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Telomerase Mutations in Families With Idiopathic Pulmonary Fibrosis

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Peer-reviewed

Research Article

Bequeathed Mutation in Telomerase Causes Defects in Echo Addition Processivity and Manifests As Familial Pulmonary Fibrosis

  • Jonathan K. Alder,
  • Joy D. Cogan,
  • Andrew F. Brown,
  • Collin J. Anderson,
  • William E. Lawson,
  • Peter Thou. Lansdorp,
  • John A. Phillips Three,
  • James E. Loyd,
  • Julian J.-50. Chen,
  • Mary Armanios

PLOS

x

  • Published: March 31, 2011
  • https://doi.org/10.1371/journal.pgen.1001352

Abstract

The telomerase opposite transcriptase synthesizes new telomeres onto chromosome ends past copying from a curt template inside its integral RNA component. During telomere synthesis, telomerase adds multiple short DNA repeats successively, a holding known as repeat addition processivity. Even so, the consequences of defects in processivity on telomere length maintenance are not fully known. Germline mutations in telomerase cause haploinsufficiency in syndromes of telomere shortening, which most commonly manifest in the historic period-related disease idiopathic pulmonary fibrosis. We identified two pulmonary fibrosis families that share two non-synonymous substitutions in the catalytic domain of the telomerase contrary transcriptase factor hTERT: V791I and V867M. The two variants fell on the same hTERT allele and were associated with telomere shortening. Genealogy suggested that the pedigrees shared a single ancestor from the nineteenth century, and genetic studies confirmed the two families had a common founder. Functional studies indicated that, although the double mutant did not dramatically affect first echo addition, hTERT V791I-V867M showed severe defects in telomere repeat add-on processivity in vitro. Our data identify an ancestral mutation in telomerase with a novel loss-of-function mechanism. They indicate that telomere repeat add-on processivity is a disquisitional determinant of telomere length and telomere-mediated disease.

Author Summary

Mutations in the essential telomerase components cause a spectrum of diseases mediated by curt telomeres. Nigh oft, these disorders manifest in the lung in an age-related disease: idiopathic pulmonary fibrosis. Telomerase synthesizes telomere repeats using a specialized reverse transcriptase, hTERT, that copies from a brusque template within its intrinsic RNA. In order to add together long telomere tracts, telomerase adds a single repeat followed past additional repeats successively. This property, known every bit repeat addition processivity, is unique to the telomerase polymerase. We identified ii families that shared two unique variants in the catalytic domain of hTERT: V791I and V867M. The variants co-segregated, indicating they are on the same allele, and were associated with short telomeres. Family history suggested the two families may accept a single ancestor, and genetic studies confirmed they had a common founder. Telomerase reconstitution indicated that, although the double mutant did not significantly affect telomerase'due south ability to add together a single telomere repeat, hTERT 791I-867M had astringent defects in repeat addition processivity. Our data identify an ancestral mutation in telomerase; this mutation possesses a unique loss-of-function mechanism. Defects in telomere addition processivity are important determinants of telomere length maintenance and of telomere-associated disease.

Commendation: Alder JK, Cogan JD, Brownish AF, Anderson CJ, Lawson Nosotros, Lansdorp PM, et al. (2011) Ancestral Mutation in Telomerase Causes Defects in Echo Improver Processivity and Manifests As Familial Pulmonary Fibrosis. PLoS Genet 7(3): e1001352. https://doi.org/10.1371/journal.pgen.1001352

Editor: Steven E. Artandi, Stanford University, Usa of America

Received: Baronial 29, 2010; Accepted: Feb 23, 2011; Published: March 31, 2011

Copyright: © 2011 Alder et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported past a grant from the National Institutes of Health (NIH KO8 CA118416) and funding from the Doris Duke Charitable Foundation to MA; a National Science Foundation CAREER Honour (MCB0642857) to JJ-LC; NIH P01 HL092870 to JDC, JAP, and JEL; and a Vanderbilt Discovery Grant to JEL. JKA received support from the Maryland Stem Jail cell and Parker B. Francis Foundations. The funders had no role in study design, information collection and analysis, decision to publish, or training of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Telomerase is a specialized DNA polymerase that synthesizes new telomere repeats onto chromosome ends [ane], [ii]. Telomerase has two essential conserved components, a catalytic reverse transcriptase, hTERT, and an RNA component, hTR [iii], [4]. The RNA component of telomerase contains a template sequence for the addition of new telomere repeats [v]. In order to synthesize long telomere tracts, hTERT copies from the RNA template once, translocates, and then iteratively adds successive repeats [half-dozen]. This property is known as telomere repeat addition processivity [7]. Functional domains inside hTR and hTERT, too as telomerase-extrinsic factors, have been implicated in repeat add-on processivity [8]–[ten]. However, whether repeat improver processivity is critical for telomere length maintenance in vivo is not fully known.

Germline mutations in the essential telomerase components hTERT and hTR pb to a clinical spectrum of syndromes of telomere shortening (reviewed in [eleven]). Affected individuals suffer from degenerative organ failure in the bone marrow, lung and liver. In adulthood, syndromes of telomere shortening most usually manifest as progressive and irreversible scarring of the lung in an age-related disorder known every bit idiopathic pulmonary fibrosis (IPF) [xi]. Mutations in hTERT or hTR underlie the inheritance in 8–xv% of familial forms of pulmonary fibrosis and 1–3% of sporadic cases [12]–[fifteen]. This mutation frequency along with the common prevalence of IPF make pulmonary disease the most mutual manifestation of germline defects in telomerase [11]. In severe forms, syndromes of telomere shortening are clinically recognized in the premature crumbling syndrome dyskeratosis congenita where aplastic anemia is the nigh common cause of bloodshed and where at that place is an increased incidence of acute myeloid leukemia (AML) [16], [17]. AML, both de novo, and in the setting of myelodysplasia, has too been reported as a first manifestation of germline mutant telomerase genes [18], [19]. A subset of pulmonary fibrosis patients and families with short telomeres endure complications from aplastic anemia and cryptogenic liver cirrhosis [12], [thirteen], [twenty], [21]. Insights into telomerase genetics equally well as their consequences on telomerase role have therefore get intimately connected with the pathophysiology of several age-related disorders.

Heterozygous mutations in hTERT and hTR cause telomere shortening through haploinsufficiency [20]–[23]. Previously identified mutations in telomerase accept been shown to cause loss-of-role due to defects in hTR stability, essential catalytic part, and ribonucleoprotein assembly, only not echo addition processivity [13], [xx], [22], [24], [25]. We identified 2 families with familial pulmonary fibrosis where each of the affected index cases carried 2 heterozygous variants that predicted non-synonymous amino acid substitutions in the reverse transcriptase domain of hTERT. Nosotros show that these two families share a common founder, identifying an ancestral mutation in telomerase. Although the double heterozygote hTERT did not drastically affect telomerase's capacity to add nucleotides within a unmarried echo, it had astringent defects in echo addition processivity and was associated with telomere shortening. Our data betoken that inherited defects in telomerase processivity may be sufficient to contribute to telomere shortening and to a familial telomere-mediated syndrome.

Results

Not-synonymous variants in the reverse-transcriptase domain of hTERT segregate with the pulmonary fibrosis phenotype

In a screen of 75 familial pulmonary fibrosis probands for telomerase mutations, nosotros identified a proband from a family unit designated number 13 who carried 2 single nucleotide variants in hTERT. The outset was a c.2371G→A transition in exon 7, and the 2nd was c.2599G→A transition in exon 10 (Figure 1A, 1B). These predicted 2 not-synonymous substitutions in the reverse transcriptase domain: V791I and V867M, respectively (Effigy 1C). The single nucleotide variants were absent in 200 ethnically matched controls, besides every bit in a multi-ethnic control console examining the hTERT factor sequence [23]. To decide whether these nucleotide substitutions were on the aforementioned allele (i.due east. in cis) and whether they were associated with the pulmonary fibrosis phenotype, we sequenced genomic DNA from affected family members and examined the segregation. hTERT V791I and V867M were e'er present together (11 of eleven individuals beyond 3 generations) suggesting that they were on the same hTERT haplotype (Figure 2). The hTERT variants predicting V791I and V867M segregated with the pulmonary fibrosis diagnosis across four generations in all the individuals nosotros examined [north = vii, five directly sequenced, 1 obligate carrier (xiii.Ii.5) and ane likely carrier (xiii.I.one), Figure 2]. The log of the odds ratio (LOD) score of the mutant hTERT allele segregating with the pulmonary fibrosis was significant at 3.3. The segregation of the mutations with the disease phenotype indicated that this double mutant hTERT was likely illness causing.

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Figure 1. Position and conservation of not-synonymous variants in hTERT shared by pulmonary fibrosis families xiii and 143 probands.

A,B. Chromatograms of single nucleotide variants predict non-synonymous amino acid substitutions. The outset was a c.2371G→A transition in exon 7 (A), and the 2d was c.2599G→A transition in exon 10 (B). C. Panel shows conserved hTERT motifs shared with other TERTs. The non-synonymous amino acid variant residues are indicated within the reverse transcriptase domain. hTERT V791 falls in the IFD domain between the A and B motifs, and hTERT V867 is adjacent to the invariant motif C aspartic acid residues which are essential for contrary transcriptase function and are indicated past *. Alignment of TERT sequence across xiv species indicates that both V791 and V867 fall within conserved motifs.

https://doi.org/ten.1371/journal.pgen.1001352.g001

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Figure 2. Four generation pedigrees of pulmonary fibrosis probands from Families 13 and 143 of the Vanderbilt Registry.

Mutation and afflicted status are indicated past symbols shown in the key and individuals in whom Deoxyribonucleic acid was available are noted past the pedigree number in bold text. In both families, the hTERT 791I and 867 M variants co-segregate, consistent with the fact that these heterozygous substitutions are on the same allele in cis. hTERT 791I-867M too segregates with the pulmonary fibrosis phenotype in all the individuals in whom DNA was available. The symbols are identified in the key, and completely filled symbols indicate clinically affected individuals who carry the double mutant TERT.

https://doi.org/x.1371/periodical.pgen.1001352.g002

Ii pulmonary fibrosis families share mutual ancestry

In an contained screen of 24 pulmonary fibrosis families, nosotros identified a second kindred, designated family unit 143, whose proband carried the identical substitutions in hTERT. In this family, hTERT V791I and hTERT V867M also co-segregated with the pulmonary fibrosis phenotype (Figure two). Since the two variants were in cis and were rare, we reasoned that Families 13 and 143 may have a single common ancestor. To address this, we carefully queried the genealogy. Independently, members of the ii families reported lineage to an individual of the same surname who was born in 1808 in the Us. According to public census records, this ancestor had grandparents who emigrated in the eighteenth century from the British Isles. The genealogy suggested that Families 13 and 143 may exist related and that the putative mutation(southward) have been present for at least 6 generations, possibly with ancestry every bit far back as the early nineteenth century. To decide whether Families xiii and 143 shared a common founder, we genotyped polymorphic microsatellite and minisatellite sequences that flank too as autumn within the hTERT cistron (Figure S1A, S1B). In all the individuals who carried the hTERT substitutions at 791 and 867 positions, we identified a shared haplotype block which was both inside and flanked hTERT (Figure S1A, S1B). These data, together with the family histories, indicated that Families 13 and 143 shared a common ancestor who carried the double mutant hTERT allele.

hTERT 791I-867M causes defects in repeat addition processivity in vitro

To determine the functional significance of the hTERT 791 and 867 variants, we first examined the evolutionary conservation of the hTERT V791 and V867 residues. hTERT V791 barbarous within the insertion in finger domain (IFD) between the A and B motifs of the contrary transcriptase domain [10], [26], a telomerase specific motif (Figure 1B, 1C). hTERT V867 fell within the universal reverse transcriptase motif C, and was adjacent to the invariant aspartic acid residues which are essential for the catalytic role of telomerase and other reverse transcriptases [4] (Effigy 1B, 1C). The sequence alignment from representative species showed that these 2 residues are mostly conserved as hydrophobic amino acids in nigh organisms, and are therefore potentially important for telomerase function.

To directly examine whether the variant hTERT affects telomerase activity, we reconstituted the mutant telomerase and measured enzyme activity in vitro. At standard assay conditions of ane mM nucleotide concentrations [eight], [12], [13], [twenty], [27], the 791I alone did not have obvious defects in activity or processivity (Figure 3A, lane 3). A minor inter-repeat interruption was present for 867M; this has been previously suggested to be due to nucleotide analogousness defects [28] (Figure 3A, lane 4). This break was also present in the 791I-867M double mutant (Effigy 3A, lane v). However, the overall activity and processivity of 867M and 791I-867M were not affected (Figure 3A, lanes iv and 5). For comparing, nosotros measured the activeness of a known hTERT mutation L55Q previously identified in a family with pulmonary fibrosis [13]. This mutant showed a significant decrease in overall action (Figure 3A, lane 2). Since the segregation and the genetic evidence supported the 791I-867M being a pathogenic allele, we assayed its function at nucleotide concentrations that are closer to the estimated Km for telomerase [29], [xxx]. The lower concentrations also more closely mimic estimates of intra-nuclear nucleotide concentrations (ten µM) [31]–[33]. Under these atmospheric condition, there was niggling effect on the synthesis of the commencement repeat compared with wildtype telomerase (Figure 3A–3D). Nosotros next measured repeat addition processivity. Notably, hTERT 867M and the double mutant hTERT had significant decreases in repeat improver processivity (Figure 3A and 3E). The decreased repeat addition processivity was not seen for the 55Q and 791I alleles (Figure 3A and 3E). This decrease was evident by the lower intensity of high molecular weight repeat products relative to the first product (Figure 3A, lane 6 compared with lanes 9 and x). For example, by the fourth repeat, both hTERT 867M and 791I-867M had approximately a 10-fold reduction in telomere product compared with wildtype telomerase (Figure 3E). The decrease in processivity was independent of the hTERT epitope tag used in affinity purification, every bit we saw the same degree of damage for 867M and 791I-867M whether a C-terminal HA or N-terminal FLAG epitope was used (not shown). To exclude the possibility that the double mutant hTERT may have dominant negative effects, we performed mixing studies of wildtype and 791I-867M and institute no additional decreases in activity or processivity (not shown). These data indicated that although hTERT 791I and 867M co-segregate with the disease phenotype, the 867M mutation appears to exist the pathogenic variant and predominantly affects telomere repeat addition processivity in vitro.

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Figure three. The ancestral mutant telomerase affects repeat add-on processivity in vitro.

A. Telomerase activity assay of non-synonymous hTERT variants identified in family thirteen and 143 probands. L55Q was previously identified in a pulmonary fibrosis family unit and known to compromise catalysis. Telomerase activity assay at loftier nucleotide (ane mM) concentrations on the left shows no defects in catalytic activeness or processivity for hTERT V791I and V867M or the double mutant. At lower nucleotide concentration (ten µM), hTERT 867M and hTERT V791I-V867M both show defects in repeat addition processivity equally evidenced past the decreased intensity of the high molecular weight products relative to the first repeat. Depression exposure image of the internal loading control is shown beneath. B. Depression exposure epitome of the gel shown in (A) is shown to visualize the +ane, +2, and +3 nucleotide bands conspicuously. C. SDS-PAGE of 35S labeled hTERT used in (A) to monitor the expression of in vitro synthesized hTERTs. D. Quantitation of showtime echo add-on as measured by the total intensity of the +1, +two, +3, and +4 nucleotide bands. Quantitation is based on 3 contained experiments. * Indicates P-value <0.01 and fault bars bespeak standard error of the mean. E. Quantitation of processivity across the first 4 repeats (R1, R2, R3 and R4) is shown past the linear regression line.

https://doi.org/10.1371/journal.pgen.1001352.g003

Mutant TERT is associated with short telomere length

Telomerase haploinsufficiency causes telomere shortening and the severity of the consistent phenotypes correlates with the telomere length [20], [34]. To examine if the mutant telomerase is associated with telomere shortening, we measured telomere length in family members using combined menses cytometry and fluorescence in situ hybridization (period-FISH) [12], [xiii]. In all cases, mutation carriers had lymphocyte telomere lengths beneath the tenth percentile of a normal distribution compared with age-matched controls (Figure four). In 6 of 9 mutation carriers, the telomere length barbarous below the 1st percentile, a range that is highly specific for the presence of a germline telomere maintenance defect [12], [13], [35] (Effigy 4, P<0.001, paired t-exam). Therefore the mutant hTERT is associated with telomere shortening in mutation carriers.

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Figure four. Telomere length in mutation carriers in families 13 and 143 have short telomeres compared to age-matched controls.

Console shows telomere length as measured in lymphocytes by menstruum-FISH compared to normal distribution of age-matched controls. Percentiles are based on telomere length information from 400 controls. Squares refer to males and circles refer to females. Individuals refer to pedigree position in Figure 2. Individual 13III.eight has brusque telomeres and was diagnosed with an overlap syndrome of emphysema and pulmonary fibrosis.

https://doi.org/10.1371/journal.pgen.1001352.g004

A spectrum of telomere-mediated disease is associated with ancestral TERT mutation

Syndromes of telomere shortening manifest as degenerative affliction in the lung, liver and os marrow, and a subset of pulmonary fibrosis families falls on this spectrum [11], [13]. To examine whether the mutant TERT leads to the full spectrum of telomere-mediated affliction, nosotros characterized the clinical phenotypes in families 13 and 143. Of the 18 genetically affected individuals in the ii pedigrees, eleven had pulmonary disease. In the majority of cases (7 of eleven, 64%), the interstitial lung disease met the criteria for usual interstitial pneumonia/IPF (Figure 5A, 5B and Table S1). In affected individuals, the onset of disease was in adulthood with a mean age of 56 (range 32–67). At that place was very subtle genetic anticipation for the age at expiry across the generations nosotros could examine (e.yard. age 61 and mean historic period 56 for generations I and 2 respectively in family thirteen, Tabular array S1). Two individuals at the age of l and 51 reported chronic liver part abnormalities that were unexplained subsequently a thorough work-upwardly. We identified subclinical cytopenias in ane individual (age 50), and one individual was diagnosed with AML at age 67 and subsequently died from complications of interstitial lung affliction (Table S1). We clinically examined the probands and their relatives for the typical mucocutaneous features of dyskeratosis congenita but did not identify whatsoever signs of skin hyperpigmentation, smash dystrophy or oral leukoplakia. Therefore the telomerase defect we identified in families 13 and 143 appears to primarily cause developed-onset phenotypes. These phenotypes are clinically well-nigh prominent in the lung, simply features of the total spectrum of a telomere syndrome manifest at lower frequency including subclinical cytopenias, liver function abnormalities and AML.

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Effigy 5. CAT scans from pulmonary fibrosis probands and non-carrier sibling with short telomeres.

A,B show lower thoracic CAT scan images from the probands in family 13 and 143. Both images show basilar honeycombing typical of idiopathic pulmonary fibrosis. C,D are apical and lower thoracic CAT scan images respectively of sibling with short telomere who does not carry the mutant hTERT (Private designated 13III.viii in Figure 2 and Figure iii). This private has apical changes consistent with centrilobular emphysema every bit well as lower thoracic footing glass changes consequent with an interstitial process.

https://doi.org/10.1371/journal.pgen.1001352.g005

Unaffected siblings of mutation carriers accept short telomeres

Telomere length is a heritable trait and parental telomere length determines offspring telomere length even when telomerase is wildtype [34], [36], [37]. In a large dyskeratosis congenita family, siblings of mutation carriers were shown to have short telomeres; nonetheless telomere-related phenotypes in these individuals accept not been previously reported [38]. Since the hTERT 791I-867M mutation was associated with brusk telomeres, we examined whether their non-mutation carrier relatives may also have short telomeres. In 4 individuals nosotros examined, the lymphocyte telomere length was beneath the tenth percentile compared with age-matched controls, and in ii individuals, the telomere length fell at or below the 1st percentile (Figure 4, P = 0.039, paired t-test). To determine whether brusk telomeres may be a run a risk factor for developing telomere-mediated illness, we examined the clinical phenotypes of the individuals who did not carry the hTERT 791I-867M allele only who had brusque telomeres. Nosotros identified one patient who presented to our clinic with shortness of breath at the historic period of 53. His history was pregnant for a lifelong history of cigarette use (greater than 50 pack-years). The patient'due south CAT scan showed a mixed picture of interstitial lung affliction with ground glass infiltrates and emphysema (Figure 5C, 5D), and lung biopsy confirmed the presence of interstitial fibrosis on the groundwork of bronchiolitis. Although cigarette fume has been shown to be associated with brusque telomeres and is known to contribute to the risk of both emphysema and pulmonary fibrosis [39], information technology is intriguing to consider the possibility that parental telomere length may accept contributed to the telomere shortening in this individual and to his risk of lung illness.

Discussion

Echo addition processivity is a unique biochemical attribute of the telomerase reverse transcriptase, and here we testify it may be disquisitional for telomere maintenance in vivo. Telomerase-intrinsic and extrinsic factors have been implicated in repeat improver processivity [8]–[10], and our study suggests that inherited defects that affect this unique property of the telomerase enzyme may contribute to telomere length heterogeneity and to telomere-mediated disease. Although individuals in the 2 kindreds we describe carried two in cis variants in hTERT, our biochemical studies suggest that hTERT 867M is likely the functionally important mutation. Equally such, the hTERT 791I rare variant may serve as a useful genetic marker and, along with the 867M, tin place other families with shared ancestry to the families nosotros report herein. Unmarried nucleotide titration studies have implicated hTERT V867 to exist important in telomerase function [28]. Studies of the Tetrahymena thermophila TERT have also implicated the orthologous residuum adjacent to V867 in echo addition processivity [forty]. These observations, along with our findings, indicate that residues within motif C of the telomerase reverse transcriptase domain are important determinants of telomere repeat addition processivity. Several mechanisms of telomerase haploinsufficiency have been previously reported for disease causing mutations including loss of hTR stability, impaired association of hTR with hTERT, and loss of catalytic function [thirteen], [20], [22], [24], [25]. In this study, the strong genetic testify linking the mutant TERT to a known telomere-mediated affliction, and the show of telomere shortening in vivo, indicate that the mutant TERT affects telomere maintenance. Our in vitro biochemical studies show that the mutant TERT is defective in echo improver processivity, pointing to this as the likely mechanism for the loss of telomerase function and the consequent organ failure.

We report on an ancestral mutation in hTERT which manifested independently in two pulmonary fibrosis families. To our noesis, hTERT 791I-867M is the most ancient telomerase mutation, and it is probable that other kindreds with familial pulmonary fibrosis and other features of telomere syndromes will be subsequently found to share ancestry with these pedigrees. In contrast to a family unit with a functionally nix hTERT mutation where genetic apprehension was hit and caused a ii decade earlier onset of disease beyond each generation [20], hTERT 791I-867M causes just subtle anticipation across the generation spans we studied. This ascertainment suggests that the extent of genetic anticipation may correlate with the degree of telomerase loss-of-part thus making genetic apprehension more than difficult to detect beyond consecutive generations in families that behave hypomorphic mutations. Telomerase preferentially elongates the shortest telomeres [41], [42], and our functional studies which prove intact single echo synthesis, point to the fact that the ancestral hTERT 791I-867M may have the capacity to add initial telomere tracts, thus healing the shortest telomeres. Nonetheless, with successive telomere repeats, telomere addition is less efficient. Loss of telomere repeat add-on processivity may therefore exist a manifestation of more than hypomorphic mutations, and therefore adult-onset phenotypes, equally the shortest telomeres may still be elongated, albeit with shorter telomere tracts.

In this multi-generation study, although pulmonary fibrosis was the most mutual manifestation in mutation carriers, several individuals had extra-pulmonary manifestations of telomere-mediated disease. One individual had avascular necrosis and macrocytosis, two individuals reported history of cryptogenic liver office abnormalities, and one patient had a history of AML prior to the diagnosis of interstitial lung disease. Bone marrow failure, avascular necrosis and cryptogenic liver cirrhosis are all known complications of dyskeratosis congenita [17], and pulmonary fibrosis families with mutant telomerase genes have been known to have an increased incidence of aplastic anemia, a common complication of dyskeratosis congenita [thirteen]. AML, often arising in the setting of myelodysplasia, has been recently reported as a first manifestation of mutant telomerase genes [18], [19], and it is possible that families with pulmonary fibrosis due to telomerase deficiency also have an increased incidence of AML. In 8 consecutive pulmonary fibrosis families with known hTR or hTERT mutations, including the ii nosotros report herein, in that location was a full of three first caste relatives of IPF probands who died with AML at ages 25, 59, 68. Dyskeratosis congenita patients are known to have an increased incidence of AML [16]. These observations highlight the fact that a subset of families with pulmonary fibrosis falls on the same spectrum as dyskeratosis congenita and that the diagnosis of telomere syndrome in these patients is relevant to their clinical work-upwards and surveillance. Pulmonary fibrosis patients should be queried most a personal or family unit history of AML, along with aplastic anemia, as part of the screening history for a telomere syndrome.

In summary, an ancestral mutation within the reverse transcriptase domain of telomerase manifests as familial pulmonary fibrosis and causes defects in telomere repeat improver processivity. Genetic factors that affect repeat addition processivity may be of import determinants of telomere length heterogeneity across populations, and can contribute to understanding the inherited ground of telomere-mediated disease.

Methods

Subjects and ideals statement

Subjects were recruited through the Vanderbilt Familial Pulmonary Fibrosis Registry and gave written informed consent [thirteen]. The report was approved by the institutional review boards of both Vanderbilt and Johns Hopkins Universities. The probands from Families 13 and 143, and the majority of mutation carrier and non-carrier relatives were clinically evaluated. Primary medical records were used to certificate the diagnoses listed in Tabular array S1. We used the consensus nomenclature to phenotype the idiopathic interstitial lung disease [43].

Genotyping and telomere length measurement

Genomic DNA was extracted from peripheral blood using standard methods. We sequenced hTERT [xiii] and confirmed variants bidirectionally. Command DNA was obtained from Corriel Repository with self-reported Northern European ethnicity, similar to the families nosotros studied. We used Merlin to calculate a single signal LOD score [44], under the supposition of autosomal dominant inheritance and a 1/10,000 population frequency of idiopathic interstitial lung illness. We determined allele size of microsatellites D5S1981 (Frontward-cctgtaccaatccatgc, Contrary-gagccatgtgagtgtcc) and D5S2005 (Frontwards-cctcaggtgggttattgac, Opposite-cccagggctttacgagt) using fluorescent labeled frontwards primers obtained from Qiagen (Valencia, CA). PCR products were analyzed on the ABI Genome Analyzer musical instrument (Applied Biosystems). Pherograms were interpreted manually to make up one's mind allele size. We amplified and genotyped mini-satellites/variable number of tandem repeats within hTERT: 2–2 (intron two) and 6–ane (intron 6) as published [45]. We determined the number of tandem repeats using gel electrophoresis. Telomere length was measured by menstruation-FISH on peripheral blood mononuclear cells [13].

Telomerase activity assay

TERT protein alignment was generated using CLUSTALW followed by BoxShade assay (v.3.21), and we used NP_937983 for the hTERT protein sequence. TERT sequences were obtained from http://telomerase.asu.edu [46]. To examination the action and processivity of the telomerase mutants, we expressed each of them in vitro and quantified part using a direct telomerase activity assay as previously described [10]. All telomerase variants were reconstituted using the TNT (transcription and translation) Quick Coupled rabbit reticulocyte lysate system (Promega) following manufacturer'due south instructions. Briefly, recombinant N-FLAG tagged hTERT was expressed in 10 µL of TNT lysate with 35S labeled methionine at 30°C for threescore minutes. To obtain active telomerase, in vitro transcribed hTR pseudoknot (nt 32–195) and CR4-CR5 (nt 239–328) fragments were each added to a concentration of 8 µM and incubated at 30°C for xxx minutes. To avoid variations in the quality of telomerase reconstituted, the wildtype and variant hTERT proteins were all expressed from the same batch of TNT lysate and the reconstituted enzymes were assayed immediately without freezing. To analysis the activity and processivity of each telomerase variant, a 10 µl reaction was carried out using 3 µl of in vitro reconstituted telomerase in the presence of 1x PE buffer (l mM Tris-HCl, pH 8.3, l mM KCl, 2 mM DTT, three mM MgCl2, and i mM spermidine) and 2 pmol of v′-32P cease-labeled (TTAGGG)3 telomere primer at 30°C for ane h. Deoxynucleotides (dATP, dTTP, and dGTP) were likewise included at concentrations of either one mM or 10 µM, as indicated. Reactions were terminated by phenol-chloroform extraction followed by ethanol precipitation earlier beingness resolved on a 10% denaturing polyacrylamide gel. To quantitate the first repeat production, we measured product intensity at depression exposure to conspicuously visualize the +i, +ii, +3, +four nucleotides (every bit shown in Figure 3B), and normalized to the amount of unpolymerized oligonucleotide loading control after subtracting background. Telomerase processivity was calculated past measuring the intensity of each repeat band, normalized to the intensity of the first echo, and plotted confronting the repeat number [ix].

Supporting Information

Figure S1.

Pulmonary fibrosis families 13 and 143 share a mutual haplotype cake flanking and within the hTERT locus. A. Pedigrees of Families 13 and 143 with numbers below indicate genotype at microsatellite D5S1981, variable number of tandem repeats (VNTR) 6–one in intron 6, and 2–2 in intron two, as well as microsatellite D5S2005, respectively from top to bottom. The shared haplotype block is shown in bold and is shared by all mutation carriers in families xiii and 143. * Refers to a mutation carrier who shares the mutual haplotype block except at D5S1981 where the allele is shorter by a unmarried dinucleotide repeat; this may be related to polymerase slippage or a recombination upshot. The symbols are identified in the cardinal, and completely filled symbols betoken clinically affected individuals who comport the double mutant TERT. B. Schema of hTERT locus indicating the location of the genotyped micro- and mini-satellites genotyped in this study. The vertical blocks inside the hTERT locus represent the 16 exons within the hTERT gene.

https://doi.org/10.1371/journal.pgen.1001352.s001

(1.51 MB EPS)

Acknowledgments

Nosotros are grateful to the family unit members who participated in this study. We are thankful to Dr. Ballad Greider for critical reading of the manuscript. We are grateful to Erin Parry for helpful discussions, Mingyi Xie for the telomerase constructs, Cheryl Markin for assist with coordinating the study, and Cate Kiefe for help with the pedigree drawings.

Author Contributions

Conceived and designed the experiments: JKA JEL JJLC MA. Performed the experiments: JKA JDC AFB CJA. Analyzed the data: JKA JDC AFB CJA WEL JEL JJLC MA. Contributed reagents/materials/analysis tools: JDC PML JAP JEL JJLC MA. Wrote the paper: MA.

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