Peripheral Neuropathy Rare Causes

Identifying the Genomic Basis of Rare Diseases David Valle 2016

Applause david valle thank you tyra and good morning everyone.It’s a pleasure to see you all here and it’s fun to be here to talk about a topic that’s near and dear to my heart.So i’m going to cover a fair amount of ground.I’m glad to answer questions, but i guess most of them are usually at the end.But if i’m not making myself clear and you just can’t wait, go ahead.So first of all my disclosures.I have no relevant financial relationships.I do i disclose that i’m a real enthusiast.

About genetics so i hope that that comes through to you.It’s a wonderful field if you’re thinking about going into it.It underlies all of biology.It is basically a hunting license to do whatever you want in biomedicine so i urge you to think about that career if you’re at that stage of your career.So i’m going to talk about some features of mendelian disease and then review the rapidly evolving field of al dna sequencing.And then i’m going to talk about disease gene discovery results and tools, and i’ll.

Focus particularly on the baylor hopkins center for mendelian genomics which is one of now four centers around the country that are charged with finding as the genes responsible for as many mendelian phenotypes as possible.So we customarily think of mendelian disease as being quite rare and yet it is becoming increasingly prominent.I see that this slide says this month.This is actually i think from january 2015 so i apologize for that error.But the point is in any month, if you look at all four issues of the new england journal, you will see a lot about mendelian.

Disease.In this particular month, there were let me see here.You can see that there were can you hear me okay you can see that, you know, there were typical mendelian disorders with onset in childhood, but look at here.Here’s one that’s adult coronary artery disease and if you looked in the editorial section, there was even an article about ethical issues about screening for genetic monogenic genetic disease.So there’s a lot of interest about mendelian disease throughout the biomedical community right now and we have i’ll talk in a minute about why that might be so.I’m going.

To talk right now about why that might be so.So first of all the genome project obviously provided a reference sequence so that made finding the relevant disease genes much easier.Obviously the availability of new sequencing technology, that dramatically decreases costs and increases throughput, also gave us many new avenues for finding genes responsible for mendelian disease.And things like the hapmap project and the 1000 genome project gave us an appreciation of the extent of normal human genetic variation, not only in north america and northern europe, but from populations around the world.So that turns out to be.

A tremendous resource.And lastly, there has been the development of genomic and genetic strategies to identify responsible variants in genes.So the first thing you might say is well when should i think of a mendelian disorder if i’m a physician seeing a patient, or i’m somewhere in the healthcare profession for many mendelian disorders, not all but for many, the phenotype includes multiple systems that are not easily related one to another.Many mendelian disorders, but not all, have relatively early age at onset, often in the first decade of life.There are of course.

the recessive ones are of course increased with consanguineous unions, and if you find multiple affected sibs andor generations then obviously that’s a pretty key clue.And if you think about it, there’s sort of an inescapable rules of biology about how genes are transmitted from one generation to the next, and we use those so called mendelian rules to really help us evaluate candidates for mendelian disorders.And it’s one sort of fundamental bedrock of genetics that whatever you find pretty much has to be put in this context.So although we do think about mendelian disorders as having their onset in childhood,.

I would submit that there are many mendelian disorders that present in adult age and that our colleagues in internal medicine, and i confess i’m a pediatrician, our colleagues in internal medicine have to be more alert to the possibility of mendelian disorders.So i just want to make that point by presenting two families to you that we’ve seen in the last few years.So the first is a man who was 34 years old and he presented to johns hopkins actually about two and a half years ago.And he had a fever, 10 day.

History of pretty high fever, really bad pharyngitis, and he’d been treated by his personal physician with antibiotics actually two different antibiotics, still febrile.And so the physician, for reasons not known to me, treated him with a large dose of steroids as well.Following that intervention, now 10 days into his illness, the man began or now eight days into his illness, the man began to develop confusion and that led to him being taken to a local emergency room where the s were smart enough to think about hyperammonemia.And.

They measured his ammonia and it was 10 times normal, 280 micromolar, and he had a mild respiratory alkalosis which in the presence of hyperammonemia suggests a urea cycle disorder because there’s no accumulation of organic acids and ammonia’s a stimulant for the central respiratory centers.So he was rapidly transferred to the johns hopkins medical intensive care unit.By the time he arrived two hours later, he was in the early stages of coma and ct scan showed mild cerebral edema, and his ammonia had already risen to 420 micromolar.For those.

Of you that are not physicians, he had about one foot and maybe three toes in the grave at this point.So he the emergency room docs or the micu docs did their thing.One of the things they did is they called genetics and so i happened to be the attending and i went with one of our residents, hans bjornsson, to see this man.So we saw him about 20 minutes after he hit the micu, and so like any good geneticist, one of the first questions we.

Asked well what is the family history we asked this of a fourth year medical student who was involved in the case, and he said what is i think the most common response to that question which is negative.So i would submit one important take home lesson from this lecture is unless the person is adopted and knows nothing about their family, the family history is never negative.You may have some pertinent negative results that help you eliminate certain things, but the family history always tells you information.But when you get the family history, you have.

To get it and think at the same time, and sometimes as you think, you’ll come up with new questions.So you have to be willing to go back and forth with the family as new ideas, new hypotheses for the diagnosis enter your mind.So this is the information we got from the medical student.So i said, what do you mean negative go out and ask the family for more detail.The family was assembling in the micu waiting room.So he went out and he came back and it turned out the family was a little bit more extensive and so there.

here’s the second version of the family history.So what you can see is that the proband indicated by the red arrow had a brother who died and he had two male twins identical twins or no, fraternal twins who also died.Now it turns out the twins died in childbirth and almost certainly had something else.The brother that the medical student i said, what do you mean negative and the brother the medical student said, well not to worry, the brother died of drowning when he was 14 years old.So what did i say so.

I said, well why did a 14 year old boy drown go back out there and find out.So he went back out there and the story was that the 14 year old brother was on an outward bound like experience and he developed a upper respiratory illness and was sick.And then his campmates reported that he began to be confused, and by confused they noted a time when he couldn’t find his hiking boots and they were right in front of him.And that night, the night before he died, they all went into their tents to go to bed.They were.

Camped by a lakeside and in the morning when they woke up, they found him floating off the end of the dock drowned.So they theorized that he got up in the middle of the night in his confused state and walked out on the end of the deck and fell off and drowned.And we actually got the autopsy because he because it was an acute death, he had to be autopsied out in west virginia someplace.And the local coroner said, you know, the strange thing about this drowning is i mean the boy clearly drowned, but he had cerebral.

Edema.And that’s a phenotype that you never see with drowning because drowning takes place very quickly.I to cut to the chase, we got a baby tooth of this boy and he also had the same urea cycle disorder that his brother presented with.So it turns out that this is late onset ornithine transcarbamylase deficiency and the we’ve studied this molecularly.One of our graduate students and ted hahn, and ted found a promoter variant never before seen in a four base highly conserved element that’s important for binding of a particular.

Hepatic a liver specific transcription factor.So we theorized, and he ted actually showed that it reduces the activity of otc and reporter assays and so forth.So we theorized that this is a promoter mutation, regulatory mutation that reduced the function of otc that the both of these boys had enough otc activity to get through early years of their life, but under conditions of severe stress this genetic vulnerability was brought out and in both cases led to their death.Now of course the geneticists in the room.

Will say well it looks like the mother must be a carrier.She’s had two affected sons, and we tested her and she was a carrier.And then we tested her sister and she was also a carrier.And we wanted to test these two boys, each of whom is at a one in two risk of having this phenotype.Both of them are young adults.Both of them are underachieving in comparison to their family.This is a quite sophisticated family and both of them refused.They live out in the midwest and they both refused to be tested.So i don’t know what.

They have, but i’m suspicious that they might have the same thing just based on their sort of performance.So here’s a mendelian disease lurking in an adult patient.The patient is just more vulnerable to particularly severe environmental stress, namely this bad infection and a dose of steroids perhaps contributing to it.Now in case you think that that’s just a one off example, a few months later hilary vernon, one of my colleagues, was asked to see this man who is a 54 year old man who presented to the cardiology with severe dilated cardiomyopathy.And they noticed that.

He seemed to have a some features of early onset dementia and the theory going theory was that perhaps because his congestive heart failure was so bad that this may just be some low level chronic cns insult, but they were worried about his b12 status and they sent homocysteine level and the methylmalonic acid level, and both of them were elevated.And it turns out that this man has a cobalamin c form of combined methylmalonic acidemia and homocystinuria.He died of his shortly thereafter he died of his cardiac his congestive.

Heart failure, but it turns out his sister also has this phenotype.She’s also middle aged.She’s also intellectually not doing as well as you might expect for the family.So here’s another late onset mendelian disorder.They’re out there, just have to look for them, think about them.So that’s all i’m going to say about the prominence of mendelian disorders.Now i want to talk briefly about finding the responsible variants in genes.So i think probably everybody in the audience knows that geneticists human geneticists since there have been human geneticists.

Have been interested in finding the genes and variants responsible for mendelian phenotypes.So archibald garrod in 1902 reported patients with alkaptonuria and noticed that the distribution of affected individuals within families was entirely consistent with what gregor mendel had described in 1865, and that he hypothesized at that time that maybe alkaptonuria disorder in tyrosine degradation was in fact one of these mendelian disorders that this monk described 30 years earlier in pea plants.And then the number of recognized human disorders began to grow and the geneticists, once we understood that the factors that were responsible were.

Actually encoded in the dna, we began to look for the genes and variants responsible.And typically we used really tedious strategies, linkage with collecting large families and doing linkage analysis or searching for a chromosomal aberration that pointed to a particular region in the genome where we might find that gene.But things changed with the genome project as i said and with the development of next generation sequencing.And i refer you if you’re interested in this to two papers.The one on top in particular is really a seminal paper in this field.So this was a paper from our colleagues at the.

University of washington, most notably mike bamshad, debbie nickerson, and jay shendure.And they were working on the development of so called next generation sequencing and genomic studying the human genome and they did a simple experiment really, but it’s very elegant.They said, you know, we’re able now to sequence the genome and particularly the exome which was about 1.5 percent of the total genome, the exome being the coding sequences the protein coding sequences, were able to sequence that pretty well and we have this reference.So what would be the chance that we if we get a patient with a particular.

Mendelian disorder, we could simply do a whole exome sequence and recognize the variant or variants that were responsible for the for the phenotype so that sounds like a straightforward hypothesis, but the problem is when you do a whole exome sequence, the problem is fundamentally that each of us differ by about 3 million single nucleotide variants from the reference genome.So you have to find which of those three million variants which single usually of the three million variants is really responsible for the phenotype.Now if you focus on the.

Exome, you cut that number way down, maybe 25,000 variants from the reference sequence in someone’s exome, but you’re still a long ways from figuring out what the responsibility gene is.So what they did and i’m not going to dwell on it, but they took a set of patients with a very well characterized mendelian phenotype, namely freemansheldon syndrome.The disease gene was already known, myh3, and they said let’s sequence one patient with freemansheldon syndrome and see if we can find the variants, and they found actually they looked only.

For severe loss of function variants, indels, splice site changes, and nonsense mutations.And sequencing that one patient, they had several hundred variants that might be candidates for this particular disease.So then they said well okay, let’s get another unrelated patient and we’ll do the same thing and we’ll look for genes that are affected in both of these two unrelated individuals.The hypothesis being since we see that they both have freemansheldon syndrome, they should have a variant in the same mutation.Notice they left out the problem of locus heterogeneity which would have killed this experiment, but.

They did very careful al phenotyping.So they sequenced the second one and they looked only for genes that had a loss of function variant in both individuals.And i forget the exact numbers, but they were down to about 100 genes at that point.Then they did.Well that looks good.Let’s do another one.They did another one and they were down to i think something like seven or eight genes, and they did a fourth and they only there was only one gene that had loss of function variants.

In all four individuals, and that was myh3, the gene that they already knew that was responsible for this phenotype.So that said unambiguously that you could use genomic technology based on next generation sequencing and what we know about the reference human genome to find the variants responsible for human disease, and you don’t have to do big timely linkage studies or anything like that.You just have to find some well characterized patients and sequence those patients either as singletons in their family or depending on the inheritance modes, you might want to take a few other people from the family and use those mendelian.

Segregation rules to help you sort through the variants as well as comparing patient’s one family to the next.So that said okay guys, this is a new age.Let’s go get them.We did a paper shortly thereafter which i like to think contributed a little bit to this effort, and that’s the reference below.And for those of you that are students in the room, i think this is a very illustrative example.We were had a speaker at hopkins, david goldstein, a great human geneticist and he was having lunch with students as we often as often happens.

And he said he was working on whole genome sequencing in this case and he was looking to see if he could solve an unsolved mendelian disorder using whole genome sequencing.Now, you know, many of us, myself included, if we were sitting around the lunch table and we heard that, we would say great and then we would forget it a couple hours later and that would be the end of it.Fortunately nara sobreira, the lead author on this paper who was at that time a human genetics graduate student, is quite persistent.And two days.

Later, she called up david goldstein and she said she had a family and she’d send him the dna, so she did.The family was provided by julie hooverfong, one of my al colleagues, and the family had something called metachondromatosis and david did the whole genome sequence in about two and a half weeks actually.Now if you do whole genome sequence as i said, you’re going to find three million single nucleotide variants compared to the reference sequence, and you’ll find some structural variants as well.So he said wow, this is really a difficult problem.What can we do.

To help us and so the only reason i mentioned this paper is because we then went back to genetics.So this first paper is all genomics.We used genetics.We said okay, we actually this family was not big enough to do convincing linkage analysis, that is to find a region of the genome that unambiguously harbored the responsible variant.But recall that linkage is actually very powerful at eliminating regions of the genome that can’t possibly have the thing have the causative gene.So we did some quick snip nucleotide linkage panels on a few other family members, very.

Cheap compared to whole genome sequencing, and we looked we quickly found six regions of the genome that could potentially harbor the responsible gene.So certainly we hadn’t narrowed it down dramatically, but actually those six regions only comprised two percent of the whole genome.So we were eliminated 98 percent of the genome using that simple genetic trick so i think of this as combining genomics with classical genetics.And sure enough, under the second linkage peak that we looked at, there was the responsible gene within an unambiguous loss of function mutation and we were able to find another family with.

The same phenotype that had a nonsense mutation in the same gene, ptpn11 so qed.And that whole exercise took about six weeks so at that time that was going pretty fast.So genomics and particularly genomics combined with genetics offers powerful reagents or tools to get at these disorders these genes.Okay, so that was a few years ago and with that sort of stimulus and because of all the other reasons that i’ve already enumerated, one of the things that’s going on in the last few years is what i call the rise of al dna sequencing.So those of you that.

See patients know that increasingly it’s possible to use molecular diagnostic tools to make to search for a precise molecular diagnosis in your patient.So i just want to review that because i find that people don’t really have not really thought through all of the approaches and what they mean.So i organized sequencing al sequencing by target.So the first is a very focused search and that’s a single disease gene, think brca1.And so you have a patient who let’s say has breast cancer, maybe a.

Positive family history, and you want to find out if that patient has breast cancer because they have a pathological variant in brca1.So you look at that single gene, one of 20,000 genes.Now a second strategy is what’s come to be called the disease gene panel.I mentioned the cardiomyopathy patient so we know of on the order of 25 to 30 genes that when that when certain variants occur in those genes, the patient will present at different age ranges with dilated cardiomyopathy.So there’s a several panels that one can send such.

Patient’s dna samples and get tested for all of those 25 or 30 genes.So it’s a collection of genes, each known to be responsible for particular disease, and you’re asking which of these genes if any is responsible for my patient’s problem.Then, whole exome sequencing, i’ve already referred to this.It’s sequencing the entire exome together with a splice sites flanking each exome.And we, by back of the envelope calculations which i believe have withstood the test of time, estimated early on that whole exome sequencing that about 85 percent of mendelian variants would be found in the.

Exome and in the flanking intronic splice sites.And i won’t go into how that comes, but there’s actually fairly good evidence for that.So this is a pretty you essentially only have to sequence one and a half percent of the genome, but you have a very high chance of finding the genes that are responsible for your patient’s problem.And then there’s whole genome sequencing that i also mentioned, the sequencing the entire genome, exomes, entrons, regulatory sequences, you know, 1.5 percent of the genome is exome.If you look at what fraction of the genome is highly conserved evolutionarily,.

It’s about five to 10 percent, maybe seven percent.That means that evolution seems to really care about seven percent of the genome so you’re still sequencing a lot of the genome that perhaps is not really very important when you do a whole genome sequence.And obviously we’re much, much, much less sophisticated in interpreting the results of variants that we discover in the noncoding part of the genome as compared to the coding part of the genome.So let me diverge briefly to just make sure everyone’s clear on the difference between.

al and research whole exome sequencing.So research whole exome sequencing typically you have a al diagnosis, but you don’t know what gene is responsible and you want to find that gene for this particular phenotype, the gene that’s responsible for this particular phenotype.So you typically sequence multiple members of a family, maybe two affected and one unaffected or maybe the proband and the two parents, depending on the inheritance model, and then what samples are available.Speed is typically not that critical so it may be months going on here.Surveys all 20 or 21,000 protein coding genes.It requires.

Validation once you find some candidate genes and variants, and we do that validation by segregation within the family to the extent that we have family members and depending on what we think the inheritance pattern is, and functional studies of the candidate genes to make sure that the variants do what we think they do.Then there’s al whole exome sequencing and there are a number now of commercial companies that provide this service.So typically a physician sees a patient in a and doesn’t know the diagnosis and says i’m rather than sort of spend a lot of time working up.

This doing various sort of classical workups, i’m just going to send the al whole exome test and see what this tells me.And you typically send the patient, and for some of the commercial operations you also send the parents or some other family member, and they may do the probands al whole exome.And then if they find something that looks interesting, they may look at that particular variant in other members of the family or they may not.And this is the key point for most al whole exome services, the genes.

They look at are the known disease genes.So in other words, right now, i’ll show you later, there are about 3,500 known disease genes out of the 20,000 in your genome.So those, although they sequenced the whole exome, they focus on those known disease genes.So in that sense the efforts for the mendelian centers and other investigators doing research, and finding and validating disease genes through research whole exomes then provide the knowledge for the commercial al services to offer their services.So i didn’t make a slide.

Of it, but i was looking at a company in europe last night on their website and it says on the website, you open it up and it says al whole exome sequencing.So i read that and i think okay, they’re surveying 20,000 genes, but then they say we give you results on 2,800 genes.So that’s they’re really only giving you results on maybe 10 or 15 percent of the genes in the genome.Now eventually as the research progresses, they’ll give a higher and higher fraction, but that’s the relationship between research whole exome.

Sequencing and al whole exome sequencing.Now one two other things that you have to be clear on when you order these kinds of tests.There are some unanticipated or if you think about it, they’re actually anticipated, consequences of large scale dna sequencing.The first is our state of knowledge right now, it’s imperfect so you will find you absolutely if you cast a broad enough net, you will absolutely find variants that you’re not sure how to interpret, and they’ve come to be called variants of unknown significance or vuss.And i’ll tell you how many you.

Find in a minute, and then you also may find incidental findings of great medical consequence.So you may, you know, be let’s say doing a al whole exome on a child who has a developmental defect or something like that and so you want to find the gene that is responsible for the developmental defect.And you do that whole exome sequencing and you find a wellknown pathological variant in brca1.Now when the family gave permission to do that test, they were not thinking about brca1 and that variant almost certainly had nothing to do with why.

The exome was ordered, but now you have a piece of information that may be very relevant to that individual’s longterm medical care.And that variant may also be in other members of the family.So you found out some information, not only about your patient, but also about family members.So the best way to deal with this possibility is to discuss it with the family before you send the test so that everybody is got their eyes wide open about what you’re doing.Medicine has always picked up incidental findings.You know, you send you think the patient.

Is anemic, you send a cbc and you discover that they have leukemia or something like that.But what’s different about these findings is that they may predict illness way down the road that have absolutely nothing to do with why you ordered the test.And they also may provide information that’s relevant to other family members who are not even your patients.Okay, so let’s look back at those four classes of sequencing approaches.So starting on the first row up here, single gene testing brca1 already mentioned costs several hundred.

Dollars to a few thousand actually a few thousand.It’s less expensive, or it’s relatively inexpensive if you’re correct.That is maybe you spend $2,500, but you get the answer.You have fewer variants of unknown significance because you’re only looking at the variants in a particular gene.And very often those genes have been pretty well studied, so you find relatively small numbers.You’ll find occasional, but relatively small numbers of variants of unknown significance, and no incidental findings because you’re only thinking about this particular gene.The second category is some sort of disease gene panel.I mentioned cardiomyopathy maybe.

25 depending on when you did the test, the numbers going up.Cost is quite similar, actually, several hundred to a few thousand dollars.It’s a broader net.It’s less expensive on a pergene basis.And, but you will find more variants of unknown significance.You won’t find incidental findings because you’re really just looking at the cardiomyopathy genes you’re not looking beyond that.Now what about a whole exome sequence a socalled al whole exome sequence so currently you can get them for around $5,000.It’s a much broader net, a bargain on the pergene basis, right it’s great.But you will find,.

Absolutely, many variants of unknown significance.So you’ll need to council the family about those variants of unknown significance or you will have to build in some approach that you’ve agreed beforehand to set those aside.And you’ll find incidental findings.I think most groups now are reporting, if you just consider these socalled 56 american college of medical genetics genes where a panel of experts decided that we that there were reportable and actionable incidental findings.And you say, how often do you find variants in those 56 genes which seem to be significant most people who are doing a lot of whole exome.

Sequencing are finding on the order of one to three percent of the people they do whole exome sequencing on will have incidental findings and that small number, 56, very solidly known disease genes.And then a whole genome sequence.Largely a research tool at this time, but several companies are beginning to suggest it.More expensive.It’s a broader net, still.It’s the broadest net we can currently cast although rnaseq will be coming down the pike.And it’s much, much, much harder to interpret.You will find variants of unknown significance.

And incidental findings galore.So one take home message is that if you’re going to use this outside of the research setting, you should we think build in a good bit of genetic counseling time for those subjects that have this to explain all this stuff.Now what is as i’ve indicated al particularly al whole exome panels, genes, and al whole exome is a growing field.So what have been the outcomes so we’re beginning to see publications now that are looking to see what has been the.

Consequence of this.So the first publication, i think, of any size was from baylor college of medicine that very quickly opened a commercial lab associated with their genetics group to provide al whole exome sequencing.So they reported in this reference on the first 2,000 samples they did 88 percent were in the pediatric age range.They made a molecular diagnosis in 25 percent of these patients.So that’s a pretty good return on a diagnostic test rate, right twentyfive percent.And, interestingly, 58 percent of a diagnostic mutations had not previously been reported.That is to say, they found a loss of function.

Allele in a gene that was known to cause a phenotype when it had loss of function.And so this is just a new loss of function variant in this known disease gene.The frequency of the various inheritance patterns are shown there for the solved cases.A key thing is that 30 percent of the diagnoses involved a disease gene that was identified in the last three years.So this gets back to this the research community, particularly, research whole exomes pumping in new disease genes and those new disease genes then can.

Add to the list of genes that the al west can interpret accurately.So it’s really going up like a rocket right now.And one interesting feature, which has been found over and over again now, it that 23 of the patients where which they got an answer or 4.6 percent actually had what they call the blended phenotype from two different mendelian disorders.So in medicine you know, it’s sort of an occam’s razor approach and you’re trying always to find a diagnosis that will explain everything about your patient.

So one of the reasons that these patients were difficult to diagnose is because they actually had two diseases two rare diseases in one.And the phenotype had features of both of these disorders.And so al geneticists were not able to recognize what it was.So very interesting.Now genedx, another private laboratory service here in rockville, maryland, does excellent work, very shortly thereafter reported 3,040 consecutive probands nearly all in the pediatric age range.They made a molecular diagnosis in 851 or 28.8 percent, roughly the same as.

The baylor lab had found.And, again, 28 of the patients or 3.3 percent had two or three mendelian disorders.And this graph, which i won’t say much about, but shows the test yield in terms of percentage of positive results by the particular systems that were involved.So actually the highest system is hearing loss, which is already known to have a huge contribution of genetic causation to isolated hearing loss.So those two studies were largely pediatric.Baylor recently reported 486 consecutive adult patients 18 or older, and they made a molecular diagnosis a little bit younger a little bit.

Less in this older group, 17.5 percent.And they found six or seven percent with two disorders.And this graph shows the diagnostic rate with the age of the patient in years.So the older the patient got, the less chance they had of finding a straightforward mendelian disorder.And this is a plot much like the genedx plot and it shows the success rate by indication and the overall diagnostic rate of 17.5 percent.So even in adult population at least young and middle aged adults suspected of having a mendelian disease, this turns out to be.

A very high yield diagnostic service.Now, for those of you that are not physicians in the room, let me just emphasize some values for having a precise diagnosis.So physicians are trying to explain the phenotype of the al problem of their patients so they can have a continuous diagnostic work up until they get the answer.So this stops that diagnostic work up it shortcuts it.It ends the uncertainty of the diagnostic odyssey.This is the term that’s been given to families or patients that keep coming back to medical attention and trying over and over again to find out.

What in the world is their problem.It turns out that if you have a child with a problem, or you, yourself, have a problem, there’s a for most affected individuals there’s a strong urge to find out exactly what you have.And that you’re not when you go to your and say, i’ve got this problem and that problem, you’re not crazy, you actually have some problem.And it provides a biological explanation for the problem.So over and over again, those of you that have been to a genetics , if.

You talk to parents who have a child with some genetic disorder, the parents will say things like, well, you know, i thought, actually, you know, three months into this pregnancy i fell on the ice.I took a bad fall.And i always thought that the reason that my baby had this problem was because i fell down.And you say, no, actually, this is a straightforward genetic disease.The fact that you fell down, or that you had a glass of wine, or you had a cold or something like that, is irrelevant to this problem.

It puts the focus on patient management.And it focuses the patient management now you know what you’re dealing with.And so you can draw from experience with other people with that problem.And it informs the family of the recurrence risk.In other words, you know, if it’s a recessive disorder, they have a one in four or 25 percent chance of having another.And i certainly have been in the i’ve had the unfortunate experience i remember a case of hurler’s syndrome which is a very high burden lysosomal storage.

Disease.Patient was referred relatively late so the patient was about 18monthsold and the family came in with this 18monthold boy that from down the hall you could tell had hurler’s syndrome.But they had a threemonthold child sitting on the mother’s knee.And i could tell that that threemonthold child also probably had that disorder.And they’ve now both of those kids have now died.But if the diagnosis had been made quickly, and the family informed, then they would not have had to go through six or eight years of very.

High burden chronic illness with those two kids.So that’s a big benefit.So i’ll give you this one example.This is a patient that i’ve been following for 36 years.He’s ‘ right now.In fact, i’m scheduled to see him two weeks from now.He had recurrent episodes of lactic acidosis from early childhood.He had diminished intellectual function for his family with an i.Q.Of 65, and cortical atrophy on his cns imaging studies.He had mild to moderate cardiomyopathy, and he had prominent dysfunction of his autonomic.

Nervous system, constipation, postural hypotension, other such things as that.And he would come in with these episodes of recurrent lactic acidosis.We would say over and over again, this is something is wrong with the function of your mitochondria.This is some sort of mitochondrial misfunction.But we’re not sure what it is.Several years ago, we finally were able to get money together to do to sequence his mitochondrial genome.And i told the mother that, you know, if his problem was as i suspected, mitochondrial, it could either be in the mitochondrial genome or the nuclear genome.At least we could check.

Out the mitochondrial genome.And it turned out to be normal.So i had to go back to her and say, well, the mitochondrial genome is normal, so i’m thinking it’s probably a mitochondrial a gene that encodes the mitochondrial protein in the nuclear genome.At that point it was out of the question, not only for that family, but just in general, to sequence, let’s say, a whole exome.But, eventually, about two years ago, we she got financial resources and insurance to actually pay for a al whole exome sequence, and.

He has a homozygous nonsense mutation to a gene called fbxl4 never heard of it before until the test was done.But it is a previously described three or four other patients mitochondrial dna depletion syndrome.The encoded protein is necessary for proper replication of mitochondrial dna.And so if you lack that protein, your mitochondria don’t have as many mitochondrial genomes as they should.The end result is your mitochondria don’t work well.So i had the pleasure, actually, of telling the mother that after 36 years, i finally had.

A diagnosis.The mother was incredibly relieved, actually, to know exactly what this is.I couldn’t i said, you know, i don’t i really don’t there’s nothing i can do about this.So it’s not that it’s going to lead to a better treatment.Maybe down the road it will, but not right now.But at least we know.And the relief of just having the knowledge of exactly what was the etiology of this boy’s problem was palpable for this woman.Really amazing.Okay, so then i want to turn to one other publication about al whole exome sequencing.

Which just came out.It’s a perspective evaluation of whole exome sequencing as a firsttier molecular test in infants with suspected monogenic disorders.It’s from the murdoch institute in australia.That’s the first author in the reference.And they did some sort of thoughtful modifications of the sort of rather than the sort of shotgun exome al whole exome diagnostic testing.So they considered using this test in 119 infants unrelated infants that met a set of criteria.They had a welldefined phenotype.Some of them had a positive family history and so forth.Of those 119 families,.

80 agreed to participate.They did a single al whole exome sequence.That is, they didn’t do any other family members and they examined in that al exome 2,830 of the 20,000 genes.And they excluded to get rid of the problem with lateonset incidental findings.They said, we’re not going to look at those.We’re not going to look at certain genes that have those incidental findings.So they excluded 122 genes.They didn’t analyze those genes.Of the 80 infants that were sequenced, 46 or 57 percent yielded a molecular a precise molecular diagnosis.And of these of the 46 32 percent had.

A significant management change based on this new diagnostic information.So it turned out to be of quite important medical significance to about a third of the patients at this point followed for a few months or a year.And, additionally, 28 couples 28 of the 80 couples that participated received high either 25 percent or 50 percent recurrence rates so they could use that information to avoid the scenario that i discussed earlier.So it will be interesting to follow these studies now and to ask, what does this.

Mean for the sort of medical economic issues was this initial investment at a rather expensive test does it not only improve the medical care, but does it reduce medical cost as the families go forward i suspect strongly that it will.But some medical economic experts need to look at this in detail so that we can get these data we desperately need those data.Okay, so that’s all i’m going to say about al sequencing.And its value and its aspects that need to be managed carefully if you’re going to use it in your .

Or with your patients.But the growth of the ability to detect mendelian disease genes and the value of detecting them led the genome institute to issue an rfa to develop centers for mendelian genomics that would use the technologies that i talked to you genomics and genetics to try to find as many genes responsible for mendelian disorders as possible.And in the initial fouryear funding period of three centers were funded udub at university of washington at seattle, debbie nickerson and mike bamshad, p.I.S yale, with the p.I.

Rick lifton.And we partnered with baylor college of medicine to form what we call the baylorhopkins center for mendelian genomics.And i’m a p.I.Along with jim lupski down at baylor.So it’s a real team effort.And there’s our website mendeliangenomics.And you’ll see me refer to it as bhcmg, baylorhopkins center for mendelian genomics.We just started our second fouryear funding period.And i just was at a meeting monday and tuesday of this week.We were sort of tooling up again for the next four year run.

At this.So it’s interesting to say, well, what is the current state of the art so we keep track of how many mendelian disease genes have been identified by using the data in online mendelian inheritance in man or omim which was started by my colleague, now deceased, victor mckusick and currently managed by my colleague ada hamosh at hopkins.And currently omim as of late last night, lists about 7,500 mendelian phenotypes.It lists 3,543 disease genes that’s about 18 percent of the total.You’ll notice that the number of phenotypes.

Is greater than the number of disease genes, that’s because, in part, that well, the next column is explained phenotypes 5,722.That number is bigger than 3,543.And that’s because some disease genes cause two different or sometimes more discreet phenotypes that ally we would have never imagined were caused by mutations in the same gene.There are some genes, lmna for example, that account for 13 or 14 discreet al phenotypes.So the average is about 1.8 phenotypes per disease gene, right now.And there’s still 1,800 unexplained phenotypes in omim and you have to realize that there are new phenotypes.

Coming into to omim all the time.They come in at a rate of about 300 new phenotypes per year.So there’s lots of mendelian disease out there that we have not yet recognized as being mendelian disease or we have not given a name to or an omim number yet.So 18 percent of the total genome number of genes in the genome, i’ve been tagged as mendelian disease genes so we have a long way to go.If depending on your view of how many genes in the genome can cause a mendelian phenotype.So let me talk about that for a.

Minute.How many mendelian disease genes are there in our genome and how close is that 3,500 to saturation so, first of all, how would i define a mendelian disease gene and i would define it as, those genes in which some fraction of variance in that gene, produce highly penetrant phenotypes.That’s sort of geneticsspeak.Penetrance means that you manifest a phenotype when you have the genetic variant.And if i ask my colleagues, how high does the penetrance have to be to call something a mendelian disease there’s no unanimity.So i arbitrarily take the set the penetrance level at.7.So.

That means that if you have the variant the genetic variant you’re chance of getting the phenotype is 70 percent or better.For example, the standard disease variants in brca1, many of them have penetrances in the range of 70 percent.So that means you’re highly likely to get the phenotype, but it also means that some people won’t.And the ones that don’t get the phenotype, geneticists refer to as, nonpenetrant.We’ll talk more about that in a minute.So with that sort of background, then you say, well, one way i might be able to get.

At how many mendelian disease genes there are, is to count the phenotypes.Well, it turns out it’s a lot harder to count phenotypes that it is to count genes.So, as i said, omim currently lists 7,500 with about 1.8 phenotypes per disease gene, and 1,800 unexplained phenotypes.So that predicts maybe 900 more disease genes a pretty small number, actually.But we know that many phenotypes are conditional and dependent on environmental variables.So think of g6pd deficiency, people with g6pd deficiency are typically entirely asymptomatic unless they happen to chow down on a plate of fava beans, in which case they.

Will have massive hemolysis and become jaundiced and perhaps severely anemic.So that’s we all think that g6pd is a mendelian disease, but if you avoid all of the environmental triggers that cause the hemolysis, you’ll never know that you have that mendelian phenotype.There are many other phenotypes of this nature.So the point is that to define all mendelian disease genes, and all variants that cause in those genes that cause mendelian disease, you have to sort of challenge the population with a variety of environmental triggers to.

See what brings out the al phenotype.Easy to do in a mouse, a little bit harder to do in a person.The other thing is that there are a vast number of unrecognized phenotypes.Remember, i said that 300 come in new phenotypes come in to omim each year.Obviously, they’re not new phenotypes they’ve been there all along.We’re just recognizing them and getting them into medical attention.And they’re vast swaths of the populations of homo sapiens around the world that don’t even sort of get access to this kind of service.So i’ve.

Recently visited the middle east and i saw my host showed me just one family after another that had genetic things that i had never seen before, but clearly based on the mendelian segregation in the family, were clearly mendelian.So they’re just waiting to be explained.So there’s sort of two schools of thought about how many mendelian disease genes are in the genome.Here’s one that says that the number of genes in the genome that when they have a certain variant could cause a highlypenetrant phenotype is substantial,.

But limited.So let’s say, arbitrarily here, i put at 30 percent.Now there’s another school of thought that says, actually if you look carefully enough across the entire population of homo sapiens, you’ll find that a large fraction 90 percent or more of genes can produce a mendelian phenotype when they have a particular class of variants in that gene.And the answer to this question is not known, okay i’m obviously i guess you would predict this.I’m greatly in favor of the red curve.I think if we look carefully.

Enough and long enough, we’ll find mendelian phenotypes for almost every gene in the genome.Now let me just give you a couple reasons why i think that’s true.So the biggest one is this, it’s evolutionary thinking.If those genes are not important for something, evolution would get rid of them, right there’s a constant mutation rate.All dna segments in the genome accumulate mutation, and if that mutation occurs in genes with important function then selection eliminates them.So the set of genes that we have right now, have.

Stood the test of time by evolutionary guidelines.And so it’s true that some of them may have been more valuable in earlier socioeconomic cultural conditions of our species.But, nevertheless, the vast majority of them are there because evolution cares about them.So, well, then you could ask, well, okay, valle, if you think the fraction of mendelian genes is so large, why are they so difficult to identify so the first answer that most people give is, well, maybe a substantial fraction of the genes in our genome are so.

Important that when there is a significant variation in those genes, it leads to earlyonset developmental lethals.And so those fetuses are only known in terms of spontaneous abortions.And it is true, that there are a fraction of our genes in the genome that are very, very, highly conserved.And by very, very, highly conserved i mean the nucleotide sequence and the amino acid sequence of the amgoda spelled phonetically protein are highly, highly conserved and that suggests that they are intolerant to variation.And we know that our species has a highfrequency of spontaneous first trimester spontaneous abortions.A large.

Fraction of those are chromosomal abnormalities, but there are other spontaneous first trimester abortions in which the karyotype appears normal.So why what’s going on there so how many of those might be mendelian disorders that affect some gene that is absolutely important for early embryonic development.And that remains to be seen.We also know that 30 percent and this statistic is often used 30 percent of the genes in the mouse genome, when made homozygous for a null allele a true knockout, lead to spontaneous fetal losses,.

Okay either perinatal or earlier in embryo.So that says, indeed a fraction of the mouse genes 30 percent of the mouse genes are absolutely necessary for normal development.So you’re not going to see so then the logic is, well, you won’t see those genes causing medical problems in later life.So i would argue that’s not the case because we know that every gene we’ve looked at there’s a spectrum of mutational events from those mutations that cause a complete loss of function to those mutations that moderately.

Decrease function, to those mutations that only mildly decrease function.So somewhere in that spectrum of functional consequence there will be some alleles for these very genes that only reduce the function of the protein product by some fraction and that allows for successful leads to viable in utero development.And then will make itself known either in infancy or later in life, depending on how the biology of that gene and the severity of that mutation.So, you know, we used to say, for example, that rett syndrome’s only seen in females.

And it’s a xlinked gene, and that it must be a developmental lethal for males.But once the gene was cloned, we did find a small number of males that survived embryonic development and have mutations in mecp2.So those are variants that are hypomorphs that make it to an extra unit in life.So i think that this mouseknockout experience and the humanknockout experience will show us genes that are really critical, but it doesn’t mean that they won’t present with mendelian disease depending on the allele.Now another reason that they’re difficult mendelian disease genes are difficult to.

Identify, is that our phenotyping is incomplete andor insensitive.So the phenotyping in humans is largely a standard medical exam and then if it’s a research project we may do some other kind of fancier testing.But we basically that phenotyping is routine phenotyping or maybe what i would call, uninformed phenotyping.You’re not thinking about a particular system when you do the phenotyping.And it’d be better to have directed phenotyping that is where we’re thinking about particular biological systems that might account for this patient problem.Or iterative even better or in addition to iterative phenotyping.

Where we go back to the patient over and over again as we learn more about their condition and look for more subtle abnormalities.Another problem with phenotyping is that we have technological limitations.We only measure certain things.And they’re big, whole systems biological systems of unequivocal importance that we don’t mention it, that we don’t really measure.So the one i like to think of is protein turnover by ubiquitination.If you’re in the , you can’t send a ubiquitin level, you don’t look at ubiquitinated proteins, we don’t really assess the protein turnover.

Pathways.We know, now, from whole genome, or whole exome sequencing, or other genomic approaches, that mutations in those pathways do cause disease.For example, certain forms of parkinsonism.So, in contrast to serum sodium or liver enzymes, we just don’t measure that biological system very well.So if we’re not measuring it, we’re not going to find those phenotypes.Except until we come at it through the genomic approach.And then the thing i’ve already mentioned, the conditional nature of some phenotypes, a patient, a person can be apparently completely normal, and then when exposed to particular.

Environmental stress, like the man we described at the beginning of this talk, the phenotype becomes apparent.And the last explanation is that biological gene products don’t work in isolation.The protein products of genes don’t work in isolation.They work in complex biological systems.And those systems have evolved to have buffering, that is, the ability to maintain homeostasis when perturbed, either by environmental variables or genetic variables.And much of that buffering and robustness comes or some of it, at least, comes from redundancy of biological systems.So you have two biological systems and they do much the.

Same thing.Now, i would submit, if you look carefully, most cases of redundancy are what i would call incomplete redundancy.They sort of cross cover one another, but you can find conditions where only one of the two systems really handles it, and other conditions where the other system handles it.So that means that there will be times when you find phenotypes in there.So, i already alluded to this mouse experience, about 30 percent are lethals.But all viable mice, nearly all viable mice, do have phenotypic features.And the point is that these mouse.

Knockouts are essentially almost all 100 percent null alleles.We don’t know much about other model systems.There’s a spectrum of phenotypic consequences, depending on the allele and the genotype, as well exemplified by mutations in a gene called lbr.Their homozygous loss of function, you get a developmental lethal, basically.If you’re homozygous for only moderate loss of functions, you may have a skeletal dysplasia.But you live a full lifespan.And, for heterozygotes, for certain alleles, all you have is an abnormality in the morphology of the nucleus of polymorphous nuclear leukocytes, called pelgerhut anomaly.So a whole span.

Of phenotypic severity, all due to different mutations at that particular locus.So, if we want to find all the mendelian genes, we have to figure out ways of casting a wide net, lots of people, lots of phenotyping, and looking carefully and rigorously.Another system, i’d just mention this, that is undoubtedly important, but we don’t phenotype it at all, is the olfactory system.We have about 1,000 olfactory receptor genes in our genome.We all know that some people have exquisitely sensitive ability to smell, and other people can’t smell anything.The men in the room probably have been told by their wife, can’t.

You smell that and you say, i can’t smell that.And so it turns out that the olfactory receptor collection is highly polymorphic.And if you study peoples’ olfactory capabilities, you find wide, wide, wide variations.We just don’t phenotype it.We sort of think, well, it’s really not that important, right but it actually has been shown to influence mate selection, it influences certain things that you do in your life.And if you look at other species, other than us, it’s critically important.For example, in mice, blind mice function.

Perfectly well, and they can’t function if they have no olfactory ability.So they live in an olfactory world, rather than a visual world.And there are few mendelian disorders of olfaction that have been discovered, but largely it’s a whole swatch of the genome we don’t pay any attention to.And i’ll just emphasize this point about conditional phenotypes with this one disorder i’ve already mentioned, g6pd deficiency.Here’s a boy that presented with seizures, hypoglycemia, and hyperammonemia, 36 hours into an episode of viral gastroenteritis when he was 18 months.

Old.He ultimately turned out to have something called mediumchain acylcoa dehydrogenase deficiency.We actually screen for it now in the neonates.He was born before the screening, in a state where the screening program was not in place.The point of importance here, though, is that this is an inborn error in the betaoxidation of fats.And it only comes to medical attention when you put stress on the beatoxidation pathway.So typically for children, that happens when they get their first bout of viral gastroenteritis.What happens the baby doesn’t feel good, doesn’t eat well.The parents put the baby to bed.

Without eating much supper.About 400 a.M.In the morning, now having fasted for 14 hours, the longest the kid has fasted in their entire life, they wake up seizing and hyperammonemic, and in metabolic crisis.Before the screening program, 25 percent of the children with this disorder, that first episode was fatal.If you simply make the diagnosis and you avoid fasting.In other words, you avoid stressing the betaoxidation system, these people do fine.And he’s not had any difficulty since the diagnosis was made.And he’s now a young.

Man with two children of his own.We, of course, did what we checked his, once we made the diagnosis, we checked his siblings.And it turns out his older brother also has mcad deficiency and had had one explained, nearly fatal illness in childhood.It was called anicteric hepatitis.But he had, that was an episode of mcad problem.So you only see it when the environment is stressed, it leads to stress on this system.So we’re going to learn a lot about this, i think, from the undiagnosed disease network.

Project that really got started here by the work of bill gahl, and in the mice knockout project.Socalled comp.And it shows the tremendous value of education.If you it’s difficult to treat the genetic disorder correct the genetic disorder, but simple education of the patient, the family, and the primary care physician, can really make a difference between life and death in this disorder.And there are other disorders.And then the buffering and the robustness really goes back to this man we heard about at the beginning of the lecture.He was able, through the robustness of biological systems.

And waste nitrogen excretion, turns out the urea cycle has tremendous buffering capacity.So that an 80 percent reduction in otc functioning probably leaves you, under most conditions, to be just fine.It’s only when you have tremendous periods of protein breakdown as he did stimulated by his illness, that you overwhelm that system.So, how many mendelian disease genes my hypothesis is that if you look carefully and across a large population, nearly all the genes in our genome will have mendelian phenotypes.Not all of my colleagues agree with this, so we’ll see.

Now, let’s come back to the centers for mendelian genomics, who are tasked with finding all of these mendelian disease genes.So, the overall strategy is to find well phenotype cases and families, perform whole exome sequencing on relevant family members, use family relationships, allele frequency data, functional predictions, model organism results, functional studies to identify the responsible genes and variants.It’s really a lot of fun.Very interesting, very exciting when you get a hit.And some things you solve right away and everyone jumps for joy.And then other things you plug away at for years and we don’t solve.And in the.

Case of centers for mendelian genomics, we have an online web tool that anybody in the world can submit their cases.And once we analyze the sequence and get an answer, if we do get an answer, we give the information back to the submitter and ask them to write the paper.So they get all that for free.You can get that service at that website.So if i’m looking for all the mendelian disease genes, i would liken it to this.I think of the world, or the entire population of homo sapiens, as our, sort of, petri dish, if you.

Will.And we’re looking around the world to try to find those families and those individuals who have very rare disorders that represent the phenotype of a particular genetic mutation.So this is sort of the ultimate genotypetophenotype connection.And we’re doing pretty well on that.These are the baylorhopkins cmg data.As of april 2016, we’ve got 9,000 consented samples and we’ve got samples from 29 countries around the world.There’s still big swaths of the population of homo sapiens, namely india and china, that we’re really not getting much access into.

We’ve developed, as i said, a webbased tool to make it easier for a healthcare professional anywhere around the world to submit a candidate case or family or cohort.These papers describe that tool.It’s called phenodb.And you can look at phenodb, it’s free, you just go in, you register with your name and your email and so forth.And then if you have a family or cohort or whatever, you can enter them in there, as long as they’re consented appropriately and so forth.And then we have a committee that meets every two weeks, looks at the submissions.

The phenodb takes all the al data in a very ordered fashion, so we can quickly review the cases and ask, is this a good family to carry through to the sequencing and analysis part of the effort this is the home page of phenodb.We have users from many, many different countries around the world.The baylorhopkins incidence of phenodb, we have data on more than 4,000 projects in there, including 53 cohorts ranging from 5 to 295 subjects, phenotypic data on more than 10,000 individuals.We have whole exome sequence.

Data on more than 6,000 samples.And there’s an analysis tool about on how to analyze the results of your whole exome sequencing in the phenodb.So it’s very convenient because you can go from the analysis back to the phenotype, back and forth.And we’re continually improving it.And the we here is largely nada subraya spelled phonetically, the same woman that took david goldstein up on his offer to do whole genome sequence.And ada hamosh who’s the director of omim.And then a really accomplished program person named francois shidikate spelled.

Phonetically.This is when you’re analyzing your data, this is the starting page.So you’ll see here, in this case, you have a pro ban and you’re going to look at the sequence of hisher parents.So you start with three anavar files, you’ve selected those.At this stage, you’re putting down the inheritance model that you want to analyze the data.And you pick filters, the frequency of the alleles that you expect.You want to eliminate common variants that are present in these databases.You may want to finetune the size of indels that you’re.

Looking for, and a variety of other variables that you can dial in.And then you just push the button and out comes a list of candidates, change, and variants.These anavar files are created as you upload the vcf, so that’s done automatically.And three standard analyses, autosomaldominant, autosomalhomozygous autosomalrecessive homozygous and compound heterozygous are generated automatically.And it automatically creates a file for pathogenic or likely pathogenic, incidental findings in the acmg 56 portable gene flavors.So it automatically goes to clinvar and asks, has this variant been seen.

And how is it classified and then comes back and gives us a dated time for the issue of whether or not there are incidental findings.And on the consent form, they check whether they want incidental findings or not.It utilizes the phenotypical info in omim and the omim algorithm to suggest possible diagnoses when the phenotypes are entered, and to flag, once you get to candidate genes, if those candidate genes have been connected to one of the phenotypes that it suggested, it will make that connection for you.And there’s an api that’s sort of.

The backend, what people call the backend, that transfers the final results, gnames, genomic coordinates and features, to genematcher.And i’ll say a word about that in a minute.It’s completely searchable on phenotypic features and genotype features.And one additional tool, so the issue of, how do you declare victory what’s the evidence, the variant you have found in a particular gene is responsible for this phenotype and it turns out that one of the most potent ways to do this is to find other patients, or model organisms that have variation in the same gene and a similar phenotype.So nada and francois and.

Ada developed this, another tool called genematcher, also free to anyone who wants to use it.And there’s the website.And it’s designed to connect investigators.So anybody can go in and put in their favorite gene.And if someone else has entered that gene into genematcher, then both of you will get an email.And then it’s up to you what you want to do with that connection.All the data is didentified, so irb is not required.It’s automated in a continuous matching.One you put it in there, it’s in there until you take it out.And so,.

If someone matches you six months later, you’ll just get an email that says, you got a match.And it’ll give you the contact information.And you can choose to collaborate or not.And also we’ve added although initially it was just matching on genes, you can click a box and decide to match on phenotypic features as well.That was put in place in october 2015.And it’s connected to this matchmaker exchange program, i’ll describe in a second.But this is the page in genematcher for your matching options.By genematch, which is required.

By diseasematch, which you can ignore or you can say, i want it.Or the location in the genome, that’s optional.Phenotypic match, that’s optional.Here’s the data from a couple days ago.We had 4,247 genes in genematcher.And there have been more than 2,000 matches.Now, we don’t know which genes are being matched.And we don’t know who matches.So i can’t tell you how many of these matches, you know, turned out to lead to productive interactions.I do know that in baylorhopkins, we certainly have solved a huge number of cases by this way.We have a strong candidate but we only.

Have one case.And we find other cases with similar phenotype and similar kinds of variants in the same gene.So currently, more than 1,500 people are using this and from 51 countries.Now, genematcher, this is the matchmaker exchange diagram of all these groups interested in rare phenotypes around the world.So we built an api that connects genematcher to decipher, and connects genematcher to phenomecentral.Phenomecentral is the careforrare program in canada, which are rare diseases in canada.Decipher does rare diseases in the u.K.And throughout europe and the world.And so, if you click a button in genematcher, you will.

Not only look in genematcher, but you’ll look at phenomecentral and decipher.So you get that added bang for your buck if you just click it there.The rest of these, some of these others are planning to come in, they just haven’t made it happen yet.And, as of april, through genematcher, we’ve made 81 matches into phenomecentral, and 74 matches into decipher.So those pipelines are working well.Now, i just thought you might be i’m near the end here but what’s the baylorhopkins summary data at four years we’ve had 9,000 and change consented samples, we’ve studies.

776 phenotypes, 56 percent of those were judged to be novel.We’ve done 6,769 exomes, we’ve found a total of 468 disease genes.Of those, 222 were novel disease genes, that is, they had not previously been connected a phenotype.And 246 were known disease genes.Now we try, in our evaluation of candidate samples, to not do things that look like they have a disease that’s already well explained.But you have to realize that for many of these mendelian phenotypes, there are only two or three people in the literature, and so the breadth of the.

Phenotype has not been really fleshed out.So the ians may look and say, well this doesn’t look like the same thing.Once we find that it’s the same gene, then we often see the overlap and realize it’s what we call ‘phenotypic expansion,’ and we’re just fleshing out the full breadth of the phenotype.So, of these known disease genes, 55 percent of them, the patients that we studied, had additional phenotypic features that were not described in the entity so far.And it’s led to 124 publications currently.Now, finding disease genes.Some immediate consequences it connects the gene to a phenotype,.

Something geneticists have been interested in since there have been geneticists, connects the phenotype to a biological system, and it tells you something about how that system works, both under normal circumstances and under perturbed circumstances.So it’s quite powerful.It unravels locus heterogeneity, which turns out to be extensive.It enables precise diagnosis and counseling, all the stuff we talked about earlier.It’s the first step in the path towards informed treatment.At least you know precisely what the problem is, so now you can begin to use rational approaches to try to find a way around it.And it’s a.

Tremendous research stimulus, from bench to bedside.So it’s very powerful.Now, some long term consequences suppose and this gets to our longterm goals suppose we had phenotypes for more than 50 percent of the genes in our genome remember i said right now, it’s about 17 or 18 percent what questions could we ask i sort of think of this as a classic forest and trees analogy, right so right now we’re getting very good at finding a particular tree in the forest.And we’re going all around it, seeing how.

Many branches it has and how tall it is, and all its individual variations.And it’s very exciting, every tree is interesting.But what i’d like to see us do, as we get farther along this, is to be able to stand back and say, each of these trees is in a forest, that is, in a human being.What are the principles we are learning not from this individual genetic disorder, but from looking at large numbers of wellexplained genetic disorders can we see new principles about how disease works what genes are important, what variants.

Are allowedtolerated and so forth so that’s just, sort of, a longterm goal that’s a broader goal.And actually, we’ve been interested in this for some time.There’s a paper we published in collaboration with lszl barabsi nearly 10 years ago.And we just simply tried to look for interactions between all the genes were known to be responsible for mendelian disorders, looking for interactions, for patterns, and so forth.Makes a nice diagram but didn’t really get us too far along.And we wanted to know, are there unappreciated principles.

Of disease and if so, what are they and what do they mean for how we think about disease at the time, we did this study, we had 1,700 disease genes.So now we’re a little over two times that number.And i think studies such as this will be redone over and over again, until we begin to really get a sense of how it all works.Here’s an example of the kinds of questions you would like to answer.So, just about networks.We talked about biological systems and networks as buffering disease.You could ask, are.

All networks equally vulnerable to mutation and if not, what are the rules we don’t really have any rules for this question, as far as i know.Or we could ask, are all components of a system equally vulnerable or if not, what are the rules that make some components more vulnerable than others can we predict the consequences of variation in one component on the behavior of the biological system in other words, if there are 30 proteins in a biological system, what happens if this particular one is reduced in function by 50.

Percent does the system still work pretty well or is it completely crippled by that change so, here’s two systems.One is the ras map kinase pathway, here is the peroxisome biogenesis pathway.Each of these systems involves about 30 genes.So they’re roughly, in terms of that parameter, the same size.This pathway has more than 15 discrete phenotypes.This pathway really has one to two phenotypes.No gene predominates in this pathway.In other words, every gene in the pathway has been pegged with mutations causing particular mendelian phenotypes.In this pathway, actually, about 65 percent of the patients with defects in.

This pathway have it in one gene, a gene called pex1.So those are two biological systems of roughly the same genetic size.But yet, they are quite different in terms of the size of the mutational target.Or the target that yields a phenotype.And the kinds of phenotypes that they produce.And we don’t really know enough about it now to look at any given biological system and be able to make meaningful predictions of that type.We should be able to and we will be able to, i would predict, as we go forward with this project.

So, i have no idea where i am here, time wise.So, we’re done close.There’s just some quick samples here.So i want to just say one word about this.I hope you get the sense of the incredible power of mendelian disease, as predicting biological things that we just didn’t notice.So this is a disorder, spondylometaphyseal dysplasia, and the patients have two features.They have a cone rod dystrophy.That is a severe visual impairment.And they’re shortstatured with skeletal dysplasia, looks sort of like achondroplasia.Now i’m sure all of you sitting.

In the audience will immediately say, well, it’s obvious what would cause that phenotype.Right connect those two different biological systems i mean, when i looked at this, i said, you know, i don’t have a clue what could possibly, what gene would possibly bring these two systems together.It’s a rare autosomal recessive trait, there’s the macular degeneration.The gene turns out to be a gene called pcyt1a.Never heard about it until we did the study.But it encodes an enzyme called phosphocholine cytidylyltransferase.Which is the enzyme that is the ratelimiting step in the major pathway for phosphatidylcholine biosynthesis.

That’s a major component of plasma membranes.Some cells, it makes up about 50 to 60 percent of the structural lipid in the plasma membrane.So it’s clearly an important molecule.So, i still don’t know the answer to why those two systems are affected, but i do know that there are cells in both those systems that have a tremendous demand on membrane biogenesis.The photoreceptors in the retina make a lot of membrane every day.And actually, the osteoblasts make a lot of membrane.They enlarge from there, sort of, resting size by about 30x.

That means they need a 10x increment in membrane.And so both of those cell types have a huge demand on membrane biogenesis.So maybe that’s why they’re the ones that show the phenotype.There an alternative pathway, but apparently that alternative pathway is not adequate, at least not for these two cell types with a big demand.So those are things we didn’t think about until this, sort of, predictive thing came along.Here’s another disorder.This is in press right now, telo2.It’s worked on by a student in my lab, jing yu.And it tags a complex called the ttd complex, never heard of it.

Before.But it’s involved in interacting with hsp 90 and the r2tp complex.And does maturation of six enzymes that are very important in the central metabolism of all cells.In fact, those socalled pik genes have already been tagged with mendelian disorders.So again, we’re getting more information about a central biological pathway that’s going to be really important, in terms of understanding brain function and other functions.The baylor group the baylor part of baylorhopkins has published this paper.They looked at 128 consanguineous families and found roughly i can’t remember the exact numbers, but.

it’s about 48 known disease genes and some of these in a subset of these families.And another 40 or so highlevel candidates in the others.They put this all together and they asked, when are these genes expressed some are expressed in the early embryonic life, some are expressed in fetal life, and so forth.So you’re, sort of, again, beginning to put these biological systems together.And these biological systems are very important for the normal morphology and functioning of the brain.So we’re beginning to move from.

An individual tree in the forest, to stand back and beginning to understand the size and the shape and the behavior of the forest itself.At least in this case, in the development of the brain.The last example, also from jim, is looking at peripheral neuropathies.Charcotmarietooth neuropathies that are now, we now know of about 65 genes, tremendous locus heterogeneity.And that monogenic disorders in each of those 65 genes can cause a charcotmarietoothlike phenotype.It turns out, however, that there is a sort of, a genetic burden principle.

So we think of these as monogenic disorders, but what jim did is score the genotype of all 65 genes in each of these patients.So it shows, in red is the distribution of loss of functional alleles in these patients versus the normal population.It looked at two different populations, these data hold up.And what they say is that you have your genotype at the disease gene locus.But that is also affected, and that causes the mendelian disease, but it’s also affected by the genotype at all these other 65 genes.And if you have additional.

Variants at some of the other genes, that will make this phenotype more severe or less severe.So you get the sense of genetic burden, the architecture of genetic disease, from these focused studies.So i’ll finish with unexpected and emerging ideas coming out of this project so far.The first is, it shouldn’t have been unexpected, but the extent and distribution of genetic variation is just really enormous.We still haven’t enumerated all of it.We’re finding tremendous locus heterogeneity.We also knew about locus heterogeneity for certain phenotypes, rp and hearing loss, stuff like that.But we’re finding it everywhere we look.There.

Are many examples of phenotypic expansion.That is, we’re fleshing out or expanding our understanding of the phenotype for particular disorders.And this turns out to be very powerful.We always, medicine, over and over again, forgets that you describe something in three patients, and then we start thinking that the phenotype, the aggregate phenotype of those three patients is that disease.And if we describe another 100 patients, we’ll find out that it’s actually quite different from the phenotype of just three.We’re finding an expectedly large role for copy number variants and de novo mutations in a lot of mendelian.

Disease, relatively high frequency of two diseases occurring in the same, difficulttodiagnose individual.And we’re learning, as i showed you on the last slide, a lot about the genetic architecture and genetic burden for disease.If you want to read about it, the project was described in sort of the first three and a half years in this paper, published in late 2015.And thanks for your attention.This is the baylorhopkins team.And i’m glad to answer questions.We’re at the end of the time, so if you want to just you can come up and see me, i’m glad to talk.

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