Download DNA copy number analysis by MAPH: molecular diagnostic

Technology Report
DNA copy number analysis
by MAPH: molecular
diagnostic applications
Edward J Hollox, Seyed M Akrami and John AL Armour†
CONTENTS
How MAPH works
Applications of MAPH
Translating MAPH from a
research to a diagnostic tool
DNA copy number variation is an important cause of genetic disease. There are several
techniques available to detect copy number changes of various sizes, each with their
limitations in resolution and cost. Here we outline the development of multiplex amplifiable
probe hybridization (MAPH) into a high-throughput diagnostic technique for detecting
copy number variation of almost any size. Its application in testing for genetic mutations
causing diseases, such as familial breast cancer, Charcot-Marie-Tooth disease Type 1A,
Duchenne/Becker muscular dystrophy and familial colorectal cancer is described, as well
as its use in identifying chromosomal changes in some individuals with mental retardation.
The analysis of the data produced by MAPH is also considered, along with its potential for
automation and development of microarray-based MAPH.
Expert Rev. Mol. Diagn. 2(4), (2002)
Expert opinion
Five-year view
KEYWORDS
breast cancer, deletion, DNA
copy number, DNA microarray,
Duchenne muscular dystrophy,
fluorescence in situ hybridization,
hybridization, subtelomeric DNA
Multiplex amplifiable probe hybridization
(MAPH) is a method that measures copy
number (also described as dosage) in genomic
DNA [1]. In humans, every somatic cell has
two copies of most genes or other loci, one
copy from its mother and one copy from its
father. One of the most familiar examples is
when one copy of chromosome 21 is inherited
from one parent and two copies from the other
parent; the result is trisomy 21 and consequently Down syndrome. Trisomy is copy
number variation of the human genome at a
very large scale (i.e., a whole chromosome) but
copy number changes involving small sections
of chromosomes, individual genes and even
single exons can cause genetic disease.
Deletion and complex rearrangements of
sections of subtelomeric DNA are associated
with mental retardation or other multiple
congenital abnormalities [2,3]. These changes
may cause the phenotype by altering the gene
copy in copy number in that section. As far as
is known, the subtelomeric DNA of any
chromosome end can be involved in such
changes and as these regions are densely
packed with genes. The specific gene or genes
that are responsible for the clinical phenotypes have not yet been identified. Studies
using fluorescent in situ hybridization (FISH)
on cohorts of patients with mental retardation suggest that at least 6% have subtelomeric deletions or rearrangements [4]. Whether
any of the remaining 94% have smaller
changes that cannot be detected by FISH is
not known.
Rearrangements within chromosomes can
cause genetic disease and can be mediated by
large low copy number chromosomal repeat
elements, or duplicons [5]. For example, Charcot-Marie-Tooth disease Type 1A (CMT1A) is
caused by duplication of a 1.4 Mb genomic
region containing the PMP22 gene and hereditary neuropathy with liability to pressure palsies (HNPP) is caused by heterozygous deletion
of the same region [6]. Pelizaeus-Merzbacher
disease is caused by duplication of the PLP1
gene [7] and Williams syndrome, in 95% of
cases, by a 1.6 Mb deletion of chromosome
7q11.23 [8].
Copy number changes of one or more exons
within specific genes can also cause genetic disease, as an alternative to point mutations creating single amino acid changes or a premature
stop codon. A duplication or deletion of an
exon can either create a premature stop codon,
if the deletion changes the reading frame of the
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89
Key issues
References
Affiliations
†Author for correspondence,
Institute of Genetics, Queen’s
Medical Centre, University of
Nottingham, NG7 2UH, UK
Tel.: +44 115 970 9398
Fax: +44 115 970 9906
[email protected]
Hollox, Akrami & Armour
mRNA, or can create a misfolded or otherwise nonfunctional
protein, if the deletion does not change the reading frame of
the mRNA. This can happen to any gene but genes that are
large or have introns rich in repeat elements are especially prone
to this type of mutation. For example, 65% of mutations at the
large DMD gene (causing Duchenne/Becker muscular dystrophy) are deletions or duplications involving one or more exons
[9], and one report testing a Dutch cohort of women suggests
that 35% of BRCA1 mutations are deletions or duplications of
one or more exons [10].
It is clear that copy number variation is an important
cause of genetic disease. However, smaller changes within
genes are often missed by conventional mutation detection
techniques and larger changes, involving several genes, are
either prohibitively slow or prohibitively expensive to test
for on a medium to large scale. MAPH has many different
advantages over the variety of different methods to detect
copy number changes (TABLE 1) and because of its low cost,
high multiplicity and flexible resolution, can be regarded as
a unifying technology applicable for detecting any copy
number change from 100 nucleotides of DNA through to
entire chromosomes.
How MAPH works
MAPH relies on the fact that probes can be quantitatively
recovered and amplified after hybridization. A diagram of the
experimental procedure is shown in FIGURE 1. The full experimental details have been published [1] with regular updates
and practical notes at our laboratory’s website [101]. Briefly,
genomic DNA is denatured and attached to a small piece of
nylon membrane. Amplifiable probes, corresponding to the
loci to be assayed for copy number, are then hybridized in
excess to this genomic DNA. Since the probes are in excess,
every site that is recognized by the probes in the genomic
DNA is occupied, so the amount of bound probe depends on
the number of available sites for that probe in the genomic
Table 1. Comparison of MAPH with other methods of DNA copy number analysis.
Resolution
Resolution limited by
Starting material
Loci per test
Major equipment
needed
PCR across rearranged 2 bp-5 kb
region, followed by gel
electrophoresis
Gel resolution, enzyme
extension ability, DNA
quality
Genomic DNA
solution
<6
None
Real-time PCR
>50 bp
Amplicon size and spacing Genomic DNA
solution
<6
Real-time PCR
machine
Quantitative multiplex
PCR
>50 bp
Amplicon size and spacing Genomic DNA
solution
<6
Fluorescent gel/
capillary sequencer
G-band cytogenetics
>2 Mb
Chromosome preparation
Metaphase
chromosomes
<1500
Microscope
CGH
>10 Mb
Chromosome preparation
Metaphase
chromosomes
<300
Fluorescence
Microscope
Array-CGH
>30 kb
Clone size and spacing
Genomic DNA
solution
~2400
Fluorescence
Microscope
Metaphase-FISH
>30 kb§
Chromosome preparation
Metaphase
chromosomes
2-3
Fluorescence
Microscope
Interphase-FISH
>30 kb
Microscope resolution/
clone spacing
Cells
2-3
Fluorescence
Microscope
Strand-FISH
>10 kb
Microscope resolution/
clone spacing
Genomic DNA in
agarose blocks
<6
Fluorescence
Microscope
Southern Blot
1 kb-2 Mb
Restriction fragment
length/gel resolution
Genomic DNA
solution (in agarose
blocks for pulse-field
gel electrophoresis)
1-2
Phosphoimager if
quantitative
Genomic DNA
solution
Up to 61§§
MAPH
>100 bp
Probe size and spacing
None if qualitative
Fluorescent gel/
capillary sequencer
§
The resolution of metaphase FISH depends on the experiment. If a presence/absence of a signal is used to detect copy number change, then the resolution is the same as
the size of the probe (usually derived from 30 kb cosmid or 100 kb BAC/PAC). If detecting copy number changes by the distance between two probes, then the resolution of
metaphase-FISH is similar to G-band cytogenetics (>2 Mb).
§§
The maximum number of loci tested simultaneously in work to date so far is 61, but the upper limit is not known.
90
Expert Rev. Mol. Diagn. 2(4), (2002)
MAPH in diagnostics
DNA. Following stringent washing of
Test DNA
the filter to remove the unbound probes,
1 µg of genomic DNA is denatured,
the bound probes are released into soludried and crosslinked on to a piece
of nylon membrane
tion by incubating at 95°C for 5 min.
Step 2
An aliquot of this solution, containing
Wash off any
the released probes, is then amplified in
unbound
amplifiable probes
the quantitative phase of PCR (20–25
Step 3
cycles) and the products run on a polyHeat at 95°c for 5 min to
acrylamide gel. Quantification of each
2-3 mm
release bound amplifiable
band of the gel allows estimation of the
probes into solution
amount of each probe, relative to the
other probes and hence estimation of
hybridise
Step 1
Analyze
Hybridize amplifiable
copy number for that locus in the
probe peak
probes with genomic
genomic DNA relative to the known
area data
DNA on filter
copy number of the other loci detected
Step 4
by the other probes.
Amplifiable probes
Amplify released probes
These are DNA probes between
using a pair of primers, one
One of the key features is that all
100 and 600 bp long, with the
fluorescently labled and
probes in an experiment (together
sequences corresponding to the
resolve each probe by size on
known as the probe set) can be amplidifference loci tested (difference
t
a fluorescent genotype gel
fied simultaneously using one pair of
colors) flanked by the same
primer-binding sequence
primers. This is because every probe is
designed to have the same primer sites
Figure 1. Principle of multiplex amplifiable probe hybridization (MAPH). Diagram illustrating the
important steps in MAPH. Amplifiable probes are shown, representing the different loci tested, with
flanking the locus-specific sequence. To
each sharing a common primer binding sequence..
enable detection and quantification on
an acrylamide gel, one of the primers is
labeled either with a radioactive isotope or, more commonly, a Applications of MAPH
fluorophore to enable detection by fluorescent-based frag- The applications of MAPH can be classified into two groups
ment detection systems. Although gels are routinely used, flu- depending on whether the variation that is being tested is
orescent-based fragment detection capillary machines, such as known a priori or not. For example, for PMP22 duplication
the ABI 3100, can also be used.
and deletion, the region that varies is known and probes correTwo gel lanes from a typical output from an ABI 377 are sponding to that region can be measured relative to reference
shown in FIGURE 2. Each MAPH probe that detects a unique probes. This can be regarded as typing known changes. For
locus at a chromosome end, is shown as a peak. The first sam- BRCA1 or subtelomeric DNA analysis, however, the exon or
ple is a heterozygous deletion of 17q (17q-) and the second is chromosome end that varies, if any, is not known. This can be
an unbalanced translocation between chromosomes 16 and 3, regarded as screening for new changes.
In the ‘typing’ class, the application of MAPH to type copy
resulting in three copies of 16q and one copy of 3p (16q+ 3p-).
These rearrangements were confirmed by subtelomeric FISH number at PMP22 has been successful [UNPUBLISHED DATA]. This
and the arrows on FIGURE 2 indicate the appropriate probes that probe set consists of 12 reference probes and seven test probes,
are at normal diploid dosage in one sample and either deleted whose values are averaged and used to diagnose a duplication
(CMT1A) or deletion (HNPP). The PMP22 assay was tested
or duplicated in the other.
Experimental values are normalized against other probe on 94 unaffected controls, followed by a blind test on 62 samresults to give a normalized ratio, where a value of 1 is ples previously tested by other methods [UNPUBLISHED DATA]. In a
expected for normal diploid dosage. A heterozygous deletion study of normal and CMT1A patients, 30 were typed as normal,
will result in a value around 0.5 and a heterozygous duplica- 31 typed as duplicate and one was typed as unknown. Sixty of
tion will give a value around 1.5. When the measured values the 62 diagnoses agreed with the referral laboratory, with one
for a probe for a series of normal diploid samples are plotted discordant result, which had in fact had conflicting diagnoses
on a histogram, the resulting distribution is similar to a nor- from different techniques, according to the previously published
mal distribution with a mean of 1. The standard deviation of study [11]. Any genetic diseases caused by a large deletion or
this distribution (the standard deviation of that probe) duplication can be typed by MAPH analysis.
describes how much variation or noise that probe shows in
The subtelomeric probe set is in the second ‘screening’ class.
MAPH. This allows any observation at the tails of the distri- Following an initial investigation into the applicability of
bution, which might be a deletion (near 0.5) or a duplication MAPH in detecting subtelomeric DNA copy number changes
(near 1.5), to be tested using standard hypothesis testing of a [12], a subtelomeric probe set consisting of 47 probes has been
normal distribution and this feature of MAPH is elaborated constructed [13]. This allowed analysis of negative and positive
in a later section of this review.
controls, as well as a cohort of patients referred to the clinic with
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91
Hollox, Akrami & Armour
120
160
200
240
280
320
360
400
440
480
known 17q- sample
140
16q two copies (normal)
17q one copy
3p two copies (normal)
120
100
80
60
Amplified
MAPH
probes
40
20
R500
marker
0
120
140
160
200
240
17q two copies (normal)
280
320
360
16q three copies
(16q+)
120
400
440
480
known 16q- 3p+ sample
3p one copy (3p-)
100
80
60
40
Amplified
MAPH
probes
20
0
R500
marker
Figure 2. Gel electropherograms of MAPH products. Two example electropherograms produced by GeneScan™ analysis of output from an ABI 377 DNA
Analyser. The x-axis shows relative fluorescence units and the y-axis shows size in base pairs. The amplified MAPH probes are shown as blue peaks, with a
R500 marker (labeled with ROX dye, Genetix plc) shown as red peaks. Two positive controls are shown, 17q- and 16q+3p-, both initially identified by FISH
analysis using subtelomeric probes. The probes that correspond to the chromosome ends under investigation and hence show the changes in copy number,
are shown by the arrows..
Fragile X syndrome but negative for the FMR1 repeat. In 83
normal individuals, three putative polymorphic changes were
found (a deletion at XpYp and duplications at 1q and XqYq),
which shows that MAPH can detect and give accurate estimates
for the allele frequency of uncommon variants in the population. The positive controls were correctly identified ‘blind’,
demonstrating the reliability of the probe set and six copy
number changes were detected in the patient cohort. This last
part of the study shows the value of screening for subtelomeric
changes in patients that would otherwise not be screened. The
higher resolution of MAPH compared with FISH (TABLE 1)
allows smaller copy number changes to be detected, especially
when a probe set with several probes per chromosome end is
available. By distinguishing different sized copy number variant
alleles and associating them with different clinical phenotypes, it
may be possible to clarify the genotype–phenotype relationship
of genes in these subtelomeric regions.
MAPH can be used as part of a mutation screen for any gene
involved in genetic disease. Our laboratory has developed
screens for BRCA1 [UNPUBLISHED DATA] and TBX5 (Holt-Oram
syndrome, [14]) and development of screens for the MLH1/
MSH2 genes (involved in hereditary nonpolyposis colorectal
92
cancer) and the DMD gene [102] are underway. More general
screening of genomic regions for loss of heterozygosity in cancer cells is possible and probe sets for scanning the whole
genome for deletion and duplication are planned.
TABLE 2 shows the MAPH probe sets constructed so far and
their applications.
Translating MAPH from a research to a diagnostic tool
Responding to a clinical need
In clinical genetics, it is increasingly common to screen PCR
products from each exon of the gene for a causative mutation.
Knowledge of the causative mutation enables prenatal screening
of offspring for the mutation and may, in the future, help with
diagnosis and treatment as different classes of mutation are found
to associate with different aspects of the disease. Methods used
for screening for substitutional mutations, such as denaturing
gradient gel electrophoresis, do not detect copy number changes.
Screening for copy number changes using MAPH will be an
important tool in the mutation detector’s inventory but especially in those genetic diseases where a large proportion of mutations are due to deletions or duplications and are therefore not
found using current methods. For example, the breast cancer
Expert Rev. Mol. Diagn. 2(4), (2002)
MAPH in diagnostics
Table 2. Current applications of MAPH to analysis of
genetic disease.
Disease/
condition
OMIM
disease
reference
Gene
Nature of
mutation
Charcot-MarieTooth disease
118220
PMP22
Duplication of
gene
Hereditary
neuropathy with
liabilty to
pressure palsies
162500
PMP22
Deletion of gene
HNPCC
114500
MLH1/
MLH2
Various,
including exon
deletion or
duplication
Holt-Oram
syndrome
142900
TBX5
Various,
including whole
gene deletion
Breast cancer
113705
BRCA1
Various,
including exon
deletion and
duplication
Duchenne/Becker 310200
muscular
dystrophy
DMD
Various,
including exon
deletion or
duplication
Mental
retardation
(subtelomeric
DNA)
Various,
unknown
Deletion or
unbalanced
translocation of
subtelomeric
DNA
Economics
gene BRCA1 and the colorectal cancer genes MLH1 and MSH2,
for which MAPH probe sets are either currently commercially
available (BRCA1) or are likely to be made available in the near
future. These are genes involved in the aetiology of two common
diseases, where large clinical mutation screening programs are
already in place in clinical centers. Diagnosis of deletions or
duplications in genes for rarer disorders is likely to remain in a
research setting for the foreseeable future.
Diagnosis of genetic diseases where the only mutation is a
change in copy number, for example PMP22, will be made
considerably easier by implementation of MAPH. Using several
different probes to detect the mutation results in negligible
false-positive and false-negative rates and can clarify uncertain
diagnoses made by other methods.
It has become increasingly clear that many cases of mental
retardation are associated with copy number changes in subtelomeric DNA at the ends of chromosomes. Indeed, it has been suggested that every patient with mental retardation that cannot
otherwise be explained should be screened for subtelomeric copy
number changes [15]. MAPH is the ideal high-throughput technique for detecting these changes, at the very least prior to detection by FISH and an initial probe set for this task has been made
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commercially available by Genetix PLC (Hampshire, UK). Since
MAPH resolution can be carefully controlled by designing
probes at appropriate intervals, it is possible, with probe sets covering subsets of chromosome ends, to map each copy number
change at high resolution and identify genes that appear to cause
the mental retardation phenotype. Since many different chromosome ends and hence many different genes, are involved, this
opens up genotype–phenotype correlation studies and a gateway
into understanding how the mutation causes the disease.
Where MAPH can be used in high-throughput screening, such
as detecting mutations in the BRCA1 gene and screening subtelomeric DNA in mental retardation patients, the cost of the
technique must be low enough and its benefits be great enough
to justify its use. This is the case in all health service systems
whether funded through private insurance or through general
taxation. The cost of screening all subtelomeric ends for one
sample using FISH is about US$150, whilst the cost of screening all subtelomeric ends by MAPH for one sample is about
US$5. This excludes cost of equipment and labor, which in
both cases will be substantially less for MAPH due to higher
throughput and decreased hands-on time. In situations where
the alternative techniques are not directly comparable, such as
screening for deletions and duplications in BRCA1 gene,
MAPH is much more cost-efficient. The standard methods for
copy number variation in BRCA1 are either multiplex quantitative PCR or junction fragment analysis by PCR. Both these
methods focus on a small number of exons that are known to
be commonly involved in copy number changes. In contrast,
MAPH screens all exons and can detect all copy number
changes, with a similar throughput level to PCR.
The cost advantages outlined above are given even greater
weight when the prevalence of the mutations is accounted for
and therefore, the proportion of the total population that may
have use for genetic diagnostics using MAPH.
Automation
Allied to the economic considerations of MAPH is the potential
for automation. The less labor and time required per test, the less
expensive the test. In its current research setting, MAPH involves
considerably less hands-on time than other techniques. It has the
advantage that the software required for analysis is standard fragment analysis software (such as GeneScan) and statistical analysis
of results is carried out using standard spreadsheets with custom
designed macros. Further customization of this analysis frontend will shorten hands-on time even more. The most labourintensive and rate-limiting steps are preparation of filters and
washing of these filters following hybridization. It is probably
possible to modify existing technology to allow these steps to be
automated. For example, DNA could be spotted onto precut filters by a robot and washing could be done by using a pump
cycling wash solution with the filters. Transfer of filters to individual tubes to allow release of bound probes into PCR buffer
would be more of a technical challenge.
93
Hollox, Akrami & Armour
Nevertheless, the high-throughput nature of the technique,
even with minimal automation, means that the absence of this
additional technology will not prevent its effective use in clinical
genetic diagnosis.
Multiplicity
A key advantage of MAPH is the large number of loci that
can be analyzed at once and the number of loci is determined
by the number of probes in the probe set. The subtelomeric
probe set has 47 probes and we have had success with a probe
set of 61 probes. However, using gel electrophoretic analysis,
each probe has to be resolved satisfactorily on a gel and is
hence the number of probes in a probe set is limited by gel
resolution. A lower limit on probe size is 100 bp, since
hybridization efficiency falls below this point and the PCR
places a practical upper limit of around 600 bp, so that allows
for around 100 probes spaced at 5 bp intervals.
Detection of MAPH products by rehybridization to
arrayed probes would overcome this limitation. In this
method, MAPH is performed as normal. However, when the
probes are recovered after hybridization, instead of separation and quantification of the probes by gel electrophoresis,
they are rehybridized back to a large excess of probe attached
to an array. Due to the large excess of probe on the array,
rehybridization will proceed to near-completion and the
amount of probe bound to the array can be quantified.
Microarray MAPH will allow detection and quantification
based on sequence, not size and is limited only by the
number of probes attached to the array and the number of
arrays that can be tested (FIGURE 3). For example, if one array
has 400 different probes immobilized to it, then 400 probes
can be included in the probe set for the initial hybridization
to genomic DNA and hence 400 separate loci can be tested.
If hybridization to multiple arrays can be performed, then
the number of loci is, theoretically, the number of arrays
x400. However, there is likely to be an upper limit to the
number of probes included in the probe set due to possible
interference between probes and hybridization kinetics of
the initial hybridization steps.
Microarray-based MAPH is currently being developed by
two groups. One group is using standard microarray technology with oligonucleotide probes arrayed on a glass slide. In our
laboratory, however, the oligonucleotide probes are arrayed on a
porous aluminium oxide substrate which not only allows faster
and more specific hybridization times but continuous monitoring of the hybridization kinetics (Pamgene BV, The Netherlands [103]). This type of microarray technology is more suited
to a high-throughput diagnostic situation.
Statistics
Gel electrophoretic analysis of MAPH products
Recovered MAPH probes amplified with one
unlabeled primer and one fluorescently
labeled primer
Run amplified products on
electrophoresis gel with
fluorescent-based fragment
detection (e.g., ABI 377).
Number of probes that can
be resolved depends on gel
resolution – no more than
approximately 100
Quantify area under each
band (peak) using standard
fragment analysis software
(e.g., Genescan and
Genotyper)
Microarray analysis of MAPH products
Recovered MAPH probes amplified
using unlabeled primers and
fluorescently label after amplification
Hybridize amplified products
back to excess of MAPH
probe immobilized in arrays
on a solid support. Number
of probes that can be
resolved depends on
number of spots on array
(400) and number of arrays
(theoretically limitless)
Quantify signal on each
array spot using packaged
array analysis software
Figure 3. Gel-based and microarray-based analysis of MAPH products. Figure illustrating the
principal differences between the two methods for analysis of MAPH products. Both methods begin
with the recovered MAPH probes after hybridization to genomic DNA immobilized on a nylon filter. In
the gel electrophoresis method, the products are labeled during amplification by a fluorescent primer,
then visualized and quantified on a standard electrophoretic gel with fluorescence detection. In the
microarray method, the products are rehybridized back to an excess of probe spotted on the array.
The amount of hybridization on each spot corresponds to the amount of probe in the solution and
hence the dosage: white, no hybridization, no probe in solution, dosage of zero; dark gray, full
hybridization, probe in solution, dosage of two; pale gray, full hybridization but half the amount of
probe, dosage of one (hemizygosity).
94
The quantitative nature of MAPH as
opposed to the qualitative nature of techniques, such as cytogenetics or FISH, enables statistical analysis of the resulting data.
Indeed, the amount of data produced by
MAPH requires statistical analysis to interpret the results correctly and to gauge the
rate of false-positives and false-negatives.
For certain applications this is straightforward. If the probe set consists of several
test probes, that show dosage change and
several reference probes, that do not show
dosage change, then each test probe can be
tested individually at a certain significance
level. An alternative approach would be to
derive a value (such as the mean or
median) taken from the test probes and
plot that value for each sample so that samples with the same dosage cluster together
and can be distinguished from clusters of
samples of other dosage. Discriminant
analysis statistics may also help diagnosis in
clinical diagnostic situations.
However, in the screening cases, such as
the subtelomeric probe set, there is no a
priori knowledge of which probes will
show dosage changes and which probes
will not. Therefore, each data point must
be tested to determine whether it shows a
Expert Rev. Mol. Diagn. 2(4), (2002)
MAPH in diagnostics
significant change from normal diploid dosage and because of
the large number of observations simultaneously generated by
MAPH, statistical analysis problems due to multiple testing
may occur. For example, a single gel’s worth of results from the
current subtelomeric probe set will produce 2115 data points
(45 samples x 47 tests per sample) and assuming a normal distribution, standard hypothesis testing using a 5% significance
level would produce over 100 apparent positives purely by
chance. This does provide a valuable level of screening, reducing the target sample from a size impractical to screen by FISH
to something more manageable. Using standard corrections for
multiple testing, such as the Bonferroni correction, will result
in fewer positive calls but risks missing a fair number of truepositives due to an increase in the false-negative rate. The balance between keeping retests/confirmations to a minimum by a
low false-positive rate (high specificity) and ensuring a low
false-negative rate (high sensitivity) is important. In a screening
situation, a reasonable rate of false-positives may be acceptable
if the false-negative rate is kept to an absolute minimum.
Where the cost of any confirmatory technique is expensive,
such as FISH, the MAPH false-positive threshold can be set so
that the number of FISH tests can be within budget.
FIGURE 4 shows the different possible consequences of changing
the false-positive and false-negative rates for one probe. FIGURE 4A
shows a normal distribution (approximately a mean of 1) of samples with normal diploid dosage and a normal distribution
(approximately 0.5) of samples with haploid dosage – one copy is
deleted. The threshold is arbitrarily set at 0.75 (corresponding to
a significance level of p = 0.006 when the standard deviation
(noise) of the probe is 10%) and the shaded areas correspond to
the false-negative samples (grey) and the false-positive samples
(black). FIGURE 4B shows that the simplest way to reduce the falsenegative rate is to increase the threshold, for example, up to 0.8
(significance level p = 0.022 when the standard deviation of the
probe is 10%). However, this has the effect of increasing the
false-positive rate. Similarly, reducing the threshold to 0.6 would
decrease the false-positive rate yet increase the false-negative rate.
The most satisfactory method of reducing false calls is to improve
the technology so that probe noise (standard deviation) is
reduced. Even a small reduction in probe noise can have large
effects on the rate of false-positive and false-negative calls
(FIGURE 4C). For example, with a false-positive rate of 1%, a reduction of the standard deviation of a probe from 10 to 9% reduces
the false-negative rate six times.
We are beginning to understand the origins of measurement error in MAPH and why some probes are ‘noisier’ than
others and this understanding will be essential for further
development of the technique.
With the current state of the technology, repeat testing is the
best method to reduce false calls and distinguish signal from
noise. The low cost and high-throughput nature of MAPH
makes duplicate and triplicate testing practical in a diagnostic situation. Duplicate testing of every sample, followed by calculating
the mean of the two independent duplicate results should produce a distribution curve with a standard deviation of σ/ 2,
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Distribution of probes Distribution of probes
for deleted loci
for normal diploid loci
a
0.50
0.75
1.00
b
0.50
0.80 1.00
c
0.50
0.75
1.00
False-negative
False-positive
Figure 4. Effect of varying threshold or standard deviation on error
rates. Diagram illustrating how false-positive or false-negative rates can
be altered by understanding the properties of the Gaussian normal
distribution curve. In each case, a normal distribution about 1.0 (for
samples showing normal diploid dosage) and a normal distribution about
0.5 (for samples showing heterozygous deletions) is shown. The area
under the curve corresponding to the false-negative rate is shown in light
gray and the area under the curve corresponding to the false-positive rate
is shown in black. False-positive and negative rates using a threshold of
0.75. Effect on false-positive and -negative rates by altering the threshold
to 0.8. Effect on false-positive and -negative rates by lowering the probe
standard deviation (noise).
where σ is the standard deviation of the curve produced by testing the samples once. Similarly, triplicate testing will result in a
standard deviation of σ/ 3. Unfortunately, taking the mean value
of multiple results is at best an approximation and may introduce
systematic bias into the data analysis. This is because multiple
results from some probes are not independent – they are correlated [13]. To account for this correlation, a multivariate analysis
approach is needed to analyze the data. A bivariate approach was
used to analyze duplicate data from the subtelomeric probe set
and we are developing these methods so that future multiple data
can be analyzed in a straightforward manner.
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Hollox, Akrami & Armour
Patent issues
MAPH is patented by the University of Nottingham
WO0053804 (2001) and limited licenses have been granted to
Genetix, PLC for commercialization of probe sets for research
[103] and to PamGene BV for microarray diagnostic analysis
[104]. The technique can be used for academic and research purposes freely but for commercial diagnostic applications the
Research Business Unit at the University of Nottingham must
be contacted [105].
Expert opinion
In its current state of development, MAPH is ideal for comprehensively screening disease genes, such as BRCA1 for copy
number variation in exons and for genotyping known copy
number variations, such as at the PMP22 gene for CMT1A and
HNPP. Applying these tests in diagnosis should be very
straightforward. We would also recommend MAPH analysis of
patients where the gene causing the disease is known but standard mutation detection techniques have found no causative
change. Heterozygous deletions and duplications are a significant proportion of genetic mutations and PCR-based detection
methods, such as DHPLC or DGGE will not detect these
mutations.
Screening subtelomeric DNA for copy number changes in
patients with mental retardation has a huge potential for routine diagnostic use instead of occasional research use but more
development is needed both in understanding and improving
the technique and in applying statistics and new software to
cope with the large amount of data it can produce. The current
rate of 6% false-positives with 2% false-negative rate by testing
in duplicate [13] is not quite sufficient to replace FISH testing,
although it is ideally suited for a screening role. When compared with a clinical checklist as a screen, where a 5% false-positive rate generates a 81% false-negative rate and even a 73%
false-positive rate generates an 11% false-negative rate [16], the
usefulness of MAPH analysis over symptom-based classification
systems is evident.
Five-year view
Developments in technology
The ability to perform MAPH analysis on very small amounts
of DNA will open a wide variety of applications, including prenatal diagnosis and analysis of paraffin-embedded specimens.
At present, 0.5 µg is the minimum amount of DNA for a reliable MAPH analysis but our laboratory is currently developing
methods to extend MAPH to nanogram amounts of DNA.
Development of MAPH on array systems has been described
above and this undoubtedly will become the standard platform
for some MAPH analyses in the future.
We must also remember that MAPH is fundamentally a lowtech method that can be aided with high-tech equipment. Since
at its simplest level, it only requires the equipment and consumables available in any molecular biology laboratory, it is an
ideal method for laboratories where funds for research and clinical diagnostics are limited. MAPH is an extremely accessible
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technology and developments to maintain and implement
MAPH in a low-tech setting will be needed over the next
few years.
Developments in applications
At the time of writing, there are currently two MAPH probe
sets commercially available, BRCA1 and subtelomeric DNA,
both marketed by Genetix Ltd. [104]. The range of ‘off-theshelf ’ probe sets for use by other laboratories will undoubtedly
increase, so that within 5-years a variety of applications, for
example typing the PMP22 gene and screening the MLH1/
MSH2 genes, will each be covered by a probe set. A new subtelomeric probe set, with more than one probe covering each
chromosome end, is a priority.
As well as being a routine part of mutation screening procedures
and subtelomeric analysis, development of a MAPH probe set for
whole genome scanning, coupled with array-based MAPH will be
of enormous benefit, especially for cancer genetics. At 1 Mb resolution, 3000 probes will be needed and at 200 kb resolution
15,000 probes will be needed. Although perfectly possible, this will
require a large amount of work with commensurate funding, probably out of the academic sector. In practice, a whole genome scanning kit will probably consist of several probe sets, although it may
not be too much of a technical challenge to screen the whole
genome at 200 kb resolution (15,000 loci/probes) in one test.
Acknowledgements
EJ Hollox is supported by The Wellcome Trust grant number
060578 and SMA is supported by the Ministry of Health of the
Islamic Republic of Iran.
Key issues
• Multiplex amplifiable probe hybridization (MAPH) is a highthroughput technique for identifying and typing copy
number variation in DNA, such as deletions and duplications
and costs an order of magnitude less than fluorescent in situ
hybridization.
• MAPH has been applied to screen BRCA1, PMP22 gene (for
Charcot-Marie-Tooth Type 1A and hereditary neuropathy
with pressure palsies), DMD (for Duchenne Muscular
Dystrophy), MLH1/MSH2 (for hereditary nonpolyposis
colorectal cancer) and subtelomeric DNA for copy number
variation.
• MAPH is currently being developed from a research tool into
a high-throughput diagnostic tool.
• MAPH provides quantitative data amenable to statistical
analysis, that can produce results on which clinical decisions
can be based. Suitable thresholds can be used so that clinical
decisions can be taken in the context of patient need and
economic restrictions.
• MAPH has the potential to be used to scan the whole
genome at very high resolution.
Expert Rev. Mol. Diagn. 2(4), (2002)
MAPH in diagnostics
References
Papers of special note have been highlighted as:
• of interest
•• of considerable interest
Armour JA, Sismani C, Patsalis PC, Cross
1
G. Measurement of locus copy number by
hybridization with amplifiable probes.
Nucleic Acids Res. 28(2), 605–609 (2000).
•• This is the ‘proof of principle’ paper
describing MAPH, patented by the
University of Nottingham WO0053804
(2001).
2
3
4
5
•
6
Knight SJ, Flint J. Perfect endings: a review
of subtelomeric probes and their use in
clinical diagnosis. J. Med. Genet. 37(6),
401–409 (2000).
Flint J, Wilkie AO, Buckle VJ et al. The
detection of subtelomeric chromosomal
rearrangements in idiopathic mental
retardation. Nature Genet. 9(2), 132–140
(1995).
Knight SJL, CM Lese, KS Precht et al., An
optimized set of human telomere clones for
studying telomere integrity and
architecture. Am. J. Hum. Genet. 67(2),
320–332 (2000).
Ji Y, Eichler EE, Schwartz S, Nicholls RD.
Structure of chromosomal duplicons and
their role in mediating human genomic
disorders. Genome Res. 10(5), 597–610
(2000).
Demonstrates the importance of low copy
number chromosomal repeats in causing
large deletions and duplications, which
may lead to genetic disease.
Lupski JR, Chance PF, Garcia CA.
Inherited primary peripheral neuropathies.
Molecular genetics and clinical implications
of CMT1A and HNPP. JAMA 270(19),
2326–2330 (1993).
www.future-drugs.com
7
Woodward K, Kendall E, Vetrie D,
Malcolm S. Pelizaeus-Merzbacher disease:
identification of Xq22 proteolipidprotein duplications and characterization
of breakpoints by interphase FISH. Am.
J. Hum. Genet. 63(1), 207–217 (1998).
8
Francke U. Williams-Beuren syndrome:
genes and mechanisms. Hum. Mol. Genet.
8(10), 1947–1954 (1999).
9
den Dunnen JT, Bakker E, van Ommen
GJ, Pearson PL. The DMD gene
analysed by field inversion gel
electrophoresis. Br. Med. Bull. 45(3),
644–658 (1989).
Petrij-Bosch A, Peelen T, van Vliet M et al.
BRCA1 genomic deletions are major
founder mutations in Dutch breast cancer
patients. Nature Genet. 17(3), 341–345
(1997).
•• Demonstrates the importance of detecting
deletions and duplications in mutation
screening of genes.
11 Rowland JS, Barton DE, Taylor GR. A
comparison of methods for gene dosage
analysis in HMSN Type 1. J. Med. Genet.
38(2), 90–95 (2001).
10
12
Sismani C, Armour JA, Flint J et al.
Screening for subtelomeric chromosome
abnormalities in children with idiopathic
mental retardation using multiprobe
telomeric FISH and the new MAPH
telomeric assay. Eur. J. Hum. Genet. 9(7),
527–532 (2001).
13
Hollox EJ, Atia T, Parkin T, Armour JAL.
(Submitted).
14
Akrami SM, Winter RM, Brook JD,
Armour JA. Detection of a large TBX5
deletion in a family with Holt-Oram
syndrome. J. Med. Genet. 38(12), E44
(2001).
•
15
16
Shows how MAPH was used to detect an
almost whole gene deletion as a cause of a
genetic disease in a family, which had been
previously missed using other mutation
detection techniques.
Zackai EH, Krantz ID, Ming JE et al.
Subtelomeric testing: don’t leave clinic
without it. Am. J. Hum. Genet. 69(4), S326
(2001).
de Vries BB, White SM, Knight SJ et al.
Clinical studies on submicroscopic
subtelomeric rearrangements: a checklist. J.
Med. Genet. 38(3), 145–150 (2001).
Websites
101
MAPH home page
www.nottingham.ac.uk/~pdzjala/maph/
maph.html
102
Leiden University Medical School
Duchenne Muscular Dystrophy pages
www.dmd.nl/
103
Pamgene BV homepage
www.pamgene.com/
104
Genetix Ltd homepage
www.genetix.com/
105
University of Nottingham Research
Business Unit
www.nottingham.ac.uk/rbu/
Affiliations
•
Edward J Hollox, PhD
Postdoctoral Research Associate,
•
Seyed M Akrami, MD
Postgraduate Research Student,
•
John AL Armour, BM, BCh, PhD
Reader in Genetics,
[email protected]
•
Institute of Genetics, Queen’s Medical Centre,
University of Nottingham, NG7 2UH, UK
Tel.: +44 115 970 9398
Fax: +44 115 970 9906
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