Clinical Chemistry 52: 1267-1275, 2006.
First published April 27, 2006; 10.1373/clinchem.2006.067967
(Clinical Chemistry. 2006;52:1267-1275.)
© 2006 American Association for Clinical Chemistry, Inc.
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Molecular Diagnostics and Genetics |
Multiplex Ligation-Dependent Probe Amplification for Rapid Detection of Proteolipid Protein 1 Gene Duplications and Deletions in Affected Males and Carrier Females with Pelizaeus–Merzbacher Disease
Ilka Warshawsky1,a,
Olga B. Chernova2,
Christian A. Hübner6,
Reinhard Stindl7,
Marco Henneke8,
Andreas Gal6 and
Marvin R. Natowicz1,3,4,5
1 Department of Clinical Pathology, 2
Brain Tumor Institute, 3
Department of Pediatrics, 4
Department of Neurology, and 5
Genomic Medicine Institute, Cleveland Clinic Foundation, Cleveland, OH.
6 Institute of Human Genetics, University Hospital Eppendorf, Hamburg, Germany.
7 Department of Human Genetics, Medical University of Vienna, Vienna, Austria.
8 Department of Pediatrics and Pediatric Neurology, Georg August University, Göttingen, Germany.
aAddress correspondence to this author at: Department of Clinical Pathology/L30, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Fax 216-445-9444; e-mail warshai{at}ccf.org.
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Abstract
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Background: Pelizaeus–Merzbacher disease is a rare X-linked neurodegenerative disorder caused by sequence variations in the proteolipid protein 1 gene (PLP1). PLP1 gene duplications account for 
50%–75% of cases and point variations for 15%–20% of cases; deletions and insertions occur infrequently. We used multiplex ligation-dependent probe amplification (MLPA) to detect PLP1 gene alterations, especially gene duplications and deletions.
Methods: We performed MLPA on 102 samples from individuals with diverse PLP1 gene abnormalities and from controls, including 50 samples previously characterized for the PLP1 gene by quantitative PCR but which were anonymized for prior results and sex.
Results: All males with PLP1 gene duplications (n = 13), 1 male with a triplication, 2 males with whole gene deletions, and all controls (n = 72) were unambiguously assigned to their correct genotype. Of 4 female carriers tested by MLPA and quantitative PCR, 3 were duplication carriers by both methods, and 1 was a triplication carrier by MLPA and a duplication carrier by quantitative PCR. For 1 sample with a partial deletion, MLPA showed exon 3 deleted but PCR showed exons 3 and 4 deleted. Sequence analysis of 2 samples with reduced dosage for exons 3 and 5 revealed point variations overlapping the annealing site for the corresponding MLPA probe. The precision of MLPA analysis was excellent and comparable to or better than quantitative PCR, with CVs of 4.3%–9.8%.
Conclusions: MLPA is a rapid and reliable method to determine PLP1 gene copies. Samples with partial PLP1 gene dosage alterations require confirmation with a non-MLPA method.
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Introduction
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Pelizaeus–Merzbacher disease (PMD)1
and the allelic disorder spastic paraplegia type 2 (SPG2) are rare X-linked recessive dysmyelination disorders caused by sequence variations in the proteolipid protein 1 (PLP1)2
gene (1)(2)(3). The PLP1 gene maps to chromosome Xq22, contains 7 exons, and spans 17 kb; together with its isoform DM20, PLP1 forms 50% of the protein content of myelin in the central nervous system(1)(2)(3)(4)(5). PLP1 gene duplication is the most common cause of PMD and accounts for 50%–75% of cases(1)(2)(3)(6)(7)(8)(9)(10). Patients with increased PLP1 dosage have variable phenotypes ranging from severe connatal PMD to mild PMD/SPG2, but most have a classic form of disease(1)(2)(3).
PLP1 gene duplications are typically arranged in a tandem head-to-tail orientation at Xq22 and are thought to form by coupled homologous and nonhomologous recombination (7)(8)(11). Each duplication event includes the entire PLP1 locus, appears unique, and varies in size from 100 kb to 4.6 Mb(7)(8)(11). Proximal duplication breakpoints vary widely between families, whereas distal breakpoints tend to cluster around low-copy repeats(7)(8)(11). Atypical PLP1 duplications, termed submicroscopic transposons(3), in which the extra copy of PLP1 is many megabases away from the Xq22 locus, have been found at Xp22.1, Xp11.4, and Xq26(3)(12).
Animal models support PLP1 gene duplication as a molecular basis for disease because neurologic symptoms and disease severity in transgenic mice correlate with PLP1 gene copy number and overexpression (13)(14). In humans, the presence of 3 or more copies of PLP1 is associated with severe disease, although patients with 3 and 5 PLP1 gene copies do not show significant differences in clinical signs(15). In 15%–20% of cases, PMD is caused by PLP1 point variations; PLP1 deletions and insertions occur infrequently. The clinical phenotypes of patients with point variations, especially those involving conserved nucleotides, are usually more severe than those of patients with duplications, and patients with a complete loss of PLP1 appear to be less severely affected(1)(2)(3)(16).
Detecting gene copy number changes is not straightforward because the assay must be quantitative (17). Many techniques have been used to identify PLP1 gene duplications(6)(7)(9)(10)(12)(18)(19)(20)(21)(22)(23). Multiplex ligation-dependent probe amplification (MLPA) is a technique for detecting duplications and deletions and is based on the ligation of 2 adjacent annealing oligonucleotides followed by quantitative PCR amplification of the ligated products(24). The authors of a recent comparison of MLPA with fluorescence in situ hybridization (FISH) using samples from 5 males with atypically severe PMD reported that MLPA is more accurate than FISH in determining specific PLP1 copy number(15); a comparative analysis of MLPA with quantitative PCR has not yet been reported. We used MLPA to measure PLP1 gene dose in 102 samples from males and females with diverse types of PLP1 gene abnormalities (duplications, triplications, deletions, point variations), individuals with sex chromosome aneuploidies, and numerous controls.
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Materials and Methods
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samples
We analyzed 102 samples by MLPA, including 50 tested previously by quantitative PCR (21), which were anonymized with respect to result and sex (12 males with a duplication, 1 male with a triplication, 2 males with a whole gene deletion, 3 female duplication carriers, 1 female triplication carrier, 19 non-PMD males, and 12 non-PMD females) and 52 additional samples (5 with PLP1 missense variations, 1 with an intragenic PLP1 deletion, 1 male with a known PLP1 duplication and his mother, 3 with sex chromosome aneuploidies, 17 control males, and 24 control females). Eight DNA samples with known PLP1 sequence variations and 3 with sex chromosome abnormalities were obtained from the Coriell Cell Repository (Camden, NJ) for use as positive controls (see Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol52/issue7/). Genomic DNA was isolated from peripheral blood leukocytes from control males (n = 17) and females (n = 24) with Puregene reagents (Gentra Systems, Inc.).
mlpa analysis
The principle for MLPA has been described previously (24). The SALSA PLP1 region test reagent set (P022; MRC-Holland) contains 32 probes, including 1 probe for each of the 7 PLP1 exons, 8 control probes on other parts of the X chromosome, 1 probe that detects a Y-chromosome sequence, and 16 probes that detect sequences on autosomes. A list of these probes is available at the MRC-Holland web site (www.mrc-holland.com). Probe sequences for each of the 7 PLP1 exons were provided by MRC-Holland. All runs included DNA from 3 unrelated control females to calibrate unknown samples. MLPA was performed according to the manufacturer’s recommendations, using 100 ng of DNA per reaction, except that reagent volumes were reduced by 50%. Reaction products were detected with an ABI 3100 Genetic Analyzer (Applied Biosystems). We used GeneScan and Genotyper software to size the PCR products and to obtain peak areas. These data were exported into a Microsoft Excel spreadsheet for gene dosage determinations.
PLP1 gene dosage determination by mlpa
We normalized each PLP1 and X-chromosome–specific locus individually to each of the 16 control autosomal loci (C1 to C16) within test samples and then performed calibration to the mean from 3 control females with the formula: [test peak area 1/test peak area C1, C2, ... C16]/[mean peak area 1 from 3 female controls/mean peak area C1, C2, ... C16 from 3 female controls]. We performed these calculations (see Table 3 in the online Data Supplement for an example) by modifying an Excel template used for BRCA1 gene dosage testing (http://leedsdna.info/science/dosage/Conventional_MLPA/Conventional_MLPA.htm). Averaging values from the 7 PLP1 exons and dividing by the mean of all X-chromosome loci allows for estimation of relative PLP1 and X-chromosome copy numbers. Expected PLP1:X ratios for non-PMD males, non-PMD females, males with a PLP1 duplication, and female carriers of a PLP1 duplication are 1, 1, 2, and 1.5, respectively.
quantitative pcr
Quantitative real-time PCR analysis of 50 samples used here was described previously (21). Briefly, PLP1 exon 4 and cystic fibrosis transmembrane conductance regulator (CFTR) exon 4 were coamplified with SYBR-Green PCR Master Mix (Applied Biosystems) in the Rotor-Gene real-time cycler (Corbett Research). PLP1 copy number was determined after normalization to CFTR and an unaffected female control. We analyzed samples in duplicate and in 2 separate experiments with values of 0.35–0.59 and 0.86–1.18, corresponding to 1 and 2 PLP1 gene copies, respectively. Quantitative real-time PCR performed by Regis et al.(20) used TaqMan probes specific for PLP1 exon 3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) exon 7 on the ABI Prism 7700 sequence detection system (Applied Biosystems). PLP1 and GAPDH were run separately and in triplicate. Absolute PLP1 gene copy numbers were determined from the ratio between PLP1 and GAPDH mean copy number with male DNA as a calibrator. Values of 0.84–1.16, 1.84–2.14, and 2.73–2.99 correspond to 1, 2, and 3 PLP1 gene copies, respectively.
fish
Metaphase chromosomes and interphase nuclei were prepared from peripheral leukocytes by standard protocols. FISH was performed as described by Woodward et al. (7) with 2 cosmids, U125A1 (PLP1) and U144A10 (control, hybridizes distal to the PLP1 locus)(7). We obtained clones from the Wellcome Trust Sanger Institute (Hinxton, United Kingdom; www.sanger.ac.uk). DNA was labeled with either biotin-16-dUTP (control) or digoxigenin-11-dUTP (PLP1) by nick translation. Labeled probes were detected by fluorescein isothiocyanate conjugated to avidin or rhodamine conjugated to anti-digoxigenin. 4',6-Diamidino-2-phenylindole, dihydrochloride (DAPI) was used as a counterstain. Cells were analyzed by fluorescence microscopy (Zeiss Axioplan 2), and images were taken with a M300 digital camera (JAI) connected to an ISIS workstation (Metasystems). Both cosmid probes were simultaneously hybridized to slides from patients, carriers, and controls. Successful hybridization to Xq22 was confirmed by inspection of red (PLP1) and green (control) signals on metaphase chromosomes. The observation of a PLP1:control ratio 
2:1 in more than 70% of the nuclei for male specimens defines PMD attributable to multiple copies of PLP1(22). Probe signals from 90–100 nuclei per slide were scored.
PLP1 pcr and sequencing
Each PLP1 exon was individually amplified by PCR in a 50-µL reaction that contained 20–100 ng of DNA, 25 µL of 2x Taq PCR master mixture (Qiagen), 10 µL of 5x Q solution (Qiagen), and 0.5 µM each of the forward and reverse primer. Q solution was omitted for exon 6. Primers (Invitrogen) flanked each exon and were designed with the human PLP1 sequence (GenBank accession no. AJ006976) and the Primer3 design program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi.) Primers were tested for specificity by National Center for Biotechnology BLAST software (http://www.ncbi.nlm.nih.gov/BLAST/). The primer sequences and expected PCR product sizes are listed in Table 2 of the online Data Supplement. PCR was performed with 1 cycle of 95 °C for 3 min followed by 45 cycles of 95 °C for 15 s, 55 °C for 15 s, and 72 °C for 15 s with a final 10-min extension at 72 °C and a 4 °C hold. PCR product sizes were verified by 2% agarose gel electrophoresis. After excess primers and nucleotides were removed from PCR products with ExoSAP-IT (USB), bidirectional DNA sequencing was performed with the same forward and reverse primers used for PCR and the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems) on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems).
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Results
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PLP1 mlpa analysis
The 32 MLPA probes in the PLP1 reagent set are summarized in Fig. 1
. Seven probes are specific to exons 1–7 of PLP1 and map to Xq22. Eight probes are specific to other X-chromosome regions. The 16 autosomal probes used to normalize gene dosage are also shown. One additional probe detects Y-chromosome sequence (Yp11). Typical electropherograms performed after MLPA of control female (Yp11 peak absent) and male (Yp11 peak present) DNA are shown in Fig. 1 of the online Data Supplement. We determined the relative PLP1 and X-chromosome copy numbers from peak areas by normalizing each PLP1 and X-chromosome–specific probe to each autosome, using normal female DNA as a calibrator. An example of how PLP1 gene dosage was calculated for a PLP1 duplication-positive male (Coriell NA11005) is shown in Table 3 of the online Data Supplement.
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Figure 1. Schematic diagram depicting the position of the MLPA probes (stars) on chromosome X (ChrX) and in the PLP1 gene.
The PLP1 exon structure is not drawn to scale. The locations of autosomal MLPA probes used for normalization are listed on the right. The Y-chromosome–specific probe (Yp11) for sex identification is also indicated.
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establishment of reference intervals
Control DNA samples were obtained from the Coriell Cell Repository (see Table 1 in the online Data Supplement). MLPA analysis using DNA from a non-PMD male, a non-PMD female, a male with a PLP1 duplication (Coriell NA11005), a female carrier of a PLP1 duplication (CH21; Fig. 3
), and a female carrier of a PLP1 triplication (CH16; Fig. 3
) was repeated in triplicate and in 3 separate assays to determine intra- and interassay variation. The PLP1:X ratios from these experiments are summarized in Fig. 2A
. Within- and between-run SDs were routinely <10% of the mean; CVs are indicated. Samples with and without gene dosage alterations were clearly distinguishable. Coriell NA11002, from the mother of a male with a PLP1 duplication (Coriell NA11005), gave PLP1:X ratios of 1.44, 1.61, 1.40 in 3 separate experiments, consistent with a female duplication carrier carrying 3 PLP1 gene copies (data not shown). Shown in Fig. 2B
are the relative copy numbers for each PLP1 exon after normalization to autosomal controls and calibration to female DNA from the replicates in Fig. 2A
. Relative copy numbers for each exon were 1 for the control female, 0.5 for the control male, 1 for the male with a PLP1 duplication, 1.5 for the female carrier of a PLP1 duplication, and 1.9 for the female carrier of a PLP1 triplication. The relative PLP1, X chromosome, and autosome copy numbers measured by use of 45,X; 47,XXY; and 47,XXX[49]/46,XX[1] DNA after normalization to normal female DNA are shown in Fig. 2C
. 45,X DNA has PLP1 and X relative copy numbers close to 0.5, consistent with 1 copy of the X chromosome; 47,XXY DNA gives PLP1 and X relative copy numbers of 0.9, close to the expected ratio of 1 for 2 copies of the X chromosome; 47,XXX [49],46,XX[1] DNA shows PLP1 and X relative copy numbers of 1.4, consistent with 3 copies of the X chromosome. The low degree of chromosomal mosaicism (2%) in this specimen did not affect gene dosage determination. Relative autosome copy numbers are 1 for 45,X; 47,XXY; and 47,XXX DNAs.
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Figure 3. PLP1:X ratios after MLPA analysis of 50 samples previously tested by quantitative PCR.
The samples consisted of 19 normal non-PMD males, 12 normal non-PMD females, 12 males with PLP1 duplication, 1 male with PLP1 triplication (CH44), 2 males with PLP1 deletions, 3 female carriers of PLP1 duplication, and 1 female carrier of PLP1 triplication (CH16).
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Figure 2. MLPA reproducibility.
(A), MLPA was performed on a normal non-PMD female, a normal non-PMD male, a male with PLP1 duplication (NA11005), a female carrier with PLP1 duplication (CH21), and a female carrier with PLP1 triplication (CH16) in triplicate and in 3 separate experiments. Columns correspond to the mean (SD; error bars) PLP1:X ratio for the indicated numbers of replicates. The interassay variability of PLP1:X ratios (SDs) and CVs from the 3 experiments are indicated. (B), mean (SD; error bars) PLP1 copy number for each PLP1 exon from samples used in panel A relative to normal female DNA. (C), MLPA analysis of 45,X; 47,XXY; and 47,XXX DNA. After normalization to female DNA, peak areas for each of the 7 PLP1 exons, the first 6 X-chromosome–specific peaks, and 16 autosome peaks were averaged. Columns correspond to mean (SD; error bars).
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The CVs for MLPA compared with 2 different quantitative PCR assays in males with 1 or 2 PLP1 gene copies and females carrying 2 or 3 PLP1 gene copies, after calibration to female [MLPA and quantitative PCR 1 (21)] or male DNA [quantitative PCR 2(20)] are shown in Table 1
. CVs for MLPA were 4.3%–9.8%, lower than or comparable to those for quantitative PCR (7.8%–14% and 3.0%–9.2% for quantitative PCR analyses 1 and 2, respectively). Ranges [mean (2 SD)] showed no overlap between cutoffs for 1, 2, and 3 PLP1 gene copies for MLPA and quantitative PCR 2 but a small degree of overlap between females with 2 (0.86–1.18) and 3 (1.15–1.99) PLP1 gene copies for quantitative PCR 1.
validation of mlpa by use of blinded samples previously characterized by quantitative pcr
For 50 samples previously analyzed by quantitative real-time PCR for PLP1 gene duplications/deletions (21), we performed MLPA to validate this assay. The mean (SD) PLP1:X ratios for these samples were 1.01 (0.066) for healthy non-PMD males (n = 19), 1.01 (0.027) for healthy non-PMD females (n = 12), and 1.89 (0.096) for duplication PMD males (n = 12; Fig. 3
). Sample CH44 showed a PLP1:X ratio of 2.88, consistent with a triplication male. Two males had all 7 PLP1 exons deleted (also see Fig. 5
). Four samples were consistent with carrier females and had PLP1:X ratios of 1.88 (CH16), 1.45, 1.44, and 1.39 (CH21). Samples CH16 and CH21 were repeated in triplicate and in 3 separate experiments (Fig. 2
, A and B) and were consistent with carriers of a triplication and duplication, respectively. We observed complete agreement between MLPA and quantitative PCR for all 50 samples except for CH16, which was a triplication carrier by MLPA and a duplication carrier by quantitative PCR. FISH was performed in an attempt to resolve this discrepancy. Selected cells from FISH analysis of CH16 as well as CH44, the son of CH16, are shown in Fig. 4
. In both samples, amplification of the PLP1 region (2–3 copies) was seen on the single X chromosome in CH44 (son) and on 1 of the 2 X chromosomes in CH16 (mother). Although several cells contained an enlarged signal (Fig. 4
, open arrows), 3 separate signals (Fig. 4
and inset, filled arrows) could be seen in only 30% of the cells. We scored 60% of the cells as a duplication, which could be a consequence of signals being so close together that they could not be resolved. This limitation of interphase FISH has been noted previously(7)(15).
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Figure 5. MLPA analysis of samples showing deletions.
(A), electropherograms after MLPA analysis of a sample with deletion of all 7 PLP1 exons (top); sample NA13434, in which PLP1 exon 3 was deleted (middle), and a wild-type male control (bottom). The positions of PLP1 exons 1–7 are boxed. Peaks on other regions of the X chromosome, autosome peaks, the Y-chromosome peak, and peak sizes (in bp) are indicated. (B), PCR analysis of samples in panel A. After PCR of each PLP1 exon, PCR products were separated by agarose gel electrophoresis. No PCR products are visible in the DNA from the sample in A with all PLP1 exons deleted (lanes 1A–7A). NA13434 (lanes 1B–7B) shows no PCR products for exons 3 and 4 (lanes 3B and 4B). PCR products are visible for all PLP1 exons in DNA from a wild-type male (lanes 1C–7C).
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Figure 4. Interphase and metaphase FISH on leukocytes of affected male CH44 and his mother, CH16.
The PLP1 cosmid U125A1 (red) and the control cosmid U144A10 (green) were used (7). (A), hybridization patterns of 3 interphase nuclei from the mother indicate amplification of PLP1 (2–3 copies) on 1 X chromosome. The nucleus on the left shows 3 adjacent red signals (filled arrow), not counting the red signal of the normal X chromosome. The other 2 nuclei (open arrows) were scored as being duplicated because of confluent signals, which could not be resolved as 3 single dots. Successful hybridization of the PLP1 cosmid (red) and the control cosmid (green) to Xq22 is shown on metaphase chromosomes. (B), interphase nucleus from the affected male. The 3 adjacent red signals (filled arrow) indicate a triplication of PLP1, which was seen in only 30% of the cells. The green signal confirms a single X chromosome.
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detection of PLP1 gene deletions by mlpa and pcr
Two samples from Fig. 3
had whole PLP1 gene deletions, and Coriell NA13434 has a partial PLP1 deletion of 49 amino acids spanning part of exon 3, intron 3, and part of exon 4 (25). MLPA analysis of 1 sample with all 7 PLP1 exons deleted is shown in the top panel of Fig. 5A
; analysis of Coriell NA13434, which shows PLP1 exon 3 deleted, is shown in the middle panel, and analysis of a wild-type male control is shown in the bottom panel. These 3 samples were also subjected to PCR and agarose gel electrophoresis using PCR primers flanking each PLP1 exon. As expected, the sample with all 7 PLP1 exons deleted, as determined by MLPA, had no PCR product for all 7 exons (Fig. 5B
, lanes 1A–7A). NA13434 had no PCR product for PLP1 exons 3 and 4 (Fig. 5B
, lanes 3B and 4B), but did have PCR products for exons 1, 2, 5, 6, and 7 (Fig. 5B
, lanes 1B, 2B, and 5B–7B). Because MLPA analysis of NA13434 showed exon 3 deleted whereas PCR showed exons 3 and 4 deleted, this suggested that the exon 4 MLPA probe(s) hybridized to a region of exon 4 that was not deleted, a theory we confirmed by comparing the MLPA exon 4 probe sequence with the deleted PLP1 sequence(25).
mlpa analysis of samples with PLP1 point variations
Relative PLP1 copy numbers for each of the 7 PLP1 exons in DNA from 5 male samples with known PLP1 missense variations are shown in Fig. 2 (lanes 1A–5A) of the online Data Supplement. Sequence confirmation of these variations (lanes 1B–5B in Fig. 2 of the online Data Supplement) along with a nonmutation control (lanes 1C–5C in Fig. 2 of the online Data Supplement) is also shown. NA13433 and N13436 are each hemizygous for an exon 2 PLP1 sequence variation (P14L; 41C>T and T42I; 125C>T, respectively), whereas NA13437 is hemizygous for an exon 3 PLP1 sequence variant (T115K; 344C>A). These samples show relative PLP1 copy numbers of 0.5 for each PLP1 exon. NA13435, which is hemizygous for an exon 3 silent sequence variant (G127G; 381C>G), and NA09546, which is hemizygous for an exon 5 variant (P215S; 643C>T), show reduced signals for exons 3 and 5, respectively. Aligning the exon 3 MLPA probe sequence with the NA13435 exon 3 sequence and aligning the MLPA exon 5 probe sequence with the NA09546 exon 5 sequence show that the probes overlap the sites of sequence variations in these 2 samples (data not shown). Thus, imperfect annealing of the probes to the target most likely accounts for the reduced exon 3 and 5 signals in these samples.
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Discussion
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Our results suggest that MLPA has important advantages over quantitative PLP1 PCR assays that determine gene copy number from analysis of 1 or 2 PLP1 exons (6)(7)(10)(20)(21). Quantitative PCR assays may miss rare PMD cases with partial PLP1 deletions if the exon being amplified is not tested. To test all exons in duplicate or triplicate by real-time quantitative PCR is tedious and expensive; if target and reference genes are not multiplexed(20), this further increases cost and reaction numbers. MLPA, in contrast, enables rapid analysis of all exons in a single tube. In addition, MLPA has been reported to be more precise and accurate than quantitative PCR: Damgaard et al.(26) reported that CVs were larger for real-time PCR than for MLPA when evaluating LDL receptor gene deletions. In the present study, we found that CVs for MLPA were similar to or lower than those for quantitative PCR. Another important advantage of the MLPA assay is that it confirms sex with a Y-chromosome–specific probe and thus may uncover sample mix-ups.
Interphase FISH can produce false-positive results if regions adjacent to, but not including, the PLP1 gene are duplicated and the FISH probe includes sequences outside the PLP1 gene. Both MLPA and quantitative PCR eliminate detection of X-chromosome duplications that are near but do not include the PLP1 locus. A recent report (15) stating that FISH is unreliable for PLP1 dosage detection above the level of a duplication was confirmed by our analysis of the PLP1 triplication status of a mother and son. Differentiating triplication from duplication is difficult by FISH if signals are too close together to resolve(7)(15). FISH, however, can identify rare PMD cases where the PLP1 gene has duplicated and integrated into a new position in the genome(3)(12).
If samples show partial deletions by MLPA, confirmation with an independent method is required to map deletion borders. Similarly, samples with reduced probe binding for a single exon should be verified with a non-MLPA method because point variations, deletions, and insertions that span where probes bind or are close to the ligation site of a pair of probes may affect probe ligation and cause reduced signals (27)(28).
In an unusual case of PMD, mosaicism in the lymphoblastoid cell lines of a PLP1 duplication carrier mother was demonstrated, but quantitative fluorescent multiplex PCR did not detect the duplication (7)(29). MLPA, like quantitative PCR, probably would not have detected this unusual event because detection of mosaicism would depend on the degree of mosaicism.
Several methods have been used to analyze MLPA dosage data (27). In global normalization, individual peak areas are divided by the combined peak area of all probes and the resulting relative peak area is divided by relative peak areas from control DNA. This has been used for genes such as dystropin(30) or BRCA1(31) because only a few exons may show deletions or duplications. However, when a large proportion of the probes have the potential for gene dosage alterations, averaging all peaks within the sample could skew results; therefore, control probes for loci elsewhere in the genome should be used. We separately normalized PLP1 exons and X-chromosome loci to autosomes because all PLP1-specific probes have the potential to be affected and males and females have different X-chromosome copy numbers.
Various methods have been used to define reference intervals for gene dosage alterations. One approach is to score duplications and deletions based on defined increases or decreases in peak areas (28)(30). A more statistical approach uses SDs for each probe. This approach enables observation of the performance of each probe, removal of probes with suboptimal performance (those with high SDs), and definition of samples as unacceptable for accurate gene dosage determination. SDs also allow assessment of the performance of individual samples used for calibration. Although the use of a premixed pool of DNA(23) could reduce the number of controls run, it does not allow scrutiny of the performance of individual DNAs in the pool. Running unknown samples in duplicate and more than 3 calibration controls may be useful because of variability during PCR, which may produce unacceptable SDs in one of the replicates or controls.
SDs can also be obtained from the mean of probe groups. When MLPA was used to detect trisomy of chromosomes 13, 18, and 21 (32), means for chromosome-specific sets of probes were determined and threshold values were set to differentiate normal from trisomy. A similar approach can be used for PLP1 dosage by use of the mean of PLP1 exons 1–7. Because partial gene dosage alterations can occur, reference intervals must be defined for each PLP1 exon.
In conclusion, we found that MLPA is a rapid and accurate method for detecting PLP1 gene duplications and deletions. The relative rapidity and ease of performance of the assay and good intra- and interassay precision are advantages over other commonly used methods of PLP1 gene copy determination.
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Acknowledgments
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We thank the Coriell Cell Repository for providing numerous samples for this study.
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Footnotes
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1 Nonstandard abbreviations: PMD, Pelizaeus–Merzbacher disease; SPG2, spastic paraplegia type 2; PLP1, proteolipid protein 1; MLPA, multiplex ligation-dependent probe amplification; and FISH, fluorescence in situ hybridization. 
2 Human genes: PLP1, proteolipid protein 1; BRCA1, breast cancer 1, early onset; CFTR, cystic fibrosis transmembrane conductance regulator; and GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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Abstract
Introduction
