>> WELCOME, I'D LIKE TO WELCOME EVERYBODY ON BEHALF OF OUR ACTING DIRECTOR JUDITH COOPER. TODAY'S TALK WILL GIVE US AN INTERESTING PERSPECTIVE ON HOW GENETIC RESEARCH IS DONE AND HOW GENES PLAY I ROLE IN CASES OF DEAFNESS. THE MISSION IS TO BRINGS NATIONAL ATTENTION TO THE DISORDERS AND DYSFUNCTIONS OF HEARING, BALANCE, TASTE, SMELL, SPEECH AND LANGUAGE. AND CONTRIBUTE TO ADVANCES IN BIOMEDICAL AND BEHAVIORAL RESEARCH. WHAT WE WISH TO DO IS TO CONTINUE TO IMPROVE THE LIVES OF MILLIONS OF PEOPLE WITH THESE DISORDERS. THE BASIC COMPONENTS OF COMMUNICATION SENSING, INTERPRETING AND RESPONDING TO THE ENVIRONMENT CAN BE CHALLENGING FOR THOSE WITH THESE DISORDERS. FOR EXAMPLE, PROBLEMS WITH HEARING LOSS CAN LEAD TO SOCIAL ISOLATION AS WELL AS DIFFICULTY IN SCHOOL, AT WORK, AND IN PERSONAL RELATIONSHIPS. THESE LECTURES ARE DESIGNED FOR THE ADMINISTRATIVE STAFF THAT SUPPORT THE NIDCD MISSION THE GOAL TO MAKE THE CONNECTION SO FOLKS UNDERSTAND THE SCIENCE THEY SUPPORT BUT WE ALSO WELCOME ANYONE INTERESTED IN THE SCIENCE OF DEAFNESS AND COMMUNICATION DISORDERS. THE SERIES IS PART OF OUR NIDCD ADMINISTRATIVE STRATEGIC PLAN WHICH IS WORKING TO IMPROVE COMMUNICATION AND ADMINISTRATIVE MANAGEMENT SERVICES THAT SUPPORT THE MISSION. NOW, I WOULD YOU LIKE TO INTRODUCE DR. ANDY GRIFFITH, NIDCD SCIENTIFIC DIRECTOR AND THE INSTITUTE'S ACTING DEPUTY DIRECTOR. HE LEADS THE NIDCD DIVISION OF INTRAMURAL RESEARCH AND HAS PLAYED AN INTEGRAL ROLE IN THE SPEAKER SERIES NOW IN ITS FIFTH YEAR AND WILL INTRODUCE TODAY'S SPEAKER. WELCOME, DR. GRIFFIN. >> OKAY. THANKS, TIM. I HAVE TO REMEMBER TO STAND CLOSE TO THE MIC BECAUSE I THINK BOTH OF US ARE TOO TALL FOR THE MIC. AND THANKS TO ALL OF YOU FOR BEING HERE. WE HAVE A FULL HOUSE. IT'S A SPECIAL PLEASURE FOR ME TO WELCOME YOU TO TODAY' TALKS ON HUMAN HEREDITARY DEAFNESS WHICH IS ALSO SOMETHING I WORK ON IN MY LAB. AS TIM MENTIONED, WE DEVELOPED THIS SPEAKER VISITOR PEOPLE WHO DON'T HAVE TRAINING OR A BACKGROUND IN SCIENCE. AND OUR GOAL IS TO TAKE WHAT WE ARE DISCOVERING IN OUR LABORATORIES AND SHARE IT WITH ALL OF YOU WHETHER OR NOT YOU'RE A SCIENTIST OR NOT. COMMUNITY MEMBERS, PATIENTS, FAMILIES, ADVOCATES, ANYONE WHO WOULD LIKE TO KNOW WHAT WE'RE DOING HERE AT NIDCD AND THE INTRAMURAL PROGRAM. AND SO WE HOPE YOU COME AWAY WITH A GREATER UNDERSTANDING OF HOW WE'RE ADVANCING THE SCIENCE AND ULTIMATELY HELPING PEOPLE WHO ARE DEAF OR HAVE OTHER COMMUNICATION DISORDERS AND THAT OFTEN INCLUDES FAMILIES. SO WE ALL PLAY A ROLE IN IMPROVING PUBLIC HEALTH AND WE ARE PRIVILEGED TO BE A PART OF THAT. SO LET ME TELL YOU ABOUT DR. FRIEDMAN IN THE SECTION ON HUMAN GENETICS AND HE IS A MICHIGAN NATIVE AND EARNED HIS BACHELORS AND Ph.D. DEGREES IN MOLECULAR GENETICS FROM THE UNIVERSITY OF MICHIGAN. AND HE THEN CAME TO NIH THE INTRAMURAL PROGRAM AND SPECIFICALLY NATIONAL INSTITUTE OF MENTAL HEALTH TO DO POST-DOCTORAL WORK. UNLIKE THESE DAYS WHERE IT CAN TAKE, FIVE, EIGHT, 10 YEARS HE DIDN'T HAVE TO SPEND SO MUCH TIME BEFORE HE RETURNED TO MICHIGAN AND EVENTUALLY LANDED AT MICHIGAN STATE UNIVERSITY. WHICH, FOR THOSE WHO KNOW ABOUT SPORTS RIVALRIES THAT'S LIKE OIL AND WATER, MICHIGAN, MICHIGAN STATE. I DON'T KNOW WHERE YOU FALL THANE PLAY? >> IN MID PAM >> HE WAS THE DIRECTOR OF THE PROGRAM IN GENETICS AND DURING THAT TIME THE HUMAN GENETICS AND DEAFNESS WAS BORNE UNDERSTANDING THE MOLECULAR BASIS AND BECAUSE HE WAS A LEADER AT THAT STAGE, DR. BATTY, OUR SCIENTIFIC DIRECTOR AT THE TIME LATER BECAME OUR DIRECTOR, RECRUITED TOM TO NIDCD IN 1996 TO JUMP START A PROGRAM INCLUDING MULTIPLE LABS ON MOLECULAR GENETICS OF DEAFNESS. LET ME TELL YOU ABOUT THAT. AT TOM'S LAB AS WELL AS OTHERS ACROSS THE GLOBE HAVE IDENTIFIED HUNDREDS OF GENES AND MUTATIONS ASSOCIATED WITH HEARING LOSS. AND THESE GENETIC FACTORS ARE THOUGHT TO BE RESPONSIBLE FOR 50% OR 70% OR 80% OF HEARING LOSS IN CHILDREN. AND TODAY, DR. FRIEDMAN'S GOING TO TALK TO YOU ABOUT HOW WE FIND THOSE GENES IN HUMAN FAMILIES THAT CAUSE DEAFNESS AND BY IDENTIFYING FAMILIES AROUND THE WORLD WHO HAVE MEMBERS LIKELY TO BE AFFECT DEAFNESS OR AFFECTED BY DEAFNESS, DR. FRIEDMAN'S LAB FINDS THE GENES AND MUTATION AND THEN USES ANIMAL MODELS TO UNDERSTAND HOW THEY CAUSE HEARING LOSS IN HUMANS. AND SO THIS IS IMPORTANT FOR TWO REASONS. ONE IS IT'S OUR ONLY HANDLE INTO UNDERSTANDING THE MOLECULAR AND CELLULAR MECHANISMS OF HEARING AND DEAFNESS BECAUSE WE ACTUALLY CAN'T BIOPSY THE INNER EAR IN HUMANS. IT JUST DOESN'T HAPPEN WITHOUT MAKING PEOPLE DEAF AND DIZZY. SO GENETICS IS THE TOOL THAT'S OPENED UP THE WHOLE WORLD OF UNDERSTANDING HOW HEARING AND BALANCE WORKS. THE OTHER REASON THIS IS IMPORTANT IS BECAUSE UP UNTIL THIS GENETIC REVOLUTION, IF A PATIENT SHOWED UP IN OUR OFFICE WHO HAD HEARING LOSS OR DEAFNESS, A CHILD, UNLESS THEY HAD SOME OBVIOUS CAUSE LIKE AN INFECTION OR A GENETIC SYNDROME WHERE THERE WERE OTHER CLINICAL FEATURES THAT HELP US PEG IT AS A GENETIC DISORDER, WE REALLY DOESN'T KNOW THE CAUSE AND THIS WAS A CAUSE OF GREAT DISTRESS TO PARENTS AND DIDN'T KNOW WHAT TO EXPECT. THEY OFTEN FELT GUILTY. WHEN I'M COMING TO IS THE IDENTIFICATION OF THE GENES AND MUTATIONS ALLOWS US NOW TO PROVIDE A DIAGNOSIS TO ALL THESE CHILDREN AND FAMILIES IN' -- IN A MAJORITY OF CASES AND HEARING AND DIAGNOSIS OF CHILDREN AND IT'S COME ON THE HEELS OF NEWBORN SCREENING. ANOTHER IMPORTANT PART OF THAT. THANK YOU FOR JOINING US TODAY. I HOPE YOU ENJOY THIS AND LEARN A LOT FROM DR. FRIEDMAN. I KNOW I ALWAYS LEARN A LOT WHEN I HEAR HIM SPEAK. [APPLAUSE] >> THANK YOU FOR COMING TODAY. SO THERE ARE ABOUT 142 DEAFNESS GENES THAT HAVE BEEN REPORTED AND THIS LIST HAS BEEN EXAMINED BY A PANEL OF EXPERTS AND THERE'S CONFLICTING OR INSUFFICIENT EVIDENCE AND I'LL PUT QUESTION MARKS BY THEM AND THERE'S THREE DEAFNESS GENES THAT ARE BEEN REFUTED SO THEY'VE GONE UP IN SMOKE. AND THERE'S LIMITED DATA FOR 24 DEAFNESS GENES ACCORDING TO THIS PANEL OF EXPERTS AND A PUT A LITTLE CLOUD UNDER THEM BECAUSE THE DATA IS DEVELOPING AND IT'S LIKELY THAT THESE ARE REAL DEAFNESS GENES AT LEAST MOST OF THEM. SO WHAT I WANT TO DO TODAY IS TO TELL YOU ABOUT HEREDITARY DEAFNESS BY DIVIDING THE TALK INTO FOUR PARTS. AND THREE OF THEM HAVE TO DO WITH THREE PIE CHARTS. SO I'M GOING TALK ABOUT THE CAUSES MUCH CHILDHOOD DEAFNESS IN BASIC GENETICS, THE PATTERNS OF TRANSMISSION OF INHERITED DEAFNESS AND SYNDROMIC OR NON-SYNDROMIC DEAFNESS AND THEN GIVE YOU AN OVERVIEW OF FOUR DEAFNESS GENES FROM STUDIES IN ANY LABORATORY, WHICH ARE INTERNATIONAL COLLABORATIONS IN ALL CASES. SO SOME BRIEF COMMENTS ABOUT HEARING LOSS. THESE ARE THINGS THAT MOST OF US KNOW BUT I JUST WANT TO EMPHASIZE THAT THERE ARE HUNDREDS OF MILLIONS OF PEOPLE THAT HAVE HEARING LOSS. TWO TO THREE IN 1,000 INFANTS HAVE HEARING LOSS AND ABOUT HALF OR 60% OF THAT IS GENETIC IDEOLOGY. AND MANY GENES ARE REQUIRED FOR NORMAL HEARING AND THAT'S NOT SURPRISING GIVING HOW COMPLEX THE HEARING PROCESS IS AND THAT TO DATE THERE'S VARIANTS OF 129 GENES LIKELY TO CAUSE DEAFNESS. AND HEREDITARY DEAFNESS IS ALSO CLINICALLY DIVERSE THOUGH I WON'T SPEAK TO THAT ISSUE VERY MUCH. WE ALL KNOW AS WE AGE, HEARING LOSS BECOMES A PROBLEM AND BEETHOVEN WAS ABLE TO WRITE THE NINTH SYMPHONY WHICH IS EXTRAORDINARY BECAUSE HE COULD HEAR EVERY NOTE IN HIS HEAD. THE FIRST PIE CHART. THERE'S GENETIC AND INFECTIOUS DISEASE AND ENVIRONMENTAL CAUSES OF DEAFNESS AND I LISTED SOME ON THE RIGHT-HAND SIDE AND I'M SURE YOU'RE FAMILIAR WITH THAT INCLUDING ME INCLUDING MEASLES AND THE NUMBERS ARE INCREASING FROM THE CDC YESTERDAY AND THERE'S ENVIRONMENTAL AGENT INCLUDING OTOTOXIC DRUGS AND THERE'S SUSCEPTIBILITY TO NOISE AND THERE'S THE ENVIRONMENT AND HEREDITARY COMPONENT THAT CAN MAKE EARS TENDER OR TOUGH. SO A LITTLE BASIC CYTOGENETICS. IN THE NUCLEUS THERE'S 46 CHROMOSOMES USUALLY AND THERE'S DISPLAYED HERE AS WHAT'S CALLED A SPECTRAL CARE CARE CARYOTITE AND THERE'S SOME VARIANCE IN THE NUMBERS OF CHROMOSOMES BUT IT'S RARE. YOU CAN IMAGINE GENES ARE LIKE HOUSES ON A STREET AND THE STREET ARE LIKE CHROMOSOMES BUT IN GENERAL GENES ARE NOT TIGHTLY PACKED TOGETHER. IT'S OFTEN THE CASE THAT GENES ARE SURROUNDED BY NINE CODING SEQUENCE. THEY CAN BE LARGE AND TINY GENES. A HUMAN CONTRIBUTOR TO DEAFNESS IS ONE PROTEIN CODING PART SO IT'S A TINY GENE. IF YOU THINK THE HUMAN GENOME IS WELL WORKED OUT AND THERE'S NO PROBLEMS THERE'S A PAPER PUBLISHED LESS THAN A YEAR AGO INDICATING ONE IN FIVE GENES STILL HAVE UNRESOLVED CODING STATUS SO THERE'S A LOT TO BE DONE IN UNDERSTANDING THE STRUCTURE AND ARRANGEMENT OF GENES AND FOR THAT MATTER THE NUMBER OF GENES. THERE'S ABOUT 22,000 GENES IN THE HUMAN GENOME. FRUIT FLIES HAVE 30,000 AND CORN HAS 30,000. FRUIT FLIES HAVE 15,000. THERE'S NO RELATIONSHIP BETWEEN ROAM -- CHROMOSOME NUMBER AND I.Q. THE CODING PORTION OF THE GENE OCCUPIES ABOUT 1% OF THE HUMAN GENOME. THE REST IS PSEUDOGENES. THESE ARE DEAD GENES. DEFECTIVE VIRAL SEQUENCES AND FRAGMENTS. DEFECTIVE TRANSPOSONS AND WHAT WON'T GET TRANSLATED AND RNAs AND LONG-CODE RNAs AND THERE'S TRANSPOSABLE AMOUNTS IN THE GENOMES THAT MOVE AROUND. LIKE TINY HOUSES ON WHEELS IF YOU WANT TO THINK OF IT THAT WAY. SO I USED THE TERM GENE BUT THE QUESTION IS WHAT'S THE DEFINITION OF A GENE? GREGOR MENDEL NEVER USED THE WORD GENE. HE TALKED ABOUT HEREDITARY DETERMINATES. IT WAS A DANISH BOTANISTS ACCOUNT THE TERM. SO GENE, GENOTYPE AND PHENOTYPE WERE COINED BY HIM AND THE TERM GENE HAS BEEN EVOLVING SINCE 1990 AND THERE'S REALLY NOT A GOOD GENETIC DEFINITION. THE ONE ON THE NATIONAL CANCER INSTITUTE'S WEBSITE IS THE BASIC UNIT OF HEREDITARY THAT OCCUPIES A SPECIFIC LOCATION ON A CHROMOSOME. THAT'S NOT A GREAT DEFINITION. I'D LIKE TO STRESS THE PHENOTYPIC EFFECTS OF ONE GENE CAN BE STRESSED BY ANOTHER GENE AND THAT'S WHAT WE REFER TO AS THE GENETIC BACKGROUND AND THE ENVIRONMENT. I WANT TO BE KNOWN FOR THE STATEMENT THE PATHWAY FROM GENE TO PHENOTYPE IS LENGTHY. SO THERE'S GREAT DIVERSITY AMONG HUMAN BEINGS THEY'RE REMARKABLY SIMILAR GENETICALLY. WE'RE ABOUT 99% SIMILAR AT THE CODING LEVEL PROTEIN CODING LEVEL. AND WILD TYPE VARIANTS CHANGES IN DNA ARE RESPONSIBLE FOR THE TYPICAL FORM OF A SPECIES AS IN NATURE. I WANT TO STRESS A NUMBER OF POINTS. THAT IS NATURAL POPULATIONS OF AN ORGANISM DO NOT HAVE A WILD TYPE. I'M NOT WILD TYPE. WE DON'T TALK ABOUT WILD TYPE HUMAN BEINGS. IN POPULATIONS THERE'S A NORMAL RANGE OF PHENOTYPIC VARIATION AND IT'S WONDERFUL. SO MOST SPECIES ARE POLYMORPHIC FOR MANY TRAITS, THAT IS FOR THE PHENOTYPE. AND THIS DIVERSITY IS MAGNIFICENT. THE PHENOTYPE CAN BE TAKEN OUT OF THE NORMAL RANGE BY PATHOGENIC VARIANTS. AND GENETICISTS HAVE BEEN USING THE WORD VARIANTS INSTEAD OF MUTATION SO I'LL TRY TO USE THE WORD VARIANTS AS A SUBSTITUTE FOR MUTATION. THIS IS AN EXAMPLE OF A CLEARLY ABNORMAL LEG BEING INHERITED WITH DEAFNESS IN THE STUDY WE DID WE SUZANNE LUAL AND THE WILD TYPE FUNCTION OF A GENE CANNOT BE IMMEDIATELY OR NECESSARILY DEDUCED DIRECTLY FROM THE PATHOLOGY. CLEARLY THIS GENE IS NECESSARY FOR NORMAL LIMB DEVELOPMENT BUT YOU CANNOT DEDUCE FROM THE PHENOTYPE WHAT THE FUNCTION OF THE GENE IS OTHER THAN AS THE NECESSARILY FOR NORMAL LEG DEVELOPMENT. I WANT TO TURN TO HUMAN PEDIGREES AND POINT OUT SOME SIMPLE PEDIGREES TO YOU. UP IN THE RIGHT HAND CORNER AND WHEN AFFECTED THEY'RE DARKENED IN. WHEN THEY'RE UNAFFECTED BUT CARRYING A RECESSIVE MUTATION OFTEN THERE'S A LITTLE DOT AND IF THEY'RE DEAD THERE'S A LINE PLACED THROUGH THEM. SO THIS IS AN EXAMPLE OF RECESSIVE INHERITANCE. BOTH PARENTS ARE CARING A VARIANT AND THEY HAVE THREE CHILDREN WHICH ARE UNAFFECTED WHICH IS WHAT YOU EXPECT FROM THE SIMPLE MELDELIUM VARIANT AND THE OTHER CARRIER IS NORMAL AND THE FATHER HAS A DIFFERENT VARIANT AND THE CHILD INHERITS TWO VARIANTS AND HAS THE PHENOTYPE. FOR AUTOSOMAL STRAITS YOU CAN HAVE THE REVERSE FROM THE MALE TO THE FEMALE DAUGHTER. THIS IS A PEDIGREE THAT DEMONSTRATES DOMINANT INHERITANCE OF A TRAIT. INFECTED FEMALE AND MALE AND THERE'S A MARRIAGE. THESE ARE SIBLINGS. AND IN THIS CASE. A DOUBLE LINE IN A COSANGINOUS MARRIAGE AND THERE'S CARRIERS IN THIS PEDIGREE. AND THIS ISN'T THE ONLY REPOSITORY OF GENETIC INFORMATION. MIG MITOCHONDRIA HAVE THEIR BETWEEN ONE OF THEIR OWN. 6,500 BASE PAIRS OF DNA AND THIS IS A PAPER FROM ANDY'S LAB. AND THERE'S A PARTICULAR MUTANT ALLELE OF THE TR GENE IN THE MITOCHONDRIAL GENOME. AND THE EGG HAS ALMOST ALL THE MITOCHONDRIA CONTRIBUTED TO THAT GENERATION. THE MOM PASSED THIS TO ALL THE CHILDREN AND THEY HAVE PROGRESSIVE HEARING LOSS AND THIS WOMAN PASSED THE MITOCHONDRIA TO HER CHILDREN AND HER SON DIDN'T CONTRIBUTE THEM TO THE NEXT GENERATION AND EVEN IF HE HAD HUNDREDS OF CHILDREN THEY'D BE UNAFFECTED. IT'S A MATERNAL TRANSMISSION OF PROGRESSIVE DEAFNESS NICELY ILLUSTRATED IN THIS PAPER. AND THERE'S VARIANTS OF THE GENES AND THEY CONTRIBUTE TO 4% OF INHERITED DEAFNESS. MOST IS AUTOSOMAL CHROMOSOMES AND 50% TO 60% OF THE INHERITED DEAFNESS WHERE AS IT DOMINANTLY INHERITED DEAFNESS IS 30% TO 40%. AND THIS DOESN'T TELL YOU MUCH ABOUT THEIR FUNCTION. ONE OF THE THINGS THAT GIVES YOU INSIGHT IS WHEN YOU DEVELOP THE PARTS LIST AND START NOTICING THE PARTS INTERACT WITH ONE ANOTHER IN THE CELL AND FIT TOGETHER INTO A MACHINE YOU BEGIN TO UNDERSTAND WHAT THE FUNCTION OF THAT GENE MIGHT BE. THE GOAL IS TO ASCERTAIN LARGE FAMILIES SEGREGATING DEAFNESS. AND BE CONFIDENT ABOUT THE PHENOTYPE. AND OFTEN IF YOU KNOW THE SEQUENCE AND THE PREDICTIVE PROTEIN AND YOU MAY NOT UNDERSTAND WHAT THE FUNCTION IS. YOU HAVE TO ENGINEER ANIMAL MODELS TO TEST HYPOTHESES ABOUT WHAT THE GENES ARE DOING AND THEY CONSISTENT OF MICE, ZEBRAFISH OR FRUIT FLIES WHERE MANY HUMAN GENES HAVE BEEN STUDIED AND WE GAINED EIGHT OF INSIGHT BY -- GAINED A LOT OF INSIGHT BY STUDYING THE SAME GENE IN A FRUIT FLY. SO WE PERFORM FUNCTIONAL STUDIES AND BEGIN THINKING ABOUT THERAPIES AND WHEN WE BEGIN UNDERSTANDING THE FUNCTION OF THE GENE, WE MAY NOTICE IN THE ANIMAL MODEL THAT THERE ARE OTHER CLINICAL FEATURES WE MISSED IN THE FAMILY. I'LL GIVE YOU SOME EXAMPLES OF THAT BUT THE MOST RECENT ONE FROM THE LAB WAS JUST PUBLISHED BY A POST-DOC IN THE LAB AND WE NAMED IT HIMS BECAUSE THE MUTATIONS, THE VARIANTS CAUSE HEARING LOSS AND INFERTILITY IN MALES NOT THE FEMALES WHO ARE DEAF. DEAF FEMALES ARE FERTILE. I'LL COME BACK TO THIS IN A COUPLE MINUTES AND TELL YOU MORE ABOUT IT. SO THE THIRD PIE CHART INDICATES OF THE GENES THAT HAVE BEEN IDENTIFIED ABOUT 36% OF THEM CAUSE SYNDROMIC DEAFNESS AND THE DEFINITION OF SYNDROMIC IS THAT THERE ARE ADDITIONAL ABNORMALITIES OF OTHER TISSUES AND ORGANS DUE TO THAT GENETIC CHANGE WHEREAS IN THE CASE OF NO NON-SYNDROMIC DEAF THERE'S NO ACCOMPANYING DISORDER YOU CAN IDENTIFY INITIALLY. I WANT TO STRESS WITH THE LIMITED CLINICAL DATA WE GET WE OFTEN MISS OTHER CLINICAL FEATURES WE WOULDN'T HAVE ASKED ABOUT AND I'LL GIVE YOU EXAMPLES IN A COUPLE MINUTES. I WANT TO POINT OUT IN THE ONLINE INHERITANCE OF MAN THERE'S ABOUT 500 DISTINCT SYNDROMES WHERE DEAFNESS IS LISTED SO THERE'S A LOT OF SYNDROMES WE HAVE NOT CHARACTERIZED AT THE GENETIC LEVEL THAT STILL NEED A LOT OF WORK. IF YOU NOTICE, I INDICATED THERE'S 57 GENES THAT ARE NOW REPORTED RESPONSIBLE FOR SYNDROMIC DEAFNESS AND 106 THAT ARE RESPONSIBLE FOR NON-SYNDROMIC DEAFNESS AND IF YOU ADD THOSE TOGETHER, IT'S MUCH MORE THAN 129. SO WHAT'S GOING ON AND THE EXPLANATION FOR THEM BEING MORE DEAFNESS GENES IN THE TWO CATEGORIES THAN VARIANTS OF PARTICULAR GENES AND THE ANSWER COMES FROM UNDERSTANDING THAT THE SAME GENE CAN -- THERE CAN BE VARIANTS THAT GIVE RISE TO SYNDROMIC AND NON-SYNDROMIC DEAFNESS. I REMEMBER BECAUSE IT WAS TRAUMATIC AT THE FIRST A.R.O. I ATTENDED, I STATED NON-SYNDROMIC DEAFNESS GENES DO NOT ENCODE AUDITORY SPECIFIC PROTEINS SPECIAL FOR HEARING. THERE'S A COUPLE EXAMPLES BUT BY AND LARGE THEY DON'T. AND I ALSO SAID THAT NON-SYNDROMIC DEAFNESS MAY BE CAUSED BY THE SAME VARIANTS THAT CAUSE SYNDROMIC DEAFNESS. IT TURNS OUT THOUGH I WAS YELLED AT LITERALLY AND THEY ENCODE PROTEINS ESSENTIAL FOR HEARING BUT THEY COULD BE IMPORTANT ELSEWHERE AS WELL BUT NOT CAUSE A DISORDER. NON-SYNDROMIC DEAFNESS IS CAUSED BY MUTATIONS OF ONE SET OF GENES AND IT'S JUST COMPLETELY WRONG. THERE ARE NOW 19 EXAMPLES WHERE VARIANTS OF THE SAME GENE GIVE RISE TO SYNDROMIC DEAFNESS AND OTHER VARIANTS GIVE RISE TO NON-SYNDROMIC DEAFNESS. SO, I WANT TO TELL YOU ABOUT THREE OF THEM. WORK IS GOING ON IN MY LAB AND THE FIRST IS THE NON-SYNDROMIC DEAFNESS CAUSE DFMB12 AND THE SYNDROME IS CHARACTERIZED BY CONGENITAL PROFOUND DEAFNESS, IN THIS CASE, THAT RP RET INITIS PIGMENTOSIS IS CHARACTERIZED AND CAN BE DETECT. THIS IS AN OLD SLIDE AND THERE'S MANY MORE EXAMPLES BUT ALL THE VARIANTS UP HERE ON THE TOP WHEN HOMO ZYGOUS ARE PRESENT IN COMPOUND HETEROZYGOUS CAUSES THE SYNDROME AND ALL THESE DOWN HERE CAUSE NON-SYNDROMIC DEAFNESS. SO JULIE SCHULTZ SAT OUT TO TRY TO ANSWER A QUESTION AND IT'S ONE OF THE MOST INTERESTING PAPER WE PUBLISHED AND TRIED TO ANSWER THE QUESTION WHAT WOULD BE THE PHENOTYPE WHEN HAVE YOU ONE USHER GENE VARIANT AND ONE DFMB12 VARIANT IN A SUBJECT AND THE ONLY WAY WE COULD ANSWER THAT QUESTION IS TO BE CERTAIN THAT THE USHER VARIED WHEN HOMO ZYGOUS GAVE RISE TO THE USHER SYNDROME AND THE NON-SYNDROMIC GENE GAVE RISE TO NON-SYNDROMIC DEAFNESS AND FIND INDIVIDUALS WITH ONE OF EACH AND IT TOOK A LONG TIME TO FIND THEM. THE QUESTION NOW IS IS THE PHENOTYPE OF THAT PHENOTYPE OF THAT USHER GENE AND NON-SYNDROMIC GENE LIL BE USHERS -- WILL BE USHERS OR IN BETWEEN OR NON-SYNDROMIC. WHAT DO YOU THINK IT WILL BE? DO YOU THINK IT WILL BE USHERS? IN BETWEEN? NON-SYNDROMIC DEAFNESS? IT TURNS OUT IF YOU INHERIT ONE OF EACH OF THESE, YOU'LL HAVE NON-SYNDROMIC DEAFNESS IN THE FAMILIES REPORTED IN THIS PAPER. A LITTLE BIT WILL PRESERVE VISION BUT NOT HEARING. IT MEANS RESTORING EVEN A LOW LEVEL OF FUNCTION OF IT MAY BE SUFFICIENT TO PRESERVE DECISION. THERE'S REASON TO BE OPTIMISTIC FOR THE INDIVIDUALS AND ONCE WE NOR MORE AND DESPITE KNOWING IT CAUSES USHER WE STILL KNOW LITTLE ABOUT THE DYSFUNCTION IN THE RETINA. I'LL GIVE YOU ANOTHER EXAMPLE AND THIS IS A COLLABORATION IN THE LAB AND WE IDENTIFIED A LARGE PAKISTANI FAMILY. I WON'T TELL YOU ABOUT A RECENT PAPER FROM A POST-DOC IN THE LAB BECAUSE I'M JUST GOING TO TELL YOU ABOUT HER SUMMARY FIGURE. FIGURE 1 BECAUSE SHE'LL HAVE A NICE STORY TO TELL THE INSTITUTE IN A FEW MONTHS. THIS IS A SUMMARY OF THE MUTATIONS AND THE VAST MAJORITY ARE FROM OTHER LABS. LOTS OF OTHER LABS. THIS WAS HER. THESE ARE ALL THE MUTATIONS DESCRIBED IN THE GENE. DON'T NEED TO KNOW ABOUT THE FUNCTION JUST LOOK AT THE VARIANTS AND THEY WERE COLOR-CODED SO THE ONES IN RED WHICH ARE MOSTLY FROM OUR LAB ARE ASSOCIATED WITH NON-SYNDROMIC DEAFNESS. THAT ONE, THAT ONE. AND THERE'S A GENE THAT CAUSED NON-SYNDROMIC DEAFNESS. THERE'S ONE THAT WAS REPORTED BY RICHARD SMITH THAT CAUSED DEAFNESS AND ALL THE OTHERS ARE RECEASIVE. AND ONE CAUSED A SYNDROME CALLED DORS THAT STANDS FOR DEAFNESS ONCODYSTROPHY AND THE R STANDS FOR RETARDATION, MENTAL RETARDATION. THEY COULD HAVE PICKED A BETTER NAME FOR THIS. BUT THE INDIVIDUALS HAVE SKELETAL ABNORMALITIES AND NAIL ABNORMALITIES AND HAVE SEIZURES AND ARE DEAF. THAT'S THE MOST EXTREME PHENOTYPE OF VARIANTS IN THIS GENE. AND WE DON'T KNOW THE FUNCTION OF THIS GENE IN THE GANGLION BUT BY SOME TIME IN THE NEAR FUTURE RISA WILL TELL YOU MORE ABOUT THIS GENE. I WANT TO BRIEFLY MENTION CDC 14A. THE MUTATIONS CAUSE NON-SYNDROMIC DEAFNESS AND HIMS BECAUSE THEY RESULT IN MALE INFERTILITY AND DEAFNESS. SO JUST TO REMIND YOU BEFORE I TELL YOU ABOUT THAT, AND CHANNELS TMC1 AND 2 UP RESPONSE TO THIS MOVEMENT AND THERE WAS SYNAPTIC TRANSMISSION. IN LIGHT OF THAT I WANT TO TELL YOU ABOUT CDC 14A. SHE'LL ALSO TELL YOU IN THE NEAR FUTURE THERE'S ADDITIONAL AXONS. IT'S MORE COMPLICATED. THIS INDICATES THE INDIVIDUAL IS DEAF. AND THERE WAS MALE INFERTILITY WE DIDN'T KNOW AT THE TIME AND THERE ARE FAMILIES SEGREGATING JUST NON-SYNDROMIC DEAFNESS. YOU NOTICE THE MALE OVER HERE IS FERTILE. MARRIED A WOMAN WHO IS DEAF AND THEY HAVE CHILDREN. THE FUNCTION WE KNOW ABOUT BECAUSE IT WAS STUDIED 30 YEARS AGO AND NECESSARY FOR EXIT FROM MITOSIS. IT WAS A PHOSPHOTASE THAT WAS REQUIRED FOR THE COMPLETION OF CELL DIVISION IN YEAST. FOR A LONG TIME AND THERE MUST BE ON THE ORDER OF 15, 20 PAPERS SAME FUNCTION IN HUMANS BUT CLEARLY IT DOESN'T. BUT IT'S A HIGHLY CONSERVED PROTEIN. IT REMOVES A PHOSPHATE WEIGHTING BUT WE DO NOT KNOW WHAT THE SUBSTRATE IS IN THE EAR AND IN BUT HIGHLY ABUNDANT IN. -- IN SPERM. I TOLD YOU TWICE ALREADY WE DIDN'T KNOW THE MALE INFERTILITY WAS ASSOCIATE WITH THE DEAFNESS. MALE INFERTILITY IS VERY COMMON SWO MADE NOTHING OF IT SOMETIME WE MADE A CROSS BETWEEN A FEMALE THAT WAS CLEARLY DEFICIENT IN THIS FOS -- PHOSPHOTASE AND THAT MOUSE IS DEAF AND THE MALES ARE FERTILE IF YOU MATE THEM WITH A WILD TYPE AND THE SAME MOUSE WE BEGAN TO THINK THE INFERTILITY MAY BE CAUSELY ASSOCIATE WITH THE CDC14A. FOEF FOEF SO IN THE CDC14A MOUSE SICILIA IS NORMAL BUT HAIR CELLS DEGENERATE AND BY P90 THERE ARE ALMOST NO HAIR CELLS LEVEL. PHOSPHATASE IS NECESSARY FOR NERVE CELLS AND FOR SPERM FUNCTION. WHEN YOU LOOK AT THE SPERM FROM THE MALE MICE THEY HAVE HEADS WITHOUT TAIL. THE TALES ARE OFTEN CROOKED AND THE TUBULES ARE OFTEN DISMORPHIC. THE PHOSPHATASE IS NECESSARY FOR MALE FERTILITY BUT NOT FEMALE FERTILITY. THIS IS A PAPER WITH A POST-DOC IN THE LAB AND IN IT WE REPORTED IF YOU MOVE XR8 FROM THE GENE CALLED TRBP AND IT MAKES A LONG PROTEIN, IT'S A LONG PROTEIN ILLUSTRATED BY THE LONG COLORFUL RECTANGLE AND MAKES TRIOBP4 AND 1 AND WE KNOW TRIO BP1 IS NECESSARY FOR VIABILITY. SO MUTATION SKILL IT THE ANIMAL DIES DURING EMBRYONIC DEVELOPMENT AND THIS THAT KILLED 5 AND 4 RESULTED IN DEAFNESS LIKE IN THE HUMAN FAMILIES WE PREVIOUSLY STUDIED. AND ONCE AGAIN THE HAIR CELLS DEGENERATIONS IN THIS MUTANT. IT TURNS OUT TRIO BP4 AND/OR 5 ARE NECESSARY SO THIS IS THE CELL MEMBRANE AND THE ROOTLET WHICH IS LIKE AN ANCHOR FOR THE CILIA DEVELOP AND IT GOES DOWN IN THE PLATE AND INTO THE STEREO CILIA ABOUT A THIRD OF THE WAY AND IN THE MUTANT WITH A DELETION OF XON8 THERE'S NO ROOTLETS. AND THE STEREOCILIA ARE FLOPPY AND DIDN'T DEGENERATE AND IT DIDN'T ANSWER THE FUNCTION OF TRIO BP5 OR 4 OR DISTRICT FROM 5 BECAUSE WE KILLED BOTH SIMULTANEOUSLY BY THE MUTATION. IN A RECENT PAPER WITH A LOT OF THE WORK WHAT WE DID WITH KILLED PART OF THE GENE THAT WOULD ONLY AFFECT TRI YO BP5 AND LEAVE 4 AND 1 AND LEFT 4 BECAUSE WE WANT TO KNOW WHAT IS IMPORTANT ABOUT THE FUNCTION OF 5 IF ANYTHING. SO WE KILLED 5 AND ONE THING ABOUT 5 IS 5 IS MAINLY IN THE RO ROOTLETS WHERE 4 IN THE PART ABOVE THE CUTICULAR PLATE. SO WHAT IS THE PHENOTYPE GOING TO BE IF YOU JUST KILL 5? THE ANSWER WAS COMPLETELY UNEXPECTED. SO ONE OF THE WAYS WE LOOKED AT THIS WAS TO USE A NOVEL TECHNIQ TECHNIQUE FOR US. IT WAS AN INSTRUMENT OUR COLLEAGUES HAVE IN JAPAN CALLED FIBSIM FOCUSSED ON MILLING. YOU BASICALLY TAKE THE TISSUE AND LOOK AT IT WITH AN ELECTRON MICROSCOPE AND MILL THE SURFACE WITH A BEAM TO SHAVE OFF A LITTLE BIT AND IMAGE IT AND DO IT AGAIN AND PUT THEM TOGETHER IN A THREE DIMENSION. HERE'S A WILD TYPE. FRAME IS MILD AND FOCUSSED AND NOW IT'S -- AND MILLED AND FOCUSSED AND ANIMATED IN A WILD TYPE WITH A MOUSE WITH NORMAL HEARING. IT GOES A THIRD OF THE WAY UP IN THE STEREOCILIA. THIS IS THE MOUSE MISSING THE TRIO BP5. YOU SEE IT HAS ROOTLETS BUT INSTEAD OF STOPPING ONE-THIRD UP THE STEREO CILIA IT GOES UP TO THE TOP AND NON-FUNCTIONAL. THE MOUSE IS DEAF. SOMEHOW IT'S CONTROLLING THE ARCHITECTURE OF THIS MOVEMENT IN A WAY WE DON'T UNDERSTAND YET. SO IN THE WILD TYPE THERE'S FOUR AND THERE'S FIVE AND IT BUNDLES THE FILAMENTS THAT MAKE UP THE ROOTLET AND IN THE ABSENCE OF 4 IT GOES WAY UP IN THE STEREOCILIUM. SO IN SUMMARY, THERE ARE MANY HUMAN GENES ASSOCIATED WITH DEAFNESS. AS I TRIED TELLING YOU AT THE BEGINNING OF THE TALK, THE STRENGTH OF THE EVIDENCE FOR HUMAN DEAFNESS GENES VARIES FROM LIMITED TO DEFINITIVE. AND PART OF THAT STRENGTH COMES FROM ANIMAL MODELS, FINDING MORE AND CONVINCING YOURSELF THERE'S A CAUSAL RELATIONSHIP BETWEEN THE VARIANT AND THE PHENOTYPE. SO ANIMAL MODELS ARE CRITICAL. THEY'RE ESSENTIAL FOR UNDERSTANDING DEAFNESS GENES AND PROVIDING THE CORROBORATING GENES AND WE ALSO KNOW FROM HUNDREDS OF FAMILIES THAT WE HAVE WHERE WE HAVE NOT IDENTIFI IDENTIFIED VARIANTS IN THE KNOWN DEAFNESS GENES THERE'S MANY MORE LIKELY TO BECOME OF INTEREST OF US AS WE DO THE GENETICS AND DEVELOP ANIMAL MODELS. SO, I'M GOING STOP THERE AND IF THERE'S ANY QUESTIONS, I'D BE HAPPY TO ANSWER THEM AND I'M LOOKING FORWARD TO ANY COMMENTS YOU MIGHT HAVE AT THIS POINT. [APPLAUSE] >> ANY QUESTIONS FROM DR. FRIEDMAN. >> YES. [OFF MIC] >> SO THE QUESTION IS ABOUT CONEXIN 26 AND THE GENE IS GJB2 AND VARIANTS OF THE TINNY GENE IS A MAJOR CONTRIBUTOR TO DEAFNESS WORLDWIDE BUT THERE'S SOME COUNTRIES WHERE YOU DON'T FIND ANY DEAF INDIVIDUALS WITH VARIANCE AND IT DEPENDS BETWEEN 20% AND 50% OF HEREDITARY DEAFNESS IN WESTERN EUROPE. ROB MORELL IDENTIFIED IT IN THE NAZI JEWISH POPULATION AND PUBLISHED IT IN THE NEW ENGLAND JOURNAL. WE HAVE BEEN VERY INTERESTED IN CONEXIN 26 IN THE PAST. >> THERE'S BEEN A LOT OF WORK ON CONEXIN 26 ACROSS THE WORLD. I'M HAPPY TO SAY WE CONTRIBUTED A LITTLE BIT TO IT BUT A LOT WORK ACROSS THE GLOBE. >> THANK YOU FOR THE QUESTION. [OFF MIC] >> THERE ARE PROTOCOLS THAT RECRUIT FAMILIES WITH SPECIFIC PHENOTYPES OR ARE OF INTEREST. MY PROTOCOL COVERS THE ASCERTAINMENT OF HEREDITARY OF DEAF FAMILIES IN PAKISTAN BUT ANDY HAS A PROTOCOL THAT COVERS DEAFNESS IN NORTH AMERICA. IT'S LIMITED IN FOCUS. DO YOU WANT TO MENTION THAT, ANDY? >> SURE. WE DO STUDY FAMILIES AND INDIVIDUALS FROM NORTH AMERICA. WE ALSO STARTED WORKING WITH OTHER PARTS OF THE GLOBE. WE STARTED WITH TOM AND COLLABORATOR IN NIGERIA AND A PROMISING STUDY OF HEARING LOSS AND DEAFNESS IN AFRICAN POPULATIONS FOR WHOM ESSENTIALLY NOTHING IS KNOWN. WE'RE BRANCHING OUT AND LIKELY FIND ADDITIONAL GENES THAT CAN CAUSE DEAFNESS BY STUDYING THOSE OTHER POPULATIONS. [OFF MIC] >> READY. >> WHY AREN'T WE DOING THAT, ANDY? >> THAT'S NOT WHY I'M STANDING HERE. I CAN TELL YOU 10 OR 15 YEARS AGO THERE WAS A WORKSHOP HELD BY THE AMERICAN COLLEGE OF GENETICS WITH THE IDEA OF SCREENING EVERY NEWBORN NOT BY CHECKING THEIR HEARING TEST BUT SEQUENCING THEIR GENES. THE DIFFICULTY AT THAT TIME WAS THERE WERE LOTS OF RESULTS IN VARIANTS WE DIDN'T KNOW HOW TO INTERPRET AND IF SOMEONE DID INHERIT ONE OF THE VARIANTS KNOWN TO CAUSE HEARING LOSS, WE DIDN'T HAVE ENOUGH INFORMATION TO COUNSEL THAT INDIVIDUAL ON WHAT TO EXPECT SO I THINK TOM'S ANSWER WAS APPROPRIATE AND MY GUESS IS THAT THERE'S STUDIES IN THE NIDCD PORTFOLIO I DON'T KNOW ABOUT THAT ARE LOOKING AT THAT VERY QUESTION. SO YOU ARE GURU OF GENOMICS IN HUMANS. DO YOU HAVE ADVISE US ABOUT? [OFF MIC] >> RIGHT. SO ONE OF THE STATISTICS, TOM MENTIONED TWO TO THREE HILDREN IN A THOUSAND IS BORN WITH HEARING LOSS. THE STATISTICS ARE GETTING MORE ACCURATE. IT TURNS OUT FOR EVERY CHILD BORN WITH HEARING LOSS BY THE AGE OF 9, ANOTHER CHILD WILL DEVELOP HEARING LOSS AND MANY WILL GET MISSED -- WELL, THEY'LL ALL GET MISSED BY THE NEWBORN HEARING SCREENING AND MANY WILL GET MISSED BY THE SCREENING PROCESSES WE HAVE IN PLACE WHICH ARE SCHOOL-BASED IN OUR COUNTRY. IT'S AN IMPERATIVE WE GET BETTER AT DETECTING THOSE CHILDREN AND GENOMI GENOMICS A LOT OF PEOPLE WOULD PREDICT IS GOING TO BE AN IMPORTANT PART OF THAT TOOL KIT. [OFF MIC] >> I DO BELIEVE THERE ARE TREATMENTS ON THE HORIZON. THE QUESTION IS AND IN A FAMILY WHERE SOMEONE DEVELOPS HEARING LOSS PROGRESSIVELY MAYBE 9 OR 10 AND THERE'S SEVERAL YEARS AT WHICH YOU CAN INTERVENE BEFORE THERE'S PROFOUND DEAFNESS AND THERE'S THERAPIES ON THE HORIZON. I CAN RELAY SOMETHING TO YOU AS A POST-DOC HERE AND ANDY MENTIONED THIS, A LONG TIME AGO, GENETIC GENE THERAPY WAS AROUND THE CORNER. IT'S STILL JUST AROUND THE CORNER. IT TAKE A LONG TIME TO DEVELOP THERAPIES. BUT THERE ARE CLINICIAN SCIENTISTS IN OUR INSTITUTE WORKING ON GENE THERAPIES USING ANIMAL MODELS AND IT'S REALLY LOOKING PROMISING. IT LOOKS EXTREMELY PROMISING. OF COURSE THERE'S THE ISSUE OF SAFETY AND ENDURANCE AND WHETHER IT WILL LAST. IT'S JUST AROUND THE CORNER. >> ANDY IS REMINDING ME THERE ARE SMALL MOLECULES. WE PUBLISHED PROBABLY THE FIRST OR SECOND SMALL MOLECULE THAT RESCUED A MOUSE WITH HEARING LOSS THAT MIMICKED A HUMAN FAMILY WE WERE STUDYING WITH PROGRESSIVE HEARING LOSS. THERE'S AN FDA APPROVED DRUG TOO THAT IS INTERESTING THAT WORKED PERFECTLY WELL IN THE MOUSE. >> SOME CAUSE DEAFNESS PARTICULARLY IN USHER SYMPTOM 1 AND FOR OTHERS IT DOESN'T SEEM TO BE SO SUSCEPTIBLE TO MUTATIONS BUT THERE'S PLENTY OF EXAMPLES WHERE YOU'RE DEAF AND DIZZY. AND THE SAME THING GOES FOR MICE TOO. ONE OF THE ONES WE STUDIED EARLY ON WAS A MOUSE THAT CIRCLED AND A MOUSE MODEL OF A HUMAN DEAFNESS GENE WE FIRST IDENTIFIED IN A VILLAGE IN BALI BUT IS THE THIRD OR FOURTH COMMON GENE WHERE VARIANTS CAUSE DEAFNESS WORLDWIDE NOW. [OFF MIC] >> IT'S HARD TO DEMONSTRATE A VARIANT IS DE NOVO AND SEPARATE IT FROM A FAMILY PROBLEM. >> OUR PROTOCOLS BECOME MORE FOCUSSED ON VERY SPECIFIC SUBSETS OF PATIENTS WE'RE STUDYING IN THE LAB. LESS FOCUSSED ON DISCOVERING LOTS OF NOVEL GENES AND IN FACT YOU CAN THINK OF OUR PROTOCOLS AS A PIPELINE FOR WHAT WE HOPE IS A FUTURE CLINICAL TRIAL AND SOMETHING DR. CHEN'S DOING. YOU'RE ASKING A GREAT QUESTION AND IT'S BEING FUNDED BY RESEARCHERS BUT NOT HERE. WHOLE EXOME WHERE IT'S BEING ANALYZED FOR ALL THE GENES IN THE GENOME ARE BECOMING QUITE COMMON. IN FACT, IT'S ALMOST THE STANDARD OF CARE. THERE'S AN EFFICIENT USE OF TAXPAYER MONEY THAT'S THE PLACE TO GO. IF YOU IDENTIFY A MUTATION THEN YOU CAN LOOK AT THE PARENTS. AS TOM POINTED OUT IT'S ALWAYS IMPORTANT TO POINT OUT THE MOTHER'S MOTHER AND THE FATHER'S THE FATHER BECAUSE IT'S NOT ALWAYS THE CASE AND THAT CAN OCCUR UP TO 10% OF CASES WHERE PATERNITY IS NOT WHAT PEOPLE THOUGHT IT WAS. AND YOU CAN DETERMINE IF IT'S A NEW MUTATION THAT AROSE IN THE GERM CELLS, THE SPERM OR THE OVA OF THE MOTHER OR FATHER. SO IN THE INTRAMURAL PROGRAM, WE'VE BEEN REALLY FOCUSSING ON RESEARCH HARD TO DO IN THE ENVIRONMENT AND IN UNIVERSITIES ACROSS THE GLOBE AND HARD TO DO WITH THE GRANT FUNDING MECHANISM. WE'RE TRYING DO STUFF AND BE MORE EFFICIENT WITH THE SYSTEM. >> ONCE AGAIN A ROUND OF APPLAUSE FOR DR. FRIEDMAN. GREAT PRESENTATION. THANK YOU ALL FOR COMING AND PARTICIPATING TODAY AND FOR THOSE PARTICIPATING ON THE VIDEOCAST, YOU MAY HAVE NOTICED THAT WE HAVE SOME FACT SHEETS BACK IN THE ROOM FOR INFORMATION. I THINK THEY'RE HERE FOR MORE INFORMATION ABOUT HEARING LOSS IF YOU'D LIKE TO LEARN MORE AND YOU WOULD HAVE FOUND AN EVALUATION SHEET ON YOUR CHAIR WHEN YOU WALKED IB -- IN. WE'D APPRECIATE FEEDBACK. THE NEXT BEYOND THE LAB FEEDBACK IS NEXT YEAR AND WE LOOK FORWARD TO HAVING YOU COME BACK AND JOIN US AGAIN. TAKE CARE, EVERYBODY.