Limbic System Associated Membrane Protein Mutation in an Iranian Family Diagnosed with Ménière’s Disease

Received: July 1, 2019, Accepted: January 26, 2020, ePublished: May 1, 2020 Abstract Background: Ménière’s disease (MD) is a common inner ear disorder which is characterized by recurrent attacks of vertigo, fluctuating sensorineural hearing loss (SNHL), tinnitus, and a sense of fullness in the affected ear. MD is a complex disorder; although six genes have been linked to familial autosomal dominant form of the disease, in many cases, the exact genetic etiology remains elusive. Methods: To elucidate the genetic causes of MD in an Iranian family, we performed exome sequencing on all members of the family: consanguineous parents and four children (two affected and two unaffected). Variant filtering was completed using a customized workflow keeping variants based on segregation with MD in autosomal recessive (AR) inheritance pattern, minor allele frequency (MAF), and in-silico prediction of pathogenicity. Results: Analysis revealed that in this family, 970 variants co-segregated with MD in AR pattern, out of which eight variants (one intergenic, four intronic, and three exonic) were extremely rare. The exonic variants included a synonymous substitution in USP3 gene, an in-frame deletion in ZBED2 gene, and a rare, highly conserved deleterious missense alteration in LSAMP gene. Conclusion: The phenotype observed in the proband described here, i.e. vertigo, poor sense of smell, tinnitus, and borderline hearing ability, may originate from aberrant changes in the cerebellum and limbic system due to a deleterious mutation in the LSAMP gene; hence, LSAMP mutation is a possible candidate for the etiology of MD in this family.


Introduction
Diseases which display phenotypic heterogeneity, pleiotropy and incomplete penetrance, often arise from a complex combination of etiological factors, including genetic and environmental components. As such, these diseases are often hard to characterize and consequently, it is difficult to elucidate the genetics and pathophysiology underlying the disease, as is the case with Ménière's disease (MD [MIM 156000]). MD is a complex disease which displays considerable clinical heterogeneity. The broad clinical spectrum of symptoms includes episodes of spontaneous vertigo lasting from 20 minutes to several hours 1,2 becoming less frequent but more severe over time. 3 Sensorineural hearing loss (SNHL) typically begins at low frequencies and fluctuates, ultimately resulting in progressive moderate to severe hearing loss. 1,3-5 Alongside vertigo and SNHL, patients often experience aural fullness and tinnitus, an imaginary but debilitating sensation of ringing, buzzing, or hissing. MD symptoms mainly affect one ear (unilateral MD); however, bilateral MD has also been reported. 1,5,6 Broadly, MD falls into two clinical categories: definite and probable, with probable being less phenotypically severe. 7 MD usually begins to manifest in the third to the seventh decades of life with a small female predominance, and only a few rare cases have been identified in younger people. 2,7,8 The prevalence of MD is estimated to be roughly 0.5-1/1000 individuals. 1,6,7,9,10 Among these, approximately 10% are familial MD (FMD), 4 in which at least one other relative (first-or second-degree) fulfills all of the criteria for definite or probable MD. 3,4,11 While FMD and sporadic MD exhibit the same phenotypes, there is often greater severity and earlier onset in FMD. 3,4,8,12 FMD has been described in families originating from the UK, Brazil, Sweden, Finland, Germany, and Spain. 3,4,6,[10][11][12][13] Outside the Caucasian population, MD is rare or virtually absent in many populations. 2,12 It is considered to be a multifactorial disorder and the clinical heterogeneity observed among patients points to the suggested etiological diversity including environmental and genetic factors, dysfunction of the immune system, and viral infections. 4,9 The fact that approximately one-third of MD patients have family members with MD-like symptoms, and different MD incidences in different populations lend credence to the role of genetics in the development of MD. 11 MD can be inherited in autosomal dominant (AD), and rarely in mitochondrial or recessive patterns. 3,4,6,12,13 Mutations in DTNA, FAM136A, COCH, DPT, SEMA3D, and PRKCB genes have been described in ADFMD 2,14-16 ; however, no gene has been identified for recessive inheritance. Here, we describe a consanguineous Iranian family with autosomal recessive (AR) FMD. After detailed genetic analysis, we conclude that a mutation of the gene encoding the limbic system associated membrane protein (LSAMP) plays a role in ARFMD.

Materials and Methods
An Iranian Lur family (L-8600471) with apparent ARFMD was selected for this study. Clinical examination, including brain MRI, was performed. Pure tone audiometry was carried out to determine air conduction thresholds at 0.25, 0.5, 1, 2, 3, 4 and 8 kHz. After obtaining informed consent, whole blood samples were collected from all members of the core family including both individuals affected with ARFMD, unaffected siblings and both parents. DNA was extracted using the conventional salting out method. 17 All procedures were approved by the human research institutional review boards at the University of Social Welfare and Rehabilitation Sciences and Iran University of Medical Sciences, Tehran, Iran.
Targeted genomic enrichment (TGE) was performed using either the Agilent SureSelect Human All Exon kit (v.5) or the Agilent SureSelect XT Human All Exon kit (v.6) (Agilent Technologies, Inc., Santa Clara, CA, USA). Captured genomic libraries underwent subsequent massively parallel sequencing (MPS) using the Illumina HiSeq2000 or the Illumina NextSeq 500 platform (Illumina Inc., San Diego, CA, USA). After sequencing, raw reads were aligned to the hg19 build of the human reference genome using Burrows-Wheeler Aligner (BWA), MEM algorithm. 18 Data processing and variant discovery (SNPs and Indels) were performed according to the best practices workflow of the Genome Analysis Toolkit (GATK). 19,20 Variants were listed in a gVCF file per sample which were then merged together. Variant annotation was performed using ANNOVAR software. 21 Annotated variants underwent filtering and prioritization based on the AR mode of inheritance. Those variants were kept for which both parents were heterozygous, both healthy siblings were wild type homozygous or heterozygous, and both affected siblings were mutated homozygous. Variants were further prioritized considering their frequencies in the 1000 Genome project, ExAC, ESP6500, genomAD, exomAD, and Iranome databases (http://iranome. com/). [22][23][24] The prioritizing cut-off started at minor allele frequency (MAF)<0.05 and then narrowed down to 0.01, 0.001, and finally 0.0001.

Results
Family L-8600471 originates from western Iran and is of Lur ethnicity with consanguineous parents (Figure 1a). Brain MRI was normal in proband IV:3. Audiological examination revealed borderline hearing in both ears of proband IV:3 ( Figure 1b). She complained of vertigo, a sense of vomiting, tinnitus, hearing impairment, and poor sense of smell, with onset around 30 years of age. After clinical examination, she was diagnosed with definite MD. The same phenotypes, apart from a poor sense of smell, were reported in the proband's sister (IV:4) with onset at 45 years of age; she also had a history of depression.

Discussion
MD is a complex disorder. It displays great inter-and intra-familial phenotypic heterogeneity and the exact pathomechanisms and genetic etiology underlying these phenotypes remain, for the most part, elusive. In this study, we utilized the power of exome sequencing on a consanguineous Iranian family with apparent ARFMD to implicate the LSAMP gene in the pathobiology of MD. For decades, families from consanguineous regions have been used to investigate the molecular genetics of diseases. [25][26][27] To this list of diseases, we add MD. We have identified a nonsynonymous variant (chr3:115561402T>C; c.673A>G; p.K225E) in the gene encoding the limbic system associated membrane protein (LSAMP) (Figure 2). Affected individuals were homozygous, and unaffected siblings and parents were heterozygous for this mutation (Figure 3). The p.K225E variant occurs in the highly conserved lysine 225 residue (Figure 4). The change from positively charged lysine to the negatively charged glutamic acid is predicted to be deleterious ( Table 2) and most likely has a negative impact on local protein folding. It is predicted that this variant perturbs wild-type function either by disrupting a site for protein interactions or by altering folding.
LSAMP is a neuronal surface adhesion glycoprotein in cortical and subcortical regions of the limbic system. It takes part in neurite outgrowth, development of neuronal connection patterns and synaptic plasticity with an essential role in regulating the formation of septohippocampal, intrahippocampal, and thalamic connections. [28][29][30][31] LSAMP is expressed in the hippocampus, cerebellum, eye, urogenital system, liver, heart, skeletal muscle, and small intestine. [31][32][33] LSAMP is a candidate in the susceptibility to familial clustering of schizophrenia, bipolar disorder, and major depressive disorder. 34 SNPs in LSAMP have been reported to have an association of genome-wide significance with ulcerative colitis in African Americans. 33 LSAMP is also suggested to be a tumor suppressor and its genomic deletion is reported in aggressive prostate cancer, osteosarcoma, clear cell renal cell carcinoma, epithelial ovarian cancer, and acute myeloid leukemia. 33,35,36 LSAMP downregulation has been shown to be associated with coronary artery disease. 37 Animal studies have shown alteration in the regulation of emotional and social behavior in a changing environment in LSAMP-deficient mice. 32,36,38 Cerebellum involvement has been established in cognitive and sensory motor processes such as maintaining balance, olfactory and auditory activities. [39][40][41][42] It has been shown that PFL and vermis regions in the cerebellum are connected to auditory centers, response to sound and involvement in tinnitus perception. 42,43 The potential role of structures in the limbic system in auditory processing and tinnitus has been suggested in various studies. [44][45][46][47][48] The hippocampus, one of the structures in the limbic system, has a bilateral connection with auditory cortices to provide and receive inputs. 46 Chan et al showed that the hippocampus coordinates brain-wide neural activities including auditory activity at slow oscillation. 44 Uchida et al suggested an association between hearing loss and hippocampal atrophy. 49 Seo et al reported a significant reduction in hippocampus volume in MD patients. 50 Tinnitus is a network problem that involves the auditory and limbic regions of the brain including the amygdala, hippocampus, and parahippocampus. [45][46][47]51,52 Chen et al showed that pharmacologically induced tinnitus implies increased connections between the auditory cortex, the cerebellum and the hippocampal gyrus. 43 Zhang et al suggested that cholinergic signaling in the hippocampus, which modifies its synaptic plasticity or excitability, may be involved in the pathophysiology of tinnitus. 46 Crippa et al observed different strengths of connections between the auditory cortex and limbic structures in patients with tinnitus. 48 It has been shown that methylamine intoxication or bilateral resection of the hippocampus  result in tinnitus development. 53,54 In summary, we have identified a promising candidate gene (LSAMP) for ARFMD segregating in an Iranian family. The phenotype observed in the proband described here, i.e. vertigo, poor sense of smell, tinnitus, and borderline hearing ability, may originate from aberrant changes in the cerebellum and limbic system due to a deleterious mutation in the LSAMP gene. Further studies, including screening of MD patients for variants in the LSAMP gene and functional studies of the pathomechanism driving the disease, are needed to fully clarify the role of LSAMP in MD.

Conflict of Interest Disclosures
None declared.

Ethical Statement
This study was approved by the human research institutional review boards at the University of Social Welfare and Rehabilitation Sciences and Iran University of Medical Sciences, Tehran, Iran.