A novel biallelic LMNB2 variant in a patient with progressive myoclonus epilepsy and ataxia: A case of laminopathy

Abstract The report of LMNB2‐related progressive myoclonus epilepsy and ataxia due to missense homozygous c.473G>T variant.


| INTRODUCTION
We report a patient presenting autosomal recessive progressive myoclonus epilepsy. Whole-exome sequencing was used, and a novel missense homozygous c.473G>T variant in the LMNB2 gene was identified in the proband. The cosegregation study confirmed the pathogenic effect of the finding in the family.
Progressive myoclonus epilepsies (PMEs) are a rare group of disorders that present with action myoclonus, tonic-clonic seizures, and progressive neurological deterioration, typically with cerebellar signs and dementia. These are disorders of gray matter, and neuroimaging reveals cerebral and cerebellar atrophy and no white matter disease. Most molecularly characterized PMEs are inherited in an autosomal recessive manner, with rare cases showing autosomal dominant or mitochondrial inheritance. 1,2 There are five main causes of PME comprising (1) Unverricht-Lundborg disease due to CSTB gene mutations; (2) Lafora disease caused by EPM2A and NHLRC1 alterations; (3) myoclonic epilepsy with ragged red fibers caused by MTTK variants; (4) neuronal ceroid lipofuscinosis as a result of a mutation in TPP1, CLN3, CLN5, and CLN6; and (5) sialidoses as a consequence of neuraminidase deficiency in leukocytes or fibroblasts that have been more accurately defined with recent advances in genetic studies. 1,3,4 PME-associated genes encode a variety of proteins, many of which are associated with the endosomal and lysosomal function 5 ; however, the association of LMNB2, a lamin-coding gene, with PME has not been reported until a variant was identified in two Palestinian patients diagnosed with PME in 2015. 6 Herein, we report a family with an affected boy who presented myoclonic seizure from consanguineous parents and exhibiting an autosomal recessive pattern of inheritance. Whole-exome sequencing was performed and identified a homozygous missense variant in the LMNB2 gene that encodes lamin B2, one of the major structural proteins in the nuclear filament. Although LMNA, another gene in the lamin family, has been reported with particular diseases such as cardiomyopathy, muscular dystrophy, lipodystrophy, and progeroid syndromes, 7 LMNB2 has been reported to cause lipodystrophy, 8 and its pathogenicity in developing disease has not been studied well.

| CLINICAL REPORT
A 5-year-old boy was referred to our clinical genetic department because of progressive wide-based ataxic gait and intractable myoclonic seizure. Following an uneventful pregnancy, he was born at 38 weeks of gestation via cesarean section to healthy consanguineous Iranian parents with a high inbreeding coefficient (F = 9.375%). His anthropometric features and Apgar score were normal at birth, and dysmorphology was unremarkable. Psychomotor development was normal until 15 months of age when he manifests unsteady gait with frequent stumbling. After three months, at age 18 months, he revealed myoclonic seizure of limbs during sleeping and awake, which evolve to intractable generalized myoclonic seizure over time.
At age three years, his brain MRI, nerve conduction velocity (NCV), electromyography (EMG), and metabolic survey were normal, where EEG was significant for generalized epileptic discharge. Initially, some improvement in myoclonic seizure was observed by using Synacthen, but seizure became worse over time even after adding multiple drugs including IVIg, pulse therapy of prednisolone, profilin, and the ketogenic diet. At age five years, in addition to his multi-AED intractable seizure, he developed progressive ataxia, intension tremor, and slurred speech.

| Patient recruitment and sampling
This study was approved by the Research Committee and Department of Molecular Genetics of Sarem Women Hospital, Tehran, Iran. We obtained informed consent, clinical data, and pedigree information from the family. Peripheral whole blood was obtained from the proband and the family. Subsequently, genomic DNA was extracted using salting-out method. 9 Then, whole-exome sequencing (WES) was performed on the proband's DNA, and data were interpreted and analyzed.

| WES and Variant prioritization
Genomic DNA was isolated from white blood cells and subjected to exome capturing using Agilent SureSelect (Santa Clara, CA), followed by next-generation sequencing on the Illumina sequencing platform HiSeq 4000 (San Diego, CA), with coverage of >80-100X. Sequence reads were mapped to the human reference genome (GRCh37/hg19) using BWA program. Variant calling was performed using a Picard and GATK-Lite toolkit (Broad Institute, Cambridge, MA). Gene annotation of variants in the generated VCF file was performed using wANNOVAR software (http://wanno var. wglab.org/). The generated file underwent filtering variants based on inheritance patterns of the disease in the pedigree. In the second step, synonymous variants and nonexonic variants that were not located within splice site regions were excluded. Then, variants with minor allele frequencies greater than 1% in 1000Genome, ExAC, and GnomAD were filtered out. Following that, remaining variants were filtered based on genotype-phenotype correlation in the proband using OMIM, GHR, GeneReviews, and Orphanet databases and publications. In step 5, remaining variants were ranked based on their probable impact on protein sequence and function, considering evolutionary conservation among orthologs across phylogeny and in silico prediction programs (such as SIFT, PolyPhen-2, MutationTaster, CADD, GERP++, and DANN). Next, remaining variants were prioritized upon clinical features of the proband and further evaluated by reviewing the existing literature. The candidate variants were confirmed in the patient's original DNA by Sanger sequencing using primers of forward (5′-CTCGGTGACCTTGTCTTTGG-3′) and reverse (5′-GAGGGTGGACTTTGCGTTTC-3′).

| RESULTS
A novel biallelic nonsynonymous missense variant NM_032737 c.473G>T (p. Arg158Leu) was identified in the LMNB2 gene that is classified as pathogenic based on ACMG guidelines. Following WES, Sanger sequencing was carried out for co-segregation analysis on the proband and the family members. The variant was confirmed in the proband, and both of the parents were heterozygote for the c.473G>T variant. Further co-segregation analysis revealed that the grandmother, but not grandfather, from the mother side, and four of the father's siblings (13 persons in the families) were carriers ( Figure 1 and Figure 2A).
The c.473G>T variant is predicted as a damaging and disease-causing variant according to in silico predictors such as SIFT, MutationTaster, and CADD. Also, the c.473G>T variant is ultra-rare (1x, allele frequency: 0.000004038) in GnomAD. In addition, bioinformatics study of the Arg158 and nearby regions exhibited high conservation among the | 3 of 7 FARAJZADEH VALILOU Et AL. species (Figure 2-B). Protein 3D modeling was carried out to assess a possible protein alteration (Figure 3). Although the alteration result was insignificant, considering the type of amino acid change from arginine-a basic amino acid with a positively charged side chain-to leucine, a nonpolar amino acid, it is highly probable that it can affect the conformation of the protein helix. In other words, the positively charged side chain of arginine is lost with the leucine substitution.

| DISCUSSION
In the current study, we described an Iranian affected offspring with indications of progressive wide-based ataxic gait and intractable myoclonic seizure with a homozygous missense c.473G>T variant in the LMNB2 gene. The LMNB2 gene encodes an intermediate nuclear filament, which together with other components of the nuclear lamina provides a scaffolding meshwork. So far, genetic alterations in the LMNB2 gene have been associated with three distinct phenotypes, such as partial acquired lipodystrophy (APLD), 8 epilepsy, progressive myoclonic 9 (EPM9), 6 and recently found autosomal dominant primary microcephaly. 10  in his carrier parents during the examination at the time of the study. It is not surprising because lamin gene alterations only affect specific types of tissues, as they have been associated with various disorders of the nervous system. 7,11,12 This tissue specificity might explain why skin keratinocytes of Lmnb1 and Lmnb2 knockout mouse are able to proliferate normally in addition to embryonic stem cells of B-type lamin knockout mouse, which are viable and self-renewable. 13,14 In contrast, lmnb2-deficient mouse dies immediately after birth because of neuronal layering defect in the cerebral cortex following interrupted neuronal migration from the ventricular zone to the cortical plate. 15 Apart from this ambiguous role of LMNB2 in lipid tissue disorders, it is obvious that LMNB 1/2 is involved in cerebral cortex development and function.
Although lamins B1 and B2 have almost 60% sequence similarity at the amino acid level, it seems that each plays unique and specific role in the earliest stages of neuronal cell development and later in its maintenance. 16 In fact, lamin B2 deficiency is not compensated by increased lamin b1 synthesis, and the disease phenotype would elicit. Recently, two heterozygote variants in the LMNB2 gene have been reported as a genetic cause of primary microcephaly and intellectual disability. 10 Table 1 compares the phenotype findings of the proband with the previously reported EPM9 patients. The proband's phenotype is compatible with previously described EPM9 patients, except in severity and some signs and symptoms. In fact, the age of onset in the proband is significantly earlier than affected individuals in Damiano JA et al's study,  at age 1 year compared to 6-7 years, respectively. This finding suggests that the novel variant can associate with more severe phenotype, at least in this case. For the first time, the His157Tyr mutation in the LMNB2 gene has been related to the progressive myoclonic epilepsy (PME). 6 In order to understand whether this mutation disrupts lamin b2 regular fibrillar formation or not, Damiano JA et al. compared wild-type and His157Tyr mutant lamin b2 by fibrillar assembly study. In conclusion, wild-type lamin b2 formed regular and extended "spikes or needles" with 24.5-nm repeat pattern. Lateral fiber arrays connected to the main spike on a regular and well-organized manner and formed extended so-called paracrystalline arrays. However, the mutant lamin b2 formed irregular and unorganized network-type structures and 24.5-nm repeat pattern was absent in the major segments and only confined to partial parts of the structures. Moreover, the His157Tyr mutation, similar to wild-type protein, did not disrupt the longitudinal assembly since it is located in a coiled coil 1b domain, instead of the a-helical central rod domain, which mediates a head-tail overlap in the longitudinal process. These findings suggesting that the coiled coil 1b domain is necessary for regular and organized lateral connections of fiber arrays in the nuclear lamina. 6 Recently, a novel variant Arg158Trp (c.472C>T) at the same amino acid codon with only one base pair difference in the cDNA position with a c.473G>T variant is identified 17 and strengthens the association of LMNB2 and PME9. Our novel candidate variant, Arg158Leu (c.473G>T), together with the previously reported pathogenic variants His157Tyr 6 and Arg158Trp 17 are located side by side in the highly conserved coiled coil 1b domain of lamin b2 protein. The presence of these two amino acids close to each other at the protein level and similar clinical outcomes of disrupted His157 and Arg158 amino acids highlight the fact that Arg158Leu mutation might affect fibrillar assembly in a similar way by disrupting regular fiber formation.
Lamin-containing protein meshwork, like a main scaffold of a bridge, connects to a bunch of proteins including nuclear pore complexes (NPCs), SUN domain proteins, and additional nuclear envelope transmembrane proteins, and makes a physical link between nucleoskeleton and cytoskeleton that helps the nuclear envelope sustain its proper shape and integrity. 18,19 In addition, the nuclear lamina plays a role in housekeeping functions as it suppresses expression of various genes in two different ways. First, it directly binds to a vast majority of genes in human cells, including nuclear lamina-associated genes, and makes them transcriptionally inactive. 20,21 Interestingly, depressed lamin-associated genes have been observed in lamin b2 knockout Drosophila, indicating the role of lamin b2 in expression controlling of the series of genes. 21,22 Second, the nuclear lamina acts as an anchoring point for chromatin and organize the global three-dimensional genome. 23,24 Chromatin detachment from the nuclear lamina due to lamin mutations can lead to the chromatin relaxation and subsequent gene expression. 25 Despite its role in sustaining nuclear shape and expression controlling of several genes, lamin b2 plays an extra nuclear role by localizing to mitochondria in axons. 26 Developing axons require a significant amount of energy to reach their distinct synaptic targets during wiring of the central nervous system. It has been demonstrated that lamin b2 is transported to mitochondria and maintain mitochondrial function to provide energy for the cell. 26 Therefore, it is required in developing axons and their subsequent maintenance. In addition to axons, LMNB2 is strongly expressed throughout oligodendrocyte developing process and plays a role in developing and maintaining of this kind of glial cells. 27 Several studies have illustrated a critical role of lamin b2 in the nervous system as it involves in neuronal migration and subsequent survival and function.
In summary, our findings confirm the genotype-phenotype correlation between the LMNB2 gene and EMP9 as a third case worldwide and suggests Arg158Leu as a pathogenic variant. However, functional studies are strongly recommended for confirmation of the true pathogenic effect of the variant prior to any genetic counseling or medical intervention.