>> WELCOME EVERYBODY TO THE FIFTH ANNUAL NHLBI MAURICE B. BURG LECTURE. SO LECTURE WAS ESTABLISHED UPON THE SWITCH, MO BURG, MENTOR AN FORMER BOSS TO EMERITUS STATUS SEVERAL YEARS AGO. AND SO THE IDEA IS TO COMMEMORATE HIS REMARKABLE CAREER WHICH BEGAN IN THE INTRAMURAL PROGRAM BACK IN THE 1950s, ACTUALLY 1957 TO BE EXACT. EARLY IN HIS CAREER MO INVENTED A METHOD FOR STUDYING RENAL FUNCTION, ISOLATED PROFUSED TUBEIAL TECHNIQUE WHICH REVOLUTIONIZED THE FIELD OF RENAL PHYSIOLOGY P THE RENAL TUBULA IS MADE UP OF SOME 14 DIFFERENT EPITHELIAL SEGMENTS EACH WITH DIFFERENT FUNCTIONS AND MO WORKED OUT A METHOD TO MICRODISSECT THE SEGMENTS FROM THE KENNEY MOUNT ON PIPETTE AS DIAGRAMMED HERE, AND STUDY THE FUNCTION OF EACH OF THESE TUBULES WHICH MIGHT CONSIST OF SOMETHING ON THE ORDER OF 500 TO A THOUSAND CELLS. SO MO WORKED OUT THE METHOD TO MICRODISSECT THESE SEGMENTS AND THIS MICRODISSECTION TECHNIQUE IS STILL A STAPLE OF RENAL PHYSIOLOGY EVEN TODAY, WE JUST GOT BACK FROM THE AMERICAN SOCIETY OF NEPHROLOGY MEETINGS AND PEOPLE ARE STILL USING THE TECHNIQUE AND MOVING FORWARD ON PHYSIOLOGICAL KNOWLEDGE. BASICALLY THE INFORMATION THAT DERIVED FROM THIS PARTICULAR TECHNIQUE HAS REWRITTEN KIDNEY PHYSIOLOGY TEXTBOOK. BEGAN THE WORK OF IDENTIFYING TRANSPORTER PROTEINS RESPONSIBLE FOR THE FORMATION OF URINE AND FOR REGULATION OF BODY COMPOSITION AS WELL AS BLOOD PRESSURE. LATER IN HIS CAREER, MO TURNED HIS ATTENTION TO OSMOTIC REGULATION. IDENTIFYING THE MECHANISMS BY WHICH CELLS OF THE KIDNEY ARE PROTECTED FROM DAMAGE BY THE EXTREMELY HIGH CONCENTRATIONS OF SALT AND UREA THAT EXIST IN THE RENAL INNER MEDULLA, ACCUMULATE AS PART OF THE CONCENTRATING MECHANISM. SO THIS WAS A COMPLETELY INDEPENDENT DIRECTION MOE TOOK SORT OF MID CAREER, WHICH IN ITSELF WOULD BE A CAREER WORTH OF RESEARCH. SO BEFORE WE INTRODUCE THIS YEAR'S BURG LECTURER RICK LIFTON, I WANT TO ACKNOWLEDGE MOE WHO IS SITTING IN THE FRONT ROW HERE WITH HIS WIFE JUDGE RUTH BURG. NOW I INVITE TO STAGE NHLBI DIRECTOR GARY GIBBONS WHO WILL INTRODUCE RICK LIFTON. >> THANK YOU, MARK. IT'S REALLY AN HONOR TO BE HERE TO PROVIDE THIS INTRODUCTION. WELCOME ALL OF YOU TO THE BURG LECTURE. RICK IS SOMEONE I HAVE KNOWN SINCE I WAS AN INTERN. THOSE WHO HAVE EXPERIENCED KNEE RATTLING SHAKING EXPERIENCE OF HAVING RESPONSIBILITY FOR TAKING CARE OF A HUMAN BEING AND FEELING TOTALLY INADEQUATE TO DO SO, TEND TO REVERE THOSE SENIOR RESIDENTS WHO ARE ABLE TO COME ALONG AND MAKE YOU FEEL A LITTLE MORE SECURE AND PROTECT YOU. RICK LIFTON WAS THAT FOR ME. BEING -- BECOMING MY CHIEF RESIDENT AS WELL AT THE BRIGHAM. SO I HAVE KNOWN DR. LIFTON FOR A NUMBER OF YEARS AND HE ALWAYS IMPRESSED ME THEN AS THE GUY WHO KNEW EVERYTHING. WHEN I WAS AN INTERN, I LATER FOUND OUT AS I HAVE TRACKED HIS CAREER HE DOES KNOW ALMOST EVERYTHING THERE IS TO KNOW, CERTAINLY ABOUT RENAL PHYSIOLOGY AND HYPERTENSION AS HIS CAREER HAS CLEARLY DEMONSTRATED. IT'S FOR THAT REASON HE'S A PARTICULARLY APPROPRIATE CHOICE FOR THE MOE BURG LECTURE. RICK HAS BEEN REAL TRAILBLAZER IN EXTENDING A LOT OF THE TRADITIONS THAT DR. BURG DID IN TERMS OF UNDERSTANDING TUBULE FUNCTION. AND FOR THOSE WHO GREW UP AS PHYSIOLOGISTS AND UNDERSTANDING OF SALT WATER METABOLISM AND CONTROL OF BLOOD PRESSURE, RICK WAS ABLE TO BRING A LOT OF POWER OF MOLECULAR GENETICS AND HUMAN MOLECULAR GENETICS TO THE FLOOR TO START TO DISSECT OUT MOLECULAR BASIS OF HYPERTENSION AND HOW THEY RELATE BACK TO ION CASH BALANCE AND MOVEMENT IN THE KIDNEY IN WAYS OF ELUCIDATING BLOOD PRESSURE REGULATION WITH IDENTIFICATION OF FAMILIAL MONOGENETIC FORMS OF HYPERTENSION. RELATED TO GORDON OR GILLMAN SYNDROME, INSIGHT THE BARTERS, ET CETERA, COULD GO ON AND ON AS HE TREKKED ALONG THE VARIOUS TUBULES, FINDING MOLECULAR BASIS. 'S THIS WORK OVER 200 PAPERS WON HONORS THROUGHOUT HIS CAREER AND RECOGNITION OF HIS TRAIL BLAZING WORK IN THE MOLECULAR GENETICS OF HYPERTENSION. IN PARTICULAR HE'S OBVIOUSLY BEEN RECEIVED HONORS OVER THE YEARS, NOTABLE FOR THOSE HE SHARES AS DR. BURG BEING ELECTED TO NATIONAL ACADEMY OF SCIENCES AS WELL AS SHARING WITH DR. BURG THE CASE SMITH AWARD IN RECOGNITION TO CONTRIBUTIONS TO OUR UNDERSTANDING OF THE KIDNEY, WITHOUT FURTHER ADIEU IT'S MY PLEASURE TO INTRODUCE RICK LIFTON PRESIDENT OF ROCKEFELLAR UNIVERSITY AND HEAD OF THE GENETICS HUMAN GENETICS AND GENOMICS LABORATORY AT ROCKEFELLAR. RICK. [APPLAUSE] THANKS VERY MUCH, GARY, FOR THAT GENEROUS INTRODUCTION, IT'S A REAL HONOR TO BE HERE TO GIVE THE MOE BURG LECTURE SUFFICE TO SAY WOULD NOT HAVE BEEN HERE TODAY IF NOT FOR MOE AND PROBABLY NOT FOR GARY ALSO, GARY IS UNDERSTATED WAY REALLY MINIMIZES WHAT BRILLIANT DOCTOR HE WAS WHEN HE SHOWED UP AT THE BRIGHAM AND HE MADE ALL THE REST OF OUR JOBS REALLY EASY. AS A CONSEQUENCE, IT'S WONDERFUL TO SEE HOW HE HAS DONE IN LEADING NHLBI AND THE NATION OBVIOUSLY RELIES ON WISDOM OF WHAT'S HAPPENING HERE AT NIH. SO I'M DELIGHTED TO TO BE HERE TODAY, BOTH TO GIVE LECTURE AND A SHORT REPRIEVE FROM MY DUTIES OTHERWISE ROCKEFELLAR ON ADMINISTRATIVE NOTE. BUT WHAT I -- MY DISCLOSURES, I'M DELIGHTED TO BE HERE TODAY TO HAVE A CHANCE TO TALK A LITTLE BIT ABOUT BLOOD PRESSURE AND FUTURES IN HUMAN MOLECULAR GENETICS AND DISEASE BIOLOGY. AS OUR GOALS IN MEDICINE WE WANT TO UNDERSTAND NORMAL HEALTHY AND DISEASE BIOLOGY WITH THE EXPECTATION THAT WE DO OUR BEST WHEN WE UNDERSTAND THE ROOT CAUSES OF DISEASE AND HOPE IT WILL ENABLE US TO PREVENT OR MITIGATE THE EFFECTS OF DISEASES BY DEVELOPING NEW APPROACHES TO PREVENTION AND TREATMENT. GENETICS TURNED OUT TO BE EXTREMELY USEFUL TECHNOLOGY FOR UNDERSTANDING DISEASES THAT HAVE NOT BECOME UNDERSTOOD BY MORE TRADITIONAL ROOTS TO UNDERSTANDING USING EPIDEMIOLOGY CELL BIOLOGY BIOCHEMISTRY AND OTHER TOOLS. I WANT TO MAKE THE POINT AT THE OUTSET WE IN A SHORT PERIOD OF TIME HAVE BEEN THROUGH THREE DISTINCT ERAS IN USE OF GENETICS AND GENOMEICS EACH DRIVEN BY SALTTORY CHANGES IN TECHNOLOGY WHERE WE HAD A NEW CAPABILITY THAT OPENED DOORS AND APPROACHES SO FIRST OF THESE WAS DEVELOPMENT OF DENSE GENETIC MAPS OF THE HUMAN GENOME THAT ENABLED US TO FOLLOW INHERITANCE OF PARTICULAR SINGLE GENE DISORDERS THROUGH FAMILIES, THERE BY MAP THE CHROMOSOMAL LOCATIONS OF THESE UNDERLYING DISEASE GENES AND BY POSITIONAL CLONING YOU IDENTIFY THE UNDERLYING MUTATIONS. THESE IDENTIFY RARE MUTATIONS WITH LARGE AFFECT THAT ARE TYPICALLY UNCOMMON IN THE POPULATION BUT CAN BE KEY TO IDENTIFYING PATHWAYS THAT UNDERLIE HUMAN DISEASE. SECOND ERA WAS USHERED IN THE LAST DECADE, WHEN WE SUDDENLY HAD THE ABILITY TO GENOTYPE HUNDREDS OF THOUSANDS TO NOW MILLIONS OF COMMON VARIANT ACROSS THE GENOME AND LOOK IN LARGE COHORTS OF INDIVIDUALS TO SEE WHICH PARTICULAR VARIANT MOST ASSOCIATED WITH PARTICULAR DISORDERS. THIS IDENTIFIED MANY VARIANT FOR VIRTUALLY EVERY COMMON DISEASE AND THESE DIFFER QUALITATIVELY WHAT WE LEARN FROM THESE RARE MENDELIAN FORMS. THIS GENOME WIDE ASSOCIATION STUDY APPROACH TYPICALLY IDENTIFIES VARIANT THAT HAVE SMALL EFFECTS ON DISEASE RISK BUT CUMULATIVELY MAY ENABLE AS WE MOVE FORWARD TO IDENTIFY PEOPLE WHO HAVE PARTICULAR RISK OF DISEASE. THAT ARE GREATER THAN THE GENERAL POPULATION. AT THE END OF LAST DECADE CONTINUING THE LAST SEVERAL YEARS WE HAVE A SALTTORY CHANGE ENABLING US TO RAPIDLY INEXPENSIVELY SEQUENCE ALL THE GENES IN THE GENOME OR COMPLETE GENOMES AND THIS AGAIN HAS LED TO A NEW APPROACH CRABBING THE FAMILY BASED APPROACHES AND SIMPLY SEQUENCING LARGE NUMBERS OF INDIVIDUALS LOOKING FOR GENES THAT ARE MUTATED MORE OFTEN THAN EXPECTED BY CHANCE. THIS WILL BE THE DEFINITIVE APPROACH, THE UNDERSTANDING THE ROLE OF GENETIC VARIATION IN HUMAN DISEASE. THE IMPORTANCE OF THESE RARE MUTATIONS WITH LARGE EFFECT IS THEY ESTABLISH UNEQUIVOCALLY CAUSAL RELATIONSHIP BETWEEN A MUTATION, A GENOTYPE AN HUMAN TRAIT, THESE PROVIDE STRONG ANCHORS YOU HAVE A GENOTYPE CAUSALLY RELATED TO A PHENOTYPE, AND ALLOWS YOU TO THEN PROCEED WITH GREAT CONFIDENCE, IF YOU UNDERSTAND THE LINK BETWEEN THOSE TWO YOU WILL UNDERSTAND THE BIOLOGY THAT IS PRODUCING THESE HUMAN DISORDERS. THAT'S VERY POWERFUL, IN PARTICULAR FOR ITS ABILITY TO IDENTIFY GENES AND PATHWAYS THAT MIGHT BE MANIPULATED FOR HEALTH BENEFIT. NOT NECESSARILY JUST IN THOSE PATIENTS WITH RARE DISEASE BUT FOR OTHERS WHO MAY SUFFER A MORE COMMON FORM OF THE SAME DISORDER. IMPORTANTLY THESE RARE MUTATIONS WITH LARGE EFFECT INDICATE HOW BIG AN EFFECT YOU MIGHT HAVE BY MANIPULATING THAT PATHWAY. AND ALSO WHAT ADVERSE EFFECTS YOU MIGHT GET FROM AS A CONSEQUENCE OF MANIPULATING THAT PATHWAY. BACK IN 2009 AS I MENTION SEQUENCING BECAME MUCH LESS EXPENSIVE OVER A SHORT PERIOD OF TIME, AND THIS HAS DRIVEN A STRATEGY THAT WE DEVELOPED IN MY LABORATORY AT THAT TIME. TO RECOGNIZING AT THE TIME THAT IT WAS STILL VERY EXPENSIVE TO SEQUENCE WHOLE GENOMES WE CAN SEQUENCE ALL 20,000 GENES IN HUMAN GENOME WHICH ONLY COME PRIDES 1% OF THE GENOME VERY INEXPENSIVELY BY HYBRID SELECTION CAPTURE AND SEQUENCING ON NEXT GENERATION SEQUENCING PLATFORMS. THIS WAS WORKED OUT BY MERV CHOY AND JOHN OVERTON IN THE LABORATORY AND WE CAN SEQUENCE THE GENES IN THE GENOME AN ANALYZE THEM FOR $200. WHICH IS PRETTY INEXPENSIVE FOR DISCOVERY PLATFORM. WE MADE THEM THE FIRST APPLICATION OF THIS TECHNOLOGY AT THAT TIME TO MAKE A DIAGNOSIS, UNEXPECTED DIAGNOSIS IN A CHILD WHO WAS THOUGHT TO HAVE A KIDNEY DISORDER, THAT RELATED TO SOME DISEASE GENES WE PREVIOUSLY IDENTIFIED. BUT SEQUENCING HAD NOT REVEALED ANY MUTATIONS IN THOSE GENES, SEQUENCING ALL THE GENES IN THE GENOME DEMONSTRATED HE HAD A MUTATION IN A DIFFERENT PATHWAY THAN ONE THAT HAD BEEN PREVIOUSLY RECOGNIZED IN THESE PATIENTS. AND LED TO THE FIRST CLINICAL DIAGNOSIS BY USE OF EXOME SEQUENCING. OVER THE LAST SIX YEARS NHLBI ALONG WITH NHGRI SUPPORTED AN EFFORT TO IDENTIFY AND EXPAND RARE MUTATIONS WITH LARGE EFFECT THAT CONTRIBUTE TO HUMAN PHENOTYPES. THERE ARE CURRENTLY FOUR GROUPS AROUND THE COUNTRY INVOLVED IN THIS EFFORT. I HAVE LED THE YALE EFFORT AND SINCE MY MOVE TO ROCKEFELLAR CONTINUE TO BE PI ON THAT PROGRAM, OVER THE SIX YEAR PERIOD 1250 NEW GENES NOT PREVIOUSLY IMPLICATED IN HUMAN DISEASE HAVE BEEN IDENTIFIED, ANOTHER 400 SHOWN TO HAVE MUTATIONS THAT GIVE A COMPLETELY DIFFERENT PHENOTYPE THAN WHAT WAS PREVIOUSLY RECOGNIZED AS A DISEASE RELATED GENE. AND IMPORTANTLY WE'RE FINDING ABOUT ONE NEW GENE FOR EVERY 30 EXOMES WE SEQUENCE. BY TYPICALLY SELECTING EXTREME PHENOTYPES WITH LARGE -- WHERE WE SUSPECT THERE MIGHT BE GENETIC CONTRIBUTION. I WILL GIVE A COUPLE OF EXAMPLES QUICKLY, TO THIS POINT. SO ONE OF THESE IS AN EXAMPLE, WITH CHRISTINE GARCIA AT UT SOUTH WESTERN WE SEQUENCED A HUNDRED PATIENTS WITH UNDIAGNOSED FORMS OF PULMONARY FIBROSIS AND SHOWED 8% OF CASES HAD THEIR DISEASE ATTRIBUTABLE TO MUTATIONS IN TWO GENES, THAT WERE IMPLICATED IN TELOMERE MAINTENANCE THAT HAD NOT BEEN PREVIOUSLY IMPLICATED IN PULMONARY FIBROSIS. AND DEMONSTRATED THESE PATIENTS HAVE SHORT TELOMERES AND THEY ALSO HAVE INCOMPLETE PENETRANTS, NOT EVERYBODY WHO GETS THESE MUTATIONS GET DISEASE. THE REASON IS ENVIRONMENTAL INTERACTION REQUIRED FOR THE PHENOTYPIC EXPRESSION. SO IN THESE CASES, MUTATION CARRIERS WHO DEVELOPED DISEASE WERE SMOKERS, CHICKEN FARMERS, BIRD BREEDERS, WELDERS, MASONS, MILLERS FARMERS, CATTLE FARMERS, OTHER CONSTRUCTION WORKERS, INDIVIDUALS INHERITED THE MUTATIONS WHO REMAIN FREE OF DISEASE WORKED IN THE OFFICE OR OTHERWISE DID NOT HAVE ENVIRONMENTAL EXPOSURE LIKE THIS. A GOOD EXAMPLE OF GENE BY ENVIRONMENT INTERACTION WHICH EXPLAINS WHY WE DIDN'T PREVIOUSLY RECOGNIZE THESE FAMILIES, A SIMPLE SEGREGATING SIMPLE MENDELIAN TRAITS. WE HAVE ALSO IN MANY CASES GONE AFTER DISORDERS WHERE WE SUSPECTED GENETIC DISTRIBUTIONS FOR A LONG TIME BUT DIDN'T HAVE TOOLS TO FIND THEM. FOR EXAMPLE, NHLBI IS FUNDED THE PEDIATRIC CARDIAC GENOMICS CONSORTIUM AND WE HAVE DONE THE SEQUENCING AND ANALYSIS FOR THIS PROJECT. THIS IS SAMIR, M.D. Ph.D. STUDENT AND PETER CHEN WHO IS A POST-DOCTORAL FELLOW IN THE LABORATORY, WE DID SEQUENCING PATIENTS WITH SEVERE CONGENITAL HEART DISEASE, WHICH COME PRIDES 1% OF LIVE BIRTHS AND SEVERERY AFFECTED CHILDREN REQUIRE -- SEVERELY AFFECTED CHILDREN REQUIRE SURGERY FIRST YEAR OF LIFE TO SURVIVE. TO DATE WE HAVE PUBLISHED ON THE SEQUENCING OF MORE THAN 2500 PARENT OFFSPRING TRIOS, HEALTHY -- HELPING PARENTS WITH SEVERELY AFFECTED OFFSPRING SUSPECTING A UNDERLYING DEFECT THAT CONTRIBUTES TO THE DISEASE. TURNS OUT 10% OF PATIENTS WILL HAVE DE NOVO MUTATIONS THAT CONTRIBUTE TO DISEASE, ANOTHER FIVE TO SEVEN PERCENT WILL HAVE POINT MUTATIONS THAT CONTRIBUTE TO DISEASE. THERE ARE SOME PATIENT -- SOME GENE FAMILIES THAT JUST JUMP OFF THE PAGE AT YOU WHEN YOU LOOK AT THE BURDEN OF MUTATIONS IN THESE PATHWAYS. SO ONE OF THESE IS CHROMATIN MODIFICATION. DNA IN THE NUCLEUS IS WRAPPED AROUND THE NUCLEOOWESOME, TURNS OUT THE TAILS OF THESE HISTOTONE PROTEINS CAN BE MODIFIED TO REGULATE GENE EXPRESSION. MIKE BRANSTEEN AND DAVID ALICE RECEIVED THE LASKER AWARD FOR THE CHROMATIN MODIFICATION TO THE REGULATION OF GENE EXPRESSION JUST THIS YEAR. TURNS OUT THE MOST FREQUENTLY MUTATED PATHWAY IN CONGENITAL HEART DISEASE ARE MUTATIONS THAT CHANGE MODIFICATION OF THESE HISTONE PROTEINS. MOST INTERESTINGLY THESE ARE HAPLO INSUFFICIENT MUTATIONS, YOU NEED ONLY ONE COPY OF THE GENE IN ORDER TO GET THE PHENOTYPE. YOU HAVE MUTATIONS THAT MODIFY METHYLATION OF HISTONE 3 LYSINE 4, HISTOTONE 3 LYSINE 27, SO FORTH. WHEN YOU STUDY YOU RECOGNIZE PREVIOUS RELATIONSHIPS WITH DISEASES. IN PARALLEL THERE'S LARGE CONSORTIUM SEQUENCING PATIENTS WITH AUTISM, ONE OF THE TWO MOST FREQUENTLY MUTATED PATHWAYS IN AUTISM IS NOT JUST THE SAME PATHWAY BUT IDENTICAL GENES IN CONGENITAL HEART DISEASE AND AUTISM. THIS TURNS OUT TO BE FAR MORE FREQUENT THAN EXPECTED BY CHANCE. WE THINK PLAYS A MAJOR ROLE IN EXPLAINING WHY SO MANY CHILDREN WITH CONGENITAL HEART DISEASE GET THEIR HEARTS CORRECTED BUT GO ON TO HAVE NEURAL DEVELOPMENTAL ABNORMALITIES AND PROVIDES INTERESTING OPPORTUNITIES FOR EARLY INTERVENTION, PATIENTS WITH AUTISM TYPICALLY APPEAR DIAGNOSED IN THEIR SECOND AND THIRD YEAR OF LIFE. PATIENTS WITH CONGENITAL HEART DISEASE, SEVERE FORM ARE DIAGNOSED AT BIRTH SO WE CAN IDENTIFY THESE PATIENTS EARLY ON AND TEST THE HYPOTHESIS THAT WE MIGHT BE ABLE TO INTERVENE IN MEANINGFUL WAYS TO PREVENT NEURO-DEVELOPMENTAL ABNORMALITIES FROM ULTIMATELY DEVELOPING. WITH THAT GENERAL COMMENT ABOUT IMPORTANT ROLES OF PROGRAMS HEART LUNG AND BLOOD IS SUPPORTING IN GENETICS AND GENOMICS, I'LL TURN TO OUR WORK ON HYPERTENSION MENT SO HYPERTENSION AFFECTS MORE THAN A BILLION PEOPLE WORLDWIDE AND BY THE WHO IS THE LEADING CAUSE OF GLOBAL DEATH. CONTRIBUTING TO MORE THAN NOW MORE THAN 13 MILLION DEATHS PER YEAR, IT'S PATHOGENESIS HAS BEEN HOTLY DEBATED. AND AS MENTIONED, ARTHUR GYTON DEFINED A WORKING MODEL FOR THE REGULATION OF BLOOD PRESSURE NOW ABOUT 45 ALMOST 50 YEARS AGO. AND THIS IS HIS WORKING MODEL FOR THE WORKING REGULATION OF BLOOD PRESSURE. FROM THE BACK ROW RECOGNIZE THAT THIS IS A PRETTY COMPLEX PATHWAY. INVOLVES A LOT OF COMPONENTS AND IS QUITE COMPLEX. 'S BEEN DEBATED WHETHER HYPERTENSION IS PRIMARY DISEASE OF BRAIN MATTER KIDNEY ADRENAL GLAND OR VASCULATURE, THIS LOOKS AS YOUNG ASSISTANT PROFESSOR STARTING OUT LIKE A PERFECT PLACE TO ASK A SIMPLE GENETIC QUESTION, ARE THERE SINGLE PLACES IN THE PATHWAY WHERE YOU MIGHT INTRODUCE A SINGLE LESION THAT WOULD GROSSLY DISRUPT THE BEHAVIOR OF THE ENTIRE SYSTEM? IF YOU CAN DO THIS REPEATEDLY, YOU MIGHT BE ABLE TO IDENTIFY BUNCH OF GENES AND FIGURE PATHWAYS THEY MIGHT LIE IN TO ENABLE YOU TO UNDERSTAND WHAT SYSTEMS ARE TONICALLY REGULATING LONG TERM REGULATION OF BLOOD PRESSURE IN HUMANS. WE SCOURED THE PLANET LOOKING FOR RARE FAMILIES THAT ARE SEGREGATING AT THE HIGH END OF THE DISTRIBUTION AND ALSO AT THE LOW END OF THE DISTRIBUTION. AND WENT AROUND THE WORLD LOOKING FOR PEOPLE WITH HIGHEST AN LOWEST BLOOD PRESSURES COMPATIBLE WITH SURVIVAL. THEN FIGURE OUT WHICH OF THESE WERE SEGREGATING GENES IN A WAY THAT SUGGESTED MENDELIAN TRANSMISSION, AND THEN USED THE THEN EMERGING TOOLS FROM THE GENOME PROJECT TO IDENTIFY UNDERLYING MUTATION. THIS IS A RECENT UP TO DATE LIST OF GENES IDENTIFY T WHERE THERE ARE RARE MUTATIONS WITH LARGE EFFECT ON BLOOD PRESSURE, 18 GENES THAT CAUSE SEVERE FORMS OF HIGH BLOOD PRESSURE AND THERE ARE 11 THAT CAUSE SEVERE FORMS OF LOW BLOOD PRESSURE. THIS COULD BE A RANDOM LIST OF GENES AND WE MIGHT NOT GAIN ANY INSIGHT FROM IT WITH THE EXCEPTION OF THE WORK OF MOE BURG. WHAT HE DID AS DESCRIBED BY MARK MEPPER IS ENABLED -- DEVELOPED THIS NOVEL TECHNOLOGY USING THE ISOLATED PROFUSED RENAL TUBULE THAT ENABLED US TO UNDERSTAND WHAT SPECIFIC PHYSIOLOGIC FUNCTIONS IN ION TRANSPORT AND PHYSIOLOGIC MEDIATORS WERE BEING MEDIATED BY EACH SEGMENT OF THE NEFRON. FROM THAT WE DEVELOPED A RELATIVELY SOPHISTICATED MODEL SHOWN IN VERY CRUDE SUMMARY HERE, OF HOW SALT AND WATER IS BEING ABSORBED ALONG THE NEPHRON. THIS IS ONE OUR MILLION NEPHRONS, WE HAVE SODIUM HYDROGEN EXCHANGE IN IN THE PROXIMAL TUBULE, SODIUM POTASSIUM CO-A TRANSPORT IN SODIUM CHLORIDE CO-TRANSPORT IN THE CONVOLUTED TUBULE AND THE ONLY ELECTROGENIC STEP IS DISTAL IN TESTIFY RON, ELECTROGENIC SODIUM REABSORPTION ACCOMPANIED BY POTASSIUM AND HYDROGEN SECRETION OR BY CHLORIDE REABSORPTION. THIS SHOWS THE ROUGH FRACTION OF THE FILTERED SODIUM LOAD THAT IS REABSORBED BY EACH OF THESE PATHWAYS ON A DAILY BASIS. NUMBER OF THAT TELLS US -- NONE TELLS US HOW IMPORTANT ANY OF THESE MIGHT BE COMPARED TO ONE ANOTHER IN THE SETTING IN WHICH ONE OF THESE IS PERTURBED. THIS PATHWAY IS KNOWN TO BE REGULATED BY THE ANGIOTENSIN SYSTEM, IN RESPONSE TO VOLUME DEPLETION, THIS PROTEASE REANYONE CLEAVES ANGIOTENSINGEN TO FORM AFFECT ANGIOTENSIN ONE AND TWO WHICH BINDS TO RECEPTOR IN ADRENAL GLOMERULUS TO STEROID AND IT BINDS TO RECEPTORS IN THE KIDNEY TO PROMOTE INCREASED REABSORPTION OF SODIUM AND CHLORIDE. SO IMPORTANTLY GENES WE IDENTIFIED TURN OUT TO FALL INTO THIS PATHWAY. SO FIRST GENES IDENTIFIED IS ONE IN WHICH ALDOSTERON,E THIS OR KNOWN THAT TELLS IT THE HANG ON TO SALT AND WART IS CONSTITUTIVELY MADE RATHER THAN REGULATE IN NORMAL FASHION. THE REASON FOR THAT IS MUTANT GENE IS ACTUALLY A GENE DUPLICATION THAT FUSES REGULATORY SEQUENCES FROM ONE GENE TO THE CODING SEQUENCES FROM ANOTHER WITH THE CONSEQUENCE THIS CHIMERIC GENE IS BEING EXPRESSED IN THE WRONG PART OF THE ADRENAL GLAND AT THE EXPENSE OF MAINTAINING NORMAL CORTISOL SECRETION THESE PATIENTS ARE ALWAYS MAKING ELDOSTERONE TELLING THE KIDNEY TO HANG ON TO SALT AND WATER, CARDIAC OUTPUT INCREASES AND BLOOD PRESSURE RISES BYOME'S LAW. THAT TURNS OFF THE ANGIOTEN SIN SYSTEM BUT FAILS TO SUPPRESS THE SECRETION AND THESE PATIENTS DEVELOP RIP ROARING HYPERTENSION. DOWNSTREAM FROM THAT IS IT RECEPTOR AND DAVID, A RENAL FELLOW IN THE LABORATORY IDENTIFIED A POINT MUTATION IN THE LIGAND BINDING DOMAIN OF THE MINERALA CORTICOID RECEPTOR THAT CAUSED CONSTITUTIVE ACTIVITY OF THE MINERALA CORTICOID RECEPTOR, ACTIVATING THIS PATHWAY, PROMOTING INCREASED SALT AND WATER REABSORPTION. THE TARGET FOR ELDOSTERONE THE EPITHELIAL SODIUM CASH BALANCE DEMONSTRATED IS MUTATED IN A DISEASE CALLED LITTLE SYNDROME, FEATURING EARLY ONSET OF SEVERE HYPERTENSION, THE MUTATIONS THAT CAUSE THIS TRAIT MODIFY THE CARBOXYTERMINUS OF THE EPITHELIAL SODIUM CHAMISO IT'S NOT ENDOCYTOSIS BY PITS SO THEY REMAIN IN SODIUM SURFACE ACTIVE IN PROMOTING INCREASED REABSORPTION OF SODIUM AND CHLORIDE, ULTIMATELY RAISING BLOOD PRESSURE. ON THE OTHER SIDE OF THE COIN, MUTATIONS AT THE EXTREME LOW END OF THE BLOOD PRESSURE DISTRIBUTION REDUCE SALT REABSORPTION BY THE KIDNEY. FOR EXAMPLE, PATIENTS WHO ARE HOMOZYGOUS NULL FOR THE SAME EPITHELIAL SODIUM CHANNEL WHICH GAIN OF FUNCTION MUTATIONS CAUSE HIGH BLOOD PRESSURE, THIS LOSS OF THIS CHANNEL, CAUSES PROFOUND SALT WAISTING WHICH ACTIVATES THE ANGIOTIEN-TSIN SYSTEM BUT DOES NOT SUFFICE TO PROMOTE SALT REABSORPTION BECAUSE THE NORMAL TARGET IS MISSING SO THESE CHILDREN MISSING 2% YOU MIGHT THINK THAT'S NOT A BIG DEAL, TURNS OUR THESE CHILDREN REQUIRE 25 TO 30 GRAMS OF SALT A DAY IN ORDER TO SIT UP AND NOT HAVE THEIR BLOOD PRESSURE BOTTOM OUT. THIS TELLS US THIS 2% CRITICAL, THE RATE LIMITING FINAL STEP IN REGULATION OF BLOOD PRESSURE AND THESE PATIENTS ARE SEVERELY IMPAIRED IF THEY DON'T HAVE SODIUM CHANNEL. IF THEY'RE HETERO HETEROZYGOUS, THE HOMOZYGOTES ARE GENERALLY LETHAL AT BIRTH UPTREATMENT WE HAVE SODIUM CHLORIDE CO-TRANSPORTER, MOST COMMONLY USED ANTI-HYPERTENSIVE MEDICATION, THIGH SIDE DIURETICS. IN THIS CASE YOU CAN COMPENSATE FOR THIS DEFECT BY ACTIVATION OF THE ANGIOTENSIN SYSTEM AN IT PROMOTES INCREASED SOLD YUM REABSORPTION AT THE EXPENSE OF LOSS OF POTASSIUM IN HYDROGEN RESULTING IN KYPERKALEMIA AND METABOLIC ALKALOSIS. THIS IS A MORE MILD DISORDER THAN THE PATIENTS MISSING EPITHEMEIAL SODIUM CHANNEL, THE INTEGRATED PHYSIOLOGY BECOMES CLEAR ONCE YOU HAVE THE HUMAN KNOCK OUT. IN THIS CASE OF HEPPEDLY THERE ARE MUTATIONS IN THE ENTRY STEP FOR SODIUM AND CHLORIDE AND THE EXIT STEP FOR CHLORIDE AND IN POTASSIUM CHANNEL REQUIRED FOR RECYCLING OF THE POTASSIUM THAT COMES IN BACK INTO THE LUMEN, IN ORDER TO DRIVE THE TOTAL PROCESS AND MUTATION IN ANY OF THESE GENES CAUSE A DISEASE CALLED BARTER SYNDROME BY FRED BARTER WHO IS HERE AT NIH AND DESCRIBED THESE DISORDERS HERE. THIS IS FIONA CARROT, PROFESSOR AT UNIVERSITY OF CAMBRIDGE AND KAREN FINBERG FACULTY MEMBER AT YALE. IN SUMMARY THESE GENES CONVERGE ON THIS FINAL COMMON PATHWAY THAT REGULATE HOW KIDNEY HANDLES SALT. DIVERSE EFFECTS ON POTASSIUM, HYDROGEN ION CALCIUM BUT IF U YOU KNOW THE VECTOR FOR SODIUM AND CHLORIDE YOU KNOW WHAT'S HAPPENING TO BLOOD PRESSURE IN THESE PATIENTS. IMPORTANTLY FIVE OF THESE GENES GAIN OF FUNCTION MUTATION DRIVES BLOOD PRESSURE TO HIGH END OF DISTRIBUTION, LOSS OF FUNCTION MUTATION IN SAME GENES DRIVES BLOOD PRESSURE LOW END OF DISTRIBUTION AND AS A CONSEQUENCE YOU CAN DRAW BLOOD PRESSURE ACROSS THE ENTIRE SPECTRUM IN THE HUMAN POPULATION BY MODULATION OF JUST THESE SINGLE GENES. TELLING YOU THE IMPORTANCE OF THIS PATHWAY IN THE REGULATION OF BLOOD PRESSURE IN HUMANS. YOU CAN ALSO DO AN INTERESTING CLINICAL INVESTIGATION ONCE YOU KNOW THESE GENES. SO I MENTION FIONA, THIS IS DINA CRUISE, NOW ON FACULTY AT UNIVERSITY OF CALIFORNIA SAN DIEGO. THEY STUDIED A MULTI-GENERATIONAL SYNDROME WITH GILLMAN SYNDROME, THESE ARE PATIENT WHOSE ARE MISSING THE THIGH SIDE SENSITIVE SEW YUM CHLORIDE CO-TRANSPORTER, THEY CHARACTERIZE THE 200 PATIENTS IN SINGLE KINDRED AND FOUND INDIVIDUALS CARRYING 0, 1, 2 MUTANT COPY OF THE GENE AND ASKED A SIMPLE QUESTION, HOW MUCH SALT DO THESE PATIENTS EAT ON A DAILY BASIS AND THEY MEASURED THIS BY MEASURING THE 24 HOUR URINARY SODIUM EXCRETION WHICH IS A GOOD PROXY FOR WHAT THEY ARE TAKING IN AT STEADY STATE. COMPARED TO THE WILE TYPE INDIVIDUALS WHO HAD NO MUTANT COPIES OF THE GENE, THEY FOUND THE INDIVIDUALS WHO TWO MUTANT COPIES OF THE GENE ARE EATING MORE SALT, HETEROZYGOTES HAD ONE MUTANT COPY OF THE THE GENE. THIS IS ABOUT THIS IN CONTEXT OF PATIENT AND CLINIC WITH HYPERTENSION WHICH GIVE DIURETIC AND SAY BY THE WAY DON'T EAT TOO MUCH SALT WHILE ON THIS DRUG. THESE PATIENTS NOBODY IS TELLING THEM WHAT TO EAT. THEY'RE SELF-SELECTING A HIGH SALT DIET BECAUSE THEIR BODY IS TELLING THEM THEIR VOLUME DEPLETED DUE TO THEIR SALT WASTING AS A CONSEQUENCE COMPENSATING FOR THAT BY EATING MORE SALT. THIS IS EXACTLY WHAT'S HAPPENED TO PATIENTS WITH DIURETIC TO. THE ANGIOTENSIN SYSTEM IS ACTIVATED AND ANGIOGENERAL SIN 2 IS ACTING THROUGH NUCLEI IN THE BRAIN THAT LIE OUTSIDE THE BLOOD BRAIN BARRIER THAT PROVIDE THE SIGNAL TO SAY GO EAT SALT, TO TRY TO STAY IN SALT BALANCE. THESE PATIENTS SELF-SELECT AD HIGH SALT DIET AS WE SUSPECT MANY PATIENTS TAKING SINGLE AGE DIURETIC AS CONSEQUENCE DIURETICS IN COMBINATION WITH DRUGS THAT BLUNT THE ANGIOTENSIN SYSTEM TURN OUT TO BE QUITE POE AT THE PRESENT TIME AND RATIONALE COMBINATION THERAPY FOR HIGH BLOOD PRESSURE. WE CAN ALSO LACK FOR EFFECTIVE HETEROZYGOTES ON BLOOD PRESSURE IN POPULATIONS LIKE THE FRAMINGHAM POPULATION. LONG TERM FUNDED BY NHLBI. WE SEQUENCED 4,000 MEMBERS OF THE COHORT FOR THESE THREE GENES IN WHICH HOMOZYGOUS LOSS OF FUNCTION RESULTS IN SEVERE FORMS OF LOW BLOOD PRESSURE AND ASK WHAT HAPPENS IF YOU'RE JUST CARRYING ONE MUTANT COPY OF THE GENE. LONGITUDINAL STUDY WE WOUND INDIVIDUALS AT -- IN EVERY AGE GROUP HAD 10-MILLIMETER LOW BLOOD PRESSURE IF THEY CARRIED ONE MUTANT COPY OF THE GENE THEN INDIVIDUALS IN THE POPULATION WHO DID NOT CARRY A MUTANT COPY OF THE GENE. THIS WAS SUFFICIENT TO PROTECT AGAINST DEVELOPMENT OF HYPERTENSION, ALSO HAD AN OUTSIDE SIZED EFFECT ON RISK CARDIOVASCULAR DISEASE AND STROKE. SO AT THE TIME OF THE STUDY, THAT'S NOT A SINGLE CARDIOVASCULAR DE AND NOT SINGLE STROKE AMONG MUTATION CARRIERS WHICH IS QUITE DIFFERENT FROM THE DISTRIBUTION IN THE REST OF THE FRAMINGHAM POPULATION. THIS ULTIMATELY LED AND CONTRIBUTED TO ESTIMATES OF THE IMPACT ON PREVENTION OF SIMPLY REDUCING DIETARY SALT INTAKE BY 25%. CHRISTINE DOMINGO AND LEE GOLDMAN MODELED THIS ON U.S. POPULATION ESTIMATED THAT 25% SMALL DECREASE IN DIETARY SALT INTAKE, WOULD REDUCE NUMBER OF STROKES ANNUALLY IN THE U.S. BY ABOUT 50,000. HEART ATTACKS BY 75,000, ALL CAUSE DEATHS BY 75,000 AND REDUCE HEALTHCARE COSTS BY ABOUT $15 MILLION, THIS WAS BACK IN 2010. SO STARTING NOW WITH A BLANK SLATE ON WHERE WE FIND CAUSAL MUTATIONS THAT AFFECT BLOOD PRESSURE IS WORTH REFLECTING ON WHAT THE WORLD WAS THINKING AT THE TIME, THE SALT INSTITUTE LONG SAID SALT HAS NO ROLE IN THE REGULATION OF BLOOD PRESSURE. NIH WAS PERHAPS A LITTLE WISER BUT NOT NECESSARILY TOO MUCH, THE IMPRESSION OBSERVATION SINCE HYPERTENSIVE PATIENTS DON'T HAVE INCREASED PLASMA VOLUME RENAL ION TRANSPORT DEFECTS ARE NOT LIKELY TO BE IMPORTANT IN THE PATHOGENESIS OF HUMAN HYPERTENSION. THESE VIEWS CHANGED OVER THE YEARS AS A CONSEQUENCE. INSTITUTE OF MEDICINE NOW THE NATIONAL ADAD MY ACADEMY OF MEDICINE ADVOCATED THAT WE CAN ACHIEVE A 25% REDUCTION IN I DO TEAR SALT INTAKE BY REDUCING SALT AND PROCESS AND RESTAURANT FOODS, THE FDA HAS PROPOSED GUIDELINES TO ACHIEVE SIMILAR GOAL IN 2016. TOM FREEDEN AT CDC IN 2017 SAID HANDS DOWN BLOOD PRESSURE CONTROL IS THE MOST IMPORTANT THING YOU CAN DO IN HEALTHCARE. WE CAN TRIGGER EFFORTS THAT SAVE 100 MILLION LIVES FOCUSING ON SODIUM REDUCTION TRANSFATS AND BLOOD PRESSURE CONTROL. WE DIDN'T SOLVE BY THIS APPROACH ALL HIGH BLOOD PRESSURE OF COURSE. WE HAD ONE PARTICULAR DISORDER THAT WE THOUGHT WAS PARTICULARLY INTERESTING FROM ITS PHYSIOLOGY. WE KNOW THE STEREOED HORMONE ELDOSTERONE IS PROAZIDOD IN RESPONSE TO VOLUME DEPLETION. SO VOLUME DEPLETION WE ACTIVATE THE RENIN ANGIOTENSIN SYSTEM, THAT STIMULATES PRODUCTION BY ADRENAL GLAND, THAT TELLS THE KIDNEY TO HANG ON TO SALT AND WATER. INTERESTINGLY HOWEVER, HIGH SERUM POTASSIUM LEVEL IS ALSO A DRIVER OF HIGH INCREASED ELDOSTERONE SECRETION, IN THIS CASE THE KIDNEY USES IT TO INCREASE POTASSIUM SECRETION. SO THIS POSES A QUESTION WHEN KIDNEY SEES ELDOSTERONE HOW DOES IT KNOW MAXIMIZING SALT REABSORPTION OR POTASSIUM SECRETION. WE THOUGHT THIS WAS A NON-TRIVIAL PATIENTS WE STUDIED WHO HAD QUITE SOME CASES VERY SEVERE HIGH BLOOD PRESSURE AND HIGH SERUM POTASSIUM LEVEL WITH SUPPRESSED RENIN ACTIVITY SUGGESTING THAT THEY WERE CONSTITUTIVELY MAKING USING ELDOSTERONE TO PROVIDE SALT REABSORPTION, INCAPABLE TO PROMOTE POTASSIUM SECRETION SUGGESTING THAT THERE WAS A YIN AND YANG RELATIONSHIP BETWEEN TWO PATHWAYS, AND YOU CAN DO ONE OR THE OTHER BUT NOT BOTH TO MAXIMUM AFFECT AT THE SAME TIME. SO RICK WILSON IN THE LABORATORY STUDIED FAMILIES THAT WERE SEGREGATING THESE TRAITS AND BACK IN 2001 HE IDENTIFIED MEMBERS OF A NOVEL FAMILY THREE ANINE SERENE KINASES THE WENT KINASES THAT UNDERLINE THIS TRAIT. SO MUTATIONS IN WINK 4 POINT MUTATIONS IN THIS ACIDIC DOMAIN OF THE PROTEIN, MUTATIONS IN WINK 1 LARGE CHRONIC DELETIONS THAT INCREASE EXPRESSION OF WINK 1. AT THAT TIME VERY LITTLE WAS KNOWN, ESSENTIALLY NOTHING WAS KNOWN ABOUT KINASES, BUT OVER THE ENSUING YEARS IT WAS -- IT BECAME TO BE APPRECIATED WINK 4 IS ACTIVATING THE THIGHZIDE SENSITIVE CO-TRANSPORTER BY PROMOTING PHOSPHORYLATION OF INTERMEDIATE AREA SPECK WHICH PHOSPHORYLATES AND ACTIVATES THIGHZIDE SENSITIVE CO-TRANSPORTER AND OLESSI AND COLLEAGUES PUBLISHED THIS IN 2005. THIS DIDN'T EXPLAIN WHAT WAS REGULATING WINK 4 WHAT THIS PATHWAY MIGHT BE DOING IN ITS NORMAL CONTEXT. ALSO WE CAME TO REALIZE MUTATIONS IN WINK 1 AND 4 EXPLAINED ONLY 10% OF FAMILIES WITH THIS PHENOTYPE OF HYPERTENSION WITH KYPERKALEMIA. IT WASN'T UNTIL THE DEVELOPMENT OF EXOME SEQUENCING THAT WE ARE ABLE TO GO BACK AND SEQUENCE THESE OTHER FAMILIES WHICH LED TO THE REALIZATION OF WHAT WAS GOING ON,ISH MENTION THIS IS MARIA LOLIATI SHE MADE BACK TRANSGENIC MICE WHERE SINGLE COPIES OF EITHER THE WILD TYPE WINK 4 OR MUTANT WINK 4 INTRODUCED TO MICE. EXPRESSED UNDER ENDOGENOUS PROMOTER AND SHOWED THAT INTERESTINGLY THIS IS IN THIS LOW POWER VIEW YOU CAN SEE THERE'S DRAMATIC HYPERPLASIA AND HYPERTROPHY OF DISTAL TUBULE, THIS IS STAINING FOR CO-TRANSPORTER. AT HIGHER POWER WINK 4 IS EXPRESSED AT MUCH HIGHER LEVEL IN THESE MICE. ADS WELL TELLING US THE SINGLE POINT MUTATIONS THAT CAUSE THIS PHENOTYPE OF HYPERTENSION AND KYPERKALEMIA IS HAVING DRAMATIC EFFECTS IN THE TUBULE. WE DID NOT UNDERSTAND HOW THESE MUTATIONS WERE ASKING. THIS IS LYNN BOYDON SEQUENCED THIS IS GENES, SEQUENCED EXOME SEQUENCING IN THIS COHORT OF PATIENTS WITH THIS PHENOTYPE OF HYPERTENSION AND KYPERKALEMIA, AND SHE DEMONSTRATED 17 OF THEM HAD DE NOVO MUTATIONS NEW MUTATIONS NOT PRESENT IN THE PARENTS AFFECTED IN THE OFFSPRING, THAT IN THE GENE CALLED COLON 3 COMPONENT OF UBIQUITIN LIGASE. THESE MUTATIONS DID THE SAME THING. THEY ALL LED TO -- THEY ALL ALTERED SPLICING OF THE GENE THEY SKIPPED EXON NINE CREATING INFRAME DELETION. IF YOU KNEW NOTHING ABOUT THE MECHANISMS OF SPLICING YOU WOULD HAVE LEARNED FROM THESE FAMILIES BECAUSE THERE WERE MUTATIONS IN SPLICE ACCEPTTOR SITES MUTATIONS IN SPLICE DONOR SITES, MUTATIONS THAT THE BRANCH POINT FOR SPLICING OF THE EXON AND ALSO EXON SPLICE ENHANCERS THAT PROMOTE SPLICING OF WEAK EXONS THESE LED TO SKIPPING OF EXON 9. THIS WAS 17 OF THE 40 ODD FAMILIES AND REMAINDER HAD MUTATIONS IN ANOTHER GENE CALLED KELCH LIKE 3 WHICH TURNS INTO A PARTNER FOR COLON 3 IN FORMATION OF UBIQUITIN LIGASE. WE HAD NOT FOUND THIS GENE PREVIOUSLY BECAUSE GENETICS WERE QUITE CONFUSING. HALF OF THESE FAMILIES HAD RECESSIVE MUTATIONS THAT LED TO SIMPLE LOSS OF FUNCTION OF THE GENE AND THESE WERE DISTRIBUTED THROUGHOUT THE PROTEIN. BUT THE OTHER HALF HAD DOMINANT POINT MUTATIONS THAT CAUSED THE DISEASE. THESE TURNED OUT TO BE CLUSTERED IN THE SURFACE OF THIS BINDING DOMAIN USED TO TARGET SUBSTRATES FOR UBIQUITYLATION AND DEGRADATION. THESE ALL CLUSTER JUST IN THE PARTS THAT ARE LIKELY TO BE INTERACTING WITH THE SUBSTRATE PROTEINS. THIS LED (INDISCERNIBLE) A TALENTED BIOCHEMIST M.D. Ph.D. FELLOW NOW AT THE UNIVERSITY OF TOKYO TO EXPLORE THIS, HE DEMONSTRATED THAT KELCH LIKE 3 NORMAL TARGET ARE THE WINK KINASES. WHEN YOU EXPRESS KELCH LIKE 3 ALONG WITH CELLS WINK 4 LEVELS DROP DRAMATICALLY, IF YOU TAKE MUTATIONS THAT HAVE MUTATIONS FOUND IN PATIENTS WITH KELCH LIKE 3, THOSE MUTATIONS YOU DON'T REDUCE LEVELS OF WINK 4. CONVERSELY THE MUTATIONS THAT CAUSE HYPERTENSION AND KYPERKALEMIA IN WEEK 4, INTRODUCE THOSE MUTATIONS, YOU ALSO ABROGATE ABILITY TO CONTRIBUTE TO UBIQUITYLATION AN DEGRADATION. HE DEMONSTRATED THEY ARE IN FACT DIRECT BINDERS BY IMMUNOPRECIPITATION. AND CURTS ET AL DEMONSTRATED THAT YOU CAN MAKE CO-CRYSTALS OF THIS BINDING DOMAIN OF KELCH LIKE 3 WITH THAT SHORT SEGMENT OF WINK 4 THAT IS MUTATED IN THIS DISEASE. YOU SEE THEY INTERACT AT THE SITES MUTATED IN THE TWO DISEASES. SO THESE ARE ALL HAVING THEIR AFFECTS BY DISRUPTING THE BINDING OF KELCH LIKE 3 TO WINK 4 AND THESE LEAD TO ELEVATED LEVELS OF WINK 4. SO WE THOUGHT ABOUT WHAT IS THIS SYSTEM LIKELY TO BE DOING? WHY ARE YOU REGULATING THIS PATHWAY? WE THOUGHT THAT KELCH LIKE 3 MUST BE REGULATING WINK 4 NORMALLY AND THERE MUST BE A MECHANISMTOR ITS REGULATION. SO WE THOUGHT WE SET OUT TO IDE IF I TARGETSES THAT MIGHT BE MODIFIED THAT UNDERLIE THIS REGULATION. SO SUBJECTED KELCH LIKE 3 BY PHOSPHORYLATION BY MASS MASS SPECTROMETRY AND IDENTIFIED SEVERAL PHOSPHORYLATION SITES AND MOST INTRIGUE ILY WAS SERINE 433, IN SERENE 433 TURNS OUT TO BE THE MOST FREQUENTLY MUTATED SITE IN PATIENTS WITH HYPERTENSION AND KYPERKALEMIA. LEADING HIM TO SERGEANT THIS PHOSPHORYLATION STEP MIGHT PREVENT THE NORMAL BINDING OF KELCH LIKE 3 TO WEEK 4. WE WENT ON TO DEMONSTRATE THAT IN FACT IS THE CASE AND THAT WHEN YOU EXPRESS WILD TYPE KELCH LIKE 3 YOU PREVENT THE -- YOU REDUCE LEVELS OF WINK 4 AND WHEN YOU INTRODUCE THE MUTANT FORM OF KELCH LIKE 3 THAT IS LOOKS LIKE CONSTITUTIVELY PHOSPHORYLATED VERSION YOU PREVENT DEGRADATION OF WINK 4. WITH THAT NOTION THERE'S A REGULATED STEP GOING ON WHAT MIGHT THAT REGULATED STEP BE? IF YOU THINK ABOUT ELDOSTERONE SIGNALING IN SETTING OF VOLUME DEPLETION AND KYPERKALEMIA YOU HAVE ELDOSTERONE IN ELEVATED CONDITIONS, WHAT IS THE FACTOR THAT DISTINGUISHES VOLUME DEPLETION AND KYPERKALEMIA AT THE PHYSIOLOGIC LEVEL, ANGIOTENSIN 2. SO IN VOLUME DEPLETION YOU HAVE AN OWE TEN SIN ELEVATED IN KYPERKALEMIA ANGIOTENSIN IS NOT ELEVATED SO SUPPOSE AN OWE TEN SIN 2 MIGHT BE THAT CRITICAL REGULATOR THAT DISTINGUISHES THESE TWO TRAITS AND SUGAR WENT ON TO TEST THAT IDEA BY ESTABLISHING ANGIOTENSIN 2 SIGNALING IN MAMMALIAN CELLS AND DEMONSTRATED THAT IN THAT SETTING IF YOU ADDED INCREASING DOSES OF ANGIOTENSIN 2, YOU COULD ALLEVIATE THE ABILITY OF KELCH LIKE 3 TO REDUCE LEVELS OF WINK 4. THIS GOES UP IN STEP WISE FASHION. IN FACT THESE PHOSPHORYLATED FORMS OF KELCH LIKE 3 HE CAN DEMONSTRATE ARE PRESENT IN THE MAMMALIAN NEPHRON AS WELL. ON THE OTHER HAND YOU WANT TO BE ABLE TO DURN THAT SIGNAL OFF -- TURN THAT SIGNAL OFF AND RECENTLY SUGAR RADIOHAS GONE ON TO DEMONSTRATE IN FACT THERE IS A PHOSPHATASE THAT NORMALLY TURNS OFF THIS PATHWAY BY LEADING TO THE DEPHOSPHORYLATION OF KELCH LIKE 3. THAT'S THE CALCINEURIN. CALCINEURIN, IONMYCIN ACTIVATES CALCINEURIN, IT LEADS TO ELIMINATION OF THE PHOSPHORYLATED 4 OF KELCH LIKE 3, IF YOU ADD BACK TO INHIBITOR OF CALCINEURIN, YOU REGAIN PHOSPHORYLATION OF THAT SITE. THIS WAS A STRONGLY SUGGESTED BY THE CLINICAL OBSERVATION CALCINEURIN INHIBITORS WHICH ARE CRITICAL AGENTS IN IMMUNOSUPPRESSION AFTER ORGAN TRANSPLANTATION SUCH AS KIDNEY OR HEART TRANSPLANTATION, HYPERTENSION AND KYPERKALEMIA IS A RELATIVELY FREQUENT SIDE EFFECT OF THAT DRUG. IT TURNS OUT THIS IS ACTING THROUGH THE SAME PATHWAY IN ESSENCE OF PHENO COPY OF MUTATION. KELCH LIKE 3. THAT'S PROBABLY NOT THE ONLY MECHANISM BY WHICH ANGIOTENSIN 2 SIGNALING CONTRIBUTES TO INCREASED ACTIVITY OF WINK KINASES. WINK 4 LEVELS GO UP WITH MUTATIONS IN KELCH LIKE 3 BUT THAT'S PROBABLY NOT SUFFICIENT TO PRODUCE THE ENTIRE PHENOTYPE. IT TURNS OUT ANGIOTENSIN SIGNALING BY PROTEIN KINASE C LEADS TO DIRECT PHOSPHORYLATION OF WINK 4 ITSELF. AN THERE ARE FOUR SITES, AT LEAST FOUR SITES IN THE PROTEI THAT ARE DIRECTLY PHOSPHORYLATED BY PROTEIN KINASE C, TWO OF THESE SITES ARE CRITICAL FOR PROMOTING PHOSPHORYLATION OF THE ACTIVATION LOOP OF WINK 4 THAT IS ESSENTIAL FOR ITS KINASE ACTIVITY AND FOR ITS ABILITY TO PHOSPHORYLATE THE THIGHZIDE SENSITIVE SODIUM CHLORIDE CO-TRANSPORTER. IN THAT SETTING ANGIOTENSIN 2 SIGNALING LEADS TO PHOSPHORYLATION OF THESE TWO SITES. LEADS TO INCREASED PHOSPHORYLATION OF THE ACTIVATION LOOP. IF YOU MUTATE THOSE SITES YOU ELIMINATE THE ABILITY TO INCREASE PHOSPHORYLATION. OF THE ACTIVATION LOOP AN THIS DIDN'T PROJECT, I'M SORRY, THIS IS MARIA WHO IS NOW AT UNIVERSITY OF -- NATIONAL UNIVERSITY OF NEW MEXICO IN IF PHYSIOLOGY DEPARTMENT -- IN THE PHYSIOLOGY DEPARTMENT THERE. IT APPEARS ANGIOTENSIN 2 IS MAKING A CRITICAL STEP IN TELLING THE DISTAL CONVOLUTED TUBULE TO ACTIVATE ELECTRONEUTRAL SODIUM CHLORIDE CO-TRANSPORT BY DIRECT EFFECTS ON KELCH LIKE 3 INHIBITING DEGRADATION OF WINK AND BY ANGIOTENSIN 2 LEADING TO DIRECT PHOSPHORYLATION OF THESE RELEASED WINK KINASES, THIS PROMOTES PHOSPHORYLATION OF SPECK WHICH LEADS TO PHOSPHORYLATION IN ACTIVATION OF THE THIGHZIDE SENSITIVE CO-TRANSPORTER. SO WE THINK THEN THE MECHANISM IS YOU'RE HANGING ON TO SODIUM AND CHLORIDE IN THE DISTAL CONVOLUTED TUBULE, TRANSMITTING LESS SODIUM AN CHLORIDE DISTALLY THERE BY PREVENTING THE EPITHELIAL SODIUM CHANNEL ACUTELY FROM BEING ABLE TO EXCHANGE SODIUM FOR POTASSIUM AND THERE BY PREVENTING A POTASSIUM SECRETION. THIS ALSO IS HAPPENING CHRONICALLY. SO IN THE CHRONIC STATE AND STEADY STATE, WHY DO THESE PATIENTS WITH HYPERTENSION AND KYPERKALEMIA NOT EVENTUALLY LOSE POTASSIUM BECAUSE ULTIMATELY IN STEADY STATE THEY MUST BE DELIVERING SODIUM DISTALLY. SO THIS GETS TO A PARTICULARLY CREATIVE PROJECT SUGAR DID IN THE LABORATORY, HE CAME TO THE LABORATORY WITH THE IDEA OF STUDYING PHOSPHORYLATION OF MINERALA CORTICOID RECEPTOR. I THOUGHT THIS WAS PRETTY BLAND IDEA, MANY NUCLEAR HORMONE RECEPTORS HAVE BEEN STUDIED, THERE WAS GREAT INSIGHT THAT COMES FROM STUDYING THE PHOSPHORYLATION OF THESE BUT HE PERSISTED AND STUDIED THIS, I'M GLAD THAT HE DID. BECAUSE HE IDENTIFIED MANY SITES OF PHOSPHORYLATION ON THE PROTEIN BUT ONE WAS PARTICULARLY INTERESTING. SERENE RESIDUE THAT WAS PHOSPHORYLATED IN THE LIGAND BINDING DOMAIN OF THE MINERALA CORTICOID RECEPTOR. THAT WAS ONE OF TWO POSITIONS IN THE MINERALA CORTICOID RECEPTOR THAT DISTINGUISH THE ACTIVITY OF THE MINERALA CORTICOID RECEPTOR FROM GLUCOCORTICOID RECEPTOR THAT HAD BEEN SHOWN PREVIOUSLY, THESE TWO GENES RECENTLY EVOLVEDED FROM A COMMON ANCESTOR. AND THIS SERINE AT POSITION 843 LIES IN THE LIGAND BINDING DOMAIN AND IS CRITICAL FOR CONNECTING THE H 6 SEGMENT TO THE H 7 SEGMENT, THIS LIES VERY CLOSE TO WHERE ELDOSTERONE IS BOUND IN THE LIGAND BINDING DOMAIN LEADING US TO WONDER WHETHER THIS PHOSPHORYLATION MIGHT ALTER ABILITY TO ACTIVATE AND TRANSMIT THE -- TRANSDUCE THE EFFECT OF THE ACTIVATED TRANSCRIPTION FACTOR. HE WENT TO SHOW THAT IN FACT IS THE CASE. IF YOU MAKE THE PHOSPHOMIMETIC MUTATION AT 843, THE GLUTAMATE SUBSTITUTION AT THIS POSITION YOU LOSE ALL ABILITY TO INDUCE TRANSCRIPTION IN THE PRESENCE OF ELDOSTERONE YOU LOOSE THE ABILITY TO BIND IT AT TOGETHER AND YOU LOSE THE ABILITY OF PHOSPHORYLATED FORM OF MINERALA CORTICOID RECEPTOR TO TRANSLOCATE TO THE NUCLEUS. SO THE WILD TYPE FORM OF THE MINERALA CORTICOID RECEPTOR, THESE ARE THE SAME CELLS, GOES STRAIGHT TO THE NUCLEUS IN HIGH ABUNDANCE IN PRESENCE OF ELDOSTERONE THE PHOSPHORYLATED FORM REMAINS IN THE CYTOPLASM. SO WHERE DOES THIS END UP BEING IN VIVO? TURNS OUT TO DATE EXPRESS IN A SINGLE CELL TYPE WHICH INITIALLY WAS VERY SURPRISING AND DISAPPOINTING. THIS IS AQUAPORIN 2 STAINING OF DISTAL NEFF FRONT, IN THAT SAME -- NEPHRON. IN THAT SAME SEGMENT OF COLLECTING DUCT THERE'S PERCOLATED CELLS, THE HOLES IN THESE CELLS, THIS IS WHERE THE PHOSPHORYLATED -- THIS IS AN ANTIBODY SPECIFIC FOR PHOSPHOSERINE AT SERINE 843 IN MINERALA CORTICOID RECEPTOR. THESE TURN OUT TO BE COMPLIMENTARY SO THESE ARE LYING ENTERPOELATED CELLS Z DEMONSTRATED BY CO-STAINING WITH AD 1 AND OTHER MARKERS OF ENTERPOELATED CELLS. THIS HANGS OUT IN THE CYTOPLASM, AGAIN COMPARED TO BULK MINERALA CORTICOID RECEPTOR, THE PHOSPHORYLATED FORM NEVER GETS INTO THE NUCLEUS. WHAT IS THE FUNCTION OF THIS PHOSPHORYLATED FORM? IF YOU GO BACK TO RENAL PHYSIOLOGY AND ASK WHAT IS HAPPENING IN THE CONNECTING TUBULE AND THE COLLECTING DUCT, YOU HAVE THE -- THIS ELECTROGENIC STEP OF ELECTROGENIC SODIUM REABSORPTION LEAVING LUMEN NEGATIVE POTENTIAL POTASSIUM SECRETION FOR HYDROGEN ION SECRETION, AND CAN ALSO BE USED TO PROMOTE CHLORIDE ABSORPTION WITH CHLORIDE COMING IN ACCOMPANYING SODIUM OUT PRODUCING WATER SO YOU END UP WITH NET SODIUM CHLORIDE REABSORPTION. SO WHERE -- WHAT IS THIS PHOSPHORYLATED FORM OF MINERALA CORTICOID RECEPTOR DOING? WHAT WE THINK THAT IT CREATES A DEAD MINERALA CORTICOID RECEPTOR THAT CAN'T DO ANYTHING IN THESE CELLS ALLOWING THIS PATHWAY OF SODIUM REABSORPTION TO BE ACCOMPANIED BY POTASSIUM SECRETION. BUT ANGIOTENSIN 2 TURNS OUT TO REGULATE THIS ACTIVITY AS DOES POTASSIUM. SO ANGIOTENSIN 2 REDUCES PHOSPHORYLATION OF THIS POSITION IN THE MINERALA CORTICOID RECEPTOR, HIGH POTASSIUM LEVELS DOES THE OPPOSITE, INCREASES PHOSPHORYLATION AT THIS SITE. IN FACT THIS IS WHAT HAPPENS ON HIGH SALT DIET ALONE, YOU HAVE HIGH LEVELS OF MINERALA CORTICOID RECEPTOR PHOSPHORYLATED ENTERPOELATED CELLS, YOU ADD ANGIOGENERAL SIN TWO, YOU LOSE MOST OF THE MINERALA -- MOST OF THE PHOSPHORYLATED FORM OF THE PROTEIN. SO YOU NOW HAVE AN INACTIVATED CORTICOID RECEPTOR THAT BECOMES ACTIVATED IN THE DOWNSTREAM OF ANGIOTENSIN 2 SIGNALING. THIS TOO IS MEDIATED BY THE WINK 4 PATHWAY. THE MUTANT FORM OF WINK 4 THAT CAUSES DISEASE PRODUCES INCREASED EXPRESSION OF PENDREN AND THIS HAPPENS AS WELL WITH ANGIOTENSIN 2 SIGNALING IN VIVO. WE THINK THE MECHANISM OF WHAT'S HAPPENING HERE IS WE NOW HAVE A SECOND NEPHRON SEGMENT THAT IS REGULATED BY ANGIOTENSIN 2 IN A WAY THAT PROMOTES MAXIMAL SODIUM CHLORIDE REABSORPTION AND MINIMAL POTASSIUM SECRETION. IN THE SETTING OF KYPERKALEMIA, YOU HAVE A PHOSPHORYLATED FORM OF MINERALA CORTICOID RECEPTOR. YOU INHIBIT SECRETION OF HYDROGEN AND CHLORIDE BICARBONATE EXCHANGE AND MAXIMIZE POTASSIUM SECRETION. IN SETTING OF VOLUME DEPLETION, ANGIOTENSIN 2 LEADS TO DEPHOSPHORYLATED OF MINERAL ACTION CORTICOID RECEPTOR, A COMPETENT MINERALA CORTICOID RECEPTOR, WHOSE ACTIVITY PROMOTES INCREASED EXPRESSION, OF PENDREN CHLORIDE BICARBONATE RECEPTOR EXCHANGE AN HYDROGEN ION, HYDROGEN PUMP IN APICAL MEMBRANE AND PROMOTES ELECTRIC SODIUM REABSORPTION TO PROMOTE CHLORIDE REABSORPTION. SO WHAT IS THE IMPORTANCE OF THIS PATHWAY? IN 1997 THERE WAS A LOT OF INTEREST IN CLINICAL STUDIES THAT SHOWED SOMETHING CALLED THE DASH DIET HAD A DRAMATIC EFFECT TO REDUCE BLOOD PRESSURE IN HUMAN POPULATION. WHEN YOU LOOK AT THE COMPOSITION OF THE DASH DIET COMPARED TO THE CONTROL DIET, LARGELY FRUITS AND VEGETABLES DIET, THE MAJOR DIFFERENCE IS THE DIETARY POTASSIUM LOAD WHERE THERE IS NEARLY THREEFOLD INCREASE IN THE TWO TO THREE FOLD DIFFERENCE IN DIETARY POTASSIUM LEVEL THAT WAS SUFFICIENT TO REDUCE BLOOD PRESSURE ABOUT THE SAME DEGREE THAT WAS ACHIEVED WITH SINGLE ANTI-HYPERTENSIVE AGENT. WHILE AN INTERESTING OBSERVATION THE MECHANISM OF THIS AFFECT WAS COMPLETELY UNKNOWN AND OBSCURE. WE BELIEVE THAT THE MECHANISM IS EXACTLY MECHANISM THAT WE HAVE BEEN TALKING ABOUT. WHERE THE KIDNEY CAN EITHER MAXIMALLY REABSORB SODIUM AND CHLORIDE, MAXIMALLY SECRETE POTASSIUM AND DOES THESE IN YIN AND YANG FASHION AND YOU CAN'T DO BOTH, IF YOU GIVE PATIENTS HIGH POTASSIUM DIET YOU OBLIGE KIDNEY TO EXTREAT POTASSIUM AND AS A CONSEQUENCE THIS IS THE MECHANISM BY WHICH BLOOD PRESSURE IS LOWERED WHICH IS THE SAME MECHANISM WHICH OTHER AGENTS ACT BY REDUCING SEW YUM CHLORIDE REABSORPTION IN THE KIDNEY, YOU REDUCE BLOOD PRESSURE. THIS HAS BEEN MODELED BY FRANK SAX WHO DID THIS WORK TO SHOW THE RELATIVE AFFECTS OF INCREASING DIETARY POTASSIUM AND DECREASING DIETARY SODIUM. AND WHAT MIGHT BE ACHIEVED BY COMBINATION OF THOSE TWO. THERE'S VERY LARGE EFFECT OF THE DASH DIET TO REDUCE BLOOD PRESSURE, SIMPLY BY INCREASING DIETARY SODIUM AND YOU CAN AUGMENT THIS FURTHER BY REDUCING DIETARY SALT INTAKE. THIS IS A MECHANISM THAT I THINK IS NOT BEEN SUFFICIENTLY EXPLORED IN THE LITERATURE. IN PUBLIC HEALTH EFFORTS. IN THE LAST FEW MINUTES I WILL TALK ABOUT OUR FURTHER EFFORTS TO UNDERSTAND SEVERE RELATIVELY COMMON FORM OF SEVERE HIGH BLOOD PRESSURE. THESE ARE ALDOSTERO NEXTE PRODUCING ADENOMAS. THESE ARE MAKING SALT AND WATER RETAINING HORMONE, FOUND IN FIVE TO TEN PERCENT OF PATIENTS WITH SEVERE HYPERTENSION, BENIGN TUMORS, THEY VIRTUALLY NEVER INVADE OR ME TAS TA SIZE, MOST ARE UNDIAGNOSED AND COME TO CLINICAL ATTENTION LATE IN CLINICAL COURSE. WE WONDERED WHETHER THERE MIGHT BE SIMPLE MECHANISMS THAT LINK CONSTITUTIVE PROLIFERATION OF TUMORS AND CONSTITUTIVE HORMONE RELEASE AND TROY AND TOBIAS LED OUR EFFORTS TO STUDY THESE TUMORS. WE SIMPLY STARTED BY SEQUENCING THE NORMAL GENOME AND THE TUMOR GENOME OF THESE PATIENTS. WE DIDN'T HAVE TO STUDY VERY MANY TUMORS, IN FACT WE HAD TO STUDY FOUR. IN ORDER TO IDENTIFY THE CAUSE OF THESE TUMORS IN 50% OF PATIENTS. TURNS OUT THAT ABOUT HALF OF THESE PATIENTS HAVE A SINGLE SOMATIC MUTATION, IN A POTASSIUM CHANNEL ENCODED BY GENE KCNJ 5 THAT ARE SUFFICIENT CAUSE OF TUMORS. THESE MUTATIONS, THERE ARE TWO MUTATIONS THAT DO THIS. ONE IS MUTATION AT GLYCENE 151. AND THE SECOND IS MUTATION AT LEUCINE 168. THE IMPORTANCE OF THESE TWO POSITIONS IS THEY BOTH ARE INVOLVED IN THE ESTABLISHMENT OF THE SELECTIVITY FILTER OF THIS POTASSIUM CHANNEL AND EVERY OTHER POTASSIUM CHANNEL IN THE BIOLOGICAL WORLD. ROD MY COLLEAGUE AT ROCKEFELLAR DETERMINED THE CRYSTAL STRUCTURE OF THE FIRST POTASSIUM CHANNEL. AND SHOWED THE MECHANISM OF EXQUISITE ION SELECTIVITY OF POTASSIUM CHANNELS IS THERE'S A GATE -- AT THE PORE DEFINED BY A GLYCENE TYROSINE GLYCENE MOTIF THAT IS PRECISELY POSITIONED TO STRIP WATR FROM POTASSIUM IONS BUT FROM NO OTHER IONS, THIS PROVIDES THE EXQUISITE SELECTIVITY OF THESE CHANNEL. THE GLYCENE MUTATED IN THESE TUMORS IS THE GATEKEEPER OF THE FIRST RESIDUE OF THE GYG MOTIF. LEUCINE 168 INTERACTS DIRECTLY WITH THE TYROSINE, Y OF THE GYG MOTIF. EITHER OF THESE TWO MUTATIONS IS SUFFICIENT TO CHANGE THE CHANNEL WHICH WE DEMONSTRATED BY ELECTROPHYSIOLOGY, CHANGES THE CHANNEL SO RATHER THAN ONLY CONDUCTING POTASSIUM, THEY CAN CONDUCT SODIUM AS WELL. THE IMPLICATION OF THAT, IS QUITE STRAIGHT FORE. STRAIGHT FORWARD. THIS IS A NORMAL ADRENAL GLOMERULOSIS CELL, IT HAS A CONSTITUTIVELY OPEN POTASSIUM CHANNEL THAT SETS MEMBRANE POTENTIAL TO A HYPERPOLARIZED POTENTIAL. AND THERE'S VOLTAGE GATED CALCIUM CHANNELS AND THESE ARE NEED TO BE OPEN IN ORDER TO PROVIDE THE SIGNAL FOR ALDOSTERONE ACUTELY AND CELL REPLICATION CHRONICALLY SO THE NORMAL MECHANISM IS ANGIOTENSIN 2 CLOSES POTASSIUM CHANNEL AND THAT LEADS TO MEMBRANE DEPOLARIZATION, ACTIVATION OF VOLTAGE GATED CALCIUM CHANNELS WHICH PROVIDE SIGNALS TO -- MAKE THE RATE MORE RATE LIMITING ENISYL SYNTHASE AND THE RIGHT AMOUNT WILL CAUSE CELL PROLIFERATION. THESE SINGLE AMINO ACID SUBSTITUTIONS IN THIS POTASSIUM CHANNEL PROVIDE SODIUM CONDUCTANCE THAT COMES IN INSTEAD OF POTASSIUM LEAVING THE CELL, THIS LEADS TO DEPOLARIZATION, ACTIVATION OF THIS SAME PATHWAY. SO THE SAME SIGNAL IS SUFFICIENT TO PRODUCE CONSTITUTIVE PRODUCTION OF HORMONE ALDOSTERONE AND CELL PROLIFERATION. WE THINK THIS PARTICULAR POTASSIUM CHANNEL IS INSIDIOUS IN HAVING THIS AFFECT BECAUSE THIS IS NORMALLY POTASSIUM CHANNEL THAT PUTS ON THE BRAKES TO PRODUCTION. THIS IS ACTIVATED BY G CUPPED PROTEIN RECEPTOR THAT TURNS OFF PRODUCTION OF ALDOSTERONE NOW PROVIDING THE GAS INSTEAD OF BRAKES YOU CAN'T BRUNT IT BY NORMAL BRAKING MECHANISM. THIS TURNS OUT TO WORK IN EVERY POPULATION AROUND THE WORLD THAT'S BEEN STUDIED. THERE IS ONE STRIKING DIFFERENCE THE POPULATION GENETICS OF THIS DISORDER. THE MUTANT CHANNELS ACCOUNT FOR ONLY 20% OF MALES BUT FOR NEARLY TWO-THIRDS OF FEMALES IN THE EUROPEAN POPULATION. IN ASIAN POPULATIONS THIS FRACTION IS HIGHER IN BOTH MALES AND FEMALES, ABOUT 70% OF BOTH. WE DON'T UNDERSTAND THE POPULATION DIFFERENCES NOR THE GENDER DIMORPHISM IN EUROPEAN POPULATION BUT EITHER TWO MUTATIONS EXPLAIN THE VAST MAJORITY OF THE CONTRIBUTION OF KCNJ 5 TO THESE TUMORS. WE THINK THEY'RE SUFFICIENT FROM THE SIMPLE GENETICS OF THESE TUMORS BECAUSE THESE ARE VERY QUIET TUMORS, THEY ONLY HAVE FOUR TO SIX SOMATIC PROTEIN ALTERING MUTATIONS BUT WE KNOW THEY'RE SUFFICIENT BECAUSE THEY'RE GERM LINE MUTATIONS THAT IN SOME CASES ARE IDENTICAL TO THE ONES FOUND IN TUMORS. THAT ARE TRANSMITTED AS A SIMPLE MENDELIAN TRAIT IN WHICH NOW EVERY CELL IN ADRENAL GLAND HARBORS MUTATIONS. CHILDREN BE THESE MUTATIONS DEVELOP RIP ROARING HYPERTENSION BECAUSE EVERY CELL IN THE ADRENAL GLAND IS TRYING GLOMERULOIS A IS TRYING TO BECOME A TUMOR. THESE CHILDREN GET ADRENAL GLOMERULOSIS THAT ARE ENORMOUSLY EXPANDED BY THE AGE OF FIVE IN VIRTUALLY ALL THESE CHILDREN REQUIRED BILATERAL ADRIENNEALECTOMY ADRENALECTOMY IN ORDER TO CONTROL BLOOD PRESSURE. SO THESE MUTATIONS ARE SUFFICIENT, SUFFICIENT CAUSE OF THIS DISORDER. USING THIS WE MANAGE TO DEVICE A APPROACH TO SEEN FOR SMALL MOLECULES THAT MIGHT SELECTIVELY INHIBIT MUTANT CHANNEL BUT NOT WILE TYPE CHANNEL. YOU CAN CONSTRUCT A SCREEN IN IN A WAY WHERE YOU CAN TURN SELECTIVELY TURN ON THE EXPRESSION OF THE MUTANT GENE IN CELLS AND WILD TYPE GENE HAS NO AFFECT ON VIABILITY TURNING ON THE MUTANT GENE INDUCES SO MUCH SODIUM ENTRY INTO THE CELL THAT SIMPLY KILLS THE CELLS. SO WE CAN LOOK FOR DRUGS OR SMALL MOLECULES THAT WOULD REVERSE THAT LETHALITY. WE IDENTIFIED SUCH DRUGS POTENTLY RESCUE LETHALITY OF BOTH EITHER PREVALENT MUTATION, NO EFFECT ON WILD TYPE CHANNEL. THESE TURN OUT TO BE MACRO LIED DRUGS. SEVERAL OF THEM ARE MARKETED ANTIBIOTICS SUCH AS CLAIRE ERYTHROMYSIN THAT INHIBIT EITHER OF THE TWO MUTANT CHANNELS BUT DON'T TOUCH WILD TYPE DRUG AND WE CAN MAKE DERIVATIVES THESE THAT LOSE ANTIBIOTIC ACTIVITY, THESE MIGHT BE USEFUL IN TRYING TO MAKE A DIAGNOSIS BY GIVING SUCH A DRUG SEEING WHERE THE LEVELS PROMPTLY DROP AS WE KNOW THEY DO IN OTHER SITUATIONS WHERE THERE'S CONSTITUTIVE PRODUCTION. WE HAVE DONE SIMILAR WORK IDENTIFYING ADDITIONAL GENES THAT REGULATE PRODUCTION OF ALDOSTERONE SEVERAL MUTATIONS IN CALCIUM CHANNELSES THAT DO THE SAME THING. THE CHLORIDE CHANNEL NOT IMPLICATED IN ANY SPECIFIC ROLE IN ADRENAL PHYSIOLOGY, BUT GAIN OF FUNCTION MUTATIONS IN CHLORIDE CHANNEL CLCN 2 LEAD TO DEPOLARIZATION AND FINAL PATHWAY THESE ARE ADDITIONAL NEW GENES FOR HIGH BLOOD PRESSURE IN HUMANS. SO STARTING WITH A BLANK SLATE WE KNOW CAUSE OF SOME OF THE LESSONS FROM THIS COMMON DISEASE EXTREME FORMS OF THIS COMMON DISEASE ARE VERY INSTRUCTIVE FOR UNDERSTANDING PATHWAYS IN IDENTIFYING PRODUCTIVE THERAPEUTIC TARGETS. THE GENETICS ESTABLISHES THE CAUSAL RELATIONSHIP BETWEEN GENOTYPES AN PHENOTYPES. AND ALLOW PRODUCTIVE EFFORTS TO DETERMINE THE BIOCHEMICAL MECHANISMS LINKING THE TWO. IMPORTANTLY I WANT TO LEAVE YOU WITH THE RECOGNITION HUMANS DEAL WITH ESSENTIALLY THE SAME DECK OF GENES AS EVERY OTHER VERTEBRATE. WE ALL SHARE THE SAME 20,000 GENES WITH SOME NOTABLE EXCEPTIONS WHERE FOR EXAMPLE PARTICULAR INFECTIOUS AGENTS SHAPED IMMUNE RESPONSES AND OTHER ASPECTS OF BRAINS EVOLVED IN DIFFERENT WAYS. BUT BY AND LARGE WE ARE DEALING WITH A VERY SIMILAR SET OF GENES OVER THE LAST 500 TO 600 MILLION YEARS OF EVOLUTION. AN THESE STRONGLY SUGGEST THE LOSS OF VIRTUALLY ALL SINGLE COPY GENES IN HUMAN GENOME HAVE CONSEQUENCES TYPICALLY NOT GOING TO BE SMALL. WE HAVE A PATH TO DETERMINE THE CONSEQUENCE OF MUTATION OF EVERY GENE IN THE HUMAN GENOME. THIS IDENTIFICATION WILL ULTIMATELY IDENTIFY THE UNDERLYING MECHANISMS OF WIDE RANGE OF HUMAN DISEASE AND CONTINUE TO IDENTIFY THOSE GENES AND PATHWAYS WITH THE GREATEST POTENTIAL FOR DEVELOPMENT OF NEW DIAGNOSTICS AND THERAPEUTICS. WE'RE MOSTLY LIMITED BY RECOGNIZING THE RIGHT PHENOTYPES TO GO AFTER AND EXPLORE. THIS IS REASON FOR EVERY PERSON WHO SEES PATIENTS TO BE RECOGNIZING THOSE RARE PATIENTS WHO HAVE DISORDER THEY SAY I HAVEN'T SEEN ANYTHING QUITE LIKE THIS BEFORE. WE NOW CAN SOLVE STRIKING FRACTION OF SUCH PATIENTS. THIS IS A PARTICULARLY EXCITING TIME TO BE EXPLORING IT. SO I'LL STOP THERE. THANK YOU VERY MUCH, MOE FOR SETTING UP MY ENTIRE CAREER AND THAT OF MANY OTHER INVESTIGATORS. THROUGH FOR YOUR ATTENTION TODAY. -- THANK YOU FOR YOUR ATTENTION TODAY. [APPLAUSE] >> IF YOU WANT TO TALK TO RICK, THERE'S A RECEPTION ACROSS THE HALL IN THE NIH LIBRARY. BUT BEFORE YOU GO TO THAT I WOULD LIKE TO PRESENT RICK WITH THIS MOMENTO OF THE FIFTH ANNUAL BURG LECTURE, A NICE PLAQUE DESIGNED BY THE STUDENTS IN MY LABORATORY. CONGRATULATIONS. >> THANKS SO MUCH. [APPLAUSE]