THANK YOU FOR ATTENDING THE MITOCHONDRIA

INTEREST GROUP MEETING TODAY.

VIDEOCAST THAT WILL BE ARCHIVED

VIDEOCAST.NIH.GOV AND IN ADDITION TO

LIVE VIDEOCAST, I AM MONITORING

WITH MY

EMAIL

STEVEN.ZULLO@NIH.GOV IF ANYONE

WANTS TO ASK QUESTIONS AND I CAN

MONITOR THE EMAIL THERE.

I'LL REMIND EVERYONE EVERY ONCE

IN A WHILE ABOUT THIS.

THOUGH IT'S WHEN YOU GET ON TO

THE VIDEOCAST.NIH.GOV SITE WHEN

YOU GET ON TO THIS IT'S LISTED

THERE.

IPT TO THANK EVERYONE FOR

ATTENDING.

WHAT WE'RE DOING TODAY IS A

ROUGHLY TWO AND A HALF HOUR

MEETING TO PRESENT CASES THAT

HAVE BEEN SEEN BY THE

UNDIAGNOSED DISEASE PROGRAM OR

BY OTHER RESEARCHERS HERE AT NIH

THAT INVOLVE MITOCHONDRIA.

THE UNDIAGNOSED DISEASE PROGRAM

NOT ONLY DEALS WITH

MITOCHONDRIAL DISEASE BUT OTHER

UNDIAGNOSED DISEASES.

AND IT'S MY PLEASURE TO BEGIN

THIS MEETING WITH THE DIRECTOR

OF THE UNDIAGNOSED DISEASE

PROGRAM, DR. BILL GAHL.

BILL.

>> THANK YOU, STEVE.

I THINK IT'S APPROPRIATE TO GIVE

A LITTLE BACKGROUND FOR THE NIH

UNDIAGNOSED DISEASE PROGRAM AND

THAT'S WHAT I'LL DO NOW.

WE DO SEE PATIENTS WHO HAVE

MITOCHONDRIAL DISORDERS BUT WE

SEE THEM WITH ALL SORTS OF OTHER

THINGS AS WELL.

IT'S PROBABLY GOOD TO SEE WHERE

THOSE MITOCHONDRIAL DISEASE

PATIENTS EMANATE FROM.

THAT WOULD BE THIS PROGRAM.

I THINK THIS PICTURE REALLY --

PAINTING TIP FIES THE CONCERN WE

HAVE WHEN WE DON'T HAVE A

DIAGNOSIS FOR OUR PATIENTS.

SO THE UNDIAGNOSED DISEASE

PROGRAM WAS ESTABLISHED

MAY 19th, 2008 SO GOING FOR

ALMOST FOUR YEARS NOW.

WE WANT TO TRY TO HELP PATIENTS

COME TO A DIAGNOSIS BUT ALSO

MAKE DISCOVERIES ABOUT NEW

DISEASES AND NEW PATHWAYS.

I'LL GIVE YOU EXAMPLES HOW WE'RE

ABLE TO DO THAT.  THE WAY THIS

WORKS IS THAT PATIENTS SUBMIT

THEIR MEDICAL RECORDS ALONG WITH

A SUMMARY LETTER FROM THEIR

DOCTOR.

SOMETIMES THAT TAKES A WHILE TO

GET THIS INFORMATION AND WE ASK

FOR SLIDES AND VIDEOS AND IMAGES

AS WELL.

I TRIAGE THE RECORDS TO CERTAIN

CONSULTANTS THAT ARE APPROPRIATE

AND THEY REVIEW THE RECORDS AND

COME BACK TO ME WITH THE

RECOMMENDATIONS.

WE PUT IT TOGETHER AND DECIDE

YAY OR NAY, ACCEPT OR REJECT.

SOMETIMES WE GIVE SOME

SUGGESTIONS WHEN WE REJECT

PATIENTS.

THE PATIENTS WE ACCEPT WE SEE A

WEEK AT THE CLINICAL CENTER

WHICH IS PICTURED HERE.

THERE'S HUGE NUMBERS OF PEOPLE

INVOLVED IN THIS, BOTH HIRED BY

THE UNDIAGNOSED DISEASE PROGRAM

AND AS CONSULTANTS FROM THE

VARIOUS ICs AND FROM THE

CLINICAL CENTER.

WE HAVE OPEN MEETINGS ONCE A

MONTH WHICH WE DISCUSS SEMINOLE

CASES WITH RADIOLOGISTS AND

PATHOLOGISTS PRESENT.

OUR NUMBERS SO FAR IS WE HAVE

INCREASE OF OVER 6,000

INDIVIDUALS.

NOW, ONLY ABOUT 2100 HAVE

ACTUALLY SUBMITTED THEIR RECORDS

AND ABOUT TWO-THIRDS OR THREE

QUARTERS ARE REJECTED.

WE HAVE ACCEPTED OVER 500

INDIVIDUALS.

AND ABOUT 400 ON OUR UNDIAGNOSED

DISEASE PROGRAM SERVICE.

HALF THE CASES ARE NEUROLOGICAL

AND THERE ARE A LOT OF PEDIATRIC

GENETIC CASES.

WE REACHED SOME DIAGNOSIS, ABOUT

25%.

ONE OF THE FEATURES OF THIS

PROGRAM IS THAT WE'RE HEAVY INTO

GENETICS.

WE DO GENETIC ANALYSES IN THREE

DIFFERENT WAYS.

ONE, WE SEEK COMMERCIAL TESTING.

ANOTHER IS WE DO MILLION SNP

ARRAYS, I'LL TELL YOU WHAT THAT

S. THEN WE DO EXONLY SEQUENCING.

-- EXOME SEQUENCING.

THE SNP ARRAYS INVOLVE CERTAIN

SOFTWARE AND CERTAIN TECHNICAL

CHIPS THAT ARE USED.

THAT'S LISTED ON HERE.

BUT AN EXPLANATION OF THIS IS

THAT THE HUMAN GENOME CONTAINS

3.2 BILLION BASES.

AND THE COMPANY HAS SELECTED A

MILLION OF THOSE RESIDUES AS

BEING POLYMORPHIC MEANING

THEY'RE EITHER A OR T OR C OR G

OR C OR T OR WHATEVER, TWO

POSSIBILITIES.

AND THOSE POSSIBILITIES ARE

PRESENT IN ROUGHLY 50/50, 60,

40, 70, 30 OR SO.

SO IT ISN'T AS ONE OF THE

POLYMORPHIC RESIDUES IS 99 TIMES

ONE, OR ONE TIME IT IS OTHER,

IT'S FAIRLY EVENLY SPLIT.

THE WAY THIS IS DETECT SECOND

DEGREE TO HAVE AN

OLIGONUCLEOTIDE SEQUENCE THAT IS

UNIFORM AT EVERY RESIDUE ACCEPT

THE ONE OF INTEREST IG LIKE

RIGHT HERE.

BUT THESE ARE ALL COMMON TO

OLIGONUCLEOTIDES THAT ARE GOING

TO HYBRIDIZE WITH THAT

PARTICULAR REGION.

SO THIS WILL -- THIS

FLUORESCENCE MARKERS ON THIS,

AND THEREFORE, ONE CAN DETECT

EACH OF THE TWO ALLELES BY THE

FLUORESCENCE INTENSITY.

IT'S BY CONVENTION THE MORE

COMMON OF THE TWO POSSIBILITIES

IS CALLED THE A ALLELE AND THE

LESS FREQUENT IS THE B ALLELE.

SINCE WE'RE ALL DIZOMIC MOST

PLACES OF OUR GENOME, WILL BE A,

A, B, B OR A B, AND ARCA AND BB

ARE HOMOZYGOUS AN A B IS

HETEROZYGOUS AND THE DEGREE OF

CORRESPONDS TO THE FLUORESCENCE

INTENSITY.

HERE WE HAVE INTENSITY OF ONE

ALLELE, THE B ALLELE AND HERE WE

HAVE INTENSITY OF THE OTHER

ALLELE.

AND IF YOU'RE DIZOMIC YOU'LL BE

EITHER BB AB OR AA.

IN OTHER WORDS HAVE THESE

DIFFERENT FLUORESCENT

INTENSITIES.

MONOSOMIC WILL BE B OR A, HAVE

ONLY THAT MUCH INTENSITY.

AND THAT SIGNIFIES THE FACT THAT

THERE WILL BE A DELETION, A

SINGLE COPY DELETION.

ONE COPY INSTEAD OF TWO.

IF YOU'RE DOUBLY DELETED YOU EEL

HAVE NO FLUORESCENT INTENSITY AT

ALL AND YOU'LL BE DOWN HERE SO

YOU CAN LOOK AT THE LOG A RATIO

AND EACH DOC HERE REPRESENTS ONE

OF THE MILLION POLYMORPHIC

MARKERS THAT ARE ON AVERAGE

ABOUT 3,000 BASES APART.

YOU CAN LOOK AT THE LOG R RATIO,

SHOULD BE ABOUT ZERO, MEANING

THERE'S HALF AND HALF.

IF YOU HAVE AN INTENSITY THAT

ORDERS OF MAGNITUDE LOWER THAN

THAT, LIKE DOWN HERE, IT

MEANIOUS EAR DOUBLY DELETED.

SO THIS PERSON IS DOUBLY DELETED

IN 32 SNPS, 32 THAT ARE WELL BOW

LOW THE MEAN FLUORESCENT

INTENSITY.

IT TURNS OUT THIS CORRESPONDS TO

A CERTAIN REGION OF THE GENOME

IN THIS CASE THE REGION THAT

ENCOMPASSES THEjK– CTNS.

THIS THEN IS THE COMMON 57,257

BASE PAIR DELETION CAUSING THE

DISEASE STENOSIS IN ABOUT HALF

THE NORTHERN EUROPEAN AN

AMERICAN INDIVIDUAL WHOSE HAVE

CYSTNOSIS.

SO THE VAJ OF THE -- ADVANTAGE

OF THE MILLION SNP ARRAY

ANALYSIS IS THE GENOME HAS BEEN

ANNOTATED BY WHERE THE GENES ARE

AND IF YOU CAN FIND DELETED

REGION IN ONE OR TWO COPIES, YOU

CAN TELL IF YOU HAVE A REGION BY

EXAMINING THE SNP ARRAY ANALYSIS

THE OTHER THING YOU CAN DO WITH

SNP ARRAYS IS TO LOOK AT

HOMOZYGOSITY.

THIS IS THE B ALLELE FREQUENCY

PLOT.

EACH ONE O THESE BLUE DOTS IS

ONE OF THE BILLION SNPS, THIS IS

ONE PARTICULAR CHROMOSOME, THIS

IS THE CENTROMERE BECAUSE IT HAS

NO SNPS AND IF YOU'RE BB, YOU'RE

UP HERE, IF AA THE SNP IS DOWN

HERE, HETEROZYGOUS, IT'S HERE.

IF YOU TAKE THE HETEROZYGOUS

REGION AND FORGET THE TOP AND

THE BOTTOM, AND PLOT A WHOLE

BUNCH OF DIFFERENT PEOPLES

HETEROZYGOUS REGIONS YOU WILL

SEE EACH ONE OF THESE IS A

DIFFERENT INDIVIDUAL.

THIS IS CHROMOSOME 10, THIS IS

THE CENTROMERE IN BETWEEN.

THIS IS BLOWN UP COMPARED TO THE

PREVIOUS IMAGE.

RIGHT HERE IS A REGION WHERE

THERE'S NO HETERO ZYGOSITY.

THIS PERSON IS HOMOZYGOUS IN

THIS REGION.

IN FACT THIS HOMOZYGOUS IN THIS

REGION.

HERE IS A 22-YEAR-OLD INDIVIDUAL

WITH ENCEPHALOPATHY FROM RUSSIA.

HE HAS ALL OF THESE DIFFERENT

REGIONS OF HOMOZYGOSITY.

TURNS OUT THAT ENDS UP TO BE

ABOUT 3%.

WELL, 3% HOMOZYGOSITY MEANS

THERE'S CONSTANT ABOUT A

CONSTANT SECOND COUSIN LEVEL.

FIRST CAN YOU SENS SHARE 1/8 OF

GENES AND SECOND COUSINS SHARE

1/32 OF THE GENES.

THIS DEGREE OF HOMOZYGOSITY IS

HELPFUL BECAUSE EACH REGION IS

ONE IN WHICH THE GENES

ASSOCIATESSED WITH THIS AREA

WOULD REQUIRE ONLY ONE MUTATION

TO HAVE OCCURRED IN A DISTANT

PAST RELATIVE THAT -- SO IF THIS

REGION WAS PASSED DOWN WITH THAT

MUTATION YOU ONLY NEED THAT ONE

PREVIOUS MUTATION AND NOW YOU

WOULD BE HOMOZYGOUS FOR THAT

MUTATION BECAUSE OF THE

CONSENGUIDITV INVOLVED SO IF

YOU'RE POSITING A DISORDER YOU

LOOK FOR THE HOMOZYGOSITY AND

THE MILLION SNP ARRAY ALLOWS YOU

TO DO THAT.

WHOLE EXOME SEQUENCING IS A

DIFFERENT MATTER.

WE HAVE 3.2 MILLION BASES.

ONLY 1.7% ENCODE GENES.

SO 1.7% OF 3.2 BILLION IS ABOUT

50 OR 60 BILLION OR SO AND WHOLE

EXONLY SEQUENCING DETERMINES THE

SEQUENCE OF ALL THOSE 60 MILLION

BASES.

WITH ABOUT 85 TO 90% COVERAGE.

SOME INACCURACIES.

INACCURACIES CAUSE ONE TO HAVE

ABOUT 20,000 VARIANTS FOR EACH

OF THE WHOLE EXOMES.

THERE'S 20,000 VARIANTS ARE

ESSENTIALLY GOING TO BE ERRORS

IN ALIGNMENT OR IN THE

SEQUENCING AND ONLY ONE OF THOSE

IS LIKELY TO BE CAUSING THE

DISEASE.

MAYBE ACTUALLY TWO IN SOME CASES

OR EVEN THREE BUT REALLY ALL THE

REST, ALL THE THOUSANDS OF REST

ARE ESSENTIALLY ERRORS OR

NON-PATHOGENIC.

IN ORDER FOR US TO DETERMINE TO

E LIMBIATE MANY OF THOSE 20,000,

WE HAVE FILTERS THAT WILL REDUCE

THE NUMBER FROM 20,000 TO 2,000

TO 200 TO 20, ET CETERA.

THEN WE HAVE A REASONABLE NUMBER

FOR US TO GO AFTER AND CONSIDER

TRUE CANDIDATE VARIANTS.

IT'S A VERY MUTATION IS THE WAY

WE LOOK AT IT.

THOSE FILTERS INCLUDE LARGELY

FAMILY MEMBERS SO IF THERE'S A

SIBLING WHO IS UNAFFECTED AND

HAS EXACTLY THE SAME VARIANT OR

SET OF VARIANTS, THEN THOSE

VARIANTS ARE NOT GOING TO BE

PATHOGENIC.

THAT'S THE WAY WE FILTER THINGS.

WE ALSO USE DATABASES OF KNOWN

BENIGN VARIANTS AND ALSO SOME

OTHER ONES THAT WE KNOW ARE NOT

CAUSING THIS PARTICULAR DISEASE

BUT MAYBE CAUSING ANOTHER ONE.

SO WE DO WHOLE EXSOME SEQUENCING

AND THE COMBINATION OF MILLION

SNP ARRAYS AND EXONLY SEQUENCING

IS BENEFICIAL.

NOW WHAT I WANT TO DO IS GIVE

YOU AN EXAMPLE OF EACH OF THE

THREE GENETIC ANALYSES THAT WE

DO.

THE FIRST ONE DOES NOT INVOLVE

THOSE MASSIVE SEQUENCING, REALLY

INVOLVES CLINICAL EXAMINATION.

ONE OF THE ADVANTAGES OF THE

UNDIAGNOSED DISEASE PROGRAM IS

THAT THE PHENOTYPING IS REALLY

EXCELLENT HERE AT THE NIH

CLINICAL CENTER.

WHERE PATIENTS DON'T HAVE TO

PAY.

WE HAVE MINDS OF RESEARCH

SCIENTISTS INVOLVED WITH GOOD

CLINICIANS.

SO WE HAVE TWO KIDS, BROTHERS

AND SISTERS, BROTHER AND SISTER

WHO HAS FACIAL DISMORE OF IMS,

DELAY, ET CETERA AND THE M RIRKS

SHOWED ATROPHY AND THE LABS WERE

NORMAL INCLUDING TRANSFERNT

FOCUSING WHICH IS A STANDARD

TEST FOR MEASURING CONGENITAL

DISORDERS OF GLYCOSYLATION OR

DETECTING THEM BECAUSE IT HAS

END LINKED SUGARS ON IT,

ACTUALLY FOUR OF THEM.

THOSE SUGARS IN ACID IS CHARGED

THEREFORE, THE NUMBER OF ACID

ALSO TELL YOU -- WILL DICTATE

HOW FAR THE PROTEIN WILL MIGRATE

ON FOCUS AND GLYCOSYLATION ARE

PROBLEMS IN PUTTING THOSE SUGARS

ON TO THE GLYCO PROTEIN.

THEREFORE, A CONGENITAL DISORDER

IS DECKED BY GLYCOSYLATION

FOCUS.

HERE ON GLYCAN SEND OUT BECAUSE

OF INDEX OFSIS SPITION BY LYNNE

WOLF WAS POSITIVE.

IN FACT IT WAS POSITIVE ON LYNNE

LAYER CHROMATOGRAPHY FOR THIS

TETRA SACCHARIDE WHICH YOU SEE

HERE.

THIS IS THIN LAYER

CHROMATOGRAPHY, THE DETECTION IS

RESOURCENAL.

THIS TETRA SACCHARIDE WAS

INVESTIGATED BY MASS SPEC AND

FOUND TO BE A GLUTE 3 MAN 1 AND

THAT SUGGESTED THAT THIS COULD

BE A GLUCOSIDASE 1.

UPON GENETIC ANALYSIS THE

PATIENTS WERE COMPOUND

HETEROZYGOTES FOR MUTATION IN

THE GLUCOSIDASE GENE MAKING THIS

GLYCOSYLATION TYPE 2B ONLY THE

SECOND AND THIRD PATIENTS IN THE

WORLD DESCRIBED WITH THIS.

AND THE AN INTERESTING INSIGHT

THAT CAME FROM THIS IS THAT

THESE INDIVIDUALS HAD HYPOGAMMA

GLOBULINEMIA.

BECAUSE THEY ARE END LINKED

GLYCO PROTEINS.

THEY HAD ENOUGH LOWERING OF

GAMMA GLOBULINS TO BE

SUSCEPTIBLE TO INFECTIONS.

YET THEY WEREN'T GETTING

INFECTIONS.

THE INTERESTING ISSUE IS THAT

THE REASON WHY IS BECAUSE

ORGANISMS ALSO REQUIRE GLYCO

PROTEINS ON THE SURFACE OF CELLS

IN ORDER TO GET INTO THOSE CELLS

AND TO BE RECOGNIZED AND TO

INVADE THE CELL.

SO THIS IS A GREAT INTEREST TO

THE THE NIAID COLLEAGUES.

IT GIVES AN EXAMPLE HOW A

PROGRAM LIKE THE UNDIAGNOSED

DISEASE PROGRAM IS ABLE TO

DISCOVER NEW PATHWAYS AND NEW

ENTITIES THAT ARE OF INTEREST TO

OUR COLLEAGUES.

SO THAT WAS AN EXAMPLE OF A

GENETIC DISEASE THAT WAS

DETECTED BY CLINICAL ACUMEN AND

STANDARD TESTING.

SPECIALIZED TESTING BUT

AVAILABLE COMMERCIALLY.

THE NEXT IS AN EXAMPLE OF A

DISORDER THAT WAS DECKED THROUGH

MILLION SNP ARRAY NAILSIS.

THE FAMILY WAS THIS RIGHT HERE

FROM THE KEN OHIO REGION WHICH

FIVE ADULTS ALL HAD THE SAME

DISORDER WHICH INVOLVED

CLOTCATION SO THEY HAD

DIFFICULTY WALKING ANY MORE THAN

ONE TO FIVE BLOCKS AND THEY WERE

ACTUALLY PRODUCTS OF PARENTS WHO

WERE THIRD COUSINS.

I'LL SHOW YOU ON THIS SLIDE WHAT

THEY HAD.

THEY HAD CALCIFICATION OF THEIR

FEMORAL AND POP TEAL ARTERIES,

AND THIS IS -- THERE'S NO

CONTRAST BEING USED HERE SO THIS

IS WHAT THEIR ARTERIES LOOKED

LIKE.

IT'S NO WONDER THEY HAVE

PROBLEMS WITH CIRCULATION.

AND WE'RE ACTUALLY SURVIVING

BASED ON COLLATERALS.

HERE IS A PA OF THAT.

HERE AGAIN IN THE PO PLA TEAL

ARTERIES.

AND THE DORSALIS PETIS AND

CALCIFICATION IN JOINT HANDS OF

THE FEEL, THE META CARPAL

>>MR. FRANCE:JEEL JOIN HOO --

PHARYNGEAL JOINT.

WE WERE LOOKING FOR A REGION OF

HOMOZYGOSITY SHARED BY FIVE

AFFECTED SIBLINGS AND NOT THE

PARENTS.

SO THAT WAS -- THERE WAS ONLY

ONE REGION IN THE ENTIRE

3.2 BILLION BASES OF THE HUMAN

GENOME, THAT WAS RIGHT HERE ON

CHROMOSOME 6 WHERE THESE FIVE

INDIVIDUALS WERE LACKING IN

HETERO ZYGOSITY AND HERE THE

PARENTS HAD HETERO ZYGOSITY SO

THIS REGION WAS A CANDIDATE

REGION.

IT HAD 92 GENES IN IT.

FORTUNATELY WE HAVE

COLLABORATORS IN HEART LUNG AND

BLOOD FAMILIAR WITH A PARTICULAR

GLEEN THAT REGION WHICH ENCODES

AN ENZYME CALLED CD 73.

CD 73 CONVERTS ADENOSINE

MONOPHOSPHATE TO ADENOSINE AND

INORGANIC PHOSPHATE.

AND IT'S PRESENT IN THE VASCULAR

ENDOTHELIAL CELLS.

SO WE'RE GOING TO TELL A STORY

HERE.

AFTER FINDING THIS REGION AND

FINDING A CANDIDATE GENE,

SEQUENCE THE GENE AND FOUND THE

FAMILY THAT I MENTIONED HAD

NON-SENSE MUTATIONS IN IT AND

HOMOZYGOUS NON-SENSE MUTATIONS

AND ITALIAN FAMILY THAT

DR. CLEDA DETECTED HAD MISSENSE

MUTATIONS, AND THERE WERE

COMPOUND HETEROZYGOUS MUTATIONS

IN A THIRD WOMAN IN A FAMILY

THAT DR. ROBERT NUSSBAUM

REFERRED TO US.

THE EXPRESSION IN THE -- OF THIS

GENE IN THE FIBROBLAST OF OUR

TWO PATIENTS WAS MUCH LOWER

COMPARED TO NORMAL.

THE AMOUNT OF PROTEIN WAS

VIRTUALLY INDISCERNIBLE LINE

HERE.

THE AMOUNT OF ENZYME ACTIVITY

WAS MUCH REDUCED.

AND THIS ENZYME ACTIVITY WAS

RESCUED BY TRANSDUCTION WITH A

VECTOR THAT CONTAINS CD 73, THE

MISSING ENZYME.

THESE AMOUNTS OF ENZYME ACTIVITY

IN THE MUTANT MUTATIONS WHEN PUT

INTO A TRANSLATION SYSTEM.

THIS WAS THE NORMAL.

SO THIS IS GOING TO BE A CD 73

EFFECT.

IN ADDITION THE FIBROBLAST IN

CULTURE COMPARED TO CONTROL, THE

MUTANT HAD INCREASED STAINING

FOR ALKALINE PHOSPHOTASE,

MITIGATED BY GIVING ADENOSINE,

IT'S THE PRODUCT OF THE MISSING

ENZYME.

DOWN HERE ARE FIBROBLASTS FROM A

CONTROL AND FIBROBLASTS FROM THE

AFFECTED INDIVIDUAL TREATED WITH

ALLO ZERIN RED TO DETECT CALCIUM

CRYSTALS.

SO THERE'S CALCIFICATION IN THE

CULTURED CELLS.

AND THAT IS ACTUALLY RESCUED BY

CD 73 TRANSDUCED IN A

LENTIVIRUS.

ALSO SOMEWHAT BY ADENOSINE AND

BY LAVAMASOL, AN ALKALINE

PHOSPHOTASE INHIBITOR.

SO WHAT COMES FROM THIS IS A

MODEL IN WHICH ORDINARILY

ADENOSINE IS PRODUCED ON THE

SURFACE OF THE VASCULAR

ENDOTHELIAL CELLS.

IT INTERACTS WITH ADEN SEEN

RECEPTORS, CAUSES SECONDARY

INTRACELLULAR MESSENGERS WHICH

INHIBIT TISSUE NON-SPECIFIC

ALKALINE PHOSPHOTASE.

IF YOU DON'T INHIBIT THE TISSUE

NON-SPECIFIC ALPHA PHOSPHOTASE,

THIS BUILDS UP AS WE SAW IN THE

STAINED FIBROBLASTS AND IT

CONVERTS PIE ROW PHOSPHATE TO

INORGANIC PHOSPHATE.

THE PIE ROW PHOSPHATE IS

NORMALLY INHIBITORY OF

CALCIFICATION AND THE INORGANIC

PHOSPHATE IS STIMULATORY OF

CALCIFICATION.

SO THAT'S HOW THESE PATIENTS GET

THEIR CALCIFICATION.

THEN ALSO MEANS THAT WE CAN

TREAT WITH ALKALINE PHOSPHOTASE

INHIBITORS INCLUDING

PHOSPHONATES AND HEART LUNG AND

BLOOD INSTITUTE HAS A PROTOCOL

TO DO THAT USING CALCIUM SCORE

IN THE VESSELS AND OUTCOME

PARAMETER.

IN THIS FASHION WE HAVE MADE

DIAGNOSES OF THAT ARE LISTED

RIGHT HERE.

SO INCREDIBLY RARE DISORDERS.

A FEW IN THE WORLD.

MAYBE 50 IN THE WORLD OR.

SO A LOT OF THESE DIFFERENT

DISORDERS.

NO WONDER THEY WEREN'T DIAGNOSED

BEFORE THEY CAME TO US.

AND AGAIN, THERE'S NO REASON WHY

PEOPLE IN THE AUDIENCE SHOULD

KNOW ANY OF THESE DISEASES.

I WILL SAY THAT IF YOU KNOW

CERTAIN NUMBER OF THESE

DISORDERS YOU'RE NOT GETTING OUT

ENOUGH.

YOU NEED TO HAVE A HOBBY.

SO MORE DISORDERS WITH CAUSATIVE

GENES.

SOME ARE MITOCHONDRIAL

DISORDERS.

YOU CAN SEE RIGHT UP THERE.

THERE WILL BE MORE AND MORE

BECAUSE REALLY MITOCHONDRIAL

DISORDERS FALL INTO THE GROUP

DIFFICULT TO DIAGNOSE AND WILL

COME TO US ON OCCASION.

THEN THERE ARE OTHER GROUPS OF

DISORDERS WHERE BECAUSE WE SEE A

SELECT GROUP OF PATIENTS WE SEE

SEVERAL WHO HAVE THE SAME

CONSTELLATION OF FINDINGS

MENTIONED HERE FACIAL ONSET

SENSORY MOTOR NEUROPATHY,

PERIODIC FEVERS WITH CSF, WITH

THERMAL ALGIA IN THE FACE,

TRANSMITTER DEFECTS.

SO THEN WE HAVE OTHER INDIVIDUAL

WHOSE REMAIN MYSTERIES.

HERE IS A LITTLE GIRL WITH

HYPOTONEIA, LOOK AT HER NERVES.

THIS RIGHT HERE IS THE SCIATIC

NERVE, AS BIG AS HER FEMUR.

THIS RIGHT HERE IS HER BRACHIAL

NERVE, AS BIG, AS TALL AS HER

VERTEBRA.

SO WE DON'T KNOW WHY THIS IS.

BUT SHE PROBABLY HAS SOME NEW

DISORDER INVOLVING THE NERVOUS

SYSTEM, IT'S OUR JOB TO FIND OUT

WHAT THAT MIGHT BE.

LAST THING I'LL MENTION BRIEFLY,

WE'RE TRYING TO ADDRESS THE

PUNITIVE MITOCHONDRIAL DISORDERS

WITH THERAPY IN SOME OF OUR

PATIENTS.

WE'RE TRYING TO DO THAT WITH

EPIPI 743, A LITTLE BIT LIKE

CO-ENZYME Q 10 BUT WITH A

DIFFERENT REDOX POTENTIAL,

SELECT ELECTRONS MORE READILY

THEN CO-ENZYME Q SO 10.

IT'S IN THE CYTOPLASM SO IT CAN

SOP UP ELECTRONS THERE INTO THE

MITOCHONDRIA.

THIS IS THROUGH EDISON

PHARMACEUTICALS AND THEY HAVE

PROVIDED THE NEXT TWO SLIDES.

SO THEY HAVE AN." ASSAY TO MEASURE

CELL VIABILITY.

AND CELL VIABILITY AS A FUNCTION

OF OXIDATIVE INJURY, SO IN OTHER

WORDS THEY GIVE CHEMICALS THAT

ARE OXIDATIVE STRESSORS AND THEY

LOOK AT THE VIABILITY AND IT

GOES DOWN IN A PATIENT WHO HAS

SURF 1 DEFECT, THAT

MITOCHONDRIAL DISORDER, IT DOSE

DOWN AT LOWER CONCENTRATION OF

OXIDATIVE INJURY THAN FOR THE

WILD TYPE CELLS.

AND NOT ONLY THAT, BUT WHEN YOU

TAKE THOSE SURF 1 CELLS AND

TREAT THEM WITH EPI743 AT THESE

DIFFERENT CONCENTRATIONS IN

NANOMOLAR, IT'S RESCUED.

THE VIABILITY INCREASES.

AND THE EC-50 IS 27 NANOMOLAR

HERE.

WHEREAS CO-ENZYME Q HAS NO

EFFECT UP TO 1,000 NANOMOLAR.

SO ONE OF OUR PATIENTS IN THIS

CASE 1166 WHO HAS INCREDIBLE

PARKINSONIAN SYMPTOMS AT AGE 7,

DIFFICULTY WALKING AND TALKING,

THIS INDIVIDUAL ALSO

DEMONSTRATES, HE'S IN YELLOW

HERE, ALSO DEMONSTRATES THE

REDUCED VIABILITY OF THE CELLS

UNDER OXIDANT STRESS AND ALSO

DEMONSTRATES THE RESCUE BY

EPI743.

SO WE HAVE A PROTOCOL THAT IS

NOW BEEN ACCEPTED BY THE

INSTITUTIONAL REVIEW BOARD WITH

STIPULATIONS, AND WILL BE

INITIATING THAT SHORTLY TO TREAT

PATIENTS IN A PLACEBO CONTROLLED

FASHION.

WITH OUTCOME MEASURES OF THE NEW

CAST L MITOCHONDRIAL SCORE.

SO WE'RE HOPEFUL THAT THIS DRUG

WHICH HAS HAD SOME EFFICACY IN

LABOR RERED TEAR MYOPATHY AND

ENCEPHALOPATHY THIS WILL BE OF

SOME BENEFIT.

SO THE WORLD BREAKS EVERYONE AND

AFTERWARDS SOME ARE STRONGER IN

THE BROKEN PLACES AND IT'S OUR

HOPE WE CAN MAKE SOME OF OUR

PATIENTS STRONGER AFTER THEY

HAVE BEEN BROKEN BY SOME OF

THESE DISORDERS.

SO WE HAVE A FAIRLY SMALL

AUDIENCE.

I'M FINISHED.

WE CAN TAKE SOME QUESTIONS,

OTHERWISE WE CAN PROCEED.

STEVE, WOULD YOU LIKE ME TO

INTRODUCE THE NEXT SPEAKER?

[APPLAUSE]

>> THANK YOU.

PEOPLE WATCHING ON ONLINE, YOU

CAN ASK QUESTIONS AND I'LL GET

THEM UP AT BREAKS.

THE NEXT SPEAKER WILL BE HARVEY

MUDD FROM THE NIMH WHO WILL TALK

TO US A DISORDER ABNORMALITIES

OF ADAMANT MITOCHONDRIAL DNA

DEPLETION DISORDERS.

HARVEY.

>> THANK YOU, STEVEN.

THIS MORNING MY TALK IS GOING TO

BE REALLY FOCUSED ON TWO

PATIENTS WITH DIFFERENT

MITOCHONDRIAL DEPLETION

DISORDERS.

THESE PATIENTS WERE REFERRED TO

ME BECAUSE I HAVE BEEN WORKING

MANY YEARS ABNORMALITIES OF

AMINO ACID METABOLISM AND I HEAR

ABOUT PATIENTS WITH ELEVATED ME

THIONEINESCH THESE PATIENTS WERE

INITIALLY FOUND TO HAVE INITIAL

METABOLIC ABNORMALITY ELEVATIONS

OF ME THIONEINE.

I'M NOT GOING TO GO INTO THE

EVIDENCE THAT THE -- OKAY.

I'M NOT GOING TO TALK ABOUT THE

EVIDENCE WHICH PROVES THEY HAVE

THESE DIFFERENT MITOCHONDRIAL

DELETIONS, THE FIRST PATIENT HAD

MPV FOR 17, THE SECOND PATIENT

DGU OK DEPLETION AND THE

EVIDENCE WAS.„ CAME ALONG LATER

AND THE EVIDENCE WAS PRODUCED BY

LEE WONG AT BAILOR UNIVERSITY

AND IF SOMEONE -- THE EVIDENCE

HAS BEEN PUBLISHED SO I WON'T GO

INTO THE DETAILS OF THE

MITOCHONDRIAL DEPLETION.

I'M JUST GOING TO TALK ABOUT THE

REFLECTIONS OF THIS

ABNORMALITIES ON THE METHIONEINE

METABOLISM.

IT MEANS WE HAVE TO TALK ABOUT

METHIONEINE METABOLISM AS A

COMPLICATED PATHWAY BUT I

SUMMARIZED IT HERE.

METHINK NEEN IS TAKEN INTO THE

DIET, CONVERTED SEQUENTIALLY TO

ADEN SEAL METHIONEINE USING A

MOIETY FROM ATP.

ADEN SIL METHIONEINE IS PROBABLY

THE MOST USEFUL VERSATILE

COMPOUND IN ALL OF BIOCHEMISTRY.

IT'S IN HUMANS, IT'S IN A METAL

DONOR FOR MAYBE 50 OR 70 ME THAT

WILL TRANSFERASES.

THE METHYL GROUP IS TRANSFERRED

TO CONVERT SOME COMPOUNDS TO A

METHYLATED COMPOUND.

THE COMMON PRODUCT OF THAT

REACTION, THOSE REACTIONS IS

ADEN ZEAL HOMOSISTINE WHICH JUST

IS THE SAME LACKING THE METHYL

GROUP.

THAT IS CLEAVED TO HOMOSISTINE

AND ADENOSINE.

HOMOSISTINE IS CONVERTED TO

METHIONEINE AND ULTIMATELY THE

CELL ENDS UP AT 15 AND SO ON.

THERE ARE PATHWAYS FOR GETTING

THE HOMOSISTINE BACK TO

METHIONEINE USING ME TAIL GROUP

FROM METHYL TETRA HYDRO FOE LICK

ACID.

THE MAJOR CONSUMERS OF -- I

SHOULD POINT OUT FOR THE

MITOCHONDRIAL AUDIENCE THAT

THERE IS A TRANSPORTER WHICH HAS

BEEN SEQUENCED AND CLONED.

WHICH TAKES ADEN SEAL

METHIONEINE INTO THE

MITOCHONDRIA.

ADENOSIL METHIONEINE BEING A BIG

MOLECULE AND MULTIPLE CHARGES

DOES NOT -- IT NEEDS A SPECIFIC

TRANSPORTER.

THERE ARE METHYL TRANSFERASES

WITHIN THE MITOCHONDRIAL, THERE

ARE SEVERAL WHICH UTILIZE IT,

CONVERT IT TO ADENSIL

HOMOSISTINE.

AND WE'LL COME BACK TO THIS

AFTER -- IN A MINUTE.

THE MAJOR CONSUMERS OF ADENOSIL

METHIONEINE IN NORMAL HUMANS ARE

THREE REACTIONS, CREATININE

SYNTHESIS, METHIONEINE PLUS

ACETATE GOING TO CREATININE AND

ADENSIL HOMOSISTINE.

THIS IS A QUANTITATIVELY --

BECAUSE CREATIN IS A

NON-ENZYMEATIC LOSS OF CREATIN

EVERY DAY IN YOUR MUSCLES, THAT

MEANS YOU HAVE TO CONTINUE TO

SYNTHESIZING SUBSTANTIAL AMOUNTS

OF CREATIN EVERY DAY, IT'S MORE

SIGNIFICANT IN MALES THAN

FEMALES BECAUSE OF THE LARGER

MUSCLE MASS.

THE SECOND AND MOST IMPORTANT

CONSUMER IS PHOSPHOTILE CHOLINE

SYNTHESIS, 3 ADENOSIL TRIENES

ARE USED TO GET THREE METHYL

TRANSFERS ETHANOL AMINE TO MAKE

PHOSPHOTILE CHOLINE.

THE THIRD REACTION WHICH IS

QUANTITATIVELY IMPORTANT AND

WHICH WILL PLAY A LARGE ROLE IN

THIS TALK IS GLYCENE CYCLE,

GLYCENE IS METHYLATED BY

ADENOSIL METHIONEINE TO FORM

METHYL GLYCENE, OTHERWISE KNOWN

AS SARCOSENE.

THIS REACTION BECOMES IMPORTANT

WHEN WE QUANTITATE -- WHEN ONE

TAKES IN MORE METHIONEINE THAN

NEEDED FOR THE -- MAJOR

REACTIONS OR IN FACT THE OTHER

THINGS.

AND IN ORDER TO DISPOSE OF THAT

METHIONEINE CONVERTED TO

ADENOSIL METHIONEINE, IN ORDER

TO BE EXPOSED WHICH WOULD BE AN

EXCESS ONE METHYLATES GLYCENE.

THIS SLIDE SHOWS IN DETAIL

ADENOSIL ME THIONEINE METHYLATES

GLYCENE TO FORM METHYL GLYCENE

OR SARK SCENE WHERE THE

SARKOSENE IS CONVERTED BACK TO A

FOLATE REACTION TO TBLIE SEEN

AND ONE CARB UNIT.

AND APPARENTLY FUTILE CYCLE BUT

THIS ALLOWS ONE TO GET RID OF

THE EXCESS ADENOSIL METHIONEINE

FORMED WHEN YOU TAKE IN MORE

METHIONEINE THAN ONE NEEDS FOR

THESE NECESSARY REACTIONS.

WE'LL COME BACK TO THE ROLE OF

THIS IN THE MITOCHONDRIAL

SITUATION THE END OF THE TALK.

THE PATIENT -- THE FIRST -- AT

THE TIME THE PATIENT NUMBER ONE

WHICH I'M GOING TO FOCUS ON WAS

REFERRED TO ME ABOUT 11 YEARS

AGO.

BECAUSE OF ELEVATED METHIONEINE,

WHAT WE KNEW AT THAT TIME ABOUT

ABNORMALITIES.

WE FOUND HE HAD AN ELEVATED

METHIONEINE AND ALSO VERY HE

ELEVATED ADENOSIL METHIONEINE,

IN THE PLASMA IS ONLY ABOUT 100

NANOMOLAR AND SO IT'S NOT

MEASURED, IT TAKES A SPECIAL

TECHNOLOGY, TECHNIQUE TO MEASURE

IT, THESE MEASUREMENTS HAVE BEEN

DONE BY MY COLLEAGUE DR. CONRAD

WAGNER AT VANDERBILT UNIVERSITY.

SO IT'S A GREAT ADVANTAGE WHEN

ONE HAS AN ELEVATED METHIONEINE

TO MEASURE THE ADENOSIL

METHIONEINE AND ALSO MEASURES

THE ADENOSIL HOMOSISTINE.

AT THAT TIME WORKING ON THIS

PATIENT WHAT WE -- THE GENETIC

PROBLEMS WE KNEW ABOUT WERE --

THESE ARE LISTED SEQUENTIALLY IN

THE ORDER CONVERT ME THIONEINE

TO ADENOSIL ME THIONEINE WHEN

ONE ACCUMULATES EXCESS

METHIONEINE WITHOUT ELEVATION OF

ADENOSIL METHIONEINE AS A NORMAL

VALUE.

THE IMPORTANCE OF THE GLYCENE

METHYL TRANSFERASE IS

ILLUSTRATED AT THIS TIME WE KNEW

ONLY -- WE HAD JUST RECENTLY

IDENTIFIED TWO ITALIAN SIBS WITH

EFFICIENCIES OF GLYCENE AND

METHYL TRANSFERASE.

THEY ACCUMULATED ADENOSIL

METHIONEINE.

AND THAT FEEDBACK INHIBITS

CONVERSION TO METHIONEINE TO

ADENOSIL METHIONEINE, ELEVATED

METHIONEINE IS ALSO ELEVATED

TESTIMONY KEY IN THESE PATIENTS

WAS THAT THERE IN SPITE OF

ELEVATED ADENOSIL METHIONEINE

THE SARKOSIL LEVELS WERE NORMAL.

THE OTHER REACTIONS WE KNEW

ABOUT AT THE TIME, WERE ADENOSIL

IN HOMOSISTINE HYDROLASE

DEFICIENCY ONE CAN BREAK DOWN

ADENOSIL HOMOSISTINE.

AND THAT THEN 'CUMULATES TO

ADENOSIL HOMOSISTINE IS

INHIBITOR OF MOST ADENOSIL ME

THIONEINE TRANSMETHYL PATIENT

REACTIONS, THAT CAUSES ELEVATION

OF ADENOSIL ME THIONEINE WHICH

THEN CAUSES THE ELEVATION OF ME

THIONEINE.

SO FIRST ONE OF THESE REACTIONS

THE SYNTHASE WHEN THERE'S A

BLOCKADE IN THE CONVERSION OF

THE HOMOSISTINE WHEN ACCUMULATES

EVERYTHING BACK UP THE LINE.

WHEN A PATIENT CAME ALONG THE

PATIENT NUMBER ONE WE FOUND HAD

AN ELEVATION OF ME THIONEINE,

ELEVATION OF ADENOSIL ME

THIONEINE BUT DOWNSTREAM

METABOLITES WERE NORMAL.

THIS IS SAME TRUE OF THE SECOND

PATIENT THAT I WILL DISCUSS WHO

CAME ALONG SEVERAL YEARS LATER.

I WON'T GO INTO AS MUCH DETAIL

BUT AT THAT POINT IN TIME

PATIENTS HAD PARENTS WHICH

SUGGESTED OR SAME ONE FINDS IN

GLYCENE AND METHYL TRANSFERASE

EFFICIENCY.

BUT NOT IN THE OTHER REACTIONS,

FURTHER ABNORMALITIES.

ONE EXCEPTION HERE HIGH SISTER

THIONEINE.

I'LL COME BACK TO THAT AT THE

END OF THE TALK.

PATIENT 1, 11 YEARS AGO SAID

THIS IS THE THIRD PATIENT IN THE

WORLD WITH GLYCENE AND METHYL

TRANSFERASE DEFICIENCY.

SO WE SEQUENCED THE METHYL

TRANSFERASE GENE AND IT TURNED

OUT TO BE ABSOLUTELY NORMAL.

PERFECTLY NORMAL.

SAME WITH PATIENT NUMBER TWO

CAME ALONG SEVERAL YEARS LATER

AT THE SAME PATTERN.

AND AGAIN, SEQUENCED GLYCENE IN

METHYL TRANSFERASE GENE TURNED

OUT TO BE ABSOLUTELY NORMAL.

SO WE WERE LEFT WITH A

LONG-STANDING PUZZLE, WE LOOKED

AT VARIOUS OTHER POSSIBILITIES

FOR PATIENT 1, COULDN'T HAVE A

VARIETY AT DIAGNOSIS.

NEXT SLIDE SHOWS A LITTLE MOFER

DETAIL T STORY ON THIS PATIENT

ONE.

YOU CAN SEE HERE THE GROWTH

MEASUREMENTS YOU CAN SEE IT WAS

PERFECTLY ALL RIGHT, ABOUT SIX

MONTHS WE HAD A DECREASE IN HIS

GROWTH RATE.

HEIGHT AND WEIGHT CELL DOWN

COMPARED TO NORMAL, THAT GOT

WORSE AND WORSE AS HE GOT, THESE

ARE AGES IN MONTHS.

THE FIRST -- HE WAS FINALLY

REFERRED FOR -- HERE FOR

METABOLIC STUDY WHEN WE FOUND AT

17 MONTHS HE HAD THIS ELEVATION

OF METHIONEINE AND ELEVATION OF

ADENOSIL METHIONEINE WHICH WE

DISCUSSED.

FOR TWO VARIOUS INVESTIGATIONS

WE COULDN'T ARRIVE AT AN ANSWER,

AS WE FOLLOWED THEM ALONG,

SEEMED TO BE SOMEWHERE AGE 3 OR

MORE YEARS, METHIONEINE AND

ADENOSIL METHIONEINE, DECREASED

MARKEDLY AND BY AGE 5 YEARS THEY

WERE PRACTICALLY DOWN TO NORMAL.

THE LAST MEASUREMENT WE HAVE

HERE IS AGE 6 1/2 YEARS.

HE WAS AT THE TIME HE WAS 7

CRERS -- WE DIDN'T KNOW WHAT THE

STORY WAS HERE.

AT AGE 7 HE WAS ABOUT TO ENTER

SECOND GRADE.

HE WAS SMALL BUT PERFECTLY

NORMALLY MENTALLY PERFECTLY

NORMAL AND INTELLIGENT CHILD.

AT AGE 7 HE SUDDENLY VOMITED

BLOOD, TURNED OUT HE HAD

HEPATOCELLULAR CARCINOMA.

HE HAD A LIVER TRANSPLANT AND

DIED AT AGE 8 1/2 YEARS, OF THE

LIVER PROBLEM.

WHAT CAUSED -- SINCE WE HAVE THE

LIVER AVAILABLE, THE REASON FOR

THINKING EVEN OF A MITOCHONDRIAL

PROBLEM HERE WAS THE ONLY OTHER

CONDITION THAT I KNOW OF AT

LEAST THAT CAUSES TRANSIENT

ELEVATIONS OF METHIONEINE WHICH

PASS AWAY IS SI TRON DEFICIENCY,

A COMMON MITOCHONDRIAL -- WHICH

IS A FAIRLY PREVALENT

MITOCHONDRIAL PROBLEM IN

ORIENTAL CHILDREN, IT'S A

TRANSPORTER PROBLEM, ASPARTATE,

BUT THAT DOES CAUSE A TRANSIENT

ELEVATION OF METHIONEINE, AS FAR

AS I KNOW NOBODY HAS MEASURED

ADENOSIL METHIONEINE, WE WOULD

LIKE TO MEASURE THAT THE IN A

CHILD WITH SITRON DEFICIENCY

WHEN THEY'RE HYPERMETHIONEINE

ANEMIC.

FURTHER SPECULATION LEE WONG

ACCEPTED TO LOOK AT THE LIVER OF

THIS CHILD AND IT TURNED OUT

THAT WE INDEED WAS ONE WITH MPV

17 DEPLEASE.

THERE'S OTHER EVIDENCE HERE

WHICH IS NOT HIGHLY SUGGESTIVE

OF A MITOCHONDRIAL PROBLEM.

THE SECOND PATIENT CAME ALONG

LATER.

HE HAD A MORE -- HIS CLINICAL

STORY WAS RETARDED, HEA AD

GROWTH IN MENTAL RETARDATION

PROBLEMS.

HE DIED AT 8 1/2 MONTHS.

HE ALSO HAD THE PATTERN THAT I

SHOWED YOU WITH ELEVATED

METHIONEINE IN ADENOSIL

METHIONEINE BUT A NORMAL GLYCENE

AND ME THEY WOULD TRANSFERASE.

ONE OF THE -- BOTH OF THESE

CHILDREN HAD SOME LIVER PROBLEMS

AND ONE OF THE THINGS THAT IS

OFTEN PUT FORWARD AS A CAUSE OF

ELEVATED METHIONEINE IS

HEPATOCELLULAR DYSFUNCTION.

HERE WHAT I WANT TO SHOW HERE IS

THESE PATIENTS HAD ELEVATIONS IN

METHIONEINE WHICH WERE

EXTRAORDINARILY HIGH.

THESE ARE REPORTS PUBLISHED

PAPER FOUND IN THE LITERATURE, I

GOT MOST BUT THERE MAYBE OTHERS.

PEOPLE WITH REPORTED METHIONEINE

CONCENTRATIONS IN PATIENTS WITH

A VARIETY OF LIVER DISEASES,

VERY SERIOUS MINOR, THESE ARE

THE MEAN THINGS.

THESE ARE STANDARD DEVIATIONS

AND THE HIGHER POINTS.

BUT YOU CAN SEE WITH LIVER

DISEASE, YES, THERE IS ELEVATION

OF METHIONEINE WHICH IS NORMALLY

ABOUT HIGH POINT OF ABOUT 30 OR

45.

BUT PATIENTS IN QUESTION WERE

MUCH MORE SEVERE ELEVATION IN

METHIONEINE THAN WE THOUGHT AND

ONE COULD PROBABLY BE ACCOUNTED

FOR BY THEIR LIVER DISEASE.

SO WHEN WE FINALLY HAVE THE THE

EVIDENCE SECOND PATIENT WAS

STUDIED BY LI WONG AND SHOWN TO

HAVE THE DGOK DEPLETION,

MITOCHONDRIAL DEPLETION

SYNDROME.

HERE IS WHAT WE THINK IS A

SITUATION, POSTULATED MECHANISM

NOW.

FIRST REMEMBER THERE'S A

TRANSPORTER ADENOSIL METHIONEINE

AND ADENSIL DEPENDENT METHYL

TRANSFERASES.

IF THE MITOCHONDRIAL DEPLETED OR

POSSIBLY DYSFUNCTION, THE ADO

ADENOSINE METHIONEINE MAY

ACCUMULATE IN THE CYTOPLASM AND

LEAK INTO THE LAS MA BECAUSE

THEY DON'T HAVE THE NORMAL HOST

IN THE MITOCHONDRIA.

THE PROBLEM WHICH KEPT US

WONDERING MANY YEARS, IF THERE'S

EXCESS -- WHY IS THE EXCESS

ADENOSIL METHIONEINE NOT

DISPOSED OF BY GLYCENE SARKOSENE

PATHWAY WHICH IT WOULD BE IN

NORMALS.

I CAN SAY THAT WE DO KNOW OF

OTHER -- IT'S -- LET'S SAY

PATIENTS WITH DEFECTS IN ACETATE

METHYL TRANSFERASE IN MAJOR

CONSUMERS OF ADENOSIL

METHIONEINE DO NOT HAVE

ELEVATIONS OF ADENOSIL

METHIONEINE.

SAME IS TRUE OF THE EXPERIMENTAL

ANIMAL RATS WITH PHOSPHOTILE

ETHANOL AMINE METHYL TRANSFERASE

THAT DO NOT BUILD UP EXCESS

ADENOSIL METHIONEINE IN PLASMA

BECAUSE THEY'RE RID OF BY THE

ACETATE BY GLYCENE AND ME THEY

WOULD TRANSFERASE DEFICIENCY.

FINALLY IT OCCURRED TO US

BELATEDLY THAT THE REASON IS

THAT THESE PATIENTS ARE LEAKING,

PROBABLY LEAKING ADENOSIL

METHIONEINE OUT OF NON-HEPATIC

TISSUES, PRIMARILY MUSCLE OR

BRAIN AND THE GLYCENE METHYL

TRANSFERASE IS EXPRESSED SOLELY,

ALMOST SOLELY IN THE LIVER TO

SOME EXTENT IN THE PANCREAS.

SO IF THE ADENOSIL ME THIONEINE

LEAKS OUT, IT'S NOT GOING --

FROM TISSUES NON-HEPATIC

TISSUES, IT'S NOT GOING TO BE

AVAILABLE FOR DISPOSITION BY THE

GLYCENE AND METHYL TRANSFERASE.

AND IT FITS WELL WITH THE

ELEVATION METHIONEINE BECAUSE IN

NON-HEPATIC TISSUES, THE THERE

ARE TWO FORMS OF METHIONEINE

ADENOSIL TRANSFERASE.

ONE IS TISSUES ARE VERY

SENSITIVE TO ADENOSIL

METHIONEINE.

THESE OUR CURRENT WORKING

HYPOTHESIS OR WHAT'S GOING ON IN

THESE PATIENTS.

AND A COUPLE OF POINTS OUT OF

THIS.

METHYL FLUX IS IN NORMAL HUMANS

HERE.

THESE ARE -- TURN OUT TO BE 15

TO 23 MILIMOLES PER DAY OF

ADENOSIL METHIONEINE CONSUMED.

DONE BY STABLE ISOTOPE STUDIES,

PROBABLY THE MOST RELIABLE

MEASUREMENT.

HOW MUCH IF ONE CALCULATES HOW

MUCH LEAKAGE THERE SHOULD BE

FROM ADENOSIL METHIONEINE TO

ACCOUNT FOR ELEVATION THAT WAS

ENCOUNTERED, IT TUSHES OUT

THERE'S NOT NECESSARILY A VERY

MAJOR -- TURNS OUT THERE'S NOT

NECESSARILY A MAJOR LEAKAGE IN

TERMS OF TOTAL CONSUMPTION OF

THE 15 TO 23 MILIMOLES A DAY.

THE ADENOSIL METHIONEINE IS

CLEARED, HAS THE SAME RENAL

CLEARANCE AS CREATININE AND THIS

CALCULATES OUT TO KEEP ADENOSIL

METHIONEINE AT 500 TO 1,000

NANOMOLAR WHICH IS WHAT THESE

PATIENTS HAD, LEAKAGE MUST BE .2

TO .3 MILIMOLES A DAY.

SO THIS DOES NOT -- THIS IS NOT

MEAN ONE HAS TO POSTULATE THE

FLUX IN THE METHIONEINE IS INTO

MITOCHONDRIA AS NECESSARILY VERY

GREAT.

AND ONE FINAL POINT OF INTEREST,

REMEMBER THE SECOND PATIENT HAS

THIONEINE ELEVATION AND WHICH

DIFFERED FROM THE PATIENT NUMBER

ONE.

MY WORKING HYPOTHESIS TO ACCOUNT

FOR THAT IS THE SECOND PATIENT

HAD SEVERE CEREBRAL SPINAL --

CNS PROBLEMS, IT WAS -- AND

TURNS OUT SISTER THIONEINE BETA,

IN NORMAL HUMANS YOU CAN FORM

CYSTA THIONEINE IN THE BRAIN BUT

THERE'S A VERY SMALL ACTIVITY TO

REMOVE CYSTA THIONEINE FROM THE

BRAIN.

THEREFORE, IF THIS CHILD WITH

CMS PROBLEMS IS FORMING CYSTA

THIONEINE AND CEREBRAL -- NEURAL

-- HE WOULD HAVE ACCUMULATED IT

AND IF -- IN THE NEURAL TISSUE

AND THE CYSTA THIONEINE WOULD

LEAK OUT AND ACCOUNT FOR HIS

HIGH CYSTA THIONEINE.

SO THAT IS THE TWO PATIENTS THAT

I CAN TALK ABOUT, THE BOTTOM

LINE I THINK IS WE HAVE NOT HAD

FURTHER PATIENTS, QUESTIONS ARE

ADENOSIL ME THIONEINE IS

MEASURED RELATIVELY INFREQUENTLY

AND IT TAKES SPECIAL TECHNIQUE.

SO WE WOULD LIKE TO HAVE OTHER

PATIENTS WITH -- TO DO

ACTIVITIES OF THE ADENOSIL

METHIONEINE AND THE CYSTA

THIONEINE IN THEIR PATIENTS TO

FIND TO ESTABLISHED HOW FREAKILY

THESE HE WILL VAGUES OCCUR AND

WHICH DISORDERS THAT ARE MOST

FREQUENTLY AND ARE THESE

TRANSIENT CHANGES WITH AGE THAT

WE SAW IN THE FIRST PATIENT, DO

THEY OCCUR IN OTHER PATIENTS.

SO I WOULD LOVE TO HAVE FURTHER

SAMPLES THAT HELP MAYBE SOMEBODY

IN THE AUDIENCE WILL HAVE SOME

AVAILABLE AN PLEASE GET IN TOUCH

WITH ME.

THANK YOU.

ANY QUESTIONS?

>> ALL RIGHT.

THANK YOU VERY MUCH, HARVEY.

I'M SURE YOU'LL GET FEEDBACK

FROM PEOPLE HERE, FROM PEOPLE

LISTENING.

THERE ARE QUITE A FEW LISTENING.

THE NEXT SPEAKER TODAY WILL BE

ELI SCORVAK WHO WILL ABOUT

TUMORIGENESIS STUDIES IN

KEARNS-SAYRE SYNDROME.

ALEX.

>> THANK YOU FOR INVITING ME.

I WOULD LIKE TO SHARE WITH YOU A

CASE STUDY WHERE MY INSTITUTE

MISSION INSTITUTE OF BIOMEDICAL

ENGINEERING WAS INVOLVED WITH A

TEAM FROM NHGRI.

I'M TALKING ABOUT THERMOGENESIS

STUDY IN KEARNS-SAYRE SYNDROME.

FIRST ABOUT IMAGES WE I PLOY IN

THE STUDY.

YOU CAN SEE (INAUDIBLE) WHICH IS

A DEVICE CAPABLE TO MEASURE THE

TEMPERATURE.

NOT MEASURE BUT IMAGE THE

DISTRIBUTION OF TEMPERATURE.

SO AS YOU RECOGNIZE IT'S REMOTE

MEASUREMENTS FROM THE DISTANCE

COMING FROM ANY LIVING TISSUE

THEY ARE CONVERTED TO THE

TEMPERATURE, CAME IN THE ORDER

OF .01-DEGREE CENTIGRADE.

THE CAMERA CAN OPERATE REMOTELY

AND CAN BE IMAGES OF 640 BY 512

PIXELS CAN BE STORE BY COMPUTER,

CONTINUOUSLY.

SO WE DIRECT THIS CAMERA FOR

DIFFERENT LIVING TISSUE AND ONE

IS AN EXAMPLE OF INTEROPERATIVE

IMAGING DURING BRAIN SURGERY.

YOU CAN SEE CAMERA ON THE RIGHT

IN MY HANDS, IN THE BACKGROUND

YOU CAN SEE THE IMAGE WHERE THE

YELLOW PIXELS SHOW THE VESSELS

OF THE HUMAN CORTEX.

AND THE EARQz YELLOW BECAUSE THESE

VESSELS ARE HOT, COMING FROM THE

CORE OF THE BODY TO SURFACE,

SURROUNDING OF THE CORTEX,

THEREFORE YOU SEE EACH

INDIVIDUAL ON THIS VERY HIGH

RESOLUTION AND IN REAL TIME.

THE OTHER EXAMPLE WE USE

INFRARED CAMERA FOR THE KIDNEY

TRANSPLANTS, YOU CAN SEE THE

KIDNEY ON THE LEFT SIDE ALREADY

PLACED IN THE RECIPIENT.

BUT IT'S NOT PURR FUSED YET.

THE SECOND THING WHICH SHOWED

THE STARTING OF THE DIFFUSION,

AND FULL SCALE PER FUSION AND

YOU CAN SEE DARK SPOT UP LEVEL

SHOWN SOME PROBLEMS PER FUSION

IN THAT AREA WHICH HAPPENS TO BE

(INAUDIBLE).

THIS IS WHY SEVERAL GROUPS NOW

USE FOR INFORMATIONAL --

INVESTIGATIONAL PURPOSES DURING

SURGERY.

ANOTHER ANOTHER EXAMPLE, MY

INSTITUTE AND NHLBI WE WERE

LOOKING FOR SIBLG CELL DISEASE

PATIENTS.

PARTICULARLY ACTION OF THE --

THIS POINT FOREARM, INFUSION OF

ACETYL CHOLINE S AND P, LMNA

TRIED TO CHALLENGE DYSFUNCTION,

THESE PATIENTS RECOGNIZES

MULTIPLE VESSELS IN REAL TIME

WHICH IS OPEN BUT NOT ALL

VESSELS ARE OPEN, THIS IS

SUBJECT OF INTEREST FOR THE

PROTOCOL AT AN AGE.

I SHOW YOU SOME EXAMPLES BUT OUR

RECENT INTEREST WAS RELATED TO

THERMO GENESIS.

NOT THE BLOOD FLOW MEASUREMENTS

THAT WAS DONE PREVIOUSLY BUT

PARTICULARLY THERMO GENIC EFFECT

WHICH IS BROWN SPOT TISSUE.

THEREFORE RECOGNIZE IN 2007

THROUGH PUBLICATIONS THAT HAVE

BROWN SPOTS ON THE BODY AND THIS

IS THE DISPOSITION OF SEVERAL

DEPOTS IN THE BODY.

RIGHT SIDE YOU CAN SEE INFRARED

IMAGE NOW DOWN HERE, THE NIH

WITH MULTIPLE TEMPERATURE

GRADIANTS AND IT'S CLEAR THAT

AREA IN SUB CLAVICLE MUCH MORE

HARDER.

AND SURROUNDING WE DECIDED TO

STIMULATE SEVERAL PATIENTS IN

NORMAL VOLUNTEERS WITH COLD

CHALLENGE TO SEE IF TEMPERATURE

IN SUB CLAVICLE AREA WILL BE

INCREASED AS IT SHOULD BE DONE.

THIS IS WHAT BROWN SPOT IS

DOING.

THIS IS ON THE LEFT SIDE YOU CAN

SEE BACKSIDE IMAGE, RIGHT SIDE

DURING BASELINE.

THIS IS A RESULT.

ON THE RIGHT SIDE.

UPPER LEVEL YOU CAN SEE THE

GRAPH.

WHERE IT SHOWS THE TEMPERATURE

EVALUATION VERSUS TIME AS

BASELINE IMAGING, SAMPLE

MUTATIONS IN TEMPERATURE BUT IN

BLUE SHOW START OF COOLING OF

TWO PATIENTS HAD IN 90-DEGREES

BATH FOR TWO MINUTES YOU CAN SEE

THE VARIATION IN THE TEMPERATURE

OF SUB CLAVICLE AND AREA AND

SURROUNDING.

WHICH IS MORE THAN TWO DEGREES

CENTIGRADE.

THIS IS BASED ON THE

UNDERSTANDING IS REACTION OF

BROWN FAT TISSUE.

SEVERAL CONTROLS FOR EXAMPLE WE

TRY TO WARM PATIENTS HAND YOU

CAN SEE SLIGHT ELEVATION BUT THE

SCALE IS ABSOLUTELY DIFFERENT,

.2-DEGREES DIFFERENT.

WE BUILD THIS POINT DEGREE

DIFFERENCE IS RELATED WITH

OPERATIVE COOLING WHEN PATIENTS

REMOVE THE HAND WET AND SLIDE

COOLING EFFECT.

SO WITH THIS BACKGROUND AND THIS

SMALL OBSERVATION, WE DECIDED TO

MOVE DIFFERENT DIRECTION TO

DETERMINE WHETHER THERMOGENESIS

IN INFRARED CLINICAL AND FIND

THE PHENOTYPES.

AND THEY SERVE AS BROWN MARKER

OF MITOCHONDRIA DISEASE.

SECOND OBJECTIVE IS VERY

AMBITIOUS BY LONG TIME, TO

DEVELOP NON-INVASIVE EASILY

REPEATABLE TESTING 2 CAPABLE TO

IDENTIFY AND MONITOR IN THE

PROGRESSION WITH (INAUDIBLE)

DISEASE IN A POPULATION OF

PATIENTS.

SO WE HAD OPPORTUNITY TO MEASURE

THE TEMPERATURE OF THESE YOUNG

(INAUDIBLE) YEARS OLD AND YOU

CAN SEE THE REPORTED SYMPTOMS.

PRIOR TO IMAGING IN COLD WEATHER

IN TARGETS TO WARM WEATHER.

NO SWEATING, CONDITIONS AND NOW

IDENTIFYING QUANTITY AND --

(INDISCERNIBLE) STUDY.

ONE NEED TO MENTION DURING

INFRARED IMAGING SESSION

PATIENTS REPORTED UNCOMFORTABLE

COLDNESS.

THIS WAS DONE IN PATIENT

BASELINE ADAPTATION AND WHERE

PATIENTS SHOULD LOOK NEAR

DIRECTING (INAUDIBLE) ON THE

LEFT SIDE, YOU CAN SEE IMAGE

HERE FRONT, RIGHT SIDE IT'S

VISIBLE LIGHT IMAGE SAME

PATIENT.

QUITE NICE SYMMETRY IN

TEMPERATURE DISTRIBUTION AS IT

SHOULD BE DONE ON THE FRONT.

AND THIS IS QUANTIFICATION OF

EACH PIXEL, ALL 64,000 PIXELS

ARE QUANTIFIED IN TERMS OF

MEASUREMENTS.

YOU ALSO CAN SEE WHAT WAS FOUND

SOME SMALLER SYMMETRY BETWEEN

LEFT AND RIGHT.

AND IT FEEDS VERY WELL TO WHAT

WAS IN THIS PATIENT POST

SURGERY.

WHAT'S HAPPENED WHEN WE DIRECTED

CAMERA ON THE BACK?

WE DO SEE VERY CLEAR TEMPERATURE

SYMMETRY BETWEEN LEFT UPPER SIDE

OF THE THORAX VERSUS RIGHT.

INITIALLY WE THOUGHT IT MIGHT BE

SOME STRANGE ARTIFACT.

WE HAVE PATIENT MOVE BACK AND

FORTH DIFFERENT TIMES TO

DIFFERENT SIDES OF THE ROOM

DURING 20 MINUTES, THE SPOT WAS

STILL OF THE SAME PLACE.

THE DIFFERENCE IS 1.0-DEGREE,

HUGE DIFFERENCE IN TERMS OF

SENSITIVITY OF OUR CAMERA AND IN

TERMS OF PHYSIOLOGY IN GENERAL.

WITH THIS FINDING, THIS IS WHAT

STUDY WAS DONE IN 2011.

BETWEEN 2011 AND 2012 SEVERAL

MONTHS, ONE YEAR EXACTLY

SIGNIFICANT CONDUCTION IN

PLACEMENT WAS MENTIONED IN THE

(INAUDIBLE).

AND PATIENT CAME BACK TO NIH AND

SEVEN MONTHS LATER SO YOU CAN

SEE ON THE LEFT SIDE

MEASUREMENTS 2011.

RIGHT SIDE THREE MONTHS AFTER

PLACEMENT, 2012 MEASUREMENTS.

AGAIN IN FRONT YOU CAN SEE SMALL

SYMMETRY.

NOW IN THE CHEST AREA, PROBABLY

BECAUSE INFLAMMATORY AREA

PACEMAKER PLACEMENT, YOU CAN SEE

34.9 DEGREES GRES VERSUS 32.6 IN

2011.

OR (INAUDIBLE) SAME

MEASUREMENTS.

SO (INAUDIBLE) SYMMETRY WAS

FOUND IN 2011 AND STILL IN 2012.

ON THE BACKSIDE IN 2012, WE

DIDN'T FIND SPOT OF TEMPERATURE

AS FOUND IN 2011.

AND OPPOSITE IN 2012 GREATER

SYMMETRY, IN FACT IN THE LIMIT

OF WHAT WAS DESCRIBEDDED IN THE

LITERATURE .2-DEGREES, .08

DECREASE OF THESE PATIENTS.

THERE IS NO SYMMETRY IN

TEMPERATURE.

HOW WE CAN INTERPRET THIS DATA?

IT WAS SEVERAL HYPOTHESIS, IS

POTENTIAL, IT'S POSITION OF THE

HEART MITOCHONDRIA IN EPICARDIAL

FAT AREA WHICH WAS REPORTED IN

THE LITERATURE.

UNUSUAL BROWN ADIPOSE TISSUE

UNUSUAL ALLOCATION PERICARDIUM,

MANY OTHER HYPOTHESIS.

WE NEED TO BE CAREFUL TO

INTERPRET THIS DATA THERE IS A

LIMITATION OF THIS, NUMBER ONE,

DIFFERENCE IN SET POINT FOR ROOM

TEMPERATURE IN 2011 AND 2012 WE

NEED TO ACCOUNT THAT.

CE CANNOT TALK ABSOLUTE

MEASUREMENTS BEFORE -- BECAUSE

THAT WAS NOT ACCOUNT

MEASUREMENTS.

IN RELATED THERMO GENESIS WAS

NOT UNDER CONTROL IN THAT STUDY.

NUMBER 3 FUNCTIONAL TEST

IDENTIFY BROWN ADIPOSE TISSUE

PRESENCE WAS DONE, I'M TALKING

AB COLD TEST WHAT SHOULD BE DONE

TO SAY IF IT'S A BROWN FAT OR

NOT BROWN FAT.

HOWEVER, IN SPITE OF THIS, WE'RE

CLAIMING 3 SMALL RESULTS.

NUMBER 1 POSTERIOR

(INDISCERNIBLE) STUDY WAS FOUND

IN THE UPPER LEFT.

NUMBER 2, DIFFERENCE IN

TEMPERATURE.

AND WE CAN TALK ABOUT

DIFFERENTIAL TEMPERATURE

INDEPENDENT ON THE TEMPERATURE

IN THE ROOM.

DIFFERENCE IN TEMPERATURE IN

2011 WAS 1.0-DEGREES.

.08-DEGREES.

THIS IS THE RESULTS.

I WOULD ALSO LIKE TO MENTION

SEVERAL ARTICLES TALKING ABOUT

THE INDICATION OF THE HEAT

DURING BROWN FOOT.

ACTIVATION WHICH WAS FOUND

SEVERAL GROUPS ALREADY.

INCLUDING USING OUR TWO WHICH IS

INFRARED IMAGING.

AND THIS IS A RESULT

MITOCHONDRIA REACH PART OF THE

BODY, OF COURSE IT'S BRAIN AND

THERE'S SUB CLAVICLE TISSUE ON

THE RIGHT SIDE AFTER IT'S

ACTIVATED WITH COLD HARD KIDNEY.

AND WE THINK THAT INFRARED

PHOTOGRAPHY CAN BE USED TO

INTENSIFY BROWN FAT MORE CLEAR,

NOT ONLY IN HUMAN BUT IN ANIMALS

TOO.

THIS IS THE SMALL STUDY DONE IN

DR. (INAUDIBLE) GROUP SCIENTISTS

WITH (INAUDIBLE).

WE TRY TO MEASURE IN MICE

ACTUALLY BROWN FAT TISSUE

ACTIVATION.

THE THIS IS AN EXAMPLE OF THE

STUDY, INFRARED IMAGE OF FIVE

SMALL MICE.

FOUND IN THE ANESTHETIC EFFECT

IN THE ROOM TEMPERATURE AND THAT

ONE IN THE COAL ROOM FOUR

DEGREES CENTIGRADE.

THE DETAIL OF THE STUDY IS

(INDISCERNIBLE) BUT THIS IS A

CLEAR AREA, WE CAN IDENTIFY

ACTIVITY ON THE BROWN FAT TISSUE

IN MICE.

IN REAL TIME RESULT IN THE

TISSUE WHAT IS REQUIRED FOR THE

TOMOGRAPHY ANALYSIS METHODS.

I ALSO WOULD LIKE TO MENTION ONE

MORE FORCE, IF YOU WISH.

WE USED TO THINK THAT

TEMPERATURE MEASUREMENTS IT'S A

SNAP SHOT MEASUREMENT, HOWEVER,

IT'S -- CONTINUES VARIABLES

WHICH HAVE NOT BEEN EXPLORED IN

CLINICAL APPLICATIONS.

SO WE MEASURE TEMPERATURE IN

HUMAN BRAIN, HUMAN KIDNEY,

INTERPRETED IN AND EVERY TIME

WHEN YOU CONTINUOUSLY MEASURE

THE TEMPERATURE YOU SEE LOW

FREQUENCY OF THIS TEMPERATURE.

WE DIDN'T KNOW WHAT TO DO WITH

THAT, THIS MATERIAL, IT'S NOT

FIT TO ANY KNOWN FOR US THEORY.

INITIALLY WE THOUGHT IT

INSTRUMENTAL NOISE WITH SOME

KIND OF CIRCADIAN RHYTHMS.

BUT RECENTLY WITH DR. CARTER AT

NHLBI WE FOUND THAT LOW

FREQUENCY ISOLATION IN THE AREA

OF .01-HERTZ IS ONE INSULATION

REGULATED IN THE MATERIAL

DYSFUNCTION.

WHEN WE START TO LOOK MORE CLOSE

THIS FOE MA'AMNA WE FOUND

TEMPERATURE (INDISCERNIBLE)

REGULATED, TEMPERATURE ELEVATION

RELATED TO THE SYMPATHETIC

ACTIVITY AND MYOGENIC ACTIVITY

AND OF COURSE HEART ACTIVITY CAN

BE MEASURED.

ALL THESE FREQUENCIES CAN BE

RECOGNIZED BY SPECTROSCOPY AND

THE PATHWAY INVOLVED IN THIS

TISSUE, TISSUE OF INTEREST CAN

BE IDENTIFIED.

THE INTRIGUING ARTICLE CAME

RECENTLY FROM THE GROUPS TO JOIN

EFFORTS IN GH AS WELL AS JOHNS

HOPKINS GROUP.

I KNOW THE KEY HERE DR. FROM

JOHNS HOPKINS THEY FOUND THAT

ISOLATED MITOCHONDRIA OF THE

CONDUCT MYOSITES EXPRESS THE

SAME FREQUENCY OF OSCILLATIONS

THAT WE FOUND WITH OUR CAMERA.

THEY HAVE ABSOLUTELY DIFFERENT

DEVICE TO MEASURE THE ISOLATIONS

IN FLUORESCENCE IMAGING BUT THEY

FOUND THAT THE HIGHLY INTENSITY

OSCILLATIONS ARE CURE OF THE

TISSUE WITH HIGH CONTENT OF

MITOCHONDRIA.

WERE FOUND THE SAME WAY.

IT'S NOT REGULATED TO SAY IT IS

THE SAME PHENOMENA BUT THE SAME

FREQUENCY.

SO WE WILL TRY TO EXPLORE THIS

IN THE FUTURE AND WE BELIEVE

WITH THIS SHOULD BE VALIDATED

MUCH MORE PROFICIENT IN MODELS

BUT WE'RE READY TO DO THIS.

ON THE LOWER LEVEL IMAGE YOU CAN

SEE THE ANALYSIS TIME VERSUS

FREQUENCY.

.01 HERE OR TEN MILIHERTZ IS

PRESENT EVERY TIME IN

MITOCHONDRIA MYOSITES.

WE KNOW HOW TO MEASURE THE

OSCILLATIONS IN HUMAN.

IN THE AN EXAMPLE OF THE SMALL

WIRELESS TEMPERATURE PATCH WE

MEASURE TEMPERATURE CONTINUOUSLY

THROUGH (INAUDIBLE) AND

CONNECTED WITH SEVERAL FORM,

LEFT SIZE A CHIP TO EMPLOY NEXT

PROTOCOL IN JUNE NHLBI, ON THE

RIGHT SIDE IT'S A (INAUDIBLE)

ELECTRONICS WHICH IS -- MIGHT BE

PLACED ON ANY PART OF THE SKIN

AND MEASURE TEMPERATURE AND

TRANSFER THIS DATA BACK TO THE

CLINICAL (INAUDIBLE).

THIS IS WHAT I WOULD LIKE TO

SHARE WITH YOU.

THANK YOU.

[APPLAUSE]

>> THANK YOU VERY MUCH, ALEX.

NEXT I WOULD LIKE TO INTRODUCE

LYNNE WOLF, NURSE PRACTITIONER

WITH THE UNDIAGNOSED DISEASES

PROGRAM.

WHO WILL TALK ABOUT WHETHER OR

NOT IT'S MITOCHONDRIAL DISEASE.

LYNNE.

THANK YOU VERY MUCH.

>> SO I THINK THIS WILL CHANGE

THE PACE A LITTLE BIT.

I WANT TO WELCOME EVERYBODY.

I'M GOING TO TALK MOSTLY ABOUT

THE CLINICAL PRESENTATION AND

TESTING OF SOME OF THE PATIENTS

WITHIN THE UNDIAGNOSED DISEASES

PROGRAM THAT WE THINK MAY HAVE A

MITOCHONDRIAL DISEASE AND SOME

THAT ACTUALLY DO AS YOU HAVE

SEEN FROM DR. GAHL.

I'M ASSUMING A LOT OF

INFORMATION ABOUT

PATHOPHYSIOLOGY OF MITOCHONDRIA

DISEASES IS WELL KNOWN TO THIS

GROUP SO I'M GOING TO REMIND

EVERYBODY BRIEFLY OF THE

CONSEQUENCES OF MITOCHONDRIAL

DYSFUNCTION WHICH IS THE BEAS

FOR MOST OF THE SYMPTOMS THAT WE

SEE IN OUR PATIENTS.

POTENTIAL LABORATORY

ABNORMALITIES ARE MANY.

WE SEE SOME IN BLOOD, SOME IN

URINE, WE SEE SOME IN OTHER

TISSUES.

AND THESE ARE THE GENERAL LIST

OF TESTS THAT WE DO IN OUR

PATIENTS.

AS WE ASSESS THEM, WE DO MUSCLE

BIOPSIES THOUGH NOT FRESH

BIOPSIES AND THERE'S LIMITATIONS

TO FRESH MUSCLE VERSUS FROZEN

MUSCLE.

WE CAN USE CLINICAL LAB TESTING

FOR MOLECULAR CONFIRMATION BUT

ALSO FIND NUCLEAR DELETIONS AND

DUPLICATIONS ON THE MILLION SNP

ARRAY AND ALSO OBVIOUSLY ON

EXONLY TESTING.

HEARSAY THE MITOCHONDRIAL GENOME

AND WE HAVE POINT MUTATIONS

DESCRIBED AND ASSOCIATED WITH

COMMON SYNDROMES.

WE CAN NOW DO COMPLETE EXOME

SEQUENCING FOR THE MITOCHONDRIAL

GENOME.

THAT IS CLINICALLY AVAILABLE.

AND IS STARTING TO IDENTIFY A

LOT MORE MUTATIONS AND DELETIONS

AND DUPLICATIONS THAT ARE NOVEL

AND WE DON'T KNOW WHETHER THOSE

ARE GOING TO BE ASSOCIATED WITH

ANY SPECIFIC SYNDROME OR

SYMPTOMS JUST YET.

I WANT TO REMIND EVERYBODY PART

OF WHAT MAKES WORKING WITH

MITOCHONDRIAL DISEASES AND

DIAGNOSING THEM SO DIFFICULT IS

WE HAVE TO HAVE COMMUNICATION

BETWEEN THE NUCLEAR GENOME AND

THE MITOCHONDRIAL GENOME AND

TOTALLY TOGETHER THAT IS ABOUT

3,000 PROTEINS DOING VARIOUS

FUNCTION, EITHER TO ASSEMBLE THE

RESPIRATORY CHAIN PROTEIN OR

TRANSFER RNAs AND RIBOSOMAL

RNAs AND OTHER ACTIVITIES.

SO THE MAJOR QUESTION IS WHAT IS

THE MITOCHONDRIAL DISEASE, THE

UNITED MITOCHONDRIAL DISEASE

FOUNDATION AND NIH RECENTLY HAD

A CONFERENCE IN WHICH WE TRIED

TO DEFINE WHAT MIGHT BE A

MITOCHONDRIAL DISEASE VERSUS

WHAT IS A SECONDARY

MITOCHONDRIAL DYSFUNCTION FROM A

COMMON DISEASE SUCH AS DIABETES

OR PARKINSON'S DISEASE OR CARDIO

MYOPATHY.

THE ORIGINAL DEFINITION TO LOOK

FOR MITOCHONDRIAL DISEASE FROM

THE UMDF WAS TO CONSIDER A

MITOCHONDRIAL DISEASE IF YOU HAD

AN ASSOCIATION OF ANY SYMPTOM IN

ANY ORGAN AT ANY TIME IN ANY

SCENARIO THAT APPEARED TO BE

PROGRESSIVE AND COULD BE

INHERITED IN MULTIPLE DIFFERENT

WAYS.

THAT'S OBVIOUSLY NOT SPECIFIC

ENOUGH FOR WHAT WE ARE FINDING

TODAY.

IN FRANCE THERE WAS A NICE TABLE

LOOKING AT 390 ADULTS AND 220

CHILDREN PRESENTED WITH

MITOCHONDRIAL DISEASE AND YOU

CAN SEE THERE ARE DIFFERENCE

SYMPTOMS THAT ARE SHOWING UP IN

THE DIFFERENT AGE GROUPS AND

THIS IS I THINK WHAT MOST

CLINICAL CENTERS ARE ALSO

SEEING.

BUT WE HAVE THE DEFINE MORE

CAREFULLY AND ASSOCIATED WITH

ENZYMATIC DEFICIENCIES.

WE TALK ABOUT GENOMIC DISORDERS,

FIRST DESCRIBED IN 2006 AND

BASICALLY WHAT HAPPENS IS YOU

HAVE A DELETION OR MULTIPLE

DEPLETION OR DELETIONS OF THE

MITOCHONDRIAL DNA COPY NUMBER

THIS IS MEASURED IN LIVER TISSUE

AS HARVEY DISCUSSED IN MPD 17

AND GUO DEFICIENCIES BUT ALSO IN

MUSCLE, BRAIN, EYE SO WE SEE

THOSE QUITE A BIT.

THEY'RE DOMINANT OR RECESSIVE OR

SPORE RADICALLY INHERITED.

THERE WAS A NICE PAPER IN 2010

THAT LOOKED AT BASIC SYMPTOMS IN

BOLD ARE THE NUCLEAR MUTATION OR

THE NUCLEAR GENES THAT ARE

ASSOCIATED WITH MITOCHONDRIAL

DEPLETION SYNDROMES AND THE

SPECIFIC TISSUES THAT THEY

AFFECT.

NOTICE THE IN TEST AND IN

PARTICULAR WE USED TO ONLY THINK

OF THIS AS MENGI FROM THYMIDINE

PHOSPHORYLASE DEFISH SU BUT NOW

A ISOLATED PHENOTYPE IS REPORTED

WITH AN ADULT PATIENT WITH RR M

2B DEFICIENCY AND ALSO AN ADULT

PATIENT WITH A PAULG-1 MUTATION.

SO WE'RE NOT REALLY SURE WHAT

WE'RE GOING TO FIND AS WE

CONTINUE TO LOOK.

THIS IS ANOTHER NICE TABLE FROM

THAT SAME PAPER LOOKING AT THE

MITOCHONDRIAL DEPLETION

SYNDROMES BY AGE.

I WILL BRIEFLY MENTION LACTIC

ACIDOSIS IS MORE COMMON IN

INFANT AND MUCH LESS COMMON IN

THE ADULTS.

AND YET WE SEE A LOT MORE ATAXIA

AND POLYNEUROPATHYS IN THE

ADULTS.

INTERESTINGLY ALSO PSYCHIATRIC

SYMPTOMS AND GI SYMPTOM.

FAILURE THE THRIVE IS MORE

COMMON IN THE KIDS SO THIS IS A

NICE WAY TO LOOK AT YOUR PATIENT

THAT U YER -- YOU'RE THINKING AB

MITOCHONDRIAL DEPLETION DISORDER

ON.

THESE ARE SOME, NOT ALL OF THE

PATIENTS WE HAVE SEEN IN THE UDP

WHERE WE EITHER THINK OR HAVE

CONFIRMATION THAT WE HAVE A

MITOCHONDRIAL DISEASE.

SO IF YOU LOOK ACROSS THE TOP OF

THIS TABLE, WE HAVE THE PRIMARY

PHENOTYPE SO WE HAVE A PATIENT

WITH A MILD ATAXIA AND ADRENAL

INSUFFICIENCY AS PRIMARY

PRESENTING FACTOR PRESENTED TO

THE UDP AT AGE 5.

WE HAVE A PATIENT THAT PRESENTED

AT NEARLY 7 YEARS WITH SEIZURES

DISTONEIA, COURTIAL ACIDOSIS AND

A PATIENT WITH THREE METHYL

CHRONIC ACIDURIA AND LOOK TICK

ACIDEMIA, WE HAVE AN UNEXPLAINED

SPINAL MEGALY SEIZURE AT 5 AND

ACQUIRED ENCEPH-- AND CORTICAL

AND SLEEP DISTURBANCE PATIENT

SEEN AT 7.

I DON'T HAVE A POINTER.

ONE OF YOU GUY VERSUS A POINTER

WE CAN USE FOR A MINUTE?

NEVER MIND.

I DON'T SEE IT.

LET ME SEE IF I CAN AIM

CORRECTLY.

IF YOU LOOK FOR MITOCHONDRIAL

DNA DISORDERS WE HAVE NOT SEEN

ANY IN THIS GROUP THAT I'M

PRESENTING TO YOU.

BUT REMEMBER DR. GAHL TALKED

ABOUT THE KEARNS-SAYRE PAIRN, A

MITOCHONDRIAL DELETION.

WE HAVE SOME INTERESTING NUCLEAR

DNA ABNORMALITIES, THIS PATIENT

HAS A KNOWN ADULT LATE ONSET

ATAXIA MUTATION.

THIS W-748S IS A COMMON IN THE

FINISH POPULATION AS A CARRIER

FREQUENCY OF ONE IN 125.

BUT THIS OTHER MUTATION IS A

NOVEL MUTATION THAT'S NEVER BEEN

SEEN BEFORE.

THIS PATIENT WITH THE CARDIO

MYOPATHY LACTIC ACIDEMIA IS

HOMOZYNOWS FOR NOVEL DNA

MUTATION, MOST SDH MUTATIONS ARE

ASSOCIATED WITH A PAIR OF GANG

GLIOMA MUTATION SO THIS IS AN

UNUSUAL PRESENTATION.

THIS PATIENT HAS ONLY ONE PAULG

ATION BUT IF ANYBODY IS

WATCHED THIS LITERATURE THIS

PARTICULAR MUTATION APPEARS TO

BE A VARIANT IN THE POPULATION

THAT REALLY DOES NOT EXPLAIN

SYMPTOMS WELL.

IF YOU CONSIDER THE

ABNORMALITIES IN THE DIAGNOSTIC

CATEGORIES THAT I TOLD YOU WE

COULD LOOK FOR, AS YOU GO DOWN

THE LIST YOU'LL SEE THAT A LOT

OF THESE ARE COMPLETELY NORMAL

EVEN IN THE PATIENTS THAT WE

KNOW HAVE MUTATIONS.

SO CONTINUING ON WITH THEIR

CLINICAL EVALUATION, YOU CAN SEE

WE ONLY HAVE ONE PATIENT THAT

ACTUALLY HAD ABNORMALITIES IN

THE CSF AND THE PATIENT WITH THE

DHA MUTATION.

OF NOTE THE KEARNS-SAYRE PATIENT

HAS CENTRAL ABNORMALITIES

INCLUDING CENTRAL LACTIC

ACIDOSIS AND ELEVATED ALANINE.

MOST PATIENTS ACTUALLY HAVE

NORMAL FIVE METHYL TETRA HYDRO

FOLATE IN THIS PARTICULAR GROUP

THAT I'M SHOWING YOU.

LOOK HOW MANY HAD NORMAL BRAIN

MRI, NORMAL ECHOCARDIOGRAMS, A

FEW HAD SOME SEIZURE ACTIVITY

AND ALMOST ALL -- WELL THE ONES

THAT HAD NERVE CONDUCTION

TESTING ACTUALLY WAS NORMAL.

SO WE HAVE THIS PATIENT WITH THE

SDH MUTATIONS WITH RETINITIS PIG

MEANTOSA AND THE OTHER PATIENTS

PRETTY MUCH WERE OKAY THIS

PATIENT HAS OPTIC -- HAS

CORTICAL BLINDNESS BUT THE OPTIC

NERVE LOOKED FINE.

SO WHEN WE LOOK AT DEFICIENCIES

OF ENZYMOLOGY WE GET REALLY ODD

FINDINGS.

SO IF THIS PATIENT HAS SDH

MUTATIONS WHICH ARE COMPLEX 2

RELATED, HOW DID WE GET A

COMPLEX ONE DEFICIENCY IN

FIBROBLASTS?

MUSCLE BIOPSY DONE AT ONE YEAR

OF AGE, VERY NON-SPECIFIC,

CONSISTENT WITH MITOCHONDRIAL

PROLIFERATION ON BOTH EM LM AN

ELECTRON TRANSPORT CHAIN

ENZYMOLOGY.

THAT'S VERY COMMON IN MOST

CHILDREN THAT HAVE BIOPSIES

UNDER 6 OR 7 YEARS OF AGE.

THIS PARTICULAR PATIENT THAT HAD

THAT ONE PAULG MUTATION

CONSIDERED A VARIANT ACTUALLY

DOES HAVE DEFICIENCIES MEASURED

WITH SEVERAL OF HER COMPLEXES.

SO HER MUTATION IS NOT

EXPLAINING HER ENZYMATIC DEFECT

AT THE MOMENT.

SO LET'S LOOK AT THIS LATE ONSET

PAULG PATIENT, A LITTLE BIT IN

MORE DETAIL JUST SO YOU KNOW THE

WORK UP THAT WE DO.

SO THE HISTORY ON THIS CHILD WAS

COMPLETELY NORMAL, MOM WAS 35,

DAD 34.

THERE E ARE OTHER SIBLINGS THAT

ARE UNAFFECTED.

MATERNAL WEIGH GAIN WAS IDEAL.

FETAL MOVEMENT WAS CONSIDERED

NORMAL.

MOM HAD OBVIOUSLY HAD OTHER

PREGNANCIES SO SHE KNEW WHAT

PREGNANCY SHOULD BE LIKE.

THIS WAS ON UNCOMPLICATED

SPONTANEOUS VAGINAL DELIVERY

WITH 38 WEEKS WITH NORMAL HEAD

AND BIRTH CIRCUMFERENCE,

DEVELOPMENTAL MILE SPHOANS WERE

MET ON TIME AND THIS CHILD

HAPPENS TO BE BILINGUAL AT HOME

SO THERE WAS CONCERN ABOUT

LANGUAGE DELAY BUT WHEN WE HAD

HIS SPEECH EVALUATED WHEN YOU

CONSIDER ALL THE WORDS HE HASN'T

TWO LANGUAGES THAT WE DOES SPEAK

HE ACTUALLY WAS ON TARGET.

PRIOR TO EVER COMING TO US HE

HAD EXTENSIVE BIOCHEMICAL AND

MOLECULAR EVALUATIONS WHICH

REVEALED ADRENAL INSUFFICIENCY

AND PAUL G COMPOUND HETERO

ZYGOSITY WHICH I TALKED ABOUT

ALREADY HIS EEG AND MRI HAD

PREVIOUSLY BEEN NORMAL.

SO THE QUESTION WAS COULD THIS

CHILD'S ADRENAL INSUFFICIENCY

AND ATAXIA BE EXPLAINED BY THE

TWO PAUL G MUTATIONS FOUND.

REPORTED PAUL G-1 DISEASE IN

CHILDREN COMES IN A COUPLE OF

FLAVORS.

IT'S USUALLY ACUTE OR SUB ACUTE

ONSET, OFTEN PRECIPITATED BY AN

ILLNESS.

THERE'S THREE SPECIFIC

PHENOTYPES OF SEVERE INTRACTABLE

EPILEPTIC ENCEPHALOPATHY WHICH

OBVIOUSLY THIS PATIENT DID NOT

HAVE.

AL PERS PHENOTYPE ESPECIALLY

WITH DEPAKOTE EXPOSURE, THE

CHILD NEVER BEEN EXPOSED TO

DEPAKOTE BUT NORMAL LIVER

FUNCTION.

THEN IN EARLY FATAL

MITOCHONDRIAL DLAN DEPLETION

WHICH OBVIOUSLY HE DID NOT HAVE.

OTHER AB NOORLTIES INCLUDE

ABNORMAL MRIs THAT CAN HAVE A

WIDE VARIETY OF GREAT OR WHITE

MATTER PROBLEMS BUT REMEMBER HIS

MRI IS COMPLETELY NORMAL.

HERE IS A RESTATEMENT OF HIS

NORMAL EVALUATION BIOCHEMICALLY.

OUR EEG WAS NOT PARTICULARLY

NORMAL.

AND MAY BE A SIGN THAT SEIZURES

MIGHT DEVELOP OVER TIME BUT

EVERYTHING ELSE HERE LOOKED

FINE.

INTEREST LIG I WANT TO DRAW

EVERYBODY'S ATTENTION TO HOW WE

EVALUATE COPY NUMBERS SO RIGHT

NOW IT'S DONE BY QUANTITATIVE

PCR BUT IF YOU LOOK AT THE

SAMPLE, AT THE AGE GROUP AND THE

SAMPLE SIZE OF CONFIRMED

MITOCHONDRIAL MUTATIONS, THESE

ARE GENES IDENTIFIED, NUMBER OF

SAMPLES, AND CONTENT THAT'S

NORMAL.

IF YOU LOOK AT WHAT WE'RE

GETTING, NOT A LOT OF SAMPLE

SIZE AND HUGE RAREIATION IN THE

MEAN.

SO THIS PATIENT HAS TWO

MUTATIONS, ONE NOVEL AND ONE

KNOWN LATE ONSET ATAXIA, MUSCLE

BIOPSY DONE AT AGE 4.

HE HAS A MITOCHONDRIAL COPY

NUMBER OF NORMAL, ACCORDING TO

THE THE INTERPRETATION, THAT

SHOULD BE A COMPLETELY NORMAL

CASE.

IF REREMUSCLE BIOPSIED HIM IN

TEN YEARS WILL THE DEPLETION BE

MORE SIGNIFICANT.

I HAVE NO IDEA.

BUT RIGHT NOW HE DOES NOT HAVE A

MITOCHONDRIAL DEPLETION SYNDROME

WE CAN PROVE BY THE CURRENT

DEFINITION.

SO TREATMENT IN GENERAL FOR

PRIMARY MITOCHONDRIAL DISEASES

IS EXTREMELY NON-SPECIFIC.

IT INCLUDES AVOIDING PHYSIOLOGIC

STRESSORS, AVOIDING

ENVIRONMENTAL TOXINS.

DIETARY ADJUSTMENTS IN SOME

SPECIFIC DISORDERS, QUITO GENIC

DIET MIGHT BE HELPFUL IN COMPLEX

1 DISORDERS, HIGH PROTEIN IN

GENERAL IS CONSIDERED REASONABLY

WELL.

VARIOUS CALORIE RESTRICTION THAT

WE CAN INDUCE IN OUR MICE THAT

MAKES THEM LIVE LONGER BUT I

DON'T WANT MY CALORIES

RESTRICTED THAT SEVERELY.

WE CAN ADD VITAMINS OR OTHER

SUPPLEMENTS AND CREATE A

MITOCHONDRIAL COCKTAIL, DR. GAHL

TALKED ABOUT EPI 743 AND OTHER

ANTIOXIDANT RESEARCH THAT'S

HAPPENING.

ENDURANCE EXERCISE AN RESISTANCE

TRAINING FOR MUSCLE

MITOCHONDRIAL DISEASE SEEMS TO

INCREASE THE WILE TYPE

MITOCHONDRIA IN THE NEW MUSCLE

CELL AS THEY'RE MADE AND BONE

MARROW TRANSPLANTATION HAS BEEN

USED NOW TO TREAT I THINK UP TO

12 MENGI PATIENTS.

BUT IN GENERAL WE HAVE

INADEQUATE DEFINITIONS FOR

PRIMARY MITOCHONDRIAL DISEASE

VERSUS SECONDARY DYSFUNCTION.

THAT CREATE AS SIGNIFICANT

AMOUNT OF DIFFICULTY I THINK IN

DEVELOPING APPROPRIATE CLINICAL

STUDY DESIGN.

THERE'S FEW NATURAL HISTORY

STUDIES FOR ANY OF THE PRIMARY

MITOCHONDRIAL DISEASES.

THERE'S ONE NICE STUDY OF 22

KEARNS-SAYRE PATIENTS FOLLOWED

OVER A 20 YEAR PERIOD OVER

FRANCE AND THE GROUP AT COLUMBIA

LAST YEAR PUBLISHED A NATURAL

HISTORY STUDY ON (INAUDIBLE)CH

BUT WE DON'T HAVE NATURAL

HISTORY STUDIES ON ALMOST ALL

OTHER MITOCHONDRIAL DNA

DISORDERS NEVER MIND ELECTRON

TRANSPORT CHAIN DEFICIENCIES OR

ANY NUCLEAR DERIVED DEPLETION

SYNDROME.

OUR CURRENT DIAGNOSTIC

STRATEGIES ARE NOT SENSITIVE OR

SPECIFIC ENOUGH TO DETECT

MITOCHONDRIAL DISEASES WHICH HAS

NEGATIVE IMPACT ON DEVELOPING

TREATMENT STRATEGIES.

SO THERE'S LOTS OF WORK FOR US

TO DO BETWEEN THE CLINICIANS AND

BASIC SCIENTISTS TO TRY AND

DEVELOP BETTER DIAGNOSTIC TOOLS

AS WELL AS TARGETED TREATMENT.

I JUST LIKE TO THANK OUR

PATIENTS, OUR FAMILIES AND

REFERRING PHYSICIANS AND

MULTIPLE PEOPLE THAT HAVE HELPED

US FROM MANY DIFFERENT

INSTITUTES.

[APPLAUSE]

>> THANK YOU, LYNNE.

I WANT TO REMIND EVERYONE

WATCHING, I KNOW THERE ARE QUITE

A FEW THAT IF YOU HAVE QUESTIONS

ALL YOU NEED TO DO IS EMAIL ME,

STEVENZULLO AT NIH.GOV AND I CAN

GET THE MESSAGES TO THE SPEAKERS

HERE.

IF YOU HAVE QUESTIONS AFTER --

WHEN WATCHING VIDEOCAST OR AFTER

IT'S ARCHIVED IN A DAY OR SO,

YOU CAN ALSO EMAIL ME AND I'LL

GET -- GIVE QUESTIONS TO THE

APPROPRIATE PERSONS.

LIKE TO FOLLOW-UP ON ON LYNNE'S

TALK WITH CAMILO TORO FROM NHGRI

WHO WILL BE TALKING ABOUT ADDING

INSULT TO INJURY, HOW A MUTATION

OF NUCLEAR ENCODER MITOCHONDRIAL

PROTEIN RUINS THE FUNCTION OF

ANOTHER.

THANK YOU VERY MUCH.

>> GOOD MORNING.

THANK YOU FOR HAVING ME

PARTICIPATE IN THIS PANEL MY

PRESENTATION TODAY IS A

PRESENTATION GEARED PRIMARILY TO

PRESENTING A CASE ORIGINATING

FROM THE UNDIAGNOSED DISEASES

PROGRAM IN WHOM WE WERE ABLE TO

ACTUALLY WITH THE HELP OF

COLLABORATORS AT NIH AND

ELSEWHERE TO CLOSE OUT AND

ATTAIN ACTUALLY REASONABLE

EXPLANATION FOR THESE PATIENTS

PHENOTYPE AND IN THE PROCESS WE

LEARN QUITE A BIT ABOUT THE WAY

HUMAN DISEASES IN A WAY INTERACT

WITH EACH OTHER AND HOW WE NEED

TO ADJUST OUR THINKING PROCESS

ABOUT NEUROLOGICAL DISEASES AND

MANY OF THESE OTHER DISEASES

ASSOCIATED WITH MITOCHONDRIA.

THE OBJECTIVE OF MY TALK IS

ACTUALLY TO GIVE CONTEXT TO

CRYPTIC TITLE TO MY TALK.

BUT I'M GOING TO START BY

DESCRIBING THIS CASE

PRESENTATION.

THIS IS A CASE OF AS A YOUNG MAN

WHO IS PROFOUNDLY DEBILITATED

NEUROLOGICALLY.

I'M GOING TO GO TO THROUGH

DETAILS OF THE COMBINATION IN

ONE SECOND BUT THIS PATIENT IS

THE PRODUCT OF CONSEGENUOUS

MARRIAGE WHERE THE PARENTS ARE

ACTUALLY FIRST COUSINS.

AND UNFORTUNATELY BOTH CHILDREN

OF THIS FAMILY ARE AFFECTED WITH

AN IDENTICAL PHENOTYPE.

THE YOUNGER PATIENT ACTUALLY HAS

PASSED AWAY AT THE AGE OF 13

FROM RESPIRATORY COMPLICATIONS

IN RELATION TO BASICALLY

INTRACTABLE SEIZURES AND SEVERE

MOTOR IMPAIRMENT.

AT THE UDP WE'RE ASKED TO

EVALUATE THE PROBAND WITH A

PHENOTYPE BASICALLY THAT IS

PROFOUNDLY DEVASTATING, THAT

INCLUDED AS I MENTIONED BEFORE

INHERITANCE PATTERN THAT WAS

RECESSIVE, A NEUROLOGICAL

SYNDROME FOR BY VERY EARLY ONSET

SYMPTOMTOLOGY THAT INCLUDED

PRIMARILY ATAXIA, MYOCLONALS,

SEVERE SEIZURES BY THEMSELVES

COMBINATION IS ACTUALLY WHAT

REMINISCENT OF MANY MITOCHONDRIA

DISEASES THAT AFFECT THE CENTRAL

NERVOUS SYSTEM.

THE PATIENT HAD EVIDENCE OF

MOTOR INS SENSORY NEUROPATHY.

THERE WERE SYMPTOMS OF

IMPAIRMENT OF OCULAR MOTOR

FUNCTION BUT EXAMINATION OF EYES

DIDN'T SHOW RETINITIS

PIGMENTOSA.

ONE OF TH THE PATIENT'S

PRESENTATIONS WAS THAT DESPITE

THE FACT THAT HE WAS VERY

IMPAIRED FROM A MOTOR POINT OF

VIEW, THE PATIENT WAS ACTUALLY

RELATIVELY INTACT IN RELATION TO

HIS COGNITIVE FUNCTIONING.

THAT WAS ACTUALLY QUITE

DISTURBING BECAUSE THE PATIENT

WAS BASICALLY IN A STATE THAT WE

IN NEUROLOGY TEND TO RELATE IN

LOCKED IN SYNDROME, THIS PATIENT

WAS FULLY AWARE OF HIS

SURROUNDINGS BUT THE DEGREE OF

HIS MOTOR IMPAIRMENT WAS SUCH

THAT HE WASN'T ABLE TO

COMMUNICATE.

OR TO MOVE IN ANY PURPOSEFUL

MANNER.

THE MUSCLE INVOLVEMENT SHOWED

SECONDARY ATROPHY TO A

PERIPHERAL NEUROPATHY AND HE HAS

NO EVIDENCE OF SYSTEMIC

INVOLVEMENT.

THIS WAS ONE OF THE FIRST CASES

FROM THE UDP THAT PROCEEDED

THROUGH THE INVESTIGATION LINE

DR. GAHL HAD THE OPPORTUNITY TO

DESCRIBE TO YOU AT THE

BEGINNING.

SO THE PATIENT AND HIS NUCLEAR

FAMILY INCLUDED THE TWO AFFECTED

CHILDREN UNDERWENT WHOLE EXONLY

SEQUENCING AND THE WHOLE EXONLY

SEQUENCING ACTUALLY DEMONSTRATED

A SERIES OF POTENTIAL CANDIDATES

AND IN THE TOP OF THE LIST WE

HAVE THIS GENE AFG CL-2.

AND THAT MUTATION WAS PRESENT IN

A HOMOZYGOUS STATE.

YOU CAN SEE HERE THE CROW MAT

GRAMS HOW BOTH PARENTS ARE

CARRIERS FOR THESE MUTATIONS AND

BOTH AFFECTED CHILDREN ARE IN A

HOMOZYGOUS STATE RESULTING IN

SUBSTITUTION OF TYROSINE FOR

SISTINE IN A CONSERVED AMINO

ACID THAT ENCODES AFG-3L-2, THE

NAME DERIVES FAMILY GENE 3 LIGHT

TWO NUCLEAR ENCODED

MITOCHONDRIAL PROTEIN THAT ALSO

GOES BY THE NAME PARAPLEGAN LIKE

PROTEIN.

THIS THIS LIKE PROTEIN

DIMINUTION WILL BECOME CLEAR AS

WE MOVE FORWARD IN THE TALK.

THIS PROTEIN HAS BEEN FOUND TO

BE RESPONSIBLE IN THE

HETEROZYGOUS STATE FOR SPINAL

SELL BELL LAR DISATAXIA TYPE 28.

IT HAS 397 AMINO ACID RESIDUES

AN MITOCHONDRIAL TARGET IN

SEQUENCE, AND HAS SOME A SEGMENT

CALLED TRIPLE A OR ATPASE

ASSOCIATED ACTIVITY OR ADENOSINE

ASSOCIATED ACTIVITY AND HE HAS

THE PROTEASE DOMAIN WHERE

MUTATION IS LOCATED.

THERE ARE A NUMBER OF REPORTED

MUTATIONS AND THIS APPEARS TO BE

THE MUTATION HOT SPOT.

BASICALLY ALL THE CASES OF SCA

28 REPORTED IF NOT MOST OF THEM

APPEAR TO BE IN THIS REGION OF

THE PROTEASE ACTIVITY.

AND THAT IS PRECISELY WHERE OUR

PATIENTS MUTATION WAS FOUND.

THESE AFD 3L-2 PROTEINS, THIS

PROTEIN IS ACTUALLY A PROTEIN

THAT ASSEMBLES INTO A COMPLEX

THAT I WILL DISCUSS IN A LITTLE

MORE DETAIL LATER.

AND ANCHORS ITSELF IN THE

INTERMITOCHONDRIAL MEMBRANE AND

IT HAS PRIMARILY AN ACTION AS

ATP DEPENDENT PROTEASE BUT THE

FUNCTIONS INCLUDE ADDITIONAL

ACTIVITIES SO HERE WE HAVE OUR

-- THIS IS OUR ASD 3L-2 ACTING

AS A PROTEASE BUT ALSO APPEARS

INVOLVED IN ACTIVATION AND

CLEAVAGE OF PROTEIN PRECURSORS

INTO THE ACTIVE FORM.

IT ALSO HAS THE CAPACITY TO

INTERVENE IN TRANSLOCATION OF

PROTEINS AN REGULAR GATE

PROGRESS TEENS IN THE MEMBRANE

AND APPEARS ALSO IN SOME OF THE

TON OF PROTEINS ACROSS THE

MITOCHONDRIAL MEMBRANES.

SO IN ESSENCE THESE PROTEINS

APPEAR VERY CRUCIAL IN

HOUSEKEEPING OF OTHER

MITOCHONDRIAL PROTEINS.

ONE INTERESTING ASPECT ABOUT

THIS AFD 3L-2 IS AFD-3L-2 HAS

ANOTHER VERY RELATED PROTEIN

THAT IS PARAPLEGAN, THAT'S

PRECISELY THE REASON IT USED TO

BE CALLED PARAPLEGAN LIKE

PROTEIN.

THIS HAS THE CAPACITY TO IN ITS

ACTIVE CONFIGURATION FORMS

BASICALLY A HOMOOLIGOMERIC

COMPLEX WITH FIXED UNITS OF

BASIC PROTEIN OR HAS THE

CAPACITY TO FORM HETERO

OLIGOMERIC COMPLEX WITH

PARAPLEGAN.

RESULTING IN THIS CONFIGURATION.

IT TURNS OUT MUTATION THAT WE

FOUND IN THIS PATIENT IS CLOSE

TO THE HINGE WHERE THE

INTERACTION OCCURS TO FORM THESE

PARTICULAR COMPLEX.

SO WE ARE IN THE SITUATION WHERE

WE HAVE THE PATIENT THAT HAS A

PROTEIN MUTATED IN HOMOZYGOUS

STATE FOR A DISEASE THAT IS

HETEROZYGOUS AND THAT IS NOT TO

RESULT IN SPINAL CEREBELLAR TYPE

28.

WE ALSO KNOW THAT AFD-3L-2 FORMS

COMPLEXES WITH PARAPLEGAN.

INTERESTINGLY ENOUGH, I SHOULD

GO BACK, PARAPLEGAN IS UNABLE

THE TO FORM COMPLEXES WITH

ITSELF.

SO ONE COULD EXPECT THAT A

MUTATION OF AFD-3L-2 IS GOING TO

SOME EXTENT INTERFERE WITH THE

FUNCTION OF THE COMPLEXES WITH

PARAPLEGIN.

BRIEFLY REVIEWING SCA-28.

SCA-28 IS A DISEASE PRIMARILY

CHARACTERIZED BY CEREBELLAR

ATAXIA.  THERE'S ATROPHY OF THE

CEREBELLUM WITH CELL LOSS.

THE INHERITANCE IS DOMINANT,

LATE ONSET DEERS, HAS --

DISEASE, HAS SLOW PROGRESSION,

MAY HAVE RARE SEIZURES AND MANY

PATIENTS SURVIVE A NORMAL LIFE

EXPECTANCY.

AND THIS IS QUITE DIFFERENT FROM

OUR CASES WHERE -- WITH A VERY

EARLY ONSET AND VERY SEVERE

PHENOTYPE.

PARAPLEGAN PRODUCES AUTOSOMAL

RECESSIVE INHERITANCE, PRODUCES

SPG-7.

IT STANDS FOR SPASTIGATE 7.

IT'S REFERRED TO AS ONE OF THE

COMPLICATED SPG DISEASES IN THAT

PATIENTS DEBT THIS AND SOMETIMES

GET ADDITIONAL SYMPTOMS.

THE DISEASE HAS EARLY ONSET

PRIMARILY CHARACTERIZED BY

SPINAL CORD ATROA FI VERY SELDOM

VERY MILD CEREBELLAR FEATURES

AND OPTIC ATROPHY.

ONE HAS TO ASK THE QUESTION IN

THE INFORMATION I PROVIDE

REDIRECT EXAMINATION THE PARENTS

AFFECTED WITH A MILD CEREBELLAR

PHENOTYPE.

WE HAD THE OPPORTUNITY ACTUALLY

OF BRINGING THE PARENTS AND

EXAMINING THEM NEUROLOGICALLY

AND BY IMAGING AND I'M

PRESENTING HERE THE INFORMATION

ON THE PROBAND, THE MOTHER AND

THE FATHER.

AND AS YOU CAN SEE, THE PROBAND

HAS PROFOUND CEREBELLAR ATROPHY

BUT IN ADDITION HAS VERY SEVERE

THINNING OF THE SPINAL CORD

ATROPHY.

THE MOTHER HAS A VERY MILD

DEGREE OF CEREBELLAR

THE FATHER APPEARS OKAY.

AND CLINICALLY IN THE PARENTS

SHOW KNOW EVIDENCE OF HAVING

SYMPTOMS OF THE DISEASE.

NOW, WITH THE HELP OF

COLLABORATORS IN GERMANY, WE

WERE ABLE TO ACTUALLY STUDY

THESE MUTATIONS IN A YEAST

SYSTEM AND AS A NEUROLOGIST I'M

GOING TO STUMBLE HERE, TRYING TO

PRESENT TO YOU A YEAST

COMPLIMENTATION STUDY BUT I'LL

DO MY BEST.

IN THIS SYSTEM, I BRING YOUR

ATTENTION TO THE PANEL WITH THE

YPG, BASICALLY IN THIS SYSTEM

THE MEDIA IS ENRICHED WITH

GLYCEROL OR WITH DEXTROSE.

OF COURSE, DEXTROSE IN THE

PRESENCE OF MITOCHONDRIA

DYSFUNCTION CAN BASICALLY LEAD

TO THE USE OF THE GLYCOLYTIC

PATHWAY AND THE YEAST WILL BE

ABLE TO SURVIVE.

IN THE PRESENCE OF GLYCEROL,

BASICALLY THE YEAST DEPENDS

EXCLUSIVELY ON MITOCHONDRIAL

BIOENERGETICS TO BE ABLE TO

SURVIVE.

SO WILD TYPE IN THE PRESENCE OF

GLYCEROL, THE YEAST IS ABLE TO

MAKE IT THROUGH.

WHEN WE PRESENT, THIS WILL BE A

DOUBLE MUTANT FOR AFD 3L-2, THE

YEAST IS UNABLE TO SURVIVE.

IN THE HETEROZYGOUS STATE AFD

3L-2 ALLOW IT IS YEAST MAKE IT

THROUGH BUT IF WE INTRODUCE

MUTATIONS INTO THE YEAST SYSTEM

THAT AFFECT INCLUDING OUR

MUTATION WE CAN SEE THAT THE

BIOENERGETICS OF THE YEAST

SYSTEM ARE BASEICALLY FAILING.

THE SAME CAN BE DONE FOR THE

CAPACITY OF PARA PLEGAN TO IN

COMBINATION WITH YEAST MUTATION,

WITH THE MUTATION USED IN THE

YEAST BASICALLY DEMONSTRATES

THAT THE PRESENCE OF THIS

PATIENT'S MUTATIONS RESULTING IN

IMPAIRED CAPACITY FOR

MITOCHONDRIAL FUNCTION DESPITE

THE FACT THAT THE AMOUNT OF

PROTEIN PRODUCED APPEARS TO BE

NORMAL.

KNOWING THAT I WAS GOING TO

STUMBLE IN THIS I DECIDED TO

MAKE IT A FIGURE THAT IS MY OWN

RESCUE.

AND WHICH I BASICALLY ILLUSTRATE

TO YOU WHAT WE CAN CONCEIVE AS A

MECHANISM FOR THE EMERGENCE OF

THESE UNIQUE PHENOTYPE IN THESE

TWO CHILDREN WHEN THEY RECEIVED

TWO COPIES OF MUTATED GENE WITH

AFD-3L-2.

THIS IS THE FIRST EVER REPORTED

CASE OF THESE PARTICULAR

INSTANCE.

SO WE CAN THINK OF THIS

COMPLEXES.

HERE WE HAVE THE HOMO COMPLEXES

HERE AND HERE THE HETERODIMER

COMPLEXES IN THE NORMAL SITION,

THE ENZYME ACTIVITY WILL BE

UNAFFECTED IN THE PRESENCE OF A

CARRIER STATE FOR SBG-7, THEN

AFD 3L-2 ACTIVITY IS UNRELATED

AND UNAFFECTED.

THE PARAPLEGAN STILL HAS

SUFFICIENT ACTIVITY TO PREVENT

DISEASE.

IN THE SPG-7 DISEASE, REMEMBER

THIS IS AN AUTOSOMAL RECESSIVE

DISEASE, THE MUTATED AFG --

PARAPLEGAN RESULTS IN

DYSFUNCTIONAL PARAPLEGAN

SPECIFICALLY, PRODUCING A

PHENOTYPE CHARACTERIZED

PRIMARILY BY SPINAL CORD

DEGENERATION, SPECIFICITY AND SO

FORTH.

WHEN WE INTRODUCED IN THIS

PARTICULAR CASE THIS WOULD BE

HETEROZYGOUS MUTATION OF AFD

3L-2 GENE, IT PRODUCES LATE

ONSET CEREBELLAR DISEASE AS

SPINAL CEREBELLAR ATAXIA, THE

COMPLEXES BECAUSE OF THE

FORMATION HEXA DIMERS BASICALLY

THIS COMPLEXES WILL RESULT IN

SOME DEGREE OF PROTEIN THAT

ASSEMBLES NORMALLY, SOME

COMPLEXES THAT ARE MIXED BETWEEN

THE MUTATED AND NORMAL PROTEIN

AND THEN WITHIN THE MUTATED

PROTEIN ONLY.

THE -- THERE IS STILL ENZYME

ACTIVITY THAT MAY EXPLAIN LATE

ONSET OF THE PHENOTYPE.

WHEREAS THEY STILL OF SPG-7

ACTIVITY THERE IS NO PLASTICITY

AS PHENOTYPE OF THIS DISEASE.

THIS IS BASICALLY THE PATIENT

THAT WE DISCUSS HERE.

THIS PATIENT HAS A MARKLY

DISRUPT FUNCTION OF AFD 3L-2, AT

THE SAME TIME BECAUSE THAT


AFD-3L-2 FORMS COMPLEXES WITH

PARAPLEGAN THE PATIENT HAS

BASICALLY A PHENOTYPE OF SPASTIC

PARAPARASAS.

SO BASICALLY KIND OF MAKES SENSE

OF MY INITIAL TITLE TO MY TALK

HOW A MUTATION IN AFD 3L-2

INDIRECTLY LEADS TO BASICALLY A

DISEASE THAT IS MANIFESTED BY A

MUTATION BUT BY A PROTEIN THAT

IS NORMAL, SPG-7.

BECAUSE OF INTERACTION WITH

AFD-3L-2.

THIS WORK HAS BEEN A COOPERATIVE

EFFORT.

THAT IS SPANS ACROSS INSTITUTES

AND ALSO ACROSS THE OCEAN.

WE HAVE NHGRI, TYLER PIERSON WAS

INSTRUMENTAL IN MOVING THIS

PROJECT FORWARD.

THE GOAL TO PROVIDE THE SPIRIT

AND ALL THE EFFORT IN GETTING

THE PROGRAM WORKING ON A DAY BY

DAY BASIS AND HE PROVIDED A LOT

OF THE DIRECTION IN THIS PAPER.

WE HAVE BIOINFORMATICS TEAM THAT

LOOKED AT THE EXOME DATA.

WE HAVE COLLABORATORS FROM

SPEASNIST, COLLABORATORS FROM

NINDS AND ALL THE CELL BIOLOGY

WORK THAT WAS PRESENTED HERE IS

ACTUALLY HARD WORK OF THE GROUP

IN GERMANY.

SO I APPRECIATE YOUR ATTENTION.

THANK YOU VERY MUCH.

[APPLAUSE]

>> I WANT TO THANK EVERYONE FOR

PARTICIPATING.

IF THERE'S ANY QUESTIONS FROM

THE AUDIENCE, ASK THEM NOW OR

CONTACT THE SPEAKERS AFTERWARDS.

THIS WILL BE ARCHIVED, AS I

MENTIONED ON VIDEOCAST.NIH.GOV

WITHIN A DAY OR TWO.

AND YOU WILL BE ABLE TO ASK

QUESTIONS AT THAT TIME ALSO.

SO ANY MORE QUESTIONS?

ANY COMMENTS?

I GUESS WE CAN QUIT EARLY.

THANK YOU ALL VERY MUCH FOR

COMING.