Canine Genetics: Alleles, Inheritance, and Testing

Basics

• Focuses on definitions, gene and protein production (transcription, translation)
• Examines gene mutations in coding and non-coding regions, regulatory mechanisms like Nonsense-Mediated mRNA Decay
• Covers modes of inheritance: autosomal recessive, dominant, X-linked, random X inactivation (lyonization), and mitochondrial inheritance
• Discusses complex traits, pharmacogenetics, and the process of identifying mutations using GWAS, DNA sequencing, and various genetic markers
• Includes genetic disease testing (limitations, sample collection, mutation detection, linked-marker tests, multiplex testing) and breeding strategies

Canine Genetics

• Dogs possess 38 pairs of autosomal chromosomes and 2 sex chromosomes
• Females are homogametic (XX), while males are heterogametic (XY), opposite of birds

Alleles and DNA

• Alleles are versions of a gene located on chromosomal DNA
• Individuals inherit two alleles (one maternal, one paternal) for each DNA segment on autosomal chromosomes
• DNA comprises four nucleotides: adenine (A), guanine (G), thymine (T), and cytosine (C)
• Complementary base pairing: A pairs with T, and C pairs with G, ensuring DNA stability and replication

Phenotype

• Result of the interaction between genetics and environment, influencing an individual's characteristics
• Includes physical traits and pathological conditions
• Background genetic effects from interacting genes can alter the disease phenotype

Penetrance

• 100% penetrance means all individuals carrying a disease allele will express the disease

Mapping Genetic Traits

• Involves identifying traits inherited in a Mendelian fashion, like dominant versus recessive alleles segregating during gamete formation
• Utilizes markers like sets of microsatellites evenly positioned across the genome
• Microsatellites are repeated nucleotide stretches acting as markers

Marker Loci and Variations

• Marker loci are located near candidate genes for inherited diseases
• Microsatellite variations arise from replication errors during meiosis
• Single-nucleotide polymorphisms (SNPs) represent single nucleotide variations at particular locations

Genes and Protein Production

• Involves two main steps; transcription and translation

Transcription

• DNA is transcribed into messenger RNA (mRNA)

Exons and Introns

• Exons are coding sequences, while introns are noncoding regions
• During mRNA formation, introns are removed through splicing, retaining only coding exons
• Alternative splicing allows different protein isoforms to be produced from a single gene

Roles of Introns

• Promoters are noncoding sequences regulating gene expression (how much and when a gene is expressed)
• Functional noncoding RNAs, including rRNA, tRNA, and microRNAs, regulate mRNA stability and translation

Short Interspersed Nuclear Elements (SINEs)

• Mobile elements propagate via a "copy and paste" mechanism
• Insertion into a gene-coding region can alter gene function
• E.g., the merle coat pattern results from SINE insertion in the SILV gene
• E.g., PRA in Tibetan Terriers/Spaniels results from SINE insertion in the FAM161A gene

Translation

• mRNA is translated into protein, with mRNA moving to ribosomes

Codons

• The DNA code after translation into mRNA is read in three-nucleotide groups called codons
• Each codon corresponds to a specific amino acid or a stop signal
• AUG is the start codon, coding for methionine, and there are also stop codons
• Multiple codons can code for the same amino acid

Gene Mutations

• Involves changes in the DNA sequence within coding regions
• Arises from external factors (radiation, mutagenic chemicals), internal factors (replication errors), or insertion of retrotransposons like SINEs

Mutation Types

• Missense mutations change one amino acid, potentially altering protein function
• Nonsense mutations convert an amino acid codon into a stop codon, causing premature termination
• Silent mutations do not affect the protein due to no change in the amino acid sequence
• Insertions and deletions can cause frameshift mutations, altering the sequence and leading to premature stop codons

Disease from Changes in Noncoding Regions

• Mutations in regulatory regions can reduce gene expression
• Intron mutations can interfere with mRNA splicing, altering exon joining
• E.g., CEP290 gene mutation results in autosomal recessive PRA in Abyssinian cats by disrupting splicing

Nonsense-Mediated mRNA Decay

• A cellular process that degrades mRNA with premature stop codons, preventing shortened or malfunctioning proteins
• More likely if the stop codon is located more than 50 nucleotides upstream from the final exon

Autosomal Recessive Inheritance

• Requires two copies of the mutated gene on autosomes for the condition to be expressed
• Heterozygotes (carriers) typically show no symptoms
• Homozygotes develop the disease
• Disease can skip generations
• Mutations leading to loss-of-function often lead to recessive conditions
• A biochemical pathway mutation where a reduced protein level in heterozygotes does not cause disease (haplosufficient)

Autosomal Dominant Inheritance

• Requires only one copy of the mutated gene on an autosome for the condition to be expressed
• Does not skip generations if the mutation is fully penetrant
• Mutation can affect structural or developmental genes, or result in dominant-negative mutations where the mutated protein affects normal function
• Haploinsufficiency, where one gene copy isn't sufficient for normal function, leads to dominant disease
• E.g., CRX mutation in Abyssinian cats

X-Linked Inheritance

• X-linked recessive inheritance affects males (XY) more than females (XX), as females need two mutated copies to express the condition
• X-linked dominant inheritance affects both males and females with one copy of the mutated gene
• In birds, males are homogametic (ZZ) and females are heterogametic (ZW)
• Males have only one X chromosome (hemizygous = only 1 copy of gene)

Random X Chromosome Inactivation (Lyonization)

• In female mammals, one X chromosome is randomly inactivated in each cell to prevent double expression of X-linked genes
• Tortoiseshell cats exhibit coat color variation due to different X chromosomes being inactivated
• Female carriers of X-linked PRA in dogs exhibit patches of retinal cells showing inactivation of the X chromosome

Mitochondrial Inheritance

• Mutations in mitochondrial DNA are inherited only from the mother, leading to maternally inherited conditions
• Leber hereditary optic neuropathy is an example

Complex Traits

• Influenced by multiple genetic and environmental factors
• PRA in Miniature Longhaired Dachshunds is influenced by RPGRIP1 mutations and additional genetic loci
• Head conformation in dogs (entropion, ectropion) involves multiple genes

Quantitative Trait Loci (QTL)

• Genetic loci contribute to traits showing a range of variation
• Traits with continuous variance like body conformation
• QTLs influence the progression of retinal diseases like PRCD

Pharmacogenetics

• Focuses on how genetic variation affects an individual's response to drugs
• Genetic sensitivity to neurotoxic drugs like ivermectin in dogs can lead to severe side effects

Phenotyping and Pedigree Analysis

• Involves careful observation and analysis of affected animals' traits and family history
• Helps determine if a condition is simple (Mendelian) or complex

Candidate Gene Screening

• Analyzes genes suspected of causing a disease based on known functions or analogies to similar diseases in other breeds/species

Genetic Markers

• Uses microsatellites or SNPs closely linked to candidate genes to identify disease status

Gene Sequencing

• Encompasses GWAS and DNA sequencing

Genome-Wide Association Studies (GWAS)

• Technology uses SNP microarrays to detect genetic variations across the genome
• Identifies associations between SNPs and diseases by comparing affected versus control animals
• High-density SNP arrays can genotype over 1 million markers across the canine genome

Sanger Sequencing

• Sequencing small DNA segments
• Ideal for confirming mutations in candidate genes

Next-Generation Sequencing (NGS)

• Allows sequencing of part or the entire genome
• Identifies genes expressed in a particular disease (RNA-seq)
• Commonly used for sequencing numerous short DNA strands

Exome Sequencing

• Sequences exons and flanking introns
• Lower cost and identifies coding region changes and intron/exon splice site mutations
• Does not identify other noncoding disease-causing variants

Targeted Sequencing

• Sequences a specific genomic region of interest
• Useful for diseases mapped to particular chromosomal areas
• Involves isolating RNA from the tissue of interest and then sequencing
• Transcriptome provides info on actively expressed genes in the tissue

Improved Sequencing Technologies

• Offer greater accuracy at lower costs, aiding in identifying disease-causing mutations
• Applied to both genomic and transcriptomic data
• Enhances our understanding of disease mechanisms and enables personalized medicine

Genetic Testing Limitations

• Limited to identifying one specific form of genetic mutation
• Genetic conditions can have multiple forms and different genetic mutations
• Tests for one form of PRA may not detect other forms
• Test results can be complicated when conditions involve more than one locus

Sample Collection

• DNA samples are usually blood or cheek swabs
• Requires proper handling and clear labeling to avoid contamination or misidentification

Mutation Detection Tests

• Identify DNA alterations causing genetic traits or diseases
• Accurate and reliable for diagnosing conditions where the mutation is known
• Only identify one form of disease

Linked-Marker Tests

• Used when the mutation causing a disease is unknown
• Detect regions surrounding the mutation
• Involves a shared region of the chromosome linked to the disease mutation
• Accuracy is affected if the marker is not closely linked or if crossover happens during meiosis

Multiplex Testing

• Uses custom genotyping arrays to detect various disease-causing mutations and genetic variations
• Cost-effective and may reveal previously unrecognized diseases in certain breeds
• Genetic counseling aids dog owners and breeders in interpreting findings and making informed decisions

Selective Breeding

• Using carriers for breeding helps preserve genetic diversity while reducing disease incidence
• Selectively breeding can separate desirable traits from disease-causing genes, preserving genetic health and breed characteristics