I'M DAN KASTNER AND IT'S MY PLEASURE TO INTRODUCE TODAY'S SPEAKER, DR. EVAN EICHLER, WHO IS A PROFESSOR OF GENOME SCIENCES AND ALSO A HOWARD HUGHES MEDICAL INSTITUTE INVESTIGATOR AT THE UNIVERSITY OF WASHINGTON, AND IS REALLY ONE OF THE PIONEERING LEADERS IN THE FIELD OF HUMAN STRUCTURAL DNA VARIANTS AND THEIR EFFECTS ON HUMAN DISEASE. DR. EICHLER WAS BORN IN CHEYENNE, WYOMING, AND SPENT HIS EARLY YEARS OF HIS LIFE IN CANADA, AND ACTUALLY GOT HIS UNDERGRADUATE DEGREE IN AN HONORS PROGRAM IN BIOLOGY AT THE UNIVERSITY OF SASKETCHEWAN IN SASKATOON, SASKATCHEWAN, AND THEN HE WENT TO ANOTHER PART OF THE WILD WEST, NAMELY HOUSTON, WHERE HE DID HIS GRADUATE WORK IN HUMAN MOLECULAR GENETICS WITH DAVID NELSON, AND THERE REALLY STARTED TO WORK IN EARNEST ON THIS QUESTION OF DNA INSTABILITY AND MECHANISMS THAT REGULATE TRIPLATE REPEAT VARIABILITY, DID POSTDOCTORAL TRAINING AT THE LAWRENCE LIVERMORE LABS AND THEN FROM 1997 TO 2004 WAS ON THE FACULTY AT CASE WESTERN AND IN 2004 TOOK HIS CURRENT POSITION AT THE UNIVERSITY OF WASHINGTON. DURING THE COURSE OF HIS CAREER HE'S REALLY ESTABLISHED HIMSELF AS ONE OF THE WORLD'S LEADING EXPERTS IN STRUCTURAL AND IN PARTICULAR COPY-NUMBER VARIATION IN EVOLUTION AND THE HUMAN GENOME, RECIPIENT OF A NUMBER OF PRESTIGIOUS AWARDS OVER THE COURSE OF HIS CAREER, NAMING THREE OF THEM, HE WAS THE RECIPIENT OF THE BASAL O'CONNOR YOUNG INVESTIGATOR AWARD FROM THE MARCH OF DIMES FOUNDATION, THE RECIPIENT OF THE KURT STERN AWARD FROM THE AMERICAN SOCIETY OF HUMAN GENETICS, AND HE GAVE THE MENDEL LECTURE OF THE EUROPEAN SOCIETY OF HUMAN GENETICS A YEAR OR SO AGO. IN 2012, HE WAS ELECTED AS A MEMBER OF THE NATIONAL ACADEMY OF SCIENCES. IN ANY CASE, IT'S REALLY JUST AN ABSOLUTE TREAT TO HAVE THE OPPORTUNITY TO HAVE HIM HERE TODAY, AND THE TITLE OF HIS TALK IS GOING TO BE "AUTISM, NEW MUTATIONS, GENES AND PATHWAYS." DR. EICHLER. [APPLAUSE] >> SO THANKS, DAN, FOR THAT INTRODUCTION. IT'S A PLEASURE TO BE HERE WITH YOU TODAY AND SHARE WITH YOU SOME OF THE MOST RECENT DATA THAT MY LAB AND OTHERS OVER THE LAST I WOULD SAY FIVE OR SIX YEARS BUT WITH A SPECIFIC FOCUS ON THE LAST COUPLE YEARS HAVE LEARNED ABOUT THE GENETICS OF AUTISM. SO I'VE APPROACHED THIS QUESTION FROM A SLIGHTLY DIFFERENT PERSPECTIVE. I SEE AUTISM PRIMARILY AS A DISEASE OF DEVELOPMENT. NEURODEVELOPMENT DISORDER, BEHAVIOR, REPETITIVE BEHAVIOR AND LANGUAGE. IF YOU BELIEVE THE CDC ESTIMATES, THE CURRENT ESTIMATES AS OF APRIL 2014 IS ON ORDER OF ONE OF EVERY 6 BOYS BORN IN THE WORLD TODAY SUFFER ARE ARE OR ARE ON AUTISM SPECTRUM DISORDER, HALF FOR GIRLS, DEVELOPMENT OF DELAY WHICH INCLUDES THINGS OUTSIDE THE AUTISM SPECTRUM, INABILITY TO MEET CERTAIN MILESTONES, AFFECTING AS MUCH AS 2 TO 3% OF THE GENERAL POPULATION. ONE OF THE INTERESTING ASPECTS OF THIS DISEASE WHICH WE HAVE NOT COMPLETELY RESOLVED, MAYBE WE NEVER WILL, IS THE GENDER BIAS. WE KNOW FOR DISEASES SUCH AS DELAY IN INTELLECTUAL DISABILITY THAT MALES ARE 1.4 TO EVERY FEMALE, THAT'S BEEN IDENTIFIED WITH THE DISEASE, IN AUTISM THE SAMPLES WE'VE ACTUALLY PARTICULARLY STUDIED, THE MALE BIAS 4-1, AND HIGH FUNCTIONING ASPERGER IT'S 6-1. DESPITE HETROGENEITY, GENETICS HAVE BEEN THEIR FOR 40 YEARS. WE KNOW THERE'S A STRONG GENETIC COMPONENT, CURRENT ESTIMATE OF HERITIBILITY PLACE IT AT 50% OR MORE FROM TERMS OF -- THIS COMES FROM FAMILY STUDIES AND IDENTICAL TWIN STUDIES. A LOT OF WORK WAS DONE WITH IDENTICAL TWINS, ESTIMATINGS OF CONCORDANCE FOR MONO ZYGOTIC IS 70-95%. IN THE 2000s, CAME UP WITH FEW LOW SIDES REPLICATED, THERE'S BEEN ARGUMENTS MUCH LARGER SAMPLE SIZES ARE NEEDED TO MAKE THIS HAPPEN. WE KNEW FROM STUDIES OF FRAGILE X SYNDROME MONOGENIC FORMS CAUSED BY A SINGLE GENE THAT WAS TRACKING WITHIN FAMILIES MIGHT ACCOUNT FOR AS MUCH AS 8 TO 10%. THE QUESTION REALLY FACING THE FIELD I WOULD SAY OF AUTISM GENETIC CIRCA 2005 IS WHERE IS THE REST? I WOULD SAY THAT A PARTICULARLY IMPORTANT BREAKTHROUGH IN THIS AREA CAME FROM THE STUDY OF LARGE COPY-NUMBER VARIATIONS, SO LARGE COPY NUMBER VARIANTS ARE LARGE-SCALE DELETION, DUPLICATIONS THAT EXIST WITHIN GENOMES, INDIVIDUALLY RARE BUT COLLECTIVELY COMMON. IT WAS FOUND IN THE SERIES OF STUDIES STARTING FIRST WITH STUDYING CHILDREN WITH DEVELOPMENTAL DELAYS, WORK DONE BY OUR GROUP, AND LATER WORK FROM MIKE'S GROUP, JOHNSON SEBAT, WHEN YOU LOOKED AT INDIVIDUALS WITH AUTISM, LATER ON DISEASES SUCH AS EPILEPSY AND ADULT SCHIZOPHRENIA THERE WAS AN EXCESS OF LARGE CNVs IN PATIENTS WHEN COMPARED TO CONTROLS. SO WHAT THIS MEANS IS THAT IN PRINCIPLE, LARGE SLOTS OF THE GENOME WENT DELETED OR DUPLICATED AND FOUND IN EXCESS OR DEFICIENCY PREDISPOSING INDIVIDUALS TO DEVELOP THESE DISEASES. OUR OWN TAKE ON THIS BEGAN AROUND 2002, THIS IS RIGHT AT THE END OR THE BEGINNING I GUESS OF THE SEQUENCING OF THE HUMAN GENOME, WHERE WE DEVELOPED A MAP OF AREAS OF THE GENOME THAT WOULD BE UNSTABLE AS A RESULT OF THEIR GENOMIC ARCHITECTURE. SHOWING HERE THE MAP AND YOU WILL THE BLUE LINES REPRESENT REGIONS OF THE GENOME THAT HAVE BEEN HISTORICALLY COPY-NUMBER VARIABLE, SO THESE REPRESENT SEGMENTAL DUPLICATIONS, IN 2002 WE MAPPED OUT IN GOLD ALL THE AREAS OF THE GENOME THAT WE THOUGHT WOULD BE PRONE TO COPY-NUMBER VARIATIONS BECAUSE THEY WERE FLANKED BY DUPLICATIONS, PULLING FROM A PLAY BOOK OF JIM AND OTHERS WHO WORKED ON CROSSING OVER THESE REGIONING BECAUSE OF GENOMIC ARCHITECTURE WOULD BE FRAGILE AND PRONE TO DELETE OR DUPLICATE EVERY TIME WE PRODUCE AS A RESULT OF CROSSING OVER. WE IDENTIFIED 111 SUCH CANDIDATE REGIONS, AT THAT TIME WE KNEW 23 OF THEM WERE ASSOCIATED WITH DEVELOPMENTAL DISORDERS, PARTICULARLY DISEASES ASSOCIATED WITH NEUROLOGIC DISEASE IN CHILDREN AND WE TARGETED THESE REGIONS USING SOME TECHNOLOGIES WHICH ARE NOW IN SOME RESPECTS OUT OF DATE TO LOOK FOR COPY NUMBER CHANGES IN PATIENTS WITH UNEXPLAINED DEVELOPMENTAL X-RAY DELAY AND AUTISM SPECTRUM DISORDER. THE APPROACH WAS SOMEWHAT SUCCESSFUL. WE IDENTIFIED ON THE ORDER OF ABOUT A DOZEN RECURRENT DELETIONS SO SITES WERE FOUND EXCLUSIVELY IN CHILDREN WITH DEVELOPMENTAL DISORDERS. A PICTURE OF CHILDREN WHICH IS NOW CALLED THE 172-2131 DELETION SYNDROME, THE SO IN 2006 WE IDENTIFIED THIS PORTION OF CHROMOSOME 17, ABOUT 500 KILOBASES IN SIZE DELETED IN THESE CHILDREN SO IN A COHORT OF 300 KIDS, WE IDENTIFIED FOUR CHILDREN THAT HAD THIS PARTICULAR SEGMENT OF DNA, ONE OF THEIR TWO CHROMOSOMES 500 KILOBASES DELETED. NONE WITH TRANSMITTED FROM A PARENT, OCCURRED IN THE PRODUCTION OF GERM CELLS OF THE PARENT, ALL THE CHILDREN HAD SIMILAR CHARACTERISTICS FACIAL FEATURES AND OTHER ASPECTS. YOU CAN SEE SIMILARITIES IN THE WAY THE KIDS LOOK, ALL UNRELATED INDIVIDUALS, THEY ALL HAVE THE CHARACTERISTIC BULBOUS NOSE, PROTRUDING TONGUE AND SOMETHING YOU CAN SEE IS THE CHILDREN HAVE BEEN GENERALLY REGARDED AS HAVING A BEHAVIORAL DISPOSITION, QUITE CONGENIAL AND HAPPY CHILDREN. SO THERE'S NOW BEEN OVER ON THE ORDER OF 300 FAMILIES IDENTIFIED WITH THIS SPECIFIC RECURRENT DELETION MEDIATED BY DUPLICATIONS, A HOT SPOT IN THE GENOME. LATER IN 2007 WE IDENTIFIED THIS REGION, THIS IS A THREE MEGABASE PAIRED COLLEAGUES ON CHROMOSOME 15, IT TAKES OUT A DOZEN GENES IN THAT REGION, SO IN THESE CHILDREN THEY HAVE ONE NORMAL COPY, ONE DELETED COPY THAT THEY HAVE INHERITED OR THEY HAVE RECEIVED FROM ONE OF THE PARENTAL GERM LINES, ALL CASES ONCE AGAIN SPORADIC, NO TRANSMISSION OF EVENTS, A DE NOVO EVENT IN ONE OF THE GERM LINES OF THE PARENTS, THEY HAD CHARACTERISTIC FACIAL FEATURES, YOU CAN SEE THE FRONTAL FOREHEAD, THOUGHT TO BE THE RESULT OF OVERGROWTH OF NEURONAL CELLS, ALMOND SHAPED EYES, WHAT YOU CAN'T SEE IS THESE KIDS ARE SHORT STATURE, 20 PERCENTILE OF GROWTH. WE DOCUMENTED THIS IN 2008, DIFFERENT FROM THE OTHER TWO, IN THE SENSE THAT NOT ALL CASES WERE SPOREATIC. SOME, 50% OF THE TIME, THE EVENTS WERE INHERITED, OFTEN FROM A MOTHER, 50% OF THE TIME THEY WERE ACTUALLY DE NOVO, NOT FOUND IN ANY OF THE PARENTS. THIS IS A DELETION OF 1.5 MEGABASES, NO CHARACTERISTIC FACIAL FEATURES WITH THESE INDIVIDUALS. HOWEVER, THESE KIDS IN ADDITION TO HAVE SUFFERING FROM MILD DEVELOPMENTAL DELAY TYPICALLY ESSENTIALLY HAVE SUSCEPTIBILITY TO SEIZURES, IN WORK I WON'T GO INTO BUT THAT WE DID WITH GROUPS FOCUSED ON EPILEPSY THIS DELETION WAS ASSOCIATED WITH 1% OF INDIVIDUALS WITH GENERALIZED EPILEPSY. AUTISM SPECTRUM DISORDER, MORE SPECIFICALLY, WHAT I'M SHOWING YOU IS A BURDEN ANALYSIS, LOOKING AT THE PROPORTION OF CHILDREN THAT COME INTO A CLINIC WITH A DIAGNOSIS OF DEVELOPMENTAL DELAY COMPARED TO CONTROLS. THE WAY TO READ THIS PARTICULAR PLOT IS WHAT'S SHOWN HERE ON X AXIS, PROPORTION OF INDIVIDUALS IN THE GENERAL POPULATION OR IN KIDS WITH DEVELOPMENTAL DELAY THAT HAVE A LARGE DELETION OR DUPLICATION GREATER THAN 500 KOLIBASES IN SIZE. 25% OF CHILDREN REFERRED TO THE CLINICS FOR UNEXPLAINED DEVELOPMENTAL DELAY HAVE A LARGE COPY-NUMBER VARIATION COMPARED TO 10% OF THE GENERAL POPULATION, USED TO SUGGEST AS MUCH AS 14% OF THE QUOTE/UNQUOTE GENETIC CAUSE OF DEVELOP MEMORIAL DELAY CAN BE EXPLAINED BY THE LARGE COPY NUMBER VARIANT. ONE OF THE INTERESTING THINGS I'M ASKED, THESE INDIVIDUALS, THESE PARTICULAR EVENTS YOU SEE, THOSE 10% OF THE GENERAL POPULATION WHICH MEANS 10% OF THE PEOPLE IN THIS ROOM HAVE A LARGE CNV, ALMOST NEVER SEEN MORE THAN ONCE OR TWICE IN A COHORT OF 10,000 NORMAL INDIVIDUALS. THE DATA SUGGESTS THOSE CNVs ARE UNDER STRONG SELECTION, BEING ELIMINATED FROM THE POPULATION. THE REASON FOR THE HIGH FREQUENCY IS THE GENOME STRUCTURED IN SUCH A WAY THAT PRODUCED THESE LARGE COPY NUMBER VARIANTS, BUT EVEN AMONG 10% OF THE GENERAL POPULATION THEY ARE CONTRIBUTING AT SOME LEVELS TO ESSENTIALLY THEIR HEALTH OR ESSENTIALLY MAYBE THEIR DEFICIENCY WITH RESPECT TO HEALTH, THEY ARE SELECTED AND NEVER REACH A PARTICULARLY HIGH FREQUENCY. THESE DATA WERE IMPORTANT BECAUSE THEY SUGGESTED TO US THAT COPY NUMBER AND IN PARTICULAR DOSAGE AND BALANCE OF GENES MUST BE A PARTICULARLY IMPORTANT MECHANISM BY WHICH WE HAVE CHILDREN WITH DEVELOPMENTAL DELAY OR AUTISM. TO BREAK THIS OUT MORE BY DISEASE, IF WE LOOK AT THE PATIENTS, CHILDREN WITH DEVELOPMENTAL -- VARIOUS FORMS OF DEVELOPMENTAL DELAY AND VARIOUS DIAGNOSES, YOU CAN SEE THE BURDEN DEPENDING UPON THE DISEASE IS DIFFERENT. SO THIS IS THE SAME PLOT I SHOWED YOU BEFORE BUT NOW BREAKING OUT TO FACIAL ABNORMALITIES, CARDIOVASCULAR, CONGENITAL HEART DEFECTS, VERSUS INDIVIDUALS WITH AUTISM AND EPILEPSY COMPARED TO CHILDREN WITH PEDIATRIC ASTHMA, YOU CAN SEE BIG DIFFERENCES IN TERMS OF BURDEN OF CNV. YOU SEE FOR HEART DEFECTS, CRANIO, LARGE BURDEN OFCNV. EPILEPSY IS INTERMEDIATE AND ASTHMA WE SEE NO DIFFERENCE IN TERMS OF BURDEN. IT'S NOT AS IF EVERY PEDIATRIC DISEASE IS ESSENTIALLY IMPINGED UPON BY A LARGE CNV BUT PARTICULARLY A SUBSET, DEVELOPMENT OF HEART, CRANIOFACIAL, DEVELOPMENT OF THE BRAIN. WE'VE ALSO HAD THE OPPORTUNITY TO LOOK AT INHERITANCE OF THESE LARGE CNVs, THIS IS DATA FROM 2500 FAMILIES WHERE WE'VE LOOKED AT LARGE CNVs AND WHAT PROPORTION ARE INHERITED, AND FROM THE MOTHER OR FATHER OR DE NOVO. YOU CAN LOOK AT THIS AND SAY THERE'S A BIG DIFFERENCE BETWEEN DELETION AND DUPLICATION, A LARGER FRACTION ARE DE NOVO, AND THIS FRACTION GETS GREATER FOR BOTH DELETIONS AND DUPLICATION AS A FUNCTION OF SIZE. THIS SHOULD MAKE SENSE, LARGER EVENTS INVOLVE MORE GENES, DELETIONS ARE GENERALLY TO BE MORE DELETERIOUS. WHEN YOU GET OUT TO THIS RANGE THE MAST MAJORITY OF EVENTS ON DE NOVO. HAVE YOU HAVE A KEEN EYE YOU SEE A DIFFERENCE BETWEEN THE MALE AND FEMALE CONTRIBUTION. IF YOU LOOK AT WHAT'S CONTRIBUTED FROM THE MOTHER AND FATHER YOU'LL SEE THE AREA UNDER THE CURVE AT LEAST FOR ESSENTIALLY THE FEMALE TRANSMISSION IS GREATER. AND THIS IS SOMETHING THAT IS BORNE OUT KNOW OVER AND OVER WHEN WE LOOK AT SEVERE EVENTS, NOT JUST LARGE CNVs, 58% OF THESE ESSENTIALLY PRIVATE LARGE EVENTS WHEN THEY ARE TRANSMITTED ARE COMING FROM MOTHERS AS OPPOSED TO FATHERS. SOME OF YOU MIGHT BE GOING, WELL, 58% DOESN'T SOUND LIKE A LARGE NUMBER OR BIG DIFFERENCE AS OPPOSED TO EXPECTED 50%, BUT THE NUMBER OF CASES, NUMBER OF TRANSMISSIONS MEANS THIS IS A HIGHLY SIGNIFICANT OBSERVATION. SO WHEN TRANSMITTED, MOTHERS ARE MORE LIKELY TO TRANSMIT OR BE CARRIERS OF EVENTS AND SOME RECENT DATA WHICH I WON'T GO INTO SUGGESTS IN FACT SONS AS OPPOSED TO DAUGHTERS MAY BE PREFERENTIALLY OBTAINING THESE CNVs IN TERMS OF MANIFESTING A DISEASED STATE. SO WE BUILT OUR INITIAL MAPS OF COPY-NUMBER VARIATIONS, STUDYING REALLY ON THE ORDER OF 10,000 CASES, 15,000 CONTROLS, NOW PUSHED THIS IN THE LAST REALLY YEAR, LOOKING ON THE ORDER OF 30,000 CASESEL DEVELOPMENTAL DELAY, 20,000 CONTROLS. WHAT WE'VE BEEN ABLE TO DO IS ESSENTIALLY BUILD A MORBIDITY MAP OF HUMAN COPY-NUMBER VARIATION BASED ON THE PA EARN OF CNV, AREAS WHERE WE SEE DELETION IN RED, OR DEVIATION IN BLUE, COMPARED TO CONTROLS, GIVE US SOME INDICATION WE'VE IDENTIFIED A REGION OF THE GENOME WHEN EITHER DELETED OR DUPLICATED RESULTS IN INCREASED RISK OF DEVELOPMENTAL DELAY. JUST TO GIVE YOU PERSPECTIVE OF WHAT I TALKED ABOUT A FEW MINUTES AGO, THESE HOT SPOT REGIONS IN YELLOW ARE SHOWN HERE, AND WHEN YOU GET TO THE LARGE NUMBERS OF 30,000 CASES VERSUS 20,000 CONTROLS, YOU BEGIN TO SEE PATTERNS EMERGING ON SPECIFIC CHROMOSOMES, WE START TO SEE EXCESS OF EVENTS OUTSIDE THE HOT SPOT REGION, WHICH FOR US IS EXCITING BECAUSE WE NOW HAVE GENOME-WIDE THAT'S NOMINALLY SIGNIFICANT IN EXCESS DELETION IN COMPARED TO CONTROLS. THIS MAP ALLOWS US TO IDENTIFY 70 REGIONS OF THE GENOME, 14 WERE NEWLY SIGNIFICANT. IT'S IMPORTANT TO CONTINUE ON THIS WORK, I THINK A LARGE PERSPECTIVE FROM THAT OF IT IS THE CLINICIAN AND PEOPLE THAT ARE DOING DIAGNOSIS, WE CONTINUE TO IDENTIFY NEW COPY NUMBER VARIANTS ASSOCIATED IN PATIENTS THAT WE'VE NEVER SEEN BEFORE, IF WE DON'T POOL LARGE NUMBERS OF DATA TOGETHER WE WON'T BE ABLE TO PROVIDE ANY REALLY RISK ASSESSMENT FOR FAMILIES IN PATIENTS THAT HAVE THESE BECAUSE THERE IS A BACKGROUND OF BENIGN SUBCLINICAL CNV WITHIN OUR GENOME. THE MOST IMPORTANT THING THAT CAME FROM STUDYING CNVs FOR SIX OR SEVEN YEARS WAS MODEL FOR THINKING OF THE GENOMIC ARCHITECTURE SUCH AS DEVELOPMENTAL DELAY OR AUTISM. THE FIRST THING WE LEARNED WAS THAT DOSAGE AND BALANCE IS VERY IMPORTANT. SO HAVING TOO MUCH OR NOT ENOUGH OF A GIVEN GENE PROBABLY EARLY IN DEVELOPMENT IS ACTUALLY CRITICAL, IF YOU THINK ABOUT THIS, WE KNOW THIS FROM DOWN SYNDROME AND SOME OF THE SYNDROMIC DISORDERS DESCRIBED EARLY ON, THIS REALLY REINFORCED IT. THE OTHER IMPORTANT POINT WAS THE MODEL HERE IS HETEROZYGOUS, SO WE'RE TALKING ABOUT EVENTS WHERE THERE'S STILL A NORMAL COPY LEFT IN THE GENOME BUT THE DEVIATION IS PREDOMINANT. THE OTHER IMPORTANT POINT, THIS GETS TO THE QUESTION OF COMMON VARIANTS VERSUS RARE VARIANTS, FOR THESE DISEASES, EACH OF THESE INDIVIDUAL CNVs IS RARE, SO AFFECTING .5%, .2%, .1% OF KIDS OF DEVELOPMENTAL DELAYS OR AUTISM SPECTRUM DISORDER, 15% OF DEVELOPMENTAL DELAY FOR CNVs, THAT'S A MINIMUM OF IMPACT, SO AUTISM, 7 TO 8% IN TERMS OF ITS IMPACT. THE FOURTH LESSON WAS GENOME FIRST. WE TOOK THINGS LIKE DEVELOPMENTAL DELAY, FOUND CNVs, RECURRENT CNVs AND STUDY PHENOTYPE TO IDENTIFY NEW SYNDROMES PREVIOUSLY UNRECOGNIZED. SO THIS IDEA OF GOING FROM A GENOTYPE WHICH IS A CNV TO ESSENTIALLY A PHENOTYPE WAS SOMETHING THAT EMERGED FROM THE STUDY OF CNVs. SO CNVs ARE ONE OF THE BIGGEST THINGS THAT CAME OUT OF THE HUMAN GENOME PROJECT BECAUSE IT ALLOWED US TO EXPLAIN TO A LOT OF FAMILIES THE CAUSE OF DEVELOPMENTAL DELAY OR AUTISM IN THEIR SPECIFIC CASES. MOST OF THE EVENTS THOUGHT TO BE PATHOGENIC ARE LARGE, SO THEY TYPICALLY AFFECT MULTIPLE GENES, USUALLY HALF A DOZEN TO A DOZEN GENES, IN MOST CASES THE ACTUAL INDIVIDUAL GENE RESPONSIBLE FOR THE DOSAGE AND BALANCE AND THEREFORE DISEASE IS NOT KNOWN. SO THIS IS WHERE I THINK EXOME SEQUENCING CAME IN TO FIT THE BILL. THE IDEA YOU COULD GO BACK AND DO THE SAME EXPERIMENT BUT NOW LOOKING FOR REALLY WITH MICROSCOPIC OR SUBMICROSCOPIC PRECISION LOOK FOR EVENTS THAT AFFECT GENES. IT'S POOR MAN'S SEQUENCING. INSTEAD OF SEQUENCING THE ENTIRE GENOME WE CONSEQUENCE THE PROTEIN CODING, REDUCING IT TO 1 1/2% OF THE GENOME AS THE TARGET. IF CNV PROVIDE A 30,000-FOOT VIEW OF THE LANDSCAPE OF GENETIC ARCHITECTURE OF DISEASE LIKE AUTISM, EXOME SEQUENCING IS THE BOTTOM UP, GO BACK TO THE FAMILIES, SEQUENCE THE MOTHER, THE CHILD, AND THE AFFECTED INDIVIDUAL IN THAT FAMILY AND TRY TO LOOK FOR THE SAME TYPE OF MUTATION, GENE-KILLING MUTATION THAT WOULD HAVE THE SAME EFFECT AS LARGE DELETION EVENT, I.E. TAKE OUT A GENE ALMOST IN ITS ENTIRETY. SO THIS WAS THE MODEL. SO FOR THE REST OF THE TALK I'M GOING TO TALK ABOUT THE WORK WE'VE DONE IN TERMS OF SEQUENCING AUTISM EXOMES. THE HYPOTHESIS WAS TO LOOK AT FAMILIES WITH THERE'S NO FAMILY HISTORY OF AUTISM, WHICH TURNS OUT TO BE ROUGHLY 70%, THERE ARE FAMILIES WITH HISTORY BUT MOST ARE SPOREATIC OR SIMPLEX, OUT OF THE BLUE AUTISM, SO ONE AFFECTED INDIVIDUAL. SEQUENCE THE MOTHER, FATHER, AFFECTED CHILD WITH AUTISM, AND IF POSSIBLE, AND IF MONEY IS AROUND, SEQUENCE THE UNAFFECTED SIBLING AS WELL. WITH ONE SIMPLE IDEA, THE IDEA WOULD BE TO COMPARE THE MUTATIONS, YOU NEED THE PARENTS FOR THAT, ASK THE QUESTION IS THERE A DIFFERENCE IN THE SIGNIFICANCE IN THE TERMS OF IMPACT OF THE MUTATIONS THAT YOU SEE IN THIS AFFECTED COMPARED TO UNAFFECTED INDIVIDUAL. SO 2012 WAS A BANNER YEAR I WOULD ARGUE FOR SEQUENCING AUTISM EXOMES, 660 FAMILIES THAT WERE SEQUENCED IN FOUR STUDIES, OUR WAS ONE, WE TARGETED 250 FAMILIES IN OUR STUDY. WE SEQUENCED THE EXOME TO 80-FOLD SEQUENCE COVERAGE, 92% OF PROTEIN CODING, IN THE SUBSET OF 600 FAMILIES A LITTLE OVER 350 AFFECTED AS WELL AS UNAFFECT THE INDIVIDUAL FROM THE FAMILY WAS SEQUENCED IN ADDITION TO THE PARENTS. SO SOME OF MY STUDENTS AND POSTDOCS WOULD KILL ME FOR SUMMARIZING THIS WITH TWO BULLET POINT BUT HERE IT GOES. THERE IS GOOD NEWS. THE GOOD NEWS WAS THIS. WHEN YOU LOOK AT THE DE NOVO MUTATIONS IN THIS INDIVIDUAL AFFECTED WITH AUTISM COMPARED TO UNAFFECTED THERE WAS A TWO-FOLD EXCESS OF GENE-KILLING MUTATION COMPARED TO UNAFFECTED. THE OVERALL RATE OF NEW MUTATIONS WAS NOT DIFFERENT BUT THEY WERE ABOUT THE SAME NUMBER OF NEW MUTATIONS, LIKE A TYPE OF MUTATION WAS DIFFERENT, SO THESE INDIVIDUALS WERE TWICE AS LIKELY TO HAVE A GENE-KILLING NEW MUTATION COMPARED TO THESE. THIS WAS IMPORTANT BECAUSE IT SUGGESTED THE MODEL OF CNVs, WHICH WAS LARGE DELETION, COULD BE APPLIED AT DE NOVO MUTATION. THE BAD NEWS IS WHEN THOSE FOUR PAPERS CAME OUT, EVERYBODY GATHERED TOGETHER AND COMPARED NOTES AND SET WHICH GENES ARE THE AUTISM GENES, THE BAD NEWS IS WE ALMOST NEVER SAW LIGHTNING STRIKE TWICE AT THE SAME GENE. SO ONLY 18 GENES WERE HIT RECURRENTLY, AND THAT IS ACTUALLY SIGNIFICANT. YOU WOULDN'T EXPECT TO SEE THAT MANY BUT WE DID SEE 18. AND IN TERMS OF LOSS OF FUNCTION FIVE GENES WERE SEEN WITH LOSS OF FUNCTION, SO INDEPENDENT GENE KILLING MUTATION IN INDEPENDENT FAMILIES. SUGGESTING 500 TO 1000 INDEPENDENT GENES WHEN MUTATED WITH LOSS OF FUNCTION MUTATION IN THE SIMPLEX MODEL COULD RESULT IN AUTISM. WHY WAS THAT BAD NEWS? THE BAD NEWS IS THAT WE COULDN'T PROVE PATHOGENICITY FOR ANY ONE OF THESE GENES, WE COULDN'T TELL ANYTHING TO FAMILIES OR CLINICIANS, AND IF YOU'VE BEEN STUDYING AUTISM FOR 30 YEARS THE CHANCES THAT YOU'VE SEEN THIS SAME GENETIC CAUSE MORE THAN TWICE IN 30 YEARS OF PRACTICE IS ALMOST ZERO. SO EVERY CHILD WITH AUTISM IS TRULY UNIQUE IN THE PERSPECTIVE A DIFFERENT MUTATION IN A DIFFERENT GENE IS GOING TO BE RESPONSIBLE FOR AUTISM. THAT'S A BIT OF A SOBERING MOMENT FOR US IN THE FIELD BECAUSE WE REALIZE THIS PROBLEM WAS -- WE HAD APPRECIATED THERE WOULD BE LOCUS HETROGENEITY, WE DIDN'T APPRECIATE IT WOULD BE THIS EXTENSIVE IN TERMS OF NUMBERS. IN OUR STUDY WE UNCOVERED OTHER THINGS. I YOU WANT TO MENTION ONE OF THE THINGS WE HAD TO DO WHEN WE STARTED CATALOGING MORE THAN 250 NEW MUTATIONS, A SMALL SUBSET, 20%, WE COULD DETERMINE IN WHOSE GERMLINE THE EVENT LIKELY OCCURRED. THE REASON IS WE WOULD HAVE A NEW MUTATION IN THE CHILD AND THEN WE WOULD HAVE IN CLOSE PROXIMITY A SNP THAT WAS INFORMATIVE THAT WE COULD SAY IF IT CAME FROM THE MOM OR DAD. WE DID THIS ANALYSIS IN 2012, WE FOUND 8 5 OF NEW MUTATIONS WERE COMING FROM THE DAD'S GERMLINE. THE THREE MONTHS AGO, LOOKING AT FULL GENOME DATA, IT WAS IDENTICAL, 80% CAME FROM THE FATHER'S GERMLINE. IF WE COMPARED A FUNCTION OF PATERNAL AGE AT TIME OF CONCEPTION WE SEE A MODEST BUT SIGNIFICANT CORRELATION WITH ADVANCED PATERNAL AGE. IN OTHER WORDS, OLDER FATHERS ARE CONTRIBUTING MORE NEW MUTATIONS, PROBABLY THROUGH THEIR GERMLINE PRODUCTION OF SPERM, TO THEIR CHILDREN AS OPPOSED TO YOUNGER MUTATIONS. THIS FITS NICELY WITH EPIDEMIOLOGICAL DATA, STUDIES OUT OF SWEDEN SUGGEST THE RISK OF HAVING A CHILD WITH AUTISM FROM A 50-YEAR-OLD FATHER IS TWICE THAT WHAT IT IS FOR A 25-YEAR-OLD FATHER. THIS IS NOT A PROPERTY JUST OF AUTISM. THIS IS A PROPERTY OF BIOLOGY. OLD MEN CONTRIBUTE MORE NEW MUTATIONS TO THEIR OFFSPRING. THE OTHER THING WHICH I HAVE TO CONFESS I WAS SKEPTICAL OFF, MY POSTDOC WAS DISAPPOINTED HE DIDN'T SEE RECURRENCES. WHAT DO YOU DO? YOU START TO LOOK AT THE GENES AND NETWORKS AND PATHWAYS. WE LOOKED AT WHAT WAS CLASSIFIED AS MOST SEVERE MUTATIONS, LIKELY GENE DISRUPTIVE MUTATIONS IN THE INITIAL 200 FAMILIES AND SEVERE SHOWN IN BLACK, BLACK REPRESENTS SEVERE. WHAT HE FOUND WAS THAT WHEN HE LOOKED AT THE PROTEIN-PROTEIN INTERACTIONS AMONG THESE HE FOUND 39% OF SEVERE MUTATIONS WERE PART OF ONE HIGHLY INTERCONNECTED PROTEIN-PROTEIN INTERACTION NETWORK. I WAS SKEPTICAL. I TELL YOU WHY I WAS SKEPTICAL, I FEEL THE DATABASE WAS LARGELY INCOMPLETE IN TERMS OF PROTEIN-PROTEIN INTERACTION, LOTS OF FALSE NEGATIVES, LOTS OF FALSE POSITIVES, BRIAN HAD TO GO THROUGH A NUMBER OF HOOPS AND IN PART COMING FROM REVIEWERS COMMENTS, HE SHOWED THIS WAS STATISTICALLY SIGNIFICANT BASED ON MODEL, HE SHOWED THAT IF YOU ACTUALLY LOOKED AT WHAT WAS DEFINED AS AUTISM GENES, THIS NETWORK WHICH HE CALLED A BETA-CATENIN WAS HIGHLY ENRICHED, GOING BACK AND LOOKING AT THE PATTERN OF DE NOVO MUTATION IN THE UNAFFECTED SIBLINGS, NOT SEEING THIS NETWORK IN THE UNAFFECTED SIBLINGS. SO HE REPORTED THIS IN 2012 AS A CHD 8 BETA-CATENIN, BECAUSE OF THE HUB POSITION OF THIS HELICASE, AND IN BETA-CATENIN WE HAD SEEN SEVERE MUTATIONS. OTHER WORK FROM OUR LABS PARTICULARLY FROM IVAN'S LAB FOUND AN ENRICHMENT WITH GENES WITH THE FMRP PROTEINS. SO FAST FORWARD NOW TWO YEARS, THREE OF THE FOUR GROUPS THAT COMPETED JOINED FORCES, TO ACTUALLY PUBLIC ONE COMPREHENSIVE ASSESSMENT OF 2517 FAMILIES, ABOUT 10,000 EXOMES SEQUENCED BETWEEN MATT, MIKE AND OUR GROUP. OF THESE WERE WERE 1900 QUADS, ONE UNAFFECTED SIBLING, 606 TRIOS. A TOTAL OF 2162 VALIDATED DE NOVO MUTATIONS, ALL THE RECURRENT AND TRUNCATED FROM VALIDATED BY STANDARD SEQUENCING. SO AS OF END OF LAST MONTH OR END OF DECEMBER WE HAD 345 GENES WHICH WE HAD ONE OR MORE DE NOVO TRUNCATED MUTATIONS, 26 GENES WITH MUST BE LOSS OF FUNCTION, DE NOVO MUTATIONS NEVER FOUND IN SIBLINGS OR IN CONTROLS. THE ESTIMATE ON THESE 26 GENES, POSSIBILITY THEY ARE CONTRIBUTING TO SIGNIFICANT RISK FOR AUTISM IS CLOSE TO 95%. WE'VE ALSO IDENTIFIED A LARGE NUMBER OF GENES WITH TWO OR MORE DE NOVO MISSENSE, SOME HAVE THREE OR MORE, 12 GENES IN PARTICULAR, GIVING US ROUGHLY 200 HIGH LIKELIHOOD, I WOULD ARGUE, CANDIDATE GENES FOR DE NOVO MUTATION ASSOCIATED WITH AUTISM. WE REVISITED THE STORY ABOUT THE EXCESS OF DE NOVO DISRUPTIVE MUTATIONS AND THE RESULT HOLDS. THIS IS DATA FOR NONSENSE MUTATIONS, SPLICE SITE MUTATIONS, AND THIS IS A COLLECTIVELY LIKELY GENE DISRUPTIVE MUTATION, A TOTAL NUMBER OF EVENTS YOU'RE SEEING IN RED COMPARED TO THE UNAFFECTED SIBLINGS, THE TWO TO ONE PATTERN IN TERMS OF PRO BANDS HAVING MORE, CALCULATE THE NUMBER OF EVENTS PER CHILD, THE NUMBER OF EVENT DE NOVO IN EXOME FOR TRUNCATING EVENTS, .12 IN SIBLINGS, DIFFERENTIAL OF .09, 42% OF TRUNCATING ARE CONTRIBUTING TO AUTISM SPECTRUM DISORDER. WHEN YOU SEQUENCE A FAMILY WITH AUTISM, ALMOST A 50% CHANCE THAT IF YOU SEE A DE NOVO TRUNCATING MUTATION IT'S CONTRIBUTING TO AUTISM. WE HAD THE LARGE NUMBER, WE SAW SIGNIFICANCE. SO WE SEE AN EXCESS OF MISSENSE MUTATION COMPARED TO UNAFFECTED SIBLINGS. NUMBER OF SYNONYMOUS MUTATIONS IS ABOUT IDENTICAL BETWEEN THE TWO. SO WE CAN DO THE SAME CALCULATIONS AND ESTIMATE 12% OF THE DE NOVO BASED ON THE DIFFERENTIAL ARE CONTRIBUTING TO AUTISM SPECTRUM DISORDER. THAT MEANS THAT NINE TIMES OUT OF TEN WHEN YOU FIND A DE NOVO MUTATION IN A CHILD WITH AUTISM WHERE THERE'S NO FAMILY HISTORY IT HAS NOTHING TO DO WITH AUTISM BUT ONE TIME OUT OF TEN IT DOES. INTERESTINGLY WHEN YOU ASK THE QUESTION HOW MUCH DOES EACH CONTRIBUTE TO THE OVERALL RISK OF AUTISM, THESE ARE ABOUT EQUAL NUMBERS, BECAUSE THERE'S SO MUCH MORE DE NOVO MISSENSE MUTATION, 20% OF AUTISM MAY BE ACCOUNTED FOR BY DE NOVO MISSENSE, LIKELY SEVERE MISTAKESES, AND DE NOVO LIKELY DISRUPTIVE MUTATIONS. ONE OTHER POINT I'LL MAKE WHICH ECHOES BACK TO THE CNV WORK WE DID AN EXTENSIVE SEARCH FOR ALL EVENTS WE SAW INCLUDING THE RECURRENT ONES FOR LOSS OF FUNCTION AND FOUND NO EVIDENCE OF COMPOUND HETEROZYGOTES, ARGUING STRONGLY THAT IT'S NOT A RECESSIVE MODEL, LIKE THE CNVs, PROBABLY A DOMINANT MODEL WITH RESPECT TO AUTISM. FOR THOSE INTERESTED IN GENES AND GENE LISTS, THESE ARE THE TOP HITTING GENES, THERE'S NOT A SINGLE CASE IN THE ESP OR IN TERMS OF OVER6000 CONTROLS SEQUENCED IN TERMS OF LOSS OF FUNCTION MUTATION. RECURRENT MISSENSE MUTATIONS, SODIUM CHANNEL GENE IS A TRANSCRIPTIONAL REGULATOR, EXCESS OF DE NOVO MUTATION. THIS IS THE PROBLEM. LOCUS HETROGENEITY, 500 TO 1000 GENES IN THE SIMPLEX MODEL, HOW ARE WE GOING TO PROVE THESETY INDIVIDUAL LOCUS LEVEL? I WAS IN A DEPARTMENT WITH JASON WAS WORKING ON NEW TECHNOLOGY TO SOLVE THIS PROBLEM. IT'S CALLED ONE OF MANY APPROACHES, THIS IS CALLED MOLECULAR INVERSION OF PROBE TECHNOLOGY, DEVELOPED IT IN GEORGE CHURCH'S LAB, PERFECTED WHEN HE CAME TO SEATTLE. THE TECHNOLOGY IS CONCEPTUALLY SIMPLE. ESSENTIALLY GLORIFIED OLIGONUCLEOTIDES, YOU DESIGN HOUSE OF THESE CORRESPONDING TO GENES YOU WANT TO SEQUENCE, PUT THEM IN WITH YOUR DNA, FROM YOUR INDIVIDUAL DNA SAMPLES, THROW IN A DNA LIGASE, A CLOSED CIRCLE, EXON NUCLEASE TREAT, REMOVE SINGLE-STRANDED DNA, THROW IN A BAR CODE, AMPLIFY, POOL THESE DNA SAMPLES, POOL 19 OR 384 AND RUN A SINGLE LANE OF ALUMINUM SEQUENCING. THE IMPORTANT POINT OF THE TECHNOLOGY IS THAT IT'S RAPID, SENSITIVE, VERY SPECIFIC, ALLOWING US TO SEQUENCE ESSENTIALLY 192 SAMPLES OR 192 INDIVIDUALS FOR 50 GENES COVERING 92% OF THE TARGET FOR ABOUT $12 A SAMPLE. SO THIS IS EXACTLY WHAT THE DOCTOR ORDERED BECAUSE IN ORDER TO PROVE ANY INDIVIDUAL GENES, YOU NOW HAVE TO RESEQUENCE NOT IN HUNDREDS OR THOUSANDS OR ULTIMATELY TENS OF THOUSANDS OF SAMPLES, THIS TECHNOLOGY ALLOWS YOU TO DO. IF EXON IS POOR MAN'S, THIS IS POOR MAN'S POOR MAN'S, IN TERMS OF GETTING AT THESE SPECIFIC GENES. SO THIS IS WHAT WE DID. WE DID A PILOT IN 2012, WE TARGETED BASICALLY 50 GENES THAT WERE OUR BEST CANDIDATES, AND WHAT WE FOUND WAS ESSENTIALLY THE FOLLOWING. WHEN WE TARGETED ESSENTIALLY INDIVIDUALS WITH AUTISM, THIS IS ROUGHLY 2000, 2500 INDIVIDUALS SEQUENCED, WE FOUND 8 DE NOVO MUTATIONS IN THE GENE CHD8, SEVEN WERE LOSS OF FUNCTION, NONE WERE SEEN IN OVER 6000 CONTROLS THAT HAD BEEN EXOME SEQUENCED. GRINDIDB, FOUR DE NOVO MUTATIONS, THREE WERE LOSS OF FUNCTION MUTATION, NONE OBSERVED, LOSS OF FUNCTION MUTATION IN CONTROL. DYRK1A, THIS IS THREE OUT OF THREE, THREE DE NOVO LOSS OF FUNCTION MUTATIONS, NEVER SEEN IN CONTROLS. AND TBR1, A MARKER OF CORTICAL DEVELOPMENT, THREE OUT OF THREE LOSS OF FUNCTION MUTATIONS. SO WE CAN COMPUTE AND BUILD A MODEL TO ESTIMATE WHAT IS THE LIKELIHOOD YOU WOULD EXPECT TO SEE THREE OR FOUR OR TWO DE NOVO MUTATIONS THAT WOULD BE LIKELY GENE DISRUPTIVE AND BASED ON DISTANCE AND SIZE OF THE GENE AND MUTATION RATE OF A LOCUS BETWEEN CHIMP AND HUMAN, WE CAN DO THAT. IN THIS MODEL WE'VE COMPUTED THE LIKELIHOOD THAT YOU WOULD EXPECT TO HAVE SEEN THAT MANY DE NOVO MUTATIONS, THAT WOULD ACTUALLY BE GENE DISRUPTIVE. WE DID THIS CALCULATION, FIVE OF THE GENES THAT WE ORIGINALLY TARGETED ACTUALLY REACHED LOCUS-SPECIFIC SIGNIFICANCE, NOW INSTEAD OF STAYING WE HAVE AN AGGREGATE WE CAN STAY SPECIFIC GENES, WHEN YOU SEE A LOSS OF FUNCTION IN THOSE GENES DE NOVO THEY ARE LIKELY CORRECTED TO AUTISM. WHILE EACH CONTRIBUTES TO A SMALL FRACTION OF THE TOTAL, COLLECTIVELY THE FIVE GENES ACCOUNT FOR 1%, OR A LITTLE UNDER 1% OF AUTISM. SO THIS IS JUST THE BEGINNING. BUT THE REALLY COOL THING IS WHEN YOU START TO GET TO THESE NUMBERS, HAVING MORE THAN TWO OR THREE PATIENTS THAT ACTUALLY HAVE A GIVEN PARTICULAR MUTATION, YOU CAN ASK SIMPLE QUESTIONS, IS THERE ANYTHING MORE SIMILAR WILL THE PATIENTS THAN LET'S SAY YOUR GENERIC AUTISM FROM WHICH THESE PATIENTS WERE DRAWN? SO I'M SHOWING YOU DATA HERE FROM ESSENTIALLY HEAD CIRCUMFERENCE DATA. SOME HAVE BEEN DESCRIBED AS HAVING LARGER HEADS, THAT'S TRUE FOR A SUBSET, A LITTLE BIT OF AN OVERSIMPLIFICATION. THIS IS A HEAD CIRCUMFERENCE, THIS IS NORMAL, THE VARIANCE IS WIDER, SOME HAVE LARGER HEADS, SOME INDIVIDUALS HAVE SMALLER HEADS. IF YOU ACTUALLY MAP THE ACTUAL PATIENTS WITH CHG8 LOSS OF FUNCTION MUTATION, SIX OUT OF EIGHT WOULD CLASSIFY AS MACRO CEPHALIC. WHEN YOU MUTATE DYRK1 AIN DROSOPHILA OR MOUSE IT RESULTS IN LOSS OF BRAIN VOLUME. THIS GENE HAS STUDIES, THOUGHT TO BE ONE OF TWO IMPORTANT FOR COGNITIVE DEFICIT ASSOCIATED WITH DOWN SYNDROME, A GAIN OF AN EXTRA COPY, THIS IS A LOSS. MUTATIONS CAN SHOW REDUCTION IN BRAIN VOLUME, DATA WOULD RECAPITULATE THAT IN HUMANS. CHD8, NOT MUCH WAS KNOWN, STILL LARGELY A BLACK BOX, IT'S A REGULATOR OF CHROMATIN, THOUGHT TO REGULATE BETA-CATENIN, IN WORK DONE BY CHRIS WALSH IF YOU EXPRESS BETA-CATENIN, YOU RESULT IN OVERGROWTH OF MOUTH BRAINS, THEY WILL DEVELOP GYRIFICATION. IF YOU HAVE A NEGATIVE REGULATOR YOU MIGHT EXPECT A MICRO CEPHALIC PHENOTYPE IF WE KNOCK THAT OUT. WE'VE IDENTIFIED THREE MUTATIONS IN BETA-CATENIN, SEVERE MUTATION DE NOVO, THESE PATIENTS PLACE WHERE YOU WOULD EXPECT ON THE OPPOSITE OF THE NEGATIVE REGULATOR OF CHD8. THIS IS JUST TO SHOW YOU DATA IN THE CONTEXT OF A FAMILY, WE HAVE THE PRO BANS HERE WITH DYR1A IN BLUE, DE NOVO, LOSS OF FUNCTION COMPARED TO THE PARENT, YOU CAN SEE THE TREND. THE FINDING IS SIGNIFICANT, CHD8 IS BORDERLINE SIGNIFICANT. INSTEAD OF DOING A FEW THOUSAND, LET'S DO 20,000. WE HAVE A LIST OF 245 GENES WE WANT TO INTERROGATE SYSTEMATICALLY, WE PUT TOGETHER 6000 AUTISM, 6000 CHILDREN WITH INTELLECTUAL DISABILITY/DELAY AND 6000 CONTROLS, SEARCHING FOR RECURRENCE, TRYING TO FIND EVIDENCE WE'VE IDENTIFIED THE LOCUS AND IF WE FIND THEM, LET'S SAY FIVE, SIX, SEVEN PATIENTS, CAN WE REASSESS THEM FOR PHENOTYPIC SIMILARITY. SO WE NOW HAVE A COHORT, 20,000 PATIENTS, INVOLVED ESSENTIALLY 10 OUT OF 13 OF THE ORIGINAL PARTICIPANTS OF THE SIMPLEX COLLECTION, TEN LABS ACROSS FOUR CONTINENTS WHERE PATIENTS HAVE BEEN COLLECTED, DNA IS ASSEMBLED, WE'RE ACTUALLY SEQUENCING IT WITH IRB APPROVAL, NO SMALL TASK, BECAUSE WE HAVE TO BREAK THE CODE AND GO BACK TO EACH INDIVIDUAL SITE TO GET IRB CONSENT TO RECONTACT. IF PATIENTS ARE FOUND WITH THE MOLECULAR LESION WE THINK IS PATHOGENIC, WE BRING THEM BACK. THIS IS THE FUNDAMENTAL DISTINCTION FOR MANY COLLECTIONS AT NIH, IS THAT WE ACTUALLY HAVE PHENOTYPICALLY RECONTACT POSSIBILITY FOR MOST SAMPLES. THESE ARE THOSE FOR YOU INTERESTED, COLLABORATIONS FROM ALL AROUND THE WORLD, A LOT FROM EUROPE IN PARTICULAR, AND THESE ARE THE RESULTS. SO THE FIRST RESULTS CAME IN REALLY THE SUMMER OF LAST YEAR. WE ACTUALLY WENT AFTER CHG8, AWAY HE NOW HAVE 20 TRUNCATING MUTATIONS IN CHD8. 11 OUT OF 12 ARE DE NOVO, OUR MOST SIGNIFICANT GENE TO DATE. IF THIS CHROMATIN HELICASE DNA BINDING PROTEIN, IN A MODEL, IT'S THOUGHT THIS AFFECTS MANY GENES DURING DEVELOPMENT, AND THE WAY IT DOES IS BETA-CATENIN INTERACTS, RECRUITING H-1, CREATING CHROMATIN TRANSCRIPTION. WHAT DO PATIENTS LOOK LIKE? WE'VE BEEN ABLE TO BRING THEM IN. MANY CAME BACK TO SEATTLE, SEEN AT THE AUTISM CENTER WHERE THEY UNDERGO A TWO-DAY EVALUATION INVOLVING A REEVALUATION OF THE DIAGNOSIS OF AUTISM TO ESSENTIALLY MEETING WITH PEDIATRICIANS DOING MRI IF THAT'S POSSIBLE AND OTHER WORKUPS. 87% HAVE A CLINICALLY DEFINED DEFINITION OF AUTISM, REMEMBER WE'RE PULLING FROM BOTH DEVELOPMENTAL DELAY AND AUTISM REDOING THIS STUDY, THIS IS A GOOD SIGN, SUGGESTING THIS IS PRIMARILY DIAGNOSIS WOULD PROBABLY BE AUTISM. LESS THAN HALF HAVE INTELLECTUAL DISABILITY, IT'S NOT AN INTELLECTUAL DISABILITY GENE. 80% HAVE MACROCEPHALY, PREDOMINANTLY AND EARLY IN DEVELOPMENT, IN THE FIRST MONTH TO TWO MONTHS OF LIFE, BASED ON LONGITUDAL RECORDS, 95th PERCENTILE AFTER THE FIRST MONTH OF LIFE. 73% HAVE SEVERE GASTROINTESTINAL FUNCTION, BOUTS OF DIARRHEA WEEKS FOLLOWED BY BOUTS OF CONSTIPATION INTO PUBERTY. MANY FAMILIES ARE REPORTING SEVERE SLEEP DYSFUNCTION IN THEIR CHILDREN. THE FIRST TWO FAMILIES THAT CAME IN, FOR EXAMPLE, REPORTED THEIR KIDS WOULD STAY UP SOMETIMES THREE DAYS CONSECUTIVELY WITHOUT SLEEPING A WINK. ONE MORE COMMENTED THEY LOCK THE DOORS AT NIGHT BECAUSE THEY WERE AFRAID THE CHILD WOULD WANDER OFF BECAUSE THE CHILD WOULD NEVER SLEEP AND THEN SUDDENLY POWER NAP FOR A LONG PERIOD OF TIME. WE'LL NEED LARGER NUMBERS TO SEE IF THIS HOLDS UP BUT POTENTIAL PURSUIT OF GENE TO THE PHENOTYPE, NEW FEATURES OF THIS FORM OF AUTISM. WE'VE MADE ATTEMPTS TO MODEL THIS IN ZEBRAFISH, EXPERIMENTS ONGOING TO DO IT IN MOUSE, THIS IS WORK OF POSTDOC, YOU CAN SEE IT'S EXPRESSED EARLY ON, AND BECOMES MORE RESTRICTED TO THE ACTUAL HEAD REGION, LATER DURING DEVELOPMENT, AND IF YOU CAN SEE HERE IT BECOMES INTENSE ALONG GASTROINTESTINAL TRACTS, THE BLACK LINE. FOLLOWED BY CRISPR WORK WE'VE BEEN ABLE TO SHOW IN FACT IN DEVELOPING ZEBRAFISH THERE'S AN INCREASE IN INTERCUSP DISTANCE CONSISTENT WITH OVERGROWTH, MARKER ANALYSIS SUGGESTS PARTICULARLY DORSAL PROGENITOR NEURONS. AND FROM GASTROINTESTINAL FUNCTION, YOU'RE STUFFING FLUORESCENT MICRO SPHERES INTO THEIR MOUTH AND AS A FUNCTION OF TIME MEASURING HOW FAR THEY TRAVERSE ALONG THE DIGESTIVE TRACT DIVIDING IN ZONES ONE, TWO, THREE, FOUR, WHEN THE MICRO SPHERES ARE EXPELLED THEY WILL COME OUT OVER HERE AT THE END. SO THESE ARE CONTROL FISH AND YOU CAN SEE AT THREE HOURS POST INJECTION SIGNIFICANT IN ZONE FOUR, BY SIX HOURS, BUT IN THE MORPHOLINO AND CRISPR FISH THEY GET HUNG UP, THIS IS A SIGNIFICANT FINDING THAT WOULD SUGGEST THIS MAY BE MIMICKING EFFECTS IN PATIENTS. INTERESTINGLY, I DON'T HAVE A GOOD EXPLANATION FOR THIS, IF YOU STAIN FOR ENTERIC NEURONS, THIS IS A NORMAL FISH, THIS IS A MORPHOLINO FISH, THERE'S A CONSISTENT DEPLETION OF ENTERIC NEURONS, THE EXACT OPPOSITE IN THE DEVELOPING FOREBRAIN OR MID-BRAIN, OVERPROLIFERATION OF NEURONAL PRECURSOR CELLS. THIS IS ANOTHER SUBTYPE OF AUTISM WE'VE BEEN CHARACTERIZING WITH OUR COLLEAGUES IN BELGIUM, THIS IS ESSENTIALLY A GENE WITH TEN LOSS OF FUNCTION MUTATIONS, IN PATIENTS, VERSUS ONE OUT OF 8000 CONTROLS, NINE OUT OF NINE OF THESE THAT WE'VE SEEN IN PATIENTS ARE ACTUALLY DE NOVO, THEY ARE NOT INHERITED FROM THE PARENT. YOU CAN SEE HERE SIMILARITIES IN THE PHENOTYPE, THESE KIDS ALL TEND TO HAVE ESSENTIALLY THIS HAIR LINE, THE DATA FROM SOME MRIs SUGGESTS THERE'S AN OVERGROWTH, PARTICULARLY OF THE NEOCORTEX IN THESE PARTICULAR CHILDREN, THE REST OF THE BRAIN SEEMS PARTICULARLY NORMAL, 100% HAVE AUTISM, 90% HAVE ALMOST COMPLETE LOSS OF EXPRESSIVE LANGUAGE DURING DEVELOPMENT, A PRETTY SEVERE FORM OF AUTISM, EVEN FROM THE PERSPECTIVE OF LANGUAGE DEVELOPMENT. WE DON'T HAVE TO MAKE A MOUSE KNOCKOUT. IT'S ALREADY BEEN MADE FOR DIFFERENT REASONS. THIS GENE ACTIVITY DEPENDENT NEUROPROTECTIVE PEPTIDE HAS BEEN STUDIED FOR MANY YEARS AS A POTENTIAL THERAPY FOR ALZHEIMER'S, PRESUMED TRANSCRIPTION FACTOR ASSOCIATING IT WITH SWI/SNF, IMPORTANT IN TUMORIGENESIS. ADNP HETEROZYGOUS MICE HAVE A NEURONAL GLIAL PATHOLOGY, INTERESTING, IDENTIFYING WHAT WAS PREVIOUSLY THOUGHT OF AS IDIOPATHIC, NOW A SYNDROMIC FORM. THE LAST EXAMPLE IS THE REGULATED KINASE, ESSENTIALLY THE DOWN SYNDROME COGNITIVE DYSFUNCTION GENE, NOW DELETED. IN A PAPER AT THE END OF THIS MONTH WE'VE CHARACTERIZED 8 TRUNCATING MUTATIONS FROM 7000 CASES, LOSS OF FUNCTION IN NONE OF THE CONTROLS THAT HAVE BEEN SEQUENCED, OR EXOME SEQUENCES AT THIS POINT. 7 OUT OF 8 ARE DE NOVO. AND THE KIDS ACTUALLY ARE QUITE INTERESTING, YOU MIGHT BE ABLE TO GUESS WHICH KIDS WERE DIAGNOSED WITH DEVELOPMENTAL DELAY AND WHICH DIAGNOSED WITH AUTISM. AUTISM ON THE TOP, DEVELOPMENTAL DELAY ON THE BOTTOM, 100% INTELLECTUAL DISABILITY, 100% MICROCEPHALY, 89% LATE ONSET EPILEPSY. THEY HAVE TYPICAL FACIAL FEATURES WHICH YOU CAN SEE HERE, IT'S THIS REDUCED JAW IN PARTICULAR, RELATIVELY POINTED NOSE THAT'S BEEN OBSERVED BY MANY CLINICIANS, THIS IS PRETTY SEVERE IN THESE PATIENTS BECAUSE SOME HAVE HAD RECONSTRUCTIVE SURGERY TO ACCOMMODATE ERUPTION OF TEETH AS THEY GET OLDER. WE CAN'T EXPLAIN THE HETROGENEITY IN THIS ONE, IT'S NOT SO STRAIGHTFORWARD BUT PRETTY CLEAR THIS IS A MICRO CEPHALIC REDUCED BRAIN VOLUME, SUPPORTED BY DATA FROM MICE AS WELL AS FROM DROSOPHILA, RESULTING IN A MUTANT CALLED MINI BRAIN, AND TROUBLE WITH REFLEXES, VARIOUS CLASSES OF BEHAVIOR. SO WE'RE GOING FORWARD DOING MANY MORE GENES. WE HAVE SIGNIFICANCE FOR NEW GENES PREVIOUSLY UNRECOGNIZED SUCH AS A GENE KNOWN AS TRIP12, PAX5, BUT THE IMPORTANT POINT HERE IS THAT I THINK GENOME SEQUENCING, IN THIS PARTICULAR CASE EXOME SEQUENCING PROVIDED A MODEL FOR GOING AFTER A SIGNIFICANT CHUNK OF GENETIC ETIOLOGY AND STUDYING THOSE INDIVIDUALS WITH COMMON GENETIC ETIOLOGY WE CAN BREAK DOWN THE UMBRELLA INTO SUBTYPES. IN SUMMARY, A COUPLE COMMENTS, THE BEGINNING PART I TALKED ABOUT CNVs, GREAT FOR MAKING US RETHINK HOW WE SHOULD APPROACH THE STUDY OF DEVELOPMENTAL DELAY IN AUTISM INSTEAD OF IT BEING AN INHERITED DISEASE THINKING NOW IN TERMS OF ACTUALLY MORE OF A SPORADIC DISEASE, PERHAPS MUTATIONS PLAYING AN IMPORTANT ROLE. EXOME SEQUENCING DATA PROVIDED INSIGHT IN TERMS OF TARGET, 42% OF DE NOVO LOSS OF FUNCTION WE BELIEVE ARE CONTRIBUTING TO AUTISM SPECTRUM DISORDER, 12 TO 13% MUTATION, COMBINED WITH CNV, SPORADIC LOSS OF FUNCTION, WE HAVE A HANDLE ON 30% OF THE GENETIC -- WE HAVE TO BE CAREFUL HERE, IN QUOTES AGAIN, "CAUSE" OF AUTISM, BUT WE HAVEN'T PROVEN THE CASE FOR MORE THAN A FEW PERCENTAGE AT THIS POINT. THAT'S AN IMPORTANT THING TO DISTINGUISH. I LOVE THE IDEA OF A GENOTYPE-FIRST APPROACH, PROPOSED NOT BY ME BUT OTHERS INCLUDING HERE AT NIH, THIS IDEA THAT YOU START WITH THE GENETICS, SORTING OUT COMMON OR INDIVIDUALS WITH COMMON GENETIC ETIOLOGY AND STUDY THEM MORE CAREFULLY TO UNDERSTAND WHETHER THERE IN FACT IS SUBTLETIES WILL THE PHENOTYPE MISSED IN TERMS OF GENERIC DIAGNOSIS SUCH AS AUTISM. SO IF I CAN JUST BASICALLY GIVE YOU MY PERSPECTIVE OF WHERE WE ARE IN THE FIELD, DEFINITELY A HALF FULL PERSPECTIVE AS OPPOSED TO HALF EMPTY. WE'VE COME A LONG WAY SINCE 2007 WHERE THE VAST MAJORITY OF GENETIC ETIOLOGY OF AUTISM WAS UNEXPLAINED. WE NOW HAVE A HANDLE, MAYBE 35%, MAYBE 40%, OF WHERE THAT MISSING QUOTE/UNQUOTE HERITIBILITY EXISTS. THE BIG UNANSWERED QUESTION, WHAT ROLE DOES ESSENTIALLY THE NON-CODING PORTION OF THE GENOME PLAY AND ABILITY TO NOW GO BACK AND LOOK AT THE FULL GENOMES OF INDIVIDUALS WHERE WE HAVEN'T FOUND CODING MUTATIONS AND LOOK AT GENES WE KNOW ARE DOSAGE SENSITIVE BECAUSE WE'VE PROVEN THEY ARE RELEVANT PROVIDES US WITH A LOGIC WE'RE BEGINNING TO TEASE APART AS OPPOSED TO BLINDLY SEQUENCING GENOMES. THIS MODEL OF A GENOTYPE FIRST APPROACH I DON'T THINK IS SPECIFIC TO AUTISM OR DEVELOPMENTAL DELAY, APPLIED MORE BROADLY. IT MAKES SENSE BUT I'M BIASED BECAUSE A GENETICIST TO START WITH GENETICS, SORT OUT A PATHOGENIC RISK AND STUDY THE PHENOTYPES OF THOSE INDIVIDUALS TO ASK IF THERE ARE COMMONALITIES BETWEEN THEM, IN SOME CASES THERE WILL BE, SOME THERE WON'T BE. PERFECT WOULD BE RECURRENCE WITH ONE PHENOTYPE AND HAVE A CORRESPONDS ONE-TO-ONE, MORE IKELY IS MANY WILL CONVERGE ON FOR EXAMPLE MACROCEPHALIC. THE IMPORTANT POINT HERE IS THIS IS THE FIRST STEP FOR LAYING DOWN WHICH PATIENTS SHOULD BE TREATED BY WHICH THERAPIES. THE IDEA YOU WOULD TREAT A CHILD WITH AUTISM WITH MICRO CEPHALY THE SAME AS CHILD WITH MACRO CEPHELY IS LUDICROUS. THEY ARE COUNTER-OPPOSING AND YOU WANT TO IDENTIFY AT A MOLECULAR LEVEL EARLY ON. LAST, GENES DO MAKE SENSE IN SOME RESPECTS, THEY ARE FALLING INTO PATHWAYS AND CLUSTERS THAT BELONG TO SPECIFIC PROCESSES, MOLECULARLY. SO WHILE 500 TO 1000 DIFFERENT GENES SOUNDS ALMOST IMPOSSIBLE TO REALLY TACKLE, THE FACT THAT THEY BOIL DOWN TO PERHAPS A DOZEN OR SO PATHWAYS IS ENCOURAGING, SO WE'RE SEEING THINGS FOR EXAMPLE LIKE WHAT I'M SHOWING IN THIS SLIDE, ESSENTIALLY THE RELATIVE SIZE OF THE CIRCLE INDICATES THE NUMBER OF DE NOVO MUTATIONS, RED LOSS OF FUNCTION YOU CAN SEE THEY ARE PART OF PROTEIN-PROTEIN AND CENTER AROUND SYNAPSE, PLASTICITY AND FUNCTION, THIS IS OUR CORE, IF YOU WILL, IF YOU ADD ALL THE LOSS OF FUNCTION MUTATIONS, ABOUT 60% CAN BE MAPPED WITH ONE STEP OR SO REMOVED FROM THE C.P.I. NETWORK. SO MY MOST DOC HAS GIVEN ME AN EDUCATION. HE'S TAUGHT ME THERE IS QUITE A BIT OF VALUE TO THINKING ABOUT THIS FROM PATHWAYS AND NETWORKS AS OPPOSED TO SIMPLY GENETICS. LASTLY, FOR THOSE WHO LIKE MYSELF HAVE FAMILY MEMBERS, THIS IS THE HOPE WE HAVE STUDYING AUTISM. GENETICS IS JUST THE BEGINNING. ET IT'S GOING TO GET US TO GENES. WHEN MUTATED, WE HAVE SOMETHING TO OFFER FOR FAMILIES, IT'S NOT A LOT BUT IT'S DIAGNOSTICS. THIS IS HOW IT BEGINS. ONCE WE IDENTIFY THE DIAGNOSTICS, SMARTER PEOPLE WILL WORK OUT BIOLOGY, NEUROBIOLOGY, CHEMISTRY AND FEED INTO NETWORKS, COLLECTIONS OF GENES CONNECTED IN TIME AND SPACE AND PERHAPS FUNCTION AND I BELIEVE WHEN THEY ARE THERAPEUTICS, WHICH ISN'T TODAY BUT HOPEFULLY IN THE FUTURE, TILL BE DIRECTED TOWARDS THE NETWORKS. SO SOME PEOPLE OFTEN ASK ME, WHY DOES IT MATTER WHETHER MY CHILD HAS TYPE 1, TYPE 2, TYPE 3 GENETICALLY, I THINK THE ANSWER IS THIS. I THINK ONCE YOU KNOW WHY YOUR CHILD HAS AUTISM, YOU WILL KNOW WHAT NETWORK ULTIMATELY THEY BELONG TO, PROTEIN-PROTEIN INTERACTION, AND IF THERE'S A THERAPY I THINK THE THERAPY WILL BE APPLIED TO THOSE SUBSETS OF PATIENTS THAT HAVE A SPECIFIC GENETIC LESION THAT MAKES MOLECULAR SENSE. THIS IS KIND OF THE MODEL OF PRECISION MEDICINE WE'RE DREAMING OF, IT CAN BE REAL FOR THINGS SUCH AS AUTISM BUT IT BEGINS BY WORKING OUT THE GENETICS, FIRST AND FOREMOST. THANKS FOR YOUR ATTENTION. THESE ARE THE FOLKS THAT REALLY DID ALL THE WORK. BRAD FOR THE CNV WORK, A POSTDOC ON THE JOB MARKET. SOME OTHER FOLKS HAVE COLLABORATED OVER THE YEARS, IN PARTICULAR LISA SHAFER AND OTHER LABS ON THE CLINICAL SIDE AND SIMON SIMPLEX CONSORTIUM AND LAST BUT NOT LEAST WE COULDN'T DO THIS WORK WITHOUT SUPPORT OF FAMILIES AND PATIENTS SO WE'RE ALWAYS INDEBTED TO THEIR CONTRIBUTIONS AND THE PARENTS HAVE BEEN FANTASTIC IN TERMS OF RECONTACT AND REALLY WORKING TOGETHER TO TRY TO WORK OUT WHY THEIR CHILD HAS AUTISM OR DEVELOPMENTAL DELAY. THANKS FOR YOUR ATTENTION. HAPPY TO TAKE QUESTIONS. [APPLAUSE] >> YOU MENTIONED THE G.I. ASSOCIATIONS AS ONE OF THE FORMS. HAS HIRSCHSTRUNGS COME UP? THAT WOULD BE A GOOD CANDIDATE TO THINK ABOUT. >> ABSOLUTELY. WE'VE DONE SOME COMPARISONS ON HIRSCHSTRUNG DISEASE WITH EARVIN AT JOHNS HOPKINS, AND WE'VE BEEN LOOK NOT AT EXOME SEQUENCING BUT CNV PATTERNS AND WE FIND IN EXCESS OF REALLY CNV, TERN TYPES OF CNV IN KIDS WITH DEVELOPMENTAL DELAY IN HIRSCHSTRUNGS. THE KIDS I DESCRIBED DO NOT HAVE A DIAGNOSIS BUT YOU'RE SUGGESTING A CONNECTION BETWEEN NEURO DEVELOPMENTAL AND G.I. WE'VE SEEN HINTS WITH HIRSCHSTRUNG ALREADY. >> THANK YOU. YEP? >> FIRST I WANT TO THANK YOU FOR YOUR WORK. ON A PERSONAL NOTE. ONE QUESTION I HAVE, WHEN YOU WERE TALKING ABOUT YOUR PATHWAYS, HAS YOUR PATHWAYS LED TO ANY METHYLATION DYSFUNCTIONS IN ANY OF THESE CHILDREN AND MY SECOND QUESTION IS DO YOU HAVE ANY POPULATION OF THE CHILDREN THAT ARE NOT DISFORMIC, THEY DON'T HAVE PROBLEMS WITH THE FACE OR FRONTAL BOSSING? >> YES, THE ANSWER TO THE FIRST QUESTION, HAVE WE SEEN METHYLATION DIFFERENCES, WE HAVE NOT LOOKED. OTHERS ARE LOOKING IN THE SAME PATIENTS BUT WE HAVE NOT COMPARED NOTES, SO WE HAVE NOT SPECIFICALLY LOOKED FOR EXAMPLE IN THE PATIENTS WITH THE CHROMATIN HELICASE MUTATION BUT IT'S A GOOD SUGGESTION. IN TERMS OF PATIENTS THAT LOOK NORMAL, IT'S FUNNY, IF YOU WOULD HAVE GRABBED THE CHD8 PATIENTS, THOSE WHO KNEW NOTHING ABOUT OUR STORIES AND LABS, WE SCRAMBLED THEM WITH OTHER PATIENTS, A CLINICIAN WOULD NEVER LOOK AT THEM AND SAY THAT'S A KID WITH CRANIOFACIAL ABNORMAL. THEY ARE WELL WITHIN THE NORMAL RANGE. THE ONLY REASON YOU SEE IT, THAT'S THE ARGUMENT WE'VE BEEN THINKING ABOUT IT, ALL DESCRIBED AS IDIOPATHIC, YOU START TO SEE SUBTLETIES. WITH DYRK1A,S HALF. WHEN I LOOK AT THE CHILDREN THEY ALL LOOK -- WELL, WHEN I SEE THE PICTURES, MANY SEEM QUITE NORMAL LOOKING TO ME. >> THANK YOU. >> THANKS FOR A WONDERFUL TALK. YOU DESCRIBE HOW THESE DE NOVO EVENTS HELP TO IDENTIFY TERRITORIES WITHIN THE SYSTEMS, NETWORKS THAT WOULD BE ENRICHED FOR RISK. YOU TO DO YOU THINK THERE'S A WAY OF PASSING NETWORKS BY USING TEST ENRICHMENT FOR ASSOCIATION OF MORE COMMON VARIANTS? >> YES, I THINK IT'S A GREAT IDEA, SOMETHING I'VE BEEN MEANING TO GO BACK TO. NOW THAT WE'VE RESEQUENCED A GENE, 8000 TIMES IN CASES SUCH AS CONTROLS, WE HAVE A WHOLE PANOPLY OF MUTATIONS WE HAVEN'T BOTHERED TO STUDY BECAUSE THEY MIGHT BE RARE OR MIGHT BE COMMON BUT INHERITED, TRANSFIXED ON FINDING THE DE NOVO KIND OF MUTATIONS LOSS OF FUNCTION, RIGHT? IT'S STRAIGHTFORWARD I THINK NOW BECAUSE WE'VE DONE THE EXPERIMENTS TO GO BACK TO DATA, REANALYZE, LOOK FOR ESSENTIALLY VERSION, WHAT YOU'RE GETTING AT, FOR THE OTHER CASES MAYBE THERE'S IN CASE SOME ADDITIONAL HITS COMPOUNDED WITH OTHER HITS IN THE GENOME THAT IT MIGHT BE CONTRIBUTING. >> I WAS WONDERING IF YOU WOULD SPECIFY WHAT CRITERIA WAS USED TO IDENTIFY A SEVERE MUTATION? >> THAT'S A GREAT ONE. WE'VE ARGUED ABOUT THIS IN THE LAB IN TERMS OF WHAT IS SEVERE AND WHAT'S NOT. WE'VE SETTLED ON THIS -- I'M NOT SURE IF YOU'RE FAMILIAR WITH THE C SCORE, CALLED THE CAD SCORE, FROM JAY'S GROUP, SO THAT ONE WE'VE USED, THE DEFINITION SPECIFICALLY IN TERMS OF A CAD SCORE BUT THE BOTTOM OR TOP I SHOULD SAY IN TERMS OF CONSERVATION, TOP 30% OF GENES. THE OTHER METRIC WE'VE BEEN USING RECENTLY, WE'RE STARTING TO PUBLIC, IS THIS R.V.IS SCORE BY DAVID GOLDSTEIN, INTOLERANCE THAT SEEMS TO BE INFORMATIVE SO WE'VE SEEN LESS THAN 50% RVIS, LESS THAN 20%. >> THANK YOU. >> DO YOU SEE ANY DIFFERENCES IN HOW THE PATIENTS GET PREGNANT? MORE IN VITRO OR ANYTHING THAT CAN HAPPEN IN THE PREGNANCY? >> MOST OF THE PATIENTS WE'RE STUDYING DON'T GET PREGNANT BECAUSE THEY HAVE CHILDREN WITH AUTISM DON'T DIVIDE EGGS. >> I MEANT THE PARENTS. >> THE PARENTS. >> YEAH. >> DO WE HAVE DATA? I CAN TELL YOU THIS. I DON'T KNOW WHAT TO MAKE OF IT. THE CHD8 PARENTS, I TALKED WITH CLINICIANS, I MAY RELATE TO MACROCEPHALY, SOMETHING LIKE 75% OF MOTHERS ACTUALLY HAD TO HAVE A C-SECTION, TREMENDOUS DIFFICULTY DURING BIRTH, MAYBE CONSISTENT WITH THE OVERGROWTH, RIGHT, WHICH WE SEE IN TERMS OF LONGITUDAL DATA, COMING ON VERY EARLY DURING DEVELOPMENT. >> NO PARTICULAR INCREASE IN VITRO FERTILIZATION? >> NOT THAT I'VE SEEN. THERE'S THE ODD RECORD HERE AND THERE, MULTIPLE MISCARRIAGES IN THE CLINICAL REPORT, BUT THERE HASN'T BEEN A SYSTEMATIC SURVEY OF THIS. >> WELL, IF THERE ARE NO OTHER QUESTIONS WE'LL CALL IT TO A CLOSE AND WE HAVE THE RECEPTION IN THE LIBRARY IN A FEW MINUTES. [APPLAUSE] >> THAT WAS TERRIFIC. THANK YOU VERY MUCH.