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A novel FKRP mutation in congenital muscular dystrophy disrupts the dystrophin glycoprotein complex
Neuromuscular Disorders 17 (2007) 285–289 www.elsevier.com/locate/nmd
Case report
A novel FKRP mutation in congenital muscular dystrophy disrupts the dystrophin glycoprotein complex Heather MacLeod a,1, Peter Pytel b,1, Robert Wollmann b, Ewa Chelmicka-Schorr c, Kenneth Silver c, Rebecca Brown Anderson d, Darrel Waggoner d, Elizabeth M. McNally a,d,* a
Department of Medicine, Section of Cardiology, The University of Chicago, Chicago, IL 60637, USA b Department of Pathology, The University of Chicago, Chicago, IL 60637, USA c Department of Neurology, The University of Chicago, Chicago, IL 60637, USA d Department of Human Genetics, The University of Chicago, Chicago, IL 60637, USA Received 9 October 2006; received in revised form 27 December 2006; accepted 8 January 2007
Abstract Mutations in the gene encoding fukutin related protein (FKRP) produce a spectrum of disease including congenital muscular dystrophy and limb girdle muscular dystrophy. FKRP is one member of a class of molecules thought to be glycosyltransferases that mediate O-linked glycosylation. The primary target of these glycosyltransferases is thought to be dystroglycan. We now report two unrelated Mexican children with congenital muscular dystrophy who each have the identical, novel 1387A > G, N463D mutation. Muscle biopsies from these children show a reduction of a-dystroglycan and also show reduction of b-dystroglycan, and a-, b-, and c-sarcoglycan, suggesting that FKRP mutations can perturb membrane associated proteins beyond dystroglycan. Ó 2007 Elsevier B.V. All rights reserved. Keywords: FKRP; Congenital muscular dystrophy; Sarcoglycan; Dystroglycan; Founder mutation
1. Introduction Fukutin-related protein (FKRP) was identified as a homolog of fukutin, the defective protein in Fukutin Muscular Dystrophy [1,2]. The primary protein sequence predicted from these gene sequences indicates homology to a class of proteins that are thought to function as glycosyltransferases, specifically mediating Olinked glycosylation. Mutations in FKRP have been Abbreviations: FKRP, fukutin related protein; LGMD, limb girdle muscular dystrophy. * Corresponding author. Tel.: +1 773 702 2672; fax: +1 773 702 2681. E-mail address: [email protected] (E.M. McNally). 1 These authors contributed equally to this work. 0960-8966/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2007.01.005
linked to variable phenotypes including congenital muscular dystrophy, also referred to as MDC1C, and limbgirdle muscular dystrophy (LGMD) 2I that may or may not be accompanied by dilated cardiomyopathy. Previous reports include descriptions of founder mutations in the European population (826C > A, L276I) and the Tunisian population (1364C > A, A455D) [1,3,4]. We now report two cases of Mexican–American girls from non-consanguineous parents with an identical homozygous (1387A > G, N463D) mutation associated with congenital muscular dystrophy. Interestingly, in addition to the expected reduction in a-dystroglycan, the muscle biopsies from these children also showed a reduction of immunoreactivity to b-dystroglycan and a-, b-, and c-sarcoglycan consistent with a more widespread effect on the dystrophin glycoprotein complex.
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H. MacLeod et al. / Neuromuscular Disorders 17 (2007) 285–289
2. Clinical information Patient 1 was a delivered at term by C-section due to breech presentation following a normal pregnancy and weighed 8 lbs 11 oz. Her parents denied consanguinity; both were from the city of San Luis Potosi, Mexico. At birth she was noted to be hypotonic, but her immediate postnatal course was otherwise unremarkable. At age 6 months, her parents noted a lag in her motor development with poor head and truncal control and the inability to bear weight on her legs. Her developmental milestones included sitting independently and rolling over at the age of 2 years. At age 5 years, she did not pull to stand nor walk. Her personal/social developments were normal. She was felt to be of normal intelligence. Her speech was mildly affected due to the weakness of her mouth and facial muscles. She receives physical, occupational and speech therapies. Her most recent physical exam at age 5 showed a head circumference of 49.5 cm. Her cranial nerves were intact save for bilateral facial weakness. Her muscle tone and muscle strength were significantly decreased proximally more than distally. She had contractures in her knees and ankles. Her deep tendon reflexes were absent. Her sensory examination was normal. She was able to reach without tremor or dysmetria. At 4 years, her serum creatine kinase (CK) was found to be 7500 U/l. An echocardiogram revealed normal heart structure and function. Her EKG demonstrated normal sinus rhythm, and non-specific ST and T wave abnormality. No imaging abnormalities were found on brain MRI. EMG done at age 3 was abnormal demonstrating evidence of myopathy. There was no evidence of abnormal neuromuscular junction transmission or motor neuron diseases. Patient 2 was born to non-consanguineous parents from Toluca, a city in central Mexico approximately 250 miles from where Patient 1 was born. The patient was noted to be hypotonic at birth and underwent evaluation for hypotonia. At age 17 months she had an episode of acute respiratory distress for which she was intubated and ventilated for 2 weeks. At this time she had myoglobinuria. Since then she has had multiple hospital admissions. At age 3 years, she was not crawling, creeping or standing. She was able to sit alone and was noted to speak in sentences with apparently normal cognition. She has not had myalgia or exercise-induced myalgia, and has tolerated general anesthesia. She had a gastrostomy tube placed for nutrition. On physical exam at age 3, her head circumference was normal. She was found to have calf hypertrophy and brachioradialis hypertrophy. She had marked myopathic facies. She had ptosis in her left eye and astigmatism. She did not have tongue hypertrophy, ophthalmoplegia, or strabismus. Her muscle weakness was more marked proximally than distally.
Her serum CK was elevated at 6607 U/l. Her EMG was significant for diffuse myopathic changes. Nerve conduction velocities, EEG and brain MRI were normal. Her EKG and echocardiogram at age 3 were both normal. 3. Pathology Muscle biopsies from both children showed chronic myopathic changes with marked variation in myofiber size as well as fatty replacement and endomysial fibrosis. Both cases showed the same pattern of abnormal immunohistochemical staining for sarcolemmal membrane proteins where reactivity for spectrin was well preserved but a-dystroglycan was absent as has been previously shown for FKRP mutations [5] (Figs. 1 and 2). Additional antibodies to the dystrophin glycoprotein complex showed a reduction of immunoreactivity for b-dystroglycan as well as a-, b-, and c-sarcoglycan. There was mild focal reduction of staining for dystrophin using antibodies directed at the N-terminus, rod or C-terminus (Supplementary Fig. 1) similar to what was seen for merosin immunostaining. All of the immunohistochemical stains were repeated three times with the same results. All stains were performed with commercially available monoclonal antibodies as follows: anti-a-sarcoglycan (Vector Laboratories, Burlingame, CA); anti-b-sarcoglycan, anti-c-sarcoglycan, anti-b-dystroglycan and the three domains of dystrophin (Novocastra, Newcastle upone Tyne, UK); anti-merosin (Chemicon, Temecula, CA); anti-spectrin (Cell Marque, Hot Springs, AR); anti-a-dystroglycan (Upstate, Lake Placid, NY). 4. Genetics Full sequencing of the FKRP gene coding region in both children identified the homozygous 1387A > G transition leading to the amino acid substitution N463D. N463 is conserved in human and mouse FKRP amino acid sequences. The parents of Patient 1 agreed to undergo testing and were both found to be heterozygous carriers 1387A > G. 5. Discussion FKRP mutations have been associated with multiple phenotypes including limb girdle muscular dystrophy and congenital muscular dystrophy with or without central nervous system involvement. Founder mutations have been noted in FKRP, and most notable is the L276I mutation that is highly prevalent in Great Britain with a significant carrier frequency [6]. It is thought that the carrier frequency of the L276I mutation has been sustained due to its comparatively later onset of disease than what is noted with congenital muscular dystrophyassociated mutations [3,7]. We now report an apparent founder mutation in the FKRP gene that is present in
H. MacLeod et al. / Neuromuscular Disorders 17 (2007) 285–289
287
Fig. 1. Muscle biopsy of Patient 1. The hematoxylin and eosin stained sections showed chronic myopathic changes with focal inflammatory infiltrates. Immunoreactivity for spectrin was preserved while staining for a-dystroglycan was reduced. Similarly reduced was the staining for b-dystroglycan, as well as a-, b-, and c-sarcoglycan, while merosin staining was only minimally decreased. The size bar is 100 lm.
individuals whose parents were born in central Mexico. The frequency of this mutation in the Mexican population is not known. This mutation is unlike the later onset LGMD-associated mutation and instead is associated with an early onset muscular dystrophy but much more consistent with a MDC1C phenotype that is without evident central nervous system involvement. The N463D mutation falls near the carboxy-terminus of FKRP,
but no definite genotype–phenotype relationship has emerged for FKRP mutations. Muscle biopsies from both of these children show an interesting pattern of immunohistochemical staining distinct from the pattern described for other FKRP mutations. Loss of a-dystroglycan is seen commonly with FKRP mutations and is also seen frequently associated with mutations in other genes thought to encode glyco-
288
H. MacLeod et al. / Neuromuscular Disorders 17 (2007) 285–289
Fig. 2. Muscle biopsy of Patient 2. Hematoxylin and eosin stained sections showed similar chronic myopathic changes as in patient 1, and a similar pattern of immunostaining was noted with preserved spectrin and reduced a-dystroglycan. Similarly reduced was b-dystroglycan, as well as a-, b-, and c-sarcoglycan, while merosin staining showed only mild focal reduction. The size bar is 100 lm.
syltransferases such as POMT1, POMT2, POMGnT1, and fukutin. This staining pattern has been attributed to likely loss of O-linked glycosylation where a-dystroglycan is the target [5]. Patchy staining for merosin (laminin a2) has also been noted and is consistent with the known interaction between a-dystroglycan and merosin. Unusual in these muscle biopsies is the secondary, patchy loss of b-dystroglycan and the reduction in a-, b-,
and c-sarcoglycan immunostaining, suggesting that unique FKRP mutations have differential effects on the dystrophin glycoprotein complex. Further biochemical studies of the enzymatic function of FKRP in conjunction with the elucidation of enzymatic targets and interacting proteins are needed to appreciate the role of the putative glycosyltransferases and their role in normal muscle and brain development.
H. MacLeod et al. / Neuromuscular Disorders 17 (2007) 285–289
Acknowledgment Supported by NIH, MDA, the Heart Research Foundation and the Burroughs Welcome Foundation (to E.M.M.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.nmd.2007.01.005. References [1] Brockington M, Yuva Y, Prandini P, et al. Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C. Hum Mol Genet 2001;10:2851–9.
289
[2] Brockington M, Blake DJ, Prandini P, et al. Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan. Am J Hum Genet 2001;69:1198–209. [3] Sveen ML, Schwartz M, Vissing J. High prevalence and phenotype–genotype correlations of limb girdle muscular dystrophy type 2I in Denmark. Ann Neurol 2006;59:808–15. [4] Louhichi N, Triki C, Quijano-Roy S, et al. New FKRP mutations causing congenital muscular dystrophy associated with mental retardation and central nervous system abnormalities identification of a founder mutation in Tunisian families. Neurogenetics 2004;5:27–34. [5] Brown SC, Torelli S, Brockington M, et al. Abnormalities in alpha-dystroglycan expression in MDC1C and LGMD2I muscular dystrophies. Am J Pathol 2004;164:727–37. [6] Poppe M, Cree L, Bourke J, et al. The phenotype of limb-girdle muscular dystrophy type 2I. Neurology 2003;60:1246–51. [7] Topaloglu H, Brockington M, Yuva Y, et al. FKRP gene mutations cause congenital muscular dystrophy, mental retardation, and cerebellar cysts. Neurology 2003;60:988–92.
Case report
A novel FKRP mutation in congenital muscular dystrophy disrupts the dystrophin glycoprotein complex Heather MacLeod a,1, Peter Pytel b,1, Robert Wollmann b, Ewa Chelmicka-Schorr c, Kenneth Silver c, Rebecca Brown Anderson d, Darrel Waggoner d, Elizabeth M. McNally a,d,* a
Department of Medicine, Section of Cardiology, The University of Chicago, Chicago, IL 60637, USA b Department of Pathology, The University of Chicago, Chicago, IL 60637, USA c Department of Neurology, The University of Chicago, Chicago, IL 60637, USA d Department of Human Genetics, The University of Chicago, Chicago, IL 60637, USA Received 9 October 2006; received in revised form 27 December 2006; accepted 8 January 2007
Abstract Mutations in the gene encoding fukutin related protein (FKRP) produce a spectrum of disease including congenital muscular dystrophy and limb girdle muscular dystrophy. FKRP is one member of a class of molecules thought to be glycosyltransferases that mediate O-linked glycosylation. The primary target of these glycosyltransferases is thought to be dystroglycan. We now report two unrelated Mexican children with congenital muscular dystrophy who each have the identical, novel 1387A > G, N463D mutation. Muscle biopsies from these children show a reduction of a-dystroglycan and also show reduction of b-dystroglycan, and a-, b-, and c-sarcoglycan, suggesting that FKRP mutations can perturb membrane associated proteins beyond dystroglycan. Ó 2007 Elsevier B.V. All rights reserved. Keywords: FKRP; Congenital muscular dystrophy; Sarcoglycan; Dystroglycan; Founder mutation
1. Introduction Fukutin-related protein (FKRP) was identified as a homolog of fukutin, the defective protein in Fukutin Muscular Dystrophy [1,2]. The primary protein sequence predicted from these gene sequences indicates homology to a class of proteins that are thought to function as glycosyltransferases, specifically mediating Olinked glycosylation. Mutations in FKRP have been Abbreviations: FKRP, fukutin related protein; LGMD, limb girdle muscular dystrophy. * Corresponding author. Tel.: +1 773 702 2672; fax: +1 773 702 2681. E-mail address: [email protected] (E.M. McNally). 1 These authors contributed equally to this work. 0960-8966/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2007.01.005
linked to variable phenotypes including congenital muscular dystrophy, also referred to as MDC1C, and limbgirdle muscular dystrophy (LGMD) 2I that may or may not be accompanied by dilated cardiomyopathy. Previous reports include descriptions of founder mutations in the European population (826C > A, L276I) and the Tunisian population (1364C > A, A455D) [1,3,4]. We now report two cases of Mexican–American girls from non-consanguineous parents with an identical homozygous (1387A > G, N463D) mutation associated with congenital muscular dystrophy. Interestingly, in addition to the expected reduction in a-dystroglycan, the muscle biopsies from these children also showed a reduction of immunoreactivity to b-dystroglycan and a-, b-, and c-sarcoglycan consistent with a more widespread effect on the dystrophin glycoprotein complex.
286
H. MacLeod et al. / Neuromuscular Disorders 17 (2007) 285–289
2. Clinical information Patient 1 was a delivered at term by C-section due to breech presentation following a normal pregnancy and weighed 8 lbs 11 oz. Her parents denied consanguinity; both were from the city of San Luis Potosi, Mexico. At birth she was noted to be hypotonic, but her immediate postnatal course was otherwise unremarkable. At age 6 months, her parents noted a lag in her motor development with poor head and truncal control and the inability to bear weight on her legs. Her developmental milestones included sitting independently and rolling over at the age of 2 years. At age 5 years, she did not pull to stand nor walk. Her personal/social developments were normal. She was felt to be of normal intelligence. Her speech was mildly affected due to the weakness of her mouth and facial muscles. She receives physical, occupational and speech therapies. Her most recent physical exam at age 5 showed a head circumference of 49.5 cm. Her cranial nerves were intact save for bilateral facial weakness. Her muscle tone and muscle strength were significantly decreased proximally more than distally. She had contractures in her knees and ankles. Her deep tendon reflexes were absent. Her sensory examination was normal. She was able to reach without tremor or dysmetria. At 4 years, her serum creatine kinase (CK) was found to be 7500 U/l. An echocardiogram revealed normal heart structure and function. Her EKG demonstrated normal sinus rhythm, and non-specific ST and T wave abnormality. No imaging abnormalities were found on brain MRI. EMG done at age 3 was abnormal demonstrating evidence of myopathy. There was no evidence of abnormal neuromuscular junction transmission or motor neuron diseases. Patient 2 was born to non-consanguineous parents from Toluca, a city in central Mexico approximately 250 miles from where Patient 1 was born. The patient was noted to be hypotonic at birth and underwent evaluation for hypotonia. At age 17 months she had an episode of acute respiratory distress for which she was intubated and ventilated for 2 weeks. At this time she had myoglobinuria. Since then she has had multiple hospital admissions. At age 3 years, she was not crawling, creeping or standing. She was able to sit alone and was noted to speak in sentences with apparently normal cognition. She has not had myalgia or exercise-induced myalgia, and has tolerated general anesthesia. She had a gastrostomy tube placed for nutrition. On physical exam at age 3, her head circumference was normal. She was found to have calf hypertrophy and brachioradialis hypertrophy. She had marked myopathic facies. She had ptosis in her left eye and astigmatism. She did not have tongue hypertrophy, ophthalmoplegia, or strabismus. Her muscle weakness was more marked proximally than distally.
Her serum CK was elevated at 6607 U/l. Her EMG was significant for diffuse myopathic changes. Nerve conduction velocities, EEG and brain MRI were normal. Her EKG and echocardiogram at age 3 were both normal. 3. Pathology Muscle biopsies from both children showed chronic myopathic changes with marked variation in myofiber size as well as fatty replacement and endomysial fibrosis. Both cases showed the same pattern of abnormal immunohistochemical staining for sarcolemmal membrane proteins where reactivity for spectrin was well preserved but a-dystroglycan was absent as has been previously shown for FKRP mutations [5] (Figs. 1 and 2). Additional antibodies to the dystrophin glycoprotein complex showed a reduction of immunoreactivity for b-dystroglycan as well as a-, b-, and c-sarcoglycan. There was mild focal reduction of staining for dystrophin using antibodies directed at the N-terminus, rod or C-terminus (Supplementary Fig. 1) similar to what was seen for merosin immunostaining. All of the immunohistochemical stains were repeated three times with the same results. All stains were performed with commercially available monoclonal antibodies as follows: anti-a-sarcoglycan (Vector Laboratories, Burlingame, CA); anti-b-sarcoglycan, anti-c-sarcoglycan, anti-b-dystroglycan and the three domains of dystrophin (Novocastra, Newcastle upone Tyne, UK); anti-merosin (Chemicon, Temecula, CA); anti-spectrin (Cell Marque, Hot Springs, AR); anti-a-dystroglycan (Upstate, Lake Placid, NY). 4. Genetics Full sequencing of the FKRP gene coding region in both children identified the homozygous 1387A > G transition leading to the amino acid substitution N463D. N463 is conserved in human and mouse FKRP amino acid sequences. The parents of Patient 1 agreed to undergo testing and were both found to be heterozygous carriers 1387A > G. 5. Discussion FKRP mutations have been associated with multiple phenotypes including limb girdle muscular dystrophy and congenital muscular dystrophy with or without central nervous system involvement. Founder mutations have been noted in FKRP, and most notable is the L276I mutation that is highly prevalent in Great Britain with a significant carrier frequency [6]. It is thought that the carrier frequency of the L276I mutation has been sustained due to its comparatively later onset of disease than what is noted with congenital muscular dystrophyassociated mutations [3,7]. We now report an apparent founder mutation in the FKRP gene that is present in
H. MacLeod et al. / Neuromuscular Disorders 17 (2007) 285–289
287
Fig. 1. Muscle biopsy of Patient 1. The hematoxylin and eosin stained sections showed chronic myopathic changes with focal inflammatory infiltrates. Immunoreactivity for spectrin was preserved while staining for a-dystroglycan was reduced. Similarly reduced was the staining for b-dystroglycan, as well as a-, b-, and c-sarcoglycan, while merosin staining was only minimally decreased. The size bar is 100 lm.
individuals whose parents were born in central Mexico. The frequency of this mutation in the Mexican population is not known. This mutation is unlike the later onset LGMD-associated mutation and instead is associated with an early onset muscular dystrophy but much more consistent with a MDC1C phenotype that is without evident central nervous system involvement. The N463D mutation falls near the carboxy-terminus of FKRP,
but no definite genotype–phenotype relationship has emerged for FKRP mutations. Muscle biopsies from both of these children show an interesting pattern of immunohistochemical staining distinct from the pattern described for other FKRP mutations. Loss of a-dystroglycan is seen commonly with FKRP mutations and is also seen frequently associated with mutations in other genes thought to encode glyco-
288
H. MacLeod et al. / Neuromuscular Disorders 17 (2007) 285–289
Fig. 2. Muscle biopsy of Patient 2. Hematoxylin and eosin stained sections showed similar chronic myopathic changes as in patient 1, and a similar pattern of immunostaining was noted with preserved spectrin and reduced a-dystroglycan. Similarly reduced was b-dystroglycan, as well as a-, b-, and c-sarcoglycan, while merosin staining showed only mild focal reduction. The size bar is 100 lm.
syltransferases such as POMT1, POMT2, POMGnT1, and fukutin. This staining pattern has been attributed to likely loss of O-linked glycosylation where a-dystroglycan is the target [5]. Patchy staining for merosin (laminin a2) has also been noted and is consistent with the known interaction between a-dystroglycan and merosin. Unusual in these muscle biopsies is the secondary, patchy loss of b-dystroglycan and the reduction in a-, b-,
and c-sarcoglycan immunostaining, suggesting that unique FKRP mutations have differential effects on the dystrophin glycoprotein complex. Further biochemical studies of the enzymatic function of FKRP in conjunction with the elucidation of enzymatic targets and interacting proteins are needed to appreciate the role of the putative glycosyltransferases and their role in normal muscle and brain development.
H. MacLeod et al. / Neuromuscular Disorders 17 (2007) 285–289
Acknowledgment Supported by NIH, MDA, the Heart Research Foundation and the Burroughs Welcome Foundation (to E.M.M.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.nmd.2007.01.005. References [1] Brockington M, Yuva Y, Prandini P, et al. Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C. Hum Mol Genet 2001;10:2851–9.
289
[2] Brockington M, Blake DJ, Prandini P, et al. Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan. Am J Hum Genet 2001;69:1198–209. [3] Sveen ML, Schwartz M, Vissing J. High prevalence and phenotype–genotype correlations of limb girdle muscular dystrophy type 2I in Denmark. Ann Neurol 2006;59:808–15. [4] Louhichi N, Triki C, Quijano-Roy S, et al. New FKRP mutations causing congenital muscular dystrophy associated with mental retardation and central nervous system abnormalities identification of a founder mutation in Tunisian families. Neurogenetics 2004;5:27–34. [5] Brown SC, Torelli S, Brockington M, et al. Abnormalities in alpha-dystroglycan expression in MDC1C and LGMD2I muscular dystrophies. Am J Pathol 2004;164:727–37. [6] Poppe M, Cree L, Bourke J, et al. The phenotype of limb-girdle muscular dystrophy type 2I. Neurology 2003;60:1246–51. [7] Topaloglu H, Brockington M, Yuva Y, et al. FKRP gene mutations cause congenital muscular dystrophy, mental retardation, and cerebellar cysts. Neurology 2003;60:988–92.