Repeat Copy Number Research Articles

Association of Short-term Change in Leukocyte Telomere Length With Cortical Thickness and Outcomes of Mental Training Among Healthy Adults

Telomere length is associated with the development of age-related diseases and structural differences in multiple brain regions. It remains unclear, however, whether change in telomere length is linked to brain structure change, and to what extent telomere length can be influenced through mental training. To assess the dynamic associations between leukocyte telomere length (LTL) and cortical thickness (CT), and to determine whether LTL is affected by a longitudinal contemplative mental training intervention. An open-label efficacy trial of three 3-month mental training modules with healthy, meditation-naive adults was conducted. Data on LTL and CT were collected 4 times over 9 months between April 22, 2013, and March 31, 2015, as part of the ReSource Project. Data analysis was performed between September 23, 2016, and June 21, 2019. Of 1582 eligible individuals, 943 declined to participate; 362 were randomly selected for participation and assigned to training or retest control cohorts, with demographic characteristics matched. The retest control cohorts underwent all testing but no training. Intention-to-treat analysis was performed. Training cohort participants completed 3 modules cultivating interoception and attention (Presence), compassion (Affect), or perspective taking (Perspective). Change in LTL and CT. Of the 362 individuals randomized, 30 participants dropped out before study initiation (initial sample, 332). Data were available for analysis of the training intervention in 298 participants (n = 222 training; n = 76 retest control) (175 women [58.7%]; mean [SD] age, 40.5 [9.3] years). The training modules had no effect on LTL. In 699 observations from all 298 participants, mean estimated changes in the relative ratios of telomere repeat copy number to single-copy gene (T/S) were for no training, 0.004 (95% CI, -0.010 to 0.018); Presence, -0.007 (95% CI, -0.025 to 0.011); Affect, -0.005 (95% CI, -0.019 to 0.010); and Perspective, -0.001 (95% CI, -0.017 to 0.016). Cortical thickness change data were analyzed in 167 observations from 67 retest control participants (37 women [55.2%], mean [SD] age, 39.6 [9.0] years). In this retest control cohort subsample, naturally occurring LTL change was related to CT change in the left precuneus extending to the posterior cingulate cortex (mean t161 = 3.22; P < .001; r = 0.246). At the individual participant level, leukocyte telomere shortening as well as lengthening were observed. Leukocyte telomere shortening was related to cortical thinning (t77 = 2.38; P = .01; r = 0.262), and leukocyte telomere lengthening was related to cortical thickening (t77 = 2.42; P = .009; r = 0.266). All analyses controlled for age, sex, and body mass index. The findings of this trial indicate an association between short-term change in LTL and concomitant change in plasticity of the left precuneus extending to the posterior cingulate cortex. This result contributes to the evidence that LTL changes more dynamically on the individual level than previously thought. Further studies are needed to determine potential long-term implications of such change in relation to cellular aging and the development of neurodegenerative disorders. No effect of contemplative mental training was noted in what may be, to date, the longest intervention with healthy adults. ClinicalTrials.gov identifier: NCT01833104.

Sensorimotor Cortical Oscillations during Movement Preparation in 16p11.2 Deletion Carriers.

Sensorimotor deficits are prevalent in many neurodevelopmental disorders like autism, including one of its common genetic etiologies, a 600 kb reciprocal deletion/duplication at 16p11.2. We have previously shown that copy number variations of 16p11.2 impact regional brain volume, white matter integrity, and early sensory responses in auditory cortex. Here, we test the hypothesis that abnormal cortical neurophysiology is present when genes in the 16p11.2 region are haploinsufficient, and in humans that this in turn may account for behavioral deficits specific to deletion carriers. We examine sensorimotor cortical network activity in males and females with 16p11.2 deletions compared with both typically developing individuals, and those with duplications of 16p11.2, using magnetoencephalographic imaging during preparation of overt speech or hand movements in tasks designed to be easy for all participants. In deletion carriers, modulation of beta oscillations (12-30 Hz) were increased during both movement types over effector-specific regions of motor cortices compared with typically developing individuals or duplication carriers, with no task-related performance differences between cohorts, even when corrected for their own cognitive and sensorimotor deficits. Reduced left hemispheric language specialization was observed in deletion carriers but not in duplication carriers. Neural activity over sensorimotor cortices in deletion carriers was linearly related to clinical measures of speech and motor impairment. These findings link insufficient copy number repeats at 16p11.2 to excessive neural activity (e.g., increased beta oscillations) in motor cortical networks for speech and hand motor control. These results have significant implications for understanding the neural basis of autism and related neurodevelopmental disorders.SIGNIFICANCE STATEMENT The recurrent ∼600 kb deletion at 16p11.2 (BP4-BP5) is one of the most common genetic etiologies of ASD and, more generally, of neurodevelopmental disorders. Here, we use high-resolution magnetoencephalographic imaging (MEG-I) to define with millisecond precision the underlying neurophysiological signature of motor impairments for individuals with 16p11.2 deletions. We identify significant increases in beta (12-30 Hz) suppression in sensorimotor cortices related to performance during speech and hand movement tasks. These findings not only provide a neurophysiological phenotype for the clinical presentation of this genetic deletion, but also guide our understanding of how genetic variation encodes for neural oscillatory dynamics.

DYNAMICS OF FREQUENCIES OF WOLBACHIA GENOTYPES IN DROSOPHILA MELANOGASTER POPULATION FROM UMAN’ UNDER INFLUENCE OF CLIMATE FACTORS

Aim. To investigate the potential influence of climate parameters on the rate of Wolbachia genotypes frequencies in the population of D. melanogaster from Uman’ for the last seven years. Methods. We have surveyed Wolbachia infection status for isofemale lines from Uman’ collected during summer – fall 2013 and 2015-2017 using polymerase chain reaction (PCR) with primers specific to the 16S rRNA and wsp (Wolbachia surface protein) genes. To determine wMel and wMelCS genotypes of Wolbachia, we have conducted PCR using polymorphic markers for the presence of the insertion sequence IS5 in WD1310 locus and the copy number of minisatellite repeats in VNTR-141 locus. Infection status of flies for 2011, 2012 and 2014 years was included from our previous studies. Data of climate factors (average seasonal temperature, dew point, and precipitation) for winter and summer of each year were obtained from weather database. Statistical analysis of Wolbachia genotypes-climate factors interaction was carried out in R version 3.5.0 using multiple linear model regression. Results. We have observed the decline of Wolbachia presence in Uman’ locality and prevalence of wMel bacteria variant. Moreover, our study documented the low persistence of rare wMelCS genotype through seven years in Uman’. wMelCS frequency has been driven by the combined influence of the average temperature and humidity in summer (p = 0.03662, R^2 = 0.9995). Conclusions. The climate variability affects frequency of wMelCS genotype of Wolbachia in the D. melanogaster natural population from Uman’.

Variable Surface Display and Post‐Translational Regulation of the Fungal Adhesin Epa1p

Infection by the fungal pathogen Candida glabrata is reliant on a collection of cell surface adhesins that play a variety of roles necessary for transmission, establishment, and cell proliferation. One particular adhesin, Epithelial Adhesin 1 (Epa1p), is responsible for binding to host tissue and biofilm formation, which is essential for fungal propagation. Epa1p structure consists of three domains: a C‐terminal GPI anchor for cell wall linkage, an N‐terminal intercellular binding domain responsible for epithelial cell binding, and a serine/threonine rich linker domain connecting these terminal domains. The linker domain contains a 40‐amino acid tandem repeat region which our lab has observed to be variable in repeat copy number between isolates from clinical sources. We expressed Epa1p with various repeat copy numbers in epa1Δ C. glabrata as well as in the related yeast, S. cerevisiae to determine how differences in repeat copy number affect Epa1p surface display and binding to human epithelial cells. Our initial data suggest that the processing of, as well as cell wall stability of, Epa1p is dependent upon the repeat copy number of Epa1p. We have also collected data, which suggest that Epa1p surface level is dependent on media growth conditions. Altogether our data demonstrate that Epa1p repeat copy number influences the properties of Epa1p and the adhesive characteristics of C. glabrata.Support or Funding Information1. Army Research Office grant W911NF16101752. Tufts Collaborates!3. 2018 ASBMB Undergraduate Research Award4. Tufts Summer ScholarsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Distribution of Mycobacterium leprae genotypes from Surabaya and Bandung Clinical Isolates by Multiple Locus Variable Number of Tandem Repeat Analysis

Multiple Locus Variable Number of Tandem Repeat (VNTR) analysis has been proposed as a means of genotyping for tracking leprosy transmission due to tandem repeats’ potential as genetic markers to differentiate M. leprae strains. However, characteristics of polymorphism can vary depending on the population. This study aimed to compare the copy number of repeats in four genetic markers: TTC, AC8a, AC9 and 6-7 in leprosy patients from Surabaya and Bandung. Twenty three patients from Dr. Soetomo General Hospital and 21 from Hasan Sadikin Hospital were recruited. Multiple locus VNTR analysis was applied using total DNA extracts from Slit Skin Smear (SSS). From Surabaya, 7 samples showed the same copy number of four genetic markers (TTC=15; AC8a=10; AC9=10 and 6-7=6) and 2 showed another (TTC=16; AC8a=10; AC9=11 and 6-7=6); as for samples from Bandung, 2 showed the same copy number (TTC=15; AC8a=8; AC9=10 and 6-7=8) and 2 showed another (TTC=16; AC8a=10; AC9=11 and 6-7=6). The multiple locus VNTR analysis showed two identical M. leprae VNTR profiles from Bandung and Surabaya which supports the use of VNTR loci for transmission studies.

Using high-resolution annotation of insect mitochondrial DNA to decipher tandem repeats in the control region

ABSTRACTIn this study, we used a small RNA sequencing (sRNA-seq) based method to annotate the mitochondrial genome of the insect Erthesina fullo Thunberg at 1 bp resolution. The high-resolution annotations cover both entire strands of the mitochondrial genome without any gaps or overlaps. Most of the new annotations were consistent with the previous annotations which had been obtained using PacBio full-length transcripts. Two important findings were that animals transcribe both entire strands of mitochondrial genomes and the tandem repeats in the control region of the E. fullo mitochondrial genome contains the repeated Transcription Initiation Sites (TISs) of the heavy strand. In addition, we found that the copy numbers of tandem repeats showed a great diversity within an individual, suggesting that mitochondrial DNA recombination occurs in an individual. In conclusion, the sRNA-seq based method uses 5′ and 3′ end small RNAs to annotate nuclear non-coding and mitochondrial genes at 1 bp resolution, and can be used to identify new steady RNAs, particularly long non-coding RNAs (lncRNAs). The high-resolution annotations of mitochondrial genomes can also be used to study the molecular phylogenetics and evolution of animals or to investigate mitochondrial gene transcription, RNA processing, RNA maturation and several other related topics. The complete mitochondrial genome sequence of E. fullo with the new annotations using the sRNA-seq based method is available at the NCBI GenBank database under the accession number MK374364. We publish our theories, methods, the high quality sRNA-seq and RNA-seq data (SRA: SRP174926) for extensive use.

Association between prenatal particulate air pollution exposure and telomere length in cord blood: Effect modification by fetal sex

IntroductionIn utero particulate matter exposure produces oxidative stress that impacts cellular processes that include telomere biology. Newborn telomere length is likely critical to an individual's telomere biology; reduction in this initial telomere setting may signal increased susceptibility to adverse outcomes later in life. We examined associations between prenatal particulate matter with diameter ≤2.5 µm (PM2.5) and relative leukocyte telomere length (LTL) measured in cord blood using a data-driven approach to characterize sensitive windows of prenatal PM2.5 effects and explore sex differences. MethodsWomen who were residents of Mexico City and affiliated with the Mexican Social Security System were recruited during pregnancy (n = 423 for analyses). Mothers’ prenatal exposure to PM2.5 was estimated based on residence during pregnancy using a validated satellite-based spatio-temporally resolved prediction model. Leukocyte DNA was extracted from cord blood obtained at delivery. Duplex quantitative polymerase chain reaction was used to compare the relative amplification of the telomere repeat copy number to single gene (albumin) copy number. A distributed lag model incorporating weekly averages for PM2.5 over gestation was used in order to explore sensitive windows. Sex-specific associations were examined using Bayesian distributed lag interaction models. ResultsIn models that included child's sex, mother's age at delivery, prenatal environmental tobacco smoke exposure, pre-pregnancy BMI, gestational age, birth season and assay batch, we found significant associations between higher PM2.5 exposure during early pregnancy (4–9 weeks) and shorter LTL in cord blood. We also identified two more windows at 14–19 and 34–36 weeks in which increased PM2.5 exposure was associated with longer LTL. In stratified analyses, the mean and cumulative associations between PM2.5 and shortened LTL were stronger in girls when compared to boys. ConclusionsIncreased PM2.5 during specific prenatal windows was associated with shorter LTL and longer LTL. PM2.5 was more strongly associated with shortened LTL in girls when compared to boys. Understanding sex and temporal differences in response to air pollution may provide unique insight into mechanisms.

Association between telomere length and completed suicide observed in 71 suicide victims – Preliminary findings

ObjectiveTelomere length has emerged as a cumulative marker for lifestyle, psychosocial stress, and cytotoxic environments. We aimed to examine the possible association between leukocyte telomere length (LTL) and completed suicide. MethodsThis study included 71 suicide completers and 117 healthy controls for whom LTL was determined by the ratio of the telomere repeat copy number to the single-copy gene copy number (T/S ratio). We compared the LTL between the suicide completers and the healthy controls and estimated the odds ratio (OR) for suicide for each age group, applying a generalized estimating equation (GEE). ResultsLTL was significantly shortened in the suicide completers as compared with the controls, overall subjects, or within-age categories (≤29 and 30–49 years). Furthermore, a longer LTL was associated with significantly decreased odds of completed suicide for those aged ≤29 years and 30–49 years (OR = 0.11, 95% confidence interval [CI] 0.03–0.37, p < .001 for the ≤29-year age group; OR = 0.54, 95% CI 0.34–0.84, p = .006 for the 30- to 49-year age group). ConclusionsThis study provides evidence regarding the relationship between shortened LTL and completed suicide, especially in those aged <50 years. Future research should further assess potential confounders and examine underlying mechanisms.

Ribosomal DNA instability and genome adaptability.

Ribosomes are large, multi-subunit ribonucleoprotein complexes, essential for protein synthesis. To meet the high cellular demand for ribosomes, all eukaryotes have numerous copies of ribosomal DNA (rDNA) genes that encode ribosomal RNA (rRNA), usually far in excess of the requirement for ribosome biogenesis. In all eukaryotes studied, rDNA genes are arranged in one or more clusters of tandem repeats localized to nucleoli. The tandem arrangement of repeats, combined with the high rates of transcription at the rDNA loci, and the difficulty of replicating repetitive sequences make the rDNA inherently unstable and particularly susceptible to large variations in repeat copy number. Despite mounting evidence suggesting extra-ribosomal functions of the rDNA, its repetitive nature has excluded it from traditional sequencing-based studies. However, more recently, several studies have revealed the unique potential of the rDNA to act as a "canary in the coalmine," being particularly sensitive to genomic stresses and acting as a source of adaptive response. Here, we review evidence uncovering mechanisms of regulation of instability and copy number variation at the rDNA and their role in adaptation to the environment, which could serve to understand the basic principles governing the behavior of other tandem repeats and their role in shaping the genome.

SU75 - ASSOCIATION OF PTSD WITH TELOMERE LENGTH IN FEMALE RAPE VICTIMS

Background Posttraumatic Stress Disorder (PTSD) is a clinical condition that occurs after experiencing a traumatic external stressful situation, such as exposure to sexual assault or rape. Previous studies suggested that PTSD may be associated with shortened Leukocyte Telomere Length (LTL), in war veterans. Telomeres are repetitive DNA sequences located at the ends of chromatids that become shorter after each cell division. Some findings proposed that stress induced by trauma is a probable cause of accelerated telomere shortening. We examined a cross-sectional association between LTL and PTSD or psychopathological measures in rape victims. Methods The study population consisted of 32 women rape victims with PTSD diagnosis (PTSD group) and 34 healthy controls women without history of sexual trauma and mental disorders (HC group), with ages between 18 and 43 years. All participants were evaluated using the Mini International Neuropsychiatric Interview (MINI) compatible with Diagnostic and Statistical Manual of Mental Disorder (DSM-V), Clinician-Administered PTSD Scale (CAPS), which assesses PTSD symptoms, and Life Events Checklist for DSM-V, which appraises the trauma exposure. DNA was extracted from peripheral blood leukocytes and telomere repeat copy number relative to single gene copy number (relative T/S ratio) was determined by quantitative real-time PCR telomere assay. We used logistic and linear regression models to assess associations between relative T/S ratio and PTSD or its symptoms, respectively. Results The relative T/S ratio was determined in all samples and we found that PTSD diagnosis was associated with longer LTL, after adjusting for age at blood draw (p=0.008; β=3.796; 95% confidence interval: 1.409 – 10.229), indicating elongation LTL in PTSD group (relative T/S ratio mean=0.336; Std Deviation=0.109) compared to HC control (relative T/S ratio mean=-0.316; Std Deviation=0.161). However, LTL was not associated to PTSD symptom severity, measured by CAPS (t=-0.993; p=329). Discussion Our study found evidence of longer telomere length in women with PTSD diagnosis compared to healthy controls. In contrast, only one study showed accelerated LTL shortening in women rape victims with PTSD diagnosis, although, this study must be interpreted with caution, as the sample size was very small (n=9). Nevertheless, the LTL was not significantly associated with CAPS scores, possibly due to our sample size. Only one study found PTSD symptoms were associated to shorter telomeres in women exposed to different traumas, include rape, interpersonal violence, sudden death of a loved one. The telomere dynamics in PTSD is unclear; few studies investigated the relationship between LTL and PTSD, in women rape victims. Different mechanisms may contribute to LTL in PTSD, including neuroprogression it was also associated with elongation of LTL in schizophrenia. Our results show that PTSD was associated to longer LTL in rape victims, perhaps, due to the action of neuroprogression in PTSD.

Chromatin Remodeling Factors Isw2 and Ino80 Regulate Chromatin, Replication, and Copy Number of the Saccharomyces cerevisiae Ribosomal DNA Locus.

In the budding yeast Saccharomyces cerevisiae, ribosomal RNA genes are encoded in a highly repetitive tandem array referred to as the ribosomal DNA (rDNA) locus. The yeast rDNA is the site of a diverse set of DNA-dependent processes, including transcription of ribosomal RNAs by RNA polymerases I and III, transcription of noncoding RNAs by RNA polymerase II, DNA replication initiation, replication fork blocking, and recombination-mediated regulation of rDNA repeat copy number. All of this takes place in the context of chromatin, but little is known about the roles played by ATP-dependent chromatin remodeling factors at the yeast rDNA. In this work, we report that the Isw2 and Ino80 chromatin remodeling factors are targeted to this highly repetitive locus. We characterize for the first time their function in modifying local chromatin structure, finding that loss of these factors decreases the fraction of actively transcribed 35S ribosomal RNA genes and the positioning of nucleosomes flanking the ribosomal origin of replication. In addition, we report that Isw2 and Ino80 promote efficient firing of the ribosomal origin of replication and facilitate the regulated increase of rDNA repeat copy number. This work significantly expands our understanding of the importance of ATP-dependent chromatin remodeling for rDNA biology.

Distribution patterns and variation analysis of simple sequence repeats in different genomic regions of bovid genomes

As the first examination of distribution, guanine-cytosine (GC) pattern, and variation analysis of microsatellites (SSRs) in different genomic regions of six bovid species, SSRs displayed nonrandomly distribution in different regions. SSR abundances are much higher in the introns, transposable elements (TEs), and intergenic regions compared to the 3′-untranslated regions (3′UTRs), 5′UTRs and coding regions. Trinucleotide perfect SSRs (P-SSRs) were the most frequent in the coding regions, whereas, mononucleotide P-SSRs were the most in the introns, 3′UTRs, TEs, and intergenic regions. Trifold P-SSRs had more GC-contents in the 5′UTRs and coding regions than that in the introns, 3′UTRs, TEs, and intergenic regions, whereas mononucleotide P-SSRs had the least GC-contents in all genomic regions. The repeat copy numbers (RCN) of the same mono- to hexanucleotide P-SSRs showed significantly different distributions in different regions (P < 0.01). Except for the coding regions, mononucleotide P-SSRs had the most RCNs, followed by the pattern: di- > tri- > tetra- > penta- > hexanucleotide P-SSRs in the same regions. The analysis of coefficient of variability (CV) of SSRs showed that the CV variations of RCN of the same mono- to hexanucleotide SSRs were relative higher in the intronic and intergenic regions, followed by the CV variation of RCN in the TEs, and the relative lower was in the 5′UTRs, 3′UTRs, and coding regions. Wide SSR analysis of different genomic regions has helped to reveal biological significances of their distributions.

Measures of Maternal Psycho-Social Stress and Biomarkers of Stress Response in the Maternal-Fetal Unit

In a cohort of 500 pregnant women recruited from two San Francisco hospitals serving economically and ethnically diverse populations, we used Targeted Minimum Loss-Based Estimation (TMLE) to evaluate the association between perceptual measures of maternal psycho-social stress and two biological measures of stress response: 1) telomere length (TL) in umbilical cord blood leukocytes collected at delivery, and 2) corticotropin-releasing hormone (CRH) in maternal plasma collected during the 2nd trimester. We evaluated ten dimensions of maternal stress, derived from questionnaires: job-related, neighborhood, community standing, experience of racism, perceived stress, acute stressful events, depression, pregnancy satisfaction, nativity, and material deprivation. All domains were identified as important predictors of TL/CRH in non-parametric variable important analyses. Therefore, CRH analyses included all stressors and were adjusted for pregnancy complications, mother&amp;#8217;s age, parity, and race/ethnicity. TL analyses were additionally adjusted for infant sex, and gestational age at birth. 30% of participants identified as Latina, 7% Black, 40% White, 16% Asian, and 7% identified as other/mixed. Less than 10% of women did not complete high-school and 60% had a bachelor&amp;#8217;s degree or higher. Biologic factors were more important at predicting TL than stress. Conversely, stress was more important at predicting CRH than biological factors. Counter-intuitively, experiencing racism (0.15, 95% CI: 0.12-0.17) and perceived stress (0.24, 95% CI: 0.21-0.27) were associated with greater TL (expressed as the relative ratio of telomere repeat copy number to single-copy gene copy number or T/S ratio). Pregnancy complications were associated with elevated CRH (0.13, 95% CI: -0.02-0.28), while pregnancy dissatisfaction was associated with lower CRH (-0.15, 95% CI: -0.30-0.01). Results suggest complex relationships between perceived stress and response biomarkers in the maternal-fetal unit.

Association between Exposure to Per- and Polyfluoroalkyl Substances (PFAS) during Pregnancy and Biomarkers of Stress Response in the Maternal-Fetal Unit

In a demographically diverse cohort of 500 pregnant women recruited from two San Francisco hospitals, we used linear regression to evaluate the association between eight PFAS with detection frequency of 50% or greater in our sample, and two biological measures of stress response: 1) telomere length (TL) in umbilical cord blood leukocytes collected at delivery, and 2) corticotropin-releasing hormone (CRH) in maternal plasma collected during the 2nd trimester. Both outcomes were log-transformed. PFAS levels (ug/L) were measured in mother&amp;#8217;s serum during the 2nd trimester, and also log-transformed. Each chemical was assessed separately since levels were highly correlated. CRH analyses were adjusted for pregnancy complications, mother&amp;#8217;s age, parity, and race/ethnicity. TL analyses were additionally adjusted for infant sex, and gestational age at birth. We also evaluated the associations separately for Latina and White women.The burden of PFAS exposure was greater among Latinas (30% of participants) than White women (40% of participants) in our study population. PFAS were not associated with TL, expressed as the relative ratio of telomere repeat copy number to single-copy gene copy number. A one unit increase in Perfluoroundecanoic acid (PFUdA) was associated with a 0.14 increase in CRH in race adjusted models (95% CI: 0.04&amp;#8211;0.23). Higher levels of Perfluorononanoic acid (PFNA; 0.25, 95% CI: 0.07-0.44), Perfluorooctanesulfonic acid (PFOS; 0.161, 95% CI: 0.003-0.32), 2-(N-Methyl-perfluorooctane sulfonamido) acetic acid (MePFOSAAchOH; 0.18, 95% CI: 0.03-0.34), Perfluorohexane sulfonic acid (PFHxS; 0.20, 95% CI: 0.02-0.38), and PFUdA (0.38, 95% CI: 0.19&amp;#8211;0.57) were all significantly associated with higher CRH among Latinas. Higher levels of Perfluorooctanesulfonamide (PFOSA; 0.07, 95% CI: 0.01-0.15) were significantly associated with higher CRH among white women. Results suggest complex relationships between prenatal PFAS exposure and stress response biomarkers.

GtTR: Bayesian estimation of absolute tandem repeat copy number using sequence capture and high throughput sequencing

BackgroundTandem repeats comprise significant proportion of the human genome including coding and regulatory regions. They are highly prone to repeat number variation and nucleotide mutation due to their repetitive and unstable nature, making them a major source of genomic variation between individuals. Despite recent advances in high throughput sequencing, analysis of tandem repeats in the context of complex diseases is still hindered by technical limitations. We report a novel targeted sequencing approach, which allows simultaneous analysis of hundreds of repeats. We developed a Bayesian algorithm, namely – GtTR - which combines information from a reference long-read dataset with a short read counting approach to genotype tandem repeats at population scale. PCR sizing analysis was used for validation.ResultsWe used a PacBio long-read sequenced sample to generate a reference tandem repeat genotype dataset with on average 13% absolute deviation from PCR sizing results. Using this reference dataset GtTR generated estimates of VNTR copy number with accuracy within 95% high posterior density (HPD) intervals of 68 and 83% for capture sequence data and 200X WGS data respectively, improving to 87 and 94% with use of a PCR reference. We show that the genotype resolution increases as a function of depth, such that the median 95% HPD interval lies within 25, 14, 12 and 8% of the its midpoint copy number value for 30X, 200X WGS, 395X and 800X capture sequence data respectively. We validated nine targets by PCR sizing analysis and genotype estimates from sequencing results correlated well with PCR results.ConclusionsThe novel genotyping approach described here presents a new cost-effective method to explore previously unrecognized class of repeat variation in GWAS studies of complex diseases at the population level. Further improvements in accuracy can be obtained by improving accuracy of the reference dataset.

Abstract 1407: Telomere length and melanoma survival

Abstract Telomeres at chromosomal ends are comprised of multiple short repeat sequences. In humans TTAGGG repeats account for 10-15 kb of double-stranded telomeres that end in single stranded G-rich tails of about 150-200 nucleotides. Telomeres in somatic cells undergo gradual shortening due to inherent limitations of DNA replication and limited levels of specialized enzyme telomerase. We previously reported an association between increased telomere length and risk of melanoma. In this study based on 1019 stage I and II cutaneous melanoma patients, we determined the effect of telomere length on patient survival. The telomere length was measured in DNA from peripheral blood cells using monochrome multiplex quantitative real-time PCR where telomere repeat copy number (T) was compared to a single-copy gene, albumin (S) and results presented as relative telomere length based on T/S ratio. A univariate Cox regression showed increased patient survival with increased telomere length (HR 0.65, 95% CI 0.42-1.00, P 0.05). With every decrease in 1 unit of telomere length the estimated odds of survival decreased by an approximate factor of 1.54 (95%CI 1.00-2.38). A model based on median distribution showed short rather than long telomere associated with poor survival with hazard ratio (HR) of 2.05 (95% confidence interval (CI) 1.33-3.16; log rank P 0.001); the model after adjustment with confounding factors including age at diagnosis showed a HR of 1.50 (95%CI 0.96-2.34). Stratification of patients based on median age showed statistically significant effect of decreased telomere length on poor survival in patients below the median age (HR 2.27 95%CI 1.09-4.27) but not in patients above the median age (HR 1.42 95%CI 0.82-2.46). Distribution of the patients into quantiles based on age showed the largest effect of decreased telomere length in patients younger than 30 years (HR 3.90 95%CI 1.28-11.93); the effect decreased with increasing age. Three single nucleotide polymorphisms, rs131178082, rs7726159 and rs6060627, previously shown to be associated with telomere length, were used to construct a weighted genetic score and paradoxically, the patients carrying the alleles associated with increased telomere length showed poor survival with shorter telomeres (HR 2.94 95%CI 1.54-5.62). Multivariate analysis that included, age, sex, outdoor life style, presence of actinic keratosis, number of nevi, tumor location, tumor stage, Breslow thickness and tumor ulceration showed a statistically significant association between composite genetic score and poor survival (HR 2.68 95%CI 1.24-5.80). Thus, the results from the study show that in contrast to association between melanoma risk and increased telomere length, the decreased telomere length associates with poor patient survival. The adverse effect on survival due to decreased telomere length was more pronounced in young rather than old patients and on the patients carrying genotypes for longer telomeres. Citation Format: Sivaramakrishna Rachakonda, Nalini Srinivas, Zaida Garcia-Casado, Celia Requena, Eduardo Nagore, Rajiv Kumar. Telomere length and melanoma survival [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 1407.

Rates and Patterns of Mutation in Tandem Repetitive DNA in Six Independent Lineages of Chlamydomonas reinhardtii.

The mutational patterns of large tandem arrays of short sequence repeats remain largely unknown, despite observations of their high levels of variation in sequence and genomic abundance within and between species. Many factors can influence the dynamics of tandem repeat evolution; however, their evolution has only been examined over a limited phylogenetic sample of taxa. Here, we use publicly available whole-genome sequencing data of 85 haploid mutation accumulation lines derived from six geographically diverse Chlamydomonas reinhardtii isolates to investigate genome-wide mutation rates and patterns in tandem repeats in this species. We find that tandem repeat composition differs among ancestral strains, both in genome-wide abundance and presence/absence of individual repeats. Estimated mutation rates (repeat copy number expansion and contraction) were high, averaging 4.3×10−4 per generation per single unit copy. Although orders of magnitude higher than other types of mutation previously reported in C. reinhardtii, these tandem repeat mutation rates were one order of magnitude lower than what has recently been found in Daphnia pulex, even after correcting for lower overall genome-wide satellite abundance in C. reinhardtii. Most high-abundance repeats were related to others by a single mutational step. Correlations of repeat copy number changes within genomes revealed clusters of closely related repeats that were strongly correlated positively or negatively, and similar patterns of correlation arose independently in two different mutation accumulation experiments. Together, these results paint a dynamic picture of tandem repeat evolution in this unicellular alga.

Assembly and annotation of a draft genome sequence for Glycine latifolia, a perennial wild relative of soybean.

Glycine latifolia (Benth.) Newell & Hymowitz (2n=40), one of the 27 wild perennial relatives of soybean, possesses genetic diversity and agronomically favorable traits that are lacking in soybean. Here, we report the 939-Mb draft genome assembly of G.latifolia (PI 559298) using exclusively linked-reads sequenced from a single Chromium library. We organized scaffolds into 20 chromosome-scale pseudomolecules utilizing two genetic maps and the Glycine max (L.) Merr. genome sequence. High copy numbers of putative 91-bp centromere-specific tandem repeats were observed in consecutive blocks within predicted pericentromeric regions on several pseudomolecules. No 92-bp putative centromeric repeats, which are abundant in G.max, were detected in G.latifolia or Glycine tomentella. Annotation of the assembled genome and subsequent filtering yielded a high confidence gene set of 54475 protein-coding loci. In comparative analysis with five legume species, genes related to defense responses were significantly overrepresented in Glycine-specific orthologous gene families. A total of 304 putative nucleotide-binding site (NBS)-leucine-rich-repeat (LRR) genes were identified in this genome assembly. Different from other legume species, we observed a scarcity of TIR-NBS-LRR genes in G.latifolia. The G.latifolia genome was also predicted to contain genes encoding 367 LRR-receptor-like kinases, a family of proteins involved in basal defense responses and responses to abiotic stress. The genome sequence and annotation of G.latifolia provides a valuable source of alternative alleles and novel genes to facilitate soybean improvement. This study also highlights the efficacy and cost-effectiveness of the application of Chromium linked-reads in diploid plant genome de novo assembly.

Low LPA gene kringle IV-2 repeat copy number association with elevated lipoprotein (a) concentration as an independent risk factor of coronary atherosclerotic heart disease in the Chinese Han population

BackgroundLipoprotein (a) [Lp(a)], which is genetically determined by the LPA gene kringle IV type 2 (KIV-2) repeat copy number, has previously been reported in different populations. However, it is uncertain if the same occurs in the Chinese Han population. This study explored the correlation of Lp(a) mass or particle concentration with KIV-2 repeat copy number and application for coronary atherosclerotic heart disease (CAHD) risk assessment.MethodsA cross-sectional study including 884 subjects was conducted. The Lp(a) level and routine risk factors of CAHD were compared. The KIV-2 copy number distribution, relationship with Lp(a), and assessment for CAHD risk were explored.ResultsThe mean of Lp(a) mass or particle concentration in the CAHD group was higher than that in the non-CAHD group, while the KIV-2 copy number in the CAHD group was lower. Lp(a) had auxiliary values in gauging the type of plaque and was significantly higher in the soft-plaque group than that in the other two groups (200 mg/L [21.5 nmol/L], 166 mg/L [18.6 nmol/L], 149 mg/L [17.1 nmol/L], respectively, P < 0.05). Kappa test indicated divergence for the same individual using two Lp(a) concentrations (kappa value was 0.536 [< 0.75]). Elevated Lp(a) was an independent CAHD risk factor, whatever mass or particle concentration, and large KIV-2 copy number was a protective factor.ConclusionLp(a) level and small KIV-2 copy number are risk factors for CAHD in the Chinese Han population; furthermore, elevated Lp(a) may gauge the type of coronary plaque.

S214. Neuropil Contraction in Relation to Complement C4 Gene Copy Number in Independent Cohorts of Adolescent-Onset and Young Adult-Onset Schizophrenia Patients

A recent report suggested Complement 4 (C4A) gene copy number repeats (CNR) as risk factors for schizophrenia. Rodent model suggested pathophysiological significance of these associations by demonstrating increased synaptic pruning. We, thus, predicted that C4A CNR would be positively correlated with increased neuropil contraction in the human brain among schizophrenia patients showing more prominent correlations in ventral regions among young adults and dorsal regions among adolescents since neuromaturation progresses dorsoventrally.