11th International Conference on Canine and Feline Genetics and Genomics HUDSONALPHA INSTITUTE FOR BIOTECHNOLOGY HUNTSVILLE, ALABAMA, USA
MAIN SPONSOR
HudsonAlpha Institute for Biotechnology shall provide equal access to and opportunity in its programs, facilities, and employment without regard to race, color, creed, religion, national origin, gender, age, marital status, disability, public assistance status, veteran status, sexual orientation, gender identity, or gender expression.
Images provided by ICCFGG, Adobe Stock, Sellars Aerials,. HudsonAlpha Archives and Unsplash artists: Alicia Gancarz, Alvan Nee, Anoir Chafik, Farid Amin, Hannah Lim, Karsten Winegeart, Taylor Kopel, and Zoe Gayah Jonker.
Canine & Feline Genetics & Genomics 2022 Program & Abstract Booklet © 2022 All rights reserved.
CONTENTS
Welcome Letters........................................................................................ 4-5 Conference Ethics.......................................................................................6–7 Sponsors...................................................................................................8–9 Location................................................................................................ 10–11 General Information.................................................................................12-13 Conference Committees.................................................................................14 Workshop Program..................................................................................15–17 Conference Program................................................................................18–21 Biosketches of Keynote Speakers.............................................................22–23 Social Events...............................................................................................25 Presentation Abstracts............................................................................26–51 Poster Abstracts....................................................................................53–106
11th International Conference on Canine and Feline Genetics and Genomics
Dear Participants,
Welcome to the 11th International Conference on Canine and Feline Genetics and Genomics at the HudsonAlpha Institute for Biotechnology in Huntsville, Alabama, October 2-5, 2022. Huntsville is known as the Rocket City for its role in putting astronauts on the moon. The Huntsville skyline even shows off a 363-foot-tall Saturn V moon rocket. Today, Huntsville sits at the intersection between its historied aerospace industry and its burgeoning biotech industry. This International Conference has occurred every two years since 2002, apart from the delay last year due to the COVID-19 pandemic. The conference alternates between Europe and the United States and has documented the many milestones reached in the fields of canine and feline genetics. Over this time, we have witnessed the development of amazing resources for genetic and genomic research coupled with wonderful studies using these tools for understan- ding canine and feline origins, diversity, biology, health, and disease. We have a very full and exciting scientific program planned. One hundred and one abstracts were submitted, and the Scientific Committee has selected 35 for oral presentations, with all other abstracts being presented at one of the two poster sessions. Ten selected posters from young scientists will additionally be highlighted in short poster flash talks. We will have a one-day Workshop on Sunday, October 2, which will feature presentations on genome assembly and annotation, large-scale genotyping/phenotyping, and updates from the 99Lives, Dog10Kgenomics consortia. We also have two very exciting keynote speakers: Heather Mefford, MD, PhD, is a faculty at St. Jude’s Research Hospital whose research aims to understand the genetic basis of neurologic and neurodevelopmental diseases, particularly severe forms of epilepsy. Jeremy Schmutz is a Faculty Investigator at HudsonAlpha Institute for Biotechnology and co-director of the HudsonAlpha Genome Sequencing Center.
The social program includes a Sunday night reception at the Jackson Center and on Tuesday, October 4, a dinner under the Saturn V Rocket at the U.S. Space and Rocket Center.
We are grateful to our main sponsor, Nestle Purina, who has supported and made this event possible for the last 20 years. We look forward to welcoming you all to Huntsville, and enjoying an exciting and collaborative scientific workshop and conference.
With best wishes,
Gregory Barsh, MD, PhD Faculty Chair and Investigator, Smith Family Chair in Genomics PloS Editor in Chief
4
5
6
Conference Ethics
The International Canine and Feline Genetics and Genomics Conference encourages scientists to present and discuss preliminary data prior to scientific publication. Presentation of research findings is a major factor for the success of the conference. In order to maintain conference integrity and participation, we stress that information presented here should be treated as preliminary work and out of respect for the authors, should only be used with the authors’ permission. In order to maintain the high quality of the conference we would like to inform all attendees of the following policies:
We ask that new information obtained at this conference will not be used, for either research or commercial purposes, without permission from the authors.
The abstracts in the conference program booklet cannot be cited.
Publication of studies presented here in peer-reviewed journals is of course highly encouraged.
Breaches of confidentiality and common sense ethics will ultimately interfere with the pre-publication free flow of information and discussion expected of our conference and endangers the spirit of the conference.
We all thank you for your understanding and attention to this matter.
7
ICCFGG 2022 Main Sponsor
REVOLUTIONIZING THE ELIMINATION DIET. AGAIN.
We started with the first truly hypoallergenic dry diet for dogs with food allergies. And now, our progressive science has led to Elemental—built with purified amino acids. It’s our most advanced elimination diet yet.
NEW FLAVOR
Explore our allergy-focused nutrition at PurinaProPlanVets.com.
Purina trademarks are owned by Société des Produits Nestlé S.A.
8
ICCFGG 2022 Additional Sponsors
9
Huntsville, Alabama
Locat ion Huntsville, Alabama
The Great Outdoors Huntsville’s location in the expansive Tennes- see Valley makes day tripping for mountain adventures easy. Venture up Round Top Mountain (also known as Monte Sano Mountain), home to Monte Sano State Park, to enjoy the tranquil Ja-
A high-tech city with a historical legacy and internatio- nal appeal. As one of the fastest growing cities in the Southeastern USA, Huntsville can aptly be described as unexpected. Huntsville seamlessly marries the rich history of southern hospitality with innovative high-tech ventures and cultural diversity. In fact, more than 100 languages and dialects are spoken in the city. A sen- se of curiosity and exploration has been rooted in the city’s foundation. The first American satellite was sent into orbit from the city in 1958. One of the “Fathers of Rocket Science,” Wernher von Braun, lived and worked in Huntsville during the 1950s and 1960s, launching the space and rocket industry still associated with the city today. Experience that same sense of awe-inspiring cu- riosity today as you explore icons like the U.S. Space and Rocket Center or head off the beaten path into Huntsvi- lle’s vibrant neighborhoods and historic districts. Art lovers shouldn’t feel left out, though The Huntsville Museum of Art beckons visitors to admire a variety of permanent and temporary exhibits, including the unique Sellars Collection of Art by Ame- rican Women. Huntsville also hosts the Panoply Arts Festival, an annual weekend celebrating the arts, music and more downtown. Art aficionados will especially love Lowe Mill ARTS & Entertainment, a former textile mill providing artists with public studio spaces. Visitors can check out galleries, watch live performances and even take creative classes including painting, sculpting and hula hooping.
USSRC
panese garden, or to hike trails blanketed with azaleas in the spring and colorful leaves in the fall. Nearby, blend your outdoor explorations with history at Burritt on the Mountain, featuring a historic mansion, open-air exhibits and nature trails. Around town, see historic antebellum homes and their gardens, or stroll through the gorgeous blooms at the Huntsville Botanical Gar- den. Whether you crave a leisurely walk or a romantic picnic, Big Spring International Park is a lakefront gem nestled downtown.
Big Spring Park
10
Locat ion
Fun, Food & Drink Unique culinary experiences abound. Dine in an old jail cell at Main Street Café, housed in a restored city hall and jail in Madison, a 15-minute drive from Huntsville. Savor Southern comfort food at G’s Coun- try Kitchen or Blue Plate Cafe, both eateries serving up meat-and-threes (a traditional dish featuring one meat with three sides – yum!). To quench your thirst, hop over to one of Huntsville’s breweries, Straight to Ale, for a Monkeynaut IPA, or check out Campus No. 805, a landmark brewing, dining and entertainment complex in the city’s urban core.
HudsonAlpha Institute for Biotechnology HudsonAlpha Institute for Biotechnology is a nonprofit institute dedicated to innovating in the field of genomic technology and sciences across a spectrum of biological challenges. Opened in 2008, its mission is three-fold: spar- king scientific discoveries that can impact human health and well-being; fostering life sciences entrepreneurship and business growth; and encouraging the creation of a genomics-literate workforce and society. The HudsonAlpha biotechnology campus consists of 152-acres nestled within Cummings Research Park, the nation’s second largest re- search park. Designed to be a hothouse of biotech economic development, HudsonAlpha’s state-of-the-art facilities co- locate nonprofit scientific researchers with entrepreneurs and educators.
Downtown Huntsville
HudsonAlpha Institute for Biotechnology
The Jackson Center The Jackson Center in the heart of Research Park describes itself as “where business class meets world class”. Conference attendees are steps away from the businesses that make up the nation’s second-largest research park. It is located on the campus of HudsonAlpha Institute for Biotechnology and its mile-plus double helix park that’s shaped like the strands of DNA. Community Information Please visit www.huntsville.org to discover what the beautiful and high-tech city of Huntsville has to offer.
Jackson Center
11
11
Delta Research
Camber Corporation
System Studies & Simulation MagnaCom
Camber Corporation
L
L3 Commun
CRP Biotech Campus
Huntsville Utilities
ICCFGG 2022
BFA Systems CNI
Colonial Center at Lakeside
State of Ala. Dept. of Revenue
SDI
Parking
Future Development
Lake 1
Quantum Research
Visitor’s parking is available in front of HudsonAlpha. Additional parking is open behind the Institute and in the Jackson Center parking lot. A map of the HudsonAlpha campus is shown below for your convenience.
United Space Alliance
D r i
Davidson Technologies
ow Road
Jackson Center
601 Genome Way, Huntsville, AL 35806
Venturi
AT&T
olumbia gh School w Century chnology gh School
ADTRAN HudsonAlpha is a smoke-free and weapons-free facility.
Boeing
Parking behind building
Lak
Lake 3
Colsa
ADTRAN
AeroVironment
ARC
Nektar Therapeutics
6001 Moquin Drive, Huntsville, AL 35806
Northrop Grumman
Shuttle Bus Transportation Schedule Shuttle buses will run between the
Pa
SAIC
QinetiQ North America Providence to Jackson Ctr. l o Sunday, October 2 Providence to Jackson Ctr. Providence to Jackson Ctr.
InfoPr
Analytical Services, Inc. 7:30 AM......................................Shuttle 1 8:15 AM......................................Shuttle 2
Digium, Inc. conference hotels listed below. Watch for the Spirit Coach buses in the joint back parking lot of your hotel. Hotels Springhill Suites – Huntsville West 320 Providence Main St NW Huntsville, AL 35806 AEgis Technologies Rockwell Collins
Research Park O ce Center This shuttle will pick up people from the hotel, come to Jackson Ctr. and people will remain on the shuttle, picking up additional people for U.S. Space and Rocket Center Lake 4 NOON.........................................Shuttle 1 Jackson Ctr. to Providence 1:30 PM......................................Shuttle 1 Providence to Jackson Ctr. 4:30 PM.....................................Shuttle 1 Jackson Ctr. to Providence 6:15 PM......................................Shuttle 1 Jackson Ctr. to Providence 8:15 PM ............................Shuttles 1 & 2 Monday, October 3 Providence to Jackson Ctr. 7:30 AM......................................Shuttle 1 Providence to Jackson Ctr. 8:00 AM......................................Shuttle 2 Providence to Jackson Ctr. NOON.........................................Shuttle 1 Jackson Ctr. to Providence 1:35 PM......................................Shuttle 1 Jackson Ctr. to Providence 4:45 PM...............................Shuttle 1 & 2 Tuesday, October 4 Providence to Jackson Ctr. 7:30 AM......................................Shuttle 1 Providence to Jackson Ctr. 8:00 AM......................................Shuttle 2 Providence to Jackson Ctr. 11:45 AM....................................Shuttle 1 Jackson Ctr. to Providence 1:20 PM......................................Shuttle 1 Providence to Jackson Ctr. 5:00pm..................................... Shuttle 1* Lake 5 Jackson Ctr. to U.S. Space & Rocket Center 5:15 PM..Shuttles 1* & 2 U.S. Space & Rocket Center to Providence 9:00 PM......Shuttles 1 & 2 SAIC BAE Systems ITT (CAS) BAE Systems Park West Center Northrop Grumman Northrop Grumman (Phase II) Northrop Grumman Phase II Decibel Old Madison Pike Eagle Drive
Future Development
Premier Pro. Systems
Quest Circle
Raytheon Company Hampton Inn Huntsville/ Village of Providence 328 Providence Main St NW, Huntsville, AL 35806
Bridge Street Town Centre
Technology Pointe
Cobham Luggage Storage Please check in with the registration desk for information on luggage storage.
Defense Acquisition University
Progress Center
DirecTV
BPI Rideshare Information Lyft and Uber
CINRAM
Aerospace Products Wednesday, October 5 Providence to Jackson Ctr. Providence to Jackson Ctr. Jackson Ctr. to Providence
7:30 AM......................................Shuttle 1 8:00 AM......................................Shuttle 2 1:35 PM.............................Shuttles 1 & 2
Highland O ce Park
Catholic High School (Future Site)
Huntsville Hospital
ARSA, P.C.
12
355 Quality Circle
565
Verizon Wireless
General Information
Exhibit Hours Please visit our sponsors in the Discovery Hall of the Jackson Center during the following times: Jackson Center atrium Poster Sessions Posters will be displayed in the HudsonAlpha Atrium throughout the duration of the conference. Registration Desk/Name Badges Registration open daily from 8:00 AM to 12:00 PM in the
Airport Information Huntsville International Airport 1000 Glenn Hearn Boulevard Huntsville, AL 35824
Phone: (256) 772-9395 Taxi Information
COMPANY
TELEPHONE NUMBER
AA Cab
(256) 536-1313 (256) 288-0909 (256) 536-3600 (256) 536-9959 (256) 534-5000
AAAA Taxi
A-1 United Deluxe Cab
DATE
TIMES
American Cab
Sunday, Oct 2
10:50 AM – 11:25 AM 3:20 PM – 3:45 PM 10:45 AM – 11:15 AM 3:00 PM – 3:30 PM 10:45 AM – 11:15 AM 2:45 PM – 3:00 PM 11:05 AM – 11:30 AM
A Plus Cab
Monday, Oct 3
Huntsville Cab Rocket City Cab Trans City Cab
(256) 539-9444 or 9445
(256) 534-4524 (256) 536-1113
Tuesday, Oct 4
Wednesday, Oct 5
Time Zone Huntsville, Alabama is located in the Central Standard Time Zone
Meals & Refreshments Lunch and breaks will be in the Jackson Center. Please visit the exhibitors. Internet Access There will be one Wi-Fi network available during the conference: ICCFGG2022 Password: Meowoof! Social Media Share your conference experience! Add #ICCFGG22 to your social media posts to join in the conversation! Mobile Telephones We ask that you kindly ensure all mobile devices are switched off during all conference sessions. Emergency Information Medical Emergency: Please call 9-1-1 or (256) 975-0961 in the case of a medical emergency. Fire/Evacuation Emergency: In the event that the fire alarm system is activated, please leave the building through the nearest exit.
13
Conference Committees
Greg Barsh HudsonAlpha & Stanford University Adam Boyko Scientific Organizing Committee
Xu Wang Auburn University Leigh Anne Clark Clemson University Leslie Lyons University of Missouri Emily Graff Auburn University Chris Kaelin Stanford University Greg Barsh HudsonAlpha and Stanford University Local Organizing Committee Conference Management Jazmine Robinson HudsonAlpha Institute for Biotechnology Email: jrobinson@hudsonalpha.org Aida El-Kholi Starling HudsonAlpha Institute for Biotechnology Email: astarling@hudsonalpha.org
Cornell University Leigh Anne Clark Clemson University Christophe Hitte University of Rennes Tosso Leeb University of Bern Hannes Lohi University of Helsinki Rondo Middleton Nestlé Purina Research Qinghong (Johnny) Li Nestlé Purina Research Ned Patterson University of Minnesota Jeffrey Schoenebeck University of Edinburg Claire Wade University of Sydney
14
SUNDAY, OCTOBER 2, 2022
WORKSHOP PROGRAM Large-scale genotype/phenotype collections: community opportunities & data-sharing Over the last decade, opportunities to expand genetic knowledge about health and normal variation in companion animals have been facilitated by availability of genotype-phenotype databases, analogous to large-scale efforts for humans such as dbGAP or the UK Biobank. Several such collections exist for companion animals carried out by direct-to-consumer genetic testing endeavors and non-profit efforts, but the approaches and opportunities to make use of these collections are a work-in-progress. This workshop will feature brief presentations by leaders of major efforts followed by a facilitated discussion on future plans, opportunities for data-sharing, standardization, and academic collaborations. Elinor Karlsson, PhD, UMass Chan Medical School, USA Reference genomes, assemblies, and annotations: applications and approaches Assemblies and their annotation are essential tools of biological research. The assembly — the DNA content of a single genome — is typically represented in haploid or diploid forms. With the evolution of 3rd generation sequencing and scaffolding techniques, production of accurate and contiguous assemblies is becoming increasingly routine. Annotation provides context to the DNA that is represented within the assembly. Transcript isoform models, DNA conservation, ChIP-, CAGE-, and ATAC-seq data are but a few examples of annotation data that enrich assemblies. In this workshop, we will discuss where the dog and cat community has come and where it must go to advance our research interests community-wide. Jeff Schoenebeck, PhD, University of Edinburg, UK, Hannes Lohi, PhD, University of Helsinki, FI, Christophe Hitte, PhD, University of Rennes, FR 99 Lives Project The current progress for the 99 Lives dataset and implementation of the new cat reference genome Fca126 will be discussed. Sub-committees will be developed for disease / trait related community projects. Needed genomic resources will be reviewed and discussed to support the cat research community. Leslie Lyons, PhD, University of Missouri, USA Dog 10K Project The Dog 10K Consortium is an international collaboration of researchers from across the globe, launched in 2019, led by Dr. Ostrander at the National Institutes for Health. The aim of the Dog 10K is to coordinate the global effort on genome sequencing in dogs and build a comprehensive resource for the canine community to increase the understanding of the evolutionary history of dogs, genetic changes associated with domestication, and relationships of genetic changes to phenotype. Elaine Ostrander, PhD, National Human Genome Research Institute (NHGRI) of the National Institutes of Health (NIH), USA
15
SUNDAY, OCTOBER 2, 2022
10:10 AM -10:20 AM Feline: “Single haplotype genome assemblies spanning the felid phylogeny” William Murphy, Texas A&M, USA
7:45 AM Breakfast 8:00 AM – 12:00 PM Registration 9:00 AM – 9:10 AM Welcome and Introduction Greg Barsh 9:10 AM – 10:40 AM & 11:15 AM – 12:15 PM Jackson Center Session: Reference genomes, assemblies, and annotations: applications and approaches
10:20 AM – 10:40 AM Assembly round table discussion
10:40 AM – 11:15 AM Break and Exibits Jackson Center B. Annotation Jackson Center 11:15 AM -11:25 AM The bioinformatics challenge to annotate the non-coding genome and predict its functions Christophe Hitte, University Rennes, France. 11:25 AM -11:35 AM The DoGA project, functional annotation of the dog genome Hannes Lohi, University of Helsinki, Finland 11:35 AM -11:55 AM How to use the DoGA data? Matthias Hörtenhuber, Karolinska Institute, Sweden (remote presentation)
Chair: Jeffrey Schoenebeck, Christophe Hitte and Hannes Lohi A. Genome Assembly 9:10 AM -9:20 AM Canine: “Use of linkage disequilibrium to assess large-scale assembly structure of canine reference genomes”. Reuben Buckley, NHGRI, USA 9:20 AM – 9:30 AM Canine: “The German Shepherd, Mischka: Uppsala University UU_Cfam_GSD_1.0, GCA_011100685.1”. Jennifer Meadows, Uppsala University, Sweden 9:30 AM – 9:40 AM Canine:“Importance of updated informatics tools for genome assembly.” Jeffrey Kidd, University of Michigan, USA 9:40 AM – 9:50 AM Canine: “Assembly, annotation, and future improvements to the Labrador Retriever assembly ROS_Cfam1.0”. Jeffrey Schoenebeck, University of Edinburgh, UK 9:50 AM-10:00 AM Canine: “De novo chromosome-length genome assembly of the Australian Alpine dingo with comparisons to the Desert Dingo and modern bred dogs” Bill Ballard, La Trobe University, Australia
11:55 AM – 12:15 PM Jackson Center Annotation round table discussion
12:15 PM – 1:30 PM Jackson Center Lunch 1:30 PM – 2:20 PM Jackson Center Session: 99 Lives Project Chair: Leslie Lyons, University of Missouri, USA 1:30 PM –1:40 PM 99 Lives Cat Genome Consortium - status and progress Leslie Lyons, University of Missouri, USA 1:40 PM -1:55 PM De novo mutation rate in the domestic cat Richard Wang, Indiana University, USA 1:55 PM -2:05 PM 99 Lives Hypertrophic Cardiomyopathy working group updates Bart Broeckx, Ghent University, Belgium
10:00 AM- 10:10 AM Feline: “Variant discovery using FelCat 9.0” Leslie Lyons, University of Missouri, USA
16
SUNDAY, OCTOBER 2, 2022
2:05 PM -2:15 PM 99 Lives Imputation working group updates Greg Barsh, HudsonAlpha Institute for Biotechnology, USA
3:55 PM – 4:05 PM A review of clinical studies at PetDx and an overview of the SPIRE program to support academic research Kristina Kruglyak, PetDx, USA 4:05 PM – 4:15 PM Canine Cancer Genomics: Prospective Clinical Studies and Data Sharing Opportunities Heather Gardner, Tufts, USA 4:15 PM – 4:25 PM Genetic epidemiology of blood type, disease and trait variants, and genome-wide genetic diversity in over 11,000 domestic cats Heidi Anderson, Wisdom Panel, Kinship, USA
2:15 PM -2:20 PM Discussion
2:20 PM–3:20 PM Jackson Center Session: Dog 10K Project
Chair: Elaine Ostrander, PhD, National Human Genome Research Institute (NHGRI) of the National Institutes
of Health (NIH) 2:20 – 2:30 PM Dog10K-A historical perspective Elaine Ostrander, NIH/NHGRI, USA 2:30 PM – 2:45 PM Approach and analysis Jeff Kidd, University of Michigan, USA
4:25 PM – 4:35 PM Round table discussion 4:35 PM Announcements and Break Greg Barsh
Keynote
5:15 PM – 6:00 PM Jackson Center Learning to love the complexity of plant genomes
2:45 PM – 3:00 PM Functional studies from Dog10K Jennifer Meadows, Uppsala University, Sweden 3:00 PM – 3:10 PM Using Dog10K data to identify and validate disease-causing variants Tosso Leeb, University Bern, Switzerland 3:10 PM – 3:20 PM Deep sequence analysis of multigenerational pedigrees to estimate mutation rates and age-related accumulation of mutations Hannes Lohi, University of Helsinki, Finland
Jeremy Schmutz, HudsonAlpha Institute for Biotechnology Co-Director HudsonAlpha Genome Sequencing Center
6:00 PM- 8:00 PM
Opening Reception
Jackson Center
3:20 PM – 3:45 PM Jackson Center Break and Exibits 3:45 PM – 4:35 PM Jackson Center
Session: Large -scale genotype/ phenotype collections: community opportunities and data-sharing Chair: Elinor Karlsson, PhD, UMass Chan Medical School 3:45 PM – 3:55 PM Genomic studies with thousands of dogs using new open-access biobanks Elinor Karlsson, Umass, USA
17
ICCFGG 2022
CONFERENCE PROGRAM
Session: Evolution and Phenotypes Jackson Center 9:45 AM – 10:05 AM A 5,000 years population history of dogs in Latin America Christophe Hitte 10:05 AM – 10:25 AM Canine lineages reveal genetic drivers of dog behavioral diversification Emily Dutrow 10:25 AM – 10:45 AM Speed versus endurance; the biological implications of the selection of sprint and distance Alaskan Sled Dogs Heather Huson 10:45 AM – 11:15 AM Break & Exibit Sessions Jackson Center Session: Single Cell Genomics Jackson Center 11:15 AM – 11:35 AM Cell type specific differences in the prefrontal cortex of young and aging tame and aggressive foxes Jennifer Johnson 11:35 AM – 11:55 AM The basis of color pattern development and diversity in cats Chris Kaelin 11:55 AM- 12:15 PM Canine Cancer Cell Atlas: A resource to advance interspecies knowledge of the tumor microenvironment Skyler Kramer 12:15 PM – 12:35 PM The microenvironment of the bone marrow niche in canine osteosarcoma McKaela Hodge
MONDAY, OCTOBER 3, 2022
7:45 AM Breakfast 8:00 AM – 12:00 PM Registration
Welcome Jackson Center 8:30 AM – 8:45 AM Welcome and introduction
Greg Barsh, HudsonAlpha Institute for Biotechnology Richard Myers, HudsonAlpha Institute for Biotechnology Qinghong (Johnny) Li, Nestlé Purina Research
Session: Insights from New Assemblies Jackson Center 8:45 AM -9:05 AM Ultracontinuous genomes elucidate complex speciation patterns within Panthera Andrew Harris 9:05 AM – 9:25 AM Comparative analysis of single haplotype genomes unmasks the role of structural variation in felid evolutionary innovation Bill Murphy 9:25 AM – 9:45 AM Evolutionary insights enabled by assembly and
annotation of the dog Y chromosome Jeffrey Schoenebeck, Wengang Zhang
18
CONFERENCE PROGRAM
12:35 PM – 1:35 PM Lunch
TUESDAY, OCTOBER 4, 2022
Session: Poster Flash Talks Jackson Center 1:35 PM – 2:00 PM #1
7:45 AM Breakfast 8:00 AM – 12:00 PM Registration
Announcements Jackson Center 8:30 AM Greg Barsh Session: Clinical Genetics 1 Jackson Center 8:45 AM – 9:05 AM Genetics of chronic superficial keratitis in the Australian racing greyhound Claire Wade 9:05 AM – 9:25 AM Revealing risk factors for Canine Atopic Dermatitis using Bayesian model and selection signature analyses Katarina Tengvall 9:25 – 9:45 Dual-species multi-omics approach suggests novel candidate genes for dilated cardiomyopathy in dog and human Julia Niskanen Session:Clinical Genetics 2 Jackson Center 9:45 AM – 10:05 AM QTL analysis of a uremic toxin indicates the xanthine dehydrogenase gene may be a risk factor for chronic kidney disease in cats. Jeffrey Brockman 10:05 AM – 10:25 AM The Canine Diabetes Genetics Partnership: whole genome and targeted sequencing of dog breeds at high and low risk of diabetes mellitus Marsha Wallace 10:25 AM – 10:45 AM A monogenic cause of calcium oxalate urolithiasis is shared by multiple dog breeds Eva Furrow 10:45 AM – 11:15 AM Break & Exhibit Sessions Keynote 11:15 AM – 12:00 PM
Genetics of Vaccine-induced Immune Response in Beagles Jeanna Blake Full-length amplicon sequencing and analysis of the class I and class II major histocompatibility complex genes in dogs Jonah Cullen A multiomic case study of feline oral squamous cell carcinoma Alana Rodney Genetics of Acral Mutilation Syndrome in Several Dog Breeds Equivalent to Human Sensory Neuropathies Anna Letko Search for Local Adaptation in Dogs Living in Chernobyl Megan Dillon
#2
#3
#4
#5
Session: Poster Session HudsonAlpha Atrium 2:00 PM – 3:00 PM Odd numbers 3:00 PM – 3:30 PM Break & Exibit Sessions
Session: Regulation of Gene Expression Jackson Center 3:30 PM – 3:50 PM Deep learning approach to predict the impact of canine regulatory mutations Camille Kergal 3:50 PM – 4:10 PM Functional annotation of the dog genome Hannes Lohi 4:10 PM – 4:30 PM Genetic control of gene expression for trait mapping in the dog Reuben Buckley 4:30 PM Adjourn Open night Enjoy Huntsville!
Epilepsy genetics and the promise of precision medicine Heather Mefford, Center for Pediatric Neurological Disease Research St. Jude Children’s Research Hospital Memphis, TN
19
TUESDAY, OCTOBER 4, 2022
2:45 PM – 3:00 PM Break & Exhibit Sessions
12:00 PM – 1:20 PM Lunch
Session: Cancer Genetics Jackson Center 1:20 PM -1:40 PM
Session: Poster Session HudsonAlpha Atrium 3:00 PM – 4:00 PM Even Numbers Session: Large Scale Resources Jackson Center 4:00 PM – 4:20 PM Dog10K: A public canid genomics resource to advance demographic and functional studies Jennifer Meadows 4:20 PM – 4:40 PM The 99 Lives Cat Genome Consortium: updates and discoveries Leslie Lyons 4:40 PM – 5:00 PM Genetic epidemiology of blood type, disease and trait variants, and genome-wide genetic diversity in over 11,000 domestic cats Heidi Anderson Conference Dinner 5:15 PM Transfer to U.S. Space & Rocket Center 5:30 PM- 6:15 PM Social Hour U.S. Space & Rocket Center 6:15 PM – 7:00 PM Dinner 7:00 PM – 9:00 PM Dancing under the rocket
Genomic characterization of somatic alterations in canine Histiocytic Sarcoma leads to the development of therapeutic options and diagnostic tools Benoit Hedan 1:40 PM – 2:00 PM Genome wide association study of Invasive Urinary Carcino- ma (iUC) in Shetland Sheepdogs and related herding breeds Heidi Parker 2:00 PM – 2:20 PM Role of PIK3CA Mutations in Chromatin Accessibility and Transcriptional Program in Canine Hemangiosarcoma Jon Kim Session: Poster Flash Talks Jackson Center 2:20 PM – 2:45 PM
#6
SH3TC2, MTMR2, and MPZ variants in Golden Retrievers with congenital hypomyelinating polyneuropathy Shawna Cook VariantscanR – an R package as a clinical tool for variant filtering of known phenotype-associated variants in domestic animals Fréderique Boeykens Genetic investigation of spontaneous harlequin coat patterning in a family of Australian Shepherds Katherine Singleton A large animal model of RDH5- associated retinopathy recapitulates important features of the human phenotype Laura Ford
#7
#8
#9
#10 Identifying genomic regions
associated with pigmentary uveitis in Golden Retrievers Nayan Bhowmik
20
WEDNESDAY, OCTOBER 5, 2022
7:45 AM Breakfast
11:05 AM – 11:30 AM Break & Exibit Sessions
Announcements Jackson Center 8:30 AM Greg Barsh Session: Retrogenes Jackson Center 8:45 AM – 9:05 AM Variable representation of duplicated sequence in recent canine genome assemblies Anthony Nguyen 9:05 AM – 9:25 AM Recently derived, full-length gene retrocopies occur frequently in dogs Kevin Batcher 9:25 AM – 9:45 AM A TUBA1B Retrogene Insertion is Associated with Dwarfism in Great Pyrenees Dogs Kari Ekenstedt Session: Diabetes and Obesity Jackson Center 9:45 AM – 10:05 AM A common canine loss-of-function variant in MC3R delays puberty and reduces both body weight and adiposity Alyce McClellan 10:05 AM – 10:25 AM Congenital idiopathic megaesophagus is associated with a variable number tandem repeat in Melanin-Concentrating Hormone Receptor 2 in German shepherd dogs Jacquelyn Evans 10:25 AM – 10:45 AM Investigating the genetics of diabetes mellitus in the domestic cat Jessica Hayward 10:45 AM – 11:05 AM A common canine POMC mutation increases obesity risk by affecting both hunger and metabolic rate Eleanor Raffan
Session: New Ideas and Strategies Jackson Center 11:30 AM- 11:50 AM Estimating Working Dog breeding values for binary health traits Joseph Thorsrud 11:50 AM – 12:10 PM
Genomics and epigenomics of the feline Prader-Willi syndrome (PWS) orthologous-domain and generation of a genome-edited in vitro model Robert D. Nicholls 12:10 PM – 12:30 PM Investigation of heritability of behavioral traits in service dogs Emma Persoon Session: Student Awards Jackson Center
12:30 PM – 12:50 PM Award Presentation
12:50 PM – 1:00 PM
Closing Remarks Jackson Center 12:50 PM – 1:00 PM Greg Barsh Qinghong (Johnny) Li 1:00 PM Lunch 1:30 PM Conference ends
Thank you for joining us for ICCFGG 2022
21
ICCFGG 2022
BIO SKETCHES
22
KEYNOTE SPEAKERS
SUNDAY, OCTOBER 2, 2022
HudsonAlpha Faculty Investigator Jeremy Schmutz is co-director of the HudsonAlpha Genome Sequencing Center (GSC), one of the few centers in the world performing original sequencing of plants. As co-director, Schmutz leads a team of experts in whole-genome sequencing, library construction, genome analysis, computational biology, evolutionary genomics, and informatics. JEREMY SCHMUTZ
Learning to love the complexity of plant genomes
TUESDAY, OCTOBER 4, 2022
Heather Mefford is a full member of the faculty at St. Jude’s Hospital in Memphis, Tennessee. She runs a research laboratory dedicated to gene discovery in pediatric disease, with a major focus on pediatric epilepsies. Her work uses cutting edge genomic technologies and has helped define the genetic landscape of developmental and epileptic encephalopathies with studies reporting novel copy number variants and numerous novel disease-causing genes. She was a co-PI for several of the Epi4K consortium projects and co-chairs the ClinGen Neurodevelopmental Disorder Clinical Domain Working Group and Epilepsy Gene Curation Working Group. HEATHER MEFFORD
Epilepsy genetics and the promise of precision medicine
23
ICCFGG 2022
24
SOCIAL EVENTS #1 Opening Reception
Kickoff this year’s conference by getting to know your peers in a relaxed setting filled with music, drinks, and hors d’oeuvres. Whether you are a first-time attendee or an eleven-year veteran, our opening night reception is a great opportunity to catch-up with friends, make new connections, and get a feel for the venue before the meetings officially begin. #2 Conference Dinner Celebrate the final evening of the conference with friends, colleagues and new acquaintances by experiencing a special evening of culture, food and live en- tertainment. You will want not to miss this one-of-kind opportunity to dine and dance under an authentic Saturn V Apollo moon rocket – a National Historic Landmark! The U.S. Space & Rocket Center, a Smithsonian affiliate, houses the most complete collection of national treasures from space exploration on the planet.
25
ICCFGG 2022
26
PRESENTATION ABSTRACTS
27
ICCFGG 2022
#1 Comparative analysis of single haplotype genomes unmasks the role of structural variation in felid evolutionary innovation William J. Murphy 1,2 *, Kevin R. Bredemeyer 1,2 , LaDeana Hillier 3 , Andrew J. Harris 1,2 , Graham Hughes 4 , Edward Rice 5 , Terje Raudsepp 1,2 , Brian W. Davis 1,2 , Stephen J. O’Brien 6,7 , Leslie A. Lyons 8 , Wesley C. Warren 5 wmurphy@cvm.tamu.edu 1 Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA, 2 Interdisciplinary Program in Genetics & Genomics, Texas A&M University, College Station, TX, USA, 3 Department of Genome Sciences, University of Washington, Seattle, WA, USA, 4 Department of Zoology, University College Dublin, Dublin, Ireland, 5 Department of Animal Science and Bond Life Science Center, University of Missouri, Columbia, MO, USA, 6 Theodosius Dobzhansky Center for Genome Bioinformatics, Saint- Petersburg State University, Saint-Petersburg, Russia, 7 Oceanographic Center, Nova Southeastern University, Fort Lauderdale, FL, USA, 8 Department of Veterinary Medicine & Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA The role of structural variation in speciation is poorly understood. The recent divergence of the cat family, Felidae, provides a tractable system to study genomic and phenotypic divergence in unprecedented detail using near-gapless genome assemblies derived from F1 interspecies hybrids. Here we describe highlights from a comparative analysis of six single haplotype genome assemblies sampled across the felid phylogeny, where greater than 99.5% of the euchromatic sequence is assembled into chromosomes. Felid genomes are highly colinear, with a paucity of autosomal chromosome rearrangements, and enrichment of microinversions on the X chromosome. We observed rapid rates of sequence evolution across large, functional X-linked satellite repeats with known roles in X chromosome inactivation and felid speciation. High resolution maps of olfactory receptor gene family dynamics reveal surprising functional variation that we interpret in the context of domestication and life history traits. Finally, we present the first insights into the structure of cat centromeres and illustrate parallels and differences with their primate counterparts.
#2 Genetics of chronic superficial keratitis in the Australian Racing Greyhound
Claire M Wade 3 * , Steven Karamatic 1 , Rebecca Goode 2 , Niruba Bageswaran 3 , Cali E Willet 4 , Georgina Samaha 4 , Ray Ferguson 5 , Hamutal Mazrier 6 claire.wade@sydney.edu.au 1 Greyhound Racing Victoria, West Melbourne, VIC 3003 AUSTRALIA; 2 Greyhound Adoption Program, Greyhound Racing Victoria, Seymour VIC 3660 AUSTRALIA; 3 Faculty of Science, School of Life and Environmental Sciences, The University of Sydney, Camperdown, NSW 2006 AUSTRALIA; 4 Sydney Informatics Hub, The University of Sydney, Camperdown, NSW 2006, AUSTRALIA; 5 Australian Greyhound Working and Sporting Dog Veterinarians, East Oakleigh Vic. 3166 AUSTRALIA; 6 Faculty of Science, Sydney School of Veterinary Sciences, The University of Sydney, Camperdown, NSW, 2006 AUSTRALIA
28
PRESENTATION ABSTRACTS
Chronic superficial keratitis (CSK) is a progressive inflammatory condition of the cornea that can cause discomfort and blindness. CSK is considered to have a genetic basis due to elevated breed dispositions in certain breeds. The occurrence of CSK can be exacerbated by exposure to ultraviolet light. The current study considered a genome-wide association analysis including 109 greyhounds: 70 with CSK and the remainder with normal phenotype at an age over four years. Three co-located variants on CFA18 near the 5’ region of the Epidermal Growth Factor Receptor (EGFR) gene were associated with genome-wide significance after multiple-test correction [BICF2P579527, CFA18: 6,068,508, praw=1.765e-07, pgenome= 0.017; BICF2P1310662, CFA18: 6,077,388, praw=4.09e-07, pgenome= 0.040; BICF2P160719, CFA18: 6,087,347, praw= 4.09e-07, pgenome= 0.040) (canFam4)]. The associated haplotype on CFA18 is protective for the CSK condition. EGFR coding variants in the associated haplotype were identified that possibly reduce the efficacy of the protein. EGFR is known to play a role in corneal healing, where it initiates differentiation and proliferation of epithelial cells that in turn signal the involvement of stromal keratocytes to commence apoptosis. We propose that the reduced risk results from a reduction in the wound healing response that is corrupted in dogs with CSK. Further validation of the putative functional variants is required prior to their use in genetic testing for breeding programs. #3 Cell type specific differences in the prefrontal cortex of young and aging tame and aggressive foxes Jennifer L. Johnson 1 , Erin E. Hecht 2 , Darya V. Shepeleva 3 , Rimma G. Gulevich 3 , Anastasiya V. Vladimirova 3 , Svetlana G. Shishkevich 3 , Anastasiya V. Kharlamova 3 , Yuri E. Herbek 3 , Lyudmila N. Trut 3 , Anna V. Kukekova 1 jjohnso@illinois.edu 1 Department of Animal Sciences, College of Agriculture, Consumer, and Environmental Sciences, University of IL Urbana-Champaign, Urbana, IL, USA, 2 Department of Human Evolutionary Biology, Harvard University, Cambridge, MA, USA, 3 Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia Genetic analysis of tame and aggressive fox populations (Vulpes vulpes), developed by 60 years of selection for behavior, previously identified SorCS1, an Alzheimer’s associated gene, as one of the main loci implicated in behavioral differences between the populations. To identify brain tran- scriptomic differences associated with behavior and aging we performed single nucleus RNAseq (snRNAseq) analysis of the prefrontal cortex (PFC) of six young (9 months) and six old (7-9 years) female foxes with an equal number of tame and aggressive individuals. After processing, we obtained an average of 5,808 nuclei per sample with an average of 3,867 genes per nucleus. The snRNAseq analysis identified cell type composition of fox PFC and assigned populations of excitatory neurons to cortex layers using single cell resources available for human and mouse. Differential expression (DE) analysis highlighted excitatory neurons in cortex layers 2/3/4 in both comparisons, between populations and between age groups. A larger number of DE genes and stronger enrichment for KEGG pathways were identified between ages than between populations. Glutamatergic and GABA signaling were among the top pathways enriched in both comparisons, in former it was driven by genes upregulated in tame samples while in latter by genes upregulated in young samples. The well-established fox populations with genetically determined differences in behavior and a life span exceeding ten years provide a promising animal model for studying tradeoffs between evolution of social behavior and brain aging.
29
ICCFGG 2022
#4 Ultracontinuous Genomes Elucidate Complex Speciation Patterns within Panthera Andrew J. Harris 1,2 , Brian W. Davis 1 , 2 , Klaus-Peter Koepfli 3,4 , Eduardo Eizirik 5 , William J. Murphy 1,2 ajharris@cvm.tamu.edu 1 Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA, 2 Interdisciplinary Program in Genetics & Genomics, Texas A&M University, College Station, TX,USA, 3 Smithsonian Con- servation Biology Institute, Center for Species Survival, National Zoological Park, Washington, DC, USA, 4 Smithsonian-Mason School of Conservation, George Mason University, Front Royal, VA, USA, 5 School of Health and Life Sciences, Pontifical Catholic University of Rio Grande do Sul, Rio Grande do Sul, Brazil Genomes are a mosaic of evolutionary histories that reflect ancient signatures of true species relationships and incomplete lineage sorting (ILS) or gene flow. Understanding how and why phylogenetic signal varies across species genomes can yield powerful insights into species’ evolutionary histories and adaptive evolution. By integrating diverse data types with local genealogies, one can differentiate genetic variation consistent with the species tree from that stemming from natural selection, ILS, or gene flow. To better resolve the complex evolutionary history of the living cat species of the genus Panthera, we aligned PacBio® HiFi genomes from the jaguar, leopard, snow leopard, lion, and Indochinese clouded leopard to a highly continuous single haplotype assembly from the tiger. We conducted a whole-genome sliding window phylogenomic analysis and used our novel phylogenomics browser Tree House Explorer to visualize genome-wide variation in evolutionary histories and genetic divergence on a chromosome-by-chromosome basis in the context of recombination rates. We identified significant differences in the distribution of phylogenetic signal along the X chromosome, where low recombining regions harbor signal of the species tree. However, a subset of these regions contain a signal of ancient introgression of the leopard with an extinct or unsampled species with an ancient origin. Whole genome evaluations of structural variation also indicate an enrichment of structural rearrangements along the X chro- mosome, which likely played a role in the unique distribution of phylogenetic signal across the X chromosome and reproductive isolation. #5 Deep learning approach to predict the impact of canine regulatory mutations Christophe Hitte 1 ,Camille Kergal 1 , Doga2 Consortium, Marie-Dominique Galibert 1 , Thomas Derrien 1 hitte@univ-rennes1.fr 1 Univ Rennes 1, CNRS, IGDR – UMR6290, F-35000 Rennes, France, 2 DogA: www.doggenomeannotation.org Deep neural networks have been recently shown to be powerful methods to predict gene expression and, in fine, to assess the impact of regulatory mutations on gene expression. Here, we used the human-based tool Basenji to train a dog-specific model of gene expression based on deep-learn- ing (DL) to predict the impact of non-coding mutations on gene expression dedicated to the dog genome. We trained the model with a comprehensive set of canine Cap Analysis of Gene Expression data representative of 37 core canine tissues. We showed that the dog-specific model reached
30
PRESENTATION ABSTRACTS
similar performance as described in humans with high correlations defined between the true expression levels and the predicted ones (Pearson correlations median =0.66). We then investigated the impact of sequence variations in the promoter sequences of 1300 dog-human orthologous cancer genes. We identifed >15500 genetic variations occuring in the promoter regions using the whole genome sequences of 1929 canids generated by the dog10K con- sortium. Using the dog-specific model, we assigned 6% of the variants a low to moderate impact and 1% a high impact on the expression levels of the cancer genes. We thus predict an evaluation and classification of variants of regulatory regions of genes that contribute to cancers in dogs. Here, we developed a deep-learning based tool (BLIMP) to predict the impact of non-coding mutations on gene expression dedicated to the dog genome. Our tool and models are freely available through GitHub: https://github.com/ckergal/BLIMP
#6 A monogenic cause of calcium oxalate urolithiasis is shared by multiple dog breeds
Eva Furrow, Katie Minor, Jonah Cullen, Steven Friedenberg and Jody Lulich furro004@umn.edu
Department of Veterinary Clinical Sciences, University of Minnesota College of Veterinary Medicine, USA Calcium oxalate (CaOx) urinary stones are a common and painful problem in dogs. While CaOx stones primarily affect middle-aged to geriatric dogs, certain breeds are predisposed to an ear- ly-onset form of the disease, including the English Bulldog. Our aim was to identify a major genetic risk factor for early-onset CaOx stones. We performed whole genome sequencing of three English Bulldogs with early-onset CaOx stones (diagnosed at ≤2 years old) and filtered variants against an internal database of 241 dogs of 42 breeds. A single variant passed filtering: a missense variant in uromodulin. Uromodulin is the most abundant protein secreted in urine. It inhibits CaOx crystalli- zation and regulates renal calcium excretion. Genotyping of 37 English Bulldogs with CaOx stones and 96 with unknown CaOx status revealed a strong association between a homozygous genotype and stones (OR = 156, p = 4x10-15). All except one homozygote (a one-year-old female) had CaOx stones, with a median age of 2.5 years at diagnosis. Genotype analysis of over 1,300 dogs revealed that the variant is present at low frequency in at least seven other breeds. In addition to risk for early-onset CaOx stones, homozygotes have increased urinary calcium and decreased uromodulin excretion. The location of the variant within the protein suggests an impact on post-translational modifications. Further analyses are underway to determine the variant effects. In conclusion, a uromodulin variant underlies risk for early-onset CaOx stones across multiple dog breeds. We are referring to this condition as Hereditary Calcium Oxalate Urolithiasis Type 1 (Hereditary CaOx1).
31
ICCFGG 2022
#7 Functional annotation of the dog genome Hannes Lohi 1 & DoGA Consortium 2 hannes.lohi@helsinki.fi 1 Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland 2 www.doggenomeannotation.org Unlike other model organisms, the promoter and regulatory regions of the dog genome are not well annotated. To address this issue, we collected fresh tissues (>5000 samples from ~120 tissues) from 14 dogs and four wolves for systematic identification and annotation of transcription starting sites (TSSs), promoters, enhancers, and tissue-specific gene expression. To capture the promoter regions and quantify their expression levels, we generated STRT2 sequencing data from nine dogs, including 482 samples from 88 tissues from 13 organs. We annotated 56,236 robust, ubiquitous and tissue-enriched promoters, of which 36,506 are novel. CAGE-sequencing identified ~55,000 promoters with ~24,000 hitherto unannotated regions and ~21,000 active enhancer regions. We found that ~9,000 promoter and ~12,000 enhancer regions were enriched in a specific tissue. Using the distance and co-expression of promoter and enhancer candidates, we found that 44% of our enhancers are linked to at least one promoter region. Genomic analysis revealed that promoter regions were almost twice as likely to be hit by an SNP compared to other regions in the genome. We compared the sequence of our dog enhancers with human enhancers and found 435 conserved enhancers. Through these efforts, we have established the most comprehensive tissue collection of dogs, a gene expression atlas with a browsable database and interactive platform, and functional annotation of the dog genome with many novel promoters and enhancers. These findings and resources will facilitate the use of the dog as a model for human disease, behavior and morphology. #8 Congenital idiopathic megaesophagus is associated with a variable number tandem repeat in Melanin-Concentrating Hormone Receptor 2 in German Shepherd dogs Jacquelyn M. Evans 1,2 , Sarah M. Bell 1 , Katy M. Evans 3,4 , Kate L. Tsai 1 , Rooksana E. Noorai 1,5 , Thom- as R. Famula 6 , Dolores M. Holle 3 , Leigh Anne Clark 1 jme255@cornell.edu 1 Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina, USA, 2 Baker Institute for Animal Health, Cornell University, Ithaca, New York, USA 3 The Seeing Eye Inc., Morristown, New Jersey, USA, 4 School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, UK, 5 Clemson University Genomics and Bioinformatics Facility, Clemson University, Clemson, South Carolina, USA, 6 Department of Animal Science, University of California, Davis, California, USA Congenital idiopathic megaesophagus (CIM) is a genetically complex gastrointestinal motility disorder found across dog breeds but occurring at a uniquely high frequency in the German shepherd dog (GSD). Reduced peristaltic activity and dilation of the esophagus impede the passage of food into the stomach, causing regurgitation of meals upon introduction of solid food. Affected puppies surviving to adulthood require lifelong management and are at risk for aspiration pneumonia. We observed that male GSDs are nearly twice as likely to be affected as females. Using records from a private breeding colony, we show that this bias is independent of body size differenc-
32
Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8 Page 9 Page 10 Page 11 Page 12 Page 13 Page 14 Page 15 Page 16 Page 17 Page 18 Page 19 Page 20 Page 21 Page 22 Page 23 Page 24 Page 25 Page 26 Page 27 Page 28 Page 29 Page 30 Page 31 Page 32 Page 33 Page 34 Page 35 Page 36 Page 37 Page 38 Page 39 Page 40 Page 41 Page 42 Page 43 Page 44 Page 45 Page 46 Page 47 Page 48 Page 49 Page 50 Page 51 Page 52 Page 53 Page 54 Page 55 Page 56 Page 57 Page 58 Page 59 Page 60 Page 61 Page 62 Page 63 Page 64 Page 65 Page 66 Page 67 Page 68 Page 69 Page 70 Page 71 Page 72 Page 73 Page 74 Page 75 Page 76 Page 77 Page 78 Page 79 Page 80 Page 81 Page 82 Page 83 Page 84 Page 85 Page 86 Page 87 Page 88 Page 89 Page 90 Page 91 Page 92 Page 93 Page 94 Page 95 Page 96 Page 97 Page 98 Page 99 Page 100 Page 101 Page 102 Page 103 Page 104 Page 105 Page 106 Page 107 Page 108 Page 109 Page 110 Page 111 Page 112Made with FlippingBook - Online magazine maker