Linked-Read Sequencing of Eight Falcons Reveals a Unique Genomic Architecture in Flux

Falcons are diverse birds of cultural and economic importance. They have undergone major lineage specific chromosomal rearrangements, resulting in greatly-reduced chromosome counts relative to other birds. Here, we use 10X Genomics linked reads to provide new high-contiguity genomes for two gyrfalcons, a saker falcon, a lanner falcon, three subspecies of peregrine falcons, and the common kestrel. Assisted by a transcriptome sequenced from 22 gyrfalcon tissues, we annotate these genomes for a variety of genomic features, estimate historical demography, and then investigate genomic equilibrium in the context of falcon-specific chromosomal rearrangements. We find that falcon genomes are not in AT-GC equilibrium with a bias in mutations towards higher AT content; this bias is predominantly driven by, but not dependent on, hypermutability of CpG sites. Small indels and large structural variants were also biased towards insertions rather than deletions. Patterns of disequilibrium were linked to chromosomal rearrangements: falcons have lost GC content in regions that have fused to larger chromosomes from microchromosomes and gained GC content in regions of macrochromosomes that have translocated to microchromosomes. Inserted bases have accumulated on regions ancestrally belonging to microchromosomes, consistent with insertion-biased gene conversion. We also find an excess of interspersed repeats on regions of microchromosomes that have fused to macrochromosomes. Our results reveal that falcon genomes are in a state of flux. They further suggest that many of the key differences between microchromosomes and macrochromosomes are driven by differences in chromosome size, and indicate a clear role for recombination and biased gene conversion in determining genomic equilibrium.

Canine Oral Melanoma Genomic and Transcriptomic Study Defines Two Molecular Subgroups with Different Therapeutical Targets

Mucosal melanoma (MM) is a rare, aggressive clinical cancer. Despite recent advances in genetics and treatment, the prognosis of MM remains poor. Canine MM offers a relevant spontaneous and immunocompetent model to decipher the genetic bases and explore treatments for MM. We performed an integrative genomic and transcriptomic analysis of 32 canine MM samples, which identified two molecular subgroups with a different microenvironment and structural variant (SV) content. The overexpression of genes related to the microenvironment and T-cell response was associated with tumors harboring a lower content of SVs, whereas the overexpression of pigmentation-related pathways and oncogenes, such as TERT, was associated with a high SV burden. Using whole-genome sequencing, we showed that focal amplifications characterized complex chromosomal rearrangements targeting oncogenes, such as MDM2 or CDK4, and a recurrently amplified region on canine chromosome 30. We also demonstrated that the genes TRPM7, GABPB1, and SPPL2A, located in this CFA30 region, play a role in cell proliferation, and thus, may be considered as new candidate oncogenes for human MM. Our findings suggest the existence of two MM molecular subgroups that may benefit from dedicated therapies, such as immune checkpoint inhibitors or targeted therapies, for both human and veterinary medicine.

Factors affecting clinical manifestation of chromosomal imbalance in carriers of segmental autosomal mosaicism: differential impact of gender

Mosaicism for unbalanced chromosomal rearrangements segmental mosaicism (SM) is rare, both in patients referred for cytogenetic testing and in prenatal diagnoses. In contrast, in preimplantation embryos SM is a frequent finding and, therefore, is even more challenging. However, there is no consistency among results of published studies on the clinical outcomes of embryos with SM, primarily due to the small number of reported cases. Moreover, there is the problem of predicting the potential for the optimal development of a mosaic embryo to a healthy individual. Therefore, we suggested comparing factors predisposing to favorable and poor prognoses, identified in postnatal and prenatal cohorts of SM carriers, with those obtained from studies on preimplantation embryos. We analyzed 580 published cases of SM including (i) postnatally diagnosed affected carriers, (ii) clinically asymptomatic carriers, (iii) prenatally diagnosed carriers, and (iv) miscarriages. We observed a concordance with preimplantation diagnoses regarding the clinical significance of the extent of mosaicism as well as a predominance of deletions over other types of rearrangements. However, there is no concordance regarding excessive involvement of chromosomes 1, 5, and 9 in unbalanced rearrangements and a preferential involvement of larger chromosomes compared to short ones. Paternal age was not found to be associated with SM in postnatally disease-defined individuals. We have identified maternal age and preferential involvement of chromosome 18 in rearrangements associated with clinical manifestations. Male predominance was found among normal pregnancy outcomes and among disease-defined carriers of rearrangements resulting in a gain of genomic material. Female predominance was found among abnormal pregnancy outcomes, among disease-defined carriers of loss and gain/loss rearrangements, and among transmitting carriers of gonadal SM, both affected and asymptomatic. According to data obtained from “post-embryo” studies, clinical manifestations of chromosomal imbalance are associated with a high proportion of abnormal cells, female gender, the type of rearrangement and involved chromosome(s), and maternal age. We believe these data are instructive in the challenging medical genetic counseling of parents faced with no option other than transfer of an embryo with segmental mosaicism.

A clinical case of severe Duchenne muscular dystrophy caused by a nonsense mutation in the DMD gene in a girl

Duchenne muscular dystrophy (DMD) is a hereditary progressive muscular dystrophy, mainly manifested in boys, is characterized by the onset at an early age, gradual symmetrical atrophy of the striated musculature of the limbs, trunk, as well as damage to the heart muscle. As a rule, girls and women inheriting a pathological mutation are classified only as its carriers and do not have clinical manifestations of the disease. Rare cases when women or girls show clinical manifestations of DMD may be due to chromosomal rearrangements involving the region of the short arm of the X chromosome (Xp21.2), deletions of this region, complete loss of the X chromosome (Shereshevsky-Turner syndrome), homogenous X chromosome dysomnia, compound heterozygous state for two pathogenic mutations in the DMD gene, nonequilibrium inactivation of the X chromosome. When female mutation carriers have DMD clinical symptoms, they usually manifest much milder than boys and young males. Descriptions of patients with the severe course and rapid progression of the disease, comparable in the rate of progression with boys, are rare. In this article, the authors share their experience of observing a girl patient who suffered from a severe form of DMD.

Dissecting supergene: evidence for recombination suppression among multiple functional loci within inversions.

Supergenes are genetic architectures associated with discrete and concerted variation in multiple traits. It has long been suggested that supergenes control these complex polymorphisms by suppressing recombination between set of coadapted genes. However, because recombination suppression hinders the dissociation of the individual effects of genes within supergenes, there is still little evidence that supergenes evolve by tightening linkage between coadapted genes. Here, combining an landmark-free phenotyping algorithm with multivariate genome wide association studies, we dissected the genetic basis of wing pattern variation in the butterfly Heliconius numata. We showed that the supergene controlling the striking wing-pattern polymorphism displayed by this species contains many independent loci associated with different features of wing patterns. The three chromosomal inversions of this supergene suppress recombination between these loci, supporting the hypothesis that they may have evolved because they captured beneficial combinations of alleles. Some of these loci are associated with colour variations only in morphs controlled by inversions, indicating that they were recruited after the formation of these inversions. Our study shows that supergenes and clusters of adaptive loci in general may form via the evolution of chromosomal rearrangements suppressing recombination between co-adapted loci but also via the subsequent recruitment of linked adaptive mutations.

Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3–5 base editing windows 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

Phenylalanine Ammonia-Lyase (PAL) Genes Family in Wheat (Triticum aestivum L.): Genome-Wide Characterization and Expression Profiling

Phenylalanine ammonia-lyase (PAL) is the first enzyme in the phenylpropanoid pathway and plays a vital role in adoption, growth, and development in plants but in wheat its characterization is still not very clear. Here, we report a genome-wide identification of TaPAL genes and analysis of their transcriptional expression, duplication, and phylogeny in wheat. A total of 37 TaPAL genes that cluster into three subfamilies have been identified based on phylogenetic analysis. These TaPAL genes are distributed on 1A, 1B, 1D, 2A, 2B, 2D, 4A, 5B, 6A, 6B, and 6D chromosomes. Gene structure, conserved domain analysis, and investigation of cis-regulatory elements were systematically carried out. Chromosomal rearrangements and gene loss were observed by evolutionary analysis of the orthologs among Triticum urartu, Aegilops tauschii, and Triticum aestivum during the origin of bread wheat. Gene ontology analysis revealed that PAL genes play a role in plant growth. We also identified 27 putative miRNAs targeting 37 TaPAL genes. The high expression level of PAL genes was detected in roots of drought-tolerant genotypes compared to drought-sensitive genotypes. However, very low expressions of TaPAL10, TaPAL30, TaPAL32, TaPAL3, and TaPAL28 were recorded in all wheat genotypes. Arogenate dehydratase interacts with TaPAL29 and has higher expression in roots. The analysis of all identified genes in RNA-seq data showed that they are expressed in roots and shoots under normal and abiotic stress. Our study offers valuable data on the functioning of PAL genes in wheat.

Comparison of Karyotypes in Two Hybridizing Passerine Species: Conserved Chromosomal Structure but Divergence in Centromeric Repeats

Changes in chromosomal structure involving chromosomal rearrangements or copy number variation of specific sequences can play an important role in speciation. Here, we explored the chromosomal structure of two hybridizing passerine species; the common nightingale (Luscinia megarhynchos) and the thrush nightingale (Luscinia luscinia), using conventional cytogenetic approaches, immunostaining of meiotic chromosomes, fluorescence in situ hybridization as well as comparative genomic hybridization (CGH). We found that the two nightingale species show conserved karyotypes with the same diploid chromosome number of 2n = 84. In addition to standard chromosomes, both species possessed a small germline restricted chromosome of similar size as a microchromosome. Just a few subtle changes in chromosome morphology were observed between the species, suggesting that only a limited number of chromosomal rearrangements occurred after the species divergence. The interspecific CGH experiment suggested that the two nightingale species might have diverged in centromeric repetitive sequences in most macro- and microchromosomes. In addition, some chromosomes showed changes in copy number of centromeric repeats between the species. The observation of very similar karyotypes in the two nightingale species is consistent with a generally slow rate of karyotype evolution in birds. The divergence of centromeric sequences between the two species could theoretically cause meiotic drive or reduced fertility in interspecific hybrids. Nevertheless, further studies are needed to evaluate the potential role of chromosomal structural variations in nightingale speciation.

Recurrent erosion of COA1/MITRAC15 exemplifies conditional gene dispensability in oxidative phosphorylation

Skeletal muscle fibers rely upon either oxidative phosphorylation or the glycolytic pathway with much less reliance on oxidative phosphorylation to achieve muscular contractions that power mechanical movements. Species with energy-intensive adaptive traits that require sudden bursts of energy have a greater dependency on glycolytic fibers. Glycolytic fibers have decreased reliance on OXPHOS and lower mitochondrial content compared to oxidative fibers. Hence, we hypothesized that gene loss might have occurred within the OXPHOS pathway in lineages that largely depend on glycolytic fibers. The protein encoded by the COA1/MITRAC15 gene with conserved orthologs found in budding yeast to humans promotes mitochondrial translation. We show that gene disrupting mutations have accumulated within the COA1 gene in the cheetah, several species of galliform birds, and rodents. The genomic region containing COA1 is a well-established evolutionary breakpoint region in mammals. Careful inspection of genome assemblies of closely related species of rodents and marsupials suggests two independent COA1 gene loss events co-occurring with chromosomal rearrangements. Besides recurrent gene loss events, we document changes in COA1 exon structure in primates and felids. The detailed evolutionary history presented in this study reveals the intricate link between skeletal muscle fiber composition and the occasional dispensability of the chaperone-like role of the COA1 gene.

Combination of Antiretroviral Drugs Zidovudine and Efavirenz Impairs Tumor Growths in a Mouse Model of Cancer

LINE1 retrotransposons, which are thought to be the remnants of ancient integrations of retrovirus-like elements, are aberrantly (re)activated in many cancer cells. Due to LINE1-induced alterations in target gene expression and/or chromosomal rearrangements, they may be important drivers of tumorigenesis. Moreover, LINE1 encoded proteins, Open Reading Frame (ORF)1 and ORF2, may have pro-oncogenic potential through inductors of oncogenic transcription factors or inhibitors of cell cycle suppressors. The current study therefore aimed to investigate in vitro and in vivo anti-tumorigenic effects of two well-known antiretroviral drugs, zidovudine, a nucleoside analogue inhibitor of RT (NRTI), and efavirenz, a non-nucleoside RT inhibitor (NNRTI). Our data demonstrate that both drugs in clinically relevant doses significantly reduced the proliferation of murine and human cancer cell lines, as well as growth of tumors in a murine subcutaneous model. Intriguingly, we found that the combination of both zidovudine and efavirenz almost entirely blocked tumorigenesis in vivo. Because both drugs are FDA-approved agents and the combination was very well tolerated in mice, the combination therapy as presented in our paper might be an opportunity to treat colorectal tumors and metastasis to the liver in an inexpensive way.