Genetics 2: Use of Genetics in Clinical Work PDF

Genetics 2: Use of Genetics in Clinical Work VETS10018/ASPL1/LEC Emi Barker BSc (hons) BVSc (hons) PhD PGCertTLHE DipECVIM-CA FRCVS RCVS Recognised and EBVS® European Veterinary Specialist in Small Animal Internal Medicine Clinical Lead in Infectious Diseases [email protected] Summary...

Genetics 2: Use of Genetics in Clinical Work VETS10018/ASPL1/LEC Emi Barker BSc (hons) BVSc (hons) PhD PGCertTLHE DipECVIM-CA FRCVS RCVS Recognised and EBVS® European Veterinary Specialist in Small Animal Internal Medicine Clinical Lead in Infectious Diseases [email protected] Summary Intended Learning Outcomes: Be able to define genotype and phenotype – and explain the  Genotype vs. difference between the two Be able to use basic coat colour as an example of differential phenotype gene expression (dominance, recessive, epistaxis,  Single nucleotide lyonization) Be able to explain what a single nucleotide polymorphisms is polymorphisms and how it can be used in genome mapping List the uses of genome mapping  Point mutations / List the types of point mutations / variants, list the variants consequences of these mutations on translation and transcription and provide examples of each  Genetic Be able to list the uses of genetic screening Be able to predict likelihood of affected / carrier in offspring, screening based on parental genotype, and parental genotype based on offspring phenotype Have a basic understanding of blood groups and why they are important Understand benefits and limitations of genotyping for feline blood type Genotype vs. phenotype  Each individual has two copies (alleles) of each gene, one inherited from each parent (= genotype) – Exception: if the gene is on the X or Y-chromosome  All somatic cells in the same animal carry identical genes – but different cells have different functions and develop inside different organs  The expressed phenotype of cells / organisms influenced by: – Genotype – the alleles present – Inherited epigenetic factors (e.g. DNA methylation) – Non-inherited environmental factors (e.g. temperature, cytokines) Genetic mutations / variants  Alterations to the genetic code can increase, modify or stop the production of a specific protein  Consequences of alterations to the coding sequence of the genetic code on the consequential amino acid sequence can be easy to predict, and to a lesser extent the effect on the individual  Consequences of alterations to the non-coding sequence are much more difficult (to impossible) to predict Basic mammalian genetics  Dominance – Dominant allele (usually) produces a functional protein  Only one copy required to affect the phenotype  May be complete or partial (incomplete) – Recessive allele (usually) produces a reduced / faulty protein  Both recessive copies required to affect the phenotype – Some alleles are ‘co-dominant’  Allele b may be recessive to allele B but dominant to third allele b’  Dominance does not indicate whether an allele is beneficial, detrimental, or neutral Example of application: Equivalent genes are found Basic coat colour in dogs in many other mammalian species  Dominance / recessive allele interaction – Black/brown – controls type of eumelanin produced  B is dominant and produces black eumelanin  b is recessive and produces brown eumelanin – 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 – i.e. gene interaction – Absence of extension – results in yellow coat but the nose colour will reflect the type of eumelanin produced  Labrador coat colour Yellow Brown Labrador Labrador (brown nose) bb, E- bb, ee Yellow Labrador Black Labrador (black nose) B-, E- B-, ee Single nucleotide polymorphisms (SNPs)  At any single position within the genome sequence nucleotide differences may occur  These differences can be passed down from one generation to the next and their presence used as (linkage) markers for the presence of nearby genes controlling traits of interest – Genome-wide association studies (GWAS) using SNPs can focus the search for target genes – Analysis of SNPs can be massively parallel, automated and high-throughput Genome mapping in animals  Identify the gene(s) responsible for a genetic trait of economic or clinical importance – Model for human disease – Map genes in animal pedigrees – Eliminate affected individuals and carriers from the gene pool  Marker-assisted selection – Use in conventional breeding programmes to increase frequency of desirable traits – Introgression from one population into another e.g. introducing fecundity genes from Chinese pigs into European breeds DNA sequencing using Sanger technique  Based on properties of dideoxynucleotide (ddNTPs) – Modified nucleotides that contain 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 thus stop the elongation of the DNA chain  Reactions containing individual ddNTPs (i.e. ddGTP, ddATP, ddTTP and ddCTP) can be run separately on a gel or labeled with different fluorophores and processed in the same reaction A C G T DNA sequencing using 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 – Two populations compared – one with target trait, the other without – Each dot signifies a SNP – Genomic coordinates are on the X-axis, with the negative logarithm of the association P- value for each SNP on the Y- axis – Investigation can then be focused on candidate genes in this region Example of application: Sensory neuropathy in the Border Collie  Region contained 27 genes  One was FAM134B (associated with hereditary sensory neuropathy in humans) = candidate gene  Sequencing of FAM134B exons (coding sequence) was unremarkable  A 6.47MB inversion was identified with breakpoints in intron 3 of FAM134B and in an upstream intergenic region  All affected dogs (no controls) were homozygous for this inversion Point mutations / variants  Insertion, deletion, or substitution of base pairs (NB: not all are SNPs)  Alteration of the exonic sequence can result in: – Silent – no change to the amino acid sequence – Missense – a different amino acid is encoded – Nonsense – a premature STOP codon is introduced – Frame shift – change in amino acid sequence from that point on  Alteration of the intronic sequence can result in: – No changes – Altered splicing sites – change in the 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 – Normal offspring from affected parents will produce normal offspring – Equal numbers of male and females will be affected – If the defect is rare, but not lethal:  Most matings that produce an affected individual will be aa x Aa  Segregation ratio will be 0.5  Examples – Feline polycystic kidney disease – nonsense point mutation in PKD1 – Scottish fold osteodystrophy (incomplete penetrance) – missense point mutation in TRPV4 Autosomal recessive  Characteristics – Defect may skip a generation – All offspring of affected parents (where both affected) will be affected – “Normal” parents of an affected individual must be a carrier – Equal numbers of male and females will be affected – If the defect is rare, but not lethal:  Most matings which produce an affected individual will be Aa x Aa  Segregation ratio will be 0.25  Examples – Hyperuricosuria in Dalmatian dogs – missense point mutation in SLC2A9 – Avermectin sensitivity in Collie dogs – frame-shift mutation (4bp deletion) in ABCB1 X-linked dominant  Characteristics – Every affected offspring has at least one affected parent – Affected males (i.e. XAY-) mated to normal females (i.e. XaXa) will transmit the defect to all their daughters and none of their sons – Unless the defect is very common:  Affected females will be heterozygous (i.e. XAXa) and therefore when mated to a normal male the defect will be transmitted to half the offspring  Example – X-linked dominant hereditary nephritis in Samoyed – nonsense point mutation in COL4A5 Sedwards63 CC BY-SA 4.0 X-linked recessive  Characteristics – May skip a generation – Incidence in male >> female – When the defect is rare:  Affected individuals will be males and will have inherited the gene from the dam  Example – Haemophilia A (Factor VIII deficiency) in Havanese dogs – SINE (short interspersed nuclear element) insert in F8 – The causative mutation of haemophilia in most other breeds (including cocker spaniels) is unknown Why perform genetic screening?  Identify carriers – e.g. recessive disorders – Gradually remove from the gene pool – Avoid mating two carriers – reduce risk of breeding affected individuals  Identify affected – e.g. with late onset presentation – Remove affected cats from the gene pool / counsel owners – Avoid mating affected / carriers – reduce risk of breeding affected cats  Select for desirable traits that are recessive / polygenetic – Direct mating to increase likelihood of obtaining desirable offspring How to perform genetic screening?  Collect a sample containing DNA from patient: – Collected by vet or owner – Cheek swab (contamination risk if suckling) or blood sample (invasive) – Might need to record microchip ID at time of sampling (official record)  Submit sample to laboratory: – Currently most tests are for individual mutations / variants  Specific to species and may be specific to breed  NB: may be more than one mutation / variant per phenotype  Some (most) mutations / variants are unknown  Interpret results Interpreting results  Results may be reported as: – Normal or homozygous ‘wild type’ alleles – Carrier (heterozygous for recessive allele) – Affected (homozygous for recessive allele; heterozygous or homozygous for dominant allele)  Forward planning – Affected individuals should be monitored for disease progression; prospective owners should be warned – Affected individuals should not be bred from (potentially difficult if heterozygous affected for dominant allele ⇒ reduced gene pool) – Carrier animals can be used cautiously in breeding programmes  NB: Whilst it is possible to say Predicting offspring… which genotypes may or may not be present and give an idea of the likely ratios it is NOT possible to be  Normal (AA) X Carrier (Aa) specific  Predicted ratios – 50% predicted be ‘normal’ A a – 50% predicted to be carriers AA Aa A AA Aa A  NB: Whilst it is possible to say Predicting offspring… which genotypes may or may not be present and give an idea of the likely ratios it is NOT possible to be  Carrier (Aa) X Carrier (Aa) specific  Predicted ratios – 25% predicted be ‘normal’ A a – 50% predicted to be carriers AA Aa – 25% predicted to be A ‘affected Aa aa a Genetic screening – unknown mutation…  Determination of carrier status of normal animal (i.e. AA or Aa) – Backcross to a known homozygous recessive (‘aa’)  If seven or more normal (A-) offspring are born >99% likelihood that the test animal is not a carrier (i.e. if Aa x aa  all Aa = 0.57 = 0.008) – Backcross to a known heterozygous carrier (‘Aa’)  If sixteen normal (A-) offspring are born  99% likelihood that the test animal is not a carrier (i.e. if Aa x Aa  all AA or Aa = 0.7516 = 0.010) – If any affected (homozygous recessive, aa) offspring are born to either combination  test animal is a carrier Blood groups  A blood type is a classification of blood – Based on the presence and absence of antibodies and inherited antigenic substances on the surface of red blood cells (RBCs) – Antigens may be proteins, carbohydrates, glycoproteins, or glycolipids (which may also be present on the surface of other cell types) – Several of these red blood cell surface antigens can stem from one allele and collectively form a blood group system  Mixing of blood with even only minor antigenic differences differences has the potential to result in significant reactions – Transfusion reactions – Neonatal isoerythrolysis Feline blood groups  AB blood group system [NB: completely independent of human ABO system] – Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) – ’B’ antigen (acetylneuraminic acid)  ’A’ antigen (glycolylneuraminic acid) – Two known alleles – A and b (null; recessive) – Rarely type AB cats – both A and B antigens expressed – Alloantibodies  All type B cats have strong anti-A antibodies (without prior exposure)  ~⅓ type A cats have weak anti-B antibodies  Mik red cell antigen – Cats are either positive or negative – Appears the be rare in the UK https://icatcare.org/advice/blood-groups-and-blood-incompatibility/ Feline blood groups prevalence  Amongst domestic shorthairs: – 77-95+% of cats are type A – permanent red blood cell sequestration, loss in 3-5 days – DEA 4 => no effect in vitro Blood groups in other species  Horse – Complex; 7 systems – A, C, D, K, P, Q and U – The two most potent antigens are Aa and Qa – Horses do not normally have alloantibodies from birth  Cattle – Complex; 12 different systems and each system can have many alleles – Groups of alleles act as multiple antigenic determinants and are called phenogroups Neonatal isoerythrolysis  Mechanism – Dam produces antibodies against the offspring’s RBC – Newborn absorbs these antibodies from the colostrum within the first 24-48 hrs following birth – Life-threatening haemolysis occurs  Mainly a problem in the horse and cat – Horse – mating a group Aa negative dam to a group Aa positive sire producing a group Aa positive foal (usually requires prior exposure) – Cat – mating a type B queen to a type A stud cat producing type A kitten(s) (NB: this can happen at first mating) Avoiding neonatal isoerythrolysis  Assess potential – blood type breeding animals if: – History of neonatal isoerythrolysis, – (In cats) if breed has a high incidence of type B (e.g. BSH; Devon Rex)  Reduce risk – avoid combinations with potential to mismatch: – Mate Aa negative stallions with Aa negative mares – Mate type B queens to type B stud cats (but… this is a faff, makes type B cats ‘less desirable’, and limits gene pool)  Assess risk – 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 – https://www.langfordvets.co.uk/diagnostic-laboratories/cat-genetic- faqs/  Veterinary Genetics Laboratory – https://vgl.ucdavis.edu/  Laboklin  https://www.laboklin.co.uk/laboklin/GeneticDiseases.jsp Summary  Genotype vs. phenotype – Basic canine coat colour as an example  Single nucleotide polymorphisms – GWAS  Point mutations / variants  Genetic screening – Predicting likelihood of affected / carrier in offspring – Blood group genotyping as an example Any Questions or Feedback? If you have any questions relating to this series of lectures – please post to the relevant padlet (chances are someone else is thinking the same thing!). Feedback is also gratefully received – things to improve, things you would like more of, things that did or didn’t work well – either anonymously on the padlet or feel free to email if you have specific queries [email protected] https://uob.padlet.org/emibarker/emi-barker-s-24-25-root-padlet-30nns46t9hq6p8n8