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We recently reported in Brain a large multi-centre study suggesting that truncating SYNE1 mutations are a recurrent cause of recessive ataxia also outside Quebec (23/434 = 5.3% of patients with unexplained early-onset ataxia) (Synofzik et al., 2016). Moreover, this study indicated that SYNE1 ataxia might commonly present with complex multisystemic phenotypes rather than pure cerebellar ataxia, including in particular motor neuron and brainstem dysfunction (Synofzik et al., 2016). However, confirmation of both the frequency estimate and the complex phenotypic spectrum is still lacking, raising the question whether these findings indeed represent systematic results rather than just exceptional or coincidental associations.
Here, we now report the mutational and phenotypic findings on SYNE1 from a second, independent ataxia series of 116 patients. These findings not only confirm the high frequency of SYNE1 ataxia and extend both the mutational spectrum (seven novel index patients, 12 novel SYNE1 mutations) and the multisystemic phenotypic spectrum, including amyotrophic lateral sclerosis (ALS)-like motor neuron features, they also indicate that muscle immunohistochemistry might provide a valuable diagnostic biomarker for clarifying the pathogenic contribution of SYNE1 missense variants. This observation may have consequences for clinical SYNE1 diagnostics, as diagnostic tests are urgently needed for clarifying the role of the ubiquitous SYNE1 missense variants with unknown clinical significance (VUS), which are frequently found in neurological and non-neurological patients and controls (Synofzik et al., 2016).
Index subjects (n = 116) with unexplained degenerative ataxia compatible with autosomal recessive inheritance (no ataxia in the parental generation) and negative for trinucleotide repeat expansions causing Friedreich’s ataxia (FRDA) were compiled from three sources: the Early Onset Ataxia Consortium (n = 88), the ataxia centre Antwerp, Belgium (n = 9), and the ataxia centre Milano, Italy (n = 19). All subjects originated from European, Middle East or Mediterranean countries. This series was sequenced after and independent from the cohort of the previous SYNE1 study (Synofzik et al., 2016). None of the subjects had been part of the previous screening cohort. Subjects were screened for SYNE1 mutations by one of the following three next-generation sequencing methods: (i) a high coverage HaloPlex gene panel kit (Agilent) including >120 known ataxia genes (n = 88); (ii) targeted exon-capture sequencing strategy (Illumina Nextera Rapid Capture Custom kit) including 107 known ataxia genes (n = 19); or (iii) whole-exome sequencing using the SureSelect Human All Exon 50 Mb kit (Agilent) (n = 9) (for technical details, filter settings, and criteria for inclusion of SYNE1 missense variants, see the online Supplementary material). All index patients carrying two pathogenic SYNE1 alleles and their affected siblings received a systematic clinical assessment, as described in detail in the previous study (Synofzik et al., 2016) (Table 1).
We identified six index patients carrying two truncating SYNE1 alleles and one index patient carrying one truncating plus one missense SYNE1 allele, thus yielding a total of seven index patients out of 116 in the total ataxia cohort (6%). In these seven index patients, we observed a total of 12 different mutations, consisting of seven frameshift, three nonsense, one splice site, and one missense mutation in the actin binding domain (Table 2 and Fig. 1A). All 12 mutations, each confirmed by Sanger sequencing, have not yet been reported in association with human disease. For all families where DNA of at least one parent was available (5/7 families), we were able to show that the respective parent carried only one of the two corresponding SYNE1 variants, supporting a biallelic localization of the variants in the index child. In one of the remaining two families, consanguinity was also suggestive of a biallelic location of the observed homozygous mutations.
The missense variant c.4732C>T; p.P1578S (observed in Patient 2-1) (i) segregated in trans with the frameshift deletion c.23767_23768delCA; p.Q7923Efs*4; (ii) had a very low minor allele frequency in ExAc (2.26E-04) and EVS6500 (7.70E-05); (iii) predicted to be damaging by three out of three in silico algorithms (Mutation Taster) (Schwarz et al., 2010; Wang et al., 2010); PolyPhen-2 HDIV (Adzhubei et al., 2010), and Likelihood Ratio Test (LRT) (Chun and Fay, 2009); (iv) highly evolutionary conserved with scores PhyloP 100way = 6.03 and PhastCons 100way = 1.0 (Pollard et al., 2010); and (v) ranked among the top 1% of all 8.6 billion single nucleotide variants in the GRCh37/hg19 (CADD score: 24.2) (Kircher et al., 2014). However, given that even rare, well-conserved missense SYNE1 variants have been shown to present a ubiquitous finding in control subjects (Synofzik et al., 2016), additional functional evidence is needed to demonstrate a pathogenic contribution. Immunohistochemistry assessment of muscle tissue in Patient 2-1 showed severely reduced SYNE1 staining (Fig. 1B; for methodological details see Supplementary material), in line with the findings seen in patients with two truncating SYNE1 mutations (Synofzik et al., 2016). This suggests that the p.P1578S missense mutation, in combination with another truncating SYNE1 variant, leads to loss of SYNE1 protein.
Clinical data were available for all eight affected subjects belonging to the seven index families. Age of disease onset was variable, ranging from 6 to 42 years (median onset: 14 years). Disease started in 4/8 patients (50%) with non-ataxia features, namely facial muscle fasciculations, speech disturbances, spasticity and cognitive deficits, respectively. At last examination (median age: 35 years), all 8/8 patients showed a ‘cerebellar ataxia plus’ phenotype, i.e. none of them showed the classical SYNE1 phenotype of pure cerebellar ataxia. Seven of eight subjects (88%) exhibited ataxia plus motor neuron disease, which involved both upper motor neuron dysfunction (bilateral positive extensor plantar reflex and/or spasticity) and lower motor neuron dysfunction muscle atrophy, including bulbar muscles (Fig. 1C) combined with reduced reflexes; fasciculations clinically or on EMG; acute denervation in EMG] in 5/8 patients (63%), and only upper motor dysfunction in 2/8 patients (25%). In 2/8 patients (one also with motor neuron disease), ataxia was complicated by additional moderate-to-severe cognitive impairment across manifold neuropsychological domains, affecting in particular processing speed, attention, memory, and executive functions (for detailed neuropsychological test results of Patient 4-1, see Supplementary material). One index patient (Patient 2-1) showed a severe and complex multisystemic phenotype, comprising of very early onset (6 years of age) ataxia, upper and lower motor neuron damage, including acute neurogenic changes with creatine kinase elevation, and unilateral diaphragm paralysis with restrictive lung function. This finding confirms that the complex early-onset phenotypes reported in three subjects in the previous report (Synofzik et al., 2016) are a recurrent manifestation of SYNE1 disease.
These findings provide evidence from an independent, second series that SYNE1 deficiency is indeed a relatively common cause of non-FRDA recessive ataxia also outside Quebec, with a frequency of 5% (Synofzik et al., 2016) to 6% (this report). Moreover, these findings further extend the mutational spectrum of SYNE1 disease by 12 novel mutations spread throughout the gene (yet sparing the KASH-domain, Fig. 1A), demonstrating the need to sequence not only particular ‘hot spot’ regions, but indeed the whole giant 146-exon gene.
These findings also help to explicate the multisystemic spectrum and disease course of SYNE1 disease. They show that multisystemic ataxia plus syndromes are not the exception, but the rule, with up to 100% of SYNE1 patients outside Canada presenting with ataxia plus syndromes, in particular complicated by upper and/or lower motor neuron disease. In up to 50% of patients, SYNE1 disease even starts with non-cerebellar features. The combination of upper plus lower motor neuron disease (seen in 63% of patients), which can include fasciculations, acute denervation and damage of bulbar motor neurons (see tongue atrophy, Fig. 1C), resembles ALS-like motor neuron features. Our findings demonstrate that such complex early-onset syndromes with upper and lower motor neuron disease and respiratory features are not a coincidental finding, but a recurrent manifestation of truncating SYNE1 mutations (Izumi et al., 2013; Synofzik et al., 2016). This contrasts the view on SYNE1 ataxia as relatively benign, slowly progressive ataxia, which was largely traced from French-Canadian SYNE1 patients (Dupre et al., 1993/2012, 2007).
Determining the pathogenicity of SYNE1 missense mutations will present a complex challenge in future clinical diagnostics, given that even rare, well conserved missense variants can be found in 5.6% of controls with unrelated disease conditions or phenotypes (Synofzik et al., 2016). Here we show that SYNE1 muscle staining might help to show a loss of SYNE1 protein in subjects carrying a SYNE1 missense variant. Muscle immunohistochemistry would thus help to corroborate the pathogenicity of at least some well selected, rare, highly conserved SYNE1 missense variants, in particular if located in trans with another truncating variant.
We are grateful to J. Reichbauer (Hertie-Institute for Clinical Brain Research, Tübingen) and Inge Bats (University of Antwerpen) for technical support.
The sequencing work of this study was supported, in part, by Actelion pharmaceuticals. This company was not involved in any parts of data acquisition, data analysis or data interpretation. This work was moreover supported by the Else Kröner-Fresenius-Stiftung (award to M.S.), the European Union [grant F5-2012-305121 ‘NEUROMICS’ to L.S., P.B., T.K., and grant PIOF-GA-2012-326681 ‘HSP/CMT genetics’ and ‘NEUROLIPID’ (01GM1408B) to R.S.], E-RARE grants of the respective national research ministries to the EUROSCAR project (to L.S., P.B., and F.T.) (grant 01GM1206), grant RF-2011-02351165 from the Italian Ministry of Health to F.T., the EUROSPA project (grant 01GM0807) (to L.S. and P.B.), and the National Institute of Health (NIH) (grants 5R01NS072248, 1R01NS075764, 5R01NS054132, 2U54NS065712 all to S.Z.). This work was supported by the Association Belge contre les Maladies Neuromusculaires (ABMM). I.M. is supported by a PhD fellowship of the agency for Innovation by Science and Technology (IWT). J.B. is supported by a Senior Clinical Researcher mandate of the Research Fund - Flanders (FWO).
Conflicts of interest: Dr Bauer is Chief Operating Officer at Centogene AG, Rostock, since January 2016. This company has no direct market-related interests in this study and was not involved in any parts of this study. Dr Synofzik received consulting fees from Actelion Pharmaceuticals Ltd. The remaining authors report no conflicts of interest.
Supplementary material is available at Brain online.
We acknowledge the following members of the Early Onset Ataxia (EOA) consortium for their contribution of ataxia patients to the screening cohort: Thomas Klopstock, Claudia Stendel (Department of Neurology with Friedrich-Baur-Institute, Ludwig-Maximilians-University, Munich, Germany); Christoph Kamm, Ales Dudesek (Department of Neurology, University of Rostock); Stefan Vielhaber, Dorothea Henkel (Department of Neurology, University of Magdeburg); Janina Gburek-Augustat (Tübingen).