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Technical Briefs |
a author for correspondence: fax 716-878-7980, e-mail gdv{at}acsu.buffalo.edu
Carnitine palmitoyltransferase II (CPT II; EC 2.3.1.21), an enzyme associated with the inner mitochondrial membrane, is important in the transport of long-chain fatty acids from the cytosol into the mitochondrial matrix for ß-oxidation (1). CPT II deficiency presents as three distinct clinical phenotypes: adult myopathic (MIM 255110), lethal neonatal (MIM 600649), and a severe infantile phenotype (2). The adult form is the most common lipid myopathy in humans and is characterized by muscle pain, stiffness, and myoglobinuria triggered by exercise, fasting, anesthesia, or other metabolic stressors (3). CPT II is a homotetramer (4) encoded by a gene (MIM 600650) on chromosome 1p32 (5) that spans 20 kb and contains five exons ranging in length from 81 to 1305 bp (6). At least 15 mutations in CPT2 are associated with the adult and infantile disorders (7)(8)(9). CPT II deficiency is an autosomal recessive disorder (3)(9); however, recent biochemical and molecular evidence suggests the existence of manifesting carriers, predicting that the prevalence of the disease may be even higher than previously believed (9).
Screening for mutations in CPT2 has been performed using DNA isolated from biopsied muscle tissue (9), venous blood (10), lymphoblasts (11), or fibroblasts (8). Isolation of DNA from these specimens is time-consuming, and mutation screening generally has been performed using restriction fragment length polymorphisms (12) or allele-specific oligonucleotide (ASO) detection (9), requiring portions of several days for completion (9). Large-scale screening has not been performed to determine the carrier frequency of the common mutations in the general population.
Although the methods for DNA isolation, PCR amplification, and ASO analysis developed previously by our laboratory are effective for detecting known mutations in the CPT2 gene (9), they were too costly and laborious for application to large family studies or population screening. We proposed that to be able to determine the prevalence of the common mutations in the CPT2 gene, we had to modify our protocol to facilitate cost-effective population screening. We decided to (a) explore dried whole blood on filter paper as a practical specimen that does not require special shipping and handling; (b) optimize the required PCR reactions to generate products of similar size and quantity; and (c) alter the ASO hybridization step to improve the signal intensity and reduce the incubation period.
We used whole blood from five healthy, unaffected individuals; two CPT II-deficient patients (A and B), and the father, mother, and sister of patient A were used for the blood-spot DNA study. Patient A is a known compound heterozygote for S113L/413 delAG-F448L (9), and patient B is a heterozygote for the R503C substitution (11). Twenty additional genomic DNA specimens were prepared from wet tissue (whole blood, muscle, lymphoblasts, or fibroblasts), previously screened for CPT2 mutations (9) and used as controls (50 mg/L) for the PCR and ASO optimization experiments. Informed consent for genetic testing was obtained from all patients who participated in this study. Whole blood specimens were spotted directly from finger pricks onto either IsoCode StixTM (12–15 µL/stick) or IsoCode CardsTM (25–30 µL/spot; Schleicher & Schuell). The collection devices are specially treated with proprietary reagents that bind inhibitors of PCR in blood, e.g., hemoglobin (13).
As per the blood card manufacturer's protocol, DNA was isolated from the blood spot samples using one triangle (8 x 6 x 6 mm) from the IsoCode Stix or using three 3-mm discs from the center of a dried blood spot on IsoCode Cards. Specimens were placed in sterile 1.5-mL microcentrifuge tubes, washed with 500 µL of deionized H2O, and heated for 30 min at 95 °C in 100 µL of fresh deionized H2O. The DNA eluates were used for PCR amplification.
The primers used for amplification and the yield of reaction products
are described in Table 1
. Control DNA samples (100 ng) were used for PCR optimization.
All PCR reactions were performed in 200-µL thin-wall strip tubes in a
thermocycler (Stratagene Robocycler Gradient 96 with Hot Top Assembly;
Stratagene). Optimal results were obtained with 25-µL reaction
volumes containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2.0
mmol/L MgCl2, 250 nmol/L each primer, 200
µmol/L deoxynucleotide triphosphates, 50 mL/L glycerol, and 0.5 U of
AmpliTaq GoldTM DNA polymerase (PE Applied
Biosystems). PCR reactions were as follows: 96 °C for 10 min to
activate the polymerase; 5 cycles of 1 min at 96 °C, 1 min at
62 °C, and 2.25 min at 72 °C; 30 cycles of 1 min at 96 °C, 1
min at 62 °C, and 2 min at 72 °C; and a final cycle at 72 °C
for 5 min.
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Blood spot eluates (2–7.5 µL) from the five healthy individuals were used to test the optimized PCR conditions; all gene regions of interest were successfully amplified. Aliquots of PCR products were mixed with one-fifth volume of gel loading buffer (150 mL/L Ficoll, 2.5 g/L bromphenol blue, and 2.5 g/L xylene cyanol) for electrophoretic analysis on a 1% agarose gel (Seakem GTG; FMC BioProducts).
PCR products in gel loading buffer were diluted 1:2 with HPLC-grade water (Sigma), and 12.5 µL of each preparation was used for dot blotting. Dot blots were prepared with a 96-well applicator (Minifold I Dot-Blot System; Schleicher & Schuell) and nylon membranes (Hybond NX; Amersham). Blots were processed for hybridization by sequential 10-min exposures to filter paper saturated with the following solutions: denaturation (0.5 mol/L NaOH, 1.5 mol/L NaCl), neutralization (1 mol/L Tris-HCl, 3 mol/L NaCl, pH 7.5), and equilibration [4x standard saline citrate (SSC): 0.06 mol/L trisodium citrate, 0.6 mol/L NaCl, pH 8.0]. The membranes were air-dried at room temperature until damp and then cross-linked under ultraviolet light (Stratalinker; Stratagene).
ASO analysis was performed using 5'-biotinylated oligonucleotides specific for the S113L, F448L, and R503C mutations, with subsequent detection using the Phototope Star Detection System (New England Biolabs) (9). Dot blots were preincubated for 15 min at 37 °C in hybridization solution [4x SSC, 40 mmol/L sodium phosphate buffer (pH 6.5), 2x Denhardt's solution, 300 mg/L sheared salmon testis DNA, and 1 g/L sodium dodecyl sulfate)], followed by a 30-min incubation at 37 °C in the hybridization solution containing 8 µmol/L of the respective biotinylated ASOs. Unbound ASO was rinsed away in 4x SSC at 37 °C for 15 min, followed by a stringent washing for 15 min in 2x SSC at 2 °C below the calculated Tm for each ASO (9). PCR-amplified DNA from a wet whole blood control known to be negative for the mutations studied was used as an assay control. The chemiluminescent signal was detected by exposure of the blots to x-ray film (Hyperfilm MP; Amersham) for 2 min at room temperature.
Plasmid DNA (1 µL; 10 ng/µL) containing previously cloned mutant or wild-type exon 3 inserts for the S113L mutation was used for amplification (9). The resulting PCR products were electrophoresed on a 0.7% low melt agarose 1x TAE gel (Qiagen). The product was gel-extracted (QIAquick Gel Extraction Kit; Qiagen), and the DNA concentration was determined at 260 nm. The extracts were diluted to a final concentration of 5 mg/L. Serial dilutions of both mutant and wild-type exon 3 extracts were made to final DNA concentrations ranging from 5 to 0.05 mg/L. Each dilution (10 µL) was dotted onto nylon membranes and screened with the appropriate ASO as outlined above. The sensitivity of each ASO was determined by comparing the signal of serially diluted dots to the background signal.
All amplicons derived from dried blood DNA samples produced
electrophoretic bands of visual intensity comparable to those from a
wet tissue control as shown, for example, with exon 2 in Fig. 1
A. Although DNA from dried blood spots was not quantifiable
because of protein carryover, 7.5 µL of the 100-µL dried blood spot
extracts contained a comparable amount of DNA template (~50–100 ng)
as found in 2 µL of the 50 mg/L wet blood DNA samples typically use
for amplification.
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Previously identified mutations in exons 3 and 4 in our patient samples
were detected in corresponding blood spot DNA samples, using the
optimized dot blot preparation and ASO hybridization protocols. Mutant
haplotypes were established for patient A's parents and sister
entirely from blood spot DNA analysis. The results of typical ASO
analyses for two of the three mutations are presented in Fig. 1B
.
DNA serial dilution studies and ASO analysis of both mutant and wild-type exon 3 extracts demonstrated that the lower limit of detection of the respective target alleles was 275 pg (data not shown). Subsequent analysis for additional mutations in the CPT2 gene indicated that this degree of sensitivity is similar for all of the ASOs used with the same molar ratio of biotinylated oligonucleotides to amplified targets (data not shown). ASO hybridization for 30 min produced comparable results and less background than the previously reported 16-h incubation (9). The cost for reagents using blood spot DNA for PCR compared with wet tissue DNA was reduced from ~$7/sample to ~$2.75/sample with ~8 h of labor. Reaction volumes and Taq polymerase were reduced by one-half from those used in previous studies (9).
We have developed a protocol that optimizes and shortens the CPT2 mutation screening procedure from specimen collection to mutation detection, producing a specific and sensitive assay using DNA from dried blood spots.
In the past, seven PCR reactions were required to amplify the entire CPT2 coding and regulatory regions (9), using different buffers and producing different yields of amplified DNA. Previously, dot blots were hybridized with ASOs overnight, and the subsequent chemiluminescent detection step produced positive signals of varying intensity, depending on the amount of product analyzed. This variability has been reduced with PCR reactions that produce similarly sized amplicons.
DNA isolated from blood spots avoids the disadvantages of wet tissue samples while producing comparable results; however, the use of untreated filter paper requires an overnight methanol fixation step to bind the PCR inhibitors from whole blood (14). The proprietary chemicals in the pretreated filter paper used in our study lyse blood cells on contact and bind the PCR inhibitors (15). Filter paper collection of blood is minimally invasive, produces a specimen that is inexpensive to ship, requires no special storage conditions (15), and is simple to process with amplifiable DNA extracted in <1 h. The isolation process costs ~60% less per sample than the DNA isolation kit currently used in our laboratory for wet tissue samples. Furthermore, only one blood spot or stick from a single blood specimen is needed for mutation analysis and provides ample material for any further studies.
Several reports have described the use of dried whole blood on filter paper for mutation screening in common heritable disorders such as cystic fibrosis (16)(17) and fragile X syndrome (18). Blood spots have also been used for screening patients at risk for mitochondrial disorders such as medium-chain acyl-CoA dehydrogenase deficiency (19)(20) and Pearson syndrome, a multisystem juvenile disorder associated with deletions in the mitochondrial genome (21). Mackey et al. (22) reported a new blood spot collection device that can be used for automated DNA isolation and amplification in a single tube. Hong et al. (23) described the pretreatment of blood cards with methanol and acetone to improve PCR amplification of various sizes of CGG repeats in fragile X syndrome analysis.
The use of dried whole blood on chemically treated collection devices and the improvements made in the PCR amplification and ASO detection will provide an inexpensive, rapid, and sensitive method for CPT2 mutation analysis applicable to large-scale population screening for determining the prevalence of the common mutations in high risk groups.
Acknowledgments
This work was supported by a grant from the Muscular Dystrophy Association (G.D.V.) and by the Children's Guild of Buffalo. We appreciate the participation of patients in this study. We thank Dr. Adrian Vladutiu for providing helpful comments.
Footnotes
State University of New York at Buffalo, 936 Delaware Ave., Buffalo, NY 14209
References
F508 in the cystic fibrosis gene using allele-specific PCR primers and time-resolved fluorometry. Genome Res 1992;2:157-162.
F508 mutation. Eur J Pediatr 1994;153:569-573.
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