Januari 07, 2017
>> our next speaker will be sharon savage.sharon was appointed in 2006 in dceg in the clinical genetics branch. she is currentlychief and senior investigator and is doing some exciting work with telomere biology andher subject is family studies, cancer etiology and telomere biology. >> okay, great. well thank you very much forthe opportunity to come in and talk about our work. it's a great honor to be here aswe recognize the many, many accomplishments of dr. fraumeni and family studies and it'sa real honor also to be a representative of our clinical genetics branch and the geneticsprogram in dceg. and i'd like to actually to start with, beyond li-fraumeni syndrome,which clearly had a huge impact. i wanted
to highlight some of the other seminal papersand topics that have resulted from the efforts in family studies and studying, the studiesby the astute clinician with bob miller and dr. fraumeni. so wilms tumor, aniridia congenitalmalformations, those first papers came out from these efforts in early dceg before itwas called dceg. we have familial chronic lymphocytic leukemia in which there are stillvery active efforts in genomics and understanding this disorder and those first papers cameout in 1969. myotonic dystrophy, now this is something that has come actually full circleand back to the clinical genetics branch, because dr. fraumeni actually wrote, and fredli and others, wrote a letter to the editor of a co-occurrence of leukemia and myotonicdystrophy in a patient. and several years
ago, mark greene in the clinical geneticsbranch, was contacted by a doctor from the myotonic dystrophy registry who said, "youknow, i think there's more cancer in these patients than we thought." and so they wenton to create a great collaboration with mark and shahinaz gadalla and the myotonic dystrophyregistry and, sure enough, these patients do have a markedly increased risk of cancercompared with the general population. and there's a large effort going on to validateand look at the molecular characteristics. but it started with these observations. wehave familial hodgkin's disease, family studies in that arena. hereditary breast and ovariancancer, six families reported with ovarian cancer which set the stage for, as we allknow, brca 1 and 2 in many different levels.
family studies of melanoma which had beeninitiated within dceg and really focused on studying these high risk families. we nowhave multiple new susceptibility genes, including pot1, a telomere gene that was just publisheda few months ago from our division. we have neurofibromatosis and its association withchildhood leukemia. chordoma, also still a very active research effort in the division,led by dilys perry and others where rose yang in dceg has recently, has been following upmany new exciting hits in this notochord developmental disorder. and so i think that this reallysets the stage and sort of shows you the breadth and depth and really how much we can learnfrom studying these rare inherited cancer syndromes. and that's exactly what i'm doingnow. and i'd like to transition and i'm going
to tell you a small piece of what we're doingnow in telomere biology disorders and dyskeratosis congenita, and we do also have a piece intelomere molecular epidemiology which i don't have time to talk about. but the combinationby being in a division that has been sort of founded in studying these rare familiesto understand biology and cancer, we've really been able to learn a lot about our patientswith dyskeratosis congenita and telomere biology. so telomeres, this is a graph of telomerelength and its decline with age. this was telomere length by flow-fish, so flow cytometrywith in situ hybridization on lymphocytes. telomere length is on the y axis, age is onthe x axis. and you can see a very sort of straight forward decline with age. this telomereis shortened with each cell division. this
is a normal decline. and these graphs aregoing to be very important in a little bit as i talk about the first percentile, thisblack line, and the 99th percentile. so 99 percent of our participants are going to bein between this normal range. telomeres are critical components of chromosomal stability,so they cap the ends of chromosomes and they are much more than just a dna repeat. theyare a long hexameric dna repeat and also a protein complex that cap the ends of eukaryoticchromosomes. now telomere are shortened with each cell division and when they get too shortcellular senescence or apoptosis and death are triggered. and that's part of the normalcell cycle. in cells that have a defective p53-dependent dna damaged checkpoint, theyactually, cells continue to divide despite
having critically short telomeres becausethey upregulate things like p53 and rb and telomerase. and they continue to divide despitehaving critically short telomeres and this results in some of those chromosome fusionsand chromosome breaks and aneuploidy that we see so often in cancer cells. and cancercells continue to divide because they upregulate telomerase or they use an alternative lengtheningof telomeres recombination base mechanism. so, now i'm going to talk only about germlinetelomere length. and when i think of germline telomere length, it's really a continuum wherewe have normal ranges of telomere length in between the first and 99th percentile andtelomere molecular epidemiology is really looking at statistical differences betweenpopulations. and there are a lot of association
studies that have looked at the associationbetween telomere length in the germline, usually in blood or buccal cells, and risk of cancer,of heart disease, and a lot of other illnesses that affect the general population. i'm notgoing to talk about that, but instead i'm going to focus on our patients who have incrediblyshort germline telomeres. and this is really at the other end of that spectrum where youhave very short germline telomeres because you have mutations in a key gene involvedin telomere biology. so this all stems from the nci�s and clinical genetics branch�sinherited bone marrow failure syndrome study. blanche alters is the pi, and the study hasbeen open for more than 12 years now. neelam giri is the key staff clinician for the study.and it's a longitudinal prospective cohort
study of cancer incidence in the more commoninherited bone marrow failure syndromes. it involves detailed questionnaires, medicalrecord review, and evaluation at the nih clinical center which is probably its greatest - notprobably, it is, its greatest strength, because we are able to bring participants here tosee not just the bone marrow failure team, but also to see all the subspecialists thatare relevant to their disease. and as a result, even our subspecialists have seen more fanconianemia, more dyskeratosis congenita patients than any other specialists in that field.and this has resulted in many different studies that have advanced our understanding of thephenotypes, including oral and dental phenotypes, lung problems, the eye problems our patientshave, endocrine problems, neuropsychiatric
problems, and many others. so, dyskeratosiscongenita is an inherited telomere biology disorder that's characterized by marked increasedrates of cancer and of bone marrow failure. patients have this classic diagnostic triadof abnormal nails, skin pigmentation and oral leukoplakia. but they can have a host of othermedical problems. it's really a true multi-systemic disease. there's a severe form of dyskeratosiscongenita called hoyeraal-hreidarsson syndrome, or hh for short, and those patients have allof these complications, plus they have cerebellar hypoplasia and severe immunodeficiency, andi'm going to talk a lot more about hh in a little bit. but what really unites this broadclinical spectrum is abnormally short telomeres and germline mutations. so our dc cohort studyto date has ascertained 90 classic dc or hh
patients. we have a subset of about 21 familieswho have a dc-like phenotype, so not quite enough for the classic diagnosis but close,and they are definitely in that spectrum. just over half of the affected individualsand their family members have been seen at the nih clinical center by our teams. andthis led to what is the first and the only study that has quantified cancer risk in ourdc patients. so blanche alter did this analysis with help from phil rosenberg in dceg wherewe looked at the cancer incidence in our patients and the bone marrow failure rates. and whatthis just shows is that by about age 50 about half of our patients will have had severebone marrow failure, the rates of solid tumors and aml are also extremely high, and the observed-to-expectedratios of especially tongue cancer in mds
are exceedingly high compared with the generalpopulation. so back 10 or 15 years ago, when all of this was getting started, we didn'thave a good diagnostic test for dc and i just told you it was very clinically heterogeneous,and it's difficult to make that diagnosis a lot of times, because patients don't developthe classic features at the same rates or at the same ages. so what we looked at waswith, in collaboration with peter lansdorp, looked at flow-fish telomere lengths, theseare those same curves, and this is our follow-up study where we showed that lymphocyte telomerelengths less than the first percentile for age were more than 95 percent sensitive andspecific for dc and this test has since become clia certified, it's the gold standard measurement,and it has really changed how we manage our
patients with dc. it's also changed how weselect our bone marrow transplant donors because we now can identify clinically silent carrierswho, if their telomeres are down here, really are not appropriate donors. and this all comesback because we didn't know all of the genes that cause dc, but we're working on that.this test, in addition to better classifying our patients and their potential bone marrowdonors, it has also led to our discovery of many new dyskeratosis congenita genes. soin 2006 this was what dc looked like from a genetic perspective. this is a telomere.we had germline mutations in the x-linked form caused by dyskerin mutations which ispart of the telomerase enzyme complex. we have dominant mutations in between five andten or 12 percent of our cases in telomerase
or terc, its rna component. and these areall key components of extending that nucleotide repeat. but about 70 percent of our patients,even those who met the classic clinical diagnosis, did not have a mutation in one of these genes.and i'm going to sort of walk you through how we got to this point and our figures keepgetting more complicated as we understand more and more of the genetic causes. so thefirst gene that we found was a gene called tin2 and that was because of this family thathad four affected individuals with classic dc and this was in the early days of the studyand blanche said, �well let's get telomere length on everyone in this family who we possiblycan.� and what we found was that the proband had two brothers who had really short telomeres.they're here in these open squares. so they
fit a molecular potential diagnosis, eventhough they didn't have the clinical phenotype. so i did a linkage scan and found that therewere germline mutations in exon six of the tinf2 gene which is a key component of theshelterin telomere protection complex. and that has since led to many other studies.and we now know that there's a hot spot in exon six that causes mutations in about 20percent, that causes about 20 percent of autosomal dominant dc. we have also done candidate genestudies. and this is a study that showed germline mutations compound heterozygous, so recessivemutations, in tcab1 which is encoded by wrap53, which notably is antisense to p53 in the genome,caused dc by causing aberrant telomerase localization in the nucleus. so telomerase is there butit can't get assembled appropriately and get
to the telomere to do its job. and then mostrecently, we've undertaken exome sequencing studies. and this was because we have developeda wonderful pipeline for exome sequencing at dceg's cancer genomics research lab, orcgr, and the team there has been instrumental in helping us understand the genetic causesof multiple cancer syndromes. so we started with these two families with individuals whohad classic dc or hh, the severe variant, and very, very short telomeres for their age.we have a specific variant filtering strategy and then we do technical validation. we foundin those two families germline mutations in rtel1. and as we kept looking and did moresequencing, we've actually got up to six families with germline mutations in rtel1. and thisis complicated because we have some families
where it looks like there's a dominant diseasewith genetic anticipation which is reported in dc, and we've seen it many times. and wealso have families with compound heterozygous autosomal recessive mutations who have moresevere disease. and we and others have found that there are, that rtel1 is a very, morecommon cause of hh than we maybe thought. rtel1 is an essential dna helicase that wasfirst studied in a mouse. it is essential for the metabolism of dna secondary structuresand for replication. and i just came back from a telomere meeting last week and rtel1was a big topic because we're really now that we understand we have mutations in differentdomains of the protein, we're better able to understand the function of this proteinand the dna replication and quadruplex formation
in telomere biology. so the other really notablething about rtel is that in addition to our dc-associated mutations that occur in thehelicase domains or in the pitbox or the pcna domains, there are also truncating mutationshere. rtel1 has snps in intron 12 and intron 17 that multiple different studies have foundassociated with increased risk of glioma. and i think that rtel is actually startingto look a little bit like tert with tert and the clptm1l locus, where that locus now hasat least ten different cancers associated with it and what we're wondering is rtel1also in this kind of type of gene where germline mutations can cause a severe disease and snpsmaybe cause increased risk of cancer because tert also causes dyskeratosis congenita. sothen this came also from a wonderful collaboration
with john petrini who is at sloan ketteringand ken offit. so ken had identified this family and john had done some sequencing andthey were trying to figure out the cause. and these two probands had hh, so the severevariants of dc. and we had this family. and i was talking to john at a meeting and, youknow, sure enough we had two families, both of ashkenazi jewish ancestry, who had theexact same homozygous mutation at p.r1264h. so we started working together on these twovery interesting families and found that our patients� cells really had a true severetelomere defect in that they didn't have, they really had loss of telomeres, which inthe controls you see these little green dots, those are the telomere staining. our patientshad very heterogeneous or telomeric loss.
and then you also have inability of the cellsto resolve and appropriately kind of protect that telomeric structure at the end. we alsodid genotyping across that locus and we found that both the affected individuals and theirparents were carriers of the exact same haplotype that, this didn't quite translate over here,but the mutation is down here and the pink haplotype is the exact same haplotype in bothfamilies. this allele frequency of r1264h was only about one in not quite 10,000 individualsfrom the publicly available databases. but this really showed us maybe there's somethingdifferent going on in the ashkenazi jewish population of which our families were bothmembers. so this was due to the persistence of the mother of our patient who said shewas participating with the dor yeshorim program
at the center for jewish genetics in new yorkand she said to them and to me, "you guys have got to work together, figure this out,what's going on in this population." and so the group genotyped about 1000 individualsof orthodox ashkenazi jewish ancestry that were all proven to be unrelated, we alwayslook for that, proven to be unrelated. and then with mount sinai genetic testing laboratoryalso genotyped the same variants in about 2200 individuals from the general ashkenazipopulation. and when they called me with the results we were all shocked in that we foundthat in the orthodox population about a one in 100 carrier frequency for heterozygotesof this r1264h mutation. and in the general ashkenazi jewish population it was about aone in 220 or so allele frequency. so when
you look back and look at what genetic testingand prenatal family planning recommends, a one in 100 frequency is actually pretty similarto what�s recommended for screening for other genetic disorders in this population,including things like bloom�s syndrome, fanconi anemia due to the founder mutation,and many others that are now screened. and so we're currently have this paper under reviewbecause we're recommending that r1264h be added to their prenatal genetic testing panel.and so with that, i'll end and say you know, we've learnt a lot about dc genetics by studyingthese special families with our outstanding clinical and genetics team in that we nowhave, others have found mutations in telomere capping proteins as a rare cause of dc. wefound tin2 which accounts for about 20 percent
of dc, telomerase trafficking due to tcab1mutations in about three percent of our patients, rtel1 mutations in about eight or so percentof our patients. and so now we're up to about 75 percent of our patients with dc who havea known mutation. and this has really helped our families understanding their disease,helping them, and helping us better manage them by selecting appropriate donors if theyneed a bone marrow transplant. but we still have a ways to go and we have several, manyother families in our exome sequencing pipeline network that we're actively working on. soi will end with thanking everyone. our patients are unbelievable. this was them at their secondfamily meeting. in 2008 we had a meeting for patients and doctors and scientists aboutdyskeratosis congenita and we helped them
form their first family support group. beforethat they had nothing. and now they've had two camp meetings. the third one is this falland we're working on their clinical guidelines book which they, again, don't have. and thenour study team, as i've mentioned throughout, especially blanche and neil, bari ballew isthe post doc who has done all of our exome sequencing analysis. and i didn't have timeto talk about our multitude of functional studies that have only been made possiblebecause of our biospecimen collections that we can do within the family studies program.so with that i'd like to thank you very much. [ applause ]
Tidak ada komentar
Posting Komentar