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An autosomal recessive limb girdle muscular dystrophy (LGMD2) with mild mental retardation is allelic to Walker–Warburg syndrome (WWS) caused by a mutation in the POMT1 gene
Neuromuscular Disorders 15 (2005) 271–275 www.elsevier.com/locate/nmd
An autosomal recessive limb girdle muscular dystrophy (LGMD2) with mild mental retardation is allelic to Walker–Warburg syndrome (WWS) caused by a mutation in the POMT1 gene Burcu Balcia,1, Go¨khan Uyanikb,1, Pervin Dincera,*, Claudia Grossc, Tobias Willerd, Beril Talime, Go¨knur Halilogluf, Gu¨lsev Kalee, Ute Hehrc, Ju¨rgen Winklerb, Haluk Topalog˘luf a
Department of Medical Biology, Hacettepe University, Faculty of Medicine, Sihhiye, 06100 Ankara, Turkey b Department of Neurology, University of Regensburg, Regensburg, Germany c Center of Gynecological Endocrinology, Reproductive Medicine and Human Genetics, Regensburg, Germany d Department of Cell Biology and Plant Physiology, University of Regensburg, Germany e Department of Pediatric Pathology, Hacettepe University, Faculty of Medicine, Ankara, Turkey f Department of Pediatric Neurology, Hacettepe University, Faculty of Medicine, Ankara, Turkey Received 21 January 2005; received in revised form 28 January 2005; accepted 31 January 2005
Abstract Mutations of the protein O-mannosyltransferase (POMT1) gene affect glycosylation of a-dystroglycan, leading to Walker–Warburg syndrome, a lethal disorder in early life with severe congenital muscular dystrophy, and brain and eye malformations. Recently, we described a novel form of recessive limb girdle muscular dystrophy with mild mental retardation, associated with an abnormal a-dystroglycan pattern in the muscle, suggesting a glycosylation defect. Here, we present evidence that this distinct phenotype results from a common mutation (A200P) in the POMT1 gene. Our findings further expand the phenotype of glycosylation disorders linked to POMT1 mutations. Furthermore, the A200P mutation is part of a conserved core haplotype, indicating an ancestral founder mutation. q 2005 Elsevier B.V. All rights reserved. Keywords: POMT1 gene; LGMD2; WWS; Hypoglycosylation of a-dystroglycan; A200P mutation
1. Introduction Congenital muscular dystrophies (CMD) with central nervous system involvement are a heterogeneous group of autosomal recessive muscular dystrophies with structural eye abnormalities and severe brain malformations. The well-defined ones are the Walker–Warburg syndrome (WWS-OMIM#236670), the muscle–eye–brain disease (MEB-OMIM#253280), and the Fukuyama congenital muscular dystrophy (FCMD-OMIM#252800) [1,2]. * Corresponding author. Tel.: C90 312 305 2541; fax: C90 312 309 6060. E-mail address: [email protected] (P. Dincer). 1 These two authors contributed equally to this study.
0960-8966/$ - see front matter q 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2005.01.013
Hypoglycosylation of a-dystroglycan (a-DG) from muscle is a common feature of these disorders, whereas the brain shows a neuronal migration defect leading to cobblestone lissencephaly (lissencephaly type II) [3,4]. Recently, the underlying genetic defects could be elucidated: mutations of the POMT1 gene are associated with WWS [5], mutations of the protein O-mannose b-1,2-N-acetylglucosaminyltransferase (POMGnT1) gene with MEB [6], and mutations of the Fukutin gene with FCMD [7], respectively. WWS is phenotypically characterized by severe cerebral malformations (regularly more severe than in FCMD and MEB) leading to lissencephaly type II, cerebellar and pontine hypoplasia as well as hydrocephalus. Patients with WWS do not survive beyond the first few years of life. Mutations in the POMT1 gene were recently reported in 20% of patients with WWS. Furthermore, WWS is
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a genetically heterogeneous disorder and likely to be associated with mutations in additional genes [5]. Since a nonsense mutation in the Fukutin gene was shown to cause a severe phenotype in a non-Japanese boy, it was hypothesized that loss of function mutations in the Fukutin gene could also lead to a WWS phenotype [8]. a-DG is a crucial component of the dystrophin– glycoprotein complex, a group of associated proteins that play a critical role in a variety of muscular dystrophies, and has been identified as one of the possible targets for POMT1. O-Mannosyl glycan chains are transferred onto aDG by a heteromeric enzyme complex comprising the POMT1 and POMT2 gene products [9,10]. These sugar moieties are not only important for the binding of laminin and other components of the extracellular matrix, but also for the maturation and proper targeting of a-DG [11]. Very recently targeted disruption of the Pomt1 gene in mice was shown to be early embryonic lethal [12]. Here, we report a cohort of Turkish patients characterized by a moderate limb girdle muscular dystrophy and mild mental retardation without any obvious structural brain abnormality caused by a unique POMT1 mutation.
2. Patients and methods 2.1. Patients The present cohort consists of five patients in part previously described with a distinct clinical phenotype of limb girdle muscular dystrophy, mild microcephaly and mental retardation [13]. Fig. 1(a) shows a typical patient (patient #1). The severe reduction of the VIA4-1 glycoepitope on a-DG in the immunohistochemistry was a common finding in their muscle biopsies. Since the onset of muscular weakness started after achieving certain motor milestones such as walking a congenital muscular dystrophy (CMD) is excluded by definition. All patients presented with difficulty in climbing stairs and being slower than their peers. Four patients were characterized by a mild muscle hypertrophy and a slow disease progression summarized in Table 1 (patients #1–4). The most severely affected patient is shown in Fig. 1(b) (patient #5) with a very prominent muscle hypertrophy of the trunk and extremities. IQs of the cohort ranged between 50 and 65. Serum CK levels were increased more than 20-fold. Four patients had normal
Fig. 1. (a) Case 1. Eight-year-old girl, mild muscle hypertrophy of thighs and calves. Walks alone long distances. (b) Case 5. Nineteen-year-old man, prominent hypertrophy of the trunk, arms and legs. Stopped walking at age 18.
B. Balci et al. / Neuromuscular Disorders 15 (2005) 271–275
273
Table 1 Clinical characteristics of patients with homozygous A200P mutation Age (age of onset) (years)
Sex
Consanguinity
Family history
Age walked (years)
Maximum motor capacity
Hypertrophy
Joint stiffness
IQ
CPK
CT/MRI
Evolution
1
7 (21⁄2 )
F
C
–
3
Walks alone
–
65
X28
N
Mild
2 3 4 5
11 10 17 19
M M M M
C C C C
– – – –
3 3 3 4
Walks alone Walks alone Walks Alone Stopped walking at age 17
Thighs, calves – – Calves Thighs, calves, trunk, arms
– – Achilles Achilles, elbows, spine, neck
50 50 55 50
X40 X20 X40
N N N.A N
Mild Mild Mild Severe
(1) (3) (3) (2)
Cases 1–3 correspond to cases 3–5 of the original publication, respectively [13].
cranial CT/MRI without any structural malformations (Table 1). Informed consent was obtained from all families according to our institutional review board. 2.2. Linkage analysis Linkage analysis was performed in five families. Highly polymorphic nine microsatellite markers located close to or within candidate genes were selected. These are: D9S64, D9S1863 and D9S179 for POMT1; D14S59 and D14S983 for POMT2; D17S925 and D17S1824 for SDF2; D22S264 and D22S539 for SDF2L1. 2.3. Sequence analysis of POMT1 gene For the analysis of the POMT1 gene the complete coding sequence including the exon–intron boundaries (20 exons) was amplified by PCR. The amplified products were screened for mutations by sequencing using an ABI PRISM w 3100-Avant (Applied Biosystems, Foster City, CA). 2.4. Restriction endonuclease enzyme analysis The sequence variation c.598GOC of the POMT1 gene abolishes a restriction site for restriction endonuclease enzyme HpyCH4V (NEBioLabs Inc., Beverly, MA). DNA samples from the patients, their parents as well as from 106 healthy Turkish control subjects were analyzed by HpyCH4V restriction digest of the exon 7 PCR product (1016 bp).
3. Results Muscle biopsies of five cases showed severe hypoglycosylation of a-DG. After candidate genes FKRP, Fukutin and POMGnT1 had been excluded previously [13], the glycosylation defect suggested that POMT1 or other glycosyltransferase related genes (POMT2, SDF2 and SDF2L1) may be involved in this distinct phenotype. No linkage was observed in all families for POMT2, SDF2 and SDF2L1. After performing linkage analysis for the POMT1 locus, however, all patients of the five consanguineous families were found to be homozygous for the tested microsatellite markers D9S64, D9S1863, and D9S179, respectively. The patients showed a homozygous G to C variation at position 598 in exon 7 resulting in the substitution of alanine by proline at amino acid residue 200 (A200P), whereas each of the parents was identified as heterozygous carrier (Fig. 2(a)–(d)). In order to delineate whether this variation reflects a polymorphism or a mutation, we performed restriction endonuclease enzyme analysis using HpyCH4V enzyme in healthy Turkish control subjects. The A200P variant was not detected in any of the 106 control subjects (212 chromosomes). Based on the 25 SNPs within the POMT1 gene, one common core haplotype was observed on the mutant chromosomes exclusively. The rare polymorphisms of the POMT1 gene (IVS11C16GOA, IVS12C98_99delCT, IVS17C48GOC, IVS17C107AOC, and IVS19C13TO C) were identified in all patients in a homozygous state. The alanine amino acid residue in position 200 is highly conserved among different species including human, mouse, and rat.
2.5. Founder haplotype analysis 4. Discussion Haplotype analysis was performed on DNA samples from patients with the A200P alleles using 25 single nucleotide polymorphisms (SNPs) within the POMT1 gene additionally to the microsatellite markers.
This form of LGMD2 is characterized by mild mental retardation, microcephaly, normal brain structure by cranial imaging, highly elevated serum CK levels and significantly
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B. Balci et al. / Neuromuscular Disorders 15 (2005) 271–275
Fig. 2. A200P mutation in POMT1 gene. Sequence analysis of (a) A200P in case 3, (b) control. (c) Restriction enzyme assay of A200P mutation in POMT1 gene (the PCR products were digested with HpyCH4V). M, molecular weight marker (pUC Mix Marker, 8); lanes 1–4, healthy controls; lanes 5–9, cases 1–5, respectively; lane 10, parental carrier (C/K); lane 11, undigested PCR product. (d) Schematic diagram of the POMT1 protein and localizations of the mutations (known mutations in light grey and A200P in dark grey) (modified from [9]).
reduced glycosylation of the a-DG in skeletal muscle [13]. It is important to note that the clinical course is characterized by a late onset of symptoms. Our genetic analysis identified a causal common missense mutation (A200P) in exon 7 of the POMT1 gene for five patients with this distinct LGMD2-MR phenotype. The mutant allele is part of a conserved core haplotype indicating an ancestral founder mutation. In general, syndromic CMDs such as WWS, MEB, and FCMD are characterized by various degrees of cerebral malformations which are usually quite severe. Recently, a novel gene encoding the Fukutin related protein (FKRP) was associated as well with a broad spectrum of CMDs with and without cerebral manifestation [14,15]. FKRP associated muscular dystrophies are also characterized by hypoglycosylation of a-DG in muscle biopsies [16]. Different mutations of this gene lead to symptoms ranging from a mild form of isolated limb girdle muscular dystrophy (LGMD2I), over pure congenital muscular dystrophy (MDC1C) to a clinical phenotype undistinguishable from MEB or WWS [8,16–18]. In mammals, a heteromeric complex of POMT1 and POMT2 initiates the biosynthesis of O-mannosyl glycans within the endoplasmic reticulum (ER) [10]. Thus far no disease causing mutation was assigned to POMT2. All missense and nonsense mutations (Q385X, G76R, Q303X, V428D, G722fs, V703fs, L421del) identified in the POMT1 protein were associated with a very severe clinical phenotype of WWS. In contrast, the herein reported amino acid exchange A200P is the first missense mutation leading to a significantly milder clinical phenotype with
predominant muscular dystrophy and mild mental retardation, but without obvious brain malformations (Fig. 2(d)). Also, this is the first mutation to be identified in a cytoplasmic oriented domain of the POMT1 protein. The milder phenotype associated with the A200P mutation suggests residual POMT1 enzyme activity, however, it is not clear yet whether the mutation directly affects the enzymatic activity of the protein or the formation of the heteromeric enzyme complex. Recently, a Japanese child with a mutation (L421del) of the POMT1 gene leading to a deletion of one single amino acid showed both a longer survival and milder cortical malformation compared to other typical POMT1 associated WWS cases [19]. This phenotype was considered as an intermediate form in terms of clinical severity and cerebral malformation. Interestingly, the oldest patient of this cohort (patient #5) presented a more severe muscle dystrophy and also may represent an intermediate form in terms of the muscle phenotype. Taking our data and recent reports together, the phenotypical spectrum of congenital glycosylation disorders linked to different POMT1 mutations have to be considered much broader than initially anticipated. Based on our findings, it is suggestive to propose that due to the reduced level of O-mannosylation neuronal migration may be affected differently by these distinct mutations leading to a broad spectrum of cortical phenotypes. In contrast to all other previously described mutations leading to cobblestone lissencephaly, it is in particular striking that this missense mutation in a highly conserved region of the POMT1 gene does not result in any obvious cortical migration defect. Similar, recently different mutations in
B. Balci et al. / Neuromuscular Disorders 15 (2005) 271–275
the human ARX gene have been shown to result in a broad spectrum of cortical abnormalities ranging from mild mental retardation without structural abnormalities to complex alterations with lissencephaly [20]. Further dissection of the signalling pathways regulating neuronal migration might point to common mechanisms and/or molecules, interacting with gene products involved in O-mannosylation.
Acknowledgements We thank our patients and their families for active participation. We appreciate the help of Drs Francesco Muntoni and Martin Brockington. This study was supported by AFM (Association Franc¸aise contre les Myopathies), France. Additionally, this study was supported in part by the ‘Regensburger Forschungsfo¨rderung der Medizinischen Fakulta¨t’ (ReForM; University of Regensburg, Germany), the ‘Volkswagen Stiftung’ (Hannover, Germany), and the ‘Fritz-Thyssen Stiftung’ (Ko¨ln, Germany).
References [1] Muntoni F, Brockington M, Blake DJ, Torelli S, Brown SC. Defective glycosylation in muscular dystrophy. Lancet 2002;360:1419–21. [2] Haliloglu G, Topaloglu H. Glycosylation defects in muscular dystrophies. Curr Opin Neurol 2004;15:521–7. [3] Hayashi YK, Ogawa M, Tagawa K, et al. Selective deficiency of alpha-dystroglycan in Fukuyama-type congenital muscular dystrophy. Neurology 2001;57:115–21. [4] Kano H, Kobayashi K, Herrmann R, et al. Deficiency of alphadystroglycan in muscle–eye–brain disease. Biochem Biophys Res Commun 2002;291:1283–6. [5] Beltran-Valero de Bernabe D, Currier S, Steinbrecher A, et al. Mutations in the O-mannosyltransferase gene POMT1 give rise to the severe neuronal migration disorder Walker–Warburg syndrome. Am J Hum Genet 2002;71:1033–43. [6] Yoshida A, Kobayashi K, Manya H, et al. Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1. Dev Cell 2001;1:717–24.
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[7] Kobayashi K, Nakahori Y, Miyake M, et al. An ancient retrotransposal insertion causes Fukuyama-type congenital muscular dystrophy. Nature 1998;394:388–92. [8] Beltran-Valero de Bernabe D, Van Bokhoven H, Van Beusekom E, et al. A homozygous nonsense mutation in the fukutin gene causes a Walker–Warburg syndrome phenotype. J Med Genet 2003;40:845–8. [9] Willer T, Valero MC, Tanner W, Cruces J, Strahl S. O-mannosyl glycans: from yeast to novel associations with human disease. Curr Opin Struct Biol 2003;13:621–30. [10] Manya H, Chiba A, Yoshida A, et al. Demonstration of mammalian protein O-mannosyltransferase activity: coexpression of POMT1 and POMT2 required for enzymatic activity. Proc Natl Acad Sci USA 2004;101:500–5. [11] Michele DE, Barresi R, Kanagawa M, et al. Post-translational disruption of dystroglycan–ligand interactions in congenital muscular dystrophies. Nature 2002;418:417–22. [12] Willer T, Prados B, Flacon JM, et al. Targeted disruption of the Walker–Warburg syndrome gene Pomt1 in mouse results in embryonic lethality. Proc Natl Acad Sci USA 2004;101:14126–31. [13] Dincer P, Balci B, Yuva Y, et al. A novel form of recessive limb girdle muscular dystrophy with mental retardation and abnormal expression of alpha-dystroglycan. Neuromuscul Disord 2003;13:771–8. [14] Topaloglu H, Brockington M, Yuva Y, et al. FKRP gene mutations cause congenital muscular dystrophy, mental retardation, and cerebellar cysts. Neurology 2003;60:988–92. [15] Brockington M, Yuva Y, Prandini P, et al. Mutations in the fukutinrelated protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C. Hum Mol Genet 2001;10:2851–9. [16] Brockington M, Blake DJ, Prandini P, et al. Mutations in the fukutinrelated protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan. Am J Hum Genet 2001;69: 1198–209. [17] Mercuri E, Brockington M, Straub V, et al. Phenotypic spectrum associated with mutations in the fukutin-related protein gene. Ann Neurol 2003;53:537–42. [18] Beltran-Valero de Bernabe D, Voit T, Longman C, et al. Mutations in the FKRP gene can cause muscle–eye–brain disease and Walker– Warburg syndrome. J Med Genet 2004;41:e61. [19] Kim DS, Hayashi YK, Matsumoto H, et al. POMT1 mutation results in defective glycosylation and loss of laminin-binding activity in alpha-DG. Neurology 2004;62:1009–11. [20] Kato M, Das S, Petras K, et al. Mutations of ARX are associated with striking pleiotropy and consistent genotype–phenotype correlation. Hum Mutat 2004;23:147–59.
An autosomal recessive limb girdle muscular dystrophy (LGMD2) with mild mental retardation is allelic to Walker–Warburg syndrome (WWS) caused by a mutation in the POMT1 gene Burcu Balcia,1, Go¨khan Uyanikb,1, Pervin Dincera,*, Claudia Grossc, Tobias Willerd, Beril Talime, Go¨knur Halilogluf, Gu¨lsev Kalee, Ute Hehrc, Ju¨rgen Winklerb, Haluk Topalog˘luf a
Department of Medical Biology, Hacettepe University, Faculty of Medicine, Sihhiye, 06100 Ankara, Turkey b Department of Neurology, University of Regensburg, Regensburg, Germany c Center of Gynecological Endocrinology, Reproductive Medicine and Human Genetics, Regensburg, Germany d Department of Cell Biology and Plant Physiology, University of Regensburg, Germany e Department of Pediatric Pathology, Hacettepe University, Faculty of Medicine, Ankara, Turkey f Department of Pediatric Neurology, Hacettepe University, Faculty of Medicine, Ankara, Turkey Received 21 January 2005; received in revised form 28 January 2005; accepted 31 January 2005
Abstract Mutations of the protein O-mannosyltransferase (POMT1) gene affect glycosylation of a-dystroglycan, leading to Walker–Warburg syndrome, a lethal disorder in early life with severe congenital muscular dystrophy, and brain and eye malformations. Recently, we described a novel form of recessive limb girdle muscular dystrophy with mild mental retardation, associated with an abnormal a-dystroglycan pattern in the muscle, suggesting a glycosylation defect. Here, we present evidence that this distinct phenotype results from a common mutation (A200P) in the POMT1 gene. Our findings further expand the phenotype of glycosylation disorders linked to POMT1 mutations. Furthermore, the A200P mutation is part of a conserved core haplotype, indicating an ancestral founder mutation. q 2005 Elsevier B.V. All rights reserved. Keywords: POMT1 gene; LGMD2; WWS; Hypoglycosylation of a-dystroglycan; A200P mutation
1. Introduction Congenital muscular dystrophies (CMD) with central nervous system involvement are a heterogeneous group of autosomal recessive muscular dystrophies with structural eye abnormalities and severe brain malformations. The well-defined ones are the Walker–Warburg syndrome (WWS-OMIM#236670), the muscle–eye–brain disease (MEB-OMIM#253280), and the Fukuyama congenital muscular dystrophy (FCMD-OMIM#252800) [1,2]. * Corresponding author. Tel.: C90 312 305 2541; fax: C90 312 309 6060. E-mail address: [email protected] (P. Dincer). 1 These two authors contributed equally to this study.
0960-8966/$ - see front matter q 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2005.01.013
Hypoglycosylation of a-dystroglycan (a-DG) from muscle is a common feature of these disorders, whereas the brain shows a neuronal migration defect leading to cobblestone lissencephaly (lissencephaly type II) [3,4]. Recently, the underlying genetic defects could be elucidated: mutations of the POMT1 gene are associated with WWS [5], mutations of the protein O-mannose b-1,2-N-acetylglucosaminyltransferase (POMGnT1) gene with MEB [6], and mutations of the Fukutin gene with FCMD [7], respectively. WWS is phenotypically characterized by severe cerebral malformations (regularly more severe than in FCMD and MEB) leading to lissencephaly type II, cerebellar and pontine hypoplasia as well as hydrocephalus. Patients with WWS do not survive beyond the first few years of life. Mutations in the POMT1 gene were recently reported in 20% of patients with WWS. Furthermore, WWS is
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a genetically heterogeneous disorder and likely to be associated with mutations in additional genes [5]. Since a nonsense mutation in the Fukutin gene was shown to cause a severe phenotype in a non-Japanese boy, it was hypothesized that loss of function mutations in the Fukutin gene could also lead to a WWS phenotype [8]. a-DG is a crucial component of the dystrophin– glycoprotein complex, a group of associated proteins that play a critical role in a variety of muscular dystrophies, and has been identified as one of the possible targets for POMT1. O-Mannosyl glycan chains are transferred onto aDG by a heteromeric enzyme complex comprising the POMT1 and POMT2 gene products [9,10]. These sugar moieties are not only important for the binding of laminin and other components of the extracellular matrix, but also for the maturation and proper targeting of a-DG [11]. Very recently targeted disruption of the Pomt1 gene in mice was shown to be early embryonic lethal [12]. Here, we report a cohort of Turkish patients characterized by a moderate limb girdle muscular dystrophy and mild mental retardation without any obvious structural brain abnormality caused by a unique POMT1 mutation.
2. Patients and methods 2.1. Patients The present cohort consists of five patients in part previously described with a distinct clinical phenotype of limb girdle muscular dystrophy, mild microcephaly and mental retardation [13]. Fig. 1(a) shows a typical patient (patient #1). The severe reduction of the VIA4-1 glycoepitope on a-DG in the immunohistochemistry was a common finding in their muscle biopsies. Since the onset of muscular weakness started after achieving certain motor milestones such as walking a congenital muscular dystrophy (CMD) is excluded by definition. All patients presented with difficulty in climbing stairs and being slower than their peers. Four patients were characterized by a mild muscle hypertrophy and a slow disease progression summarized in Table 1 (patients #1–4). The most severely affected patient is shown in Fig. 1(b) (patient #5) with a very prominent muscle hypertrophy of the trunk and extremities. IQs of the cohort ranged between 50 and 65. Serum CK levels were increased more than 20-fold. Four patients had normal
Fig. 1. (a) Case 1. Eight-year-old girl, mild muscle hypertrophy of thighs and calves. Walks alone long distances. (b) Case 5. Nineteen-year-old man, prominent hypertrophy of the trunk, arms and legs. Stopped walking at age 18.
B. Balci et al. / Neuromuscular Disorders 15 (2005) 271–275
273
Table 1 Clinical characteristics of patients with homozygous A200P mutation Age (age of onset) (years)
Sex
Consanguinity
Family history
Age walked (years)
Maximum motor capacity
Hypertrophy
Joint stiffness
IQ
CPK
CT/MRI
Evolution
1
7 (21⁄2 )
F
C
–
3
Walks alone
–
65
X28
N
Mild
2 3 4 5
11 10 17 19
M M M M
C C C C
– – – –
3 3 3 4
Walks alone Walks alone Walks Alone Stopped walking at age 17
Thighs, calves – – Calves Thighs, calves, trunk, arms
– – Achilles Achilles, elbows, spine, neck
50 50 55 50
X40 X20 X40
N N N.A N
Mild Mild Mild Severe
(1) (3) (3) (2)
Cases 1–3 correspond to cases 3–5 of the original publication, respectively [13].
cranial CT/MRI without any structural malformations (Table 1). Informed consent was obtained from all families according to our institutional review board. 2.2. Linkage analysis Linkage analysis was performed in five families. Highly polymorphic nine microsatellite markers located close to or within candidate genes were selected. These are: D9S64, D9S1863 and D9S179 for POMT1; D14S59 and D14S983 for POMT2; D17S925 and D17S1824 for SDF2; D22S264 and D22S539 for SDF2L1. 2.3. Sequence analysis of POMT1 gene For the analysis of the POMT1 gene the complete coding sequence including the exon–intron boundaries (20 exons) was amplified by PCR. The amplified products were screened for mutations by sequencing using an ABI PRISM w 3100-Avant (Applied Biosystems, Foster City, CA). 2.4. Restriction endonuclease enzyme analysis The sequence variation c.598GOC of the POMT1 gene abolishes a restriction site for restriction endonuclease enzyme HpyCH4V (NEBioLabs Inc., Beverly, MA). DNA samples from the patients, their parents as well as from 106 healthy Turkish control subjects were analyzed by HpyCH4V restriction digest of the exon 7 PCR product (1016 bp).
3. Results Muscle biopsies of five cases showed severe hypoglycosylation of a-DG. After candidate genes FKRP, Fukutin and POMGnT1 had been excluded previously [13], the glycosylation defect suggested that POMT1 or other glycosyltransferase related genes (POMT2, SDF2 and SDF2L1) may be involved in this distinct phenotype. No linkage was observed in all families for POMT2, SDF2 and SDF2L1. After performing linkage analysis for the POMT1 locus, however, all patients of the five consanguineous families were found to be homozygous for the tested microsatellite markers D9S64, D9S1863, and D9S179, respectively. The patients showed a homozygous G to C variation at position 598 in exon 7 resulting in the substitution of alanine by proline at amino acid residue 200 (A200P), whereas each of the parents was identified as heterozygous carrier (Fig. 2(a)–(d)). In order to delineate whether this variation reflects a polymorphism or a mutation, we performed restriction endonuclease enzyme analysis using HpyCH4V enzyme in healthy Turkish control subjects. The A200P variant was not detected in any of the 106 control subjects (212 chromosomes). Based on the 25 SNPs within the POMT1 gene, one common core haplotype was observed on the mutant chromosomes exclusively. The rare polymorphisms of the POMT1 gene (IVS11C16GOA, IVS12C98_99delCT, IVS17C48GOC, IVS17C107AOC, and IVS19C13TO C) were identified in all patients in a homozygous state. The alanine amino acid residue in position 200 is highly conserved among different species including human, mouse, and rat.
2.5. Founder haplotype analysis 4. Discussion Haplotype analysis was performed on DNA samples from patients with the A200P alleles using 25 single nucleotide polymorphisms (SNPs) within the POMT1 gene additionally to the microsatellite markers.
This form of LGMD2 is characterized by mild mental retardation, microcephaly, normal brain structure by cranial imaging, highly elevated serum CK levels and significantly
274
B. Balci et al. / Neuromuscular Disorders 15 (2005) 271–275
Fig. 2. A200P mutation in POMT1 gene. Sequence analysis of (a) A200P in case 3, (b) control. (c) Restriction enzyme assay of A200P mutation in POMT1 gene (the PCR products were digested with HpyCH4V). M, molecular weight marker (pUC Mix Marker, 8); lanes 1–4, healthy controls; lanes 5–9, cases 1–5, respectively; lane 10, parental carrier (C/K); lane 11, undigested PCR product. (d) Schematic diagram of the POMT1 protein and localizations of the mutations (known mutations in light grey and A200P in dark grey) (modified from [9]).
reduced glycosylation of the a-DG in skeletal muscle [13]. It is important to note that the clinical course is characterized by a late onset of symptoms. Our genetic analysis identified a causal common missense mutation (A200P) in exon 7 of the POMT1 gene for five patients with this distinct LGMD2-MR phenotype. The mutant allele is part of a conserved core haplotype indicating an ancestral founder mutation. In general, syndromic CMDs such as WWS, MEB, and FCMD are characterized by various degrees of cerebral malformations which are usually quite severe. Recently, a novel gene encoding the Fukutin related protein (FKRP) was associated as well with a broad spectrum of CMDs with and without cerebral manifestation [14,15]. FKRP associated muscular dystrophies are also characterized by hypoglycosylation of a-DG in muscle biopsies [16]. Different mutations of this gene lead to symptoms ranging from a mild form of isolated limb girdle muscular dystrophy (LGMD2I), over pure congenital muscular dystrophy (MDC1C) to a clinical phenotype undistinguishable from MEB or WWS [8,16–18]. In mammals, a heteromeric complex of POMT1 and POMT2 initiates the biosynthesis of O-mannosyl glycans within the endoplasmic reticulum (ER) [10]. Thus far no disease causing mutation was assigned to POMT2. All missense and nonsense mutations (Q385X, G76R, Q303X, V428D, G722fs, V703fs, L421del) identified in the POMT1 protein were associated with a very severe clinical phenotype of WWS. In contrast, the herein reported amino acid exchange A200P is the first missense mutation leading to a significantly milder clinical phenotype with
predominant muscular dystrophy and mild mental retardation, but without obvious brain malformations (Fig. 2(d)). Also, this is the first mutation to be identified in a cytoplasmic oriented domain of the POMT1 protein. The milder phenotype associated with the A200P mutation suggests residual POMT1 enzyme activity, however, it is not clear yet whether the mutation directly affects the enzymatic activity of the protein or the formation of the heteromeric enzyme complex. Recently, a Japanese child with a mutation (L421del) of the POMT1 gene leading to a deletion of one single amino acid showed both a longer survival and milder cortical malformation compared to other typical POMT1 associated WWS cases [19]. This phenotype was considered as an intermediate form in terms of clinical severity and cerebral malformation. Interestingly, the oldest patient of this cohort (patient #5) presented a more severe muscle dystrophy and also may represent an intermediate form in terms of the muscle phenotype. Taking our data and recent reports together, the phenotypical spectrum of congenital glycosylation disorders linked to different POMT1 mutations have to be considered much broader than initially anticipated. Based on our findings, it is suggestive to propose that due to the reduced level of O-mannosylation neuronal migration may be affected differently by these distinct mutations leading to a broad spectrum of cortical phenotypes. In contrast to all other previously described mutations leading to cobblestone lissencephaly, it is in particular striking that this missense mutation in a highly conserved region of the POMT1 gene does not result in any obvious cortical migration defect. Similar, recently different mutations in
B. Balci et al. / Neuromuscular Disorders 15 (2005) 271–275
the human ARX gene have been shown to result in a broad spectrum of cortical abnormalities ranging from mild mental retardation without structural abnormalities to complex alterations with lissencephaly [20]. Further dissection of the signalling pathways regulating neuronal migration might point to common mechanisms and/or molecules, interacting with gene products involved in O-mannosylation.
Acknowledgements We thank our patients and their families for active participation. We appreciate the help of Drs Francesco Muntoni and Martin Brockington. This study was supported by AFM (Association Franc¸aise contre les Myopathies), France. Additionally, this study was supported in part by the ‘Regensburger Forschungsfo¨rderung der Medizinischen Fakulta¨t’ (ReForM; University of Regensburg, Germany), the ‘Volkswagen Stiftung’ (Hannover, Germany), and the ‘Fritz-Thyssen Stiftung’ (Ko¨ln, Germany).
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