Genetics
Personalised medicine comes of age
Diagnosis emerges from complete sequencing of patient’s genes
For the first time, scientists
have diagnosed a genetic
disease by completely
sequencing all of a patient’s
genes. Using high-throughput
DNA sequencing technology,
Howard Hughes Medical
Institute (HHMI) researchers
successfully identified a gene
mutation that was responsible
for the patient’s disease, but
had not been suspected based
on clinical observations.
Starting with DNA from a
blood sample from the patient
– an infant in Turkey who was
persistently dehydrated and
failing to gain weight – the
team found in 10 days a gene
mutation known to affect electrolyte
transport in the intestines
and cause a condition
called congenital chloride diarrhea.
Doctors in Turkey
confirmed the diagnosis clinically
and were able to provide
a treatment tailored to the
disease.
Instead of searching the
patient’s complete 3-billionbasepair
genome for the
disease-causing mutation, the
team, led by HHMI investigator
Richard Lifton at the
Yale School of Medicine,
focused only on the small fraction
of DNA that encodes
proteins. The success demonstrates
that this strategy is a
viable and efficient means of
diagnosing genetic disease.
“I think in the coming years
we’re going see a dramatic
increase in the use of this kind
of technology,” Lifton says.
“This is going to be a very
powerful technology for
disease-gene discovery and
clinical application.”
Lifton and his colleagues
reported their method and diagnosis in an advanced
online publication of the
Proceedings of the National
Academy of Sciences on 19
October 2009.
Molecular diagnostics
“This is the first volley in what
I think is going to be an important
era in molecular diagnostics,”
he notes. “Up to now
we’ve been guessing what
genes might be mutated and
selectively sequencing a
handful of genes to try to find a
mutation. This paper demonstrates
a new capability to
capture, ostensibly, all of the
genes. Instead of sequencing
ten or a hundred or a thousand
genes to try to find a mutation,
we are now capable of
sequencing all of the genes and
making clinical sense of the
resulting data.”
Only about one percent of
the human genome serves as
blueprints for protein production.
Mutations in these
protein-coding regions can alter
a protein’s function or prevent
it from being made at all – often
with significant effects on
health. For this reason, 85% of
mutations known to have major
clinical effects lurk in this small
portion of the genome. The
remaining 99% of the human
genome contains valuable
information – such as when as
where genes should be turned
on – and errors in “non-coding”
regions can also be problematic.
But when searching for an
unknown disease-causing mutation, Lifton and his colleagues
reasoned, it made the most
sense to start with the one
percent of DNA where those
mutations most commonly
occur.
Exome
That protein-coding portion of
the genome is also known as
the “exome”, because segments
of protein-coding DNA are
called exons. For the past six
months, Lifton and Murim
Choi, a postdoctoral fellow in
his lab, have been developing a
method for completely and
accurately sequencing the
exome.
The first step of their
approach is to separate the exome from the vast amounts
of DNA that do not encode
protein. To do this, the team
employs a microarray chip that
has on its surface 180,000 short
pieces of DNA – bits of the
coding-regions of 18,673
human genes. When a sample
of DNA is applied to the chip,
protein-coding regions pair
with their matching segments
and stick. Researchers can
wash away the unstuck DNA,
then shear the coding-regions
away from the chip and
collect them for further
analysis. With the proteincoding
DNA collected, the
next step is to sequence it.
Today’s sequencing technology
makes it possible to do
this rapidly and efficiently,
Lifton says.
Once Lifton and his
colleagues were satisfied that
their method was accurate and
sensitive enough to find mutations
hidden in the 34 million
base pairs that make up a
complete exome, they turned
their attention to the patient
referred to the lab with an
undiagnosed condition.
Doctors suspected a rare
disease called Bartter
syndrome, which affects the
kidney – but wanted Lifton’s
team to determine whether
genetics confirmed their suspicion.
“This was an opportunity
to try our technique in a case
where we didn’t know what
the answer was,” Lifton says.
Using a sample of the
patient’s blood, Lifton and
Choi’s colleague at Yale,
Shrikant Mane, sequenced
the exome. Within a few
hours of receiving the
sequencing data, Choi found
the mutations that caused
congenital chloride diarrhea
and were responsible for the
patient’s symptoms.
“There has been concern
that [this sequencing strategy]
would be so involved and
generate data that would be so
difficult to handle that you
couldn’t do this without a team
of a dozen informatics people.
This is really not the case,” Lifton observes. “This is really
quite straightforward to
perform and interpret.”
Additionally, he notes,
because the cost of DNA
sequencing has come down
dramatically in the last ten
years, exome sequencing is
currently very affordable in a
research setting. Lifton says it
will be important to continue
to drive the cost down so that
the method can see wide clinical
use.
Gene mutations
Outside of clinical use, Lifton
says exome sequencing offers a
means to speed the discovery
of disease genes, particularly
in cases where gene mutations
are not passed on from the
parents. “In genetics now
we’re quite good at finding
mutations where we are able
to pinpoint the location of a
disease-causing gene because
it is present in many family
members, but we haven’t had
a good method to find mutations
that arise anew in an
affected child of unaffected
parents,” he says. “The ability
to sequence all the genes will
enable one to find these new
mutations and link them to
disease. We anticipate this
will be relevant to a range of
diseases such as autism and
congenital heart disease.” 
Date
of upload: 26th Jan 2010
