Mild Pomgnt1 Mutations Underlie a Novel Limb-Girdle Muscular Dystrophy Variant

OBSERVATION Mild POMGnT1 Mutations Underlie a Novel Limb-Girdle Muscular Dystrophy Variant

Emma M. Clement, MB, ChB; Caroline Godfrey, BSc; Jenny Tan, PhD; Martin Brockington, BSc; Silvia Torelli, PhD; Lucy Feng, PhD; Susan C. Brown, PhD; Cecilia Jimenez-Mallebrera, PhD; Caroline A. Sewry, PhD; Cheryl Longman, MD; Rachael Mein, BSc; Steve Abbs, PhD; Jiri Vajsar, MD; Harry Schachter, MD, PhD; Francesco Muntoni, MD

Background: Mutations in protein-O-mannose-␤1,2-N- Results: A homozygous POMGnT1 missense mutation acetylglucosaminyltransferase 1 (POMGnT1) have been (c.1666GϾA, p.Asp556Asn) was identified. Enzyme stud- found in muscle-eye-brain disease, a congenital muscu- ies of the patient’s fibroblasts showed an altered kinetic lar dystrophy with structural eye and brain defects and profile, less marked than in patients with muscle-eye- severe mental retardation. brain disease and in keeping with the relatively mild phenotype in our patient. Objective: To investigate whether mutations in POMGnT1 could be responsible for milder allelic vari- Conclusions: Our findings widen the spectrum of dis- ants of muscular dystrophy. orders known to result from mutations in POMGnT1 to include limb-girdle muscular dystrophy with no mental Design: Screening for mutations in POMGnT1. retardation. We propose that this condition be known as LGMD2M. The enzyme assay used to diagnose muscle- Setting: Tertiary neuromuscular unit. eye-brain disease may not detect subtle abnormalities of POMGnT1 function, and additional kinetic studies must Patient: A patient with limb-girdle muscular dystrophy phenotype, with onset at 12 years of age, severe myo- be carried out in such cases. pia, normal intellect, and decreased ␣-dystroglycan immunolabeling in skeletal muscle. Arch Neurol. 2008;65(1):137-141

HE DYSTROGLYCANOPA- hypoplasia, flattened brain stem, and ven- thies are a group of hetero- tricular dilatation.3-5 More recently Author Affiliations: Dubowitz geneous autosomal reces- POMGnT1 mutations have been found in Neuromuscular Unit, Department of Paediatrics, sive disorders characterized patients with phenotypes resembling 5 Hammersmith Hospital, by reduced glycosylation of Walker-Warburg syndrome, a more se- Imperial College London ␣-dystroglycan (␣-DG) on the sarco- rious disorder that develops prenatally and T 1 (Drs Clement, Torelli, lemma of skeletal muscle fibers . These dis- is associated with absent motor develop- Feng, Brown, orders share the common clinical feature ment and severe structural brain and eye Jimenez-Mallebrera, Sewry, of muscular dystrophy with a variable neu- defects. Walker-Warburg syndrome is usu- Longman, and Muntoni), rologic and ophthalmic phenotype. ally fatal within the first year of life.4,6 Ms Godfrey, Mr Brockington, Protein-O-mannose-␤1,2-N-acetylglu- POMGnT1 is a glycosyltransferase gene and DNA Laboratory, Genetics cosaminyltransferase 1 (POMGnT1; On- involved in the glycosylation of ␣-DG Centre, Guy’s Hospital (Mss Godfrey and Mein and line Mendelian Inheritance in Man data- and is responsible for the transfer of Dr Abbs), London, and Centre base 606822) mutations were first detected N-acetylglucosamine (GlcNAc) to man- 2,7 for Inherited Neuromuscular in 2001 in patients with a typical muscle- nose. Quantifying the pathologic conse- Disorders, Robert Jones and eye-brain (MEB) disease phenotype.2 quenceassociatedwithPOMGnT1mutations Agnes Hunt Orthopaedic Muscle-eye-brain disease is a severe form has been facilitated by the development of Hospital, Oswestry (Dr Sewry), of congenital muscular dystrophy typi- a functional assay of POMGnT1 enzyme ac- England; and Department of cally presenting with weakness at birth or tivity. This assay enables the detection of Structural Biology and in the first few months of life and ocular pathogenic POMGnT1 alterations in indi- Biochemistry (Drs Tan and abnormalities including myopia, retinal de- viduals with MEB and the identification of Schachter) and Division of fects, and congenital glaucoma. Patients carriersofthesemutations,andhasbeenused Neurology (Dr Vajsar), Hospital 8,9 for Sick Children, and Faculty invariably have mental retardation and as a successful adjunct to diagnosis. of Medicine, University of typically have absent expressive lan- Herein we report a homozygous Toronto (Drs Vajsar and guage. Magnetic resonance imaging brain POMGnT1 mutation in a patient with a Schachter), Toronto, scans show abnormalities including fron- novel form of limb-girdle muscular dys- Ontario, Canada. toparietal cortical pachygyria, cerebellar trophy without mental retardation, ex-

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50 µm 50 µm

C D

Figure 1. Muscle biopsy specimen. A, Note dystrophic changes and granular fibers with vacuoles (hematoxylin-eosin). B, Immunolabeling for ␤-dystroglycan. C and D, Labeling for ␣-dystroglycan using an antibody to the glycosylated epitope IIH6 (C) and the core protein (D).

panding the spectrum of diseases derived from muta- ␣-dystroglycan. Immunostaining was performed as previ- tions of this gene. This mutation led to a subtle ously described by Godfrey et al.11 abnormality in POMGnT1 enzyme activity not typical of that seen in MEB. SINGLE-SECTION WESTERN BLOT AND OVERLAY ASSAYS

METHODS Proteins from control and patient muscle biopsy specimens were extracted from sections using the method published by PATIENT Cooper et al.12 In brief, sections were collected in sample buffer consisting of 4% sodium dodecylsulfate, 125mM Tris A patient with limb-girdle muscular dystrophy was studied. The (pH 8.8), 40% glycerol, protease inhibitor cocktail, 100mM finding of abnormal glycosylation of ␣-DG in a muscle biopsy dithiothreitol, and bromophenol blue, and were boiled for specimen was the criterion for inclusion in this study, which 3 minutes. After centrifugation to sediment particulate mate- was approved by the Hammersmith Hospital Trust Ethics Com- rial, the supernatant was used for Western blot analysis and mittee (Research Ethical Committee 2000/5802). for the laminin overlay. Samples were loaded into a NuPAGE Pre-Cast Gel System (4%-12% Bis–Tris gel; Invitrogen Corp, HISTOLOGIC ANALYSIS Carlsbad, California), then transferred electrophoretically to AND IMMUNOHISTOCHEMISTRY nitrocellulose membrane. Nitrocellulose strips were blotted with ␣-dystroglycan IIH6 antibody (Upstate Biotechnology, Frozen 10-µm muscle sections were stained with hematoxylin- Waltham, Massachusetts) and ␤-dystroglycan antibody eosin using a standard technique.10 Frozen 7-µm sections were (Novocastra Laboratories Ltd) as described by Longman incubated with mouse monoclonal antibodies to spectrin and et al.13 The laminin overlay assay was performed as described dystrophin (spectrin DYS1, DYS2, and DYS3; Novocastra Labo- by Longman et al.13 ratories Ltd, Newcastle upon Tyne, England); laminin-␣2 (MAB1922; Chemicon International Inc, Temecula, Califor- MUTATION ANALYSIS nia); the 80-kDa C-terminal fragment; ␤-dystroglycan (43DAG; Novocastra Laboratories Ltd); and an ␣-dystroglycan, IIH6; and Genomic DNA was isolated from total blood samples using with a sheep polyclonal antibody to the core protein of chick standard extraction protocols. The complete coding regions of

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©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 FKRP, POMT1, POMT2, POMGnT1, LARGE, and fukutin were 11 screened as described by Godfrey et al (GenBank Accession A B numbers: FKRP, NM_024301; POMT1, NM_007171.2; Control Patient Control Patient POMT2, NM_013382.3; POMGnT1, NM_017739.1; fukutin, muscle muscle muscle muscle NM_006731.1; and LARGE, NM_133642.2).

Laminin overlay KINETIC ANALYSIS OF POMGnT1 α-DG ENZYME ACTIVITY

Kinetic analysis of POMGnT1 was carried out by a modifica- tion of a previously reported method.8,14 Fibroblasts from a control subject without congenital muscular dystrophy and β-DG from the patient were scraped off dishes without trypsin. Cells containing about 1 mg of protein were homogenized at 4°C in 0.2 mL of homogenizing buffer (2.0% Triton X-100, 0.2M Figure 2. Single-section skeletal muscle analysis. A, Western blot analysis sodium chloride in phosphate-buffered saline solution) con- using the IIH6 antibody (␣-DG) shows that expression and molecular weight taining one-fourth of a tablet of protease inhibitor cocktail of patient muscle were similar to those of control muscle. ␤-Dystroglycan (Roche Boehringer Mannheim Diagnostics, Basel, Switzer- expression (␤-DG) was similar to control and demonstrates equal loading ␣ land). The assay incubations contained, in a total volume of between samples. B, Laminin overlay assay. The patient’s -DG shows the same ability to bind laminin compared with the control muscle. ␣-DG and 0.020 mL, 0.015 mL of fibroblast extract (approximately 0.05- ␤-DG indicate ␣- and ␤-dystroglycan, respectively. 0.07 mg of protein per assay), 75mM 2-(N-morpholino) ethanesulfonic acid buffer (pH 6.0), 10mM manganese chloride, 5mM adenosine monophosphate, 0.2M GlcNAc, and HISTOLOGIC ANALYSIS 8 combinations of donor and acceptor concentrations: (1) 0.25, 0.5, 0.75, and 1.0mM uridine diphosphate (UDP)– AND IMMUNOHISTOCHEMISTRY (3H)GlcNAc (New England Nuclear Corp, Boston, Massachu- setts; 125 000 dpm/nmol) and 62.5mM Man␣1-O-benzyl The muscle biopsy specimen exhibited dystrophia with (Toronto Research Chemicals Inc, North York, Ontario, abnormal variation in fiber size, necrosis, increased en- Canada), and (2) 5, 10, 15, and 20mM Man␣1-O-benzyl and domysial connective tissue and fat, and basophilic fi- 0.5mM UDP–(3H)GlcNAc. Incubations were carried out at bers, some of which were granular and had vacuoles 37°C for 2 hours. SepPak C18 cartridges (Waters Corp, Mil- (Figure 1A). Immunolabeling of ␤-spectrin, dystro- ford, Massachusetts) were used to determine the amount of phin, and laminin-␣2 yielded normal findings (data not 15,16 radioactive product as previously described. Control shown). A percentage of these fibers were also weaker assays were routinely carried out in the absence of an accep- with the anti-␤-dystroglycan antibody and may corre- tor, and the value obtained was subtracted from all assays. All assays were carried out in duplicate. spond to basophilic fibers (Figure 1B). Labeling with IIH6 antibody to the glycosylated epitope of ␣-DG was variable between fibers (Figure 1C). Some fibers showed a RESULTS mild reduction; others were brightly labeled. Similarly, labeling with the core antibody was weaker on some small fibers (Figure 1D). Further examination of the biopsy CLINICAL DESCRIPTION specimen demonstrated normal findings at Western blot analysis of dystrophin, sarcoglycans, laminin-␣2, caveo- Our patient is one of 5 children born to healthy non- lin, emerin, calpain 3, dysferlin, and telethonin. consanguineous Irish parents. She first developed proximal limb muscle weakness at the age of 12 years, SINGLE-SECTION WESTERN BLOT with difficulty rising from a sitting position and climb- AND OVERLAY ASSAYS ing stairs. Her early motor milestones were normal. The weakness progressed rapidly, and by age 14 years was Western blot analysis of skeletal muscle showed an ex- more proximal than distal, with the neck, hip girdle, pression of ␣-DG similar to that in the control sample and shoulder abductor muscles particularly affected. using an antibody that recognizes a glycosylated epi- Her Gower sign was positive. There was hypertrophy of tope (IIH6; Figure 2A). ␤-Dystroglycan expression was the calves and quadriceps and wasting of the hamstring normal and demonstrates equal protein loading. The lami- and deltoid muscles. She had a lordotic stance and poor nin overlay assay showed that ␣-DG ability to bind lami- heel strike because of Achilles tendon tightening. Facial nin was similar to that in the control sample (Figure 2B). expression was normal. Progressive weakness resulted in loss of ambulation at age 19 years after a leg fracture. MUTATION ANALYSIS Her general health remains excellent. She has myopia (ϩ6); at the age of 6 years, she underwent surgery to A novel homozygous point mutation, c.1666GϾA, was correct a convergent squint. Her intellect is normal, and detected in exon 20 of POMGnT1. This change is pre- she is attending a university and is 21 years old. Results dicted to result in the substitution of a conserved aspar- of initial investigation included serum creatine kinase tic acid at amino acid 556 to asparagine (p.Asp556Asn) concentration consistently elevated between 5000 and in the POMGnT1 protein. No other sequence variations 12 000 U/L and electromyographic findings suggestive were detected in the other known dystroglycanopathy of a myopathic process. genes (FKRP, POMT1, POMT2, fukutin, or LARGE), and

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10 0.10

5 nmol/h/mg v, 0.05 1/(v, nmol/h/mg) 1/(v,

0.00 –103 1 2450155 10 20 1/(UDP-GlcNAc, mmol/L) Manα1-O-Benzyl, mmol/L

Figure 3. Kinetic analysis of protein-O-mannose-␤1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) enzyme activity. A, Reciprocal plot of enzyme rate vs uridine diphosphate–N-acetylglucosamine (UDP-GlcNAc) concentration (0.25, 0.5, 0.75, and 1.0 mmol/L) at 62.5 mmol/L of Man␣1-O-benzyl for control (circles) and

patient (squares) fibroblasts. The plots are linear, showing classic Michaelis-Menten behavior. The apparent Michaelis-Menten constant (Km) and maximum velocity (Vmax) values are, respectively, 2.2 and 6.1 mmol/L (highly significant difference) and 3.4 and 2.5 nmol/h/mg (difference not statistically significant) for control and patient fibroblasts. Standard deviation bars are shown. B, Plot of enzyme rate vs Man␣1-O-benzyl concentration (5, 10, 15, and 20 mmol/L) at 0.5 14 mmol/L of UDP-GlcNAc for control and patient fibroblasts. The plot for the control fibroblasts is linear, as previously described, indicating relatively large Km and Vmax values. The plot for patient fibroblasts is hyperbolic. The reciprocal plot (not shown) is linear, yielding apparent Km and Vmax values of 9 mmol/L and 0.1 nmol/h/mg, respectively. The slopes of the 2 reciprocal plots are significantly different at the 95% confidence interval. v indicates velocity.

linkage to the MDC1B locus on 1q42 was excluded.17 Both been reported.2,5 One of the unanswered questions about parents were found to be carriers of the mutation, and the genotype-phenotype correlation for POMGnT1 mu- none of the 4 unaffected siblings tested were homozy- tations is whether it can cause mild as well as severe phe- gous for this change. This alteration was not detected in notypes, as noted in other dystroglycanopathy genes, most more than 100 disease control samples (data not shown). notably FKRP18,19 but more recently also fukutin.11 The p.Asp556Asn mutation reported herein is pre- KINETIC ANALYSIS OF POMGnT1 dicted to be located in the substrate-specific region of the ENZYME ACTIVITY catalytic domain. Several lines of evidence indicate that this alteration is pathogenic. First, the patient’s skeletal Kinetic analysis of POMGnT1 activity at 4 different con- muscle biopsy specimen demonstrated findings consis- centrations of UDP-GlcNAc (Figure 3A) showed ap- tent with a muscular dystrophy and suggestive of dys- parent Michaelis-Menten constant (Km) values of 2.2 and troglycanopathy. No mutations were detected in the 5 6.1 mmol/L for control and patient fibroblasts, respec- other dystroglycanopathy genes screened. The reduc- tively. The respective apparent maximum velocity (Vmax) tion of ␣-DG labeling was subtle at immunocytochem- values were 3.4 and 2.5 nmol/h/mg of protein (differ- istry, and no substantial reduction in molecular weight ence not statistically significant). It is impossible to ob- was observed at Western blot analysis. This highlights ␣ tain accurate Km and Vmax values for Man 1-O-benzyl with the difficulty in detecting mild ␣-DG abnormalities and normal POMGnT1 because these parameters are both so has been previously observed with mild FKRP muta- large that a plot of enzyme rate vs acceptor concentrations in limb-girdle muscular dystrophy type 2I.20 Sec- tion shows a straight line through the origin, up to the ond, the mutation segregated with the disease in this large maximum soluble concentration of 60 mmol/L of Man␣1- 14 family. Third, the mutation causes a reversal of amino O-benzyl. However, a similar analysis of patient fibro- acid polarity in the substrate-specific domain of blasts (Figure 3B) showed a qualitative change from a POMGnT1, which may be expected to have functional straight line to a classic hyperbolic Michaelis-Menten plot consequences. In addition, the detailed enzyme studies (apparent Km and Vmax values of 9 mmol/L and 0.1 nmol/ offer support for this theory. The POMGnT1 activity is h/mg of protein, respectively). observed but differs in its kinetics from that of control samples. The apparent Km for UDP-GlcNAc is substan- ␣ COMMENT tially higher and the apparent Vmax for Man 1-O-benzyl is substantially lower than in the control sample. Appli- To date, the identification of POMGnT1 mutations has cation of the POMGnT1 assay conditions that were pre- been restricted to patients with congenital muscular dys- viously used for analysis in patients with MEB (1 mmol/L trophy and brain abnormalities. Most of these patients of UDP-GlcNAc and 62.5 mmol/L of Man␣1-O-benzyl)8 have a phenotype consistent with MEB, although some to our patient with limb-girdle muscular dystrophy yields cases resembling Walker-Warburg syndrome have also a POMGnT1 rate that is significantly higher than the val-

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©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 ues found in patients with MEB. Reliable diagnosis in pa- Service (NSCAG) provided support to the Dubowitz Neu- tients such as ours, therefore, requires determination of romuscular Centre, Hammersmith Hospital, Imperial Col- kinetic parameters. lege London. Kate Bushby, MD, FRCP, of the Limb Girdle This mutation had no effect on the subcellular local- Muscular Dystrophies NSCAG Centre in Newcastle upon ization or expression of a recombinant form of POMGnT1 Tyne, England, performed the Western blot analysis of when overexpressed in C2C12 myotubes (data not proteins involved in our patient with limb-girdle mus- shown). However, the p.Asp556Asn mutation intro- cular dystrophy. The ␣-dystroglycan IIH6 was a gift of duces a potential N-glycosylation site (Asn-X-Ser) into Kevin Campbell, University of Iowa, Iowa City, and the the protein, which is predicted to have no other such sites. sheep polyclonal antibody to the core protein of chick The presence of an N-glycan at this position may cause ␣-dystroglycan was a gift of Stephan Kroger, University defective enzyme folding that results in an active but in- of Mainz, Mainz, Germany. efficient enzyme. REFERENCES CONCLUSION 1. Muntoni F, Voit T. The congenital muscular dystrophies in 2004: a century of exciting progress. Neuromuscul Disord. 2004;14(10):635-649. This article substantially expands the spectrum of dis- 2. Yoshida A, Kobayashi K, Manya H, et al. Muscular dystrophy and neuronal mi- orders associated with POMGnT1 mutations, and our pa- gration disorder caused by mutations in a glycosyltransferase, POMGnT1. Dev tient has the mildest POMGnT1 deficiency described. The Cell. 2001;1(5):717-724. finding of normal POMGnT1 activity at conventional en- 3. Santavuori P, Somer H, Sainio K, et al. Muscle-eye-brain disease (MEB). Brain zyme assay but the identification of altered kinetic prop- Dev. 1989;11(3):147-153. 4. Cormand B, Pihko H, Baye´s M, et al. Clinical and genetic distinction between Walker- erties of the mutant enzyme highlight the importance of Warburg syndrome and muscle-eye-brain disease. Neurology. 2001;56(8): careful interpretation of functional data, especially when 1059-1069. studying atypical clinical findings. 5. Taniguchi K, Kobayashi K, Saito K, et al. Worldwide distribution and broader clini- This finding reinforces the presence of glycosyltrans- cal spectrum of muscle-eye-brain disease. Hum Mol Genet. 2003;12(5):527- 534. ferase mutations among the limb-girdle muscular dys- 6. Dobyns WB, Pagon RA, Armstrong D, et al. Diagnostic criteria for Walker- trophies. We propose to name this new phenotype Warburg syndrome. Am J Med Genet. 1989;32(2):195-210. LGMD2M. 7. Manya H, Sakai K, Kobayashi K, et al. Loss-of-function of an N-acetylglucosaminyltransferase, POMGnT1, in muscle-eye-brain disease. Biochem Biophys Res Commun. 2003;306(1):93-97. Accepted for Publication: May 2, 2007. 8. Zhang W, Vajsar J, Cao P, et al. Enzymatic diagnostic test for muscle-eye-brain Correspondence: Francesco Muntoni, MD, Dubowitz Neu- type congenital muscular dystrophy using commercially available reagents. Clin romuscular Unit, Department of Paediatrics, Hammersmith Biochem. 2003;36(5):339-344. 9. Vajsar J, Zhang W, Dobyns WB, et al. Carriers and patients with muscle-eye- Hospital, Imperial College London, Du Cane Road, London brain disease can be rapidly diagnosed by enzymatic analysis of fibroblasts and W12 ONN, England ([email protected]). lymphoblasts. Neuromuscul Disord. 2006;16(2):132-136. Author Contributions: Dr Clement and Ms Godfrey con- 10. Dubowitz V, Sewry CA. Muscle Biopsy: A Practical Approach. 3rd ed. Philadel- tributed equally to the manuscript. Study concept and de- phia, PA: WB Saunders Co; 2007. sign: Clement, Godfrey, Abbs, and Muntoni. Acquisition 11. Godfrey C, Escolar D, Brockington M, et al. Fukutin gene mutations in steroid- responsive limb girdle muscular dystrophy. Ann Neurol. 2006;60(5):603- of data: Clement, Godfrey, Tan, Brockington, Torelli, Feng, 610. Jimenez-Mallebrera, Sewry, Longman, Mein, Abbs, and 12. Cooper ST, Lo HP, North KN. Single section Western blot: improving the Schachter. Analysis and interpretation of data: Clement, molecular diagnosis of the muscular dystrophies. Neurology. 2003;61(1): Godfrey, Tan, Torelli, Feng, Brown, Jimenez-Mallebrera, 93-97. 13. Longman C, Brockington M, Torelli S, et al. Mutations in the human LARGE gene Sewry, Mein, Abbs, Vajsar, and Schachter. Drafting of the cause MDC1D, a novel form of congenital muscular dystrophy with severe men- manuscript: Clement, Godfrey, Tan, Brockington, Torelli, tal retardation and abnormal glycosylation of alpha-dystroglycan. Hum Mol Genet. Feng, Jimenez-Mallebrera, Sewry, Longman, Mein, Abbs, 2003;12(21):2853-2861. Schachter, and Muntoni. Critical revision of the manu- 14. Zhang W, Betel D, Schachter H. Cloning and expression of a novel UDP-GlcNAc: script for important intellectual content: Clement, Godfrey, alpha-D-mannoside beta1,2-N-acetylglucosaminyltransferase homologous to UDP- GlcNAc:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I. Bio- Brown, Jimenez-Mallebrera, Mein, Abbs, Vajsar, and chem J. 2002;361(pt 1):153-162. Muntoni. Statistical analysis: Tan and Schachter. Ob- 15. Palcic MM, Heerze LD, Pierce M, Hindsgaul O. The use of hydrophobic synthetic tained funding: Abbs and Muntoni. Administrative, techni- glycosides as acceptors in glycosyltransferase assays. Glycoconj J. 1988;5 cal, and material support: Clement, Godfrey, Brockington, (1):49-63. doi:10.1007/BF01048331. 16. Sarkar M. Expression of recombinant rabbit UDP-GlcNAc: alpha 3-D-mannoside Feng, Brown, Jimenez-Mallebrera, Sewry, Longman, Mein, beta-1,2-N-acetylglucosaminyltransferase I catalytic domain in Sf9 insect cells. Abbs, Vajsar, and Schachter. Study supervision: Clement, Glycoconj J. 1994;11(3):204-209. Godfrey, Mein, Abbs, and Muntoni. 17. Brockington M, Sewry CA, Herrmann R, et al. Assignment of a form of congen- Financial Disclosure: None reported. ital muscular dystrophy with secondary merosin deficiency to chromosome 1q42. Funding/Support: This work was supported by grants Am J Hum Genet. 2000;66(2):428-435. 18. Brockington M, Yuva Y, Prandini P, et al. Mutations in the fukutin-related pro- PC3143 from the Muscular Dystrophy Campaign (MDC) tein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic (Dr Muntoni) and MT-3285 from the Canadian Insti- variant of congenital muscular dystrophy MDC1C. Hum Mol Genet. 2001;10 tutes of Health Research (Dr Schachter). Dr Clement and (25):2851-2859. Ms Godfrey received MDC research fellowships PO0916 19. Silan F, Yoshioka M, Kobayashi K, et al. A new mutation of the fukutin gene in a non-Japanese patient. Ann Neurol. 2003;53(3):392-396. and RA3/734, respectively. 20. Brown SC, Torelli S, Brockington M, et al. Abnormalities in alpha-dystroglycan Additional Contributions: The National Specialist Com- expression in MDC1C and LGMD2I muscular dystrophies. Am J Pathol. 2004; missioning Advisory Group Diagnostic and Advisory 164(2):727-737.

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