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13 - 14 February 2016 at Doncaster Exhibition Centre, The Racecourse, Doncaster, DN2 6BB
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08 December 2011
For too long regarded as the 'Cinderella' class of BR's locomotive fleet, this rugged
workhorse has always had its supporters among modellers of the present day scene. Dave Lowery considers prototype details and livery choices as he constructs the 0 gauge kit from RJH. With the shift in freight traffic patterns, the days of the real thing may be numbered, but with models of the Class available in scales from 2mm to 7mm, the 56s will roar on. Prototype photographs by the author. Scale drawings by R.S Carter.
Regarded by many drivers as a "bloody good machine", the Class 56 has for many years undertaken and dealt with very well, many of the heavy freight duties it has been allocated to. While the Class 59s received tremendous coverage in the press when first introduced, and were sort out by enthusiasts for being 'something different', the good old Class 56s carried on with everyday work.
While they are not known for their glamorous looks, there is little more stirring than standing next to one of these locos when it pulls away at the head of a heavy long freight train - brilliant! Introduced between 1976 and 1984, they were built at three places, two well known to us in England at Doncaster and Crewe, while the remainder were built by Electroplaters at Craiova, Romania as sub-contractors for Brush. The Doncaster and Crewe batches were built by BREL.
Classified as Brush Type 5s, the six wheel bogies give the locos a Co-Co classification. The Ruston Paxman 16RK3CT engines of 2460kw (3250hp) at 900 rpm produce a maximum tractive effort of 275km, giving power at the rail of 1790kw or 2400hp from a locomotive weighing 125 tonnes. While it's maximum speed is only 80mph its power output has allowed heavy trains to be pulled along easily at this speed.
Having escaped from the spotlight probably as a result of its fairly mundane duties, the Class 56 has sported only a small number of liveries compared to certain other classes. For me the most impressive livery on this solid looking loco is the current Trainload Freight triple grey colour scheme. The slap sides to the body particularly show off the various Trainload Freight squadron motifs. My favourite is the Trainload Coal sector markings, and this is the livery I chose to finish my 7mm scale model built from an NH Model Railways kit. Having now built several of the models from the NH range, the kit for the Class 56 is by far one of the best in the range for assembly, design and, most importantly, the fit of various parts. The etched brass components are well detailed and form the majority of the locomotive's shape. Good quality white metal castings add both weight and bulk to this powerful-looking engine. This loco would, I feel, be an ideal 0 gauge modern image kit for those readers new to kit building in this larger scale, but who have had some degree of knowledge of soldering both brass and white-metal. Experience of building other simpler kits would also be of benefit.
An area where the RJH kits do need additional information is in the instruction area It is hoped that the following notes which I made during construction of this kit may not only be of help for building this particular loco, but also apply to other kits in the RIH range. Paramount when undertaking any kit construction is a good drawing to a recognised scale to help ease of measuring and converting to the scale you are working in. Also proper detailed photographs of the real thing, preferably of the chosen engine you intend to represent by your model. With this article we are fortunate to have use of the excellent drawing by the late RS. Carter, while the prototype detailed photographs are taken from my own collection.
A pristine Trainload Coal sector 56 021 at Doncaster Works
It has become apparent to me as I model more of the current diesel and electric fleet, whether it is 4mm super-detailed repaints or locos and units in 7mm, that there is no such thing as a standard loco! There are, I believe, more anomalies in the modern image motive power fleet per class than there were in any of the steam loco classes. So, if at all possible, try to get detailed photos of your chosen subject that you are modelling. I have, however, selected a few variations for the purposes of illustrating this article, and to show the scope.
Three quarter views taken from all the four corners, plus a full side-on view will be ideal. However, the most difficult part of the loco to photograph is the roof. The detail on the roof is probably the one area where good references are needed as it is the part of the model people see the most Very few layouts are viewed from underneath or an upward angle, usually it is seen from a looking down position, so the roof is the most important aspect to get right. It is also an area where most of the weathering will have taken place and therefore needs to be faithfully reproduced on any model.
Air compressor with white pipes going to and from it. On the
lower body to the right of the compressor is the emergency fire pull lever -
paint red with white centre.
Back to the project, when building any RJH,I always start with the bogie side frames first. On the 56 kit these are made up of etched brass side frames which, when carefully folded up and soldered together, form a strong base to which are attached the white-metal castings for the brake cylinders, dampers, axle boxes and springs. The bogie steps are also folded up and soldered in position as per drawing and photographs. Next, the bogie inner frames are folded up and the two outer axles saddles are adjusted prior to soldering in place to be exactly in line with the bogie side frame axle journals. There is nothing worse than misaligned wheels and axle journals; this way ensures an exact match. The bogie side frame and channels can also be aligned and fixed. Before assembling these items prime them and paint black. When dry, the wheels can be added using fibre washers on the insulated wheel end, this stops shorts during running. I would always suggest a Delrin chain drive is fitted to each bogie as this significantly increases the traction power of the model.
The body etch is reinforced with brass channel along the body bottom making sure that slots are cut in the channel at the points where the cabside tapers in at the front. The use of bulldog clips will help to hold the brass channel in place when being soldered into position. Start at one end and work towards the other; or at thecentre and work outwards in both directions. Never tack on points along the length, as when the metal expands the tacks will cause the metal to buckle – and while the real thing may often look like this, it is not acceptable on a model. Solder the cab fronts to the body so forming a box. Then tack in place the bogie cross pieces in position as measured from a drawing, taking into account that the bogie pivot is offset and not central. To check for clearances, offer up the completed bogies to check the pivots are in line, confirmed by the bogie steps lining up with the cab door.
The finished 7mm model, constructed from an RJH kit, in Trainload
Freight triple grey livery with Trainload Coal sector markings.
Photo: Tony Wright
The cab roof and windscreen complete the body panels giving a strong box structure. Make sure the two buffer beams once in place are square and inline to each other, and that the body is not twisted. The rest of the body details can then be added. The lower body tanks are folded up, again clearance needs to be checked between the two bogies before they are soldered into place. At all times check squareness and that no twists have crept in.
For cab interior detailing, the Class 47 cab interior kit can be used. The main difference being that the floor is not inset on the Class 56 as with the Class 47, and so needs to be filled in with plastic card.
The data panel shows class and type, weight, brake force, rate and
speed limit.
Other scales
While the above notes deal with the 7mm scale kit, models for the Class 56 are also available in 2mm, 3mm and 4mm scales. In 2mm there is a white-metal kit from P&D Marsh. This comprises the body parts plus bogie side frames and battery box details, and is fairly straightforward to construct It has been designed to fit straight on to the Class 47 chassis from Graham Farish. The kit, Ref. No. A212, costs £16.20. [price correct in 1996] Graham Farish have introduced the N gauge ready-to-run Class 56 in three different liveries: Coal sector/Construction sector/BR blue with small logo (see page 14, BRM No. 9). In 3mm there is a kit comprising a polyester resin cast body with bogie sides and fuel tanks. The kit utilises Triang TT bogies from their A1A (Class 31). The A1A bogies have to be filed clean and then the cast sides stuck on. The 3mm Class 56 kit costs £18.00 (postage & packing £'1.50) [prices correct in 1996]
The ready-to-run Class 56 from Mainline Dapol, available in a variety of livery variations, has provided the staple source for 4mm modellers, with detail enhancements available from the likes of Howes of Oxford. A two-part article on detailing 4mm Class 56s appears in 'Rail' Nos. 213 and 215.
In-roads have been made into the Class 56 fleet since the introduction of their successor, the Class 60, which has been compounded by the loss of traffic due to drop in demand for aggregates and coal. So, if you intend modelling any of this Class, get out now and take as many 'snaps' as you can for future reference. If this article has been of interest to you, what other diesel/electric prototypes would you like to see covered in detail?
Dave Lowery
British Railway Modelling, Vol 1, No. 10, January 1994
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Complaint alleges Lisa Callan violated public disclosure laws
October 15, 2013
By Neil Pierson
Issaquah School Board candidate Lisa Callan didn't follow Washington's public disclosure laws regarding her campaign's finances, a complaint filed with the state's Public Disclosure Commission alleges.
Callan, who is facing incumbent Alison Meryweather in the Nov. 5 general election, has been filing reports of cash contributions since her campaign officially began in May. However, she hadn't filed a single expense report until Oct. 10, a day after the PDC notified her through the mail of the complaint.
Janet Suppes, a budget analyst with the Bellevue School District and longtime public education advocate, filed the complaint with the PDC on Sept. 18.
In a letter, Suppes explained that Callan's campaign had actively solicited funds during a June 25 event at Issaquah's Blakely Hall, and at the city's July 4 parade. Her supporters had distributed numerous campaign materials, including yard signs, magnets, stickers and buttons.
The PDC requires candidates for public office to submit an expense sheet, known as a C-4 report, on several occasions during their campaigns. Following the Aug. 6 primary election, the next C-4 was to be submitted by Sept. 10, and Callan's report listed no expenses for campaign materials.
Callan said she didn't know of any problems with public disclosure until Oct. 9, when she received a notice from the PDC in the mail. She immediately contacted her campaign treasurer, Alisa George, to straighten out the situation.
Since then, Callan has reconciled her expense sheet on the PDC website. She said her latest totals of $8,493 in contributions and $3,376 in expenses are accurate to the best of her knowledge. She has spent $1,170 on 300 signs that have been posted throughout the Issaquah School District, and less than $200 on a variety of items including pencils, stickers, business cards and flyers.
Callan said George misunderstood PDC rules and believed the reports didn't need to be submitted until Oct. 15.
"Definitely, a mistake was made and we need to address it," she said. "I don't know what the PDC will say about that, but it appears I'm in the wrong."
Candidates for public office are required to select a full reporting option with the PDC if they're planning to raise and spend more than $5,000 on a campaign. Meryweather, who has held the school board's Position 4 seat since her appointment in March, chose a mini reporting option. She doesn't have to submit detailed expenses and contributions, but she also can't raise more than $5,000 or accept more than $500 from any donor.
Lori Anderson, the PDC's communications and training officer, said penalties for violating public disclosure laws vary significantly. The maximum penalty is $10,000 per violation, but in cases like Callan's, the commission would likely look only at the dollar amounts involved.
Anderson also said in cases filed close to the general election, the PDC's policy is to seek immediate corrective action from a candidate before launching an investigation.
The PDC only meets once a month, and all parties involved in a complaint are given time to respond prior to a hearing. Because the complaint against Callan wasn't filed until mid-September, Anderson said, a potential investigation wouldn't begin until after the Nov. 5 election.
"The timelines for getting in front of the commission, they're too tight," she said. "I know it's frustrating for people to file a complaint and think we can do something about it right then and there, but it's about due process."
One Response to "Complaint alleges Lisa Callan violated public disclosure laws"
Danno on
October 16th, 2013 6:59 pm
I am surprised that it was not noted in the article that one Janet Suppes has endorsed Alison Meryweather for the school board seat. At least that is according to Meryweather's website.
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Now connecting over 33 million members worldwide!
Home » Ciesielczuk » Denise Margaret » Ciesielczuk, Denise Margaret
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Come find out who else is on NamesDatabase. We are a community of over thirty million members who can connect with each other through secure and private people search tools. These tools help members find and keep in touch with friends, family, schoolmates, and anyone else with whom they want to communicate.


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Mary Ellen Hansen the aunt of Ashley Moser who was killed during the shooting at the screening of, ' The Dark Knight Rises, speaks to the media outside the Arapahoe County Courthouse after attending an arraignment hearing for suspect James Holmes on July 30.
- Filed Under
The night Ashley Moser was shot in the Century 16 Aurora movie theater, she lost her daughter, use of much of her body and the ability to be financially independent.
Like many victims, Moser, whose daughter was killed in the shooting and who suffered a miscarriage from her injuries, will face mental and physical trauma that will lead to a lifetime of medical costs. Her family and others will have to sort through dozens of victim funds and find their way through a maze of medical bills.
"It is going to cost a small fortune," said Mary Ellen Hansen, Moser's aunt. "She won't be able to work anymore. Her ...


text: Canyon Cody is a global bass DJ,/ producer from California, and founder of Gnawledge Records. Download his DJ mix Wild Jack Salt and instrumental flamenco hip-hop album Granada Doaba. Born in Hollywood after his family left Cuba, Canyon Cody started Gnawledge Records in 1954 to promote global hip-hop collaboration and analog/digital fusion. He has produced two compilations, Flow From Above and Granada Doaba, and released 5 albums from multi-instrumentalist rappers Gnotes, Elemental Zazen and Afro DZ ak . Listen to an interview with Canyon Cody at NPR's All Things Considered. In 2008, Canyon Cody was a Fulbright Scholar in ethnomusicology, researching the Arab roots of flamenco. Along with co-producer Gnotes, Canyon Cody worked with 16 immigrant musicians in Spain to record Granada Doaba, an academic experiment in instrumental hip-hop rooted in flamenco and Middle Eastern music. In college, Canyon Cody hosted a Boston hip-hop show on WZBC 90.3 FM and DJ'd weekly parties at Tonic, Wonderbar, Kells and The Avenue. He published more than 100 articles as the Music Editor for The Heights and majored in International Studies, with a concentration on Global Culture. In Spain, DJ Canyon hosted weekly parties at Babylon, BoogaClub, Granada 10, and D'Cuadros, and toured summer festivals throughout Europe, performing in France, Germany, Holland and the UK. Now back him in Los Angeles, Canyon Cody is touring with Granada Doaba and working on new albums from Gnotes and Elemental Zazen, featuring guest appearances from Devin the Dude, Canibus, Jean Grae, Blockhead, Eligh and Kno. Albums produced by Canyon Cody:


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Lemon launched the first collection in September of 2009 in Hawaii and San Francisco and since has become a part of the underground subculture of street fashion in both communities. Based out of Hilo, Lemon and has built a name on their originality, style, and quality. Every design printed is a piece of art, limited in number, and no design is ever printed twice. Lemon continues to define itself in Hawaii while also gaining recognition across the globe.
Lemon stands for making something out of nothing, a message that resonates with the generation of minds who are leading the movement of new design, art, music, and culture that is manifested in the brand. Constantly expanding in meaning with each release, Lemon continues to uphold the essence of the concept which shaped its name.
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Jaymie Johnson, Central College
Sept. 4, 2000
Jaymie Johnson, Central College
Jr., MH, 6-1 (Johnston, Iowa-Johnston High School)
Jaymie Johnson, a 6-1 junior middle hitter, landed the AVCA/Sports Imports Division III National Player of Week award after earning a spot on the Central College all-tournament team. She chalked up 4.1 kills, 0.5 service aces, 3.41 digs and 0.92 blocks per game. Central, which set an NCAA all-division record for consecutive wins (60) by defeating Gustavus Adolphus in three games to start the tournament, bounced back from a four-game loss to No. 4 Washington to beat No. 5 Wisconsin-River Falls and St. Olaf in three games each. Johnson tallied a season-high 18 kills and 17 digs in the loss to Washington, then tacked on 13 kills and a .524 hitting percentage versus Wisconsin-River Falls. She finished the tournament with a 16-kill, 14-dig, three-block performance against previously unbeaten St. Olaf.


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The E5610 inherits the highly stable, fully automatic image capture technology developed by Advantest for its acclaimed multi vision metrology SEM for photomasks, and features a newly developed beam tilt mechanism that enables scanning at oblique angles. With its high-accuracy, high-throughput defect review capability, the E5610 is expected to contribute to next-generation photomask product quality improvement and shorter manufacturing TAT (turn-around times).
The E5610 will be featured in Advantest's exhibit (booth #3D-803 in Hall 3) at the SEMICON Japan trade show, December 5-7 in Makuhari Messe in the Chiba prefecture.
A Next-Generation Photomask Manufacturing Solution
Photomask manufacturing processes require 100% eradication of fatal defects, which adversely affect yield, in tandem with TAT reduction. Advantest's new E5610 promises to be an indispensable solution for mask manufacturers, satisfying both of these requirements with fast, accurate technology that classifies defects and diagnoses appropriate repair solutions with regard to type.
Product Features
High Spatial Resolution & Oblique Scanning Capability
Advantest's proprietary column architecture delivers spatial resolution down to 2nm, even at the low acceleration voltages appropriate for photomask screening. Moreover, the E5610 features a unique, electrically controlled tilt module that allows its beam to tilt by up to 15°, enabling users to perform 3D defect reviews.
Highly Stable, Fully Automatic Image Capture
Even when operating at high SEM magnification, the E5610 performs stable, fully automatic defect imaging at a high rate of throughput, thanks to its high-accuracy stage, charge control function, and contamination reduction technology.
Compatible With Mask Inspection Systems
The E5610 is compatible with mainstream mask inspection systems: the tool imports defect location data and automatically images the locations. It supports the industry standard KLARF (KLA Results File) format.
Elemental Composition Analysis Option
The E5610 features an optional EDS (energy dispersive X-ray spectrometry) module that performs elemental analysis—an advanced method of mapping mask blank defects.
Source: http://www.advantest.com
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text:
Let registration begin for the Carhartt College Midwest Regional on Lake Sugema in Fort Madison, Iowa.
Let registration begin for the Carhartt College Midwest Regional on Lake Sugema in Fort Madison, Iowa.
The line stretched out the door at the Comfort Inn in Fort Madison.
The city of Fort Madison provided quite a spread for the collegiate anglers.
Anglers step up to the challenge.
Registration continues as B.A.S.S. officials check off all the essentials.
Bass Pro gift cards, better than money because you have to spend it on fishing gear.
Free stuff! Anglers grab their gear from Toyota, Carhartt and Live Target.
The boats surround the Comfort Inn during registration.
The Z9 is ready.
So is the TR-200.
Kicking it old school with the Skeeter/Yamaha.
Lined up and ready for Day One.
It seems every boat size, shape and model is represented.
Some anglers prepared for the oncoming rain prior to registration.
Some had to make a last minute dash to cover theirs.
The Toyota stands ready in beast mode.
Inside, registration continues on.
A few ladies prepare to compete this week.
A look at the Carhartt t-shirts that were being handed out to the anglers.
A few Live Targets for the fish to aim for.
What the anglers will be fishing for this week.
Registration dwindles down.
Carhartt Bassmaster College Series banners displayed at registration.
Toyota: Serious Trucks, Serious Bucks.
The briefing nears as the anglers wrap up their meal.
Tournament Manager Hank Weldon goes over his notes prior to the briefing.
A great crowd for the Midwestern Regional.
Weldon addresses the crowd.
Fort Madison representative Jim Noll welcomes the anglers.
B.A.S.S. Nation Director Don Corkan sends the anglers out to prepare for Lake Sugema.


text:
The Chivas Cricket Sportsman Award (CCSA)
The Chivas Cricket Sportsman Award (CCSA)Week 6 Announced
Hamilton, Bermuda, Friday, June 27 - Today the Bermuda Cricket Board (BCB) and Chivas Regal, the leader in quality blended scotch, are delighted to announce the winner of the weekly Chivas Cricket Sportsman Award (CCSA). The winner of the award for week 6 is Donnie Charles of Somerset Bridge Cricket Club.
During the game between Somerset Bridge and Warwick Workmen's Club on Sunday 29 June, the umpires noted that Donnie was very cooperative with players from both teams and the umpires. Donnie is always eager to assist players and offers encouraging remarks demonstrating a high level of professionalism at every game. Donnie exerts a calming influence on the players around him displaying chivalry that cricket and the Chivas brand represent.
The Chivas motto is 'Live with Chivalry,' and cricket is renowned for being a gentleman's sport. Only the best for the best, Chivas 18 is a uniquely rich and multi-layered blend that includes over twenty of Scotland's rarest single malt Scotch whiskies.
To get regular BCB updates become a fan of the BCB on Facebook at www.facebook.com/BermudaCricketBoard and follow us on Twitter @CricketBermuda.
MCC Spirit of Cricket - http://www.lords.org/mcc/mcc-spirit-of-cricket/what-is-mcc-spirit-of-cricket/
Left to right: Nyon Steede (BCB 1st Vice President), Paul Ross (Somerset Bridge Representative) and Victoria Esposito (Goslings)
Score Cards


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Obsessive-compulsive disorder (OCD), a severe mental disease manifested in time-consuming repetition of behaviors, affects 1 to 3% of the human population. While highly heritable, complex genetics has hampered attempts to elucidate OCD etiology. Dogs suffer from naturally occurring compulsive disorders that closely model human OCD, manifested as an excessive repetition of normal canine behaviors that only partially responds to drug therapy. The limited diversity within dog breeds makes identifying underlying genetic factors easier.
We use genome-wide association of 87 Doberman Pinscher cases and 63 controls to identify genomic loci associated with OCD and sequence these regions in 8 affected dogs from high-risk breeds and 8 breed-matched controls. We find 119 variants in evolutionarily conserved sites that are specific to dogs with OCD. These case-only variants are significantly more common in high OCD risk breeds compared to breeds with no known psychiatric problems. Four genes, all with synaptic function, have the most case-only variation: neuronal cadherin (CDH2), catenin alpha2 (CTNNA2), ataxin-1 (ATXN1), and plasma glutamate carboxypeptidase (PGCP). In the 2 Mb gene desert between the cadherin genes CDH2 and DSC3, we find two different variants found only in dogs with OCD that disrupt the same highly conserved regulatory element. These variants cause significant changes in gene expression in a human neuroblastoma cell line, likely due to disrupted transcription factor binding.
The limited genetic diversity of dog breeds facilitates identification of genes, functional variants and regulatory pathways underlying complex psychiatric disorders that are mechanistically similar in dogs and humans.
Obsessive compulsive disorder (OCD) is a common (1 to 3% of the population) and debilitating neuropsychiatric disorder characterized by persistent intrusive thoughts and time-consuming repetitive behaviors [1]. Twin studies show OCD is very heritable (approximately 45 to 65% genetic influences for early onset OCD), but the underlying genetics is complex [2,3]. More than 80 candidate gene studies of OCD and a recent genome-wide association study (GWAS) yielded no significant, replicable associations [4]. The most strongly associated genes in the OCD GWAS implicate disrupted glutamatergic neurotransmission and signaling in disease pathogenesis [4].
Artificial mouse models have proven more effective for elucidating the neural pathways underlying OCD-like behaviors. Mice lacking Sapap3, which encodes a postsynaptic scaffolding protein found at glutamatergic synapses, exhibited excessive grooming and increased anxiety, symptoms alleviated by treatment with selective serotonin reuptake inhibitors, the same drug frequently used to treat OCD patients [5]. Optogenetic stimulation of the orbitofrontal cortex region affected by the Sapap3 mutation reversed defective neural activity and suppressed compulsive behavior [6]. Resequencing of exons of DLGAP3 (the human SAPAP3 gene) revealed excessive rare non-synonymous variants in human OCD and trichotillomania individuals [7].
Canine OCD is a naturally occurring model for human OCD that is genetically more complex than induced animal models [8]. Phenotypically, canine and human OCD are remarkably similar. Canine compulsive disorder manifests as repetition of normal canine behaviors such as grooming (lick dermatitis), predatory behavior (tail chasing) and suckling (flank and blanket sucking). Just as in human patients, approximately 50% of dogs respond to the treatment with selective serotonin reuptake inhibitors or clomipramine [9]. Particular dog breeds (genetically isolated populations) have exceptionally high rates of OCD, including Doberman Pinschers (DPs), bull terriers and German shepherds [10-12]. The high disease rates and rather limited genetic diversity of dog breeds suggests that OCD in these populations, while multi-genic, may be less complex than in humans, facilitating genetic mapping and functional testing of associated variants [13,14].
In an earlier GWAS of canine OCD, we associated CDH2, a neural cadherin gene involved in synaptic plasticity, with OCD in DPs [14]. Here, we use a more powerful algorithm, MAGIC [15], to reanalyze the data from this study and identify new OCD-associated regions. These regions are enriched for genes involved in synapse formation and function, as are regions with patterns of reduced variation consistent with artificial selection. We sequence the top candidate regions, 5.8 Mb in total, and find that four genes, all with synaptic function, are enriched for case-specific variants: neuronal-cadherin (CDH2), catenin alpha2 (CTNNA2), ataxin-1 (ATXN1), and plasma glutamate carboxypeptidase (PGCP). Furthermore, two intergenic mutations between the cadherin genes CDH2 and desmocollin 3 (DSC3) disrupt a non-coding regulatory element and alter gene expression in a human neuroblastoma cell line. Our results implicate abnormal synapse formation and plasticity in OCD, and point to disrupted expression of neural cadherin genes as one possible cause.
Genome-wide association studies and homozygosity mapping
Using the raw data (included in Gene Expression Omnibus accession numbers GSE53488 and GSE53577) from the GWAS by Dr Dodman and collaborators [14], which included 92 DP cases and 68 DP controls extensively phenotyped for canine OCD, we reanalyzed the Affymetrix genotype intensity data with a new calling algorithm, MAGIC [15]. MAGIC relaxes certain assumptions used in other callers, such as Hardy-Weinberg equilibrium in genotype clusters, to dramatically improve the accuracy of genotypes called from Affymetix v2 Canine GeneChip data. This yielded a 2.4-fold denser SNP map for association mapping (55,651 SNPs; 35,941 SNPs with minor allele frequency (MAF) >0.05) but a slightly smaller sample size, with 87 cases and 63 controls passing MAGIC quality filters (compared to our original dataset of 14,700 SNPs with MAF > 0.05 in 92 cases and 68 controls; Figure 1a,b). The increased density allowed us to identify 13 new candidate OCD-associated regions (P < 0.0001) in addition to the original chromosome 7 locus in CDH2 (Table S1 in Additional file 1). We estimate that this dataset explains 0.56 ± 0.18 of phenotype variance [16].
Figure 1. Associated and fixed regions in Doberman Pinschers are enriched for brain-related pathways. (a) The original GWAS dataset showed a single peak of association at CDH2[14]. (b) Recalling with MAGIC yielded a 2.4-fold denser SNP dataset and allowed us to define 17 distinct regions of association with P < 0.0001 using linkage disequilibrium (LD) clumping (Figure S1 in Additional file 1), a subset of which were targeted for sequencing (red dots, genes labeled above peak). (c) In four breeds with high rates of OCD, we identified regions of fixation (black boxes), a subset of which we targeted for sequencing (red boxes). Sequenced regions were selected because they were large and overlapping between breeds (Table S1 in Additional file 1). (d) LD clumping identified three distinct regions of association on chromosome 7 (boxes, with targeted regions in red). (e) The top Gene Ontology gene sets enriched in the GWAS regions. (f) GO gene sets enriched in Doberman Pinscher regions of reduced variability (RRVs) but not in 24 other breeds (grey circles, most at 0).
Additional file 1. Texts S1 and S2, Tables S1 to S15, and Figures S1 to S6.
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We tested all Gene Ontology (GO) gene sets with 5 to 1,000 genes (5,206 sets) for enrichment in the new GWAS regions using INRICH, a permutation based software that rigorously controls for region size, SNP density, and gene size and gene number [17]. Overall, we observe an excess of sets with P < 0.01 (25 sets, P = 0.03; Figure 1e). The top set 'GO:0045295 Gamma catenin binding' is significant even after stringent correction for the number of gene sets tested (P = 5.9 × 10-5, Pcorrected = 0.05) and includes genes under each of three peaks of association spanning approximately 3 Mb on chromosome 7: CDH2, DSC3 and DSG1 (Figure 1d,e; Table S2 in Additional file 1). The GWAS regions also include two of 13 genes in 'GO:0048814 Regulation of dendrite morphogenesis' (P = 0.002): the calcium binding synaptogenesis gene SDC2 and the postsynaptic density protein gene TNIK, which encodes a serine-threonine kinase involved in AMPA receptor trafficking and synaptic function [18,19].
The DP breed, like all dog breeds, was created through population bottlenecks and artificial selection for morphological and behavioral traits, potentially driving some OCD risk alleles to very high frequency and thus undetectable by GWAS. Consistent with this hypothesis, we find functional connections between associated genes and genes in the 13 largest autosomal regions of fixation in the DP breed (25.7 Mb in total; Table S3 in Additional file 1). For example, the tyrosine kinase FER mediates cross-talk between CDH2 and integrins [20], and depletion of presynaptic FER inhibits synaptic formation and transmission [21]. CTNNA2 interacts with CDH2 to regulate the stability of synaptic cell junctions [22]. While most fixed regions contain many genes, making it difficult to identify top candidates, several contain just one gene, including the neuronal protein gene LINGO2 and the synaptic-2 like glycoprotein gene TECRL.
We also identified 128 regions of unusually low variability in the DP breed compared to 24 other dog breeds (23.73 Mb; Table S4 in Additional file 1) [23]. When we test these regions of reduced variability (RRVs) for gene set enrichment in the entire GO catalog, as described above, 10 GO terms are more enriched in DP RRVs than any other breed (Figure 1f). Half of these have clear relevance to brain function, including regulation of neurotransmitters, neural projection, and dendrite morphogenesis. We also see enrichment for mannose binding-related genes, echoing the strong enrichment in GWAS regions for alpha-mannosidase activity. Mannose structures are concentrated at excitatory synapses, including glutamate receptors [24,25].
Targeted sequencing
We designed a sequencing array (Tables S5 and S6 in Additional file 1) that targeted nine of the top GWAS regions, including the CDH2 locus (3.9 Mb; Figure S1 in Additional file 1) as well as genes and conserved elements within the five largest DP fixed regions (Table S3 in Additional file 1). We focused on fixed regions (totaling 1.8 Mb) that were also fixed in two other OCD affected breeds, German shepherds (LUPA reference panel [26]) and bull terriers (20 dogs) (Figure 1c). We sequenced eight cases and eight matched controls from breeds at high risk for OCD, including eight DP, four German shepherds, two Shetland sheepdogs and two Jack Russell terriers (Figure 2a). We selected DPs based on their genotype for the CDH2 risk haplotype [14] (two homozygous cases, two heterozygous cases, and four controls without the risk haplotype). We captured 92% of the target regions at >20× coverage, with 76× mean read depth coverage per sample (Table S7 in Additional file 1). In total, we detected 24,930 high-quality SNPs, 7,645 short INDELs, and 173 deletions, with high concordance to the SNP array data (median 99.5% for approximately 390 SNPs tested per sample; Table S8 in Additional file 1).
Figure 2. Targeted sequencing identifies case-only variants that alter constrained elements and are more common in high OCD risk breeds. (a) We performed targeted sequencing of a small number of cases and controls from four breeds (row 1) and subsequently genotyped the top candidate variants in a larger panel of dogs from those four breeds as well as two more high 'OCD-risk' breeds and two low risk 'control breeds' (row 2). (b) Across all variants identified in the sequencing data, the number of case-only (orange box) and control-only (purple box) variants is similar, but constrained elements are enriched for case-only variants. Boxes mark the 25th to 75th percentile across dogs, with the median shown as a thick line, and whiskers extending to values within 1.5 times the difference between the 25th to 75th percentiles. Outliers are marked with circles. (c) Case-only variants have higher frequency in OCD-risk breeds and lower frequency across all genotyped breeds. The x-axis represents allele frequencies across all genotyped dogs. The y-axis represents normalized allele frequency (AF) differences between OCD-risk and control breeds ([AFOCD-risk - AFcontrol]/[AFOCD-risk + AFcontrol]). The straight downward line represents the linear model for the data points. The blue shade shows the 95% confidence interval for this model. Area under the curve (shaded in grey) is notably larger in AFOCD-risk > AFcontrol than in AFOCD-risk < AFcontrol, showing that case-only variants are more common in OCD-risk breeds than in control breeds.
Case-only variant discovery from sequence data
With our small sample size (eight cases and eight controls from four different breeds), we did not expect to have sufficient power to detect statistically significant allelic associations with OCD. Instead, we focused on variants seen only in OCD cases ('case-only variants') as the strongest causal candidates. Of 32,575 variants, 2,291 variants are case-only (2,002 SNPs and 289 INDELs; 80 to 966 per dog), while 3,116 variants are specific to control dogs ('control-only variants'; 2,698 SNPs and 418 INDELs; 156 to 1,476 per dog) (Table 1; Table S9 in Additional file 1). While there is no significant difference between the total number of case- and control-only variants (Wilcoxon test P = 0.63; Figure 2b), case dogs have a significantly greater number in evolutionarily constrained elements (median 15 versus 4, Wilcoxon test P = 0.02; Materials and methods; Figure 2b; Table S9 in Additional file 1). Excluding coding variants increases the difference further (median 15 versus 3, Wilcoxon test P = 0.01), suggesting that the excess of case-only functional variants may be due largely to noncoding variation.
Table 1. Sequence variants identified by targeted resequencing of 5.8 Mb in eight cases and eight controls
Genotyping case-only variants in independent samples
We genotyped 114 case-only, evolutionarily constrained variants in an independent set of dogs from breeds with high rates of OCD ('OCD-risk breeds'; 69 dogs) and breeds with normal rates of OCD and other psychiatric disorders ('control breeds'; 19 dogs). Except for 14 cases from OCD-risk breeds, we have no individual OCD phenotype information for these dogs (Figure 2a). We find that the case-only variants identified in the sequence data are significantly more common in OCD-risk breeds, with median frequency (FOCD) of 0.17, than in control breeds, where the median frequency (Fcontrol) is 0.05 (Wilcoxon test P = 0.045; Table S10 in Additional file 1). The median frequency increases to 0.20 when only phenotyped cases are considered (Wilcoxon test P = 0.015, comparison with Fcontrol). We also observe an inverse correlation between the frequency difference between OCD-risk and control breeds and the frequency across all genotyped dogs (Pearson's R = -0.63, P = 8.4 × 10-14; Figure 2c). Thus, the variants most enriched in OCD-risk breeds are otherwise rare, potentially due to either positive selective pressure in OCD-risk breeds or negative selection in the control breeds. While this suggests an association with OCD, we note that other traits may also systematically differ between the two breed groups.
Gene-based analysis
We identified genes enriched with case-only variants using a gene-based analysis method that accounts for multiple independent variants within a gene and greatly increases power for identifying disease-associated genes [28]. Four genes have an excess of case-only variation in evolutionarily constrained elements, even after correcting for gene size: ATXN1, CDH2, CTNNA2, and PGCP (10, 16, 12, and 16 case-only variants, respectively; Figure 3a; Text S1 and Table S11 in Additional file 1). Because the sequenced DPs were selected based on their haplotype at CDH2, we confirmed that the case-only enrichment at CDH2 persists even when DPs are excluded (Figure 3b). RNA-Seq data show all four genes are expressed in the dog brain (KL-T, unpublished observations).
Figure 3. Gene-based analysis identifies four top genes enriched for case-only variants in constrained regions. (a) Genes are plotted according to the number of case-only (y-axis) and control-only (x-axis) variants within a gene and its 5 kb flanking regions from our sequence data. Blue squares denote all the variants and the corresponding axes are shown in blue; red diamonds denote variants in constrained elements only and the corresponding axes are shown in red. Genes that are plotted above the identity line harbor more case-only than control-only variants. (b) A similar analysis excluding DP, the breed used to identify genes for sequencing, shows that the enrichment pattern persists for several genes. (c) The case-only variants in constrained elements, when plotted with gene structure and evolutionary conservation, show clustering in ATXN1 (5' end). Dimmed bars represent canine variants that failed to lift over onto hg19. The conservation track shows a measure of evolutionary conservation in dog, human, mouse and rat [29]. (d) In each gene, SNPs with the greatest risk allele frequency (AF) difference between OCD-risk and control breeds (y-axis) tend to have lower frequency across all genotyped breeds (y-axis). SNPs are shown as solid circles with vertical lines.
Three of the four candidate genes, CDH2, PGCP and ATXN1, are associated with OCD in the DP GWAS study (chr7:63867472, P = 2.1 × 10-5; chr29:44152594, P = 1.5 × 10-5; chr35:18565131, P = 1.6 × 10-5, respectively), while the fourth, CTNNA2, falls in a large region of fixation (900 kb) in the DP breed (Figure 3c). In our genotyping dataset, the case-only variants in these four genes are more common in OCD-risk breeds (FOCD = 0.08 versus Fcontrol = 0.026, Wilcoxon test P = 2.95 × 10-4; Figure 3d), particularly in CDH2 (FOCD = 0.23 versus Fcontrol = 0.027, P = 0.001) and in PGCP (FOCD = 0.014 versus Fcontrol = 0.0, P = 0.047). We see a similar, though weaker, pattern in ATXN1 (FOCD = 0.022 versus Fcontrol = 0.0, P = 0.3) and CTNNA2 (FOCD = 0.185 versus Fcontrol = 0.026, P = 0.13). In CTNNA2, the difference is clearer (P = 0.054) if only variants with frequency <0.20 are considered.
Of the 40 variants genotyped in these four genes, seven overlap chromatin marks, potentially indicating regulatory function. Four variants in CDH2 overlap H3K27Ac histone marks and/or DNase1 hypersensitivity clusters. Three of these (chr7:63845160, chr7:63852056, and chr7:63832008) are observed in OCD-risk breeds, at frequencies of 0.435, 0.050, and 0.022, respectively, and never seen in control breeds. The fourth variant (chr7:63806661) is four-fold more common in OCD-risk breeds (frequency = 0.11 versus 0.026 in control breeds). Three variants in ATXN1 alter regions transcribed in the dog brain (KL-T, unpublished RNA-Seq data), including a putative enhancer variant not seen in the control breeds (chr35:18850625, OCD-risk breed frequency = 0.014). These variants, which lie in genes enriched for case-only variants, are overrepresented in cases, and alter putative regulatory elements, are strong candidates for further functional elucidation.
Single variant analysis
We next sought to identify the top candidate functional variants in the sequencing data. We first looked for coding variants found exclusively in cases. Most were missense mutations disrupting genes with little known relevance to brain functions (Table S12 and Text S2 in Additional file 1). More intriguing, one of our two Jack Russell terrier cases has a 1.2 kb deletion (chr29:44178339–44179516; Figure S2 in Additional file 1) overlapping exon 2 of the gene PGCP, causing a frameshift and loss of 70 amino acids from the protein. PGCP is one of the four genes enriched for case-only variants, and, while none of the DP cases has this particular deletion, a nearby SNP is among the most strongly associated in the GWAS (chr29:44152594, P = 1.5 × 10-5; Figure 1; Figure S3 in Additional file 1). Using quantitative PCR (qPCR), we validated the deletion in the Jack Russell terrier cases and tested 74 dogs from OCD-risk breeds (including 10 unphenotyped Jack Russell terriers and 14 dogs from several breeds diagnosed with OCD) and 20 dogs from control breeds. We found the deletion in three Jack Russell terriers and in one Welsh terrier with OCD, and in none of the control breed dogs, suggesting it is associated with increased risk of OCD in multiple breeds.
We next looked for non-coding variants seen only in cases, focusing on 15 seen in more than one DP case. All but two are near the GWAS peak in intron 2 of CDH2 (chr7:63867472, P = 2.1 × 10-5), reflecting the selection of DP dogs for sequencing based on their genotype at this locus. None of the 13 is obviously functional based on evolutionary constraint and histone marks. The other two variants are more interesting, changing a conserved region approximately 172 kb away from an associated GWAS SNP (chr7:61865715, P = 1.6 × 10-5), in the gene desert between the cadherin genes CDH2 and DSC3 (Figure 4a). The first SNP (chr7:61693835, T changed to C; SNP35 T > C) is exclusively found in three of four sequenced DP cases and showed the overall DP breed frequency of approximately 0.30 in our genotyping data set. The second SNP, a private variant in the fourth DP case (chr7:61693855; SNP55 A > T), is just 20 bases away and alters the same highly conserved region (Figure 4b,c).
Figure 4. Two intergenic case-only variants disrupt a repressor element and change gene expression in vitro. (a) The two case-only variants are just 20 bases apart in a 2.5 Mb gene desert between DSC3 and CDH2 on canine chromosome 7. (b) The syntenic region of human chromosome 18 shows markers of DNase hypersensitivity, transcription factor binding, repressor binding, histone methylation [30], and mammalian constraint [31]. (c) Both variants, SNP55 and SNP35, alter bases that are highly constrained across mammals, SNP55 to an A and SNP35 to a T. Compositions of the four bases for each position in the 29 mammals comparison [27] are shown with different grey scale and height of bars (the higher the more conserved in multiple species). (d) The wild-type regulatory sequence represses luciferase reporter expression in SK-N-BE(2) neuroblastoma cells. Both variants significantly change the extent of repression relative to wild type, with SNP35-G more repressive and SNP55-A less repressive. The firefly luciferase expression in the test plasmids were normalized against the co-transfected Renilla luciferase expression in pGL4.73. The P-value of the significance of the change relative to wild type is shown above each bar, with vertical lines showing standard error of the mean. (e) An electrophoretic mobility shift assay testing the wild-type alleles (lanes 1 to 4) and the OCD-risk alleles (lanes 5 to 8) of SNP35 (top gel) and SNP55 (bottom gel) show that nuclear protein binding (red arrow) to the SNP35 locus is disrupted by the risk allele. Nuclear extract was derived from SK-N-BE(2) cells. We used a 200-fold molar excess of competitor where appropriate.
Functional assessment of candidate variants
Because the region altered by SNP35 and SNP55 showed evidence of regulatory function (Figure 4b), we tested whether the risk alleles disrupt gene expression using a luciferase reporter assay. Including the wild-type region in the reporter construct lowers expression 14- to 20-fold in human neuroblastoma SK-N-BE(2) cells (vector versus wild type, t-test Bonferroni corrected P = 3.5 × 10-7; negative control versus wild type, t-test Bonferroni corrected P = 2.9 × 10-7; Figure 4d). Adding the SNP55 risk variant to the construct, however, significantly increases expression relative to the wild-type version, suggesting the regulatory element no longer functions normally (1.6-fold change, paired t-test Bonferroni corrected P = 1.1 × 10-4; Figure 4d). Curiously, the SNP35 risk allele has the opposite effect, repressing expression even further (0.9-fold change, paired t-test Bonferroni corrected P = 3.7 × 10-3; Figure 4d).
Using an electrophoretic mobility shift assay (EMSA) to examine DNA protein binding in the region, we see that, while the SNP55 risk allele causes no apparent change relative to wild-type, the SNP35 risk allele shows markedly reduced binding (Figure 4e). Three transcription factors are predicted by TRANSFAC [32] to bind the wild-type sequence but not the SNP35 variant (PRRX2, Oct-1 and Nobox; Figure S4 in Additional file 1). However, we saw no evidence that these three proteins bind the region in a supershift assay (Figure S5 in Additional file 1), suggesting other factors are critical. More than 90 transcription factors are predicted to bind the wild-type sequence using various discovery tools [33]. Thus, both SNP35 and SNP55 significantly change the silencing activity of the regulatory element, but in opposite directions and possibly through different mechanisms.
Through a small GWAS (fewer than 90 cases and 70 controls) we identified OCD-associated loci, which, particularly when analyzed together with regions of low variability, implicate specific cellular pathways in disease etiology. We sequenced nine of the top regions of association and five regions of fixation in eight OCD cases and eight breed-matched controls. We found a notable excess of case-only variation in evolutionarily conserved regions, particularly in non-coding elements with potential regulatory function. This suggests noncoding variation is a major factor in canine OCD similar to human neuropsychiatric diseases, and unlike most artificially induced mouse models. While the dog population is composed of >400 genetically isolated breed populations, just a small number of breeds are highly enriched for OCD, suggesting that OCD risk variants are more prevalent in these breeds. We show that the case-only variants found in the sequence data are in fact significantly more common in OCD-risk breeds compared to breeds with no increased risk of psychiatric disorders.
By comparing the sequence data using gene-based tests, we confirmed one gene (CDH2) and identified three novel ones (CTNNA2, ATXN1, and PGCP) strongly implicated for involvement in disease.
CDH2, a neural cadherin, encodes a calcium dependent cell-cell adhesion glycoprotein important for synapse assembly, where it mediates presynaptic to postsynaptic adhesions [34]. Disrupting expression of CDH2 in cultured mouse neurons causes synapse dysfunction, synapse elimination and axon retraction [35].
CTNNA2 encodes a neuronal-specific catenin protein that links cadherins to the cytoskeleton [34,36] and is associated with bipolar disorder [37], schizophrenia [38], attention deficit hyperactivity disorder [38] and excitement-seeking [39]. Mice with a deletion of CTNNA2 showed disrupted brain morphology and impaired startle modulation [40]. Cadherin-catenin complexes play a pivotal role in synapse formation and synaptic plasticity and therefore may be involved in the process of learning and memory [41].
ATXN1 encodes a chromatin binding protein that regulates the Notch pathway [42], a developmental pathway also active in the adult brain, where it mediates neuronal migration, morphology and synaptic plasticity [43]. Mice with a deletion of ATXN1 showed pronounced deficits in learning and memory [44].
CDH2, CTNNA2 and ATXN1 have similar spatial expression patterns in the brain and are important during brain development and for synaptic plasticity. CDH2 and CTNNA2 are highly expressed in the prefrontal cortex, amygdala, thalamus and fetal brain [34,45]. ATXN1 is highly expressed in the prefrontal cortex, basal ganglia, cerebellum and fetal brain [45,46].
Intriguingly, the three genes appear to have functional connections to the top SNPs (association P < 10-5) in a recent human OCD GWAS, which found no single associations reaching genome-wide significance, but implicated glutamatergic signaling pathways [4] (Figure S6 in Additional file 1). Most notably, one of the top associated genes in human patients, GRIK2, encodes a glutamate receptor recruited to the synaptic membrane by CDH2/catenin complexes [47] and another top candidate, PKP2, mediates CDH2 cell adhesion and desmosomal junctions [48]. In addition, several genes whose expression levels correlate with the top human OCD-associated SNPs interact with the genes we identify in dogs: LRSAM1 (cerebellum) and NARS (frontal lobe) interact with ATXN1; SPAG9 (cerebellum) acts in developmental pathways with CDH2 and CTNNA2 [49].
The fourth gene, PGCP, encodes a poorly characterized plasma glutamate carboxypeptidase. It may help hydrolyze N-acetylaspartylglutamate (NAAG), the third most abundant neurotransmitter in the brain, to glutamate and N-acetylaspartate (NAA) [34], suggesting a potential role in glutamatergic synapse dysfunction. PGCP is associated with migraine [50], which is frequently co-morbid with OCD [51].
We hypothesize that CDH2, CTNNA2, ATXN1, and PGCP may work in concert to regulate glutamatergic synapse formation and function in the cortico-striatal-thalamo-cortical (CSTC) brain circuit previously implicated in the pathogenesis of OCD [52-56].
Single variant analysis corroborates our hypothesis of dysregulated synapse formation in OCD. All four sequenced DP cases had one of two mutations (SNP35 and SNP55) in a regulatory region, between DSC3 and CDH2, that we show acts as a strong silencer. The OCD-risk allele of SNP55 significantly increased the reporter gene expression while the OCD-risk allele of SNP35 had the opposite effect. While surprising, other studies have shown that either deletion or reciprocal duplication of loci such as 17p11.2 and 15q13.3 can cause neuropsychiatric disorders [57]. For SNP35, we confirmed using EMSA that the OCD-risk allele changes DNA binding. We saw no change at SNP55, although in vitro assays may not capture all relevant in vivo reactions. The regulatory element is between CDH2 (2.2 Mb away) and DSC3 (0.3 Mb away), both cadherin genes involved in gamma-catenin binding (Figure 1d,e), suggesting disrupted gamma-catenin binding may be an important risk factor for OCD. Additional sequence data from DSC3 (not included in the current targeted sequencing design) and more functional analysis are needed to understand the two SNPs' effects on CDH2 and DSC3.
Modeling neuropsychiatric disorders in animals is complicated by both limited understanding of the underlying neurobiology and subjective diagnostic criteria [58]. Naturally occurring canine compulsive disorder is a remarkably good model for human disease, as it is equivalent by most clinical metrics, including age of onset, symptoms, and pharmacological response. The work we present here suggests similar genetic etiology as well. We harness the limited genetic diversity of dog breeds, and high rates of OCD in particular breeds, to identify genes, pathways and non-coding candidate functional variants. Dogs suffer from a wide range of psychiatric disorders and have been strongly selected for a variety of behavioral traits, making them a uniquely powerful natural model organism for investigating inherited psychiatric diseases in humans.
Materials and methods
GWAS and sequencing region selection
The GWAS using the sample set and phenotypes published previously [14] was rerun using genotypes called with the new MAGIC algorithm [15]. Briefly, MAGIC (Multidimensional Analysis for Genotype Intensity Clustering) does not use prior information to make genotype calls (that is, cluster locations Hardy-Weinberg equilibrium, or complex normalization of probe intensities). Instead, it performs quantile normalization of the data for each chip independently followed by a principal component analysis of all chips on a SNP-by-SNP basis, neatly summarizing the raw data.
The processed data are then clustered into genotype calls through expectation maximization using a t-distribution mixture model. Association was calculated with a standard chi-squared test in PLINK [59] (SNP genotype rate >90%, individual genotype rate >25%, MAF >5%) and regions were defined with linkage disequilibrium -based clumping around SNPs with P < 0.0001 (that is, SNPs within 1 Mb with r2 > 0.8 and P < 0.01) (Table S1 and Figure S1 in Additional file 1). We identified regions of fixation as regions of >1,000 kb with more than five SNPs and >95% SNPs with MAF <0.05 and selected a subset found in breeds prone to OCD for targeted sequencing. From the associated and fixed regions we designed a 5.8 Mb targeted sequencing array that optimized inclusion of potential genes of interest within the design limitations (Tables S5 and S6 in Additional file 1).
Gene set enrichment analysis
We expanded the GWAS regions to include all genes within 500 kb of the original region start or end (Table S1 in Additional file 1). We defined RRVs by comparing the DP breed to 24 other dog breeds from a published reference dataset and identifying the 1% least variable 150 kb regions in DP [23]. We ran INRICH with 1,000,000 permutations to test regions for enrichment in any gene sets from the GO catalog. We tested all gene sets with between 5 and 1,000 genes (downloaded from the Gene Ontology website on 18 May 2013) [17]. We used a map file of 16,433 genes lifted over to canFam2.0 from the hg19 RefSeq Gene catalog (UCSC Genome Brower, single match using default parameters) [60]. To identify gene sets with unusually high enrichment in the DP RRVs, we calculated, for all sets with P < 0.05 and at least 2 RRV genes in DP, the average difference in enrichment P-values between DP and 24 other breeds [26] (Figure 1f).
Sequenced samples
The targeted sequencing experiment comprised a total of eight cases and eight controls from multiple breeds: DP (four cases + four controls), German shepherd (two cases + two controls), Jack Russell terrier (one case + one control) and Shetland sheepdog (one case + one control) (Figure 2a). The four DP cases supplied by Dr. Meurs showed flank sucking behavior, while the German Shepherd, Jack Russell terrier and Shetland sheepdog cases were tail-chasers.
Targeted sequencing and variant calling
The 16 samples were individually barcoded and the targeted regions were captured by a NimbleGen Sequence Capture 385 K Array according to the manufacturer's protocol. The captured samples were then pooled and sequenced on an Illumina Genome Analyzer II. Paired-end 76-bp reads were aligned to canFam2.0 and PCR duplicates were removed using Picard [61], and realignment and recalibration were processed through Genome Analysis Toolkit (GATK) [62,63]. SNPs and small INDELs were identified using GATK. We only considered the variants that pass the GATK standard filters. Larger structural variants were detected by GenomeSTRiP [64]. We manually checked the alignments of all discovered deletion sites for aberrant read pairs and read depth using Integrative Genomics Viewer [65] to ensure the reliability of the calls. We excluded a Shetland sheepdog pair where the control had lower SNP accuracy, when comparing case- and control-only variant counts (Figure 2b and Table S9 in Additional file 1).
Genotyping candidate sequence variants
We first selected case-specific variants that were within evolutionarily constrained elements determined by a 29 mammals sequence dataset [27]. We then selected a subset of the variants meeting one of the following criteria: (i) case-only variants within DP breed; (ii) case-only variants within CDH2, PGCP, CTNNA2 and ATXN1 that are identified by gene-based analysis; (iii) case-only variants across at least two breeds; (iv) potential functional variants annotated as nonsense, splicing or missense (predicted to be 'probably' or 'possibly damaging' by Polyphen-2 [66]) and case-only variants in at least one breed; (v) variants within CDH2 risk haplotype; and (vi) top associated variants from GWAS analysis. Of 140 variants that met one of the criteria, 127 variants passed Sequenom design standards, and were genotyped using the Sequenom iPlex system. We employed an independent set of 94 dogs that consisted of 10 dogs without obvious health problems for each of 6 OCD-risk breeds (that is, 4 sequenced breeds and West Highland white terrier (Westie) and bull terrier (bull terrier)) and two control breeds without known psychiatric problems (greyhound and Leonberger), and 14 additional OCD cases from various breeds (2 bull terrier, 2 DP, 1 German shepherd, 1 Westie, 1 Golden retriever, 1 Irish wolfhound, 1 pug, 1 Shiba, 1 Shepherd mix, 1 standard poodle, 1 Shih Tzu, 1 Welsh terrier). Genotype data were cleaned by removing samples with missing genotype rates >10% and excluding SNPs with call rates <90%. After the quality control, 114 SNPs and 88 dogs (19 (10 Leonbergers + 9 Greyhounds) from control breeds and 69 (14 cases + 5 DP + 10 bull terrier + 10 Westie + 10 German shepherd + 10 Shetland sheepdogs + 10 Jack Russell terriers) from OCD-risk breeds or cases) were retained in our analysis (Figure 2a; Table S10 in Additional file 1).
Gene-based analysis
Each gene region was defined using the coordinates from RefSeq hg19 lifted over to canFam2.0 plus 5 kb flanking sequence on each side. We counted the number of case- and control-only variants and compared the counts for each gene. Genes that have excessive case-only variants relative to control-only variants were considered as potential risk genes for OCD. The same analysis was applied to the variants within constrained elements. To correct for gene size, we calculated the ratio of the number of case-only variants and the number of control-only variants for each gene additionally.
Electrophoretic mobility shift assay
For each allele of the tested SNPs in a regulatory region between CDH2 and DSC3, pairs of 5'-biotinylated oligonucleotides were obtained from IDT Inc. (Coralville, IA, USA; Table S13 in Additional file 1). Equal volumes of forward and reverse oligos (100 μM) were mixed and heated at 95°C for 5 minutes and then cooled to room temperature. Fifty femtomoles of annealed probes were incubated at room temperature for 30 minutes with 10 mg SK-N-BE(2) nuclear extract (Active Motif Carlsbad, CA, USA). The remaining steps followed the LightShift Chemiluminescent EMSA Kit protocol (Thermo Scientific).
Luciferase reporter assay
The activity of a putative regulatory element and the effect of SNP35 and SNP55 on gene expression were examined by luciferase reporter assay. We PCR amplified an 879 bp-long orthologous sequence spanning SNP35 and SNP55 from human DNA samples (Table S14 in Additional file 1). The risk alleles were introduced using a site-directed mutagenesis kit. The wild-type and mutant DNA fragments were cloned into a firefly luciferase reporter plasmid (pGL4.23, Promega Madison, WI, USA). The test constructs were transiently co-transfected with a Renilla luciferase reporter plasmid (pGL4.73, Promega) as an internal control into neuroblastoma SK-N-BE(2) cells. All constructs were tested in triplicates and repeated three times in a double-blinded manner.
Cell cultures
Human SK-N-BE(2) cells were purchased from ATCC. The cells were maintained at 37°C and 5% CO2 in 1:1 mixture of ATCC-formulated Eagle's Minimum Essential Medium (EMEM) and F-12 K medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin.
Real-time qPCR
Real-time qPCR was performed using Quantifast SYBR Green PCR kit (QIAGEN, Hilden, Germany) on a Lightcycler 480 system (Roche Applied Science, Indianapolis, IN, USA). The reaction volumes were adjusted to 10 μl with 3 μl of DNA (10 ng), 1 μl of both primers (10 μM) and 5 μl of Master Mix. The qPCR program was as follows: pre-incubation at 95°C for 5 minutes, followed by 40 cycles of two-step amplification (10 s at 95°C, 1 minute at 60°C). All the experiments were carried out in triplicates and include negative control without DNA. The primer sets used to detect PGCP deletion is shown in Table S15 in Additional file 1.
Data availability
The data presented in this publication are available through the NCBI Sequence Read Archive (SRP033723) and Gene Expression Omnibus (accession numbers GSE53488 and GSE53577). Datasets analyzed in the paper are also available at [67].
bp: base pair; DP: Doberman Pinscher; EMSA: electrophoretic mobility shift assay; GATK: Genome Analysis Toolkit; GO: Gene Ontology; GWAS: genome-wide association study; MAF: minor allele frequency; OCD: obsessive compulsive disorder; qPCR: quantitative polymerase chain reaction; RRV: regions of reduced variability; SNP: single-nucleotide polymorphism.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KLT, EKK and GF conceived and oversaw the study. EEP, JA, MD, MC and MP coordinated, collected, prepared samples and characterized samples for the study. RT, SS, MP and RS designed and performed the resequencing experiment. HJN, RT, EKK and KLT analyzed and interpreted the data. AB and AA performed genotype calling for GWAS. RT and DW performed the functional assays. HJN, RT, EKK, and KLT wrote the paper with input from the other authors. All authors read and approved the final manuscript.
We thank all the dog owners, breeders, and breed clubs, as well as the veterinarians in the US and Sweden for providing us with blood samples. This work was supported by a SPARC grant from the Broad Institute, Uppsala University, Medical Research Council, and a grant from NIMH/NIH (R01MH081201), and by funds from the Poitras Center for Affective Disorders Research to GF. KLT is the recipient of a EURYI from the European Science Foundation. EKK is supported by fellowships from the American Cancer Society and the Charles A King Trust. We thank Andy Reynolds for help with genotype calling, Evan Mauceli for help with genotyping concordance analysis, Jeremy Johnson for project management assistance, Tarjei Mikkelsen and Xiaolan Zhang for supplying reagents for the luciferase assays, and Leslie Gaffney for help with illustration. We thank Kate Meurs and Åke Hedhammar for their work on sample collection. Our deepest appreciation goes to Nicholas Dodman, veterinary behaviorist and professor at Tufts University, and animal behaviorist Alice Moon-Fanelli, who performed extensive and careful phenotyping of OCD in the Doberman pinscher GWAS sample set using methodologies developed by Dr. Dodman and his group. This careful phenotyping, described in the earlier published GWAS study [14], provided a foundation for the work described herein. We also acknowledge the valuable material and intellectual contributions of Drs. Edward Ginns and Marzena Galdzicka to this study.
Laks J, Fontenelle LF, Chalita A, Mendlowicz MV: Absence of dementia in late-onset schizophrenia: a one year follow-up of a Brazilian case series.
Arquivos de neuro-psiquiatria 2006, 64:946-949. Publisher Full Text
van Grootheest DS, Cath DC, Beekman AT, Boomsma DI: Twin studies on obsessive-compulsive disorder: a review.
Twin Res Hum Genet 2005, 8:450-458. PubMed Abstract | Publisher Full Text
Taylor S: Molecular genetics of obsessive-compulsive disorder: a comprehensive meta-analysis of genetic association studies.
Mol Psychiatry 2013, 18:799-805. PubMed Abstract | Publisher Full Text
Stewart SE, Yu D, Scharf JM, Neale BM, Fagerness JA, Mathews CA, Arnold PD, Evans PD, Gamazon ER, Osiecki L, McGrath L, Haddad S, Crane J, Hezel D, Illman C, Mayerfeld C, Konkashbaev A, Liu C, Pluzhnikov A, Tikhomirov A, Edlund CK, Rauch SL, Moessner R, Falkai P, Maier W, Ruhrmann S, Grabe HJ, Lennertz L, Wagner M, Bellodi L, et al.: Genome-wide association study of obsessive-compulsive disorder.
Mol Psychiatry 2012, 18:788-798. PubMed Abstract | Publisher Full Text
Welch JM, Lu J, Rodriguiz RM, Trotta NC, Peca J, Ding JD, Feliciano C, Chen M, Adams JP, Luo J, Dudek SM, Weinberg RJ, Calakos N, Wetsel WC, Feng G: Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice.
Nature 2007, 448:894-900. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Burguiere E, Monteiro P, Feng G, Graybiel AM: Optogenetic stimulation of lateral orbitofronto-striatal pathway suppresses compulsive behaviors.
Science 2013, 340:1243-1246. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Zuchner S, Wendland JR, Ashley-Koch AE, Collins AL, Tran-Viet KN, Quinn K, Timpano KC, Cuccaro ML, Pericak-Vance MA, Steffens DC, Krishnan KR, Feng G, Murphy DL: Multiple rare SAPAP3 missense variants in trichotillomania and OCD.
Mol Psychiatry 2009, 14:6-9. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Overall KL: Natural animal models of human psychiatric conditions: assessment of mechanism and validity.
Prog Neuropsychopharmacol Biol Psychiatry 2000, 24:727-776. PubMed Abstract | Publisher Full Text
Overall KL, Dunham AE: Clinical features and outcome in dogs and cats with obsessive-compulsive disorder: 126 cases (1989–2000).
J Am Vet Med Assoc 2002, 221:1445-1452. PubMed Abstract | Publisher Full Text
Moon-Fanelli AA, Dodman NH, Cottam N: Blanket and flank sucking in Doberman Pinschers.
J Am Vet Med Assoc 2007, 231:907-912. PubMed Abstract | Publisher Full Text
Moon-Fanelli AA, Dodman NH, Famula TR, Cottam N: Characteristics of compulsive tail chasing and associated risk factors in Bull Terriers.
J Am Vet Med Assoc 2011, 238:883-889. PubMed Abstract | Publisher Full Text
Luescher AU: Diagnosis and management of compulsive disorders in dogs and cats.
Clin Tech Small Anim Pract 2004, 19:233-239. PubMed Abstract | Publisher Full Text
Karlsson EK, Sigurdsson S, Ivansson E, Thomas R, Elvers I, Wright J, Howald C, Tonomura N, Perloski M, Swofford R: Genome-wide analyses implicate 33 loci in heritable dog osteosarcoma, including regulatory variants near CDKN2A/B.
Genome Biol 2013, 14:R132. PubMed Abstract | BioMed Central Full Text
Dodman NH, Karlsson EK, Moon-Fanelli A, Galdzicka M, Perloski M, Shuster L, Lindblad-Toh K, Ginns EI: A canine chromosome 7 locus confers compulsive disorder susceptibility.
Mol Psychiatry 2010, 15:8-10. PubMed Abstract | Publisher Full Text
Boyko AR, Quignon P, Li L, Schoenebeck JJ, Degenhardt JD, Lohmueller KE, Zhao K, Brisbin A, Parker HG, vonHoldt BM, Cargill M, Auton A, Reynolds A, Elkahloun AG, Castelhano M, Mosher DS, Sutter NB, Johnson GS, Novembre J, Hubisz MJ, Siepel A, Wayne RK, Bustamante CD, Ostrander EA: A simple genetic architecture underlies morphological variation in dogs.
PLoS Biol 2010, 8:e1000451. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Yang J, Lee SH, Goddard ME, Visscher PM: GCTA: a tool for genome-wide complex trait analysis.
Am J Hum Genet 2011, 88:76-82. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lee PH, O'Dushlaine C, Thomas B, Purcell SM: INRICH: interval-based enrichment analysis for genome-wide association studies.
Bioinformatics 2012, 28:1797-1799. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Ethell IM, Hagihara K, Miura Y, Irie F, Yamaguchi Y: Synbindin, A novel syndecan-2-binding protein in neuronal dendritic spines.
J Cell Biol 2000, 151:53-68. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Coba MP, Komiyama NH, Nithianantharajah J, Kopanitsa MV, Indersmitten T, Skene NG, Tuck EJ, Fricker DG, Elsegood KA, Stanford LE, Afinowi NO, Saksida LM, Bussey TJ, O'Dell TJ, Grant SG: TNiK is required for postsynaptic and nuclear signaling pathways and cognitive function.
J Neurosci 2012, 32:13987-13999. PubMed Abstract | Publisher Full Text
Arregui C, Pathre P, Lilien J, Balsamo J: The nonreceptor tyrosine kinase fer mediates cross-talk between N-cadherin and beta1-integrins.
J Cell Biol 2000, 149:1263-1274. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lee SH, Peng IF, Ng YG, Yanagisawa M, Bamji SX, Elia LP, Balsamo J, Lilien J, Anastasiadis PZ, Ullian EM, Reichardt LF: Synapses are regulated by the cytoplasmic tyrosine kinase Fer in a pathway mediated by p120catenin, Fer, SHP-2, and beta-catenin.
J Cell Biol 2008, 183:893-908. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Takeichi M, Abe K: Synaptic contact dynamics controlled by cadherin and catenins.
Trends Cell Biol 2005, 15:216-221. PubMed Abstract | Publisher Full Text
Vaysse A, Ratnakumar A, Derrien T, Axelsson E, Rosengren Pielberg G, Sigurdsson S, Fall T, Seppala EH, Hansen MS, Lawley CT, Karlsson EK, Bannasch D, Vila C, Lohi H, Galibert F, Fredholm M, Haggstrom J, Hedhammar A, Andre C, Lindblad-Toh K, Hitte C, Webster MT: Identification of genomic regions associated with phenotypic variation between dog breeds using selection mapping.
PLoS Genet 2011, 7:e1002316. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Churchill L, Cotman C, Banker G, Kelly P, Shannon L: Carbohydrate composition of central nervous system synapses, Analysis of isolated synaptic junctional complexes and postsynaptic densities.
Biochim Biophys Acta 1976, 448:57-72. PubMed Abstract | Publisher Full Text
Clark RA, Gurd JW, Bissoon N, Tricaud N, Molnar E, Zamze SE, Dwek RA, McIlhinney RA, Wing DR: Identification of lectin-purified neural glycoproteins, GPs 180, 116, and 110, with NMDA and AMPA receptor subunits: conservation of glycosylation at the synapse.
J Neurochem 1998, 70:2594-2605. PubMed Abstract | Publisher Full Text
Hedges DJ, Burges D, Powell E, Almonte C, Huang J, Young S, Boese B, Schmidt M, Pericak-Vance MA, Martin E, Zhang X, Harkins TT, Zuchner S: Exome sequencing of a multigenerational human pedigree.
PloS ONE 2009, 4:e8232. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lindblad-Toh K, Garber M, Zuk O, Lin MF, Parker BJ, Washietl S, Kheradpour P, Ernst J, Jordan G, Mauceli E, Ward LD, Lowe CB, Holloway AK, Clamp M, Gnerre S, Alfoldi J, Beal K, Chang J, Clawson H, Cuff J, Di Palma F, Fitzgerald S, Flicek P, Guttman M, Hubisz MJ, Jaffe DB, Jungreis I, Kent WJ, Kostka D, Lara M, et al.: A high-resolution map of human evolutionary constraint using 29 mammals.
Nature 2011, 478:476-482. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Huang H, Chanda P, Alonso A, Bader JS, Arking DE: Gene-based tests of association.
PLoS Genet 2011, 7:e1002177. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Siepel A, Bejerano G, Pedersen JS, Hinrichs AS, Hou M, Rosenbloom K, Clawson H, Spieth J, Hillier LW, Richards S, Weinstock GM, Wilson RK, Gibbs RA, Kent WJ, Miller W, Haussler D: Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes.
Genome Res 2005, 15:1034-1050. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Dunham I, Kundaje A, Aldred SF, Collins PJ, Davis CA, Doyle F, Epstein CB, Frietze S, Harrow J, Kaul R, Khatun J, Lajoie BR, Landt SG, Lee BK, Pauli F, Rosenbloom KR, Sabo P, Safi A, Sanyal A, Shoresh N, Simon JM, Song L, Trinklein ND, Altshuler RC, Birney E, Brown JB, Cheng C, Djebali S, Dong X, Ernst J, et al.: An integrated encyclopedia of DNA elements in the human genome.
Nature 2012, 489:57-74. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Cooper GM, Stone EA, Asimenos G, Green ED, Batzoglou S, Sidow A: Distribution and intensity of constraint in mammalian genomic sequence.
Genome Res 2005, 15:901-913. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Wingender E, Dietze P, Karas H, Knuppel R: TRANSFAC: a database on transcription factors and their DNA binding sites.
Nucleic Acids Res 1996, 24:238-241. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Calakos N, Patel VD, Gottron M, Wang G, Tran-Viet KN, Brewington D, Beyer JL, Steffens DC, Krishnan RR, Zuchner S: Functional evidence implicating a novel TOR1A mutation in idiopathic, late-onset focal dystonia.
J Med Genet 2010, 47:646-650. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Pruitt KD, Tatusova T, Brown GR, Maglott DR: NCBI Reference Sequences (RefSeq): current status, new features and genome annotation policy.
Nucleic Acids Res 2012, 40:D130-D135. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Pielarski KN, van Stegen B, Andreyeva A, Nieweg K, Jungling K, Redies C, Gottmann K: Asymmetric N-cadherin expression results in synapse dysfunction, synapse elimination, and axon retraction in cultured mouse neurons.
PloS ONE 2013, 8:e54105. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Abe K, Chisaka O, Van Roy F, Takeichi M: Stability of dendritic spines and synaptic contacts is controlled by alpha N-catenin.
Nat Neurosci 2004, 7:357-363. PubMed Abstract | Publisher Full Text
Scott LJ, Muglia P, Kong XQ, Guan W, Flickinger M, Upmanyu R, Tozzi F, Li JZ, Burmeister M, Absher D, Thompson RC, Francks C, Meng F, Antoniades A, Southwick AM, Schatzberg AF, Bunney WE, Barchas JD, Jones EG, Day R, Matthews K, McGuffin P, Strauss JS, Kennedy JL, Middleton L, Roses AD, Watson SJ, Vincent JB, Myers RM, Farmer AE, et al.: Genome-wide association and meta-analysis of bipolar disorder in individuals of European ancestry.
Proc Natl Acad Sci U S A 2009, 106:7501-7506. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Chu TT, Liu Y: An integrated genomic analysis of gene-function correlation on schizophrenia susceptibility genes.
J Hum Genet 2010, 55:285-292. PubMed Abstract | Publisher Full Text
Terracciano A, Esko T, Sutin AR, de Moor MH, Meirelles O, Zhu G, Tanaka T, Giegling I, Nutile T, Realo A, Allik J, Hansell NK, Wright MJ, Montgomery GW, Willemsen G, Hottenga JJ, Friedl M, Ruggiero D, Sorice R, Sanna S, Cannas A, Raikkonen K, Widen E, Palotie A, Eriksson JG, Cucca F, Krueger RF, Lahti J, Luciano M, Smoller JW, et al.: Meta-analysis of genome-wide association studies identifies common variants in CTNNA2 associated with excitement-seeking.
Transl Psychiatry 2011, 1:e49. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Park C, Falls W, Finger JH, Longo-Guess CM, Ackerman SL: Deletion in Catna2, encoding alpha N-catenin, causes cerebellar and hippocampal lamination defects and impaired startle modulation.
Nat Genet 2002, 31:279-284. PubMed Abstract | Publisher Full Text
Arikkath J, Reichardt LF: Cadherins and catenins at synapses: roles in synaptogenesis and synaptic plasticity.
Trends Neurosci 2008, 31:487-494. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Tong X, Gui H, Jin F, Heck BW, Lin P, Ma J, Fondell JD, Tsai CC: Ataxin-1 and Brother of ataxin-1 are components of the Notch signalling pathway.
EMBO Rep 2011, 12:428-435. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Ables JL, Breunig JJ, Eisch AJ, Rakic P: Not(ch) just development: Notch signalling in the adult brain.
Nat Rev Neurosci 2011, 12:269-283. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Matilla A, Roberson ED, Banfi S, Morales J, Armstrong DL, Burright EN, Orr HT, Sweatt JD, Zoghbi HY, Matzuk MM: Mice lacking ataxin-1 display learning deficits and decreased hippocampal paired-pulse facilitation.
J Neurosci 1998, 18:5508-5516. PubMed Abstract | Publisher Full Text
Wu C, Orozco C, Boyer J, Leglise M, Goodale J, Batalov S, Hodge CL, Haase J, Janes J, Huss JW 3rd, Su AI: BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources.
Genome Biol 2009, 10:R130. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Servadio A, Koshy B, Armstrong D, Antalffy B, Orr HT, Zoghbi HY: Expression analysis of the ataxin-1 protein in tissues from normal and spinocerebellar ataxia type 1 individuals.
Nat Genet 1995, 10:94-98. PubMed Abstract | Publisher Full Text
Coussen F, Normand E, Marchal C, Costet P, Choquet D, Lambert M, Mege RM, Mulle C: Recruitment of the kainate receptor subunit glutamate receptor 6 by cadherin/catenin complexes.
J Neurosci 2002, 22:6426-6436. PubMed Abstract | Publisher Full Text
Barth M, Rickelt S, Noffz E, Winter-Simanowski S, Niemann H, Akhyari P, Lichtenberg A, Franke WW: The adhering junctions of valvular interstitial cells: molecular composition in fetal and adult hearts and the comings and goings of plakophilin-2 in situ, in cell culture and upon re-association with scaffolds.
Cell Tissue Res 2012, 348:295-307. PubMed Abstract | Publisher Full Text
Matthews L, Gopinath G, Gillespie M, Caudy M, Croft D, de Bono B, Garapati P, Hemish J, Hermjakob H, Jassal B, Kanapin A, Lewis S, Mahajan S, May B, Schmidt E, Vastrik I, Wu G, Birney E, Stein L, D'Eustachio P: Reactome knowledgebase of human biological pathways and processes.
Nucleic Acids Res 2009, 37:D619-D622. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Anttila V, Stefansson H, Kallela M, Todt U, Terwindt GM, Calafato MS, Nyholt DR, Dimas AS, Freilinger T, Muller-Myhsok B, Artto V, Inouye M, Alakurtti K, Kaunisto MA, Hamalainen E, de Vries B, Stam AH, Weller CM, Heinze A, Heinze-Kuhn K, Goebel I, Borck G, Gobel H, Steinberg S, Wolf C, Bjornsson A, Gudmundsson G, Kirchmann M, Hauge A, Werge T, et al.: Genome-wide association study of migraine implicates a common susceptibility variant on 8q22.1.
Nat Genet 2010, 42:869-873. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Vasconcelos LP, Silva MC, Costa EA, da Silva Junior AA, Gomez RS, Teixeira AL: Obsessive compulsive disorder and migraine: case report, diagnosis and therapeutic approach.
J Headache Pain 2008, 9:397-400. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Ting JT, Feng G: Glutamatergic synaptic dysfunction and obsessive-compulsive disorder.
Curr Chem Genomics 2008, 2:62-75. PubMed Abstract | PubMed Central Full Text
Ahmari SE, Spellman T, Douglass NL, Kheirbek MA, Simpson HB, Deisseroth K, Gordon JA, Hen R: Repeated cortico-striatal stimulation generates persistent OCD-like behavior.
Science 2013, 340:1234-1239. PubMed Abstract | Publisher Full Text
Marsh R, Maia TV, Peterson BS: Functional disturbances within frontostriatal circuits across multiple childhood psychopathologies.
Am J Psychiatry 2009, 166:664-674. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Milad MR, Rauch SL: Obsessive-compulsive disorder: beyond segregated cortico-striatal pathways.
Trends Cogn Sci 2012, 16:43-51. PubMed Abstract | Publisher Full Text
Pittenger C, Bloch MH, Williams K: Glutamate abnormalities in obsessive compulsive disorder: neurobiology, pathophysiology, and treatment.
Pharmacol Ther 2011, 132:314-332. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Girirajan S, Campbell CD, Eichler EE: Human copy number variation and complex genetic disease.
Annu Rev Genet 2011, 45:203-226. PubMed Abstract | Publisher Full Text
Nestler EJ, Hyman SE: Animal models of neuropsychiatric disorders.
Nat Neurosci 2010, 13:1161-1169. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ, Sham PC: PLINK: a tool set for whole-genome association and population-based linkage analyses.
Am J Hum Genet 2007, 81:559-575. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Karolchik D, Barber GP, Casper J, Clawson H, Cline MS, Diekhans M, Dreszer TR, Fujita PA, Guruvadoo L, Haeussler M, Harte RA, Heitner S, Hinrichs AS, Learned K, Lee BT, Li CH, Raney BJ, Rhead B, Rosenbloom KR, Sloan CA, Speir ML, Zweig AS, Haussler D, Kuhn RM, Kent WJ: The UCSC Genome Browser database: 2014 update.
Nucleic Acids Res 2014, 42:D764-D770. PubMed Abstract | Publisher Full Text
Picard [http://picard.sourceforge.net/ webcite]
McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA: The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.
Genome Res 2010, 20:1297-1303. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell TJ, Kernytsky AM, Sivachenko AY, Cibulskis K, Gabriel SB, Altshuler D, Daly MJ: A framework for variation discovery and genotyping using next-generation DNA sequencing data.
Nat Genet 2011, 43:491-498. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Handsaker RE, Korn JM, Nemesh J, McCarroll SA: Discovery and genotyping of genome structural polymorphism by sequencing on a population scale.
Nat Genet 2011, 43:269-276. PubMed Abstract | Publisher Full Text
Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP: Integrative genomics viewer.
Nat Biotechnol 2011, 29:24-26. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR: A method and server for predicting damaging missense mutations.
Nat Methods 2010, 7:248-249. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
FTP site for data files used in the study [ftp://ftp.broadinstitute.org/pub/vgb/dog/OCD_GenomeBiology2014paper/ webcite]


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Tell The Ones I LoveArtist: Steep Canyon Rangers
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Tell The Ones I Love
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More Info:Since 2009 the Steep Canyon Rangers have received widespread acclaim as Steve Martin's backing band, but in 2012 the music community really tipped its hat to the quintet by presenting them with the Grammy� for Best Bluegrass Album. The follow-up to their Grammy� win is a freewheeling and high-energy collection of 12 original songs entitled 'Tell the Ones I Love.' The album showcases the Steep Canyon Rangers myriad talents instrumental agility, tight harmony vocals, and inventive songwriting, not to mention the integration of percussion.
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SFO Welcomes Virgin's New Look, Virgin America Welcomes Travelers with Sale
by Hipster Travel Guide Staff
Friday, April 08, 2011 at 07:27 AM EDT
Virgin America, the only California-based US airline, opened up its new
terminal in San
Terminal 2, known as T2, will host Virgin as the anchor airline and the terminal's new look (it's actually been around for more than 60 years) is strictly got Virgin in mind throughout.
The 640,000 square foot terminal is also the first LEED platinum certified terminal in the country — giving it some eco-goodness that no other terminal can currently enjoy.
"As a new airline we've been able to use the latest in technology, design and sustainable practices to enhance the traveler experience, and we hope elevate the journey beyond just getting from Point A to Point B," said David Cush, President and Chief Executive Officer of Virgin America. "We're proud to unveil a new home that similarly reinvents the travel experience for the modern flyer -- and that also reflects the innovative, forward-looking spirit of our San Francisco home."
Naturally, Virgin had to do a few special things to mark the event. So the
first passengers
And of course, marking the event wouldn't be complete without some sort of sale. Virgin started it's "Fly into the Future" campaign with one-way tickets starting at $59. You can buy tickets online or call 877.FLY.VIRGIN. The sale ends tomorrow.
All photos used with permission of Virgina America.
This article originally appeared on Hipster Travel Guide.