Dear Editor,
Accelerated shortening of telomeres can induce premature cell senescence that can
clinically manifest as bone marrow failure (BMF), idiopathic pulmonary fibrosis (IPF),
cryptogenic cirrhosis, nodular regenerative hyperplasia, vascular malformations, immunodeficiency,
and structural brain abnormalities, all of which are included under the umbrella term
of short telomere syndromes (STSs)
1
. Two main criteria used to diagnose STS include the documentation of shortened telomere
lengths (TLs) by a Clinical Laboratory Improvement Amendments (CLIA)-certified flowFISH
(fluorescence in situ hybridization) assay
2
and the presence of pathogenic variants in genes related to telomere maintenance identified
through next-generation sequencing (NGS). These genes include, but are not limited
to, hTERT (human telomerase reverse transcriptase) and hTERC (human telomerase RNA
component), the two main components of the telomerase holoenzyme complex that is responsible
for creating new telomeric DNA (Supplementary Fig. 1)
1
.
In our experience, a pathogenic variant in the coding sequence of a telomere-associated
gene can be identified in only 40% of STS cases using current sequencing approaches,
with the remaining cases either having no identifiable variant or possessing a variant(s)
of uncertain significance (VUS)
1,3
. In addition, in some of these cases, the TL values were not conclusively shortened
(i.e., not < first percentile in lymphocytes and/or granulocytes documented by a CLIA-certified
FlowFISH assay) complicating the diagnosis. At Mayo Clinic, we established a dedicated
BMF clinic in collaboration with the center for Individualized Medicine and the division
of Hematology, so as to leverage the latest NGS technologies and functional assays
to assist patients with unexplained BMF syndromes, including STS-related marrow failure
3,4
. We have developed a systematic algorithmic approach to assess STS patients that
includes TL testing by flowFISH and genomic assessment of STS-related gene mutations
using an in-house designed targeted NGS panel (Supplementary Table 1). Through this
effort, we have identified 32 patients with an STS phenotype and TLs at or below the
tenth percentile. Twenty-two patients (69%) did not have detectable pathogenic variants
(in spite of a clinical phenotype) or were found to carry a VUS in STS-related genes
(ten patients; 31%). In the latter group, the VUS were located in hTERT (four variants),
hTERC (one patient), RTEL1 (regulator of telomere elongation helicase 1; two variants),
TINF2 (TERF1-interacting nuclear factor 2; two variants), and NAF1 (nuclear assembly
factor 1 ribonucleoprotein; one variant). Patients with hTERT VUS (Table 1 and Supplemental
Fig. 2) were selected for three-dimensional (3D) computational modeling and functional
interrogation, largely due to the availability of a well-described functional assays,
namely the telomerase repeat amplification protocol (TRAP), which can be used to test
telomerase activity (Supplemental Methods)
5,6
.
Table 1
Table summarizing clinical features, variants, and testing results for patients with
a clinical phenotype of a STS.
Case no.
Age/sex
Clinical features
Significant family history (yes/no)a
FlowFISH TL (centile length in granulocytes/ lymphocytes)
Gene
cDNA change
Protein change
Protein region
In silico predictions (SIFT/PolyPhen)
CADD score
3D model prediction
Conclusion from TRAP assay
ACMG classification (at the clinical report/after research testing)
1b
23/M
Premature graying of hair, macrocytosis, thrombocytopenia, bilateral hip avascular
necrosis
Yes
<1st/<1st
TERT
c.2768 C > T
p.Pro923Leu
Reverse-transcriptase domain
Deleterious/probably damaging
24.2
Destabilizes structure
NA
Pathogenic/pathogenic
2
69/F
Premature graying of hair, IPF, anemia, leukopenia
No
10th/10th
TERT
c.3362 C > T
p.Pro1121Leu
C-terminal extension
Deleterious/probably damaging
22.9
Destabilizes structure
Absent telomerase function
VUS/likely pathogenic
3
27/F
Macrocytosis, neutropenia
No
<1st/1st
TERT
c.1765A > C
p.Ile589Leu
None described
Tolerated/benign
5.08
Neutral
Decreased telomerase function
VUS/likely pathogenic
4
19/M
Macrocytosis
No
<1st/<1st
TERT
c.1885G > A
p.Gly629Arg
Reverse-transcriptase domain
Deleterious/probably damaging
23.1
NA
Decreased telomerase function
VUS/pathogenic
5
47/M
IPF, pancytopenia
No
<1st/<1st
TERC
n.238 G > C
NA
NA
NA
NA
Changes TERC organization
NA
VUS/pathogenic
FISH fluorescence in situ hybridization, IPF idiopathic pulmonary fibrosis, NA not
applicable, STS short telomere syndrome, TL telomere length, TRAP telomerase repeated
amplification protocol.
aSignificant family history was defined as the presence of one or more first- or second-degree
relatives with one or more clinical features characteristic of STSs, such as premature
onset of hair graying (age < 30 years), IPF, cryptogenic cirrhosis or nodular regenerative
hyperplasia, or unexplained cytopenias.
bClinical history of this case were previously published; however, functional testing
results are new.
All patients (Table 1) had BMF with additional STS features (IPF in patients 2 and
5, and premature graying of hair in patients 1 and 2). FlowFISH testing indicated
a TL below the first centile in both lymphocytes and granulocytes in all patients,
except in patient 2 were the TL was at the tenth centile range (Fig. 1a). NGS testing
uncovered hTERT variants in patients 1–4 and a hTERC variant in patient 5. To increase
our interpretive resolution for hTERT variants, we created a computational 3D protein
model of the human telomerase complex that we used to assess the intra- and inter-molecular
interactions within the complex. Our current model includes all amino acids of hTERT
and a contiguous section of hTERC with the bound DNA heteroduplex. This model was
generated using homology-based methods
7,8
and the experimentally solved structure of TERT from Tetrahymena thermophila [PDB
6d6v
9
]. The human Reverse Transcriptase thumb domain of telomerase was experimentally solved
[5ugw
10
] and added to our model using threading. Genomic variants were assessed in their
3D context using FoldX
11
. Recent model fitting to a cryogenic electron microscopy map of human telomerase
core particle was published
12
and was used here in comparison to our current model of full-length TERT.
Fig. 1
Functional validation of variants of uncertain significance in hTERT/hTERC.
a Mean telomere length in lymphocytes and granulocytes from blood samples measured
by flowFISH. All patients presented clinical signs of STS and genetic variants in
the holozenzyme telomerase. While most patients (1, 3, 4, and 5) presented clearly
shortened telomeres, patient 2 showed fringe values closer to the 10th centile. b
Recent studies have resolved human telomerase core components, which can be combined
with additional data to generate a more specific and comprehensive model for interpreting
the effects of telomerase variation. We used molecular modeling to develop a finer
resolution of interpretation for telomerase variants identified through clinical genomics
sequencing. The nucleotide backbones of RNA and DNA are colored in orange and gold,
respectively, and each protein domain is colored distinctly with the intervening loops
demarcated in white. Our current model of human telomeres consists of all amino acids
of hTERT and a large contiguous section of hTERC. This model was generated based off
of homology relationships and energetic refinement. The 3D relationships among amino
acids of TERT and nucleotides of TERC enable us to make specific predictions for the
impact and effects of genomic variants. c Blood protein extracts from patients carrying
TERT variants were tested using the TRAP assay for telomerase activity. In comparison
to healthy individuals used as controls (second lane in each panel), the results indicated
absent (patient 2) or diminished (patients 3 and 4) telomerase activity in all samples
tested, suggesting pathogenicity of the variants.
When indicated, we used our 3D model to suggest mechanisms of pathogenicity for these
variants as follows
1
: hTERT c.2768 C > T; p.Pro923Leu (patient 1) is located near the helix required for
oligomerization and is adjacent to p.Arg901 and p.Lys902—sites where bona fide loss-of-function
mutations have been described (Fig. 1b)
2
. hTERT c.3362 C > T; p.Pro1121Leu (patient 2) is present at the end of the reverse-transcriptase
thumb domain at a position where the peptide chain crosses back against residues 999–1003.
Further, hTERC wraps around one side of this thumb and the DNA winds across the other
side, so that any changes to the internal organization/arrangement of this thumb may
significantly alter function (Fig. 1b)
3
. The third variant hTERT c,1765A > C; p.Ile589Leu (patient 3) affects a residue located
in the helix near the hTERC interface possibly affecting stability of this interaction
(Fig. 1b)
4
. The fourth variant, hTERT c.1885G > A; p.Gly629Arg is located in a loop between
the DNA strand of the heteroduplex, and Lys626 and Arg631, both of which make contact
with the DNA strand (Fig. 1b).
In addition to this information, further functional testing was pursued for these
variants with the exception of hTERT c.2768 C > T; p.Pro923Leu (Patient 1), where
we considered the evidence already available, including a published functional report,
to be strong enough to consider this variant as being pathogenic
13
(more information in ref.
3
). For the remaining patients with hTERT variants, we evaluated their telomerase activity
using the TRAP assay
5
. TRAP semi-quantitatively measures the capacity to elongate a telomere-imitating
oligonucleotide via hTERT processing in a patient’s samples over several amplification
cycles, akin to a quantitative PCR reaction. The commercially available TRAPeze Telomerase
Detection Kit (Supplemental Methods) was employed in peripheral blood mononuclear
cells from patients 2–4 and the results indicated decreased telomerase activity in
all samples compared to age-matched patient controls (Fig. 1c). This data, together
with our 3D-prediction models, allowed us to re-classify these variants as pathogenic
or likely pathogenic (Table 1).
The hTERC VUS in patient 5 (n.238 G > C) was located in the stem loop in the CD4/5
region and our 3D model analysis indicated a likely reorganization of the stem loop
as a consequence of this change, thereby altering the secondary structure of the RNA.
Additionally, a previous report had indicated decreased telomerase activity in patients
carrying this variant when tested by the TRAP assay
14
. We felt that given the clinical context, this information was sufficient enough
to classify this variant as pathogenic without the need for additional testing.
In conclusion, we demonstrate the importance and feasibility of using 3D molecular
modeling and functional assays to classify variants identified in the hTERT holoenzyme.
By using these methods, we were able to provisionally re-classify five VUS identified
in patients with STS, an important step forward in mutational nomenclature. Importantly,
pathogenic variants in hTERT/hTERC comprise 52% of STS-related mutations described
in the Human Gene Mutation Database (https://portal.biobase-international.com/hgmd/).
For the remaining 48% of mutations that encompass several genes involved in TL regulation,
there currently are no reliable functional assays (e.g., assessment of RTEL1 abnormalities
by T-circle detection demonstrates considerable variability). Thus, variants identified
in our clinic impacting RTEL1, TINF2, and NAF1 still remain unclassifiable and their
causal impact on TL in these patients remains unknown. TRAP assays also demonstrate
inherent variabilities between tissue samplings and between individuals within the
same age range, limiting the routine implementation of these assays in clinic. The
development of cell line-based assays utilizing genetic engineering of variants and
assessing their impact on TL could be one potential way of overcoming these limitations.
In summary, while we were able to assess the pathogenicity of h
TERT/h
TERC variants using 3D modeling and the TRAP assay, a lot more work is needed to help
develop accurate in silico approaches and functional testing for variants in other
genes regulating TL.
Supplementary information
Supplemental Figures legends
Supplemental Methods
Supplemental Table 1
Supplemental Figure 1
Supplemental Figure 2