Abstract
Objective: To characterize the clinical phenotype of LGMD2C in gypsies.
Background: Limb-girdle muscular dystrophy (LGMD) in gypsies of Western Europe is caused by a homozygous C283Y mutation on the same haplotype, suggesting a founder effect.
Methods: We performed clinical, laboratory, and muscle imaging studies of 40 patients.
Results: Mean age at onset was 5.3 years. One half of the patients had loss of ambulation by the age of 12; 13% still could walk after age 16. Calf hypertrophy, scapular winging, macroglossia, and lumbar hyperlordosis were common. Girdle, trunk, and proximal limb flexor muscles had earlier and more severe involvement. Cardiomyopathy was not observed. Five patients in the third decade of life required mechanical ventilation. Scoliosis was common in the nonambulatory stage.
Conclusions: LGMD2C in gypsy patients with C283Y mutation presents a rather homogeneous phenotype, characterized by an initial Duchenne-like progressive course followed by a more prolonged survival rate possibly due to the absence of early respiratory impairment and cardiac failure.
Sarcoglycanopathies are autosomal recessive limb-girdle muscular dystrophies (LGMD) resulting from mutations in any of the four (α, β, γ, δ) sarcoglycan genes already identified, corresponding respectively with the following morbid loci: LGMD2D at 17q12, LGMD2E at 4q12, LGMD2C at 13q12, and LGMD2F at 5q33.1 It has been estimated that sarcoglycanopathies account for at least 11% of patients with Duchenne-like and limb-girdle muscular dystrophy with normal dystrophin.2,3 The clinical spectrum of sarcoglycanopathies varies from severe Duchenne-like to late-onset mild and slowly progressive muscular dystrophy, and the molecular basis of this variability is unclear.4 It may depend on the gene involved and for each gene on the type of mutation. Various different mutations have already been described in the four sarcoglycan genes,5 representing a major difficulty in genotype–phenotype correlation studies. In addition, considerable intrafamilial phenotypic variation has been reported,6 particularly for missense mutations of the α-sarcoglycan gene.7,8
Western European gypsies present LGMD due to a G→A mutation in the 283rd codon of the γ-sarcoglycan gene, causing the replacement of a conserved cysteine in the extracellular domain of the protein by a tyrosine.9 All the patients are homozygous for this mutation, which is carried on the same haplotype, indicating a founder effect.9 The same mutation was subsequently reported in other gypsy families,10-12 confirming that the mutation is specific to this endogamic (the custom of marrying only within one’s tribe) population, and that the disorder may be widespread among gypsies in Europe.
Here we present the results of a joint study aiming to characterize the clinical phenotype of a large cohort of 40 gypsy patients belonging to 24 families living in six different European countries, sharing the same “private” C283Y mutation in γ-sarcoglycan gene.
Patients and methods.
A total of 40 patients (20 male and 20 female) were examined. All the patients were homozygous for the C283Y mutation. The patients originated from 24 gypsy families including the seven already briefly reported.9 Altogether, four families came from France, four from Spain, two from Germany, two from Portugal, two from Italy, and 10 from Bulgaria.
The age at examination varied from 3 to 37 years. Each patient was clinically evaluated by one of the authors. Examining physicians were asked to complete a standardized clinical information sheet including sex, age at examination, consanguinity between parents, age at onset, age at loss of ambulation, cognitive function, functional clinical grade,13 manual muscle test,14 presence of scapular winging, calf hypertrophy, macroglossia, contractures, lumbar hyperlordosis, scoliosis, serum creatine kinase (CK), respiratory function, cardiac function, and muscle CT or MRI studies.
Creatine kinase.
CK was reported as a multiple of the upper value of normal.
Mutation detection.
The C283Y mutation in the γ-sarcoglycan gene creates a new RsaI restriction site, easily detectable by Rsa1 digestion of the PCR amplified exon eight.9
Muscle CT.
Five standard scans at shoulder, lumbar, hip, midthigh, and leg were performed for the evaluation of the principal muscles.15 The CT changes were assessed by one of the authors (L.M.) and graded according to the relative proportion between decreased and normal density muscle areas: mild (hypodensity areas less than 50% of normal looking muscle areas), moderate (hypodense areas between 1/2 and 2/3 of normal muscle looking areas), and severe (the muscle is atrophic or more than 2/3 of it is replaced by low density tissue). The muscle MRI was evaluated in the same way.
Statistical analysis.
We used mean and SD to resume continuous data; one-way ANOVA with the Scheffè post hoc test was used to determine what differences exist among the means of two or more groups. The Levene test for homogeneity of variance was used to test the assumption that groups came from populations with equal variance. Mann-Whitney and Kruskal-Wallis tests were used to determine if two or more populations are equivalent in location. We used the χ2 test for categorical comparisons of data. Survival analysis with the Kaplan-Meier estimation method was used to evaluate data on the age at loss of ambulation. We reported regression analysis with R2 to establish whether the value of a variable depends on one or more other variables. A p value of <0.05 was considered to indicate statistical significance; all tests were two-tailed.
Results.
Age at onset and pattern of muscle weakness. The first symptoms were walking difficulty, frequent falls, toe walking, and difficulty in running, climbing stairs, and raising from the floor. The average age at onset was (mean ± SD) 5.3 ± 2.3 years (range 1 to 10) (figure 1). In boys it was 4.8 ± 1.5 years and in girls 5.9 ± 2.7 years (p = 0.116). Muscle weakness was initially detectable in the pelvic girdle and, after age 6, in the shoulder with scapular winging. Soon after that, trunk weakness became evident with abdominal prominence and lumbar hyperlordosis. At this stage, hypertrophy of calves and macroglossia were frequently reported (figure 2). Ankle contractures were noted around the age of 8 years. At this stage muscle wasting was diffuse, involving most of the pelvic, shoulder, and limb muscles, predominating in glutei, psoas, sacrospinalis, periscapular, and trapezius muscles. In the proximal limbs there was an early selective involvement of the flexors (biceps brachii and hamstrings) with a comparable good preservation of the extensors (triceps brachii and quadriceps). Of the 13 patients who were still able to walk, 12 were between 2 to 13 years and one was 22 years old.
Figure 1. Correlation between age at loss of ambulation and age at onset of walking difficulty in 27 gypsy patients with limb-girdle muscular dystrophy due to the C283Y mutation in the γ-sarcoglycan gene. Note that all the patients with early onset (1 to 7 years of age) lost the ability to walk between 10 and 14 years of age.
Figure 2. Gypsy patients with LGMD2C due to C283Y mutation. (A) A 14-year-old patient with evident macroglossia. (B) A 23-year-old patient who lost ambulation at the age of 18 years. Note the wasting of shoulder girdle and proximal limb muscles, particularly of the biceps, that is atrophic; the lordotic posture; and the marked abdominal protrusion due to severe weakness of abdominal muscles.
Creatine kinase.
Mean CK was 42 ± 23 times normal (range 5 to 100 times normal), correlating inversely with age and degree of disability (Walton scale).
Muscle biopsy.
Protein analysis showed a complete deficiency of γ-sarcoglycan with some preservation of the other sarcoglycans (data not shown).
Progression.
Half of the patients became unable to walk by the age of 12 years (figure 3) and 79% before age 15. In our series, 27 patients out of 40 had lost the ability to walk at an average of 12.7 ± 2.9 years of age (range 10 to 22; median 12). The 14 men were wheelchair-bound at 11.9 ± 2.2 years of age on average and the 13 women at 13.5 ± 3.4 years (p = 0.189). There was a very significant correlation between age at onset of hip or lower limb weakness and age when the patients stopped walking (R = 0.69; p < 0.001). Equally significant was the correlation coefficient between age and degree of disability measured with the Walton scale (R = 0.70).
Figure 3. Accumulative frequency of age with preserved ambulation in a group of 40 gypsy patients with limb-girdle muscular dystrophy due to the C283Y mutation in the γ-sarcoglycan gene. Note that the probability of remaining ambulant at the age of 12 years is 51%, but only 13% at age 17.
Most of the patients who were no longer able to walk developed a scoliosis, progressive in some of them, with marked rigidity of the neck in the later stages. The treatment of scoliosis was with corsets; none of the patients had spine surgery. Vital capacity slowly deteriorated with age (figure 4). Five patients were on mechanical ventilation that started between the ages of 22 and 28 years. Intrafamilial variability in the severity of scoliosis and respiratory impairment was observed in one family.
Figure 4. Correlation between forced vital capacity (% of predicted) and age in gypsy patients with limb-girdle muscular dystrophy due to the C283Y mutation in the γ-sarcoglycan gene. Patients up to the age of 14 years had a forced vital capacity comparable with normal subjects in the same age range.
Muscle imaging.
Muscle CT scans were performed in 12 patients and muscle MRI in two. The degree and extent of muscle hypodensity or atrophy varied according to age and functional ability (walking versus nonwalking). Glutei (medius and minimus) were minimally involved in very young patients still able to climb stairs without aid; moderately involved in 10- to 12-year-old ambulant patients; severely involved in patients of the same age group who were unable to walk; and completely substituted in older, wheelchair-bound patients. In a given compartment, some muscles were selectively involved but others were not (figure 5). For instance, subscapularis and trapezius were already minimally involved in younger patients, whereas the deltoid looked normal; severely involved around the age of 12 to 14, when deltoids were minimally to moderately involved; and completely substituted in older patients, when there were still traces of the deltoids. Selectively affected muscles in 14 patients aged between 5 and 27 years included the glutei, adductors, hamstrings, abdominals, spinalis, subscapularis, and soleus. During the wheelchair stage, most muscles were completely substituted or atrophic. However, also at this stage, there was some sparing of the deltoid, quadriceps, sartorius, gracilis, and gastrocnemius.
Figure 5. (A) Schematic section of the right midthigh shows the contours of the various muscles. (B) In a 19-year-old patient, wheelchair-bound since age 17, muscle CT shows a discrete preservation of rectus femoris (RF), vastus lateralis (VL), sartorius (S), and gracilis (G). On the contrary, ischiocruralis (BF, ST, SM), adductors (ADD), vastus intermedius (VI), and medialis (VM) are completely substituted. (C) In a 27-year-old patient, wheelchair-bound since age 14, RF, VL, and G are partially preserved compared with the complete disappearance of the other thigh muscles.
Other features.
No patient had clinically relevant cardiac abnormalities. No dilated cardiomyopathy was found on echocardiogram. Intellect was normal.
Discussion.
Genetically isolated populations are an important tool of research into hereditary disorders.16 The gypsy population is a closed and endogamic, thus genetically isolated, society not confined to a small territory but spread throughout Europe, where it is estimated to comprise 7 to 9 million people.
Our observations suggest that LGMD2C gypsy patients with the C283Y mutation present a rather homogeneous phenotype and distinctive clinical features. Initial symptoms are related to weakness in pelvic girdle muscles. Calf hypertrophy, scapular winging, and lumbar hyperlordosis were constantly present in the early stages. Macroglossia has been frequently noted.
Clinical examination and muscle imaging showed an early selective involvement of the glutei, adductors, hamstrings, spinalis, abdominals, subscapularis, and soleus, and long-lasting sparing of quadriceps. The scapular winging and relative sparing of quadriceps have also been observed in calpain muscular dystrophy17 but not in Duchenne muscular dystrophy.18 Detection of distinctive patterns of muscle involvement by CT or MRI might be helpful for the diagnosis of different forms of LGMD.
Loss of ambulation before the 13th birthday defines the category of “severe progression” that characterizes the Duchenne phenotype; ambulant beyond 16 years characterizes the Becker phenotype; and loss of ambulation between 13 and 16 years pertains to an intermediate phenotype.19,20 Within this definition, 49% of the gypsy patients with the C283Y mutation had the same severe progression as the Duchenne patients, 38% of patients had the intermediate phenotype, and the remaining 13% of patients had the Becker phenotype. This difference in severity, for the first time documented in a large cohort of patients sharing the same homozygous mutation, has relevant implications. It might be due to different levels of the remaining sarcoglycans,21 as found in the Tunisian LGMD2C.22 Alternatively, a modifier gene or environmental factor may play a role.23
LGMD2C due to the C283Y missense mutation has a more severe presentation than that caused by the North African out of frame del521T mutation.6 Twenty percent of the Tunisian patients lost the ability to walk before age 15,6 as compared to 79% of the gypsy patients we studied. The phenotype in gypsy and Tunisian patients is similar apart from no macroglossia and more frequent cardiomyopathy in the latter.6 It is now recognized that in the Tunisian population, apart from the prevalent del521T mutation,24 other defects are occasionally present, at least in the α-8 and in the β-sarcoglycan genes.25
Another feature distinct from Duchenne muscular dystrophy is the fact that neither mental retardation nor cardiomyopathy are observed in gypsy LGMD2C, whereas mental retardation and dilated cardiomyopathy are common in dystrophinopathies. Surprisingly, in contrast with the C283Y γ-sarcoglycanopathy, mice in which the γ-sarcoglycan gene has been knocked out exhibit a lethal cardiomyopathy.26
Life span is limited in Duchenne muscular dystrophy, with only 5% of patients living longer than 26 years without mechanical ventilation.27 In contrast, 25% of LGMD2C gypsy patients with the C283Y missense mutation were already over 26 at the time of examination. It seems that although almost 79% of the LGMD2C gypsy patients present an initial Duchenne-intermediate progressive course, most of them enjoy a more prolonged survival rate, possibly due to the absence of early respiratory impairment and cardiac failure.
Acknowledgments
Acknowledgment
The authors thank E. Pignotti for assistance with data analysis and J. Miller for help with the English language.
- Received March 27, 1999.
- Accepted November 19, 1999.
References
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