Multiplex Quantitative PCR Method Research Articles

The relation between sensory processing sensitivity and telomere length in adolescents.

BackgroundIn the present study, we investigated the association between sensory processing sensitivity (SPS) and telomere length (TL), which is considered a biomarker of cellular aging. SPS is an individual characteristic describing increased perception and procession of inner or outer stimuli, and is positively related to self‐perceived stress.MethodsWe recruited 82 healthy adolescents aged 13–16 from secondary schools in Germany. SPS was measured with the Highly Sensitive Person Scale, and TL was determined by a multiplex quantitative PCR method.ResultsOur results show that students with higher values of SPS are likely to have shorter telomeres (β = 0.337, p = .001), when adjusting for sex, socioeconomic status, age, and body mass index. These findings are also independent of the negative impact of stress students might have perceived shortly before data collection.ConclusionsOur analysis suggests that students who struggle with low sensory threshold are likely to have shorter telomeres.

Assessment of a Multiplex PCR for the Simultaneous Diagnosis of Intestinal Cryptosporidiosis and Microsporidiosis: Epidemiologic Report from a French Prospective Study

Microsporidiosis and cryptosporidiosis are associated with chronic diarrhea in immunocompromised patients. The objectives of this study were to: i) assess a multiplex quantitative PCR assay targeting Cryptosporidium spp and the microsporidian Enterocytozoon bieneusi and Encephalitozoon spp, and ii) provide an update on the epidemiology of these pathogens. A prospective study was conducted from January 2017 to January 2019. Performance of the assay was assessed, and all cryptosporidia and microsporidia isolates were genotyped. The sensitivity of the multiplex PCR method reached 1 copy/μL for each targeted pathogen. The sensitivity of co-proantigen testing in the diagnosis of cryptosporidiosis was 73%. The sensitivity of microscopy in the diagnosis of cryptosporidiosis was 64%, and microsporidiosis, 50%. Among the 456 patients included, 14 were positive for Cryptosporidium spp (4 different species); 5, for E. bieneusi; and 2, for Encephalitozoon intestinalis. The overall prevalence of cryptosporidia was 3.1%, and of microsporidia, 1.5%; in kidney transplant recipients (n= 82), corresponding values were 7.3% and 2.4% (6 and 2 patients), respectively. Two cases of E. intestinalis infection were diagnosed in children who had traveled to the tropics. This study is the first to assess a multiplex quantitative PCR method for the simultaneous diagnosis of intestinal microsporidiosis and cryptosporidiosis. The highest prevalences of both pathogens were observed in kidney transplant recipients.

OR18-2 Higher Plasma Estradiol Concentration Is Independently Associated with Lower Biological Age, Assessed as Leucocyte Telomere Length, in Older Men

Telomeres are essential DNA-protein complexes comprising TTAGGG repeats binding specific proteins, which protect the physical ends of chromosomes from fusion and degradation. Attrition of telomeres results in cellular senescence. Leucocyte telomere length (LTL) reflects lengths of telomeres in various tissues, and shorter LTL is a marker of advancing biological age. Previous work has associated bioactive metabolites of T, dihydrotestosterone (DHT) and estradiol (E2) with LTL in a population of predominantly middle-aged men [1]. However, the relationship of these hormones to biological age in older men was unclear. We aimed to clarify associations of sex hormones with LTL in a cohort of 2,913 community-dwelling men aged 70-89 years. Early morning blood samples were assayed for T, DHT and E2 using mass spectrometry, and for sex hormone-binding globulin (SHBG) using immunoassay. LTL was measured using a multiplex quantitative PCR method and expressed as the amount of telomeric DNA relative to beta-globin, a single copy control gene (T/S ratio). Cross-sectional analyses utilised multivariable linear regression. Mean (±SD) age was 76.7±3.2 years. The average difference per decade of age was T -0.46 nmol/L, DHT -0.11 nmol/L, E2 -7.5 pmol/L, SHBG +10.2 nmol/L, and LTL (T/S ratio) -0.065. E2 correlated with T/S ratio (r=0.038, p=0.039). After excluding highest and lowest 1% of values, the correlation between E2 and T/S ratio was largely unchanged (r=0.039, p=0.037). SHBG was inversely correlated with T/S ratio (r=-0.053, p=0.004), also unchanged in the trimmed analysis (r=-0.055, p=0.004.) After adjusting for age, BMI, cardiovascular disease, diabetes, alcohol, smoking, physical activity, lipids and hypertension, E2 remained associated with T/S ratio (per 1 SD increase E2: coefficient 0.011, p=0.043). When E2 and SHBG were simultaneously included in the multivariate model, E2 remained positively associated with T/S ratio (coefficient 0.014, p=0.014) and SHBG inversely associated (coefficient -0.013, p=0.037). The magnitude of increase in T/S ratio associated with a 1 SD higher plasma E2 concentration was comparable with having a BMI 3.6 kg/m2 lower, and two thirds that associated with being 3.6 years younger. T, DHT and LH were not associated with LTL in multivariate analyses. To conclude, in older men, neither T nor DHT are associated with LTL while E2 is independently associated with LTL and SHBG is inversely associated. These findings associate activity of the gonadal axis with lower biological age in older men. However, causality cannot be inferred from an observational, cross-sectional study, thus additional research is necessary to determine whether sex hormone exposure modulates male biological ageing. Reference: [1] Yeap BB, et al. J Clin Endocrinol Metab 2016; 101: 1299-1306.

Telomere shortening in blood leukocytes of patients with posttraumatic stress disorder

Telomeres are protective fragments on chromosome ends involved in maintaining genome stability, preventing chromosomal fusions, regulation of cell division. It was shown that telomere attrition rate is accelerated in age-related diseases, as well as in response to physiological and psychosocial stress. The aim of this study was to evaluate relative leukocyte telomere length (LTL) in patients with post traumatic stress disorder (PTSD), as well as to investigate association of functional SNPs of telomerase TERC and TERT genes with LTL and PTSD. The relative LTL was measured by multiplex quantitative PCR method; genotyping of TERC rs12696304, TERT rs7726159 and rs2736100 was performed by PCR with sequence specific primers. Comparison of LTL in diseased and healthy subjects showed that PTSD patients had shorter average LTL than controls. Also, the frequency and the carriage rate of the TERT rs2736100*T allele was higher in PTSD patients compared to controls. Overall our results are in line with previous research in different populations. Furthermore, we have demonstrated that rs2736100 of TERT gene was significantly associated with PTSD and the minor allele of this polymorphism may be considered as a risk factor for PTSD in the Armenian population.

Abstract 1470: Relative telomere length in tumor tissue and adjacent mucosa of colorectal cancer patients

Abstract Colorectal cancer (CRC) represents a major public health problem, being the fourth leading cause of cancer related deaths mainly due to the late diagnosis and a lack of proper prognostic markers. According to the current knowledge, shortening of telomeres is important contributor in cancer onset. Telomeres, repetitive TTAGGG sequences and boud proteins, are structures at the end of all linear eukaryotic chromosomes. Telomeres are double-edged sword regarding to cancer. On one hand, they serve as a potent tumor-supressor mechanism limiting the cells ability of uncontroled growth. However, on the other hand, shortening of telomeres can lead to the formation of anaphase bridges, initiation of breakage-fusion-bridges cycles and eventually to the induction of chromosome instability phenotype. Telomeric regions are composed of microsatellite repeats and DNA mismatch repair (MMR) defects should directly affect telomere length. Studies looking into the relationship between microsatellite instability (MSI) and telomere length have been sparse. We hypothesize that CRC patients with MSI should have better prognosis, longer survival, also due to the MMR defects in telomere regions replication failure. The aim of our present study was to investigate relative telomere length (RTL) in tumor tissue and adjacent mucosa of 650 CRC patients; it is the largest study on RTL in target tissue so far. Moreover, we measured RTL in peripheral blood lymphocytes obtained from 150 cancer patients. Tumor and adjacent non-affected tissue samples were obtained from patients at surgical resection. RTL was measured using monochrome multiplex quantitative PCR method. Despite the analysis is still running, our preliminary results showed significantly shorter RTL in tumor tissues as compared to adjacent mucosa. The shortest RTL was discovered in right, ascendent part of colon (0.68±0.59, 25.5% of cases), followed by left, descendent colon (0.77±0.47, 37.6%) and the longest RTL was in rectal tumors (0.92±0.69, 36.9%). We recorded gradually increasing RTL ratio (RTL in tumor tissue/ RTL in adjacent mucosa) in later stages according to TNM staging system. Interestingly, patients with RTL ratio > 1 had also worse survival. RTL of CRC tissue and adjacent mucosa may provide a valuable prognostic tool in patients treatment management. This study was supported by GACR 15-14789S and by the National Sustainability Program I (NPU I) Nr. LO1503 provided by the Ministry of Education Youth and Sports of the Czech Republic Citation Format: Michal Kroupa, Vaclav Liska, Krishna Rachakonda, Marketa Urbanova, Michaela Schneiderova, Katerina Jiraskova, Ondrej Vycital, Ludmila Vodickova, Rajiv Kumar, Pavel Vodicka. Relative telomere length in tumor tissue and adjacent mucosa of colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1470.

Accumulative effects of indoor air pollution exposure on leukocyte telomere length among non-smokers

Indoor air pollution is an important environmental factor that contributes to the burden of various diseases. Long-term exposure to ambient air pollution is associated with telomere shortening. However, the association between chronic indoor air pollution from household fuel combustion and leukocyte telomere length has not been studied. In our study, 137 cancer-free non-smokers were recruited. Their exposure levels to indoor air pollution from 1985 to 2014 were assessed using a face-to-face interview questionnaire, and leukocyte telomere length (LTL) was measured using a monochrome multiplex quantitative PCR method. Accumulative exposure to solid fuel usage for cooking was negatively correlated with LTL. The LTL of residents who were exposed to solid fuel combustion for three decades (LTL = 0.70 ± 0.17) was significantly shorter than that of other populations. In addition, education and occupation were related to both exposure to solid fuel and LTL. Sociodemographic factors may play a mediating role in the correlation between leukocyte telomere length and environmental exposure to indoor air pollution. In conclusion, long-term exposure to indoor air pollution may cause LTL dysfunction.

Electrocardiogram QRS duration and associations with telomere length: A cross-sectional analysis in Australian rural diabetic and non-diabetic population

Prolonged electrocardiogram QRS durations are often present in aging populations. Shorter telomere length is considered a biomarker of cellular aging. Decreased telomere length has been associated with coronary artery risk, and ventricular remodeling. However, the association between telomeres and cardiac conduction abnormalities, such as increased QRS duration are not well understood. A retrospective cross-sectional population was obtained from the CSU Diabetes Screening Research Initiative database where 273 participants had both ECG-derived QRS duration and DNA to permit leukocyte telomere length (LTL) determination. Telomere length was determined using the monochrome multiplex quantitative PCR method to measure mean relative LTL. Resting 12-lead electrocardiograms were obtained from each subject using a Welch Allyn PC-Based ECG system. Relative LTL was moderately negatively associated with QRS duration in type 2 diabetes mellitus (T2DM) patients (R2=0.055), compared to controls (R2=0.010). In general linear models with no adjustments a significant interaction between QRS duration and LTL is observed for a combined population of T2DM and non-diabetics. When we compared T2DM to non-diabetics, we found that T2DM increased the effect size for relative LTL on QRS duration in comparison to controls. Hence, for each 0.1 unit of relative LTL attrition, QRS duration in T2DM patients increased by 3.24ms (95% CI, −63.00 to −1.84), compared to 1.65ms in controls (95% CI, −40.44 to 7.40). In summary we have observed an association between LTL in a rural aging mixed population of T2DM and non-diabetes. We have observed an unadjusted association between QRS duration and LTL in T2DM. We noted that the control group demonstrated no such association. This highlights the complexity of T2DM when exploring disease phenotype–telomere interactions.

Heterozygous RTEL1 Variants Are Associated with Bone Marrow Failure and Abnormal Clinical Phenotype

IntroductionHeterozygous RTEL1 mutations have recently been described in familial pulmonary fibrosis (PF) but are not known to be associated with cytopenias or bone marrow failure (BMF), in contrast to heterozygous mutations in other telomere maintenance genes TERT, TERC and TINF2. Constitutional BMF syndromes typically present with less severe pancytopenia and it is often unclear if they have hypocellular MDS (hypoMDS) or non-severe AA (NSAA) morphologically.MethodsWe screened 284 patients with idiopathic AA or uncharacterised BMF and 172 patients with MDS or acute myeloid leukemia (AML) for TL and RTEL1 variants, and for the other currently known telomere gene complex (TGC) mutations, after excluding patients with Fanconi anemia, DBA or other known inherited BMF syndrome. TL was measured using a monochrome multiplex quantitative PCR method on peripheral blood mononuclear cells. Illumina Nextera-amplicon sequencing was used to screen exons of the DC genes (DKC1, TERC, TERT, RTEL1, CTC1, NHP10, NOP2, USB1, WRAP53, TINF2, PARN and ACD) by MiSeq platform. Constitutional DNA was also analysed in 10 patients (skin 9, buccal swab 1) with RTEL1 variants. A targeted gene panel of 24 genes of an Illumina Tru-Seq Custom Amplicon workflow and platform was used to identify genes frequently mutated in MDS/AML. Impact of mutations was predicted based on 3D structure information from comparative modelling for the helicase domain, comprising the HD1 and HD2 subdomains, a Fe-S cluster and an ARCH domain, and for two harmonin-like (HML) domains and a RING finger domain, located in the C-terminal regulatory region of RTEL1.ResultsHeterozygous RTEL1 variants were identified in 20 (4.4%) patients. RTEL1 variant allele frequency (VAF) was 45-70% consistent with heterozygous inheritance in all cases. TL was short in 18 (90%) patients, being < 1st centile in 15 and <10th centile in 3. 2 patients had normal TL, <20th centile and >50th centile, respectively. Median age was 35 years (range 18-73). 15/20 (75%) had a hypocellular BM (7 hypoMDS, 5 non-severe AA, 3 ICUS), and 1 each with RAEB1, RAEB2, CMML1, AML and isolated macrocytosis. 3 patients had abnormal karyotype: +8 (hypoMDS), -Y,+1,del(1) (hypoMDS), del7q (RAEB1). 2 other patients with hypoMDS had somatic mutations: U2AF1 (30% VAF) with ASXL1 (27% VAF); U2AF1 (43% VAF). Lung abnormalities were early PF (1), interstitial lung disease (1), and abnormal lung function with reduced TLCO (1) and an obstructive picture (1). Liver fibrosis with portal hypertension and varices and reticulate skin pigmentation were present in the patient with ILD, 2 patients had dystrophic nails, and 1 unexplained mild hepato-splenomegaly. 2 patients had familial MDS, 5 had a family history of cancers affecting first-degree relatives, and 2 had skeletal and cartilage anomalies, associated with learning difficulties in 1 patient. 8/15 patients with hypocellular BM required no treatment (5 hypoMDS and 3 NSAA), one hypoMDS had CR with ciclosporin and another underwent successful unrelated donor stem cell transplant; for NSAA, 2 received ATG with CSA, with PR followed by relapse in one, the other was lost to follow up, and 1 was androgen responsive. 16/20 (80%) patients are alive; 3 patients with RAEB or AML died of progressive disease and 1 patient with ICUS and severe constitutional features died from lymphoma 10 years after presentation.Mutations were spread throughout the entire RTEL1 sequence (summarised in Figure). 3D structure analysis predicted the missense RTEL1 mutations would result in disturbance of the FeS cluster and/or interfere with DNA binding, destabilisation of the HD1, HD2 or the ARCH sub-domains of the helicase domain, or destabilisation of inter-domain interactions. One HML1 mutation occurred in a loop opposite the putative ligand binding site and the rest in the variable regions outside the conserved domains. RTEL1 variants were associated with TERT mutations in 4 patients, of which 3 were known pathogenetic and 1 novel TERT mutation with low telomerase activity on TRAP assay confirming its pathogenetic nature.ConclusionsWe show for the first time that heterozygous RTEL1 mutations occur in 4.4% of patients, most commonly in young patients with a hypocellular BM, and often a family history of BMF/malignancy, and less often with high risk MDS/AML. Abnormal clinical features were present in a third of patients, some similar to but others distinct from dyskeratosis congenita. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interactions between UCP2 SNPs and telomere length exist in the absence of diabetes or pre-diabetes.

Mitochondrial uncoupling protein 2 (UCP2) can affect oxidative stress levels. UCP2 polymorphisms are associated with leukocyte telomere length (LTL) in Type 2 Diabetes, which also induces considerable background oxidative stress. The effects of UCP2 polymorphisms on LTL in populations without diabetes have not been well described. Our aims are to evaluate the interaction between LTL and UCP2 polymorphisms in 950 subjects without diabetes. The monochrome multiplex quantitative PCR method was used to measure relative LTL. Taqman SNP genotyping assay was applied to genotypes for UCP2 rs659366 and rs660339. We found shorter LTL associated with increased age (P < 0.001) and triglyceride levels (P = 0.041). After adjustment for cardiovascular risk factors, rs659336 GG genotype carriers demonstrated a shorter LTL (1.257 ± 0.186), compared to GA carriers (1.288 ± 0.230, P = 0.022) and AA carriers (1.314 ± 0.253, P = 0.002). LTL was shorter in the CC rs660339 genotype (1.254 ± 0.187) compared to TT (1.297 ± 0.242, P = 0.007) and CT carriers (1.292 ± 0.229, P = 0.016). The T allele of rs660339 is associated with a longer LTL of approximately 0.04 compared to CC homozygotes. Thus, UCP2 rs659366 A allele and rs660339 T allele are both related to longer LTL in subjects without diabetes, independent of cardiovascular risk factors.

Reduced T-Cell Thymic Export Reflected by sj-TREC in Patients with Coronary Artery Disease.

Immunologic dysfunction was recently found to be one of the most important mechanisms underlying the initiation and development of atherosclerosis. Thymus involution can contribute to immune disturbance and disequilibrium of T-cell subsets. This study aimed to explore whether recent thymic emigration (RTE) is impaired in patients with coronary artery disease (CAD). Content of signal-joint T cell receptor excision circles (sj-TREC) in T lymphocytes, a molecular marker of RTE, was assessed among CAD patients and age-matched controls. Monochrome multiplex quantitative PCR method was used to assess the samples' telomere length in order to exclude the potential influence of T cell proliferation on the dilution of sj-TREC. Patients were grouped according to Gensini score (GS) (low, GS ＜18; intermediate, GS 18-41; high, GS ＞41). Ordinary logistic regression models were used to determine potential risk factors for CAD and GS tertiles. Average copy numbers of sj-TREC per 10(6) T lymphocytes among patients with unstable angina, stable angina, and controls were 726±429, 1213±465, and 1795±838, respectively (P＜0.001). However, there was no significant difference in telomere length among groups. Moreover, the content of sj-TREC in the high GS group was most significantly reduced than the low GS group (P＜0.001). Multivariate logistic regression analysis revealed that lower sj-TREC was independently associated with the progression of CAD (OR=0.44, P＜0.001) and higher GS (OR=0.4, P＜0.001). Impaired RTE could be partly responsible for CAD development. Mechanisms may be involved in the disturbance of T lymphocyte compartment and interruption of maintained immune tolerance resulting from thymus involution.

Leukocyte telomere length and left ventricular function after acute ST-elevation myocardial infarction: data from the glycometabolic intervention as adjunct to primary coronary intervention in ST elevation myocardial infarction (GIPS-III) trial.

BackgroundTelomere length has been associated with coronary artery disease and heart failure. We studied whether leukocyte telomere length is associated with left ventricular ejection fraction (LVEF) after ST-elevation myocardial infarction (STEMI).Methods and resultsLeukocyte telomere length (LTL) was determined using the monochrome multiplex quantitative PCR method in 353 patients participating in the glycometabolic intervention as adjunct to primary percutaneous coronary intervention in STEMI III trial. LVEF was assessed by magnetic resonance imaging. The mean age of patients was 58.9 ± 11.6 years, 75 % were male. In age- and gender-adjusted models, LTL at baseline was significantly associated with age (beta ± standard error; −0.33 ± 0.01; P < 0.01), gender (0.15 ± 0.03; P < 0.01), TIMI flow pre-PCI (0.05 ± 0.03; P < 0.01), TIMI flow post-PCI (0.03 ± 0.04; P < 0.01), myocardial blush grade (−0.05 ± 0.07; P < 0.01), serum glucose levels (−0.11 ± 0.01; P = 0.03), and total leukocyte count (−0.11 ± 0.01; P = 0.04). At 4 months after STEMI, LVEF was well preserved (54.1 ± 8.4 %) and was not associated with baseline LTL (P = 0.95). Baseline LTL was associated with n-terminal pro-brain natriuretic peptide (NT-proBNP) at 4 months (−0.14 ± 0.01; P = 0.02), albeit not independent for age and gender.ConclusionOur study does not support a role for LTL as a causal factor related to left ventricular ejection fraction after STEMI.

Abstract 4160: Telomere length in white blood cell DNA and lung cancer: a pooled analysis of three prospective cohorts

Abstract Background: There is growing evidence that longer telomere length is associated with higher risk of lung cancer. We investigated this association in the prospective Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial, which was conducted in the United States. We also combined these data with two previously published prospective cohorts: the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) trial conducted among ever smoking males in Finland and the Shanghai Women's Health Study (SWHS) which comprised primarily of never-smoking women, resulting in a pooled analysis on a total of 847 cases and 847 controls matched by age, sex and study. Methods: Blood samples were collected prior to diagnosis of lung cancer and telomere length was measured using the same monochrome multiplex quantitative PCR method in all three studies. We used conditional logistic regression models to calculate odds ratios (OR) and their 95% confidence intervals (CI) for the association between telomere length and lung cancer risk adjusted for age, to account for residual confounding, and pack-years of smoking as continuous variables. Analyses by telomere length quartile retaining the initial categorization used in each study, and using categorization based on telomere length in pooled controls, produced similar findings and results are presented for the former. Results: In the PLCO Trial, increasing telomere length was significantly associated with lung cancer risk (adjusted OR [95% CI] by quartile: 1.00; 1.11 [0.65-1.92]; 1.20 [0.66-2.15]; and 1.83 [1.05-3.19]; P-trend = 0.011), consistent with results from the ATBC and SWHS studies. In the pooled analyses, the adjusted OR (95% CI) by quartile was 1.00; 1.24 (0.90-1.72); 1.27 (0.91-1.77); and 1.87 (1.33-2.63); P-trend = 0.000022. This positive association was particularly evident for adenocarcinoma cases, especially those diagnosed more than 6 years after blood collection (n=115; adjusted OR [95% CI] by quartile: 1.00; 2.48 [0.85-7.23]; 2.05 [0.81-5.15]; and 3.59 [1.38-9.34]; P-trend = 0.0027). Conclusion: Telomere length in white blood cell DNA may be an important biomarker of future increased risk of lung cancer in diverse populations. Citation Format: Wei Jie Seow, Richard Cawthon, Mark Purdue, Wei Hu, Yu-Tang Gao, Wen-Yi Huang, Stephanie J. Weinstein, Bu-Tian Ji, Jarmo Virtamo, Dean Hosgood, Bryan Bassig, Xiaoou Shu, Qiuyin Cai, Yongbin Xiang, Shen Min, Wong-Ho Chow, Sonja Berndt, Christopher Kim, Unhee Lim, Demetrius Albanes, Neil E. Caporaso, Stephen Chanock, Wei Zheng, Nathaniel Rothman, Qing Lan. Telomere length in white blood cell DNA and lung cancer: a pooled analysis of three prospective cohorts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4160. doi:10.1158/1538-7445.AM2014-4160

Placental telomere length decreases with gestational age and is influenced by parity: A study of third trimester live-born twins

BackgroundIn contrast to the postnatal period, little is known about telomere length (TL) during prenatal life. The decrease in placental TL remains unknown, although intra uterine growth retardation and preeclampsia are associated with shorter placental TL. The aim of this study is to assess the decrease of placental TL during the third trimester of gestation and to explore the role of potential “growth influencing factors”. MethodsThe study sample consisted of 329 live-born twins from the East Flanders Prospective Twin Survey. TL was determined using a multiplex quantitative PCR method. Gestational age, sex, birth order, placental characteristics, parity, maternal and paternal age, diabetes, hypertension, smoking, alcohol use, and socio economic status (SES) were considered “growth influencing factors”. Bivariable multilevel regression analysis with “growth influencing factors” was performed. ResultsPlacental TL ranged from 4.3 kbp to 84.4 kbp with a median of 10.8 kbp. Ln(TL) decreased in a linear fashion with an estimated TL decreasing from 13.98 kbp at 28 weeks to 10.56 kbp at 42 weeks. The regression coefficient of gestational age became smaller if considered together with SES (b = −0.017; p = 0.08) or diabetes (b = −0.018; p = 0.07) and bigger if considered together with parity (b = −0.022; p = 0.02), indicating that part of the association between gestational age and telomere length is explained by these three confounding factors. ConclusionPlacental TL decreases during the third trimester of gestation of live-born twins with approximately 25% indicating that telomere shortening may play a role in aging of the placenta.

Longer Telomeres Are Associated with Cancer Risk in MMR-Proficient Hereditary Non-Polyposis Colorectal Cancer

Aberrant telomere length measured in blood has been associated with increased risk of several cancer types. In the field of hereditary non-polyposis colorectal cancer (CRC), and more particularly in Lynch syndrome, caused by germline mutations in the mismatch repair (MMR) genes, we recently found that cancer-affected MMR gene mutation carriers had shorter telomeres and more pronounced shortening of telomere length with age than controls and unaffected MMR gene mutation carriers. Here we evaluate blood telomere length in MMR-proficient hereditary non-polyposis CRC, i.e. familial CRC type X (fCRC-X). A total of 57 cancer-affected and 57 cancer-free individuals from 34 Amsterdam-positive fCRC-X families were analyzed and compared to the data previously published on 144 cancer-affected and 100 cancer-free MMR gene mutation carriers, and 234 controls. Relative telomere length was measured using a monochrome multiplex quantitative PCR method, following strict measures to avoid sources of bias and adjusting by age. Despite the retrospective nature of our study, the results show that longer telomeres associate with cancer risk in fCRC-X, thus identifying different patterns of telomere length according to the status of the MMR system.

Telomere Length and Genetic Anticipation in Lynch Syndrome

Telomere length variation has been associated with increased risk of several types of tumors, and telomere shortening, with genetic anticipation in a number of genetic diseases including hereditary cancer syndromes. No conclusive studies have been performed for Lynch syndrome, a hereditary colorectal cancer syndrome caused by germline mutations in the DNA mismatch repair genes. Here we evaluate telomere length in Lynch syndrome, both as a cancer risk factor and as a mechanism associated with anticipation in the age of cancer onset observed in successive generations of Lynch syndrome families. Leukocyte telomere length was measured in 244 mismatch repair gene mutation carriers from 96 Lynch syndrome families and in 234 controls using a monochrome multiplex quantitative PCR method. Cancer-affected mutation carriers showed significantly shorter telomeres than cancer-free mutation carriers. In addition, cancer-affected carriers showed the most pronounced shortening of telomere length with age, compared with unaffected carriers. The anticipation in the age of cancer onset observed in successive generations was not associated with telomere shortening, although, interestingly, all mother-son pairs showed telomere shortening. In conclusion, cancer-affected mismatch repair gene mutation carriers have distinct telomere-length pattern and dynamics. However, anticipation in the age of onset is not explained by telomere shortening. Pending further study, our findings suggest that telomere attrition might explain the previously reported dependence of cancer risk on the parent-of-origin of mismatch repair gene mutations.

Telomere length measurement by a novel monochrome multiplex quantitative PCR method

The current quantitative polymerase chain reaction (QPCR) assay of telomere length measures telomere (T) signals in experimental DNA samples in one set of reaction wells, and single copy gene (S) signals in separate wells, in comparison to a reference DNA, to yield relative T/S ratios that are proportional to average telomere length. Multiplexing this assay is desirable, because variation in the amount of DNA pipetted would no longer contribute to variation in T/S, since T and S would be collected within each reaction, from the same input DNA. Multiplexing also increases throughput and lowers costs, since half as many reactions are needed. Here, we present the first multiplexed QPCR method for telomere length measurement. Remarkably, a single fluorescent DNA-intercalating dye is sufficient in this system, because T signals can be collected in early cycles, before S signals rise above baseline, and S signals can be collected at a temperature that fully melts the telomere product, sending its signal to baseline. The correlation of T/S ratios with Terminal Restriction Fragment (TRF) lengths measured by Southern blot was stronger with this monochrome multiplex QPCR method (R2 = 0.844) than with our original singleplex method (R2 = 0.677). Multiplex T/S results from independent runs on different days were highly reproducible (R2 = 0.91).