Petit Basset Griffon Vendéen Club of America does not provide specific medical advice, but rather provides users with information to help them better understand health and disease. Please consult with a qualified health care professional for answers to medical questions.
By Dr. Cathryn Mellersh, Animal Health Trust
November 2011 (Reprint with permission from Dr. Mellersh)
What Is a Carrier?
‘Carrier’ is the term given to an individual (of any species) that carries a single copy of a recessive mutation that is associated with a specific inherited condition, usually an inherited disorder. An individual will only suffer from a recessive disorder if it inherits two copies of the causal mutation, one from each parent. If it inherits a single copy of the mutation it will remain healthy but will pass the mutation on to about half of its offspring.
Breeding with Carriers
Once a specific disease mutation has been identified a DNA test can be developed that enables the identification of non-symptomatic carriers. Knowing which dogs carry the mutation and which don’t (the so-called ‘clear’ dogs) enables breeders to make sensible choices about the dogs they mate together. All dogs can be safely bred with provided at least one of the mating pair is clear of the mutation (see Table below). Breeding dogs that will never develop the condition should obviously be the priority for all conscientious breeders and the desire to eliminate a disease-associated mutation from a breed should therefore be the long-term goal. But the instinct to choose only clear dogs to breed from, as soon as a DNA test becomes available, may not always be a sensible choice and the rest of this document discusses why.
If carriers are prevented from breeding, the opportunity to pass the rest of their genetic material to the next generation is also lost and the genetic diversity of the remaining population is thus reduced. It is worth remembering that there is a clear and well-established link between the genetic diversity of a population and its overall health, and that breeding closely related individuals tends to lead to the accumulation of deleterious recessive mutations in the population. This is due to the fact that an individual is more likely to inherit two identical copies of a mutation if its parents share common ancestors than if they are unrelated, and the more common ancestors the parents share the greater that chance is.
It is also worth remembering that the disease mutation for which there is a DNA test is not the only mutation a carrier has. Every human, on average, carries about 50 recessive mutations and there is no reason to believe the average dog won’t carry a similar number. So the only real difference between a clear and a carrier is the single mutation that can be tested for. Both dogs will both carry around 49 other mutations that the breeder doesn’t know about and can’t test for. If carriers are not bred from and clear dogs are used extensively then there is a real risk that other mutations will increase in frequency in the breed and new inherited disease(s) could emerge.
There is no reason why the eventual elimination of a disease mutation from a breed shouldn’t be the goal, once a DNA test for that mutation becomes available. But, providing all breeding dogs are tested for the mutation prior to mating, the breeders can take their time and ensure that desirable traits are not eliminated along with the disease mutation and that the genetic diversity of the breed is not reduced.
The speed with which the mutation can be eliminated depends on several factors, including the frequency of the mutation, the population structure and the rate of inbreeding for that breed. The more frequent the mutation is the more slowly it should be eliminated. Calculating the true frequency of a mutation is not trivial, and requires a random subset of a breed be screened. Dogs that are tested once a commercial DNA test becomes available are not always representative of the breed as a whole, and similarly cohorts of dogs that have been sampled by a research institute during development of the DNA test are also rarely characteristic of the breed.
The frequency of a mutation is typically expressed as the fraction of chromosomes in a population that carry the mutation. For example, if the frequency of a mutation is described as 0.1, this means that 10% of the chromosomes in that breed carry the mutation and the remaining 90% carry the normal copy of DNA. If 10% of the chromosomes carry the mutation then just under 20% of dogs are expected to be carriers and about 1% of dogs will be affected.
Carriers should always be included in the first one to two generations that follow the launch of a DNA test for a recessive mutation, regardless of the frequency of the mutation, to give breeders the opportunity to capture desirable traits, such as breed type and temperament, before they start to select for dogs that are clear of the mutation. Specific breeding policy for future generations should be breed-dependent and ideally formulated after consideration of factors such as the population structure and rate of inbreeding. But in general terms, carriers should only be removed from the breeding population if the frequency of the mutation is below 0.01 (1%), as this will mean only around 2% of dogs will be prevented from breeding. Avoiding carriers of a mutation that is more frequent will result in a greater number of dogs being prevented from breeding and could lead to a detrimental loss of diversity for the breed.
(PBGVCA does not provide specific medical advice, but rather provides users with information to help them better understand health and disease. Please consult with a qualified health care professional for answers to medical questions.)
Kennel Club Genetics Centre, Animal Health Trust, Newmarket Suffolk, CB8 7UU, United Kingdom,
Department of Clinical Science & Services, Royal Veterinary College, University of London, Hawkshead Lane, Hatfield, Hertfordshire, AL9 7TA, United Kingdom, 3 Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, Veterinary Medical Center, 736 Wilson Road, East Lansing, MI, 48824–1314, United States of America
Closed breeding populations in the dog in conjunction with advances in gene mapping and sequencing techniques facilitate mapping of autosomal recessive diseases and identification of novel disease-causing variants, often using unorthodox experimental designs. In our investigation we demonstrate successful mapping of the locus for primary open angle glaucoma in the Petit Basset Griffon Vendéen dog breed with 12 cases and 12 controls, using a novel genotyping by exome sequencing approach. The resulting genome-wide association signal was followed up by genome sequencing of an individual case, leading to the identification of an inversion with a breakpoint disrupting the ADAMTS17 gene. Genotyping of additional controls and expression analysis provide strong evidence that the inversion is disease causing. Evidence of cryptic splicing resulting in novel exon transcription as a con- sequence of the inversion in ADAMTS17 is identified through RNAseq experiments. This investigation demonstrates how a novel genotyping by exome sequencing approach can be used to map an autosomal recessive disorder in the dog, with the use of genome sequencing to facilitate identification of a disease-associated variant.
It is well documented that population structure in the purebred dog can help to facilitate genome-wide association study (GWAS) approaches . The development of most modern breeds within the last 200 years from small numbers of founding individuals has led to high levels of linkage disequilibrium (LD) within breeds. These high levels of LD lead to very strong signals of association being produced from GWASs for autosomal recessive diseases, even with very modest sample numbers . Closed breeding populations, high levels of inbreeding and the extensive use of popular sires (dogs that closely fit the standard for a particular breed) can lead to rapidly emerging autosomal recessive disorders, as rare deleterious alleles are rapidly amplified. An example of an emerging autosomal recessive disorder is primary open angle glaucoma (POAG) in the Petit Basset Griffon Vendéen (PBGV).
The first recognised case of POAG in the PBGV was identified in the United Kingdom in 1996 and recent survey work completed in 2014 has demonstrated a 10.4% prevalence for the disease (personal communication, Peter Bedford). The initial clinical features of POAG are usually seen in 3 to 4 year old dogs of either sex, the disease being characterised by a small, sustained rise in intraocular pressure (IOP) and lens subluxation. In approximately one third of affected dogs phacodonesis and the appearance of the aphakic crescent associated with lens subluxation are seen before a noticeable rise in IOP (Fig 1). There is no pectinate ligament abnormality and the iridocorneal angle remains open until the late stages of the disease, when globe enlargement has developed. Retinal degeneration and a cupping deformation of the optic papilla are only seen in late disease. Pain is not a feature and the quiet, chronic clinical nature of this disease means that often owners only become aware of the presence of POAG when either the globe enlargement or a vision problem becomes noticeable.
As POAG is an autosomal recessively inherited disease, mapping of which are facilitated by the high levels of LD described, we designed a novel GWAS approach using genotyping by exome sequencing methodology with 12 cases and 12 controls with the dual aim of identifying both the disease-associated locus and causal variant for POAG through a single experiment.
Results Genome-wide association study by exome sequencing (POAG)
Exome sequencing was carried out using a commercially available human exome capture kit to capture the exomes of 12 POAG cases and 12 breed matched control dogs. Illumina sequencing produced a 15.0 Gb dataset of 250 bp paired-end reads (sufficient for low coverage of ~5x). Alignment to the canine reference sequence CanFam3.1 and variant calling across all 24 individuals identified a total of 841,115 SNP and indel calls (variants). After filtering variants with a minor allele frequency (MAF) of less than 5% and genotyping frequency (GF) of less than 80%, 61,977 remained.
Basic allele association analysis identified a single signal of genome-wide significance on canine chromosome 3 (praw = 6.15×10-10)(Fig 2). The genomic inflation factor (based on median chi-squared) was 1.34. Correction for the effects of population substructure was performed using a mixed model approach (EMMAX)  and the strong single signal on chromo-some 3 remained (p = 1.34×10-9)(S1 Fig). The adjusted genomic inflation factor (based on median chi-squared) was 1.04.
Visual analysis of the raw genotyping data revealed a disease associated interval of chr3:40,153,292–47,300,360 based on the CanFam3.1 genome build (Fig 3). All cases were homozygous for the disease-associated haplotype. The disease-associated interval contained 28 genes, including ADAMTS17, a potential glaucoma candidate gene. A list of interval genes can be found in S1 Table. As all cases were homozygous for the disease-associated haplotype the exome sequencing datasets were combined for all cases to increase read depth for interrogation of the disease-associated interval. As the human kit was used for target enrichment, capture of canine exons was incomplete (approximately 80%). For ADAMTS17 additional exon resequencing was performed to cover all exons, in three POAG cases and three controls, although no coding or splice site variants were identified.
The SNP with the lowest p-value from the GWAS (top SNP) was a non-synonymous SNP in the SYMN gene (chr3:41,599,598). Conservation analysis across vertebrate species showed weak conservation of this residue, with a number of naturally occurring amino acids at this position. The variant is also predicted to be tolerated by SIFT. In total, 2,696 SNPs and indels were identified across the disease-associated interval, including 12 non-synonymous variants, although none segregated fully with disease status (i.e. homozygotes for the non-reference allele were present in both case and control sets). A list of non-synonymous variants with consequent predictions is shown in S2 Table.
The disease-associated interval was further investigated by genome resequencing of a single POAG case. To consider intronic, exonic and intergenic regions in detail, sequence read align-ments were visually scanned using the Integrative Genomics Viewer (IGV) . Sequence read alignments indicative of a 4.96 Mb inversion were identified with breakpoints in intron 12 of ADAMTS17 (chr3:40,812,274) and a downstream intergenic region (chr3:45,768,123) (Fig 4).
To gauge whether the inversion had an impact on gene expression, limited qRT-PCR experiments were performed. Tissues for RNA extraction were selected based on the availability of suitable case and control material and assessment of
expression levels of ADAMTS17 using RNAseq data generated in previous studies (data not shown). In a comparison of retinal cDNA from one POAG case against one control, results suggested a 2.4 fold increase in ADAMTS17 expression upstream of the inversion for the POAG case relative to the control. No ADAMTS17 expression was detected downstream of the inversion for the POAG case. (Full results are shown in S1 Dataset).
RNAseq data generated from retinal RNA of one POAG case, showed concordance with the results of qPCR analysis. Expression of novel exons as the result of cryptic splicing was observed after the final normally transcribed exon of ADAMTS17 before disruption by the inversion. An example of a novel exon established through a cryptic splicing event is shown in Fig 5. A schematic diagram of ADAMTS17 exon arrangement is shown in Fig 6.
Table 1. Genotyping of an extended PBGV sample set for the POAG-associated inversion.
Both of the sequences of the two independent novel exons contained stop codons after an aberrant sequence of amino acids (S1 File).
In this investigation we have demonstrated a novel GWAS approach using exome sequencing variants calls as the genotyping dataset. The major potential advantage of this approach is that a causal variant for a single gene disease could be identified in a single step, and would theoretically be the most strongly disease segregating variant with the lowest associated p-value (top SNP). Although the causal variant for POAG was not directly found by this method, due to the non-exonic location, a genome-wide significant locus was identified, enabling a disease-associated interval to be determined.
The causal mutation for POAG was eventually identified through a genome sequencing approach. Genome sequencing is an increasingly cost effective method of following up disease-associated intervals identified through GWAS. In contrast to other approaches such as exome sequencing and target enrichment, coverage is near to 100%, enabling identification of causal mutations in repetitive regions of the genome. Genome sequencing has recently been adopted to directly identify causal mutations in the dog [5–7] and is being used in human studies through projects such as the 100,000 genomes project, studying cancers and rare diseases . However, structural variants still often present a challenge to smaller laboratories using genome sequencing as part of an investigation, making GWAS in many cases the most appropriate method of locus identification for single gene diseases, before using genome sequencing or other approaches to interrogate disease-associated intervals.
Although the ADAMTS17 gene was majorly disrupted by the inversion, qPCR analysis on a limited number of samples showed little evidence of nonsense mediated decay, as expression of exons outside of the inverted region was not majorly affected. Analysis of RNAseq data revealed novel exon expression for ADAMTS17 due to cryptic splicing occurring 3′ of the exons located immediately upstream of the inversion event. The unavailability of suitable anti-bodies targeting the 5’ region of ADAMTS17 prevented western blot analysis to determine whether protein is still produced from the modified transcript sequence. Visual analysis of RNAseq data aligned to the genes flanking and within the inversion region suggested gene expression for these genes was not affected. We speculate that a mobile element (SINE) which spans the 3′ inversion breakpoint is likely to have been involved in the inversion mechanism, although there is no similar mobile element in the 5′ region. There is one previous report of a gene rearrangement involving ADAMTS17 found in a patient with pregnancy-related acute promyelocytic leukemia . This rearrangement resulted in a novel transcription product with the insertion of exon 15 of ADAMTS17 between the PML and RARA genes. As the breakpoint within ADAMTS17 identified in this study was within intron 12, it is unlikely that the mutation mechanism was shared between these two independent events.
Genotyping of an extended sample set was used to determine whether the identified inver-sion was fully concordant with POAG. Of the 212 PBGV assayed for the POAG associated inversion there was one discordant case. A single dog was reported as having clinical signs of POAG but was homozygous for the reference sequence. It could be speculated that POAG for this case is due to another clinical or genetic cause, or is a secondary form of glaucoma.
A Weill-Marchesani like syndrome (WMS) in humans was the first disease phenotype to be associated with mutations in the ADAMTS17 gene. Clinical signs of WMS include glaucoma, ectopia lentis (lens luxation), lenticular myopia, spherophakia, and short stature . It is interesting to speculate that as the PBGV is a miniature version of the Grand Basset Griffon Vendéen, artificial selection pressure for small size may have contributed to an increase in mutant allele frequency, assuming mutations in ADAMTS17 could be contributing to small stature. An ADAMTS17 splice donor site mutation has been associated with hereditary primary lens luxation in several breeds of dog [11, 12]. The range of phenotypes associated with ADAMTS17 mutations suggests that the exact phenotypic presentation is dependent on the positioning and nature of the mutations within the ADAMTS17 gene. The ADAMTS17 gene is a member of the ADAMTS family of extracellular proteases . Domains which are characteristic of the ADAMTS family include an N terminal protease domain and a C-terminal ancillary domain . The inversion in intron 12 of ADAMTS17 is within the proteolytic domain and is therefore likely to disrupt the enzymatic function of the protein. Phenotypic similarities between ADAMTS associated disease and Fibrillin1 (FBN1) associated disease, and functional evidence, suggest ADAMTS family members have a role in microfibril assembly and function . It could be speculated that disruption of the maintenance of microfibrils in the eye are likely to lead to development of disease phenotypes such as lens luxation and glaucoma . The role of the ADAMTS family members in glaucoma pathogenesis is further highlighted by the identification of a mutation in ADAMTS10 as the cause of glaucoma in the Beagle and Norwegian Elkhound dog breeds [17, 18]. Functional work would be required to further understand the roles of the ADAMTS family in glaucoma disease pathogenesis.
In summary, we have used a novel GWAS approach and genome sequencing to identify an inversion associated with POAG in the PBGV dog breed. The approach further highlights how the increasing availability and cost effectiveness of massively parallel sequencing are facilitating studies into inherited disease in the dog.
Materials and Methods Ethics Statement
Collection of DNA samples was performed by buccal swabbing, which is a non-invasive technique that does not require a United Kingdom Home Office License. Only pet dogs were used in the study with full owner consent. Tissue samples for the study into primary open angle glaucoma were obtained after enucleation of a glaucoma case by a veterinary ophthalmologist on welfare grounds due to the severity of clinical signs (carried out in accordance with the Veterinary Surgeons Act 1966 and under the auspices of the RCVS). Full owner consent was obtained. As the techniques used were either non-invasive or in the case of eye surgery, were required to alleviate animal suffering rather than for research purposes, no ethics committee approval was required.
Diagnosis of POAG cases, and sample set selection
All cases of POAG were diagnosed by a specialist veterinary ophthalmologist. The basic clinical examination for POAG involves the use of slit lamp biomicroscopy, both indirect and direct ophthalmoscopy, applanation tonometry and gonioscopy. Tonometry and gonioscopy are completed before inducing mydriasis using 1% tropicamide, slit lamp biomicroscopy utilised pre and post mydriasis and ophthalmoscopy post mydriasis. Vision assessment is based on history, the menace and dazzle reflexes and, where possible, maze performance.
DNA extraction and genotyping
DNA was extracted from buccal swabs using the QIAamp Midi kit (Qiagen). Genotyping of the POAG associated inversion was performed by analysis of a fragment length polymorphism generated by PCR. Primers for PCR were as follows: POAG_F, 6FAM-AGGCTCAGAGGAGGG TGACT; POAG_R1, ACAAGGACAAAGCTGTCTGTGA; POAG_R2, ACACAAAGCACCCATGAC AG. PCRs were carried out in 12 ul volumes consisting of 1.5 mM dNTPs, 1x Qiagen PCR buffer, 0.5 µM of each primer, 0.6U of Qiagen HotStarTaq polymerase and template DNA. Thermal cycling consisted of 5 minutes at 95°C, followed by 35 cycles of 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds, with a final elongation stage of 72°C for 5 minutes. Products of PCR were analysed using the fragment analysis module of an ABI3130xl genetic analyser. Sequencing of the ADAMTS17 gene was carried out as previously described .
Exome sequencing and allelic association analysis
Libraries for exome sequencing of 12 POAG cases and 12 breed matched controls were made using the Illumina Nextera Exome Enrichment kit according to the manufacturer’s instructions. Libraries were quantified by qPCR using the KAPA library quantification kit. Sequencing of libraries was performed using two runs on the Illumina MiSeq platform, generating paired-end reads of 250 bp in length. The sequence reads generated were aligned to the canine reference genome build, CanFam3.1 using BWA . Variants were called using the GATK . Variants were converted to PLINK format for use in allelic association analysis using VCFtools . Allelic association analyses were carried out using the whole genome data analysis toolset, PLINK . Genome sequencing was outsourced to the Wellcome Trust Centre for Human Genetics, Oxford. Sequencing data can be found in the European Nucleotide Archive, study accession number PRJEB11835.
RNA was extracted from retina using the Qiagen RNeasy Midi kit, and included an on column DNase treatment. Isolation of mRNA from total RNA was performed using Sera-Mag oligo-dT beads. Libraries for RNAseq were generated using NEBNext Ultra RNA Library Prep Kit for Illumina sequencing. Sequencing was performed on an Illumina MiSeq generating a dataset of 75 bp paired-end reads, using one run per RNAseq library. Approximate dataset sizes were 4 Gb.
Synthesis of cDNA for qPCR expression analysis was performed using the Qiagen Quantitech reverse transcription kit. Expression analysis by qPCR was performed in 8 µl reactions, containing 1x primer- probe mix, 1x KAPA Probe Fast qPCR Master Mix and 2 µl template cDNA. Standard curves were generated over a seven point, two fold dilution. Standard curves for all qPCR assays had an r2 of greater than 0.99 and all assays had an efficiency of greater than 95%. Primers for qPCR, reaction efficiencies and full datasets with calculations can be found in S1 Dataset.
S1 Dataset. qRT-PCR primer sequences, reaction efficiencies and calculations.(XLSX)
S1 Fig. Association analysis using a Mixed Model approach correcting for population structure for the POAG GWAS.
S1 File. Sequence information for novel ADAMTS17 exons.(DOCX)
S1 Table. Genes in the disease-associated interval for POAG.(DOCX)
S2 Table. Non-synonymous SNP variants in the POAG disease-associated interval.(DOCX)
We are thankful to all owners, breeders and clinicians that have contributed samples to this study. We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics for the generation of the sequencing data.
Conceived and designed the experiments: OPF CM. Performed the experiments: OPF LP. Ana-lyzed the data: OPF. Contributed reagents/materials/analysis tools: OPF AMK PB CM. Wrote the paper: OPF LP AMK PB CM.
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(PBGVCA does not provide specific medical advice, but rather provides users with information to help them better understand health and disease. Please consult with a qualified health care professional for answers to medical questions.)