Functional requirements for fukutin-related protein in the Golgi apparatus

Two forms of congenital muscular dystrophy (CMD), Fukuyama CMD and CMD type 1C (MDC1C) are caused by mutations in the genes encoding two putative glycosyltransferases, fukutin and fukutin-related protein (FKRP). Additionally, mutations in the FKRP gene also cause limb-girdle muscular dystrophy type 2I (LGMD2I), a considerably milder allelic variant than MDC1C. All of these diseases are associated with secondary changes in muscle alpha-dystroglycan expression. To elucidate the function of FKRP and fukutin and examine the effects of MDC1C patient mutations, we have determined the mechanism for the subcellular location of each protein. FKRP and fukutin are targeted to the medial-Golgi apparatus through their N-termini and transmembrane domains. Overexpression of FKRP in CHO cells alters the post-translational processing of alpha- and beta-dystroglycan inhibiting maturation of the two isoforms. Mutations in the DxD motif in the putative active site of the protein or in the Golgi-targeting sequence, which cause FKRP to be inefficiently trafficked to the Golgi apparatus, did not alter dystroglycan processing in vitro. The P448L mutation in FKRP that causes congenital muscular dystrophy changes a conserved amino acid resulting in the mislocalization of the mutant protein in the cell that is unable to alter dystroglycan processing. Our data show that FKRP and fukutin are Golgi-resident proteins and that FKRP is required for the post-translational modification of dystroglycan. Aberrant processing of dystroglycan caused by a mislocalized FKRP mutant could be a novel mechanism that causes congenital muscular dystrophy.     

Asymptomatic carriers for homozygous novel mutations in the FKRP gene: the other end of the spectrum

Autosomal recessive limb-girdle muscular dystrophy linked to 19q13.3 (LGMD2I) was recently related to mutations in the fukutin-related protein gene (FKRP) gene. Pathogenic changes in the same gene were detected in congenital muscular dystrophy patients (MDC1C), a severe disorder. We have screened 86 LGMD genealogies to assess the frequency and distribution of mutations in the FKRP gene in Brazilian LGMD patients. We found 13 Brazilian genealogies, including 20 individuals with mutations in the FKRP gene, and identified nine novel pathogenic changes. The commonest C826A European mutation was found in 30% (9/26) of the mutated LGMD2I alleles. One affected patient homozygous for the FKRP (C826A) mutation also carries a missense R125H change in one allele of the caveolin-3 gene (responsible for LGMD1C muscular dystrophy). Two of her normal sibs were found to be double heterozygotes. In two unrelated LGMD2I families, homozygous for novel missense mutations, we identified four asymptomatic carriers, all older than 20 years. Genotype-phenotype correlation studies in the present study as well as in patients from different populations suggests that the spectrum of variability associated with mutations in the FKRP gene seems to be wider than in other forms of LGMD. It also reinforces the observations that pathogenic mutations are not always determinant of an abnormal phenotype, suggesting the possibility of other mechanisms modulating the severity of the phenotype that opens new avenues for therapeutic approaches.     

Limb-girdle muscular dystrophy 2I: phenotypic variability within a large consanguineous Bedouin family associated with a novel FKRP mutation

Limb-girdle muscular dystrophies (LGMDs) represent a group of diseases characterized mainly by muscle wasting of the upper and lower limbs, with a wide range of clinical severity. The clinical heterogeneity is paralleled by molecular heterogeneity; each of the 10 forms of autosomal-recessive LGMD recognized to date is caused by mutations in a distinct gene. In a large consanguineous Bedouin tribe living in northern Israel, 15 individuals affected by LGMD demonstrate an autosomal recessive pattern of inheritance. A genome-wide screen followed by fine mapping in this family revealed linkage to a region on chromosome 19 harboring the fukutin-related protein gene (FKRP), with a maximal LOD score of 4.8 for D19S902. FKRP, encoding a putative glycosyltransferase, has been implicated in causing congenital muscular dystrophy 1C (MDC1C), and has recently been shown to be mutated in LGMD2I. We identified a novel missense mutation in exon 4 of the FKRP gene in all the patients studied. Although all affected individuals were homozygous for the same mutation, a marked phenotypic variability was apparent within the family. This finding may suggest a role of modifier genes and environmental factors in LGMD2I. Moreover, the demonstration that an identical, novel mutation in the FKRP gene can cause a muscle disease of either a congenital onset or of a later onset within a single family provides clinical support to the molecular evidence, suggesting that MDC1C and LGMD2I are overlapping ends of one and the same entity.     

Abnormalities in alpha-dystroglycan expression in MDC1C and LGMD2I muscular dystrophies

We recently identified mutations in the fukutin related protein (FKRP) gene in patients with congenital muscular dystrophy type 1C (MDC1C) and limb girdle muscular dystrophy type 2I (LGMD2I). The sarcolemma of these patients typically displays an immunocytochemical reduction of alpha-dystroglycan. In this report we extend these observations and report a clear correlation between the residual expression of alpha-dystroglycan and the phenotype. Three broad categories were identified. Patients at the severe end of the clinical spectrum (MDC1C) were compound heterozygote between a null allele and a missense mutation or carried two missense mutations and displayed a profound depletion of alpha-dystroglycan. Patients with LGMD with a Duchenne-like severity typically had a moderate reduction in alpha-dystroglycan and were compound heterozygotes between a common C826A (Leu276Ileu) FKRP mutation and either a missense or a nonsense mutation. Individuals with the milder form of LGMD2I were almost invariably homozygous for the Leu276Ile FKRP mutation and showed a variable but subtle alteration in alpha-dystroglycan immunolabeling. Our data therefore suggest a correlation between a reduction in alpha-dystroglycan, the mutation and the clinical phenotype in MDC1C and LGMD2I which supports the hypothesis that dystroglycan plays a central role in the pathogenesis of these disorders.     

Biochemical and ultrastructural evidence of endoplasmic reticulum stress in LGMD2I

Limb girdle muscular dystrophy type 2I (LGMD2I) is due to mutations in the fukutin-related protein gene (FKRP), encoding a putative glycosyltransferase involved in alpha-dystroglycan processing. To further characterize the molecular pathogenesis of LGMD2I, we conducted a histological, immunohistochemical, ultrastructural and molecular analysis of ten muscle biopsies from patients with molecularly diagnosed LGMD2I. Hypoglycosylation of alpha-dystroglycan was observed in all FKRP-mutated patients. Muscle histopathology was consistent with either severe muscular dystrophy or myopathy with a mild inflammatory response consisting of up-regulation of class I major histocompatibility complex in skeletal muscle fibers and small foci of mononuclear cells. At the ultrastructural level, muscle fibers showed focal thinning of basal lamina and swollen endoplasmic reticulum cisternae with membrane re-arrangement. The pathways of the unfolded protein response (UPR; glucose-regulated protein 78 and CHOP) were significantly activated in LGMD2I muscle tissue. Our data suggest that the UPR response is activated in LGMD2I muscle biopsies, and the observed histopathological and ultrastructural alterations may be related to sarcoplasmic structures involved in FKRP and alpha-dystroglycan metabolism and malfunctioning.     

Post-Natal knockdown of fukutin-related protein expression in muscle by long-termRNA interference induces dystrophic pathology [corrected

Limb-girdle muscular dystrophy 2I (LGMD2I) is caused by mutations in the fukutin-related protein (FKRP) gene. Unlike its severe allelic forms, LGMD2I usually involves slower onset and milder course without defects in the central nervous system. The lack of viable animal models that closely recapitulate LGMD2I clinical phenotypes led us to use RNA interference technology to knock down FKRP expression via postnatal gene delivery so as to circumvent embryonic lethality. Specifically, an adeno-associated viral vector was used to deliver short hairpin (shRNA) genes to healthy ICR mice. Adeno-associated viral vectors expressing a single shRNA or two different shRNAs were injected one time into the hind limb muscles. We showed that FKRP expression at 10 months postinjection was reduced by about 50% with a single shRNA and by 75% with the dual shRNA cassette. Dual-cassette injection also reduced a-dystroglycan glycosylation and its affinity to laminin by up to 70% and induced α-dystrophic pathology, including fibrosis and central nucleation, in more than 50% of the myofibers at 10 months after injection. These results suggest that the reduction of approximately or more than 75% of the normal level of FKRP expression induces chronic dystrophic phenotypes in skeletal muscles. Furthermore, the restoration of about 25% of the normal FKRP level could be sufficient for LGMD2I therapy to correct the genetic deficiency effectively and prevent dystrophic pathology.     

The most common mutation in FKRP causing limb girdle muscular dystrophy type 2I (LGMD2I) may have occurred only once and is present in Hutterites and other populations

Limb girdle muscular dystrophy (LGMD) is common in the Hutterite population of North America. We previously identified a mutation in the TRIM32 gene in chromosome region 9q32, causing LGMD2H in approximately two-thirds of the 60 Hutterite LGMD patients studied to date. A genomewide scan was undertaken in five families who did not show linkage to the LGMD2H locus on chromosome 9. A second LGMD locus, LGMD2I, was identified in chromosome region 19q13.3, and the causative mutation was identified as c.826C>A (L276I), a missense mutation in the FKRP gene. A comparison of the clinical characteristics of the two LGMD patient groups in this population reveals some differences. LGMD2I patients generally have an earlier age at diagnosis, a more severe course, and higher serum creatine kinase (CK) levels. In addition, some of these patients show calf hypertrophy, cardiac symptoms, and severe reactions to general anesthesia. None of these features are present among LGMD2H patients. A single common haplotype surrounding the FKRP gene was identified in the Hutterite LGMD2I patients. An identical core haplotype was also identified in 19 other non-Hutterite LGMD2I patients from Europe, Canada, and Brazil. The occurrence of this mutation on a common core haplotype suggests that L276I is a founder mutation that is dispersed among populations of European origin.     

Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C.

The limb girdle and congenital muscular dystrophies (LGMD and CMD) are characterized by skeletal muscle weakness and dystrophic muscle changes. The onset of symptoms in CMD is within the first few months of life, whereas in LGMD they can occur in late childhood, adolescence or adult life. We have recently demonstrated that the fukutin-related protein gene (FKRP) is mutated in a severe form of CMD (MDC1C), characterized by the inability to walk, leg muscle hypertrophy and a secondary deficiency of laminin alpha2 and alpha-dystroglycan. Both MDC1C and LGMD2I map to an identical region on chromosome 19q13.3. To investigate whether these are allelic disorders, we undertook mutation analysis of FKRP in 25 potential LGMD2I families, including some with a severe and early onset phenotype. Mutations were identified in individuals from 17 families. A variable reduction of alpha-dystroglycan expression was observed in the skeletal muscle biopsy of all individuals studied. In addition, several cases showed a deficiency of laminin alpha2 either by immunocytochemistry or western blotting. Unexpectedly, affected individuals from 15 families had an identical C826A (Leu276Ileu) mutation, including five that were homozygous for this change. Linkage analysis identified at least two possible haplotypes in linkage disequilibrium with this mutation. Patients with the C826A change had the clinically less severe LGMD2I phenotype, suggesting that this is a less disruptive FKRP mutation than those found in MDC1C. The spectrum of LGMD2I phenotypes ranged from infants with an early presentation and a Duchenne-like disease course including cardiomyopathy, to milder phenotypes compatible with a favourable long-term outcome.     

Hutterite brothers both affected with two forms of limb girdle muscular dystrophy: LGMD2H and LGMD2I

Limb girdle muscular dystrophy (LGMD) is very common in the Hutterite population of the North American Prairies. We have recently reported the homozygous c.1459G>A mutation in TRIM32 associated with LGMD2H. We have also identified Hutterite patients with LGMD2I, homozygous for the common c.826C>A mutation in FKRP. To date, all Hutterites with LGMD have been shown to be homozygous for either the TRIM32 or FKRP mutation. We now report a Hutterite family in which both parents and five sons were all found to be homozygous for the TRIM32 mutation. The father had slowly progressive proximal muscle weakness, whereas three sons and their mother, all currently asymptomatic, had normal physical examinations. The remaining two sons (7 and 10 years old), presented with mild decrease in stamina, had normal neuromuscular examinations and were found to be homozygous for the FKRP mutation in addition to the TRIM32 mutation. These two boys do not differ in age at or mode of presentation, physical findings, or serum CK levels compared to age-matched individuals affected with LGMD2I alone. This suggests that the effects of these two mutations are not acting synergistically at this time. It remains to be seen whether there will be signs of interaction between these two mutations as the patients get older.     

Diagnosis and cell-based therapy for Duchenne muscular dystrophy in humans,mice, and zebrafish

The muscular dystrophies are a heterogeneous group of genetically caused muscle degenerative disorders. The Kunkel laboratory has had a longstanding research program into the pathogenesis and treatment of these diseases. Starting with our identification of dystrophin as the defective protein in Duchenne muscular dystrophy (DMD), we have continued our work on normal dystrophin function and how it is altered in muscular dystrophy. Our work has led to the identification of the defective genes in three forms of limb girdle muscular dystrophy (LGMD) and a better understanding of how muscle degenerates in many of the different dystrophies. The identification of mutations causing human forms of dystrophy has lead to improved diagnosis for patients with the disease. We are continuing to improve the molecular diagnosis of the dystrophies and have developed a high-throughput sequencing approach for the low-cost rapid diagnosis of all known forms of dystrophy. In addition, we are continuing to work on therapies using available animal models. Currently, there are a number of mouse models of the human dystrophies, the most notable being the mdx mouse with dystrophin deficiency. These mice are being used to test possible therapies, including stem-cell-based approaches. We have been able to systemically deliver human dystrophin to these mice via the arterial circulation and convert 8% of dystrophin-deficient fibers to fibers expressing human dystrophin. We are now expanding our research to identify new forms of LGMD by analyzing zebrafish models of muscular dystrophy. Currently, we have 14 different zebrafish mutants exhibiting various phenotypes of muscular dystrophy, including muscle weakness and inactivity. One of these mutants carries a stop codon mutation in dystrophin, and we have recently identified another carrying a mutation in titin. We are currently positionally cloning the disease-causative mutation in the remaining 12 mutant strains. We hope that one of these new mutant strains of fish will have a mutation in a gene not previously implicated in human muscular dystrophy. This gene would become a candidate gene to be analyzed in patients which do not carry a mutation in any of the known dystrophy-associated genes. By studying both disease pathology and investigating potential therapies, we hope to make a positive difference in the lives of people living with muscular dystrophy.     

Two endoplasmic reticulum-associated degradation (ERAD) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II)

Dysferlin is a type-II transmembrane protein and the causative gene of limb girdle muscular dystrophy type 2B and Miyoshi myopathy (LGMD2B/MM), in which specific loss of dysferlin labeling has been frequently observed. Recently, a novel mutant (L1341P) dysferlin has been shown to aggregate in the muscle of the patient. Little is known about the relationship between degradation of dysferlin and pathogenesis of LGMD2B/MM. Here, we examined the degradation of normal and mutant (L1341P) dysferlin. Wild-type (wt) dysferlin mainly localized to the ER/Golgi, associated with retrotranslocon, Sec61alpha, and VCP(p97), and was degraded by endoplasmic reticulum (ER)-associated degradation system (ERAD) composed of ubiquitin/proteasome. In contrast, mutant dysferlin spontaneously aggregated in the ER and induced eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation and LC3 conversion, a key step for autophagosome formation, and finally, ER stress cell death. Unlike proteasome inhibitor, E64d/pepstatin A, inhibitors of lysosomal proteases did not stimulate the accumulation of the wt-dysferlin, but stimulated aggregation of mutant dysferlin in the ER. Furthermore, deficiency of Atg5 and dephosphorylation of eIF2alpha, key molecules for LC3 conversion, also stimulated the mutant dysferlin aggregation in the ER. Rapamycin, which induces eIF2alpha phosphorylation-mediated LC3 conversion, inhibited mutant dysferlin aggregation in the ER. Thus, mutant dysferlin aggregates in the ER-stimulated autophagosome formation to engulf them via activation of ER stress-eIF2alpha phosphorylation pathway. We propose two ERAD models for dysferlin degradation, ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II). Mutant dysferlin aggregates on the ER are degraded by the autophagy/lysosome ERAD(II), as an alternative to ERAD(I), when retrotranslocon/ERAD(I) system is impaired by these mutant aggregates.     

The common missense mutation D489N in TRIM32 causing limb girdle muscular dystrophy 2H leads to loss of the mutated protein in knock-in mice resulting in a Trim32-null phenotype

Mutations in tripartite motif protein 32 (TRIM32) are responsible for several hereditary disorders that include limb girdle muscular dystrophy type 2H (LGMD2H), sarcotubular myopathy (STM) and Bardet Biedl syndrome. Most LGMD2H mutations in TRIM32 are clustered in the NHL β-propeller domain at the C-terminus and are predicted to interfere with homodimerization. To get insight into TRIM32's role in the pathogenesis of LGMD2H and to create an accurate model of disease, we have generated a knock-in mouse (T32KI) carrying the c.1465G > A (p.D489N) mutation in murine Trim32 corresponding to the human LGMD2H/STM pathogenic mutation c.1459G > A (p.D487N). Our data indicate that T32KI mice have both a myopathic and a neurogenic phenotype, very similar to the one described in the Trim32-null mice that we created previously. Analysis of Trim32 gene expression in T32KI mice revealed normal mRNA levels, but a severe reduction in mutant TRIM32 (D489N) at the protein level. Our results suggest that the D489N pathogenic mutation destabilizes the protein, leading to its degradation, and results in the same mild myopathic and neurogenic phenotype as that found in Trim32-null mice. Thus, one potential mechanism of LGMD2H might be destabilization of mutated TRIM32 protein leading to a null phenotype.     

Myopathy phenotype of transgenic mice expressing active site-mutated inactive p94 skeletal muscle-specific calpain, the gene product responsible for limb girdle muscular dystrophy type 2A

A defect of the gene for p94 (calpain 3), a skeletal muscle-specific calpain, is responsible for limb girdle muscular dystrophy type 2A (LGMD2A), or 'calpainopathy', which is an autosomal recessive and progressive neuromuscular disorder. To study the relationships between the physiological functions of p94 and the etiology of LGMD2A, we created transgenic mice that express an inactive mutant of p94, in which the active site Cys129 is replaced by Ser (p94:C129S). Three lines of transgenic mice expressing p94:C129S mRNA at various levels showed significantly decreased grip strength. Sections of soleus and extensor digitorum longus (EDL) muscles of the aged transgenic mice showed increased numbers of lobulated and split fibers, respectively, which are often observed in limb girdle muscular dystrophy muscles. Centrally placed nuclei were also frequently found in the EDL muscle of the transgenic mice, whereas wild-type mice of the same age had almost none. There was more p94 protein produced in aged transgenic mice muscles and it showed significantly less autolytic degradation activity than that of wild-type mice. Although no necrotic-regenerative fibers were observed, the age and p94:C129S expression dependence of the phenotypes strongly suggest that accumulation of p94:C129S protein causes these myopathy phenotypes. The p94:C129S transgenic mice could provide us with crucial information on the molecular mech-anism of LGMD2A.     

Loss of calpain-3 autocatalytic activity in LGMD2A patients with normal protein expression

The diagnosis of limb girdle muscular dystrophy (LGMD) type 2A (due to mutations in the gene encoding for calpain-3) is currently based on protein analysis, but mutant patients with normal protein expression have also been identified. In this study we investigated 150 LGMD patients with normal calpain-3 protein expression, identified gene mutations by an allele-specific polymerase chain reaction test, and analyzed the mutant calpain-3 catalytic activity. Four different mutations were found in eight patients (5.5%): a frame-shifting deletion (550 A del) and three missense (R490Q, R489Q, R490W). Patients with normal calpain-3 protein expression on Western blot are a considerable proportion (20%) of our total LGMD2A population. While in control muscle the calpain-3 Ca(++)-dependent autocatalytic activity was evident within 5 minutes and was prevented by ethylene diaminetetraacetic acid, in all mutant patient samples the protein was not degraded, indicating that the normal autocatalytic function had been lost. By this new functional test, we show that conventional protein diagnosis fails to detect some mutant proteins, and prove the pathogenetic role of R490Q, R489Q, R490W missense mutations. We suggest that these mutations impair protein activity by affecting interdomain protein interaction, or reduce autocatalytic activity by lowering the Ca(++) sensitivity.     

Mutations that impair interaction properties of TRIM32 associated with limb-girdle muscular dystrophy 2H

TRIM32 belongs to a large family of proteins characterized by a tripartite motif, possibly involved in the ubiquitination process, acting as an E3 ligase. In addition, TRIM32 has six NHL repeats with putative interaction properties. A homozygous mutation at the third NHL repeat (D487N) has been found in patients with limb girdle muscular dystrophy 2H (LGMD2H). This mutation was only identified in the inbred Manitoba Hutterite or their descendants. Interestingly, a mutation in the B-box domain of TRIM32 cosegregates with Bardet-Biedl syndrome type 11 (BBS11). The signs of BBS11 include obesity, pigmentary and retinal malformations, diabetes, polydactyly, and no muscular dystrophy, suggesting an alternative disease mechanism. We aim to ascertain whether D487N is the only pathological LGMD2H allele, limited to Hutterites. We studied the TRIM32 gene in 310 LGMD patients with no mutations at the other known loci. We identified four patients with novel mutated alleles. Two mutations were homozygous and missing in controls. These mutations also clustered at the NHL domain, suggesting that a specific (interaction) property might be abolished in LGMD2H patients. No mutations were found at the B-box region where the BBS11 mutation is found. We tested TRIM32 and its mutants by yeast-two-hybrid assay, developing an interaction test to validate mutations. All LGMD2H mutants, but not the BBS11, lost their ability to self-interact. The interaction of TRIM32 mutants with E2N, a protein involved in the ubiquitination process, was similarly impaired. In conclusion, the mutations here reported may cause muscular dystrophy by affecting the interaction properties of TRIM32.     

Glyc-O-genetics of Walker-Warburg syndrome

Walker-Warburg syndrome (WWS) is the most severe of a group of multiple congenital anomaly disorders known as the cobblestone lissencephalies. These are characterized by congenital muscular dystrophy in conjunction with severe brain malformation and ocular abnormalities. In the last 3 years, important progress has been made towards the elucidation of the genetic causes of these disorders. Mutations in three genes, POMT1, fukutin and FKRP, have been described for WWS, which together account for approximately 20% of patients with Walker-Warburg. It has become evident that some of the underlying genes may cause a broad spectrum of phenotypes, ranging from limb girdle muscular dystrophy type 2I to WWS. In some cases, a genotype-phenotype correlation can be recognized. In line with the known or proposed functions of the resolved genes, all patients with cobblestone lissencephaly show defects in the O-linked glycosylation of the glycoprotein alpha-dystroglycan. Perhaps, the missing genes underlying the remainder of the unexplained WWS patients have also to be sought in the pathways involved in O-linked protein glycosylation.     

Transgenic mice expressing the myotilin T57I mutation unite the pathology associated with LGMD1A and MFM

Myotilin is a muscle-specific Z-disc protein with putative roles in myofibril assembly and structural upkeep of the sarcomere. Several myotilin point mutations have been described in patients with limb-girdle muscular dystrophy type 1A (LGMD1A), myofibrillar myopathy (MFM), spheroid body myopathy (SBM), three similar adult-onset, progressive and autosomal dominant muscular dystrophies. To further investigate myotilin's role in the pathogenesis of these muscle diseases, we have characterized three independent lines of transgenic mice expressing mutant (T57I) myotilin under the control of the human skeletal actin promoter. Similar to LGMD1A and MFM patients, these mice develop progressive myofibrillar pathology that includes Z-disc streaming, excess myofibrillar vacuolization and plaque-like myofibrillar aggregation. These aggregates become progressively larger and more numerous with age. We show that the mutant myotilin protein properly localizes to the Z-disc and also heavily populates the aggregates, along with several other Z-disc associated proteins. Whole muscle physiological analysis reveals that the extensor digitorum longus muscle of transgenic mice exhibits significantly reduced maximum specific isometric force compared with littermate controls. Intriguingly, the soleus and diaphragm muscles are spared of any abnormal myopathology and show no reductions in maximum specific force. These data provide evidence that myotilin mutations promote aggregate-dependent contractile dysfunction. In sum, we have established a promising patho-physiological mouse model that unifies the phenotypes of LGMD1A, MFM and SBM.