Serum pyruvate-kinase (PK) and creatine-phosphokinase (CPK) in female relatives and patients with X-linked muscular dystrophies (Duchenne and Becker)

Journal of the Neurological Sciences, 1980, 46: 267-279

267

© Elsevier/North-Holland BiomedicalPress

S E R U M P Y R U V A T E - K I N A S E (PK) A N D C R E A T I N E - P H O S P H O K I N A S E (CPK) I N F E M A L E R E L A T I V E S A N D P A T I E N T S W I T H X - L I N K E D M U S C U L A R DYSTROPHIES ( D U C H E N N E A N D BECKER)

MAYANA ZATZ1'3, LARRY J. SHAPIROl, DAVID S. CAMPION2, MICHAEL M. KABACK1and PAULO A. OTTO3 I Department of Pediatrics, Division of Medical Genetics, U.C.L.A. School of Medicine, Harbor General Hospital Campus, Torrance, CA, 2Department of Medicine, U.C.L.A. School of Medicine, Center for the Health Sciences, Los Angeles, CA and 3Laborat6rio de GenOtica Humana, Instituto de Bioci~ncias, Cidade Universit6ria, Caixa Postal 11.461, S6o Paulo (Brazil)

(Received 30 August, 1979) (Revised, received 13 December, 1979) (Accepted 8 January, 1980)

SUMMARY Determination of serum creatine phosphokinase (CPK) activity is often used in efforts to detect carriers of X-linked muscular dystrophies. We have recently demonstrated that another serum enzyme, pyruvate-kinase (PK) may also be of use in the diagnosis of patients affected with a variety of neuromuscular disorders. To evaluate the usefulness of this assay for carrier detection, a comparative study of serum PK and C P K activity was performed in 74 female relatives of patients affected with Duchenne ( D M D ) and Becker (BMD) muscular dystrophies. For obligate carriers of the D M D gene, 10 of 14 had elevated CPK's, 11 of 14 had elevated PK's and 12 of 14 had abnormal results for either of the two enzymes. Three of 16 mothers of isolated cases had increased serum CPK activity and 6 of 16 had increased PK activity (7 had elevation of at least one enzyme). These preliminary data suggest that the use of PK may enhance the capability to discriminate carriers for these X-linked recessive genes.

This work was supported in part by a grant from Funda~,~o de Amparo ft. Pesquisa do Estado de S~.oPaulo, Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico(CNPq), The National Institutes of Health Research Training Grant, NIGMS IT32MO7414-01,and the National FoundationMarch of Dimes Genetic Center Grant, C-114.

268 INTRODUCTION

The ability to identify heterozygotes for X-linked deleterious alleles is of major importance for genetic counseling in families where such disorders have occurred. The severe and relatively common Duchenne muscular dystrophy (DMD) and the more rare but also more benign Becker dystrophy (BMD) are usually inherited as X-linked recessive traits. In such pedigrees there may be many females at risk. In some families, the propositus may represent the only known affected individual. It is important, in this instance, to be able to discriminate between those families where the affected individuals represent new mutations from those in which the patients have inherited the gene from their heterozygous mothers. Elevated serum CPK in carriers of the Duchenne gene was first reported by Ebashi et al. (1959) and since then, many reports have confirmed the value of serum CPK determinations for carrier detection (Aebi et al. 1961 ; Pearce et al. 1964; Emery 1965; Milhorat and Goldstone 1965; Dreyfus et al. 1966; Thompson et al. 1967; Hausmanowa-Petrusewicz et al. 1968; Hetnarska 1968; Radu et al. 1968; D6mos 1969, Gardner-Medwin et al. 1971; Zatz et al. 1976). This test, although detecting only about 2/3 of the carriers of the DMD gene and 1/2 of the carriers of the BMD gene (Mabry et al. 1965; Emery et al. 1967; Skinner et al. 1975; Emery and Skinner 1976; Zatz et al. 1976) is still the only one generally employed in most centers providing genetic counseling for the muscular dystrophies. In the last decade, several new methods for DMD carrier detection have been reported. Increased protein synthesis in polyribosome preparations derived from muscle biopsies from DMD patients and carriers has been described (Ionasescu et al. 1971 ; Ionasescu et al. 1973). An increase in the mean "peak II" phosphorylation in erythrocyte membranes from mothers of DMD patients (from pedigrees with isolated and multiple cases) also has been proposed as a sensitive method for carrier detection (Roses et al. 1976). Another study reports increased serum haemopexin in heterozygotes for the Duchenne gene, with detection rates greater than with CPK alone (Danielli and Angelini 1976). Since some of these methods require an invasive procedure (biopsy), and/or complex biochemical analyses, they have received only limited application to date. In 1973, Harano and his group reported elevated serum pyruvate-kinase activity (PK) in patients with different types of muscular dystrophy. Other laboratories (Alberts and Samaha 1974; Hardy et al. 1977 ; Zatz et al. 1978) have confirmed these results. Alberts and Samaha first used serum PK in the evaluation of DMD heterozygotes. They found increased serum PK activity in 5 of 7 obligate carriers and 3 of 4 probable carriers. Only 4 among these 11 had elevated serum CPK activities. These data suggest that serum PK determination might be a more sensitive test than CPK for detecting carriers of the X-linked Duchenne gene. Subsequently, 3 other groups (Hardy et al. 1977; Yamuna et al. 1977 and Smith and Thomson 1977) have compared the utility of serum PK and CPK determinations in the detection of DMD carriers. In one report (Hardy et al. 1977) increased PK levels were found in only 5 of 17 obligate carriers, while 11 (includ-

269 ing the same 5) showed high CPK activity. A second study (Yamuna et al. 1977) found elevated PK in 2 of 17 definite carriers and 2 of 20 possible heterozygotes. Twenty of these 37 women had increased CPK activities. In the third investigation (Smith and Thomson 1977) 9 of 11 DMD carriers had an increase in both PK and CPK levels. In summary, then, conflicting data exist regarding the usefulness of serum PK determinations for DMD carrier detection. Recently, our group has investigated the diagnostic applicability of serum PK determinations in 138 patients afflicted with a variety of neuromuscular disorders (Zatz et al. 1978). We found this measurement to be of equal or greater value than CPK in the diagnosis of several conditions, including both Duchenne and Becker dystrophies. For this reason we have elected to evaluate further the usefulness of PK determination for heterozygote detection in these X-linked conditions. MATERIALAND METHODS Sixty-four female relatives of patients with DMD and 10 female relatives of patients with BMD were included in this study. The affected patients were attending the University of California, Los Angeles - Clinic for Neuromuscular Dystrophy. Diagnoses of affected males were determined by clinical examination, mode of inheritance and by electromyography and/or muscle biopsy. All of them had grossly elevated serum CPK and PK activities. Obligate carriers for the DMD gene were defined as: (a) a woman with one affected brother (or maternal uncle) and at least 1 affected son; (b) a mother of at least 2 affected sons; and (c) a woman with 1 affected son and 1 affected grandson through a carrier daughter. In addition to the 3 categories above, daughters of Becker dystrophy patients were also considered obligate carriers. The control group consisted of 62 normal adults (43 females and 19 males) and 9 healthy children with no history of neuromuscular diseases in their families. Blood samples were drawn by venipuncture without any anticoagulant and centrifuged shortly after collection. Sera that showed any sign of hemolysis were discarded. None of the individuals had undergone vigorous exercise 24 hours prior to collection. Most of the PK and CPK determinations were performed in fresh sera within 3-6 h of venipuncture. If not assayed the same day, the samples were aliquoted and frozen at - 2 0 °C until assay, the next day. Other aliquots were kept frozen for extended periods of time to evaluate the stability of enzyme activity. All PK and CPK determinations were done in duplicate. Whenever a subject was tested on different occasions, the mean activity of serum PK and CPK was used. Assay of total PK activity was performed at 21 °C by measuring the decrease in absorbance of reduced nicotinamide adenine dinucleotide at 340 nm with a Gilford recording spectrophotometer. The assay was similar to that used by Valentine and Tanaka (1966) and modified by Alberts and Samaha (1974). The only methodologic difference introduced in our laboratory was that normal saline was used to adjust the reaction volume instead of distilled water. All enzyme activities are reported as lamoles/ml serum/h. CPK activities were determined with Sigma kits in the same

270 sera as those used for PK determinations. The colorimetric method used is described in Sigma Bulletin 520-C (1976). The results are expressed in Sigma units. For statistical analysis a logarithmic transformation was used on all data since some distributions differed significantly from normality; the transformed data could then be treated as Gaussian, as shown by K o l m o g o r o v - S m i r n o v goodness of fit tests, and all statistical tests were performed on these data. Back-transformed data were used for reporting the means and their confidence intervals. Since the variances in the two groups of comparison (normal females versus heterozygous women) were greatly different and the size of the latter group was small (n = 14) simplified methods were used for calculating the discriminating power of combined PK and C P K determinations (that is, we added the probability ratios in logarithmic form obtained for each enzyme separately, without taking into account the correlation of 0.60 IP = 0.021 found between P K and C P K for heterozygous women). A more formal model of discriminant analysis will be applied in a larger sample now being collected in Brazil. RESULTS In Tables 1, 2, 3 and 4 are summarized the results on serum PK and C P K activities in all groups which were studied, including the correlation coefficients between the two enzymes.

(A ) Normal controls The mean P K activity in the adult control group was significantly lower in males than in females (t = 2.416; d.f. = 60; P = 0.019). The C P K levels were significantly higher in normal men than in normal women (t' = 3.332; d.f. = 24; P = 0.003) as described previously (Hughes 1961; Rosalki 1967; Meltzer 1971). In 9 normal children (1 girl and 8 boys) aged 3-14 years, P K levels were significantly higher than in normal adults (t = 8.677; d.f. = 26; P = 4 × 10 -9) and decreased with increasing age (r = - 0 . 8 9 3 ; t = 5.400; d.f. = 7; P = 0.001). However no correlation was found between P K or C P K and age in 34 adult female controls whose ages were recorded. Therefore PK levels in suspected carriers under 15 could not be compared with the adult controls and were not taken into account for statistical purposes. The CPK activities in the 9 normal children did not differ significantly from the adult group. F o r the adult women the upper limit of normal was considered to be 2.1 ~tmoles/ml/h for PK and 8.4 Sigma units for CPK. For the 9 children under 15 that we have tested these limits were 13.3 Sigma units for C P K and 5.2 p,moles/ml/h for PK.

(B) Obligate carriers for the DMD gene Fourteen women from 10 pedigrees with multiple D M D affected patients were included in this category. The mean P K and C P K for the group were significantly greater than in the control group (t = 7.044; d.f. = 55; P = 3 x 10 9 and t' = 5.709; d.f. = 16; P = 3 x 10 ~. respectively. No correlation was found between PK or C P K

271 and age. Eight of the 14 had the PK and CPK estimations repeated on 3 different occasions. Of these 8, 2 had abnormal serum P K activities on all 3 occasions, 5 on 2 of 3 determinations and 1 was normal on all 3 tests. For CPK, 5 showed abnormal values in all 3 examinations and 3 had only one abnormal value among the 3 determinations. Two individuals had 2 separate determinations and 4 were tested only once. Using for each individual the mean value of the determinations performed, w e - f o u n d that 11 had abnormal PK levels (above 2.1 lamoles/ml/h) and 10 had abnormal CPK activities (above 8.4 Sigma units). One had elevated CPK and normal PK. Therefore, only 2 of the 14 were normal for both enzymes (Table 1). Six of the 16 mothers of isolated cases had elevated PK activities in their sera and 3 had increased C P K (Table 1). Seven had an increase in either I of the 2 enzymes. In addition, 2 among those with normal PK and CPK had at least 1 daughter with abnormal CPK activity. If we consider that an elevated CPK activity in the sister of an isolated patient indicates tha~ their mother is a carrier for the D M D gene, we would have 9 carriers among the 16 mothers of isolated cases. (C) Sisters o f affected patients with D M D The results in younger sisters of affected patients are difficult to analyse because we have not tested the PK values in a large sample of normal young girls. However, if we would tentatively compare the sisters under 15 years of age with the 9 normal children that we have tested and those 15 or older with the normal adult females we observed that 4 of 9 daughters of obligate carriers were abnormal in both PK TABLE 1 SERUM PK AND CPK IN FEMALE RELATIVESOF PATIENTSWITH X-LINKED DYSTROPHIES Subjects

Individuals tested

(A ) Normal controls adult females

43

(B) Femalerelatives of DMD patients obligate carriers mothers of isolated cases daughters of obligate carriers sisters of isolated patients other at-risk female relatives

14 16 9 14 9

affected possible carriers (C) Femalerelatives of BMD patients obligate carriers possible carriers

2

5 5

Numberwith elevated PK

CPK

PK or CPK

0

3

3

11 10 6 3 4 4 5 3 2 3 1 borderline 2 2

12 7 4 6 3

2 3 1 borderline 1 2

3

2

2

272 and CPK activities. Among the 14 sisters of isolated patients, 5 would have elevated PK and 3 had abnormal CPK. Two with abnormal PK had normal CPK levels. In addition, 1 with abnormal CPK had normal PK activity. Therefore, 6 of the 14 sisters of isolated cases would be abnormal for either 1 of the 2 enzymes studied (Table 1).

(D) Clinically affected carriers Two possible carriers of the D M D gene, aged 10 and 11 years, had clinical symptoms of muscular dystrophy (marked proximal and moderate distal muscular weakness), as well as abnormal electromyographic examinations and muscle biopsies characteristic of Duchenne dystrophy. Both had very high serum PK (67.53 and 77.71 ~tmol/ml/h) and CPK activities (580,0 and 660,0 Sigma units) which were typical of affected boys (Table 2). Chromosomal analyses were performed since abnormal karyotypes have been previously reported in girls affected with Duchenne dystrophy (Walton 1956 ; Ferrier et al. 1965; Jalbert et al. 1966; De Meyer et al. 1977 ; Greenstein et al. 1977). Both of them had a normal 46,XX female karyotype. Therefore the best explanation for these findings is a preferential inactivation of the X chromosome bearing the normal allele.

(E) Other at-risk female relatives In this group we included 2 first cousins, 4 nieces (of the proband) and 3 grandmothers all related to affected patients through maternal lines (Table 1). Serum P K activities were increased in l of 4 nieces and 1 grandmother had borderline values. CPK was abnormal in the same 2 subjects and in an additional grandmother. Three among the 9 that were tested in this group were abnormal for either 1 of the 2 serum enzyme activities.

(F) Female relatives of Jilatients affected with BMD Two of 5 obligate carriers had grossly elevated PK levels and 1 was borderline. CPK activities were abnormal in the same 3 individuals (Table t). Among 4 sisters of affected patients, 1 had abnormal P K and CPK, 1 was borderline for PK with normal CPK and the remaining 2 were normal for both enzyme levels. One mother of an isolated case had borderline PK and abnormal CPK activities. Her daughter had grossly elevated PK and CPK.

(G) Stability of serum PK Serum samples that had been stored f o r extended periods of time showed a progressive loss of serum PK activity. In sera that had been frozen for 2 months at - 2 0 °C, there was a loss of approximately 50~o in serum P K activity. Therefore, the apparent stability of M-type PK as suggested previously (Harano et al. 1973) was not confirmed in this study.

19 43 9

14 16 23 9 14 13 10 9 26

5 5

2.25 2.27 5.79

0.00-177.57 0.76- 7.04

1.07 4.86 0.7% 3.78 0.4% 13.53 0.49- 15.80 0.27- 14.47 0.68- 20.11 0.75- 5.48 0.37- 9.92 2.59 212.69

0.250.611.77-

9 5 ~ confidence interval of the distribution

5.08 2.77

2.49 1.91 3.65 4.01 3.43 4.96 2.36 2.86 26.71

1.02 1.30 3.34

X

0.34-26.58 1.68- 4.29

2.03- 3.00 1.57- 2.29 2.66- 4.89 2.35- 6.50 2 . 1 ~ 5.19 3.20- 7.46 1.73- 3.14 1.73- 4.46 17.56-40.36

0.81- 1.25 0.61- 2.27 2.73- 4.04

95~o confidence interval o f the mean (X)

21.00 9.35 15.29

0.00-2470.15 2.72- 37.32

3.56- 34.62 1.85- 22.78 0.77- 60.61 1.63 75.10 1.18- 45.84 1.12- 83.96 2.14- 27.52 2.75 25.74 38.74-1722.42

2.352.403.15-

9 5 ~ confidence interval o f the distribution

CPK

The extremely wide interval is due probably to the small n u m b e r o f studied subjects and the large variation in age.

obligate carriers a possible carriers

Subjects from [amilies with BMD patients

obligate carriers isolated patients' mothers patients' sisters obligate carriers' daughters isolated patients' sisters sisters younger than 15 yr old sisters 15 yr old or older other at-risk females affected patients

Subjects from families with DMD patients

adult males adult females children

Normal con trois

n

PK

7.59 4.94 7.22

23.47 10.95

11.75 7.24 9.43 13.16 9.10 12.41 8.47 9.01 260.73

~

S E R U M PK A N D C P K IN N O R M A L C O N T R O L S A N D SUBJECTS F R O M F A M I L I E S W I T H D M D A N D B M D A F F E C T E D

TABLE 2

9.66 5.46 9.33

2.11-191.78 6.09- 19.12

8.69 15.78 5.32- 9.74 6.20- 14.11 7.08 23.80 5.70- 14.22 7.04- 21.38 5.68 12.42 6.21 12.89 179.86-377.74

5.92 4.455.54-

95~,, confidence interval o f the m e a n (X)

PATIENTS

t,J --O t~

274 DISCUSSION AND CONCLUSIONS

Serum PK determination is a relatively simple and inexpensive test. If it provided relevant information, it could be a very useful adjunct to CPK for carrier detection. In our sample of 14 obligate carriers of the D M D gene 10 had elevated serum CPK (71%) which is very similar to the 2/3 detection rate reported in many laboratories working with CPK determinations (Pearce et al. 1964; Milhorat and Goldstone 1965; Dreyfus et al. 1966; Thompson et al. 1967; HausmanowaPetrusewicz et al. 1968; D6mos 1969; Gardner-Medwin et al. 1971). With concomitant use of serum PK and CPK we were able to identify 12 of 14 (86~) of the definite carriers. In addition, the proportion of 4 of 9 daughters of obligate carriers with an enzyme abnormality on combined enzymatic study is in accordance with the 1/2 expected proportion. It would appear that multiple enzyme determinations enhance carrier detection. Others have previously emphasized the importance of repeated sampling for this purpose (Perry and Fraser 1973). In our studies of 8 obligate carriers whose serum PK and CPK were tested on multiple occasions, only 5 would have been considered abnormal after the first CPK assay, However, all 8 were correctly identified following 3 independent PK and CPK determinations. Clearly a larger data base for normal females tested with both P K and CPK on multiple occasions is required. It is important that PK determinations be performed within 24 h of venipuncture in order to ensure maximum activity. Instability of serum PK was observed by Smith and Thomas (1977) and might explain the small proportion of D M D carriers with elevated serum PK activity recently reported by others. In one study (Hardy et al. 1977) PK was assayed after 10 months of storage at - 2 0 ° C . In a second report (Yamuna et al. 1977) enzyme determinations were performed in fresh serum or after storage at - 7 0 °C for an unspecified time. As previously stated, we have observed a 50°0 decrease in serum P K activity after storage of serum for 2 months at - 2 0 ° C , and the instability of serum PK was recently confirmed by Percy et al. (1979). An interesting and controversial problem, with serious implications for genetic counseling of mothers of isolated cases, is the mutation rate in DMD. According to classical theory (Haldane 1935; Morton and Chung 1959) 1/3 of the total cases of Duchenne patients should arise through new mutations and therefore from noncarrier mothers. Recently, however, it has been suggested (Roses 1976; Roses et al. 1976) that new mutations are rare in D M D and most of the cases are, in fact, inherited. Our previous studies on CPK and genetic data from 146 pedigrees with patients affected with D M D lend support to the classical model (Zatz et al. 1976; Zatz et al. 1977). In addition, if most of the mothers of isolated cases were carriers, one would expect to find a much higher proportion of individuals with increased serum CPK among the mothers of isolated patients (as comparable to that among the obligate carriers). However, most investigators that have tested a large number of female relatives of D M D subjects have reported that the percentage of mothers of isolated patients with high serum CPK activity is significantly lower than the proportion

275 found in the group of obligate carriers (Hughes 1962; Pearce et al. 1964; Milhorat and Goldstone 1965; Dreyfus et al. 1966; Thompson et al. 1967; HausmanowaPetrusewicz et al. 1968; Hetnarska 1968 ; Radu et al. 1968; D6mos 1969; GardnerMedwin et al. 1971 ; Zatz et al. 1976). The studies reported here lend further support to the classical model. Among the 26 pedigrees, 10 had multiple affected patients and 16 had an isolated case. In 9 of the families with a single affected individual, an enzyme abnormality was found in the mother and/or one of the sisters of the proband. Hence, there were 7 families with isolated cases in which no enzyme elevation was found in any of the at-risk female relatives tested. If we conclude that these probands were the result of new mutations, then 7 of 26 affected patients represent offspring of non-carrier mothers. This proportion does not differ significantly from the 1/3 expected according to classical theory. Furthermore, if 2/3 of the total probands' mothers were carriers of the D M D gene, one would expect an enzyme elevation in approximately 1/3 of all female siblings. In the present data, 10 among 23 patients' sisters tested were found to have increased PK and/or CPK activity. Again, this does not differ significantly from the expected 1/3 proportion. A valuable method for estimating the risk of heterozygosity of a suspected carrier is to combine the results of laboratory tests with data obtained from the pedigree through Bayesian calculations (Murphy 1968; Murphy and Mutalik 1969). TABLE 3 C O R R E L A T I O N COEFFICIENTS BETWEEN SERUM CPK A N D PK ACTIVITIES IN N O R M A L CONTROLS A N D INDIVIDUALS B E L O N G I N G TO FAMILIES WITH D M D A N D BMD PATIENTS Group

n

r

t

P

43 9 19

+0.10106 -0.10019 -0.02319

26 14 16 23 9 14 13 10 9

+0.89666** +0.60604* +0.58022* +0.86715** +0.95030** +0.82703** + 0.95602** +0.62571 +0.51192*

9.92205 2.63930 2.66555 7.97885 8.07556 5.09621 10.81026 2.26879 2.48586

3 x 10-10 0.02160 0.01846 0.0000001 0.00009 0.00026 0.0000003 0.05299 0.04185

5 5

+ 0.98983* +0.16824

12.05281 0.29561

0.00123 0.78681

(A) Normal controls (1) adult females (2) children (3) adult males

0.65046 0.26642 0.09565

0.51902 0.79759 0.92492

(B) Subjects from families with DMD patients (1) affected patients (2) obligate carriers (3) isolated patients' mothers (4) patients' sisters (5) obligate carriers' daughters (6) isolated patients' sisters (7) patients' sisters younger than 15 (8) patients' sisters 15 or older (9) other at-risk female relatives

(C) Subjects from families with BMD patients (1) obligate carriers (2) possible carriers * P < 0.05; ** P < 0.001.

276 TABLE 4 A G E × C O N C E N T R A T I O N (PK, CPK) IN N O R M A L C O N T R O L S A N D I N D I V I D U A L S B E L O N G I N G TO F A M I L I E S W I T H D M D A N D B M D P A T I E N T S

Groups

n

r

t

P

34 34 11 11 9 9

-0.09921 +0.05758 -0.77321 +0.19861 -0.89800 +0.27594

0.56400 0.32628 3.65789 0.60793 5.39987 0.75955

0.57669 0.74634 0.00525* 0.55826 0.00101" 0.47232

26 26 14 14 16 t6 9 9 14 14 13 13 10 10 23 23 9 9

-0.87829 -0.74035 +0.50772 + 0.27560 + 0.14446 +0.47120 -0.65062 -0.49582 -0.51639 -0.09250 -0.06858 -0.19241 -0.79800 -0.13156 -0.57247 -0.28981 -0.82941 -0.14876

8.99904 5.39551 2.04152 0.99316 0.54624 1.99891 2.26675 1.51057 2.08891 0.32181 0.22800 0.65031 3.74521 0.37538 3.19953 1.38765 3.92813 0.39801

0.000000003* 0.00002* 0.06382 0.34024 0.59350 0.06542 0.05775 0.17465 0.05869 0.75313 0.82383 0.52884 0.00566* 0.71714 0.00431* 0.17979 0.00569* 0.70248

5 5 5 5

-0.70403 -0.65312 -0.96484 + 0.03781

1.71709 1.49387 6.35813 0.06554

0.18446 0.23206 0.00787* 0.95187

Normal controls adult contr, q P K adult contr, q CPK adult contr, o" PK adult contr, o" CPK children contr. PK children contr. C P K

Suhjeets./i'om,lamilies with D MD patients affected patients D M D PK affected patients D M D C P K obligate carriers PK obligate carriers CPK isolated patients' mothers PK isolated patients' mothers CPK obligate carriers' daughters PK obligate carriers" daughters CPK isolated patients' sisters P K isolated patients" sisters CPK sisters younger than 15 PK sisters younger than 15 CPK sisters 15 or older PK sisters 15 or older CPK all sisters PK all sisters CPK other at-risk females relatives D M D P K other at-risk females relatives D M D CPK

Suhjects.ti'om.families with BMD patients obligate obligate possible possible

carriers carriers carriers carriers

Becker Becker Becker Becker

PK CPK PK CPK

* Statistically significant P < 0.01.

Previous reports (Emery and Morton 1968; Zatz et al. 1975; Emery and Holloway 1977) have shown that density functions, of serum c P K levels, in a large sample of obligate carriers and normal female controls (in a formula based on Bayes' theorem) are very helpful in the calculation of the relative probability of heterozygosity in a suspected carrier. This method allows the geneticist to assign a probability value to each enzyme level instead of artificially classifying it as normal or abnormal. Because of the small number of obligate carriers included in this study such a combined approach would not be statistically informative. Once a large sample of obligate carriers is ascertained the construction of a table of density functions of

277 serum P K activity for a sample of normal controls and heterozygotes for the D M D gene might be very useful. An interesting finding is the observed correlation coefficients between the 2 serum enzyme activities in the different group ofheterozygotes (Table 3). In the group of older women (obligate carriers and mothers of isolated cases) these coefficients were 0.60 and 0.58, respectively. In the group of younger "possible carriers" (sisters of patients below 15 years old) this coefficient was 0.95. A possible explanation for these findings could be a differential age effect of the 2 enzyme activities. It is apparent that serum C P K decreases in carriers with increasing age (Moser and Vogt 1974) but no such data are available for serum PK. If, for example, serum PK was not age-dependent in carriers or decreased less than C P K with age, one would expect to find in young carriers (sisters of probands) high levels of both enzymes and therefore a high correlation coefficient between them. If with increasing age the serum P K did not decrease in parallel with the serum C P K (in carriers of the D M D gene) the correlation coefficients between the 2 enzymes would progressively diminish. A study of the discriminant power of P K and C P K determinations showed 5.26~, 12.28~ and 7.02~ misclassification of subjects when PK, C P K or P K + C P K determinations were performed, respectively. In spite of the small number of obligate carriers included in the present report, it seems that the P K test alone discriminates better than C P K because of the lower frequency of misclassifications in both directions. In addition, the combined use of the 2 enzymes, although detecting more heterozygous females, does not enhance significantly the capacity of carrier detection by PK alone. ACKNOWLEDGEMENTS The authors are thankful for the helpful suggestions and comments of Dr. Marie-Louise Lubs, from the University of Miami School of Medicine, and to express their gratitude to Miriam Hooper, Cindy Hashin, and D o n n a Ray Smith for their valuable help in contacting the families with affected patients, to all our colleagues who donated their blood as controls, and to Laetitia Wotta and Juraci Giareta for their secretarial assistance in the preparation of this manuscript. REFERENCES Aebi, U., R. Richterich, J.P. Colombo and E. Rossi (1961) Progressive muscular dystrophy, Part 2 Biochemical identification of the carrier state in the recessive sex-linked juvenile (Duchenne) type by serum phosphokinase determinations, Enzymol. Biol. Clin., 1: 61-74. Alberts, M.C. and F.J. Samaha (1974) Serum pyruvate-kinase in muscle disease and carrier states, Neurology ( Minneap. ) , 5: 462-464. Danielli, G.A. and C. Angelini (1976) Duchenne carrier detection, Lancet, 2: 90. De Meyer, R., C.H. Verellen, M. Freund, R. De Meyer, C.H. Latterre, B. Scholberg and J. Frederic (1977) Progressive muscular dystrophy of the Duchenne type in a young girl associated with an aberration of chromosome X. In: J.W. Littlefield (Ed.), 5th Int. Conf. on Birth Defects (Int. Congress Series, No. 426), Excerpta Medica, Amsterdam, Abstracts, p. 42.

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