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Rare reports on patients with congenital myopathy with excess of muscle spindles (CMEMS), hypertrophic cardiomyopathy and variable features resembling Noonan syndrome have been published, but the genetic basis of this condition is so far unknown.
We analysed PTPN11 and RAS genes in five unrelated patients with this phenotype, and found HRAS mutations in four of them. Two disease‐associated mutations, G12V and G12S, have previously been observed in patients with Costello syndrome (CS), and two other mutations, E63K and Q22K, are novel. All four mutations are predicted to enhance downstream HRas signalling, suggesting that CMEMS is a developmental consequence of sustained HRas activation in skeletal muscle.
This type of myopathy may represent a previously unrecogned manifestation of CS. However, some patients carrying HRAS mutations may exhibit prominent congenital muscular dysfunction, although features of CS may be less obvious, suggesting that germline HRAS mutations may underlie some cases of otherwise unclassified neonatal neuromuscular disorders.
Several groups have shown that Noonan syndrome (NS; OMIM 163950) and the related disorders cardio‐facio‐cutaneous syndrome (CFC; OMIM 115150] and Costello syndrome (CS; OMIM 218040) are caused by activating mutations in genes of the Ras–MAPK–ERK signalling cascade (reviewed by Gelb and Tartaglia1). These disorders share a common pattern of congenital anomalies, including typical heart defects, craniofacial dysmorphism, short stature and skeletal anomalies.2,3,4 Although NS, CFC and CS represent separate entities, they have overlapping features that may hamper the clinical distinction between these three syndromes, particularly in young infants.
There have been some reports describing newborns or young infants with congenital myopathy with excess of muscle spindles (CMEMS) associated with hypertrophic cardiomyopathy and variable NS‐like facial features.5,6,7 Therefore, we conducted a study to identify the genetic basis of this condition.
This study was approved by the University of Erlangen‐Nuremberg Ethics Committee, and informed consent for genetic analyses was obtained from parents. Our study population consisted of four previously reported patients5,6,7 and one newly ascertained case. All patients presented with congenital muscular weakness.
A muscle biopsy was taken from each patient, which revealed an excess of muscle spindles with otherwise minor and non‐specific myopathic changes (fig 1A1A).). Two patients were originally recogned as having NS‐like facial features; in the other three patiens, nonspecific facial anomalies were recorded. The clinical findings are summared in table 11.
DNA samples were collected from blood, buccal swabs, fibroblasts and/or frozen or paraffin‐embedded tissues. Mutational screening of PTPN11, NRAS, KRAS and HRAS was carried out as described previously.8 Primer pairs and PCR conditions are available on request. Sequence analysis was performed by bidirectional sequencing using a commercial sequencing kit (ABI BigDye Terminator Sequencing Kit V2.1; Applied Biosystems, Weiterstadt, Germany) and an automated capillary sequencer (ABI 3730; Applied Biosystems).
We discovered heterozygous missense HRAS mutations in four of five patients (fig 1B1B,, table 11).). The respective mutation was detected in different tissue samples in three patients: patient 1 (blood, muscle biopsy), patient 3 (muscle, liver, thymus) and patient 5 (buccal cells, muscle biopsy), thus demonstrating that the mutations probably occurred in the germline. The G12V and G12S mutations have been described previously in patients with CS: G12V was reported in a single case with a severe fatal disease course9 and G12S has been identified as the most common mutation in patients with CS, representing approximately 85% of disease‐associated alleles.9,10,11,12 Two novel mutations were identified: Q22K and E63K in exons 2 and 3, respectively. Both affect evolutionarily conserved amino acid residues identical in all Ras proteins (data not shown) and are de novo mutations. Notably, a Q22K exchange in K‐Ras, the G domain of which is almost identical to HRas in its sequence and function, has been found in tumors (COSMIC database; http://www.sanger.ac.uk/genetics/CGP/cosmic/) and previously shown to transform NIH3T3 cells.13 The HRas mutant, E63K, has been demonstrated to abolish guanosine triphosphatase reaction stimulated by guanosine triphosphatase activating protein14 and to potentiate cellular transformation.15,16 Together, these data indicate that all four mutations lead to constitutive activation of HRas. Structural analysis suggests that these gain of function effects are mediated by different mechanisms (supplementary information online; available online at http://jmg.bmj.com/supplemental).
We conclude that activating HRAS mutations cause the syndrome of CMEMS, hypertrophic cardiomyopathy and variable NS‐like facial features. The morphological changes with an excess of muscle spindles probably reflect developmental consequences of sustained HRas activation in skeletal muscle. This muscular phenotype possibly represents a previously unrecogned manifestation of CS and may, at least in part, be responsible for the muscular weakness and congenital contractures often observed in CS patients. However, skeletal muscle morphology has yet not been studied systematically in CS patients. Nevertheless, a review of clinical data on our patients found that they have some features consistent with the diagnosis of CS, such as the craniofacial findings (originally quoted as NS‐like5), hypertrophic cardiomyopathy, lymphoedema and neuroblastoma6 (table 11).). Additionally, dysmorphic features of CS were less obvious, possibly due to the young age of the patients. Skin abnormalities, such as increased palmar and plantar skin, were not noted. Other symptoms known as typical but less specific early manifestations of CS, such as polyhydramnios and feeding difficulties, are readily explained by the severe neurological phenotype. In comparison with a “classic” CS phenotype, however, our patients displayed more prominent muscular problems, including marked weakness, extended congenital contractures, neonatal ventilatory insufficiency, severe myocardial involvement and early lethality attributable to their underlying muscular disease. We therefore speculate that, particularly in early lethal cases, clinicians may classify this disorder as a neuromuscular disease rather than a variant of CS.
One patient of our small cohort (patient 2) did not have an HRAS mutation, suggesting genetic heterogeneity. This patient had a similar phenotype, but in contrast to the other four cases, parental consanguinity was reported,5 thus raising the speculation that there might be an autosomal recessive form of this disorder.
Notably, when selecting patients for this particular type of myopathy, we detected two HRAS mutations that have not been encountered in any of the >100 reported cases of CS. In addition, the mutation G12V was previously described in only one child, who had an early lethal course of CS.9 In contrast, the G12S mutation largely predominating in CS9,10,11,12 was found only in one patient of our cohort, thus indicating that there might be a different mutational spectrum in patients with this phenotype compared with CS patients in general. However, whether this neonatal myopathic phenotype is a distinct entity caused by specific HRAS alleles is undetermined at this time.
Our observations, together with the high frequency of cardiomyopathy and rhabdomyosarcoma in CS patients and in vitro studies showing that Ras activation perturbs both the biochemical and morphological differentiation of myoblasts,17,18,19 highlights the particular role of HRas in striated muscle development. Moreover, transgenic mice overexpressing human HRas (rasH2 mice) have been found to exhibit a skeletal myopathy.20 It remains to be clarified whether the excess of muscle spindles itself plays the primary pathogenetic role in the condition or it represents the most prominent morphological sign of a more complex disorder of skeletal muscle development. Our patients did not exhibit consistent findings other than muscular weakness that would allow us to draw conclusions about the nature of the myopathic process (no elevated CK, no specific electromyographic abnormalities).5,6,7
Muscle spindles are stretch receptors that consist of specialed encapsulated groups of myofibres (intrafusal muscle fibres) innervated by γ and β motor neurons from the anterior horn and receiving Ia and II sensory afferents. There are indications that unique populations of myogenic cells are destined to become intrafusal fibres.21 Muscle spindle formation is also dependent on trophic factors released by sensory Ia afferent neurons in a subpopulation of myotubules that receive sensory myoneural contacts and are followed by γ innervation. The exact mechanisms of muscle spindle development are incompletely understood, but there is evidence that growth factors such as neurotrophin‐3,22 neuregulin‐1,23,24 and glial cell‐derived neurotrophic factor25 are involved in the process. Notably, signalling induced by these molecules has been shown to be at least partly dependent upon Ras.26,27,28 These processes may be perturbed in the presence of constitutively active HRas during skeletal muscle differentiation and eventually lead to excess muscle spindle formation. Further experimental studies are needed to elucidate the precise underlying mechanisms.
Rather than defining a distinct syndrome or separate entity, we propose that the skeletal muscle phenotype referred to as CMEMS is a variant of CS and thus belongs to the spectrum of abnormalities caused by activating mutations of HRAS. The finding of muscle spindle excess in a patient with a congenital myopathy should prompt an analysis of the HRAS gene. Whether Ras pathway inhibitors that are currently being developed as anti‐cancer agents may have beneficial effects for patients with germline mutations of HRAS and life‐threatening symptoms such as myopathy is an important research question.
Supplementary information can be viewed on the JMG website at http://jmg.bmj.com/supplemental
We are grateful to the patients and families who participated in this study. We thank Dr Mwe Mwe Chao for help with the preparation of the manuscript. Mohammad Reza Ahmadian recived a DFG grant (AH 92/1‐3).
CFC - cardio‐facio‐cutaneous syndrome
CMEMS - congenital myopathy with excess of muscle spindles
CS - Costello syndrome
NS - Noonan syndrome
Competing interests: None declared.
Supplementary information can be viewed on the JMG website at http://jmg.bmj.com/supplemental