Congenital Heart Disease: Many Genes Lead to a Broken Heart

Congenital Heart Disease: Many Genes Lead to a Broken Heart


>>GOOD AFTERNOON, EVERYONE. WELCOME TO THE WEDNESDAY
AFTERNOON LECTURE WE ANTICIPATE SOME SORT OF A SNOW STORM BUT I’M GLAD YOU ALL ARE HERE AND THOSE WATCHING ON THE WEB PROBABLY IN GREAT NUMBERS BECAUSE WE HAVE A SPECIAL SPEAKER TODAY, DR. CHRISTINE SEIDMAN KNOWN TO ALL OF US WHO KNOW KNOW HER WELL AS FRIEND AS KRICKET. IN MEDICINES AND W. SMITH GENETICS AT HARVARD, ALSO THE GENETICS DIRECTOR AT BRIGHAM AND INVESTIGATOR AT HOWARD HUGHES MEDICAL INSTITUTE. SHE DID HER UNDERGRADUATE WORK AT HARVARD. GOT HER MD AT GEORGE WASHINGTON UNIVERSITY. DID RESIDENCY AT JOHNS HOPKINS WHERE SHE WAS INFLUENCED PARTICULARLY BY ONE VICTOR McCUESICK IN TERMS OF HOW TO BRING SCIENCE AND MEDICINE TOGETHER IN INTERESTING WAYS. AND BOY HAS SHE LIVED UP TO THAT EXHORTATION BY DR. McCUEBIK. SUBSEQUENT TO THAT SHE DID RESEARCH FELLOWSHIP IN MEDICINE IN THE CARDIAC UNIT IN HARVARD AND SHE’S BEEN CONTINUING SINCE THAT TIME IN BOSTON AS I MENTIONED NOW AS PROFESSOR IN BRIGHAM AND WOMEN’S HOSPITAL IN HARVARD MEDICAL SCHOOL. SHE, MORE THAN ANYONE ELSE, HAS REALLY DEFINED THE CARDIOVASCULAR GENETICS FIELD AS IT RELATES TO CARDIAC FAILURE, BUT ALSO TO CARDIAC DEVELOPMENTAL ABNORMALITIES AND TAKEN A FIELD LARGELY DESCRIPTIVE AND CAUSED IT TO BECOME MOLECULAR WITH MANY CONSEQUENCES BOTH FOR DIAGNOSIS AND INTERVENTIONS TO ASSIST PEOPLE WHO’S FAMILIES ARE AFFLICTED BY THE DISORDERS. IT’S A GREAT PLEASURE TO HAVE THE OPPORTUNITY TO HAVE HER HERE FOR THIS WEDNESDAY AFTERNOON LECTURE. PLEASE WELCOME KRICKET SEIDMAN. [APPLAUSE]>>DR. COLLINS, THANK YOU SO VERY, VERY MUCH AND TO ALL OF YOU WHO STOOD WITH ME AGAINST THIS NASTY STORM THAT IS COMING FROM THE SOUTH AND HOPEFULLY NOT GOING ALL THE WAY TO BOSTON, I REALLY APPRECIATE YOU COMING THIS AFTERNOON. IT’S AN INCREDIBLE HONOR TO BE HERE. SOME OF YOU MAY KNOW THAT I SPENT A VERY IMPORTANT YEAR IN MY LIFE AFTER LEAVING JOHNS HOPKINS IN NICHD BUILDING 6 WHEN IT WAS A LITTLE BITTY SMALL PLACE AND IT’S ALWAYS FUN TO SEE HOW MUCH THIS HAS GROWN AND PARTICULARLY FUN TO SEE HOW MANY INCREDIBLY TALENTED, BRIGHT, SMART, AND OFTEN FEMALE SCIENTISTS ARE ARE HERE TODAY. THANK YOU FOR HAVING ME COME BACK. WHAT I’D LIKE TO DO IN THE NEXT 45 MINUTES OR SO IS TO TELL YOU A LITTLE BIT ABOUT HOW WE’RE REALLY USING GENETICS TO UNDERSTAND CAUSES OF HEART DISEASES THAT OCCUR IN RARE FAMILIAR FORMS AND ALSO IN POPULATIONS, AND EQUALLY IMPORTANT, HOW WE ARE SEEING THE COMPLEXITY OF USING THIS NEW TECHNOLOGY TO TRY AND UNDERSTAND HOW SIMILAR PHENOTYPES THAT ARISE IN A SINGLE INDIVI MIGHT BE INTERPRETED USING GENOME SEQUENCING. FINALLY N THE LAST LITTLE BIT, I HOPE TO BE ABLE TO TELL YOU A LITTLE BIT ABOUT HOW THERE ARE NEW STRATEGIES. I’M GOING TO TELL YOU ABOUT ONLY ONE BUT MANY ARE ARE STILL UNDER DEVELOPMENT, TO TRY AND USE THAT GENETIC INFORMATION TO ACTUALLY USE AND CREATE GENETICS THEY WERE P PI. A STUDY UNIT F UH YOU WILL, IS GOING TO BE AS DR. COLLINS SAID, ON CARDIO MYOPATHY OF SOMETHING THAT I’VE BEEN INTERESTED IN FOR A VERY LONG TIME. I WILL REMIND ALL OF YOU THAT THIS IS BY FAR AND AWAY THE MOST BEAUTIFUL ORGAN IN YOUR BODY. NOT ONLY DOES IT FUNCTION 24/7 BUT FOR THOSE OF YOU WITH A STETHOSCOPE YOU KNOW IT SING TOSS YOU. IF EVER YOU’RE UNSURE OF WHETHER YOU WANT TO CONTINUE IN YOUR PATH OF MEDICINE OR NOT, JUST GO LISTEN TO A HEART WITH A GREAT MURMUR AND I’M SURE YOU’LL COME ALONG TO THE CARD AWAYSIDE OF THINGS. UNFORTUNATELY FACED WITH THE MANY INSULTS WE THROW TO OUR HEARTS AND ENTIRE BODIES, THIS ORGAN OFTEN UNDERGO A LOT OF REMODELLING, BUT UNLIKE MANY OTHER DIFFERENT TISSUES, IT REALLY TAKES ONE OF TWO DISTINCT COURSES. EITHER RESULTS IN CARDIAC HYPERTROPHIES WITH THE WALLS — AND CAVITY SIZE IS DIMINISHED. THIS CAN BE A GENETIC CONDITION OR SPONTANEOUSLY OCCURRING CONDITION OR THE WALLS BECOME THIN AND THE INTRAVASCULAR VOLUME IS INCREASED. FOR YEARS WE’VE KNOWN THAT LOT OF SECONDARY CAUSES OF THIS MIGHT BE DUE TO HYPER TENSION, VALVE HEART DISEASE OR MYOCARDIAL ISCHEMIA BUT IN MANY INDIVIDUALS WE’VE KNOWN THAT THESE ARISE SPONTANEOUSLY AND OFTEN IN YOUNG PEOPLE WHICH REPRESENTS FINGERPRINTS OF GENETIC DISEASES. OVER THE PAST DECADES THERE HAVE BEEN THE DISCOVERY OF MANY GENES THAT GIVE ARISE TO EACH OF THESE AND WE CALL THESE INTRINSIC CARDIO MYOPATHIES. THOSE CAUSING REMODELLING I’VE SHOWN IN THE UPPER LEFT. THE MOST IMPORTANT OF THESE ARE MUTATIONS IN THE SASHGMINUTE GENE. SIMILARLY, WE HAV IDENTIFIED MANY OTHER GENES THAT CAN GIVE RISE TO PRIMARY VENTRICULAR DILATION. ONE HAS BECOME QUITE A STAND OUT AND THAT’S [INDISC]. I’D LIKE TO TELL YOU A LITTLE BIT MORE ABOUT THESE TWO DIFFERENT CLASSES OF PROTEINS AND ARE REMODELLING STRUCTURES. THE WAY IN WHICH WE KNOW THAT THESE ARE DEFINITIVE CARDIO MYOPATHY GENES REALLY IS BASED ON LOTS OF DISORDERS IN WHICH INDIVIDUALS WHO ARE KNOWN TO HAVE A GENETIC RELATIONSHIP, THEIR SISTERS, BROTHERS, AUNTS, UNCLES, HAVE BEEN IDENTIFY BY ROUTINE IMAGING STUDIES SOMETIMES ASSOCIATED WITH UNFORTUNATELY SUDDEN CARDIAC DEATH EVENTS WHICH WE CAN SEE CARDIO MORPHOLOGY AND THIS ENA ABLES VERY LARGE PED DRI DEBRISES. THIS LED TO GENETIC LINKAGE ANALYSES. REASON WE KNOW THIS IS SO DEFINITIVELY TRUE IS BECAUSE THE STATISTICAL POWER OF A VERY LARGE FAMILY IS QUITE REMARKABLE AND A LOT OF YOU CAN PREDICT HOW RANDOMLY SUCH A COINHERITANCE WOULD OCCUR ALL IN THE 40 PEOPLE INFECTED ON THIS SCREEN AS COMPARED TO THOSE WHO ARE NOT. AS YOU CAN IMAGINE THAT STATISTIC SHOWS THAT THAT WOULD BE EXTRAORDINARILY UNLIKELY TO OCCUR BY CHANCE. THINK ABOUT WHERE WE ARE TODAY. TODAY, INSTEAD OF USING THAT ROVING CIRCLE THAT TAKES SEVERAL POSTDOCTORAL YEARS TO THIN, WE CAN GO DIRECTLY TO ACTUALLY SEQUENCING NOT ONLY XOEMS BUT INCREASINGLY GENOMES WITH ONE BOX THAT CAN SIT ON YOUR TABLE TOP. WE KNOW THE THIS HIGH CAPACITY NOT ONLY GIVES US THE ACTUAL GATC SEQUENCE BUT IT ALLOWS US TO REALLY ROBUSTLY INTERROGATE WHETHER THE NUMBER OF EACH OF THOSE SPACE PAIRS ARE APPROPRIATELY TWO COPIES, ONE FOR EACH OF THE AUTOSOMES OR IF THERE ARE A COPY NUMBER OF INVARIANTS IN GENES A NEW SOURCE OF HIDDEN PATHOLOGIES. IF WE HAVE AN INDIVIDUAL WHO’S XOEM OR GENOME WE CHOOSE TO SEQUENCE FROM A PEDIGREE SUCH AS THIS AND FIND A VERY RARE MUTATION, WE MIGHT HAVE A FAIR DEGREE OF CONFIDENCE THAT THAT MIGHT CAUSE THE DISEASE. IT BECOMES ALL THE MORE POWERFUL IF WE LOOK AT ANOTHER DISTANTLY RELATED INDIVIDUAL WHICH SHARED ONLY ONE 32nd OF HIS GENOME WITH HIS COUSIN OVER HERE AND THAT MAKES IT HIGHLY UNLIKELY THAT THESE SINGLE RARE VARIANCES IDENTIFIED IN BOTH OF THESE INDIVIDUALS WOULD HAVE ARISEN BY CHANCE. SORT OF THE XOEM E — WHAT HAPPENS WHEN WE DON’T HAVE NO FAMILY HISTORY OF DISEASE AND A SINGLE PATIENT WHO COMES IN? CAN WE MAKE THOSE SAME PREDICTIONS THAT VARIANT WE IE E DENT FIE IN A GENE, EVEN ONE KNOWN TO CAUSE DMZ THE CONTEXT OF FAMILIES, DO WE KNOW THAT ACCOUNTS FOR THE DISEASE IN THIS INDIVIDUAL? I WANT TO BRIEFLY REMIND YOU THAT IN ADDITION TO HAVING VARIANCE WE INHERIT, WE ALSO HAV THE OPPORTUNITY TO ACQUIRE OUR OWN PERSONAL VARIANCE. IT’S ESTIMATED THAT THERE WAS APPROXIMATELY ONE RARE DE NOVO VARIANT IN EACH AND EVERYONE OF US THAT ABOUT ONE IN 50 OF US WILL CARRY A COPY NUMBER OF VARIANTS AS WELL AND IF WE LOOK NOT JUST AT XOEM SEQUENCES BUT INDEED ACROSS THE GENOME, WE SEE THERE ARE UH NEW VARIANCE ARISEN IN US. THE SIGNIFICANCE BECOMES DIFFICULT TO INTERPRET WITHOUT FAMILY MEMBERS AND WITHOUT KNOWING A WHOLE LOT MORE ABOUT THE GENE. HOW DO WE TAKE OUR ABILITY TO KNOW GENETIC CAUSES OF DISEASE AND BRING IT TO POPULATIONS AND PATIENTS IN A CLINIC? HOW CAN WE DISCERN THE SIGNIFICANCE DIFFERENCE IN EACH AND EVERY PATIENT BEFORE US? THAT’S GOING TO REQUIRE A VERY, VERY BIG DATABASE SO I HOPE THAT NCBI WILL GET MORE FUNDING, DR. COLLINS. I THINK THAT CERTAINLY THAT’S GOING TO REQUIRE A SUBSTANTIAL HARMONIZATION OF PHENOTYPE SO THAT WE KNOW WHEN WE SAY THAT THIS VARIANCE IS FOUND IN THIS COLLECTION OF PHENOTYPES THAT WE’RE ALL TALKING ABOUT EXACTLY THE SAME THING, AND REMEMBER THAT A LOT OF OUR PHENOTYPES ARE PRETTY NON-SPECIFIC. A CHEST X-RAY PROBABLY SEEMS VERY SIMILAR LIS TICK BUT IT’S NOT THAT VERY DISSIMILAR FROM DOING AN ECHO OR MRI OF THE HEART. IT’S NOT EXACTLY HIS-SELF LOGIC ANALYSIS. SIMILARLY WE’LL COLLECT ALL OF THE DEFINITIVE MUTATION AND COMPARE THEM TO THE VARIANCE IN THE SAME GENE. ADD IN WHAT HAPPENS WHEN YOU HAVE DIFFERENT LIFESTYLES, EXPOSURES, ENVIRONMENTS, AND ULTIMATELY COME OUT WITH SOME KIND OF INTEGRATED ANALYSES. FIRST PART OF THIS TALK I’D LIKE TO TELL YOU SOME OF THE STRATEGIES WE’RE TRYING TO USE TO TRY AND OVERCOME THESE VERY BIG OBSTACLES AND IN A SECOND PART HOPEFULLY CONVINCE YOU IT’S WORTHWHILE BECAUSE I CAN LEARN SOME INTERESTING BIOLOGY. SO WHEN WE THINK ABOUT THE CONTRACTILE APPARATUS, MANY OF YOU MAY BE REMEMBERING ON THE SLIDE TO THIS IN WHICH YOU KNOW THAT THE MOTOR PROTEINS IN ALL MUSCLE CELLS ARE COMPOSED OF A HEAVY CHAIN — COOKING, EXCUSE THIN FILAMENT AND SLIDE ACROSSH E KNOWN FOR A LONG TIME THAT. MUTATIONS IN THE EIGHT MAJOR COMPONENTS OF THE MOLECULAR MOTORS OF CARDIAC CELLS CAN GIVE RISE PRIMARILY TO HYPER TROPHIC CARDIO MYOPATHY. THESE ARE FROM FAMILIAR STUDIES — AND WE HAVE ROBUST STATISTICS TO SAY THESE ARE DEFINITIVE DISEASE GENES. WE ALSO KNOW THAT CARDIAC HYPERTROPHY THAT REMODELLING I SHOWED YOU ON THE FIRST SLIDE IS FOUND IN GENERAL POPULATION. 4% OF THE GENERAL POPULATION HAS UNEXPLAINED CARDIAC HIGH PERCH FI AND LARGELY ATTRIBUTED TO DIABETES, INCREASE IN BLOOD PRESSURE AND LOTS OF OH COMMON CARDIOVASCULAR DISORDER. WE THOUGHT IT’D BE INTERESTING TO KNOW IF SASHG MERE — THIS STUDY A LOLY COMMUNITY BEING BURIED IN SNOW AND AS ALL OF YOU KNOW IT’S HAD ABOUT 50 YEARS OF LONGITUDINAL STUDY OF OVER THOUSANDS OF SUBJECTS WHO HAVE KINDLY PARTICIPATED IN SHOWING US LOTS OF IMPORTANT PIECES OF INFORMATION OVER MORE THAN EIGHT, NOW, DIFFERENT SERIAL EXAMINATIONS. WE’VE LOOKED AT THE JACKSON HEART STUDY, AT LEAST NOW ONE-AND-A-HALF ROBUST PROFILES IN WHICH WE HAVE CARDIAC IMAGES AND LOTS OF ANCILLARY PHENOTYPES AS WELL. WHEN YOU TAKE THESE THREE THOUSAND INDIVIDUALS AND YOU SAY LET’S JUST SEQUENCE ALL OF THOSE EIGHT MAJOR SASHG MERE GENES THAT CAUSE MICRO TROPICAL MYOPATHY, WE HAD EXPECTED WE WOULD FIND SOME VARIANCE AND THAT THAT WOULD CORRELATE WITH THEIR DEGREE OF HIGH PERCH FI. WE FOUND 11% OF THE GENERAL POPULATION HAS A CHANGE IN THE SEQUENCE THAT ALTERS THE PROTEIN OF THESE EIGHT SASHG MERE GENES AMONGST THESE TWO POPULATIONS. MOREOVER, BECAUSCE THE DISCOVER ARE RI OF THESE GENES AS CAUSING HYPOTROPHIC CARDIO MYOPATHY, A LOT OF CRITERIA HAS BEEN DEVELOPED TO ACTUALLY DISCERN WHICH ARE PATHOGENIC ONES AND WHICH ARE COMMON VARIANTS. SO WE APPLIED THOSE TO APPROVE CLASSIFICATION AND WE COULD RESTRICT THOSE EIGHT RARE VARIANCE DOWN TO ABOUT 8.6 WHO WOULD BE DEEMED PATHOGENIC. THAT MAY NOT SEEM LIKE A VERY BIG NUMBER TO YOU BUT THAT’S ABOUT THREE TIMES MORE PREVALENT THAN ALL GIVEN REPORTS THE DISEASE INCIDENCE IS IN A GENERAL POPULATION WHICH WOULD MAKE YOU CONCLUDE ONE OF TWO THINGS. ONE, [INDISCERNIBLE] VARIANT, OR TWO, WE’RE MISSING A LOT OF THE DIAGNOSTIC CRITERIA IN THE SO WHEN WE LOOKED IN DETAIL AT THE PHENOTYPES OF THESE VARIANT CARRIERS. WE FOUND ONLY ABOUT ONE IN TEN OF THEM HAD ANY CRITERIA, EVEN THE [INDISCERNIBLE] OF HAVING PHENO TIPIC MANIFESTATION. IT’S NOT AS IF THESE PEOPLE ARE LIKELY TO DEVELOP IT OVERTIME. IT’S REALLY IMPLIES THAT WE’RE NOT CALLING ALL OF THE VARIANCE IN A CORRECT MANNER. IT DOESN’T MEAN HOWEVER THAT THESE VARIANCE ARE COMPLETELY SILENT BECAUSE AGAIN, CAPITALIZING ON LONGITUDINAL PHENOTYPES WHEN WE LOOKED AT ADVERSE MYOCARDIAL EVENTS THAT OCCURRED IN CARRIERS IN COMPARISON TO THOSE IN FRAMING HAM WHO DID NOT CARRY THE VARIANT WE SAW ALMOST AN INCREASE IN EVENTS. THIS IS AFTER ACCOUNTING THE USUAL RISK FACTORS. SO TAKEN TOGETHER WHAT AYE TRIED TO TELL YOU IS THAT WE’VE KNOWN FROM DECADES NOW OF RESEARCH RAL NICK PATHOGENIC NETRANT MUTATIONS AND THIS OCCURS IN GENES WE CAN BEGIN TO STUDY IN GENERAL POPULATION. WE FIND LOTS OF RARE VASHTS BUT THE PENETRANTS AND [INDISCERNIBLE] APPEARS TO BE SIGNIFICANTLY LOWER. IT’S NOT ABSENT, IT’S JUST LOWER. ONE OF THE BIG CHALLENGES THEN BEGINS TO SAY WHAT ARE THOSE MODIFIERS THAT ARE INFLUENCING THE EXPRESSIVITY; GENETIC? BACKGROUND? EXPOSURE? CAN WE USE THAT INFORMATION TO TAKE AND MODIFY THE IMPACT OF THESE PATHOGENIC MUTATIONS? THAT’S A LONG WAY INTO THE FUTURE RIGHT NOW, BUT I THINK IT’S SOMETHING THAT’S GOING BE IMPORTANT FOR US TO BEGIN TO GRAPPLE WITH. I’M GOING SHIFT GEARS NOW AND TELL YOU ABOUT OTHER FORM OF CARDIAC REMODELLING. I WANT TO INTRODUCE WHAT IS BECOMING THE MOST POPULAR GENE IN MY PARTICULAR LABORATORY, TITAN. TITAN IS NOT TIE-TAN, IT IS THE LARGEST GENE AND LARGEST PROTEIN IN THE HUMAN BODY, BUT IT’S A TITIN AND IT’S HUGE IN TERMS OF SIZE. IT ACTUALLY IS APPROXIMATELY A HUNDRED THOUSAND BASE PAIRS LONG FOR THE PROTEIN ENCODING SEQUENCES. THE MOLECULE IS ALMOST ONE MICRO METER AND SPANS ONE HALF OF THE ENTIRE [INDISCERNIBLE]. THIS IS TITIN HAS BEEN LONG KNOWN TO BE CRITICAL — IT’S BELIEVED TO PARTICIPATE IN DEVELOPING AND SERVING AS A SCAFFOLD FOR THE DEVELOPMENT OF THE SASHG MERE DURING EMBRYOGENESIS. IT CLEARLY IS IMPORTANT NOT PARTICULARLY IN FOURTH GENERATION BUT IN FOURTH TRANSMISSION AND BECAUSE IT’S BEEN RECOGNIZED THAT THIS PURPLE DOMAIN HERE DESIGNATED M, THE M BAND, HAS KINASE ACTIVITY UH BUT IT MAY ALSO BE INVOLVED IN SIGNALING. AT THE FIVE CRIME END OF THE MOLECULE, THE Z BAND IS INTERACTING REGION ANOTHER AREA KNOWN TO BE CRITICAL FOR ACTUALLY TRANSDUCING SIGNALS. SEVERAL YEARS AGO WE BEGAN TO ASK WHETHER WE COULD FIND CAUSES OF ID OWE E PATHIC DIELTED REMODELLING BY LOOKING AT SOME SMALL SMALLIES AND INCREASINGLY BECAUSE THERE WERE NOT LARGE LARGE KINDRE FOR ANALYSES TO ICE LATED PATIENTS. WE FOUND RARE FAMILIES THAT WE COULD IDENTIFY FEW MUTATIONS IN THE GIANT PROTEIN TITIN. WE THEN ASKED SINCE WE’VE NOEP THAT THIS IS A SINGLE GENE THAT IS VERY BIG BUT MAYBE WE COULD ALSO BEGIN TO GLEAN STRATEGIES TO INTERPRET VARIANTS IN SINGLE INDIVIDUALS BY DOING A DEEP DIVE OF SEQUENCING THIS GIANT MOLECULE IN LARGE COHORTS OF PATIENTS. AS YOU CAN IMAGINE, WE TOOK THE SAME STRATEGY I TOLD YOU ABOUT BEFORE WITH A LITTLE EXCEPTION. FIRST OF ALL, WE TOOK ABOUT FIVE HUNDRED INDIVIDUALS WITH DIELTED CARDIO MYOPATHY WHO HAS BEEN EXCLUSIVELY SEEN IN TIME. WE’VE HAD EXPENSIVE MRI WHICH HAS HELPED TO INFORM SOME OF THE FEE KNOW TIP I CAN MANIFESTATIONS. WE BEGAN SEQUENCE BOTH FRAMING HAM AND JACKSON OVER THREE THOUSAND INDIVIDUALS AND BECAUSE WE KNOW THAT THERE’S A VERY RARE FORM OF PEDIATRIC ONSET WE HAD A CHANCE TO LOOK AT A SMALL SUBSET OF PATIENTS. WE DID IS DO ROBUST SEQUENCING USING AN ALUMINUM PLATFORM AND USE THE STANDARD PIPELINE FOR ANALYSES. WHEN WE USE THIS AND THEN BEGAN A VARIANT CALLING, WE HAD THE UNFORTUNATE CIRCUMSTANCE OF RECOGNIZING THAT THE NUMBER OF VARIANTS IN THIS GENE ALONE ARE ABSOLUTELY ASTRONOMICAL. THERE IS ONE SIS SENT VARIANT THAT IS RARE, NOT IDENTIFIED IN THE VAST MAJORITY OF DATABASES OR OCCURRING IN LESS THAN 1% OF THE POPULATION IN EACH AND EVERYONE OF US. WITHOUT A FAMILY HISTORY, WITHOUT THE CAPACITY TO DEEPLY EXAMINE THE CONSEQUENCES OF EACH ONE OF THOSE, THERE ARE GOING BE A LOT OF VARIANTS THAT ARE OF UNKNOWN SIGNIFICANCE IN THIS GENE FOR A VERY LONG TIME AHEAD. WE’VE RESTRICTED OUR ANALYSES TO THOSE VARIANCE WHO APPEAR TO HAVE A MORE PROFOUND CAPACITY TO CHANGE WHAT TITIN DOES. THOSE ARE THE ONES THAT ACTUALLY PREMATURELY TERMINATE THE ENCODED PROTEIN EITHER BECAUSE OF A PREMATURE SEQUENCE, A NONSENSE, OR AN ESSENTIAL SPLICE SITE MUTATION THAT RESULTS IN FRAME SHIFT AND ALTERNATIVE SPLICING. WHEN WE EMPLOYED ONLY THE ANALYSES ON THOSE TRUNCATING MUTATIONS, WE FOUND VERY INTERESTING RESULTS AS SHOWN HERE. FOR THOSE PATIENTS WITH N SAGE REALLY PROFOUND HEART FAILURE — THESE ARE INDIVIDUALS WHO HAVE BEEN REFERRED FOR TRANSPLANTATION OR WHO ARE UNDERGOING BAG TREATMENTS WE FOUND SIGNIFICANT ENRICHMENT IN THE TITIN TRUNCATION MUTATIONS FOUND NAH N THAT POPULATION AS COMPARED TO HEALTHY FOLLOW VOLUNTEERS AND FRAMING HAM IN JACKSON. WE’RE NOT YET PROGRESSED TO HEART FAILURE, THEY WERE ALSO SIGNIFICANTLY ENRICHED IN COMPARISON TO THIS OTHER POPULATION. BUT AS YOU’LL SEE, THERE WERE NOT ZERO PATHOGENIC SOUNDING OR LOOKING MUTATIONS IN THE GENERAL POPULATION. THEY WERE CERTAINLY PRESENT ARE, ALBEIT SOMEWHERE BETWEEN 1-3%. THAT MAY NOT SEEM LIKE A VERY HIGH E PERCENTAGE, BUT IF IT WAS YOUR HEART, I THINK SOME OF U.S. MIGHT BE WORRIED ABOUT IT. WE BEGAN TO ASK WHETHER THERE WERE OTHER STRATEGIES THAT WE COULD USE TO KEEP OUT WHICH ARE THE MEANINGFUL VARIANTS AND WHICH ARE NOT. WE TRIED TO ASK WHETHER THERE WAS A DISTRIBUTION OF THESE VARIANTS ALONG THE PROTEIN THAT MIGHT BE INFORMA SHOWN HERE ON THE TOP ARE THE AMBULATORY SEVERE PATIENTS IN THE JACKSON FRAMING HEART DISTRIBUTED ALONG THIS REGION. IT’S NOT A VERY IMPRETIVE CLUSTERING. SO THE NEXT THING THAT WE ASKED WAS WHETHER THERE WERE DIFFERENCES IN THE LOCATION OF THESE VARIANCE NOT ALONG THE LONGITUDINAL DISTRIBUTION OF THIS LARGE MOLECULE BUT THE DEGREE WITH WHICH THE EXXON IN EACH OF THOSE ARE EXPRESSED. LIKE MANY GREAT PROTEINS WITH CAPACITIES TO PERFORM DIFFERENT PROCESSES, TITIN UNDERGOES LOTS OF DIFFERENT ISOFORM SWITCHES — NOT ISOE FORMS, EXCUSE ME — EXXON USES. SO WE RESTRICTED OUR ANALYSIS TO ASK WHICH EXXONS ARE HIGHLY EXPRESSED AS COMPARED TO THOSE LOWLY EXPRESSED. THE HIGHER THE GRADE PEAK, THE HIGHER OF THE LEVEL OF EXPRESSION. WHAT YOU’LL SEE IS THAT ALONG THIS REGION HERE, THERE’S CONSIDERABLE DROPOUT BECAUSE THESE EXXONS HAS MUCH, MUCH LESS EXPRESSION WITHIN THE HUMAN HEART. YOU’LL ALSO NOTE THAT A LOT OF THE VARIANTS THAT ARE NOT FOUND IN OUR DILATED IN — SO IF WE ELIMINATE THOSE FROM ONLY THE LOWLY EXPRESSED EXXON YOU GET A FAR MORE CLUSTERING KIND OF APPROACH WITH SUBSTANTIAL ENRICHMENT ALONG THE A BAND, AND THAT IS SIGNIFICANTLY ENRICHED AMONG THOSE WITH DCM COMPARED TO GENERAL POPULATION WHERE THOSE THAT ARE ENRICHED IN THE GENERAL POPULATION TURNED OUT TO BE IN THE LOWLY EXPRESSED EXXON. SO THIS IS IDIOPATH DIELTED HEART IN WHICH THERE IS A GLOBAL VENTRICULAR DECREASE IN FUNCTION BUT THERE’S NO REGIONAL WALL ABNORMALITITY, NOTHING PARTICULARLY REMARKABLE ABOUT THE INTENSITY OF THE IMAGE QUALITY AROUND HERE AND THE HIS TOE PATHOLOGY IS WHAT I LIKE TO CALL QUITE VANILLA. IT DOESN’T TELL YOU EITHER THE IDEOLOGY OR WHETHER IT’S GOING TO PROMOTE THE ARRHYTHMIAS IN A PATIENT FROM WHOM IT’S OBTAINED OR NOT. WE ASKED WHETHER KNOWING THIS MUTATION WOULD ACTUALLY INFORM PHENOTYPE OUTCOMES BASED ON DETAILED INFORMATION WE HAD ABOUT THEM. FROM OUR PATIENT WITH MRI WE LOOKED AT THOSE WHO HAD THE M SHOWN HERE VERSUS THOSE WHO DID NOT. YOU SEE A SLIGHT DECREASE IN LEFT VENTRICULAR EJECTION FRACTION. PERHAPS MORE IMPRESSIVE TO ME WAS THE FACT THAT THEIR WALL THICKNESS WAS DECREAS COMPARISON TO OTHER FORMS OF DIELTED CARDIO MYOPATHY AND SADLY THE NUMBER OF ARRHYTHMIAS THEY HAD WERE SUBSTANTIALLY INCREASED. THIS IS PROVIDED TO US FOR THE FIRST TIME THAT WE’RE BEGINNING TO TEASE OUT A PHENOTYPE THAT MAY BE GENOTYPE CHECK SLEKTIVE IN THE DIELTED POPULATION. DILATED POPULATION. MOREOVER, WE HAD THE SENSE FROM THE DISTRIBUTION OF MUTATIONS AND WHONS THAT ARE HIGHLY EXPRESSED IN THAT CLUSTERING THAT THOSE WERE MORE LIKE TO OCCUR IN INDIVIDUALS WHO HAS HAD THE SWER — NOT SURPRISING THAT DISTRIBUTION ALWAYS IMPLIES THEY WOULD HAVE THE POOREST EJECTION FRACTION. THE FURTHER THE TRUNCATION OCCURRED TOWARDS THE THREE PRONG END OF THE MOLECULES T WORST THE VENTRICULAR FUNK. THAT WAS TRUE IF YOU HAD DIELTED MYOPATHY OR IF YOU WERE A FRAMING HAM HEART SUBJECT AND SIMPLY CARRIED ONE OF THESE MUTATIONS SO YOU HAD A DECREASE IN YOUR FRACTION IN COMPARISON TO THOSE WHO DID NOT AND IT WAS WORST THE MORE THREE PRONG TO THE GENE OR TO THE MORE CAR BOTTLE END OF THE MOLECULE THAT BECAME. WE’VE DONE THOSE SAME KIND OF ANALYSES IN CHILDREN WHO HAVE PEDIATRIC ONSET OF CARDIO MYOPATHY BEFORE THE AGE OF 16. THIS IS A DEVASTATED DIS ORDER THAT LEADS TO RAPID DEATH IF THEY HAVEN’T DONE THEIR TRANSPLANTATION. WE’VE NOW LOOKED AT ABOUT 84 GENES THAT HAVE BEEN IDENTIFIED FROM FAMILY STUDIES IN PATIENT COHORT AND SUFFICE IT TO SAY THAT A FEW MUTATIONS THAT HAVE BEEN IDENTIFIED, NOT VERY MANY. THERE’S A LOT MORE TO DO IN THIS COHORT OF PATIENT. MOST IMPORTANTLY, WE SEE VERY FEW MUTATIONS WITHIN TITIN, SIGNIFICANTLY LESS THAN WE DO WITHIN THE ADULT POPULATION. TAKEN TOGETHER, I THINK WHAT THIS INFORMATION HAS GOWN TEASE OUT IN TERMS OF INFORMATION THAT’S BEEN RELEVANT TO INTERPRETING TITIN VARIANTS IS THAT MOST OF THESE APPEAR TO TRUNCATE THE PEPTIDE T PATHOGENIC ONES, THAT THEY OCCUR IN HIGHLY EXPRESSED EXXONS WITHIN THE LEFT VENN TRI CALL. THEY ARE MORE REGIONALLY LOCATED THAN RANDOMLY DISTRIBUTED ACROSS THE MOLECULE, AND THOSE THAT ARE CLUSTERED THE FOR AB END OF THE MOLECULE ARE CLEARLY ASSOCIATED WITH MORE SEVERE DISEASE, POORER LEFT VENN TRICK CALL EJECTION ACTION AND THEY ARE UNLIKELY IF YOU FIND THEM IN A PEDIATRIC ONSET DIELTED CARDIO MY MY OP THINMYOPOTHY, — THE FIRST
THING TO DO IS UNDERSTAND MECHANISM. WHEN WE THINK ABOUT A POLYPEPTIDE THAT’S TRUNCATED WE OFTEN ASSUME IT HAD A DIMINISHED CAPACITY TO PARTICIPATE IN ITS USUAL ROLES. THAT MAY BE SO PROFOUND THAT IT’S NOT INCORPORATED AND THAT WE ARE HAVE LOSS OF ONE OF THE FUNCTIONAL GENE COPIES OF THE MOLECULE OR HAVE ONE INSUFFICIENCY. IF WE HAD — BY THE MECHANISM BY WHICH TITIN TRUNCATION CAUSED DIELTED CARDIO MYOPOTHY, WE WOULD NOT EXPECT A SELECTED DISTRIBUTION OF DCM MUTATIONS ARRIVING AT THE THREE-WRONG PRONGED END OF THE MOLECULE. RATHER THEY SHOULD BE ABLE TO TRUNCATE ANYWHERE A ALONG THE CHAIN. I’VE TRIED TO CONVINCE YOU THAT THOSE AT THE AMY KNOW TERMINUS FAIR FOOR BE FAR LESS DEVASTATING. THAT RAISED THE CHANCE THAT THIS IS — BUT RATHER IT SUGGESTS SOMETHING ELSE AND THAT HAS A LOT TO DO WITH THE GIANT SIZE OF THIS PEPTIDE. THIS IS A VERY, VERY LARGE TRANSCRIPT AND CHANCES ARE THE FIVE-PRONGED SEQUENCES ARE ARE TO BE TRANSL AND ACTUALLY BEGIN TO BE INCORPORATED INTO THE SAR KOE MERE EVEN BEFORE THE THREE-PRONGED SEQUENCES HAVE BEEN GENERATE INTO PEPTIDES. MOREOVER, WE KNOW THAT FROM VERY RARE FAMILIES WHERE THERE ARE TRUNCATION MUTATIONS AT THE VERY END BAND T MOST CAR BOTTLE END OF THE PROTEIN THAT IMMUNE CHEMICAL STUDIES REVEAL THAT THOSE PROTEINS ARE INCORPORATED INTO THE SASHG MERE. THIS IS A PROFILE OF THESE KINDS OF PATIENTS. ANTIBODIES THAT ARE SELECTED FOR THE Z DISK REGION OF TITIN, THE A BANDS REGION OF TITINS SHOW ROBUST EXPRESSION SUGGESTING THAT THESE PEPTIDES ARE SUFFICIENTLY STABLE, THAT THEY ACTUALLY ARE INCORPORATED. I THINK THIS PROVIDES THE MOST PROFOUND EVIDENCE THAT THESE ARE LIKELY TO OPERATE BY A DOMINANT NEGATIVE MECHANISM. SO IF THEY’RE DOMINANT NEGATIVE, WHAT DO THEY DO? WE HAVEN’T HAD A A CHANCE TO DO A WHOLE LOT OF WORK ON THIS YET. I CAN TELL YOU THAT SOME OF THE HYPOTHESES WE CAN PUT TO REST. NAMELY THAT THEY MIGHT DISRUPT FORMATION OF NORMAL SASHG MEEZ STRUCTURE. WE’VE DONE LOTS OF ELECTRON MICROSCOPY AND AS BEST WE CAN SEE STRUCTURE IS COMPLETELY NORMAL DESPITE HAVING THESE PATHOGENIC MUTATIONS. WHAT WE DO SEE IS LOTS OF INTERESTING CHANGES IN NUCLEAR MOREOLOGY, SOMETHING THAT HINTS THAT THERE MIGHT BE SIGNIFICANT ABNORMALITIES. WE KNOW THAT A LOT MORE WORK WILL HAVE TO BE DONE TO TRY AND BUILD ROBUST MODELS TO DO THIS, I WANT TO OUTLINE FOR YOU HOW WE COULD USE THIS INFORMATION, BUT IN THIS SENSE, SORRY A TRUNCATION OF TITIN IN IS ACTING IN A DOMINANT NEGATIVE MODE OF ACTION MIGHT PROVIDE A NEW WAY FOR TREATING. THE FIRST THING IS TO DEVELOP
MODELS IN WH
ICH WE CAN ROBUSTLY LOOK FOR SUFFICIENCY VERSUS DOMINANT NEGATIVE. WE CAN DO ALL SORTS OF TRANSCRIPTIONAL PROFILING THAT IDENTIFY — IN PARTICULAR BECAUSE OF THE INBAND KINASE — AND THEN IDENTIFY CANDIDATE MOLECULES. SEE HOW THEY PERTURB CARDIAC BIOLOGY AND PHYSIOLOGY AND THEN SEE IF ANY OF THESE THINGS MIGHT PLAY OUT AS POTENTIALLY BENEFICIAL TO OUR PATIENT. WE’VE DONE A LOT OF THIS ALREADY IN THE MUTATIONS THAT CAUSE HYPOTROPHIC CARDIO MYOPATHY BECAUSE WE’VE BEEN AWARE OF THE BIOPHYSICAL PROCESSES FOR SEVERAL YEARS NOW. WE’VE MADE THE MICE AND DONE THE STUDIES, AND I JUST WANT TO TELL YOU LATE BUILT ABOUT WHAT THOSE STUDIES HAVE INFORMED US. I WANT TO REMIND YOU THESE ARE IN THE CONTRACT HALL APPARATUS THAT ARE GENERATING THESE SACOMERE POWER AND FORCE BECAUSE THEY OCCUR IN MYOSIN, ACTIN, [INDISCERNIBLE] AND THEY RESULT IN A VERY REMARKABLE HIS TOE PATHOLOGY THAT IS QUITE DIFFERENT FROM DILATED CARDIO MYOPATHY. THEY’RE ENLARGED, SEPARATED BY MATERIAL — THIS KIND OF THIS MORPHOLOGY ULTIMATELY RESULTS IN [INDISCERNIBLE] DYSFUNCTION AND PRODUCES THE [INDISCERNIBLE] WE SEE IN PATIENTS. HOW COULD A MYOSIN MUTATION DO THIS? THE FIRST THING WE NEED TO DO IS UNDERSTAND WHAT DOES THE SIGH SIN MUTATION DO TO SACOMERE PERFORMANCE? W THE UNIVERSITY OF VERMONT AND STANFORD, WE’VE BEEN ABLE TO ENGINEER HUMAN MUTATIONS EITHER INTO CELLS AND HAVE THEM PRO BUSTLY EXPRESSED IN VITRO OR, INDEED, FROM MOUSE MODELS IN WHICH WE CAN ISOLATE THE MUTANTS FROM THOSE ANIMAL HEARTS. AND WHAT WE’VE SEEN IS THAT QUITE SURPRISINGLY, MUTATIONS THAT RESULT IN HYPOTROPHIC CARDIO MYOPOTHY DOES NOT DECREASE THE CAPACITY OF MYOSIN TO PERFORM. RATHER IT INCREASES IT. SHOWN HERE WITH THE SLIDING VELOCITY OF ACTIN AND FOURTH GENERATION IN A WILD TYPE COMPARED TO HYPERTROPHY MUTATION. ONE OF THE MORE PROFOUND USES OF HYPOTROPHIC REMODELLING. WE SEE TR’S ACTUALLY INCREASE IN SLIDING VELOCITY ALONG ACTIN AND APP A ACE AS WELL ANOTHER MUTATION HAS SOMEWHAT DIFFERENT DISTRIBUTIONS IN WHICH OF THOSE BIOPHYSICAL PROPERTIES ARE RETURNED, BUT BOTH OF THESE WILL DO EXACTLY THE SAME THING. THEY WILL INCREASE THE POWER OF THE CONTRACTILE APPARATUS, NOT DECREASE IT. WELL IF THAT’S THE CASE F WE ARE INCREASING POWER, HOW ARE WE PRODUCING THOSE HISTONE CHANGES THAT WE SEE? WHAT WE THINK IS GOING ON RIGHT NOW IS FROM THE INCREASE IN POWER FROM THE SACOMERE GENE MUTATION THAT WE ARE RESULTING IN AN INCREASE OF SIGNALING THAT IS TRANSMITTED TO THE NON-MYOCYTE POPULATION, NON-MYOCYTE BEING THE FIBER ONES THAT ARE RESIDENT WITHIN ALL OF OUR HEART CELLS AND THE BIOPHYSICAL — ARE ACTUALLY TRANSDUCING SIGNALS IN THOSE NON-MYOCYTE CELLS. THIS IS BREASTING ‘CUZ THESE NON-MYOCYTE CELLS DON’T EVEN CONTAIN THE MUTATION, BUT THE ACTIVATION OF SIGNALING ON THEM IS SO POWERFUL THAT WE SEE INCREASES IN CALCIUM, HOMEOSTAS SIS, INCREASED IN TGF BETA SIGNALING THAT LEADS TO A PROFOUND INCREASE IN THE [INDISCERNIBLE] AND THAT OVERTIME RESULTS IN DEPOSITION OF THE EXTRA CELLAR MATRIX THAT WE SEE IN HUMAN HEARTS. WE CAN CHALLENGE THIS KIND OF PATHWAY WITH LOTS OF FARM LOGIC INTERVENTION, EITHER THOSE THAT DECREASE MECHANICAL POWER OR MORE DIRECTLY THOSE THAT INHIBIT TGF BETA SIGNALING WITH ANTIBODIES OR SOME OTHER PHARMACOLOGIC AGENT. WE’RE ABLE TO DIMINISH — THAT’S WELL AND GOOD BUT THAT’S A SECONDARY CONSEQUENCE TO THE ACTUAL OR PROBLEM OF THE HEART, NAMELY THE MYOSIN MUTATION, ITSELF. SO ONE OF THE THINGS WE’VE ASKED IS WHETHER WE COULD HARNESS NEWER TECHNOLOGIES THAT REALLY GRAPPLE WITH THE CLOSER APPROXIMATE MALL PROBLEMS THE ACTUAL GENE MUTATION, WHETHER WE COULD IN FACT SILENT THE MYOSIN MUTATIONS THAT IS ONE OF TWO COPIES WITHIN THESE ANIMALS. TO DO THAT, WE’VE EMPLOYED A SYSTEM OFFING A KNOW ASSOCIATED VIRUSES, IN PARTICULAR TYPE 9 WHICH IS KNOWN TO BE CARDIO TROPIC. WE HAVE MANIPULATED THAT SO THAT WE HAVE IT GRIFFIN UNDER THE EXPRESSION OF [INDISCERNIBLE] T SO THAT THERE’S EXQUISITE — — WHAT WE CAN SEE IS GENERATE LOVELY GREEN HEARTS THAT ARE NOT RESULTING IN INCREASE OF EGFP EXPRESSION IN OTHER TISSUES AND I’LL TELL YOU THAT LIST LASTS FOR AT LEAST FIVE MONTHS PERIOD OF TIME AFTER A SINGLE INJECTION OF THE AAD VIRUS ON DAY ONE OF BIRTH. IF NOW INSTEAD OF JUST HAVING NON-SPECIFIC VECTOR SUCH AS GREEN PROTEIN WE USE RNAI WE THOUGHT THIS MIGHT PROVIDE US CAPACITY TO DECREASE MUTANT EXPRESSION. IF WE’RE GOING TO DO THAT, WE HAVE TO BE PRETTY CAREFUL BECAUSE THIS IS A DOMINANT MUTATION. SINGLE NUKE YEAH TIDE CHANGE IN ONE OF TWO ALLELES AND WOULD NOT BE BENEFICIAL TO THE MOUSE AND CERTAINLY NOT A HUMANS IF WE KNOCKED OUT BOTH COPIES OF MICE IN EXPRESSION. WE KNOW THAT’S LETHAL PHENOTYPE FROM KNOCKOUT MICE. WE ASKED WHETHER WE COULD SELECTIVELY KNOCK DOWN THE MUTANT EXPRESSION WITH SHRNA THAT IN PARTICULAR TAGGED THE PARTICULAR MUTATION AND WHETHER THAT WOULD BE SPECIFIC TO DISCRIMINATE BETWEEN MUTANT AND WILD TYPE. WHICH N COMPARISON TO SCRAMBLE HERE THAT OUR MUTANT SELECTIVE SHRNA WAS WHOAFULLY INACCURATE. THIS IS DIFFERENT FROM THE WILD TYPE ALLELE BY SINGLE NEWICALLY TIDE. SO IF WE ADD ONE MORE VARIANT IN THE SHRNA SO THAT WE ARE NOW TWO NUCLEOTIDES DIFFERENT FROM WILD TYPE AND ONE FROM THE DIFFERENT ALLELE, WE RECHIEFED A REMARKABLE DIFFERENT RESULT WITH SUPPRESSION TO ABOUT 25% OF THE PRIOR LEVEL OF MUTANT RNA EXPRESSION. IS THAT SUFFICIENT TO DO MUCH? WELL, WE INJECTED THESE SHRNAs INTO OUR HEARTS OF TO OUR MICE, RATHER, AND EVALUATED THEM APPROXIMATELY EIGHT WEEKS AFTER THE INJECTION AND THE RESULT WAS TRULY ASTONISHING. RECALL NOW THAT WE’RE REDUCING AT MOST BY 25% THE AMOUNT OF MUTANT RNA EXPRESSION AND THIS IS A SERIES OF CARDIAC EVALUATION. HERE’S THE WILD TYPE AND WHAT YOU CAN SEE IS THE LEFT VENTRICULAR WALL THICK SNESZ ABOUT .7 MILLIMETERS. IF WE REDUCE THE LEVEL OF EXPRESSION OF THE MU ABOUT THE ALLELE CONSIDERABLY, WE GET IT BACK TO NORMAL L. THAT’S A NICE NON-SPECIFIC PHENOTYPE, THE IMNEW NIS TICK ARE ARE FAR MORE IMPRESSIVE. THIS IS ANIMAL TREATED ARE [INDISCERNIBLE] I HOPE YOU CAN SEE THAT THERE’S DISARRAY AND ACCUMULATION OF FIBROSIS. IN THE ANIMAL TREATED AFTER EIGHT WEEKS WITH THE SH RSHG NA, SEE NONE OF THAT. SIMILARLY, THE KINDS OF INCREASE EXPRESSION AND MARKERS OF HYPERTROPHY ARE ENORMOUSLY REDUCED WHEN WE HAVE MAXIMUM VIRAL DELIVERY. THE AMOUNT OF FIBROSIS IS SIMILARLY REDUCED. WE KNOW THAT WHEN YOU HAVE A A HYPOTROPHIC REMODELLING, YOU HAVE LOTS OF EKG CHANGES AND WE ALSO SEE THOSE IN OUR MOUSE PARTS. SHOWN IS A WIDENING THAT OCCURS QUITE COMMONLY IN THE HYPOTRO IFIC MOUSE. WE CAN NOW BEGIN TO KNOW SOME OF THE MUTATIONS THAT ARE DEFINITIVE CAUSES OF CARDIO MYOPATHY. WE CAN BEGIN TO SAY THAT THESE ARE LIKELY TO BE ACTING THROUGH A DOMINANT MECHANISM, AND THAT WE HAVE A STRATEGY TO POTENTIALLY SILENCE THEM IN AN ALLELE-SPECIFIC WAY IN MICE. COULD WE BRING THIS TO THE HUMAN POPULATION? I WISH IT WAS FRIDAY AFTERNOON BECAUSE IN BOSTON THAT’S A TIME WHEN THE LABS ALL GATHER TOGETHER AND HAVE A BEER AND ENJOY EACH OTHERS COMPANY AND IT MAKES YOU DREAM A LITTLE BIT AND WE CARDIOLOGIES ARE SOMETIMES VERY BIG DREAMERS. THINGS.OT AFRAID TO TACKLE WHAT I’M SHOWING HERE IN THIS SCHIZOPHRENIC ECHOCARDIOGRAM IS AN IMAGE OF A HUMAN HEART IN WHICH THE HEAD OF THE INDIVIDUAL’S HERE, FEET OVER HERE. ECHO IS IS SHOWING THE BLOOD GOING FROM THE LEFT ATRIUM TO LEFT VENN TRI CALL AND THIS BIG CYST AREA OVER HERE IS A HIGHLY INTERVENTRICULAR ACCEPT TIM. IT ACCOUNTS THE FOR ENORMOUS AMOUNT OF SYMPTOMS AND CAN INCREASE RISK OF SUDDEN DEATH IN INDIVIDUAL WHO IS HAVE PROFOUND — WE KNOW THIS IS SO PROFOUND THAT WE TAKE TWO HEROIC MEASURES TO GET RID OF IT. FIRST IS TO DO OPEN HEART SURGE RAND TO REMOVE THAT HYPERTROPHY SEPTUM. LESS INFAY VA SIEVE WAY IS THE PASS CATHETER INTO THE AROR TA FROM THE LEG AND RETROGRADE TO CAN LATE THE VESSELS THAT SPECIFICALLY FEED THE INTERVENTRICULAR SEPTUM, THE ONE SHOWN RIGHT HERE CALLED THE INTERVENTRICULAR ACCEPT TALL — AND TO INJECT ALCOHOL IN IT SO THAT WE’VE GIVEN THE INDIVIDUAL A HEART ATTACK TO HAVE THAT HIGH PERT THE FI SET AND DIE. I WOULD SUGGEST TO YOU THAT I DON’T THINK TOO FAR OFF TO THINK ABOUT RATHER THAN KILLING THE MYOCARDIUM, TO USE THESE TO POTENTIALLY FUND MUTATION. IF WE COULD ACHIEVE LONG-TERM SILENTING OF EVEN 25-30%, WE MIGHT SUBSTANTIALLY REDUCE THE HYPERTROPHY IN THAT REGION AND SPARE THE MYOCARDIUM FROM DEATH. HOW MANY PEOPLE HAVE THAT PARTICULAR MUTATION? PRIVATE MUTATIONS ARE THE NAME OF THE GAME IN LOTS OF IMPORTANT DISEASES INCLUDING ALL THE CARDIO MYOPATHY. THEY’RE IN NOW MORE THAN A THOUSAND DIFFERENT MUTATIONS THAT HAVE BEEN IDENTIFIED TO CAUSE HYPOTROPHIC CARDIO MYOPATHY. THERE WILL BE AN EQUAL NUMBER THAT CAUSE DILATED CARDIO MYOPATHY. WHAT WE CAN DO IS CAPITALIZE ON EXTENSIVE KNOWLEDGE THAT’S COME BEGIN TO ASK WHETHER WE COULDND INSTEAD OF TARGETING THE PRECISE MUTATION, TO TARGET A NEARBY VARIANT THAT IS IN LINKAGE ASSOCIATION WITH THAT MUTATION. IT’S ON THE NAME ALLELE, IT ACTUALLY IS COINHERIT BD W THE MUTATION. TO SEE IF THAT WAS EVEN FEASIBLE, WE WENT TO A FAVORITE SOURCE OF US, THE NHLBI LOOKING AT MYOSIN HEAVY CHAIN GENE AND WE SEE HERE THAT THIS IS A VARIANCE BUT THAT ONE TURNS OUT TO BE IN AN INTRON WHICH WOULD BE HEART TO SILENT FROM THE RNA POINT OF VIEW BUT HERE’S A CODING SYNONYMOUS VARIANT AND HERE’S ANOTHER ONE OVER HERE. SO EVERYWHERE IF — IF WE WERE TO TEST THAT STRATEGY, WE COULD IMBRED MOUSE, BREED IT INTO A DIFFERENT BACKGROUND AND NOW TRY AND SILENT NOT THE MUTATION BUT THE NEARBY POLYMORPHIC [INDISCERNIBLE] WHEN WE DO SO THE RESULTS ARE ESSENTIALLY THE SAME. WHAT I’VE TRIED TO TELL YOU IS THAT WHAT WE THINK BY ACTUALLY CONSIDERING THE USE OF NEARBY SNPs THAT ARE SIGHTLY LINKED TO MU TAN ATE A LEGALS THAT WE COULD STILL A BATTERY OF APPROACHES TO SILENCE MUTATION IN A VERY SPECIFIC WAY THAT WOULD LEAVE THE NON-MOW TATED ALLELE TO MAKE THE PROTEIN AND WHILE IT WOULD BE UNLIKELY THAT WE COULD COMPLETELY REDUCE THE MUTANT LEVEL TO ZERO THAT WE MIGHT HAVE THE CAPACITY TO REDUCE IT SIGNIFICANTLY TO ATTENUATE THE REMODEL THAT OCCURS. WHERE ARE WE GOING WITH ALL THIS? WELL, I HOPE I’VE CONVINCED YOU THAT THERE ARE LOTS OF CARDIOVASCULAR DISEASE GENES. THEY’VE BEEN POWERFUL IMPORTANT IN TEACHING ABOUT SACOMERE FUNCTION AND BIOLOGY. I THINK THEY’RE INCREASINGLY GOING TO BECOME VERY USEFUL IN UNDERSTANDING PHENO TIP PICK VARIANCE IN CLINICAL SETTING. THERE’S A LOT MORE FOR US TO BE ABLE DO THAT IN A VERY ROBUST FASHION, BUT WITH THE INCREASE USE OF GENETIC TESTING I’M CERTAIN WE’LL GET THERE. SHRNA IS ONLY THE BEGINNING OF TRYING TO SILENCE MUTATION IN SEMATIC TISSUES. I THINK THERE’S ENORMOUS CAPACITY TO USE GENETIC ANTEDOTES FOR TREATING NOT ONLY HEART DISEASE BUT A VARIETY OF OTHER CONDITIONS, AND I WOULD ENCOURAGE ALL OF YOU YOUNG PEOPLE IN THE AUDIENCE TO THINK ABOUT GETTING INVOLVED IN THIS REALLY, REALLY EXCITING AREA OF SCIENCE. I HAVE TO ACKNOWLEDGE THIS AMAZING TEAM I WORK WITH, IN PARTICULAR ALEX THICK, DAN HERMAN, PAULA KEY, BASIC RESEARCHERS, AND ALSO ND Ph.D. STUDENTS, CAR LYNN HULL ARK WHOLE TEAM OF SEQUENCING COLLEAGUE FROM THE BROAD, FROM THE JACKSON AND FRAMING HAM HEART COHORTS AND MY COLLEAGUE FROM THE UK, STEWART COOK. THANK YOU VERY MUCH. [APPLAUSE]>>THAT WAS TERRIFIC. PLEASE DO USE THE MICROPHONES TO POSE QUESTIONS SO PEOPLE LISTENING ON THE WEB CAN HEAR AS WELL. GO AHEAD.>>THANK YOU VERY MUCH. WHEN THOSE [INDISCERNIBLE] [LOW AUDIO].>>I WILL.>>THIS IS SA QUESTION ABOUT [LOW AUDIO] LINK TO [INDISCERNIBLE] — >>TO THE QUESTION IS, CAN WE REVERSE THE ESTABLISHED HIGH P PERT FI USING SHRNA APPROACH RIGHT NOW? THOSE ARE EXPERIMENTS THAT WE HAVE DONE ONLY IN A SHORT-TERM SEQUENCE RIGHT NOW. WE HAVE NOT DONE THEM IN A FULL EIGHT-WEEK TO 12-WEEK PERIOD OF TIME AND WE THINK THOSE ARE IMPORTANT TO DO FOR A PROTRACTED PERIOD OF TIME. WHEN WE DO A ACCEPT L OBLATION, IT TAKES MONTHS FOR THE MYOCARDIAL TO REMODEL AND THAT’S AFTER KILLING THE HEART MUSCLE. WE WOULD ANTICIPATE IT COULD TAKE MANY MONTHS TO REMODEL HYPERTROPHY MOUSE HEART. DO WE THINK IT’LL WORK, YEAH, I THINK IT WILL. WE WOULD EXPECT THERE TO BE INCREASED AMOUNT OF WILD TYPE ALLELE SHOULD ALLOW FOR THAT INCREASE TO BE REACHED OVER TIME. SECOND REASON I THINK THAT IS ANYBODY WHO’S TREATED A PATIENT WITH HYPER TENSION AND HIGH P PERT FI, IT TAKES TIME BUT OVER TIME THAT LEFT VENTRICULAR WILL RESOLVE. WE JUST DON’T HAVE THE RESULTS YET.>>BASIC QUESTION ABOUT THE TITIN PART OF YOUR TALK. IN AN INDIVIDUAL WHO HAS HETERO PSI GUS TRUNCATING MUTATION, ARE THE TWO ALLELES TRANSLATED IN 50/50 PORTION OR DOES THE TRUNCATED HAVE AN ADVANTAGE?>>THAT’S A VERY GOOD QUESTION. WE BEGUN TO LOOK AT THAT BY THE ONE GOOD NEWS ABOUT TITIN IS THERE’S SO MANY VARIANCE IN THERE THAT YOU CAN LOOK TO SEE FROM RNA SEEK ANALYSIS IF BOTH IN FACT ARE EXPRESSED. WE LOOKED AT THAT AND WE SEE NO INCREASE NOR DECREASE IN ALLELEIC-SPECIFIC SNPs AS WE MARCH ALONG THE TITIN MOLECULE. SO WE DON’T SEE AN ADVANTAGE NOR A DISADVANTAGE.>>[INDISCERNIBLE] HOW LONG [INDISCERNIBLE] WHERE DOES THE OUTCOME COME FROM CARDIAC [INDISCERNIBLE]?>>SO ALCOHOL ACCEPT TALL OWE BLAGS IS JUST SO WE’RE CLEAR NOT SOMETHING THAT I ROUTINELY DO BUT MANY OF MY COLLEAGUES DO AND IT IS USUALLY RECOMMENDED FOR INDIVIDUALS WHO HAVE FIRST AND FOREMOST A LOT OF SYMPTOMS THAT HAVE NOT RESPONDED TO PHARMACOLOGIC INTERVENTION, AND FOR REASONS THAT THE PATIENT OFTEN IS INVOLVED IN OR BECAUS THEY’RE A A CLINICAL INDICA THAT, PERSON IS NOT APPROPRIATE FOR REFERRAL TO SURGERY TO OPEN HEART SURGERY. IT’S INCREASINGLY BEING USED BECAUSE OF PATIENT PREFERENCE. IT USUALLY TAKES A PERIOD OF SIX WEEKS OR LONGER TO SEE REMODELLING AND REGRESSION OF THE INTERVENTRICULAR SEPTUM SO THAT THE OUTFLOW TRACK BECOMES MORE OPENED. FROM THE LONG-TERM FOLLOW-UP WE DO NOT SEE A GROWING BACK. WHETHER THAT WILL HAPPEN IN FIVE YEARS, I DON’T KNOW, BUT RIGHT NOW WE HAVE NOT SEEN IT.>>[INDISCERNIBLE] LIGATION [LOW AUDIO] — >>I’M SORRY. OH, IT’S ONE-TIME, YES. I WILL MENTION FOR PEOPLE WHO ARE NOT INTERESTED IN THIS CLINICALLY, THE ADVANTAGE OF THIS AV THING IS IT’S ALSO ONE-TIME USE AND YOU GET FIVE MONTHS OF SILENCING THE GENE OF YOUR CHOICE FOR ONE-TIME INJECTION IN YOUR MOUSE. THAT’S A GOOD EXPERIMENTAL SYSTEM WHICH WE’RE USING FOR LOTS OF OTHER INVESTIGATION.>>THANKS.>>JUST WONDERING ABOUT THE PART OF THE TALK TITINS, THE DIFFERENTIAL EXPRESSION OF THE TITINS [LOW AUDIO] THOUGHT TO BE PROTECTIVE AND HAVE YOU DEALT WITH IT TO THE GENETIC SEQUENCES MIGHT BE?>>I’M SORRY.>>WITHIN THE TITIN GENE, YOU SHOWED DIFFERENT EXPRESSION PATTERN INS AND DIFFERENT PARTS MORE THAN OTHERS AND THAT THERE’S CORRELATION WHICH — [LOW AUDIO] COMPARED TO THE EXPRESSION PATTERN. JUST WONDER IS THAT EXPRESSION PATTERN DIFFERENT HYPOTHESIZE BEING PROTECTED?>>THE REGIONS THAT WHERE THE NUMBER L POPULATION HAS ACCUMULATED SOME VARIANCE THAT TRUNCATE THE MOLECULES ARE IN PARTS POF, CAN BE INVOLVED IN MAKING TITIN MOLECULES THAT ARE VERY, VERY LOW-LEVEL EXPRESSION. SO THEIR ALTERNATIVE EXXONS THAT ARE SPLICED IN AND SPLICED OUT BECAUSE THERE’S A LOT OF DIFFERENT TITINS IN EACH AND ERE HEART, BUT THE ABUNDANCE OF THEM IS VERY, VERY LOW, AND INDEED THE ONES THAT ARE INCORPORATED INTO THE SACOMERE ALMOST BY DEFINITION BECAUSE THEY STAND FROM THE [INDISCERNIBLE] ARE WHAT WE CALL FULL-LENGTH TITINS REE FERED TO AND IT DOES NOT INCLUDE THOSE LOWLY EXPRESSED EXXONS. THERE’S ALSO ANOTHER FOR THOSE, CALLED THE KNOW VEX THREE ISOFORM WHICH IS A VERY, VERY SHORT FIVE THOUSAND BASE PAIR TRANSCRIPT AND NO ONE ACTUALLY KNOWS WHAT THAT DOES. IF IT DOES ANYTHING.>>WHOLE TITIN STORY GETS MORE INTERESTING ALL THE TIME. I GUESS YOU’RE REALLY CAUSING MANY OF US TO DEPEND UPON NOT JUST MEDIATED DECAY TO PREDICT THE CONSEQUENCE OF ONSETS OR FRAMESET MUTATION TO QUESTION WHETHER WE SHOULD DEPEND ON THAT IF THE GENE IS ANYTHING OTHER THAN HEMOGLOBIN ‘CUZ IT SOUNDS AS IF MAYBE BECAUSE THE COUPLE OF TRANSLATION, SPLICING THAT YOU ALREADY PASSED THE POINT OF NOT HAVING A PROTEIN BEFORE YOU HIT THAT STOP CODE. I ASSUME THOUGH THAT THERE WOULD BE SOME RELATIONSHIP — WE’RE TALKING ABOUT THIS A MINUTE AGO — IN TERMS OF ABUNDANCE OF THE MUTANT ALLELE DEPENDING ON WHERE THE TRUNCATING MUTATION WAS AND THAT IT WAS CLEARLY EARLY ON THAT YOU WOULD GET AMOUNTS OF TRANSCRIPT — PART OF THE QUESTION I WANTED TO ASK IS WHAT IS THE PHENOTYPE OF A TRUE APP LOW INSUFFICIENT TITIN ALLELE AND IF IT’S NOT BAD DOES IT SAY THAT WHAT YOU’RE DOING FOR THESE HIGH PROTROPHIC CARDIO MYOPATHIES MIGHT ALSO WORK FOR TITIN MUTATION AND CAUSE DIELTED CARDIO MYOPATHY OR TRYING TO DIAL?>>SO THE MOST DEFINITIVE EVIDENCE, I THINK, OF HUMAN PATIENTS — NOT A MOUSE — WITH A TITIN INSUFFICIENT MUTATION ANNUL HAS COME FROM SOME RARE RE RECESSIVE FAMILIES. IT’S AT THAT THREE PRIME, IT’S THOSE M BAN MUTATIONS. THERE HAS BEEN NO REPORTS OF A HOMO SIGH GUS OR HETERO PSI GUS WITH OVER — >>SO IT MIGHT BE [LOW AUDIO].>>AT THE VERY, VERY AMY KNOW TERMINAL SEQUENCE, THAT’S CORRECT. AND WHEN WE SEE WHERE WE FIND THOSE CLUSTERS IN OUR RANDOM PEOPLE, THEY’RE MORE LIKELY TO BE AT THAT AMY KNOW TERM NANT. SO THERE MAY BE NO ABNORMALITITY OF THE HETERO PSI GUS LOSS OF FUNCTION. WHEN WE SHIFT TO MICE NOW AND THERE IS ONE COMPLE MOUSE MODEL THAT WAS A KNOCKOUT BUT IT ALSO INTRODUCED A SIMULTANEOUS MUTATION. SUFFICE IT TO SAY THAT IT APPEARS THAT THE HOMO SIGH GUS IS LETHAL. THE HETERO SIGH GUS IS FINE. SO WE DO THINK THAT IF IT IS A DOMINANT NEGATIVE MODE OF ACTION SILENTING TITINS WOULD BE BENEFICIAL.>>WE HAVE PLENTY OF WAYS TO DO THAT GIVEN THE TITIN SUCH A HUGE NUMBER OF VARIANTS AND ALMOST EVERYBODY’S GOING BE HETERO PSI GUS — >>RIGHT. CORRECT. WE WOULD LIKE TO HAVE THOSE THAT ARE COMMON SO WE DIDN’T NEED TO MAKE A THOUSAND OF THEM, JUST A COUPLE HUNDRED.>>ONE MORE QUESTION.>>I READ AT THE BEGINNING WHAT IS THE PERCENTAGE OF CARDIO MYOPATHIES [INDISCERNIBLE] JE NE SIX AND WHAT’S THE [INDISCERNIBLE] LIFESTYLE OR ENVIRONMENT FACTORS OR — >>OR ALL OF THE ABOVE, RIGHT? [LAUGHTER] IF IF YOU TAKE INDIVIDUALS THAT HAVE ONE FAMILY MEMBER WHO WAS ALSO AFFECTED WITH A HYPER TROPHIC PHENOTYPE, 75% OF THOSE ARE GENETIC. THAT’S HIGH. IF YOU TAKE INDIVIDUALS WHO HAVE NO FAMILY HISTORY OF CARDIAC HYPERTROPHY AND THEY HAVE ALL OF THE CLASSIC MANIFESTATIONS OF HYPER TRO IF I CAN CARDIO MYOPATHY BUT NO FAMILY HISTORY, 35% ARE GENETIC AT TOPS. IF WE DO THE SAME EXPERIMENT WITH DIELTED CARDIO MYOPATHY, THE BEST EVEN IN A FAM MILL Y’ALL CASE WE’RE DOING IS ABOUT 50% AND THAT’S INCLUDING TITIN WHERE THAT’S 25%. THAT’S THE BIGGEST PIECE OF THE PIE RIGHT NOW AND EVERYTHING ELSE IS A THOUSAND OTHER LITTLE GENES. SO THERE’S STILL MORE TO DISCOVER THERE. AND WHEN YOU TAKE AN ISOLATED INDIVIDUAL SIT OWE PA THICK CARDIO PAFK AND INCLUDE TITIN, WE’RE AT ABOUT 15%.>>WELL, DR. SEIDMAN WOULD BE WILLING TO CONTINUE THIS LIBRARY.ION AT A A RECEPTION IN MEANWHILE, PLEASE JOIN ME IN OUR SPEAKER AGAIN.