Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome

Epigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence data generated from hair shafts of a 4000-yr-old Paleo-Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery of the expected signals at promoter regions, exon/intron boundaries, and CTCF sites. The top-scoring nucleosome calls revealed distinct DNA positioning biases, attesting to nucleotide-level accuracy. The ancient methylation levels exhibited high conservation over time, clustering closely with modern hair tissues. Using ancient methylation information, we estimated the age at death of the Saqqaq individual and illustrate how epigenetic information can be used to infer ancient gene expression. Similar epigenetic signatures were found in other fossil material, such as 110,000- to 130,000-yr-old bones, supporting the contention that ancient epigenomic information can be reconstructed from a deep past. Our findings lay the foundation for extracting epigenomic information from ancient samples, allowing shifts in epialleles to be tracked through evolutionary time, as well as providing an original window into modern epigenomics.Ancient DNA research started in the mid-1980s with the successful cloning and sequencing of a short mitochondrial DNA fragment from the quagga zebra, a species that became extinct in the early 20th century (Higuchi et al. 1984). Soon after, the invention of PCR unlocked access to this fragmented and degraded DNA material (Pääbo 1989), making it possible to amplify short gene markers of interest and compare their sequence to that from extant organisms. This illuminated a range of topics ranging from the reconstruction of the evolutionary origins of several now-extinct iconic mammals (Orlando et al. 2003; Krause et al. 2006), the evaluation of the possible role played by major past climatic changes in driving mega-fauna extinctions (Shapiro et al. 2004; Campos et al. 2010; Lorenzen et al. 2011), to the identification of the pathogens responsible for massive historical outbreaks (Taubenberger et al. 1997).However, before the advent of next-generation sequencing (NGS) platforms, the amount of ancient sequence information one had access to was limited to several tens of thousands of nucleotides at best (Noonan et al. 2005, 2006), and until very recently, sequencing whole ancient mitochondrial genomes was considered a major achievement (Cooper et al. 2001; Krause et al. 2006). Parallel sequencing of millions to billions of short DNA fragments has revolutionized ancient DNA research, and today a series of ancient genomes has been reconstructed from humans (Rasmussen et al. 2010, 2011; Keller et al. 2012; Raghavan et al. 2013), archaic hominins (Green et al. 2010; Reich et al. 2010; Meyer et al. 2012), the woolly mammoth (Miller et al. 2008), and several microbial pathogens (Bos et al. 2011; Martin et al. 2013; Schuenemann et al. 2013; Yoshida et al. 2013). Those mainly date back to recent historical periods or the Late Pleistocene, but most recently, the characterization of a 560,000- to 780,000-yr-old horse draft genome revealed that genomic information could be retrieved over much longer evolutionary time scales, probably up until the last million years (Orlando et al. 2013).Ancient genomes have provided important new insights into human evolution and dispersals (Rasmussen et al. 2010, 2011; Keller et al. 2012; Raghavan et al. 2013), revealing an admixture between contemporary human ancestors and archaic hominins (Green et al. 2010; Reich et al. 2010; Meyer et al. 2012) and multiple early human expansions into both Asia and North America (Rasmussen et al. 2010, 2011). The information gained from these samples has largely been limited to nucleotide polymorphisms. Unlike mutations, epigenetic modifications do not alter the underlying DNA sequence, but can be inherited across cell divisions and from parents to offspring and can control gene expression by reshaping cytosine methylation landscapes, nucleosome organization, and histone modification patterns. The range of biological processes that depend on some level of epigenetic regulation is diverse and includes imprinting (Bird 2002), transposition (Hollister and Gaut 2009), cell differentiation (Meissner et al. 2008), and cancer (Teschendorff et al. 2011). In this study, we use the Saqqaq genome that was retrieved from an ∼4000-yr-old tuft of hair of a Paleo-Eskimo from Greenland and sequenced to an average depth of 20× (Rasmussen et al. 2010). We demonstrate that NGS data can be used in the absence of bisulfite or further experimental treatment to directly infer genome-wide nucleosome organization and regional methylation levels, thereby providing the first survey of an ancient epigenome.     

Diagnostic yield,interpretation, and clinical utility of mutation screening of sarcomere encoding genes in Danish hypertrophic cardiomyopathy patients and relatives

The American Heart Association (AHA) recommends family screening for hypertrophic cardiomyopathy (HCM). We assessed the outcome of family screening combining clinical evaluation and screening for sarcomere gene mutations in a cohort of 90 Danish HCM patients and their close relatives, in all 451 persons. Index patients were screened for mutations in all coding regions of 10 sarcomere genes (MYH7, MYL3, MYBPC3, TNNI3, TNNT2, TPM1, ACTC, CSRP3, TCAP, and TNNC1) and five exons of TTN. Relatives were screened for presence of minor or major diagnostic criteria for HCM and tracking of DNA variants was performed. In total, 297 adult relatives (>18 years) (51.2%) fulfilled one or more criteria for HCM. A total of 38 HCM‐causing mutations were detected in 32 index patients. Six patients carried two disease‐associated mutations. Twenty‐two mutations have only been identified in the present cohort. The genetic diagnostic yield was almost twice as high in familial HCM (53%) vs. HCM of sporadic or unclear inheritance (19%). The yield was highest in families with an additional history of HCM‐related clinical events. In relatives, 29.9% of mutation carriers did not fulfil any clinical diagnostic criterion, and in 37.5% of relatives without a mutation, one or more criteria was fulfilled. A total of 60% of family members had no mutation and could be reassured and further follow‐up ceased. Genetic diagnosis may be established in approximately 40% of families with the highest yield in familial HCM with clinical events. Mutation‐screening was superior to clinical investigation in identification of individuals not at increased risk, where follow‐up is redundant, but should be offered in all families with relatives at risk for developing HCM. Hum Mutat 0,1–8, 2008. © 2008 Wiley‐Liss, Inc.     

Effects of preterm birth and fetal growth retardation on cardiovascular risk factors in young adulthood

Background

The association between low birth weight (LBW) and increased risk of obesity, hypertension and cardiovascular disease later in life is well documented in epidemiological studies. However, clinical follow-up studies of LBW populations have only partly supported this.

Aims

Evaluate associations between LBW and body fat, blood pressure (BP), lung and endothelial function, and maximal oxygen uptake (VO_2max) in 18 year old young adults.

Subjects

Thirty-seven subjects born prematurely with birth weight < 1501 g (VLBW group), 47 born at term with low weight (< 10th centile) for gestational age (SGA group) and 63 controls with normal birth weight participated in the study.

Outcome measures

Anthropometric measurements, BP, endothelial function, lung function and VO_2max were recorded.

Results

Both LBW groups were shorter, lighter, had smaller head circumference and higher subscapular-to-triceps skinfold-ratio than controls. Systolic and mean arterial BP was higher in the VLBW compared with the control group, whereas there were no differences between the groups in endothelial function. The VLBW group had reduced dynamic lung volumes lower carbon monoxide transfer factor and lower VO_2max compared with controls. In particular young adults born VLBW who were also growth retarded in utero had higher indices of central body fat, higher BP and lower VO_2max.

Conclusion

We found that very preterm birth, but not growth retardation at term, was associated with higher BP and a less favourable fat distribution. In particular, the young adults born VLBW who were also growth retarded in utero had less favourable outcomes.     

Low birth weight infants and Calmette-Guérin bacillus vaccination at birth: community study from Guinea-Bissau

BACKGROUND: In developing countries, low birth weight (LBW) children are often not vaccinated with Calmette-Guérin bacillus (BCG) at birth. Recent studies have suggested that BCG may have a nonspecific beneficial effect on infant mortality. We evaluated the consequences of not vaccinating LBW children at birth in Guinea-Bissau. METHODS: Between 1989 and 1999, 7138 children born at the central hospital had a birth weight registered. We assessed BCG coverage until 3 years of age. Data on tuberculin skin test (TST) for 297 children and BCG scar for 1319 children in the study population were reanalyzed for differences between normal birth weight (NBW) children and LBW children. We assessed the effect of early BCG vaccination on mortality to 12 months of age. RESULTS: Among LBW children there were 1.5- to 3-fold more unvaccinated individuals than among NBW children up to 4 months of age. There was no overall difference between LBW and NBW children in TST or BCG scarring; LBW children vaccinated early may have had slightly reduced reactions to tuberculin. Among 845 LBW children, 182 had received BCG within the first week of life. Controlling for background factors and censoring at first diphtheria-tetanuspertussis vaccination, measles vaccination or at 6 months of age (whichever came first), the mortality rate ratio for BCG-vaccinated versus -unvaccinated LBW children was 0.17 (95% confidence interval, 0.06-0.49), with an even stronger effect for LBW children vaccinated in the first week of life (mortality rate ratio, 0.07; 95% confidence interval, 0.01-0.62). CONCLUSIONS: The policy of not vaccinating with BCG at birth had a negative impact on vaccination coverage for LBW children. Early BCG vaccination had no large negative impact on TST and BCG scarring. Mortality was lower for BCG-vaccinated than for unvaccinated LBW children controlling for available background factors. BCG vaccination of LBW children may have a beneficial effect on survival that cannot be explained by protection against tuberculosis. Future studies should examine possible adverse effects from equalizing BCG policy for LBW and NBW children.     

Contractile properties of in situ perfused skeletal muscles from rats with congestive heart failure

We hypothesized that in congestive heart failure (CHF) slow-twitch but not fast-twitch muscles exhibit decreased fatigue resistance in the sense of accelerated reduction of muscle force during activity. Experiments were carried out on anaesthetized rats 6 weeks after induction of myocardial infarction or a sham operation (Sham). Animals with left ventricular end-diastolic pressure (LVEDP) > 15 mmHg under anaesthesia were selected for the CHF group. There was no muscle atrophy in CHF. Force generation by in situ perfused soleus (Sol) or extensor digitorum longus (EDL) muscles was recorded during stimulation (trains at 5 Hz for 6 s (Sol) or 10 Hz for 1.5 s (EDL) at 10 or 2.5 s intervals, respectively) for 1 h in Sol and 10 min in EDL at 37 °C. Initial force was almost the same in Sol from CHF and Sham rats, but relaxation was slower in CHF. Relaxation times (95–5 % of peak force) were 177 ± 55 and 131 ± 44 ms in CHF and Sham, respectively, following the first stimulation train. After 2 min of stimulation the muscles transiently became slower and maximum relaxation times were 264 ± 71 and 220 ± 45 ms in CHF and Sham, respectively (   P < 0.05  ). After 60 min they recovered to 204 ± 60 and 122 ± 55 ms in CHF and Sham, respectively (   P < 0.05  ). In CHF but not in Sham rats the force of contraction of Sol declined from the second to the sixtieth minute to 70 % of peak force. The EDL of both CHF and Sham fatigued to 24–28 % of initial force, but no differences in contractility pattern were detected. Thus, slow-twitch muscle is severely affected in CHF by slower than normal relaxation and significantly reduced fatigue resistance, which may explain the sensation of both muscle stiffness and fatigue in CHF patients.     

Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis

Pseudomonas aeruginosa causes a chronic infection in the lungs of cystic fibrosis (CF) patients by establishing an alginate-containing biofilm. The infection has been studied in several animal models; however, most of the models required artificial embedding of the bacteria. We present here a new pulmonary mouse model without artificial embedding. The model is based on a stable mucoid CF sputum isolate (NH57388A) with hyperproduction of alginate due to a deletion in mucA and functional N-acylhomoserine lactone (AHL)-based quorum-sensing systems. Chronic lung infection could be established in both CF mice (Cftr(tmlUnc-/-)) and BALB/c mice, as reflected by the detection of a high number of P. aeruginosa organisms in the lung homogenates at 7 days postinfection and alginate biofilms, surrounded by polymorphonuclear leukocytes in the alveoli. In comparison, both an AHL-producing nonmucoid revertant (NH57388C) from the mucoid isolate (NH57388A) and a nonmucoid isolate (NH57388B) deficient in AHL were almost cleared from the lungs of the mice. This model, in which P. aeruginosa is protected against the defense system of the lung by alginate, is similar to the clinical situation. Therefore, the mouse model provides an improved method for evaluating the interaction between mucoid P. aeruginosa, the host, and antibacterial therapy.     

An integrative approach to CTL epitope prediction: a combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions

Reverse immunogenetic approaches attempt to optimize the selection of candidate epitopes, and thus minimize the experimental effort needed to identify new epitopes. When predicting cytotoxic T cell epitopes, the main focus has been on the highly specific MHC class I binding event. Methods have also been developed for predicting the antigen-processing steps preceding MHC class I binding, including proteasomal cleavage and transporter associated with antigen processing (TAP) transport efficiency. Here, we use a dataset obtained from the SYFPEITHI database to show that a method integrating predictions of MHC class I binding affinity, TAP transport efficiency, and C-terminal proteasomal cleavage outperforms any of the individual methods. Using an independent evaluation dataset of HIV epitopes from the Los Alamos database, the validity of the integrated method is confirmed. The performance of the integrated method is found to be significantly higher than that of the two publicly available prediction methods BIMAS and SYFPEITHI. To identify 85% of the epitopes in the HIV dataset, 9% and 10% of all possible nonamers in the HIV proteins must be tested when using the BIMAS and SYFPEITHI methods, respectively, for the selection of candidate epitopes. This number is reduced to 7% when using the integrated method. In practical terms, this means that the experimental effort needed to identify an epitope in a hypothetical protein with 85% probability is reduced by 20-30% when using the integrated method.The method is available at http://www.cbs.dtu.dk/services/NetCTL. Supplementary material is available at http://www.cbs.dtu.dk/suppl/immunology/CTL.php.     

Geographical and temporal conservation of antibody recognition of Plasmodium falciparum variant surface antigens

The slow acquisition of protection against Plasmodium falciparum malaria probably reflects the extensive diversity of important antigens. The variant surface antigens (VSA) that mediate parasite adhesion to a range of host molecules are regarded as important targets of acquired protective immunity, but their diversity makes them questionable vaccine candidates. We determined levels of VSA-specific immunoglobulin G (IgG) in human plasma collected at four geographically distant and epidemiologically distinct localities with specificity for VSA expressed by P. falciparum isolates from three African countries. Plasma levels of VSA-specific IgG recognizing individual parasite isolates depended on the transmission intensity at the site of plasma collection but were largely independent of the geographical origin of the parasites. The total repertoire of immunologically distinct VSA thus appears to be finite and geographically conserved, most likely due to functional constraints. Furthermore, plasma samples frequently had high IgG reactivity to VSA expressed by parasites isolated more than 10 years later, showing that the repertoire is also temporally stable. Parasites from patients with severe malaria expressed VSA (VSASM) that were better recognized by plasma IgG than VSA expressed by other parasites, but importantly, VSASM-type antigens also appeared to show substantial antigenic homogeneity. Our finding that the repertoire of immunologically distinct VSA in general, and in particular that of VSASM, is geographically and temporally conserved raises hopes for the feasibility of developing VSA-based vaccines specifically designed to accelerate naturally acquired immunity, thereby enhancing protection against severe and life-threatening P. falciparum malaria.     

Effort reward imbalance, and salivary cortisol in the morning

Background

Effort reward imbalance (ERI) is suggested to increase risk for stress and is hypothesized to increase cortisol levels, especially the awakening cortisol response, ACR.

Methods

In 2006 and 2008, 480 individuals collected saliva samples at awakening and 30 min post-awakening. Mixed effects models with subject as a random effect and appropriate covariates were used to evaluate associations between the Effort Reward Model, and salivary cortisol at awakening (S0), and ACR.

Results

ERI was negatively and significantly associated with S0 for women and positively associated with ACR. S0 and ACR increased significantly from 2006 to 2008.

Conclusion

ERI was significantly associated with cortisol levels at awakening (inverse association) for women, and positively associated with ACR. The population experienced a significant increase in morning cortisol levels and ACR from 2006 to 2008, which may originate in a re-organization of the included work places.