SERUM. CREATTNE PHOSPHOKINASE ACTIVITY AS A FUNCTION OF AGE TN POSSiBLE CARMERS 0F DUCHENNE M-USCULAR DYSTROPHY Thesisfor the Degree of M. S. , MICHIGAN STATE UNIVERSITY . SUSAN L. REIMER 1973 LIBRARY Michigan Sme University g: ” Bl m? HMS & SBNS' 300K BINDERY IND. LTBRA 3V B'NDERS s=- : :Icmcn ABSTRACT SERUM CREATINE PHOSPHOKINASE ACTIVITY AS A FUNCTION OF AGE IN POSSIBLE CARRIERS OF DUCHENNE MUSCULAR DYSTROPHY BY Susan L. Reimer It is well documented that CPK levels of persons affected with Duchenne type muscular dystrophy are highest in early childhood but gradually fall in the advanced di- sease. This study investigates an age distribution of CPK levels such as this in the case of carriers of the gene for Duchenne muscular dystrophy. From a total of 191 sisters of dystrophic boys analyzed in this study, 58 sisters showed abnormal increases in their CPK values (30%). The wide vari— ation of CPK levels seen in these 58 presumed carriers was then related to the factor of age. No significant differ- ence was found in the mean CPK levels of carriers under or over the age of 12. However, it was shown that the detec— tion rate among possible carriers is significantly higher in infancy and childhood, suggesting the possibility that early age may be a factor in the elevated serum CPK of some carriers. A hypothesis is proposed that as carrier females Susan L. Reimer grow older, and especially as they enter puberty, a certain percentage of them (those whose CPK levels were only slight- 1y elevated in young childhood) may fall back into the nor— mal range while other carriers (those whose CPK levels were considerably elevated in young childhood) may remain ele— vated into adulthood. SERUM CREATINE PHOSPHOKINASE ACTIVITY AS A FUNCTION OF AGE IN POSSIBLE CARRIERS OF DUCHENNE MUSCULAR DYSTROPHY By Susan LI Reimer A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1973 ACKNOWLEDGMENTS I would like to express special thanks to my pro- fessor, Dr. James V. Higgins, for suggesting this project and for his help and guidance during the investigation. I also wish to express my appreciation to Dr. Margaret Jones, Dr. Herman Slatis, and Dr. Leonard Robbins for their expert suggestions during the course of the study. Further thanks goes to Dr. Joseph Mann and the tech- nologists at Butterworth Hospital for their cooperation and interest. I am also indebted to the directors and staff mem- bers of the 3 muscular dystrophy clinics which participated in this study; special gratitude is reserved for Dr. Robert Puite, Dr. Richard Zarling and Dr. Henry Peters for their interest and encouragement The friendship and encouragement of the following people in the genetics laboratory made my work at Michigan State University enjoyable and rewarding: Gary Marsiglia, Lou Betty Richardson, Carola Wilson, Rachel Rich, Robert Pandolfi, Frankie Brown, Astrid Mack, Habib Fakhrai, and Mike Abruzzo. ii Special thanks is also reserved for my parents and for my sister and brother-in-law, Sally and Chad Cullen, all of whom, in their own ways, aided and encouraged me in my graduate work. And for my husband, Ron, to whom this study is ded- icated, my feelings of appreciation and thanks are best ex— pressed by the following quote: "For this I bless you most; You give much and know not that you give at all... Wise men have come to you to give you of their wisdom. I came to take of your wisdom: And behold I have found that which is greater than wisdom." from The Prophet by Kahil Gibran iii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES . . . . . INTRODUCTION . LITERATURE REVIEW MATERIALS AND METHODS RESULTS . . . . . . . . . . . DISCUSSION . LIST OF REFERENCES APPENDIX . iv Page vi 38 45 67 86 93 LIST OF TABLES Table Page 1. Classification of the muscular dystrophies . . . . . . . . . . . . . . . . . . S 2. Summary of results on detection of carriers using CPK . . , . . . . . . . . . . 46 3. Corrected percentage of genetic carriers among possible carriers . . . . . . . 47 4. Mean CPK values for the different age groups in the controls . . . . . . . . . . 51 5. Mean CPK elevation for the different age groups of carrier sisters . . . . . . . . . 55 6. Serum creatine phosphokinase activity in carrier sisters . . . . . . . . . . . . . . S6 7. Percentage of elevated CPK levels among possible carrier sisters by age . . . . . S8 8. Mean CPK levels of all possible carrier sisters by age . . . . . . . . . . . . 60 9. Comparison of mother-daughter CPK levels . . . . . . . . . . . . . . . . . . . . 66 10. Detection of carriers with CPK by various authors . . . . . . . . . . . . . . . . 68 11. Ages and CPK values of possible carriers reported by various authors . . . . . . . . . . . . . . . . . . . . 76 Figure 1. 10'. 11. LIST OF FIGURES Gower's maneuver Muscular dystrophy in man . . . . Schematic representation of the CPK reaction Serum creatine phosphokinase activity in 82 cases of progressive muscular dystrophy and neuromuscular diseases Activity of the serum kinase in cases of the Duchenne type of muscular dystrophy charted against the patients' ages 0 I O I O O O O O O O O O C O 0 Distribution of CPK activities in two groups of known carriers (under and over 40 years of age) . . . . . Values for serum creatine kinase in carrier females of different ages, expressed in units (micromoles of creatine formed per m1 of serum at 37°C) . . . . . . . . . . . Distribution of CPK values in controls and possible carrier sisters . . CPK values as a function of age in the control group . . . . . . . . . CPK values as a function of age in carrier sisters . . . . . . . Distribution of CPK activities in two groups of carriers (under and over 12 years of age) . . . . . . . . vi Page 18 22 23 24 36 37 49 SO 52 53 Figure Page 12. Cumulative percentage curve of CPK values in two groups of carriers (under and over 12 years Of age) 0 O O O l O O O O O O O O O O O I O 0 0 54 13. CPK values in individual carriers over a period of years . . . . . . . . . . . . 61 14. Family correlation . . . . . . . . . . . . . . 64 15. CPK values among possible carriers as a function of age . . . . . . . . . . . . . 77 vii INTRODUCTION There is new ample evidence that in 90 percent of cases of muscular dystrophy of the Duchenne type the condi- tion is inherited by means of a sex-linked recessive 44’67’72 In the remaining 10 percent of cases inheri- gene. tance appears to be by means of an autosomal recessive mech- anism. In the case of sex-linked recessive inheritance, the gene responsible for the dystrophy is carried on one of the two X chromosomes of a "carrier" female; thus, statistically, half of the sons of a carrier female are likely to be af- fected and half of the daughters are likely to ke carriers. It is obvious that a reliable test to identify carrier women would thus be invaluable. A good deal of evidence has accumulated in recent years suggesting that biochemical, electrophysiological, and histopathological methods may be of some value in detecting the female carrier. Most promising of these methods is the determination of the serum creatine phosphokinase (CPK) ac- tivity, since accumulated evidence shows that from two- thirds to three—fourths of all carriers have an increased level of this enzyme. In order that the maximum use may be made of the serum CPK level as a criterion for detecting carriers it is important to possess adequate data concern- ing variations occurring in normal individuals as well as variations occurring in carriers. Very few attempts have been made to relate the wide variation of CPK levels seen in carriers to factors such as age or the degree of histo- pathological or electrophysiological changes often seen in carriers. It is well documented that CPK levels of affected persons are highest in children six months of age or less and that the levels remain high as the weakness and atrophy progress. As the extensive loss of muscle mass intervenes, however, the level gradually falls until in the advanced di- sease it may be normal or only slightly elevated (2—5X nor- mal).9’ 21, 36, 41 An age distribution of CPK levels such as this has never been fully investigated in the case of carriers of the disease. The purpose of this study is to determine the ef- fect of age on CPK levels of carriers of Duchenne type mus— cular dystrophy. This will be done by pooling the results of three laboratories working on the detection of carriers and comparing the results with a group of control females. The problem of correlating values from three laboratories, each using a different method for CPK determinations, will be overcome by expressing all values in terms of mean and standard deviations. The more desirable method to attack this problem and one which was able to be used in a few cases, is to follow the serum CPK levels in the same persons for a long time. Such a study has important genetic counseling im- plications; if evidence suggests a diminution in the level of CPK activity in the carriers with increasing age, then it would be advisable to test CPK levels of possible car- riers (female siblings of an affected boy) at a very early date. Furthermore, such data would be important in the in- terpretation of a normal or only slightly elevated CPK val- ue of a possible carrier who is much older; such a value may be reflecting a correlation between age and serum CPK level or it may be indicating that this person is not a car- rier. Also, if evidence reveals a progressive decrease in CPK activity with the age of the carrier, it would explain some failures of this method to detect carriers among older women . LITERATURE REVIEW The term muscular dystrophy is a general designa- tion for a group of chronic diseases, whose most prominent characteristic is the progressive degeneration and atrophy of the skeletal or voluntary muscles, after a latent period of apparently normal development and function. Despite this common denominator, it has been repeatly shown that the dystrophies are a group of clinically, genetically, and biochemically distinct entities, each with its own charac- teristic physical findings, initial muscle groups affected, typical age of onset, rate of progression and inheritance 7’44’58’72’25’68’45’27 However, the classification pattern. of the dystrophies has proven difficult since the pathogen- esis is still largely unknown. A 1966 classification sys- tem by John N. Walton and R. J. T. Pennington based on clin- ical and genetic evidence represents the most widely accepted subdivision of the major groups (Table 1). This paper will be limited to a discussion of the Duchenne type muscular dystrOphy. Within the subdivision called Duchenne muscular dystrophy (also known as childhood, pseudohypertrophic, progressive, or Leyden and Moebius mus- cular dystrophy) there are now recognized to be at least Table 1. Classification of the muscular dystrophies. l. The ”pure" muscular dystrophies (a) The Duchenne type muscular dystrophy Sex-linked recessive variety (1) Severe (2) Less severe of later onset Autosomal recessive variety (b) Limb-girdle muscular dystrophy Autosomal recessive variety Occasional sporadic cases (possibly due to expres- sion in the heterozygote) (c) Facioscapulohumeral muscular dystrOphy Autosomal dominant (rarely recessive) (d) Distal muscular dystrophy (e) Ocular myopathy and its oculopharyngeal subvariety (f) Congenital muscular dystrophy 2. The Myotonic syndrome (a) Myotonia congenita (b) Dystrophic myotonica (c) Paramyotonia congenita (showing relationships with adynamia episodica hereditaria and myotonic periodic.paralysis three varieties separable on a clinical and/or genetic ba- sis. Early attempts at classification gave differing opin- ions on what constitutes muscular dystrophy of the Duchenne type, largely due to a failure to recognize these different varieties. Stevenson (1953) laid down rigid criteria for his "Duchenne-type rapidly progressive muscular dystrophy of young boys," defining it as a disease with "(a) expression solely in the male (b) onset mainly in the first two years of life (c) high frequency of pseudohypertrophy of the calves (d) universal affection of the gluteal, thigh adduc- tors, and later scapulohumeral muscules, with (e) rapid progression to inability to walk usually before the age of 12 (f) inheritance via a sex-linked receggive gene with a relatively high mutation rate.” Walton and Nattrass (1954), while accepting the sex-linked recessive mode of inheritance, give the disease a broader definition in an attempt to explain the apparent occurrence in a few females and the later onset and slower progression observed in a minority of cases. Now widely accepted is the approach, as outlined in Table l, of recognizing three distinct kinds of muscular dystrophy under the general Duchenne title. According to this classification system the predominant type, responsible for approximately 80 per- cent of all cases that fall within the Duchenne grouping, is the severe sex-linked variety.67 In patients with this type, neonatal development is apparently normal with the on- set of symptoms usually occurring between the first and sixth years.*44’72 The most common presenting symptoms are a waddling gait and frequent falling or clumsiness while the 67,72 child is walking or running. On physical examination * However the biochemical identification of preclin- ical cases and the histological changes that have been seen in muscle biopsies obtained from preclinical cases indicate that the dystrophic process is active even in early infancy, long before clinical weakness is manifested. (Pearce et al., 1964; Pearson et al., 1961). Pearce et a1. hypothesize—that the disease praaess commences in fetaI_life, since the changes observed in early infancy appear too advanced to have developed in the brief interval since birth. pseudohypertrophy is seen in approximately 90 percent of patients, caused by interstitial infiltration of mature fatty tissue, perhaps as a replacement phenomenon for the wasting muscle tissue.44’67’72 However, pseudohypertrophy is not specific for this form of dystrophy, being seen oc- casionally in the facioscapulohumeral, limb-girdle and myo— tonic dystrOphies as well as the more unusual forms of mus- cular dystrophy.67’72 The most useful diagnostic feature for this variety appears to be the characteristic pattern of muscle involvement: axial and proximal girdle muscles are involved before distal girdle muscles and the sternal head of the pectoralis and lower fibers of the trapezius are involved before the remainder of those muscles.22’72 Thus, on early examination, there is weakness of the pel- vic, quadriceps, and abdominal muscles leading to lordosis, a waddling gait and difficulty in ascending stairs and ris- ing from the floor. Gower's maneuver describes the charac- teristic fashion in which these children rise from the floor (Figure l). The child rolls to his abdomen, places hands and feet on the floor and advances his feet toward his hands to raise the buttocks. The hands are then brought toward the feet and up the legs to complete the maneuver. Accord- ing to Tyler and Stephens this maneuver is not specific for Duchenne muscular dystrophy, being seen in a variety of 60 other diseases in which the pelvic girdle muscles are weak. The progression of the disease is rapid, with no remission Gower's maneuver. Figure 1. and the progression appears to be roughly inversely propor- 44 Most of these children are tional to the age at onset. unable to walk by about nine to twelve years of age and they are confined to wheelchairs. Death usually occurs within ten to fifteen years after clinical onset so that approxi— mately three-fourths are dead by the age of twenty. The mor- tality rate decreases sharply beyond that age so that 5 per- cent of patients remain alive at the age of 50. Death usu- ally results from inanition, respiratory infection, or car- diac failure.“’67 It has been well established from numerous pedigrees that the inheritance of this type of Duchenne muscular dys- trophy is sex-linked recessive.* Among the children of a carrier female, therefore, half of the boys are dystrophic and half the girls are carriers; the other half of the chil- dren on the average would be normal. In most studies, one- third to two-thirds of the patients give no family histories and are thus referred to as sporadic cases. These patients are clinically indistinguishable from those in pedigrees show- ing clear-cut sex-linked recessive transmission. They have * Philip and Walton in 1956 were able to demonstrate crossing-over between the benign form of sex-linked Duchenne muscular dystrophy and color blindness. In 1966 Emery re- ported the only study of linkage between color blindness and the severe Duchenne type muscular dystrophy, which indicated that the loci for deutan and protan color vision are not closely linked to the gene for Duchenne muscular dystrophy. It also seems that the gene for this type of muscular dys- trophy is not closely linked to the X blood group gene (Clark 33 11;, 1963; Blyth g}; 2.1-: 19%5). 10 been regarded as new mutants, but Stephens and Tyler point out that to account for all the observed cases in this fash- ion requires a mutation rate higher than is usually found in human genetic material. Boyer and Fainer (1963) hypoth- esized that the metabolic pathway involved in this situation contains a long chain of enzymatically controlled steps and that defective synthesis at any one of these steps could be expected to affect the end product similarly.4 Thus, many different mutations would express themselves by the same anatomic and physiologic abnormality. In that case what is really being determined by the mutation rate is a composite of many mutations occurring separately but having indistin- guishable end results. Tyler and Stephens (1951) estimated the mutation rate for the severe type of Duchenne muscular dystrophy to be 9.5 x 10’5, Walton (1955) estimated it to IDe 4.3 X 10-5, and Stevenson (1953) found the mutation rate tC) be 5.4-7.2 X 10-5. Knudson (1965) was least specific in :hcis calculations, merely estimating the mutation rate to be 5 . . mutat1ons to the rece551ve in the range of 5 to 10 x 10' ‘aJllele per gamete per generation. Another possible explana- tlion of the "sporadic cases" is that they are phenocopies E11nd thus the clinical abnormalities, though appearing the SSame, are not genetically caused. A theory put forth by Becker (1957, 1962) suggests that there exists a set of at least three alleles on the X chromosome for Duchenne type muscular dystrophy: a dominant 11 allele for normal musculature; a recessive for late onset of the disease; and a recessive for early onset. Becker's theory was set forth to explain the occurrence of a milder X-linked recessive type of muscular dystrophy which has been included under the name of Duchenne. As illustrated in Table 1, an autosomal recessive type has also been re- ported with the same name. According to reports in the lit- erature severe X-linked, mild X-linked and autosomal reces- sive types occur in the proportions of roughly 27: 3: 5 (Lam and de Grouchy, 1954; Walton, 1955, 1956; Milhorat, 1961; Becker, 1964a). Becker and Kiener (1955) defined the severe form as having an oldest age of onset of ten years, inability to walk usually by the age of eleven, and death by age of twenty, while the mild form usually has onset age aafter ten years (range 2-35 years), ability to walk in ma- tnarity, and prolongation of the life expectancy with death éiifter the age of twenty-five and sometimes as late as the Scixth decade. Because the milder X-linked muscular dystro- I>}1y form was recognized first by Becker (1955, 1957, 1962) ift.is often called Becker's muscular dystrophy. More re- ‘3E3nt1y it has been claimed that there may be at least three 'tarpes of benign X-linked muscular dystrophy differing in age of onset and clinical symptoms (Emery and Walton, 1967). Since the severe and mild forms of X-linked muscular dys- 'trophy never occur in the same family (Blyth and Pugh, 1959) it seems that the two diseases are distinct entities. From 12 the X-linked pedigrees of Walton (1955, 1956) it would seem that about 9 out of 10 pedigrees represent the rapidly pro- ,gressive Duchenne type with onset before age ten years. 'Fhe autosomal recessive type which represents a small mi- Iuarity of the cases of Duchenne muscular dystrophy, is dis- txinguishable from the severe X-linked recessive type mainly b)r the genetic history, although there is a later age of orlset and slower progression reported. It does, however, oiifer one explanation for the forty reported cases of fe- nuiles affected with the disease. Other hypotheses have beeen.suggested to explain these cases. First, the patient C(yuld have Turner's syndrome with the one X chromosome car- r)'ing the dystrophy trait (this seems to be the explanation 111 the case described by Walton in 1956). Similarly, mo- Salics such as XO/XX and XO/XX/XY would be expected to pro- ‘illce atrophy in those cells with the XDO karyotype (XD being the X chromosome carrying the dystrophy trait). Sec- <3r1d” mating of a carrier female, or of a normal female who hadproduced an ovum with a mutant XD, with a normal male ‘V}Lo had produced a sperm carrying a mutant XD chromosome ‘Vcruld also account for an affected female. The mating of a. carrier female with an affected male could also lead to affected females, but it is most unlikely that affected nHales could reproduce. The above discussion of Duchenne muscular dystrophy indicates that there are still difficulties in differentiation 13 and diagnosis which await clear-cut, objective tests. In the past years much work has been done with histopatholog- ical, electrophysiological, and biochemical methods for r diagnosing myopathies in general and specific types of nuascle disease in particular appears to be the changes in 'bcady fluids which accompany the muscle diseases. Review (Di? this subject will include discussion of changes in body fluids which are common to many of the myopathies but will ‘Zcrncentrate on those substances, particularly the serum eIlzyme CPK, which are specifically useful in diagnosing DLlchenne muscular dystrophy and the carrier state. One of the earliest discovered characteristics of In)Vopathies was a marked increase in creatinine excretion, ‘S<3en in almost all types of muscle disease (Levene and Kiriszteller, 1909). It is now generally accepted that cre- atinuria is a nonespecific manifestation of muscle atrophy :and is caused by the muscle wastage and the consequent de- creased ability of the muscle to take up creatine, which then becomes concentrated in the blood and excreted by the 15 kidneys.28’44 A contributory cause to the creatinuria seen in muscular dystrophy may be an inability of the muscle to retain creatine normally (Fitch and Sinton, 1964). Tyler (1964) postulated that the abnormality in the skeletal mus- <:1e is one of membrane permeability, rather than a primary (lisorder in the regulation of creatine synthesis or renal llandling of creatine. Creatinuria is almost universal in patients with Iluchenne's dystrophy and in most of the patients with limb- ggirdle dystrophy. It is quite irregular in patients with :Eacioscapulohumeral dystrophy and myotonic dystrophy, and Inay be entirely absent even in advanced disease with exten- ESive muscle atrophy and reduction in total muscle mass suf- fficient to lower creatinine excretion. In patients who llave muscle atrophy as the result of inanition or certain (Jther processes, such as myotonic dystrophy, no creatinuria C>ccurs. However, in patients with polymyositis and derma- 1:0myositis, and to a lesser degree in patients with neural Eitrophy of muscle, creatinuria does occur and may be as se- \rere as in Duchenne's dystrophy. Thus creatinuria is not ESpecific to dystrophy, nor is it uniform in all the various Icinds of dystrophy. An enhanced excretion of some, if not all, amino acids has also been reported in muscular dystrophy pa- tients.44’45 Hurley and Williams (1955) found elevated leucine, taurine, and possibly threonine and valine, while 16 Konieczy 33 El: (1958) detected arginine, threonine, and proline in the urine of muscular dystrophy patients. A few ;preliminary studies done by Pearson in 1963 indicate that ‘two dipeptides, anserine and carnosine, may be found in :fairly large amounts in cases of muscular dystrophy. The exxcretion of hydroxyproline is decreased in patients with nulscular dystrophy (Kibrick 35 al., 1964). It can be con- cxluded from these reports that muscular dystrophy may be axzcompanied by an increased amino-aciduria perhaps result- irlg from the protein breakdown associated with the muscle wasting. Of the serum proteins, Oppenheimer and Milhorat (1.961) found an increase in the éZ—globulin fraction in all types of myopathy. The change in J-Z-globulin probably is a. nonspecific response to injury. It is found commonly in Inelny chronic diseases. In particular, sialic acid, a con- £5t1ituent of some glycoporteins, was found in high levels ill the serum of muscular dystrOphy cases, while protein- IDound hexose was found to be decreased. However, none of 'tlle changes in serum proteins seem likely to be of any use— 35111 diagnostic value because of the lack of specificity of Clhanges and because the average alterations are usually not Zlarge compared with the known variations in the normal val- lies. At present the serum enzymes promise to be most use- ful in diagnosing specific muscle diseases. It appears that 17 many of the enzymes which normally reside in striated mus- cle fibers leak out of the fibers as a result of the dys- trOphic process and are thus found in increased quantities in the serum. Pearce £3 31. (1964) hypothesize that the dystrophic process, in addition to producing degeneration and necrosis of muscle cell cytoplasm and nuclei, selec- tively damages the muscle cell membrane; this process ap- pears to be most marked in the most rapidly destructive variety of muscular dystrophy, the Duchenne type.41 Aldolase (l:6-diphosphofructoaldolase) was the first serum enzyme found elevated in dystrophy in 1949 by Sibley and Lehninger. Aldolase, an intracellular glyco- 1ytic enzyme which is particularly abundant in muscle, ca- talyses the cleavage of fructose 1,6-diphosphate to glycer- aldehyde 3-phosphate and dihydroxyacetone phosphate. While there may be a rise in serum aldolase in adult types of mus- cular dystrophy and in myotonic dystrophy, the most strik- ing elevations of serum aldolase occur in the Duchenne type 799911921936344’45’48’58’72 Figure of muscular dystrOphy. 2 shows, in graph form, the aldolase levels that Chung, Morton and Peters found for various types of myopathies. The normal range is delimited by two broken lines above which 2.5 percent of normals are expected to lie. Of the 66 Duchenne cases examined in this study, all but two are above the normal limits, and these exceptions are very ad- vanced cases of the disease. As can be seen in this graph 18 Serum Aldolase Figure 2. Muscular dystrophy in man. Key: A Duchenne o Limb-girdle o Facioscapulohumeral IOther neuromuscular disease 19 and as has been verified elsewhere (Thomson gt al., 1960) the elevation is most marked in young patients in whom the disease has been of short duration. Thomson gt_al. attempt to explain this phenomenon as follows: "It has been shown experimentally that, after the intra- venous injection of known amounts of crystalline a1- dolase in animals the rate of clearance of the aldolase is proportional to its initial activity in the serum (Sibley, 1958). In the early stages of the disease, therefore, when a large bulk of muscle is steadily lib- erating large amounts of aldolase, the serum clearance mechanisms are probably saturated and high serum activ- ities of aldolase result; later when the muscle bulk has diminished and with it the amount of aldolase enter- ing the serum, the accelerated clearance mechanisms may be better able to meet the demands now made and the ac- tivity of serum aldolase falls rapidly. Finally, when there is little muscle left and a minimal liberation of aldolase, the stimulus of serum activity towards clear- ance of aldolase falls steadily away as does the remain- ing muscle bulk, and thereafter the passage of time has little effect on the serum aldolase activity which re- mains now only slightly raised above the upper limit of normal." Schapira E£.§l¢ (1953) and Dreyfus and Schapira (1955) have shown that many cases of muscular dystrophy man- ifest abnormally high activities of other serum enzymes, in- cluding phosphohexoisomerase, lactic dehydrogenase, and the transaminases. The transaminases are intracellular enzymes found abundantly in muscle, and also appear in the serum.58 They catalyze the reversible exchange of'A-amino groups. Lactic dehydrogenase (LDH), a glycolytic enzyme that catal- yses the interconversion of pyruvate and lactate, exists in five electrophoretically distinct molecular varieties in nearly all tissues. In contrast to normal adult muscle 20 which contains a preponderance of LD4 and LD5 isoenzymes, several investigations have shown that some patients with muscular dystrophy lack LD5 completely and have lesser quan- 44’69 The quantitative increase in serum LDH titles of LD4. activity seen in cases of muscular dystrophy is due primar- ily to the great increase in LDl, but also, to a lesser de- gree, to an increase in the anodal hydrids LD2 and LD3. Thus there is a shift in concentration of the isoenzymes toward those which migrate to the anodal pole in cases of dystrophy. All the above mentioned enzymes have been found to be elevated in the serum of patients with progressive mus- cular dystrophy (most notably the Duchenne type), but not in those with muscular atrophy due to disorders of the cen- 58 Thus the assay of tral or peripheral nervous system. these serum enzymes is useful in the diagnosis of muscular dystrophy and in the clinical investigation of muscle weak- ness and atrophy of obscure origin. However, one drawback is that elevated levels of the transaminases, LDH, and phos- Phohexoisomerase are found in other conditions besides mus- CUlar dystrophy, such as myocardial infarctions and malig- nant cachexia.58 It appears, at the present time, that the enzyme CII‘eatine phosphokinase (CPK) is the most sensitive index for the diagnosis of muscular dystrophy. It was first in- tI‘oduced for this purpose in 1959 by Ebashi gt £1: The 21 enzyme CPK catalyzes the reversible reaction: Creatine + ATPa======éCreatine phosphate + ADP Figure 3 shows a schematic representation of the transfer (Jf the phosphate group from ATP to creatine, catalyzed by Inuscle CPK. Adg_represents the adenosine protion of ATP. 'The upper left diagram shows the disposition of all groups at the instant of binding of creatine and ATP. The lower left diagram represents completion of the reaction just prior to departure of creatine phosphate and ADP from the eulzyme surface. The enzyme CPK, found principally in skel- ertal and heart muscle cells and also, in lesser amounts, in Irrain tissue, is elevated almost exclusively in myocardial iIlfarction, hypothyroidism, and diseases of skeletal mus- 41,47,50 Cite. From the original study, and many others fol- lxywing it, it has been shown that CPK is more specific than tlle previously mentioned enzymes for muscle disease (Figure 4) .21’22’24’36’44’51 Among the many types of muscular dys- tI‘Qphy, it has been shown that the highest values of this erlzyme are detected in patients with the Duchenne type, and tile lowest values in patients with the facioscapulohumeral type.*7,21,48 *However, a preliminary communication by B. P. 'Hnghes (1971) has revealed that estimation of serum CPK may provide a useful screening procedure for early detection and for assessing the status of relatives of a patient with facioscapulohumeral dystrophy. 22 M890 ‘ 5° 6' r /a'\ 5‘0 ' CH, [4H2 0‘ [O (I? '1 ‘P' . . Figure 3. Schematic representation of the CPK reaction. 23 A 59$0 95214 59744 240. 2674 24¢ -4 440.9 220 ZOOP Serum creatine phosphokinase units/m1 H r-I H H H 4:- O‘ 00 O N b O\ OO O O O O O O O O r I l fl U I l I O O N O i O . O 0 3.3.3.51 L—o—h. 3 i m 32:. :: 1 2 3 4 5 6 Figure 4. Serum creatine phosphokinase activity in 82 cases of progressive muscular dystrophy and neuro« muscular diseases: (1) Progressive muscular dystrophy, (2) Motor neuron disease, (3) Anterior poliomyelitis, (4) Polyneuritis, (5) Miscella- neous, (6) Normal subjects. 24 Many investigators have noted a definite inverse cxarrelation between the age and the level of CPK in patients vvith Duchenne type of muscular dystrophy. (Figure 5)9’21’36’41 1000, 900’ 800- 700. 600, 500. 400- 300- 200. 100- CPK (units) Age (Years) Iiigure 5. Activity of the serum kinase in cases of the Duchenne type of muscular dystrophy charted against the patients' ages. Idle elevation of CPK seems to be maximum in the youngest pa- tlients with disease of shortest duration. This is in agree- “Nint with the findings for serum aldolase, which were men- 'tioned earlier. In the case of CPK the levels remain high as the weakness and atrOphy progress, but as the extensive loss of muscle mass intervenes, the level gradually falls until in the advanced disease it may be normal or only slightly elevated. The increased amounts of CPK appearing 25 111 the blood originate largely or entirely in the muscle 1:issue itself, leaking out of the fibers as the latter are (listurbed and damaged by the disease.45 In conclusion, it can be said that the magnitude of 'the elevations found in serum enzymes, when related to the <:linical and genetic findings, is of great value in distin- guishing between the various types of muscular dystrophy :Ind in separating them from the acquired myopathies and at- 'rophies of neurogenic origin. In the latter cases no con- sistent elevation of serum enzymes has been reported. Serum enzymes may also be helpful in detecting the czarrier state of Duchenne muscular dystrophy. In many ge- lletic diseases it has long been recognized that clinically Ilormal carriers of the abnormal gene may show, to a minor (legree, biochemical abnormalities characteristic of the di- ssease. Two well-known examples of this are seen in the lleterozygous carriers of sickle cell anemia and galactosemia. III the case of the Duchenne type muscular dystrophy, the fe- Huale carriers have reportedly been identified by a variety th methods: clinical, histological, electrophysiological, and biochemical . There have been several reports (Dubowitz, 1963; Dawidenkow and Kryschowa, 1960; Emery, 1963; Walton, 1964; 'Milhorat and Goldstone, 1965) of female carriers observed to have an enlargement of one or both calves, resembling the pseudohypertrophy of the Duchenne patients. In several 26 ssuch instances, these women complained of cramps and/or eveakness in the calves upon exertion. Dr. Robert Puite (Personal Communications) reports that he has three cases :in his files of female carriers (the carrier state confirmed 13y other methods) showing clinical symptoms of the disease. A number of investigators have reported abnormali- ‘ties in the histology of muscle biopsy specimens in a pro- ]portion of carriers (Dubowitz, 1963; Emery, 1963, 1965; IPearson g£__l., 1963; Stephens and Lewin, 1965; Kowalewski SEE al., 1966; Pearce et_al,, 1966; Smith §t_§1,, 1966). 'The most consistent findings, although often minimal, were 'random variations of the fiber size and shape, focal areas (of degeneration and phagocytosis, an increase in sarcolemmal Iluclei, proliferation of endomysial connective tissue and/ ()r interstitial infiltration of mature fatty tissue partic- lllarly in relation to atrophic fibers. The general conclu- ESion is that a muscle biopsy, along with other tests, may 136 a valuable aid in carrier detection but is not reliable hfllen used alone (Emery, 1965; Pearce £3 31., 1966; Smith a a_1_., 1966). In 1963 two reports were published in which elec- 'tromyograms were recorded from carrier females and found to have a higher incidence of abnormalities than in the female control subjects. VandenBosch reported that he had found an excess of polyphasic potentials in EMGs recorded from eleven possible carriers of the Duchenne type muscular 27 (lystrophy while Barwick reported that when he used an arbi’ 1rrary numerical score to assess the degree of myopathic <:hange in the EMG, he found a higher incidence of short du- :ration potentials and polyphasic potentials in carrier fe- Inales than in a group of control women of comparable age. 1\ 1964 study by Davey and Woolf revealed no significant dif- ference in the duration and amplitude of the potentials re- <:orded from the biceps brachii of carriers but the next year 'they published an additional report in which they had found six out of twenty-two definite and possible carriers to have :an increased incidence of polyphasic potentials. These au- ‘thors concluded that in occasional cases the EMG might help in detection of the carrier state when serum enzymes had Taeen negative. In 1965 Caruso and Buchthal made an exten- sive study which showed no difference in the mean action po- ‘tential duration and amplitude measurements in carriers and <:ontrols but they agreed with Davey and Woolf that a percen- ‘tage of polyphasic potentials in excess of normal was ob- trained from their carriers. However, according to these in- Vestigators the more successful method of carrier detection \vas by means of recording the absolute refractory period of IhUscle, which they found to be significantly reduced in a high proportion of the carriers. Subsequent studies have revealed that although abnormalities may be detected subjec- tively in the EMGs of a small percentage of carriers, elec- tromyography carried out on carriers is of no value unless 28 quantitative techniques are used (Hausmanowa-Petrusewicz, 1965; Smith gg gl., 1966; Walton and Pennington, 1966) and measurement of the absolute refractory period of muscle, as described by Caruso and Buchthal, are too difficult and time- consuming for routine use (Walton and Pennington, 1966). A reduction in the peripheral circulation time has been described in some carriers (Demos g3 g1., 1962) although actual limb flow appeared to be normal (Emery and Schalling, 1965). However, adequate data is not available to compare this with other methods of carrier detection. A reduction in the proportion of the more slowly mi- grating isoenzymes of muscle lactic dehydrogenase has been described in patients with Duchenne muscular dystrophy (Dreyfus E£.§lr: 1962; Wieme and Herpol, 1962) and in some carriers (Emery, 1964; Mannucci gg.gl., 1965; Johnston gg gl., 1966; Pearson and Kar, 1966). This is useful, diag- nostically, in differentiating muscular dystrophy from other diseases in which increased LDH is seen. However, negative results have also been reported using LDH to detect the car- rier state (Brugsch, 1960). As in the case of muscle his- tology, muscle LDH isoenzyme studies may be useful only as a confirmatory test in detecting carriers and are not reli- able alone. A new approach for carrier detection in Duchenne muscular dystrophy was described by Ionasessu, Zellweger, and Conwau in 1971. They examined human muscle polyribosomes 29 ()f carriers in respect to their distribution and ability to ijacorporate amino acids ig_giggg, Polyribosomes from pa— txients with Duchenne muscular dystrophy had earlier been sdlown to have increased activity (Monckton and Nihei, 1969). Ftight of their ten carriers (all of the definite carriers, alfil of the probable carriers, and four of the six possible cerrriers) showed a significant increase in protein synthe- 5145, while the CPK levels were elevated in only five cases (tnno probable carriers and three possible carriers) and dys- 1:rophic histological findings were positive in three of tfllese five. They concluded that incorporation of amino zuzids by muscle polyribosomes, ig.yi£gg, appears to be a nubre sensitive test of the carrier state of Duchenne muscu- lar-dystrophy than either histological or serum enzyme stud- iesw Many investigators have examined the question of \Mhether serum enzyme levels may be helpful in detecting the o pom Moscow mpofispmo czocx mo munchm 03» ca mofipw>fluom xmu mo soapsnwhumfln .o answem mead: goo NH «a m w u o m w m N H _ __ _ _ . _ . . , H_ r cw v hove: - . — . 1H .N I in v mowed gag ad t . 4H . . a m a e m a m N a d 1 J u u d OVA H®>O .H -N .m .v Figure 7. Serum CPK (units) 37 °51.8 25F o 20 ' 15. o 10 - 5- 0°00 o o 00 oo o 1L4 14 L11 10 20 30 40 50 60 70 80 Age of carrier in years Values for serum creatine kinase in carrier females of different ages, expressed in units (micromoles of creatine formed per ml of serum at 37°C). MATERIALS AND METHODS Assay of Serum Creatine Kinase Creatine phosphokinase is the common name for ATP: creatine phosphotransferase (Report of the Commission, 1961) which catalyzes the reaction: Creatine phosphate + ADP 1W Creatine + ATP Assays of the enzyme have been based on either the forward or backward reaction. In the forward reaction either the creatine or the ATP is measured. In the backward reaction the creatine phosphate is measured as phosphate or as cre- atine in a one-enzyme system or the ADP is estimated by means of two additional enzyme systems (Tanzer and Gilvarg Method, 1959): ADP + phosphoenolpyruvategL—uégs-d pyruvate + ATP PYTuVate + DPNH sling" “2:9: ' r0 ends; DPN + lactate The end point of this three enzyme reaction is based on the oxidation of DPNH as indicated by the decrease in the opti- cal density of the asSay solution at 340 pm. At this time the data still seems inadequate to establish the superiority of any of the various assay procedures for CPK. The forward reaction occurs about ten times faster than the backward re- action so it is theoretically more suitable for serum enzyme 38 39 assays. However, Rotthauwe g£_gl. (1961) and Vassella gg g1. (1965) have spoken against the forward reaction assays based on the formation of creatine (called the Schapira Method: Schapira g£_gl,, 1960; Dreyfus g£.gl., 1960). The latter group also pointed out that the forward reaction with ATP production as the end point (Oliver Method, 1955) may yield false low values as a result of ATPase present in ser- um. Vassella E£.§ln have also criticized the one stage back- ward reaction (Ebashi Method, 1959) as being insensitive and nonspecific but the method has been defended by Danowski and his colleagues (1968). Because this study involved the pool- ing of data from three laboratories, several differing meth- ods of CPK determination were used, as explained below. Control Subjects For the control group, serum CPK determinations were performed on seventy-one healthy female staff members and hospitalized females suffering from diseases in which normal CPK values have been reported.4’8’zz’34’36 The latter group included patients with diabetes, cancer (including two cases of leukemia), pneumonia, kidney infections, asthma, cystic fibrosis, anemia, bronchitis, and pre-operative patients hos- pitalized for tonsilectomies and corrective surgery. Pa- tients suffering from myocardial infarctions, hypothyroidism, chronic alcoholism, trauma, epilepsy, cardioversion or any 40 of the myopathies were strictly excluded because of reports indicating some elevation of CPK levels in such cases.18’22’ 41’47’50 The blood samples were taken by venipuncture and all the subjects had been resting at least one-half hour be- fore the drawing to avoid any enzyme changes due to physical activity. The control group samples were taken from the var- ious age groups to determine the variation. All controls were females to match the carriers. Once drawn the blood samples from the control group were allowed to clot and then centrifuged. The CPK activity was estimated immediately by the enzymatic technique de- scribed by Rosalki in 1966. The reagents and technique have been standardized and prepared in kit form by the California Corporation for Biochemical Research. In this method the primary reaction is coupled to two subsequent reactions: Creatine phosphate 4' ADP ;——c-P-K—==éCreatine + ATP ATP + glucose MDALfiglucose-mphosphate + ADP glucose‘G-phoSPHa‘l’e Glucose-6-phosphate + NADP-AW) NADPH + 6-phosphogluconate 0.1 ml of each serum tested was added to a cuvet containing 3 m1 of the activitated substrate, creatine phosphate and ADP, plus glucose, NADP and the enzymes hexokinase and glu- cose-6-phosphate dehydrogenase. The conversion of NADP to NADPH results in a decrease in absorbance of the solution measured at a wave length of 340 pm. The initial absorbance 41 (A0) was read six minutes after placing the cuvet in the Beckman spectrophotometer while the final absorbance (A5) was read exactly five minutes after the initial reading. Thus AA, the change in absorbance, is obtained by subtract- ing the initial absorbance from the final: AA = As-Ao The CPK in mu/ml is then calculated as follows: CPK = AA X 1000 X F X D where the 1000 is the conversion factor needed to obtain CPK values in international milliunits per milliter of serum at 30°C, the "F” is a temperature factor (when the assay is not carried out at 30°C), and the "D" is a dilution factor which always equaled one in the determinations because 100 microliters of sample were used. Because the temperature is critical in this assay, it was kept constant during the reaction at 30°C 1 0.2°C. Normal values for human serum at 30°C were given in the brochure as: females---S-40 mu/ml Patient Material Information on carriers of the gene for Duchenne mus- cular dystrophy was obtained from three separate muscular dystrophy clinics. All of these females were mothers or sis- ters of one or more boy affected with the severe type, early- onset Duchenne muscular dystrophy, as verified by clinical findings, genetic history, biochemical tests and muscle bi- opsy if necessary. Only those females whose exact age at 42 the time of the CPK test was known were included in this study. The subjects were divided into two groups: 1. Possible carriers--these consisted of two subgroups a. Mothers with only one affected son and no family history b. Female siblings of patients 2. Known carriers-~these were mothers with two or more affected sons or one affected son and a definitive history (an affected brother or maternal uncle) From the Minneapolis Muscular DystrOphy Clinic un- der the direction of Dr. Richard Zarling, results of CPK determinations of seventy-six sisters, fifty-one mothers with one affected son, and ten mothers with two or more sons and/or a positive family history were gathered. The method for CPK determination used in their laboratory is based on the Tanzer and Gilvarg (1959) method but with the addition of one micromole of cysteine per ml. of incubation mixture. They found that the use of cysteine not only increases the CPK values in fresh serum but allows the serum to be stored in the frozen state for at least a month with no loss of ac- tivity provided that the cysteine is added immediately be- fore the test is carried out. From the Madison, Wisconsin Muscular DystrOphy Clin- ic headed by Dr. Henry A. Peters came information on fifty- four sisters of muscular dystrophy patients, fourteen mothers with one dystrophic son and eight mothers with one or more affected sons and/or a positive family history. On all CPK determinations prior to October of 1970, the Madison 43 1aboratory followed the Tanzer-Gilvarg method, not activated, which has been described previously. The normal values for their laboratory were considered to be 0-1.7 I.U. From No- vember of 1970 to the present the Rosalki or Oliver (1955) procedure has been used to assay for CPK activity in the mus- cular dystrophy carriers. This method uses the forward re- action and measures, optically, the ATP formed using the hexokinase and glucose-6-phosphate dehydrogenase system. The normal value for women using this method was determined by the laboratory to be up to 85 I.U. The final laboratory which cooperated in this study was the Grand Rapids, Michigan Muscular Dystrophy Clinic un- der the direction of Dr. Robert Puite. Through this clinic, CPK values had been determined on fifty-six sisters of af- fected boys, thirty-nine mothers with only one dystrophic son, and nineteen mothers with one or more affected sons and/or a positive family history. Those who had been tested after January of 1970 were excluded from the study because the CPK determinations from that time on have been done by the Technican Method (on the SMA 12) and data is not avail- able to correlate this method with the other methods. Prior to 1970 this laboratory used the standardized reagents put out by the Sigma Chemical Company. In this procedure the phosphocreatine which is formed in the backward reaction is hydrolyzed to yield inorganic phosphorus which is then mea- sured colorimetrically as an index of the CPK activity: 44 1) ATP + CreatinegéL—é—‘ADP + Phosphocreatine 2) Phosphocreatine—MiL—ACreatine + Inorganic P The inorganic P is measured colorimetrically by the Fiske and SubbaRow procedure. Phosphorus values can then be con- verted to CPK activity by a Table which the laboratory com- piles. The normal range suggested by Sigma and followed by the Grand Rapids laboratory was 0-10 Sigma units per ml. RESULTS Carrier Detection Table 2 summarizes the pooled results on detection of carriers using CPK from the three muscular dystrOphy clinics participating in this study. Of 37 mothers defined as known carriers (having more than one male patient in the family), 25 displayed elevated values of the serum enzyme CPK, e.g., 68 percent. There were 104 mothers of isolated cases classified as possible carriers and of these, 50 had high CPK values, e.g., 48 percent. Among the S7 sisters of dystrophic boys whose mothers are known carriers, a total of 25 showed elevated CPK levels, e.g., 43 percent. Of the 134 sisters whose mothers are possible carriers, 33 dis- played elevated values of the serum enzyme, e.g., 25 per- cent. Using the data from both the known carrier mothers and the sisters with known carrier mothers, it is possible to calculate a detection efficiency based on the method of maximum likelihood. Looking at Table 2, one can see that the two estimates of the detection rate of carriers differ: 68 percent detection efficiency among known carrier mothers and 86 percent detection efficiency (43 percent of the 45 46 expected 50 percent) among sisters with known carrier moth- ers. The appendix contains details of the statistical pro- cedure used here, showing that the best estimate of the de- tection rate is really 0.725 (~73%). Comparing the data from the two groups one finds no significant difference from the expected based on this estimate. 'Table 2. Summary of results on detection of carriers using CPK. Number having Percent Number increased CPK increased CPK Controls 71 l 1.4 IKnown Carrier Mothers 37 25 68 IPossible Carrier Mothers 104 50 48 Sisters with known carrier mothers S7 25 43 Sisters with possible carrier mothers 134 33 25 E On the basis of this 73 percent detection rate of (:arriers it is possible to recalculate the percentage of (ietection of the mothers of isolated cases and the sisters :in my series and arrive at a corrected percentage of car- trier detection (Table 3). For example, the 50 mothers of isolated cases who were determined as having elevated CPK ‘levels, actually represent only about 73 percent of all the true genetic carriers in that group. By taking into account 47 the carriers who cannot be detected by this method, it can be determined that approximately 65 percent of all the pos- sible carrier mothers are true carriers: 50 68 ll 1; (N >< >< III III 65% In the same manner, approximately 59 percent of all sisters with known carrier mothers should be true genetic carriers, and approximately 34 percent of the sisters with possible carrier mothers should be true carriers even though some are not detectable by the CPK method. Table 3. Corrected percentage of genetic carriers among possible carriers. Corrected Overall Percent percent of results detection detection Possible Carrier Mothers 50/104 48 65 Sisters with known carrier mOthers 25/57 43 59 Sisters with possible lcarrier mothers 33/134. 25 34 Figure 8 shows the superimposed graphs of my con- trol group and my group of sisters of dystrophic boys, il- lustrating the distribution of the CPK values. As would be expected, the greater percentage of these sisters fall in a pattern similar to that of the controls. In this 48 group are the "normal" sisters who have not inherited the dystrophy gene from their mothers as well as the approxi- mately 27 percent of carriers which are not detected using CPK. However the graph also shows a group of sisters with CPK values elevated to various degrees, and it is assumed that these sisters are the carriers of the gene for Duchenne type muscular dystrophy. As can be seen from the graph in Figure 8, the elevation in these carrier sisters varies from the 2nd standard deviation above the mean of the controls to the 82nd standard deviation above that mean. It is this ex- treme variation in CPK values of carrier sisters that was then investigated to determine its correlation to the factor of age of the carrier. Control Group First, information was gathered on seventy-one con- trol subjects to determine any affect of age on the CPK val- ues of normal persons. Figure 9 shows a scattergram of the CPK values of the control group as a function of age. From this graph it appears that there is a greater percentage of high CPK values among the younger than among the older con- trols. The mean CPK value for each age group is listed in Table 4, with 0-40 being the normal range. From this data it was determined by use of Scheffe's test that the apparent difference in the CPK levels of children from 0-12 years as compared with persons aged 13 and older is not statistically 30% r 25% P 459 62nd 82nd Slst 36th — — — — — Controls Sisters L 0" 60 O In N H stenprArput 12101 50 luaolad 10% ' 5% l_L L H192 H152 H172 P122 ‘ PUZZ - lsIZ . H102 ~ H161 - H181 ‘ HJLI I ~ H151 ‘ H171 - 91:1 ‘ 9121 . quit - 9201 ‘ H16 - use d 919 « 915 l qlv 919I qll CPK Values (standard deviations) "PJE notaernap plepueis PUZ uorlerAap plepuels 151 . ueaw uoriernap ‘ plepuels 181 uoraeraap - plepuels PUZ Distribution of CPK values in controls and possible carrier sisters. Figure 8. 50 VHN O... O. .QDOHM fichucou may a“ 0mm mo :oflgucsm m we mosfim> Mao ONIBH flmhmoxv mom< OHTmH - Nata N wto bl o .m opswfim .oN rem rwm .Nm .om roe .wv twv (rm/um) SQHIBA ydg significant. 51 found in the appendix, page 95. Details of this statistical procedure can be Table 4. Mean CPK values for the different age groups in the controls. No. of Age persons CPK Mean Range 0-4 18 21.4 mu/ml 1-40 mu/ml 5-8 10 25.4 mu/ml 15-38 mu/ml 9-12 9 26.1 mu/ml 11-44 mu/ml 13—16 10 15.8 mu/ml 0-34 mu/ml 17-20 4 17.0 mu/ml 10-27 mu/ml 21 8 Over 19 17.6 mu/ml 2-37 mu/ml Effect of Age on Possible Carrier Sisters Figure 10 shows a scattergram of all the elevated (ZPK values of my group of sisters as a function of age. It will be seen that there is no suggestion in this diagram <3f any differences in the levels of CPK between the younger zand.the older carriers. The mean CPK elevation, expressed :in standard deviations above the mean of the control group, for each age group is listed in Table 5. The six extremely 'high values shown in Figure 10 were rejected as unrepresen- tative and were not used in calculating the means. Figures 11 and 12 show the comparison of two groups of carriers, under and over 12 years of age. Figure 11 shows comparatively the distribution of the CPK values while 52 .mhopmflm Hofihpmo cw mum we soapocsm m we mosfim> xmu nmpmozv om< .OH ohsmflm VHN owina Carma NHum wsm «so . p . h P _ o o D a u . m M m .N m O a o O o o o u o A O O 1V 9 0 I o o o o n 9 o 0 no . . m, o o Lw 0m I..u p . .Smm 0 9p . -Smw. HA .6 1W. o .VHMI. It. 0 3H lOHJS 0 n9 . .. Mm. 3 A 9 ION 1+ U; 9 o INN m 9 m o r em Mflo? 0b.: O «2.. m7... .3... 8+ q 3 53 .Homm mo whom» NH po>o paw powcsv mpoHapmu Ho mmsoam 03p :H moHpH>Huom gnu Ho coHuanapmHm HNNsNH omcmpv NH ho>o macaw Hoepcou on“ mo memos onp o>onm mcoHpmH>ow wamwcmumu moHuH>Huum ado omAmeNnN 3 mm emmm NNHN om mHmH :oH mHeHmHNH HHOH m ms 0 m e m N . .1::::_:;::,_ __:_. r + HHH . .05 e omcmuv NH have: macaw Hopycou may HkomoE may o>oam msoHuwH>o© vpmecmumv moHuH>Huow Mao omM mmwm NNON vam mNNN HNON meHKHOH mHeHMHNHHH S m w H. o a .J-W-__fiu¢-#_fiaqdu— 44 m llg Lr.m _In l WNOU‘JVMN \0 LO V M N H 5959:) JO .quumN .HH oaswfla 5958: go laqmnN 54 .Howm mo memo» NH ho>o paw howcav mHoHHhmu mo mascam ozu :H moon> MAO Ho o>pso ommpcoohom o>HumHSEso Homes on» o>onm m:0HpmH>ow wumecmumv moHpH>.uum Mmu omA.vN MN NN HN ON mH wH NH oH mH «H mH NH HH OH m w H o m w m NH po>o llllll NH poem: % laqmnu [Biol JO 55 Table 5. Mean CPK elevation for the different age groups of carrier sisters. Age No. of carriers CPK mean Range 0-4 14 4.8 2-13 5-8 11 5.2 2-24 9-12 8 5.4 2-18 13-16 10 5.0 2-14 17-20 6.8 2-22 21 8 Over 4.0 2-8 Figure 12 shows a cumulative percentage curve of the two groups as a function of CPK values. No correlation between CPK activities and age is apparent from these graphs. Due to the fact that each of the three laboratories participat- ing in this study used different methods for their CPK de- terminations, it was necessary to statistically analyze each separately for any significant age correlation. Table 6 groups by laboratory the serum CPK values for all the car- rier sisters in this study who had elevated CPK values. Using the Student's t-Distribution Test on this data it was determined that no significant difference in the mean CPK levels of muscular dystrophy carriers under or over the age of 12 exists. The appendix, pages 96-99, contains details of these statistical procedures. It was then questioned whether different percentages of elevated CPK activities might be found among the possible 56 Table 6. Serum creatine phosphokinase activity in carrier sisters. Laboratory Case no. Age CPK activity Madison, Wis. (prior to 1970) 1 18 2.7 2 20 4.3 3 7 3.8 4 16 24.4 5 9 1.9 6 8 4.9 7 7 17.9 8 4 1.7 9 16 2.5 10 12 8.3 11 10 2.9 Normal Activity onm m:0HumH>oe pampcwpmv Ho>oH gnu A o 15 10 Age (years) 5 P) D n b P n h 2m8642 Hades HQMHSOU o>onm mcoHpmH>op phmpdmpmv Ho>oH umu o o ,6 4. 72 m Ho>oH v30 We 10 15 20 Age (years) 5 E .r 1 AU . AI 100 O .6 14 12 p n n p P L 00 6 4 2 n a e M memos H0ppcou o>onm mcoHumH>ow eaeeeoomv Ho>oH gag P S 2 Ho>oH xmu 50H 1 1 9 8 7 6 3 4 5 Age (years) 2 1 CPK values in individual carriers over a period of years. Figure 13. 62 P I nu? 8642 Mean- Ho>oH ans 8 10 12 14 4 6 2 P I b 4 F F 8 6 4 2 Mean. Hence Hahucou o>onm :oHumH>om whmvcmumv Ho>oH any Age (years) {Age (years) _l. O L . 1 . i J j r p u n — Ru ,0 .4 “A n a e M” Hades Honucou o>onm :OHpmH>oe pudendumv Ho>oH age "u o . b b b p r 0 0 0 0 0 8 7 6 S 4 H0>0H MAG 15 16 17 18 19 20 21 9 10 11 12 8 Age (years) Age (years) (Continued) Figure 13. 63 have results which fit no specific pattern. The fact that five of the nine carriers showed decreased CPK values over a period of years may be suggestive, as was the previous data, of an inverse correlation between age and CPK values among some carriers. It is interesting to note that the subjects in graphs B, C, and F all have CPK values which are only slightly elevated (2-4X normal) at the first test- ing but which fall into the normal range at a later age. This observation supports the hypothesis mentioned above that as carrier females grow older a certain percentage of them (those whose CPK levels were only slightly elevated at a younger age) may fall back into the normal range. It might be significant that in three of the five graphs show- ing decreasing CPK values with increasing age (C, F, and H) the decrease takes place around the time of puberty (9-16 years). In another subject (D) her CPK value is falling as she approaches puberty. These observations correspond fa- vorably to the data above which demonstrates a higher per- centage of elevated CPK levels in possible carriers under the age of nine. Family Studies The data was examined with reference to family stud- ies to detect any relationship between level of CPK eleva- tion and family membership. Figure 14 is a graph of seven families in which two or more sisters had elevated CPK .aoHumHohhoo HHHEmm .VH opsmHm Hmpmosv ow< NN HNONmeH: onHvaHNHHHo.Hm m N. o m e m N H o 64 HHEmm 0 o . «No .328 4 5% 228* .3.“ 32am o 5%. xHHEwm O .wa based 1 so remm e. K .2. m 65 levels. It was thought that perhaps the extremely high CPK levels seen in certain carriers might "run in the fam- ily," suggesting that there might be different types of the disease which are evident in the degree to which het- erozygotes are affected. Each of the carrier sisters is identified by symbol as belonging to a certain family and her CPK level is then graphed as a function of her age to determine any family correlation. Using Spearman's Coeffi- cient of Rank Correlation Test on these data it was deter- mined that there is no significant correlation between the sisters' CPK levels. There is no significant difference in variance within sibships compared to that between unre- lated carrier sisters. (See appendix, page 104 for the statistical procedure.) Information was also available to compare the CPK levels of mothers to their daughters who had demonstrated high CPK activity. It was thought that perhaps extremely high levels in certain carrier daughters might be related to high levels in their mothers. Data was available on the CPK activities of the mothers of 38 possible carrier sisters who had shown elevated CPK levels (Table 9). Spearman's Coefficient of Rank Correlation Test was used on these data to detect any significant correlation between the mother-daughter CPK values. The coefficient of rank correlation was computed to be .13, showing that no signif- icant correlation exists (see appendix, page 105 for de- tails of this statistical procedure). 66 Table 9. Comparison of mother—daughter CPK levels. Daughter's CPK Mother's CPK Family (standard deviations (standard deviations in No. above control mean) relation to control mean) 1 3 1 Below Mean 2 5 8 3 4 Mean 4 2 Mean 5 82 1/2 6 35 8 7 10 1/2 8 2 Mean 6 Mean 24 Mean 2 Mean 9 3 2 1/2 11 2 1/2 10 18 1/2 65 1/2 11 3 1/2 1/2 12 4_1/2 7 1/2 13 8 2 2 2 14 2 5 15 3 1/2 12 16 3 2 17 2 7 18 2 2 l9 2 3 20 3 4 21 12 12 22 3 1/2 2 23 2 1/2 1 Below Mean 24 3 1/2 1 25 3 2 26 2 2 27 22 2 1/2 28 2 1/2 7 29 2 1 Below Mean 30 6 Mean 31 7 15 32 13 2 DISCUSSION Serum CPK measurement is of value in the diagnosis of the carrier state in Duchenne dystrophy, a conclusion agreed on by many authors, and is confirmed by the present data (Tables 2 and 3). These data point out that it is possible to detect a large proportion of the heterozygous carriers of this sex-linked disease by this method. This is of great practical importance since it provides a basis for genetic counseling in this disease. Since the frequency of carriers of this severe, sex-linked form of the disease is about 16 per 1000 births, the method may even prove suit- able for the screening of selected populations. However, the frequency of carrier detection in the present groups is somewhat less than reported by 7 of 9 other authors, as sum- marized in Table 10. Possible reasons to account for a de- tection rate of less than 100 percent among known carriers include: 1) Inclusion of some cases, indistinguisable by the pedigree and clinical findings, of autosomal recessive in- heritance. 2) The possibility that multiple alleles or genes at more than one locus can identically result in the disease 67 68 NmNNN OHHNee eeHNVNH aBenz Hosea aHNe QMNVH mm\mm mesa ..HI m1_mamsoae NNm momH .Hoeoaz mmeHOXO houwm e\e-e\o soda .eflosa one meoeaoom NNNON soda ..HI mm coma“: oa\e wHNe eHNMH soda .Hxasoaasog one osaoeooom mm\mH mH\OH meafi ..HI mm ooaeoa oNNNN meaH .1H1.mm eoaaooaowm HH\N HHNe mHNNH meafl .aoewo: NH\m N\a QNe mesa .aomeeoa mkmumwm mEHOH woumHOMH mhmflhhmu ¢3OGVH whoaufids .mhonusm monhm> zn mac nqu mMlohmo mo :oHuoouom .OH oHan 69 but differ in the degree to which heterozygotes are affected and thus detectable by this method. 3) The possibility that one or more of the several methods of CPK determinations used in this study were not as sensitive as other methods; this would also explain the somewhat lower than average detection rate reported in this study. The existence of an increase of serum CPK in the majority of carriers may also help to solve experimentally the problem of determining the number of mutations computed now from purely genetic considerations. Considering the re- sults tabulated in Table 3, one obtains for the mothers of isolated cases a figure of 65 percent true carriers. There- fore, 35 percent of the isolated cases probably arose from new mutations. Considering the ratio of isolated to famil- ial cases in this study (approximately 2/3's) one concludes that 23 percent of cases of Duchenne type muscular dystrOphy arise as mutations: .35 (2/3) = .23. This value is somewhat lower than the 30 percent mutants computed by Chung and Morton (1959) from genetic considerations. However, it com- pares very closely with the 24 percent mutants that Milhorat and Goldstone (1965) calculated from their families. Apart from the practical value, serum CPK determin- ations may some day be able to give an insight into the mechanism of the events happening in the tissues of the car- riers and the patients. An increased permeability of the 70 cell has been postulated to explain the increase of serum CPK and is supported by Zierler's research (1958) which demonstrated that the efflux of aldolase is greater in the muscles of dystrophic than of control mice when suspended in the same buffer. However, from information available on increased serum enzyme levels in various diseases it seems that such increases are a nonspecific result of tis- sue damage; almost certainly the leakage of muscle enzymes into the serum represents a secondary effect of muscle cell dysfunction, and is not of primary etiological significance. The ability of the muscle fibers to retain many of the in— tracellular compounds is a very delicate characteristic, quickly imparied by unfavorable conditions. Further stud- ies are needed to fully understand the mechanisms of per- meability and its role in this disease. Other investigators suggest that circulatory dis- turbances may be responsible for serum enzyme increases and/or pathological symptoms. This hypothesis arises from the Demos gg g1. study (1962) which reported an inverse correlation in carriers between serum aldolase and the cir- culation time in the arm. Much more study is needed to de- termine the relationship between serum CPK levels and the etiology of the disease. In order that the maximum use may be made of the serum CPK level as a criterion for detecting carriers it is important to possess adequate data concerning variations 71 occurring in normal individuals under a variety of physio- logical conditions. The data on the CPK levels in normal control persons (Figure 9, and Table 4) indicate that there is no significant difference in the CPK values of females in the different age groups. This finding agrees with the results of three previous studies. Pearce gg_gl. (1964) found that the level of serum CPK is significantly higher in normal infants and in children under the age of five years but saw no significant differences in the other age groups. Griffens (1964) found no difference between the levels in adults and children, though he too reported that the CPK level was raised in the neonatal period. Wilson g3 g1. (1965) also found no significant difference between the levels in girls and adult women. The subjects in this study do not appear to differ in any respect from those of the other studies. In all cases the subjects were taken from hospital staff members and hospitalized patients not suffering from myopathic disorders. Previous studies have shown that the ingestion of a large meal, pregnancy, dif- ferent stages of the menstrual cycle or time of day do not 40 Thus these factors affect the CPK level significantly. were ignored in the control subjects. It is possible that the factor of exercise could cause significant variation in the serum enzyme levels of normal persons since many con- flicting reports have been published on the subject (Tessari and Parrini, 1961; Pearce g£_gl., 1964; Hughes, 1963; 72 Swaiman and Awad, 1964; Vejjajeva and Teasdale, 1965). The Hughes, Swaiman, and Pearce groups did not find any signif- icant increase in the CPK level after moderate exercise. However, the study by Vejjajeva and Teasdale revealed a con- sistent elevation of the enzyme after severe physical exer- tion. They suggest alteration in the permeability of the cell membrane from physical activity or the release of en- zyme due to anoxia as possible explanations. It is now gen- erally concluded that light or moderate exercise has no ef- fect on CPK activity, whereas severe physical exercise does. All the control persons in this study as well as those in the three previous studies had been resting at least half an hour before the blood sample was taken. It has been re- ported that intramuscular drug injections and muscle trauma (e.g., biopsy, surgery, fractures, D 8 C, etc.) may also produce transient elevations of CPK. These factors were not taken into account in the control group, many of whom were hospitalized patients susceptible to intramuscular in- jections and/or muscle trauma of the kinds mentioned. How- ever, all age groups wOuld be equally "at risk" for the af- fects of injection and trauma (each age group contained ap— proximately the same number of hospitalized subjects). The graph of the distribution of CPK values among the control subjects (dotted line in Figure 8) shows that only about 2 percent of them have CPK values over two stan- dard deviations above the mean. Values at this point and 73 over were taken as elevated among the carrier females. Ap- proximately 27 percent of known carriers were below this limit. Thus the finding of a high value in a possible car- rier indicates with a considerable degree of certainty that the person is a carrier while a low value does not so cer- tainly exclude her from the carrier state. Many investigators have noted that the elevations of CPK levels among carrier females vary to a considerable degree. As can be seen from the graph in Figure 8, the elevation in the group of carrier sisters varies from the second standard deviation above the mean of the control group to the eighty-second standard deviation above that mean. Very few attempts have been made to relate this wide variation of CPK levels in carriers to factors such as age or the degree of histopathological or electrophysiological changes as has been done with the affected children them- selves. Figures 10, 11, and 12 and Table 5 show the re- sults of correlating the wide variation of CPK levels seen in the group of carrier sisters with the factor of age. The results were somewhat obscured by six very high values which occurred at no particular age or age group and there- fore increased the standard deviation. These values were significantly enough "unlike" the others to make it statis- tically valid to discard them in the analysis. The statis- tical analysis of the data (done separately for each labo- ratory's data) revealed no significant age distribution 74 pattern of CPK levels. It is difficult to compare this re- sult with results of other investigators because none has analyzed the CPK activities in carrier girls between the ages of 0 and 22 for an age correlation as was done in the present study. A 1966 study conducted by Dreyfus gg g1. concluded, from a comparison of two groups of women under and over the age of forty, that the percentage of low CPK activities is greater in the older mothers (see Figure 6). It is difficult to correlate this finding with the present results since each study was concerned with a different population; however, it is entirely possible that even though no diminution in the level of CPK activity among the carriers from ages 0-22 was detected, there may be a dimi- nution in carriers older than 22. In a study conducted by Walton and Pennington (1966) they presented in graphic form the CPK determinations of sixteen known or probable carriers of the gene for severe type Duchenne muscular dystrophy as a function of age (Fig- ure 7). They failed to demonstrate any significant age- dependent difference in the CPK levels among these people. Once again it is difficult to compare this conclusion with the present findings because all of the carriers in their small series were between the ages of thirty and eighty. A thorough search of the literature revealed that other studies have listed the ages and CPK values of their possible carriers using one of the same methods of enzyme 75 determinations as was used in the present study or one in which the mean and standard deviation of normal persons is given. Table 11 summarizes these findings. Figure 15 shows a scattergram of all the elevated CPK values among these groups of possible carriers as a function of age. As in the previous data the results here are obscured by very high values which apparently can occur at any age. However, by ignoring these very high values it will be seen that there is no suggestion from this diagram of any pro- gressive fall in the level of CPK in the older carriers. This finding is compatible with the previous results. While no correlation was found between mean CPK ele- vation and age of the carrier, Table 7 indicates that there may indeed be some relationship between CPK level and age. All possible carrier sisters were divided into six groups on the basis of age and the percentage in each group who had elevated CPK levels was determined. The table shows that 56 percent of all possible carrier sisters aged 4 years or younger had elevated CPK levels; this percentage keeps drop- ping until only 21 percent of all possible sisters over 17 years had elevated CPK values. Thus the detection rate for carriers is significantly higher in infancy and young child- hood, suggesting the possibility that early age is a factor in elevating the serum CPK (but not to any specific level). This data and the results of Table 5 and Figures 10, 11 and 76 Table 11. Ages and CPK values of possible carriers reported by various authors. Age of CPK Standard deviations Author possible carrier value above mean of controls Pearce §£.§l°* 33 6.7 7 ' ‘ 3 4.9 4 6 5.7 5 5 3.9 2 33 4.4 3 24 3.6 2 17 2.2 1 31 5.6 5 32 1.9 Mean 27 1.8 Mean 26 99.0 120 27 26.0 30 15 45.0 55 32 5.3 4 17 11.0 12 6 2.2 l 10 2.2 l 26 1.9 Mean 14 31.0 37 2 2.1 Mean 35 2.0 Mean 36 3.4 2 34 2.5 l 34 33.3 40 36 4.8 4 36 1.9 Mean 34 2.2 1 40 20.5 24 37 2.1 Mean 38 2.7 l 30 60.0 74 42 3.4 2 47 0.9 1 Below Mean 36 0.9 1 Below Mean 16 11.0 12 Ionasescu et a1.* “_ __ 6 7.45 2 16 3.17 Mean 18 32.25 l6 18 1.20 1 Below Mean 27 1.87 1/2 Below Mean 40 28.60 14 a Possible carriers defined here as female sibs of an affected male and women with one affected son and no other affected male relatives. 77 34_ (55)”‘ (120)“; (37)" (40): 4 a. (74) DSZV o 5.. cm3o. H C 328- 4.) 8 026’ o 1824~ ' LH 022. : U>820— a>E 3o «3:18L >+J Wild 0 Us 0314. 0 U) 512+ o o u-l ‘5 ".410? > o 'o 8- 'E o to 6b . a m + ' : 2. : 5 0-4 5-8 9-12 13-16 17-20 21< Age (years) Figure 15. CPK values among possible carriers as a function of age. 78 10, 11 and 12 support the hypothesis that as carrier fe- males grow older, a certain percentage of them (probably those whose CPK levels were only slightly elevated in young childhood) fall back into the normal range, while other carriers (probably those whose CPK levels were con- siderably elevated in young childhood) remain elevated in adulthood so that no difference in the mean CPK levels of elevated carriers of different ages is detected. A dif- ference should, therefore, be detected in the overall CPK means of all possible carrier sisters grouped according to age. As shown in Table 8, there is a decrease in mean CPK level among possible carrier sisters as they age. Two other studies gave results which SUpport these findings and the proposed hypothesis. The first, reported by Milhorat and Goldstone (1965) found that in their group of younger possible carrier sisters 14 out of 20 or 67 percent had elevated serum CPK levels, whereas only 2 out of 12 or 17 percent of the subjects older than fourteen had elevated serum enzyme activity. They conclude that the high incidence of elevated CPK levels in these young subjects as contrasted to the low incidence in the older ones, suggests the possibility that early age is a factor in elevating the serum CPK. The second study whose find- ings correspond to those in the present report was pub- lished in 1971 by Moser g3 g1. Out of 44 possible carriers of Duchenne muscular dystrophy, 60 percent of those below 79 twenty years of age were identified as carriers by elevated CPK values, while only 45 percent of the older possible carriers had elevated serum CPK activities. They suggest that the detection rate for heterozygosity might be higher in infancy and childhood. The ideal way to determine what affect, if any, age has upon the CPK levels of carriers would be to follow the serum CPK activities in the same persons for a long time. As shown in Figure 13, information was obtained on nine possible carrier females who had been tested at least twice over a period of years. While five of the nine showed de- creased CPK values as their age increased, the remaining four showed a conquing variety of results. A point made clear by graphs G and H, and one which could well account for all the varying results obtained from the many investi- gators working with CPK determinations, is that there is significant individual variations in CPK levels from day to day. The girl whose CPK levels are plotted in graph G had been measured twice at the ages of three and four, each time a few months apart. At the age of three her CPK ele— vation varied from the third to the fourth standard devia- tion above the mean of the control group while at four years of age the elevation varied from the fourth to the seventh standard deviation above the control mean. In graph H the carrier female is seen to have extremely high CPK elevations at all ages tested; however even at these extreme levels it 80 can be seen that the elevation varies from the fiftieth to the sixtyvfifth standard deviation above the control mean at the age of ten years. These findings question the va- lidity of any conclusions based on single CPK determina- tions. Although it was impossible to obtain multiple CPK determinations from the females in the present study, most of whom were tested years ago, any future study should cor— rect for this individual variation by using the average of at least three sequential CPK levels determined under iden- tical conditions. It is evident that further investiga- tions of the type shown in Figure 13 are needed. The fact that five of the nine carriers showed decreased CPK values over a period of years supports the idea that in certain carriers the CPK level drops into the normal range as a function of age while in other carriers it remains elevated over a period of years. In three individuals (B, C, and F) the CPK values actually did fall into the normal range. It may also be significant that in three of the five graphs showing decreasing CPK values with age (C, F, and H) the decrease takes place around the time of puberty (9-16 years). This observation supports the finding in Table 7 that there is a higher incidence of elevated CPK values in possible carriers under the age of nine. The wide variation of CPK elevations seen among carrier women bears some similarity to the varying degrees of aminoaciduria seen in the heterozygotes of cystinuria. 81 It is now known that there are at least three distinct types of cystinuria (probably allelic) which differ in the degree to which heterozygotes are affected. Families of cystinuria Type I patients have no abnormal urinary amino acid excre- tion. Type II and Type III heterozygous individuals excrete excessive amounts of cystine and the dibasic amino acids in their urine; however, the quantities excreted are consis- tently higher in the Type II heterozygotes. Type II and Type III cystinurias are said to be incompletely recessive. One wonders whether there may be, in the same manner, two or more types of severe, early-onset Duchenne dystrophy in which the carrier females differ as to degree of pathology and thus degree of serum CPK elevations. Such a finding would explain not only the wide variation of CPK elevations seen among carriers but also the abnormal clinical and his- tologic findings found in certain carriers. To explore such a possibility, the data presented in this report was reexam- ined with reference to family studies. Seven families were found in which two or more female siblings had been identi- fied as carriers by elevations in their CPK levels. Each family was identified by a symbol and the CPK level of each carrier was then graphed as a function of age to determine any family correlation (Figure 14). From the graph it will be seen that there is no suggestion of any relationship be- tween level of CPK elevation and family membership. Statis- tical analysis revealed no significant sister—sister 82 correlation. Similarly, an attempt was made to relate the variability of serum CPK elevations in carrier girls to the CPK level in their mothers (Table 8). It was thought that perhaps extremely high levels in certain carrier daughters might be related to high levels in their mothers, support- ing the idea of different types of the disease which are evident only in the degree to which heterozygotes are af- fected. However, in only one of nine cases with a daughter demonstrating a CPK elevation of over 10 standard deviations above the control mean, did the mother also show such an ex- tremely high CPK level. Statistical analysis confirmed the fact no correlation exists between mother-daughter CPK ele- vations. A search of the literature revealed that no other studies of this nature have been done in the case of muscu- lar dystrophy. Further investigation using a larger number of families seems warranted. Another theory has been more commonly evoked to ex- plain the wide variation in biochemical as well as histo- logical and clinical findings in female carriers of the gene for Duchenne type muscular dystrophy. Until 1960 when Mary Lyon first proposed her theory of mosaicism of the X chromo- some there was no reasonable explanation for female carriers of a sex-linked recessive disease who showed certain abnor- malities of the disease. According to classical genetic theory, a female carrier for an X-linked recessive disease such as Duchenne muscular dystrophy should show no evidence 83 of the disease because she would have a dominant gene for normal musculature on her other X chromosome. However, to explain the mottled coats of female mammals heterozygous for X-linked genes for coat color, Lyon (1961, 1962) sug- gested that in a proportion of cells in the females one of the X chromosomes is inactivated early in development. Either of the X chromosomes in a cell might be inactivated, and all its descendant cells would then carry the same pat- tern. Autonomous genes could, according to this hypothesis, produce a mosaic effect in the heterozygote. This hypothe- sis has been applied to humans and is strongly supported by the study of glucose-6-phosphate dehydrogenase in the red cells of women heterozygous for this enzyme deficiency (Beutler gg’g1., 1962; Dreyfus gg gl., 1963). Since the Duchenne type of muscular dystrophy is sex-linked, the hy- pothesis of mosaicism of the muscle cells has been invoked by many investigators. It was developed by Pearson (1963) and was supported by biochemical (Emery, 1964a) and histo- logical (Pearson, 1963; Dubowitz, 1963) observations. According to the applied Lyon hypothesis, after fer- tilization of an ovum carrying the dystrophy gene by a sperm with a normal X chromosome, one X chromosome is inactivated early in embryonic life. This leaves the normal X active in some cells and the Xd (X chromosome carrying the gene for Duchenne muscular dystrophy) active in others. As the indi- vidual ages, atrophy presumably proceeds in the Xd-carrying 84 cells just as in the male sibling, but the cells in which the normal paternal X is active develop normally. If this explanation is the correct one, then one might anticipate that greater biochemical abnormalities would be seen in younger carriers and these would become less striking as the age increased. This hypothesis explains the occurrence of clinical signs of muscular dystrophy in some female carriers by assum- ing that the proportion of their cells in which the active X chromosome carries the mutant gene is greater in number than in the majority of carriers. Furthermore, the variabil- ity of the magnitude of the changes in serum CPK activity and the patchy changes observed in biopsy studies are expli- cable on the basis of the Lyon hypothesis. The female car- rier, heterozygous for this gene, will exhibit variations in her tissues dependent upon the relative proportions of active normal X chromosomes and active Xd chromosomes ex- pressed. A carrier with a preponderance of normal cells may exhibit only a slight elevation of serum CPK activity while a carrier with a proponderance of mutant X chromosome cells may exhibit a high serum CPK activity, abnormal muscle bi- cpsy findings, and even clinical evidence of muscular weak- ness. This offers an explanation for the negative results in trying to detect greater biochemical abnormalities (i.e., higher serum CPK levels) in younger carriers. Perhaps the overwhelming factor in determining variability in serum CPK 85 activity is the relative proportion of active versus mutant- bearing X chromosomes expressed. The ideal study to solve the problem would be to test individual carriers over a per- iod of years to detect any decreases in their CPK levels. As yetthere is no direct evidence that the Lyon hy- pothesis is even applicable in this case. The indirect evi- dence now available does not allow definitive conclusions for two reasons. Firstly, some parts of the X chromosome do not seem to display mosaicism (e.g., that part which bears the Xg blood group locus). Secondly, muscle tissue is peculiar in that each cell contains many nuclei. It is not known what the consequences would be of both normal and abnormal genes in different nuclei in the same fiber. The hypothesis of mosaicism is far from being proven and this problem awaits further study. LI ST OF REFERENCES LIST OF REFERENCES Barnett, Henry L. (ed.), Pediatrics, New York, Meredith Corporation, 1972. Bhahd, William H., Cassen, Benedict, and Lederer, Marrianne, "Decreased Body Potassium In Nondystro- phic Relatives of Patients With Muscular DystrOphy: A Biochemical Trait," New Eng. J. Med., 270:197-198, 1964. Blyth, Helen and Pugh, R. J., "Muscular Dystrophy In Childhood. 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Zierler, Kenneth, "Muscle Membrane as a Dynamic Struc- ture and Its Permeability to Aldolase," Ann. N.Y. Acad. Sci., 75:227, 1958. 72. Zundel, Wayne and Tyler, Frank, "The Muscular Dystro- phies," New Eng. J. Med., 273:537-543, 1965. APPENDIX APPENDIX STATISTICAL PROCEDURES USED ON THE DATA 1. Best Estimate of the Detection Efficiency Based on the Method of Maximum Likelihood L likelihood of observing what was actually observed (f)25(l-f)12(1/2f)25(1-1/2f)32, where "f" is the L detection efficiency based on the results of carrier detectlon among known carrier mothers and sisters w1th known carrier mothers 1nL = 251n(f) + 121n(1-f) + 251n(1/2f) + 321n(1-1/2f) 2 d 1nL = s _ 12 + 25 _ 16 d If) f‘ I-f f" I-I72f a £9 - .3 - _19_ f l-f 1-1/2f 50(1—1/2£)(1.£) - 12£(l;1/2£) - 16f(1-f) f(1-f)(1—1/2f) so - 75(f) + 25(t3) a 12(f) + o(£2) . 16(f) + 16(f2) f(l-f)(1-1/2f) 50 — 103(f) + 47(5314 = o f(l-f)(l-1/2f) f s 103 t JQlos)z - 4(50)(47) 94 ruh II 0.725 93 94 A Chi-Square Test was then used to detect any significant difference between the results observed in known carrier mothers and sisters with known carrier mothers and the ex- pected based on this estimate: Carriers Non-Carriers l 12 Known Carrier Mothers 25 12 Sisters with Known 21 22 Carrier Mothers 25 32 Expected11 = 26.8 Expected12 = 10.2 EXpected21 = 20.7 Expected22 = 36.3 2 (Observedij Expectedij) Expectedij (25 - 26.8)2 + (12 - 10.2)2 + (25 - 20.7)2 + (32 - 36.3)2 26.8 10.2 20.7 36.3 1.8 2 x 0.05,1 = 3.841 Therefore, there is no significant difference between the observed and the expected based on the estimate of (f) = 0.725 II. Effect 95 of Age on the CPK Values of the Control Subjects Scheffe's Test, a method for judging all contrasts in the analysis of variance, was used to determine any sig- nificant difference in the mean CPK values in controls 12 years old and younger as compared with controls 13 years and older. q=(h+iz+fi)-O4+fi+ip = (21.4 + 25.4 + 26.1) - (15.8 + 17.0 + 17.6) = 72.9 - 50.4 = 22.5 v(q) = £(ci/ni) ,Mean Square Error = (1/18 + 1/10 + 1/9 + 1/10 + 1/4 + 1/19)MSError The Mean Square Error was computed as follows: 2 (0-4 age group), compute [Zyi - 53.)...) = SS1 18 2 (5-8 age group), compute 22y2 - (ZY) = SS2 2 10 " = SS6 ZSSi = 6,057.90 ZSSi = SSError, MSError = (SSError/64) = 94.65 95% confidence interval: q t /(t-l)f v(q) Where "t" is the total number 0°05’5°64 ’ Of groups (9) and f0.05 5 64 = _ 2.37 and v(q) was computed to q 3 75(2.37)(63.4) be 63.4 22.5 + 27.4 Since zero is included, then the difference is not signifi- cant . 96 111. Effect of Age on the CPK Values of the Carrier Sisters Hypothesis: Mean $12 = Mean >12 A. Wisconsin Data (prior to 1970) ylj y2j j=l 3.8 j=l 2.7 i=2 1.9 j=2 4.3 n2 = 3 j=3 4.9 j=3 2.5 = j=4 17.9 n1 7 j=5 1.7 j=6 8.3 j=7 2.9 _ 2 2 _ 2 _ z , 2 551 - zylj - (Zy1.) ss2 - Zij £_:311_ n n 1 2 = 442.71 - 244.85 = 197.86 = 32.03 - 30.08 = 1.95 82 = 851 + SS2 111 + 112 - 2 = 197.86 + 1.95 = 24,93 7 + 3 - 2 t=yl-y2 2 1 l 7% (51 + n2) = 5.91-3.16 _ .82 /' l l 24.98(7 + 3 ) .82 vs. t0.05,8 = 2.31 Not significant 97 B. Wisconsin Data (1970rpresent): Not enough data to analyze C. Minneapolis Data (1963v1966) ylj ‘< N j i=7- 1‘3 UoUoUoUoUo II II II II [I m-hMNt-I NNNm-h o o o o o NONQ‘D :3 II U1 H II U1 MMMNNNchNN \ONmO-FhmION-PVO‘ 2 2 = _ 2 _2-2. 881 Zyij czylj) ss 2Y2; C YzJ) n1 n2 114.36 - 110.09 = 4.27 51.38 - 45.00 = 6.38 2 551 + 352 S a: = 4.27 + 6.38 = 0.76 11 + 5 - 2 t = 91 ' 92 2 1 1 V/S (— + —-) n1 D2 = 3.16 - 3.00 = 0.34 1 .34 vs. t0.05, 14 2 2.14 Not significant 98 D. Minneapolis Data (1967-present) SS Y1- i=1 5‘2 5 j=3 i=4 5‘5 13 13,850 -10,396.8 J 26 95 32 25 50 2 XY-- - (2y .)2 11 n1 Y2- .__;L___ j=l 68 j-z 29 j=3 107 n2 = 5 j=4 37 j=5 159 2 2 = z _ SSZ yzj (Zyzj) n2 = 3,453 2 = 43,564 -32,000 = 11,564 a 551 + 532 n1 + nz - 2 = 3,453.2 + 11,564 = 1877.1 5 + 5 — 2 3.7-21-92 2 l l S( *— v/r Hi n2) = 45.6 - 80.0 = 1.25 ‘ 1 1 1877.1 — + — / c5 5) 1.25 vs. t0.05,8 ~ 2.31 Not significant 99 E. Grand Rapids Data (1966-1969) ylj ij j=l ll j=1 15 j=2 23 j=2 29 j=3 26 j=3 15 = j=4 19 j=4 25 n = 6 n1 7 j=5 14 j=5 10 2 j=6 43 j=6 12 a 2 _ 2 _ 2 z . 2 $51 Zylj (Zylj) ss2 - Zyzj - C YzJ) 11 n1 2 = 4,021 _ 3,344 a 677.00 - 2,160 - 1,872.6 = 287.40 S2 = SS1 + SS2 111 + n2 '- 2 = 677.00 + 287.40 = 87.67 7 + 6 - 2 t = X1 ' 92 2 1 1 S C— + — ) / .1 .2 = 21.8 - 17.6 = .808 0/87.67( .803 VS- 1:0.0591 2 2.20 Not significant 1 +6) \IIH 100 IV. Percentage of Elevated CPK Levels Among Possible Car— rier Sisters by Age A. A Chi-Square test of a 2 X 6 contingency table was used to detect any significant deviation of the data in Table 7 from the expected Elgvated Not Elevated 0-4 11 12 14 ,//// 11 25 L 5-8 21 22 12 24 36 31 32 9’12 ///l 10 ,////1 28 38 41 42 13‘16 ll 30 41 51 52 17-20 7 26 33 _4 6 62 21 6 over /}/7 4 15 19 58 134 192 Expected 11 = 25 58 = 7.55 192 Expected12 = (25311342: 17.45 192 Expected21 = (361(58) = 10.8 192 Expected = (36)(134) = 25.2 22 192 Expected31 = (33 53 = 11.5 Expected32 = 38 134 = 26.5 = (41)1§8) = Expected41 192 12.4 = 41 134 = Expected42 ( %é2 J 28.6 101 33 58) _ Expected51 - - 9.96 Expected52 = (33 134) = 23.0 Expected = (19 58) = 5.7 61 Ex ected (1911134) = 13.2 p 62 192 (Observed.. - Expected..)2 q = x 13 13 Expected.. 13 2 2 2 2 = (l4-7.55) + (ll-17.45) + (12-10.8) + (24—25.2) 7.557 17.45 10.8 25.2 (10 ll 5)2 + 2 2 2 2 + - . ( 8-26.5) + (ll-12.4), + (30-28.6) 11.5 26.5 12.4 28.6 (7-9.96)2 + (26-23.0)2 + (457)2 + (ls-13.2)2 9.96 23.0 5.7 13.2 = 11.12 2 X 0.05,5 = 11.07 Therefore, a significant difference in the observed data and the expected data does exist 102 Chiquuare test of a 2 X 2 contingency table: B. Hypothesis: The percentage of elevated CPK levels among the 0-4 aged sisters is the same as that of the other age groups Elevated Not Elevated ll 12 0 - 4 14 ll 25 21 2 >4 44 123 167 58 134 192 _ 25 58 = Expected11 - (T92-)(I§2')192 7.55 Expected12 = (253(134) = 17.45 192 Expected21 = 167 53 = 50.45 Expected22 = 167 134 = 116.55 _ 2 q = (Observedij Expectedij) Expectedij = (14—7.55)2 + (ll-17.45)2 + (44-so.45)2 + (123-116.55)2 7.55 17.45 50.45 116.55 = 9.08 x2 = 3 841 0.05,1 ° x2 = 6 635 o.01,1 ° Therefore, a highl significant percentage of elevated CPK levels exists in c ildren under the age of five C. Hypothesis: 103 The percentage of elevated CPK levels among the 5—8 aged girls is the same as that of the older age groups Elevated Not Elevated 11 1 5-8 ‘12 24 36 21 2 >8 32 99 131 44 123 167 a 36 44 = Expected11 9.5 a 36 123 = Expected12 £-—%%fir—JL 26.5 Expected = (13;)(45) = 34.5 21 167 Ex ected = (131)(123)= 96.5 p 22 167 q = 2 (ObservediJ - Expectedij)2 Expectedij r 2 2 2 2 = (12-9.5) + (24-26.S) + (32-34.5) + (99-96.5) 9.5 26.5 34.5 96.5 = 1.14 x2 = 3.841 Therefore, the hypothesis holds true and there is no signifi- cant difference in the percentage of elevated CPK levels in the 5-8 age group. 104 V. Correlation of CPK Values Among Family Members A. Sister—Sister Correlation Spearman's Coefficient of Rank Correlation test was used on the data in Figure 14. Each younger sister and each older sister were assigned a number corresponding to where her CPK level ranked among all the others in her group. For those sisters with the same CPK values and thus identical ranking, averages were used. YD or the difference in the rank values between each sister-sister pair was then determined: Family Younger Older YD Sister's Rank Sister's Rank A 12 4 8 B 12 2 10 12 5.5 6.5 12 12.5 - .5 3.5 5.5 -2.0 3.5 12.5 -9.0 8 12.5 -4.5 C S 9 -4.0 D 1 3 -2.0 E 12 12.5 - .5 F 6.5 l 5.5 6.5 9 -2.5 2 9 -7.0 G 9 7 -2.0 6(n 2 s _ _ 2 Y.) R12 1 D1 9 highest correlation = l ‘_—7F_‘_' lowest correlation = 0 n(n -l) 1; YD]? = 421.50, 61421.59) ... .93 1 14(196-1) Rf, = 1 - .93 = .07 With this value an approximate test was used to test the hypothesis that the correlation is zero: 105 t = S R 12 , t g .07 t /l—(R§2)2 J1-(.07)2 (n—2) 12 t0.05,12 = 1'78 .24 < 1.78, so accept the hypothesis of no significant correlation between sister-sister CPK values. .24 106 B. Mother-Daughter Correlation Spearman's Coefficient of Rank Correlation test was used on the data in Table 8. Each daughter and mother were assigned a number corresponding to where her CPK level ranked among all the others in her group (e.g., 1st, 2nd, 11th, etc.). For those with the same CPK values and thus identical rank- ing, averages were used. YD or the difference in the rank values between each mother-daughter pair was then determined: Family No. Daughter's Rank Mother's Rank YD 1 24 37 ~13 2 15 4.5 10.5 3 17 32 -15 4 33 5 32 1 5 5 1 26 -25 6 3 4.5 - l 5 7 10 26 -16 8 33 5 32 l 5 14 32 -18 4 32 -28 33 5 32 1.5 9 24 13 ll 9 13 - 4 10 6 26 -20 2 26 -24 11 19.5 26 - 6 5 12 16 6 10 13 11 18 5 - 7 5 33.5 18 5 15 14 33.5 9 24 5 15 19.5 2 5 l7 16 24 18 5 5 5 17 33.5 7 5 26 0 18 33.5 18 5 15 19 33.5 11 22 5 20 24 10 14 21 8 2.5 5.5 22 19.5 18.5 1.0 23 27.5 37 - 9.5 24 19.5 23 - 3.5 25 24 18.5 5.5 26 33.5 18.5 15 27 5 l3 - 8 28 27.5 7 5 20 29 33.5 37 - 3 5 30 13 32 -19 31 12 1 ll 107 n 2 s _ 6 Z Y .) R12 ’ 1 ” C D1 , highest correlation = 1 2 lowest correlation = 0 n(n . 1) 38 2 12 YDi - 8041 8041§6) _ .87 38(38 - 1) R5 = 1 - .87 = .13 With this value an approximate test was used to test the hypothesis that the correlation is zero: t = 5 R12 5 2 1'(R12) (n—Z) t = .13 = .78 v/(l-.0169) 36 t0.os,36 = 1'69 .78 < 1.69, so accept the hypothesis of no significant correlation between mother-daughter CPK values. “A 111111111111105 ”'1me