Publications by authors named "Michael Buckley"

150 Publications

Pathogenic variants in CDH11 impair cell adhesion and cause Teebi hypertelorism syndrome.

Hum Genet 2021 Apr 3. Epub 2021 Apr 3.

Center for Applied Genomics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.

Teebi hypertelorism syndrome (THS; OMIM 145420) is a rare craniofacial disorder characterized by hypertelorism, prominent forehead, short nose with broad or depressed nasal root. Some cases of THS have been attributed to SPECC1L variants. Homozygous variants in CDH11 truncating the transmembrane and intracellular domains have been implicated in Elsahy-Waters syndrome (EWS; OMIM 211380) with hypertelorism. We report THS due to CDH11 heterozygous missense variants on 19 subjects from 9 families. All affected residues in the extracellular region of Cadherin-11 (CHD11) are highly conserved across vertebrate species and classical cadherins. Six of the variants that cluster around the EC2-EC3 and EC3-EC4 linker regions are predicted to affect Ca binding that is required for cadherin stability. Two of the additional variants [c.164G > C, p.(Trp55Ser) and c.418G > A, p.(Glu140Lys)] are also notable as they are predicted to directly affect trans-homodimer formation. Immunohistochemical study demonstrates that CDH11 is strongly expressed in human facial mesenchyme. Using multiple functional assays, we show that five variants from the EC1, EC2-EC3 linker, and EC3 regions significantly reduced the cell-substrate trans adhesion activity and one variant from EC3-EC4 linker results in changes in cell morphology, focal adhesion, and migration, suggesting dominant negative effect. Characteristic features in this cohort included depressed nasal root, cardiac and umbilical defects. These features distinguished this phenotype from that seen in SPECC1L-related hypertelorism syndrome and CDH11-related EWS. Our results demonstrate heterozygous variants in CDH11, which decrease cell-cell adhesion and increase cell migratory behavior, cause a form of THS, as termed CDH11-related THS.
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http://dx.doi.org/10.1007/s00439-021-02274-3DOI Listing
April 2021

Advances in the Formulation and Assembly of Non-Cationic Lipid Nanoparticles for the Medical Application of Gene Therapeutics.

Nanomaterials (Basel) 2021 Mar 23;11(3). Epub 2021 Mar 23.

Department of Surgery, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway Box U-11, Knoxville, TN 37920, USA.

Lipid nanoparticles have become increasingly popular delivery platforms in the field of gene therapy, but bench-to-bedside success has been limited. Many liposomal gene vectors are comprised of synthetic cationic lipids, which are associated with lipid-induced cytotoxicity and immunogenicity. Natural, non-cationic PEGylated liposomes (PLPs) demonstrate favorable biocompatibility profiles but are not considered viable gene delivery vehicles due to inefficient nucleic acid loading and reduced cellular uptake. PLPs can be modified with cell-penetrating peptides (CPPs) to enhance the intracellular delivery of liposomal cargo but encapsulate leakage upon CPP-PLP assembly is problematic. Here, we aimed to identify parameters that overcome these performance barriers by incorporating nucleic acid condensers during CPP-PLP assembly and screening variable ethanol injection parameters for optimization. CPP-PLPs were formed with R8-amphiphiles via pre-insertion, post-insertion and post-conjugation techniques and liposomes were characterized for size, surface charge, homogeneity, siRNA encapsulation efficiency and retention and cell associative properties. Herein we demonstrate that pre-insertion of stearylated R8 into PLPs is an efficient method to produce non-cationic CPP-PLPs and we provide additional assembly parameter specifications for a modified ethanol injection technique that is optimized for siRNA encapsulation/retention and enhanced cell association. This assembly technique could provide improved clinical translation of liposomal based gene therapy applications.
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http://dx.doi.org/10.3390/nano11030825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004789PMC
March 2021

Selection preferences for animal species used in bone-tool-manufacturing strategies in KwaZulu-Natal, South Africa.

PLoS One 2021 1;16(4):e0249296. Epub 2021 Apr 1.

School of Natural Sciences, Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom.

Animal symbolism is a prominent feature of many human societies globally. In some cases, these symbolic attributes manifest in the technological domain, influencing the decision to use the bones of certain animals and not others for tool manufacture. In southern Africa, animals feature prominently in the cosmogenic narratives of both hunter-gatherer and Bantu-speaking farmer groups. Whenever these two culturally distinct groups came into contact with each other there would be an assimilation of cosmogenic concepts of power and the adoption of certain symbolically important animals. In this paper, we report on which animals were selected to make bone tools during the first millennium AD contact period in KwaZulu-Natal Province, South Africa, and explore the extent to which this selection may have been influenced by the symbolic associations of specific animals. Our results show selective targeting of specific animals for tool manufacture at some sites, with a narrowing of the range of selected species during the first millennium AD contact period. Certain antelope tribes, such as Aepycerotini, Cephalophini and Antilopini, appear to have been deliberately avoided, thus arguing against opportunistic selection. Nor does the range of selected animals appear to show any obvious mechanical considerations, as has been noted in similar studies. We highlight the potential of ZooMS for understanding the dynamics of animal symbolism in the past.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0249296PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016335PMC
April 2021

Comparing the Use of Magnetic Beads with Ultrafiltration for Ancient Dental Calculus Proteomics.

J Proteome Res 2021 Mar 17;20(3):1689-1704. Epub 2021 Feb 17.

School of Natural Sciences, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.

Over the past two decades, proteomic analysis has greatly developed in application to the field of biomolecular archaeology, coinciding with advancements in LC-MS/MS instrumentation sensitivity and improvements in sample preparation methods. Recently, human dental calculus has received much attention for its well-preserved proteomes locked in mineralized dental plaque which stores information on human diets and the oral microbiome otherwise invisible to other biomolecular approaches. Maximizing proteome recovery in ancient dental calculus, available only in minute quantities and irreplaceable after destructive analysis, is of paramount importance. Here, we compare the more traditional ultrafiltration-based and acetone precipitation approaches with the newer paramagnetic bead approach in order to test the influence of demineralization acid on recovered proteome complexity obtained from specimens as well as the sequence coverages matched for significant proteins. We found that a protocol utilizing EDTA combined with paramagnetic beads increased proteome complexity, in some cases doubling the number of unique peptides and number of proteins matched, compared to protocols involving the use of HCl and either acetone precipitation or ultrafiltration. Although the increase in the number of proteins was almost exclusively of bacterial origin, a development that has implications for the study of diseases within these ancient populations, an increase in the peptide number for the dairy proteins β-lactoglobulin and casein was also observed reflecting an increase in sequence coverage for these dietary proteins of interest. We also consider structural explanations for the discrepancies observed between these two key dietary proteins preserved in archaeological dental calculus.
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http://dx.doi.org/10.1021/acs.jproteome.0c00862DOI Listing
March 2021

Proteome Variation with Collagen Yield in Ancient Bone.

J Proteome Res 2021 Mar 2;20(3):1754-1769. Epub 2021 Feb 2.

Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.

Isotope analyses are some of the most common analytical methods applied to ancient bone, aiding the interpretation of past diets and chronology. For this, the evaluation of "collagen yield" (as defined in radiocarbon dating and stable isotope research) is a routine step that allows for the selection of specimens that are deemed adequate for subsequent analyses, with samples containing less than ∼1% "collagen yield" normally being used for isotopic analysis but discounted for radiocarbon dating. The aims of this study were to use proteomic methods of MALDI-TOF (matrix assisted laser desorption ionization time-of-fligh mass spectrometry) and LC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) to investigate the endogeneity of the dominant proteinaceous biomolecules within samples that are typically considered to contain poorly preserved protein. Taking 29 archaeological samples, we evaluated the proteome variability between different acid-soluble fractions removed prior to protein gelatinization and considered waste as part of the radiocarbon dating process. We then correlated these proteomes against the commonly used "collagen yield" proxy for preservation. We found that these waste fractions contained a significant amount of both collagenous and noncollagenous proteins (NCPs) but that the abundance of these was not correlated with the acquired "collagen yield". Rather than a depleted protein load as would be expected from a low "collagen yield", the variety of the extracted NCPs was comparable with that commonly obtained from ancient samples and included informative proteins useful for species identification, phylogenetic studies, and potentially even for isotopic analyses, given further method developments. Additionally, we did not observe any correlation between "collagen yield" and peptide mass fingerprint success or between the different fractions taken from the same sample but at different radiocarbon pretreatment stages. Overall, these findings highlight the value in retaining and analyzing sample fractions that are otherwise discarded as waste during the radiocarbon dating process but more importantly, that low "collagen yield" specimens that are often misinterpreted by archaeologists as being devoid of protein can still yield useful molecular sequence-based information.
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http://dx.doi.org/10.1021/acs.jproteome.0c01014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944572PMC
March 2021

Relative Protein Abundances and Biological Ageing in Whole Skeletal Elements.

J Proteome Res 2021 01 22;20(1):538-548. Epub 2020 Oct 22.

Department of Earth and Environmental Sciences, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.

Establishing biological age is an integral part of forensic investigations, currently achieved through morphological methods with varying degrees of accuracy. Furthermore, biological ageing is much easier in juveniles than in adults, at which point traditional ageing methods struggle. Therefore, biomolecular approaches are considered of great interest, with several protein markers already recognized for their potential forensic significance. However, previous studies have typically relied on subsampling different parts of skeletal elements. Here, we attempt to evaluate the proteome of complete elements using a rat model. In the analysis of specimens spanning beyond adulthood (1 week to 1.5 years), we observed 729 unique proteins across 33 samples (three for each sex for each of the five (female) or six (male)), five of which represent newly identified proteins in relation to age estimation: vimentin, osteopontin, matrilin-1, apolipoprotein A-I, and prothrombin. Most of these follow the trend of decreasing abundance through age, with the exception of prothrombin that increases. We consider the combined use of these relative abundances, along with those of previously noted fetuin-A, biglycan, albumin, and chromogranin-A signatures, as being of potential value to the development of an age estimation tool worthy of further evaluation in forensic contexts.
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http://dx.doi.org/10.1021/acs.jproteome.0c00555DOI Listing
January 2021

Biogeographic problem-solving reveals the Late Pleistocene translocation of a short-faced bear to the California Channel Islands.

Sci Rep 2020 09 16;10(1):15172. Epub 2020 Sep 16.

Department of Anthropology, University of Oklahoma, Norman, OK, USA.

An accurate understanding of biodiversity of the past is critical for contextualizing biodiversity patterns and trends in the present. Emerging techniques are refining our ability to decipher otherwise cryptic human-mediated species translocations across the Quaternary, yet these techniques are often used in isolation, rather than part of an interdisciplinary hypothesis-testing toolkit, limiting their scope and application. Here we illustrate the use of such an integrative approach and report the occurrence of North America's largest terrestrial mammalian carnivore, the short-faced bear, Arctodus simus, from Daisy Cave (CA-SMI-261), an important early human occupation site on the California Channel Islands. We identified the specimen by corroborating morphological, protein, and mitogenomic lines of evidence, and evaluated the potential natural and anthropogenic mechanisms of its transport and deposition. While representing just a single specimen, our combination of techniques opened a window into the behavior of an enigmatic species, suggesting that A. simus was a wide-ranging scavenger utilizing terrestrial and marine carcasses. This discovery highlights the utility of bridging archaeological and paleontological datasets to disentangle complex biogeographic scenarios and reveal unexpected biodiversity for island systems worldwide.
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http://dx.doi.org/10.1038/s41598-020-71572-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494929PMC
September 2020

Ancient Beringian paleodiets revealed through multiproxy stable isotope analyses.

Sci Adv 2020 Sep 4;6(36). Epub 2020 Sep 4.

Department of Anthropology, University of Alaska Fairbanks, Fairbanks, AK, USA.

The earliest Native Americans have often been portrayed as either megafaunal specialists or generalist foragers, but this debate cannot be resolved by studying the faunal record alone. Stable isotope analysis directly reveals the foods consumed by individuals. We present multi-tissue isotope analyses of two Ancient Beringian infants from the Upward Sun River site (USR), Alaska (~11,500 years ago). Models of fetal bone turnover combined with seasonally-sensitive taxa show that the carbon and nitrogen isotope composition of USR infant bone collagen reflects maternal diets over the summer. Using comparative faunal isotope data, we demonstrate that although terrestrial sources dominated maternal diets, salmon was also important, supported by carbon isotope analysis of essential amino acids and bone bioapatite. Tooth enamel samples indicate increased salmon use between spring and summer. Our results do not support either strictly megafaunal specialists or generalized foragers but indicate that Ancient Beringian diets were complex and seasonally structured.
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http://dx.doi.org/10.1126/sciadv.abc1968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473743PMC
September 2020

Double Mandibular Osteotomy for Access to High-Carotid Pathology.

Ann Vasc Surg 2021 Jan 28;70:202-212. Epub 2020 Aug 28.

Division of Vascular Surgery, University of Tennessee, Knoxville, TN.

Background: Anecdotal experience demonstrates the existence of patients with superiorly located carotid stenosis, neoplasms, or aneurysms where the mandible obstructs effective surgical access using standard techniques. As carotid pathology extends anatomically beyond the limits of standard operative technique, additional exposure becomes paramount to safely and effectively address the lesion. Double mandibular osteotomy (DMO) is one of several techniques to obtain additional exposure to high-carotid pathology; however, there is no large series to address the outcomes of patients undergoing this procedure.

Methods: A retrospective case series was performed for all patients undergoing surgery for carotid pathology from 2011-2019 that could not be approached with standard cervical incision. The primary predictor variable was high-anatomic carotid pathology necessitating DMO. The primary outcome variable was early and late complications sustained by patients.

Results: Fifteen patients met study criteria and underwent 16 DMOs to access high-carotid pathology including carotid stenosis (n = 8 patients), carotid aneurysm (n = 2 patients), and carotid body tumor (n = 8 patients). Two patients had dual ipsilateral pathology with one patient having both carotid artery stenosis and aneurysm, and the other patient diagnosed with carotid artery stenosis and carotid body tumor. One patient had bilateral carotid artery stenosis, each requiring high anatomic exposure for treatment. Early complications occurred in 8 patients. Five patients experienced significant dysphagia requiring enteral feeding, and 2 patients developed malocclusion directly related to the double mandibular osteotomy. One patient experienced contralateral cortical watershed infarcts. Late complications included one patient developing osteomyelitis of the mandible, and this patient also developed distal mandibular segment screw exposure. The comparison of the outcome groups for categorical predictor variables using Fisher's exact test detected no statistically significant differences for gender, hypertension, hyperlipidemia, type 2 diabetes, chronic obstructive pulmonary disease, tobacco use, chronic kidney disease, or cerebrovascular disease. For the continuous variable comparisons, independent-samples t-tests detected no difference between the complication groups for age, operative time, or years of follow-up. No significant differences were found between the groups for body mass index or intraoperative blood loss.

Conclusions: The double mandibular osteotomy provides excellent exposure and surgical access to the distal internal carotid artery for repair of vascular pathology with acceptable outcomes and long-term complications compared with previously reported techniques. Because of the early complications realized with the DMO, we recommend the procedure for symptomatic patients with a high risk of failing medical therapy alone and not appropriate for endovascular treatment as well as those patients with tumors requiring surgical intervention.
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http://dx.doi.org/10.1016/j.avsg.2020.08.093DOI Listing
January 2021

Soil Fungal Communities Investigated by Metabarcoding Within Simulated Forensic Burial Contexts.

Front Microbiol 2020 24;11:1686. Epub 2020 Jul 24.

Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom.

Decomposition of animal bodies in the burial environment plays a key role in the biochemistry of the soil, altering the balance of the local microbial populations present before the introduction of the carcass. Despite the growing number of studies on decomposition and soil bacterial populations, less is known on its effects on fungal communities. Shifts in the fungal populations at different post-mortem intervals (PMIs) could provide insights for PMI estimation and clarify the role that specific fungal taxa have at specific decomposition stages. In this study, we buried pig carcasses over a period of 1- to 6-months, and we sampled the soil in contact with each carcass at different PMIs. We performed metabarcoding analysis of the mycobiome targeting both the internal transcribed spacer (ITS) 1 and 2, to elucidate which one was more suitable for this purpose. Our results showed a decrease in the fungal taxonomic richness associated with increasing PMIs, and the alteration of the soil fungal signature even after 6 months post-burial, showing the inability of soil communities to restore their original composition within this timeframe. The results highlighted taxonomic trends associated with specific PMIs, such as the increase of the Mortierellomycota after 4- and 6-months and of Ascomycota particularly after 2 months, and the decrease of Basidiomycota from the first to the last time point. We have found a limited number of taxa specifically associated with the carrion and not present in the control soil, showing that the major contributors to the recorded changes are originated from the soil and were not introduced by the carrion. As this is the first study conducted on burial graves, it sets the baseline for additional studies to investigate the role of fungal communities on prolonged decomposition periods and to identify fungal biomarkers to improve the accuracy of PMI prediction for forensic applications.
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http://dx.doi.org/10.3389/fmicb.2020.01686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393272PMC
July 2020

Global reference mapping of human transcription factor footprints.

Nature 2020 07 29;583(7818):729-736. Epub 2020 Jul 29.

Altius Institute for Biomedical Sciences, Seattle, WA, USA.

Combinatorial binding of transcription factors to regulatory DNA underpins gene regulation in all organisms. Genetic variation in regulatory regions has been connected with diseases and diverse phenotypic traits, but it remains challenging to distinguish variants that affect regulatory function. Genomic DNase I footprinting enables the quantitative, nucleotide-resolution delineation of sites of transcription factor occupancy within native chromatin. However, only a small fraction of such sites have been precisely resolved on the human genome sequence. Here, to enable comprehensive mapping of transcription factor footprints, we produced high-density DNase I cleavage maps from 243 human cell and tissue types and states and integrated these data to delineate about 4.5 million compact genomic elements that encode transcription factor occupancy at nucleotide resolution. We map the fine-scale structure within about 1.6 million DNase I-hypersensitive sites and show that the overwhelming majority are populated by well-spaced sites of single transcription factor-DNA interaction. Cell-context-dependent cis-regulation is chiefly executed by wholesale modulation of accessibility at regulatory DNA rather than by differential transcription factor occupancy within accessible elements. We also show that the enrichment of genetic variants associated with diseases or phenotypic traits in regulatory regions is almost entirely attributable to variants within footprints, and that functional variants that affect transcription factor occupancy are nearly evenly partitioned between loss- and gain-of-function alleles. Unexpectedly, we find increased density of human genetic variation within transcription factor footprints, revealing an unappreciated driver of cis-regulatory evolution. Our results provide a framework for both global and nucleotide-precision analyses of gene regulatory mechanisms and functional genetic variation.
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http://dx.doi.org/10.1038/s41586-020-2528-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410829PMC
July 2020

Index and biological spectrum of human DNase I hypersensitive sites.

Nature 2020 08 29;584(7820):244-251. Epub 2020 Jul 29.

Altius Institute for Biomedical Sciences, Seattle, WA, USA.

DNase I hypersensitive sites (DHSs) are generic markers of regulatory DNA and contain genetic variations associated with diseases and phenotypic traits. We created high-resolution maps of DHSs from 733 human biosamples encompassing 438 cell and tissue types and states, and integrated these to delineate and numerically index approximately 3.6 million DHSs within the human genome sequence, providing a common coordinate system for regulatory DNA. Here we show that these maps highly resolve the cis-regulatory compartment of the human genome, which encodes unexpectedly diverse cell- and tissue-selective regulatory programs at very high density. These programs can be captured comprehensively by a simple vocabulary that enables the assignment to each DHS of a regulatory barcode that encapsulates its tissue manifestations, and global annotation of protein-coding and non-coding RNA genes in a manner orthogonal to gene expression. Finally, we show that sharply resolved DHSs markedly enhance the genetic association and heritability signals of diseases and traits. Rather than being confined to a small number of distal elements or promoters, we find that genetic signals converge on congruently regulated sets of DHSs that decorate entire gene bodies. Together, our results create a universal, extensible coordinate system and vocabulary for human regulatory DNA marked by DHSs, and provide a new global perspective on the architecture of human gene regulation.
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http://dx.doi.org/10.1038/s41586-020-2559-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7422677PMC
August 2020

Feasibility of Ultra-Rapid Exome Sequencing in Critically Ill Infants and Children With Suspected Monogenic Conditions in the Australian Public Health Care System.

JAMA 2020 06;323(24):2503-2511

Australian Genomics Health Alliance, Parkville, Australia.

Importance: Widespread adoption of rapid genomic testing in pediatric critical care requires robust clinical and laboratory pathways that provide equitable and consistent service across health care systems.

Objective: To prospectively evaluate the performance of a multicenter network for ultra-rapid genomic diagnosis in a public health care system.

Design, Setting, And Participants: Descriptive feasibility study of critically ill pediatric patients with suspected monogenic conditions treated at 12 Australian hospitals between March 2018 and February 2019, with data collected to May 2019. A formal implementation strategy emphasizing communication and feedback, standardized processes, coordination, distributed leadership, and collective learning was used to facilitate adoption.

Exposures: Ultra-rapid exome sequencing.

Main Outcomes And Measures: The primary outcome was time from sample receipt to ultra-rapid exome sequencing report. The secondary outcomes were the molecular diagnostic yield, the change in clinical management after the ultra-rapid exome sequencing report, the time from hospital admission to the laboratory report, and the proportion of laboratory reports returned prior to death or hospital discharge.

Results: The study population included 108 patients with a median age of 28 days (range, 0 days to 17 years); 34% were female; and 57% were from neonatal intensive care units, 33% were from pediatric intensive care units, and 9% were from other hospital wards. The mean time from sample receipt to ultra-rapid exome sequencing report was 3.3 days (95% CI, 3.2-3.5 days) and the median time was 3 days (range, 2-7 days). The mean time from hospital admission to ultra-rapid exome sequencing report was 17.5 days (95% CI, 14.6-21.1 days) and 93 reports (86%) were issued prior to death or hospital discharge. A molecular diagnosis was established in 55 patients (51%). Eleven diagnoses (20%) resulted from using the following approaches to augment standard exome sequencing analysis: mitochondrial genome sequencing analysis, exome sequencing-based copy number analysis, use of international databases to identify novel gene-disease associations, and additional phenotyping and RNA analysis. In 42 of 55 patients (76%) with a molecular diagnosis and 6 of 53 patients (11%) without a molecular diagnosis, the ultra-rapid exome sequencing result was considered as having influenced clinical management. Targeted treatments were initiated in 12 patients (11%), treatment was redirected toward palliative care in 14 patients (13%), and surveillance for specific complications was initiated in 19 patients (18%).

Conclusions And Relevance: This study suggests feasibility of ultra-rapid genomic testing in critically ill pediatric patients with suspected monogenic conditions in the Australian public health care system. However, further research is needed to understand the clinical value of such testing, and the generalizability of the findings to other health care settings.
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http://dx.doi.org/10.1001/jama.2020.7671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312414PMC
June 2020

Collagen Sequence Analysis Reveals Evolutionary History of Extinct West Indies Nesophontes (Island-Shrews).

Mol Biol Evol 2020 10;37(10):2931-2943

Interdisciplinary Centre for Ancient Life, School of Natural Sciences, University of Manchester, Manchester, United Kingdom.

Ancient biomolecule analyses are proving increasingly useful in the study of evolutionary patterns, including extinct organisms. Proteomic sequencing techniques complement genomic approaches, having the potential to examine lineages further back in time than achievable using ancient DNA, given the less stringent preservation requirements. In this study, we demonstrate the ability to use collagen sequence analyses via proteomics to assist species delimitation as a foundation for informing evolutionary patterns. We uncover biogeographic information of an enigmatic and recently extinct lineage of Nesophontes across their range on the Caribbean islands. First, evolutionary relationships reconstructed from collagen sequences reaffirm the affinity of Nesophontes and Solenodon as sister taxa within Solenodonota. This relationship helps lay the foundation for testing geographical isolation hypotheses across islands within the Greater Antilles, including movement from Cuba toward Hispaniola. Second, our results are consistent with Cuba having just two species of Nesophontes (N. micrus and N. major) that exhibit intrapopulation morphological variation. Finally, analysis of the recently described species from the Cayman Islands (N. hemicingulus) indicates that it is a closer relative to N. major rather than N. micrus as previously speculated. This proteomic sequencing improves our understanding of the origin, evolution, and distribution of this extinct mammal lineage, particularly with respect to the approximate timing of speciation. Such knowledge is vital for this biodiversity hotspot, where the magnitude of recent extinctions may obscure true estimates of species richness in the past.
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http://dx.doi.org/10.1093/molbev/msaa137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530613PMC
October 2020

Mutation Distribution and Type Suggests Genetic Differences between the Etiology of Orofacial Clefting and Gastric Cancer.

Genes (Basel) 2020 04 3;11(4). Epub 2020 Apr 3.

New South Wales Health Pathology, Prince of Wales Hospital, Randwick, Sydney 2031, Australia.

Pathogenic variants in , encoding epithelial cadherin (E-cadherin), have been implicated in hereditary diffuse gastric cancer (HDGC), lobular breast cancer, and both syndromic and non-syndromic cleft lip/palate (CL/P). Despite the large number of mutations described, the nature of the phenotypic consequence of such mutations is currently not able to be predicted, creating significant challenges for genetic counselling. This study collates the phenotype and molecular data for available variants that have been classified, using the American College of Medical Genetics and Genomics criteria, as at least 'likely pathogenic', and correlates their molecular and structural characteristics to phenotype. We demonstrate that variant type and location differ between HDGC and CL/P, and that there is clustering of CL/P variants within linker regions between the extracellular domains of the cadherin protein. While these differences do not provide for exact prediction of the phenotype for a given mutation, they may contribute to more accurate assessments of risk for HDGC or CL/P for individuals with specific variants.
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http://dx.doi.org/10.3390/genes11040391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7231129PMC
April 2020

Climate stability and societal decline on the margins of the Byzantine empire in the Negev Desert.

Sci Rep 2020 01 30;10(1):1512. Epub 2020 Jan 30.

Zinman Institute of Archaeology, University of Haifa, 199 Aba-Hushi Avenue, Haifa, 3498838, Israel.

Understanding past human settlement of inhospitable regions is one of the most intriguing puzzles in archaeological research, with implications for more sustainable use of marginal regions today. During the Byzantine period in the 4 century CE, large settlements were established in the arid region of the Negev Desert, Israel, but it remains unclear why it did so, and why the settlements were abandoned three centuries later. Previous theories proposed that the Negev was a "green desert" in the early 1 millennium CE, and that the Byzantine Empire withdrew from this region due to a dramatic climatic downturn. In the absence of a local climate archive correlated to the Byzantine/Early Islamic transition, testing this theory has proven challenging. We use stable isotopic indicators of animal dietary and mobility patterns to assess the extent of the vegetative cover in the desert. By doing so, we aim to detect possible climatic fluctuations that may have led to the abandonment of the Byzantine settlements. The findings show that the Negev Desert was not greener during the time period under investigation than it is today and that the composition of the animals' diets, as well as their grazing mobility patterns, remained unchanged through the Byzantine/Early Islamic transition. Favoring a non-climatic explanation, we propose instead that the abandonment of the Negev Byzantine settlements was motivated by restructuring of the Empire's territorial priorities.
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http://dx.doi.org/10.1038/s41598-020-58360-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992700PMC
January 2020

The Practice of Pharmaceutics and the Obligation to Expand Access to Investigational Drugs.

J Med Philos 2020 03;45(2):193-211

Lehman College, City University of New York, Bronx, New York, USA.

Do pharmaceutical companies have a moral obligation to expand access to investigational drugs to patients outside the clinical trial? One reason for thinking they do not is that expanded access programs might negatively affect the clinical trial process. This potential impact creates dilemmas for practitioners who nevertheless acknowledge some moral reason for expanding access. Bioethicists have explained these reasons in terms of beneficence, compassion, or a principle of rescue, but their arguments have been limited to questions of moral permissibility, leaving for future research the question of whether expanded access is morally obligatory. We take up this further question and argue that pharmaceutical companies have a moral obligation to expand access. Our defense is not based on beneficence, compassion, or rescue, but instead on a reciprocal moral expectation resulting from existing social commitments that help ensure a robust pharmaceutical practice within the broader healthcare system. Our aim is to give this obligation, along with several others, a coherent and plausible structure within the wider clinical trial process so that one might better explain the sources of the dilemmas and their possible resolutions.
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http://dx.doi.org/10.1093/jmp/jhz038DOI Listing
March 2020

Preserved collagen reveals species identity in archaeological marine turtle bones from Caribbean and Florida sites.

R Soc Open Sci 2019 Oct 30;6(10):191137. Epub 2019 Oct 30.

Manchester Institute of Biotechnology, School of Earth and Environmental Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.

Advancements in molecular science are continually improving our knowledge of marine turtle biology and evolution. However, there are still considerable gaps in our understanding, such as past marine turtle distributions, which can benefit from advanced zooarchaeological analyses. Here, we apply collagen fingerprinting to 130 archaeological marine turtle bone samples up to approximately 2500 years old from the Caribbean and Florida's Gulf Coast for faunal identification, finding the vast majority of samples (88%) to contain preserved collagen despite deposition in the tropics. All samples can be identified to species-level with the exception of the Kemp's ridley () and olive ridley () turtles, which can be separated to genus level, having diverged from one another only approximately 5 Ma. Additionally, we identify a single homologous peptide that allows the separation of archaeological green turtle samples, spp., into two distinct groups, which potentially signifies a difference in genetic stock. The majority of the archaeological samples are identified as green turtle ( spp.; 63%), with hawksbill (; 17%) and ridley turtles ( spp; 3%) making up smaller proportions of the assemblage. There were no molecular identifications of the loggerhead turtle () in the assemblage despite 9% of the samples being morphologically identified as such, highlighting the difficulties in relying on morphological identifications alone in archaeological remains. Finally, we present the first marine turtle molecular phylogeny using collagen (I) amino acid sequences and find our analyses match recent phylogenies based on nuclear and mitochondrial DNA. Our results highlight the advantage of using collagen fingerprinting to supplement morphological analyses of turtle bones and support the usefulness of this technique for assessing their past distributions across the Caribbean and Florida's Gulf Coast, especially in these tropical environments where DNA preservation may be poor.
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http://dx.doi.org/10.1098/rsos.191137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6837194PMC
October 2019

Missense variants in TAF1 and developmental phenotypes: challenges of determining pathogenicity.

Hum Mutat 2019 Oct 23. Epub 2019 Oct 23.

Institute for Basic Research in Developmental Disabilities (IBR), Staten Island, NY, USA.

We recently described a new neurodevelopmental syndrome (TAF1/MRXS33 intellectual disability syndrome) (MIM# 300966) caused by pathogenic variants involving the X-linked gene TAF1, which participates in RNA polymerase II transcription. The initial study reported eleven families, and the syndrome was defined as presenting early in life with hypotonia, facial dysmorphia, and developmental delay that evolved into intellectual disability (ID) and/or autism spectrum disorder (ASD). We have now identified an additional 27 families through a genotype-first approach. Familial segregation analysis, clinical phenotyping, and bioinformatics were capitalized on to assess potential variant pathogenicity, and molecular modelling was performed for those variants falling within structurally characterized domains of TAF1. A novel phenotypic clustering approach was also applied, in which the phenotypes of affected individuals were classified using 51 standardized Human Phenotype Ontology (HPO) terms. Phenotypes associated with TAF1 variants show considerable pleiotropy and clinical variability, but prominent among previously unreported effects were brain morphological abnormalities, seizures, hearing loss, and heart malformations. Our allelic series broadens the phenotypic spectrum of TAF1/MRXS33 intellectual disability syndrome and the range of TAF1 molecular defects in humans. It also illustrates the challenges for determining the pathogenicity of inherited missense variants, particularly for genes mapping to chromosome X. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/humu.23936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187541PMC
October 2019

A case study of vertebral fusion in a 19-century horse from Serbia.

Int J Paleopathol 2019 12 21;27:17-23. Epub 2019 Sep 21.

Manchester Institute of Biotechnology, School of Earth and Environmental Sciences, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. Electronic address:

Objective: To evaluate the etiology of skeletal changes noted in equid vertebrae from a 19- century context near Belgrade, Serbia.

Materials: A vertebral column consisting of 15 fused thoracic vertebrae (T2-T16), with right ribs fused to T4 and T5 and small remnants of ribs articulating with T4, T5 and T6 on the left side were accidentally recovered during industrial exploitation of sand.

Methods: The specimen was subjected to morphological analysis and collagen fingerprinting by mass spectrometry for species identification. In order to determine the absolute temporal context, radiocarbon dating was employed. Pathological changes were analyzed macroscopically and then underwent X-ray and (CT) imaging.

Results: Species identification indicates that the vertebral column belonged to a domestic horse (Equus caballus) living in the early 19 century. Pathological changes included exuberant bone proliferation, fusion of small articulations, enthesopathy formation, complete fusion between the vertebral bodies, and ossification of the anterior longitudinal ligament.

Conclusions: Pathological changes represent signs of an advanced stage of vertebral fusion consistent with diffuse idiopathic skeletal hyperostosis (DISH).

Significance: This case study provides a clear distinction between diffuse idiopathic skeletal hyperostosis (DISH) and other vertebral column diseases in equids. It also presents a new and significant contribution to the nascent discipline of animal paleopathology in present-day Serbia.

Limitations: Given that only 15 thoracic vertebrae were discovered, the impact of this disease on other parts of the horse skeleton remains unknown, as does the archaeological context of the remains.

Suggestions For Further Research: Research into the frequency of DISH in equids, as well as the historical context of equine husbandry in Serbia will allow greater insight into the causes and effects of this pathological condition.
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http://dx.doi.org/10.1016/j.ijpp.2019.07.007DOI Listing
December 2019

Fetal diagnosis of Mowat-Wilson syndrome by whole exome sequencing.

Am J Med Genet A 2019 10 19;179(10):2152-2157. Epub 2019 Jul 19.

Neuroscience Research Australia (NeuRA), Sydney, New South Wales, Australia.

Mowat-Wilson syndrome (MWS) is a complex genetic disorder associated with heterozygous variation in ZEB2. It is mainly characterized by moderate-to-severe intellectual disability, facial dysmorphism, epilepsy, and various malformations including Hirschsprung disease, corpus callosum anomalies, and congenital heart defects. It is rarely diagnosed prenatally and there is limited information available on the prenatal phenotype associated with MWS. Here we report the detection of a heterozygous de novo nonsense variant in ZEB2 by whole exome sequencing in a fetus with microphthalmia in addition to cardiac defects and typical MWS facial dysmorphism. As the prenatal phenotypic spectrum of MWS expands, the routine addition of fetal genomic testing particularly in the presence of multiple malformations will increase both the sensitivity and specificity of prenatal diagnostics.
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http://dx.doi.org/10.1002/ajmg.a.61295DOI Listing
October 2019

Dietary supplementation with Zyflamend poly-herbal extracts and fish oil inhibits intimal hyperplasia development following vascular intervention.

Nutr Res 2019 08 8;68:34-44. Epub 2019 Jun 8.

University of Tennessee Graduate School of Medicine, Department of Surgery, Knoxville, TN. Electronic address:

The polyherbal blend Zyflamend™ has been shown to have anti-inflammatory properties and attenuate inflammatory-modulated pathologies. Fish oils have also been shown to have cardioprotective properties. However, the beneficial effects of their combination have not been investigated. Intimal hyperplasia (IH), a pathological remodeling response of a vessel to injury, is heavily regulated by an immune-mediated reaction. The objective of this study was to determine if dietary supplementation with Zyflamend and/or Wholemega could affect inflammatory-dependent vascular remodeling mechanisms when provided at human equivalent doses. Based on their anti-inflammatory properties and protective benefits demonstrated in previous pre-clinical studies, we hypothesized administration of these supplements would prevent IH in an animal model of vascular injury. The diets of aged male rats were supplemented with human equivalent doses of Zyflamend (Zyf) and/or Wholemega (WMega) or placebo (Plac) for 1wk prior to balloon angioplasty (BA)-induced injury of the left carotid artery. At 28d post-injury morphometric analysis of carotid tissue revealed IH was decreased in Zyf + WMega animals compared to placebo, while Zyf or WMega independently had no significant effect. Serum cytokine screening indicated injury-induced interleukin family isoforms, interferon-γ, and macrophage inflammatory proteins were downregulated by Zyf + WMega. Immunohistochemical staining for monocyte/macrophage phenotypic markers revealed that while overall monocyte/macrophage vessel infiltration was not affected, Zyf + WMega limited the alternative differentiation of M2 macrophages and reduced the presence of myofibroblasts in the injured vessel wall. In summary, dietary supplementation with Zyf + WMega attenuated the acute inflammatory response following vascular injury and inhibited IH development in vivo.
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http://dx.doi.org/10.1016/j.nutres.2019.06.001DOI Listing
August 2019

Methylome and transcriptome maps of human visceral and subcutaneous adipocytes reveal key epigenetic differences at developmental genes.

Sci Rep 2019 07 2;9(1):9511. Epub 2019 Jul 2.

Epigenetics Research Laboratory, Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, 2010, New South Wales, Australia.

Adipocytes support key metabolic and endocrine functions of adipose tissue. Lipid is stored in two major classes of depots, namely visceral adipose (VA) and subcutaneous adipose (SA) depots. Increased visceral adiposity is associated with adverse health outcomes, whereas the impact of SA tissue is relatively metabolically benign. The precise molecular features associated with the functional differences between the adipose depots are still not well understood. Here, we characterised transcriptomes and methylomes of isolated adipocytes from matched SA and VA tissues of individuals with normal BMI to identify epigenetic differences and their contribution to cell type and depot-specific function. We found that DNA methylomes were notably distinct between different adipocyte depots and were associated with differential gene expression within pathways fundamental to adipocyte function. Most striking differential methylation was found at transcription factor and developmental genes. Our findings highlight the importance of developmental origins in the function of different fat depots.
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http://dx.doi.org/10.1038/s41598-019-45777-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606599PMC
July 2019

Mutations in GDF11 and the extracellular antagonist, Follistatin, as a likely cause of Mendelian forms of orofacial clefting in humans.

Hum Mutat 2019 10 18;40(10):1813-1825. Epub 2019 Jun 18.

New South Wales Health Pathology, Prince of Wales Hospital, Randwick, New South Wales, Australia.

Cleft lip with or without cleft palate (CL/P) is generally viewed as a complex trait with multiple genetic and environmental contributions. In 70% of cases, CL/P presents as an isolated feature and/or deemed nonsyndromic. In the remaining 30%, CL/P is associated with multisystem phenotypes or clinically recognizable syndromes, many with a monogenic basis. Here we report the identification, via exome sequencing, of likely pathogenic variants in two genes that encode interacting proteins previously only linked to orofacial clefting in mouse models. A variant in GDF11 (encoding growth differentiation factor 11), predicting a p.(Arg298Gln) substitution at the Furin protease cleavage site, was identified in one family that segregated with CL/P and both rib and vertebral hypersegmentation, mirroring that seen in Gdf11 knockout mice. In the second family in which CL/P was the only phenotype, a mutation in FST (encoding the GDF11 antagonist, Follistatin) was identified that is predicted to result in a p.(Cys56Tyr) substitution in the region that binds GDF11. Functional assays demonstrated a significant impact of the specific mutated amino acids on FST and GDF11 function and, together with embryonic expression data, provide strong evidence for the importance of GDF11 and Follistatin in the regulation of human orofacial development.
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http://dx.doi.org/10.1002/humu.23793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6764866PMC
October 2019

Glibenclamide treatment in a Cantú syndrome patient with a pathogenic ABCC9 gain-of-function variant: Initial experience.

Am J Med Genet A 2019 08 7;179(8):1585-1590. Epub 2019 Jun 7.

Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, St. Louis, Missouri.

Cantú syndrome (CS), characterized by hypertrichosis, distinctive facial features, and complex cardiovascular abnormalities, is caused by pathogenic variants in ABCC9 and KCNJ8 genes. These genes encode gain-of-function mutations in the regulatory (SUR2) and pore-forming (Kir6.1) subunits of K channels, respectively, suggesting that channel-blocking sulfonylureas could be a viable therapy. Here we report a neonate with CS, carrying a heterozygous ABCC9 variant (c.3347G>A, p.Arg1116His), born prematurely at 32 weeks gestation. Initial echocardiogram revealed a large patent ductus arteriosus (PDA), and high pulmonary pressures with enlarged right ventricle. He initially received surfactant and continuous positive airway pressure ventilation and was invasively ventilated for 4 weeks, until PDA ligation. After surgery, he still had ongoing bilevel positive airway pressure (BiPAP) requirement, but was subsequently weaned to nocturnal BiPAP. He was treated for pulmonary hypertension with Sildenafil, but failed to make further clinical improvement. A therapeutic glibenclamide trial was commenced in week 11 (initial dose of 0.05 mg kg day in two divided doses). After 1 week of treatment, he began to tolerate time off BiPAP when awake, and edema improved. Glibenclamide was well tolerated, and the dose was slowly increased to 0.15 mg kg day over the next 12 weeks. Mild transient hypoglycemia was observed, but there was no cardiovascular dysfunction. Confirmation of therapeutic benefit will require studies of more CS patients but, based on this limited experience, consideration should be given to glibenclamide as CS therapy, although problems associated with prematurity, and complications of hypoglycemia, might limit outcome in critically ill neonates with CS.
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http://dx.doi.org/10.1002/ajmg.a.61200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899598PMC
August 2019

Pathogenic variants in result in a Stickler syndrome-like connective tissue disorder with vascular complications.

J Med Genet 2019 09 25;56(9):629-638. Epub 2019 May 25.

NSW Health Pathology East Laboratory, Prince of Wales Private Hospital, Randwick, New South Wales, Australia.

Background: Pathogenic variants cause a connective tissue disorder (CTD) that has been described rarely. We further characterise this CTD and propose a clinical diagnostic label to improve recognition and diagnosis of -related disease.

Methods: Reported phenotypes were compared with known CTDs utilising data from three further individuals from a consanguineous family with a homozygous c.809C>T; p.(Pro270Leu) variant. PLOD3 mRNA expression in the developing embryo was analysed for tissue-specific localisation. Mouse microarray expression data were assessed for phylogenetic gene expression similarities across CTDs with overlapping clinical features.

Results: Key clinical features included ocular abnormalities with risk for retinal detachment, sensorineural hearing loss, reduced palmar creases, finger contractures, prominent knees, scoliosis, low bone mineral density, recognisable craniofacial dysmorphisms, developmental delay and risk for vascular dissection. Collated clinical features showed most overlap with Stickler syndrome with variable features of Ehlers-Danlos syndrome (EDS) and epidermolysis bullosa (EB). Human lysyl hydroxylase 3/ expression was localised to the developing cochlea, eyes, skin, forelimbs, heart and cartilage, mirroring the clinical phenotype of this disorder.

Conclusion: These data are consistent with pathogenic variants in resulting in a clinically distinct Stickler-like syndrome with vascular complications and variable features of EDS and EB. Early identification of variants would improve monitoring for comorbidities and may avoid serious adverse ocular and vascular outcomes.
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http://dx.doi.org/10.1136/jmedgenet-2019-106019DOI Listing
September 2019

Integrated epigenomic profiling reveals endogenous retrovirus reactivation in renal cell carcinoma.

EBioMedicine 2019 Mar 1;41:427-442. Epub 2019 Mar 1.

Department of Pathology, University of Washington, Seattle, WA 98195, United States; Kidney Research Institute, Seattle, WA 98104, United States. Electronic address:

Background: Transcriptional dysregulation drives cancer formation but the underlying mechanisms are still poorly understood. Renal cell carcinoma (RCC) is the most common malignant kidney tumor which canonically activates the hypoxia-inducible transcription factor (HIF) pathway. Despite intensive study, novel therapeutic strategies to target RCC have been difficult to develop. Since the RCC epigenome is relatively understudied, we sought to elucidate key mechanisms underpinning the tumor phenotype and its clinical behavior.

Methods: We performed genome-wide chromatin accessibility (DNase-seq) and transcriptome profiling (RNA-seq) on paired tumor/normal samples from 3 patients undergoing nephrectomy for removal of RCC. We incorporated publicly available data on HIF binding (ChIP-seq) in a RCC cell line. We performed integrated analyses of these high-resolution, genome-scale datasets together with larger transcriptomic data available through The Cancer Genome Atlas (TCGA).

Findings: Though HIF transcription factors play a cardinal role in RCC oncogenesis, we found that numerous transcription factors with a RCC-selective expression pattern also demonstrated evidence of HIF binding near their gene body. Examination of chromatin accessibility profiles revealed that some of these transcription factors influenced the tumor's regulatory landscape, notably the stem cell transcription factor POU5F1 (OCT4). Elevated POU5F1 transcript levels were correlated with advanced tumor stage and poorer overall survival in RCC patients. Unexpectedly, we discovered a HIF-pathway-responsive promoter embedded within a endogenous retroviral long terminal repeat (LTR) element at the transcriptional start site of the PSOR1C3 long non-coding RNA gene upstream of POU5F1. RNA transcripts are induced from this promoter and read through PSOR1C3 into POU5F1 producing a novel POU5F1 transcript isoform. Rather than being unique to the POU5F1 locus, we found that HIF binds to several other transcriptionally active LTR elements genome-wide correlating with broad gene expression changes in RCC.

Interpretation: Integrated transcriptomic and epigenomic analysis of matched tumor and normal tissues from even a small number of primary patient samples revealed remarkably convergent shared regulatory landscapes. Several transcription factors appear to act downstream of HIF including the potent stem cell transcription factor POU5F1. Dysregulated expression of POU5F1 is part of a larger pattern of gene expression changes in RCC that may be induced by HIF-dependent reactivation of dormant promoters embedded within endogenous retroviral LTRs.
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http://dx.doi.org/10.1016/j.ebiom.2019.01.063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441874PMC
March 2019

Integrated Functional Genomic Analysis Enables Annotation of Kidney Genome-Wide Association Study Loci.

J Am Soc Nephrol 2019 Feb 13. Epub 2019 Feb 13.

Department of Anatomic Pathology,

Background: Linking genetic risk loci identified by genome-wide association studies (GWAS) to their causal genes remains a major challenge. Disease-associated genetic variants are concentrated in regions containing regulatory DNA elements, such as promoters and enhancers. Although researchers have previously published DNA maps of these regulatory regions for kidney tubule cells and glomerular endothelial cells, maps for podocytes and mesangial cells have not been available.

Methods: We generated regulatory DNA maps (DNase-seq) and paired gene expression profiles (RNA-seq) from primary outgrowth cultures of human glomeruli that were composed mainly of podocytes and mesangial cells. We generated similar datasets from renal cortex cultures, to compare with those of the glomerular cultures. Because regulatory DNA elements can act on target genes across large genomic distances, we also generated a chromatin conformation map from freshly isolated human glomeruli.

Results: We identified thousands of unique regulatory DNA elements, many located close to transcription factor genes, which the glomerular and cortex samples expressed at different levels. We found that genetic variants associated with kidney diseases (GWAS) and kidney expression quantitative trait loci were enriched in regulatory DNA regions. By combining GWAS, epigenomic, and chromatin conformation data, we functionally annotated 46 kidney disease genes.

Conclusions: We demonstrate a powerful approach to functionally connect kidney disease-/trait-associated loci to their target genes by leveraging unique regulatory DNA maps and integrated epigenomic and genetic analysis. This process can be applied to other kidney cell types and will enhance our understanding of genome regulation and its effects on gene expression in kidney disease.
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http://dx.doi.org/10.1681/ASN.2018030309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405142PMC
February 2019

Age estimates for hominin fossils and the onset of the Upper Palaeolithic at Denisova Cave.

Nature 2019 01 30;565(7741):640-644. Epub 2019 Jan 30.

Oxford Radiocarbon Accelerator Unit, Research Laboratory for Archaeology and the History of Art, University of Oxford, Oxford, UK.

Denisova Cave in the Siberian Altai (Russia) is a key site for understanding the complex relationships between hominin groups that inhabited Eurasia in the Middle and Late Pleistocene epoch. DNA sequenced from human remains found at this site has revealed the presence of a hitherto unknown hominin group, the Denisovans, and high-coverage genomes from both Neanderthal and Denisovan fossils provide evidence for admixture between these two populations. Determining the age of these fossils is important if we are to understand the nature of hominin interaction, and aspects of their cultural and subsistence adaptations. Here we present 50 radiocarbon determinations from the late Middle and Upper Palaeolithic layers of the site. We also report three direct dates for hominin fragments and obtain a mitochondrial DNA sequence for one of them. We apply a Bayesian age modelling approach that combines chronometric (radiocarbon, uranium series and optical ages), stratigraphic and genetic data to calculate probabilistically the age of the human fossils at the site. Our modelled estimate for the age of the oldest Denisovan fossil suggests that this group was present at the site as early as 195,000 years ago (at 95.4% probability). All Neanderthal fossils-as well as Denisova 11, the daughter of a Neanderthal and a Denisovan-date to between 80,000 and 140,000 years ago. The youngest Denisovan dates to 52,000-76,000 years ago. Direct radiocarbon dating of Upper Palaeolithic tooth pendants and bone points yielded the earliest evidence for the production of these artefacts in northern Eurasia, between 43,000 and 49,000 calibrated years before present (taken as AD 1950). On the basis of current archaeological evidence, it may be assumed that these artefacts are associated with the Denisovan population. It is not currently possible to determine whether anatomically modern humans were involved in their production, as modern-human fossil and genetic evidence of such antiquity has not yet been identified in the Altai region.
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http://dx.doi.org/10.1038/s41586-018-0870-zDOI Listing
January 2019

Metabarcoding to investigate changes in soil microbial communities within forensic burial contexts.

Forensic Sci Int Genet 2019 03 12;39:73-85. Epub 2018 Dec 12.

Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. Electronic address:

The estimation of the time elapsed since death (post-mortem interval, or PMI) is one of the key themes that forensic scientists have to address frequently. However, the estimation of PMI still suffers from poor accuracy and biases especially when decomposition stages are prolonged, so further improvements in methods for PMI estimation are desirable. Soil microbial communities associated with decomposing bodies have been shown to be good candidates for the estimation of the PMI of exposed bodies. Nevertheless, further research is required to better understand the bacterial succession associated with decomposition of buried carcasses in order to test its reliability and applicability for the estimation of PMI and to better understand the dynamics involved with decomposition within this particular scenario. Therefore we explored the succession of soil microbial communities associated with four decomposing pig carcasses (from one to six months PMI) using a metabarcoding approach. The sequencing of the bacterial 16S rRNA variable region 4 (V4) revealed trends linking particular microbial taxa with specific PMIs, and notably an increase in Proteobacteria, Firmicutes and Bacteroidetes at specific PMIs as well as a decrease in Acidobacteria. Our results, in accordance with previous studies conducted on exposed bodies of different mammalian species (including humans), also showed a general reduction of the taxonomic richness from two months PMI onwards, as well as an incomplete re-establishment of the starting soil microbial conditions after six months PMI. We also found specific mammal-derived taxa, such as Bacteroides spp., being still present in the soil after six months PMI. As such, this study serves as a baseline for additional research to allow the characterisation of biomarkers associated with specific PMIs. Due to the similarity between the results presented here and those reported in other types of decomposition studies we believe that the metabarcoding approach has considerable potential in the estimation of the PMI, particularly to clarify cases involving heavily skeletonised bodies or for the investigation of clandestine graves in which the carcass has been moved from its original place of deposition.
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http://dx.doi.org/10.1016/j.fsigen.2018.12.002DOI Listing
March 2019