Genetics 2: Use of Genetics in Clinical Work

Genetics 2: Use of Genetics in Clinical Work

• Lecturer: Emi Barker, BSc (Hons), BVSc (hons), PhD, PGCertTLHE, DipECVIM-CA, FRCVS

• Course: VETS10018/ASPL1/LEC

• Specialization: RCVS Recognised and EBVS® European Veterinary Specialist in Small Animal Internal Medicine, Clinical Lead in Infectious Diseases

Genotype vs. Phenotype

• Genotype: Two copies (alleles) of each gene inherited from parents
• Exception: Genes on the X or Y chromosome
• Phenotype: The expressed traits of cells/organisms
• Influenced by:
  • Genotype - the alleles present
  • Inherited epigenetic factors (e.g., DNA methylation)
  • Non-inherited environmental factors (e.g., temperature, cytokines)
  • All somatic cells in the same animal carry identical genes, but different cells have different functions and develop in different organs

Genetic Mutations/Variants

• Alterations to genetic code: Can increase, modify, or stop the production of a specific protein
• Coding sequence alterations: Easy to predict consequences on amino acid sequence and to a lesser extent on the individual
• Non-coding sequence alterations: More challenging, or impossible, to predict consequences

Basic Mammalian Genetics

• Dominance:
  • Dominant allele (usually) produces a functional protein.
  • Only one copy is needed to affect the phenotype.
  • Can be complete or partial (incomplete).
  • Recessive allele(usually) produces a reduced/faulty protein.
  • Both recessive copies needed to affect the phenotype.
  • Some alleles are "co-dominant".
  • Allele b may be recessive to allele B but dominant to allele b'.
• Dominance does not indicate: Whether an allele is beneficial, detrimental, or neutral

Example of Application: Basic Coat Color in Dogs

• Dominance/Recessive Allele Interaction:
  • Black/brown - controls type of melanin produced.
  • B is dominant and produces black eumelanin.
  • b is recessive and produces brown melanin.
  • Extension - controls the proportion of eumelanin and pheomelanin.
  • E extends the amount of eumelanin into the hair.
  • e reduces eumelanin and increases pheomelanin (yellow/red).
• Epistasis: Gene interaction where absence of extension results in yellow coat, but nose color reflects the type of melanin produced

Labrador Coat Color

• Black Labrador: B-, E-
• Brown Labrador: bb, E-
• Yellow Labrador (brown nose): bb, ee
• Yellow Labrador (black nose): B-, ee

Single Nucleotide Polymorphisms (SNPs)

• Definition: Sequence nucleotide differences at a single position within the genome.
• Inheritance: Passed down from one generation to the next.
• Applications:
  • Genome-wide association studies (GWAS) for detecting genes controlling traits.
  • Linkage markers to search for target genes.
  • Analysis of SNPs can be massively parallel, automated, and high-throughput.

Genome mapping in animals

• Purpose: Identify genes responsible for economic/clinical traits, model human disease
• Applications: Map genes in animal pedigrees, and eliminate affected individuals and carriers to avoid breeding. Marker-assisted selection use in conventional breeding, increasing the frequency of desirable traits, introgression from one population to another (e.g. Chinese pigs to European breeds).

DNA Sequencing (Sanger Technique)

• Based on the properties of dideoxynucleotides (ddNTPs).
• Modified nucleotides, containing a hydrogen group on the 3' carbon instead of a hydroxyl group (OH).
• When integrated into a DNA sequence, they prevent the addition of further nucleotides.
• Reactions with individual ddNTPs (ddGTP, ddATP, ddTTP, ddCTP), labelled with different fluorophores, are run separately on a gel.

DNA sequencing (Pyrosequencing)

• Combination of biochemistry, physics optics, and phenomenal computing power.
• Illumina MiSeq sequencer

Example of Application: Sensory Neuropathy in the Border Collie

• GWAS study: Manhattan plot used to compare two populations (one with trait, one without).
• Dots signify SNPs, with genomic coordinates on X-axis and negative logarithm of P values on Y-axis.
• Investigation focuses on candidate genes in the region.

Example of application: Sensory neuropathy in the Border Collie (cont.)

• Region containing: 27 genes were identified, one (FAM134B) is associated with hereditary sensory neuropathy.
• Sequencing of FAM134B exons: was unremarkable indicating a genomic inversion.
• 6.47MB inversion: located in intron 3 of FAM134B and an upstream intergenic region, and present in all affected dogs (no controls), which were homozygous for this inversion

Point Mutations/Variants

• Definition: Insertion, deletion, or substitution of base pairs. (Note: not all are SNPs).
• Consequences of exonic mutations:
  • Silent: No change in amino acid sequence.
  • Missense: different amino acid is encoded. -Nonsense: premature STOP codon is introduced. -Frame shift: Change in amino acid sequence from that point on.
• Consequences of intronic mutations:
  • No changes.
  • Altered splicing sites: Change in amino acid sequence
  • Altered gene expression (increased or decreased).

Autosomal Dominant

• Characteristics: Defect seen in every generation; every affected offspring has at least one affected parent; equal numbers of males and females affected.
• Segregation ratio: 0.5
• Examples: Feline polycystic kidney disease and Scottish fold osteodystrophy.

Autosomal Recessive

• Characteristics: Defect may skip a generation.
• "Normal" parents of an affected individual must be carriers. Equal numbers of affected males and females.
• Segregation ratio: 0.25
• Examples: Hyperuricosuria in Dalmatian dogs, and Avermectin sensitivity in Collie dogs

X-linked Dominant

• Characteristics: Every affected offspring has at least one affected parent; Affected males transmit the defect to all their daughters; Affected females will be heterozygous.
• Examples: X-linked dominant hereditary nephritis in Samoyeds

X-linked Recessive

• Characteristics: Defect may skip a generation; Incidence in males is higher than in females when the defect is rare.
• Affected individuals: Will be males who inherited the gene from the dam
• Examples: Haemophilia A in Havanese dogs

Why Perform Genetic Screening?

• Identify carriers: By gradually removing affected/carrier individuals from the gene pool.
• Identify affected animals: In cases of late-onset diseases and to counsel owners.
• Select desirable traits: In cases of recessive/polygenic traits, via directed matings of animals with desirable traits.

How to Perform Genetic Screening?

• Sample collection: Cheek swab (less invasive), blood sample (invasive).
• Laboratory submission: Microchip ID recording for official records, specific tests for breeds.
• Interpreting results: Interpreting results may be reported as normal/homozygous or carrier/Affected.

Interpreting Results

• Reporting results: Can be in terms of normal/homozygous 'wild type' alleles, carrier (heterozygous for recessive allele) or Affected(homozygous for recessive/heterozygous or homozygous for dominant allele).
• Planning with Results: Affected individuals should be monitored, for disease progression and owners should be warned if affected animals should not be bred from, but if heterozygous for dominant allele, breeding programs can be used cautiously, affected individuals should not be bred from.

Predicting Offspring: Normal (AA) x Carrier (Aa)

• Predicted Ratios: 50% predicted to be 'normal', 50% predicted to be carriers

Predicting Offspring: Carrier (Aa) x Carrier (Aa)

• Predicted Ratios: 25% predicted to be 'normal', 50% predicted to be carriers, 25% predicted to be 'affected'

Genetic Screening - Unknown Mutation...

• Determination of carrier status:
• Backcross known homozygous recessive (aa) or to a carrier (Aa).
• Probability calculation of if no affected offspring, then the test animal is likely not a carrier.
• If affected offspring are born it indicates the test animal is a carrier.

Blood Groups

• Definition: Classification of blood based on the presence/absence of inherited antigenic substances on red blood cells. Antigens can be proteins, carbohydrates, glycoproteins, or glycolipids; Several of these antigens form a blood group system.

Feline Blood Groups

• System: AB blood group system (independent of human ABO system).
• Alleles: Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). B antigen (N-acetylneuraminic acid) and A antigen (glycolylneuraminic acid).
• Alloantibodies: Type B cats have strong anti-A antibodies. Type A cats may have weak anti-B antibodies.
• Blood Types Prevalence: Varies between breeds and countries.

Canine Blood Groups

• Types: 13 different loci, eight DEA (Dog Erythrocyte Antigen) types.
• Naturally occurring antibodies: against DEA 3, 5, and 7 types.
• Antibody-antigen interactions: DEA 1 - acute hemolytic transfusion reactions; DEA 3, 5, and 7 - Permanent red blood cell sequestration; loss in 3-5 days; DEA 4 - No effect in vitro.

Blood Groups in Other Species

• Horses: Complex system of 7 systems (A, C, D, K, P, Q, and U). The two most potent antigens are Aa and Qa.
• Cattle: Complex system of 12 different systems. Groups of alleles act as multiple antigenic determinants and are called phenogroups.

Neonatal Isoerythrolysis

• Mechanism: Dam produces antibodies against offspring RBCs, absorbed from colostrum within 24-48 hours after birth. Life-threatening hemolysis occurring.
• Problem in Horses and Cats: Primarily a concern in horses and cats. Maternal antibodies against offspring blood type, can cause life-threatening hemolysis/red blood cell destruction, primarily in newborn animals.

Avoiding Neonatal Isoerythrolysis

• Assessment: History/Blood types, breeds known to have high incidences of type B cats.
• Reduction risk: Avoid mixing mismatched blood types (e.g. Aª negative dams and Aª positive sires in horses).
• Test offspring at birth: Safe offspring treated as normal; at risk offspring hand-reared for 24-48 hours or fostered.

Online Resources for Genetic Testing

• Langford Vets - Cat Genetics: website link.
• Veterinary Genetics Laboratory: website link.
• Laboklin: website link.

Summary (slide 34)

• Genotype vs. Phenotype: Basic canine coat color as an example.
• Single nucleotide polymorphisms (SNPs): GWAS.
• Point mutations/variants.
• Genetic screening: Predicting likelihood of affected/carrier offspring.
• Blood group genotyping: Provided as an example.

Test your understanding of key concepts in genetics and inheritance with this quiz. It covers topics such as genetic coding, dominant and recessive alleles, X-linked disorders, and the implications of genetic alterations. Perfect for students studying genetic principles in biology.