genetics in its modern sense can be traced to the beginning of the 20th
Century, and to just two key individuals. The
first was the Austrian physician Karl Landsteiner who in 1900 discovered the
ABO blood group system, the first example of a variable human characteristic
that was inherited according to the simple rules of Mendel. The second was the
English physician Sir Archibald Garrod whose work at St Bartholomew’s
Hospital on the inherited disorder Alkaptonuria provided, in 1902, the first
example of a human inherited disease which similarly showed Mendelian
two discoveries really defined the subsequent two great pathways of research
in human genetics that continue to this day. The
first is the study of normal genetic variation in humans, initially through
blood group variation and subsequently in other classes of protein using
biochemical techniques. These studies
provide fundamental information on the genetic variability of the human
species and on the diversification of mankind.
The second great theme has been understanding how inherited defects in
our chromosomes can cause inherited disease. Both
approaches were however severely limited by the lack of large numbers of
genetic markers, and only fully blossomed in the wake of the DNA revolution.
THE RISE OF GENOMICS
announcement by Watson and Crick in 1953 of the structure of DNA signalled the
beginning of a molecular revolution that is still with us.
Recombinant DNA technology, first developed in 1973, allowed complex
genomes to be manipulated, dissected and sequenced.
The first human gene was isolated in 1977 and today we now have the
final draft of the entire human genome sequence covering most of the
3,000,000,000 base pairs that constitute the human book of life.
new science of genomics, exploring gene and chromosome organisation, however,
tells us nothing about genetics, defined as the study of inherited variation.
Genomic technologies have proved critical in exploring variation between
individuals in our precise DNA sequences and it is this theme that has
fascinated me ever since I first moved to Leicester in 1977.
INHERITED VARIATION IN HUMAN DNA
in 1977, the only tools we had available for exploring human DNA variability
were probes which enabled us to detect, by nucleic acid hybridisation,
specific regions of DNA in human chromosomes, and restriction enzymes that
allowed us to cut DNA at predetermined sites.
combining these two approaches, we showed that humans do indeed show variation
from person to person in the way that restriction enzymes cut their DNA and
that these variations in the genetic material were inherited in a simple
so-called restriction fragment length polymorphisms allowed us to estimate for
the first time just how variable human DNA was from person to person; the
answer, at the molecular level, was not very great, with perhaps one base pair
in every 300 showing polymorphic variation between people.
However, at the level of the whole genome, the variation was immense
with perhaps 10,000,000 different positions in our chromosomes showing
problem of limited numbers of genetic markers was therefore, in principle at
least, solved. The drawback was that
these single nucleotide polymorphisms were at the time rather difficult to
detect and assay, and were in any event genetically not very informative.
therefore started in the early 1980s a quest for far more variable regions in
human DNA that could provide highly informative markers for the mapping of
by accident, we stumbled across one of the first examples of a human
minisatellite, a region of DNA consisting of 30 or so base pairs repeated over
and over again for tens or hundreds of times. Not
only were some of these minisatellites extraordinarily variable between
people, with variation arising through inherited differences in the number of
repeats, but they also seemed to share a similar DNA sequence.
therefore reasoned that it should be possible to detect, by nucleic acid
hybridisation, many minisatellites simultaneously by making use of this shared
sequence motif. This approach worked
remarkably well, not only displaying large numbers of minisatellites, but also
providing for the first time a truly individual-specific pattern of human DNA.
Thus was DNA fingerprinting born.
FORENSIC DNA – AN UNEXPECTED DIVERSION
was blindingly obvious that DNA fingerprinting not only had generated large
numbers of highly informative genetic markers for medical genetic research,
but also had accidentally solved another major problem in human genetics,
namely the issue of biological identification and the establishment of family
relationships in forensic and legal medicine.
months of developing the first DNA fingerprint, we had applied this approach
to resolving an immigration case and shortly afterwards a paternity dispute,
the first cases of their kind ever to be tackled using molecular genetics.
Using both DNA fingerprinting and the more refined DNA profiling
technology, we carried out our first forensic investigation in 1986 in the
Enderby murder case. It was this case
that triggered the application of molecular genetics to criminal
investigations worldwide, with DNA profiling remaining the standard technology
well into the 1990s.
next revolution came with the development of the polymerase chain reaction (PCR)
invented by Kary Mullis, Henry Erlich and others.
This exquisitely elegant and sensitive technology enabled minute
amounts of DNA to be copied over and over again in the test tube, allowing
traces of biological evidence found for example at the scene of crime to be
amplified and typed. It also allowed
the development of highly variable markers much shorter than minisatellites
that could be easily amplified and typed.
used these short markers, called microsatellites, in 1989 in an investigation
of skeletal remains exhumed from a gravesite in Brazil and believed to be
those of Dr Josef Mengele, the notorious Auschwitz concentration camp doctor.
By comparing bone DNA profiles with living relatives of Mengele, it was
possible to establish that the remains were indeed those of the war criminal,
allowing a major investigation that had proceeded for decades to be brought to
microsatellite typing has been greatly refined and largely automated, allowing
in 1995 the development in Britain of the first national criminal DNA
intelligence database which currently holds over 2,000,000 DNA profiles,
mainly from convicted criminals but also from unsolved casework.
This database is proving to be extraordinarily effective at identifying
repeat offenders and in establishing links between different unsolved crimes.
Indeed, it has been so successful that momentum is now gathering for the possible databasing of the entire UK population. Such a global database would unquestionably prove to be an enormously powerful tool in the fight against crime, but does raise major social, legal and ethical issues that have yet to be adequately addressed.
THE ORIGINS OF DNA VARIATION
science of DNA forensics having been largely solved, we turned our attention
to more fundamental issues of human DNA variation, in particular trying to
understand the mechanisms which result in heritable changes in our DNA.
are two key processes at work here. The
first is mutation whereby spontaneous, or possibly environmentally-induced,
alterations in our DNA become established and transmitted to subsequent
generations; unfortunately, mutation occurs over most of our DNA at a
frequency so low as to be undetectable in human families.
second process is recombination or crossing over, whereby maternal and
paternal (homologous) chromosomes pair up at meiosis and exchange genetic
material before being separated into individual sperm or eggs.
Recombination is fundamentally important in reshuffling patterns of
variation in human chromosomes, greatly increasing the genetic diversity of
mankind and exposing novel combinations of genetic markers to the forces of
our knowledge of processes of recombination in human chromosomes is severely
limited by the very low rate, at the molecular level, at which this process
occurs. Our challenge therefore has
been to develop new approaches to the very high resolution analysis of
mutation and recombination in an attempt to study the evolution of human DNA
in real time.
have proved to be a superb system for developing these technologies.
These regions of DNA are not only extremely variable but can also
mutate (change their number of repeats) at a phenomenal rate, with in some
cases as high as 1 in 10 sperm or eggs carrying a new mutation, compared with
perhaps 1 in 1,000,000 for mutation in conventional genes.
Thus it is possible to see minisatellites mutating in families.
better, it is possible to use PCR to detect new mutations directly in sperm
DNA, enabling for the first time unlimited numbers of germline mutants to be
recovered from any appropriate man. In
addition, we can analyse the internal structures of
minisatellites in great detail, and by comparing the structures before
and after mutation, we have shown, rather surprisingly, that minisatellite
mutation is in fact driven by the process of crossing over at meiosis; this
shows that for this class of DNA at least,
mutation and recombination are not different and distinct processes.
most cases minisatellites mutate by the copies (alleles) on homologous
chromosomes pairing, followed by information being copied and transferred from
one allele into the other, resulting in a recombinant mutant but without
crossover as such. However, using new very powerful single sperm techniques it
has been possible to study true crossovers at very high resolution.
has shown that minisatellites are located at the boundaries of intense
crossover hotspots, with current evidence suggesting that it is these hotspots
that drive instability at minisatellites, and not vice versa.
This in turn suggests that minisatellites are hotspot parasites that
have succeeded in engaging the recombination machinery to propagate
discovery of crossover hotspots near minisatellites raises major issues about
the distribution of crossover events along human chromosomes, long thought to
be fairly random. To see whether these
hotspots are the rules rather than the exceptions in human chromosomes, we
have recently commenced an analysis of a region within the Major
Histocompatibility Complex, a giant complex of genes intimately involved in
the function of the immune system.
region has long been studied in families, in particular in relation to tissue
matching in transplantation surgery, and these family studies have provided
provisional evidence that crossovers may be significantly non-randomly
distributed in this region of the human genome.
We have therefore used very high-resolution sperm analysis to begin to
dissect the molecular distribution of crossovers in this region.
the evidence to date suggests that the highly localised hotspots associated
with minisatellites are not the exception in the human genome, but are instead
the rule. This in turn implies
that crossovers in human chromosomes are tightly regulated, resulting in heavy
clustering into highly localised regions of intense activity.
are now extending these findings to other parts of the human genome, as well
as trying to understand the rules that govern why hotspots exist and how they
are distributed in human chromosomes. This
is not just an academic quest; the clustering of crossovers into hotspots will
profoundly influence the way in which variation is distributed along human
chromosomes, with major implications for the search for genes involved in
human inherited disease.
has long been known that environmental agents such as ionising radiation can
induce inherited mutation in the DNA of experimental organisms.
Surprisingly, there is no evidence for such a phenomenon in human
populations, in for example individuals exposed to radiation following the
Hiroshima and Nagasaki atom bombs.
problem lies in the very low mutation rates associated with traditional
germline monitoring systems, which require the monitoring of hundreds of
thousands, or indeed millions, of families to be able to detect induced
inherited mutation. My colleague Dr
Yuri Dubrova and I have therefore used highly unstable minisatellites as a
novel approach to mutation monitoring, in particular to see whether they
respond to environmental agents such as ionising radiation.
experiments are of course very difficult to perform in humans.
We have therefore focused our attention on populations accidentally
exposed to radioactivity. In a major
study on the Chernobyl disaster, we have shown that populations in Belarus
living in contaminated regions show an unusually high mutation rate in their
children, which appears to correlate with the degree of radioactive fallout in
provides the first direct evidence for radiation-induced inherited mutation in
humans, but must be treated with considerable caution since there may be other
confounding environmental factors responsible for shifting mutation rates.
Dr Dubrova has observed a similar phenomenon in populations exposed to
radioactive fallout in the vicinity of the Soviet nuclear weapons testing
facility at Semipalatinsk in the former Soviet Union of Kazakhstan.
Two studies on the Chernobyl liquidators, the men sent in to clean up
the power station following the disaster, have produced conflicting results,
one showing evidence for radiation-induced mutants appearing in their
offspring and the other showing no effect.
minisatellite mutation studies on Japanese atom bomb survivors and their
families have shown no evidence for radiation-induced mutation, nor is there
any evidence for radiation inducing sperm mutation in men undergoing lower
abdominal radiotherapy. Clearly, much
more work is needed to see whether human minisatellites do respond to
radiation in the remarkable fashion seen for repeat DNA in mice.
If radiation-induced mutation in humans is verified, then it clearly
has major implications both for understanding the interaction between
radioactivity and the germline and for radiobiological protection.
the human genome sequence largely completed, human genetics is poised to move
rapidly in two major directions.
functional genomics, namely an attempt to understand the functions of the tens
of thousands of genes present in our chromosomes.
The second challenge is to gain a far more global picture of diversity
in the human genome and to use this to understand our evolutionary origins and
to begin to dissect the many and varied processes of mutation, recombination
and natural selection that drive variability into human populations.
the latter respect, we have made our first few steps using minisatellites as
model systems. The challenge for the
future is to extend this work to other classes of rarer but potentially more
biologically relevant inherited mutation.
Finally, I would like to thank all my colleagues both in my research group and in the Department of Genetics at the University of Leicester, together with many other friends both within and outside the University, who have contributed so much to all our research efforts.
Last updated: 18 June 2003 10:55
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