Alan H. Beggs, PhD
Associate Professor of Pediatrics
Harvard Medical School/Children's Hospital
Department of Pediatrics (Genetics)

MRRC Project(s)

K02 AR02026-04
Alpha Actinins in Normal and Diseased Muscle

R01 AR44345-05
Sarcomeric Proteins in Normal and Diseased Muscle

P01 NS40828-01 (Pending)
Gene Expression in Normal and Diseased Muscle During Development - PI, Project 3: Gene Expression and Biochemical Analysis of Muscle Development in Myotubular Myopathy

The cytoskeleton plays a major role in determining cell structure and function in the nervous system and other tissues. Many cytoskeletal elements have cognate proteins that perform related functions within the muscle sarcomere. The alpha-actinins represent good example of this phenomenon; the human genome encodes four closely related alpha-actinin genes, two for sarcomeric skeletal muscle isoforms and two for non-muscle cytoskeletal forms. This laboratory is taking two complementary approaches to understanding the structure and functions of sarcomeric and cytoskeletal proteins associated with the alpha-actinins. The first involves identification and characterization of new alpha-actinin genes and genes for new proteins that interact with alpha-actinin. Simultaneously, studies are underway to determine if any of these genes are mutated in human neuromuscular or other diseases. These approaches converge by allowing correlation of biochemical and cell biological data on protein function and subcellular localization with pathophysiologic phenomena observed in disorders caused by abnormalities in these proteins.

Research Description

Highlights of Major Accomplishments

• Cloning and characterization of three new human genes for alpha-actinins.

• Demonstration of expression of alpha-actinin-2 in postsynaptic densities of dendrites of cerebral cortical neurons and in association with NMDA receptors.

• Identification of defects in three genes for thin filament proteins, alpha-tropomyosin-3, nebulin and alpha-actin, in patients with nemaline myopathies.

Major Results

Three new human genes for alpha-actinins have been cloned and characterized. The ACTN2 and ACTN3 genes (encoding alpha-actinin-2 and –3 isoforms, respectively) encode sarcomeric proteins that anchor the actin-thin filaments in skeletal and cardiac muscle. ACTN4 encodes a cytoskeletal isoform expressed in all tissues. Remarkably, up to 20% of humans are homozygous for a nonsense mutation of ACTN3 and do not express any detectable alpha-actinin-3 in their skeletal muscle fast fibers. This observation indicates that ACTN3 is genetically redundant. Most likely, its function is replaced by expression of ACTN2, which in humans is expressed in all fiber types. Although absence of ACTN3 is not associated with any obvious clinical phenotype, it remains possible that this mutation acts as a genetic modifier, either of other neuromuscular disease, or possibly accounting for some of the natural variability in human athletic performance.

Interestingly, alpha-actinin-2 is also expressed in the CNS. In the rat cerebral cortex, alpha-actinin-2 immunoreactivity is highest in layers 2/3 and 5 and weakest in layers 4 and 6. The protein is prominent in scattered interneurons and in pyramidal neurons where it is present extensively at postsynaptic densities in the dendritic processes but absent in the cell bodies. In contrast, alpha-actinin-2 staining is virtually absent in the cerebellum. In cultured neurons, alpha-actinin-2 localizes specifically to glutamatergic synapses. A screen of brain cDNAs by our collaborator, Dr. Morgan Sheng, identified alpha-actinin-2 as a binding partner for the NMDA receptor. Both proteins colocalize with PSD-95 and actin in dendritic spines of rat cortical layer-5 pyramidal neurons. Interestingly, binding of alpha-actinin-2 to NMDA receptors was directly antagonized by calmodulin in a Ca²⁺-dependent manner, suggesting that Ca²⁺-dependent inactivation of NMDA receptors may be mediated via effects on alpha-actinin-2-NMDA receptor binding. We are currently evaluating ACTN2 as a candidate gene for Walker-Warburg syndrome, muscle-eye-brain disease, and other congenital muscular dystrophies with CNS involvement.

In an exciting recent development, we have collaborated with Dr. Martin Pollak to identify mutations of ACTN4 in several families with isolated idiopathic focal segmental glomerulosclerosis (FSGS). Dr. Pollak’s group had mapped an autosomal dominant FSGS –1 locus to human chromosome 19q13.1 near to where we mapped ACTN4. Western blot data indicated the presence of alpha-actinin-4 protein in kidney lysates, thereby supporting the hypothesis that ACTN4 was a candidate gene for FSGS. rtPCR and direct sequencing of ACTN4 lymphocyte transcripts from patients from three families revealed unique heterozygous non-conservative amino acid substitutions in affected members from all three of these families. Remarkably, these changes (K228E, T232I and S235P), none of which was seen in 214 normal chromosomes, were all in close proximity to each other in exon 8 between the actin binding domain and the first rod repeat of the protein. Co-sedimentation assays of these mutant actinins with F-actin revealed that each co-sedimented to a greater degree than with wild type alpha-actinin-4, indicating that these mutations may alter the affinity of alpha-actinin-4 for actin under certain conditions. It remains unclear why mutations of this ubiquitous protein cause such a tissue-specific phenotype.

The nemaline myopathies are a group of related congenital myopathies characterized by weakness and the presence of nemaline rods in affected skeletal muscles. Work in our laboratory over the past few years has now identified in patients with nemaline myopathies of varying severities mutations in three genes for thin filament proteins: alpha-tropomyosin-3, nebulin and alpha-actin. At least one, or more, additional gene(s) must exist, as each of these loci have been ruled out in some families with nemaline myopathies. Current studies are aimed at identification of these new genes as well as at understanding the basis for extensive clinical and pathological heterogeneity in this group of myopathies through large scale gene expression studies.

Finally, a series of new alpha-actinin-bindings proteins have been identified through the use of yeast two-hybrid assays. One of these, myozenin, is a new skeletal muscle Z-line protein whose function is currently under study. Each of these new genes is a potential candidate to be mutated in human neuromuscular disease and genetic studies to address this question are underway.

Publications

North KN, Miller G, Iannaccone ST, Clemens PR, Chad DA, Bella I, Smith TW, Beggs AH, Specht LA. Cognitive dysfunction as the major presenting feature of Becker's Muscular Dystrophy. Neurology 1996; 46:461-465.

Zhu J, Guo S-Z, Beggs AH, Maruyama T, Santarius T, Dashner K, Olson N, Wu JK, Black PM. Microsatellite instability analysis of primary human brain tumors. Oncogene 1996; 12:1417-1423.

North KN, Specht LA, Sethi RK, Shapiro F, Beggs AH. Congenital muscular dystrophy associated with merosin deficiency. J Child Neurol 1996; 11:291-295.

North KN, Beggs AH. Deficiency of a skeletal muscle isoform of a-actinin (a-actinin-3) in merosin-positive congenital muscular dystrophy. Neuromusc Disorders 1996; 6:229-235.

Scharf JM, Damron D, Frisella A, Bruno S, Beggs AH, Kunkel LM, Dietrich WF. The mouse region syntenic for human spinal muscular atrophy lies within the Lgn1 critical interval and contains multiple copies of Naip exon 5. Genomics 1996; 38:405-417.

Engle EC, Gumnerov BC, McKeown CA, Schatz M, Johns DR, Porter JD, Beggs AH. Oculomotor nerve and muscle abnormalities in congenital fibrosis of the extraocular muscles. Ann Neurol 1997; 41:314-325.

Wyszynski M, Lin J, Rao A, Nigh E, Beggs AH, Craig AM, Sheng M. Competitive binding of a-actinin and calmodulin to the NMDA receptor. Nature 1997; 385:439-442.

Engle EC, Castro A, Macy M, Knoll, JHM, Beggs AH. A gene for isolated congenital ptosis maps to a 3 cM region within 1p32-p34.1. Am J Hum Genet 1997; 60:1150-1157.

De Girolami U, Beggs AH. Skeletal Muscle. In: Silverberg SG, DeLellis RA, Frable WJ. eds. Principles and Practice of Surgical Pathology and Cytopathology, 3rd ed. NY: Churchill Livingstone, 1997; 943-994.

Beggs, AH. Dystrophinopathy, the expanding phenotype: Dystrophin abnormalities in X-linked dilated cardiomyopathy. Editorial in Circulation 1997; 95:2344-2347.

Vainzof M, Costa CS, Marie SK, Moreira ES, Reed U, Passos-Bueno MR, Beggs AH, Zatz M. Deficiency of a-actinin-3 (ACTN3) occurs in different forms of muscular dystrophy. Neuropediatrics 1997; 28:223-228.

Khurana TS, Specht LA, Beggs AH, Tome FMS, Letureq F, Chevallay M, Chafey P, Kunkel LM. The concomitant use of dystrophin and utrophin/dystrophin related protein antibodies to reduce misdiagnosis of Duchenne/Becker muscular dystrophy. Biochem Biophys Res Comm 1997; 241:232-235.

Duggal P, Vesely MR, Wattanasirichaigoon D, Villafane J, Kaushik V, Beggs AH. Mutation of the gene for IsK associated with both Jervell and Lange-Nielsen and Romano Ward forms of long QT syndrome. Circulation 1998; 97:142-146.

Wyszynski M, Kharazia V, Shanghvi R, Rao A, Beggs AH, Craig AM, Weinberg R, Sheng M. Differential regional expression and ultrastructural localization of a-actinin-2, a putative NMDA receptor anchoring protein, in rat brain. J Neurosci 1998; 18:1383-1392.

Satler CA, Vesely MR, Duggal P, Ginsburg GS, Beggs AH. Multiple different missense mutations in the pore region of HERG in patients with long QT syndrome. Hum Genet 1998; 102:265-272.

Wallgren-Pettersson C, Beggs AH, Laing NG. Workshop report: 51st ENMC International Workshop: Nemaline myopathy. 13-15 June 1997, Naarden, The Netherlands. Neuromusc Dis 1998; 8:53-56.

Chan Y-M, Tong H-Q, Beggs AH, Kunkel LM. Human skeletal muscle-specific a-actinin-2 and -3 isoforms form homodimers and heterodimers in vitro and in vivo. Biochem Biophys Res Comm 1998; 248:134-139.

Wang SM, Zwaan J, Mullaney PB, Jabak MH, Al-Awad A, Beggs AH, Engle EC. Congenital fibrosis of the extraocular muscles type 2 (CFEOM2), an inherited exotropic strabismus fixus, maps to distal 11q13. Am J Hum Genet 1998; 63:517-525.

Wattanasirichaigoon D, Beggs AH. Molecular genetics of long-QT syndrome. Curr Opin Peds 1998; 10:628-634.

Pelin K, Hilpelä P, Donner K, Sewry C, Akkari PA, Wilton SD, Wattanasirichaigoon D, Centner T, Hanefeld H, Odent S, Fardeau M, Urtizberea JA, Muntoni F, Dubowitz V, Beggs AH, Laing NG, Labeit S, de la Chapelle A, Wallgren-Pettersson C. Mutations in the nebulin gene associated with autosomal recessive nemaline myopathy. Proc Natl Acad Sci USA 1999; 96:2305-2310.

Rossignol M, Beggs AH, Pierce EA, Klagsbrun M. Human neuropilin-1 and neuropilin –2 map to 10p12 and 2q34, respectively. Genomics 1999; 57:459-460.

North KN, Yang N, Wattanasirichaigoon D, Mills M, Easteal S, Beggs AH. A common nonsense mutation results in a-actinin-3 deficiency in the general population: evidence for genetic redundancy in humans. Nature Genet 1999; 21:353-354.

Hance JE, Fu SY, Watkins SC, Beggs AH, Michalak M. Alpha-actinin-2 is a new component of the dystrophin glycoprotein complex. Arch Biochem Biophys 1999; 365:216-222.

Wattanasirichaigoon D, Vesely MR, Duggal P, Levine JC, Blume ED, Wolff GS, Edwards SB, Beggs AH. Sodium channel abnormalities are infrequent in patients with long QT syndrome: Identification of two novel SCN5A mutations. Am J Med Genet 1999; 86:470-476.

Nowak KJ, Wattanasirichaigoon D, Goebel HH, Wilce M, Pelin K, Donner K, Jacob RL, Hubner C, Oexle K, Anderson JR, Verity CM, North KN, Iannaccone ST, Muller CR, Nurnberg P, Muntoni F, Sewry C, Hughes I, Stuphen R, Lacson AG, Swoboda KJ, Vigneron J, Wallgren-Pettersson C, Beggs AH, Laing NG. Mutations in the skeletal muscle "a-actin gene in patients with actin myopathy and nemaline myopathy. Nature Genet 1999; 23:208-212.

Wallgren-Pettersson C, Pelin K, Hilpela P, Donner K, Porfirio B, Graziano C, Swoboda KJ, Fardeau M, Urtizberea JA, Muntoni F, Sewry C, Dubowitz V, Iannaccone S, Minetti C, Pedemonte M, Seri M, Cusano R, Lammens M, Castagna-Sloane A, Beggs AH, Laing NG, de la Chapelle A. Clinical and genetic heterogeneity in autosomal recessive nemaline myopathy. Neuromusc Dis 1999; 9:564-572.

Kaplan JM, Kim SH, North KN, Rennke H, Correia LA, Tong H-Q, Mathis BJ, Rodriguez-Perez J-C, Allen PG, Beggs AH, Pollak MR. Mutations in ACTN4, encoding "a-actinin-4, cause familial focal segmental glomerulosclerosis. Nature Genet 2000; 24:251-256.

Schwartz PJ, Priori SG, Spazzolini C, Moss AJ, Vincent GM, Napolitano C, Denjoy I, Guicheney P, Breithardt G, Keating MT, Towbin JA, Beggs A, Brink P, Wilde A, Toivonen L, Zareba W, Robinson J, Timothy K, Corfield V, Wattanasirichaigoon D, Corbett C, Haverkamp W, Schulze-Bahr E, Lehmann M, Schwartz K, Coumel P, Bloise B. Genotype-phenotype correlation in the long-QT syndrome : Gene-specific triggers for life-threatening arrhythmias. Circulation 2001;103:89-95.

Takada F, Vander Woude D, Tong H-Q, Thompson T, Watkins S, Kunkel L, Beggs A. Myozenin: An a-actinin and g-filamin-binding protein of skeletal muscle Z lines. PNAS USA 2001;98:1595-1600.

Gazda H, Lipton J, Willig T-N, Ball S, Niemeyer C, Tchernia G, Mohandas N, Daly M, Ploszynska A, Orfali K, Vlachos A, Glader B, Rokicka-Milewska R, Ohara A, Baker D, Pospisilova D, Weber A, Viskochil D, Nathan D, Beggs A, Sieff C. Evidence for linkage of familial Diamond-Blackfan anemia to chromosome 8p23.3-p22 and for non-19q non-8p disease. Blood 2001;97:2145-2150.

Mizuno Y, Puca A, O'Brien K, Beggs A, Kunkel L. Genomic organization and single-nucleotide polymorphism map of desmuslin, a novel intermediate filament protein on chromosome 15q26.3. BMC Genetics 2001;2:8.

Mills M, Yang N, Weinberger R, Vander Woude D, Beggs A, Easteal S, North K. Differential expression of the actin binding proteins, a-actinin-2 and -3 in different species: implications for the evolution of functional redundancy. Hum Molec Genet 2001;10:1335-1346.

Ryan M, Schnell C, Strickland C, Shield L, Morgan G, Iannaccone S, Laing H, Beggs A, North K. Nemaline myopathy: a clinical study of 143 cases. Ann Neurol 2001;50:312-320.

Sanoudou D, Beggs A. Clinical and genetic heterogenaity in nemaline myopathy, a disease of sarcomeric thin filaments. Trends in Molec Med 2001;7:362-368.

Takada F, Beggs A. a-Actinins. In: Creighton T, editor. Encyclopedia of Molecular Medicine. NY: Wiley & Sons, Inc.; 2002, p. 122-127.

Splawski I, Timothy KW, Tateyama M, Clancy CE, Malhortra A, Beggs AH, Cappuccio FP, Sagnella GA, Kass RS, Keating MT. Variant of SCN5A sodium channel implicated in risk of cardiac arrhythmia. Science 2002;297:1333-1336.

Vainzof M, Moreira ES, Suzuki OT, Faulkner G, Valle G, Beggs AH, Carpen O, Ribeiro AF, Zanoteli E, Gurgel-Gianneti J, Tsanaclis AM, Silva HC, Passos-Bueno MR, Zatz M. Telethonin protein expression in neuromuscular disorders. Biochim Biophys Acta 2002;1588:33-40.

Zervos A, Hunt KE, Tong HQ, Avallone J, Morales J, Friedman N, Cohen BH, Clark B, Guo S, Gazda H, Beggs AH, Traboulsi EI. Clinical, genetic and histopathologic findings in two siblings with muscle-eye-brain disease. Eur J Ophthalmol 2002;12:253-61.

Wattanasirichaigoon D, Swoboda KJ, Takada F, Tong HQ, Lip V, Iannaccone ST, Wallgren-Pettersson C, Laing NG, Beggs AH. Mutations of the slow muscle alpha-tropomyosin gene, TPM3, are a rare cause of nemaline myopathy. Neurology 2002;59:613-7.

Splawski I, Timothy KW, Tateyama M, Clancy CE, Malhotra A, Beggs AH, Cappuccio FP, Sagnella GA, Kass RS, Keating MT. Variant of SCN5A sodium channel implicated in risk of cardiac arrhythmia. Science 2002;297(5585):1333-6.

Haslett JN, Sanoudou D, Kho AT, Bennett RR, Greenberg SA, Kohane IS, Beggs AH, Kunkel LM. Gene expression comparison of biopsies from Duchenne muscular dystrophy (DMD) and normal skeletal muscle. PNAS USA 2002;99:15000-5.

Greenberg SA, Sanoudou D, Haslett JN, Kohane IS, Kunkel LM, Beggs AH, Amato AA. Molecular profiles of inflammatory myopathies. Neurology 2002;59(8):1170-82.

Bonnemann CG, Thompson TG, van der Ven PFM, Goebel HH, Warlo I, Vollmers B, J. R, Herms J, Gautel M, Takada F, Beggs AH, Furst DO, Kunkel LM, Hanefeld F, Schroder R. Filamin C accumulation is a strong but nonspecific immunohistochemical marker of core formation in muscle. J Neurol Sci 2003;206:71-78.

Sparrow JC, Nowak KJ, Durling HJ, Beggs AH, Wallgren-Pettersson C, Romero N, Nonaka I, Laing NG. Muscle disease caused by mutations in the skeletal muscle alpha-actin gene (ACTA1). Neuromuscul Disord 2003;13(7-8):519-31.

Yang N, MacArthur DG, Gulbin JP, Hahn AG, Beggs AH, Easteal S, North K. ACTN3 genotype is associated with human elite athletic performance. Am J Hum Genet 2003;73:627-31.

Zanoteli E, Lotuffo RM, Oliveira AS, Beggs AH, Canovas M, Zatz M, Vainzof M. Deficiency of muscle alpha-actinin-3 is compatible with high muscle performance. J Mol Neurosci 2003;20:39-42.

Ryan MM, Ilkovski B, Strickland CD, Schnell C, Sanoudou D, Midgett C, Houston R, Muirhead D, Dennett X, Shield LK, De Girolami U, Iannaccone ST, Laing NG, North KN, Beggs AH. Clinical course correlates poorly with muscle pathology in nemaline myopathy. Neurology 2003;60(4):665-73.

Nimgaonkar A, Sanoudou D, Butte AJ, Haslett JN, Kunkel LM, Beggs AH, Kohane IS. Reproducibility of gene expression across generations of Affymetrix microarrays. BMC Bioinformatics 2003;4:27.

Haslett JN, Sanoudou D, Kho AT, Han M, Bennett RR, Kohane IS, Beggs AH, Kunkel LM. Gene expression profiling of Duchenne muscular dystrophy skeletal muscle. Neurogenetics 2003;4(4):163-71.

Sanoudou D, Haslett JN, Kho AT, Guo S, Gazda HT, Greenberg SA, Lidov HG, Kohane IS, Kunkel LM, Beggs AH. Expression profiling reveals altered satellite cell numbers and glycolytic enzyme transcription in nemaline myopathy muscle. PNAS USA 2003;100(8):4666-71.

Tomczak KK, Marinescu VD, Ramoni MF, Sanoudou D, Montanaro F, Han M, Kunkel LM, Kohane IS, Beggs AH. Expression profiling and identification of novel genes involved in myogenic differentiation. FASEB J Exp 2004;18:403-405.

Sanoudou D, Kang PB, Haslett JN, Han M, Kunkel LM, Beggs AH. Transcriptional profile of postmortem skeletal muscle. Physiol Genomics 2004;16:222-228.

Sanoudou D, Frieden LA, Haslett JN, Kho AT, Greenberg SA, Kohane IS, Kunkel LM, Beggs AH. Molecular classification of nemaline myopathies: "non-typing" specimens exhibit unique patterns of gene expression. Neurobiol Dis 2004, in press.

See Dr. Beggs' publications via PubMed

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