MUSCULAR DYSTROPHY encompasses

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1 CASE REPORTS Loss of Podocyte Dysferlin Expression Is Associated With Minimal Change Nephropathy Hassane Izzedine, MD, PhD, Isabelle Brocheriou, MD, PhD, Bruno Eymard, MD, PhD, Monique Le Charpentier, MD, Norma Beatriz Romero, MD, Gilles LeNaour, MD, Edward Bourry, MD, and Gilbert Deray, MD We report a case of limb-girdle muscular dystrophy type 2B (LGMD2B) associated with minimal change disease. Immunohistochemical examination of quadriceps muscle showed a deficiency in dysferlin in sarcolemma, and dysferlin gene analysis showed 3370 G missense mutation, leading us to the diagnosis of LGMD2B. The patient also developed glomerular proteinuria. We also explored urinary protein levels in 3 other patients with dysferlinopathy and found microalbuminuria with albumin excretion of 0.14 to 0.18 g/d in 2 patients. Renal abnormalities during LGMD2B and kidney dysferlin expression have never been reported. Renal biopsy showed a lack of glomerular dysferlin expression compared with a positive immunohistochemical marking in patients with idiopathic minimal change nephropathy and healthy controls. We therefore suggest that dysferlin is present in glomeruli and may be associated with glomerular permeability. Am J Kidney Dis 48: by the National Kidney Foundation, Inc. INDEX WORDS: Dysferlin; podocyte; minimal change nephropathy. MUSCULAR DYSTROPHY encompasses a diverse group of inherited muscle diseases characterized by wasting and weakness of skeletal muscle. 1 During the last 15 years, an increasing number of genes have been identified that cause different forms of muscular dystrophy (Table 1). Dysferlin is a member of a recently defined class of homologous proteins called ferlins. Other members of this group are Caenorhabditis elegans spermatogenesis factor fer-1, 2 the human FER-1 like protein, 3 otoferlin, 4 and myoferlin. 5 The dysferlin gene product, a 230-kd membrane-associated protein residing on chromosome 2p13, is mutant in the autosomal recessive disorders limb-girdle muscular dystrophy (LGMD) type 2B (LGMD2B; Miyoshi myopathy [MM]: ) and MM (MM: ). 2,6 Bansal et al 7 showed that disruption of the muscle membrane repair machinery is responsible for dysferlindeficient muscle degeneration, but function of the dysferlin protein and damage and regression of muscle fiber that occur in its absence are still under investigation. Characteristic clinical features include proximal or distal muscular dystrophy and dramatic increase in serum creatine kinase levels, typically to more than 20 times the normal level. A thorough MEDLINE search revealed that renal abnormalities and kidney dysferlin expression during MM and LGMD have not been reported before. We describe a case of LGMD2B associated with minimal change nephropathy (MCN). We also explored urinary protein levels in 3 other patients with dysferlinopathy and compared renal dysferlin expression in this patient versus normal kidney tissue and an idiopathic MCN biopsy specimen. CASE REPORT A 30-year-old African man was admitted to the Department of Internal Medicine at Pitie-Salpetriere Hospital (Paris, France) in August 2001 with a history of progressive muscle wasting and weakness in the lower limbs and proximal portion of the upper limbs that had lasted for several months. The patient was the second son of consanguineous parents. His elderly parents are still alive and without specific neuromuscular diseases. The patient was of short stature (155 cm, 55.3 kg) with relative preservation of these muscles and marked atrophy of the back muscles and hamstrings. Manual muscle testing showed 3/5 to 4( )/5 in the proximal and 4/5 to 4( )/5 in the distal part of the upper extremities, with 3/5 to 2/5 in the pelvic girdle and 1/5 to 0/5 in the knee and From the Departments of Nephrology and Pathology and Myopathy Research Unit, Pitie-Salpetriere Hospital, Paris, France. Received February 24, 2006; accepted in revised form April 18, Originally published online as doi: /j.ajkd on May 30, Support: None. Potential conflicts of interest: None. Address reprint requests to Hassane Izzedine, MD, PhD, Department of Nephrology, Pitie-Salpetriere Hospital 83, Blvd de l Hôpital, 75013, Paris, France. hassan. izzedine@psl.ap-hop-paris.fr 2006 by the National Kidney Foundation, Inc /06/ $32.00/0 doi: /j.ajkd American Journal of Kidney Diseases, Vol 48, No 1 (July), 2006: pp

2 Table 1. Muscular Dystrophy and Gene Locations 144 Disease Name Mode of Inheritance Clinical Features Gene Locus Gene Product Muscle Only: LGMD LGMD1A ADI LGMD affecting mainly the proximal limb 5q31 Myotilin (TTID; ) muscles and sparing the face; distal muscle weakness may occur later LGMD1C ADI Calf hypertrophy and mild to moderate proximal 3p25 1q41 Caveolin-3 (CAV3; ) RMD1 (600332) muscle weakness with a positive Gowers sign; rippling muscle syndrome (RSMD2); distal myopathy LGMD2A ARI Leyden-Moebius muscular dystrophy 15q15.1-q21.1 Calpain 3 (CAPN3; ) LGMD2B ARI MM, distal myopathy 2p13.3-p13.1 Dysferlin (DYSF; ) LGMD2H ARI Proximal muscle weakness, creatine kinase levels 4 times the upper limit of normal 9q Tripartite motif-containing protein-32 (TRIM32; ) LGMD2J ARI Tibial muscular dystrophy 2q24.3 Titin (TTN; ) Extensive Muscular Dystrophy: Merosin-absent CMD ARI Partial deficiencies lead to milder disease, heart may be affected LGMD2I ARI Spine rigidity, early restrictive lung disease; creatine kinase level elevated Rigid spine disease (RSMD1) ARI Spine rigidity, early restrictive lung disease; creatine kinase level normal Ullrich myopathy Genetic heterogeneity Very early contractures including arthrogryposis, distal hyperlaxicity, flat feet Ocular and Brain Involvements: Fukuyama CMD RI Myopia, cataract, and optic nerve atrophy in Japanese population; cobblestone cortex malformation, cerebellar and brainstem hypoplasia; severe mental retardation Muscle-eye-brain disease Myopia, noncongenital cataract, ganglion cell and optic nerve atrophy in Finland population; cobblestone cortex, pachygyria/ agyria, cerebellar and brainstem hypoplasia, mild hydrocephalus; severe mental retardation (Continued) 6q22-q23 Merosin, 2 chain of laminin (LAMA2; ) 19q13.3 Fukutin-related protein (FKRP; ). 1p36-p35 Selenoprotein N (SEPN1, ) 21q22.3 2q37 Coll VI (COL6A1,A2) Coll VI (COL6A3) 9q31-33 Fukutin (FCMD; ) 1p34-p33 O-Mannose beta-1,2-n-acetylglucosaminyltransferase (POMGNT1; ) IZZEDINE ET AL

3 Disease Name Table 1 (Cont d). Muscular Dystrophy and Gene Locations Mode of Inheritance Clinical Features Gene Locus Gene Product Walker Warburg syndrome ARI Retinal abnormality, myopia, noncongenital cataract, and optic nerve atrophy, Peters anomaly; cobblestone lissencephaly, severe hydrocephalus, abnormal white matter, polymicrogyria, cerebellar and brainstem hypoplasia, midline fusion, abnormal corpus callosum; severe mental retardation 9q34.1 9q31 O-Mannosyltransferase (POMT1; ) Cardiac Involvement: DMD/BMD XLR Progressive proximal muscular dystrophy with Xp21.2 Dystrophin (DMD; ) characteristic pseudohypertrophy of the calves; isolated cardiomyopathy; massive elevation of creatine kinase levels; abnormal retinal neurotransmission LGMD1B* ADI Dilated cardiomyopathy and cardiac conduction 1q11-21 Lamin A/C (LMNA ) system defect; Emery-Dreifuss muscular dystrophy LGMD1C** ADI Moderate cardiomyopathy, 3p25 Caveolin 3 (CAV3; ) hypercreatinekinaseemia, distal myopathy LGMD2C ARI Severe cardiomyopathy, LGMD 13q12 -Sarcoglycan (SGCG; ) LGMD2D ARI LGMD, moderate cardiomyopathy 17q Sarcoglycan (SGCA; ) LGMD2E ARI LGMD, severe cardiomyopathy 4q13 -Sarcoglycan (SGCB; ) LGMD2F ARI LGMD, severe cardiomyopathy 5q Sarcoglycan (SGCD; ) LGMD2G ARI LGMD, severe cardiomyopathy 17q11-12 Telethonin (TCAP; ). Abbreviations: ADI, autosomal dominant inheritance; ARI, autosomal recessive inheritance; XLR, X-linked recessive; DMD/BMD, Duchenne muscular dystrophy/becker muscular dystrophy; LGMD1B*, Duningan-type familial partial lipodystrophy; LGMD1C**, rippling muscle syndrome. DYSFERLINOPATHY AND MINIMAL CHANGE NEPHROPATHY 145

4 146 ankle. Grip powers were 25.0:30.0 kg with approximately 50% reduction in his third decade. Deep tendon reflexes generally were negative without myotonia or fasciculation. Intellectual and cranial nervous systems were intact, as were sensory and autonomic functions. Bone studies throughout the spine and joints showed moderate to marked limitation of flexion, especially in the ankle joints. Routine laboratory data were unremarkable, except for high levels of serum creatine kinase (14,500 IU/L compared with normal levels of 195 IU/L), with 98% of creatine kinase MB isoform; lactic dehydrogenase (1,601 IU/L compared with normal levels of 190 to 390 IU/L); myoglobin (706 ng/ml versus 10 ng/ml in urine of healthy individuals); and aldolase (221 ng/ml compared with normal levels of 7.6 ng/ml). There were no abnormal findings on a chest radiogram or repeated electrocardiograms. Additional physical examination, including ultrasonic cardiogram and magnetic nuclear resonance cardioimaging, alimentary system surveys, and autoantibodies, showed normal results. A computed tomographic scan of skeletal muscle showed selective atrophy with fatty replacement that was most prominent in the posterior component of femoral muscles and anteromedial compartment of calf muscles. Moderate atrophy also was noted in the posterior compartment of shoulder girdle muscles and paravertebral muscles. Electrophysiological examinations showed that nerve conduction was unremarkable. Needle electromyography showed typical myogenic patterns corresponding to the grade of muscle atrophy shown by computed tomographic scanning, consisting of occasional positive sharp waves at rest, whereas at mild contraction, they were mainly myogenic. In addition, there was variation in the severity and selectivity of muscle involvement, but no myotonic discharge was detected. A muscle biopsy was performed in the left quadriceps muscle; immunohistochemical and immunoblot analyses showed a lack of dysferlin labeling such that a diagnosis of dysferlinopathy was made. Three years later, the patient experienced selective glomerular proteinuria with protein of 3.5 g/24 h, serum albumin level of 3.6 g/dl (36 g/l), and microscopic hematuria. Blood pressure was controlled by using diuretic therapy (115/68 mm Hg). Renal function was normal (serum creatinine, 0.82 mg/dl [72 mol/l]; creatinine clearance, 90 ml/min [1.50 ml/s]), and urinalysis showed microscopic hematuria. At no time were nonsteroidal anti-inflammatory drugs administered. A percutaneous kidney biopsy was performed. Twenty-four hour urinary protein testing performed in 3 of our 10 patients with dysferlinopathy showed microalbuminuria with albumin excretion of 140 and 180 mg/d in a 38-year-old woman and 61-year-old man, respectively. Results were negative in a 21-year-old man. Muscle Tissue For pathological and immunohistochemical analyses, serial frozen sections of the biopsied muscle were stained with hematoxylin and eosin, modified Gomori trichrome, and a series of routine histochemical stains that include nicotinamide adenine dinucleotide tetrazolium reductase, succinate dehydrogenase, adenosine triphosphatase, acid phosphatase, cytochrome-c oxidase, periodic acid Schiff, oil red O, alkaline phosphatase, acetylcholine esterase, neuron-specific enorase, phosphorylase, phosphofructokinase, and adenosine monophosphate deaminase. For immunohistochemical studies, specimens were reacted with antibodies to dysferlin. Immunoblot analysis was performed using a mini-multiplex Western blotting system. 8,9 Molecular genetic analysis was performed after obtaining informed consent. Genomic DNA was extracted from peripheral-blood lymphocytes of the patient and used as a template for polymerase chain reaction amplification for single-strand conformation polymorphism analysis. Primer pairs and conditions for polymerase chain reaction for each 55 exons of the dysferlin gene have been described previously. 10 Direct sequence analyses of mutant alleles detected by means of single-strand conformation polymorphism analysis were performed on polymerase chain reaction fragments. Kidney Tissue Renal biopsy specimens, cut at 2 m or less, were processed by a skilled technologist. Renal biopsy specimens were processed for light microscopy and immunofluorescence according to standard techniques. At least 11 serial sections were stained with hematoxylin and eosin and saffron, periodic acid Schiff, Masson trichrome stain, and Jones methenamine silver stain. Routine immunofluorescence analyses were performed on 3- m cryostat sections by using polyclonal fluorescein isothiocyanate-conjugated antibodies to immunoglobulin G (IgG), IgM, IgA, C3, C1q,,, fibrinogen, and albumin (Dako Corp, Carpinteria, CA). Routine immunoperoxidase analyses of kidney serial frozen sections were performed with monoclonal dysferlin antibodies (1/80 Novocastra Laboratories, Newcastle upon Tyne, UK, using a ventana kit [Ventana Medical Systems, Illkirch Cedex, France]). Finally, electron microscopy was performed. RESULTS IZZEDINE ET AL Muscle Tissue Hematoxylin and eosin staining showed a moderate variation in fiber size, measuring from 20 to 120 m in diameter. Few necrotic fibers were seen, but there were many regenerating fibers and moderate endomysial fibrosis with centrally placed nuclei (comprising 5% of all fibers). On modified Gomori trichrome, no ragged red fibers were seen. Nicotinamide adenine dinucleotide tetrazolium reductase and succinate dehydrogenase staining showed lobulated fibers, but there were no strongly succinate dehydrogenase-reactive blood vessels. On adenosine triphosphatase staining, fiber-type groupings were not seen. Types 1, 2A, 2B, and 2C fibers comprised 60%, 17%, 13%, and 10% of fibers, respectively. Staining of other enzymes showed no abnormalities. These findings were consistent with muscular dystrophy and, in particular, suggestive of LGMD.

5 DYSFERLINOPATHY AND MINIMAL CHANGE NEPHROPATHY 147 Immunohistochemical examination showed that sections were labeled positively with all antibodies similar to normal muscle, except for dysferlin. Immunoblot analysis showed a lack of dysferlin labeling, but other dystrophy-relating antibodies were detectable (antibodies to dystrophin, calpain 3, -sarcoglycan, -dystroglycan, and caveolin 3), confirming dysferlin deficiency in this patient. Sequence analysis of the dysferlin gene showed a compound heterozygote for a frameshift (G3016 1A) in exon 25 and a missense (G3370T, causing amino acid replacement of cysteine by tryptophan at position 999) mutation in exon 28, again confirming dysferlin deficiency. Kidney Tissue Renal biopsy showed normal renal parenchyma containing 15 glomeruli: 3 glomeruli were sclerotic and 12 glomeruli had normal architecture (Fig 1A). Tubular, interstitial, and vascular compartments also were normal, except for some nephroangiosclerosis lesions. No staining was found by using immunofluorescence. These pathological findings suggested MCN. By electron microscopy, the most characteristic lesion is the disappearance of slit diaphragms with effacement of foot processes. No immunetype electron-dense deposits were seen. Peripheral glomerular basement membranes were normal in thickness, texture, and contour. Major ultrastructural findings were marked hypertrophy of visceral epithelial cells with increased organella content, intracytoplasmic transport vesicles, and extensive foot-process fusion involving more than 95% of the total glomerular capillary surface area (original magnification 10,000; Fig 1B). Immunohistochemically, sections were labeled positively with dysferlin antibodies in cytoplasm of renal epithelial tubular cells within normal renal tissue (Fig 2A and B), idiopathic MCN kidney tissue (Fig 2C and D), and tissue from our patient (Fig 2E). However, there was a lack of dysferlin labeling in glomeruli in the biopsy sample from our patient (Fig 2E and F) compared with positive dysferlin labeling in podocyte cytoplasm in renal tissue from a patient with isolated hematuria (Fig 2A and B) and another patient with idiopathic MCN (Fig 2C and D), suggesting a relationship between dysferlin deficiency and MCN in our patient. Furthermore, immunohistochemical studies of renal tissue from 1 other patient presenting with only idiopathic MCN showed the same labeling pattern for dysferlin as noted in our control case. Fig 1. (A) Glomerulus shows no proliferative or sclerotic lesions. (Periodic acid Schiff stain; original magnification 40.) (B) Electron microscopy shows extensive foot-process fusion involving more than 95% of the total glomerular capillary surface area. (Original magnification 10,000.)

6 148 IZZEDINE ET AL Fig 2. Immunohistochemical staining for dysferlin on normal kidney tissue shows (A) tubular and (A, B) glomerular positive cytoplasmic staining in epithelial cells and podocytes, as in (C, D) kidney tissue from a patient with idiopathic MCN. Immunohistochemical staining for dysferlin on kidney tissue of our patient shows similar positive cytoplasmic staining of (E) epithelial tubular cells as in normal control and idiopathic MCN tissue, but (E, F) loss of staining in dysferlin podocytes. (Original magnification: [A, C] 200, [B, D, F] 400, [E] 100.)

7 DYSFERLINOPATHY AND MINIMAL CHANGE NEPHROPATHY 149 DISCUSSION Our results show that dysferlin is located in renal epithelial tubular cells and podocyte cytoplasm in normal renal and idiopathic MCN tissue and seems to provide some evidence for its lack of expression in MCN associated with dysferlinopathy. MCN is a renal disease characterized by heavy glomerular proteinuria occurring in the absence of cellular glomerular infiltrates or immunoglobulin deposits. Electron microscopy showed that changes are focused in glomerular epithelial cells in the form of foot-process effacement. Almost all cases are idiopathic, but a small percentage of cases ( 10% to 20%) may have an identifiable cause, such as drugs (eg, nonsteroidal anti-inflammatory drugs, lithium), toxins (eg, mercury), infection (eg, human immunodeficiency virus), and tumor (eg, Hodgkin lymphoma). Dysferlin was associated with the calciumdependent protease calpain 3 and the sarcolemma protein caveolin 3. 10,11 Modulation of calpain activity contributes to muscular dystrophies by disrupting cell-regulatory mechanisms, whereas caveolin 3 organizes lipid and protein constituents in plasma membrane as part of a vesicular transport mechanism. Calpain 3 messenger RNA in skeletal muscle is 1,000% more abundant than messenger RNA of ubiquitous calpains 1 and The caveolin family consists of caveolin 1, 2, 3 and their isoforms; caveolin 1 and caveolin 2 are coexpressed and form a heterooligomeric complex in many cell types, whereas caveolin 3 is expressed mainly in muscle. Caveolin 1 immunogold localization in podocytes is often, but not exclusively, found at podocyte filtration slits. Filtration slit proteins, including nephrin, CD2-associated protein, 13 and podocin, 14 are associated with lipid rafts. 15 These filtration slit proteins are all required to maintain the glomerular barrier to protein filtration because deletion of nephrin, CD2-associated protein, and podocin result in congenital proteinuria and progressive focal and segmental glomerular sclerosis. In addition, dual labeling with antinephrin and anticaveolin 1 antibodies results in partial colocalization of antibodies around the capillary loops of glomeruli, further increasing the evidence for nephrin and caveolin 1 association. In skeletal muscle, many of the genes implicated in the LGMD2B and MM group of diseases encode proteins that make up the dystrophin-glycoprotein complex, which straddles the sarcolemma and crosslinks structural components inside the cell and extracellular environment. 16 Mutations in any component of the dystrophin-glycoprotein complex lead to secondary loss of other components and eventual breakdown of the sarcolemma. Furthermore, in MCN, the density of -dystroglycan expression is significantly decreased to 25% of that in controls, and that of -dystroglycan, to approximately 50% of that in controls. 17 In our 4 patients with dysferlinopathy, 1 patient presented with nephrotic-range proteinuria, whereas 2 patients had only microalbuminuria and no proteinuria was found in the last patient. One might ask whether there is a link between the expressed renal phenotype and underlying specific genotype of dysferlinopathy. In other words, is there a relation between a specific genotypic subpopulation and degree of proteinuria? Here, we show that dysferlin is expressed on normal tubular and glomerular epithelial cells. Dysferlinopathy perhaps might be added to the list of diseases that can induce MCN. Additional studies are warranted to establish the role of dysferlin in glomerular permeability. Although no cause-and-effect relationship can be deduced from this report, careful monitoring of renal function and proteinuria might be needed in patients with dysferlinopathy. REFERENCES 1. Cohn RD, Campbell KP: Molecular basis of muscular dystrophies. Muscle Nerve 23: , Bashir R, Britton S, Strachan T, et al: A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B. Nat Genet 20:37-42, Britton S, Freeman T, Vafiadaki E, et al: The third human FER-1-like protein is highly similar to dysferlin. Genomics 68: , Yasunaga S, Grati M, Cohen-Salmon M, et al: A mutation in OTOF, encoding otoferlin, a FER-1-like protein, causes DFNB9, a nonsyndromic form of deafness. Nat Genet 21: , Davis DB, Delmonte AJ, Ly CT, McNally EM: Myoferlin, a candidate gene and potential modifier of muscular dystrophy. Hum Mol Genet 9: , Liu J, Aoki M, Illa I, et al: Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy. Nat Genet 20:31-36, 1998

8 150 IZZEDINE ET AL 7. Bansal D, Miyake K, Vogel SS, et al: Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature 423: , Tagawa K, Ogawa M, Kawabe K, et al: Protein and gene analysis of dysferlinopathy in a large group of Japanese muscular dystrophy patients. J Neurol Sci 211: 23-25, Ho M, Gallardo E, McKenna-Yasek D, De Luna N, Illa I, Brown RH Jr: A novel, blood-based diagnostic assay for limb girdle muscular dystrophy 2B and Miyoshi myopathy. Ann Neurol 51: , Matsuda C, Hayashi YK, Ogawa M, et al: The sarcolemmal proteins dysferlin and caveolin-3 interact in skeletal muscle. Hum Mol Genet 10: , Anderson LV, Harrison RM, Pogue R, et al: Secondary reduction in calpain 3 expression in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy (primary dysferlinopathies). Neuromuscul Disord 10: , Sorimachi H, Toyama-Sorimachi N, Saido TC, et al: Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle. J Biol Chem 268: , Shih NY, Li J, Cotran R, Mundel P, Miner JH, Shaw AS: CD2AP localizes to the slit diaphragm and binds to nephrin via a novel C-terminal domain. Am J Pathol 159: , Schwarz K, Simons M, Reiser J, et al: Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin. J Clin Invest 108: , Simons M, Schwarz K, Kriz W, et al: Involvement of lipid rafts in nephrin phosphorylation and organization of the glomerular slit diaphragm. Am J Pathol 159: , Durbeej M, Campbell KP: Muscular dystrophies involving the dystrophin-glycoprotein complex: An overview of current mouse models. Curr Opin Genet Dev 12: , Regele HM, Fillipovic E, Langer B, et al: Glomerular expression of dystroglycans is reduced in minimal change nephrosis but not in focal segmental glomerulosclerosis. J Am Soc Nephrol 11: , 2000