Variants In TERT Research Articles

Genetic variants associated with white blood cell count amongst individuals with sickle cell disease.

Sickle cell disease (SCD) is a Mendelian disorder characterized by a point mutation in the β-globin gene that leads to sickling of erythrocytes. Several studies have shown that absolute neutrophil count is strongly associated with clinical severity of SCD, suggesting an apparent role of white blood cells (WBC) in SCD pathology. However, the mechanism by which genetic variants lead to WBC count differences in SCD patients remains unclear. Genome-wide association (GWA) analyses were carried out amongst a cohort of 2409 Brazil SCD participants. Association of WBC count and genetic markers were investigated in homozygous sickle cell anaemia participants and compound heterozygous sickle cell haemoglobin C participants. GWA analysis showed that variants in genes TERT, ACKR1, and FAM3C are associated with WBC count variation. The well-studied association between WBC count and Duffy null phenotype (variant in ACKR1) in healthy populations was replicated, reinforcing the influence of the SNP rs2814778 (T>C) in WBC count. Genetics plays an important role in regulating WBC count in patients with SCD. Our results point to possible mechanisms involved in WBC count variation and as increased WBC count is associated with more severe SCD, these results could suggest potential therapeutic targets for individuals with SCD.

Determinants of mosaic chromosomal alteration fitness

Clonal hematopoiesis (CH) is characterized by the acquisition of a somatic mutation in a hematopoietic stem cell that results in a clonal expansion. These driver mutations can be single nucleotide variants in cancer driver genes or larger structural rearrangements called mosaic chromosomal alterations (mCAs). The factors that influence the variations in mCA fitness and ultimately result in different clonal expansion rates are not well understood. We used the Passenger-Approximated Clonal Expansion Rate (PACER) method to estimate clonal expansion rate as PACER scores for 6,381 individuals in the NHLBI TOPMed cohort with gain, loss, and copy-neutral loss of heterozygosity mCAs. Our mCA fitness estimates, derived by aggregating per-individual PACER scores, were correlated (R2 = 0.49) with an alternative approach that estimated fitness of mCAs in the UK Biobank using population-level distributions of clonal fraction. Among individuals with JAK2 V617F clonal hematopoiesis of indeterminate potential or mCAs affecting the JAK2 gene on chromosome 9, PACER score was strongly correlated with erythrocyte count. In a cross-sectional analysis, genome-wide association study of estimates of mCA expansion rate identified a TCL1A locus variant associated with mCA clonal expansion rate, with suggestive variants in NRIP1 and TERT.

Oxidative Damage and Telomere Length as Markers of Lung Cancer Development among Chronic Obstructive Pulmonary Disease (COPD) Smokers.

Lung cancer (LC) constitutes an important cause of death among patients with Chronic Obstructive Pulmonary Disease (COPD). Both diseases may share pathobiological mechanisms related to oxidative damage and cellular senescence. In this study, the potential value of leucocyte telomere length, a hallmark of aging, and 8-OHdG concentrations, indicative of oxidative DNA damage, as risk biomarkers of LC was evaluated in COPD patients three years prior to LC diagnosis. Relative telomere length measured using qPCR and serum levels of 8-OHdG were determined at the baseline in 99 COPD smokers (33 with LC and 66 age-matched COPD without LC as controls). Of these, 21 COPD with LC and 42 controls had the biomarkers measured 3 years before. Single nucleotide variants (SNVs) in TERT, RTEL, and NAF1 genes were also determined. COPD cases were evaluated, which showed greater telomere length (p < 0.001) and increased serum 8-OHdG levels (p = 0.004) three years prior to LC diagnosis compared to the controls. This relationship was confirmed at the time of LC diagnosis. No significant association was found between the studied SNVs in cases vs. controls. In conclusion, this preliminary study shows that longer leucocyte telomere length and increased 8-OHdG serum levels can be useful as early biomarkers of the risk for future lung cancer development among COPD patients.

Identification of influential rare variants in aggregate testing using random forest importance measures.

Aggregate tests of rare variants are often employed to identify associated regions compared to sequentially testing each individual variant. When an aggregate test is significant, it is of interest to identify which rare variants are "driving" the association. We recently developed the rare variant influential filtering tool (RIFT) to identify influential rare variants and showed RIFT had higher true positive rates compared to other published methods. Here we use importance measures from the standard random forest (RF) and variable importance weighted RF (vi-RF) to identify influential variants. For very rare variants (minor allele frequency [MAF]<0.001), the vi-RF:Accuracy method had the highest median true positive rate (TPR=0.24; interquartile range [IQR]: 0.13, 0.42) followed by the RF:Accuracy method (TPR=0.16; IQR: 0.07, 0.33) and both were superior to RIFT (TPR=0.05; IQR: 0.02, 0.15). Among uncommon variants (0.001<MAF<0.03), the RF methods had higher true positive rates than RIFT while observing comparable false positive rates. Finally, we applied the RF methods to a targeted resequencing study in idiopathic pulmonary fibrosis (IPF), in which the vi-RF approach identified eight and seven variants in TERT and FAM13A, respectively. In summary, the vi-RF provides an improved, objective approach to identifying influential variants following a significant aggregate test. We have expanded our previously developed R package RIFT to include the random forest methods.

Association of germline variants in telomere maintenance genes (POT1, TERF2IP, ACD, and TERT) with spitzoid morphology in familial melanoma: A multi-center case series

Spitzoid morphology in familial melanoma has been associated with germline variants in POT1, a telomere maintenance gene (TMG), suggesting a link between telomere biology and spitzoid differentiation. To assess if familial melanoma cases associated with germline variants in TMG (POT1, ACD, TERF2IP, and TERT) commonly exhibit spitzoid morphology. In this case series, melanomas were classified as having spitzoid morphology if at least 3 of 4 dermatopathologists reported this finding in ≥25% of tumor cells. Logistic regression was used to calculate odds ratios (OR) of spitzoid morphology compared to familial melanomas from unmatched noncarriers that were previously reviewed by a National Cancer Institute dermatopathologist. Spitzoid morphology was observed in 77% (23 of 30), 75% (3 of 4), 50% (2 of 4), and 50% (1 of 2) of melanomas from individuals with germline variants in POT1, TERF2IP, ACD, and TERT, respectively. Compared to noncarriers (n=139 melanomas), POT1 carriers (OR=225.1, 95% confidence interval: 51.7-980.5; P<.001) and individuals with TERF2IP, ACD, and TERT variants (OR=82.4, 95% confidence interval: 21.3-494.6; P<.001) had increased odds of spitzoid morphology. Findings may not be generalizable to nonfamilial melanoma cases. Spitzoid morphology in familial melanoma could suggest germline alteration of TMG.

Idiopathic Pulmonary Fibrosis Is Associated with Common Genetic Variants and Limited Rare Variants.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a rare, irreversible, and progressive disease of the lungs. Common genetic variants, in addition to nongenetic factors, have been consistently associated with IPF. Rare variants identified by candidate gene, family-based, and exome studies have also been reported to associate with IPF. However, the extent to which rare variants, genome-wide, may contribute to the risk of IPF remains unknown. Objectives: We used whole-genome sequencing to investigate the role of rare variants, genome-wide, on IPF risk. Methods: As part of the Trans-Omics for Precision Medicine Program, we sequenced 2,180 cases of IPF. Association testing focused on the aggregated effect of rare variants (minor allele frequency ⩽0.01) within genes or regions. We also identified individual rare variants that are influential within genes and estimated the heritability of IPF on the basis of rare and common variants. Measurements and Main Results: Rare variants in both TERT and RTEL1 were significantly associated with IPF. A single rare variant in each of the TERT and RTEL1 genes was found to consistently influence the aggregated test statistics. There was no significant evidence of association with other previously reported rare variants. The SNP heritability of IPF was estimated to be 32% (SE = 3%). Conclusions: Rare variants within the TERT and RTEL1 genes and well-established common variants have the largest contribution to IPF risk overall. Efforts in risk profiling or the development of therapies for IPF that focus on TERT, RTEL1, common variants, and environmental risk factors are likely to have the largest impact on this complex disease.

P730: GENETIC LANDSCAPE OF SOMATIC MYELOID MUTATIONS IN THE PRESENCE OF RARE TERT VARIANTS AND THEIR RELATION WITH MYELOID NEOPLASIA

Background: Genetic variants in TERT, the gene that encodes telomerase reverse transcriptase, can result in accelerated telomere attrition. Impaired telomere maintenance is implicated in the pathogenesis of myelodysplastic syndromes (MDS), with mechanisms remaining to be elucidated. Aims: To explore the clinical and genetic associations of TERT variants in an unselected cohort of patients screened with a myeloid mutation next generation sequencing panel due to clinical suspicion of a myeloid neoplasia. Methods: Using a research protocol approved by the Mayo Clinic Institutional Review Board, we performed a retrospective chart review to identify a cohort of unselected individuals within all historical data at our institution that had undergone somatic mutational analysis for suspected myeloid neoplasia using our insitutional amplicon based next generation sequencing panel (42 genes, including exons 2 to 16 of TERT, with a read depth of at least 250X). TERT variants were classified into those with CADD score >20 and <20 in line with previous literature to identify variants within the top 1% according to their predicted deleteriousness. Additionally, we identified within our cohort individuals that received a diagnosis of MDS, clinical and survival information. Results: We identified 55 different TERT variants (46 missense, 3 in canonical splice sites, 4 synonymous and 2 small inframe deletions) in 148 individuals tested from April of 2016 to November of 2021 (Fig. 1A&B). The five most recurrent variants were c.1234C>T (48 patients), c.1323_1325del (29 patients), c.3164C>T (5 patients), c.604G>A (4 patients) and c.969G>A (4 patients), all with CADD<20. None of these patients were diagnosed with a bona fide telomere biology disorder and no clinical information about telomere length status was available. All variants were considered germline since their allele frequency was near 50%, although no germline confirmation was performed. All variants were clinically classified as uncertain significance according to the American College of Medical Genetics guidelines. Of the 148 total patients, 17 (12%) carried TERT variants with CADD>20 that were distributed thorough all the gene domains with no clustering or presence of hotspots (Fig.1A&B). 59% (10 patients) of CADD>20 variants carriers were diagnosed with MDS: 5 patients with <5% bone marrow blasts (4 with normal karyotype) and 5 patients with 5-19% blasts, all with complex karyotype. This percentage was significantly higher compared to CADD<20 variant carriers (34%, 49 patients, p=0.048 by Chi-square test, Fig. 1C). However, no major differences were found between the two groups in terms of age at diagnosis (median 70 vs 73 years old, p = 0.2305) or AML-free survival (median 10 vs 15.5 months since diagnosis, p=0.4674). Similarly, the percentage of patients presenting additional somatic variants in other myeloid-related genes was similar between CADD<20 and CADD>20 TERT variant carriers (60% vs 64%). The two most frequenly mutated genes in CADD>20 carriers were TP53 (22%), TET2 (18%), RUNX1 (9%), U2AF1 (9%) and BCOR (9%) while in the CADD<20 carriers were TET2 (14%), ASXL1 (11%), SRSF2 (9%), TP53 (7%) and JAK2 (6%). Our data showed that TP53 pathogenic variants were significantly enriched in CADD>20 TERT variant carriers compared to CADD<20 carriers. (22.7% vs 7.4%, p=0.0169, Fig. 1D). Image:Summary/Conclusion: Carriers of TERT variants with CADD score of >20 were more likely to be associated with a diagnosis of MDS and with somatic TP53 mutations in comparison to those with CADD score <20, findings that we plan to validate functionally and prospectively.

Clinical Presentation of Patients with Adult Late-Onset Telomere Biology Disorders - Results from the Aachen Telomeropathy Registry

Clinical Presentation of Patients with Adult Late-Onset Telomere Biology Disorders - Results from the Aachen Telomeropathy Registry

The Genetic Architecture of Gliomagenesis-Genetic Risk Variants Linked to Specific Molecular Subtypes.

Genome-wide association studies have identified 25 germline genetic loci that increase the risk of glioma. The somatic tumor molecular alterations, including IDH-mutation status and 1p/19q co-deletion, have been included into the WHO 2016 classification system for glioma. To investigate how the germline genetic risk variants correlate with the somatic molecular subtypes put forward by WHO, we performed a meta-analysis that combined findings from 330 Swedish cases and 876 controls with two other recent studies. In total, 5,103 cases and 10,915 controls were included. Three categories of associations were found. First, variants in TERT and TP53 were associated with increased risk of all glioma subtypes. Second, variants in CDKN2B-AS1, EGFR, and RTEL1 were associated with IDH-wildtype glioma. Third, variants in CCDC26 (the 8q24 locus), C2orf80 (close to IDH), LRIG1, PHLDB1, ETFA, MAML2 and ZBTB16 were associated with IDH-mutant glioma. We therefore propose three etiopathological pathways in gliomagenesis based on germline variants for future guidance of diagnosis and potential functional targets for therapies. Future prospective clinical trials of patients with suspicion of glioma diagnoses, using the genetic variants as biomarkers, are necessary to disentangle how strongly they can predict glioma diagnosis.

MUC5B variant is associated with visually and quantitatively detected preclinical pulmonary fibrosis

BackgroundRelatives of patients with familial interstitial pneumonia (FIP) are at increased risk for pulmonary fibrosis. We assessed the prevalence and risk factors for preclinical pulmonary fibrosis (PrePF) in first-degree relatives...

Telomere length and aging-related outcomes in humans: A Mendelian randomization study in 261,000 older participants.

Inherited genetic variation influencing leukocyte telomere length provides a natural experiment for testing associations with health outcomes, more robust to confounding and reverse causation than observational studies. We tested associations between genetically determined telomere length and aging‐related health outcomes in a large European ancestry older cohort. Data were from n = 379,758 UK Biobank participants aged 40–70, followed up for mean of 7.5 years (n = 261,837 participants aged 60 and older by end of follow‐up). Thirteen variants strongly associated with longer telomere length in peripheral white blood cells were analyzed using Mendelian randomization methods with Egger plots to assess pleiotropy. Variants in TERC, TERT, NAF1, OBFC1, and RTEL1 were included, and estimates were per 250 base pairs increase in telomere length, approximately equivalent to the average change over a decade in the general white population. We highlighted associations with false discovery rate‐adjusted p‐values smaller than .05. Genetically determined longer telomere length was associated with lowered risk of coronary heart disease (CHD; OR = 0.95, 95% CI: 0.92–0.98) but raised risk of cancer (OR = 1.11, 95% CI: 1.06–1.16). Little evidence for associations were found with parental lifespan, centenarian status of parents, cognitive function, grip strength, sarcopenia, or falls. The results for those aged 60 and older were similar in younger or all participants. Genetically determined telomere length was associated with increased risk of cancer and reduced risk of CHD but little change in other age‐related health outcomes. Telomere lengthening may offer little gain in later‐life health status and face increasing cancer risks.

Abstract 3105: Serial profiling of ctDNA identifies clinically actionable genomic evolution in high-risk neuroblastoma

Abstract Background: Analysis of circulating tumor DNA (ctDNA) provides a noninvasive method to profile tumor-associated genomic aberrations, heterogeneity and evolution. Methods: Peripheral blood from 5 newly diagnosed and 33 relapsed high-risk neuroblastoma patients was serially profiled for ctDNA (n=1-7 samples/patient; total n=122 samples) with the FoundationACT assay that utilizes hybrid capture-based genomic profiling of 62 genes. Samples were sequenced to a median unique coverage depth of at least 3168x and variants were evaluated and compared with temporally-matched tumor sequencing and imaging evaluations. Results: Ninety-three percent (114/122) of peripheral blood samples yielded suitable cell-free DNA for sequencing. ctDNA was detected in 68% (78/114) of samples (maximum somatic allele frequency [MSAF] &amp;gt;0; median MSAF=0.53%; range MSAF 0-79%). At least 1 pathogenic genomic alteration (genomic short-variant or amplification) was found in 54% (61/114) of samples (range 1-6 alterations). Fifty-four percent (13/24) of detected ctDNA genomic-short variants were not present in temporally matched tumor samples at diagnosis or relapse (collected within 3 months of each other; n=23 patients), including pathogenic variants in ALK, TP53, TERT, NF1, FLT3, PTPN11, and PIK3CA. There was 100% concordance between the detection of MYCN (6/6) and ALK (3/3) amplification in paired ctDNA/tissue samples. For example, a newly diagnosed patient with stage 4 neuroblastoma had MYCN amplification noted on both tumor and ctDNA sequencing, however had 3 separate ALKmutations (R1275Q, F1245L, and F1174L) uniquely identified in ctDNA. Twenty-eight patients had multiple ctDNA samples sequenced (range 2-7 samples) and 50% (14/28) had alterations emerge or regress across serial samples. For example, pathogenic variants in ALK, BRCA2, NRAS, PTEN, TP53, CDKN2A, PTPN11, ABL1,CDH1, MET, ERRFl1 and ERBB2 appeared in subsequent ctDNA sequencing that were not present in the initial ctDNA or tumor sequencing. Overall, serial ctDNA profiling identified additional pathogenic variants in driver cancer genes beyond that derived from tumor sequencing in 45% (17/38) of cases, including identifying targetable ALK-RAS-MAPK pathway alterations uniquely in ctDNA in 21% (8/38) of cases. For the 18 patients that had 3 or more ctDNA samples, 33% (6/18) developed ctDNA unique ALK-RAS-MAPK pathway mutations during therapy. Finally, in most cases ctDNA profiling was complementary to standard imaging surveillance, however in 3 cases rising ctDNA allele frequencies occurred prior to clinical or imaging signs of disease relapse; additional correlation of ctDNA data and disease evaluations is ongoing. Conclusions: Sequencing of ctDNA from neuroblastoma patients identified clinically actionable tumor-associated genetic aberrations emerging under the selective pressure of standard and targeted therapies. Citation Format: Kristopher R. Bosse, Samantha Buongervino, Maria Lane, Adam Hyman, Maria Gemino-borromeo, Jennifer Saggio, Alana Fitzsimmons, Brady Forcier, Anne Murphy, John Wick, Matthew Cooke, Jennifer Webster, Russell Madison, Alley Welsh, Vincent A. Miller, Siraj M. Ali, John M. Maris, Yael P. Mosse. Serial profiling of ctDNA identifies clinically actionable genomic evolution in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3105.

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

BackgroundTelomeres 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.MethodsThis 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.ResultsForty-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.ConclusionNovel 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.

MON-321 Metastatic Melanoma with Unknown Primary Site in a Patient with Multiple Endocrine Neoplasia Type 1

Background: MEN1 is a rare hereditary tumor syndrome caused by germline inactivating mutations of the tumor suppressor gene MEN1 and is characterized by a predisposition to endocrine tumors primarily of parathyroid, enteropancreatic, and anterior pituitary origin, as well as nonendocrine neoplasms. Cutaneous manifestations mainly include lipomas, angiofibromas, and collagenomas. Melanoma has been observed in MEN1 with 9 cases described in the literature, including 2 cases with unknown primary site (Nord et al. 2000; Baldauf et al. 2009; Brown et al. 2015). Clinical Case: A 45 year old man with MEN1 syndrome manifesting as primary hyperparathyroidism, metastatic pancreatic neuroendocrine tumor (NET), Zollinger-Ellison syndrome, nonfunctional pituitary microadenoma, anterior mediastinal mass and skin angiofibromas presented with a left neck mass that developed over 1 year. CT neck confirmed a 2.2 cm left lateral neck level V mass. Further evaluation revealed stable NET disease with the exception of an enlarging 1.3 cm anterior mediastinal nodule. 18F-FDG PET/CT demonstrated more activity in the left neck mass and mediastinal mass than 68Ga-DOTATATE PET/CT. Biopsy of the left neck mass revealed a high grade malignant neoplasm with epithelioid features and markedly elevated MIB1. Tumor cells were positive for CD56, SOX-10 and S-100 protein, but negative for CD20, CD3, CD5, chromogranin, synaptophysin, AE1/AE3, CAM5.2, C-Kit and p53. Molecular analysis showed pathogenic variants in BRAF, TERT, PIK3C, and RB1, a likely pathogenic variant in FBXW7, and a variant of uncertain significance in PMS2. Given these histologic and molecular findings, the diagnosis of metastatic melanoma was favored. Ophthalmological evaluation, skin examination and biopsy of suspicious cutaneous lesions did not reveal the primary site of melanoma. PCR did not reveal MEN1 loss of heterozygosity (LOH) of the neck mass, though there was insufficient material to evaluate for large MEN1 deletions. Conclusion: Of the 9 cases of melanoma previously reported in MEN1, only 1 was evaluated for MEN1 LOH, which was not demonstrated. MEN1 LOH has been described in sporadic melanoma and in vitro and in vivo studies in mice suggest a role for MEN1 as a tumor suppressor gene in melanoma (Gao et al. 2011, Fang et al. 2013). Whether melanoma in MEN1 is related to the germline MEN1 mutation or is sporadic is still unclear. Evaluation of melanomas for MEN1 LOH initially with targeted sequencing of the germline mutation followed by LOH for markers in and near the MEN1 gene at 11q13, may help further elucidate the role of MEN1 in melanoma.

Lung Transplant Outcomes in Patients With Pulmonary Fibrosis With Telomere-Related Gene Variants

Pulmonary fibrosis (PF) is the most common disease indication for lung transplantation. Our recent work implicated an excess of rare genetic variants in the telomere-related genes TERT, RTEL1, and PARN in PF disease risk. The impact of such variants on posttransplant outcomes is uncertain. The objective of this study was to determine if patients with these PF-associated variants have altered rates of posttransplant acute rejection (AR), chronic lung allograft dysfunction (CLAD), and survival. The study cohort consisted of 262 PF lung transplant recipients previously genetically characterized by whole exome sequencing. Thirty-one patients (11.8%) had variants in TERT, RTEL1, or PARN, whereas 231 (88.2%) did not. Multivariate Cox proportional hazards models adjusted for relevant clinical variables were used to assess the outcomes of death and CLAD. The AR burden was quantified and compared over the first posttransplant year. Patients with PF with disease-associated variants in TERT, RTEL1, or PARN had a significantly higher risk of death (adjusted hazard ratio [HR], 1.82; 95%CI, 1.07-3.08; P= .03) and CLAD (adjusted HR, 2.88; 95%CI, 1.42-5.87; P= .004) than patients without these variants. There was no difference in AR burden or rates of grade 3 primary graft dysfunction between the two groups. Rare variants in the telomere-related genes TERT, RTEL1, or PARN are associated with poor posttransplant outcomes among PF lung transplant recipients. Further research is needed to understand the biological mechanisms by which telomere-related variants increase the risk for death and CLAD.

Analysis of protein-altering variants in telomerase genes and their association with MUC5B common variant status in patients with idiopathic pulmonary fibrosis: a candidate gene sequencing study

Idiopathic pulmonary fibrosis (IPF) risk has a strong genetic component. Studies have implicated variations at several loci, including TERT, surfactant genes, and a single nucleotide polymorphism at chr11p15 (rs35705950) in the intergenic region between TOLLIP and MUC5B. Patients with IPF who have risk alleles at rs35705950 have longer survival from the time of IPF diagnosis than do patients homozygous for the non-risk allele, whereas patients with shorter telomeres have shorter survival times. We aimed to assess whether rare protein-altering variants in genes regulating telomere length are enriched in patients with IPF homozygous for the non-risk alleles at rs35705950. Between Nov 1, 2014, and Nov 1, 2016, we assessed blood samples from patients aged 40 years or older and of European ancestry with sporadic IPF from three international phase 3 clinical trials (INSPIRE, CAPACITY, ASCEND), one phase 2 study (RIFF), and US-based observational studies (Vanderbilt Clinical Interstitial Lung Disease Registry and the UCSF Interstitial Lung Disease Clinic registry cohorts) at the Broad Institute (Cambridge, MA, USA) and Human Longevity (San Diego, CA, USA). We also assessed blood samples from non-IPF controls in several clinical trials. We did whole-genome sequencing to assess telomere length and identify rare protein-altering variants, stratified by rs35705950 genotype. We also assessed rare functional variation in TERT exons and compared telomere length and disease progression across genotypes. We assessed samples from 1510 patients with IPF and 1874 non-IPF controls. 30 (3%) of 1046 patients with an rs35705950 risk allele had a rare protein-altering variant in TERT compared with 34 (7%) of 464 non-risk allele carriers (odds ratio 0·40 [95% CI 0·24-0·66], p=0·00039). Subsequent analyses identified enrichment of rare protein-altering variants in PARN and RTEL1, and rare variation in TERC in patients with IPF compared with controls. We expanded our study population to provide a more accurate estimation of rare variant frequency in these four loci, and to calculate telomere length. The proportion of patients with at least one rare variant in TERT, PARN, TERC, or RTEL1 was higher in patients with IPF than in controls (149 [9%] of 1739 patients vs 205 [2%] of 8645 controls, p=2·44 × 10-8). Patients with IPF who had a variant in any of the four identified telomerase component genes had telomeres that were 3·69-16·10% shorter than patients without a variant in any of the four genes and had an earlier mean age of disease onset than patients without one or more variants (65·1 years [SD 7·8] vs 67·1 years [7·9], p=0·004). In the placebo arms of clinical trials, shorter telomeres were significantly associated with faster disease progression (1·7% predicted forced vital capacity per kb per year, p=0·002). Pirfenidone had treatment benefit regardless of telomere length (p=4·24 × 10-8 for telomere length lower than the median, p=0·0044 for telomere length greater than the median). Rare protein-altering variants in TERT, PARN, TERC, and RTEL1 are enriched in patients with IPF compared with controls, and, in the case of TERT, particularly in individuals without a risk allele at the rs35705950 locus. This suggests that multiple genetic factors contribute to sporadic IPF, which might implicate distinct mechanisms of pathogenesis and disease progression. Genentech, National Institutes of Health, Francis Family Foundation, Pulmonary Fibrosis Foundation, Nina Ireland Program for Lung Health, US Department of Veterans Affairs.

Valproic acid treatment response in vitro is determined by TP53 status in medulloblastoma.

Histone deacetylate inhibitors (HDACi), as valproic acid (VA), have been reported to enhance efficacy and to prevent drug resistance in some tumors, including medulloblastoma (MB). In the present study, we investigated VA role, combined to cisplatin (CDDP) in cell viability and gene expression of MB cell lines. Dose-response curve determined IC50 values for each treatment: (1) VA single, (2) CDDP single, and (3) VA and CDDP combined. Cytotoxicity and flow cytometry evaluated cell viability after exposure to treatments. Quantitative PCR evaluated gene expression levels of AKT, CTNNB1, GLI1, KDM6A, KDM6B, NOTCH2, PTCH1, and TERT, before and after treatment. Besides, we performed next-generation sequencing (NGS) for PTCH1, TERT, and TP53 genes. The most effective treatment to reduce viability was combined for D283MED and ONS-76; and CDDP single for DAOY cells (p < 0.0001). TERT, GLI1, and AKT genes were overexpressed after treatments with VA. D283MED and ONS-76 cells presented variants in TERT and PTCH1, respectively and DAOY cell line presented a TP53 mutation. MB tumors belonging to SHH molecular subgroup, with TP53MUT, would be the ones that present high risk in relation to VA use during the treatment, while TP53WT MBs can benefit from VA therapy, both SHH and groups 3 and 4. Our study shows a new perspective about VA action in medulloblastoma cells, raising the possibility that VA may act in different patterns. According to the genetic background of MB cell, VA can stimulate cell cycle arrest and apoptosis or induce resistance to treatment via signaling pathways activation.

1239 Genome-wide scans found variants in TERT associated with pigmented spots on hands/arms but not with other skin aging signs

Skin aging is a health topic of growing concern to the general public. Researchers have been identifying extrinsic and intrinsic factors affecting skin aging. So far, genetic studies on skin aging have mainly focused on facial pigmentations and wrinkles. Here we are presenting the first genome-wide study on skin aging signs also including pigmentations and wrinkles on hands/arms (according to SCINEXATM). By sampling 2959 individuals from the Taizhou longitudinal cohort in China, we found pigmented spots on hands/arms strongly associated with genetics variants in TERT (p=1.51×10-28). This association was independently replicated in SALIA cohort in Germany. An associated haplotype near the TERT promoter region was identified by fine mapping. Luciferase reporter gene assay was perform to confirm that the haplotype is associated with modified expression level of TERT (P<0.01). We also showed that sun exposure is the main confounding factor that facial pigmented spots were not found to be associated with variants in TERT. Interestingly, even after controlling all the potential confounding factors, the variants are not associated with other skin aging signs, such as wrinkle and sagging, indicating that TERT is not affecting skin aging in a global aging mechanism, but rather affecting pigmented spots in a more specific yet unknown mechanism.

An Exome Sequencing Study to Assess the Role of Rare Genetic Variation in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an increasingly recognized, often fatal lung disease of unknown etiology. The aim of this study was to use whole-exome sequencing to improve understanding of the genetic architecture of pulmonary fibrosis. We performed a case-control exome-wide collapsing analysis including 262 unrelated individuals with pulmonary fibrosis clinically classified as IPF according to American Thoracic Society/European Respiratory Society/Japanese Respiratory Society/Latin American Thoracic Association guidelines (81.3%), usual interstitial pneumonia secondary to autoimmune conditions (11.5%), or fibrosing nonspecific interstitial pneumonia (7.2%). The majority (87%) of case subjects reported no family history of pulmonary fibrosis. We searched 18,668 protein-coding genes for an excess of rare deleterious genetic variation using whole-exome sequence data from 262 case subjects with pulmonary fibrosis and 4,141 control subjects drawn from among a set of individuals of European ancestry. Comparing genetic variation across 18,668 protein-coding genes, we found a study-wide significant (P < 4.5 × 10-7) case enrichment of qualifying variants in TERT, RTEL1, and PARN. A model qualifying ultrarare, deleterious, nonsynonymous variants implicated TERT and RTEL1, and a model specifically qualifying loss-of-function variants implicated RTEL1 and PARN. A subanalysis of 186 case subjects with sporadic IPF confirmed TERT, RTEL1, and PARN as study-wide significant contributors to sporadic IPF. Collectively, 11.3% of case subjects with sporadic IPF carried a qualifying variant in one of these three genes compared with the 0.3% carrier rate observed among control subjects (odds ratio, 47.7; 95% confidence interval, 21.5-111.6; P = 5.5 × 10-22). We identified TERT, RTEL1, and PARN-three telomere-related genes previously implicated in familial pulmonary fibrosis-as significant contributors to sporadic IPF. These results support the idea that telomere dysfunction is involved in IPF pathogenesis.

The hypomorphic TERT A1062T variant is associated with increased treatment-related toxicity in acute myeloid leukemia.

Hypomorphic germline variants in TERT, the gene encoding the reverse transcriptase component of the human telomerase complex, occur with a frequency of 3-5% in acute myeloid leukemia. We analyzed the clinical and prognostic impact of the most common TERT A1062T variant in younger patients with acute myeloid leukemia intensively treated within two prospective multicenter trials. Four hundred and twenty patients (age 17-60years) were analyzed for the TERT A1062T variant by direct sequencing. Fifteen patients (3.6%) carried the TERT A1062T variant. Patients with the TERT A1062T variant had a trend towards less favorable and more intermediate 2/adverse karyotypes/genotypes according to the European Leukemia Net classification. In univariate and multivariate analysis, patients with the TERT A1062T variant had a significantly inferior overall survival compared to wild-type patients (6-year overall survival 20 vs. 41%, p=0.005). Patients with the TERT A1062T variant showed a high rate of treatment-related mortality: 5/15 (33%) died during induction therapy or in complete remission as compared to 62/405 (15%) of the wild-type patients. In patients with the TERT variant, 14/15 (93%) suffered from non-hematological/non-infectious grade 3/4 adverse events (mostly hepatic and/or mucosal) as compared to 216/405 (53%) wild-type patients (p=0.006). In multivariate analysis, the TERT A1062T variant was an independent risk factor predicting for adverse events during induction chemotherapy. In conclusion, the TERT A1062T variant is an independent negative prognostic factor in younger patients with acute myeloid leukemia and seems to predispose those patients to treatment-related toxicity.