LIMS2 mutations are associated with a novel muscular dystrophy, severe cardiomyopathy and triangular tongues

Transcription

1 Clin Genet 2015: 88: Printed in Singapore. All rights reserved Short Report 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: /cge LIMS2 mutations are associated with a novel muscular dystrophy, severe cardiomyopathy and triangular tongues Warman Chardon J., Smith A.C., Woulfe J., Pena E., Rakhra K., Dennie C., Beaulieu C., Huang L., Schwartzentruber J., Hawkins C., Harms M.B., Dojeiji S., Zhang M., FORGE Canada Consortium, Majewski J., Bulman D.E., Boycott K.M., Dyment D.A. LIMS2 mutations are associated with a novel muscular dystrophy, severe cardiomyopathy and triangular tongues. Clin Genet 2015: 88: John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2015 Limb girdle muscular dystrophy (LGMD) is a heterogeneous group of genetic disorders leading to progressive muscle degeneration and often associated with cardiac complications. We present two adult siblings with childhood-onset of weakness progressing to a severe quadriparesis with the additional features of triangular tongues and biventricular cardiac dysfunction. Whole exome sequencing identified compound heterozygous missense mutations that are predicted to be pathogenic in LIMS2. Biopsy of skeletal muscle demonstrated disrupted immunostaining of LIMS2. This is the first report of mutations in LIMS2 and resulting disruption of the integrin linked kinase (ILK) LIMS parvin complex associated with LGMD. Conflict of interest No potential conflicts of interests declared. Jodi Warman Chardon a,d, A.C. Smith b, J. Woulfe c,d,e.pena e, K. Rakhra d,e, C. Dennie d,e,f, C. Beaulieu b, Lijia Huang b, J. Schwartzentruber g, C. Hawkins h, M.B. Harms i, S. Dojeiji j, M. Zhang d, FORGE Canada Consortium b,j. Majewski k, D.E. Bulman b,l, K.M. Boycott a,b and D.A. Dyment a,b a Department of Genetics, Children s Hospital of Eastern Ontario, Ottawa, Ontario, Canada, b Children s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Ontario, Canada, c Department of Pathology, The Ottawa Hospital, Ottawa, Ontario, Canada, d Ottawa Hospital Research Institute, Ottawa, Ontario, Canada, e Department of Medical Imaging, The Ottawa Hospital, Ottawa, Ontario, Canada, f University of Ottawa Heart Institute, Ottawa, Ontario, Canada, g McGill University and Genome Quebec Innovation Center, Montreal, Quebec, Canada, h Department of Laboratory Medicine & Pathobiology, The Hospital for Sick Children, Toronto, Ontario, Canada, i Department of Neurology and Hope Center for Neurological Disorders, Washington University, Saint Louis, MO, USA, j The Ottawa Hospital Rehabilitation Center, Ottawa, Ontario, Canada, k Department of Human Genetics, McGill University, Montreal, Quebec, Canada, and l Department of Pediatrics, Children s Hospital of Eastern Ontario, Ottawa, Ontario, Canada Key words: cardiomyopathy exome sequencing limb girdle muscular dystrophy LIMS2 Corresponding author: Jodi Warman Chardon, MSc, MD, FRCPC, Children s Hospital of Eastern Ontario, Regional Genetics Program, 401 Smyth Road, Ottawa, Ontario, Canada K1H 8L1. 558

2 LIMS2 mutations associated with LGMD Tel.: x2826; fax: Received 30 November 2014, revised and accepted for publication 9 January 2015 Limb girdle muscular dystrophy (LGMD) refers to a group of genetic conditions that present with weakness in a proximal distribution from child to adulthood. The LGMDs are progressive in nature, and patients can develop severe quadriparesis. The underlying genetic cause can be difficult to elucidate due to significant genetic heterogeneity and general lack of clinically defining features that might suggest a particular gene is responsible. Given that certain LGMD subtypes are associated with atrioventricular conduction blocks, dilated cardiomyopathy, and sudden death (1), a precise molecular diagnosis is an important step in the care of these patients. At present, LGMD may result from alteration in one of over two dozen genes (2). The traditional approach of Sanger sequencing each individual candidate gene can be a costly and time consuming endeavor; despite best efforts, a large proportion of patients remain molecularly undiagnosed (3). Variant identification by targeted next-generation sequencing (NGS) has become an important diagnostic tool for disorders that are characterized by significant genetic heterogeneity, such as LGMD (2), Charcot Marie Tooth disease (4), and ataxia (5, 6). When this approach does not identify the causative variant(s) in known genes, the likelihood that the phenotype is caused by a previously unidentified disease gene is relatively high. In this regard, sequencing the exome (the coding sequence of the genome) has proven to be a highly effective method for novel gene discovery (7). Subjects and methods Patients A brother and sister presented to the regional Neurogenetics clinic for evaluation with a previous clinical diagnosis of LGMD. Charts, clinical records, and pathology slides were reviewed, and the affected individuals were examined. All participants provided written informed consent, and all study procedures were approved by the ethics committee at the Children s Hospital of Eastern Ontario as part of the Finding of Rare Disease Genes (FORGE) Canada project. DNA from the patients and unaffected family members was extracted using standard procedures. Muscle biopsy Fragments of the muscle biopsy were flash-frozen in liquid nitrogen at 80 C. Other fragments were immersed in 4% buffered paraformaldehyde and submitted for paraffin embedding. From the frozen muscle, sections were cut at 5 μm on a cyrotome and stained using hematoxylin and eosin, modified Gomori trichrome, periodic acid Schiff, van Gieson, oil red O, and Masson trichrome. Additional sections were stained histochemically for the demonstration of NADH, lactate dehydrogenase, acid phosphatase, and myophosphorylase and myofibrillar ATPase (developed at ph 4.3, 4.6, and 9.4). Sections of the frozen muscle were immunostained using antibodies to the N- and C-terminal and rod domains of dystrophin and glycosylated alpha-dystroglycan. Sections of the paraffin-embedded muscle biopsy were cut at 4μm, processed for antigen retrieval by microwaving in sodium citrate buffer ph 6.0, and stained immunohistochemically for PINCH 2 (Abcam, Cambridge, MA; goat polyclonal 1:100) using a rabbit anti-goat biotinylated secondary antibodies (1:200; Vector, Burlingame, CA). Detection of bound secondary antibody was achieved using the avidin-biotin peroxidase technique and diaminobenzidine as the chromogen. Exome sequencing Exome capture and high-throughput sequencing of DNA from the two siblings and unaffected mother were performed at McGill University and Genome Québec Innovation Centre (Montréal, Canada). Exome target enrichment was performed using the Agilent SureSelect, Santa Clara, CA, 50 Mb All Exon Kit, and sequencing (Illumina HiSeq, San Diego, CA) generated 9 11 Gbp of 100 bp paired-end reads per sample. Mean coverage of coding sequence regions, after accounting for duplicate reads, was over 80 for each of the affected siblings. An in-house annotation pipeline was used to call and annotate coding and splice-site variants and has been described previously (8). Candidate variants were confirmed by Sanger sequencing and segregated in the family. Results Clinical features The siblings were born to nonconsanguineous parents of Northern European ancestry. Both sibs met early motor milestones. At age 5 years, they both developed proximal weakness, with calf hypertrophy and macroglossia documented on examination. Their weakness progressed to severe quadriparesis; both siblings required a manual wheelchair by approximately age 12 years, and motorized wheelchair by age 19 years. The brother was diagnosed with a dilated cardiomyopathy 559

3 Warman Chardon et al. A B C D E F Fig. 1. Clinical features associated with limb girdle muscular dystrophy (LGMD) in these patients. (a) Photograph of the brother showing the triangular shape to his tongue at 36 years. Macroglossia was less evident at the tip, giving the appearance of a triangular tongue with a broad based root and small tongue tip. (b) Late gadolinium-enhanced magnetic resonance (MR) image in short axis view demonstrates subepicardial enhancement involving the inferior and inferolateral wall of the left ventricle (arrows) indicating fibrosis that is similar in both siblings with regional hypokinesis. Biventricular dilation and systolic dysfunction on cardiac MRI was noted in both siblings. Muscle MRI from the affected sister 33 years of age. Axial images through (d) the level of the proximal humeri and (b) upper lumbar spine and (c) coronal images at the hips demonstrate diffuse, bilateral, symmetric and severe muscle atrophy and fat infiltration consistent with longstanding muscular dystrophy. with an echocardiogram at age 35 years, demonstrating an ejection fraction of 32% and moderate global hypokinesis. The sister had asymptomatic, moderate, dilated cardiomyopathy. Both siblings performed at a high academic level in university and currently hold professional level employment. The brother was more severely affected. At age 36 years, neurological exam demonstrated moderate to severe quadriparesis as follows: 0/5 proximally with 1 2/5 in finger flexors, extensors and biceps. In the lower extremities, there was hypotonia, 0/5 muscle grading and severe equinovarus contractures. Macroglossia was observed that was less evident at the tip, giving the appearance of a triangular tongue with a broad based root and small tongue tip (Fig. 1a). The younger sister demonstrated 2/5 power in finger and wrist flexors/ extensors, biceps and triceps; she also had a triangular tongue. Serum creatine kinase values were approximately 5000 IU at age of 5 years and 500 IU in the third decade. Cardiac magnetic resonance imaging (MRI) demonstrated a region of subepicardial delayed enhancement involving the inferior and inferolateral wall of the left ventricle that was similar in both siblings (Fig. 1b). Cardiac arrhythmias were not seen on Holter monitor. Muscle MRI of the affected sister at age 33 demonstrated diffuse, bilateral, symmetric and severe muscle atrophy and fat infiltration consistent with longstanding muscular dystrophy (Fig. 1c e); these findings were similar for both siblings. Clinical genetic testing Genetic testing performed on clinical basis was unrevealing and included targeted NGS for the LGMD genes ANO5, CAPN3, DYSF, FKRP, FKTN, POMT1, POMT2, SGCA, SGCB, SGCD, SGCG, TCAP, TRIM32 and TTN. Exome sequencing results Given that a molecular diagnosis was not forthcoming, exome sequencing of both siblings and their mother was undertaken. There were no rare pathogenic variants seen in any other known myopathy-related genes. Exome data were analyzed under an autosomal recessive model of inheritance, and only variants in LIMS2 were both rare (<1%) and present in trans. In both affected siblings, we identified two variants in the same exon (c.342c>g; p.asn114lys) and (c.356c>t; p.pro119leu) and a third variant (c.1034 T>C; p.leu345pro) in the LIMS2 gene (NM_ ), none of which are seen in the 1000 genomes or NHLBI exome datasets. Sanger 560

4 LIMS2 mutations associated with LGMD Fig. 2. Pedigree and Sanger sequencing pedigree (a) indicating the affected siblings. Sanger sequencing (b) confirmed that the LIMS2 variants were present in the affected siblings. The father and unaffected brother carried only the c.1034 T>C variant in exon 11. The mother carried the two variants in exon 5 (c.342c>g and c.356c>t), confirming that both of these variants were in cis. sequencing confirmed that the LIMS2 variants were present in the affected siblings (Fig. 2a,b). The father and unaffected brother carried only the c.1034 T>C variant. The mother carried the two variants in the same exon (c.342c>g and c.356c>t), confirming they were in cis. In silico analyses of the variants with Polyphen2, SIFT and GERP showed evidence for pathogenicity for the three variants; however, the programs are unable to differentiate the degree of pathogenicity of the two variants in exon 5 inherited from the mother. Muscle biopsy Exome sequencing results led to re-analysis of the original muscle biopsy. A second biopsy was considered but deemed unlikely to be helpful given the marked diffuse muscle wasting. The muscle biopsy of the deltoid muscle at age 6 years in the brother demonstrated dystrophic features. Interestingly, these appeared to be focal and consisted of variation in fiber size with scattered necrotic fibers invaded by macrophages (myophagocytosis) and a proliferation of endomysial and perimysial connective tissue (Fig. 3a). Immunostaining for Pinch2 in control muscle revealed intense immunoreactivity of z-discs (Fig. 3b), consistent with its previously described subcellular localization. There was a marked reduction in this pattern of staining in the patient muscle (Fig. 3c), despite the preservation of z-discs as revealed in sections of patient muscle stained for desmin (Fig. 3d). In addition, Pinch1 (Fig. 3e) and Pinch2 (Fig. 3f) immunostaining in muscle fibers is localized to z-discs in a muscle sample of a patient with LGMD2I related to homozygous Fukutin-related protein gene (FKRP) mutations. Discussion Exome sequencing of two siblings with severe LGMD revealed compound heterozygous mutations in LIMS2. Ongoing functional studies may determine which of the two exon 5 variants inherited from the mother is the causative mutation, and there is a possibility that both may be required. However, the presence of the mutations inherited from each parent highlights LIMS2 as the disease gene. These siblings had the hallmark features of an LGMD with the additional distinctive feature of a triangular shaped tongue. On cardiac MRI, both siblings also demonstrated a subepicardial rim of delayed enhancement predominantly in the inferolateral wall, likely representing fibrosis. Enhancement in this distinctive location has been hypothesized to represent myocardial damage in response to mechanical stress in patients with muscular dystrophies (9). The assessment of systolic dysfunction and myocardial enhancement on cardiac magnetic resonance in both patients, even in the younger sibling asymptomatic from a cardiac perspective, highlights the role of this imaging modality in characterizing the cardiac dysfunction in patients with muscular dystrophy. LIMS2, also known as PINCH2 (particularly interesting new cysteine-histidine rich protein 2), has 5 LIM domains each with unique sequences and 2 zinc finger motifs important for protein interaction and is expressed in cardiac and skeletal muscle (10). LIMS is an evolutionarily conserved protein (Fig. 4), critical for muscle attachment (11). LIMS, along with integrin linked kinase (ILK) and parvin, is part of a ternary complex known as ILK Pinch Parvin (IPP), a core component of the integrin actin cytoskeleton and functions as a signaling mediator that transmits mechanical signals to downstream effectors (12). In humans, mutations in this gene have not been previously identified in the IPP complex. The gold standard for novel gene discovery would be identifying additional families with a similar phenotype and pathogenic mutations in the LIMS2 gene. We have presented results of a single family and are looking to identify additional families with a similar clinical presentation and mutation in LIMS2. In lieu of a second family, model systems provide additional evidence for novel gene pathogenicity. We have identified mutations in the LIM2 (c.342c>g; p.asn114lys) and (c.356c>t; p.pro119leu) and LIM5 domains 561

5 Warman Chardon et al. A B C D E F Fig. 3. Muscle biopsy. (a) Hematoxylin and eosin-stained section of the frozen muscle biopsy specimen shows focal dystrophic features with variability in fiber size, necrotic fibers with myophagocytosis (arrows), and increased endomysial and perimysial connective tissue (asterisks). (b) Pinch2 immunostaining in non-diseased muscle fibers is localized to z-discs (arrows in inset, lower left). Reduced Pinch2 (c), relative to desmin (d) immunostaining of z-discs in patient muscle (insets showing higher magnification lower left). Pinch 1 (e) and Pinch 2 (f) and immunostaining in musclefibers is localized to z-discs in LGMD2I. Scale bars = 100 μmina, 10μMinb-f. (c.1034t>c; p.leu345pro) of LIMS2. Loss of full length protein of any of the IPP components, including PINCH2/LIMS2, affects efficient assembly of the IPP complex into focal adhesions in vertebrate cell culture (12). Mutations in the LIMS ortholog in Caenorhabditis elegans cause paralysis characterized by muscle detachment (13). In Drosophilia, LIMS the protein localizes with ILK/integrins at muscle attachment and flies lacking the protein show detachment of muscles from the body wall (14). In humans, the two LIMS paralogs, LIMS1 and LIMS2, demonstrate 82% homology (15), although PINCH2 contains an 11 amino acid extension on the C-terminal tail. Overexpression of LIMS2 inhibits ILK PINCH1 interactions and decreases cell migration and spreading, suggesting that PINCH2 regulates ILK PINCH1 interactions (15). Both LIMS proteins localize to sarcomeric Z disks and costameres in heart and skeletal muscle and inactivation leads to dilated cardiomyopathy in zebrafish (16). LIMS2 knockout mice have not been reported to exhibit a LGMD-like phenotype, which may reflect a redundant role for the two LIMS paralogs in the mouse myocardium (10), alternatively, the weakness and cardiomyopathy may develop later. In keeping with the latter hypothesis, the absence of either LIMS1 or LIMS2 in myocardium leads to aggravated cardiac injury and cardiomyopathy after myocardial infarction in mice (10). Loss of function or null mutations in the other members of the IPP complex also result in muscle detachment (11). Taken together, these findings support that mutations in LIMS2 cause the LGMD and cardiomyopathy phenotype in this family. In conclusion, exome sequencing is continuing to provide novel pathogenic insights into LGMD (17). Here, we present a novel clinical syndrome characterized by early-onset proximal greater than distal muscular dystrophy, calf hypertrophy and macroglossia progressing to severe LGMD with cardiomyopathy and triangular tongues in siblings. We identified rare compound heterozygous variants in LIMS2 and the observed human phenotype mirrors the observations in model organisms. 562

6 LIMS2 mutations associated with LGMD Fig. 4. LIMS 2 conservation and LIMS2 variant protein modeling. (a) LIMS2 variants are perfectly conserved among vertebrates. (b) PyMOL structure of exon 2 variants of LIMS2 (p.asn114lys; p.pro119leu); there is no available three dimensional structure for exon 5 variant (p.leu345pro) and therefore unable to predict effect of this change on protein structure. This is the first report of defective LIMS2 associated with a human neuromuscular disease in a single family. Our findings suggest disruption of the IPP complex as a novel mechanism of muscular dystrophies. Acknowledgements The authors would like to thank the study participants and their families first without their participation, this work would not have been possible. This work was funded in part by the Government of Canada through Genome Canada, the Canadian Institutes of Health Research (CIHR), and the Ontario Genomics Institute (OGI-049). Additional funding was provided by Genome Quebec and Genome British Columbia. J. W. C. was in part supported by a fellowship from the Children s Hospital of Eastern Ontario. We would like to acknowledge the contribution of the high-throughput sequencing platform of the McGill University and Génome Québec Innovation Centre, Montréal, Canada. This work was selected for study by the FORGE Canada Steering Committee, consisting of K. Boycott (University of Ottawa), J. Friedman (University of British Columbia), J. Michaud (University of Montreal), F. Bernier (University of Calgary), M. Brudno (University of Toronto), B. Fernandez (Memorial University), B. Knoppers (McGill University), M. Samuels (University of de Montreal), and S. Scherer (University of Toronto). References 1. Chrestian N, Valdmanis PN, Echahidi N et al. A novel mutation in a large French-Canadian family with LGMD1B. Can J Neurol Sci 2008: 35 (3):

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