS cases described here, the mutation impacting telomerase function was found to lie within TERT. The hTERT protein
consists of 4 structural domains: the telomerase essential N-terminal
domain, the telomerase RNA binding domain, the catalytic reverse
transcriptase (RT) domain, and the C-terminal extension.21 The RT
domain is responsible for the addition of nucleotides onto the 39 end
of the chromosome by reverse transcribing the telomeric complementary template contained within hTR.22 In addition, telomerases
from many species, including human, have the ability to repetitively
copy the template after a single binding event, resulting in the
synthesis of multiple telomeric repeats prior to telomerase’s release
from the DNA substrate. This property of repeat addition processivity
(referred to hereafter as processivity) requires 3 successive steps: (1)
alignment of the 39 end of the telomeric DNA with the 39 portion of
the template region of hTR, (2) reverse transcription of the RNA
template adding nucleotides to the DNA 39 end until the 59 boundary
of the template is reached, and (3) template translocation, which
involves DNA/RNA strand separation and realignment of DNA to
the 39 portion of template, followed by repositioning of the DNA/
RNA hybrid within the enzymatic active site.21,23 All 4 structural
domains of hTERT contribute to processivity.24-26 Moreover,
processivity can be enhanced by interactions with TPP1-POT1
via the telomerase essential N-terminal and C-terminal extension domains.27,28
The T motif, which lies within the telomerase RNA binding
domain, was the first region of hTERT recognized as being both
highly conserved and telomerase specific,2 as well as the first non-RT
motif shown to be required for telomerase activity.29 The T motif is
encoded by amino acids 547–594, within which lies a near-universally
conserved sequence motif, FYXTE. Targeted mutagenesis of residues
within this motif result in varying degrees of reduced telomerase
activity,29 as well as significantly increased telomere extension rates,
without affecting either hTR binding or measured processivity.30 Only
1 naturally occurring mutant in the T motif has been described to date;
it was found in a multigenerational family manifesting blood diseases
from macrocytosis to AML but without other clinical features of DC.31
Use of the telomerase repeat amplification protocol (TRAP) to
functionally analyze the mutated residue (p.K570N) demonstrated
a drastic reduction in telomerase enzymatic activity, correlating with
severely shortened telomere length.31
Recently, 2 families with an ancestral TERT mutation were
reported as having a predominantly adult-onset pulmonary fibrosis
phenotype over multiple generations, with only 4 of 18 mutation
carriers developing other characteristics of a telomere biology
disorder, such as subclinical cytopenias, liver function abnormalities,
and AML.32 This was striking because a notable feature of both
heterozygous TERT and TERC mutations is disease anticipation,
resulting in an increasingly severe phenotype and earlier onset across
successive generations, correlating with progressive telomere shortening.7,33 In the reported families, however, although telomere length
was consistently below the 10th percentile, with the majority falling
below the first percentile, there was no evidence for evolution to
a more severe DC clinical presentation with this ancestral mutation;
the absence of the classical mucocutaneous features of DC was
notable. Disease phenotypes in these families tracked with a double
heterozygous mutation in the hTERT RT domain, p.V791I/V867M,
REDUCED TELOMERASE PROCESSIVITY RESULTING IN HHS
3587
that, when reconstituted in vitro at physiologic nucleotide concentrations, had little impact on telomerase catalytic activity but
demonstrated a significant decrease in processivity. A second study
revealed that the hTERT RT domain mutation p.R865H, also
associated with familial adult-onset pulmonary fibrosis and telomere
shortening,34 impacted processivity without affecting telomerase
catalytic activity.35 These findings have led to the proposal that a
selective defect in processivity, while limiting the extent of telomere
lengthening, allows for sufficient replenishment of telomeric repeats
on the shortest telomeres, thereby preventing severe, early-onset
telomere-related disease.32
Here, we describe 2 cases of an inherited DC-spectrum disease
resulting from a sequence variant within the hTERT T motif, just C
terminal of the critical FYXTE region. Functional analysis revealed
this variant to be associated with significantly reduced processivity.
Notably, this patient and his similarly affected sister carried a
homozygous mutation and manifested a phenotype consistent with
HHS. Thus, we show that reduced processivity alone may drastically
impair telomere maintenance and result in a severe clinical phenotype.
Materials and methods
Human subjects
The Baylor College of Medicine Institutional Review Board approved this
study. Informed consent was obtained from the participants in accordance
with the Declaration of Helsinki.
Telomerase genotyping
Telomerase genotyping was performed by bidirectional polymerase chain
reaction (PCR)-based double-stranded automated sequencing of the exons
and flanking intronic regions (Ambry Genetics).
Telomere length
Telomere flow fluorescence in situ hybridization (FISH) was carried out as
part of clinical testing as previously described.36 Telomere length analysis
on the proband was performed by the Universitat Bern (Bern, Switzerland)
and on the parents of the proband at Repeat Diagnostics (Vancouver,
British Columbia).
Telomerase in vivo reconstitution
In vivo reconstitution of telomerase was carried out in 293FT cells (Invitrogen)
by cotransfection of pcDNA-hTERT and pBS-U1-hTR vectors. Substitutions
K570N and T567M were introduced into pcDNA-hTERT by site-direct
mutagenesis using overlapping PCR.37 Cell transfection and lysate preparation
were carried out as previously described.23
Northern blotting
Total RNA was isolated from transfected 293FT cells using TRI Reagent
(Molecular Research Center) following the manufacturer’s instructions.
Five micrograms of total RNA was resolved on a 4% polyacrylamide/8M
urea denaturing gel, followed by electroblotting to a Hybond-XL membrane
(GE Healthcare) and UV crosslinking. The membrane was hybridized with
riboprobes targeting 5S rRNA and hTR as described previously.38
Western blotting
Thirty micrograms of protein for each lysate was heated at 95°C for
5 minutes in 13 sodium dodecyl sulfate-polyacrylamide gel electrophoresis
loading buffer, resolved on an 8% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis gel, and electrotransferred onto a polyvinylidene
difluoride membrane. The membrane was blocked in 5% nonfat milk/13
TTBS buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05%
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3588
BLOOD, 2 MAY 2013 x VOLUME 121, NUMBER 18
GRAMATGES et al
Table 1. Naturally occurring TERT variants resulting in a missense change associated with results from 4 algorithms predicting functional
impact, telomerase activity, and presence or absence in a family pedigree
AA
position
Reference
nucleotide
Alternative
nucleotide
Reference
AA
Alternative
AA
PhyloP SIFT
PolyPhen
2
Total
LRT MutationTaster score
Telomerase
activity, %
Present in
‡2 relatives Reference
567
G
A
T
M
C
T
P
N
NDe
0.5
60
Yes
This study
33
G
A
P
S
NC
T
B
N
NDe
0
80
Yes
34
55
A
T
L
Q
C
D
P
N
De
2.5
Yes
13,44
144
C
T
V
M
NC
D
D
N
De
3
75
Yes
34
202
C
T
A
T
NC
T
B
N
NDe
0
,1
Yes
14,45
35–40
31,44,46,47
279
C
T
A
T
NC
T
P
N
NDe
0.5
81–100
No
412
G
A
H
Y
C
T
B
N
De
1
50–91
Yes
14,44
486
G
A
R
C
C
T
D
N
De
2
45
Yes
34
570
C
G
K
N
C
D
B
De
De
3
,1
No
31
682
C
T
G
D
C
T
P
De
De
2.5
,1
No
31,48
694
C
T
V
M
NC
D
D
N
De
3
,1
Yes
704
G
A
P
S
C
T
P
N
De
1.5
13
No
46,49
716
G
A
A
V
C
D
P
De
De
3.5
14
No
46
721
G
C
P
R
C
D
D
De
De
4
100
Yes
31,47
726
G
A
T
M
NC
T
B
N
NDe
0
100
No
31,48
772
T
C
Y
C
C
T
D
N
De
2
,1
Yes
14
811
G
A
R
C
C
D
D
N
NDe
2
,50
Yes
19
846
T
C
Y
C
NC
D
D
De
De
4
10
No
49
865
C
T
R
H
C
D
D
De
De
4
28
Yes
34
876
G
C
H
Q
C
T
P
De
NDe
1.5
10
No
49
901
G
A
R
W
NC
D
B
N
NDe
1
,25
Yes
19
902
C
G
K
N
C
D
P
De
De
3.5
,1
Yes
7,31
979
G
A
R
W
NC
D
P
N
De
2.5
100
Yes
31,45
1015
A
G
C
R
C
T
P
De
De
2.5
9
No
46
1062
C
T
A
T
NC
T
B
N
NDe
0
100
No
44,46
1090
C
T
V
M
NC
D
B
N
NDe
1
,1
No
14
14
B, benign; C, conserved; D, damaging; De, deleterious; N, neutral; NC, nonconserved; NDe, nondeleterious; P, possibly damaging; T, tolerated.
Tween-20), followed by incubation with anti-hTERT goat polyclonal antibody L-20 (Santa Cruz Biotechnology) or anti–glyceraldehyde-3-phosphate
dehydrogenase mouse monoclonal antibody 6C5 (Life Technologies) in 5%
nonfat milk/13 TTBS for 1 hour. After washing 3 times with 13 TTBS
buffer, the membrane was incubated with the HRP-conjugated donkey–
anti-goat antibody (Santa Cruz Biotechnology) or goat–anti-mouse antibody (Bio-Rad) in 5% nonfat milk/13 TTBS for 1 hour at room
temperature. Following 3 washes with 13 TTBS, the membrane was
developed and analyzed as previously described.23
Telomerase direct primer-extension assay
The direct primer-extension assay was performed using 1 mL of transfected
293FT cell lysate at 1.5 mg protein/mL as previously described.23 Telomerase
activity was quantitated and normalized to the loading control and the expression level of hTR, which is limiting in the in vivo reconstitution system.
Telomerase processivity was quantitated as previously described.38
TRAP assay
A 2-tube TRAP assay was performed using 1 mL of diluted cell lysate with
3 ng/mL and 0.6 ng/mL of protein concentrations as previously described, with
minor modifications.39 Cell lysate was mixed with 0.5 mM of TS primer
(59-AATCCGTCGAGCAGAGTT-39) in 13 primer extension buffer (50 mM
Tris-HCl, pH 8.3, 2 mM DTT, 0.5 mM magnesium chloride, and 1 mM
spermidine) containing 20 mM dATP, 20 mM dTTP, and 10 mM dGTP,
followed by 1 hour incubation at 30°C. The TS primer was purified by phenol/
chloroform extraction and ethanol precipitation. The telomerase-extended
products were PCR amplified in a 25-mL reaction consisting of 13 Taq buffer
(NEB), 50 mM of dNTP, 0.4 mM of 32P end-labeled TS primer, 0.4 mM of
ACX primer (59-GCGCGGCTTACCCTTACCCTTACCCTAACC-39), 0.4
mM of NT primer (59-ATCGCTTCTCGGCCTTTT-39), 2 3 10213 M of
TSNT primer (59-CAATCCGTCGAGCAGAGTTAAAAGGCCGAGAAG
CGATC-39), and 1 unit of Taq (NEB). Samples were denatured at 94°C for
2 minutes, followed by 25 cycles at 94°C for 25 seconds and at 59°C for
30 seconds. PCR products were resolved on a 10% polyacrylamide/2%
glycerol gel. The gel was dried, exposed to a phosphor storage screen, and
analyzed with a Bio-Rad FX-Pro molecular imager. Total activity was
quantitated and normalized to the intensity of an internal control band.
TERT variant analysis
Data for the TERT variant table (Table 1) were compiled using dbNSFP version
light 1.3 (December 2011), incorporating recent updates to both PolyPhen-2 and
SIFT. Of the 34 TERT mutations listed in the Telomerase Database (http://
telomerase.asu.edu/), the 6 insertions or deletions were excluded because they
could not be evaluated by the algorithms used by dbNSFP. An additional 3
mutations were excluded because the specific change was not included in the
dbNSFP database. Because the H412Y variant has reported telomerase activity
of between 50% and 91%, it was excluded for ease of interpretation. Therefore,
24 previously described mutations were included (Table 1), in addition to the
novel variant described here. The “total predicted damaging” score was
calculated by assigning 1 point to each of the 4 algorithms for prediction of either
“damaging” or “deleterious.” A half-point was added for a prediction of
“possibly damaging” from PolyPhen-2. Therefore, the scores ranged from 0,
indicating prediction as benign or nondeleterious by all 4 algorithms, to 4,
indicating prediction as deleterious or damaging by all 4 algorithms.
“Telomerase activity” and “Present in > relatives” columns were summarized
from published literature describing these variants (see Reference column) in
terms of functional analysis and presence in at least 2 members of a pedigree.
Results
An HHS phenotype is associated with a homozygous TERT
p.T567M mutation within the T motif
The male patient presented at age 30 months as a referral from the
Middle East, with pancytopenia, developmental delay, ataxia, and
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REDUCED TELOMERASE PROCESSIVITY RESULTING IN HHS
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Figure 1. Clinical manifestations. (A) Brain magnetic
resonance imaging. (B) Chest computed tomography.
cerebellar hypoplasia. Pancytopenia, with 50% bone marrow
cellularity, was first noted at age 20 months, following a viral
illness. He was born at term with evidence of intrauterine growth
retardation (birth weight 1.9 kg) and hospitalized for the first 2
weeks of life with respiratory and swallowing difficulties. His
weight was in the fifth percentile and his height was below the third
percentile. His exam was notable for oral leukoplakia, ataxia,
hypotonia, and decreased reflexes. No pigmentation abnormalities
were evident, and nails were thin but not dystrophic. His speech
and gross and fine motor development were significantly delayed,
with inability to walk independently and to dress or feed himself.
He spoke no intelligible words, but was able to follow simple
1-step commands.
Brain magnetic resonance imaging demonstrated multiple
findings including profound atrophy of the cerebellar vermis
(Figure 1A). A chest computed tomography revealed diffuse, illdefined ground-glass nodules and bilateral pleural blebs (Figure 1B).
Liver function test results were within normal limits.
Given his clinical findings, telomere length testing was performed
at age 28 months using the telomere flow FISH assay (University
Hospital, Bern).36 His telomere lengths were well below the first
percentile in all white blood cell (WBC) subsets (Figure 2). Sequencing of TINF2, NHP2, and NOP10 revealed no variants (Ambry
Genetics). Sequencing of DKC1 and TERC was not performed as
these were found to be without variants in his similarly affected
sibling (see below). TERT sequencing revealed a homozygous variant
of unknown significance, p.T567M (c.1700C.T), in exon 3, mapping to the T motif (Figure 3).
The patient was diagnosed with HHS. Given his progressive,
severe cytopenias and bone marrow hypocellularity (,5% to 10% to
20%), he received a bone marrow transplant from a 9/10 matched
unrelated donor. The combination of fludarabine, melphalan, and
alemtuzumab was used as a conditioning regimen, and cyclosporine
and prednisone were used as graft-versus-host-disease prophylaxis.
He engrafted on day 14, and subsequent chimerism studies revealed
100% donor cells. His immediate post transplant course was
Figure 2. Homozygosity of TERT p.T567M results in dramatic telomere shortening. Telomere lengths determined by telomere flow FISH analysis (courtesy of University
Hospital, Bern and Repeat Diagnostics, Vancouver). Reference percentiles indicated in the top left graph obtained from the determination of telomere length in 400 normal
controls.14 The proband’s telomere length is indicated by the diamond, the mother’s by the square, and the father’s by the circle.
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GRAMATGES et al
BLOOD, 2 MAY 2013 x VOLUME 121, NUMBER 18
Figure 3. Family pedigree demonstrating consanguinity between the proband’s parents. The homozygous proband and deceased sibling are indicated by
the completely filled circle/square. The heterozygous
parents as well as obligate heterozygous grandfathers
are indicated by the half-filled circles/ half-filled squares.
complicated by transaminitis, engraftment syndrome, and adenovirus enteritis. At the time of manuscript preparation, he continues to
be in remission from his marrow failure.
Biallelic inheritance of TERT p.T567M (c.1700C>T) results in
severe disease and dramatic telomere shortening, whereas
heterozygosity for the variant has minimal clinical consequence
The patient’s sister had clinical features that were similar to those
of her brother, also presenting at an early age with ataxia and BMF.
At age 3 years, she underwent matched unrelated cord blood
transplantation. However, a complicated post transplant course,
including idiopathic pulmonary syndrome and persistent cytopenias, resulted in her death 1 month following transplant. She did not
receive telomere length testing; however, post-mortem sequencing
confirmed homozygosity for TERT p.T567M (GeneDx).
The parents, who were first cousins, were found to be
heterozygous for TERT p.T567M (Figure 3). They were healthy
and reported no clinical manifestations consistent with a telomere
biology disorder. Their complete blood counts and indices were
normal. Telomere flow FISH analysis revealed their telomere
lengths near or just below the first percentile for all leukocyte
subsets (Figure 2). Pedigree analysis indicated that the paternal and
maternal grandfathers, who were brothers, were obligate carriers,
with the mutation being inherited from one of their parents. The
maternal grandfather reported having a low WBC count for the
past 6 to 7 years. Otherwise, there was no reported family history
of other blood disorders, cancers, or hepatic or pulmonary disease.
hTERT p.T567M is associated with reduced
telomerase processivity
Given the disease severity in the proband, we explored the impact of
this mutation on telomerase activity and processivity. We analyzed
the effect of the mutation on in vivo reconstituted telomerase using
the TRAP assay. We found total activity was only modestly reduced
to 69% of wild-type (WT) levels (Figure 4A), which was surprising
given the extreme telomere shortening in the proband. As the TRAP
assay does not directly measure processivity, we then analyzed the
same lysates using a direct primer extension assay, which allows
quantitative assessment of processivity.21 In this assay, the T567Mmutant telomerase exhibited a marked decrease in processivity,
specifically showing a dramatic decrease of the longer length
products corresponding to 122R4 and above bands relative to WT
(Figure 4B). These differences could not be accounted for by
differences in hTERT or hTR levels (Figure 4C-D). A similar
processivity defect was observed using telomerase reconstituted in
vitro (supplemental Figure 1), thus indicating an inherent defect in
telomerase processivity. Although the total activity was reduced
(48% of WT), this was accounted for mostly by the loss of the
multiple repeat addition products, ie, defective processivity.
Given the impact of the TERT p.T567M mutation on telomerase
processivity, we addressed whether the other disease-causing motif T
mutation K570N31 had a similar impact. Remarkably, in the direct
primer extension assay, the in vivo reconstituted K570N-mutant
telomerase was nonprocessive, producing only the 14R1 product
(Figure 4B). In the TRAP assay, the K570N-mutant telomerase showed
7% of WT activity (Figure 4A). However, since the PCR amplification
step of TRAP does not favor the single-repeat product, the activity in the
TRAP assay does not correctly represent the actual activity or processivity of this mutant. Thus, as revealed by the direct assay, the effect
of this mutation in vivo is likely a consequence of defective processivity.
Position 567 in hTERT is not an evolutionarily conserved residue
As noted in Figure 5, although a threonine is present at the position
aligning with human 567 in 6 of the 14 orthologs examined, the
remainder contains either a methionine or valine at this position.
This was surprising, as the substitution in our case, which resulted
in a profound defect in processivity, was a methionine. Moreover,
among the species with a methionine at this position, Gallus gallus
and Takifugu rubripes have telomerases that are processive in
vitro,40,41 whereas Xenopus laevis telomerase is known to be
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BLOOD, 2 MAY 2013 x VOLUME 121, NUMBER 18
REDUCED TELOMERASE PROCESSIVITY RESULTING IN HHS
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Figure 4. Telomerase activity analyses of TERT
p.K570N and p.T567M mutants. (A) Telomerase TRAP
assay of TERT mutants. Two concentrations of lysate
(3 ng and 0.6 ng of protein) are analyzed in a 2-step
TRAP reaction. Control reactions with CHAPS lysis
buffer in place of lysate (buffer only) and a telomerase
positive cell lysate (positive) are included. (B) Telomerase direct assay of TERT p.K570N and p.T567M mutant
telomerase reconstituted in vivo. Cell lysates (1.5 mg of
protein) of 293FT cells transfected with TERC and
TERT-WT (lane 1), T567M (lane 2), or K570N (lane 3)
are analyzed by direct primer extension assays. Transfections of TERC (lane 4) or TERT-WT (lane 5) alone
are included as controls. A 32P end-labeled 18-mer
oligonucleotide is included as the loading control (L.C.).
(C) Western blot of ectopic TERT expression. The
glyceraldehyde-3-phosphate dehydrogenase protein is
used as the internal control. (D) Northern blot analysis of
ectopic TERC expression. The endogenous 5S rRNA is
probed to ensure equal loadings.
nonprocessive, producing at most 2 repeats when analyzed by
conventional assays.42 No reports of telomerase activity and
processivity in Coturnix japonica were found. Thus, while human
telomerase contains threonine at position 567 for an optimal
processivity, other species adapt other amino acids for maintaining
processivity or simply tolerate a lower processivity.
Computational predictions of effect of variants on protein
function are inconsistent with measured telomerase activity
and/or observed clinical phenotype
Several algorithms have been developed to predict the impact of an
amino acid substitution on protein function. Among the commonly
used algorithms, SIFT (http://sift.jcvi.org/), PolyPhen-2 (http://
genetics.bwh.harvard.edu/pph2/), MutationTaster (http://www.
mutationtaster.org), and LRT43 predicted no effect of the hTERT
p.T567M substitution on protein function, reflecting, in part, the
lack of evolutionary conservation of T567. However, as the proband’s
phenotype was so severe and the functional analyses demonstrated
definitive impact on processivity, we hypothesized that these algorithms might be limited in their capacity for predicting the effect
of missense changes on telomerase function. To investigate this
hypothesis, we extracted missense changes associated with a clinical phenotype found in the Telomerase Database4 and correlated
each with its reported telomerase activity, as well as whether the
mutation was observed in more than 1 family member with a
telomere biology disorder phenotype. In addition, we used dbNSFP
(https://sites.google.com/site/jpopgen/dbNSFP) to compile predictions for each of these mutations and generated a cumulative
predictive score for deleterious effect across all 4 algorithms. As
outlined in Table 1, the predictions generated by these programs
were often inconsistent with both clinical observation and measured telomerase function. Table 2 summarizes these findings in
terms of the number of variants with cumulative predictive scores
of 0 to 1, 1.5 to 2, 2.5 to 3, and 3.5 to 4, categorized by telomerase
functional analysis (<80% or .80%) and presence of the variant
Figure 5. Sequence alignment of the TERT T motif
across species. Colors represent amino acids with
similar properties. Residue 567 is outlined in blue.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
3592
BLOOD, 2 MAY 2013 x VOLUME 121, NUMBER 18
GRAMATGES et al
Table 2. Association between cumulative scores predicting consequence of a missense change and telomerase functional analysis as well
as association with a hereditary clinical phenotype
Variant characteristics
Score 0 to 1
Score 1.5 to 2
Reported in pedigree (2 or more relatives)
3
Telomerase activity no higher than 80%
1
Number of variants reported in pedigree and with
4
5
Sum of variants with
scores of 0 to 2
Score 2.5 to 3
Score 3.5 to 4
3
3
2
2
3
2
6
4
1
9
Sum of variants with
scores of 2.5 to 4
10
reduced telomerase activity
Not reported in pedigree
0
0
1
Telomerase activity more than 80%
3
0
0
0
Number of variants not reported in a pedigree and
3
0
1
1
3
2
with normal telomerase activity
in 2 or more affected individuals within a pedigree. By dividing
this table into 4 quadrants, we found that of the 12 variants
predicted to be deleterious (cumulative score of 2.5 to 4), only 2
were associated with normal telomerase activity in vitro.
Conversely, of the 12 variants predicted to be benign (cumulative
score of 0 to 2), 9 were associated with significantly reduced
telomerase activity in vitro. Thus, these algorithms exhibited a poor
sensitivity for telomerase defects.
Discussion
Here we report a novel TERT variant with marked effects on
processivity, resulting in significantly shortened telomere length and
severe clinical manifestations when inherited as a homozygous
mutation. This mutation represents the second naturally occurring
mutation reported within the T motif of hTERT. This motif, although
both highly conserved and known to be required for normal telomerase catalytic activity, had thus far not been implicated in
promoting processivity. We demonstrate that both of the diseaseassociated T-motif variants, T567M and K570N, impact processivity,
underscoring the T motif’s role in this aspect of telomerase function.
Within this pedigree, we noted the absence of a telomere biology
disorder phenotype in the proband’s parents, both heterozygous for
the mutation described, despite having telomere lengths near the
first percentile in all leukocyte subsets. The proband’s grandfathers,
who were obligate carriers, lacked a significant history as well,
except for the maternal grandfather who had a several-year history
of low WBC count. However, this mutation was associated with
marked telomere shortening and a severe HHS phenotype when
both alleles were affected in the parents’ offspring. Telomere
lengths on the sister were not measured; however, the proband’s
telomere lengths fell well below the first percentile for his age, as
would be expected in this severe form of DC. As has been noted in
familial pulmonary fibrosis cohorts, individuals with heterozygous
mutations affecting processivity may benefit from telomerase’s
natural ability to preferentially elongate the shortest telomeres.32
These heterozygous individuals, though incapable of producing
telomeres of normal length, are able to maintain lengths above the
critical threshold for senescence, thus minimizing the potential for
manifestation of a telomere biology disorder. However, in the
absence of a functional copy of TERT, the moderate processivity
defect of the T567M mutation is able to result in a severe phenotype.
The direct primer-extension assay shows that the T567M mutation
moderately reduces processivity, whereas the K570N mutation abolishes telomerase processivity (Figure 4B). Telomerase processivity
mutants have higher probability of enzyme dissociation from the
DNA products and could, thus, increase enzyme turnover in the
reaction.38 This effect is observed with the p.T567M mutant, which
generates more initial telomere repeat products, ie, the 14R1 and
110R2, than WT, presumably due to enzyme turnover. In comparison, the nonprocessive p.K570N mutant generates the 14R1
repeat product at the WT level, suggesting a low enzyme turnover.
However, telomerase processivity likely plays a more important role
than enzyme turnover in telomere lengthening in cells where the
telomere substrate and telomerase concentration are low. Notably,
the heterozygous TERT p.K570N mutation tracked with various
blood disorders across a 4-generation pedigree.31 As in the familial
pulmonary fibrosis cohort, classical manifestations of DC were not
observed, again contrasting the effects of inheritance of a heterozygous vs homozygous processivity mutant.
We also note that the hTERT p.T567M substitution does not occur
within an evolutionarily conserved residue. In addition, species with a
naturally occurring methionine at the position cognate to human TERT
567 do not consistently demonstrate reduced telomerase processivity,
suggesting the presence of alternative factors that are influencing
telomerase processivity, eg, coevolution of TERT and TERC. Last, our
survey of known disease-associated TERT mutations demonstrates that
algorithms commonly used to predict functional impact are unreliable
in many cases, particularly when a missense change is predicted to be
benign. A possible explanation for this discrepancy is failure of the algorithm to consider the effect of a residue substitution on RNA–protein
interactions. Therefore, we recommend reliance on measured in vitro
functional analysis, including processivity assays, when determining
the impact of TERT variants on telomerase function, including rare
variants predicted to be benign.
Acknowledgments
The authors thank the described family for their participation. The
authors also thank Vinayaka Prasad, Albert Einstein School of
Medicine, and Joachim Lingner, Swiss Institute for Experimental
Cancer Research, for the pNFLAG-hTERT, and pcDNA-hTERT
and pBS-U1-hTR plasmids, respectively.
This work was supported by grants from the National Institutes
of Health (5K12CA090433-10 to M.M.G. and R01GM094450 to
J.J.-L.C.) and from the Cancer Prevention Research Institute of
Texas (RP120076 to A.A.B.).
Authorship
Contribution: A.A.B. and J.J.-L.C. were the principal investigators
and take primary responsibility for the paper; M.M.G. drafted the
manuscript and performed the research related to the computational
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 2 MAY 2013 x VOLUME 121, NUMBER 18
REDUCED TELOMERASE PROCESSIVITY RESULTING IN HHS
algorithms; X.Q. performed the telomerase functional analyses;
G.S.S. and A.A.B. were involved directly in the patient’s care,
obtained consents for the research study, and collected samples and
clinical information; and all authors analyzed the data, critically
revised the manuscript, and agreed upon the final version.
3593
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Alison A Bertuch, Baylor College of Medicine, 1102 Bates St, Suite 1200, Houston, TX 77030; e-mail:
[email protected].
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2013 121: 3586-3593
doi:10.1182/blood-2012-08-447755 originally published
online March 28, 2013
A homozygous telomerase T-motif variant resulting in markedly reduced
repeat addition processivity in siblings with Hoyeraal Hreidarsson
syndrome
Maria M. Gramatges, Xiaodong Qi, Ghadir S. Sasa, Julian J.-L. Chen and Alison A. Bertuch
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