ZMAT2 in Humans and Other Primates: A Highly Conserved and Understudied Gene.

Recent advances in genetics present unique opportunities for enhancing our understanding of human physiology and disease predisposition through detailed analysis of gene structure, expression, and population variation via examination of data in publicly accessible genome and gene expression repositories. Yet, the vast majority of human genes remain understudied. Here, we show the scope of these genomic and genetic resources by evaluating ZMAT2, a member of a 5-gene family that through May 2020 had been the focus of only 4 peer-reviewed scientific publications. Using analysis of information extracted from public databases, we show that human ZMAT2 is a 6-exon gene and find that it exhibits minimal genetic variation in human populations and in disease states, including cancer. We further demonstrate that the gene and its encoded protein are highly conserved among nonhuman primates and define a cohort of ZMAT2 pseudogenes in the marmoset genome. Collectively, our investigations illustrate how complementary use of genomic, gene expression, and population genetic resources can lead to new insights about human and mammalian biology and evolution, and when coupled with data supporting key roles for ZMAT2 in keratinocyte differentiation and pre-RNA splicing argue that this gene is worthy of further study.

The Zmat2 gene in non-mammalian vertebrates: Organizational simplicity within a divergent locus in fish.

ZMAT2 is among the least-studied of mammalian proteins and genes, even though it is the ortholog of Snu23, a protein involved in pre-mRNA splicing in yeast. Here we have used data from genomic and gene expression repositories to examine the Zmat2 gene and locus in 8 terrestrial vertebrates, 10 ray-finned fish, and 1 lobe-finned fish representing > 500 million years of evolutionary diversification. The analyses revealed that vertebrate Zmat2 genes are similar to their mammalian counterparts, as in 16/19 species studied they contain 6 exons, and in 18/19 encode a single conserved protein. However, unlike in mammals, no Zmat2 pseudogenes were identified in these vertebrates, although an expressed Zmat2 paralog was characterized in flycatcher that resembled a DNA copy of a processed and retro-transposed mRNA, and thus could be a proto-pseudogene captured during its evolutionary journey from active to inert. The Zmat2 locus in terrestrial vertebrates, and in spotted gar and coelacanth, also shares additional genes with its mammalian counterparts, including Histidyl-tRNA synthetase (Hars), Hars2, and others, but these are absent from the Zmat2 locus in teleost fish, in which Stem-loop-binding protein 2 (Slbp2) and Lymphocyte cytosolic protein 2a (Lcp2a) are present instead. Taken together, these observations argue that a recognizable Zmat2 was present in the earliest vertebrate ancestors, and postulate that during chromosomal tetraploidization and subsequent re-diploidization during modern teleost evolution, the duplicated Zmat2 gene was retained and the original lost. This study also highlights how information from genomic resources can be leveraged to reveal new biologically significant insights.

Revisiting the Population Genetics of Human Height.

CONTEXT: Recent advances in genetics and genomics present unique opportunities for enhancing knowledge of human physiology and disease susceptibility. An outstanding example of these new insights may be seen in the study of human height, of which it has been estimated that approximately 80% is genetically determined. Over the past decade, large-scale population analyses have led to the identification of novel variation in genes and loci individually associated with changes in adult height of as much as 2 cm. OBJECTIVE: To assess these same variants in the genomes of 213 158 individuals compiled by the Genome Aggregation Database (GnomAD) consortium, representing different population groups from around the world. RESULTS: The majority of these height-changing alleles are substantially less prevalent in GnomAD than found previously in other cohorts, with 4 of 5 amino acid substitution variants with the largest impact on adult height being more frequent in the European population than in other groups. CONCLUSIONS: A larger-scale analysis of individuals from diverse backgrounds will be necessary to ensure a full and accurate understanding of the genetic underpinnings of human height throughout the world, and additional studies will be needed to discern the biochemical and molecular mechanisms governing the physiological processes that explain how these variant proteins might selectively impact the biology of the growth plate. Broader understanding of the genetics of height also should set the stage for more comprehensive investigation into the causes of prevalent polygenic human diseases.

Structure and expression of the long noncoding RNA gene MIR503 in humans and non-human primates.

Recent technical and other advances in genomics provide unique opportunities to improve our understanding of human physiology and disease predisposition through a detailed analysis of gene structure and expression by examining data in public genome and gene-expression repositories. Yet, the vast majority of human genes remain understudied. This is particularly true of genes for long noncoding RNAs (lncRNAs). Here, we describe the detailed characterization of MIR503HG, a lncRNA gene found on the X chromosome in humans. Using information extracted from public databases, we show that human MIR503HG is a 5-exon gene, and that it is highly conserved among 5 non-human primates spanning over 85 million years ago of evolutionary diversification. MIR503HG is transcribed and processed into multiple distinct RNAs in each of these species through differential exon use and alternative RNA splicing, with a higher abundance of transcripts being found in reproductive tissues, especially during the early stages of ovary and testis development, indicating a possible role in reproductive biology. Furthermore, in select reproductive system cancers, MIR503HG transcripts are downregulated, with higher levels of RNA expression being associated with clinical outcomes. Collectively, these investigations show how the use of genomic, gene expression, and other genetic resources can lead to new insights about human biology and disease, and argue that MIR503HG is worthy of additional study.

Regulation of gene expression by growth hormone.

Growth hormone (GH) plays a pivotal role in many physiological processes in humans, and in other mammalian and non-mammalian vertebrate species, through actions on somatic growth, tissue development and repair, and intermediary metabolism. This review will focus on mechanisms of GH actions on gene expression, primarily from the perspective of the genes that encode proteins stimulated by GH to regulate somatic growth, especially insulin-like growth factor 1 (IGF-I), but also others that are induced or repressed by GH. Topics to be discussed will include a brief overview of GH-mediated signal transduction pathways and how these cascades alter the functions of responsive transcription factors, with a specific focus on STAT5B, a key member of the signal transducers and activators of transcription family, characterization of essential GH-regulated genes, and elucidation of mechanisms of their regulation from biochemical, genetic, and genomic perspectives.

Zmat2 in mammals: conservation and diversification among genes and Pseudogenes.

BACKGROUND: Recent advances in genetics and genomics present unique opportunities for enhancing our understanding of mammalian biology and evolution through detailed multi-species comparative analysis of gene organization and expression. Yet, of the more than 20,000 protein coding genes found in mammalian genomes, fewer than 10% have been examined in any detail. Here we elucidate the power of data available in publicly-accessible genomic and genetic resources by querying them to evaluate Zmat2, a minimally studied gene whose human ortholog has been implicated in spliceosome function and in keratinocyte differentiation. RESULTS: We find extensive conservation in coding regions and overall structure of Zmat2 in 18 mammals representing 13 orders and spanning ~ 165 million years of evolutionary development, and in their encoded proteins. We identify a tandem duplication in the Zmat2 gene and locus in opossum, but not in other monotremes, marsupials, or other mammals, indicating that this event occurred subsequent to the divergence of these species from one another. We also define a collection of Zmat2 pseudogenes in half of the mammals studied, and suggest based on phylogenetic analysis that they each arose independently in the recent evolutionary past. CONCLUSIONS: Mammalian Zmat2 genes and ZMAT2 proteins illustrate conservation of structure and sequence, along with the development and diversification of pseudogenes in a large fraction of species. Collectively, these observations also illustrate how the focused identification and interpretation of data found in public genomic and gene expression resources can be leveraged to reveal new insights of potentially high biological significance.

The insulin-like growth factor 2 gene in mammals: Organizational complexity within a conserved locus.

The secreted protein, insulin-like growth factor 2 (IGF2), plays a central role in fetal and prenatal growth and development, and is regulated at the genetic level by parental imprinting, being expressed predominantly from the paternally derived chromosome in mice and humans. Here, IGF2/Igf2 and its locus has been examined in 19 mammals from 13 orders spanning ~166 million years of evolutionary development. By using human or mouse DNA segments as queries in genome analyses, and by assessing gene expression using RNA-sequencing libraries, more complexity was identified within IGF2/Igf2 than was annotated previously. Multiple potential 5' non-coding exons were mapped in most mammals and are presumably linked to distinct IGF2/Igf2 promoters, as shown for several species by interrogating RNA-sequencing libraries. DNA similarity was highest in IGF2/Igf2 coding exons; yet, even though the mature IGF2 protein was conserved, versions of 67 or 70 residues are produced secondary to species-specific maintenance of alternative RNA splicing at a variable intron-exon junction. Adjacent H19 was more divergent than IGF2/Igf2, as expected in a gene for a noncoding RNA, and was identified in only 10/19 species. These results show that common features, including those defining IGF2/Igf2 coding and several non-coding exons, were likely present at the onset of the mammalian radiation, but that others, such as a putative imprinting control region 5' to H19 and potential enhancer elements 3' to H19, diversified with speciation. This study also demonstrates that careful analysis of genomic and gene expression repositories can provide new insights into gene structure and regulation.

Characterizing the complexity of Australian marsupial insulin-like growth factor 1 genes.

Insulin-like growth factor 1 (IGF1) actions are essential for somatic growth and tissue repair. IGF1 gene regulation is controlled by many inputs, with growth hormone playing a major role. In most mammals, the 6-exon IGF1/Igf1 gene produces multiple transcripts via independent activity of its promoters plus alternative RNA splicing and differential polyadenylation. Here, by analyzing public genomic and RNA-sequencing repositories, I have characterized three Australian marsupial IGF1 genes. Koala, Tasmanian devil, and wallaby IGF1 are more complicated than other mammals, as they contain up to 11 exons, and encode multiple mRNAs and predicted protein precursors, including potentially novel isoforms. Moreover, just two of multiple growth hormone-stimulated transcriptional enhancers found in other IGF1/Igf1 loci are detected in these species. These observations define Australian marsupial IGF1 genes and demonstrate that comprehensive interrogation of genomic and RNA-sequencing resources is an effective strategy for characterizing genes and gene expression in otherwise experimentally intractable organisms.

Variation in the repulsive guidance molecule family in human populations.

Repulsive guidance molecules, RGMA, RGMB, and RGMC, are related proteins discovered independently through different experimental paradigms. They are encoded by single copy genes in mammalian and other vertebrate genomes, and are ~50% identical in amino acid sequence. The importance of RGM actions in human physiology has not been realized, as most research has focused on non-human models, although mutations in RGMC are the cause of the severe iron storage disorder, juvenile hemochromatosis. Here I show that repositories of human genomic and population genetic data can be used as starting points for discovery and for developing new testable hypotheses about each of these paralogs in human biology and disease susceptibility. Information was extracted, aggregated, and analyzed from the Ensembl and UCSC Genome Browsers, the Exome Aggregation Consortium, the Genotype-Tissue Expression project portal, the cBio portal for Cancer Genomics, and the National Cancer Institute Genomic Data Commons data site. Results identify extensive variation in gene expression patterns, substantial alternative RNA splicing, and possible missense alterations and other modifications in the coding regions of each of the three genes, with many putative mutations being detected in individuals with different types of cancers. Moreover, selected amino acid substitutions are highly prevalent in the world population, with minor allele frequencies of up to 37% for RGMA and up to 8% for RGMB. These results indicate that protein sequence variation is common in the human RGM family, and raises the possibility that individual variants will have a significant population impact on human physiology and/or disease predisposition.

Quantifying promoter-specific Insulin-like Growth Factor 1 gene expression by interrogating public databases.

The actions of insulin-like growth factor 1 (IGF1), a small, secreted protein, are essential for normal somatic growth in children and are important for tissue regeneration and repair in adults. Similar functions are conserved in other mammalian species. IGF1 gene regulation is complicated in mammals, with transcription being controlled by different hormonal, nutritional, and tissue-specific inputs. Quantifying IGF1 gene expression in different organs and tissues also has been difficult because of the variable contributions of its two promoters and because of the lack of standard platforms for analysis. Here, I have taken advantage of the wealth of information found in publicly accessible RNA-sequencing libraries to measure steady-state levels of IGF1 mRNAs from human and macaque, species chosen because they are not readily tractable experimental organisms, yet retain similar IGF1 gene organization. Results demonstrate that IGF1 transcripts are highly expressed in fat and liver in both species, and are induced during human adipocyte differentiation. IGF1 mRNAs also are increased in macaque skeletal muscle after selected dietary manipulations. In the organs and tissues examined, IGF1 promoter 1 appears to be far more active than promoter 2. Collectively, these observations show that interrogating large-scale public genomic resources is an effective strategy for quantifying gene expression across different tissues and species.

Gene Mapping by RNA-sequencing: A Direct Way to Characterize Genes and Gene Expression through Targeted Queries of Large Public Databases.

Recent advances in genomics present new opportunities for enhancing knowledge about gene regulation and function across a wide spectrum of organisms and species. Understanding and evaluating this information at the individual gene level is challenging, and not only requires extracting, collating and interpreting data from public genetic repositories, but also recognizing that much of the information has been developed through implementation of computationally based exon-calling algorithms, and thus may be inaccurate. Moreover, as these data usually have not been validated experimentally, results also may be incomplete and incorrect. This has created a quality-control problem for scientists who want to use individual gene-specific information in their research. Here, I describe a simple experimental strategy that takes advantage of the large amounts of untapped primary experimental data for characterizing gene expression that have been deposited in the Sequence Read Archive of the National Center for Biotechnology Information. The approach consists of a readily adaptable pipeline that may be used to confirm exons, to define 5' and 3' un-translated regions and the beginnings and ends of individual genes, and to quantify alternative RNA splicing. The series of experimental strategies described offers effective replacements for older molecular biological methods, and can rapidly and reproducibly resolve major gene mapping problems.

The insulin-like growth factor 2 gene and locus in nonmammalian vertebrates: Organizational simplicity with duplication but limited divergence in fish.

The small, secreted peptide, insulin-like growth factor 2 (IGF2), is essential for fetal and prenatal growth in humans and other mammals. Human IGF2 and mouse Igf2 genes are located within a conserved linkage group and are regulated by parental imprinting, with IGF2/Igf2 being expressed from the paternally derived chromosome, and H19 from the maternal chromosome. Here, data retrieved from genomic and gene expression repositories were used to examine the Igf2 gene and locus in 8 terrestrial vertebrates, 11 ray-finned fish, and 1 lobe-finned fish representing >500 million years of evolutionary diversification. The analysis revealed that vertebrate Igf2 genes are simpler than their mammalian counterparts, having fewer exons and lacking multiple gene promoters. Igf2 genes are conserved among these species, especially in protein-coding regions, and IGF2 proteins also are conserved, although less so in fish than in terrestrial vertebrates. The Igf2 locus in terrestrial vertebrates shares additional genes with its mammalian counterparts, including tyrosine hydroxylase (Th), insulin (Ins), mitochondrial ribosomal protein L23 (Mrpl23), and troponin T3, fast skeletal type (Tnnt3), and both Th and Mrpl23 are present in the Igf2 locus in fish. Taken together, these observations support the idea that a recognizable Igf2 was present in the earliest vertebrate ancestors, but that other features developed and diversified in the gene and locus with speciation, especially in mammals. This study also highlights the need for correcting inaccuracies in genome databases to maximize our ability to accurately assess contributions of individual genes and multigene families toward evolution, physiology, and disease.

Insulinlike Growth Factor 1 Gene Variation in Vertebrates.

IGF1-a small, single-chain, secreted peptide in mammals-is essential for normal somatic growth and is involved in a variety of other physiological and pathophysiological processes. IGF1 expression appears to be controlled by several different signaling mechanisms in mammals, with GH playing a key role by activating an inducible transcriptional pathway via the Jak2 protein kinase and the Stat5b transcription factor. Here, to understand aspects of Igf1 gene regulation over a substantially longer timeline than is discernible in mammals, Igf1 genes have been examined in 21 different nonmammalian vertebrates representing five different classes and ranging over ∼500 million years of evolutionary history. Parts of vertebrate Igf1 genes resemble components found in mammals. Conserved exons encoding the mature IGF1 protein are detected in all 21 species studied and are separated by a large intron, as seen in mammals; the single promoter contains putative regulatory elements that are similar to those functionally mapped in human IGF1 promoter 1. In contrast, GH-activated Stat5b-binding enhancers found in mammalian IGF1 loci are completely absent, there is no homolog of promoter 2 or exon 2 in any nonmammalian vertebrate, and different types of "extra" exons not present in mammals are found in birds, reptiles, and teleosts. These data collectively define properties of Igf1 genes and IGF1 proteins that were likely present in the earliest vertebrates and support the contention that common structural and regulatory features in Igf1 genes have a long evolutionary history.

Similarity and variation in the insulin-like growth factor 2 - H19 locus in primates.

Insulin-like growth factor 2 (IGF2), a small, secreted protein, is critical for fetal and prenatal growth in humans and other mammals. The IGF2 gene and its mouse homolog comprise part of a conserved linkage group that is regulated by parental imprinting, with IGF2/ Igf2 being expressed from the paternal chromosome, and the adjacent H19 gene from the maternal chromosome. By using information extracted from public genomic and gene expression databases, I have now analyzed this locus in nine nonhuman primate species representing over 60 million years of evolutionary divergence from a common progenitor. Both IGF2 and H19 genes and the entire locus have been conserved among these primates. Each primate IGF2 gene except for gibbon and marmoset is composed of 10 exons and contains five potential promoters, each with distinctive 5'-untranslated exons. Similarly, except for marmoset and mouse lemur, H19 consists of six exons and has two promoters. DNA sequence conservation is high, not only in orthologous exons and promoters, but also in a putative imprinting control region located 5' to H19 and in multiple potential distal enhancer elements found 3' to H19. Collectively, these results support the hypothesis that common regulatory processes shaped the IGF2 - H19 locus before the onset of primate speciation more than 85 million years ago. This study also leads to the conclusion that inaccuracies in data presentation in genetic repositories could limit our ability to develop novel insights about roles of individual genes and multigene loci in mammalian physiology and disease.

The complex genetics of human insulin-like growth factor 2 are not reflected in public databases.

Recent advances in genetics present unique opportunities for enhancing knowledge about human physiology and disease susceptibility. Understanding this information at the individual gene level is challenging and requires extracting, collating, and interpreting data from a variety of public gene repositories. Here, I illustrate this challenge by analyzing the gene for human insulin-like growth factor 2 (IGF2) through the lens of several databases. IGF2, a 67-amino acid secreted peptide, is essential for normal prenatal growth and is involved in other physiological and pathophysiological processes in humans. Surprisingly, none of the genetic databases accurately described or completely delineated human IGF2 gene structure or transcript expression, even though all relevant information could be found in the published literature. Although IGF2 shares multiple features with the mouse Igf2 gene, it has several unique properties, including transcription from five promoters. Both genes undergo parental imprinting, with IGF2/Igf2 being expressed primarily from the paternal chromosome and the adjacent H19 gene from the maternal chromosome. Unlike mouse Igf2, whose expression declines after birth, human IGF2 remains active throughout life. This characteristic has been attributed to a unique human gene promoter that escapes imprinting, but as shown here, it involves several different promoters with distinct tissue-specific expression patterns. Because new testable hypotheses could lead to critical insights into IGF2 actions in human physiology and disease, it is incumbent that our fundamental understanding is accurate. Similar challenges affecting knowledge of other human genes should promote attempts to critically evaluate, interpret, and correct human genetic data in publicly available databases.

Diversification of the insulin-like growth factor 1 gene in mammals.

Insulin-like growth factor 1 (IGF1), a small, secreted peptide growth factor, is involved in a variety of physiological and patho-physiological processes, including somatic growth, tissue repair, and metabolism of carbohydrates, proteins, and lipids. IGF1 gene expression appears to be controlled by several different signaling cascades in the few species in which it has been evaluated, with growth hormone playing a major role by activating a pathway involving the Stat5b transcription factor. Here, genes encoding IGF1 have been evaluated in 25 different mammalian species representing 15 different orders and ranging over ~180 million years of evolutionary diversification. Parts of the IGF1 gene have been fairly well conserved. Like rat Igf1 and human IGF1, 21 of 23 other genes are composed of 6 exons and 5 introns, and all 23 also contain recognizable tandem promoters, each with a unique leader exon. Exon and intron lengths are similar in most species, and DNA sequence conservation is moderately high in orthologous exons and proximal promoter regions. In contrast, putative growth hormone-activated Stat5b-binding enhancers found in analogous locations in rodent Igf1 and in human IGF1 loci, have undergone substantial variation in other mammals, and a processed retro-transposed IGF1 pseudogene is found in the sloth locus, but not in other mammalian genomes. Taken together, the fairly high level of organizational and nucleotide sequence similarity in the IGF1 gene among these 25 species supports the contention that some common regulatory pathways had existed prior to the beginning of mammalian speciation.

Variation in Akt protein kinases in human populations.

The three Akt kinases are related proteins that are essential for normal growth and metabolic regulation and are implicated as key signaling mediators in many physiological and pathophysiological processes. Each Akt is activated by common biochemical signals that act downstream of growth factor and hormone receptors via phosphatidylinositol-3 kinase, and each controls several downstream pathways. The importance of Akt actions in human physiology is strengthened by the rarity of modifying mutations in their genes and by the devastating impact caused by these mutations on growth and development and in disorders such as cancer. Recent advances in genomics present unique opportunities for enhancing our understanding of human physiology and disease predisposition through the lens of population genetics, and the availability of DNA sequence data from 60,706 people in the Exome Aggregation Consortium has prompted this analysis. Results reveal a cohort of potential missense and other alterations in the coding regions of each AKT gene, but with nearly all changes being uncommon. The total number of different alleles per gene varied over an approximately threefold range, from 52 for AKT3 to 158 for AKT2, with variants distributed throughout all Akt protein domains. Previously characterized disease-causing mutations were found rarely in the general population. In contrast, a fairly prevalent amino acid substitution in AKT2 appears to be linked to increased predisposition for type 2 diabetes. Further analysis of variant Akt molecules as identified here will provide opportunities to understand the intricacies of Akt signaling and actions at a population level in human physiology and pathology.

The New Genomics: What Molecular Databases Can Tell Us About Human Population Variation and Endocrine Disease.

Major recent advances in genetics and genomics present unique opportunities for enhancing our understanding of human physiology and disease predisposition. Here I demonstrate how analysis of genomic information can provide new insights into endocrine systems, using the human growth hormone (GH) signaling pathway as an illustrative example. GH is essential for normal postnatal growth in children, and plays important roles in other biological processes throughout life. GH actions are mediated by the GH receptor, primarily via the JAK2 protein tyrosine kinase and the STAT5B transcription factor, and inactivating mutations in this pathway all lead to impaired somatic growth. Variation in GH signaling genes has been evaluated using DNA sequence data from the Exome Aggregation Consortium, a compendium of information from >60,000 individuals. Results reveal many potential missense and other alterations in the coding regions of GH1, GHR, JAK2, and STAT5B, with most changes being uncommon. The total number of different alleles per gene varied by ~threefold, from 101 for GH1 to 338 for JAK2. Several known disease-linked mutations in GH1, GHR, and JAK2 were present but infrequent in the population; however, three amino acid changes in GHR were sufficiently prevalent (~4% to 44% of chromosomes) to suggest that they are not disease causing. Collectively, these data provide new opportunities to understand how genetically driven variability in GH signaling and action may modify human physiology and disease.

Large-scale analysis of variation in the insulin-like growth factor family in humans reveals rare disease links and common polymorphisms.

The insulin-like growth factors IGF1 and IGF2 are closely related proteins that are essential for normal growth and development in humans and other species and play critical roles in many physiological and pathophysiological processes. IGF actions are mediated by transmembrane receptors and modulated by IGF-binding proteins. The importance of IGF actions in human physiology is strengthened by the rarity of inactivating mutations in their genes and by the devastating impact caused by such mutations on normal development and somatic growth. Large-scale genome sequencing has the potential to provide new insights into human variation and disease susceptibility. Toward this end, the availability of DNA sequence data from 60,706 people through the Exome Aggregation Consortium has prompted the analyses presented here. Results reveal a broad range of potential missense and other alterations in the coding regions of every IGF family gene, but the vast majority of predicted changes were uncommon. The total number of different alleles detected per gene in the population varied over an ∼15-fold range, from 57 for IGF1 to 872 for IGF2R, although when corrected for protein length the rate ranged from 0.22 to 0.59 changes/codon among the 11 genes evaluated. Previously characterized disease-causing mutations in IGF2, IGF1R, IGF2R, or IGFALS all were found in the general population but with allele frequencies of <1:30,000. A few new highly prevalent amino acid polymorphisms were also identified. Collectively, these data provide a wealth of opportunities to understand the intricacies of IGF signaling and action in both physiological and pathological contexts.