Exam 3

What is the physiology of double muscling? Specifically in cattle?

• High number of muscle fibers

• Less marbling (less fat)

• Reduced fertility, high embryo mortality rate

• Cattle: DM in cattle contains less connective tissue, show sings of fatigue faster (reduced blood circulation)

What are the genetics of double muscling?

• Caused by the myostatin (MSTN) gene mapped on chr. 2 (highly conserved)

• Myostatin is responsible for skeletal muscle growth → mutations lead to muscle hypertrophy

• Six mutations: deletions, insertions, point mutations

How did the backcross design in mapping myostatin work?

Microsatellites

Naturally existing polymorphisms. Short sequences of DNA repeated (tandem arrays → TGTGTG = TG3)

Comparative mapping approach

Method for establishing the relative genomic positions of orthologous (genes from common ancestor that retained similar function in different species) gene pairs in two species (human/mouse)

Positional Candidate Gene Approach

Combines genetic mapping techniques with targeted selection/function analysis of candidate genes to identify genetic factors linked to a specific trait or disease.

MSTN mutation affect on protein function within dogs

A 2-bp deletion leads to a premature stop codon instead of producing regular cysteine. The loss of cysteine results in lack of formation of disulfide dimers required for protein function (lack thereof causes double muscling).

Myostatin is a negative regulator of muscle growth

• Controls total number of muscle fibers by regulating myoblast proliferation

• Absence of functional protein (mutations) leads to more muscle fibers made

What factors affect genetic diversity?

1. Founder effect

2. Genetic drift

3. Population Sire effect

4. Population bottleneck

Founder Effect

Most breeds were established using a few dogs that possessed certain desirable traits

Genetic Drift

The use of small number of founders caused fluctuations in allele frequencies and the loss of rare alleles

Population Sire effect

A dog with desirable traits is used more frequently for breeding than other males in population

Population bottleneck

A sudden reduction in numbers. Recovery of the breed depends on a small number of individuals, resulting in genetic drift and increased homogeneity

Transposable Elements (TE)

1. DNA Transposons → move using cut-and-paste mechanism

2. Retrotransposons → move using copy-and-paste mechanism (duplicates the element into a new genomic location via RNA intermediate)

   1. SINE and LINES, use copy n paste to invade genomic regions

3. DNA methylation mechanism represses activation of TE

SINEs and LINEs

SINEs have an internal promotor for transcription but depend upon enzymes produced by LINEs for reverse transcription (RNA → cDNA) and integration into genome.

• Once integrated, SINEs/LINEs passed from generation to generation.

What attributes have been linked to an insertion of a SINE element?

• Merle Coat pattern: caused by insertion at the intron 10/11

• Extreme piebald: insertion upstream of MITF

• Saddle Tan: insertion in intron 1 of ASIP (agouti signaling protein)

Genome-wide Association Studies (GWAS)

• Used for identification of genetic variants underlying both simple and complex traits.

• Utilizes unrelated cases/controls to compare between cases for identification of associations

GWAS for a neurological disorder in Cavalier King Charles Spaniels

Strong association with the phenotype on chromosome 7 led to identifying the causative mutation. Extent of linkage disequilibrium (LD) in dogs is 100x longer than humans bc of long stretches of homozygosity.

What makes dogs a good animal model for studying human diseases?

• Large litter sizes

• a short gestational period

• accelerated aging/disease progression

• Large population have dogs making data widely available

• Share similar environments

• Diseases are commonly shared between dogs and humans

• similar organ size

• Fewer regulatory guidelines than human medicine

Importance of Merle Phenotypes

• Cryptic Merle → dog with merle genotype but does not have merle phenotype (very rare) and produces merle offspring

• All breeds → double merle genotype can be sublethal and associated with multiple skeletal, cardiac, and repro abnormalities

Linkage Disequilibrium Analysis

1. Two loci in close proximity may be in high LD

2. Two loci in high LD typically are in close proximity

LD = the nonrandom association of alleles at two or more loci

Whole Genome Association Study

1. Sample drawn from general population

2. Phenotypes with discrete classes (disease)

   1. case (affected) vs control (unaffected)

3. Phenotypes for continuous traits

   1. sample from tails of the distribution

Linkage Disequilibrium Analysis

1. Genotype LARGE number of SNPs

2. Test association between phenotype and marker genotype

3. Association between causative polymorphism and linked markers diminished for all but most closely linked

4. Assumption: disease predisposition due to common allelic variants at multiple loci (compared to rare variants with HUGE effects)

Meta-analyses (combining data across studies)

• Identified associated regions that failed to pass significance thresholds in original studies

  • Follow-up by identifying causal variant associated with SNP-associated (can be difficult)

Lessons Learned with conducting a GWAS

• Need large sample

• Must account for multiple stat tests

• Replication of results is essential

• accurate assessment of phenotype, narrowly defined

Lessons learned - results of conducting a GWAS

• Complex traits are highly polygenic

• Pleiotropy is common (a single gene affects multiple, seemingly unrelated traits or characteristics)

How do you identify causative variants?

1. Is there a candidate gene coding sequence change? If not..

   1. Combine independent sources of info (multi-omics)

   2. Results from GWAS where: phenotype is trait, gene expression, or protein level.

   3. Results from genomic analysis of epigenetic mods

   4. Results from identification of regions with open chromatin

Applications for causative variants (how is GWAS relevant to human or veterinary medicine)

• Drug choice/dosage

• Identify personal disease risk (polygenic risk score)

• Drug targets

• Genomic selection for livestock

Ovine Progressive Pneumonia (OPP)

Caused by ovine lentivirus infection. Slow progression (2+ yrs).

• Loss of body condition (but normal appetite)

• Increased breathing effort at rest

• Common for second bacterial infection → fever, cough, lethargy, nasal discharge

• Hard bag (enlarged, firm udder, no milk)

How is OPP spread?

• Respiratory in adults → consumption of infected colostrum or milk

• Integrates into host genome

• Infected never clear virus → no treatment

• Manage through reducing prevalence (testing, remove infected, increase genetic resistance)

Sensitivity vs Specificity

• Sensitivity- proportion of patients with disease that test positive

• Specificity- proportion of patients without disease that test negative

Porcine Reproductive and respiratory syndrome (PRRS)

• Causes repro, respiratory, and reduced growth problems

• Caused by single-stranded RNA virus (Arterivirus)

• Different breed susceptibilities suggest genetic component

GWAS study for PRRS

1. Pigs donated from companies challenged with virus

   1. Eliminate exposure questions

2. Dedicated facility for pig housing

3. Study conducted in replicates of 200 pigs

4. Pigs tested for viral growth and load

PRRS results

Strongest signals for viremia found on SSC4 and SSCX

What was the characterization of the positional candidate gene (SSC4)

• Several GBP family members in region (all candidates) → mediate proinflammatory immune response

• GBP5 → thought to function in uncovering virus in endosome (splice site mutation, frameshift mutation, low expression of wild-type)

How does understanding PRRS help us?

• Wild-type less common → greater opportunity to improve population by selection

• Wild-type is dominant → no need for homozygous genotyping

Bovine Respiratory Disease (BRD)

• Caused by variety of pathogens, viral and bacterial

• Symptoms: fever, loss of appetite, shallow breathing, coughing, salvation, watery/pus/bloody nasal/eye discharge

How was BRD in a GWAS studied?

• Scored values visually

• Samples cultured to determine pathogen

• genotyped cases and matching controls (herd, gender, pen, location)

Results from BRD study

• no single genomic region of large effect

• Multiple regions with small to modest effect

Microphthalmia

Abnormal eye development, prominent third eyelids, small eyes, blind, partial deafness

Melanin Synthesis

Extension Locus (E)

• E → normal extension. Allows black pigment to be expressed

• e → recessive red. Coat is red or yellow instead of black

• E^M → Black mask around muzzle (boxer)

• E^m → larger black mask (pug)

• E^G → grizzle (pale face with a widow’s peak above the eyes i.e. afghan hounds)

• Dominance: E^M > E^G > E > e

• MC1R

Important Concepts

1. Genes and their functions are conserved across species

2. Specific mutations differ btwn species

3. phenotypic effect of mutations may be similar

Important Concepts #2

1. One gene may affect multiple phenotypes

   1. pleiotropy

   2. MC1R allelic variants in humans effect: skin/hair color, freckling, risk for skin cancer, pain sensitivity

Important concept #3

• Multiple genes can have complex interactions in determining phenotype

• Epistasis → phenotype dependent on combination of genotypes at multiple loci

Tyrosinase Function (involved with B locus)

• If tyrosinase low and cysteine high → Phaeo Melanogenesis (reddish yellow color)

• If tyrosinase high and cysteine low → Eumelanogenesis (Brownish/black color)

Brown (B) Locus

• B → dominant black. Dogs with at least 1 B with express black

• b → brown. Dogs with two bb will only have brown coat with no black

• TYR1P

Agouti (A) locus

• ay → fawn or sable. Dominant to other alleles

• aw → Wild-type agouti. Produces banded hairs, resulting in a wolf-grey or sable appearance

• at → tan point pattern. Causes tan or cream pattern specific to areas of the body.

• a → recessive black. Causes solid black coat color

  • OTHER: A^DY > A^SY > A^AG > A^BS > A^BB > A^a

    • DY (dom yellow)

    • SY (shaded yellow)

    • AG (agouti)

    • BS (black saddle)

    • BB (black black)

    • A (recessive black)

• ASIP

K Locus

• KB → dominant black. Dogs with at least 1 KB will express black

• ky → non-solid black. causes brindle, tan points, or other patterns. Requires 1 E to express

• kbr → brindle (needs two kbr)

• k → allows expression of other alleles at other loci, red or dilute

• Dominance: k^B > k^br > k^y

• CBD103

Dilution (D) locus

• D → Full pigmentation. No dilution of color

• d → dilution. Causes dilution of black pigment, resulting in blue (from black) or isabella (from brown)

• Causes from mutations in melanophilin (MLPH gene)

Spotting (S) locus

• S → no white spotting. Solid coat

• sp → piebald. causes white spotting on coat in specific patterns

• si → irish spotting. Results in white markings on the face, chest, belly, and feet.

• sw → extreme white (deaf and blind)

• MITF

Interaction of E and K loci

• If Ee or EE and k^br/k^br or k^br/k^y → will be brindle with the color of the phaeomelanin part of the brindling in turn affect by agouti alleles

• If Ee or EE and k^y/k^y → the distribution of eumelanin and phaeomelanin will be determined solely by the agouti alleles

Becker’s Muscular Dystrophy (BMD)

In-frame deletions in the DMD gene that allow synthesis of an internally truncated, but functional protein

Duchenne Muscular Dystrophy (DMD)

Mutations that cause premature translation termination (nonsense or frameshift) → loss of functional DMD gene product

• Dystrophin gene responsible

• recessive/fatal (X-linked) → affects males

What is the path of different types of muscular dystrophy in dogs?

Symptoms of MD in dogs, what causes different variations?

• Symptoms: chewing but dropping food, muscle stiffness, pneumonia, cardiac failure, enlargement of the tongue (hypertrophy)

• Why variation? Different mutations with different effects, gene x environment interactions.

Dystrophin

Maps to chrm. X → needed for mechanical stability of muscle cells

RNA splicing

Original mRNA copy of all exons and introns → before RNA moves from nucleus to cytoplasm → introns are removed and exons are spliced together

GT-AG rule

Introns begin with a GT and end with a AG. Mutations near either end of an intron may cause a change in splicing exons and produce abnormal protein

List the process for a frameshift mutation creating a stop codon in exon * of dystrophin in dogs

Base substitution in the splice-acceptor site of intron 6 of the dystrophin gene (AG to GG) → exon 7 is spliced along with intron 6 and intron 7 → deletion of exon 7 → stop codon in 8 → result: truncated transcript of dystrophin

“Treatments for MD”

Molecular: gene therapy/gene correction, gene editing using CRISP/cas9, Cellular stem cell transplants into muscle, pharmacologic compounds that reduce inflammation.

Exon Skipping therapy

Small pieces of DNA called antisense oligonucleotides (AOs) (molecular patches) → used to mask the exon that you want to skip → ignored in protein production.

Gene Editing types and challenges?

• Non-homologous end joining (NHEJ) or homology directed recombination (HDR)

• Germline editing not feasible → ethical issues, many mutations make it impractical to use for everyone, components must be delivered efficiently in vivo to skeletal muscles and heart

Cas9 Gene editing

Cas9 can cut DMD gene to have deletion or insertion of a few nucleotides to restore correct alignment of triplet codons

Ringo!

• Born with another mutation that protected him from MD!

  • Jagged1 plays important role in muscle regeneration 

  • Mutation in the promoter region caused upregulation of the gene in ringo

  • Overexpression of Jagged1: normal muscle phenotype

MHC Class 1 Formation Process

degradation of cell proteins by proteasomes into peptide fragments → fragments transport into lumen of ER where they are complexed with MHC 1 receptors → peptide-receptor complex is then transported to plasma membrane for antigen presentation to CD8+ T cells

MHC class 2 Formation process

Foreign peptides taken up by antigen presenting cells and degraded into small fragments → fragments complexed with MCH class 2 receptors and presented to t-helper cells → T-helper recognize the MHC-peptide complex which stimulates adaptive immune response.

MHC class 1: in infected vs uninfected cells

• Uninfected: peptides only derived from host cell proteins

• Infected: foreign peptide-MHC complex activates t-cytotoxic cells → T-cyto produce cytokines and other proteins to trigger apoptosis of the infected host cell

Peptides are not all equal which means…

Different shapes and amino acid sequences determine peptide binding → 2 different MHC alleles may bind to different peptide fragments → different individuals respond differently to same pathogen

Dendritic cells

Antigen-presenting cells of mammalian immune system. Function is to process antigen material and present it on the surface to T cells

MHC Class 1 vs MHC class 2

• Class 1:

  • present on cell surface of all nucleated cells

  • consists of 2 non-identical chains: alpha (chrm 6) and beta (chr 15).

  • Responsible for presentation of peptides to T-cells

• Class 2:

  • Present on the surface of antigen-presenting cells (B cells, macrophages, dendritic cells)

  • Consists of 2 identical chains (both on chr. 6)

  • Responsible for presentation of peptides to T-cells

Role of MHC in immune response process

MHC Present T cells a small fragment of pathogen → fragment held within a groove

Characteristics of the immune system

• Specificity: each antibody is specific for one antigenic determinant (epitope)

• Diversity: immune system can recognize >10 million different epitopes

• Memory: once exposed to antigen, the immune system remembers that antigen

• Distinguishing self from non-self

Immunoglobulins

Produced by B-cells: either found on the surface of B-cells (b-cell receptor or antigen receptor) OR secreted into extracellular fluid (antibodies)

Structure of Immunoglobulin G (IgG) molecule

The light chains have three domains: V (variable), J (joining), and C (constant). Heavy chains have 4 domains: V, D, J, C

Sources of immunoglobulin diversity

Gene rearrangements, class switching, nucleotide insertion/deletion, receptor editing, gene conversion, somatic hypermutation

Gene Conversion

Involves the transfer of genetic (sequence) info from one gene to another (basically copy and paste)

Myxomatosis info

Infects only rabbits. Caused by myxoma virus. Introduced into Australia to control rabbit infestation. Innocent rabbits are imported → CSIRO releases virus → almost all rabbits died due to high susceptibility and virulent pathogen → rabbits become resistant and virulence decreases → CSIRO releases new strains.

Trypanosomiasis

“Sleeping sickness.” caused by a parasite named Trypanosome. Become infected from bite of infected tsetse fly. Effects humans and animals and decreases production and fertility.

Trypanosomiasis early stages vs late stages

• Early: Onset occurs 1-3 weeks after bite. Fever, headache, general weakness, and weight loss occur. Multiple organs (liver, spleen, skin, eyes, etc) affected.

• Late: start to psychotic behaviors (violence, mania, suicide, etc.), motor, sensory, sleep abnormalities (wanting to sleep all the time and changes to sleep cycle).

Trypanosomiasis late stages

• Psychiatric, motor, sensory abnormalities, sleep disturbances.

• Psychiatric: personability changes, headache, violence, hallucinations, suicidal tendencies, mania

• Sleep disturbances: tiredness, distractibility, spontaneous urges to sleep, reversal of the normal sleep-wake cycle

Trypanosome Biology

Animals are reservoir for parasite. Flies become carrier after feeding on effected host, parasites undergo morphological and biochemical changes in fly’s anterior midgut.

Antigenic Variation

Parasite encapsulated by glycoprotein coat. Antigenic variation: the parasite changes its variant surface glycoprotein (VSG) coat to evade the immune system. Keeps parasite one step ahead. →Unexpressed VSGs are scattered among the different chromosomes.

→All expressed are located near telomeres.

→ telomeres contain expression sites (ES) under control of promoter, but only one ES can be active at a time

→ trypanosomes are preprogrammed to produce sequence of antigens (spontaneous)