BMD Patients Research Articles

Cardiac Involvement in Patients With Muscular Dystrophies

Muscular dystrophy (MD) connotes a heterogeneous group of inherited disorders characterized by progressive wasting and weakness of the skeletal muscles. In several forms of MD, cardiac dysfunction occurs, and cardiac disease may even be the predominant manifestation of the underlying genetic myopathy. Cardiologists may be unfamiliar with these diseases owing to their low incidence; also, significant advances in respiratory care have only recently unmasked cardiomyopathy as a significant cause of death in MD.1 Early detection of MD-associated cardiomyopathy is important, because institution of cardioprotective medical therapies may slow adverse cardiac remodeling and attenuate heart failure symptoms in these patients.2–6 Although ECG and echocardiography are typically advocated for screening,7,8 cardiovascular magnetic resonance (CMR) has shown promise in revealing early cardiac involvement when standard cardiac evaluation is unremarkable.9,10 This review will focus on 4 groups of skeletal muscle disease most commonly associated with cardiac complications (the Table): (1) dystrophin-associated diseases such as Duchenne and Becker (DMD and BMD, respectively), (2) Emery-Dreifuss MD (EDMD), (3) limb-girdle MD (LGMD), and (4) myotonic dystrophy (DM). View this table: Table. Characteristics of the Types of MD ### Molecular and Genetic Features DMD and BMD are X-linked disorders affecting the synthesis of dystrophin, a large, sarcolemmal protein that is absent in DMD11 and reduced in amount or abnormal in size in BMD patients.12 Dystrophin provides the connection between a large, multimeric complex of glycoproteins in the muscle cell membrane (termed the dystrophin-glycoprotein complex) and intracellular actin filaments (Figure 1), thereby transmitting forces generated by sarcomere contraction to the extracellular matrix.13,14 Correlations between dystrophin mutations and the onset of cardiomyopathy have been noted15; some mutations result in only cardiomyopathy without skeletal myopathy.16 Other proteins not shown in Figure 1 that are particularly involved in both inside-out and outside-in transmission …

Cardiovascular magnetic resonance imaging evaluation of two families with Becker muscular dystrophy

In this study we evaluated two families with Becker muscular dystrophy and although the patients were completely asymptomatic and with normal ECG and echocardiogram, their left ventricular function was abnormal and the presence of subepicardial scar tissue was identified in the majority of them. The latter was also documented in one of the mothers, who had normal systolic function and was free of symptoms. Cardiac evaluation with cardiovascular magnetic resonance is sensitive enough to detect abnormal findings in BMD patients, missed by conventional echocardiography. Due to its ability to perform tissue characterization non-invasively, cardiovascular magnetic resonance can detect silent myocardial lesions in patients with Becker muscular dystrophy.

G.P.13.09 Muscle biopsy mRNA-based analysis of point mutations in DMD gene in Spanish patients

Duchenne and Becker muscular dystrophies (DMD/BMD) are the most common X-linked disorders, causing progressive weakness. Both muscular disorders are produced by mutations in the dystrophin gene. The most common mutations are large intragenic deletions and duplications that account for 75% of all cases. In DMD patients the majority of cases the DMD deletions or duplications are frameshift type, and in BMD patients the deletions or duplications are in-frame type. The remaining cases are due to small mutations consisting in nonsense mutations, missense mutations, splicing mutations, frameshift small deletions or insertions and midintronic insertions.

Genotype-phenotype analysis in 2,405 patients with a dystrophinopathy using the UMD-DMD database: a model of nationwide knowledgebase

UMD-DMD France is a knowledgebase developed through a multicenter academic effort to provide an up-to-date resource of curated information covering all identified mutations in patients with a dystrophinopathy. The current release includes 2,411 entries consisting in 2,084 independent mutational events identified in 2,046 male patients and 38 expressing females, which corresponds to an estimated number of 39 people per million with a genetic diagnosis of dystrophinopathy in France. Mutations consist in 1,404 large deletions, 215 large duplications, and 465 small rearrangements, of which 39.8% are nonsense mutations. The reading frame rule holds true for 96% of the DMD patients and 93% of the BMD patients. Quality control relies on the curation by four experts for the DMD gene and related diseases. Data on dystrophin and RNA analysis, phenotypic groups, and transmission are also available. About 24% of the mutations are de novo events. This national centralized resource will contribute to a greater understanding of prevalence of dystrophinopathies in France, and in particular, of the true frequency of BMD, which was found to be almost half (43%) that of DMD. UMD-DMD is a searchable anonymous database that includes numerous newly developed tools, which can benefit to all the scientific community interested in dystrophinopathies. Dedicated functions for genotype-based therapies allowed the prediction of a new multiexon skipping (del 45-53) potentially applicable to 53% of the deleted DMD patients. Finally, such a national database will prove to be useful to implement the international global DMD patients' registries under development.

Screening of deletions and RFLP analysis in Turkish DMD/BMD families by PCR.

We have screened 76 DMD and 5 BMD patients for deletions, using two separate Multiplex gene amplification systems. The use of both systems together revealed deletions in 52% of the cases in the Turkish population. The majority of these deletions (33/37) were found to be localized within the central region of the dystrophin gene. The remaining deletions were mapped to the proximal hotspot. Deletion end-points were identified by PCR and/or by Southern blot analysis with cDNA probes, and exceptions to the Open Reading Frame (ORF) hypothesis are discussed. PCR-based techniques to screen the pERT87.15/XmnI, pERT87.15/BamHI, and pERT87.8/TaqI polymorphisms were used for linkage analysis in the Turkish DMD/BMD families, and approximately 70% of the mothers at risk were found to be informative for at least one of these polymorphisms studied.

PTH Circadian Rhythm and PTH Target-Organ Sensitivity Is Altered in Patients With Adult Growth Hormone Deficiency With Low BMD

AGHD is associated with osteoporosis. We examined PTH circadian rhythmicity and PTH target-organ sensitivity in 23 patients with AGHD with low BMD and 20 patients with AGHD with normal BMD. Patients with low BMD had a blunted nocturnal rise in PTH concentration and reduced PTH target-organ sensitivity compared with patients with normal BMD; these factors may be important in the pathogenesis of AGHD-related osteoporosis. Adult growth hormone deficiency (AGHD) is associated with decreased BMD. Reduced parathyroid gland sensitivity to changes in calcium and reduced PTH target-organ sensitivity may underlie the pathogenesis of AGHD-related osteoporosis. A blunted nocturnal PTH rise has been reported in AGHD and may contribute to the reduction in BMD. We examined the difference in PTH concentration and markers of bone metabolism in patients with AGHD with normal and low BMD. Forty-three patients with AGHD consented to the study. Twenty-five patients were growth hormone (GH) naïve (GH-N, 13 had BMD femoral neck or lumbar spine T-score < -1.0), and 18 patients had received GH for >2 yr (GH-R, 10 had BMD T-score < -1.0). Patients were hospitalized for 24 h, where blood samples were collected every 0.5 h and urine samples were collected every 3 h for PTH, calcium, phosphate, NcAMP, 1,25-dihydroxyvitamin D [1,25(OH)(2)D], type-I collagen beta C-telopeptide (betaCTX), and procollagen type-I amino-terminal propeptide (PINP). Serum calcium was adjusted for albumin (ACa). Low BMD GH-N and GH-R patients exhibited a reduced nocturnal rise in PTH concentration compared with patients with normal BMD (p < 0.001). GH-N low BMD patients had significantly higher 24-h mean PTH (p < 0.001) than GH-N normal BMD patients, with significantly lower 24-h mean NcAMP, ACa, and 1,25(OH)(2)D (p < 0.01), suggesting a reduction in renal PTH sensitivity. GH-R low BMD patients had significantly lower 24-h mean PTH, NcAMP, ACa, and 1,25(OH)(2)D (p < 0.01) than GH-R normal BMD patients, suggesting reduced renal PTH action. Lower PTH concentration in the presence of lower ACa may reflect reduced sensitivity of the parathyroid calcium-sensing receptor to changes in ACa concentration in the GH-R low BMD patients. Low BMD in GH-N and GH-R AGHD patients may be a consequence of abnormalities in PTH circadian rhythmicity together with reduced parathyroid gland and target-organ sensitivity. Further studies are needed to determine the potential benefit of therapeutic manipulation of PTH rhythmicity and sensitivity on BMD.

Postmenopausal women with osteoporosis asymptomatic vertebral fracture prevalence and vitamin D deficit

Marked suppression of bone turnover by bisphosphonates is associated with increased bone microdamage accumulation in animal models. The purpose of this study was to test the hypothesis that long-term treatment with alendronate (ALN) results in accumulation of microdamage in bone in women after menopause. Sixty-six postmenopausal women with osteoporosis (mean age of 68.0 years and mean BMD T-score of − 1.7 at total hip and − 2.8 at lumbar spine; 62% with prevalent fractures) were evaluated in this cross-sectional analysis. Thirty-eight had been treated previously with ALN (10 mg/day or 70 mg/week for a mean duration of 63.6 months) while twenty-eight were treatment naive (TN). Without adjustments, crack surface density (Cr.S.Dn) and crack density (Cr.Dn) were not different between ALN and TN patients. After adjustment for potential confounders (age, prevalent fractures, femoral neck BMD, activation frequency and center), Cr.Dn was elevated in ALN patients (P = 0.028 and P = 0.069 for Cr.S.Dn). In ALN patients only, lower femoral neck BMD (Cr.S.Dn, r = − 0.58, P = 0.003; Cr.Dn, r = − 0.54, P = 0.005) and increased age (Cr.S.Dn, r = 0.43, P = 0.03; Cr.Dn, r = 0.43, P = 0.03) were associated with microdamage accumulation. Among potential confounders, femoral neck BMD was the only independent predictor for these correlations (P = 0.04 for Cr.Dn and P = 0.03 for Cr.S.Dn). We conclude that increased microdamage accumulation may occur in low BMD patients treated with alendronate.

Low bone mineral density is associated with bone microdamage accumulation in postmenopausal women with osteoporosis

Marked suppression of bone turnover by bisphosphonates is associated with increased bone microdamage accumulation in animal models. The purpose of this study was to test the hypothesis that long-term treatment with alendronate (ALN) results in accumulation of microdamage in bone in women after menopause. Sixty-six postmenopausal women with osteoporosis (mean age of 68.0 years and mean BMD T-score of −1.7 at total hip and −2.8 at lumbar spine; 62% with prevalent fractures) were evaluated in this cross-sectional analysis. Thirty-eight had been treated previously with ALN (10 mg/day or 70 mg/week for a mean duration of 63.6 months) while twenty-eight were treatment naive (TN). Without adjustments, crack surface density (Cr.S.Dn) and crack density (Cr.Dn) were not different between ALN and TN patients. After adjustment for potential confounders (age, prevalent fractures, femoral neck BMD, activation frequency and center), Cr.Dn was elevated in ALN patients (P=0.028 and P=0.069 for Cr.S.Dn). In ALN patients only, lower femoral neck BMD (Cr.S.Dn, r=−0.58, P=0.003; Cr.Dn, r=−0.54, P=0.005) and increased age (Cr.S.Dn, r=0.43, P=0.03; Cr.Dn, r=0.43, P=0.03) were associated with microdamage accumulation. Among potential confounders, femoral neck BMD was the only independent predictor for these correlations (P=0.04 for Cr.Dn and P=0.03 for Cr.S.Dn). We conclude that increased microdamage accumulation may occur in low BMD patients treated with alendronate.

Molecular deletion patterns in Duchenne and Becker muscular dystrophy patients from KwaZulu Natal

There exists much phenotypic heterogeneity in Duchenne muscular dystrophy and its allelic variant, Becker muscular dystrophy. The molecular findings on 53 patients with Duchenne and 15 patients with Becker type muscular dystrophy in KwaZulu Natal, South Africa are reported. Multiplex PCR was performed using primers targeting 18 hot-spot exons throughout the dystrophin gene. Analysis of the multiplex PCR data revealed that 39/68 (57.0%) patients included in the study showed a deletion (33 DMD and 6 BMD patients). Twenty-five patients were Black, 4 were White and 10 were Indian. Using the Chamberlain and Beggs multiplex PCR assays, the region of the genome most frequently affected by a deletion includes exons 47–51. The distal region of the dystrophin gene was most frequently affected by the deletion in both Black and Indian patients. There were too few White patients for conclusions to be drawn concerning the most frequently affected part of the gene. Although the numbers are insufficient to determine whether ethnic differences are present, the Chamberlain and Beggs multiplex PCR assays detect deletions with the same frequency in South African DMD/BMD patients as that reported in the literature.

Exploring the Frontiers of Therapeutic Exon Skipping for Duchenne Muscular Dystrophy by Double Targeting within One or Multiple Exons

Through antisense-induced single-, double-, and multiexon skipping, we have previously demonstrated restoration of dystrophin expression in Duchenne muscular dystrophy (DMD) patient-derived muscle cells in vitro. In this study we further explored the frontiers of this strategy by using specific combinations of 2′-O-methyl phosphorothioate antisense oligonucleotides (AONs) targeting either one or multiple exons. We show that skipping efficiencies may indeed be improved by targeting two putative splicing regulatory sequences within one exon. In particular, such double targeting was effective for the thus far “unskippable” exons 47 and 57. We previously reported the feasibility of multiexon skipping spanning exon 45 to exon 51, using a combination of AONs targeting both outer exons (45 and 51). This would be applicable to 13% of all DMD patients. We here explored the frontiers of multiexon skipping both to increase the number of patients that can be treated with the same set of AONs and to mimic large deletions found in relatively mildly affected BMD patients. We aimed at inducing larger multiexon-skipping stretches, such as exons 17–51, exons 42–55, and exons 45–59. However, this appeared complicated and may be dependent on cotranscriptional splicing and the size of the flanking introns.

Nitric oxide synthase in muscular dystrophies: a re-evaluation

Duchenne and Becker muscular dystrophies (DMD and BMD) are associated with decreased total nitric oxide (NO). However, mechanisms leading to NO deficiency with consequent muscle-cell degeneration remain unknown. To address this issue, we examined skeletal muscles of DMD and BMD patients for co-expression of NO synthase (NOS) with nitrotyrosine and transcription factor CREB, as well as with enzymes engaged in NO signaling. Employing immunocytochemical labeling, Western blotting and RT-PCR, we found that, in contrast to the most commonly accepted view, neuronal NOS was not restricted to the sarcolemma and that muscles of DMD and BMD patients retained all three NOS isoforms with an up-regulation of the inducible NOS isoform, CREB and nitrotyrosine. We suggest that enhanced nitrotyrosine immunostaining in muscle fibers as well as in the vasculature of DMD and BMD specimens reflects massive oxidative stress, resulting in withdrawal of NO from its regular physiological course via the scavenging actions of superoxides.

Analysis of Dystrophin Gene Deletions Indicates that the Hinge III Region of the Protein Correlates with Disease Severity

We have investigated the frequency of deletions in the dystrophin gene in 108 unrelated Duchenne and Becker muscular dystrophy (DMD/BMD) patients from southern Italy (DMD, n. 47; BMD, n. 61) and identified 89 deletions. The de novo mutation rate (about 30%), and the preferentially maternal origin of deletional mutations, analysed in families in which the maternal grandparents were available or their haplotypes could be unequivocally reconstructed, are in agreement with data reported for other populations. The correlation between BMD phenotype and type of deletion suggests that, in the distal rod domain region, the deletion size may not be as crucial as the particular combination of missing exons. In fact, we provide immunohistochemical and clinical evidence that in-frame deletion of the hinge III region in the distal rod domain results in a milder phenotype as compared with shorter deletions that do not include the hinge III region. Our data obtained in BMD patients, by confirming inferences arising from minigene transfection experiments in mdx mice, represent an important contribution to gene therapy approaches.

Analysis of Dystrophin Gene Deletions Indicates that the Hinge III Region of the Protein Correlates with Disease Severity

SummaryWe have investigated the frequency of deletions in the dystrophin gene in 108 unrelated Duchenne and Becker muscular dystrophy (DMD/BMD) patients from southern Italy (DMD, n. 47; BMD, n. 61) and identified 89 deletions. The de novo mutation rate (about 30%), and the preferentially maternal origin of deletional mutations, analysed in families in which the maternal grandparents were available or their haplotypes could be unequivocally reconstructed, are in agreement with data reported for other populations. The correlation between BMD phenotype and type of deletion suggests that, in the distal rod domain region, the deletion size may not be as crucial as the particular combination of missing exons. In fact, we provide immunohistochemical and clinical evidence that in‐frame deletion of the hinge III region in the distal rod domain results in a milder phenotype as compared with shorter deletions that do not include the hinge III region. Our data obtained in BMD patients, by confirming inferences arising from minigene transfection experiments in mdx mice, represent an important contribution to gene therapy approaches.

Duchenne's and Becker's muscular dystrophy: analysis of phenotype-genotype correlation in 28 patients]

Duchenne's and Becker's muscular dystrophy (DMD & BMD) is a X linked disease caused by mutations in the dystrophic gene. DMD is the malign form of the disease, which significantly shortens the lifetime of the patient, while BMD has late onset with slow progression. Sixty five percent of DMD and BMD cases are caused by deletion of one or more exons in the dystrophic gene, while duplications cause these diseases in 6 to 7% of the cases. There are two hot spots for deletions and duplications. These are exons in the proximal part of the gene (3rd to 18th) and exons of a distal part of the gene (45th to 52nd). The remaining 30% of DMD and BMD cases are caused by point mutations, small deletions or inversions in the dystrophic gene. The correlation between the severity of the disease and the position of deletion shows that most of the out of frame deletions cause DMD phenotype, while in frame deletions result in BMD phenotype. We report on the results of 28 non-related DMD and BMD patients. In 57% of cases deletions were detected and all were found in the distal hot spot of the gene. These results suggest that in most of the cases, out of frame deletions produce DMD phenotype while in frame deletions result in BMD phenotype. This is in compliance with data from literature.

Bone turnover in hyperthyroidism before and after thyrostatic management.

Hyperthyroidism is associated with enhanced osteoblastic and osteoclastic activity, and patients frequently have low bone mineral density and high bone turnover. The aim of this study was to examine the bone formation and resorption markers trend in 12 female patients, before and after normalization of thyroid activity. The following measurements were made at baseline and 1 and 6 months after hormone normalization induced by methimazole treatment: total alkaline phosphatase (ALP), bone alkaline phosphatase (BALP), collagen type C-terminal propeptide (PICP), osteocalcin (BGP), telopeptide (ICTP), urinary-hydroxyproline/urinary creatinine (uOHP/uCreat), urinary calcium/urinary creatinine (uCa/uCreat) and deoxypyridinoline crosslinks (D-Pyr). Compared with controls, all of these parameters were significantly increased (ALP p = 0.014; BALP p = 0.0001; PICP p = 0.013; BGP p = 0.009; ICTP p = 0.0001; uOHP/uCreat p = 0.002; uCa/uCreat p = 0.044; crosslinks p = 0.0001). After treatment the values of ALP, BALP and PICP in hyperthyroid patients showed an initial slight increase and then a significant downwards trend (ALP p = 0.008, BAP p = 0.001, PICP p = 0.026). Furthermore, resorption markers showed a significant decrease (uOHP/ uCreat p < 0.005 and D-Pyr p < 0.008). As regards lumbar BMD patients, measurements were significantly reduced in comparison with the control group (p = 0.005). Six months after serum thyroid hormones level normalization, we observed a significant increase (p=0.014 vs baseline). Both neoformation and resorption markers are useful to assess pathological bone turnover and bone involvement in hyperthyroidism. They could also be employed to monitor the effect of antithyroid treatment on bone and to indicate if bone antiresorption therapy should be considered.

Muscular dystrophy: from gene to patient.

Altered expression of dystrophin led to a reduction in the myocardial PCr to ATP ratio in MD carriers and BMD patients. Decreased myocardial PCr was also observed in the mouse model of MD, themdx mouse, which had decreased myocardial glucose uptake in response to insulin and during ischaemia, with increased susceptibility to ischaemic damage. However, utrophin transgene expression corrected the functional and metabolic abnormalities in the dystrophin-deficient mouse heart. To our knowledge, this is the first report of beneficial effects resulting from the expression of a dystrophin-related transgene in heart, making the findings important for the use of gene therapy in the treatment of cardiomyopathy in dystrophic patients.

Detection of gene deletions in Chinese patients with Duchenne/Becker muscular dystrophy using cDNA probes and the polymerase chain reaction method

One hundred thirty-eight patients with Duchenne/Becker muscular dystrophy (DMD/BMD) were screened with complete cDNA probes and the multiplex polymerase chain reaction (mPCR) amplification of 18 pairs of oligonucleotide primers. Eighty-six deletions and 4 duplications were detected, the deletion frequency being 62.3%, Eighty-two deletions were detected with the two sets of primers described by Chamberlain et al. and Beggs et al, which was 95.4% of deletions detected by complete cDNA probes. Consistent with the deletion locations described previously, the deletions of dystrophin gene in Chinese individuals are clustered mainly in two high-frequency deletion regions of exons 44–52 (68.6%) of 3′ side of the gene central regions and exons 1–19 (26.7%) in the 5′ side. The distribution of deletions in dystrophin gene is associated with the phenotype of DMD/BMD. In the 25 cases with in-frame deletions, 15 deletions located in the region of exons 2–47 were milder BMD and intermediate patients, as the location of deletions was not the important region of the dystrophin gene.

Mutation analysis of the dystrophin gene in Southern French DMD or BMD families: from Southern blot to protein truncation test.

Data from 6 years of experience in molecular diagnosis of Duchenne (DMD) and Becker (BMD) muscular dystrophy in Southern France are reported. DMD and BMD patients have been extensively analyzed for deletions and for point mutations in the dystrophin gene. By scanning the whole coding sequence as reverse-transcribed from lymphocytes or muscular RNA by the protein truncation test, we have reached a minimum of an 86% detection rate for point mutations responsible for DMD; these mutations consist of nonsense, frameshifting, and splicing mutations. Four of 12 small alterations identified in our sample are novel and described in this study. We also present an improved protocol for the automated detection of fluorescently labeled duplex polymerase chain reactions of six known intragenic microsatellites (Dys II, TG 15, STRs 44, 45, 49, and 50). Accurate sizing of the alleles at each locus was performed, and we elucidated the sequence of several repeat units. Allele frequencies at each of the six microsatellite loci and at one restriction fragment length polymorphism site (intron 16/TaqI) were defined in a sample of normal, DMD, and BMD X chromosomes from Southern France. The determination of the grandparental origin of either deletions or point mutations revealed differences depending on the type of the mutation, with most of the deletions occurring in oogenesis and most of the point mutations occurring in spermatogenesis.

How the magnitude of clinical severity and recurrence risk affects reproductive decisions in adult males with different forms of progressive muscular dystrophy.

The reproductive history of 177 male patients affected with Becker (BMD) (n=69), limb-girdle (LGMD) (n=54), and facioscapulohumeral (FSHMD) (n=54) muscular dystrophy (MD) was analysed according to severity of the disease...

Novel mutations and polymorphisms in the human dystrophin gene detected by double-strand conformation analysis.

Recently we reported the possibility of detectingthe presence of single base substitutions in PCRamplicons by double-stranded conformation anal-ysis (DSCA) (Saad et al., 1994). The method ex-ploits the property of double-strand DNA in aqueoussolution to show an intrinsic curvature, which is de-pendent on its nucleotide sequence (Bolshoy et al.,1991). A single base substitution induces a modifi-cation of the curvature of the double-helix and aconsequent variation in the friction encountered bythe DNA in a porous gel. This, in turn, would pro-duce an electrophoretic mobility shift in polyacryla-mide nondenaturing gels. DSCA already proved tobe able to detect new mutations in the humandystrophin gene (Saad et al., 1994). By using thismethod, a mutation screening was performed onDNAs from 50 DMD and 28 BMD patients in whichno intragenic alterations were detected, and on 20DNAs from subjects showing hyperCKemia.