Copy number variation of LINGO1 in familial dystonic tremor

Objective To elucidate the genetic cause of a large 5 generation South Indian family with multiple individuals with predominantly an upper limb postural tremor and posturing in keeping with another form of tremor, namely, dystonic tremor. Methods Whole-genome single nucleotide polymorphism (SNP) microarray analysis was undertaken to look for copy number variants in the affected individuals. Results Whole-genome SNP microarray studies identified a tandem duplicated genomic segment of chromosome 15q24 present in all affected family members. Whole-genome sequencing demonstrated that it comprised a ∼550-kb tandem duplication encompassing the entire LINGO1 gene. Conclusions The identification of a genomic duplication as the likely molecular cause of this condition, resulting in an additional LINGO1 gene copy in affected cases, adds further support for a causal role of this gene in tremor disorders and implicates increased expression levels of LINGO1 as a potential pathogenic mechanism.

Tremor is a common movement disorder, and in recent years, it has become clear that essential tremor (ET) may be a group of diseases or a syndrome with clinical features that overlap with dystonia and dystonic tremor (DT). 1 Both may be associated with isolated upper limb postural and kinetic tremor, although DT has different characteristics and is often associated with posturing or other evidence of dystonia. 2,3 However, the considerable overlap in symptoms and signs has led to misdiagnosis of each with the other. 4 This phenotypic heterogeneity is a complicating factor when interpreting the results of genetic studies of familial tremor. The lack of ET-or DT-specific serum, or imaging biomarkers, or defining neuropathologic features mean that clinical assessment is required to distinguish between the 2.
The LINGO1 gene (leucine-rich repeat and Ig domain containing Nogo receptor interacting protein 1) is selectively expressed in the CNS. [5][6][7] Previous studies have identified LINGO1 as a notable genetic risk factor displaying significant association between intragenic single nucleotide polymorphism (SNP) rs9652490 and familial ET, and the same LINGO1 SNP was replicated in independent case-control studies of ET as well as Parkinson disease. [8][9][10][11][12][13] In the current study, we report our investigation of a large family from Southern India with multiple individuals presenting with an early-onset, bilateral, postural tremor of the upper limbs with some associated dystonic features, suggestive of DT, associated with a tandem duplication of the chromosome 15 genomic region encompassing the entire LINGO1 gene.

Clinical studies
The investigated family is from Kerala, a Southern state of India, with a total of 11 affected individuals from 5 generations (figure, A) recruited with informed written consent, including permission to publish photographs. Six participants with a history of tremor (figure, A: III:5, III:9, II:9, IV:1, IV:2, and V:1) underwent a general medical and neurologic examination by the regional consultant neurologist, and a structured videotaped neurologic examination as well as Archimedes spirals were assessed by senior neurologists specializing in movement disorders (table). The videotaped neurologic examination included assessments of gait, tremor at rest, dystonia, postural tremor of the arms, and with each hand the finger-nose maneuver, the drawing of a spiral, and pouring of water. The other 5 individuals with tremor (II:1, II:6, II:10, III:16, and IV:8) had their affected status confirmed with an examination conducted by the local consultant neurologist. Four other family members (II:4, II:11, III:11, and IV:5) were examined by the same consultant neurologist and confirmed to have no evidence of tremor or dystonia.

Microarray analysis and fluorescent in situ hybridization
Venous blood was collected in EDTA and PAXgene Blood RNA tubes (PreAnalytiX), and skin biopsy was performed in 3 cases (III:5, IV:2, and II:9) for fluorescent in situ hybridization (FISH) studies. Genomic DNA and RNA samples were extracted from peripheral blood following standard protocols. Genome-wide SNP genotyping was undertaken using Illumina HumanCytoSNP-12 v2.1 SNP microarrays, and image data were processed using Illumina GenomeStudio software to generate genotype calls, B allele frequency, and logR ratio values. These were further analyzed for copy number variant (CNVs) using Illumina's KaryoStudio software. To minimize falsepositive CNV calls, filtering approaches were applied to exclude smaller repeats (<100 kb) and common CNVs from Database of Genomic Variants (dgv.tcag.ca/dgv/app/home). For FISH analysis, BlueFISH probe RP11-114H24 was used to confirm chromosomal duplication in individuals II:9, III:5, and IV:2.
Genomic library preparation Genomic DNA (;3 μg) was fragmented by sonication using a Bioruptor (UCD-200; Diagenode, Seraing, Liege, Belgium) to an average size of ;400 base pairs (bp), and DNA was purified using 1.2 volumes Ampure XP (Agencourt). End repair and dA tailing were carried out using NEBNext modules (New England Biolabs, Hertfordshire, United Kingdom), with DNA purification using 1.8 volumes Ampure after each step. DNA fragments were then ligated to paired-end adapters for Illumina sequencing using Epicentre Fast-Link DNA ligation kit (Cambio, Cambridgeshire, United Kingdom). The entire ligation reaction was separated on a 1.2% agarose gel, and DNA fragments in the size range ;400-450 bp were excised. DNA was extracted from the gel slice using the QIAquick gel extraction kit (Qiagen, Manchester, United Kingdom) and analyzed on a high-sensitivity chip for the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Adapter-ligated DNA (50 ng) was amplified for 6 cycles using Herculase II Fusion DNA Polymerase (Agilent Technologies) and Illumina PE_PCR primers 1 and 2, then diluted for sequencing. Sequencing was carried out by the Exeter Sequencing Service (School of Biosciences, University of Exeter, Exeter, United Kingdom) on an Illumina HiSeq2500 using 100-bp paired-end reads in rapid run mode, yielding a total of 23.8 gigabases (Gb) of sequence. Reads were aligned to the human reference genome (build GRCh37/hg19) using the BWA alignment tool, and duplicate reads were removed using Picard, yielding an average coverage depth of ;7.5X reads per base. Glossary CNV = copy number variant; DT = dystonic tremor; ET = essential tremor; FISH = fluorescent in situ hybridization; NgR1 = Nogo-66 receptor.
Standard protocol approvals, registrations, and patient consents The study was approved and performed under the ethical guidelines issued by our institutions for clinical studies, with written informed consent obtained from all participants for genetic studies.

Clinical studies
The extended pedigree of the family is presented in figure A, and clinical details of the tremor and salient neurologic features are presented in table, including age at onset. The  BlueFish probe RP11-114H24; chr15:78146252-78322027, figure, C). Targeted next-generation sequencing of an affected patient (III:16) was then used to precisely map the chromosomal breakpoints of the duplication. This identified read pairs mapping ;550 kb apart in reverse-forward rather than forward-reverse orientation, indicative of a tandem duplication event (figure, B; figure e-1A, links.lww.com/NXG/ A139). The exact coordinates of the rearrangement event (chr15:77775483-78331797dup [hg19]) were identified in reads spanning each breakpoint (figure e-1B, links.lww.com/ NXG/A139), located in genes HMG20A and TBC1D2B, respectively. The read count across the region indicates an average coverage increase from approximately 7.5X to 10X, broadly consistent with an expected ;50% increase in the number of reads for a heterozygous duplication. The 14 genes located within the duplicated region (figure, B) were investigated for candidacy, which identified only a single standout candidate with a role in brain development or function (LINGO1).
PCR primers were positioned to produce an amplicon specific to the genetic sequence created at the boundary between the tandem duplications, generating a product of 1,184 bp arising from this de novo event. Dideoxy sequencing of this PCR product confirmed the location of the duplication event to chr15:77775487-78331797 [hg19]. This facilitated the genotyping of family members using a simple PCR-based strategy to identify family members who have inherited the rearragnement. PCR analysis on all individuals from the pedigree confirmed co-segregation of the rearrangement in affected family members (figure, A), as well as its absence from 100 age-matching healthy controls from the same geographical region.

Discussion
Here, we investigated an extended Indian family with multiple individuals affected by a movement disorder involving tremor. The presence of abnormal upper limb postures in all 11 affected individuals along with the presence of only mild tremor is most consistent with DT rather than ET in this family. 1 As noted above, previous genome-wide association studies have demonstrated association between DNA sequence variants in the LINGO1 gene and ET. With the case we report now, the potential involvement of a duplication involving the LINGO1 gene may suggest a similar genetic and molecular mechanistic basis to some cases of ET and DT. LINGO1 protein is known to interact with Nogo-66 receptor (NgR1) and p75 neurotrophin receptor (p75 NTR ) or TROY, to form an NgR1 complex, which binds to inhibitory molecules such as Nogo-A. 14,15 The NgR1 complex Nogo-A activates RhoA as a negative regulator for neuronal survival, axonal regeneration, oligodendrocyte maturation, and neuronal myelination. [15][16][17][18][19][20][21] Notably, the p75NTR-sortilin (SORT1) receptor complex has previously been implicated in tremor phenotypes via a p.Gly171Ala SORT1 missense variant, which impaired expression of both protein members None of the sortilin-p75 NTR complex. 22 As such, LINGO1 represents a strong candidate gene for involvement in movement disorders such as DT and ET, and consistent with this, previous genome-wide association studies have indeed indicated an association between variants in LINGO1 and ET. [9][10][11][12][13] However, all identified risk variants are located in LINGO1 intronic regions, and subsequent sequencing of LINGO1 coding exons in ET patients have failed to identify putative pathogenic sequence variants. [9][10][11]23,24 The studies reported here define a previously undescribed duplication event encompassing the LINGO1 gene present in multiple individuals of this extended Indian family. While it is not possible to exclude involvement of other genes in the duplicated region, LINGO1 represents the only stand-out functional candidate in the region. This finding lends us to speculate that increased transcription and ensuing gene activity deriving from the additional (trisomic) copy of LINGO1 are the likely pathogenic cause of this condition. This indicates that the previously identified intronic LINGO1 gene variants displaying association with ET may result in the condition by directly influencing (or being in linkage disequlibrium with variants that directly influence) native gene transcription. Consistent with this notion, a previous study detected increased levels of LINGO1 in the cerebellum ET patients. 25 Thus, while the outcome of duplication of the other genes located within the genomic region defined in our study requires further exploration, our data, combined with existing studies of LINGO1 in ET, indicate that hypermorphic mutation leading to increased transcriptional or protein activity of LINGO1 represents a likely pathogenic cause. This may manifest itself via an increased density of basket cell processes generated by an increased LINGO1 dosage effect, leading to an inhibitory effect on Purkinje cell GABAergic neurones. Decreased or inhibited cerebellar inhibitory output has been demonstrated to cause postural, kinetic tremor as well as motor incoordination in GABAA α1 knockout mice. 24 In the olivary animal models of action tremor, it has also been shown that such action tremor is a primarily electrophysiologic entity caused by abnormal olivary-cerebellar excitatory output. 26 Thus, it is tempting to speculate that this may result from an imbalance of excitatory-inhibitory interneuronal connection secondary to the dosage effect of LINGO1, and it would be of interest to observe the effect of LINGO1 protein agonists on the olivary animal models of ET to investigate this hypothesis. The data from this study are consistent with this notion and demonstrate LINGO1 copy number gain in familial postural tremor, suggestive of a LINGO1 dosage disease mechanism.