Genetic analyses of aplastic anemia and idiopathic pulmonary fibrosis patients with short telomeres, possible implication of DNA-repair genes

Patients with severe telomere shortening (percentile < 10) who presented dyskeratosis congenita (DC), aplastic anemia (AA) or pulmonary fibrosis (PF) (sporadic and familial forms) were included. These patients were diagnosed, treated and followed in Spanish reference health care provider centres for these rare diseases. DC was diagnosed in patients with the characteristic mucocutaneous symptoms, nail dystrophy, abnormal skin pigmentation, oral leukoplakia and bone marrow failure. The diagnosis of AA was based on bone marrow and blood cells counts. The diagnosis of PF was established in accordance to international guidelines [19]. The more relevant characteristics of the patients are summarized in Additional file 1: Table S1. All samples were collected after obtaining informed consent for genetic analysis, including for research. Clinical and demographic data were collected.

The existence of sequence variants in the TERT, TERC and DKC1 genes was determined in six patients by PCR amplification of exons, high resolution melting analyses (HRM) and sequencing of candidate exons as previously described [20]. Later on, genetic analysis of 41 patients was made by massive parallel sequencing using a panel of genes related to haematological disorders, as shown in Additional file 1: Table S2 (MBFSv1.1 panel). Detailed protocols are presented in Additional file 1: Supplemental Material.

Telomere length was determined by two different methods, Southern blots of enzymatically digested DNA (Additional file 1: Figure S1) and quantitative PCR. Both methods have been previously used for determination of telomere length from patients with inherited bone marrow failure [21]. Detailed protocols are presented in Additional file 1: Supplemental Material.

All the participants in this study provided written informed consent for genetic analysis. The investigation was conducted in accordance with the ethical standards of the Declaration of Helsinki and the guidelines of the concerned hospitals.

The possible genetic bases of the diseases were studied in patients that presented symptoms related to dyskeratosis congenita (DC), either mucocutaneous symptoms or, more often, aplastic anemia (AA) or sporadic or familial cases of pulmonary fibrosis (PF). Patients with these diseases present significant telomere shortening. According to previous reports [12, 22], patients with telomere length below the 1 % of the age-mated population in the case of DC and < 10% in AA and PF where included in this study. Beside clinical symptoms, DC/AA and PF patients differed at the age of presentation of the disease, 12.2 + 12.2 years for DC/AA and 58.3 + 10.2 years for PF (Additional file 1: Table S1).

First analyses were made in six patients by PCR amplification of exons of the DKC, TERT and TERC genes using the previously described High-resolution melting and DNA sequencing method [20] (Patients indicated by an asterisk in Tables 1, 4 and Additional file 1: Table S1). Subsequently, taking advantage of newer technologies, DNA samples of 41 patients were analyzed by massive sequencing of a panel of genes related to haematological disorders including all the genes previously associated to telomere-related diseases (Additional file 1: Table S2). Out of these patients, 23 were diagnosed of DC/AA and 18 of PF.

Nucleotide variants were further analysed when their frequency in the general population was lower than one in a thousand. The results obtained for genes previously related to telomere shortening are shown in Table 1. These studies allowed the identification of single nucleotide variants (SNVs) and indels at TERT, TERC, RTEL1, ACD and CTC1 genes in heterozygosis. Histograms of the Sanger sequencing reaction confirming these variants are shown in Additional file 1: Figure S2.

TERT variants were found in eight patients (Table 1). AA patient 129 presented two missense variants, p.P65T and p.C931R. The p.P65T variant was inherited from the mother of the patient, which also presented short telomeres. The p.C931R variant was not present in the parents of the patient. AA patient 196 presented in heterozygosis a deletion of two nucleotides at positions c.475_476 that produced a frame shift mutation after the amino acid residue 158. One stop codon will be found 31 residues downstream, resulting in a 189 amino acids-long protein. This variant was inherited from the father of the patient, which presented PF. A son of the patient that also presented this variant, died before birth. AA patient 263 carried in heterozygosis the TERT p.V84E variant. His father also carries the variant in heterozygosis and presents PF and short telomeres. A sister that carries the variants is presently unaffected at the age of 33 years (Fig. 1).

PF patient F075 presented two missense variants at TERT, p.R447C and p.R742H. The variant p.R742H has been already reported [23] while the p.R447C variant is described for the first time. The mother and a brother of this patient died of PF. PF patient F185 presented the p.S602 L amino acid variant. PF patient F219 presented the p.R671W variant, reported to be pathogenic [24]. PF patient F220 presented an in-frame deletion at amino acids 671_672 that has not been previously described. A variant in a splicing consensus site was found in patient F400.

Variants in TERC were found in three patients (Table 1). AA patient 156 presented in heterozygosis a deletion of two bases (r.96_97delCU) that has been previously described [25]. The mother of the patient died with PF and liver cirrhosis, a brother carrying this variant also presented liver cirrhosis and short telomeres. However, a sister that carries the variant is presently healthy at the age of 40 years even if her telomeres are short. PF patient F120 presented the r.269G > A variant, that has not been previously reported. PF patient F253 presented the undescribed r.23G > C variant. PF patient F219 presented the p.P722S variant in CTC1 that has not been previously described.

Four patients presented rare variants in RTEL1 in heterozygosis (Table 1). AA patient 90 presented the p.R998* variant recently reported as pathogenic [26]. The father and one sister of the patient carried this variant, and presented short telomeres (Fig. 1). A representative Southern blot of telomeres including relatives of patient 90 is shown in Additional file 1: Figure S1. In addition, patient 90 and mother carried the p.Q929del variant, recently described [26]. AA patient 211 presented the new pS483I variant and the described p.C1244R variant [27]. In addition, patient 211 presented the amino acid variant p.F818S in PALB2 protein, also involved in telomere maintenance, as will be discussed later. The RTEL1 variant p.C1244R was inherited from the father and the RTEL1 variant p.S483I and the PALB2 variant from the mother and both parents presented average telomere length (Fig. 1). PF patient F001 presented the p.R998* pathogenic variant. This RTEL1 variant was present in the three analyzed relatives of patient F001, all with short telomeres (Fig. 1). PF patient F020 presented the described p.F988 L [15] and the non-described p.R574C variants.

Massive sequencing identified a number of rare SNVs (frequency lower than one in a thousand) in genes coding for proteins involved in DNA repair by homologous recombination, as shown in Table 2. Among these genes are those coding for the protein kinases ATM (Ataxia-telangiectasia mutated) and ATR (Ataxia telangiectasia and Rad3-related), mutated in the genetic-instability syndromes Ataxia Telangiectasia and Seckel syndrome, respectively. Rare variants in these proteins were found in 35% of the DC/AA and 17% of the PF patients (Table 2). Fanconi Anemia is also due to cytogenetic instability, hypersensitivity to DNA crosslinking agents and defective DNA repair. This disease can be caused by mutation in genes that code for components of a protein complex involved in DNA repair. Rare variants in several of these genes (FANCA, FANCD2, FANCF, FANCL, FANCM, FANCU) were found in 22% of the DC/AA and PF patients (Table 2). The DNA- associated proteins BRCA1 and BRCA2 (Breast Cancer 1 and 2) also play an important role in DNA break repair and recombination in association with PALB2 (Partner and Localizer of BRCA2). Rare variants in the BRCA1, BRCA2 or PALB2 genes were found in 26% of the DC/AA and 17% 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 patients, respectively. Variants in the gene coding for the structure-specific-endonuclease subunit SLX4, involved in DNA repair and replication, were found in 22% of the DC/AA and 6% of the PF patients (Table 2). Mutations in SLX4 have been associated with Fanconi Anemia (group P). Finally, single rare variants were found in other genes coding for proteins involved in DNA repair such as WRN, BLM, RAD51, RAD51C and NBN in 9 and 17% of the DC/AA and PF patients, respectively. The variants found in these genes for each patient are shown in Table 3. Some of these variants are described for the first time as indicated (“Not described” in Table 3).

The variants present in each patient and their clinical evolution from diagnosis are summarized in Table 4. Two patients with previously described DKC1 mutations, 26 and 36 [20], are included. This table includes some variants whose frequency is slightly higher than one in a thousand (indicated in bold letters). One of them is a variant in the gene POT1 (pQ301H) present at a frequency of 0.002 in the ExAC database. The change is possibly damaging and is present in patient F106. A second variant in NHP2 (p.R101Q) is present at a frequency of 0.002 at the ExAC database. The change is possibly damaging, and has been detected in three patients (F200, F120 and 223). Among the proteins involved in DNA repair, two patients (F20, 109) present the p.T287A variant at the RAD51C gene (frequency of 0.003 in the ExAC database). Other two patients (26 and 134) present the p.A492D variant at the MRE11A gene (frequency 0.002 in ExAC).

The possible correlation between the presence of rare variants in proteins involved in DNA repair or replication, telomere length and clinical manifestation was investigated in the family of several patients (Fig. 2). AA patient 134 carries one variant in the ATR gene not previously described. The variant is inherited from his mother and grandmother, both relatives have short telomeres. The father and maternal grandfather, that do not carry this variant, present an average telomere size.

AA patient 198 presents a novel variant in ATM and a rare variant in RAD50. The ATM variant is inherited from the mother and the RAD50 p.R850Q variant from the father (Fig. 2). The father present telomeres of average size and the mother of size bellow the average. However, the son presents relatively shorter telomeres than the parents.

The PF patient F106 carries a rare variant in BRCA1 and a relatively rare variant in POT1 (frequency 0.002). The patient has four brothers and three have been also diagnosed of PF. Two of them carry the same variants in POT1 and BRCA1 and short telomeres. A sister carries only the BRCA1 variant and very short telomeres although is presently asymptomatic. Two of the brothers carry exclusively the POT1 mutation and present longer telomeres (25–50% of the population) although one of them has developed pulmonary fibrosis. The patient has a daughter, which inherited the BRCA1 variant and present very short telomeres. Two nephews and a niece carry the BRCA1 variant and have short telomeres. One niece inherited the POT1 change and presents short telomeres.