values graphed in Fig. 1C after log10 transformation. Gaussian by jrskeirwta


									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.                                        

                                                                                     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
(                                     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

The enzyme catalase (EC 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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