Available online at www.sciencedirect.com
ScienceDirect Neuromuscular Disorders 23 (2013) 1010–1015 www.elsevier.com/locate/nmd
Case report
Novel mutations in the fukutin gene in a boy with asymptomatic hyperCKemia Chiara Fiorillo a,⇑, Francesca Moro a, Guja Astrea a, Maria Aurora Morales b, Jacopo Baldacci a, Maria Marchese a, Sara Scapolan c, Claudio Bruno c, Roberta Battini a, Filippo M. Santorelli a a
Molecular Medicine and Neuromuscular Lab, IRCCS Stella Maris, Pisa, Italy b CNR Clinical Physiology Institute, Pisa, Italy c Center of Myology, Pediatric Neurology Unit, Department of Neuroscience and Rehabilitation, IRCCS G. Gaslini, Genoa, Italy
Abstract Mutations in the fukutin gene were first identified in Japanese patients with classic Fukuyama congenital muscular dystrophy, a severe form of congenital muscular dystrophy associated with cobblestone lissencephaly and ocular defects. Patients of different ethnicities and with milder phenotypes, including limb girdle muscular dystrophy and cardiomyopathy without brain impairment, have also been reported. The hallmark of this disorder, regardless of the clinical outcome, is moderate-to-severe hypoglycosylation of alpha-dystroglycan in muscle sections. We describe the case of a boy harboring two novel mutations in fukutin gene and presenting a five-year history of asymptomatic hyperCKemia, without overt muscle, brain or ocular involvement. Genetic investigations, guided by the presence of moderate myopathic changes on muscle biopsy with loss of immunodetectable alpha-dystroglycan, led to a definitive diagnosis. Cardiac and echocardiographic examinations at follow-up disclosed low normal left ventricular function but no active cardiovascular symptoms. We suggest that fukutin mutations should be sought in asymptomatic hyperCKemia and subclinical heart dysfunction. Ó 2013 Elsevier B.V. All rights reserved. Keywords: Fukutin gene; Mutations; HyperCKemia; Alpha-dystroglycan; Cardiomyopathy
1. Introduction The dystroglycanopathies are a group of muscular dystrophies that share the common pathological feature of aberrant alpha-dystroglycan (a-DG) glycosylation. To date, defects in at least 11 putative glycosyltransferases, accessory proteins, or proteins influencing O-mannosylation of a-DG have been reported [1]. The associated clinical phenotypes range from congenital onset and severe structural brain abnormalities, such as those seen in Walker–Warburg syndrome (WWS, [MIM 236670]),
⇑ Corresponding author. Address: IRCCS Stella Maris, Via dei Giacinti 2, 56028 Pisa, Italy. Tel.: +39 050886311; fax: +39 050886247. E-mail address: chiara.fi[email protected] (C. Fiorillo).
0960-8966/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nmd.2013.09.010
muscle–eye–brain disease (MEB, [MIM 253280]) and Fukuyama congenital muscular dystrophy (FCMD, [MIM 253800]), to forms with infantile myopathy and mild/ moderate brain involvement (MDC1C [MIM 606612] and MDC1D [MIM 608840]). Patients can also present with adult-onset limb girdle muscular dystrophy (LGMD) [1]. FCMD is the most common form of a-dystroglycanopathy in Japan and most patients carry the founder insertion of a 3-kb retrotransposon element in the 30 untranslated region of the fukutin gene (FKTN) [2]. FCMD patients display dystrophic changes in skeletal muscle, structural brain malformations, and severe ocular abnormalities and have a reduced life expectancy [3,4]. There are descriptions of Japanese and non-Japanese FKTN-mutated patients presenting with limb girdle
C. Fiorillo et al. / Neuromuscular Disorders 23 (2013) 1010–1015
muscle weakness without ocular or brain defects (LGMD type 2M, MIM 253600) [5–9], as well as occasional reports of association with isolated dilated cardiomyopathy [10]. As in more typical a-dystroglycanopathies, all fukutin patients, regardless of their clinical presentation, show elevated serum creatine kinase (CK) and depleted a-DG on skeletal muscle biopsy. Interestingly, residual levels of a-DG do not seem to correlate with disease severity [11]. HyperCKemia is considered a hallmark of hereditary neuromuscular disorders. However, several acquired conditions (infections, alcoholism, drugs, intramuscular injections, hypothyroidism, etc.) may also cause a rise in this enzyme, and the number of apparently healthy individuals with persistent hyperCKemia has increased considerably since the CK test started to be commonly included in routine blood workup. Therefore, the criteria for the diagnosis of isolated hyperCKemia have been revised in the past decade, with the emphasis shifting to thorough analysis of muscle biopsy [12]. We describe the case of a boy with persistent hyperCKemia without overt muscle weakness or pain, in whom the finding of marked a-DG deficiency on muscle biopsy targeted genetic analyses and led to the identification of two novel mutations in FKTN. 2. Case report A 12-year-old boy was first referred for evaluation at the age of 7 years after a routine blood test revealed a raised CK level (1081 U/L, normal values < 190 U/L). His family history was negative for neuromuscular disorders and his parents were unrelated; their CK levels were in the normal range. The patient had developed normally and had never complained of muscle weakness, cramps or fatigue after exercise, or myoglobinuria. His general physical examination was normal. Neurological examination did not show muscle weakness or central nervous system impairment. He had neither calf hypertrophy nor contractures. The patient could get up from floor without support and was able to run and jump normally for his age. Electrocardiogram and ultrasound recordings, performed elsewhere, were reportedly normal. Electromyography, nerve conduction studies, and respiratory function were also normal. The patient was not taking any drugs or receiving intramuscular injections. Screening for metabolic disorders and blood lactic acid levels were normal. Spine X-ray did not show scoliosis. Brain MRI and ophthalmological assessments were unremarkable. Over a five-year follow up the boy remained free from significant clinical complaints, even though his CK levels remained persistently elevated (ranging from 400 U/L to 3700 U/L). Liver enzymes were also increased (AST 80 U/L, normal < 40). Mutations in DYS, CAV3, and FKRP were excluded. Muscle MRI of the lower limbs did not show signs of muscle damage. At 12 years, the
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boy’s motor performances were comparable to those of his peers. He presented only a narrow chest, but no weakness in the upper or lower girdle muscles (Supplementary Fig. 1). He was profitably attending junior high school. At the same age, a muscle biopsy was obtained from the vastus lateralis muscle with written parental informed consent (Fig. 1A). Histological and histochemical investigations, including staining for myophosphorylase, phosphofructokinase and myoadenylate deaminase, were performed using standard methods. Moderate myopathic changes were noted including variability of fiber size (from 30 lm to 110 lm in diameters), few grouped hypotrophic fibers (Fig. 1Aii), several internal nuclei, without necrosis and endomysial fibrosis. Slight thickening of the connective tissue was observed in some areas with limited cell infiltration (Fig. 1Aiii). Minicores and a “moth-eaten” appearance of fibers were observed on NADH staining and considered non-specific features. A slight predominance of type 1 fibers was also noted, with type I fibers being smaller in average. There was no evidence of lipid or glycogen accumulation and no areas tested positive for acid phosphatase. Immunohistochemistry revealed a marked reduction of glycosylated a-DG in respect to control whereas merosin, b-dystroglycan, dystrophin and sarcoglycans were normally expressed (Fig. 1Aiv–vi). Protein analyses of muscle homogenates performed using Western blotting with monoclonal antibodies revealed abnormal expression of glycosylated a-DG in the patient (Fig. 1B). Analyses of the coding exons of known genes associated with hypoglycosylated a-DG in blood DNA [1] did not show mutations in POMT1, POMT2, POMGNT1, LARGE, ISPD, or DAG1. A novel heterozygous nonsense mutation (c.766C>T/p.R256X) and a novel splice-site variant (c.1045–6C>G/ p.V349Cfs22) were identified in FKTN (Fig. 1C). The latter variant affects pre-mRNA processing via activation of a novel acceptor splice site and leads to insertion of 28 bp in muscle cDNA with predictable frameshift and subsequent premature stop. Both variants segregated independently in the healthy parents, whereas the elder healthy brother did not carry any of the mutant alleles. Because of the genetic findings and previous reports of dilated cardiomyopathy associated with dysfunctional fukutin [10], the patient, despite having no clinical complaints, underwent a follow-up cardiac examination. Echocardiography showed a slightly enlarged left ventricle (Supplementary Fig. 2) and a left ventricular ejection fraction in the lower range of control values (52%, calculated according to the biplane Simpson’s method). 3. Discussion To date, at least 55 different FKTN mutations have been described (Leiden Muscular Dystrophy pages, www. dmd.nl) and the associated phenotypes embrace the entire
1012
C. Fiorillo et al. / Neuromuscular Disorders 23 (2013) 1010–1015
Fig. 1. (A) Muscle biopsy of the patient at 12 years of age taken from the vastus lateralis muscle and Western blot of muscle homogenate. (i) NADH and (ii) ATP pH 4.6 staining showing variation in fiber size and a group of smaller type I fibres. Non-specific abnormalities of myofibrillar pattern appear as “moth eaten” fibers and pale areas can also be seen; (iii) Modified Gomori Trichrome staining showing slight thickening of connective tissue and limited cell infiltration; (iv) immunofluorescence for a-dystroglycan with IIH6C4 antibody showing virtually absent binding; (v) immunofluorescence for adystroglycan in a control case and (vi) immunofluorescence for laminin-a2 of patient, showing normal protein expression. (B) Western blot with monoclonal antibody anti-a-Dystroglycan, clone VIA4, (Millipore, Temecula, CA) showing different expression profile of glycosilated a-dystroglycan. In control case (C) a broader band at about 150 kDa is detected together with two bands sized roughly 110 kDa and < 90 kDa (arrows). In the patient (P) the expression of glycosilated a-dystroglycan is reduced. b-Tubulin expression is used to control for protein loading. (C) cDNA amplification from muscle showing, in addition to the wild-type 1154 base pair (bp) fragment, an upper band of about 1182 bp. The two fragments (arrows) are separated on a 1% agarose gel. A DNA molecular marker is also shown (D) cDNA sequence of the agarose-purified fragment of 1154 bp (lower band) displaying the c.766C>T/p.R256X mutation (arrow) and absence of the 28 bp insertion with normal exon 8–exon 9 boundaries. (E) cDNA sequence of the agarosepurified fragment of 1182 bp (upper band) showing the insertion of 28 bp (flanked by vertical bars) of intron 8 between the sequences of exons 8 and 9. The c.766 residue in exon 6 (arrow) is wild type in this cDNA fragment.
Table 1 A schematic overview of clinical features from patients harboring mutations in FKNT and presenting with relatively milder phenotypes. The last column summarizes the patient in this work. Numbers refer to references. M, men; F, women. (a) Reference Mutation Gender/Age Onset
(b) Reference Mutation Gender/Age Onset CK Distribution of muscle weakness and wasting Max motor function Muscle hypertrophy Joint contractures Other symptoms Heart
Godfreyl 2006 [5] c.920G>A + c.1167dupA M/10 Motor developmental delay
Saredi 2009 [6] c.42Gdel fsX1 + c.920G>A F/4 Delayed motor development
857–9951 UI/L Proximal and distal upper extremity weakness. Periscapular atrophy.
Godfrey 2006 [5] c.920G>A + c.1167dupA F/7 Truncal weakness and hypotonia at 4 months 13,000 IU/L Distal and proximal weakness, with the legs more affected than the arms.
862–17,000 IU/L Truncal and proximal weakness more pronounced in lower limbs. Diffuse wasting
Puckett 2009 [7] c.340G>A hom M/5 13 months: elevated CK, failue to thrive 1865–12,131 IU/L Pectus excavatum, modified Gowers’ maneuver and decreased deep tendon reflexes
Walk for short distance
Ambulant
Ambulant
Ambulant with waddling gait
Ambulant
Triceps, thighs, and calves
Lateral gastrocenemius
Lateral gastrocenemius
Calf
Calf
Not reported
Not reported
Not reported
Not reported
Not reported
Response to steroid treatment
Response to steroid treatment
Worsening during febrile illness
Not reported
Not reported Not reported Mild ventriculomegaly at 8 years Dystrophic picture with severely reduced a-dystroglycan. Inflammatory changes
Not reported Not reported Normal Dystrophic pattern with infiltrating cells
Response to steroid treatment. Worsening during illness Not reported Not reported Frontal subarachnoid cyst Necrotic fibers, virtually absent a-dystroglycan
Normal Normal Normal Increased connective tissue, variation in fiber size, central nuclei. No apparent fiber degeneration or inflammation
Normal Normal Normal Small numbers of necrotic and regenerating myofibers, mild endomysial fibrosis
Puckett 2009 [7] c.340G>A hom M/7 Mild hypotonia at 4 years
Vuillaumier-Barrot 2009 [8] c.920G>A hom F/19 Motor delay, equinovarus feet at 2 years 4940 IU/L Predominant weakness in lower extremities, axial and proximal limbs Diffuse wasting Loss of ambulation at 11 years Normal
Vuillaumier-Barrot 2009 [8] c.736A>G + c.139C>T M/7 15 months: HyperCKaemia 6 y: post-exercise myalgia 1500 IU/L Proximal arms and legs.
Yis 2011 [9] c.842T>C + c.1045–22A>G M/20 20 months: hypotonia diagnosis of myositis during viral infection 1608 IU/L Pes cavus and protracted shoulders
This patient c.766C>T + c.1045–6C>G M712 Elevated CK at 4 years
Walking, with normal gait
Can walk 1 mile
Calf
Not reported
Walking, with normal gait and running Normal
Not reported
Not reported
Normal
Not reported
Early distal (ankle). Late hip, knee, elbow Not reported
Exercise myalgia
Normal
Normal
Normal
Not reported
Myositis during viral infection. Exercise intolerance Bicuspid aortic valve
4068 and 9955 IU/L Pectus excavatum, Gowers’ maneuver. Lumbar hyperlordosis
Ambulant with waddling gait Not reported Not reported
Left ventricle remodeling 1013
Line missing
400–3700 IU/L Narrow chest, mild scapular winging
C. Fiorillo et al. / Neuromuscular Disorders 23 (2013) 1010–1015
CK Distribution of muscle weakness and wasting Max motor function Muscle hypertrophy Joint contractures Other symptoms Heart Eyes Brain Muscle biopsy
Godfrey 2006 [5] c.1167dupA + c.1363delG F/11 Hypotonia 10 months. Diagnosis of polymyositis at 18 months. 800–60,000 IU/L Proximal weakness of lower limb
Variation of fiber size, few internal nuclei. No necrosis, no fibrosis. Severe reduction of a-dystroglycan
Normal
C. Fiorillo et al. / Neuromuscular Disorders 23 (2013) 1010–1015
Mild changes, few necrotic fibers and foci of inflammatory infiltrates; fibrosis almost absent Not reported Muscle biopsy
Marked fiber size variability, variable necrotic and regenerative fibers, and increased connective tissue
Not reported Not reported Brain
Normal
Not reported Eyes
Normal
Not reported
Strabismus concomitans convergens alternans IQ at the lower limit. Mild polymicrogyria of frontal basal lobe. Retrocerebellar arachnoid cysts Increased interstitial connective and fat tissue, and evidence of recent dystrophic changes
Normal
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range of pathologies linked to low/absent a-DG. Clinical manifestations other than those of typical FCMD are increasingly being described [13]. In particular, an LGMD phenotype characterized by normal intelligence and unremarkable brain MRI (LGMD2M) does not seem to be particularly rare [1] (Table 1), although the genotype does not seem to predict the degree of clinical severity. On the basis of the neuromuscular manifestations observed in our patient, it remains debatable whether he should be deemed to fall into the clinical category of early LGMD2M, classified as a case of subclinical cardiomyopathy, or, more reasonably, considered as asymptomatic hyperCKemia. Clinical nomenclature issues aside, we believe, on the basis of several considerations, that our findings are significant. First, we detected (by means of Western blotting and immunohistochemistry) a reduction in a-DG glycoepitope expression in muscle from a boy with long-term benign hyperCKemia, and found this to be associated with two novel loss-of-function changes in FKTN that predicted a prematurely truncated protein. Neither variant has previously been annotated in the dbSNP131, 1000 genome and “HapMap” databases, nor in a large exome database of 6500 exomes (NHLBI Exome Sequencing Project, evs.gs.washington.edu/EVS/), and in a sample of 200 in-house normal Italian chromosomes. Fukutin is implicated in the a-DG processing pathway because its mutations disrupt a-DG glycosylation [14]; however, neither the complete a-DG O-mannose structure nor the site of FKTN activity is known. While we do not yet know precisely what the activity of FKTN in the a-DG processing pathway is, we presume that the degree of a-DG dysfunction and the disruption of fukutin activity will prove to be positively correlated with the FKTN mutation, just as POMT1 and POMGnT1 enzyme activity has been shown to be in MEB patient cells [15]. Often, as in our case, the magnitude of residual muscular a-DG glycosylation is hard to reconcile with the disease phenotype, making it legitimate to consider other factors. One possibility is that modifier genes or proteins (as yet unknown) might influence the specific output of patient phenotype [16]. An additional important consideration is that our patient, the second reported in Italy [6], provides a further reminder of the possible association of fukutin mutations with severe or latent heart disease. Unlike previously reported cases, showing progressive cardiac insufficiency due to dilated cardiomyopathy [10], in the case we describe only a targeted echocardiography was able to unveil initial signs of left ventricular remodeling. In clinical practice, the patient will now modify his physical activities and will undergo six-monthly cardiac monitoring to allow early detection and, if necessary, treatment of any heart dysfunction. Finally, the patient’s muscle biopsy showed minimal foci of inflammatory infiltrates (Fig. 1Aiii), a finding already
C. Fiorillo et al. / Neuromuscular Disorders 23 (2013) 1010–1015
seen in abnormal fukutin [5] and also described in conjunction with mutations in POMT2 and a milder phenotype [17]. Thus, we suggest that minimal inflammation can be considered a red flag to suspect a milder clinical form of dystroglycanopathy. Acknowledgements This study was supported in part by the Italian Ministry of Health, by a grant from the Regione Toscana-Bando Salute 2009 (to C.B.), and by Fondazione Telethon Italy (Grant GUP11001D to F.M.S., GUP11001E to C.B.). The authors thank Catherine J. Wrenn for her editorial assistance and Dr Anna Maria Valleriani for the imaging studies. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.nmd.2013.09.010. References [1] Mercuri E, Muntoni F. The ever-expanding spectrum of congenital muscular dystrophies. Ann Neurol 2012;72:9–17. [2] Kobayashi K, Nakahori Y, Miyake M, et al.. An ancient retrotransposal insertion causes Fukuyama-type congenital muscular dystrophy. Nature 1998;394:388–92. [3] Kondo-Iida E, Kobayashi K, Watanabe M, et al.. Novel mutations and genotype–phenotype relationships in 107 families with Fukuyama-type congenital muscular dystrophy (FCMD). Hum Mol Genet 1999;8:2303–9. [4] Bertini E, D’Amico A, Gualandi F, Petrini S. Congenital muscular dystrophies: a brief review. Semin Pediatr Neurol 2011;18:277–88. [5] Godfrey C, Escolar D, Brockington M, et al.. Fukutin gene mutations in steroid-responsive limb girdle muscular dystrophy. Ann Neurol 2006;60:603–10.
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[6] Saredi S, Ruggieri A, Mottarelli E, et al.. Fukutin gene mutations in an Italian patient with early onset muscular dystrophy but no central nervous system involvement. Muscle Nerve 2009;39:845–8. [7] Puckett RL, Moore SA, Winder TL, et al.. Further evidence of Fukutin mutations as a cause of childhood onset limb-girdle muscular dystrophy without mental retardation. Neuromuscul Disord 2009;19:352–6. [8] Vuillaumier-Barrot S, Quijano-Roy S, Bouchet-Seraphin C, et al.. Four Caucasian patients with mutations in the fukutin gene and variable clinical phenotype. Neuromuscul Disord 2009;19:182–8. [9] Yis U, Uyanik G, Heck PB, et al.. Fukutin mutations in nonJapanese patients with congenital muscular dystrophy: less severe mutations predominate in patients with a non-Walker–Warburg phenotype. Neuromuscul Disord 2011;21:20–30. [10] Murakami T, Hayashi YK, Noguchi S, et al.. Fukutin gene mutations cause dilated cardiomyopathy with minimal muscle weakness. Ann Neurol 2006;60:597–602. [11] Jimenez-Mallebrera C, Torelli S, Feng L, et al.. A comparative study of alpha-dystroglycan glycosylation in dystroglycanopathies suggests that the hypoglycosylation of alpha-dystroglycan does not consistently correlate with clinical severity. Brain Pathol 2009;19:596–611. [12] Morandi L, Angelini C, Prelle A, et al.. High plasma creatine kinase: review of the literature and proposal for a diagnostic algorithm. Neurol Sci 2006;27:303–11. [13] Brown SC, Winder SJ. Dystroglycan and dystroglycanopathies: report of the 187th ENMC Workshop 11–13 November 2011, Naarden, The Netherlands. Neuromuscul Disord 2012;22:659–68. [14] Lynch TA, Lam Le T, Man Nt, Kobayashi K, Toda T, Morris GE. Detection of the dystroglycanopathy protein, fukutin, using a new panel of site-specific monoclonal antibodies. Biochem Biophys Res Commun 2012;424:354–7. [15] Vajsar J, Zhang W, Dobyns WB, et al.. Carriers and patients with muscle–eye–brain disease can be rapidly diagnosed by enzymatic analysis of fibroblasts and lymphoblasts. Neuromuscul Disord 2006;16:132–6. [16] Beedle AM, Turner AJ, Saito Y, et al.. Mouse fukutin deletion impairs dystroglycan processing and recapitulates muscular dystrophy. J Clin Invest 2012;122:3330–42. [17] Biancheri R, Falace A, Tessa A, et al.. POMT2 gene mutation in limb-girdle muscular dystrophy with inflammatory changes. Biochem Biophys Res Commun 2007;363:1033–7.
ScienceDirect Neuromuscular Disorders 23 (2013) 1010–1015 www.elsevier.com/locate/nmd
Case report
Novel mutations in the fukutin gene in a boy with asymptomatic hyperCKemia Chiara Fiorillo a,⇑, Francesca Moro a, Guja Astrea a, Maria Aurora Morales b, Jacopo Baldacci a, Maria Marchese a, Sara Scapolan c, Claudio Bruno c, Roberta Battini a, Filippo M. Santorelli a a
Molecular Medicine and Neuromuscular Lab, IRCCS Stella Maris, Pisa, Italy b CNR Clinical Physiology Institute, Pisa, Italy c Center of Myology, Pediatric Neurology Unit, Department of Neuroscience and Rehabilitation, IRCCS G. Gaslini, Genoa, Italy
Abstract Mutations in the fukutin gene were first identified in Japanese patients with classic Fukuyama congenital muscular dystrophy, a severe form of congenital muscular dystrophy associated with cobblestone lissencephaly and ocular defects. Patients of different ethnicities and with milder phenotypes, including limb girdle muscular dystrophy and cardiomyopathy without brain impairment, have also been reported. The hallmark of this disorder, regardless of the clinical outcome, is moderate-to-severe hypoglycosylation of alpha-dystroglycan in muscle sections. We describe the case of a boy harboring two novel mutations in fukutin gene and presenting a five-year history of asymptomatic hyperCKemia, without overt muscle, brain or ocular involvement. Genetic investigations, guided by the presence of moderate myopathic changes on muscle biopsy with loss of immunodetectable alpha-dystroglycan, led to a definitive diagnosis. Cardiac and echocardiographic examinations at follow-up disclosed low normal left ventricular function but no active cardiovascular symptoms. We suggest that fukutin mutations should be sought in asymptomatic hyperCKemia and subclinical heart dysfunction. Ó 2013 Elsevier B.V. All rights reserved. Keywords: Fukutin gene; Mutations; HyperCKemia; Alpha-dystroglycan; Cardiomyopathy
1. Introduction The dystroglycanopathies are a group of muscular dystrophies that share the common pathological feature of aberrant alpha-dystroglycan (a-DG) glycosylation. To date, defects in at least 11 putative glycosyltransferases, accessory proteins, or proteins influencing O-mannosylation of a-DG have been reported [1]. The associated clinical phenotypes range from congenital onset and severe structural brain abnormalities, such as those seen in Walker–Warburg syndrome (WWS, [MIM 236670]),
⇑ Corresponding author. Address: IRCCS Stella Maris, Via dei Giacinti 2, 56028 Pisa, Italy. Tel.: +39 050886311; fax: +39 050886247. E-mail address: chiara.fi[email protected] (C. Fiorillo).
0960-8966/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nmd.2013.09.010
muscle–eye–brain disease (MEB, [MIM 253280]) and Fukuyama congenital muscular dystrophy (FCMD, [MIM 253800]), to forms with infantile myopathy and mild/ moderate brain involvement (MDC1C [MIM 606612] and MDC1D [MIM 608840]). Patients can also present with adult-onset limb girdle muscular dystrophy (LGMD) [1]. FCMD is the most common form of a-dystroglycanopathy in Japan and most patients carry the founder insertion of a 3-kb retrotransposon element in the 30 untranslated region of the fukutin gene (FKTN) [2]. FCMD patients display dystrophic changes in skeletal muscle, structural brain malformations, and severe ocular abnormalities and have a reduced life expectancy [3,4]. There are descriptions of Japanese and non-Japanese FKTN-mutated patients presenting with limb girdle
C. Fiorillo et al. / Neuromuscular Disorders 23 (2013) 1010–1015
muscle weakness without ocular or brain defects (LGMD type 2M, MIM 253600) [5–9], as well as occasional reports of association with isolated dilated cardiomyopathy [10]. As in more typical a-dystroglycanopathies, all fukutin patients, regardless of their clinical presentation, show elevated serum creatine kinase (CK) and depleted a-DG on skeletal muscle biopsy. Interestingly, residual levels of a-DG do not seem to correlate with disease severity [11]. HyperCKemia is considered a hallmark of hereditary neuromuscular disorders. However, several acquired conditions (infections, alcoholism, drugs, intramuscular injections, hypothyroidism, etc.) may also cause a rise in this enzyme, and the number of apparently healthy individuals with persistent hyperCKemia has increased considerably since the CK test started to be commonly included in routine blood workup. Therefore, the criteria for the diagnosis of isolated hyperCKemia have been revised in the past decade, with the emphasis shifting to thorough analysis of muscle biopsy [12]. We describe the case of a boy with persistent hyperCKemia without overt muscle weakness or pain, in whom the finding of marked a-DG deficiency on muscle biopsy targeted genetic analyses and led to the identification of two novel mutations in FKTN. 2. Case report A 12-year-old boy was first referred for evaluation at the age of 7 years after a routine blood test revealed a raised CK level (1081 U/L, normal values < 190 U/L). His family history was negative for neuromuscular disorders and his parents were unrelated; their CK levels were in the normal range. The patient had developed normally and had never complained of muscle weakness, cramps or fatigue after exercise, or myoglobinuria. His general physical examination was normal. Neurological examination did not show muscle weakness or central nervous system impairment. He had neither calf hypertrophy nor contractures. The patient could get up from floor without support and was able to run and jump normally for his age. Electrocardiogram and ultrasound recordings, performed elsewhere, were reportedly normal. Electromyography, nerve conduction studies, and respiratory function were also normal. The patient was not taking any drugs or receiving intramuscular injections. Screening for metabolic disorders and blood lactic acid levels were normal. Spine X-ray did not show scoliosis. Brain MRI and ophthalmological assessments were unremarkable. Over a five-year follow up the boy remained free from significant clinical complaints, even though his CK levels remained persistently elevated (ranging from 400 U/L to 3700 U/L). Liver enzymes were also increased (AST 80 U/L, normal < 40). Mutations in DYS, CAV3, and FKRP were excluded. Muscle MRI of the lower limbs did not show signs of muscle damage. At 12 years, the
1011
boy’s motor performances were comparable to those of his peers. He presented only a narrow chest, but no weakness in the upper or lower girdle muscles (Supplementary Fig. 1). He was profitably attending junior high school. At the same age, a muscle biopsy was obtained from the vastus lateralis muscle with written parental informed consent (Fig. 1A). Histological and histochemical investigations, including staining for myophosphorylase, phosphofructokinase and myoadenylate deaminase, were performed using standard methods. Moderate myopathic changes were noted including variability of fiber size (from 30 lm to 110 lm in diameters), few grouped hypotrophic fibers (Fig. 1Aii), several internal nuclei, without necrosis and endomysial fibrosis. Slight thickening of the connective tissue was observed in some areas with limited cell infiltration (Fig. 1Aiii). Minicores and a “moth-eaten” appearance of fibers were observed on NADH staining and considered non-specific features. A slight predominance of type 1 fibers was also noted, with type I fibers being smaller in average. There was no evidence of lipid or glycogen accumulation and no areas tested positive for acid phosphatase. Immunohistochemistry revealed a marked reduction of glycosylated a-DG in respect to control whereas merosin, b-dystroglycan, dystrophin and sarcoglycans were normally expressed (Fig. 1Aiv–vi). Protein analyses of muscle homogenates performed using Western blotting with monoclonal antibodies revealed abnormal expression of glycosylated a-DG in the patient (Fig. 1B). Analyses of the coding exons of known genes associated with hypoglycosylated a-DG in blood DNA [1] did not show mutations in POMT1, POMT2, POMGNT1, LARGE, ISPD, or DAG1. A novel heterozygous nonsense mutation (c.766C>T/p.R256X) and a novel splice-site variant (c.1045–6C>G/ p.V349Cfs22) were identified in FKTN (Fig. 1C). The latter variant affects pre-mRNA processing via activation of a novel acceptor splice site and leads to insertion of 28 bp in muscle cDNA with predictable frameshift and subsequent premature stop. Both variants segregated independently in the healthy parents, whereas the elder healthy brother did not carry any of the mutant alleles. Because of the genetic findings and previous reports of dilated cardiomyopathy associated with dysfunctional fukutin [10], the patient, despite having no clinical complaints, underwent a follow-up cardiac examination. Echocardiography showed a slightly enlarged left ventricle (Supplementary Fig. 2) and a left ventricular ejection fraction in the lower range of control values (52%, calculated according to the biplane Simpson’s method). 3. Discussion To date, at least 55 different FKTN mutations have been described (Leiden Muscular Dystrophy pages, www. dmd.nl) and the associated phenotypes embrace the entire
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C. Fiorillo et al. / Neuromuscular Disorders 23 (2013) 1010–1015
Fig. 1. (A) Muscle biopsy of the patient at 12 years of age taken from the vastus lateralis muscle and Western blot of muscle homogenate. (i) NADH and (ii) ATP pH 4.6 staining showing variation in fiber size and a group of smaller type I fibres. Non-specific abnormalities of myofibrillar pattern appear as “moth eaten” fibers and pale areas can also be seen; (iii) Modified Gomori Trichrome staining showing slight thickening of connective tissue and limited cell infiltration; (iv) immunofluorescence for a-dystroglycan with IIH6C4 antibody showing virtually absent binding; (v) immunofluorescence for adystroglycan in a control case and (vi) immunofluorescence for laminin-a2 of patient, showing normal protein expression. (B) Western blot with monoclonal antibody anti-a-Dystroglycan, clone VIA4, (Millipore, Temecula, CA) showing different expression profile of glycosilated a-dystroglycan. In control case (C) a broader band at about 150 kDa is detected together with two bands sized roughly 110 kDa and < 90 kDa (arrows). In the patient (P) the expression of glycosilated a-dystroglycan is reduced. b-Tubulin expression is used to control for protein loading. (C) cDNA amplification from muscle showing, in addition to the wild-type 1154 base pair (bp) fragment, an upper band of about 1182 bp. The two fragments (arrows) are separated on a 1% agarose gel. A DNA molecular marker is also shown (D) cDNA sequence of the agarose-purified fragment of 1154 bp (lower band) displaying the c.766C>T/p.R256X mutation (arrow) and absence of the 28 bp insertion with normal exon 8–exon 9 boundaries. (E) cDNA sequence of the agarosepurified fragment of 1182 bp (upper band) showing the insertion of 28 bp (flanked by vertical bars) of intron 8 between the sequences of exons 8 and 9. The c.766 residue in exon 6 (arrow) is wild type in this cDNA fragment.
Table 1 A schematic overview of clinical features from patients harboring mutations in FKNT and presenting with relatively milder phenotypes. The last column summarizes the patient in this work. Numbers refer to references. M, men; F, women. (a) Reference Mutation Gender/Age Onset
(b) Reference Mutation Gender/Age Onset CK Distribution of muscle weakness and wasting Max motor function Muscle hypertrophy Joint contractures Other symptoms Heart
Godfreyl 2006 [5] c.920G>A + c.1167dupA M/10 Motor developmental delay
Saredi 2009 [6] c.42Gdel fsX1 + c.920G>A F/4 Delayed motor development
857–9951 UI/L Proximal and distal upper extremity weakness. Periscapular atrophy.
Godfrey 2006 [5] c.920G>A + c.1167dupA F/7 Truncal weakness and hypotonia at 4 months 13,000 IU/L Distal and proximal weakness, with the legs more affected than the arms.
862–17,000 IU/L Truncal and proximal weakness more pronounced in lower limbs. Diffuse wasting
Puckett 2009 [7] c.340G>A hom M/5 13 months: elevated CK, failue to thrive 1865–12,131 IU/L Pectus excavatum, modified Gowers’ maneuver and decreased deep tendon reflexes
Walk for short distance
Ambulant
Ambulant
Ambulant with waddling gait
Ambulant
Triceps, thighs, and calves
Lateral gastrocenemius
Lateral gastrocenemius
Calf
Calf
Not reported
Not reported
Not reported
Not reported
Not reported
Response to steroid treatment
Response to steroid treatment
Worsening during febrile illness
Not reported
Not reported Not reported Mild ventriculomegaly at 8 years Dystrophic picture with severely reduced a-dystroglycan. Inflammatory changes
Not reported Not reported Normal Dystrophic pattern with infiltrating cells
Response to steroid treatment. Worsening during illness Not reported Not reported Frontal subarachnoid cyst Necrotic fibers, virtually absent a-dystroglycan
Normal Normal Normal Increased connective tissue, variation in fiber size, central nuclei. No apparent fiber degeneration or inflammation
Normal Normal Normal Small numbers of necrotic and regenerating myofibers, mild endomysial fibrosis
Puckett 2009 [7] c.340G>A hom M/7 Mild hypotonia at 4 years
Vuillaumier-Barrot 2009 [8] c.920G>A hom F/19 Motor delay, equinovarus feet at 2 years 4940 IU/L Predominant weakness in lower extremities, axial and proximal limbs Diffuse wasting Loss of ambulation at 11 years Normal
Vuillaumier-Barrot 2009 [8] c.736A>G + c.139C>T M/7 15 months: HyperCKaemia 6 y: post-exercise myalgia 1500 IU/L Proximal arms and legs.
Yis 2011 [9] c.842T>C + c.1045–22A>G M/20 20 months: hypotonia diagnosis of myositis during viral infection 1608 IU/L Pes cavus and protracted shoulders
This patient c.766C>T + c.1045–6C>G M712 Elevated CK at 4 years
Walking, with normal gait
Can walk 1 mile
Calf
Not reported
Walking, with normal gait and running Normal
Not reported
Not reported
Normal
Not reported
Early distal (ankle). Late hip, knee, elbow Not reported
Exercise myalgia
Normal
Normal
Normal
Not reported
Myositis during viral infection. Exercise intolerance Bicuspid aortic valve
4068 and 9955 IU/L Pectus excavatum, Gowers’ maneuver. Lumbar hyperlordosis
Ambulant with waddling gait Not reported Not reported
Left ventricle remodeling 1013
Line missing
400–3700 IU/L Narrow chest, mild scapular winging
C. Fiorillo et al. / Neuromuscular Disorders 23 (2013) 1010–1015
CK Distribution of muscle weakness and wasting Max motor function Muscle hypertrophy Joint contractures Other symptoms Heart Eyes Brain Muscle biopsy
Godfrey 2006 [5] c.1167dupA + c.1363delG F/11 Hypotonia 10 months. Diagnosis of polymyositis at 18 months. 800–60,000 IU/L Proximal weakness of lower limb
Variation of fiber size, few internal nuclei. No necrosis, no fibrosis. Severe reduction of a-dystroglycan
Normal
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Mild changes, few necrotic fibers and foci of inflammatory infiltrates; fibrosis almost absent Not reported Muscle biopsy
Marked fiber size variability, variable necrotic and regenerative fibers, and increased connective tissue
Not reported Not reported Brain
Normal
Not reported Eyes
Normal
Not reported
Strabismus concomitans convergens alternans IQ at the lower limit. Mild polymicrogyria of frontal basal lobe. Retrocerebellar arachnoid cysts Increased interstitial connective and fat tissue, and evidence of recent dystrophic changes
Normal
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range of pathologies linked to low/absent a-DG. Clinical manifestations other than those of typical FCMD are increasingly being described [13]. In particular, an LGMD phenotype characterized by normal intelligence and unremarkable brain MRI (LGMD2M) does not seem to be particularly rare [1] (Table 1), although the genotype does not seem to predict the degree of clinical severity. On the basis of the neuromuscular manifestations observed in our patient, it remains debatable whether he should be deemed to fall into the clinical category of early LGMD2M, classified as a case of subclinical cardiomyopathy, or, more reasonably, considered as asymptomatic hyperCKemia. Clinical nomenclature issues aside, we believe, on the basis of several considerations, that our findings are significant. First, we detected (by means of Western blotting and immunohistochemistry) a reduction in a-DG glycoepitope expression in muscle from a boy with long-term benign hyperCKemia, and found this to be associated with two novel loss-of-function changes in FKTN that predicted a prematurely truncated protein. Neither variant has previously been annotated in the dbSNP131, 1000 genome and “HapMap” databases, nor in a large exome database of 6500 exomes (NHLBI Exome Sequencing Project, evs.gs.washington.edu/EVS/), and in a sample of 200 in-house normal Italian chromosomes. Fukutin is implicated in the a-DG processing pathway because its mutations disrupt a-DG glycosylation [14]; however, neither the complete a-DG O-mannose structure nor the site of FKTN activity is known. While we do not yet know precisely what the activity of FKTN in the a-DG processing pathway is, we presume that the degree of a-DG dysfunction and the disruption of fukutin activity will prove to be positively correlated with the FKTN mutation, just as POMT1 and POMGnT1 enzyme activity has been shown to be in MEB patient cells [15]. Often, as in our case, the magnitude of residual muscular a-DG glycosylation is hard to reconcile with the disease phenotype, making it legitimate to consider other factors. One possibility is that modifier genes or proteins (as yet unknown) might influence the specific output of patient phenotype [16]. An additional important consideration is that our patient, the second reported in Italy [6], provides a further reminder of the possible association of fukutin mutations with severe or latent heart disease. Unlike previously reported cases, showing progressive cardiac insufficiency due to dilated cardiomyopathy [10], in the case we describe only a targeted echocardiography was able to unveil initial signs of left ventricular remodeling. In clinical practice, the patient will now modify his physical activities and will undergo six-monthly cardiac monitoring to allow early detection and, if necessary, treatment of any heart dysfunction. Finally, the patient’s muscle biopsy showed minimal foci of inflammatory infiltrates (Fig. 1Aiii), a finding already
C. Fiorillo et al. / Neuromuscular Disorders 23 (2013) 1010–1015
seen in abnormal fukutin [5] and also described in conjunction with mutations in POMT2 and a milder phenotype [17]. Thus, we suggest that minimal inflammation can be considered a red flag to suspect a milder clinical form of dystroglycanopathy. Acknowledgements This study was supported in part by the Italian Ministry of Health, by a grant from the Regione Toscana-Bando Salute 2009 (to C.B.), and by Fondazione Telethon Italy (Grant GUP11001D to F.M.S., GUP11001E to C.B.). The authors thank Catherine J. Wrenn for her editorial assistance and Dr Anna Maria Valleriani for the imaging studies. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.nmd.2013.09.010. References [1] Mercuri E, Muntoni F. The ever-expanding spectrum of congenital muscular dystrophies. Ann Neurol 2012;72:9–17. [2] Kobayashi K, Nakahori Y, Miyake M, et al.. An ancient retrotransposal insertion causes Fukuyama-type congenital muscular dystrophy. Nature 1998;394:388–92. [3] Kondo-Iida E, Kobayashi K, Watanabe M, et al.. Novel mutations and genotype–phenotype relationships in 107 families with Fukuyama-type congenital muscular dystrophy (FCMD). Hum Mol Genet 1999;8:2303–9. [4] Bertini E, D’Amico A, Gualandi F, Petrini S. Congenital muscular dystrophies: a brief review. Semin Pediatr Neurol 2011;18:277–88. [5] Godfrey C, Escolar D, Brockington M, et al.. Fukutin gene mutations in steroid-responsive limb girdle muscular dystrophy. Ann Neurol 2006;60:603–10.
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[6] Saredi S, Ruggieri A, Mottarelli E, et al.. Fukutin gene mutations in an Italian patient with early onset muscular dystrophy but no central nervous system involvement. Muscle Nerve 2009;39:845–8. [7] Puckett RL, Moore SA, Winder TL, et al.. Further evidence of Fukutin mutations as a cause of childhood onset limb-girdle muscular dystrophy without mental retardation. Neuromuscul Disord 2009;19:352–6. [8] Vuillaumier-Barrot S, Quijano-Roy S, Bouchet-Seraphin C, et al.. Four Caucasian patients with mutations in the fukutin gene and variable clinical phenotype. Neuromuscul Disord 2009;19:182–8. [9] Yis U, Uyanik G, Heck PB, et al.. Fukutin mutations in nonJapanese patients with congenital muscular dystrophy: less severe mutations predominate in patients with a non-Walker–Warburg phenotype. Neuromuscul Disord 2011;21:20–30. [10] Murakami T, Hayashi YK, Noguchi S, et al.. Fukutin gene mutations cause dilated cardiomyopathy with minimal muscle weakness. Ann Neurol 2006;60:597–602. [11] Jimenez-Mallebrera C, Torelli S, Feng L, et al.. A comparative study of alpha-dystroglycan glycosylation in dystroglycanopathies suggests that the hypoglycosylation of alpha-dystroglycan does not consistently correlate with clinical severity. Brain Pathol 2009;19:596–611. [12] Morandi L, Angelini C, Prelle A, et al.. High plasma creatine kinase: review of the literature and proposal for a diagnostic algorithm. Neurol Sci 2006;27:303–11. [13] Brown SC, Winder SJ. Dystroglycan and dystroglycanopathies: report of the 187th ENMC Workshop 11–13 November 2011, Naarden, The Netherlands. Neuromuscul Disord 2012;22:659–68. [14] Lynch TA, Lam Le T, Man Nt, Kobayashi K, Toda T, Morris GE. Detection of the dystroglycanopathy protein, fukutin, using a new panel of site-specific monoclonal antibodies. Biochem Biophys Res Commun 2012;424:354–7. [15] Vajsar J, Zhang W, Dobyns WB, et al.. Carriers and patients with muscle–eye–brain disease can be rapidly diagnosed by enzymatic analysis of fibroblasts and lymphoblasts. Neuromuscul Disord 2006;16:132–6. [16] Beedle AM, Turner AJ, Saito Y, et al.. Mouse fukutin deletion impairs dystroglycan processing and recapitulates muscular dystrophy. J Clin Invest 2012;122:3330–42. [17] Biancheri R, Falace A, Tessa A, et al.. POMT2 gene mutation in limb-girdle muscular dystrophy with inflammatory changes. Biochem Biophys Res Commun 2007;363:1033–7.
