patients with multiple deletions or depletion of mitochondrial DNAby a dHPLC-based assay. Eur J Hum Genet 2006;14:917–922.

18. Agostino A, Valletta L, Chinnery PF, Ferrari G, Carrara F, TaylorRW, et al. Mutations of ANT1, Twinkle, and POLG1 in sporadic pro-gressive external ophthalmoplegia (PEO). Neurology 2003;60:1354–1356.

19. Hudson G, Amati-Bonneau P, Blakely EL, Stewart JD, He L, SchaeferAM, et al. Mutation of OPA1 causes dominant optic atrophy withexternal ophthalmoplegia, ataxia, deafness and multiple mitochon-drial DNA deletions: a novel disorder of mtDNA maintenance. Brain2008;131:329–337.

20. Horvath R, Hudson G, Ferrari G, Futterer N, Ahola S, Lamantea E,et al. Phenotypic spectrum associated with mutations of the mito-chondrial polymerase gamma gene. Brain 2006;129:1674–1684.

21. Longley MJ, Graziewicz MA, Bienstock RJ, Copeland WC. Conse-quences of mutations in human DNA polymerase gamma. Gene2005;354:125–131.

22. Tzoulis C, Engelsen BA, Telstad W, Aasly J, Zeviani M, Winterthun S,et al. The spectrum of clinical disease caused by the A467T and W748SPOLG mutations: a study of 26 cases. Brain 2006;129:1685–1692.

23. Chan SS, Longley MJ, Copeland WC. The common A467T mutationin the human mitochondrial DNA polymerase (POLG) compromisescatalytic efficiency and interaction with the accessory subunit. J BiolChem 2005;280:31341–31346.

24. Hakonen AH, Davidzon G, Salemi R, Bindoff LA, Van Goethem G,DiMauro S, et al. Abundance of the POLG disease mutation in Europe,Australia, New Zealand, and the United States explained by single an-cient European founders. Eur J Hum Genet 2007;15:779–783.

NON-LETHAL NEONATAL NEUROMUSCULAR VARIANT OFGLYCOGENOSIS TYPE IV WITH NOVEL GBE1 MUTATIONSCARLA FERNANDEZ, MD, PhD,1 CECILE HALBERT, MD,2 ANDRE MAUES DE PAULA, MD,1 VALERIE LACROZE, MD,3

ROSELINE FROISSART, MD,4 DOMINIQUE FIGARELLA-BRANGER, MD, PhD,1

BRIGITTE CHABROL, MD,2 and JEAN-FRANCOIS PELLISSIER, MD1

1 Laboratoire d’Anatomie Pathologique et Neuropathologie, Hopital de la Timone Adultes, 264 rue Saint-Pierre, 13385 MarseilleCedex 05, France

2 Service de Neuropediatrie, Hopital de la Timone Enfants, Marseille, France3 Service de Medecine Neonatale, Hopital de la Conception, Marseille, France4 Service des Maladies Hereditaires du Metabolisme, Centre de Biologie et de Pathologie Est, Bron, France

Accepted 2 July 2009

ABSTRACT: We report a recent case of the severe congenitalvariant of glycogen storage disease type IV with prolonged sur-vival. The patient was found to be a compound heterozygote fortwo novel mutations, a missense mutation in exon 5 (p.H188P,c.563A>C) and a severe mutation in intron 5 (c.691þ2T>C).We propose that the genotype and the quality of medical caremay account for the severe but non-lethal phenotype.

Muscle Nerve 41: 269–272, 2010

Glycogen storage disease type IV (GSD-IV), orAndersen disease, is an autosomal-recessive disorderdue to mutations in the GBE1 gene that cause defi-ciency of the glycogen branching enzyme (GBE).1

The typical form of GSD-IV presents as a rapidly pro-gressive liver disease with liver cirrhosis and death inearly childhood.1 Many variants, including severalneuromuscular forms based on age of onset (perina-tal, congenital/infantile, juvenile, and adult) havebeen reported.2–15 The perinatal form is character-ized by fetal akinesia deformation sequence, multi-ple contractures, polyhydramnios, fetal hydrops,and early death.4,10 The congenital variant is a rare,severe infantile neuromuscular disease that resem-bles Werdnig–Hoffman disease; patients present atbirth as ‘‘floppy infants’’ with severe hypotonia, mus-cle atrophy, respiratory insufficiency, and sometimesdilated cardiomyopathy or neuronal involvement,generally without hepatic failure.2,3,5–9,11–15 Death

generally occurs before the third month of agebecause of cardiac and respiratory failure. The juve-nile and adult forms generally present as an isolatedmyopathy, but cardiac or nervous system involve-ment may occur16; adults can present with centraland peripheral nervous system involvement (adultpolyglucosan body disease, or APBD).

GBE catalyzes the last step in glycogen biosynthe-sis by attaching short glycosyl chains (about six gly-cosyl units in length) in a-1,6-glucosidic links to na-ked peripheral chains of nascent glycogen.1,6,8,12

GBE deficiency leads to abnormal glycogen with lon-ger chain length and fewer branch points, resultingin a structure that resembles amylopectin; this sub-stance may accumulate in all tissues, particularlyliver, skeletal muscle, heart, skin, and the centralnervous system. It is intensely positive to periodicacid–Schiff (PAS) stain and partially resistant to dia-stase digestion. Ultrastructurally, it consists of fila-mentous and finely granular material.1,6,8,12

Only a few cases of perinatal and congenital/in-fantile neuromuscular variants of GBE deficiencyhave been reported and characterized geneti-cally.3,5–15 We report a recent case of the severecongenital variant of GSD-IV, with typical pathologi-cal findings but an unusually long survival that maywell be related to the genotype as well as the qualityof neonatal intensive care and medical care.

CASE REPORT

Clinical and Paraclinical Data. An infant girl wasthe first child of healthy, non-consanguineousparents. She was born at 39 weeks of gestation. Dur-ing pregnancy the mother experienced decreased

Abbreviations: APBD, adult polyglucosan body disease; CK, creatine ki-nase; EMG, electromyography; GBE, glycogen branching enzyme; GSD-IV, glycogen storage disease type IV; MRI, magnetic resonance imaging;PAS, periodic acid–Schiff

Correspondence to: C. Fernandez; e-mail: carla.fernandez@ap-hm.fr

VC 2009 Wiley Periodicals, Inc.Published online 7 October 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mus.21499

Key words: Andersen disease; fetal akinesia; floppy infant; glycogenstorage disease type IV; glycogen branching enzyme; muscle biopsy;amylopectin

Glycogenosis Type IV MUSCLE & NERVE February 2010 269

fetal movements. At birth her weight was 1920 g(<5th percentile), her length was 48 cm, and herhead circumference was 32 cm. Apgar scores were0, 2, and 2 at 1, 5, and 10 minutes, respectively. Theinfant had severe hypotonia, hyporeflexia, absenceof movements, multiple joint contractures (Fig. 1),and no breathing activity or sucking. She was imme-diately intubated and mechanically ventilated. Me-chanical ventilation was continued for 4 days, fol-lowed by continuous positive airway pressure. Sherequired enteral feeding via a nasogastric tube.There was no organomegaly. Liver function studieswere normal. Creatine kinase (CK) levels wereslightly elevated. Karyotype and genetic testing formyotonic dystrophy and the SMN1 gene were nor-mal. An echocardiogram showed no evidence ofcardiac dysfunction. On electromyography (EMG)there was evidence of possible myopathy, with spon-taneous myotonic discharges. Brain magnetic reso-nance imaging (MRI), performed at day 9, showedbilateral enlargement of the subarachnoid spacesand lateral ventricles. At 23 days of age, muscle bi-opsy of the quadriceps was performed.

Muscle Biopsy. For light microscopy, the musclespecimen was snap frozen in liquid nitrogen–cooled isopentane, and serial sections were pre-pared using conventional histologic stains. Forelectron microscopy, a small muscle sample wasfixed in phosphate-buffered 2.5% glutaraldehyde,postfixed in 1% osmium tetroxide, and embeddedin Araldite; ultrathin sections were obtained.

Muscle biopsy (Fig. 2) showed increased vari-ability in muscle fiber diameter and discrete fibro-sis. There was no evidence of regenerating fibers,inflammation, or neurogenic atrophy. Numeroustype I and type II muscle fibers contained coarsecytoplasmic vacuoles with pale and ground-glassinclusions suggestive of abnormal storage material.

This material was strongly positive on PAS stainingand was diastase-resistant, suggestive of abnormalglycogen. Rare fibers showed increased acid phos-phatase activity, one of which contained a rimmedvacuole. Ultrastructural examination revealed thepresence of sharply circumscribed non–membrane-bound aggregates of filamentous and finely granu-lar amylopectin-like material highly suggestive ofGSD-IV.

Biochemical and Genetic Studies. GBE activity incultured fibroblasts was markedly deficient (20%of control value), confirming the diagnosis ofGSD-IV. The patient was found to be a compoundheterozygote for two novel mutations, a missensemutation in exon 5 (p.H188P, c.563A>C) and amutation that affected splicing in intron 5(c.691þ2T>C). Both parents were found to be het-erozygous for one of the mutations.

Outcome. At 10 months of age, she had littlespontaneous limb movement and major axial hypo-tonia, and she continued to require enteral feed-ing via a nasogastric tube. At 17 months of age,swallowing disorders and cough led to placementof a gastrostomy tube. At 23 months of age, signifi-cant hypotonia persisted, but she began sittingunsupported. Limb movements improved slightly

FIGURE 1. Clinical features. At 3 months of age, the infant had

severe muscle hypotonia and multiple joint contractures, espe-

cially of the lower limbs, with bilateral involvement of hips,

knees, ankles, and feet. As shown, she required non-invasive

ventilation.

FIGURE 2. Muscle biopsy. (A, B) Hematoxylin–eosin stain

shows ground-glass inclusions present in numerous muscle

fibers. (C, D) The storage material is PAS-positive (C) and dia-

stase-resistant (D). (E) Semithin sections show sharply circum-

scribed aggregates of abnormal material. (F) Electron

microscopy shows filamentous and finely granular material con-

sistent with amylopectin. Original magnifications: (A) �100; (B–

D) �300; and (E) �600. In (F), scale bar ¼ 1 lm.

270 Glycogenosis Type IV MUSCLE & NERVE February 2010

with the help of physical therapy, but she wasunable to pull to stand or walk. Neck retractionswere observed. She was fed via gastrostomy tubeand required non-invasive ventilation during sleep.She seemed curious and bright and began to speaksome words. An echocardiogram was unremark-able. CK and liver enzymes were normal.

DISCUSSION

The infantile neuromuscular form of GSD-IV isextremely rare. To date, only about 20 cases havebeen characterized genetically.3,5–15 This diagnosisshould be included in the differential diagnosis offetal akinesia and neonatal hypotonia with or with-out congenital contractures. Our case was diag-nosed on the basis of characteristic histologicalfindings, and evaluation of GBE activity as well asgenetic analysis of the GBE1 gene confirmed thediagnosis. It is of utmost importance for neuropa-thologists to recognize amylopectin-like inclusionsof GSD-IV, because these provide the key to the di-agnosis. They appear as pale or faintly basophilic,ground-glass, PAS-positive/diastase-resistant inclu-sions that do not resemble other glycogenic stor-age and ultrastructurally consist of filamentous andgranular material. In previous studies, the abnor-mal material has been found in skin, liver, muscle,heart, and the central nervous system, but thequantity varied considerably among the differenttissues.2,6–8,13,15

Current data support a relatively good geno-type–phenotype correlation in GSD-IV. All casescharacterized by perinatal death or fatal neonatalhypotonia have been associated with a nearly com-plete absence of GBE activity and null muta-tions.7,9,12,13 Conversely, non-lethal phenotypes,such as our case, have residual enzyme activity athigher than 5% and usually harbor at least onemissense mutation.9 Our patient was a compoundheterozygote for two novel mutations. The mis-sense mutation p.H188P (c.563A>C) is presumedto be pathogenic, as it affects an amino acid that ishighly conserved between species and is replacedwith a residue of different conformation. Themutation c.691þ2T>C is predicted to be severe, asit affects a consensus splice site (splice donor siteof intron 5). In total, as our patient exhibited lowbut not absent GBE activity and was considered tobe a compound heterozygote missense/null muta-tion, she may exemplify a slightly less severe formof congenital GSD-IV, with prolonged survival andthe need for permanent medical care.

Recently, Burrow et al. reported a child withnon-lethal congenital hypotonia without hepaticor cardiac involvement due to GSD-IV.11 Geneticanalysis revealed the presence of two missensemutations in the GBE1 gene. At 1 year of age,

she was able to sit unsupported. At 2 years of ageshe was able to crawl, and at 2.5 years of age shehad a stable myopathy. Her gross motor skillshad plateaued to the level of development of a10-month-old child, and she moved about withthe aid of a motorized wheelchair.11 The childhad a slightly less severe form of the disease thanour patient. Bruno et al. also described two chil-dren with early presentation. One of theirpatients had arthrogryposis and hepatopathyrequiring liver transplantation, but both are stillalive at 3 and 4 years of age, respectively, withmyopathic symptoms.9 These two patients werecompound heterozygotes for one missense andone nonsense mutation. These data support theconcept that GSD-IV is a clinical continuum ratherthan a clear-cut variant, with different degrees ofinvolvement for each organ system,11–13 age ofonset, and evolution, depending on GBE activityand mutation severity.

In conclusion, GSD-IV is a rare glycogen stor-age disorder for which accurate interpretation ofhistological findings is essential and makes it possi-ble to guide the biochemical and genetic studies.Our patient was a compound heterozygote for amissense and a null mutation in the GBE1 genethat likely accounts for her severe, but non-lethal,phenotype. Prenatal diagnosis will be possible bydetermination of the glycogen branching enzymeactivity or GBE1 gene analysis on chorionic villoussampling, thus preventing recurrence in the samefamily. In the near future, better knowledge of thisdisease should help to identify treatmentstrategies.

REFERENCES

1. Moses SW, Parvari R. The variable presentations of glycogen storagedisease type IV: a review of clinical, enzymatic and molecular studies.Curr Mol Med 2002;2:177–188.

2. Tang TT, Segura AD, Chen YT, Ricci LM, Franciosi RA, SplaingardML, et al. Neonatal hypotonia and cardiomyopathy secondary totype IV glycogenosis. Acta Neuropathol 1994;87:531–536.

3. Bao Y, Kishnani P, Wu JY, Chen YT. Hepatic and neuromuscularforms of glycogen storage disease type IV caused by mutations in thesame glycogen-branching enzyme gene. J Clin Invest 1996;97:941–948.

4. Cox PM, Brueton LA, Murphy KW, Worthington VC, Bjelogrlic P,Lazda EJ, et al. Early-onset fetal hydrops and muscle degeneration insiblings due to a novel variant of type IV glycogenosis. Am J MedGenet 1999;86:187–193.

5. Nambu M, Kawabe K, Fukuda T, Okuno TB, Ohta S, Nonaka I, et al.A neonatal form of glycogen storage disease type IV. Neurology2003;61:392–394.

6. Tay SK, Akman HO, Chung WK, Pike MG, Muntoni F, Hays AP,et al. Fatal infantile neuromuscular presentation of glycogen storagedisease type IV. Neuromuscul Disord 2004;14:253–260.

7. Janecke AR, Dertinger S, Ketelsen UP, Bereuter L, Simma B, MullerT, et al. Neonatal type IV glycogen storage disease associated with‘‘null’’ mutations in glycogen branching enzyme 1. J Pediatr 2004;145:705–709.

8. Giuffre B, Parini R, Rizzuti T, Morandi L, van Diggelen OP, BrunoC, et al. Severe neonatal onset of glycogenosis type IV: clinical andlaboratory findings leading to diagnosis in two siblings. J InheritMetab Dis 2004;27:609–619.

9. Bruno C, van Diggelen OP, Cassandrini D, Gimpelev M, Giuffre B,Donati MA, et al. Clinical and genetic heterogeneity of branchingenzyme deficiency (glycogenosis type IV). Neurology 2004;63:1053–1058.

Glycogenosis Type IV MUSCLE & NERVE February 2010 271

10. L’Hermine-Coulomb A, Beuzen F, Bouvier R, Rolland MO, FroissartR, Menez F, et al. Fetal type IV glycogen storage disease: clinical, en-zymatic, and genetic data of a pure muscular form with variable andearly antenatal manifestations in the same family. Am J Med GenetA 2005;139A:118–122.

11. Burrow TA, Hopkin RJ, Bove KE, Miles L, Wong BL, Choudhary A,et al. Non-lethal congenital hypotonia due to glycogen storage dis-ease type IV. Am J Med Genet A 2006;140A:878–882.

12. Bruno C, Cassandrini D, Assereto S, Akman HO, Minetti C, DiMauro S. Neuromuscular forms of glycogen branching enzyme defi-ciency. Acta Myol 2007;26:75–78.

13. Assereto S, van Diggelen OP, Diogo L, Morava E, Cassandrini D, Car-reira I, et al. Null mutations and lethal congenital form of glycogen

storage disease type IV. Biochem Biophys Res Commun 2007;361:445–450.

14. Raju GP, Li HC, Bali DS, Chen YT, Urion DK, Lidov HG, et al. Acase of congenital glycogen storage disease type IV with a novelGBE1 mutation. J Child Neurol 2008;23:349–352.

15. Nolte KW, Janecke AR, Vorgerd M, Weis J, Schroder JM. Congenitaltype IV glycogenosis: the spectrum of pleomorphic polyglucosanbodies in muscle, nerve, and spinal cord with two novel mutations inthe GBE1 gene. Acta Neuropathol 2008;116:491–506.

16. Lossos A, Barash V, Soffer D, Argov Z, Gomori M, Ben- Nariah Z,et al. Hereditary branching enzyme dysfunction in adult polygluco-san body disease: a possible metabolic cause in two patients. AnnNeurol 1991;30:655–662.

SPORADIC LATE ONSET NEMALINE MYOPATHY RESPONSIVE TO IVIGAND IMMUNOTHERAPYMARGHERITA MILONE, MD, PhD,1 AMIRAM KATZ, MD,2 ANTHONY A. AMATO, MD,3 CARL A. SODERLAND, MD, MPH,4

MIRUNA SEGARCEANU, MD,5 NATHAN P. YOUNG, DO,1 and H. ROYDEN JONES, MD6

1Department of Neurology, Mayo Clinic, Rochester, Minnesota, USA2Department of Neurology, Yale University School of Medicine, New Haven, Connecticut, USA3Department of Neurology, Brigham and Women’s Hospital, Boston, Massachusetts, USA4Department of Internal Medicine, Lahey Clinic, Burlington, Massachusetts, USA5Department of Neurology, Dartmouth-Hitchcock Manchester, Manchester, New Hampshire, USA6Department of Neurology, Lahey Clinic, 41 Mall Road, Burlington,Massachusetts 01805, Harvard Medical School, Boston, Massachusetts, USA

Accepted 7 July 2009

ABSTRACT: Sporadic late onset nemaline myopathy(SLONM) is a progressive myopathy of indeterminate etiologyand poor outcome. If associated with a monoclonal gammop-athy, SLONM carries a more unfavorable prognosis. Immuno-therapy was unsuccessful. We report two HIV-negative SLONM/monoclonal gammopathy patients who improved following intra-venous immunoglobulin (IVIg) treatment alone or in combinationwith immunosuppressant agents. This favorable response totreatment suggests that a dysimmune mechanism is operativein some SLONM individuals. We suggest that IVIg deserves ini-tial consideration for SLONM therapy.

Muscle Nerve 41: 272–276, 2010

Sporadic late onset nemaline myopathy (SLONM)is a rare idiopathic myopathy initially describedindependently by A.G. Engel1 and W.K. Engel.2

Clinically it is characterized by subacute muscleweakness that can variably affect proximal, distal,or axial muscles.3–6 Clusters of rods, often fillingatrophic fibers, are the pathological hallmark.Traditionally, non-HIV SLONM is resistant topharmacological treatment. A concomitant mono-clonal gammopathy of undetermined significance(MGUS) leads to a very poor prognosis.6 In con-trast, HIV-associated SLONM does respond toimmunotherapy.7–10 However, recently two non-HIV SLONM/MGUS patients were successfullytreated with melphalan and stem cell transplanta-tion.11,12 This has provided a hope for this severemyopathy.13

We describe two non-HIV SLONM/MGUSpatients with clear and maintained response tointravenous immunoglobulin (IVIg) treatment,one in isolation, and the other concomitant withcorticosteroids and mycophenolate mofetil.

CASE REPORTS

Patient 1. A 61-year-old man presented to theLahey Clinic in 2003 for insidiously progressivepainless proximal leg weakness superimposed on2 years of vitiligo and IgG kappa MGUS. Serumcreatine kinase (CK) was normal. Needle electro-myography (EMG) demonstrated low amplitude,short duration motor unit potentials (MUPs) withnumerous fibrillation potentials only in the L5 par-aspinal muscles. Biopsy of the homologous contra-lateral paraspinal muscles demonstrated scattereddegenerating muscle fibers. A few rod-like struc-tures were detected only by electron microscopy. Arepeat muscle biopsy of the quadriceps showednonspecific myopathic findings. During the next 2years his clinical status continued to worsen signifi-cantly; he developed severe proximal and axialweakness causing head drop and prominent lum-bar lordosis. He could barely squat and rise. Mus-cle strength testing showed mild bifacial weaknessand moderate to severe proximal and axial weak-ness (Table 1). CK values continued to be normal.HIV antibodies and inflammatory markers werenegative; thyroid stimulating hormone was normal.The MGUS persisted; a skeletal survey was normal,and a bone marrow biopsy was unremarkable.Repeat EMG demonstrated that the myopathicMUPs and fibrillation potentials were now found inproximal muscles. Biopsy of the biceps brachii was

Abbreviations: CK, creatine kinase; EMG, electromyography; IVIg, intra-venous immunoglobulin; MGUS, monoclonal gammopathy of undeter-mined significance; MUP, motor unit potential; SLONM, sporadic lateonset nemaline myopathy

Correspondence to: H.R. Jones; e-mail: Royden.H.Jones@lahey.org

VC 2009 Wiley Periodicals, Inc.Published online 22 October 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mus.21504

Key words: nemaline myopathy; IVIg; monoclonal gammopathy

272 Nemaline Myopathy and IVIg MUSCLE & NERVE February 2010