Tip:
Highlight text to annotate it
X
All right, weíre going to start. Our next speaker is David Page from the Whitehead Institute
who will be talking about sex chromosome evolution and medicine. David.
[low audio]
David Page: OK, thank you Eric [spelled phonetically]
and thank you very much for the invitation to speak at this terrific symposium on a wonderful
occasion. And I would like to begin by thanking NIH and NIHGRI for 21 years of support of
our studies of a chromosome that was in need of some respect. Studies that have allowed
us to completely rethink the Y-chromosome structure, function, evolution, and medical
significance. And what I want to do today -- so yeah, I should say that Iíve spent
the better part of my career pondering the wonders of this pair of chromosomes. On the
left, the stately and upright X-chromosome, and to its right somewhat downtrodden, even
demure Y-chromosome. I spent the better part of my career defending the honor of the Y-chromosome.
Thankfully, with the support of the support of NIHGRI in the face of innumerable insults
to its character and its future prospects. So thereís so much Iíd like to tell you
today, but Iíve decided to be compact in my messages and perhaps dive into a little
bit more detail. What Iíd like to say today, Iíd like to make three points.
First, I want to tell you that sequencing the human Y-chromosome has yielded completely
unanticipated insights, including as Iíll explain today, a possible mechanism of origin
for the chromosomal defect in Turner syndrome, which as you know exclusively effects girls
and women. Second, I will point out that due to the structural complexity of the Y-chromosome,
sequencing it required some unorthodox strategies, strategies that would allow us to differentiate
between repeated or -- repeated sequences or paralogues that are more similar than alleles.
That is, we had to travel beyond what I might call the ìallelic limitî. And third, I will
recommend that a similar strategy be combined with the latest sequencing technologies to
scrutinize about 160 of the most structural complex U-chromatic sights on the human X
and autosomes. These are about 160 sites that likely account for a disproportionate burden
of disease and I will suggest that this investment will be returned each of the -- I think we
heard this morning -- million times or did Eric say billion times that the genome is
resequenced. OK.
So, let me first give you a crash course then in how we think about sex chromosomes evolution.
What weíve learned over the last 15 years is that the human X and Y chromosomes evolved
from a perfectly ordinary and unsuspecting pair of autosomes. So as Iím sure you discuss
when you get together on family occasions, youíll recall that 300 million years ago
when we were reptiles, we had no sex chromosomes. We had only ordinary autosomes. And what happened
beginning about 200 or 300 million years ago was that one of the perfectly ordinary pairs
of autosomes sustained a mutation that would begin to give rise to what lives on today
as SRY, the sex determining gene, on the Y-chromosome. And what transpired -- and I should say, this
is work -- this understanding of the sex chromosomes is based completely on genomic analyses over
the last 15 years -- what then transpired was that first in the immediate vicinity of
SRY and then over a larger region of what was becoming a Y-chromosome, crossing over
between the X and the Y was suppressed. And it was actually this suppression of X, Y crossing
over that allowed the X and the Y to differentiate from a perfectly ordinary and identical pair
of autosomes. Now, *** recombination is a good thing for the health of genes in the
long run. And so the X-chromosome retained the rejuvenating benefits of *** recombination
due to crossing over in female meiosis during Oogenesis. But the Y was now left to reproduce
clonally without the benefit of *** recombination and so it began to lose genes. And in this
expanding blue region, the Y-chromosome lost genes. If you take this out, if you sort of
extrapolate this out to infinity, it does not bode well for the Y-chromosome.
[laughter]
And thinking of our timing, about 10 years ago -- actually nine years ago -- this was
taken to its logical extreme in of course, a journal that we seem to be making frequent
reference to today, this obscure one called Nature and in an editorial somewhat grandly
entitled ìThe Future of Sexî, what I just told you was pointed out, that itís the Y-chromosome
that is particularly vulnerable against the ravages of evolutionary time because it doesnít
have a matching partner with which to swap genes so it cannot retrieve genetic information
by recombination. And then came the very devastating conclusion in this editorial. The present
rate of decay, the Y-chromosome will self-destruct, 10 million years. Well, a graduate student
came running into my lab having read this, tears streaming down his face and we held
an emergency lab meeting and resolved to pick up the pace of our research.
[laughter]
So thankfully we were already supported by NIHGRI. So by the next year, we together with
the sequencing center at Wash U [spelled phonetically] had completed the sequences of Y-chromosome.
And I think itís fair to say that itís purely and simply genomics that has saved the Y-chromosome
from the intellectual wrecking ball. So let me briefly summarize some of the things that
we learned having studied the sequence for some time now. First, give you a crash course,
a little overview of the Y-chromosome. So, the centromere is over here. and so we got
the short arm, the long arm, and Iím gonna emphasize -- out at the green bits out on
the end are the so-called pseudoautosomal regions where XY crossing over is a normal
and frequent event in male meiosis. In between lies the portion of the Y-chromosome that
differs from all other nuclear chromosomes in two respects. Itís specific to one sex
and itís the only part of the nuclear genome that does not ordinarily participate in crossing
over with a homologue. The distal long arm of the Y-chromosome consists of rather simple,
monotonous repeats, heterochromatic and sadly, no molecular geneticist has entered the hetero-chromatic
region of the Y and returned alive. This, Iím sure is why we as a community have made
-- have steered clear in all of our sequencing efforts, have steered clear of centromeres
in heterochromatic regions. That was actually intended to be a joke, but we actually have
some challenges ahead. And I will readily admit that though I just said we sequenced
Y-chromosomes, we havenít even touched this half of the Y-chromosome heterochromatic,
which is emblematic of what we have yet to -- the challenges that lie ahead in the rest
of the genome. So, weíve instead focused on the U-chromatic portion of the Y-chromosome,
which comprises about one percent of the human genome. And I will very quickly summarize
some of the largest changes in our thinking then and now about the Y-chromosome.
Well, so the notion was the Y was a genetic wasteland. We now understand it in codes at
least 27 distinct proteins. Thereís a larger number of protein coding genes because many
of these proteins are encoded by families with virtually identical members and thereís
a clear specialization in *** production of the most of the genes on the Y-chromosomes.
The view that the Y was merely a rotting copy of an ancient autosome, weíve learned that
many genes during the evolution of the Y-chromosome have been imported from the X-chromosome or
from autosomes, especially during primate evolution. And many of these have been amplified
once arrived at the Y-chromosome. So much of the Y-chromosomeís gene kind is relatively
recent invention. Iíve been told for years that the Y is nothing but junky repeats. What
I will show you is that the Y-chromosome carries features, gene rich palindromes of unprecedented
scale and precision. The notion that the Y does not participate in crossing over doesnít
have produced recombination was the explanation for years as to why all the genes must be
disintegrating.
What we actually found is that an alternative form of recombination within the Y, gene conversion,
probably sustains many of the genes of the Y-chromosomes across evolutionary periods.
And yes, the Y is said to be headed for extinction. Well, it turns out thank you very much that
even single copies genes on the Y look to be persevered through natural selection even
in the absence of *** recombination. And finally, the greatest of medical insults
-- of insults in this age on focus of translation, that the Y would have no medical significance.
Itís clear now that actually deletions on the Y-chromosomes are the most common known
genetic cause of male infertility. The somatic genetic failure is ultimately implicated in
*** cancer, and as will point out to you today, may actually play believe it or not
a central role in the ideology of Turner syndrome. So, I will focus now the rest of my remarks
on the gene rich palindromes and a possible connection to Turner syndrome.
OK, so let me tell you briefly about the structure of Y-chromosomeís palindromes. You all know
that palindromes are sequences of letters that read the same backwards and forwards
like madam Iím Adam. These palindromes on the Y are much, much larger than those you
would encounter in literature. They show very amazing degrees of left arm to right arm identity
as high as 99.997 percent or conversely they show less than .06 percent diversions. I already
want to begin to plant the seed in your mind that is less than the difference between alleles
in the human population at [unintelligible]. These are big. The biggest of palindromes
on the X and Y-chromosome has an arm length of almost one and half megabases or a wingspan
of three megabases. That one palindrome is one thousandth of the human genome. And these
arenít just pretty structures, they actually carry genes. The great majority of the genes
required for somatogenesis on the human Y-chromosome are born on palindromes. And this means that
-- well, the one thing that we knew before any of this work, the one thing that we knew
was the central, biological reality of the Y-chromosome was that its genes did not have
partners on a [unintelligible]. But what we came to realize was the genes on the Y-chromosome
carry their partners on the opposite arm of the palindrome where they exist as mere image
copies. And it turns out that these palindromes, and there are about eight of them in total
on the human Y-chromosome, they comprise 25 percent of the business part of the Y. They
carry all of the intact copies of the long arms test to specific gene families. Over
on the left, I list eight gene families and on the shown across are the doublets or couplets,
the diodes of genes that occur in palindromic locations. It turns out that knowledge of
this hall of mirrors, if it is the Y-chromosome, has lead to many predictions. It turns out
even if that structure is very complicated, you know it and you know a little bit about
the principles of homologous recombination, you can predict variation and you can predict
mutations and even predict, in some sense, what their phenotypic consequences might be.
Many such predictions over the last 10 years have been fulfilled. For example, there are
now six known highly reproducible deletions that occur [unintelligible] but repeatedly
and those are collectively the most common known genetic cause of somatagenetic failure
in our species.
What I want to do is tell you about one particularly outrageous prediction based on the knowledge
of sequences of palindromes and some of the simple principles of homologous recombination.
So we made the speculative prediction the palindrome, palindrome crossing over might
occasionally produce a mirror image Y-chromosome, an isodicentric one. So what if two copies
of the Y-chromosome, two sister chromatids for example were to align in opposite orientation.
Palindromes would be equally find templates for crossing over in either orientation. If
a cross over occurred, you might produce a mirror image chromosome. Well, this might
sound a little fantastic, but it turns out to be the case. So, here Iíll just show you
that by C2 hibernazation [spelled phonetically] with a probe -- actually the red probe is
for XRY sex determining gene, the green probe for the centromere -- this is a probe pattern
you would see on a normal Y-chromosome. This is what you see on some isodicentric Yís
and this is what you see on some bigger isodicentric Yís, which had as their target for crossing
over a different palindrome. So Iím summarizing here a study that carried out just published
a year ago, a year and a half ago, which we reexamined the Y-chromosomes of over 2,000
patients who we studied because they had a discordance between sex-chromosome constitution
and their anatomy, or they had a microscopically detectable anomaly of the Y, or they were
men with little or no *** production. And out of those, we identified 60 individuals
who had isodicentric or iso Y-chromosomes that had arisen by the mechanism I just showed
you. 50 of these -- 51 one of these I should say arose from crossing over the palindromes
and essentially all of the palindromes, each of the palindromes that we knew about on the
Y-chromosome was hit as a target in one or more of these individuals. This turns out
to be a significant cause of somatogenic failure in our species.
And then Iíd like to just point out to you just sort of go a little bit more into depth
into one particularly interesting aspect of the correlation with the phenotype. And that
is that 20 of these individuals who carried mere image Y-chromosomes were anatomically
female. So I want you to think about how that could be the case. Well, perhaps some of these
isochromosomes had deleted or lacked SRY, the sex determining gene. Well that turns
out to explain two of these cases. SRY is not present on the isodicentric, but that
leaves 18 cases where SRY is actually present not once but on both ends of the isodicentric.
So, again appealing to systems -- to studies from for example yeast, we came up with a
hypothesis. And that is that these chromosomes, with two copies of SRY, they have two centromeres.
This is not a happy formula for going into mitosis. The mitotic instability of these
isodicentric Y-chromosomes could have given rise in parts of the developing embryo, specifically
to the existence of XO cells in the embryonic *** and this could lead to feminization
of the external genitalia, anatomic feminization despite the presence in skin and blood of
the isodicentric Y-chromosome. This lead to a prediction. It turns out the farther these
centromeres are apart, the greater the distance between the centromeres, the greater predictably
the mitotic instability, the greater the probability of anatomic feminization. This turned out
to be the case. So simply plotted for the 60 individuals. Here are plotted the 40 individuals
who are anatomically male out of the 60 and here each of these dots represent the location
of the targeted palindrome and here are the 18 anatomically feminized individuals, which
tended as you see to target more distal palindromes. Just to translate that that translates into
intercentromiric distances distances between the centromeres being actually on average
about twice the intercentromiric distance in the feminized 18 as in the masculinized
40, fitting the prediction. So pointing to the existence, the very likely existence of
XO cells -- well to take this even further -- I should say in human beings with isodicentric
Y-chromosomes, the more Y DNA you have, the greater the likelihood that you will be a
female. Lead to further speculation, this is where I get to Turner syndrome.
Could mitotic instability of isodicentric Y-chromosome in zygotes be a significant cause
of the XO state in girls and women with Turner syndrome? Now, this should sound crazy to
you right? XO, isnít that due to meiotic non-disjunction? No. It turns out there is
no maternal age effect in XO Turner syndrome unlike trisome 21. In other words, the mothers
of girls with Turner syndrome do not have elevated ages. And it turns out that if you
look at who contributed the X-chromosome that XO girls retained? In three quarters of cases,
the X-chromosome is of maternal origin. Which is to say, the missing sex chromosome is dadís
chromosome. Now, since theyíre female, you would immediately leap to the conclusion its
dadís X thatís missing, but it could easily be dadís Y or an isodicentric Y. So I would
like to suggest that Y palindrome, palindrome recombination could be a very significant
cause of XO Turner syndrome. And I should say that none of this would we be able to
make any sense of except for the availability of a very precise, highly accurate sequence
across the most structurally complex parts of the Y-chromosome. So thank you [unintelligible]
for helping make that possible.
Well, so how is the sequencing done anyway? Well first of all, it required the dedicated
efforts of not surprisingly, women. Iíd like to particularly highlight the key -- the really
passionate contributions of Temuco [spelled phonetically] and Helen [spelled phonetically]
here. Some men contributed to the effort, among others Bob Waterston [spelled phonetically],
Dennis Washu [spelled phonetically], and with the work continuing with Rick Wilson [spelled
phonetically] there. Well, how is it done? I wonít go through the details but Iíll
just highlight a couple of principles which I think have more general applicability. So
how would you sequence a large palindrome whose arms are more similar than alleles?
Well, turns out then that allelic differences, if you were to choose clones from two or more
men, allelic differences would absolutely confound assembly. So you avoid allelic differences
by sequencing one and only one chromosome, sequence just one manís chromosome.
Now of course, we didnít know there were going to be palindromes when we started this
project, but thinking there might be some trouble ahead, we chose to sequence one manís
Y-chromosome. And it turns out that in this case, the left versus the right arm differ
by only occasionally nucleotide substitutions. To actually work your way across, the only
way to do it is to capture a few such differences in large insert clones like backs derived
from one man. So thatís what we did. And then we used these left versus right nucleotide
substitutions as unique markers. They become your landmarks. These occasional nucleotide
substitutions become the landmarks with which you pursue the growing of back [unintelligible].
So you find additional such markers as you iterate. And so I would just call this in
some sense haploid iterative mapping and sequencing.
Just to give you an example. So here are two Y-chromosome backs from one man. And theyíre
typically sized backs. These come from the famous Mr. RP 11 on whom most of the reference
sequence of the genome was constructed. And you will see this is a particularly inefficient
use of NRGI funds because these two backs overlap by 105 kilobases. We should have been
taken out and shot at that point. But it turned out that within these 105 kilobases of overlap,
there are 11 nucleotide substitutions. Or 99.99 percent identities. Now if these backs
had come from two different men, youíd say oh this is allelic difference, in fact, itís
barely allelic difference. But it turns out these two backs come from one man. And these
two backs turn out to [unintelligible] where the initial seeding of the two arms of a palindrome
on the Y-chromosome. So, and so on.
Well, we know that such structurally complex regions exist elsewhere in the genome and
that was recognized in Eric Green [spelled phonetically] and colleagues article just
published yesterday where they said that structurally complex regions which are known to have --
and I might add the word disproportionate -- role in human disease remain inherently
difficult to sequence even with the new DNA sequencing technologies. Went on to say additional
technological improvements for example, much longer reed lengths are needed to sequence
such complex regions. I couldnít agree more. New technologies, which I hope actually appear
on the scene maybe all tell me that theyíre just around the corner. I agree that longer
reed lengths would be a tremendous asset in the pursuit particularly of those naughty
private parts of the genome required for replicating it, like centromeres and kilomeres [spelled
phonetically] that weíre not even prepared to talk about entering yet. Those longer reeds
will be absolutely needed there. But in addition, thereís something else that Iíve already
mentioned thatís needed.
So one of the key impediments in resolving the complexity of these regions is the diploid
and polymorphic nature of the human genome, what I might call the allelic limit. In the
past, the distinction between allelic versus polymorphic variation has been successfully
circumvented by the use of genetic material of haploid complexity. This is not from the
paper just published yesterday. The final sequence in the assembly of the Y was achieved
in large part due to the fact that all of the back clones came from one manís Y-chromosome.
And so sequence assembly was not impaired by polymorphism and all sequence variants
represented distinct copies of paralogues.
Now, this was actually written in 2002 not by me but by Evan Eichler and colleagues in
proposing the use of some unusual human conceptions that end up having a haploid representation
of the human genome. And in fact, NHGRI made considerable investment in resources based
on these hydatidiform moles. And I would like to suggest that the opportunity is ripe to
take advantage of those resources made some years back now with NHGRI resources with NHGRI
funding in combination with the newest sequencing technologies to extend the notion of haploid
iterative mapping and sequencing to the approximately 160 structurally complex U-chromatic sites
on the X and the autosomes that are sort of crying out for a little closer scrutiny. I
think again that the investment will be returned many times over. And so with that, Iíd like
to stop and thank you very much for your attention. Maybe we have time for questions. Thanks.
[applause]
Male Speaker: Questions for David? Iíll ask one. So to
your knowledge, has anybody taken a hydatitiform mold and taken its genome and just tried to
hold genome shotgun using the new technology?
David Page: I think that is in the works as we speak to
give it a good look.
Male Speaker: But your proposal would be to take back clones
that have been derived from those and through the painstaking work of developing the maps
across the structurally complex regions.
David Page: To go right to these regions, which I think
actually are pretty much identifiable today.
Male Speaker: But not yet mapped.
David Page: But not yet mapped in this way.
Male Speaker: And admittedly the technologies for making
those maps havenít advanced substantially in the last five, eight years. So it would
still be pretty painful to make.
David Page: I think it would actually be far more efficient
today. Because using sort of back pooling strategies. I donít think it would actually
be that hard or be that tremendously difficult.
Male Speaker: Max one more question.
Male Speaker: David, I have a quick question regarding the
Y-chromosome heterochromatic. I remember when we did chromosomes of medical students the
male medical students always seemed to like it when they had longer Y-chromosomes than
the ones with the shorter Y-chromosomes that are a little jealous. But I really wonder
is what do you think, whatís the difference, what do you think these repeat sequences could
really mean because I donít think they are just junk?
David Page: Yeah males always like to say that mine is
bigger than yours when it comes to the Y-chromosome. And so, there is there actually Max is a great
question. One of the -- ironically if we go back now 50 years, one of the first documented
polymorphisms in the human genome was variation in the size of the Y-chromosome. This is a
very hot topic of publication in about 1959 and it turns out there is to my knowledge
no known phenotypic correlate of variation in the size of the Y-chromosome. And in fact,
there are men who are essentially missing the heterochromatic region who appear perfectly
healthy, including their fertility. So, the way I think about it, the Y-chromosome minus
that heterochromatic region would be by far the smallest chromosome. With the heterochromatic
region, it joins the smaller autosomes in size. So, maybe itís the kite, maybe itís
the tail that helps that kite sail better. I donít know. Not a very satisfactory answer,
but thank you for asking.
Male Speaker: OK, weíre going to move on. Thank you David.