Comparison of different techniques for detecting 17p12 duplication in CMT1A

Neuromuscular Disorders 15 (2005) 488–492 www.elsevier.com/locate/nmd

Comparison of different techniques for detecting 17p12 duplication in CMT1A Alessandra Patituccia,1, Maria Mugliaa,1, Angela Magarielloa, Anna Lia Gabrielea, Giuseppina Pelusoa, Teresa Sprovieria, Francesca Luisa Confortia, Rosalucia Mazzeia, Carmine Ungaroa, Francesca Condinoa, Paola Valentinob, Franco Bonob, Carmelo Rodolicoc, Anna Mazzeoc, Antonio Toscanoc, Giuseppe Vitac, Aldo Quattronea,b,* a

Institute of Neurological Sciences, National Research Council, Piano Lago di Mangone, Cosenza, Italy b Institute of Neurology, University ‘Magna Graecia’, Catanzaro, Italy c Department of Neurology, University of Messina, Messina, Italy Received 21 December 2004; received in revised form 25 March 2005; accepted 20 April 2005

Abstract Charcot–Marie-Tooth type 1A is caused by a 1.5 Mb DNA duplication in the 17p12 chromosomal region encompassing the peripheral myelin protein 22 gene. In the present study, we compared the Real-Time PCR with the other methods currently used for the diagnosis of Charcot–Marie-Tooth. By using a combination of junction fragment PCR, analysis of microsatellite markers, and pulsed field gel electrophoresis, we identified 76 unrelated patients with 17p12 duplication. In these patients, junction fragment PCR detected 63% of cases of duplication, the microsatellite markers method revealed 74%, while the combined use of microsatellite markers and junction fragment PCR revealed 91% of cases of Charcot–Marie-Tooth type 1A. Pulsed field gel electrophoresis detected 100% of the cases with duplication, even in presence of atypical 17p12 duplication. Real-Time PCR detected 100% of the cases with Charcot–Marie-Tooth type 1A and was comparable to pulsed field gel electrophoresis. However, in contrast to pulsed field gel electrophoresis, Real-Time PCR does not need fresh blood, minimizes diagnosis time and cost, and thus can be easily used for the molecular diagnosis of Charcot–Marie-Tooth type 1A. q 2005 Elsevier B.V. All rights reserved. Keywords: CMT1A; PFGE; Real-Time PCR

1. Introduction Charcot–Marie-Tooth (CMT) disease, also known as hereditary motor and sensory neuropathy (HMSN), includes a group of hereditary neuropathies affecting the peripheral nervous system. It is characterized by slowly progressive weakness and atrophy of the distal limb muscles [1]. The prevalence has been estimated at one in 2500 [2]. Two major clinical forms have been identified, CMT1 and CMT2, on the basis of electrophysiological studies. The most common form, * Corresponding author. Address: Cattedra ed U.O. di Neurologia, Facolta` di Medicina e Chirurgia, Universita` ‘Magna Graecia’, Via Tommaso Campanella, 88100 Catanzaro, Italy. Tel.: C39 0961775322; fax: C39 0961777775. E-mail address: [email protected] (A. Quattrone). 1 Both authors contributed equally to the manuscript.

0960-8966/$ - see front matter q 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2005.04.006

CMT1, is characterized by pes cavus, reduced or absent deeptendon reflexes, and hypertrophic nerves. The nerve conduction velocities (NCV) are reduced in both motor and sensor nerves; in particular, a NCV of the motor median nerve ! 38 m/s is currently deemed to be the crucial criterion for differentiating CMT1 and CMT2. This latter form displays normal to near normal NCVs (O38 m/s) associated with absence of demyelination/remyelination and evidence of axonal dysfunctions. CMT1 is genetically heterogeneous and five subtypes have been identified so far: CMT1A, CMT1B, CMT1C, CMT1D, and CMT1X [3–7]. The most frequent form, CMT1A, is caused by a 1.5 Mb DNA duplication in the 17p12 chromosomal region, arising after unequal crossingover between repeated sequences called CMT1A-REPs, flanking the 1.5 Mb unit. A 3.2 kb recombination hot spot has been defined, resulting in a junction fragment between EcoRI (distal CMT1A-REP) and SacI (proximal CMT1AREP) [8]. This region encompasses the peripheral myelin

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protein 22 gene (PMP22). Also, point mutations in PMP22 have been identified in non-duplicated CMT1A patients, confirming the direct role of the gene in the CMT1A disease process [9,10]. Currently, routine diagnosis of CMT1A is mainly performed with direct allele-specific amplification of the junction fragment by PCR or by using microsatellite markers within the 1.5 Mb region flanking the PMP22 gene [11–13]. Southern blot, PFGE, and Fluorescence in situ hybridization (FISH) are used as well [14–16]. In this study, we report the results obtained using RealTime PCR and a comparison of this technique with the other methods currently used for the diagnosis of CMT1A.

2. Patients and methods 2.1. Patients DNA analysis was carried out on 76 patients with CMT1A and 79 normal controls. Genomic DNA was extracted from peripheral blood samples using phenol–chloroform extraction. The presence of the duplication was first investigated by amplification of the 3.2 kb junction fragment by PCR analysis followed by restriction analysis with EcoRI. In all the patients, the duplication was also investigated using fluorescent PCR of microsatellite markers RM11, D17S1358, D17S1357, D17S1356, D17S921, D17S839, and D17S955. PCR reaction was performed on Perkin Elmer 9600 and the analysis was carried out using an ABI377 automatic DNA sequencer and Genescan 372 software (Applied Biosystems, Inc., Foster City, CA). In seven patients, PFGE has been performed isolating high molecular weight DNA from leucocytes embedded in agarose blocks and digested with restriction enzyme SacII. The DNA fragments were separated by PFGE in 1% agarose gel in 0.5! Tris–borate buffer at 14 8C using Bio-rad CHEF MAPPER XA. After agarose gel electrophoresis, the DNA fragments were transferred to a nylon membrane (Hybond NC, Amersham, England) and hybridized with radiolabelled probes PNEA1 and pVAW409R3a.

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were selected having melting temperatures between 51.4 and 50.8 8C in the exon 4 of PMP22 gene. The sequences of the primers and probe are: PMP22 exon 4 forward TCCCCCTGGCCCTTCTC; PMP22 exon 4 reverse CTGGGCGCCTCATTCG; PMP22 exon 4 TaqMan probe: 5 0 - 6 FAM-CGGTGTCATCTATGTGATCTTGCGGAAATAMRA. Probe and primers were stored in aliquots at K20 8C. The RNase P kit is provided by Applied Biosystems. Before starting our experiment we had to optimize the primers and probe concentrations: the optimal concentration provides the lowest threshold cycle (Ct) value and the highest increase of the fluorescence compared to the background. The concentration of the primers must be optimized by spanning an initial concentration range of 50–900 nM, while the concentration of the probe is in a range of 50–250 nM. Our results showed an optimal concentration of 200 and 300 nM for the PMP22 exon 4 TaqMan probe and each primer, respectively. To use the comparative Ct method (DDCt), a validation experiment must be run to show that the efficiencies of the target and the endogenous control amplifications are approximately equal. Then, we proceeded to analyze the samples and calibrate in triplicates and in parallel for PMP22 exon 4 and RNase P. 2.4. Quantitative analysis

The OD 260/280 method on a photometer was used to determine the appropriate DNA concentration. DNA was diluted in TE buffer (10 mM Tris–HCl 1 mM EDTA pH 8.0 made with HPLC-H2O) to a final concentration of 20 ng/ml, stored at 4 8C and mixed immediately before use.

PCR was carried out in a 384-well optical plate closed by adhesive cover using ABI Prism 7900 Sequence Detection System (Applied Biosystems) with a final reaction volume of 20 ml. All samples, prepared from the same master mix, were run in triplicate. For PMP22 assay, each well contained 20 ng/ml of DNA, 2! TaqMan PCR Master Mix (Applied Biosystems), 200 nM probe PMP22 exon 4, 300 nM of each primer, and HPLC pure water. In the TaqMan universal PCR master mix, there is a passive reference dye (ROX) included in the solution that does not participate in the reaction, but provides an internal reference for background fluorescence emission. This reference is used to normalize the reporter dye signal. RNaseP reference locus assay was prepared in parallel and in the same run; each well contains 20 ng/ml of DNA, 2! PCR Master Mix, 20! RNaseP, and HPLC pure water. Thermal cycling conditions included a pre-run of 2 min at 50 8C and 10 min at 95 8C. Cycle conditions were 40 cycles at 95 8C for 15 s and 60 8C for 1 min according to the TaqMan Universal PCR Protocol (Applied Biosystems); run time 1 h and 30 min. Calculation of the gene copy number was performed using the DDCt method that requires a healthy control sample (diploid) as a calibrator in all amplifications.

2.3. Probes and primers

2.5. Data analysis

TaqMan probe and primers were designed using the Primer Express software (Applied Biosystems, Weiterstadt) following the criteria indicated in the program. Primer pairs

Data evaluation was carried out using the ABI Prism Detection Software and Microsoft Excel. The threshold cycle number (Ct) was determined for all wells. The Ct

2.2. DNA concentration for Real-Time PCR

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represents the cycle number (DRn) at which the fluorescence emission of the reporter dye passed a fixed threshold. The threshold was automatically set at 10 standard deviations (SD) above the mean baseline emission. For all samples, including the calibrator, the same threshold and baseline were set. The starting gene copy number of the unknown samples was determined comparing it to the copy number of the calibrator sample applying the following formula previously described [17]: DDCtZ[DCt RNaseP (calibrator sample)KDCt PMP22 exon 4 (calibrator sample)]K[DCt RNaseP (unknown sample)KDCt PMP22 exon 4 (unknown sample)]. The relative gene copy number was calculated by the expression 2KDDCt. 2.6. Statistics The Mann–Whitney U-test was used to compare DDCt values between CMT1A patients and controls. Sensitivity, specificity, and Positive Predictive Value (PPV) for differentiating CMT1A patients from controls were calculated for all the used methods.

3. Results By using a combination of junction fragment PCR, analysis of microsatellite markers, and PFGE, we identified 76 unrelated patients with CMT1A. In 48 of 76 (63%) subjects, PCR detected the presence of a 3.2 kb junction fragment, the microsatellite markers method revealed 56 of 76 (74%), while the combined use of a junction fragment PCR and microsatellite markers revealed duplication in 69 out of 76 (91%) patients. The duplication in the samples by microsatellite analysis was inferred when at least one of the seven microsatellites showed the presence of three different

Table 1 DDCt values in examined samples

CMT1A patients (nZ76) Controls (nZ79)

Mean

SD

Min

Max

1.61 0.98

0.26 0.11

1.25 0.75

2.50 1.21

alleles. Seven of 76 (9%) patients did not show either 3.2 band or three different alleles, but they showed an allelic ratio indicative of duplication. Namely, three patients had an abnormal gene dosage with all used markers, while the remaining subjects showed an altered dosage with markers RM11 and D17S1357. In order to confirm the presence of the duplication in this group of patients, we performed PFGE in all these patients. PFGE was performed using SacII restriction enzyme, and pNEA1 and pVAW409R3a probes. By hydridization with pNEA1 in three patients we identified the additional 500 kb CMT1A fragment; the remaining four subjects showed a normal pattern (720, 600, and 550 kb). The hybridization with the pVAW409R3a probe revealed the 500 kb fragment in the first three patients, and about a 400 kb SacII fragment in the remaining samples (Fig. 1). Our findings indicated a smaller size of the duplication in this group of patients, as suggested by previous results obtained using microsatellite markers. All samples were also analyzed by Real-Time PCR. Quantitative PCR analysis showed that CMT1A patients had DDCt values of 1.61G0.26 SD (range 1.25–2.50), while control subjects had DDCt values of 0.98G0.11 SD (range 0.75–1.21) (Table 1). No overlap in DDCt values was found between patients and controls (Fig. 2). The current findings demonstrate that quantitative PCR analysis was able to detect 17p12 duplication in all the CMT1A patients. 2.5

∆∆Ct

2.0

1.5

1.0

0.5

Controls Fig. 1. PFGE analysis after hybridization with probe pVAW409R3a: DNA of a patient carrying a small size duplication (w400 kb) (lane 1), DNA of a CMT1A patient with the typical 1.5 Mb duplication (lane 2), and a normal sample (lane 3).

CMT1A

Fig. 2. On the y-axis are reported the DDCt values in control subjects and CMT1A patients calculated as described in Section 2. The controls ranged from 0.75 to 1.21 DDCt values (mean 0.98G0.11 SD), whereas the CMT1A patients ranged from 1.25 to 2.50 DDCt values (mean 1.61G0.26).

A. Patitucci et al. / Neuromuscular Disorders 15 (2005) 488–492 Table 2 Validity measures of DDCt, junction fragment PCR, and microsatellite markers Validity measures (%) CMT1A vs controls

DDCt

Junction fragment PCR

Microsatellite markers

Junction fragment PCRC Microsatellite markers

Sensitivity Specificity PPV

100 100 100

63 100 100

74 100 100

91 100 100

Seven patients who did not show 17p12 duplication on junction fragment PCR and microsatellite marker analysis underwent PFGE, which detects duplication.

The sensitivity, specificity and PPV for all used method are shown in Table 2.

4. Discussion The routine molecular test for diagnosis of CMT1A is based on the detection of the duplication on chromosome 17p12. Many methods have been used such as junction fragment PCR, microsatellite markers, Southern blotting, PFGE, and FISH. The detection of a recombinant hotspot by PCR analysis is accurate, rapid, and specific, with the results available within 24 h. This method is convenient to use because no radioisotope or hybridization reaction is needed. Our results, obtained in 76 patients with 17p12 duplication, showed that this technique detected duplication in 48 of 76 subjects (63%). The method of analysis by using the microsatellite markers detected 56 of 76 subjects (74%) and added information for detecting 17p12 duplication, because the combined use of the junction fragment PCR and microsatellite markers detected duplication in 91% of cases. The microsatellite marker is also a very fast procedure but, however, it is dependent on the informative ability of markers located in the 1.5 Mb genomic region. Seven patients who did not show evidence of duplication by junction fragment PCR or microsatellite analysis underwent PFGE that confirmed the duplication in all the cases. It is well known that FISH and PFGE can detect 100% of cases, even in presence of atypical duplication, but they have some disadvantages. Both of them are technically demanding, they need fresh blood or lymphoblastoid cells and may take about one week or more for the results. The recent advent of the Real-Time PCR technique has been applied for many investigations, including pathogen detection, gene expression and regulation, and allelic discrimination. Previous papers have used Real-Time PCR for the diagnosis of CMT1A. Some authors [18] investigated four CMT1A patients previously diagnosed with Southern Blot and microsatellite analysis by using the Real-Time PCR based on the combination of the competitive approach with Real-Time fluorescence PCR technology. In the meantime,

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a study by Wilke et al. [19] reported the development of a Real-Time PCR method for the diagnosis of eight CMT1A patients using an absolute method. In both studies, however, the number of examined patients was too small for having conclusive information on the validity of Real-Time PCR procedures for the diagnosis of CMT1A. A following study by Thiel et al. [20] was performed using the comparative Ct method to determine the PMP22 gene copy number. These authors analyzed 47 subjects with CMT1A previously investigated by polymorphic markers and only in one subject with FISH. The authors considered the subjects to be positive for the duplication when they showed at least three alleles in one marker and one significant dosage difference in at least one other marker or a dosage difference in at least two independent markers. All subjects had 17p12 duplication on Real-Time PCR. In the above mentioned study, however, the Real-Time PCR was compared to microsatellite analysis, a technique that lacks high specificity for detecting 17p12 duplication, mainly when gene dosage is used [21]. Differing from the Thiel’s report, in the present study, the patients with CMT1 underwent detailed molecular analysis for detecting duplication including combined procedures based on junction fragment PCR, microsatellite markers, and PFGE. Our findings demonstrate that Real-Time PCR was able to detect duplication in all the patients. Indeed, all the CMT1A patients had DDCt values significantly higher than those observed in normal samples, and no overlap in DDCt values was found between patients and controls. RealTime PCR was also useful for detecting duplication in seven cases (9%) that could not be diagnosed by rapid tests such as microsatellite markers and junction fragment PCR, in which the molecular diagnosis was obtained using only a complicated procedure like PFGE that is time consuming and labor intensive. Real-Time PCR was also able to detect duplication in four of these seven patients who had an atypical duplication on PFGE. This finding further supports the usefulness of Real-Time PCR for diagnosis of CMT1A because, in the presence of a small size duplication, the microsatellite method could give a misdiagnosis when the markers located very near to the PMP22 gene (RM11 and D17S1357) are not informative and the additional ones have a normal gene dosage. In conclusion, Real-Time PCR is a valid and sensitive method for detecting 17p12 duplication. Moreover, this technique does not need fresh blood and minimizes diagnosis time and cost.

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