dystrophin deficiency-induced changes in porcine …
TRANSCRIPT
Dan Nonneman, Ph.D. Research Molecular Biologist
U.S. Meat Animal Research Center USDA, Agricultural Research Service
DYSTROPHIN DEFICIENCY-INDUCED CHANGES IN PORCINE SKELETAL
MUSCLE
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Dystrophin Deficiency-Induced Changes in Porcine Skeletal Muscle
! Brief Introduction to Porcine Stress Syndrome ! Identification of a Novel Porcine Stress Syndrome ! Characterization of some skeletal muscle changes due to
dystrophin deficiency
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Transport Stress and Dead and Downer Pigs
! Nonambulatory pigs result from injury, fatigue, heat and stress ! Downer pigs arriving at the packing plant range at 0.25-0.75% ! DOAs are about 0.26% ! Downers can be as high as 10% for individual loads ! The incidence has increased from 0.08 to 0.3% in the last decade ! The incidence is aggravated by stressful handling; electric prods, crowding,
heat, motion sickness ! Other risks include Paylean, high-lean genetics ! These losses cost the industry ~$50-$150 million/year ! Losses are both an economic and a welfare concern
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! PSS was first described in 1968 ! Open-mouth breathing ! Blotching of skin ! Hyperthermia ! Muscle rigidity ! Loss of mobility or death ! Induced by transport, stress or
halothane anesthesia ! Caused by a mutation in RYR1
located on chromosome 6
Porcine Stress Syndrome (PSS)
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Porcine Stress Syndrome and PSE ! In 1968 in South Africa, while developing a model for liver transplantation, 6
Landrace pigs die under halothane anesthesia – “malignant hyperthermia” ! In 1978 halothane was used to test stress susceptibility in piglets ! In 1991, Fujii identified a ryanodine receptor 1 (RYR1) mutation (C1843T) in a
halothane-sensitive Pietrain pig ! This R615C mutation is associated with PSE (pale, soft, exudative pork) ! The incidence of PSS has been dramatically reduced by halothane testing and
marker assisted selection against the RYR1 mutation ! Today over 97% of downer pigs are RYR1 mutation free ! About 23 - 48% of pigs that don’t carry the RYR1 mutation respond abnormally to
halothane anesthesia (limb rigidity, blotching skin, tremors; Allison et al., 2005) ! Pigs that are RYR1 normal, but are sensitive to halothane have higher drip loss
and lower pH (Bates et al., 2012)
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Porcine Stress Syndrome Pigs
! After being transported two barrows (siblings) died with apparent symptoms of PSS, another was euthanized – our herd is “stress” free
! We thought this may be a malignant hyperthermia due to other alleles of RYR1 or other MH genes
! We repeated the original mating and mated the boar to his daughters and produced additional litters
! At 8 weeks the pigs were challenged with 3% isoflurane anesthesia for 3 minutes
Normal Affected
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Identification of Stress Pigs ! Stress response or death during handling or transport ! Stress response to isoflurane challenge ! Heart rate, pulse and ECG monitored during anesthesia challenge ! Plasma creatine phosphokinase was measured ! Body temperature and physical appearance
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Electrocardiograms during Isoflurane Anesthesia
-5
0
5
20 22 24 26 28 30 Seconds
-5
0
5
20 22 24 26 28 30 Seconds
Normal Pig
“Stress” Pig
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Plasma Creatine Phosphokinase Activity
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! We challenged 242 pigs; 32 litters, 13 boars and 21 sows ! Response to isoflurane was bradycardia, metabolic stress and death ! Of 49 affected pigs, 18 pigs died during anesthesia ! Eight affected pigs died during transport later ! Affected animals were predominantly male ! Pigs are resistant to stress as they get older ! The stress syndrome was not linked to RYR1 ! Other hyperthermia loci SCN4, CPT2, CACNA1S, CACNA2D1 and
RYR2 were not associated with the stress syndrome
Summary
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Illumina PorcineSNP60 Beadchip Association
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SNP Associations with Stress on Chromosome X
DMD
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Dystrophin (DMD)
! Dystrophin is the largest mammalian gene ~ 2.4 Megabases ! Mature mRNA is 14 kb and takes ~16 hours to transcribe ! 79 exons code for 3679 amino acids, 427 kDa protein ! Deletions/insertions cause 60-65% of Duchenne (DMD) Muscular Dystrophy ! Milder mutations cause Becker (BMD)
Muscular Dystrophy ! Dystrophin connects the cytoskeleton
to the ECM (costamere)
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Immunoblot of Pig Dystrophin
Muscle Heart
Normal Stress Normal Stress Normal Stress Normal Stress
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Immunoblot of Pig Heart Dystrophin
Carrier Affected Normal Affected Normal Affected Normal Affected
427-
260-
140-
71-
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Normal Normal Carrier Affected Normal Carrier Affected Normal Affected Carrier
Heart
Retina
Immunoblot Blot of Pig Dystrophin
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Dystrophin Protein on Immunoblots of Heart
n=4
**
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Dystrophin RT-‐qPCR of Affected Heart cDNA
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Histopathology of Dystrophic Heart Muscle
! Muscle fiber degeneration ! Fragmentation and fibrosis ! Loss of cross striation and myofibrils ! Aggregation of lymphocytes
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Evaluation of the Dystrophin (DMD) Gene
! Sequenced exons and intron boundaries from genomic DNA ! Sequenced the complete cDNA in overlapping fragments ! Found no splice variants or exon skipping ! Found five non-synonymous SNPs ! Sequenced promoter regions - no obvious mutations
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Marker Nucleotide Polymorphism aa Δ Location -Log(p)
84000_82 1349 CRA R396G Exon 11 2.96
84002_411 1629 CAS H489Q Exon 12 3.92
84339_200 2102 ARC N647S Exon 16 0.38
84339_333 2235 ACR T691T Exon 16 0.24
85890_783 6034 YGG R1958W Exon 41 23.20
ALGA0099513 Intron 44 22.56
ALGA0099414 Intron 44 22.56
85904_613 7274 CYG L2371P Exon 49 2.66
Amino Acid Changes in Pig Dystrophin
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! An R1958W mutation in dystrophin was associated with the porcine stress syndrome, but was also found in “normal” unrelated animals
! We did not have a more predictive marker or haplotype for the syndrome ! The identification of the mutation will allow us to determine the prevalence
(or absence) in commercial populations and the affect on performance ! The objective was to obtain a comprehensive sequence of porcine
dystrophin from affected and normal pigs ! A library of >47,000 120 bp overlapping RNA capture baits was designed ! Libraries were constructed from 8 affected pigs, 6 normal pigs and 2 pools
(Duroc/Large White and Landrace) and sequenced on a MiSeq
Next-generation Sequencing of Dystrophin
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! Reads were assembled to the masked reference ! Variation was identified under different criteria ! Average read depth was 25-35x/library and 200-268x/pool for ~1M reads
Sequence Assembly of Capture Libraries
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! Variants were detected for affected and normal libraries ! Variation from reference and among individuals (reads)
Sequence Variant Analysis in Dystrophin
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Runs of Homozygosity Among Affected
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Histopathology of Dystrophic Muscle
! Varia<on in muscle fiber diameter ! Disorganized fibrosis ! FaEy infiltra<on
Normal Affected Normal Affected Normal Affected
Diaphragm Psoa Longissimus
H&
E T
richr
ome
Hollinger et al., 2014. FASEB J 28:1600-‐9
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Hollinger et al., 2014. FASEB J. 28:1600-9
Dystrophin Protein Expression
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Hollinger et al., 2014. FASEB J. 28:1600-‐9
Dystrophin mRNA Expression
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Singh et al., 2010. PNAS 107:15069-74
Dystrophin and the Dystrophin Glycoprotein Complex (DGC)
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Reduction of α-Sarcoglycan in Dystrophic Muscle
Hollinger et al., 2014. FASEB J 28:1600-9.
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Fortunato et al., 2014. PLOS 9:e97567
Reduction of α-Dystroglycan in Dystrophic Muscle
Affe
cted
N
orm
al
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Desmin and Laminin Protein Expression in Dystrophic Muscle
Hollinger et al., 2014. FASEB J 28:1600-‐9
Desmin Laminin
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Utrophin Protein Expression in Dystrophic Muscle
Hollinger et al., 2014. FASEB J 28:1600-‐9
! Utrophin is over expressed in DMD and mdx mouse muscle
! It can bind to actin and the DGC ! Has a protective affect on
dystrophic muscle
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Muscle Glycogen is Increased in Dystrophic Pigs
! Glucose intolerance ! Hyperinsulinemia ! Increased muscle and
decreased liver glycogen in mdx mouse and DMD patients
! Increased synthesis and decreased glycogen breakdown in muscle n = 7/group
n = 7/group
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Meat Quality in Dystrophic Pigs
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Meat Color in Dystrophic Pigs
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! A porcine stress syndrome was identified that is associated with a R1958W mutation in dystrophin
! The syndrome is associated with dramatic reduction in dystrophin ! This reduction is not enough to cause a severe muscular dystrophy ! Dystrophin deficiency causes a collapse of the dystrophin-glycoprotein
complex
Summary
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! Current animal models for dystrophinopathies are either mild or variable and these pigs could provide a more appropriate biomedical model for human MD therapies – stem cell replacement, oligonucleotide directed exon skipping/splicing, micro genes, drug therapies
! This deficiency could provide a model for muscle cell degeneration and regeneration
! May provide insight to the role of dystrophin in muscle biology
Conclusions
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Acknowledgements
Iowa State University ! Josh Selsby ! Jason Ross ! Katrin Hollinger
USMARC Scientists ! Gary Rohrer ! Tami Brown-Brandl ! Shuna Jones ! Ralph Wiedmann ! Andy King ! Steven Shackelford ! Tommy Wheeler
University of Georgia ! Aaron Beedle ! Marisa Fortunato