- Open Access
Severe dystonia, cerebellar atrophy, and cardiomyopathy likely caused by a missense mutation in TOR1AIP1
© Dorboz et al.; licensee BioMed Central. 2014
- Received: 21 July 2014
- Accepted: 28 October 2014
- Published: 26 November 2014
Dystonia, cerebellar atrophy, and cardiomyopathy constitute a rare association.
We used homozygosity mapping and whole exome sequencing to determine the mutation, western blot and immunolabelling on cultured fibroblasts to demonstrate the lower expression and the mislocalization of the protein.
We report on a boy born from consanguineous healthy parents, who presented at three years of age with rapidly progressing dystonia, progressive cerebellar atrophy, and dilated cardiomyopathy. We identified regions of homozygosity and performed whole exome sequencing that revealed a homozygous missense mutation in TOR1AIP1. The mutation, absent in controls, results in a change of a highly conserved glutamic acid to alanine. TOR1AIP1 encodes lamina-associated polypeptide 1 (LAP1), a transmembrane protein ubiquitously expressed in the inner nuclear membrane. LAP1 interacts with torsinA, the protein mutated in DYT1-dystonia. In vitro studies in fibroblasts of the patient revealed reduced expression of LAP1 and its mislocalization and aggregation in the endoplasmic reticulum as underlying pathogenic mechanisms.
Conclusions and relevance
The pathogenic role of TOR1AIP1 mutation is supported by a) the involvement of a highly conserved amino acid, b) the absence of the mutation in controls, c) the functional interaction of LAP1 with torsinA, and d) mislocalization of LAP1 in patient cells. Of note, cardiomyopathy has been reported in LAP1-null mice and in patients with the TOR1AIP1 nonsense mutation. Other cases will help delineate the clinical spectrum of LAP1-related mutations.
- Cerebellar atrophy
- Nuclear membrane
Dystonia, cerebellar atrophy and cardiomyopathy constitute a very rare association, observed in rare mitochondrial disease and organic acidemia. We report on a boy with early onset dystonia, for whom work-up performed in several institutions failed to reach a diagnosis.
We performed homozygosity mapping in the patient by using the Human Omni2.5 array (Illumina), and whole-exome sequencing (IntegraGen), using the SureSelect V4 capture kit (Agilent) and the HighSeq2000 sequencer (Illumina) .
For cell cultures work up, skin fibroblasts were maintained in DMEM (Life Technologies) with 10% FBS and penicillin/streptomycin. Cells were either plated on glass coverslips for immunofluorescence or pelleted for protein analysis. Cells were imaged using an Olympus FV-1000 confocal microscope, and electron microscopy was performed as described ,.
For Protein extraction and immunoblotting, Cell pellets were lysed in Protein Lysis Buffer. Proteins (20 μg) were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were blocked with blocking buffer and incubated with primary or secondary antibodies diluted in the same buffer  (secondary Alexa-conjugates antibodies: Life Technologies; HRP-conjugated antibodies: Jackson ImmunoResearch).
Written informed consent was obtained from the patient’s legal guardian(s) for gene analysis and publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
Prior to death, brain MRIs showed progressive global cerebellar atrophy (Figure 1B). Monovoxel MR spectroscopy of the left basal ganglia revealed a reduced NAA/Cr ratio indicative of neuronal loss without iron accumulation. Brain PET scans, electroencephalographic recordings, somatosensory evoked potentials, audition and fundus examination, electroneurography, liver and kidney echographies were unremarkable. Muscle biopsy, performed at the age of 6, revealed no abnormalities or biochemical deficits. Glucose, proteins, lactate, blood cell count, and neurotransmitters levels in the CSF were normal. Analyses for each of the following, performed at least once, were normal: blood cell count, ASAT, ALAT, CK, urea, creatinine, cholesterol, triglyceride, arterial lactate and pyruvate levels, ceruloplasmin, cupremia and cupruria, alpha fetoprotein, very long chain fatty acids and long chain fatty acids, biopterin, urine creatine and guanidinoacetate, amino acid (blood and urine) and organo acid (urine) chromatography, high-resolution caryotype, glucocerebrosidase, galactocerebrosidase, β-galactosidase, α-N-acetylgalacosaminidase, aryl sulfatase A, hexosaminidase A and B, α-glucosaminidase, β-glucuronidase, α-mannosidase, β-mannosidase, α-neuraminidase, acid sphingomyelinidase mucopolysaccharidoses and oligosaccharidoses, and sialotransferrin. No acanthocytosis was present on any of several blood smears.
No mutations were identified in DYT1, PANK2, PLA2G6, SCA17, or APTX, genes known to be involved in neurodegenerative diseases with cerebellar atrophy and/or dystonia. Given consanguinity in the family, we assumed a recessive mode of inheritance and predicted the causative variant would fall in a region of homozygosity.
Homozygosity mapping revealed six homozygous regions greater than 2 Mb. Whole exome sequencing produced 142 million reads, 98% of which could be aligned to the targeted sequence. Mean coverage of the targeted sequence was 77 fold. 56 687 SNVs and 7 265 indels were called. Only nine homozygous variants with frequency below 1% in internal (IntegraGen) and public databases (dbSNP 132; HapMap; 1,000 Genomes Project) were filtered out of these. Sequencing in the patient and in family members (primers available upon request) reduced the number of candidates to four genes, present at the homozygous state in the patient, and at the heterozygous state in the parents: ALMS1, B3GALT1, ZNF804B, and TOR1AIP1.
The A>C missense variant (c.1448A>C) at position 179,887,067 on chromosome 1q25.1-1q25.3 in TOR1AIP1 (NM_015602), located in a 6.8-Mb homozygosity region, resulted in replacement of a highly conserved glutamic acid with alanine at amino acid 482 (GERP++ score 5.96; PhyloP score 2.285) (Figure 1C,D). Furthermore, pathogenicity predictions were deleterious in Align GVGD, Polyphen-2, SIFT, and MutationTaster analyses. On the contrary, the variants in ALMS1 (NM_015120; c.2202T>A/p.S732R), B3GALT1 (c.192A>T/p.E64D, NM_020981) and ZNF804B (c3118C>A/p.L1040I, NM_181646), were predicted to be benign by at least three of the above-mentioned programs. GERP++ and PhyloP scores were lower for the ZNF804B variant (GERP++ score 4.15, PhyloP score 1.467), and even negative for the ALMS1 and B3GALT1 variants. There was thus a strong bioinformatic convergence towards the pathogenic character of the TOR1AIP1 variant only. In addition, the phenotype of this patient was divergent from that of Alström syndrome (OMIM #203800) patients who have mutations in ALMS1. TOR1AIP1 encodes LAP1, a type II transmembrane protein. LAP1 interacts with torsinA (encoded by TOR1A gene), which is mutated in autosomal dominant dystonia (DYT1; OMIM #12810) . The amino acid mutated in our patient is located in the luminal domain, which interacts with torsinA. This domain is common to the three isoforms and has significant homology with LULL1, another protein that interacts with torsinA. This variant was not observed in any of 100 ethnically matched controls and was absent from >6500 exomes at the Exome Variant Server.
We have searched TOR1AIP1 mutation in 10 additional patients with dystonia during childhood: Five with primary dystonia associated with cerebellar atrophy and 5 with primary cerebellar ataxia with progressive cerebellar atrophy and severe dystonia. We did not find mutations, which seems to indicate that TOR1AIP1 mutation is not a common cause of dystonia of unknown origin.
To our knowledge, cerebellar atrophy, dystonia, and dilated cardiomyopathy are very rarely associated, except in some mitochondrial disease or organic acidemia. Our patient did not suffer from organic acidemia and mitochondrial disorders were unlikely given normal blood and CSF lactate levels, NMR spectroscopy, and muscular biopsy. Cerebellar atrophy and dystonia are observed in neurodegenerative diseases, such as Wilson disease, acanthocytosis, gangliosidosis , and Niemann Pick type C  and in some cases of spinocerebellar ataxias, particularly SCA17 . These were ruled out in the patient. TUBB4A mutations have been recently involved in patients with dystonia, cerebellar and basal ganglia atrophy and hypomyelinating leukodystrophies (HABC syndrome) . In addition to the absence of white matter abnormalities in our case, exome analysis ruled out TUBB4A mutations. In the clinical work up, other causes of dystonia (such as DYT1 and PANK2 mutation) or cerebellar atrophy (such as PLA2G6 mutation) were also ruled out. Much older patients who have presented with the association of a less severe dystonia and a cerebellar atrophy have been described . Extreme painfull dystonia was one of the primary clinical features observed when we first evaluated the child at the age of 11. Absence of effect of any usual oral medications tried by us and others, and the dramatic improvement upon administration of nabiximols, a cannabinoid derived from cannabis plant , remains very enigmatic but as been reported in adults with central pain and paroxysmal dystonia .
LAP1, encoded by TOR1AIP1, induces the ATPase activity of torsinA , the protein mutated in DYT1, an autosomal dominant dystonia with purely neurological phenotype. LAP1 is the only protein with such function at the nuclear envelope described so far, and hence its mislocalization likely leads to a dramatic loss of function of torsinA and probably of other torsin family proteins . Mice lacking LAP1 have abnormal nuclear membranes that form blebs visible by electron microscopy in all cell types . In mice lacking torsinA, blebs are only present in neurons, probably because of the high level of torsinB in non-neuronal cells . Such blebs were not observed in patient’s fibroblasts. The mutated LAP1 expressed by the patient may retain some functions required for the structure of the nuclear envelope. Our case appears much more severe than DYT1, not only because of the severity of dystonia itself, but because of the cardiac involvement, similar to the phenotype of Tor1a ∆E/∆E mice  and with disorders of nuclear membrane such as laminopathies . Recently, cardiomyopathy has been demonstrated in a tissue-specific mutant lacking LAP1 .
One Turkish family with a homozygous nonsense mutation was recently reported . Interestingly, affected patients also present with a cardiomyopathy, although less severe than that observed in our patient, and associated with a limb-girdle muscular dystrophy.
The present observation is unique, and, therefore, it cannot be completely ruled out that the phenotype is not related to a mutation in a region not covered by exome sequencing. However, we believe that the association between the previously unreported phenotype of the patient and the mutation is very likely. First, clinical evaluations failed to identify another likely cause. Second, the mutation affects a highly conserved amino acid, is predicted to be pathogenic, is absent in 100 ethnically matched controls and in >6500 exomes, and affects a protein that interacts with a protein involved in dystonia. Third, the patient phenotype correlates with that of mice lacking LAP1 , and functional explorations of patient cells revealed reduced LAP1 expression and mislocalization. It is known that adequate localization of LAP1 is crucial for its function . Finally, another family has been reported with nonsense mutation leading to cardiomyopathy . For these different reasons, we believe that sequencing of TOR1AIP1 should be performed in patients with similar peculiar phenotypic associations.
All supporting data are available within the limits of patient’s confidentiality.
The authors thank the patient and his family for their strong support to report this case, Alexandra Durr for helpful comments, William T. Dauer for the anti-LAP1 antibody, the DNA and Cell Bank of the Institut du Cerveau et de la Moelle épinière, and the Fond National de la Recherche Scientifique for the fellowship attributed to M. C. (aspirant FNRS). This work was financially supported by the French National Agency for Research (ANR), the Institut national de la santé et de la recherche médicale (INSERM), the Université Paris Diderot-Sorbonne Paris Cité, DHU PROTECT and the ELA Fondation (post doc fellowship for I.D.), the Université Pierre et Marie Curie Paris 06, the Centre National de la Recherche Scientifique (CNRS), the Association Française contre les Myopathies (AFM), the Verum Foundation, the Fondation Roger de Spoelberch, and the program “Investissements d’avenir” ANR-10-IAIHU-06.
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