Download Direct DNA Sequencing in the Clinical Laboratory

CLIN. CHEM. 41/10, 1407-1409 (1995)
Direct DNA Sequencing in the Clinical Laboratory
The article by Berg et al. (1) in this issue touches on
of issues investigators
are currently
dealing
with in seeking
robust application
of direct DNA sequencing to clinical specimens.
These include automation, optimal
methods
to obtain
high-quality
data,
efficient
multiplex
amplification
strategies,
quality
control, and procedures
to deal with very small samples. Not discussed
but inherent
in cost-effective
application of sequencing
technology
are efficient means for
handling
the primary
sequence
data generated.
Direct DNA sequencing
will be a routine procedure
in specialized
clinical laboratories
within 10 years. The
pace of discovery for disease-causing
genes and underlying mutations
is accelerating,
and a number of autosomal genes, such as major ones associated
with familial breast cancer (BRCA1, BRCA2,
and possibly ATM),
cumulatively
affect large numbers
of individuals
in the
general
population
(2-4). Significant
challenges
confront the transition
of DNA sequencing
from the research to clinical laboratory,
and this editorial
focuses
on background
and general issues in this area.
Single nucleotide
substitutions
leading to amino acid
changes (missense
mutations),
translation
stop signals
(nonsense
mutations),
or aberrant
exonhintron
splicing
are responsible
for most disease-causing
alterations
of
genes. Whereas
the latter
two types of change
are
usually
pathogenic,
single amino acid changes
range
from a host of innocent
polymorphisms
without
functional effect on the gene product to devastating
instability as wrought by the glutamic acid-to-valine
change
between
hemoglobins
A and S. In disorders
in which
one or relatively
few known mutations
cause disease,
forward and reverse dot-blot hybridization,
allele-specific amplification,
amplification
with restriction
endonuclease
digestion,
or heteroduplex
analyses
are used
as relatively
simple and rapid methods
to look for
specific changes (5, 6). Otherwise,
screening
for single
nucleotide
changes and less frequent
frameshift
losses
or gains of one to a few nucleotides
is most easily
accomplished
by direct DNA sequencing.
Sequencing
generally
begins with polymerase
chain
reaction
(PCR) amplification
of a patient’s
DNA directed at gene regions of interest,
including exonhintron
boundaries,
followed by sequencing
reactions
on the
PCR products.
RNA may also be used as starting
material
in some circumstances.
Attractive
targets for
direct sequencing
include genes such as the 13chain of
class II histocompatibility
molecules,
in which numerous nucleotide
changes occur in several hundred nudeotides of exon 2 (7). A larger region encompassed
by
one or perhaps
two exons, such as connexin
32 in
a range
X-linked Charcot-Marie--Tooth
disease (8), is also attractive because much information
is obtained
from a
few sequencing
reactions.
Next in order of appeal are
genes with clinical significance,
such as p53, in which
many mutations
occur in a relatively
defined area of
the gene. The large number
of mutations
eliminates
practical
forward or reverse hybridization
approaches
to detection.
DNA sequencing
may require PCR amplification
of several
exons. There is more work and
modest additional
expense associated
with sequencing
such genes, but multiplex
amplification
schemes such
as that described by Berg et al. (1 ) frequently
allow this
to be done economically.
Least attractive
in terms of effort are relatively
large
genes in which disease-causing
mutations
are spread
throughout
the gene, usually
over many exons. Sequencers usually approach candidate
samples for these
mutations
first by screening
for nucleotide
substitutions by a method such as single-strand
conformation
polymorphism
analysis or denaturing
gradient
gel electrophoresis.
Protein-truncation
approaches
are becoming more popular to screen for nonsense
and frameshift
mutations
(9). Here, cDNA for the gene of interest
is
generated
from sample mRNA PCR-amplified
for different areas of the gene and translated
in vitro into
peptides;
peptides
of sizes less than expected indicate
the region and approximate
location within the gene of
a mutation.
Despite
their greater
complexity,
these
more-complex
genes are likely to be a major group
analyzed
in the future,
as many important
diseases
will fall into this category,
including
familial
breast
cancer (10), neurofibromatosis
type 1 (11), Duchenne/
Becker muscular
dystrophy
(12), and hereditary
nonpolyposis colon cancer (HNPCC) (13).
With good sequence
data-and
methods
must be
adequate
to detect heterozygotes-sample
interpretation is generally
straightforward
for nonsense or splicesite mutations.
Missense
nucleotide
changes
in genes
in which many mutations
may occur are another
matter. Are these polymorphisms
or disease-causing
mutations?
Rules based on conservative
vs nonconservative amino acid changes
will almost
certainly
need
clinicopathologic
confirmation
for individual
disorders.
For an inherited
disease in a large pedigree,
it may be
possible to examine whether
a putative
mutation
segregates as expected to affected and unaffected
individuals, although this may raise difficult individual
issues
of privacy, consent, and family loyalty if analysis
constitutes
presymptomatic
testing.
For spontaneous
or
acquired
mutations,
as in neoplasms,
the significance
CLINICAL
CHEMISTRY,
Vol. 41,
No.
10, 1995
1407
of missense
mutations
will be particularly
problematic
until we have a larger database
of information.
It is essential
for clinical laboratories
that sequencing methods
be efficient
and robust, with few or no
repeat analyses
required,
since considerable
time goes
into sample handling,
sequencing
gels, and data analysis with both standard
and automated
sequencing.
That is the utility of studies such as that of Berg et al.
(1), involving
a-thio-dNTPs
or other methods
as general strategies
or to approach
difficult-to-sequence
areas. Improvements
in thermal
cycler-based
sequencing, which improve accuracy and eliminate
the need for
strand
separation,
are also being studied.
The prospects for non-gel-based
DNA sequencing
by hybridization to oligonucleotide
arrays on silicon chips (14) is
particularly
appealing
because of the potential
to design disease-specific
sequencing
cassettes
and the elimination of time-consuming
sequencing
gels. The ability
to apply direct sequencing
to a variety of sample types,
including small areas of tumor samples (15) or archival
paraffin tissue blocks using multiplex
approaches
such
as those described
by Berg et al. (1), is also an important feature of robust clinical methods.
The impetus of
the Human
Genome
Project has also spurred
many
technical
improvements
in DNA sequencing
(16) for
research,
including
the development
of high throughput, automated
DNA sequencing
protocols, instrumentation, and interfaced
data analysis
programs,
which
may eventually
find applicability
in clinical laboratories. The ability
to effectively
manage
the large
amounts
of raw information
produced
by direct sequencing is crucial to the efficient adaptation
of direct
sequencing
to the clinical
laboratory.
One program
currently
being evaluated
for HLA allele assignment
clinically
shows promise
but may require
significant
time for manual checking (17).
Given the implications
for patient
management
along with direct repercussions
on individual
lifestyle
and long-term
and reproductive
planning,
direct sequencing
must prove itself a reliable technique.
Current studies in histocompatibility
analysis and familial
cancer mutations
will provide important
benchmarks
on the promise and possible pitfalls of real-time
direct
DNA sequencing
for clinical analysis.
Laboratory
protocols will clearly need to incorporate
rigorous qualitycontrol
stratagems,
perhaps
sequencing
both DNA
strands as for Berg’s p53 studies (1), as well as possible
sequencing
of the same region with different
primer
sets or even cross-confirming
methodologies.
Concurrent sequencing
of identity genes such as HLA alleles
(1) is an interesting
idea to monitor possible sample
contamination
or carryover,
particularly
in laboratories in which large numbers
of cancer specimens
are
analyzed
and DNA sequence
data are used for immediate therapeutic
and prognostic
decisions.
In addition
to many newly cloned genes and associated disease
mutations
each month,
several
clinical
applications
highlight
the potential
benefit of direct
DNA sequencing.
High-resolution
HLA typing appears
to be beneficial
in allogeneic
bone marrow transplan1408 CLINICAL CHEMISTRY, Vol. 41,
No.
10,
1995
tation, a technique
becoming more and more common
for treating
cancer and certain inherited
diseases (18).
Mutations
associated
with HNPCC and familial breast
and ovarian
cancer also appear to occur in sporadic
cases of these neoplasms
(19, 20). Direct sequencing
of
genes with broad mutational
heterogeneity
obviates
the need for familial linkage studies (which may sometimes be uninformative)
and permits
prenatal
and
direct carrier
detection
for near and distant relatives.
Databases
of such information
offer potentially
important prognostic
and therapeutic
information
to other
patients
by better defining the biology of a particular
disorder
(21). Selected patients
who lack common diagnostic
changes
in the genome but exhibit clinical
features
strongly
suggestive
of a particular
inherited
disorder
may benefit from direct DNA sequencing
of
particular
genes known
to be associated
with that
disorder
(22). Sequence
information
from specific
genes may also be useful in certain clinical contexts to
predict the likely phenotype
and course of individuals
with craniofacial
dysmorphic
syndromes
(23) in the
case of fibroblast
growth-factor
receptor
mutations.
Similarly,
a diverse set of outcomes,
from conditions
such as Hirschsprung
disease to aggressive
hereditary
cancers,
appear associated
with specific mutations
of
the RET protooncogene
(24). Direct sequencing
of specific genes in mycobacterial
isolates to detect antimicrobial resistance
properties
offers significant
benefit
in choosing appropriate
therapy
(25).
The relevance
of direct sequencing
is thus clear and
certain
to grow. Its full impact in the near term on
clinical samples may depend on providers
of managed
care. Direct sequencing
is moderately
expensive
now, a
combination
of instrument,
reagent,
and personnel
costs along with limited sample volumes. On the other
hand, current
US Medicare
reimbursement
rates for
molecular
Current
Procedural
Terminology
codes are
unrealistically
low (26) and are often used as guidelines by other payors. Organizational
efficiencies
and
future methodologic
advances could lead to substantial
savings,
particularly
for personnel.
However,
studies
documenting
the financial value of direct sequencing
in
overall patient
management
are sorely needed, since
this technology
lacks the encrusted
precedent
that
many other laboratory
tests enjoy. Given that patients
have shown a willingness
to pay out-of-pocket
for
genetic tests and may exhaust
all possibilities
when
cancer is the issue, there may be some wild cards in the
financial
equation.
I would echo the call by many
others for timely legislation
to prevent
discrimination
and to protect individual
privacy for genetic information derived from such testing.
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Jeffrey
A. Kant
Molecular
Diagnosis
Laboratory
Depart ment of Pathology
and Laboratory
Medicine
University
of Pennsylvania
Medical
Center
3400 Spruce St.
Philadelphia,
PA 19104-4283
Fax 215-662-7529
CLINICAL CHEMISTRY, Vol. 41, No. 10, 1995
1409