Clinical Chemistry 47: 838-843, 2001;
(Clinical Chemistry. 2001;47:838-843.)
© 2001 American Association for Clinical Chemistry, Inc.
New Polymorphic Short Tandem Repeats for PCR-based Charcot-Marie-Tooth Disease Type 1A Duplication Diagnosis
Jose L. Badano1,
Ken Inoue1,
Nicholas Katsanis1 and
James R. Lupski1,2,3a
Departments of
1
Molecular and Human Genetics and
2
Pediatrics, Baylor College of Medicine, Houston, TX 77030.
3
The Texas Children’s Hospital, Houston, TX 77030.
aAddress correspondence to this author at: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Rm 609E, Houston, TX 77030. Fax 713-798-5073; e-mail
jlupski{at}bcm.tmc.edu.
 |
Abstract
|
|---|
Background: Charcot-Marie-Tooth disease type 1A (CMT1A) accounts
for 70–90% of cases of CMT1 and is most frequently caused by the
tandem duplication of a 1.4-Mb genomic fragment on chromosome 17p12.
Molecular diagnosis of CMT1A has been based primarily on pulsed-field
electrophoresis, fluorescence in situ hybridization, polymorphic allele
dosage analysis, and quantitative PCR. We sought to improve the
fidelity and applicability of PCR-based diagnosis by developing a panel
of novel, highly polymorphic short tandem repeats (STRs) from within
the CMT1A duplicated region.
Methods: We used a recently available genomic sequence to
identify potentially polymorphic simple repeats. We then amplified
these sequences in a multiethnic cohort of unaffected individuals and
assessed the heterozygosity and number of alleles for each STR. Highly
informative markers were then tested in a set of previously diagnosed
CMT1A duplication patients, and the ability to identify the genomic
duplication through the presence of three bands was assessed.
Results: We identified 34 polymorphic markers, 15 of which were
suitable for CMT1A diagnosis on the basis of high heterozygosity in
different ethnic groups, peak uniformity, and a large number of
alleles. On the basis of the fluorescent dye and allele range of each
marker, we developed two panels, each of which could be analyzed
concurrently. Panel 1, which comprised 10 markers, detected 37 of 39
duplications, whereas panel 2, which comprised the remaining 5 markers,
identified 21 of 39 duplications. Through the combination of both
panels, we identified 39 of 39 duplications in previously diagnosed
CMT1A patients.
Conclusions: The newly developed 15-marker set has the capability
of detecting >99% of duplications and thus is a powerful and
versatile diagnostic tool.
|
Introduction
|
|---|
Charcot-Marie-Tooth disease
(CMT;1
MIM 118220) is a common Mendelian disorder with a frequency of
1 in 2500 (1). CMT has been separated historically into two
distinct clinical/pathological categories, CMT1 and CMT2, which are
further subdivided according to genetic mapping criteria into -A, -B,
-C, and so forth. Overall, CMT exhibits substantial genetic
heterogeneity with at least 15 loci identified to date (2).
Of those, CMT1A is the most common form of the disease, accounting for
70–90% of CMT1 patients (2). A tandem duplication of a
1.4-Mb genomic fragment on chromosome 17p12 is responsible for most
CMT1A cases (3)(4). The CMT1A region is flanked
by a set of 24-kb, low-copy number repeats (CMT1A-REPs)
(5)(6), and >99% of the CMT1A duplication is
mediated by unequal crossing-over between the proximal and distal
CMT1A-REPs [Ref. (2), and references therein]. The gene
encoding peripheral myelin protein 22 (PMP22) maps in this
interval, and several lines of evidence have indicated that alterations
in gene dosage of PMP22 are responsible for the pathogenesis
of CMT1A [reviewed in Refs. (7)(8)]. A
clinically distinct hereditary neuropathy with liability to pressure
palsy (HNPP) has been found allelic to CMT1A, in which deletion of the
same 1.4-Mb region is responsible for the disease. Additional molecular
studies revealed that CMT1A and HNPP result from a reciprocal
interchromosomal recombination event (9).
Several methods have been used in clinical laboratories for the
molecular diagnosis of CMT1A and HNPP (10). Conventional
Southern hybridization was used initially to visualize the difference
in dosage by densitometric measurement using a region-specific probe
(3). Subsequently, pulsed-field gel electrophoresis (PFGE)
has been used to detect recombination-specific junction fragments
(3)(5)(9)(11). More recently, interphase fluorescence in
situ hybridization (FISH) analysis was developed for the CMT1A/HNPP
diagnosis, which directly visualizes the gain or loss of the
PMP22 signal (12)(13), as well as
real-time fluorescent PCR, which measures gene dosage (14).
PCR-based methods using short tandem repeats (STRs) (3)(15)(16), quantification of gene dosage (17)(18), and
detection of the unique junction fragment of the CMT1A/HNPP
recombination (19) have also been reported. STR-PCR methods
detect three different alleles in CMT1A duplication in combination with
semiquantitative dosage measurement. Because of its advantages in cost,
amount of DNA sample required, labor, and turnaround time, the STR-PCR
method has been widely used for molecular diagnosis of CMT1A. There is,
however, a limitation in sensitivity because of the low number of
polymorphic markers available in the 1.4-Mb duplicated region.
Initially, only one marker, D17S122 (RM11-GT)
(3) was used for CMT1A PCR-based diagnosis, but its reduced
informativeness allowed detection of three alleles in only 46% of
CMT1A cases (20). Two studies subsequently identified
additional STRs in this region, improving the fidelity of this type of
testing to 85% (15)(16).
We recently constructed a phage P1 artificial chromosome (PAC) and
bacterial artificial chromosome (BAC) contig of the 1.4-Mb CMT1A region
and described the complete genome sequence between the CMT1A-REPs
(21). In the present study, we used this sequence to
identify several STRs that are potentially polymorphic. We hypothesized
that new, highly informative STRs may improve our ability to detect
genomic rearrangements associated with CMT1A/HNPP, thus enhancing the
sensitivity of PCR-based testing and rendering it a more sensitive and
informative diagnostic method. Tri-, tetra-, and pentanucleotide
repeats could be particularly useful in this process because they
typically generate substantially reduced or no stutter peaks. We thus
evaluated 34 novel STRs and developed a robust multimarker diagnostic
test for CMT1A genomic rearrangements that may be able to detect
duplications unambiguously in >99% of the patients.
|
Materials and Methods
|
|---|
dna samples
To establish the polymorphic potential of newly identified STRs
from the CMT1A/HNPP genomic region, we used DNA from 96 unrelated
Caucasians collected previously in our laboratory. An additional 36
samples of African-American, 36 Asian, and 24 Hispanic individuals were
obtained from the Baylor College of Medicine Human Polymorphism
Resource. We ascertained the ability to detect genomic rearrangements
by genotyping 39 individuals diagnosed with CMT1A. Duplication of the
CMT1A region was established independently by densitometric analyses of
restriction fragment length polymorphisms from the region, STR analysis
with the (CA)n polymorphic marker
D17S122 (RM11-GT), detection of a junction
fragment with PFGE, or FISH with a PMP22 probe. DNA was
extracted either from venous blood or from transformed lymphoblastoid
cells by a salting-out process (Puregene; Gentra Systems).
informed consent
We obtained informed consent from all individuals participating in
this study in accordance with protocols approved by the Baylor College
of Medicine Institutional Review Board.
identification of polymorphic repeats
We analyzed the complete 1.4-Mb genomic sequence of the CMT1A
region for STRs with the Sequencher sequence analysis program
(GeneCodes Corp.), RepeatMasker
(http://repeatmasker.genome.washington.edu/cgi-bin/RepeatMasker),
and the GCG software package (Ver. 9; University of Wisconsin).
Fluorescently labeled primers were designed with the Primer Ver. 3
program
(www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi).
The 5' end of the unlabeled primer was tagged with the sequence GTGTCTT
to minimize band stuttering, and primers were obtained from MWG
Biotech. All DNA samples were adjusted to 30 ng/µL, and PCR reactions
were carried out on an MJR Tetrad or a MWG Primus thermocycler with the
True Allele PCR premix (Perkin-Elmer). Amplification conditions were as
follows: 95 °C for 5 min, followed by 10 cycles of a touchdown
sequence, in which the annealing temperature was reduced by
1 °C/cycle from 66 °C to 56 °C, and then 25 cycles at 95 °C
for 30 s, 56 °C for 30 s, and 72 °C for 30 s. A
final step of 72 °C for 5 min was also included. All markers were
amplified individually and then pooled according to the panel to which
they were binned, with ratios determined by the fluorescent label on
each marker [6-carboxy-fluorescein (FAM), 1µL;
hexachloro-6-carboxyfluorescein (HEX), 2 µL;
4,7,2',7'-tetrachloro-6-carboxyfluorescein (TET), 2 µL]. Products
were resolved on an ABI 377 automated sequencer (Applied Biosystems),
and alleles were assigned with GENESCAN (Ver. 2.3) and GENOTYPER (Ver.
2.1) software (Applied Biosystems) as described previously
(22).
|
Results
|
|---|
identification of novel STRs from the cmt1a region on
17p12
We identified a total of 42 novel potential polymorphic STRs
throughout the 1.4-Mb CMT1A/HNPP genomic interval. Subsequent
alignments using unique sequence flanking each repeat confirmed that
these STRs did not overlap with any existing genetic markers. We did,
however, eliminate eight STRs from further analyses because these were
embedded in Alu- and LINE1-rich regions, which precluded the
design of suitable amplification primers.
str evaluation and characterization
We selected 34 STRs, including 20 di-, 1 tri-, 10 tetra-, and 3
pentanucleotide repeats. We first ascertained the polymorphic potential
of all markers on a panel of eight control DNA samples. STRs were
evaluated for the following criteria: (a) heterozygosity
65%; (b) identification of more than four alleles;
(c) allele-peak uniformity; and (d) minimal
interference by shadow alleles.
At the conclusion of this analysis, we selected 15 STRs from across the
CMT1A region that best fit the above criteria (Fig. 1
and Table 1
) and evaluated them further by genotyping in 96 unrelated
Caucasian control DNAs. We identified >10 alleles in 13 of 15 markers;
1 marker had 7 alleles (Table 1
; D17S2228), whereas some
markers had as many as 16 alleles (Table 1
; D17S2219). The
heterozygosity values obtained were likewise suggestive of high
informativeness because they were 59–93% (Table 2
). To ascertain the potential global applicability of these
markers, we also assessed the same criteria in a cohort of
African-American, Asian, and Hispanic individuals. In most cases, we
found no significant variation in allele frequencies among the
different ethnicities (Table 2
). However, in a few cases, including
D17S2222, D17S2228, and D17S2230, such
differences were evident, suggesting that the ethnicity of the patient
may become relevant to the diagnostic applicability of some STRs.
View larger version (15K):
[in this window]
[in a new window]
|
Figure 1. Physical map and large-insert clone contig of the CMT1A
genomic region on human 17p12.
Gray vertical boxes indicate the position of the
CMT1A-REPs. The positions of previously described microsatellites are
depicted with gray arrowheads along the upper
horizontal line, as are some genes for landmarks. The locations
of the new STRs are shown with black arrowheads on the
lower horizontal line. The bottom of the
figure shows the genomic clones from which the genome sequence was
derived (21).
|
|
identification of duplications in patients with cmt1a
Given the above data, we hypothesized that this panel of 15
markers would detect duplications accurately in the patients with
CMT1A. To test this hypothesis, we genotyped 39 unrelated individuals
who had previously been diagnosed with CMT1A duplication by multiple
criteria, including densitometry of restriction fragment length
polymorphism bands, detection of junction fragments with PFGE, and
identification of three alleles with microsatellite RM11-GT,
and/or FISH. No individual marker detected three alleles in every
patient. However, the combination of all markers identified
duplications (i.e., three alleles) in 39 of 39 samples (Table 3
). Furthermore, there were only 2 cases in which the duplication
was detected by a single marker; genomic rearrangements in the
remaining 37 patients were clearly visible by a minimum of 3 and a
maximum of 11 STRs (Fig. 2
).
View larger version (32K):
[in this window]
[in a new window]
|
Figure 2. Microsatellites detecting three alleles in patients
previously diagnosed with CMT1A.
(A), representation of genotypes from patients
previously diagnosed with CMT1A. Duplications were detected by the
presence of three alleles and could be clearly visualized with
dinucleotide (D17S2221, D17S2222),
trinucleotide (D17S2228), tetranucleotide
(D17S2224), and pentanucleotide
(D17S2227, D17S2230) markers.
(B), plot of the number of patients with duplications
(y axis) against the number of markers that detected
three alleles (x axis). With the exception of 2 patients
who were diagnosed by only one marker, duplications in all other 37
patients were detectable by three or more STRs.
|
|
|
Discussion
|
|---|
Recent advances in the Human Genome Project will not only impact
the identification of disease genes and further our understanding of
basic cellular processes, but are also likely to substantially improve
our ability to diagnose genetic disorders accurately, inexpensively,
and expeditiously. In this work, we used sequence information from the
CMT1A/HNPP genomic region (21) to identify and characterize
novel microsatellites. We also demonstrated that the combinatorial use
of a panel of 15 of these markers was able to detect duplications in
all 39 CMT patients who had been diagnosed previously to harbor such a
duplication. It is therefore possible to apply this marker set for
CMT1A diagnostic testing. Until now, CMT1A diagnosis has relied heavily
on densitometric analyses of restriction fragment length polymorphisms,
PFGE data, FISH, and quantitative determination of dosage, primarily
because of the reduced availability of polymorphic markers spanning the
CMT1A genomic region. Initial STR-based studies identified only 46% of
duplications (20), which was later improved to 80–90%
(12)(13). Despite these improvements, a
substantial fraction of CMT1A duplication cases remained undetectable,
necessitating the application of more time-consuming and expensive
techniques. The new STR panel presented here will substantially improve
the feasibility of the PCR-based diagnosis of CMT1A/HNPP because it can
potentially detect >99% of duplication cases. Furthermore, the
allele-size ranges of this new marker set were selected to allow the
construction of two panels of markers that are amenable to
multiplexing. Panel 1 alone, consisting of 10 markers, was sufficient
to identify 37 of 39 duplications in our cohort. We therefore recommend
that panel 2 needs to be used only in the absence of three alleles from
any panel 1 marker. This will allow high-throughput analysis and is
likely to reduce the cost of testing.
PCR-based data of HNPP deletions are typically less robust because they
do not allow direct detection of the deletion, but only infer the
deletion by detecting a single allele for any marker tested. The
availability of a large set of highly informative markers will improve
the ability to diagnose deletions because the probability that 15
highly polymorphic loci will be homozygous, rather than hemizygous in
any given patient sample, is small.
Our data are also relevant to understanding the mechanism by which the
duplication (CMT1A) and deletion (HNPP) are generated. Such genomic
rearrangements are the result of unequal crossing-over between a set of
low copy repeats flanking the 1.4-Mb CMT1A region. The finding of three
alleles in all duplicated patients indicates that homologous
recombination between chromosomes (interchromosomal homologous
recombination) is the more common cause of this rearrangement, rather
than intrachromosomal homologous recombination (23). The
latter would give rise to two copies of the allele, a phenomenon
detectable only through calculation of the ratios of the surface area
of each allele peak. Because this is less accurately detectable, the
presence of rare cases of intrachromosomal recombination in de novo
duplication patients is a potential error in informative,
polymorphic-allele PCR-based approaches and should be considered when a
third allele is not seen with any marker. In such cases, PFGE or FISH
would be the recommended method of choice for fully informative
analysis, but quantification of peak heights for multiple heterozygous
STR alleles may suffice.
|
Acknowledgments
|
|---|
We thank the members of the families that participated in this
study for their cooperation. We also thank Dr. John Belmont and the
Baylor Human Polymorphism Resource for providing DNA samples, Dr.
Cornelius Boerkoel for assistance with patient diagnosis, and the
members of the Lupski laboratory for critical evaluation of the
manuscript. K.I. is a recipient of a postdoctoral fellowship from the
Charcot-Marie-Tooth Association. This study was supported in part by
the National Eye Institute, NIH (N.K.); the National Institute for
Neurological Disorders and Stroke, NIH (J.R.L.); and the Muscular
Dystrophy Association (J.R.L.).
|
Footnotes
|
|---|
1 Nonstandard abbreviations: CMT, Charcot-Marie-Tooth disease; CMT1A-REP, CMT1A repeat; PMP22, peripheral myelin protein 22; HNPP, hereditary neuropathy with liability to pressure palsy; PFGE, pulsed-field gel electrophoresis; FISH, fluorescence in situ hybridization; STR, short tandem repeat; FAM, 6-carboxy-fluorescein; HEX, hexachloro-6-carboxyfluorescein; and TET, 4,7,2',7'-tetrachloro-6-carboxyfluorescein. 
|
References
|
|---|
-
Skre H. Genetic and clinical aspects of Charcot-Marie-Tooth’s disease. Clin Genet 1974;6:98-118.[ISI][Medline]
[Order article via Infotrieve]
-
Lupski JR, Garcia CA. Charcot-Marie-Tooth peripheral neuropathies and related disorders. Scriver CR Beaudet AL Sly WS Valle D Childs B Kinzler KW Vogelstein B eds. The metabolic and molecular bases of inherited disease, 8th ed 2000:5759-5788 McGraw-Hill Columbus, OH. .
-
Lupski JR, de Oca-Luna RM, Slaugenhaupt S, Pentao L, Guzzetta V, Trask BJ, et al. DNA duplication associated with Charcot-Marie-Tooth disease type 1A. Cell 1991;66:219-232.[ISI][Medline]
[Order article via Infotrieve]
-
Raeymaekers P, Timmerman V, Nelis E, De Jonghe P, Hoogendijk JE, Baas F, et al. Duplication in chromosome 17p11.2 in Charcot-Marie-Tooth neuropathy type 1a (CMT1a). Neuromuscul Disord 1991;1:93-97.[Medline]
[Order article via Infotrieve]
-
Pentao L, Wise CA, Chinault AC, Patel PI, Lupski JR. Charcot-Marie-Tooth type 1A duplication appears to arise from recombination at repeat sequences flanking the 1.5 Mb monomer unit. Nat Genet 1992;2:292-300.[ISI][Medline]
[Order article via Infotrieve]
-
Reiter LT, Murakami T, Koeuth T, Gibbs RA, Lupski JR. The human COX10 gene is disrupted during homologous recombination between the 24 kb proximal and distal CMT1A-REPs. Hum Mol Genet 1997;6:1595-1603.[Abstract/Free Full Text]
-
Boerkoel CF, Inoue K, Reiter LT, Warner LE, Lupski JR. Molecular mechanisms for CMT1A duplication and HNPP deletion [Review]. Ann N Y Acad Sci 1999;883:22-35.[ISI][Medline]
[Order article via Infotrieve]
-
Lupski JR. Charcot-Marie-Tooth disease: a gene-dosage effect [Review]. Hosp Pract 1997;32:83-122.
-
Chance PF, Abbas N, Lensh MW, Pentao L, Roa BB, Patel PI, Lupski JR. Two autosomal dominant neuropathies result from reciprocal DNA duplication/deletion of a region on chromosome 17. Hum Mol Genet 1994;3:223-228.[Abstract/Free Full Text]
-
Lupski JR. DNA diagnostics for Charcot-Marie-Tooth disease and related inherited neuropathies [Editorial]. Clin Chem 1996;42:995-998.[Free Full Text]
-
Roa BB, Ananth U, Garcia CA, Lupski JR. Molecular diagnosis of CMT1A and HNPP Charcot-Marie-Tooth disease type 1A; and hereditary neuropathy with liability to pressure palsy [Review]. Lab Med Int 1995;12:22-25.
-
Shaffer LG, Kennedy GM, Spikes AS, Lupski JR. Diagnosis of CMT1A duplications and HNPP deletions by interphase FISH: implications for testing in the cytogenetics laboratory. Am J Med Genet 1997;69:325-331.[ISI][Medline]
[Order article via Infotrieve]
-
Kashork CD, Lupski JR, Shaffer LG. Prenatal diagnosis of Charcot-Marie-Tooth disease type 1A by interphase fluorescence in situ hybridization. Prenat Diagn 1999;19:446-449.[ISI][Medline]
[Order article via Infotrieve]
-
Ruiz-Ponte C, Loidi L, Vega A, Carracedo A, Barros F. Rapid real-time fluorescent PCR gene dosage test for the diagnosis of DNA duplications and deletions. Clin Chem 2000;46:1574-1582.[Abstract/Free Full Text]
-
Cudrey C, Chevillard C, Le Paslier D, Vignal A, Passage E, Fontes M. Assignment of microsatellite sequences to the region duplicated in CMT1A (17p12): a useful tool for diagnosis. J Med Genet 1995;32:231-233.[Abstract/Free Full Text]
-
Blair IP, Kennerson ML, Nicholson GA. Detection of Charcot-Marie-Tooth type 1A duplication by the polymerase chain reaction. Clin Chem 1995;41:1105-1108.[Abstract/Free Full Text]
-
Young P, Stögbauer F, Wiebusch H, Löfgren A, Timmerman V, Van Broeckhoven C, et al. PCR-based strategy for the diagnosis of hereditary neuropathy with liability to pressure palsies and Charcot-Marie-Tooth disease type 1A. Neurology 1998;50:760-763.[Abstract/Free Full Text]
-
Poropat RA, Nicholson GA. Determination of gene dosage at the PMP22 and androgen receptor loci by quantitative PCR. Clin Chem 1998;44:724-730.[Abstract/Free Full Text]
-
Stronach EA, Clark C, Bell C, Lofgren A, McKay NG, Timmerman V, et al. Novel PCR-based diagnostic tools for Charcot-Marie-Tooth type 1A and hereditary neuropathy with liability to pressure palsies. J Peripher Nerv Syst 1999;4:117-122.[ISI][Medline]
[Order article via Infotrieve]
-
Wise CA, Garcia CA, Davis SN, Heju Z, Pentao L, Patel PI, Lupski JR. Molecular analyses of unrelated Charcot-Marie-Tooth (CMT) disease patients suggest a high frequency of the CMT1A duplication. Am J Hum Genet 1993;53:853-863.[ISI][Medline]
[Order article via Infotrieve]
-
Inoue K, Dewar K, Katsanis N, Reiter LT, Lander ES, Devon
KL, et al. The 1.4 Mb CMT1A duplication/HNPP deletion genomic region
reveals unique genome architectural features and provides insights into
the recent evolution of new genes. Genome Res 2001;in press..
-
Katsanis N, Lewis RA, Stockton DW, Mai PMT, Baird L, Beales PL, et al. Delineation of the critical interval of Bardet-Biedl syndrome 1 (BBS1) to a small region of 11q13, through linkage and haplotype analysis of 91 pedigrees. Am J Hum Genet 1999;65:1672-1679.[ISI][Medline]
[Order article via Infotrieve]
-
Lopes J, Vandenberghe A, Tardieu S, Ionasescu V, Levy N, Wood N, et al. Sex-dependent rearrangements resulting in CMT1A and HNPP. Nat Genet 1997;17:136-137.[ISI][Medline]
[Order article via Infotrieve]
The following articles in journals at HighWire Press have cited this article:
|
|
|
|
 
A. De Vos, K. Sermon, M. De Rijcke, V. Goossens, P. Henderix, N. Van Ranst, P. Platteau, W. Lissens, P. Devroey, A. Van Steirteghem, et al.
Preimplantation genetic diagnosis for Charcot-Marie-Tooth disease type 1A
Mol. Hum. Reprod.,
July 1, 2003;
9(7):
429 - 435.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. G.B. Leonard
Improved Method for Diagnosis of Charcot-Marie-Tooth Type 1A: Patent Pending?
Clin. Chem.,
May 1, 2001;
47(5):
807 - 808.
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
