Details of relevant genes in Bota 19 and Hosa 17.
There are interesting similarities and differences when comparing the histopathology of bovine marbling and human muscular dystrophy. At the simplest level, both conditions are characterized by genetically controlled and more or less inexorable replacement of muscle fibers with fat cells. At issue is whether an improved understanding of these two processes can lead to better outcomes for patients. There are many forms of dystrophy that differ in their genetics and their histopathology. There are also many forms of “marbling” ranging from the coarse to fine, epimysial, perimysial to endomysial and even to total replacement or steatosis. A detailed examination of marbling will provide a framework for further investigation of human dystrophy. Ultimately, the many genetic factors involved can be addressed through a better understanding of the metabolic pathways involved in marbling.
- muscular dystrophy
- bovine marbling
The purpose of this review is to compare the genetics and histopathology of bovine marbling and human muscular dystrophy. Surprisingly, in spite of similarities, the literature suggests that marbling is a function of extreme adipogenesis whereas dystrophy is a consequence of fundamental defects in muscle itself. In fact, completely independent studies, as summarized here, reveal that similar genes have been implicated in some selected situations. Further, it is clear that the histopathology of some forms of dystrophy can resemble some forms of bovine marbling.
Marbling is the term used to describe the presence of macroscopically visible fat within muscle (Figures 1 and 2). Coarse marbling refers to white areas of fat through and around muscle bundles, generally as continuous bands arising from the subcutaneous adipose tissue. By contrast, fine or “snowflake” marbling is characterized by more even white flecks resulting in pink rather than red muscle.
These two forms may coexist but can be distinguished and quantified by skilled observers. Fine marbling is associated with improved taste and tenderness . Further, it has been shown to relate to a preferred fatty acid profile. Accordingly, there is copious funding and now a substantial understanding of the environmental and genetic factors which favor fine rather than coarse.
3. Interspecies translation
Interspecies translation from cattle to man has unrecognized potential. Firstly, cattle are close to humans in evolutionary time and fall within that window of 50–100 million years of separation (or last common ancestor) which is characterized by very similar proteins but vastly different regulations of expression. The same window may explain the fact that the two species have synergized over some 40,000 years of contact and at least 7000 years of domestication. As one example, infections can be similar and, in some cases, are transmissible from one to the other, but close exposure to cattle is generally innocuous implying some form of immunity. As for example in the case of pox and tuberculosis. We argue that cattle are both relevant and relatively safe for translational studies.
Secondly, domestic cattle are well maintained, closely observed, and very well understood. There are huge databases and DNA banks which have been in existence for 50 years. Innumerable breeds can be compared often under different environmental conditions. Many of these breeds have been closed for hundreds of years and then intentionally crossed with each other. There is great potential for meaningful studies of population genetics and family and haplotype associations and, even more so, for structure-function genomics. Metabolic and inflammatory pathways are relatively well understood and are supported by inestimable funding available to ensure future supplies of meat, milk, cheese, butter, leather, and fertilizer.
Thirdly, cattle are plentiful and even more so than humans. Because the generation time and life expectancy are much shorter, there are excellent opportunities to study and treat genetically determined diseases prospectively .
4. Other instances of translation
White muscle disease or selenium/vitamin E deficiency occurs quite commonly in livestock raised on leached soils. The pathology resembles dystrophy in some respects. A mutation in the selenoprotein N gene (SEPN1) is responsible for some types of congenital muscular dystrophies and myopathies . Kakulas  demonstrated that dystrophy-like changes explained the weakness observed in quokkas on Rottnest Island. Importantly, the condition could be corrected by treating the deficiency raising the possibility that human dystrophies could be reversible if the basic defect could be corrected.
5. Genomic approach
The term genome is used here to refer to the architecture of DNA sequences, whereas others have come to use the term in the context of single-nucleotide polymorphisms wherever they occur. The difference is fundamental to the discovery of gene clusters with coherent cis and trans interactions between conserved sequences known as ancestral haplotypes [5, 6, 7, 8, 9]. Many studies have shown that the SNP approach in livestock and humans fails to identify these critical sequences and can be misleading at best . SNPs are neutral markers of parentage rather than functionally important .
One major benefit of ancestral haplotypes as opposed to SNPs is that it is possible to use interspecies translation. During mammalian evolution, polymorphic frozen blocks have diverged to some extent although the functionally important sequences tend to be conserved.
As shown in Figure 3 and Table 1, there are similarities between genomic regions on Hosa 17 and Bota 19. Although there have been architectural changes such as insertions and transversions, the gene content has been preserved.
|Gene location||Description||Human muscular dystrophy||Meat quality trait|
Bota chr19: 30.13Mb
|MYH2 encodes the myosin heavy chain isoform that is expressed in fast type 2A muscle fibers||Proximal myopathy and ophthalmoplegia is caused by heterozygous, compound heterozygous, or homozygous mutation in MYH2 leading to a lack of type 2a fibres ||In pork, IMF, water holding capacity, and meat color |
Bota chr19: 33.35Mb
|Peripheral myelin protein-22||Duplication of peripheral myelin protein 22 causes Charcot-Marie- Tooth disease type 1A |
Bota chr19: 33.816Mb
|Transient receptor potential cation channel, V2: responds to heat and cations||Muscular dystrophy is ameliorated in dystrophin-deficient mdx mice by dominant-negative inhibition of TRPV2 |
Bota chr19: 35.23Mb
|Sterol regulatory element-binding protein-1 controls cholesterol homeostasis by stimulating transcription of sterol-regulated genes||Mutations of LMNA that cause Emery-Dreifuss muscular dystrophy (EDMD2-AD) and familial partial lipodystrophy (FPLD2) result in less binding of lamin A to SREBP1 ||SREBF1 is involved in adipogenesis and polymorphisms are associated with fatty acid composition of Japanese Black Cattle |
Bota chr19: 35.557Mb
|Myosin phosphatase rho-interacting protein targets myosin phosphatase to regulate the phosphorylation of myosin light chain ||Haplotypes diffentiated by polymorphsims in MRIP are associated with differences in intramuscular fat development in Wagyu |
Bota chr19: 37.11Mb
Sarcoglycans form part of the dystrophin-glycoprotein complex
|Mutations in SGCA cause limb-girdle muscular dystrophy type 2D. SGCB, SGCD, and SGCG are associated with LGMD types 2E, 2F, and 2C, respectively |
Bota chr19: 40.69Mb
|Titin-cap (telethonin) is a sarcomeric protein localized to the periphery of Z discs that define the border of the sarcomere as a structural anchor and signaling center||Limb-girdle muscular dystrophy type 2G (LGMD2G) is caused by mutations in the TCAP gene ||A polymorphism of TCAP is associated with IMF content and fatty acid composition of beef [13, 28]|
Bota chr19: 43.14Mb
|Cavin is an essential factor in the biogenesis of caveolae||Congenital generalized lipodystrophy, type 4; (CGL4) is caused by mutations in CAVIN1 that result in CAV 3 deficiency |
Bota chr19: 43.47Mb
|Beclin-1 participates in the regulation of autophagy||Expression of BECN1 was reduced in patients with muscular dystrophies BTHLM1 and UMCD1 which were caused by COL6A1 mutations ||Involved in proteolysis and beef aging |
|Growth Hormone||A polymorphism of growth hormone is associated with fatty acid composition of Wagyu beef |
|Fatty Acid Synthase the key enzyme of de novo lipogenesis to produce saturated fatty acids||Fatty Acid Synthase is highlighted in GWAS for fatty acid content and composition of Wagyu and Hanwoo beef [33, 34]|
|A receptor abundant in heart and pancreas and responsive to Urotensin II which has potent vasoactive properties||A polymorphism of UTS2R is associated with IMF content of Wagyu x Holstein beef |
Hosa 17 was chosen for comparison because it contains some of the same genes such as TCAP. Further analysis revealed an extraordinary degree of preservation or synteny in spite of an evolutionary separation time of at least 50 million years and therefore millions of generations. Implicit is that there are functional reasons for similarities in genomic architecture.
Yet further analysis suggests some explanations for the co-location of similar genes. Irrespective of cis and trans interactions between the protein products, there is evidence of co-regulation (see, e.g., SREBP). In this context, we conclude that, although products and their regulating transcription factors are preserved, separation has permitted the insertion of species-specific elements, which control the quantitative differences between humans and cattle.
Importantly, as shown in Figure 3 and Table 1, Hosa 17 contains multiple candidates for involvement in human muscular dystrophy. There is even more complexity in explaining the multiple candidates as shown in Tables 2 and 3.
|Gene location||Description||Human muscular dystrophy||Meat quality trait|
Bota chr2: 6.21Mb
|Myostatin||Muscle hypertrophy was caused by a homozygous mutation in myostatin ||Mutations in myostatin cause double muscling in several cattle breeds |
Bota chr10: 37.8Mb
|Calpains are nonlysosomal intracellular cysteine proteases. CAPN3 is a muscle-specific large subunit||Limb-girdle muscular dystrophy type 2A (LGMD2A) is caused by homozygous or compound heterozygous mutation in CAPN3||SNPs within CAPN3 are associated with tenderness in Bos Indicus cattle |
Bota chr29: 44.06Mb
|m-Calpain||Two CAPN1 genetic markers are associated with tenderness in Brahman beef |
Bota chrX: 115.34Mb
|Dystrophin maintains the structural integrity of myofibrils||Duchene muscular dystrophy|
Bota chr9: 67.96Mb
|LAMA2 gene encodes the alpha-2 chain of laminin-2|
Laminin-2 (merosin) is the main laminin found in muscle fibers
|Congenital merosin-deficient muscular dystrophy type 1A; MDC1A|
Bota chr7: 50.94Mb
|Myotilin directly binds F-actin and efficiently cross-links actin filaments and prevents filament disassembly||LGMD1A is caused by heterozygous mutation in the MYOT. It is characterized by adult-onset muscle weakness, progressing from the hip to the shoulder girdle||SNPs in MYOT correlate with loin muscle area and intramuscular fat in Qinchuan cattle|
Bota chr22: 17.83Mb
|Caveolin 3||Muscular dystrophy, limb-girdle, type 1C; LGMD1C|
Bota chr7: 69.59Mb
|Sarcoglycan, delta is expressed in skeletal and heart muscles and to a lesser extent in smooth muscle. Delta-sarcoglycan is localized at the sarcolemma||Muscular dystrophy, limb-girdle, type 2F; LGMD2F|
Bota chr4: 11.84Mb
|Epsilon-sarcoglycan||Myoclonus-dystonia is a genetically heterogeneous disorder characterized by myoclonic jerks affecting mostly proximal muscles|
Bota chr6: 69.53Mb
Bota chr12: 34.92Mb
|Collagen, type VI, alpha-1, and alpha-2|
Members of the collagen VI family form distinct networks of microfibrils in connective tissue and interact with other extracellular matrix components
|Ullrich congenital muscular dystrophy 1|
Bethlem myopathy 1
|COLLAGEN, TYPE VI, ALPHA-3||Ullrich congenital muscular dystrophy 1, Bethlem myopathy 1|
|The alpha-7 integrin is a specific cellular receptor for the basement membrane proteins laminin-1, laminin-2, and laminin-4. The alpha-7 subunit is expressed mainly in skeletal and cardiac muscles and may be involved in differentiation and migration processes during myogenesis||Congenital muscular dystrophy|
|Emerin is found along the nuclear rim of many cell types and is a member of the nuclear lamina-associated protein family||Emery-dreifuss muscular dystrophy 1, X-LINKED; EDMD1|
|Calcium-transporting ATPase lower cytoplasmic Ca(2+) concentration by pumping Ca(2+) to luminal or extracellular spaces. ATP2A1 is the ATPase type found in fast twitch muscles||Brody myopathy|
|Desmin is the muscle-specific member of the intermediate filament (IF) protein family. It is one of the earliest myogenic markers, both in the heart and somites, and is expressed in satellite stem cells and replicating myoblasts||Myopathy, myofibrillar, 1|
|Plectin-1 is one of the largest polypeptides known and is believed to provide mechanical strength to cells and tissues by acting as a cross-linking element of the cytoskeleton||Epidermolysis bullosa with muscular dystrophy|
Limb-girdle type 2Q
|Absent protein||Dystrophy type||Gene location|
|Dystrophin||Xp21 muscular dystrophies (Duchenne, Becker)||DMD|
Bota chrX: 115,342,323-117,606,340
|Sarcoglycans||Limb-girdle muscular dystrophies 2C-F||SGCA Hosa 17q21.33 Bota 19|
SGCB Bota chr6
SGCD Hosa 5q33.2 Bota 7
SGCE Hosa 7q21.3 Bota 4
SGCG Bota chr12
|Dysferlin||Limb-girdle muscular dystrophy 2B||DYSF Hosa 2p13.2|
|Caveolin-3||Limb-girdle muscular dystrophy 1a, rippling muscle disease, hyperCKemia||CAV3 Hosa 3p25.3 Bota 22|
CAVIN1 Hosa 17q21.2 Bota 19
|Telethonin||Limb-girdle muscular dystrophy 2G||TCAP Hosa 17q12 Bota 19|
|Laminin a2||MDC1A (“merosin”-deficient congenital muscular dystrophy)||LAMA2 Hosa 6q22.33, Bota 9|
|Collagen VI||Ullrich congenital muscular dystrophy||COL6A1&2 Hosa 21q22.3|
COL6A3 Hosa 2q37.3
|Integrin alpha7||Mild congenital dystrophy/myopathy||ITGA7 Hosa 12q13.2|
|Calpain-3 (easier to assess on immunoblots than sections)||Limb-girdle muscular dystrophy 2A||CAPN3 Hosa 15q15.1 Bota 10|
|Emerin||X-Linked emery-dreifuss muscular dystrophy||EMD Hosa Xq28|
|SERCA 1||Brody disease||ATP2A1 Hosa 16p11.2|
|Plectin||Epidermolysis bullosa with muscular dystrophy, limb-girdle dystrophy 2Q||PLEC Hosa 8q24.3|
|LAMP-2||Danon disease||LAMP2 Xq24|
|Accumulating protein||Dystrophy type||Gene location|
|Actin||Congenital actin myopathy/nemaline myopathy||ACTA1 Hosa 1q24.13|
TPM3 Hosa 1q23
|Myosin||Myosin storage myopathy||MYH7 Hosa 14q11|
|Myotilin||Myotilin-related myofibrillar myopathy||MYOT Hosa 5q31.2|
|Desmin||Desmin myopathy||DES Hosa 2q35|
SEPN1 Hosa 1p36
Thus, syntenic analysis has suggested a novel approach to identification of operative elements in marbling and in some forms of dystrophy.
6. Histopathological approach
The substantial range of changes found in the human dystrophies is illustrated in the study of Dubowitz et al. .
We are fortunate in having histological muscle samples from cattle with degrees of marbling . Some of these changes are illustrated in Figures 4, 5, 6, 7, 8 from three animals (M508, M621, and M129) fed a standard ration for 471, 443, and 481 days respectively. The macroscopic measure of marbling (MSA MB) ranged from high to moderate (1100, 920 and 820, respectively) as expected in high content Wagyu (88, 75, and 63%, respectively). A common feature is the invasion of adipose tissue between intact muscle fascicles (Figure 4). For the most part, the process extends along the perimysium leading to variation in fiber size, staining of myofibers (Figures 5 and 6), and the formation of residual islands of myofibers (Figure 7), which suggests an explanation for fine (see Figure 1) rather than coarse (see Figure 2) marbling; fine is due to more aggressive invasion reflecting quantitative differences in gene regulation.
In some fields, there are collections of nuclei including intracytoplasmic (Figure 8).
These observations have led us to the conclusion that the extent and type of marbling is a function of the aggressive extension of the advancing adipocytes with secondary loss of myocytes.
As in human dystrophies, there can be different degrees depending upon the muscle group and the field selected. Here, we focus on Sacrocaudalis dorsalis medialis, because it is convenient to biopsy, whereas the loin can only be accessed readily post-mortem.
Accordingly, it will be possible to undertake detailed time course studies so as to monitor sequential changes and eventually responses to therapy. Future studies should also address bovine steatosis. The pathology [16, 17] is different from marbling. Adipocytes occur within rather than around fascicles (Figure 10) suggesting that the process may be a function of differentiation of stem cells, rather than invasion .
In spite of similarities in pathology and genomics, there is more to learn before precise translation is possible. However, there are strong indications that such approaches could have important implications for human dystrophies and other muscle diseases. Moreover, a better understanding of the control factors and signals responsible for determining the relative proportions of muscle and adipose tissue in bovine muscles, and how they are coordinated, is fundamental and will be crucial to understanding more fully the significance of adipose tissue replacement in human dystrophies and to developing new therapeutic strategies for these diseases.
This research was supported by the CY O’Connor ERADE Village Foundation as publication number MS1803. The authors acknowledge the earlier contributions of J F Williamson and would like to thank JR Dawkins and Melaleuka Stud for access to the cattle used in this study.
Conflict of interest
Collectively, the authors associated with the CY O’Connor ERADE Village Foundation have an interest in the work described in this manuscript as it forms part of the foundation’s intellectual property.