Articles, Blog

Positional cloning of genes for monogenic disorders

August 12, 2019

Welcome back to the fourth week of this NPTEL
course on human molecular genetics. This week also happens to be the final week, wherein
we will be discussing more recent developments in the area of human molecular genetics. What
you have seen in the last first, second and third week lectures is how the genetic material
is, you know, sort of understood with regard to its contribution to human disorders. We
looked into, for example the central dogma and how the information is processed in biological
system. Then, we moved on and understood the concept behind recessive and dominant characters
and variations therein and then we looked at pedigrees and looking into phenotype that
segregate in the human population to arrive at a conclusion whether the genes that is
causing a particular phenotype is dominant or recessive in nature and then, we looked
into some of the complications with regard to the Mendelian segregation. Then, we moved on and looked into some of
the molecular biology techniques people use in characterising what are the defects that
are there in the gene and how that may contribute to the disease condition. Then we discussed
some of the issues about model systems and how model systems are used to understand disease
pathology to validate some of the hypothesis and finally, to use it for some therapeutic
applications. So that, as these are the topics that we covered so far and in this, we are
going to look into the approach we use to identify the disease causing genes. So, this is going to be a challenging topic,
because it is going to have, I really, you know, expect you to have the fundamentals
clear, otherwise it would be difficult for you to follow. So, even if you are having
some difficulty in understanding this lecture, I would encourage you to go back and watch
the, week II and week III lectures and come back and revise and, and then see whether
you can understand. If you have any issues in understanding, you can always write to
us and we will try to help you and of course, we are going to have hangout sections at the
end of the course, wherein I could also, help you with some of the concepts, if you still
are unable to understand some. So, let us look into what is the topic that
we are going to study in this week. The title is, from, you know, pedigree to defective
genes. So, we have looked into the pedigree analysis, we have looked into the gene structure
and how defects cause particular disease. But, what you are going to see today is how
we use pedigree to identify the gene that causes the disease. So, the disorder that
we look into in the human population can be categorized into two groups that have the
genetic origin. One we call as a monogenic disorder, because here the, the defects lies
in one particular gene, therefore you have the phenotype. The other group is polygenic disorders, wherein
the disease phenotype results from a combination of variations in more than one gene, may be
4, 5, 6 depending on what is the complexity. So, the approach that we used to identity
the genes that contribute to the disease varies. If you are using a monogenic disorder to identify
a disease gene, we use a very different kind of approach. If it is a polygenic disorder,
we use a very different approach. So, what we are going to do is to cover these two along
with some other advancements, like for example in the last two decades we have seen tremendous
advancement in molecular genetics, molecular biology. The human genome has been sequenced.
Now, we are trying to understand all the variations that are there in the human population. So
we will, what we will do is that we will combine the approaches with how the advancement has
really helped us to arrive at different approaches, right, to identify the genes. So, that would
be the focus of, you know, this weeks lecture. Let us look into the pedigree. So, we will
start with the pedigree. So, this is something that we should be familiar with. Now, looking
at it you will be able to say this is a autosomal recessive inheritance and the phenotype is
because of autosomal recessive conditions. So, we have seen all these things. The question
that we are going to address today is what is the current method or how people really
used what approach they have used to identify the genes? So, when you say autosomal recessive
disease, we are talking about a gene that is located in one of the 22 pairs of autosomes.
So, we are excluding X and Y, because they show very different kind of inheritance. Now,
that is all that we can do with the pedigree analysis. You have looked at the pedigree
you have, you know, you come up with the model, which suggest it could be autosomal recessive.
But, now it is going to be a daunting task as to which one of the 22 chromosome that
you see in this slide harbours the gene, right? So, that is going to be extremely difficult
task to see. So, it is going to be a daunting task to come
up with kind of a valid proof to say in which one of the 22 pairs of the autosome that we
have, the gene could be located. So, what we are going to discuss is some of the conventional
ways people used to identify human disease genes. So, let us look into what are the ways
people have identified at least in the past, the disease genes. One of the initial discoveries
on genes that are causing human genetic disorders have come from understanding the, knowing
the functions of the gene or using functional assays. So, in other words, you know a physiological
pathway that is defective in a disorder and you know that disorder is caused by some genetic
defect, because it runs in the family. So, now you use your understanding on the
physiology to identify the gene that could possibly contribute to the disease. So, this
is called as a functional cloning, because you identified the gene, because of the known
functions of the gene. So, that is why you have gone on to identify them. So, let us
look into some of those examples. One of the famous example is the bleeding
disorder, what you call as a haemophilia that was, you know, discovered in the family of
Queen Victoria, the then Queen of the British kingdom and this is one of those rare photographs
that you can find in the website about the family. This is a very happy family; at least
in the picture it looks so. But, there are many unfortunate incidences
in this family. So, as you can see here, this is a kind of pedigree that is shown and you
have some individuals that are identified with a red colour filled in symbols for male
and female. These are the people who are affected with this bleeding disorder. It has a very
very severe condition, because in this condition the blood will not clot. As a result, you
know, unless or otherwise you are very careful, we are going to have extremely, you know,
high volume of blood being lost, because of some injury or cut or anything. So, you can
see here that the Queen herself was not affected. She was all right, but she is expected to
be a carrier and it is sort of perceived that the mutant gene could have come from her father
and you can see in her next generation, one of her sons, he was affected and we know now
that this particular disorder is caused by a gene that is located on the X chromosome.
So it is X-linked inheritance. So, let us look in how they have really identified.
So, this is a kind of symbol that I have shown, but let us not worry about it. What is, what
has been understood is that by then people have understood the biochemical basis of how
the blood clot happens. So, it is known that we have a large number of factors, proteins
that are there in your blood and which, you know, upon activation, for example when it
gets in contact with the air, oxygen or atmosphere, because of an injury or whatever, then it
sets in a cascade which results in the blood clotting. So, there are many different factors
that are listed here on the right side, the left side of the screen, like you can see
here. So, you can see that these are the factors that are involved in the blood clotting. What
you find in all of us who are very normal that you have all these factors are very active.
So, if we do the biochemical assay for these factors, you will find that it is working. So, what they have done is, very similar kind
of approach they have used to identify which particular factor could possibly be abnormal
in this family, you know, those who have the haemophilia and they found was this particular
factor, at least in this family, that factor was defective. In these individuals, you do
not see factor VIII being active. So, that is how they understood probably this is what
is deficient. It could be some genetic mutation that results in the deficiency of factor VIII. So, with that understanding they went on to
identify the gene. So, the approach that they used then was that they purified the factor
from the blood. So, it is, when you say factor it is a protein and then they sequenced this
protein to obtain what is called as amino acid sequence, right. So, they have now the
amino acid sequence for the protein and then they predicted what could be the coding sequence.
So, you know what are the different codons for each amino acid and with that you can
come up with multiple options as to what would be the coding sequence and based on that sequence,
they have designed DNA, short stretch of DNA using lab machines. Now we have what is called
as a DNA synthesizer. We can add those, it is a kind of chemistry by which you will be
able to add certain bases to get kind of sequence that you want and they have designed this
coding region and then they went and screened what is called as a cDNA library. Remember, we discussed about that that we
convert the messenger RNA into a single stranded DNA and then to double stranded DNA, we clone
them in the vectors and then they are stored and you can use any kind of a probe what you
call here for example, the DNA coding region for factor VIII,. you can use it as a probe
and identify the cDNA that codes for this factor. That is how they cloned gene and then
they went on to say what is the defect in that particular individual. So, that is how
they have done. So, this approach that led to the discovery
of the gene is really really is time consuming and you have lots of challenges. I know there
are few such examples, wherein people identified the protein product that is defective and
then went on to identify the, the gene that codes for that protein. So, there are challenges.
Some of them are listed in the screen. Not all genes are categorized. In fact, we do
not know all the genes that our human genome contains and even if you know the gene, we
do not know what is the function of the gene. So, you may know this is the protein that
it codes for; that is all you can predict, but still you do not know what is the function
of this protein in the organism. For example, you may say it is an enzyme present in a cell,
but how does it really, you know, change the individual, right, that is not known. So, the physiological functions of most of
the genes are not known and therefore, since you do not know the function of the gene or
the protein, you cannot, come up with assays to test whether it is active or inactive,
how mutations could have affected and so on. A protein could have multiple different functions
and how are you going to come up with all sorts of assays? For example, you are talking
about their mental retardation as a condition, right? So, wherein your intellectual ability
is not that good, your IQ is low. So, what kind of assays you will do to understand how
the gene defect affected your intellectual abilities. It is going to be extremely difficult.
So, even designing assays for some of the genes of some of the phenotype that you are
looking at are going to be extremely difficult. So, therefore even predicting, I mean these
are the candidate genes, therefore I can go and identify mutation, this is also, is difficult,
but still there are some examples. For example, epilepsy, one of the conditions
wherein people get what you call as fits. You know, they thought that it could be resulting
from abnormal neuronal function. Therefore, people thought it is the channels that regulate
the, what you call as the neuronal functions in terms of its membrane potential, how the
electricity is passed on from one to the other, these are regulated by channels and people
thought the channel could be involved and they went ahead and sequenced many of the
channels. There were some success, they were able to find some mutation, but still it is
extremely difficult approach to identify the genes based on the functions of the protein
that you know. So, therefore it is very very, the success has been, very very limited. The other approach people have used is to
look at animal models, you know, when you talk about disorders, disorders are caused
by defect in a gene. So, we have genes, likewise every living species have got the genes and
defect there in should also result in some abnormality. But more often what we do is,
we breed domesticate animals. We breed them and we have selected for some good characters.
If the character that comes in a generation that are not desirable, more often that are
not, you know, selected by the human. Therefore, the gene gets lost, but still there are many
condition that you see wherein, there are animals that developed some symptoms, some
phenotype that resemble some human disorders and when they have identified such, then they
have located the gene for that using human, human, the sorry, using the mouse species
or any other model that they have used and once you know this is the gene that causes
a phenotype in this animal, then you can go and look at the mutations in that equivalent
gene in the human. So, this is also has been some of the approaches. I will show you one such example wherein,
so this is a kind of approach people have used looking at the function of the gene product.
See, the other approach people have used to identify a gene is based on structural changes
in the chromosome. So, as we discussed, the chromosomes, at times there are exchange between
the chromosomes. As a result, you have rearrangements and the rearrangements are known to result
in certain disorders, right? So, therefore if a given translocation and what I mean is
that a part of the chromosome goes and attaches to some other chromosome, you have a disease.
If you are getting the same disease and the translocation is seen in many individual because
of recurrent events, then you would assume that the gene involved in the onset of the
given disease is located very close to where the chromosome is broken, right? So, that
is the approach, so they have used and there are many examples. This is something I am showing that we discussed
already that is the Philadelphia chromosome associating with a type of blood cancer is
resulting from translocation between chromosome 9 and chromosome 22 and we know that the break
point where the chromosome break happens, here and here, you know, harbours genes ABL
and BCR and because of the fusion, you have a new sequence and one gene gets activated,
it would normally should not be expressed and then you have the disease. So now you
know, this is in the, for example ABL or BCR is involved in, you know, this particular
cancer. So, this is another way by which, you know, by looking at chromosomal changes,
translocations, people have, they are able to identify the disease causing genes, right?
So, again later they have understood what is the function; so this is the, one of the
ways by which we can do. So, the third, approach people have used is
to characterize animal models. There are animals that we use for studying genetics and many
of these animals also have, genes that are very, very similar to us. For example, if
you look into the mammalian species, whether it is a rat, mouse or any other model system
that you study have got all the genes that we also have. Therefore, if there are some
defect that we expect to cause a genetic condition in humans, is likely that the same gene, gene
defect in the mouse or rat and rabbit should also be causing this similar phenotype. That
we have discussed already in the gene knockout lecture, wherein you are able to create deletions,
create mutations in the gene that cause disorder in the human. So, you create similar mutations in the animal
and then look at whether the animal develops and if it develops, then you understand what
is the pathology. But people also looked at animals, which some how inherited a natural
mutation. As a result, they have certain defect and resulting in a phenotype and if the phenotype
resembles some of the disorder that you see in humans, then you know that the gene that
is mutated in mouse is also likely to be mutated in the humans. So, you identify what is the
gene that is defective in mouse and look at the corresponding gene in the human and see
whether that has defect. There are some success stories there too.
What I am showing is a condition called as Waardenburg syndrome and this particular syndrome
is caused by a gene called SOX10. This is a transcription factor. So, what is this disease?
This is a congenital anomaly, there are multiple developmental problems. One of the phenotype
is also something to do with pigmentation, whether, the colourations. So, what you can
see on the top is that this particular mouse, you know, this naturally developed a mutation
resulting in many developmental abnormality, as well as pigmentation you can see here in
the fore head that the hair colour, it is very very unique, you do not have the colouration
and also the other condition is the distance between the eyes, between the two eyes also
is enlarged. We have more flat face and that there are other problems, there are a number
of problems. What is interesting is that very similar phenotype
has also been seen in human. For example, you can see here the baby; she has got premature
grey, you know, grey hairs and then there are problems with eye and there are problem
with, you know, heart development and many, many different conditions. What is , you know,
important to understand from this particular course point of view is that they identified,
they did a mapping for the mouse gene as to what is the gene that causes this phenotype
in the mouse and they identified the gene called as SOX10 and then they looked at that
there is a deletion that it causes and so on. Once they understood the gene, then immediately
what they did? They did not do any mapping for the humans, straight away they looked
at whether SOX10 is a gene that is mutated and indeed that was the case. You know, they
went ahead and then shown that it is similar gene mutation, similar phenotype in the humans
as well. So, there are handful of examples wherein
animal models which developed, one or the other this condition, because of spontaneous
natural mutation led to the discovery of genes that causes the disease in the human as well.
Again, these are limited. The challenges, we do not have animal models for all the disorders. That is, it is not possible, because of reason
that most of the model system that we use for our experimental purpose are in built
and maintained in the colony. Normally, look at whether the animals are normal, they have
a healthy phenotype, if not we remove them. Therefore, we know that whatever experiments
we do, whatever effect you see is because of the experimental change, but not because
of something, not linked to the experiment that you do. Therefore, you normally maintain
very healthy animals in their lab settings. Therefore, you will not find any natural mutations
coming in. Rarely you see that and lab animals do not have variations or mutation like, what
you see in human. All of us look different, all of us behave
different and all of us are exposed to very different kind of environment, whereas lab
animals are in bred, meaning they have very very similar genetic make up; variations is
very very limited and then, therefore you keep selecting for a, you know, the good phenotype.
Therefore, you do not really allow them to sort of acquire in the population, some abnormal
mutation or variation in the DNA and even if there were mutations resulting in the phenotype
in the lab animals, mapping them is going to be extremely difficult. It is more difficult
than what you can do with humans, because normally you do mapping based on the sequence
variation that you have in the genome. That is what we are going to discuss now. But you know, the sequence variations if you,
look into the lab animals, they are very, very limited, that is what I was explaining
that they are what is called as isogenic line, meaning you minimize all the variations at
the genetic level, at the environmental level and only you look at changes you bring in,
because of your experimental condition and the phenotype that you see in. Therefore, it is extremely difficult to use,
model animals to identify human genetic, or, or the defective genes for human disorders.
So, what is the approach that was very successfully used to identify a majority of the genes that
cause monogenic disorders? The approach that people used very successfully, thousands of
genes have been identified in the last two decades is called as positional cloning approach,
meaning you look at region of a chromosome that is associated with the disease. Without really bothering about what is a gene,
what is the function, what is the pathophysiology, you only look at a region of chromosome that
is likely to be contributing to the disease phenotype and then look at what are the genes
that are there and then look at what variations there results in the disease condition. So,
that is called as, you know, positional cloning approach and there are two different approaches.
In the post genomic era, where we now know the human genome sequence, the approach now
you use is somewhat different as compared to the approaches that were used in the, when
we were not knowing the human genome sequence. So, we will talk about, the approaches that
were used before the human genome sequence was understood and then how that is really
not necessary now, once you know the human genome sequence, so that is what we are going
to discuss. So, these are the different steps in identifying
the defective gene using the approach what you called as positional cloning. We call
it as conventional approach, because this is the approach that was used before the human
genome sequence was made available. So, what you do? You define the candidate chromosomal
region, you sort of say that is the region of the chromosome, a particular chromosome
that is causing a particular disease likely and then up time get the genomic segments
representing that chromosome and then look into just just block it here. So, let us look into the conventional ways
to identify the human disease genes. So, what you call as a positional cloning approach,
wherein we identify a region of a chromosome that is associating with a particular disease,
then go on to identify the gene that are located in that region. So, what are the steps? So,
the first step is, you have to identify the candidate chromosomal region that is something
that is shown here, right? So this is called as identify; so, identify the chromosomal
region that is associating with the disease. That is the very first step. Once you identified,
then you have to get, DNA fragments representing that region of the chromosome, you go to the
genomic DNA library and identify all the fragment that represents the region of the chromosome
and then you have to go and next look into genes that are located in that region where
you thought, that defective gene could be present and then based on some features like,
for example whether the gene is expressed in the tissue that is normally affected in
the disease condition. You prioritize your gene list and look for
variations or mutation in the gene and with this approach by repeating one gene after
the other you will be able to identify possibly the gene that causes the disease condition.
So, again you will be looking at some variations and you will be assuming that this is the
variation that causes the disease and eventually what you have to do is you have to validate.
You have to use some animal models or some other systems to validate that the changes
that you have seen indeed, affect the way the cell functions, tissue functions and organism
functions. So this, as you can see is a laborious process. It takes years to identify that defective
gene and to characterize using different model systems. So, first let us look into this particular
aspect that how do you define the candidate chromosomal region, because the rest of them
we have already discussed. We have discussed how you get the genes from cDNA library, we
discussed how you do mutation screening, we discussed how you create animal models for
a given gene. So, let us look into the first two topics that is define the candidate chromosomal
region and then up time the DNA fragment representing that particular region and then the rest of
them you would be able to follow. Let us look into the pedigree that is shown
on the lower side of the, the lower panel. This is an autosomal recessive disorder. You
can see that affected persons parents are not affected and you have consanguinity, there
is a affected person in the previous generation. Because it is autosomal recessive, we know
that the disorder, the gene that is defective should be in the autosome, the point that
we have already discussed and we have excluded X and Y. So, how will you really identify
which chromosome and then which part of that particular chromosome possibly has got the
defective gene. So, let us look in how we people do? This is what we sort of, we are going to look
at some puzzle that would try to sort of help you to understand the concepts. So, what we
are trying to say is that the, right, so what we are going to say is that this particular
individual who is a, you know, individual that are born, that is born to the male and
female, he has got mixture of the DNA that he inherited from his father and mother. So,
you have, 50% of your genome is derived from your father and 50% of the genome is derived
from mother. To represent that we are showing it as a mixture of these two colours, but
it is not necessary that these two are exclusive, I mean is not that chromosome 1 has come from
father and chromosome 2 has come from mother. For every chromosome we have two copies one
that has come from father, one that has come from mother. So, this is what we expect, right?
So, let us look into a puzzle. This puzzle is, you know, you have, you must
have played this kind of puzzles during your childhood days. So, you have a identical piece,
but they differ in the colour. For example, motherÕs puzzle pieces are red in colour,
fatherÕs puzzle pieces are blue in colour. So, what you are expecting is that there is
a mixture of the mother and father DNA and that is what you, you know, in the next generation;
how will you, this is the concept. So, what we are talking about, each puzzle for example
we can say it is a gene and the colour represent, for example the, let us say the dominant allele,
right, for that particular piece, for example this piece here that you have seen, it could
be a colour represent a different allele of a gene, let us say that way, right? Let us go on to look into, so how you have,
you know, you can use this information to identify the gene. This is a kind of a, you
know, a kind of a test, how quickly you can identify a puzzle piece and colour. Let us
see this pedigree and this pedigree obviously represent a dominant disorder, because in
every generation you find individuals are affected, transmitted by either a male or
female and, and both male and female are affected, right? So, how do you really find? So, assuming
that what is shown here is the genome, right and all the, each one of the piece are the
different genes that the genome has, you need to identify which piece of this particular
puzzle could possibly be carrying the gene that is defective and causing the disease So, how do you go about doing it? The correct
puzzle piece must be present in every individual affected with the disorder, the correct puzzle
piece must be absent in every unaffected individual, because you are talking about a dominant disorder
and we are assuming here it is a 100% penetrant, you would expect that if I carry the mutant
allele, I would definitely show the symptoms, the phenotype. So, if I am not showing the
phenotype that means I am not carrying the defective gene. So you have to use this logic to identify
which is the puzzle piece. So, this is the genotype that is given, the genotype given
for each individual that are numbered here. So, 1,2, 3 and so on. So, you have father,
mother that are identified here and then you can look into how the whole thing segregates So, if we can spend time and look at this
slide, probably you will be able to identify which is the puzzle piece. Again you have
to go back and look at that the individual who is affected should have, all the individuals
that are affected in the family must have a particular puzzle piece with particular
colour that is the gene and the mutation therein, because that piece is going to be there in
every individual, because you, all of us have the gene, but only a some of us will have
mutation and therefore you will have the phenotype and all the unaffected individual should not
be carrying that particular colour of that particular piece. So, if you look in this
and you will be able to identify. So I have given the link for this particular site. This
is something that I have downloaded from a popular teaching tool that is available on
the net. So this is, sort of helps you to understand how we can identify the disease
gene. So, if you really go through this link and
you spend time, you will be able to identify that piece number 36 and colour red should
be the one that is causing the disease, right, because this is absent in every individual
that is not having the disease and invariably present in individuals that are having the
disease, this particular piece and red colour. So, you can go and verify by looking into
this site. So that is the approach indeed that we used to identify that, you know, the
disease gene. So, what is the equivalent of that, you know,
the pÕs and the colour in the human genome? These are nothing but the repeat loci. So
you have in, your genome, in large number of the regions are having repeat sequences
and these repeat sequences vary from one individual to the other and we use this as the piece
as well as the colour and that is how we identify the disease gene. Let us look at how we use
this. See, in all our chromosomes we have a large
number of repeats, as I said. So, these are called as microsatellite repeats. They vary
in size and, and how the variation in the population, for example it could have just
a CA repeat, repeated many a times. So, for example, it could be 7 times repeated, 27
times repeated, 8 times repeated, varies in the population. It could be GATA, it could
be GTC, it could be TA and you can see that from this telomere to that top telomere, every
chromosome has got these repeats spread over the chromosome, right and this is what we
use as a marker to track the segregation across generation. So, you can really say which part
your chromosome has come from which grand parents. That means, you know, your fatherÕs
father or your fatherÕs mother who contributed that small piece, although it came through
mother. So, we can really really look at and then decipher that. How do you do this is something that is shown
here. So, you have repeat loci. We are looking at a particular region, particular, let us
for example, you are looking that region, let us say. So, you have variations. In certain
individuals you may have an allele which is 10, , meaning 10 times repeated CA, in some
it is 11, some it is 12, it varies depending on you may be homozygous or heterozygous.
You may, at the maximum, for a given loci, you may have two alleles. So, if you look
into a pedigree that is shown on this side, what you see is that this individual is a
carrier or carries two alleles for this gene carrying 10 and 11, these are the repeats.
So, a particular marker we are talking about, not the gene., This marker has got 10 repeat,
the other homologue has got 11 repeat, this individual as got 12 and 13. Now, we can see
in the next generation that there is a combination, for example from father 10 has come, from
mother 13 has come. Here, the other allele, from father 11 had come, 12 had come from
mother, 10 had come from father, 12 had come from mother. So, you can really track the segregation of
each of the alleles from the parents, right and this is vary, although it is very similar
to the mutation that you have seen. So, we have looked at point mutation and we have
looked at how it segregates in the population, you know in a family. The difference is for
a region of a gene, where your particular base that is mutated, at the maximum you may
have, four different alleles that is possible, one of the four bases. So, but more often
you have only two alleles, the wild type and then mutant. You do not see many more, but
when you are looking at microsatellite markers like what is shown here, the CA repeat for
example, there may be 20 or 30 or 40 different alleles in the population. So, that makes
it much more powerful to identify which region has come from mother and father. For example, if you have only two alleles
like what you see here GG and mother is TG, heterozygous and you have GG. Now, you have
many ways to tell which G had come from which parent, whether it is from father or it is
from mother, we cannot say. Likewise for this individual, so it becomes extremely difficult.
That is where the microsatellite markers are powerful, because they have a large number
and the, the probability that you would have same allele in both the parents is less as
compared to this kind of mutant allele that you see, where one base is changed with the
other. So, how do you really type them, how do you see this segregation? So, this is something that I have shown you
that you have the repeats. In a repeats that you see here, it is CA repeat. What is shown
on the top strand, this is a complimentary strand and you have, you call this allele
as CA5, because it has got 5 times repeated, you have another allele that is 7 times repeated.
So, how do you really type? Therefore, I can tell this is the one that had come here. So,
how do you do that, you know, typing? If we do it by the conventional PCR, that is called
as polymerase chain reaction, routine PCR, wherein we design two primers, one is forward
and the reverse. Now, the primers as you see, have come from the flanking sequence. You
have the repeat here, but this is what you call as the flanking sequence. These are identical
whether it is 5 repeat or whether it has got 7 repeat, the flanking sequence are identical.
Therefore, I can use the same set of primer to amplify any number of alleles, it becomes
very very easy; that is one particular point. So, let us look into how you type the allele,
which allele is the one that you are, you know, looking at? So, when you do a PCR, you
will be able to see a difference for each allele, the reason being your primers are
coming from the flanking sequence, unique sequence. So, you can calculate what is the
size of your amplified product and then you will be able to tell which allele. For example
the two primers that you see the two primers have a definite length that is 9 bases. Therefore,
when you add them, it becomes 18 bases and then you have the repeats. The repeats are
dinucleotide repeats. You have two nucleotide unit that is repeating CA, CA and so on. So,
how many times it is repeated that would tell you what is the length. For example, if you
have 5 repeat, then it is going to be 10, so plus if you add 10, plus 18, which is this
sequence of the primer, now you are going further 5 allele it is going to be 28 bases;
for 7 allele for example, it is going to be 32 bases. By looking at the size difference in a gel
and calculating what is the size of that particular fragment, we will be able to tell what is
the allele that you are looking at. So, this is the approach people use and when you, obviously
when you run these products in a gel you are going to find distinct fragment, DNA fragments
you can expect the size and then calculate. So here is a is a pedigree which shows a autosomal
dominant disorder. We can see that individuals in every generation is affected and what you
have done is, you have looked at a marker, a marker by PCR and what is shown here is
the, how it migrated in a gel. So, each of the number that you are showing, either below
an individual or in the gel represent the particular allele; 25 is an allele, 22 is
an allele and so on, so forth. So, you are looking at an autosomal marker. Therefore,
individual would be having two copies that is what you are seeing here. This individual
in the first generation is having 22 and 25 as you can see here and she has got 27 and
35, again that is shown here. Likewise, you show for every individual what is the genotype
that you see. So, basically you look at the gel and then you derive what is the allele
that is got. Now, you can clearly map it. For example, this 35 has come from this individual
and this 22 has come from this individual and so on, you can map. Now, how do they really help you to identify
the gene? So, you can see there are certain allele markers, now that are highlighted and
these are the markers that are invariably present in the affected individual. If you
can go back and connect with the puzzle that you have seen, so you have looked at a piece
and you have looked at a colour. So, what you have looked at that particular piece having
a particular colour should invariably present in the affected that means that is the piece
that possible is likely to have the defective gene. So, that is exactly the same thing we
are doing. We have used markers and you are looking at
which allele of a given marker is invariably present in the affected and whether it is
invariably absent in the unaffected, assuming 100% penetrance. You can see here this 35
marker is present in all the affected individuals and it is not present in any of the unaffected
individual, suggesting this marker could possibly present close to a gene which is defective,
because this marker itself is not causing the disease, because these are repeats which
are not part of any genome, any gene. So, they are present randomly and they may not
have any functional significance. That is why you see a large number of variation in
the population, but they may be present close to a gene that is what shown here. For example, this is a marker that let us
say that has got number 35, right? The allele that causes the disease and close to this
marker in the chromosome very near by you have a gene that causes a disease which you
call as HD here and because they are present close to each other, now they are going to
go together. They will be not be separated by a recombination; they will not be separated
by a recombination. Therefore, whenever you see the individual is affected, you will have
that marker, like you can see affected and the marker is present. In those individual
who are not affected, this particular allele of a given marker is not present. So, that
is what you do. You basically look at a given marker, an allele of it that is co-segregating
that is present in all the affected and not present in the unaffected, if the penetrance
that you are talking about is 100%. So, this is the way you go about and identify
a small chromosomal region that could possibly be contributing to the disease. So, how do you really calculate? Because you
are going to look at thousands of markers to begin with, because you are looking at
all the chromosome, when we talk about autosome, you are having 22 chromosomes, each chromosome
you may have 100 markers you are going to look into a large number of markers. So, you
need, you know, here we are doing it with a some statistics and then try to sort of
guess whether this is the region that could possibly harbour the gene. So, that is called
as a likelihood, the possibility. So, the likelihood that a marker will be inherited
together with the affected gene is measured as a log of odds ratio. So, this is a statistical
approach people use that you find a marker co-segregating with a phenotype, because it
is present in close proximity to a gene that is defective and is not happening by chance.
It is not random, you know, probability, right? It happens likely, because it is linked, closely
linked to that particular affected gene. So, that is what you calculate by using a statistical
method and that is what depicted as LOD score, LOD that normally we call. So, if the LOD
score is higher, then it is likely that the gene is present very close to that marker.
If the LOD score is very low for a particular marker in your analysis, then it is not likely
that the gene is present close to that region. So, this is how you come up with, for example
what is shown here is, there are, each square here is a marker that was tested for the so
called linkage analysis. That is what shown here that you have done a pedigree and done
the genotyping for all the individuals in that pedigree and then you are calculating
the LOD score and you find that a small region of the chromosome you have all the marker
showing very high LOD score, like that what you see here. That suggest likely that the
gene is present somewhere in this particular region, say for example it is present here.
So, from all the 22 chromosomes we have come to a small region of a particular chromosome
by looking at markers. They themselves are not genes, but because they are present somewhere
close to a gene that is defective they would co-segregate with a particle phenotype. So,
that is how you are able to narrow down the gene. So, that’s how you narrow down a region,
which possibly could have the gene. So let us see how from that segment, that small segments
that you identified by this linkage analysis, how do you now go and find the gene? So, what
is the second step? The second step is that the region that you
identified narrowed down what you call here you need to look at large number of markers
in that. So, that is what the next step is. Now, you do not really worry about the rest
of the chromosomes or even the rest of the regions of this particular chromosome. You
know this segment you go and look at large number of markers. If markers not available
you generate markers and then look at again, analysize, you know, you do this, you know,
genotyping and then you narrow down further. We will talk about how do you narrow down
and then finallyonce you have done that this is the smallest region that I can define,
then, you go about looking at what are the genes that are present there. Say, suppose
you found only one gene, then go about checking for mutations in the gene and if that is having
a mutation, then you have found the gene. So, this is the step that you go about doing
it. That is what shown here in this cartoon as
well. So, you start with just a recap, because you are getting into a problem that you, I
would help you to solve and later you may have some assignments given on that. But,
what we have done is that we have used a pedigree approach to identify whether it is a autosomal
or sex linked or X or Y or dominant, recessive. Then, you have done the genetic mapping, you
have used large number of markers and then you are able to say this region of the chromosome,
possibly the gene is located and then you go about characterising that region of the
chromosome to find the genes, right? What is shown here and then look at whether a given
gene is having a mutation something like what you have seen. So, in a patient if there is
a mutation resulting in stop codon, then you know that you have gotten the gene that you
are looking for. So, in this process what you have done is
you really did not bother about what is the gene, what is the function, whether it is
expressed, what kind of protein it codes, you did not bother about that. You looked
at what is a chromosome that is linked to the disease, what are the genes that are present
and then, whether any of the gene carries any mutation that could be causing the disease.
So this is an approach based on the position of the gene. Therefore, it is called as positional
cloning, because more often this led to a discovery of a new gene, more often that is
the case and therefore, it is called as cloning, because you identified a new gene. So, that
is why it is called as positional cloning. So, let us see, you know, in this, in this
particular slide what I am saying is that if you have identified and narrow down a small
segment of chromosome you need to look at the genomic region. So, now you have to carefully
look at that genomic region, what are the genes that are present there, right? For that
you need to go to the library, what you call as genomic library and get fragments that
represent that region of the chromosome, arrange them and then, you know, characterize that
region as to what are the genes that are present. How do you really do this? This process is
called as physical mapping. What do you do in this case is there for example, this is
the region you narrowed down, you have markers and then you go and use this marker to screen
the genomic library and then once you screen the genomic library you would get a DNA fragment
from a library that represent somewhere here, you know, a fragment of that particular chromosome. Now you use this genomic DNA fragment that
you got from the library to get the clones, different fragments that overlap with each
other and representing the region which could possibly harbour the gene. So you have to,
you have to take pieces of the DNA that are there in your genomic library, stitch them
together to get the DNA representing that region of the chromosome. So, this is called
as physical mapping. Sometimes it is called as chromosome walking, because it is a very
slow process; you get one, one step, get the other clone that is the second step and then
the third clone that is third step. So, you slowly move on either side to get all the
clones that represent that region. The question is why should you do this? Because,
when you do this kind of exercise, you would have a fragment representing a part of the
chromosome and that results in the identification of another piece, both of which may share
some region. That is the way you are able to say, this follows this. So, this overlap
is very very critical. If you can recall our discussion on how do you create genomic library,
we said that we do either a partial digestion wherein all the sides are not cut or we use
two different enzymes to create fragments that overlap with each other. That is the
way you will be able to connect one with the other. That is how they are done earlier.
So in each, you know, junction, you can find there are small segments that overlap with
each other and you are able to create what is called as overlapping clones, which in
colour in a, in a term in genomics called as contig. So, how do you really use this contig? So,
what is the purpose of the contig? One of the reasons is that the microsatellite markers
people used them to screen for chromosomal regions. We know that these markers are coming
from this region of the chromosome, but the exact physical order, order of these markers
were not known. The reason being the approach that they used for localising a given marker
is very different, because then you do not, you know, that time even the fragments were
not identified like the way now we are talking about that there is a physical, you know,
mapping or chromosome walking. Only in this process you will be able to arrange the markers
in the physical order. We know that the markers are present here, but exact order of the markers
are not very, very clear and they are very, very essential for mapping studies, like we
are going to see now. How do you really arrange the markers? So,
basically what you do is, so you have markers which are nothing, but, you know, the region
of the chromosome that you can amplify by PCR. So, once you have got, for example, clones,
genomic clones either from yeast artificial chromosome or bacterial artificial chromosome
or of phage library, these are the fragments. Now, what you do is, you, each DNA fragment
you isolate and then do a PCR and test whether a given marker, you know, is located on this
piece. If it amplifies that segment that means it is present there. If it does not amplify
that marker is not present. So, as you can see here, you have, different primer pairs,
like for example 1, 2, 3, 4, 5, 6 and so on and what you have done for each primer pair,
you have used the DNA either from the clone A, B or C and then, you have seen that for
some there is a product, for some there is no product. Based on this information, you are able to
place it for example PCR primer 1, you know, that amplified only from this particular clone
that is clone A. That means it is located only in clone A, not in any other. On the
other hand, some of them are, some of the fragments, some of the PCR products were,
you are able to amplify from more than one. So, this information helps you to say the
5 and 3 probably are present in a region that overlap between A and C. Likewise, primer
4, you know, is located in a region that overlaps between C and B, whereas 2 is unique to B,
6 is unique to B and likewise 1 is unique to B. So, this is how you are able to position
in their relative order. Still you are unable to solve this 5 and 3, which is the order,
but if you have had some other clone, probably you will be able to still place them. So, this is how you position them and once
you are done, you are going to look at something like this, which is called as a contig map.
So, all that are shown on the top are markers and each of this line that you see here are
the different clones of, for example bacterial artificial chromosome. You can see that these
markers are present and these markers are present and so on and you are able to position
this is the order of the marker, right? It starts from 11, ends with 30 and so on. So,
this is what called as contig. So to just to summarize what we have discussed,
you have looked at a region that you have mapped on to your chromosome likely to have
a gene and you want to identify the gene. How do you go about doing it? So, you have
done what is called as genomic library screening, identified several overlapping contigs, clones
and you have tested the markers that you have used for the physical order. So, what you
have done? You have done PCR using each one of the clones that you have got and you are
able to, using this information you are able to say, this is the order of all the markers.
This is the correct order. So, you know, all the markers are located within this segment,
but you are not sure about the order. Now, you are able to get the order, right? This
is the physical order. So, how this particular physical order helps you to identify the gene? This is a real life situation. This, you know,
pedigree that is from one of the papers, publications describing an autosomal dominant disorder,
right? You can see here there are three markers. You can see that D22S117, these three are
the markers and whatever number that you see here are the allele numbers. For example,
this is number 1, this is 2 and this is 3 and these are the three markers. In every
individual it is then typed and what you can see here is that there is a particular order
of the markers. For example, you have an individual here who is affected and this individual has
got 2 1 3, you know, 2 allele for the first marker, 1 allele for the second marker, 3
allele for the third marker and this combination 2 1 3, because these are in the physical order
that is also represented by, you know, by the black bar there, is invariably present
in every individual that are affected, right? So, that is, you know, what you are able to
get. By now, you are able to clearly look at a combination of alleles that are located
to each other what you call as haplotype, you know, the physical order of certain alleles
as a block not separated by a recombination is called as haplotype and this haplotype,
you can see that goes from this generation to this generation and likewise, you know,
from this generation to here and from here to here and so on. So, this you are able to
track. Suppose your order was not accurate, you do not know the order, you may miss out
this particular information. There were marker somewhere in between these two, you would
assume this is not the haplotype. So, for you to identify a haplotype, the physical
order is extremely important. Therefore, it is not simply the information as to where
is this allele or marker is located, but the physical order of the markers are very, very
important in successfully identifying the regions that could have the disease, right? Now, let us see how the haplotype helps us
to identify the gene. This is a schematic again to show how do you use these markers
to identify the disease gene. Now, you are using a autosomal recessive condition. You
have a male, female and we have shown, each box here represent the different alleles,
right, of, you know, given marker and here is a gene. This is the gene that you are seeing
in between and here is an individual who is heterozygous having a muted allele and she
marries a normal individual and she is a carrier and then, they have got two sons, they marry,
you know, of course and then in the next generation, his, you know, this particular parentÕs grand
son and grand daughter they marry together, right? It is common, happens in many societies
and as a result, this individual who was a carrier for the disease linked allele mutation,
now she is able to give this allele to both her son and daughter who are heterozygous,
asymptomatic normal. But now, when there is a marriage between
these two individuals, what you see is that, you know, these two alleles could come together
in the next generation. When it happens, you become, you know, a homozygous for the mutation,
therefore you develop symptoms. Now, you can identify this region, because it remains homozygous,
by looking at the markers that are flanking here. You see that these are like the puzzle
piece. We have given a different colour. The allele green is one allele, the blue is another
allele, red is another allele and you can see that in every generation there, at times
there are recombination but you can see around the gene you invariably have the green allele,
meaning they are not separated by, you know, the recombination. So, if you are able to look at the allele,
which allele, you know, segregates in this phenotype, in this family and whether there
is a homozygosity for a given allele, invariably every affected is homozygous for that particular
allele. That would give you a clue that a gene could be located here and that is what
called as homozygous mapping, when you look at a recessive disorders, because it is normally
as is shown here, it is a same defective allele, when it comes in the homozygous condition
you end up having the disease, because most, most often these are consanguineous marriages,
marriages within related and that is a result you see here and that is again shown here
with a real life situation for one of the disorders. Let us not worry about what disease it is.
These are the markers, right and then what you are showing is that there is a homozygous
region. This is a large family. You can see there are four affected and there are consanguinity,
recessive disease and you see a region that is shown with a box and you will find homozygosity,
right? These are affected and this 1 1, 2 2, 1 1, 2 2, 1 1. Again, see homozygosity
2 2, 1 1. So, you can see that 211 is present invariably in homozygous condition. That means
your gene should be located somewhere within this segment. So, this is how you arrive at
the gene. So, that is what you call as homozygous mapping. 1: So we are going to look at one such problem.
So, I help you with, you know, solving this puzzle and then we may give of couple of examples
for you to work out on your own. So, let us find a disease, we name it as XY disease,
nothing to do with X and Y chromosome, some name and this is the family. It is clear from
here that is a recessive disease, because you have an affected, you have consanguinity,
affected parents are not affected and of course, there are relatives who are affected here,
right and we have already done the whole genome, large number of markers and we are able to
narrow down a region of a chromosome which has got a gene for this particular disease,
right? What we need to do? We need to narrow down the region, we need to identify more
markers, you know, make them in physical order and look at the homozygosity, as to, because
this is a recessive disease. That is what you are going to do. So, these are the markers,
right? 1: The markers are A G S E N H, right? Some names
that that you need not really worry about, right now and these are the markers and what
you do? 1: You have done genotyping. For every marker,
you have done a PCR and you are able to identify the alleles and that is what shown here. You
can see that quickly. There is no large extended homozygosity or any thing. This is a kind
of thing that you see that that you are a, you are getting alleles from both the generations,
going to the next generation and so on. 1: So, what you need to do is that you need to
identify the correct physical order of these markers. So, what do you do? You have to go
for the genomic library and then identify clones that represent this region of the chromosome
and do PCR for each of the clones and then identify the order, right? So, what you have
done? That is what you have done. So, you have done that, you have, so these are the
markers A G S E N H and you have done a screening in the genomic library. You are able to get
five clones, say these are bacterial artificial chromosomes; so, therefore you call as a BACS
B1, B2, B3, B4, B5. Here, when you say ÒminusÓ that means the PCR did not work, meaning that
marker is not present in this clone, whereas S marker and E marker and H marker are present
in B5. So, that is the indication, so plus amplified in PCR did not amplify, right? So,
this is what you use, right? Now, this is the information that you have. Now, based on this you have to arrange the
order of this particular set of markers. How do you do it? So, let us say I am going to
put is that, I am going to say this is B1clone. The B1 clone is positive for, as you can see
here that it is positive for marker A, G and N. B2 is positive for A and N. Therefore,
I would assume it is, G is away from A, it is not in between A and N, therefore it is
away from it. So, let me say that A and N are close to each other. So, I would, you
know, put a line and you would say that here it is, this is A and this is N. Let us look into B2. B2 is again positive
for A and N and I would say this is B2. Now, let us look into A again. So, sorry, let us,
let us look into the B1. B1 is also positive for G and no other marker has got that G marker,
right? So, then I would put it on the extreme left, because they are not present in any
other. So, I would say this is present here. Let us go to B3. B3 is positive for E and
N. So, N is already there. I would say E is here and then B4 that is positive for S and
E. So, E is here, therefore, S should be here and that is like this. This is B, this is
B3, this is B4 and then you have B5, which has got S, E and H. So, what is missing? H;
it has got everything. So, when you order, you are able to get an order which also convey
some meaning. For example, it is my name. So, it is jumbled up when you order, it, it
gives you the, so that sort of tells you what is the physical order of these markers. Now,
why should I do this? How does it really help me? That is what you are going to see. 1: So, you know, by, by doing all these things
I am able to make a contig, which clearly tells me that this is the order of the marker
that is GANESH, right? That is the contig, right, we made it. 1: Now, how do they really help me? So, this
is what the genotyping that I had. For every marker, I have done the amplification. This
is what it is. Now, this was before, the haplotype was before the physical ordering was done.
Now, we know that this is not the correct order. We have changed it, right? So, now
what I have done? I have rearranged everything based on this particular marker, right? Now, what you need to look at? Because it
is a recessive disease, you have to carefully look at the homozygous region. So, if you
look into these two individual that are affected and you will find markers A, N and E, you
know, they remain in homozygous condition. You see that 4 4, 5 5, 4 4, 4 4, 5 5, 4 4
and you do not find that homozygosity in any of the affected individuals, sorry unaffected
individuals. So that, such is that this is a region likely, you know, having the disease
causing the, you know, disorder. This is the region that is likely to have the gene that
causes the disorder and if you look in here, you know, when you have not done the homozygosity
mapping, when you have not done the correct ordering of the markers, they are not really
explaining what is the real scenario, which region is showing the homozygous. That is
why the physical ordering is very, very important. 1: Now, how does it really help you? This is
the contig region, homozygous region that you have seen that is A N E, here. This is
a region that remains homozygous. How does it really help you in identifying the gene?
The way it helps us is by screening for genes. Now, you need to look into what are the genes
that are located here, right? So, I know there are three genes G1, G2 and G3. These are the
three genes. What did I do? I did screening for each of the gene, whether it is, this
gene is located on B1 or B2 or B3, B4, B5 and likewise, it is amplified here, amplified
here, this particular gene amplified in two different BACS. Now, you had used this information
to locate again the gene BAC in the contig and that is what you see here. G1 amplified
only in B1. That means it is present in a region that are not shared by any other BACS.
Likewise, G2 is located only in B5. Therefore, their position is a region that is not shared
by any other BAC clone, whereas G3 amplified by both B2 and B3, but not by any other clone
and this is the BAC and you know, this is the only region, you know, which is not present
in other BACS. 1: Therefore, that is where the G3 is and likely,
because that is also the region where you have homozygosity and therefore, you will
be able to confidently tell and this is likely the gene that could cause the disease and
is involved in a disease that you are looking at and now you go on sequence. If you are
lucky, you can find mutations in the affected and that is how you identify the disease gene. So, that, you know, pretty much ends the first
lecture of week IV and wherein we have looked at the approach people use to identify the
disease gene in monogenic disorders. We have looked at how markers help us, how you use
markers to identify the haplotype, how do you narrow down, for example in case of recessive
disease for a homozygous region and then locate a gene and then identify a novel gene which
may cause a particular disease. So, this is the approach people take to identify the disease
gene and with that we will end the first lecture and in the next lectures we are going to look
into the other aspects like, how the other approach, what are the other approaches because
of the post genomic era, meaning you are, you now know the human genome sequence, how
that has changed the approach. It has become much more easier now. So, that is something
that we are going to look into, in the next lecture.


  • Reply Mahwish Adnan August 15, 2018 at 5:49 pm

    Thank u for ur lecture it helped me alot

  • Reply citizenJonathan December 28, 2018 at 10:35 am

    excellent excellent excellent !

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