Summary

Compound heterozygous or homozygous variants in the STAC3 gene have been identified in patients presenting with a common set of myopathic features. This disorder was originally called Native American Myopathy, but similar phenotypes in individuals of different ancestries suggest the disorder should be renamed Stac3 myopathy. In all cases reported to date, inheritance is autosomal recessive.  In some cases, an association with MH has been noted, but insufficient clinical details, and what appears as a complete lack of follow up in vitro contracture testing, casts some doubt on a bona fide association of MH with STAC3variants.  Investigations of Stac3 structure, cellular location and function, place the protein in the cytosol positioned in close proximity to both the DHPR and RyR1 and importantly, Stac3 is one of five proteins that together represent the minimal unit for functional EC-coupling. A direct interaction between the DHPR and Stac3 has been demonstrated but insufficient evidence is available to confirm a similar interaction between RyR1 and Stac3. Most studies suggest that homozygous STAC3 variants have little effect on Ca2+ release via RyR1 agonists, but there is general agreement that there are significant loss of function effects on EC-coupling.  MH is generally considered to be inherited in an autosomal dominant manner, with associated genetic variants producing a gain of function phenotype in terms of hypersensitivity to RyR1 agonists.10  RYR1 variants that result in MH-susceptibility and/or other myopathies, exhibit a range of different functional consequences on both EC-coupling and Ca2+ release form the SR.  These include, leaky, uncoupled and unresponsive to agonist when expressed in heterologous systems or myotubes. Given the complexity of the MH phenotype and associated genotypes, as well as its cellular roles, Stac3 should be considered a potential candidate for MH-susceptibility.  However, the paucity of clinical information surrounding the MH (or MH-like) episodes noted in patients with STAC3 variants, the range of associated myopathies and the lack of robust experimental evidence in favour of STAC3 variants resulting in a phenotype consistent with MH-susceptibility, it would be premature to include STAC3 variants as diagnostic for MH-susceptibility per se.  Further research needs to be carried out to clearly define the phenotypic and pathogenic consequences of STAC3 variants before they can be appropriately assessed for a role in MH-susceptibility.  In addition, the clinical reactions to volatile anaesthetics observed in patients with STAC3 variants need to be more effectively investigated and documented and also followed up with in vitro contracture testing to assess relevance to MH-susceptibility.  In the meantime, given the possible association of STAC3 variants with MH or MH-like reactions to volatile anaesthetics, MH-susceptibility should be considered if a rare homozygous or compound heterozygous STAC3 variant is identified in any genetic investigation associated with a myopathy. As with patients diagnosed with central core disease, any patient with a phenotype consistent with NAM should, for the present, be considered at risk for MH and be given non-triggering anaesthesia. 

Background

The first association between a STAC3 variant and MH-susceptibility was reported in Native American (Lumbee) Indians in 2013 with the identification of the homozygous NM_145064.2:c851G>C variant corresponding to the amino acid change p.Trp284Ser in the Stac3 protein.1  Native American myopathy (NAM) was first associated with MH-susceptibility in Lumbee Indians in 1987 and 1988.2, 3  These two original case reports describe the phenotype presenting at birth with congenital abnormalities, including weakness, cleft palate and multiple skeletal anomalies. The mode of inheritance was autosomal recessive.  Of six children (three of whom were related) in one study, 2 presented with MH symptoms during cleft palate repair using halothane for anaesthesia. Dantrolene was not administered and no details were provided to assess the severity of the suspected MH reactions.3  The second report detailed two anaesthetic events using halothane in one case.  The first at 7 weeks of age was uneventual, while the second at 3 months resulted in an MH reaction where the baby became “extremely rigid”, and the temperature rose to 38oC. The clinical parameters measured did not appear to be unusually abnormal and the condition improved as soon as halothane was withdrawn.  Dantrolene was administered (1 mg/kg) and after 36 hours the surgery was completed without the use of halothane.2  A further study of 14 Lumbee Indians with a phenotype consistent with NAM reported 4 cases of MH during surgery which resolved with dantrolene administration. There was one report of an anaesthetic-related death in one family, but no details were provided. Thirty-nine exons of RYR1 were screened in one patient, with no potentially pathogenic variants identified. No clinical parameters were provided and no subsequent in vitro contracture tests were carried out.4  This study was followed by a genome-wide 10K single-nucleotide screen which identified a linked locus on chromosome 12q13.13-14.1.5  The STAC3 gene was positioned at this locus and the NAM-associated NM_145064.2:c851G>C variant subsequently identified by Sanger sequencing of all exons and surrounding splice sites of the STAC3gene.1  

The same NM_145064.2:c851G>C variant has since been identified in 18 individuals in 12 families presenting with congenital myopathy with a range of dysmorphic features similar to those in NAM patients.6Variant identification was either by Sanger sequencing of exon 10 of STAC3 or in 4 individuals, targeted sequencing of a panel of known neuromuscular genes.  Seventeen of the individuals were homozygous for the NM_145064.2:c851G>C variant, while one was a compound heterozygote with the NM_145064.2:c851G>C variant as well as a splice variant in the STAC3 gene which would result in an in-frame deletion in exon 12. The families were of African, Afro-Caribbean, Comorian, Middle Eastern or South American Origin. Adverse reactions to anaesthetics were reported in 10 patients after general anaesthesia and two were labelled MH-like. The MH clinical grading scale did not appear to have been used and no in vitro contracture tests were reported.6  An independent case report detailed a 24 month girl with dysmorphic features, hypotonia and poor respiratory effort. Targeted sequencing using a neuromuscular panel of 78 genes revealed no potential pathogenic variants. Subsequent whole exome sequencing identified the homozygous NM_145064.2:c851G>C variant. The parents of the girl were of remote Cherokee and Shawnee heritage.7  Another study reports the homozygous NM_145064.2:c851G>C variant in a Puerto Rican proband with Moebuis syndrome with associated MH.8  The NM_145064.2:c851G>C variant also occurred together with a STAC3 deletion resulting in a frameshift.  This was in a proband from Qatar who presented with Carey-Fineman-Ziter syndrome and a history of MH in the probands sister, also carrying the same STAC3 variants.  No details were provided about the MH episodes. Phenotypically these three individuals appeared to have similar facial and skeletal features as described for Native American Myopathy and the mode of inheritance was autosomal recessive. The authors note however, that while the three disorders exhibit clinical overlap, they have important differences and STAC3 variants have not been identified in a large cohort of people with Carey-Fineman-Ziter or Moebuis syndromes. Other STAC3 variants associated with a phenotype consistent with NAM, were identified in a patient of Turkish ancestry.  In this case compound heterozygosity was observed with the STAC3 variants NM_145064.2:c.862A>T (p.Lys288*) and NM_145064.2:c.432+4A>T which resulted in a premature stop codon and two different splice variants, respectively. Potential protein products were not investigated, although premature termination or nonsense-mediated decay were predicted. No association with MH in this patient was reported.9

In summary, homozygous or compound heterozygous STAC3 variants appear to cause the NAM myopathic phenotype, which is present at birth and appears to overlap with a range of other myopathies. In this there are parallels with RYR1 variants which can be associated with MH-susceptibility as well as myopathies including central core disease, multiminicore disease, and King-Denborough syndrome.10  Some RYR1variants linked to these disorders appear to exhibit recessive inheritance and some result in altered Ca2+release from intracellular stores, while others do not.

Cellular function of Stac3 protein

STAC3, one of a family of three genes encoding adaptor proteins, is predominantly expressed in skeletal muscle and is named for its protein domains, Src homology three (SH3) and cysteine rich (C1) domain 3. The amino acid sequences of the three isoforms exhibit a high degree of identity but STAC and STAC2appear to be largely expressed in neuronal tissue. Three independent studies described potential functions of the Stac3 protein in skeletal muscle. The first of these reported a STAC3 knockout in mice. 11  STAC3 -/- caused perinatal lethality in mice, whereas STAC3+/- mice were viable, fertile and grew normally as their wild type littermates.  The STAC3-/- mice had abnormal body curvature, with abnormal myofibres which were disorganized, such that muscle would be unlikely to function. This study was the first to report a role for the Stac3 protein in skeletal muscle development and function.11  The second study used a forward genetic screen in zebrafish to identify new genes involved in excitation-contraction (EC) coupling.  One mutation was autosomal recessive with mutants dying as larvae and with defective motor behaviours at early stages of development. This mutation was mapped to the zebrafish STAC3 gene, which was similar to the murine STAC3 gene.1 

Functional analysis

Functional analysis in the zebrafish system showed that mutant (STAC3 knockout) and wildtype sibling muscles both respond in the same way when exposed to caffeine, suggesting the contractile apparatus was intact.1 In addition, the STAC3-/- muscle formed normal triad junctions.  Ca2+ transients were however, greatly reduced in both mutant slow and fast twitch fibres suggesting a defect in EC-coupling. These experiments identified Stac3 as a new component of EC-coupling in skeletal muscle.  The STAC3NAM variant was then expressed in the STAC3-/- zebrafish.  Unlike the wildtype rescue, expression of STAC3NAM did not rescue touch-induced swimming although some Stac3NAM localized to triads. Stac3NAMwas able to rescue Ca2+ transients in slow twitch but not fast twitch muscle fibres. A normal response of muscle fibres from STAC3 knockout mice to 4-cmc, but lack of response to depolarization supported a role for Stac3 in EC-coupling, and confirmed normal function of the RyR1 Ca2+ channel.12,13  Using fluorescently labelled recombinant proteins, Stac3 was shown to participate in delivery of Cav1.1 to the plasma membrane of tsA201 cells, a cell type that does not normally express Cav1.1.  Stac3 also conferred functional expression of Cav1.1 in tsA201 cell line as shown by whole cell patch-clamping.14  Stac3 can be incorporated into a skeletal muscle triad in the absence of the dihydropyridine receptor (DHPR) as it has been shown to colocalize with RyR1 in dysgenic (Cav1.1-/-) myotubes.15  In the absence of Stac3, Cav1.1 did not produce Ca2+ currents or membrane-bound charge movements. However, in the presence of Stac3, there were no significant differences between tsA201 cells expressing Cav1.1 and dysgenic myotubes (RYR1-/-) in the rate of Ca2+ current activation.14  When the NAM variant was introduced into mouse STAC3and expressed in murine STAC3 knockout myotubes, a reduced amount of membrane expression of Cav1.1 was observed compared to rescue by wildtype STAC3.16  This study also showed that while the Stac3 NAM variant caused a partial recovery of L-type Ca2+ currents in STAC3 knockout myotubes, there was very little restoration of EC-coupled Ca2+ release.  Similar results were observed in a study expressing the STAC3NAMvariant in STAC3-/- zebrafish.17  In addition to the defect in EC-coupling, this study showed that myofibers expressing the STAC3NAM variant showed a significantly higher Ca2+ release to saturating [caffeine] compared to either STAC3-/- or STAC3 wildtype rescued myofibres, which correlated with a higher SR [Ca2+] in Stac3NAM myofibers. The authors suggested that the increased response to caffeine may be due to an increased SR Ca2+ store and possible RyR1 regulation by luminal Ca2+.17

Another study using tsA201 cells established a minimal set of proteins required to confer EC-coupling that resembles that in muscle.18  This study showed that co-expression in tsA201 cells of five proteins Cav1.1, RyR1, b1a (the auxiliary cytoplasmic subunit of the DHPR), Stac3 and junctophilin 2 was sufficient to recapitulate EC-coupled Ca2+ release by using voltage-clamp techniques. 

Myotubes from one patient with a homozygous NM_145064.2:c851G>C variant were assessed for Ca2+release in response to 4-cmc and K+. EC50 values, resting or store [Ca2+] were not significantly different to a control but considerably less Ca2+ was released at maximal concentrations of either 4-cmc or K+. Levels of STAC3, DHPR-b1a, and Cav1.1 were measured by immunoblotting in three cases and three controls, with no differences observed. Co-immunoprecipitation of Stac3 protein with Cav1.1 showed no differences between cases and controls.  The reciprocal co-immunoprecipitation was not carried out. 6  

Functions of Stac3 protein domains

By using fluorescently-labelled truncated forms of the protein as well as site-directed mutants Stac3 has since been shown to interact with Cav1.1 via its C1 domain (towards the N-terminus) and more specifically, a binding pocket containing residues Val104 and Tyr133 was necessary for targeting Cav1.1 to the membrane.19  The NAM variant resides in the SH3 domain (towards the C-terminus) which suggests the SH3 domain is responsible for Stac3 function in EC-coupling. These observations are supported by another study which used isothermal calorimetry to study protein-protein interactions.20  This provided strong biophysical evidence that the Cav1.1 II-III loop (amino acids 728 to 775) binds to the SH3-1 domain of Stac3 between amino acids 245 and 364, the position of the NAM variant. A crystal structure of the Stac2 isoform in complex with Cav1.1 (residues 747-760) at 1.73 A resolution revealed that Trp329 (analogous to Stac3 Trp 284) is in a binding pocket with a side chain nitrogen forming a hydrogen bond with the main chain of the peptide and an additional cation-pi interaction with Arg757 of Cav1.1. By mutating three Cav1.1 residues that interact with the Stac2 binding pocket, binding was not detectable by isothermal calorimetry.  Similarly, by introducing the Trp284Ser or Trp329Ser mutation into Stac2 and Stac3, respectively, isothermal calorimetry could not detect an interaction with the Cav1.1 II-III loop. In support of a functional role for the interaction between the Stac3 SH3-1 domain and the Cav1.1 II-III loop, the authors reconstituted dysgenic (Cav1.1-/-) myotubes with wildtype Cav1.1 or Cav1.1 containing alanine substitutions for the three interacting residues.  Very few of the mutant myotubes responded to electrical stimulation, and those that did exhibited significantly weaker Ca2+ transients compared to wildtype. The decay of the transients was not affected by the II-III loop mutants suggesting that overall Ca2+ handling was not affected. A similar approach using expression of various forms of Cav1.1 and Stac proteins in either  tsA201 cells or Stac-/- myotubes  confirmed  a functional interaction between the Cav1.1 II-III loop and Stac3.21 A weak interaction between Stac3 and RyR1 has been observed using immunoprecipitation with RyR1 antibodies, however the amounts of Stac3 and RyR1 observed in the immunoblot were barely above background and not particularly convincing.1 The reciprocal immunoprecipitation using Stac3 antibodies was not reported. To date, a direct interaction between Stac3 and RyR1 has not been corroborated, however, endogenous Stac3 has been shown to be present in triads of Cav1.1-/- muscle, suggesting that Stac3 can be correctly targeted without the DHPR.15 Models describing a direct interaction between Stac3 and RyR1 have been proposed, but further experimental evidence is required to provide support for these models.22

References

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