Acta Neurol. Scandinav. 54, 442-452, 1976
Departments of Pediatrics and Biostatistics, College of hledicine, University of Iowa, Iowa City, Iowa, U.S.A.
DETECTION O F CARRIERS AND GENETIC COUNSELING IN DUCHENNE MUSCULAR DYSTROPHY BY RIBOSOMAL PROTEIN SYNTHESIS VICTORIONASESCU, HANSZELLWEGER and LEONBURMEISTER ABSTRACT The in vitro protein synthesis by polyribosomes extracted from biopsied muscle (vastus lateralis) was studied in 47 known carriers, 87 possible carriers and in 60 normal females. A significant increase in specific activity of monomeric ribosomes, total polyribosomes and collagen synthesis was found in 46 (97.8 per cent) known carriers and 47 (54 per cent) possible carriers of Duchenne muscular dytrophy. The latter showed an increase in ribosomal protein synthesis in 10 (52.6 per cent) of 19 mothers of isolated cases, 31 (53.3 per cent) of 58 sisters, and 6 (60 per cent) of 10 other female relatives. Serum creatine phosphokinase was increased in 30 (63.8 per cent) of 47 known carriers.
T h e most widely used method for identification of carriers in Duclienne niuscular dystrophy (DMD) so f a r has been serum creatine phospholtinase. T h e elevated level of this enzyme is, however, found only in 60 to 70 per cent of known carriers (Richterich et al. 1963, Milhorat & Goldstone 1965, E m e r y 1969, W a l t o n & Gardner-Medwin 1974, Duboiuitz 1975). In a previous study (lonasescu et al. 1973), we showed that i n uitro amino acid incorporation of polyribosonies extracted froin biopsied muscle of 11 known carriers revealed i n all of them a n increase in the rate of noncollagen and collagen synthesis with inlcrmcdiatc values between the controls and patients with DRID. The present study, performed on a larger number of cases, shows consistent findings with This study was supported in part by the Muscular Associations of America, Inc., National Institutes of Health (NS09283), USPHS Clinical Research Center Grant M01-FR-59 f o r patient services, and State Services for Crippled Children, Special Project Budget MR12, IIEW Children’s Bureau, Clinical Research in Mental Retardation and Genetic Study.
-
443 our earlier results and illustrates the way we are using the ribosomal protein test (RPS) in genetic counseling of this disease at thc present time. MATERIALS AND METHODS Our material includes 134 female relatives of patients with Duchenne hlD and GO age-matched normal female controls. Geographically 12 of the suspected carriers were from Canada, one from Sweden, 36 from other centers i n the United Statcs and 85 from the state of Iowa. The suspcctcd carriers were grouped in Itnown carriers (47) and possible carriers (87) using both genealogical criteria and serum creatine phosphokinasc (CPK) findings. The known carriers included : 1. Genealogically proven carriers : 1) definite carriers, in which the mother has a n affected son and also has a n affected brother o r some other affected male o n the maternal side such as a maternal uncle o r a cousin via a maternal aunt o r a nephew via a sister (Dubowitz 1975) ; 2 ) probable carriers, i n which a mother has more than one affected son. For practical purposes these carriers are always grouped with definite carriers. 2. Mothers w i t h normal serum CPK and a n affected son and one o r several daughters, who were screened as carriers by high serum CPIL 3. Female relatives on t h e maternal side of Duchenne patients (mothers, sisters, aunts, cousins) with high serum CPK unrelated to other disease. The possible carriers included mothers of one affected son, o r other female relatives of Duchenne patients (sisters, aunts, cousins) with normal serum CPK. Muscle specimens of t h e left vastus lateralis were obtained from both suspcrterl carriers and controls. Muscle specimens were shipped to u s in liquid nitrogen cot>tainers when the muscle biopsies were done i n other neuromuscular centers. The procedure for t h e extraction of muscle polyribosomes and for in v i f r o amino acid incorporation were done as previously rcported (lonasesczc e f al. 1970, 1971). The assay f o r collagen synthesis was based o n the action of purified Closfritliiim histolgficum collagenase on t h e labeled proteins made in uitro. The difference i n the radioactivity between t h e duplicate samples served as a measure of the amount of collagen peptides solubilized b y the added collagenase. Determination of the noncollagen protein i n the muscle homogenate was done by t h e method of IJoiur!\, Rosebroiigh and Farr (1951) with bovine serum albumin as standard. The method of Aosalski (1967) was used for the determination of serum CPK. 4 statistical analysis of t h e d a t a was done by t h e estimation of Fischcr’s t cocfficient.
. RESULTS
Ribosome content: All classes of ribosomes showed normal values for known and possible carriers of DhfD. Distribulion of ribosomes on sucrose density gradienfs: Normal pattern was found for all carriers studied by us. Ribosomal protein synthesis: Table 1 contains the mean values with SD and range for in uitro amino acid incorporation of monomeric ribosomes, total polyribosomes and collagen synthesis of heavy poly-
*
44.9
> 0.05 28.1 < 0.01
< 20.7
>
40.4
49.6
26.8 0.05 28.9 0.01
30.3
10-117
64.7
141.7
< 0.01
54.1
< 0.05
Departments of Pediatrics and Biostatistics, College of hledicine, University of Iowa, Iowa City, Iowa, U.S.A.
DETECTION O F CARRIERS AND GENETIC COUNSELING IN DUCHENNE MUSCULAR DYSTROPHY BY RIBOSOMAL PROTEIN SYNTHESIS VICTORIONASESCU, HANSZELLWEGER and LEONBURMEISTER ABSTRACT The in vitro protein synthesis by polyribosomes extracted from biopsied muscle (vastus lateralis) was studied in 47 known carriers, 87 possible carriers and in 60 normal females. A significant increase in specific activity of monomeric ribosomes, total polyribosomes and collagen synthesis was found in 46 (97.8 per cent) known carriers and 47 (54 per cent) possible carriers of Duchenne muscular dytrophy. The latter showed an increase in ribosomal protein synthesis in 10 (52.6 per cent) of 19 mothers of isolated cases, 31 (53.3 per cent) of 58 sisters, and 6 (60 per cent) of 10 other female relatives. Serum creatine phosphokinase was increased in 30 (63.8 per cent) of 47 known carriers.
T h e most widely used method for identification of carriers in Duclienne niuscular dystrophy (DMD) so f a r has been serum creatine phospholtinase. T h e elevated level of this enzyme is, however, found only in 60 to 70 per cent of known carriers (Richterich et al. 1963, Milhorat & Goldstone 1965, E m e r y 1969, W a l t o n & Gardner-Medwin 1974, Duboiuitz 1975). In a previous study (lonasescu et al. 1973), we showed that i n uitro amino acid incorporation of polyribosonies extracted froin biopsied muscle of 11 known carriers revealed i n all of them a n increase in the rate of noncollagen and collagen synthesis with inlcrmcdiatc values between the controls and patients with DRID. The present study, performed on a larger number of cases, shows consistent findings with This study was supported in part by the Muscular Associations of America, Inc., National Institutes of Health (NS09283), USPHS Clinical Research Center Grant M01-FR-59 f o r patient services, and State Services for Crippled Children, Special Project Budget MR12, IIEW Children’s Bureau, Clinical Research in Mental Retardation and Genetic Study.
-
443 our earlier results and illustrates the way we are using the ribosomal protein test (RPS) in genetic counseling of this disease at thc present time. MATERIALS AND METHODS Our material includes 134 female relatives of patients with Duchenne hlD and GO age-matched normal female controls. Geographically 12 of the suspected carriers were from Canada, one from Sweden, 36 from other centers i n the United Statcs and 85 from the state of Iowa. The suspcctcd carriers were grouped in Itnown carriers (47) and possible carriers (87) using both genealogical criteria and serum creatine phosphokinasc (CPK) findings. The known carriers included : 1. Genealogically proven carriers : 1) definite carriers, in which the mother has a n affected son and also has a n affected brother o r some other affected male o n the maternal side such as a maternal uncle o r a cousin via a maternal aunt o r a nephew via a sister (Dubowitz 1975) ; 2 ) probable carriers, i n which a mother has more than one affected son. For practical purposes these carriers are always grouped with definite carriers. 2. Mothers w i t h normal serum CPK and a n affected son and one o r several daughters, who were screened as carriers by high serum CPIL 3. Female relatives on t h e maternal side of Duchenne patients (mothers, sisters, aunts, cousins) with high serum CPK unrelated to other disease. The possible carriers included mothers of one affected son, o r other female relatives of Duchenne patients (sisters, aunts, cousins) with normal serum CPK. Muscle specimens of t h e left vastus lateralis were obtained from both suspcrterl carriers and controls. Muscle specimens were shipped to u s in liquid nitrogen cot>tainers when the muscle biopsies were done i n other neuromuscular centers. The procedure for t h e extraction of muscle polyribosomes and for in v i f r o amino acid incorporation were done as previously rcported (lonasesczc e f al. 1970, 1971). The assay f o r collagen synthesis was based o n the action of purified Closfritliiim histolgficum collagenase on t h e labeled proteins made in uitro. The difference i n the radioactivity between t h e duplicate samples served as a measure of the amount of collagen peptides solubilized b y the added collagenase. Determination of the noncollagen protein i n the muscle homogenate was done by t h e method of IJoiur!\, Rosebroiigh and Farr (1951) with bovine serum albumin as standard. The method of Aosalski (1967) was used for the determination of serum CPK. 4 statistical analysis of t h e d a t a was done by t h e estimation of Fischcr’s t cocfficient.
. RESULTS
Ribosome content: All classes of ribosomes showed normal values for known and possible carriers of DhfD. Distribulion of ribosomes on sucrose density gradienfs: Normal pattern was found for all carriers studied by us. Ribosomal protein synthesis: Table 1 contains the mean values with SD and range for in uitro amino acid incorporation of monomeric ribosomes, total polyribosomes and collagen synthesis of heavy poly-
*
44.9
> 0.05 28.1 < 0.01
< 20.7
>
40.4
49.6
26.8 0.05 28.9 0.01
30.3
10-117
64.7
141.7
< 0.01
54.1
< 0.05
