Rachita Yadav

The genome of a Late Pleistocene human from a Clovis burial site in western Montana

Clovis, with its distinctive biface, blade and osseous technologies, is the oldest widespread archaeological complex defined in North America, dating from 11,100 to 10,700 14C years before present (bp) (13,000 to 12,600 calendar years bp). Nearly 50 years of archaeological research point to the Clovis complex as having developed south of the North American ice sheets from an ancestral technology. However, both the origins and the genetic legacy of the people who manufactured Clovis tools remain under debate. It is generally believed that these people ultimately derived from Asia and were directly related to contemporary Native Americans. An alternative, Solutrean, hypothesis posits that the Clovis predecessors emigrated from southwestern Europe during the Last Glacial Maximum. Here we report the genome sequence of a male infant (Anzick-1) recovered from the Anzick burial site in western Montana. The human bones date to 10,705 ± 35 14C years bp (approximately 12,707–12,556 calendar years bp) and were directly associated with Clovis tools. We sequenced the genome to an average depth of 14.4× and show that the gene flow from the Siberian Upper Palaeolithic Mal’ta population into Native American ancestors is also shared by the Anzick-1 individual and thus happened before 12,600 years bp. We also show that the Anzick-1 individual is more closely related to all indigenous American populations than to any other group. Our data are compatible with the hypothesis that Anzick-1 belonged to a population directly ancestral to many contemporary Native Americans. Finally, we find evidence of a deep divergence in Native American populations that predates the Anzick-1 individual.

A genome-wide association study identifies CDHR3 as a susceptibility locus for early childhood asthma with severe exacerbations

Asthma exacerbations are among the most frequent causes of hospitalization during childhood, but the underlying mechanisms are poorly understood. We performed a genome-wide association study of a specific asthma phenotype characterized by recurrent, severe exacerbations occurring between 2 and 6 years of age in a total of 1,173 cases and 2,522 controls. Cases were identified from national health registries of hospitalization, and DNA was obtained from the Danish Neonatal Screening Biobank. We identified five loci with genome-wide significant association. Four of these, GSDMB, IL33, RAD50 and IL1RL1, were previously reported as asthma susceptibility loci, but the effect sizes for these loci in our cohort were considerably larger than in the previous genome-wide association studies of asthma. We also obtained strong evidence for a new susceptibility gene, CDHR3 (encoding cadherin-related family member 3), which is highly expressed in airway epithelium. These results demonstrate the strength of applying specific phenotyping in the search for asthma susceptibility genes.

Novel variation and de novo mutation rates in population-wide de novo assembled Danish trios

Building a population-specific catalogue of single nucleotide variants (SNVs), indels and structural variants (SVs) with frequencies, termed a national pan-genome, is critical for further advancing clinical and public health genetics in large cohorts. Here we report a Danish pan-genome obtained from sequencing 10 trios to high depth (50 × ). We report 536k novel SNVs and 283k novel short indels from mapping approaches and develop a population-wide de novo assembly approach to identify 132k novel indels larger than 10 nucleotides with low false discovery rates. We identify a higher proportion of indels and SVs than previous efforts showing the merits of high coverage and de novo assembly approaches. In addition, we use trio information to identify de novo mutations and use a probabilistic method to provide direct estimates of 1.27e−8 and 1.5e−9 per nucleotide per generation for SNVs and indels, respectively.

Transcriptome profiling of brown adipose tissue during cold exposure reveals extensive regulation of glucose metabolism

We applied digital gene expression profiling to determine the transcriptome of brown and white adipose tissues (BAT and WAT, respectively) during cold exposure. Male C57BL/6J mice were exposed to cold for 2 or 4 days. A notable induction of genes related to glucose uptake, glycolysis, glycogen metabolism, and the pentose phosphate pathway was observed in BAT from cold-exposed animals. In addition, glycerol-3-phosphate dehydrogenase 1 expression was induced in BAT from cold-challenged mice, suggesting increased synthesis of glycerol from glucose. Similarly, expression of lactate dehydrogenases was induced by cold in BAT. Pyruvate dehydrogenase kinase 2 (Pdk2) and Pdk4 were expressed at significantly higher levels in BAT than in WAT, and Pdk2 was induced in BAT by cold. Of notice, only a subset of the changes detected in BAT was observed in WAT. Based on changes in gene expression during cold exposure, we propose a model for the intermediary glucose metabolism in activated BAT: 1) fluxes through glycolysis and the pentose phosphate pathway are induced, the latter providing reducing equivalents for de novo fatty acid synthesis; 2) glycerol synthesis from glucose is increased, facilitating triacylglycerol synthesis/fatty acid re-esterification; 3) glycogen turnover and lactate production are increased; and 4) entry of glucose carbon into the tricarboxylic acid cycle is restricted by PDK2 and PDK4. In summary, our results demonstrate extensive and diverse gene expression changes related to glucose handling in activated BAT.

Inherited coding variants at the CDKN2A locus influence susceptibility to acute lymphoblastic leukaemia in children

There is increasing evidence from genome-wide association studies for a strong inherited genetic basis of susceptibility to acute lymphoblastic leukaemia (ALL) in children, yet the effects of protein-coding variants on ALL risk have not been systematically evaluated. Here we show a missense variant in CDKN2A associated with the development of ALL at genome-wide significance (rs3731249, P=9.4 × 10−23, odds ratio=2.23). Functional studies indicate that this hypomorphic variant results in reduced tumour suppressor function of p16INK4A, increases the susceptibility to leukaemic transformation of haematopoietic progenitor cells, and is preferentially retained in ALL tumour cells. Resequencing the CDKN2A–CDKN2B locus in 2,407 childhood ALL cases reveals 19 additional putative functional germline variants. These results provide direct functional evidence for the influence of inherited genetic variation on ALL risk, highlighting the important and complex roles of CDKN2A–CDKN2B tumour suppressors in leukaemogenesis.

TIMP-1 increases expression and phosphorylation of proteins associated with drug resistance in breast cancer cells.

Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a protein with a potential biological role in drug resistance. To elucidate the unknown molecular mechanisms underlying the association between high TIMP-1 levels and increased chemotherapy resistance, we employed SILAC-based quantitative mass spectrometry to analyze global proteome and phosphoproteome differences of MCF-7 breast cancer cells expressing high or low levels of TIMP-1. In TIMP-1 high expressing cells, 312 proteins and 452 phosphorylation sites were up-regulated. Among these were the cancer drug targets topoisomerase 1, 2A, and 2B, which may explain the resistance phenotype to topoisomerase inhibitors that was observed in cells with high TIMP-1 levels. Pathway analysis showed an enrichment of proteins from functional categories such as apoptosis, cell cycle, DNA repair, transcription factors, drug targets and proteins associated with drug resistance or sensitivity, and drug transportation. The NetworKIN algorithm predicted the protein kinases CK2a, CDK1, PLK1, and ATM as likely candidates involved in the hyperphosphorylation of the topoisomerases. Up-regulation of protein and/or phosphorylation levels of topoisomerases in TIMP-1 high expressing cells may be part of the mechanisms by which TIMP-1 confers resistance to treatment with the widely used topoisomerase inhibitors in breast and colorectal cancer.

Global gene expression profiling of brown to white adipose tissue transformation in sheep reveals novel transcriptional components linked to adipose remodeling.

BackgroundLarge mammals are capable of thermoregulation shortly after birth due to the presence of brown adipose tissue (BAT). The majority of BAT disappears after birth and is replaced by white adipose tissue (WAT).ResultsWe analyzed the postnatal transformation of adipose in sheep with a time course study of the perirenal adipose depot. We observed changes in tissue morphology, gene expression and metabolism within the first two weeks of postnatal life consistent with the expected transition from BAT to WAT. The transformation was characterized by massively decreased mitochondrial abundance and down-regulation of gene expression related to mitochondrial function and oxidative phosphorylation. Global gene expression profiling demonstrated that the time points grouped into three phases: a brown adipose phase, a transition phase and a white adipose phase. Between the brown adipose and the transition phase 170 genes were differentially expressed, and 717 genes were differentially expressed between the transition and the white adipose phase. Thirty-eight genes were shared among the two sets of differentially expressed genes. We identified a number of regulated transcription factors, including NR1H3, MYC, KLF4, ESR1, RELA and BCL6, which were linked to the overall changes in gene expression during the adipose tissue remodeling. Finally, the perirenal adipose tissue expressed both brown and brite/beige adipocyte marker genes at birth, the expression of which changed substantially over time.ConclusionsUsing global gene expression profiling of the postnatal BAT to WAT transformation in sheep, we provide novel insight into adipose tissue plasticity in a large mammal, including identification of novel transcriptional components linked to adipose tissue remodeling. Moreover, our data set provides a useful resource for further studies in adipose tissue plasticity.

Sequencing and de novo assembly of 150 genomes from Denmark as a population reference

Hundreds of thousands of human genomes are now being sequenced to characterize genetic variation and use this information to augment association mapping studies of complex disorders and other phenotypic traits. Genetic variation is identified mainly by mapping short reads to the reference genome or by performing local assembly. However, these approaches are biased against discovery of structural variants and variation in the more complex parts of the genome. Hence, large-scale de novo assembly is needed. Here we show that it is possible to construct excellent de novo assemblies from high-coverage sequencing with mate-pair libraries extending up to 20 kilobases. We report de novo assemblies of 150 individuals (50 trios) from the GenomeDenmark project. The quality of these assemblies is similar to those obtained using the more expensive long-read technology. We use the assemblies to identify a rich set of structural variants including many novel insertions and demonstrate how this variant catalogue enables further deciphering of known association mapping signals. We leverage the assemblies to provide 100 completely resolved major histocompatibility complex haplotypes and to resolve major parts of the Y chromosome. Our study provides a regional reference genome that we expect will improve the power of future association mapping studies and hence pave the way for precision medicine initiatives, which now are being launched in many countries including Denmark.

Genomic profiling of thousands of candidate polymorphisms predicts risk of relapse in 778 Danish and German childhood acute lymphoblastic leukemia patients

Childhood acute lymphoblastic leukemia survival approaches 90%. New strategies are needed to identify the 10–15% who evade cure. We applied targeted, sequencing-based genotyping of 25 000 to 34 000 preselected potentially clinically relevant single-nucleotide polymorphisms (SNPs) to identify host genome profiles associated with relapse risk in 352 patients from the Nordic ALL92/2000 protocols and 426 patients from the German Berlin–Frankfurt–Munster (BFM) ALL2000 protocol. Patients were enrolled between 1992 and 2008 (median follow-up: 7.6 years). Eleven cross-validated SNPs were significantly associated with risk of relapse across protocols. SNP and biologic pathway level analyses associated relapse risk with leukemia aggressiveness, glucocorticosteroid pharmacology/response and drug transport/metabolism pathways. Classification and regression tree analysis identified three distinct risk groups defined by end of induction residual leukemia, white blood cell count and variants in myeloperoxidase (MPO), estrogen receptor 1 (ESR1), lamin B1 (LMNB1) and matrix metalloproteinase-7 (MMP7) genes, ATP-binding cassette transporters and glucocorticosteroid transcription regulation pathways. Relapse rates ranged from 4% (95% confidence interval (CI): 1.6–6.3%) for the best group (72% of patients) to 76% (95% CI: 41–90%) for the worst group (5% of patients, P<0.001). Validation of these findings and similar approaches to identify SNPs associated with toxicities may allow future individualized relapse and toxicity risk-based treatments adaptation.

Asparaginase-associated pancreatitis: a study on phenotype and genotype in the NOPHO ALL2008 protocol.

Asparaginase (ASP)-associated pancreatitis (AAP) occurs during acute lymphoblastic leukemia treatment. Among 1285 children (1.0–17.9 years) diagnosed during July 2008–December 2014 and treated according to the Nordic/Baltic ALL2008 protocol, 86 (cumulative incidence=6.8%) developed AAP. Seventy-three cases were severe (diagnostic AAP criteria persisting >72 h) and 13 mild. Cases were older than controls (median: 6.5 vs 4.5 years; P=0.001). Pseudocysts developed in 28%. Of the 20 re-exposed to ASP, 9 (45%) developed a second AAP. After a median follow-up of 2.3 years, 8% needed permanent insulin therapy, and 7% had recurrent abdominal pain. Germline DNA on 62 cases and 638 controls was genotyped on Omni2.5exome-8-v1.2 BeadChip arrays. Overall, the ULK2 variant rs281366 showed the strongest association with AAP (P=5.8 × 10−7; odds ratio (OR)=6.7). Cases with the rs281366 variant were younger (4.3 vs 8 years; P=0.015) and had lower risk of AAP-related complications (15% vs 43%; P=0.13) compared with cases without this variant. Among 45 cases and 517 controls <10 years, the strongest associations with AAP were found for RGS6 variant rs17179470 (P=9.8 × 10−9; OR=7.3). Rs281366 is located in the ULK2 gene involved in autophagy, and RGS6 regulates G-protein signaling regulating cell dynamics. More than 50% of AAP cases <10 years carried one or both risk alleles.