DYSTROPHIN DEFICIENCY-INDUCED CHANGES IN PORCINE …

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

American Meat Science Association AMSA American Meat Science Association AMSA American Meat Science Association AMSA American Meat Science Association AMSA American Meat Scie

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


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


!  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)


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)


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


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


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


Plasma Creatine Phosphokinase Activity


!   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


Illumina PorcineSNP60 Beadchip Association


SNP Associations with Stress on Chromosome X

DMD


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)


Immunoblot of Pig Dystrophin

Muscle Heart

Normal Stress Normal Stress Normal Stress Normal Stress


Immunoblot of Pig Heart Dystrophin

Carrier Affected Normal Affected Normal Affected Normal Affected

427-

260-

140-

71-


Normal Normal Carrier Affected Normal Carrier Affected Normal Affected Carrier

Heart

Retina

Immunoblot Blot of Pig Dystrophin


Dystrophin Protein on Immunoblots of Heart

n=4

**


Dystrophin RT-‐qPCR of Affected Heart cDNA


Histopathology of Dystrophic Heart Muscle

! Muscle fiber degeneration ! Fragmentation and fibrosis ! Loss of cross striation and myofibrils ! Aggregation of lymphocytes


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


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


!   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


!   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


!   Variants were detected for affected and normal libraries !   Variation from reference and among individuals (reads)

Sequence Variant Analysis in Dystrophin


Runs of Homozygosity Among Affected


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


Hollinger et al., 2014. FASEB J. 28:1600-9

Dystrophin Protein Expression


Hollinger et al., 2014. FASEB J. 28:1600-‐9

Dystrophin mRNA Expression


Singh et al., 2010. PNAS 107:15069-74

Dystrophin and the Dystrophin Glycoprotein Complex (DGC)


Reduction of α-Sarcoglycan in Dystrophic Muscle

Hollinger et al., 2014. FASEB J 28:1600-9.


Fortunato et al., 2014. PLOS 9:e97567

Reduction of α-Dystroglycan in Dystrophic Muscle

Affe

cted

N

orm

al


Desmin and Laminin Protein Expression in Dystrophic Muscle


Desmin Laminin


Utrophin Protein Expression in Dystrophic Muscle


! 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


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


Meat Quality in Dystrophic Pigs


Meat Color in Dystrophic Pigs


!   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


!   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


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

DYSTROPHIN DEFICIENCY-INDUCED CHANGES IN PORCINE …

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