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Pathogenic TERT promoter variants in telomere diseases

Pathogenic TERT promoter variants in telomere diseases ARTICLE © American College of Medical Genetics and Genomics 1,2 2 2 Fernanda Gutierrez-Rodrigues, PhD , Flávia S. Donaires, PhD , André Pinto, BSc , 1 1 2 2 Alana Vicente, MD , Laura W. Dillon, PhD , Diego V. Clé, MD, PhD , Barbara A. Santana, PhD , 1 1 1 Mehdi Pirooznia, MD, PhD , Maria del Pilar F. Ibanez, MSc , Danielle M. Townsley, MD , 1 1 1 Sachiko Kajigaya, PhD , Christopher S. Hourigan, MD, DPhil , James N. Cooper, MD , 2 1 Rodrigo T. Calado, MD, PhD and Neal S. Young, MD Purpose: The acquisition of pathogenic variants in the TERT treatment. However, it was specific for patients with telomeropa- promoter (TERTp) region is a mechanism of tumorigenesis. In thies, more frequently co-occurring with TERT germline variants nonmalignant diseases, TERTp variants have been reported only in and associated with aging. patients with idiopathic pulmonary fibrosis (IPF) due to germline Conclusion: We extend the spectrum of nonmalignant diseases variants in telomere biology genes. associated with pathogenic TERTp variants to marrow failure and Methods: We screened patients with a broad spectrum of liver disease due to inherited telomerase deficiency. Specificity of telomeropathies (n = 136), their relatives (n = 52), and controls pathogenic TERTp variants for telomerase dysfunction may help to (n = 195) for TERTp variants using a customized massively parallel assess the pathogenicity of unclear constitutional variants in the amplicon-based sequencing assay. telomere diseases. Results: Pathogenic −124 and −146 TERTp variants were Genetics in Medicine (2019) 21:1594–1602; https://doi.org/10.1038/s41436- identified in nine (7%) unrelated patients diagnosed with IPF 018-0385-x (28%) or moderate aplastic anemia (4.6%); five of them also presented cirrhosis. Five (10%) relatives were also found with these variants, all harboring a pathogenic germline variant in telomere Keywords: somatic TERT promoter variants; telomere diseases; biology genes. TERTp clone selection did not associate with bone marrow failure peripheral blood counts, telomere length, and response to danazol INTRODUCTION functionally compensate the deleterious impact of disease- The TERT gene encodes the catalytic component of the causing germline TERT variants by increasing telomerase telomerase complex required to elongate telomeres in stem activity and cell proliferation. 1,2 and progenitor cells. TERT is epigenetically silenced in Germline variants in telomere-related genes are etiologic in normal somatic and nonproliferative cells, but aberrantly a broader spectrum of diseases collectively named telomere 3–6 14 expressed in many human cancers. Acquisition of diseases or telomeropathies, including IPF but also affecting pathogenic TERT promoter (TERTp) variants located other organs, such as the bone marrow (aplastic anemia [AA] upstream of the translation initiation site at positions and dyskeratosis congenita [DC]) and the liver (cirrhosis and −124C>T (chr5:1,295,228), −146C>T (chr5:1,295,250), and nonalcoholic steatohepatitis). We investigated the distribution −57A>C (chr5:1,295,161) has been described as a mechanism of somatic pathogenic TERTp variants in a large cohort of 3,6–9 of tumorigenesis in cancer cells. These variants increase patients with a spectrum of telomere diseases using a TERT expression and promote cell proliferation through customized low-cost massively parallel sequencing assay recruitment of the transcription factor GABPA to the mutant optimized for identification and quantification of hemato- 10–12 allele. Somatic TERTp variant clones recently have been poietic clones bearing the pathogenic −124 and −146 TERTp found in a few patients with idiopathic pulmonary fibrosis variants. We further assessed the association of these TERTp (IPF) caused by pathogenic germline variants in telomere variants with telomere length (TL) and peripheral blood biology genes. These pathogenic TERTp variants appear to counts of patients. 1 2 Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, MD, USA; Department of Internal Medicine, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto, SP, Brazil. Correspondence: Rodrigo T. Calado (rtcalado@usp.br) Last authors equally contributed: Rodrigo T. Calado and Neal S. Young. Submitted 4 September 2018; accepted: 16 November 2018 Published online: 7 December 2018 1594 Volume 21 Number 7 July 2019 GENETICS in MEDICINE | | | GUTIERREZ-RODRIGUES et al ARTICLE MATERIALS AND METHODS regardless of symptoms or evidence of disease. Enrolled Cohort individuals were seen in the Hematology Branch clinic of In this retrospective study, we screened blood leukocytes from National Heart, Lung, and Blood Institute (NHLBI) or the 136 patients with telomeropathies (median age, 29 years; bone marrow failure clinic in the Hospital das Clínicas, range, 1–76), 52 relatives (median age, 40 years; range, 8–72), Ribeirão Preto School of Medicine, University of São Paulo and 195 controls for the pathogenic −124 and −146 TERTp (USP) between 2004 and 2017 (Supplementary Tables S2–S3). variants (Table 1). Patients were primarily diagnosed with DC TL was measured by Southern blot (SB), quantitative (n = 21), AA (n = 86), IPF with or without another polymerase chain reaction (qPCR), or flow fluorescent in situ telomeropathy-related phenotypes (n = 18), or other pheno- hybridization (flow-FISH) according to protocols previously types (n = 11; Supplementary Table S1). Clinical diagnosis of described. TL measurements by qPCR were confirmed by 2,15,16 DC and AA was defined according to previous criteria. SB or flow-FISH at the time of this study. Patients with Briefly, DC patients had at least two of three manifestations of acquired immune AA (n = 70), IPF without evidence of the clinical triad (dystrophic nails, patchy skin hyperpigmen- inherited disease and a telomere-related germline pathogenic tation, and oral leukoplakia) and TL below the 1st percentile variant (n = 12), other inherited bone marrow failure for age-matched controls, whereas AA patients presented with syndromes (IBMFS; Diamond–Blackfan anemia, n = 4; pancytopenia and hypocellular bone marrow without any chronic neutropenia, n = 3), and acute myeloid leukemia evidence for myelodysplasia, myelofibrosis, or leukemia. (AML; n = 106) were studied as controls (Supplementary Inclusion criteria were based on molecular diagnosis: TL Table S2-S3). below the 10th percentile of age-matched controls or a Approval was obtained from the Institutional Review Board germline variant in a telomere biology gene classified as of NHLBI and from the Comitê de Ética em Pesquisa do pathogenic, likely pathogenic, or variant of uncertain Hospital das Clínicas de Ribeirão Preto. Samples were collected significance (VUS) by the Sherloc criteria, a framework that according to the Declaration of Helsinki and written consent incorporates the American College of Medical Genetics and was obtained from all participants or their legal guardians. 17,18 Genomics (ACMG) criteria (Supplementary Tables S2– S3). The Sherloc criteria refined the ACMG guidelines to Massively parallel amplicon-based sequencing assay for comprehensively assess variants’ pathogenicity based on both detection of TERTp variants clinical and functional evidence, attributing points to score We customized a massively parallel amplicon-based sequen- each variant for pathogenicity. The point score thresholds for cing assay that targeted the TERTp region in which pathogenic and likely pathogenic variants are four (4P) and pathogenic −124 and −146 TERTp variants were located five points (5P), respectively (Supplementary Table S4). (Supplementary Figure S1). Although primers were not Patients’ relatives were only studied if they harbored the optimized to cover the −57A>C TERTp position, we also same germline variant as the proband or had short telomeres, detected this variant in some of our samples. TERTp variant Table 1 The cohort screened for pathogenic TERT promoter variants by massively parallel amplicon-based sequencing assay Telomeropathies Family members Control group DC AA IPF (with or Other (n = 52) AML Acquired AA, (n = 21) (n = 86) without AA, phenotypes (n = 106) IPF or other MDS, and (n = 11) IBMFS (n = 89) cirrhosis) (n = 18) Median age (range) 13 (1–59) 28 (5–73) 54 (27–76) 27 (3–69) 40 (8–72) 50 (2–86) 29 (1–88) Females/males 4/9 40/46 7/11 3/8 30/22 51/55 44/45 Patients with somatic TERTp (%) 0 4 (4.6%) 5 (28%) 0 5 (9.6%) 0 0 Patients with a germline TERT 337 14 5 34 00 variant Patients with a somatic TERTp and03 5 0 5 0 0 a germline TERT variant Patients with somatic TERTp 0 8.1 36 0 14.7 0 0 variants from the total of patients with TERT germline variants (%) TERTp pathogenic TERT. In this study, we screened 136 patients with telomeropathies that presented dyskeratosis congenita (DC, n = 21), aplastic anemia (AA, n = 86), idiopathic pulmonary fibrosis (IPF, n = 18), and other phenotypes that included myelodysplastic syndrome (MDS) or hypoplastic MDS (HypoMDS, n = 7), isolated thrombocytopenia (n = 3), and thrombocythemia (n = 1). Control group was composed of patients with acquired AA (n = 70; median age = 28 years), IPF (n = 12; median age = 62 years), other inherited bone marrow failure syndromes (IBMFS; Diamond–Blackfan anemia, n = 4; chronic neutropenia, n = 3), and acute myeloid leukemia (AML, n = 106; Supplementary Tables S2–S3). GENETICS in MEDICINE Volume 21 Number 7 July 2019 1595 | | | 1234567890():,; ARTICLE GUTIERREZ-RODRIGUES et al screening was performed in patients, relatives, and controls ddPCR was performed according to a protocol previously using peripheral blood leukocytes collected at the time of first optimized with minor modifications to transfer the assay to clinical evaluation. Whenever possible, testing was also the RainDance platform (RainDance). To evaluate the performed in granulocytes separated by gradient centrifuga- linearity and limit of detection of the ddPCR assay, a variant tion in parallel with the respective leukocyte samples. control sequence (CCCCTTCCGG) was serially diluted into Library preparation consisted of two rounds of PCR to 300 ng of sheared normal human genomic DNA to have an amplify the TERTp region. A first PCR was designed to expected variant target copy number. A mean frequency amplify the targeted region and a second PCR for addition abundance of the variant template was plotted versus the of Illumina adapters (Illumina, San Diego, CA, USA) into target copies input to generate a standard curve. Linearity of fragments. In the first PCR, we used a set of four different the assay was high (R = 0.99; Supplementary Figure S3a) and forward and reverse primers (total of eight primers) that lower limit of detection was 0.17%. The highest mean were pooled in equimolar amounts to amplify the TERTp frequency abundance obtained when genomic DNA from region (Supplementary Table S5). PCR products were then negative controls were used was 0.5%, and this value was used subjected to a second PCR round for the addition of the full as the negative cut-off. The customized sequencing assay i5/i7 Illumina adapter/index sequences into the DNA accurately detected pathogenic TERTp variants confirmed by fragments. ddPCR but did not identify false positives. The correlation Up to 96 libraries were pooled in equimolar amounts and between sequencing and ddPCR in quantification of TERTp pair-end sequenced in 300 cycles on the MiSeq platform variant clones was high (R = 0.97; Supplementary Fig- (Illumina). Median coverage depth on targets was 104×. Reads ure S3b). Agreement between techniques was evaluated by were aligned to the human genome reference (hg19) using Bland–Altman analysis, a statistical tool to compare clinical 20 26 Burrows–Wheeler Aligner (BWA) and data quality was assays. Bland–Altman analysis evidenced a good agreement assessed using FastQC. Sequences were trimmed to remove between these two methods (Supplementary Figure S3c), as the adapters as well as low-quality bases (-q 15 –minimum- the mean difference of VAF measurements was 0.95 and no length 35). Variants were called using VarDict and the measurements exceed the 95% confidence interval (CI) limits following filtering criteria: -f 0.005 -v -c 1 -S 2 -E 3 -g 4 -th 8 of agreement. The standard deviation (SD) between assays 21 22 (ref. ). For comparison, we also used SAMTools and was 2.1 and 95% limits of agreement ranged from 5.15 to Genome Analysis Toolkit (GATK) to call the variants. −3.25. Detailed protocols and primer sequences are available While VarDict called all TERTp variants that were further in Online Supplementary Data. validated by droplet digital PCR (ddPCR; RainDance Technologies, Billerica, MA, USA), GATK and SAMTools RESULTS only called variants with allele frequency >20% and >8%, Pathogenic TERTp variants associated with different respectively. Variants were annotated using Annovar and phenotypes from the spectrum of telomeropathies and variant allele frequency (VAF) was calculated by a ratio of patients’ ages variant minor allele counts and total reads. Of 136 cases, nine unrelated patients (7%; median age, 39 years; range, 24–65; Fig. 1) were found with TERTp variants. Droplet digital PCR Nine patients had the pathogenic −124C>T variant, including In samples that were available, the −124C>T and −146C>T two patients who also had the −57A>C TERTp variant TERTp variants identified by sequencing were validated by (NIH37 and NIH93; Table 2). Patients were clinically ddPCR (Supplementary Figure S2a), except for a single case diagnosed with IPF (5/18; 28%) and moderate AA (MAA; that was confirmed by Sanger sequencing (Supplementary 4/86; 4.6%) (Table 1 and Fig. 1). Of note, eight of them also Figure S2b). TERTp variant clones were also tracked over time presented other phenotypes related to telomeropathies using this technique. In patient NIH46, the TERTp variant (Table 3); co-occurrence with cirrhosis and MAA was was quantified in the following blood cells’ subpopulations by frequent (Tables 2–3). Five relatives (10%; median age, 63 ddPCR: leukocytes after whole-blood ammonium chloride years; range, 17–72) had the −124C>T (n = 4) or the potassium (ACK) lysis, granulocytes separated by gradient −146C>T (n = 1) TERTp variants: three were asymptomatic centrifugation using Ficoll–Hypaque, peripheral mononuclear and two were diagnosed with MAA or a DC-like phenotype. cells separated by gradient centrifugation using (Table 2). Within the same family, a somatic TERTp variant + − + Ficoll–Hypaque, CD14 CD16 monocytes, CD3 T cells, was not observed in more than one subject (Supplementary and mononuclear fraction depleted for CD3 and Figure S4), suggesting that acquisition of these variants was + - + - CD14 CD16 . The CD14 CD16 monocytes were isolated not due to a genetic susceptibility caused by the telomere- from mononuclear cells using immunomagnetic negative related germline pathogenic variant identified in the family. selection (the EasySep™ Human Monocyte Isolation kit, The frequency of pathogenic TERTp variants was much Stemcell Technologies, Cambridge, MA, USA) and CD3 higher in IPF patients compared with AA cases (28% vs. 4.6%; T cells were isolated from mononuclear cells by immuno- Fisher’s exact test, P = 0.007; Table 1). Because some patients magnetic positive selection (the EasySep™ Human CD3 presented different phenotypes from the spectrum of Positive Selection kit II, Stemcell Technologies). telomeropathies, we then investigated whether pathogenic 1596 Volume 21 Number 7 July 2019 GENETICS in MEDICINE | | | GUTIERREZ-RODRIGUES et al ARTICLE TERT germline variant TERC germline variant MAA IPF Asymptomatic IPF MAA Relatives 08 50 100 Patients and relatives screened (%) Fig. 1 Clinical association of somatic pathogenic TERT promoter (TERTp) variants and telomere diseases. Frequency of pathogenic TERTp variants in patients and relatives screened in the study. The size of the TERTp clone is represented by the variant allele frequency (VAF) and shown for each individual screened according to their primary diagnosis: idiopathic pulmonary fibrosis (IPF), moderate aplastic anemia (MAA), and relatives. Of 188 patients and relatives, 14 had the −124 or −146 TERTp variants (8%). An additional graph shows in detail the pathogenic TERTp clone size, initial diagnosis, and the telomere biology gene in which a germline variant was identified from the patients/relatives with pathogenic TERTp variants. Four relatives were asymptomatic. TERTp variants were more frequent in the setting of ddPCR. Overall, TERTp clones were more frequent in pulmonary disease compared with marrow failure or liver individuals with germline TERT variants (12/14 cases); only disease. No difference in frequency of disease phenotypes (IPF two patients harbored a germline variant in TERC. The vs. marrow failure or liver disease) was observed among germline variants identified in telomere biology genes were patients with a pathogenic TERTp variant (36% vs. 50% or classified as pathogenic or likely pathogenic (n = 8), and VUS 42%, respectively; χ test, P> 0.05; Table 3), suggesting that (n = 6; Table 2) and, except for the TERT R696C found in these variants occurred in all these clinical presentations. USP26, they were heterozygous. All variants classified as VUS Pathogenic TERTp variant clones positively correlated with had some evidence for being pathogenic (all had a Sherloc age, as they were only present in individuals older than 18 score of 3.5P) but insufficient to meet Sherloc criteria for years old and more frequent in those 60 to 80 years old pathogenicity; all were predicted as deleterious in silico, (Fig. 2a). Six of 86 individuals ranging in age from 21 to 40 absent in control populations, and associated with a family (7%), 3 of 44 individuals ranging in age from 41 to 60 years history and phenotype of telomere diseases (Supplementary (6.8%), and 5 of 18 patients older than 61 years (27.8%) had Table S4). pathogenic TERTp variants. The median age of patients with Pathogenic TERTp variant clone sizes varied from 1.2% to TERTp variants and a primary diagnosis of IPF and MAA was 50% in total leukocytes and clones were found at higher allele 57 and 27 years, respectively. frequencies in the granulocytic fractions in four patients Pathogenic TERTp variants were found in telomeropathy (Table 2 and Fig. 2b). In NIH93, the −124C>T TERTp was patients who had a germline variant in telomere biology genes identified in total leukocytes and granulocytes by both but not in controls (median age, 29 years; range, 1–88) or in sequencing and ddPCR at VAF as low as 6%. However, in patients with very short telomeres without a germline variant NIH61, pathogenic TERTp clones were only detected in in telomere biology genes (median age, 25 years; range, 5–63; granulocytes by sequencing due to very low VAF in total Table 1). We also confirmed the absence of pathogenic leukocytes. Results were similar when peripheral blood cell TERTp variants in the granulocytic fractions from controls in subpopulations from NIH46 were separated by magnetic which materials were available (Supplementary Table S2–S3). selection for screening of the −124C>T TERTp variant by ddPCR. The −124C>T TERTp variant was found enriched in Pathogenic TERTp variants more frequently co-occurred the granulocyte fraction and mononuclear cells depleted for + + with germline TERT variants in myeloid cells CD3 and CD14 cells (Fig. 2b). Clonal dominance was not The customized sequencing assay detected pathogenic TERTp observed in most individuals with pathogenic TERTp variants; clones at VAF as low as 1.2%, which was confirmed by nine had pathogenic TERTp clones at VAF lower than 10%. GENETICS in MEDICINE Volume 21 Number 7 July 2019 1597 | | | NIH98 NIH53 NIH93 NIH61 NIH46 USP79 NIH34 NIH31 NIH20 NIH37 USP26 USP41 NIH07 NIH95 Pathogenic TERTp VAF (%) Pathogenic TERTp VAF (%) ARTICLE GUTIERREZ-RODRIGUES et al 1598 Volume 21 Number 7 July 2019 GENETICS in MEDICINE | | | Table 2 Mutational and clinical profile of patients with telomeropathies and somatic pathogenic TERT promoter variants Age TL Clinical Germline Sherloc Somatic TERTp Leukocyte Bone marrow Hb (g/ MCV Plt ANC WBC 3 3 3 diagnosis variant germline variant VAF (%) cellularity dL) (fL) (×10 / (×10 /µL) (×10 /µL) variant µL) classification Patients USP79 46 <1st IPF/cirrhosis TERT, T937A VUS −124C>T 50 NA 16.9 107 117 2 3.5 c.2809A>G NIH20 61 <1st IPF/cirrhosis TERT, V170L P − 124C>T 6.6 40% 13.5 103 117 3.9 7 c.508G>A NIH31 57 <10th IPF/MAA/ TERT, A1009G VUS −124C>T 50 30–40% 9 99 55 1.37 2.3 cirrhosis c.3026C>A NIH37 65 <1st IPF TERT, P59S VUS − 124C>T 5 40–50% 13 111 104 3.0 5.4 c.175C>T − 57C>T 4.5 NIH43 24 <1st MAA TERT, V1025F P −124C>T 3 <10% 8.7 89 21 0.86 1.7 c.3073G>T NIH46 39 <1st IPF/MAA TERC, r.433G>C VUS −124C>T 21 <5% 8.9 107 65 1.32 2.7 NIH53 30 <1st MAA/cirrhosis TERC, r.107G>C P −124C>T 4.4 <5% 9.2 92 31 0.72 2.5 b d NIH93 38 <1st MAA/cirrhosis TERT, A130V VUS −124C>T 5/6 <20% 11.4 101 27 0.32 0.6 c.389C>T −57C>T 6/5 NIH98 25 Normal MAA TERT, R537C VUS −124C>T 20 10% 10.6 112 83 1.0 3.3 c.1609C>T Relatives USP26 18 <1st Asymptomatic TERT, R696C P −124C>T 1.2 NA 10.4 86 87 2.0 4.0 but with DC-like c.2086C>T phenotype USP41 63 Normal Asymptomatic TERT, R865H P −124C>T 1.8 NA 15.4 105 163 2.8 6.2 c.2594G>A NIH07 71 <1st Asymptomatic TERT, K570N P −124C>T 13.5 NA 12.1 85 305 4.0 4.8 c.1710G>C NIH95 72 <1st Asymptomatic TERT, R1084P P −124C>T 7 NA 12.6 104 245 2.0 4.7 c.3251G>C b e NIH61 44 <1st MAA TERT, L864P LP −146C>T 3 15% 6.7 110 39 0.3 1.6 c.2591T>C a b Patients who underwent danazol treatment and were off study or responders. The only pathogenic germline variant in TERT or TERC identified in homozygosity. TERTp clones were detected in both leukocyte and granulocytic fractions. Clone sizes in granulocytes are described after the VAF observed in leukocytes. TERTp VAF in the granulocytic fraction. Clones were not detected in leukocytes. ANC absolute neutrophil count, DC dyskeratosis congenita, Hb hemoglobin, IPF idiopathic pulmonary fibrosis, LP likely pathogenic, MAA moderate aplastic anemia, MCV mean corpuscular volume, NA not available, P pathogenic, Plt platelets, TERTp pathogenic TERT promoter, TL telomere length, VAF variant allele frequency, VUS variant of uncertain significance, WBC white blood cell count. TLs below the first percentile of age- matched controls (<1st) were considered very short and below the tenth percentile (<10th) were considered short. GUTIERREZ-RODRIGUES et al ARTICLE Table 3 Spectrum of phenotypes observed in patients with telomeropathies and pathogenic TERTp variants Age Sex TL Primary diagnosis Spectrum of phenotypes related to telomeropathies Pulmonary fibrosis Marrow failure Liver disease Isolated cytopenia Patients USP79 46 M <1st IPF x x NIH20 61 M <1st IPF x x NIH31 57 F <10th IPF x x x NIH37 65 F <1st IPF x NIH43 24 F <1st MAA x NIH46 39 M <1st IPF x x x NIH53 30 M <1st MAA x x NIH93 38 M <1st MAA x x NIH98 25 F Normal MAA x Relatives USP26 17 M <1st Asymptomatic x USP41 63 M Normal Asymptomatic NIH07 71 M <1st Asymptomatic NIH95 72 F <1st Asymptomatic NIH61 44 F <1st MAA x Frequency (%) 36 50 42 7 AA aplastic anemia, DC dyskeratosis congenita, F female, IPF idiopathic pulmonary fibrosis, M male, MAA moderate AA, TL telomere length below the first (<1st) or tenth (<10th) percentile of age-matched controls. Patient with mild steatosis. (VAF of 50%) and NIH37 had two different variants at VAFs Pathogenic TERTp variant clones expanded over time but <5% (Table 2). did not associate with telomere elongation or response to danazol treatment Chronological analysis of pathogenic TERTp variants The clonal dynamics of pathogenic TERTp variants were during androgen treatment was assessed using serial assessed in serial samples from five patients over a period as samples that were available for NIH46 and NIH61 (Fig. 2d). long as four years; three had a pathogenic or likely In both, thesizeof pathogenic TERTp clones decreased pathogenic germline variant, and two had a germline VUS. during danazol treatment while blood counts improved. In all cases, the TERTp variant clone size expanded over time After treatment, the VAF of these pathogenic TERTp (Fig. 2c), suggesting a selective growth advantage in variant clones increased. These data suggest that hemato- comparison with unmutated hematopoietic cells. logicresponseobservedinthese patients was mostly Pathogenic TERTp variants were not associated with attributed to danazol and not to the presence of pathogenic changes in patients’ TLs or improvement in blood counts; TERTp variants, which appeared diluted in peripheral blood most subjects with a pathogenic TERTp variant, which is by treatment response. Indeed, pathogenic TERTp var- known to upregulate TERT expression, nevertheless had iants did not predict response to danazol, because patients short or very short telomeres (12/14 individuals). Two with and without these somatic variants responded to individuals presented with normal TL (NIH98 and USP26). treatment at 3–6 months and were not off-study (with Despite her normal TLs, NIH98 had short 3’ overhangs as TERTp variants vs. without TERTp variants, 3/5 vs. 8/12; previously reported. Fisher’sexact test, P> 0.5). Twenty-one patients and three relatives from the cohort were enrolled in a clinical trial for treatment with danazol for DISCUSSION two years (Supplementary Table S3) (ref. ). Five had We have expanded the spectrum of nonmalignant diseases pathogenic TERTp variant clones at diagnosis; three were associated with pathogenic TERTp variants to MAA and responders and two were off-study after 3–6 months (Table 2). cirrhosis. Our data indicated that the emergence of patho- NIH93 and NIH61 achieved a hematologic response genic TERTp variants correlates with chronologic aging and at 3 months of treatment and NIH46 showed a response at may be clonal evidence of a telomere disease in 8% of patients 6 months. In these patients, an average size of pathogenic and relatives presenting these clinical phenotypes. TERTp clone in myeloid cells was 4%, except for NIH46 who Pathogenic TERTp variants have been reported only in 4,5 had a clone of 21%. Patients NIH31 and NIH37 responded to cancer or IPF but are rare in hematologic malignancies. danazol at 3 months of treatment but were withdrawn from Indeed, we did not find these variants in 106 AML patients the study due to organ complications not involving the screened by our sequencing assay. Lack of association between marrow. NIH31 had a large pathogenic TERTp variant clone pathogenic TERTp activation and hematologic diseases has GENETICS in MEDICINE Volume 21 Number 7 July 2019 1599 | | | ARTICLE GUTIERREZ-RODRIGUES et al ab NIH46 –124C>T TERTp Leukocytes Granulocytes Mononuclear CD3– CD14– Total mononuclear cells CD14+ CD3+ 010 20 30 40 50 Age range VAF (%) cd NIH46 NIH61 –124C>T TERTp –146C>T TERTp 70 60 Danazol Danazol NIH31 Granulocytes 60 Leukocytes NIH46 –1 0 0.5 1 1.5 2 3 4.5 0 0.5 1 1.5 2.5 4.5 20 30 30 3 NIH07 14 Platelets (10 /µL) 11 8 Hb (g/dL) NIH37 4 7 4 NIH61 5 WBC (10 /µL) 4 2 Reticulocytes (%) 0 0.5 1 24 1 Neutrophils (10 /µL) 0 –1 Years 0 0.5 1 1.5 2 3 4.5 0 0.5 1 1.5 2.5 4.5 Years Years Fig. 2 Molecular and clinical characteristics of individuals with pathogenic TERT promoter (TERTp) variants. (a) Frequency of pathogenic TERTp variants in the different groups classified by age range. Pathogenic TERTp variant frequency increased with aging. (b) The −124C>T TERTp variant allele frequency (VAF) in blood cells’ subpopulations from patient NIH46 quantified by droplet digital PCR (ddPCR). (c) Chronological pathogenic TERTp variant dynamics detected by ddPCR. In serial samples, clones bearing the −146C>T or −124C>T pathogenic TERTp variants expanded over time for all patients evaluated. (d) Chronological analysis of the pathogenic TERTp VAF and hematologic blood counts from two patients during danazol treatment. During the androgen therapy, TERTp clone sizes decreased in both total leukocytes and granulocytes as blood counts improved, especially the platelet counts and hemoglobin levels. A blue bar represents the time frame in which patients were under the danazol treatment. Pathogenic TERTp clones were tracked by ddPCR and dashed lines represent the lower limit of detection (0.5%) of this technique. Hb hemoglobin, WBC white blood cell count. been attributed to constitutively high TERT expression in lower cost and a known subset of genes recurrently mutated 4,12 hematopoietic stem cells. Consistent with this hypothesis, in these diseases. However, an accurate interpretation of clones bearing pathogenic TERTp variants in our study were genetic testing is often complicated by the heterogeneity in positively selected in the bone marrow when patients had an telomere diseases’ penetrance and presentation. In this work, inherited telomerase deficiency. No pathogenic TERTp seven patients had a germline VUS; all were novel and found variants were found in our control group or previously in TERT and TERC genes, both commonly mutated genes in reported cohorts including more than 2,500 healthy subjects telomere diseases and not tolerable to either missense or loss- (mean age of 50 ± 11 years) and 132 elderly individuals of-function variants according to ExAC metrics (http://exac. (range, 98–108 years). broadinstitute.org/). Despite the strong clinical evidence, the Germline pathogenic variants in 12 genes that impair identified VUS did not meet criteria for pathogenicity using telomere biology have been linked to telomere diseases. TL either ACMG or Sherloc classifications due to lack of measurement and genetic screening have been used for segregation data and specific functional assays. Specificity of differential diagnosis of telomeropathies. Both commercial pathogenic TERTp variants for telomerase dysfunction may and in-house targeting sequencing panels are preferred due to be additional evidence for pathogenicity of germline VUS and 1600 Volume 21 Number 7 July 2019 GENETICS in MEDICINE | | | 0–17 18–40 41–60 61–80 Pathogenic TERTp VAF (%) Pathogenic TERTp VAF (%) Blood cell counts Pathogenic TERTp VAF (%) GUTIERREZ-RODRIGUES et al ARTICLE may help identify patients with telomeropathies that lack wild-type allele due to insufficiency of clinical samples. classical phenotypes. Although these somatic variants have been described In cancer cells, current models propose that pathogenic preferentially in cis with the wild-type allele, one of our TERTp acquisition only occurs in cells with very short patients with a homozygous pathogenic TERT germline telomeres due to selective pressure for cell immortaliza- variant also harbored the pathogenic −124 TERTp variant 6,28–30 tion. However, two of our patients with pathogenic that was not associated with clinical worsening. Lack of serial TERTp variants had normal TLs (NIH98 and USP41) in which and myeloid cell–enriched samples also hampered analysis of the germline TERT variant pathogenicity is supported by TERTp variant clones’ chronological dynamics and detection eroded 3’ overhangs, functional assays, and segregation of pathogenic TERTp variants at low allele frequency in 27,31 data. We did not observe longer TL in patients with leukocytes. Second, the natural history of these variants in pathogenic TERTp variants nor telomere elongation in telomeropathies and the clonal dynamics under androgen screened serial samples. In agreement with our findings is the treatment need to be assessed by functional studies. Never- report that TERT upregulation driven by TERTp pathogenic theless, the mechanism for TERT reactivation by androgens variants does not prevent telomere erosion but rather sustains appears to be different from pathogenic TERTp variants, cell proliferation. Also, a pathogenic TERTp variant increased because the hormone does not bind to the region in which the 10,39 cell proliferation and telomerase activity in Epstein–Barr virus somatic TERTp variants are located. Based on our (EBV)-transformed lymphoblastoid cells derived from an IPF preliminary results, cells without pathogenic TERTp variants patient despite his very short telomeres. In fibroblasts from may be more responsive to danazol compared with patho- DC patients, TERT expression prevents premature senescence genic TERTp variant clones, which would lead to a dilution of and extends cells' proliferative lifespan but is insufficient to TERTp variant clones during treatment. Also, these variants 32,33 maintain TL. In different cancers, TERT reactivation has may not affect patients’ responses to androgen therapy, not been widely associated with telomere elongation but rather because patients responded to treatment regardless of 8,34,35 with sustained proliferative capacity. pathogenic TERTp clones. The TERTp variant most commonly identified in our cohort was the pathogenic -124C>T (C228T) variant. Conclusion However, we found rare −124 (n = 2) and −146 (n = 1) Our findings indicate that pathogenic TERTp clones were TERTp variants with a C>A transition instead of the positively selected in nonmalignant diseases, in the setting of frequently found C>T substitution in two DC patients telomerase-deficiency bone marrow cells. TERTp clones’ (NIH39 and USP02) and one IPF patient (NIH34; Supple- selection appeared to be age-dependent and random, and mentary Figure S5). The two DC patients harbored patho- clones might be selected as an attempt to restore telomerase genic germline variants in RTEL1 or DKC1 whereas the IPF activity in patients with telomere diseases. Pathogenic TERTp patient harbored a germline VUS in TERT. The −124 C>A variants may be good evidence of an inherited bone marrow 11,36 also creates a putative ETS motif and has been previously failure driven by telomerase impairment; although 36,37 observed in melanoma and meningioma. The −146 C>A these somatic variants have been found in only 8% patients has been previously found in a single case of thyroid cancer and relatives, the addition of TERTp region in targeted panels but fails to increase TERT expression in luciferase reporter may further support the pathogenic role of germline variants assays and does not create a novel ETS motif. Even though identified in telomere biology genes. these variants have been reported in other studies, the −124C>A and −146C>A variants identified by our sequencing assay could be artifacts because they were not ELECTRONIC SUPPLEMENTARY MATERIAL validated by either ddPCR or Sanger sequencing: the ddPCR The online version of this article (https://doi.org/10.1038/s41436- probes were specific for the C>T transition; clones were very 018-0385-x) contains supplementary material, which is available small to be detected by Sanger; and amounts of patients’ to authorized users. samples were limited. In our study, the frequency of pathogenic TERTp variants in IPF patients was higher than previously observed in a cohort ACKNOWLEDGEMENTS of 200 IPF patients with heterozygous germline variants in The authors would like to thank Olga Rios for assistance in TERT, TERC, PARN,or RTEL1 (28% vs. 5%) (ref. ). This obtaining patients’ samples, Valentina Giudice and Zhijie Wu for difference may be explained by the distinct methodologies their advice and helpful discussion, the Bioinformatics and used to detect the TERTp variants. Our customized amplicon- Computational Core Facility at the NHLBI for data analysis, and based sequencing assay detected clones as small as 1.2% VAF, the DNA sequencing and Genomics Core Facility at NHLBI. This as opposed to Sanger sequencing, which detects clones at VAF work was funded by the Intramural Research Program of the of 20% or higher. Also, our IPF cohort was relatively small National Heart, Lung, and Blood Institute/NIH, and by the Coordi- (n = 18). nation of Improvement of Higher Education Personnel (CAPES) Our study has limitations. First, we did not evaluate and the São Paulo Research Foundation (FAPESP); grants 2014/ whether pathogenic TERTp variants were in cis or trans to the 27294-7, 2015/19074-0, and 2013/08135-2. 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Standards and guidelines for the Attribution-NonCommercial-ShareAlike 4.0 International interpretation of sequence variants: a joint consensus recommendation License, which permits any non-commercial use, sharing, adaptation, of the American College of Medical Genetics and Genomics and the distribution and reproduction in any medium or format, as long as you give Association for Molecular Pathology. Genet Med. 2015;17:405–424. appropriate credit to the original author(s) and the source, provide a link to the 18. Nykamp K, Anderson M, Powers M, et al. Sherloc: a comprehensive Creative Commons license, and indicate if changes were made. If you remix, refinement of the ACMG-AMP variant classification criteria. Genet Med. transform, or build upon this article or a part thereof, you must distribute your 2017;19:1105–1117. contributions under the same license as the original. The images or other third 19. Gutierrez-Rodrigues F, Santana-Lemos BA, Scheucher PS, Alves-Paiva RM, party material in this article are included in the article’s Creative Commons Calado RT. Direct comparison of flow-FISH and qPCR as diagnostic tests license, unless indicated otherwise in a credit line to the material. If material is not for telomere length measurement in humans. PLoS ONE. 2014;9: included in the article’s Creative Commons license and your intended use is not e113747. permitted by statutory regulation or exceeds the permitted use, you will need to 20. Li H, Durbin R. Fast and accurate short read alignment with Burrows- obtain permission directly from the copyright holder. To view a copy of this Wheeler transform. Bioinformatics. 2009;25:1754–1760. license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/. 21. Lai Z, Markovets A, Ahdesmaki M, et al. VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research. Nucleic Acids Res. 2016;44:e108. © The Author(s) 2018 22. Li H, Handsaker B, Wysoker A, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25:2078–2079. 1602 Volume 21 Number 7 July 2019 GENETICS in MEDICINE | | | http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genetics in Medicine Springer Journals

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Biomedicine; Biomedicine, general; Human Genetics; Laboratory Medicine
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Abstract

ARTICLE © American College of Medical Genetics and Genomics 1,2 2 2 Fernanda Gutierrez-Rodrigues, PhD , Flávia S. Donaires, PhD , André Pinto, BSc , 1 1 2 2 Alana Vicente, MD , Laura W. Dillon, PhD , Diego V. Clé, MD, PhD , Barbara A. Santana, PhD , 1 1 1 Mehdi Pirooznia, MD, PhD , Maria del Pilar F. Ibanez, MSc , Danielle M. Townsley, MD , 1 1 1 Sachiko Kajigaya, PhD , Christopher S. Hourigan, MD, DPhil , James N. Cooper, MD , 2 1 Rodrigo T. Calado, MD, PhD and Neal S. Young, MD Purpose: The acquisition of pathogenic variants in the TERT treatment. However, it was specific for patients with telomeropa- promoter (TERTp) region is a mechanism of tumorigenesis. In thies, more frequently co-occurring with TERT germline variants nonmalignant diseases, TERTp variants have been reported only in and associated with aging. patients with idiopathic pulmonary fibrosis (IPF) due to germline Conclusion: We extend the spectrum of nonmalignant diseases variants in telomere biology genes. associated with pathogenic TERTp variants to marrow failure and Methods: We screened patients with a broad spectrum of liver disease due to inherited telomerase deficiency. Specificity of telomeropathies (n = 136), their relatives (n = 52), and controls pathogenic TERTp variants for telomerase dysfunction may help to (n = 195) for TERTp variants using a customized massively parallel assess the pathogenicity of unclear constitutional variants in the amplicon-based sequencing assay. telomere diseases. Results: Pathogenic −124 and −146 TERTp variants were Genetics in Medicine (2019) 21:1594–1602; https://doi.org/10.1038/s41436- identified in nine (7%) unrelated patients diagnosed with IPF 018-0385-x (28%) or moderate aplastic anemia (4.6%); five of them also presented cirrhosis. Five (10%) relatives were also found with these variants, all harboring a pathogenic germline variant in telomere Keywords: somatic TERT promoter variants; telomere diseases; biology genes. TERTp clone selection did not associate with bone marrow failure peripheral blood counts, telomere length, and response to danazol INTRODUCTION functionally compensate the deleterious impact of disease- The TERT gene encodes the catalytic component of the causing germline TERT variants by increasing telomerase telomerase complex required to elongate telomeres in stem activity and cell proliferation. 1,2 and progenitor cells. TERT is epigenetically silenced in Germline variants in telomere-related genes are etiologic in normal somatic and nonproliferative cells, but aberrantly a broader spectrum of diseases collectively named telomere 3–6 14 expressed in many human cancers. Acquisition of diseases or telomeropathies, including IPF but also affecting pathogenic TERT promoter (TERTp) variants located other organs, such as the bone marrow (aplastic anemia [AA] upstream of the translation initiation site at positions and dyskeratosis congenita [DC]) and the liver (cirrhosis and −124C>T (chr5:1,295,228), −146C>T (chr5:1,295,250), and nonalcoholic steatohepatitis). We investigated the distribution −57A>C (chr5:1,295,161) has been described as a mechanism of somatic pathogenic TERTp variants in a large cohort of 3,6–9 of tumorigenesis in cancer cells. These variants increase patients with a spectrum of telomere diseases using a TERT expression and promote cell proliferation through customized low-cost massively parallel sequencing assay recruitment of the transcription factor GABPA to the mutant optimized for identification and quantification of hemato- 10–12 allele. Somatic TERTp variant clones recently have been poietic clones bearing the pathogenic −124 and −146 TERTp found in a few patients with idiopathic pulmonary fibrosis variants. We further assessed the association of these TERTp (IPF) caused by pathogenic germline variants in telomere variants with telomere length (TL) and peripheral blood biology genes. These pathogenic TERTp variants appear to counts of patients. 1 2 Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, MD, USA; Department of Internal Medicine, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto, SP, Brazil. Correspondence: Rodrigo T. Calado (rtcalado@usp.br) Last authors equally contributed: Rodrigo T. Calado and Neal S. Young. Submitted 4 September 2018; accepted: 16 November 2018 Published online: 7 December 2018 1594 Volume 21 Number 7 July 2019 GENETICS in MEDICINE | | | GUTIERREZ-RODRIGUES et al ARTICLE MATERIALS AND METHODS regardless of symptoms or evidence of disease. Enrolled Cohort individuals were seen in the Hematology Branch clinic of In this retrospective study, we screened blood leukocytes from National Heart, Lung, and Blood Institute (NHLBI) or the 136 patients with telomeropathies (median age, 29 years; bone marrow failure clinic in the Hospital das Clínicas, range, 1–76), 52 relatives (median age, 40 years; range, 8–72), Ribeirão Preto School of Medicine, University of São Paulo and 195 controls for the pathogenic −124 and −146 TERTp (USP) between 2004 and 2017 (Supplementary Tables S2–S3). variants (Table 1). Patients were primarily diagnosed with DC TL was measured by Southern blot (SB), quantitative (n = 21), AA (n = 86), IPF with or without another polymerase chain reaction (qPCR), or flow fluorescent in situ telomeropathy-related phenotypes (n = 18), or other pheno- hybridization (flow-FISH) according to protocols previously types (n = 11; Supplementary Table S1). Clinical diagnosis of described. TL measurements by qPCR were confirmed by 2,15,16 DC and AA was defined according to previous criteria. SB or flow-FISH at the time of this study. Patients with Briefly, DC patients had at least two of three manifestations of acquired immune AA (n = 70), IPF without evidence of the clinical triad (dystrophic nails, patchy skin hyperpigmen- inherited disease and a telomere-related germline pathogenic tation, and oral leukoplakia) and TL below the 1st percentile variant (n = 12), other inherited bone marrow failure for age-matched controls, whereas AA patients presented with syndromes (IBMFS; Diamond–Blackfan anemia, n = 4; pancytopenia and hypocellular bone marrow without any chronic neutropenia, n = 3), and acute myeloid leukemia evidence for myelodysplasia, myelofibrosis, or leukemia. (AML; n = 106) were studied as controls (Supplementary Inclusion criteria were based on molecular diagnosis: TL Table S2-S3). below the 10th percentile of age-matched controls or a Approval was obtained from the Institutional Review Board germline variant in a telomere biology gene classified as of NHLBI and from the Comitê de Ética em Pesquisa do pathogenic, likely pathogenic, or variant of uncertain Hospital das Clínicas de Ribeirão Preto. Samples were collected significance (VUS) by the Sherloc criteria, a framework that according to the Declaration of Helsinki and written consent incorporates the American College of Medical Genetics and was obtained from all participants or their legal guardians. 17,18 Genomics (ACMG) criteria (Supplementary Tables S2– S3). The Sherloc criteria refined the ACMG guidelines to Massively parallel amplicon-based sequencing assay for comprehensively assess variants’ pathogenicity based on both detection of TERTp variants clinical and functional evidence, attributing points to score We customized a massively parallel amplicon-based sequen- each variant for pathogenicity. The point score thresholds for cing assay that targeted the TERTp region in which pathogenic and likely pathogenic variants are four (4P) and pathogenic −124 and −146 TERTp variants were located five points (5P), respectively (Supplementary Table S4). (Supplementary Figure S1). Although primers were not Patients’ relatives were only studied if they harbored the optimized to cover the −57A>C TERTp position, we also same germline variant as the proband or had short telomeres, detected this variant in some of our samples. TERTp variant Table 1 The cohort screened for pathogenic TERT promoter variants by massively parallel amplicon-based sequencing assay Telomeropathies Family members Control group DC AA IPF (with or Other (n = 52) AML Acquired AA, (n = 21) (n = 86) without AA, phenotypes (n = 106) IPF or other MDS, and (n = 11) IBMFS (n = 89) cirrhosis) (n = 18) Median age (range) 13 (1–59) 28 (5–73) 54 (27–76) 27 (3–69) 40 (8–72) 50 (2–86) 29 (1–88) Females/males 4/9 40/46 7/11 3/8 30/22 51/55 44/45 Patients with somatic TERTp (%) 0 4 (4.6%) 5 (28%) 0 5 (9.6%) 0 0 Patients with a germline TERT 337 14 5 34 00 variant Patients with a somatic TERTp and03 5 0 5 0 0 a germline TERT variant Patients with somatic TERTp 0 8.1 36 0 14.7 0 0 variants from the total of patients with TERT germline variants (%) TERTp pathogenic TERT. In this study, we screened 136 patients with telomeropathies that presented dyskeratosis congenita (DC, n = 21), aplastic anemia (AA, n = 86), idiopathic pulmonary fibrosis (IPF, n = 18), and other phenotypes that included myelodysplastic syndrome (MDS) or hypoplastic MDS (HypoMDS, n = 7), isolated thrombocytopenia (n = 3), and thrombocythemia (n = 1). Control group was composed of patients with acquired AA (n = 70; median age = 28 years), IPF (n = 12; median age = 62 years), other inherited bone marrow failure syndromes (IBMFS; Diamond–Blackfan anemia, n = 4; chronic neutropenia, n = 3), and acute myeloid leukemia (AML, n = 106; Supplementary Tables S2–S3). GENETICS in MEDICINE Volume 21 Number 7 July 2019 1595 | | | 1234567890():,; ARTICLE GUTIERREZ-RODRIGUES et al screening was performed in patients, relatives, and controls ddPCR was performed according to a protocol previously using peripheral blood leukocytes collected at the time of first optimized with minor modifications to transfer the assay to clinical evaluation. Whenever possible, testing was also the RainDance platform (RainDance). To evaluate the performed in granulocytes separated by gradient centrifuga- linearity and limit of detection of the ddPCR assay, a variant tion in parallel with the respective leukocyte samples. control sequence (CCCCTTCCGG) was serially diluted into Library preparation consisted of two rounds of PCR to 300 ng of sheared normal human genomic DNA to have an amplify the TERTp region. A first PCR was designed to expected variant target copy number. A mean frequency amplify the targeted region and a second PCR for addition abundance of the variant template was plotted versus the of Illumina adapters (Illumina, San Diego, CA, USA) into target copies input to generate a standard curve. Linearity of fragments. In the first PCR, we used a set of four different the assay was high (R = 0.99; Supplementary Figure S3a) and forward and reverse primers (total of eight primers) that lower limit of detection was 0.17%. The highest mean were pooled in equimolar amounts to amplify the TERTp frequency abundance obtained when genomic DNA from region (Supplementary Table S5). PCR products were then negative controls were used was 0.5%, and this value was used subjected to a second PCR round for the addition of the full as the negative cut-off. The customized sequencing assay i5/i7 Illumina adapter/index sequences into the DNA accurately detected pathogenic TERTp variants confirmed by fragments. ddPCR but did not identify false positives. The correlation Up to 96 libraries were pooled in equimolar amounts and between sequencing and ddPCR in quantification of TERTp pair-end sequenced in 300 cycles on the MiSeq platform variant clones was high (R = 0.97; Supplementary Fig- (Illumina). Median coverage depth on targets was 104×. Reads ure S3b). Agreement between techniques was evaluated by were aligned to the human genome reference (hg19) using Bland–Altman analysis, a statistical tool to compare clinical 20 26 Burrows–Wheeler Aligner (BWA) and data quality was assays. Bland–Altman analysis evidenced a good agreement assessed using FastQC. Sequences were trimmed to remove between these two methods (Supplementary Figure S3c), as the adapters as well as low-quality bases (-q 15 –minimum- the mean difference of VAF measurements was 0.95 and no length 35). Variants were called using VarDict and the measurements exceed the 95% confidence interval (CI) limits following filtering criteria: -f 0.005 -v -c 1 -S 2 -E 3 -g 4 -th 8 of agreement. The standard deviation (SD) between assays 21 22 (ref. ). For comparison, we also used SAMTools and was 2.1 and 95% limits of agreement ranged from 5.15 to Genome Analysis Toolkit (GATK) to call the variants. −3.25. Detailed protocols and primer sequences are available While VarDict called all TERTp variants that were further in Online Supplementary Data. validated by droplet digital PCR (ddPCR; RainDance Technologies, Billerica, MA, USA), GATK and SAMTools RESULTS only called variants with allele frequency >20% and >8%, Pathogenic TERTp variants associated with different respectively. Variants were annotated using Annovar and phenotypes from the spectrum of telomeropathies and variant allele frequency (VAF) was calculated by a ratio of patients’ ages variant minor allele counts and total reads. Of 136 cases, nine unrelated patients (7%; median age, 39 years; range, 24–65; Fig. 1) were found with TERTp variants. Droplet digital PCR Nine patients had the pathogenic −124C>T variant, including In samples that were available, the −124C>T and −146C>T two patients who also had the −57A>C TERTp variant TERTp variants identified by sequencing were validated by (NIH37 and NIH93; Table 2). Patients were clinically ddPCR (Supplementary Figure S2a), except for a single case diagnosed with IPF (5/18; 28%) and moderate AA (MAA; that was confirmed by Sanger sequencing (Supplementary 4/86; 4.6%) (Table 1 and Fig. 1). Of note, eight of them also Figure S2b). TERTp variant clones were also tracked over time presented other phenotypes related to telomeropathies using this technique. In patient NIH46, the TERTp variant (Table 3); co-occurrence with cirrhosis and MAA was was quantified in the following blood cells’ subpopulations by frequent (Tables 2–3). Five relatives (10%; median age, 63 ddPCR: leukocytes after whole-blood ammonium chloride years; range, 17–72) had the −124C>T (n = 4) or the potassium (ACK) lysis, granulocytes separated by gradient −146C>T (n = 1) TERTp variants: three were asymptomatic centrifugation using Ficoll–Hypaque, peripheral mononuclear and two were diagnosed with MAA or a DC-like phenotype. cells separated by gradient centrifugation using (Table 2). Within the same family, a somatic TERTp variant + − + Ficoll–Hypaque, CD14 CD16 monocytes, CD3 T cells, was not observed in more than one subject (Supplementary and mononuclear fraction depleted for CD3 and Figure S4), suggesting that acquisition of these variants was + - + - CD14 CD16 . The CD14 CD16 monocytes were isolated not due to a genetic susceptibility caused by the telomere- from mononuclear cells using immunomagnetic negative related germline pathogenic variant identified in the family. selection (the EasySep™ Human Monocyte Isolation kit, The frequency of pathogenic TERTp variants was much Stemcell Technologies, Cambridge, MA, USA) and CD3 higher in IPF patients compared with AA cases (28% vs. 4.6%; T cells were isolated from mononuclear cells by immuno- Fisher’s exact test, P = 0.007; Table 1). Because some patients magnetic positive selection (the EasySep™ Human CD3 presented different phenotypes from the spectrum of Positive Selection kit II, Stemcell Technologies). telomeropathies, we then investigated whether pathogenic 1596 Volume 21 Number 7 July 2019 GENETICS in MEDICINE | | | GUTIERREZ-RODRIGUES et al ARTICLE TERT germline variant TERC germline variant MAA IPF Asymptomatic IPF MAA Relatives 08 50 100 Patients and relatives screened (%) Fig. 1 Clinical association of somatic pathogenic TERT promoter (TERTp) variants and telomere diseases. Frequency of pathogenic TERTp variants in patients and relatives screened in the study. The size of the TERTp clone is represented by the variant allele frequency (VAF) and shown for each individual screened according to their primary diagnosis: idiopathic pulmonary fibrosis (IPF), moderate aplastic anemia (MAA), and relatives. Of 188 patients and relatives, 14 had the −124 or −146 TERTp variants (8%). An additional graph shows in detail the pathogenic TERTp clone size, initial diagnosis, and the telomere biology gene in which a germline variant was identified from the patients/relatives with pathogenic TERTp variants. Four relatives were asymptomatic. TERTp variants were more frequent in the setting of ddPCR. Overall, TERTp clones were more frequent in pulmonary disease compared with marrow failure or liver individuals with germline TERT variants (12/14 cases); only disease. No difference in frequency of disease phenotypes (IPF two patients harbored a germline variant in TERC. The vs. marrow failure or liver disease) was observed among germline variants identified in telomere biology genes were patients with a pathogenic TERTp variant (36% vs. 50% or classified as pathogenic or likely pathogenic (n = 8), and VUS 42%, respectively; χ test, P> 0.05; Table 3), suggesting that (n = 6; Table 2) and, except for the TERT R696C found in these variants occurred in all these clinical presentations. USP26, they were heterozygous. All variants classified as VUS Pathogenic TERTp variant clones positively correlated with had some evidence for being pathogenic (all had a Sherloc age, as they were only present in individuals older than 18 score of 3.5P) but insufficient to meet Sherloc criteria for years old and more frequent in those 60 to 80 years old pathogenicity; all were predicted as deleterious in silico, (Fig. 2a). Six of 86 individuals ranging in age from 21 to 40 absent in control populations, and associated with a family (7%), 3 of 44 individuals ranging in age from 41 to 60 years history and phenotype of telomere diseases (Supplementary (6.8%), and 5 of 18 patients older than 61 years (27.8%) had Table S4). pathogenic TERTp variants. The median age of patients with Pathogenic TERTp variant clone sizes varied from 1.2% to TERTp variants and a primary diagnosis of IPF and MAA was 50% in total leukocytes and clones were found at higher allele 57 and 27 years, respectively. frequencies in the granulocytic fractions in four patients Pathogenic TERTp variants were found in telomeropathy (Table 2 and Fig. 2b). In NIH93, the −124C>T TERTp was patients who had a germline variant in telomere biology genes identified in total leukocytes and granulocytes by both but not in controls (median age, 29 years; range, 1–88) or in sequencing and ddPCR at VAF as low as 6%. However, in patients with very short telomeres without a germline variant NIH61, pathogenic TERTp clones were only detected in in telomere biology genes (median age, 25 years; range, 5–63; granulocytes by sequencing due to very low VAF in total Table 1). We also confirmed the absence of pathogenic leukocytes. Results were similar when peripheral blood cell TERTp variants in the granulocytic fractions from controls in subpopulations from NIH46 were separated by magnetic which materials were available (Supplementary Table S2–S3). selection for screening of the −124C>T TERTp variant by ddPCR. The −124C>T TERTp variant was found enriched in Pathogenic TERTp variants more frequently co-occurred the granulocyte fraction and mononuclear cells depleted for + + with germline TERT variants in myeloid cells CD3 and CD14 cells (Fig. 2b). Clonal dominance was not The customized sequencing assay detected pathogenic TERTp observed in most individuals with pathogenic TERTp variants; clones at VAF as low as 1.2%, which was confirmed by nine had pathogenic TERTp clones at VAF lower than 10%. GENETICS in MEDICINE Volume 21 Number 7 July 2019 1597 | | | NIH98 NIH53 NIH93 NIH61 NIH46 USP79 NIH34 NIH31 NIH20 NIH37 USP26 USP41 NIH07 NIH95 Pathogenic TERTp VAF (%) Pathogenic TERTp VAF (%) ARTICLE GUTIERREZ-RODRIGUES et al 1598 Volume 21 Number 7 July 2019 GENETICS in MEDICINE | | | Table 2 Mutational and clinical profile of patients with telomeropathies and somatic pathogenic TERT promoter variants Age TL Clinical Germline Sherloc Somatic TERTp Leukocyte Bone marrow Hb (g/ MCV Plt ANC WBC 3 3 3 diagnosis variant germline variant VAF (%) cellularity dL) (fL) (×10 / (×10 /µL) (×10 /µL) variant µL) classification Patients USP79 46 <1st IPF/cirrhosis TERT, T937A VUS −124C>T 50 NA 16.9 107 117 2 3.5 c.2809A>G NIH20 61 <1st IPF/cirrhosis TERT, V170L P − 124C>T 6.6 40% 13.5 103 117 3.9 7 c.508G>A NIH31 57 <10th IPF/MAA/ TERT, A1009G VUS −124C>T 50 30–40% 9 99 55 1.37 2.3 cirrhosis c.3026C>A NIH37 65 <1st IPF TERT, P59S VUS − 124C>T 5 40–50% 13 111 104 3.0 5.4 c.175C>T − 57C>T 4.5 NIH43 24 <1st MAA TERT, V1025F P −124C>T 3 <10% 8.7 89 21 0.86 1.7 c.3073G>T NIH46 39 <1st IPF/MAA TERC, r.433G>C VUS −124C>T 21 <5% 8.9 107 65 1.32 2.7 NIH53 30 <1st MAA/cirrhosis TERC, r.107G>C P −124C>T 4.4 <5% 9.2 92 31 0.72 2.5 b d NIH93 38 <1st MAA/cirrhosis TERT, A130V VUS −124C>T 5/6 <20% 11.4 101 27 0.32 0.6 c.389C>T −57C>T 6/5 NIH98 25 Normal MAA TERT, R537C VUS −124C>T 20 10% 10.6 112 83 1.0 3.3 c.1609C>T Relatives USP26 18 <1st Asymptomatic TERT, R696C P −124C>T 1.2 NA 10.4 86 87 2.0 4.0 but with DC-like c.2086C>T phenotype USP41 63 Normal Asymptomatic TERT, R865H P −124C>T 1.8 NA 15.4 105 163 2.8 6.2 c.2594G>A NIH07 71 <1st Asymptomatic TERT, K570N P −124C>T 13.5 NA 12.1 85 305 4.0 4.8 c.1710G>C NIH95 72 <1st Asymptomatic TERT, R1084P P −124C>T 7 NA 12.6 104 245 2.0 4.7 c.3251G>C b e NIH61 44 <1st MAA TERT, L864P LP −146C>T 3 15% 6.7 110 39 0.3 1.6 c.2591T>C a b Patients who underwent danazol treatment and were off study or responders. The only pathogenic germline variant in TERT or TERC identified in homozygosity. TERTp clones were detected in both leukocyte and granulocytic fractions. Clone sizes in granulocytes are described after the VAF observed in leukocytes. TERTp VAF in the granulocytic fraction. Clones were not detected in leukocytes. ANC absolute neutrophil count, DC dyskeratosis congenita, Hb hemoglobin, IPF idiopathic pulmonary fibrosis, LP likely pathogenic, MAA moderate aplastic anemia, MCV mean corpuscular volume, NA not available, P pathogenic, Plt platelets, TERTp pathogenic TERT promoter, TL telomere length, VAF variant allele frequency, VUS variant of uncertain significance, WBC white blood cell count. TLs below the first percentile of age- matched controls (<1st) were considered very short and below the tenth percentile (<10th) were considered short. GUTIERREZ-RODRIGUES et al ARTICLE Table 3 Spectrum of phenotypes observed in patients with telomeropathies and pathogenic TERTp variants Age Sex TL Primary diagnosis Spectrum of phenotypes related to telomeropathies Pulmonary fibrosis Marrow failure Liver disease Isolated cytopenia Patients USP79 46 M <1st IPF x x NIH20 61 M <1st IPF x x NIH31 57 F <10th IPF x x x NIH37 65 F <1st IPF x NIH43 24 F <1st MAA x NIH46 39 M <1st IPF x x x NIH53 30 M <1st MAA x x NIH93 38 M <1st MAA x x NIH98 25 F Normal MAA x Relatives USP26 17 M <1st Asymptomatic x USP41 63 M Normal Asymptomatic NIH07 71 M <1st Asymptomatic NIH95 72 F <1st Asymptomatic NIH61 44 F <1st MAA x Frequency (%) 36 50 42 7 AA aplastic anemia, DC dyskeratosis congenita, F female, IPF idiopathic pulmonary fibrosis, M male, MAA moderate AA, TL telomere length below the first (<1st) or tenth (<10th) percentile of age-matched controls. Patient with mild steatosis. (VAF of 50%) and NIH37 had two different variants at VAFs Pathogenic TERTp variant clones expanded over time but <5% (Table 2). did not associate with telomere elongation or response to danazol treatment Chronological analysis of pathogenic TERTp variants The clonal dynamics of pathogenic TERTp variants were during androgen treatment was assessed using serial assessed in serial samples from five patients over a period as samples that were available for NIH46 and NIH61 (Fig. 2d). long as four years; three had a pathogenic or likely In both, thesizeof pathogenic TERTp clones decreased pathogenic germline variant, and two had a germline VUS. during danazol treatment while blood counts improved. In all cases, the TERTp variant clone size expanded over time After treatment, the VAF of these pathogenic TERTp (Fig. 2c), suggesting a selective growth advantage in variant clones increased. These data suggest that hemato- comparison with unmutated hematopoietic cells. logicresponseobservedinthese patients was mostly Pathogenic TERTp variants were not associated with attributed to danazol and not to the presence of pathogenic changes in patients’ TLs or improvement in blood counts; TERTp variants, which appeared diluted in peripheral blood most subjects with a pathogenic TERTp variant, which is by treatment response. Indeed, pathogenic TERTp var- known to upregulate TERT expression, nevertheless had iants did not predict response to danazol, because patients short or very short telomeres (12/14 individuals). Two with and without these somatic variants responded to individuals presented with normal TL (NIH98 and USP26). treatment at 3–6 months and were not off-study (with Despite her normal TLs, NIH98 had short 3’ overhangs as TERTp variants vs. without TERTp variants, 3/5 vs. 8/12; previously reported. Fisher’sexact test, P> 0.5). Twenty-one patients and three relatives from the cohort were enrolled in a clinical trial for treatment with danazol for DISCUSSION two years (Supplementary Table S3) (ref. ). Five had We have expanded the spectrum of nonmalignant diseases pathogenic TERTp variant clones at diagnosis; three were associated with pathogenic TERTp variants to MAA and responders and two were off-study after 3–6 months (Table 2). cirrhosis. Our data indicated that the emergence of patho- NIH93 and NIH61 achieved a hematologic response genic TERTp variants correlates with chronologic aging and at 3 months of treatment and NIH46 showed a response at may be clonal evidence of a telomere disease in 8% of patients 6 months. In these patients, an average size of pathogenic and relatives presenting these clinical phenotypes. TERTp clone in myeloid cells was 4%, except for NIH46 who Pathogenic TERTp variants have been reported only in 4,5 had a clone of 21%. Patients NIH31 and NIH37 responded to cancer or IPF but are rare in hematologic malignancies. danazol at 3 months of treatment but were withdrawn from Indeed, we did not find these variants in 106 AML patients the study due to organ complications not involving the screened by our sequencing assay. Lack of association between marrow. NIH31 had a large pathogenic TERTp variant clone pathogenic TERTp activation and hematologic diseases has GENETICS in MEDICINE Volume 21 Number 7 July 2019 1599 | | | ARTICLE GUTIERREZ-RODRIGUES et al ab NIH46 –124C>T TERTp Leukocytes Granulocytes Mononuclear CD3– CD14– Total mononuclear cells CD14+ CD3+ 010 20 30 40 50 Age range VAF (%) cd NIH46 NIH61 –124C>T TERTp –146C>T TERTp 70 60 Danazol Danazol NIH31 Granulocytes 60 Leukocytes NIH46 –1 0 0.5 1 1.5 2 3 4.5 0 0.5 1 1.5 2.5 4.5 20 30 30 3 NIH07 14 Platelets (10 /µL) 11 8 Hb (g/dL) NIH37 4 7 4 NIH61 5 WBC (10 /µL) 4 2 Reticulocytes (%) 0 0.5 1 24 1 Neutrophils (10 /µL) 0 –1 Years 0 0.5 1 1.5 2 3 4.5 0 0.5 1 1.5 2.5 4.5 Years Years Fig. 2 Molecular and clinical characteristics of individuals with pathogenic TERT promoter (TERTp) variants. (a) Frequency of pathogenic TERTp variants in the different groups classified by age range. Pathogenic TERTp variant frequency increased with aging. (b) The −124C>T TERTp variant allele frequency (VAF) in blood cells’ subpopulations from patient NIH46 quantified by droplet digital PCR (ddPCR). (c) Chronological pathogenic TERTp variant dynamics detected by ddPCR. In serial samples, clones bearing the −146C>T or −124C>T pathogenic TERTp variants expanded over time for all patients evaluated. (d) Chronological analysis of the pathogenic TERTp VAF and hematologic blood counts from two patients during danazol treatment. During the androgen therapy, TERTp clone sizes decreased in both total leukocytes and granulocytes as blood counts improved, especially the platelet counts and hemoglobin levels. A blue bar represents the time frame in which patients were under the danazol treatment. Pathogenic TERTp clones were tracked by ddPCR and dashed lines represent the lower limit of detection (0.5%) of this technique. Hb hemoglobin, WBC white blood cell count. been attributed to constitutively high TERT expression in lower cost and a known subset of genes recurrently mutated 4,12 hematopoietic stem cells. Consistent with this hypothesis, in these diseases. However, an accurate interpretation of clones bearing pathogenic TERTp variants in our study were genetic testing is often complicated by the heterogeneity in positively selected in the bone marrow when patients had an telomere diseases’ penetrance and presentation. In this work, inherited telomerase deficiency. No pathogenic TERTp seven patients had a germline VUS; all were novel and found variants were found in our control group or previously in TERT and TERC genes, both commonly mutated genes in reported cohorts including more than 2,500 healthy subjects telomere diseases and not tolerable to either missense or loss- (mean age of 50 ± 11 years) and 132 elderly individuals of-function variants according to ExAC metrics (http://exac. (range, 98–108 years). broadinstitute.org/). Despite the strong clinical evidence, the Germline pathogenic variants in 12 genes that impair identified VUS did not meet criteria for pathogenicity using telomere biology have been linked to telomere diseases. TL either ACMG or Sherloc classifications due to lack of measurement and genetic screening have been used for segregation data and specific functional assays. Specificity of differential diagnosis of telomeropathies. Both commercial pathogenic TERTp variants for telomerase dysfunction may and in-house targeting sequencing panels are preferred due to be additional evidence for pathogenicity of germline VUS and 1600 Volume 21 Number 7 July 2019 GENETICS in MEDICINE | | | 0–17 18–40 41–60 61–80 Pathogenic TERTp VAF (%) Pathogenic TERTp VAF (%) Blood cell counts Pathogenic TERTp VAF (%) GUTIERREZ-RODRIGUES et al ARTICLE may help identify patients with telomeropathies that lack wild-type allele due to insufficiency of clinical samples. classical phenotypes. Although these somatic variants have been described In cancer cells, current models propose that pathogenic preferentially in cis with the wild-type allele, one of our TERTp acquisition only occurs in cells with very short patients with a homozygous pathogenic TERT germline telomeres due to selective pressure for cell immortaliza- variant also harbored the pathogenic −124 TERTp variant 6,28–30 tion. However, two of our patients with pathogenic that was not associated with clinical worsening. Lack of serial TERTp variants had normal TLs (NIH98 and USP41) in which and myeloid cell–enriched samples also hampered analysis of the germline TERT variant pathogenicity is supported by TERTp variant clones’ chronological dynamics and detection eroded 3’ overhangs, functional assays, and segregation of pathogenic TERTp variants at low allele frequency in 27,31 data. We did not observe longer TL in patients with leukocytes. Second, the natural history of these variants in pathogenic TERTp variants nor telomere elongation in telomeropathies and the clonal dynamics under androgen screened serial samples. In agreement with our findings is the treatment need to be assessed by functional studies. Never- report that TERT upregulation driven by TERTp pathogenic theless, the mechanism for TERT reactivation by androgens variants does not prevent telomere erosion but rather sustains appears to be different from pathogenic TERTp variants, cell proliferation. Also, a pathogenic TERTp variant increased because the hormone does not bind to the region in which the 10,39 cell proliferation and telomerase activity in Epstein–Barr virus somatic TERTp variants are located. Based on our (EBV)-transformed lymphoblastoid cells derived from an IPF preliminary results, cells without pathogenic TERTp variants patient despite his very short telomeres. In fibroblasts from may be more responsive to danazol compared with patho- DC patients, TERT expression prevents premature senescence genic TERTp variant clones, which would lead to a dilution of and extends cells' proliferative lifespan but is insufficient to TERTp variant clones during treatment. Also, these variants 32,33 maintain TL. In different cancers, TERT reactivation has may not affect patients’ responses to androgen therapy, not been widely associated with telomere elongation but rather because patients responded to treatment regardless of 8,34,35 with sustained proliferative capacity. pathogenic TERTp clones. The TERTp variant most commonly identified in our cohort was the pathogenic -124C>T (C228T) variant. Conclusion However, we found rare −124 (n = 2) and −146 (n = 1) Our findings indicate that pathogenic TERTp clones were TERTp variants with a C>A transition instead of the positively selected in nonmalignant diseases, in the setting of frequently found C>T substitution in two DC patients telomerase-deficiency bone marrow cells. TERTp clones’ (NIH39 and USP02) and one IPF patient (NIH34; Supple- selection appeared to be age-dependent and random, and mentary Figure S5). The two DC patients harbored patho- clones might be selected as an attempt to restore telomerase genic germline variants in RTEL1 or DKC1 whereas the IPF activity in patients with telomere diseases. Pathogenic TERTp patient harbored a germline VUS in TERT. The −124 C>A variants may be good evidence of an inherited bone marrow 11,36 also creates a putative ETS motif and has been previously failure driven by telomerase impairment; although 36,37 observed in melanoma and meningioma. The −146 C>A these somatic variants have been found in only 8% patients has been previously found in a single case of thyroid cancer and relatives, the addition of TERTp region in targeted panels but fails to increase TERT expression in luciferase reporter may further support the pathogenic role of germline variants assays and does not create a novel ETS motif. Even though identified in telomere biology genes. these variants have been reported in other studies, the −124C>A and −146C>A variants identified by our sequencing assay could be artifacts because they were not ELECTRONIC SUPPLEMENTARY MATERIAL validated by either ddPCR or Sanger sequencing: the ddPCR The online version of this article (https://doi.org/10.1038/s41436- probes were specific for the C>T transition; clones were very 018-0385-x) contains supplementary material, which is available small to be detected by Sanger; and amounts of patients’ to authorized users. samples were limited. In our study, the frequency of pathogenic TERTp variants in IPF patients was higher than previously observed in a cohort ACKNOWLEDGEMENTS of 200 IPF patients with heterozygous germline variants in The authors would like to thank Olga Rios for assistance in TERT, TERC, PARN,or RTEL1 (28% vs. 5%) (ref. ). This obtaining patients’ samples, Valentina Giudice and Zhijie Wu for difference may be explained by the distinct methodologies their advice and helpful discussion, the Bioinformatics and used to detect the TERTp variants. Our customized amplicon- Computational Core Facility at the NHLBI for data analysis, and based sequencing assay detected clones as small as 1.2% VAF, the DNA sequencing and Genomics Core Facility at NHLBI. This as opposed to Sanger sequencing, which detects clones at VAF work was funded by the Intramural Research Program of the of 20% or higher. Also, our IPF cohort was relatively small National Heart, Lung, and Blood Institute/NIH, and by the Coordi- (n = 18). nation of Improvement of Higher Education Personnel (CAPES) Our study has limitations. First, we did not evaluate and the São Paulo Research Foundation (FAPESP); grants 2014/ whether pathogenic TERTp variants were in cis or trans to the 27294-7, 2015/19074-0, and 2013/08135-2. 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Nucleic Acids Res. 2016;44:e108. © The Author(s) 2018 22. Li H, Handsaker B, Wysoker A, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25:2078–2079. 1602 Volume 21 Number 7 July 2019 GENETICS in MEDICINE | | |

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