LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectable WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes.

Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of TP53 and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic TP53 mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment.

Mutations in the TP53 tumor suppressor gene are common in ovarian carcinoma (OC) but their impact on outcomes is controversial. We sought to define the relationship of TP53 mutations to cancer outcomes and their interactions with co-occurrent BRCA1 or BRCA2 (BRCA) mutations, comparing three different TP53 mutation classification schemes.

BAC arrays were used to evaluate genomic instability along the colon of patients with ulcerative colitis (UC). Genomic instability increases with disease progression and biopsies more proximal to dysplasia showed increased instability. Pan-colonic field copy number gain or loss involving small (<1Mb) regions were detected in most patients and were particularly apparent in the UC progressor patients who had dysplasia or cancer. Chromosomal copy gains or losses affecting large regions were mainly restricted to dysplastic biopsies. Areas of significant chromosomal losses were detected in the UC progressors on chromosomes 2q36, 3q25, 3p21, 4q34, 4p16.2, 15q22, and 16p13 (p-value⩽0.04). These results extend our understanding of the dynamic nature of pan-colonic genomic instability in this disease.

Werner syndrome (WS) is the canonical adult human progeroid ('premature aging') syndrome. Patients with this autosomal recessive Mendelian disorder display constitutional genomic instability and an elevated risk of important age-associated diseases including cancer. Remarkably few analyses of WS patient tissue and tumors have been performed to provide insight into WS disease pathogenesis or the high risk of neoplasia. We used autopsy tissue from four mutation-typed WS patients to characterize pathologic and genomic features of WS, and to determine genomic features of three neoplasms arising in two of these patients. The results of these analyses provide new information on WS pathology and genomics; provide a first genomic characterization of neoplasms arising in WS; and provide new histopathologic and genomic data to test several popular models of WS disease pathogenesis.

All species should invest in systems that enhance longevity; however, a fundamental adult life-history trade-off exists between the metabolic resources allocated to maintenance and those allocated to reproduction. Long-lived species will invest more in reproduction than in somatic maintenance as they age. We investigated this trade-off by analyzing correlations among telomere length, reproductive effort and output, and basal corticosterone in Magellanic penguins (Spheniscus magellanicus). Telomeres shorten with age in most species studied to date, and may affect adult survival. High basal corticosterone is indicative of stressful conditions. Corticosterone, and stress, has been linked to telomere shortening in other species. Magellanic penguins are a particularly good model organism for this question as they are an unusually long-lived species, exceeding their mass-adjusted predicted lifespan by 26%. Contrary to our hypothesis, we found adults aged 5 years to over 24 years of age had similar telomere lengths. Telomeres of adults did not shorten over a 3-year period, regardless of the age of the individual. Neither telomere length, nor the rate at which the telomeres changed over these 3 years, correlated with breeding frequency or investment. Older females also produced larger volume clutches until approximately 15 years old and larger eggs produced heavier fledglings. Furthermore, reproductive success (chicks fledged/eggs laid) is maintained as females aged. Basal corticosterone, however, was not correlated with telomere length in adults and suggests that low basal corticosterone may play a role in the telomere maintenance we observed. Basal corticosterone also declined during the breeding season and was positively correlated with the age of adult penguins. This higher basal corticosterone in older individuals, and consistent reproductive success, supports the prediction that Magellanic penguins invest more in reproduction as they age. Our results demonstrate that telomere maintenance may be a component of longevity even with increased reproductive effort, investment, and basal corticosterone.

Microarrays and high-throughput sequencing methods can be used to measure the expression of thousands of genes in a biological sample in a few days, whereas PCR-based methods can be used to measure the expression of a few genes in thousands of samples in about the same amount of time. These methods become more costly as the number of biological samples increases or as the number of genes of interest increases, respectively, and these factors constrain experimental design. To address these issues, we introduced 'vertical arrays' in which RNA from each biological sample is converted into multiple, overlapping cDNA subsets and spotted on glass slides. These vertical arrays can be queried with single gene probes to assess the expression behavior in thousands of biological samples in a single hybridization reaction. The spotted subsets are less complex than the original RNA from which they derive, which improves signal-to-noise ratios. Here, we demonstrate the quantitative capabilities of vertical arrays, including the sensitivity and accuracy of the method and the number of subsets needed to achieve this accuracy for most expressed genes.

Although somatic mutations in colorectal cancer are well characterized, little is known about the accumulation of cancer mutations in the normal colon before cancer. Here, we have developed and applied an ultrasensitive, single-molecule mutational test based on CRISPR-DS technology, which enables mutation detection at extremely low frequency (<0.001) in normal colon from patients with and without colorectal cancer. This testing platform revealed that normal colon from patients with and without colorectal cancer carries mutations in common colorectal cancer genes, but these mutations are more abundant in patients with cancer. Oncogenic KRAS mutations were observed in the normal colon of about one third of patients with colorectal cancer but in none of the patients without colorectal cancer. Patients with colorectal cancer also carried more TP53 mutations than patients without cancer and these mutations were more pathogenic and formed larger clones, especially in patients with early-onset colorectal cancer. Most mutations in the normal colon were different from the driver mutations in tumors, suggesting that the occurrence of independent clones with pathogenic KRAS and TP53 mutations is a common event in the colon of individuals who develop colorectal cancer. These results indicate that somatic evolution contributes to clonal expansions in the normal colon and that this process is enhanced in individuals with cancer, particularly in those with early-onset colorectal cancer.

For all species, finite metabolic resources must be allocated toward three competing systems: maintenance, reproduction, and growth. Telomeres, the nucleoprotein tips of chromosomes, which shorten with age in most species, are correlated with increased survival. Chick growth is energetically costly and is associated with telomere shortening in most species. To assess the change in telomeres in penguin chicks, we quantified change in telomere length of wild known-age Magellanic penguin (Spheniscus magellanicus) chicks every 15 days during the species' growth period, from hatching to 60 days-of-age. Magellanic penguins continue to grow after fledging so we also sampled a set of 1-year-old juvenile penguins, and adults aged 5 years. Telomeres were significantly shorter on day 15 than on hatch day but returned to their initial length by 30 days old and remained at that length through 60 days of age. The length of telomeres of newly hatched chicks, chicks aged 30, 45 and 60 days, juveniles, and adults aged 5 years were similar. Chicks that fledged and those that died had similar telomere lengths. We show that while telomeres shorten during growth, Magellanic penguins elongate telomeres to their length at hatch, which may increase adult life span and reproductive opportunities.

After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.

Current screening methods for ovarian cancer (OC) have failed to demonstrate a significant reduction in mortality. Uterine lavage combined with TP53 ultra-deep sequencing for the detection of disseminated OC cells has emerged as a promising tool, but this approach has not been tested for early-stage disease or non-serous histologies. In addition, lavages carry multiple background mutations, the significance of which is poorly understood. Uterine lavage was collected preoperatively in 34 patients undergoing surgery for suspected ovarian malignancy including 14 patients with benign disease and 20 patients with OC (6 non-serous and 14 high grade serous-like (serous)). Ultra-deep duplex sequencing (~3000x) with a panel of common OC genes identified the tumor mutation in 33% of non-serous (all early stage) and in 79% of serous cancers (including four early stage). In addition, all lavages carried multiple somatic mutations (average of 25 mutations per lavage), more than half of which corresponded to common cancer driver mutations. Driver mutations in KRAS, PIK3CA, PTEN, PPP2R1A and ARID1A presented as larger clones than non-driver mutations and with similar frequency in lavages from patients with and without OC, indicating prevalent somatic evolution in all patients. Driver TP53 mutations, however, presented as significantly larger clones and with higher frequency in lavages from individuals with OC, suggesting that TP53-specific clonal expansions are linked to ovarian cancer development. Our results demonstrate that lavages capture cancer cells, even from early-stage cancers, as well as other clonal expansions and support further exploration of TP53 mutation burden as a potential OC risk factor.