Available online at www.sciencedirect.com Neuromuscular Disorders 22 (2012) 534–540 www.elsevier.com/locate/nmd Rippling muscle disease and facioscapulohumeral dystrophy-like phenotype in a patient carrying a heterozygous CAV3 T78M mutation and a D4Z4 partial deletion: Further evidence for “double trouble” overlapping syndromes Giulia Ricci a,⇑, Isabella Scionti c, Greta Alı` b, Leda Volpi a, Virna Zampa d, Marina Fanin e, Corrado Angelini e, Luisa Politano f, Rossella Tupler c, Gabriele Siciliano a a Department of Neuroscience, University of Pisa, Pisa, Italy b Department of Surgery, University of Pisa, Pisa, Italy c Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy d Department of Radiology, University of Pisa, Pisa, Italy e Department of Neurosciences, University of Padua, Padua, Italy f Department of Experimental Medicine, Cardiomyology and Medical Genetics 2nd University of Naples, Naples, Italy Received 8 August 2011; received in revised form 11 November 2011; accepted 1 December 2011 Abstract 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. Ó 2011 Elsevier B.V. All rights reserved. Keywords: Rippling muscle disease; Caveolinopathy; Facioscapulohumeral dystrophy; Limb girdle muscular dystrophy type 1C 1. Introduction [5]. All these disorders are usually transmitted as an auto- somal dominant trait and are caused by mutations in the Caveolinopathies, clinically heterogeneous neuromuscu- CAV3 gene (OMIM 601253, gene map locus 3p25), which lar and/or cardiac diseases, mainly present as limb girdle encodes for caveolin-3 (Cav-3), the myocyte-specific iso- muscular dystrophy type 1C (LGMD-1C) or inherited rip- form of caveolin proteins. Caveolins are the main protein pling muscle disease (RMD), characterized by electrically components of caveolae, vesicular invaginations of the silent percussion-induced muscle mounding. However, plasma membrane involved in several biological processes these disorders also include isolated hyperCKemia, distal such as cellular vesicular trafficking, endocytosis, choles- myopathy (MD) [1,2], familial hypertrophic cardiomyopa- terol homeostasis, and signal transduction [6,7]. Functional thy (HCM) [3], arrhythmogenic long QT syndrome (LQTS) studies of different CAV3 missense mutations have shown [4] and some cases of sudden infant death syndrome (SIDS) that mutant Cav-3 can be held in the Golgi apparatus and rapidly degraded by the ubiquitin–proteasome system, ⇑ Corresponding author. exerting a dominant negative effect on the wild-type protein E-mail address: [email protected] (G. Ricci). [8–10], thereby explaining the dominance of these 0960-8966/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2011.12.001 G. Ricci et al. / Neuromuscular Disorders 22 (2012) 534–540 535 mutations. The same mutation can be associated with dif- The observed phenotype was consistent with these combined ferent phenotypes, sometime overlapping, in different indi- genetic mutations in determining an “overlapping” syndrome. viduals, suggesting that additional unknown loci affect the disease phenotypes. However, muscle impairment and car- 2. Case description diac dysfunction rarely coexist in a patient, probably due to the involvement of distinct pathogenic molecular pathways The proband, a 55-year-old Caucasian male, born to [11,12]. non-consanguineous parents, was delivered at term after Facioscapulohumeral muscular dystrophy (FSHD) is an an uneventful pregnancy. He achieved normal developmen- autosomal dominant neuromuscular disorder, character- tal motor milestones. His remote pathological anamnesis ized by progressive weakness of the facial, shoulder girdle was unremarkable. At the age of 45, the patient began to and upper limb muscles, often involving peroneal and pel- complain of limb girdle fatigability and muscle weakness vic girdle muscles. The molecular bases of this disease are with difficulty in some motor tasks, such as raising the unknown, but the disorder is associated with DNA rear- upper limbs or climbing stairs. Since the age of 53, he rangements of a polymorphic array of tandemly repeated had also been experiencing muscle cramps, pain, stiffness 3.3 kb DNA segments (named D4Z4 repeats) located at induced by exercise and the occurrence of localized mound- the subtelomeric region of chromosome 4q [13]. It has been ing and rapid contractions of lower limb muscles, generally established that normal subjects carry p13E-11 EcoRI caused by muscle percussion. Neurological examination digested alleles larger than 50 kb (P11 D4Z4 repeats) revealed a hyperlordotic posture, bilateral mild winged originating from chromosome 4, while the majority of scapula (Fig. 1), a partial limitation of arms abduction, either de novo or familial FSHD patients carries alleles of up to 90°, and a moderate hyposthenia at the pelvic girdle 35 kb, corresponding to 8 D4Z4 units, or shorter [14]. muscle level (score 4.5 at MRC grading Hammersmith Genotype–phenotype correlation studies have revealed an Hospital motor scale [23]). Mechanical percussion of thigh inverse correlation between the number of D4Z4 repeats muscle bellies induced a transient local mounding phenom- and the severity of the disease. Nevertheless, the variability enon, resembling RMD [24]. The deep tendon reflexes were of clinical outcomes has resulted to be more pronounced mildly reduced. No signs of facial weakness were present. than expected [15,16], even within the same family The family history was inconsistent, although his father [17,18]. In addition, subjects carrying reduced D4Z4 allele was reported to have difficulties in raising arms in his sev- without signs of the disease have also described enties and his mother, who died at age of 70 years from [15,16,19]. To explain the clinical variability and the pres- congestive heart failure, was reported to have heart com- ence of non-penetrant carriers, recent studies have sug- plaints since age of 35 years, after her last delivery (medical gested that reduction of D4Z4 repeats on chromosome reports are not available) (Fig. 2). 4q35 is pathogenic only in certain “permissive” chromo- Blood creatine kinase (CK) level was mildly increased somal backgrounds, such as polymorphisms that map at (up to 1000 U/L; n.v. <190); the routine blood tests and the distal region of chromosome 4q and chromosome 10q thyroid hormones were in the normal range. The ischemic [20–22]. forearm exercise test for lactate dosage showed a delayed Here we report on a patient with rippling myopathy, recovery in lactate kinetics after-exercise. Needle electro- limb girdle muscle weakness and some phenotypic features myography revealed myopathic signs in the four limbs; reminiscent of FSHD, who proved to be a carrier of a het- the involuntary rolling muscle contractions were electri- erozygous CAV3 mutation and a D4Z4 FSHD-sized allele. cally silent. In order to exclude an immunogenic form of Fig. 1. Proband with bilateral mild winged scapula and hyperlordotic posture. 536 G. Ricci et al. / Neuromuscular Disorders 22 (2012) 534–540 Fig. 2. Pedigree of the proband family. Arrow indicates the proband. Proband’s father was reported to have difficulties in raising arms in his seventies;he died at the age of 83 years for Parkinson’s disease. Proband’s mother, affected by congestive heart failure, died at the age of 70 years (medical reports are not available). The brother, the sister and the two daughters of the proband resulted completely asymptomatic. RMD, such as associated with myasthenia gravis [25,26], levels of anti-acetylcholine receptor-antibodies were deter- mined with a negative result; thoracic computed tomogra- phy was inconsistent. Cardiac assessment was normal. Thigh and leg muscle magnetic resonance imaging (MRI) revealed an asymmetric muscle pattern involve- ment, with marked fatty infiltration and atrophy of long adductor of the left thigh and medial head of the left gas- trocnemius (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 bilat- eral 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 vari- ability in both fiber types, scattered round shaped atrophic fibers, rare degenerative fibers and a considerable endomy- sial inflammatory infiltrate (Fig. 4). Immunostaining, per- formed for dystrophin (dilution 1:20), laminin (dilution 1:50), alfa-sarcoglycan (dilution 1:100), dysferlin (dilution Fig. 3. T1-weighted skeletal muscle MRI of the lower limbs: (a) at thigh 1:20) and emerin (dilution 1:20) (mouse monoclonal anti- level, marked fatty infiltration and atrophy of