1198 Technical Briefs values graphed in Fig. 1C after log10 transformation. 15. Anonymous. ACMG statement. Statement on storage and use of genetic materials. American College of Medical Genetics Storage of Genetic Mate- Gaussian distribution of values within each given cluster rials Committee. Am J Hum Genet 1995;57:1499 –500. was observed. Parsimonious construction of a similarity tree through the join procedure in SYSTAT (SPSS, Inc.), using several quantitative algorithms, correctly revealed the existence of four data clusters: (a) no DNA added, (b) wild type, (c) heterozygous, and (d) homozygous mutant Ischemic Exercise Testing in Suspected McArdle Dis- for FVL. Kmeans analysis demonstrated adequate cluster ease, Zahur Zaman1*and Stefaan De Raedt2 (1 Department of separation to distinguish 97.6% of the 256 results (P Laboratory Medicine, University Hospitals Leuven, Here- 0.01); the remaining 6 samples were not outliers in a straat 49, B-3000 Leuven, Belgium; 2 Clinical Laboratory, repeat PCR and visualization. We believe this demon- St. Elizabeth Hospital, B-2300 Turnhout, Belgium; * au- strates the accuracy and laboratory utility of this micro- thor for correspondence: fax 32-16-34-70-42, e-mail well plate-based genotyping method. email@example.com) Ischemic exercise testing is used in evaluation of patients We thank C.S. Chiang, Peter Noce, Wayne Grody, Su- with suspected McArdle disease, also known as glycogen zanne Cheng, John Griffin, Dan Farkas, David Gjertson, storage disease type V (1 ). Lack of an increase in the blood Glen Palomaki, and Stephen Lappin for helpful com- lactate concentration during exercise is indicative of a ments. In addition, we thank Jeffrey Kant of the Univer- defect in conversion of glycogen (or glucose) to lactate, sity of Pittsburgh, Geoff O’Connor, then of the Scripps consistent with the deficiency of skeletal muscle phos- Immunology Research Laboratory, and Jeffrey Wisotzkey phorylase in this disease. Other glycogen storage diseases, of York Hospital in York, Pennsylvania for furnishing such as deficiencies of phosphofructokinase and de- validation samples. branching enzyme, would also yield an abnormal isch- emic exercise response. In anticipation of performing the ischemic exercise test Tables showing (a) cluster analysis, (b) cost analysis, and on a suspected case, one of us (S.D.R.) carried out the test (c) a FORTRAN program to analyze data are available as on a healthy subject according to the method of Threatte a supplement at the Clinical Chemistry Online Web site (http://www.clinchem.org/content/vol46/issue8/). and Henry (2 ), who describe the procedure as follows: “Laboratory diagnosis of McArdle disease is made by References applying a blood pressure cuff on an exercising forearm 1. Gregg JP, Yamane AJ, Grody WW. Prevalence of the factor V-Leiden mutation and sampling blood lactate one minute after the exercise in four distinct American ethnic populations. Am J Med Genet 1997;73: has begun”. Thus, after a butterfly catheter was inserted 334 – 6. 2. Griffin JH, Evatt B, Wideman C, Fernandez JA. Anticoagulant protein C into the antecubital vein, a pre-exercise blood specimen pathway defective in majority of thrombophilic patients. Blood 1993;82: was drawn and the blood pressure cuff was inflated to 50 1989 –93. mmHg above the systolic pressure. The arm was exer- 3. Koster T, Rosendaal FR, de Ronde H, Briet E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated cised vigorously by opening and closing the fist for 1 min, protein C: Leiden thrombophilia study. Lancet 1993;342:1503– 6. and a blood sample was taken for lactate measurement. 4. Svensson PJ, Dahlback B. Resistance to activated protein C as a basis for With the cuff still inflated, additional blood samples were venous thrombosis. N Engl J Med 1994;330:517–22. 5. Sun X, Evatt B, Griffin JH. Blood coagulation factor Va abnormality associ- taken 2 and 3 min after cessation of the exercise. ated with resistance to activated protein C in venous thrombophilia. Blood The pre-exercise lactate was 1.5 mmol/L, and the 1994;83:3120 –5. 6. Griffin JH. Control of coagulation reactions. In: Beutler E, ed. Williams postexercise concentrations were 1.4 mmol/L at 1 min, 1.5 hematology, 6th ed. New York: McGraw-Hill, 2000:in press. mmol/L after 2 min, and 1.5 mmol/L after 3 min. The test 7. Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, was repeated on two healthy subjects and gave similar et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994;369:64 –7. results. According to these results, all three healthy test 8. Ridker PM, Miletich JP, Hennekens CH, Buring JE. Ethnic distribution of subjects had a glycogen or glucose metabolism defect. factor V Leiden in 4047 men and women. Implications for venous thrombo- This was patently false. Therefore, the test was repeated embolism screening. JAMA 277:1305–7. 9. Kalafatis M, Rand MD, Mann KG. The mechanism of inactivation of human on other healthy subjects with the modification that after factor V and human factor Va by activated protein C. J Biol Chem 1994;269: 31869 – 80. 10. Ridker PM, Hennekens CH, Lindpaintner, Stampfer MJ, Eisenberg PR, Miletich JP. Mutation in the gene coding for coagulation factor V and the risk Table 1. Ischemic test results of two healthy subjects. of myocardial infarction, stroke, and venous thrombosis in apparently Lactate, mmol/L healthy men. N Engl J Med 1995;332:912–7. 11. Lee DH, Henderson PA, Blajchman MA. Prevalence of factor V Leiden in a Subject A Subject B Canadian blood donor population. Can Med Assoc J 1996;155:285–9. 12. Solymoss S. Factor V Leiden. Who should be tested? Can Med Assoc J Pre-exercise 1.4 1.4 1996;155:296 – 8. Postexercise 1 min 4.5 4.7 13. Zehnder JL, Benson RC, Cheng S. A microplate allele-specific oligonucleo- (cuff deflated) 2 min 4.9 5.7 tide hybridization assay for detection of factor V Leiden. Diagn Mol Pathol 1997;6:347–52. 3 min 4.1 5.5 14. Stanley CJ, Johannsson A, Self CH. Enzyme amplification can enhance both 4 min 3.7 5.0 the speed and the sensitivity of immunoassays. J Immunol Methods 5 min Not done 4.1 1985;83:89 –95. Clinical Chemistry 46, No. 8, 2000 1199 exercise the blood pressure cuff was deflated to reestab- deletion in exon 4 have been detected in five Japanese lish circulation. The results of two subjects are shown in patients (6 – 8 ). Table 1. Both are normal responses in that the basal lactate We have reported on nine hypocatalasemic families for was 2 mmol/L and that this was followed by an the first time in Hungary (9 ). The frequency of inherited increase to 2.5 mmol/L in the first 4 –5 min post hypocatalasemia is 0.18% in Hungary (9 ). The syndrome- exercise, the peak being between 1 and 3 min. causing mutations detected in Japanese patients (6 – 8 ) The ischemic exercise test is not a frequently performed have not been found in the Hungarian hypocatalasemic test, and the procedure for it is not described in easily patients (10, 11 ). available clinical chemistry textbooks, e.g., Tietz Textbook We report here on a new catalase mutation that caused of Clinical Chemistry (1999) and Kaplan and Pesce’s Clinical hypocatalasemia in three (M, D, and G) Hungarian hy- Chemistry (1989), or in The Metabolic and Molecular Bases of pocatalasemic families. For this mutation, we amplified Inherited Disease (1 ). This makes it all the more important all exons and exon-intron junctions of the catalase gene by to emphasize that to avoid false-positive results and PCR. These PCR products were screened for mutations by unnecessary traumatic repetitions of the test, the blood a simple heteroduplex detection method. The mutation pressure cuff must be deflated before the postexercise was determined by nucleotide sequence analysis. specimens are taken. Genomic DNA was isolated from 23 hypocatalasemic Since performing this study, we have discovered that and 25 normocatalasemic members of six Hungarian the procedure for the ischemic exercise test is correctly hypocatalasemic families. The DNA extraction was made described by Thomas (3 ). However, his prescription of by a QIAamp Blood Kit (QIAGEN). The PCR amplifica- ischemic exercise for 2 min (rather than 1 min) can be tion was performed in a total volume of 10.5 L, contain- difficult for many people and appears to be unnecessary. ing 1 L of genomic DNA (0.2 g/ L), 1.6 L of four dNTPs (1.25 mmol/L each), 1 L of each primer (10 References mol/L), 0.5 L of 5 U/ L Taq polymerase, 1 L of 8.3 1. Chen Y-T, Burchell A. Glycogen storage diseases. In: Scriver CR, Beaudet AL, mmol/L MgCl2, and 1 L of buffer. PCR reagents were Sly MS, Valle D, eds. The metabolic and molecular bases of inherited disease, 7th ed. New York: McGraw-Hill, 1995:935– 65. purchased from Pharmacia. Thirty cycles of amplification 2. Threatte GA, Henry JB. Carbohydrates. In: Henry JB, ed. Clinical diagnosis at 94, 55, and 72 °C for 0.5, 0.5, and 1 min, respectively, and management by laboratory methods, 19th ed. Philadelphia: WB Saun- were performed in a DNA thermal cycler (TC 1; Perkin- ders, 1996:194 –207. Elmer Cetus). Oligonucleotide primers were synthesized 3. Thomas L. Lactate. In: Thomas L, ed. Clinical laboratory diagnostics, 1st ed. Frankfurt/Main, Germany: TH-Books Verlagsgesellschaft, 1998:160 – 6. by Pharmacia, according to the sequences reported by Kishimoto et al. (7 ). Heteroduplex analysis was performed according to the Hydrolink protocol (AT Biochem). PCR product (2 L) was heated to 94 °C, cooled down slowly, and then loaded A Simple PCR-Heteroduplex Screening Method for De- onto a Hydrolink gel (280 180 0.75 mm). DNA bands tection of a Common Mutation of the Catalase Gene in Hungary, Laszlo Goth,1* Andras Gorzsas,1 and Tibor Kalmar2 ´ ´ ´ ´ ´ ´ (1 Department of Clinical Biochemistry and Molecular Pathology, Medical School, University of Debrecen, PO Box 40, H-4012 Debrecen, Hungary; 2 Department of Genetics, Biological Research Institute, PO Box 521, H-6701 Szeged, Hungary; * author for correspondence: fax 36-52-417-631, e-mail firstname.lastname@example.org) The enzyme catalase (EC 184.108.40.206) has a predominant role in controlling the concentration of hydrogen peroxide in human erythrocytes (1 ). Hydrogen peroxide is involved in physiological processes, but its increased concentration may contribute to the pathogenesis of various diseases, such as diabetes and atherosclerosis. Human erythrocytes with high catalase content provide a general defense against toxic concentrations of hydrogen peroxide (2, 3 ). Hypocatalasemia is the heterozygous state of the acata- lasemia gene and is inherited as an autosomal, recessive Fig. 1. Pedigree (top), heteroduplex pattern (middle), and nucleotide trait without any characteristic clinical sign. The fre- sequence analysis (bottom) of hypocatalasemic family G. quency of hypocatalasemia in East Asia is 0.2– 0.4%, (Top), , hypocatalasemic female; m, hypocatalasemic male; , normocata- whereas in two Iranian populations it is 0.5% (4, 5 ). There lasemic male. (Middle), the heteroduplex pattern A represents a wild-wild are only limited data available on the disease-causing homoduplex, B represents a mutant-mutant homoduplex, and C and D represent wild-mutant heteroduplexes. (Bottom), the nucleotide sequence analyses show mutations. The splicing mutation (guanine-to-adenine nucleotides 127–138 for the wild type and 127–140 for the mutant. The GA substitution) at the fifth position of intron 4 and the 358T repeats are numbered above the nucleotide sequence.
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