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Plenary Paper
HEMATOPOIESIS AND STEM CELLS
Inherited bone marrow failure associated with germline mutation of
ACD, the gene encoding telomere protein TPP1
Yiran Guo,1 Melissa Kartawinata,2,3 Jiankang Li,4 Hilda A. Pickett,2,3 Juliana Teo,5 Tatjana Kilo,5 Pasquale M. Barbaro,2,3,5
Brendan Keating,1,6 Yulan Chen,4 Lifeng Tian,1 Ahmad Al-Odaib,3,7,8 Roger R. Reddel,2,3 John Christodoulou,3,8 Xun Xu,4
Hakon Hakonarson,1,6 and Tracy M. Bryan2,3
1
Center for Applied Genomics, The Children’s Hospital of Philadelphia, Philadelphia, PA; 2Children’s Medical Research Institute, Westmead, NSW,
Australia; 3University of Sydney, Sydney, NSW, Australia; 4Beijing Genomics Institute-Shenzhen, Shenzhen, China; 5Haematology Department, Children’s
Hospital Westmead, Westmead, NSW, Australia; 6Department of Pediatrics, The Perelman School of Medicine, University of Pennsylvania, Philadelphia,
PA; 7Department of Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia; and 8Western Sydney Genetics Program,
Children’s Hospital Westmead, Westmead, NSW, Australia
Key Points
• Bone marrow failure can
derive from germline mutation
of the telomere protein TPP1
that is involved in recruiting
telomerase to telomeres.
• The observed mutation
disrupts recruitment of
telomerase to telomeres and
segregates with short telomere
length and disease phenotype.
Telomerase is a ribonucleoprotein enzyme that is necessary for overcoming telomere
shortening in human germ and stem cells. Mutations in telomerase or other telomeremaintenance proteins can lead to diseases characterized by depletion of hematopoietic
stem cells and bone marrow failure (BMF). Telomerase localization to telomeres requires an
interaction with a region on the surface of the telomere-binding protein TPP1 known as the
TEL patch. Here, we identify a family with aplastic anemia and other related hematopoietic
disorders in which a 1-amino-acid deletion in the TEL patch of TPP1 (DK170) segregates
with disease. All family members carrying this mutation, but not those with wild-type TPP1,
have short telomeres. When introduced into 293T cells, TPP1 with the DK170 mutation
is able to localize to telomeres but fails to recruit telomerase to telomeres, supporting
a causal relationship between this TPP1 mutation and bone marrow disorders. ACD/TPP1
is thus a newly identified telomere-related gene in which mutations cause aplastic anemia
and related BMF disorders. (Blood. 2014;124(18):2767-2774)
Introduction
Telomeres are the repetitive DNA–protein complexes at the ends
of linear chromosomes, which shorten with every cell division in
human somatic cells.1 Telomere shortening eventually leads to the
activation of a DNA damage response and cellular senescence or
apoptosis.2-4 Germ cells, stem cells, and cells in the developing
human embryo overcome telomere shortening by expression of
the ribonucleoprotein enzyme telomerase.5-8 Telomerase catalyzes
addition of telomeric DNA repeats to chromosome ends by reverse
transcription of a template sequence within its integral RNA subunit
(hTR in humans9). The core active human telomerase complex consists of hTR, the reverse-transcriptase protein subunit (hTERT),10
and the RNA-binding protein dyskerin.11,12 Telomerase interacts
with many other proteins during its biogenesis and transport to the
telomere, such as the protein TCAB1 (also known as WDR79 and
WRAP53), which is responsible for trafficking telomerase to nuclear
Cajal bodies13,14 and from there to the telomere.15
Individuals who inherit extremely short telomeres are at risk of
developing one of a group of disorders collectively termed “short
telomere syndromes.”16,17 The first of these diseases linked to
short telomeres was dyskeratosis congenita (DC),11 defined by a
mucocutaneous triad of abnormal skin pigmentation, oral leukoplakia, and nail dystrophy and characterized by progressive bone marrow
failure due to depletion of functional hematopoietic progenitor and
stem cells.18 Patients with DC, or its more severe variants Revesz
syndrome and Hoyeraal-Hreidarsson syndrome, harbor deleterious
mutations in the genes encoding dyskerin,19 hTR,20 hTERT,21
TCAB1,22 the dyskerin-associated proteins Nop1023 and Nhp2,24 or
the telomere-binding proteins TIN2,25,26 CTC1,27,28 and RTel1.29-32
Patients with acquired or familial aplastic anemia, but lacking the
other clinical features of DC, can also carry short telomeres and
mutations in the genes encoding hTR33 or hTERT.21,34
The telomere-binding protein TPP1 (encoded by the gene adrenocortical dysplasia homolog [mouse] [ACD] and previously known
as PTOP, PIP1, or TINT2) is critical for telomere stability and length
regulation.35-38 TPP1 binds to the telomere via interaction with TIN2
and in turn tethers the single-stranded DNA binding protein Pot1
to telomeres.36-38 TPP1 is required for Pot1 to perform its role in
protecting telomeres from being recognized as DNA damage.39-41
The Pot1/TPP1 heterodimer also has a separate role in promoting the
processivity of telomerase, ie, its ability to synthesize long tracts of
Submitted August 19, 2014; accepted September 1, 2014. Prepublished
online as Blood First Edition paper, September 9, 2014; DOI 10.1182/blood2014-08-596445.
There is an Inside Blood Commentary on this article in this issue.
Y.G., M.K., and J.L. contributed equally to this study.
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.
The online version of this article contains a data supplement.
© 2014 by The American Society of Hematology
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GUO et al
BLOOD, 30 OCTOBER 2014 x VOLUME 124, NUMBER 18
Figure 1. ACD/TPP1 mutation and telomere length in family with history of anemia and cancer. (A) Pedigree and clinical diagnoses of family. The genotypes of the
proband and 4 of her family members are shown (1/1 for homozygous WT and 1/DK170 for heterozygous deletion). Numbering of individuals corresponds with numbering in
supplemental Table 1. (B-C) Telomere lengths of 5 family members were measured by Flow-FISH (B) or qPCR (C). Red symbols indicate individuals with the DK170 mutation,
and blue symbols indicate WT individuals. Curves represent the 1st, 10th, 50th, 90th, and 99th percentiles of telomere lengths in ;240 healthy individuals. diag., diagnosed.
telomeric DNA.42 A region on the surface of TPP1 known as the
TEL patch mediates this interaction with telomerase and is
necessary for the recruitment of telomerase to telomeres. 43-46
Here, we identify a family with 3 generations of individuals with
short telomeres and aplastic anemia or other related blood disorders,
all of whom carry a heterozygous mutation in the ACD/TPP1 gene.
This mutation results in an in-frame deletion of an amino acid in
the TEL patch of TPP1, and we demonstrate that the mutated TPP1
protein is able to localize to telomeres but fails to recruit telomerase
to telomeres. These data support a causal relationship between this
TPP1 mutation and disease in this family.
Methods
Subjects
The proband presented to Children’s Hospital Westmead, with severe
thrombocytopenia and macrocytosis and has been diagnosed with aplastic
anemia. Her family history includes other cases of bone marrow failure (BMF)
as well as oral carcinoma and leukemia (Figure 1A and Table 1). Peripheral
blood DNA was available from the proband, her parents, and her maternal
grandparents. Informed consent was obtained from all participating
individuals, in accordance with the Declaration of Helsinki, and the studies
were approved by the Human Research Ethics Committee of the Sydney
Children’s Hospitals Network.
Whole-exome sequencing and bioinformatics
The procedure was described elsewhere.47 Briefly, exonic regions were
captured using Agilent SureSelect Human All Exon kit, and pair-end
sequencing was carried out on Illumina HiSeq 2000 machines. The Illumina
pipeline at default settings was applied to process raw image files for base
calling and generating raw sequencing read files in fastq format. Subsequently, 2 independent analysis workflows were used to perform sequencing read alignment, variant calling, and variant annotation (supplemental
Table 1, available on the Blood Web site). In the first pipeline, BurrowsWheeler alignment was used to align fastq files to the human reference genome
(University of California, Santa Cruz [UCSC] hg19).48 Then, variants were
called using the Genome Analysis Tool Kit (GATK, version 1.4)49 followed by
functional annotation with Annovar50 and SnpEff.51 In the second pipeline,
alignment to the human reference genome (UCSC hg19) was performed by
Short Oligonucleotide Analysis Package (SOAP, version 2.21).52 We used
SOAPsnp (version 1.05)53 to detect single-nucleotide variants (SNVs) and
GATK to call insertion-deletions (indels). Finally, annotated by BGI’s selfdeveloped scripts, the variants were grouped into different categories.
Multiple filtering steps and Mendelian genetic analysis
Under an autosomal-dominant inheritance, we applied the following mutational filtering steps in both pipelines described above. We first selected
heterozygous variants shared by all 3 affected individuals and required that
those variants should not be found in the proband’s unaffected grandfather.
Then, we eliminated variants that met either one of the following criteria:
(1) out of exonic regions, (2) synonymous changes, and (3) with frequency
.0.5% in dbSNP135,54 1000 Genomes Project (n 5 1092 genotyped samples),55
HapMap Project (n 5 1301 genotyped samples),56 NHLBI Exome Sequencing
Project (n 5 6500 exomes) (http://evs.gs.washington.edu/EVS/), CAG-CHOP
(n 5 334 exomes), or BGI (n 5 1414 control exomes) databases. We paid
special attention to variants close to splicing sites and frameshift indels.
In the next step, we considered evolutionary conservation and filtered the
nonconserved regions with PhyloP57 value ,0.95, then retained variants
with “deleterious/damaging” prediction of pathogenicity by PolyPhen58
and SIFT.59 Finally, we took into account biological and clinical relevance of
the identified variants (supplemental Table 2).
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Table 1. Hematologic features of affected family members
Age (y)
Hb (g/L)
MCV (fL)
Neutrophils
(3 109/L)
Platelets
(3 109/L)
HbF (%)
Bone marrow
BM
cytogenetics
Diagnosis
Outcome
Proband V:1
8
70
121
0.6
18
31
Aplastic
Normal
Severe aplastic anemia
Alive, transfusion
Mother IV:2
33
126
120
1.7
33
3.5
Hypocellular
Normal
BMF, mild
Alive, stable
Maternal
55
104
117
1.6
102
N/A
Hypocellular
N/A
Aplastic anemia, oral
Deceased (61 y)
dependent
hematology
g/m III:6
carcinoma
g/m, grandmother; Hb, hemoglobin; HbF, fetal hemoglobin; MCV, mean corpuscular volume; N/A, not available.
PCR and Sanger sequencing
The deletion in ACD/TPP1 (RefSeq: NM_001082486.1) identified through
whole-exome sequencing (WES) was confirmed by Sanger sequencing of
DNA from peripheral blood of all available family members, including the 3
affected individuals and clinically unaffected father and maternal grandfather.
The primers TPP1-SeqF and TPP1-SeqR (supplemental Table 3) were used
for polymerase chain reactions (PCRs) as reported previously.60 Purified
PCR products were sequenced by the Australian Genome Research Facility
(AGRF Ltd., Sydney, Australia). The sequencing traces were analyzed
using BioEdit Sequence Alignment Editor.61
Patient telomere length analysis (Flow-FISH)
Telomere flow-fluorescence in situ hybridization (Flow-FISH) was performed with a published protocol.62 Approximately 10 mL of lithium-heparin
peripheral blood was collected, and mononuclear cells were isolated by Ficoll
density centrifugation (1077 Ficoll Histopaque; Sigma-Aldrich). Duplicate
samples of 2 3 106 cells were used for FISH analysis. A known cell line with
long telomeres (human tetraploid T-cell lymphoblastic line CCRF-CEM,
GM03671C; Coriell Institute for Medical Research, Camden, NJ) served
as an internal reference standard for telomere length. Equal numbers of
CCRF-CEM cells were mixed with patient cells prior to hybridization with
a fluorescein isothiocyanate–conjugated (CCCTAA)3 peptide nucleic acid
probe (Panagene, Daejeon, Korea) at 0.3 mg/mL. Unstained duplicate tubes
were run in parallel to determine autofluorescence. After denaturation,
hybridization was performed at 4°C overnight, and excess telomere probe
was removed with washing. Flow cytometry was performed on a FACS
CANTO II (BD Biosciences, San Jose, CA) instrument and data displayed
and analyzed with BD FACSDiva software (BD Biosciences). Calculation
of relative telomere length of the patient’s mononuclear cells was performed
by comparing the fluorescence of these cells with the tetraploid CCRFCEM cell line and expressed as a percentage. Values obtained from 240
healthy individuals show the normal percentiles for different age groups
(Figure 1B).
Patient telomere length analysis (qPCR)
Genomic DNA from peripheral blood samples was used in telomere-length
analysis by quantitative PCR (qPCR), performed on the Rotor-Gene Q
(Qiagen), using Rotor-Gene SYBR Green PCR Master Mix (Qiagen). The
assay was similar to that previously reported.63,64 Briefly, telomeric (T) DNA
was amplified using Tel1b and Tel2b primers (supplemental Table 3),
whereas the single copy gene (S), used to normalize the amount of input DNA,
was amplified using the HBG1 and HBG2 primers (supplemental Table 3).
A standard curve was used to convert the cycle threshold into nanograms
of DNA and relative telomere length calculated as the T/S ratio from the mean
of triplicate samples. Values obtained from 240 healthy individuals show the
normal percentiles for different age groups.
Detection of endogenous TPP1 messenger RNA
RNA was extracted from cells using the RNeasy Mini Kit (Qiagen),
followed by DNA digestion with DNase I (Invitrogen). Complementary
DNA synthesis was performed with the SuperScript III First Strand Synthesis
System for RT-PCR (Invitrogen). To detect expression of the mutant allele,
a 400-bp region of ACD/TPP1 spanning the mutation was amplified by PCR
using primers TPP1-Fw1 and TPP1-Rv1 (supplemental Table 3). Amplification was performed using Platinum Taq DNA Polymerase (Life
Technologies) in an Eppendorf Mastercycler gradient at 95°C for 5 minutes,
followed by 36 cycles of 95°C for 30 seconds, 60°C for 45 seconds, and 72°C
for 45 seconds. An aliquot of the PCR products was visualized with 1%
agarose gel electrophoresis and the remainder purified using a Qiagen PCR
Purification Kit and digested with the restriction enzyme XmnI, followed
by 1% agarose gel electrophoresis.
For quantitative reverse-transcription PCR (qRT-PCR) of endogenous
TPP1 messenger RNA (mRNA) levels, the primers targeting the 59 untranslated
region (UTR) of TPP1 (ie, specific for expression of the endogenous gene)
were TPP1-Fw2 and TPP1-Rv2 (supplemental Table 3). Glyceraldehyde-3phosphate dehydrogenase was used as the internal control (supplemental
Table 3). qRT-PCR was performed using SYBR Green Master Mix (Invitrogen)
in a LightCycler 96 (Roche) with 40 cycles of 95°C for 30 seconds, 60°C for
45 seconds, and 72°C for 45 seconds.
Site-directed mutagenesis of TPP1 expression plasmid
An expression construct for mutant DK170 of TPP1 was constructed in
pCMV6-Myc-DDK vector containing wild-type (WT) TPP1 (ACD) (RC204381;
Origene) as the template. The deletion of lysine 170 was achieved using the
Stratagene QuikChange II XL site-directed mutagenesis kit.
Cell culture, transfections, and cell-cycle synchronizations
Human embryonic kidney 293T cells were grown in Dulbecco’s modified
Eagle’s medium supplemented with 10% (vol/vol) fetal bovine serum in a
humidified 37°C incubator with 5% CO2. Cells were transfected with 1 mg
each of expression constructs of WT and mutant DK170 TPP1 using
X-tremeGENE 9 (Roche) for 9 hours. After medium change, 90 pmol
of silencing RNA (siRNA) targeting the 59 UTR of TPP1 (Hs_ACD_9
FlexiTube siRNA, Qiagen) was transfected with RNAiMax (Life Technologies) for 24 hours. All Stars Negative Control siRNA (Qiagen) was used
as the negative control. Cells were then synchronized at G1/S phase by adding
2 mM thymidine (Sigma-Aldrich) for 15 hours, releasing for 9 hours, and
adding 0.5 mg/mL aphidicolin (Sigma-Aldrich) for 16 hours. Cells were
harvested 3 hours after release and confirmed to be in mid-S phase by analysis
of DNA content by flow cytometry on a FACSDiva (Becton Dickinson).
FISH of hTR and telomeric DNA
Simultaneous FISH against hTR and telomeric DNA was performed as
described previously.15 Briefly, harvested synchronized 293T cells were
cytospun onto slides, fixed with formaldehyde, and permeabilized. Slides
were incubated with 5 Alexa Fluor 488–labeled anti-hTR oligonucleotides
(Sigma-Aldrich; supplemental Table 3) and a Texas red–labeled telomere
probe (Sigma-Aldrich; supplemental Table 3) in FISH hybridization
buffer.15 FISH staining was visualized on a Zeiss AxioImager M1 microscope
with a Plan-Apochromat 633 oil objective (numerical aperture 1.4) and an
AxioCam MR digital camera (Carl Zeiss) with consistent exposure times
between experiments. For presentation purposes, pixel intensity histograms
were adjusted in ZEN (Carl Zeiss), equally across all figure panels, and
images were cropped in Adobe Photoshop. Colocalizing hTR and telomere
foci were manually counted in 100 cells per treatment, and data were analyzed
by Student t test for pairwise comparison.
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GUO et al
Chromatin immunoprecipitation (ChIP) of telomeric DNA
15
Telomeric ChIP was conducted as described previously. Briefly, nuclei
from synchronized cells were crosslinked with formaldehyde. Nuclei were
lysed and sonicated, and the chromatin was clarified by centrifugation. An
aliquot of chromatin was used as an “input” sample for normalization of
recovered chromatin, and the remaining chromatin was incubated with antiMyc antibody (Myc-tag [9B11] mouse monoclonal antibody, #2276S; Cell
Signaling Technology). Samples were immunoprecipitated and DNA
purified using Qiagen PCR Purification Kit and applied to Hybond XL
membrane (GE Healthcare Life Sciences). The membrane was hybridized
overnight with a (CCCTAA)3 telomeric DNA oligonucleotide probe end
labeled with [g-32P]ATP. The amount of DNA recovered was visualized on
a Typhoon TRIO Imager (GE Healthcare Life Sciences) and quantified with
ImageQuant (GE Healthcare Life Sciences). Standard curves based on the
signal of the input samples were used to normalize the amount of telomeric
DNA recovered.
Cell lysis and western blot
Harvested synchronized cells were lysed using 43 lithium dodecyl sulfate
buffer (106 mM Tris-HCl, 141 mM Tris-base, 2% lithium dodecyl sulfate,
40% glycerol, 0.075% SERVA Blue G50, and 0.025% phenol red) containing
2% b-mercaptoethanol (Sigma-Aldrich) and Benzonase nuclease (0.5 U/mL;
Novagen). The cells extracts were incubated at 68°C for 10 min prior to
loading 70 000 cell equivalents onto precast NuPAGE 4% to 12% Bis-Tris
gels (Life Technologies) and electrophoresis with MES buffer (Life Technologies) for 50 minutes at 200 V. The proteins were transferred onto
Hybond ECL membrane (GE Healthcare Life Sciences) at 100 V for 60
minutes at 4°C with transfer buffer (25 mM Tris-base and 192 mM Glycine).
The membrane was washed and blocked with 5% skim milk in TBS buffer
(15 mM Tris-HCl [Sigma], 4.6 mM Tris-base, and 137 mM NaCl) and 0.1%
Tween-20 (TBST) for 1 hour at room temperature. TPP1 antibody (a-ACD
MaxPAB, H00065057-B01P, Abnova) was added to 5% skim milk in TBST
(1:2500) and incubated with the membrane overnight at 4°C. The membrane was washed 5 times for 5 minutes with TBST and incubated with
secondary antibody (Dako goat anti-mouse; 1:5000) conjugated to horseradish
peroxidase in 5% skim milk-TBST for 1 hour and then washed as before.
Horseradish peroxidase signal was detected using Supersignal West Pico
Chemiluminescent Substrate (#34080; Pierce) and visualized with a FujiFilm
LAS 4000 Imager.
Results
Disease phenotype of proband and family
The 18-year-old proband (Figure 1A V:1), her mother, and her maternal grandmother presented with BMF of varying severity, and
their decreasing ages of presentation in successive generations suggested disease anticipation (Table 1). The proband is the only child of
nonconsanguineous white parents. She presented at 8 years of age
with increasing pancytopenia, having been previously healthy with
normal growth and development. Pancytopenia was associated with
marked elevation in fetal hemoglobin (31%) and macrocytosis
(Table 1). The bone marrow was markedly hypocellular without
morphological dysplasia or cytogenetic abnormality, consistent with
aplastic anemia. There was no associated facial dysmorphism or
skeletal, cardiac, or urogenital tract anomalies. There were also no
mucocutaneous manifestations, and none developed over the next
10 years. The screen for Fanconi anemia was negative, with no increase
in chromosomal breakages on exposure to mitomycin C and
diepoxybutane. Immunosuppressive therapy (4 courses of antithymocyte globulin and corticosteroids) was trialed without response.
She responded to androgen therapy (oxymetholone) between the
ages of 9 and 14 years, maintaining hemoglobin between 100 and
120 g/L, neutrophils 1.0 to 2.0 3 109/L, and platelets 35 to 50 3 109/L.
However, androgen therapy was complicated by virilization, leading to
variable compliance, and it was ceased at 14 years of age allowing
normal pubertal progression; pancytopenia promptly recurred, and
the proband became transfusion dependent for erythrocytes and
platelets.
Although the proband’s mother (Figure 1A, IV:2) was diagnosed
with myelodysplasia in her early 20s following investigations for
thrombocytopenia, she most likely has mild BMF (Table 1). She has
remained asymptomatic without disease progression or malignant
transformation into her mid-40s. She has 1 unaffected healthy male
sibling.
The proband’s maternal grandmother (Figure 1A, III:6) was
investigated for mild macrocytic anemia with variable thrombocytopenia associated with hypocellular bone marrow at 47 years
of age. A diagnosis of possible aplastic anemia/myelodysplasia
was made, and she was monitored without therapy as cytopenia
was mild and there were minimal symptoms. Earlier blood counts
are not available, but parameters at 55 years are shown in Table 1.
She developed carcinoma of the tongue at 59 years of age despite
having been a lifelong nonsmoker and teetotaler; this was treated
with surgery and local radiotherapy only. Over the ensuing
12 months, there was increasing pancytopenia requiring erythrocyte transfusions. Her death at 61 years was attributed to aplastic
anemia. This patient’s paternal grandfather died of leukemia in
his 30s; there were, however, no clinical or hematologic data
available on this individual.
The proband was screened for mutations in DC genes with
autosomal-dominant inheritance at the age of 14 years, and no
mutations were detected in TERC, TERT, and TINF2. She was later
retested using WES (performed by the Dyskeratosis Congenita
Registry, London), and no significant variants in any of the 9 known
DC genes were detected. A blood sample referred in 2012 to Repeat
Diagnostics (Vancouver, BC, Canada) for telomere-length testing
showed an average telomere length of less than the 1st percentile
(lymphocytes and granulocytes) by Flow-FISH, supporting the
diagnosis of DC or a related disorder of telomere length in the
proband (J.T. and Repeat Diagnostics, unpublished data). The
proband and her mother were retested when an in-house Flow-FISH
assay was available; mononuclear telomere length was less than the
1st percentile for the proband and around the 5th percentile for her
mother (Figure 1B). qPCR analysis of peripheral blood telomere
length63 confirmed these findings and revealed short telomeres in the
affected grandmother, but not in the unaffected father or grandfather
(Figure 1C). Thus, short telomeres segregate with disease phenotype in
this family.
Genomic variants identified by WES and bioinformatics
We performed WES on the proband (V:1), her mother (IV:2), and
her maternal grandparents (III:5 and III:6) (Figure 1A), with each
individual’s exome covered at least 62 times (supplemental
Table 1). On average, ;44 000 SNVs and ;7000 small indels
were called for each exome by pipeline 1 (see “Methods”),
whereas ;87 000 SNVs and ;6000 indels were called by pipeline
2. After filtering, over 16 000 SNVs and ;600 indels were retained
as qualified for further analysis in pipeline 1 and over 17 000 SNVs
and ;700 indels were left in pipeline 2. We followed an autosomaldominant inheritance, and each pipeline generated a list of candidate
variants (supplemental Table 2). We noticed the overlapping variant of a 3-bp deletion in gene ACD, which encodes the human
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Expression of the mutant allele in fibroblasts derived from the
proband was confirmed by RT-PCR (Figure 2B). The primers used
for this PCR generate a product of 400 bp spanning the mutated
region, and deletion of the codon encoding K170 removes an XmnI
restriction site in the center of the PCR product, enabling us to
distinguish mutant from WT mRNA. A cell line with WT ACD/TPP1
produced a band of 400 bp that was cleaved in two with Xmn1,
whereas approximately half of the RT-PCR product from the
patient’s cells was not digested with this enzyme (Figure 2B). This
demonstrates that the WT and DK170 TPP1 alleles are expressed
equally at the mRNA level in cells from the patient.
TPP1 mutant DK170 localizes to telomeres in human cells
Lysine 170 is localized on the surface of the TPP1 protein known as
the TEL patch, between 2 amino acids shown to be vital for binding
to telomerase, recruitment of telomerase to telomeres, and telomere
length maintenance.44 Substitution of K170 with alanine resulted
in a modest reduction in the ability of telomerase to colocalize
with TPP1.45 To determine whether deletion of this residue could be
Figure 2. Genomic sequencing and mRNA expression of the DK170 mutant
allele. (A) Sanger sequencing validates segregation with disease of a 3-bp deletion in the ACD gene, encoding the protein TPP1. The deletion (c.499_501del;
p.Lys170del) was detected in the proband, mother, and maternal grandmother.
The deletion was not present in the father and maternal grandfather. (B) RTPCR of a 400-bp region across the DK170 region of TPP1 (left). RNA was
isolated from fibroblasts of the proband (DK170) and the cell line HCT116 as
a positive control for WT TPP1 expression (WT). Plasmids encoding WT or
DK170 TPP1 sequence (pWT and pDK170) were amplified as positive controls.
XmnI digestion of the PCR products from the left panel (right). WT sequence is
digested into 2 pieces of 212 and 188 bp, whereas the DK170 sequence
remains uncut.
TIN2- and Pot1-interacting protein (TPP1); this variant could only be
found in the 3 affected samples and is absent from any public
databases (supplemental Table 2). Sanger sequencing confirmed the
heterozygous 3-bp deletion (c.499_501del; p.Lys170del) in the
genome of the affected individuals, including the proband, her
mother, and her maternal grandmother (Figure 2A). The deletion is
predicted to result in an in-frame deletion of amino acid lysine 170
(K170) of the TPP1 protein. The proband’s unaffected father and
maternal grandfather were homozygous WT, demonstrating that the
mutation segregates perfectly with both telomere length and disease
phenotype.
Figure 3. TPP1 with DK170 mutation localizes to telomeres. (A) Western blot of
WT or DK170 TPP1, overexpressed in 293T cells in the presence of siRNA against
endogenous TPP1. Vector: empty plasmid transfection only. (B) Levels of endogenous TPP1 mRNA, measured by qRT-PCR using primers in the 59 UTR of the ACD/TPP1
gene. 293T cells were transfected with plasmids encoding WT or DK170 TPP1,
followed by transfection with siRNA targeting the 59 UTR of ACD/TPP1 or control
siRNA. Vector: empty plasmid transfection only. (C) Telomere ChIP of myc-tagged,
siRNA-resistant TPP1, expressed in 293T cells in the presence of siRNA to reduce
endogenous TPP1 levels. Shown are 5%, 10%, and 20% of the input chromatin used
in each immunoprecipitation (top). Immunoprecipitation with an anti-myc antibody, in
duplicate (bottom). Blot was probed for telomeric DNA. (D) Quantitation of the amount
of telomeric DNA recovered as a percentage of input, in three independent transfections and ChIP experiments (data shown as mean 6 standard error of the mean).
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Figure 4. TPP1 with DK170 mutation does not recruit telomerase to telomeres. (A) FISH using probes against telomeres and hTR in 293T cells treated with siRNA
against endogenous TPP1 and expressing either WT or mutant TPP1 or a vector-only control. (B) Quantitation of the number of hTR/telomere colocalizations in 3 independent
transfections and FISH experiments (100 cells [300-1000 colocalizations] counted for each sample in each experiment; data shown as mean 6 standard error of the mean;
control had 7.8 6 1.2 colocalizations; *P 5 .024).
causing short telomeres and disease in the family of interest, we
analyzed the functional impact of deletion of K170 in immortal
human cells in culture.
Nearby mutations in TPP1 are known to be “separation of function”
mutations, because they disrupt telomerase interactions without
affecting the ability of TPP1 to localize to and protect telomeres.44
To test the hypothesis that DK170 TPP1 could localize to telomeres
as efficiently as WT TPP1, we transiently transfected plasmids encoding Myc-tagged versions of both proteins into the human
embryonic kidney cell line 293T, in the presence of siRNA directed
against the 59 UTR of TPP1 to reduce competition from endogenous
TPP1. Western blot analysis of cells harvested 3 days later confirmed
equal expression and stability of WT and DK170 TPP1 (Figure 3A),
and quantitative RT-PCR confirmed knockdown of the endogenous
gene (Figure 3B). We then carried out ChIP using an anti-Myc
antibody, measuring the amount of recovered telomeric DNA as a
measure of TPP1 presence at the telomere (Figure 3C). When normalized against the starting amount of telomeric DNA (“input”), the
amounts recovered with WT and DK170 TPP1 were equal (Figure 3D).
This indicates that WT and DK170 TPP1 load onto telomeres at the
same density.
TPP1 mutant DK170 fails to recruit telomerase to telomeres
To test the hypothesis that DK170 TPP1 is unable to recruit
telomerase to telomeres despite its telomeric localization, we analyzed
the presence of telomerase at the telomere using FISH against hTR
and telomeres simultaneously.15 This was performed in immortal
293T cells expressing endogenous levels of telomerase, because
we have found that telomerase overexpression can perturb some
telomerase-recruitment pathways.15 The transfected 293T cells were
synchronized in S phase using thymidine and aphidicolin prior to FISH
(supplemental Figure 1). As expected, knockdown of endogenous
TPP1 reduced the level of telomerase recruitment to telomeres,43 and
this was rescued by expression of siRNA-resistant WT TPP1,
confirming that the observed recruitment defect was due to lack of
TPP1 (Figure 4). Importantly, the levels of endogenous TPP1 were
reduced by ;50% by our siRNA (Figure 3B), demonstrating that a
telomerase-recruitment defect is observable even in the presence of
remaining WT protein, as would be the case in the heterozygous patients.
As predicted, expression of siRNA-resistant DK170 TPP1 was completely unable to rescue telomerase recruitment to telomeres (Figure 4).
Discussion
The role of the TEL patch of TPP1 in mediating the interaction of
this protein with telomerase is well established.44,45 This interaction is necessary for the recruitment of telomerase to telomeres
but dispensable for the function of TPP1 in protecting telomeres.44
Our data confirm that this surface of TPP1 is vital for telomerase
recruitment and provide evidence that interference with this pathway
through TPP1 mutation can lead to telomere shortening in humans.
This conclusion is compellingly supported by the finding that mutations
in hTERT linked to the short telomere disease idiopathic pulmonary
fibrosis have been shown to abrogate interaction with TPP1.45
These data collectively support the hypothesis that a defect in
TPP1 renders telomerase unable to maintain telomeres during development and hematopoiesis in the affected family members, leading
to short telomeres and progressive BMF and eventual aplastic
anemia. The increasing severity and younger age of onset of
symptoms over several generations of this family is consistent with
the phenomenon of genetic anticipation, leading to more severe
disease in each successive generation.65 Curiously, however, telomere
lengths did not continue to shorten over the 3 generations of affected
females, relative to expected telomere lengths for age (Figure 1C).
This is consistent with a previous observation of shorter telomeres in
the offspring of fathers, but not mothers, with hTERT mutations,66
suggesting an intriguing paternal influence on telomere length.
In addition to hematologic disorders, the clinical history of this
family includes oral carcinoma and leukemia. This is supportive
of a link between telomere attrition and increased risk of cancer67
and is consistent with previous reports of leukemia and other
malignancies, including oral carcinoma, in families with aplastic
anemia due to mutations in telomerase components.68,69 Families
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 30 OCTOBER 2014 x VOLUME 124, NUMBER 18
ACD/TPP1 MUTATION IN INHERITED BONE MARROW FAILURE
with inherited idiopathic pulmonary fibrosis, short telomeres, and
hTERT mutations also frequently contain members with leukemia and other malignancies.66,70,71 It is likely that inherited short
telomeres can lead to chromosome fusions and genetic instability,
which predisposes to malignancy.
This study represents the first identification of ACD/TPP1 as a
disease-causing gene in humans, bringing the number of genes
linked to BMF disorders to 10. Seven of these genes encode
proteins or RNA that are either part of the telomerase complex (hTR,
hTERT, dyskerin, Nop10, and Nhp2) or involved in telomerase
transport to the telomere (TCAB1 and TPP1), and the remaining 3
proteins (TIN2, CTC1, and RTel1) are known to be involved in
telomere protection. This strongly supports the causative role of
telomere shortening or deprotection in the etiology of bone marrow
disorders in humans.
Academic and Training Affairs at King Faisal Specialist Hospital
and Research Center and the Ministry of Higher Education (Riyadh,
Saudi Arabia), project support from the Kids Cancer Alliance (NSW),
a National Health and Medical Research Council postgraduate medical/
dental scholarship (P.M.B.), program grant PG11-08 from Cancer
Council NSW (R.R.), and a Cancer Institute NSW Career Development and Support Fellowship and project grant RG10-02 from
Cancer Council NSW (T.M.B).
Acknowledgments
The authors are grateful to the patient and her family for their willing
involvement in this study. The authors thank Dr Monica Bessler and
her laboratory for their generous assistance in setting up the telomere
length Flow-FISH assay and Prof Inderjeet Dokal and Dr Tom
Vulliamy for sequencing of known DC-associated genes.
This work was supported by an Institutional Development
Award to the Center for Applied Genomics from The Children’s
Hospital of Philadelphia, an Australian Awards Scholarship (M.K.),
a Cancer Institute New South Wales (NSW) Career Development
Fellowship (H.A.P.), a scholarship to A.A.-O. provided by the
2773
Authorship
Contribution: Y.G., J.L., B.K., Y.C., L.T., X.X., and H.H.
designed and performed WES and bioinformatic analysis; M.K.
and H.A.P. designed, performed, and analyzed experiments; J.T.
collected clinical data and samples; T.K. and P.M.B. performed
patient telomere-length analysis; A.A.-O. performed Sanger
sequencing; H.A.P., R.R.R., J.C., and T.M.B. planned the study,
organized the research, and contributed to the interpretation of
results; T.M.B. wrote the manuscript; and all of the authors
edited the manuscript.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: Tracy M. Bryan, Children’s Medical Research
Institute, 214 Hawkesbury Rd, Westmead, NSW 2145, Australia;
e-mail: [email protected]; and Hakon Hakonarson, The Children’s Hospital of Philadelphia, 3615 Civic Center Blvd, Philadelphia,
PA 19104; e-mail: [email protected].
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2014 124: 2767-2774
doi:10.1182/blood-2014-08-596445 originally published
online September 9, 2014
Inherited bone marrow failure associated with germline mutation of ACD
, the gene encoding telomere protein TPP1
Yiran Guo, Melissa Kartawinata, Jiankang Li, Hilda A. Pickett, Juliana Teo, Tatjana Kilo, Pasquale M.
Barbaro, Brendan Keating, Yulan Chen, Lifeng Tian, Ahmad Al-Odaib, Roger R. Reddel, John
Christodoulou, Xun Xu, Hakon Hakonarson and Tracy M. Bryan
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