Genome instability is a consequence of transcription deficiency in patients with bone marrow failure harboring biallelic ERCC6L2 variants.

Biallelic variants in the ERCC excision repair 6 like 2 gene (ERCC6L2) are known to cause bone marrow failure (BMF) due to defects in DNA repair and mitochondrial function. Here, we report on eight cases of BMF from five families harboring biallelic variants in ERCC6L2, two of whom present with myelodysplasia. We confirm that ERCC6L2 patients' lymphoblastoid cell lines (LCLs) are hypersensitive to DNA-damaging agents that specifically activate the transcription coupled nucleotide excision repair (TCNER) pathway. Interestingly, patients' LCLs are also hypersensitive to transcription inhibitors that interfere with RNA polymerase II (RNA Pol II) and display an abnormal delay in transcription recovery. Using affinity-based mass spectrometry we found that ERCC6L2 interacts with DNA-dependent protein kinase (DNA-PK), a regulatory component of the RNA Pol II transcription complex. Chromatin immunoprecipitation PCR studies revealed ERCC6L2 occupancy on gene bodies along with RNA Pol II and DNA-PK. Patients' LCLs fail to terminate transcript elongation accurately upon DNA damage and display a significant increase in nuclear DNA-RNA hybrids (R loops). Collectively, we conclude that ERCC6L2 is involved in regulating RNA Pol II-mediated transcription via its interaction with DNA-PK to resolve R loops and minimize transcription-associated genome instability. The inherited BMF syndrome caused by biallelic variants in ERCC6L2 can be considered as a primary transcription deficiency rather than a DNA repair defect.

ERCC6L2 mutations link a distinct bone-marrow-failure syndrome to DNA repair and mitochondrial function.

Exome sequencing was performed in three index cases with bone marrow failure and neurological dysfunction and whose parents are first-degree cousins. Homozygous truncating mutations were identified in ERCC6L2 in two of the individuals. Both of these mutations affect the subcellular localization and stability of ERCC6L2. We show here that knockdown of ERCC6L2 in human A549 cells significantly reduced their viability upon exposure to the DNA-damaging agents mitomycin C and Irofulven, but not etoposide and camptothecin, suggesting a role in nucleotide excision repair. ERCC6L2-knockdown cells also displayed H2AX phosphorylation, which significantly increased upon genotoxic stress, suggesting an early DNA-damage response. Intriguingly, ERCC6L2 was seen to translocate to the mitochondria and the nucleus in response to DNA damage, and ERCC6L2 knockdown induced intracellular reactive oxygen species (ROS). Treatment with the ROS scavenger N-acetyl cysteine attenuated the Irofulven-induced cytotoxicity in ERCC6L2-knockdown cells and abolished ERCCGL2 traffic to the mitochondria and nucleus in response to this DNA-damaging agent. Collectively, these observations identify a distinct bone-marrow-failure syndrome due to mutations in ERCC6L2, a gene implicated in DNA repair and mitochondrial function.

Constitutional mutations in RTEL1 cause severe dyskeratosis congenita.

Dyskeratosis congenita (DC) and its phenotypically severe variant, Hoyeraal-Hreidarsson syndrome (HHS), are multisystem bone-marrow-failure syndromes in which the principal pathology is defective telomere maintenance. The genetic basis of many cases of DC and HHS remains unknown. Using whole-exome sequencing, we identified biallelic mutations in RTEL1, encoding a helicase essential for telomere maintenance and regulation of homologous recombination, in an individual with familial HHS. Additional screening of RTEL1 identified biallelic mutations in 6/23 index cases with HHS but none in 102 DC or DC-like cases. All 11 mutations in ten HHS individuals from seven families segregated in an autosomal-recessive manner, and telomere lengths were significantly shorter in cases than in controls (p = 0.0003). This group had significantly higher levels of telomeric circles, produced as a consequence of incorrect processing of telomere ends, than did controls (p = 0.0148). These biallelic RTEL1 mutations are responsible for a major subgroup (∼29%) of HHS. Our studies show that cells harboring these mutations have significant defects in telomere maintenance, but not in homologous recombination, and that incorrect resolution of T-loops is a mechanism for telomere shortening and disease causation in humans. They also demonstrate the severe multisystem consequences of its dysfunction.

Exome sequencing identifies autosomal-dominant SRP72 mutations associated with familial aplasia and myelodysplasia.

Aplastic anemia (AA) and myelodysplasia (MDS) are forms of bone marrow failure that are often part of the same progressive underlying disorder. While most cases are simplex and idiopathic, some show a clear pattern of inheritance; therefore, elucidating the underlying genetic cause could lead to a greater understanding of this spectrum of disorders. We used a combination of exome sequencing and SNP haplotype analysis to identify causative mutations in a family with a history of autosomal-dominant AA/MDS. We identified a heterozygous mutation in SRP72, a component of the signal recognition particle (SRP) that is responsible for the translocation of nascent membrane-bound and excreted proteins to the endoplasmic reticulum. A subsequent screen revealed another autosomal-dominant family with an inherited heterozygous SRP72 mutation. Transfection of these sequences into mammalian cells suggested that these proteins localize incorrectly within the cell. Furthermore, coimmunoprecipitation of epitope-tagged SRP72 indicated that the essential RNA component of the SRP did not fully associate with one of the SRP72 variants. These results suggest that inherited mutations in a component of the SRP have a role in the pathophysiology of AA/MDS, identifying a third pathway for developing these disorders alongside transcription factor and telomerase mutations.

Aberrant 3' oligoadenylation of spliceosomal U6 small nuclear RNA in poikiloderma with neutropenia.

The recessive disorder poikiloderma with neutropenia (PN) is caused by mutations in the C16orf57 gene that encodes the highly conserved USB1 protein. Here, we present the 1.1 Å resolution crystal structure of human USB1, defining it as a member of the LigT-like superfamily of 2H phosphoesterases. We show that human USB1 is a distributive 3'-5' exoribonuclease that posttranscriptionally removes uridine and adenosine nucleosides from the 3' end of spliceosomal U6 small nuclear RNA (snRNA), directly catalyzing terminal 2', 3' cyclic phosphate formation. USB1 measures the appropriate length of the U6 oligo(U) tail by reading the position of a key adenine nucleotide (A102) and pausing 5 uridine residues downstream.We show that the 3' ends of U6 snRNA in PN patient lymphoblasts are elongated and unexpectedly carry nontemplated 3' oligo(A) tails that are characteristic of nuclear RNA surveillance targets. Thus, our study reveals a novel quality control pathway in which posttranscriptional 3'-end processing by USB1 protects U6 snRNA from targeting and destruction by the nuclear exosome. Our data implicate aberrant oligoadenylation of U6 snRNA in the pathogenesis of the leukemia predisposition disorder PN.

Mutations in the telomere capping complex in bone marrow failure and related syndromes.

Dyskeratosis congenita and its variants have overlapping phenotypes with many disorders including Coats plus, and their underlying pathology is thought to be one of defective telomere maintenance. Recently, biallelic CTC1 mutations have been described in patients with syndromes overlapping Coats plus. CTC1, STN1 and TEN1 are part of the telomere-capping complex involved in maintaining telomeric structural integrity. Based on phenotypic overlap we screened 73 genetically uncharacterized patients with dyskeratosis congenita and related bone marrow failure syndromes for mutations in this complex. Biallelic CTC1 mutations were identified in 6 patients but none in either STN1 or TEN1. We have expanded the phenotypic spectrum associated with CTC1 mutations and report that intracranial and retinal abnormalities are not a defining feature, as well as showing that the effect of these mutations on telomere length is variable. The study also demonstrates the lack of disease-causing mutations in other components of the telomere-capping complex.

Mutations in C16orf57 and normal-length telomeres unify a subset of patients with dyskeratosis congenita, poikiloderma with neutropenia and Rothmund-Thomson syndrome.

Dyskeratosis congenita (DC) is an inherited poikiloderma which in addition to the skin abnormalities is typically associated with nail dystrophy, leucoplakia, bone marrow failure, cancer predisposition and other features. Approximately 50% of DC patients remain genetically uncharacterized. All the DC genes identified to date are important in telomere maintenance. To determine the genetic basis of the remaining cases of DC, we undertook linkage analysis in 20 families and identified a common candidate gene region on chromosome 16 in a subset of these. This region included the C16orf57 gene recently identified to be mutated in poikiloderma with neutropenia (PN), an inherited poikiloderma displaying significant clinical overlap with DC. Analysis of the C16orf57 gene in our uncharacterized DC patients revealed homozygous mutations in 6 of 132 families. In addition, three of six families previously classified as Rothmund-Thomson syndrome (RTS-a poikiloderma that is sometimes confused with PN) were also found to have homozygous C16orf57 mutations. Given the role of the previous DC genes in telomere maintenance, telomere length was analysed in these patients and found to be comparable to age-matched controls. These findings suggest that mutations in C16orf57 unify a distinct set of families which clinically can be categorized as DC, PN or RTS. This study also highlights the multi-system nature (wider than just poikiloderma and neutropenia) of the clinical features of affected individuals (and therefore house-keeping function of C16orf57), a possible role for C16orf57 in apoptosis, as well as a distinct difference from previously characterized DC patients because telomere length was normal.

Marked genetic heterogeneity in familial myelodysplasia/acute myeloid leukaemia.

The myelodysplastic syndromes (MDS) are heterogeneous and can evolve into acute myeloid leukaemia (AML). Rare familial cases are reported in which five disease genes have been identified to date (RUNX1, CEBPA, TERC, TERT and GATA2). Here we report the genetic categorization of 27 families with familial MDS/AML. All of these families were screened for RUNX1, CEBPA, TERC, TERT and GATA2 as well as TET2 and NPM1. Five of the 27 families had telomerase mutations; one had a RUNX1 mutation, while none were found to have TET2, CEBPA or NPM1 mutations. We identified four families with heterozygous GATA2 mutations, each associated with a different phenotype. While one of these mutations is novel, three have been previously reported: one has been described in dendritic cell, monocyte, B and NK lymphoid (DCML) deficiency and one is in a family that has been reported in a series with primary lymphoedema with a predisposition to AML (Emberger syndrome). In summary, genetic characterization was shown in 10 (four GATA2, three TERT, two TERC, one RUNX1) of these families; however 17 remain uncharacterized, highlighting marked genetic heterogeneity in familial MDS/AML and the scope for further functional pathways that could give rise to this group of disorders.

Cigarette smoke and platelet-activating factor receptor dependent adhesion of Streptococcus pneumoniae to lower airway cells.

BACKGROUND: Exposure to cigarette smoke (CS) is associated with increased risk of pneumococcal infection. The mechanism for this association is unknown. We recently reported that the particulate matter from urban air simulates platelet-activating factor receptor (PAFR)-dependent adhesion of pneumococci to airway cells. We therefore sought to determine whether CS stimulates pneumococcal adhesion to airway cells. METHODS: Human alveolar (A549), bronchial (BEAS2-B), and primary bronchial epithelial cells (HBEpC) were exposed to CS extract (CSE), and adhesion of Streptococcus pneumoniae determined. The role of PAFR in mediating adhesion was determined using a blocker (CV-3988). PAFR transcript level was assessed by quantitative real-time PCR, and PAFR expression by flow cytometry. Lung PAFR transcript level was assessed in mice exposed to CS, and bronchial epithelial PAFR expression assessed in active-smokers by immunostaining. RESULTS: In A549 cells, CSE 1% increased pneumococcal adhesion (p<0.05 vs control), PAFR transcript level (p<0.01), and PAFR expression (p<0.01). Pneumococcal adhesion to A549 cells was attenuated by CV-3988 (p<0.001). CSE 1% stimulated pneumococcal adhesion to BEAS2-B cells and HBEpC (p<0.01 vs control). CSE 1% increased PAFR expression in BEAS2-B (p<0.01), and in HBEpC (p<0.05). Lung PAFR transcript level was increased in mice exposed to CS in vivo (p<0.05 vs room air). Active smokers (n=16) had an increased percentage of bronchial epithelium with PAFR-positive cells (p<0.05 vs never smokers, n=11). CONCLUSION: CSE stimulates PAFR-dependent pneumococcal adhesion to lower airway epithelial cells. We found evidence that CS increases bronchial PAFR in vivo.

Exome sequencing identifies MPL as a causative gene in familial aplastic anemia.

The primary cause of aplastic anemia remains unknown in many patients. The aim of this study was to clarify the genetic cause of familial aplastic anemia. Genomic DNA of an affected individual from a multiplex consanguineous family was hybridized to a Nimblegen exome library before being sequenced on a GAIIx genome analyzer. Once the disease causing homozygous mutation had been confirmed in the consanguineous family, this gene was then analyzed for mutation in 33 uncharacterized index cases of aplastic anemia (<13 years) using denaturing HPLC. Abnormal traces were confirmed by direct sequencing. Exome sequencing identified a novel homozygous nonsense mutation in the thrombopoietin receptor gene MPL. An additional novel homozygous MPL mutation was identified in the screen of 33 aplastic anemia patients. This study shows for the first time a link between homozygous MPL mutations and familial aplastic anemia. It also highlights the important role of MPL in trilineage hematopoiesis.

Adhesion of Streptococcus pneumoniae to human airway epithelial cells exposed to urban particulate matter.

BACKGROUND: Epidemiologic studies report an association between pneumonia and urban particulate matter (PM) less than 10 microns (µm) in aerodynamic diameter (PM(10)). Streptococcus pneumoniae is a common cause of bacterial pneumonia worldwide. To date, the mechanism whereby urban PM enhances vulnerability to S pneumoniae infection is unclear. Adhesion of S pneumoniae to host cells is a prerequisite for infection. Host-expressed proteins, including the receptor for platelet-activating factor (PAFR), are co-opted by S pneumoniae to adhere to lower airway epithelial cells. OBJECTIVES: To define whether inhalable urban PM enhances the adhesion of S pneumoniae to airway epithelial cells. METHODS: A549 cells were cultured with PM(10) from Leicester (United Kingdom [UK]) and PM(10) and PM less than 2.5 µm in aerodynamic diameter (PM(2.5)) from Accra (Ghana), then infected with S pneumoniae strain D39. Pneumococcal adhesion to human primary bronchial epithelial cells was also assessed. Bacterial adhesion was determined by quantitative culture and confocal microscopy. The role of oxidative stress was assessed by N-acetyl cysteine, and the role of PAFR was assessed by mRNA transcript level, receptor expression, and receptor blocking. RESULTS: PM(10) (UK) increased S pneumoniae adhesion to both A549 airway epithelial cells and human primary bronchial epithelial cells. PM(10) (Ghana) and PM(2.5) (Ghana) also increased adhesion. Culture of A549 cells by PM(10) (UK) increased PAFR mRNA transcript level and PAFR expression. PM(10) (UK)-stimulated adhesion to A549 cells was attenuated by a PAFR blocker and N-acetyl cysteine. CONCLUSION: Urban PM increases adhesion of S pneumoniae to human airway epithelial cells. PM-stimulated adhesion is mediated by oxidative stress and PAFR.

Dyskeratosis congenita and the DNA damage response.

Dyskeratosis congenita (DC) is a heterogeneous bone marrow failure disorder with known mutations in components of telomerase and telomere shelterin. Recent work in a mouse model with a dyskerin mutation has implicated an increased DNA damage response as part of the cellular pathology, while mouse models with Terc and Tert mutations displayed a normal response. To clarify how these contradictory results might apply to DC pathology in humans, we studied the cellular phenotype in primary cells from DC patients of several genetic subtypes, focussing on T lymphocytes to remain close to the haematopoietic system. We observed novel cell cycle abnormalities in conjunction with impaired growth and an increase in apoptosis. Using flow cytometry and confocal microscopy we examined induction of the DNA damage proteins γ-H2AX and 53BP1 and the cell cycle protein TP53 (p53). We found an increase in damage foci at telomeres in lymphocytes and an increase in the basal level of DNA damage in fibroblasts, but crucially no increased response to DNA damaging agents in either cell type. As the response to induced DNA damage was normal and levels of global DNA damage were inconsistent between cell types, DNA damage may contribute differently to the pathology in different tissues.

Mutations in the telomerase component NHP2 cause the premature ageing syndrome dyskeratosis congenita.

Dyskeratosis congenita is a premature aging syndrome characterized by muco-cutaneous features and a range of other abnormalities, including early greying, dental loss, osteoporosis, and malignancy. Dyskeratosis congenita cells age prematurely and have very short telomeres. Patients have mutations in genes that encode components of the telomerase complex (dyskerin, TERC, TERT, and NOP10), important in the maintenance of telomeres. Many dyskeratosis congenita patients remain uncharacterized. Here, we describe the analysis of two other proteins, NHP2 and GAR1, that together with dyskerin and NOP10 are key components of telomerase and small nucleolar ribonucleoprotein (snoRNP) complexes. We have identified previously uncharacterized NHP2 mutations that can cause autosomal recessive dyskeratosis congenita but have not found any GAR1 mutations. Patients with NHP2 mutations, in common with patients bearing dyskerin and NOP10 mutations had short telomeres and low TERC levels. SiRNA-mediated knockdown of NHP2 in human cells led to low TERC levels, but this reduction was not observed after GAR1 knockdown. These findings suggest that, in human cells, GAR1 has a different impact on the accumulation of TERC compared with dyskerin, NOP10, and NHP2. Most of the mutations so far identified in patients with classical dyskeratosis congenita impact either directly or indirectly on the stability of RNAs. In keeping with this effect, patients with dyskerin, NOP10, and now NHP2 mutations have all been shown to have low levels of telomerase RNA in their peripheral blood, providing direct evidence of their role in telomere maintenance in humans.

Dyskeratosis congenita, stem cells and telomeres.

Dyskeratosis congenita (DC) is a multi-system disorder which in its classical form is characterised by abnormalities of the skin, nails and mucous membranes. In approximately 80% of cases, it is associated with bone marrow dysfunction. A variety of other abnormalities (including bone, brain, cancer, dental, eye, gastrointestinal, immunological and lung) have also been reported. Although first described almost a century ago it is the last 10 years, following the identification of the first DC gene (DKC1) in 1998, in which there has been rapid progress in its understanding. Six genes have been identified, defects in which cause different genetic subtypes (X-linked recessive, autosomal dominant, autosomal recessive) of DC. The products of these genes encode components that are critical for telomere maintenance; either because they are core constituents of telomerase (dyskerin, TERC, TERT, NOP10 and NHP2) or are part of the shelterin complex that protects the telomeric end (TIN2). These advances have also highlighted the connection between the more "cryptic/atypical" forms of the disease including aplastic anaemia and idiopathic pulmonary fibrosis. Equally, studies on this disease have demonstrated the critical importance of telomeres in human cells (including stem cells) and the severe consequences of their dysfunction. In this context DC and related diseases can now be regarded as disorders of "telomere and stem cell dysfunction".

TINF2 mutations result in very short telomeres: analysis of a large cohort of patients with dyskeratosis congenita and related bone marrow failure syndromes.

Dyskeratosis congenita (DC) is a multisystem bone marrow failure syndrome characterized by a triad of mucocutaneous abnormalities and a predisposition to cancer. The genetic basis of DC remains unknown in more than 60% of patients. Mutations have been identified in components of the telomerase complex (dyskerin, TERC, TERT, NOP10, and NHP2), and recently in one component of the shelterin complex TIN2 (gene TINF2). To establish the role of TINF2 mutations, we screened DNA from 175 uncharacterised patients with DC as well as 244 patients with other bone marrow failure disorders. Heterozygous coding mutations were found in 33 of 175 previously uncharacterized DC index patients and 3 of 244 other patients. A total of 21 of the mutations affected amino acid 282, changing arginine to histidine (n = 14) or cysteine (n = 7). A total of 32 of 33 patients with DC with TINF2 mutations have severe disease, with most developing aplastic anaemia by the age of 10 years. Telomere lengths in patients with TINF2 mutations were the shortest compared with other DC subtypes, but TERC levels were normal. In this large series, TINF2 mutations account for approximately 11% of all DC, but they do not play a significant role in patients with related disorders. This study emphasises the role of defective telomere maintenance on human disease.

Defining the pathogenic role of telomerase mutations in myelodysplastic syndrome and acute myeloid leukemia.

The primary pathology in many cases of myelodysplasia (MDS) and acute myeloid leukemia (AML) remains unknown. In some cases, two or more affected members have been identified in the same family. To date, mutations in two genes have been directly implicated: the hematopoietic transcription factors RUNX1 (runt-related transcription factor 1) and CEBPA (CCATT-box enhancer binding protein alpha). However, there are also other familial cases of MDS/AML where the genetic basis remains unknown. Both MDS, and to a lesser extent AML, have been observed in cases of the bone marrow failure syndrome dyskeratosis congenita, in which telomerase mutations have been identified. Recently, an increased incidence of telomerase reverse transcriptase mutations has been reported in a series of de novo AML. We have now identified novel mutations in the telomerase RNA (TERC) or telomerase reverse transcriptase component (TERT) within 4 of 20 families presenting with familial MDS/AML. Functional analysis has demonstrated that all mutations adversely impact on telomerase activity in vitro, and affected individuals have short telomeres. These families, in conjunction with a review of previously published cases, help to further define the pathological role of telomerase mutations in MDS/AML and have implications for the biology, treatment and screening regimen of de novo cases.

Single-molecule analysis of the human telomerase RNA.dyskerin interaction and the effect of dyskeratosis congenita mutations.

It has been proposed that human telomerase RNA (hTR) interacts with dyskerin, prior to assembly of the telomerase holoenzyme. The direct interaction of dyskerin and hTR has not been demonstrated and is an experimentally challenging research problem because of difficulties in expressing and purifying dyskerin in quantities that are useful for biophysical analysis. By orthogonally labeling dyskerin and hTR, we have been able to employ single-molecule two-color coincidence detection (TCCD) to observe directly the formation of a dyskerin.hTR complex. By systematic deletion of hTR subdomains, we have gained insights into the RNA sites required for interaction with dyskerin. We then investigated mutated forms of hTR and dyskerin that are associated with dyskeratosis congenita (DC), on the basis of clinical genetics studies, for their effects on the dyskerin.hTR interaction. Dyskerin mutations associated with X-linked DC resulted in significant impairment of the dyskerin.hTR interaction, whereas mutations in hTR associated with autosomal dominant (AD) DC did not affect the interaction. We propose that disruption of the dyskerin.hTR interaction may contribute to X-linked DC.

Exogenous TERC alone can enhance proliferative potential, telomerase activity and telomere length in lymphocytes from dyskeratosis congenita patients.

Dyskeratosis congenita (DC) is an inherited multi-system disorder characterised by muco-cutaneous abnormalities, bone marrow failure and a predisposition to malignancy. Bone marrow failure is the principal cause of mortality and is thought to be the result of premature cell death in the haematopoietic compartment because DC cells age prematurely and tend to have short telomeres. DC is genetically heterogeneous and patients have mutations in genes that encode components of the telomerase complex (DKC1, TERC, TERT, NOP10 and NHP2), and telomere shelterin complex (TINF2), both important in telomere maintenance. Here, we transduced primary T lymphocytes and B lymphocyte lines established from patients with TERC and DKC1 mutations with wild type TERC-bearing lentiviral vectors. We found that transduction with exogenous TERC alone was capable of increasing telomerase activity in mutant T lymphocytes and B lymphocyte lines and improved the survival and thus overall growth of B-lymphocyte lines over a prolonged period, regardless of their disease mutation. Telomeres in TERC-treated lines were longer than in the untreated cultures. This is the first study of its kind in DC lymphocytes and the first to demonstrate that transduction with TERC alone can improve cell survival and telomere length without the need for exogenous TERT.

Functional characterization of novel telomerase RNA (TERC) mutations in patients with diverse clinical and pathological presentations.

BACKGROUND AND OBJECTIVES: Functional characterization of heterozygous TERC (telomerase RNA component) and TERT (telomerase reverse transcriptase) mutations found in autosomal dominant dyskeratosis congenita (DC) and aplastic anemia (AA) shows that telomerase function is defective and that this is associated with short telomeres. This leads to reduced cell longevity with maximal impact on tissues with high proliferate potential. The aim of this study was to establish the role of TERC in the pathophysiology of uncharacterized patients with AA with some features of DC. DESIGN AND METHODS: The TERC gene was screened for mutations by denaturing high performance liquid chromatography. To determine the functional significance of TERC mutations telomerase activity was assessed in an in vitro (TRAP) assay and telomere length of patients' samples was determined using Southern blot analysis. RESULTS This study led to the identification of four novel TERC mutations (G178A, C180T, D52-86 and G2C) and a recurrent TERC mutation (D110-113GACT). INTERPRETATION AND CONCLUSIONS: Two of the de novo TERC mutations (G178A and C180T) found uniquely produce a clinical phenotype in the first generation, differing from previously published cases in which individuals in the first generation are usually asymptomatic. Curiously these mutations are located near the triple-helix domain of TERC. We also observed that the recurrent D110-113GACT can present with AA, myelodysplasia or leukemia. The D52-86 is associated with varied phenotypes including pulmonary disease (pulmonary fibrosis) as the first presentation. In summary, this study reports the functional characterization of several novel TERC mutations associated with varied hematologic and extra-hematologic presentations.

Relative telomere lengths in tumor and normal mucosa are related to disease progression and chromosome instability profiles in colorectal cancer.

Telomeric dysfunction is linked to colorectal cancer (CRC) initiation. However, the relationship of normal tissue and tumor telomere lengths with CRC progression, molecular features and prognosis is unclear. Here, we measured relative telomere length (RTL) by real-time quantitative PCR in 90 adenomas (aRTL), 419 stage I-IV CRCs (cRTL) and adjacent normal mucosa (nRTL). Age-adjusted RTL was analyzed against germline variants in telomere biology genes, chromosome instability (CIN), microsatellite instability (MSI), CpG island methylator phenotype (CIMP), TP53, KRAS, BRAF mutations and clinical outcomes. In 509 adenoma or CRC patients, nRTL decreased with advancing age. Female gender, proximal location and the TERT rs2736100 G allele were independently associated with longer age-adjusted nRTL. Adenomas and carcinomas exhibited telomere shortening in 79% and 67% and lengthening in 7% and 15% of cases. Age-adjusted nRTL and cRTL were independently associated with tumor stage, decreasing from adenoma to stage III and leveling out or increasing from stage III to IV, respectively. Cancer MSI, CIMP, TP53, KRAS and BRAF status were not related to nRTL or cRTL. Near-tetraploid CRCs exhibited significantly longer cRTLs than CIN- and aneuploidy CRCs, while cRTL was significantly shorter in CRCs with larger numbers of chromosome breaks. Age-adjusted nRTL, cRTL or cRTL:nRTL ratios were not associated with disease-free or overall survival in stage II/III CRC. Taken together, our data show that both normal mucosa and tumor RTL are independently associated with CRC progression, and highlight divergent associations of CRC telomere length with tumor CIN profiles.