Carrier detection and genetic counselling in Duchenne muscular dystrophy: a follow-up study ELAINE M. HUTTON,* PH D MARGARET W. THOMPSON,t PH D

Assay of serum creatine kinase activity is useful in the detection of carriers of the X-linked gene for Duchenne muscular dystrophy (DMD). For genetic counselling this assay has been used in conjunction with pedigree analysis to improve estimates of the risk that a female relative of a DMD patient is a carrier. To measure the impact of the program, follow-up information was obtained from women who had received genetic counselling for DMD. Their responses showed that the risk of producing an affected son had been a major factor in their attitude toward family planning, and their reproductive performance correlated inversely with their genetic risk. The decision by the majority of proven carriers to prevent the birth of further male offspring was reflected in a recent decline in the frequency of a known family history of DMD among newly ascertained cases. Le test d'activlt6 de Ia cr6atine kinase serique est utile pour detecter les porteurs du gene situe sur le chromosome X responsable de Ia dystrophie musculaire de Duchenne (DMD). Pour fin d'orientation genetique ce test a ete associe a une analyse des antecedents familiaux dans le but d'ameliorer l'appreciation du risque qu'une parente d'un patient atteint de DMD soit porteur de ce gene. Afin de mesurer l'influence de ce programme, on a recueilli de l'information sur le devenir des femmes qui avaient requ une orientation genetique en regard de Ia DMD. Leurs reponses ont montre que le risque de donner naissance a un fils atteint de cette maladie avait constitue un facteur determinant dans leur attitude face au contr6le des naissances, et leur fecondite a ete inversement proportionnelle au risque genetique encouru. La decision de Ia majorite des porteurs demontres de prevenir Ia naissance d'autres enfants mAles s'est refletee dans une baisse recente de Ia fr6quence d'une From the departmellt of genetics, Hospital for Sick Children, Toronto 6Re.arch associate, department of genetics, Hospital for Sick Children tSenior staff geneticist, Hospital for Sick Children, and professor of medical genetics, faculty of medicine, University of Toronto Reprint requests to: Dr. EM. Hutton, Department of genetics, Hospital for Sick Children, 555 University Ave., Toronto, ON M5G 1X8

histoire familiale connue de DMD parmi les cas nouvellement constates. Duchenne muscular dystrophy (DMD) is a severe, X-linked, genetically lethal condition that affects boys, usually before the age of 5. The disease is characterized by pseudohypertrophy of the calf muscles and weakness of mainly the proximal groups of muscles. The muscular disability progresses steadily, leading to inability to walk within 10 years of onset.' In X-linked recessive inheritance any son of a female carrier has a 50% chance of being affected and any daughter has a 50% chance of being a carrier. Thus the risk that a carrier will produce an affected male in a given pregnancy is 25 %. However, when an X-linked disorder is a "genetic lethal" - that is, when affected males do not reproduce - not all mothers of affected sons are heterozygous. Theoretically, in one third of all cases of DMD and other X-linked lethal conditions the disease results from a new mutation in an ovum of a genetically normal mother, and female relatives in such "new mutant" cases do not have an increased risk of producing affected sons. Thus tests for carrier detection can be valuable to female relatives of DMD patients in their reproductive planning. Genetic counselling has been available to DMD families for over 20 years through the department of genetics at the Hospital for Sick Children, Toronto, but the enzyme assay used for the detection of carriers has been available only since 1964. In this paper we examine the impact of genetic counselling on the reproductive decisions of families during the 10-year period (196574) in which carrier testing has been part of the genetic counselling program. Methods Carrier detection by, creatine kinase assay The measurement of serum creatine kinase (CK) activity is the simplest and most specific test for carrier identification. Extremely high values of this enzyme are found in the serum of affected males, and less strikingly elevated values in about 70% of known carrlers. 2,3 The major problem in applying the test is that almost one third of carriers have enzyme activity within

the normal range - that is, less than two standard deviations above the control mean, expressed as logarithms. We have used both the Hughes method,4 in which the creatine formed is converted into a coloured complex by means of the a-naphthol diacetyl reaction, and the modified Rosalki method,5 in which CK is assayed spectrophotometrically by coupled enzymatic reactions. Currently we use the Calbiochem* kit for performing the modified Rosalki method. For identification of carriers two blood samples (or preferably three) are obtained from the subject at 1-month intervals. Both age and previous exercise may influence CK activity.6-9 In our assessment of carrier status of female relatives of affected individuals, CK activity is compared with the range obtained for "normal" controls of a similar age. If possible, assessment is postponed until the subject is at least 5 years old. To avoid the effect of exercise on enzyme activity, both normal controls and female relatives to be tested are asked to avoid strenuous exercise for 48 hours before blood samples are obtained. A female relative is designated as a carrier in any of the following situations: if her son as well as some other maternal male relative is affected; if she has passed the gene to at least two of her children (that is, if she has two affected sons or an affected son and a carrier daughter); or if her blood sample shows elevated CK activity on more than one occasion. In a few women the enzyme value is borderline or is elevated on only one occasion; their carrier status is designated as "doubtful". No precise risk figure can be calculated for these women bui we consider them likely to be carriers. A female relative with CK activity consistently within the normal range is designated as a "possible" carrier. The probability that such a woman is a carrier is calculated by Bayesian statistical methods on the basis of the pedigree and the CK values of her female relatives.'0 Bayesian analysis, a method of assessment of the relative probability of each of two alternative possibilities (that is, that the subject is a carrier or is not a carrier), is especially useful for assessing genetic risks in severe Xlinked recessive conditions such as * 10933 N Torrey Pines Rd., La Jolla, CA 92037, USA

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DMD in which some of the mothers One commonly used measure is in are not carriers. The risk is interpreted terms of subsequent reproductive decito the possible carrier in terms of her sions and action. In the present study risk of producing an affected son. The we classified women into four caterisk is classified as low if less than 5% gories (high, doubtful, moderate and and moderate if above S % but below low) with respect to their risk of 25%. producing an affected son, then examined the effect of carrier detection and Genetic counselling counselling on reproductive attitudes When the family of a patient with and behaviour among women in the DMD is referred for genetic investiga- different risk groups. Questionnaires were mailed in 1973 tion and counselling, a detailed family history is obtained and the mother is to all female relatives of childbearing requested to provide a blood sample on age whose carrier status had been deat least two occasions for enzyme de- termined during our research. Each termination. She is asked to inform her subject was asked what decision had maternal female relatives about their been made about family planning 1 potential risk and to encourage them year after the genetic counselling interto have enzyme determinations and view and whether further pregnancies occurred. genetic counselling. To determine whether the carrier deIt is the responsibility of the genetic counsellor to determine the recurrence tection and genetic counselling program risk and interpret the risk to the in- has had an effect on the incidence of dividual or family. Parents or potential new cases with a known family history, parents must understand fully the prog- information was also recorded for all nosis in order to make rational deci- typical cases of DMD ascertained at sions about reproductive planning.1' the Hospital for Sick Children in the Some couples may consider surgical 10-year period 1965-74. sterilization. Others may prefer to use a reliable method of contraception in the hope that an accurate prenatal test Results may eventually be developed, or may Follow-up of female relatives decide to have future pregnancies monSeventy-six percent of the women itored by amniocentesis for determination of fetal sex, with the possibility (256 of 336) responded to questions of termination of pregnancy if the fetus about their understanding of genetic information and family planning deciis found to be male. If a possible carrier decides to pro- sions. From the 256 respondents a ceed with a pregnancy, she is encour- group of 122 was selected for this aged to have her sons tested by estima- study, the remainder being excluded tion of CK activity approximately 1 because of marital status (single, sepmonth after birth. Although detection arated or divorced), because further of an affected son at an early age may reproduction was impossible owing to cause anguish for the parents, they are infertility or surgical sterilization of one alerted promptly to the fact that the of the spouses, or because they indicated that their family size was sufmother is a carrier. ficient at the time they had participated Follow-up in the research program. The family planning decisions of the A follow-up interview is desirable because new developments in carrier 105 women who responded to a quesdetection and prenatal diagnosis may tion about family planning are shown enable couples to alter their decision in Table I. Although a few women inabout family planning. They may also dicated that their decision to have no want to reassess the risk if their own attitude or family situation changes. Sisters of patients often require further information when they marry and they may wish to have their husbands fully informed about the situation. Our follow-up of the immediate family is usually carried out during the twicemonthly muscular dystrophy clinic; however, information about more distant family members must be obtained by special contact. Measurement of the impact of genetic counselling Methods for evaluation of genetic counselling are still under development. 750 CMA JOURNAL/OCTOBER 23, 1976/VOL. 115

future offspring was made because of such reasons as marital incompatibility and ill health, the majority based their decision on their genetic risk. In the calculation of the percentage of women deterred by their genetic risk, only female relatives of affected individuals who desired to limit their family because of fear of producing affected sons or carrier daughters or both were included. Of the 34 carriers who decided to have no future offspring 23 were concerned about the risk of having either affected sons or carrier daughters, 10 were concerned about the risk of having affected sons, and 1 was concerned only about the risk of having carrier daughters. The majority of proven carriers (81%) did not plan to have further offspring and in 55% of cases either the carrier or her husband obtained surgical sterilization without producing another liveborn child. Among the women with a low risk of producing an affected child, only one indicated that she was deterred by her genetic risk and so obtained surgical sterilization. In this family the affected brother was an isolated case, and both the subject and her sister had normal enzyme values. Their mother, however, refused to be tested, and this probably increased the anxiety of her daughter. The reproductive performance of these women, in terms of pregnancies initiated after the first carrier detection test, is recorded in Table II. (Women married less than 1 year were omitted.) Some women indicated that they were pregnant or attempting pregnancy; their numbers were included for determining the percentage that appeared to be undeterred by their genetic risk. Fourteen of 45 carriers had further pregnancies, and 8 of these highrisk women did not obtain amniocentesis or have the pregnancy terminated. Their pregnancies produced only two liveborn males, one of whom was affected. Of five carriers who obtained therapeutic abortions, four did so without prior determination of fetal sex by

amniocentesis, and one had two terminations in two consecutive pregnancies after the fetus was found to be male. Of the women with moderate risk, 18 of 33 became pregnant, only 2 chose therapeutic abortion and none of the 12 liveborn males was affected. Of the women with a low risk, 22 of 29 became pregnant (or indicated they were attempting pregnancy) and of their 12 liveborn males 1 was affected. Incidence of new cases In the 10-year interval 1965-74, 135 cases of DMD in 115 kindreds were ascertained (Table III). The annual number of patients remained constant, although a decline might have been expected because of the decrease in the birth rate and because of the failure of most carriers to reproduce. The constant rate may be due to increasing awareness of the facilities of the muscular dystrophy clinic for accurate diagnosis and supportive therapy. Since the majority of carriers indicated that although they desired more children they had taken steps to prevent pregnancies, there should have been a resulting decrease in the num-

ber of cases with a known family history of DMD. The percentage of new cases in which the mother indicated she was aware at the onset of her pregnancy of a maternal male relative affected with DMD is shown in Table III. Since there is an average interval of 6 years between birth and ascertainment of DMD a decrease in the incidence of such new cases would not have been expected before 1970. From 1965 to 1969 a family history of DMD was known to the mother in 20 of 64 cases (31%) compared with 12 of 71 cases (17%) in 1970-74; during 197374 less than 10% of the families knew of another affected male in the kindred. In 5 of the 71 cases ascertained since 1970 a family history was already on record. Only two mothers had received carrier detection tests and counselling; one of these was found to be a carrier but decided to take a chance with just one pregnancy, and the other was an undetected carrier. No counselling had been given to the other three mothers; in one case pregnancy had occurred before carrier detection was available, in a second case the mother refused counselling, and in the third case either

the mother was not informed by other family members or perhaps she was discouraged by the travelling distance to Toronto. Discussion With the introduction of the assay of CK activity for carrier detection in 1965 and more efficient and acceptable methods of contraception (including a more permissive attitude to surgical sterilization and termination of pregnancy), most families are able to implement their family planning decisions. In a follow-up of 455 couples who received counselling for a variety, of genetic disorders, Carter and colleagues12 observed that two thirds of parents of patients with diseases that carried high recurrence risks (that is, equal to or greater than 1 in 10) were deterred from further reproduction. Therefore, in a condition with a longterm disability such as DMD, one would expect to find that proven carriers would desire to prevent subsequent pregnancies, or to have any further pregnancies monitored by amniocentesis with the option of selective abortion. In our study 81 % of carriers who desired (more) children indicated they were deterred by the genetic risk, and in more than half the couples either the husband or wife was sterilized without producing further liveborn offspring. Similarly, in a follow-up study of female relatives of individuals with DMD, Emery, Watt and Clack11 found that only 2 of 41 women at high risk of producing an affected son disregarded the risks and intentionally became pregnant, and approximately one half of the women obtained surgical sterilization after genetic counselling. The number of women discouraged by their genetic risk decreased directly as their risk of producing an affected son decreased, and this attitude was confirmed by their reproductive per-

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DaImane. Roche (fi urazepam) Rx summary: Indications Useful in all types of insomnia characterized by difficulty in falling asleep, frequent nocturnal awakenings and/or early morning awakening. Dalmane' can be administered effectively for short-term and intermittent use in patients with recurring insomnia and poor sleeping habits; however, the safety and efficacy of long-term use has not been established. Cont raindicat ions Known hypersensitivity to the drug and, because of lack of sufficient clinical experience, in children under fifteen years of age. Warnings Safety in women who are or may become pregnant has not been established; hence as with all medication, Dalmane should be given only when the potential benefits have been weighed against possible hazard to mother and child. Precautions Use in fhe Elderly: In elderly and debilitated patients, it is recommended that the dosage initially be limited to 15 mg to preclude the development of oversedation, dizziness and/or ataxia reported in some patients. Use in Emotional Disorders: The usual precautions are indicated for severely depressed patients or those in whom there is any evidence of latent depression, particularly the recognition that suicidal tendencies may be present and protective measures may be necessary. Pofontiation of Drug Effects: Since Dalmane has a central nervous system depressant effect, patients should be advised against the simultaneous ingestion of alcohol and other central nervous system depressant drugs during Dalmane therapy. Physical and Psychological Dependence: As with any hypnotic, caution must be exercised in administering to individuals known to be addiction-prone or those whose history suggesfs they may increase the dosage on their own initiative. General: All patients should be cautioned against engaging in activities requiring precision and complete mental alertness such as in operating machinery or driving a motor vehicle shortly after ingesting the drug. Should 'Dalmane be used repeatedly, periodic blood counts and liver and kidney function tests should be performed. The usual precautions should be observed in patients with impaired renal or hepatic function. Patients' reactions will be modified to a varying extent depending on dosage and individual susceptibility. Adverse Effects Dizziness, drowsiness, lightheadedness and ataxia may occur. These adverse effects are particularly common in elderly and debilitated patients. (See Precautions). Severe sedation, lethargy, disorientation, probably indicative of drug intolerance or overdose, have been reported. Dosage Dosage should be individualized for maximal beneficial effects. The usual adult dosage is 30 mg before retiring. In elderly and/or debilitated patients, it is recommended that therapy be initiated with 15 mg until individual responses are determined. Moderate to severe insomnia may require 15 or 30 mg. Supply Dalmane 15, capsules (orange and ivory) 15 mg, 100 and 500. Dalmane' 30, capsules (red and ivory) 30 mg, 100 and 500. Complete prescribing information available on request. *Reg. Trade Mark

Hoffmann-La Roche Limited XIuIIIaIIIj/ Vaudreull, Quebec

formance. Most female relatives of affected individuals (76%) with a low risk (less than 5%) of producing an affected male have attempted one or more pregnancies, whereas a small percentage (18%) of proven carriers appeared to be undeterred by their risk. The fact that their reproductive attitude and performance correlate directly with their risk of producing an affected son indicates that most of the women understood their risk and planned their families accordingly. Factors such as maternal age, number of previous children and religious affiliation may have influenced the decision of those who disregarded their risk; these factors are under study. The attitude of the female relatives in our study is reflected in the low incidence in the past few years of new cases with a family history of DMD. Similarly, Hurse and Kakulas13 have observed that the incidence of DMD in Western Australia has decreased from 21 per 100 000 live male births in 1960-64 to 7 per 100000 in 1965-69. They attribute the decrease to early recognition and improved diagnostic methods, as well as reliable methods of contraception and genetic counselling (personal communication, 1974). On the basis of the recurrence risks for the 122 women in our study, and assuming a minimum of one pregnancy per woman (although many desired more than one), approximately 15 affected males would be expected and only 2 were born. In addition to preventing the birth of some potentially affected males, carrier detection and genetic counselling have enabled many female relatives who might have* been discouraged by Mendelian risks to plan their families with greater confidence. However, there is evidence that our genetic counselling program has failed to reach many of the persons for whom it is designed; 20 of the 32 mothers who were aware of a family history of DMD had never been referred for counselling. Carrier detection tests can be helpful only to families in which an affected individual has already been recognized. Isolated cases of DMD will probably never be completely eliminated because one third of all cases are expected to be produced by a new mutation in an ovum of the mother. Isolated cases of DMD occur for two other reasons. The patient's mother may be a "new mutant" carrier who received the DMD gene from a normal parent; none of her sisters or nieces will be at risk of being carrie.rs but her daughters will have a, 50% risk of carrying the gene and many of these carrier daughters can be detected by testing. Alternatively, the mother in an

752 CMA JOURNAL/OCTOBER 23, 1976/VOL. 115

isolated case may be a carrier of a gene that has been in her family for one or more generations, the male descendants having escaped it purely by chance; in this instance a number of female relatives should be detectable as carriers. The present carrier detection program seems to have been effective in reducing the incidence of DMD in families in which this disease has occurred previously. Unfortunately it can have no effect on the incidence of cases due to a new mutation. Mass screening procedures for carrier females or for prenatal diagnosis of DMD, neither of which is feasible at present, are needed if the large proportion of cases of DMD with no known family history are to be prevented. We are grateful for the cooperation of Dr. E.G. Murphy and the staff of the muscular dystrophy clinic, and for the assistance of Mrs. H. Johnson, Mrs. I. Oss and Mrs. B. Youson of the department of genetics, Hospital for Sick Children. Financial support was provided by the Muscular Dystrophy Association of Canada. References 1. WALTON JN, GARDNER-MEDWIN D: Progressive muscular dystrophy and the myotonic disorders, in Disorders of Voluntary Muscle, 3rd ed, WALTON JN (ed), London, Churchill Livingstone, 1974, p 561 2. THOMPSON MW, MURPHY EG, MCALPKNE PJ: An assessment of the creatine kinase test in the detection of carriers of Duchenne muscular dystrophy. J Pediatr 71: 82, 1967 3. EMERY AEH: The use of serum creatine kinase for detecting carriers of Duchenne muscular dystrophy, in Exploratory Concepts in Muscular Dystrophy and Related Disorders, Intl Congr Ser no 147, New York, Excerpta Medics, 1967, pp 90-7 4. HUGHES BP: A method for the estimation of serum creatine kinase and its use in comparing creatine kinase and aldolase activity in normal and pathological sera. Clin Chim Acta 7: 597, 1962 5. Ros#.uu SB: An improved procedure for serum creatine phosphokinase determination.

I Lab Clin Med 69: 696, 1967 6. ZELLWEGER H, HANSON JW, MARKOWITZ E: Age- and sex-dependent differences of serum enzymes in normal controls, in Muscle Diseases, WALTON JN, CANAL N, SCARLATO J

(eds), Amsterdam, Excerpts Medics, 1970, pp 445-9 7. HILL JG, CHERIAN AG: Percentile estimate of reference values for creatine kinase, aspartate transaminase and alkaline phosphatase in children (abstr). Clin Biochem 6: 325, 1973 8. RICHrERIcH R, RosiN 5, AEBI V, et al: Progressive muscular dystrophy. V. The identification of the carrier state in the Duchenne type by serum creatine kinase determination.

Am / Hum Genet 15: 133, 1963

9. GRIFFITHS PD: Serum levels of ATP: creatine phosphotransferase (creatine kinase). The normal range and effect of muscular

activity. Clin Chim Acta 13: 413, 1966

10. MURPHY EA, MUTALIK GS: The application of Bayesian methods in genetic counselling.

Hum Hered 19: 126, 1969 11. EMERY AEH, WATT MW, CLACK ER: The effects of genetic counselling in Duchenne muscular dystrophy. Clin Genet 3: 147, 1972

12. CARThR CO, FRASER ROBERTS JA, EVANS KA,

et al: Genetic clinic: a follow-up. Lancet 1: 281, 1971 13. HURSE PV, KAKULAS BA: Genetic counselling in inherited muscle diseases in Western Australia, in Ilird International Congress on Muscle Disease, Intl Congr Ser no 334, Excerpts Medics, abstr no 205, 1974, p 88