HLA Antigenes and Diseases
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Lindau Nobel Laureate Meetings174 / 340
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Computer animation
Transcript: English(auto-generated)
00:22
Dear colleagues, dear friends, I am here a newcomer in this Lindo meeting and I would like to say how much I appreciate to have been invited by our host and how much I appreciate the charming city and the climate, the spirit of this gathering and the stimulating
00:52
contact with my colleague and with the students. I would like today to present to you the exciting perspective which has been recently
01:05
opened in predictive and preventive medicine by the use of molecular biology technology applied to the disease which are linked or associated with the main histocompatibility
01:21
complex, the HLA complex, and possibly later to extend this model to other parts of the human genome. The aim of medicine has for centuries been to treat and try to cure disease, but now
01:41
we have the possibility to detect on the population the individuals susceptible to be affected by such or such disease. Of course, prevent is better than to treat. In the first part of my talk, I shall briefly summarize our present knowledge on the HLA
02:05
complex at the gene level. Then I shall show you that with the modern technology, we are able to define an extraordinarily extensive new polymorphisms in the DNA of the HLA genes, both in the entrance on the
02:27
exons. And in the third part, I shall take an example, an important disease, the juvenile diabetes that you heard this morning by Professor Yaloff, in which these new markers will allow
02:46
to detect in the population or even before birth the susceptible individuals. It is well known that the HLA complex code for molecules which are on the surface on
03:01
most cells. Because they influence greatly the survival and the rejection of a graft organ, they have been called transplantation antigen. But, in fact, their natural function is much more general since they are deeply involved
03:24
in the defense of the organisms, in the recognition of the self and the non-self. One distinguished two classes of product, class one present in all nucleated cells involve mainly in the defense against viruses.
03:45
Class two present on the surface of immunocompetent cells, mostly B cells, B lymphocytes, involved in the defense against all kind of foreign antigens. The first slide, please, you will see this product.
04:07
Here are the molecule of class one. Here are the molecule of class two on the surfaces of 1B lymphocytes. You see that class one is comprised three domains, one, two, three, on the heavy chain
04:26
and light chain, which is a beta two microglobulin. And class two is composed by two chain with two domain, beta one and beta two, alpha one and alpha two. The next slide is the general organization of the gene of the HLA complex.
04:48
This is the H2 in the mouse, the HLA chromosome in man. And just I want to show you that there is a region for class one with the ABC loci,
05:02
class two region with other loci, and class three, which are governing for the complement component, and Professor Porter will certainly speak of that this morning. So in the next slide, I will give detail of this region.
05:23
Next slide, please. This is the D region, which is the most important in disease. And this is composed of three sub-regions. The SB now called DP, composed of two alpha and two beta genes.
05:45
The DC now called DQ with two alpha and two beta genes. And the DR region with three beta and one alpha. The next slide, you will see the genes themselves of class one heavy chain, class
06:03
two alpha and class two alpha and class two beta. And you see that this gene, like in all eukaryote cells, are composed of exon, which are the square, and inton, which are between the exons.
06:22
So only the exons are expressed on the cell surface, as you know. You see that each domain of the molecule, first domain, second domain, and third domain, correspond to three exons. It is the same for the other genes.
06:42
Next slide. So we are now able to work on the HLA genes, thanks to the molecular biology technology. And this is just to remind you how it's working.
07:01
The DNA of an individual can be extracted from the cell. And you know that the DNA is a long molecular chain. And this chain can be cut by various restriction enzymes. And the DNA chain is cut at various sites,
07:29
giving the appearance of fragments of various lengths. These fragments are sprayed by electrophoresis.
07:44
Of course, the longest one remains at the origin, and the light ones are going far from the origin. And now we can visualize where are the HLA genes by a probe.
08:01
A probe is nothing else than a copy of the genes that is made possible by transcriptase reverse. And with a radioactive probe, you can visualize these fragments of the DNA's genes.
08:22
Next slide, please. So here is to show you that the fragments that we obtain by this technique are of valued lengths. This one, for example, with ECHOR1, is cut when the sequence G-A-A-T-T-C
08:44
is found in the DNA molecule. And you have a long fragment of 10 kilobases here, which involve the exon and the introns.
09:04
In another situation, you have a smaller fragment of 6 kilobars, which is only found in the exon. Next slide. Next slide, please. So with this technique, you observe in the laboratory
09:24
these lanes. Each lane is one individual. This is here 30 individuals, which has been studied with a probe of class one gene. And you see that some of the genes are present in some individual and not in the other.
09:42
There are the demonstration of a polymorphism in the HLA genes very clearly here. And this polymorphism correlates with the HLA-B8 specific antigen. Next slide.
10:02
In the next slide, you see that these fragments are segregating in family with the HLA haplotype. For example, here you have the father with the A and B haplotype and the mother with a C and D haplotype. And if you look at this fragment present only
10:26
in the mother, it's segregating with a C haplotype in the children. And I want also to show you that some children have a spot much more intense than the other. These show that they are homozygotes for this fragment.
10:46
By this way, it is possible to assign all the fragments to the HLA chromosome. Next one. So with this technique, we have studied many normal individuals with class one probe.
11:02
And we observe that many, 55% of these probes are correlating with the HLA-known serologically defined alleles. 17% only for HLA-B and 8% for HLA-C. And 18% are not correlating at all with A and B and C.
11:25
This is probably due to the other class one genes that we don't yet know in human, equivalent of the QA and TL in mouse. Next slide is just to impress you, as said Professor Yalov. This is on the vertical line, the presence or absence
11:46
of the fragment. And the horizontal line is the haplotype, each individual HLA haplotype. And you can see that these fragments are very well correlating with the HLA-A alleles.
12:06
But there are many fragments we are not correlating very well. And these fragments are those we are probably the most interesting in HLA and disease that we are speaking today. Next slide. And when we made the same study with the class two probe,
12:28
beta DQ probe, we were very impressed by the fact that working with four enzyme, there are never two identical haplotype.
12:41
And this is also valid even when only two enzyme are used. And this is on a very small part of the HLA chromosome. So if the same study would have been done for all the gene of the HLA complex,
13:03
no one individual will be, no one haplotype will be identical. Next slide. This is also the distribution of this fragment in the population. And they are correlating well with the DR.
13:22
But they are also correlating with other series that it is too specific and specialized to speak on that today. Next slide. Next slide, please. So what are the practical implications of these new DNA polymorphism detected by these probes
13:46
in class one and class two? I will begin by this second point, that it is now possible to make the HLA type by the DNA, more or less. But I think that it can be very useful.
14:04
For example, for the earlier prenatal diagnostic of a disease which is HLA linked, which is called 21OH deficiency or seronal hyperplasia. This can be used also, and we already
14:21
use it for choosing an HLA identical bone marrow donor. In the case of the patients who have no HLA antigen on their lymphocyte surfaces. But I would like to underline the practical implication
14:44
for a better detection of susceptible individuals in the population or in family. Next slide. And I would like to recall you very quickly the association
15:01
which exists between HLA and disease. This is the associated disease with the locus A, acute lymphocytic leukemia, Hodgkin disease, and idiopathic hemochromatosis. On the next slide, you will see the association
15:22
with the HLA-B locus. There is the best set disease, the disease that I just spoke about, the 21OH deficiency, and the ankylosing spondyloarthritis. This is the most closely linked association.
15:41
As you will see, the relative risk is one hundred more in the individuals which are B27 than in those which are not B27.
16:00
Reiter syndrome, anterior vitis, subacuteeroiditis. And next slide. This is the association with the C locus, the psoriasis. Next slide. Next slide, please. And the most important is the association with the DR and all the D regions.
16:22
There are many autoimmune disease which are associated with DR3 and DR4 and DR5, celiac disease, multiple sclerosis, best disease, diabetes, and so on. You can read all these diseases
16:41
which are very important in our countries. And the next slide will speak now about only one disease which is the juvenile diabetes you heard already this morning.
17:01
Very important disease which is very frequent, one in one thousand births in our countries. And the susceptibility to juvenile diabetes is marked by the HLA DR3 and 4.
17:21
You see that when you are DR3 and 4, the relative risk is much higher than when you are only DR3 or DR4, 47 against 3 and 6. But also, the resistance to the disease has a marker which is DR2.
17:41
You see that the DR2 patients are very rare. Also, the other feature is that it exists an excess of affected HLA identical sibling, meaning that the disease is governed at least by two genes in each of the HLA chromosome.
18:01
Next slide. So we made a very, this is also to impress you, we make a very large study of 35 diabetic patients and we compare these DNA fragments obtained in this patient with the DNA fragments obtained
18:23
in the same number of DR match control, DR match control. And we observe a great number of significant differences. Next slide. One is shown here, for example, in the very rare,
18:44
I told you that the TR2 are very rare. And you see that we never observe a given fragment, ECOR122, in the diabetes and always in the control and the P is very high.
19:01
We have others, but I just wanted to give you this example. More important, next slide, more important, next slide please, is that the fact that we were able to find specific patterns of bands of fragments
19:21
of DNA fragments in these patients. For example, if there is this fragment is absent, this one is present, this pattern is very much, is absent in almost absent one out of eight
19:47
in juvenile diabetes, but is present always in the match control DR1. So this is a pattern that we observe in DR1 patient. Next slide is another pattern, next slide,
20:03
is another pattern that we observe in DR3 and DR4 patient. As you can see here, the pattern in the 12 patient is almost the same with two exception. And in the control, there is six different patterns,
20:22
and never the pattern which is observed in a diabetic patient. And these two bands are the most important to define the susceptibility to diabetes. Next slide, next slide, when you take the pattern
20:46
that I told you with the two important bands, which you can see that the relative risk of the patients of the individuals, which are DR3 and DR4,
21:05
and possess this pattern is now over that 400. So you see that with the DNA pattern, we are able to detect in the population the susceptible individuals much better than with only
21:24
the serological markers. Next slide. We did the same study for multiple sclerosis. And you know that multiple sclerosis is correlated with DR2. And the DR2, all the DR2 are susceptible to MS.
21:45
And as I show you, the DR2 is specific for the resistance for juvenile diabetes. So there is a contrasting distribution between the two diseases.
22:01
I wanted to underline the importance of these detection of diabetic patients because diabetes is a disease which can be treated and there is preventively.
22:23
There is hope recently that with immunosuppression, it is possible to avoid the appearance of the diabetes and to avoid the destruction of the beta cell of the pancreas.
22:44
And also, I would like to insist on the fact that with this study of the DNA polymorphism, we are for the first time studying not only the gene, but the non-coding region of the DNA.
23:04
That means the flanking region and the intron. This is the only way at the present time to study this part of the genome. And this is important in diseases because it is known that the flanking region are
23:21
the site of the regulatory mechanism for the genes. So with this technique, we are maybe able to detect some trouble, some disorder in the regulatory mechanism of the genes.
23:42
On the next slide, you will see that the prenatal diagnosis of the HLA-linked disease is now feasible by this DNA technique. At present, the prenatal diagnosis is done by taking the fibroblast by amniocentesis
24:05
at the 20th week with psychological consequence for the mother and with physical risk for the fetus. But now there is a new technique which is very promising that you can take some little part of the velocity by a biopsy done
24:27
very early at the eighth week of pregnancy, which is, of course, much lower risk for the mother and the fetus.
24:40
On the next slide, you will see this possibility to take by the vaginal way by biopsy a small part of the velocity of the placenta in the trophoblast of the fetus. This is enough to work with the DNA. The light, please.
25:01
The light, please. One can foresee that such a systematic detection will be rapidly used, at least in the family at risk, in which the first child is already affected. The prenatal diagnostic of HLA-linked disease
25:24
will be greatly facilitated by this new technique, and especially to detect the serenal hyperplasty, which is linked to HLA. And this technique is already used
25:44
for the diagnostic of thalassemia in some country, like in Sardinia, and, of course, can be used for many other diseases, raising, of course, important and difficult ethical problems.
26:04
I took the HLA-associated disease as a model, but now we can generalize. HLA is only one marker of the genome. There are many other markers with the numerous new DNA
26:24
probes. There will be hundreds or thousands of new markers. Thanks to this new technology, it can be awfully expected that new polymorphisms,
26:40
markers of susceptibility and resistance to disease, will be found, which will present strong positive or negative association with many diseases HLA-linked or not. These studies are only just beginning, and will very quickly spread to new diseases.
27:05
It is thus hoped that it will be possible to screen, more accurately than at present in the general population, those individuals susceptible to contracting a serious illness, so as to be able to prescribe a preventive treatment if it
27:22
exists, or at least to institute an early treatment. This is, therefore, a new powerful means of preventive and predictive medicine, permitting a more effective and less onerous personalized medicine.
27:41
But the ultimate goal should remain the understanding of the pathogenesis of the diseases. Isolation and characterization of the gene or genes involved will be a decisive step in this direction.
28:00
The sequence of the gene would allow to construct the corresponding molecule. Likewise, the transfection of the gene in a somatic cell would permit its expressions, leading to the knowledge of its function. When the function will be known,
28:22
one can foresee that the therapeutic is not far away. I hope that I have shown to the young students and scientists the extraordinary perspective opened by this new technology in epidemiology and in public health.
28:41
And I am sure that their enthusiasm and dedication, they will very rapidly exploit this vein. Thank you very much.