Boot Camp: How to Order and Interpret Genetic Data


SO THE SECOND PART OF THE TALK IS GOING TO BE REALLY FOCUSED ON GENETIC TESTING OF MONOGENIC AUTOINFLAMMATORY DISEASES. SO THE FIRST GENE IDENTIFIED IN AUTOINFLAMMATORY DISEASE WAS ACTUALLY GENES THAT IS FOUND IN PATIENTS OR MUTATED IN PATIENTS WITH — AND THIS GENE IS KNOWN AS M — 3. BASICALLY TODAY THERE ARE MORE THAN 30 GENES THAT ARE ASSOCIATED WITH DIFFERENT MONOGENIC AUTOINFLAMMATORY DISEASES, AND PART OF THIS MAJOR PROGRESS IN IDENTIFICATION OF ALL THESE DISEASE-CAUSING GENES, STILL MUTATIONS IN THESE GENES ACCOUNT ONLY FOR ABOUT 40% OF THE PATIENTS THAT ARE CLINICALLY DIAGNOSED WITH AUTOINFLAMMATORY DISEASES. MOST GENES WILL BE DISCOVERED IN THE NEAR FUTURE WITH THE USE OF NEXT GEN SEQUENCING TECHNOLOGIES WITH IMPROVEMENT OF THE ALGORITHMS THAT WE USE FOR DATA ANALYSIS AND ALSO HAVING BETTER OF VARIANT FREQUENCIES IN DIFFERENT POPULATIONS WHICH ARE VERY IMPORTANT FOR GENE DISCOVERY PROCESS AND ALSO GENETIC DIAGNOSIS OF THESE PATIENTS. SO HERE SHOWN ARE A LIST OF DOMINANTLY INHERITED MONOGENIC INFLAMMATORY DISEASES AND BASICALLY WHAT I WOULD LIKE JUST TO SAY HERE IS THAT THESE PATIENTS HAVE SINGLE OR HETEROZYGOUS MUTATIONS IN RESPECTIVE GENES, AND THESE MUTATIONS ARE PASSED FROM ONE TO ANOTHER GENERATION. PATIENTS THAT HAVE VERY SEVERE PHENOTYPE AS IS THE CASE, FOR EXAMPLE, WITH PATIENTS THAT HAVE — DISEASE, THEY HAVE WHAT WE CALL DE NOVO MUTATINS. SO THESE DE NOVO MUTATIONS ARE NOT FOUND IN PARENT. HERE IS SHOWN A LIST OF RECESSIVELY INHERITED MONOGENIC AUTOINFLAMMATORY DISEASES, AND THESE PATIENTS HAVE TWO MUTATIONS IN A GIVEN GENE. WE CALL THEM — TYPICALLY THESE MUTATIONS ARE LOSS OF FUNCTION — FOR MOST OF THESE DISEASES EXCEPT FOR THE GENE OR MUTATIONS THAT ARE ASSOCIATED WITH FAMILIAL MEDITERRANEAN FEVER, THEY ARE RECESSIVELY INHERITED BUT CONSIDERED TO BE AGAIN FUNCTION MUTATIONS. THERE ARE MORE GENES LINKED TO AUTOINFLAMMATORY PHENOTYPE IN THE CONTEXT OF BROADER IMMUNE DYSREGULATION AND THIS IS THE CONCEPT THAT HAS BEEN INCREASINGLY RECOGNIZED DURING THE PAST FEW YEARS. SO IN THE PAST, WE TEND TO GROUP THE PATIENTS INTO PATIENTS WITH AUTOIMMUNITY OR AUTO INFLAMMATION OR PRIMARY IMMUNODEFICIENCY. SO WHAT WE ARE TALKING MORE ABOUT THESE DAYS IS THERE IS THIS SPECTRUM OF AMEUN DYSREGULATION OF PATIENTS THAT HAVE IMMUNE DISEASES SO THAT IN ADDITION TO AUTOIMMUNITY, PATIENTS MAY PRESENT WITH SOME FEATURES OF AUTO INFLAMMATION OR THERE ARE PATIENTS, FOR EXAMPLE, THAT HAVE PRIMARY NEURODEFICIENCY, THEY ARE SUSCEPTIBLE TO VARIOUS INFECTION AS IS THE CASE — HOWEVER, THEY MAY ALSO HAVE COMPONENT OF AUTO INFLAMMATION, SO THEY PRESENT, FOR EXAMPLE, WITH GI INFLAMMATION. SO TO CONFIRM A SPECIFIC DIAGNOSIS IN AN INDIVIDUAL PATIENT OR A FAMILY AND TO EXCLUDE ALL OTHER CONDITIONS THAT PRESENT WITH OVERLAPPING PHENOTYPES. SO THIS GENETIC TESTING IS NOT SIMPLE PROCESS FOR THE REASON THAT INHERITANCE OF THESE AUTOINFLAMMATORY DISEASES IS NOT STRAIGHTFORWARD. SO HOW — HOWEVER IN ADDITION TO THESE TYPE OF INHAIR — ALSO
KNOWN AS CANDLES. SO THESE PATIENTS HAVE TWO MUTATIONS BUT INSTEAD OF HAVING TWO MUTATIONS IN ONE GENE, THEY HAVE TWO MUTATIONS IN TWO DIFFERENT GENES AND TYPICALLY THESE GENES ENCODE THE PROTEINS THAT MAKE UP OR COMPOSE A LARGE MULTI-PROTEIN COMPLEX. SO MOST PATIENTS THAT HAVE — OR CANDLE HAVE TWO MUTATIONS IN THE PSMBA GENE, HOWEVER THERE ARE SOME PATIENTS WHO MAY HAVE ONE MUTATION IN ONE GENE AND ANOTHER IN ANOTHER GENE ENCODES FOR PROTEIN THAT ALSO MAKES UP THIS IMMUNO — COMPLEX. SO BASICALLY ESSENTIALLY IT MEANS IF YOU HAVE A PATIENT WITH SUSPECTED CANDLE DISEASE, AT THIS POINT WE ARE SCREENING FOR MUTATION IN SIX GENES BUT IT’S ALSO INDICATED TO SCREEN FOR MUTATIONS FOR ALL THE 14 GENES THAT ARE INVOLVED IN THE MAKING UP OF THIS COMPLEX OR THAT MAKE UP THIS COMPLEX. THERE IS ALSO EVIDENCE FOR OLIGOGENIC TYPE OF INHERITANCE AND THIS IS BASICALLY A TYPE OF INHERITANCE WHICH INVOLVES SEVERAL LOW FREQUENCY VARIANTS. SO THESE ARE VARIANTS THAT ARE FOUND IN THE ALLELE FREQUENCY BETWEEN 1 TO 5% IN GENERAL POPULATION. SO PATIENTS THAT HAVE VERY MONOGENIC DISEASES, THEY HAVE EITHER NOVEL OR VERY RARE VARIANTS. PATIENTS THAT HAVE DISOLIGOGENIC TYPE OF INHERITANCE, THEY HAVE BASICALLY SEVERAL OF THESE LOW FREQUENCY MUTATIONS AND PATIENTS THAT HAVE WHAT WE CALL COMPLEX OR POLYGENIC DISEASES, THEY BASICALLY HAVE COINHERITANCE OF SEVERAL COMMON ALLELE VARIANTS THAT ARE FOUND AT THE FREQUENCY OF 30 OR 40% IN THE GENERAL POPULATION AND FOR THESE PATIENTS SUCH AS PATIENTS WITH RA OR LUPUS OR DUE SHETS DISEASE BASICALLY WE SAY THEY HAVE PRESENCE OF SEVERAL WHAT WE CALL SUSCEPTIBILITY ALLELE MUTATIONS BUT NOT REAL THROUGH PATHOGENIC VARIANTS BECAUSE THEY ARE FOUND IN UNAFFECTED PEOPLE AS WELL. SO BECAUSE THESE VARIANTS ARE LOW FREQUENCY VARIANTS, THEY ARE OFTEN FOUND IN UNAFFECTED PARENTS OF THESE PATIENTS AND TYPICALLY THESE PATIENTS PRESENT WITH NONSPECIFIC AND MILDER INFLAMMATORY PHENOTYPES COMPARED TO PATIENTS WHO CARRY NOVEL OR RARE VARIANTS IN THE SAME GENE. THE HYPOTHESIS IS SEVERAL OF THESE — BASICALLY MAY HAVE SOME SORT OF SYNERGISTIC EFFECT ON SOME INFLAMMATORY PATHWAY TO INDUCE INFLAMMATION IN THESE PATIENTS, HOWEVER, THAT HAS NOT BEEN DEMONSTRATED BY ANY FUNCTIONAL STUDIES. EXAMPLES OF SUCH VARIANTS THAT ARE VERY COMMONLY — B46 PERMUTATION, TNF RECEPTOR 1 GENE OR SEVERAL VARIANTS THAT ARE IDENTIFIED IN THE NLRP3 GENE. SO LOW FREQUENCY VARIANTS ARE BASICALLY WHAT WE CALL NON-CONFIRMATORY GINO TYPES, SO
GENOTYPES. SO DISEASES ASSOCIATED WITH SOMATIC MUTATIONS, TYPICALLY PATIENTS WITHOUT INFLAMMATORY DISEASES HAVE MUTATIONS IN THE MYELOID CELLS AND THEY CAN EXPAIN OR ACCOUNT TO UP TO 30% OF THE PATIENTS THAT ARE DIAGNOSED WITH CAPS. THERE ARE ALSO REPORTS OF SOMATIC MUTATIONS IN PATIENTS WITH SAVI, WITH NLRC4 ASSOCIATED MAS AND ALSO HAPLOINSUFFICIENCY OF A20. HAL ALSO MENTIONED THERE IS EVIDENCE FOR GONADAL MOSAICISM. YOU HAVE TWO AFFECTED SIBLINGS, YOU LOOK AT THE PARENTS, THEY DON’T HAVE SYMPTOMS OR THEY HAVE VERY MILD SYMPTOMS, BUT BASICALLY THERE ARE TWO AFFECTED SIBLINGS IN THIS FAMILY SUGGESTS THERE IS A GONADAL MOSAICISM IN THE FAMILY OR IN ONE OF THE PARENTS. TO PROVE THE PRESENCE OF SOMATIC MUTATIONS BASICALLY REQUIRES SINGLE OR TARGETED GENE PANELS OR WHOLE GENOME SEQUENING, WE CAN TEST FOR THE PRESENCE IN MYELOID CELLS HOWEVER WE CANNOT REALLY TEST FOR THE GONADAL MOSAICISM. SO WHY DO WE DO GENETIC TESTIN FOR SAIDs? THERE IS A LOT OF OVERLAP IN CLINICAL MANIFESTATIONS AND SIGNIFICANT PHENOTYPIC HETEROGENEITY. BY THAT I MEAN MUTATIONS IN ONE GENE CAN LEAD TO VERY DIFFERENT PHENOTYPES AS I WILL DEMONSTRATE IN THE FOLLOWING SLIDES. SO NEUTROPHILIC DETERMINE PTOSIS, SKIN RASH WHICH IS SEEN IN TWO PATIENTS, AND PRESENTATION, CLINICAL PRESENTATION IS VERY SIMILAR, NEVERTHELESS THESE PATIENTS HAD MUTATIONS IN TWO DIFFERENT GENES. SO THIS PATIENT BASICALLY HAS A MUTATION IN THE — GENE AND HAS A DISEASE WHICH IS KNOWN AS PAAND, WHILE THE SECOND PATIENT PSTPIP1 MUTATION — ARE INTERACTING PROTEINS AND BASICALLY THEY ARE INVOLVED IN REGULATION OF THE SAME INFLAMMATORY PATHWAY, NAMELY, IL-1 BETA SIGNALING PATHWAY. HERE SHOWN ARE ALSO PATIENTS THAT ARE DIAGNOSED WITH ANOTHER RARE INFLAMMATORY DISEASE, AND THAT IS PRAID, I’M NOT DOWNPLAYING THE ROLE OF CLINICAL MANIFESTATIONS WHICH ARE IMPORTANT FOR THE CLINICAL DIAGNOSIS BUT NEVERTHELESS, THEY ALSO PRESENT WITH THE FEATURES — SEVERE FEATURES OF SKIN INFLAMMATIONS, AND FINALLY HERE IS SHOWN PATIENT THAT PRESENT WITH SERIAL SKIN ULCERS AND THIS PATIENT IS KNOWN TO HAVE A MUTATION IN THE TNFAIP TNFAIP3 GENE, SO OTHER EXAMPLES, PATIENTS THAT PRESENT WITH EARLY ONSET MONOGENIC VASCULITIS OF SMALL VESSELS, THESE PATIENTS BASICALLY PRESENT SOMETIMES WITH VERY SIMILAR CLINICAL FEATURES AS SHOWN HERE, SKIN ULCERATIONS, TYPE OF SKIN RASH, NEVERTHELESS, THEY HAVE MUTATIONS IN TWO DIFFERENT GENES THAT ARE INHERITED IN DIFFERENT WAYS. SO PATIENTS THAT HAVE SAVI, THEY HAVE — DISTINCT PROTEIN BASICALLY INTERFERON SIGNALING PATHWAY WHILE PATIENTS WITH OTHER TWO HAVE RECESSIVELY INHERITED LOSS OF FUNCTION MUTATION IN THE ADA2 GENE AND PROTEIN. FURTHER EVIDENCE FOR GENETIC HETEROGENEITY PATIENTS, COLD-INDUCED AUTO IP FLAM TRI DISEASE, THE FIRST DESCRIBED WITH THIS PHENOTYPE WAS BASICALLY FCAS1 OR MUTATION IN THE NLRP3 GENE, TODAY THERE ARE AT LEAST FIVE LINKED TO COLD-INDUCED AUTOINFLAMMATORY DISEASE. WE HAVE TO NOW CONSIDER SCREENING FOR MUTATIONS ALL THESE OTHER GENES. SO HERE SHOWN IS ANOTHER FAMILY, TWO SIBLINGS THAT WERE PROVISIONALLY DIAGNOSED WITH EARLY ONSET LUPUS, MONOGENIC TYPE OF LUPUS BECAUSE THEY PRESENTED WITH SPECIFIC SKIN RASHES, SUCH AS THIS RASH SEEN ON THE COMEEX OF THIS CHEEKS OF
THIS PATI ENT AND OTHER TYPE OF SKIN RASHES. ONE OF THESE TWO SIBLINGS ALSO HAD OR PRESENTED WITH STROKES AND THAT WAS THE REASON WHY THE CLINICIANS CONSIDERED POSSIBLE DIAGNOSIS OF DADA2. AND BASICALLY THESE TWO SIBLINGS WERE PROVEN TO HAVE DADA — PRO TEENGS FUNCTIONAL TESTING AND ALSO MOLECULAR DIAGNOSIS. SO AGAIN, AUTOIMMUNE PHENOTYPE IS DESCRIBED IN THE SPECTRUM OF DADA 2 PHENOTYPE OTHERWISE IT COULD BE MISLEADING, MORE SUGGESTIVE OF MONOGENIC LUPUS THAN OF DADA2. SO THERE IS ALSO EXAMPLES OF MUTATIONS IN THE SAME GENE THAT CAN CAUSE TWO VERY DIFFERENT PHENOTYPES AND THAT’S CERTAINLY THE CASE WITH MUTATIONS IN THE PLC GAMMA 2 GENE LINKED TO PLAID AND APLAID. PATIENTS THAT HAVE APLAID WHICH IS QUITE DIFFERENT CLINICALLY, QUITE DIFFERENT PHENOTYPE HAVE SINGLE BASE SCWEUTIONS OR MISSENSE MUTATIONS IN THE SAME GENE. BOTH TYPE OF THESE DELETIONS AFFECT THE AUTO INHIBITORY DOMAIN OF THIS PROTEIN AND BASICALLY ACT AS A GAIN OF PROTEIN FUNCTION MUTATION IN THIS PATHWAY AND THEY UPREGULATE ACTIVITY OF MANY DOWNSTREAM MOLECULES. SO THERE ARE ALSO EXAMPLES THAT MUTATIONS IN THE SAME GENE COULD BE INHERITED IN DIFFERENT WAYS OR THEY MAY HAVE DIFFERENT INHERITANCE PATTERN, AND WE’LL ILLUSTRATE THAT ON THE EXAMPLE OF PAIRING ASSOCIATED DISEASES. SO MOST OF YOU KNOW FAMILIAL MEDITERRANEAN FEVER OR FMF LINKED TO RECESSIVE MUTATIONS IN THE C TERMINAL DOMAIN OF THIS PROTEIN IN MOST PATIENTS HAVE TWO MUTATIONS IN THIS DOMAIN. THERE ARE SOME EXAMPLES OF PATIENTS THAT HAVE ONE MUTATION AND WE BELIEVE THAT THESE PATIENTS PROBABLY HAVE SUSCEPTIBILITY ALLELE — FORMATION IN OTHER GENES SO THERE ARE SOME RARE EXAMPLES OF PATIENTS THAT HAVE ONLY ONE MUTATION IN THIS PROTEIN DOMAIN. HOWEVER, THERE ARE PATIENTS THAT HAVE CLEARLY DOMINANTLY INHERITED MUTATIONS IN OTHER DOMAINS OF THIS PROTEIN AND THESE PATIENTS HAVE DISEASE WHICH ARE KNOWN AS PAAND ADD
AND SEVERE INFLAMMATORY SYMPTOM THAT USED TO BE CALLED FMF-LIKE BUT THEY HAVE MUCH MORE SEVERE PHENOTYPES. SO WHAT ES A THE EXPLANATION FOR THAT? BASICALLY THESE FMF ASSOCIATED MUTATIONS ARE SORT OF — WE CALL THEM GAIN OF FUNCTION MUTATIONS BUT ESSENTIALLY THEY ARE HYPER MORPHIC MUTATIONS BECAUSE THEY AFFECT THE FUNCTION OF THIS AUTO IB HIB TRI AUTO INHIBITORY DOMAIN AND THEY BASICALLY REDUCE THE THRESHOLD FOR THE ACTIVATION OF THIS PROTEIN, WHILE THESE OTHER MUTATIONS INHERIED AS A DOMINANT TRAIT, THEY AFFECT THE RESIDUES WHICH ARE BASICALLY LEADING TO MUTATIONS IN THESE RESIDUES LEAD TO THE — IN THESE PROTEIN SO IN THE CASE OF PAAND PATIENTS — FOR RELATION OF PROTEIN. SO THERE ARE THREE TYPES OF CLINICAL GENETIC TESTING, SINGLE GENE TESTING, — BASICALLY INVOLVES REPORTING ONLY CLINICALLY SIGNIFICANT VARIANTS BUT ESSENTIALLY IS DONE WITH WHOLE EXOME SEQUENCING OR WHOLE GENOME SEQUENCING. I WOULD ALSO LIKE TO MENTION TODAY A NEW PAPER THAT IS PUT TOGETHER BY THE INTERNATIONAL TEAM OF GENETICISTS WHO ARE EXPERTS FOR THE GENETICS OF AUTOINFLAMMATORY DISEASES WHICH BASICALLY PROPOSED BEST PRACTICE GUIDANCE FOR THE GENETIC DIAGNOSIS OF THESE EIGHT AUTOINFLAMMATORY DISEASES, FMF, MKD, TRAPS, CAPS, BLAU/IBD, PAPA, DADA2 AND HA20. SO WHAT IS SHOWN IN THIS PAPER OR WHAT WE PROPOSED IN THIS PAPER IS THAT IF THE PATIENT PRESENTS WITH CLINICAL PHENOTYPE HIGHLY SUGGESTIVE OF SPECIFIC S AD, THEY PRESENT WITH FEVERS AND COME FROM — POPULATION AS IS THE CASE WITH PATIENTS WITH FMF WHICH COME FROM THE MEDITERRANEAN POPULATION OR IF THERE’S A POSITIVE FUNCTIONAL TEST THAT ARE DONE IN PATIENTS THAT ARE SUSPECT THE SUSPECTED
TO HAVE DA DA2 OR — KINASE DEFICIENCY, WE SUGGEST TESTED BY SINGLE GENE, SANGER BASICALLY SEQUENCING FOR MUTATIONS EITHER IN SPECIFIC EXON OR ALL THE EXONS OF A GIVEN GENE. SO IF THIS SINGLE GENE SEQUENCING IS NOT INFORMATIVE, THEN THE NEXT STEP TO DO IS FOR PATIENTS THAT HAVE, FOR EXAMPLE, DADA2 AND — KINASE DEFICIENCY AND — TO SEARCH FOR — THERE ARE REPORTS OF PATIENTS THAT BASICALLY HAVE — NUMBER VARIATIONS AS THE DISEASE-CAUSING MUTATIONS. IN PATIENTS THAT PRESENT WITH CAPS LIKE BLAU OR TRAPS LIKE DISEASE, IF THE SINGLE GENE SEQUENCE IS NEGATIVE OR WE DON’T FIND CONFIRMATORY GENOTYPE, THE NEXT STEP WOULD BE TO SEARCH FOR MOSAIC MUTATIONS BECAUSE THERE ARE REPORTS OF MULTIPLE PATIENTS WHO CARRY MOSAIC MUTATIONS IN — CELLS LEADING TO RESPECTIVE PHENOTYPES. SO IF THIS SEARCH IS AGAIN NON-CONFIRMATORY, THE NEXT THING WILL BE TO RUN TARGETED GENE PANELS FOR DID GENES OR MORE GENES IF AVAILABLE, AND IF THIS TESTING IS NEGATIVE, THEN THE NEXT STEP WOULD BE TO DO WHOLE EXOME SEQUENCING OR WHOLE GENOME SEQUENCING. PATIENTS THAT PRESENT WITH NONSPECIFIC PHENOTYPE SHOULD BE TESTED EITHER BY TARGETED GENE PANEL SEQUENCING OR SHOULD BE TESTED BY –. SO HERE YOU MAY WONDER WHY DO WE GO THROUGH ALL THESE STEPS, YOU KNOW, INSTEAD OF DOING STRAIGHT AND DOING TARGETED PANEL SEQUENCING OR WHOLE GENOME SEQUENCING? THE ADVANTAGE OF DOING SINGLE GENE SEQUENCING IS JUST IT’S FASTER. FOR EXAMPLE, WE CAN DO RESULTS IN COUPLE OF DAYS IF NECESSARY, WE CAN HAVE THE GENETIC TESTING RESULT REPORTED. AND IT’S ALSO LESS EXPENSIVE THAN DOING WHOLE EXOME SEQUENCING OR WHOLE GENOME SEQUENCING, ALTHOUGH THIS TECHNOLOGY HAS BEEN INCREASINGLY NOW USED AT NIH AND MANY OTHER INSTITUTES AND HOSPITALS. SO HERE I WOULD LIKE TO GIVE YOU EXAMPLE OF WHAT WE HAD TO DO IN ONE FAMILY TO BASICALLY PROVE THAT THIS FAMILY HAS A DADA2. SO HERE SHOWN IS THE FAMILY FROM INDIA WITH UNUSUAL INHERITANCE PATTERN. SO BASICALLY THERE ARE TWO AFFECTED SIBLINGS IN THIS FAMILY. THE FATHER IS ALSO AFFECTED SO YOU THINK DOMINANTLY INHERITED DISEASE, HOWEVER, THERE IS — IN THIS FAMILY WHICH WOULD THEN SUGGEST THEY’RE MORE LIKELY TO HAVE — MUTATIONS IN SOME GENE. THIS FAMILY WAS PROVISIONALLY DIAGNOSED WITH DIAMOND-BLACKFAN ANEMIA, NO VARIANTS WERE IDENTIFIED. BECAUSE SOME PATIENTS WITH DADA2
MAY PRESE NT ALSO WITH D/B/A-LIKE CLINICAL MANIFESTATIONS, THEY WERE SUSPECTED MAYBE TO HAVE DADA2. SO WE DID SEQUENCING AND DIDN’T IDENTIFY ANY PATHOGENIC VARIANTS. TESTING FOR DADA2 COULD ALSO BE DONE WITH — PROTEIN TESTS AND BASICALLY SHOW THESE TWO AFFECTED SIBLINGS AND FATHER HAVE ABSENCE OF ADA2 ACTIVITY. SO THIS IS A PROTEIN TEST WHICH IS UNFORTUNATELY NOT AVAILABLE FOR MANY AUTOINFLAMMATORY DISEASES BUT IN CASE OF DADA2, IT IS AVAILABLE. SO WHY DO WE NEED TO GO A STEP FURTHER, WHY DO WE NEED TO DO NOW SEQUENCING OF THIS GENE? BASICALLY THE IMPORTANCE OF THAT IS REALLY FOR GENETIC COUNSELING IN THIS FAMILY. SO WHAT WE DID INITIALLY WAS TO SCREEN BY SANGER SEQUENCING, HOWEVER A NUMBER OF VARIATIONS ALSO REPORTED AS CAUSE OF DISEASE DADA2, SO FOR THAT REASON, WE DEVELOPED ANOTHER ASSAY NOW IN A LAB WHICH IS CALLED MLPA ASSAY, SHOWING BOTH AFFECTED FATHER BASICALLY WERE HOMOZYGOUS FOR DUPLICATION OF EXOME 7 IN THIS GENE. SO YOU CAN SEE WE HAD TO DO MULTIPLE LEVELS OF TESTING IN ORDER TO BASICALLY FIND WHAT IS THE CAUSE OF THE DISEASE IN THIS FAMILY. SO TARGETED GENE PANEL SEQUENCING HAVE ALREADY TALKED ABOUT THEM SO THEY’RE TIME-EFFECTIVE, TYPICALLY YOU CAN GET YOUR RESULTS IN A COUPLE OF WEEKS RELATIVE TO WHOLE GENOME OR WHOLE EXOME SEQUENCING, THEY’RE COST-EFFECTIVE, THEY COST LESS MONEY THAN DOING WHOLE EXOME OR WHOLE GINO SEQUENCING, THEY HAVE HIGHER ACCURACY — THAT’S WHY THEY ARE MORE INDICATED TO FIND SOMATIC MUTATIONS COPY NUMBER VARIATIONS. THE OTHER ADVANTAGE OF DOING TARGETED GENE PANEL SEQUENCING FOR OUR — FOR GENETICISTS IS THAT BASICALLY THERE IS LOWER LIKELIHOOD TO IDENTIFY VARIANTS OF UNKNOWN SIGNIFICANCE THAT ARE HARD FOR US TO IPT PRET,
INTERPRET, ALSO CHALLENGING FOR PATIENTS. SO THE RECOMMENDED COVERAGE FOR CLINICAL SEQUENCES — OVER SINGLE NUCLEOTIDE SO THE NEXT QUESTION IS WE HAVE ALL THESE BEAUTIFUL TECHNOLOGIES AND ACCESS TO PUBLIC DATABASES, SOMETHING WE COULDN’T EVEN DREAM OF 20 YEARS AGO OR 30 YEARS AGO WHEN I STARTED DOING GENETIC STUDIES, SO WHAT IS THE DIAGNOSTIC YIELD AND CLINICAL UTILITY OF TARGETED GENE PANEL SEQUENCING? UNFORTUNATELY FOR MOST DISEASES, WITH EXCEPTION FOR SOME INTELLECTUAL DISABILITIES AND SOME NEURODEVELOPMENTAL DISEASES, IT’S NOT HIGHER THAN 30%. THERE IS CERTAINLY SITUATION WITH MONOGENIC AUTOINFLAMMATORY DISEASES SO WHAT ARE THE POSSIBLE EXPLANATIONS? WHY WE DON’T HAVE A VECTOR, WHY WE DON’T HAVE A VECTOR DIAGNOSTIC — SO WE NEED BETTER ALGORITHMS FOR INSILICO ANALYSIS, POPULATION-SPECIFIC ALLELE FREQUENCY DATABASES. — BASED ON THE FREQUENCY OF THESE VARIANTS MOSTLY IN CAUCASIAN POPULATION, SO WE ARE STILL MISSING THIS POPULATION DATABASES IN OTHER, YOU KNOW, ANCESTRIES OR OTHER POPULATIONS. SO THERE IS THIS ISSUE OF REDUCED PENETRANCE, WHICH IS COMPLICATING INTERPRETATION OF THESE RESULTS, THERE ARE ALSO CELL-SPECIFIC SOMATIC MUTATIONS THAT MAY NOT BE DETECTED IN PRIF PERIPHERAL BLOOD SAMPLES, WE HAVE TO DO SEQUENCING IN MYELOID CELLS, FOR EXAMPLE, AND THIS TECHNOLOGY ALSO DOES NOT CAPTURE NON-CODING VARIANTS AND DOES NOT CAPTURE LARGE COPY NUMBER VARIATINS BY DATA, LARGE DELETIONS LIKE 100 MEGA BASES OR SOMETHING LIKE THAT, THEY ARE NOT GOING TO BE DETECTED BY THESE TARGETED PANEL GENE SEQUENCING. SO HERE I WILL JUST SHOW YOU A COUPLE OF STUDIES THAT REPORTED WHAT IS THE YIELD OF TARGETED SEQUENCING FOR AUTOINFLAMMATORY DISEASES. SO THIS STUDY WAS DONE IN THE U.K. AND BASICALLY THEY EVALUATED 55 PATIENTS WITH UNDIAGNOSED AUTO INFLATION AND VASCULITIS AND THEIR DIAGNOSTIC YIELD WAS ONLY 12%, SO SIX OUT OF 50 PATIENTS HAD AT LEAST ONE CLEARLY PATHOGENIC VARIANT CLASS 5 VARIANT. THREE OUT OF SIX WERE DEFINITE CASES, THE OTHER THREE PATIENTS WERE PRESUMABLY DIAGNOSED BUT ACTUALLY NOT COMPLETELY, THEY DIDN’T HAVE CONFIRMATORY GENOTYPE BECAUSE TWO OF THESE PATIENTS HAD MONO ALE LICK PATHOGENIC VARIANT IN THIS GENE WHICH IS ASSOCIATED WITH HLH, IN ORDER TO CONFIRM DIAGNOSIS OF HLH WHICH WAS NOT THEIR CLINICAL PRESENTATION TO BEGIN WITH, ONE WOULD REALLY HAVE TO FIND TWO MUTATIONS IN THIS GENE AND ANOTHER PATIENT WHO WAS A FEMALE CHARACTER FOR X-LINKED — SYNDROME MUTATION. SO THEY ALSO CLAIM THAT 22% OF PATIENTS IN THIS COHORT HAD AT LEAST ONE LIKELY PATHOGENIC VARIANT. SO THE OVERALL YIELD WAS NO HIGHER THAN 30%. DIAGNOSTIC YIELD WAS NOT HIGHER THAN 30%. HERE IS EXAMPLE OF ANOTHER STUDY WHICH NOTHING TO DO WITH AUTO INFLAMMATION BUT THESE ARE PATIENTS WITH PRIMARY IMMUNODEFICIENCY TESTED FOR — 171 GENES AND CONCLUSIVE DIAGNOSIS WAS MADE ONLY IN 60% OF THESE PATIENTS. ANOTHER GOOD OUT COME OF THE STUDY IS THEY IDENTIFIED THREE PATIENTS THAT PRESENTD WITH ATYPICAL FEATURES AND BASICALLY THEY CONFIRMED THEIR DIAGNOSIS BY IDENTIFYING MUTATIONS IN RESPECTIVE GENES. SO HOW — TO INTERPRET THE
GENETIC TESTING? WE HAVE TO DO FILTERING FOR LOW COVERAGE, SYNONYMOUS VARIANTS, ALTHOUGH THEORETICALLY THEY COULD ALSO BE DISEASE-CAUSING VARIANTS, WE ALSO FILTER OUT VARIANTS THAT HAVE ALLELE FREQUENCIES HIGHER THAN 1%. I PREFER TO DO FILTERING EVEN WITH A LOWER NUMBER, I FILTER VARIANTS THAT HAVE FREQUENCY HIGHER THAN 1 IN 1,000 BECAUSE THEY’RE LESS LIKELY TO BE PATHOGENIC. FORTUNATELY WE HAVE ACCESS TO THESE LARGE DATABASES THAT ARE SHOWN HERE, AND AS I MENTIONED, THIS POPULATION/ANCESTRY SPECIFIC DATABASES OF VARIANTS ARE ALSO VERY IMPORTANT. IF THEY’RE THE NOT AVAILABLE, THE NEXT STEP IS ALWAYS TO DO TESTING OF THE PARENTS. WE DON’T TEST ALWAYS — PARENTS ARE VERY IMPORTANT ESPECIALLY FOR PATIENTS THAT COME FROM SPECIFIC ANCESTRIES. WE ALSO HAVE ACCESS TO INFEVERS DATABASE OF GENE VARIANTS FOUND IN PATIENTS WITH SAIDs AND THIS DATABASE WHICH IS VERY USEFUL FOR GENETICISTS BASICALLY HAS BEEN RECENTLY CURATED BY EXPERT GENETICISTS FOR MUTATIONS IN AID GENES — IF YOU GO NOW TO THIS INFEVERS DATABASE, THERE IS A COLUMN THERE WHICH WILL CLEARLY SAY WHETHER THIS MUTATION IS PATHOGENIC, LIKELY PATHOGENIC OR BENIGN. AND IF IN DOUBT, IT’S ALWAYS GOOD TO DO SEGREGATION ANALYSIS IN A NEAM, AND FAMILY AND WE
ALSO RELY ON PREDICTION TOOLS WHICH ARE SHOWN HERE, POLYPHEN, CADD, SIFT, ET CETERA. HOWEVER WE HAVE TO BE RESERVED WHEN RELYING ON THESE INSILICO PREDICTION TOOLS FOR EXAMPLE FMF MUTATIONS THAT HAVE BEEN KNOWN FOR A NUMBER OF YEARS SHOWN IN THOUSANDS OF PATIENTS ARE PREDICTED TO BE BENIGN. ON THE OTHER HAND, YOU CAN SEE A VARIANT THAT HAS VERY HIGH CUT SCORE BUT THEY ARE VERY COMMON IN THE GENERAL POPULATION. SO THEY ARE HELPFUL BUT THEY’RE NOT ABSOLUTE. SO HERE IS SHOWN THE STANDARDIZED DNA VARIANT SCORING SYSTEM THAT WAS DEVELOPED FOR MONOGENIC — HUMAN DISEASES ALSO USED IN PATIENTS WITH MONOGENIC AUTOINFLAMMATORY DISEASES SO THIS IS WHAT YOU’RE GOING TO SEE ON THE REPORT FROM DIAGNOSTIC COMPANIES. IT’S EITHER PATHOGENIC LIKE, PATHOGENIC VARIANT OF UNCERTAIN SIGNIFICANCE, LIKELY BENIGN OR BENIGN. SO THE MOST COMPLICATED, OF COURSE, ARE THESE VARIANT OF UNKNOWN OR UNCERTAIN CLINICAL SIGNIFICANCE. SO WHAT DOES IT TAKE TO MAKE CONFIRMATION OF CLINICAL DIAGNOSIS OF A PATIENT WITH SUSPECTED MONOGENIC AUTOINFLAMMATORY DISEASE? SO CONFIRMATION OF A COME NANT DISEASE REQUIRES PRESENCE OF PATHOGENIC OR LIKELY PATHOGENIC VARIANT. CLASS 4, CLASS 5 VARIANT. CONFIRMATION OF RECESSIVE DISEASE REQUIRES TWO PATHOGENIC OR PATHOGENIC-LIKE VARIANTS AND THEY SHOULD BE SHOWN TO BE INHERITED FROM TWO DIFFERENT PARENTS. COMBINATION IN PATHOGENIC OR LIKELY PATHOGENIC VARIANT WITH A RARE OR NOVEL VOUS SHOULD BE REPORTED AS CONSISTENT WITH THE CLINICAL DIAGNOSIS BECAUSE WE ARE NOT SURE WHETHER THIS NOVEL VOUS IS REALLY FUNCTIONALLY A RELEVANT VARIANT. IDETIFICATION OF A DE NOVO VARIANT MAY FURTHER SUPPORT ITS GENOTYPE PATHOGEN
PATHOGENICITY. A COMMON VOUS IS NOT REGARDED AND A CONFIRMATORY GENOTYPE AND ONLY NOVEL AND RARE VOUS, CLASS 4 AND CLASS 5 VARIANTS SHOULD BE REPORTED. PREDICTIVE DIAGNOSIS. FOR DISEASES WITH A RISK OF IRREVERSIBLE DAMAGE SUCH AS SEVERE NLRP3-AID, PREDICTIVE TESTING OF ASYMPTOMATIC FAMILY MEMBERS MAY BE JUSTIFIED. SO FOLLOW UP OF THESE AT RISK INDIVIDUALS MAY AVOID OCCURRENCE OF LIFE-THREATENING COMPLICATIONS, WHETHER SUCH CASES SHOULD BE GIVEN PROPHYLACTIC TREATMENT REMAINS CONTROVERSIAL. SO OUR PRACTICE HERE AT NIH IS TO TREAT PATIENTS WITH MOLECULARLY CONFIRMED DADA2 — WITH COMPLICATIONS OF DISEASE BUT THAT IS NOT THE CASE IN MANY OTHER HOSPITALS AND INTS TEUTS IN INSTITUTES IS VERY EXPENSIVE. SO GENETIC TEST SOMETHING NOW AN IMPORTANT PART OF RHEUMATOLOGY, ALLERGY, IMMUNOLOGY PRACTICE. CLINICAL DIAGNOSIS OF SAIDs IS HAMPERED BY LACK OF CLINICAL CRITERIA AND FUNCTIONAL TESTS FOR MOST OF THESE DISEASES. I DON’T WANT TO OVERESTIMATE THE IMPORTANCE OF GENETIC TESTING, OBVIOUSLY THAT’S WHAT I DO, I DO MOLECULAR DIAGNOSTIC TESTING, AND I DON’T WANT TO UNDERESTIMATE THE IMPORTANCE OF CLINICAL DIAGNOSIS BUT IT IS REALLY, YOU KNOW, BECOMING VERY, VERY IMPORTANT FOR DIAGNOSTIC PURPOSES. GENETIC TESTING IS CONSIDERED NOT ONLY DIFFERENTIAL DIAGNOSIS OF SAID BUT CAN ALSO ASSIST CLINICIANS IN THE PROCESS OF CHOOSING AN APPROPRIATE TREATMENT. SO IF YOU GET YOUR RESULT AND YOU SEE MUTATION IN NLRP3 YOU KNOW THIS PATIENT SHOULD BE TREATED WITH IL-1 INHIBITORS IF YOU GET A RESULT THAT SAYS THE PATIENT HAS A MUTATION IN 173 YOU KNOW TO GO AND USE JAK INHIBITORS, ET CETERA. ADVANCES IN GENETIC TESTING TECHNOLOGY HAVE INCREASED DETECTION OF PATHOGENIC VARIANTS BUT ALSO COMPLEEXITY OF DATA TO ANALYZE. AND WE CANNOT IGNORE THE ROLE OF ENVIRONMENTAL AND EPIGENETIC INFLUENCES AND FACTORS THAT MODULATE THE LIN CL CAL PENOTYPE AND IT SHOULD BE TAKEN INTO ACCOUNT FOR GENETIC COUNSELING OF PATIENTS AND FAMILIES. SO GOING BACK TO THESE TWO CASES THAT WERE PRESENTED EARLIER IN THIS TALK, SO BASICALLY THE RESULT OF TARGETED GENE PANEL SEQUENCING OF CASE ONE WAS NOT VERY INFORMATIVE SO BASICALLY THIS PATIENT WAS FOUND TO HAVE SIX VARIANTS OF UNKNOWN SIGNIFICANCE, INCLUDING R92Q AND Q703K MUTATION AND SO WHAT DOES HE HAVE, WHAT DO YOU DO WITH A PATIENT LIKE THIS? OBVIOUSLY ONE CAN THINK OF DOING RVGS WHICH WOULD BE EVEN MORE COMPLICATED FOR ANALYSIS AND MORE EXPENSIVE, ET CETERA, BUT IF THIS PATIENT PRESENTS ONLY WITH FEVER, WHAT I WOULD DO PERSONALLY, I WOULD JUST FOLLOW UP THIS PATIENT FOR A LITTLE WHILE TO SEE, YOU KNOW, WHETHER HE MAY DEVELOP SOME OTHER MORE SPECIFIC, YOU KNOW, MANIFESTATIONS, CLINICAL MANIFESTATIONS AND THAT CAN HELP ALSO A LOT EVEN IF YOU GET HIS — DATA WITH DATA ANALYSIS, SO WE CAN FOCUS ON DIFFERENT PATHWAYS MAYBE AND NOT TO LOOK THE ENTIRE EXOME DATA. SECOND PATIENT WAS WHAT WE SAY SOFT CASE SO THIS PATIENT WAS BASICALLY SHOWN TO HAVE A SOMATIC MUTATION IN THE NLRP3 GENE, AND THIS MUTATION WAS FOUND IN 10% OF BLOOD CELLS THAT WERE TESTED. SO I THINK I WENT A LITTLE BIT OVER THE TIME THAT WAS ALLOCATED FOR THIS TALK AND I APOLOGIZE TO THE NEXT SPEAKER. I DON’T THINK WE HAVE TIME TO TAKE ANY QUESTIONS BUT YOU ARE MORE THAN WELCOME TO COME TO ME AND IF YOU WANT TO TALK ABOUT GENETIC DIAGNOSIS OR IF YOU HAVE SOME INTERESTING PATIENTS OR WHATEVER, I WOULD BE MOST HAPPY TO TALK TO YOU DURING THE LUNCHTIME OR LATER. THANK YOU FOR YOUR ATTENTION.

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