Isoform Evolution in Primates through Independent Combination of Alternative RNA Processing Events.

Recent RNA-seq technology revealed thousands of splicing events that are under rapid evolution in primates, whereas the reliability of these events, as well as their combination on the isoform level, have not been adequately addressed due to its limited sequencing length. Here, we performed comparative transcriptome analyses in human and rhesus macaque cerebellum using single molecule long-read sequencing (Iso-seq) and matched RNA-seq. Besides 359 million RNA-seq reads, 4,165,527 Iso-seq reads were generated with a mean length of 14,875 bp, covering 11,466 human genes, and 10,159 macaque genes. With Iso-seq data, we substantially expanded the repertoire of alternative RNA processing events in primates, and found that intron retention and alternative polyadenylation are surprisingly more prevalent in primates than previously estimated. We then investigated the combinatorial mode of these alternative events at the whole-transcript level, and found that the combination of these events is largely independent along the transcript, leading to thousands of novel isoforms missed by current annotations. Notably, these novel isoforms are selectively constrained in general, and 1,119 isoforms have even higher expression than the previously annotated major isoforms in human, indicating that the complexity of the human transcriptome is still significantly underestimated. Comparative transcriptome analysis further revealed 502 genes encoding selectively constrained, lineage-specific isoforms in human but not in rhesus macaque, linking them to some lineage-specific functions. Overall, we propose that the independent combination of alternative RNA processing events has contributed to complex isoform evolution in primates, which provides a new foundation for the study of phenotypic difference among primates.

Revising the human mutation rate: implications for understanding human evolution.

It is now possible to make direct measurements of the mutation rate in modern humans using next-generation sequencing. These measurements reveal a value that is approximately half of that previously derived from fossil calibration, and this has implications for our understanding of demographic events in human evolution and other aspects of population genetics. Here, we discuss the implications of a lower-than-expected mutation rate in relation to the timescale of human evolution.

Context characterization of amino acid homorepeats using evolution, position, and order.

Amino acid repeats, or homorepeats, are low complexity protein motifs consisting of tandem repetitions of a single amino acid. Their presence and relative number vary in different proteomes, and some studies have tried to address this variation, proteome by proteome. In this work, we present a full characterization of amino acid homorepeats across evolution. We studied the presence and differential usage of each possible homorepeat in proteomes from various taxonomic groups, using clusters of very similar proteins to eliminate redundancy. The position of each amino acid repeat within proteins, and the order of co-occurring amino acid repeats were also addressed. As a result, we present evidence about the unevenly evolution of homorepeats, as well as the functional implications of their relative position in proteins. We discuss some of these cases in their taxonomic context. Collectively, our results show evolutionary and positional signals that suggest that homorepeats have biological function, likely creating unspecific protein interactions or modulating specific interactions in a context dependent manner. In conclusion, our work supports the functional importance of homorepeats and establishes a basis for the study of other low complexity repeats. Proteins 2017; 85:709-719. © 2016 Wiley Periodicals, Inc.

Natural Selection in the Great Apes.

Natural selection is crucial for the adaptation of populations to their environments. Here, we present the first global study of natural selection in the Hominidae (humans and great apes) based on genome-wide information from population samples representing all extant species (including most subspecies). Combining several neutrality tests we create a multi-species map of signatures of natural selection covering all major types of natural selection. We find that the estimated efficiency of both purifying and positive selection varies between species and is significantly correlated with their long-term effective population size. Thus, even the modest differences in population size among the closely related Hominidae lineages have resulted in differences in their ability to remove deleterious alleles and to adapt to changing environments. Most signatures of balancing and positive selection are species-specific, with signatures of balancing selection more often being shared among species. We also identify loci with evidence of positive selection across several lineages. Notably, we detect signatures of positive selection in several genes related to brain function, anatomy, diet and immune processes. Our results contribute to a better understanding of human evolution by putting the evidence of natural selection in humans within its larger evolutionary context. The global map of natural selection in our closest living relatives is available as an interactive browser at http://tinyurl.com/nf8qmzh.

Core promoter short tandem repeats as evolutionary switch codes for primate speciation.

Alteration in gene expression levels underlies many of the phenotypic differences across species. Because of their highly mutable nature, proximity to the +1 transcription start site (TSS), and the emerging evidence of functional impact on gene expression, core promoter short tandem repeats (STRs) may be considered an ideal source of variation across species. In a genome-scale analysis of the entire Homo sapiens protein-coding genes, we have previously identified core promoters with at least one STR of ≥ 6-repeats, with possible selective advantage in this species. In the current study, we performed reverse analysis of the entire Homo sapiens orthologous genes in mouse in the Ensembl database, in order to identify conserved STRs that have shrunk as an evolutionary advantage to humans. Two protocols were used to minimize ascertainment bias. Firstly, two species sharing a more recent ancestor with Homo sapiens (i.e. Pan troglodytes and Gorilla gorilla gorilla) were also included in the study. Secondly, four non-primate species encompassing the major orders across Mammals, including Scandentia, Laurasiatheria, Afrotheria, and Xenarthra were analyzed as out-groups. We introduce STR evolutionary events specifically identical in primates (i.e. Homo sapiens, Pan troglodytes, and Gorilla gorilla gorilla) vs. non-primate out-groups. The average frequency of the identically shared STR motifs across those primates ranged between 0.00005 and 0.06. The identified genes are involved in important evolutionary and developmental processes, such as normal craniofacial development (TFAP2B), regulation of cell shape (PALMD), learning and long-term memory (RGS14), nervous system development (GFRA2), embryonic limb morphogenesis (PBX2), and forebrain development (APAF1). We provide evidence of core promoter STRs as evolutionary switch codes for primate speciation, and the first instance of identity-by-descent for those motifs at the interspecies level.

Exceptional expansion and conservation of a CT-repeat complex in the core promoter of PAXBP1 in primates.

Adaptive evolution may be linked with the genomic distribution and function of short tandem repeats (STRs). Proximity of the core promoter STRs to the +1 transcription start site (TSS), and their mutable nature are characteristics that highlight those STRs as a novel source of interspecies variation. The PAXBP1 gene (alternatively known as GCFC1) core promoter contains the longest STR identified in a Homo sapiens gene core promoter. Indeed, this core promoter is a stretch of four consecutive CT-STRs. In the current study, we used the Ensembl, NCBI, and UCSC databases to analyze the evolutionary trend and functional implication of this CT-STR complex in six major lineages across vertebrates, including primates, non-primate mammals, birds, reptiles, amphibians, and fish. We observed exceptional expansion (≥4-repeats) and conservation of this CT-STR complex across primates, except prosimians, Microcebus murinus and Otolemur garnettii (Fisher exact P<4.1×10(-7)). H. sapiens has the most complex STR formula, and longest repeats. Macaca mulatta and Callithrix jacchus monkeys have the simplest STR formulas, and shortest repeat numbers. CT≥4-repeats were not detected in non-primate lineages. Different length alleles across the PAXBP1 core promoter CT-STRs significantly altered gene expression in vitro (P<0.001, t-test). PAXBP1 has a crucial role in craniofacial development, myogenesis, and spine morphogenesis, properties that have been diverged between primates and non-primates. To our knowledge, this is the first instance of expansion and conservation of a STR complex co-occurring specifically with the primate lineage.

Proximal metatarsal articular surface shape and the evolution of a rigid lateral foot in hominins.

This study quantifies the proximal articular surface shape of metatarsal (MT) 4 and MT 5 using three-dimensional morphometrics. Humans and apes are compared to test whether they have significantly different shapes that are skeletal correlates to comparative lateral foot function. In addition, shod and unshod humans are compared to test for significant differences in surface shape. The MT 4 fossils OH 8, Stw 628, and AL 333-160, and the MT 5 fossils AL 333-13, AL 333-78, OH 8, and Stw 114/115 are compared with humans and apes to assess whether they bear greater similarities to humans, which would imply a relatively stable lateral foot, or to apes, which would imply a flexible foot with a midfoot break. Apes have a convex curved MT 4 surface, and humans have a flat surface. The MT 4 fossils show greater similarity to unshod humans, suggesting a stable lateral foot. Unshod humans have a relatively flatter MT 4 surface compared with shod humans. There is much overlap in MT 5 shape between humans and apes, with more similarity between humans and Gorilla. The fossil MT 5 surfaces are generally flat, most similar to humans and Gorilla. Because of the high degree of shape overlap between humans and apes, one must use caution in interpreting lateral foot function from the proximal MT 5 surface alone.

Homo sapiens as physician and patient: a view from Darwinian medicine.

Medicine's cardinal diagnostic and therapeutic resource is the clinical encounter. Over the last two centuries and particularly over the last five decades the function of the clinical encounter has been eroded to the point of near irrelevance because of the atomized and atomizing influence of technology and microspecialization. Meanwhile, over the past five decades the exceptionalist view of Homo sapiens inherent in the social and religious traditions of the West has similarly undergone radical changes. H. sapiens is now best understood as a microecosystem integrated into a much broader ecosystem: the biosphere. That human microecosystem is composed of constituents derived from the archaeal, bacterial, and eukaryan domains via endosymbiotic, commensalistic and mutualistic interactions. This amalgamation of 100 trillion cells and viral elements is regulated by a composite genome aggregated over the 3.8 billion years of evolutionary history of organic life. No component of H. sapiens or its genome can be identified as irreducibly and exclusively human. H. sapiens' humanity is an emergent property of the microecosystem. Ironically as H. sapiens is viewed by evolutionary science in a highly integrated manner medicine approaches it as a balkanized, deaggregated entity through the eye of 150 different specialties. To effectively address the needs of H sapiens in its role as patient by the same species in its role as physician the disparate views must be harmonized. Here I review some conceptual elements that would assist a physician in addressing the needs of the patient in integrum, as a microecosystem, by the former address the latter as a historical gestalt being. The optimal way to recover the harmony between patient and physician is through a revitalization of the clinical encounter via an ecological and Darwinian epistemology.

Increased expression of cell-cell signaling genes by stimulated mononuclear leukocytes in patients with previous atherothrombotic stroke. A whole genome expression profile study.

BACKGROUND/AIMS: Inflammation plays an important role in atherosclerosis and stroke. Acute infections are recognized as trigger factors for ischemic stroke. METHODS: In this whole genome expression profile study of 15 patients and 15 control subjects, we tested the hypothesis that patients with a history of atherothrombotic stroke show enhanced transcription of inflammatory genes in circulating leukocytes. RNA from unstimulated or lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) was analyzed with Affymetrix U133A GeneChips using a pooling design. Expression of single genes and functional groups of genes was analyzed by global statistical tests. RESULTS: A total of 10,197 probe sets showed positive calls. After correction for multiple testing no single probe set revealed significant differences either without or with LPS stimulation. However, significant global expression differences were found upon LPS stimulation for the group of genes that are involved in cell-cell signaling. CONCLUSION: LPS stimulation of PBMCs, a condition mimicking bacterial infection, induces differential expression of a group of cell-cell signaling genes in patients with previous atherothrombotic stroke. This finding can be caused by genetic differences between both groups, but acquired risk factors, medication and technical factors may also have contributed to the result.

How and why genetic linkage has not solved the problem of psychosis: review and hypothesis.

OBJECTIVE: The author examined the chromosomal linkage method as an approach to the genetic basis of schizophrenia and bipolar disorder. METHOD: Comparisons were conducted of recent meta-analyses of genome scans of schizophrenia and bipolar disorder and of the three largest (N>300) sibling pair studies of schizophrenia and schizoaffective disorder and a comparable study of bipolar illness. RESULTS: Recent meta-analyses have not identified consistent sites of linkage. The three largest studies of schizophrenia fail to agree on a single locus, no commonality with bipolar illness has been demonstrated, and there is no replicable support for any of the current candidate genes. DISCUSSION: An alternative to the concept that DNA sequence variation lies in "multiple genes of small effect" is the hypothesis that the variation is epigenetic but related to the genetic transition ("the speciation event") that separated Homo sapiens from a prior hominid species. This hypothesis draws attention to the chromosomal rearrangement (the Xq21.3/Yp translocation) that occurred some 6 million years ago in the hominid lineage and subsequent rearrangements, including a paracentric inversion, that have taken place within the translocated segment. Here it is argued that the most recent of these events is relevant to specifically human characteristics, including language. The gene pair protocadherin X and Y within this region is under new selective pressure and is in a novel (epigenetic) situation with respect to X inactivation. CONCLUSIONS: Epigenetic variation associated with chromosomal rearrangements that occurred in the hominid lineage and that relates to the evolution of language could account for predisposition to schizophrenia and schizoaffective and bipolar disorder and failure to detect such variation by standard linkage approaches.

Penetrance and the Healthy Elderly.

The variable penetrance of pathogenic variants (PVs) represents a major challenge to the field of human genetics, often complicating clinical decision-making and risk management. Nonpenetrance, the detection of PVs in the absence of disease manifestation, is a common phenomenon, yet, we know very little about the underlying factors, which may protect some individuals and not others. Placing a new focus on the genomic study of the healthy elderly may be pivotal for advancing our understanding of penetrance. Studying those who remain unaffected late into life, despite harboring known genetic risk variants, could provide important insights into disease mechanisms and ultimately inform clinical care, yet, it has received relatively little attention as a research strategy. The ever increasing use of sequencing technology is further driving the requirement to understand the penetrance of ascertained variants. The ASPREE Biobank of Healthy Ageing provides a unique opportunity to address this area of need. DNA has been collected from a cohort of over 14,000 healthy elderly individuals aged 70 years or older enrolled in an aspirin clinical trial. The ASPREE cohort represents a healthy reference population ascertained without the typical biases of a genetic study. The cohort is depleted of expressed monogenetic disease, yet will contain hundreds of elderly individuals with known PVs in clinically actionable genes. Investigating this population along with other cohorts of the healthy elderly will provide critical new knowledge into the penetrance of actionable variants as a foundation for informing clinical care.

Evaluation of the relationship between CD36 and MARCO single-nucleotide polymorphisms and susceptibility to carotid atherosclerosis in a Chinese Han population.

OBJECTIVE: This study analyzed the genetic association between two scavenger receptors single nucleotide polymorphisms (CD36 rs1761667, MARCO rs12998782) and carotid atherosclerosis in a Chinese Han population. METHODS: Samples of genomic DNA collected from patients (n=215) and healthy control subjects (n=252) were analyzed by the polymerase chain reaction with high-resolution melting analysis. Odds ratios and 95% confidence intervals were used to evaluate the association between the two SNPs and carotid atherosclerosis. RESULTS: There was no difference between the SNPs regarding their association with the frequency of carotid atherosclerosis in the case and control groups or in the male case group and control group. Female patients of genotype GA for CD36 rs1761667 and CT for MARCO rs12998782 were at an increased risk for carotid atherosclerosis. The presence of rs1761667 GA and rs12998782 CT may increase the risk for carotid atherosclerosis among postmenopausal females. CONCLUSIONS: CD36 and MARCO are associated with the susceptibility of Chinese Han females to carotid atherosclerosis. Menopausal status may affect the association between gene polymorphisms and carotid atherosclerosis in the female Chinese Han population.

The shape of human gene family phylogenies.

BACKGROUND: The shape of phylogenetic trees has been used to make inferences about the evolutionary process by comparing the shapes of actual phylogenies with those expected under simple models of the speciation process. Previous studies have focused on speciation events, but gene duplication is another lineage splitting event, analogous to speciation, and gene loss or deletion is analogous to extinction. Measures of the shape of gene family phylogenies can thus be used to investigate the processes of gene duplication and loss. We make the first systematic attempt to use tree shape to study gene duplication using human gene phylogenies. RESULTS: We find that gene duplication has produced gene family trees significantly less balanced than expected from a simple model of the process, and less balanced than species phylogenies: the opposite to what might be expected under the 2R hypothesis. CONCLUSION: While other explanations are plausible, we suggest that the greater imbalance of gene family trees than species trees is due to the prevalence of tandem duplications over regional duplications during the evolution of the human genome.

Efficient transfection of primary cells relevant for cardiovascular research by nucleofection.

Cell types that are important for cardiovascular research, e.g., cardiomyocytes, endothelial cells, or adult stem cells, are often hard to isolate, culture, and transfect. Low-transfection efficiencies are a major limitation because, in many cases, results achieved with surrogate model cell lines, if any at all are available for the primary cell type of interest, do not reflect the situation in the primary cell. We have demonstrated that unprecedented transfection results are achieved with primary cells when novel electroporation conditions are combined with a treatment of the cells in specific solutions that help stabilize the cells in the electrical field. This led to the development of the new proprietary transfection technology nucleofection. Nucleofection has proved to be successfully applicable to a variety of primary cells and other hard-to-transfect cell lines, and, thus, opens unique perspectives for novel experimental setups as therapeutic strategies. Herein we present protocols for the efficient nucleofection of human umbilical vein endothelial cells, human coronary artery endothelial cells, smooth muscle cells (e.g., pig vascular smooth muscle cells), neonatal rat cardiomyocytes, and human mesenchymal stem cells and depict some results obtained with such transfected cells.

Detection of human adaptation during the past 2000 years.

Detection of recent natural selection is a challenging problem in population genetics. Here we introduce the singleton density score (SDS), a method to infer very recent changes in allele frequencies from contemporary genome sequences. Applied to data from the UK10K Project, SDS reflects allele frequency changes in the ancestors of modern Britons during the past ~2000 to 3000 years. We see strong signals of selection at lactase and the major histocompatibility complex, and in favor of blond hair and blue eyes. For polygenic adaptation, we find that recent selection for increased height has driven allele frequency shifts across most of the genome. Moreover, we identify shifts associated with other complex traits, suggesting that polygenic adaptation has played a pervasive role in shaping genotypic and phenotypic variation in modern humans.

Partial nucleotide sequences and expression patterns of frog (Rana pipiens) ephrin-A2 and ephrin-A5 mRNA.

We have generated 362 bp and 547 bp partial sequences for Rana pipiens ephrin-A2 and ephrin-A5 mRNA, respectively. Translation homologies for the comparable segments of cDNA of chicken, mouse and human are 90.8, 86.9 and 84.4% for the ephrin-A2 sequence and 85.7, 85.0 and 85.0% for the ephrin-A5 sequence. Digoxigenin-labeled riboprobes were prepared and applied by means of in situ hybridization to whole-mounts of the brains of mature adults and expression patterns in tadpoles were also explored. The RNA probes revealed similar posterior (high) to anterior (low) expression gradients in the adult tectum, demonstrating that both ephrin-As are expressed in the adult Ranid frog tectum. Only the ephrin-A2 probe was tested on tadpole brain, yielding an appropriately graded expression pattern similar to the adult.

Comparative analysis of cell cycle regulated genes in eukaryotes.

We compared microarray experiments on cell cycle of three model eukaryotes: budding and fission yeast and human cells. Only 112 orthologous groups were cyclic in the three model organisms. The common set of cyclic orthologs includes many taking part in the cell cycle progression, like cyclin B homologs, CDC5, SCH9, DSK2, ZPR1. Proteins involved in DNA replication included histones, some checkpoint kinases and some proteins regulating DNA damage and repair. Conserved cyclic proteins involved in cytokinesis included myosins and kinesins. Many groups of genes related to translation and other metabolic processes were also cyclic in all three organisms. This reflects rebuilding of cellular components after the replication and changes of metabolism during the cell cycle. Many genes important in cell cycle control are not cyclic or not conserved. This includes transcription factors implicated in the regulation of budding yeast cell cycle. The partially overlapping roles of regulatory proteins might allow the evolutionary substitution of components of cell cycle.