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We report the first case of a heterozygous T78M mutation in the caveolin-3 gene (CAV3) associated with rippling muscle disease and proximal myopathy. The patient displayed also bilateral winged scapula with limited abduction of upper arms and marked asymmetric atrophy of leg muscles shown by magnetic resonance imaging. Immunohistochemistry on the patient’s muscle biopsy demonstrated a reduction of caveolin-3 staining, compatible with the diagnosis of caveolinopathy. Interestingly, consistent with the possible diagnosis of FSHD, the patient carried a 35 kb D4Z4 allele on chromosome 4q35. We discuss the hypothesis that the two genetic mutations may exert a synergistic effect in determining the phenotype observed in this patient.
Caveolinopathies, clinically heterogeneous neuromuscular and/or cardiac diseases, mainly present as limb girdle muscular dystrophy type 1C (LGMD-1C) or inherited rippling muscle disease (RMD), characterized by electrically silent percussion-induced muscle mounding. However, these disorders also include isolated hyperCKemia, distal myopathy (MD) [1,2], familial hypertrophic cardiomyopathy (HCM) , arrhythmogenic long QT syndrome (LQTS)  and some cases of sudden infant death syndrome (SIDS) . All these disorders are usually transmitted as an autosomal dominant trait and are caused by mutations in the CAV3 gene (OMIM 601253, gene map locus 3p25), which encodes for caveolin-3 (Cav-3), the myocyte-specific isoform of caveolin proteins. Caveolins are the main protein components of caveolae, vesicular invaginations of the plasma membrane involved in several biological processes such as cellular vesicular trafficking, endocytosis, cholesterol homeostasis, and signal transduction [6,7]. Functional studies of different CAV3 missense mutations have shown that mutant Cav-3 can be held in the Golgi apparatus and rapidly degraded by the ubiquitin–proteasome system, exerting a dominant negative effect on the wild-type protein [8–10], thereby explaining the dominance of these mutations. The same mutation can be associated with different phenotypes, sometime overlapping, in different individuals, suggesting that additional unknown loci affect the disease phenotypes. However, muscle impairment and cardiac dysfunction rarely coexist in a patient, probably due to the involvement of distinct pathogenic molecular pathways [11,12].
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder, characterized by progressive weakness of the facial, shoulder girdle and upper limb muscles, often involving peroneal and pelvic girdle muscles. The molecular bases of this disease are unknown, but the disorder is associated with DNA rearrangements of a polymorphic array of tandemly repeated 3.3 kb DNA segments (named D4Z4 repeats) located at the subtelomeric region of chromosome 4q . It has been established that normal subjects carry p13E-11 EcoRI digested alleles larger than 50 kb (11 D4Z4 repeats) originating from chromosome 4, while the majority of either de novo or familial FSHD patients carries alleles of 35 kb, corresponding to 8 D4Z4 units, or shorter . Genotype–phenotype correlation studies have revealed an inverse correlation between the number of D4Z4 repeats and the severity of the disease. Nevertheless, the variability of clinical outcomes has resulted to be more pronounced than expected [15,16], even within the same family [17,18]. In addition, subjects carrying reduced D4Z4 allele without signs of the disease have also described [15,16,19]. To explain the clinical variability and the presence of non-penetrant carriers, recent studies have suggested that reduction of D4Z4 repeats on chromosome 4q35 is pathogenic only in certain “permissive” chromosomal backgrounds, such as polymorphisms that map at the distal region of chromosome 4q and chromosome 10q [20–22].
Here we report on a patient with rippling myopathy, limb girdle muscle weakness and some phenotypic features reminiscent of FSHD, who proved to be a carrier of a heterozygous CAV3 mutation and a D4Z4 FSHD-sized allele. The observed phenotype was consistent with these combined genetic mutations in determining an “overlapping” syndrome.
The proband, a 55-year-old Caucasian male, born to non-consanguineous parents, was delivered at term after an uneventful pregnancy. He achieved normal developmental motor milestones. His remote pathological anamnesis was unremarkable. At the age of 45, the patient began to complain of limb girdle fatigability and muscle weakness with difficulty in some motor tasks, such as raising the upper limbs or climbing stairs. Since the age of 53, he had also been experiencing muscle cramps, pain, stiffness induced by exercise and the occurrence of localized mounding and rapid contractions of lower limb muscles, generally caused by muscle percussion. Neurological examination revealed a hyperlordotic posture, bilateral mild winged scapula (Fig. 1), a partial limitation of arms abduction, up to 90°, and a moderate hyposthenia at the pelvic girdle muscle level (score 4.5 at MRC grading Hammersmith Hospital motor scale ). Mechanical percussion of thigh muscle bellies induced a transient local mounding phenomenon, resembling RMD . The deep tendon reflexes were mildly reduced. No signs of facial weakness were present. The family history was inconsistent, although his father was reported to have difficulties in raising arms in his seventies and his mother, who died at age of 70 years from congestive heart failure, was reported to have heart complaints since age of 35 years, after her last delivery (medical reports are not available) (Fig. 2).
Blood creatine kinase (CK) level was mildly increased (up to 1000 U/L; n.v. <190); the routine blood tests and thyroid hormones were in the normal range. The ischemic forearm exercise test for lactate dosage showed a delayed recovery in lactate kinetics after-exercise. Needle electromyography revealed myopathic signs in the four limbs; the involuntary rolling muscle contractions were electrically silent. In order to exclude an immunogenic form of RMD, such as associated with myasthenia gravis [25,26], levels of anti-acetylcholine receptor-antibodies were determined with a negative result; thoracic computed tomography was inconsistent. Cardiac assessment was normal.
Thigh and leg muscle magnetic resonance imaging (MRI) revealed an asymmetric muscle pattern involvement, with marked fatty infiltration and atrophy of long adductor of the left thigh and medial head of the left gastrocnemius (Fig. 3), mild signs of edema in thigh muscles (right femoral biceps and left gracile muscles) and in leg muscles (lateral head of the right gastrocnemius and bilateral tibial anterior).
The patient underwent biopsy at the right quadriceps femoris muscle. The frozen muscle specimen was processed for routine histology and immunohistochemistry analysis. Muscle biopsy showed moderate myopathic changes including increase in connective tissue and diameter variability in both fiber types, scattered round shaped atrophic fibers, rare degenerative fibers and a considerable endomysial inflammatory infiltrate (Fig. 4). Immunostaining, performed for dystrophin (dilution 1:20), laminin (dilution 1:50), alfa-sarcoglycan (dilution 1:100), dysferlin (dilution 1:20) and emerin (dilution 1:20) (mouse monoclonal antibodies; Novocastra Ltd., United Kingdom) using a Benchmark immunostainer (Ventana Medical System, Tucson, AZ), was normal.
Genetic analysis for CAV-3 gene was performed. High molecular weight genomic DNA was extracted from peripheral blood lymphocytes with standard procedures. Using polymerase chain reaction (PCR), denaturing high-performance liquid chromatography, and direct DNA sequencing, the open reading frame/splice site mutational analysis on CAV3 revealed n.233C > T (T78M) missense mutation in exon 2, involving a highly conserved residue in the Cav-3 transmembrane domain . Subsequently skeletal muscle immunostaining for Cav-3 (monoclonal antibodies diluted 1:100 in 1% BSA-PBS; Transduction Laboratories, Lexington, KY) was carried out and resulted markedly reduced at the sarcolemma in almost all fibers (Fig. 5), compatible with diagnosis of caveolinopathy .
Based on some phenotypic features of the patient, such as bilateral mild winged scapula and upper limb girdle weakness as well as muscle MRI pattern, facioscapulohumeral muscular dystrophy (FSHD) was also considered. The molecular test for FSHD, Southern blot hybridization of EcoRI/BlnI-digested DNA resolved by pulsed field gel electrophoresis detected with probe p13-E11 , revealed an EcoRI restriction fragment of 35 kb on chromosome 4q35. Polymorphisms flanking the D4Z4 repeat array were also investigated. The 35 kb D4Z4 allele was associated with 4qA variant, 161 Simple Sequence Length Polymorphism (SSLP) and the permissive Single Nucleotide Polymorphism (SNP) ATTAAA [20–22].
Molecular analysis for CAV3 gene and FSHD in other family members (brother, sister and daughters), all completely asymptomatic, was not performed because they declined to undergo genetic testing.
We report the first case of a heterozygous CAV3 T78M mutation associated with rippling muscle disease and proximal weakness in which immunohistochemical analysis of skeletal muscle confirmed reduced Cav-3 immunolabeling. The heterozygous n.233C > T missense mutation in CAV3 observed in our case is responsible of a threonine to methionine substitution at codon 78 (T78M), a highly conserved residue of Cav-3. This mutation has originally been reported in three unrelated individuals with long QT syndrome (LQTS) . In particular, one patient, a 14-year-old girl with non-exertional syncope and a ‘seizure-like’ presentation, carried biallelic digenic mutations, a A913V mutation in the LQT2-associated KCNH2 gene as well as the T78M mutation. In contrast, the other two patients with T78M mutations, an 8-year-old boy with non-exertional syncope and marked sinus bradycardia, and an asymptomatic 40-year-old male, resulted negative for mutations in known LQTS genes. A positive family history was reported in all three patients, but molecular analysis in other family members was not performed. Subsequently, Cronk et al. (2007)  has identified the same mutation in heterozygosis in a 2-month-old black female infant who died of SIDS. As this patient presented neither primary cardiomyopathy nor skeletal muscle impairment, it was speculated that Cav-3 might have a role in cardiac excitability. Indeed, Cav-3 co-localizes with beta2-adrenoreceptors in ventricular and sinoatrial myocytes, thus supporting the idea that disruption of caveolae can affect the beta-adrenergic responsiveness and the excitation–contraction coupling of cardiac myocytes [27–29]. The CAV3 T78M mutation has also been found in a subject with idiopathic hyperCKemia, although in this case immunohistochemical analysis of Cav-3 in a muscle biopsy was normal . More recently, Traverso and coworkers (2008)  have described homozygous T78M mutation in CAV3 in a 58-year-old woman born to consanguineous parents, affected by dilated cardiomyopathy and limb girdle muscular dystrophy. The patient’s daughter, heterozygous for the mutation, was asymptomatic and had normal CK, suggesting an autosomal recessive mode of inheritance for this trait. In vitro studies showed that T78M mutated Cav-3 impairs the ability of the Cav-3 hydrophobic domain to assembly Cav-3 homo-oligomers units for the formation of caveolae in muscle cells. Thus it was concluded that the T78M mutation acts through a loss-of-function mechanism [31–33]. This hypothesis was consistent with the fact that the patient carrying a homozygous CAV3 T78M substitution developed a severe muscular phenotype, whereas her heterozygous daughter was asymptomatic.
Collectively, published data [4,5,10,30] do not fully support the hypothesis that CAV3 T78M mutation is pathogenic in the skeletal muscle in the heterozygous state. Instead, it can be hypothesized that the CAV3 T78M mutation may exert a pathogenic effect in association with other genetic factors Indeed, genotype–phenotype correlation studies suggest that in caveolinopathies genetic modifiers may contribute to determine Cav-3 deficiency phenotype [1,10,12,34].
In our case, the clinical diagnosis of caveolinopathy was suggested by the presence of rippling phenomenon and proximal muscle weakness resembling a LGMD phenotype and was supported by the discovery of CAV3 T78M mutation. The histochemical analysis performed on skeletal muscle biopsy revealed reduced immunolabeling for Cav-3, compatible with diagnosis of caveolinopathy . However, the shoulder girdle involvement presenting with scapular winging observed in our case is unusual in caveolinopathies, although these disorders are associated with a broad spectrum of clinical phenotypes [12,35]. Beyond the bilateral winged scapula and the limitation of upper arms abduction, the patient also displayed other clinical features reminiscent of FSHD, such as marked asymmetry at MRI imaging of muscle involvement in the legs [36,37] and the inflammatory infiltration observed in muscle biopsy . Consistent with these clinical observations we detected a 35 kb D4Z4 allele on chromosome 4q35, which is generally reported in patients with a mild FSHD phenotype . Further, this was also associated with the 161A-SNP ATTAAA permissive haplotype , recently proposed as the genetic marker for FSHD . Nevertheless, weakness of the pelvic girdle muscles, as complained by our patient, is not usually present at onset in FSHD  and the rippling phenomenon, specific sign of caveolinopathy, has never been reported associated with D4Z4 reduced allele.
The extensive use of the molecular analysis as diagnostic test in human hereditary myopathies has led to the identification of an increasing number of atypical phenotypes. This is particularly true for FSHD, in which various clinical features have been found in subjects carrying FSHD-sized D4Z4 alleles, including a facial-sparing form of FSHD (SHD) , limb-girdle muscular dystrophy , distal myopathy , asymmetric brachial weakness , chronic progressive external ophthalmoplegia , asymptomatic hyperCKemia , hypertrophic cardiomyopathy , adult-onset distal myopathies with rimmed vacuoles , isolated axial myopathy with camptocormia and bent spine syndrome [49,50]. In addition, overlapping FSHD phenotypes in which 4q35 deleted D4Z4 mutation is associated with other pathogenic mutations in other genes, have been reported in cases of patients with mitochondrial myopathy/FSHD , Becker dystrophy/FSHD , Duchenne dystrophy/FSHD [53,54], Leber’s hereditary optic neuropathy/FSHD , suggesting a synergistic effect of those simultaneous mutations in reaching disease threshold and determining overlapping phenotypes.
In conclusion, our case may represent a further example of such atypical phenotypes in which D4Z4 deletion contributes to pathogenicity of heterozygous CAV3 T78M mutation for the observed myopathic phenotype, resulting in an overlapping syndrome.
Grant support was received from Telethon GUP08004 GS (RT), AFM 14339 (RT), NIH-NINDS R01 047584 (RT).