An assessment of the creatine kinase test in the detection of carriers of Duchenne muscular dystrophy

82

July, 1967 T h e Journal o[ P E D I A T R I C S

An assessment of the creatine kinase test in the detection of carriers of Duchenne muscular dystrophy Serum creatine kinase activity has been utilized to evaluate the probability that female relatives of a patient with Duchenne muscular dystrophy are heterozygous. Each possible carrier was classified genetically on the basis o[ whether the risk that she is heterozygous is known or unknown. Although only 65 per cent of definite carriers could be identified by elevated creatine kinase activity, there was close agreement between the number o[ possible carriers with elevated creatine kinase and the ,number expected to be heterozygous. These observations could be explained on the basis of age differences in creatine kinase activity in heterozygotes, and suggest that carrier detection studies should be performed at an early age. Comparison of creatine kinase activity in mothers who are definite carriers and mothers o[ isolated cases shows that approximately fifty per cent of mothers o[ isolated cases are heterozygous.

Margaret W. Thompson, Ph.D., * E. G. Murphy, M.B., and Phyllis J. McAlpine, M.A. TORONTO,

ONTARIO,

CANADA

A T T FIE P R E S E N T T I M E t h e only means of reducing the incidence of Duchenne muscular dystrophy (DMD) is by the genetic approach, that is, through restriction of reproduction by heterozygous female carriers. In order to provide genetic prognosis in the families of Duchenne patients, accurate identification of carriers is essential.

From the Neurological Service and Department of Genetics, The Hospital for Sick Children, and the Faculty of Medicine, University of Toronto. Supported by grants #ore The Muscular Dystrophy Association o[ Canada. ~Address, Department o[ Genetics, Hospital [or Sick Children, 555 University Ave., Toronto 2B, Canada, Vol. 71~ No. 1, pp. 82-93

DMD is distinguished from other common forms of muscular dystrophy by: (1) an X-linked recessive pattern of inheritance, so that typically only male subjects are affected; (2) by early onset, with steady and rapid progression; and (3) by enlargement of calf muscles, at least in the early stages of the disease?, 2 Because the Duchenne gene has a high mutation rate and affected male subjects do not reproduce, many cases are sporadic, due to new mutations? According to some recent classification, at least two other forms of muscular dystrophy may be included in the Duchenne category: the relatively less severe X-linked form,

Volume 71 Number 1

with later onset and longer survival (now frequently termed the "Becker" type4) ; and a type clinically similar to the Duchenne type, which, however, shows strong evidence of autosomal recessive inheritance, including parental consanguinity- in some pedigrees2 -7 The discordance between the genetic and clinical classifications of D M D complicates carrier identification and genetic prognosis. Conditions determined by the same mutant gene (and thus having the same basic molecular defect) could be expected to show similar expression in carriers. Conditions that are genetically heterogenous, though clinically similar, might affect heterozygotes differently. For carrier investigation it appears necessary to select a uniform group of cases phenotypically resembling the severe Xlinked type; even so, there is no proof that severe X-linked childhood dystrophies may not be produced by more than one mutant gene. Emery and Dreifuss s have recently suggested that there may be at least four distinct types, three of which are less severe than the Duchenne type. For an X-linked recessive gene, the genetic prognosis in a given family depends upon whether the mother of the propositus is a carrier, or has a normal genotype but has transmitted a new mutation. Though a typical X-linked pattern of transmission demonstrates that the mother is a carrier, absence of demonstrable X-linkage does not prove her to be a noncarrier. She may be heterozygous for a mutation that arose in an X chromosome of her father or mother, or in an earlier generation in the maternal line. With small family size, it is quite possible for an X-linked recessive mutation to be transmitted for several generations by heterozygous female subjects without being expressed in hemizygous male subjects. The observation that activity of certain serum enzymes is elevated in D M D patients suggested the utilization of serum enzyme measurements in the identification of carriers. Of the various serum enzymes known to be elevated in these patients, creatine kinase (CK) is the most sensitive and specific for the Duchenne type. 9, 10 The activity of C K

Creatine kinase test in D M D

83

was shown by Dreyfus and his associates 9' 11 to be elevated in some mothers of Duchenne patients, and this observation has been repeatedly confirmed for mothers and other female relatives. 12-as It is now clear that the majority of heterozygotes have elevated levels of C K activity; in all studies, however, a large minority (one quarter to one third of all heterozygotes tested) have C K activity within the normal range. The obsel-vation of such a high proportion of "false negatives" limits the reliability of C K measurements for genetic prognosis. The present report describes a study of serum C K activity in 200 relatives of 98 patients in an attempt to evaluate the reliability of the C K test in the detection of carriers of the Duchenne gene. MATERIAL

AND METHODS

Subjects. The subjects were mothers, sisters, and other female maternal relatives (all at least five years of age) of boys who have attended the Muscular Dystrophy Clinic here during the last three years. T h e diagnosis in the propositi has been made on clinical grounds and confirmed by biochemical investigation, electromyography, and muscle biopsy. Patients with atypical features have been rigorously excluded. The age of onset of the disease in the propositi has been under five years, usually much earlier, so that the less severe Becker type of muscular dystrophy has been excluded. Insofar as can be determined, autosomal recessive cases of severe childhood muscular dystrophy have also been excluded, but it is not always possible to be certain that an isolated case or a family with two affected sibs only is not an example of autosomal recessive rather than X-linked recessive inheritance. In one family there was distant parental consanguinity, but the elevated C K levels in the mother and two sisters of the propositus fit the usual pattern of X-linked recessive inheritance. Ninety adult female hospital workers served as controls. Ascertainment. This clinic receives referrals from an area of Southern Ontario with a population of approximately four million.

8 4 Thompson, Murphy, and McAlpine

Morton and Chung 3 have estimated the prevalence of D M D as 66 living patients per million living male subjects and the incidence as 279 patients per million male births. If these estimates are accurate, there should be about 132 patients in this area, and the 90,000 annual births should produce about 12.5 new patients per year. Rough calculation suggests that the clinic roster of 98 patients includes the great majority of cases of D M D in the area. There are a number of reasons, however, for suspecting that ascertainment is less than complete. Diagnosis may be made accurately at an earlier age if the disease is already known in the family; consequently, the series may include an excess of cases with a family history. On the other hand, if parents familiar with the relentless course of the disease are less willing than other parents to bring affected children to the clinic, there may be a deficit of cases with a family history. Finally, the age distribution of the patients attending the clinic is not typical of the age distributio.n of the disease, since the progressive nature of the handicap excludes many older, more severely involved patients. Some mothers of isolated cases (i.e., patients with no known family history of D M D ) are heterozygous and others genotypically normal. In either instance, the mother's genotype could not conceivably affect the probability that her son would be ascertained. Therefore the proportion of heterozygotes among mothers of ascertained isolated cases should be representative of the proportion of heterozygotes among mothers of isolated cases in the general population. Genetic classification of relatives. Some of the discrepancies in the published literature concerning the proportion of carriers identifiable by measurement of serum C K activity could result from lack of uniformity in the genetic classification of the various groups of relatives. For brevity, the tenTt "relative" is used in this report to denote a member of a kindred who for genetic reasons has some probability of carrying the same mutant gene as the propositus; for an X-linked recessive mutant, these members would be the moth-

The ]ournal of Pediatrics July 1967

er's mother, mother's sister, and so forth. Family members with no probability of carrying the mutant gene (such as father's family and mother's father's family) are not included. In the present study, the scheme of classification has been: Definite carrier. A woman who has at least one affected son and another affected relative, usually a brother but occasionally a more distant relative in a pattern of kinship concordant with X-linked recessive inheritance. Probable carrier. A woman with two or more affected sons but no other known affected relative. Probable carriers are distinguished from definite carriers because the family history could be compatible with autosomal recessive inheritance, or with germinal mosaicism. The former probability has been ruled out insofar as selection is limited to patients in whom the phenotype is of the severe Duchenne type, and the latter (germinal mosaicism) is exceptionally rare in man; thus the most probable explanation for the occurrence of the disease in two brothers is that their mother is heterozygous. Possible carrier, type A. A female related to a definite carrier and thus having a known statistical risk of being a carrier herself. Daughters, but not other female relatives, of probable carriers have been included in this group. Possible carrier, type B. A mother, sister, or other female relative of an isolated case, or a female relative (other than daughter) of a probable carrier. T h e members of this group may be carriers, but, unlike the previous category, the statistical risk is not known. The four different classes of relatives are shown in abbreviated pedigree form in Fig. 1. Laboratory techniques. Creatine kinase is an enzyme of nervous and muscular tissue that catalyzes the reaction: creatine phosphate + ADP ~ ~

c re a t i ne + A T P , t

Its activity in serum is normally low, and ~ADP, adenoslnediphosphat e. tATP, adenosine triphosphate.

Volume 71 Number 1

Creatine kinase test in D M D

when elevated is believed to reflect loss of the enzyme from degenerating tissue. I n the present investigation, C K activity in serum has been determined according to the technique of Hughes, z9 with minor modification. By the Hughes method, the amount of creatine liberated from creatine phosphate in the reaction per unit of time is measured

.? Definite carrier

Probable carrier

,? Possible carrier, Type B (unknown risk of heterozygosity)

Possible carrier, Type A (known risk of heterozygosity)

Fig. 1. Genetic classification of carrier status of the subjects used in the present investigation. F o r details, see text.

spectrophotometrically, and the activity is expressed in units representing micromoles of creatine formed per milliliter of serum per hour at 37 ~ C. This unit can readily be converted to the international unit (micromoles of creatine formed per liter of serum per minute at 37 ~ C.) by multiplication by 16.67. T h e reaction is linear within the range of normal C K activity, but m a y not be linear in serum with elevated activity; therefore, if the final optical density of the test serum greatly exceeds that of the blank, the serum must be appropriately diluted and the test repeated. Blood samples (8 to 10 ml.) were obtained by venipuncture under ordinary ambulant conditions, without regard to previous exercise, stage of menstrual cycle, pregnancy, or other physiological variables. T h e serum was separated and stored at - 4 ~ C. in 0.5 ml. aliquots for later determination. T h e first determination was made within the first week of storage. Repeated tests after periods ranging from one m o n t h to two years in frozen storage have shown some decline in activity, but the decline has in no case been sufficient to permit a formerly elevated level to be classified as normal. Each sample was tested in duplicate at each determination, and two separate determinations were made, Whenever possible, the test was repeated on separate samples of serum from each subject two or three times, at six-month intervals. I n subjects who had an elevated C K value, the activity

Table I. Typical values in possible carriers

Subject 1

2 3 4 5 6 7 8 9 10 11 '12

I

Test 1 0.4 1.9 4.3 8.8 0.5 1.9 1.7 9.8 4.8 11.5 41.7 2.9

[

Test 2 0.7 2.8 6.6 9.7 0.6 1.9 1.8 15.7 2.7 6.7 124.2 4.7

I

85

Test S -- ---

--1.6 37.8 5.0 - -

42.2 14.6

[

Mean 0.6 2.4 5.5 9.3 0.6 1.9 1.7 21.1 4.2 9.1 69.4 7.4

[

Classification Low

Elevated Elevated Elevated Low Doubtful Doubtful Elevated Elevated Elevated Elevated Elevated

86

Thompson, Murphy, and McAlpine

The Journal o[ Pediatrics July 1967

upper part of Fig. 2. T h e mean C K level for the group was 0.9 units, and the standard deviation + 0.4 units. W h e n a normal curve is fitted to the data, it is seen that the distribution is essentially normal, but skewed slightly to the left, and truncate at the lower edge of the range since obviously no values below zero, are encountered. There is some question as to what point should be selected as the upper limit of the normal range. N o values above 2.0 units were observed in the control series, and 99 per cent of the subjects had C K levels equal to or less than 1.8 units. For genetic prog-

varied considerably between successive tests, but remained above the upper limit of the normal range. I n subjects with low C K activity, the activity was more constant and remained low on repeated tests. I n the few subjects with doubtful values., the activity was likely to remain questionable in each of several tests. T h e value used is the m e a n of the tests performed on successive samples (Table I ) . RESULTS

Controls. T h e range and distribution of C K levels in the 90 controls is shown in the

Con|rols 17.5 - n : 9 0 roll :1% 15.0 -

125 Frequency 10.0 % 7.5-

5.0-

2.5-

0

0.5

1.0

1.5

2.0

2.5

Serum Creatine )inase Actlvity ,urn./mr./hr.

Definite and Probable Carriers 20.0-

n=37

Range

J=65%

6.0-90 units

17.5 -

Frequency

10.O-

% 7.5

5.0

2.5 0

0.5

1D

1.5

2.0

2.5

3.0

3.5

4.0

4.5

1

1

5.0

5.5

60

Serum Creatine Kinase Activity /um./ml /hr.

Fig. 2. Distribution of creatine kinase activity in 90 controls (above) and in 37 definite and probable carriers (below). Creatine kinase activity is expressed as micromoles of creatine phosphate converted to creatine per milliliter of serum per hour at 37 ~ C.

Volume 71 Number 1

Creatine kinase test in D M D

nosis, we have considered a value of 1.8 or less to be in the normal range and a value above 1.8 to. be elevated. Definite and probable carriers. The range and distribution of C K levels in 22 definite and 15 probable carriers is shown in the lower part of Fig. 2. Elevated C K levels were found in 14 of the 22 definite carriers and in 10 of the 15 probable carriers. Because C K levels were similar in definite and probable carriers, the two groups have been regarded as genetically uniform and have been combined for further investigation. The distribution of C K levels in the combined group is obviously not normal, but has a long tail to the right, reflecting the finding

of extremely high levels, as high as 90 units, in a few carriers9 T h e mean value was 7.1 units, and the standard deviation _+ 15.7 units9 As Fig. 2 shows, 24 (65 per cent) of the 37 carriers had elevated C K levels, but the remaining 13 (35 per cent) could not be distinguished from controls on this basis9 In other words, if elevated C K is regarded as an indicator of carrier status, about one third of the carrier mothers had falsely negative values9 Effect of age on C K level. To investigate the possibility that in carriers, as in affected boys, C K activity is high at earlier ages but returns to normal or near-normal levels with

Range

Possible Carriers, Type A J(known risk of heterozygosity) 20.0 -'J n=64 l i =43%

6.0-260 units

I

/

17.5-~ Frequency 10.0% 7.5-

I-I

5.02.5-

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6,0

Serum Creatine Kinase Activity

.um./ml./hr. Possible Carriers, Type B (unknown risk of heterozygosity) n=99 IR =32%

100 -

Range

FI

Frequency

%

6.o?'.

7.55.025-

0.5

1.O

1.5

2.0

2,5

87

3,0

35

4.0

4.5

S.0

5.5

6.0

Serum Creatlne Kinase Activity tam./ml./hr.

Fig. 3. Distribution of creatine kinase activity in possible carriers. Above: distribution in 64 possible carriers of type A (having a known risk of heterozygosity). Below: distribution in 99 possible carriers of type B (having an unknown risk of heterozygosity).

88

Thompson, Murphy, and McAlpine

The Journal o[ Pediatrics July 1967

increasing age, the ages of the mothers with elevated C K and those with normal C K have been compared. The mean age was 39.4 years for the mothers with elevated C K and 44.2 years for mothers with normal CK, but the difference of 4.8 years was not statistically significant (0.10 < p < 0.20). Electromyographic and histological observations. Three definite carriers in whom the C K activity was within the normal range on repeated tests were examined electromyographically and by muscle biopsy. In none of the three was any electromyographic or histopathological myopathic change observed. Application of observations in controls and carriers to the classification of possible carriers. The data for the control and carrier groups were t h e n applied to the detection of carrier status in other relatives of the Duchenne patients. T h e C K activity in the two groups of possible carriers (type A, in which each member has a definite statistical risk of heterozygosity, and type B, in which the risk is unknown) is shown in Fig. 3. The possible carriers of type A included 51 relatives (daughters, sisters, motllers, and nieces) of definite carriers, and 13 daughters of probable carriers. Daughters of probable carriers are included because the distribution of C K levels in the probable carriers suggested that they were heterozygous; other female relatives of probable carriers have an unknown risk of heterozygosity (because the family members in whom the mutation first arose are not known), thus are classified as type B. Elevated C K activity was observed in 22

of 51 relatives of definite carriers, and in 5 of 13 daughters of probable carriers. The distribution is shown in the upper part of Fig. 3. Because the statistical risk of heterozygosity is known for each member of this group, the expected total number of heterozygotes can be calculated and compared with the observed (Table I I ) . There is close agreement between the expected number of heterozygotes and the number of subjects in whom high C K activity was observed. Among 44 daughters, high CK was found in 17 (22 expected), and among 20 other relatives, high C K was found in 10 (6.75 expected). The data are statistically in agreement with expectation. For these possible carriers, the data support the validity of the C K level as an indication of carrier status. Some caution is in order, since for a sample of this size a deficiency of up to 8 carriers would not be a "statistically significant" deviation from expectation; but the close agreement of the observed and expected numbers permits a degree of confidence that all or the great majority of the carriers have been detected. The mean age of the subjects in this group was 21.2 years; again there is an indication, but no proof, that the C K test is more accurate in younger persons than in older ones. Possible carriers, type B. The possible carviers in whom the risk of heterozygosity cannot be statistically evaluated include the mothers and other female relatives of isolated cases, and relatives (other than daughters) of probable carriers. The range and distribution of C K levels in this group is shown in the lower part of Fig. 3. T h e range

Table I I

Category Daughters of definite carriers Daughters of probable carriers Sisters of definite carriers Nieces or granddaughters of definite carriers Total

Number of sub,jects 31 13 7 13 64

Risk of heterozy.gosity 0.50 0.50 0.50 0.25

Number of heterozygotes expected I5.50 6.50 3.50 3.25

Number with high CK observed 12 5 2 8

28.75

27

Volume 71 Number 1

is from 0.3 to 74 units, and elevated C K levels were observed in 32 of the 99 individuals tested. Thirty-nine mothers of isolated cases were examined, and 13 of these were found to have elevated CK. Since only two thirds of the "definite carrier" mothers were found to have elevated CK, it is assumed that falsely negative values have also been obtained in a proportion of these mothers. If the proportion identified is similar in the two groups of mothers, only two thirds of the carriers among the mothers of isolated cases have been identified, and probably about 6.5 additional carriers are included in the 26 mothers with normal C K values. Thus approximately 6.5/26 or 25 per cent of the C K levels in the normal range are "falsely negative." It has been possible to identify some of the heterozygous mothers with normal C K levels by investigating the C K levels of their daughters. Of the mothers who had normal C K levels, 3 had 1 or more daughters with elevated CK. On this basis, the 3 have been classified as carriers, raising the total number of carriers identified among the 39 mothers to 16 (41 per cent). The remaining 23 mothers may include approximately 3.5 additional carriers. The estimate of 19.5 carriers among 39 mothers of isolated cases suggests that the risk that any mother of an isolated case is a carrier is approximately 50 per cent. DISCUSSION

The data presented here agree with those of previous workers in demonstrating that most, but not all, carriers of the Duchenne gone have elevated creatine kinase activity. Elevated C K activity is exceptional in normal female subjects? 4, 20 Abnormally high C K levels have been described in young children, in women late in pregnancy, and after prolonged violent physical exercise, ~~ though Pearce and associates 2~ did not find any effect of exercise. Exceptionally high values are practically pathognomonic of the early stages of Duchenne muscular dystrophy; less striking increases are found in other muscular dystrophies. The level may be high in

Creatine kinase test in D M D

89

polymyositis, especially during the active disease processY 1 In the present series, the narrow range and essentially normal distribution of C K activity in control subjects suggests that falsely positive findings rarely occur. Consequently, elevation of C K activity in a clinically normal female relative of a Duchenne patient is taken to be a reliable sign of heterozygosity for the Duchenne gone. Carriers of the X-linked "Becker" gone are similar in C K activity to carriers of the Duchenne gene, whereas carriers of the gone for the autosomal recessive limb-girdle type are indistinguishable from controls, lr This observation can probably be best explained on the basis of the Lyon principle of gone action on the X chromosome? ~, 2a Although a high level of C K activity is a reliable index of heterozygosity for the Duchenne gene, the converse is not the case, since in the present series 35 per cent of definite carriers had normal C K levels. There are at least four possible explanations for the failure of some carriers to show elevated CK: technical difficulties, changes with age, the heterozygote variability consequent to inactivation of one X chromosome, and genetic heterogeneity. With respect to technique, review of the several surveys reported in the literature shows that the average detection rate falls between 70 and 75 per cent. (Uncertainty as to whether some cases have been reported in more than one paper makes it impossible to ascertain the exact detection rate.) There seems to be no significant difference in detection rate between the technique used in the present study and the alternative technique of Tanzer and Gilvarg, 2~ which uses the backward reaction and auxiliary systems. All reports agree in failure to identify carriers with 100 per cent accuracy. Wilson and co-workers 17 have attempted to calculate a "relative probability" that a particular woman is a carrier by comparing her genetic risk and C K level with the C K levels of controls and carriers. T o test this approach, our data for C K levels have been plotted cumulatively (Fig. 4). A log scale is used to reduce the range of C K values

90

Thompson, Murphy, and McAIpine

The Journal o/ Pediatrics July 1967

Controls ~-

---

Carriers

~, IOO c a) o

75

u,.

.g"~

50 -~

d

25

0.1

I

I

1,0

10

100

IJm,/ml./hr. Fig. 4. Cumulative distribution of creatine kinase activity in carriers as compared with controls. Serum Creatine Kinose Activity

in carriers. As an example of the use of this graph, consider a niece of a definite carrier in w h o m the C K level is 1.5 units. T h e genetic risk of heterozygosity is 1/4. T h e graph shows that 6 per cent of controls have C K values as high or higher, whereas 22 per cent of carriers have values as low or lower. T h e n the relative risk that the niece is a carrier is 0.25 x 0.22 (0.25 x 0.22) + (0.75 x 0.06) or approximately 55 per cent. T h e proportion of heterozygotes in the general population, if the gene frequency q is 279 per million, is 2 ( l - q ) q , or approximately 1 in 5,000. Thus in a control female subject with a C K level of 1.5, the relative risk of heterozygosity is 0.0002 x 0.22 (00002 x 0.22) + (0.9998 x 0.06) or 1 in 1,364. Calculation of relative probabilities has not been used routinely in the present investigation because the range and distribution of C K levels in controls and carriers has appeared to, be distinctive enough to allow accurate interpretation in most instances, but in doubtful cases it m a y be useful.

T h e question of whether or not creatine kinase levels decline with age, after early childhood, remains open. Griffiths 2~ and Wilson and associates 17 found no, significant difference with age in normal subjects. In Duchenne patients, however, the C K activity is particularIy high in the early stages of the disease, and declines hyperbolically as the total functional muscle mass is reduced by the dystrophic process. Pearce and his colleagues ~-1 have shown that the activity levels off by about the age of ten years, though it remains above normal; Richterich and co-workers 1~ have also found elevated values, even in very advanced cases. As yet there has been no systematic study of possible changes in C K activity with increasing age in carriers, though Richterich and associates 1~ have postulated that activity is higher in younger carriers. In the present series, values in different carriers varied from less than one to ninety units and fluctuated in the same carriers o.n repeated tests, especially if the C K activity was high (see examples). Hence, it has not been possible to establish that C K activity declines with increasing age. Nevertheless, the possibility of such a decline remains; the lower mean age of definite carriers with elevated CK, as c o m p a r e d with definite carriers with normal

Volume 71 Number 1

CK, and the higher detection rate in younger subjects are suggestive but do not constitute proof. It should be noted that whereas CK values in normal individuals do not fluctuate appreciably under normal physiological conditions, the same may not be true of carriers; indeed it is more likely that they resemble patients in the sensitivity of their C K levels to exercise and other variables. EmerylS, 27 has described histopathological changes in muscle fibers in carriers of Duchenne muscular dystrophy. These changes include swelling, variation in diameter, hyalinization, necrosis, increase in number of nuclei, central nuclei, and increase in connective tissue, sometimes with fatty infiltration. Slight but significant electromyographic changes have also been described in carriers (Caruso and Buchthal, 28 Smith and associates ~4) Our failure to demonstrate either histopathological or electromyographic abnormalities in three definite carriers with CK activity in the normal range may signify that, at least at the time of study, no dystrophic changes were taking place in the muscle of these carriers. Several workers have evoked the Lyon principle22, 2a to explain the normal CK levels found in some heterozygotes. In particular, Pearson and associates is have proposed that female X chromosome mosaicism, with an excessive number of active normal X chromosomes, could produce totally normal or nearly normal female carriers. Emery 29 has postulated that random inactivation of one X chromosome per cell, followed by fusion of many uninucleate myoblasts to form muscle fibers, could produce fibers in which there might be a spectrum of severity, from normal to severely dystrophic, in a single subject. An additional factor in the production of variability in heterozygous girls might be the size of the muscle stem cell population at the time that inactivation of one chromosome takes place. Voluntary muscle is derived from the myotome, a limited part of the somite in higher vertebrates. At the time of X chromosome inactivation (believed to be the sixteenth day of embryonic life or earlier3~ the myotome

Creatine kinase test in D M D

9 1

might include very few cells destined to give rise to muscle. The smaller the cell population at that time, the greater the possibility for significant heterozygote variability. If only a few cells are involved in the random process of X-inactivation, variability of expression in carriers and mosaicism are both fully in accord with expectation. On this basis, failure to find any abnormality in a proportion of carriers is not a discrepancy, but the expected observation. The view that all heterozygous carriers are affected, though usually to a subclinical extent, may explain the occasional observation of a woman in a Duchenne kindred with muscle weakness, markedly elevated creatine kinase activity, and electromyographic and histological abnormalities. Several such cases, occurring in a single kindred, have been described from this institution? 1 Though much of the variation in the severity and rate of progression of DMD may be explained by variations in muscle mass and strength, genetic heterogeneity is a probable explanation of at least part of the clinical variability. It is virtually impossible to prove that DMD is a single disease entity rather than a number of clinically indistinguishable but genetically heterogeneous entities. Boyer and Fainer a2 postulated that genetic heterogeneity may be the rule in any "type" of muscular dystrophy, and that variable success in carrier detection may be expected. SUMMARY

Creatine kinase activity has been investigated in 200 female subjects related to 98 patients with Duchenne muscular dystrophy, and in 90 controls. All subjects were over five years of age. Elevated creatine kinase activity was found in 24 (65 per cent) of 37 definite and probable carriers. Among 64 possible carriers in whom the risk of heterozygosity could be calculated, 27 subjects were identified as carriers, in close agreement with the expectation of 28.75. Among 99 other possible carriers, in whom the risk of heterozygosity could not be determined, 32 carriers were identified. Of the 39 mothers of sporad-

92

Thompson, Murphy, and McAlpine

ic cases included in this group, high C K activity was f o u n d in 13, a n d 3 others had daughters with high CK. By comparison with the identification rate in definite carriers, probably about three of the r e m a i n i n g 23 were actually carrier's who had false negative values. For genetic prognosis, a n elevated C K value in a relative of a D u e h e n n e p a t i e n t is regarded as reliable evidence of heterozygosity, b u t if the C K value is within the n o r m a l range there is an appreciable risk that it is a "false negative" a n d that the subject is actually a carrier. Such risk may be low in younger subjects b u t is as high as 35 per cent in older ones. Approximately half the mothers of isolated cases are heterozygotes. Possible causes of "false negative" observations are technical difficulties, decline of C K levels with increasing age, heterozygote variability consequent to Lyonization of an X chromosome, a n d genetic heterogeneity. The technical assistance of Mrs. Irina Oss and the financial support of the Muscular Dystrophy Association of Canada are appreciated.

The Journal of Pediatrics July 1967

10.

I1.

12.

!3. 14.

15.

16.

REFERENCES

t. Walton, J. N., and Nattrass, F. J.: On the classification, natural history, and treatment of the myopathies, Brain 77: 169, 1954. 2. Chung, C. S., and Morton, N. E.: Discrimination of genetic entities in muscular dystrophy, Am. J. Human Genet. 11: 339, 1959. 3. Morton, N. E., and Chung, C. S.: Formal genetics of muscular dystrophy, Am. J. Human Genet. 11: 360, 1959. 4. Becket, P. E.: Two new families of benign sex-linked muscular dystrophy, Rev. Canad. Biol. 2t: 551, 1962. 5. Kloepfer, H. W., and Talley, C.: Autosomal recessive inheritance of Duchenne type muscular dystrophy, Ann. Human Genet. 22: 138, 1958. 6. Jackson, C. E., and Carey, J. H.: Progressive muscular dystrophy: Autosomal recessive type, Pediatrics 28: 77, 1961. 7. Skyring, A., and McKusick, V. A.: Clinical, genetic, and electrocardiographic studies in childhood muscular dystrophy, Am. J. M. Sc. 242: 534, I961. 8. Emery, A. E. H., and Dreifuss, F. E.: Unusual type of benign X-linked muscular dystrophy, J. Neurol. Neurosurg. & Psychiat. 29: 338, 1966. 9. Dreyfus, J. C., Schapira, G., and Demos, J.: l~tude de la cr6atine-kinase ~erique chez les

17.

18. 19.

20.

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