Rapid detection of a recombinant hotspot associated with Charcot--Marie--Tooth disease type IA a duplication by a PCR-based DNA test.
Charcot-Marie-Tooth (CMT) disease is a group of genetically heterogeneous disorders that comprise motor and sensory neuropathies.  Type 1 CMT (CMT1) disease, the demyelinating or hypertropic form, is the most common form among the subtypes of CMT disease [1-3]. CMT patients have slowly progressive motor weakness and muscle atrophy, mainly in the distal limbs, and sensory deficits. CMT1 is associated with reduced nerve conduction velocity due to demyelination. CMT1A disease, which is the most frequent type, is linked to chromosome 17p11.2 [4,5]; CMT1B disease, caused by the gene coding for the myelin protein [P.sub.0], is linked to 1822 [6-9]. The abnormally decreased nerve conduction velocity observed in CMT1A patients, though genetically heterogeneous, correlates directly with the stable inheritance of a 1.5-Mb duplication on chromosome 17p11.2-12 [10-12]. Approximately 70% of unrelated patients and 90% of sporadic cases demonstrate the CMT1A duplication, which is caused by an unequal crossing-over event between misaligned flanking CMT1A repeat sequences (REPs) [11-15]. The 1.5-Mb duplication region also contains the dosage-sensitive gene coding for the peripheral myelin protein 22, mutations that are responsible for the phenotype in CMT1 patients without duplication [16-20].
Several methods are used to detect the CMT1A duplication, such as detection of a junction fragment by pulsed-field gel eletrophoresis (PFGE)  measurement of the dosage difference between restriction fragment length polymorphic (RFLP) alleles or analysis of polymorphic [(GT).sub.n] repeats  detection of three unique alleles by analyzing a set of polymorphic [(AC).sub.n] repeat markers , and fluorescence in situ hybridization (FISH) [11,18]. These methods require radioisotopes or complicated procedures, and they are time consuming and labor intensive. Moreover, the RFLP dosage test and polymorphic repeat markers are uninformative if the patient is homozygous and an alternative test is required.
Through the detection of novel junction fragments from the recombinant CMT1A-REPs in CMT1A patients, a 3.2-kb recombination hotspot within the approximately 30-kb CMT1A-REPs was identified [22-25]. These recombination events or CMT1A duplications were directly detected by Southern transfer analysis [22,26] and the 3.2-kb recombination hotspot accounted for more than three-quarters of the cases of CMT1A-REP duplication among our patients. In this study, we developed a PCR-based method that is simpler, quicker, and more convenient than those previously mentioned methods in diagnosing CMT1A duplication [11,13, 21]. Locus-specific PCR amplification was performed, and then the PCR products were cut by a restriction enzyme and visualized in agarose gel, giving a clear and specific pattern for CMT1A patients who have a recombinant CMT1A-REP formed by misalignment of the distal and proximal CMT1A-REPs and crossing-over in the 3.2-kb recombination hotspot area. This method may be used routinely for clinical diagnosis as a rapid screening test, but it will not detect all CMT1A duplications; rather it detects only those within the recombination hotspot.
Materials and Methods
Blood samples from 31 patients with CMT1A and their families and 50 healthy individuals over 65 years of age as healthy controls were analyzed. DNA was extracted from blood by the standard method [271. The diagnosis of CMT1A was based on the clinical presentation, abnormal nerve conduction velocities (<40 m/s), analysis of ratios obtained by Southern transfer between proximal CMT1A-REP and distal CMT1A-REP  and the allelic numbers of RM11-GT [131. All 31 cases showed evidence of CMT1A duplication.
PCR AMPLIFICATION OF THE 3.2-KB HOTSPOT REGION AND SEQUENCING ANALYSIS
The novel 3.2-kb junction fragments were amplified from CMT1A patients with two junction-specific primers. Two locus-specific primers were used for PCR; only one of these two primers having the correct 3'-sequence of distal or proximal CMT1A-REP will be extended by the polymerase. Therefore, only those CMT1A patients who had a 3.2-kb hotspot duplication demonstrate PCR products (Fig. 1). The primers were as follows: upstream primer A: 5'-GGAGCCCTCAATCAGTGGAA-3' (distal CMT1A-REP bases 1785-1806) (GenBank #41165), the A of the last base of the 3'-end of the primer making the base unable to bind the corresponding base G of the proximal CMT1A-REP specifically, so the primer will be extended only with the distal CMT1A-REP by the Taq polymerase [27,28]; downstream primer B: 5'-ACAGATGGAATAGTAGAGCTCACTC-3' (proximal CMT1A-REP bases 50695093) (GenBank # 41166), which has a six-base difference at the 3'-end of the primer in comparison with distal CMT1A-REP. These changes will make the primer be specifically extended only with the proximal CMT1A-REP by the Taq polymerase [221. The PCR conditions were as follows: 0.5 [micro]g of genomic DNA was mixed with 100 ng of each primer and 200 [micro]mol/L of each dNTP in 100 [micro]L of reaction buffer containing 20 mmol/L Tris-HCI (pH 8.5), 16 mmol/L [([NH.sub.4]).sub.2]S[O.sub.4], 1 mmol/L Mg[Cl.sub.2], and 4 U of Taq polymerase (Angewandte Gentechnologie System). The PCR procedure involved denaturation at 94 [degrees]C for 2 min, annealing at 56 [degrees]C for 2 min, and extension at 72 [degrees]C for 4 min. A total of 45 cycles was run in a PCR. After electrophoresis and visualization of the PCR products on 1% agarose gel, the products were subjected to direct sequencing or T-vector subcloning with a commercial kit (pT7 Blue T-vector kit, Novagen) and sequencing by the cycling sequencing method (AmpliCycle Sequencing kit, Perkin-Elmer).
[FIGURE 1 OMITTED]
PCR DETECTION OF THE SUBTYPES OF THE 3.2-KB HOTSPOT BREAKPOINTS
After sequencing the 3.2-kb hotspot regions of CMT1A patients, two pairs of primers were used to differentiate the subgroups of the hotspot breakpoints. For detection of the type 1 breakpoint, the upstream primer was identical to the above-mentioned primer A, which amplified the distal CMT1A-REP region specifically, and the downstream common primer C was 5'-ACAAGAGTTCAAGTAACTTGC-3' (distal CMT1A-REP bases 3751-3771) (GenBank #41165) (Fig. 1) . The PCR product was subjected to restriction enzyme Nsil digestion. For detection of the type 2 breakpoint, the upstream common primer D was 5'-TGCTTCGTTGAGAGAAACAG-3' (proximal CMT1A-REP bases 3489-3509) (GenBank #41166) (Fig. 1)  and the downstream primer was identical to the above-mentioned primer B, which amplified the proximal CMT1A-REP region specifically (Fig. 1B and C). The PCR products were subjected to digestion by the restriction enzymes Accl and Nsil. The PCR conditions were performed as described previously , except that the annealing temperature was 56 [degrees]C for both reactions. Amplified DNA fragments were digested with different restriction enzymes according to the conditions recommended by the manufacturers (New England Biolabs and Boehringer Mannheim).
SOUTHERN TRANSFER ANALYSIS OF CMTIA PATIENTS AND HEALTHY CONTROLS
Three to 10 [micro]g of genomic DNA was digested with restriction enzymes EcoRI, Nsil, and Sacl according to the manufacturer's instructions, electrophoresed in 0.8% agarose gel, Southern transferred to Hybond N+ membrane (Amersham), and hybridized with a random-primer [sup.32]P-labeled probe (DECA primer 228 11 DNA labeling kit, Ambion), which corresponded to the 3.2-kb hotspot region (the above-mentioned subcloned 3.2-kb fragment that was cloned from our 3.2 kb PCR product) (Fig. 1A). Loss or gain of restriction fragments was assessed by visual comparison of hybridization signals.
PCR DETECTION OF THE 3.2-KB HOTSPOT BREAKPOINTS
Thirty-one CMT1A patients, their families, and 50 healthy controls were studied. The results showed that 27 of the 31 CMT1A patients had a 3.2-kb product in the hotspot crossover breakpoint (one of the 27 was false negative in the first PCR test), whereas none of these patients' healthy family members nor any of the 50 controls had this 3.2-kb PCR product (data not shown). The upstream primer reacted only with the distal CMT1A-REP, and the downstream primer reacted only with the proximal CMT1A-REP; hence, only the CMT1A patients who had a 3.2-kb hotspot crossover demonstrated the 3.2-kb PCR product. The PCR products were subsequently sequenced, and revealed two types of breakpoints. The type 1 breakpoint was located at the distal CMT1A-REP around the PmeI site (base 3092, distal CMT1A-REP) region. There were two subtypes of the type 1 breakpoint: one subtype was located between base 2792 and the Pmel site; the other subtype was located between the PmeI site and base 3169. For the upstream base 2792, or for 3169 for the second subtype, the sequences were identical to the distal CMT1A-REP; otherwise, they were identical to the proximal CMT1A-REP (Fig. 1B). This type of crossover was located proximal to the restriction enzymes Nsil restriction site (base 3555, proximal CMT1A-REP), so there was no Nsil site in the normal distal CMT1A-REP. However, an Nsil site was present in the crossover of CMT1A duplication patients. The type 2 breakpoint was located on the distal CMT1A-REP downstream base 3625. Proximal to this base, the sequences were identical to the distal CMT1A-REP; otherwise, they were identical to the proximal CMT1A-REP (Fig. 1C). This type of crossover was located after the restriction enzyme Nsil cutting site; hence, there was an Nsil site in the normal proximal CMT1A-REP, but no Nsil site in the crossover of CMT1A duplication patients.
[FIGURE 2 OMITTED]
We also found several base polymorphisms in comparison with the reported sequences of Reiter et al. X22]. Base 3169 of the distal CMT1A-REP has T polymorphism, and bases 3158, 3933, and 4472 of the proximal CMT1A-REP have T, A, and C polymorphisms, respectively. We had tested five different cases; one of them had the above polymorphisms.
PCR DETECTION OF THE SUBTYPES OF THE 3.2-KB HOTSPOT BREAKPOINTS
The results for PCR detection of the type 1 breakpoint are shown in Fig. 2B. Twenty-four of the 27 cases that had a 3.2-kb hotspot crossover showed an abnormal 1789-bp band after Nsil digestion. There was also an undigested 1986-bp band after Nsil digestion for the normal distal CMT1A-REP, but no PCR product for the normal proximal CMT1A-REP, due to the fact that the primer specifically amplified only the distal CMT1A-REP (Fig. 2B).
The results for PCR detection in the type 2 breakpoint are shown in Fig. 3B. Three of the 27 cases that had the 3.2-kb hotspot crossover showed an abnormal 336-bp band after Nsil and Acct digestion. There was also a 265-bp band after Nsil and Accl digestion for the normal proximal CMT1A-REP, but no PCR product for the normal distal CMT1A-REP, due to the fact that the primer specifically amplified only the proximal CMT1A-REP (Fig. 3B). Because the change in size after Nsil digestion was only 71 bp, we simultaneously used the restriction enzyme Accl with a common cutting site for both distal and proximal CMT1A-REP. Using this kind of approach, we could differentiate the type 2 breakpoint easily.
[FIGURE 3 OMITTED]
Fifty healthy controls in this series were also tested by the above method, and there were no false-positive results.
SOUTHERN TRANSFER ANALYSIS OF THE 3.2-KB HOTSPOT BREAKPOINTS
The results of Southern transfer analysis are shown in Figs. 2A and 3A. Twenty-four of the 27 cases that had the 3.2-kb hotspot crossover showed an additional 1.7-kb band in comparison with the healthy controls. All these cases belonged to the type 1 crossover (Fig. 2A). Three of the 27 cases who had the 3.2-kb hotspot crossover showed an additional 3.2-kb band after comparison with the healthy controls. All these cases belonged to the type 2 crossover (Fig. 3A).
We have demonstrated that PCR detection of hotspot breakpoints in CMT1A duplication is accurate, rapid, and specific, with the results available within 24 h. These methods correlate very well with Southern transfer analysis, and are much more convenient to use. We only detected three-quarters of the CMT1A duplication in our patients. PFGE and FISH can detect almost 100% of cases of duplication [11,18]. The method of analyzing the dosage difference between polymorphic alleles [131 and the PCR method to detect three distinguishable alleles in duplication microsatellite loci can both detect about 85% of cases of duplication . Our method has some advantages: No radioisotope or hybridization reaction is needed, and the method is able to detect the duplication hotspot even if all the polymorphic markers are homozygous. However, up to 30% of cases with a CMT1 phenotype who have undergone a strand exchange event outside of the hotspot region may remain undetected by our method. Therefore, we suggest the use of our method as a screening approach to detect patients with crossovers in the hotspot, then follow with PGFE or FISH to identify the remaining 30% of patients with crossovers outside of the hotspot.
To subtype the 3.2-kb hotspot breakpoints, we designed a locus-specific primer to amplify distal or proximal CMT1A-REP specifically. By taking advantage of the Nsil site (base 3555) in the proximal CMT1A-REP and the hotspot crossover point just in front of or after this site, we used the specific primer and other polar common primers to perform the PCR, followed by subsequent digestion of the PCR products at the Nsil site to subgroup the 3.2-kb hotspot breakpoints. This kind of approach is an excellent method for the amplification and differentiation of highly homologous areas. We have previously used this kind of approach to detect the [-alpha]4.2 deletion in [alpha]-thalassemia-2, which is due to a misalignment between two highly homologous x boxes of the a globin gene cluster .
To amplify the 3.2-kb hotspot breakpoint successfully, we used more Taq polymerase and more amplification cycles (45 cycles instead of 35 cycles). We have tested several types of Taq polymerase and found that some types did not work well. Because the amplified fragment was quite long, the PCR required more cycles and much more Taq polymerase (4 U of Taq used in this series, rather than the 1 to 2.5 U normally used). We also tested a long PCR kit (Expand Long Template PCR System, Boehringer Mannheim), and the long PCR also worked very well for the detection of the 3.2-kb hotspot breakpoints. The template DNA quality is also very important. Because the detection of the 3.2-kb hotspot breakpoint depends on the presence of a 3.2-kb PCR product, there was a false-negative result (one of 27) due to inappropriate PCR reactions. Because false-negative results will be obtained for CMT1A cases where the breakpoints are not within the 3.2-kb hotspot, patients who test negative may be retested by other diagnostic methods. Moreover, we recommend the use of our PCR-based method for subtyping, because it may be more reliable since there should be PCR products in healthy persons or CMT1A duplication patients that are differentiated by digestion with the restriction enzyme Nsil. We believe that this kind of approach is much more accurate than the method involving amplification of the 3.2-kb hotspot breakpoints directly.
In this study, we used Chinese CMT1A patients to develop a PCR method for the detection of CMT1A duplication. We found that most Chinese CMT1 patients have the duplication of CMT1A-REP. This is similar to other ethnic groups [13,14, 22-26]. We also found that there is a higher incidence of the type 1 crossover breakpoint, which is similar to the findings of Reiter et al. . This work was supported in part by grants from the National Science Council of Taiwan (NSC 86-2314-B-196-002M02) and Taipei Municipal Jen-Ai Hospital (TMJA 86-1).
Received March 11, 1997; revision accepted October 16, 1997.
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JAN-GOWTH CHANG, (1,6)* YUH-JYH JONG, (2) WEN-PIN WANG, (3) JYH-CHWAN WANG, (1,6) CHAUR-JONG HU,  MAN-CHI Lo,  and CHIH-PENG CHANG 
(1) Division of Molecular Medicine, Department of Medical Research, Mackay Memorial Hospital, 92, Sec. 2, Chung Shan N. RD., Taipei, Taiwan.
(2) Division of Pediatric Neurology, Kaohsiung Medical College, Kaohsiung, Taiwan.
(3) Department of Molecular Biology, Tzu-Chi College of Medicine, Hualien, Taiwan.
Departments of (4) Neurology and (6) Molecular Medicine, Taipei Municipal Jen-Ai Hospital, Taipei, Taiwan.
(5) Department of Neurology, Changhua Christian Hospital, Changhua, Taiwan.
* Address correspondence to this author at the first address given. Fax 886-2-704-6952; e-mail fl037437@msS.Nnet.net. 7 Nonstandard abbreviations: CMT1A, Charcot-Marie -Tooth disease type 1A; REP, repeat sequence; PFGE, pulsed-field gel electrophoresis; RFLP, restriction fragment length polymorphism; and FISH, fluorescence in situ hybridization.
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|Title Annotation:||Molecular Pathology and Genetics|
|Author:||Jan-Gowth Chang, Yuh-Jyh Jong, Wen-Pin Wang, Jyh-Chwan Wang, Chaur-Jong Hu, Man-Chi Lo, Chih-Peng Ch|
|Date:||Feb 1, 1998|
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