Evolutionary Selection and Constraint on Human Knee Chondrocyte Regulation Impacts Osteoarthritis Risk.

During human evolution, the knee adapted to the biomechanical demands of bipedalism by altering chondrocyte developmental programs. This adaptive process was likely not without deleterious consequences to health. Today, osteoarthritis occurs in 250 million people, with risk variants enriched in non-coding sequences near chondrocyte genes, loci that likely became optimized during knee evolution. We explore this relationship by epigenetically profiling joint chondrocytes, revealing ancient selection and recent constraint and drift on knee regulatory elements, which also overlap osteoarthritis variants that contribute to disease heritability by tending to modify constrained functional sequence. We propose a model whereby genetic violations to regulatory constraint, tolerated during knee development, lead to adult pathology. In support, we discover a causal enhancer variant (rs6060369) present in billions of people at a risk locus (GDF5-UQCC1), showing how it impacts mouse knee-shape and osteoarthritis. Overall, our methods link an evolutionarily novel aspect of human anatomy to its pathogenesis.

Molecular mechanism of synovial joint site specification and induction in developing vertebrate limbs.

The vertebrate appendage comprises three primary segments, the stylopod, zeugopod and autopod, each separated by joints. The molecular mechanisms governing the specification of joint sites, which define segment lengths and thereby limb architecture, remain largely unknown. Existing literature suggests that reciprocal gradients of retinoic acid (RA) and fibroblast growth factor (FGF) signaling define the expression domains of the putative segment markers Meis1, Hoxa11 and Hoxa13. Barx1 is expressed in the presumptive joint sites. Our data demonstrate that RA-FGF signaling gradients define the expression domain of Barx1 in the first presumptive joint site. When misexpressed, Barx1 induces ectopic interzone-like structures, and its loss of function partially blocks interzone development. Simultaneous perturbations of RA-FGF signaling gradients result in predictable shifts of Barx1 expression domains along the proximo-distal axis and, consequently, in the formation of repositioned joints. Our data suggest that during early limb bud development in chick, Meis1 and Hoxa11 expression domains are overlapping, whereas the Barx1 expression domain resides within the Hoxa11 expression domain. However, once the interzone is formed, the expression domains are refined and the Barx1 expression domain becomes congruent with the border of these two putative segment markers.

The Notch-mediated circuitry in the evolution and generation of new cell lineages: the tooth model.

The Notch pathway is an ancient, evolutionary conserved intercellular signaling mechanism that is involved in cell fate specification and proper embryonic development. The Jagged2 gene, which encodes a ligand for the Notch family of receptors, is expressed from the earliest stages of odontogenesis in epithelial cells that will later generate the enamel-producing ameloblasts. Homozygous Jagged2 mutant mice exhibit abnormal tooth morphology and impaired enamel deposition. Enamel composition and structure in mammals are tightly linked to the enamel organ that represents an evolutionary unit formed by distinct dental epithelial cell types. The physical cooperativity between Notch ligands and receptors suggests that Jagged2 deletion could alter the expression profile of Notch receptors, thus modifying the whole Notch signaling cascade in cells within the enamel organ. Indeed, both Notch1 and Notch2 expression are severely disturbed in the enamel organ of Jagged2 mutant teeth. It appears that the deregulation of the Notch signaling cascade reverts the evolutionary path generating dental structures more reminiscent of the enameloid of fishes rather than of mammalian enamel. Loss of interactions between Notch and Jagged proteins may initiate the suppression of complementary dental epithelial cell fates acquired during evolution. We propose that the increased number of Notch homologues in metazoa enabled incipient sister cell types to form and maintain distinctive cell fates within organs and tissues along evolution.

Survival analysis on subchondral bone length for total knee replacement.

OBJECTIVE: Use subchondral bone length (SBL), a new MRI-derived measure that reflects the extent of cartilage loss and bone flattening, to predict the risk of progression to total knee replacement (TKR). METHODS: We employed baseline MRI data from the Osteoarthritis Initiative (OAI), focusing on 760 men and 1214 women with bone marrow lesions (BMLs) and joint space narrowing (JSN) scores, to predict the progression to TKR. To minimize bias from analyzing both knees of a participant, only the knee with a higher Kellgren-Lawrence (KL) grade was considered, given its greater potential need for TKR. We utilized the Kaplan-Meier survival curves and Cox proportional hazards models, incorporating raw and normalized values of SBL, JSN, and BML as predictors. The study included subgroup analyses for different demographics and clinical characteristics, using models for raw and normalized SBL (merged, femoral, tibial), BML (merged, femoral, tibial), and JSN (medial and lateral compartments). Model performance was evaluated using the time-dependent area under the curve (AUC), Brier score, and Concordance index to gauge accuracy, calibration, and discriminatory power. Knee joint and region-level analyses were conducted to determine the effectiveness of SBL, JSN, and BML in predicting TKR risk. RESULTS: The SBL model, incorporating data from both the femur and tibia, demonstrated a predictive capacity for TKR that closely matched the performance of the BML score and the JSN grade. The Concordance index of the SBL model was 0.764, closely mirroring the BML's 0.759 and slightly below JSN's 0.788. The Brier score for the SBL model stood at 0.069, showing comparability with BML's 0.073 and a minor difference from JSN's 0.067. Regarding the AUC, the SBL model achieved 0.803, nearly identical to BML's 0.802 and slightly lower than JSN's 0.827. CONCLUSION: SBL's capacity to predict the risk of progression to TKR highlights its potential as an effective imaging biomarker for knee osteoarthritis.

Epigenetics as a mediator of genetic risk in osteoarthritis: role during development, homeostasis, aging, and disease progression.

The identification of genomic loci that are associated with osteoarthritis (OA) has provided a starting point for understanding how genetic variation activates catabolic processes in the joint. However, genetic variants can only alter gene expression and cellular function when the epigenetic environment is permissive to these effects. In this review, we provide examples of how epigenetic shifts at distinct life stages can alter the risk for OA, which we posit is critical for the proper interpretation of genome-wide association studies (GWAS). During development, intensive work on the growth and differentiation factor 5 (GDF5) locus has revealed the importance of tissue-specific enhancer activity in controlling both joint development and the subsequent risk for OA. During homeostasis in adults, underlying genetic risk factors may help establish beneficial or catabolic "set points" that dictate tissue function, with a strong cumulative effect on OA risk. During aging, methylation changes and the reorganization of chromatin can "unmask" the effects of genetic variants. The destructive function of variants that alter aging would only mediate effects after reproductive competence and thus avoid any evolutionary selection pressure, as consistent with larger frameworks of biological aging and its relationship to disease. A similar "unmasking" may occur during OA progression, which is supported by the finding of distinct expression quantitative trait loci (eQTLs) in chondrocytes depending on the degree of tissue degradation. Finally, we propose that massively parallel reporter assays (MPRAs) will be a valuable tool to test the function of putative OA GWAS variants in chondrocytes from different life stages.

Detection of Neanderthal Adaptively Introgressed Genetic Variants That Modulate Reporter Gene Expression in Human Immune Cells.

Although some variation introgressed from Neanderthals has undergone selective sweeps, little is known about its functional significance. We used a Massively Parallel Reporter Assay (MPRA) to assay 5,353 high-frequency introgressed variants for their ability to modulate the gene expression within 170 bp of endogenous sequence. We identified 2,548 variants in active putative cis-regulatory elements (CREs) and 292 expression-modulating variants (emVars). These emVars are predicted to alter the binding motifs of important immune transcription factors, are enriched for associations with neutrophil and white blood cell count, and are associated with the expression of genes that function in innate immune pathways including inflammatory response and antiviral defense. We combined the MPRA data with other data sets to identify strong candidates to be driver variants of positive selection including an emVar that may contribute to protection against severe COVID-19 response. We endogenously deleted two CREs containing expression-modulation variants linked to immune function, rs11624425 and rs80317430, identifying their primary genic targets as ELMSAN1, and PAN2 and STAT2, respectively, three genes differentially expressed during influenza infection. Overall, we present the first database of experimentally identified expression-modulating Neanderthal-introgressed alleles contributing to potential immune response in modern humans.

Human and African ape myosin heavy chain content and the evolution of hominin skeletal muscle.

Humans are unique among terrestrial mammals in our manner of walking and running, reflecting 7 to 8 Ma of musculoskeletal evolution since diverging with the genus Pan. One component of this is a shift in our skeletal muscle biology towards a predominance of myosin heavy chain (MyHC) I isoforms (i.e. slow fibers) across our pelvis and lower limbs, which distinguishes us from chimpanzees. Here, new MyHC data from 35 pelvis and hind limb muscles of a Western gorilla (Gorilla gorilla) are presented. These data are combined with a similar chimpanzee dataset to assess the MyHC I content of humans in comparison to African apes (chimpanzees and gorillas) and other terrestrial mammals. The responsiveness of human skeletal muscle to behavioral interventions is also compared to the human-African ape differential. Humans are distinct from African apes and among a small group of terrestrial mammals whose pelvis and lower limb muscle is slow fiber dominant, on average. Behavioral interventions, including immobilization, bed rest, spaceflight and exercise, can induce modest decreases and increases in human MyHC I content (i.e. -9.3% to 2.3%, n = 2033 subjects), but these shifts are much smaller than the mean human-African ape differential (i.e. 31%). Taken together, these results indicate muscle fiber content is likely an evolvable trait under selection in the hominin lineage. As such, we highlight potential targets of selection in the genome (e.g. regions that regulate MyHC content) that may play an important role in hominin skeletal muscle evolution.

Single Cell Omics for Musculoskeletal Research.

PURPOSE OF REVIEW: The ability to analyze the molecular events occurring within individual cells as opposed to populations of cells is revolutionizing our understanding of musculoskeletal tissue development and disease. Single cell studies have the great potential of identifying cellular subpopulations that work in a synchronized fashion to regenerate and repair damaged tissues during normal homeostasis. In addition, such studies can elucidate how these processes break down in disease as well as identify cellular subpopulations that drive the disease. This review highlights three emerging technologies: single cell RNA sequencing (scRNA-seq), Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), and Cytometry by Time-Of-Flight (CyTOF) mass cytometry. RECENT FINDINGS: Technological and bioinformatic tools to analyze the transcriptome, epigenome, and proteome at the individual cell level have advanced rapidly making data collection relatively easy; however, understanding how to access and interpret the data remains a challenge for many scientists. It is, therefore, of paramount significance to educate the musculoskeletal community on how single cell technologies can be used to answer research questions and advance translation. This article summarizes talks given during a workshop on "Single Cell Omics" at the 2020 annual meeting of the Orthopedic Research Society. Studies that applied scRNA-seq, ATAC-seq, and CyTOF mass cytometry to cartilage development and osteoarthritis are reviewed. This body of work shows how these cutting-edge tools can advance our understanding of the cellular heterogeneity and trajectories of lineage specification during development and disease.

Variation in mouse pelvic morphology maps to locations enriched in Sox9 Class II and Pitx1 regulatory features.

Variation in pelvic morphology has a complex genetic basis and its patterning and specification is governed by conserved developmental pathways. Whether the mechanisms underlying the differentiation and specification of the pelvis also produce the morphological covariation on which natural selection may act, is still an open question in evolutionary developmental biology. We use high-resolution quantitative trait locus (QTL) mapping in the F34 generation of an advanced intercross experiment (LG,SM-G34 ) to characterize the genetic architecture of the mouse pelvis. We test the prediction that genomic features linked to developmental patterning and differentiation of the hind limb and pelvis and the regulation of chondrogenesis are overrepresented in QTL. We find 31 single QTL trait associations at the genome- or chromosome-wise significance level coalescing to 27 pleiotropic loci. We recover further QTL at a more relaxed significance threshold replicating locations found in a previous experiment in an earlier generation of the same population. QTL were more likely than chance to harbor Pitx1 and Sox9 Class II chromatin immunoprecipitation-seq features active during development of skeletal features. There was weak or no support for the enrichment of seven more categories of developmental features drawn from the literature. Our results suggest that genotypic variation is channeled through a subset of developmental processes involved in the generation of phenotypic variation in the pelvis. This finding indicates that the evolvability of complex traits may be subject to biases not evident from patterns of covariance among morphological features or developmental patterning when either is considered in isolation.

Biological clocks and incremental growth line formation in dentine.

Dentine- and enamel-forming cells secrete matrix in consistent rhythmic phases, resulting in the formation of successive microscopic growth lines inside tooth crowns and roots. Experimental studies of various mammals have proven that these lines are laid down in subdaily, daily (circadian), and multidaily rhythms, but it is less clear how these rhythms are initiated and maintained. In 2001, researchers reported that lesioning the so-called master biological clock, the suprachiasmatic nucleus (SCN), halted daily line formation in rat dentine, whereas subdaily lines persisted. More recently, a key clock gene (Bmal1) expressed in the SCN in a circadian manner was also found to be active in dentine- and enamel- secretory cells. To probe these potential neurological and local mechanisms for the production of rhythmic lines in teeth, we reexamined the role of the SCN in growth line formation in Wistar rats and investigated the presence of daily lines in Bmal1 knockout mice (Bmal1-/- ). In contrast to the results of the 2001 study, we found that both daily and subdaily growth lines persisted in rat dentine after complete or partial SCN lesion in the majority of individuals. In mice, after transfer into constant darkness, daily rhythms continued to manifest as incremental lines in the dentine of each Bmal1 genotype (wild-type, Bmal+/- , and Bmal1-/- ). These results affirm that the manifestation of biological rhythms in teeth is a robust phenomenon, imply a more autonomous role of local biological clocks in tooth growth than previously suggested, and underscore the need further to elucidate tissue-specific circadian biology and its role in incremental line formation. Investigations of this nature will strengthen an invaluable system for determining growth rates and calendar ages from mammalian hard tissues, as well as documenting the early lives of fossil hominins and other primates.

Correction to: Assessment of knee pain from MR imaging using a convolutional Siamese network.

The original version of this article, published on 13 February 2020, unfortunately contained a mistake.

Assessment of knee pain from MR imaging using a convolutional Siamese network.

OBJECTIVES: It remains difficult to characterize the source of pain in knee joints either using radiographs or magnetic resonance imaging (MRI). We sought to determine if advanced machine learning methods such as deep neural networks could distinguish knees with pain from those without it and identify the structural features that are associated with knee pain. METHODS: We constructed a convolutional Siamese network to associate MRI scans obtained on subjects from the Osteoarthritis Initiative (OAI) with frequent unilateral knee pain comparing the knee with frequent pain to the contralateral knee without pain. The Siamese network architecture enabled pairwise learning of information from two-dimensional (2D) sagittal intermediate-weighted turbo spin echo slices obtained from similar locations on both knees. Class activation mapping (CAM) was utilized to create saliency maps, which highlighted the regions most associated with knee pain. The MRI scans and the CAMs of each subject were reviewed by an expert radiologist to identify the presence of abnormalities within the model-predicted regions of high association. RESULTS: Using 10-fold cross-validation, our model achieved an area under curve (AUC) value of 0.808. When individuals whose knee WOMAC pain scores were not discordant were excluded, model performance increased to 0.853. The radiologist review revealed that about 86% of the cases that were predicted correctly had effusion-synovitis within the regions that were most associated with pain. CONCLUSIONS: This study demonstrates a proof of principle that deep learning can be applied to assess knee pain from MRI scans. KEY POINTS: • Our article is the first to leverage a deep learning framework to associate MR images of the knee with knee pain. • We developed a convolutional Siamese network that had the ability to fuse information from multiple two-dimensional (2D) MRI slices from the knee with pain and the contralateral knee of the same individual without pain to predict unilateral knee pain. • Our model achieved an area under curve (AUC) value of 0.808. When individuals who had WOMAC pain scores that were not discordant for knees (pain discordance < 3) were excluded, model performance increased to 0.853.

Disentangling Immediate Adaptive Introgression from Selection on Standing Introgressed Variation in Humans.

Recent studies have reported evidence suggesting that portions of contemporary human genomes introgressed from archaic hominin populations went to high frequencies due to positive selection. However, no study to date has specifically addressed the postintrogression population dynamics of these putative cases of adaptive introgression. Here, for the first time, we specifically define cases of immediate adaptive introgression (iAI) in which archaic haplotypes rose to high frequencies in humans as a result of a selective sweep that occurred shortly after the introgression event. We define these cases as distinct from instances of selection on standing introgressed variation (SI), in which an introgressed haplotype initially segregated neutrally and subsequently underwent positive selection. Using a geographically diverse data set, we report novel cases of selection on introgressed variation in living humans and shortlist among these cases those whose selective sweeps are more consistent with having been the product of iAI rather than SI. Many of these novel inferred iAI haplotypes have potential biological relevance, including three that contain immune-related genes in West Siberians, South Asians, and West Eurasians. Overall, our results suggest that iAI may not represent the full picture of positive selection on archaically introgressed haplotypes in humans and that more work needs to be done to analyze the role of SI in the archaic introgression landscape of living humans.

Exercise-induced loading increases ilium cortical area in a selectively bred mouse model.

OBJECTIVES: Little is known about how ilium cortical bone responds to loading. Using a mouse model, this study presents data testing the hypothesis that iliac cross-sectional properties are altered in response to increased activity. MATERIALS AND METHODS: The sample derives from lines of High Runner (HR) mice bred for increased wheel-running activity. Four treatment groups of female mice were tested: non-selected control lines housed without (N = 19) and with wheels (N = 20), and HR mice housed without (N = 17) and with wheels (N = 18) for 13 weeks beginning at weaning. Each pelvis was µCT-scanned, cross-sectional properties (cortical area-Ct.Ar, total area-Tt.Ar, polar moment of area, and polar section modulus) were determined from the ilium midshaft, and robusticity indices (ratio of the square root of Ct.Ar or Tt.Ar to caudal ilium length) were calculated. Mixed models were implemented with linetype, wheel access, and presence of the mini-muscle phenotype as fixed effects, replicate line nested within linetype as a random effect, and body mass as a covariate. RESULTS: Results demonstrate that the mouse ilium morphologically resembles a long bone in cross section. Body mass and the mini-muscle phenotype were significant predictors of iliac cross-sectional properties. Wheel access only had a statistically significant effect on Ct.Ar and its robusticity index, with greater values in mice with wheel access. DISCUSSION: These results suggest that voluntary exercise increases cortical area, but does not otherwise strengthen the ilium in these mice, corroborating previous studies on the effect of increased wheel-running activity on femoral and humeral cross-sectional properties in these mice.

Complex Phenotypes: Mechanisms Underlying Variation in Human Stature.

PURPOSE OF REVIEW: The goal of the review is to provide a comprehensive overview of the current understanding of the mechanisms underlying variation in human stature. RECENT FINDINGS: Human height is an anthropometric trait that varies considerably within human populations as well as across the globe. Historically, much research focus was placed on understanding the biology of growth plate chondrocytes and how modifications to core chondrocyte proliferation and differentiation pathways potentially shaped height attainment in normal as well as pathological contexts. Recently, much progress has been made to improve our understanding regarding the mechanisms underlying the normal and pathological range of height variation within as well as between human populations, and today, it is understood to reflect complex interactions among a myriad of genetic, environmental, and evolutionary factors. Indeed, recent improvements in genetics (e.g., GWAS) and breakthroughs in functional genomics (e.g., whole exome sequencing, DNA methylation analysis, ATAC-sequencing, and CRISPR) have shed light on previously unknown pathways/mechanisms governing pathological and common height variation. Additionally, the use of an evolutionary perspective has also revealed important mechanisms that have shaped height variation across the planet. This review provides an overview of the current knowledge of the biological mechanisms underlying height variation by highlighting new research findings on skeletal growth control with an emphasis on previously unknown pathways/mechanisms influencing pathological and common height variation. In this context, this review also discusses how evolutionary forces likely shaped the genomic architecture of height across the globe.

Heads, Shoulders, Elbows, Knees, and Toes: Modular Gdf5 Enhancers Control Different Joints in the Vertebrate Skeleton.

Synovial joints are crucial for support and locomotion in vertebrates, and are the frequent site of serious skeletal defects and degenerative diseases in humans. Growth and differentiation factor 5 (Gdf5) is one of the earliest markers of joint formation, is required for normal joint development in both mice and humans, and has been genetically linked to risk of common osteoarthritis in Eurasian populations. Here, we systematically survey the mouse Gdf5 gene for regulatory elements controlling expression in synovial joints. We identify separate regions of the locus that control expression in axial tissues, in proximal versus distal joints in the limbs, and in remarkably specific sub-sets of composite joints like the elbow. Predicted transcription factor binding sites within Gdf5 regulatory enhancers are required for expression in particular joints. The multiple enhancers that control Gdf5 expression in different joints are distributed over a hundred kilobases of DNA, including regions both upstream and downstream of Gdf5 coding exons. Functional rescue tests in mice confirm that the large flanking regions are required to restore normal joint formation and patterning. Orthologs of these enhancers are located throughout the large genomic region previously associated with common osteoarthritis risk in humans. The large array of modular enhancers for Gdf5 provide a new foundation for studying the spatial specificity of joint patterning in vertebrates, as well as new candidates for regulatory regions that may also influence osteoarthritis risk in human populations.

Impact of broad regulatory regions on Gdf5 expression and function in knee development and susceptibility to osteoarthritis.

OBJECTIVES: Given the role of growth and differentiation factor 5 (GDF5) in knee development and osteoarthritis risk, we sought to characterise knee defects resulting from Gdf5 loss of function and how its regulatory regions control knee formation and morphology. METHODS: The brachypodism (bp) mouse line, which harbours an inactivating mutation in Gdf5, was used to survey how Gdf5 loss of function impacts knee morphology, while two transgenic Gdf5 reporter bacterial artificial chromosome mouse lines were used to assess the spatiotemporal activity and function of Gdf5 regulatory sequences in the context of clinically relevant knee anatomical features. RESULTS: Knees from homozygous bp mice (bp/bp) exhibit underdeveloped femoral condyles and tibial plateaus, no cruciate ligaments, and poorly developed menisci. Secondary ossification is also delayed in the distal femur and proximal tibia. bp/bp mice have significantly narrower femoral condyles, femoral notches and tibial plateaus, and curvier medial femoral condyles, shallower trochlea, steeper lateral tibial slopes and smaller tibial spines. Regulatory sequences upstream from Gdf5 were weakly active in the prenatal knee, while downstream regulatory sequences were active throughout life. Importantly, downstream but not upstream Gdf5 regulatory sequences fully restored all the key morphological features disrupted in the bp/bp mice. CONCLUSIONS: Knee morphology is profoundly affected by Gdf5 absence, and downstream regulatory sequences mediate its effects by controlling Gdf5 expression in knee tissues. This downstream region contains numerous enhancers harbouring human variants that span the osteoarthritis association interval. We posit that subtle alterations to morphology driven by changes in downstream regulatory sequence underlie this locus' role in osteoarthritis risk.

Fossil hominin shoulders support an African ape-like last common ancestor of humans and chimpanzees.

Reconstructing the behavioral shifts that drove hominin evolution requires knowledge of the timing, magnitude, and direction of anatomical changes over the past ∼6-7 million years. These reconstructions depend on assumptions regarding the morphotype of the Homo-Pan last common ancestor (LCA). However, there is little consensus for the LCA, with proposed models ranging from African ape to orangutan or generalized Miocene ape-like. The ancestral state of the shoulder is of particular interest because it is functionally associated with important behavioral shifts in hominins, such as reduced arboreality, high-speed throwing, and tool use. However, previous morphometric analyses of both living and fossil taxa have yielded contradictory results. Here, we generated a 3D morphospace of ape and human scapular shape to plot evolutionary trajectories, predict ancestral morphologies, and directly test alternative evolutionary hypotheses using the hominin fossil evidence. We show that the most parsimonious model for the evolution of hominin shoulder shape starts with an African ape-like ancestral state. We propose that the shoulder evolved gradually along a single morphocline, achieving modern human-like configuration and function within the genus Homo. These data are consistent with a slow, progressive loss of arboreality and increased tool use throughout human evolution.

Human-specific loss of regulatory DNA and the evolution of human-specific traits.

Humans differ from other animals in many aspects of anatomy, physiology, and behaviour; however, the genotypic basis of most human-specific traits remains unknown. Recent whole-genome comparisons have made it possible to identify genes with elevated rates of amino acid change or divergent expression in humans, and non-coding sequences with accelerated base pair changes. Regulatory alterations may be particularly likely to produce phenotypic effects while preserving viability, and are known to underlie interesting evolutionary differences in other species. Here we identify molecular events particularly likely to produce significant regulatory changes in humans: complete deletion of sequences otherwise highly conserved between chimpanzees and other mammals. We confirm 510 such deletions in humans, which fall almost exclusively in non-coding regions and are enriched near genes involved in steroid hormone signalling and neural function. One deletion removes a sensory vibrissae and penile spine enhancer from the human androgen receptor (AR) gene, a molecular change correlated with anatomical loss of androgen-dependent sensory vibrissae and penile spines in the human lineage. Another deletion removes a forebrain subventricular zone enhancer near the tumour suppressor gene growth arrest and DNA-damage-inducible, gamma (GADD45G), a loss correlated with expansion of specific brain regions in humans. Deletions of tissue-specific enhancers may thus accompany both loss and gain traits in the human lineage, and provide specific examples of the kinds of regulatory alterations and inactivation events long proposed to have an important role in human evolutionary divergence.

Screening of reproduction-related single-nucleotide variations from MeDIP-seq data in sheep.

Extensive variation in reproduction has arisen in Chinese Mongolian sheep during recent domestication. Hu and Small-tailed Han sheep, for example, have become non-seasonal breeders and exhibit higher fecundity than Tan and Ujumqin breeds. We therefore scanned reproduction-related single-nucleotide variations from methylated DNA-immunoprecipitation sequencing data generated from each of those four breeds to uncover potential mechanisms underlying this breed variation. We generated a high-quality map of single nucleotide variations (SNVs) in DNA methylation enriched regions, and found that the majority of variants are located within non-coding regions. We identified 359 SNVs within the Sheep Quantitative Trait Locus (QTL) database. Nineteen of these SNVs associated with the Aseasonal Reproduction QTL, and 10 out of the 19 reside close to genes with known reproduction functions. We also identified the well-known FecB mutation in high-fecundity sheep (Hu and Small-tailed Han sheep). When we applied these FecB finding to our breeding system, we improved lambing rate by 175%. In summary, this study provided strong candidate SNVs associated with sheep fecundity that can serve as targets for functional testing and to enhance selective breeding strategies. Mol. Reprod. Dev. 83: 958-967, 2016 © 2016 Wiley Periodicals, Inc.