A novel ANO3 variant identified in a 53-year-old woman presenting with hyperkinetic dysarthria, blepharospasm, hyperkinesias, and complex motor tics
© The Author(s). 2016
Received: 10 June 2016
Accepted: 24 November 2016
Published: 5 December 2016
Cervical dystonias have a variable presentation and underlying etiology, but collectively represent the most common form of focal dystonia. There are a number of known genetic forms of dystonia (DYT1-27); however the heterogeneity of disease presentation does not always make it easy to categorize the disease by phenotype-genotype comparison.
In this report, we describe a 53-year-old female who presented initially with hand tremor following a total hip arthroplasty. The patient developed a mixed hyperkinetic disorder consisting of chorea, dystonia affecting the upper extremities, dysarthria, and blepharospasm. Whole exome sequencing of the patient revealed a novel heterozygous missense variant (Chr11(GRCh38): g.26525644C > G; NM_031418.2(ANO3): c.702C > G; NP_113606.2. p.C234W) in exon 7 in the ANO3 gene.
ANO3 encodes anoctamin-3, a Ca+2-dependent phospholipid scramblase expressed in striatal-neurons, that has been implicated in autosomal dominant craniocervical dystonia (Dystonia-24, DYT24, MIM# 615034). To date, only a handful of cases of DYT-24 have been described in the literature. The complex clinical presentation of the patient described includes hyperkinesias, complex motor movements, and vocal tics, which have not been reported in other patients with DYT24. This report highlights the utility of using clinical whole exome sequencing in patients with complex neurological phenotypes that would not normally fit a classical presentation of a defined genetic disease.
KeywordsANO3 Anoctamin-3 Dystonia-24 DYT24 Craniocervical dystonia
Dystonias are a heterogeneous group of movement disorders with both primary genetic and secondary environmental etiologies . Over the last few decades, several novel disease associated genes (DYT1-27) have been identified in dystonic syndromes, but the underlying genetic diagnosis remains elusive in most patients . Inherited isolated craniocervical dystonias are rare, and most commonly caused by pathogenic variants in THAP1 (Dystonia-6, DYT6, MIM# 602629) and GNAL (Dystonia-25, DYT25, MIM# 615073) and have adolescent to late adult onset with variable penetrance . To date, targeted clinical gene testing has been performed with limited success, however with the advent of next generation sequencing technologies in the clinic, we are beginning to unravel the complex genetic landscape of primary dystonias.
In this report we describe a patient with atypical craniocervical dystonia presenting with chorea and complex motor tics with a novel variant (Chr11(GRCh38): g.26525644C > G; NM_031418.2(ANO3): c.702C > G; NP_113606.2. p.C234W) in ANO3 that was identified using whole exome sequencing (WES).
Additional file 1: Video S1. Patient history and tremor. Video of patient tremor with diffuse abnormal choreiform movements affecting her upper extremities and torso. (WMV 19181 kb)
Additional file 2: Video S2. Patient gait and complex motor tics. Video of patient performing movements while walking, such as clapping her hands, which reflect complex motor tics. The patient’s lower extremities were unaffected. (M4V 3657 kb)
Additional file 3: Video S3. Motor impersistence with tongue protrusion. With tongue protrusion. (WMV 15275 kb)
Initial workup of the patient for secondary causes of dystonia was unrevealing and included complete blood count (CBC) with peripheral smear (no acanthocytes seen), normal sedimentation rate, vitamin B12, methylmalonic acid, electrolytes, ammonia, ceruloplasmin, copper, liver function tests, thyroid-stimulating hormone (TSH), paraneoplastic profile, tests of connective tissue disorders, sera rapid plasma reagin (RPR), negative anti-thyroid peroxidase (TPO) antibodies and tissue transglutaminase antibodies. Additionally, the patient’s local physician reported genetic testing for Huntington’s disease that was normal.
The patient had no history of diplopia, oscillopsia, or history of previous ophthalmologic disease processes. The patient’s neuro-ophthalmologic examination revealed normal visual acuity and color vision. The patient was noted to have constant bilateral forcible eyelid closure and it was difficult to demonstrate any definite apraxia of eyelid opening or eye closure. The patient was noted to have bilateral upward deviation of the eyes that was consistent with physiologic Bell’s phenomenon. The patient’s ocular motility was full with lateral gaze intact. Occasionally she developed an esotropia with constriction of the pupils compatible with spasm of the near reflex. The patient did not have nystagmus. Cranial nerves V and VII were intact except for occasional abnormal facial movements and frequent eye closure.
The patient’s voice was hypophonic, with high-pitch and strained stuttering speech (Additional file 1: Video S1). She repeated consonants at the beginning of some sentences and had some elongated vowel sounds as well. The patient understood what was said to her and despite her challenges with speech, she was able to communicate her ideas although she exhibited echopraxia. She also had several episodes of spontaneous crying that were suggestive of a pseudobulbar component to her disease. The patient exhibited perceptual evidence of a moderate dysarthria, with clinical features that appear compatible with a hyperkinetic dysarthria. The patient’s receptive and expressive language skills were unimpaired, but her writing legibility and reading from computer screen were affected due to uncontrolled upper extremity movements, as well as visual sensitivity.
The EEG was moderately abnormal due to the presence of excessive myogenic activity. The patient was tense and experienced a number of abnormal movements (tremor, jerks) that were not associated with epileptiform activity. These were only associated with movement and myogenic artifacts but baseline activity was maintained. The background activity was predominantly around 7 Hz and was intermixed with beta activity that was symmetrical and reactive. There was excessive beta activity that was thought to be due to medication effect as the patient was taking benzodiazepine medication. The background activity was mainly in theta frequency band and was thought to represent either medication effect or a more organic pathology such as encephalopathy.
Clinical WES was performed by GeneDX (XomeDxPlus), which also included mitochondrial DNA sequencing. Briefly, genomic DNA was extracted from blood from the proband and her mother. As described in the clinical testing methodology by GeneDX, the SureSelectXT Clinical Research Exome (Agilent) capture kit was used for exome enrichment and sequencing was done on an Illumina HiSeq 2000 that generates 100 bp paired-end reads. Bi-directional sequence was assembled, aligned to reference gene sequences based on human genome build GRCh37/UCSC hg19, and analyzed for sequence variants using a proprietary analysis tool (Xome Analyzer, GeneDx) as previously described . Sanger sequencing was used to confirm all potentially pathogenic variants identified in this individual and in the parental sample . Sequence alterations were reported according to the Human Genome Variation Society (HGVS) nomenclature guidelines. The exome was covered to a mean depth of 97×, with a quality threshold of 95.7%.
Clinically reportable variants found within the patient by whole exome sequencing or mitochondrial DNA sequencing
NCBI accession number
Amino acid change
c.702C > G
c.473 T > C
In this report we describe a 53-year-old female patient with a novel heterozygous missense variant of uncertain significance (VUS) (Chr11(GRCh38): g.26525644C > G; NM_031418.2(ANO3): c.702C > G; NP_113606.2. p.C234W) in ANO3 who had a late and precipitous onset of disease. The patient shares many of the same clinical and pathological features as patients described with autosomal dominant craniocervical dystonia, including initial manifestation in the form of a progressive tremor, with development of a dystonia affecting the upper extremities, dysarthria, and blepharospasm. However, the patient also has mixed hyperkinesias manifesting as chorea, as well as simple and complex motor and vocal tics, which have not been observed in other patients with DYT24. Potentially complicating the patient’s phenotype is the fact that she was on promethazine for several months following a total right hip arthroplasty. Her hyperkinesias became evident while she was on the promethazine thus, leading to speculation that some of her hyperkinesias could have a tardive etiology.
The p.C234W ANO3 variant described in this patient is classified as a VUS by clinical report, is predicted to be damaging by in silico analysis, and has never been reported in any publically available databases. Given the good phenotypic overlap, we posit that this variant may contribute to the patient’s disease. However, a recent study identified a neighboring c.704A > G (p.Y235C) missense mutation in 4 of 4300 European American individuals within the NHLBI-ESP cohort ; the variant is also reported 35 times (out of 114122 alleles) in ExAC database. As yet there is no report of the p.Y235C variant linked with disease, even though it is rare and predicted to be damaging . It remains possible that with the noted reduced penetrance and later onset of tremor observed in some families, that the more benign manifestations of DYT24 could go undetected in a seemingly healthy control population.
To date, 8 pathogenic missense variants in ANO3 have been identified including: c.2540A > G (p.Y847C), c.1480A > T (p.R494W), c.1470G > C (p.W490C), c.161C > T (p.T54I), c.2053A > G (p. S685G), c.2586G > T (p.K862N), c.2190C > T, c.2497A > G (p.I833V), c.2917G > C (p.G973R) (Table 1) . These variants fall within the transmembrane spanning alpha helices, within the intracellular and extracellular loops, and within the N and C-termini of ANO3 (Fig. 1). While these variants do not implicate a hotspot, their spatial relationships within the 3D protein structure are unknown (Fig. 1). Future work investigating the 3D structure could shed light on common mechanisms of alteration by each variant. No nonsense or frame-shift mutations in ANO3 have been reported in association with DYT24, however there are several rare frameshift and nonsense variants present in ExAC, suggesting that there may be additional phenotypes associated with this gene such as autism spectrum disorders .
The ANO3 p.C234W substitution in the patient under study is located within the N-terminus of the protein (amino acids #1-403). The only other variant described in the N-terminus (NM_031418.2, exon 2, c.161C > T, p.T54I) was in a patient who was diagnosed with familial essential tremor (Table 1) . The function of the N-terminal region of ANO3 and other anoctamin family members remains poorly described, but may be involved in dimerization or interactions with other proteins such as calmodulin, as has been demonstrated in ANO1 (TMEM16A) .
Only the c.1470G > C (p.W490C) variant has been evaluated using functional studies, with patient fibroblasts showing reduced ATP- and thapsigargin-induced calcium signal compared to controls, that was thought to be due to a smaller calcium pool in the endoplasmic reticulum . ANO3 is expressed throughout the central and peripheral nervous system. In one study, rats were shown to have high Ano3 expression in a subset of nociceptive neurons in dorsal root ganglia (DRG) [6, 18]. Ano3 knockout rats (Ano3 −/− ) were hypersensitive to high temperatures and electrophysiological measurement from DRG neurons from these animals showed action potential broadening and lower threshold for action potential firing [6, 18]. Interestingly, Na+-activated K+ current was also strongly reduced in Ano3 −/− rats [6, 18]. Colocalization experiments showed that Ano3 directly interacts with Kcnt1 (Slack), a sodium-activated potassium channel implicated in infantile epileptic encephalopathy-14 (EIEE14, MIM# 614959) [6, 18]. Ano3 may enhance the activity of Kcnt1, which in turn helps regulate the excitability of nociceptive neurons [6, 18].
As we see an increase in the utilization of whole-exome and -genome sequencing in the clinic, there will be an ever-increasing demand for methods of determining disease relevance and pathogenicity. In this case report we identified a novel mutation of likely pathogenicity in a gene known to present with a similar phenotype. For rare protein variants such as ANO3 p.C234W, clinical genetic studies may not be sufficient to prove pathogenicity, rather additional functional studies will likely be needed. However with this in mind, it is critical that robust functional assays are developed that truly reflect the underlying disease mechanisms occurring, that is to say not all functional effects are created equally. As we gain a better understanding of the pathways and mechanisms underlying DYT24, and dystonia in general, clarification of rare variants will better direct targeted drug design and clinical trials.
Complete blood count
Dorsal root ganglia
Probability of loss-of-function intolerant value
Rapid plasma regain
Variant of uncertain significance
Whole exome sequencing
We would like to thank the patient and her family for participating in this study. We would also like to thank the Mayo Clinic Center for Individualized Medicine (CIM) for supporting this research through the CIM Investigative and Functional Genomics Program.
The authors would like to thank the Mayo Clinic Center for Individualized Medicine for supporting this research.
Availability of data and materials
The patient variant identified in this case report has been deposited in ClinVar under submission accession SCV000299087.2. This entry can be accessed at: https://www.ncbi.nlm.nih.gov/clinvar/variation/254028/
PRB, JAV, and PSA designed the study and wrote the manuscript. PRB, JMG, MAC, NJB, OAR, and EWK helped with data collection and provided critical review of the manuscript. MTZ helped with the generation of figures and writing of the manuscript. PWB, JAV, KGH, and PSA collected the clinical data. All authors approved the final version of the manuscript for publication.
The authors declare that they have no competing interests.
Consent for publication
Written informed consent was obtained from the patient for publication and accompanying images and video. A copy of the written consent is available for review by the editor.
Ethics approval and consent to participate
The patient provided consent for sample collection and subsequent analysis under a protocol approved by the institutional review board of the Mayo Clinic. The patient provided an additional consent for publication of this case report as well as accompanying images and video.
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