The incidence of elevated serum creatine kinase (CK) and pyruvate kinase (PK) activities was compared in 20 definite carriers of Duchenne muscular dystrophy (DMD), 47 possible carriers, and 42 female controls. When adult age was not regarded as a variable, 70% of the definite carriers had elevated PK, 55% had elevated CK, and 75% had elevated PK or elevated CK or both; 38% of the possible carriers had elevated PK, 19% had elevated CK, and 40% had elevated PK or elevated CK or both. The detection efficiency of the CK test was influenced by the age of the subjects: the upper normal limit of serum CK in the adult controls was at the minimum between 21 and 35 years of age, and CK activity in some carriers declined from elevated to normal levels with increasing age. With these considerations, 70% of definite carriers had elevated CK and 80% had elevated PK and/or CK; 34% of the possible carriers had elevated CK and 43% had elevated PK and/or CK. On the basis of the PK and CK measurements, only 16 of 24 possible carrier mothers were likely to be DMD carriers, implying that the other 8 were noncarrier mothers of new mutant sons. MUSCLE & NERVE

2:329-339

1979

SERUM CREATINE KINASE AND PYRUVATE KINASE IN DUCHENNE MUSCULAR DYSTROPHY CARRIER DETECTION MAIRE E. PERCY, PhD, LEBE S. CHANG, MSc, E. GORDON MURPHY, MB, IRlNA OSS, BA, CHRISTINE VERELLEN-DUMOULIN, MD, and MARGARET W. THOMPSON, PhD

Elevated serum creatine kinase (CK) (ATP: creatine phosphotransferase, EC 2.7.3.2) activity was first described in patients with progressive muscular dystrophies by Ebashi et a1 in 1959.5 Subsequently, the measurement of serum CK has been used extensively in diagnosis-particular1 y for the X-linked fornis of muscular dystrophy, Duchenne (DMD) and Becker (BMD), and in DMD carrier detection. On the average, about two-thirds of female carriers of DMD have elevated serum CK; the remainder have CK activities within the normal range.21,33 In 1973, Harano et a19 reported that serum pyruvate kinase (PK) (ATP: pyruvate phosphotransferase, EC 2.7.1.40) was greatly elevated in patients with DMD and in those with limb girdle muscular dystrophy. Subsequently, serum PK was found to be elevated in patients with facioscapulohunieral dystrophy, in those with myotonic dystrophy,' and in those with BMD.39 In addition, serum PK was elevated in a higher proportion of Duchenne rarriers than was serum CK, which suggests that the former criterion may be a more efficient index of the carrier state than the 1atter.l One group of in-

Serum CK and PK Levels in DMD

v e s t i g a t o r ~has ~ ~ since published data supporting this proposal. However, results from another laboratory37 suggest that in DMD carriers the frequency of elevations in CK and PK activity may be the same, and that concurrent determinations of both serum CK and PK might detect carriers that would escape identification using either test alone.

From the Department of Genetics and the Muscular Dystrophy Clinic, The Hospital for Sick Children, Toronto, Canada. Acknowledgments: This work was supported by grants for research and service from the Muscular Dystrophy Association of Canada. The authors are grateful to Mrs. Patricia Fulford, RN, for her assistance with obtaining blood samples, and lo Dr. E. M. Hutton for her contributions to the collection of pedigree data. A brief presentation of this material was made at the lVth International Congress on Neuromuscular Disease, Montreal, Canada, September 1721, 1978. Address reprint requests to Dr. Percy at the Department of Genetics, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1x8, Canada. Received for publication February 1, 1979; revised manuscript accepted

for publication May 7, 1979. 01 48-639X1020510329 $00.0010 @

1979 Houghton Mifflin Professional Publishers

MUSCLE & NERVE

SepiOct 1979

329

maintain that a serum CK Two other test is superior tor DMD carrier detection. To extend these findings and t.o attempt to clarify the conflicting observations, we measured the corresponding CK and PK activities in individual serum samples from 42 adult female controls and from 20 definite and 47 possible adult DMD carriers, as well as from 9 rnalc children affected with DMD. Serum C K activity was also measured in 485 other normal females, ranging in age from 2 t o 70 years. In this report, we describe the CK arid PI( assays, establish upper normal limits for activities of both serum enzymes, and compare the efficiency of the two assays in reflecting the carrier state. Enzyme activities were considered to be elevated if' they were equal to or exceeded the normal mean value plus t b v o standard deviations (i.e., 97.5% confidence levels). In the case of the CK assay, detection efficiencies were deterrriiried both with and without consideration of the age of the control arid carrier subjects. This is the first of a series of studies designed to evaluate simple methods that have potential value in DMD carrier detection. MATERIALS AND METHODS

Between January 1974 and October 1978, berum C K activit) h a \ measured in 527 healthy, active females ranging in age from 2 to 70 years. Between October 1977 arid October 1978 serum PK activity was determined in 42 of these individuals, who ranged in age from 17 to 60 1ears.

Control Subjects.

The group of definite carriers who participated in this study included 12 obligate carriers (mothers of one or more affected sons who had clearly, on the basis of pedigree analysis, inherited the DMD gene in an X-linked recessive fashion) and 8 probable carriers (mothers of two or more affected sons). Bayesian analyses indicate that the latter are virtually certain to be carriers." 'I'he group ol' possible carriers included 24 mothers with one affected son and no family history of the disease, and 23 other adult females considered to be at risk, of whom 5 were maternal grandmothers, 10 were maternal aunts, 7 were sisters, and one was a first cousin (daughter of a maternal aunt) of an affected male.

Carrier Subjects.

Serum was obtained by centrifugation (750 x g, 5 min, at room temperature) of venous blood that had been allowed to clbt at room temperature for 45 min. Samples

Processing of Serum Samples.

330

Serum CK and PK Levels in DMD

were further clarified by a second ccni rifiigation. 'l'hesc \\;ere then chilled on ice for at least one-half hour and assayed for PK activity within four hours, or frozen at -20°C: in 0.2 ml aliquots for assay of CK activity at more convenient times. We agree with others that all modalities of the PK isozymes progressively lose activity upon freezing, whereas CK activity is esscntially stable for about two months under the same conditions.""'" In adults, blood samples were taken between 9 am and 3 pm without previous restriction of diet or activity. In children, some samples were taken as late as 7 pm. Hemolyzed serum sarriplcs were not assayed. Although strenuous exercise is knmvn to cause increases in serum CK activit.y in both normal individuals and female carriers of DMD,12 none of the donors had taken part in unusually strenuous forms of exercise in the preceding 48 hours. The effect of exercise on serum PK activity has not yet been assessed. There are three main CK isozymes in human tissues: the M M form (muscle type), the BB form (brain type), and the M H form (hybrid type).14 Mitochondria1 forms have also been described.26I n normal serum, the M M form predominates. MM activity is greatly elevated in the serum of DMD patients arid is significantly elevated in many DMD carriers. MB activity is also increased in the serum of DMD patients and of some carriers. Recently, a CK enzyme with electrophoretic mobility intermediate between M M and MB has been identified in DMD patient serum, but its relationship to the known CK isozymes has hot yet been e~tablished.~ In this study, serum CK activity was determined using the Calbiochern Super Stat Pack (Calbiochem-Behring Corp., San Diego, CA) at 30°C. This method issimilar t o that used by Rosalki;2sit incorporates adenosine monophosphate (AMP) to inhibit aderiylate kinase derived from erythrocytes, and a siilfhydryl compound to restore the activity of CK inactivated during storage. The forward reaction of the enzyme is measured by recording the absorbance increase at 340 nm via the following reactions:

CK Assay.

c-rcarinc phosphate

A T P + glu(xise

+ .2Dt' c (:c I( trcatinc

-

-t

ATP

HIi

glucose-fi-t)tiospharr t A D P glucose-6-ph(ispl~~te + S.4I)P (;6Pu S A U I ' t I + 6-phc~sph(iglllc.onate

-

(Abbreviations: ADP = adenosine diphosphate; ATP = adenosine triphosphate; HK = hexokinase; GGPD = glucose-6-phosphate dehydrogenase.)

MUSCLE & NERVE

SepiOct 1979

Serum samples were usually assayed after one to three days of storage at -20°C. They were kept in an ice bath after removal from the frec7er and warmed to 30”Cjust prior to the a5say; 2 0 pl patient sera and 100 pl each of carrier arid normal sera were used per 3 ml of assay mixture. In mammalian tissues, two groups o f PK isozymes have been identified. T h e first group consists of an I, type, which is found in liver, and an L-L precursor complex, which is found in erythrocytes in addition to the L form.’: T h e second group, the M type, consists of four subtypes: M1, found in skeletal muscle arid brain, and predominant in heart; M2, found in lung, adrenal gland, thyroid, fat cells, leukocytes, platelets, spleen, and skin; and M 3 and M4, found in smooth muscle, testicle, kidney, and other organs.9 Zatz et a139recently reported that the major serum isozyrne in DMD patients is the M 1 form derived from skeletal muscle. Using electrophoresis on Titan I11 cellulose acetate plates (Helena Laboratories, Beaumont, -1.X) in conjunction wit.h t h e buffer s~’: ,\tern of Susor and Rutter,” we have confirmed this observation. but we consistently find a second PK isozyme corresponding to the major erythrocyte form in all normal, carrier, and DMD patient sera. Although PK isozymes in serum can be distinguished by this technique, quantitation on the acetate plates is difficult. A spectrophotometric method for activity measurements is far more rapid arid reliable. T h e bl isozymes are characterized by a low Km for phosplioenolpyruvate (PEP) (less than 0.1 x M), whereas the 1, form has a much higher Km for PEP (0.4-1 x M).’Thus, by choosing an appropriately low PEP concentration, we can measure the M form selectively. In our study, P K activity was determined at 25°C on fresh serum samples, using the method of Valentine and Tanaka.:j6 at a final PEP concentration of 140 p M . Under these conditions, activities of the M and I . forms are about 95% and 60% maximal, respectively. I n initial experiments, PK activity was measured at 1 2 different PEP concentrations, ranging from 10 pbf t o 6 mkl, using ADP concentrations of 0.4 mb1 and 2 mM. Empirically, a PEP concentration of 140 pM and an ADP concentration of 0.4 riiM provided the best discrimination betiveen norrnal and carrier sera. ‘The following reaction Lvas measured by r-ecording the absorbance decreasc at 340 nlll:

PK Assay.

PE:P

+

XI)”

1’ li

p ) i ~ i \ ‘ l t ct

+

p r ~ i ~ ~~ . -w\ ~ ) ~ - ~ l t r . l t c

Serum CK and PK Levels in DMD

+

.A 11’

S.W.

+

H

(Abbreviations: NADH = reduced form of nicotinaniide adenine dinucleotide; LD = lactate dehydrogenase.) l‘he assay mixture contained 2 nil distilled water, 0.1 ml 0.24 M MgSO,, 0.1 rnl 2.25 M KCl, 0.2 rril 0.006 M ADP, 0.1 rnl LD diluted lvith water to contain 540 enzyme units/ml, 0.4 ml NADH solution (2 mg dissolved in 2 in1 0.05 M triethanolamine-HC1 TEA) buffer.. p~ 7.5), 50 p~ serum, and 0.1 ml 0.0042 M PE,P in 0.05 M .l’EA buffer. The solution was mixed after the addition of the first four componcnts and after the addition of each subsequent component. DMD patient sera, but not control or carrier scra. \\ere diluted 1:20 with 0.05 M TEA buffer. Sera were kept in an icebath arid were added to the reaction mixture without prior incubation at room temperature. LAD (rabbit muscle, esstmtially free of pyruvate kinase, ammonium sulfate slurry), the tricyclohexylamrnoniurn salt of PEP, the disodium salt of ADP. and the disodium salt of P-NADH in preiveighed vials containing 2 mg each were purchased from Sigma Chemical Co. (St. Louis, MO). Fresh PEP and ADY solutions were made \veekly and stored frozen at -20°C in small aliquots. The N A D I 1 solution \$.as prepared,just before addition to the assay mixture. The LD solution was made on the day of use and kept in an ice bath. T h e serum samples were chilled on ice for at least 30 rnin prior to assay t o prevent activation of the allosteric pr-operties of the L isoqmes.’ Concentrations of ADY higher than that specified in the assay should not be used unless the buffering capacity of the system is increased. T h e addition of a larger amount of ADP radically lowers the pH, causing spontaneous oxidation of NADH without the addition of PEP or serum (unpublished observations). .I’hc CK and PI( assays were performed in duplicate. arid for most comparisons o n l y one serum sample from each indik-idual was analyzed. However, all o f the definit.c carriers had had preL’ ‘10LlS C K tests so that their classification as a low CK-, borderliric C K - , or high CK-carrier \\as firmly established. CK and P K activities were calculated from the linear reactions that occur after the initial lag period (2-10 min), and the results are reported as I U (pmoles of‘ NAD or NADPH converted to NADH o r NADP. respectively. per minute pcr liter of serum). The absorbance readings at 340 nm Fvere recorded automatically using a Gilford model 2400s spectrophotometer (Gilford Instrument Labs, Inc., Oberlin. OH) with a thermostatically regulated, 4-chamber cuvette selector.

MUSCLE & NERVE

Sep:Oct 1979

331

20

15

n

1.0

1.2

1.6

1.4

1.8

2.0

Figure 7 D/stnbution of serum CK act/wbes /n 343 normal female controls aged 17 to 70 years (39 8 f 15 4, mean 5 SD) When percent frequency rn subjects is plotted against log CK activity, the data (histogram) can be represented by a theorebcal log normal distribution (st/pp/edarea) with a mean of 1 57 and a standard dewation of 0 183 The 95% conf/dence brnits (mean f 2 SO) are designated with arrows The upper normal l/mit of this /og normal d/stribution IS 89 IU (antdog mean 2 SD)

2.2

LOG CK ACTIVITY (I U )

+

1

RESULTS Distribution of Serum CK Activity in Adult Female Controls. T h e distribution of serum C K activity was

examined in 343 normal female controls aged 39.8 15.4 (mean ? SD) years. As shown in figure 1, the distribution was essentially log normal and could be represented by a theoretical normal distributiofi, with a mean of 1.57 and a standard deviation of 0.183. The tail in the high CK range of this distribution was omitted in the calculation of these parameters. The upper normal limit of this distribution-(i.c., antilog [mean 2 SDI-is 89 I U (97.5% confidence level). Theoretically, only 2.5% of this normal population would have higher serum CK activities. T h e upper normal limit, calculated in this fashion, was virtually identical to that determined from a cumulative frequency plot (97.5%confidence level).8

5

+

Distribution of Serum PK Activity in Adult Female

Serum PK activity was also measured in 42 of the controls, aged 28.7 2 6.2 years. As shown in figure 2, the distribution of- PK activity in this group could be represented by a theoretical normal distribution with a mean of 13.1 IU and a standard deviation of 2.4. The upper normal limit of serum PK activity (mean + 2 SD; 973% confidence

Controls.

332

Serum CK and PK Levels in DMD

level) was 17.9 IU. The corresponding serum CK and PK activities for these 42 women are shown in figure 3 (small solid circles). Relationship between Serum PK and Serum CK Activities in DMD Patients, DMD Carriers, and Control Subjects. PK activity in serum samples from the

patients with DMD, from the definite and possible carriers, and from thr: female controls have been compared with the corresponding CK activity in figure 3. In this figure, the upper normal limit of serum CK (89 IU) and serum PK (17.9 IU) in adult female controls of random age have been indicated by intersecting dotted lines. PK arid CK activity was greatly elevated in the DMD patients and significantly correlated (correlation coefficient, r = 0.67), and both enzymes tended to decrease i n activity with increasing age (fig. 4A and B). In both the definite arid the possible carriers, elevated ~ a l ues ol' PK were not always associated with elevated values of C K , although the activities of these erizymes were significantly correlated: definite carriers, r = + 0.68; possible carriers designated as heterozygotes, r = 0.57. There was no significant correlation between CK and PK in the control group (r = 0.24). Of the 20 definite carriers, 10 of 11 with ele-

+

+

+

MUSCLE & NERVE

SepIOct 1979

5

10

15 PK ACTIVITY (I U )

vated CK had elevated PK. Of 9 definite carriers with normal C K , 4 had elevated PK. Therefore, 15 of the 20 definite carriers had elevated CK or elevated PK or both; l l and 14, respectively, had elevated CK or elevated PK. Of the 47 possible carriers, 8 had elevated CK and elevated YK, 10 had normal CK but elevated PK, and 28 had normal CK and normal PK. Nineteen of the 47 had elevated CK or elevated PK or both, while 9 and 18, respectively, had elevated C:K or elevated PK. Effect of Age on the Detection Efficiency of the CK Assay. We considered the possibility that the upper

normal limit of CK activity might vary with age, and calculated means and upper normal liniits for several different age ranges within our control population of 527 normal females (table 1). The mean CK activity was highest between 6 and 10 years of age, lowest between 16 and 25, arid increased slightly with increasing age thereafter. The upper normal limits of CK activity were lowest (about 70 1U) between the ages of 2 1 and 35. Since an elevation in CK activity is used t o classify a subject as a DMD carrier, it is clear from the data in table 1 that the efficiency of the CK test in detecting carriers is affected by the age range of the population used as a control.

Serum CK and PK Levels in DMD

20

25

Ffgure 2. Distribution of PK achvities in 42 normal female controls aged 17 to 60 years (28.7 ? 6.2; mean -c SD).The empirical data can be represented by a theoretical normal distribution (stippled area) with a mean of 73.I IU and a standard deviation of 2.4. The 95% confidence limits (mean ? 2 SD) are designated with arrows. The upper normal limit of this distribution (mean SD)is 17.9 IU.

+

Listed in table 2 are the age and the corresponding PK and CK activities in the definite and possible carriers who currently have serum CK activity below 89 1U (i.e., within the normal range in 527 adult female controls of random age) but elevated or borderline-normal PK activity. When the CK levels of these women were compared with those of age-matched controls (table l ) , the CK levels of one 01' the definite carriers (No.956) arid four of the possible carriers (Nos. 945, 985, 986, and 952) were reinterpreted as elevated (see footnote e, table 2). In previous carrier studies, serum CK activity has not been considered to be age-dependent in normal adults. MeltzeP found differences similar to those reported in table 1, but they were striking only in black males. Satapathy and Skinner,29however, recently reported that CK levels in normal females tended to increase with age, being lowest in children under 15. T h e latter observation is not consistent wit.h the data in table 1, but the discrepancy may arise from the fact that the blood samples used for that study were taken from children who were inactive because they were hospitalized, whereas our samples wcre taken from subjects who were leading a normal, more active existence. This issue must be resolved if a method is to be

MUSCLE & NERVE

Sep/Oct 1979

333

14

-

0

30-

0 0 0

26

-

0

-

0

3

+

22-

t

L c

0

V

X

4

K (3 9

0

18-

.

0

0 4

0

14-

10

12

Id

16

I8

I

20

22

24

26

established for ietermining the carrier state during childhood.33 The effect of carrier age on the detection efficienryaf the CK assay was next taken into consideration, since two g r o ~ p s lreported ~ , ~ ~ that in some carriers serum CK activity falls from a significantly elevated level to within normal limits with increasing age. Two of the definite carriers (Nos. 949 and 880) and two of the possible carriers (Nos. 588 and 948) were reclassified because they had had elevated CK in previous years, although the levels were currently within normal limits (see footnote d, table 2). Seven of these eight women who now were considered to have elevated CK levels also had elevated PK. Even when these age effects were taken into consideration, however, there were still several women with elevated PK but normal CK (definite carriers 902, 996; possible carriers 1026, 619, 662, 585, 1020, and 647) (see footnote c, table 2). The average age of the possible

334

28

30

32

LOG CK ACTIVITY (I U )

Serum CK and PK Levels in DMD

34

Figure 3. The relationship between serum PK and serum CK activities in D M D patients, D M D carriers, and control subjects. 0 = D M D patients; 0 = definite camers (obligate and probable); X = possible carriers; = normal female controls. The intersecting dotted lines represent the normal upper limits of the serum enzymes /n adult female controls of random age: CK, 89 lU; PK, 17.9 lU (figs. 1 and 2).

carriers with elevated PK but low or borderline C K was 30.6 9.3 years, whereas that of the control group was 28.7 f 6.2 years. Since these two groups are essentially age-matched it seems unlikely that the elevations in PK reflect age differences o n thP auerage, rather than carrier state. In summary, when the dual effect of subject age was considered in the C K assay, 14 of the 20 definite carriers had elevated CK and 15 of the 20 had elevated CK and/or PK. Of the 47 possible carriers, 16 had elevated CK and 20 had elevated CK and/or PK.

*

Age-Dependenceof Serum PK Activity and CK activity in DMD Patients, DMD Carriers, and Control Subjects. Another question of importance is whether

serum PK activity is age-dependent. T h e logarithms of serum PK activity and the corresponding serum CK activity in each DMD patient and carrier as a function of subject age are plotted in figure 4.

MUSCLE & NERVE

SepiOct 1979

3.44

g

2.2-

5 +

-

V

Q:

K

(3

9

1.8-

A

0

0 0

X

x

X

e

x X

x

e x

-

X

1.4-

0

l

10

l

l

20

l

l

l

0.

1

30 40 AGE (YEARS)

l

l

50

1

l

60

1

1

70

B

% 1A ]

0 0

0

Clearly, PK and CK activities in both groups tend to decrease with increasing age, although possibly not in identical fashions. It is of interest that in both u s e s there is no apparent discontinuity between the DMD patient arid DMD carrier data, and

Serum CK and PK Levels in DMD

Figure 4. Age-dependence of serum PK activity (A} and CK activity (8) in DMD patients, carriers, and controls. Enzyme activities were measured in 33 serum samples from 23 definite carriers and in single samples from the DMD patients and possible carriers. 0 = D M D patients; 0 = definite carriers with elevated serum CK in one or more of 3 tests; 0 = definite carriers with serum C K in the normal range; X = possible carriers with elevated serum C K (age-matched controls used as reference in all cases). The upper normal limits (97.5% confidence levels) of the enzyme activities in female controls of various age ranges have been indicated by horizontal dashed lines. The limits for CK were obtained from table 1 . Those for PK should be regarded as preliminary because of the limited data available (N = 3, 5 to 13 years; N = 25 in each of the other age groups).

that some female heterozygotes have enzyme activities as high as those of DMD patients in the same age range yet are not debilitated by the disease. Although our data are limited and should be regarded as preliminary, the upper normal limit

MUSCLE & NERVE

SepiOct 1979

335

Table 1. Relationship of serum creatine kinase (CK) activity to age in normal female controls Age (Yd

No tested

0-5 6-10 11-15 16-20 21-25 26-30 31-35 36-45 46-55 56-70

20 34 44 45 86 78 43 63 80 34

Mean log CK activity (IU)a

Upper normal limitb (IU)

1 773 2 0 130

108 155 168 83 6a 73 75 80 110 102

1 868 1 691 1 507 1 516 1 557 1 581 1559 1610 1 602

"Mean log CK actw\ty (lU) bAntilog (mean -C 2 SO)

?

0 161 t 0 268 t 0 195 t 0 158 ? 0 152 +- 0 146 t 0 171 i. 0 2 1 7 5 0 203 ?

SD

Table 2. Carriers with borderline-normal or normal creatine kinase (CK) activity and elevated or borderline-normal pyruvate kinase (PK) activity.a Category and code nob Definite carriers 902(P) 949(0) 880(0) 956(0) 996(0) Possible carriers 1006(M) 948(M) 588(M) 945(M) 1040(M) 1029(M) 1026(M) 954(M) 985(MS) 986(MS) 619(S) 952(M) 662(M) 585(M) 1020(S) 647(MSD)

Age (Yr)

PK activity (IU)

CK activity (IU)

44 43 39 31 20

22.0" 15.5 40 I c 20.0c 23.W

28 7ad 77d 78' 65

54 47 43 42

16.7' 13.4 26.6c 18.9" 16.7 1 7.0b 18.9" 17.4 21 .gC 25.OC 23.7c 19.9" 18.9" 29.9" 19.6" 25.4c

26 82d 72d u7e 75 50 25 37 79" 74e 61

40

39 38 35 33 29 27 26 25 25 20 17

"Carriers were included in this table if their serum CK activity was !ess than 89 lU and their PK actwity was elevated or borderline normal (15.5 lU or greater) bietters in brackets after the code number indicate the exact status of the carrier 0 = obligate carrier, P = probable carrier, M = mother of one affected son and no previous family history of the disease, MS = maternal aunt. S = sister of an affected DMD pabent. MSD = first cousin (daughter of a maternal aunt). cPK activity exceeded fhe normal upper limit. aSubject previously bad an e!evated serum CK, but / t fell with increasing age to a normal level. 'CK activity was elevated compared to age-matched controls (see table I ) . 'PK activity was elevated on a second testing.

336

Serum CK and PK Levels in DMD

80e 47 52 50 28

of serum PK activity in normal females may show an age-dependence similar to that of CK (dashed horizontal lines, fig. 4A and B); 98%1 confidence levels determined from cumulative plots of data in figure 2 and from data obtained more recently were somewhat lower for 26 to 35-year-olds than for those aged 15 to 25 or 36 to 60, and several children between the ages of 5 and 13 had serum PK activities approximately twice as high as those in adults. This possible age-dependence of PK in normals does not signihcantly af-Sect our interpretation of the PK data in table 2; the only consequence is that the PK levels of three possible carriers (Nos. 945, 1026, and 662) become "borderline normal" rather than "marginally elevated." We are continuing to collect data to establish reliable means and ranges of corresponding serum C K and P K activities for various age groups. Evidence that Some Affected Boys Are New Mutants.

To consider the question whether some mothers of affected boys are noncarrierq, we examined the CK and PK levels in 24 possible carriers who were mothers with one affected son but had no family history of the disease. Eleven of these 24 had significantly elevated serum CK and/or PK. Four others had borderline PK values, but CK values within the normal range (possible carriers 954, 1006, 1029, and 1040 in table 2); on retesting, the PK values were significantly elevated in two of these four women (Nos. 1006 and 1029). If we assumc. that mothers with elevated PK and/or CK levels are carriers, then a total of 13 mothers were detected as carriers using the two tests (see footnote c, table 2). Thus, if the combined efficiency of the PK and CK detection tests is the same in the possible and definite carrier groups (i.e., about 80%) a maximum of 16 mothers are likely to be carriers of the DMD gene, and the remaining 8 are noncarrier mothers of mutant sons. DISCUSSION

The efficiencies of the serum CK and PK assays in reflecting the DMD carrier state are summarized in table 3. In the group of definite carriers, the PK test appeared to be a more sensitive indicator (70% detection efficiency) of carrier state than the CK lest (55% detection efficiency) when subject age was not considered as a variable. Subject age, however, influenced the detection efficiency of the CK assay in two ways. First, the upper normal limit of serum CK activity varied in adults, being lowest between the ages of 21 and 35 (table 1). Second, in agreement with results published earlier,'9335 CK

MUSCLE & NERVE

SepiOct 1979

activity in some carriers declined with increasing age (table 2). When these age effects were taken int.o consideration, the efficiency of the CK test in the group of definite carriers was the same (70%) as that of the PK test (table 3). Used together, hobvever, the two tests appeared to detect a higher proportion (80%) of definite carriers than either test alone. The detection efficiencies of the two tests might be expected to be very similar since the correlation between CK and PK activity in DMD patients and carriers is so high (fig. 3). In the group of possible carriers, the PK test appeared to be somewhat more efficient than the C K test even when age effects were considered, and in combination the two tests again appeared to detect a higher proportion (43%) than either test alone. It was anticipated that the efficiency of' the CK test would not be as high for this group as for the definite carrier group, because most of the possible carriers had only one seruni analysis whereas all of the definite carriers had had previous CK tests. To maximize detection efficiencies, we suggest that each person be tested on at least three separate occasions over a period of several months, and that the individual test results rather than their average be scored as elevated or normal relative to established control distributions. The observation that PK and CK activities in carriers decline with increasing age, whereas in normal adults they appear to increase after the age of 35, suggests that both tests are more efficient in younger persons and that carrier testing should begin at as early an age as possible (fig. 4). Although the PK test appears to be more sensitive when subject age is not taken into consideration, the CK test is more convenient because CK does not lose act.ivity irreversibly upon freezing. As in other situations,' elevated PK or CK levels must be interpreted with care. Elevated CK and PK are known to occur in myocardial infarction and in the other skeletal muscle diseases men-

tioned previously,!' as well as in DMI). There are also statistical considerations. Therorctically, 2.5% of the elevations in either enzyme in definite or possible carriers (tables 2 and 3 ) may be false positive r e s u h , because the borderline between normal and elevated has been arbitrarily drawn at the 97.5% level of confidence. I t may be possible to improve the classification by combining information from the two tests and using a more sophisticated statistical procedure such as discriminant analysis. The probability that the subject is a carrier must also be considered. In a control female, an elevated CK or PK level has a low probability of indicating the carrier state, since the prior risk that she is a carrier is low; if the incidence of DMD is about 1:3,100,6 the incidence of carriers is only about 1:2,300 (413 x the incidence of affected males). In contrast, in a DMI) pedigrce, a mother or other female relative in the X-linked line of descent has a much higher prior probability of being a carrier. A statistical method for combining the results of serum enzyme tests with prior genetic risks determined from pedigree analysis Ivould improve the discrimination between normal subjects and carriers in doubtful cases. The results of this study strongly suggest that the combined use of- serum PK and serum CK measurements will be of value in the classification of possible carriers who do not have an unequivocally high serum CK activity. The reliability of this approach should be assessed on a larger scale and by other laboratories. The data presented in table 3 are basically in agreement with the controversial proposal of Alberts and Samaha' and Zatz et who suggested that elevations of serum PK activity were more common than elevations of serum CK in DMD carriers (age-dependence was not considered). The data are in disagreement with those of Hardy et all' and Smith and T h o m ~ o nwho , ~ ~rcgard the CK assay as superior for DMD carrier detection. One

Table 3. Detection efficiencies of the PK and CK tests a Category Deftntte carriers Possible carriers Female controls

No. tested

Elevated PK alone

Elevated CK alone

Elevated PK and/or CK

20 47 42

70%

38%

55% (70%) 19% (34%) 0

75% (80%) 40% (43%) 2 3%

2 3%

'Adult age was not considered to be a variabie in the calculation of the unbracketed detection efficiencies As explained in M e text, the detection efficiency of the CK test IS influenced by the age of both the control group and the carriers, this age dependence was taken info consideration in calculating the bracketed defection efficiencies

Serum CK and PK Levels in DMD

MUSCLE & NERVE

Sep/Oct 1979

337

major factot- contributing to this apparent coritroversj. is the use of "upper normal limits" to define the bordcrline tlettveen normal and elevated. As shown in this study, the u p p e r riorrnal limit o f CK (and possibly PK) activity is age-dependent, a n d failure to consider the age of controls or carriers could bias the number of "elevated" levels in o n e direction o r the other. Another possible cause is that different assay terns are used in different laboixtories to measure CK and PI( activities. Detection efficiencies rnay ivell depend on the specific assay used, because the biochemical bases for these differ arid different assays probably will measure activities of' the rnuscle isozynies in serum with differing ckgrees o f selectivity.1310330 Finally, PK progressively loses activity upon storage at -2o"C;, whereas C K actil-ity is relatively stable.3"Ji N o t all laboratories have measured PK activities in fresh serum sample^.'^ In families ivith X-linked disorders, the affected boy either has inherited the gene from his carrier mother or is a new mutant. Until there is ;I test that can distinguish carriers frotn noncarriers with certainty. one must assume a prior probability to calculate risks. According to classical genetic theory, the prior probability that a mother with ari affected s o n is a carrier is 2:3,'" although Roses et a P and Pickarti et a12' have suggested that it is much closer to 1 : 1, implying that a very high proportion of all mothers of DMI) patients are carriers, and that w r y few of the boys are new mut;ints. O n the basis of o u r combined C K a n d PK tests, we estimatccl thai ttvo-thirds of the 24 possible r a r 1-ier rriothers (tvilh one affected son and no previous famil). history) were likely t o be c:arric:rs o f the Duchenne gene, the remainder being normal individuals with new mutant sons. Although our sarriplc si7e is sinall, these data are similar to those previous1 y reported b y Thompson et al,32,3'Mort o n arid Ghurig,IXa n d Ilavie and Emery,4 and suggest that a substantial proportion of DMD patients have rioricarrier mothers. Many differ-enl methods a r e currently being employed in DMD carrier detection." These iriclude complex studies of muscle biop~ies,'~."red cells,26 peripheral blood l y r r ~ p h o c y t e s ,electro~~ tnyography,l' and measurement of enLynie activity or cor1c:eritrations of other proteins in serum." We have been assessing several serum tests (CK, PK, LD. and h e m ~ p e x i n " , " * ~in ~ )combination f o r the diagnosis of 11MD carriers, since these a r e easy to p e r f o r m , convenient., low in cost, a n d can be used to screen large numbers of possible DMD t-arriers.22 T h e results of the present study are en-

338

Serum CK and PK Levels in DMD

couraging, since the combined use of only the serum PK and CK measurements under the given assay conditions appears to increase detection efficiency over that afforded by the conventional CK test alone. T h e ultimate goal of being able to discriminate between carriers arid noncarriers with a high degree of' certainty, ~vithinthe limits imposed by random X-inactivation, may be reached in the not-toodistant futiire. Carrier detection efficiency in DMD continues to increase, as do the number o f different carrier tests. With the variety of methods available for carrier detection, t.he risk of obtaining false-negative results is bound to be minimized. REFERENCES 1. Alberts MC. Samalia FJ: Serum pyriivate kiriase in rriuscle disease and carrier states. h'wiokop (Minneap) 24:462-464, 1974. 2. Casscells M;, Schoeriberger A , Graboys T B : Interpretation by physicians of clinical Iahoratory results. .2: Engl J .Wed 299:YYYL 1001, 1979. 3. Danieli GA, Angelini C: Duc heririe carrier detection. LnrirPt ii:80, 1976. 4. Davit: AM, Emery AEII: Estimation of proportion of new inritants among cases of D u c I i e ~ i ~ muscular i~ dystrophy. J .Wrd G ~ n d1.5:339-345, 1978. 5 . Ehashi S Y , Toyokura H M , Sugita H: High creatine phosphokinase activity of sera of progressive muscular dystrophy paLier1ts.J Bzoch~m( T o k y o ) 46: 103- 104, 1959. 6. Gardner-Medwin 1): Mutation rate in Ducheririe type of niuscular dystrophy.,/ M i 4 GrnPt 7:334-337, 1970. 7. Goeddc H W , Christ I , Benkniann HG, Beckrrian R, Larig H: Creatine kiriase isoen7yme pattcrns in Duchenne mu5cular dy-strophy. (:/zn Gcnd 14:257-260, 1978. 8. Griffiths PD: Serum lebels of ATP: creatine phosphotransfcrasc (creatine kinase). T h e normal range and effect of muscular activity. Clin Chinz Actu 13:413-420. 1966. 9. Harano Y , Adair R, Vignos PJ J I , Miller h4, Kowal J : Pyruvate kinase isoenzynies in progressive muscular dystrophy and in acute myocardial infarction. ;~frtubolisrn22:493501, 1973. L), Pcmiington R,]l':Serum 10. Hard? MF. Gai-dnei--R.Ied\~,in p! ruvate kinase in carriers o f Duthenrie muscular dystrophy.,/.\'eurol Sci 32:137- 139, 1977. 11. Hausinanowa-Pctrusewicl I , Kichi-qj-Ihbosz I, Borkowska ,J. Lukasik E, Liszewska-Heifer D: Carrier detectiori in Duchenne dysti-ophy. I n Rowland LP (Editor): Pnthogmaz.q o/ Humarc ;Muscdnr D y t r o p h w Amsterdam, Exccrpta Medica, 19i7, pp 32-41. 12. Huttori EM: Duclieririe rriuscular dystrophy: genetic and biochemical studies of' the female can-ici-s and their families. PhD Thesis, University o f Toronto. 1970. 13. Ionasescu V , Zellweger H, Burmeister L: Detection 01 carriers and genetic counselling in h c h e i m e muscular d p rrophy hy ribosomal protein synthesi 34:442-452, 1976. 14. Kuby SA, Keutel HJ, Okabe K, Jacobs f i K , Ziter F , Gerber D, Tyler FH: Isolation of thc human ATP-creatine transphosphorylases (creatine phosphokiriase) from tissues of patients with Diichenne muscular dystrophy. J B i d Chem 252:8382-8390, 1977. 15. Marie J , Garreau H , Kahn A: Evidence for a postsynthetic proteolyrir transformation of human rrythrocyte pyruvate kinase into L-type enzyme. FEBS Lett 78:91-97, 1977.

MUSCLE & NERVE

SeplOct 1979

16. Meltzer HY: Factors affecting serum creatine phosphokinase levels in the general population: the I-isk of I-ace, activity aud age. Clin Chin8 Arta 93: 163- 172, 197 I . 17. Monckton C: Incorporarinn of 3€I(C)L-leucine into single muscle fibres in Ducheririe dystrophy and Charcot-MarieTooth Disease. IVth International Congress on Neuromuscular Diseases, Montreal, 1978, .4bstract 259. 18. Morton KE. Chung CS: Formal gerietics of inusrular dystrophy. Am J H u m C h i p t 11:360-379. 1959. 19. Munsar I’L, Raloh R, Pearson CM, Fowler 1%’: Serum cn7yme alterations in rieuromuscular disorders. J An/ :\,fed AJsor 226: 1536- 1.543, 1975. 20. Murphy EA, Chase GA: hlendeliari conditions: the grnotypes of the consultants are unknown. 11. All positite information is posterior. I n Murphy E l l , Chase GA (Edirors): Principles ofCPnP/ic Counrdling. Chicago. Year Book Medical Publishers, 1975, pp 137- 162. 21. Pennington KIT: Serum enqrnes. In Walton .IN (Editor): f h o r d m OJ‘ Volurrtnq i2lust.k. Edinburgh, Churchill Livingstone, 1974, p p 488-516. 22. Percy ME, Chang L, Oss I, Pitt MA, Vei-rllen C:, Thompson MW: An improved method for carrier detection in Ducheririe muscular dystrophy-. Proceedings of the AmPnrnn Sociefy nf Human G P ~ P / ~29th c s , Annual Meeting, Vancouver, 1978, p 63A. 23. Percy ME, Chang L, Verellen C , l’hotnpsun M\V: Serum hernopexin in Duchennc muscular dystrophy. Proceedings of the Can Fed B i d Soc, Lotidon, Ontario, 1978, p 92. 24. Pickard NA, Gruerner H-D, Vcrrill HL, Isaacs ER, Robinow M, Nance WE, Myers EC. Goldsrnith B: Systemic membrane defect in the proximal muscular dystrophies. iV EngI J M p d 299:841-846, 1978. 25. Rosalki SB: An improved procedure for serum creatine phosphokinase detcrmination.J Lab Clin i M ~ dfi9:696-705, 1967. 26. Roses AD: Erythrocytes in dystrophies. In Rowland 1.P (Editor): Palhogenrsis vf Human iWiurular DyJtruphies. Amsterdam, Excerpta Medica, 1977, p p 648-658. 27. Rosm AD, Roses YJ, Nicholson GA, Roe CK: Lactate dehydrogeiiase isoenzyrue 5 in detecting carriers of DLIchenne muscular dystrophy. ,VPurolog (Minneap) 27:414421, 1977.

Serum CK and PK Levels in DMD

28. Saks \’A, Scppet EK, Lyulina NV: Comparative irivestigation of the role of creatine phosphokiriase i s o e r q tries in energy metabolism of skeletal muscles and myocardium. Bzukkirniia 42:579-588, 1977. 29. Satapathy RK, Skinner R: Seruni creatine kiriase levels in normal females./ M r d G P 30. Smith 1, Thonison WHS: wssive (Duchenne) muscular dystrophy: pyruvate kinace isoenrynies and creatine phosophokiriase in serum and blood cells. Clzn Chrm A c t o 78:439-451, 1977. 3 1. S u s o n W A , Rutter h’J:Method for the detection of pyruvate kiriaae. aldolase, and other pyridine nuclcotide linked enzyme activities after electrophoresis. ,4na[ Bzochpm 13:147155,1971. 32. ’I‘honipson MW’, Hutton EM: The occurrence of new mutants in X-linked recessive lethal disorders. Vth International Congress of Hurnan Genetics, Mexico, 1976. .Ihstract 3 15. 33. Thompson MW-, Murphy EC,. McAlpine Pj: A n assessment of thr c x a t i n e kinase test in the detection of cari-iers of Duchenne muscular dystrophy. / Prdiatr 7 I :82-93. 1967. 34. Thompson MW, Oss 1, Hutton EM, Murphy EG: T h c carrier status of mothers of Duchenne muscular dystrophy patients. IVth International Congress on Kcuromuscular Discascs, Montreal, 1978, Abstract 486. 35. Thorrisori WHS, Sweetin JC, Elton KA: ‘1 he neurogenic and myogenic hypotheses in human (Ductienne) muscular dystrophy. R’aturr 249: 151- 152, 1974. 36. Valentine WN. Tanaka K K : Qruvate kinase: clinical aspects. M ~ h r l Enzjrrml s 9:468-473, 1966. 37. Weinstork IM, Hehrendt J , Wiltshire HE, Jr, Kelemen J , Louis S: Pyruvate kinase: diagnostic value in neuromuscular disease. Clin (:him Acta 80:415-122, 1977. 38. Zata M. Sltapiro LJ, Campion DS, Kaback M M : Improved diagnosis and cari-ier- detection for the muscular dystrophies by the combined use of Serum pyriivate-kinase ( P K ) arid creatine-phosphokinasc (CPK). Conkreiice on Birth Defects, Montreal, 1977, Abstract 167. 39. ZatL M, Shapiro L.J. Campion DS, Oda E, Kaback Mhl: Serum pyruvate-kinase (PK) arid creatine-phosphokinase (CPK) in progressive muscular dystrophies. J Neurol Srz 36:349-362. 1978.

MUSCLE & NERVE

Sep!Oct 1979

339

Serum creatine kinase and pyruvate kinase in Duchenne muscular dystrophy carrier detection.

The incidence of elevated serum creatine kinase (CK) and pyruvate kinase (PK) activities was compared in 20 definite carriers of Duchenne muscular dys...
914KB Sizes 0 Downloads 0 Views