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Clinical features and genetic findings in patients with Charcot Marie Tooth Disease Type 2 (CMT2) due to LRSAM1 mutation
Abstracts of the 10th Neuromuscular Translational Research Conference / Neuromuscular Disorder 27S1 (2017) S5–S44
the flies had severely reduced life span and locomotor activity, phenocopying the patients’ pathology. These features could be rescued by an ester of riboflavin. Conclusions: Overall our findings confirm the pathogenetic role of SLC52A2 and SLC52A3 in BVVLS, and thus have important clinical and therapeutic implications. PN04 Functional validation of non-coding variants of GJB1 in patients with CMTX1 A. Cortese1, A. Manole2, B. Ashokkumar2, R. Simone3, P.J. Tomaselli1, A.M. Rossor1, M. Laurá1, M. Skorupinska1, J.M. Polke4, R. Poh4, H. Houlden2, M.M. Reilly1 1 MRC Centre for Neuromuscular Diseases, National Hospital for Neurology and Neurosurgery, UCL Institute of Neurology, Queen Square, London, UK; 2 Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK; National Hospital for Neurology and Neurosurgery, Queen Square, London, UK; 3Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London, UK; 4Department of Neurogenetics, The National Hospital for Neurology and Neurosurgery, UCL Institute of Neurology, London, UK E-mail: [email protected]. Background: Changes in the cis-regulatory sequences of the untranslated regions (UTR), by influencing expression of specific genes at the level of translation, are increasingly recognised as a genetic cause of inherited disease in humans. In particular, variants in the non-coding region of GJB1 account for 10% of all mutation in GJB1 in our cohort of CMTX patients. One of the biggest challenges of analysing non-coding variants is to identify the diseasecausing mutations and to distinguish them from polymorphisms. Aims: Aim of this study is to implement a reliable method for in-vitro functional validation of pathogenicity of non-coding variants in the promoter and 3’UTR regions of GJB1. Methods and Preliminary Results: In our cohort of CMTX patients we previously identified 6 mutations (c.-592_591insT, c.-570G>A, c.-529T>G, c.-529T>C, c.-527G>C, c.-459C>T) in the promoter region and 1 novel mutation in the 3’UTR (c.876C>T), which were considered likely to be pathogenic based on the clinical features of patients and segregation of the mutation in affected family members, as well as one previously reported polymorphism (c.-713G>A). We generated a luciferase-based gene reporter system and optimised it in multiple cell line (Hela, HEK293 and glial H4) in order to account for tissuedependent variability of GJB1 gene expression. Mutations in the promoter region were generated by site-directed mutagenesis using a commercially available GJB1 promoter clone (Genecopoeia). The functional impact of the c.876C>T 3’UTR mutation will be tested by cloning the patient and healthy control DNA into pmirGLO vector (Promega). Discussion: Firstly, we plan to assess the effect of variants on GJB1 transcriptional activity. Secondly, mutations associated with a reduced luciferase activity will be further characterized in terms of their possible role in altering SOX10 and EGR2 binding to the promoter region and influencing splicing, miRNA binding to the 3’UTR or reducing GJB1 mRNA stability. If successful, our study will provide a useful tool for validation of mutations in non-coding regions of GJB1. Moreover, it will constitute a proof-of-principle method which could be adapted for the functional validation of non-coding variants in different genes, which will represent one of the major challenges in the forthcoming whole-genome sequencing era.
S25
PN05 Clinical features and genetic findings in patients with Charcot Marie Tooth Disease Type 2 (CMT2) due to LRSAM1 mutation A. Cortese1, M. Laurá1, J.M. Polke2, A.M. Rossor1, P.J. Tomaselli1, J. Blake1, R. Poh2, M.P. Lunn1, H. Houlden3, M.M. Reilly1 1 MRC Centre for Neuromuscular Diseases, National Hospital for Neurology and Neurosurgery, UCL Institute of Neurology, Queen Square, London, UK; 2Department of Neurogenetics, The National Hospital for Neurology and Neurosurgery, UCL Institute of Neurology, London, UK; 3Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK, and National Hospital for Neurology and Neurosurgery, Queen Square, London, UK Background: Recessive and dominant mutations in leucin-rich repeat and sterile alpha-motif-containing 1 (LRSAM1) have been associated with CMT2P. LRSAM1 is an ubiquitin E3 ligase containing a RING domain in its C-terminal, which is crucial for correct protein folding and ubiquitination activity. So far most of the mutations reported alter a major portion of the RING-domain by either frameshift or insertion of additional amino-acids, but point mutations have also been identified. Aim: To report the prevalence, clinical features and genetic findings in CMT2P patients due to LRSAM1 mutation. Methods: Review of genetic, clinical and neurophysiologic data in patients with LRSAM1 mutations. Results: By targeted next-generation sequencing of genes responsible for CMT2 in 57 genetically undiagnosed CMT2 patients, we identified 6 cases (10.5%) from 6 unrelated families with heterozygous mutations in LRSAM1. Identified mutations included frameshift insertions and deletions, non-frameshift deletion, non-sense and missense point mutations. All of the mutations were novel and were all located in the RING domain or in flanking regions. The average age of disease onset was in the 3rd decade but an earlier onset was reported in some cases. Four had a positive family history in keeping with an autosomal dominant disease. Symptoms at presentation were heterogeneous and encompassed distal numbness, unsteadiness, distal weakness of upper or lower limbs and foot deformities. Positive sensory symptoms, including tingling and shooting pains, and cramps were also frequently reported. Neurological examination showed mild to moderate distal atrophy and weakness, with early ankle plantar flexion involvement in three of them. Loss of vibration and reduced position sense was usually prominent in the lower limbs and appeared to be disproportionate to the degree of weakness and to the milder impairment of pinprick sensation. Ankle jerks were absent but knee and upper limb reflexes could be normal or brisk. After an average disease duration of 20 years, all but one patient were still able to walk independently. Nerve conduction studies showed a sensory and motor axonal neuropathy with normal conduction velocities. Discussion: Mutations in LRSAM1 are a relevant cause of CMT2 and are associated with prominent large fibre sensory loss. PN06 Mitochondrial dysfunction and abnormal calcium handling in cell models of Hereditary Sensory Neuropathy type I E. Wilson1,2, B. Kalmar1, M. Kugathasan1,2, A. Abramov3, M.M. Reilly2, L. Greensmith1,2 1 Sobell Department of Motor Neuroscience and Movement Disorders, 2MRC Centre for Neuromuscular Diseases, 3Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square House, Queen Square, London, UK E-mail: [email protected] Background: HSN-1 is a peripheral neuropathy most frequently caused by missense mutations in the SPTLC1 or SPTLC2 genes, which code for two subunits of the enzyme serine palmitoyltransferase (SPT). SPT catalyzes the first and rate limiting step of de novo sphingolipid synthesis. It has been shown that mutations in SPT cause a change in enzyme substrate specificity which results in the production of two atypical sphinganines, deoxysphinganine (DSp) and deoxymethylsphinganine (DMSp), rather than the normal enzyme product, sphinganine (Sp). Levels of DSp and DMSp are elevated in the blood of HSN-1 patients and this has been shown to cause the peripheral nerve damage characteristic of the disease, which affects both sensory as well as motor axons.
the flies had severely reduced life span and locomotor activity, phenocopying the patients’ pathology. These features could be rescued by an ester of riboflavin. Conclusions: Overall our findings confirm the pathogenetic role of SLC52A2 and SLC52A3 in BVVLS, and thus have important clinical and therapeutic implications. PN04 Functional validation of non-coding variants of GJB1 in patients with CMTX1 A. Cortese1, A. Manole2, B. Ashokkumar2, R. Simone3, P.J. Tomaselli1, A.M. Rossor1, M. Laurá1, M. Skorupinska1, J.M. Polke4, R. Poh4, H. Houlden2, M.M. Reilly1 1 MRC Centre for Neuromuscular Diseases, National Hospital for Neurology and Neurosurgery, UCL Institute of Neurology, Queen Square, London, UK; 2 Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK; National Hospital for Neurology and Neurosurgery, Queen Square, London, UK; 3Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London, UK; 4Department of Neurogenetics, The National Hospital for Neurology and Neurosurgery, UCL Institute of Neurology, London, UK E-mail: [email protected]. Background: Changes in the cis-regulatory sequences of the untranslated regions (UTR), by influencing expression of specific genes at the level of translation, are increasingly recognised as a genetic cause of inherited disease in humans. In particular, variants in the non-coding region of GJB1 account for 10% of all mutation in GJB1 in our cohort of CMTX patients. One of the biggest challenges of analysing non-coding variants is to identify the diseasecausing mutations and to distinguish them from polymorphisms. Aims: Aim of this study is to implement a reliable method for in-vitro functional validation of pathogenicity of non-coding variants in the promoter and 3’UTR regions of GJB1. Methods and Preliminary Results: In our cohort of CMTX patients we previously identified 6 mutations (c.-592_591insT, c.-570G>A, c.-529T>G, c.-529T>C, c.-527G>C, c.-459C>T) in the promoter region and 1 novel mutation in the 3’UTR (c.876C>T), which were considered likely to be pathogenic based on the clinical features of patients and segregation of the mutation in affected family members, as well as one previously reported polymorphism (c.-713G>A). We generated a luciferase-based gene reporter system and optimised it in multiple cell line (Hela, HEK293 and glial H4) in order to account for tissuedependent variability of GJB1 gene expression. Mutations in the promoter region were generated by site-directed mutagenesis using a commercially available GJB1 promoter clone (Genecopoeia). The functional impact of the c.876C>T 3’UTR mutation will be tested by cloning the patient and healthy control DNA into pmirGLO vector (Promega). Discussion: Firstly, we plan to assess the effect of variants on GJB1 transcriptional activity. Secondly, mutations associated with a reduced luciferase activity will be further characterized in terms of their possible role in altering SOX10 and EGR2 binding to the promoter region and influencing splicing, miRNA binding to the 3’UTR or reducing GJB1 mRNA stability. If successful, our study will provide a useful tool for validation of mutations in non-coding regions of GJB1. Moreover, it will constitute a proof-of-principle method which could be adapted for the functional validation of non-coding variants in different genes, which will represent one of the major challenges in the forthcoming whole-genome sequencing era.
S25
PN05 Clinical features and genetic findings in patients with Charcot Marie Tooth Disease Type 2 (CMT2) due to LRSAM1 mutation A. Cortese1, M. Laurá1, J.M. Polke2, A.M. Rossor1, P.J. Tomaselli1, J. Blake1, R. Poh2, M.P. Lunn1, H. Houlden3, M.M. Reilly1 1 MRC Centre for Neuromuscular Diseases, National Hospital for Neurology and Neurosurgery, UCL Institute of Neurology, Queen Square, London, UK; 2Department of Neurogenetics, The National Hospital for Neurology and Neurosurgery, UCL Institute of Neurology, London, UK; 3Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK, and National Hospital for Neurology and Neurosurgery, Queen Square, London, UK Background: Recessive and dominant mutations in leucin-rich repeat and sterile alpha-motif-containing 1 (LRSAM1) have been associated with CMT2P. LRSAM1 is an ubiquitin E3 ligase containing a RING domain in its C-terminal, which is crucial for correct protein folding and ubiquitination activity. So far most of the mutations reported alter a major portion of the RING-domain by either frameshift or insertion of additional amino-acids, but point mutations have also been identified. Aim: To report the prevalence, clinical features and genetic findings in CMT2P patients due to LRSAM1 mutation. Methods: Review of genetic, clinical and neurophysiologic data in patients with LRSAM1 mutations. Results: By targeted next-generation sequencing of genes responsible for CMT2 in 57 genetically undiagnosed CMT2 patients, we identified 6 cases (10.5%) from 6 unrelated families with heterozygous mutations in LRSAM1. Identified mutations included frameshift insertions and deletions, non-frameshift deletion, non-sense and missense point mutations. All of the mutations were novel and were all located in the RING domain or in flanking regions. The average age of disease onset was in the 3rd decade but an earlier onset was reported in some cases. Four had a positive family history in keeping with an autosomal dominant disease. Symptoms at presentation were heterogeneous and encompassed distal numbness, unsteadiness, distal weakness of upper or lower limbs and foot deformities. Positive sensory symptoms, including tingling and shooting pains, and cramps were also frequently reported. Neurological examination showed mild to moderate distal atrophy and weakness, with early ankle plantar flexion involvement in three of them. Loss of vibration and reduced position sense was usually prominent in the lower limbs and appeared to be disproportionate to the degree of weakness and to the milder impairment of pinprick sensation. Ankle jerks were absent but knee and upper limb reflexes could be normal or brisk. After an average disease duration of 20 years, all but one patient were still able to walk independently. Nerve conduction studies showed a sensory and motor axonal neuropathy with normal conduction velocities. Discussion: Mutations in LRSAM1 are a relevant cause of CMT2 and are associated with prominent large fibre sensory loss. PN06 Mitochondrial dysfunction and abnormal calcium handling in cell models of Hereditary Sensory Neuropathy type I E. Wilson1,2, B. Kalmar1, M. Kugathasan1,2, A. Abramov3, M.M. Reilly2, L. Greensmith1,2 1 Sobell Department of Motor Neuroscience and Movement Disorders, 2MRC Centre for Neuromuscular Diseases, 3Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square House, Queen Square, London, UK E-mail: [email protected] Background: HSN-1 is a peripheral neuropathy most frequently caused by missense mutations in the SPTLC1 or SPTLC2 genes, which code for two subunits of the enzyme serine palmitoyltransferase (SPT). SPT catalyzes the first and rate limiting step of de novo sphingolipid synthesis. It has been shown that mutations in SPT cause a change in enzyme substrate specificity which results in the production of two atypical sphinganines, deoxysphinganine (DSp) and deoxymethylsphinganine (DMSp), rather than the normal enzyme product, sphinganine (Sp). Levels of DSp and DMSp are elevated in the blood of HSN-1 patients and this has been shown to cause the peripheral nerve damage characteristic of the disease, which affects both sensory as well as motor axons.