Satya R. Vemula

Mutations in CIZ1 cause adult-onset primary cervical dystonia

Primary dystonia is usually of adult onset, can be familial, and frequently involves the cervical musculature. Our goal was to identify the causal mutation in a family with adult onset, primary cervical dystonia.

Role of Gα(olf) in familial and sporadic adult-onset primary dystonia

The vast majority of patients with primary dystonia are adults with focal or segmental distribution of involuntary movements. Although ~10% of probands have at least one first- or second-degree relative to dystonia, large families suited for linkage analysis are exceptional. After excluding mutations in known primary dystonia genes (TOR1A, THAP1 and CIZ1), whole-exome sequencing identified a GNAL missense mutation (c.682G>T, p.V228F) in an African-American pedigree with clinical phenotypes that include cervical, laryngeal and hand-forearm dystonia. Screening of 760 subjects with familial and sporadic primary dystonia identified three Caucasian pedigrees with GNAL mutations [c.591dupA (p.R198Tfs*13); c.733C>T (p.R245*); and c.3G>A (p.M1?)]. These mutations show incomplete penetrance. Our findings corroborate those of a recent study which used whole-exome sequencing to identify missense and nonsense GNAL mutations in Caucasian pedigrees of mixed European ancestry with mainly adult-onset cervical and segmental dystonia. GNAL encodes guanine nucleotide-binding protein G(olf), subunit alpha [Gα(olf)]. Gα(olf) plays a role in olfaction, coupling D1 and A2a receptors to adenylyl cyclase, and histone H3 phosphorylation. African-American subjects harboring the p.V228F mutation exhibited microsmia. Lymphoblastoid cell lines from subjects with the p.V228F mutation showed upregulation of genes involved in cell cycle control and development. Consistent with known sites of network pathology in dystonia, immunohistochemical studies indicated that Gα(olf) is highly expressed in the striatum and cerebellar Purkinje cells, and co-localized with corticotropin-releasing hormone receptors in the latter.

Genotype–phenotype correlations in THAP1 dystonia: Molecular foundations and description of new cases

An extensive variety of THAP1 sequence variants have been associated with focal, segmental and generalized dystonia with age of onset ranging from 3 to over 60 years. In previous work, we screened 1114 subjects with mainly adult-onset primary dystonia (Neurology 2010; 74:229-238) and identified 6 missense mutations in THAP1. For this report, we screened 750 additional subjects for mutations in coding regions of THAP1 and interrogated all published descriptions of THAP1 phenotypes (gender, age of onset, anatomical distribution of dystonia, family history and site of onset) to explore the possibility of THAP1 genotype-phenotype correlations and facilitate a deeper understanding of THAP1 pathobiology. We identified 5 additional missense mutations in THAP1 (p.A7D, p.K16E, p.S21C, p.R29Q, and p.I80V). Three of these variants are associated with appendicular tremors, which were an isolated or presenting sign in some of the affected subjects. Abductor laryngeal dystonia and mild blepharospasm can be manifestations of THAP1 mutations in some individuals. Overall, mean age of onset for THAP1 dystonia is 16.8 years and the most common sites of onset are the arm and neck, and the most frequently affected anatomical site is the neck. In addition, over half of patients exhibit either cranial or laryngeal involvement. Protein truncating mutations and missense mutations within the THAP domain of THAP1 tend to manifest at an earlier age and exhibit more extensive anatomical distributions than mutations localized to other regions of THAP1.

Pathogenic variants in TUBB4A are not found in primary dystonia

Objective: To determine the contribution of TUBB4A, recently associated with DYT4 dystonia in a pedigree with “whispering dysphonia” from Norfolk, United Kingdom, to the etiopathogenesis of primary dystonia. Methods: High-resolution melting and Sanger sequencing were used to inspect the entire coding region of TUBB4A in 575 subjects with primary laryngeal, segmental, or generalized dystonia. Results: No pathogenic variants, including the exon 1 variant (c.4C>G) identified in the DYT4 whispering dysphonia kindred, were found in this study. Conclusion: The c.4C>G DYT4 mutation appears to be private, and clinical testing for TUBB4A mutations is not justified in spasmodic dysphonia or other forms of primary dystonia. Moreover, given its allelic association with leukoencephalopathy hypomyelination with atrophy of basal ganglia and cerebellum and protean clinical manifestations (chorea, ataxia, dysarthria, intellectual disability, dysmorphic facial features, and psychiatric disorders), DYT4 should not be categorized as a primary dystonia.

Clinical and genetic features of cervical dystonia in a large multicenter cohort

Objective: To characterize the clinical and genetic features of cervical dystonia (CD). Methods: Participants enrolled in the Dystonia Coalition biorepository (NCT01373424) with initial manifestation as CD were included in this study (n = 1,000). Data intake included demographics, family history, and the Global Dystonia Rating Scale. Participants were screened for sequence variants (SVs) in GNAL, THAP1, and Exon 5 of TOR1A. Results: The majority of participants were Caucasian (95%) and female (75%). The mean age at onset and disease duration were 45.5 ± 13.6 and 14.6 ± 11.8 years, respectively. At the time of assessment, 68.5% had involvement limited to the neck, shoulder(s), and proximal arm(s), whereas 47.4% had dystonia limited to the neck. The remaining 31.5% of the individuals exhibited more extensive anatomical spread. A head tremor was noted in 62% of the patients. Head tremor and laryngeal dystonia were more common in females. Psychiatric comorbidities, mainly depression and anxiety, were reported by 32% of the participants and were more common in females. Family histories of dystonia, parkinsonian disorder, and tremor were present in 14%, 11%, and 29% of the patients, respectively. Pathogenic or likely pathogenic SVs in THAP1, TOR1A, and GNAL were identified in 8 participants (0.8%). Two individuals harbored novel missense SVs in Exon 5 of TOR1A. Synonymous and noncoding SVs in THAP1 and GNAL were identified in 4% of the cohort. Conclusions: Head tremor, laryngeal dystonia, and psychiatric comorbidities are more common in female participants with CD. Coding and noncoding variants in GNAL, THAP1, and TOR1A make small contributions to the pathogenesis of CD.

A rare sequence variant in intron 1 of THAP1 is associated with primary dystonia

Although coding variants in THAP1 have been causally associated with primary dystonia, the contribution of noncoding variants remains uncertain. Herein, we examine a previously identified Intron 1 variant (c.71+9C>A, rs200209986). Among 1672 subjects with mainly adult‐onset primary dystonia, 12 harbored the variant in contrast to 1/1574 controls (P < 0.01). Dystonia classification included cervical dystonia (N = 3), laryngeal dystonia (adductor subtype, N = 3), jaw‐opening oromandibular dystonia (N = 1), blepharospasm (N = 2), and unclassified (N = 3). Age of dystonia onset ranged from 25 to 69 years (mean = 54 years). In comparison to controls with no identified THAP1 sequence variants, the c.71+9C>A variant was associated with an elevated ratio of Isoform 1 (NM_018105) to Isoform 2 (NM_199003) in leukocytes. In silico and minigene analyses indicated that c.71+9C>A alters THAP1 splicing. Lymphoblastoid cells harboring the c.71+9C>A variant showed extensive apoptosis with relatively fewer cells in the G2 phase of the cell cycle. Differentially expressed genes from lymphoblastoid cells revealed that the c.71+9C>A variant exerts effects on DNA synthesis, cell growth and proliferation, cell survival, and cytotoxicity. In aggregate, these data indicate that THAP1 c.71+9C>A is a risk factor for adult‐onset primary dystonia.

Motor phenotypes and molecular networks associated with germline deficiency of Ciz1.

A missense mutation in CIZ1 (c.790A>G, p.S264G) was linked to autosomal dominant cervical dystonia in a large multiplex Caucasian pedigree (OMIM614860, DYT23). CIZ1 is a p21((Cip1/Waf1)) -interacting zinc finger protein, widely expressed in neural and extra-neural tissues, and plays a role in DNA synthesis at the G1/S cell-cycle checkpoint. The role of CIZ1 in the nervous system and relative contributions of gain- or loss- of function to the pathogenesis of CIZ1-associated dystonia remain indefinite. Using relative quantitative reverse transcriptase-PCR, cerebellum showed the highest expression levels of Ciz1 in adult mouse brain, over two fold higher than liver, and higher than striatum, midbrain and cerebral cortex. Overall, neural expression of Ciz1 increased with postnatal age. A Ciz1 gene-trap knock-out (KO) mouse model (Ciz1(-/-)) was generated to examine the functional role(s) of CIZ1 in the sensorimotor nervous system and contributions of CIZ1 to cell-cycle control in the mammalian brain. Ciz1 transcripts were absent in Ciz1(-/-) mice and reduced by approximately 50% in Ciz1(+/-) mice. Ciz1(-/-) mice were fertile but smaller than wild-type (WT) littermates. Ciz1(-/-) mice did not manifest dystonia, but exhibited mild motoric abnormalities on balance, open-field activity, and gait. To determine the effects of germline KO of Ciz1 on whole-genome gene expression in adult brain, total RNA from mouse cerebellum was harvested from 6 10-month old Ciz1(-/-) mice and 6 age- and gender- matched WT littermates for whole-genome gene expression analysis. Based on whole-genome gene-expression analyses, genes involved in cellular movement, cell development, cellular growth, cellular morphology and cell-to-cell signaling and interaction were up-regulated in Ciz1(-/-) mice. The top up-regulated pathways were metabolic and cytokine-cytokine receptor interactions. Down-regulated genes were involved in cell cycle, cellular development, cell death and survival, gene expression and cell morphology. Down-regulated networks included those related to metabolism, focal adhesion, neuroactive ligand-receptor interaction, and MAPK signaling. Based on pathway analyses, transcription factor 7-like 2 (TCF7L2), a member of the Wnt/β-catenin signaling pathway, was a major hub for down-regulated genes, whereas NF-κB was a major hub for up-regulated genes. In aggregate, these data suggest that CIZ1 may be involved in the post-mitotic differentiation of neurons in response to external signals and changes in gene expression may compensate, in part, for CIZ1 deficiency in our Ciz1(-/-) mouse model. Although CIZ1 deficiency was associated with mild motor abnormalities, germline loss of Ciz1 was not associated with dystonia on the C57BL/6J background.

Whole-exome sequencing for variant discovery in blepharospasm

Blepharospasm (BSP) is a type of focal dystonia characterized by involuntary orbicularis oculi spasms that are usually bilateral, synchronous, and symmetrical. Despite strong evidence for genetic contributions to BSP, progress in the field has been constrained by small cohorts, incomplete penetrance, and late age of onset. Although several genetic etiologies for dystonia have been identified through whole‐exome sequencing (WES), none of these are characteristically associated with BSP as a singular or predominant manifestation.

Blepharospasm in a multiplex African-American pedigree

BACKGROUND Isolated blepharospasm (BSP) is a late-onset focal dystonia characterized by involuntary contractions of the orbicularis oculi muscles. Genetic studies of BSP have been limited by the paucity of large multiplex pedigrees. Although sequence variants (SVs) in THAP1 have been reported in rare cases of BSP, the genetic causes of this focal dystonia remain largely unknown. Moreover, in the absence of family history and strong in silico or in vitro evidence of deleteriousness, the pathogenicity of novel SVs in THAP1 and other dystonia-associated genes can be indeterminate. METHODS A large African-American pedigree with BSP was phenotypically characterized and screened for mutations in THAP1, TOR1A and GNAL with Sanger sequencing. Whole-exome sequencing of the proband was used to examine other dystonia-associated genes for potentially pathogenic SVs. In silico and co-segregation analyses were performed for a novel THAP1 SV identified in the proband. RESULTS Seven family members exhibited increased blinking and/or stereotyped bilateral and synchronous orbicularis oculi spasms with age of onset ranging from early childhood to late adult life (7 to 54 years). The proband was found to harbor a novel THAP1 SV (c.314T>C, p.L105S). However, the p.L105S SV did not co-segregate with blepharospasm in the pedigree. Moreover, in silico analyses suggest that p.L105S is benign. No pathogenic or likely pathogenic SVs in other dystonia-associated genes were identified with whole-exome sequencing. CONCLUSIONS Blepharospasm can be familial and may be hereditary in African-Americans. A comprehensive array of in silico tools, and, if possible, co-segregation analysis should be used to classify SVs in dystonia-associated genes.

Consequences of Cre‐mediated deletion of Ciz1 exon 5 in mice

CIZ1 plays a role in DNA synthesis at the G1/S checkpoint. Ciz1 gene‐trap null mice manifest motor dysfunction, cell‐cycle abnormalities, and DNA damage. In contrast, it has previously been reported that mouse embryonic fibroblasts derived from presumed Ciz1 knock‐out mice (Ciz1tm1.1Homy/tm1.1Homy) generated by crossing Cre‐expressing mice with exon 5‐floxed mice (Ciz1tm1Homy/tm1Homy) do not exhibit evidence of enhanced DNA damage following γ‐irradiation or cell‐cycle defects. Here, we report that Ciz1tm1.1Homy/tm1.1Homy mice show loss of Ciz1 exon 5 but are neurologically normal and express abnormal transcripts (Ciz1ΔE5/ΔE5 mice) that are translated into one or more proteins of approximate wild‐type size. Therefore, Ciz1tm1.1Homy/tm1.1Homy mice (Ciz1ΔE5/ΔE5) lose residues encoded by exon 5 but may gain function from novel amino acid sequences.