Advances in genetic mapping and sequencing techniques: a demonstration using the domestic dog model

Over the past ten years huge advances have been made in the field of genetics and genomics. Genetic mapping has evolved from laborious linkage and homozygosity based approaches to high-throughput genome-wide association studies using whole genome SNP array technology. Through massively parallel sequencing technology, gigabases of sequencing data can now be produced in a single experiment. The domestic dog has been increasingly recognised as a model for human disease and mapping of inherited disease in the domestic dog is facilitated by fixed and genetically isolated populations. The aims of this thesis were to demonstrate advances in mapping and sequencing techniques by investigating the genetics of five inherited disorders, representing significant welfare issues in the purebred dog. An additional aim was to develop diagnostic DNA tests to identify affected individuals and asymptomatic carriers. A parallel mapping approach was used to map two autosomal recessive conditions in the Cavalier King Charles Spaniel. The use of a single common set of controls for two independent genome-wide association studies was demonstrated as an efficient mapping strategy when studying two conditions affecting a single breed. Newly available target enrichment and massively parallel sequencing methodology was used to simultaneously sequence both disease-associated loci, with one condition acting as a control for the other. A genome-wide homozygosity mapping approach using microsatellite markers was used to investigate spinocerebellar ataxia in the Italian Spinone. The disorder was successfully mapped to a single chromosome using six cases and six controls, and fine mapped with additional microsatellite markers. Subsequently, a progression of sequencing techniques were used to identify the disease-associated mutation, with the study highlighting the potential difficulties of using massively parallel sequencing technologies. Spinocerebellar ataxia (or late onset ataxia) in the Parson Russell Terrier was investigated using a genome-wide association study followed by a target enriched massively parallel sequencing approach. Further sequencing was performed to reduce the large number of potential causal variants, with the entire workflow achieved in-house. The final experimental chapter describes the use of a genome-wide mRNA sequencing (mRNA-seq) approach as a method of candidate gene sequencing of a single case of neonatal cerebellar cortical degeneration in a Beagle dog. The mRNA-seq approach demonstrates a simple, fast and cost effective method of targeted resequencing of expressed genes when a suitable tissue resource is available. For all five disorders under investigation, disease-associated mutations were identified leading to the development of diagnostic tests. Three of the mutations were in genes not previously associated with similar conditions in humans or other model organisms

Becoming a System of Professional Learning: Conceptualizing Improvement As a Throughline of Learning

This white paper introduces the “Throughline of Learning” (Throughline) model developed by the Bank Street Education Center. The model builds on the concept of the instructional core and demonstrates how focusing on system-wide adult learning needs can help districts successfully reimagine their approach to instructional improvement.https://educate.bankstreet.edu/bsec/1002/thumbnail.jp

Are lower rates of surgery amongst older women with breast cancer in the UK explained by co-morbidity?

Background: Around 60% of women greater than or equal to 80 years old, in the UK do not have surgery for their breast cancer (vs<10% of younger age groups). The extent to which this difference can be accounted for by co-morbidity has not been established. Methods: A Cancer Registry/Hospital Episode Statistics-linked data set identified women aged greater than or equal to 65 years diagnosed with invasive breast cancer (between 1 April 1997 and 31 March 2005) in two regions of the UK (n=23 038). Receipt of surgery by age was investigated using logistic regression, adjusting for co-morbidity and other patient, tumour and treatment factors. Results: Overall, 72% of older women received surgery, varying from 86% of 65–69-year olds to 34% of women aged greater than or equal to 85 years. The proportion receiving surgery fell with increasing co-morbidity (Charlson score 0=73%, score 1=66%, score 2+=49%). However, after adjustment for co-morbidity, older age still predicts lack of surgery. Compared with 65–69-year olds, the odds of surgery decreased from 0.74 (95% CI: 0.66–0.83) for 70–74-year olds to 0.13 (95% CI: 0.11–0.14) for women aged greater than or equal to 85 years. Conclusion: Although co-morbidity is associated with a reduced likelihood of surgery, it does not explain the shortfall in surgery amongst older women in the UK. Routine data on co-morbidity enables fairer comparison of treatment across population groups but needs to be more complete

Canine genome assembly correction facilitates identification of a MAP9 deletion as a potential age of onset modifier for RPGRIP1-associated canine retinal degeneration.

Retinal degeneration (RD) in the Miniature Long Haired Dachshund (MLHD) is a cone-rod dystrophy resulting in eventual blindness in affected individuals. In a previous study, a 44-nucleotide insertion (ins44) in exon 2 of RPGRIP1 was associated with RD. However, results on an extended population of MLHD revealed a variable RD onset age for ins44 homozygous dogs. Further investigations using a genome-wide association study comparing early onset and late onset RD cases identified an age of onset modifying locus for RD, approximately 30 Mb upstream of RPGRIP1 on chr15. In this investigation, target enriched sequencing identified a MAP9 deletion spanning approximately 22 kb associated with early RD onset. Identification of the deletion required correction to the CanFam3.1 genome build as canine MAP9 is part of a historic tandem duplication, resulting in incomplete assembly of this genome region. The deletion breakpoints were identified in MAP9 intron 10 and in a downstream partial MAP9 pseudogene. The fusion of these two genes, which we have called MAP9 EORD (microtubule-associated protein, early onset retinal degeneration), is in frame and is expressed at the RNA level, with the 3' region containing several predicted deleterious variants. We speculate that MAP9 associates with α-tubulin in the basal body of the cilium. RPGRIP1 is also known to locate to the cilium, where it is closely associated with RPGR. RPGRIP1 mutations also cause redistribution of α-tubulin away from the ciliary region in photoreceptors. Hence, a MAP9 partial deficit is a particularly attractive candidate to synergise with a partial RPGRIP1 deficit to cause a more serious disease.This is the author accepted manuscript. The final version is available from Springer via http://dx.doi.org/10.1007/s00335-016-9627-

Parallel Mapping and Simultaneous Sequencing Reveals Deletions in BCAN and FAM83H Associated with Discrete Inherited Disorders in a Domestic Dog Breed

The domestic dog (Canis familiaris) segregates more naturally-occurring diseases and phenotypic variation than any other species and has become established as an unparalled model with which to study the genetics of inherited traits. We used a genome-wide association study (GWAS) and targeted resequencing of DNA from just five dogs to simultaneously map and identify mutations for two distinct inherited disorders that both affect a single breed, the Cavalier King Charles Spaniel. We investigated episodic falling (EF), a paroxysmal exertion-induced dyskinesia, alongside the phenotypically distinct condition congenital keratoconjunctivitis sicca and ichthyosiform dermatosis (CKCSID), commonly known as dry eye curly coat syndrome. EF is characterised by episodes of exercise-induced muscular hypertonicity and abnormal posturing, usually occurring after exercise or periods of excitement. CKCSID is a congenital disorder that manifests as a rough coat present at birth, with keratoconjunctivitis sicca apparent on eyelid opening at 10–14 days, followed by hyperkeratinisation of footpads and distortion of nails that develops over the next few months. We undertook a GWAS with 31 EF cases, 23 CKCSID cases, and a common set of 38 controls and identified statistically associated signals for EF and CKCSID on chromosome 7 (Praw 1.9×10−14; Pgenome = 1.0×10−5) and chromosome 13 (Praw 1.2×10−17; Pgenome = 1.0×10−5), respectively. We resequenced both the EF and CKCSID disease-associated regions in just five dogs and identified a 15,724 bp deletion spanning three exons of BCAN associated with EF and a single base-pair exonic deletion in FAM83H associated with CKCSID. Neither BCAN or FAM83H have been associated with equivalent disease phenotypes in any other species, thus demonstrating the ability to use the domestic dog to study the genetic basis of more than one disease simultaneously in a single breed and to identify multiple novel candidate genes in parallel

Late Quaternary aeolian dynamics, pedostratigraphy and soil formation in the North European Lowlands – new findings from the Baruther ice-marginal valley

The construction of dunes in central Europe reflects ample sediment supply during the last deglacial hemicycle. A Quaternary inland dune complex in southern Brandenburg, Germany, was studied to determine the duration of recent pedogenesis, from two outcrops, which show buried paleosols. An integrative approach, which combined geomorphological, sedimentological, (paleo-)pedological and chronological methods was used to identify aeolian deposition events, ensuing pedogenesis and anthropogenic remobilization. At the outcrops, which were situated approximately 2 km apart from each other, in total twelve samples of the aeolian sands were dated using optically stimulated luminescence (OSL) and six using 14C dating. Although the dunes have similar morphological features, these forms have a different history of aeolian sand deposition and pedogenesis. At the older dune (Gl 1) the surface soil is a well developed Podzol, whereas soil development of the younger dune (Gl 2) is clearly in an initial state. The two dunes also differ in grain size distribution and in the presence of buried soils, thereby indicating a climatic impact on aeolian remobilization

Whole Genome Sequencing of Giant Schnauzer Dogs with Progressive Retinal Atrophy Establishes NECAP1 as a Novel Candidate Gene for Retinal Degeneration

Canine progressive retinal atrophies (PRA) are genetically heterogeneous diseases characterized by retinal degeneration and subsequent blindness. PRAs are untreatable and affect multiple dog breeds, significantly impacting welfare. Three out of seven Giant Schnauzer (GS) littermates presented with PRA around four years of age. We sought to identify the causal variant to improve our understanding of the aetiology of this form of PRA and to enable development of a DNA test. Whole genome sequencing of two PRA-affected full-siblings and both unaffected parents was performed. Variants were filtered based on those segregating appropriately for an autosomal recessive disorder and predicted to be deleterious. Successive filtering against 568 canine genomes identified a single nucleotide variant in the gene encoding NECAP endocytosis associated 1 (NECAP1): c.544G>A (p.Gly182Arg). Five thousand one hundred and thirty canids of 175 breeds, 10 cross-breeds and 3 wolves were genotyped for c.544G>A. Only the three PRA-affected GS were homozygous (allele frequency in GS, excluding proband family = 0.015). In addition, we identified heterozygotes belonging to Spitz and Dachshund varieties, demonstrating c.544G>A segregates in other breeds of German origin. This study, in parallel with the known retinal expression and role of NECAP1 in clathrin mediated endocytosis (CME) in synapses, presents NECAP1 as a novel candidate gene for retinal degeneration in dogs and other species

A Novel Genome-Wide Association Study Approach Using Genotyping by Exome Sequencing Leads to the Identification of a Primary Open Angle Glaucoma Associated Inversion Disrupting ADAMTS17

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 consequence 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