>> WELCOME BACK TO OUR WORKSHOP ON GENETIC MODIFIERS LESSONS FROM NERVOUS SYSTEM DISORDERS. TODAY'S TOPIC IS MODIFIER CHARACTERIZATIONS AND MECHANISMS AND THE SESSION IS CHAIRED BY [INDISCERNIBLE], AS LIKE YESTERDAY, WE WILL BROADER IMPACTS HAVE A BREAK AT 11:10 FOR OUR POSTER SESSION AND WE WILL RETURN TO THE TOPIC OF CHARACTERIZATION AND MECHANISMS AT 12:30 AND IT WILL HAVE AN OPEN GROUP DIG CUSHION AT 1 TIENL TIME 30--DISCUSSION AT 1:30 AND THEN THERE WILL BE A HAPPY HOUR WITH STUDENTS AND PRESENTERS AND CHAIRS AND NIH AND THE PROGRAM STAFF. SO I WILL TURN IT OVER TO DR. --I MUST THANK ALL THE NIH STAFF FOR THIS SPECIAL SYMPOSIUM AND SPECIAL THANKS TO ALL THE CHAIR. --THEY CAN COME FROM MANY DIFFERENT APPROACHES, THEY CAN COME FROM INDIVIDUALS OR THIS ANIMAL MODELS AND THE DIVERSITY OF ANIMAL MODELS. BUT WHAT'S REALLY INTERESTING IS THAT, WHILE IN THE OF GENETIC MODIFIERS THAT MAY AFFECT THE FUNCTION OF THE TARGET DISEASE PROTEIN, WE ARE QUICKLY LEARNING THAT THE LANDSCAPE OF GENETIC MODIFIERS IS EXTREMELY BROAD AND CAN START FROM ANYWHERE WHERE THE MODIFIER AFFECT THE ACTUAL DNA OR SPECIFIC TYPE OF MUTATION CAUSING DISEASE, NOT JUST THE PROTEIN FUNCTION. THAT'S SOMETHING WE LEARNED ABOUT FROM HUNTING TON, WE WILL LEARN ABOUT EVEN MORE TODAY ALL THE WAY TO AFFECTING THE FUNCTION OF THE PROTEIN. SO TODAY'S SESSION IS REALLY A FINE EXAMPLE OF THAT. TODAY'S SESSION IS A SESSION WHERE YOU WILL SEE WORK FROM ANIMAL MODELS BUILDING ON HUMAN DATA AND SOMETIMES INFORMING HUMAN DATA THAT SHOWS US THE VARIOUS, IF YOU WILL, REGIONS OR MOLECULES THAT CAN AFFECT A DISEASE CHAIN. WE WILL HEAR ABOUT DNA, THE AFFECTS ON THE DNA OF GENE, WE WILL ALSO HEAR ABOUT REGULATORY ELEMENTS OF THE GENE CONTRIBUTING TO MODIFICATIONS AND MOVING FROM DNA, WE WILL LEARN ABOUT VARIOUS ROLES OF RNA IN THIS PROCESS WHETHER IT'S EFFECTS OF RNA SPLICING, DIFFERENT RNA ISOFORMS IN A CELL, TO PERHAPS NONCODING RNA WHICH OPEN UP A WHOLE NEW AREA OF RESEARCH BY NOW AS WE LOOK THESE GENES AND REGULATORY TRANSCRIPTION, AND PROTEINS AND EVEN POST TRANSLATIONAL MODIFICATIONS NOT ONLY INVOLVING TYPICAL PROTEINS BUT SOMETIMES INVOLVING LIPIDS AND SOMETIMES INVOLVING THE MELUE OF THE CELL, ALL THAT TOGETHER WITH AN EYE TOWARDS SPECIFIC CELL CHANGES AND WE WILL HEAR ABOUT SOME OF THEM. I THINK WHAT YOU WILL TAKE AWAY FROM THIS SESSION TODAY, IS REALLY THE BEAUTIFUL INTERFACE BETWEEN HUMAN DATA AND THE MODEL ORGANISMS WHETHER IT'S A FRUIT FLY OR MOUSE OR RPGHT, I THINK WE WILL HEAR ABOUT THAT TODAY AND YOU WILL SEE THE POWER OF THIS CROSS SPECIES STUDY. I WOULD SAY TYPICALLY MODIFIERS THAT ARE PROTECTED AND CONSISTENT AMONG SPECIES ARE SOME OF THE MOST ROBUST MODIFIERS AND I HOPE TODAY WILL ILLUSTRATE THAT POINT. SO WITH THAT I WOULD LIKE TO WELCOME I WOULD LIKE TO WELCOME MY CO-CHAIR SUSAN ACKERMAN WHO WILL LEAD THE DISCUSSION AND Q&A FOR THE DISCUSSION BEFORE THE BREAK AND I WILL TAKE AFTER THE BREAK AND LOOK FORWARD TO A VERY EXCITING AND DYNAMIC DISCUSSION WITH ALL OF YOU TODAY. >> HI, EVERYBODY, I WANT TO WELCOME YOU TO THIS SESSION TODAY AND IT'S MY PLEASURE TO INTRODUCE OUR FIRST SPEAKER DR. VANESSA WHEELER WHO'S A ASSOCIATE PROFESSOR OF NEUROLOGY AT MASS GENERAL AND HARVARD MEDICAL SCHOOL AND VANESSA'S RESEARCH HAS FOCUSED ON HUNTINGTON'S DISEASE AND REALLY HER WORK HAS CONTRIBUTED TO OUR UNDERSTANDING GREATLY OF THE INTERPLAY BETWEEN DNA REPAIRED GENES AND CAG TRIPLET REPEAT LENGTH AND IN THIS DISEASE AND TODAY SHE'S GOING TO TELL US HOW SHE'S USING GENOME EDITING AND MICE TO TEST CANDIDATE HUNTINGTON MODIFIER GENES. WELCOME, VANESSA. >> THANK YOU. THANK YOU VERY MUCH VERY MUCH FOR THE INTRODUCTION AND THE INVITATION TO SPEAK. LET ME JUST SHARE MY SCREEN. CAN EVERYBODY SEE THAT? >> YES. >> SO IT'S A REAL PLEASURE TO BE AT THIS EXCITING WORKSHOP AND THIS IS MY DISCLOSURE SLIDE. THIS OF COURSE PROVIDED THE BASIS FOR THE GENOME WIDE ASSOCIATION STUDY THAT JIM TALKED ABOUT YESTERDAY AND THE REASON FOR THE DIFFERENT INHERITED REPEAT LENGTHS IS BECAUSE THE REPEAT IS UNSTABLE UPON GERMLINE TRANSMISSION. AND THE REPEAT IS ALSO UNSTABLE IN SOMATIC CELLS AND A NUMBER OF YEARS AGO WE SHOWED USING A SUBSET OF INDIVIDUALS FOR THESE AS EARLY OR LATE ONSET, THEY RESEARCH MATCHED FOR CAG REPEAT LEG INHERITED CAG REPEAT LENTHD THAT THE EXPANSIONS IN THE BRAIN WERE ASSOCIATED WITH EARLIER AGE OF DISEASE ONSET AND THAT SUGGESTED THAT AT LEAST SOME OF THE DIFFERENCES OF THE AGE OF ONSET OF INDIVIDUALS MIGHT BE EXPLAINED BY DIFFERENT PROPENSITIES OF THE REPEAT TO EXPAND AND OF COURSE AND OF COURSE THE GENOME WIDE ASSOCIATION AGENCY, I'M SHOWING THIS AGAIN HERE, THAT HIGHLIGHTED THE MANHATTAN PLOT HIGHLIGHTING THE GENES HIGHLIGHTING A NUMBER OF DNA REPAIR GENES THAT MODIFYS THE ONSET AND THE GENES IN THE MISMATCH REPAIR. AND WE AND OTHERS HAVE PREVIOUSLY SHOWN THE IMPORTANCE OF MANY OF THESE GENES IN REPEAT INSTABILITY IN MOUSE MODELS AND SO THIS OF COURSE WAS A VERY EXCITING FINDING THESE GENES BACK TO DISEASE ONSET IN HUMANS. AND AS JIM HIGHLIGHTED YESTERDAY, THE GWAS PROVIDES SUPPORT FOR THIS 2 STEP HD PATHOGENESIS, WHERE 1 WAS REPEAT EXPANSION, IT COULD BE MODIFIED BY THE REPEAT AND OTHER FACTORS, THERE'S ALSO CELL TYPE DEPENDENT AND TOXICITY COMPONENT AND BOTH OF THESE ARE NEEDED FOR PATHOGENESIS OF THE DISEASE. AND SO REALLY A FULL UNDERSTANDING OF HD PATHOGENESIS WILL ENTAIL SIGNIFY DETECTING BOTH OF THESE COMPONENTS AND BOTH OF THESE COMPONENTS PROVIDE TARGETS FOR THE THERAPIES. IN MY LAB HAS BEEN PARTICULARLY INTERESTED FOR MANY YEARS IN UNDERSTANDING THE SOMATIC EXPANSION COMPONENT AND THIS WILL PROVIDE PROXIMAL TARGETS FOR THERAPEUTIC INTERVENTION AND LIKELY APPLICABLE TO BE APPLICABLE ACROSS MANY EXPANSION DISEASES. SO, TO IDENTIFY GENETIC MODIFIERS OF REPEAT INSTABILITY, WE'VE PREVIOUSLY BEEN DOING KNOCK OUT EXPERIMENTS IN MICE IN EXTENSIVE BREEDING AND THIS DOESN'T LEND TO ANY SORT OF HIGHER THROUGH PUT APPROACH SO IF THEN TRYING TO DEVELOP A HIGHER THROUGH PUT APPROACH TO IDENTIFY GENETIC MODIFIERS AND I WOULD LIKE TO PARTICULARLY ACKNOWLEDGE MY COLLEAGUE AT THE PREVIOUS FORMER POST DOC IN MY LAB WHO SPEARHEADED THIS WORK. AND OUR APPROACHES TO USE AVA CARING EXPRESSING 9 RNAs OF INTEREST TO THE LIVERS OF THE Q111 HD KNOCK-IN MICE CONSISTENTLY EXPRESSING CAS9. AND THE LIVER SHOULD HIGH LEVELS OF REPEAT INSTABILITY AND REEL KEEPSAKES TYPHLY EN--STRATEGIESATIVE READ OUT OF INSTABILITY IN A SHORT PERIOD OF TIME. AND WE CAN QUANTIFY EXPANSION USING GENE PRACTICING MENTORSHIP SKILL BASE ANALYSIS OF THE REPEAT CONTAINING PC R PRODUCTS. WE WANTED TO UNDERSTAND THE SENSITIVITY OF THE FOCUSED ON DETECT EXPANSION MODIFYS, SO WE DID A MIXING EXPERIMENT OF STABLE AND UNSTABLE DNAs IN DIFFERENT PROPORTIONS AND USE THESE AS SUBSTRATES IN OUR CAG EXPANSION INDEX ASSAY AND INDICATED TO US IF WE WERE ABLE TO INJECT MICE IN AT 6 WEEKS OF AGE AND FIND IT STABLE, BY 12 WEEKS WE WOULD HAVE A SENSITIVITY TO DETECT AND EXPECT ON EXPANSION IF THE REPEAT WAS SUPPRESSED IN 50% OF THE CELLS AND ANY AS LOW AS 25% OF THE CELLS SO WE HAD A SENSITIVE ASSAY TO DETECT MODIFIERS OF INSTABILITY. AND USING THIS PARADIGM OF INJECTING AT 6 WEEKS AND ANALYZING AT 12 WEEKS WE WERE ABLE TO DO VIRAL TRANSDAKS. AND AND 60 TO 70% IN TD LIVER FOR A NUMBER OF THEEN GENES ON THE RIGHT RIGHT HERE. LET GENES ARE MOST LIKELY MODIFIED GENES--AND BASED ON BIOLOGICAL FUNCTIONS AS YOU CAN SEE HERE, A NUMBER OF THESE GENES ARE CAREERLY IMPLICATED IN REPEAT INSTABILITY BY THEIR FUNCTIONS AND OTHERS ARE LESS SO. SEVERAL OF THESE GENES HAVE ALREADY BEEN TESTED AND SHOWN TO BE IMPORTANT IN REPEAT INSTABILITY BASED ON THE EFFECT OF CONSTITUTIONAL NONMUTATIONS IN Q111 MICE AND IT'S WORTH POINTING OUT THAT THE MISMATCH REPAIR GENES IN FUNCTION AND DISCIPLINARY MERRIC COMPLEXES SHOWN HERE ON THE RIGHT CALLED MUTE S AND MUTE L DIMERS AND A NUMBER OF COMPONENTS, BUT IN ALL OF THE COMPONENTS OF THESE DIMERS HAVE EMERGED AS MODIFIER ONSET GENES. SO THIS SHOWS IT SHOWS EXPANSION WE SEE IN UNTREATED MICE AT 6, 12 AND 24 WEEKS WITH AN ANTIVECTOR TREATED MICE. AND THEN ANALYZING AT 12 WEEKS, TALKING TO MSH2, 3, MLH1, AND PS1 AND MLH 3 PRESSES EXPANSION DISTINGUISHABLE FROM THOSE IN 6 WEEK UNTREATED MICE. TARGETING RM2 B SLIGHTLY SUPPRESSIVE EXPANSIONS TARGETS MSH 6, PMS 2 OR FAN 1 PROMOTES EXPANSIONS AND WE DIDN'T FIND AN EFFECT OF TARGETING ANY OF THE OTHER GENES AND THE TABLE ON THE RIGHT AND DETECTING STRONG MODIFIER EFFECTS. SO THESE DATA WOULD INDICATE THAT SOME SUPPORT THE IDEA REALLY THAT SOME OF THE ONSET MODIFIER GENES LIKE THE ACTING PATIENTS AT THE LEVEL OF CAG INSTABILITYS WHERE OTHERS MIGHT BE MORE LIKELY TO ACT THE TOXICITY COMOPPOSITE BEHAVIORIAL PHENOTYPENT OF THE PATHOGENIC PROCESS, HOWEVER, IT IS IMPORTANT TO NOTE THAT REALLY THIS IS 1 WAY OF PROBING THESE GENES AND THE LACK OF AN EFFECTIVENESS IN THE SYSTEM DOES NOT NECESSARILY RULE OUT A ROLE IN CAG EXPANSION IN PATIENTS. I THINK LIG1 IS A GOOD EXAMPLE OF THIS, THE BIOLOGICAL FUNCTION OF LIG1 WOULD IMPLICATE THIS GENE IN REPEAT INSTABILITY AND THIS IS A SUBJECT OF FURTHER INVESTIGATION. AND CONVERSELY AN IMPACT ON EXPANSION DOES NOT NECESSARILY AT MODIFICATION SO THIS IS A FIRST PART SCREEN OF PUTTING THESE INTO DIFFERENT CATEGORIES. SO FAN 1 IS NOT A PROTEIN, SO ITS ROLE IS QUITE INTERESTING, IT'S BEEN FOUND TO INTERACT WITH MLH 1 AND NUMBER OF MISMATCHED REPAIR PROTEINS AND WE WERE INTERESTED TO UNDERSTAND MORE ABOUT THE ROLE OF FAMILIES 1 IN REPEAT INSTABILITY. AND SO ON TO DO THIS WE DID IN COLLABORATION WITH JACOB LUKE AND MCDONALD'S LAB, WE DID A LEVEL 1 ACROSS OF MLH1, AND FOUND 1 NULL MICE. THE FOUND OF THE FAN 1 NULL IS TO PROMOTE EXPANSION IN THE STRIATUM AND THE LIVER, HOWEVER IN THE ABSENCE, WE DON'T SEE THE EFFECT OF FAN 1 KNOCK OUT SO THIS TELLS US THAT IT'S A DESTABILIZING EFFECT OF FAN 1'S LOSS. THAT WE DISCOVERED ON THE CRSPR SYSTEM, SO THIS SHOWED THE ABILITY TO DO THIS WHERE CO INJECTIONS OF 2 AAVs TARGETING A DIFFERENT GENE DOES NOT RESULT IN THE PRECCABLE DIFFERENCE IN THE EDITING FREQUENCIESS BY THOSE TARGETING EACH OF THOSE GENES ALONE. SO WE'VE GONE ON TO TEST VARIOUS COMBINATIONS OF MODIFIER GENES IN THIS SYSTEM AND THIS SHOWS THE TEST OF THE GENETIC INTERACTIONS WE HAVE DONE SO THOSE ARE INDICATED BILET GENE SQUARES. AND HERE WE FOCUSED ON INTERACTIONS BETWEEN FAN 1 AND MISMATCHED REPAIR PATHWAY GENES AND INTERACTIONS BETWEEN THE MISMATCHED REPAIR PATHWAY GENES THAT DEFINE THE DIFFERENT MUTE S DIMERS AND THE DIFFERENT MUTL DIMERS SO THE SLIDE SHOWS TESTING THESE GENETIC INTERACTIONS. THIS PANEL ON THE LEFT SHOW SAYS A RESULT OF TARGETING EACH OF THOSE GENES. TAU SUPPRESSES EXPANSION SO IN COMBINATION, FIRST OF ALL WE SEE THE KNOCK OUT EXPERIMENT, SO THIS SHOWS WE'RE ABLE TO USE THIS SYSTEM TO TEST THESE GENETIC INTERACTIONS, YOU WILL ALSO SEE THAT THE AGENT OF FAN 1 KNOCK OUT TO PROMOTE EXPANSION WILL DEPEND UPON EACH OF THESE OTHER MISMATCHED REPAIR GENES HERE. SIMILARLY HERE, THE ABILITY OF MS 2 OR MSH 6 IS DEPENDENT ON THESE MISMATCHED REPAIR GENES. ON THIS 1 WE SEE WHAT HAPPENS WHEN WE SUPPRESS 2 INDIVIDUALLY AND KNOCK OUT EXPANSION BECAUSE WE DON'T SEE ANY NEW NOVEL PHENOTYPES THAT WE WERE WERE ABLE TO DISCERN BETWEEN THESE SUBUNIT GENES. AND HERE WE TESTED COMBINATIONS OF GENES THAT INDIVIDUALLY WHEN KNOCKED OUTLET PROMOTE EXPANSION AND THIS REVEALS QUITE INTERESTING GENETIC INTERACTION SO THE KNOCK OUT OF MSH 6 AND FAN 1 RESULTS IN A PHENOTYPE THAT IS BETWEEN THAT OF THE MSH 6 AND THE FAN 1 KNOCK OUT, THE KNOCK OUT OF PMS 2 AND FAN 1 RESULTS IN A PHENOTYPE OF INDISTINGUISHABLE OF THE FAN 1 KNOCK OUT ALONE AND THE KNOCK OUT OF PMS6 AND MH2 INDIVIDUALLY PROMOTED EXPANSION, RESULTS IN NO DIFFERENT EXPANSION FROM THE EMPTY VECTOR CONTROL. SO THIS REALLY INDICATES THAT WE HAVE SOME QUITE COMPLEX GENETIC INTERACTIONS AND SAYS THAT THESE GENES REALLY ACTED IN A COMMON PATHWAY TO TO MODIFY INSTABILITY AND I INDICATED THE GENETIC ENHANCERS ON THE LEFT AND THE GENETIC SUPPRESSORS ON THE RIGHT AND THE INTERACTIONS BETWEEN THEM, ALL HERE FOR IN THE CONTEXT OF THE CANNONICLE MISMATCH REPAIR DIMERS FOR THE INTERACTIONS BETWEEN THE COMPONENTS ARE INDICATED WITH LINES SO THERE'S QUITE A LOT OF COMPLEXITY GOING ON HERE AND THERE ARE A NUMBER OF CONSIDERATIONS AND IMPLICATIONS THAT WE NEED TO THINK ABOUT. ONE IS THAT BECAUSE MANY OF THESE MISMATCH REPAIR GENES ACT IN DISCIPLINARY MERRIC COMPLEXES, MULTIPLE DISCIPLINARY MERRIC COMPLEXES, THE KNOCK OUT MY HAVE EFFECTS OF AFFAIRS TEAM LEADERRERRING THE BALANCE OF THESE DIFFERENT SM-MUTE AND SML DIMERS. ANOTHER THING TO THINK ABOUT IS THAT THIS IS A VERY DIFFERENT PROCESS TO THE CANNONICLE MISMATCH REPAIR AGHT PATHWAY WHERE THEY MAY BE DIFFERENT AND THE DISEASE HAVE BEEN IN CANCER AND DNA REPLICATION AND HERE WE HAVE THE EFFECTS OF GENES AND QUITE DIFFERENT OF THAT PROCESS, SO THE IRCHT ACTIONS OF COMPLEXES THAT WE SEE IN THIS PROCESS MAY DIFFER FROM THOSE OF THE DEFINED BY THE CANNONICLE MISMATCH REPAIR, CANNONICLE REPAIR PATHWAYS AND WE SHOULDN'T BE CONSTRAINED BY OUR KNOWLEDGE OF THOSE PATHWAYS AND THE KEY EXAMPLE OF THIS IS FAN 1 AND ITS ROLE IN THIS MECHANISM. I ULTIMATELY COMBINING THIS WITH CHEMICAL DATA WILL BE WHAT WE NEED TO DISSECT THE MECHANISM. AND THE OTHER SMLICATION OF THIS DAT IS INTERACTIONS OF MODIFIERS GENES WE SEE IN THE MOUSE SUGGEST MODIFY INTERACTIONS BETWEEN MODIFIERS AND HUMANS WILL BE LIKELY. SO JUST TO HIGHLIGHT ON A FEW SUMMARY SLIDES, THE CRSPR EDITING IN THERE, THE SIZE HAS ALLOWED US TO RAPIDLY TEST AND IDENTIFY NOVEL INSTABILITY MODIFIERS INVIVO. IT'S A SYSTEM THAT PROVIDES THE FIRST PART OF THE SCREEN INDICATING POTENTIAL ROLES OF THE MODIFIER GENES THAT COME OUT OF HUMAN GENETIC STUDIES IN THE HUMAN EXPANSION PROCESS OF THE PATHOGENESIS BECAUSE WE HAVE KNOWLEDGE OF CONSTITUTIONAL KNOCK OUTS WE WERE ABLE TO BENCHMARK EFFECTS AGAINST KNOWN MOBILITY MODIFIERS REALLY ALLOWING US TO IDENTIFY NOVEL GENES AND THE STRENGTH OF THOSE NOVEL GENES AND IT TURNS OUT TO BE QUITE A POWERFUL FORM TO TEST GENETIC INTERACTIONS. HOWEVER, THIS IS FAR FROM THE WHOLE PICTURE AND OF COURSE GENE KNOCK OUTS DO NOT CAPTURE THE COMPLEXITY OF HUMAN MODIFY EROZANS. SO WE NEED TO DO MUCH MORE, SO GENETIC KNOCK OUTS FOR EXAMPLE, MAY NOTLY AND PROBABLY DON'T RECAPITULATE THE EFFECTS OF FUNCTIONAL MUTATIONS, FOR EXAMPLE, PROTEIN CODING SNPs THAT ALTER SPECIFIC FUNCTIONS THAT MAY NOT BE RECAPITULATED BY A KNOCK OUT AND OF COURSE REGULATORY SNPs THAT ALTER EXPRESSION OF THE TARGET GENE. AND MODIFIER GENES OR SNPs AND/OR SNPs, MAY HAVE TISSUE EFFECTS AND WE'RE NOT SPECIFICALLY CAPTURING BY LOOKING AT TISSUES IN THE LIVER, SO GOING FORWARD WE'RE DOING WORK TO REALLY. --THEN PATIENT BASED DATA SUPPORT EXPANSION IS THE FIRST COMPONENT OF THE HD PATHOGENIC PROCESS AND A COMPELLING PROXIMAL TARGET FOR THERAPEUTIC INTERVENTION, THE INVIVO CRISPR GENE EDITING PROVIDES A POWERFUL MEANS TO IDENTIFY MODIFIER OF CAG INABILITY AND THEIR GENETIC IRPT ACTIONS AND FURTHER FUNCTIONAL DISSECTION WILL BE NEEDED TO UNDERSTAND THE MECHANISM AND REALLY OPTIMAL THERAPEUTIC APPROACHES AS WELL. AND WITH THAT I WOULD LIKE TO ACKNOWLEDGE MANY PEOPLE, SO MY LABS OF PEOPLE WHO HAVE DONE THE WORK I TALKED ABOUT TODAY AS WELL AS CURRENT PEOPLE IN THE LAB AND PEOPLE IN THE LAB IN GENERAL AND MY COLLEAGUES AT THE CENTER FOR GENOMIC MEDICINE AND OTHER COLLABORATORS INVOLVED IN MANY STUDIES DONE IN THE LAB. THANK YOU. >> OKAY, THANK YOU VERY MUCH FOR THE GREAT TALK AND WE ALREADY HAVE QUESTIONS, POPPING IN, HERE, SO THIS IS GREAT. SO THE FIRST QUESTION IS FROM JOSE DOMINGO BARRE-PAEZ AND HE SAYS GOOD MORNING, I WAS WONDERING HOW MUCH THERAPEUTICAL POTENTIAL HAS TARGETED THE DNA REPAIR PATHWAY TO IMPROVE THE PATIENT'S CLINICAL OUTCOME. I AM PARTICULARLY CONCERNED ABOUT THE POSSIBLE UPTARGET EFFECTS OF SUCH APPROACHES. >> YEAH, SO, I THINK THERE ARE A NUMBER OF GOOD TARGETS. AND SO OBVIOUSLY 1 CONCERN IS THAT THESE GENES ARE INVOLVED IN DNA REPAIR AND THERE'S A POTENTIAL FOR FOR TUMORIGENESIS SO I THINK WE HAVE TO BE CAREFUL IN THINKING ABOUT WHAT THE TARGETS ARE. BUT A NUMBER OF THESE--A NUMBER OF THESE GENES ARE NOT PRIMARY DRIVERS OF CANCER FOR EXAMPLE, MSH3 IS A GOOD EXAMPLE AND I THINK WOULD BE A VERY GOOD CANDIDATE FOR THERAPEUTIC TARGETS SO I THINK THERE ARE TARGETS AND I THINK THE MORE WE CAN UNDERSTAND ABOUT THE SPECIFIC MECHANISM AS WELL, WHICH MAY BE DIFFERENT, THE MORE WE CAN THINK ABOUT HOW SPECIFICALLY TARGETING THIS PROCESS. SO 1 APPROACH WOULD BE TO REDUCE LEVELS OF THESE GENES AND THAT'S 1 THAT IS 1 APPROACH IF WE CAN REALLY UNDERSTAND GOING FORWARD HOW WE INTERPLAY AND REALLY WHAT THE MECHANISM IS, MAYBE THERE'S A WAY TO INTERVENE MORE SPECIFICALLY IN THIS PROCESS. WITHOUT AFFECTING GLOBALLY GENOME WIDE EFFECTS. >> OKAY, WONDERFUL. THE NEXT QUESTION IS FROM MIRIAM MEISLER, HI, MIRIAM, DO YOU THINK THERE THERE MAY BE PATIENTS WITH HD CLINICALLY DUE TO SOMATIC EXPANSION OF REPEATS IN THE NORMAL RANGE? >> YEAH, THAT'S A GOOD QUESTION. SO IN THE NORMAL RANGE, THE REPEAT IS QUITE STABLE. THERE IS SOME VARIATION SO WE'VE DONE SOME--IF YOU DO SINGLE MOLECULE TYPE ANALYSIS, CAN YOU SEE A VERY SMALL AMOUNT OF VARIATION AND IT GOES UP IN A REPEAT LENGTH DEPENDENT MANNER. I THINK WHERE THAT BECOMES VERY INTERESTING IS IN THE HIGH NORMAL RAINCHL AND THE INCOMPLETE BEN TRANT RANGE WHERE YOU COULD HAVE HIGH LEVELS OF SOMATIC EXPANSION IN THE INCOMPLETELY PENETRANT RANGE THAT COULD PUSH YOU TO CLINICAL ONSET. AND SO THAT'S SOMETHING THAT WOULD BE OBVIOUSLY REALLY INTERESTING TO THINK ABOUT. >> OKAY, JOES LYNN PEARL ASKS, GREAT PRESENTATION AND EXCITING DATA. IS YOUR LAB PLANNING ON EVALUATING SOME OF THESE TARGETS USING AN APPROACH BESIDES GENE KNOCK OUT? PERHAPS ACTIVATION OR REPRESSION OR CDNA OVEREXPRESSION LIKE DR. HEIMAN SPOKE ABOUT YESTERDAY? >> YEAH, FOR SURE. SO SOME OF THESE--SOME OF THE HUMAN MODIFIER ALLELES THAT WERE ASSOCIATED WITH ALTERED EXPRESSION AND SOME ARE ALTERED WITH AND SOCIALITY WIDE INCREASED EXPRESSION SO I THINK THIS PROVIDES AN OPPORTUNITY TO TAKE THIS INTO THE MOUSE AND NOT JUST KNOCK IT OUT. BUT TO MODIFY EXPRESSION. SO WE'RE WORKING ON DEVELOPING CRISPR ACTIVATION FOR EXAMPLE TO TRY AND UPREGULATE SOME OF THESE GENES IN THE MOUSE. >> OKAY, AND I'M GOING TO TAKE 1 FROM DAN [INDISCERNIBLE] ALONG THE SAME LINES HERE. >> NOT THAT WE KNOW ABOUT. >> OKAY, AND DOUGLAS SNYDER WANTS THE LAST SLIDE AGAIN. I'M ASSUMING THIS IS YOUR-- >> THE ACKNOWLEDGE SLIDE. >> YEAH. >> ONE SECOND. >> WE'RE TRYING TO TAKE THEM DOWN SO WE CAN SEE EVERYBODY, BUT I DON'T SEE IT. >> OKAY, HOLD ON A SECOND, SORRY. YOU WILL HAVE TO SHARE AGAIN. WELL IF WE CAN'T DID THIS DOUGLAS--OH, I'M NOT SURE. SUMMARY, MAYBE ACKNOWLEDGMENTS. I HOPE THIS HELPS HIM. AND OKAY, WE ARE--THANKS. WE ARE GOING TO MOVE ON NOW TO THE SECOND TALK AND I WOULD LIKE TO INTRODUCE TUULI LAPP A LAINEN WHO'S A CORE MEMBER OF THE NEW YORK GENOME CENTER AND ALSO AT KTH ROYAL INSTITUTE OF TECHNOLOGY AND SCILIFE LAB IN STOCKHOLM, AND TUULI HAS BEEN STUDYING REGULATORY VARIANTS AS POTENTIAL MODIFIERS OF CODING VARIANT PEN TRANCE, LARGELY IN GENE EXPRESSION BUT NOT SOLELY BUT TODAY TUULI WILL SHOW US THAT REGULATORY VARIANTS CAN MODIFY PENETRANTS OF CODING VARIANTS. WELCOME TUULI. >> THANKS SUSAN AND THANKS FOR HAVING ME HERE AND GIVING ME THE OPPORTUNITY TO PRECEPT SOME OF THE WORK THAT MY LAB HAS BEEN DOING IN THIS VERY INTERESTING AND VERY IMPORTANT TOPIC OF MODIFIERS. SO JUST A JUMP AHEAD OKAY, HERE WE GO. SO THIS IS A PROBLEM THAT'S BEEN DISCUSSED MANY TIMES OVER SO I WON'T SPEND TOO MUCH TIME DISCUSSING THE VERY PERSISTENT PROBLEM, OF INCOMPLETE PEN TRANCE AND THAT IS FOR PRECISION MEDICINE WHERE EVEN THIS VERY SAME GENE IS DISRUPTING VARIANT AND RESULT IN A VERY DIFFERENT PHENOTYPIC OUTCOMES AND HOW THIS--THE SORT OF MECHANISMS OF HOW THIS ACTUALLY HAPPENS IS VERY UNCLEAR AND THIS IS OF COURSE IT'S A PROBLEM IN--IN ACTUAL CLINICAL PRACTICE AND IT'S ALSO 1 OF THOSE KIND OF FUNDAMENTAL BIOLOGICAL PROBLEMS THAT IS LIKE A VERY, VERY CLASSICAL DILEMMA THAT PEOPLE HAVE BEEN WORKING ON FOR A VERY, VERY LONG TIME AND SO IN ORDER TO ADDRESS THIS QUESTION, MY LAB HAS BEEN SORT OF WORKING ON REGULATORY VARIATION OOSKTING GENE EXPRESSION AND I WILL TALK ABOUT THAT IN A SECOND. BEFORE I GET TO THAT, I WANT TO KIND OF JUST CLARIFY THE TERMINOLOGY HERE, SO I USE--I TALK CONSISTENTLY ABOUT PENETRANTS HERE AS COMBINED TERM FOR BOTH PENETRANTS AND EXPRESSIVITY BECAUSE FROM A BIOLOGICAL PERSPECTIVE, THE DIFFERENCE BETWEEN THE 2 AND RATHER ARBITRARY AND I HAVE LAB STUDIES GENE EXPRESSION A LOT SO THEN YOU TALK ABOUT EXPRESSIVITY IT GETS QUITE CONFUSING VERY EASILY SO I'M BASICALLY REFERRING TO BOTH OF THEM AS PENETRANTS. SO 1 OF THE POTENTIAL MECHANISMS FOR VARIABLE PENETRANT SYSTEM VARIATION IN GENE DOSAGE AND CONCAN ACTUALLY KIND OF THINK ABOUT THIS JUST STARTING FROM THE VERY CLASSIC MODELS OF DOMINANCE AND THINKING ABOUT HOW THAT ACTUALLY LINKS GENE DOZAGE TO DISEASE, RISK AND SEVERITY AND WE HAVE A SITUATION WHERE FOR EXAMPLE, LOSS OF HALF OF THE GENE DOZAGE BY CLOSES OF FUNCTION HETEROZYGOUSOUS AND PATIENT OR A VARIANT HAS VERY DIFFERENT DISEASE CONSEQUENCES IN RECESSIVE GENES IN DOMAIN NABT HAPLO INSUFFICIENT GENES AND IN PARTICULAR WE ARE INTERESTED IN SITUATIONS WHERE THERE IS SOME DEGREE OF CO-DOMINANCE AND DOSAGE GENES WHERE IT TAKES YOU LIKE SOMEWHERE IN BETWEEN THIS OF THIS RANGE. IT INCREASES YOUR DISEASE RISK BUT IT DOESN'T NECESSARILY LEAD TO A DISEASE. AND 1 CAN SORT OF EXTEND THIS THINKING BY CONSIDERING THE SORT OF PATTERN OW IN THE DOSE SENSITIVE GENE, EVEN RELATIVELY DOSAGE CHANGES THAT DOSAGE AND HETEROZYGOUSOUS INDIVIDUALS CAN HAVE, CAN ACTUALLY RELATE TO A RELATIVELY LARGE DIFFERENCE IN DOWN STREAM FUNCTION AND DISEASE. --AND YOU'RE ALREADY IN A PRECARIOUS STATE WHERE EVEN SMALL MODIFICATIONS MIGHT BE VARIABLE. SO THEN THE QUESTION IS WHAT WOULD BE GENE EXPRESSERS IN THESE HETEROGENEOUS ROW SWRIENOUS INDIVIDUALS. THIS INCLUDES ENVIRONMENTAL MODIFIERS AND IN HUMAN THIS IS QUITE DIFFICULT TO STUDY. AND THE SAME LARGELY APPLIES TO ALSO OTHER GENETIC VARIANTS ACROSS THE GENOME, SO BASICALLY TRANSREGULATORY VARIANTS THAT ARE TYPICALLY UNKNOWN FOR MOST GENES AND GENERALLY DIFFICULT TO MAP IN GENERAL WITH IMPORTANT EXPRESSIONS OF COURSE. BUT THE PROBLEM MY LAB SAW AS A TRACTABLE SPACE TO LOOK FOR GENETIC MODIFIERS OF CODING VARIANTS WAS LOOKING AT GENETIC VARIANTS THAT AFFECT GENE REGULATION IN STASEIS, SO THOSE ARE IN THE PROXIMAL REGULATORY REGION AND THEN ASSOCIATED GENE EXPRESSION, AND BEFORE JUMPING INTO THE SORT OF HUMAN DATA AND ANALYSIS THAT WE'VE BEEN DOING, I WILL MENTION THIS BASIC THINKING OF GENE DOSAGE AS THE MODIFIER PENETRANT AND THE CRUCIAL LINK TO GENE FUNCTION HAS BEEN DESCRIBED WITH VERY ELEGANT DATA FROM MODEL ORGANISMS AND ALSO IN SPECIFIC DISEASE EXAMPLES FOR EXAMPLE, GENE AND REGULATORY CONTRIBUTORS TO DISEASE RISK. SO WHEN WE ARE THINKING ABOUT GENETIC REGULATORY VARIANTS IN HUMANS, THERE IS 1 VERY IMPORTANT KEY SOURCE FOR THESE VARIANTS AND THIS IS THE GENOTYPE TISSUES OR GTEX STUDY THAT IS KNOWN ALREADY, BUT VERY RECENTLY ENDED NIH CONSORTIUM THAT OVER THE PAST 10 YEARS HAS BUILD A VERY LARGE CATALOGY OF REGULATORY VARIANTS AMONG TISSUES, IDENTIFYING 10S OF THOUSANDS OF GENES WITH THIS TISSUE THAT HAVE THIS REGULATORY VARIANCE, AFFECTING BOTH GENE EXPRESSION AND THEN ALSO SPLICING AND I WILL TALK TODAY ABOUT BOTH OF THEM. SO, THE BASIC MECHANISM THAT WE KIND OF STARTED TO STUDY WAS BASED ON THE THINKING THAT IF WE HAVE AN INDIVIDUAL THAT IS HETEROZYGOUSOUS FOR A CODING VARIANT IN SUCH A WAY THAT THE HEALTHY COPY OF THE GENE HAS A HIGHER EXPRESSING REGULATORY OOH LEGAL, THIS MIGHT DRIVE HIGHER EXPRESSION OF THE FUNCTIONAL ALLELE OF THE PROTEIN AND LEAD TO A BETTER PHENOTYPIC OUTCOME THAN THE OPPOSITE, UNFORTUNATE CASE WHERE THE INDIVIDUAL HAS LOW EXPRESSION LEVEL OF THE HEALTHY ALLELE AND THUS SORT OF AT HIGHER DISEASE RISK. SO THESE WORK THAT I'M DESCRIBING IN THE NEXT COUPLE OF SLIDES WERE PUBLISHED A COUPLE YEARS AGO AND I WILL END IN THE END DESCRIBE SOME UNPUBLISHED WORK. AND HERE THE IMPORTANT THING IS TO KIND OF LIKE ACKNOWLEDGE THAT WE'RE MOSTLY FOCUSING ON THE LOSS OF FUNCTION, SORT OF DOSAGE SENSITIVITY KIND OF HYPOTHESIS WHICH I THINK IS MOST COMMONLY GOING TO BE--GOING TO BE THE SITUATION WHERE IT'S REALLY KIND OF THE DOSAGE OF THE HEALTHY ALLELE, WHERE THE LOW DOSAGE OF THE FUNCTIONAL PROTEIN AND LEADS TO HIGHER PENETRANTS OF THE CODING VARIANT AND THERE MAY BE LITTLER MODELS AS WELL, BUT WE FOCUS LESS HERE ON THOSE. SO IN THIS PAPER, WE POSE THIS QUESTION FROM A COUPLE OF DIFFERENT ANGLES AND A COUPLE OF VERY ORTHOGONAL KIND OF DAT A. WE FIRST OF ALL GENERALIZED IN THE POPULATION IF THERE'S A PATTERN WHERE NATURAL SELECTION IS FAVORING HAPLO TYPE THESE OF INDIVIDUALS THAT THEN THERE'S CODING VARIANTS. WE ALSO ANALYZED IN A COUPLE DISEASE COHORTS IF PATIENTS HAVE A ENRICHMENT OF HIGH PENETRANT COMBINATIONS OF THE OPPOSITE PATTERN AND HERE WE'RE NOT ROLELY FOCUSING ON ANY SPECIFIC DISEASES WE ARE INTERESTED IN THIS QUESTION OF SORT OF LIKE A [INDISCERNIBLE] PERSPECTIVE AND THEN WE DID EXPERIMENTAL VALIDATION WITH CRISPR WHICH I WILL TALK ABOUTED IT. BUT JUST TO DISCUSS A COUPLE OF KEY FINDINGS BRIEFLY, WHEN WE HAVE DATA SETS LIKE GTEX WHERE WE HAVE DATA WE CAN DIRECTLY MEASURE WHAT IS THE RELATIVE EXPRESSION LEVEL IN A CODING VARIANT, HETEROZYGOUS OF THE 2 ALLELES BY LEAKINGA AT EXPRESSIONS AND LOOKING AT HOW THE ALLELES ARE PRESENTED IN RNA SEQUENCING, AND HERE WE CAN SEE VERY CLEARLY THAT RARE PATHOGENIC VARIANTS ARE UNDER REPRESENTED IN THE SORT OF HIGHER EXPRESS IN HAPLOTYPES SUGGESTING THERE IS AN ENRICHMENT BEING MORE OFTEN ACCUMULATED WITH THE LOWER EXPRESSING HAPLOTYPE OF THE HIGHER EXPRESSION COPY OF THE GENE. BUT SADLY IN MOST COHORTS WE DON'T HAVE BOTH RNA SEQUENCING AND DNA SEQUENCING DATA WHICH KIND OF LIKE FORCES US TO BE ABLE TO STUDY THESE IN DATA SETS WHERE WE HAVE GENETIC DATA ONLY. AND THE WAY WE DO AND BASICALLY ASKED THE QUESTION, ARE THOSE RARE CODING VARIANTS REPRESENTED RANDOMLY IN THE DIFFERENT EQTL, OR IS THE BIAS IN A PARTICULAR DIRECTION? AND ANALYZING THESE PATTERNS IN DISEASE COHORTS, WE WERE INTERESTED IN 2 DISEASE COHORTS, 1 AN AUTISM COHORTS, A SIM PLEOF THE COHORT AND THEN ALSO LOOKING AT BASICALLY GERMLINE CANCER BASED BY LOOKING AT TCGA AND TUMOR SUPPRESSOR GENES AND HERE WE ARE REALLY FOCUSING ON AUTISM IMPLICATED GENES AND TUMOR SUPPRESSIVE GENES AND ASKING DO THE PATIENTS IN THIS COHORT HAVE AN ENRICHMENT OF HIGH PEN TRANCE COMBINATIONS AND THERE'S A COUPLE OF THINGS GOING ON IN THE SLIDE BUT TO HIGHLIGHT A COUPLE OF SORT OF KEY MOST IMPORTANT DATA SORT OF NUGGETS HERE IS THAT WHAT DO WE LOOK AT THESE DISEASE RELEVANT GENES AND THE COMBINATIONS OF THE HAPLOTYPE REG IEWLGTZS AND CODING VARIANTS? WE SEE IN NOSE VARIANTS WHERE WE ARE LOOKING AT THESE HAPLOTYPE COMBINATIONS IN CASES AND VARIANTS THAT ARE CODING VARIANTS THAT ARE UNIQUE TO CASES, WE SEE THE VARIOUS BIAS TOWARDS INCREASED PENETRANTS AND INTERESTINGLY, WE LOOK AT VARIANTS THAT ARE SHARED BETWEEN CASES AND CONTROLS THAT COMBINE AND INCLUDE A COMBINATION OF DISEASE CAUSING NEUTRAL VARIANTS, WE SLEEP APNEA AND OBESITYY THAT IN CONTROLS THERE, IS ACTUALLY AN ENRICHMENT OF THE DECREASED PENETRANTS KIND OF A HAPLOTYPE COMBINATION PATTERN SUGGESTING THAT THESE ARE ABLE TO SORT OF EXIST IN THE CONTROLS AND THE CONTROLS ARE STILL CONTROLS WITHOUT THE DISEASE BECAUSE THE REGULATORY VARIANT REDUCES THE PENETRANTS OF THESE VARIANTS AND THERE ARE A PUNCH OF CONTROLS THAT WE HAVE BENIGN VARIANTS AND SHOWING THIS PATTERN IS REALLY UNIQUE TO THIS PATTERN OF DISEASE. BUT TO JUMP FORWARD ON UNPUSHLISHED DATA THAT A STUDENT IN MY LAB HAS BEEN WORKING ON, WE WANTED TO EXTEND THIS ANALYSIS TO THINKING ABOUT SPLICING QTLs, SO BASICALLY COMMON GENETIC ARRAIANTS THAT AFFECT SPLICING WITH THE IDEA THAT IS ACTUALLY QUITE OBVIOUS THAT IF YOU HAVE DELETERIOUS LOSS OF FUNCTION CODING THERE IN ANAXON THAT IS ON HAPLOTYPE THAT SKIPS THATAXON DUE TO A COMMON REGULATORY VARIANT THAT MODIFYS THE SPLICING OF THAT AXON THIS COULD LEAD TO BASICALLY MORE PRODUCTION OF FUNCTIONAL PROTEIN THAN IN THE SITUATION WHERE THAT LOSS OF FUNCTION VARIANT CONTAINING AXON IS FULLY ENCLUEDED IN THE TRANSCRIPT AND THEN KIND OF KILLS THAT TRANSCRIPT. AND I WANT TO HIGHLIGHT THAT HERE THERE ARE MANY DIFFERENT WAYS THAT SPLICING CAN MODIFY THE PENETRANTS AND THERE IS ANOTHER PATTERN WHERE WE'RE NOT ANALYZING HERE WHERE LOSS OF FUNCTION VARIANTS IN DIFFERENT AXONS THAT HAVE REALLY DIFFERENT USAGE LEVELS BUT LIKE SOME AXONS BEING INSTITUTED AND SOME EXONS LESS SO, THEY COULD HAVE DIFFERENT LEVELS BECAUSE OF GENERAL EXON USAGE IN THE POPULATION. BUT WAY WOO ARE INTEREST--WE ARE INTERESTED IN IS VARIANTS IN DIFFERENT INDIVIDUALS GENETICALLY DRIVEN DIFFERENCES IN EXON USAGE AND SIMILARLY WITH THE QTLs WE NEEDED A SET OF SPLICING QTLs SO WE HAD TO REDO SPLICING MAPPING WITH GTEX DATA FINDING OVER 5000 VARIANTS THAT ARE ASSOCIATED TO SPLICING AND LEVERAGING HERE TOP MED DATA THAT IS A REALLY, REALLY LARGE WHOLE GENOME SEQUENCE DATA EXPRESSING THEIR VARIANTS IN TOP MED INDIVIDUALS, OVER 60,000 INDIVIDUALS WITH EUROPEAN ANCESTRY AND THEN BASICALLY ANALYZES OVER 164,000 HAPLOTYPES OF SPLICING VARIANTS AND RARE VARIANTS AND ANALYZING THE PATTERNS OF ARE THESE RARE VARIANTS RANDOMLY DISTRIBUTED ON THESE QTL HAPLOTYPES OR IS THERE A BIAS? AND TOP MED IN A HORMONE OR LESS HEGHTY COHORT WE WOULD EXPECT TO SEE THAT IN DELETERIOUS VARIANTS AND DOSE SENSITIVE GENES THEY WOULD BE AN UNDERREPRESENTATION OF HIGH PENETRANTS COMBINATIONS. AND THIS IS INDEED WHAT WE SEE. SO HERE, THE SORT OF PUTATIVELY DELETERIOUS RARE VARIANTS ARE IN RED AND WE SEE THERE IS GENERALLY A SHIFT TOWARDS THEM, THEM BEING MORE OFTEN PRESENT IN THE TOP MED DATA IN THE DECREASED HAPLOTYPE COMBINATION WITH SPLICING QTLs, SUGGESTING THAT THEY MAY MODIFY THESE QTLs AND WE CAN SEE THE CONSEQUENCES IN THE HAPLOTYPE IN THE GENERAL POPULATION. WE HAVE SOME ONGOING ANALYSIS TO LOOK INTO THESE PATTERNS IN AUTISM AND--WHERE THEY HAVE DONE CAREFUL ANNOTATION OF REALLY NASTY DISEASE CAUSING VARIANTS THAT SHOULD NOT BE OBSERVED IN HEALTHY INDIVIDUALS BUT ARE STILL THERE AND SEEING IF SOME OF THESE PATTERNS COULD EXPLAIN THE PRESENCE OF AND THE LOW PEN TRANCE OF THOSE VARIANTS IN THE POPULATION. AND WITH THAT I WANT TO WRAP UP AND HIGHLIGHT THAT THE WORK WE ARE DOING HERE IS IN SOME WAYS COMBINING 2 DISTINCT AREAS OF HUMAN GENETICS WHERE THERE IS THE SORT OF RARE DISEASE FOCUS COMMUNITY THAT IS FOCUSED AND RARE CODING VARIANTS AND EXOME SEQUENCING AND MENDELIAN DISEASE, ET CETERA AND THEN THERE'S THE COMMON COMPLEX DISEASE COMMUNITY, THE GWAS COMMUNITY THAT IS FOCUSED ON ANALYSIS OF COMMON REGULATORY VARIANTS AND NONCODING SPACES. SO WE ARE PUTTING THOSE VARIANTS ITTH AND THINKING ABOUT THEIR EFFECTS ON GENE DOSAGE AND HOW IT SKTAS DISEASE RISK AND WE'RE ACTUALLY NOW EXPLORING THE IDEA OF SORT OF HOW TO LINK GENE DOSAGE TO FUNCTION AND DISEASE RISK IN A MORE SOPHISTICATED WAY ALSO USING NULL CRISPR DOSAGES AND TITRATION IN GENE SIMILAR MODELS AND THUS GET THE ADDITIONAL RESOLUTION INTO THE MATTER. SO SOME OF THE KIND OF PERSPECTIVES LIKE LOOKING AHEAD INTO THESE TYPES OF ANALYSIS, I KIND OF LIKE--ACTUALLY WE THOUGHT SOME OF THIS,AINALIS THAT I TALKED ABOUT HERE WOULD BE A BIT EASIER, LIKE WE'RE SORT OF LIKE IN SOME WAYS GOING AFTER THE LOWER HANGING FRUIT, LOOKING AT THESE REGULATORY VARIANTS COMPARED TO COMPLEX VECTORS THAT MAY AFFECT GENE EXPRESSION DOZAGE BUT THERE IS A LOT OF ANALYSIS ARE NOT EXACTLY SUPER STRAIGHT FORWARD, THERE'S A LOT OF BIASES THAT STEM FROM POPULATION STRUCTURE AND THERE'S BLUEPRINT ERRORS AND BEING ABLE TO NARROW THE DOSES DOWN TO DOSAGE-SENSITIVE GENES CAN BE DIFFICULT AT TIMES. IT'S GOOD TO HAVE LARGE POPULATION DATA, REGULATORY VARIANTS, TOP MED IS FANTASTIC WITH THE SIZE AND COMPREHENSIVENESS IN CAPTURING THE DIFFERENT TYPE OF GENETIC VARIANTS AND HAVING THE DISEASE SORT OF SPECIFIC COHORTS IS ALSO VERY IMPORTANT TO BE ABLE TO CAPTURE THOSE PATTERNS IN PATIENTS AND I THINK THERE IS A LOT OF INTERESTING BIOLOGY, THAT WE NEED TO EXPLORE AND HOW DOES GENE DOSAGE RELATE TO GENE FUNCTION, IF WE WANTED TO TAKE THESE THINGS FROM A LITTLE BIT MORE FROM A PROOF OF CONCEPT TO CLINICALLY MEANINGFUL ESTIMATES AND ACTUALLY SORT OF LIKE SHOW SPECIFIC MECHANISMS IN INDIVIDUAL GENES, INDIVIDUAL PATIENTS, INDIVIDUAL DISEASES, I THINK THERE'S A LOT OF WORK TO DO, JUST TO FIND THE BURDEN OF PROOF AND I THINK THIS WORKSHOP IS A WONDERFUL VENUE TO DO SOME OF THAT THINKING. BUT WITH THAT I WOULD LIKE TO ACKNOWLEDGE MY LAB ESPECIALLY [INDISCERNIBLE], WHO HAVE WORKED ON THIS, THIS WORK IN MY LAB AND FRANCOIS, OF AND ALSO NIGMS WHO FUNDED A GRANT RELATED TO SPECIFICALLY THAL WORK AND I WILL ALSO MENTION I AM RECRUITING POST DOCS TO BOTH OF MY NEW YORK LAB AND STOCKHOLM LAB, OR A COMBINATION OF THE 2 SO IF YOU ARE INTERESTED PLEASE REACH OUT. I AM HAPPY TO TAKE YOUR QUESTIONS. >> OKAY, WONDERFUL TALK AND WE ALREADY HAVE A COUPLE QUESTIONS HERE. THE FIRST IS FROM ROGER WHITTLE, HE SAYS HI, TUULI, GREAT TALK, HAVE YOU THOUGHT ABOUT MUTATIONS THAT CAUSE AN EXON TO BE EXPRESSED IN THE CELL OR TISSUE WHERE IT WOULD NOT NORMALLY BE PRESENT? THERE COULD BE A GAIN OF FUNCTION EFFECT, INTERACTIONS NOT INTENDED IN THAT SOUR TISSUE TYPE OR OTHER EFFECTS? >> YEAH, NO, THAT'S AN INTERESTING IDEA. WE VBT LOOKED INTO THAT. AND IT'S FOR SOMETHING LIKE WE'RE STILL IN THE PROCESS OF LIKE BUILDING THE CATALOGS OF WHAT ARE THE CELLS WHERE DIFFERENCE EXCELLENCE IS EXPRESSED CONSIDER POST CELLS DON'T CAPTURE FULL TRANSCRIPT SO KRAPTURING SPLICING PATTERN SYSTEM COMPLICATED IN SINGLE CELL DAT AWE BI THINK THAT'S A REALLY KIND OF YEAH, AN INTERESTING IDEA TO EXPLORE IN THE FUTURE. >> GREAT AND THE NEXT QUESTION FROM DAN DAWTERTY, FANTASTIC WORK TUULI, THE STUDIES YOU DESCRIBE FOCUS ON LOSS OF FUNCTION, CODING VARIANTS. IS IT POSSIBLE TO LOOK AT THE EFFECTS OF REGULATORY VARIANTS ON DISEASE-ASSOCIATED GAIN OF FUNCTION CODING VARIANTS? AND HAS ANYONE DONE THESE TYPE OF ANALYSIS? WOULD YOU EXPECT EFFECTS OPPOSITE TO THE LOSS OF FUNCTION VARIANTS. >> YES,--TYPICALLY WE DON'T HAVE A GREAT ANNOTATION OF WHAT IS LOSS OF FUNCTION, WHAT IS GAIN OF FUNCTION IN A DISEASE GENE SO 1 WOULD NEED TO LIKE REALLY GO WITH A MORE SORT OF GENE AND DISEASE SPECIFIC APPROACH PROBABLY RATHER THAN THIS GENOME WIDE PAN-DISEASE TYPE OF THINGS THAT WE TEND TO DO IN THE LAB. AND THEN ALSO I THINK IT'S GOING TO DEPEND VERY MUCH ON WHAT IS THE SPECIFIC MECHANISM OF THE GAIN OF FUNCTION OF LIKE HOW DOSE SENSITIVE IT IS, IT WILL BE LIKE A GAIN OF FUNCTION THAT PRODUCES LIKE A TOXIC PROTEIN THAT DOES TERRIBLE THINGS IN SMALL DOSES THAN MAYBE THE DOSAGE OF THE FUNCTION OF MUTATION, IT DOESN'T MATTER, MAYBE SOMETHING THAT'S MORE A COMPLEX OF MULTIPLE SUBUNITS AND THE IMBALANCE THERE IS THE PROBLEM AND MAYBE IT WOULDN'T MATTER, SO IT'S A QUESTION THAT IS SUPER INTERESTING TO EXPLORE IT. PROBABLY IN A CANCER CONTEXT, ABOUT SOMATIC MUTATIONS WOULD BE VERY INTERESTING, BUT WE--WE HAVEN'T GONE THERE. >> JAMES DOWELING SAYS GREAT TALK. CAN YOU WALK THROUGH SOME OF YOUR THOUGHTS OR IDEAS ABOUT VALIDATION, THIS SEEMS KEY? >> YEAH, IT IS ABSOLUTELY KEY. AND I THINK, SO THERE IS THE SORT OF LIKE--LET'S SAY VALIDATION IN THE SENSE OF LIKE, LET'S SAY IF YOU HAVE LARGE DEEP FAMILIES WITH MENDELIAN DISEASE WHERE 1 MIGHT BE ABLE TO FOLLOW LIKE SPECIFIC MUTATIONS AND SORT OF ESTABLISH POTENTIAL MODIFIERS IN THE SORT OF SLIGHTLY MORE CLASSICAL GENETIC ANALYSIS, AND WITH MORE SPECIFIC HYPOTHESIS, I THINK CRISPR TECHNOLOGY OFFERS EXCITING OPPORTUNITIES TO VALIDATE THINGS SO IN THE NATURE GENETICS PAPER, WE KIND OF LIKE BASICALLY INTRODUCED CODING VARIANT IN A CELL LINE IN A GENE THAT ALREADY--THAT ARE ALREADY HETEROZYGOUSOUS FOR THIS GENETIC VARIANT AND SHOWING THIS GENETIC IS VARIANT DEPENDING ON WHICH HAPPEN LOW TYPE IT ENDS ON AND WE'VE BEEN WANTING TO DO THAT IN A MORE SCALABLE MANNER BUT THAT IS TILL--CRISPR IS TRICKY SO NOW WE'VE SHIFTED TO CHARACTERIZE THE DOSAGE TO THE CURVE FOR DIFFERENT GENES, SPECIALLY FOR GENES THAT ARE DISEASE SPECIFIC IS I THINK 1 WAY TO KIND OF LIKE IF THAT IS THE CASE, IF YOU CAN SORT OF LIKE SHOW THE DOSAGE TO FUNCTION RELATIONSHIP, THEN YOU COULD POTENTIALLY JUST ACTUALLY KIND OF PUT YOUR DIFFERENT COMBINATIONS OF GENETIC VARIANTS ON ON TO THAT CURVE AND KIND OF APPROACH IT IN THAT WAY. BUT I THINK THERE IS ALSO GOING TO BE A SPECTRUM OF OTHER APPROACHES TO VALIDATE. THANKS. >> AND THE LAST QUESTION IS FROM SEMITA [INDISCERNIBLE], IS IT ALSO POSSIBLE TO INCLUDE IMPRINTED GENES IN THIS TYPE OF ANALYSIS? >> YEAH, THAT'S A REALLY INTERESTING QUESTION LIKE WE ALREADY WORKED ON IMPRINTING, IT WAS ONLY LIKE 6 YEARS AGO, WE PUBLISHED A BIG PAPER IN PRINT NOTHING GTEX DATA. I THINK IT'S ABSOLUTELY IN PRINCIPLE POSSIBLE WHICH JUST HAVEN'T DONE THERE. THERE ARE CERTAIN APPROACHES FOR EXAMPLE OF ALLELIC EXPRESSION THAT WOULDN'T NECESSARILY WORK ON IMPRINTED GENES BUT THE SORT OF ANALYSIS ON MAYBE LEAKY IMPRINTING AND HOW THAT MIGHT AFFECT PEN TRANCE, ET CETERA, IT'S GENETIC GOING FORWARD. YEAH, INTERESTING QUESTION, WE HAVEN'T LOOKED INTO IT. >> GREAT. THANK YOU VERY MUCH. WONDERFUL TALK. AND WE'RE GOING TO MOVE ON TO OUR LAST SPEAKER OF THE MORNING SESSION, OR THIS EARLY MORNING PART OF THE SESSION AND I'D LIKE TO INTRODUCE AARON GITLER WHO'S A PROFESSOR AT STANFORD UNIVERSITY, HOWEVER, I THINK HE'S GOING TO BE SPEAKING TO US FROM SHANGHAI THIS MORNING AND AARON HAS PERFORMED REALLY WONDERFUL STUDIES THAT BEGAN IN YEAST AND HAVE EVOLVED FROM THERE BUT TO IDENTIFY MODIFIERS OF ALS CAUSING MUTATIONS AND THE IDENTIFICATION OF THESE MODIFIERS AND THE STUDIES THAT AARON HAS DONE ON THESE MODIFIERS HAVE REALLY INFLUENCED THE ALS FIELD. SO, I THINK WE'RE GOING TO HEAR ABOUT A NEW MODIFIER THIS MORNING AND WELCOME AARON. >> THANK YOU VERY MUCH. I'M A RESEARCHER AT STANFORD AND AS DR. ACKERMAN MENTIONED, RIGHT NOW I'M LIVING IN SHANGHAI. I WOULD LIKE TO THANK THE NIH STAFF FOR ORGANIZING THIS WORKSHOP. I WILL START WITH MY DISCLOSURES HERE, AND I'D LIKE TO GET STARTED. SO THE FOCUS OF MY LABORATORY IS TO STUDY MECHANISMS OF HUMAN NEURODEGENERATIVE DISEASES AND THESE DISEASES ARE ALL INCREASING IN PREVALENCE AS OUR POPULATION AGES AND THEY HAVE VERY DIFFERENT CLINICAL PRESENTATIONS, SOME OF THEM AFFECT MEMORY WHERE OTHER 1S AFFECT MOVEMENT AND BUT DESPITE THOSE DIFFERENCES IN CLINICAL PRESENTATION, THERE'S A UNIFYING FEATURE AND ALL OF THESE NEURODEGENERATIVE DISEASES ARE ASSOCIATE WIDE MISFOLDING, THEY MISFOLD AND ACCUMULATE IN THE BRAIN OF PATIENTS WITH THESE DISORDERS AND THEY CAN ACCUMULATE OUTSIDE OF NEURONS OR INSIDE NEURONS, THEY CAN ACCUMULATE INTO THE NUCLEUS LIKE HUNTING TON'S DISEASE AND THE MISSION OF MY LABORATORY IS TO DEFINE THE CELLULAR PATHWAY AFFECTED FOR EACH OF THESE PROTEINS MISFOLDS AND AGGREGATE. IT'S ALSO AN INTERESTING BASIC CELL BIOLOGY QUESTION BECAUSE THESE PROTEINS IN THE GENES THAT ENCODES THEM ARE EXPRESSED THROUGHOUT THE BRAIN AND SOME ARE EVEN UBIQUITOUSLY EXPRESSED, YET THE DISEASES ARE ASSOCIATED WITH SELECTED VULNERABILITIES AND SO WE HYPOTHESIZE THAT THESE DISEASES THOUGH COMPLICATED ARE DEEPLY ROOT INDEED BASIC CELL BIOLOGY AND THESE PROTEINS ARE GOING TO INHIBIT THESE CELLULAR PATHWAYS THAT SOME NEURONS ARE MORE SUSCEPTIBLE TO DO THAN OTHERS. TODAY I'M JUST GOING TO BE TALKING ABOUT 1 NEURODEGENERATIVE DISEASE, A MY O TROAPIC LATERAL SCLEROSIS OR ALS, IT'S ASSOCIATE WIDE PROTEIN MISFOLDING. ABOUT 2% OF THESE ARE CAUSED BY SOD1 MUTATION IN THE GENE THIS, IS THE PROTEIN THAT AGGREGATES SOD 1, SO FOR THE OTHER CASES THAT DO NOT HAVE THE SOD 1 MUTATIONS, ALSO PROTEINS THAT AGGREGATE BUT NOT SOD 1, IT'S A DIFFERENT PROTEIN AND THIS PROSEEN IS AN RNA BINDING PROTEIN CALLED BDP43, THIS IS THE UNIVERSAL PATHOLOGY IN VIRTUALLY ALL ALS CASES AS WELL AS A LARGE NUMBER OF RELATED DISORDER CALL TD FRONTAL TEMPORAL DEMENTIA AND TDP-FREE AND LOCALIZED TO THE NUCLEUS AND THIS SLIDE ILLUSTRATES THE PROBLEM. THESE ARE 2 SECTIONS OF SPINAL CORD THEY STAINED WITH AN ANTIBODY OF TDP-43, YOU SEE THE LEFT NORMAL SITUATION, THIS IS THE MOTOR NEURON, YOU CAN SEE THE NUCLEUS, YOU CAN SEE IMAGINE, IT'S PROBABLY BINDING MANY DIFFERENT RNAs AND REGULATING ALTERNATIVE SPLICING AND OTHER ASPECTS OF R NA-METABOLISM. BUT IN THE DISEASE SITUATION ON THE RIGHT, CAN YOU SEE A NUMBER OF THINGS ARE GOING ON, FIRST YOU SEE THESE LARGE AGGREGATES OF THESE LARGE ROUND LOUIS BODIES LIKE AGGREGATES. SO THESE AGGREGATIVES MIGHT BE CAUSING SOME SORT OF TOXIC GAIN OF FUNCTION, PERHAPS INTERFERING WITH RNA AND RNA BINDING PROTEINS AND CAUSING TOXICITY IN THE CYTOPLASM. AT THE SAME TIME YOU SEE A LOSS OF TDP-FREE AND DEPLETED FROM THE NUCLEUS, SO NOW THIS IS NO LONGER ABLE TO IMPORT ALL THESE THAT IT'S NORMALLY REGULATING SO THERE CAN BE A LOSS OF FUNCTION AND A GAIN OF FUNCTION. OF COURSE THESE 2 ARE NOT MUTUALLY EXCLUSIVE BUT WE SHOULD CONSIDER THOSE ALONG THE WAY. SO WORK IN MY LAB LABORATORY WAS TO GAIN THIS, WE USED A SIMPLE MODEL SYSTEM, STARTING WITH A YEAST CELL, STARTER CELLS BUDDING AND EXPRESS TDP-43 HIGH LEVELS AND IN HUMANS AND SEE SEE THE ACCUMULATIONS OF THIS AND WE USE THIS VERY SIMPLE MODEL SYSTEM AS A BASIS TO FORM GENETIC MODIFIERS AND WE IDENTIFIED A YEAST GENE AND HAD A HUMAN MEMORY CLONE O LOG OF TAX 2 AND WE ARE LUCKY TO IDENTIFY PATIENTS IN THE GENE AND NOT AS LONG AS THE OTHERS BUT INCREASED 1 FOR ALS AND SUBSEQUENT WORK FOR MY LAB RAARE TOY DESCRIBED THESE INCREASE IN ATAXIN-2 AND THIS MOTIVATED US TO PROFORM EXPERIMENTS IN MOUSE WHERE WE USE MOUSE KNOCK OUTS TO SHOW WE CAN EXTEND SURVIVAL OF SIGNIFICANT EXTENSION IN SURVIVAL BY LOWERING 1 COPY OF ATAXIN,-2, AND THIS IS MOTIVATED BY PHARMACEUTICALS TOGETHER WITH BIOGEN TO JUST THE LAST MONTH OR 2 PHASE 1 CLINICAL TRIAL OF THE HUMAN TARGETING ATTACKS INTO ALS PATIENTS. BUT MEAN WHILE THERE'S ANOTHER ASPECT OF TDP-43 PATHOLOGY THAT I WANT TO TALK TO BUT NOW. --TALK TO YOU ABOUT NOW. WHEN TDP-43 WAS FIRST IDENTIFIED AS THE MAJOR PATHOLOGY, THERE WAS A RACE TO FIGURE OUT WHAT IT DOES, WHAT ARE THE RNAs THAT IT BINDS TOO? AND THE FIRST HINT OF THIS CAME FROM A STUDY FROM DONNA CLEVELAND AND GENE YTOGETHER WITH [INDISCERNIBLE] WHERE THEY USED GENOME WIDE APPROACHES TO IDENTIFY ALL OF THE TARGETS OF TDP-43 IN MOUSE BRAIN AND THEY FOUND THAT TDP-43 REGULATE THIS SPLICING OF GENES CONTAINING LONG INTRONS, AT THE THIS TIME IN HUMAN BRAIN AND NEURONS AND FOUND THE BINDING SITES THAT TPD-43 REGULATES. AND THEN IN A REALLY -- LANDMARK PAPER IN THE FIELD, PHIL WRONG'S LABORATORY FOUND THAT A MAJOR FUNCTION OF TPD-43 IS AS A REPRESSOR OF CRITIC AXONS. THESE ARE SEQUENCES THAT ARE IN INTRONS THAT SHOULD NOT BE INCLUDED INTO THE MATURE MESSENGER RNA. THERE ARE TPD-43 BINDING SITES RIGHT NEAR THESE. AND IT SITS THERE AND REPRESSES THESE CRITIC EXONS. WHEN YOU LOSE THE FUNCTION, THEY SNEAK INTO THE MATURE RNA AND CAUSE PROBLEMS FOR A NUMBER OF REASONS. DISRUPTION FOR SHIFTS. THEY CAN CAUSE DECAY. THEY CAN INTRODUCE NOVEL PEPTIDES. THESE ARE NONCONSIDERED. IT HAPPENS IN MOUSE AND HUMANS, BUT SPECIFIC GENES, AND CRITIC EXONS ARE DIFFERENT FROM MOUSE THAN HUMAN. LAST YEAR TWO LABS, DON AND LUTEAR TOOK WITH KEVIN DISCOVERED THAT A HUMAN TARGET OF TPD-43 NEURONS IS THE NEURONAL PROTEIN 2. THIS IS -- CRITIC SLICING START OF TPD-43. WE WERE JUST WONDERING COULD THERE BE MORE OF THESE OUT THERE? COULD BE FIND THEM? SO, THIS IS A NEW AND PUBLISHED COLLABORATIVE PROJECT I WOULD LIKE TO SCARE WITH YOU TODAY, I THINK IT REALLY ILLUSTRATES THE CONCEPT OF GENETIC MODIFIERS. THIS IS A COLLABORATION WITH MY LABORATORY AND LEN'S LABORATORY AT THE MAYO CLINIC. WE COLLABORATED WITH BILL AT UCSF AND WORK WITH A STARTUP COMING IN THE AREA THERAPEUTIC WHOSE SCIENTISTS, CREATIVITY AND SKILL ARE WITHOUT EQUAL. SO THE START OF THE SHOW IS -- A BRILLIANT GRADUATE STUDENT AT STANFORD. BY BRILLIANT, I MEAN, REALLY IN A LITERAL DEFINITION. AND THIS IS A PROJECT THAT STARTED A YEAR AND A HALF AGO DUE TO COVID. WE WERE ALL HOME, YOU COULDN'T BE IN THE LAB. WE COULD READ PAPERING ANALYZE DATA, WHAT ARE WE SUPPOSED TO DO ANYWAY, IF YOU THINK ABOUT IT? WHAT ROSA DID, SHE READ THIS PAPER FROM EDDIE'S LABORATORY IN WHICH HE TOOK FTD ALS STAINED THEM WITH AN ANTIBODY AND TPD-43 IN THE NUCLEUS AND WITHOUT AND DO RNA SEQUENCES WITH GREEN EXPRESSION CHANGES WITH LOSS OF TPD-43. HE FOUND VERY INTERESTING THINGS AND PUBLISHED THEM. BUT ROSA WAS NOT INTERESTED IN CHANGING THE GENE EXPRESSION. SHE WANTED TO SEE IF SHE COULD USE THAT VALUABLE DATASET AND ANALYZE IT IN A DIFFERENT WAY TO FIND NOVEL CRITIC SLICING TARGETS. SHE USED TWO PIPELINES TO REANALYZE THE DATA AND IDENTIFIED 65 CRYPTIC ALTERNATIVE SPLICING CHAINS THAT WERE TPD-43 LOSS DEPENDENT IN BRAINS OF PATIENTS. SHE FOUND 2 AND 3, THESE WERE VALIDATED TARGETS OF TPD-43. BUT THEN THE ONE THAT IMMEDIATELY CAUGHT HER EYE WAS 13A. FIRST, IT WAS ONE OF THE TOM ALTERNATIVE SLICING CHANGES THAT SHE FOUND, BUT IT CAUGHT HER EYE, BECAUSE THIS IS ONE OF THE TOP, IF YOU DON'T INCLUDE, THE TOP -- FOR ALS. IT'S, IT'S -- CONSTANTLY REPLICATED IN STUDIES FOR TERRIFIC ALS, AND FTD WITH ALS. IT IS THE TOP HIT. IT IS ALWAYS IN THE TABLES AND REVIEW ARTICLES THAT WE READ ABOUT THE GENETICS OF ALS, BUT NO ONE KNOWS HOW THE VARIANCE IN UNC13A CONTRIBUTE TO RISK. UNTIL NOW. THIS IS THE DATA THAT ROSA ANALYZED AT THE PAPER. THESE ARE RNA SEQUENCES ON THE TOP ARE ONE WITH TPD-43 IN THE NUCLEUS, AND THE BOTTOM TPD-43 NEGATIVE. YOU CAN SEE BETWEEN EXONS 20 AND 21, THIS IS A CRITIC EX-CON. SO IN ALL THREE OF THE SAMPLES THE CRYPTIC EXON IS IN THERE. SHE LOOKS AT THE TABLE OF THE PAPER YOU CAN SEE TPD-43 BINDING SITES IN THE INTRON HARBORING THIS CRYPTIC EXON. SO IT COULD HAVE BEEN THERE INDIRECTLY BECAUSE OF DEGENERATION. SHE FIRST DID A EXPERIMENT IN NEURO PLASTTOMA LINE. AND YOU CAN SEE WHEN YOU KNOCKDOWN TPD-43 YOU SEE A INCREASE IN CRYPTIC EXONS AND YOU SEE A, SEE A -- A DECREASE IN THE, THE CONICAL TRANSCRIPT AND ALSO IN TPD-43. SO KNOCKDOWN TPD-43 YOU SEE AN INCREASE IN THE CRYPTIC EXONS THAT YOU NEVER SEE WITH CELLS EXPRESSING TPD-43. THERE WAS ALSO A DISTRESS IN UNC13A PROTEIN IN THE CELLS. SO, UNC13A IS A CRITICAL SYNAPTIC PROTEIN AND BELONGS TO A FAMILY OF PROTEINS FOUND IN THE UNCORONATED PHENOTYPE OF THE MUTANT WITH NEUROTRANSMITTER RELEASED. UNC13A IN MOUSE IS REQUIRED FOR SYNAPTIC TRANSMISSION AND INVOLVED IN THE PRIMING STEP OF VESICLES. THIS IS FOR Y CELLS. AND ROSA LOOKED IN MOTONEURONS TO DIFFERENTIATE FROM HUMAN NEURONS. KNOCKDOWN TPD-43, YOU SEE AN INCREASE IN CRYPTIC EXONS AND UNC13A PROTEIN. THIS IS A CRITICAL NEUROPROTEIN THAT IS PRACTICALLY ELIMINATED BECAUSE OF INCREASE -- ASSOCIATED WITH AN INCREASE OF KIPTIC EXON WITH THE LOSS OF ENCK. AND ROSA TEXT WANTED TO EXTEND HER STUDIES FROM THE SEVEN RNA SEQUENCES SHE ANALYZED. SO SHE ANALYZED WITH HER COLLEAGUES AT THE MAYO CLINIC, EVERY SAMPLE AND SHE CAN ROBUSTLY DETECT THE CRYPTIC EXONS. THE MAYO CLINIC BRAIN BANK HAS WONDERFUL PATHOLOGICAL FEATURES, INCLUDING TPD-43, AND -- YOU CAN SEE THE STRONG CORRELATION WITH THE ABUNDANCE OF THE CRYPTIC EXONS AND ANOTHER GENOME CENTER RESOURCES AND RECAPITULATES THE CRYPTIC EXONS IN THE APPROPRIATE BRAIN REGIONS. BUT WHERE SHE DOESN'T FIND IT IS MORE IMPORTANT. SHE DOESN'T FIND IT IN ALS MUTATIONS, AND OF COURSE, NEVER IN THE NEGATIVE CONTROL. SO THE CRYPTIC EXONS ARE A SPECIFIC FACET RELATED TO TPD-43 PATHOLOGY. SO, RNA-SEQ IS ONE THING, BUT WE REALLY WANTED TO COME UP WITH A WAY OF VISUALIZING THE AXONS IN THE BRAINS OF PATIENTS WITH TPD-43 PATHOLOGY. SO WE DEVELOPED A FACE SCOPE PROBE, A SINGLE MOLECULE FISH PROBE, BUT HAD TO DO IT IN A DIFFERENT WAY THEN THE COMPANY WANTED US TO. WE HAD TO DESIGN ONE WITH THE CRYPTIC EXON CE AND THEN THE CONICAL EXON. HERE IS A STAIN OF AN FTD BRAIN WITH THE TPD-43 ANTIBODY AND THE PROBE. YOU CAN SEE TPD-43 IS IN THE NUCLEUS, NO CRYPTIC EXONS, BUT IN EVERY ONE OF THE NUCLEI THAT LACKS TPD-43, YOU CAN SEE BOOM, BOOM, BOOM, CRYPTIC EXONS IN EVERY CASE AND NEURON WE LOOK AT, AND NEVER IN THE SITUATION WITH TPD-43 IN THE NUCLEUS. YOU DON'T SEE THE CRYPTIC EXONS. WE ALSO HAVE A CONTROL PROBE. THIS IS ONE THAT IS EXON 20, EXON 21, YOU CAN SEE IT DETECTING THE ARE BUST HERE. WE HAVE AN INKLING THERE IS A DECREASE IN THE CONCHAL TRANSCRIPTION WHEN YOU HAVE TPD-43 PATHOLOGY. THEN WE WERE, REMEMBER I MENTIONED UNC13A IS THE TOP HIT FOR ALS? SO, WE WERE JUST SAYING, LET'S TAKE A LOOK AT THE LOCUST. AND THIS IS THE LOCUST ZOOM PLOT. AND THE TOP HIT IS RIGHT -- RIGHT IN THE CRYPTIC EXON ITSELF. SO, THE UNC13A IS AN ENORMOUS GENE, SO IT DIDN'T HAVE TO BE THAT WAY. BUT RIGHT THERE. AND THEN THE OTHER ONE, THE SECOND TOP ONE IS IN THE HARBORING THE CRYPTIC EXON, THIS SUGGESTED A HYPOTHESIS, THE SNIPS THAT INCREASE RISK FOR ALS DO SO BY PROMOTING CRYPTIC EXON SLICING IN THE FACE OF TPD-43 PATHOLOGY. SO, TO TEST THAT HYPOTHESIS, WE FIRST WENT BACK AND ROSA ANALYZED THE RNA-SEQ SAMPLES SHE HAD. FOR SOME OF THEM, SHE WAS LUCKY, INDIVIDUALS WERE HETEROZYGOUS. THEY ARE A RISK ALLELE AND REFERENCE ALLELE. AND FOR THOSE, THERE WERE AN IMBALANCE. AND THEN DID IT SYSTEMATICALLY WITH MAYO CLINIC DATA. AND YOU CAN SEE THE INDIVIDUALS WITH 1 OR 2 COPIES OF THE RISK ALLELE. AND THEN THIS IS, I THINK, QUITE REMARKABLE. I DON'T WANT TO OVER, I SHOULDN'T OVERINTERPRET IT, BUT I WILL JUST SAY, YOU CAN SEE THIS IS THE SURVIVAL DATA OF PATIENTS, FTD PATIENTS THAT HAVE ZERO RISK ALLELES, ONE RISK ALLELE, OR TWO RISK ALLELE. YOU SEE A DRAMATIC SURVIVAL HAVING THESE. SO I DON'T WANT TO OVERINTERPRET THIS, BUT TO ME, THIS SUGGESTS WE COME UP WITH SOME THERAPY THAT CAN CHANGE THE CRYPTIC SLICING FROM HERE-TO-HERE WILL HAVE A DRAMATIC THERAPEUTIC BENEFIT. BECAUSE THERE ARE PLENTY OF CRYPTIC EXONS IN TPD-43 IN THE BRAIN THERE MIGHT BE A BETTER RETENTION. IT IS VERY HARD IN HUMAN GENETICS, AS IT WAS JUST TAUGHT US TO GO FROM A VARYUMENT TO THE FUNCTIONING BECAUSE OF EQUILIBRIUM. SO WE THINK THIS SET RIGHT HERE IS HAVING AN EFFECT. BUT THERE ARE MANY ONES LINKED TO IT. SO WE STARTED TO TEASE THIS OUT USING MANY GREEN REPORTER ASSAYS. WE CAN CLONE PART OF THE GENE AWAY FROM THE ASSAY AND SEE THE EFFECM IN THIS WE HAVE THE CRITICAL EXON WITH OR WITHOUT, THE RISK HAPLOTYPE, WE CAN SEE IN THE REPORTER ESSAY, WE USE TPD-43, WE SEE SLICING DEFECTS, INCREASED CRYPTIC SLICING AND THE HAPLOTYPE AS A MORE PRONOUNCED EFFECT THEN THE INDIVIDUAL VARIANCE. SO I THINK THAT WE ARE IDENTIFIED YET A FEW FACET TO TPD-43 PATHOLOGY. THE ACCUMULATION OF UNC13A CRYPTIC EXONS. WE HAVE, WE HYPOTHESIS WE MIGHT BE ABLE TO FIND ADDITIONAL POISON EXON PROFILES, THAT'S A NAME WE CAME UP FOR THEM. THAT MIGHT REPRESENT DIFFERENT SUBSITES OF ALS OUR FTD. IT IS A FASCINATING ACTUALLY PICTURE OF THE DISEASES, YOU CAN HAVE TPD-43 PATHOLOGY AND GET FTD AND ALS OR BOTH. IT MIGHT BE INTERESTING THAT DIFFERENT COMBINATIONS OF THESE MAY ACTUALLY, DIFFERENT BRAIN REGIONS MAY CONTRIBUTE TO THAT WE ARE INTERESTED IN STUDYING THAT. OTHER GENETIC VARIATIONS THAT ACT AS MODIFIERS? IN THE FACE OF TPD-43 DYSFUNCTION? UNC13A IS THE FIRST ONE. COULD THERE BE MORE OUT THERE? SO THE RESOURCE THAT DOCTOR MENTIONED, THIS RESOURCE, IF YOU LOOK ON THERE YOU DON'T SEE THE CRYPTIC EXON, WE THINK IT IS A TRUE GENETIC MODIFIER. IT IS ONLY ACTIVE IN THE PRESENCE OF TPD-43 PATHOLOGY. WE THINK OF THESE AS AN ACHILLES' HEAL. SO ACHILLES' HEAL WAS A -- WAS A -- WAS A -- A -- GREEK MYTHOLOGY A WARRIOR WHO WAS RESISTANT TO BATTLE BECAUSE OF THE PROTECTION HE HAD, HIS FIELD WAS VULNERABLE. AND THAT IS STRUCK, HE, HE, HE DIED IN BATTLE. SO WE THINK OF THESE SNIPS AS WORKING THROUGHOUT THE GENOME, NOT CAUSING PROBLEMS ON THEIR OWN, BUT WITH TPD-43 PATHOLOGY IT KICKS OFF THE CASCADES. AND FINALLY, THERAPEUTIC STRATEGY IS TO BROCK THIS EXON. WE KNOW WHERE THE SPLICE ACCEPTOR SITE AND DESIGNED AL GOSE, AND THIS COULD BE THERAPEUTIC. WE DEPOSITED DESCRIBING THE RESULTS THE SAME DAY, AND ANOTHER TEAM DESCRIBED SIMILAR RESULTS USING A DIFFERENT APPROACH. SO PLEASE CHECK THESE OUT. THANK YOU. >> SUSAN: OKAY. WONDERFUL. WE'RE ALMOST OUT OF TIME. BUT I'M GOING TO TAKE THE QUESTION THEN. I'M WONDERING -- SO, SO THE STORY OF STAFF MAN 2, YOU KNOW, LEADS OFF WITH THIS, NOW YOU HAVE REALLY SHOWN THERE, IS I DON'T KNOW, 60-SOMETHING GENES THAT COULD BE MISREGULATED BY TPD-43 MISREGULATION. SO, HOW IS IT GOING TO BE POSSIBLE TO DO A THERAPEUTIC INTERVENTION DOWNSTREAM OF TPD-43? WHEN ALL OF THESE MIGHT BE CONTRIBUTING TO SOME EXTENT? >> AARON: YEAH, THAT'S A GREAT POINT. THEY MIGHT NOT ALL BE EQUAL. THERE MIGHT BE KEY ONES, WE THINK BASED ON THE HUMAN GENETICS. OF COURSE, UNC13A IS ONE OF THE KEY ONES. BUT, WE'RE, WE'RE INTERESTED IN FIGURING -- OF TARGETING TPD-43 SPLICING FUNCTION OR BOOSTING THE REPRESSOR FUNCTION IN GENERAL SO WE CAN HIT MANY OF THEM. OR MAYBE WE CAN FIND NODE HUB GENES THAT REGULATE MANY OTHER ONES OR COLLABORATORS OF TPD-43. >> SUSAN: YEAH. OKAY. WONDERFUL TALK. THANK YOU, AARON. WE ARE OUT OF TIME, BUT YOU SHOULD LOOK IN THE CHAT BOX. THERE'S A COUPLE MORE QUESTIONS FOR YOU. AND I'M SURE SOME WILL COME UP ON THE Q & A, TOO. SO WE'RE RUNNING A LITTLE BIT WAIT, DO YOU WANT TO GO AHEAD AND TAKE A FIVE-MINUTE BREAK? >> I THINK WE WILL SKIP THE BREAK AND GO STRAIGHT INTO THE VIRTUAL POSTER SESSION TO PICK UP A LITTLE BIT OF TIME. BUT THANKS FOR THE SPEAKERS SO FAR. I WILL TURN IT OVER TO CRYSTAL LEE TO MODERATE THE POSTER SESSION. >> HI, EVERYONE. SO MY NAME IS CRYSTAL LEE, I'M A HEALTH PROGRAM SPECIALIST AT NINDS. I WILL MODERATE THIS SESSION. WE ARE VERY PLEASED TO HIGHLIGHT THE RESEARCH FROM GRADUATE STUDENTS, POSTDOCS AND EARLY-STAGE INVESTIGATORS IN THESE VIRTUAL POSTER SESSIONS. SO, 5 TALKS REPRESENTED YESTERDAY. AND NINE WILL BE PRESENTED TODAY IN THIS SESSION. THESE 14 OUTSTANDING ABSTRACTS WERE CHOSEN BY THE COCHAIRS TO GIVE SHORT PRERECORDED TALKS THAT HIGHLIGHT GENETIC MODIFIERS RESEARCH. ALL OF THE SUBMITTED ABSTRACTS ARE AVAILABLE TO VIEW ON THE WEBSITE. THE PRESENTERS OF EACH TALK WILL BE AVAILABLE FOR A LIVE Q & A SESSION FOLLOWING THEIR PRERECORDED TALK. PLEASE RAISE YOUR HAND IN ZOOM, OR TYPE YOUR QUESTIONS IN THE Q & A BOX. I WILL CALL ON YOU OR READ YOUR QUESTION OUT LOUD. LET'S GET STARTED. FIRST UP WE HAVE DR. MICHAEL FROM THE UNIVERSITY OF CALIFORNIA, SAN DIEGO. GREG, PLEASE GO AHEAD AND PLAY THE FIRST RECORDING. >> HI. MY NAME IS MIKE. I'M A POSTDOC SUSAN ACKERMAN'S LAB AT THE UNIVERSITY OF CALIFORNIA. I'M GOING TO TALK ABOUT MY WORK THAT WE PUBLISHED IN SCIENCE IN 2014 WITH AN INTERSECTION BETWEEN GTPBP2 AND N-TR20 TO CAUSE SELL TYPE SPECIFIC NEURODEGENERATION IN MICE. THIS WAS DISCOVERED IN A FORWARD SCREEN THAT ISOLATED AN DECAY OF GTPBP2 AND CAUSES NEURODEGENERATION IN MOUSE GENERALLER CELLS BUT WITH A SPECIFIC MUTATION IN THE tRNA. THIS CAUSES IMPAIRED PROCESSES OF MATURE tRNA FORM AND SUBSEQUENT LOSS OF THE MAJORITY OF THE POOL FOR THE tRNA FAMILY CODING FOR THE UCU ANTICODON. THERE ARE FIVE tRNA IN THE CODON, THE OTHER FOUR ARE LISTED HERE. AND THIS GENERATION ACTUALLY OCCURS BECAUSE OF STALLED RIBOSOMES AT THE AGA CODONS DUE TO THE DEPLETED ANTICODON POOL. HERE IN THE INITIAL IDENTIFICATION OF THE MODIFIER NTR20 MODIFIER, THEY USE OTHER MOUSE INBRED LINES TO ACTUALLY MAP THIS MUTATION. HOWEVER, THEY NOTICED ABOUT 25% OF THE BACKGROUND MUTANT F2 ANIMALS ACTUALLY HAD ENHANCED NEURODEGENERATION THAT WAS SUGGESTIVE OF YET ANOTHER AND SECOND MODIFIER ON THE MOUSE INBRED LINE BACKGROUND. WE WERE ABLE TO ISOLATE THIS TO CHROMOSOME 3 IN A 25 MEGA BASE REGION THAT ACTUALLY CONTAINED 22. THE MATURE SEQUENCE OF NTR22 LINKED TO THE LINE AND THE LINE USED TO MAP THE NTR20 MUTATION WAS ACTUALLY THE SAME FOR THE MATURE NTR22 SEQUENCE, BUT I DID FIND TWO SNIPS AT THE NINTH BASE PAIR AND 12th BASE PAIR UPSTREAM OF THE NTR22 SEQUENCE THROUGH GENETIC BREEDING AND CRISPR KNOCKIN, THE SNIPS ARE FOR THE PHENOTYPE AS QUANTIFIED HERE WITH THE GRANULAR CELL LOSS. THROUGH CRISPR KNOCKIN, I WAS ALSO ABLE TO SEPARATE THESE TWO MUTATIONS AND SHOW THAT KATRINAULAR CELL LOSS STILL OCCURS WHEN YOU HAVE JUST ONE OF THE MUTATIONS. AND IT ALSO OCCURS, BUT IT IS NOT AS SEVERE WITH JUST THE 12-BASED PAIR MUTATION UPSTREAM AS WELL, WHICH IS AN INTERESTING RESULT. THEY CAN CONTRIBUTE UNIQUELY TO THIS PHENOTYPE. WHAT I DON'T HAVE TIME TO ACTUALLY SHOW, I HAVE ALSO BEEN ABLE TO SHOW THAT EACH SNIP DIFFERENTIALLY REGULATES 22 AND THE MECHANISM ACTION HERE IS THAT THE SHIPS UPSTREAM OF NTR22 ACTUALLY REGULATE RNA POLYMERASE 3 BINDING TO THE ACTUAL tRNA GENE. THIS IS INTERESTING BECAUSE ENERGETIC VARIANCE UPSTREAM OF tRNA CAN HAVE A MOLECULAR PHENOTYPE AND HAVE EMSTATIC INTERACTIONS WITH OTHER GENES. THIS IS IMPORTANT WITHIN HUMANS BECAUSE SEQUENCE DIVERSITY UPSTREAM OF HUMAN tRNA ACTUALLY VERY QUITE WIDELY, IT WAS PUBLISHED BACK IN 2018 IN THE PAPER THAT SHOWED THE UPSTREAM OF HUMAN tRNA CONTAINED MUTATION RATES AND HIGHLY POLYMORPHIC BECAUSE OF HIGHLY EXPRESSED tRNA NOW WITH MY WORK IN MICE, THEY CAN HAVE EPISTATIC INTERACTIONS WITH OTHER GREENS TO PRODUCE LOW MOLECULAR PHENOTYPES. I WILL TAKE ANY QUESTION. >> THANK, MICHAEL, ARE YOU HERE LIBE WITH US TODAY. >> YES, I AM. >> OKAY. GIVE IT A COUPLE OF SECONDS TO SEE IF ANY QUESTIONS POP UP FOR YOU IN THE Q & A BOX. I DON'T SEE QUESTIONS YET. HERE IT GOES. SO, THIS IS FROM -- JUNE. WONDERFUL WORK. HAS ANY SIGNALS BEEN MAPPED TO tRNA PROMOTERS? >> I THINK I HAVE ACTUALLY BEEN ASKED THIS BEFORE. I DON'T KNOW IF I, I SAID I WAS GOING TO GO AND LOOK AND I NEVER DID, BECAUSE I FORGOT ABOUT. BUT I DON'T KNOW SPECIFICALLY, BUT IT WOULD BE PROBABLY PRETTY EASY TO GO AND FIND THAT. I DON'T ACTUALLY KNOW THOUGH. >> OKAY. I DON'T SEE ANYMORE QUESTIONS IN THE CHAT BOX RIGHT NOW. BUT CONTINUE TO ASK YOUR QUESTIONS AND MICHAEL CAN RESPOND TO YOU THERE. BUT I HAVE A QUESTION FOR YOU. COULD YOU COMMENT ON IF ANY OF THESE tRNA MODIFYING MUTATIONS HAVE BEEN IDENTIFIED IN HUMAN DISEASES OR THE tRNA YOU'RE STUDYING IS EXPRESSED IN HUMANS. >> tRNA, LIKE SPECIFICALLY WITHIN THE MATURE SEQUENCE, ESPECIALLY NOT THE FLANKING REGIONS HAVE NOT. BUT USUALLY THE HUMAN ONES ARE IN THE SYNTHETASES. SO, DIRECTLY tRNA IS NO. BUT THERE IS, I THINK, MAYBE, HOPEFULLY, GOING TO BE SOME TYPE OF EMERGENCE OF THAT, ONE PART THAT IS KIND OF UNDERPLAYED IN THIS IS THE CELL TYPE SPECIFICITY ROLE IN tRNA, THAT EVERYBODY THINKS OF THEM AS UBIQUITOUS, YOU HAVE MUTATIONS FLANKING THEM OR WITH tRNA THEMSELVES, BUT IT IS VERY PREVALENT IN HUMANS. THAT IS ACTUALLY BE MORE TARGETED TO SPECIFIC CELL TYPES IN HUMANS AND ALSO AS, EXPLORING IN MICE. >> GREAT. THANK YOU, MICHAEL. LET'S MOVE ONTO THE NEXT PRESENTATION FROM MEGAN FROM THE BAYLOR COLLEGE OF MEDICINE. >> MY NAME IS MEGAN I'M AT BAYLOR COLLEGE OF MEDICINE AND RESEARCHING AGING AND NEURODEGENERATION IN THE LAB. SO, AGING IS THE SINGLE HIGHEST RISK FACTOR FOR THE DEVELOPMENT OF NEURODEGENERATION DISEASE. DESPITE THIS, NOT EVERYONE WHO REACHES OLD AGE GOES ON TO DEVELOP DEMENTIA. CERTAIN INDIVIDUALS THAT ARE GENETICALLY PREDISPOSED TO A LONG LIFE ARE ABLE TO DELAY OR AVOID NEURODEGENERATION ENTIRELY. THERE ARE A NUMBER OF LIFESTYLE AND GENETIC FACTORS THAT PLAY A ROLE IN IT NEURODEGENERATION DISEASE ONSET, BUT WE ALSO KNOW THE BRAIN IS NATURAL RESILIENT AGAINST DISEASE, INJURY, AND AGING. AND THE BRAIN USES TO COMBAT AGAINST THE OFFENSES. IN THE LAB, I'M INVESTIGATING WHICH GENETIC VARIANCE OF PATHWAYS PLAY A ROLE IN PROTECTING THE BRAIN FROM COMMINATIVE DECLINE AS A RESULT OF DISEASE AND IN A. I'M USING TWO UNIQUE METHODOLOGIES TO APPROACH THE QUESTION. FIRST, THERE IS A HYDROBOTIC INSTRUMENTS TO STOP DYSFUNCTION USING BEHAVIORAL READOUTS. IMPORTANTLY, TO ASSESS THE IMPACT OF AGE, DYSFUNCTION IS ASSESSED LONGITUDABLY. USING THIS DATA WE IDENTIFIED OVER 1200 DISEASE PHENOTYPES. AND TANDEM WITH OUR BEHAVIOR ESSAYS, I'M USING AGING AT A SYMPTOMS BIOLOGY LEVEL. THE GENETIC FACTORS CONTRIBUTING TO HUMAN LIFE SPAN ARE NOT FULLY CHARACTERIZED. BUT AGING PROCESS IS IN ORGANISM. AGING IS A WELL-PRESERVED PROCESS ACROSS EVOLUTION, WE CAN UTILIZE DATA TO EXPLORE THE PATHWAYS FOUND IN AGING AS WELL AS INFER WHICH GENES AND PATHWAYS ARE RELEVANT TO HUMAN BIOLOGY. UTILIZING LIFESPAN ASSAY DATA, WE HAVE CONSTRUCTED GENES IN YEAST, FLIES, SEA ITEMS, AND FLIES. WE ARE FORMED A CONSENSUS NETWORK. WHEN WE OVERLAPPED THIS CROSS-SPECIES NETWORK PICTURED HERE WITH EDGES REMOVED FOR VISIBILITY, WITH OUR LARGE NEURODEGENERATION SUPPRESSOR DATASET WE FOUND THAT 23% OF THESE NODES MATCH EITHER A GENE THAT IS KNOWN TO EXPRESS NEURODEGENERATION DISEASE PHENOTYPES. INVERSELY, WE FOUND A QUARTER OF THE GENES WITHIN OUR DATASET ARE INDICATED IN LIFESPAN EXCEPTION IN ONE MODEL ORGANISM. AND GENES IMPLEMENTED IN ALL FOUR MODEL ORGANISMS ACT AS SUPPRESSERS OF NEURODEGENERATION DISEASES AND OUR DISEASE MODEL. USING THE INDUCIBLE EXPRESS SYSTEM WE ARE ABLE TO MANIPULATE OF THE EXPRESSION OF THE GENES IN DROSOPHILA. USING THIS METHODOLOGY, WE FOUND THAT CERTAIN NETWORK GENES CAN ALSO EXTEND HEALTH SPAN OF THE CENTRAL NERVOUS SYSTEM. FOR EXAMPLE, THE INSULIN RECEPTOR GENE IS IMPLICATED IN LIFESPAN EXPRESSION IN ALL GREENS. AND IT ACTS AS NEURODEGENERATION DISEASE IN OUR DROSOPHILA. WHEN EXPRESSION OF THIS GENE IS REDUCED IN THE NEURONS OF HEALTHY AIMING FLIES THERE IS A REDUCTION IN THE AGE BEHAVIORAL DECLINE. WE ARE SCREENING OR GENES IN THIS MATTER. AND WE FOUND CNS HEALTH SPAN WITH AGE. THIS ANALYSIS WILL HELP US BETTER UNDERSTAND THE LINK BETWEEN LONG LIFESPAN AND PROTECTION AGAINST NEURODEGENERATION DISEASE AND HELP IN THE IDENTIFY OF NEW THERAPIES TO IMPROVE BRAIN SPANS WITH AGE. I WOULD LIKE TO THANK MY LAB AND THE TEAM THAT MAKES THIS EXPERIMENTATION POSSIBLE. THANK YOU VERY MUCH. >> THANKS, MEGAN. ARE YOU HERE? OH, I SEE YOU. SEE IF THERE ARE ANY QUESTIONS FOR YOU. SO WHILE WE'RE WAITING, SO I WAS VERY IMPRESSIVE HIGH THROUGH SCREENING TECHNIQUE IN FLIES. I WAS WONDERING IF YOU CAN TALK ABOUT THAT A LITTLE BIT MORE. WHAT SORT OF BEHAVIORAL READOUTS ARE BEING MAJORED. JUST CLIMBING ASSAYS? >> YEAH. WE PRIMARILY USED CLIMBING ASSAYS WE MEASURED THE TURNING EVENTS OF THE FLIES, AND TUMBLING EVENTS AND THE SPEED AND VERTICAL DISTANCE THAT THE FLIES CLIMB. LIKE WALKING BETWEEN YOUR HOUSE AND FAVORITE RESTAURANT, IF YOU'RE EXPERIENCING COGNITIVE DECLINE, YOU WILL NOT WALK AS QUICKLY AND EFFECTIVELY, YOU MIGHT TURN AROUND A LITTLE BIT MORE OFTEN. THAT IS THE SAME SORT OF BEHAVIOR WE'RE READING IN THE FLIES. WE'RE ABLE TO MEASURE THAT LONGITUDINALLY. WE ARE ABLE TO RECREATE THE SAME SORT OF AGE-RELATED DECLINE OVER TIME IN BOTH NEURODEGENERATION DISEASE MODELS, BUT ALSO IN HEALTHY AGING FLIES. >> VERY GOOD. SO HERE'S A QUESTION FOR YOU IN THE BOX. FROM KLEMENT. AND HE SAYS, WHAT ARE SOME OF THE FUNCTIONAL CATEGORIES IN THE MOST CONSERVED MODIFIERS? AND ARE THERE CATEGORIES UNIQUE TO EACH SPECIES? >> YEAH. SO SOME OF THE CATEGORIES THAT WE'RE SEEING IN THE WELL-CONSERVED UNITS WITHIN THE NETWORK IS A LOT TO DO WITH LIKE MTOR PATHWAYS, AND INSULIN PATHWAYS AND THEN SOME OF THE BIOLOGY FOR SOME OF THOSE MORE SPECIFIC TO A SPECIFIC ORGANISM, STRANGELY, YOU ACTUALLY STILL GET SOME OF THOSE MORE CONSERVED PATHWAYS. SO LIKE I SAID, INSULIN AND MTOR. HOWEVER, OVERWHELMINGLY, EVEN THOUGH THOSE LOOK LIKE THEY'RE SPECIFIC TO A SPECIFIC MODEL ORGANISM, OVERWHELMINGLY IT IS JUST BECAUSE WE DON'T HAVE DATA FOR IT IN ANOTHER MODEL ORGANISM. THEY ARE JUST NOT BEEN TESTED YET. I WOULD IMAGINE MANY OF THOSE SPECIES-SPECIFIC MODULES YOU SAW WITHIN THE NETWORK, ARE ACTUALLY NOT TRULY SPECIES-SPECIFIC. ONCE YOU BRANCH OUT AND GET MORE DATA IN OR MODELS THEY WOULD START TO MERGE WITH THE OTHER MODULES WITHIN THE NETWORK. >> GREAT. THANKS VERY MUCH, MEGAN. >> THANK YOU. >> OUR NEXT TALK IS FROM DR. JONATHAN, FROM VANDERBILT UNIVERSITY MEDICAL CENTER. >> MY NAME IS JONATHAN MERIT. I AM A POSTDOC IN DR. JEFFREY'S LAB AT THE VANDERBILT UNIVERSITY MEDICAL CENTER. I WILL BE TALKING ABOUT OUR EFFORTS TO IDENTIFY GENETIC MODIFIERS OF RETT SYNDROME. THE MAJORITY OF CASES ARE CAUSED IN 2, AND THE POPULATION LEVEL OF ASSOCIATED WITH DIFFERENTIAL CLINICAL SEVERITY. ON THE RIGHT, THEY SHOW A GENERAL MORE MILD CLINICAL PRESENTATION THEN WITH LARGE DELETIONS. HOWEVER, THERE ARE OUTLIERS TO THIS GENOTYPE AND PHENOTYPE RELATIONSHIPS. THIS SUGGESTS THERE ARE ADDITIONAL FACTORS OF THE DISORDER. ULTIMATELY WE ARE INTERESTED THE MODIFIERS FOR PREDICTING CLINICAL OUTCOMES AND IDEALLY TO REVEAL NOVEL TARGETS OF THERAPEUTIC DEVELOPMENT. CURRENTLY WE ARE SURGING FOR CANDIDATE MODIFIERS AND SEEKING INDIVIDUALS WITH MILD OR CAREER CLINICAL PHENOTYPE. AND GREAT UTILITY IN IDENTIFYING MODIFIERS WITH OTHER DISORDERS, MAINLY MICHAEL'S GROUP WORK WITH INDIVIDUALS WITH CYSTIC FIBROSIS. THE IDEA HERE WITH THE EXTREMES OF THE CLINICAL POPULATION, YOU CAN OBTAIN SUFFICIENT POWER TO DETECT CAUSAL GENES FROM A LIPID SAMPLE SIZE. TO IDENTIFY INDIVIDUALS FITTING THE STUDY DESIGN, WE LOOKED AT DISTRIBUTIONS FOR COLLEGE MUTATION TYPES AND OVER A THOUSAND GIRLS AND WOMAN WITH RETT SYNDROME. WE SELECTED CANDIDATES FROM THOSE IN THE 20% TAILS OF THESE DISTRIBUTIONS WHO HAVE RANDOM X CHROMOSOME AND ACTIVATION STATUS TO MITIGATE EXPRESSION ATTACKS ON CLINICAL SEVERITY. AT THIS TIME WE HAVE COLLECTED AND IDENTIFIED 55 INDIVIDUALS. OVER THE PAST SUMMER WE STARTED WHOLE GOO GNOME SEQUENCING OF 112 INDIVIDUALS. I HAVE A BREAK DOWN OF OUR STUDY BELOW TO HAVE A BALANCED REPRESENTATION BETWEEN THE DIFFERENT SEVERITY CLASSES ACROSS MUTATIONS. THE BULK OF ANALYSIS AT THIS POINT IS FOCUSED ON THE XM SEQUENCE DATASET. THIS MIGHT BE EXPECTED GIVEN OUR SMALL SAMPLE SIZE, WE DIDN'T FIND ANY SINGLE VARIOUS WITH RETT SYNDROME, SO WE RESTRICTED OUR ANALYSIS TO THOSE DAMAGING TO PROTEIN FUNCTION AND COLLAPSE THESE WITHIN THE GENE LEVEL TO IDENTIFY GENES THAT SHOW ENRIPMENT OF DAMAGING VARIANCE IN EITHER FINTYPIC GROUPING. FROM THE TESTS WE HAVE THREE CANDIDATES THAT SHOWED ASSOCIATION WITH INCREASED DISEASE SEVERITY. AND CASTING A BROADER NET, WE EXPLORED A PATHWAY ANALYSIS AND NETWORK PROPAGATION TO COMBINE VARIANCE ACROSS RELATED AND INTERACTING GENES. WE HAVE REVEALED A POSSIBLE ASSOCIATION OF DAMAGING VARIANCE IN THE BIOSYNTHESIS PATHWAY WITH RETT SYNDROME SEVERITY. WE ARE EXCITED TO SEE IF THESE CANDIDATES REMAIN VALID FOLLOWING ANALYSIS OF THE WHOLE GENOME SEQUENCING DATA. ASIDE FROM THE ANALYSIS, THE NEXT MAJOR STEM WILL BE FOCUSED ON INTRODUCING THE CANDIDATE MODIFIERS AND ASSESSING THE IMPACT ON PHENOTYPES WITH DYSFUNCTION. ADDITIONALLY, WE WILL INTERROGATE THE ASSOCIATION WITH THE SPECIFIC FOCUS ON CANDIDATES REGULATORY ELEMENTS IN PREDICTED ENHANCER REGIONS. WITH THAT, I WOULD LIKE TO THANK THE LAB FOR THEIR CONTINUED HELP WITH THEIR PROJECT. AND FUNDING THROUGH THE RETT SYNDROME RESEARCH TRUST AND INTERNATIONAL RETT SYNDROME FOUNDATION FOR THE EXCELLENT SEQUENCING AND WHOLE GENOME SEQUENCING RESPECTTLELY. AND MY OWN FUNDING THROUGH THE VANDERBILT PROGRAM AND GENOMICS. >> THANKS, JONATHAN. SO WE HAVE A QUESTION FOR YOU. >> YES. >> THIS IS FROM JINN. AND HIS QUESTION IS ANY OVERLAP BETWEEN THE CANDIDATES AND THE GENES PATHWAYS IDENTIFIED BY MONICA IN MICE? >> SO WE DON'T IS SEEING ANY SIGNIFICANT VARIANCE, BUT ONE THING THAT IS INTERESTING THE BIOSYNTHESIS PATHWAY, THERE IS ENRICHING DAMAGING VARIANCE THAT IS UPSTREAM IN THE CHOLESTEROL BIOSYNTHESIS. SO IT IS EXCITING THAT WE'RE SEEING SOME CONVERGENCE BETWEEN THESE. WE STILL HAVE TO LOOK AT THE WHOLE GENOME DATA AND SEES IF IT IS HOLDS UP. WE GOT THE DATA BACK AT THE BEGINNING OF SEPTEMBER AND NOT HAVE BEEN ABLE TO HASH IT OUT YET. >> GREAT. SO ADDITIONAL QUESTION. THIS IS FROM -- IT SAYS VERY NICE TALK. ANY FUNCTIONAL RELATIONSHIP BETWEEN THE THREE CANDIDATES? >> SO, ONE OF THEM IS INVOLVE IN THE BIOSYNTHESIS PATHWAY. THE OTHER TWO ARE ACTUALLY LARGELY UNCHARACTERIZED GENES. THIS IS SOMETHING COMMON IN THESE SORTS OF STUDIES. YOU PULL OUT AN EXCITING CANDIDATE, BUT THERE IS NO PREDICATIVE FUNCTION FOR IT AT THIS TIME. >> SO I WILL SEE THREE MORE QUESTIONS. I WILL TRY TO LET YOU ANSWER THEM. >> OKAY. >> ONE IS FROM -- AND HE SAYS, COOL PROJECT. IT SOUNDS LIKE YOU STRATIFIED YOUR ANALYSIS BY LOOKING AT PATIENTS WITH TRUNCATING VARIANCE. IS THERE A WAY TO ADJUST FOR PREDICTED SEVERITY SO YOU CAN INCLUDE MORE OF YOUR PATIENTS? >> YEAH. IF WE COULD GO BACK TO THAT SLIDE THERE, RECURRENT MUTATIONS THAT ARE SEEN IN OVER 70% OF INDIVIDUALS. FOUR OF THOSE ARE TRUNCATING MUTATIONS. SO WE DO HAVE REPRESENTATION IN OTHER GENETIC LESIONS. ONE THING THAT WE HAVE, WE INTRODUCED A COVARIANT IN THE ANALYSIS WE'RE DOING AT THIS TIME, WHETHER IT IS PREDICTED TO BE A TOTAL LOSS OF FUNCTION TO THE PROTEIN OR ANOTHER MECHANISM OF DAMAGE. >> THIS QUESTION IS FROM -- MAY WE KNOW MORE ABOUT THE TOP THREE CANDIDATE GENES? >> SO, ONE OF THEM IS INVOLVED IN BIOSYNTHESIS, AGAIN THE OTHER TWO ARE LARGELY UNCHARACTERIZED. AND AT THIS TIME WE'RE NOT REALLY COMFORTABLE SHARING WHAT THEY ARE SINCE WE DON'T HAVE THE GENOME DATA AND WE HAVE THOUGHT DETERMINED WHETHER OR NOT THEY WILL HOLD UP WITH THE HIGHER SAMPLE NUMBERS. >> OKAY. SO LAST QUESTION FOR YOU. THIS IS FROM REBECCA. AND SHE SAYS, COULD YOU ELABORATE ON HOW YOU CONTROLLED FOR SCREWED X AND ACTIVATION IN YOUR POPULATION? >> SO THERE'S TWO WAYS OF DOING IT. ALL OF THESE SAMPLES AT THE TIME OF DNA COLLECTION WE SEND A SAMPLE OF THE DNA FOR X CHROMOSOME ANALYSIS WHICH INVOLVES LOOKING AT THE ANTIGEN RECEPTOR METHLOLOGY STATUS IN THE REPEATS YOU'RE ABLE TO SEE WHETHER THERE IS A BIAS. NONE OF THIS ASSAY IS BASED ON IF YOU HAVE 80% ONE WAY OR THE OTHER OR LESS THAT IS CONSIDERED RANDOM. SO WE ALSO HAVE INTRODUCED A COVARIANCE LOOKING AT THE REPEAT LENGTH AND SEE THE RELEVANT SKEWING. AGAIN THESE SAMPLES ARE COLLECTED FROM BLOOD AND IT SHOULD TRANSLATE TO THE BRAIN FROM PREVIOUS WORK. BUT WE DO HAVE METHODS TO CONTROL FOR IT. >> THANK YOU, JONATHAN. WE WILL MOVE ONTO OUR NEXT TALK. THIS IS FROM CAROLINE FROM UNIVERSITY OF MINNESOTA. >> HI, ALL, I'M A GRADUATE STUDENT AT THE UNIVERSITY OF MINNESOTA, WELCOME TO MY TALK. A ROLE -- SHERIFF MINGLING IN HOMEOSTASIS. THIS LITTLE BOY HE HAS CONGENITAL HYDRO CEPHALOUS, THIS IS DUE TO THE PRODUCTION, FLOW, OR ABSORPTION OF CEREBRAL SPINAL FLUID. THIS CAN LEAD TO DEATH. THE ULTRASOUND IMAGE HERE SHOWS A FETUS WITH HYDROCEPHALUS. THERE IS NO CLEAR, AND TREATMENTS ARE REALLY LIMITED IN LARGE PART DUE TO OUR UNDERSTANDING ABOUT THE UNDERLYING PHYSIOLOGICAL MECHANISMS. WE KNOW HYDROCEPHALUS CAN ARISE DUE TO ENVIRONMENTAL OR GENETIC CAUSES. AND THE MOST COMMONLY MUTATED SINGLE GENE IS THE MOLECULE COLLETED L1. L1 FUNCTIONS IN THE NORMAL DEVELOPMENT AND MAINTENANCE WITH EXON GUIDANCE AND SYNAPSE DEVELOPMENT. AND THE LOSS OR IMPAIRED FUNCTION OF L1 CAUSES L1 SYNDROME. A GROUP OF CONDITIONS THAT CAUSES HYDROCEPHALUS. THERE IS INCOMPLETE AND STUDIES IN MICE HAVE SHOWN THIS. IN THESE SLICES OF MICE BRAINS HERE, ALL OF THE MICE HAVE THE SAME L1 MUTATION, BUT IN DIFFERENT GENETIC BACKGROUNDS, THAT EFFECTS THE SEVERITY OF THEIR PHENOTYPE, IT MAY INFLUENCE THE SPECTRUM OF PHENOTYPES WE SEE. OUR OBJECTIVE IS TO UNCOVER THE GENETIC INTERACTIONS THAT ARE L1 HEIGHT ROCEPHALOUS. WE RECENTLY DISCOVERED THAT L1 INTERACTS WITH THE SIGNALING PATHWAY. AND ANIMALS WITH LOSS OF L1. WE HAVE DEFECTS. SO, ANIMAL LOOKS TRANSPARENT, CLEAR, AND EXPLODED FROM ITS CORE. AND THE FIGURE YOU CAN SEE SEVERAL MORE IMAGES. THESE LARGE VACUOLES AND THE PROCEEDING PORE THERE. SO, IN THE QUANTITATIVE DATA ON THE RIGHT, YOU CAN SEE OVER TIME. AND THE BLUE AND THE ORANGE LINE ARE SHOWING THE DOUBLE MUTANTS WHICH ARE MUCH MORE LIKELY TO DIE. THE DARK BLUE LINE HERE IS SHOWING SUPPRESS KNOCKING OUT KSR1 WHICH IS A SCAFFOLD PROTEIN WITH ERK PROTEIN. SO WHAT IS CAUSING THE ANIMALS TO APPEAR CLEAR, SWELL UP AND DIE. THE CLEAR PHENOTYPE IS CAUSED BY THE REGULATION DEFECT. THE SYSTEM IN THE WORM HERE IS KNOWN TO BE THE ORGANISM THAT REGULATES FLUID HOMEOSTASIS. SO IF L1 IS FUNCTIONING IN THE SYSTEM. THE SO WHERE. OUR APPROACH TO TEST THIS WAS TO KNOCKOUT L1 IN SPECIFIC ISSUES USING THE SYSTEM. YOU CAN SEE THE ALLELE WAS ASSIGNED TO THIS HERE. WE GOT REALLY INTERESTING RESULTS WHEN WE KNOCKED OUT L1 IN NEURONS, AND THE CLEAR ONE, YOU CAN SEE THE AMOUNT IS TO THE DOUBLE MUTANTS. THIS FINDING THAT L1 FUNCTIONS IN NEURONS AND THEN A CAN NEURONS A PAIR OF NEURONS IN HOMEOSTASIS AND IT IS VARIABILITY IS KNOWN. SO WE ARE NOW USING OUR SYSTEM TO SPECIFICALLY KNOCKOUT L1 IN THE CAN NEURONS. AND OUR FINDINGS IN WORMS ARE PARTICULARLY EXCITING. WE ARE GOING TO INVESTIGATE ON A HYPOTHESIS IN HUMAN SEQUENCING DATA FROM HUMAN PATIENTS. WE ARE ABLE TO CHARACTERIZE SIGNALING AS THE GENETIC MODIFIER OF L1 FUNCTION, THAT WOULD PROVIDE A POSITIVE THERAPEUTIC TARGET AND ADDITIONAL AVENUES TO UNCOVER THE MECHANISMS WITH L1 ASSOCIATED HYDROCEPHALUS. THANK YOU FOR COMING TO MY TALK. AND THE FUNDING OF THE PROJECT IS BY THE UNIVERSITY HERE. >> THANKS, CAROLINE. JUST WAITING TO SEE IF YOU GET ANY QUESTIONS. SO I DON'T SEE ANY QUESTIONS FOR YOU. BUT IF ANY DO COME IN, YOU CAN GO AHEAD AND ANSWER THEM IN THE Q & A BOX. THANK YOU. SO OUR NEXT TALK IS FROM DR. WENXI FROM THE UNIVERSITY OF MINNESOTA. >> HELLO, EVERYONE. MY NAME IS WENXI. OUR LAB IS -- GABRA2 MUTATIONS. THIS IS A SEVERE FORM OF OFTENTIMES CHARACTERIZED BY EARLY ONSET AND REFRACTIVE SEIZURES. THE FUNCTIONING MUTATIONS OF SCN8A, WITH PREMATURE CHANNEL OPENING OR INCOMPLETE CHANNEL OPENING CAUSES SEIZURES. OUR LAB GENERATED PATIENTS WITH DOUBLING. AND SEE THE RED CURVE IS REPRESENTED THE MUTANT MOUSE SPRING AND THESE MICE HAVE EARLY SEIZURE ONSET AND VERY SHORT LIFESPAN. HOWEVER, WHEN THIS IS CROSSED WITH THE WILD-TYPE, THEY WILL HAVE DECAYED SEIZURE ONSET AND EXTENDED SURVIVAL. THE FIGURE ON THE RIGHT IS SHOWING SURVIVAL OF THE BLACK 6 MICE. YOU CAN SEE THE BLACK 6 MICE MOSTLY DIED BETWEEN 20 TO 30 DAYS. HOWEVER, THE F2 MICE HAVE A WIDER VARIETY OF LIFESPAN. SOME DIE EARLY, LIKE THE BLACK SIX MICE, SOME HAVE INTERMEDIAN LIFESPAN. THIS SUGGESTS THAT THE GENETIC BACKGROUND IS PROVIDING MODIFIER GENES FOR THIS MUTATION. SO, BASED ON THE SURVIVAL, OF THE 2 MICE, AND THE SNIP THAT WAS IN QTL ANALYSIS. EAR IS A PLOT REPRESENTING THE RESULTS. THE X AXIS IS REPRESENTING THE SNIP ACROSS ALL OF THE CHROMOSOMES AND THE Y HAMPUS WITH LONGER SURVIVAL. AND SEE, THERE ARE IS MAIN PEAK THAT IS SIGNIFICANCE IN THE CHROMOSOME 5 REGION. WE ARE SEEING THIS LOCUST ARE ABOUT 80 GENES. AMONG THE 80 GENES, GABRA2 IS A VERY INTERESTING CANDIDATE GENE. BECAUSE GABRA2 CAUSES THE ALPHA-2 OF THE GABA RECEPTOR WHICH FUNCTIONS TO REDUCE NEURONAL EXCITABILITY. IT IS KNOWN THAT MUTATIONS OF THE HUMAN GABRA2 BETWEEN CAN CAUSE -- GENERALIZE, AND GABRA2 HAS SUBCELLULAR LOCALIZATION SIGNAL. DISRUPTION OF THE SIGNAL LEADS TO SEIZURES AND MORTALITY IN MOUSE MODELS. SO, WE DID A CLOSER LOOK INTO GABRA2 AND DISCOVERED THERE IS PREVIOUS STUDY THAT DISCOVERED THE SINGLE LESION THAT SUPPLIES EXON 5, YOU CAN SEE THERE ARE 3 KEY IN THE B6 GENE. IN THE SJL STRAIN THERE ARE 14. THIS MUTATION IS CAUSING A FOURFOLD INCREASE IN THE GABRA2 TRANSCRIPT. AND UPON THIS, WE WANT TO VERIFY IF GABRA2 IS THE MODIFIER GENE IN OUR STUDY. WE REVISITED OUR SURVIVAL DATA OF THE MICE AND RECHARACTERIZED THE DATABASED ON THE MICE GABRA2 GENOTYPE. YOU CAN SEE THAT MOUSE, MICE WITH HOMOZYGOUS, B6 ALLELE HAVE SHORT LIFESPAN, ABOUT 50 DAYS. AND THE MICE THAT CARRIED ONE OR TWO ALLELE OF THE SJL GABRA2 GENE HAVE EXTENDED SURVIVAL. TO VERIFY THIS IS THE MODIFIER GENE, WE FIXED ONE OF THE ALLELES. YOU CAN SEE THAT PROVIDING ONE OF THE CORRECTIONS, THE CORRECTION, ONE OF THE ALLELE, IT CAN EXTEND THEIR SURVIVAL FROM 21 DAYS TO 72 DAYS. THIS COUNTRY FIRMED GABRA2 IS THE MODIFIER GENE. THIS ALSO REPRESENTS THAT PHARMACOLOGICAL GABRA2 OF ALLELE HAS THE POTENTIAL TO BE A THERAPEUTIC METHOD. THIS WORK IS SUPPORTED BY NIH GRANTS. THANKS. >> ALL RIGHT. THANKS. >> HELLO. >> SO I SEE THAT THERE'S A QUESTION FOR CAROLINE. IF SHE WANTS TO COME BACK ON AND ANSWER, THAT WILL BE FINE. I WILL GIVE YOU A FEW MINUTES TO ASK WENXI QUESTIONS. BUT I DON'T KNOW IF CAROLINE IS STILL THERE. HI, CAROLINE. SO A QUESTION FOR YOU TOM DAN. AND HE SAYS INTERESTING WORK, CAROLINE. THERE ARE MANY PATHOGENIC VARIANCE IN L1 CAP, HOW DO YOU PLAN TO ACCOUNT FOR PHENOTYPIC VARIABLE DUE TO THE L1 CAP VARIABILITY IN YOUR STUDIES. >> YEAH, THE HUMAN PROJECT WE'RE WORKING ON IS USING WHOLE-EXOME SEQUENCING FROM A COHORT OF PATIENTS WITH NO SYNDROMIC PHENOTYPE. THEY DON'T HAVE AN L1 SYNDROME FEATURES. ONE OF THE HYPOTHESES WE WILL BE TESTING IS WHETHER THESE PATIENTS HAVE A MORE MILD MUTATION IN L1 AS IN COMBINATION WITH A DOUBLE HIT SORT OF SEQ IN THE PATHWAY. IF THE TWO MUTATIONS TAKEN TOGETHER CAUSE SYNERGY THAT THEN CAN CAUSE THE HYDROCEPHALUS TO KIND OF LIKE WHAT WE HAVE SEEN IN WORMS. THAT IS WHAT WE WILL BE FOCUSING ON WITH THAT STUDY. >> THANKS, CAROLINE FOR REJOINING US. >> YEAH. >> NO OTHER QUESTIONS FOR YOU. SO WE WILL MOVE ON. SO WE HAVE A QUESTION. IT ASKS, THERE IS A SUBSET OF THE F2 MICE THAT ARE SURVIVING QUITE LONG. EVEN LONGER THAN THE SSF2. ANY IDEAS WHAT IS GOING ON HERE? >> YEAH, THAT'S A GOOD QUESTION. I THINK THE GABRA2 IS THE MAJOR MODIFIER IN THIS STUDY. BUT IT IS POSSIBLE, LIKE, THEIR ADDITIONAL MODIFIERS THAT PROVIDE MAYBE MINOR EFFECT. SO THAT'S PROBABLY WHY THERE'S LIKE FROM THE FIGURE THERE'S LIKE FOUR OR FIVE OF THE BS MICE THAT LIVE LONGER THAN THE SS MICE. AND ACTUALLY, THE DATA IS SUGGESTING THERE ARE OTHER MODIFIERS THAT MIGHT CONTRIBUTE TO THE EXTENT OF SURVIVAL. IF YOU LOOK AT THE PLOT, THERE'S SOME MINOR PEAKS IN OTHER CHROMOSOME REGIONS EVEN THOUGH THEY WERE NOT AS SIGNIFICANT IN THE MAJOR PEAK IN THE CHROMOSOME FIVE. I THINK THAT IS PROBABLY BECAUSE OF OTHER MODIFIERS. AND ALSO, IF YOU CHECK THE, BB MICE IN THE F2, THE AVERAGE SURVIVAL IS LIKE AROUND 50 DAYS. THERE'S A BIG VARIATION. BUT IF YOU FIX ONE OF THE ALLELES IN ONLY THE B6 MICE, THE AVERAGE SURVIVAL IS QUITE, SORRY, THE VARIATION IS QUITE SMALL. THERE ARE ONLY LIKE AROUND 23 BASE. SO INDICATING IN THE F2 MICE, EVEN THE OBB, THE SJL IS PROVIDING OTHER MINOR MODIFIERS TO MAKE THE VARIATION. SO I THINK TO ANSWER THE QUESTION, THERE ARE OTHER MINOR MODIFIER GENES WE HAVEN'T FOUND YET. >> SO THIS IS A QUESTION FROM JOHN. NICE WORK. HOW DO GABRA2 IN SCN8A INTERACT WITH EACH OTHER AT THE CELLULAR LEVEL? >> THAT'S A GOOD QUESTION. WE DID SOME RESEARCH AND IT SUGGESTS THE, IT IS SUGGESTED THAT THE ALPHA-2, SO I'M GOING TO CALL IT GABRA2 HAS THE SUBCELLULAR LOCALIZATION SIGNAL THAT ACTUALLY DIRECTIONS LOCAL GABRA2 RECEPTOR TO THE SYNAPSE LOCALIZED TO THE EXON INITIAL SACRAL IN THE NEURON. AND ACTUALLY, THE 8A IS VERY HIGHLY CONCENTRATED. ALSO, AT THE EXON INITIAL SEGMENT. SO GUESS, MAYBE THE -- THE GABRA2, SORRY -- THE GABA RECEPTOR HAS SPECIFIC ROLE IN TUNING DOWN THE ACTIVITY -- ACTION POTENTIALS ESPECIALLY IN THE, YEAH, AS -- REGION. BECAUSE IF YOU ONLY DISRUPT THE SUBSET AND LOCALIZATION SIGNAL WITHOUT AFFECTING THE EZMATIC FUNCTION OF GABRA2, IT STILL CAUSES SEIZURES IN THE MOUSE MODEL. SO I THINK THAT IS PROBABLY WHY. MAYBE THEY HAVE -- WHAT'S THAT CALLED UNDER SPATIALLY, THEY LOCALIZE TO THE SAME PLACE. YEAH. >> SO, YOU HAVE LOTS OF QUESTIONS. BUT WE ONLY HAVE TIME FOR ONE MORE. SO WILL LET YOU ANSWER ONE MORE. PLEASE DO ANSWER THE REST IN THE Q & A BOX. SO THIS IS FROM JIM. AND HE SAYS, WHAT IS KNOWN ABOUT THE GABA MODIFYING ANTIEPILEPTICS IN TERMS OF THEIR EFFECTIVENESS WITH PATIENTS OR IN YOUR MICE? >> OH, THAT'S A GOOD QUESTION. SO WE KNOW THERE ARE DRUGS THAT CAN AUGMENT THE GABA RECEPTOR AT FUNCTION. LIKE-- BUT WE HAVEN'T REALLY PURSUED THAT IN OUR MOUSE MODELS. SO I CAN'T ANSWER THAT QUESTION. FOR THE PATIENT PART, I'M NOT -- 100% SURE. BUT I THINK THERE ARE, THE, THE RESULTS ARE MIXED. SO IT IS NOT VERY -- CLEAR. OR-- I CAN'T -- MAKE A -- YOU KNOW? VERY CLEAR ANSWER TO THIS QUESTION, BECAUSE I'M NOT FULLY SURE ABOUT THAT. YEAH, BUT IT IS GOOD DIRECTION TO GO IN. >> THANKS VERY MUCH. >> UH-HUH. >> SO THE NEXT TALK IS FROM DR. ALEXIS FROM THE NORTHWESTERN UNIVERSITY. >> HI. MY NAME IS ALEXA FROM NORTHWESTERN UNIVERSITY, TODAY I WILL BE TELLING YOU ABOUT TARGETING LATENT TGT BETA PROTEIN 4 FOR THE TREATMENT OF MUSCULAR DYSTROPHY. THERE ARE A NUMBER OF DISORDERS THAT MAP TREATMENT. SO, WE MAPPED MODIFIER AS THERAPEUTIC TARGETS. WE BRED MICE ON TWO BACKGROUNDS WITH MUSCULAR DYSTROPHY AND HAD QUANTIFIED TRAIT MAPPING AND FULL GENOME SEQUENCING. WE IDENTIFIED CHROMOSOME SEVEN. THIS REGION CONTAINS LTB4, WHICH WAS KNOWN TO REDUCED TGF-BETA SIGNALING. AND THREE INDEPENDENT COHORTS IT WAS CORDILATING WITH AMBIINATION STATUS. IN THIS EXAMPLE PATIENTS WITH THE PROACTIVE IAAL FORM AS WELL AS THE CHRONIC STEROIDS SHOWED BY THE RED LINE WALKED A NUMBER OF YEARS LONGER THAT THOSE WHO DID NOT. IN THE MILD STRAIN, THE MICE HAD A INSERTION POLYMORPH. I IN THE RISK REGION. THIS REDUCED THE RESENTABLE TO PROTEASE CLEAVAGE. BE DESIGNED A POLYCLONAL ANTIBODY AGAINST THE HINGE REGION AND SHOWED IN VITRO THAT THE ANTIBODY WAS SUFFICIENT TO BLOCK PROTEASE CLEAVAGE AND PREVENT RELEASE OF TGF-BETA. WE THEN GENERATED A HIGH-AFFINITY MONOCLONAL HUMAN ANTIBODY. THIS ANTIBODY LOCALIZED IN THE PATTERN ON THE EXTERIOR OF THE MYOFIBER. WHEN MDX MICE WERE TREATED ONCE WEEKLY WITH LTBP4, THERE WAS A REDUCTION IN FORCE LOSS. WHEN MICE WERE COTREATED WITH LTBP4 AND PEDESTRIAN ZONE THERE IS SUGGESTING STABILIZATION OF THE MUSCLE. WE ISOLATED MYOFIBERS AND SUBJECTED THEM TO INDUCED LASER INJURY. MYOFIBRILS THAT WERE TREATED WITH LTBP4 HAD A MORE SARCOLEMMA. IN VIVO, WE SAW A SIMILAR EFFECT WHEN MUSCLE WAS PRETREATED AND INJURY WOULD CARDIOTOXIN, THERE WAS A MORE STABLE MEMBRANE. THIS CORRELATED WITH REDUCED INJURY AREA SEVEN DAYS AFTER CARD TOXIN INJURY. WITH MICE TREATED FOR SIX MONTHS, WE SAW A REDUCTION IN MUSCLE FIBROSIS VISUALIZED BY REDUCED STAGING AND RED. IT WAS EVALUATED AS A MEASURE OF COLLAGEN CONTENT. AND NIS ALSO WAS REDUCED. MUSCLE FORCE AN IMPROVED SHOWN BY A MORE NORMALIZED AGE VALUES OF THE DIAPHRAGM AS WELL AS INCREASED SPECIFIC FOREST OF THE TIBIALIS ANTERIOR MUSCLE. SO TODAY I SHOWED YOU THAT LTBP4 WAS A MODIFIER, AND TREATMENT WITH ANTIBODY IMPROVED MUSCLE FORCE AND REDUCED FIBROSUS. I WOULD LIKE TO THANK EVERYONE WHO CONTRIBUTED AS WELL AS OUR FUNDING AGENTS. >> ALL RIGHT. ALEXIS. VERY NICE TALK. >> THANK YOU. >> SO WE HAVE A QUESTION FOR YOU FROM -- CHRISTIAN. WHAT HAPPENS IF YOU REMOVE, BUT NOT BLOCK THE HINGE DOMAIN OF TBP4. >> WE ACTUALLY HAVEN'T TRIED THAT, I DON'T KNOW IF IT WILL CREATE A STABLE PROTEIN, BUT THAT IS A THOUGHT. >> HERE'S A QUESTION FROM JIM. GREAT WORK. WHAT ARE YOUR THOUGHTS ON PATH 2 TRANSLATION FOR ANTITPBP4. >> I THINK HE MEANS. >> YEAH. >> WE'RE CURRENTLY TRYING TO OPTIMIZE OUR DOSING. WE ARE DEFINITELY TRYING TO EXPAND TO OTHER DYSTROPHIES AND LOOK AT THE HEART AS WELL. WE'RE REALLY HOPING TO MOVE THIS FORWARD IN THE NEXT COUPLE OF YEARS. >> QUESTION FROM GLENN. NICE TALK. DOES TREATMENT WITH LTBP4 ANTIBODY ALTER LTBF4 EXPRESSION LEVELS? >> OUR PRELIMINARY ANALYSIS SOWING IT IS NOT INCREASING OR REDUCING EXPRESSION LEVELS. >> THERE'S TWO MORE QUESTIONS. I WILL LET YOU ANSWER THEM REALLY QUICK. ANY MORE, ALEXIS WILL ANSWER THEM AFTER. >> SOUNDS GOOD. >> FROM KRISTINA, MAYBE I MISSED >> RIGHT. SO LTBP4 IS KNOWN TO BIND ALL THREE TGF-BETAS IN THE C-TERMINUS. BY BINDING SEQUESTERS AND PREVENTS THEM FROM DOWNSTREAM NEGATIVE EFFECTS. IT ALSO BINDS MYOSTATIN ON THE N-TERMINUS, POTENTIAL FOR INCREASED MUSCLE GROWTH AS WELL. >> ONE LAST QUESTION FROM CHRISTIAN. A FOLLOW-UP QUESTION. HAVE YOU ASSAYED THE MYOSTATIN SIGNALING AXIS AS WELL IN ADDITION TO TGF-BETA SIGNALING? >> SO FAR THE ONLY THING WE'VE LOOKED AT IS SIGNALING, SO WE DO SEE THIS TREATMENT IN OUR FOUR-WEEK INJURY STUDY, THE ADMINISTRATION OF ANTIBODY DOES REDUCE SIGNALING IN MUSCLE. >> THANKS VERY MUCH, ALEXIS. >> THANK YOU. >> THE NEXT TALK WE HAVE SOPHIE FROM THE UNIVERSITY OF MICHIGAN. >> HI, EVERYONE. MY NAME IS SOPHIE HILL, FOURTH YEAR NEUROSCIENCE Ph.D. STUDENT AT THE UNIVERSITY OF MICHIGAN. TODAY I'M GOING TO TELL BUT A SERIES OF GENETIC INTERACTIONS THAT HAVE IMPLICATIONS FOR ANTI-EPILEPTIC THERAPY, A SERIOUS PROBLEM, A THIRD OF PEOPLE WITH EPILEPSY DON'T RESPOND TO CURRENTLY AVAILABLE TREATMENT. OUR LAB IS INTERESTED IN SCN8A, INCLUDING A SODIUM CHANNEL, GAIN OF FUNCTION MUTATIONS CAUSE SEIZURES, AND THIS IS CONCENTRATED IN THE SEGMENT SHOWN ON THE BOTTOM, RESPONSIBLE FOR THE FIRING ACTION POTENTIAL. WE THINK SCN8A IS A GOOD TARGET, HE ESPECIALLY FOR THOSE CAUSED BY MUTATIONS OF OTHER PROTEINS CONCENTRATED AT THE AXON SEGMENT. WE THINK BY REDUCING EXPRESSION OF THE TRANSCRIPT WE CAN COUNTERACT EXCITABILITY AND HOPEFULLY PREVENT SEIZURES. WE COLLABORATED TO DEVELOP AN ANTISENSE OLIGONUCLEOTIDE BY RECRUITING RNA H1. IN PREVIOUSLY PUBLISHED WORK WE'VE ADMINISTERED IN TWO MODELS. ON THE LEFT TREATED MICE WITH GAIN OF FUNCTION MUTATIONS AND SCN8A, POSTNATAL DAY TWO, MICE THAT RECEIVED AT DAY 2 AND POSTNATAL DAY 30 LIVED UNTIL NINE WEEKS OF AGE, TWO AND A HALF TIMES LONGER THAN CONTROL MICE. ON THE RIGHT WE INVESTIGATED EFFECT OF ASO WITH LOSS OF FUNCTION, ENCODES ANOTHER VOLTAGE GATED SODIUM CHANNEL, LOSS OF FUNCTION IN INHIBITOR NEURONS. UNTREATED MUTANT MICE DIED BETWEEN THREE AND FOUR WEEKS OF AGE, A SINGLE ADMINISTRATION AT P2 INVESTED UNTIL SIX MONTHS WITH NO SEIZURES OR DEATH IN TREATED MICE. THESE RESULTS SUGGEST REDUCTION OF SCN8A IS THERAPEUTIC, AND THIS LED TO US ASK WHETHER OTHERS NOT CAUSED BY SODIUM CHANNEL DYSFUNCTION COULD ALSO BE RESCUED. AND WE CHOSE TWO MODELS TO WORK WITH. FIRST ENCODES KV 1.1, IN HUMANS, HETEROZYGOUS FUNCTIONS CAUSE ATAXIA TYPE 1 WHICH CAN INCLUDE SEIZURES. WE TREATED HOMOZYGOUS WITH THE ASO STARTING AT POSTNATAL DAY 2 AND FOUR WEEK INTERVALS SHOWN IN RED. THE ASO PROLONGED TO 16 WEEKS, COMPARED TO 3 WEEKS IN CONTROL TREATED ANIMALS. SO WE CAN CONCLUDE THE SCN8A ASO ASK EFFECTIVE AND MIGHT BE USEFUL FOR TREATING SEIZURES IN PEOPLE WITH EPISODIC ATAXIA. SECOND MU TAKES IS KCNQ2, ANOTHER POTASSIUM CHANNEL, AND HUMANS HETEROZYGOUS LOSS OF FUNCTION MUTATIONS CAUSE A RANGE OF PHENOTYPES. WE TESTED IN Q2, CONTROL MICE IN BLACK EXHIBIT SPONTANEOUS SEIZURES AND DEATH ONSET 3 WEEKS OF AGE. MICE TREATED WITH SCN8AASO AT POSTNATAL DAY 2 AND 8 WEEKS HAVEN'T HAD ANY DEATHS SO FAR, OLDEST ARE AROUND 12 WEEKS OLD. THIS APPEARS TO BE EFFECTIVE IN THIS EPILEPSY MODEL SUGGESTING REDUCING EXPRESSION CAN BE THERAPEUTIC IN AT LEAST FOUR TIMES OF EPILEPSY CAUSED BY MUTATION OF PROTEINS. I'D LIKE TO THANK THE FUNDING SOURCE AND FOR YOUR ATTENTION AND LOOK FORWARD TO YOUR QUESTIONS. THANKS. >> HI, SOPHIE. THANKS FOR BEING HERE. SO, I HAVE A QUESTION FROM JENNIFER KIERNEY. ANY THOUGHTS ON WHY YOU LOSE EFFECT WITH THE SCN8A ASO IN OLDER MICE DESPITE CONTINUED ADMINISTRATION? >> YEAH, THE SHORT ANSWER IS THAT WE DON'T KNOW. STAY TUNED. HOPEFULLY WE'LL FIGURE IT OUT. >> SPECTACULAR, HAVE THERE BEEN MODELS WHERE THIS ASO DIDN'T WORK? >> THANKS. SO, SO FAR WE HAVE NOT FOUND A MODEL WHERE THE ASO DIDN'T WORK WE HAVE AN EXAMPLE WHERE IT'S MUCH LESS EFFECTIVE, TREATING LGF MICE, THAT WASN'T RELATED TO POTASSIUM CHANNEL AND EXTENDED LIFESPAN BY ONE WEEK BUT IT WAS REALLY, REALLY CONSISTENT. OUR P-VALUE WAS LESS THAN .001. >> MATTHEW ASKS, NICE TALK, COULD YOU PLEASE COMMENT ON POTENTIAL OFF-TARGET EFFECTS OF ASOs? >> SO, THAT'S ONE REASON WE WANTED TO WORK WITH ASOs IN THE FIRST PLACE, THEY HAVE LOW POTENTIAL FOR OFF-TARGET EFFECTS BASED ON SEQUENCE. THAT'S ONE MAJOR PROBLEM WITH TREATING SODIUM CHANNEL EPILEPSY, THEY ARE SIMILAR, HARD TO FIND A DRUG SPECIFIC FOR ONE. ASO IS SEQUENCE SPECIFIC, TARGETING THREE PRIME UTR CAN BE SPECIFIC BUT POSSIBLE YOU'RE GOING TO GET TOXICITY FROM ASO OR SOMETHING YOU CAN'T PREDICT BASED ON THE SEQUENCE. AT THE MOMENT WE HAVE SORT OF OBSERVED THAT WITH THIS ASO, WE CAN'T GET ABOVE A CERTAIN DOSE OR THE MICE DIE. BUT BECAUSE THIS ASO WOULDN'T BE USED IN HUMANS WE DON'T THINK THAT'S A HUGE ISSUE AT THE MOMENT. >> SCOTT ASKS DO YOU HAVE EGG DATA ON ASO STUDIES? >> YES, PREVIOUSLY PUBLISHED PAPER, ANNALS, WE HAVE EEG DATA. THE SCN8A MICE, WHAT DELAYING SEIZURES, VERY SEVERE FORM OF SCN8A MUTATIONS, THEY USUALLY DIE AT THE FIRST SEIZURE. WE RUN EEG, THEY DIDN'T HAVE SEIZURES UNTIL THEY DIED MUCH LATER THAN THEY NORMALLY WOULD. IT WAS EXCITING, WE DIDN'T SEE SEIZURES IN THE MICE UP TO FIVE MONTHS BY EEG, SO DOES SEEM LIKE A COMPLETE RESCUE. FOR THE KCN MODELS WE'VE NOT DONE EEG YET BUT THAT'S IN THE WORKS. >> LAST QUESTION, SOPHIE, FROM HUDA. EXCITING RESULTS, SOPHIE. DID YOU LOOK FOR ANY OTHER PHENOTYPES OR NEURAL PHYSIOLOGICAL RESPONSES BEYOND SURVIVAL? >> THAT'S ANOTHER THING WE'D LIKE TO DO ESPECIALLY TO LOOK AT ELECTROPHYSIOLOGY OF THE TREATED NEURONS. BUT WE HAVEN'T DONE IT YET YEAH, SO NOT YET. >> THANKS, SOPHIE. NEXT TALK IS FROM REBECCA MacPHERSON, CLEMSON UNIVERSITY. >> HELLO. MY NAME IS REBECCA MacPHERSON, GRADUATE STUDENT IN THE MACKAY LABORATORY AT THE CLEMSON UNIVERSITY. TODAY I WILL TALK ABOUT MY PROJECT USING DROSOPHILA MELANOGASTER TO IDENTIFY LOCI IN COFFIN-SIRIS SYNDROME. THESE PATIENTS PRESENT WITH SOME DEGREE OF INTELLECTUAL DISABILITY, AS WELL AS FACIAL AND DIGIT ABNORMALITY AS SHOWN IN PICTURES. THE SYNDROME IS ASSOCIATED WITH MANY GENES, ALL OF WHICH ARE PART OF A COMPLEX. WE CAN'T INVESTIGATE WHY THERE'S A HIGH DEGREE OF CLINICAL VARIABILITY IN HUMANS DUE TO STATISTICAL AND MONETARY COMPLAINTS, CAN USE A FLY TO INVESTIGATE. GENES ARE CONSERVED ACROSS TAXA. YOU'LL SEE A CARTOON SCHEMATIC OF THE STRUCTURE OF THE SYI-SNF COMPLEX, AND ON THE RIGHT SIDE NOTE FOR THE GENE PAIR MUTATIONS ARE NOT SOLELY ASSOCIATED WITH COFFIN-SIRIS AND ARE ASSOCIATED WITH AUTISM SPECTRUM DISORDER. WE DECIDED TO USE THE FLY MODEL, TO KNOCK DOWN EXPRESSION OF ORTHOLOGOUS GENES IN FLIES AND ASSESS BEHAVIORAL EFFECTS. I WILL ONLY BE SHOWING YOU RESULTS FROM KNOCK DOWN OF OSA, AS WELL AS KNOCKOUT OF SNR 1, WHICH CORRESPONDS TO THE HUMAN GENE SMARCB1. MALES ARE GREEN. IN THE FLY MODELS WE FOUND STRONG GENE INSECT SPECIFIC EFFECTS, PARTICULARLY HIGHLIGHTED IN STARTLE RESPONSE. WE SAW TRENDS TOWARDS DECREASED NIGHT SLEEP AND INCREASED ACTIVITY IN FLY MODELS. WE PERFORMED RNA SEQUENCING ON THESE KNOCKDOWN MICE TO IDENTIFY GENES IN A ARE CO-REGULATED. THESE CO-REGULATED GENES MAY SERVE AS POTENTIAL MODIFIERS. WE FOUND SEVERAL HUNDRED IF NOT A FEW THOUSAND DIFFERENTIALLY EXPRESSED GENES THAT ARE POTENTIAL CANDIDATES WE WERE ABLE TO NARROW DOWN TO A FEW, BASED ON ORTHOLOG STATUS, DEGREE OF DIFFERENTIAL EXPRESSION, AVAILABILITY OF REAGENTS. WE KNOCKED DOWN THESE GENES, EXACT SAME UBIQUITOUS DRIVER IN THE FLY MODELS. GENES SHOWN HERE HAVE MANY TIES TO METABOLISM AS WELL AS DEVELOPMENT, ALTHOUGH THE TWO CGG GENES HAVE AN UNKNOWN FUNCTION. AGAIN, WE FOUND GENE INSPECT SPECIFIC EFFECTS IN PHENOTYPES TESTED. TOGETHER THESE RESULTS SHOW WE CAN USE THE FLY TO IDENTIFY POTENTIAL MODIFIERS FOR THE SYNDROME. WE HOPE TO PURSUE LARGE SCALE GENETIC MODIFIER SCREENS FOR THESE ASSOCIATED FLY GENES USING DROSOPHILA GENETIC PANEL, OUTLINED YESTERDAY IN A TALK. TO CLOSE I'D LIKE TO THANK FORMER MEMBERS OF THE LABORATORY. THANK YOU. >> THANKS, REBECCA. ARE YOU HERE WITH US TODAY? >> YES. >> GREAT. I DON'T SEE ANY QUESTIONS IN THE CHAT BOX YET. SO, THE CSS IS A DEVELOPMENTAL DISORDER, CAN YOU COMMENT IF THE FLY MODIFIES THE DISEASE WELL, ARE YOU DOING ASSAYS IN THE LARVAL STAGE OR ADULT STAGE OR BOTH? >> SURE. SO, WE ARE MODELING COFFIN-SIRIS IN THE ADULT FLY, IT'S HARD TO MODEL DIGIT ABNORMALITIES SO WE'RE FOCUSING ON NEURODEVELOPMENTAL SIDE OF THE DISORDER IN ADULT FLIES. >> OKAY, GREAT. >> SO I DON'T SEE ANY OTHER QUESTIONS FOR YOU. IF THEY DO COME PLEASE DO ANSWER THEM IN THE Q&A BOX. >> SOUNDS GOOD. THANK YOU. >> THANKS, REBECCA. OUR LAST TALK FROM THIS SESSION IS FROM PAUL FROM BAYLOR COLLEGE OF MEDICINE. >> MY NAME IS PAUL, A POSTDOCTORAL FELLOW. I'LL PRESENT LOSS OF IRF2BPL IMPAIRS NEURONAL MAINTENANCE. THE SCREENING CENTER FOR THE NIH CENTER, WITH OUR CLINICAL COLLABORATORS FOUND DE NOVO TRUNCATIONS CAUSES SEVERE DISORDER CHILDREN REACH MILE STONES UNTIL 5 WHEN THEY DEVELOP PROGRESSIVE ATAXIA, SEIZURE DISORDER, BRAIN ATROPHY. SINCE THE INITIAL COHORT OVER 25 PATIENTS WITH WHAT NOW HAS BEEN TERMED NEDAMSS HAVE BEEN REPORTED. IN THE FIRST REPORT I USED DROSOPHILA. IT'S STILL UNCLEAR HOW LOSS OF IR 2BPL LEADS TO DYSFUNCTION. I CONTINUED MY WORK, THE ESSENTIAL GENE IN FLIES, BUT THE SELECTIVE REDUCTION IN NEURONS LEADS TO PROGRESSIVE MOTOR DEFICITS OBSERVED ONLY IN OLDER FLIES. AS WELL AS AGE-DEPENDENT AXONAL LOSS IN THE PERIPHERAL NERVE, CRITICAL FOR DEVELOPE AND LONG-TERM NEURONAL MAINTENANCE. I CONDUCTED GAIN OF FUNCTION STUDIES, FOUND OVEREXPRESSION OF PITS CAUSES ADULT WING MARGIN LOSS, AND HAVE SHOWN THIS LOSS IS DUE TO INHIBITION OF WNT EXPRESSION AND DOWNSTREAM SIGNALING. I ALSO FOUND THAT FULL LENGTH IRF2BPL IS REQUIRED, TRUNCATION ACT OF LOSS OF FUNCTION, WE USE THIS AT PLATFORM TO FIND A STRONG ANTAGONISTIC GENETIC RELATIONSHIP BETWEEN THE TWO. WHAT ABOUT LOSS OF FUNCTION AND WHAT ABOUT ESSENTIAL NERVOUS SYSTEM? WHEN WE KNOCK DOWN PITS IN NEURONS WE SEE INCREASE IN THE WNT LIGAND PROTEIN AND WERE ALSO ABLE TO OBSERVE THIS INCREASE IN WNT1 AND SIGNALING IN THE CNS OF MUTANT ZEBRAFISH AND INCREASED WNT 1 IN PATIENT CELLS, USING INHIBITORS FROM CANCER FIELD ABLE TO SUPPRESS SOME OF THE PHENOTYPES WE FOUND IN BOTH FLIES AND IN THE FISH UPON LOSS OF PITS FROM IRF2BPL. WE OVEREXPRESSED IN FLY NEURONS AND OBSERVED DEFICITS AND AXONAL LOSS IN AGED FLIES ONLY, TWO PHENOTYPES THAT WERE REMINISCENT TO THE LOSS OF PITS IN NEURONS. IN SUMMARY, WE'VE SHOWN THAT LOSS OF PITS LEADS TO INCREASED WNT IN MULTIPLE MODELS AND INCREASED PITS LEADS TO DECREASE WNT AND DOWNSTREAM SIGNALING. DRUGS THAT INHIBIT WNT SIGNALING SUPPRESS NEUROBEHAVIORAL PHENOTYPES WHEN PITTS/IRF2BPL IS REDUCED. THERE IS A CONSERVED PHYSICAL INTERACTION WITH THE COMPLEX MEMBER KC 1 ALPHA KNOWN TO INHIBIT WNT SIGNALING IN CYTOSOL, IR 2BPL, A NUCLEAR PROTEIN, CO-LOCALIZES IN THE NUCLEUS OPENING QUESTIONS OF EXACTLY HOW THIS INTERACTION LEADS TO INHIBITION OF WNT. TOGETHER WE'VE IMPLICATED WNLT SIGNALING PATHWAY AS MODIFIER OF NEURODEVELOPMENTAL AND NEURODEGENERATIVE DISEASE. I THANK MY COLLABORATORS AND FUNDING SOURCES, IN PARTICULAR THE NIH AND PLEASE FEEL FREE TO MESSAGE ME WITH ANY OF YOUR QUESTIONS. >> WELCOME BACK, EVERYONE. WE'RE CONTINUING THE SESSION ON MECHANISMS AND CHARACTERIZATION MODIFIERS, THIS PART WILL BE MODERATED BY DR. HUDA ZOGBE. >> THANK YOU, GLEN. IT'S MY PLEASURE TO INTRODUCE MY COLLEAGUE DR. HUGO BELLEN, CHARLES DARWIN PROFESSOR AT BAYLOR COLLEGE OF MEDICINE, DUNCAN NEUROLOGICAL RESEARCH INSTITUTE AT CHILDREN'S HOSPITAL. DROSOPHILA TO UNDERSTAND SYNAPTIC TRANSMISSION, NEURODEGENERATIVE DISORDERS, DEVELOP AMAZING TOOLS HE SHARED WITH THE DROSOPHILA COMMUNITY TO MANIPULATE FRUIT FLIES. CURRENT RESEARCH IS FOCUSING ON DISCOVERY OF DISEASE GENES, AS YOU KNOW SEQUENCE AND REVEALING VARIANTS IS CHALLENGING TO FIND THE ACTUAL CULPRIT GENE, WORKING WITH UNDIAGNOSED DISEASE NETWORK FUNDED BY NIH HUGO ESTABLISHED MODEL ORGANISM SCREENING CENTER AND SOLVED MANY, MANY HUMAN DISORDERS USING THE FRUIT FLIES. TODAY HE'S GOING TO SHARE HIS WORK ON DIFFERENT ASPECT OF THE LAB AND UNDERSTANDING MECHANISM OF DISORDERS, SPHINGOLIPIDS AND CERAMIDES. >> THIS WORK WAS DONE BY A POSTDOC IN THE LAB. I WANT TO ARGUE HERE THAT RARE DISEASE CAN PROVIDE US WITH A LOT OF INFORMATION ABOUT MUCH MORE COMMON DISEASE, AND IN THIS CASE I'LL TALK ABOUT LYSOSOMAL STORAGE DISEASES AND RELATIONSHIP TO PARKINSON'S DISEASE. SO, SINGLE LIPID METABOLISM IS QUITE COMPLICATED. I THOUGHT I COULD START MAYBE WITH, YOU KNOW -- LET ME PUT THE POINTER. I COULD STARRED BY SAYING THAT IN THE E.R., THROUGH A SERIES OF ENZYMES LEADS TO CERAMIDE IN THE ER, TRANSFERRED TO CERAMIDE TRANSFER PROTEINS TO THE GOLGI WHERE A LOT OF MODIFICATIONS CAN OCCUR, GLUCOSE CAN BE ADDED, AND EXPANDED UPON AND LEAD TO A WHOLE SET OF REALLY MOLECULES SUGAR MODIFIED, THEIR ROLE IS NOT WELL DEFINED. MANY ENZYMES HAVE BEEN ASSOCIATED WITH NEUROLOGICAL DISORDERS, BUT WE DON'T REALLY KNOW PRECISELY WHAT THESE CERAMIDE DIVERSITY ACTIVES DO. WHAT WE DO KNOW IS THAT CERAMIDE CAN BE TRANSFERRED TO PLASMA MEMBRANE CAN BE RETURNED. UPON ENDOCYTOSIS OF THE PLASMA MEMBRANES THESE LIPIDS END UP IN THE LYSOSOME, AND CAN BE DEGRADED AND CAN BE RECYCLING PATHWAY HERE. SO THIS IS A COMPLEX PATHWAY, AND THERE'S QUITE A FEW DISEASES ASSOCIATED WITH ENZYMES INVOLVED WITH THESE PATHWAYS. ONE OF THE PATHOGENIC MECHANISMS WHEN CERAMIDE LEVELS ARE ALTERED, YOU'RE GOING TO AFFECT MEMBRANE FLUID, CERAMIDES ARE IMPORTANT MEMBRANE COMPONENTS, PLAY AN IMPORTANT ROLE IN SIGNALING, AND ILL-DEFINED PROCESS TO BE HONEST. TWO HIGH LEVELS OF CERAMIDE DECREASE FLUIDITY AND CURVATURE, AND COMPARING ESSENTIALLY VESICLE FORMATION, AND BLOCKING MEMBRANE. WHEN PEOPLE INCREASE CERAMIDE LEVELS IN ARTIFICIALLY CREATED VESICLES, MEMBRANES STIFFEN UP AND DECREASE. SIMILAR TO HIGH LEVEL, TOO LOW LEVELS CAUSE A PROBLEM BECAUSE MEMBRANES BECOME TOO FLUID. THAT'S BEEN SHOWN ELEGANTLY IN DROSOPHILA BY KNOCKING OUT CERAMIDE TRANSFER PROTEIN SHOWING THIS REDUCES CERAMIDE LEVELS, THESE FLIES ARE ALSO SHORT-LIVED AND HAVE CLEARLY INCREASE IN MEMBRANE FLUIDITY. SO BOTH LOSS OF FUNCTION MUTATIONS THAT LEAD TO INCREASE IN CERAMIDE OR DECREASE IN CERAMIDE HAVE SEVERELY REDUCED LONGEVITY IN FLIES. I WILL TALK TODAY ABOUT A GENE CALLED INAD, AND THIS PHENOTYPE IS CAUSED BY MUTATIONS IN THE GENE CALLED PARK 14 BECAUSE THERE IS AN EARLY ONSET WHICH IS CAUSING, AND LATER ONSET SHOW DYSTONIA AND PARKINSONISM, BEFORE IT WAS CLONED IT WAS CALLED PARK 4D. KIDS ARE FINE FOR SIX MONTHS, A YEAR, START DEVELOPING LOSS OF MOTOR SKILLS, THEY HAVE MENTAL AND PHYSICAL DISABILITIES, SEVERE VISION LOSS, MANY OF THEM HAVE SEIZURES. THEY ACCUMULATE MEMBRANES, VESICULAR STRUCTURES, CONTAINING SYNUCLEIN, DISRUPT THE MEMBRANE, A LATE PROCESS. SO, INAD IS REFERRED TO AS PARKINSON'S FOR KIDS, AND WE WERE INTERESTED IN STUDYING THIS GENE BECAUSE WE WERE INTERESTED IN LIPID METABOLISM, AND AS THE NAME INDICATES IT SHOULD BE A PHOSPHO-LIGASE. THE FLIES ARE SHORT-LIVED, THEY LIVED FOR 25 DAYS, SOMETIMES 30 DAYS. WE STARTED LOOKING AT THE PHENOTYPES THAT WERE ASSOCIATED WITH THE LOSS OF THIS IS A2G6. WE NOTED THE LYSOSOMES EXPAND NOT ONLY IN THE NERVOUS SYSTEM HERE, YOU CAN SEE THE NUMBER AND SIZE OF THE LYSOSOMES ESPECIALLY EXPANDS. WE STARTED DOING TRANSMISSION ELECTRON MICROSCOPY STUDIES IN THESE FLIES, AND KNOW QUICKLY IN THE PHOTORECEPTORS WHICH IS OUR FAVORITE SUBSTRATE TO DO TRANSMISSION ELECTRON MICROSCOPY, THERE WAS INCREASE IN NUMBER AND SIZE, QUANTIFIED HERE, PHENOTYPE IS PROGRESSIVE EARLY ON. THERE IS VERY LITTLE INCREASE BUT BY DAY 15 THERE'S MODEST INCREASE, BY DAY 30 MASSIVE INCREASE YOU CANS REMINISCENT OF A LYSOSOMAL STORAGE DISEASE. THE COMMON PHENOTYPE, IT WAS ALSO OBVIOUS BY AFTER DAY 20 STARTED SEEING CLEAR PHENOTYPES IN THE MITOCHONDRIA, AND THERE'S A LOT OF ABNORMAL MITOCHONDRIA BY DAY 30. FINALLY WE LOOKED AT LIPIDS, AND LIPID THAT'S MOSTLY INCREASED IN THESE MUTANTS THAT PLAY ANOTHER ROLE IN GOSHEA'S. WE'RE LOOKING AT A SECTION IN THE PHOTORECEPTORS, STRUCTURES LABELED IN RED, SURROUNDED BY GLIAL CELLS THAT SOUND THESE PHOTORECEPTORS CALLED PIGMENT CELLS. WHEN YOU LOOK WITH LIGHT MICROSCOPY AT SECTIONS YOU CAN SEE IN THE WHOLE THERE IS NO CERAMIDE BUT IN THE MUTANT THAT LACKS THIS PROTEIN THERE'S ACCUMULATION. YOU SEE THE SAME LEVELS OF GLUCOSYLCERAMIDES. THIS KIND OF SPARKED OUR INTEREST. WE'VE BEEN STUDYING THIS PATHWAY EXTENSIVELY. I WANTED TO GIVE THE TAKEHOME MESSAGE. TURNS OUT IN ADDITION TO ITS FUNCTION AS ENZYME, AS A PHOSPHO-LIPASE BINDS TO THESE PROTEINS. THESE ARE PROTEINS THAT PLAY CRITICAL ROLES IN THE RETROMER. IN THIS PATHWAY, WE ARGUE THAT IT IS ENDOCYTOSED, WHEN IT'S IN THE ENDOSOME, IT'S RETRIEVED, YOU HAVE A CYCLE SO YOU DON'T NEED TO SYNTHESIZE CERAMIDE ALL THE TIME, YOU CAN RETRIEVE AND RECYCLE. LET'S LOOK AT THE PATHWAY. TRANSFER THROUGH THE GOLGI, FORM SPHINGOMYELINS, THE MEMBRANES ARE ENDOCYTOSED IN ALL CELLS AND RETROMER PLAYS A CRITICAL ROLE. THIS GENE IS SHOWN TO CAUSE PARKINSON'S DISEASE, 35 IS ASSOCIATED WITH PARKINSON'S DISEASE. NOW, OBVIOUSLY THE ENDOSOME TRAVELS, FORMS CERAMIDE. WHAT WE THINK IS HAPPENING UNDER PATHOLOGICAL CIRCUMSTANCES, THAT IS WHEN THIS PROTEIN IS LOST IS THAT CERAMIDE IS PRODUCED, TRANSFERRED TO THE PLASMA MEMBRANE, BUT BECAUSE THE RETROMER IS NOT FUNCTIONING PROPERLY, TOO MUCH OF THE CERAMIDE ENDS UP IN THE LYSOSOME, AND THERE IS INCREASE IN CERAMIDE, DISRUPTING LYSOSOMAL FUNCTION LEADING TO EXPANSION OF THE LYSOSOME SO IT BECOMES A LYSE OH SOME STORAGE DISEASE PHENOTYPE. WE'VE SHOWN WHEN YOU LOSE PHOSPHO-LIPAS YOU DESTABILIZE 35 AND 26, AND BY DESTABILIZES THESE PROTEINS YOU IMPAIR PROGRESSIVELY MORE AND MORE RETROMER FUNCTION. INTERESTINGLY, GENETICALLY, WE CAN SUPPRESS THESE PHENOTYPES BY DOING A VARIETY OF MANIPULATIONS, OVEREXPRESS AND SUPPRESS THE PHENOTYPE. CLEARLY SHOWING THE RETROMER PLAYS AN IMPORTANT ROLE. WE CAN LOWER THE SYNTHESIS OF CERAMIDE BY TAKING ENZYMES THAT ARE INVOLVED IN CERAMIDE SYNTHESIS AND LOWERING THEIR LEVEL OR USING DRUG TO LOWER THE LEVEL THAT SUPPRESSES THE PHENOTYPE OF THE MUTANT. FINALLY WE CAN USE A DRUG THAT'S USED IN THE CLINIC, DESIPRAMINE, NOT USED IN PATIENTS IN MUTATIONS, AND BLOCK THE SPHINGO MILENASE, SUPPRESSING THE PHENOTYPE. WE HAVE DIFFERENT GENETIC DRUGS THAT CAN IMPROVE THE PATHWAY BY UNDERSTANDING HOW THE PATHWAY WORKS. NOW, YOU COULD SAY IS THIS PATHWAY CONSERVED? WE COLLECTED PATIENT CELLS, FIBROBLASTS, TURNED THEM INTO NPCs, IN COLLABORATION WITH AN ORGANIZATION IN NEW YORK CITY, AND THE PATIENT HERE HAS R70X MUTATION, STOP CODON. WE LOOKED IN THE NPCS AND LOOKED AT LAMP2, THE MUTATION WHEN CORRECTED BACK TO WILD TYPE AND LOOKING AT THESE CELLS, LYSOSOMES, BUT MANY FEWER LYSOSOMES THAN IN THE UNCORRECTED PATIENT CELLS. SIMILARLY LOOKING AT MITOCHONDRIA, THE NETWORK THAT IS PRESENT IN THE CONTROL, CORRECTED NPC, IS DRAMATICALLY DIFFERENT IN THE MUTANT IPCs. IN THE CERAMIDE, THE CORRECTED HAS RELATIVELY LOW LEVELS, THE MUTANT HAS HIGH LEVELS. SINCE THERE'S A LINK WITH PARKINSON'S, LET'S OVEREXPRESS SYNUCLEIN, SHOWN IN NUMEROUS CELLS IN THE BRAINS OF PARKINSON'S DISEASE PATIENTS. AND AGAIN WHEN WE OVEREXPRESS SYNUCLEIN WE SEE SIMILAR DECREASE AS IN LOSS OF RETROMER PROTEINS, INCREASE OF THE SIZE AND NUMBER OF THE LYSOSOMES WHEN WE KNOCK DOWN IN THESE VERTEBRATE CELLS, OVEREXPRESSING SYNUCLEIN AND SEE INCREASE OF CERAMIDES. WHEN YOU OVEREXPRESS SYNUCLEIN YOU GET SAME HALLMARKS. LET'S LOOK AT THE FLY, OVEREXPRESSING SYNUCLEIN, DEGENERATIVE PHENOTYPE, AGAIN DECREASED WHEN YOU TREAT MYRIOCIN, SYNTHESIS PATHWAY PLAYING AN IMPORTANT ROLE IN THE PATHOLOGY. WHAT ABOUT THESE OBSERVATIONS AND WHY DID WE START WORKING ON GOSHEA'S? PARP 14 CAUSES PARKINSONISM, LOSS AFFECTS PARK17, ANOTHER LOCUS VPS35, OF SYNUCLEIN, CAUSING SIMILAR PHENOTYPE PROMPTING US TO LOOK AT GAUCHER'S DISEASE, AND ELEVATED LEVELS OF GLUCOSYLCERAMIDES. GOSHEA DISEASE LEADS TO RELATION OF GLUCOSYLCERAMIDES. THERE'S A SEVERE CASE AND ABNORMALITIES IN SOME BUT ONE KEY FEATURE IS HETEROZYGOUS HAVE FIVE-FOLD INCREASE FOR RISK OF DEVELOPING PARKINSON'S DISEASE. SO IF THE GENE MANIFESTS ITSELF DIFFERENT PHENOTYPES, OFTEN ASSOCIATED WITH PARKINSON'S DISEASE. AGAIN ARGUING ELEVATION OF GLUCOSYLCERAMIDES CAUSES PHENOTYPE. SO WE INTEGRATED AND KILLED GBA1B GENE IN THE FLY INSERTING GAL 4, THE EXPRESSION PATTERN CAN BE USED TO DETERMINE WHERE THE GENE IS EXPRESSED. TO OUR SURPRISE THE GENE IS ONLY EXPRESSED SHOWN HERE, GREEN ON TOP, NEURONS. RED IS GBA1B. AT THE BOTTOM CO-LABEL WITH GLIAL CELLS, THERE'S A PERFECT CO-LOCALIZATION SO THIS GENE IS NOT EXPRESSED IN NEURONS, EXPRESSED SPECIFICALLY IN GLIA. WE'VE DONE MANY EXPERIMENTS TO CONFIRM THIS. SO LET'S NOW LOOK EXPLAINING MORPHOLOGY. LOOK AT THE PHOTORECEPTORS, GLIA SURROUND THESE PHOTORECEPTOR CELLS, AND YOU CAN SEE WHEN YOU LABEL WITH ANTIBODY FOR GLUCOSYLCERAMIDES THERE'S VERY NICE LABEL. WHEN YOU EXPOSE THE FLIES TO LIGHT. IF YOU DON'T THERE'S NO ACCUMULATION OFFER IS OF CERAMIDES. OVER TIME, THIS LEADS TO MASSIVE DEGENERATION. NOW, YOU CAN ARGUE ABOUT IT'S IN THE GLIA, WHAT ABOUT STOPPING THE SYNTHESIS OF GLUCOSYLCERAMIDES IN THE PHOTORECEPTORS. HERE WE DO THAT BY DRIVING IN THE PHOTORECEPTORS RNAi AGAINST LUCIFERASE OR ENZYME INVOLVED IN SYNTHESIS OF GLUCOSYLCERAMIDES. WHEN YOU BLOCK SYNTHESIS, IN THE PHOTORECEPTORS, HAVE YOU NO ACCUMULATION OF GLUCOSYLCERAMIDES HERE, BUT IF YOU KNOCK IT OUT IN THE GLIAL CELLS NOTHING CHANGES. SO THIS CLEARLY SHOWS GLUCOSYLCERAMIDES IS SYNTHESIZED IN PHOTORECEPTORS, TRANSFERRED TO THE GLIA. SO LIGHT INDUCES THE ACTIVITY OF THE PRODUCTION OF GLUCOSYLCERAMIDES, AND THIS IS TRANSPORTED TO GLIAL CELLS, IN THE LYSE LYSOSOME OF THE GLIAL CELLS TO FORM CERAMIDE. I'LL SHOW YOU IN A FEW SLIDES THIS IS REALLY RELYING ON NEW PROCESS WE DISCOVERED, THOSE ARE EXTRACELLULAR VESICLES, GLUCOSYLCERAMIDES PRODUCED HERE TO THE GLIAL CELLS, IN THE GLIAL CELLS THIS EXTRA VESICULAR STRUCTURES ARE TAKEN BY THE 'EM BRAIN, ENDOCYTOSED, INFUSES TO DECORATE THE GLUCOSYLCERAMIDES. AND SO FIRST SHOWING YOU THE HUMAN GENE FUNCTION SLIDE, THE FLY GENE IF YOU KNOCK OUT THE FLY GENE AND LOOK AT PHOTORECEPTORS, FUNCTIONING, YOU HAVE THIS FUNCTION OF THE PHOTORECEPTORS AND YOU CAN FULLY RESCUE THE HUMAN GENE. THAT SHOWS THE HUMAN GENE IS CONSERVES AND FUNCTIONS IN THE FRUIT FLIGHT. WE DID A SET OF ASSAYS SHOWING NEURONS IN VERTEBRATE CULTURED NEURONS AND GLIA THERE'S CLEARLY MUCH HIGHER LEVEL OF GBA THAN IN NEURONS, AND CULTURED AND LABELED THEM, CAN YOU SEE THE GLUCOSYLCERAMIDES IS TAKEN UP BY THE CELLS, AFTER 30 MINUTES STARTS TO BE ENDOCYTOSED INTO THE NEURONS. ON TOP OF THE NEURONAL CULTURES YOU PUT NEURONS, NOTHING WILL CHANGE. IT WILL ADD THE SAME PICTURE. IF YOU PUT ON TOP OF THIS GLEEING, THE GLUCOSYLCERAMIDES DISPERIODS. AND TRANSPORTED FROM NEURONS TO THE GLIA. AND I WANT TO SUMMARIZE DATA SHE RECENTLY ACQUIRED. NEURON GLIAL CO-CULTURE REDUCES GLUCOSYLCERAMIDES IN NEURONS, A NEURON-NEURON CO-CULTURE DOESN'T DO ANYTHING. IF YOU KNOCK DOWN LEADS TO ACCUMULATION OF GLUCOSYLCERAMIDES IN THE GLIA. AND THE GLIA MEDIUM IS SUFFICIENT TO INDUCE SECRETION OF NEURONS. FINALLY, TGF INDUCES TRANSFER. AND THAT TRANSFER OCCURS THROUGH EXOSOMES WHEN YOU LOOK AT THAT CULTURED MEDIA. NEURONS PRODUCE GLUCOSYLCERAMIDES, TRANSFERRED FROM THE GLIA UPON SIGNAL, THAT STIMULATES SEE VESICLES TAKEN UP. >> WE'VE USED UP YOUR TIME AND FOR QUESTIONS. WE'LL HAVE TO SKIP QUESTIONS TO THE DISCUSSION, MAYBE BECAUSE WE'RE OVER. >> NO PROBLEM. >> MY PLEASURE TO INTRODUCE SUSAN ACKERMAN, CHAIR OF THE BIOLOGY AND PROFESSOR OF NEUROBIOLOGY AT UNIVERSITY OF CALIFORNIA SAN DIEGO, ALSO THE DEAN OF RESEARCH, A LEADING AUTHORITY IN THE USE OF MOUSE GENETICS TO IDENTIFY GENES IDENTIFYING NEW PATHWAYS AND NETWORKS INVOLVED IN DISEASES, AND DEATH OF NEURONS. MORE RECENTLY HER EXCITING WORK SHOWN CHANGES IN TRANSLATIONAL FIDELITY CAN PUT THE BRAIN AT RISK, STUDIED MECHANISM OF TRANSLATAL PROOF READING,. MAKE SURE YOU'RE UNMUTED. YOU'RE MUTED. CAN YOU UNMUTE? YOU'RE STILL MUTED. >> OKAY. SORRY ABOUT THAT. OKAY. THANKS. SORRY. I SHOULD KNOW HOW TO DO THIS BY NOW. SO TODAY WHAT I'D LIKE TO TELL YOU ABOUT IS HOW WE USE GENETIC DIVERSITY OF INBRED STRAINS TO IDENTIFY MODIFIER GENES. I'M GOING TO FOCUS ON OUR EFFORTS TO IDENTIFY MODIFIERS OF PRIMARY MUTATIONS THAT CAUSE SUBTLE CHANGES IN mRNA TRANSLATION, AND THESE WHILE SUBTLE, THESE CHANGES ARE ENOUGH TO DISRUPT HOMEOSTASIS. FIRST HOW WE IDENTIFIED USING THIS APPROACH, A MODIFIER OF tRNA AND PUT THESE UP YET, AND THEIR ROLE IN DISEASE. THESE ENZYMES ARE MAJOR CONTRIBUTORS TO TRANSLATIONAL FIDELITY, THEY NEED TO BE ABLE TO ACCURATELY DETECT AND BIND TO THEIR COG NATURE tRNAs AS WELL AS COG NATURE AMINO ACIDS, WHICH THEN THEY ACTIVATE AND TRANSFER ONTO THE PROPER tRNAs, BUT MISTAKES CAN HAPPEN. AND IN THE CASE OF THESE, THE MISTAKES ARE USUALLY AT THE LEVEL OF THE AMINO ACID, IF THE WRONG AMINO ACID IS ACTIVATED AND TRANSFERRED TO A tRNA THIS OF COURSE CAN LEAD TO IMPROPER READING OF GENETIC CODE WITH WRONG AMINO ACID BEING INCORPORATED INTO NASCENT POLYPEPTIDE. BUT MANY OF THE SYNTHETASE CONTAIN FUNCTIONS TO DEAL WITH SUCH MISTAKES. AND WE FIRST GOT INTERESTED IN tRNA SYNTHETASE EDITING WHEN STUDYING THE MOUSE KNOWN AS STICKY, DEGENERATION WAS ACCOMPANIED BY PROGRESSIVE ATAXIA, YOUR AND WE COULD GO AND SHOW THAT THE MOLECULAR DEFECT IN THESE MICE WAS A POINT MUTATION IN THE ETADI NEXT DOMAIN OF SYNTHETASE BUT WHAT WE NOTICED IS WHEN WE CROSSED THESE MICE TO DIFFERENT INBRED STRAINS OF MICE ONE STRAIN LARGELY RESCUED DEGENERATION AS SHOWN HERE. AND THIS IS THE E I STRAIN. USING SNP ANALYSIS AND RECOMBINATION MAPPING, WE WERE ABLE TO IDENTIFY THIS SNP THAT WAS CAUSAL IN MOST STRAINS, FOR SENSITIVITY TO NEURODEGENERATION, SO MOST STRAINS HAVE AN INTRONIC SNP, PRIOR TO THIS STUDY HAD NO KNOWN FUNCTION. AND WHEN THIS INTRONIC SNP IS USED IN THE ALTERNATIVE FASHION, IT LEADS TO INCORPORATION OF STOP CODON LEADS TO PARTIAL LOSS OF EXPRESSION, PARTIAL BUT TRAUMATIC, ABOUT 80% DOWN IN EXPRESSION. WE PERFORMED MANY MECHANISTIC STUDIES, AND ULTIMATELY ARE LEFT WITH THIS MODEL. IT APPEARS THAT 16 INTERACTS WITH tRNA SYNTHETASE TO SERVE AS CO-REGULATOR FOR tRNA SYNTHETASE EDITING AND IT DOES THIS BY DIRECTLY ACCEPTING MISACTIVATED SERINE, THE MISTAKE MADE ON LYSINES FOUND IN 16, YOU CAN THINK OF THIS AS A SPONGE, IF YOU WILL. THIS WAS EXCITING THEN. THE MODIFIER REALLY GAVE US NEW BIOLOGY, THIS WAS THE FIRST CO-REGULATOR FOUND FOR tRNA SYNTHETASE EDITING WHICH PRIOR WAS THOUGHT TO BE TOTALLY INTRINSIC TO SYNTHETASE ITSELF. I THINK THERE'S A VERY EXCITING SECOND STORY THAT COMES OUT OF THIS, AND THAT IS THE LEVEL OF THE MODIFIER EXPRESSION OF THIS MODIFIER SEEMS TO CONFER NEURONAL SUSCEPTIBILITY TO THE LRS MUTATION. WE HEAR ALL THE TIME, WHY DOES A POINT MUTATION OR COMPLETE LOSS OF FUNCTION IN A YOUR BIC WITTOUS EXPRESSED GENE GIVE A LIMITED PHENOTYPE. IN THIS CASE WE THINK IT'S DUE TO THE EXPRESSION OF THE MODIFIER, PURKINJE CELLS, AND THEY UNDERGO DEATH IN THE NEURONAL MODEL. HIPPOCAMPAL NEURONS OR CORTICAL NEURONS HAVE MUCH HIGHER LEVELS OF WITH 16, CORRELATING WITH SURVIVAL, SUGGESTING IN THAT PURKINJE CELLS ARE ON THE BRINK OF NOT DEALING WITH MISTRANSLATION THAT IS CONFERRED BY THE LRS MUTATION, AND OTHER NEURONS, LEVELS HAVE GONE DOWN BECAUSE OF THE SNP, THEY STILL EXPRESS ENOUGH 16 TO SURVIVE. WE WENT AHEAD, TO TEST THIS, AND KNOCKED OUT THE CONDITION MAL MANNER, 16 GENE ENTIRELY, AND SURE ENOUGH WHEN WE LOOK AT SURVIVING HIPPOCAMPAL OR CORTICAL NEURONS WE SEE THESE NEURONS DEGENERATE, AND WHAT I'M NOT IN THE PRESENCE OF THE STICKING MUTATION, ALSO FORM YOUR UBIQUINATED PROTEIN AGGREGATES. ANOTHER STORY GIVES A SIMILAR PICTURE THE EXPRESSION LEVEL OR EXPRESSION AT THE PLACE THAT THIS MODIFIER IS EXPRESSED REALLY CAN CONFER SOME OF THE TISSUE SPECIFICITY. AND WE WORKED ON INDUCE THE MUTATION THAT LED TO PROGRESSIVE ATAXIA BUT IN THIS CASE A DRAMATIC LOSS OF GRANULE NEURONS IN THE CEREBELLUM AND NEURODEGENERATION IN OTHER PLACES. THE MUTATION IN THIS CASE WAS ON BLACK 6J BACKGROUND. WHEN WE CROSSED TO DIFFERENT STRAINS NEURODEGENERATION WAS ATTENUATED. AS SHOWN HERE. AND WE COULD MAP THE RESCUE MODIFIER, THE SUPPRESSOR, TO THE DISTAL REGION OF CHROMOSOME 1. NOW, WE WERE REALLY SURPRISED TO FIND OUT THAT THIS WAS A NUCLEAR ENCODED tRNA, AN ARGININE UCU tRNA. AND WE WERE CONFUSED BY THE FACT THAT THERE WERE FOUR OTHER FUNCTIONAL COPIES OF UCU tRNAs IN THE GENOME. THIS LED US TO LOOK AT THE EXPRESSION OF NTR 20 AND WE FOUND SO SURPRISINGLY THAT IT WAS EXPRESSED ONLY IN NEURAL TISSUES BUT THE MOUSE AND IN HUMAN, AND NOT ONLY WAS THIS EXPRESSION PATTERN VERY SPECIFIC AND I CAN TELL YOU IT'S IN NEURONS SPECIFICALLY, I DON'T HAVE TIME TO SHOW YOU THAT, BUT IT'S ALSO A VERY PROMINENT MEMBER OF THIS GENE FAMILY OF AGA ARGININE tRNAs, SINGLE SNP THAT OCCURS IN THE BLACK 6 J MOUSE IN THE tRNA WHICH LEADS TO A PROCESSING DEFECT, AND LOSS OF MATURE LEVELS DRAMATICALLY LOWERS THE POOL, IF YOU WILL, OF tRNA COMING FROM THESE GENES. SO, IN THIS CASE WE AGAIN HAVE A PRIMARY MUTATION THAT IS GTPBP 2, SHOWED FROM THIS WORK, A RESCUE FACTOR, BUT UBIQUITOUSLY EXPRESSED, AND PHENOTYPE WAS STRICTLY NEURONAL, AND THIS IS CONFERRED AT LEAST IN PART BY THE EXPRESSION LEVEL, THE EXPRESSION, SORRY, PLACE OF EXPRESSION OF NTR 20. SO, AFTER WE COULD SHOW THIS WAS A MODIFIER OF NEURODEGENERATION WE REALLY WONDERED, COULD LOSS OF A SINGLE MULTI-COPY tRNA LEAD TO ANY NEUROPHYSIOLOGICAL PHENOTYPES? AND WE NOW THERE WERE STUDIES FROM MOUSE THAT POINTED TO DISTAL CHROMOSOME 1 AS A BASIS FOR MANY BEHAVIORAL PHENOTYPES, BUT OF COURSE THIS QTL WAS BROAD AND CONTAINED 140 GENES. NONETHELESS, WE INVESTIGATED NTR 20 AND LOOKED AT DATA FROM HIPPOCAMPAL NEURONS SLICE CULTURE THAT HAD A LOSS OF FUNCTION. AND WE FOUND THAT JUST THE LOSS OF N-TR20 CAN INCREASE INHIBITION IN CA1 OF THE HIPPOCAMPUS AND IT ALSO LEADS TO INCREASE IN SEIZURE THRESHOLD, SO INCREASE IN SEIZURE RESISTANCE. INTERESTINGLY, THIS HAD BEEN KNOWN IN BLACK 6 J FOR SOME TIME THESE MICE WERE RESISTANT SEIZURES SUGGESTING TO US THIS TRNA MAY MODIFY MUTATIONS THAT PERHAPS CHANGE A NEUROPHYSIOLOGY AND SYNAPTIC TRANSMISSION, AND WE TURNED TO A POINT MUTATION IN THE GAP A RECEPTOR GAMMA2 THAT IN PATIENTS HAD BEEN ASSOCIATED WITH CHILDHOOD EPILEPSY AND FEBRILE SEIZURES, BUT QUITE INTERESTING TO US BECAUSE IT WAS INCOMPLETELY PENETRANT. ONLY 20% OF KIDS WITH THIS HETEROZYGOUS FOR THIS MUTATION HAVE SPIKE WAVE DISCHARGES, AND ONLY 65% HAVE FEBRILE SEIZURES. SO TO INVESTIGATE WHAT THEIR EXPRESSION LEVELS OF N-TR20 CAN ALTER, SEIZURE APPEARANCE IN THESE MICE, BEARING THIS MUTATION, WE EXPRESS NORMAL LEVELS OF N-TR20 AND COULD ININCREASE RELATIVE TO THE ORIGINAL MODEL MADE ON BLACK 6J. SO, FROM THIS WORK ON N-TR20 WE'VE COME DOWN TO A WORKING MODEL THINKING ABOUT tRNAs AS DOSE SENSITIVE MODIFIERS. SO, A NUCLEAR tRNAs ARE GENERALLY ENCORRODED BY MULTIPLE GENES, AND WE BELIEVE THAT THE AMOUNT OF THE LEVEL OF EXPRESSION FROM A GIVEN GENE AND WILL REALLY DICTATE ITS IMPACT AS POTENTIAL MODIFIER. FOR EXAMPLE, N-TR20 IS EXPRESSED HIGHLY IN NEURONS, WHEN IT'S DELETED IT LEADS TO REDUCED NEURONAL POOL OF THESE UCU tRNAs, WHICH IN TURN CAUSES RIBOSOME STALLING, AND LEADS TO TWO SIGNALING PATHWAYS CHANGES. ONE IS AN INCREASE IN ACTIVATION IN THE INTEGRATED STRESS RESPONSE, AND THE SECOND IS A DOWNREGULATION OF mTOR 1 ACTIVITY, BOTH OF WHICH WILL THEN FEEDBACK TO CHANGE TO DECREASE RIBOSOME INITIATION ON TRANSLATING mRNAs. BUT WE REALLY WANT TO EXTEND THIS WORK AND LOOK AT OTHER VARIANTS IN NUCLEAR ENCODED -- HUMAN NUCLEAR ENCODED tRNAs. WE KNOW THAT THERE'S MANY VARIANTS THROUGHOUT THE BODY OF THESE tRNAs, AND AS MICHAEL SHOWED THIS MORNING WE'VE BEEN ABLE TO FIND THAT UPSTREAM SNPs ALSO CAN CHANGE THE EXPRESSION LEVEL OF THESE tRNAs, AND THEY SERVE AS MODIFIERS. WE'RE STUCK WITH THIS PROBLEM. IN HUMANS AND MICE THERE'S FOUR TO FIVE HUNDRED NUCLEAR ENCODED tRNAs. AND HOW WILL WE KNOW WHICH ONES ARE EXPRESSED WHERE? THIS IS SOMETHING THAT WE'VE REALLY BEEN TACKLING IN A DISTINCT WAY, AND WE THINK IT'S IMPORTANT TO HAVE AN ALTERNATIVE APPROACH, I'M GOING TO TELL YOU HERE, BECAUSE RNA SEQUENCING ISN'T QUANTITATIVE ON tRNAs, LARGELY BECAUSE OF THE MANY MODIFICATIONS, BUT MANY tRNAs ARE IDENTICAL OR NEARLY IDENTICAL. SO IT'S GOING TO BE IMPOSSIBLE TO MAP READS BACK TO SPECIFIC GENES THAT MAY HAVE VARIANTS. SO WHAT WE'VE DONE NOW IS MADE A CONDITIONAL EPITOPE TAGGED MOUSE WITH FLAG-tag ON POL3 ALLOWING CHIP-seq AS SURROGATE FOR tRNA EXPRESSION. AND I JUST WANT TO END WITH THIS SLIDE SHOWING YOU THE INTERESTING DATA WE'RE GETTING FROM THESE EXPERIMENTS, FIRST OF ALL IF WE LOOK AT tRNA FLAG LARGE TCT, UCU FAMILY, YOU CAN SEE THAT THE N-TR20 GENE SHOWN HERE IN GREEN IS EXPRESSED ON THE GRANULE CELLS, NOT IN ASTROCYTES OR MICROGLIA. YOU CAN ALSO SEE BY LOOKING AT SOME OF THESE OTHER FAMILIES THAT THE LEVEL OF EXPRESSION OF THESE tRNAs VARIES DEPENDING ON CELL TYPE. NOTABLY I'LL POINT YOU HERE TO PURKINJE CELLS THAT SUPPRESS MANY MORE FAMILY MEMBERS OF tRNAs WITH CTG OR CAC CODONS. OUR HOPE IS TO TAKE THIS ATLAS THAT WE'RE DEVELOPING OF tRNA EXPRESSION AND OVERLAY IT WITH VARIANTS, AND LOOK AT THE EFFECTS OF THESE VARIANTS. WITH THAT I WANT TO ACKNOWLEDGE THOSE IN THE LAB AND DID MUCH OF THE WORK I SHOWED YOU, AS WELL AS FORMER POSTDOCS AND FORMER GRADUATE STUDENT, MARCUS TERRY, WHO WORKED ON THE 16 PROJECT. >> THANK YOU, SUSAN. BEAUTIFUL TALK. MAYBE IF YOU WOULD TAKE THE SLIDES DOWN AND WE HAVE A COUPLE MINUTES FOR QUESTIONS UNTIL WE HEAR FROM OTHERS, I HAVE ACTUALLY A QUESTION. SINCE YOU STARTED A COUPLE OF THOSE, IS THERE EXPRESSION PATTERN CONSERVED BETWEEN SPECIES? RIGHTS. >> OR CODON USAGE IS SLIGHTLY ALSO DIFFERENT BETWEEN SPECIES. >> IF WE LOOK AT N-TR20, IT APPEARS AMAZINGLY CONSERVED, IT'S ONLY EXPRESSED IN THE BRAIN OF HUMANS. WE HAVEN'T -- WE ALSO KNOW IT'S EXPRESSED IN iPSCs, WHEN THEY ARE DIFFERENTIATED INTO NEURONS, NOT BEFORE. SUGGESTING THAT IT'S NEURONAL. IT'S ALSO EXPRESSED IN THE BRAIN OF CHICKEN. SO IT LOOKS LIKE IT'S VERY CONSERVED. AND THIS IS SOMETHING WE'RE NOW DOING USING HUMAN iPSC NEURONS TO FURTHER CHECK OTHER GENES. >> GREAT. ALL RIGHT. I THINK WE PROBABLY WILL TAKE THE DISCUSSION A LITTLE BIT MORE SINCE WE ONLY HAVE 50 SECONDS TO THE END OF THE SESSION. THERE'S ONE QUESTION. LET'S TRY TO ANSWER IT REAL QUICK. IT'S FROM JOSE DOMINGO. GOOD AFTERNOON. DO YOU HAVE ANY BIOLOGICAL EXPLANATION FOR ALL THE SURPRISINGLY HETEROGENEOUS tRNA EXPRESSION? >> AH, WE DO NOT HAVE A BIOLOGICAL EXPLANATION NOW. WE CAN CATALOG IT, ONE POSSIBILITY, FOR EXAMPLE, IN PURKINJE CELLS WE'D LIKE TO INVESTIGATE ARE DIFFERENT tRNAs COMPARTMENTALIZED IN DIFFERENT REGIONS OF THESE CELLS OR WE'RE LOOKING AT PURKINJE CELLS AS A WHOLE POPULATION. WE KNOW PURKINJE CELLS, OTHER GENES ARE EXPRESSED IN SPECIFIC SUBSETS, SOMETHING WE NEED TO GO BACK AND LOOK AT. >> JENNIFER, VERY INTERESTING TALK. DO YOU HAVE THOUGHTS ABOUT IMPLICATIONS FOR tRNAs THERAPEUTICS? >> WELL, I THINK ONE NEEDS TO THINK ABOUT WHETHER YOU'RE EXPRESSING tRNAs OR KNOCKING THEM DOWN, AND THIS IS A DISCUSSION WE SHOULD CERTAINLY CONTINUE IN THE HAPPY HOUR. >> SOUNDS GREAT. THANK YOU SO MUCH, SUSAN. AND FOR OUR LAST SPEAKER FOR TODAY'S PRESENTATION, PROFESSOR ROHAN D'SILVA, IN THE FIELD OF NEURODEGENERATION. SUSAN, IF YOU COULD TAKE YOUR -- ROHAN, GO AHEAD AND SHARE THE SCREEN. DO THE SLIDES. GREAT. PROFESSOR OF MOLECULAR NEUROAS SOON AS, UNIVERSITY COLLEGE LONDON, INSTITUTE OF NEUROLOGY, DEEPLY ROOTED IN RESEARCH IN NEURODEGENERATION AND WAS INVOLVED IN THE IDENTIFICATION OF EARLY ALZHEIMER'S DISEASE, CHARACTERIZATION OF SYNUCLEIN AND STUDIED TAU, SYNUCLEIN AND OTHER NEURODEGENERATIVE PROCESSES THROUGHOUT THE YEARS, CURRENTLY HIS GROUP HAS BEEN FOCUSED ON DIFFERENT FACTORS THAT REGULATE PRODUCTION OF TAU PROTEIN. AND THROUGH THAT WORK, A VERY EXCITING DISCOVERIES ABOUT TAU. WHEN I PRESENTED YOUR PAPER, ROHAN, I WAS TOTALLY BLOWN AWAY BY THE BEAUTIFUL WORK. WE'RE ALL LOOKING FORWARD TO HEARING FROM YOU TODAY. >> THANK YOU, HUDA. CAN YOU SEE MY SCREEN AND HEAR ME? >> WE DON'T SEE YOUR SLIDES. CAN YOU SHARE YOUR SCREEN ONE MORE TIME? >> SORRY, YEAH. CAN YOU SEE THAT? >> NOW WE SEE IT. JUST PUT IT IN FULL SLIDE. PERFECT. >> OKAY. PERFECT. THANKS A LOT. THANK YOU, HUDA. THANKS TO THE ORGANIZERS OF THIS AMAZING WORKSHOP, AND ALSO IT'S A GREAT PRIVILEGE TO BE IN THE COMPANY OF SO MANY LEADING LIGHTS IN THE FIELD. SO, AS WE'RE TALKING GENETIC MODIFIERS, LONG CODING RNA GENE THAT OVERLAPS IN ANTISENSE AND REGULATING TAU TRANSLATION. AND AS WE ALL KNOW TAU IS AN IMPORTANT PLAYER IN A VARIETY OF NEURODEGENERATIVE DISORDERS INCLUDING ALZHEIMER'S DISEASE, AND WE TAUOPATHIES ARE CHARACTERIZED BY NEURAL AND/OR GLIAL INCLUSION CONSISTING OF TAU PROTEIN. TAU, LIKE SEVERAL OTHER PROTEINS, WHOSE AGGREGATION IS ASSOCIATED WITH NEURODEGENERATION, IS A DISORDERED PROTEIN, CLUES CAUSATIVE MUTATIONS IN FRONTOTEMPORAL DEMENTIA, POST-TRANSLATIONAL MODIFICATION, TAU UNDERGOES PATHOLOGICAL CONVERSION, AND THESE IN PRION-LIKE FASHION TEMPLATE CONVERSION OF HEALTHY TAU RESULTING IN CASCADE-LIKE SPREAD OF PATHOLOGY IN THE BRAIN, AS DISEASE PROGRESSES. SO AND THIS IS WELL DESCRIBED IN EARLY DAYS OF ALZHEIMER'S DISEASE, WITH STAGING SHOWN PATHOLOGY AS THE DISEASE PROGRESSES. SO, DURING OUR WORK ON TAU GENE WE IDENTIFIED LONG NON-CODING GENE OVERLAPPING, THE FIRST NON-CODING TAU PATHOLOGY GENE MAPT, AND IN THE OPPOSITE DIRECTION, AND IF I CAN ACKNOWLEDGE A VERY TALENTED AND DEDICATED, A MAJOR PART THANKS TO HIS HERCULEAN EFFORTS AND CREATION. ALTERNATIVE SPLICING IDENTIFIED THREE TRANSCRIPTS THAT WE CALL MAPT 1 AND MAP 2, NATURAL ANTISENSE TRANSCRIPTS. THESE TRANSCRIPTS ARE ENRICHED AND UPREGULATED, AND UPREGULATE DURING NEURONAL DIFFERENTIATION OF iPS CELLS. BASED ON OVERLAP WITH TAU PROMOTER, WE SURMISE TRANSCRIPTS CAN AFFECT TAU TRANSCRIPTION, SO HOWEVER WITH STABLE OVEREXPRESSION OF THE TAU, MAPT 1 AND 2, KNOCK DOWN OF ENDOGENOUS TRANSCRIPTS WE SAW NO CHANGE IN TAU TRANSCRIPT LEVELS AS YOU CAN SEE HERE WITH OVEREXPRESSION OF MAPT 1 AND 2, ALSO KNOCKDOWN OF TRANSCRIPTS, YOU CAN SEE NO EFFECT ON THE TAU TRANSCRIPT. HOWEVER, WITH KNOCKDOWN OF ENDOGENOUS MAPT WERE SURPRISED TO SEE INCREASES IN TAU PROTEIN LEVELS, FOR EXAMPLE HERE IN NEUROBLASTOMA CELLS, KNOCKED DOWN RNA, YOU CAN SEE INCREASES IN TAU PROTEIN LEVEL COMPARED TO THESE. IN THESE iPSC MULTI-NEURONS, ONCE AGAIN YOU CAN SEE INCREASED TAU LEVELS DUE TO KNOCKDOWN OF THE TRANSCRIPT, OF THE NON-CODING TRANSCRIPTS, FLUORESCENCE. IN CONCLUSION FROM THESE DATA IT IS DESPITE VERY LOW LEVELS THESE MAPT CAN REPRESS TAU TRANSLATION IN SUB-STOICHIOMETRIC MANNER. THE FIRST PLACE, WITH MAPT 1 WE HAVE THIS REGION WHICH OVERLAPS WITH THE CO-PROMOTER. THEN WE HAVE THIS DISTAL SHARED BY TRANSCRIPTS, THIS RED REGION HERE, THIS IS OFF MAMMALIAN FIRST REPEAT CLASS. AND I'LL COME BACK TO THIS LATER. TO IDENTIFY THE ESSENTIAL DOMAINS OF THESE TRANSCRIPTS, OVEREXPRESSED VARIOUS DELETION AND MUTATION CONSTRUCTS, VARIANTS OF THESE TRANSCRIPTS, INCLUDING DELETED NEAR ELEMENTS, AND HE FLIPPED THIS REGION OF ANTISENSE OVERLAP TO SEE IF COMPLEMENT HAS ANYTHING TO DO WITH THE FUNCTION. LONG STORY SHORT, IN THE FIRST PLACE YOU CAN SEE TRANSCRIPTS, ROBUST DOWNREGULATION OF TAU PROTEIN LEVELS, WITH FULL LENGTH OVEREXPRESSION OF FULL LENGTH TRANSCRIPT IN NEUROBLASTOMA CELLS BUT IF YOU REMOVE EITHER THE MIR ELEMENT OR FLIP THE REGION OF OVERLAP WITH FIVE PRIME REGION OF TAU TRANSCRIPT YOU CAN SEE REVERSAL OF THIS, IT GOES IN OPPOSITE DIRECTION, INCREASED LEVELS OF TAU PROTEIN. SO, INTEREST, WHEN WE JUXTAPOSED JUST THESE TWO ESSENTIAL REGIONS, REGION OF ANTISENSE OVERLAP AND MIR ELEMENT, THIS REPRESSION, THE EFFECT IN SOME CASES, EFFECT IS STRONGER THAN THE FULL LENGTH TRANSCRIPT. SO IN SUMMARY, THE REGION, THIS REGION OF FIVE PRIME OVERLAP AND MIR ELEMENTS ARE ESSENTIAL FOR ACTIVITY. TO FURTHER DEMONSTRATE THE TRANSLATIONAL LEVEL IN COLLABORATION WITH COLLEAGUES, ROBERTO SHOWED RIBOSOMAL PROFILING IN CELLS THAT OVEREXPRESS LEFT TRANSCRIPTS THERE'S REDUCED RECRUITMENT OF TAU HERE SHOWN IN THE SOLID BLUE AND SOLID RED BARS HERE. THERE'S REDUCED RECRUITMENT OF TAU MESSAGE WITH OVEREXPRESSION OF THE FULL LENGTH MAPT 1 AND MAPT, AND THIS REDUCTION IS REVERSED WITH DELETION OF THE MIR LIMIT AND HERE CELLS OVEREXPRESSING. THIS HEAVY ELEMENT, THIS HEAVY POLY SOMAL FRACTION REPRESENTS THE FRACTION WHERE ACTIVE PROTEINATION IS TAKING PLACE. SO THIS INDICATES MAPT 1 AND 2 IMPAIR RECRUITMENT OF TAU MESSENGER RNA TO THE RIBOSOME AND THIS RELIES ON THE MIR DOMAIN. SO THIS MIR ELEMENT, IT IS ONE OF THOSE TRANSPOSABLE ELEMENTS COMING IN THE HUMAN GENOME, AND IN ITSELF MAKES UP ABOUT 2.6% OF THE HUMAN GENOME, AND SO -- YEAH, GO TO THE MESSAGE, I'M SORRY. IT'S A RETROTRANSPOSAL USUALLY 260 NUCLEOSIDES IN LENGTH BUT THEY HAVE THIS 60 HIGH NUCLEOTIDE SEQUENCE, AND THIS IS CONSERVED IN ALL PRIMATES. SO THIS SUGGESTS FUNCTIONAL ELEMENTS. SO, POSSIBLE MECHANISM, THIS IS QUITE A COMPLICATED SET OF PICTURES. FIRST CASE, THE REGION OF THE TAU TRANSCRIPT CONTAINS THIS STRUCTURED INTERNAL RIBOSOME ENTRY SITE, IRES, ALLOWS TRANSLATION OF TAU THROUGH DIRECT RECRUITMENT TO THE RIBOSOME. USING ALIGNMENT OF CONSERVED REGION I MENTIONED BEFORE ROBERTO IDENTIFIED TWO MOTIFS, ONE AND TWO HERE. COMPLEMENTARITY AND IDENTITY, SEVERAL SEQUENCES IN RIB RIBOSOMAL RNA AND IRES. THREES OF EITHER OF THESE TWO MOTIFS, ONCE AGAIN, REMOVES THIS TRANSFERATIONAL REPRESSION, AT LEAST PARTIALLY, NOT AS STRONG AS FULL LENGTH BUT YOU CAN SEE INCREASE IN TAU PROTEIN COMPARED TO FULL LENGTH. BASED ON THIS EVIDENCE, ROBERTO PROPOSED THIS COMPLEMENTARITY, AND BINDING TO THE TAU IRES, SO NAT1 CAN AFFECT RECRUITMENT BINDING TO MOTIFS, BE A TENSE OF NAT1, TO THE RIBOSOME AND TRANSLATION. I'D LIKE TO POINT OUT H1 H2 H2 POLYMORPHISM THAT AFFECT SEVEN MOTIFS, AND THIS POLYMORPHISM IS STRONGLY ASSOCIATED WITH PARKINSON'S DISEASE, AND ITS PROTECTED ALLELE COULD REDUCE INTERACTION AND TRANSLATION. IN FACT, SEVERAL SEPARATE STUDIES SHOWED THESE DECREASED LEVELS OF PROTEIN TRANSCRIPTS, IN SUBSUBSTANTIA NIGRA OF PARKINSON'S DISEASE PATIENTS, AND ALSO INCREASED TAU PROCESS, WITH LIMITED NUMBERS. SO FURTHERMORE, ROBERTO LOOKED AT BRAIN EXPRESSION DATA FROM WHO LARGE POSTMORTEM BRAIN COLLECTIONS AND SHOWED FIRSTLY THE EXPECTED -- SORRY, I SKIPPED. HE SHOWED EXPECTED INCREASES IN THE PATHOLOGICALLY PHOSPHORYLATED TAU LEVELS WITH INCREASES THAT I MENTIONED EARLIER. AND ALSO INCREASING BRAAK STAGE REDUCTION IN TRANSCRIPT LEVEL OF ANTISENSE GENE, SO THIS SUGGESTS DYSREGULATION OF TAU EXPRESSION. SO, WHAT IS THE RELEVANCE OF THE MIR-CONTAINING AND NON-CODING GENES? TOGETHER WITH TAU WE'RE PROBABLY LOOKING AT LARGER CLASS OF THESE GENES, AND THEY HAVE PARTICULAR EVENTS IN REGULATION OF GENES IN THE CENTRAL NERVOUS SYSTEM, AND THOSE INVOLVED IN NEURODEGENERATION AND ALSO PROTEIN STASIS OF INTRINSICALLY DISORDERED PROTEINS, SO I'D LIKE TO REITERATE ONCE AGAIN TRANSCRIPTS OF NON-CODING GENES THAT OVERLAP ANTISENSE WITH CODING GENES, SO IN THE FIRST PLACE MORE THAN TWO-THIRDS OF NON-CODING TRANSCRIPTS CONTAIN EMBEDDED TRANSPOSABLE ELEMENTS, AND OF THESE NEARLY 6%, 1200 GENES CONTAIN MIR ELEMENTS. AND OF THESE MIR-NATs, WE CALL THEM, 41% OVERLAP WITH FIVE PRIME REGION OF THEIR GENES, PROTEIN CODING GENES, AND 37% OVERLAP THREE PRIME REGION, AND THE REST OVERLAP WITH CODING SEQUENCES. WHAT I FIND MOST CURIOUS IS THIS BIAS OF THE 41% OF THESE GENES THAT OVERLAP WITH FIVE PRIME UNTRANSLATED REGION, CODING GENES ARE SIGNIFICANTLY LOW, MORE HIGHLY EXPRESSED IN THE BRAIN IN GREEN, AND ALSO THESE CODING GENES ARE ASSOCIATED WITH DEMENTIA AND PARKINSON'S DISEASE, MYOTROPIC LATERAL SCLEROSIS, NEURONAL PROJECTION. ROBERTO SHOWED 446 MIR-NATs ARE DIFFERENTIALLY EXPRESSED IN ARIZONA DISEASE, AND AT LEAST 40% OF THESE GENES CODE FOR INTRINSICALLY DISORDERED PROTEINS, WITH GREATER THAN 90% INTRINSICALLY DISORDERED REGIONS. AND ALSO MIR-NATs HAD CODING GENE PRODUCTION, FORMING EXTENSIVE PROTEIN-PROTEIN INTERACTION NETWORK, OVER 40% OF DISEASE ARE INTRINSICALLY DISORDERED PROTEINS SHOWN BY RED CIRCLES HERE, AND THESE GENES ARE HIGHLY ENRICHED. AND THIS IS PARTICULARLY THE CASE WITH THE HUB PROTEINS, THE ONCE 40 INTERACTORS. 90% OF INTRINSICALLY DISORDERED. PROTEINS ASSOCIATED WITH NEURODEGENERATIVE DISORDERS ARE OVERREPRESENTED IN THESE HUBS, AND HERE WE HAVE A VIEW OF THIS CENTRAL HUB, CENTRAL CLUSTER, AND YOU CAN NOTE FAMILIAR CULPRITS IN THIS LIST. NOTABLY, THE MAJORITY OF GENES HAVE RESIDENCE OVERLAPPING FIVE PRIME UNTRANSLATED REGION. FINALLY IN ORDER TO TEST IF OTHERS SHOWED SIMILAR, BASED ON SIMILAR TOPOLOGY, SEQUENCE MOTIFS INTERACTIONS WITH RIBOSOMAL SUBUNITS, WHAT IS SHOWED IS OVEREXPRESSION OF THE FULL LENGTH TRANSCRIPT CAUSED REDUCTION IN PROTEIN LEVELS, EXCLUSION OF MIR ELEMENTS REMOVED THIS EFFECT. SO, ALL OF IT -- SORRY. IT REMAINS TO BE SEEN IF ALL MIR-NATs WORK BY THE SAME MECHANISM. I WOULD LIKE TO CONCLUDE WITH THE INTRIGUING POSSIBILITY THAT THESE MIR-NATs PRESENT THIS ADDITIONAL LAYER FOR THE TIGHT REGULATION OF PROTEOSTASIS OF INTRINSICALLY DISORDERED PROTEINS, PARTICULARLY CENTRAL NERVOUS SYSTEM. AND ANY SURPLUS OF THESE PROTEINS BY VIRTUE OF THEIR ABILITY TO TAKE ON TRANSIENT STRUCTURE RELEVANCE, THE DIRECTIONS, THE MORE STABLE PROTEINS IN NEURODEGENERATIVE DISORDERS, ALSO COME TO THE GWAS STUDIES AND FREQUENT IDENTIFICATION, IT'S POSSIBLE SOME MAY AFFECT ONE CODE LONG CODING GENES AND INCREASE DISEASE RISK. ALSO NOTED, SPECIFICITY OF THESE MIR-NATs ESPECIALLY BASED ON OVERLAP WITH FIVE PRIME REGION OF THE CODING GENES COULD BE USED FOR THERAPEUTIC TARGETING OF THESE GENES, PARTICULARLY WITH DOWNREGULATION AND WE'VE BEEN TALKING ABOUT THIS. WITH THAT I'D LIKE TO CONTRIBUTE THIS SLIDE OF ALL OUR COLLABORATORS FROM LONDON, ITALY AND STOCKHOLM. AND THANK YOU FOR LISTENING. >> THANK YOU VERY MUCH. WE CAN TAKE DOWN THE SLIDES. WE HAVE ONE QUESTION AND WE'LL MOVE TO THE NEXT SECTION BECAUSE WE RAN OUT OF TIME. FROM MATTHEW, GREAT TALK. ARE THERE ELEMENTS WITHIN THE MAPT TAU PROMOTER THAT COULD REGULATION TRANSCRIPTION? AS FAR AS WE KNOW, NO. >> MAYBE I'LL KICK OFF THE DISCUSSION. IT'S INTERESTING, FIRST OF ALL, THAT YOU FOUND SO MANY ENRICHMENT PROTEINS, DISORDERED PROTEIN, AND YOUR POINT THESE COULD BE HARNESSED AS POTENTIAL THERAPEUTIC, CAN YOU TELL ME ABOUT YOUR THOUGHTS HERE? >> IN THE FIRST PLACE, I'M A TAU PERSON. WITH TAU THERE HAVE BEEN -- WE ALREADY HAVE ANTISENSE THERAPIES IN THE CLINIC. WE HAVE ALL THESE PROTEINS, IT SEEMS THAT REDUCTION OF THIS SUBSTRATE, REALLY FOR AGGREGATION, IS BENEFICIAL WITHOUT, AND IN MOUSE CAN KNOCK DOWN TAU QUITE SUBSTANTIALLY WITHOUT CAUSING ANY DELETERIOUS EFFECT. SO I THINK IN TERMS OF REDUCTION OF EXPLOITING GENES TO REDUCE TAU, I GUESS WE NEED TO HAVE GENE THERAPY APPROACH BECAUSE IT'S QUITE EASY TO DELIVER, DELIVER RNAs, SO, YEAH. >> GREAT. THANK YOU. THERE'S ONE MORE QUESTION. IS THERE ANY REASON TO HAVE SEVERAL NATs? >> SO, YEAH, THAT'S A GOOD QUESTION BECAUSE -- AND REVIEWERS TO OUR PAPER ASKED THE SAME QUESTION. SOME NATs DON'T QUITE OVERLAP, I DIDN'T EXPLAIN THAT PROPERLY. INTERNAL ENTRY SITES, I SKIPPED THAT. BUT THE FIVE PRIME OVERLAP IS EITHER CO-PROMOTER OR DOWNSTREAM SEQUENCES, SO WE HAVE NOT ANSWERED THAT QUESTION YET. >> WELL, SUSAN, I INVITE YOU TO JOIN ME SO WE CAN HAVE A BROADER DISCUSSION. I WOULD LIKE TO THANK ALL THE SPEAKERS TODAY. REALLY, REALLY BEAUTIFUL DAY, A LOT OF INSIGHT, THE POWER OF GENETIC STUDIES FROM HUMAN ALL THE WAY TO MULTIPLE ORGANISMS, AND BIG SHOUT OUT TO THE TRAINEES WHO I MUST SAY WERE THE ONLY ONES REALLY ON TIME, AND IN SPITE OF THE VERY LIMITED PLATFORM THEY DELIVERED THEIR MESSAGES WONDERFULLY. GREAT THANKS FOR THE TRAINEES. NOW WE HAVE ABOUT 20 MINUTES OR SO TO REALLY DISCUSS SOME ELEMENTS OF THE DAY, AND SOME IDEAS, WE DIDN'T HAVE A CHANCE TO PERHAPS COVER DEEPLY. SUSAN, I DON'T KNOW IF YOU HAVE ANY THOUGHTS. I HAVE A COUPLE FOLLOW-UP QUESTIONS OR THOUGHTS FOR SOME PANELISTS. BUT I'LL LET YOU MAYBE GO FIRST, IF YOU HAVE COMMENTS. >> I WANTED TO FOLLOW UP WITH HUGO. I THINK THAT THE IDEA THAT CERAMIDES, LYSOSOMES, ET CETERA, PLAY SUCH AN IMPORTANT ROLE IN NEURONAL SURVIVAL, AS EXCITING AND CONTINUES TO DEVELOP. I'M WONDERING, DO YOU HAVE ANY THOUGHTS ABOUT NEURONAL SPECIFICITY OF THESE DISEASES? >> THAT'S A GOOD QUESTION. WHEN WE LOOK IN FRUIT FLIES THEY SEEM TO AFFECT MANY DIFFERENT NEURONAL POPULATIONS. WE USE THE PHOTORECEPTORS BECAUSE PHOTORECEPTORS HAVE A HUGE ADVANTAGE. YOU CAN CONTROL LIGHT. THEREFORE YOU CAN CONTROL NEURONAL ACTIVITY. AND GIVEN THE TRANSMISSION OF ELECTRON MICROSCOPY IN THE EYE IS STANDARD AND VERY EASY TO IDENTIFY EVERY CELL, THAT'S OUR FAVORITE SUBSTRATE. EVERY TIME WE'VE LOOKED AT OTHER CELLS, NOT REGIONAL SPECIFIC FOR MOST OF THEM. SO, FOR ALZHEIMER'S DISEASE MODELS WE SEE ACCUMULATION OF LIPID DROPLETS IN GLIAL CELLS. AND WE'VE SEEN THIS REPEATEDLY. AND, YOU KNOW, LIPIDS GET TRANSFERRED TO GLIAL CELLS, OBVIOUS IN MANY GLIAL CELLS, AGAIN WE USED PHOTORECEPTORS. ALL OF WHAT WE'VE DONE WE VERIFY WITH VERTEBRATE CELLS AND TAKE PRIMARY NEURONS FROM RATS TO VERIFY WHAT WE SEE IN THE FLY IS IMMEDIATELY TRANSLATABLE TO PRIMARY CELLS. NEURONS FROM RATS OR MICE OR EVEN CULTURED CELLS LIKE I SHOWED FOR, YOU KNOW, SO I DON'T KNOW HOW MUCH SPECIFICITY THERE IS BUT IN GENERAL IT SEEMS TO BE TRANSLATABLE JUST IN CELL CULTURE EXPERIMENTS. >> OKAY. >> I HAVE A FOLLOW-UP QUESTION ALSO TO HUGO. SO, CLEARLY THE STABILITY OF THE PROTEINS IS REALLY CRUCIAL, RIGHT, IN THOSE DISORDERS. I'M CURIOUS HAS VPS 26 OR 29 BEEN IMPLICATED IN ANY NEURODEGENERATIVE DISORDERS? >> NO. 35 FOR PARKINSON'S DISEASE, BUT VPS 26 I'M NOT AWARE, ALSO VPS 29 NOT AWARE >> I GUESS THEY WOULD BE CANDIDATES. THE QUESTION FOR YOU, THEY OBVIOUSLY WORK AS A COMPLEX BUT HAS THERE BEEN EXPERIMENTATION TO SEE IF YOU UPREGULATE ONE YOU COULD MAKE UP FOR LOSS OF ANOTHER? >> NOT FOR VPS 29 BUT FOR 26 AND 35, IF YOU OVEREXPRESS 36, 35 GOES UP. IF YOU OVEREXPRESS 35, 26 GOES UP. THEY ARE IN SYNC. ONE GOES DOWN, THE OTHER GOES DOWN. ONE GOES UP, THE OTHER GOES UP. THERE'S A COMPLETE SYNCHRONY BETWEEN THOSE TWO. >> SO, MAYBE THIS IDEA OF HAVING THE PARALOGS TOGETHER AND MAYBE IF WE CAN UPREGULATE, WHEN DEFICIENT, I WANT TO TAKE THIS TRACK. MAYBE WE HAVE TO THINK OF STRATEGY, OF STRATEGIES THAT WE CAN UPREGULATE SOME OF THOSE GENES IN THE CASE OF DEFICIENCY OF ONE. SO, TONY, I'D LIKE TO HEAR FROM YOU. WOULD WE EVER BE SUCCESSFUL IN THAT REGARD? >> YEAH, NO, I DON'T SEE WHY NOT. THAT IS ONE SORT OF POTENTIAL INTERVENTION SORT OF MECHANISM. AND I THINK THAT'S IDENTIFIED THE BEST WAY TO DO THAT, HAS A LOT OF POTENTIAL. IT'S OF COURSE A QUESTION OF LIKE IF IT'S VERY MUCH ON THE SPECIFIC SORT OF MECHANISMS OF A GIVEN GENE AND GIVEN DISEASE IF YOU NEED A TARGET GENE OR IF IT'S ENOUGH TO -- LET'S SAY THERE'S A PATHWAY, WHATEVER ITS FUNCTION IS, THE DOWNSTREAM FUNCTION, BUT YEAH. >> WHILE I'VE GOT YOU, HAVE THERE BEEN EFFORTS TO TAKE LARGE DATASET, GENETIC INFORMATION PUT ON GENOME SEQUENCING, AND RNA EXPRESSION IN THE SAME INDIVIDUALS, TO BEGIN TO CORRELATE REGULATORY ELEMENTS WITH, YOU KNOW, THE EFFECT ON DOWNSTREAM TRANSCRIPT? AND I DON'T KNOW IF U.K. BIOBANK FOR EXAMPLE OR SOME OR MORE LARGER SEQUENCING EFFORTS, I THINK WITH GENOMES INCORPORATED GENOME EXPRESSION STUDIES, ARE YOU AWARE OF THAT? >> YEAH, YEAH. NO, THAT'S BEING DONE, CHOP MED HAS 20 SAMPLES WITH RNA SEQUENCING DATA SORT OF COMING UP, MOST FROM BLOGS, SO TISSUE AVAILABILITY, EVENTUALLY STRONG TRADEOFFS, BUT THERE'S DATA AND ALSO WHAT PEOPLE HAVE BEEN DOING FOR THE STUDIES, GENETIC GENE EXPRESSION AND ASSOCIATES THAT PREDICTED EXPRESSION, TWAS APPROACH, IF WE -- AS WE BUILD MAPS OF VARIANTS IN THE LABS FURTHER. >> TUULE, I HAD A QUESTION TOO COMING BACK TO THIS IDEA, THE QUESTION THAT HUDA ASKED CAN WE UPREGULATE THESE GENES. SO WHEN YOU DO -- WE KNOW THAT IN MANY CASES HETEROZYGOUS KNOCKOUT DOESN'T EXPRESS 50% OF THE mRNA, RIGHT? AND SO CAN WE LEARN, I DON'T KNOW ENOUGH ABOUT HOW THAT COMPENSATORY UPREGULATION OF THE WILD TYPE ALLELE NORMALLY HAPPENS BUT THIS IS IN THE CASE WITH NO SNPs AT ALL, EVEN IN A MOUSE. CAN WE TAKE ANYTHING THAT WE'VE LEARNED FROM THIS SORT OF COMPENSATION AND USE THAT AS A WAY TO UPLEG RATE THESE GENES? AND THE SECOND PART TO THAT, DO WE KNOW IF THIS KIND OF COMPENSATION FROM THE WILD TYPE ALLELE OCCURS IN SPECIFIC CLASSES OF GENES? SUCH AS RNA-BINDING PROTEINS OR IS IT WIDESPREAD? >> WE DON'T SEE COMPENSATORY MECHANISMS THAT WIDELY, LIKE WHEN WE LOOK AT SORT OF THE VARIANTS, WE LOOK AT THE THE EXPRESSION, TWO ORTHOGONAL METRICS TO MEASURE EFFECT ON TOTAL EXPRESSION AND THEY TEND TO DO IT WELL. THERE'S SITUATIONS WHERE THERE IS COMPENSATION AND ONE COULD ALSO IMAGINE WHEN WE LOOK AT THESE DATA WE LOOK AT GENOME WIDE, WE LOOK AT THIS INCLUDES GENES, ONE COULD IMAGINE THIS IS SPECIFIC, SENSITIVE GENES MIGHT HAVE EVOLVED TO HAVE COMPENSATORY MECHANISMS. BUT WE HAVE BEEN DISCUSSING A NUMBER OF POSTDOCS, HEY, WHEN WE CORRELATE THE TWO THINGS AND SEE THERE ARE EXCEPTIONS WE MIGHT BE ABLE TO FIND OUT WHAT IS ACTUALLY DRIVING THE COMPENSATORY MECHANISMS BUT I HAVE DISCOURAGING GOING AFTER THAT BECAUSE COULD INCLUDE INTERESTING COMPENSATORY CASES AND ALL KINDS OF TECHNICAL BIASES, AND IT'S GOING TO BE BUSY, HOPEFULLY A COUPLE NUGGETS OF GOLD. BUT WE DIDN'T WANT TO SIFT THROUGH THE GARBAGE OF TECHNICAL BIAS. >> I HAVE RUN COMPENSATORY MECHANISM, WHERE ESSENTIAL GENE WILL RESULT IN 50%, PROTEIN IS NEVER 50%. I'VE RUN INTO THAT SITUATION SO THAT CAN ACTUALLY HAPPEN. >> YEAH, WE HAD A PROTEIN LEVEL, I WOULD EXPECT THAT. AND IT'S IN SOME WAYS LET'S SAY GENES NOT SO SENSITIVE MIGHT APPEAR NOT SO SENSITIVE, AT SOME POINT APPROACHING LEVELS OR AT VERY MIXED DOWN STREAM IMPACT LEVEL THERE'S A COMPENSATION HAPPENING. >> I HAVE A QUESTION FOR ROHAN. SO, YOU KNOW, IT'S REALLY INTERESTING TO SEE THE TRANSCRIPTS AND HOW THEY PLAY A ROLE. I DIDN'T FOLLOW AT THE END, HOW MANY GENES IN THE HUMAN GENOME HAVE THESE TRANSCRIPTS? >> VERTICAL CLASS, ABOUT A THOUSAND TWO HUNDRED GENES. >> A THOUSAND TWO HUNDRED GENES. >> YEAH. EXPRESSING, YEAH, OF THE NON-CODING GENES. >> AND IN THE CASE WHERE WE'RE THINKING ABOUT THERAPIES, ONE COULD OBVIOUSLY -- IN CASES WHERE THE GENES ARE MUTATED AND HAPLOINSUFFICIENT, TO UPREGULATE THE GOOD COPY YOU COULD DO AN ASO AND UPREGULATE THE GOOD COPY OF THE GENE. HAS THAT BEEN TRIED? >> WE HAVE INTRIGUING DATA THAT SUGGESTS IT HAS THE OPPOSITE EFFECT. YES, IT IS POSSIBLE TO TRY AND INCREASE, SO WE HAVE TWO CLASSES, TWO APPROACHES, EITHER INCREASE OR DECREASE GENE EXPRESSION. >> BECAUSE FOR THE GENES THAT ARE HAPLOINSUFFICIENT, IF THERE ARE 1200 GENES, SOME OF THESE MIGHT BE INTERESTING TO DEVELOP. >> YEAH. >> IT SOUNDS TO ME FROM WHAT ROHAN DESCRIBED IS THOSE PROTEINS ARE DISORDERED PROTEINS, MAYBE MAKE THE AGING BRAIN FOR VULNERABLE, THAT'S WHAT I SURMISED, LOOKING AT THE LIST. I'M CURIOUS, ROHAN, TWO QUESTIONS FOR YOU. OF THE 1200 WAS THERE ENRICHMENT FOR DISEASE GENE IN THIS GROUP? AND WAS ENRICHMENT FOR GENETIC DISEASES FOR CHILDHOOD EARLY DEVELOPMENTAL DISORDERS I'M CURIOUS IF YOU LOOK AT IT THAT WAY. >> THAT CERTAINLY WAS NUMBER ONE IN ENRICHMENT FOR ESPECIALLY THE ONES THAT HAVE THE FIVE PRIME OVERLAP. THERE'S ENRICHMENT FOR NEURONAL GENES, AND FOR THOSE INVOLVED IN NEURODEGENERATION. >> JUST FOR ME TO UNDERSTAND YOU SAID THE MIR, IT WOULD ACT AS A GAIN OF FUNCTION? I DIDN'T QUITE GET THAT. DOWNREGULATED OR UPREGULATED? EITHER WAY, ONE REDUCES, ONE ELEVATES, INCREASES GENE EXPRESSION. WE HAVEN'T TESTED. WE HAVE SOME PRELIMINARY DATA BUT WE HAVEN'T TESTED IT. THIS IS ONE THING WE'D LIKE TO DO, IT CAN ALSO BE A TOOL FOR ADJUSTING THE GENE EXPRESSION. >> YEAH. >> THAT'S WHAT I WAS THINKING. >> THERE'S A COMMENT. SHE SAYS VERY INFORMATIVE PRESENTATIONS, WE'VE LEARNED ABOUT A WIDE RANGE OF MODEL SYSTEMS ALL OF WHICH PROVIDED GREAT INSIGHT INTO COMPLEX QUESTIONS. WOULD YOU ELABORATE A BIT ABOUT THE PROCESS OF CHOOSING THE BEST MODEL SYSTEM FOR YOUR EXPERIMENTS? SO -- >> I CAN START IF YOU WANT TO, BRIEFLY. >> SURE. >> WE HAVE A FORMALIZED PROCESS IN THE ORGANISM SCREENING CENTER WHEN PHYSICIANS SUBMIT VARIANTS TO US, FROM UNDIAGNOSED DISEASE PATIENTS. AND WE HAVE A GROUP OF TWO PHYSICIANS, FOUR BASIC SCIENTISTS AT WORK ON SQUIRREL, FLY, ZEBRAFISH, AND THEY, YOU KNOW, MINE THE DATA IN MARVEL AND DO CAREFUL ANALYSIS. IN A NUTSHELL WHEN GENES ARE NOT CONSERVING WORMS AND FLIES THEY GO TO ZEBRAFISH. THERE'S A HOMOLOG IN FLIES OR WORMS, IT'S ONE OR THE OTHER. AND IN FLIES, ABOUT 85% OF THE GENES THAT ARE SUBMITTED, HUMAN DISEASE, HAVE AN ORTHOLOG. AND SO 85%. THE WORM MODELS GENES SUPER COULD BE SERVED WHERE AMINO ACIDS ARE CONSERVED, FLIES ARE MOST OF THE GENES NOT SUPER CONSERVED, BUT QUITE CONSERVED. AND SO IN GENERAL, THAT SYSTEM HAS WORKED WELL, AND WE CAN SYSTEMATICALLY TRACK MOST OF THE HUMAN DISEASE GENES. >> IF I COULD ALSO HAVE A QUESTION ABOUT THIS MODEL. THIS MIR ELEMENT IS CONSERVED IN PRIMATES, IN ABOUT -- IN PRIMATES. THE MOUSE, WE TRIED TESTING TO THE MOUSE AND DON'T SEE ANY EFFECTS. SO, FOR US THE BEST MODEL IS HUMANS, HUMAN CELLS. AND THAT'S QUITE A STUMBLING BLOCK FOR US. >> WOULD IT BE POSSIBLE, ROHAN, TO JUST, YOU KNOW, RECREATE, IF YOU WILL, THE GENOMIC PORTION FROM THE HUMAN INTO THE MOUSE? >> WE'VE BEEN TRYING. WE WANT TO NEURONNIZE THE MODEL, BUT IT'S DIFFICULT TO SAY BECAUSE THE MOUSE DOESN'T HAVE THESE CONSERVED MOTIFS FOR BINDING TO THE RIBOSOMAL RNA. SO, THE MECHANISM IS CONSERVED, THIS WOULD DISCONNECT THERE. >> GOT IT. ELLEN? >> YEAH, A QUESTION FOR DR. BELLEN, VERY EXCITED ABOUT HIS RESULTS BUT I'M STILL NOT SURE HOW APPLICABLE THE FLY RESULTS ARE TO MAMMALIAN SYSTEMS IN PARKINSON'S DISEASE. DO YOU KNOW WHETHER -- THE ENZYME RELEASED IN LINEAGE, YOU SEE IN GLIA AND NEURONS, CAN YOU EXPLAIN HOW THIS MODEL MIGHT WORK? >> THE MODEL IN DROSOPHILA WORKS, RECAPITULATED CAN CELLS. IF THEY PRODUCE GLUCOSYLCERAMIDES, IF YOU CULTURED AND ON TOP MILLIMETER AWAY PUT NEURONS NOTHING HAPPENS. PUT GLIAL CELLS, IT GETS DEGRADED, JUST LIKE IN DROSOPHILA. THAT'S THE FACT. GLIAL CELLS, VERTEBRATE GLIAL CELLS SECRETE TGF-BETA, IF YOU NAUGHT IN THE MEDIUM ALL THE GLUCOSYLCERAMIDES GOES AWAY. I THINK THE WHOLE MODEL AS FAR AS WE CAN TELL IS COULD CONSERVED. IT ALL HAPPENS IN GLIAL CELLS. >> JIM, DO YOU HAVE A QUESTION? THANK YOU FOR BEING PATIENT. >> SURE. AND IT'S JUST ADDING A BIT TO A QUESTION RELATING TO HUMANIZATION AND MODEL SYSTEMS BECAUSE IT SEEMS LIKE WE'RE DISCOVERING REGULATORY ELEMENTS OR VARIATION OUTSIDE CODING GENOME, AND HOW WE'RE GOING TO BEST MODEL THOSE AND OBVIOUSLY HUMAN-DERIVED CELL LINES OR iPS CELLS IS ONE MODEL, GREAT IF WE COULD USE MODEL ORGANISMS, THERE WAS ALREADY ONE COMMENT I THINK FROM ROHAN ABOUT NON-SEATABILITY FOR DIFFERENCES BUT MAYBE FOR MOUSE PEOPLE HOW MUCH CAN WE USE THE MOUSE AS A WAY OF STUDYING THESE THINGS. >> THAT'S A GREAT POINT. ACTUALLY IN OUR CASE FOR THE RETT SYNDROME CAUSING PROTEIN THE MOST WAS PERFECT, WE USED AND HAS REGULATORY -- IDENTIFIED THEM IN THE MOUSE, CRISPR MUTATION, FOUND THE DOSAGE OF RNA AND PROTEIN IN EITHER DIRECTION, WENT BACK TO HUMAN NEURONS AND IT HELD THROUGH PRECISELY. NOT ONLY THE REGION THAT WE DELETED IN THE HUMAN BUT ALSO DEGREE OF THE CHANGE AND LEVEL OF THE PROTEIN, WITH THE HUMAN NEURONS, SO I THINK IT'S GOING TO BE SOME GENES HIGHLY CONSERVED, AND MOUSE AND HUMAN REGULATORY ELEMENTS WORK TOGETHER BEAUTIFULLY THERE. BUT THINGS ARE UNIQUELY HUMAN. THOSE WILL BE CHALLENGING. AND I THINK FOR FUNCTIONALITY AND MECHANISM I SEE THE FLY AS THE MOST POWERFUL. AGAIN FROM THE UDN WORK MANY DISEASES, OVER 40 DISEASES WITH SOLVED IN THE LAST TWO TO THREE YEARS. YOU HAD NO WAY OF KNOWING THE CULPRIT GENE UNTIL YOU PUT IT IN FUNCTION AND STUDIED EFFECT OF FRUIT FLIES. I THINK THE ANSWER, ALL MODEL ORGANISMS ARE VALUABLE. WE JUST HAVE TO -- >> I THINK THIS IS TRUE. WE HAVE WORKSHOPS, NIH, AND LOTS OF PEOPLE AND LOTS OF MODELING OR -- ORGANISM WERE PRESENTED WITH A FEATURE FOR A FUTURE DISEASE OR ALLOWS TO PROBE MECHANISMS MUCH FASTER THAN ANOTHER SPECIES, BUT THE CONCLUSION WE SHOULD ALL COLLABORATE. WE SHOULD ALL WORK TOGETHER AND DRIVE WHAT WE CAN IN THE SIMPLEST MODEL ORGANISMS THE BEST WE CAN AND COLLABORATE, TO BE ZEBRAFISH AND MOUSE, WORK OUR WAY UP THE EVOLUTIONARY SCALE. MINIMAL EXPERIMENTS WE CAME TO VERIFY IN THE FEATURES THAT WERE DISCOVERED IN THE MORE SIMPLE MODEL ORGANISM, BOOTSTRAP OUR WAY EVENTUALLY TO PRIMATES. >> THANK YOU. ON THAT VERY POSITIVE NOTE WE SHOULD COLLABORATE ACROSS SPECIES. GLEN TURNED HIS CAMERA ON. WE NEED TO WRAP THIS UP. BACK TO YOU, GLEN. >> THANKS TO ALL THE SPEAKERS TODAY, IN THE CHARACTERIZATION AND MECHANISMS SESSION AS WELL AS ALL THE POSTER PRESENTERS. THANKS TO SUSAN AND HUDA FOR MODERATING THIS SESSION. VERY EXCITING SET OF TALKS. SO NEXT WHAT WE WILL HAVE IS THE HAPPY HOUR, IT'S INTENDED FOR STUDENTS AND FELLOWS AND NEW INVESTIGATORS TO MEET WITH THE SPEAKERS AND CO-CHAIRS AND NIH STAFF. TO MAKE IT EASIER TO TRANSITION TO THE BREAKOUT ROOM TODAY YOU SHOULD HAVE RECEIVED AN E-MAIL DURING THE 12:20 OR SO AT THE BREAK TODAY THAT CONTAINS A LINK TO GET YOU THERE AND THAT LINK IS PROVIDED HERE, HOPEFULLY EASY FOR EVERYBODY TO MOVE ON OVER WHO WANTS TO PARTICIPATE. IF YOU'RE NOT GOING TO PARTICIPATE IN THE HAPPY HOUR, THEN WE'LL SEE YOU TOMORROW FOR THE FINAL SESSION OF THE WORKSHOP WHICH WILL BE TRANSLATIONAL RESEARCH, FACILITATED BY GENETIC MODIFIER STUDIES, IT WILL BE, AGAIN, 10:00 EASTERN, LOOK FORWARD TO ANOTHER SET OF GREAT PRESENTATIONS. >> THANK YOU SO MUCH.