Elena Pegoraro

Mutations in the sarcoglycan genes in patients with myopathy.

BACKGROUNDnSome patients with autosomal recessive limb-girdle muscular dystrophy have mutations in the genes coding for the sarcoglycan proteins (alpha-, beta-, gamma-, and delta-sarcoglycan). To determine the frequency of sarcoglycan-gene mutations and the relation between the clinical features and genotype, we studied several hundred patients with myopathy.nnnMETHODSnAntibody against alpha-sarcoglycan was used to stain muscle-biopsy specimens from 556 patients with myopathy and normal dystrophin genes (the gene frequently deleted in X-linked muscular dystrophy). Patients whose biopsy specimens showed a deficiency of alpha-sarcoglycan on immunostaining were studied for mutations of the alpha-, beta-, and gamma-sarcoglycan genes with reverse transcription of muscle RNA, analysis involving single-strand conformation polymorphisms, and sequencing.nnnRESULTSnLevels of alpha-sarcoglycan were found to be decreased on immunostaining of muscle-biopsy specimens from 54 of the 556 patients (10 percent); in 25 of these patients no alpha-sarcoglycan was detected. Screening for sarcoglycan-gene mutations in 50 of the 54 patients revealed mutations in 29 patients (58 percent): 17 (34 percent) had mutations in the alpha-sarcoglycan gene, 8 (16 percent) in the beta-sarcoglycan gene, and 4 (8 percent) in the gamma-sarcoglycan gene. No mutations were found in 21 patients (42 percent). The prevalence of sarcoglycan-gene mutations was highest among patients with severe (Duchenne-like) muscular dystrophy that began in childhood (18 of 83 patients, or 22 percent); the prevalence among patients with proximal (limb-girdle) muscular dystrophy with a later onset was 6 percent (11 of 180 patients).nnnCONCLUSIONSnDefects in the genes coding for the sarcoglycan proteins are limited to patients with Duchenne-like and limb-girdle muscular dystrophy with normal dystrophin and occur in 11 percent of such patients.

Familial Skewed X Inactivation: A Molecular Trait Associated with High Spontaneous-Abortion Rate Maps to Xq28

We report a family ascertained for molecular diagnosis of muscular dystrophy in a young girl, in which preferential activation (> or = 95% of cells) of the paternal X chromosome was seen in both the proband and her mother. To determine the molecular basis for skewed X inactivation, we studied X-inactivation patterns in peripheral blood and/or oral mucosal cells from 50 members of this family and from a cohort of normal females. We found excellent concordance between X-inactivation patterns in blood and oral mucosal cell nuclei in all females. Of the 50 female pedigree members studied, 16 showed preferential use (> or = 95% cells) of the paternal X chromosome; none of 62 randomly selected females showed similarly skewed X inactivation was maternally inherited in this family. A linkage study using the molecular trait of skewed X inactivation as the scored phenotype localized this trait to Xq28 (DXS1108; maximum LOD score [Zmax] = 4.34, recombination fraction [theta] = 0). Both genotyping of additional markers and FISH of a YAC probe in Xq28 showed a deletion spanning from intron 22 of the factor VIII gene to DXS115-3. This deletion completely cosegregated with the trait (Zmax = 6.92, theta = 0). Comparison of clinical findings between affected and unaffected females in the 50-member pedigree showed a statistically significant increase in spontaneous-abortion rate in the females carrying the trait (P < .02). To our knowledge, this is the first gene-mapping study of abnormalities of X-inactivation patterns and is the first association of a specific locus for recurrent spontaneous abortion in a cytogenetically normal family. The involvement of this locus in cell lethality, cell-growth disadvantage, developmental abnormalities, or the X-inactivation process is discussed.

Laminin α2 muscular dystrophy: Genotype/phenotype studies of 22 patients

Objective: To determine the number of primary laminin α2 gene mutations and to conduct genotype/phenotype correlation in a cohort of lamininα2-deficient congenital muscular dystrophy patients. Background: Congenital muscular dystrophies (CMD) are a heterogenous group of muscle disorders characterized by early onset muscular dystrophy and a variable involvement of the CNS. Laminin α2 deficiency has been reported in about 40 to 50% of cases of the occidental, classic type of CMD.1,2 Laminin α2 is a muscle specific isoform of laminin localized to the basal lamina of muscle fibers, where it is thought to interact with myofiber membrane receptor, such as integrins, and possibly dystrophin-associated glycoproteins.3,4 Methods: Seventy-five CMD patients were tested for laminin α2 expression by immunofluorescence and immunoblot. The entire 10 kb laminin α2 coding sequence of 22 completely laminin α2-deficient patients was screened for causative mutations by reverse transcription (RT)-PCR/single strand conformational polymorphisms (SSCP) analysis and protein truncation test(PTT) analysis followed by automatic sequencing of patient cDNA. Clinical data from the laminin α2-deficient patients were collected. Results: Thirty laminin α2-negative patients were identified (40% of CMD patients tested) and 22 of them were screened for laminin α2 mutations. Clinical features of laminin α2-deficient patients were similar, with severe floppiness at birth, delay in achievement of motor milestones, and MRI findings of white matter changes with normal intelligence. Loss-of-function mutations were identified in 95% (21/22) of the patients studied. SSCP analysis detected laminin α2 gene mutations in about 50% of the mutant chromosomes; PTT successfully identified 75% of the mutations. A two base pair deletion mutation at position 2,096-2,097 bp was present in 23% of the patients analyzed. Conclusions: Our data suggest that the large majority of laminin α2-deficient patients show laminin α2 gene mutations.

Familial prion disease with a novel 144-bp insertion in the prion protein gene in a Basque family

Three members of a Basque family carrying a novel six R2 octapeptide repeat 144-bp insertion in the prion protein gene (PRNP) showed a slowly progressive dementia associated with cerebellar signs, myoclonic jerks, and seizures. Although postmortem examination revealed only focal and minimal spongiform degeneration in one subject with a 4-year course, significant astrogliosis and neuronal loss were associated with pronounced spongiform degeneration in the patient with a duration of symptoms of 10 years. Prion protein (PrP)-immunoreactive patches with a unique morphology were present in the molecular layer of the cerebellum in both subjects. Western blot analysis demonstrated the presence of protease-resistant prion protein (PrPres) with the same characteristics (size and ratio of the three differently glycosylated isoforms) of that found in typical sporadic Creutzfeldt-Jakob disease (CJD129M/M, PrPres type 1). The amount of PrPres correlated with presence and severity of spongiform degeneration in the cerebral cortex. The findings suggest that a relatively low rate of PrPres deposition is the cause of the lack of spongiform degeneration in subjects carrying a 144-bp insertion in PRNP. The presence of PrP-immunoreactive patches with unique morphology in the molecular layer of the cerebellum is a hallmark of certain prion encephalopathies with insertional mutations and is useful in the diagnosis of this subtype of human prion disease.

Genetic and biochemical normalization in female carriers of Duchenne muscular dystrophy: Evidence for failure of dystrophin production in dystrophin-competent myonuclei

Article abstract-We studied 19 symptomatic female carriers of the Duchenne muscular dystrophy (DMD) gene. Most of these dystrophinopathy patients had had an erroneous or ambiguous diagnosis prior to dystrophin immunofluorescence testing. We assessed clinical severity by a standardized protocol, measured X-chromosome inactivation patterns in blood and muscle DNA, and quantitated the dystrophin protein content of muscle. We found that patients could be separated into two groups: those showing equal numbers of normal and mutant dystrophin genes in peripheral blood DNA (random X-inactivation), and those showing preferential use of the mutant dystrophin gene (skewed X-inactivation). In the random X-inactivation carriers, the clinical phenotype ranged from asymptomatic to mild disability, the dystrophin content of muscle was >60% of normal, and there were only minor histopathologic changes. In the skewed X-inactivation patients, clinical manifestations ranged from mild to severe, but the patients with mild disease were young (5 to 10 years old). The low levels of dystrophin (<30% on average) and the severe symptoms of the older patients suggested a poor prognosis for those with skewed X-inactivation, and they all showed morphologic changes of dystrophy. The random inactivation patients showed evidence of biochemical normalization, with higher dystrophin content in muscle than predicted by the number of normal dystrophin genes. Seventy-nine percent of skewed X-inactivation patients (11/14) showed genetic normalization, with proportionally more dystrophin-positive nuclei in muscle than in blood. In 65% of the skewed X-inactivation patients, dystrophin was not produced by dystrophin-positive nuclei; an average of 20% of myofiber nuclei were genetically dystrophin-positive but did not produce stable dystrophin. Biochemical normalization seems to be the main mechanism for rescue of fibers from dystrophin deficiency in the random X-inactivation patients. In the skewed X-inactivation patients, genetic normalization is active, but production failure of dystrophin by dystrophin-normal nuclei may counteract any effect of biochemical normalization. In the skewed X-inactivation patients, the remodeling of the muscle through cycles of degeneration and regeneration led to threefold increase in the number of dystrophin-competent nuclei in muscle myofibers (3.3 +-4.6), while dystrophin content was on the average 1.5-fold less than expected (-1.54 +-3.38). Our results permit more accurate prognostic assessment of isolated female dystrophinopathy patients and provide important data with which to estimate the potential effect of gene delivery (gene therapy) in DMD. NEUROLOGY 1995;45: 677-690

Myotonia and the muscle chloride channel Dominant mutations show variable penetrance and founder effect

The delayed relaxation or sustained contraction of skeletal muscle-myotonia--is frequently seen in myotonic dystrophy and sodium channelopathies (hyperkalemic periodic paralysis, paramyotonia congenita). Many cases of congenital myotonia without other clinical symptoms have been associated with mutations in the muscle chloride channel gene. Most cases reported to date show a recessive inheritance pattern, with loss of function of the corresponding protein. Six families have been reported with dominantly inherited myotonia and mutations of the chloride channel gene. Here we report clinical and molecular data on 38 family members from four new families with dominantly inherited myotonia congenita. Three families show a previously characterized G230E mutation, and we show that these three share a common affected ancestor despite living in different regions of the United States (linkage disequilibrium). One Italian family is shown to have a novel dominant mutation--I290M. This is the sixth mutation identified in Thomsens myotonia. Genotype/phenotype correlations in these four families showed that both of the dominant mutations resulted in a mild clinical picture in 90% of the patients, and no symptoms in 10% of mutation-positive patients. The EMG was the clinical feature that most closely correlated with mutation data; however, 3 of 16 (19%) mutation-positive patients tested negative by electromyography at least once, and 1 (6%) tested negative despite multiple tests. Only about half (55%) of the mutation-positive patients tested positive for percussion myotonia. Most of the clinically symptomatic individuals stated that cold temperatures and stress substantially worsened their myotonia. Our data show that dominantly inherited Thomsens myotonia is most often a very mild disorder that shows considerable clinical heterogeneity. NEUROLOGY 1996;47: 963-968

Frequency of LGMD gene mutations in Italian patients with distinct clinical phenotypes.

Background: The frequency of various limb-girdle muscular dystrophy (LGMD) molecular diagnoses has previously been investigated only in cohorts of patients presenting LGMD phenotype. Methods: A total of 550 muscle biopsies underwent multiple protein screening (including calpain-3 functional assay) and extensive gene mutation analysis to examine the frequency of LGMD subtypes in patients with distinct clinical phenotypes (severe childhood-onset LGMD, adult-onset LGMD, distoproximal myopathy, and asymptomatic hyperCKemia). Results: The percentage of molecularly ascertained cases directly relates with the degree of clinical involvement: 60% of total LGMD (77% of childhood-onset, 46% of adult-onset, 66% of distoproximal myopathy) and 14% of hyperCKemia. The higher number of molecular diagnoses in severe phenotypes might suggest that genes selected for our screening are those more frequently associated with severe LGMD, and that the hyperCKemia group includes heterogeneous diagnoses. The probability of obtaining a molecular diagnosis increases when a protein defect is found in a muscle biopsy: in such cases, we diagnosed 87% of LGMD and 76% of hyperCKemia. Conclusions: Diagnosing 77% of childhood-onset limb-girdle muscular dystrophy (LGMD) and 60% of total LGMD is an important result. The missing identification of gene mutations in about 40% of patients with typical LGMD phenotype suggests that unknown genetic or nongenetic etiologies are still to be recognized. Dysferlin, caveolin-3, and emerin protein defects invariably corresponded to primary disorders (100%), whereas a lower correlation was found for sarcoglycans (77%) and calpain-3 (84%). The different efficiency of genetic diagnosis after the identification of a protein defect in the various disorders is possibly due to different pathogenetic effects of mutations.

Cardiac transplantation in a Duchenne muscular dystrophy carrier

We report here for the first time the case of a symptomatic DMD carrier, who had a heart transplant for a severe dilated cardiomyopathy. Dystrophin immunohistochemistry, western blot and analysis of X-chromosome inactivation on leucocytes, and skeletal and cardiac muscle biopsies on the explanted heart were performed. The patient was a heterozygote for exons 50-52 deletion in the dystrophin gene. The number of dystrophin-deficient fibres in the heart was much higher than in skeletal muscle. On the other hand, the explanted heart showed a non-skewed pattern of X-chromosome inactivation, as in leukocytes and skeletal muscle. The adverse cardiac course may be explained by the absence of regeneration among cardiomyocytes.

X‐inactivation patterns in female Leber's hereditary optic neuropathy patients do not support a strong X‐linked determinant

Lebers hereditary optic neuropathy (LHON) accounts for about 3% of the cases of blindness in young adult males. The underlying mitochondrial pathogenesis of LHON has been well studied, with specific mitochondrial DNA (mtDNA) mutations of structural genes described and well characterized. However, enigmatic aspects of the disease are not explained by mutation data, such as the higher proportion of affected males, the later onset of the disease in females, and the presence of unaffected individuals with a high proportion of mutant mtDNA. A hypothesis which has been put forward to explain the unusual disease expression is a dual model of mtDNA and X-linked nuclear gene inheritance. If a nuclear X-linked modifier gene influences the expression of the mitochondrial-linked mutant gene then the affected females should be either homozygous for the nuclear determinant, or if heterozygous, lyonization should favor the mutant X. In order to determine if an X-linked gene predisposes to LHON phenotype we studied X-inactivation patterns in 35 females with known mtDNA mutations from 10 LHON pedigrees. Our results do not support a strong X-linked determinant in LHON cause: 2 of the 10 (20%) manifesting carriers showed skewing of X-inactivation, as did 3 of the 25 (12%) nonmanifesting carriers.

Steroids in muscular dystrophy: Where do we stand?

1. IntroductionThe promise of gene therapy for Duchenne musculardystrophy (DMD) is awaiting future developments in thefield of molecular biology. Moreover, gene replacement orrestitution of dystrophin by means of this technique may notrepresent a cure for the disease. Gene therapy in the treat-ment of muscular dystrophy has met a number of obstacles,including immune reaction to viral vectors, difficulty inpenetrating through the basal lamina and limited spread ofaction to a relatively small number of muscle fibres.Meanwhile, the utilisation of drugs capable of producinga benefit by slowing down the course of the disease seems toafford an alternative path that clinical research should fol-low over the next few years. So far, the only drugs shown toproduce a slowing in the downhill course of the disease havebeen the steroids.Since the first observations by Drachman [1], whenimprovement was originally documented, several reportshave provided positive data on the use of glucocorticoidsin DMD and have already been reviewed up to 1991 in aneditorial paper in this journal [2]. Later reports are includedand summarised in Tables 1, 2 and 3, but a critique of eachstudy is beyond the scope of this current clinical practicereview.More recently, during the 47th International ENMCWorkshop experiences on the use of diverse steroid regimeswere presented by authors from seven countries [16]. Themore relevant conclusions were (a) that there was evidenceof potential value of steroids in DMD treatment, (b) thatfurther multicentric studies following agreed protocolswere essential and (c) that a wide diversity of specific ques-tions had to be answered. Some aspects related to currentknowledge, experience and opinions concerning the use ofsuch therapeutic agents in Duchenne dystrophy are the sub-ject of this clinical-practice review.The mechanisms through which steroids produce clinicalbenefit have not yet been definitively established but thoserelated to protein synthesis and turnover in the muscle fibre,or even those involved in immunological regulation deserveconsideration.Although we have accumulated interesting experiencewith the use of corticosteroids in the last few years, severalquestions regarding the common practice of this treatmentremain unanswered. For some authors, the risks involved inthe long-term use of steroids still preclude their administra-tion, for others, the option is clear but some issues are still amatter of discussion, such as the best patient age to start thetreatment, when withdrawal is appropriate and whether it isworth using the medication for wheel-chair-bound patients.2. Mechanisms involved in steroid action in musculardystrophyOn the basis of the cumulative data summarised in Table1, 2 and 3, there seems to be little doubt about the usefulnessof steroids in DMD but the mechanisms of action involvedin clinical benefit are still a matter of speculation. Fromwhat has been observed in clinical trials, steroids may in-crease muscle mass, since there is an increase in creatinineexcretion [5] in prednisone-treated patients.Further studies on prednisone-treated DMD/Becker mus-cular dystrophy (BMD) patients have suggested that bene-ficial drug effect on strength appears to be related to aninhibition of muscle proteolysis rather than stimulation ofprotein synthesis. In treated patients, the 3-methylhisti-dine–creatinine excretion ratio decreased by 26% [17].