Arias-Salgado et al. Orphanet Journal of Rare Diseases (2019) 14:82 https://doi.org/10.1186/s13023-019-1046-0 RESEARCH Open Access Genetic analyses of aplastic anemia and idiopathic pulmonary fibrosis patients with short telomeres, possible implication of DNA-repair genes Elena G. Arias-Salgado1,2, Eva Galvez3, Lurdes Planas-Cerezales4, Laura Pintado-Berninches1,2, Elena Vallespin5, Pilar Martinez5, Jaime Carrillo1, Laura Iarriccio1,2, Anna Ruiz-Llobet6,7, Albert Catalá6,7, Isabel Badell-Serra8, Luis I. Gonzalez-Granado9, Andrea Martín-Nalda10, Mónica Martínez-Gallo10, Ana Galera-Miñarro11, Carmen Rodríguez-Vigil12, Mariana Bastos-Oreiro13, Guiomar Perez de Nanclares14, Virginia Leiro-Fernández15, Maria-Luz Uria10, Cristina Diaz-Heredia10, Claudia Valenzuela16, Sara Martín4, Belén López-Muñiz17, Pablo Lapunzina5,18, Julian Sevilla3,18, María Molina-Molina4,19, Rosario Perona1,18† and Leandro Sastre1,18*† Abstract Background: Telomeres are nucleoprotein structures present at the terminal region of the chromosomes. Mutations in genes coding for proteins involved in telomere maintenance are causative of a number of disorders known as telomeropathies. The genetic origin of these diseases is heterogeneous and has not been determined for a significant proportion of patients. Methods: This article describes the genetic characterization of a cohort of patients. Telomere length was determined by Southern blot and quantitative PCR. Nucleotide variants were analyzed either by high-resolution melting analysis and Sanger sequencing of selected exons or by massive sequencing of a panel of genes. Results: Forty-seven patients with telomere length below the 10% of normal population, affected with three telomeropathies: dyskeratosis congenita (4), aplastic anemia (22) or pulmonary fibrosis (21) were analyzed. Eighteen of these patients presented known pathogenic or novel possibly pathogenic variants in the telomere-related genes TERT, TERC, RTEL1, CTC1 and ACD. In addition, the analyses of a panel of 188 genes related to haematological disorders indicated that a relevant proportion of the patients (up to 35%) presented rare variants in genes related to DNA repair or in genes coding for proteins involved in the resolution of complex DNA structures, that participate in telomere replication. Mutations in some of these genes are causative of several syndromes previously associated to telomere shortening. Conclusion: Novel variants in telomere, DNA repair and replication genes are described that might indicate the contribution of variants in these genes to the development of telomeropathies. Patients carrying variants in telomere-related genes presented worse evolution after diagnosis than the rest of patients analyzed. Keywords: Telomere, Dyskeratosis congenita, Pulmonary fibrosis, Aplastic anemia, DNA repair, Telomeropathies * Correspondence: lsastre@iib.uam.es † Rosario Perona and Leandro Sastre are joint senior authors of this work. 1 Instituto de Investigaciones Biomedicas CSIC/UAM, IDIPaz, Arturo Duperier, 4, 28029 Madrid, Spain 18 CIBER de enfermedades raras (CIBERER), Madrid, Spain Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Arias-Salgado et al. Orphanet Journal of Rare Diseases (2019) 14:82 Page 2 of 12 Background those of dyskeratosis congenita (DC) or the related Chromosome ends are protected by nucleoprotein struc- Hoyeraal-Hreidarsson, Resvesz and Coats plus syndromes tures, the telomeres. In humans, telomere DNA is com- [11]. Other diseases may appear later such as Aplastic posed by multiple repetitions of the TTAGGG Anemia, (AA) (20–30 years) [12] or pulmonary fibrosis hexanucleotide [1]. The 3′ end of telomeres is formed by a (40–60 years) (PF) [13]. Mutations in 13 different genes G-rich single stranded extension that invades the upstream have been identified as causative of these diseases but a double-stranded region to form telomeric loops (T-, large number of patients remain genetically undiag- D-loop). Telomere DNA is bound by a protein complex nosed [14]. named shelterin [2] and this nucleo-protein structure pro- Variants in the gene coding for one helicase, RTEL1 tects chromosome ends from degradation and is critical for (OMIM 608833) are also present in patients with telo- genome stability (recently reviewed in [3]). meropathies [15]. Others helicases and nucleases, like Telomeres cannot be completely synthesized during SLX4, BLM, WRN and RecQL4, have been shown to DNA replication, which is known as the end-replication participate in telomere preservation [16]. Actually, muta- problem and results in progressive shortening of the telo- tions in some of these genes also result in telomere meres as cells proliferate. In most organisms, telomere shortening [16, 17]. The term secondary telomeropathies DNA is elongated after each round of DNA replication by has been recently proposed for diseases caused by muta- a specialized complex with reverse transcriptase activity tions in this group of genes [18]. known as telomerase [4]. The enzymatic activity of the The reduced number of patients of these rare diseases complex is provided by the TERT (telomerase reverse and the number of putative candidate genes makes transcriptase) protein while the RNA component TR (tel- their molecular diagnosis challenging. In this article, omerase RNA, encoded by TERC) is used as template. Es- the molecular analysis of 47 patients treated in Spanish sential components of the complex are also DKC hospitals was performed. Twenty-six of them were di- (Dyskeratosis congenita), NOP10, NHP2 and GAR1 pro- agnosed of DC or AA and twenty-one of PF. The re- teins, which bind and stabilize TR and are required for tel- sults obtained identified possibly pathogenic variants at omerase complex assembly [5]. the telomere-associated genes TERT, TERC, ACD, Telomere replication, elongation and protection are CTC1 and RTEL1 in heterozygosis. In addition, a sig- impaired by the strong secondary and tertiary structure nificant proportion of patients presented rare variants of their DNA [6]. The high content of Guanines of the in genes coding for proteins involved in DNA repair telomeres makes them prone to form secondary struc- and in the resolution of complex DNA structures. tures such as G-quadruplexes. In addition, the T- and D-loops impair the access of the replicative machinery. Methods These structures are solved by helicases and structure- Patients specific endonucleases. Patients with severe telomere shortening (percentile < 10) In the absence of a functional telomerase complex, who presented dyskeratosis congenita (DC), aplastic telomeres are progressively shortened. One of the main anemia (AA) or pulmonary fibrosis (PF) (sporadic and fa- functions of telomere structure is to avoid the recogni- milial forms) were included. These patients were diag- tion of telomeres as damaged DNA which is achieved by nosed, treated and followed in Spanish reference health inhibitory interactions between shelterin components care provider centres for these rare diseases. DC was diag- and DNA-repair proteins [7]. When several telomeres nosed in patients with the characteristic mucocutaneous reach critically short length the structure of the nucleo- symptoms, nail dystrophy, abnormal skin pigmentation, protein complex can no longer be maintained. In that oral leukoplakia and bone marrow failure. The diagnosis case, telomeres are recognized as damaged DNA and a of AA was based on bone marrow and blood cells counts. response is triggered that can result in cell-cycle arrest, The diagnosis of PF was established in accordance to cell apoptosis or senescence [8]. international guidelines [19]. The more relevant character- The presence of functional mutations in the genes cod- istics of the patients are summarized in Additional file 1: ing for proteins of the shelterin and telomerase complexes Table S1. All samples were collected after obtaining in- and auxiliary proteins causes a number of diseases named formed consent for genetic analysis, including for re- telomeropathies or telomere biology disorders that may search. Clinical and demographic data were collected. affect different organs [9, 10]. Telomeropathies are very heterogeneous diseases depending on the gene mutated Genetic analyses and the specific mutations, their penetrance and the exist- The existence of sequence variants in the TERT, TERC and ence of anticipation effects. Therefore, patients with the DKC1 genes was determined in six patients by PCR ampli- same mutation can present different manifestations. Some fication of exons, high resolution melting analyses (HRM) patients present severe symptoms at an early age such as and sequencing of candidate exons as previously described Arias-Salgado et al. Orphanet Journal of Rare Diseases (2019) 14:82 Page 3 of 12 [20]. Later on, genetic analysis of 41 patients was made by studies allowed the identification of single nucleotide vari- massive parallel sequencing using a panel of genes related ants (SNVs) and indels at TERT, TERC, RTEL1, ACD and to haematological disorders, as shown in Additional file 1: CTC1 genes in heterozygosis. Histograms of the Sanger Table S2 (MBFSv1.1 panel). Detailed protocols are pre- sequencing reaction confirming these variants are shown sented in Additional file 1: Supplemental Material. in Additional file 1: Figure S2. TERT variants were found in eight patients (Table 1). Telomere length determination AA patient 129 presented two missense variants, p.P65T Telomere length was determined by two different methods, and p.C931R. The p.P65T variant was inherited from the Southern blots of enzymatically digested DNA (Additional mother of the patient, which also presented short telo- file 1: Figure S1) and quantitative PCR. Both methods have meres. The p.C931R variant was not present in the parents been previously used for determination of telomere length of the patient. AA patient 196 presented in heterozygosis a from patients with inherited bone marrow failure [21]. De- deletion of two nucleotides at positions c.475_476 that pro- tailed protocols are presented in Additional file 1: Supple- duced a frame shift mutation after the amino acid residue mental Material. 158. One stop codon will be found 31 residues down- stream, resulting in a 189 amino acids-long protein. This Ethical statement variant was inherited from the father of the patient, which All the participants in this study provided written in- presented PF. A son of the patient that also presented this formed consent for genetic analysis. The investigation variant, died before birth. AA patient 263 carried in hetero- was conducted in accordance with the ethical standards zygosis the TERT p.V84E variant. His father also carries the of the Declaration of Helsinki and the guidelines of the variant in heterozygosis and presents PF and short telo- concerned hospitals. meres. A sister that carries the variants is presently un- affected at the age of 33 years (Fig. 1). Results PF patient F075 presented two missense variants at TERT, Single nucleotide variants in genes related to telomere p.R447C and p.R742H. The variant p.R742H has been biology already reported [23] while the p.R447C variant is described The possible genetic bases of the diseases were studied for the first time. The mother and a brother of this patient in patients that presented symptoms related to dyskera- died of PF. PF patient F185 presented the p.S602 L amino tosis congenita (DC), either mucocutaneous symptoms acid variant. PF patient F219 presented the p.R671W vari- or, more often, aplastic anemia (AA) or sporadic or fa- ant, reported to be pathogenic [24]. PF patient F220 pre- milial cases of pulmonary fibrosis (PF). Patients with sented an in-frame deletion at amino acids 671_672 that these diseases present significant telomere shortening. has not been previously described. A variant in a splicing According to previous reports [12, 22], patients with consensus site was found in patient F400. telomere length below the 1 % of the age-mated popula- Variants in TERC were found in three patients (Table 1). tion in the case of DC and < 10% in AA and PF where AA patient 156 presented in heterozygosis a deletion of included in this study. Beside clinical symptoms, DC/AA two bases (r.96_97delCU) that has been previously de- and PF patients differed at the age of presentation of the scribed [25]. The mother of the patient died with PF and disease, 12.2 + 12.2 years for DC/AA and 58.3 + 10.2 liver cirrhosis, a brother carrying this variant also presented years for PF (Additional file 1: Table S1). liver cirrhosis and short telomeres. However, a sister that First analyses were made in six patients by PCR ampli- carries the variant is presently healthy at the age of 40 years fication of exons of the DKC, TERT and TERC genes even if her telomeres are short. PF patient F120 presented using the previously described High-resolution melting the r.269G > A variant, that has not been previously re- and DNA sequencing method [20] (Patients indicated by ported. PF patient F253 presented the undescribed r.23G > an asterisk in Tables 1, 4 and Additional file 1: Table S1). C variant. PF patient F219 presented the p.P722S variant in Subsequently, taking advantage of newer technologies, CTC1 that has not been previously described. DNA samples of 41 patients were analyzed by massive Four patients presented rare variants in RTEL1 in het- sequencing of a panel of genes related to haematological erozygosis (Table 1). AA patient 90 presented the p.R998* disorders including all the genes previously associated to variant recently reported as pathogenic [26]. The father telomere-related diseases (Additional file 1: Table S2). and one sister of the patient carried this variant, and pre- Out of these patients, 23 were diagnosed of DC/AA and sented short telomeres (Fig. 1). A representative Southern 18 of PF. blot of telomeres including relatives of patient 90 is shown Nucleotide variants were further analysed when their in Additional file 1: Figure S1. In addition, patient 90 and frequency in the general population was lower than one in mother carried the p.Q929del variant, recently described a thousand. The results obtained for genes previously re- [26]. AA patient 211 presented the new pS483I variant lated to telomere shortening are shown in Table 1. These and the described p.C1244R variant [27]. In addition, Arias-Salgado et al. Orphanet Journal of Rare Diseases (2019) 14:82 Page 4 of 12 Table 1 Mutations found in telomere-related genes *Patients analyzed by exon amplification and Sanger sequencing **The prediction shown is a summary of the ones made by the programs SIFT ensemble 66, Polyphen-2 v2.2.2, MutationAssessor, release 2, FATHMM v2.3, CADD v1.3 and dbscSNV1.1. patient 211 presented the amino acid variant p.F818S in that reports several relatives affected of DC. Patients 109 PALB2 protein, also involved in telomere maintenance, as and 145 presented chromosome 7 monosomy. will be discussed later. The RTEL1 variant p.C1244R was inherited from the father and the RTEL1 variant p.S483I Single nucleotide variants in genes related to DNA repair and the PALB2 variant from the mother and both parents and homologous recombination presented average telomere length (Fig. 1). PF patient F001 Massive sequencing identified a number of rare SNVs (fre- presented the p.R998* pathogenic variant. This RTEL1 vari- quency lower than one in a thousand) in genes coding for ant was present in the three analyzed relatives of patient proteins involved in DNA repair by homologous recombin- F001, all with short telomeres (Fig. 1). PF patient F020 pre- ation, as shown in Table 2. Among these genes are those sented the described p.F988 L [15] and the non-described coding for the protein kinases ATM (Ataxia-telangiectasia p.R574C variants. mutated) and ATR (Ataxia telangiectasia and Rad3-related), Nucleotide variants in ACD, coding for the TPP1 shel- mutated in the genetic-instability syndromes Ataxia Tel- terin protein, were observed in two patients. AA patient angiectasia and Seckel syndrome, respectively. Rare variants 145 presented the p.P16Q amino acid variant and AA in these proteins were found in 35% of the DC/AA and patient 109 the p.V94I variant, both in heterozygosis. 17% of the PF patients (Table 2). Fanconi Anemia is also The variant of patient 145 is inherited from the mother due to cytogenetic instability, hypersensitivity to DNA Arias-Salgado et al. Orphanet Journal of Rare Diseases (2019) 14:82 Page 5 of 12 Fig. 1 Familiar inheritance of variants in telomere-related genes in patients with aplastic anemia and pulmonary fibrosis. The family of four affected patients is represented in panels (a-d). The probands (indicated by arrows) of families (a-c) are affected of aplastic anemia and that of family (d) of pulmonary fibrosis. The number of the family and the variants analyzed are shown in the upper part of each diagram. In the cases where several variants were studied, they are shown in the heading in the same relative order and colour as in the diagrams. The presence of the variants is represented as colour-filled squares or circles and that of the presence of the reference allele as empty figures. The number assigned to each patient or relative is indicated in the upper, right part of each symbol. The age of each individual, the determined telomere size, in kb, and the corresponding percentage of the age-matched population are shown under each symbol. Relatives affected by pulmonary fibrosis are indicated in the upper left part of their symbol (PF). Patient F001 in panel (d) had a deceased identical twin brother as indicated by joining them with a broken line crosslinking agents and defective DNA repair. This disease associated proteins BRCA1 and BRCA2 (Breast Cancer 1 can be caused by mutation in genes that code for compo- and 2) also play an important role in DNA break repair and nents of a protein complex involved in DNA repair. Rare recombination in association with PALB2 (Partner and variants in several of these genes (FANCA, FANCD2, Localizer of BRCA2). Rare variants in the BRCA1, BRCA2 FANCF, FANCL, FANCM, FANCU) were found in 22% of or PALB2 genes were found in 26% of the DC/AA and 17% the DC/AA and PF patients (Table 2). The DNA- of the PF patients (Table 2). RecQL4 is a DNA helicase (RecQ-like, type 4) involved in DNA damage repair and rare variants were found in 17 and 11% of DC/AA and PF Table 2 Frequency of rare variants in genes coding for proteins patients, respectively. Variants in the gene coding for the involved in DNA-Repair structure-specific-endonuclease subunit SLX4, involved in Dyskeratosis/ Pulmonary DNA repair and replication, were found in 22% of the DC/ Anemia Fibrosis AA and 6% of the PF patients (Table 2). Mutations in SLX4 ATM/ATR 8/23 (35%) 3/18 (17%) have been associated with Fanconi Anemia (group P). Fi- BRCA1, BRCA2, PALB2 6/23 (26%) 3/18 (17%) nally, single rare variants were found in other genes FANC-A, −D2, −F, −L, −M, −U 5/23 (22%) 4/18 (22%) coding for proteins involved in DNA repair such as RECQL4 4/23 (17%) 2/18 (11%) WRN, BLM, RAD51, RAD51C and NBN in 9 and SLX4 5/23 (22%) 1/18 (6%) 17% of the DC/AA and PF patients, respectively. The variants found in these genes for each patient WRN, BLM, NBN, RAD51, RAD51C 2/23 (9%) 3/18 (17%) are shown in Table 3. Some of these variants are Arias-Salgado et al. Orphanet Journal of Rare Diseases (2019) 14:82 Page 6 of 12 Table 3 Variants in DNA repair genes *The prediction shown is a summary of the ones made by the programs SIFT ensemble 66, Polyphen-2 v2.2.2, Mutation Assessor, release 2, FATHMM v2.3, CADD v1.3 and dbscSNV1.1. Arias-Salgado et al. Orphanet Journal of Rare Diseases (2019) 14:82 Page 7 of 12 Table 4 Genes coding for proteins involved in telomere Table 4 Genes coding for proteins involved in telomere biology and DNA repair in which rare variants were found in biology and DNA repair in which rare variants were found in the different patients and clinical evolution the different patients and clinical evolution (Continued) Patient Telomere DNA repair Clinical Patient Telomere DNA repair Clinical biology genes genes evolution biology genes genes evolution Pulmonary Fibrosis 90 RTEL1 BRCA2 HSC transplant F001 RTEL1 BRCA2 Deceased 109 ACD SLX4, Rad51C HSC transplant F010 Stable 121 ATM HSC transplant F020 RTEL1 FANCM, RAD51C Lung 129* TERT N.D. Deceased transplant 134 ATR, Mre11A Stable F021 FANCM Deceased 145 ACD BRCA2, SLX4 Deceased F025 Lung transplant 156* TERC N.D. Deceased 169 FANCM Deceased F044 ATM; RECQL4 Disease progressing 181 RECQL4 Stable F075* TERT N.D. Deceased 196* TERT N.D. Deceased F106 POT1 BRCA1 Lung 198 ATM, FANCL, Disease transplant PALB2, RAD50 progressing F120 TERC, NHP2 SLX4 Deceased 204 ATM, RECQL4 Stable F179 BRCA2 Disease 205 Stable progressing 211 RTEL1 PALB2 HSC transplant F185* TERT N.D. Deceased 223 NHP2 ATM Stable F188 RECQL4 Disease progressing 263 TERT, RTEL1 BRCA2, SLX4 HSC transplant F200 NHP2 ATR Deceased 282 SLX4 N.D. N.D. Not determined. Boldface: genes that present variants with a frequency F213 Disease between 0.001 and 0.003 in the ExAC database. progressing F219 TERT, CTC1, RTEL1 FANCU Deceased described for the first time as indicated (“Not F220 TERT FANCA Lung described” in Table 3). transplant The variants present in each patient and their clinical F242 Lung transplant evolution from diagnosis are summarized in Table 4. Two patients with previously described DKC1 muta- F244 ATR Disease progressing. tions, 26 and 36 [20], are included. This table includes some variants whose frequency is slightly higher than F253 TERC, CTC1 RAD51, RAD51C Deceased one in a thousand (indicated in bold letters). One of F382 NBN Disease progressing them is a variant in the gene POT1 (pQ301H) present at a frequency of 0.002 in the ExAC database. The change F400* TERT N.D. Waiting transplant is possibly damaging and is present in patient F106. A second variant in NHP2 (p.R101Q) is present at a fre- Dyskeratosis/ Aplastic anemia quency of 0.002 at the ExAC database. The change is 26 DKC1Ref 19 Mre11A Deceased possibly damaging, and has been detected in three pa- Ref 19 tients (F200, F120 and 223). Among the proteins in- 36 DKC1 ATR, BRCA2 Disease progressing volved in DNA repair, two patients (F20, 109) present the p.T287A variant at the RAD51C gene (frequency of 44 FANCF Stable 0.003 in the ExAC database). Other two patients (26 and 47 ATR, FANCD2, Recovered FANCL, RECQL4, SLX4 134) present the p.A492D variant at the MRE11A gene (frequency 0.002 in ExAC). 48 WRN, N.D. The possible correlation between the presence of rare 52 FANCM, BLM HSC transplant variants in proteins involved in DNA repair or replica- 62 SLX4 HSC transplant tion, telomere length and clinical manifestation was in- 69 ATM Stable vestigated in the family of several patients (Fig. 2). AA 86 RECQL4 Stable patient 134 carries one variant in the ATR gene not pre- viously described. The variant is inherited from his Arias-Salgado et al. Orphanet Journal of Rare Diseases (2019) 14:82 Page 8 of 12 Fig. 2 Familiar inheritance of variants in genes related to DNA repair and replication in patients with aplastic anemia and pulmonary fibrosis. Schematic representation of four families of patients (indicated by arrows) affected by aplastic anemia (a-c) or pulmonary fibrosis (d). The number of each family and the variants studied are shown in the upper part of each panel. The presence of the rare variant is indicated as coloured square or circles and the presence of the reference variant by open figures. In panels (c and d) where several variants were studied, their relative position in the diagrams and colour are indicated in the upper part. The number of each family member and its possible pulmonary fibrosis diagnosis (PF) is indicated in the upper part of each symbol. The age, telomere length (kb) and the corresponding percentage of the age-matched population are indicated in the lower part of each symbol mother and grandmother, both relatives have short telo- AA patient 181 present a variant in RECQL4 not de- meres. The father and maternal grandfather, that do not scribed previously. This variant is present in the father carry this variant, present an average telomere size. with very short telomeres. The mother does not carry Arias-Salgado et al. Orphanet Journal of Rare Diseases (2019) 14:82 Page 9 of 12 this variant and has longer telomeres even that below Several of the variants found had been reported previ- the average of the population (10%). ously as pathogenic. The TERT variants p.R671W and AA patient 198 presents a novel variant in ATM and a p.R742H have been described associated to PF [23, 24]. rare variant in RAD50. The ATM variant is inherited The TERC variant r.96_97delCU was described in a DC from the mother and the RAD50 p.R850Q variant from patient transmitted with a dominant pattern of inheritance the father (Fig. 2). The father present telomeres of aver- [25]. The RTEL1 variants p.Q929del [26] pF988 L [29], age size and the mother of size bellow the average. How- p.R998* [26] and p.C1244R [27] were described in DC pa- ever, the son presents relatively shorter telomeres than tients. The RTEL1 p.R574C variant has been described as the parents. a clinical variant. The other variants found in this study The PF patient F106 carries a rare variant in BRCA1 have not been previously reported and according to the and a relatively rare variant in POT1 (frequency 0.002). actual guidelines should be considered as variants of un- The patient has four brothers and three have been also di- known significance (VUS). However, we speculate that agnosed of PF. Two of them carry the same variants in some of them could be pathogenic because they are POT1 and BRCA1 and short telomeres. A sister carries closely related to described pathogenic variants, as sche- only the BRCA1 variant and very short telomeres although matically shown in Fig. 3. This is the case of TERT p.P65T is presently asymptomatic. Two of the brothers carry ex- variants since the p.P65A variant has been found in pa- clusively the POT1 mutation and present longer telomeres tients of acute myeloblastic leukemia (AML) [26]. The (25–50% of the population) although one of them has de- TERT p.V84E variant is close to the p.R83P variant, found veloped pulmonary fibrosis. The patient has a daughter, in patients with AA and myelodisplastic syndrome [30]. In which inherited the BRCA1 variant and present very short addition, the V84 residue is conserved through evolution telomeres. Two nephews and a niece carry the BRCA1 and the V > E change is considered as damaging. Other variant and have short telomeres. One niece inherited the variants produced changes potentially damaging in con- POT1 change and presents short telomeres. served residues located in functional domains of the pro- teins like the TERT variants p.F159fsX32 and p.S602 L that affect residues located in the conserved N-terminal Discussion domain (Fig. 3). The p.F159fsX32 variant would produce a The genetic variants present in patients with symptoms of truncated protein and a situation of functional hemizygo- DC, AA or PF have been studied in a population of patients sis. The variants p.R672del and pC931R are located in the from Spanish hospitals. Heterozygous variants were found reverse transcriptase and C-terminal domains of TERT, re- in genes previously related to telomeropathies in 17 pa- spectively (Fig. 3). In the case of the RTEL1 gene, the tients, 8 diagnosed of DC/AA and 9 of PF (Table 1). Two pS483I variant in a conserved residue is close to the variants in ACD were found in the only two AA patients p.P484L variant associated to PF [31]. that also presented chromosome 7 monosomy, previously The gene panel used for the analyses of the DNA of associated to the presence of short telomeres in AA pa- 41 patients by massive sequencing detected the presence tients as a consequence of chromosomal instability [28]. of numerous variants in genes coding for proteins Fig. 3 Schematic representation of the location of TERT variants on the conserved and functional domains of the protein. Conserved regions of TERT are represented as filled boxes and coloured according to their participation in the protein functional domains (shown in the lower part of the scheme). Regions of the N-terminal extension, involved in RNA binding, are coloured in blue. Regions of the reverse transcriptase domain (RT-domain) are shown in cyan and those of the C-terminal extension in green. The location of the new amino acid variants described in the article is shown in the upper part of the figure. Two previously described variants close to new variants (P65A and R83P) are shown in the lower part of the figure. The location of two variants found in this project and previously described as pathogenic are shown in the lower part inside boxes Arias-Salgado et al. Orphanet Journal of Rare Diseases (2019) 14:82 Page 10 of 12 involved in DNA repair such as ATM, ATR, BRCA1, The existence of overlapping symptoms in diseases BRCA2 and several genes of the FANC complex, mu- caused by mutations in genes coding for proteins involved tated in Fanconi Anemia. Variants in genes coding for in telomere-maintenance and in DNA-repair and replica- DNA helicases and nucleases involved in the resolution tion proteins have been recently reviewed [18]. In this art- of DNA complex structures, such as SLX4, RECQL4, icle we describe the presence of rare variants in many of WRN, BLM, RAD51, RAD51C or NBN were also found. these genes in patients that present short telomeres and Variants in these genes have not been related to telo- telomeropathies such as DC, AA and PF. We consider the meropathies previously, with the exception of SLX4 [32]. possibility that the presence of one or more variants in The proteins ATM and ATR play a signalling role in these genes in heterozygosis, some times in addition to DNA double-strand break repair pathways and their variants in telomere maintenance genes, could have a cu- continued activation can result in cell cycle arrest, repli- mulative pathogenic effect. This association could be ex- cative senescence or apoptotic cell death. However, spe- tensive to some more frequent variants in genes involved cific interactions with shelterin protein, ATM/TRF2, and in primary telomeropathies, as those observed in NHP2 or ATR/TRF1, inhibit their activation to preserve telomere POT1. This possible pathogenic association need to be structure. Therefore, it is conceivable that functional confirmed by functional studies and analyzing more ex- variants at ATM and ATR could affect telomere struc- tensive cohorts of patients. ture and stability. Indeed, cells of Ataxia telangiectasia The small number of patients analyzed prevents a stat- patients with homozygous mutations in ATM present istical analysis of the possible genotype-phenotype cor- telomere shortening and increase frequency of chromo- relation. There seems to be, however poor outcome in some end-to-end fusions (CEFs) [33]. patients that carry variants in genes related to telomere BRCA1 and BRCA2 proteins also participate in DNA biology, as shown in Table 4. For example, nine AA pa- damage repair by homologous recombination and non- tients carried variants in telomere genes and five of them homologous end joining [34] and play a protective role at died in the course of the study and the other four re- telomeres [35]. BRCA1/2 mutations result in shortening quired hematopoietic stem cell transplantation (HSCT). of the telomere single-stranded overhang and increase In contrast, only one patient died and three required telomere length variability [36]. BRCA2 heterozygous cell HSCT of the sixteen that presented short telomeres but lines present frequently CEFs [37]. Germline mutations in not rare variants in telomere related genes. Similar evo- BRCA1 and BRCA2 are associated with greatly increased lution was observed in PF patients. Nine carried muta- frequency of breast and ovarian cancers and telomere tions in telomere genes and four died and four required shortening was associated with genetic anticipation in her- lung transplant. Of the 12 patients without mutations in editary breast cancer [38]. these genes, two died and three required lung transplant. Proteins mutated in Fanconi Anemia (FA) patients are in- In particular, the two patients that carried two TERT var- volved in repair of DNA-inter-strand crosslinks and have iants evolved very quickly and both died in a few months been also involved in telomere maintenance (reviewed by after the diagnosis of the disease. One of these patients Sarkar and Liu [39]). FA patients are reported to present in- was diagnosed of AA and the other of PF. Among the creased CEFs and overall shorter telomeres [40, 41]. patients with worst evolution without mutations in tel- Replication of telomeric DNA presents specific challenges omerase genes, three presented variants in FANCM (two due to structure of the DNA with unconventional regions died and one required HSCT). However, the analysis of including T loop and G quadruplexes and requires the par- a larger cohort of patients will be required to confirm or ticipation of a large number of proteins (reviewed by Marti- discard these proposals. nez and Blasco [6]). Among them are proteins with DNA helicase activity, like RTEL1, BLM, WRN or RecQL4, endo- nuclease complexes, like the one formed by SLX4, SLX1, Conclusions MU881 and XPF and recombinases like Rad51 and Rad51C. Using massive sequencing of a panel of genes and Mutations in these genes cause diseases with some symp- Sanger sequencing of selected exons we identified novel toms resembling those of telomeopathies, as mentioned in variants in telomere biology genes in a series of patients the Background section. Many of these diseases show telo- of DC, AA and PF form Spanish hospitals. In additions mere fragility [42], telomere loss and/or increased CEFs we describe the existence of frequent rare variants in [43]. These proteins are located to telomeres through inter- genes involved in DNA damage response and the reso- actions with TRF1 of TRF2 shelterin proteins [44–48] . In lution of complex DNA structures in this population of addition, TERT expression in cells isolated from patients of patients. Achieving a molecular diagnosis from patients these diseases rescue some of their alterations [49, 50]. Var- of these telomere biology diseases would be important iants in SLX4 have been found with increased frequency in for accurate genetic counselling and effective treatment AA patients [32]. of the patients. Arias-Salgado et al. Orphanet Journal of Rare Diseases (2019) 14:82 Page 11 of 12 Additional file Hospitalario Universitario de Vigo, NeumoVigoI+i Research Group, Vigo Biomedical Research Institute (IBIV), Barcelona, Spain. 16Hospital de La Princesa, Madrid, Spain. 17Hospital Infanta Leonor, Madrid, Spain. 18CIBER de Additional file 1: Supplemental methods. Table S1A. Clinical characteristics enfermedades raras (CIBERER), Madrid, Spain. 19CIBER of Respiratory diseases of the Dyskeratosis congenita, Aplastic anemia patients. Table S1B. Clinical (CIBERES), Barcelona, Spain. characteristics of Pulmonary fibrosis patients. Table S2. Genes included in the panel used for massive sequencing. Table S3. Description of the control Received: 31 October 2018 Accepted: 12 March 2019 population used for the estimation of normal mean, telomere length and percentile for each age-range. Figure S1. Representative Southern blot used for telomere length determination. Figure S2. Histograms of the sequences obtained by Sanger sequencing of the regions of genes related to telomere biology that presented SNVs or indels. (DOC 3474 kb) References 1. McElligott R, Wellinger RJ. The terminal DNA structure of mammalian chromosomes. EMBO J. 1997;16(12):3705–14. Acknowledgments 2. de Lange T. 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