Myopathy caused by HRAS germline mutations: implications for disturbed myogenic differentiation in the presence of constitutive HRas activation

Abstract

Background

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.

Methods and results

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.

Conclusion

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.

PATIENTS AND METHODS

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 1A). 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 1.

Figure 1 Myopathy with excess of muscle spindles: histological presentation and genetic alterations. (A) Representative section from the quadriceps femoris muscle of patient 5 showing an excess of muscle spindles (white circles) (H&E staining, magnification ×100). (B) Genomic representation of the HRAS gene and location of disease‐associated HRAS mutations. A schematic drawing showing exon–intron structure of the HRAS gene is shown on the top of the panel. Exons are represented by filled boxes. Coding (parts of) exons are depicted in blue and non‐coding regions in grey. Exon 5 (light blue) is alternatively spliced, generating two HRas isoforms. Electropherograms showing HRAS mutations (red arrows) in exons 2 and 3 are presented in the lower part of the figure, together with the normal sequence in letters and electropherograms from a control. Red lettering indicates the codon affected by the respective mutation.

Table 1 Clinical features of patients with congenital myopathy and excess of muscle spindles (CMEMS).

Patient no		1		2		3		4		5
Reference		de Boode et al,5 case 1		de Boode et al,5 case 2		Selcen et al6		Stassou et al7		This report
Age at last follow‐up		3 weeks		10 months		14 months		7 months		13 months
Sex		Male		Female		Male		Female		Male
Birth weight		1938 g (75th centile), preterm infant		3370 g (40th centile)		Not available		2460 g (75th centile), preterm infant		2765 g (<3rd centile)
Congenital heart defect		Progressive HOCM		Severe HOCM		HOCM, chaotic atrial tachycardia		HOCM		Mild (transient) HCM
Facial anomalies		Hypertelorism, epicanthal folds, broad nasal bridge, low‐set, posteriorly rotated ears		Hypertelorism, broad nasal bridge, low‐set, posteriorly rotated and dysplastic ears		Bifrontal hollowing, full cheeks, triangular mouth, high‐arched palate		Hypertelorism, low‐set ears		Low‐set ears, triangular mouth
Short stature		3rd–10th centile		10th centile		Not available		<10th centile		<1st centile
Relative macrocephaly		Yes (98th centile)		No		Increased brain weight on autopsy		Yes (>90th centile)		No
Short/webbed neck		Webbed neck		Short neck		No		Short, webbed neck		Short neck
Lymphoedema		Fetal hydrops		No		No		Congenital generaled oedema		No
Ophthalmologic problems		Cataracta incipiens		No		No		No eye contact at age 3 months		Nystagmus
Neurological signs/development		Generaled hypotonia, absence of spontaneous movements		None mentioned		Generaled muscle weakness, areflexia, joint contractures, bilateral club feet		Flaccid quadriplegia, absent reflexes, contractures of hands, bilateral talipes equinovarus		Generaled hypotonia, delayed motor development
EMG		No abnormalities		Not available		No abnormalities		Myopathic pattern		Not available
Creatine kinase		Normal		Normal		Normal		Normal		Normal
Muscle histology		Excess of small fibres (type IIC), packed or centrally located nuclei, excess of muscle spindles*. Heart: myocardial disarray		Excess of muscle spindles, excess of type IIC fibres		Fibre atrophy with rounded, small‐diameter muscle fibres, excess of formations resembling muscle spindles		Abnormally numerous muscle spindles, increased fibre diameter variation		Excess of muscle spindles; fibre atrophy with rounded small‐diameter muscle fibres; type II fibre predominance
Others		Polyhydramnios, hepatosplenomegaly, periventricular haemorrhage with consecutive hydrocephalus, respiratory insufficiency		Polyhdramnios, feeding difficulties, parental consanguinity		Ventilatory dependence by age 3 months, mild enlargement of cerebral ventricles neonatal neuroblastoma		Polyhydramnios, postnatal respiratory insufficiency, osteopenia, poor sucking		Intrauterine growth delay, postnatal transitory respiratory insufficiency, poor sucking requiring tube feeding
Outcome		Died at age 3 weeks		Died at age 10 months from HOCM		Died at age 14 months from cardiorespiratory failure		Died at age 7 months from respiratory failure		Alive (14 months)
HRAS mutation		c.35‐36GC→TT (G12V)		Negative		c.34G→A (G12S)		c.187G→A (E63K)		c.64C→A (Q22K)

H(O)CM, hypertrophic (obstructive) cardiomyopathy; ASD, atrial septal defect.

*Small fibres and excess of type IIC fibres were reported to be consistent with immaturity and hypotonia.

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).

RESULTS AND DISCUSSION

We discovered heterozygous missense HRAS mutations in four of five patients (fig 1B, table 1). 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 1). 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.

ELECTRONIC DATABASE INFORMATION

• http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db = gene for HRAS cDNA (accession numbers NM_005343 and NM_176795.2) and HRas amino acid (accession numbers NP_005334 and NP_789765) sequences.

• Catalogue of somatic mutations in cancer (COSMIC) at http://www.sanger.ac.uk/genetics/CGP/cosmic/ for somatic mutations observed in HRAS and KRAS.

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

KEY POINTS

• We identified two known activating and two novel germline HRAS mutations in four patients with an unusual form of congenital myopathy histologically charactered by an excess of muscle spindles.

• This myopathic disorder is probably a variant of Costello syndrome (CS), and musculoskeletal problems frequently observed in patients with CS may have the same underlying pathogenesis.

• These observations provide evidence that hyperactive HRas perturbs skeletal muscle development and function in a specific manner.

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

Abbreviations

CFC - cardio‐facio‐cutaneous syndrome

CMEMS - congenital myopathy with excess of muscle spindles

CS - Costello syndrome

NS - Noonan syndrome