Germline mutations in RYR1 are associated with foetal akinesia deformation sequence/lethal multiple pterygium syndrome

Foetal akinesia deformation sequence syndrome (FADS) is characterised by a variable combination of foetal akinesia, prenatal growth restriction, developmental defects (including cleft palate, cryptorchidism, cystic hygroma, heart abnormalities, intestinal malrotation and lung hypoplasia), arthrogryposis and, in some cases, limb pterygia, so that there is phenotypic overlap between FADS and severe cases of multiple pterygium syndrome (MPS) [1]. Clinically MPS can be divided into the severe lethal form (LMPS) and the milder non-lethal Escobar type (EVMPS). MPS is most commonly inherited as an autosomal recessive trait though autosomal dominant and X-linked cases are described [2]-[4]. Both MPS and FADS are genetically heterogeneous and although, in some cases, a diagnosis of a specific primary myopathy, metabolic or neurodevelopmental disorder can be made by clinical and pathological investigations, the underlying aetiology is unknown in the majority of cases [5]. Previously, we and others have reported that germline mutations in genes encoding specific components of the acetylcholine receptor (AChR) complex at the neuromuscular junction may present with autosomal recessively inherited forms of FADS, LMPS and EVMPS [6],[7]. Thus mutations in CHRNG (which encodes the foetal gamma subunit of the acetylcholine receptor) have been associated with FADS, LMPS and EVMPS and mutations in genes that encode other subunits that make up the foetal acetylcholine receptor (CHRNA1, CHRND) or regulators of AChR function (e.g. RAPSN and DOK7) have been described in FADS/LMPS [8]-[10]. Interestingly, mutations in CHRNA1, CHRND, RAPSN and DOK7 can also cause congenital myasthenia syndrome (CMS), a milder disorder that is characterised by muscle fatigability and, rarely, arthrogryposis [11]-[13].

After undertaking genetic linkage studies in a consanguineous family with recurrent FADS/LMPS we mapped a large candidate autozygous region to chromosome 19 and then proceeded to identify a homozygous nonsense mutation in the ryanodine receptor 1 (RYR1) gene. RYR1 encodes the largest known ion channel and the 2.3 MDa homotetrameric ryanodine receptor 1 structure is formed from the 565 kDa RYR1 component proteins. Though the large size (15.3 kb coding sequence in 106 exons) of the RYR1 gene makes mutation analysis challenging, RYR1 mutations have previously been described in a variety of human disease phenotypes. Thus initially RYR1 mutations were described in individuals susceptible to malignant hyperthermia (MHS) [17],[18]. RYR1 mutations associated with MHS are typically heterozygous missense substitutions [19]-[21]. Subsequently RYR1 mutations were described in the context of a variety of histological subtypes of congenital myopathies including central core disease, minicore/centronuclear myopathy with external ophthalmoplegia, centronuclear myopathy and congenital fibre-type disproportion [22]-[24]. Both dominant and recessive forms of RYR1-related congenital myopathies have been described and genotype-phenotype correlations have provided insights into likely clinical-functional relationships. Thus, in a large cohort of RYR1-associated myopathies, dominant mutations tended to be associated with milder phenotypes whereas recessively inherited cases had an earlier onset and a more severe course [25]-[27]. In addition, though the age at presentation of recessive RYR1 myopathies is variable, in a recent series all presented before age 10 years. Whereas RYR1 mutations in dominantly inherited disease tend to cluster in specific protein domains e.g. C-terminal region (amino acids 4,550-4,940) in central core disease and N-terminal regions (amino acids 35–614 and 2,163-2,458) with MHS, recessively inherited mutations are widely distributed throughout the protein [25],[27]. Typically the mutations found in patients with recessively inherited RYR1-myopathies are a combination of null mutation with a missense mutation, though two missense mutations can occur. Thus in a series of 118 patients with RYR1-related recessively inherited myopathies from four recent reports [25],[27]-[30], 61.5% of the cases had a truncating/in frame deletion/splice site mutation in combination with a missense mutation and 38.1% harboured two missense mutations. It is therefore striking that we identified a homozygous null mutation in affected individuals from Family MPS001. This observation is consistent with (a) our previous observation for RAPSN that homozygosity for a null mutation can cause FADS/LMPS other mutation combinations with only a single null mutation can cause a milder phenotype, (b) that mice homozygous for a Ryr1 mutation die in the perinatal period with gross abnormalities of skeletal muscle [31] and (c) a history of foetal akinesia may be found with early onset autosomal recessive RYR1-related congenital myopathies [25]. In addition, Romero et al. [32] reported seven foetuses/infants from six unrelated families affected by central core disease in whom there was a history of foetal akinesia [33]. Four cases from three families were found to harbour RYR1 mutations: three cases (from two families) were compound heterozygotes for RYR1 missense mutations and in one case only a heterozygous missense mutation was detected. Three of the four cases presented at birth and though in one case the foetus died at 32 weeks gestation (following termination of pregnancy after a previously affected sibling). Thus the phenotype in these cases was less severe than we observed and our findings demonstrate that the association between RYR1 mutations and foetal akinesia extends to severe early onset lethal FADS and that histopathological evidence of central core disease is not a prerequisite for molecular investigation of RYR1 in foetal akinesia.

The overall frequency of RYR1-related disease in our FADS/LMPS/EVMPS cohort was 4.5% (3/66; 95% CI: 0 to 9.5%) and 8.3% (3/36; 95% CI 0 to 19.5%) in our FADS/LMPS cohort. The case for pathogenicity of the two in frame deletions is supported by their absence from large repositories of genetic variation in control individuals, segregation with disease within the relevant families and evolutionary conservation of the mutated/deleted amino acid residues. RYR1 is a key component of the excitation-coupling process in skeletal muscle such that opening of RYR1 channels result in release of Ca²⁺ from the sarcoplasmic reticulum and initiation of muscle contraction. The novel in-frame deletion of 27 nucleotides (c.2097_2123del p.(Glu699_Gly707del)) detected in Family MPS002 is predicted to result in a missense substitution (p.E699N) followed by a deletion of 9 amino acids (GWGGNGVGD) within the SPRY2 predicted protein-protein interaction motif [32] (highlighted in Figure 4). Previously missense substitutions within or adjacent to this deletion (c.2113G > C; p.Gly705Arg and p.Asp708Asn) have been reported in recessively inherited myopathies [25]. The exon 45 in-frame deletion (c.7043delGAG) identified in Family MPS003 was predicted to result in loss of a glutamic acid residue at codon 2347. A missense mutation at a nearby residue (p.Arg2355Trp) has been reported in both dominantly and recessively inherited myopathies [20] and it is interesting that this deletion was previously described in the heterozygous state, in two unrelated families with malignant hyperthermia [34]. p.Glu2347 is contained within the MHS/CCD mutation hotspot in N-terminal region 2 (stippled box Figure 4), [16]. Though no history of malignant hyperthermia syndrome was reported in Family MPS003, incomplete penetrance is well recognised in malignant hyperthermia and mutation carriers may not have been exposed to trigger events.

Though our findings establish recessive RYR1 mutations as a cause FADS/LMPS, further work is required to fully establish the frequency of RYR1 mutations in FADS/LMPS cohorts and to address how novel missense or in-frame deletions/insertions might be reliably interpreted in a clinical diagnostic setting. In a recent review of congenital myopathies treated at a single referral centre, a genetic diagnosis was established in two-thirds of cases and almost 60% of those with a genetic diagnosis had a RYR1-related myopathy [27]. Our findings suggest that RYR1-related neuromuscular disease may be a significant cause of FADS/LMPS. Though recessively inherited RYR1-related myopathies have been associated with certain histopathological subtypes such as minicore, centronuclear and congenital fibre-type disproportion myopathies, RYR1 mutations may be associated with other histological subtypes or only nonspecific myopathic features [27]. Extrapolating from these observations, we suggest that, in cases of FADS/LMPS, RYR1 mutation should be performed as part of a multigene diagnostic strategy (e.g. by second generation sequencing analysis) rather than being specifically targeted to cases with histopathological features that are considered characteristic of a RYR1-associated myopathy. The identification of RYR1 mutations as a cause of familial LMPS/foetal akinesia enables accurate reproductive risk prediction and reproductive options including prenatal diagnosis and pre-implantation diagnosis but also might lead to the identification of relatives at risk of malignant hyperthermia.

Arthur B McKie, Atif Al-Saedi, Julie Vogt, Kyra E Stuurman joint first authorship.