The epigenetic clock, biological age, and chronic diseases

GOOD AFTERNOON, WELCOME TO THE AUDITORIUM STEVE HORVATH GOT A PH.D. IN MATHEMATICS, AND HE REMAINED A MATHEMATICIAN AND IN FACT, THE WAY HE APPROACHED THE PROBLEM, HE WAS NOT HAPPY ABOUT IT, SO HE ALSO DECIDED THAT HE NEEDED A PH.D IN BIOSTATISTICS, AND HE GOT THE BIOSTATISTICS PH.D. AT HARVARD, WHERE HE WORKED ON FAMILY STUDIES AND REALLY DEVELOPED SOFTWARE THAT IS USED FOR LINKAGE NOWADAYS HIS CLAIM TO FAME IS THE DEVELOPMENT OF THE EPIGENETIC CLOCK AND I WILL TAKE TWO MINUTES TO DIVERSION MANY YEARS AGO, IN THE 80s, WHEN I WAS STARTING TO COME AT NIH, VISITING FROM ITALY, I REMEMBER IN AN AFTERNOON I HAD A MEETING WITH ANDREA — ONE OF THE PRINCIPAL INVESTIGATORS OF THE WOMEN’S HEALTH INITIATIVE WE WERE DISCUSSING ABOUT THE DREAMS FOR SOMEBODY WAS WORKING ON THE AGING FIELD, AND WHAT SHE WAS TELLING ME IS THAT WHAT WE REALLY NEED IS THE ROBUST MY BYE OWE MARKER OF BIOLOGICAL AGING THAT WE COULD USE TO UNDERSTAND WHY SOME PEOPLE TEND TO LOOK LIKE THEY’RE AGING FASTER AND SOME PEOPLE LOOK LIKE THEY’RE AGING SLOWER SLOWER — AS CLOSE AS WE ARE NOW TO THAT DREAM, AND YOU WILL SEE THAT TSH USING METHYLATION DATA A BIOMARKER THAT AAPPROXIMATES AGE VERY, VERY CLOSELY ALSO INDEPENDENT OF AGE, IS ABLE TO PREDICT MORTALITY AND OTHER HEALTH OUTCOMES I DON’T WANT TO STEAL MORE OF HIS IDEAS AND RESULTS, BUTLY JUST CONCLUDE TO SAY THAT WE WORK TOGETHER ON SOME PROJECTS, IT’S ALWAYS AN INCREDIBLE PLEASURE TO DISCUSS WITH HIM, HE’S VERY, VERY OPEN TO DISCUSSION AND TO, YOU KNOW, ADDRESS QUESTIONS, SO GIVEN THESE PREMISES, PLEASE HELP ME IN WELCOMING STEVEN HORVATH TO OUR WEDNESDAY AFTERNOON LECTURE >> THANK YOU, LUIGI, FOR THE VERY KIND AND GENEROUS INTRODUCTION I ALSO WANT TO THANK DR. HODES AND FRANCIS COLLINS FOR INVITING ME IT’S A TREMENDOUS PRIVILEGE TO STABBED IN FRONT OSTAND IN FRONT OF YOU AN D TO DESCRIBE SOME OF THE LATEST WORK WHICH I SUM UP BY THE EPIGENETIC CLOCK AND WITHOUT GIVING YOU MUCH DETAILS, THESE FIGURES ILLUSTRATE PROPERTIES OF THE CLOCK ON THE RIGHT-HAND SIDE, YOU CEELSEELEONARDO DAVINCI THAT DEPICT THE PHYSICAL DIMENSIONS OF THE HUMAN BODY NOW WE AIM TO DEVELOP CLOCKS THAT ADD A TIME ACCESS TO THE HUMAN BODY THAT TELL US WHAT SHOULD BE THE AGE OF THE HEART OF A 40-YEAR-OLD, OR WHAT SHOULD BE THE AGE OF THE BREAST OF A 40-YEAR-OLD? SO THIS EPIGENETIC CLOCK APPLIES TO ALL TISSUES, ORGANS AND CELLS THAT CONTAIN DNA WITH THE EXCEPTION OF SPERM AND ALTHOUGH IT’S BASED ON METHYLATION LEVELS, THAT IN PRINCIPLE COMPRISE HUNDREDS IF NOT MILLIONS OF VARIABLES, THE OUTPUT OF THE METHOD IS A NUMBER, AN INTUITIVE NUMBER, WHICH IS JUST THE AGE SO FOR EXAMPLE, FOR A MIDDLE AGED MAN, I COULD SAY HIS HEART IS 50 YEARS OLD, HIS LUNG IS 30 YEARS OLD AND SO ON THE LEFT PANEL SHOWS ANOTHER PROPERTY OF THE EPIGENETIC CLOCK WHICH IS THAT IT APPLIES TO THE WHOLE LIFESPAN HERE I TALK, OF COURSE, TO AN AUDIENCE OF AGING RESEARCHER, HOWEVER, BE AWARE OF IT THAT THE

EPIGENETIC CLOCK ALSO APPLIES TO DEVELOPMENT IT APPLIES TO PRENATAL SAMPLES HERE’S MY ONE MINUTE PITCH WHY AGING RESEARCH MATTERS SO THIS IS AN ARTICLE FROM DANA GOLDMAN AND JAY OLSHANSKI, WHO CALCULATED THAT IF WE CAN SLOW DOWN BIOLOGICAL AGING BY AS LITTLE AS 20%, THE U.S. WILL SAVE $3 TRILLION IN MAJOR ENTITLEMENT SPENDING OVER THE NEXT 50 YEARS HUGE BENEFIT, IF WE CAN JUST TWEAK THE AGING A LITTLE BIT COMPARE THAT TO DELAYING THE ON SEPTEMBEONSET OF CANCER, WHICH IS THE BLUE CURVE AGAIN, A VERY WORTHWHILE GOAL, BUT IF WE DELAY THE ONSET OF CANCER BY 20%, WE ONLY SAVE LESS THAN $5 BILLION. SO THERE ARE TREMENDOUS BENEFITS TO BE HAD IF WE CAN SLOW BIOLOGICAL AGING NOW THERE IS GOOD NEWS IN PRINCIPLE, IT’S FAIRLY STRAIGHTFORWARD TO FIND INTERVENTIONS THAT SLOW HUMAN AGING IF YOU HAVE A BIOMARKER SO HERE’S MY THREE-STEP APPROACH FOR FINDING INTERVENTION FOR HUMAN SUBJECTS STEP 1, DEVELOP A BIOMARKER OF BIOLOGICAL AGE THAT’S, OF COURSE, THE TOPIC OF THIS TALK STEP NUMBER 2, FIND INTERVENTIONS THAT AFFECT THE BIOMARKER, FOR EXAMPLE, IN A DISH OR IN A MODEL AND WE CAN BRING TO BEAR ALL THE THE — — IN STEP THREE, OF COURSE, WE NEED TO CON DCT HUMAN CLINICAL TRIALS AND AGAIN, IF YOU HAVE A SUITABLE BIOMARKER THAT CAN BE USED AT THE SURROGATE OUTCOME OF AGING, YOU CAN SAVE TREMENDOUSLY BECAUSE INSTEAD OF SHOWING THAT AN INTERVENTION REDUCES MORTALITY, AND YOU WOULD NEED TO SHOW IT BY FOLLOWING PEOPLE FOR MAYBE 10, 20 YEARS, IF INSTEAD YOU CAN SAY THAT AN INTERVENTION MOVES ITS BIOMARKER IN THESE SUBJECTS, THEN POSSIBLY YOU CAN SPEED THROUGH THE RECOVERY PROCESS SO THERE IS OF COURSE A MAJOR CHALLENGE, HOW DO WE DEFINE A BIOMARKER OF BIOLOGICAL AGE? AND THAT’S A CHALLENGING PROBLEM FOR MANY REASONS, BUT ONE IS THAT THERE’S NO CLEAR DEFINITION OF WHAT IS BIOLOGICAL AGE SEEMINGLY EVERYBODY HAS THEIR OWN DEFINITION AS A FORMER STATISTICIAN, I WOULD CALL BIOLOGICAL AGE A LAY AT THE PRESENT TIME VARIABLE THAT IS NOT WELL-DEFINED AND THAT HAS TO BE DEFINED THROUGH INDICES AND OF COURSE INTUITIVELY, THERE’S ONE PROPERTY ABOUT BIOLOGICAL AGE THAT WE CAN REALLY LEVERAGE, AND THAT IS BIOLOGICAL AGE WILL BE HIGHLY CORRELATED WITH CHRONOLOGIC AGE AND WHY DO I KNOW IT? WELL, BECAUSE THE WHOLE INSURANCE INDUSTRY IS BUILT ON THAT CONCEPT THE OLDER YOU ARE, THE HIGHER YOUR PREMIUM AND SO, THEREFORE, TO THEN FIND BIOMARKERS OF BIOLOGICAL AGE, ONE CAN FOLLOW THIS PATH FIRST, YOU FIND MOLECULAR BIOMARKERS THAT STRONGLY CORRELATE WITH CHRONOLOGICAL AGE AND YOU USE CHRONOLOGIC AGE REALLY AS A PROXY OF BIOLOGICAL AGE ONCE YOU HAVE THESE BIOMARKERS, THEN YOU VALIDATE THEM IN VARIOUS COHORT STUDIES TO SHOW THAT THEY PREDICT OR RELATE TO MANY AGE-RELATED PHENOTYPES SO I TALKED ABOUT BIOMARKERS OF AGING, BUT YOU REMEMBER THAT I ALSO TALK ABOUT THE EPIGENETIC CLOCK, SO WHAT IS A CLOCK? I HAVE A PERSONAL DEFINITION I CALL SOMETHING AN AGING CLAK IF IT’S AN EXTREMELY ACCURATE BIOMARC OF AGING FOR EXAMPLE, GRAY HAIR WOULD NOT BE CALLED AN AGING CLOCK, IT’S SIMPLY NOT ACCURATE ENOUGH HOWEVER, IN MY PERSONAL DEFINITION, IF YOU HAVE AN ESTIMATOR OF AGE THAT HAS A CORRELATION OF .8, WITH THE TRUE KROCHRONOLOGIC AGE IN SUBJECTS THAT ARE AGED BETWEEN ZERO AND 100, IF THAT CAN BE ACHIEVED, I FEEL COMFORTABLE THAT YOU CALL IT AN AGING CLOCK IT’S WHY A CLOCK IS SIMPLY VERY ACCURATE AND I DO WANT TO MENTION THAT IT’S ABSOLUTELY CRITICAL TO VALIDATE ALL CLAIMS AND INDEPENDENT DATASETS, BECAUSE IT’S VERY EASY TO COME UP WITH A MULTIVARIATE MODEL IN ONE TRAINING SET THAT IS VERY ACCURATE, BUT IT SIMPLY DOESN’T VALIDATE AN INDEPENDENT DATA

SO IN PRINCIPLE, I’M COMING BACK TO DEVELOPING BIOMARKERS OF AGING WE HAVE WONDERFUL CANDIDATE AGING CLOCK CLEARLY TELOMERE LENGTH MEASURES A LOT IN HEALTH AND DISEASE THEN, OF COURSE, TRANSCRYPTOMIC DATA, AND THE FOURTH TYPE, THE DNA METHYLATION DATA OR EPIGENETIC DATA ARE THE FOCUS OF THIS TALK THE TRUTH IS I’M NOT AWARE OF ANY BIOMARKER THAT MEETS MY STRINGENT DEFINITION OF AN AGING CLOCK APART FROM METHYLATION-BASED BIOMARKERS, AND THERE ARE SEVERAL OUT THERE SO LESS THAN THREE YEARS AGO, I PUBLISHED A MULTI-TISSUE BIOMARKER OF AGING, WHICH IS BASED ON DNA METHYLATION LEVELS TOE TIME WHEN AT THE TIME I PUBLISHED IT, IT WAS NOT CLEAR WHETHER THIS ACTUALLY CAPTURES BIOLOGICAL AGE THE TALK TODAY IS VERY MUCH ABOUT SHOWING THE PROPERTIES OF THIS MULTI-TISSUE PREDICTER SO BEFORE I GO THERE, LET ME REMIND YOU THAT WHAT IS DNA METHYLATION? SO DNA METHYLATION IS REALLY AN EPIGENETIC MODIFICATION OF THE DNA IT’S AN ADDITIONA AN ADDITION OF THE METHY L GROUP OF ALL OF MY RESULTS WERE BASED ON THE ILLUMINA ARRAY, THE EPIGENETIC CLOCK IS ACTUALLY PLATFORM INDEPENDENT YOU CAN APPLY IT TO THE LATEST PLATFORM, THE EPIC CHIP, YOU CAN APLAY TO THE OLDER PLATFORM, THE INFINIUM 4050K AND THE INFINIUM27K RATE THESE MARKERS ARE CALLED CPGs AND FOR EACH CPG, YOU GET A NUMBER BETWEEN ZERO AND 1, AND THIS NUMBER ROUGHLY CORRESPONDS TO THE PROPORTIONAL CHROMOSOMES THAT ARE METHYLATED AT THIS LOCATION SO TO DEVELOP THE EPIGENETIC CLOCK, I DOWNLOADED 82 PUBLICLY AVAILABLE DNA METHYLATION DATASETS THAT HAD BEEN DEPOSITED IN GENE EXPRESSION OMNIBUS IN THE CANCER GENOME AT LAS AND OTHER VARIOUS DATA REPOSITORIES THEY WERE ALL IN REPOSITORIES THAT WERE FUNDED SO PEOPLE WHO WANTED TO PROVIDE OPEN ACCESS AND COMPLETELY FREE ACCESS TO THE DATA IN THE MANY OF THE DATASETS, THE INVESTIGATORS WERE SO KIND TO INCLUDE CLINICAL VARIABLES AND DEMOGRAPHIC VARIABLES SUCH AS CHRONIC LOGIC AGE SO YOU RETBRES A TRANSFORMED VERSION OF CHRONOLOGIC AGE ON ALL THE CPGs, AND YOU CAN USE WHAT IS KNOWN AS AN ELASTIC NET REGRESSION MODEL, AND THIS MODEL AUTOMATICALLY TAKES THE BEST COVARIANTS, THE BEST CPGs, FOR PREDICTING THE OUTCOME AND THE RESULTING EPIGENETIC CLOCK METHOD IS RATHER STRAIGHTFORWARD SO YOU CAN IMPLEMENT IT IN THREE STEPS FIRST, YOU WANT TO MEASURE THE METHYLATION LEVELS OF 353 CPGs IN STEP NUMBER TWO, YOU FORM A WEIGHTED AVERAGE OF THE CPG, AND YOU GET A SINGLE NUMBER BUT THIS NUMBER IS NOT YET IN UNITS OF YEARS SO NEXT, WE TRANSFORM THIS NUMBER SO THAT IS AS THE UNIT APPEARS, AND THE RESULT IS THEN LITERALLY AN AGE ESTIMATE, A NUMBER, AND THAT AGE ESTIMATE IS IT REFERRED TO AS EPIGENETIC AGE, SO FOR EXAMPLE, OR THE DNA METHYLATION AGE SO WHAT MAKES THIS ALGORITHM SO NOTEWORTHY IS THAT IT IS THE IDENTICAL ALGORITHM FOR EVERY TISSUE AND CELL TYPE NOWHERE DO I INPUT NOW YOU’RE DEALING WITH BLOOD, NOW YOU’RE DEALING WITH BRAIN TISSUE IT JUST RECOGNIZES IT AUTOMATICALLY, AND THAT’S THE SURPRISING THING IT’S SURPRISING BECAUSE CELL TYPES HAVE VERY DIFFERENT METHYLATION PATTERN A BLOOD CELL IS COMPLETELY DIFFERENT FROM A NEURON WHEN YOU LOOK AT METHYLATION LEVELS HOWEVER, THERE IS SOMETHING INTRINSIC THAT’S REALLY PRESERVED ACROSS ALL TISSUES AND CELL TYPES SO LET ME GO BACK, SO REMEMBER IN STEP NUMBER 2 OF THIS ALGORITHM, YOU GET A WEIGHTED AVERAGE IT’S A WEIGHTED AVERAGE OF THE 353 CPGs AND I WILL SHOW HOW THIS

WEIGHTED AVERAGE DEPENDS ON CHRONOLOGIC AGE SO HERE THE Y AXIS SHOWS THE WEIGHTED AVERAGE AND WHAT DO WE SEE? EACH IS A HUMAN SUBJECT. SO WHAT WE SEE IS THAT BETWEEN AGE ZERO AND 20, THERE’S A SHARP INCREASE IN THIS WEIGHTED AVERAGE, AND AFTER AGE 20, THERE’S AN ALMOST LINEAR RELATIONSHIP AND SO I INTERPRET THIS BEHAVIOR THEN AS SAYING THAT THE EPIGENETIC CLOCK TAKES VERY QUICKLY AS THE ORGANISM GROW, BUT AFTER AGE 20, WHEN YOU’VE REACHED ADULTHOOD, IT STARTS TO TAKE ON A CONSTANT LEVEL SO THE EPIGENETIC CLOCK IS EXTREMELY ACCURATE, AS ONE CAN SEE FROM THE EVALUATIONS AND TEST DATASETS, SO THE UPPER LEFT PANEL SHOWS HOW CHRONOLOGIC AGE CORRELATES WITH METHYLATION AGE IN INDEPENDENT TEST DATA, AND THERE THE CORRELATION IS .96 THE OTHER PANELS REPORT FIND NGS DIFFERENT TISSUE TYPES IN THE MIDDLE PANEL, IN PANEL H, YOU SEE THE EXAMPLE OF BREAST TISSUE, AND THE BLACK SOLID LINE SHOWS THE AXIS Y EQUALS X, WHICH INDICATES PERFECT CALIBRATION AND WHAT WE SEE IS THERE’S A SYSTEMATIC SHIFT, SO THE CLOCK ALWAYS OVERESTIMATES THE AGE OF BREAST TISSUE AND LET’S MAKE A NOTE OF IT, BECAUSE IT MIGHT SUGGEST THEN THAT POSSIBLY FEMALE BREAST TISSUE IS OLDER THAN THE REST OF THE BODY THAT’S ONE INTERPRETATION ANOTHER INTERPRETATION IS THE CLOCK SIMPLY ISN’T WELL CALIBRATED HERE SO IN THIS APPLICATION, I ANALYZED CADAVER, A FEMALE CADAVER AND THE MALE CADAVER, AND THE EPIGENETIC AGES OF DIFFERENT BODY PARTS, AND THE ONE BODY PART THAT AGAIN DIDN’T FIT THE MOLD WAS THE FEMALE BREAST TISSUE HERE IT HAD AN AGE ESTIMATE OF 67 YEARS, WHEREAS THE REST OF THE BODY HAD AN AVERAGE AGE OF 44 THAT’S, OF COURSE, JUST AN ANECDOTE SO RECENTLY, WE REVISITED THAT QUESTION ABOUT THE AGE OF BREAST TISSUE, USING HEALTHY FEMALE BREAST TISSUE SAMPLES FROM THE KOMEN TISSUE BANK, AND FROM THE VERY SAME WOMEN, WE ALSO HAD BLOOD SAMPLES COLLECTED ON THE VERY SAME DAY AND THE UPPER LEFT PANEL SHOWS HOW METHYLATION AGE ON THE Y AXIS RELATES TO CHRONOLOGIC AGE, AND YOU SEE THE RED DOTS ARE ALWAYS ABOVE THE BLACK DOTS, AND THE BLACK DOTS ARE BLOOD — WE SEE BREAST TISSUE IS ALWAYS OLDER THAN THE BLOOD TISSUE FROM THE SAME WOMAN, WHICH IN CERTAIN WAYS VALIDATES THIS FINDING SO TO ME, THAT’S INTERESTING BECAUSE IT PROBABLY TEACHES ME SOMETHING ABOUT THE EPIGENETIC CLOCK I THINK HORMONE EXPOSURE SOMEHOW RELATES TO EPIGENETIC AGING I WANT TO MOVE TO ANOTHER APPLICATION WHERE WE ANALYZED THE EFFECT OF OBESITY ON VARIOUS HUMAN TISSUES THE UPPER PANEL SHOW THE ACCURACY OF THE CLOCK IN LIVER TISSUE SAMPLES, ADIPOSE TISSUE SAMPLES, MUSCLE TISSUE SAMPLES AND BLOOD SAMPLES AND YOU NOTICE I PUT A REGRESSION LINE THROUGH THESE POINTS AND LET’S THINK ABOUT THE INTERPRETATION IF THERE’S A LIVER SAMPLE THAT LIES ABOVE THE REGRESSION LINE, THEN IT’S ESTIMATED TO BE OLDER THAN IT SHOULD BE, AND IF THE SAMPLE IS BELOW THE LINE, THEN IT’S ESTIMATED TO BE YOUNGER THAN IT SHOULD BE SO THE RESIDUALS OR THE DELTAS TO THE REGRESSION LINE CAN BE USED TO DEFINE A MEASURE OF WHAT I CALL AGE ACCELERATION IF IT’S POSITIVE, YOUR LIVER IS OLDER, IF IT’S NEGATIVE, IT’S YOUNGER THAN IT SHOULD BE SO THIS MEASURE OF EPIGENETIC AGE ACCELERATION CAN THEN BE CORRELATED TO BODY MASS INDEX, AND THAT’S DONE IN THE LOWER PANEL AND WHAT DO WE SEE? WE SEE A STRIKING CORRELATION IN LIVER TISSUE, CORRELATION .42 WHICH BY THE WAY HAS BEEN VALIDATED IN INDEPENDENT DATA SO THE HIGHER YOUR BMI, THE OLDER THE AGE OF YOUR LIVER IN CONTRAST, WE DON’T SEE SUCH A DIFFERENCE IN ADIPOSE TISSUE, —

OR BLOOD THAT ALREADY TEACHES US SOMETHING STRESS FACTORS THAT AI AGE YOU PROBABLY WILL AGE NEW YOU IN A TISSUE-SPECIFIC MANNER WE REVISITED THIS USING MUCH LARGER DATA FROM THE WOMEN’S HEALTH INITIATIVE, AND BY NOW WE DO SEE A CORRELATION BETWEEN BODY MASS INDEX AND AGE ACCELERATION IN BLOOD TISSUE, BUT THE CORRELATION IS ONLY .1 SO THE BIG QUESTION IS, OF COURSE, DOES THE EPIGENETIC CLOCK RELATE TO BIOLOGICAL AGE? AGAIN, IT’S HARD TO DEFINE BIOLOGIC AGE, BUT I THINK MOST PEOPLE WOULD AGREE WITH THE ROUGH BIOLOGIC DEFINITION OF AGE, WHICH IS THAT WHATEVER ESTIMATES YOU HAVE, IT SHOULD PREDICT ALL-CAUSE MORTALITY, EVEN AFTER YOU ADJUST FOR CHRONOLOGIC AGE AND MANY OTHER COVARIANTS THE SECOND CRITERION IS IT SHOULD PROBABLY BE DECREASED IN THE OFFSPRING OF CENTENARIANS IF YOU’RE SO LUCKY TO HAVE A PARENT WHO LIVED UNTIL THE AGE 100, CHANCES ARE YOU INHERIT GOOD AGING GENES AND, THEREFORE, THE EPIGENETIC AGE OF YOUR BLOOD IS PROBABLY YOUNGER THAN IT SHOULD BE THE THIRD CRITERION IS, OF COURSE, AN ESTIMATE OF BIOLOGIC AGE SHOULD REALLY RELATE TO MANY AGE-RELATED DISEASES AND MULTI-MORBIDITY, IDEALLY, IT SHOULD PREDICT THEM AND THE EXCITING NOTION REALLY THAT THE EPIGENETIC CLOCK SATISFIES ALL OF THESE CRITERIA METHYLATION AGE SHOULD PREDICT ALL CAUSE MORTALITY BY NOW THERE’S THREE PUBLICATIONS OUT THERE WHERE PEOPLE HAVE SHOWN EXACTLY THAT, THE METHYLATION AGE OF BLOOD IS PREDICTIVE OF HOW LONG YOU LIVE THE FIRST STUDY THAT SHOWED IT WAS THE COLLABORATION WIT WHERE WE ANALYZED FOUR DIFFERENT COHORTS AND SHOWED THAT THESE MEASURES PREDICT MORTALITY BUT I WANT TO SHOW YOU SOME UNPUBLISHED FINDINGS WHERE WE REVISITED THIS QUESTION, AND IN THIS STUDY, WE CARRIED OUT A VERY COMPREHENSIVE META-ANALYSIS WE ANALYZED 13 DIFFERENT COHORTS FROM THE WOMEN’S HEALTH INITIATIVE, NORMATIVE AGING STUDY, MANY OTHERS WE ANALYZED OVER 13,000 SUBJECTS AND FOR EACH SUBJECT, WE HAD METHYLATION LEVELS COLLECTED IN THE PATH OF SAY 1990 AND WE HAD MAYBE 10, 15 YEARS OF FOLLOW-UP SO WE COULD ASSESS DOES THE EPIGENETIC AGE ACCELERATION PREDICT MORTALITY HERE ARE THE RESULTS SO THESE ARE META-ANALYSIS PLOTS, AND EACH PANEL CORE SPONTS TO A DIFFERENT MEASURE OF AGE ACCELERATION I WON’T TORTURE YOU WITH THE TECHNICAL DETAILS, BUT WE NOW DISTINGUISH INTRINSIC AGE ACCELERATION FROM SOMETHING CALLED EXTRINSIC AGE ACCELERATION ALL MEASURES WE CONSIDERED, ALL MEASURES HAVE VERY SIGNIFICANT PREDICTIVE ASSOCIATIONS WITH MORTALITY P VALUES RANGE FROM 10 TO THE MINUS 9 TO 10 TO THE MINUS 43 IN THIS UNIVARIATE MODEL BUT WHAT ABOUT IF WE ADJUST FOR CLINICAL VARIABLES? WHAT ABOUT IF WE ADJUST ALSO FOR BODY MASS INDEX, EDUCATION, ALCOHOL, SMOKING, PRIOR HISTORY OF DIABETES, CANCER, HYPERTENSION, PHYSICAL ACTIVITY? AFTER WE ADJUST FOR ALL OF THESE COVARIANTS, WE STILL END UP WITH VERY SIGNIFICANT P VALUES RANGING FROM 10 TO THE MINUS 4 TO 10 TO THE MINUS 19 SO I FEEL VERY CONFIDENT THAT THIS STUDY ESTABLISHES ONCE AND FOR ALL THAT THE EPIGENETIC CLOCK AT LEAST CAPTURES ASPECTS OF BIOLOGIC AGE I MENTIONED TO YOU THE FINDING IN OFFSPRING OF CENTENARIANS SO WE SHOWED THAT PBMCs FROM THE OFFSPRING OF ITALIAN SUPERCREPT NAIRIANCENTENARIANS OR CENTENARIANS HAVE AN AGE SIGNIFICANTLY YOUNGER OF THOSE OF AGE MATCHED CONTROL HOWEVER, THIS FINDING WAS ONLY IDENTIFIED IN AN ITALIAN POPULATION, AND WE OF COURSE HOPE THAT OTHERS WILL VALIDATE IT IN OTHER POPULATIONS AS WELL TALKING ABOUT THIS ASPECT OF CAPTURING MANY AGE-RELATED PHENOTYPES, SO BY NOW, THERE’S MANY PHENOTYPES HAVE BEEN LINKED

TO THE EPIGENETIC CLOCK, AND I ARRANGE THEM HERE IN ALPHABETICAL ORDER IF YOU DON’T MIND, I READ IT THROUGH, AND THE OTHER CALL — WHAT OTHER TISSUES IT HAS BEEN LINKED SO FOR EXAMPLE, ALZHEIMER’S DISEASE IS ASSOCIATED WITH INCREASED EPIGENETIC AGING IN PREFRONTAL CORTEX SIMILARLY, AMYLOID LOAD IS RELATED, BODY MASS INDEX, CALORIE RESTRICTION, CANCER, CELL PATHOGEN, CENTENARIAN STATUS, COGNITIVE PERFORMANCE, DIET HAS A WEAK EFFECT, DOWN SYNDROME HAS A STRONG EFFECT, GESTATIONAL AGE, GRIP STRENGTH, HAYFLICK LIMIT, SENESCENT CELLS HAVE AN OLDER AGE HIV STATUS HUNTINGTON’S DISEASE, VARIOUS LIPID LEVELS, FOR EXAMPLE, TRY GLITRIGLYCERIDES, MENOPAUSE, MORTALITY, NEWER PATHOLOGICAL VARIABLES, OBESITY, OSTEOARTHRITIS, PARKINSON’S DISEASE AND SEX, MEN ARE SLIGHTLY OLD NER WOMEN IN CERTAIN TISSUE, BLOOD AND BRAIN, FOR EXAMPLE SLEEP HAS A WEAK EFFECT, AND FINALLY WALKING SPEED SO I WILL FLESH OUT THESE RESULTS IN THE COMING SLIDES LET’S COME TO A DIFFERENT TOPIC WHAT ABOUT PROGERIOD SYNDROME HE RANKED SEGMENTAL PROGERIOD SYNDROME ACCORDING TO VARIOUS CRITERIA AND INTERESTINGLY, THE ULTIMATE IS ACTUALLY DOWN SYNDROME ACCORDING TO THIS ANALYSIS, FOLLOWED BY WERNER SYNDROME AND HUTCHINSON GILFORD SYNDROME AND MANY OTHERS SO IN COLLABORATION I ANALYZED BLOOD AND BRAIN TISSUE IN DOWN SYNDROME SUBJECTS AND SURE ENOUGH, WE FIND IN BOTH TISSUES, THERE’S A SIGNIFICANT ACCELERATED AGING EFFECT I’M VERY INTERESTED IN TESTING OTHER POSTMORTEM TISSUES AS WELL TO SEE TO WHAT EXTENT THIS GENERALIZES TO OTHER TISSUES WHAT ABOUT HUTCHINSON’S PRO JEER YA? HERE I SHOW YOU SOME VERY PRELIMINARY DATA WHERE WE IN COLLABORATION WITH OTHERS ANALYZED DERMAL FIBROBLASTS, THE DOTS ARE COLORED BY PROGERIA STATUS, SO RED AND MAGENTA ARE PROGERIA, AND WHAT DO WE SEE? WE BASICALLY SEE NOTHING OR I DON’T SEE ANYTHING I JUST DON’T SEE THAT PROGERIA IS OLDER, YOU KNOW, THAN CONTROL SAMPLES, AT LEAST IN THE DERMAL FIBROBLASTS I’M NOT YET CONFIDENT ENOUGH IN THIS FINDING, AND NOW WE GENERATE A SECOND DATASET BUT I JUST WANTED TO SHOW IT TO YOU THE GREAT — IS OF COURSE ALZHEIMER’S DISEASE WE HAVE RECENTLY SHOWN IN COLLABORATION WITH MORGAN LE VEEP, WHO ILEVINE, A POSTDOC, AND DAVID BENNETT, WE HAVE SHOWN THAT EPIGENETIC AGE IN THE PREFRONTAL CORTEX RELATES TO NEUROPATH VARIABLES AND MORE AND THESE ARE 700 DORSO LATERA LATERAL SAMPLES FROM THE ROS STUDY AND ALSO THE RUSH MEMORY AND AGING PROJECT, AND WE THEN ANALYZED EPIGENETIC AGE ACCELERATION IN THE PREFRONTAL CORTEX AND RELATED IT TO THESE NEUROPATH VARIABLES AND IT RELATES TO ALL OF THEM AMYLOID LOAD, NEURITIQUE PLAQUES PLAQUES, NEUROFIBRILLARY TANGLES, PHFTAU TANGLE WE FIND MUCH STRONGER ASSOCIATION BUT THEY ARE THERE SO IT’S AN IMPORTANT PROOF OF CONCEPT TO ME, IT SUGGESTS THAT, YES, THE EPIGENETIC CLOCK MEASURES IN CERTAIN WAYS THE BIOLOGIC AGE OF THE BRAIN AND EVEN MORE EXCITINGLY, THE EPIGENETIC AGE ACCELERATION ALSO CORRELATES TO MEASURES OF COGNITIVE FUNCTIONING THAT WERE ASSESSED PREMORTEM OBVIOUSLY, AND SO THERE ARE SIGNIFICANT — WITH GLOBAL COGNITIVE FUNCTIONING, EPISODIC MEMORY AND SO ON SO THAT’S THE TOPIC AGING I WANT TO BRIEFLY MENTION APPLICATIONS TO PRENATAL

SAMPLES, BECAUSE IT TEACHES US SOMETHING ABOUT THE UNDERLYING BIOLOGY SO HERE THEY ANALYZED 179 PRENATAL BRAIN SAMPLES, AND THEY APPLIED THE EPIGENETIC CLOCK ALGORITHM AND ON THE Y AXIS, YOU SEAT MEASURE, AND YOTHE MEASURE AND YOU NO TICE IT’S A NEGATIVE NUMBER THAT INDICATION A PRENATAL SAMPLE SO ON THAT LEVEL, THE CLOCK ALREADY WORKS BUT MORE IMPORTANTLY, THERE IS A WEAK CORRELATION EVEN OF .15, THAT SUGGESTS THAT ALREADY GESTATIONAL AGE AND BRAIN RELATED TO THE EPIGENETIC AGE, AND I SHOULD MENTION THAT WE REVISITED THESE ANALYSIS IN BLOOD AND NOT YET DISCOVERED A CORRELATION BETWEEN GESTATIONAL AGE AND EPIGENETIC AGE IN BLOOD TISSUE BLOOD MAY BE FOR WHATEVER REASON MORE DIFFICULT SO WHY DO I HIGHLIGHT THIS RESULT? BECAUSE PEOPLE OFTEN THINK OF EPIGENETIC AS A MEDIATOR OF ENVIRONMENT ON THE ORGANISM, RIGHT? HOWEVER, EASY PAY IN MY WORD PLAYS OF COURSE ANOTHER CRUCIAL ROLE, WHICH IS DEVELOPMENT, IT DISTINGUISHES CELL TYPES, AND IN MY WORK, IT WAS VERY INTERESTING THAT WE ALREADY SEE AN APPLICATION IN PRENATAL SAMPLES BECAUSE MAYBE WE CAN INTERPRET THEN AGING AS A CONTINUATION OF DEVELOPMENT, WHICH IS, OF COURSE, AN OLD IDEA I WANTED TO REPORT AN ANALYSIS OF A CURIOUS DISORDER, WHICH IS KNOWN AS SYNDROME X AND OCCASIONALLY SEE IT ON TELEVISION SO THERE ARE CHILDREN WHO SIMPLY NEVER DEVELOP THEY ALWAYS REMAIN TODDLERS THEY NEVER LEARN HOW TO WALK, THEY NEVER LEARN HOW TO TALK YOU HAVE TO CHANGE THEIR DIAPER UNTIL THEY’RE AGE 20 OR WHENEVER SO HERE’S A GIRL FOR EXAMPLE WHO AT AGE 2, 4 AND 6 I WAS VERY INTERESTED IN STUDYING WHETHER THESE CHILDREN ARE IMMORTAL, WHETHER THEY NEVER AGE AND SO I WORKED WITH RICHARD WALKER ON THIS ANALYSIS OF BLOOD SAMPLE FROM THESE CHILDREN, AND THE FINDING WAS, THESE CHILDREN, AT LEAST THEIR AGE — THEIR BLOOD AGE IS PERFECTLY NORMALLY SO ACCORDING TO THE BLOOD ANALYSIS, THESE CHILDREN ARE MORTAL PEOPLE HAVE LOOKED AT PRENATAL AND EARLY LIFE INFLUENCES ON EASEPIGENETIC AGE, IF YOU’RE INTERESTED IN THAT I INVITE YOU TO READ THE PAPER FROM ANDREW SIMPKIN AND OTHERS IN THIS PAPER, WE LOOKED AT MANY FACTORS, BUT THE ONE FACTOR THAT REALLY STOOD OUT TO ME WAS GENDER BECAUSE BOYS WERE ALREADY OLDER THAN GIRLS, ACCORDING TO THE EPIGENETIC CLOCK SO THAT FEMALE ADVANTAGE STARTS EARLY ON WE OF COURSE ARE ALL INTERESTED IN INTERVENTIONS THAT AFFECT THE EPIGENETIC CLOCK, SO I JUST WANT TO GIVE YOU AN OVERVIEW OF WHAT I LEARNED OVER THE LAST THREE YEARS SO FIRST OF ALL CELL PASSAGING OR POPULATION DOUBLING LEVEL IS NOT THE SAME AS EPIGENETIC AGE THESE ARE DIFFERENT CONCEPTS, HOWEVER, THEY ARE CORRELATED AND RECENTLY, THEY VALIDATED THIS FINDING BY LOOKING AT ENDOTHELIAL CELLS AND AGAIN THEY FOUND REPLICATIVE SENESCENCE AND ALSO SHOWED ONCOGENE-INDUCED SENESCENCE ARE ACCOMPANIED BY INCREASED ERG AGE EPIGENETIC AGE BUT THERE IS A THIRD WAY, AND THAT’S RADIATION STRIKSTRIKINGLY, RADIATION DOES NOT INCREASE EPIGENETIC AGE AND I’LL TALK ABOUT IT LATER HERE’S ANOTHER QUESTION PEOPLE ASK, IS THERE A CERTAIN TIE ET THAT WILL KEEP ME YOUNG MANY COLLABORATED FROM THE WOMEN’S HEALTH INITIATIVE AND ALSO IMPORTANTLY LUIGI AND BRIAN CHEN AND TOSHIKO TAN A/K/A AND OTHERS, WE POOLED OUR EFFORT TO ASSESS WHAT DIETS AFFECT THE EPIGENETIC AGE OF BLOOD LET ME ALREADY COME UP WITH THE MAIN MESSAGE: DIET HAS ONLY A VERY WEAK EFFECT ON THE

EPIGENETIC AGE OF BLOOD SO WHATEVER I SHOW YOU, TAKE IT WITH A GRAIN OF SALT YOU REALLY NEED THOUSANDS OF SAMPLES TO DETECT THESE EFFECTS, BUT HERE WE GO SO THIS SHOWS A PLOT WHERE ON THE Y AXIS, I REPORT THE PERCENTAGE OF PROTEIN IN THE DIET AND ON THE X AXIS, I SHOW THE PERCENTAGE OF CARBOHYDRATES AND RED MEANS HIGH INTRINSIC EPIGENETIC AGE IN THE WOMEN’S HEALTH INITIATIVE, AND WHAT WE SEE IS THAT IN THE LOWER RIGHT CORNER IS INCREASED AGING AND I WANTED TO CALL THIS LOWER RIGHT-HAND CORNER THE COOKIE DIET, LOTS OF SUGAR, UNTIL I DID A GOOGLE SEARCH ON IT UNTIL I LEARNED THERE IS SUCH A THING AS THE COOKIE DIET SO I DIDN’T WANT TO OFFEND ANYBODY, ANYWAY, THAT’S ONE FINDING SO HERE IS A GRAPHIC OF THIS STUDY, AND AS I MENTIONED EARLIER, WE DID DISTINGUISH INTRINSIC EPIGENETIC AGING FROM EXTRINSIC AGING SO THE EXTRINSIC AGING MEASURE WHICH INCIDENTALLY IS BASED ON AN EPIGENETIC AGE ESTIMATED, THIS MEASURE IS AFFECTED BY ANYTHING YOU WOULD THINK OF IF YOU EAT A LOT OF FISH, FRUITS, HIGH EDUCATION, IF YOU EX-AREXERCISE, IT SLOWS THE EXTRINSIC AGING HIGH BMI, INSULIN LEVELS, TRIGLYCERIDE LEVEL, HOMOCYSTEINE LEVELS, INCREASE THE AGING CLOCK AGAIN, WEAK EFFECT LET’S NOW COME TO AN INTERVENTION THAT HAS A HUGE EFFECT SO IF YOU TAKE A DERMAL FIBROBLAST, FOR EXAMPLE, AND YOU TURN IT INTO AN INDUCED FLEUR PLURIPOTENT STEM CELL, THAT RESETS THE EPIGENETIC AGE COMPLETELY SO IPS CELLS HAVE A NEGATIVE AGE, INDICATING A PRENATAL AGE, OR AN AGE CLOSE TO ZERO AND THAT SIGNAL IS EXTREMELY ROBUST I SEE IT IN EVERY CELL SIMILARLY, EMBRYONIC STEM CELLS HAVE AN AGE THAT IS NEGATIVE OR ZERO LET’S NOW COME TO INTERVENTIONS THAT DO NOT AFFECT THE EPIGENETIC CLOCK SO HERE IS AN UNPUBLISHED STUDY BY ANDREW, CHRISTINE AND OTHERS, WHERE ANDREW CONVERTED FIBROBLASTS INTO NEURONS USING WHAT IS KNOWN AS DIRECT CONVERSION OR TRANSDIFFERENTIATION SO THIS IS AN MICRO RNA BASED METHOD FOR TURNING THE FIBROBLAST OF AN ADULT INTO A NEURON AND SO HE STARTED WITH THE FIBROBLAST, ADDED CERTAIN MICRO RNAs BY TRANSDUCING THEM AND ENDED UP WITH THESE NEURONS, AND THE LOWER PANEL SHOWS THE AGE ESTIMATE BEFORE AND AFTER CONVERSION THE X FACTOR SHOWS THE METHYLATION AGE OF THE FIBROBLAST BEFORE CONVERSION, THE Y AXIS SHOWS THE AGE OF THE RESULTING NEURON AFTER CONVERSION AND WHAT DO WE SEE? WE SEE A CORRELATION OF .91, YOU KNOW, SO IN OTHER WORDS, THAT PROCEDURE, MEDIATED DIRECT CONVERSION, DOES NOT CHANGE THE EPIGENETIC AGE DIFFERENT FROM WHAT IS OBSERVED IN IPS CELL I WANTED TO FLESH OUT MY EARLIER COMMENT THAT ANOTHER THING THAT DOES NOT ACCELERATE EPIGENETIC AGING IS RADIATION SO HERE RADIATED ENDOTHELIAL CELLS OVER VARIOUS TIME POINTS AND WE JUST NEVER SAW ANY CHANGE IN EPIGENETIC AGE WHAT ABOUT WEIGHT LOSS? SO WE HAD LIVER SAMPLE FROM PEOPLE WHO UNDERWENT BARIATRIC SURGERY, AND AS YOU KNOW, BARIATRIC SURGERY DRASTICALLY REDUCES THE WEIGHT, AND THE UPPER PANEL SHOWS HOW THE BODY MASS INDEX DECREASES UP TO NINE MONTHS IN THE SAME SAMPLE, WE ASSESSED THE METHYLATION AGE AND THERE WAS NO DIFFERENCE IN THE NINE MONTHS OF FOLLOW-UP SO THEREFORE THE DEPRESSING NEWS IS THAT ONCE YOUR LIVER IS ACCELERATED AGING DUE TO OBESITY, PROBABLY AN INTERVENTION WILL NOT RESET THE AGE AT LEAST NOT IN THE SHORT TERM YOU KNOW OF COURSE THAT NEEDS TO BE REVISITED WITH MUCH LONGER FOLLOW-UP STUDIES IN THE LOWER PANEL, I SHOW YOU AN EXAMPLE WHERE I ANALYZED

PUBLICLY AVAILABLE DATA, WHERE THE OFFICE CARRIED OUT A SIX MONTH PHYSICAL EXERCISE INTERVENTION, AND THEN MEASURED METHYLATION LEVELS IN ADIPOSE TISSUE AND AGAIN I SAW NO DIFFERENCE SO IF YOU EXERCISE A LOT, IT PROBABLY DOESN’T REJUVENATE YOUR FAT TISSUE THAT’S THE MESSAGE BUT CLEARLY IT WOULD BE VERY EXCITING TO REVISIT THAT NOW IN OTHER TISSUES, MUSCLE TISSUE WOULD BE AN OBVIOUS QUESTION ANOTHER NEGATIVE FINDING IS REALLY RELATIONSHIP TO TELOMERE LENGTH SO THERE IS NO RELATIONSHIP BETWEEN TELOMERE LENGTH AND EPIGENETIC AGE IN BLOOD OR ADIPOSE TISSUE AFTER YOU CORRECT FOR CHRONOLOGIC AGE, RIGHT? CHRONOLOGIC AGE CONFOUNDS THE TWO VARIABLES SO IF YOU HAVE PEOPLE OF THE SAME AGE, THE BIRTH COHORT, AND YOU JUST MEASURE THE TELOMERE LENGTH AND EPIGENETIC AGE, YOU WILL NOT SEE A RELATIONSHIP AND THERE IS A PUBLICATION FROM A GERMAN GROUP WHO SHOW THAT FRAILTY IS ASSOCIATED WITH THE EPIGENETIC CLOCK BUT NOT WITH TELOMERE LENGTH IN A LARGE GERMAN COHORT MANY OTHERS REVISITED MORTALITY PREDICTION AND THEY FOUND THAT TELOMERE LENGTH AND EPIGENETIC AGE INDEPENDENTLY PREDICTED MORTALITY, WHICH IS EXCITING NEWS, BECAUSE CLEARLY TELOMERE LENGTH IS A VERY IMPORTANT BIOMEDICAL, BIOMARKER, AND IT JUST SHOWS THAT THESE MARKERS COMPLEMENT EACH OTHER LET’S NOW TALK ABOUT ANOTHER ANGLE OF GENETICS SO REMEMBER YOU HAVE A MEASURE OF EPIGENETIC AGE ACCELERATION, WHICH SL A NUMBER, AND YOU CAN USE THAT QUANTITATIVE READOUT AS A PHENOTYPE IN A QTL ANALYSIS TO FIND SNPs THAT RELATE TO AGE ACCELERATION AND WHY WOULD WE DO THAT? FIRST, WE WANT TO UNDERSTAND THE MECHANISM UNDERLYING THE EPIGENETIC CLOCK, SO YOU WANT TO FIND KEY GENES, BUT ALSO WE CLEARLY WANT TO FIND AGING GENES WE USE AGE ACCELERATION AS AN ENDOPHENOTYPE FOR RELATED PATHOLOGY HERE I WANT TO SHOW YOU RESULTS WHERE WE ANALYZED THE CEREBELLAR SAMPLES, AND WE FOUND GENETIC VARIANTS NEAR TWO GENES AFFECTING THE EPIGENETIC AGE OF THE CEREBELLUM I WANT TO SHOW YOU A MANHATTAN PLOT THAT THE GENETICISTS AMONG YOU WILL RECOGNIZE SO HERE WE HAVE THE P VALUES VERSUS CHROMOSOMAL LOCATIONS AND WE FIND VERY SIGNIFICANT FIND NGS TWO LOCATIONS BUT WHAT IS STRIKING ABOUT THIS MANHATTAN PLOT IS THAT IT’S ACTUALLY BASED ON ONLY 500 SAMPLES BECAUSE WHEN YOU TYPICALLY SEE THESE TYPES OF MANHATTAN PLOTS, THEY ARE BASED ON THOUSANDS IF NOT TENS OF THOUSANDS OF SAMPLES, BUT ARGUABLY IF YOU HAVE A RIGHT ENDOPHENOTYPE, YOU REALLY ENRICH THE SIGNAL AND YOU GET AWAY WITH MUCH SMALLER SAMPLE SIZES ONE OF THE GENES THAT’S INTERESTING TO AGING RESEARCHERS RESEARCHERS, IT’S A KEY COMPONENT OF BOTH MTOR COMPLEXES, WHICH, OF COURSE, PLAY AN IMPORTANT ROLE IN MODEL ORGANISM AGE SO I WANT TO PRESENT — I SHOULD SAY THESE STUDIES ARE ONGOING, WE NOW ANALYZE MANY DIFFERENT BRAIN REGIONS, WE ANALYZE BLOOD SAMPLE, WE HAVE SOME EXCITING HITS AND JUST STAY TUNED TO IT I WANT TO NOW SHIFT GEARS AND PRESENT SOME APPLICATIONS SO HIV IS UNFORTUNATELY OFTEN ACCOMPANIED BY AGE-RELATED PHENOTYPES, AND SO WE WERE INTERESTED IN TESTING WHETHER HIV INFECTION ACCELERATES THE AGE OF BLOOD AND ALSO BRAIN TISSUE AND THE ANSWER IS IT REALLY DOES, IN BOTH TISSUE, WE FOUND SIGNIFICANT ACCELERATED AGE BEING EFFECTS IT’S INTERESTING TO ME HOW THAT COULD COME ABOUT WE LACK A ME CAN NIS TISH MECHANISTIC UNDERSTANDING BUT THE FINDING IS CLEAR THIS IS ANOTHER APPLICATION IN COLLABORATION WITH OTHERS WHERE WE ANALYZE SUPERCENTENARIANS SO A SUPERCENTENARIAN IS A PERSON WHO IS 110 YEARS OLD OR OLD E AND IN THIS APPLICATION, WE ANALYZED IN THE UPPER PANEL A LADY WHO WAS 112 YEARS OLD, AND WE ANALYZED 30 DIFFERENT PARTS

OF HER BODY ALL PARTS THAT I COULD THINK OF AND WE MEASURED THE EPIGENETIC AGES AND THE ONE BODY PART THAT HAD THE YOUNGEST AGE WAS THE CEREBELLUM NOARND, BONE TISSUE AND BLOOD WAS OLDER THAT’S ON THE RIGHT-HAND SIDE AND THIS FINDING OF THE YOUNG CEREBELLUM WAS SUBSEQUENTLY VALIDATED IN THE LOWER PANELS, WHICH SHOW BRAIN OF OTHER CENTENARIANS AND SUPERCENTENARIANS, ALWAYS THE CEREBELLUM IS THE YOUNGEST BRAIN REGION FURTHER, WE ANALYZED HALF A KOZ DATASETS WHERE WE ANALYZED BRAINS FROM 80 YEARS OLD AND AGAIN FIND IT SO I’M VERY CONFIDENT IN THAT STATEMENT THAT THE CEREBELLUM AGES SLOWLY ACCORDING TO THE EPIGENETIC CLOCK AND OF COURSE THE QUESTION IS WHY IS THAT THE CASE? IT’S AN INTERESTING RESULT BECAUSE IF WE CAN FIGURE OUT WHY THE CEREBELLUM STAYS YOUNG, MAYBE WE FIND A WAY TO KEEP THE REST OF THE BRAIN YOUNG AS WELL I WANT TO SHIFT GEARS AGAIN AND MENTION AN APPLICATION TO BREAST CANCER TODAY I WON’T TALK MUCH ABOUT CANCER, BUT I DO WANT TO MENTION THAT BREAST CANCER TYPES HAVE VERY DIFFERENT AGES YOU MAY HAVE HEARD OF LUMINAL TYPE VERSUS BASAL TYPE, AND LUMINAL CANCERS PRESENT WITH CANCER TISSUES THAT HAVE SIGNIFICANT POSITIVE AGE ACCELERATION IN CONTRAST, THE BASAL TYPE, THE TRIPLE NEGATIVE CANCER, IS ACCOMPANIED WITH NEGATIVE AGE ACCELERATION SO IN GENERAL, CANCER CAN BOTH INCREASE THE METHYLATION AGE OF THE AFFECTED TISSUE AND DECREASE THE AGE SO THE EPIGENETIC CLOCK IS BROKEN IN CANCER TISSUE I WANT TO ALSO MENTION ANOTHER FINDING THAT’S COMING OUT SOON BY MORGAN LEVINE AND OTHERS WHERE WE EVALUATED THE EFFECT OF MENOPAUSE ON EPIGENETIC AGING, AND THE FINDING IS CLEAR SO WE LOOKED AT SEVERAL COHORTS, WHI, PARKINSONS, AND WE FOUND THAT WOMEN WHO ENTER MENOPAUSE LATE, THEIR EPIGENETIC AGE IN BLOOD IS YOUNGER SO DELAYED ONSET OF MENOPAUSE IS ACCOMPANIED WITH LOWER AGING AND THAT HOLDS EVEN AFTER YOU ADJUST FOR ALL SORTS OF COVARIANTS I WANT TO MENTION SOME FUTURE DIRECTIONS CLEARLY WE NEED TO EXTEND THE EPIGENETIC CLOCK TO MODEL ORGANISMS HERE I SHOW YOU AN ONGOING STUDY UNPUBLISHED WHERE WE’RE NOW BUILDING AN ER EPIGENETIC CLOCK FOR DOGS AND YOU CAN CLEARLY DO IT THAT’S IN BLOOD SO IN CONCLUSION, THEN, I FEEL THAT THE EPIGENETIC CLOCK IS AN ATTRACTIVE BIOMARC OF AGING, IT’S HIGHLY ROBUST, I ALWAYS TELL THE STORY THAT I COLLECTED BLOOD TUBES IN LOS ANGELES AND THEN THE PERSON WHO TRANSPORTED THEM KEPT THEM IN HIS CAR, AND THEY MELTED BECAUSE HE DIDN’T HAVE AN AIR CONDITIONERRER, SO WE ANALYZED THE BLOOD SAMPLES FROM THE COAGULATED BLOOD AND WE STILL GOT PERFECT AGE ESTIMATES I COULD TELL YOU LOTS OF THESE STORIES SO METHYLATION LEVELS ARE SURPRISINGLY ROBUST IT’S A VERY ACCURATE MEASURE OF TISSUE AGE, IT’S ASSOCIATED WITH MANY AGE-RELATED CONDITIONS, IT’S PROGNOSTIC OF MORTALITY, AND IT ALLOWS YOU TO CONTRAST THE AGES OF DIFFERENT TISSUES SO I FEEL MOST STUDY THRAS LOOK — SUCH AS TELOMERE LENGTH COULD BE REVISITED, BUT I DO WANT TO MENTION THAT IT WILL BE IMPORTANT TO LOOK AT TISSUES BEYOND JUST BLOOD FOR EXAMPLE, SOME OF THE MOST STRIKING FINDINGS CAN BE FOUND IN BUCCAL EPITHELIUM AS OPPOSED TO BLOOD SO REMEMBER THE FINDING FOR OBESITY, STRONG EFFECT IN LIVER BUT NOT IN OTHER TISSUE FINALLY, I MENTIONED THERE IS PRFREE USER-FRIENDLY SOFTWARE ON MY WEBPAGE SO I INVITE YOU TO TRY THAT OUT I WANT TO ACKNOWLEDGE THE ABOVE ALL THE NATIONAL INSTITUTE ON AGING AND NIH FOR ALL THE WONDERFUL SUPPORT FOR CREATING WONDERFUL DATA REPOSITORIES, I WANT TO THANK THE MANY RESEARCHERS WHO ANSWERED MY EMAILS AND FREELY SHARED THEIR METHYLATION DATA IN THESE PUBLIC REFOS TRIES I THINK I ALREADY MENTIONED MANY OF THESE PEOPLE DURING MY TALK

SO THANK YOU VERY MUCH [APPLAUSE] QUESTIONS? >> THANK YOU FOR YOUR WONDERFUL AND CHALLENGING PRESENTATION THIS IS OPEN TO QUESTIONS >> THANK YOU MY QUESTION IS ABOUT ESLs — WHEN YOU DIFFERENTIATE THEM IN VITRO, DO THEY UNDERGO VERY RAPID AGING OR IS THE AGING PURELY CHRONOLOGICAL? >> ALL RIGHT SO LET’S — LET ME MENTION AN UNPUBLISHED FINDING THAT I HOPE ANSWER YOUR QUESTION SO WHEN YOU TAKE SAY AN IPSL AND YOU TURN IT INTO — YOU DIFFERENTIATE IT INTO A NEURAL PRECURSOR IN VARIOUS STAGES, VERY IMPORTANT QUESTION IS, IS THAT ACCOMPANIED WITH INCREASES IN EPIGENETIC AGE? SO I ANALYZED SUCH A DATASET AND THE ANSWER IS YES IT IS STATISTICALLY VERY SIGNIFICANT IN ACTUAL UNITS, IT’S PROBABLY NOT THAT MUCH, AN INCREASE OF, I DON’T KNOW, SEVERAL MONTHS, BUT NO DOUBT IT WAS ASSOCIATED >> I HAVE TWO QUESTIONS YOU SAID THAT DIFFERENT — MAY HAVE DIFFERENT BIOLOGICAL AGE SO THE HEART MAY BE 50, THE LIVER MAY BE 30, SO WE ONE IT IS ACTUALLY THE AGE? IS IT THE SCORE, IS IT A FORMULA, WHICH ONE IS THE AGE? >> THAT’S A VERY GOOD QUESTION LET ME GIVE YOU MY OPINION I THINK IF YOU HAVE A PERFECTLY HEALTHY PERSON, COMING BACK TO THE TRUE — THE IDEAL HUMAN BODY, I THINK ALL PARTS HAVE THE IDENTICAL AGE, YOU KNOW? IT’S CALIBRATED CHRONOLOGIC AGE, ALL PARTS MOVE AT THE SAME SPEED BUT VARIOUS STRESS FAK, TO FACTOR, INTERNAL OR GENETIC, WILL THEN LEAD TO DIVERGENCES, SO AGAIN, IF EURO BEES YOU’RE OBESE, YOUR LIVER WILL BE OLDER THAN THE OTHER PARTS OF YOUR BODY BUT MAY BE UNAFFECTED PEOPLE OFTEN ASK ME, DOES SMOKING ACCELERATE AGING? THE ANSWER IS NOT IN BLOOD CERTAINLY YOUR BLOOD IS YOUNG, YOU KNOW, BUT I DON’T KNOW WHETHER SMOKING ACCELERATES THE AGE OF YOUR LUNG TISSUE OR THE HEART, YOU KNOW SO IN ANY EVENT, COMING TO YOUR QUESTION, WHAT IS THE ONE SUMMARY MEASURE OF THE BODY THEN, AND I MEAN, AS A MATHEMATICIAN, I WOULD ANSWER FOCUS ON THE WEAKEST LINK, RIGHT, YOU TAKE THE MINIMUM IF YOUR HEART IS 20 YEARS OLDER THAN IT SHOULD BE, YOU WILL PROBABLY BE IN TROUBLE BY THE WAY, THESE STATEMENTS NEED TO BE TAKEN WITH A GRAIN OF SALT BECAUSE WE HAVE NOT YET VALIDATED THAT THE EPIGENETIC AGE OF HEART TISSUE HAS ANY CLINICAL RELEVANCE CLEARLY THAT’S A VERY IMPORTANT RESEARCH DIRECTION TO UNDERSTAND TO WHAT EXTENT THE EAS EPIGENETIC AGES OF DIFFERENT ORGANS RELATE TO PATHOLOGY >> UNDERSTAND AND BEING A NUTRITIONIST AND ONCE EXPLAINED THAT NUTRITION IS NOT AS IMPORTANT, HOW COME YOU KEEP MENTIONING THE GRAIN OF SALT? [LAUGHTER] >> TAKE EVERYTHING WITH A GRAIN OF SALT >> I HAVE A SECOND QUESTION SO MY SECOND QUESTION IS, I’M VERY INTERESTED IN GAS MICROBIOTA AND GAS MICROBIOME MICROBIOTA CHANGES WITH THE AGE >> YES >> HAVE YOU LOOKED INTO A POSSIBILITY OF MODULATING OR IT IS A SPOBILITY I POSSIBILITY IN YOUR OPINI ON OF MODULATING THE AGING PROCESS THROUGH MODULATING THE MICROBIOME? >> I THINK IT’S A SUPER INTERESTING QUESTION I JUST DON’T HAVE ANY DATA THAT WOULD LINK THE COMPOSITION OF THE MICROBIOME TO EPIGENETIC AGING BUT FOR ALL THE REASONS YOU MENTIONED, I THINK IT’S AN IMPORTANT QUESTION >> THANK YOU >> IN YOUR EPIGENETIC METHYLATION ANALYSIS OF THE PRENATAL BRAIN, I’M ASSUMING YOU GOT THAT FROM AUTOPSY MATERIAL? >> YES >> OKAY IF YOU’RE GETTING IT FROM AUTOPSY MATERIAL, MY EXPERIENCE IS THAT MOST OF THOSE ARE GOING TO BE FROM ABORTIONS IF THEY’RE FROM ABORTIONS, IT’S A HUGELY HETEROGENEOUS GROUP IT COULD BE A VASCULAR INSULT, IT COULD BE SOME KIND OF CHROMOSOMAL THING, SO THAT WOULD KIND OF EXPLAIN THAT AWAY IF YOU HAD THE GOOD LUCK TO RECEIVE MONO ZYGOTIC TWINS OR TRIPLETS OR EVEN QUINN IT UP LETS, THAT WOULD CERTAINLY GIVE

YOU A BETTER ANSWER TO WHAT YOU’RE LOOKING AT YOU COULD KIND OF CORRELATE THOSE THE OTHER QUESTION IS, IN TERMS OF — YOU SEEM TO HAVE SKIPPED PLACENTA, WHICH WOULD SEEM TO BE THE BEST CONTROL TISSUE OF THEM ALL IT’S HUMAN, IT LIVES FOR 8 TO 9 MONTHS, AND YOU COULD SAY, GEE, OUR EPIGENETIC METHYLATION REALLY WORKS OR IT DOESN’T >> THANKS FOR THROWIN DRAWING MY ATTENTION TO IT SO I CERTAINLY EVALUATED PLACENTA, AND IT TURNS OUT THAT THE EPIGENETIC AGE OF PLACENTAL SAMPLES IS BASICALLY ZERO OR NEGATIVE, MEANING AGAIN, VERY YOUNG SO THE EPIGENETIC CLOCK — WHAT IS THE TRUE AGE OF PLACENTA DO YOU TAKE THE CHRONOLOGIC AGE OF THE MODEL OR DO YOU TAKE THE GESTATIONAL AGE OF THE CHILD SO FAR I COULD NOT FIND ANY — THERE’S CERTAINLY NO RELATIONSHIP BETWEEN THE PLACENTAL AGE BASED ON THE EPIGENETIC CLOCK AND THE CHRONOLOGIC AGE OF THE MOTHER THAT I KNOW WHEN I APPLI THE ALGORITHM TO THE PLA SET TA PLACENTA, IT’S AGE ZERO >> THANK YOU >> THANKS VERY MUCH FOR SPEAKING I’M NOT A — MY QUESTION IS, DID YOU SAY BOTTOM LINE THAT GENETICS IS THE LIFESPAN — YOU CAN HAVE PARENTS THAT LIVE TO BE ELDERLY, IN THEIR 90s, WHAT’S THE PERCENTAGE OF CHANCE YOU HAVE? >> THERE’S NO DOUBT THAT THE AGE OF YOUR PARENTS IS A VERY SIGNIFICANT PREDICTER OF HOW LONG YOU WILL LIVE I DON’T HAVE THE EXACT NUMBERS, BUT CAN SOMEONE HELP ME? I WANT TO SAY HERITABILITY 40% MAYBE? 30% — LUIGI HAS VERY OLD PARENTS SO HE’S BLESSED BUT YEAH, I CANNOT ANSWER BEYOND THAT BUT YES, GENETICS MATTERS A LOT >> WHAT PERCENT DID YOU SAY? >> 30% >> THAT’S ALL? >> YEAH AND ALSO THE DISSH WELL — WELL, I THINK IT’S A LOT, BECAUSE THINK ABOUT IT, IF YOU HAVE THE PERFECT GENES, HOWEVER, YOU SMOKE AND HAVE POOR LIFESTYLE CHOICE, YOU KNOW, THAT WILL DO YOU IN ALSO THE EPIGENETIC AGE ACCELERATION ALSO IS QUITE GENETIC, MAYBE 40% OF THE VARIATION IS UNDER GENETIC INFLUENCE >> I HAVE ANOTHER QUESTION WHAT ABOUT TELOMERE GENE, THAT AGING GENE? >> SO TEL TELOMERE LENGTH SHORTENS AS WE AGE, THAT’S A VERY WELL STUDIED MOLECULAR — IN CERTAIN WAYS, AFTER YOUR TELOMERES BECOME SHORT, IT TRIGGERS CELLULAR SENESCENCE, BUT INTERESTINGLY, SHORTENING VEILY NOT RELATED TO THE EPIGENETIC CLOCK, WHICH IS EXCITING BECAUSE AFTER 20 YEARS OF RESEARCH, WE KNOW TELOMERE SHORTENING IS NOT THE ROOT CAUSE OF AGING IT’S A CONTRIBUTOR TO AGING BUT ARGUABLY WE ARE OPEN FOR NEW IDEAS TO FIND NOVEL MECHANISMS >> THANK YOU >> THANK YOU >> YOU TRIED IONIZING RADIATION AND FOUND THAT THAT DOES NOT ACCELERATE >> YES >> SO WHICH MEANS DOUBLE STRANDED DNA BREAKS ARE NOT THE DRIVING FORCE >> YES >> HOW ABOUT ROS OXIDATIVE STRESS? >> I DON’T THINK I HAVE — I CAN’T THINK OF A STUDY RIGHT NOW THAT INVESTIGATED IT BUT IT’S CLEARLY AN EXCITING QUESTION I HAVE CERTAINLY EVIDENCE THAT MITOCHONDRIA PLAY A ROLE, YOU KNOW, SO IN THIS OBESITY PAPER, WE HAVE METHYLATION LEVELS FROM LIVER SAMPLES BUT WE ALSO ARE TRAN SCRIP TOE MIC DATA FROM THE VERY SAME LIVER, SO WE COULD CORRELATE GENE TRANSCRIPTS THAT RELATE TO THE AGE OF THE LIVER, AND SURE ENOUGH, NUCLEAR MITOCHONDRIAL GENES REALLY POPPED UP SO MEANING IF YOU EXPRESS NUCLEAR MITOCHONDRIAL GENES, THEN THE CORRESPONDING LIVER SAMPLES ARE YOUNGER THAN EXPECTED >> THIS YOU CHECKED MITOCHONDRIAL DNA CONTENT AND HOW IT CORRELATES WITH EPIGENETIC AGING? >> NO, NOT YET BUT IT’S ON MY TO-DO LIST, WAY UP ON MY TO-DO LIST, BUT MAYBE OTHERS CAN LOOK AT IT I THINK IT’S A VERY PROMISING HYPOTHESIS THAT THERE IS A RELATIONSHIP >> BECAUSE WE LOSE MITOCHONDRIAL COPY NUMBER AS WE — >> YES, BUT MANY OTHER PROPERTIES OF MITOCHONDRIA, VARIOUS WAYS OF — THEIR HEALTH, IT WOULD BE VERY EXCITING >> DR. HORVATH, THANK YOU VERY MUCH FOR A VERY INTERESTING TALK >> THANK YOU >> I WANT TO RETURN TO THE BEGINNING OF IT, WHERE YOU

DESCRIBED THE DEVELOPMENT OF THE EPIGENETIC CLOCK AND MENTIONED THAT THE METHYLATION IS AFFECTED BY DOZENS OF FACTORS, ALMOST ANYTHING CAN HAPPEN, SMOKING, ANY EXOGENOUS INFLUENCE, AND YET YOU WERE ABLE TO EXTRACT A STABLE BIOMARKER, AND I’M WONDERING IF YOU HAVE THOUGHT ABOUT WHAT IT IS IN THE BIOMARKER OR WHAT IT MIGHT TELL YOU ABOUT THE BIOLOGY OF METHYLATION ARE THERE CERTAIN POSITIONS THAT ARE AGE-RELATED PRESUMABLY AND OTHERS WHICH ARE JUST HIGHLY VARIABLE AND LABILE? >> JUST TO TELL THERE YOU IS A VAST LITERATURE ON STUDYING THE EFFECT OF AGE ON METHYLATION LEVELS IN VARIOUS TISSUES OVERALL, SIGNIFICANT TISSUE-SPECIFIC EFFECT SO FOR EXAMPLE, CPGs HAVE CORRELATED AGE AND BLOOD WILL IN GENERAL BE DIFFERENT FROM CPG THATHAT CORRELATES WITH AGE IN MUSCLE TISSUE HOWEVER, THE STRIKING AND SURPRISING THING IS ACTUALLY YOU CAN IDENTIFY A SUBSET OF CPGs THAT ARE — ENCODE SOME SORT OF A UNIVERSAL PROGRAM THAT IS TISSUE-INDEPENDENT BUT COMING TO YOUR QUESTION, I THINK IN ORDER TO LEARN THE BIOLOGY UNDERNEATH IT, ONE SHOULD NOT LOOK AT MY 353 CPG, BECAUSE THEY ARE JUST REPRESENTATIVES OF A GLOBAL IF PHENOMENON SO AGE AFFECTS PROBABLY HUNDREDS OF THOUSANDS OF CPGs, YOU KNOW, AND SO — AND PEOPLE HAVE CERTAINLY CHARACTERIZED WHICH CPGs GET HYPERMETHYLATED AS WE AGE THESE ARE CPGs THAT TEND TO BE LOCATED IN CPG ISLANDS AND PROMOTERS, WHEREAS CPGs THAT TEND TO LOSE METHYLATION TEND TO BE LOCATED IN OUTSIDE OF CPG ISLANDS THE OTHER KEY ITEM IS WHAT WE AND MANY OTHERS HAVE FOUND BEFORE IT WAS THAT REGIONS THAT ARE KNOWN AS POLYCON GROUP TARGETS, THESE REGIONS TEND TO GET HYPERMETHYLATED WITH AGE IN MY OPINION, THAT’S ONE OF THE UNDERLYING CAUSES WHY SUCH AN AGING CLOCK IS POSSIBLE, BECAUSE THAT’S PROPERTY REALLY PRESERVED ACROSS MANY TISSUE THIS POLYCAN CON PROTEIN PLAY A VERY IMPORTANT ROLE IN MAINTAINING — OF THE STEM CELLS AND WHEREVER THEY BIND, THESE REGIONS ALSO GET HYPERMETHYLATED SO THAT’S SOME ANSWER, BUT WE’RE ONLY AT THE BEGINNING I THINK IT’S ONE OF THE GRAND CHALLENGES, REALLY, TO UNDERSTAND THE EXACT MECHANISM OF THIS AGING CLOCK THIS CLOCK WAS FOUND BY A MACHINE LEARNING ALGORITHM WHICH LED TO AN EXTREMELY ACCURATE CLOCK, BUT THIS CAME TO THE EXPENSE — MANY OTHERS AND I WORK ON IT CERTAINLY TO KIND OF DISSECT THE UNDERLYING MECHANISM >> WELL, THANK YOU VERY MUCH FOR STAYING AND FOR A GREAT PRESENTATION FR YOU WANT TO JOIN US AT THE NIH LIBRARY FOR A RECEPTION AND YOU CAN CONTINUE YOUR CONVERSATION WITH STEVE HORVATH