Molecular diagnosis of medium-chain acyl-CoA dehydrogenase deficiency by oligonucleotide ligation assay

¹ Department of Clinical Chemistry, Kuopio University Hospital, P.O. Box 1777, FIN-70211 Kuopio, Finland.

² Department of Biochemistry and Biotechnology, University of Kuopio, FIN-70211 Kuopio, Finland.
^a Author for correspondence. Fax 358-17-173186;

	Abstract

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Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a recessively inherited defect in the mitochondrial ß-oxidation of fatty acids. A single nucleotide change, the A₉₈₅G transition, in the MCAD gene accounts for ~90% of all the disease-causing mutations in the patients. We have used PCR to amplify a segment of the human MCAD gene and typed the allelic sequence variation at base 985 by a colorimetric oligonucleotide ligation assay (OLA). PCR/OLA provides a technique that permits differentiation of the homozygotes, heterozygotes, and normals for the A₉₈₅G allele in the MCAD gene. Genotyping of 1908 random Finnish DNA samples by OLA identified 10 carriers of the mutant allele, but no homozygotes were found. The calculated carrier frequency for the A₉₈₅G mutation was 1:191 (95% confidence limits, 1:118–1:501), and the calculated frequency for the A₉₈₅G homozygotes was 1:147 000 (95% confidence limits, 1:56 000–1:1 004 000).

	Introduction

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Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (McKusick 201450) is the most common currently known disorder of ß-oxidation of fatty acids (1). More than 200 patients have been identified, most being of northwestern European origin. The estimated frequency of MCAD deficiency among Caucasians ranges from 1:6 400 to 1:18 800 (2)(3)(4)(5). Furthermore, ~80% of the patients are homozygous for a single mutation, the A₉₈₅G transition in the MCAD gene (6)(7).

Clinical manifestations of the disease vary markedly, ranging from clinically asymptomatic through attacks of hypoketotic hypoglycemia to permanent brain damage or death (1). Treatment includes long-term diet therapy to provide adequate caloric intake and intravenous glucose infusion for acutely ill patients. Neonatal screening for MCAD deficiency has been proposed because of its high incidence, available therapy, and potentially serious outcome (8).

The high prevalence of a single mutation responsible for the MCAD deficiency has led to the possibility of developing diagnostic methods based on molecular analysis (5)(6)(7)(9). We have now combined PCR with a colorimetric oligonucleotide ligation assay (OLA) to detect the A₉₈₅G transition in the MCAD gene. OLA uses two adjacent oligonucleotides that are hybridized to a PCR-amplified target DNA. Thermostable DNA ligase covalently joins these oligonucleotides in those cases where complete complementarity between the oligonucleotides and the template DNA sample are present. Temperature cycling results in a linear increase in the amount of the product, which is detected by ELISA (10)(11). PCR/OLA technology has proved to be a rapid, nonisotopic method with a high signal/noise ratio that can be automated for handling a large number of samples, e.g., in population screening (11)(12).

	Materials and Methods

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Cell lines.
Two fibroblast cell lines from MCAD deficiency patients, who were homozygous for the A₉₈₅G mutation (GM04488 and GM08768), and one fibroblast cell line from a MCAD deficiency patient, who was heterozygous for the A₉₈₅G mutation (GM08684), were from the Coriell Cell Repositories, Coriell Institute for Medical Research (Camden, NJ).

Primer reagents.
Oligonucleotide primers for PCR and ligase reactions were synthesized by using standard phosphoramidite chemistry. The allele-specific ligation primers were modified with a 5'-biotin group during the synthesis. 5'-Phosphorylated reporter primers were labeled with dUTP-digoxigenin (Boehringer Mannheim) according to the manufacturer's directions. The target DNA template in the human MCAD gene was amplified with primers 5'-AGCACCAAGCAATATCAT-3' and 5'-TGGCATCCCTCATTAGTT-3' (13). The nucleotide sequence of the allele-specific oligonucleotide for the normal MCAD gene structure was 5'-ATTCTAGCTAGTTCAACTTT-3', and that for the A₉₈₅G mutation was 5'-ATTCTAGCTAGTTCAACTTC-3'. The sequence of the reporter probe was 5'-CATTGCCATTTCAGCCAGCA-3'. Allele-specific and reporter probes were diluted to a final concentration of 5 µmol/L.

PCR amplification.
Genomic DNA was isolated from peripheral blood or from cultured fibroblasts by the sodium dodecyl sulfate–proteinase K/phenol–chloroform extraction method. Reactions involved 25 ng of DNA, 7.5 pmol of each amplification primer, 6 nmol of each of the four deoxynucleotide triphosphates, 0.25 U of DyNAZyme II thermostable DNA polymerase (Finnzymes), and 1x PCR buffer [per liter, 10 mmol of Tris-HCl (pH 8.8 at 25 °C), 1.5 mmol of MgCl₂, 50 mmol of KCl, and 1 mL of Triton X-100] in a total volume of 30 µL. The DNA polymerase was added after the initial denaturing step. Thermal cycling was performed in microtiter plates on an MJ Research (Watertown, MA) PTC-100 thermal cycler programmed for an initial temperature step cycle of 96 °C (5 min), 84 °C (for the time needed to add the enzyme), 51 °C (45 s), and 72 °C (60 s), followed by a cycle of 96 °C (60 s), 51 °C (45 s), and 72 °C (60 s). A total of 30 cycles were performed, with final steps at 51 °C for 90 s and 72 °C for 10 min. After the amplification, the samples were diluted 1:10 with 10 mL/L Triton X-100.

OLA.
Ligation reactions were assembled for both alleles. Reactions involved 10 µL of diluted PCR-amplified samples, 0.15 pmol of allele-specific probe, 0.15 pmol of reporter probe, 1.2 U of thermostable ligase (New England Biolabs), and 1x NEBuffer [per liter, 20 mmol of Tris-HCl (pH 7.6 at 25 °C), 20 mmol of potassium acetate, 10 mmol of magnesium acetate, 10 mmol of dithiothreitol, 1 mmol of NAD, and 1 mL of Triton X-100] in a total volume of 20 µL. The reactions were placed in the thermal cycler and heated, 97 °C for 30 s and 60 °C for 2 min (10 times). After cycling, the reaction was stopped with 10 µL of 0.1 mol/L EDTA in 1 mL/L Triton X-100. The entire reaction mixtures were transferred into the wells of a streptavidin-coated, bovine serum albumin-blocked microtiter plate, and the products were allowed to accumulate for 30 min at room temperature. The wells were washed once with 0.01 mol/L NaOH containing 0.5 mL/L Tween 20 and five times with wash buffer (140 mmol of NaCl, 0.5 mL of Tween 20, and 10 mmol of Tris-HCl, pH 7.4, per liter). To the wells was added 30 µL of 1:2500 diluted anti-digoxigenin-AP (Boehringer Mannheim) and the samples were incubated for 2 h at room temperature. After six washes with the wash buffer, the presence of the covalently linked reporter probe was detected by the ELISA Amplification system (Gibco BRL).

Verification of assay results.
Each DNA sample that showed the presence of the A₉₈₅G mutation in the OLA assay was reanalyzed for this mutation by another method based on PCR, StyI digestion, and agarose gel electrophoresis (6). Additionally, 5% of the samples that showed a normal genotype in OLA during the analysis of 1908 DNA specimens were randomly selected for similar verification of the genotype.

	Results

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The OLA scheme in which a normal template DNA is probed for the A₉₈₅G polymorphism responsible for MCAD deficiency is shown in Fig. 1 . After amplification of the template DNA by PCR, the target DNA is denatured and hybridized with two oligonucleotides in two different ligation reactions. In reaction A, the biotinylated oligonucleotide complements the normal allele, and in reaction B, the biotinylated probe complements the mutated DNA sequence. After the ligation, the biotinylated oligonucleotide is captured on immobilized streptavidin, and unligated digoxigenin-labeled oligonucleotides are washed away. When ligation occurs, a color signal appears in the alkaline phosphatase-catalyzed ELISA reaction for digoxigenin. The oligonucleotide mismatch between the template DNA and the allele-specific oligonucleotide in reaction B precludes the ligation, and no color is formed. In a normal individual (Fig. 2 , sample 1), the test results in a positive signal for the normal allele 1 with an absorbance of 2.500 to >3.000 at 510 nm and a negative signal for the mutant allele 2 has an absorbance reading of 0.060–0.090. In an individual heterozygous for the mutant allele (Fig. 2 , sample 2), a positive signal is detected for both alleles. In an individual homozygous for the mutant allele (Fig. 2 , sample 3), the positive signal for allele 2 and the negative signal for allele 1 are shown. A fourth possibility, a negative result with both reactions, usually indicates a failure of the PCR reaction. A simple positive/negative readout of the test result because of the high signal/noise ratio (30:1–60:1) enables rapid genotyping visually or by calculating the ratio of the absorbance for each allele after spectrophotometric analysis.

View larger version (19K): [in this window] [in a new window]   Figure 1. Principle of the OLA for detecting A985G transition responsible for MCAD deficiency: step 1, PCR amplification of the target DNA sequence; step 2, its denaturation to generate a single-stranded template; step 3, analysis of the target nucleotide sequence with digoxigenin (D)- and biotin (B)-labeled oligonucleotides; and step 4, capture of the biotin (B)-labeled oligonucleotides on streptavidin-coated microplate wells and analysis for digoxigenin by ELISA procedure. Normal target DNA (A985) was tested for the presence of normal sequence (Reaction A) and the A985G mutation (Reaction B). Complementarity of the oligonucleotides to the target sequence leads to ligation and a colored product in ELISA (Reaction A), whereas a single-nucleotide mismatch precludes the ligation (Reaction B).

View larger version (141K): [in this window] [in a new window]   Figure 2. Analysis of 47 amplified DNA samples by OLA for the presence of the A985G mutation in the MCAD gene, where allele 1 represents the normal allele and allele 2 represents the mutant allele. Sample 1 is a control DNA from a normal individual, sample 2 is from a heterozygous carrier of the mutation, and sample 3 is from a MCAD deficiency patient homozygous for the mutation. Specimens 4–47 are from blood samples of individuals with unknown carrier status. Samples 29 and 42 are the carriers of the A985G mutation detected by OLA. Sample 48 is a negative control with no DNA in the amplification reaction.

We analyzed 1908 random Finnish DNA samples by OLA for the frequency of the MCAD A₉₈₅G mutation and detected 10 carriers. OLA results from 44 individuals are shown in Fig. 2 (samples 4–47). Samples 29 and 42 in this assay series are from previously unknown carriers of the A₉₈₅G transition. The last sample in the microtiter plate (sample 48) represents a negative control analysis, which was carried out without added DNA. Restriction enzyme digestion of PCR-amplified DNA was used to verify the OLA results (6). StyI digestion of DNA from the 10 heterozygous individuals produced the full-length 63-bp product, as well as a 43-bp fragment and a 20-bp fragment resulting from the cleavage at the StyI-site created by the AG transition. No such cleavage was detected in any of 100 random DNA samples that had indicated normal genotype in OLA. The results suggest a carrier frequency of 1:191 (95% confidence limits, 1:118–1:501 [14]) of the MCAD A₉₈₅G mutant allele in the given population.

	Discussion

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Diagnosis of MCAD deficiency usually requires analysis of disease-specific metabolites such as acylcarnitines in plasma or urinary acylglycines by mass spectrometry, measurement of MCAD activity, or molecular analysis to detect the prevalent A₉₈₅G mutation, which is found in 90% of those patients with the MCAD deficiency (1)(15). Molecular analysis clearly differentiates homozygotes, heterozygotes, and normals for the A₉₈₅G allele. Both molecular and metabolite analyses are needed to assure the detection of compound heterozygotes of this mutation, which may account for 20% of the MCAD patients. Approximately 2% of the MCAD patients do not carry the A₉₈₅G allele (7) and would be missed by any molecular technique directed to that allelic region.

Molecular analysis by PCR combined with restriction enzyme digestion and electrophoresis (RFLP), single-strand conformation polymorphism analysis, and, recently, hybridization with allele-specific oligonucleotides have been used to detect the A₉₈₅G mutation (5)(6)(7)(9). Compared with these methods, PCR/OLA provides a rapid, reliable, nonisotopic method with a high signal/noise ratio and convenient assessment of the analysis result by the naked eye or spectrophotometry. Because PCR/OLA can be automated, it is suitable for handling a large number of samples, e.g., in population screening. In this study, verification by RFLP assigned the same genotype as OLA to all heterozygotes and normal individuals tested, indicating better identification of the different genotypes by OLA than by hybridization with allele-specific oligonucleotides (6).

In conclusion, OLA is well suited for large-scale population studies. We used it to analyze 1908 Finnish DNA samples of unknown MCAD genotype. Ten carriers for the A₉₈₅G mutation were found, indicating a carrier frequency of 1:191, which is lower than in a previous small-scale study, in which 4 carriers of the mutation were found among 200 DNA samples from southern Finland (16). On the basis of the present results, the calculated frequency of homozygotes in the Finnish population would be 1:147 000 (95% confidence limits, 1:56 000–1:1 004 000), which is lower than the range of MCAD deficiency from 1:6 400 to 1:18 800 reported in several studies among non-Finnish Caucasians (2)(3)(4)(5). We conclude that automated PCR/OLA provides a rapid, sensitive, high-throughput system for the analysis of the MCAD mutation. It could clearly facilitate diagnosis of affected patients, screening either at-risk members of families or the general population.

	Acknowledgments

 
This research has been supported by grants from The Savo Foundation for High Technology (to E.-L.R.), Pediatric Research Foundation, and the Sigrid Juselius Foundation (to I.M.). We thank Ulla Korhonen for technical assistance.

	References

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