MENU- THE PIG PROTEOME
- THE PIG ATLAS
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Pigs in Biomedical ResearchPigs are one of the most attractive large animal models for biomedical research and preclinical toxicologic testing of pharmaceuticals. They are similar to humans in metabolism, physiology and anatomy. The first draft of pig reference genome was completed in 2012 by the Swine Genome Sequencing Consortium (SGSC) Groenen MA et al. (2012). The genome-wide mapping of gene expression in all pig tissues and organ is essential for the broad utility of pigs in biomedical research and pharmaceutical applications. Examples of pigs used in biomedical research include human disease modeling and organ transplantation. As part of the Pig RNA Atlas, we compare expression specificity of 3,432 or 3,695 disease-associated genes between pig and human. These genes are implicated in 1,894 human diseases, grouped into 69 disease types and 14 disease systems, including nervous system (850 genes), endocrine and metabolic system (214 genes), respiratory system (50 genes), cardiovascular system (310 genes), musculoskeletal system (145 genes), digestive system (80 genes), urinary system (65 genes), reproductive system (75 genes), skin (95 genes), immune system (270 genes), congenital disorders of metabolism (658 genes), congenital malformations (912 genes), other congenital disorders (80 genes), and cancers (218 genes). The comparative disease gene analysis can facilitate the utility of pigs in biomedical research and disease modeling in the future. For example, alteration of the NPPA gene expression is linked to the development of type 6 atrial fibrillation (AF), a cardiovascular disease that affects over 30 million individuals worldwide. NPPA is specifically expressed in both human and pig heart atrium and also validated by antibody-based tissue profiling (IHC image of similar). The highly conserved expression between pig and human suggesting that pigs could be used as an excellent model for AF6 modeling and development of treatments. Nervous systemNervous system diseases include neurodegenerative diseases, epilepsy, eye diseases, ear diseases, and other sensory system diseases. The Pig Atlas contains 850 genes associated to nervous system diseases. Genetic mutations play an important role in the development of nervous system diseases, exemplified by the HTT gene, which has maximum expression in the brain of both pig and human. HTT Pig models have been successfully generated to recapitulate the pathogenesis of Huntington’s Disease in humans. Another example of nervous system associated disease genes are genes involved in retinitis pigmentosa, such as the rhodopsin gene (RHO) and retinol-binding protein 3 gene (RBP3). The expression specificity of these two genes is conserved between humans and pigs, and enriched in the eye. Endocrine and metabolic systemEndocrine and metabolic diseases are disorders that affect the endocrine system and disrupt metabolism in tissues such as pineal gland, pituitary gland, pancreas, ovaries, testes, thyroid gland, parathyroid gland, hypothalamus and adrenal glands. The Pig Atlas includes 214 genes related to diseases of the endocrine and metabolic system. Examples of such diseases include diabetes, hypothalamus and pituitary gland diseases, thyroid gland diseases, adrenal gland diseases, parathyroid diseases and gonadal diseases. For example, the cholesterol side-chain cleavage enzyme, encoded by gene CYP11A1, converts cholesterol to pregnenolone and is expressed in adrenal cortex, as well as steroidogenic tissues such as testis and ovary. Mutations of CYP11A1 have previously been linked to the development of adrenal insufficiency (al Kandari H et al. (2006)). The expression profile is similar between pigs and human with highest expression in the adrenal gland. Respiratory systemRespiratory diseases are pathological conditions that affect the respiratory tracts such as the lung, trachea and bronchi. The development of respiratory diseases can be due to environmental and/or genetic factors. The Pig RNA Atlas includes the comparison of 50 human respiratory disease-causing genes. Examples include the SFTPB and SFTPC genes, for which the expression is highly enriched in the lung in both pig and human. SFTPB and SFTPC are expressed by alveolar type II cells and are associated with the development of Congenital pulmonary alveolar proteinosis. Cardiovascular systemCardiovascular diseases (CVD) are diseases involving the heart and blood vasculature system, and is the leading cause of death worldwide. Both genetic and environmental factors are known to increase risk of developing CVD. The Pig Atlas includes 310 genes associated with CVD, involved in cardiac, vascular, hematologic and hypertensive diseases.
Immunohistochemical labeling targeting NPPA in pig tissues (from left to right: heart atrium, heart ventricle, skeletal muscle and skin) using HPA064007. An example is NPPA, a gene involved in atrial fibrillation (AF), a cardiovascular disease that affects over 30 million individuals worldwide. AF type 6 is caused by mutations of the NPPA gene, which is expressed primarily in atrial myocytes. The gene NPPA is specifically expressed in both human and pig heart muscle, and this is confirmed by antibody-based tissue profiling showing detection of NPPA exclusively in heart atrium of both species. Musculoskeletal systemMusculoskeletal diseases are disorders that affect the musculoskeletal systems, such as joints, bones, and muscles. More than 150 conditions have been reported and genetic risk factors have been found to be involved in the susceptibility and development of musculoskeletal diseases. The Pig RNA Atlas compared body wide expression specificity of 145 musculoskeletal disease genes between pig and human. For example, the skeletal muscle enriched gene KLHL40 is associated with the development of nemaline myopathy in human. Similar to human, the expression of KLHL40 is enriched in pig skeletal muscle. Another example is the filamin encoding gene FLNC, which is commonly enriched in the skeletal muscle of pig and human. The FLNC gene is associated with the development of distal myopathy in human. Digestive systemDigestive system diseases are disorders of the digestive system, including the mouth, dental system, gastrointestinal tract (GI), liver, pancreas, and gallbladder. 80 genes have been found with genetic perturbations and associated with the development of digestive system diseases. Examples of such genes include the progressive familial intrahepatic cholestasis associated gene NR1H4, which is expressed in the liver; the congenital diarrhea associated gene DGAT1 categorized as intestinal enriched; and the congenital bile acid synthesis defect associated gene ACOX2, which is liver enriched. Conserved expression specificity for all these three genes are found in both pig and human. Urinary systemUrinary system diseases are disorders affecting the urinary system, including the kidney and urinary bladder. The Pig RNA Atlas include body-wide expression comparison of 65 genes with genetic perturbations involving the development of urinary system diseases. One such example is the GPI anchored glycoprotein encoding gene UMOD which can cause autosomal dominant tubulointerstitial kidney disease in human. The expression of UMOD is elevated in the kidney of both pig and human. Reproductive systemReproductive system diseases are caused by disorders occurring in the reproductive systems, such as the endometrium, fallopian tube, ovary, bladder, penis, testis. The Pig RNA ATLAS include comparison of 75 reproductive system disease genes between pig and human. For example, the Phospholipase C Zeta 1 encoding gene PLCZ1 is associated with development of spermatogenic failure. The expression of PLCZ1 is enriched in testis in both pig and human. An example of species differences is the bone morphogenetic protein 15 (BMP15), which regulates the development of primordial follicles into pre-ovulatory follicles. BMP15 mutation in human is related to ovulation defects, and implicated in several reproductive diseases such as primary ovarian insufficiency and polycystic ovary syndrome. The expression profile of BMP15 in pig is restricted to the ovary, while human expression is not observed in ovary, but instead fallopian tube and testis. SkinSkin diseases are conditions that affect the functions and physiology of the skin. Many skin diseases are caused by genetic predispositions such as psoriasis and familial progressive hyperpigmentation. 95 genes associated with the development of skin diseases are included for this comparison. One example is the CARD14 gene, which encodes a caspase recruitment domain-containing protein 14, also known as CARD- containing MAGUK protein 2 (Carma2) involved in the development of psoriasis. The expression of CARD14 is enhanced in the skin of both pig and human. Immune systemThe Immune system diseases are disorders caused by dysregulated activity in the immune system (either low or overactive), including allergies, autoimmune disease, primary immunodeficiency and other immune system diseases. The lymphatic system is the major part of the immune system. The Pig RNA atlas includes five immune tissues: bone marrow and lymphoid tissues (lymph node, spleen, thymus and tonsil) and include 270 genes with evidence supporting their association with the development of immune diseases. Combined immunodeficiency is an immune disorder caused by multiple components of the immune system, and one well characterized genetic cause of this disease is autosomal recessive deficiency in the T lymphocyte-specific protein tyrosine kinase (LCK). LCK is a key component of the TCR signaling machinery Hauck F et al. (2012) and its expression is enriched in lymphoid tissues of both pig and human. Congenital disorders of metabolismCongenital disorders of metabolism, or inborn errors of metabolism, are rare genetic diseases present at birth and caused by defects in specific enzymes involved in the metabolism of nutritional molecules, such as carbohydrates, lipid/glycolipids, glycan/glycoproteins, amino acids, nucleotides and cofactor/vitamins. Defects in genes involved in the ion transport, lysosomal storage, peroxisome and mitochondria functions are also found to cause congenital disorders of metabolism. The Pig Atlas includes a collection of 658 genes associated with congenital disorders of metabolism. One example is the glycogen synthase GYS2 which causes hepatic glycogen storage disease. The expression of GYS2 is enriched in both pig and human liver. Another example where expression differs between pig and human is the moonlighting protein dicarbonyl and L-xylulose reductase (DCXR), which plays a role both in converting L-xylulose to xylitol, and breakdown of toxic compounds. In human, DCXR is highly expressed in liver, while pig shows highest expression in thyroid gland. Congenital malformationsCongenital malformations are physical defects present in babies at birth. The malformations can have a genetic cause and involve many different tissues/organs of the body. The Pig Atlas contains a collection of 912 genes, of which one is the MYBPC1 gene which is associated with malformations of the musculoskeletal system. This gene is also classified as skeletal muscle enriched in both pig and human. CancersCancer is caused by abnormal and uncontrollable cell growth and spreading into surrounding tissues. Genetic factors, such as mutations occurring in tumor suppressor genes and proto-oncogenes, play a major role in the development of cancers. Epigenetic and chromosomal alterations, which leads to expression changes in genes e.g. regulating cell proliferation, apoptosis, DNA damage repair, can also lead to the development and progression of cancers. The Pig Atlas includes a collection of 218 genes found to be associated with the development of different cancers . By comparing the specificity of cancer gene expression between pig and human, we aim to provide valuable insights into cancer genetics and modeling of human cancers in pigs. For example, overexpression of homeobox protein CDX-2, encoded by the CDX2 gene is associated with the development of gastric cancers in humans, and also categorized as intestine enriched in both pig and human. OtherThe categories of other diseases (80 genes) and other congenital disorders (208 genes) include genes which are associated with other diseases not included in other categories, and genes that are involved in rare genetic diseases such as mental and behavioral disorders. Facts on Pig GeneticsSimilar to the human genome (3 Gb), the size of pig genome is approximately 2.7 Gb. According to the latest assembly (Sscrofa11.1, Ensembl build 99), two de novo sequencing studies applying deep shot gun sequencing (100 X coverage) and long read technologies have significantly resolved gaps and missing protein-coding genes in the early assembly Sscrofa10.2 Li M et al. (2017).
Using Pig for Human Genetic Disease ModellingThe human genetic diseases are caused mainly or partially by mutations in genes or by chromosomal alterations. Depending on the mutation types, the function of genes, location in chromosomes and the complexity of diseases, genetic diseases can be caused by single gene mutations (monogenic diseases) or by a combination of mutations in multiple genes and environmental factors (multifactorial inheritance diseases). To understand the innate complexity of human genetic diseases, genetically engineered animal models carrying the corresponding genetic mutation(s) have been created. These genetically engineered animal models successfully mimicking the human genetic disorder have been proven useful for the understanding of disease pathogenesis, as well as for the development of diagnosis and treatments. Due to the complexity of human diseases, rodent models frequently fail to recapitulate the full spectrum of disease phenotypes and pathogenesis in human. Thus, the genetically, anatomically, physiologically and metabolically closer large animal model, the pig, is considered an excellent preclinical animal model to bridge the gap between rodent and human. Genetic engineering approaches in animal modelsThree major genetic engineering methods are used for generating genetically tailored animal models for human diseases: Approach 1: Overexpressing the corresponding disease-causing mutation human gene, which is known as transgenesis, and is typically used for modeling genetic diseases with dominant phenotypes.
Approach 2 Introducing the corresponding mutation to the pig ortholog disease gene by homology directing repair (also known as knockin). This is a better complementary strategy to the approach 1, and can diminish expression of the wildtype allele. However, this strategy is greatly hampered by its low efficiency.
Approach 3 Disrupting the corresponding disease-causing gene (ortholog) in pigs by homologous recombination or by gene editing. This approach is typically used for modeling human genetic diseases caused by loss of function.
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