Background
Limb girdle muscular dystrophies (LGMDs) are a heterogeneous group of inherited progressive muscle disorders characterized by progressive shoulder and pelvic girdle muscle weakness variably associated with cardiac, respiratory, and cognitive involvement [
1]. The number of genes involved in these disorders has exponentially increased in recent years, and now up to 30 different forms have been described, inherited both with autosomal dominant (7 forms) and autosomal recessive (23 forms) patterns [
2].
In particular, a high number of genes involved in α-dystroglycan (α-DG) glycosylation have been associated with LGMD. α-DG is a highly glycosylated core component of the dystrophin glycoprotein complex, and forms a link between the sarcolemma and the extracellular matrix [
3]. To date, mutations in 14 genes [
4‐
18], all coding for putative or demonstrated glycosyltransferase, have been associated with muscular dystrophies (referred to as secondary dystroglycanopathies) [
19], while only a couple of cases have been associated with mutations in
DAG1, the gene that encodes both α-dystroglycan and β-dystroglycan [
20,
21]. Dystroglycanopathies are characterised by a broad variety of clinical phenotypes, ranging from congenital muscular dystrophy (CMD), with or without brain and eye involvement as Walker-Warburg syndrome (WWS) and muscle eye brain disease (MEB), to LGMD, as summarised by Godfrey et al. [
22]. Mutations in most of these genes are mainly associated with severe or congenital conditions, with few notable exceptions:
FKRP mutations account for a variable proportion of LGMD depending on ethnic background (from 6 % in the Italian population [
1,
23] to 38 % in the Danish population [
24]). On the other hand, LGMD phenotypes caused by mutations in
POMT1 (LGMD2K) [
25],
FKTN (LGMD2L) [
26],
POMT2 (LGMD2N) [
27],
POMGNT1 (LGMD2O) [
22],
DAG1 [
21], and
DPM3 [
10] have been reported in a very limited number of patients.
ISPD, a gene located on chromosome 7p21, encodes the Isoprenoid synthase domain-containing protein and has been implicated in the initial step of the O-mannosylation of α-DG. Mutations in this gene were first identified within the most severe spectrum of dystroglycanopathies, WWS and MEB cases [
13,
14], although more recently they have also been associated with milder phenotypes [
28,
29]. In a paediatric cohort of dystroglycanopathies with British and Turkish background,
ISPD mutations have been found to cause LGMD in seven probands, including four LGMD cases with normal cognitive development (LGMD – no MR); two LGMD cases with cerebellar involvement (LGMD-CRB); and one case of LGMD with mental retardation, but without structural brain abnormalities (LGMD-MR) [
28].
ISPD mutations were also detected in two Italian LGMD families that presented with disease onset during the first two decades of life, late motor impairment, and no functional or structural brain involvement [
28]. Muscle biopsy revealed dystrophic features and α-DG reduction at immunohistochemistry [
28,
29]. According to this description, forms of LGMD caused by mutations in
ISPD are described as LGMD2S [
2]. Intra-familial variability has also been described [
30].
ISPD mutations account for 9–11 % of the most severe dystroglycanopathy variants (comprising CMD, WWS, and cobblestone lyssencephaly) in three large cohorts from different ethnic backgrounds [
13,
14,
17]. The prevalence of
ISPD mutations has not yet been estimated in LGMD cohorts: at this time, only 12 patients with
ISPD mutation and this phenotype have been described. Similar considerations apply to disease phenotypes caused by
GMPPB mutations, initially shown to be causally linked to MEB/FCMD-like syndrome [
16,
31], and more recently to a wider phenotypic spectrum that includes infantile phenotypes with mental retardation [
16] and adult-onset LGMD with normal cognition [
32].
The aim of this study is to establish the prevalence of ISPD and GMPPB mutations within an Italian cohort of LGMD patients.
Methods
Patient selection and characterization
From a cohort of 174 Italian LGMD patients (140 families), all followed at a single neuromuscular centre, we selected 41 patients (39 families) without a molecular diagnosis. Written informed consent was obtained (and preserved in original) from all patients or their caregivers at first evaluation, with explicit consent to future use for research purposes, in accordance with the Declaration of Helsinki. This protocol was approved by the Research Ethics Board of IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico. The patients have been previously screened for the following genes: MYOT, LMNA, CAV3, DNAJB6, and TNPO3, if autosomal dominant transmission was supported by family history; CAPN3, DYSF, SGCA, SGCB, SGCG, SGCD, FKRP, ANO5, FKTN, and LAMA2 in sporadic or autosomal recessive cases.
LARGE, POMT1, POMT2, POMGnT1 were also screened in selected cases.
Patients were defined as affected with LGMD if they fulfilled the following criteria: clinical phenotype characterised by progressive muscle weakness and wasting affecting primarily the shoulder girdle and pelvic muscles, in keeping with the diagnostic criteria for LGMD [
33]; and dystrophic features at muscle biopsy. All patients had undergone systematic clinical characterisation, including comprehensive neurological [Medical Research Council (MRC) and functional scales], cardiac (electrocardiogram and echocardiogram), and respiratory (spirometry and nocturnal saturimetry) assessments. Data about clinical and familial history were also collected. All specimens were obtained from the Skeletal Muscle, Peripheral Nerve, DNA and Cell Line Bank of the Neuromuscular Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan. Written informed consent was obtained and preserved in the original form from all individuals or their caregivers when primary diagnostic procedures were performed, with explicit consent for future use for research purposes, according to the Declaration of Helsinki.
Muscle biopsy analysis
All probands had previously undergone a skeletal muscle biopsy during the period between 1975 and 2014. Muscle samples were frozen in isopentane, cooled in liquid nitrogen, and stained histochemically according to standard procedures [
34]. We reviewed muscle biopsies from cases without a genetic characterization, which included data about α-DG immunohistochemical (IHC) analysis (performed with the clone VIA4-1; Merck Millipore, UK). In muscle samples in which α-DG IHC had not been conducted previously, this study was performed using the antibody cited above if a muscle tissue sample was available.
Molecular analysis
Genomic DNA was extracted from peripheral blood samples according to standard procedures (Flexi Gene DNA Handbook, Qiagen).
ISPD and
GMPPB analysis were performed in patients who exhibited α-DG deficiency at muscle IHC and in patients who had LGMD inclusion criteria, but whose muscle sample was unavailable at the time of this investigation. Mutations in FKRP, the most common LGMD gene involved in α-DG glycosylation [
1,
32,
35], were also ruled out for the first group of patients.
All exons and flanking intronic regions were directly sequenced using an ABI PRISM 3100 XL Genetic Analyzer (Applied Biosystems) and previously published primer [
13,
16].
The pathogenic nature of new mutations was confirmed by screening 100 healthy control subjects. The intronic mutation that leads to abnormal mRNA splicing was investigated through transcript analysis. The parental origin of each mutation was assessed through analysis of parental genomic DNA, when available. Amino acid conservation was confirmed by comparison with sequences from different species.
We isolated mRNA from muscle tissue using Eurozol; the cDNA was produced through reverse transcription polymerase chain reaction (Ready-To-Go RT-PCR kit, Amersham Pharmacia) and analyzed by amplification, cloning, and sequencing. Mutations were named according to the Leiden Muscular Dystrophy database (
www.dmd.nl).
Discussion
Mutations in genes involved in α-DG glycosylation have been associated with a broad spectrum of disorders ranging from severe CMD to milder LGMD phenotypes [
36]. An increasing number of genes involved in these disorders have been discovered recently, enlarging the spectrum of molecular heterogeneity of dystroglycanopathies. Among them,
ISPD and
GMPPB appear to follow
FKRP as relevant genes in the LGMD population, according to earlier reports [
16,
28].
It should be kept in mind that variations in relative frequency for a gene may be population-dependent. This is the case for the
FKRP gene, which accounts for approximately 6 % of LGMD in Italian patients [
1,
23], compared with 19 % among the British population [
37] and 38 % among the Danish population [
24]. Regarding other genes responsible for secondary dystroglycanopathies, only sporadic cases with LGMD phenotype have been reported thus far [
10,
16,
21,
25‐
28]. In particular,
ISPD mutations appear to be responsible for a relatively high proportion of dystroglycanopathies within the most severe clinical spectrum [
13,
14,
17], although they have also been described in a few LGMD cases [
28‐
30].
We analysed a large cohort of Italian LGMD patients in order to estimate the frequency of ISPD and GMPPB mutations and their associated clinical picture. In our LGMD sample, only a small proportion of undiagnosed cases (3/27, 11 %) exhibited reduction of α-DG staining. This finding suggests that if we exclude mutations in the FKRP gene, the other forms associated with defects of α-DG glycosylation are much more rare. Forms of LGMD caused by mutations in ISPD were also rare overall in our cohort, as they represent 0.9 % of genetically defined cases. This proportion increases to 4 % if we consider the group of patients with onset before 10 years of age (which is mainly patients with sarcoglycanopathies, calpainopathies, and LGMD2I). Interestingly, GMPPB mutations were absent from our cohort.
Overall, our patient carrying
ISPD mutations presented a very mild LGMD phenotype compared with other cases described in the literature. He presented with early onset at 5 years of age with abnormal gait on tiptoe, and complained of his first motor limitation (impairment of his ability to run) at 14 years of age. Muscle weakness demonstrated a slowly progressive course with preserved independent ambulation at 42 years of age. Motor performances in the previously reported
ISPD-mutated cases were variable, ranging from supported standing to independent running [
29]; however, loss of independent ambulation (or ambulation for very short distances) has been reported universally in patients who had their last follow-up in adult age [
28,
29]. In particular, the four cases of LGMD without central involvement described by Cirak et al. [
29] presented a more severe Duchenne-like phenotype: they have early onset (1.5 to 3 years), higher creatine kinase levels, and severe progression (3 of 4 were non-ambulant at 12 years of age). Regarding cardiac impairment in patients with mutated
ISPD, one adult patient exhibited a cardiac conduction defect in a likely history of previous myocardic ischemia [
28], and three of four LGMD children described by Cirak et al. exhibited decreased contractility without any conduction defects [
29]. Respiratory impairment has been described in a minority of patients affected with CMD with α-dystroglycan deficiency [
36,
38]; however, decreased pulmonary volumes have been detected among both paediatric and adult patients with
ISPD mutations [
28,
29]. Interestingly, our patient did not exhibit any cardiac or respiratory involvement and he was fully ambulant in his forties, featuring the mildest symptoms on the
ISPD-mutated spectrum reported thus far. Furthermore, our case confirms that absence of cognitive impairment is common in patients with
ISPD mutations and LGMD phenotype [
28,
29].
ISPD pathogenic mutations are generally located in the first exons of the gene. Furthermore, LGMD phenotypes are generally associated with milder mutations, such as missense and in-frame mutations, compared with CMD presentations.
In our patient, we detected two heterozygous mutations located in the first exons, namely one missense substitution and one intronic change that caused alteration of splicing and production of an out-of-frame transcript. The missense mutation had also been described in a foetal presentation with cobblestone lissencephaly, in association with a large deletion of three exons (exons 4 through 6) [
17]. We can argue that our patient’s relatively mild phenotype is correlated with the compensatory action of other enzymes that have been implicated in α-DG glycosylation.
Immunohistochemical analysis revealed a complete absence of α-DG staining in our case. Among dystroglycanopathies, good correlation between α-DG staining and disease course was demonstrated in only a few forms (
POMT1,
POMT2, and
POMGnT1-mutated cases), and is absent in patients with mutations in
FKRP and
FKTN [
39]. As previously reported, α-DG labelling was severely reduced or absent in all patients with mutations in
ISPD, irrespective of clinical severity [
13,
14,
28,
29]. Our case, in whom clinical phenotype did not correlate with the absence of α-DG, further supports this lack of correlation.
Conclusion
Overall, ISPD mutations are a rare cause of LGMD in the Italian population, and account for approximately 1 % of our entire cohort of genetically characterised LGMD (in comparison, FKRP mutations are responsible for up to 8 %). If we consider patients with paediatric onset, the frequency of LGMD2S increases to 4 % of molecularly diagnosed forms. At the time of this writing, no cases with adult onset have been reported. GMPPB mutations were absent in our cohort. Furthermore, reduction of α-DG staining is not frequent among LGMD cases; it accounts for only 11 % of biopsies from genetically undiagnosed patients. However, considering the increasing number of genes involved in α-DG glycosylation and the genetic overlap between congenital muscular dystrophies and LGMD, α-DG IHC analysis should be always performed in cases of undiagnosed LGMD in order to detect reduction of the protein level, which can then be investigated further.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
FM and IC defined the study design and drafted the manuscript. RD conducted the molecular genetic studies. PC conducted the muscle biopsy analysis and immunoassays. RB, MS, and SP participated in patient selection. SC, MM, and NB revised the manuscript. GPC conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.