Dystrophin immunofluorescence pattern in manifesting and asymptomatic carriers of Duchenne's and Becker muscular dystrophies of different ages.

In order to investigate if the same apparent decrease in dystrophin negative fibers with aging observed in mouse mdx female heterozygotes also occurs in carriers of the DMD and BMD gene, we have studied the muscle of 29 DMD carriers (19 adults and 10 young daughters of obligate carriers, including 3 manifesting carriers) and 5 adult asymptomatic heterozygotes for Becker dystrophy (BMD). All young DMD possible carriers and 11 of 24 adult DMB/BMD heterozygotes had increased serum enzymes activities. A population of dystrophin negative fibers, more evident with the use of the C-terminal antibody, was seen in the three manifesting and in a 9-yr-old possible DMD carrier. In the remaining females, a positive immunohistochemical pattern of dystrophin, which did not differ from normal controls, was observed. Our results suggest that: (1) the increased population of dystrophin negative fibers reported in young mdx female heterozygotes was not seen in young DMD carriers, aged 6-17 yr; and (2) abnormalities in dystrophin immunostaining are not easily observed and are more frequent in manifesting carriers, when the muscle is grossly altered.

A novel stop codon mutation in the PMP22 gene associated with a variable phenotype.

The most frequent inherited peripheral neuropathy is the peripheral myelin protein 22 (PMP22) gene related disease. Duplication, deletion, and point mutations in that gene are associated with phenotypic variability. Here we report a family carrying a novel mutation in the PMP22 gene (c. 327C>A), which results in a premature stop codon (Cys109stop). The family members who carry this mutation have a Charcot-Marie-Tooth type 1 variable phenotype, ranging from asymptomatic to severely affected. These findings suggest that the fourth transmembrane domain of the PMP22 gene may play an important role, although the intrafamilial clinical variability reinforces the observation that pathogenic mutations are not always phenotype determinant and that other factors (genetic or epigenetic) modulate the severity of the clinical course.

Sarcolemmal distribution of abnormal dystrophin in Xp21 carriers.

Some Becker muscular dystrophy carriers, related to patients with specific DNA deletions, demonstrate both normal and abnormally sized dystrophin bands through qualitative Western blot analysis. The purpose of the present investigation was to assess the sarcolemmal distribution of the altered dystrophin in such carriers. Fibres expressing the normal or deleted dystrophin were identified using specific antibodies which reacted with epitopes from within the deleted region. No negative fibres or patchy immunostaining could be seen when sections from four carriers were labelled with either antibodies (C-terminal and corresponding to the deleted region), although a significant amount of abnormal dystrophin was present in their muscle (as seen on blots). Thus, we were able to confirm that in a proportion of the myonuclei, the defective allele was present on the active X chromosome. Our results suggest that the two types of nuclei were randomly distributed, resulting in normal and abnormal dystrophin molecules which were so intimately mixed that dystrophin-incompetent fibres could not be distinguished in the skeletal muscle from the Xp21 carriers.

Dysferlin protein analysis in limb-girdle muscular dystrophies.

Dysferlin is the protein product of the DYSF gene mapped at 2p31, which mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. To date, nine autosomal recessive forms (AR-LGMD) have been identified: four genes, which code for the sarcoglycan glycoproteins, are associated with both mild and severe forms, the sarcoglycanopathies (LGMD2C, 2D, 2E and 2F). The other five forms, usually causing a milder phenotype are LGMD2A (calpain 3), LGMD2B (dysferlin), LGMD2G (telethonin), LGMD2H (9q31-11), and LGMD21 (19q13.3). We studied dysferlin expression in a total of 176 patients, from 166 LGMD families: 12 LGMD2B patients, 70 with other known forms of muscular dystrophies (LGMD2A, sarcoglycanopathies, LGMD2G), in an attempt to assess the effect of the primary gene-product deficiency on dysferlin. In addition, 94 still unclassified LGMD families were screened for dysferlin deficiency. In eight LGMD2B patients from five families, no dysferlin was observed in muscle biopsies, both through immunofluorescence (IF) and Western blot methodologies, while in two families, a very faint band was detected. Both patterns, negative or very faint bands, were concordant in patients belonging to the same families, suggesting that dysferlin deficiency is specific to LGMD2B. Myoferlin, the newly identified homologue of dysferlin was studied for the first time in LGMD2B patients. Since no difference was observed between patients mildly and severely affected, this protein do not seem to modify the phenotype in the present dysferlin-deficient patients. Dystrophin, sarcoglycans, and telethonin were normal in all LGMD2B patients, while patients with sarcoglycanopathies (2C, 2D, and 2E), LGMD2A, LGMD2G, and DMD showed the presence of a normal dysferlin band by Western blot and a positive pattern on IF. These data suggest that there is no interaction between dysferlin and these proteins. However, calpain analysis showed a weaker band in four patients from two families with intra-familial concordance. Therefore, this secondary deficiency of calpain in LGMD2B families, may indicate an interaction between dysferlin and calpain in muscle. Dysferlin was also present in cultured myotubes, in chorionic villus, and in the skin. Dysferlin deficiency was found in 24 out of a total of 166 Brazilian AR-LGMD families screened for muscle proteins (approximately 14%), thus representing the second most frequent known LGMD form, after calpainopathy, in our population.

Genetic counseling for childless women at risk for Duchenne muscular dystrophy.

The aim of the present study was to assess the impact of genetic counseling in young women at risk to have Duchenne muscular dystrophy (DMD) children prior to childbearing. A total of 263 potential DMD carriers, who had had genetic counseling and were given different genetic risks, were included in this investigation. Their reproductive outcome and future plans as well as their requests for DNA tests (for carrier detection and prenatal diagnosis) were analyzed according to genetic risk magnitude, comprehension of genetic counseling is- sues, family and personal history, socio-educational level, and subjective opinion about selective abortion. We noted that genetic risk magnitude had no significant influence on reproductive plans or outcome nor on the request for additional DNA testing, even considering only those clients with good comprehension and retention of issues discussed during genetic counseling. On the other hand, counselees who had more than one affected or at least one deceased DMD case in their family understood genetic counseling significantly better, suggesting that "learning with life" has a stronger impact than genetic counseling.

Nocturnal rhythm of growth hormone in Duchenne patients: effect of different doses of mazindol and/or cyproheptadine.

Human growth hormone (hGH) inhibition may be beneficial for Duchenne muscular dystrophy (DMD) patients and slow the rate of progression of the disease. The purpose of the present investigation was 1) to assess, before any therapeutic trial, the natural growth hormone (GH) rhythm during physiological sleep in DMD patients and in normal control boys of comparable age; 2) to evaluate the effect of different doses of two potential GH inhibitors on nocturnal GH secretion in DMD patients receiving mazindol (1-4 mg), cyproheptadine (4-8 mg), or both drugs. The results from the present investigation showed 1) wide variability in nocturnal GH secretion before medication; 2) no correlation between nocturnal GH concentration and height, age, bone age, L-dopa provocative test, or Tanner staging; and 3) no consistent effect on GH release after mazindol, cyproheptadine therapy, or combined therapy.

The facioscapulohumeral muscular dystrophy (FSHD1) gene affects males more severely and more frequently than females.

We investigated 52 families of patients with facioscapulohumeral muscular dystrophy (FSHD1), including 172 patients (104 males and 68 females). Among 273 DNA samples which were analyzed with probe p13E-11, 131 (67 males and 64 females) were shown to carry an EcoRI fragment smaller than 35 kb; 114 among them were examined clinically and neurologically. Results of the present investigation showed that: a) there is no molecular evidence for autosomal or X-linked recessive inheritance of FSHD1; b) an excess of affected males, which is explained by a significantly greater proportion of females than males among asymptomatic cases and a significantly greater proportion of affected sons than daughters observed in the offspring of asymptomatic mothers; c) the penetrance of the FSHD1 gene until age 30 was estimated as 83% for both sexes but was significantly greater for males (95%) than for females (69%); d) new mutations occur significantly more frequently in females than males among somatic/germinal mosaic cases; and e) severely affected cases originated more often through new mutations or were transmitted through maternal than through paternal lines including somatic/germinal mothers. These observations have important implications for understanding the molecular mechanisms responsible for FSHD1 and for genetic and prognostic counseling according to the gender of the affected patient.

Screening of male patients with autosomal recessive Duchenne dystrophy through dystrophin and DNA studies.

Previously we estimated that about 2.5-4% of isolated male patients diagnosed as Duchenne dystrophy (DMD) may have the autosomal recessive form (AR-DMD). Such cases can be distinguished from X-linked DMD through the analysis of dystrophin. Fifty DMD patients from 47 families were investigated for dystrophin and DNA deletions. Based on our results, we estimate that the frequency of AR-DMD may be about 8-12% among male patients diagnosed as DMD in whom X-linked inheritance could not be confirmed through pedigree data, serum enzymes in female relatives or DNA studies. Such an estimate must be confirmed in a larger sample; however, it shows the importance of assessing dystrophin in all patients diagnosed as DMD in whom X-linked inheritance cannot be proved, since the distinction between these 2 forms has implications for genetic counseling.

Severe nonspecific X-linked mental retardation caused by a proximally Xp located gene: intragenic heterogeneity or a new form of X-linked mental retardation?

X-linked mental retardation (XLMR) can be subdivided into syndromic and nonsyndromic or nonspecific. Patients with non-syndromal XLMR show no characteristic manifestations, biochemical defects, or distinct fragile sites. Nevertheless, nonspecific XLMR seems to be heterogeneous. To determine the number and location of the genes responsible for XLMR, linkage studies in large pedigrees have to be performed. Here we report the data of linkage analysis in a large Brazilian family with 7 patients affected by a severe form of XLMR, with no other associated malformations. All the obligate carriers are normal. A close linkage without recombination (lod scores 1.95 and 3.25) was found between the disease locus and polymorphic DNA loci DXS255 (Xp11.22), DXS14 (Xp11.21). These results suggest that the gene responsible for the disease in this family maps in the Xp11-cent of the X chromosome. Positive lod scores in this region have also been reported for other XLMR genealogies, but with a much milder phenotype. The possibility of intragenic or locus heterogeneity is discussed.

Is dystrophin always altered in Becker muscular dystrophy patients?

The differential diagnosis between autosomal recessive limb-girdle (LGMD) and X-linked Becker muscular dystrophy (BMD) is very important for genetic counseling. It has been hypothesized that all BMD patients would have dystrophin alterations and dystrophin analysis could identify the Xp21 MD. Qualitatively abnormal dystrophin is easily detectable, but it is generally associated with in-frame DNA deletions or duplications. In patients with no detectable DNA deletions, in which X-linked inheritance cannot be proved, dystrophin quantification is still the only available test for differential diagnosis. In order to assess the accuracy of dystrophin quantification test in delineating Becker patients, we analyzed dystrophin abundance in BMD patients with a positive history of X-linked inheritance and no DNA detectable mutation, as compared to patients from families with LGMD. We observed that patients from 2 among the 5 BMD families have nearly normal dystrophin, while alteration in dystrophin content was observed in patients from 2 among the 7 LGMD families studied (probably as a secondary effect of alteration in the whole dystrophin-glycoproteins complex). These results suggest that dystrophin quantification, as an isolated test is not helpful for differential diagnosis between BMD and LGMD.

Dystrophin immunostaining in muscles from patients with different types of muscular dystrophy: a Brazilian study.

The localization of the protein dystrophin was studied using the immunofluorescence method, in muscle biopsies from 74 patients affected by different types of muscular dystrophy and 4 normal controls. In 15 patients with limb-girdle muscular dystrophy (LGMD) the pattern was indistinguishable from normal. Among 42 Duchenne patients (DMD), 3 were totally negative and 39 showed a variable proportion (4-30%) of partially labelled fibers. With one exception 17 Becker dystrophy patients (BMD), showed a positive sarcolemmal reaction. A diffuse reaction inside the fibers, which was not observed in normal controls, was seen in the majority of DMD and also in some of the BMD patients. Based on these observations it is suggested that in DMD, a small quantity of protein is still present or there is a cross-reaction with other proteins which share some homology with dystrophin. The present results suggest that it is possible to make a differential diagnosis between DMD and BMD through dystrophin immunohistochemistry. However, to distinguish between patients with BMD and LGMD phenotypes, or DMD and outliers, complementary immunoblot studies and quantitative determination of dystrophin are necessary.

Immunofluorescence dystrophin study in Duchenne dystrophy through the concomitant use of two antibodies directed against the carboxy-terminal and the amino-terminal region of the protein.

Dystrophin immunohistochemical studies in muscle from Duchenne patients (DMD) have shown a population of fibers with partial labelling. In order to determine whether this is related to a cross reaction or to the presence of dystrophin. 22 DMD patients were studied immunohistochemically, through the concomitant use of antibodies from the N-terminal and the C-terminal regions of the protein. In 2, the reaction was negative while in 2 others 17 and 25% of fibers were positive with both antibodies. In the remainder, a population of partially stained fibers was seen: 11 were positive with both antibodies and in 7 only with the N-terminal one. Apparently, there is no correlation between the proportion of positive fibers and clinical progression, or the presence and pattern of DNA deletions in the central part of the gene. These observations led us to suggest that some truncated protein, intermediate synthesis or degradation products of dystrophin are present in muscle from Duchenne patients.

Assessment of the 50-kDa dystrophin-associated glycoprotein in Brazilian patients with severe childhood autosomal recessive muscular dystrophy.

Recently, we have demonstrated the specific deficiency of the 50-kDa dystrophin-associated glycoprotein (50DAG) in severe childhood autosomal recessive muscular dystrophy with Duchenne-like phenotype (SCARMD or AR-DLMD), a disease first reported in Tunisia and now presumed to be prevalent in North Africa and the Middle East. Here we demonstrate the deficiency of the 50DAG in one caucasoid and 5 negroid Brazilian patients with severe muscular dystrophy, which confirms that AR-DLMD with the 50DAG deficiency is not confined to the Arab populations. Without the analysis of both dystrophin and 50DAG, isolated male patients with this condition could be undiagnosed or misdiagnosed as having Duchenne or severe Becker muscular dystrophy. We also report, for the first time, the normal expression of the 50DAG and other dystrophin-associated proteins in one negroid and 2 caucasoid Brazilian patients with a phenotype indistinguishable from that of AR-DLMD with 50DAG deficiency. This is consistent with the genetic heterogeneity for the phenotype of AR-DLMD.

Sarcoglycanopathies are responsible for 68% of severe autosomal recessive limb-girdle muscular dystrophy in the Brazilian population.

Sarcoglycanopathies (SGPs) constitute a subgroup of limb-girdle muscular dystrophies (LGMD), where the primary defect in one sarcoglycan (SG) glycoprotein (alpha-SG, beta-SG, gamma-SG or delta-SG) results in a deficiency of the whole complex. Four genes, at 17q, 4q, 13q and 5q, encode the four glycoproteins, and mutations in these genes cause diseases called LGMD2D, 2E, 2C and 2F. To estimate the prevalence, relative proportions and clinical features of SGPs, we have studied the SG proteins in muscle biopsies of 140 patients (from 115 unrelated Brazilian families) with a clinical diagnosis of LGMD. Alpha-SG immunofluorescence analysis showed a positive staining pattern in 70% (80/115) of the families, a patchy pattern in 14% (16/115) and a negative pattern in 16% (19/115) of the families. All the 19 alpha-SG negative, and four of the 16 alpha-SG patchy patients were also deficient for the other three SG proteins, confirming the diagnosis of SGP in 20% of the LGMD families. None of the positive alpha-SG patients were deficient for any of the other three SG proteins, supporting the view that the SG complex functions as a unit. DNA analysis for the four sarcoglycan genes showed that alpha-SG mutations accounted for 47%, beta-SG for 16%, gamma-SG for 16% and delta-SG for 21% of the cases. SG abnormalities were observed in only 8.5% of patients with milder LGMD forms, but were present in 68% of patients with a severe Duchenne-like course. The relatively high frequency of SGP among Brazilian people with LGMD may be due to the disproportionally high frequency of African Brazilian SGP patients with the same mutation (particularly among LGMD2C and 2F patients), suggesting a founder effect. Consanguinity is also common in our SGP families.

Milder course in Duchenne patients with nonsense mutations and no muscle dystrophin.

Duchenne muscular dystrophy (DMD), a severe and lethal condition, is caused by the absence of muscle dystrophin. Therapeutic trials aiming at the amelioration of muscle function have been targeting the production of muscle dystrophin in affected Duchenne patients. However, how much dystrophin is required to rescue the DMD phenotype remains an open question. We have previously identified two exceptional golden retriever muscular dystrophy (GRMD) dogs with a milder course despite the total absence of muscle dystrophin. Here we report two unusual patients carrying nonsense mutations in the DMD gene and dystrophin deficiency but with an unexpectedly mild phenotype. Three reported polymorphisms, respectively in genes LTBP4, SPP1 and ACTN3 were excluded as possible DMD genetic modifiers in our patients. Finding the mechanisms that protect some rare patients and dogs from the deleterious effect of absent muscle dystrophin is of utmost importance and may lead to new avenues for treatment. Importantly, these observations indicate that it is possible to have a functional large muscle even without dystrophin.